SARS冠状病毒S蛋白C端片段基因的克隆及其DNA疫苗的免疫学研究

Cloning of SARS Sc gene and its effect on the immune response of mouse elicted by DNA vaccine pcDNA-Sc

目的:构建抗原基因为SARS冠状病毒(Severe acute respiratory syndrome coronavirus,SARS-CoV)S蛋白C端片段基因的DNA疫苗,采用肌注和滴鼻的方法接种小鼠后观察肌肉注射途径和黏膜途径免疫使机体产生免疫应答的情况。方法:将S蛋白C端片段克隆至真核表达载体pcDNA3．1(-),随之将重组质粒进行小鼠肌肉和黏膜免疫,定期检测外周血中抗 SARS-CoV的病毒特异性抗体水平,流式细胞仪观察其淋巴细胞表型变化,免疫组化检测抗原表达分布,脾细胞增殖实验评价 CTL效果。结果:疫苗注射后15天就能在血清中检测出病毒特异性抗体,随着时间的延续,抗体水平逐步升高,至30天后达到稳定,以CTL为主的CD8+T淋巴细胞的百分比含量增加极显著,引起强大的体液免疫和细胞免疫;疫苗滴鼻后45天可在鼻黏膜检测到抗原表达;而空载体质粒对照组未检测出明显的特异性免疫应答。结论:以S蛋白C端片段基因为抗原基因的 DNA疫苗通过肌注和滴鼻能诱导小鼠针对SARS病毒强大的免疫应答。

Objective：To construct DNA vaccine against C region gene of SARS-CoV S protein, and observe the immune response induced by DNA vaccine injection and mucosal immunization. Methods：A recombinant DNA vaccine plasmid was constructed by cloning Sc gene fragment into pcDNA3. 1 （ - ）, which could induce anti-SARS-CoV specific antibodies in mouse. After mice immunized with the recombinant DNA vaccine by injection and mucosal immunization,the SARS-CoV antibodies in the sera of mice were detected by ELISA, the lymphocyte phenotypes were detected by FCM, the specific antigen expressed in tissues was detected with immunohistochemical analysis. Detect spleen and MLN lymphocyte proliferation and CD8^＋ CTL response in vitro. Results：The titer of anti-Sc IgG was much higher in the mouse injected pcDNA3.1-Sc than that of control group （P 〈 0. 05 ） ;it can induce potent humoral and cellular immune response in mice,there were dramatic leaps in the quantity of CD8^＋T cells;as for mucosal immunity,45 days after immunity ,the expression of antigen can be detected in nose mucous membrane. No immune response could be observed in the control group. Conclusion： Sc gene fragment of SARS-CoV can be used as a candidated antigen of DNA vaccine to induce SARS-CoV specific antibodies,it can induce potent humoral and cellular immune response in mice.