人类SIRT6蛋白硝基化位点突变体的构建  

Construction of a mutant recombinant of human SIRT6 at nitrated sites

作　　者：曾凡强[1] 王德广[1] 

机构地区：[1]徐州医学院解剖学教研室,江苏徐州221002

出　　处：《徐州医学院学报》2014年第12期871-873,共3页Acta Academiae Medicinae Xuzhou

基　　金：江苏省高校自然科学基金重点项目（10KJA310053）;江苏省“六大人才高峰”项目（2013-SWYY-024）;江苏省“青蓝工程”项目资助课题

摘　　要：目的构建人类SIRT6蛋白硝基化位点突变体，用于其活性调节机制的研究。方法以野生型pCD—NA3．1-SIRT6-FLAG真核表达重组体为模板，用定点突变PCR的方法获得SIRT6的酪氨酸突变体；突变产物转化大肠杆菌DH5α进行筛选、序列测定；野生型和突变型重组体转到293H细胞，Westernblot鉴定SIRT6的表达。结果①经PCR法得到了SIRT6的突变重组体；②SIRT6突变重组体测序图谱与预期结果完全一致；③Westernblot检测野生和突变型SIRT6蛋白能表达。结论获得了能真核表达硝基化位点的突变型SIRT6蛋白的重组体。Objective To establish a mutant recombinant of human SIRT6 at nitrated sites in order to investigate the mechanism by which its activity is regulated. Methods A wild eukaryotic expression recombinant pCDNA3.1 - SIRT6 - FLAG was adopted as templates to produce SIRT6 mutants at tyrosines by the site - directed mutagenesis PCR. Then, the mutant products were transformed into E. coll. DHScL for screening and sequencing. Both wild and mutant recomhi- nants were transfected into 293H cells. The expression of SIRT6 was measured by Western blot. Results The SIRT6 mutant recombinant was obtained by PCR, whose sequence was consistent with those in GenBank. The expression of SIRT6 in both the wild and mutant recombinants was determined by Western blot. Conclusion The recombinant which can express mutant SIRT6 protein is established.

关 键 词：SIRT6 硝基化位点突变体 真核表达 

分 类 号：R743.31[医药卫生—神经病学与精神病学]