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

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DsbC-pp150-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。

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

ObjectiveTo obtain epitopes of pp150-gp52 fusion protein of HCMV expression in solubility in prokaryotic system and to establish IgM antibody capture-ELISA test．MethodsThe gene sequence of antigen superiority segment of HCMV pp150-gp52 was obtained by overlap PCR from HCMV genome，which was cloned into pET-DsbC vector．After expression and purification，the antigenicity and specificity of the fusion protein were examined by Western Blot and ELISA．ResultsThe fusion protein DsbC-pp150-gp52 showed a strong antigenicity 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%．ConclusionThe 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 serodiagnostic reagent for early clinical detection of HCMV infection．