SARS-CoV S蛋白受体结合区的重组表达及免疫原性分析

发布时间：2018-06-06 05:46

本文选题：严重急性呼吸窘迫综合征病毒(SARS-CoV) + 刺突蛋白(S蛋白)　；参考：《中国病毒学》2005年05期

【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。
[Abstract]:In order to express the receptor binding region of S protein of SARS-CoV and analyze its immunogenicity, the receptor binding region of S protein was amplified by PCR and cloned into prokaryotic expression plasmid pET-32a and expressed in Escherichia coli. Western-blot was used to identify the expressed target protein, and then the protein was coated with enzyme linked plate as diagnostic antigen to detect 20 sera of SARS patients and 28 healthy people. The results showed that the expressed S protein could react with the serum of SARS patients. This suggests that the expressed S recombinant protein has good antigenicity. The purified recombinant protein and renatured protein were immunized subcutaneously respectively. The antiserum was collected one week after the third immunization. The antibody and neutralizing antibody activity were determined by ELISA. Mice immunized with denatured antigens had no neutralization activity, while mice immunized with renatured proteins produced neutralizing antibodies. The results showed that the production of neutralizing antibodies was induced by conformational epitopes. The results suggest that this protein may be used in the study of subunit vaccine.
【作者单位】：军事医学科学院微生物流行病研究所病原微生物生物安全国家重点实验室军事医学科学院微生物流行病研究所病原微生物生物安全国家重点实验室军事医学科学院微生物流行病研究所病原微生物生物安全国家重点实验室军事医学科学院微生物流行病研究所病原微生物生物安全国家重点实验室军事医学科学院微生物流行病研究所病原微生物生物安全国家重点实验室河北医科大学免疫学教研室军事医学科学院微生物流行病研究所病原微生物生物安全国家重点实验室
【基金】：北京科委中英SARS免疫病理学研究课题(H030230100130)
【分类号】：R392;

【参考文献】