小鼠白细胞介素-18的克隆及在原核细胞中的表达

Cloning and Prokaryotic Expression of Mouse Interleukin-18 Gene

目的克隆小鼠白细胞介素-18(mIL-18)全长cDNA,并使其在大肠杆菌中表达。方法利用RT-PCR和巢式-PCR,从活化小鼠腹腔巨噬细胞中扩增成熟IL-18的全长cDNA。经双酶切,将该cDNA片断插入表达载体pRSET-C,构建含T7启动子的原核表达载体pRSET-mIL-18。测序鉴定后,将重组体转化大肠杆菌BL21(DE3),在IPTG和/或乳糖的诱导下,使重组质粒获得表达。结果重组质粒读码框及mIL-18的序列与预期一致,表达产物经SDS-PAGE和Western blot分析获得证实。结论已成功构建了原核表达载体pRSET-mIL-18,并使其在大肠杆菌中获得了融合表达。

Objective To clone the full length cDNA of mouse IL-18 for expression in E. coli.Methods Amplify the full length cDNA of mature IL-18 from activated peritoneal macrophages of mice by RT-PCR and nest PCR. Digest the amplified cDNA with restriction enzyme and insert into plasmid pRSET-C to construct a prokaryotic expression vector pRSET-mIL-18 with a T7 promoter. Identify the recombinant plasmid by sequencing and transform to E. coli BL21(DE3) for expression under induction of IPTG and/or lactose. Results The murine IL-18 cDNA cloned into the plasmid was in a correct read frame and with expected sequence. SDS-PAGE and Western blot proved that the expressed protein was mIL-18. Conclusion A prokaryotic expression vector pRSET-mIL-18 was successfully constructed, and mIL-18 fusion protein was expressed in E. coli.