【作者】 李艳红；

【导师】 于源华；

【作者基本信息】 长春理工大学 ， 化学， 2015， 硕士

【摘要】 类固醇药物在世界医药市场的发展地位中呈显著的上升态势,激素成为新兴的环境污染物已经得到证实,激素对环境的污染程度的不断加深及对人们健康产生的不利影响,使人们对甾体激素生物降解及降解基因的表达调控途径的研究产生了浓厚的科研兴趣和引起了高度重视。睾丸酮丛毛单胞菌是革兰氏阴性菌的一种,在科研中此菌对环境的修复有很重要的作用,其中甾体激素就被当作唯一的碳源和能源从而被降解。本文利用基因克隆、表达、酶联吸附免疫试验、电转化、基因敲除技术、高效液相色谱等技术研究LysR基因对3,17β-羟基类固醇脱氢酶(3,17β-HSD)的表达调控机制。本文通过聚合酶链式反应(PCR),以睾丸酮丛毛单胞菌基因组DNA为模板,克隆3,17β-HSD基因,构建原核表达载体,转化大肠杆菌BL21(DE3),建立IPTG原核表达体系,经Ni柱纯化,获得纯度高的3,17β-HSD蛋白。利用自制的高纯度3,17β-HSD蛋白作为抗原,制备兔抗3,17β-HSD多克隆抗体,得到了高效价(1:8000)的兔抗3,17β-HSD多克隆抗体,并建立了测定菌株中3,17β-HSD的酶联吸附免疫方法(ELISA)。本文利用启动子元件在线分析软件对睾丸酮丛毛单胞菌3,17β-HSD基因上游5Kb左右序列进行分析,发现在3,17β-羟基类固醇脱氢酶(3,17β-HSD)基因序列上游存在两段类似启动子序列,分别克隆连接到带有报告基因(3,17β-HSD基因)的质粒pK-3,17β-HSD中,利用ELISA测定3,17β-HSD表达量,确定了3,17β-HSD的启动子。研究结果表明3,17β-HSD启动子序列片段长度为216bp,位于3,17β-HSD基因上游-1242bp位置。采用基因敲除,成功构建了LysR基因缺失突变菌株MLysR。与野生型菌株ATCC11996进行对比分析,提出了LysR基因对睾丸酮丛毛单胞菌3,17β-HSD的表达调控模型,类似于“色氨酸操纵子模型”。更多还原

【Abstract】 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".更多还原