结核杆菌含信号肽的Mtb8.4/hIL12嵌合基因真核表达质粒的构建及鉴定

目的：克隆含信号肽的Mtb8．4（MS）／hIL12嵌合基因，构建其真核表达质粒，并进行鉴定。方法：以peDNA3．1（＋）-含信号肽的Mtb8．4（pMS）质粒为模板，经聚合酶链反应（PCR）扩增出MS-linker基因，与pCIneo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hIL12基因，将hIL12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序列测定等方法进行鉴定。结果：MS／hIL12嵌合基因重组真核表达质粒经证实构建成功。结论：MS／hIL12嵌合基因重组真核表达质粒的成功构建，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。

CONSTRUCTION AND IDENTIFICATION OF THE CHIMERIC MS/HIL12 EUKARYOTIC EXPRESSION PLASMID

Objective:To construct and identify the chimeric MS/hIL12 eukaryotic expression plasmid.Methods:Firstly MS-linker was amplified by PCR and cloned into the single Nhe I and Mil I cloning sites of pCI-neo,so pCI-neo-MS-linker(pMSL)plasmid was constructed.Secondly hIL 12 was amplified by PCR and cloned into the single Mlu I and Sal I colning sites of pMSL.Finally correct pCI-neo-MS/hIL12(pMSI)plasmid was identified by PCR,RE digestion and DNA seguencing.Results:The accuracy of pMSI plasmid construction was confirmed by a number of molecular biological technique.Conclusion:The construction of MS/hIL12 chimera by linkage of M.tuberculosis Mtb8.4 gene with the signal peptide(MS)to human IL12 gene provided the possibility for investigating a new tuberculosis vaccine and research on its immune protection.