细粒棘球绦虫Eg95抗原基因疫苗体外瞬时表达及对小鼠诱导的免疫应答

目的　检测细粒棘球绦虫Eg95抗原基因疫苗(pcDNA3 Eg95)体外瞬时表达,探讨其诱导小鼠的体液和细胞免疫效果。　方法　pcDNA3 Eg95经脂质体转染HeLa细胞瞬时表达。逆转录聚合酶链反应(RTPCR)检测Eg95抗原信使RNA(mRNA)在HeLa细胞中的表达,酶联免疫吸附测定(ELISA)和蛋白质印迹法(Westernblot ting)检测Eg95蛋白的瞬时表达情况。pcDNA3 Eg95基因疫苗肌肉注射免疫BALB/c小鼠,ELISA检测IgG和IgG2a水平,四甲基偶氮唑盐试验(MTT法)检测免疫小鼠T淋巴细胞增殖反应。　结果　RTPCR检测结果显示,pcD NA3 Eg95瞬时表达组有Eg95抗原基因mRNA表达,ELISA和Westernblotting检测结果表明,可在HeLa细胞中特异性表达Eg95蛋白。用pcDNA3 Eg95基因疫苗免疫BALB/c小鼠,第3周出现特异性IgG免疫应答,持续升高至第10周,显著高于对照组。从第2周开始,小鼠血清IgG2a应答即为阳性,且长时间(至第10周 )维持较高水平,与pcD NA3空质粒组比较,其差异具有非常显著性意义(P<0.01)。用原核表达的Eg95重组蛋白刺激免疫小鼠脾细胞,有明显的T细胞增殖反应。pcDNA3 Eg95基因疫苗免疫组刺激指数明显高于pcDNA3空质粒组(P<0.01)。结论　pcDNA3 Eg95基因疫苗可诱发小鼠产生特异的体液免疫和细

Transient Expression of Echinococcus granulosus Eg95 DNA Vaccine and Induction of Immune Response in Mice

OBJECTIVE: To detect the in vitro expression of pcDNA3-Eg95 and to observe the immunological effect of the Eg95 DNA vaccine in mice. METHODS: The eukaryotic recombinant plasmid pcDNA3-Eg95 was transfected into HeLa cells with liposome-mediated method. RT-PCR, ELISA and Western blotting were used to analyze the expression of Eg95 mRNA and Eg95 protein, respectively. The BALB/c mice were immunized by pcDNA3-Eg95. Anti-Eg95 IgG and IgG2a in murine serum were determined by ELISA. The proliferation activity of spleen T lymphocytes was tested using MTT assay. RESULTS: Using RT-PCR method, the expression of Eg95 mRNA was confirmed in vitro. The results of ELISA and Western blotting showed that there was a specific Eg95 protein, which can be specifically recognized by anti-sera of Eg95-prokaryotic-expressing protein in pcDNA3-Eg95 transfected HeLa cell lysis. The specific IgG was induced during the 3rd week and continued to increase until week 10. IgG2a was detected after 2 weeks and maintained a higher level till week 10. There was a significant difference of IgG2a level between pcDNA3-Eg95 immunized group and pcDNA3 control (P<0.01). In spleen T cell proliferation response, the stimulation index (SI) in pcDNA3-Eg95 group was higher than that of vector control (P<0.01). CONCLUSION: Eg95 DNA vaccine can induce significant cellular and humoral immune response in mice.