人PPARγ2 cDNA的克隆及其在大肠杆菌中的表达纯化  

Cloning and expression of human PPARγ2 cDNA in Escherichia coli

作　　者：曹廷兵[1] 叶治家[1] 彭家和[1] 巩燕[1] 黄刚[1] 

机构地区：[1]第三军医大学基础医学部生物化学与分子生物学教研室,重庆400038

出　　处：《第三军医大学学报》2004年第1期57-60,共4页Journal of Third Military Medical University

基　　金：国家自然科学基金资助项目 ( 30 0 70 358) ;重庆市科技攻关资助项目 ( 2 0 0 12 6 )~~

摘　　要：目的　克隆中国人的PPARγ2cDNA ,并在大肠杆菌中进行表达纯化。方法　从正常人面部脂肪组织分离总RNA ,采用RT PCR及序列测定等方法获得人的PPARγ2cDNA ,然后克隆至原核表达载体pET2 8a ,转化至大肠杆菌BL2 1(DE3 ) ,IPTG进行诱导表达 ;在N末端融合了 6×His纯化标签的表达产物进行Westernblot分析和用Ni2 + NTA离子交换树脂进行纯化 ;纯化蛋白进行SDS PAGE纯度分析。结果　获得了正常中国人的PPARγ2cDNA ,并成功构建了高效原核表达质粒pET2 8a PPARγ2 ;Westernblot结果表明 ,经IPTG诱导 ,在大肠杆菌中表达了分子量为 5 6× 10 3 的PPARγ2目的蛋白 ;表达产物经Ni2 + NTA离子交换树脂纯化 ,PPARγ2蛋白的纯度大于 90 %以上。结论　克隆得到正常中国人PPARγ2cDNA的序列与Genebank报道的序列一致 ,并且在E .coli中成功表达和纯化了PPARγ2蛋白。Objective To clone and express human PPARγ2 cDNA in Escherichia coli ( E. coli ). Methods PPARγ2 cDNA was obtained from total RNA isolated from adipose tissue of normal human face by RT PCR and sequencing method, and then PPARγ2 cDNA was inserted into prokaryotic expression vector pET28a for the IPTG induced expression in E. coli BL21(DE3). The expression product fused with 6×His at N terminal was analyzed by Western blotting, and purified by using Ni 2+ NTA ion exchange resin. The purity of PPARγ2 protein was analyzed by SDS PAGE. Results Normal human PPARγ2 cDNA was obtained, and the expression plasmid pET28a PPARγ2 was constructed successfully. Western blot analysis showed that human PPARγ2 with 56×10 3 molecular weight was expressed in E.coli at 20 ℃. The purity of the recombined PPARγ2 was more than 90% after purification using Ni 2+ NTA ion exchange resin. Conclusion The sequence of Chinese PPARγ2 cDNA shows no difference to the known sequence from the Genbank. PPARγ2 induced and expressed in E. coli has been purified successfully.

关 键 词：PPARΓ CDNA 克隆 表达 

分 类 号：R378.21[医药卫生—病原生物学]