【摘要】 目的　克隆人 3种类型载脂蛋白E (apolipoproteinE ,apoE)cDNA ,表达并纯化 3种apoE融合蛋白。 方法　采用RT PCR方法从肝组织中克隆得到apoE3cDNA ,以此为基础通过定点突变得到apoE2和apoE4的cDNA。将上述 3种cDNA亚克隆到含有His标签的融合表达载体pT7 PL中 ,表达的蛋白经镍离子柱亲和层析纯化。结果　序列分析表明 ,获得的apoE2、apoE3、apoE4与GenBank中的一致。表达并纯化了N端带有 6个组氨酸的apoE2、apoE3、apoE4融合蛋白。结论　成功地获得了人 3种类型apoEcDNA和 3种apoE融合蛋白。更多还原

【Abstract】 Objective To clone three isoforms of apolipoprotein E3 (apoE3) cDNA, to express and purify the corresponding proteins. Methods The cDNA encoding apoE3 was cloned by the method RT PCR from total human liver RNA. Site directed mutagenesis was used to obtain the cDNA encoding apoE2 and apoE4 isforms. These three cDNAs were subcloned into pT7 PL expressing the target protein as a (His)6 tagged fusion. The purified proteins were gained by Ni NTA column. Results These three sequences were confirmed by comparison with GenBank. 6×His fusion proteins (apoE2, apoE3 and apoE4) were correctly expressed and purified. Conclusion The cDNA encoding apoE2, apoE3 and apoE4 have been obtained, and their fusion proteins being purified successfully as well.更多还原