Construction of bait plasmid containing SIGIRR cytoplasmic tail and detection of self-activation in yeast two hybrid system
10.3969/j.issn.1671-8348.2014.10.004
- VernacularTitle:SIGIRR胞内区酵母双杂交诱饵质粒的构建及自激活作用检测
- Author:
Xuxin CHEN
;
Huasong FENG
;
Yunyou DUAN
;
Xueling WU
;
Guisheng QIAN
- Publication Type:Journal Article
- Keywords:
single immunoglobin interleukin-1 receptor related protein;
two-hybrid system techniques;
bait plasmid;
self-activation
- From:
Chongqing Medicine
2014;(10):1164-1167
- CountryChina
- Language:Chinese
-
Abstract:
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 .
