摘要
为了解睾丸酮丛毛单胞菌(Comamonastestosteroni,C.testosteroni)ATCC11996的激素降解原理,研究了睾丸酮丛毛单胞菌中三种调控蛋白LysR familytranscriptionalregulator、LuxR family transcriptional regulator、TetR family transcriptional regulator(LysR、LuxR、TetR)对短链脱氢酶Short-chain dehydrogenase/reductase(SDRx)的生物学作用。SDRx作为SDR家族中的一员对甾体激素降解具有重要作用,利用基因同源重组及移码突变原理构建SDRx敲除株,并检测其在五种不同甾体激素培养条件下降解量,基因敲除对甾体激素降解能力影响程度为睾丸酮>甲睾酮>孕酮>雌酮>雌二醇;构建SDRx蛋白表达载体并进行原核表达,得到纯化目的蛋白浓度116.66μg/mL;制备特异性小鼠多克隆抗体,ELISA方法测定抗体效价为1∶8000;分别构建了含有三种调控蛋白与SDRx启动子共转化菌株,用ELISA方法检测了其SDRx的表达量,LuxR和TetR调控蛋白在两种激素条件下对SDRx脱氢酶的表达起到促进作用,且睾丸酮诱导条件下的促进作用尤为显著,其中睾丸酮条件下TetR的促进作用更明显大于LuxR。
In order to understand the principle of hormonal degradation of Comamonastestosteroni(C.testosteroni)ATCC11996,the subject of three regulatory proteins LysR family transcriptional regulator,LuxR family transcriptional regulator,TetR family transcriptional regulator(LysR,LuxR,TetR)on the short-chain dehydrogenase Short-chain dehydrogenase/reductase(SDRx)biological effects was studied.As a member of the SDR family,SDRx played an important role in steroid hormone degradation.The principle of gene homologous recombination and frameshift mutation were used to construct SDRx knockout strains;and the degradation amount under five different steroid hormone culture conditions,genes was measured.The effect of knockout on the degradation ability of steroid hormones was testosterone>methyltestosterone>progesterone>estrone>estradiol;the SDRx protein expression vector was constructed and expressed prokaryotically;and the purified target protein concentration was 116.66μg/mL;the preparation specificity Mouse polyclonal antibody;the antibody titer determined by ELISA method was 1∶8000;three strains were co-transformed with the SDRx promoter and the expression of SDRx was detected by ELISA.LuxR and TetR regulatory proteins.This hormone promoted the expression of SDRx dehydrogenase under the conditions of testosterone;and the effect of testosterone induction was particularly significant.Among them,the effect of TetR under testosterone was more significant than LuxR.
作者
张海鸥
杨鑫蕾
尉建
嵇冶
ZHANG Hai-ou;YANG Xin-lei;YU Jian;JI Ye(School of Life Science and Technology,Changchun University of Science and Technology,Changchun 130022)
出处
《长春理工大学学报（自然科学版）》
2021年第5期115-121,共7页
Journal of Changchun University of Science and Technology(Natural Science Edition)
基金
吉林省科技发展计划项目(20180311133YY)。
