| Canine coronavirus(CCoV)is a common pathogen that causes gastrointestinal diseases in dogs.The mortality rate of dogs was significantly increased when coinfection with CCo V and intestinal pathogens,which seriously affected the healthy development of the dog industry.In recent years,cases of highly pathogenic coronavirus infectionhave increased significantly,and the positive rate of antibodies has also increased year by year.The structural proteins of the canine coronavirus genome mainly include membrane protein M,spike protein S,small envelope protein E,nucleocapsid protein N.Among them,S protein is a structural protein that can induce host immune cells to produce neutralizing antibodies,has the regulating role of binding to host cell receptors and triggering the fusion of viruses and cell membrane,which is an important determinant of cell orientation and pathogenicity.The S protein is relatively large and the overall expression is difficult.In this study,the recombinant plasmid of the multi-epitope gene of the canine coronavirus S protein antigen was constructed,and the recombinant S protein with strong neutralizing activity.Thus,an indirect ELISA(r SP-ELISA)method was developed to monitor the level of neutralizing antibodies in dogs,laying the foundation for the prevention and treatment of canine coronavirus disease,the monitoring of antibody levels,and subsequent related research.The B cells and T cell linear epitopes of the S protein of CCo V were analyzed by bioinformatics methods,and 4 dominant B cell epitopes and 1 T cell epitope were screened out.The dominant epitopes were recombined by gene shuffling,and four different combinations were obtained.Different epitopes were linked by linker and spacer flexible peptide,and the codons were optimized for full gene synthesis.Then the sequences were cloned into the pET-28? prokaryotic expression vector,respectively.Four multiepitope recombinant plasmids were successfully constructed,named pET28?-S1,pET28?-S2,pET28?-S3 and pET28?-S4.They were transformed into E.coli BL21(DE3)competent cells,and induced with1mmol/L IPTG for 8 hours at 37 ?.The 4 recombinant plasmids all successfully expressed the recombinant proteins.The four recombinant proteins were purified by Ni-NTA affinity tomography columns and the correct expression of the protein was confirmed by Western blot and liquid phase mass spectrometry proteins.The recombinant protein was emulsified and immunized to 6-week-old Balb/c female mice.Blood sample was collected 14 days after the thirdly immunization to prepare mouse derived multi-anti-serum.The results showed that the serum neutralization titer of pET28?-S1 protein group was the highest,reaching 1:178,can better induce the host to produce neutralizing antibodies.Using purified pET28?-S1 protein to coat 96-well plate,an indirect ELISA antibody detection method(r SP-ELISA)for canine coronavirus was established.The optimal antigen coating concentration of this method is 2 ?g/m L.The best dilution of the serum was 1:200.The dilution of the enzyme-labeled secondary antibody is 1:10000,the incubation time is 60 min,the best blocking solution is 5% skim milk,and the best substrate color time is 10 min.The calculation confirms that when S/P?0.2395,it is judged as positive.The results of the repeatability test showed that the coefficient of variation of the intra-assay and inter-assay of r SP-ELISA method was less than 10%,indicating that the test method has good repeatability.The specificity results showed that the method does not have specific reaction with CDV,CPV and FCV positive serum.The sensitivity test results showed that the sensitivity of this method for detecting positive serum can reach 1:800.This method was used to detect 64 dog serums collected clinically and the results showed that the positive rate was 82.81%,and the coincidence rate with immunotomographic method was 98.44 %. |
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