A novel DNA vaccine against Streptococcus bovis: study on multi-epitope DNA antigen based on RodA gene and its specific IgY antibody

A novel DNA vaccine against Streptococcus bovis: study on multi-epitope DNA antigen based on RodA gene and its specific IgY antibody | 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 A novel DNA vaccine against Streptococcus bovis: study on multi-epitope DNA antigen based on RodA gene and its specific IgY antibody Ge Liang, Yanrong Zhang, Xiya Yan, Lifa Fu, Jiayan Huang, Zhihui Tang, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6213136/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 Streptococcus bovis is one of the leading causes of infective endocarditis and is associated with colon cancer. It can also cause rumen acidosis in ruminants and cause pigeon sepsis. The prevalence of pathogens among susceptible animals not only poses a serious threat to human health but also causes losses to animal husbandry. Its harm cannot be ignored. Vaccination can effectively control and prevent infection. In this study, the T cell and B cell dominant epitope gene sequences screened from the Rod A gene of Streptococcus bovis were tandem with the mucosal immune adjuvant cholera toxin B subunit ( CTB ) gene. The codon was optimized as CTB-RodA-RodA ( CRR ) gene sequence. After artificial synthesis, the CRR gene was inserted into the eukaryotic expression vector pVAX1. The multi-epitope DNA vaccine pVAX1-CRR was successfully constructed. The pVAX1-CRR and immune adjuvant CTB were combined to immunize laying hens. The specific IgY in eggs was extracted by salting out method and named CRR-IgY. Preliminary exploration of pVAX1-CRR immunogenicity showed that the titer of CRR-IgY was as high as 1: 6400. The in vitro antibacterial effect of the CRR-IgY on Streptococcus bovis was detected. It was found that 10 mg/ml CRR-IgY could significantly inhibit the growth of Streptococcus bovis isolates. In summary, this study successfully screened, constructed and expressed the multi-epitope vaccine pVAX1-CRR of Streptococcus bovis. It produces a high level of antibodies and a good antibacterial effect. Biological sciences/Immunology/Vaccines/Dna vaccines Biological sciences/Microbiology/Vaccines/Dna vaccines Biological sciences/Biological techniques/Genetic engineering Streptococcus bovis DNA vaccines Recombinant protein IgY Immunization Adjuvants Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Streptococcus bovis is a facultative anaerobic bacteria. It belongs to group D streptococcus, Gram-positive, non-β-hemolytic Streptococcus. It is a normal flora in the human gastrointestinal tract and herbivores' digestive tract. It is common in the gastrointestinal tract and feces (Herrera et al. 2009 ;Jans et al. 2015 )[ 1,2 ]. In recent years, Streptococcus bovis has been associated with many human and animal diseases. It causes human infecting bacteremia, sepsis, infective endocarditis (Dekker et al. 2016;Pernow et al. 2022 ;Öberg et al. 2022 ;Corredoira et al. 2023 )[ 3–6 ] and colon cancer(Deng et al. 2020 ;Gupta et al. 2010 ;Boltin et al. 2015 ) [ 7–9 ]. It causes rumen acidosis in ruminants, pigeon sepsis and acute deaths (Russell et al. 2001;Cheng et al. 1998 ;De Herdt et al. 1994 )[ 10–12 ]. The incidence of Streptococcus bovis infection and its drug resistance rate has recently increased (Pompilio et al. 2019 ;Coffey et al. 2012 ;Corredoira et al. 2015 )[ 13–15 ]. The prevalence of pathogens among susceptible animals seriously threatens human health and causes losses to animal husbandry. The harm cannot be ignored. Developing and designing a vaccine with Streptococcus bovis has excellent clinical value. The new generation of vaccines, such as recombinant subunit and DNA vaccines have received considerable attention as alternatives to traditional vaccines. The multi-epitope vaccine is a research hotspot of new vaccines in human medicine and animal husbandry medicine. It contains multiple dominant B / T cell epitopes of pathogens and has the advantages of high safety and strong induction of specific immune responses(Gao et al. 2022 ) [ 16 ]. DNA vaccines encode a particular protein of antigen of exogenous gene and eukaryotic expression vector after recombination directly into the body, activating the host to produce an immune response. DNA vaccines has shown strong potential for application in preventing and treating various diseases (Jeon et al. 2002 )[ 17 ]. Epitope-based DNA vaccines have been tested as potential candidates for preventing bacterial, fungal, parasitic and viral infections (Silveira et al. 2021 ;Carvalho et al. 2010 ;Lowrie 2006 ;Rivas-Santiago et al. 2014;Zhang et al. 2016)[ 18–22 ]. Designing a multi-epitope DNA vaccine of Streptococcus bovis is conducive to ensuring human health, reducing the threat of Streptococcus bovis to susceptible animals and developing animal husbandry. In this study, the rod shape-determining protein ( RodA ) of Streptococcus bovis was screened as the target protein of the multi-epitope vaccine. RodA is very important in the growth and development of bacteria and is relatively conserved in different genera(Uehara et al. 2008) [ 23 ]. Structural analysis shows that RodA constitutes a suitable antigen epitope (Sjodt et al. 2018 )[ 24 ]. Therefore, RodA can be used as a potential target for the design of Streptococcus bovis vaccine. In this study, the T cell and B cell dominant epitope gene sequences screened from the RodA gene of Streptococcus bovis were connected in series with the mucosal immune adjuvant Cholera toxin subunit B ( CTB ) gene to CTB - RodA- RodA ( CRR ) gene sequence. Based on this, a recombinant Streptococcus bovis eukaryotic expression vector pVAX1-CRR was constructed and identified. The specific Immunoglobulin of yolk (IgY) produced by immunizing the hens with pVAX1-CRR was characterized. Materials and Methods Main strains, cells, and animals Streptococcus bovis ATCC 33317 was purchased from China Ningbo Mingzhou Biological Company. The clinical strain of Streptococcus bovis was isolated from the feces samples of yaks in Naqu, Tibet, China. Our laboratory provided HEK293T cells. The laying hens are provided by Professor Suizhong Cao of Sichuan Agricultural University. The breed is White Leghorn Provided by Experimental Animal Center of Sichuan Agricultural University.Feeding, experimental procedures, euthanasia methods and biosafety precautions have been approved by the Medical Ethics Review Committee of Sichuan University in China and are in line with relevant guidelines and regulations.All experiments were conducted in accordance with the ARRIVE guidelines and regulations. Design of multi-epitope antigen CRR The RodA of Streptococcus bovis was selected as the target of the DNA vaccine. The amino acid sequence and corresponding gene sequence of RodA of Streptococcus bovis were selected on National Center for Biotechnology Information ( NCBI ). The T / B cell epitopes of the RodA protein were predicted using the Immune Epitope Database ( IEDB ). The T and B cell epitopes were screened. The selected antigen epitopes were connected in series ( it was connected twice to amplify the signal ). The epitopes were screened in series with cholera toxin subunit B ( CTB ) at the N-terminal to improve the body's immunoreactivity. Finally, the fusion protein sequence was CTB-RodA-RodA ( CRR ). JCat online software was used for reverse translation and codon optimization of CRR. The BamH I restriction site was introduced at the 5 ′ end, and the EcoR I restriction site and protective sequence were introduced at the 3 ′ end. Shenggong Biotechnology synthesized the sequence. Prediction of antigenicity and physicochemical properties of the CRR protein VaxiJenv2.0 was used to predict the antigenicity of the CRR protein, and ExPASy ProtParam was used to evaluate its physicochemical properties. Structure prediction of the CRR protein The SOPMA online program predicted the CRR protein's secondary structure and the SWISS-MODEL online tool predicted its tertiary structure. Homology analysis of the CRR protein The CRR protein( first remove the CTB sequence and then perform BLAST analysis ) was subjected to BLAST homology analysis in common biological populations and target biological genomes. The biological categories added to the BLAST interface biologist are : Homo sampiens ( taxid : 9606 ), rodents ( taxid : 9989 ), Enterobacteriaceae and related endosymbionts ( taxid : 91347 ), Bacillus / Lactobacillus / Streptococcus group ( taxid : 91061 ), oxen, cattle ( taxid : 9903 ), birds ( taxid : 8782 ). Construction of pVAX1-CRR The synthesized CRR gene sequence was inserted into the pVAX1 vector by BamH I and EcoR I restriction sites and transformed into Escherichia coli(E.coli) DH5α. The positive clones were screened for kanamycin resistance, and BamH I and EcoR I were digested and identified. After identification, it was delivered to Sangon Biotechnology for DNA sequencing confirmation. The recombinant plasmid pVAX1-CRR is the DNA vaccine constructed in this study. pVAX1-CRR transfected in HEK293T cells. This experiment set up three groups: pVAX1-CRR transfection group, empty vector pVAX1 transfection group, and untransfected blank control group. Three holes were set up in each group. Transfection was performed according to the instructions of the PEI transfection reagent and Lipofectamine 2000. Before transfection, HEK293T cells were inoculated into 24-well plates at 0.5x105 per well. The cells were transfected when covered about 60% of the bottom of the well. One hour before transfection, the cells were replaced with fresh serum-free and antibiotic-free DMEM medium, 0.5mL / well. Lipofectamine 2000, or PEI, was used as a transfection reagent. For each well of cells, 4 µL of target DNA and 1 µL of transfection reagent were diluted with 100 µL serum-free medium and incubated at 37 ° C for 20 min to form transfection 105 ul working solution. The 105 µL transfection working solution was added to the 24-well plate drop by drop, and the blank control was added with 105 µL serum-free medium. The cells were cultured in 37°C, 5% CO2 incubator. After 8 hours of culture, the medium was changed to a complete medium ( DMEM medium with 10% FBS + 1% penicillin + 1% streptomycin ) and continued to be cultured for 40 hours. Immunofluorescence analysis of transfection rate HEK293T cells were fixed with 4% paraformaldehyde for 20 min at 48 h after transfection, washed three times with PBS, added 0.3% TritonX-100, permeabilized for 15 min, added 3% BSA, blocked for 15 min, incubated with mouse anti-CTB monoclonal antibody ( 1: 100 ) / mouse anti-streptococcal polyclonal antibody ( 1: 500 ), washed three times with PBS, and overnight at 4°C. Alexa Fluor ® 488 labeled goat anti-mouse IgG ( H + L ) ( 1: 200 ) was incubated for 1.5 h and washed three times with PBS. DAPI was added and incubated at room temperature for 15 min. Add an anti-fluorescence quenching agent to seal the tablets. The transfection rate was observed by fluorescence microscope. Western blot The pVAX1-CRR transfection group was set up, and the blank control was the pVAX1 plasmid transfection group. The protein lysate ( RIPA: PMSF = 50: 1 ) was incubated on ice for 10 min, 4°C, 14000 r / min, and centrifuged for 15 min to collect protein supernatant. Western blot was used to detect the specific expression of the fusion protein. Hen immunization and preparation of IgY In this experiment, three groups were set up: the DNA antigen group pre-immunized with CTB, the DNA antigen group without pre-immunization, and the inactivated antigen of Streptococcus bovis group pre-immunized with CTB. The pVAX1-CRR was introduced into E.coli DH5α for enrichment culture, and the DNA antigen preparation was adjusted to 500ng / µL by plasmid extraction kit. The ultrasonically disrupted streptococcus bovis ( ATCC33317 ) antigen was prepared into an antigen solution with a final 1 mg / mL protein concentration for later use. Seven days before immunization, the immune adjuvant CTB was used for pre-immunization. The three groups of antigens were used to immunize one laying hen during the formal immunization. The hen's wings, legs, and back muscles were multi-point immunized. Eggs were collected before and after immunization. The immunization plan for laying hens is shown in Table 1 . Eggs were collected for 10 consecutive weeks, and antibodies were extracted by the salting out method. IgY antibodies induced by pVAX1-CRR ( CRR-IgY )with or without CTB adjuvant and IgY antibodies induced by Streptococcus bovis ( Streptococcus bovis-IgY ) with CTB adjuvant were obtained. Table 1 Immunization program of laying hens Immunization and egg collection period group immunized 1 preimmune(0 days) CTB 600ug normal saline 600ug CTB 600ug 2 First immunization(7-9days) Streptococcus bovis DNA antigen 150µL Streptococcus bovis DNA antigen 150µL Inactivated Streptococcus bovis antigen 300ug 3Second immunization(14–21 days) Streptococcus bovis DNA antigen 150µL Streptococcus bovis DNA antigen 150µL Inactivated Streptococcus bovis antigen 300ug 4Third immunizations (28–52 days) Streptococcus bovis DNA antigen 150µL Streptococcus bovis DNA antigen 150µL Inactivated Streptococcus bovis antigen 300ug 5Fourth Immunization (62–72 days) Streptococcus bovis DNA antigen 150µL Streptococcus bovis DNA antigen 150µL Inactivated Streptococcus bovis antigen 300ug Enzyme-Linked Immunosorbent Assay(ELISA) The broken antigen of Streptococcus bovis was diluted to 100 µg / mL with the coating solution, and 100 µL was added to each well and blocked with 3% BSA. The yolk antibodies ( CRR-IgY and Streptococcus bovis-IgY ) were added to the sample to be tested, and HRP-rabbit anti-chicken IgY was used as the secondary antibody. After color development, the OD450 nm value of the sample was detected by a microplate reader. When the OD value of the detected immune IgY / negative IgY was ≥ 2.1, and the OD value of the immune IgY itself was greater than 0.21, the maximum dilution was the titer of the antibody IgY. Plate colony counting experiment The standard strain of Streptococcus bovis ( ATCC33317 ), Streptococcus bovis clinical strains, and E.coli BL21 were adjusted to a concentration of OD600 = 0.1 bacterial solution with LB. The standard strain of Streptococcus bovis was incubated with specific antibody CRR-IgY ( 10 mg/ml, 5 mg/ml, 2 mg/ml), non-specific antibody IgY antibodies induced by BL21 (BL21-IgY )( 10 mg/ml, 5 mg/ml, 2 mg/ml), positive control kanamycin ( 50µg / mL ) and control ( Pbs ) for 3h, then diluted 10 3 times, 100µL coated plate, 3 parallels in each group, cultured at 37°C for 12 h. The number of colony clones was observed and recorded. The inhibitory effect of CRR-IgY on the activity of clinical strains of Streptococcus bovis in vitro was detected. The clinical strains were incubated with specific antibody CRR-IgY ( 10 mg/ml), non-specific antibody BL21-IgY ( 10 mg/ml), kanamycin ( 50µg / mL ) and control ( Pbs ) for 3h, respectively. Other steps are the same as above. Escherichia coli was incubated with CRR-IgY ( 10mg / ml ), kanamycin ( 50µg / mL ), and control ( Pbs ) for 3h. Other steps are the same as above. statistical analysis SPSS 17.0 statistical software was used for statistical analysis. The Student's t-test evaluated the significance of the difference between groups. Each experiment was repeated more than 3 times. P < 0.05 was considered statistically significant. Results Design of multi-epitope antigen CRR The obtained Streptococcus bovis ATCC 33317 is one of the standard strains of group D Streptococcus bovis-Streptococcus equi complex. The following epitopes were screened from the RodA(NCBI GenBank, KFN86186.1) amino acid sequence. B cell epitopes: KNDWKL, GISWWII, HGKDIFYSLGMDTYQIN, WLDPFSYAKSIAY. T cell epitopes: ALITLPVMI, LQKDLGTAM, HGKDIFYSL, YQINRISAW, QQTQGMISI. In addition, to improve the body's immune response, the cholera toxin B subunit ( CTB ) can also be inserted into the N-terminal of the above epitope. The sequence is as follows ( NCBI GenBank, LC427969.1 ) : MIKLKFGVFFTVLLSSAYAHGTPQNITDLCAEYHNTQIHTLNDKILSYTESLAGKREMAIITFKNGETF QVEVPGSQHIDSQKKAIERMKDTLRIAYLTEAKVEKLCVWNNKTPHAIAAISMAN.The above epitopes were connected in series. The Streptococcus bovis RodA was connected in series twice to amplify the signal. Finally, the fusion protein sequence CTB-RodA-RodA ( CRR ) was obtained. Its amino acid sequence is: MIKLKFGVFFTVLLSSAYAHGTPQNITDLCAEYHNTQIHTLNDKILSYTESLAGKREMAIITFKNGETFQVEVPGS QHIDSQKKAIERMKDTLRIAYLTEAKVEKLCVWNNKTPHAIAAISMANGGGGSGGGGSGGGGSKNDWKLKK GISWWIIKKHGKDIFYSLGMDTYQINKKWLDPFSYAKSIAYKKALITLPVMIKKLQKDLGTAMKKHGKDIFYS LKKYQINRISAWKKQQTQGMISIKKKNDWKLKKGISWWIIKKHGKDIFYSLGMDTYQINKKWLDPFSYAKSIA YKKALITLPVMIKKLQKDLGTAMKKHGKDIFYSLKKYQINRISAWKKQQTQGMISI. JCat online software was used for reverse translation and codon optimization of CRR. The BamH I restriction site was introduced at the 5 ' end. The EcoR I restriction site and protective sequence were introduced at the 3 ' end. A multi-epitope vaccine cDNA sequence ( 1080 bp ) was generated. Prediction of antigenicity and physicochemical properties of the CRR protein The antigenicity score of the CRR protein was 0.5278, evaluated by VaxiJenv2.0, which was higher than the default threshold of 0.4 and had good antigenicity. The prediction results of physicochemical properties showed that the CRR protein contained 349 amino acids, the molecular weight was 40 kD, the isoelectric point was 9.97, the half-life was 30 h, the fat coefficient was 83.35, and the total average hydrophobic index was − 0.434, indicating that the CRR protein had good hydrophilicity and was soluble in water. The stability index is 29.15, indicating that the CRR protein has good stability. Structure prediction of the CRR protein The secondary structure of the CRR protein predicted by SOMPA showed that α-helix, extended strand, and random coil accounted for 43.27%, 22.64%, and 34.10%, respectively ( Fig. 1 ). The SWISS-MODEL online tool was used to predict the tertiary structure of the CRR protein ( Fig. 2 ). The CRR protein's global model quality estimate ( GMQE ) value was 0.3. The Lagrangian diagram was used to evaluate the quality of the tertiary structure. The results show that most of the dotted structures in the figure fall within the allowable range. The Z score plot was used to evaluate the quality of the entire tertiary structure. The Z score of the CRR protein was 0.72, indicating that it was within the characteristic range of natural proteins. Therefore, the credibility of the three-level structure is high. Homology analysis of the CRR protein The results showed that the fusion protein had high species specificity, and there was no cross between the prevention target organisms ( Streptococcus ) in humans, rodents, birds, cattle, intestinal flora, Bacillus / Lactobacillus / Streptococcus, and other biological categories. Construction and identification of eukaryotic expression vector pVAX1-CRR The synthesized CRR gene sequence was inserted into the pVAX1 vector by BamH I and EcoR I restriction sites. About 3000 and 1000 fragments were identified by agarose gel electrophoresis, consistent with the expected results ( Fig. 3 ). The sequencing results showed that the sequence was 100% consistent with the design sequence, and the recombinant plasmid pVAX1-CRR was constructed correctly. HEK293T cells expressed pVAX1-CRR successfully The recombinant expression plasmid pVAX1-CRR was transfected into HEK293T cells. The transfection rate was detected by immunofluorescence. The immunofluorescence staining showed apparent green fluorescence in HEK293T cells transfected with pVAX1-CRR ( Fig. 4 A / B ). In contrast, no green fluorescence was observed in cells transfected with pVAX1 ( Fig. 4 C ) and untransfected cells ( Fig. 4 D ), suggesting that the construction of pVAX1-CRR is effectively expressed in HEK293T cells. Image J analysis showed that the transfection efficiency was about 44–63%.The protein is mainly located in the cytoplasm. Western blot analyzes the CRR protein expressed in HEK293T cells. The expression of the CRR protein was detected by mouse anti-CTB monoclonal antibody ( 1: 100 ) ( Fig. 5 A ) and mouse anti-Streptococcus bovis polyclonal antibody ( 1: 500 ) ( Fig. 5 B ). The results showed that the protein expressed in HEK293T cells could specifically bind to two antibodies. The fusion protein showed a band at about 40KD ( Fig. 5 ), and the control did not, indicating that the expression of the CRR protein was successful. PVAX1-CRR induced a high concentration of IgY antibody. ELISA was used to continuously detect the titer of IgY in egg yolks 10 weeks after immunization. The particular antibody appeared about 2 weeks after vaccination, and the titer of the antibody increased gradually with the immunization time. The delay of the antibody in the yolk may be because the antibody was first produced in the serum, then transferred and accumulated in the yolk. After four DNA vaccine immunizations, the CRR-IgY titer gradually increased and peaked about 7 weeks after vaccination. The peak titer of CRR-IgY without CTB pre-immunization was finally stabilized at about 1: 4500, and the CRR-IgY with CTB pre-immunization was stabilized at 1: 6400 for more than 2 weeks, indicating that pre-immunization with CTB protein as an adjuvant increases the DNA immune effect of CRR-IgY by 40%. The titer of CRR-IgY after pre-immunization was close to that of Streptococcus bovis immunization ( 1: 8000 )( Fig. 6 ). CRR-IgY has an antibacterial effect on both standard and clinical strains of Streptococcus bovis. The antibacterial effect of IgY on Streptococcus bovis standard strain, clinical strain, and Escherichia coli BL21 was detected by plate colony counting experiment. Compared with the control group, 2 mg/ml CRR-IgY had no inhibitory effect on the growth of a standard strain of Streptococcus bovis. 5 mg/ml and 10 mg/ml CRR-IgY and kanamycin had an apparent inhibitory effect on the growth of a standard strain of Streptococcus bovis. Non-specific BL21-IgY had no inhibitory effect on the standard strain of Streptococcus bovis ( Fig. 7 A ). Compared with the control group, both CRR-IgY and kanamycin could significantly inhibit the growth of clinical strains of Streptococcus bovis. In contrast, non-specific BL21-IgY had no inhibitory effect on clinical strains of Streptococcus bovis ( Fig. 7 B ). Compared with the control group, CRR-IgY could not inhibit the growth of E.coli BL21, and kanamycin could significantly inhibit the growth of E.coli BL21 ( Fig. 7 C ). Therefore, the 10 mg/ml antibody CRR-IgY has an obvious antibacterial effect on standard strains and clinical strains of Streptococcus bovis and has no antibacterial effect on Escherichia coli. The non-specific antibody BL21-IgY had no antibacterial effect on the standard strain and clinical strain of Streptococcus bovis. It shows that the antibacterial effect of CRR-IgY against standard strains and clinical strains of Streptococcus bovis is specific and effective. Discussion Streptococcus bovis is a common pathogen affecting the health of cattle, which brings serious harm to the breeding and production of cattle. The fecal dissemination of pathogens and the persistence of pathogens in the environment make it more challenging to prevent and control pathogens in pastoral areas. It seriously threatens people's health in pastoral areas and causes losses to the breeding industry. In recent years, the resistance rate of Streptococcus bovis infection has increased. In 2005, Leclercq R et al.reported that tetracycline resistance was widespread in Streptococcus bovis, and the incidence of erythromycin resistance was also high(Leclercq et al. 2005 ) [ 25 ]. In 2019, Pompilio A et al.reported that the resistance rate of Streptococcus bovis to tetracycline was 36–77%, to erythromycin was 8.9–78%, and to clindamycin was 10.6–62% (Pompilio et al. 2019 )[ 13 ]. Currently, no efficient and convenient method for preventing and treating this pathogen exists. People have tried to control the growth of Streptococcus bovis in the rumen by vaccinating cattle to produce specific antibodies against the bacteria and have achieved some success. Gill et al. ( 2000 )[ 26 ] described that immunizing animals with Streptococcus bovis induces specific immune responses and reduces the excessive production of lactic acid in the rumen by calling specific anti-Streptococcus bovis antibodies. Shu et al. ( 1999 )[ 27 ] proved that intramuscular injection of live Streptococcus bovis vaccine effectively reduces acute lactic acidosis in animals fed a cereal-based diet. Developing multi-epitope vaccines with the continuous development of new vaccines is undoubtedly effective. Epitope vaccines have many advantages, such as high antigenicity, non-allergic, non-toxic, and simple molecular structure. They directly stimulate the body to produce specific immunity, which makes it challenging to produce autoimmune suppression or immune response (Devarakonda et al. 2023 ;Jyotisha et al. 2023)[ 28, 29 ]. Many studies have shown that DNA vaccines are safe, non-toxic, and effective and are one of the most promising immune technologies for pathogen prevention (Pagliari et al. 2023 ;Ghaffarifar 2018 )[ 30, 31 ]. Based on this, this study constructed an epitope-based DNA vaccine to prevent and control the infection of Streptococcus bovis. Rod shape-determining protein ( RodA ) was first identified as determining bacteria's short rod shape. It was later classified into the Shape, Elongation, Division, and Sporulation ( SEDS ) protein family, essential for bacterial growth and development. It is widely present in Gram-positive and Gram-negative bacteria but is relatively conserved in different genera (Uehara et al. 2008;Sjodt et al. 2018 )[ 23,24 ]. In 2008, Uehara et al.showed that the central role of RodA is not only related to the morphology of bacteria but also closely related to the synthesis and degradation of peptidoglycan in life processes such as bacterial elongation. In 2018, Sjodt et al.reported the crystal structure of RodA, which contains three huge outer rings. The outer ring contains many functional essential residues, while the outer ring of bacterial membrane proteins often constitutes a suitable B-cell epitope. In addition, there are also reports that there are highly conserved peptides in RodA. The induction of mutations in these conserved peptides will lead to changes in bacterial morphology and may lead to bacterial lysis. Therefore, using RodA as a target for Streptococcus bovis vaccine may produce better results. In this study, the key dominant epitopes of RodA of Streptococcus bovis were screened for fusion antigen design, which was connected in series with the intramolecular adjuvant CTB. At the same time, the epitopes screened by RodA were connected in series twice to enhance the immunogenicity and stability of the recombinant protein. After reverse translation and codon optimization, the gene sequence CRR was obtained. The recombinant Streptococcus bovis eukaryotic expression vector pVAX1-CRR was constructed. Immunofluorescence and Western blot verified the successful expression of the fusion protein. The specific IgY antibody against Streptococcus bovis extracted from eggs immunized with the pVAX1-CRR. It was found that the antibody level changed significantly after 2 weeks of immunization. The antibody level reached 1: 6400 after 7 weeks of vaccination and could be maintained for over 2 weeks. The titer of CRR-IgY was close to that of whole bacteria immunization, similar to that of Streptococcus bovis. Wang et al. (2019)[ 32 ] found that anti-K88 fiber-IgY could significantly inhibit the growth of E.coli K88, block the adhesion of bacteria in the intestinal mucosa, reduce diarrhea caused by E.coli in weaned piglets, and achieve the effect of prevention and treatment by co-culturing IgY with E.coli. The production of antibodies effectively reduces bacterial infection and protects the host. In this study, the in vitro antibacterial test showed that 10 mg/ml CRR-IgY could inhibit the growth of standard and clinical strains of Streptococcus bovis. The antibacterial effect of CRR-IgY on standard strains and clinical strains of Streptococcus bovis was specific and compelling. IgY induced by pVAX1-CRR produces practical bacteriostatic effects in vitro. This study hopes to achieve the purpose of efficient bacteriostasis by studying the bacteriostatic effect of anti-Streptococcus bovis. Further studies are needed to evaluate the impact of IgY in vivo on treating Streptococcus bovis infection. Previous studies have shown that the cholera toxin B subunit ( CTB ) is a classic mucosal immune adjuvant that enhances the immune effect of vaccines and can be used as a compelling candidate adjuvant for DNA vaccine inoculation (Hou et al. 2014 ;Verjan Garcia et al. 2023 )[ 33,34 ]. In this study, after four times of DNA vaccine immunizations, the peak titer of CRR-IgY without CTB pre-immunization was finally stabilized at about 1: 4500, and that of CRR-IgY with CTB pre-immunization was stabilized at 1: 6400. After 7 days of pre-immunization with CTB protein as an adjuvant, the immune effect of CRR-IgY could be effectively improved by 40%. This study successfully constructed a multi-epitope DNA vaccine, pVAX1-CRR, of Streptococcus bovis, expressed in eukaryotic cells. Hens immunized with this pVAX1-CRR produce specific IgY antibodies that inhibit the growth of Streptococcus bovis in vitro. It provides ideas and references for developing a Streptococcus bovis vaccine to prevent or treat Streptococcus bovis infection. The follow-up application promotion research intends to explore the possibility of pVAX1-CRR as a vaccine further. Conclusion In this experiment, the recombinant eukaryotic expression vector pVAX1-CRR was constructed. The successful expression of the CRR protein was verified. The CRR-IgY obtained by immunizing laying hens has good immunogenicity and significantly inhibits the growth of Streptococcus bovis isolates. These results indicate that pVAX1-CRR may be used as a promising DNA vaccine to prevent and treat Streptococcus bovis infection, which requires further experiments to explore. Abbreviations CTB Cholera toxin subunit B DMEM Dulbecco's modified Eagle medium E.coli Escherichia coli ELISA Enzyme-Linked Immunosorbent Assay GMQE global model quality estimate IgY Immunoglobulin of yolk Kan Kanamycin OD Optical density RodA Rod shape-determining protein SEDS The Shape, Elongation, Division, and Sporulation Declarations Acknowledgements We are very grateful to Professor Suolang sizhu of Tibet Agricultural and Animal Husbandry College for providing us with Tibetan yak feces samples. We are very grateful to Professor Suizhong cao of Sichuan Agricultural University for providing us with laying hens. Funding This work was supported by the key technology research project of Tibet Streptococcus bovis nucleic acid rapid detection kit and new prevention and control preparations. ( Grant numbers [No. : 2023 YFQ0051] ) and the key R & D program of Tibet Autonomous Region. ( Grant numbers [No. : XZ202201ZY0009N]) Ethics declarations Ethics approval and consent to participate All animal procedures were approved by the Animal Ethics Committee of Sichuan University. Conflict of interest The authors declare no financial or personal conflicts of interest. The authors declare no competing interests. Authors' contributions B. W. is responsible for design and resource support. G.L. is responsible for Streptococcus bovis gene analysis and construction, immune experiments, data collection and writing. L.F. is responsible for Streptococcus bovis reset gene design, DNA immunization. J.H. is responsible for the collection of Tibetan samples and the isolation and culture of Streptococcus bovis, the purchase and culture of standard strains, the immunization of laying hens, the extraction and purification of IgY, the determination of titer, and the determination of in vitro antibacterial experiments. Z.T. ,X.Y. and Y.Z. carried out the result analysis. C.Z. ,Y.Y. and X.D. reviewed and edited it. All authors reviewed the manuscript. Data availability statements The datasets generated and analysed during the current study are available in the [NCBI] repository. Streptococcus equinus rodA gene for rod shape-determining protein, partial cds, strain: ATCC 33317（NCBI GenBank, AB441161.1）rod shape-determining protein, partial [Streptococcus equinus ATCC 33317]（NCBI GenBank, BAH03909.1）Cholera Toxin B subunit, CTB（NCBI GenBank, LC427969.1） References Herrera, P., Kwon, Y. M. & Ricke, S. C. Ecology and pathogenicity of gastrointestinal Streptococcus bovis. 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DNA vaccines against COVID-19: Perspectives and challenges. Life sciences, 267, 118919. (2021). https://doi.org/10.1016/j.lfs.2020.118919 Carvalho, J. A., Rodgers, J., Atouguia, J., Prazeres, D. M. & Monteiro, G. A. DNA vaccines: a rational design against parasitic diseases. Expert Rev. Vaccines . 9 (2), 175–191. https://doi.org/10.1586/erv.09.158 (2010). Lowrie, D. B. DNA vaccines for therapy of tuberculosis: where are we now? Vaccine 24 (12), 1983–1989. https://doi.org/10.1016/j.vaccine.2005.11.010 (2006). Rivas-Santiago, B. & Cervantes-Villagrana, A. R. Novel approaches to tuberculosis prevention: DNA vaccines. Scand. J. Infect. Dis. 46 (3), 161–168. https://doi.org/10.3109/00365548.2013.871645 (2014). Zhang, H. & El Zowalaty, M. E. DNA-based influenza vaccines as immunoprophylactic agents toward universality. Future Microbiol. 11 (1), 153–164. https://doi.org/10.2217/fmb.15.110 (2016). Uehara, T. & Park, J. T. Growth of Escherichia coli: significance of peptidoglycan degradation during elongation and septation. J. Bacteriol. 190 (11), 3914–3922. https://doi.org/10.1128/JB.00207-08 (2008). Sjodt, M. et al. Structure of the peptidoglycan polymerase RodA resolved by evolutionary coupling analysis. Nature 556 (7699), 118–121. https://doi.org/10.1038/nature25985 (2018). Leclercq, R., Huet, C., Picherot, M., Trieu-Cuot, P. & Poyart, C. Genetic basis of antibiotic resistance in clinical isolates of Streptococcus gallolyticus (Streptococcus bovis). Antimicrob. Agents Chemother. 49 (4), 1646–1648. https://doi.org/10.1128/AAC.49.4.1646-1648.2005 (2005). Gill, H. S., Shu, Q. & Leng, R. A. Immunization with Streptococcus bovis protects against lactic acidosis in sheep. Vaccine 18 (23), 2541–2548. https://doi.org/10.1016/s0264-410x(00)00017-7 (2000). Shu, Q. et al. Immunisation against lactic acidosis in cattle. Res. Vet. Sci. 67 (1), 65–71. https://doi.org/10.1053/rvsc.1998.0284 (1999). Devarakonda, Y., Reddy, M. V. N. J., Neethu, R. S., Chandran, A. & Syal, K. Multi epitope vaccine candidate design against Streptococcus pneumonia. J. Biomol. Struct. Dyn. 41 (22), 12654–12667. https://doi.org/10.1080/07391102.2023.2167123 (2023). Jyotisha, Qureshi, R. & Qureshi, I. A. Development of a multi-epitope vaccine candidate for leishmanial parasites applying immunoinformatics and in vitro approaches. Front. Immunol. 14 , 1269774. https://doi.org/10.3389/fimmu.2023.1269774 (2023). Pagliari, S., Dema, B., Sanchez-Martinez, A., Zurbia-Flores, M., Rollier, C. S. & G., & DNA Vaccines: History, Molecular Mechanisms and Future Perspectives. J. Mol. Biol. 435 (23), 168297. https://doi.org/10.1016/j.jmb.2023.168297 (2023). Ghaffarifar, F. Plasmid DNA vaccines: where are we now? Drugs of today (Barcelona, Spain: 1998), 54(5), 315–333. (2018). https://doi.org/10.1358/dot.2018.54.5.2807864 Zhou, X., Wang, P., Chen, Y. & Ma, S. Y. Intact anti-LPS IgY is found in the blood after intragastric administration in mice. FEBS open. bio . 9 (3), 428–436. https://doi.org/10.1002/2211-5463.12571 (2019). Hou, J. et al. Cholera toxin B subunit acts as a potent systemic adjuvant for HIV-1 DNA vaccination intramuscularly in mice. Hum. vaccines immunotherapeutics . 10 (5), 1274–1283. https://doi.org/10.4161/hv.28371 (2014). Verjan Garcia, N., Celis, S., Dent, I. C., Matoba, N. & M., & Characterization and utility of two monoclonal antibodies to cholera toxin B subunit. Sci. Rep. 13 (1), 4305. https://doi.org/10.1038/s41598-023-30834-2 (2023). Additional Declarations No competing interests reported. Supplementary Files WESTERNBLOTORIGINALIMAGES.doc 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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14:53:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6213136/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6213136/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":82002333,"identity":"0bf15b7d-5345-41c4-bb20-bfb6a7efa97d","added_by":"auto","created_at":"2025-05-05 20:12:38","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":229734,"visible":true,"origin":"","legend":"\u003cp\u003ePrediction of the CRR protein secondary structure\u003c/p\u003e\n\u003cp\u003eh,Alpha helix;e,Extendedstrand;c,Randomcoil\u003c/p\u003e","description":"","filename":"image1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6213136/v1/b0b4c4971d63e5ceead8f47f.jpeg"},{"id":82002639,"identity":"d4693c80-5835-4877-99c3-52ba697bbe38","added_by":"auto","created_at":"2025-05-05 20:20:38","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":122240,"visible":true,"origin":"","legend":"\u003cp\u003eTertiary structure of the CRR protein and model quality assessment maps\u003c/p\u003e\n\u003cp\u003eA: tertiary structure B: Laspeyres plot. The dark green area is the allowable area, the light green area is the maximum permissible area, and the blank area is the non-allowable area C: Z score plot. The normalized QMEAN4 Score is the standardized qualitative model energy analysis score, and Protein Size ( Residues ) is the size of protein( residue ).\u003c/p\u003e","description":"","filename":"image2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6213136/v1/4c187ae74936ada14566575f.jpeg"},{"id":82002640,"identity":"54724085-7d01-4269-8865-a20f8f15a1aa","added_by":"auto","created_at":"2025-05-05 20:20:38","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":117025,"visible":true,"origin":"","legend":"\u003cp\u003eConstruction and identification of pVAX1-CRR\u003c/p\u003e\n\u003cp\u003eA : pVAX1-CRR vector ; B : Restriction enzyme digestion of recombinant plasmid pVAX1-CRR M : Marker ; 1 : pVAX1 ; 2 : pVAX1-CRR digested by BamH I and Hind III ;\u003c/p\u003e","description":"","filename":"image3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6213136/v1/9f5fda2e7fd900957e57ca84.jpeg"},{"id":82002337,"identity":"6f6c072d-61c4-4fe6-9d7d-4ef0b62dc69f","added_by":"auto","created_at":"2025-05-05 20:12:38","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":227009,"visible":true,"origin":"","legend":"\u003cp\u003eTransfection efficiency of pVAX1-CRR in HEK293T cells（400×）\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA: \u003c/strong\u003epVAX1-CRR (anti-CTB antibody)；B\u003cstrong\u003e：\u003c/strong\u003epVAX1-CRR (anti-streptococcus bovis antibody)；C\u003cstrong\u003e：\u003c/strong\u003epVAX1 (anti-CTB antibody)；D\u003cstrong\u003e：\u003c/strong\u003eHEK293T (anti-CTB antibody)\u003c/p\u003e","description":"","filename":"image4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6213136/v1/3ec5f3483a6a6143163e5593.jpeg"},{"id":82003236,"identity":"a9fdec81-b98b-4c2d-a3c8-e6f3e21de753","added_by":"auto","created_at":"2025-05-05 20:36:38","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":137505,"visible":true,"origin":"","legend":"\u003cp\u003eDetection of intracellular expression of the CRR protein\u003c/p\u003e\n\u003cp\u003eAnti-CTB antibody (A) and anti-streptococcus bovis antibody (B) were used as primary antibodies, and Goat anti-mouse IgG was used as the secondary antibody. \u003cstrong\u003eM:\u003c/strong\u003eProtein molecular mass standards;\u003cstrong\u003e1:the \u003c/strong\u003eCRR protein（PEI transfection reagent）；\u003cstrong\u003e2\u003c/strong\u003e:the CRR protein（Lipofectamine 2000 transfection reagent）;\u003cstrong\u003eNC\u003c/strong\u003e: Control.\u003c/p\u003e","description":"","filename":"image5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6213136/v1/da4badbc776de7ac1eff7202.jpeg"},{"id":82002335,"identity":"e9cfa5a9-c44d-4fb0-a0e5-8153e27a9697","added_by":"auto","created_at":"2025-05-05 20:12:38","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":65655,"visible":true,"origin":"","legend":"\u003cp\u003eELISA determination of lgY in egg yolks during the immunization period\u003c/p\u003e","description":"","filename":"image6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6213136/v1/8f2bc0ae71d8d2446c054bfe.jpeg"},{"id":82002339,"identity":"942e0848-06ca-4ca9-9f00-0fb58866b86d","added_by":"auto","created_at":"2025-05-05 20:12:38","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":360368,"visible":true,"origin":"","legend":"\u003cp\u003eAntibacterial effect of IgY\u003c/p\u003e\n\u003cp\u003eA: The antibacterial effect of IgY on the standard strain of Streptococcus bovis ( ATCC33317 ). B: The antibacterial effect of IgY on clinical strains of Streptococcus bovis; C: The antibacterial effect of IgY on Escherichia coli BL21\u003c/p\u003e","description":"","filename":"image7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6213136/v1/827064dce5c1b7dd7d07bbf7.jpeg"},{"id":90063244,"identity":"f4b45b22-98f3-49a6-ba4c-a0df38964679","added_by":"auto","created_at":"2025-08-28 04:16:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2262656,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6213136/v1/e003f19f-3849-4901-b1b6-1d0a8073665a.pdf"},{"id":82003008,"identity":"db1f8ab8-93a2-45cb-8564-b80a5def0730","added_by":"auto","created_at":"2025-05-05 20:28:38","extension":"doc","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":773120,"visible":true,"origin":"","legend":"","description":"","filename":"WESTERNBLOTORIGINALIMAGES.doc","url":"https://assets-eu.researchsquare.com/files/rs-6213136/v1/aa5189a693b0e3ddb0e440a8.doc"}],"financialInterests":"No competing interests reported.","formattedTitle":"A novel DNA vaccine against Streptococcus bovis: study on multi-epitope DNA antigen based on RodA gene and its specific IgY antibody","fulltext":[{"header":"Introduction","content":"\u003cp\u003eStreptococcus bovis is a facultative anaerobic bacteria. It belongs to group D streptococcus, Gram-positive, non-β-hemolytic Streptococcus. It is a normal flora in the human gastrointestinal tract and herbivores' digestive tract. It is common in the gastrointestinal tract and feces (Herrera et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2009\u003c/span\u003e;Jans et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2015\u003c/span\u003e)[ 1,2 ]. In recent years, Streptococcus bovis has been associated with many human and animal diseases. It causes human infecting bacteremia, sepsis, infective endocarditis (Dekker et al. 2016;Pernow et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e;\u0026Ouml;berg et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022\u003c/span\u003e;Corredoira et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2023\u003c/span\u003e)[ 3\u0026ndash;6 ] and colon cancer(Deng et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e;Gupta et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2010\u003c/span\u003e;Boltin et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) [ 7\u0026ndash;9 ]. It causes rumen acidosis in ruminants, pigeon sepsis and acute deaths (Russell et al. 2001;Cheng et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1998\u003c/span\u003e;De Herdt et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1994\u003c/span\u003e)[ 10\u0026ndash;12 ]. The incidence of Streptococcus bovis infection and its drug resistance rate has recently increased (Pompilio et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e;Coffey et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2012\u003c/span\u003e;Corredoira et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2015\u003c/span\u003e)[ 13\u0026ndash;15 ]. The prevalence of pathogens among susceptible animals seriously threatens human health and causes losses to animal husbandry. The harm cannot be ignored. Developing and designing a vaccine with Streptococcus bovis has excellent clinical value. The new generation of vaccines, such as recombinant subunit and DNA vaccines have received considerable attention as alternatives to traditional vaccines. The multi-epitope vaccine is a research hotspot of new vaccines in human medicine and animal husbandry medicine. It contains multiple dominant B / T cell epitopes of pathogens and has the advantages of high safety and strong induction of specific immune responses(Gao et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) [ 16 ]. DNA vaccines encode a particular protein of antigen of exogenous gene and eukaryotic expression vector after recombination directly into the body, activating the host to produce an immune response. DNA vaccines has shown strong potential for application in preventing and treating various diseases (Jeon et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2002\u003c/span\u003e)[ 17 ]. Epitope-based DNA vaccines have been tested as potential candidates for preventing bacterial, fungal, parasitic and viral infections (Silveira et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e;Carvalho et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2010\u003c/span\u003e;Lowrie \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2006\u003c/span\u003e;Rivas-Santiago et al. 2014;Zhang et al. 2016)[ 18\u0026ndash;22 ]. Designing a multi-epitope DNA vaccine of Streptococcus bovis is conducive to ensuring human health, reducing the threat of Streptococcus bovis to susceptible animals and developing animal husbandry.\u003c/p\u003e \u003cp\u003eIn this study, the rod shape-determining protein ( RodA ) of Streptococcus bovis was screened as the target protein of the multi-epitope vaccine. RodA is very important in the growth and development of bacteria and is relatively conserved in different genera(Uehara et al. 2008) [ 23 ]. Structural analysis shows that RodA constitutes a suitable antigen epitope (Sjodt et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2018\u003c/span\u003e)[ 24 ]. Therefore, RodA can be used as a potential target for the design of Streptococcus bovis vaccine. In this study, the T cell and B cell dominant epitope gene sequences screened from the RodA gene of Streptococcus bovis were connected in series with the mucosal immune adjuvant Cholera toxin subunit B ( CTB ) gene to CTB - RodA- RodA ( CRR ) gene sequence. Based on this, a recombinant Streptococcus bovis eukaryotic expression vector pVAX1-CRR was constructed and identified. The specific Immunoglobulin of yolk (IgY) produced by immunizing the hens with pVAX1-CRR was characterized.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMain strains, cells, and animals\u003c/h2\u003e \u003cp\u003eStreptococcus bovis ATCC 33317 was purchased from China Ningbo Mingzhou Biological Company. The clinical strain of Streptococcus bovis was isolated from the feces samples of yaks in Naqu, Tibet, China. Our laboratory provided HEK293T cells. The laying hens are provided by Professor Suizhong Cao of Sichuan Agricultural University. The breed is White Leghorn Provided by Experimental Animal Center of Sichuan Agricultural University.Feeding, experimental procedures, euthanasia methods and biosafety precautions have been approved by the Medical Ethics Review Committee of Sichuan University in China and are in line with relevant guidelines and regulations.All experiments were conducted in accordance with the ARRIVE guidelines and regulations.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDesign of multi-epitope antigen CRR\u003c/h3\u003e\n\u003cp\u003eThe RodA of Streptococcus bovis was selected as the target of the DNA vaccine. The amino acid sequence and corresponding gene sequence of RodA of Streptococcus bovis were selected on National Center for Biotechnology Information ( NCBI ). The T / B cell epitopes of the RodA protein were predicted using the Immune Epitope Database ( IEDB ). The T and B cell epitopes were screened. The selected antigen epitopes were connected in series ( it was connected twice to amplify the signal ). The epitopes were screened in series with cholera toxin subunit B ( CTB ) at the N-terminal to improve the body's immunoreactivity. Finally, the fusion protein sequence was CTB-RodA-RodA ( CRR ). JCat online software was used for reverse translation and codon optimization of CRR. The BamH I restriction site was introduced at the 5 \u0026prime; end, and the EcoR I restriction site and protective sequence were introduced at the 3 \u0026prime; end. Shenggong Biotechnology synthesized the sequence.\u003c/p\u003e\n\u003ch3\u003ePrediction of antigenicity and physicochemical properties of the CRR protein\u003c/h3\u003e\n\u003cp\u003eVaxiJenv2.0 was used to predict the antigenicity of the CRR protein, and ExPASy ProtParam was used to evaluate its physicochemical properties.\u003c/p\u003e\n\u003ch3\u003eStructure prediction of the CRR protein\u003c/h3\u003e\n\u003cp\u003eThe SOPMA online program predicted the CRR protein's secondary structure and the SWISS-MODEL online tool predicted its tertiary structure.\u003c/p\u003e\n\u003ch3\u003eHomology analysis of the CRR protein\u003c/h3\u003e\n\u003cp\u003eThe CRR protein( first remove the CTB sequence and then perform BLAST analysis ) was subjected to BLAST homology analysis in common biological populations and target biological genomes. The biological categories added to the BLAST interface biologist are : Homo sampiens ( taxid : 9606 ), rodents ( taxid : 9989 ), Enterobacteriaceae and related endosymbionts ( taxid : 91347 ), Bacillus / Lactobacillus / Streptococcus group ( taxid : 91061 ), oxen, cattle ( taxid : 9903 ), birds ( taxid : 8782 ).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eConstruction of pVAX1-CRR\u003c/h2\u003e \u003cp\u003eThe synthesized CRR gene sequence was inserted into the pVAX1 vector by BamH I and EcoR I restriction sites and transformed into Escherichia coli(E.coli) DH5α. The positive clones were screened for kanamycin resistance, and BamH I and EcoR I were digested and identified. After identification, it was delivered to Sangon Biotechnology for DNA sequencing confirmation. The recombinant plasmid pVAX1-CRR is the DNA vaccine constructed in this study.\u003c/p\u003e \u003cp\u003e \u003cb\u003epVAX1-CRR transfected in HEK293T cells.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThis experiment set up three groups: pVAX1-CRR transfection group, empty vector pVAX1 transfection group, and untransfected blank control group. Three holes were set up in each group. Transfection was performed according to the instructions of the PEI transfection reagent and Lipofectamine 2000. Before transfection, HEK293T cells were inoculated into 24-well plates at 0.5x105 per well. The cells were transfected when covered about 60% of the bottom of the well. One hour before transfection, the cells were replaced with fresh serum-free and antibiotic-free DMEM medium, 0.5mL / well. Lipofectamine 2000, or PEI, was used as a transfection reagent. For each well of cells, 4 \u0026micro;L of target DNA and 1 \u0026micro;L of transfection reagent were diluted with 100 \u0026micro;L serum-free medium and incubated at 37 \u0026deg; C for 20 min to form transfection 105 ul working solution. The 105 \u0026micro;L transfection working solution was added to the 24-well plate drop by drop, and the blank control was added with 105 \u0026micro;L serum-free medium. The cells were cultured in 37\u0026deg;C, 5% CO2 incubator. After 8 hours of culture, the medium was changed to a complete medium ( DMEM medium with 10% FBS\u0026thinsp;+\u0026thinsp;1% penicillin\u0026thinsp;+\u0026thinsp;1% streptomycin ) and continued to be cultured for 40 hours.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eImmunofluorescence analysis of transfection rate\u003c/h3\u003e\n\u003cp\u003eHEK293T cells were fixed with 4% paraformaldehyde for 20 min at 48 h after transfection, washed three times with PBS, added 0.3% TritonX-100, permeabilized for 15 min, added 3% BSA, blocked for 15 min, incubated with mouse anti-CTB monoclonal antibody ( 1: 100 ) / mouse anti-streptococcal polyclonal antibody ( 1: 500 ), washed three times with PBS, and overnight at 4\u0026deg;C. Alexa Fluor \u0026reg; 488 labeled goat anti-mouse IgG ( H\u0026thinsp;+\u0026thinsp;L ) ( 1: 200 ) was incubated for 1.5 h and washed three times with PBS. DAPI was added and incubated at room temperature for 15 min. Add an anti-fluorescence quenching agent to seal the tablets. The transfection rate was observed by fluorescence microscope.\u003c/p\u003e\n\u003ch3\u003eWestern blot\u003c/h3\u003e\n\u003cp\u003eThe pVAX1-CRR transfection group was set up, and the blank control was the pVAX1 plasmid transfection group. The protein lysate ( RIPA: PMSF\u0026thinsp;=\u0026thinsp;50: 1 ) was incubated on ice for 10 min, 4\u0026deg;C, 14000 r / min, and centrifuged for 15 min to collect protein supernatant. Western blot was used to detect the specific expression of the fusion protein.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eHen immunization and preparation of IgY\u003c/h2\u003e \u003cp\u003eIn this experiment, three groups were set up: the DNA antigen group pre-immunized with CTB, the DNA antigen group without pre-immunization, and the inactivated antigen of Streptococcus bovis group pre-immunized with CTB. The pVAX1-CRR was introduced into E.coli DH5α for enrichment culture, and the DNA antigen preparation was adjusted to 500ng / \u0026micro;L by plasmid extraction kit. The ultrasonically disrupted streptococcus bovis ( ATCC33317 ) antigen was prepared into an antigen solution with a final 1 mg / mL protein concentration for later use. Seven days before immunization, the immune adjuvant CTB was used for pre-immunization. The three groups of antigens were used to immunize one laying hen during the formal immunization. The hen's wings, legs, and back muscles were multi-point immunized. Eggs were collected before and after immunization. The immunization plan for laying hens is shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Eggs were collected for 10 consecutive weeks, and antibodies were extracted by the salting out method. IgY antibodies induced by pVAX1-CRR ( CRR-IgY )with or without CTB adjuvant and IgY antibodies induced by Streptococcus bovis ( Streptococcus bovis-IgY ) with CTB adjuvant were obtained.\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\u003eImmunization program of laying hens\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\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=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eImmunization and egg collection period\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003egroup immunized\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1 preimmune(0 days)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCTB 600ug\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003enormal saline 600ug\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCTB 600ug\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2 First immunization(7-9days)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eStreptococcus bovis DNA antigen 150\u0026micro;L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eStreptococcus bovis DNA antigen 150\u0026micro;L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eInactivated Streptococcus bovis antigen 300ug\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3Second immunization(14\u0026ndash;21 days)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eStreptococcus bovis DNA antigen 150\u0026micro;L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eStreptococcus bovis DNA antigen 150\u0026micro;L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eInactivated Streptococcus bovis antigen 300ug\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4Third immunizations\u0026nbsp;(28\u0026ndash;52 days)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eStreptococcus bovis DNA antigen 150\u0026micro;L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eStreptococcus bovis DNA antigen 150\u0026micro;L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eInactivated Streptococcus bovis antigen 300ug\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5Fourth Immunization\u0026nbsp;(62\u0026ndash;72 days)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eStreptococcus bovis DNA antigen 150\u0026micro;L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eStreptococcus bovis DNA antigen 150\u0026micro;L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eInactivated Streptococcus bovis antigen 300ug\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eEnzyme-Linked Immunosorbent Assay(ELISA)\u003c/h2\u003e \u003cp\u003eThe broken antigen of Streptococcus bovis was diluted to 100 \u0026micro;g / mL with the coating solution, and 100 \u0026micro;L was added to each well and blocked with 3% BSA. The yolk antibodies ( CRR-IgY and Streptococcus bovis-IgY ) were added to the sample to be tested, and HRP-rabbit anti-chicken IgY was used as the secondary antibody. After color development, the OD450 nm value of the sample was detected by a microplate reader. When the OD value of the detected immune IgY / negative IgY was \u0026ge;\u0026thinsp;2.1, and the OD value of the immune IgY itself was greater than 0.21, the maximum dilution was the titer of the antibody IgY.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003ePlate colony counting experiment\u003c/h2\u003e \u003cp\u003eThe standard strain of Streptococcus bovis ( ATCC33317 ), Streptococcus bovis clinical strains, and E.coli BL21 were adjusted to a concentration of OD600\u0026thinsp;=\u0026thinsp;0.1 bacterial solution with LB. The standard strain of Streptococcus bovis was incubated with specific antibody CRR-IgY ( 10 mg/ml, 5 mg/ml, 2 mg/ml), non-specific antibody IgY antibodies induced by BL21 (BL21-IgY )( 10 mg/ml, 5 mg/ml, 2 mg/ml), positive control kanamycin ( 50\u0026micro;g / mL ) and control ( Pbs ) for 3h, then diluted 10\u003csup\u003e3\u003c/sup\u003e times, 100\u0026micro;L coated plate, 3 parallels in each group, cultured at 37\u0026deg;C for 12 h. The number of colony clones was observed and recorded. The inhibitory effect of CRR-IgY on the activity of clinical strains of Streptococcus bovis in vitro was detected. The clinical strains were incubated with specific antibody CRR-IgY ( 10 mg/ml), non-specific antibody BL21-IgY ( 10 mg/ml), kanamycin ( 50\u0026micro;g / mL ) and control ( Pbs ) for 3h, respectively. Other steps are the same as above. Escherichia coli was incubated with CRR-IgY ( 10mg / ml ), kanamycin ( 50\u0026micro;g / mL ), and control ( Pbs ) for 3h. Other steps are the same as above.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003estatistical analysis\u003c/h2\u003e \u003cp\u003eSPSS 17.0 statistical software was used for statistical analysis. The Student's t-test evaluated the significance of the difference between groups. Each experiment was repeated more than 3 times. P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003eDesign of multi-epitope antigen CRR\u003c/h2\u003e\n \u003cp\u003eThe obtained Streptococcus bovis ATCC 33317 is one of the standard strains of group D Streptococcus bovis-Streptococcus equi complex. The following epitopes were screened from the RodA(NCBI GenBank, KFN86186.1) amino acid sequence. B cell epitopes: KNDWKL, GISWWII, HGKDIFYSLGMDTYQIN, WLDPFSYAKSIAY. T cell epitopes: ALITLPVMI, LQKDLGTAM, HGKDIFYSL, YQINRISAW, QQTQGMISI.\u003c/p\u003e\n \u003cp\u003eIn addition, to improve the body\u0026apos;s immune response, the cholera toxin B subunit ( CTB ) can also be inserted into the N-terminal of the above epitope. The sequence is as follows ( NCBI GenBank, LC427969.1 ) :\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMIKLKFGVFFTVLLSSAYAHGTPQNITDLCAEYHNTQIHTLNDKILSYTESLAGKREMAIITFKNGETF\u003cbr\u003eQVEVPGSQHIDSQKKAIERMKDTLRIAYLTEAKVEKLCVWNNKTPHAIAAISMAN.The above epitopes were connected in series. The Streptococcus bovis RodA was connected in series twice to amplify the signal. Finally, the fusion protein sequence CTB-RodA-RodA ( CRR ) was obtained. Its amino acid sequence is: MIKLKFGVFFTVLLSSAYAHGTPQNITDLCAEYHNTQIHTLNDKILSYTESLAGKREMAIITFKNGETFQVEVPGS\u003cbr\u003eQHIDSQKKAIERMKDTLRIAYLTEAKVEKLCVWNNKTPHAIAAISMANGGGGSGGGGSGGGGSKNDWKLKK\u003cbr\u003eGISWWIIKKHGKDIFYSLGMDTYQINKKWLDPFSYAKSIAYKKALITLPVMIKKLQKDLGTAMKKHGKDIFYS\u003cbr\u003eLKKYQINRISAWKKQQTQGMISIKKKNDWKLKKGISWWIIKKHGKDIFYSLGMDTYQINKKWLDPFSYAKSIA\u003cbr\u003eYKKALITLPVMIKKLQKDLGTAMKKHGKDIFYSLKKYQINRISAWKKQQTQGMISI. JCat online software was used for reverse translation and codon optimization of CRR. The BamH I restriction site was introduced at the 5 \u0026apos; end. The EcoR I restriction site and protective sequence were introduced at the 3 \u0026apos; end. A multi-epitope vaccine cDNA sequence ( 1080 bp ) was generated.\u003c/p\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003ePrediction of antigenicity and physicochemical properties of the CRR protein\u003c/h2\u003e\n \u003cp\u003eThe antigenicity score of the CRR protein was 0.5278, evaluated by VaxiJenv2.0, which was higher than the default threshold of 0.4 and had good antigenicity. The prediction results of physicochemical properties showed that the CRR protein contained 349 amino acids, the molecular weight was 40 kD, the isoelectric point was 9.97, the half-life was 30 h, the fat coefficient was 83.35, and the total average hydrophobic index was \u0026minus;\u0026thinsp;0.434, indicating that the CRR protein had good hydrophilicity and was soluble in water. The stability index is 29.15, indicating that the CRR protein has good stability.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003eStructure prediction of the CRR protein\u003c/h2\u003e\n \u003cp\u003eThe secondary structure of the CRR protein predicted by SOMPA showed that \u0026alpha;-helix, extended strand, and random coil accounted for 43.27%, 22.64%, and 34.10%, respectively ( Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e ). The SWISS-MODEL online tool was used to predict the tertiary structure of the CRR protein ( Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). The CRR protein\u0026apos;s global model quality estimate ( GMQE ) value was 0.3. The Lagrangian diagram was used to evaluate the quality of the tertiary structure. The results show that most of the dotted structures in the figure fall within the allowable range. The Z score plot was used to evaluate the quality of the entire tertiary structure. The Z score of the CRR protein was 0.72, indicating that it was within the characteristic range of natural proteins. Therefore, the credibility of the three-level structure is high.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n \u003ch2\u003eHomology analysis of the CRR protein\u003c/h2\u003e\n \u003cp\u003eThe results showed that the fusion protein had high species specificity, and there was no cross between the prevention target organisms ( Streptococcus ) in humans, rodents, birds, cattle, intestinal flora, Bacillus / Lactobacillus / Streptococcus, and other biological categories.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n \u003ch2\u003eConstruction and identification of eukaryotic expression vector pVAX1-CRR\u003c/h2\u003e\n \u003cp\u003eThe synthesized CRR gene sequence was inserted into the pVAX1 vector by BamH I and EcoR I restriction sites. About 3000 and 1000 fragments were identified by agarose gel electrophoresis, consistent with the expected results ( Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e ). The sequencing results showed that the sequence was 100% consistent with the design sequence, and the recombinant plasmid pVAX1-CRR was constructed correctly.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\n \u003ch2\u003eHEK293T cells expressed pVAX1-CRR successfully\u003c/h2\u003e\n \u003cp\u003eThe recombinant expression plasmid pVAX1-CRR was transfected into HEK293T cells. The transfection rate was detected by immunofluorescence. The immunofluorescence staining showed apparent green fluorescence in HEK293T cells transfected with pVAX1-CRR ( Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA / B ). In contrast, no green fluorescence was observed in cells transfected with pVAX1 ( Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC ) and untransfected cells ( Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eD ), suggesting that the construction of pVAX1-CRR is effectively expressed in HEK293T cells. Image J analysis showed that the transfection efficiency was about 44\u0026ndash;63%.The protein is mainly located in the cytoplasm.\u003c/p\u003e\n \u003cp\u003eWestern blot analyzes the CRR protein expressed in HEK293T cells. The expression of the CRR protein was detected by mouse anti-CTB monoclonal antibody ( 1: 100 ) ( Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA ) and mouse anti-Streptococcus bovis polyclonal antibody ( 1: 500 ) ( Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB ). The results showed that the protein expressed in HEK293T cells could specifically bind to two antibodies. The fusion protein showed a band at about 40KD ( Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e ), and the control did not, indicating that the expression of the CRR protein was successful.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003ePVAX1-CRR induced a high concentration of IgY antibody.\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eELISA was used to continuously detect the titer of IgY in egg yolks 10 weeks after immunization. The particular antibody appeared about 2 weeks after vaccination, and the titer of the antibody increased gradually with the immunization time. The delay of the antibody in the yolk may be because the antibody was first produced in the serum, then transferred and accumulated in the yolk. After four DNA vaccine immunizations, the CRR-IgY titer gradually increased and peaked about 7 weeks after vaccination. The peak titer of CRR-IgY without CTB pre-immunization was finally stabilized at about 1: 4500, and the CRR-IgY with CTB pre-immunization was stabilized at 1: 6400 for more than 2 weeks, indicating that pre-immunization with CTB protein as an adjuvant increases the DNA immune effect of CRR-IgY by 40%. The titer of CRR-IgY after pre-immunization was close to that of Streptococcus bovis immunization ( 1: 8000 )( Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e ).\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eCRR-IgY has an antibacterial effect on both standard and clinical strains of Streptococcus bovis.\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eThe antibacterial effect of IgY on Streptococcus bovis standard strain, clinical strain, and Escherichia coli BL21 was detected by plate colony counting experiment. Compared with the control group, 2 mg/ml CRR-IgY had no inhibitory effect on the growth of a standard strain of Streptococcus bovis. 5 mg/ml and 10 mg/ml CRR-IgY and kanamycin had an apparent inhibitory effect on the growth of a standard strain of Streptococcus bovis. Non-specific BL21-IgY had no inhibitory effect on the standard strain of Streptococcus bovis ( Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eA ). Compared with the control group, both CRR-IgY and kanamycin could significantly inhibit the growth of clinical strains of Streptococcus bovis. In contrast, non-specific BL21-IgY had no inhibitory effect on clinical strains of Streptococcus bovis ( Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eB ). Compared with the control group, CRR-IgY could not inhibit the growth of E.coli BL21, and kanamycin could significantly inhibit the growth of E.coli BL21 ( Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eC ). Therefore, the 10 mg/ml antibody CRR-IgY has an obvious antibacterial effect on standard strains and clinical strains of Streptococcus bovis and has no antibacterial effect on Escherichia coli. The non-specific antibody BL21-IgY had no antibacterial effect on the standard strain and clinical strain of Streptococcus bovis. It shows that the antibacterial effect of CRR-IgY against standard strains and clinical strains of Streptococcus bovis is specific and effective.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eStreptococcus bovis is a common pathogen affecting the health of cattle, which brings serious harm to the breeding and production of cattle. The fecal dissemination of pathogens and the persistence of pathogens in the environment make it more challenging to prevent and control pathogens in pastoral areas. It seriously threatens people's health in pastoral areas and causes losses to the breeding industry. In recent years, the resistance rate of Streptococcus bovis infection has increased. In 2005, Leclercq R et al.reported that tetracycline resistance was widespread in Streptococcus bovis, and the incidence of erythromycin resistance was also high(Leclercq et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2005\u003c/span\u003e) [ 25 ]. In 2019, Pompilio A et al.reported that the resistance rate of Streptococcus bovis to tetracycline was 36\u0026ndash;77%, to erythromycin was 8.9\u0026ndash;78%, and to clindamycin was 10.6\u0026ndash;62% (Pompilio et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e)[ 13 ]. Currently, no efficient and convenient method for preventing and treating this pathogen exists. People have tried to control the growth of Streptococcus bovis in the rumen by vaccinating cattle to produce specific antibodies against the bacteria and have achieved some success. Gill et al. (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2000\u003c/span\u003e)[ 26 ] described that immunizing animals with Streptococcus bovis induces specific immune responses and reduces the excessive production of lactic acid in the rumen by calling specific anti-Streptococcus bovis antibodies. Shu et al. (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1999\u003c/span\u003e)[ 27 ] proved that intramuscular injection of live Streptococcus bovis vaccine effectively reduces acute lactic acidosis in animals fed a cereal-based diet. Developing multi-epitope vaccines with the continuous development of new vaccines is undoubtedly effective. Epitope vaccines have many advantages, such as high antigenicity, non-allergic, non-toxic, and simple molecular structure. They directly stimulate the body to produce specific immunity, which makes it challenging to produce autoimmune suppression or immune response (Devarakonda et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e;Jyotisha et al. 2023)[ 28, 29 ]. Many studies have shown that DNA vaccines are safe, non-toxic, and effective and are one of the most promising immune technologies for pathogen prevention (Pagliari et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2023\u003c/span\u003e;Ghaffarifar \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2018\u003c/span\u003e)[ 30, 31 ]. Based on this, this study constructed an epitope-based DNA vaccine to prevent and control the infection of Streptococcus bovis.\u003c/p\u003e \u003cp\u003eRod shape-determining protein ( RodA ) was first identified as determining bacteria's short rod shape. It was later classified into the Shape, Elongation, Division, and Sporulation ( SEDS ) protein family, essential for bacterial growth and development. It is widely present in Gram-positive and Gram-negative bacteria but is relatively conserved in different genera (Uehara et al. 2008;Sjodt et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2018\u003c/span\u003e)[ 23,24 ]. In 2008, Uehara et al.showed that the central role of RodA is not only related to the morphology of bacteria but also closely related to the synthesis and degradation of peptidoglycan in life processes such as bacterial elongation. In 2018, Sjodt et al.reported the crystal structure of RodA, which contains three huge outer rings. The outer ring contains many functional essential residues, while the outer ring of bacterial membrane proteins often constitutes a suitable B-cell epitope. In addition, there are also reports that there are highly conserved peptides in RodA. The induction of mutations in these conserved peptides will lead to changes in bacterial morphology and may lead to bacterial lysis. Therefore, using RodA as a target for Streptococcus bovis vaccine may produce better results. In this study, the key dominant epitopes of RodA of Streptococcus bovis were screened for fusion antigen design, which was connected in series with the intramolecular adjuvant CTB. At the same time, the epitopes screened by RodA were connected in series twice to enhance the immunogenicity and stability of the recombinant protein. After reverse translation and codon optimization, the gene sequence CRR was obtained. The recombinant Streptococcus bovis eukaryotic expression vector pVAX1-CRR was constructed. Immunofluorescence and Western blot verified the successful expression of the fusion protein. The specific IgY antibody against Streptococcus bovis extracted from eggs immunized with the pVAX1-CRR. It was found that the antibody level changed significantly after 2 weeks of immunization. The antibody level reached 1: 6400 after 7 weeks of vaccination and could be maintained for over 2 weeks. The titer of CRR-IgY was close to that of whole bacteria immunization, similar to that of Streptococcus bovis.\u003c/p\u003e \u003cp\u003eWang et al. (2019)[ 32 ] found that anti-K88 fiber-IgY could significantly inhibit the growth of E.coli K88, block the adhesion of bacteria in the intestinal mucosa, reduce diarrhea caused by E.coli in weaned piglets, and achieve the effect of prevention and treatment by co-culturing IgY with E.coli. The production of antibodies effectively reduces bacterial infection and protects the host. In this study, the in vitro antibacterial test showed that 10 mg/ml CRR-IgY could inhibit the growth of standard and clinical strains of Streptococcus bovis. The antibacterial effect of CRR-IgY on standard strains and clinical strains of Streptococcus bovis was specific and compelling. IgY induced by pVAX1-CRR produces practical bacteriostatic effects in vitro. This study hopes to achieve the purpose of efficient bacteriostasis by studying the bacteriostatic effect of anti-Streptococcus bovis. Further studies are needed to evaluate the impact of IgY in vivo on treating Streptococcus bovis infection.\u003c/p\u003e \u003cp\u003ePrevious studies have shown that the cholera toxin B subunit ( CTB ) is a classic mucosal immune adjuvant that enhances the immune effect of vaccines and can be used as a compelling candidate adjuvant for DNA vaccine inoculation (Hou et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2014\u003c/span\u003e;Verjan Garcia et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2023\u003c/span\u003e)[ 33,34 ]. In this study, after four times of DNA vaccine immunizations, the peak titer of CRR-IgY without CTB pre-immunization was finally stabilized at about 1: 4500, and that of CRR-IgY with CTB pre-immunization was stabilized at 1: 6400. After 7 days of pre-immunization with CTB protein as an adjuvant, the immune effect of CRR-IgY could be effectively improved by 40%.\u003c/p\u003e \u003cp\u003eThis study successfully constructed a multi-epitope DNA vaccine, pVAX1-CRR, of Streptococcus bovis, expressed in eukaryotic cells. Hens immunized with this pVAX1-CRR produce specific IgY antibodies that inhibit the growth of Streptococcus bovis in vitro. It provides ideas and references for developing a Streptococcus bovis vaccine to prevent or treat Streptococcus bovis infection. The follow-up application promotion research intends to explore the possibility of pVAX1-CRR as a vaccine further.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this experiment, the recombinant eukaryotic expression vector pVAX1-CRR was constructed. The successful expression of the CRR protein was verified. The CRR-IgY obtained by immunizing laying hens has good immunogenicity and significantly inhibits the growth of Streptococcus bovis isolates. These results indicate that pVAX1-CRR may be used as a promising DNA vaccine to prevent and treat Streptococcus bovis infection, which requires further experiments to explore.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eCTB\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCholera toxin subunit B\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eDMEM\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDulbecco's modified Eagle medium\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eE.coli\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eEscherichia coli\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eELISA\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eEnzyme-Linked Immunosorbent Assay\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eGMQE\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eglobal model quality estimate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eIgY\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eImmunoglobulin of yolk\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eKan\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eKanamycin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eOD\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eOptical density\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eRodA\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRod shape-determining protein\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eSEDS\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eThe Shape, Elongation, Division, and Sporulation\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are very grateful to Professor Suolang sizhu of Tibet Agricultural and Animal Husbandry College for providing us with Tibetan yak feces samples. We are very grateful to Professor Suizhong cao of Sichuan Agricultural University for providing us with laying hens.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the key technology research project of Tibet Streptococcus bovis nucleic acid rapid detection kit and new prevention and control preparations. ( Grant numbers [No. : 2023 YFQ0051] ) and the key R \u0026amp; D program of Tibet Autonomous Region. ( Grant numbers [No. : XZ202201ZY0009N])\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal procedures were approved by the Animal Ethics Committee of Sichuan University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no financial or personal conflicts of interest. The authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eB. W. is responsible for design and resource support. G.L. is responsible for Streptococcus bovis gene analysis and construction, immune experiments, data collection and writing. L.F. is responsible for Streptococcus bovis reset gene design, DNA immunization. J.H. is responsible for the collection of Tibetan samples and the isolation and culture of Streptococcus bovis, the purchase and culture of standard strains, the immunization of laying hens, the extraction and purification of IgY, the determination of titer, and the determination of in vitro antibacterial experiments. Z.T. ,X.Y. and Y.Z. carried out the result analysis. C.Z. ,Y.Y. and X.D. reviewed and edited it. All authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and analysed during the current study are available in the [NCBI] repository. Streptococcus equinus rodA gene for rod shape-determining protein, partial cds, strain: ATCC 33317（NCBI GenBank, AB441161.1）rod shape-determining protein, partial [Streptococcus equinus ATCC 33317]（NCBI GenBank, BAH03909.1）Cholera Toxin B subunit, CTB（NCBI GenBank, LC427969.1）\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHerrera, P., Kwon, Y. M. \u0026amp; Ricke, S. C. Ecology and pathogenicity of gastrointestinal Streptococcus bovis. \u003cem\u003eAnaerobe\u003c/em\u003e \u003cb\u003e15\u003c/b\u003e (1\u0026ndash;2), 44\u0026ndash;54. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.anaerobe.2008.11.003\u003c/span\u003e\u003cspan address=\"10.1016/j.anaerobe.2008.11.003\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJans, C., Meile, L., Lacroix, C. \u0026amp; Stevens, M. J. 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Rep.\u003c/em\u003e \u003cb\u003e13\u003c/b\u003e (1), 4305. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41598-023-30834-2\u003c/span\u003e\u003cspan address=\"10.1038/s41598-023-30834-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2023).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"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":"Streptococcus bovis, DNA vaccines, Recombinant protein, IgY, Immunization, Adjuvants","lastPublishedDoi":"10.21203/rs.3.rs-6213136/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6213136/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eStreptococcus bovis is one of the leading causes of infective endocarditis and is associated with colon cancer. It can also cause rumen acidosis in ruminants and cause pigeon sepsis. The prevalence of pathogens among susceptible animals not only poses a serious threat to human health but also causes losses to animal husbandry. Its harm cannot be ignored. Vaccination can effectively control and prevent infection. In this study, the T cell and B cell dominant epitope gene sequences screened from the Rod A gene of Streptococcus bovis were tandem with the mucosal immune adjuvant cholera toxin B subunit ( CTB ) gene. The codon was optimized as CTB-RodA-RodA ( CRR ) gene sequence. After artificial synthesis, the CRR gene was inserted into the eukaryotic expression vector pVAX1. The multi-epitope DNA vaccine pVAX1-CRR was successfully constructed. The pVAX1-CRR and immune adjuvant CTB were combined to immunize laying hens. The specific IgY in eggs was extracted by salting out method and named CRR-IgY. Preliminary exploration of pVAX1-CRR immunogenicity showed that the titer of CRR-IgY was as high as 1: 6400. The in vitro antibacterial effect of the CRR-IgY on Streptococcus bovis was detected. It was found that 10 mg/ml CRR-IgY could significantly inhibit the growth of Streptococcus bovis isolates. In summary, this study successfully screened, constructed and expressed the multi-epitope vaccine pVAX1-CRR of Streptococcus bovis. It produces a high level of antibodies and a good antibacterial effect.\u003c/p\u003e","manuscriptTitle":"A novel DNA vaccine against Streptococcus bovis: study on multi-epitope DNA antigen based on RodA gene and its specific IgY antibody","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-05 20:12:34","doi":"10.21203/rs.3.rs-6213136/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":"93187ea4-7677-4350-86c4-efee67562727","owner":[],"postedDate":"May 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":48041997,"name":"Biological sciences/Immunology/Vaccines/Dna vaccines"},{"id":48041998,"name":"Biological sciences/Microbiology/Vaccines/Dna vaccines"},{"id":48041999,"name":"Biological sciences/Biological techniques/Genetic engineering"}],"tags":[],"updatedAt":"2025-08-28T04:08:33+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-05 20:12:34","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6213136","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6213136","identity":"rs-6213136","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}