Molecular Epidemiology Investigation Of PRRSV In South China And Preparation Of Monoclonal Antibodies Against N Protein

Porcine reproductive and respiratory syndrome is a serious infectious diseases toswine industry, causing pregnancy sow abortion, stillbirth, weak young, mummy andpiglets respiratory disease. Porcine reproductive and respiratory syndrome has caused hugeeconomic losses to breeding industry in China.Porcine reproductive and respiratorysyndrome virus was mainly divided into two genotypes, the European, or type Ⅰ virus,alsoknown as Lelystad virus (LV), and the American, or type Ⅱ virus.At present, the American, or type2virus is popular in our country, but the isolated strainsin China is different from the American type (VR-2332). The rapid change of PRRSVgenome and immunosuppressive properties brought great difficulties for the prevention andcontrol of PRRSV. Therefore, the objectives of this study were to investigate the changes ofthe complete ORF5gene and part of NSP2gene current PRRSV isolates in southern Chinaby phylogenetic analysis and infection status. In order to a preliminary understanding ofPRRSV variant, the gene sequence of XF strains compared with both at home and abroad toalignment. At the same time, ORF7gene was expressed using prokaryotic expressionvector and purification of recombinant protein for preparing of monoclonal antibodies forthe purpose of the molecular epidemiology of the disease diagnosis and prevention toprovide the reference.1The genetic variation analysis of NSP2and ORF5gene of Porcine reproductive andrespiratory syndrome virus in south ChinaIn order to understand the porcine reproductive and respiratory syndrome virus(PRRSV) situation of genetic variation and infection, NSP2and GP5genes of fourteenstrains PRRSV isolated were amplified by RT-PCR, sequenced and analyzed byCLUSTALW DNAstar and MegAlign software.The results showed that the homology ofNSP2and ORF5genes among fourteen strains were83.3%-100%and95%-100% rspectively in nucleotide sequence and69.5%-99.3%,62.9%-99%nucleotide homologywith the domestic and foreign isolated. Genetic evolution analysis of the NSP2show thatthe14PRRSV isolated are of PRRSV American genotype. Among them14PRRSVisolates belong to the same branch with JXA1. It showed that mutant strains were stillwidely popular in south China.2.Porcine reproductive and respiratory syndrome virus XF strains of cloning andpurification and genome analysisIn order to further study the characteristics of recently popular PRRSV strains insouth China, this test use the plaque purification XF strain. We designed primers by primier5.0software for RT-PCR amplification product and joined together.Finally we get fullgenetic sequence of PRRSV XF strain.Then nucleotide sequence analysis of separatedstrains compared with at home and abroad. The results found that comparing with thedomestic and foreign and vaccine strains isolates,the nucleotides homology was between60.3%and99.2%. Sequence analysis showed that complete gene shared99.2%、60.3%、99.0%-99.1%、89.4%and94.9%nucleotide homologies compared with other highpathogenic PRRSV strains JXA1,EuroPRRSV,three guangdong strains,RespPRRS MLVand CH-1a,respectiveiy. It suggested that XF strains was still variants,sharing more closelyrelationship with JXA1.3The prokaryotic expression of the PRRSV N protein and monoclonal antibodypreparationOur laboratory cloned N protein gene form the strains using plaque purification, and itis successfully connected to the pET-32α(+).The recombinant expression plasmid was thenused to transform competent cells of Escherichia coli B121(DE3)and the expression wasinduced by IPTG．SDS—PAGE electrophoresis analysis showed was in the form of soluble,in the meantime,western-bloting testing proved the fusion protein have goodantigenicity.We purified fusion protein by Ni2+-NTA resin affinity chromatography andimmune Balb/c mice four times.After cells fusion and screening,three hybridoma cellswhich produced McAbs steadily were screene by ELISA, named B7D3.IFA assays showedthat the monoclonal antibody reacted with PRRSV N protein specialIy．The titer of serumwas1:8000by ELISA identification. The McAb belong to IgG2b.Finally, the pmcaryotic expression and purification of recombinant N protein and its highly specific monoclonalantibody were successfully achieved.This sudy laid the foundation for the analysis of Nprotein．...