Porin protein could be a vaccine candidate against Riemerella anatipestifer for ducks

Porin protein could be a vaccine candidate against Riemerella anatipestifer for ducks | 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 Original article Porin protein could be a vaccine candidate against Riemerella anatipestifer for ducks Hui Yang, Haiwen Zhang, Chenghong Liao, Jianguo Zhao, Jinhua Wang, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-58801/v2 This work is licensed under a CC BY 4.0 License Status: Posted Version 2 posted You are reading this latest preprint version Show more versions Abstract such as domestic ducks and geese. Young birds have a high mortality rate after infection. The resistance caused by the abuse of antibiotics is also getting worse. Since there are 25 serotypes of RA , and the cross-immunization between serotypes is weak, it is necessary to find a vaccine that has cross-immunization against multiple serotypes of RA . In this article, the bioinformatics analysis of RA Proin protein was conducted, and it was speculated that it has the potential of a subunit vaccine. The protein was recombinantly expressed and purified, and immunized with Cherry Valley Duck. The results show that the serum antibodies of the Porin protein immunized group were positive at 1:24300 for the porin protein and RA strains CH1. The serum could improve the killing effect of complement and phagocytic cells on RA. After the challenge, the survival rate of Cherry Valley Duck can be increased by 80%. Animal Science RA subunit vaccine bioinformatics analysis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Key Points There are 21 serotypes of RA , but the cross immunoprotective effect between each serotype is extremely weak. The development of subunit vaccines is a powerful way to prevent the infection of multiple serotypes of RA . Through bioinformatics analysis, it was found that Porin protein is conserved in RA and also has abundant epitopes. It is speculated that this protein has the potential to develop a subunit vaccine. The recombinant Porin protein was used to immunize Cherry Valley Duck. The serum produced had a cross-immunization effect on serum type 1 and serum type 3 RA , and enhanced the clearance of pathogens by complement and phagocytes. After challenge, the survival rate of Cherry Valley was improved. Introduction RA is a contact infectious bacterium that infects domestic ducks, geese(Chen et al.; Shu-Chuan et al., 1999), turkeys(Rubbenstroth et al.) and a variety of birds(Hinz et al., 1998). Its infection symptoms are fibrinous pericarditis, fibrinous perihepatitis, fibrinous air sacs, encephalitis, Arthritis, etc. The disease was first reported in Long Island, New York, USA in 1932, and subsequently in Europe, South Asia, and Oceania. At present, the occurrence and prevalence of this disease is extremely extensive, and the disease is prevalent in almost all places where ducks are raised. All kinds of ducks in poultry can be infected. Under natural conditions, ducks of all ages are susceptible, and the incidence rate is about 90%(Zhu et al., 2017). Drug treatment is one of the important measures to prevent and treat the disease, mainly antibiotics and horizontal amines. The disease can occur in the whole stage of duck breeding, and birds can be infected repeatedly, resulting in higher cost of medication. Clinically, drug abuse, poor therapeutic effects, and even treatment failures often occur. Although RA is sensitive to many antibiotics, it is highly susceptible to drug resistance(Sun et al., 2019; Xing et al., 2015). Different regions have different susceptibility to antibacterial drugs in different regions, which brings difficulties to the actual treatment work. Therefore, immunization is an effective way to prevent and control this disease. The vaccines currently used to prevent this disease mainly include inactivated vaccines, attenuated vaccines, and subunit vaccines. Due to the complexity of RA serotypes and the lack of cross-protection between different serotypes, both attenuated and inactivated vaccines are effective against specific serotypes but are not protective against other serotypes of RA . Gram-negative outer membrane proteins (Omps) not only play an important role in maintaining the integrity of the cell envelope and transmembrane transport of the substances, but also in the the adhesion of bacteria to invading cells and bacteria to serum resistance. Outer membrane proteins can be divided into major proteins and microproteins according to their copy number in cells. Among them, Porin protein, also known as channel-forming protein, has a transmembrane domain with only 10 to 12 amino acid residues, forming a β-sheet structure, and antiparallel β-sheets interact to form a non-specific transmembrane. The channel allows small molecules with a relative molecular mass of less than 10,000 D to pass freely and is associated with bacterial resistance. Studies have shown that porin protein has good immunoprotective effects against strains such as E. coli (Fox et al., 2009), Salmonella (Dodd et al., 2011) and Pseudomonas aeruginosa (Bahey-El-Din et al., 2020). According to the genome sequencing results of RA (Wang, et al., 2012), it was found that porin was also present in RA . At present, the research of RA porin is still in the prediction stage, and there are no reports of expression and immune protection analysis. In this paper, the conservation, structural properties and immunogenicity of porin protein were analyzed by bioinformatics, and the duck was immunized by recombinant expression of the protein to verify the cross-immunoprotective effect and immune mechanism of the protein. Materials and methods Bacterial strains and culture conditions RA strain ATCC 11845 were purchased from Shanghai yiyan biological technology Co. Ltd (ShangHai, China). Serotype 1 RA strain CH-1 (RA-CH1) was isolated from sick ducks in HaiKou, China. RA strains were grown in tryptic soy broth medium (TSB) and at 37°C. E. coli strain DH5α and BL21 (DE3) strains were purchased from TransGen Biotech, Inc. (Beijing, China). E. coli strains were grown on Luria-Bertani (LB) agar. Animals 30 One-day-old Cherry Valley ducklings obtained from the Hainan Tianyuan Industrial Co., Ltd.(Haikou, China). The ducks were housed in cages, and provided water and food adlibitum during the study. The animal experiments were conducted in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the Institutional Animal Care and Use Committee guidelines set by HanNan University. Signal peptide prediction Signal peptide cleavage site was predicted by SignalP 5.0 (http://www.cbs.dtu.dk/services/SignalP/). Homology analysis and phylogenetic tree construction The RA Porin protein (Uniprot NO. E4TDA8) was blasted by UniProtKB database with parameters including E-Threshold 10, Matrix BLOSUM-62 and no filtering in Uniprot ( https://www.uniprot.org/blast/ ). The number of returned alignments was 50. The blasted data was aligned with ClustalW and phylogeny tree was constructed with maximum likelihood statistical method by MEGAX. Transmembrane structure prediction Mature peptide sequence of RA Porin protein was pasted into PRED-TMBB web server (http://biophysics.biol.uoa.gr/PRED-TMBB/input.jsp)(Bagos et al., 2004), which is for predicting the topology of beta-barrel outer membrane proteins. Viterbi (VI), N-best (NB) and Posterior Decoding (DY) method were applied and the common part of the three analyses was as prediction results. Tertiary structure prediction The tertiary structure of the Porin protein sequence from which the signal peptide was removed was predicted by the swissmodel online server (https://swissmodel.expasy.org/). Antigenic prediction The B cell epitopes were predicted by ElliPro web server (http://tools.iedb.org/ellipro/) based based upon structural protrusion. The predicted results were screened to retain only epitopes outside the membrane. Expression and purification of poring protein of RA The genome of RA strain ATCC 11845 is extracted by the genome extraction kit (TIANGEN Biotech(Beijing)Co.,Ltd.,BeiJing, China), and the porin gene (sequence accession number in genebank:NC_017045.1, from 1940759 to 1942012 ) is amplified by primer pairs porin F: 5’-GGATCCGTACCAGATACTATTATCGTTC-3’ and porin R: 5’-GCGGCCGCAATTCCTATTTCTACTTGAAATCT-3’, with Restriction site Bam H 1and Not 1. The gene was then ligated into the pET 28a vector, and the recombined vectors were transformed into BL21 competent cells. These successfully transformed BL21 E. coli cells were induced to express the target protein by auto-inducing medium(Studier, 2014). The expressed product was subjected to cleavage, separation, denaturation, renaturation, dialysis, and finally purification through a nickel column based on the previous protocol(Saleem et al., 2012). All proteins were determined by 12 % sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS–PAGE). The protein was then lyophilized with the ALPHA 1-2 LD plus freeze dryer (Christ, Germany) and kept in −20 °C. Immunization protocols The Cherry Valley ducks were randomly divided into 3 groups of 10 animals each injected with PBS (PBS group), PBS + adjuvant (PBS + adjuvant group), PBS + adjuvant + Porin protein (Porin group). The ducks were first immunized at 7 days of age. The Porin protein was mixed with Freund's complete adjuvant in a 1:1 oily form and injected subcutaneously. For the second immunization 7 day post the first immunization, Porin protein was mixed with Freund's incomplete adjuvant by 1:1 to form an oil emulsion. Both immunization doses were 0.2 mg/duck. The days of blood collection was 1, 7, 14, 21 and 28 days post the first immunization. Indirect enzyme-linked immunosorbent assay (iELISA) for Porin protein and RA cells detection 96-well plates were coated at a concentration of 2 μg/ml protein antigen with 100 μl/well, overnight at 4 ° C, and washed 4 times with 100 μl PBST wash for 5 min each. The plates were added with 200 μl/well blocking solution and incubaed at 37 ° C for 2 hours. Then the plates were washed 4 times with each time 5 min. The first well was diluted 1:100, diluted 1:3 in a gradient, 100 μl/well. The plates were then incubated at 37 °C for 1.5 h, and washed 4 times for 5 min each time. Horseradish-labeled mouse anti-duck IgG (baiaolaibo, Beijing) was diluted 1:5000, 100 μl/well, incubated at 37 ° C for 20 to 30 min, and washed 4 times for 5 min each time. The plates were added with 1×TMB at 100 ul/well, and then incubated for 15-30 min at 37 °C. Finally, stop solution (2M H 2 SO 4 ) was added into the plates at 50 μl/well. The OD value of each well was measured at a single wavelength of 450 nm, and the ratio (Positive/Ngetive, P/N) of the OD value to the negative control well was greater than 2.1 as a critical point judged to be positive or negtive. 96 well plates were added with 150 μl 0.1 M NaHCO 3 plus 2.5 % glutaraldehyde, incubated for 1 h at 37 °C, and washed four times with sterile water. The plates were then coated with 10 7 CFU/100 μl RA cells per well and incubated at 37 °C until dry. Subsequent steps from antigen blocking were carried out in accordance with the above iELISA procedure. Serum bactericidal assay (SBA) The serum 7 days after the second immunization was used for SBA. A single colony of the purely cultured strain was picked, inoculated into TSB medium, and cultured to the early stage of growth (OD 600 = 1.5 or so). After washing with PBS, it was diluted to 10 8 cfu/ml.Duck serum was diluted to a range of concentrations of 100%, 50%, 25%, 12.5%, 5% with PBS. Inactivated 100% duck serum and PBS were used as controls. 10 uL of the prepared bacterial solution was mixed with 190 uL of different concentrations of duck serum and incubated at 37 ° C for 30 min. The number of bacteria survived was counted by plate counting. Opsonophagocytosis assay The serum 7 days after the second immunization was used for Opsonophagocytosis assay. RA single colonies were picked, inoculated into TSB liquid medium, and cultured to the early stage of growth (OD 600 = 1.5 or so). After washing with PBS, it was diluted to 5 x 10 4 CFU/ml. 20 μl of the test serum was added to the 96-well cell plate and incubate for 20 min at 37 °C. 10 μl of duck complement and 40 μl of duck blood was add into the plates and incubate for 1 hour at 37 °C with shaking. The number of bacteria survived was counted by plate counting. Challenge assay 30 one-day-old cherry valley ducks were randomly divided into three groups. Porin protein with adjuvant, PBS with adjuvant and PBS were separately immunized according to the previous method. After 14 days of secondary immunization, 10 times and a half lethal dose (LD50) RA CH1 was intraperitoneally injected for 1 week, and survival rate after challenge was recorded. Statistical analysis The SigmaPlot software (version 12) and graphpad prism 8.0.2 were used for all statistical analyses. One-way repeated analysis of variance (ANOVA) and the Mann–Whitney rank test were used to evaluate differences between groups. Differences were considered significant at p < 0.05. Results Signal peptide prediction Cleavage site of Porin protein was between pos. 18 and 19: LKS-QE, and probability of prediction was 0.6038. Homology analysis The top 50 sequences with the highest similarity to the Porin protein in the Uniprot database were extracted for comparison. The identity of Porin protein in RA was above 94.2%, blew 68% with other strains.The evolutionary history was inferred by using the Maximum Likelihood method based on the JTT matrix-based model(Jones et al., 1992). The result was shown in Figure 1. The tree with the highest log likelihood (-11905.39) is shown. The analysis involved 50 amino acid sequences. There were a total of 451 positions in the final dataset. Evolutionary analyses were conducted in MEGA X(Kumar et al., 2018). The Porin protein was conservative in RA . Transmembrane structure prediction Transmembrane structure of Porin protein was predicted in figure 2A. The predicted results of VI and NB were consistent and slightly different from the DY prediction results. The same parts of the three prediction structures were plotted and the results are shown in Figure 3. There were 10 extramembranous fragments, 20 transmembrane fragments, and 2B fragments located in the cytoplasmic space. Tertiary structure prediction 50 templates were found and descended by GMQE score. The template name 2o4v.1.A was used for modeling. Structure assessment of the model performed by Ramachandran Plots was shown in Figure 3A. Ramachandran Favoured rate was 89.04%. Modeling results was downloaded and named PorinM, then viewed by Swiss-PdbViewer 4.1, which was shown in Figure 3B. The homo-trimer consisted of alpha helix, a beta sheet and some irregularities, forming a barrel-like protein dominated by beta sheet, which is a typical structure of the outer membrane protein of Gram-negative bacteria. Antigenic prediction The PDB file PorinM was used to analyze the linear and spatial epitopes of the porin protein. There were 14 linear epitopes and 6 spatial epitopes were initially predicted. After screening non-membrane epitopes, only 9 linear epitopes (Table 1) and 2 spatial epitopes (Table 2) remained. Expression and purification of poring protein of RA Expression and purification of RA was shown in Figure 4. Porin gene was cloned, as shown in figure 4A. Agarose gel electrophoresis showed that the gene was located between 1000bp and 2000bp, which was in line with the expected electrophoretic band position. The recombinant protein contains his tag, which is recombinantly expressed, cleaved, denatured and renatured, and then purified by a nickel column. SDS-PAGE showed that the molecular weight of the protein was between 43kd and 55kd (Figure 4B), which was in line with the expected results. The purity of the purified protein is about 90%. Strong antibody induced by immunization with Porin protein. After the first immunization and the second immunization, the serum antibodies of the Porin protein immunized group were positive at 1:24300 for the porin protein and RA strains CH1 (P/N values were higher than 2.1), while PBS and PBS+ adjuvant group serum were negative (Figure 5). Bactericidal activity and phagocytosis of the anti-porin sera against RA Bactericidal activity was shown in Fig.6. Compared with 0% serum concentration, when the serum concentrations were 12.5%, 25%, 50%, and 100%, the colonies of the Porin group decreased by 6.43%, 14.46%, 16.25%, and 22.95%, respectively, while the other two groups had no significant difference. This indicated that the serum immunized with Proin protein may activate complement sterilization in vitro. Opsonophagocytosis assay was shown in Fig.7. Compared with the PBS control group, the number of viable bacteria in the PBS + adjuvant group and the Porin group decreased by 7% (the difference was not significant) and 58% (the difference was extremely significant), as shown in Fig.7. The results show that serum antibodies can effectively enhance the phagocytosis of phagocytes after Porin protein immunized ducks. Protective efficacy of the Porin sera against RA in vivo The survival rates of the Porin group and the PBS group after challenge were shown in Fig. 8. Porin group ducks died on the second day after challenge, and the survival rate remained at 80% as of the seventh day. Ducks in the PBS group died on the first day after challenge, and all died on the fourth day. Discussion RA mainly infects ducks, geese and turkeys, et al, and young animals have a higher mortality rate after infection. Antibiotic treatment has led to increasing resistance to RA . The current vaccines against RA are mainly inactivated vaccines(Liu et al.) and subunit vaccines(Chu et al., 2015). However, there are many RA serotypes, and the cross-immunoprotective effect between each serotype is weak. Currently reported serotypes with cross-immunoprotection are serotypes 1 and 2(Zhai et al., 2013). So, it is particularly important to develop subunit vaccines that have immune protection against multiple serotypes of RA . The Porin protein has been reported to be immunoprotective in other strains. However, the protein was only presumed to be present in RA by sequencing, and has not been analyzed by recombinant expression, nor has it been verified whether it has immune protection against RA . The protein was firstly analyzed using bioinformatics tools. By blast comparison and Phylogenetic tree analysis, it can be seen that the protein is highly conserved in RA and has low similarity to Proin protein of other strains. It could be speculated that if the protein has immunoprotective effect on serotype 1 RA , then it may also have immunoprotective effect on other serotype RA . Our previous research also showed that even if the conserved antigen exists in different bacterial species, the antigen will provide cross-immunity protection(Guan et al., 2015; Guan et al., 2016). The transmembrane structure and epitope of Porin protein in RA were also predicted. Only epitopes located outside the membrane have the potential to develop vaccines. After screening, 9 linear epitopes and 2 spatial epitopes were considered to be effective. According to the analysis, this protein is conserved in RA and has abundant epitopes, which may have the potential to develop vaccines. Bioinformatics analysis also showed that the protein may be expressed as inclusion bodies in E. coli. Therefore, the expressed protein needs to be refolded and renatured. The membrane protein contains a transmembrane portion and a hydrophilic portion. In order to keep the protein as natural as possible, a mild detergent needs to be added to the refolding buffer. Serum antibody affinity was analyzed by iELISA. 28 days after the initial immunization, duck serum antibodies still have a strong affinity for Porin protein and serotype 1 RA strains. SBA and opsonization phagocytosis also prove that serum antibodies can improve the elimination of RA by the immune system. Compared with the control group, the survival rate of Porin protein immunized group after challenge increased, indicating that the protein has the potential to be developed into a vaccine. Declarations The article is original, has been written by the stated authors who are all aware of its content and approve its submission, and has not been published previously. It is not under consideration for publication elsewhere, no conflict of interest exists. If accepted, the article will not be published elsewhere in the same form, in any language, without the written consent of the publisher. Ethics approval and consent to participate The experimental protocol was established, according to the ethical guidelines of Hainan University Institutional Animal Use and Care Committee and was approved by the international bioethics committee. Written informed consent was obtained from individual or guardian participants. Consent for publication Not applicable. Availability of data and material All data generated or analysed during this study are included in this published article. Competing interests The authors declare that they have no competing interests. Funding This study was supported by Hainan Provincial Natural Science Foundation of China (317030) and Hainan Science and Technology Project (ZDYF2018085). Authors' contributions QG and QH conceived and designed research. HY and HZ conducted experiments. KW contributed new reagents or analytical tools. CL, JZ and JW analyzed data. QG wrote the manuscript. All authors read and approved the manuscript. Acknowledgements This study was supported by Hainan Science and Technology Project (ZDYF2018085),Hainan Provincial Natural Science Foundation of China (317030). References Bagos, P. G., Liakopoulos, T. D., Spyropoulos, I. C., & Hamodrakas, S. J. (2004). PRED-TMBB: a web server for predicting the topology of beta-barrel outer membrane proteins. 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Evaluation of the effects of a commercially available Salmonella Newport siderophore receptor and porin protein vaccine on fecal shedding of Salmonella bacteria and health and performance of feedlot cattle. Am J Vet Res, 72(2), 239-247. Fox, J. T., Thomson, D. U., Drouillard, J. S., Thornton, A. B., & Nagaraja, T. G. (2009). Efficacy of Escherichia coli O157:H7 Siderophore Receptor/Porin Proteins–Based Vaccine in Feedlot Cattle Naturally Shedding E. coli O157. Foodborne Pathog Dis, 6(7), 893-899. Guan, Q., Wang, X., Wang, X., Teng, D., Mao, R., Zhang, Y., & Wang, J. (2015). Recombinant outer membrane protein A induces a protective immune response against Escherichia coli infection in mice. Appl Microbiol Biotechnol, 99(13), 5451-5460. Guan, Q., Wang, X., Wang, X., Teng, D., & Wang, J. (2016). In silico analysis and recombinant expression of BamA protein as a universal vaccine against Escherichia coli in mice. Appl Microbiol Biotechnol, 100(11), 5089-5098. Hinz, K. H., Ryll, M., & Köhler, B. (1998). Detection of acid production from carbohydrates by Riemerella anatipestifer and related organisms using the buffered single substrate test. Vet Microbiol, 60(2–4), 277-284. Jones, D. T., Taylor, W. R., & Thornton, J. M. (1992). The rapid generation of mutation data matrices from protein sequences. Comput Appl Biosci, 8(3), 275-282. Kumar, S., Stecher, G., Li, M., Knyaz, C., & Tamura, K. (2018). MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms. Mol Biol Evol, 35(6), 1547-1549. Liu, H., Wang, X., Ding, C., Han, X., Cheng, A., Wang, S., & Yu, S. Development and Evaluation of a Trivalent Riemerella anatipestifer -Inactivated Vaccine. Clin Vaccine Immunol, 20(5), 691-697. Rubbenstroth, D., Ryll, M., Behr, K. P., & Rautenschlein, S. (2009). Pathogenesis of Riemerella anatipestifer in turkeys after experimental mono-infection via respiratory routes or dual infection together with the avian metapneumovirus. 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Zhai, Z., Li, X., Xiao, X., Yu, J., Chen, M., Yu, Y., Wu, G., Li, Y., Ye, L., Yao, H., Lu, C., & Zhang, W. (2013). Immunoproteomics selection of cross-protective vaccine candidates from Riemerella anatipestifer serotypes 1 and 2. Vet Microbiol, 162(2-4), 850-857. Zhu, T. F., Chen, F. F., & Li, J. C. (2017). A strain of pathogenic Bacillus subtilis results in brain damage in ducklings when co-infected with Riemerella anatipestifer . Pol J Vet Sci , 20(4), 803-809. Tables Table 1 linear epitopes outside of outer membrane in RA . No. Start End Peptide Number of residues 1 52 67 YYNFGKGVGITSPDSLF 16 2 86 94 NRSTEYKAA 9 3 125 133 GEIKEGNPL 9 4 196 202 NNTKANE 7 5 235 246 YPLGAFKNNGEF 12 6 278 287 RGKELLESRD 10 7 314 329 SNPLTHNTDFSKVIPV 16 8 359 368 NKSIQNLIPK 10 9 392 406 SKNNFLYIDGRKDNN 15 Table 2 spatial epitopes outside of outer membrane in RA No. Residues Number of residues 1 A:S271, A:N272, A:F273, A:Q275, A:G276, A:G279, A:K280, A:E281, A:L282, A:L283, A:E284, A:S285, A:R286, A:D287, A:S314, A:N315, A:P316, A:L317, A:T318, A:H319, A:N320, A:T321, A:D322, A:F323, A:S324, A:K325, A:V326, A:I327, A:P328, A:V329, A:K360, A:S361, A:I362, A:Q363, A:N364, A:L365, A:I366, A:P367, A:K368, A:L397, A:Y398 41 2 A:N77, A:L79, A:E80, A:A81, A:D82, A:F83, A:N84, A:D85, A:N86, A:S88, A:T89, A:E90, A:Y91, A:K92, A:A93, A:A94, A:P236, A:L237, A:G238, A:A239, A:F240, A:K241, A:N242, A:N243, A:G244, A:E245, A:F246, A:K393, A:N395, A:F396, A:I399, A:D400, A:G401, A:R402, A:K403, A:D404, A:N405, A:N406 38 Cite Share Download PDF Status: Posted Version 2 posted You are reading this latest preprint version Show more versions Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-58801","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Original article","associatedPublications":[],"authors":[{"id":1740504,"identity":"28e67190-9ef0-4267-ab2b-fdc98eeeaa4a","order_by":0,"name":"Hui Yang","email":"","orcid":"","institution":"Hainan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hui","middleName":"","lastName":"Yang","suffix":""},{"id":1740505,"identity":"fefb1b49-abea-4917-b442-e714ea2cece2","order_by":1,"name":"Haiwen Zhang","email":"","orcid":"","institution":"Hainan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Haiwen","middleName":"","lastName":"Zhang","suffix":""},{"id":1740506,"identity":"3bd3aa75-9db6-46da-a26c-9c7f232ba4f8","order_by":2,"name":"Chenghong Liao","email":"","orcid":"","institution":"Hainan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Chenghong","middleName":"","lastName":"Liao","suffix":""},{"id":1740507,"identity":"ee7cbc20-fb50-41c7-9cf5-4e22dd55673c","order_by":3,"name":"Jianguo Zhao","email":"","orcid":"","institution":"Hainan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jianguo","middleName":"","lastName":"Zhao","suffix":""},{"id":1740508,"identity":"a2459cb4-a6f7-42ed-93ef-b7d1f2d2427f","order_by":4,"name":"Jinhua Wang","email":"","orcid":"","institution":"Hainan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jinhua","middleName":"","lastName":"Wang","suffix":""},{"id":1740509,"identity":"750dca12-b714-4571-a49e-7ae181c18506","order_by":5,"name":"Kebang Wu","email":"","orcid":"","institution":"Hainan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Kebang","middleName":"","lastName":"Wu","suffix":""},{"id":1740510,"identity":"30afce3b-bdac-4df9-bcd4-2ec68397f652","order_by":6,"name":"Qian Han","email":"","orcid":"","institution":"Hainan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Qian","middleName":"","lastName":"Han","suffix":""},{"id":1740511,"identity":"f2a05706-8cbf-4963-b14c-7baa3fba00b9","order_by":7,"name":"qingfeng guan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzElEQVRIiWNgGAWjYBACfobDBww/GNjU97M3EKlFsvFYQrFERRrjzJ4DRGoxOHzG4APPmcOMG24kEOuyY2cMN0i2HWaWnPl44w2GGptogjoYe44VGxS2pbPxS6cVWzAcS8ttIKSFWeLwNgPJNmseydk5ZhKMDYcJa2GTf2D+g7eNWcLg5hkitfAwHDEw4DnjbGBwg4dILRIMxxKMgYGcINkD9EsCMX6xPwCJygR+9sMbb3yosSGsBRkYSCSQohyihVQdo2AUjIJRMDIAAJIyQ7dIajmeAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0003-0567-2650","institution":"Hainan University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"qingfeng","middleName":"","lastName":"guan","suffix":""}],"badges":[],"createdAt":"2020-08-13 11:21:00","currentVersionCode":2,"declarations":"","doi":"10.21203/rs.3.rs-58801/v2","doiUrl":"https://doi.org/10.21203/rs.3.rs-58801/v2","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":2164045,"identity":"98f7a059-6bd5-442c-b1e9-030cf590f665","added_by":"auto","created_at":"2020-08-31 14:12:38","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":92053,"visible":true,"origin":"","legend":"Molecular Phylogenetic analysis by Maximum Likelihood method","description":"","filename":"fig1.JPG","url":"https://assets-eu.researchsquare.com/files/rs-58801/v2/fig1.JPG"},{"id":2164046,"identity":"ef2d41a2-4b2c-48e9-ad6e-08ecd1a0d2c4","added_by":"auto","created_at":"2020-08-31 14:12:38","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":93635,"visible":true,"origin":"","legend":"Transmembrane structure prediction of Porin protein. A: Prediction by VI, NB and DY methods. B: common elements of the prediction in three methods.","description":"","filename":"fig2.JPG","url":"https://assets-eu.researchsquare.com/files/rs-58801/v2/fig2.JPG"},{"id":2164047,"identity":"89262408-6847-4521-9146-9922e5ac6b0c","added_by":"auto","created_at":"2020-08-31 14:12:38","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":73087,"visible":true,"origin":"","legend":"Tertiary structure prediction of Proin protein. A: Structure assessment of the model by Ramachandran Plots. B: 3D structure of PorinM.","description":"","filename":"fig3.JPG","url":"https://assets-eu.researchsquare.com/files/rs-58801/v2/fig3.JPG"},{"id":2164048,"identity":"4d240703-e59d-4dbb-9e26-7d96d03f1ee0","added_by":"auto","created_at":"2020-08-31 14:12:38","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":37927,"visible":true,"origin":"","legend":"Electrophoresis of Porin gene cloning and protein purification. 4A: Agarose gel electrophoresis of Porin gene. 4B:SDS-PAGE of Porin protein. M: 180 kd protein ruler. M1: Trans 2K DNA marker. 1: BL21 strain. 2: BL21 strain with pET 28a vector. 3: BL21 strain with Porin gene. 4: Purified Porin. 5: Porin gene.","description":"","filename":"fig4.JPG","url":"https://assets-eu.researchsquare.com/files/rs-58801/v2/fig4.JPG"},{"id":2164049,"identity":"445a9475-4bf3-40f9-8076-0277988b5022","added_by":"auto","created_at":"2020-08-31 14:12:38","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":47078,"visible":true,"origin":"","legend":"IELISA for Porin immunization serum.","description":"","filename":"fig5.JPG","url":"https://assets-eu.researchsquare.com/files/rs-58801/v2/fig5.JPG"},{"id":2164050,"identity":"210e08f3-1734-4463-97b3-02d54ce70cff","added_by":"auto","created_at":"2020-08-31 14:12:38","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":60123,"visible":true,"origin":"","legend":"Colonies change in different dilution of surum.","description":"","filename":"fig6.JPG","url":"https://assets-eu.researchsquare.com/files/rs-58801/v2/fig6.JPG"},{"id":2164051,"identity":"6fb7a099-4dc2-44cf-a6c7-f0eff2d8bea2","added_by":"auto","created_at":"2020-08-31 14:12:38","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":44183,"visible":true,"origin":"","legend":"Phagocytosis of the rBamA sera in vitro.","description":"","filename":"fig7.JPG","url":"https://assets-eu.researchsquare.com/files/rs-58801/v2/fig7.JPG"},{"id":2164052,"identity":"5e043811-e280-4c58-80af-bb92099b7874","added_by":"auto","created_at":"2020-08-31 14:12:38","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":34446,"visible":true,"origin":"","legend":"Survival percent of the mice immunized with Porin or PBS.","description":"","filename":"fig8.JPG","url":"https://assets-eu.researchsquare.com/files/rs-58801/v2/fig8.JPG"},{"id":15668693,"identity":"16576364-8900-4ed0-9a9f-23610151a7a5","added_by":"auto","created_at":"2021-11-18 13:49:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":830249,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-58801/v2/21a3594c-fb11-4569-a535-18f23d165f10.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003ePorin protein could be a vaccine candidate against \u003cem\u003eRiemerella anatipestifer \u003c/em\u003efor ducks\u003c/p\u003e","fulltext":[{"header":"Key Points","content":"\u003cul\u003e\n\u003cli\u003eThere are 21 serotypes of\u003cem\u003e RA\u003c/em\u003e, but the cross immunoprotective effect between each serotype is extremely weak. The development of subunit vaccines is a powerful way to prevent the infection of multiple serotypes of\u003cem\u003e RA\u003c/em\u003e.\u003c/li\u003e\n\u003cli\u003eThrough bioinformatics analysis, it was found that Porin protein is conserved in\u003cem\u003e RA\u003c/em\u003e and also has abundant epitopes. It is speculated that this protein has the potential to develop a subunit vaccine.\u003c/li\u003e\n\u003cli\u003eThe recombinant Porin protein was used to immunize Cherry Valley Duck. The serum produced had a cross-immunization effect on serum type 1 and serum type 3\u003cem\u003e RA\u003c/em\u003e, and enhanced the clearance of pathogens by complement and phagocytes. After challenge, the survival rate of Cherry Valley was improved.\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"Introduction","content":"\u003cp\u003eRA is a contact infectious bacterium that infects domestic ducks, geese(Chen et al.; Shu-Chuan et al., 1999), turkeys(Rubbenstroth et al.) and a variety of birds(Hinz et al., 1998). Its infection symptoms are fibrinous pericarditis, fibrinous perihepatitis, fibrinous air sacs, encephalitis, Arthritis, etc. The disease was first reported in Long Island, New York, USA in 1932, and subsequently in Europe, South Asia, and Oceania. At present, the occurrence and prevalence of this disease is extremely extensive, and the disease is prevalent in almost all places where ducks are raised. All kinds of ducks in poultry can be infected. Under natural conditions, ducks of all ages are susceptible, and the incidence rate is about 90%(Zhu et al., 2017).\u003c/p\u003e\n\u003cp\u003eDrug treatment is one of the important measures to prevent and treat the disease, mainly antibiotics and horizontal amines. The disease can occur in the whole stage of duck breeding, and birds can be infected repeatedly, resulting in higher cost of medication. Clinically, drug abuse, poor therapeutic effects, and even treatment failures often occur. Although\u003cem\u003e RA \u003c/em\u003eis sensitive to many antibiotics, it is highly susceptible to drug resistance(Sun et al., 2019; Xing et al., 2015). Different regions have different susceptibility to antibacterial drugs in different regions, which brings difficulties to the actual treatment work. Therefore, immunization is an effective way to prevent and control this disease. The vaccines currently used to prevent this disease mainly include inactivated vaccines, attenuated vaccines, and subunit vaccines. Due to the complexity of\u003cem\u003e RA \u003c/em\u003eserotypes and the lack of cross-protection between different serotypes, both attenuated and inactivated vaccines are effective against specific serotypes but are not protective against other serotypes of\u003cem\u003e RA\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eGram-negative outer membrane proteins (Omps) not only play an important role in maintaining the integrity of the cell envelope and transmembrane transport of the substances, but also in the the adhesion of bacteria to invading cells and bacteria to serum resistance. Outer membrane proteins can be divided into major proteins and microproteins according to their copy number in cells. Among them, Porin protein, also known as channel-forming protein, has a transmembrane domain with only 10 to 12 amino acid residues, forming a \u0026beta;-sheet structure, and antiparallel \u0026beta;-sheets interact to form a non-specific transmembrane. The channel allows small molecules with a relative molecular mass of less than 10,000 D to pass freely and is associated with bacterial resistance. Studies have shown that porin protein has good immunoprotective effects against strains such as \u003cem\u003eE. coli\u003c/em\u003e(Fox et al., 2009), \u003cem\u003eSalmonella\u003c/em\u003e(Dodd et al., 2011) and \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e(Bahey-El-Din et al., 2020). According to the genome sequencing results of\u003cem\u003e RA \u003c/em\u003e(Wang, et al., 2012), it was found that porin was also present in\u003cem\u003e RA\u003c/em\u003e. At present, the research of\u003cem\u003e RA \u003c/em\u003eporin is still in the prediction stage, and there are no reports of expression and immune protection analysis.\u003c/p\u003e\n\u003cp\u003eIn this paper, the conservation, structural properties and immunogenicity of porin protein were analyzed by bioinformatics, and the duck was immunized by recombinant expression of the protein to verify the cross-immunoprotective effect and immune mechanism of the protein.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003eBacterial strains and culture conditions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eRA \u003c/em\u003estrain ATCC 11845 were purchased from Shanghai yiyan biological technology Co. Ltd (ShangHai, China). Serotype 1 \u003cem\u003eRA \u003c/em\u003estrain CH-1 (RA-CH1) was isolated from sick ducks in HaiKou, China. \u003cem\u003eRA \u003c/em\u003estrains were grown in tryptic soy broth medium (TSB) and at 37\u0026deg;C. \u003cem\u003eE. coli\u003c/em\u003e strain DH5\u0026alpha; and BL21 (DE3) strains were purchased from TransGen Biotech, Inc. (Beijing, China).\u003cem\u003e E. coli\u003c/em\u003e strains were grown on Luria-Bertani (LB) agar.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimals \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e30 One-day-old Cherry Valley ducklings obtained from the Hainan Tianyuan Industrial Co., Ltd.(Haikou, China). The ducks were housed in cages, and provided water and food adlibitum during the study. The animal experiments were conducted in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the Institutional Animal Care and Use Committee guidelines set by HanNan University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSignal peptide prediction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSignal peptide cleavage site was predicted by SignalP 5.0 (http://www.cbs.dtu.dk/services/SignalP/).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHomology analysis and phylogenetic tree construction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe\u003cem\u003e RA\u003c/em\u003e Porin protein (Uniprot NO. E4TDA8) was blasted by UniProtKB database with parameters including E-Threshold 10, Matrix BLOSUM-62 and no filtering in Uniprot (\u003ca href=\"https://www.uniprot.org/blast/\"\u003ehttps://www.uniprot.org/blast/\u003c/a\u003e). The number of returned alignments was 50. The blasted data was aligned with ClustalW and phylogeny tree was constructed with maximum likelihood statistical method by MEGAX.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTransmembrane structure prediction \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMature peptide sequence of\u003cem\u003e RA\u003c/em\u003e Porin protein was pasted into PRED-TMBB web server (http://biophysics.biol.uoa.gr/PRED-TMBB/input.jsp)(Bagos et al., 2004), which is for predicting the topology of beta-barrel outer membrane proteins. Viterbi (VI), N-best (NB) and Posterior Decoding (DY) method were applied and the common part of the three analyses was as prediction results.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTertiary structure prediction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe tertiary structure of the Porin protein sequence from which the signal peptide was removed was predicted by the swissmodel online server (https://swissmodel.expasy.org/).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAntigenic prediction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe B cell epitopes were predicted by ElliPro web server (http://tools.iedb.org/ellipro/) based based upon structural protrusion. The predicted results were screened to retain only epitopes outside the membrane.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExpression and purification of poring protein of \u003cem\u003eRA\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe genome of \u003cem\u003eRA\u003c/em\u003e strain ATCC 11845 is extracted by the genome extraction kit (TIANGEN Biotech(Beijing)Co.,Ltd.,BeiJing, China), and the porin gene (sequence accession number in genebank:NC_017045.1, from 1940759 to 1942012 ) is amplified by primer pairs porin F: 5\u0026rsquo;-GGATCCGTACCAGATACTATTATCGTTC-3\u0026rsquo; and porin R: 5\u0026rsquo;-GCGGCCGCAATTCCTATTTCTACTTGAAATCT-3\u0026rsquo;, with Restriction site \u003cem\u003eBam\u003c/em\u003eH 1and \u003cem\u003eNot\u003c/em\u003e1. The gene was then ligated into the pET 28a vector, and the recombined vectors were transformed into BL21 competent cells. These successfully transformed BL21 \u003cem\u003eE. coli\u003c/em\u003e cells were induced to express the target protein by auto-inducing medium(Studier, 2014). The expressed product was subjected to cleavage, separation, denaturation, renaturation, dialysis, and finally purification through a nickel column based on the previous protocol(Saleem et al., 2012). All proteins were determined by 12 % sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS\u0026ndash;PAGE). The protein was then lyophilized with the ALPHA 1-2 LD plus freeze dryer (Christ, Germany) and kept in \u0026minus;20 \u0026deg;C.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunization protocols\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Cherry Valley ducks were randomly divided into 3 groups of 10 animals each injected with PBS (PBS group), PBS + adjuvant (PBS + adjuvant group), PBS + adjuvant + Porin protein (Porin group). The ducks were first immunized at 7 days of age. The Porin protein was mixed with Freund's complete adjuvant in a 1:1 oily form and injected subcutaneously. For the second immunization 7 day post the first immunization, Porin protein was mixed with Freund's incomplete adjuvant by 1:1 to form an oil emulsion. Both immunization doses were 0.2 mg/duck. The days of blood collection was 1, 7, 14, 21 and 28 days post the first immunization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIndirect enzyme-linked immunosorbent assay (iELISA) for Porin protein and\u003cem\u003e RA\u003c/em\u003e cells detection \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e96-well plates were coated at a concentration of 2 \u0026mu;g/ml protein antigen with 100 \u0026mu;l/well, overnight at 4 \u0026deg; C, and washed 4 times with 100 \u0026mu;l PBST wash for 5 min each. The plates were added with 200 \u0026mu;l/well blocking solution and incubaed at 37 \u0026deg; C for 2 hours. Then the plates were washed 4 times with each time 5 min. The first well was diluted 1:100, diluted 1:3 in a gradient, 100 \u0026mu;l/well. The plates were then incubated at 37 \u0026deg;C for 1.5 h, and washed 4 times for 5 min each time. Horseradish-labeled mouse anti-duck IgG (baiaolaibo, Beijing) was diluted 1:5000, 100 \u0026mu;l/well, incubated at 37 \u0026deg; C for 20 to 30 min, and washed 4 times for 5 min each time. The plates were added with 1\u0026times;TMB at 100 ul/well, and then incubated for 15-30 min at 37 \u0026deg;C. Finally, stop solution (2M H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e) was added into the plates at 50 \u0026mu;l/well.\u003c/p\u003e\n\u003cp\u003eThe OD value of each well was measured at a single wavelength of 450 nm, and the ratio (Positive/Ngetive, P/N) of the OD value to the negative control well was greater than 2.1 as a critical point judged to be positive or negtive.\u003c/p\u003e\n\u003cp\u003e96 well plates were added with 150 \u0026mu;l 0.1 M NaHCO 3 plus 2.5 % glutaraldehyde, incubated for 1 h at 37 \u0026deg;C, and washed four times with sterile water. The plates were then coated with 10\u003csup\u003e7\u003c/sup\u003e CFU/100 \u0026mu;l\u003cem\u003e RA\u003c/em\u003e cells per well and incubated at 37 \u0026deg;C until dry. Subsequent steps from antigen blocking were carried out in accordance with the above iELISA procedure.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSerum bactericidal assay (SBA)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe serum 7 days after the second immunization was used for SBA. A single colony of the purely cultured strain was picked, inoculated into TSB medium, and cultured to the early stage of growth (OD 600 = 1.5 or so). After washing with PBS, it was diluted to 10\u003csup\u003e8\u003c/sup\u003e cfu/ml.Duck serum was diluted to a range of concentrations of 100%, 50%, 25%, 12.5%, 5% with PBS. Inactivated 100% duck serum and PBS were used as controls. 10 uL of the prepared bacterial solution was mixed with 190 uL of different concentrations of duck serum and incubated at 37 \u0026deg; C for 30 min. The number of bacteria survived was counted by plate counting.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOpsonophagocytosis assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe serum 7 days after the second immunization was used for Opsonophagocytosis assay.\u003cem\u003e RA\u003c/em\u003e single colonies were picked, inoculated into TSB liquid medium, and cultured to the early stage of growth (OD 600 = 1.5 or so). After washing with PBS, it was diluted to 5 x 10\u003csup\u003e4\u003c/sup\u003e CFU/ml. 20 \u0026mu;l of the test serum was added to the 96-well cell plate and incubate for 20 min at 37 \u0026deg;C. 10 \u0026mu;l of duck complement and 40 \u0026mu;l of duck blood was add into the plates and incubate for 1 hour at 37 \u0026deg;C with shaking. The number of bacteria survived was counted by plate counting.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChallenge assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e30 one-day-old cherry valley ducks were randomly divided into three groups. Porin protein with adjuvant, PBS with adjuvant and PBS were separately immunized according to the previous method. After 14 days of secondary immunization, 10 times and a half lethal dose (LD50)\u003cem\u003e RA\u003c/em\u003e CH1 was intraperitoneally injected for 1 week, and survival rate after challenge was recorded.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe SigmaPlot software (version 12) and graphpad prism 8.0.2 were used for all statistical analyses. One-way repeated analysis of variance (ANOVA) and the Mann\u0026ndash;Whitney rank test were used to evaluate differences between groups. Differences were considered significant at p \u0026lt; 0.05.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eSignal peptide prediction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCleavage site of Porin protein was between pos. 18 and 19: LKS-QE, and probability of prediction was 0.6038.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHomology analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe top 50 sequences with the highest similarity to the Porin protein in the Uniprot database were extracted for comparison. The identity of Porin protein in\u003cem\u003e RA\u003c/em\u003e was above 94.2%, blew 68% with other strains.The evolutionary history was inferred by using the Maximum Likelihood method based on the JTT matrix-based model(Jones et al., 1992). The result was shown in Figure 1. The tree with the highest log likelihood (-11905.39) is shown. The analysis involved 50 amino acid sequences. There were a total of 451 positions in the final dataset. Evolutionary analyses were conducted in MEGA X(Kumar et al., 2018). The Porin protein was conservative in\u003cem\u003e RA\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTransmembrane structure prediction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTransmembrane structure of Porin protein was predicted in figure 2A. The predicted results of VI and NB were consistent and slightly different from the DY prediction results. The same parts of the three prediction structures were plotted and the results are shown in Figure 3. There were 10 extramembranous fragments, 20 transmembrane fragments, and 2B fragments located in the cytoplasmic space.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTertiary structure prediction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e50 templates were found and descended by GMQE score. The template name 2o4v.1.A was used for modeling. Structure assessment of the model performed by Ramachandran Plots was shown in Figure 3A. Ramachandran Favoured rate was 89.04%. Modeling results was downloaded and named PorinM, then viewed by Swiss-PdbViewer 4.1, which was shown in Figure 3B. The homo-trimer consisted of alpha helix, a beta sheet and some irregularities, forming a barrel-like protein dominated by beta sheet, which is a typical structure of the outer membrane protein of Gram-negative bacteria.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAntigenic prediction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe PDB file PorinM was used to analyze the linear and spatial epitopes of the porin protein. There were 14 linear epitopes and 6 spatial epitopes were initially predicted. After screening non-membrane epitopes, only 9 linear epitopes (Table 1) and 2 spatial epitopes (Table 2) remained.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExpression and purification of poring protein of \u003cem\u003eRA\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExpression and purification of\u003cem\u003e RA\u003c/em\u003e was shown in Figure 4. Porin gene was cloned, as shown in figure 4A. Agarose gel electrophoresis showed that the gene was located between 1000bp and 2000bp, which was in line with the expected electrophoretic band position. The recombinant protein contains his tag, which is recombinantly expressed, cleaved, denatured and renatured, and then purified by a nickel column. SDS-PAGE showed that the molecular weight of the protein was between 43kd and 55kd (Figure 4B), which was in line with the expected results. The purity of the purified protein is about 90%.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStrong antibody induced by immunization with Porin protein.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter the first immunization and the second immunization, the serum antibodies of the Porin protein immunized group were positive at 1:24300 for the porin protein and \u003cem\u003eRA\u003c/em\u003e strains CH1 (P/N values were higher than 2.1), while PBS and PBS+ adjuvant group serum were negative (Figure 5).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBactericidal activity and phagocytosis of the anti-porin sera against\u003cem\u003e RA\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBactericidal activity was shown in Fig.6. Compared with 0% serum concentration, when the serum concentrations were 12.5%, 25%, 50%, and 100%, the colonies of the Porin group decreased by 6.43%, 14.46%, 16.25%, and 22.95%, respectively, while the other two groups had no significant difference. This indicated that the serum immunized with Proin protein may activate complement sterilization in vitro.\u003c/p\u003e\n\u003cp\u003eOpsonophagocytosis assay was shown in Fig.7. Compared with the PBS control group, the number of viable bacteria in the PBS + adjuvant group and the Porin group decreased by 7% (the difference was not significant) and 58% (the difference was extremely significant), as shown in Fig.7. The results show that serum antibodies can effectively enhance the phagocytosis of phagocytes after Porin protein immunized ducks.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProtective efficacy of the Porin sera against\u003cem\u003e RA\u003c/em\u003e in vivo\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe survival rates of the Porin group and the PBS group after challenge were shown in Fig. 8. Porin group ducks died on the second day after challenge, and the survival rate remained at 80% as of the seventh day. Ducks in the PBS group died on the first day after challenge, and all died on the fourth day.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eRA mainly infects ducks, geese and turkeys, et al, and young animals have a higher mortality rate after infection. Antibiotic treatment has led to increasing resistance to\u003cem\u003e RA\u003c/em\u003e. The current vaccines against\u003cem\u003e RA\u003c/em\u003e are mainly inactivated vaccines(Liu et al.) and subunit vaccines(Chu et al., 2015). However, there are many\u003cem\u003e RA\u003c/em\u003e serotypes, and the cross-immunoprotective effect between each serotype is weak. Currently reported serotypes with cross-immunoprotection are serotypes 1 and 2(Zhai et al., 2013). So, it is particularly important to develop subunit vaccines that have immune protection against multiple serotypes of\u003cem\u003e RA\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eThe Porin protein has been reported to be immunoprotective in other strains. However, the protein was only presumed to be present in\u003cem\u003e RA\u003c/em\u003e by sequencing, and has not been analyzed by recombinant expression, nor has it been verified whether it has immune protection against\u003cem\u003e RA\u003c/em\u003e. The protein was firstly analyzed using bioinformatics tools. By blast comparison and Phylogenetic tree analysis, it can be seen that the protein is highly conserved in\u003cem\u003e RA\u003c/em\u003e and has low similarity to Proin protein of other strains. It could be speculated that if the protein has immunoprotective effect on serotype 1\u003cem\u003e RA\u003c/em\u003e, then it may also have immunoprotective effect on other serotype\u003cem\u003e RA\u003c/em\u003e. Our previous research also showed that even if the conserved antigen exists in different bacterial species, the antigen will provide cross-immunity protection(Guan et al., 2015; Guan et al., 2016). The transmembrane structure and epitope of Porin protein in\u003cem\u003e RA\u003c/em\u003e were also predicted. Only epitopes located outside the membrane have the potential to develop vaccines. After screening, 9 linear epitopes and 2 spatial epitopes were considered to be effective. According to the analysis, this protein is conserved in\u003cem\u003e RA\u003c/em\u003e and has abundant epitopes, which may have the potential to develop vaccines.\u003c/p\u003e\n\u003cp\u003eBioinformatics analysis also showed that the protein may be expressed as inclusion bodies in \u003cem\u003eE. coli.\u003c/em\u003e Therefore, the expressed protein needs to be refolded and renatured. The membrane protein contains a transmembrane portion and a hydrophilic portion. In order to keep the protein as natural as possible, a mild detergent needs to be added to the refolding buffer.\u003c/p\u003e\n\u003cp\u003eSerum antibody affinity was analyzed by iELISA. 28 days after the initial immunization, duck serum antibodies still have a strong affinity for Porin protein and serotype 1 \u003cem\u003eRA\u003c/em\u003e strains. SBA and opsonization phagocytosis also prove that serum antibodies can improve the elimination of\u003cem\u003e RA\u003c/em\u003e by the immune system. Compared with the control group, the survival rate of Porin protein immunized group after challenge increased, indicating that the protein has the potential to be developed into a vaccine.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eThe article is original, has been written by the stated authors who are all aware of its content and approve its submission, and has not been published previously. It is not under consideration for publication elsewhere, no conflict of interest exists. If accepted, the article will not be published elsewhere in the same form, in any language, without the written consent of the publisher.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe experimental protocol was established, according to the ethical guidelines of Hainan University Institutional Animal Use and Care Committee and was approved by the international bioethics committee. Written informed consent was obtained from individual or guardian participants.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this published article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by Hainan Provincial Natural Science Foundation of China (317030) and Hainan Science and Technology Project (ZDYF2018085).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eQG and QH conceived and designed research. HY and HZ conducted experiments. KW contributed new reagents or analytical tools. CL, JZ and JW analyzed data. QG wrote the manuscript. All authors read and approved the manuscript.\u003c/p\u003e\n\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eThis study was supported by Hainan Science and Technology Project (ZDYF2018085),Hainan Provincial Natural Science Foundation of China (317030).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBagos, P. G., Liakopoulos, T. D., Spyropoulos, I. C., \u0026amp; Hamodrakas, S. J. (2004). PRED-TMBB: a web server for predicting the topology of beta-barrel outer membrane proteins. Nucleic Acids Res, 32(Web Server issue), W400-404.\u003c/li\u003e\n\u003cli\u003eBahey-El-Din, M., Mohamed, S. A., Sheweita, S. A., Haroun, M., \u0026amp; Zaghloul, T. I. (2020). Recombinant N-terminal outer membrane porin (OprF) of \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e is a promising vaccine candidate against both \u003cem\u003eP. aeruginosa\u003c/em\u003e and some strains of \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e. Int J Med Microbiol, 310(3), 151415.\u003c/li\u003e\n\u003cli\u003eChen, Y. P., Lee, S.-H., Chou, C.-H., \u0026amp; Tsai, H.-J. Detection of florfenicol resistance genes in \u003cem\u003eRiemerella anatipestifer\u003c/em\u003e isolated from ducks and geese. Vet Microbiol, 154(3-4), 325-331.\u003c/li\u003e\n\u003cli\u003eChu, C. Y., Liu, C. H., Liou, J. J., Lee, J. W., \u0026amp; Cheng, L. T. (2015). Development of a subunit vaccine containing recombinant \u003cem\u003eRiemerella anatipestifer\u003c/em\u003e outer membrane protein A and CpG ODN adjuvant. Vaccine, 33(1), 92-99.\u003c/li\u003e\n\u003cli\u003eDodd, C. C., Renter, D. G., Thomson, D. U., \u0026amp; Nagaraja, T. G. (2011). Evaluation of the effects of a commercially available \u003cem\u003eSalmonella Newport\u003c/em\u003e siderophore receptor and porin protein vaccine on fecal shedding of Salmonella bacteria and health and performance of feedlot cattle. Am J Vet Res, 72(2), 239-247.\u003c/li\u003e\n\u003cli\u003eFox, J. T., Thomson, D. U., Drouillard, J. S., Thornton, A. B., \u0026amp; Nagaraja, T. G. (2009). Efficacy of \u003cem\u003eEscherichia coli\u003c/em\u003e O157:H7 Siderophore Receptor/Porin Proteins\u0026ndash;Based Vaccine in Feedlot Cattle Naturally Shedding \u003cem\u003eE. coli\u003c/em\u003e O157. Foodborne Pathog Dis, 6(7), 893-899.\u003c/li\u003e\n\u003cli\u003eGuan, Q., Wang, X., Wang, X., Teng, D., Mao, R., Zhang, Y., \u0026amp; Wang, J. (2015). Recombinant outer membrane protein A induces a protective immune response against \u003cem\u003eEscherichia coli\u003c/em\u003e infection in mice. Appl Microbiol Biotechnol, 99(13), 5451-5460.\u003c/li\u003e\n\u003cli\u003eGuan, Q., Wang, X., Wang, X., Teng, D., \u0026amp; Wang, J. (2016). In silico analysis and recombinant expression of BamA protein as a universal vaccine against \u003cem\u003eEscherichia coli\u003c/em\u003e in mice. Appl Microbiol Biotechnol, 100(11), 5089-5098.\u003c/li\u003e\n\u003cli\u003eHinz, K. H., Ryll, M., \u0026amp; K\u0026ouml;hler, B. (1998). Detection of acid production from carbohydrates by \u003cem\u003eRiemerella anatipestifer\u003c/em\u003e and related organisms using the buffered single substrate test. Vet Microbiol, 60(2\u0026ndash;4), 277-284.\u003c/li\u003e\n\u003cli\u003eJones, D. T., Taylor, W. R., \u0026amp; Thornton, J. M. (1992). The rapid generation of mutation data matrices from protein sequences. Comput Appl Biosci, 8(3), 275-282.\u003c/li\u003e\n\u003cli\u003eKumar, S., Stecher, G., Li, M., Knyaz, C., \u0026amp; Tamura, K. (2018). MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms. Mol Biol Evol, 35(6), 1547-1549.\u003c/li\u003e\n\u003cli\u003eLiu, H., Wang, X., Ding, C., Han, X., Cheng, A., Wang, S., \u0026amp; Yu, S. Development and Evaluation of a Trivalent \u003cem\u003eRiemerella anatipestifer\u003c/em\u003e-Inactivated Vaccine. Clin Vaccine Immunol, 20(5), 691-697.\u003c/li\u003e\n\u003cli\u003eRubbenstroth, D., Ryll, M., Behr, K. P., \u0026amp; Rautenschlein, S. (2009). Pathogenesis of \u003cem\u003eRiemerella anatipestifer\u003c/em\u003e in turkeys after experimental mono-infection via respiratory routes or dual infection together with the avian metapneumovirus. Avian Pathol, 38(6), 497-507.\u003c/li\u003e\n\u003cli\u003eSaleem, M., Moore, J., \u0026amp; Derrick, J. P. J. M. i. M. B. (2012). Expression, Purification, and Crystallization of \u003cem\u003eNeisserial\u003c/em\u003e Outer Membrane Proteins. 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Plos One, 10(6), e0131078.\u003c/li\u003e\n\u003cli\u003eZhai, Z., Li, X., Xiao, X., Yu, J., Chen, M., Yu, Y., Wu, G., Li, Y., Ye, L., Yao, H., Lu, C., \u0026amp; Zhang, W. (2013). Immunoproteomics selection of cross-protective vaccine candidates from \u003cem\u003eRiemerella anatipestifer\u003c/em\u003e serotypes 1 and 2. Vet Microbiol, 162(2-4), 850-857.\u003c/li\u003e\n\u003cli\u003eZhu, T. F., Chen, F. F., \u0026amp; Li, J. C. (2017). A strain of pathogenic Bacillus subtilis results in brain damage in ducklings when co-infected with \u003cem\u003eRiemerella anatipestifer\u003c/em\u003e. Pol J Vet Sci , 20(4), 803-809.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 linear epitopes outside of outer membrane in\u003cem\u003e RA\u003c/em\u003e.\u003c/p\u003e\n\u003ctable border=\"1\" width=\"554\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd width=\"35\"\u003e\n\u003cp\u003eNo.\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e\u003cstrong\u003eStart\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"53\"\u003e\n\u003cp\u003e\u003cstrong\u003eEnd\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"227\"\u003e\n\u003cp\u003e\u003cstrong\u003ePeptide\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"179\"\u003e\n\u003cp\u003e\u003cstrong\u003eNumber of residues\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"35\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e52\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"53\"\u003e\n\u003cp\u003e67\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"227\"\u003e\n\u003cp\u003eYYNFGKGVGITSPDSLF\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"179\"\u003e\n\u003cp\u003e16\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"35\"\u003e\n\u003cp\u003e2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e86\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"53\"\u003e\n\u003cp\u003e94\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"227\"\u003e\n\u003cp\u003eNRSTEYKAA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"179\"\u003e\n\u003cp\u003e9\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"35\"\u003e\n\u003cp\u003e3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e125\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"53\"\u003e\n\u003cp\u003e133\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"227\"\u003e\n\u003cp\u003eGEIKEGNPL\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"179\"\u003e\n\u003cp\u003e9\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"35\"\u003e\n\u003cp\u003e4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e196\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"53\"\u003e\n\u003cp\u003e202\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"227\"\u003e\n\u003cp\u003eNNTKANE\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"179\"\u003e\n\u003cp\u003e7\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"35\"\u003e\n\u003cp\u003e5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e235\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"53\"\u003e\n\u003cp\u003e246\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"227\"\u003e\n\u003cp\u003eYPLGAFKNNGEF\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"179\"\u003e\n\u003cp\u003e12\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"35\"\u003e\n\u003cp\u003e6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e278\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"53\"\u003e\n\u003cp\u003e287\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"227\"\u003e\n\u003cp\u003eRGKELLESRD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"179\"\u003e\n\u003cp\u003e10\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"35\"\u003e\n\u003cp\u003e7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e314\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"53\"\u003e\n\u003cp\u003e329\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"227\"\u003e\n\u003cp\u003eSNPLTHNTDFSKVIPV\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"179\"\u003e\n\u003cp\u003e16\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"35\"\u003e\n\u003cp\u003e8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e359\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"53\"\u003e\n\u003cp\u003e368\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"227\"\u003e\n\u003cp\u003eNKSIQNLIPK\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"179\"\u003e\n\u003cp\u003e10\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"35\"\u003e\n\u003cp\u003e9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e392\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"53\"\u003e\n\u003cp\u003e406\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"227\"\u003e\n\u003cp\u003eSKNNFLYIDGRKDNN\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"179\"\u003e\n\u003cp\u003e15\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eTable 2 spatial epitopes outside of outer membrane in\u003cem\u003e RA\u003c/em\u003e\u003c/p\u003e\n\u003ctable border=\"1\" width=\"554\"\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003ctd width=\"35\"\u003e\n\u003cp\u003eNo.\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"394\"\u003e\n\u003cp\u003eResidues\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003eNumber of residues\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"35\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"394\"\u003e\n\u003cp\u003eA:S271, A:N272, A:F273, A:Q275, A:G276, A:G279, A:K280, A:E281, A:L282, A:L283, A:E284, A:S285, A:R286, A:D287, A:S314, A:N315, A:P316, A:L317, A:T318, A:H319, A:N320, A:T321, A:D322, A:F323, A:S324, A:K325, A:V326, A:I327, A:P328, A:V329, A:K360, A:S361, A:I362, A:Q363, A:N364, A:L365, A:I366, A:P367, A:K368, A:L397, A:Y398\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003e41\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"35\"\u003e\n\u003cp\u003e2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"394\"\u003e\n\u003cp\u003eA:N77, A:L79, A:E80, A:A81, A:D82, A:F83, A:N84, A:D85, A:N86, A:S88, A:T89, A:E90, A:Y91, A:K92, A:A93, A:A94, A:P236, A:L237, A:G238, A:A239, A:F240, A:K241, A:N242, A:N243, A:G244, A:E245, A:F246, A:K393, A:N395, A:F396, A:I399, A:D400, A:G401, A:R402, A:K403, A:D404, A:N405, A:N406\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003e38\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003c/table\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":"RA, subunit vaccine, bioinformatics analysis","lastPublishedDoi":"10.21203/rs.3.rs-58801/v2","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-58801/v2","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003esuch as domestic ducks and geese. Young birds have a high mortality rate after infection. The resistance caused by the abuse of antibiotics is also getting worse. Since there are 25 serotypes of\u003cem\u003e RA\u003c/em\u003e, and the cross-immunization between serotypes is weak, it is necessary to find a vaccine that has cross-immunization against multiple serotypes of\u003cem\u003e RA\u003c/em\u003e. In this article, the bioinformatics analysis of\u003cem\u003e RA\u003c/em\u003e Proin protein was conducted, and it was speculated that it has the potential of a subunit vaccine. The protein was recombinantly expressed and purified, and immunized with Cherry Valley Duck. The results show that the serum antibodies of the Porin protein immunized group were positive at 1:24300 for the porin protein and \u003cem\u003eRA\u003c/em\u003e strains CH1. The serum could improve the killing effect of complement and phagocytic cells on RA. After the challenge, the survival rate of Cherry Valley Duck can be increased by 80%.\u003c/p\u003e","manuscriptTitle":"Porin protein could be a vaccine candidate against Riemerella anatipestifer for ducks","msid":"","msnumber":"","nonDraftVersions":[{"code":2,"date":"2020-08-31 14:10:20","doi":"10.21203/rs.3.rs-58801/v2","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}},{"code":1,"date":"2020-08-19 17:22:08","doi":"10.21203/rs.3.rs-58801/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":"83db9017-cda5-4784-8dac-ead76fa77d29","owner":[],"postedDate":"August 31st, 2020","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":397136,"name":"Animal Science"}],"tags":[],"updatedAt":"2020-12-08T23:25:24+00:00","versionOfRecord":[],"versionCreatedAt":"2020-08-31 14:10:20","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v2","identity":"rs-58801","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-58801","identity":"rs-58801","version":["v2"]},"buildId":"FbvkV6FR0MCFSLy54lSbu","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}