CAJ | 학술논문

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。
목적：구건단면역구단백백세포개소-1수체상관단백（SIGIRR）포내구효모쌍잡교유이질립，병검측기시부존재자격활작용。방법취합매련반응（PCR）확증인SIGIRR포내구기인편단（480～1230 bp），병장차기인편단중조입pSos재체중，구건유이질립pSos-SIGIRR ，경매절급측서감정구건정학후，장중조질립여대조질립공전화감수태효모균cdc25H ，접충우25℃SD/Glucose（-UL）화SD/Galactose（-UL）평판이급37℃ SD/Glucose（-UL）화SD/Galactose（-UL）평판상，련속관찰6 d효모균생장상황；용Western blot법검측목적단백표체정황。결과정학구건료인SIGIRR포내구효모쌍잡교유이질립pSos-SIGIRR ， pSos-SIGIRR재효모쌍잡교계통중무자격활급독성작용。Western blot결과현시，목적단백이170×103적융합단백형식표체。결론유이질립pSos-SIGIRR가응용우효모쌍잡교계통중，위재인폐호보탈양핵당핵산（cDNA ）문고중심조여SIGIRR상호작용단백전정료중요기출。
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 .