基于全基因组的ncRNA 序列分析发掘结核分枝杆菌和巨噬细胞的互作机制

项目名称： 基于全基因组的ncRNA 序列分析发掘结核分枝杆菌和巨噬细胞的互作机制

项目编号： No.31472219

项目类型： 面上项目

立项/批准年度： 2015

项目学科： 水产学、兽医学

项目作者： 郭爱珍

作者单位： 华中农业大学

项目金额： 88万元

中文摘要： 非编码RNA（ncRNA）包括宿主miRNA和病原体sRNA，是一类发挥转录后调控的小RNA，其在感染和抗感染斗争中的作用是一个新型领域，感染状态下结核分枝杆菌（MTB）sRNA研究才起步。本研究利用MTB/BCG的巨噬细胞感染模型，从sRNA和miRNA鉴定、ncRNA和mRNA互作分析角度揭示MTB与巨噬细胞的作用机制。分别对感染体系中的细菌（MTB、BCG）和巨噬细胞进行mRNA和小RNA高通量测序、对mRNA和sRNA进行整合性分析，推测MTB-巨噬细胞全局性互作机制；利用qRT-PCR、Northern blot等验证序列，选择与持续感染和免疫逃避相关的1-2种miRNA和sRNA，利用过表达、抑制和共表达等手段，确定其在感染和抗感染中的作用；检测结核病人血清中的miRNA/sRNA，验证其与MTB感染发病的相关性。结果对阐明MTB致病机制和发现新型药物疫苗诊断靶标具有重要意义。

中文关键词： 结核分枝杆菌；非编码RNA；转录组；致病机理；相互作用

英文摘要： Non-coding RNA(ncRNA)includes host miRNA and pathogen sRNA. It is a group of small RNAs which play a regulatory role posttranscription. Their role in pathogen infection and host anti-infection has launched a new area for study. The research on sRNA of Mycobacterium tuberculosis(MTB) and BCG during infection has just begun.This project is aimed to discover genome-wide ncRNA of MTB and macrophage and investigate the association between mRNA and ncRNA to excavate new mechanism underlying interaction between MTB and macrophage. At different timepoints after infection, the mRNAs and sRNA of the bacilli (MTB,BCG) and host cells were sequenced with RNA-seq. The integrated analysis of mRNA-sRNA for both bacilli and macrophage will be performed, and the global interaction mechanism of MTB and macrophages will be deduced. The sequences of ncRNAs (miNRAs and sRNAs) and mRNA of bacilli and host cells will be verified by qRT-PCR, Northern blot, and RNA in situ hybridization. One or two transcripts from each group of miRNAs and sRNAs potentially related to MTB persistent infection and immune evasion are selected to determine their functions. The techniques including overexpression, inhibition of expression by inhibitors or gene deletion, and co-expression of ncRNA and their deduced target genes (fragment covering interaction sites) will be used, and their parameters such as bacterial growth properties, expression of target genes, response effect on infection and anti-infection will be detected. Furthermore, the presence of miRNAs/sRNAs will be checked in sera of tuberculosis (TB) patients and the association between certain miRNAs/sRNAs and TB development or MTB persistence will be determined. In conclusion,the results will play an important role in elucidating the mechanism of MTB pathogenesis, and provide potential targets for development of novel new drugs, vaccines and diagnotic techniques for tuberculosis prevention and treatment.

英文关键词： Mycobacterium tuberculosis;non-coding RNA;transcriptom;pathogenesis;interaction