| Porcine epidemic diarrhea(PED),caused by porcine epidemic diarrhea virus( PEDV), is an acute and highly contagious disease in swine, which has produced a great loss to the swine industry.Epidemiological and clinical symptoms of this disease are very similar to the porcine transmissible gastroenteritis(TGE), porcine rotavirus(Po RV). To investigate the molecular epidemiological features and dynamic trends of PEDV and commit to develop a better diagnostic method we conducted this study mainly including three parts as described below:1.Genetic variation analysis of the main structural protein of PEDVDuring 2013 and 2014,we selected five representative samples from different farms in five provinces in the occurrence of PEDV and then M,N,S gene were amplified, cloned and sequenced. Nucleotide sequence alignment and genetic variation are based on the three genes. The results indicated that compared with domestic vaccine strain(CV777), four strains’ amino acid sequence showed a significant diversity in S protein, and the most mutations were in S1 domain. There are a discontinuous 5-amino-acid insertions(59QGVN62 and 140N) and two continuous amino acid deletions(163NI164) existed in the S1 domain of epidemic strains. Besides, there were seven mutations in two neutralizing epitopes(499-638 aa and 764-771 aa). Amino acid sequences of M and N protein are highly conserved. Genetic evolution analysis results showed that 4 epidemic strains had low sequence homology(93.8%~94.7%)with Asia’s main vaccine strains(Chinese vaccine strain CV777, South Korean attenuated vaccine strain DR13 and Japanese attenuated vaccine strain 83P-5), while they shared high sequence homology(96.0%~99.6%) with South Korean strain in the year of 2007~2009, Japanese strain of the year 2011 and Chinese strains of recent years.2.The expression of M,N and S1 fusion proteinM, N and S1 gene were cloned to prokaryotic expression vector p GEX-6p-1, and then were transformed into Ecoli.BL21. SDS-PAGE showed that the three kinds of recombinant Ecoli.BL21 could express M, N and S1 recombinant protein. The molecular weights of three recombinant proteins—M, N and S1 were 50k Da, 80 k Da and 110 k D respectively. The main expression form was inclusion body. The detection result of Western blot showed that all the three recombinant proteins can react with PEDV positive serum of swine. The result suggested that the expression of three kinds of recombinant protein had good antigenicity and could be used as diagnostic antigen to detect PEDV infection specifically.3.Development and application of an indirect ELISA for detecting antibodies against PEDVAn indirect ELISA method was established with the purified PEDV N protein as the coated antigen after optimization of reaction conditions. The optimal antigen concentration and serum samples dilution were set at0.5mg/m L and 1:400. The coated time was 2h at 37°C followed by 4°C over night. The sealing buffer was5% skim milk and the sealing time was 3h at 37°C. The serum samples were incubated for lh at 37°C. The dilution of the conjugate was defined as 1:40,000 and the reaction time was 1h at 37°C. It was judged as positive when the cutoff value OD450nm30.40,as negative when OD450nm <0.4. It had good inter-and intra-batch reproducibility.The results in this study indicated that this method could be used for PEDV epidemiological surveys and diagnosis in the future.Changes in gene sequence and genetic evolution of PEDV’s structural genes--M, N, S were analysed in this study. It provided a theoretical reference for variation research of PEDV strains; recombinant protein M, N and S1 have been expressed successfully, which had good reactogenicity and lay a good foundation to establish the indirect ELISA method for PEDV; development of a sensitive and specific indirect ELISA method for detections of PEDV antibodies, which provides an easy means of serological diagnosis for PEDV. |
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