结核分枝杆菌Ag85B-MPT64融合基因的构建及其在大肠杆菌中的表达

目的　构建结核分枝杆菌早期分泌蛋白Ag85B和MPT6 4融合基因 (AM )及其原核表达质粒并进行表达。方法　采用 geneSOEing法将ag85b和mpt6 4用疏水甘氨酸接头 (Gly4Ser) 3 融合 ,经限制性内切酶切后克隆入原核表达载体pET32a中 ,进行酶切、PCR鉴定和DNA序列测定。将重组质粒转染Ecoli BL2 1,经过IPTG诱导后其表达产物进行SDS -PAGE和免疫印迹分析。结果　AM融合基因定向克隆入 pET32a中 ,双向测序验证碱基突变率为 0 11% (2 / 170 7) ,均为无意义突变。SDS -PAGE和免疫印迹分析结果均显示在约 73kDa的位置可见明显的蛋白条带。结论　成功地构建了ag85b -mpt6 4融合基因及其原核表达质粒 ,重组质粒能够在大肠杆菌在中表达。

Construction and expression of the fusion gene encoding Mycobacterium tuberculosis Ag85B and MPT64 in E.coli BL21

To construct fusion gene and prokaryotic expression plasmid encoding Mycobacterium tuberculosis Ag85B and MPT64 and to investigate its expression it in Ecoli.BL21.By using gene SOEing method,fusion gene encoding Ag85B and MPT64 of tubercle bacilli was linked with the (Gly 4Ser) 3 linker.The fusion gene was cloned into pET32a.The recombinant plasmid was introduced into Ecoli.BL21,after induction with IPTG,and its expression product was analysed by SDS-PAGE and Western-blot.The fusion gene had been inserted into pET32a and confirmed by restriction endonuclease digestion,and its sequence was same as that of expected.The expressed 73Kda fusion protein was confirmed by SDS-PAGE and Western-blot.It concludes the fusion gene has been constructed successfully based on the gene encoding Ag85B and MPT64 respectively and cloned into pET32a.The fusion gene can be expressed in E.coli.BL21.