TAZ原核表达载体的构建及在大肠杆菌中的表达  

Construction of Prokaryotic Expression Vector of TAZ and Its Expression in E. coli

作　　者：许会静[1] 王宁宁[1] 刘磊[1] 董媛[1] 王会岩[1] 

机构地区：[1]吉林医药学院,吉林吉林132013

出　　处：《中国医药工业杂志》2015年第12期1301-1304,1329,共5页Chinese Journal of Pharmaceuticals

基　　金：国家自然科学基金项目(81273421);吉林省大学生创新创业训练计划项目(2015029)

摘　　要：通过PCR方法扩增心磷脂酰基转移酶全长基因tafazzin,将其插入到原核表达载体p EASY中。经菌落PCR筛选阳性克隆,并通过DNA测序确定原核表达载体p EASY-TAZ构建成功。将p EASY-TAZ分别转入大肠杆菌(E.coli)感受态细胞BL21(DE3)、BL21(DE3)pLys S和Transetta(DE3)中,以不同浓度的IPTG在不同温度条件下诱导TAZ蛋白表达,选择蛋白表达量较高的Transetta(DE3)菌株作为工程菌。结果显示,重组蛋白表达量随IPTG浓度的提高而增加;温度对TAZ蛋白原核表达有重要影响,低温条件(16℃)可以提高重组蛋白的可溶性。经Western blot试验证实重组蛋白TAZ表达成功。Tafazzin, the full-length gene of cardiolipin acyltransferase, was amplified by polymerase chain reaction （PCR） and inserted into expression vector pEASY. The positive clones were screened by colony PCR, and the construction of the prokaryotic expression vector pEASY-TAZ was confirmed by DNA sequencing, pEASY-TAZ was transformed into E. coil competent cells BL21 （DE3）, BL21 （DE3）pLysS and Transetta （DE3）, respectively. Recombinant TAZ proteins were induced at different IPTG concentrations and temperatures. As a result, Transetta （DE3） was selected as the engineering bacteria for its higher expression amount. The results showed that the expression of the recombinant protein increased accordingly as the IPTG concentration raised. Temperature played an important role in TAZ expression. The expression of soluble recombinant proteins could be improved at the low temperature conditions （16 ℃）. A Western blot analysis was conducted and it confimled that TAZ protein was successfully expressed.

关 键 词：心磷脂酰基转移酶 TAFAZZIN 原核表达 重组蛋白表达 

分 类 号：Q78[生物学—分子生物学]