LysR Gene On Analysis Of The Regulation Of 3,17 Hydroxysteroid Dehydrogenase In Comamonas Testosteroni

Steroids has a upward trend in the pharmaceutical market, hormone has environmental pollutants, which is deepening and adverse effects on people’s health.People have a strong research interest and attracted great attention on study the expression regulation of the steroid hormone biological degradation.Comamonas testosterone is a gram negative bacterium has an important role on repair environment, including steroid hormones were degradation as carbon and energy. To research LysR gene on 3,17 beta-HSD expression and regulation mechanism by gene cloning, expression, elisa, electroporation, gene knockout, HPLC and so on in this paper.Cloning 3, 17 beta-HSD gene by PCR and Comamonas testosteroni genomic DNA as template. To construct prokaryotic expression vector transformed into E.coli BL21(DE3), to establish IPTG prokaryotic expression system, purified by Nicolumn, high purity 3,17 beta-HSD protein.3,17 beta-HSD protein homemade as antigen and gain rabbit polyclonal antibody.High titer(1:8000) which was established enzyme immunoassay method combined adsorption(ELISA) for the determination of 3,17 beta-HSD strains.In this paper, two promoter sequences were found in 3, 17 beta-HSD gene upstream about 5Kb sequencean analysis by the analysis softwere of Comamonas testosterone using the promoter element online. gene sequence were cloned and connected with report gene of the plasmid pK-317 beta –HSD.3,17beta-HSD be measured to identified 3, 17 beta-HSD promoter by ELISA.Study show that 3,17 beta-HSD promoter sequence fragment length is 216 bp, located 3,17 beta-HSD gene upstream of-1242 bp position.LysR gene deletion mutant strain MLysR is constructed successfully by gene knockout and compared with the wild type strain ATCC11996.poposed to Expression Regulation Model on LysR gene 3,17 beta –HSD, similar to the "tryptophan operon model".