结核分枝杆菌新抗原Mtb8.4基因的克隆与真核表达  

Cloning and eukaryotic expression of the gene encoding the new antigen Mtb8.4 of Mycobacterium tuberculosis

作　　者：李晖[1] 李榕 钟森[1] 任红[3] 罗月贝 

机构地区：[1]泸州医学院附属医院感染病科,泸州646000 [2]江西省南昌市血站,南昌330008 [3]重庆医科大学病毒性肝炎研究所,重庆400010

出　　处：《中国人兽共患病杂志》2005年第11期949-952,共4页Chinese Journal of Zoonoses

基　　金：四川省青年科技基金资助(批准号:川青科基[2002]1号);四川省重点学科重点建设项目资助(批准号:SZD0241)

摘　　要：目的克隆结核杆菌新抗原Mtb8.4基因,导入真核表达载体pcDNA3.1(+),构建成重组质粒pcDNA3.1(+)-Mtb8.4,并在真核细胞中进行表达。方法提取人结核杆菌H37Rv株的基因组作为模板,进行PCR扩增,获得Mtb8.4目的基因后,与pcDNA3.1(+)载体进行连接重组,用限制性内切酶消化、PCR及DNA序列分析等多种方法进行鉴定;重组质粒转染COS-7细胞48h后,用RT-PCR方法鉴定Mtb8.4在转录水平的表达情况。结果pcDNA3.1(+)-Mtb8.4真核表达载体构建成功;转染COS-7细胞后,Mtb8.4在转录水平成功表达。结论pcDNA3.1(+)-Mtb8.4真核表达质粒的构建成功以及Mtb8.4在COS-7细胞中的成功表达,为进一步研究该真核表达质粒的免疫保护效果及制备结核病pcDNA3.1(+)-Mtb8.4DNA疫苗奠定了基础。To construct and express the eukaryotic expression plasrnid pcDNA3.1 （ ＋ ）-Mtb8.4, the genornic DNA was extracted from Mycobacterium tuberculosis （ Mtb ） and used as template in the PCR amplification. The target gene obtained was cloned into the unique HindIII and EcoR I cloning sites of pcDNA3.1（ ＋ ）. and the correct pcDNA3.1（ ＋ ）-Mtb8.4 recombinant plasmid was subjected to PCR, RE digestion and DNA sequencing. Meanwhile, the COS-7 ceils were transfected with pcDNA3.1 （ ＋ ）- Mth8.4 construct by cationic llposome 48 hours later, and the mRNA of the target genes was determined hy RT-PCR. It was demonstrated that the accuracy of plasmid pcDNA3.1 （ ＋ ）-Mt6b8.4 construct was confirmed by a series of molecular biology techniques Transfection of the CO8-7 cells with plasmid pcDNA3, i（ ＋ ）-Mtb8.4 caused a transient expression of the Mtb8.4 protein. It is concluded that the construction and expressionof plasmid pcDNA3.1-Mtb8.4 would provide the possibility to investigate the immunogenicity of the recombinant plasrnid and to prepare a new type of vaccine for tuberculosis.

关 键 词：结核杆菌 MTB8.4 PCR 基因克隆 真核表达 

分 类 号：R378.91[医药卫生—病原生物学]