人促甲状腺素受体膜外区蛋白在原核系统表达及其生物活性鉴定

为研究重组人促甲状腺素受体 (hTSHR)膜外区表达产物及其生物活性与免疫活性 ,将hT SHR膜外区编码基因 (编码第 3～ 4 2 0位氨基酸 )重组到表达型质粒pGEX 4T 3上 ,测序结果表明序列正确 ,未改变读码框架 .然后转入E .coliAd4 94进行诱导表达 .纯化后的表达产物经SDS PAGE、Western印迹及放射受体法分别检测其分子量、免疫活性和生物活性 .重组TSHR3～ 4 2 0 膜外区蛋白 (简称TSHR3～ 4 2 0 )产率为 15 9～ 2 0 2 μg L培养基 ,分子量为 4 8.9kD ;融合蛋白 (简称GST TSHR3～ 4 2 0 )分子量为 75 .4kD .两种表达产物都可与TSHRAb反应 ;TSHR3～ 4 2 0 可与12 5I TSH结合 .

Expression of Soluble Extracellular Domain Proteins of Human Thyrotropin Receptor in E. coli and Its Bioactivities

To study the bioactivities and the immunoactivities of recombinant extracellular region proteins of hTSH receptor expressed in E. coli , the encoding fragment of extracellular domain gene of hTSH receptor was inserted into expression vector pGEX 4T 3, its sequence was verified and the reading frame proved to be correct. The recombinant plasmid was transformed into E. coli Ad494. The molecular mass, bioactivities and immunoactivities of the purified expression product were determined by SDS PAGE, Western blot and radioreceptor assay. The recombinant hTSHR 3～420 products was 159 to 202 μg/L medium. The molecular mass of hTSHR 3～420 and GST TSHR 3～420 were 48.9 kD and 75.4 kD, respectively. Both products could react with TSHRAb and hTSHR 3～420 was characterized by binding to 125 I hTSH.