GPI修饰的ESAT-6核酸疫苗的构建及其免疫功能初探  

Construction of GPI-modified ESAT-6 nucleic acid vaccine and preliminary investigation of its immune function

作　　者：何向锋[1] 王净[2] 余方流[1] 赵枫姝[1] 张洪义[1] 陈峻崧[1] 陈登宇[1] 曹文虎[1] 窦骏[1] 

机构地区：[1]东南大学医学院病原生物学与免疫学系,南京210009 [2]东南大学医学院附属中大医院妇产科

出　　处：《免疫学杂志》2011年第9期737-740,746,共5页Immunological Journal

基　　金：国家自然基金资助项目(81071769)

摘　　要：目的构建糖基化磷脂酰肌醇(GPI)修饰的结核杆菌早期分泌性抗原靶(ESAT-6)核酸疫苗,初步分析其免疫功能。方法利用重叠PCR法构建pIRES-ESAT-6-gpi重组体,转染B16F10细胞,G418筛选阳性克隆,用RT-PCR、免疫荧光检测转染细胞的ESAT-6抗原表达情况;采集ESAT-6核酸疫苗免疫鼠血清,分别检测抗体滴度和CDC效应,免疫磁珠法分选CD4+和CD8+T细胞,CFSE/7-AAD细胞毒实验分析CTL细胞毒活性。结果测序正确的重组体pIRES-ESAT-6-gpi转染细胞后,ESAT-6表达于细胞膜表面。ESAT-6核酸疫苗免疫血清和CD8+T细胞分别通过CDC效应和细胞毒作用杀伤膜表达ESAT-6的B16F10细胞。结论构建的GPI修饰的ESAT-6核酸疫苗能够诱导免疫鼠产生体液和细胞免疫反应,杀伤膜表达ESAT-6的B16F10细胞,为进一步基于该法构建B16F10瘤苗的抗肿瘤免疫效应及机制研究奠定了基础。We aim to construct the glycosyl phosphatidyl inositol（GPI）-modified 6 kDa early secretory antigen target（ESAT-6） nucleic acid vaccine and investigate its immune function.The recombinant plasmid pIRES-ESAT-6-gpi was constructed by using overlap PCR,and then transfected into B16F10 cells.The expression of GPI-modified ESAT-6 on the membrane of B16F10 cells was detected by using RT-PCR and immunofluorescence.Sera from the mice immunized with nucleic acid vaccine were analyzed for antibody titre and complement dependent cytotoxicity（CDC） assay.The phenotypes of CD4＋ and CD8＋ T cells were selected by magnetic microbeads,and then used for the flow cytometric CFSE/7-AAD cytotoxicity assay.The results showed that the recombinant plasmid pIRES-ESAT-6-gpi was successfully constructed,and the expression of GPI-modified ESAT-6 target molecule was detected on the surface of transfected B16F10 cells.B16F10-ESAT-6-gpi cells were killed through CDC effect and cytotoxicity of CD8＋ T cells,respectively.In conclusion,we have successfully constructed the GPI-modified ESAT-6 nucleic acid vaccine and it could induce mice to generate a specific humoral and cellular immune responses for killing B16F10 cells expressing GPI-modified ESAT-6,which could be a foundation for further research on the immunity of B16F10 tumor vaccine cells.

关 键 词：B16F10 ESAT-6 GPI 核酸疫苗 

分 类 号：R392.11[医药卫生—免疫学]