CD44对口蹄疫病毒感染的影响

发布时间：2018-03-09 11:01

本文选题：口蹄疫病毒　切入点：CD44　出处：《长江大学》2017年硕士论文　论文类型：学位论文

【摘要】：口蹄疫(foot-and-mouth disease,FMD)是由口蹄疫病毒(foot-and-mouth disease virus,FMDV)引发的能够感染偶蹄动物的一种高致病性传染病。该病在全球范围内流行,对畜牧业造成巨大的经济损失。研究FMDV的致病机理,对于开发新型有效的FMD防控方法具有重要作用。本课题前期利用SILAC定量蛋白质组学方法,发现在FMDV感染早期,CD44的表达量显著上调。为了进一步研究CD44在FMDV感染中的作用机理,本研究主要做了以下几方面的工作:为确定CD44对FMDV感染的影响,本研究利用Asia1型FMDV感染BHK-21细胞,利用Western blot和RT-PCR方法检测内源性CD44的表达变化,并利用激光共聚焦显微镜观察CD44与FMDV结构蛋白的共定位,结果发现FMDV感染能促进细胞内源性CD44的表达,并且激光共聚焦显微镜观察发现FMDV可以与细胞膜表面的CD44发生共定位。为了进一步研究FMDV与CD44的关系,本研究利用MβCD抑制剂和siRNA干扰CD44的表达,利用Western blot、TCID50和RT-PCR方法分别检测FDMV结构蛋白表达、病毒滴度变化和病毒基因组拷贝变化,发现抑制CD44表达能够抑制FMDV复制,此外,利用构建真核表达载体过表达CD44后,利用Western blot、TCID50和RT-PCR方法检测发现,过表达CD44能够促进FMDV增殖,这些结果表明CD44参与FMDV的感染过程。先前研究表明,FMDV利用巨胞饮途径进入宿主细胞,FMDV入侵细胞早期,首先结合并激活受体酪氨酸激酶RTKs,RTKs活化后,可激活细胞内的肌动蛋白调节蛋白和其他一些蛋白因子,包括p-αPAK、FGF、FLT3、Rac1,导致细胞内的肌动蛋白发生瞬时性解聚,并在细胞膜表面形成皱褶。为了研究CD44是否参与FMDV的巨胞饮作用,本研究将CD44干扰或过表达,利用Western blot方法研究发现p-αPAK、FGF、FLT3、P-Rac1、Rac1蛋白的表达变化,结果发现,CD44促进p-αPAK、FGF、FLT3、P-Rac1、Rac1蛋白的表达,表明CD44表达与p-αPAK、FGF、FLT3、P-Rac1、Rac1蛋白的表达具有协同作用。为了进一步验证CD44是否参与RTKs通路,通过巨胞饮途径感染宿主细胞,本研究利用特异性的RTKs抑制剂genistein(Gen)处理BHK-21细胞,Western blot检测CD44的表达变化,结果表明CD44参与RTKs通路,通过巨胞饮途径感染宿主细胞。总之,本研究通过探索CD44在口蹄疫病毒感染中的作用,进一步揭示了FMDV的感染机制,为开发阻断FMDV入侵的药物和高效疫苗提供新靶点。
[Abstract]:Foot-and-mouth disease (FMDV) is a highly pathogenic infectious disease caused by foot-and-mouth disease virus (FMDV). The disease is prevalent in the world and causes huge economic losses to animal husbandry. It was found that the expression of CD44 was significantly up-regulated in the early stage of FMDV infection by using SILAC quantitative proteomics method. In order to further study the mechanism of CD44 in FMDV infection, it was found that the expression of CD44 was significantly up-regulated in the early stage of FMDV infection. In order to determine the effect of CD44 on FMDV infection, we used Asia1 type FMDV to infect BHK-21 cells, and used Western blot and RT-PCR methods to detect the expression of endogenous CD44. The co-localization of CD44 and FMDV structural proteins was observed by laser confocal microscopy. The results showed that FMDV infection could promote the expression of endogenous CD44. In order to further study the relationship between FMDV and CD44, we used M 尾 CD inhibitor and siRNA to interfere the expression of CD44. The expression of FDMV structural protein, viral titer and viral genome copy changes were detected by Western blottit TCID50 and RT-PCR, respectively. It was found that inhibition of CD44 expression could inhibit FMDV replication. In addition, eukaryotic expression vector was used to express CD44. Using Western blottir TCID50 and RT-PCR methods, it was found that overexpression of CD44 could promote the proliferation of FMDV, which suggested that CD44 was involved in the process of FMDV infection. After the activation of the receptor tyrosine kinase, RTKsN RTKs, actin regulatory proteins and other protein factors, including p- 伪 PAKN FGFFGFFLT3 Rac1, were activated, which led to transient depolymerization of actin in cells. In order to study whether CD44 is involved in the giant cell drink of FMDV, CD44 was interfered or overexpressed by Western blot method. The expression of p- 伪 PAKN FGFFGF3, P-Rac1Rac1 was detected by Western blot method. The results showed that CD44 promoted the expression of p- 伪 PAKT FGFFLT3P-Rac1Rac1 protein, and the expression of pPAK- 伪 PAKG FGFFLT3P-Rac1Rac1 protein was also detected by Western blot method, and the results showed that p- 伪 PAKT FGFFLT3P-Rac1Rac1 protein was increased. The results indicated that the expression of CD44 was synergistic with the expression of p- 伪 PAKGF-FLT3FLT3, P-Rac1Rac1 protein. In order to further verify whether CD44 participated in the RTKs pathway, the host cells were infected by the megalocytic pathway. In this study, BHK-21 cells were treated with specific RTKs inhibitor genistein Genin to detect the expression of CD44 by Western blot. The results showed that CD44 participated in the RTKs pathway and infected host cells through the megalocytic pathway. By exploring the role of CD44 in the infection of foot-and-mouth disease virus (FMDV), this study further revealed the mechanism of FMDV infection, and provided a new target for the development of drugs to block the invasion of FMDV and efficient vaccine.
【学位授予单位】：长江大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.65

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