结核分枝杆菌新抗原Mtb8.4基因的克隆与真核表达

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48k后，用RT-PCR方法鉴定Mth8.4在转录水平的表达情况。结果pcDNA3．1（＋）．Mth8．4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。

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

To construct and express the eukaryotic expression plasmid pcDNA3.1(+)-Mtb8.4,the genomic 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 cells were transfected with pcDNA3.1(+)-Mtb8.4 construct by cationic liposome 48 hours later,and the mRNA of the target genes was determined by 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 COS-7 cells with plasmid pcDNA3.1(+)-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 plasmid and to prepare a new type of vaccine for tuberculosis.