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目的制备特异性靶向的嵌合DNA疫苗,并探讨其诱导机体产生免疫应答的效果。方法用基因重组技术构建细胞毒T淋巴细胞抗原4(CTLA4)与戊肝病毒抗原HEVE2嵌合基因的真核表达质粒,同时构建不带CTLA4的pHEVE2作为平行对照质粒,体外转染COS-7细胞,Westernblotting法检测细胞培养上清表达产物;于BALB/c小鼠皮内注射,每隔2周1次,共免疫3次,100μg/次;于免疫的第3、5和10周ELISA检测抗体效价。结果获得pHEVE2和pCTLA4-HEVE2嵌合基因真核表达质粒,测序证实各连接点阅读框序列正确;体外转染COS-7细胞,证明真核质粒以分泌形式表达;接种pCTLA4-HEVE2小鼠产生高滴度的特异性抗HEVE2抗体,比接种pHEVE2的对照组高50~100倍;pCTLA4-HEVE2诱导高水平的IgG2a、IgG2bTh1型抗体和IgG1Th2型抗体应答,pHEVE2则以IgG1Th2型抗体应答为主。结论CTLA4-HEVE2嵌合DNA疫苗有效地增强动物对HEVE2抗原的免疫应答,为进一步研究抗原靶向性的嵌合DNA疫苗奠定基础。
Objective To prepare specific targeted chimeric DNA vaccine and explore its effect of inducing immune response in the body. Methods The eukaryotic expression plasmid of cytotoxic T lymphocyte antigen 4 (CTLA4) and hepatitis E virus antigen HEVE2 chimeric gene was constructed by gene recombination technique. At the same time, pHEVE2 without CTLA4 was constructed as a parallel control plasmid and transfected into COS-7 cells in vitro Western blotting was used to detect the expression of the cell culture supernatant; BALB / c mice were injected intradermally once every 2 weeks for three times with 100 μg / potency. Results The eukaryotic expression plasmids of pHEVE2 and pCTLA4-HEVE2 chimeric genes were obtained. The sequencing of the reading frame of each junction was confirmed. The transfection of COS-7 cells in vitro proved that the eukaryotic plasmids were secreted. The pCTLA4-HEVE2 mice inoculated with pCTLA4- The titer specific anti-HEVE2 antibody was 50 to 100 times higher than that of the control group inoculated with pHEVE2. The high level IgG2a, IgG2bTh1 antibody and IgG1Th2 antibody were induced by pCTLA4-HEVE2, while the IgG1Th2 antibody response was the major factor of pHEVE2. Conclusion The CTLA4-HEVE2 chimeric DNA vaccine effectively enhances the immune response of HEVE2 antigen in animals, which lays the foundation for further research on antigen-targeted chimeric DNA vaccine.