癌基因iASPP的克隆、表达与纯化  

Cloning,Expression and Purification of Oncogene iASPP

作　　者：刁世勇[1] 张新伟[1] 饶青[1] 邢海燕[1] 陈森[1] 王建祥[1] 王敏[1] 

机构地区：[1]中国医学科学院/中国协和医科大学血液学研究所血液病医院实验血液学国家重点实验室,天津300020

出　　处：《中国生物工程杂志》2005年第5期36-40,共5页China Biotechnology

基　　金：国家杰出青年基金资助项目 (3 0 0 2 5 0 19) ;国家自然科学基金资助项目 (3 0 3 70 5 93 ) ;天津市自然科学基金资助项目 (0 43 60 73 11)

摘　　要：iASPP是新近发现的高度保守的p5 3相关基因,其蛋白产物定位于细胞核内,具有结合NF κBp65亚基和p5 3功能,进而抑制NF κB的转录调节和p5 3对凋亡的调节功能。利用RT PCR方法从人白血病细胞系U 937中克隆出癌基因iASPP ,将其克隆至原核表达载体pET2 8a( + )中,成功构建iASPP表达载体PIAF ,重组质粒读码框和序列与预期一致。在IPTG诱导下,重组载体大肠杆菌Rosetta(DE3)菌株,表达产物经SDS PAGE和Westernblot方法分析证实系iASPP融合蛋白。利用Ni离子鳌合层析的方法纯化iASPP融合蛋白,经SDS PAGE鉴定其纯度超过80 %。iASPP is a novel evolutionarily conserved p53 related gene. Its protein product locates in the nuclear and is capable of binding to NF-κB p65 subunit and p53 The iASPP protein can inhibit the transcriptional activity of NF-κB p65 subunit and prevent p53-induced apoptosis. The oncogene iASPP from human leukemic cell line U937 was cloned by RT-PCR and the expression vector PIAF was constructed successfully by cloning the gene into pET28a(+) vector. The open reading frame and sequence are identical with the sequences predicted. The recombinant vector can express the iASPP fusion protein as inclusion body in E.coli Rosetta(DE3)strain induced by IPTG.The product was identified as iASPP fusion protein by SDS-PAGE and Western blot analysis. The fusion protein purified by only one step of Ni NTA chromatography, and the purity of the fusion protein was over 80% analyzed by SDS-PAGE.

关 键 词：癌基因 IASPP 克隆 表达 纯化 P53 细胞凋亡 抗体 

分 类 号：R730.51[医药卫生—肿瘤] Q78[医药卫生—临床医学]