猪繁殖与呼吸综合征病毒非结构蛋白Nsp9基因的克隆与原核表达

Cloning and prokaryotic expression of non-structural protein Nsp9 geneof Porcine Reproductive and Respiratory Syndrome Virus

通过PCR方法从重组质粒pSK-B2扩增得到非结构蛋白Nsp9的基因片段。将基因克隆至原核表达载体pET-28a(+),得到重组表达载体pET-28-Nsp9,转化Escherichia coliBL21(DE3)细胞。经IPTG诱导,Nsp9蛋白以包涵体形式表达,SDS-PAGE分析表明重组蛋白的分子量约为27.3 kD,表达量占菌体蛋白的43.2%。Western-Blotting结果表明重组蛋白可被PRRSV阳性血清所识别。表达的重组蛋白为进一步研究PRRSV的免疫特性和分子生物学功能奠定了基础。

The non-structural protein Nsp9 gene sequence was successfully amplified from recombinant plasmids pSK-B2 by PCR and cloned into the prokaryotic expression vector pET-28a （＋）. The obtained recombinant expression vector pET-28-Nsp9 was transformed into Escherichia coli BL21 （DE3） . Induced by IPTG, the recombinant protein was highly expressed as the form of inclusion body. Analyzed by SDS- PAGE, it showed that the molecular weight of recombinant protein was 27.3kD and could amount to 43. 2% of the total bacterial protein. Western-Blotting analysis indicated that the purified recombinant protein Nsp9 could be recognized by the positive serum from the pig infected with PRRSV. The result provided fundamental data and materials for the further investigation on immunogenicity and molecular biological function of Nsp9 of PRRSV.