Cloning And Expression Of 3α-HSD Gene

3α-HSD (3α-hydroxysteroid dehydrogenase) is one variety of steroid dehydrogenase is excreted from Comamonas/Pseudomonas testosteroni. Comamonas/Pseudomonas testosteroni is strictly needed oxygen ,non-zymogenic Gram-negative rod,that can grow on steroids as its only scarce of carbon.When it growed in nutritive medium that contained steroid ,could generate a variety of steroid dehydrogenase.One of specieses is 3α-HSD,total length of 3α-HSD gene is 774bp,encodes 3α-HSD proteinum of 258 amino acids,about relative molecular mass is 26.4×103. 3α-HSD can utend sorts of ground substance,reversibly catalyze C19-27 oxidation-reduction. Bile acid is one effectiveness substrate of 3α-HSD, 3α-HSD is as instrument determines TBA(total bile acids) concertration in Human serum in clinically.At present, 3α-HSD is straightly extracted from Comamonas/Pseudomonas testosteroni in determination of TBA, complicated determination artwork , needed depurate through laminar analysises and preparation electrofocusing eletrophoretic technique ,enzyme activity was lost in course of depuration,the yield of apoferment was low,the segregation was hard between 3α-HSD andβ-HSD,so the cost that natural 3α-HSD straightly segregated from bacterium is expensive ,confined the determination TBA gemeralization in clinic.So,we segregated Comamonas/ Pseudomonas testosteroni in nature,construct its prokaryotic expression system,got higher activity fusion protein,establish the base of establishing enzymatic cycling method.To check strain that was Comamonas/Pseudomonas testosteronisegregated 3α-HSD,according to 3α-HSD gene order designed primer, the follow board on segregated strain gene DNA carry out PCR amplification,obtained about 800bp fragment.By means of pET15b plasmid as carrier,succeedly constructed 3α-HSD prokaryotic expression system,processed enzyme cut characteriza- tion and sequencing,confirmed fragment that amplified certainty was 3α-HSD,Recombinant plasmid was induced expression by IPTG in E.coli.BI21(DE3)pLysS,determine bacterium crude extract clear supernatant liquid and sediment,confirmed recombination protein to be solubility expression in cell,don't form inclusion,so need not process degeneration renaturation complex process on downstreanm purification ,depleted apoferment degradation and activity loss. 10%SDS-PAGE eletrophoresis showed,the recombination protein relative molecular mass was 29×103 ,explained relative molecular mass was 29×103 interest protein progressed induction and expression in E . coli . BI21(DE3)pLysS .More confirmed segregated strain was Comamonas/Pseudomonas testosterone that generated 3α-HSD.Recombinant plasmid pET28a(+)-3α-HSD was shifted in parasitifer bacterium BL21(DE3)by the motherd of Calcium chloride,by preliminary screening,positive clone bacterium could identify correctly expressing 3α-HSD proteinum that molecular weight was 34Kb by IPTG induction expression and SDS-PAGE eletrophoretic analysis.Bile acid is the metabolin that cholesterin decomposed in liver and enterohepatic circulation,when liver function was damaged or enterohepatic circulation generated disorder,TBA density was advanced in blood .But TBA density was lower in normal blood serum(Ixmol/L level),was not esay todetect,so sensibility of detection method needed be higher.Since 70 age,after high punity 3α-HSD was extracted from Comamonas/Pseudomonas testosteroni, TBA enzymic method determination was developed step by step.At 70 age,first generation TBA enzymology assay mothod was ultraviolet method, its sensibility was not good,needed blood was many.At 80 age in the early days,developed second filial generation TBA colonimetry by NTB indicator system,the method was still lower,its production polluted pipe line of biochemistry analysator and colonimetric cylinder.Third TBA enzymology assay method was on the base of second filial generation inleted△4DH,sensibility was elevated,but didn't solve reaction product pollution .Quartus generation TBA enzymology assay method was enzyme circulation assay.Utilize substrate and coferment repeat reaction,quantity of measured was increased by time extended,elevated detection sensibility,and reaction product was not pollution,was very application perspective assay method.At present,instrument enzyme 3α-HSD on bile acid determinion was straightly purified from Comamonas/Pseudomonas testosterone, the yield of apoenzyme was low,cost was expensive, confined TBA determinion spreaded in clinical,Molecular bilolgy technique progressed, enormously impulsed protein biosynthesis development.Now established 3α-HSD fusion protein prokaryotic expression, succeedly progressed high performance in E.coli.,activity of fusion protein was elementary identical with natual 3α-HSD,but enzymology assay by fusion protein as instrument enzyme wasn't established now.We got Comamonas/Pseudomonas testosteroni that produced 3α-HSD from nature,utilized molecular biology technique, impulsed thedevelopment of protein biosynthesis.At present established 3α-HSD fusion protein prokaryotic expression system,but didn't establish blood serum TBA enzymology assay with recombinate .Utilze molecular biology technique ,established expression activity high,easy to purify, high performance fision protein expression system,clinical application value was great.