结核杆菌热休克蛋白70与绿色荧光蛋白基因真核共表达质粒的构建  

Construction of the eukaryotic coexpression vector containing Mycobacterium tuberculosis heat shock protein 70 and green fluorescent protein

作　　者：曾曙光[1] 张积仁[2] 章锦才[1] 吴世卿[1] 刘启才[3] 艾伟健[1] 薛国初[1] 

机构地区：[1]南方医科大学附属口腔医院/广东省口腔医院,广东广州510280 [2]南方医科大学珠江医院肿瘤中心,广东广州510282 [3]广州医学院实验医学研究中心,广东广州510182

出　　处：《南方医科大学学报》2009年第3期433-436,445,共5页Journal of Southern Medical University

基　　金：广东省社会发展基金(2008B030301190);广东省医学科研基金(A2007109)

摘　　要：目的利用pIRES-EGFP载体构建含有人巨细胞病毒(cytomegaloviru,CMV)启动子调控表达的结核杆菌热休克蛋白70(mtHSP70)与增强型绿色荧光蛋白(EGFP)基因双顺反子真核表达载体pCMV-hsp70-IRES-EGFP。方法根据mtHSP70基因全长cDNA的特异引物PCR扩增获得mtHSP70基因序列。将PCR产物进行T载体克隆获得重组质粒PMD-18T-mtHSP70,测序分析选定序列正确的克隆提取质粒,用限制性内切酶Xho I和EcoR I酶切pMD18T-TK和pCMV-IRES-EGFP质粒。将mtHSP70基因定向亚克隆至pCMV-IRES-EGFP载体,酶切鉴定,构建获得含mtHSP70和EGFP基因的重组质粒。Lipofectamine介导下转染B16细胞,并分别在荧光显微镜下观察EGFP的表达和以Westernblot检测mtHSP70蛋白表达情况。结果酶切见特异酶切图谱,该载体在瞬时表达时获得mtHSP70/EGFP的良好表达。结论含mtHSP70和EGFP基因双顺反子真核表达载体pCMV-mtHSP70-IRES-EGFP构建成功,为研究基于以GFP为报告基因的mtHSP70的肿瘤DNA疫苗对肿瘤的治疗作用及机制奠定了实验基础。Objective To construct an eukaryotic coexpression vector containing Mycobacterium tuberculosis heat shock protein 70 （mtHSP70） and enhanced green fluorescent protein （EGFP） controlled by cytomegalovirus promoter using plRES-EGFP vector. Methods The mtHSP70 gene fragment was amplified by PCR from pVAX-mtHSP70-HSV2gD using specific primers. The PCR product was cloned into the vector pMD 18-T vector, and the correct clone was selected according to DNA sequence analysis. The interested mtHSP70 gene fragment was subcloned into pCMV-IRES-EGFP vector with XhoI and EcoR I digestion. The recombinant plasmid was transfected into mouse melanoma B16 cell line, and the green fluorescent cells were detected by fluorescence microscopy and mtHSP70 expression was detected by Western blotting. Results The recombinant plasmid obtained was confirmed by enzyme digestion. The transfected mouse melanoma B16 cells exhibited green fluorescence under fluorescence microscopy and expressed mtHSP70 protein as demonstrated by Western blotting. Conclusion The eukaryotic coexpression vector PCMV-mtHSP70-IRES-EGFP has been established to allow further investigation of the role ofmtHSP70 gene in tumor immunotherapy.

关 键 词：结核杆菌 热休克蛋白70 增强型绿色荧光蛋白 真核表达 免疫治疗 肿瘤 

分 类 号：R378[医药卫生—病原生物学]