摘要
为了验证rv3229c是否为结核分枝杆菌(MTB)的转录调控蛋白Rv3295的调控靶点,并研究哪些小分子对rv3229c与Rv3295蛋白的结合有影响,本研究通过PCR扩增MTB rv3295基因后构建了重组表达载体pET-22b-rv3295,经诱导后得到重组蛋白Rv3295。凝胶迁移试验(EMSA)和染色质免疫沉淀(ChIP)试验结果显示,Rv3295与rv3229c(desA3)启动子在体外与体内均能够结合。DNA大沟小沟结合试验表明,Rv3295与rv3229c DNA的大沟结合。通过EMSA小分子筛选,结果显示,小分子(VB1、VB3、VB6、VC)和金属离子(Fe3+和Zn2+)在体外均可降低Rv3295与rv3229c启动子区域的结合,而氨基酸及其它小分子对该结合无影响。分子对接试验表明Rv3295的Glu 67和Glu 105氨基酸残基在该蛋白质与小分子及金属离子的相互作用中起重要作用。以上试验结果表明rv3229c为TetR家族蛋白Rv3295新的调控靶点。本研究首次揭示了MTB中的Rv3295调控蛋白可直接调控rv3229c(desA3)的表达,进而参与油酸合成的调控。
In order to verify whether rv3229c is the regulatory target of transcriptional regulatory protein Rv3295 and screening which small molecules have an effect on the binding of rv3229c to Rv3295 protein.In this study,the expression vector pET-22b-rv3295 was constructed by PCR amplification of the rv3295 gene,and the recombinant protein Rv3295 was expressed and purified.We found that Rv3295 bound to the promoter of rv3229c(desA3)both in vitro and in vivo using electrophoretic mobility shift assays(EMSA)and Chromatin immunoprecipitation(ChIP)assays.DNA major-minor groove binding test show that Rv3295 bind to DNA major groove.After EMSA screening,small molecules VB1,VB3,VB6,VC,and metal irons Fe3+and Zn2+decreases the binding affinity between Rv3295 and the rv3229c promoter region,however,amino acids and other tested small molecules did not have an impact on binding.Additionally,molecular docking predicted that residues 67(Glu)and 105(Glu)of Rv3295 could play an important role in protein interactions with small molecules and metal irons.The above results indicate that rv3229c is a new regulatory target of TetR family protein Rv3295.This study firstly indicated that Rv3295 in MTB directly regulate rv3229c(desA3)expression and oleic acid biosynthesis.
作者
吕明月
崔莹莹
唐祎依
党光辉
崔子寅
刘思国
宋宁宁
LYU Ming-yue;CUI Ying-ying;TANG Yi-yi;DANG Guang-hui;CUI Zi-yin;LIU Si-guo;SONG Ning-ning(College of Animal Science and Veterinary Medicine,Heilongjiang Bayi Agricultural University,Daqing 163000,China;State Key Laboratory of Veterinary Biotechnology,Harbin Veterinary Research Institute,ChineseAcademy ofAgricultural Sciences,Harbin 150069,China)
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2020年第7期643-649,共7页
Chinese Journal of Preventive Veterinary Medicine
基金
国家重点研发计划(2017YFD0500906、2017YFD0500304)
国家自然科学基金(31873014、31772767)
黑龙江省青年科学基金(QC2018034)。
