From In Silico to In Vivo: Characterizing Ag-RBDN331-V524 for Effective COVID-19 Vaccine Development

From In Silico to In Vivo: Characterizing Ag-RBDN331-V524 for Effective COVID-19 Vaccine Development | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article From In Silico to In Vivo: Characterizing Ag-RBDN331-V524 for Effective COVID-19 Vaccine Development Elmira Ranjbar Zeidi, Solaleh Javadi, Pouya Farokhi, Vahid Siavashi, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5388446/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract COVID-19 has become a challenge of the century to the healthcare system. One of the best targets to produce the COVID-19 vaccine is the receptor binding domain (RBD) which is located in the Spike protein of Coronavirus. This domain is extremely conserved among different variants of COVID-19. In this study, the most potent region of RBD was selected to design a vaccine against COVID-19 in E. coli as an expression system. The shuttle vector was applied to express the vaccine construct in E. coli . The primary vector is a backbone plasmid pBluescriptIISK (+) and a Tn5 transposon is a secondary vector of 1657 bp inside. The C-phycocyanin operon including the gene cassette was embedded in Tn5. The quantitation of total protein was done by Bradford assay. Then, SDS-PAGE and Western Blot were carried out to characterize and confirm recombinant protein expression. Affinity chromatography was performed for the purification of recombinant protein. The molecular weight of the RBD protein was 34 kDa which is compatible with western blot results. The aim of this study was expression of RBD domain in E. coli which could apply in a future study to the production of vaccine against COVID-19 based on a host that has ideal C-phycocyanin expression. The selected RBD sequence has a complete identity to the newest variant. The short length of the sequence selected in this study leads to increased solubility and decreased allergenicity. On the contrary, this trait has led to a decrease in the probability of mutation, which can cover new variants of this virus. Biological sciences/Drug discovery Biological sciences/Genetics Biological sciences/Immunology Biological sciences/Molecular biology SARS-CoV-2 COVID-19 RBD Vaccine Recombinant Protein E. coli Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus-2) which is well-known as COVID-19 has been introduced by the World Health Organization (WHO) as the causative agent of the COVID-19 pandemic. The COVID-19 epidemic began in December 2019 in Wuhan, Hubei Province, China, when patients with pneumonia were diagnosed with symptoms such as fever, cough, dyspnea, and myalgia (He, Deng and Li 2020 ). Statistics show by 11 February 2022 404,910,528 confirmed cases of COVID-19, including 5,783,776 deaths reported to WHO (Zheng, Shao et al. 2022). In recent years, before the recent pandemic, two outbreaks have been caused by coronaviruses. In 2002, the coronavirus caused the SARS disease in China and 28 countries (Cherry 2004 ). Also in 2012, another coronavirus which is called MERS-CoV (Middle East Respiratory Syndrome-related Coronavirus) was first reported in Saudi Arabia (Chan, Lau and Woo 2013 ). COVID-19 is a member of the genus Beta coronavirus which is recognized as a positive-sense, enveloped, and single-stranded RNA virus (Artika, Dewantari and Wiyatno 2020 ). The genome of the COVID-19 virus is 79% similar to SARS and 50% similar to MERS-CoV (Lu, Zhao et al. 2020). The COVID-19 virus is transmitted from person to person through direct contact or respiratory droplets (Li, Guan et al. 2020). Coronavirus is composed of four structural proteins including nucleocapsid (N), membrane (M), envelope (E), and spike (S) proteins (Deng, Li et al. 2021 ). The COVID-19 virus enters the cell by binding Spike (S) glycoproteins to the ACE2 (angiotensin-converting enzyme2). The ACE2 receptors are abundant in human alveolar epithelial cells (AECs), which is why the lungs are considered one of the most vulnerable organs in the body when the COVID-19 coronavirus enters (Gibson, Evans et al. 2020 ). Glycoprotein S is responsible for the entry of virus particles through attachment to the host cell membrane and fusion. This protein is composed of two subunits, S1 and S2, which are involved in binding to the receptor and fusion to the cell membrane, respectively (Belouzard, Millet et al. 2012 ). Attachment of the S1subunit to the ACE2 as the receptor is carried out through the receptor binding domain (RBD) of the S1 subunit. SARS-CoV-2 RBDs a key part of a virus located on its spike domain that allows it to bind to ACE2 with an affinity in the low nanomolar range to gain entry into cells and lead to infection (Tian, Li et al. 2020 , Walls, Park et al. 2020). S2 subunit of the S protein participates in the fusion of the virus membrane with the host cell. Therefore, S protein is a target in the development of antibodies, inhibitors, and vaccines (Gangadevi, Badavath et al. 2021). Researchers have developed different vaccine platforms to prevent COVID-19, including live attenuated virus, inactivated virus, subunit vaccines, mRNA, viral vectors, recombinant DNA, and protein vaccines (Belete 2020 ). As using live, inactivate or subunit vaccine of COVID-19 are usually associated with immune problems, finding the most potent region of virus which could activate immune response against different variants of Covid-19 is massively being investigated. Data showed that RBD or recombinant RBD region could effectively induce immune response without any side-effect compared to using fully length if s protein or recombinant form which caused liver damage and enhance infection (Du, He et al. 2009). Also, Y. He & Jiang ( 2005 ) reported multiple neutralizing antibodies against RBD region in SARS which means that this region is highly potent to activate immune response (He and Jiang 2005 ). Du et al. 2009 found a concave region in RBD region of SARS which contains 14 amino acids and could interact with ACE2 receptor (Du, He et al. 2009). Many items affect the yields of every recombinant protein production. Promoters are one of the key regulatory elements which control the level of recombinant protein expression in the host (Abdali, Kazaemitabar et al. 2022). The phycocyanin-specific promoter sequence, which is the strongest promoter in Spirulina ( Arthrospira platensis ) and 20–50% of the spirulina proteins weight, is considered in this study. So, we selected a highly conserved region of RBD protein to expressed in E. coli expression system. After purification of this protein by chromatography, we analyzed the efficacy of this selected region by using western blot. The aim of this study was expression of RBD domain in E. coli which could apply in a future study to the production of vaccine against COVID-19 based on a host that has ideal C-phycocyanin expression. 2. Materials and Methods 2.1. Selecting a region of the RBD domain As the RBD region of SARS-CoV2 is the most potent region of spike protein which plays a critical role in interaction with neutralized antibodies, this region was selected to design a vaccine construct. 2.1.1. Antigenicity, allergenicity, and physicochemical properties assessment The antigenicity of the vaccine construct was evaluated by the VaxiJen webserver ( https://www.ddg-pharmfac.net/vaxijen/VaxiJen/VaxiJen.html ) with a threshold of 0.4. The allergenicity was predicted by Algpred ( http://crdd.osdd.net/raghava/algpred/ ) and Allertop ( https://www.ddg-pharmfac.net/AllerTOP/method.html ) webserver. The Algpred webserver used different algorithms such as mapping IgE epitope, searching MEME/MAST allergen motifs using MAST, SVM modules by applying amino acid or dipeptide composition and Blast search against 2890 allergen-representative peptides (ARPs) to illustrate the potential of protein allergenicity. The Allertop webserver anticipated the allergenicity of protein sequence based on the auto cross-covariance (ACC) method. The physicochemical properties of the construct were evaluated by Protparam ( https://web.expasy.org/protparam/ ) webserver. The stability of the construct was determined by an instability index value. An instability index of less than 40 means the stability of the protein sequence. The aliphatic index showed thermo stability of the protein sequence. More aliphatic index value indicates more thermo stability of a protein. The Gravy index illustrated the hydropathicity of protein. Less Gravy index means more hydrophilicity of protein. 2.1.2. Protein solubility assessment The solubility of the vaccine construct is essential which increases the vaccine accessibility to the immune system. For this reason, several protein solubility web servers were applied to check design vaccine construct solubility compared to the RBD domain. The protein-sol webserver estimates the solubility of protein based on a sequence of the protein (Hebditch, Carballo-Amador et al. 2017 ). This web server calculates protein solubility by 35 sequence features. Proteins that have a solubility value over 0.45 predicts to have a higher solubility than the average solubility of E. coli protein from the experiment dataset. Soluprot web application uses a machine learning algorithm and employs 96 features of protein to predict the solubility of the protein in the E. coli expression system by protein sequence (Hon, Marusiak et al. 2021). A value of more than 0.5 indicates good solubility of the protein. SoDoPE (Soluble Domain for Protein Expression) is a web tool for the solubility prediction of proteins by applying Solubility-Weighted Index (SWI) which is derived from crystallographic B-factors (Bhandari, Gardner and Lim 2020 ). The web server used a sequence of the protein and designates solubility by the score. The higher solubility value shows better solubility of the protein. Camsol webserver could predict the solubility of protein by protein structure with three different algorithms (Sormanni, Amery et al. 2017 ). The Camsol value larger than 1 donates higher soluble regions while the Camsol value smaller than − 1 specifies lower solubility. Aggrecan web server predicts the potency of globular protein to aggregate by computing the aggregation propensity of each amino acid. A lower Aggrescan value means less potential to aggregate and better water solubility. 2.1.3. Homology modeling As the tertiary structure of vaccine construct is essential in inducing humoral immunity, the structure of selected RBD region was predicted by Robetta webserver ( https://robetta.bakerlab.org/ ). 2.2. Sequence optimization and constructs design To optimize the expression of selected RBD region of COVID-19 in E. coli , codon optimization was performed based on the Biomatik Codon Optimization Analysis platform, which can optimize multiple critical parameters to stabilize DNA fragments and improve gene expression efficiency. These parameters include but are not limited to Codon usage bias, GC content, Premature polyA sites, Internal chi sites, ribosomal binding sites, RNA instability motif (ARE), mRNA secondary structure, CpG dinucleotides content, repeat sequences (direct repeat, inverted repeat, and dyad repeat), Codon usage bias. The shuttle vector was designed by the composition of the primary vector as a backbone plasmid (pBluescriptIISK (+)) with a secondary vector of 1657 bp inside. The construct was synthesized by Biomatik Company (Canada) with its sequence confirmation. 2.3. Transformation The E. coli TOP10 competent cell was prepared using optimized CaCl 2 method of Sambrook 1989. The final competent cells were kept at -80 ºC until use (Sambrook, Fritsch and Maniatis 1989 ). To transformation, melting component was mixed with added 5µl of plasmid. Then, one cooling step: 30 min on ice, then one heating step: 2 min in water bath at 42°C and again one cooling step: 5 min on ice were performed, respectively. After that, 600µl of LB Broth was added to the sample and incubated at 37°C with 150 rpm for 1 hour. The solution containing the transgenes was relocated to LB agar with Ampicillin in dilutions, 1/10, 1/100, 1/1000 and 1/10000. Plates were incubated at 37°C for 18–24 hours. 2.4. Quantitation of total protein and purified RBD The Bradford assay was used for the measurement of the quantitation of total protein (Bradford 1976). The confirmation of expression of the protein was carried out initially by SDS-PAGE and subsequently western blot. 2.4.1. Western blot analysis The bacteria were lysed by using a lysis Buffer solution (1% Triton X-100, 50 mM Tris-HCl, 150 mM NaCl, 0.25% sodium deoxycholate, 1 mM Ethylene glycol tetraacetic acid, and 1 mM NaF). Bradford (Cat No: DB0017, DNAbiotech; Iran) assay was conducted for measurement of protein quantitation. The lysates were boiled for 5 min. then, 25 µg lysates were applied to SDS-PAGE 12%. The protein band was then transferred to a 0.2 µm Immune-Blot™ polyvinylidene difluoride (PVDF) membrane (Cat No: 162-017777; Bio-Rad Laboratories, CA, USA). The 5% BSA with 0.1% Tween 20 (Cat No: A-7888; Sigma Aldrich, MO, USA) was utilized to block the membranes for 1 h. The membranes were then incubated together with HRP Anti-6X His tag® (Cat. No: ab1187 -Abcam) as a secondary antibody at room temperature for 1 h. Ultimately, the membranes were located in an incubator with enhanced chemiluminescence (ECL) for 1–2 minutes (Asadian, Siavashi et al. 2018, Asadian, Alibabrdel et al. 2019, Abdali, Kazaemitabar et al. 2022). 2.4.2. Affinity Chromatography The recombinant protein’s recovery and purification were conducted using affinity chromatography through its hexa histidine tag. The protocols were optimized, based on the QIA expressionist ™ (QIAGEN 2003 , Katalani, Ahmadian et al. 2020, Katalani, Nematzadeh et al. 2020, Abdali, Kazaemitabar et al. 2022). 3. Results 3.1. Selected RBD region Multiple sequence alignment of the RBD region among COVID-19 and its variants revealed that the RBD region is highly conserved among its variants. To improve the potency of antigenicity of the RBD region, some amino acid sequences were removed from both the N and C terminals of protein (Fig. 1 ). 3.1.1. Physiochemical, aggregation and, solubility assessment The antigenicity and allergenicity of the construct compared to the RBD region were analyzed by the VaxiJen webserver. Data showed that construct had a high antigenicity value (0.5822). Allergenicity assessment with Allertop webserver illustrated the construct had no allergenicity compared to RBD domain. Although, construct showed probable allergenicity by the Algpred web server but its allergenic effect was lower than RBD region (Table 1 ). Physicochemical properties assessment of the construct showed that although the construct had more instability index (29.91) than the RBD region while it has a more aliphatic index which means this construct had more thermos-stability than the RBD region. No significant changes were observed based on hydrophilicity by Gravy index. The half-life analysis illustrated the half-life of construct increased in three different expression systems compared to the RBD region especially, in E. coli (Table 2 ). Table 1 Antigenicity and allergenicity prediction of construct compared to RBD by Algpred webservers Model VaxiJen* Allertop Algpred algorithms Prediction by mapping of IgE epitope MAST Prediction by SVM method based on amino acid composition** Prediction based on SVM method based on dipeptide composition ** * Blast RBD 0.4952 probable allergen non-allergen non-allergen potential allergen score = 0.96 allergen score = 0.066 non-allergen Construct 0.5822 non-allergen non-allergen non-allergen potential allergen score = 0.83 allergen score = 0.058 non-allergen *Threshold more than 0.4 means potential of antigenicity **Threshold is −0.4 ***Threshold was −0.2 Table 2 Physicochemical assessment of construct compared to RBD of SARS-Cov2. Model MW Protparam Instability index Aliphatic index Gravy Half-life in mammalian Half-life in yeast Half-life in E.coli PI RBD 25098 22.69 71.61 -0.259 1 h 2 min 2 min 8.91 Construct 22115 29.91 74.85 -0.216 1.4 h 3 min > 10 h 8.79 3.1.2. Solubility assessment The solubility of the construct was assessed by the different web servers. Data showed that the solubility of the RBD domain is slightly better than Construct by Protein-sol and Soluprot while according to Camsol result, construct had better water solubility in a wide range of pH compared to the RBD domain. No difference was observed between the two proteins by the SoDoPE webserver. Aggregation analysis of protein based on sequence and protein structure by Aggrescan webserver revealed that although aggregation assessment based on protein sequence showed less potential of aggregation by RBD while a result of aggregation based on protein structure did not show any differences between these two protein structures (Table 3 ). Table 3 Evaluation of solubility of construct and RBD by a different web server Model Protein-sol Soluprot SoDoPE Camsol in pH range Aggrescan sequence structure sequence structure RBD 0.538 0.76 0.24 -0.87 -0.87 5.1 -76.8 Construct 0.461 0.67 0.2 -1.07 -1.07 8.1 -76.7 *Higher value means higher water solubility ** Less value means more solubility and less aggregation 3.1.3. Tertiary structure analysis To find out if this amino acid sequence reduction in construct led to no changes in RBD tertiary structure, homology modeling by Robetta webserver was performed (Fig. 2 (A)). structure alignment of design construct with S protein of Covid-19 and the RBD region of SARS-CoV2 variant Omicron showed that the construct have not illustrated any changes in the tertiary structure (Fig. 2 (B)). Also, structure alignment of design construct with Omicron RBD region which in interaction with the Antibody illustrated no structure changes in the designed construct compared to the RBD domain. This result means that the designed construct could potentially interact with neutralized antibody (Fig. 2 (C)). 3.2. Codon optimization and Vector design Codon usage optimized based on E. coli as cloning host. The optimization significantly improves the expression levels of genes by strategically optimizing the underlying DNA sequence. The native gene has several features which may lead to poor expression. The gene has been improved by applying a set of carefully selected design criteria which improve the expression. The codon bias has been adapted to the preferred codon usage of the target organism, E. coli. The squared difference of the actual codon frequency vs. the preferred codon frequency has been changed from 3.60 to 5.87. The GC content throughout the sequence has been homogenized, in order to increase the half-life of the mRNA. The mRNA secondary structure has been reduced. The codon optimizing resulted in changing some factors like GC content, Secondary structure, etc. (Fig. 3 & Table 4 ) Table 4 Results of some variation in sequence of genes after codon usage optimizing GENES Changing Original sequence Optimized sequence RBD Amino acid position *CAI: 0.76 Squared difference: 3.60 CAI: 0.96 Squared difference: 5.87 GC content adjustment 35% 43% Secondary structure: hairpin stem motifs 554 476 GFP Amino acid position *CAI: 0.73 Squared difference: 3.19 CAI: 0.97 Squared difference: 5.12 GC content adjustment 38% 41% Secondary structure: hairpin stem motifs 649 602 * The distribution of relative adaptiveness (where the most frequent codon scores 100%) along the length of the gene sequence. A CAI of 1.0 is considered to be perfect in the desired expression organism, and a CAI of > 0.9 is regarded as very good, in terms of high gene expression level Vaccine construct was inserted in the secondary vector from 5' to 3' in position where EcoRI was cleavage site, ME (mosaic end) left sequence, c-Phycocyanin operon (C-PC promotor, Ribosomal Binding Site (RBS) sequence, respectively, gene locus 1 (RBD-Spike-COVID-19 sequence), gene locus 2 (GFP molecular marker) and terminator sequence), ME Right sequence and HindIII cleavage site (Fig. 4 ). Moreover, enzymatic digestion based on two EcoRI and HindIII sites was performed to reconfirm the synthesized structures. 3.3. Transformation The transformation was initially evaluated by culturing transformed E. coli in an Amp + medium. 3.3.1. Purification of expressed protein A C-phycocyanin promoter was used to express a recombinant protein in E. coli . The best time and temperature for purification were determined based on GFP expression at different times and temperatures under the blue light of fluorescent microscope was applied (Fig. 5 ). Data showed that the best temperature was 37 ο C and 16 hours after transformation. 3.3.2. SDS-PAGE and Western blot SDS-PAGE was applied to initially proof the expression of construct in transgenic E. coli . Western blot was done to approve the RBD expression in transgenic bacteria based on anti-His-Tag antibody (Fig. 6 & Fig. 7 ). The prediction of Ag-RBD size was 22 kDa. In SDS-PAGE there was a band at 22 kDa for inclusion body phase, while in soluble phase of SDS-PAGE and in both solution phase and inclusion phase in western blot analysis the band was located in 34 kDa. 3.3.3. Total protein solubility The results of Bradford assay demonstrated that the concentration of total protein in the soluble phase was 2 to 3 times more than that of the inclusion body phase (1265 µg/ml in the soluble phase vs. 720 µg/ml in the inclusion body phase). (Table 5 ) Table 5 Total protein concentration in solution phase (Rs) and inclusion phase (R) in lysed samples prepared for chromatography Sample Protein concentration quantitation (µg/ml) R 720 Rs 1265 4. Discussion The COVID-19 virus spread a pandemic that affected many people all over the world. Statistics shows that millions of people are infected and die because of COVID-19. Also, there is a lot of pressure on the health care system (Ciotti, Ciccozzi et al. 2020). The use of various drug therapies to cure patients with COVID-19 has not been very successful due to the lack of effective drug therapy (Malek, Bill and Vines 2021 ). Therefore, scientists sought to develop an effective vaccine to create immunity against this disease. Various types of vaccine platforms was developed such as live attenuated virus, inactivated virus, subunit vaccines, viral vectors, recombinant DNA, and protein vaccines (Dutta 2020 ). S-protein of COVID-19 virus is an important part of virus which involved in the attachment of virus to the host cell. The RBD is the main region of S-protein which directly bind to receptor of host cell in order to entrance to cell. Immunity problems are present with the models of live or inactivated or subunit viruses, because they may elicit inflammatory or immune responses. Among all the studied regions of the COVID-19 genome, the best region for vaccine design is the RBD domain (Wang, Wang et al. 2021 ). Considering the start of this project in the first days of the pandemic, RBD was selected in the first step based on the compatibility of the available sequences from SARS (NP_828851.1) and COVID-19 (YP_009724390.1). As the second step, articles on the allergen status of SARS Subunit vaccines were reviewed (He and Jiang 2005 ). In the next step, the prediction of allergens and identification of epitopes were checked on the AlgPred website. The exact position of the sequence containing the RBD was finalized (RBD N331−V524 ) based on proper folding. The purpose of this study was expression of recombinant protein RBD to design an effective vaccine against COVID-19. Evaluation showed that removing some amino acids from N and C terminal parts of RBD region improved antigenicity and reduce its allergenicity without changing its solubility and the half-life of designed construct became higher in E. coli . The tertiary structure prediction showed no structural changes in the RBD-Ag. According to the results of Western blot analysis, a special band in size of 34kDa was indicated. While as predicted by the software, the size of the RBD-Ag of S protein should normally be about 12KDa. Consistent with Yang et al. 2020 , the molecular weight of the RBD protein was determined around 34 kDa, which was about a quarter more than the molecular weight estimated using the RBD amino acid sequence alone (~ 27 kDa). They suggested that the RBD is densely glycosylated(Yang, Wang et al. 2020). Although this explanation was acceptable considering the protein expression host, which was insect cells, the expression host in the present study is bacteria. So, this justification becomes questionable. The difference in MW ,in eukaryotes, is attributed to chemical modifications of the protein, especially glycosylation which impress gel mobility shift (Goldstein, Scheid et al. 1975, Stanley 2011 ), but here RBD could not be glycosylated in E.coli and this rules out the possible contribution of glycosylation to the observed MW difference. It was investigated that amino acid composition can cause the difference between the predicted and western blot displayed MW (Guan, Zhu et al. 2015). In another similar study, the expression of the S-RBDN318-V510 protein was investigated in E. coli , the results of showed that the molecular weight of the S-RBDN318-V510 protein was approximately 31 KDa. They indicated the overall yields of approximately 1.5 mg of pure S-RBD N318−V510 per liter .(Márquez-Ipiña, González-González et al. 2021). Our data showed that the concentration of total protein in the soluble phase is more than that of the inclusion body phase. Compared to the aforementioned study, our expression, based on the area and intensity of the Western blot band, is low. Although in the present research, codon usage was optimized for E.coli , the expression level remains low, similar to our previous study that focused on the expression of the hepatitis B antigen under the C-phycocyanin promoter in bacteria (Abdali, Kazaemitabar et al. 2022). In fact, it can be claimed that the low expression in the previous study, which had been attributed to codon usage, is not accurate. The results of the present study and the previous one indicate the low efficiency of this algal promoter in bacteria. 5. Conclusion The result indicated although the c-phycocyanin promoter is considered a super-promoter in cyanobacteria, particularly in Spirulina , it does not exhibit strong performance in bacteria. Taken together, this study showed that the RBD region was successfully expressed in E. coli . This means that this vaccine construct could apply in a future study to order to production of vaccine against COVID-19. On the other hand, the size of the antigen protein RBD according to our observation and the results of SDS-PAGE and Western blot analysis was 34 kDa. Hence, the expression of RBD protein was successful in E. coli which could apply in a future study to order to production of vaccine against COVID-19. Abbreviations RBD receptor binding domain C-PC C-phycocyanin SARS-CoV-2 Severe Acute Respiratory Syndrome Coronavirus-2 ACE2 angiotensin-converting enzyme2 O.L.PHB Oral cell-loaded PHB. PHB that is loaded with Spirulina for oral administration O.U.PHB Oral unloaded PHB I.Vac. Injected Vaccine. SpikoGen™ COVID-19 vaccine administered via injection C- The negative control group without any treatment ALT Alanine aminotransferase AST Aspartate aminotransferase IFN-γ Interferon-gamma IL-4 Interleukin 4 MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] reduction assay Declarations Author Contribution All authors contributed to the study. Elmira Ranjbar Zeidi: Writing – original draft, Methodology, Investigation, Data curation. Solaleh Javadi: Writing – original draft, Methodology, Investigation, Data curation. Pouya Farokhi: Writing – original draft, Methodology, Investigation, Data curation. Vahid Siavashi: Writing – review & editing, Funding acquisition. Ali Ahmari: Writing – review & editing, Methodology, Investigation. Ladan Mafakher: Writing – review & editing, Supervision, Formal analysis. Camellia Katalani: Formal analysis, Data curation. Alaleh Maleki: Formal analysis, Data curation. Nargess Abdali: Writing – review & editing, Visualization, Validation, Supervision, Software, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. Reza Tabaripour: Writing – review & editing, Visualization, Validation, Supervision, Software, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. Conflict of Interest The authors declare no conflict of interest, financial or otherwise. Funding statement No funding was received for conducting this study. Ethical Approval This declaration is “not applicable” with headings. Data Availibility All data generated or analyzed during this study are included in this published article. References Abdali, N. et al. Evaluation of Phycocyanin Promoter Function in Bacteria by Investigating the Expression of HBsAg. Egypt. J. Veterinary Sci. 53 (1), 99–109 (2022). Artika, I. M., Dewantari, A. K. & Wiyatno, A. 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Pharmacother. 144 , 112276 (2021). Márquez-Ipiña, A. R. et al. and G. Trujillo-de Santiago Serological test to determine exposure to SARS-CoV-2: ELISA based on the receptor-binding domain of the spike protein (S-RBDN318-V510) expressed in Escherichia coli. Diagnostics 11(2): 271. (2021). QIAGEN. The QIA expressionist ™ - A handbook for high-level expression and purification of 6xHis-tagged proteins. (2003). Sambrook, J., Fritsch, E. F. & Maniatis, T. Molecular cloning: a laboratory manual (Cold Spring Harbor Laboratory Press, 1989). Sormanni, P., Amery, L., Ekizoglou, S., Vendruscolo, M. & Popovic, B. Rapid and accurate in silico solubility screening of a monoclonal antibody library. Sci. Rep. 7 (1), 1–9 (2017). Stanley, P. Golgi glycosylation. Cold Spring Harb Perspect. Biol. 3 (4). (2011). Tian, X. et al. Potent binding of 2019 novel coronavirus spike protein by a SARS coronavirus-specific human monoclonal antibody. Emerg. microbes infections . 9 (1), 382–385 (2020). Walls, A. C. et al. Structure, function, and antigenicity of the SARS-CoV-2 spike glycoprotein. Cell 181 (2), 281–292 (2020). e286. Wang, Y., Wang, L., Cao, H. & Liu, C. SARS-CoV‐2 S1 is superior to the RBD as a COVID‐19 subunit vaccine antigen. J. Med. Virol. 93 (2), 892–898 (2021). Yang, J. et al. A vaccine targeting the RBD of the S protein of SARS-CoV-2 induces protective immunity. Nature 586 (7830), 572–577 (2020). Zheng, C. et al. Real-world effectiveness of COVID-19 vaccines: a literature review and meta-analysis. Int. J. Infect. Dis. 114 , 252–260 (2022). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5388446","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":390683676,"identity":"fe28690d-0c55-434f-af06-b13b7ca66492","order_by":0,"name":"Elmira Ranjbar Zeidi","email":"","orcid":"","institution":"Razi Herbal Medicines Research Center, Lorestan University of Medical Science","correspondingAuthor":false,"prefix":"","firstName":"Elmira","middleName":"Ranjbar","lastName":"Zeidi","suffix":""},{"id":390683677,"identity":"d024fb63-0e57-4cee-94a0-4ef70a259d97","order_by":1,"name":"Solaleh Javadi","email":"","orcid":"","institution":"Department of Biotechnology, Advanced Science and Technology, Tehran Medical Sciences, Islamic Azad University","correspondingAuthor":false,"prefix":"","firstName":"Solaleh","middleName":"","lastName":"Javadi","suffix":""},{"id":390683678,"identity":"f9d65251-3679-4bed-ba58-e34216a67976","order_by":2,"name":"Pouya Farokhi","email":"","orcid":"","institution":"Pharmaceutical Ward, Laleh Hospital","correspondingAuthor":false,"prefix":"","firstName":"Pouya","middleName":"","lastName":"Farokhi","suffix":""},{"id":390683679,"identity":"015d8349-3ef8-453f-97bf-52a4ab8d28ce","order_by":3,"name":"Vahid Siavashi","email":"","orcid":"","institution":"Department of Clinical Pathology, Faculty of Veterinary Medicine, University of Tehran","correspondingAuthor":false,"prefix":"","firstName":"Vahid","middleName":"","lastName":"Siavashi","suffix":""},{"id":390683680,"identity":"afde4ace-9a06-4f86-8a55-3f486b6536d3","order_by":4,"name":"Abolfazl Nikpour","email":"","orcid":"","institution":"Department of Microbial Biotechnology, Amol University of Special Modern Technologies","correspondingAuthor":false,"prefix":"","firstName":"Abolfazl","middleName":"","lastName":"Nikpour","suffix":""},{"id":390683681,"identity":"15387ceb-32b3-4293-bab6-79e3e74fbedb","order_by":5,"name":"Ladan Mafakher","email":"","orcid":"","institution":"Thalassemia \u0026 Hemoglobinopathy Research Center, Health Research Institute, Ahvaz Jundishapur University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Ladan","middleName":"","lastName":"Mafakher","suffix":""},{"id":390683682,"identity":"ce151e12-0eaf-45c6-b4b6-e29fc2dadef8","order_by":6,"name":"Camellia Katalani","email":"","orcid":"","institution":"Department of Plant Breeding \u0026 Biotechnology, Sari Agriculture Science and Natural Resource University","correspondingAuthor":false,"prefix":"","firstName":"Camellia","middleName":"","lastName":"Katalani","suffix":""},{"id":390683683,"identity":"005f7ae2-bb3b-4c5c-a70e-0a1ae1142e93","order_by":7,"name":"Alaleh Maleki","email":"","orcid":"","institution":"Department of Cellular and Molecular Biology, Islamic Azad University, Babol Branch","correspondingAuthor":false,"prefix":"","firstName":"Alaleh","middleName":"","lastName":"Maleki","suffix":""},{"id":390683684,"identity":"68c668d1-4a0f-4d1d-87df-b42f668dcb5b","order_by":8,"name":"Nargess Abdali","email":"data:image/png;base64,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","orcid":"","institution":"Razi Herbal Medicines Research Center, Lorestan University of Medical Science","correspondingAuthor":true,"prefix":"","firstName":"Nargess","middleName":"","lastName":"Abdali","suffix":""},{"id":390683685,"identity":"832ab3db-111d-4b1f-9030-d498887898a0","order_by":9,"name":"Reza Tabaripour","email":"","orcid":"","institution":"Comprehensive health research center, Babol branch, Islamic Azad University","correspondingAuthor":false,"prefix":"","firstName":"Reza","middleName":"","lastName":"Tabaripour","suffix":""}],"badges":[],"createdAt":"2024-11-04 13:23:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5388446/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5388446/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":71730813,"identity":"de506f12-d446-4187-b5e6-f52d05c4f7b6","added_by":"auto","created_at":"2024-12-18 06:48:40","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":720085,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Multiple sequence alignment of COVID-19 (SARS-CoV2) and its concern variants according to WHO. (B) Schematic view of RBD domain with high potent RBM (Receptor binding motif) and the region which was selected for construct.\u003c/p\u003e","description":"","filename":"image1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5388446/v1/b64f069e8e5c1fd8a0da4f03.jpeg"},{"id":71730807,"identity":"12bb2fae-1e10-4d6c-9526-dee62c4a2aa4","added_by":"auto","created_at":"2024-12-18 06:48:39","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":643245,"visible":true,"origin":"","legend":"\u003cp\u003eTertiary structure and Structure alignment of a construct with RBD and S protein of SARS-CoV-2. (A) Tertiary structure of construct (B) Structural alignment of S protein of SARS-CoV2 (magenta) with RBD domain of SARS-CoV2, RBD domain of Omicron variant (cyan) and design construct (yellow). Data shows no significant structural difference between these structures. (C) Structure alignment of design construct (yellow) with RBD domain of Omicrom variant of SARS-CoV2 (cyan) in interaction with antibody (magenta). The result shows that no structural differences in the binding site of these two structures which were colored in red in construct and blue in the RBD domain of Omicron were observed.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-5388446/v1/ad2f2043d8396b4faaae0e97.png"},{"id":71732111,"identity":"a4864a5a-cce8-415e-b820-0e9a9265e285","added_by":"auto","created_at":"2024-12-18 06:56:40","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":212037,"visible":true,"origin":"","legend":"\u003cp\u003eCodon usage for RBD expression in \u003cem\u003eE. coli\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-5388446/v1/7512979bc34b9a49e77d8ba7.png"},{"id":71730809,"identity":"23a6f341-6788-491b-9e2b-50013ecd377d","added_by":"auto","created_at":"2024-12-18 06:48:40","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":87010,"visible":true,"origin":"","legend":"\u003cp\u003eShuttle vector that its primary vector is a backbone plasmid (pBluescriptIISK (+)) with a Tn5 as a secondary vector.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-5388446/v1/5f13cc953603a37a9fccf5ad.png"},{"id":71730820,"identity":"6668b17b-6707-4dac-b35a-511fbdecdde1","added_by":"auto","created_at":"2024-12-18 06:48:40","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":541529,"visible":true,"origin":"","legend":"\u003cp\u003eEvaluation of GFP expression to find the best time and temperature for purification. (A) 37\u003csup\u003eο\u003c/sup\u003eC for 10 hours, (B) 37\u003csup\u003eο\u003c/sup\u003eC for 16 hours, (C) Negative control (bacteria without GFP).\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-5388446/v1/d74e954ebba61b3184f5d517.png"},{"id":71730808,"identity":"24e6e83d-8c15-4cea-a927-0fc1dae464b1","added_by":"auto","created_at":"2024-12-18 06:48:40","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":44919,"visible":true,"origin":"","legend":"\u003cp\u003eThe expression of a protein in the soluble phase (Rs well) and Inclusion bodies (R well) by SDS-PAGE. (L: Ladder Trmo26616, R: Purified antigen of COVID-19 in the inclusion body phase, Rs: Purified COVID-19 antigen in the soluble phase)\u003c/p\u003e","description":"","filename":"image6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5388446/v1/3b9aaf2c37f4836fab21793b.jpeg"},{"id":71730830,"identity":"3e3a7849-71f9-4179-a1c7-95be5775ac4f","added_by":"auto","created_at":"2024-12-18 06:48:41","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":65404,"visible":true,"origin":"","legend":"\u003cp\u003eWestern blot results from solution phase (Rs well) and inclusion phase (R well)\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-5388446/v1/850c153cf5b94e693d863a8d.png"},{"id":71734003,"identity":"33bfa07e-f15f-4ff0-9e3e-dd5d09d39ef2","added_by":"auto","created_at":"2024-12-18 07:12:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3162100,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5388446/v1/43081fb2-5b63-4c8e-aed8-000af73d7919.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"From In Silico to In Vivo: Characterizing Ag-RBDN331-V524 for Effective COVID-19 Vaccine Development","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eSARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus-2) which is well-known as COVID-19 has been introduced by the World Health Organization (WHO) as the causative agent of the COVID-19 pandemic. The COVID-19 epidemic began in December 2019 in Wuhan, Hubei Province, China, when patients with pneumonia were diagnosed with symptoms such as fever, cough, dyspnea, and myalgia (He, Deng and Li \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Statistics show by 11 February 2022 404,910,528 confirmed cases of COVID-19, including 5,783,776 deaths reported to WHO (Zheng, Shao et al. 2022). In recent years, before the recent pandemic, two outbreaks have been caused by coronaviruses. In 2002, the coronavirus caused the SARS disease in China and 28 countries (Cherry \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Also in 2012, another coronavirus which is called MERS-CoV (Middle East Respiratory Syndrome-related Coronavirus) was first reported in Saudi Arabia (Chan, Lau and Woo \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). COVID-19 is a member of the genus Beta coronavirus which is recognized as a positive-sense, enveloped, and single-stranded RNA virus (Artika, Dewantari and Wiyatno \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The genome of the COVID-19 virus is 79% similar to SARS and 50% similar to MERS-CoV (Lu, Zhao et al. 2020). The COVID-19 virus is transmitted from person to person through direct contact or respiratory droplets (Li, Guan et al. 2020). Coronavirus is composed of four structural proteins including nucleocapsid (N), membrane (M), envelope (E), and spike (S) proteins (Deng, Li et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The COVID-19 virus enters the cell by binding Spike (S) glycoproteins to the ACE2 (angiotensin-converting enzyme2). The ACE2 receptors are abundant in human alveolar epithelial cells (AECs), which is why the lungs are considered one of the most vulnerable organs in the body when the COVID-19 coronavirus enters (Gibson, Evans et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Glycoprotein S is responsible for the entry of virus particles through attachment to the host cell membrane and fusion. This protein is composed of two subunits, S1 and S2, which are involved in binding to the receptor and fusion to the cell membrane, respectively (Belouzard, Millet et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Attachment of the S1subunit to the ACE2 as the receptor is carried out through the receptor binding domain (RBD) of the S1 subunit. SARS-CoV-2 RBDs a key part of a virus located on its spike domain that allows it to bind to ACE2 with an affinity in the low nanomolar range to gain entry into cells and lead to infection (Tian, Li et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Walls, Park et al. 2020). S2 subunit of the S protein participates in the fusion of the virus membrane with the host cell. Therefore, S protein is a target in the development of antibodies, inhibitors, and vaccines (Gangadevi, Badavath et al. 2021). Researchers have developed different vaccine platforms to prevent COVID-19, including live attenuated virus, inactivated virus, subunit vaccines, mRNA, viral vectors, recombinant DNA, and protein vaccines (Belete \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). As using live, inactivate or subunit vaccine of COVID-19 are usually associated with immune problems, finding the most potent region of virus which could activate immune response against different variants of Covid-19 is massively being investigated. Data showed that RBD or recombinant RBD region could effectively induce immune response without any side-effect compared to using fully length if s protein or recombinant form which caused liver damage and enhance infection (Du, He et al. 2009). Also, Y. He \u0026amp; Jiang (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2005\u003c/span\u003e) reported multiple neutralizing antibodies against RBD region in SARS which means that this region is highly potent to activate immune response (He and Jiang \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Du et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2009\u003c/span\u003e found a concave region in RBD region of SARS which contains 14 amino acids and could interact with ACE2 receptor (Du, He et al. 2009).\u003c/p\u003e \u003cp\u003eMany items affect the yields of every recombinant protein production. Promoters are one of the key regulatory elements which control the level of recombinant protein expression in the host (Abdali, Kazaemitabar et al. 2022). The phycocyanin-specific promoter sequence, which is the strongest promoter in \u003cem\u003eSpirulina\u003c/em\u003e (\u003cem\u003eArthrospira platensis\u003c/em\u003e) and 20\u0026ndash;50% of the spirulina proteins weight, is considered in this study.\u003c/p\u003e \u003cp\u003eSo, we selected a highly conserved region of RBD protein to expressed in \u003cem\u003eE. coli\u003c/em\u003e expression system. After purification of this protein by chromatography, we analyzed the efficacy of this selected region by using western blot. The aim of this study was expression of RBD domain in \u003cem\u003eE. coli\u003c/em\u003e which could apply in a future study to the production of vaccine against COVID-19 based on a host that has ideal C-phycocyanin expression.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Selecting a region of the RBD domain\u003c/h2\u003e \u003cp\u003eAs the RBD region of SARS-CoV2 is the most potent region of spike protein which plays a critical role in interaction with neutralized antibodies, this region was selected to design a vaccine construct.\u003c/p\u003e \u003cdiv id=\"Sec4\" class=\"Section3\"\u003e \u003ch2\u003e2.1.1. Antigenicity, allergenicity, and physicochemical properties assessment\u003c/h2\u003e \u003cp\u003eThe antigenicity of the vaccine construct was evaluated by the VaxiJen webserver (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ddg-pharmfac.net/vaxijen/VaxiJen/VaxiJen.html\u003c/span\u003e\u003cspan address=\"https://www.ddg-pharmfac.net/vaxijen/VaxiJen/VaxiJen.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) with a threshold of 0.4. The allergenicity was predicted by Algpred (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://crdd.osdd.net/raghava/algpred/\u003c/span\u003e\u003cspan address=\"http://crdd.osdd.net/raghava/algpred/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and Allertop (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ddg-pharmfac.net/AllerTOP/method.html\u003c/span\u003e\u003cspan address=\"https://www.ddg-pharmfac.net/AllerTOP/method.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) webserver. The Algpred webserver used different algorithms such as mapping IgE epitope, searching MEME/MAST allergen motifs using MAST, SVM modules by applying amino acid or dipeptide composition and Blast search against 2890 allergen-representative peptides (ARPs) to illustrate the potential of protein allergenicity. The Allertop webserver anticipated the allergenicity of protein sequence based on the auto cross-covariance (ACC) method. The physicochemical properties of the construct were evaluated by Protparam (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://web.expasy.org/protparam/\u003c/span\u003e\u003cspan address=\"https://web.expasy.org/protparam/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) webserver. The stability of the construct was determined by an instability index value. An instability index of less than 40 means the stability of the protein sequence. The aliphatic index showed thermo stability of the protein sequence. More aliphatic index value indicates more thermo stability of a protein. The Gravy index illustrated the hydropathicity of protein. Less Gravy index means more hydrophilicity of protein.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.1.2. Protein solubility assessment\u003c/h2\u003e \u003cp\u003eThe solubility of the vaccine construct is essential which increases the vaccine accessibility to the immune system. For this reason, several protein solubility web servers were applied to check design vaccine construct solubility compared to the RBD domain. The protein-sol webserver estimates the solubility of protein based on a sequence of the protein (Hebditch, Carballo-Amador et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). This web server calculates protein solubility by 35 sequence features. Proteins that have a solubility value over 0.45 predicts to have a higher solubility than the average solubility of \u003cem\u003eE. coli\u003c/em\u003e protein from the experiment dataset. Soluprot web application uses a machine learning algorithm and employs 96 features of protein to predict the solubility of the protein in the \u003cem\u003eE. coli\u003c/em\u003e expression system by protein sequence (Hon, Marusiak et al. 2021). A value of more than 0.5 indicates good solubility of the protein. SoDoPE (Soluble Domain for Protein Expression) is a web tool for the solubility prediction of proteins by applying Solubility-Weighted Index (SWI) which is derived from crystallographic B-factors (Bhandari, Gardner and Lim \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The web server used a sequence of the protein and designates solubility by the score. The higher solubility value shows better solubility of the protein. Camsol webserver could predict the solubility of protein by protein structure with three different algorithms (Sormanni, Amery et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The Camsol value larger than 1 donates higher soluble regions while the Camsol value smaller than \u0026minus;\u0026thinsp;1 specifies lower solubility. Aggrecan web server predicts the potency of globular protein to aggregate by computing the aggregation propensity of each amino acid. A lower Aggrescan value means less potential to aggregate and better water solubility.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.1.3. Homology modeling\u003c/h2\u003e \u003cp\u003eAs the tertiary structure of vaccine construct is essential in inducing humoral immunity, the structure of selected RBD region was predicted by Robetta webserver (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://robetta.bakerlab.org/\u003c/span\u003e\u003cspan address=\"https://robetta.bakerlab.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Sequence optimization and constructs design\u003c/h2\u003e \u003cp\u003eTo optimize the expression of selected RBD region of COVID-19 in \u003cem\u003eE. coli\u003c/em\u003e, codon optimization was performed based on the Biomatik Codon Optimization Analysis platform, which can optimize multiple critical parameters to stabilize DNA fragments and improve gene expression efficiency. These parameters include but are not limited to Codon usage bias, GC content, Premature polyA sites, Internal chi sites, ribosomal binding sites, RNA instability motif (ARE), mRNA secondary structure, CpG dinucleotides content, repeat sequences (direct repeat, inverted repeat, and dyad repeat), Codon usage bias. The shuttle vector was designed by the composition of the primary vector as a backbone plasmid (pBluescriptIISK (+)) with a secondary vector of 1657 bp inside. The construct was synthesized by Biomatik Company (Canada) with its sequence confirmation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Transformation\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eE. coli\u003c/em\u003e TOP10 competent cell was prepared using optimized CaCl\u003csub\u003e2\u003c/sub\u003e method of Sambrook 1989. The final competent cells were kept at -80 \u0026ordm;C until use (Sambrook, Fritsch and Maniatis \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1989\u003c/span\u003e). To transformation, melting component was mixed with added 5\u0026micro;l of plasmid. Then, one cooling step: 30 min on ice, then one heating step: 2 min in water bath at 42\u0026deg;C and again one cooling step: 5 min on ice were performed, respectively. After that, 600\u0026micro;l of LB Broth was added to the sample and incubated at 37\u0026deg;C with 150 rpm for 1 hour. The solution containing the transgenes was relocated to LB agar with Ampicillin in dilutions, 1/10, 1/100, 1/1000 and 1/10000. Plates were incubated at 37\u0026deg;C for 18\u0026ndash;24 hours.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Quantitation of total protein and purified RBD\u003c/h2\u003e \u003cp\u003eThe Bradford assay was used for the measurement of the quantitation of total protein (Bradford 1976). The confirmation of expression of the protein was carried out initially by SDS-PAGE and subsequently western blot.\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.4.1. Western blot analysis\u003c/h2\u003e \u003cp\u003eThe bacteria were lysed by using a lysis Buffer solution (1% Triton X-100, 50 mM Tris-HCl, 150 mM NaCl, 0.25% sodium deoxycholate, 1 mM Ethylene glycol tetraacetic acid, and 1 mM NaF). Bradford (Cat No: DB0017, DNAbiotech; Iran) assay was conducted for measurement of protein quantitation. The lysates were boiled for 5 min. then, 25 \u0026micro;g lysates were applied to SDS-PAGE 12%. The protein band was then transferred to a 0.2 \u0026micro;m Immune-Blot\u0026trade; polyvinylidene difluoride (PVDF) membrane (Cat No: 162-017777; Bio-Rad Laboratories, CA, USA). The 5% BSA with 0.1% Tween 20 (Cat No: A-7888; Sigma Aldrich, MO, USA) was utilized to block the membranes for 1 h. The membranes were then incubated together with HRP Anti-6X His tag\u0026reg; (Cat. No: ab1187 -Abcam) as a secondary antibody at room temperature for 1 h. Ultimately, the membranes were located in an incubator with enhanced chemiluminescence (ECL) for 1\u0026ndash;2 minutes (Asadian, Siavashi et al. 2018, Asadian, Alibabrdel et al. 2019, Abdali, Kazaemitabar et al. 2022).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.4.2. Affinity Chromatography\u003c/h2\u003e \u003cp\u003eThe recombinant protein\u0026rsquo;s recovery and purification were conducted using affinity chromatography through its hexa histidine tag. The protocols were optimized, based on the QIA expressionist \u0026trade; (QIAGEN \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2003\u003c/span\u003e, Katalani, Ahmadian et al. 2020, Katalani, Nematzadeh et al. 2020, Abdali, Kazaemitabar et al. 2022).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1. Selected RBD region\u003c/h2\u003e\n \u003cp\u003eMultiple sequence alignment of the RBD region among COVID-19 and its variants revealed that the RBD region is highly conserved among its variants. To improve the potency of antigenicity of the RBD region, some amino acid sequences were removed from both the N and C terminals of protein (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e\n \u003ch2\u003e3.1.1. Physiochemical, aggregation and, solubility assessment\u003c/h2\u003e\n \u003cp\u003eThe antigenicity and allergenicity of the construct compared to the RBD region were analyzed by the VaxiJen webserver. Data showed that construct had a high antigenicity value (0.5822). Allergenicity assessment with Allertop webserver illustrated the construct had no allergenicity compared to RBD domain. Although, construct showed probable allergenicity by the Algpred web server but its allergenic effect was lower than RBD region (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Physicochemical properties assessment of the construct showed that although the construct had more instability index (29.91) than the RBD region while it has a more aliphatic index which means this construct had more thermos-stability than the RBD region. No significant changes were observed based on hydrophilicity by Gravy index. The half-life analysis illustrated the half-life of construct increased in three different expression systems compared to the RBD region especially, in \u003cem\u003eE. coli\u003c/em\u003e (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n \u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eAntigenicity and allergenicity prediction of construct compared to RBD by Algpred webservers\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth rowspan=\"2\" align=\"left\"\u003e\n \u003cp\u003eModel\u003c/p\u003e\n \u003c/th\u003e\n \u003cth rowspan=\"2\" align=\"left\"\u003e\n \u003cp\u003eVaxiJen*\u003c/p\u003e\n \u003c/th\u003e\n \u003cth rowspan=\"2\" align=\"left\"\u003e\n \u003cp\u003eAllertop\u003c/p\u003e\n \u003c/th\u003e\n \u003cth colspan=\"5\" align=\"left\"\u003e\n \u003cp\u003eAlgpred algorithms\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePrediction by mapping of IgE epitope\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMAST\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePrediction by SVM method based on amino acid composition**\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePrediction based on SVM method based on dipeptide composition ** *\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eBlast\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eRBD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.4952\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eprobable allergen\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003enon-allergen\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003enon-allergen\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003epotential allergen score\u0026thinsp;=\u0026thinsp;0.96\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eallergen\u003c/p\u003e\n \u003cp\u003escore\u0026thinsp;=\u0026thinsp;0.066\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003enon-allergen\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eConstruct\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.5822\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003enon-allergen\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003enon-allergen\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003enon-allergen\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003epotential allergen\u003c/p\u003e\n \u003cp\u003escore\u0026thinsp;=\u0026thinsp;0.83\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eallergen\u003c/p\u003e\n \u003cp\u003escore\u0026thinsp;=\u0026thinsp;0.058\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003enon-allergen\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"8\"\u003e*Threshold more than 0.4 means potential of antigenicity\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"8\"\u003e**Threshold is \u0026minus;0.4\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"8\"\u003e***Threshold was \u0026minus;0.2\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n \u003cdiv class=\"colspec\" align=\"char\"\u003e\u0026nbsp;\u003c/div\u003e\n \u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003ePhysicochemical assessment of construct compared to RBD of SARS-Cov2.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth rowspan=\"2\" align=\"left\"\u003e\n \u003cp\u003eModel\u003c/p\u003e\n \u003c/th\u003e\n \u003cth rowspan=\"2\" align=\"left\"\u003e\n \u003cp\u003eMW\u003c/p\u003e\n \u003c/th\u003e\n \u003cth colspan=\"7\" align=\"left\"\u003e\n \u003cp\u003eProtparam\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eInstability index\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAliphatic index\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGravy\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eHalf-life in mammalian\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eHalf-life in yeast\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eHalf-life in \u003cem\u003eE.coli\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePI\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eRBD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e25098\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e22.69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e71.61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-0.259\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2 min\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2 min\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8.91\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eConstruct\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e22115\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e29.91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e74.85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-0.216\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.4 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3 min\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt;\u0026thinsp;10 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8.79\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e\n \u003ch2\u003e3.1.2. Solubility assessment\u003c/h2\u003e\n \u003cp\u003eThe solubility of the construct was assessed by the different web servers. Data showed that the solubility of the RBD domain is slightly better than Construct by Protein-sol and Soluprot while according to Camsol result, construct had better water solubility in a wide range of pH compared to the RBD domain. No difference was observed between the two proteins by the SoDoPE webserver. Aggregation analysis of protein based on sequence and protein structure by Aggrescan webserver revealed that although aggregation assessment based on protein sequence showed less potential of aggregation by RBD while a result of aggregation based on protein structure did not show any differences between these two protein structures (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n \u003cdiv class=\"colspec\" align=\"char\"\u003e\u0026nbsp;\u003c/div\u003e\n \u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eEvaluation of solubility of construct and RBD by a different web server\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr style=\"height: 35px;\"\u003e\n \u003cth style=\"height: 70px;\" rowspan=\"2\" align=\"left\"\u003e\n \u003cp\u003eModel\u003c/p\u003e\n \u003c/th\u003e\n \u003cth style=\"height: 70px;\" rowspan=\"2\" align=\"left\"\u003e\n \u003cp\u003eProtein-sol\u003c/p\u003e\n \u003c/th\u003e\n \u003cth style=\"height: 70px;\" rowspan=\"2\" align=\"left\"\u003e\n \u003cp\u003eSoluprot\u003c/p\u003e\n \u003c/th\u003e\n \u003cth style=\"height: 70px;\" rowspan=\"2\" align=\"left\"\u003e\n \u003cp\u003eSoDoPE\u003c/p\u003e\n \u003c/th\u003e\n \u003cth style=\"height: 35px;\" colspan=\"2\" align=\"left\"\u003e\n \u003cp\u003eCamsol in pH range\u003c/p\u003e\n \u003c/th\u003e\n \u003cth style=\"height: 35px;\" colspan=\"2\" align=\"left\"\u003e\n \u003cp\u003eAggrescan\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr style=\"height: 35px;\"\u003e\n \u003cth style=\"height: 35px;\" align=\"left\"\u003e\n \u003cp\u003esequence\u003c/p\u003e\n \u003c/th\u003e\n \u003cth style=\"height: 35px;\" align=\"left\"\u003e\n \u003cp\u003estructure\u003c/p\u003e\n \u003c/th\u003e\n \u003cth style=\"height: 35px;\" align=\"left\"\u003e\n \u003cp\u003esequence\u003c/p\u003e\n \u003c/th\u003e\n \u003cth style=\"height: 35px;\" align=\"left\"\u003e\n \u003cp\u003estructure\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr style=\"height: 35px;\"\u003e\n \u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eRBD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"height: 35px;\" align=\"char\"\u003e\n \u003cp\u003e0.538\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"height: 35px;\" align=\"char\"\u003e\n \u003cp\u003e0.76\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"height: 35px;\" align=\"char\"\u003e\n \u003cp\u003e0.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"height: 35px;\" align=\"char\"\u003e\n \u003cp\u003e-0.87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"height: 35px;\" align=\"char\"\u003e\n \u003cp\u003e-0.87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"height: 35px;\" align=\"char\"\u003e\n \u003cp\u003e5.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"height: 35px;\" align=\"char\"\u003e\n \u003cp\u003e-76.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr style=\"height: 35.3032px;\"\u003e\n \u003ctd style=\"height: 35.3032px;\" align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eConstruct\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"height: 35.3032px;\" align=\"char\"\u003e\n \u003cp\u003e0.461\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"height: 35.3032px;\" align=\"char\"\u003e\n \u003cp\u003e0.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"height: 35.3032px;\" align=\"char\"\u003e\n \u003cp\u003e0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"height: 35.3032px;\" align=\"char\"\u003e\n \u003cp\u003e-1.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"height: 35.3032px;\" align=\"char\"\u003e\n \u003cp\u003e-1.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"height: 35.3032px;\" align=\"char\"\u003e\n \u003cp\u003e8.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"height: 35.3032px;\" align=\"char\"\u003e\n \u003cp\u003e-76.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr style=\"height: 15px;\"\u003e\n \u003ctd style=\"height: 15px;\" colspan=\"8\"\u003e*Higher value means higher water solubility\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr style=\"height: 15px;\"\u003e\n \u003ctd style=\"height: 15px;\" colspan=\"8\"\u003e** Less value means more solubility and less aggregation\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e\n \u003ch2\u003e3.1.3. Tertiary structure analysis\u003c/h2\u003e\n \u003cp\u003eTo find out if this amino acid sequence reduction in construct led to no changes in RBD tertiary structure, homology modeling by Robetta webserver was performed (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e(A)). structure alignment of design construct with S protein of Covid-19 and the RBD region of SARS-CoV2 variant Omicron showed that the construct have not illustrated any changes in the tertiary structure (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e(B)). Also, structure alignment of design construct with Omicron RBD region which in interaction with the Antibody illustrated no structure changes in the designed construct compared to the RBD domain. This result means that the designed construct could potentially interact with neutralized antibody (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e(C)).\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2. Codon optimization and Vector design\u003c/h2\u003e\n \u003cp\u003eCodon usage optimized based on \u003cem\u003eE. coli\u003c/em\u003e as cloning host. The optimization significantly improves the expression levels of genes by strategically optimizing the underlying DNA sequence. The native gene has several features which may lead to poor expression. The gene has been improved by applying a set of carefully selected design criteria which improve the expression. The codon bias has been adapted to the preferred codon usage of the target organism, \u003cem\u003eE. coli.\u003c/em\u003e The squared difference of the actual codon frequency vs. the preferred codon frequency has been changed from 3.60 to 5.87. The GC content throughout the sequence has been homogenized, in order to increase the half-life of the mRNA. The mRNA secondary structure has been reduced. The codon optimizing resulted in changing some factors like GC content, Secondary structure, etc. (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e \u0026amp; Table \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n \u003ctable id=\"Tab4\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eResults of some variation in sequence of genes after codon usage optimizing\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGENES\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eChanging\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eOriginal sequence\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eOptimized sequence\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eRBD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAmino acid position\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e*CAI: 0.76\u003c/p\u003e\n \u003cp\u003eSquared difference: 3.60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCAI: 0.96\u003c/p\u003e\n \u003cp\u003eSquared difference: 5.87\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGC content adjustment\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e35%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e43%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSecondary structure: hairpin stem motifs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e554\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e476\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eGFP\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAmino acid position\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e*CAI: 0.73\u003c/p\u003e\n \u003cp\u003eSquared difference: 3.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCAI: 0.97\u003c/p\u003e\n \u003cp\u003eSquared difference: 5.12\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGC content adjustment\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e38%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e41%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSecondary structure: hairpin stem motifs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e649\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e602\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\"\u003e* The distribution of relative adaptiveness (where the most frequent codon scores 100%) along the length of the gene sequence. A CAI of 1.0 is considered to be perfect in the desired expression organism, and a CAI of \u0026gt; 0.9 is regarded as very good, in terms of high gene expression level\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eVaccine construct was inserted in the secondary vector from 5\u0026apos; to 3\u0026apos; in position where EcoRI was cleavage site, ME (mosaic end) left sequence, c-Phycocyanin operon (C-PC promotor, Ribosomal Binding Site (RBS) sequence, respectively, gene locus 1 (RBD-Spike-COVID-19 sequence), gene locus 2 (GFP molecular marker) and terminator sequence), ME Right sequence and HindIII cleavage site (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eMoreover, enzymatic digestion based on two EcoRI and HindIII sites was performed to reconfirm the synthesized structures.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3. Transformation\u003c/h2\u003e\n \u003cp\u003eThe transformation was initially evaluated by culturing transformed \u003cem\u003eE. coli\u003c/em\u003e in an Amp\u0026thinsp;+\u0026thinsp;medium.\u003c/p\u003e\n \u003cdiv id=\"Sec19\" class=\"Section3\"\u003e\n \u003ch2\u003e3.3.1. Purification of expressed protein\u003c/h2\u003e\n \u003cp\u003eA C-phycocyanin promoter was used to express a recombinant protein in \u003cem\u003eE. coli\u003c/em\u003e. The best time and temperature for purification were determined based on GFP expression at different times and temperatures under the blue light of fluorescent microscope was applied (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e). Data showed that the best temperature was 37\u003csup\u003e\u0026omicron;\u003c/sup\u003eC and 16 hours after transformation.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec20\" class=\"Section3\"\u003e\n \u003ch2\u003e3.3.2. SDS-PAGE and Western blot\u003c/h2\u003e\n \u003cp\u003eSDS-PAGE was applied to initially proof the expression of construct in transgenic \u003cem\u003eE. coli\u003c/em\u003e. Western blot was done to approve the RBD expression in transgenic bacteria based on anti-His-Tag antibody (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e \u0026amp; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e). The prediction of Ag-RBD size was 22 kDa. In SDS-PAGE there was a band at 22 kDa for inclusion body phase, while in soluble phase of SDS-PAGE and in both solution phase and inclusion phase in western blot analysis the band was located in 34 kDa.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec21\" class=\"Section3\"\u003e\n \u003ch2\u003e3.3.3. Total protein solubility\u003c/h2\u003e\n \u003cp\u003eThe results of Bradford assay demonstrated that the concentration of total protein in the soluble phase was 2 to 3 times more than that of the inclusion body phase (1265 \u0026micro;g/ml in the soluble phase vs. 720 \u0026micro;g/ml in the inclusion body phase). (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e)\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab5\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eTotal protein concentration in solution phase (Rs) and inclusion phase (R) in lysed samples prepared for chromatography\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSample\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eProtein concentration quantitation (\u0026micro;g/ml)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eR\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e720\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eRs\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1265\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe COVID-19 virus spread a pandemic that affected many people all over the world. Statistics shows that millions of people are infected and die because of COVID-19. Also, there is a lot of pressure on the health care system (Ciotti, Ciccozzi et al. 2020). The use of various drug therapies to cure patients with COVID-19 has not been very successful due to the lack of effective drug therapy (Malek, Bill and Vines \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Therefore, scientists sought to develop an effective vaccine to create immunity against this disease. Various types of vaccine platforms was developed such as live attenuated virus, inactivated virus, subunit vaccines, viral vectors, recombinant DNA, and protein vaccines (Dutta \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). S-protein of COVID-19 virus is an important part of virus which involved in the attachment of virus to the host cell. The RBD is the main region of S-protein which directly bind to receptor of host cell in order to entrance to cell. Immunity problems are present with the models of live or inactivated or subunit viruses, because they may elicit inflammatory or immune responses. Among all the studied regions of the COVID-19 genome, the best region for vaccine design is the RBD domain (Wang, Wang et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eConsidering the start of this project in the first days of the pandemic, RBD was selected in the first step based on the compatibility of the available sequences from SARS (NP_828851.1) and COVID-19 (YP_009724390.1). As the second step, articles on the allergen status of SARS Subunit vaccines were reviewed (He and Jiang \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). In the next step, the prediction of allergens and identification of epitopes were checked on the AlgPred website. The exact position of the sequence containing the RBD was finalized (RBD\u003csub\u003eN331\u0026minus;V524\u003c/sub\u003e) based on proper folding. The purpose of this study was expression of recombinant protein RBD to design an effective vaccine against COVID-19. Evaluation showed that removing some amino acids from N and C terminal parts of RBD region improved antigenicity and reduce its allergenicity without changing its solubility and the half-life of designed construct became higher in \u003cem\u003eE. coli\u003c/em\u003e. The tertiary structure prediction showed no structural changes in the RBD-Ag.\u003c/p\u003e \u003cp\u003eAccording to the results of Western blot analysis, a special band in size of 34kDa was indicated. While as predicted by the software, the size of the RBD-Ag of S protein should normally be about 12KDa. Consistent with Yang et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, the molecular weight of the RBD protein was determined around 34 kDa, which was about a quarter more than the molecular weight estimated using the RBD amino acid sequence alone (~\u0026thinsp;27 kDa). They suggested that the RBD is densely glycosylated(Yang, Wang et al. 2020). Although this explanation was acceptable considering the protein expression host, which was insect cells, the expression host in the present study is bacteria. So, this justification becomes questionable. The difference in MW ,in eukaryotes, is attributed to chemical modifications of the protein, especially glycosylation which impress gel mobility shift (Goldstein, Scheid et al. 1975, Stanley \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), but here RBD could not be glycosylated in \u003cem\u003eE.coli\u003c/em\u003e and this rules out the possible contribution of glycosylation to the observed MW difference. It was investigated that amino acid composition can cause the difference between the predicted and western blot displayed MW (Guan, Zhu et al. 2015). In another similar study, the expression of the S-RBDN318-V510 protein was investigated in \u003cem\u003eE. coli\u003c/em\u003e, the results of showed that the molecular weight of the S-RBDN318-V510 protein was approximately 31 KDa. They indicated the overall yields of approximately 1.5 mg of pure S-RBD\u003csub\u003eN318\u0026minus;V510\u003c/sub\u003e per liter .(M\u0026aacute;rquez-Ipi\u0026ntilde;a, Gonz\u0026aacute;lez-Gonz\u0026aacute;lez et al. 2021). Our data showed that the concentration of total protein in the soluble phase is more than that of the inclusion body phase. Compared to the aforementioned study, our expression, based on the area and intensity of the Western blot band, is low. Although in the present research, codon usage was optimized for \u003cem\u003eE.coli\u003c/em\u003e, the expression level remains low, similar to our previous study that focused on the expression of the hepatitis B antigen under the C-phycocyanin promoter in bacteria (Abdali, Kazaemitabar et al. 2022). In fact, it can be claimed that the low expression in the previous study, which had been attributed to codon usage, is not accurate. The results of the present study and the previous one indicate the low efficiency of this algal promoter in bacteria.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThe result indicated although the c-phycocyanin promoter is considered a super-promoter in cyanobacteria, particularly in \u003cem\u003eSpirulina\u003c/em\u003e, it does not exhibit strong performance in bacteria. Taken together, this study showed that the RBD region was successfully expressed in \u003cem\u003eE. coli\u003c/em\u003e. This means that this vaccine construct could apply in a future study to order to production of vaccine against COVID-19. On the other hand, the size of the antigen protein RBD according to our observation and the results of SDS-PAGE and Western blot analysis was 34 kDa. Hence, the expression of RBD protein was successful in \u003cem\u003eE. coli\u003c/em\u003e which could apply in a future study to order to production of vaccine against COVID-19.\u003c/p\u003e"},{"header":"Abbreviations","content":" \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eRBD\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003ereceptor binding domain\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eC-PC\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eC-phycocyanin\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eSARS-CoV-2\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eSevere Acute Respiratory Syndrome Coronavirus-2\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eACE2\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eangiotensin-converting enzyme2\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eO.L.PHB\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eOral cell-loaded PHB. PHB that is loaded with \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSpirulina\u003c/span\u003e for oral administration\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eO.U.PHB\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eOral unloaded PHB\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eI.Vac.\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eInjected Vaccine. SpikoGen\u0026trade; COVID-19 vaccine administered via injection\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eC-\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eThe negative control group without any treatment\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eALT\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eAlanine aminotransferase\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eAST\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eAspartate aminotransferase\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eIFN-γ\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eInterferon-gamma\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eIL-4\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eInterleukin 4\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eMTT\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] reduction assay\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003cbr/\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the study.\u003c/p\u003e\n\u003cp\u003eElmira Ranjbar Zeidi: Writing \u0026ndash; original draft, Methodology, Investigation, Data curation.\u003c/p\u003e\n\u003cp\u003eSolaleh Javadi:\u0026nbsp;Writing \u0026ndash; original draft, Methodology, Investigation, Data curation.\u003c/p\u003e\n\u003cp\u003ePouya Farokhi:\u0026nbsp;Writing \u0026ndash; original draft, Methodology, Investigation, Data curation.\u003c/p\u003e\n\u003cp\u003eVahid Siavashi: Writing \u0026ndash; review \u0026amp; editing, Funding acquisition.\u003c/p\u003e\n\u003cp\u003eAli Ahmari: Writing \u0026ndash; review \u0026amp; editing, Methodology, Investigation.\u003c/p\u003e\n\u003cp\u003eLadan Mafakher: Writing \u0026ndash; review \u0026amp; editing, Supervision, Formal analysis.\u003c/p\u003e\n\u003cp\u003eCamellia Katalani: Formal analysis, Data curation.\u003c/p\u003e\n\u003cp\u003eAlaleh Maleki: Formal analysis, Data curation.\u003c/p\u003e\n\u003cp\u003eNargess Abdali: Writing \u0026ndash; review \u0026amp; editing, Visualization, Validation, Supervision, Software, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization.\u003c/p\u003e\n\u003cp\u003eReza Tabaripour: Writing \u0026ndash; review \u0026amp; editing, Visualization, Validation, Supervision, Software, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest, financial or otherwise.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo funding was received for conducting this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis declaration is \u0026ldquo;not applicable\u0026rdquo; with headings.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availibility\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbdali, N. et al. 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Dis.\u003c/em\u003e \u003cb\u003e114\u003c/b\u003e, 252\u0026ndash;260 (2022).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"SARS-CoV-2, COVID-19, RBD, Vaccine, Recombinant Protein, E. coli","lastPublishedDoi":"10.21203/rs.3.rs-5388446/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5388446/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCOVID-19 has become a challenge of the century to the healthcare system. One of the best targets to produce the COVID-19 vaccine is the receptor binding domain (RBD) which is located in the Spike protein of Coronavirus. This domain is extremely conserved among different variants of COVID-19. In this study, the most potent region of RBD was selected to design a vaccine against COVID-19 in \u003cem\u003eE. coli\u003c/em\u003e as an expression system. The shuttle vector was applied to express the vaccine construct in \u003cem\u003eE. coli\u003c/em\u003e. The primary vector is a backbone plasmid pBluescriptIISK (+) and a Tn5 transposon is a secondary vector of 1657 bp inside. The C-phycocyanin operon including the gene cassette was embedded in Tn5. The quantitation of total protein was done by Bradford assay. Then, SDS-PAGE and Western Blot were carried out to characterize and confirm recombinant protein expression. Affinity chromatography was performed for the purification of recombinant protein. The molecular weight of the RBD protein was 34 kDa which is compatible with western blot results. The aim of this study was expression of RBD domain in \u003cem\u003eE. coli\u003c/em\u003e which could apply in a future study to the production of vaccine against COVID-19 based on a host that has ideal C-phycocyanin expression. The selected RBD sequence has a complete identity to the newest variant. The short length of the sequence selected in this study leads to increased solubility and decreased allergenicity. On the contrary, this trait has led to a decrease in the probability of mutation, which can cover new variants of this virus.\u003c/p\u003e","manuscriptTitle":"From In Silico to In Vivo: Characterizing Ag-RBDN331-V524 for Effective COVID-19 Vaccine Development","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-18 06:48:34","doi":"10.21203/rs.3.rs-5388446/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d1286390-56d1-4c1f-b231-215e4e81b76e","owner":[],"postedDate":"December 18th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":41636731,"name":"Biological sciences/Drug discovery"},{"id":41636732,"name":"Biological sciences/Genetics"},{"id":41636733,"name":"Biological sciences/Immunology"},{"id":41636734,"name":"Biological sciences/Molecular biology"}],"tags":[],"updatedAt":"2025-02-06T09:53:39+00:00","versionOfRecord":[],"versionCreatedAt":"2024-12-18 06:48:34","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5388446","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5388446","identity":"rs-5388446","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}