含信号肽的Mtb8.4/hIL12嵌合基因真核表达质粒的构建及在COS-7细胞中的表达  

CONSTRUCTION OF THE CHIMERIC MS/hIL12 EUKARYOTIC EXPRESSION PLASMID AND ITS EXPRESSION IN COS-7 CELL

作　　者：李榕 李晖[2] 钟森[3] 任红[4] 

机构地区：[1]江西南昌市血站,南昌330006 [2]军事医学科学院野战输血研究所一室 [3]成都中医药大学附属医院 [4]重庆医科大学第二临床学院感染科

出　　处：《现代预防医学》2007年第17期3227-3230,共4页Modern Preventive Medicine

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

摘　　要：[目的]克隆结核分枝杆菌含信号肽的Mtb8.4(MS)/hIL12嵌合基因,导入真核表达载体pCI-neo,并在COS-7细胞中进行表达。[方法]首先以pcDNA3.1(+)-含信号肽的Mtb8.4(pMS)为模板,经聚合酶链反应(PCR)扩增出MS-linker基因,与pCI-neo载体进行连接重组,构建成pCI-neo-MS-linker(pMSL)重组质粒,然后以pORF-hIL12质粒为模板,经PCR扩增出hIL-12基因,将hIL-12基因与pMSL质粒进行连接重组,构建成MS/hIL12嵌合基因真核表达质粒,用限制性内切酶消化、PCR及DNA序列测定等多种分子生物学方法进行鉴定;重组嵌合质粒转染COS-7细胞后,用RT-PCR方法鉴定MS/hIL12嵌合基因在转录水平的表达情况。[结果]MS/hIL12嵌合基因重组真核表达质粒经证实构建成功;转染COS-7细胞后,MS/hIL12嵌合基因在转录水平成功表达。[结论]MS/hIL12嵌合基因重组真核表达质粒的成功构建及在COS-7细胞中的成功表达,为进一步研究其免疫保护效果及制备结核病MS/hIL12嵌合基因疫苗奠定了基础。[Objocticve] To construct and express the chimeric MS/hIL12 eukaryotic expression plasmid. [ Methods] Firstly MS-linker waa amplified by PCR and cloned into the single Nhe Ⅰ and Mlu Ⅰ cloning sites of pCI-neo, recombinant plasmid of pCI-neo-MS-linker （pMSL） had been constructed. Secondly hIL-12 was amplified by PCR and cloned into the single Mlu Ⅰ and Sal Ⅰ cloning sites of pMSL to construct chimeric MS/hIL12 eukaryotic expression plasmid. Thirdly recombinant plasmid of pCI-neo-MS/hIL12 （pMSI） waa identified by PCR, restriction enzyme （RE） digestion and DNA sequencing. Finally COS-7 cells were tmnsfected with pMSI by cationic liposome. 48 hours later, the mRNA level of MS/hIL12 chimeric gene was detected by RT-PCR. [Results] The accuracy of plasmid construction was confirmed by a series of molecular biological techniques. Tranafection of COS-7 cells with plaamlds pMSI leaded to transient expression of fusion proteins. [Conclusions ] The construction and expression of MS/hIL12 chimera by recombination of M. tuberculosis MS gene and human IL12 gene provides the possibility of preparing a new kind of MS/hIL12 chimera vaccine for tuberculosis.

关 键 词：结核病 MS hIL-12 嵌合基因 

分 类 号：Q789[生物学—分子生物学]