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

【摘要】： 人類8型皰疹病毒(human herpesvirus 8，HHV-8)可導(dǎo)致卡波西肉瘤及多種腫瘤，是最常見(jiàn)的艾滋病相關(guān)病毒之一。隨著艾滋病人的日益增多，HHV-8的研究越來(lái)越受到重視。HHV-8感染有潛伏和裂解兩個(gè)狀態(tài)，由其ORF50基因編碼的復(fù)制與轉(zhuǎn)錄激活蛋白(replication and transcription activator，RTA)的表達(dá)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(zhuǎn)換。RTA能與靶基因啟動(dòng)子上的RTA應(yīng)答元件(RTA response element，RRE)序列直接結(jié)合，激活靶基因的轉(zhuǎn)錄表達(dá)，也能通過(guò)與細(xì)胞內(nèi)蛋白的相互作用間接與靶序列結(jié)合，激活下游基因的表達(dá)。干擾素是治療病毒性疾病的常用藥物之一，其在體內(nèi)的表達(dá)主要受干擾素調(diào)節(jié)因子(interferon regulatory factors，IRFs)調(diào)控。干擾素調(diào)節(jié)因子7(interferon regulatoryfactors 7，IRF-7)是IRFs家族中的一個(gè)重要成員，，能控制Ⅰ型干擾素依賴的免疫反應(yīng)。病毒感染細(xì)胞后，IRF-7將被磷酸化入細(xì)胞核，啟動(dòng)下游靶基因的轉(zhuǎn)錄，發(fā)揮抗病毒效應(yīng)。IRF-7還可直接作用于病毒啟動(dòng)子，抑制病毒的激活。研究表明，IRF-7可以競(jìng)爭(zhēng)性結(jié)合HHV-8 ORF57啟動(dòng)子上的RTA結(jié)合位點(diǎn)，從而抑制RTA激活ORF57啟動(dòng)子。但I(xiàn)RF-7是否可以抑制RTA激活其它啟動(dòng)子，影響病毒基因表達(dá)目前尚未有報(bào)道。 本研究表達(dá)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(dá)質(zhì)粒和ORF57報(bào)告質(zhì)粒共轉(zhuǎn)染293T細(xì)胞，觀察到IRF-7能抑制RTA對(duì)ORF57啟動(dòng)子的激活功能，且抑制作用呈現(xiàn)出劑量依賴效應(yīng)。將IRF-7與RTA的表達(dá)質(zhì)粒和PAN報(bào)告質(zhì)粒共轉(zhuǎn)染293T細(xì)胞，發(fā)現(xiàn)IRF-7能抑制RTA對(duì)PAN啟動(dòng)子的激活功能，且抑制作用呈現(xiàn)出劑量依賴效應(yīng)。將野生性IRF-7和賴氨酸突變型IRF-7分別與RTA的表達(dá)質(zhì)粒和ORF57報(bào)告質(zhì)粒共轉(zhuǎn)染293T細(xì)胞，發(fā)現(xiàn)不同賴氨酸突變IRF-7抑制RTA激活ORF57啟動(dòng)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(dá)質(zhì)粒和PAN報(bào)告質(zhì)粒共轉(zhuǎn)染293T細(xì)胞，發(fā)現(xiàn)不同賴氨酸突變型IRF-7抑制RTA對(duì)PAN啟動(dòng)子的激活功能也不同；而且不同突變對(duì)RTA激活ORF57與PAN啟動(dòng)子的抑制作用的趨勢(shì)也不盡相同。我們還將RTA以及IRF-7的真核表達(dá)質(zhì)粒共轉(zhuǎn)染293T細(xì)胞，利用所制備的抗體通過(guò)蛋白質(zhì)免疫印跡實(shí)驗(yàn)技術(shù)，發(fā)現(xiàn)RTA能減少IRF-7的蛋白產(chǎn)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(duì)PAN等基因的激活；某些賴氨酸點(diǎn)突變后改變IRF-7的修飾使其對(duì)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(guān)疾病的預(yù)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(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2009
【分類號(hào)】：R373

【相似文獻(xiàn)】

相關(guān)博士學(xué)位論文 前3條

1 秦宇;干擾素調(diào)節(jié)因子7對(duì)人類8型皰疹病毒基因表達(dá)的影響[D];天津醫(yī)科大學(xué);2009年

2 何方平;人類8型皰疹病毒組合抗原血清檢測(cè)方法的建立及新疆地區(qū)流行病學(xué)調(diào)查[D];新疆醫(yī)科大學(xué);2006年

3 王星;新疆人類8型皰疹病毒的致病機(jī)制研究及在獻(xiàn)血者中流行病學(xué)調(diào)查[D];新疆醫(yī)科大學(xué);2009年

相關(guān)碩士學(xué)位論文 前1條

1 康曉靜;新疆Kaposi肉瘤組織內(nèi)人類8型皰疹病毒DNA的熒光原位PCR檢測(cè)[D];新疆醫(yī)科大學(xué);2001年

本文編號(hào)：2046506