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

肾上腺皮质束状细胞中内氮平的表达和功能的研究。

基本信息

批准号：
05670104
负责人：
OHNO Yuji
金额：
$ 1.34万
依托单位：
THE JIKEI UNIVERSITY SCHOOL OF MEDICINE
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1994
项目状态：
已结题

项目摘要

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.

通过聚合酶链式反应（PCR），我们阐明了内氮平在牛肾上腺皮质束状细胞中表达。然后合成了用于内氮平粘端克隆的两个引物。在BamHI位点和内氮平编码区之间，上引物具有编码被因子X识别和切割的氨基酸的核苷酸序列。将内氮平cDNA的PCR产物用BamHI和EcoRI双酶切并插入到pGEX-2T的质粒载体中。胆固醇转运并转化为孕烯醇酮或不转化。将蛋白质载体(pGEX/EDZ)转染至大肠杆菌。转染的大肠杆菌被诱导产生GST-内氮平融合蛋白。融合蛋白可用谷胱甘肽-Sepharose 4B亲和柱纯化。该融合蛋白可被X因子裂解为GST和内氮平。随后我们尝试制备针对该融合蛋白的单克隆抗体。我们通过腹腔注射将蛋白质注射给五只小鼠。所有小鼠都产生了针对该蛋白质的抗体。我们从脾脏中制备了免疫细胞，并用聚乙二醇（PEG）1500制备了杂交瘤细胞。我们利用有限稀释的单细胞克隆技术。我们还检测了克隆的内氮平在大鼠肾上腺皮质线粒体中的类固醇生成活性。用ACTH（0.5IU/大鼠）和放线菌酮（10mg/大鼠）处理的大鼠制备的线粒体在线粒体外膜中积累了胆固醇。已知有一种蛋白质可以加速胆固醇从线粒体外膜到内膜的转运。我们检查了克隆的内氮平是否可以促进独立刺激的孕烯醇酮的产生。