建立基于RNA免疫共沉淀技术的鼠疫耶尔森菌Hfq蛋白相关sRNA的体内验证方法

摘要

Objective: To establish the RNA-binding immunoprecipitation(RIP) method for identification of Yer-sinia pestis small non-coding RNA(sRNA) associated with Hfq protein in vivo. Methods: The Flag peptide-coding gene was fused downstream of the hfq gene. These method involve immunoprecipitation of endogenously formed complexes of RNA-binding protein Hfq by the antibody of Flag and isolation of RNA associated with Hfq protein. Every part of Hfq protein was confirmed by Western blot, then the interest sRNA, RyhB1 and RyhB2, were detect-ed by Northern blot. Results: The plasmid of RNA-binding protein Hfq with the Flag was successfully construct-ed, this carrier transformed into hfq deletion mutant did not have any significant difference in the growth curve of wild-type Y.pestis. Two Hfq-binding sRNA, RyhB1 and RyhB2, were identified in Y.pestis by RNA-binding protein immmunoprecipitation. Conlusion: RNA-binding protein immmunoprec ipitation has been established for identifica-tion of Y.pestis sRNA-binding Hfq protein, which was a high sensitivity method and provided an powerful tool for validation of bacterial sRNA, function studies and protein interaction with RNA studies in vivo.%目的：：建立RNA免疫共沉淀方法，为鼠疫耶尔森菌Hfq蛋白相关非编码小RNA（sRNA）提供体内验证方法。方法：首先在RNA结合蛋白Hfq下游加入Flag标签，用Flag标签抗体进行免疫共沉淀，获得蛋白-RNA复合物，然后从沉淀的蛋白-RNA复合物中分离得到纯化的RNA；通过Western印迹检测各步骤Hfq蛋白的表达，再利用Northern印迹检测目的sRNA--RyhB1和RyhB2。结果：构建了带有Flag标签的RNA结合蛋白Hfq的载体，此载体转导入hfq缺失株后与鼠疫菌野生株的生长曲线无明显差异；通过RNA-蛋白免疫共沉淀技术鉴定出已知与鼠疫菌Hfq蛋白结合的2个sRNA--RyhB1和RyhB2。结论：建立了利用RNA-蛋白免疫共沉淀鉴定与鼠疫菌Hfq蛋白结合的sRNA的技术，为细菌sRNA的验证、功能研究和体内蛋白质与RNA相互作用研究提供了有利工具。