Cloning And Expression Of CDNA Of Rat Liver 3a-Hydroxysteroid Dehydrogenase

Objects: Rat liver 3a-HSD [EC1. 1. 1.50] has the activity of dihydrodiol dehydrogenase, we can make use of this activity to detect the level of total bile acids in serum. Jn.order to provide a technique basic feature for diagnosis and therapy of liver disease in future. The project was involved in cloning of cDNA fragment of rat liver 3a-HSD, constructing expression vector pET-30a-c(+)/r3 a -HSD DNA, expression and purification of recombinant r3 a-HSD from E. col i transformed by pET-30a-c (+) /r3 a-HSD cDNA, assessing r3a-HSD biologcal activity. Methods: 1. Rat liver 3a-HSD cDNA was obtained by RT-PCR method with total RNA extracted from rat hepatic tissue, then was cloned into pGEM-T vector, and subcloned into expression vector pET-30a-c(+) to construct the recombinant expression plasmid pET-30a-c(+) /r3 a-HSD cDNA. 2. After proved to be correct by DNA sequencing, the recombinant expression plasmid pET-30a-c(+)/r3 a-HSD cDNA was transformed into E. coli BL21-Gold(DE3)pLysS to induce the expression of fusion protein pET-30a-c(+)/r3 a -HSD under optimal condition. The fusion protein pET-30a-c(+) /r3 a -HSD was isolated by His-Bind Resin affinity chromatography. 3. Enzymatic assays-The dihydrodiol dehydrogenase activity was determined spectrophometrically at 37C by NADH production at 340 nm. Results and Disscussions: 1. The total RNA extracted from the rat hepatic tissue was integrity and homogeneous. The concentration of total RNA was 2.88ug/ul by ultraviolet spectrophotometry. Sequence analysis on cDNA showed T-C mutation at the 814th base, the directed point mutation wasn't corrected. 2. The recombinant fusion protein pET-30a-c(+)/r3a-HSD was expressed from E.coli BL21-Gold(DE3)pLysS cells transformed by pET-30a-c(+)/r3a-HSD eDNA plasmid, under lmmol/L IPTG induce at 32℃ for 4 hour in LB medium. The recombinant fusion protein was 37. 9% of total protein in bacterial lysate. There was a single band at 42KD molecular weight as expected as pET-30a-c(+)/r3 a -HSD molecular weight in SDS-PAGE. The recombinant pET-30a-c(+) /r3 a -HSD expressed in cytoplasma was soluble. All the procedures of purification to release free were easy and simple, and the protocal of preparation involved in this project could be used for a large scale of preparation in the future. 3. The assays of the activity of r3a -HSD suggested fusion protein pET-30a-c(+)/r3a -HSD existed 3a -HSD activity...