猪生殖与呼吸综合征病毒宁夏分离株ORF5基因的克隆表达及其表达产物的活性分析

参考已发表的猪生殖与呼吸综合征病毒(PRRSV)ORF5基因序列,设计合成了1对覆盖完整ORF5基因区段的引物,克隆了PRRSV NX/HY株的ORF5基因(登录号为EU600287),并进行了序列比较.将该基因克隆到原核表达栽体pGEX-6P-1中,构建了融合表达质粒pGEX-6P-1-5并转化BL21,对表达蛋白进行Western-blot分析.结果表明,NX/HY分离株ORF5基因与PRRSV JX1、CH-la株的核苷酸序列同源性分别为99.2%和95.2%,推导的氨基酸序列的同源性分别为98.5%和92.5%;层析扫描分析显示,目的蛋白的含量约占菌体总蛋白的30%,且具有良好的反应原性.GP5蛋白的表达为建立相应的血清学诊断方法奠定了基础.

Cloning and expression of ORF5 gene of porcine reproductive and respiratory syndrome virus NX/HY strain and analysis of activity of the expressed product

According to the ORF5 gene sequence(GenBank accession No. EU200961) of porcine repro-ductive and respiratory syndrome virus(PRRSV) JX-3 strain,a pair of primers was designed and synthe-sized. The complete ORF5 gene of PRRSV NX/HY strain was amplified,cloned into pMD18-T vector,se-quenced and compared with those of other PRRSV strains. Then, the ORF5 gene was subcloned into the ex-pression vector pGEX-6P-1 to construct expression plasmid pGEX-6P-1-5. The pGEX-6P-1-5 was trans-formed into Escherichia coli BL21(DE3) and induced by IPTG. Sequencing analysis results showed that the ORF5 gene shared 99. 2% and 95. 2% identity with that of PRRSV JX1 and CH-1a strains respectively,and the deduced amino acid sequence from the ORF5 gene shared 98. 5% and 92. 5% similarity with that of PRRSV JX1 and CH-1a strains respectively. SDS-PAGE and Western-blot analyses showed that the GP5 protein was expressed about 30% in total proteins and had good reactionogenicity. The results provided a basis for establishment of serological method for diagnosis of PRRS.