论文部分内容阅读
目的构建SARS冠状病毒棘突糖蛋白(S蛋白)的两个片段S1和S2的真核表达质粒pVAC-S1和pVAC-S2,检测它们在哺乳动物细胞中的表达,并观察它们在小鼠中诱导的体液和细胞免疫应答。方法采用PCR方法体外扩增SARS冠状病毒棘突糖蛋白的两个片段S1和S2的基因片段,定向克隆入真核表达载体pVAC,构建pVAC-S1和pVAC-S2重组质粒。并通过Lipofectamine 2000将它们转染到HEK293细胞,RT-PCR和Western-blot鉴定重组质粒在真核细胞中的转录和表达。以构建的重组质粒直接免疫小鼠,ELISA检测小鼠血清特异性抗体以及抗体亚类,流式细胞术检测小鼠脾脏T淋巴细胞亚群分布。结果成功构建了重组真核表达质粒pVAC-S1和pVAC-S2,并可在HEK293细胞中获得表达。免疫小鼠三次后,抗体滴度达到了1∶3 200。pVAC-S1诱导了高滴度的IgG2a,IgG2b和IgG1,pVAC-S2刺激产生高滴度IgG2a,IgG2b和较高滴度的IgG3。脾脏T淋巴细胞亚群分析示pVAC-S1和pVAC-S2两免疫组小鼠CD3+和CD8+T细胞明显升高。结论构建的真核表达质粒pVAC-S1和pVAC-S2能在哺乳动物细胞中表达,免疫小鼠后诱导了明显的细胞和体液免疫应答。
Objective To construct eukaryotic expression plasmids pVAC-S1 and pVAC-S2 of two fragments S1 and S2 of the spike protein of SARS coronavirus (S protein) and to detect their expression in mammalian cells and to observe their expression in mice Induced humoral and cellular immune responses. Methods The gene fragments of S1 and S2 of SARS coronavirus were amplified by PCR in vitro and cloned into eukaryotic expression vector pVAC to construct recombinant plasmid pVAC-S1 and pVAC-S2. The recombinant plasmids were transfected into HEK293 cells by Lipofectamine 2000, and the expression and transcription of the recombinant plasmids were confirmed by RT-PCR and Western-blot. The recombinant plasmids were used to directly immunize mice. Serum specific antibodies and antibody subtypes were detected by ELISA. The distribution of T lymphocyte subsets in spleen was detected by flow cytometry. Results The recombinant eukaryotic expression plasmids pVAC-S1 and pVAC-S2 were successfully constructed and expressed in HEK293 cells. After three immunizations, the antibody titer reached 1: 300. pVAC-S1 induced high titers of IgG2a, IgG2b and IgG1, and pVAC-S2 stimulation produced high titers of IgG2a, IgG2b and higher titers of IgG3. Analysis of spleen T-lymphocyte subsets showed that the CD3 + and CD8 + T cells of mice immunized with pVAC-S1 and pVAC-S2 were significantly increased. Conclusion The constructed eukaryotic expression plasmids pVAC-S1 and pVAC-S2 can be expressed in mammalian cells. The immunized mice induced obvious cellular and humoral immune responses.