人IL-23R胞外区基因的克隆及蛋白表达研究  

Cloning of the human interleukin-23 receptor extracellular region gene and its expression in Escherichia coli

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,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：《中国病原生物学杂志》2009年第5期340-342,358,共4页Journal of Pathogen Biology

基　　金：国家自然科学基金项目(No.30671944)

摘　　要：目的克隆人IL-23R(hIL-23R)胞外区基因,并对其在Escherichia coliBL21(DE3)中的表达进行鉴定分析。方法通过PCR获得hIL-23R胞外区基因片段,将其克隆至原核表达质粒pGEX-4T-1中,构建重组表达质粒pGEX-4T-1-hIL-23R(O)。重组质粒经双酶切鉴定及序列测定后,转化E.coliBL21(DE3),经IPTG诱导蛋白表达,通过SDS-PAGE和Western blot对目的蛋白进行检测分析。结果获得全长为990 bp的hIL-23R胞外区基因,以构建的重组质粒pGEX-4T-1-hIL-23R(O)转化E.coliBL21(DE3)后,SDS-PAGE显示表达蛋白分子质量单位约为64 ku,Westernblot检测该蛋白能被兔抗GST多克隆抗体识别。结论成功构建了hIL-23R胞外区基因的原核表达载体pGEX-4T-1-hIL-23R(O),并在E.coli中表达出重组蛋白,为进一步研究hIL-23R的功能奠定了实验基础。Objective To clone the human interleukin-23 receptor（hIL-23R） extracellular domain gene and express the gene in Escherichia coli BL21 （DE3）. Methods The hIL-23R extracellular fragment was amplified by PCR. The recombinant plasmid pGEX-4T-1-hIL-23R （O） was constructed by cloning the extracellular fragment of hIL-23R into the prokaryotic expression vector pGEX-4T-1. After the recombinant plasmid was identified by restriction endonuclease digestion analysis and DNA sequencing, pGEX-4T-1-hIL-23R （O） was transformed into E. coli BL21 （DE3） through IPTG induction to express the target protein. The expressed protein was analyzed by SDS-PAGE and Western blot. Results A 990 bp extracellular fragment of the hIL-23R gene was obtained by PCR, inserted into the prokaryotic expression vector pGEX-4T-1-hIL-23R （O） and expressed in E. coli BL21 （DE3）. SDS PAGE showed the molecular weight of the fusion protein to be 64 ku, and Western blot showed that the fusion protein could be recognized by the specific rabbit anti-GST antibody. Conclusion The hIL-23R extracellular domain recombinant expression plasmid pGEX-4T-1-hIL-g3R（O） was successfully constructed, and the fusion protein GST-hIL-23R was successfully expressed in E. coli BL21. This research lays the foundation for further functional study of hIL-23R.

关 键 词：hIL-23R 胞外区基因 克隆 蛋白表达 

分 类 号：R392[医药卫生—免疫学]