结核分枝杆菌Ag85B-MPT64融合基因的构建表达

Construction and expression of fusion gene encoding Mycobacterium tuberculosis Ag85B-MPT64 in eukaryotic cell

目的 :构建结核分枝杆菌 (H37Rv)Ag85B MPT6 4融合基因并在真核细胞中表达 .方法 :采用序贯PCR(geneSOEing)法将Ag85B和MPT6 4编码基因用疏水甘氨酸接头(Gly4Ser) 3经PCR扩增融合 ,定向克隆入 pcDNA3.1(+)中 .采用脂质体转染法将pcDNA/Ag85B MPT6 4 (pcDNA/AM )转染COS 7细胞 ,用RT PCR、ELISA和斑点印迹法检测其表达 .结果 :Ag85B MPT6 4融合基因经双向DNA序列测定 ,碱基突变率为 0 .11%(2 /170 7) ,突变为无意义突变 ;重组质粒转染COS 7细胞后经检测证实 ,该融合基因能在真核细胞中表达 .结论 :成功地构建了Ag85B MPT6 4融合基因并在真核细胞中表达 。

AIM: To construct fusion gene encoding Mycobacterium tuberculosis Ag85B and MPT64 and to express fusion protein in COS 7 cell line. METHODS: Using gene SOEing method, fusion gene encoding H37Rv strain tubercle Ag85B MPT64 was linked with the(Gly4Ser)3 linker. The fusion gene was cloned into pcDNA3.1(+) and the recombinant plasmid pcDNA Ag85B MPT64 was transfected into COS 7 cells. The fusion protein expressed in COS 7 cells was determined by RT PCR, ELISA and dot blotting. RESULTS: Sequencing of Ag85B MPT64 fusion gene revealed that the mutation rate was 0.11%(2/1707) and the base mutation was nonsense. Expression of fusion gene in transfected COS 7 cells was justified.CONCLUSION: Fusion gene encoding Ag85B MPT64 was constructed successfully and the good antigenicity of fusion protein expressed in COS 7 cells was confirmed.