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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: To express the extracellular domain of glycoprotein E (gE) gene of chicken zoster virus (VZV) by using prokaryotic expression system and to purify New Zealand rabbits. Methods The prokaryotic expression plasmid gE-p ET-32a (+) was constructed and transformed into E. coli BL21 after induced by isopropyl thio-β-D-galactoside (IPTG) to obtain VZV gE fusion protein. SDS-PAGE electrophoresis and Western blot were used to identify the specificity of gE protein. Purified gE protein was purified by Ni2 + -NTA column and immunized New Zealand rabbits to obtain anti-VZV gE serum. Immunofluorescence and Western blot The ELISA method was used to detect the specificity and titer of the serum. Results The fusion protein VZV g E was induced successfully. SDS-PAGE analysis indicated that the fusion protein was mainly expressed in inclusion bodies at a concentration of about 3.01 mg / ml. Purification of protein by Ni2 + -NTA column, the purity of about 90%. Western blot showed that the denatured, refolded fusion protein has good immunoreactivity. Rabbit anti-VZV gE serum was obtained after immunizing New Zealand rabbits with this protein. ELISA analysis showed that the rabbit antiserum titer was 1: 400. Conclusion The prokaryotic expression system for high expression of VZV g E protein was successfully constructed and g E protein with high purity was obtained. As a candidate antigen of VZV subunit vaccine, a rabbit anti-VZV g E Polyclonal antibodies, VZV infection for the clinical diagnosis and treatment provides an important experimental tool.