结核分枝杆菌Ag85B-MPT64融合基因的构建表达

目的：构建结核分枝杆菌(H37Rv)Ag85B-MPT64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MPT64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—MPT64(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-MPT64融合基因经双向DNA序列测定，碱基突变率为0．11％(2／1707)，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-MPT64融合基因并在真核细胞中表达，融合蛋白具有良好的抗原性。

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

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.