单纯疱疹病毒2型衣壳支架蛋白ICP35原核表达载体的构建及其表达特性分析

Construction of prokaryotic expression vector of herpes simplex virus type 2 capsid scaffolding protein ICP35 and analysis of its expression characteristics

本研究旨在构建单纯疱疹病毒2型(herpes simplex virus type 2,HSV-2)衣壳支架蛋白ICP35的原核表达载体,并分析其在HSV-2增殖中的表达特性。以HSV-2基因组DNA为模板,采用聚合酶链反应(polymerase chain reaction,PCR)扩增HSV-2 ICP35的编码基因UL26.5,并克隆至原核表达载体pET-32a(+),转化大肠埃希菌BL21(DE3)进行诱导表达,将纯化的重组ICP35免疫家兔后获得抗体,采用免疫荧光检测ICP35在HSV-2增殖中的表达特性。结果显示,HSV-2 UL26.5基因的PCR产物大小约为1065 bp,原核表达重组蛋白的相对分子质量约为6.3×10^(4),免疫家兔的血清抗体可特异性识别HSV-2 ICP35。免疫荧光检测结果显示,HSV-2 ICP35可在感染后8 h出现于细胞核周围,12 h表达量进一步增加并向细胞核内聚集,16 h后在细胞核、细胞质内均可检测到聚集成斑点状的衣壳支架蛋白。结果表明,HSV-2 ICP35原核表达载体的成功构建及对其在HSV-2增殖中表达特性的分析,将为研究UL26.5基因的功能、蛋白相互作用、病毒与宿主的相互作用及筛选药物靶标等奠定基础。

The aim of this study is to construct the prokaryotic expression vector of capsid scaffolding protein ICP35 of herpes simplex virus type 2(HSV-2)and to analyze its expression characteristics during HSV-2 proliferation.The UL26.5 gene of HSV-2 encoding capsid scaffolding protein ICP35 was amplified by polymerase chain reaction(PCR)and cloned into the prokaryotic expression vector pET-32a(+).Then the recombinant plasmid was transformed into Escherichia coli(E.coli)BL21(DE3)to express capsid scaffolding protein ICP35 which was induced by isopropylβ-D-thiogalactoside(IPTG).The purified ICP35 recombinant protein was used as antigen to immunize rabbits for the preparation of polyclonal antibody specific for ICP35 protein,and the expression characteristics of capsid scaffolding protein ICP35 during HSV-2 proliferation was analyzed by immunofluorescence detection.The results of agarose gel electrophoresis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)analysis respectively showed that the PCR target band appeared around 1065 bp,the relative molecular weight of recombinant protein target band was about 6.3×10^(4),which was in accordance with the expectation.In addition,the serum antibody of immunized rabbit was able to specifically recognize HSV-2 capsid scaffold protein ICP35.Immunofluorescence assay results showed that the HSV-2 capsid scaffold protein ICP35 appeared around the nucleus as early as 8 h post infection.At 12 h post infection,the expression level of ICP35 increased further and ICP35 began to migrate into the nucleus.The fluorescence of ICP35 gradually converged into spots and could be detected in both cytoplasm and nucleus at 16 h post infection.The successful construction of prokaryotic expression vector of HSV-2 capsid scaffold protein ICP35 and analysis of its expression characteristics during HSV-2 infection will be helpful to study the function of UL26.5 gene,the interaction of proteins,the interaction of virus-host and the screening for antiviral drug targets.