| Porcine epidemic diarrhea(PED), caused by porcine epidemic diarrhea virus (PEDV), is an acute and highly contagious disease in swine. Infected pigs present with clinical symptoms of watery diarrhea,vomiting,dehydration, and recovered pigs develop lifelong latent infection. In China, PED was first reported in1976. Since the end of2010, the intensity and epidemic area of PED have continuously enlarged, causing a significantly high mortality of suckling piglets and serious economic losses to the world’s pig industry. At present, epidemiological and clinical symptoms of this disease is very similar to the porcine transmissible gastroenteritis, porcine rotavirus and pocine colibacillosis. There are difficulties in differential diagnosis. In this study, by using purified PEDV or recombinant N protein, two indirect ELISA were developed for detecting antibody to PEDV and used for epidemiological survey of PEDV. Meanwhile, the Nsp2of PEDV was expressed prokaryotic expression system and monoclonal antibodies were prepared which laid a foundation for the further study of diagnosis and pathogenic mechanism of PEDV.The main contents were as following:1. Evelopment and application of an indirect ELISA for detecting antibodies against PEDVAn indirect ELISA method was established with the purified PEDV as the coated antigen after optimization of reaction conditions.The optimal antigen concentration and serum samples dilution were set at3μg/mL and1:100. The coated time was2h at37℃. The sealing buffer was1.5%BSA-PBST, and the sealing time was3h at37℃. The serum samples were incubated for1h at37℃. The dilution of the conjugate was defined as1:10,000and the reaction time was30min at37℃. It was judged as positive when the cutoff value OD450nm≥0.306, as negative when OD450nm≤0.268, and as suspicious between0.268and0.306. It could not react with the positive sear of other five diseases (PRRS、PCV、CSFV、PR、FMDV). And it had good inter-and intra-batch reproducibility. Total587clinical serum samples obtained from pig farms in Jiangsu Shanghai Zhejiang Anhui province were detected. The positive rate was56.76%. The diagnostic accuracy of the ELISA were91.3%, compared with TSZ ELISA kit. It indicated that this method could be used for PEDV epidemiological surveys and diagnosis in the future.2. Evelopment and application of an indirect ELISA for detecting antibodies against N protein of PEDVProkaryotic expression pET-28a-N. An indirect ELISA method was developed using purified recombinant N protein as coating antigen after optimization of reaction conditions. The optimal antigen concentration and serum samples dilution were set at0.8μg/mL and1:100. The coated time was2h at37℃then overnight at4℃. The sealing buffer was1%BSA-PBST, and the sealing time was3h at37℃. The serum samples were incubated for1h at37℃The dilution of the conjugate was defined as1:15,000and the reaction time was45min at37℃. It was judged as positive when the cutoff value OD450nm≥0.312, as negative when OD450nm≤0.265, and as suspicious between0.265and0.312. It could not react with the positive sear of other five diseases (PRRS、PCV、CSFV、PRV、FMDV). And it had good inter-and intra-batch reproducibility;Total458clinical serum samples obtained from pig farms in Jiangsu Shanghai Zhejiang Anhui province were detected. The positive rate was69.21%. The diagnostic accuracy of the ELISA were89.13%and90%, compared with TSZ ELISA kit and an indirect ELISA method for detection of antibody to porcine epidemic diarrhea virus. It indicated that this method could provided an available tool for antibody detection and serological survey of PEDV.3. Expression and purification of Nsp2of porcine epidemic diarrhea in prokaryotic expression and Preparation of monoclonal antibodies against Nsp2proteinIn order to express the nonstructural protein Nsp2of porcine epidemic diarrhea virus, Nsp2gene was amplified by PCR and cloned into a prokaryotic expression vector pET-32a. A recombinant plasmid named pET-32a-Nsp2was constructed and identified with restriction enzyme digestion and sequencing. Protein was then expressed in E.coli BL21(DE3) cells by induced with IPTG.SDS-PAGE analysis reviewed expressed protein was about50KDa. Correctness and antigenicity of expressed target protein were identified by Western-blotting analysis,and recombinant protein Nsp2was purified by molecular sieve chromatography;The monoclonal antibodies (MAbs) were prepared by fusing mouse myloma cells (SP2/0) with spleen cells from BALB/c mice immunized with purified recombinant Nsp2protein of Porcine epidemic diarrhea. Three hybridoma cell lines secreting MAbs against Nsp2protein were screened by indirect enzyme-linked immunosorbent assay (ELISA)and named as6B6. The titers in supernatant and asicite were1:3200and1:2.408×105respectively.Western-blot results showed that the MAbs (6B6) reacted specifically with recombinant Nsp2protein expressed in Prokaryotic cell.In summary, this study has developed a simpler, time-saving, sensitive and specific indirect ELISA method for the detection of PEDV antibodies, at the same time,monoclonal antibodies were prepared of Nsp2protein,which being suitable for large scale monitor of PEDV infection at low cost and helpful for epidemiological survey of PEDV and evaluation of vaccine immune effect. This study have laid a foundation of study on structure and function of Nsp2protein. |
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