VZV糖蛋白E基因胞外域的原核表达及其兔抗血清的制备  

Prokaryotic expression of varicella zoster virus glycoprotein E extracellular domain and preparation of antiserum

作　　者：伊兴旭[1] 陈敬贤[2] 甘霖[2] 王明丽[2] 

机构地区：[1]安徽医科大学附属巢湖医院检验科,合肥238000 [2]安徽医科大学微生物学教研室,合肥230032

出　　处：《安徽医科大学学报》2016年第2期151-155,共5页Acta Universitatis Medicinalis Anhui

基　　金：国家自然科学基金(编号:30872253);安徽省科技厅科技攻关项目(编号:08010302179);校企合作实践教育基地(编号:2012sjjd014)

摘　　要：目的采用原核表达系统表达水痘-带状疱疹病毒(VZV)糖蛋白E(g E)基因的胞外域,将纯化后的目的蛋白免疫新西兰家兔,制备该蛋白特异性抗血清。方法构建原核表达质粒g E-p ET-32a(+),测序后将其转化至E.coli BL21,以异丙基硫代β-D-半乳糖苷(IPTG)诱导,获得VZV g E融合蛋白。采用SDS-PAGE电泳、Western blot法鉴定其特异性,利用Ni2+-NTA柱对g E蛋白进行纯化,复性后免疫新西兰家兔,获得兔抗VZV g E血清;采用间接免疫荧光、Western blot法和ELISA法检测该血清的特异性和效价。结果成功诱导表达出VZV g E融合蛋白,SDS-PAGE鉴定提示其主要为包涵体表达,浓度约为3.01 mg/ml。蛋白经Ni2+-NTA柱纯化后,纯度约为90%。Western blot法显示,经变性、复性后的该融合蛋白有良好的免疫反应性。用该蛋白免疫新西兰家兔后获得兔抗VZV g E血清。ELISA分析显示,该兔抗血清的效价为1∶6 400。结论成功构建了高效表达VZV g E蛋白的原核表达系统,获得较高纯度的g E蛋白,可以作为VZV亚单位疫苗的候选抗原,制备了特异性及效价均较高的兔抗VZV g E多克隆抗体,为VZV感染的临床诊断及治疗提供了重要的实验工具。Objective Varicella-zoster virus （VZV） glycoprotein E （gE） extracellular domain was cloned and ex- pressed in E. coll. New Zealand rabbit was immunized with the recombinant proteins to produce anti-VZV gE polyclonal antibody. Methods Prokaryotic expression plasmid gE-pET-32a （ ＋ ）, which contained the coding sequence for the extracellular domain of VZV gE. The authenticity of the insert was confirmed by DNA sequencing before it was transformated into E. coli BL21 strain. The fusion proteins were analyzed by Western blot and purified with Ni^2＋ -NTA column. The New Zealand rabbit was immunized with the recombinant gE protein. Antibody titers were determined by ELISA. Results Using prokaryotic expression system, we were able to successfully induce the expression of VZV gE fusion proteins in E. coll. The fusion proteins reached 3.01 mg/ml in cell lysate and the purity was nearly 90%. The titer of the rabbit anti-VZV gE polyclonal antibody was 1 ： 6 400. Conclusion The extracellular domain of VZV gE proteins are successfully expressed in E. coli, and could be a candidate for VZV vaccine. Highly specific polyclonal anti-VZV gE antibodies are obtained by immunization in New Zealand rabbit, which could be used to develop a method for VZV laboratory diagnosis.

关 键 词：水痘-带状疱疹病毒 原核表达 融合蛋白 兔抗血清 

分 类 号：R373.1[医药卫生—病原生物学] R392.7[医药卫生—基础医学]