日本脑炎病毒WHe株NS1基因的克隆、测序及表达

以日本脑炎病毒(Japanese encephalitis virus,JEV)WHe株的基因组RNA为模板,采用RT-PCR技术,克隆了JEV WHe株的NS1基因,并对其进行了测序和序列分析。构建了pET28b-NS1表达载体,转化表达宿主大肠杆菌BL21(DE3),并对其进行了诱导表达,对表达产物进行检测。结果表明,NS1基因全长1 145 bp,其核酸序列与JEV P3株同源性为99．4％,与SA14和SA14-14-2等27个JEV毒株的核苷酸序列同源性为98％,表明NS1基因的保守性很高;pET28b-NS1表达产物的相对分子质量约为43 ku,大小与预期结果相符。NS1基因克隆表达成功。

Cloning,sequencing and expression of nonstructural protein NS1 of Japanese encephalitis virus strain WHe

Genomic RNA was separated from JEV WHe strain, and used as template for cDNA synthesis of NS1 gene. Then NS1 gene (1 145 bp) was amplified by RT-PCR. The analysis of sequence showed the nucleotide sequence of NS1 had the 99. 4% identities with that of JEV P3 strain, and 98% identities with the NS1 reported in other 27 strains. Then the NS1 was cloned into the expression vector pET 28b ( ) to construct a recombinant prokaryotic expression plasmid, pET 28b( ) NS1,and the recombinant plasmid was then transfected into E. coliBL21(DE3) . SDS PAGE electrophoresis showed the relative molecular weight of the expressed protein was about 43 ku in accordance with the presupposition.