人白介素24cDNA克隆、突变纠正和原核表达  

Prokaryotic expression,point mutation correction and cloning of human interleukin-24 cDNA

作　　者：魏丽丽[1] 李成华[1] 袁成福[1] 陈济[1] 丁嵩涛[1] 刘革力[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：《西安交通大学学报（医学版）》2007年第2期130-133,共4页Journal of Xi’an Jiaotong University（Medical Sciences）

摘　　要：目的获取人白介素24(IL-24)cDNA,构建原核表达载体以表达含有谷胱甘肽S转移酶(GST)的融合蛋白GST-IL-24。方法以人外周血单核细胞(PBMC)总RNA为模板,RT-PCR扩增IL-24 cDNA,克隆入原核表达载体pGEX-4T-2,测序结果显示在IL-24 cDNA第223位G→A,370位T→C,从而导致其编码蛋白的氨基酸发生改变:A75T,H124Y,通过二次PCR将其纠正,重组载体pGEX-IL-24转化大肠杆菌BL21(DE3),异丙基--βD-硫代半乳糖苷(IPTG)诱导表达,SDS-PAGE,Western blot检测显示有融合蛋白GST-IL-24表达。结果通过RT-PCR及二次PCR得到人IL-24 cDNA,构建其原核表达载体,经IPTG诱导在相对分子量约为50 000处有融合蛋白表达。结论IL-24的重组载体在大肠杆菌BL21(DE3)可以表达GST-IL-24融合蛋白。Objective To obtain human interleukin-24（IL-24）cDNA and construct the prokaryotic expression vector of IL-24 for fusion protein GST-IL-24 expression. Methods IL-24 cDNA was amplified by RT-PCR from human PBMC and then was cloned to the vector of pGEX-4T-2. The mutants, （G→A, T→C in IL-24 cDNA）, resulted in the change of IL-24 protein （A75T, H124Y）. Correction of mutations was performed by a two-step PCR reaction. The correct recombinant vector was transformed into E. coli BL21 （DE3） and induced to express fusion protein GST-IL-24 with isopropyl β-D-thiogalactopyranoside. The expression of GST-IL-24 was detected by SDSPAGE and Western blotting. Results By RT-PCR and two-step PCR, we obtained human IL-24 cDNA. The prokaryotic expression plasmid pGEX-IL-24 was constructed, GST-IL-24 molecular mass was approximately 50 000. Conclusion GST-IL-24 fusion protein was expressed in E. coli BL21 （DE3） which was transformed from the recombinant vector of IL-24.

关 键 词：白介素24 二次PCR 突变纠正 融合蛋白 克隆 原核表达 

分 类 号：R730.5[医药卫生—肿瘤]