人巨细胞病毒pp150-gp52蛋白原核可溶性表达与IgM捕获ELISA方法建立和应用  

Soluble Expression of HCMV pp150-gp52 Protein in Prokaryotic System and Establishment of IgM Capture-ELISA

作　　者：孙卫国[1] 孙雯娜[1] 侯江厚[1] 杨秉芬[1] 张灵霞[1] 

机构地区：[1]解放军第309医院、结核病研究所、全军结核病防治重点实验室,北京100091

出　　处：《现代检验医学杂志》2015年第5期1-4,共4页Journal of Modern Laboratory Medicine

基　　金：国家传染病重大项目（2013ZX-10003-006）.

摘　　要：目的 原核系统内表达并纯化巨细胞病毒pp150-gp52融合蛋白优势抗原表位,建立IgM捕获ELISA方法并应用.方法 通过重叠PCR技术扩增获得巨细胞病毒pp150-gp52优势片段核酸序列,在原核系统内可溶性表达DsbCpp150-gp52融合蛋白并纯化,用Western blot和ELISA检测融合蛋白的特异性和应用价值.结果 纯化获得的融合蛋白DsbC-pp150-gp52经酶标记建立IgM捕获ELISA方法,检测60份临床阳性血清和60份健康人血清.其中以酶标记DsbC-pp150-gp52蛋白建立的捕获ELISA法阳性检出率96.7％,阴性检出率100％,初步验证DsbC-pp150-gp52融合肽具有非常好的抗原特异性.结论 融合蛋白DsbC-pp150-gp52在大肠埃希菌中以可溶性表达形式存在,获得的高纯度重组融合蛋白具有抗原性和特异性强的特点,采用IgM捕获ELISA的实验方法,可开发检测试剂盒用于风疹病毒的早期检测.Objective To obtain epitopes of pp150-gp52 fusion protein of HCMV expression in solubility in prokaryotic sys- tem and to establish IgM antibody eapture-ELISA test. Methods The gene sequence of antigen superiority segment of HC- MV pplS0-gp52 was obtained by overlap PCR from HCMV genome, which was cloned into pET-DsbC vector. After expres- sion and purification,the antigenicity and specificity of the fusion protein were examined by Western Blot and ELISA. Results The fusion protein DsbC-ppl50-gp52 showed a strong antigenieity when examined by Western Blot. Detecting 60 HCMV positive sera and 60 negative sera by IgM capture ELISA assay showed that the positive rate was 98.3% and negative rate was 100%. Conclusion The results suggested that the fusion protein DsbC-pp150-gp52 could express solubly in Escherichia coli. The recombinant protein with high purity may be a potential candidate of serodiagnostie reagent for early clinical detec- tion of HCMV infection.

关 键 词：巨细胞病毒 pp150蛋白 gp52蛋白 可溶性表达 

分 类 号：R373[医药卫生—病原生物学] Q503[医药卫生—基础医学]