摘要
利用分子克隆的方法扩增与克隆睾丸酮丛毛单胞菌ATCC 11996的3α-HSD基因,构建以pET-15b为载体的表达工程菌,IPTG诱导蛋白表达,通过优化表达菌的最佳表达条件得到最大表达量的目的蛋白。诱导后将工程菌裂解,通过硫酸铵沉淀,Ni-NTA亲和层析,分子筛分离进行纯化,并测定纯化后蛋白的酶活性。结果表明:1)克隆获得了睾丸酮丛毛单胞菌3α-HSD基因并实现了异源表达;2)工程菌的最佳表达条件为:37℃培养,0.6mmol/L IPTG诱导4h,培养基中Zn2+终浓度为0.1mmol/L;3)表达工程菌裂解后用60%硫酸铵沉淀,亲和层析和分子筛分离后得到了纯度高达99%的目的蛋白,纯化后的蛋白保持酶活性。本试验成功克隆表达了睾丸酮丛毛单胞菌3α-HSD基因,建立了3α-HSD的高效表达与纯化方法,为相应抗体的制备与水资源中的类固醇激素检测方法的建立提供基础。
3α-HSD gene was amplified and cloned into the expression vector pET-15b. The constructed plasmid was over expressed in the host bacterial of E. coil BL21 after IPTG induction. Expression of 3α-HSD was optimized by bacterial culture temperature,concentration of IPTG and induction time. The engineering bacteria was lysed, and purified by ammounium sulfate precipitation, Ni-NTA affinity purification and molecular sieve separation. Enzymatic activity of expressed 3α-HSD was detected. Comamonas testosteroni 3α-HSD gene was cloned and expressed in the prokaryotic system. To reach the optimal expression, the recombinant E. coil need to be cultured at 37 ℃ and induced by 0.6 mmol/L IPTG for 4 h,while the medium containing 0.1 mmol/L Zn2＋ . The protein was purified through 60% ammonium sulfate precipitation, Ni- NTA affinity chromatography, and molecular sieve. A target protein with 99% purity was obtained which keeping enzyme activity. 3a-HSD gene was cloned and highly expressed. It will build the foundation of antibody preparation and steroid detection in the water resource.
出处
《中国农业大学学报》
CAS
CSCD
北大核心
2013年第1期142-146,共5页
Journal of China Agricultural University
基金
质检公益性行业科研专项(201110018)
作者简介
第一作者：肖琳，硕士研究生，E—mail：xiaolin871018@163．com
通讯作者：薛强，副研究员，博士，主要从事检验检测新方法开发研究，E-mail：xueq@caiq．gov.cn.
