本文關鍵詞：馬TRIM5α激活AP-1信號通路的分子機制研究　出處：《中國農業(yè)科學院》2016年碩士論文　論文類型：學位論文

  更多相關文章： 馬TRIM5α AP-1信號通路 天然免疫 抗病毒活性

【摘要】：TRIM5α是TRIM家族的一個成員,具有典型的RBCC結構(RING、B-box和Coil-Coiled)和一個C端SPRY結構域。TRIM5α是存在于多種宿主細胞內的抗逆轉錄病毒天然免疫限制因子,可通過SPRY結構靶向降解逆轉錄病毒衣殼蛋白(CA)和激活天然免疫來發(fā)揮抗病毒功能。我們前期研究證明,相對于其它動物而言,馬TRIM5α具有獨特的分子特征,在進化過程中缺失了重要的C末端結構域而失去降解EIAV的CA蛋白功能,但過表達后仍明顯限制馬傳染性貧血病病毒(EIAV)的感染。為了進一步確定這個結果,本論文利用慢病毒表達載體建立了穩(wěn)定表達馬TRIM5α和恒河猴TRIM5α的HEK293細胞系,并進行了抗病毒實驗。結果顯示馬TRIM5α表現(xiàn)出明顯的限制EIAV感染作用。同時,利用AP-1和NF-κB熒光素酶報告系統(tǒng)檢測馬TRIM5α激活天然免疫的活性,結果證明了馬TRIM5α可以很好地激活AP-1和NF-κB信號通路。上述結果說明由于馬TRIM5α沒有完整的SPRY結構導致其不能靶向降解EIAV的Gag蛋白,但是馬TRIM5α仍可以發(fā)揮抗病毒功能并激活天然免疫信號,因此這提示我們馬TRIM5α可能是通過激活天然免疫來抵抗病毒感染。靈長類TRIM5α發(fā)揮天然免疫激活功能依賴完整的SPRY結構域,而馬TRIM5α蛋白僅包含五分之一SPRY結構,仍能顯著激活天然免疫信號通路。這提示不同種屬TRIM5α發(fā)揮激活天然免疫功能的分子基礎與分子機制不同。為了確定馬TRIM5α激活AP-1信號通路的關鍵氨基酸,本研究對馬TRIM5α從C端開始進行截短表達,構建了截短表達突變體(eqTRIM5-333、eqTRIM5-328、eqTRIM5-324、eqTRIM5-318、eqTRIM5-310、eqTRIM5-303、eqTRIM5-282),利用雙熒光素酶報告系統(tǒng)初步確定了馬TRIM5α激活AP-1的關鍵氨基酸在第303位至第310位氨基酸這7個氨基酸之間。因此在eqTRIM5-310的基礎上對這7個氨基酸逐一進行了點突變,構建了重組質粒(eqTRIM5-310-303、eqTRIM5-310-304、eqTRIM5-310-305、eqTRIM5-310-306、eqTRIM5-310-307、eqTRIM5-310-308、eqTRIM5-310-309),確定了馬TRIM5α激活AP-1的關鍵氨基酸是第303位蘇氨酸和第304位亮氨酸,并且這兩個氨基酸對其激活NF-κB也是重要的。為進一步研究馬TRIM5α激活AP-1信號通路的分子機制,我們將馬TRIM5α及其突變體分別和TAB2、TAK1共轉293T細胞,結果表明馬TRIM5α及其突變體可以降解TAB2,但是失去激活作用的突變體均失去了與TAK1的相互作用。通過將馬TRIM5α及其突變體、TAK1、泛素(Ub)共轉293T細胞,結果表明馬TRIM5α可能是通過泛素化TAK1進而激活下游的AP-1和NF-κB信號通路。本研究揭示了馬TRIM5α激活AP-1信號通路的分子機制,提示馬TRIM5α可能是通過激活天然免疫從而限制病毒感染,這為進一步理解宿主細胞天然免疫分子進化及與病毒相互適應的機理提供了研究基礎,并且馬TRIM5α可作為潛在的廣譜抗病毒新型分子,在開發(fā)新型抗病毒制劑和藥物等方面具有應用前景。
[Abstract]:TRIM5 alpha is a member of the TRIM family and has a typical RBCC structure (RING, B-box and Coil-Coiled) and a C end SPRY domain. TRIM5 alpha is an antiretroviral natural immune limiting factor existing in various host cells. It can play the antiviral function by targeting the degradation of retroviral capsid protein (CA) by SPRY structure and activating innate immunity. Our previous studies have demonstrated that compared with other animal, horse TRIM5 alpha has unique molecular characteristic, in the evolutionary process of the deletion of C terminal domain important without degradation of EIAV CA protein function, but still significantly restricted expression of equine infectious anemia virus (EIAV) infection. In order to further confirm this result, a HEK293 cell line stably expressing horse TRIM5 alpha and Ganges RIver monkey TRIM5 alpha was established by Lentivirus Expression Vector, and antiviral experiments were carried out. The results showed that the TRIM5 alpha showed a significant effect on EIAV infection. Meanwhile, AP-1 and NF- kappa B luciferase reporter system were used to detect the activity of natural immunization activated by horse TRIM5 TRIM5, which proved that horse TRIM5 alpha can activate AP-1 and NF- kappa B signaling pathway. The results show that because the horse TRIM5 alpha no complete structure of the SPRY it can not lead to targeted degradation of EIAV Gag protein, but the horse TRIM5 alpha can still play antiviral function and activate innate immune signal, so this suggests that Ma TRIM5 alpha may be through the activation of innate immunity against viral infection. Primate TRIM5 alpha exerts natural immune activation function and relies on a complete SPRY domain, while the horse TRIM5 alpha protein contains only 1/5 SPRY structure, which still can significantly activate the innate immune signaling pathway. This suggests that different species of TRIM5 alpha play a different molecular basis and molecular mechanism to activate the natural immune function. In order to determine the key amino acid horse TRIM5 alpha activation of the AP-1 pathway, the study of Ma TRIM5 alpha began expression of truncated from the C, constructed the expression of truncated mutants (eqTRIM5-333, eqTRIM5-328, eqTRIM5-324, eqTRIM5-318, eqTRIM5-310, eqTRIM5-303, eqTRIM5-282), using the dual luciferase reporter system initially identified the key amino acid horse TRIM5 alpha activation AP-1 between 303rd to 310th amino acid of the 7 amino acids. Therefore, on the basis of the eqTRIM5-310 of these 7 amino acids by point mutations, to construct recombinant plasmid (eqTRIM5-310-303, eqTRIM5-310-304, eqTRIM5-310-305, eqTRIM5-310-306, eqTRIM5-310-307, eqTRIM5-310-308, eqTRIM5-310-309), to determine the key amino acids of TRIM5 alpha horse activation AP-1 is 303rd bit and 304th bit leucine and threonine, these two amino acids of the the activation of NF- K B is important. The molecular mechanism of activation of AP-1 signaling pathway for the further study of horse TRIM5 alpha, we will ma TRIM5 alpha and TAB2, TAK1 and its mutants were co transfected 293T cells. The results showed that the horse TRIM5 alpha and its mutants can degrade TAB2, but lost activation mutants lost the interaction with TAK1. Through the transformation of horse TRIM5 alpha and its mutant, TAK1 and ubiquitin (Ub) into 293T cells, the results indicate that TRIM5 TRIM5 may activate downstream AP-1 and NF- kappa B signaling pathway through ubiquitination. This study reveals the molecular mechanism of TRIM5 alpha activation of the AP-1 pathway, suggesting that TRIM5 may be through the activation of alpha horse natural immunity to limit virus infection, it may provide a basis for the further understanding of the host innate immune cells and molecular evolution mechanism to adapt to each other with the virus, and the horse can be used as a broad-spectrum antiviral TRIM5 a new molecular potential and has the application prospect in the development of new antiviral agents and drugs etc..
【學位授予單位】：中國農業(yè)科學院
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：S821

【參考文獻】

相關期刊論文 前2條

1 王翠輝;那雷;王曉鈞;;TRIM蛋白參與天然免疫信號轉導的研究進展[J];中國預防獸醫(yī)學報;2016年02期

2 蔡偉剛;那雷;劉荻，

本文編號：1338360