利用MS2识别序列标签纯化技术富集猪链球菌小RNA结合蛋白  

A purification technique for adding MS2 recognition sequence tag to RNA to enrich Streptococcus suis small RNA binding proteins

作　　者：张寿明 唐欢宇[1] 吴宗福[1] ZHANG Shouming;TANG Huanyu;WU Zongfu(College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China)

机构地区：[1]南京农业大学动物医学院

出　　处：《畜牧与兽医》2018年第6期68-73,共6页Animal Husbandry & Veterinary Medicine

基　　金：国家自然科学基金项目(31572544)

摘　　要：为建立理想的猪链球菌RNA结合蛋白筛选方法,以该菌小RNA rss04和rss06为研究对象,通过纯化MS2序列识别蛋白MS2-MBP,构建表达MS2融合RNA（MS2-rss04与MS2-rss06）的质粒p SET2-MS2-rss04、pSET2-MS2-rss06及阴性对照质粒p SET2-MS2-negative,将上述质粒分别电转化至猪链球菌小RNA缺失株Δrss04、Δrss06及野生株P1/7,通过MS2识别序列标签纯化,收集洗脱液,提取RNA。结果显示：表达MS2融合RNA的样品,rss04和rss06分别富集16倍和110倍,而阴性对照组未检测到rss04和rss06的富集。结果表明：MS2识别序列纯化体系成功建立,可用于猪链球菌RNA结合蛋白的筛选,为深入研究猪链球菌小RNA功能提供技术平台。Bacterial RNA-binding proteins not only play a key role in regulation of small RNAs,but also participate in regulation of bacterial virulence. However,reports on identification and function of bacterial RNA-binding proteins are rare in gram-positive bacteria. In order to establish an ideal method for screening Streptococcus suis RNA-binding proteins,S. suis small RNAs rss04 and rss06 were selected. The MS2-MBP that binds to the MS2 sequence was purified; the plasmids p SET2-MS2-rss04 and pSET2-MS2-rss06,expressing the fusion RNAs MS2-rss04 and MS2-rss06 respectively,as well as the negative control plasmid pSET2-MS2-negative,were constructed and then electroporated into the S. suis small RNA deletion mutants Δrss04,Δrss06,and wild strain P1/7,respectively. Then,the MS2 recognition sequence tag purification technology was used,and RNA was extracted from eluent. Quantitative real-time PCR results showed that rss04 and rss06 were significantly enriched by 16 folds and 110 folds,respectively,expressing fusion RNAs in the samples,whereas rss04 and rss06 were not enriched in the negative control group. The above results indicated that the MS2 purification system was successfully established and could be used for screening S. suis RNA-binding proteins,providing a technical platform for in-depth study on the function of S. suis small RNA.

关 键 词：猪链球菌 RNA结合蛋白 小RNA MS2标签 

分 类 号：S852.6[农业科学—基础兽医学]