Establishment of immunochemical determination of human cholesterol 7 alpha-hydroxylase

利用含有大肠杆菌lacZ基因3′端附近人胆固醇7 α -羟化酶cDNA的框架内融合基因制备融合蛋白，并建立抗人胆固醇7 α -羟化酶单克隆抗体。利用该抗体建立免疫化学测定方法。为了阐明肝再生过程中胆固醇- 7 -羟化酶的酶水平，我们测定了肝切除术后大鼠的酶水平。我们还测定了mRNA水平和酶活性。肝切除术后12h， mRNA水平、酶活性和酶蛋白质量平行下降，逐渐恢复正常水平。这些结果表明，这种酶蛋白在细胞分裂过程中减少，并受到翻译前调控。目前正在准备大鼠和家兔胆管结扎后胆固醇- 7 -羟化酶基因表达的物种差异稿件。在项目进行过程中，我们研究了与胆固醇7 α -羟化酶相关的酶，3 - β -羟基- delta ^5-C_<27>-类固醇脱氢酶和25-羟基维生素D_3 - 1 α -羟化酶，并发表在期刊上。

A fusion protein was prepared from an in-frame fusion gene containing the cDNA for human cholesterol 7alpha-hydroxylase near the 3' terminus of the lacZ gene of Escherichia coli and monoclonal antibody angainst human cholesterol 7alpha-hydroxylase was established. Using this antibody, immunochemical determination was established.To clarify the enzyme level of cholesterol 7alpha-hydroxylase during liver regeneration, we determined the enzyme level after hepatectomy in the rats. We also determined mRNA level and enzyme activity. 12hrs after hepatectomy, the level of mRNA,enzyme activity and enzyme protein mass decreased in parallel and gradually returned to normal level. These results suggest that this enzyme protein decreased during cell division and is under pretranslational regulation. Now we are preparing the manuscript concerning the species difference of gene expression of cholesterol 7alpha-hydroxylase after bile duct ligation in the rats and rabbits.In the course of pursuing the project, we have investigated the enzymes, 3beta-hydroxy-DELTA^5-C_<27>-steroid dehydrogenase and 25-hydroxyvitamin D_3 1alpha-hydroxylase, which are related to the cholesterol 7alpha-hydroxylase and published in the journals.