Epidemiological Investigation Of Porcine Reproductive And Respiratory Syndrome Virus In Jiangsu And Prokaryotic Expression Of Nucleocapsid Protein Gene Of PRRSV

Porcine Reproductive and Respiratory Syndrome(PRRS),characterized by severe reproductive disorders in sows and respiratory diseases in young pigs,was first recognized in the US in 1987.Since its first appearance, PRRS has been causing immense economic loss in swine industry all over the world.The"high fever"syndrome, characterized by high fever, and high-mortality, occurred in the pigs in main pig-producing provinces in China in 2006. It was demonstrated that the highly pathogenic PRRSVmutants were the major pathogens of the"high fever"syndrome by lots of research.1. Establishment of RT-PCR detection method and the sequence analysis of Nsp2 in Jiangsu ProvinceA pair of primers was designed based on the Nsp2 gene sequences of classical PRRSV and the highly pathogenic PRRSV isolates. The DNA fragments of sizes of 734bp and 644bp for classical PRRSV and high pathogenic PRRSV respectively were amplified by reverse transcription-polymerase chain reaction (RT-PCR).The results of RT-PCR for clinical samples detection showed that the positive rate of PRRSV was up to 65% in Jiangsu province, and the pathogen was the high pathogenic PRRSV. No European-like PRRSV was detected in the clinical samples with the primers designed according to PRRSV LV strain.Nsp2 genesof eighteen PRRSV from Jiangsu pig farm were sequenced and analyzed. The results showed that the fragment sizes of all Nsp2 genes were 644bp. The amino acids sequences showed 57.5%-68.7% identity with American reference strain VR-2332. And the amino acids sequences showed 82.2%-97.6%,80.4%-95.3% identity with JXA1 and WUH1. A discontinuous deletion of 30 amino acids in Nsp2 was found. It shared the same location of deletion with the JXA1.2. Isolation and identification of the high pathogenic PRRSVThe positive samples in RT-PCR was selected and homogenated. After centifuge the supernatant was inoculated in Marc-145 cells, Cell pathogenic effect (CPE) after two passages was clearly observed.The cells infected with PRRSV showed aggregation, clustering, and finally lysis.The virus in the cells was conformed by RT-PCR, with a 644bp band. The cells infected with the virus could be recognized by monoclonal antibody against PRRSV N protein in IFA analysis. The green fluorescence in infected cells wsa found. It indicated that the PRRSV wsa isolated in this study and named as PRRSV RD2007.3. Complete genome analysis of the high pathogenic PRRSV RD2007The complete genome of RD2007 was amplified by RT-PCR with 13 pairs of truncated overlap primers and sequenced. The most important character is a discontinuous deletion of 30 amino acids in Nsp2, which shared the same location of deletion with the JXA1, high pathogenic PRRSV. The genome sequences showed 99.1% identity with PRRSV JXA1. A full-length genome phyogenetic tree revealed that RD2007was belonged to North American genotype and displayed a close relationship and the same origin with other high pathogenic PRRSV in China.4. Clone and prokaryotic expression of the PRRSV nucleocapsid protein geneBaseing to the nucleotide sequence of ATCC VR-2332, a pair of primers was designed to amplify Ngene of RD2007 strain PRRSV by RT-PCR.The fragment obtained was cloned into pGEM-T Easy Vector and sequenced and then subcloned into expression plasmid pET32a to produce plasmid pET32a-N. After that the plasid pET32a-N was transformed into E.coli BL21.The positive recombinant bacteria pET32a-N was induced with 0.8 mMol/L IPTG at 28℃for 5 hour. SDS-PAGE and Western-blot analysis results indicated that the nucleocapid protein of PRRSV was expressed in E.coli. The molecular weight of the recombinant protein was 35kDa, a soluble protein.