乙型脑炎病毒SA14-14-2株NS1基因片段的克隆、测序与表达

Cloning,Sequencing And Expression of NS1 Gene of Japanese Encephalitis Virus(JEV)Strain SA14-14-2

以流行性乙型脑炎病毒减弱毒株SA14 14 2的基因组RNA为模板 ,采用RT PCR技术扩增其 NS1基因的cDNA ,将其克隆到 pMD18 T载体中 ,得到克隆质粒pMD18 T NS1。pMD18 T NS1经 EcoRI和SalI酶切后 ,回收NS 1片段克隆到原核表达载体 pET 2 8a(+) EcoRI/SalI位点 ,构建了重组原核表达质粒 pET 2 8a(+) NS1。转化大肠杆菌BL2 1(DE3) ,IPTG诱导获得高效表达 ,产物经SDS PAGE电泳结果显示 ,表达产物分子量约为 45kD。

Genomic RNA was separated from JEV attenuated strain SA14 14 2,and was used as template for cDNA synthesis of NS1 gene.The cDNA of NS1 gene was amplified by RT PCR and cloned into the pMD18 T.The cloned fragment was digested with EcoRⅠ and SalⅠ,then cloned into the expression vector pET 28a(+).A recombinant prokaryotic expression plasmid,pET 28a(+) NS1,was constructed.The recombinant plasmid was then transfected into E.coli BL21(DE3).SDS PAGE and Western blotting were performed to detect the NS1 gene product.NS1 was highly expressed with molecular weight about 45kD.The protein was specific to antisera against JEV by Western blotting.