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.
出处
《现代检验医学杂志》
CAS
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）.
