家蚕二分浓核病毒NS1蛋白的表达及定位

Expression and subcellular localization of Bombyx mori bidensovirus nonstructural protein NS1

[目的]在Bm N细胞中表达家蚕二分浓核病毒(Bombyx mori bidensovirus,Bm BDV)非结构蛋白NS1,并分析其亚细胞定位。[方法]在病毒非结构蛋白NS1基因5'端加上kozak序列、3'端融合Flag标签序列;将重组序列克隆至昆虫细胞表达载体pIBV5/His上,转染BmN细胞,通过Western blot和免疫荧光检测NS1蛋白的表达和亚细胞定位。[结果]PCR和酶切鉴定显示重组表达载体构建正确;Western blot检测到一条大小约37 kDa的特异条带,免疫荧光分析显示表达的蛋白主要定位于细胞核中。[结论]构建的真核表达质粒能在BmN细胞中稳定表达NS1蛋白,该蛋白主要定位在细胞核中。

[Objective]To express Bombyx mori bidensovirus（Bm BDV） NS1 in BmN cells and characterize the subcelluar localization of NS1.[Methods]The kozak sequence and the Flag tag were fused with NS1 gene at the 5＇ end and at the 3＇ end,respectively.The resulting sequence was cloned into the expression vector pIB-V5/His to construct the expression vector of NS1.The recombinant vector was transfected into Bm N cells and the expression of NS1 was detected by Western blot and immunofluorescence.[Results]Analysis of enzyme digestion and PCR indicated that the recombinant expression vector of NS1 was generated correctly.A specific protein about 37 kDa was detected by Western blot.Immunofluorescence showed that NS1 was predominantly located in the nucleus of Bm N cell.[Conclusion]NS1 was successfully expressed in Bm N cells using a recombinant vector and the NS1 was mainly located in nucleus of Bm N cell.