沉默调节蛋白6-谷胱甘肽巯基转移酶融合蛋白的克隆表达  

Expression and purification of sirtuin6-glutathione S-trausferase fusion protein

作　　者：夏天[1] 刘建湘[1] 姚景宏[2] 吴太鼎 刘子建[3] 杨述华[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院骨科,武汉430022 [2]华中科技大学同济医学院附属协和医院感染科,武汉430022 [3]华中科技大学同济医学院解剖学系

出　　处：《中华实验外科杂志》2012年第2期318-320,共3页Chinese Journal of Experimental Surgery

基　　金：湖北省卫生厅资助项目（JX4809）

摘　　要：目的构建含有人沉默调节蛋白6（hSIRT6）基因的重组原核表达载体，获得高效表达hSIRT6蛋白的基因工程菌，以及较高产量的SIRT6蛋白。方法取205ngHEK293细胞总RNA逆转录后合成的cDNA为模板，聚合酶链反应（PCR）扩增hSIRT6的编码序列，并克隆至原核表达载体pGEx-4T-3中，构建重组的质粒pGEX-4T-3／SIRT6；将重组质粒pGEX-4T-3／SIRT6转化感受态细菌BL21（DE3），在含100mg／L氨苄西林的LB平板37℃培养过夜，以pGEx-4T．3空载体作为对照，在相同条件下转化并异丙基-β-D-硫代半乳糖苷（IPTG）诱导表达谷胱甘肽巯基转移酶（GST）蛋白。产物经SDS．PAGE电泳及Westernblot鉴定。结果PCR产物大小及双酶切鉴定证明所克隆的基因是hSIRT6，DNA测序进一步证实与GeneBank序列完全一致。获得高表达及纯化的SIRT6-GST融合蛋白，该可溶蛋白的相对分子质量为72×10^3，经Westernblot鉴定证实为目的蛋白。结论成功构建了基因重组体pGEX-4T-3／hSIRT6．并制备出可溶性SIRT6-GST融合蛋白。Objective To construct a recombinant prokaryotic expression vector and obtain gene engineering bacteria which can efficiently express human sirtuin6. Methods 205 ng SIRT6 gene obtained from HEK293 cells by using reverse transcription-polymerase chain reaction （RT-PCR） was cloned into vector pGEX-4T-3 to generate the recombinant plasmid named as pGEX-4T-3/SIRT6. The recombinant plasmid was transformed into E. coli BL21 under the 37℃cultivation overnight on the 100 mg/L ampicillin plate. After inducing expression with isopropyl-13-D-thiogalactopyranoside （IPTG）, the SIRT6-glutathione S-transferases （GST） fusion protein was purified by Glutathione Sepharose, and then analyzed by SDS PAGE and Western blotting. Results The cloned gene was determined as human SIRT6 via detection. Highly expressed and purified SIRT6-GST fusion protein was obtained. The specificity of fusion protein was verified by SDS PAGE and Western blotting. The relative molecular weight of target protein was 72 x 10^3. Conclusion The recombinant plasmid pGEX-4T-3/SIRT6 was successfully constructed. SIRT6-GST fu- sion protein was successfully exoressed and ourified.

关 键 词：沉默调节蛋白6 谷胱甘肽-S-转移酶 融合蛋白 纯化 

分 类 号：Q78[生物学—分子生物学]