本文選題：TRIM22 + TRIM14�。� 參考：《北京協(xié)和醫(yī)學(xué)院》2016年碩士論文

【摘要】：流感病毒(influenza virus)是一種流行廣泛、傳染性極強(qiáng)的負(fù)鏈病毒,可在高危人群中造成嚴(yán)重疾病和死亡。每年流感在世界范圍內(nèi)的流行造成約300萬至500萬例嚴(yán)重疾病和約25萬至50萬例死亡。流感病毒分為A、B、C三型,其中A型流感病毒(influenza A virus, IAV)致病性最強(qiáng)。目前治療流感的藥物有NA的抑制劑扎那米韋、奧司米韋,及針對(duì)M2的抑制劑金剛烷胺,但是流感病毒通常會(huì)對(duì)藥物產(chǎn)生耐藥性。雖然疫苗能夠預(yù)防病毒感染,但流感病毒每年流行的毒株都不一樣而且病毒的突變性很強(qiáng)容易導(dǎo)致疫苗錯(cuò)配,單純靠疫苗預(yù)防感染并不現(xiàn)實(shí)。因此,有必要尋找新的抑制流感病毒的機(jī)制,為新藥物的研發(fā)提供線索。在固有免疫對(duì)抗流感病毒感染過程中干擾素發(fā)揮了重要作用。干擾素能夠誘導(dǎo)表達(dá)ISG(interferon-stimulated gene)。ISG通過改變細(xì)胞的代謝和內(nèi)環(huán)境來調(diào)節(jié)機(jī)體免疫反應(yīng)或者與病毒的某些組分發(fā)生直接的相互作用來抑制病毒的復(fù)制。這些ISG中包括較多的TRIM蛋白,TRIM蛋白家族是由N端到C端以RING、B-box、 Coiled-coil結(jié)構(gòu)域依次排列為結(jié)構(gòu)特征的一類蛋白,TRIM14和TRIM22屬于TRIM (tripartite motif)家族成員。目前已經(jīng)發(fā)現(xiàn)一些TRIM蛋白能夠抑制HBV、HIV、 IAV等多種病毒,但TRIM14與流感病毒的關(guān)系還不清楚。本研究以TRIM22為研究對(duì)象來驗(yàn)證BiLC系統(tǒng)能否快速有效的檢測ISGs蛋白與流感蛋白相互作用；同時(shí)建立了穩(wěn)定表達(dá)細(xì)胞系的構(gòu)建方法及快速檢測流感病毒復(fù)制的系統(tǒng)。與文獻(xiàn)報(bào)道一致,TRIM22能有效抑制流感病毒的復(fù)制,并且BiLC系統(tǒng)檢測到TRIM22與NP蛋白有特異的結(jié)合�；谏鲜鰧�(shí)驗(yàn)方法,我們研究了TRIM14與流感病毒的關(guān)系。發(fā)現(xiàn)TRIM14屬于ISG并且主要被I型干擾素誘導(dǎo)高表達(dá)。過表達(dá)TRIM14和敲除內(nèi)源TRIM14細(xì)胞的病毒擴(kuò)增實(shí)驗(yàn)都證明TRIM14對(duì)流感病毒具有抑制作用。利用高效BiLC系統(tǒng)檢測發(fā)現(xiàn)TRIM 14可能通過與M2、NP、PA、NS1等蛋白相互作用來抑制流感病毒復(fù)制。上述研究的完成,為快速研究ISG與流感病毒的關(guān)系和機(jī)制提供了新的方法。豐富了固有免疫對(duì)流感病毒抑制方式的認(rèn)識(shí),同時(shí)也為研究抗流感病毒藥物提供了新的視角。
[Abstract]:Influenza virus Influenza virus (Influenza virus) is a widespread and highly infectious negative chain virus, which can cause serious disease and death in high risk population. A worldwide pandemic causes about 3 million to 5 million serious diseases and 250000 to 500000 deaths each year. Influenza virus can be classified into three types, type A influenza virus influenza A virus (IAV) is the most pathogenic. Current treatments for influenza include anamivir, an inhibitor of na, osmevir, and amantadine, an inhibitor of M2, but influenza viruses are often resistant to drugs. Although the vaccine can prevent the infection of the virus, the influenza virus each year is different, and the mutation of the virus is very easy to cause the vaccine mismatch, it is not realistic to rely on the vaccine to prevent the infection. Therefore, it is necessary to find new mechanisms to inhibit influenza virus and provide clues for the development of new drugs. Interferon plays an important role in innate immunity against influenza virus infection. Interferon can induce the expression of ISG interferon-stimulated gene. ISG inhibits the replication of the virus by changing the metabolism and internal environment of the cells to regulate the immune response or to interact directly with some components of the virus. These ISGs include more trim protein family, which are members of tritrite motif family from N-terminal to C-terminal with RING-B-box. Coiled-coil domain is arranged in order of structural characteristics. Some trim proteins have been found to inhibit HIV, IAV and other viruses, but the relationship between TRIM14 and influenza virus is not clear. In this study, TRIM22 was used to verify the ability of BiLC system to detect the interaction between ISGs protein and influenza protein, and to establish a stable expression cell line and a system for rapid detection of influenza virus replication. As reported in the literature, TRIM22 can effectively inhibit the replication of influenza virus, and the specific binding of TRIM22 to NP protein was detected by BiLC system. Based on the above experimental method, we studied the relationship between TRIM14 and influenza virus. It was found that TRIM14 belongs to ISG and is mainly induced by type I interferon. Both overexpression of TRIM14 and knockout of endogenous TRIM14 cells showed that TRIM14 had inhibitory effect on influenza virus. Highly efficient BiLC detection revealed that TRIM14 may inhibit influenza virus replication by interacting with proteins such as M2, NPN, PANS1 and so on. The above research provides a new method to study the relationship and mechanism between ISG and influenza virus. It not only enriches the understanding of innate immunity to the inhibition of influenza virus, but also provides a new perspective for the study of anti-influenza drugs.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R373.13

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1 陳志高;TRIM家族蛋白：TRIM22、TRIM14抑制流感病毒的功能和機(jī)制的初步研究[D];北京協(xié)和醫(yī)學(xué)院;2016年

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本文編號(hào)：2032313