The Biological Effects Of Multiple Proteins And SDRx In C.Testosteroni

Comamonas testosteroni is a Gram-negative bacterium capable of degrading steroids and it has 20 enzymes involved in its metabolism.In recent years,it has bioremediation and enzyme gene expression of C.testosteroni.Regulation has become a hot spot for many scholars.SDRx short-chain dehydrogenase is an important hormone-degrading enzyme in C.testosteroni.In this paper,the biological effects of three regulatory proteins(LysR,LuxR,TetR)in C.testosteroni on SDR were studied by recombinant plasmid construction,enzyme-linked immunosorbent assay,high performance liquid chromatography and plasmid co-transformation.The SDRx gene protein expression vector was constructed;the E.coli BL21(DE3)plysS strain was used for prokaryotic expression and purification of the target protein;the purified protein was used to prepare mouse polyclonal antibody,and the antibody titer was determined by ELISA method and a standard curve was drawn;The SDRx knockout mutant of C.testosteroni was constructed by the principle of homologous recombination and frameshift mutation,and the remaining hormone content in LB medium under five different hormone culture conditions was detected by HPLC;The recombinant plasmid containing the three regulatory proteins was co-transformed into the HB101 strain with the SDRx recombinant plasmid containing the 107 promoter,and the total protein of the co-transformed strain was induced and extracted by hormone,and the expression of SDRx in the co-transformed strain was detected by ELISA.The results showed that the SDRx gene was successfully cloned,expressed and purified,and the protein concentration was 116.66 ?g/mL.The polyclonal antibody against SDRx mice with high titer was successfully prepared,and the titer reached 1:8000.The SDRx gene affected the testosterone cluster.The ability of the bacterium to decompose hormones,the degree of influence of testosterone > progesterone > methyltestosterone > estrone > estradiol;LysR regulatory protein in C.testosteroni has almost no effect on SDRx dehydrogenase.The LuxR and TetR regulatory proteins inhibit SDRx dehydrogenase,and the progesterone-induced conditions have a stronger inhibitory effect on TetR-regulated proteins,which is double that of LuxR-regulated proteins.The experimental results in this paper provide experimental evidence for the biodegradation and gene regulation of steroid hormones in C.testosteroni in nature.