Isolation Of Porcine Epidemic Diarrhea Virus (Pedv),and The Development Ofindirct Elisa With Recombinant Protein

Porcine Epidemic Diarrhea (PED) is a worldwide porcine disease. Since1978, PED was firstly reported in England and Belgium, later it was reported in different countries of Europe and Asia inducing an important economic loss. Currently, the incidence of PED is highly reported in co-infection with other porcine diseases (Porcine transmissible gastroenteritis, Porcine rotavirus, Porcine circovirus type2etc.) leading researchers to pay more attention on it. This study focused more on the PED virus isolation and identification, molecular epidemiological investigation and development of an indirect ELISA diagnostic method to furthermore characterize the Guangxi epidemic PED virus strain in the prospective of developing a diagnostic kit and suitable vaccine as basic settlement to prevent and control PED.In order to study the current PEDV prevalence of guangxi, Two hundred and forty diarrheic samples collected from piglets in11cities of Guangxi between Feb,2011and Jan,2013were firstly analyzed by RT-PCR for PEDV antigen detection. There were positive rate of41.7%(100/240). PEDV could be detected through the whole year; amongst them, high rate was recorded during the spring (49%) and the winter (46.2%). Results indicate that PED prevedent in most pigs populations of Guangxi. M gene and ORF3genes from positive samples were cloned、sequenced and analyzed by DNAStar, MEGA software. Reseults showed a significant divergence between the obtained M,ORF3gene sequences and the domestic or exotic homolog’s gene sequences. Phylogenetic tree analysis showed that the Guangxi isolate was extremely homologous with the Guangdong, Fujian, Jiangsu and Jiangxi isolates in year2010and closely related to the Thailand and Korea isolates in2007. In the contrary, the isolate was distantly related with2006China isolates, CV777strain, and attenuated strains isolated in Japan and Korea. Moreover, within the cloned sequences, one segment of M gene sequence and CV777attenuated strain are very close; there are consistant deletions of49nt in three segments of the ORF3sequence, they are similer to these strains of CV777and attenuated-DR13. These results prompt us to make further investigdte on PEDV and to undertake a valuable prevention and control measures. In this study, it is the first time to estabilish a profound molecular epidemiology for the current epidemic PEDV strain in Guangxi.To further understand the biological characteristics of pandemic strain of guangxi, One of40positive samples was successfully isolated in the VERO cells, we one stable, sub-cultured PEDV strain which induced vacuolization, lysis and cell fusion and so on. The isolate was named PEDV-LB and its S, ORF3, N and M genes were cloned and sequenced. Compared with exotic homolog ous genes sequences, we discovered that PEDV-LB, ORF3and M genes were close to the CV777attenuated strain; N gene was close to CH/GDS/09sequence and was not in the same sub-group with the CV777. S gene mutation was relatively significant and close to the Chinese2011epidemic strain, but distantly related with CV777. Refer to the CV777complete genome sequence we designed primers to amplify and clone PEDV-LB strain’s complete genome, The results obtained most past nucleotide sequences (a total of25094bp) of the whole PEDV genes, excepted5’-N,3’-C and and part of the Pol gene.To establish diagroustic ELISA method fordetecting PEDV, specific primers ware designed to amplify the N gene partial hydrophilic antigenic determinant region (position135-319amino-acid) NB. Recombinant prokaryotic plasmid (NB-PET32a-BL21) was constructed for expressing partial N protein fused with His-tag. Through IPTG induction we constructed a recombinant N protein. It was identified and analyzed by SDS-PAGE and Western-Blot, results show that the expressing was soluble. Besied, the recombinant protein could be recognized by field PEDV positive serum. Recombinant N protein was purified and coated on ELISA plate and indirect ELISA method for PEDV was established through optimizing reacted condition. In order todetect the reliahitity of this method,704clinical samples which from12pig farms of Guangxi were tested and the positive rate were31.4%～92.9%. This study has the advantages of simple operation, rapid, accurate and can be applied to the clinical monitoring of PEDV antibody levels, and can provide a theoretical guidance for further development of gene vaccine and ELISA diagnostic kit.