结核杆菌Ag85A/GM-CSF嵌合表达质粒的构建及表达

目的：探讨对结核病DNA疫苗进行基因修饰的新方法。方法：以结核杆菌H37Rv株基因组DNA为模板，通过PCR扩增获得结核杆菌Ag85A抗原蛋白基因的成熟肽编码区，以经PHA诱导后的小鼠脾组织总RNA为模板，通过RT－PCR获得小鼠粒细胞－巨噬细胞集落刺激因子的cDNA，将二者定向克隆入质粒pBK-CMV中构建含嵌合目的基因的重组质粒，转染培养的COS7细胞后检测嵌合目的基因的表达。结果：成功构建了小鼠粒细胞－巨噬细胞集落刺激因子cDNA与结核杆菌Ag85A抗原基因的真核嵌合表达质粒，重组质粒能在COS7细胞中进行稳定表达。结论：本研究首次将细胞因子基因与结核杆菌免疫保护性抗原基因嵌合构建嵌合DNA疫苗，为进一步研究其在动物体内的免疫原性和免疫保护性奠定了基础。

Construction of a recombinant plasmid expresses Mycobacterium tuberculosis A g85A fu sed to murine GM-CSF and its expression in COS7 cells

Objective To find a new way of genetic modification for t uberculosis DNA vaccine.Methods The gene fragment encoding My cobacterium tuberculosis Ag85 A was obtained by using PCR and the cDNA encoding murine granulocyte macrophage clone stimulating factor(GM CSF)was obtained by using RT PCR , the two gene f rag ments were inserted into plasmid vector to construct recombinant plasmid , the r ecombinant plasmid was transferred into COS7 cells by using cationic liposomes. The fused expression product was determined by Western blotting analysis.Results A Recombinant plasmid which can express Mycobacterium tuber culosis Ag85A fuse d to murine Granulocyte Macrophage clone stimulating factor was successfully co n structed.Conclusion All these results provided the basis for fu rther study the immunogenicity and protective immunity of the recombinant plasmi d. [