Production of African Swine Fever Virus p54 ectodomain and p30 in an E. coli system and their potential application in immunodetection

Production of African Swine Fever Virus p54 ectodomain and p30 in an E. coli system and their potential application in immunodetection | 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 Research Article Production of African Swine Fever Virus p54 ectodomain and p30 in an E. coli system and their potential application in immunodetection Bunyarit Meksiriporn, Puey Ounjai, Kampon Kaeoket, Tanapati Phakham, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5017399/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 African swine fever (ASF) is a lethally infectious viral disease caused by the African swine fever virus (ASFV), leading to a high mortality of almost 100% in domestic pigs worldwide. ASFV has significantly impacted the global swine industry and food security. Highly effective vaccines are in high demand; however, no current vaccines provide effective immunity against ASFV. Therefore, there is an urgent need to develop reliable immunodetection assays to prevent the spread of ASFV. Traditionally, ASFV antigens are produced using mammalian expression systems, which are labor-intensive, costly, time-consuming, and challenging to scale up. In this study, two ASFV structural proteins associated with viral infection, p30 and the p54 ectodomain from genotype II ASFV, were recombinantly expressed in E. coli BL21(DE3). The results demonstrated that recombinant p54 ectodomain and p30 were highly expressed in E. coli BL21(DE3) using the pET28a system. Both recombinant p54 ectodomain and p30 were then validated for their ability to serve as antigens to detect anti-ASFV antibodies in an indirect ELISA platform. The p54 ectodomain/p30-based indirect ELISA was validated using serum from ASFV-infected pigs and serum from ASFV-uninfected pigs. Both p54 ectodomain and p30 demonstrated binding ability in the serum from ASFV-infected pigs, while no binding was observed in the serum from ASFV-uninfected pigs. Collectively, our recombinant p30 and p54 ectodomain were successfully expressed in E. coli and can be used as antigens to develop an indirect ELISA-based detection assay for anti-ASFV antibodies. Animal Science African swine fever African swine fever virus p54 p30 Indirect ELISA Ectodomain Figures Figure 1 Figure 2 Figure 3 Introduction African swine fever (ASF) is an extremely infectious viral disease caused by the African swine fever virus (ASFV) and has a high mortality rate of almost 100% in the swine industry worldwide 1 , 2 . The discovery of ASF was first reported in the 1920s in Montgomery in Kenya in 1921 as a new disease leading to high lethality in in domestic pigs ( Sus scrofa domesticus ). Currently, ASF outbreak has spread across the globe leading to epidemics not merely in other African countries, but also in Europe, the Caribbean, South America, and Asia 3 , 4 . The invasion of ASF has caused huge losses in the swine industry’s global economy and food security 5 . Accordingly, there is an urgent demand to develop an effective ASFV vaccine to combat the widespread of the disease. However, the generation of an effective ASF vaccine to has been under investigation. The development of ASF vaccine has largely been hindered because of restricted understanding of viral complexity and virulence factors involved in a pivotal role in immune protection. To address these shortcomings due to the absence of a safe and effective ASF vaccine, there is an urgent demand to develop reliable immunodetection assays for early detection of ASFV to prevent the widespread transmission of ASFV in swine farms. The clinical symptoms of ASF infection can range widely, from sudden deaths to undetectable cases, and are associated with various pathological changes. Therefore, laboratory tests must detect both the virus itself and ASFV-specific antibodies. (Blome et al., 2013; M. C. Gallardo et al., 2015; Nah et al., 2022). ASFV is the sole member of the family Asfarviridae with a large double-stranded DNA virus in which genome size is ranging from 170 to 193 kbp long varying according to the strain and encodes 150–160 ORFs playing critical roles in virus replication, host interactions, viral entry and immune evasion of host defenses; however, many proteins have not been identified 10 – 13 . So far, it has been reported that up to 68 structural proteins have been characterized from the virion alone 14 . In comparison to other viruses, ASFV possesses a larger and more complex structure than most other viruses, and its mechanistic understanding regarding defense and neutralization process has not been completely comprehended 2 , 9 . Several ELISA-based serological tests include p72 and p30 antigens which currently used as the bases for commercial assays of anti-ASFV antibodies 15 – 19 . In addition to p30 and p72, another promising target is p54, which is the 183 amino acid structural protein, a product of the E183L gene, localized in the inner envelope virion. The p54 antigen is a type II membrane protein consisting of the cytoplasmic inner N-terminus of 60 amino acids, a transmembrane domain, and C-terminal ectodomain of 131 amino acids 18 . In combination with p30, p54 involves in the binding of the virion to target cells 20 . An indirect ELISA-based detection assay of anti-ASFV antibodies demonstrated 98% sensitivity and 97% specificity compared to the OIE ELISA 21 . Furthermore, the ASFV antibody-positive samples remained positive for p54 reactivity after storage for one month at 37°C. Therefore, p54 could represent an ideal target for the detection of ASFV antibodies 22 . Currently, ASFV antigens are mainly produced using mammalian expression systems, such as the baculovirus expression system, which is laborious, expensive, time-consuming, and difficult to scale up. To ameliorate the challenging problems due to mammalian expression system, E. coli systems have been employed as an alternative platform; however, the expression of the p54 antigen in E. coli has shown low expression and solubility due to the p54 structure itself, which includes a hydrophobic transmembrane domain that tends to have a high aggregation propensity. Here, to overcome the bottleneck, we sought to improve the expression and solubility of p54 in E. coli system by selectively expression of p54 ectodomain and validated the ASFV-specific antibody detection ability of our recombinant p54 ectodomain expressed in E. coli platform. Additionally, we expressed p30 antigen which is a highly expression ASFV antigen in E. coli as a benchmark. Our recombinant p54 ectodomain and p30 were able to selectively bind to their specific antibodies in ASFV-infected serum, while no binding ability was observed in ASFV-noninfected serum. Collectively, our recombinant p54 ectodomain and p30 could be highly expressed in E. coli with high production yield which render savings in time and resources. At the same time, this provides a reliable tool for generation of indirect ELISA assay against ASFV. Results Expression of recombinant antigens p54 ectodomain and p30 in E. coli system. To efficiently express the p54 antigen in E. coli , the cytosolic domain and transmembrane region of p54 were excluded. Only the p54 ectodomain region (amino acids 61–178) was used in this study, while the amino acid sequence of p30 remained unchanged. Recombinant p54 ectodomain and p30 were heat-shock transformed and expressed in E. coli BL21(DE3) using the pET28a system. Detection of protein expression was performed via a 6xhistidine tag at the N-terminus of both antigens. The 6×His-p54 ectodomain and 6×His-p30 proteins demonstrated molecular masses of 17.0 kDa and 30.0 kDa, respectively, on SDS-PAGE analysis (Fig. 1 ). The sizes of p30 and p54 ectodomain were consistent with the calculated values. According to western blot analysis, recombinant p54 ectodomain and p30 were successfully expressed in E. coli BL21(DE3). Protein purification of recombinant p54 ectodomain and p30 in E. coli system. Encouraged by the high expression of recombinant p54 ectodomain and p30 in E. coli , we next attempted to purify both recombinant antigens to validate their potential as ASFV antibody immuno-detection agents. Protein purification was performed using His-tag protein purification with IMAC (Immobilized Metal Affinity Chromatography), and the results showed that the purity of both proteins was higher than 95%, as visualized by UVP GelStudio PLUS (Analytik Jena GmbH). The sizes of the purified proteins were in agreement with the previously calculated values. The protein bands of p30 and p54 ectodomain were confirmed by SDS-PAGE and western blot analysis, as illustrated in Fig. 2 . The yield of purified proteins was determined by total protein assay and is provided in Table 1 . Table 1 Characterization of purified p30 and p54 ectodomain. Protein Purity (%) Yield (mg/ml) p30 > 95% 10.47 p54 ectodomain > 95% 10.38 Data availability statement All data generated or analyzed in this study are provided within the manuscript. Indirect ELISA detection of Anti-ASFV antibodies by recombinant p30 and p54 ectodomain. To validate the potential for immunodetection using our recombinant p30 and p54 ectodomains, we evaluated the reactivity of these purified ASFV antigens against anti-ASFV antibodies. This evaluation was conducted using serum samples from both ASFV-infected and ASFV-non-infected pigs. The ASFV antigens (p30 and p54 ectodomains) were immobilized on 96-well ELISA plates. The binding ability of each antigen to anti-ASFV antibodies was determined using serum from ASFV-infected pigs, with serum from ASFV-non-infected pigs serving as a negative control. The results from the indirect ELISA demonstrated clear reactivity in the antigen-immobilized wells probed with ASFV-infected serum, while no binding response was observed with ASFV-non-infected serum, as illustrated in Fig. 3 . These findings indicate that our recombinant p30 and p54 ectodomains, expressed in E. coli , can be used as antigens to detect anti-ASFV antibodies. Discussion African Swine Fever is a lethally contagious swine disease that spreads rapidly and affects domestic and wild swine worldwide. Although it does not pose a threat to human health, ASF outbreaks have devastated the global swine industry, food supply, and food security. ASF is currently affecting several regions, including African countries, Europe, the Caribbean, South America, and Asia 2 – 4 . Currently, there are no safe and effective vaccines to provide protective immunity against ASFV. In this context, early detection of ASFV is a critical step for disease containment to prevent the widespread transmission of ASFV 17 . During ASFV pandemic situations, recovered ASFV carrier pigs and persistently infected wild pigs pose the biggest challenge to disease control. The presence of ASFV antibodies is directly associated with ASFV infection; therefore, detecting these antibodies is invaluable for diagnosis. Consequently, laboratory assays must include the detection of both ASFV and ASFV-specific antibodies (Blome et al., 2013; M. C. Gallardo et al., 2015; Nah et al., 2022). Therefore, a simpler, reliable, and cost-effective detection method must be developed based on efficient, low-cost specimen collection, accurate diagnostic testing, and non-infectious material 17 . ASFV antibody-based detection approaches using serum as a test specimen and live virus as an antigen have been approved by the World Organization for Animal Health (OIE). However, using live virus requires level 3 biosafety facilities for propagation and handling of the antigen. Therefore, antibody-based immunodetection using recombinant ASFV antigens could avoid the risks and difficulties associated with handling live virus 17 , 23 – 26 . Previous reports suggest that ASFV proteins, including p30, p54, and p72, could be used as diagnostic antigens to detect anti-ASFV antibodies in sera. However, producing these recombinant antigens mainly relies on mammalian production systems, including insect cells, which are expensive, laborious, and time-consuming 23 , 25 , 27 . In this study, we aimed to produce recombinant p30 and p54 ectodomains in an E. coli system with high production yield and validate their potential application as antigens to detect ASFV antibodies using an indirect ELISA platform. The native structure of the p54 antigen consists of an N-terminal cytosolic domain, a transmembrane domain, and a C-terminal ectodomain. We hypothesized that the N-terminal cytosolic domain and transmembrane domain might have a high propensity for aggregation; therefore, only the C-terminal ectodomain was selected to generate recombinant p54 antigen in this study. According to western blot analysis, our recombinant p30 and p54 ectodomains were highly expressed in the soluble fraction with the expected sizes. Building on successful expression in E. coli , the recombinant p30 and p54 ectodomains were further purified by Ni-column chromatography. In a single-step purification process, p30 and p54 ectodomains demonstrated high production yields of 10.47 mg/ml and 10.38 mg/ml, respectively. The purified recombinant antigens clearly demonstrated their binding ability against ASFV antibodies in serum derived from ASFV-infected pigs, with no binding observed in serum from ASFV-non-infected pigs. These results confirmed that ASFV antigens expressed in the E. coli platform could still retain their binding ability against their counterpart antibodies in ASFV-infected pig serum. These results corroborate previous reports on the utility of p30 and p54 expressed in E. coli for early detection of ASFV antibodies in serum 17 , 18 . Overall, the results showed that our recombinant p30 and p54 could be successfully expressed in E. coli system with high yields and demonstrated their binding ability against their ASFV-specific antibodies in ASFV-infected pig serum. Hence, our recombinant antigens represent a simpler alternative to existing mammalian expression systems, offering savings in time and resources, and could be potentially used to develop a reliable immunodetection tools against ASFV antibodies. Conclusion In this study, we successfully achieved the recombinant expression of two ASFV structural proteins, p30 and the p54 ectodomain from genotype II ASFV, in E. coli BL21(DE3). Both proteins demonstrated strong selective binding ability in serum from ASFV-infected pigs, with nearly undetectable binding observed in serum from uninfected pigs, confirming their high selectivity and effectiveness. These findings highlight the potential of our recombinant p30 and p54 ectodomains as robust antigens for developing an indirect ELISA-based detection assay for anti-ASFV antibodies. The successful expression and functionality of these proteins in E. coli present a simpler, more reliable, and cost-effective alternative to mammalian expression systems. Materials and Methods Construction of recombinant proteins. The amino acid sequences of p30 and p54 proteins from the ASFV Georgia 2007/1 strain were retrieved from GenBank (GenBank Accession number FR682468.1). The cytosolic domain and transmembrane region of p54 were omitted to generate recombinant p54 ectodomain encompassing amino acids 60–178. The amino acid sequence of p30 retrieved from ASFV Georgia 2007/1 strain remained unchanged. The amino acid sequences of recombinant p30 and p54 were reverse-translated to nucleotide sequences and codon-optimized for E. coli expression. An optimized nucleotide sequence encoding p30 and p54 was synthesized and separately cloned into pET-21a(+) and pET-28b(+) expression vector, respectively (Novagen, San Diego, CA, USA). A hexa-histidine tag was added at N-terminal for detection and purification purposes through double enzyme digestion with NdeI and XhoI (New England Biolabs, Ipswich, MA, USA). Expression of recombinant antigens p54 ectodomain and p30 and Western blot analysis. To prepare whole cell lysates for western blot analysis, recombinant pET28b-6×His-p54 ectodomain and pET21a-6×His-p30 plasmids were chemically transformed into E. coli BL21(DE3) cells to generate recombinant E. coli for p54 ectodomain and p30 protein expression. The transformed bacteria were grown overnight at 37°C on Luria Bertani (LB) agar supplemented with kanamycin (50 µg/ml) for p54 or ampicillin (100 µg/ml) for p30. A single colony of each transformant was inoculated into LB broth supplemented with the respective selective antibiotic and cultured with shaking at 37°C overnight. The overnight E. coli culture (1% vol) was transferred into 50 ml of LB medium supplemented with the selective antibiotic in a 250 ml Erlenmeyer flask and incubated at 37°C with shaking until an optical density (OD) of 0.5 at 600 nm was reached. Protein expression was induced by adding IPTG to a final concentration of 0.1 mM and incubating for an additional 4 h at 37°C with shaking. The induced culture was pelleted at 5,000×g for 10 min at 4°C, washed with 20 ml PBS buffer, and harvested by centrifugation. Cell pellets were resuspended in ice-cold PBS buffer, followed by sonication on ice for 8 cycles (15 sec on and 45 sec off) at 40% amplitude using a Cole-Parmer ultrasonic homogenizer (CPX750). The lysate was then spun down at 16,000×g for 20 min at 4°C. The supernatant was collected as the soluble whole-cell lysate fraction and used for western blot analysis. Proteins (4 µg total per well) were mixed with SDS-loading dye containing dithiothreitol (DTT) and heated at 95°C for 5 min. Proteins were separated by 15% SDS-PAGE using 130 Volts constant for 90 min (BioRad), and western blot analysis was performed according to standard protocols. Briefly, protein samples were transferred onto polyvinylidene fluoride membranes using the Trans-Blot® Turbo™ transfer system (BioRad) in turbo mode (mixed MW program). After blocking with 5% skim milk in Tris-buffered saline with 0.1% Tween (TBST) buffer and washing with TBST buffer, membranes were probed with mouse anti-His-HRP (SouthernBiotech) diluted at 1:20,000 in TBST buffer to detect the His-tagged proteins, p30 and p54. Target proteins were visualized using SuperSignal™ West Femto maximum sensitivity substrate (Thermo Scientific™) for the HRP enzyme, and chemiluminescence signals were captured using Chemiluminescence mode by UVP GelStudio PLUS (Analytik Jena GmbH). Protein Purification. Induced bacterial cell cultures (1L) were pelleted by centrifugation. After washing with ice-cold PBS buffer, cell pellets were weighed and resuspended in binding buffer at a ratio of 1 g cell wet weight per 20 ml binding buffer containing 20 mM sodium phosphate, 500 mM NaCl, and 20 mM imidazole (pH 7.4). DNase I solution (Thermo Fisher Scientific) was added to the cell suspensions before cell disruption to reduce viscosity. Phenylmethylsulfonyl fluoride (PMSF) was added to the cell suspension at a final concentration of 0.5 mM to inhibit protease activities during cell disruption. The cell suspensions were disrupted using a continuous flow cell disrupter (CF1 Model, Constant Systems) at 36,000–38,000 psi at 4°C by passing five times and centrifuged at 22,000×g for 30 min at 4°C. The clarified lysate was filtered through a 0.2-µm PES syringe filter before sample loading. The sample was initially loaded through a 1 ml HisTrap HP column (Cytiva) using an ÄKTA Pure protein purification system at 1 ml/min. Non-specific binding proteins were washed with a washing buffer containing 20 mM sodium phosphate, 500 mM NaCl, and 60 mM imidazole, pH 7.4. The captured protein was eluted with an elution buffer containing 20 mM sodium phosphate, 500 mM NaCl, and 250 mM imidazole, pH 7.4. The pooled eluent was desalted into HEPES buffer containing 100 mM HEPES, 150 mM NaCl, and 200 mM arginine, pH 8.0, and concentrated using a 3-kDa molecular weight cut-off ultrafiltration spin filter (Amicon Ultra, Merck). The final purity of the proteins was evaluated by SDS-PAGE and Coomassie staining. Protein concentration was quantified using a BCA protein assay kit (Thermo Fisher Scientific). Antibody-binding activity by Indirect ELISA. Recombinant p30 and p54 ectodomain (100 µg/ul) in coating buffer (0.1 M Carbonate buffer, pH 9.4) were immobilized on 96-well plates overnight at 4°C, then washed twice with PBS (pH 7.4) with 0.05% Tween-20. The plates were then blocked with PBS (pH 7.4) with 5% (w/v) BSA for 3 h at room temperature and washed three times with PBS (pH 7.4) with 0.05% Tween-20. To measure binding activity between recombinant proteins and ASFV-antibodies, different titers of sera (ASFV-infected and ASFV non-infected) ranging from 1/400 to 1/25 dilution were applied to wells coated with either p30 or p54 for 1 h at room temperature. Binding interaction was detected by goat anti-pig IgG-HRP conjugate (SPC RT CO., LTD) at 1/2500 dilution in PBS (pH 7.4) with 0.05% Tween-20. After 1 h of incubation at room temperature, plates were washed and then incubated with TMB (Sigma) for 30 min in the dark. The reaction was quenched with 3 M H2SO4, and the absorbance of the wells was measured at 650 nm. Declarations Author contributions. B.M. designed all research, performed all research, analyzed data, and wrote the paper. P.O design all research, performed protein expression, western blot, ELISA experiments, and analyzed data. K.K. performed gene construction and analyzed data. T.P., P.S., T.W., and T.P. performed protein expression and purification. N.N. conceptualized the project, designed research, analyzed data, and wrote the paper. Acknowledgements. We really appreciated Faculty of Veterinary Science, Mahidol University and other supportive staffs for providing us with materials and kind assistance. Additional Information - competing financial interests. The authors declare no competing financial interests. Data availability statement All data generated or analyzed in this study are provided within the manuscript. References Cisek AA, Dąbrowska I, Gregorczyk KP, Wyżewski Z (2016) African Swine Fever Virus: a new old enemy of Europe. Ann Parasitol 62:161–167 Dixon LK, Stahl K, Jori F, Vial L, Pfeiffer DU (2020) African Swine Fever Epidemiology and Control. Annu Rev Anim Biosci 8:221–246 Guinat C et al (2016) Transmission routes of African swine fever virus to domestic pigs: current knowledge and future research directions. 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J Clin Microbiol 44:3114–3121 Neilan JG et al (2004) Neutralizing antibodies to African swine fever virus proteins p30, p54, and p72 are not sufficient for antibody-mediated protection. Virology 319:337–342 Additional Declarations The authors declare no competing interests. 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-5017399","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":348326442,"identity":"ef928d06-5f71-4155-9613-44e6982bf13d","order_by":0,"name":"Bunyarit Meksiriporn","email":"","orcid":"","institution":"Department of Biology, Faculty of Science, King Mongkut’s Institute of Technology Ladkrabang, Bangkok, Thailand","correspondingAuthor":false,"prefix":"","firstName":"Bunyarit","middleName":"","lastName":"Meksiriporn","suffix":""},{"id":348326443,"identity":"e23fbffb-cf61-4330-a4b4-06dbd4fcf94f","order_by":1,"name":"Puey 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Pisitkun","email":"","orcid":"","institution":"Center of Excellence in Systems Biology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand","correspondingAuthor":false,"prefix":"","firstName":"Trairak","middleName":"","lastName":"Pisitkun","suffix":""},{"id":348326449,"identity":"87ecf769-d74e-4dd9-81ae-4315d4d1183c","order_by":7,"name":"Natharin Ngamwongsatit","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAElEQVRIiWNgGAWjYHACNiCWgDA/MDAksDEkMB5gYGDGo4MZoYVxBkQLAzFaoEweoBYGQlrM2c8fe/Bzj0Ueg3T7xc+2OXZ5fOzJBw4wVFgnNuDQYtmTzG7Y80yimEHmTLF07rbkYjaeZwkHGM6k49RicCCZTYLngERig0ROAlALc2KbRI7BAca2w7i1nH/MJvkHoiX5t+W2eqiWf3i03Ehmk4bYkn5MmnHbYaiWBnxaHptJywC1AFWyWfZuO57YBvJLwrF0Y9wOS3wm+eZAXWK/RPrjGz+3VSfOb08++OBDjbUsLi1wwMbAY4DgJRBSDgHsD4hTNwpGwSgYBSMOAAAZzFymZRMy7gAAAABJRU5ErkJggg==","orcid":"","institution":"Department of Clinical Sciences and Public Health, Faculty of Veterinary Science, Mahidol University","correspondingAuthor":true,"prefix":"","firstName":"Natharin","middleName":"","lastName":"Ngamwongsatit","suffix":""}],"badges":[],"createdAt":"2024-09-02 10:08:53","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":true,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":true},"doi":"10.21203/rs.3.rs-5017399/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5017399/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":64004323,"identity":"c37207ff-df71-45ec-ad84-a18090c914c5","added_by":"auto","created_at":"2024-09-04 21:21:12","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":9475,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eWestern blot analysis of whole cell lysate derived from \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eE. coli\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e BL21(DE3) cells harboring either pET28b-6×His-p54 ectodomain or pET21a-6×His-p30 plasmid.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5017399/v1/a7bb7b75370dac12fee19855.jpg"},{"id":64004322,"identity":"8088c0a6-6ec7-4fe4-a357-3929f42f3723","added_by":"auto","created_at":"2024-09-04 21:21:12","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":11739,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProtein purification of p30 and p54 in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eE. coli\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eBL21(DE3). \u003c/strong\u003e(a) Representative Coomassie-stained SDS-PAGE gels loaded with p30 and p54 following expression and purification. The purity of all proteins was estimated to be \u0026gt;95%. Molecular weight (MW) markers are loaded in lane 1 of each gel. (b) Representative immunoblots of purified p30 and p54.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5017399/v1/ed645c566d7b341ad2db84f7.jpg"},{"id":64004321,"identity":"a06e0f4a-9370-4b7d-9087-63eda24fdf67","added_by":"auto","created_at":"2024-09-04 21:21:12","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":21700,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eELISA binding activity for purified p30 and p54 against ASFV-infected serum and ASFV-infected serum collected from pig. \u003c/strong\u003eAll data represent the average of three replicates, with error bars indicating the standard deviation (SD).\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5017399/v1/bbab1a2fabb7fad1057141d3.jpg"},{"id":64004719,"identity":"9aab4eb3-341a-4541-8b81-8006fa9e209f","added_by":"auto","created_at":"2024-09-04 21:29:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":539797,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5017399/v1/43402fac-7c70-48fc-ba76-267dc5b9204e.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eProduction of African Swine Fever Virus p54 ectodomain and p30 in an \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eE. coli\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e system and their potential application in immunodetection\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAfrican swine fever (ASF) is an extremely infectious viral disease caused by the African swine fever virus (ASFV) and has a high mortality rate of almost 100% in the swine industry worldwide \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. The discovery of ASF was first reported in the 1920s in Montgomery in Kenya in 1921 as a new disease leading to high lethality in in domestic pigs (\u003cem\u003eSus scrofa domesticus\u003c/em\u003e). Currently, ASF outbreak has spread across the globe leading to epidemics not merely in other African countries, but also in Europe, the Caribbean, South America, and Asia \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. The invasion of ASF has caused huge losses in the swine industry\u0026rsquo;s global economy and food security \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Accordingly, there is an urgent demand to develop an effective ASFV vaccine to combat the widespread of the disease. However, the generation of an effective ASF vaccine to has been under investigation. The development of ASF vaccine has largely been hindered because of restricted understanding of viral complexity and virulence factors involved in a pivotal role in immune protection. To address these shortcomings due to the absence of a safe and effective ASF vaccine, there is an urgent demand to develop reliable immunodetection assays for early detection of ASFV to prevent the widespread transmission of ASFV in swine farms. The clinical symptoms of ASF infection can range widely, from sudden deaths to undetectable cases, and are associated with various pathological changes. Therefore, laboratory tests must detect both the virus itself and ASFV-specific antibodies. (Blome et al., 2013; M. C. Gallardo et al., 2015; Nah et al., 2022).\u003c/p\u003e \u003cp\u003eASFV is the sole member of the family \u003cem\u003eAsfarviridae\u003c/em\u003e with a large double-stranded DNA virus in which genome size is ranging from 170 to 193 kbp long varying according to the strain and encodes 150\u0026ndash;160 ORFs playing critical roles in virus replication, host interactions, viral entry and immune evasion of host defenses; however, many proteins have not been identified \u003csup\u003e\u003cspan additionalcitationids=\"CR11 CR12\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. So far, it has been reported that up to 68 structural proteins have been characterized from the virion alone \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. In comparison to other viruses, ASFV possesses a larger and more complex structure than most other viruses, and its mechanistic understanding regarding defense and neutralization process has not been completely comprehended \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eSeveral ELISA-based serological tests include p72 and p30 antigens which currently used as the bases for commercial assays of anti-ASFV antibodies \u003csup\u003e\u003cspan additionalcitationids=\"CR16 CR17 CR18\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. In addition to p30 and p72, another promising target is p54, which is the 183 amino acid structural protein, a product of the E183L gene, localized in the inner envelope virion. The p54 antigen is a type II membrane protein consisting of the cytoplasmic inner N-terminus of 60 amino acids, a transmembrane domain, and C-terminal ectodomain of 131 amino acids \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. In combination with p30, p54 involves in the binding of the virion to target cells \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. An indirect ELISA-based detection assay of anti-ASFV antibodies demonstrated 98% sensitivity and 97% specificity compared to the OIE ELISA \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Furthermore, the ASFV antibody-positive samples remained positive for p54 reactivity after storage for one month at 37\u0026deg;C. Therefore, p54 could represent an ideal target for the detection of ASFV antibodies \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Currently, ASFV antigens are mainly produced using mammalian expression systems, such as the baculovirus expression system, which is laborious, expensive, time-consuming, and difficult to scale up. To ameliorate the challenging problems due to mammalian expression system, \u003cem\u003eE. coli\u003c/em\u003e systems have been employed as an alternative platform; however, the expression of the p54 antigen in \u003cem\u003eE. coli\u003c/em\u003e has shown low expression and solubility due to the p54 structure itself, which includes a hydrophobic transmembrane domain that tends to have a high aggregation propensity.\u003c/p\u003e \u003cp\u003eHere, to overcome the bottleneck, we sought to improve the expression and solubility of p54 in \u003cem\u003eE. coli\u003c/em\u003e system by selectively expression of p54 ectodomain and validated the ASFV-specific antibody detection ability of our recombinant p54 ectodomain expressed in \u003cem\u003eE. coli\u003c/em\u003e platform. Additionally, we expressed p30 antigen which is a highly expression ASFV antigen in \u003cem\u003eE. coli\u003c/em\u003e as a benchmark. Our recombinant p54 ectodomain and p30 were able to selectively bind to their specific antibodies in ASFV-infected serum, while no binding ability was observed in ASFV-noninfected serum. Collectively, our recombinant p54 ectodomain and p30 could be highly expressed in \u003cem\u003eE. coli\u003c/em\u003e with high production yield which render savings in time and resources. At the same time, this provides a reliable tool for generation of indirect ELISA assay against ASFV.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eExpression of recombinant antigens p54 ectodomain and p30 in\u003c/b\u003e \u003cb\u003eE. coli\u003c/b\u003e \u003cb\u003esystem.\u003c/b\u003e To efficiently express the p54 antigen in \u003cem\u003eE. coli\u003c/em\u003e, the cytosolic domain and transmembrane region of p54 were excluded. Only the p54 ectodomain region (amino acids 61\u0026ndash;178) was used in this study, while the amino acid sequence of p30 remained unchanged. Recombinant p54 ectodomain and p30 were heat-shock transformed and expressed in \u003cem\u003eE. coli\u003c/em\u003e BL21(DE3) using the pET28a system. Detection of protein expression was performed via a 6xhistidine tag at the N-terminus of both antigens. The 6\u0026times;His-p54 ectodomain and 6\u0026times;His-p30 proteins demonstrated molecular masses of 17.0 kDa and 30.0 kDa, respectively, on SDS-PAGE analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The sizes of p30 and p54 ectodomain were consistent with the calculated values. According to western blot analysis, recombinant p54 ectodomain and p30 were successfully expressed in \u003cem\u003eE. coli\u003c/em\u003e BL21(DE3).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eProtein purification of recombinant p54 ectodomain and p30 in\u003c/b\u003e \u003cb\u003eE. coli\u003c/b\u003e \u003cb\u003esystem.\u003c/b\u003e Encouraged by the high expression of recombinant p54 ectodomain and p30 in \u003cem\u003eE. coli\u003c/em\u003e, we next attempted to purify both recombinant antigens to validate their potential as ASFV antibody immuno-detection agents. Protein purification was performed using His-tag protein purification with IMAC (Immobilized Metal Affinity Chromatography), and the results showed that the purity of both proteins was higher than 95%, as visualized by UVP GelStudio PLUS (Analytik Jena GmbH). The sizes of the purified proteins were in agreement with the previously calculated values. The protein bands of p30 and p54 ectodomain were confirmed by SDS-PAGE and western blot analysis, as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The yield of purified proteins was determined by total protein assay and is provided in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCharacterization of purified p30 and p54 ectodomain.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\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 \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProtein\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePurity (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eYield (mg/ml)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ep30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;95%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10.47\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ep54 ectodomain\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;95%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10.38\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003eData availability statement\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003eAll data generated or analyzed in this study are provided within the manuscript.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eIndirect ELISA detection of Anti-ASFV antibodies by recombinant p30 and p54 ectodomain.\u003c/b\u003e To validate the potential for immunodetection using our recombinant p30 and p54 ectodomains, we evaluated the reactivity of these purified ASFV antigens against anti-ASFV antibodies. This evaluation was conducted using serum samples from both ASFV-infected and ASFV-non-infected pigs. The ASFV antigens (p30 and p54 ectodomains) were immobilized on 96-well ELISA plates. The binding ability of each antigen to anti-ASFV antibodies was determined using serum from ASFV-infected pigs, with serum from ASFV-non-infected pigs serving as a negative control.\u003c/p\u003e \u003cp\u003eThe results from the indirect ELISA demonstrated clear reactivity in the antigen-immobilized wells probed with ASFV-infected serum, while no binding response was observed with ASFV-non-infected serum, as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. These findings indicate that our recombinant p30 and p54 ectodomains, expressed in \u003cem\u003eE. coli\u003c/em\u003e, can be used as antigens to detect anti-ASFV antibodies.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAfrican Swine Fever is a lethally contagious swine disease that spreads rapidly and affects domestic and wild swine worldwide. Although it does not pose a threat to human health, ASF outbreaks have devastated the global swine industry, food supply, and food security. ASF is currently affecting several regions, including African countries, Europe, the Caribbean, South America, and Asia \u003csup\u003e\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Currently, there are no safe and effective vaccines to provide protective immunity against ASFV. In this context, early detection of ASFV is a critical step for disease containment to prevent the widespread transmission of ASFV \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. During ASFV pandemic situations, recovered ASFV carrier pigs and persistently infected wild pigs pose the biggest challenge to disease control. The presence of ASFV antibodies is directly associated with ASFV infection; therefore, detecting these antibodies is invaluable for diagnosis. Consequently, laboratory assays must include the detection of both ASFV and ASFV-specific antibodies (Blome et al., 2013; M. C. Gallardo et al., 2015; Nah et al., 2022). Therefore, a simpler, reliable, and cost-effective detection method must be developed based on efficient, low-cost specimen collection, accurate diagnostic testing, and non-infectious material \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eASFV antibody-based detection approaches using serum as a test specimen and live virus as an antigen have been approved by the World Organization for Animal Health (OIE). However, using live virus requires level 3 biosafety facilities for propagation and handling of the antigen. Therefore, antibody-based immunodetection using recombinant ASFV antigens could avoid the risks and difficulties associated with handling live virus \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan additionalcitationids=\"CR24 CR25\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Previous reports suggest that ASFV proteins, including p30, p54, and p72, could be used as diagnostic antigens to detect anti-ASFV antibodies in sera. However, producing these recombinant antigens mainly relies on mammalian production systems, including insect cells, which are expensive, laborious, and time-consuming \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn this study, we aimed to produce recombinant p30 and p54 ectodomains in an \u003cem\u003eE. coli\u003c/em\u003e system with high production yield and validate their potential application as antigens to detect ASFV antibodies using an indirect ELISA platform. The native structure of the p54 antigen consists of an N-terminal cytosolic domain, a transmembrane domain, and a C-terminal ectodomain. We hypothesized that the N-terminal cytosolic domain and transmembrane domain might have a high propensity for aggregation; therefore, only the C-terminal ectodomain was selected to generate recombinant p54 antigen in this study. According to western blot analysis, our recombinant p30 and p54 ectodomains were highly expressed in the soluble fraction with the expected sizes. Building on successful expression in \u003cem\u003eE. coli\u003c/em\u003e, the recombinant p30 and p54 ectodomains were further purified by Ni-column chromatography. In a single-step purification process, p30 and p54 ectodomains demonstrated high production yields of 10.47 mg/ml and 10.38 mg/ml, respectively. The purified recombinant antigens clearly demonstrated their binding ability against ASFV antibodies in serum derived from ASFV-infected pigs, with no binding observed in serum from ASFV-non-infected pigs. These results confirmed that ASFV antigens expressed in the \u003cem\u003eE. coli\u003c/em\u003e platform could still retain their binding ability against their counterpart antibodies in ASFV-infected pig serum. These results corroborate previous reports on the utility of p30 and p54 expressed in \u003cem\u003eE. coli\u003c/em\u003e for early detection of ASFV antibodies in serum \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOverall, the results showed that our recombinant p30 and p54 could be successfully expressed in \u003cem\u003eE. coli\u003c/em\u003e system with high yields and demonstrated their binding ability against their ASFV-specific antibodies in ASFV-infected pig serum. Hence, our recombinant antigens represent a simpler alternative to existing mammalian expression systems, offering savings in time and resources, and could be potentially used to develop a reliable immunodetection tools against ASFV antibodies.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, we successfully achieved the recombinant expression of two ASFV structural proteins, p30 and the p54 ectodomain from genotype II ASFV, in \u003cem\u003eE. coli\u003c/em\u003e BL21(DE3). Both proteins demonstrated strong selective binding ability in serum from ASFV-infected pigs, with nearly undetectable binding observed in serum from uninfected pigs, confirming their high selectivity and effectiveness. These findings highlight the potential of our recombinant p30 and p54 ectodomains as robust antigens for developing an indirect ELISA-based detection assay for anti-ASFV antibodies. The successful expression and functionality of these proteins in \u003cem\u003eE. coli\u003c/em\u003e present a simpler, more reliable, and cost-effective alternative to mammalian expression systems.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e \u003cb\u003eConstruction of recombinant proteins.\u003c/b\u003e The amino acid sequences of p30 and p54 proteins from the ASFV Georgia 2007/1 strain were retrieved from GenBank (GenBank Accession number FR682468.1). The cytosolic domain and transmembrane region of p54 were omitted to generate recombinant p54 ectodomain encompassing amino acids 60\u0026ndash;178. The amino acid sequence of p30 retrieved from ASFV Georgia 2007/1 strain remained unchanged. The amino acid sequences of recombinant p30 and p54 were reverse-translated to nucleotide sequences and codon-optimized for \u003cem\u003eE. coli\u003c/em\u003e expression. An optimized nucleotide sequence encoding p30 and p54 was synthesized and separately cloned into pET-21a(+) and pET-28b(+) expression vector, respectively (Novagen, San Diego, CA, USA). A hexa-histidine tag was added at N-terminal for detection and purification purposes through double enzyme digestion with \u003cem\u003eNdeI\u003c/em\u003e and \u003cem\u003eXhoI\u003c/em\u003e (New England Biolabs, Ipswich, MA, USA).\u003c/p\u003e \u003cp\u003e \u003cb\u003eExpression of recombinant antigens p54 ectodomain and p30 and Western blot analysis.\u003c/b\u003e To prepare whole cell lysates for western blot analysis, recombinant pET28b-6\u0026times;His-p54 ectodomain and pET21a-6\u0026times;His-p30 plasmids were chemically transformed into \u003cem\u003eE. coli\u003c/em\u003e BL21(DE3) cells to generate recombinant \u003cem\u003eE. coli\u003c/em\u003e for p54 ectodomain and p30 protein expression. The transformed bacteria were grown overnight at 37\u0026deg;C on Luria Bertani (LB) agar supplemented with kanamycin (50 \u0026micro;g/ml) for p54 or ampicillin (100 \u0026micro;g/ml) for p30. A single colony of each transformant was inoculated into LB broth supplemented with the respective selective antibiotic and cultured with shaking at 37\u0026deg;C overnight. The overnight \u003cem\u003eE. coli\u003c/em\u003e culture (1% vol) was transferred into 50 ml of LB medium supplemented with the selective antibiotic in a 250 ml Erlenmeyer flask and incubated at 37\u0026deg;C with shaking until an optical density (OD) of 0.5 at 600 nm was reached. Protein expression was induced by adding IPTG to a final concentration of 0.1 mM and incubating for an additional 4 h at 37\u0026deg;C with shaking. The induced culture was pelleted at 5,000\u0026times;g for 10 min at 4\u0026deg;C, washed with 20 ml PBS buffer, and harvested by centrifugation. Cell pellets were resuspended in ice-cold PBS buffer, followed by sonication on ice for 8 cycles (15 sec on and 45 sec off) at 40% amplitude using a Cole-Parmer ultrasonic homogenizer (CPX750). The lysate was then spun down at 16,000\u0026times;g for 20 min at 4\u0026deg;C. The supernatant was collected as the soluble whole-cell lysate fraction and used for western blot analysis. Proteins (4 \u0026micro;g total per well) were mixed with SDS-loading dye containing dithiothreitol (DTT) and heated at 95\u0026deg;C for 5 min. Proteins were separated by 15% SDS-PAGE using 130 Volts constant for 90 min (BioRad), and western blot analysis was performed according to standard protocols. Briefly, protein samples were transferred onto polyvinylidene fluoride membranes using the Trans-Blot\u0026reg; Turbo\u0026trade; transfer system (BioRad) in turbo mode (mixed MW program). After blocking with 5% skim milk in Tris-buffered saline with 0.1% Tween (TBST) buffer and washing with TBST buffer, membranes were probed with mouse anti-His-HRP (SouthernBiotech) diluted at 1:20,000 in TBST buffer to detect the His-tagged proteins, p30 and p54. Target proteins were visualized using SuperSignal\u0026trade; West Femto maximum sensitivity substrate (Thermo Scientific\u0026trade;) for the HRP enzyme, and chemiluminescence signals were captured using Chemiluminescence mode by UVP GelStudio PLUS (Analytik Jena GmbH).\u003c/p\u003e \u003cp\u003e \u003cb\u003eProtein Purification.\u003c/b\u003e Induced bacterial cell cultures (1L) were pelleted by centrifugation. After washing with ice-cold PBS buffer, cell pellets were weighed and resuspended in binding buffer at a ratio of 1 g cell wet weight per 20 ml binding buffer containing 20 mM sodium phosphate, 500 mM NaCl, and 20 mM imidazole (pH 7.4). DNase I solution (Thermo Fisher Scientific) was added to the cell suspensions before cell disruption to reduce viscosity. Phenylmethylsulfonyl fluoride (PMSF) was added to the cell suspension at a final concentration of 0.5 mM to inhibit protease activities during cell disruption. The cell suspensions were disrupted using a continuous flow cell disrupter (CF1 Model, Constant Systems) at 36,000\u0026ndash;38,000 psi at 4\u0026deg;C by passing five times and centrifuged at 22,000\u0026times;g for 30 min at 4\u0026deg;C. The clarified lysate was filtered through a 0.2-\u0026micro;m PES syringe filter before sample loading. The sample was initially loaded through a 1 ml HisTrap HP column (Cytiva) using an \u0026Auml;KTA Pure protein purification system at 1 ml/min. Non-specific binding proteins were washed with a washing buffer containing 20 mM sodium phosphate, 500 mM NaCl, and 60 mM imidazole, pH 7.4. The captured protein was eluted with an elution buffer containing 20 mM sodium phosphate, 500 mM NaCl, and 250 mM imidazole, pH 7.4. The pooled eluent was desalted into HEPES buffer containing 100 mM HEPES, 150 mM NaCl, and 200 mM arginine, pH 8.0, and concentrated using a 3-kDa molecular weight cut-off ultrafiltration spin filter (Amicon Ultra, Merck). The final purity of the proteins was evaluated by SDS-PAGE and Coomassie staining. Protein concentration was quantified using a BCA protein assay kit (Thermo Fisher Scientific).\u003c/p\u003e \u003cp\u003e \u003cb\u003eAntibody-binding activity by Indirect ELISA.\u003c/b\u003e Recombinant p30 and p54 ectodomain (100 \u0026micro;g/ul) in coating buffer (0.1 M Carbonate buffer, pH 9.4) were immobilized on 96-well plates overnight at 4\u0026deg;C, then washed twice with PBS (pH 7.4) with 0.05% Tween-20. The plates were then blocked with PBS (pH 7.4) with 5% (w/v) BSA for 3 h at room temperature and washed three times with PBS (pH 7.4) with 0.05% Tween-20. To measure binding activity between recombinant proteins and ASFV-antibodies, different titers of sera (ASFV-infected and ASFV non-infected) ranging from 1/400 to 1/25 dilution were applied to wells coated with either p30 or p54 for 1 h at room temperature. Binding interaction was detected by goat anti-pig IgG-HRP conjugate (SPC RT CO., LTD) at 1/2500 dilution in PBS (pH 7.4) with 0.05% Tween-20. After 1 h of incubation at room temperature, plates were washed and then incubated with TMB (Sigma) for 30 min in the dark. The reaction was quenched with 3 M H2SO4, and the absorbance of the wells was measured at 650 nm.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions. \u003c/strong\u003eB.M. designed all research, performed all research, analyzed data, and wrote the paper. P.O design all research, performed protein expression, western blot, ELISA experiments, and analyzed data. K.K. performed gene construction and analyzed data. T.P., P.S., T.W., and T.P. performed protein expression and purification. N.N. conceptualized the project, designed research, analyzed data, and wrote the paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements. \u003c/strong\u003eWe really appreciated Faculty of Veterinary Science, Mahidol University and other supportive staffs for providing us with materials and kind assistance.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional Information - competing financial interests. \u003c/strong\u003eThe authors declare no competing financial interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed in this study are provided within the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCisek AA, Dąbrowska I, Gregorczyk KP, Wyżewski Z (2016) African Swine Fever Virus: a new old enemy of Europe. 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Virology 319:337\u0026ndash;342\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":"Mahidol University","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":"African swine fever, African swine fever virus, p54, p30, Indirect ELISA, Ectodomain","lastPublishedDoi":"10.21203/rs.3.rs-5017399/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5017399/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAfrican swine fever (ASF) is a lethally infectious viral disease caused by the African swine fever virus (ASFV), leading to a high mortality of almost 100% in domestic pigs worldwide. ASFV has significantly impacted the global swine industry and food security. Highly effective vaccines are in high demand; however, no current vaccines provide effective immunity against ASFV. Therefore, there is an urgent need to develop reliable immunodetection assays to prevent the spread of ASFV. Traditionally, ASFV antigens are produced using mammalian expression systems, which are labor-intensive, costly, time-consuming, and challenging to scale up. In this study, two ASFV structural proteins associated with viral infection, p30 and the p54 ectodomain from genotype II ASFV, were recombinantly expressed in E. coli BL21(DE3). The results demonstrated that recombinant p54 ectodomain and p30 were highly expressed in E. coli BL21(DE3) using the pET28a system. Both recombinant p54 ectodomain and p30 were then validated for their ability to serve as antigens to detect anti-ASFV antibodies in an indirect ELISA platform. The p54 ectodomain/p30-based indirect ELISA was validated using serum from ASFV-infected pigs and serum from ASFV-uninfected pigs. Both p54 ectodomain and p30 demonstrated binding ability in the serum from ASFV-infected pigs, while no binding was observed in the serum from ASFV-uninfected pigs. Collectively, our recombinant p30 and p54 ectodomain were successfully expressed in E. coli and can be used as antigens to develop an indirect ELISA-based detection assay for anti-ASFV antibodies.\u003c/p\u003e","manuscriptTitle":"Production of African Swine Fever Virus p54 ectodomain and p30 in an E. coli system and their potential application in immunodetection","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-09-04 21:21:07","doi":"10.21203/rs.3.rs-5017399/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":"b16680b0-97a5-4490-a6fc-e00251ad1a3d","owner":[],"postedDate":"September 4th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":36952735,"name":"Animal Science"}],"tags":[],"updatedAt":"2024-09-04T21:21:07+00:00","versionOfRecord":[],"versionCreatedAt":"2024-09-04 21:21:07","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5017399","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5017399","identity":"rs-5017399","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}