干扰素调节因子7对人类8型疱疹病毒基因表达的影响

发布时间：2018-06-21 01:14

本文选题：人类8型疱疹病毒 + 复制与转录激活蛋白　；参考：《天津医科大学》2009年博士论文

【摘要】： 人类8型疱疹病毒(human herpesvirus 8，HHV-8)可导致卡波西肉瘤及多种肿瘤，是最常见的艾滋病相关病毒之一。随着艾滋病人的日益增多，HHV-8的研究越来越受到重视。HHV-8感染有潜伏和裂解两个状态，由其ORF50基因编码的复制与转录激活蛋白(replication and transcription activator，RTA)的表达为病毒重激活所必需，并足以完成HHV-8从潜伏态到裂解态的转换。RTA能与靶基因启动子上的RTA应答元件(RTA response element，RRE)序列直接结合，激活靶基因的转录表达，也能通过与细胞内蛋白的相互作用间接与靶序列结合，激活下游基因的表达。干扰素是治疗病毒性疾病的常用药物之一，其在体内的表达主要受干扰素调节因子(interferon regulatory factors，IRFs)调控。干扰素调节因子7(interferon regulatoryfactors 7，IRF-7)是IRFs家族中的一个重要成员，，能控制Ⅰ型干扰素依赖的免疫反应。病毒感染细胞后，IRF-7将被磷酸化入细胞核，启动下游靶基因的转录，发挥抗病毒效应。IRF-7还可直接作用于病毒启动子，抑制病毒的激活。研究表明，IRF-7可以竞争性结合HHV-8 ORF57启动子上的RTA结合位点，从而抑制RTA激活ORF57启动子。但IRF-7是否可以抑制RTA激活其它启动子，影响病毒基因表达目前尚未有报道。本研究表达和纯化了RTA、ORF57、IRF-7和GST蛋白，并制备其多克隆抗体。蛋白免疫印迹显示所制备的多克隆抗体具有较高的滴度和良好的特异性。将IRF-7与RTA的表达质粒和ORF57报告质粒共转染293T细胞，观察到IRF-7能抑制RTA对ORF57启动子的激活功能，且抑制作用呈现出剂量依赖效应。将IRF-7与RTA的表达质粒和PAN报告质粒共转染293T细胞，发现IRF-7能抑制RTA对PAN启动子的激活功能，且抑制作用呈现出剂量依赖效应。将野生性IRF-7和赖氨酸突变型IRF-7分别与RTA的表达质粒和ORF57报告质粒共转染293T细胞，发现不同赖氨酸突变IRF-7抑制RTA激活ORF57启动子的效果不同。将野生型IRF-7和赖氨酸突变型IRF-7分别与RTA的表达质粒和PAN报告质粒共转染293T细胞，发现不同赖氨酸突变型IRF-7抑制RTA对PAN启动子的激活功能也不同；而且不同突变对RTA激活ORF57与PAN启动子的抑制作用的趋势也不尽相同。我们还将RTA以及IRF-7的真核表达质粒共转染293T细胞，利用所制备的抗体通过蛋白质免疫印迹实验技术，发现RTA能减少IRF-7的蛋白产量。本研究表明IRF-7除能抑制HHV-8 RTA激活ORF57之外，还可以抑制RTA对PAN等基因的激活；某些赖氨酸点突变后改变IRF-7的修饰使其对RTA激活功能的抑制作用也随之改变。本文还为探讨RTA与IRF-7的相互作用积累了前期材料，为HHV-8相关疾病的预防和治疗提供新的理论依据。
[Abstract]:Human herpesvirus 8 (HHV-8) can cause Kaposi's sarcoma and many kinds of tumors, and is one of the most common AIDS-related viruses. With the increasing number of AIDS patients, more and more attention has been paid to the study of HHV-8 infection. HHV-8 infection has both latent and lytic states. The expression of replication and transcription activator RTAs encoded by its ORF50 gene is necessary for virus reactivation. HHV-8 can be transformed from latent state to lytic state. RTA can directly bind to the RTA response element RRER sequence on the target gene promoter and activate the transcription expression of the target gene. The expression of downstream genes can also be activated by indirect binding with target sequences through interaction with intracellular proteins. Interferon is one of the commonly used drugs for the treatment of viral diseases. Its expression in vivo is mainly regulated by interferon regulatory factors (IRFs). Interferon regulatory factor 7(interferon regulatoryfactors 7 (IRF-7) is an important member of the IRFs family, which can control the immune response of type I interferon dependent. After virus infection, IRF-7 will be phosphorylated into the nucleus and activate the transcription of downstream target gene. IRF-7 can also directly act on the virus promoter and inhibit the activation of the virus. It has been shown that IRF-7 can competitively bind to the RTA binding site on the HHV-8 ORF57 promoter, thereby inhibiting RTA activation of the ORF57 promoter. However, whether IRF-7 can inhibit RTA activation of other promoters has not been reported. In this study, RTAF57 IRF-7 and GST proteins were expressed and purified, and their polyclonal antibodies were prepared. Western blot showed that the polyclonal antibody had high titer and good specificity. IRF-7 and RTA expression plasmid and ORF57 reporter plasmid were co-transfected into 293T cells. It was observed that IRF-7 could inhibit the activation of ORF57 promoter by RTA, and the inhibitory effect was dose-dependent. The expression plasmids of IRF-7 and RTA and pan reporter plasmids were co-transfected into 293T cells. It was found that IRF-7 could inhibit the activation of pan promoter by RTA in a dose-dependent manner. Wild IRF-7 and Lysine mutant IRF-7 were co-transfected with RTA expression plasmid and ORF57 reporter plasmid into 293T cells. It was found that different Lysine mutant IRF-7 inhibited RTA activation of ORF57 promoter. Wild type IRF-7 and lysine mutant IRF-7 were co-transfected with RTA expression plasmid and pan reporter plasmid into 293T cells. It was found that different lysine mutant IRF-7 inhibited the activation of pan promoter by RTA. The inhibitory effects of different mutations on RTA activated ORF57 and pan promoters were also different. We also cotransfected 293T cells with RTA and IRF-7 eukaryotic expression plasmids. By Western blot, we found that RTA can reduce the protein production of IRF-7. In addition to inhibiting the activation of ORF57 by HHV-8 RTA, IRF-7 can also inhibit the activation of pan and other genes by RTA, and the modification of IRF-7 after some Lysine point mutations changes the inhibitory effect of IRF-7 on the activation of RTA. This paper also provides a new theoretical basis for the prevention and treatment of HHV-8 related diseases by accumulating materials for the study of the interaction between RTA and IRF-7.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R373

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