目的:构建单免疫球蛋白白细胞介素-1受体相关蛋白(SIGIRR)胞内区酵母双杂交诱饵质粒,并检测其是否存在自激活作用。方法聚合酶链反应(PCR)扩增人SIGIRR胞内区基因片段(480~1230 bp),并将此基因片段重组入pSos载体中,构建诱饵质粒pSos-SIGIRR ,经酶切及测序鉴定构建正确后,将重组质粒与对照质粒共转化感受态酵母菌cdc25H ,接种于25℃SD/Glucose(-UL)和SD/Galactose(-UL)平板以及37℃ SD/Glucose(-UL)和SD/Galactose(-UL)平板上,连续观察6 d酵母菌生长状况;用Western blot法检测目的蛋白表达情况。结果正确构建了人SIGIRR胞内区酵母双杂交诱饵质粒pSos-SIGIRR , pSos-SIGIRR在酵母双杂交系统中无自激活及毒性作用。Western blot结果显示,目的蛋白以170×103的融合蛋白形式表达。结论诱饵质粒pSos-SIGIRR可应用于酵母双杂交系统中,为在人肺互补脱氧核糖核酸(cDNA )文库中寻找与SIGIRR相互作用蛋白奠定了重要基础。
Objective To construct the bait plasmid of pSos-single immunoglobin IL-1 receptor related protein (SIGIRR) in Cy-toTrap yeast two hybrid system ,and to test its self-activation .Methods The cDNA fragments of SIGIRR(480 -1 230 bp) were amplified from pReceiver-LV19-SIGIRR and ligated into the bait plasmid pSos to generate the plasmid pSos-SIGIRR .The pSos-SI-GIRR was identified by DNA sequencing and dual-site endonuclease digestion .Then the recombinant plasmid and control plasmid were introduced into the yeast cell cdc25H .The transformants were inoculated on plates of 25 ℃ /SD/Glucose(-UL) ,25 ℃/SD/Ga-lactose(-UL) ,37 ℃ /SD/Glucose(-UL) and 37 ℃ /SD/Galactose ,respectively and the proliferation ability of transformant was ob-served for 6 d .The Western blot was adopted to detect the expression of target protein .Results The pSos-SIGIRR vector was cor-rectly constructed and proved of no self-activation and toxic action .The Western blot showed that the target protein was expressed in a form of fusion protein of 170KD .Conclusion The bait plasmid containing SIGIRR cytoplasmic tail can be applied to the yeast two-hybrid system and lays the important foundation for seeking the interacting protein with SIGRR from the human lung cDNA li-brary in .