| Japanese Encephalitis virus (JEV) is a single-strand RNA virus that can cause orchitis boar and sow abortion, is a zoonotic disease virus infected pigs after bited by mosquito,then transmited the virus to humans. JEV major proteins include core protein C, membrane protein PrM/M and envelope protein E, and seven non-structure proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5). In the translation process, NS2A genes produce derivatives fused with NS1 protein C-terminal cause of -1 ribosomal frameshift. With G changed to A at 66 nucleotide in NS2A gene, JEV attenuated strain JEV14-14-2 couldn’t produce NS1’ protein without the frameshift structure.In theory, antibody against NS1’ indicate the JEV infection of field virus. Indirect ELISA method utilizes synthetic NS1’44aa as detection antigen was established to detect Japanese encephalitis virus antibody of swine.With the method, we investigated the herd epidemiological in East China in 2013. This study prepared the recombinant EDⅢ-Cap proteins by genetic recombination technology and identified the recominant protein by anti EDⅢ polyclonal antibody and anti Cap polyclonal antibody. Meanwhile, based on clinical and laboratory animal model test data for analysis of NS1’ peptides detected objects discovered the existence of cross-epitope Cap protein. Details are as follows:1.Development and application of indirect ELISA for detecting frameshift protein antibody of JEV in swine serumAn indirect ELISA method was developed using synthetic NS1’44aa peptides as coating antigen after optimization of reaction conditions. The optimal antigen concentration and serum samples dilution were set at 0.5μg/mL and 1:100. The coated time was 2h at 37℃ then overnight at 4℃.The plates were blocked by 1% BSA incubated at 37℃ for 2h. The conjugate was diluted at 1:5000,incubated at 37℃ for lh.It was judged as positive when the cutoff value OD450nm≥0.286,as negative when OD450nm≤0.238, and as suspicious between 0.286 and 0.238.1t did not react with the swine serum antibodies to pseudorabies, porcine reproductive and respiratory syndrome, porcine circovirus type 2, swine fever and foot-mouth disease virus.And it had good inter-and intra-batch reproducibility.According to the guideline of veterinary biological products technical, the method was used to detect the antibody of mice immunized with JEV NJ08 strain. It was compared with the JEV EDIII indirect ELISA method to verify the feasibility of detecting clinical animal serum. Total 2292 clinical serum samples obtained from eastern China were detected,and the positive rate was 73.1%. For sows,132 copies were positive out of 154 copies,positive rate is 86.1%.For commercial pigs,432 copies were positive out of 638 copies, positive rate is 67.8%.The method lay a good foundation for JEV diagnostic and epidemiological investigation. Therefore, the indirect ELISA to detect frameshift exists nonspecific reaction, indicating a cross epitope between poplprotein of JEV and NS1’ protein.2.Series Expression and identification of EDIII gene and Capsid gene of JEVBased on software analysis, the core protein of JEV may have a cross epitope with NS1’protein.With different plasmid vector and host bacteria, we didn’t get the whole core protein. The 1-81bp and 241-347bp of JEV Capsid gene were amplified by PCR and cloned into a prokaryotic expression vector pET-32a. PCR and restriction endonuclease analysis and sequencing were directed first to identified recombinant plasmid pET32a-Cap-a and pET32a-Cap-b. Protein was then expressed in E.coli Rosseta cells by induced with IPTG. The pET32a-Cap-b protein exists in the supenrnatan after ultrasounding. After SDS-PAGE electrophoresis,cut the tape containing the pET32a-Cap-b protein.After triturated with PBS, mixed with synthetic Capl-27aa peptides, JEV Cap polyclonal antibody was prepared by immunizing New Zealand white rabbit with the mixture, the polyclonal antibody was identified by IFA. After IPTG induction, the inclusion bodies were purified and triturated with PBS, mixed with Freund’s complete adjuvant and Freund’s incomplete adjuvant, JEV EDIII polyclonal was prepared by immunizing New Zealand white rabbit with the mixture, the polyclonal antibody was identified by IFA.JEV EDⅢ gene and Capsid gene were amplified by SOE-PCR and cloned into a prokaryotic expression vector pET-28a. PCR and restriction endonuclease analysis and sequencing were directed first to identified recombinant plasmid pET28a-EDⅢ-Cap. Protein was then expressed in E.coli Rosseta cells by induced with IPTG. SDS-PAGE analysis reviewed expressed protein was about 29KDa.EDⅢ-cap protein was washed by the gradient of urea for one-step purification and refolding, the refolded protein EDⅢ-cap can react with His-Tag monoclonal antibody, JEV EDIII polyclonal antibody and JEV Cap polyclonal antibody by Western-blotting analysis.In summary, the study prepared JEV ED III protein polyclonal antibodies using recombinant EDⅢ,prepared JEV Cap polyclonal antibody using recombinant Cap protein C-terminal and synthetic N-terminal. The refolded EDIII-cap can react with JEV EDIII polyclonal antibody and JEV Cap polyclonal antibody, with good reactogenicity.3.Analysis of cross epitope beween JEV NS1’and capsid proteinWith G-66-A in NS2A gene, JEV SA14-14-2 can not form the essential structure of ribosomal frameshift, therefore lost the ability to create NS1’. Theologically, the NS1’ indirect ELISA is supposed to be differentiate the vaccinated antibody from field virus. According to the data of chapter one, there is no difference between the NS1’indirect ELISA and commercial kit. And the high positive rate of 73.1% also make us question the chances of pig natural infected with JEV. To detect the serum antibody of mice immunized with JEV SA14-14-2 by time points with the NS1’ indirect ELISA and EDIII indirect ELISA, we got the consistent result. Based on the above data,we believe that the NS1’ indirect ELISA can not detect the NS1’ antibody resulted by wild virus infection. With the bioinformatics comparison,we found that the JEV NS5 region exsit amino acids homology to NS1’44aa peptide. With different mutations in the NS1’ 44aa peptide,we got three new synthesized peptide, named NS1’-1,NS1’-2 and NS1’-3. However, the test results are consistent with NS1’.With the EDIII-Cap protein as detection antigen, we found one NS1’ monoclonal antibody reacts with specific band in western blotting analysis, meanwhile, no reaction with EDIII protein. It suggests that there exists one cross epitope between NS1’ and capsid protein, also an explanation to the high positive rate of NS1’ antibody in chapter one. |
