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目的 :克隆结核分枝杆菌mtb8.4 /hIL12嵌合基因 ,构建其真核表达质粒 ,并进行鉴定。方法 :采用基因工程技术 ,首先以 pcDNA 3.1(+) -mtb 8.4为模板 ,经聚合酶链反应 (PCR)扩增出mtb 8.4 -linker基因 ,与pCI-neo载体进行连接重组 ,构建成pCI-neo -mtb 8.4 -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mtb 8.4 /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。结果 :mtb 8.4 /hIL12嵌合基因重组真核表达质粒经证实构建成功。结论 :mtb 8.4 /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mtb 8.4 /hIL12嵌合基因疫苗奠定了基础
Objective: To clone the mtb8.4 / hIL12 chimeric gene of Mycobacterium tuberculosis and construct its eukaryotic expression plasmid and identify it. Methods: The mtb 8.4 -linker gene was amplified by polymerase chain reaction (PCR) using pcDNA 3.1 (+) -mtb 8.4 as a template and then ligated with pCI-neo vector to construct pCI- neo-mtb 8.4-linker (pML) recombinant plasmid, and then hIL-12 gene was amplified by PCR using pORF-hIL12 plasmid as a template. The hIL-12 gene and pML plasmid were ligated and recombined to construct mtb 8.4 / hIL12 The eukaryotic expression plasmids were identified by restriction enzyme digestion, PCR and DNA sequencing. Results: The recombinant eukaryotic expression plasmid of mtb 8.4 / hIL12 chimeric gene was successfully constructed. Conclusion: The successful construction of mtb 8.4 / hIL12 chimeric gene recombinant eukaryotic expression plasmid lays the foundation for further study on its immunoprotection and preparation of tuberculosis mtb 8.4 / hIL12 chimeric gene vaccine