人白介素24基因克隆表达、纯化及活性检测  

Procaryotic expression, purification and activity analysis of human IL-24

作　　者：魏丽丽[1] 李成华[1] 杨俊霞[1] 石华[1] 丁嵩涛[1] 马永平[1] 易发平[1] 宋方洲[1] 

机构地区：[1]重庆医科大学生物化学和分子生物学教研室,重庆400016

出　　处：《中国现代医学杂志》2007年第22期2710-2713,共4页China Journal of Modern Medicine

摘　　要：目的克隆人IL-24基因,构建原核表达载体,表达纯化GST-IL-24融合蛋白,检测其活性。方法用密执毒素诱导HeLa细胞表达IL-24 mRNA,通过RT-PCR获取IL-24cDNA,将其克隆至原核表达载体pGEX-4T-2,IPTG诱导表达,经GSTrap TMF Fcolumn亲合纯化,SDS-PAGE和Westernblot检测融合蛋白的表达,MTT法检测GST-IL-24对宫颈癌CaSKi细胞的抑制作用。结果克隆得到人IL-24的cDNA,序列与GenBank公布序列完全一致,SDS-PAGE电泳可见50kD大小的融合蛋白表达。GST-IL-24以剂量依赖的方式抑制宫颈癌CasKi细胞的生长。结论成功构建人IL-24基因原核表达载体,表达纯化得到GST-IL-24,该融合蛋白对宫颈癌CasKi细胞具有抑制作用。[Objective] To clone human IL-24 gene and construct its prokaryotic expression vector, to express purifmg the fusion protein GST-IL-24, and then to study its activity. [Methods] IL-24 mRNA was expressed in HeLa cells which was induced by mezerein. IL-24 eDNA was amplified by RT-PCR and then was subcloned to vector pGEX-4T-2. After induced by IPTG, the GST-IL-24 fusion protein was purified by GSTrapTM FF column and identified by SDS-PAGE and Western blot, and its inhibitory effect was detected by Mrl＇r assay. [Results] The sequence of IL-24 gene was identical with that in GenBank. GST-IL-24 molecular mass was nearly 50 kD. GST-IL- 24 caused a dose-dependend growth suppression. [Conclusion] The prokaryotic expression system of hlL-24 was successfully established and GST-IL-24 fusion protein was gained, which suppressed CasKi cell growth.

关 键 词：人IL-24基因 克隆 原核表达纯化 MTT 

分 类 号：R730.5[医药卫生—肿瘤]