鼠伤寒沙门菌转录调控因子HilD的蛋白纯化及与hilA启动子DNA复合物晶体培养  

Purification of transcription regulator HilD protein from Salmonella typhimurium and complex crystal culture with hilA promoter DNA

作　　者：刘晴 谢荣现 范丙乾 李冰清 贾海红 LIU Qing;XIE Rongxian;FAN Bingqian;LI Bingqing;JIA Haihong(School of Clinical and Basic Medicine&Institute of Basic Medicine,Shandong First Medical University&Shandong Academy of Medical Sciences,Jinan 250062,China)

机构地区：[1]山东第一医科大学(山东省医学科学院)临床与基础医学院(基础医学研究所),山东济南250062

出　　处：《中国病原生物学杂志》2024年第3期270-274,共5页Journal of Pathogen Biology

基　　金：国家自然科学基金项目(No.31900124,32170034)。

摘　　要：目的本研究将获取鼠伤寒沙门菌毒力转录调控因子HilD的蛋白,培养HilD蛋白晶体,为进一步分析HilD功能机制和靶向胞内沙门菌药物研发奠定基础。方法以鼠伤寒沙门菌转录调控因子HilD生物信息学分析为基础,使用鼠伤寒沙门菌14028S菌株作为模板进行分子克隆,构建hilD-pGl01重组质粒,使用原核体系表达HilD,通过镍柱亲和层析柱和透析获取纯化蛋白。EMSA实验验证HilD活性。使用晶体培养试剂盒进行晶体培养。结果成功构建了HilD原核表达系统,HilD是一种碱性蛋白,在蛋白提取过程中需要将缓冲液的pH值维持在7.0左右以维持HilD蛋白的稳定性,还需添加2%甘油和2%蔗糖进行保护。hilA能够稳定HilD蛋白,有利于晶体的生长。结论成功获取HilD纯化蛋白,实验证明具有生物活性,培养出HilD与hilA启动子DNA的复合物晶体,复合物的培养为HilD结构解析奠定了基础。Objective HilD is a transcriptional regulator of Salmonella typhimurium virulence.In this study,we will obtain the protein and culture the crystal structure of HilD,This study laid a foundation for further research on the functional mechanism of HilD and the development of drugs targeting intracellular salmonella.Methods Based on the bioinformatics analysis of the transcriptional regulatory factor HilD of S.typhimurium,the recombinant plasmid hilDpGl01was constructed using S.typhimurium 14028S as a template,and hilD was expressed in prokaryotic system.The purified protein was obtained by nickel affinity chromatography and dialysis.HilD activity was verified by EMSA assay.Crystal culture was performed using a crystal culture kit.Results The prokaryotic expression system of HilD was successfully constructed.HilD is a kind of basic protein,and the pH value of buffer should be maintained at about 7.0 to maintain the stability of HilD protein during protein extraction.In addition,it is necessary to add 2%glycerin and 2%sucrose for protection.hilA can stabilize HilD protein and facilitate crystal growth.Conclusion The purified HilD protein was successfully obtained and proved to be biologically active.The complex crystal of HilD and hilA promoter DNA was cultured.The culture of the complex laid a foundation for the structure analysis of HilD.

关 键 词：鼠伤寒沙门菌 HilD 原核表达 晶体培养 

分 类 号：R378[医药卫生—病原生物学]