Cloning And Prokaryotic Expression Of The ORF7 Gene Of Henan PRRSV Then Immunogenicity Analysis Of N Protein

Porcine Reproductive and Respiratory Syndrome (PRRS) which is caused by Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) deeply damages global swine industry. PRRSV includs two genostypes:European type and American type. The genetic variation between the two genotypes is high, especially between the American type. So, it is difficult to develop a kind of effective vaccine based on a single stain. N protein, encoded by ORF7, is nucleocapsid protein. There are 123 and 128 amino acids respectively in American type and European type PRRSV N proteins. The content of N protein is the highest and the immunogenicity is the strongest in all of the virus protein, it can induce specific antibodies but not neutralization the earlist. So, N protein has important meaning in the early diagnosis and pathogenic or immune mechanism study.In order to diagnose and detect PRRSV, we used RT-PCR and gene sequencing technology. Two sets of primers (S1R1 and S2R2) were designed according to the sequence of the nucleocapsid protein (N) gene of reference strain:LV and IAF-exp91. The primers S1R1 were referred to as the specific primers, since they could amplify only sequences from American type PRRSV and not from European type PRRSV; On the other hand, the primer pair S2R2 was common to both strains of PRRSV. We amplified two Henan isolated strain by primers(S1R1 and S2R2), Results showed that: two sets of primers both cloned corresponding base sequences, and the length of ORF7 of the two strain were both 372bp and the sequences was same two each other, encoding 123 amino acids. We analysised ORF7 sequences of Henan PRRSV and 24 strain of PRRSV selected from Genbank by DNAStar. Results showed that:the homology of ORF7 sequence between HN07-1,HN07-2 PRRSV and European and America type PRRSV were 65.1%—66.9% and 92.2%—99.7%.Used primers(P1P3 and P2P3) designed according to the sequence of the Nsp2 gene we successfully cloned 439bp and 217bp base sequences. All the Results demonstrated that two isolates we used were American type and high pathogenic strain of PRRSV. In order to supply material foundation to N protein research we firstly cloned ORF7 sequences of two Henan isolates and log them into Genebank. This research has successfully constructed expression plasmid pGEX-6P-1-N of Henan PRRSV, exprssed and purified soluble N protein successfully and analysised the immunogcnicity of Nptotein being expressed.