Identification of an activator which specifically binds to thyroid hormone receptor b

我们试图利用酵母双杂交系统，鉴定能特异性结合甲状腺激素受体（TR） b的新蛋白。{方法}使用酵母双杂交配对系统3 （Clontech, Palo Alto， CA， USA）筛选新的tr相互作用蛋白。将人trb全长cDNA克隆到含有GAL4 DNA结合域的pGBKT7质粒载体上。以人TR cDNA为诱饵，在酵母双杂交系统中筛选人肝脏cDNA文库（Clontech）。{结果}筛选的交配效率和无性系数与厂家说明书上的数值相符。1)第一次筛选，在添加T3 （+T3）的培养皿上获得131个克隆。另外的筛选是在+T3和-T3板上同时进行酵母克隆条带化。91个无性系以T3依赖方式生长。对这些酵母克隆的质粒进行了纯化，并对插入物进行了测序。鉴定出两个未知序列；No. 60和93.2)用RT-PCR从人肝脏总RNA中克隆出2个全长克隆。在肝脏中发现了3个不同的60号克隆mRNA和1个93号克隆mRNA。为了确定TR是否与克隆60或克隆93的产物发生物理相互作用，构建了GST融合蛋白的质粒。

We tried to identified novel proteins which can specifically bind to thyroid hormone receptor (TR) b using yeast two-hybrid system.{Method}Yeast two-hybrid Matchmaker System 3 (Clontech, Palo Alto, CA, USA) was used to screen for novel TR-interacting proteins. A human TR b full length cDNA was cloned into pGBKT7 plasmid vector which contained GAL4 DNA binding domain. We screened a human liver cDNA library (Clontech) using the human TR cDNA as bait in the yeast two hybrid system.{Results}The mating efficiency and number of clones screened corresponded to the values indicated in the manufacture's instructions.1) In the 1st screening we got 131 clones on the plates supplemented with T3 (+T3). The additional screening was yeast clones were streaked both on the +T3 and -T3 plates. Ninety one clones grew in the T3 dependent manner. The plasmids of these yeast clones were purified and the inserts were sequenced. Two unknown sequences were identified ; No. 60 and 93.2) The full length of 2 clones were cloned from human liver total RNA by RT-PCR. Three different mRNAs of the clone No. 60 and a mRNA of No.93 were found in the liver. To determine whether TR physically interact with either the products of clone 60 or 93, the plasmid for GST fusion proteins were constructed.