BVDV非结构蛋白NS4B的结构分析及其蛋白表达与鉴定

Structural Analysis of BVDV Non-structural Protein NS4B and Protein Expression and Identification

NS4B是牛病毒性腹泻病毒(Bovine viral diarrhea virus,BVDV)的非结构蛋白,为研究BVDV在病毒侵染宿主细胞过程中的作用,对ns4b进行了结构分析、表达载体构建及蛋白表达。采用Prot-Param、TExpasy和SignalP-4.1等软件分析NS4B蛋白的氨基酸组成、疏水性和空间结构。根据Genebank上公布的BVDVns4b基因序列设计引物,利用PCR方法获得ns4b基因片段,连接到p3×Flag-CMV10表达载体上,构建重组质粒p3×Flag-CMV10-ns4b,经双酶切与测序鉴定正确后,将p3×FlagCMV10-ns4b转染到HEK-293T细胞中,利用Western blot检测NS4B蛋白的表达。结果表明BVDV NS4B蛋白分子量为38.4 kDa,不存在信号肽,有49个磷酸化位点和6个糖基化位点,是一种主要由α-螺旋和无规则卷曲结构组成的疏水性稳定蛋白。成功构建了重组质粒p3×Flag-CMV10-ns4b,并且p3×Flag-CMV10-ns4b在HEK-293T细胞中成功表达,为今后研究NS4B蛋白在BVDV感染宿主细胞及其免疫逃逸机制奠定了基础。

NS4B is a nonstructural protein of Bovine viral diarrhea virus(BVDV).In order to study its function in the process of BVDV infecting host cells,we analyzed the structure of ns4b,constructed the expression vector and expressed NS4B protein.The amino acid composition,hydrophobicity and spatial structure of BVDV ns4b were analyzed by Prot-Param,TExpasy and SignalP-4.1.The ns4b gene was obtained by PCR using primers designed according to the sequence of BVDV ns4b gene published in Genebank,the recombinant plasmids p3×Flag-CMV10-ns4b were constructed and identified by restriction enzyme digestion and sequencing.The recombinant plasmids p3×Flag-CMV10-ns4b were transfected into HEK-293T cells,and the expression of NS4B protein was detected by Western blot.The results showed that the molecular weight of BVDV NS4B protein was 38.4 kDa,there was no signal peptide,but 49 phosphorylation sites,6 glycosylation sites,α-helix and random coil structure about NS4B as a hydrophobic protein.In addition,the recombinant plasmids p3×Flag-CMV10-ns4b were successfully constructed and expressed in HEK-293T cells.These results provided an important foundation for the further research of BVDV infection and immune escape mechanism mediated by NS4B protein.