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

通过聚合酶链反应（PCR）的方法，我们证实了endozepine在牛肾上腺皮质束状细胞中的表达。然后合成了两条粘端克隆引物。在BamHI位点和endozepine编码区之间插入能被X因子识别和切割的氨基酸序列。PCR产物经BamHI和EcoRI双酶切后插入pGEX-2 T质粒载体中，观察胆固醇是否转运并转化为胆固醇烯醇酮。将表达载体pGEX/EDZ转染到大肠杆菌中，表达产物经SDS-PAGE电泳和Western blot分析，证明表达产物为EDZ。转染后的大肠杆菌经诱导表达GST-endozepine融合蛋白。融合蛋白经谷胱甘肽-Sepharose 4 B亲和层析柱纯化。该融合蛋白能被X因子切割成GST和内分泌蛋白，并制备了抗融合蛋白的单克隆抗体。通过腹腔注射，我们给五只小鼠注射了这种蛋白质。所有小鼠都产生了针对该蛋白的抗体。我们从脾脏中制备免疫细胞，用聚乙二醇（PEG）1500制备杂交瘤细胞。我们利用有限稀释法建立了单细胞克隆技术，并检测了克隆的endozepine在大鼠肾上腺皮质线粒体中的类固醇生成活性。用ACTH（0.5IU/rat）和放线菌酮（10 mg/rat）处理的大鼠制备的线粒体在线粒体外膜上积累胆固醇。已知一种蛋白质可以加速胆固醇从线粒体外膜向线粒体内膜的转运。我们研究了克隆的endozepine是否可以促进endently刺激的双烯醇酮的产生。

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.