摘要
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。
To construct the eukaryotic expression plasmid for two gene fragments of the S-protein of SARS coronavirus （S1 and S2） to detect the expression of these recombinant plasmid in vitro, and to observe the immune responses in BALB/c mice vaccinated, the specific gene fragments of S1 and S2 were amplified by PCR, and were subcloned to eukaryotic expression plasmid pVAC. Then the recombinant plasmids were transfected into HEK293 cell line with lipofectamine 2000, and the transcription and expression of S1 and S2 were detected by RT-PCR and Western blotting respectively. The plasmids were injected into the right leg of each mouse at a dosage of 50 μg three times at two weeks intervals. Meanwhile, the titers of antibodies and the isotypes of IgG were detected by ELISA and percentage of the splenic T ceils was determined by flow cytometry analysis. It was demonstrated that the recombinant plasmid pVAC-S1/pVAC-S2 was successfully constructed, and the expressions of S1 and S2 were also detected in vitro. Mice vaccinated three times could produce high titers of antibodies with the highest titer up to 1 ： 3200, in which the titers of IgG2a, IgG2b and IgG1 antibodies were high in mice immunized with pVAC-S1, while those of IgG2a, IgG2b and IgG3 were high in mice immunized with pVAC-S2. In comparison with the pVAC control group of mice, the percentages of the splenic CD3^＋ and CD8^＋ T ceils increased, and that of CD4^＋ T cells showed no change. It is concluded that the recombinant pVAC-S1 and pVAC-S2 could be expressed in mammalian ceils with induction of both humoral and cell mediated immune responses in mice.
出处
《中国人兽共患病杂志》
CAS
CSCD
北大核心
2005年第12期1042-1046,共5页
Chinese Journal of Zoonoses
