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

利用聚合酶链反应（PCR）证实了内啡肽在牛肾上腺皮质束状细胞中的表达。然后合成了两种用于内啡肽粘端克隆的引物。在BamHI位点和endozepine编码区之间，上部引物含有编码被x因子识别和切割的氨基酸的核苷酸序列，用BamHI和EcoRI酶切得到endozepine cDNA的PCR产物，并插入pGEX-2T质粒载体。胆固醇是否转运并转化为孕烯醇酮。将载体（pGEX/EDZ）转染大肠杆菌。转染后的大肠杆菌被诱导产生gst - endoepine融合蛋白。融合蛋白可通过谷胱甘肽- sepharose 4B亲和柱纯化。该融合蛋白可被x因子裂解为GST和endozepine，并尝试制备针对该融合蛋白的单克隆抗体。我们对5只小鼠进行了腹腔注射。所有小鼠都产生了针对这种蛋白质的抗体。用聚乙二醇（PEG） 1500制备脾脏免疫细胞和杂交瘤细胞。我们利用限制稀释的单细胞克隆技术。我们还检测了克隆的内啡肽在大鼠肾上腺皮质线粒体中的类固醇生成活性。ACTH （0.5IU/大鼠）和环己亚胺（10mg/大鼠）处理后，大鼠线粒体外膜积累胆固醇。已知一种蛋白质可以加速胆固醇从线粒体外膜向内膜的运输。我们研究了克隆的endoepine是否能促进独立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.