人IL-23R胞内区基因的克隆与原核表达  

Cloning and Prokaryotic Expression of Gene Encoding Cytoplasmic Domain of Human IL-23 Receptor

作　　者：车昌燕[1] 张国华[1] 刘力[2] 

机构地区：[1]山西医科大学汾阳学院检验系,山西汾阳032200 [2]北京协和医学院基础学院病原生物学系,北京100005

出　　处：《中国生物制品学杂志》2009年第6期571-573,582,共4页Chinese Journal of Biologicals

基　　金：国家自然科学基金资助项目(30671944)

摘　　要：目的克隆人IL-23R(hIL-23R)胞内区基因,并在大肠杆菌中表达融合蛋白。方法通过PCR获得hIL-23R基因的胞内区片段,克隆至载体pGEX-4T-1中,构建重组原核表达质粒pGEX-4T-1-hIL-23R(I),转化感受态大肠杆菌BL21(DE3),IPTG诱导表达,表达产物经SDS-PAGE及Westernblot进行鉴定。结果hIL-23R胞内区基因的PCR扩增产物经琼脂糖凝胶电泳分析可见约760bp的目的片段。重组原核表达质粒经双酶切及测序证明构建正确。表达的融合蛋白相对分子质量约为55000,在低温(20℃)、低IPTG浓度(0.5mmol/L)诱导条件下能以可溶形式表达,可溶性蛋白的表达量约占菌体可溶性蛋白总量的16.1%,且可被兔抗GST多克隆抗体识别。结论已成功克隆了hIL-23R胞内区基因,并在大肠杆菌中表达了可溶性的GST融合蛋白。Objective To clone the gene encoding cytoplasmic domain of human IL-23 receptor （IL-23R） and express fusion protein in E. coli. Methods The gene encoding cytoplasmic domain of human IL-23R was amplified by PCR and cloned into vector pGEX-4T- 1. The constructed recombinant plasmid pGEX-4T- 1-hIL-23R （I） was transformed to E. coli BL21 （ DE3 ） for expression under induction of IPTG. The expressed product was identified by SDS-PAGE and Western blot. Results Agarose gel electrophoresis proved that the target gene fragment at a length of about 760 bp was amplified by PCR. Both restriction analysis and sequencing proved that recombinant plasmid pGEX-4T-l-hIL-23R（I） was constructed correctly. The fusion protein with a relative molecular mass of about 55 000 was expressed in a soluble form under induction of 0. 5 mmol / L IPTG at 20℃. The expressed product contained about 16. 1% of total soluble somatic protein and was recognized with rabbit anti-GST polyclonal antibody. Conclusion The gene encoding cytoplasmic domain of human IL-23R was successfully cloned, and soluble GST fusion protein was expressed in E. coll.

关 键 词：人IL-23受体 胞内区 基因克隆 原核表达 

分 类 号：Q785[生物学—分子生物学] Q786