SARS-CoV S蛋白受体结合区的重组表达及免疫原性分析

为了表达SARS-CoV的S蛋白的受体结合区并对其免疫原性进行分析，用PCR方法扩增S蛋白的受体结合区基因片段，克隆至原核表达质粒pET-F32a＋并在大肠杆菌中表达，应用Western—blot鉴定表达的目的蛋白，而后以该蛋白作为诊断抗原包被酶联卡反来检测20份SARS病人血清和28份健康人血清，结果原核表达的S蛋白能够和所用的SARS病人血清反应。这提示表达的S重组蛋白具有良好的抗原性。将变性纯化的重组蛋白和复性蛋白分别皮下免疫小鼠，第三次免疫一周后收集抗血清，用ELISA测定抗体和同时测定中和抗体活性。用变性的抗原免疫的小鼠血清均无中和活性；而用复性的蛋白免疫的小鼠产生了中和抗体。实验表明，S蛋白受体结合区无线性中和表位，中和抗体的产生是由构象表位诱导的。提示该蛋白有可能应用于亚单位疫苗的研究。

Expression and Immunogenicity Analysis of the Receptor-binding Region of SARS-CoV Spike Protein

In order to express the recombinant protein of SARS-CoV S receptor-binding region and to study its immunogenicity,the gene encoding SARS-CoV S receptor-binding region was cloned to pET32a+ and expressed in E.coli.The expression of the recombinant protein was confirmed by Western blot analysis.The S purified protein as diagnostic antigen coated the microtiter plates to detect 20 SARS sera and 28 healthy sera.The results showed that the S purified recombinant protein was able to react with all sera of SARS patients.BALB/c mice were immunized with the denatured and refolding protein respectively.Sera were collected after the third immunization for detection of neutralizing activity against SARS virus.The denatured recombinant S receptor-binding region protein could not induce neutralizing antibodies,whereas refolding recombinant S-RBD could elicit neutralizing antibodies.These results indicated that the receptor-binding region of the S protein could be used for vaccine development.