Expression and construction of “bait plasmid” containing human Foxp3 gene in yeast two-hybrid system
- VernacularTitle:Foxp3酵母双杂交诱饵表达载体的构建、鉴定和其毒性及自激活效应检测
- Author:
Lina ZHOU
;
Jun WU
;
Gaoxing LUO
;
Weifeng HE
;
Xiwei CHEN
;
Ganping BAI
;
Dongwen SHI
;
Qinghong WANG
;
Shunzong YUAN
;
Xiaorong ZHANG
;
Xiaohong HU
- Publication Type:Journal Article
- Keywords:
gene;
clone;
Foxp3;
yeast two hybrid
- From:Journal of Third Military Medical University
2003;0(08):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct a bait vector containing human Foxp3 gene in yeast two-hybrid system in order to screen the cDNA library of T lymphocyte. Methods RT-PCR was used to amplify the Foxp3 gene fragment from the peripheral blood mononuclear cells (PBMC) with the primers designed in accordance with the sequence in GenBank. The product was inserted into pMD18-T vector. After verified with restriction endonuclease digestion of EcoRⅠ and SalⅠ, the vector was inserted into the “bait plasmid” pGBKT7 (named as pGBKT7-Foxp3). After confirmation with restricted endonuclease digestion and sequence analysis, the plasmid was transformed into the yeast cell AH109, and its toxicity and transcriptional activation was tested by both the phenotype assay and the color assay. Results The amplified product of 1 203 bp was inserted into PMD18-T vector and proven correctly by double restriction enzyme digestion. Sequence analysis revealed that the fragment was correctly inserted into pGBKT7 with a right reading frame and its expression in yeast was verified. Conclusion The bait plasmid pGBKT7-Foxp3 constructed expresses correctly, and can not activate the transcription of reporter gene alone in yeast two-hybrid system
