人白介素24cDNA克隆、突变纠正和原核表达

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。

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

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 SDS-PAGE 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.