Prokaryotic Expression Of Nucleocapsid Protein Gene Of Porcine Reproductive And Respiratory Syndrome Virus And Development Monoclonal Antibodies Against The Product

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.As the most abundant protein of Porcine Reproductive and Respiratory Syndrome virus(PRRSV),the nucleocapsid protein is highly antigenic.That makes it be a suitable candidate for diagnosis and detection.In this study, the nucleocapsid protein gene of North American PRRSV SY strain isolated from JiangSu was cloned, identificated and expressed in Escherichia Coli.On the base of it,the monoclonal antibodies against N protein was successfully prepared, which supplied good substantial elements for the establishment of detection methords and the research of the structure protein.1. Clone and prokaryotic expression of the PRRSV nucleocapsid protein geneAccording to the nucleotide sequence of ATCC VR-2332, a pair of primers was designed to amplify Ngene of SY strain PRRSV by reverse transcription-PCR.The fragment obtained was cloned into pGEM-T Easy Vector and sequenced.The result showed that the N gene of SY strain PRRSV was 372bp and encoded 123 amino acids. The fragment was subcloned into expression plasmid pGEX-6p-1,and transformed into competent E.coli BL21.The positive recombinant bacteria pGEX-6p-1-N/BL21 screened out by resistance screening and restraction endonuclease analysis was induced by IPTG and N protein fused to GST protein was successfully expressed.The analysis of SDS-PAGE and Western-blot indicated that the molecular weight of the recombinant protein was 36.5kDa and it had a good antigenic specificity.The optimized condition of induction is 0.8 mM IPTG and 4 hour.The protein expressed appeared as inclusion bodies. After abstersion, the recombinant protein was purified roughly.2. Development and identification of monoclonal antibodies against N Protein of PRRSVThe BALB/c mice were immunized subcutaneously with recombinant protein GST-N and PRRSV respectively. The later was multiplicated in Marc-145 and concentrated by polyethylene glycol. Spleen cells from immunized mice were fused with SP2/0-Ag-14 myeloma cells. Indirect enzyme linked immunosorbent assay (ELISA) was used to screen hybridoma cells, and limiting dilution methord was performed to subclone positive hybridoma cells. Ten positive clones were obtained, designated as P1A1, P1A2, P1C5, P2D3, P2G7, P5E5, P6G7, P7F2, P7E8 and N1H4. All the McAbs belonged to IgG1 subgroup except N1H4, which was subtyped to IgM. These McAbs were specific for PRRSV and didn't have the mutual reaction with other porcine viruses. The result of Western-blot furtherly approved that the McAbs were against the nucleocapsid protein of PRRSV and presented high specificity to it. Preliminarily determining by Plus-ELISA, P6G7 and N1H4 strains had different binding sites to antigen with other McAbs. With McAbs, Indirect-ELISA, Dot-ELISA and IFA were performed to detect PRRSV, which made a foundion for furtherly establishing a PRRS-testing methord, that was specific, sensitive and convenient.