抗结核MPT64-Ag85B/katG/GNLY-PRF1重组质粒DNA疫苗的构建与表达  

Construction and expression of a plasmid DNA vaccine MPT64-Ag85B/katG/GNLY-PRF1 against Mycobacterium tuberculosis

作　　者：李晓倩[1] 曹广如[2] 王英全[1] 王苗[1] 敖骏 蔡玉强[2] 岳昌武[1,3] 

机构地区：[1]遵义医学院贵州省微生物资源及药物开发特色重点实验室,贵州遵义563099 [2]遵义医学院附属医院脊柱外科,贵州遵义563099 [3]遵义医学院第三附属医院中心实验室,贵州遵义563002

出　　处：《遵义医学院学报》2016年第6期577-583,共7页Journal of Zunyi Medical University

基　　金：贵州省科技厅社发攻关项目(NO:黔科合SY字2015[3036]);遵义医学院基础-临床合作项目(NO:F-688)

摘　　要：目的构建抗结核MPT64-Ag85B/katG/GNLY-PRF1重组质粒DNA疫苗。方法利用DNA重组技术,将激活细胞免疫和体液免疫的MPT64/Ag85B优势抗原基因和野生型katG基因编码区以及穿孔素/颗粒溶素整合进串联真核表达载体pIRES2-EGFP中,构建抗结核重组质粒MPT64-Ag85B/katG/GNLY-PRF1;电转化方法将质粒重组耻垢分枝杆菌制备菌苗;通过脂质体Lipofectamine 2000介导将重组质粒转染293T细胞。应用qRT-PCR技术在mRNA水平检测靶基因体外表达效果并用Western blot方法检测转染细胞是否表达目的蛋白。结果 PCR证实重组质粒MPT64-Ag85B/katG/GNLYPRF1构建成功并制备重组耻垢分枝杆菌菌苗;qRT-PCR分析表明重组质粒的靶基因MPT64/Ag85B、katG、GNLY/PRFl基因转染293T细胞后均有表达,免疫印迹分析重组质粒的融合蛋白MPT64/Ag85B在相对分子质量72 kDa处有明显蛋白表达条带,且融合蛋白MPT64/Ag85B可分泌表达;融合蛋白GNLY/PRF在相对分子质量75 kDa处有明显蛋白表达条带。结论成功构建抗结核MPT64-Ag85B/katG/GNLY-PRF1重组质粒DNA疫苗,为进一步研究该疫苗的免疫作用提供依据。Objective Construction and expression of a plasmid DNA vaccine MPT64-Ag85B/katG/GNLYPRF1 against Mycobacterium tuberculosis.Methods A polyvalent DNA vaccine was constructed by integrated the artificially fused gene with the coding regions of Ag85B/MPT64,GNLY/PRF1 and the coding region of kat G into pIRES2-EGFP.After the vaccine was transfected into 293 T cells mediated with lipofectamine 2000 for 48 h,the protein was examined by Western blotting techniques.Results MPT64/Ag85 B,GNLY/PRF1 and katG genes were successfully amplified by PCR.The results of Western blotting demonstrated that the protein was expressed in293 T cells and fusion protein MPT64/AG85 B was expressed.Conclusion MPT64-Ag85B/katG/GNLY-PRF1 vaccine of Mycobacterium tuberculosis was constructed,which may the provide basis for development of new antituberculosis vaccine.

关 键 词：耻垢分枝杆菌 疫苗 耐药结核分枝杆菌 多价疫苗 表达分析 

分 类 号：R186[医药卫生—流行病学]