Biological Functions Of Short-chain Dehydrogenase In C.testosterone

Comamona stestosteroni(CT)ATCC11996,a Gram-negative bacterium that uses steroid hormones as its sole carbon source,and uses steroid hormones as its carbon source and energy substance to conduct metabolic activities.In recent years,many researchers have paid attention to its degradation and the regulation factors that affect its hormone degradation.The genome sequence of CT has been reported in previous studies,and the reaction process of several steroid metabolic enzymes and steroid binding has been described and characterized.The process of bacterial decomposition of steroid hormones by CT bacteria is intricate and complicated.A total of dozens of enzymes have participated in its metabolic process.SDR short chain dehydrogenase is one of many metabolic enzyme families.Through the analysis of transcriptome data,it was found that the SDRy gene has a certain up-regulation effect on hormone degradation.A series of studies were performed on this gene.In this paper,four regulatory proteins(Lys R,Tet R,Lux R1,Lux R2)in C.testosterone were tested by the construction of a knockout strain,high performance liquid chromatography(HPLC),plasmid co-transformation,and enzyme-linked immunosorbent(ELISA).)A series of studies on the biological effects of SDRy have been performed.First,the SDRy gene knockout strain(M-CT)was constructed,and five different hormones were induced.The remaining amount of hormone was detected by high performance liquid chromatography to further prove that SDRy has a certain effect on hormone degradation.Then,the SDRy protein was constructed.The expression vector and prokaryotic expression were obtained to obtain purified target protein and prepare mouse polyclonal antibody.Finally,four co-transformed strains with different regulatory proteins and SDRy including the suspected front end promoter part(QSDRy)were constructed.Qualified by Western Blot and ELISA Detect its regulatory effect.The experimental results show that: 1.Through the gene knockout experiment,under the testosterone-induced conditions,the difference between the hormone degradation of wild type and the knockout strain was 61%,followed by the difference between progesterone and estradiol of 13%,9 %,While the degradation amounts of estrone and methyltestosterone are not much different,it can be inferred that SDRy plays an important role in the degradation of testosterone by CT bacteria;2.The target protein was successfully obtained at a concentration of 9.75 mg / m L,using immunological methods The titer of mouse polyclonal antibody was 1: 25600,and the polyclonal antibody standard curve R2> 0.995 was drawn,and the linear relationship was good;3.p K-QSDRy,p UCLys R / Tet R / Lux R1 / Lux R2 recombinant plasmids were successfully constructed,and Cotransformed into HB101 for expression,and the expression levels were compared by ELISA method.It was concluded that the Tet R regulatory protein showed a significant positive promotion effect under the induction of two steroid hormones,and the effect was significantly higher than that induced by testosterone.Induction,Lux R1 and Lux R2 regulatory proteins also showed a positive promotion effect under the induction of two hormones,but the expression was only slightly increased,the effect was not obvious,and Lys R regulatory proteins were not significantly changed under the induction of both hormones It is speculated that there is no direct regulatory effect on SDRy.The experimental results in this article indicate that SDRy has a certain effect on the degradation of steroid hormones.Tet R regulatory protein has a positive effect on the expression of SDRy.This provides a theoretical and experimental basis for studying the degradation mechanism of Testosterone Biodegradation and gene regulation of steroid hormones in nature provide experimental evidence.