摘要
为研究睾丸酮丛毛单胞菌(Comamonas Testosteroni,C.T.)ATCC11996降解甾体类激素过程中关键蛋白质的相互作用关系,本文利用基因同源重组及移码突变原理构建睾丸酮降解关键酶3,17β-HSD基因敲除突变株,在回收率95%条件下高效液相检测野生株与突变株睾丸酮降解率,突变株对睾丸酮降解能力明显低于野生株;原核表达3,17β-HSD获得浓度2.4 mg/mL纯化蛋白,制备特异性鼠源多克隆抗体,ELISA检测抗体效价1∶320000;将多克隆抗体与经睾丸酮诱导的野生型C.T.细胞裂解液蛋白质复合物进行免疫共沉淀,质谱测定蛋白质相互作用组,结果表明短链脱氢酶SDRy(WP_003078287.1)为睾丸酮降解过程中关键酶3,17β-HSD的主要相互作用蛋白。
In order to understand protein-protein interaction of the key enzyme 3,17β-HSD for steroid degradation in Comamonas testosteroni(C.testosteroni)ATCC11996,reveal steroid degradation mechanism in the future.Constructed 3,17β-HSD gene knockout mutant byhomologous recombination and frameshift mutation.Under the condition of 95%testosterone recovery rate,detected testosterone degradation by wild-type and 3,17β-HSD gene mutantC.testosteronis-trains with HPLC.The ability for steroid degradationof mutant strain was significantly lower than the wild-type strain.Expression and purification 3,17β-HSD in E.coli system to obtain purified protein,the concentration was 2.4 mg/mL.Preparation specific mouse polyclonal antibody with 3,17β-HSD protein,ELISA detected antibody titer was 1:320000.Co-immunoprecipitation(Co-IP)was performed with 3,17β-HSD polyclonal antibody and protein complexes from cell lysates of testosterone induced wild type C.testosteroni.Detected protein-protein interaction with mass spectrometer.The results showed that short chain dehydrogenase SDRy(WP_003078287.1)was the major interacting protein of 3,17β-HSD for testosterone degradation in C.testosteroni.
作者
于璇
史翔予
赵艳阳
高乐
张昊
YU Xuan;SHI Xiangyu;ZHAO Yanyang;GAO Le;ZHANG Hao(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）》
2024年第2期84-91,共8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(20220402038GH,20210101025JC)。
