产气肠杆菌组成型启动子的筛选及其活性测定

Screening and Identification of Constructive Promoters from the Ge-nome of Enterobacter Aerogenes

目的：从产气肠杆菌基因组中筛选组成型启动子，并检测其活性。方法用Sau3 AⅠ限制性内切酶将产气杆菌的基因组酶切到500 bp大小，连接到pCMR8 a载体上，转化DH5α感受态后涂布氯霉素和卡那抗生素的双抗平板上筛选启动子，经SDS－PAGE分析启动子的蛋白表达能力，筛选较强启动子并对其进行截短和顺反性实验，最后利用截短启动子表达不同的蛋白。结果成功筛选到了5个启动子序列，获得到了表达能力最强启动子的截短核心序列，验证了其具有反向启动外源蛋白表达的能力，成功表达了其他外源蛋白。结论本实验通过大肠埃希菌表达系统成功的筛选并验证了产气杆菌的一种组成型启动子，具有较强的启动外源蛋白表达能力，对工业上蛋白的生产及实验室表达某种蛋白具有重要意义。

Objective To screen constitutive promoters from the genome of Enterobacter aero-genes and to detect their activities.Methods The genome of Enterobacter aerogenes was digested in-to about 500-bp fragments with restriction endonuclease Sau3 A I.The fragments were cloned into pC-MR8a vector and then the constructs were transformed into competent E.coli DH5α.The positive clones were screened by growing on the LB agar plate with both chloramphenicol and kanamycin, and by restriction digestion analysis and DNA sequencing.SDS-PAGE and Western blotting were used to analyze the foreign protein expression levels and the promoter activity.The core region, di-rection and the ability to initiate protein expression was detected in the promoter with the highest ac-tivity.Results Five promoters were successfully obtained from the genome of Enterobacter aero-genes.The truncated core region of the promoter with the highest activity was well characterized.The reverse direction of the promoter was confirmed to have the ability to enhance protein expres-sion.Other foreign proteins were expressed by this promoter in E.coli.Conclusion This study suc-cessfully screened out and verified the constructive promoters from the genome of Enterobacter aero-genes, which have strong abilities of initiating heterologous protein expression.The constructive pro-moters are of great significance in the production of a certain type of protein in the laboratory and protein preparation in the industry.