酵母双杂交技术构建重组人雌激素受体基因酵母

应用酵母双杂交技术构建重组人雌激素受体基因酵母,用以检测类/抗雌激素化合物和环境样品的类/抗雌激素活性.采用多聚酶链反应(PCR)法扩增人雌激素受体(hER)基因,构建诱饵质粒pGBKT7-ER LBD;分别提取并纯化两种含有ER共激活因子基因的靶质粒pGAD424-GRIP1和pGAD424-SRC1;诱饵质粒和靶质粒同时转染酵母细胞Y187,于营养缺陷型培养基(SD/-Trp/-Leu)上筛选阳性菌落,分别构建两种ER双杂交酵母ER+GRIP1和ER+SRC1.考察双杂交酵母与不同激素:17β-雌二醇(E2)、二氢睾酮(DHT)、孕酮(PG)以及三碘甲状腺原氨酸(T3)的结合情况,并考察了雌激素受体拮抗剂4-羟基他莫昔芬(4-OHT)与酵母的相互作用.结果表明:ER+GRIP1和ER+SRC1酵母均能够专一性的和E2结合,并存在显著的剂量-效应关系,E2对ER+GRIP1和ER+SRC1酵母β-半乳糖苷酶活性诱导的EC50值分别为7.3×10-11mol·L-1和1.5×10-10mol·L-1,其中ER+GRIP1酵母细胞诱导产生的酶活性值明显高于ER+SRC1酵母细胞.4-OHT能够抑制E2诱导ER+GRIP1酵母细胞产生的酶活性,并存在显著的剂量-效应关系,IC50值为1.0×10-7mol·L-1.表明重组人雌激素受体基因酵母可以用于检测化合物和环境样品的类/抗雌激素活性.

Construction the Recombinant Human Estrogen Receptor (hER)Gene Yeast Using Two-Hybrid Yeast Technique

The recombinant human estrogen receptor(hER)gene yeast was constructed using two-hybrid yeast technique. It can be used to screen the environmental compounds with hER ant/agonistic activity. The yeast expression plasmid,pGBKT7-ER LBD,was constructed by PCR amplifying,cloning the ER gene. Other two yeast expression plasmids were extracted and purificated,including pGAD424-GRIP1 coding for ER coactivator,GRIP1,and pGAD424 SRC1 coding for SRC1,another ER coactivator. The yeast cells Y187 were cotransformed with the pGBKT7-ER LBD and pGAD424 GRIP1 or pGAD424 SRC1,and selected by growth on synthetic dextrose(SD)medium(lacking tryptophan and leucine)added agar. Then,the two-hybrid ER+GRIP1 yeast and ER+SRC1 yeast were constructed,respectively. We tested the binding of two-hybrid yeast to different hormones including 17β-estrogen(E2),dihydrotestosterone(DHT),progesterone(PG),and 3,3′,5-triiodo-L-thyronine(T3). Furthermore,the interaction of two-hybrid yeast with known ER antagonist,4-hydroxytamoxifen(4-OHT),was also investigated. The results showed that ER+GRIP1 yeast and ER+SRC1 yeast had specific binding with E2 and the activity of recombinant gene yeast induced by E2 was demonstrated in a concentration-depended manner. The EC50 values of E2 for ER+GRIP1 yeast and ER+SRC1 yeast were 7.3×10-11mol·L-1and 1.5×10-10mol·L-1,respectively. In addition,the β-galactosidase activity induced by ER+GRIP1 was higher than the activity induced by ER+SRC1. We also observed that 4-OHT inhibited the β-galactosidase activity induced by E2 in a concentration dependent manner,tested by ER+GRIP1 yeast assay. And the IC50 value was 1.0×10-7mol·L-1. All of the results support that the recombinant hER gene yeast can be used for screen chemicals and environmental samples with ant/agonistic activity.