Expression And Humoral Immunogenity Of A And B Epitope Of GP5 Protein Of Porcine Reproductive And Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome(PRRS) is a highly contact and infectious disease with worldwide distribution, and has caused severe economic losses to the swine industry, which is characteristed both reproductive failure in sows and a respiratory illness tract in nursery-age pigs. Porcine reproductive and respiratory syndrome virus(PRRSV) is a member of the Arteriviridae in Nidovirales, whose genome is a non-segmented single-strand positive-sense RNA.The GP5 glycoprotein is one of the major structure protein of PRRSV. It can induce neutralization antibody to PRRSV and provide protective efficacy in pigs. An highly conserved neutralizing epitope B (between amino acid 37-45)(HLQLIYNL)and an highly variably immunodominant nonneutralizing epitope A were identified in the ectodomain of GP5 protein.The core of epitope A (between amino acid 27 and 31) (A/VLAN) is located seven amino acid residues ahead of the neutralizing epitope B.In order to study on the immunity of the neutralizing epitope and nonneutralizing epitope of GP5, one pair of specific primers which have the restriction enzyme site BamH I, EcoR I were designed, according to the GP5 gene sequence of S1 strain of porcine reproductive and respiratory syndrome(PRRSV). The 141 bp double strands DNA of the A, B epitope of the GP5 coding gene was amplified by PCR and was inserted into prokaryotic expression plasmid pGEX-6p-1 and eukaryotic expression plasmid pcDNA3, respectively. The two recombinant plasmids were identified by restriction enzymes and sequencing. The result showed that the GP5AB gene was cloned into pGEX-6p-1 and pcDNA3 in the correct open reading frame. After transformating the prokaryotic recombinant plasmid into E.coli competent cells BL21 (DE3) , the fusion protein was expressd by isopropyl-β-D-thiogalactoside(IPTG) 18℃, and purified through GSTrap FF column. It...