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  

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

作　　者：林仁勇[1] 丁剑冰[1] 卢晓梅[1] 王笑峰[1] 阿孜古丽[1] 魏晓丽[2] 王俨[1] 温浩[1] 

机构地区：[1]新疆医科大学第一附属医院 [2]新疆医科大学基础医学院免疫学教研室,乌鲁木齐830054

出　　处：《中国寄生虫学与寄生虫病杂志》2004年第4期204-208,共5页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：国家自然科学基金 (No 30 1 60 0 81 );新疆科技厅基金(No 2 0 0 2 2 1 1 0 1 )~~

摘　　要：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 (RT PCR)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　RT PCR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 0 1)。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 0 1)。结论　pcDNA3 Eg95基因疫苗可诱发小鼠产生特异的体液免疫和细?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.

关 键 词：免疫小鼠 PCDNA3 瞬时表达 抗原基因 体外 基因疫苗 细粒棘球绦虫 白刺 RT-PCR检测 诱导 

分 类 号：R392[医药卫生—免疫学] R512[医药卫生—基础医学]