摘要
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-32a+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板来检测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。
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 Spurified 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.
出处
《中国病毒学》
CSCD
2005年第5期472-475,共4页
Virologica Sinica
基金
北京科委中英SARS免疫病理学研究课题(H030230100130)
