结核杆菌Mtb8.4/hIL12嵌合基因真核表达质粒的构建与表达

Construction and identification of the chimeric Mtb8.4/hIL12 eukaryotic expression plasmid

目的 克隆结核分枝杆菌Mtb8.4 /hIL12嵌合基因 ,导入真核表达载体pCI neo ,并在真核细胞中进行表达。方法 采用基因工程技术 ,首先以 pcDNA3.1(+) -Mtb8.4为模板 ,经聚合酶链反应 (PCR)扩增出Mtb8.4 linker基因 ,与 pCI neo载体进行连接重组 ,构建成pCI neo Mtb8.4 linker(pML)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 pML质粒进行连接重组 ,构建成Mtb8.4 /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Mtb8.4 /hIL12嵌合基因在转录水平的表达情况。结果 Mtb8.4 /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 ,Mtb8.4 /hIL12嵌合基因在转录水平成功表达。结论 Mtb8.4 /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Mtb8.4

To construct and express the chimeric Mtb8.4/hIL12 eukaryotic expression plasmid, the Mtb8.4-linker was firstly amplified by PCR and then cloned into the single NheⅠand MluⅠcloning sites of pCI-neo, to construct the recombinant plasmid of pCI-neo-Mtb8.4-linker (pML). Later on the hIL-12 gene was amplified by PCR and cloned into the single MluⅠand SalⅠcloning sites of pML and correct pCI-neo-Mtb8.4/hIL12 (pMI) recombinant plasmid was identified by PCR, RE digestion and DNA (sequencing). Finally COS-7 cells were transfected with pcDNA3.1(+)-Mtb8.4 constructs by cationic liposom. Forty-eight hours later, mRNA of targets gene were detected by RT-PCR. The accuracy of pMI plasmid construction was confirmed by a number of molecular biological techniques. Transfection of COS-7 cells with plasmids pMI could lead to transient expression of fusion proteins. It concludes that the construction and expression of Mtb8.4/hIL12 chimera by linkage of M.tuberculosis Mtb8.4 gene to human IL12 gene provides the possibility for investigating a new tuberculosis vaccine.