本文選題：人類8型皰疹病毒 + 復制與轉錄激活蛋白　； 參考：《天津醫(yī)科大學》2009年博士論文

【摘要】： 人類8型皰疹病毒(human herpesvirus 8，HHV-8)可導致卡波西肉瘤及多種腫瘤，是最常見的艾滋病相關病毒之一。隨著艾滋病人的日益增多，HHV-8的研究越來越受到重視。HHV-8感染有潛伏和裂解兩個狀態(tài)，由其ORF50基因編碼的復制與轉錄激活蛋白(replication and transcription activator，RTA)的表達為病毒重激活所必需，并足以完成HHV-8從潛伏態(tài)到裂解態(tài)的轉換。RTA能與靶基因啟動子上的RTA應答元件(RTA response element，RRE)序列直接結合，激活靶基因的轉錄表達，也能通過與細胞內蛋白的相互作用間接與靶序列結合，激活下游基因的表達。干擾素是治療病毒性疾病的常用藥物之一，其在體內的表達主要受干擾素調節(jié)因子(interferon regulatory factors，IRFs)調控。干擾素調節(jié)因子7(interferon regulatoryfactors 7，IRF-7)是IRFs家族中的一個重要成員，，能控制Ⅰ型干擾素依賴的免疫反應。病毒感染細胞后，IRF-7將被磷酸化入細胞核，啟動下游靶基因的轉錄，發(fā)揮抗病毒效應。IRF-7還可直接作用于病毒啟動子，抑制病毒的激活。研究表明，IRF-7可以競爭性結合HHV-8 ORF57啟動子上的RTA結合位點，從而抑制RTA激活ORF57啟動子。但IRF-7是否可以抑制RTA激活其它啟動子，影響病毒基因表達目前尚未有報道。 本研究表達和純化了RTA、ORF57、IRF-7和GST蛋白，并制備其多克隆抗體。蛋白免疫印跡顯示所制備的多克隆抗體具有較高的滴度和良好的特異性。將IRF-7與RTA的表達質粒和ORF57報告質粒共轉染293T細胞，觀察到IRF-7能抑制RTA對ORF57啟動子的激活功能，且抑制作用呈現(xiàn)出劑量依賴效應。將IRF-7與RTA的表達質粒和PAN報告質粒共轉染293T細胞，發(fā)現(xiàn)IRF-7能抑制RTA對PAN啟動子的激活功能，且抑制作用呈現(xiàn)出劑量依賴效應。將野生性IRF-7和賴氨酸突變型IRF-7分別與RTA的表達質粒和ORF57報告質粒共轉染293T細胞，發(fā)現(xiàn)不同賴氨酸突變IRF-7抑制RTA激活ORF57啟動子的效果不同。將野生型IRF-7和賴氨酸突變型IRF-7分別與RTA的表達質粒和PAN報告質粒共轉染293T細胞，發(fā)現(xiàn)不同賴氨酸突變型IRF-7抑制RTA對PAN啟動子的激活功能也不同；而且不同突變對RTA激活ORF57與PAN啟動子的抑制作用的趨勢也不盡相同。我們還將RTA以及IRF-7的真核表達質粒共轉染293T細胞，利用所制備的抗體通過蛋白質免疫印跡實驗技術，發(fā)現(xiàn)RTA能減少IRF-7的蛋白產量。 本研究表明IRF-7除能抑制HHV-8 RTA激活ORF57之外，還可以抑制RTA對PAN等基因的激活；某些賴氨酸點突變后改變IRF-7的修飾使其對RTA激活功能的抑制作用也隨之改變。本文還為探討RTA與IRF-7的相互作用積累了前期材料，為HHV-8相關疾病的預防和治療提供新的理論依據(jù)。
[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.
【學位授予單位】：天津醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2009
【分類號】：R373

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