miRNAs在基于TLR5配体亚单位疫苗免疫应答中的调控作用研究

Although the TLR5 ligand-based subunit vaccines have a solid immune effect and the immune response mechanism have been analyzed preliminarily, but the underlying regulatory mechanisms remain unclear. MicroRNAs (miRNAs) are a class of endogenous non-coding small RNAs which play an important role in the regulation when TLR ligands activate dendritic cells (DCs). On the basis of previous work, a swine-origin influenza subunit vaccine candidate, a recombinant protein HA1-2-FljB is chosen to induce the mouse BMDCs activation related miRNAs through miRNA microarray screening and the miRNAs targeted molecules based on the TLR5/MyD88 signaling pathway is identified, therefore, the molecular basis of its function will be explained. In addition, the effect of miRNAs in the process of DCs activation when the cells are stimulated with the recombinant protein will be evaluated in vitro. Also in vivo, DCs-targeted recombinant adenovirus constructs are designed to analyze the influence of miRNAs in innate and acquired immune response to the recombinant protein. The objective of this study is to demonstrate that miRNAs regulate the DC's activation when stimulated with the recombinant protein, and to verify the hypothesis that miRNAs play a regulatory role in the immune response. Thus, herein we provide a new theory and approach to design the novel TLR5 ligand-based immune adjuvant molecules and genetic engineering vaccines.

基于TLR5配体的亚单位疫苗免疫效应确实，其免疫应答机理亦获得初步解析，但该类疫苗免疫应答的调控机制尚不清楚。而microRNAs(miRNAs)对TLR配体活化树突状细胞（DCs）具有调节功能。本项目在前期工作的基础上，选用猪源流感亚单位疫苗候选株HA1-2-FljB这一重组融合蛋白作为研究对象，拟通过miRNA microarray 筛选出重组蛋白活化小鼠BMDCs相关的miRNAs；以TLR5/MyD88信号通路为基础，鉴定miRNAs的靶标分子；通过体外试验，评价miRNAs在重组蛋白活化DCs中的功能；构建靶向DCs的重组腺病毒，以体内试验分析miRNAs对重组蛋白固有免疫和获得性免疫应答的影响，以论证miRNAs通过对重组蛋白活化DCs的调节，进而对免疫应答具有调控作用的假说，并揭示其作用的分子基础，以期为基于TLR5配体的高效基因工程疫苗研制提供新理论和新途径。

基于TLR5配体的亚单位疫苗免疫效应确实，其免疫应答机理亦获得初步解析，但该类疫苗免疫应答的调控机制尚不清楚。而microRNAs （miRNAs）对TLR配体活化树突状细胞（DCs）具有调节功能。本项目在前期工作的基础上，利用miRNA microarray 筛选出鞭毛蛋白活化小鼠BMDCs过程中相关差异的miRNAs；经qRT-PCR验证鞭毛蛋白活化BMDCs过程中共有2个差异表达的miRNA，mmu-miR-193-5p和mmu-miR-5112，其均为下调表达。选取变化差异最显著的miR-5112为研究对象。通过体内外实验均证明了鞭毛蛋白在活化小鼠脾脏DCs过程中，miR-5112的表达水平发生下调。利用miR-5112 模拟物通过体内外实验均证明了miR-5112可以抑制鞭毛蛋白诱导的炎性因子的分泌。以TLR5/MyD88信号通路为基础，通过生物信息学方法和荧光报告载体实验证明了miR-5112的靶基因为IKKγ，揭示了miR-5112作用的分子基础；表明miR-5112可以通过靶向抑制IKKγ的表达间接调控鞭毛蛋白诱导的炎性反应。以鞭毛蛋白与OVA联合免疫小鼠，miR-5112降低了鞭毛蛋白诱发的炎性反应，且可调控鞭毛蛋白的佐剂效应，为安全有效的鞭毛蛋白佐剂（低炎性损伤高佐剂活性鞭毛蛋白）的获得提供了新的思路和方法。此外，以链霉素预处理的小鼠为模型，发现了miR-5112也可抑制沙门菌诱导的炎性反应，延缓沙门菌诱导的死亡；以DSS诱导的结肠炎小鼠为模型，证实了miR-5112可减缓DSS诱导的结肠炎，降低其炎性应答水平；显示miR-5112作为炎性应答的重要调节因子，可为炎性疾病的治疗提供新的思路和靶点。