摘要
目的:建立副溶血弧菌QsvR的染色质免疫共沉淀实时定量PCR(ChIP-qPCR)实验方法。方法:通过热激转化和转化结合将标记2×Flag标签的qsvR片段转入qsvR突变株(ΔqsvR)中,阿拉伯糖诱导表达QsvR-2×Flag蛋白,通过甲醛与基因组DNA进行交联形成2×Flag-QsvR-DNA复合物,将基因组DNA超声裂解为100~1000 bp大小不等的片段,通过特异性抗原抗体反应将蛋白质-DNA复合物沉淀下来,高盐和加热解交联,回收DNA进行qPCR定量DNA,分析副溶血弧菌体内QsvR与靶基因的结合情况。结果:成功构建出实验菌株ΔqsvR/pBAD33-qsvR-2×Flag,以不加阿拉伯糖诱导为对照,经阿拉伯糖诱导菌株的aphA、opaR、exsB和vtrA的免疫共沉淀量明显较高,表明在副溶血弧菌体内QsvR与aphA、opaR、exsB和vtrA具有结合作用。结论:成功建立了副溶血弧菌QsvR的ChIP-qPCR实验方法,可用于原核生物体内蛋白质-DNA相互作用的研究。
Objective:To establish the chromatin immunocoprecipitation-real-time quantitative PCR(ChIP-qPCR)assay for Vibrio parahemolyticus QsvR.Methods:The QsvR fragments labeled with 2×Flag tag were transferred intoΔqsvR by heat shock transformation and transformation binding.Arabinose induced the expression of QsvR-2×Flag protein,which was crosslinked with genomic DNA through formaldehyde to form 2×Flag-QsvR-DNA complex.The genomic DNA was ultrasonically cleaved into fragments ranging in size from 100—1000 bp,and the protein-DNA complex was precipitated by specific antigen-antibody reaction.The cross-linked DNA was de-crosslinked by high salt and heating,and the DNA was recovered for qPCR quantitative DNA to analyze the binding of QsvR and target genes in V.parahemolyticus.Results:The experimental strainΔqsvR/pBAD33-qsvR-2×Flag was successfully constructed.The IP expression of aphA,opaR,exsB and vtrA of the strain induced by arabinose was significantly higher than that of the control strain.The results indicated that QsvR could bind aphA,opaR,exsB and vtrA in V.parahaemolyticus.Conclusion:The ChIP-qPCR assay of V.parahemolyticus QsvR was established successfully,which can be used to study protein-DNA interaction in prokaryotes.
作者
张苗苗
李雪
陆仁飞
张义全
周敏
ZHANG Miaomiao;LI Xue;LU Renfei;ZHANG Yiquan;ZHOU Min(Department of Clinical Laboratory,Affiliated Nantong Hospital 3 of Nantong University,Nantong Jiangsu 226006;School of Medicine,Jiangsu University,Zhenjiang Jiangsu 212013;Department of Microbiology,Nantong Center for Disease Control and Prevention,Nantong Jiangsu 226007,China)
出处
《江苏大学学报（医学版）》
CAS
2023年第6期516-520,527,共6页
Journal of Jiangsu University：Medicine Edition
基金
南通市卫生健康委员会科研课题(QA2020026,QN2022044)。
