SARS病毒的S1蛋白基因的克隆、表达及其ELISA检测方法的建立

Cloning,Expression and Establishment of the ELISA Detection Method of the S1 Protein of SARS

为了建立方便、敏感和特异的SARS病毒血清学诊断方法，利用PCR扩增，克隆了SARS冠状病毒S蛋白基因中从第68～918位碱基的基因序列（S1蛋白）．并通过pGEX-4T1表达系统在大肠杆菌BL21中高效表达了SARS病毒S1蛋白，用超声波破碎菌体及用不同浓度的尿素洗涤包涵体，纯化了S1蛋白，纯度可达75％以上．以在E．coli高效表达的S1蛋白为抗原，以辣根过氧化物酶（HRP）标记的羊抗人IgG为二抗，建立了检测SARS抗体的间接ELISA方法．经检测，筛选出最佳反应条件为5μg／孔．用纯化的E．coli表达的抗原包被酶标板，用5g／L的小牛血清进行封闭，以正常E．coli裂解上清液稀释待检血清．实验表明，应用表达的蛋白作为诊断SARS抗原具有特异性高、抗原易纯化和成本低等特点．

Serological testing of SARS cases is very important for diagnosis and treatment of clinical patients, for control of SARS epidemics and the epidemiological study. A highly sensitive, specific and convenient indirect enzyme-linked immunosorbent assay （ELISA） was established by using the S1 protein of SARS coronavirus as the antigen. The S1 proteins were highly expressed independently using pGEX-4T1 expression system, and the target protein was purified by the inclusion bodies. The inclusion bodies were washed,lysed, refolded to a purity of 75 %. An indirect ELISA for the detection of antibodies against people was developed using the recombinant S1 protein expressed highly in E. coli as antigen. The best condition which is filtrated through experimentation of veaction is 5 μg per hole. Using the antigen expressed by purifying E. coli cover the board of enzyme-linked and obturate it with bovine serum then dilute uninspected serum with liquor of E. coll. The results show that the assay is characterized by its specificity, simplicity and economical cost.