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

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

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载体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-1a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。

According to the ORF5 gene sequence（GenBank accession No. EU200961） of porcine reproduct ive and respiratory syndrome virus（PRRSV） JX-3 strain, a pair of primers was designed and synthesized. The complete ORF5 gene of PRRSV NX/HY strain was amplified, cloned into pMD18-T vector, sequenced and compared with those of other PRRSV strains. Then, the ORF5 gene was subcloned into the expression vector pGEX-6P-1 to construct expression plasmid pGEX-6P-1-5. The pGEX-6P-1-5 was transformed 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.