HSV-1衣被蛋白UL7基因的克隆及其在病毒增殖中的表达分析  

Cloning of UL7 protein of HSV-1 and analysis of the expression of UL7 protein during the prolifera-tion of HSV-1

作　　者：周洁楠 徐兴丽[1] 高有[1] 张莹[1] 王晶晶[1] 刘龙丁[1] 李琦涵[1] 

机构地区：[1]中国医学科学院北京协和医学院医学生物学研究所,云南省重大传染病疫苗研发重点实验室,昆明650118

出　　处：《中华微生物学和免疫学杂志》2016年第4期241-246,共6页Chinese Journal of Microbiology and Immunology

基　　金：973计划（2012CB518901）;国家自然科学基金（31100127,31300143）

摘　　要：目的：原核表达并纯化单纯疱疹病毒1型（HSV-1）UL7蛋白,免疫小鼠制备多克隆抗体,初步分析UL7蛋白在HSV-1增殖过程中的表达特点。方法 PCR方法扩增UL7基因,构建原核表达质粒pGEX-5X-1-UL7,转化到E. coli BL21（DE3）中,经IPTG诱导表达,获得并纯化GST-UL7重组蛋白,将其作为抗原免疫ICR小鼠制备抗UL7多克隆抗体。间接法ELISA检测抗体效价,Western blot检测抗体的特异性,使用该多克隆抗体对HSV-1病毒感染Vero细胞后不同时间点的UL7蛋白表达量进行检测。结果 GST-UL7重组蛋白在E. coli BL21（ DE3）中高效表达,间接法ELISA检测抗UL7抗体效价可达1∶105以上。 Western blot试验证明抗UL7多克隆抗体可以特异性识别UL7蛋白。在HSV-1病毒感染Vero细胞后的不同时间点均检测到了UL7蛋白的表达。结论成功获得GST-UL7融合蛋白,且制备了抗UL7蛋白的多克隆抗体,利用该抗体初步确定了UL7蛋白在HSV-1增殖过程中的不同时间点均有表达。Objective To express and purify the recombinant UL7 protein of herpes simplex virus 1 （HSV-1）, to prepare the corresponding UL7-specific polyclonal antibody and to preliminarily analyze the expression of UL7 protein during the proliferation of HSV-1. Methods The UL7 gene was amplified by PCR and then cloned into the pGEX-5X-1 vector for expression of UL7 protein in the prokaryotic expression system. The constructed expression plasmid, pGEX-5X-1-UL7, was transformed into E. coli BL21 （DE3） to induce the expression of UL7 protein by IPTG. The purified GST-UL7 fusion protein was used as antigen to inject the ICR mouse for the preparation of polyclonal antibody specific for UL7 protein. The titer and speci-ficity of the polyclonal antibody were analyzed by using indirect ELISA and Western blot assay, respectively. The UL7 protein-specific polyclonal antibody was used to detect the expression of UL7 protein at different time points after infecting Vero cells with HSV-1. Results The GST-UL7 fusion protein was efficiently ex-pressed in E. coli BL21 （DE3）. The UL7 protein-specific polyclonal antibody was prepared with high titer （1 ∶ 105） and high specificity as indicated by the indirect ELISA and Western blot assay. The expression of UL7 protein was detected at different time points after infecting Vero cells with HSV-1. Conclusion The GST-UL7 fusion protein was obtained successfully and the UL7 protein-specific polyclonal antibody was pre-pared. Accompany with the proliferation of HSV-1, the expression of UL7 protein was detected at different time points by using the polyclonal antibody.

关 键 词：单纯疱疹病毒1型(HSV-1) UL7 原核表达 多克隆抗体 

分 类 号：R373.11[医药卫生—病原生物学]