人白细胞介素23受体基因的克隆和原核表达

Cloning and prokaryotic expressing of human interleukin-23 receptor

目的:从外周血单个核细胞(PBMC)中克隆人白细胞介素23受体(hIL-23R)编码区序列,构建原核表达载体并在大肠杆菌中进行表达。方法:分离人外周血单个核细胞(PBMC),提取PBMC的总RNA,应用RT-PCR技术,以PBMC的cDNA为模版,扩增出hIL-23R的编码区(1890bp),将其克隆至pMD 19-T载体,经酶切和测序鉴定后,亚克隆于pGEX-4T-1中构建原核表达载体pGEX-4T-1-hIL-23R,转化大肠杆菌感受态细胞BL-21(DE3),异丙基硫代-β-D-半乳糖苷(IPTG)诱导后收集菌体蛋白,经Western-blot对融合蛋白进行鉴定。结果:获得了hIL-23R编码区序列,构建原核表达载体并在大肠杆菌中表达,表达的融合蛋白主要以包涵体形式存在,其相对分子质量Mr为97KDa。结论:成功构建hIL-23R原核表达载体并在大肠杆菌中表达hIL-23R融合蛋白。

Objective ：To clone the encoding sequence of human interleukin - 23 receptor （ hlL - 23R） from PB-MC, to construct prokaryotic expression vector and to express hlL-23R in E. coli. Methods.Using the total RNA isolated from human PBMC as template, the 1890bp eDNA of hlL - 23R was amplified by reverse transcription - polymerase chain reaction（ RT - PCR）. The PCR product was cloned into pMD - 19 T vector, digested by restriction enzyme and sequenced, then subcloned into pGEX - 4T - 1 to form prokaryotic expression vector pGEX - 4T - 1 - hlL -23 R. After transforming with pGEX -4T- 1 -hlL- 23 R, the E. coli BL21 （DE3） cells were induced by Isopropanol -13- D -galactoside（IPTG）. Western blot was performed to identify the expression of hlL -23R fusion protein. Results：The full length of hlL -23 R eDNA was cloned, constructed prokaryotic expression vector pGEX -4T - 1 -hlL -23R and expressed in E. coli. The recombinant protein mainly exist as inclusion bodies and the Mr of fusion protein was 97KDa. Conclusion：The prokaryotic expression vector pGEX -4T - 1 - hlL -23R is successfully constructed and the fusion protein is expressed in E. eoli BL21 （DE3） cells.