3α-HSD基因的克隆和表达

发布时间：2018-04-11 03:25

本文选题：3α-HSD + 基因克隆　；参考：《吉林大学》2007年硕士论文

【摘要】： 3α-羟类固醇脱氢酶(3α-hydroxysteroid dehydrogenase, 3α-HSD)是睾酮丛毛单胞菌分泌的一种类固醇脱氢酶,临床上用3α-HSD作为工具酶来测定人血清中的总胆汁酸(TBA)浓度。本实验首先从睾酮丛毛单胞菌中得到DNA,经PCR获得大量3α-HSD DNA,将目的基因插入原核表达载体pET28a(+)的启动子下游,构建了原核表达质粒pET28a(+)-3α-HSD。重组质粒pET28a(+)-3α-HSD用氯化钙法转化入宿主菌BL21(DE3)中,筛选出含重组质粒的基因工程菌。工程菌经IPTG诱导表达和酶活性测定,证明原核重组表达质粒在大肠杆菌BL21(DE3)中表达了目的蛋白,经IPTG诱导后,其菌的裂解液具有3α-羟类固醇脱氢酶的活性。
[Abstract]:3 伪 -hydroxysteroid dehydrogenase (3 伪 -HSDs) is a steroid dehydrogenase secreted by Trichotomonas testosterone. In clinic, 3 伪 -HSD is used as a tool enzyme to determine the total bile acid TBAs in human serum.In this experiment, a large number of 3 伪 -HSD DNAs were obtained from testosterone tuomonas. The target gene was inserted into the prokaryotic expression vector pET28a (pET28a()) downstream, and the prokaryotic expression plasmid pET28a- 伪 -HSD-SD-1 was constructed.The recombinant plasmid pET28a- 伪 -HSD was transformed into the host strain BL21 (DE3) by calcium chloride method, and the recombinant plasmid was screened out.The recombinant prokaryotic expression plasmid expressed the target protein in Escherichia coli BL21 (DE3) by IPTG induced expression and enzyme activity test. After IPTG induction, the lytic solution of the strain had the activity of 3 伪 -hydroxysteroid dehydrogenase.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R346

【引证文献】