论文部分内容阅读
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 extracellular domain of human IL-23R (hIL-23R) and identify its expression in Escherichia coli BL21 (DE3). Methods The extracellular region of hIL-23R gene was amplified by PCR and cloned into prokaryotic expression vector pGEX-4T-1 to construct recombinant plasmid pGEX-4T-1-hIL-23R (O). The recombinant plasmid was identified by double restriction enzyme digestion and sequenced. The recombinant plasmid was transformed into E. coli BL21 (DE3) and induced by IPTG. The recombinant protein was detected by SDS-PAGE and Western blot. Results The full-length hIL-23R extracellular region was obtained. The recombinant plasmid pGEX-4T-1-hIL-23R (O) was transformed into E.coli BL21 (DE3) The unit is about 64 ku, Western blot detection of the protein can be rabbit anti-GST polyclonal antibody recognition. Conclusion The prokaryotic expression vector pGEX-4T-1-hIL-23R (0) of hIL-23R extracellular region gene was successfully constructed and expressed in E.coli, which laid the foundation for further study on the function of hIL-23R basis.