SARS病毒S蛋白受体结合区DNA疫苗及免疫效果研究

目的 研究SARS冠状病毒(SARS-CoV)靶细胞受体结合区所构建之DNA疫苗的免疫效果,为进一步的SARS-CoV免疫机理研究及疫苗研制奠定基础.方法 选取SARS-CoV S基因包含靶细胞受体结合区和S1亚单位C端2个基因片段作为目的基因,构建真核表达质粒pVAX-RBD(receptor binding domain)、pVAX-S1C作为DNA疫苗免疫BALB/c小鼠,检测其特异性体液免疫及细胞免疫情况.结果 体液免疫方面,以SARS全病毒裂解产物和原核表达的RBD蛋白作为诊断抗原,用ELISA均可检测到高滴度的小鼠血清抗体IgG的产生.而且,血清中和试验显示pVAX-RBD质粒激发了小鼠保护性中和抗体的产生.通过流式细胞分析和酶联免疫斑点实验(ELISPOT)检测,pVAX-RBD和pVAX-S1C两组质粒均诱导免疫小鼠产生了特异性细胞免疫反应.结论 证明SARS-CoV S蛋白受体结合区上中和表位的存在;体液免疫在抗SARS-CoV感染方面起到重要作用.

Construction of a DNA vaccine encoding the receptor-binding domain of SARS-CoV spike protein and its immune effects

Objective To investigate immune response of the DNA vaccine encoding receptor-binding domain of SARS-coronavirus (SARS-CoV) and its immune effects for exploring the immune mechanisms of SARS-CoV and developing vaccines. Methods Two kinds of truncated S protein gene fragments were cloned into eukaryotic expression domain (RBD) and S1 subunit C terminal (S1C), respectively. Balb/c mice were immunized with the two DNA vaccines pVAX-RBD and pVAX-S1C and the specific humoral and cell-mediated immune responses were detected by flow cytometry (FCM) and enzyme-linked immunospot (ELISPOT). Results Mouse sera IgG antibodies with high titer were detected by enzyme-linked immunosorbent assay (ELISA), with E.coli-expressed protein RBD or SARS-CoV lysate as diagnostic antigen. Furthermore, neutralizing antibodies were also detected in mice vaccinated with the plasmid pVAX-RBD containing the receptor-binding domain. The two expression plasmids could induce some specific cell-mediated immune responses. Conclusion It is proved that the existence of neutralizing epitope in S protein receptor-binding domain; in addition, humoral immunity plays a significant role in preventing virus infection, while the effect of cell-mediated immune is uncertain and need to be further study.