糖皮质激素受体配体结合区的克隆及酵母双杂交诱饵载体的构建和表达

Expression and construction of "bait plasmid" containing human GR ligand-binding domain in yeast two-hybrid system

目的 用糖皮质激素受体配体结合区 (GR LBD)片段构建酵母双杂合系统中的诱饵载体。方法 根据GenBankGR LBD序列设计引物 ,以人胎肝cDNA文库为模板 ,PCR扩增GR LBD片段 ,该片段与pGEM TVectorSystems连接 ,SmaⅠ SalⅠ双酶切鉴定。回收相应连接片段 ,与酵母表达载体pGBKT7连接 ,重组质粒命名为pGBKT7 GRLBD ,送大连宝生物公司测序。用醋酸锂法转化酶母菌AH10 9,在选择性培养基上观察pGBKT7 GRLBD在AH10 9中的表达情况 ,酵母双杂交检测GR与已知阳性蛋白SRC 1的结合 ,鉴定转化酵母株表型及表达。结果 PCR扩增出的DNA片段约 893bp ,大小正确 ,并成功连接到T 载体 ,释放出所需片段893bp。构建与Gal4 BD融合表达载体pGBKT7 GRLBD ,测序结果表明序列完全正确 ,融合区域的读码框正确。pGBKT7 GRLBD转化感受态AH10 9,在SD Trp板上生长良好 ,AH10 9[pGBKT7 GRLBD]与Y187[pTD1 1]杂交后在SD Trp、 Leu、 Trp Leu板上生长而不能在 His Leu Trp、 Ade His Leu Trp板上生长 ,说明AH10 9[pGBKT7 GRLBD]转化成功但并未自主激活报告基因 ,酵母双杂交测定GRLBD与SRC 1有结合作用。结论 获得了在酵母细胞中正确表达且未自主激活报告基因的融合表达载体pGBKT7-GRLBD ,可作为酵母双杂合系统中的“诱饵”。

Objective To construct a bait vector containing human glucocorticoid receptor(GR) ligand binding domain (LBD) in yeast two hybrid system in order to screen its cDNA library Methods PCR was used to amplify GR LBD fragment from the fetal liver cDNA library with the primers designed in accordance with the sequence in GenBank GR LBD The product was inserted into pGEM(r) T Vector Systems After verified with restriction endonuclease digestion of SmaⅠ/SalⅡ, the vector was inserted into the 'bait plasmid' pGBKT7 (named as pGBKT7 GR LBD) After confirmation with restricted endonuclease digestion and sequence analysis, the plasmid was transformed into the yeast cell AH109, and the transformants were selected on SD/ Trp plates The interaction between GR and SRC 1 was tested by β gal activity with yeast two hybrid system Results The amplified product of 893 bp was inserted into pGEM(r) T Vector and proven to be successful with double restriction enzyme digestion Sequence analysis revealed that the sequence was correctly inserted into pGBKT7 with a right reading frame AH109 [pGBKT7 GR LBD] grew on SD/ Trp plates, but not on the other selective media GR could interact with SRC 1 Conclusion The bait plasmid pGBKT7 GR LBD constructed expresses GR LBD correctly, and can't activate the transcription of reporter gene alone in yeast two hybrid system