树突状细胞表面新型热休克蛋白Hsp70L1相关受体的寻找和确证

发布时间：2018-02-16 02:40

本文关键词： 树突状细胞佐剂 GST Pull Down 受体克隆表达 TLR4 CD40 Lox-1 TLR2　出处：《第二军医大学》2009年硕士论文　论文类型：学位论文

【摘要】： Hsp70L1是我们从人树突状细胞(Dendritic cell, DC)cDNA文库中发现的一个新分子,与HSP70家族成员具有高度同源性。重组Hsp70L1蛋白能诱导DC成熟并表达IL-10p70,MIP-1α,MIP-1β,RANTES,TNF-α和IL-1β等细胞因子和趋化因子;同时具有佐剂样效应,增强抗原特异性的细胞免疫应答。本研究旨在发现并鉴定DC表面Hsp70L1蛋白相关受体,通过研究Hsp70L1与相关受体作用的分子机制来阐明其发挥佐剂效应的原理。首先我们表达纯化了GST-Hsp70L1融合蛋白,通过GST Pull-Down技术,寻找能与其结合的DC膜蛋白,Western-Blot结果表明TLR2、TLR4和Lox-1能与Hsp70L1在体外结合;在此基础上,通过受体表达克隆,筛选出稳定表达HSP70蛋白家族受体CD40,CCR5,Lox-1的HEK293细胞表达株,观察与Hsp70L1蛋白的结合情况。结果显示TLR4和CD40能与Hsp70L1结合,CCR5和LOX-1不与HSP70L1结合;随后研究了TLR4与Hsp70L1蛋白之间相互作用的生物学效应,结果表明: Hsp70L1可以诱导野生型小鼠,但不能诱导TLR4缺陷小鼠DC的表型改变和炎性因子TNF-α和IL-1β表达,HSP70L1可以在野生小鼠模型,但不能在TLR4小鼠模型上介导CEA特异性细胞免疫应答增强;采用人TLR4抑制剂可以明显抑制HSP70L1介导的CEA抗原负载的人DC诱导特异细胞免疫应答的能力。HSP70L1与TLR4作用后的信号转导的研究表明其活化了MAPK和NF-κB级联信号通路。从而证实了TLR4是Hsp70L1诱导DC成熟表型改变和炎性因子表达以及诱导特异性的T细胞应答的一个关键的功能受体。这些结果为Hsp70L1的免疫佐剂功能的研究提供了理论基础。
[Abstract]:Hsp70L1 is a novel molecule found in human dendritic cells (DC)cDNA) library, which has high homology with members of HSP70 family. Recombinant Hsp70L1 protein can induce DC maturation and express cytokines such as IL-10p70, MIP-1 伪, MIP-1 尾, RANTESN TNF- 伪 and IL-1 尾. The aim of this study was to identify and identify the receptor associated with Hsp70L1 protein on the surface of DC, and to enhance the antigen-specific cellular immune response. By studying the molecular mechanism of the interaction of Hsp70L1 with related receptors, the mechanism of its adjuvant effect is elucidated. Firstly, we express and purify the GST-Hsp70L1 fusion protein, and then we express and purify the GST-Hsp70L1 fusion protein by GST Pull-Down technique. The results of Western-Blot showed that TLR2TLR4 and Lox-1 could bind to Hsp70L1 in vitro, and on this basis, the HEK293 cell lines expressing HSP70 family receptor CD40, CCR5, Lox-1, were screened through receptor cloning, and the results showed that TLR2TLR4 and TLR4 could bind to Hsp70L1 in vitro. The results showed that TLR4 and CD40 could bind to Hsp70L1, CCR5 and LOX-1 could not bind to HSP70L1. The biological effects of TLR4 interaction with Hsp70L1 protein were studied. The results showed that Hsp70L1 could induce wild-type mice. However, the phenotypic changes of DC and the expression of TNF- 伪 and IL-1 尾 in TLR4 deficient mice could not be induced by HSP70L1 in wild mice, but the CEA specific cellular immune response could not be enhanced in TLR4 mice. Human TLR4 inhibitor could significantly inhibit the ability of HSP70L1 mediated CEA antigen-loaded human DC to induce specific cellular immune response. Studies on the signal transduction of HSP70L1 interacting with TLR4 showed that HSP70L1 activated MAPK and NF- 魏 B cascade signaling pathway. It is confirmed that TLR4 is a key functional receptor for Hsp70L1 induced mature phenotypic changes, inflammatory factor expression and specific T cell response of DC. These results provide a theoretical basis for the study of the immune adjuvant function of Hsp70L1.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392

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