本文選題：3α-HSD + 基因克隆　； 參考：《吉林大學(xué)》2007年碩士論文

【摘要】： 3α-羥類固醇脫氫酶(3α-hydroxysteroid dehydrogenase, 3α-HSD)是睪酮叢毛單胞菌分泌的一種類固醇脫氫酶,臨床上用3α-HSD作為工具酶來測(cè)定人血清中的總膽汁酸(TBA)濃度。 本實(shí)驗(yàn)首先從睪酮叢毛單胞菌中得到DNA,經(jīng)PCR獲得大量3α-HSD DNA,將目的基因插入原核表達(dá)載體pET28a(+)的啟動(dòng)子下游,構(gòu)建了原核表達(dá)質(zhì)粒pET28a(+)-3α-HSD。重組質(zhì)粒pET28a(+)-3α-HSD用氯化鈣法轉(zhuǎn)化入宿主菌BL21(DE3)中,篩選出含重組質(zhì)粒的基因工程菌。工程菌經(jīng)IPTG誘導(dǎo)表達(dá)和酶活性測(cè)定,證明原核重組表達(dá)質(zhì)粒在大腸桿菌BL21(DE3)中表達(dá)了目的蛋白,經(jīng)IPTG誘導(dǎo)后,其菌的裂解液具有3α-羥類固醇脫氫酶的活性。
[Abstract]:3 偽 -hydroxysteroid dehydrogenase (3 偽 -HSDs) is a steroid dehydrogenase secreted by Trichotomonas testosterone. In clinic, 3 偽 -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 偽 -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- 偽 -HSD-SD-1 was constructed.The recombinant plasmid pET28a- 偽 -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 偽 -hydroxysteroid dehydrogenase.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R346

【引證文獻(xiàn)】

相關(guān)期刊論文 前1條

1 連國(guó)軍;趙長(zhǎng)容;劉文涵;方芳;曹建明;;熒光猝滅法研究NADH、Thio-NAD~+與3α-HSD的相互作用[J];光譜實(shí)驗(yàn)室;2011年05期

相關(guān)碩士學(xué)位論文 前1條

1 張倩;有機(jī)氯化物的生物降解技術(shù)研究[D];武漢理工大學(xué);2007年

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本文編號(hào)：1734221