STUDIES ON EXPRESSION AND FUNCTION OF ENDOZEPINE IN ADRENOCORTICAL FASCICULATA CELLS.

通过聚合酶链式反应(PCR)，我们证实了牛肾上腺皮质束状细胞中表达了多西平。在此基础上，合成了两个用于克隆内齐平粘端的引物。在BamHI酶切位点和Endozepine编码区之间，上端有编码编码X因子识别和切割的氨基酸的核苷酸序列。用BamHI和EcoRI双酶切后，将Endozepine基因的扩增产物插入到pGEX-2T载体中，观察胆固醇是否转运并转化为孕烯醇酮。将表达载体(pGEX/EDZ)转入大肠杆菌。诱导表达GST-Endozepine融合蛋白。融合蛋白可通过谷胱甘肽-Sepharose4B亲和层析柱纯化。融合蛋白可被因子X切割成GST和Endozepine，并尝试制备抗融合蛋白的单抗。在腹膜内，我们将这种蛋白质注射到五只小鼠身上。所有小鼠都产生了针对该蛋白的抗体。从小鼠脾中提取免疫细胞，用聚乙二醇1500制备杂交瘤细胞。我们开发了有限稀释的单细胞克隆技术，并检测了克隆的内齐平在大鼠肾上腺皮质线粒体中的类固醇生成活性。ACTH(0.5IU/只)和放线菌酮(10 mg/只)处理的大鼠线粒体在线粒体膜外积聚胆固醇。已知有一种蛋白质可以加速胆固醇从线粒体膜外向内的运输。我们检查了克隆的endzepine是否可以促进依赖刺激孕烯醇酮的产生。

By means of polymerase chain reaction (PCR), we have clarified endozepine was expressed in bovine adrenocortical fasciculata cells. Then two primers for sticky end cloning of endozepine were synthesized. Between BamHI site and coding region of endozepine, the upper primer had nucleotide sequences encoding amino acids that recognized and cleaved by Factor X.The PCR product of endozepine cDNA was digested with BamHI and EcoRI and inserted into a plasmid vector of pGEX-2T.the cholesterol transport and transform into pregnenolone or not. The protein could dosedThe vector (pGEX/EDZ) was transfected to E.coli. The transfected E.coli were induced the production of GST-endozepine fusion protein. The fusion protein could purified by the glutathione-Sepharose 4B affinity column. The fusion protein could cleaved into GST and endozepine by Factor X.Then we have tried to make monoclonal antibodies against the fusion protein. Intraperitoneally, we injected the protein to five mice. All mice made antibody against the protein. We prepared immune cells from spleen and made hybridoma cells by polyethylene glycol (PEG) 1500. We exploited single cell cloning technique by limiting dilution.We also checked the steroidogenic activity of cloned endozepine in rat adrenocortical mitochondria. The mitochondria prepared from rats treated with ACTH (0.5IU/rat) and cycloheximide (10mg/rat) accumulated cholesterol in outer mitochondrial membrane. A protein was known that could accelerate the cholesterol transport from outer to inner mitochondrial membrane. We examined whether the cloning endozepine could facilitateependently stimulated pregnenolone production.