Prokaryotic Expression Of ORF7 Gene Of PRRSV Isolates From Shandong And Detection Of PRRSV By RT-PCR

Porcine reproductive and respiratory syndrome(PRRS) is a new infectious disease,which is caused by porcine reproductive and respiratory syndrome virus(PRRSV).Some epitopes of nucleocapsid protein are identical to the two virus types,whereas some are different and only conservative to the European type or the American type.Type-specific epitopes of the two type virus were expressed in the two trials as follows,aiming to provide the test antigen for the differentiation of the two type in shandong.(Ⅰ) A local strain of PRRSV was isolated from lungs and lymph nodes of piglets suffered hematosepsis in Shandong swinefarms.The isolate was high virulent and the affected piglets display serious respiratory disorder.The isolate could be propogated in Marc-145 cell and cause the cell crumble ,wrinkle and breaking apart,but not do so in other cell lines.According to the complete sequence of PRRSV VR2332 from the GeneBank,a gene encoding a polypeptide composed of the N protein epitopes was constructed artificially.This gene was sub-cloned into pMD18-T plasmid. A recombinant plasmid pMD18-T-N was constructed and transformed into the DH5α..The amplified fragments were cloned and sequenced.The length of N gene fragments is 376 base pairs.Comparison of the homology among VR-2332 and Lelystad virus by DNAStar software,the results showed that the homology of nucleotide sequences of nucleotide sequences of VR-2332 and Lelystad virus were 99.9%,58%,revealed that Shandong strains perhaps have a close relation to VR-2332.(Ⅱ)According to the complete sequence of PRRSV VR-2332,a pair of primers that have restrict enzyme sites EcoR I and BamH I were designed to amply the American type-specific epitopes of N protein.A 366bp gene fragment was amplified from the virus RNA by RT-PCR and inserted into the pGEX-6P-1 plasmid.A recombinant plasmid pGEX-6P-1-N was constructed and transformed into the BL21(DE3).SDS-PAGE showed that a fusion...