摘要
目的:利用大肠杆菌BL21(λDE3)的表达系统,表达7个有活性的、与鼠疫耶尔森菌(鼠疫菌)传播及致病密切相关的调控子蛋白,并对这些蛋白与DNA的结合活性进行分析,为构建鼠疫菌毒力基因转录调控网络建立分子生化实验平台。方法:通过分子克隆技术构建鼠疫菌调控子蛋白的表达菌株,所得菌株经IPTG诱导后能分别表达鼠疫菌CRP、Fur、PhoP、OxyR、OmpR、RcsB和RovA带His标签的融合蛋白;对这些蛋白与DNA的结合基序进行生物信息学预测;通过体外凝胶迁移实验验证上述蛋白与靶DNA的结合活性。结果:表达了7种有活性的鼠疫菌调控子蛋白,这些蛋白与靶基因启动子区具有体外结合活性。结论:表达的7种调控子蛋白在鼠疫菌的传播致病中有重要作用,这些调控子蛋白与DNA体外结合实验平台的建立,是构建鼠疫菌毒力基因转录调控网络的基础。
Objective： A total of seven regulator proteins involved in Yersinia pestis pathogenicity and/or transmission were expressed using the Escherichia coli BL21（λDE3） protein expression system,and the DNA-binding activity of them was characterized.Methods： The entire coding region of each of the crp,fur,phoP,oxyR,ompR,rcsB,and rovA genes was amplified by PCR from Y.pestis strain 201,and then cloned into the vector pET28a.The recombinant plasmids were transformed into E.coli BL21（λDE3）,respectivley.Over-expression of His-tagged proteins in the LB medium was induced by addition of 1 mmol/L IPTG.The over-expressed proteins were purified under native conditions with nickel loaded HiTrap Chelating Sepharose columns（Amersham）.Based on computational promoter analysis,the electrophoretic mobility shift assays were carried out to analyze the DNA-binding activity of each His-tagged protein in vitro.Results： The purified His-tagged proteins had the ability to bind to the upstream DNA regions of their target genes of Y.pestis.Conclusion： The purified His-tagged proteins were able to bind to target DNA fragments,suggesting that they would regulate the transcription of relevant genes in Y.pests.
出处
《生物技术通讯》
CAS
2011年第4期468-473,共6页
Letters in Biotechnology
基金
国家自然科学基金(30930001
30900823
30771179)
国家重点基础研究发展计划(2009CB522600)
