發(fā)布時間：2018-04-23 09:34

  本文選題：雞鋅指抗病毒蛋白 + J亞群禽白血病病毒　； 參考：《山東農(nóng)業(yè)大學》2015年碩士論文

【摘要】：CCCH型鋅指抗病毒蛋白(ZAP)為宿主防御因子之一,具mRNA結(jié)合特性,在RNA病毒感染時,會通過與病毒mRNA作用,而使之失去感染性,可對抗突變的病毒。J亞群禽白血病病毒(ALV-J)為致瘤RNA病毒,由于其自身的超突變性以及引起疾病的復雜性,至今未研制出有效的疫苗或藥物,嚴重危害了我國的養(yǎng)禽業(yè)。因此,CCCH型ZAP有望成為下一代抗ALV-J的藥物,然而對于禽CCCH型ZAP的基因特征、組織分布及抗病毒效果等問題,目前尚未有研究。本實驗室前期證明,雞膽汁外泌體(exosome)具有抑制ALV-J復制的功能,對其進行蛋白組學分析時發(fā)現(xiàn)雞膽汁exosome內(nèi)出現(xiàn)新型蛋白,即CCCH型鋅指抗病毒蛋白(ZAP),因此推測可能是CCCH型ZAP具有抑制ALV-J復制的作用�；谏鲜霭l(fā)現(xiàn),我們首先對雞體內(nèi)的chZAP基因序列的保守性進行了分析。分別從正常SPF雞的肝臟、脾臟、腎臟及DF-1細胞中獲得了雞CCCH型鋅指抗病毒蛋白(chZAP)基因。序列比對分析發(fā)現(xiàn),雞不同組織器官及DF-1細胞的chZAP基因序列完全一致;而不同物種(禽、鼠、人)之間的鋅指抗病毒蛋白具有種屬差異性,但其四個CCCH型鋅指模體卻具有保守性。序列比對結(jié)果表明CCCH型鋅指模體在動物進化過程中被完整保留具有超保守性,為我們研究其抗病毒特性奠定了理論基礎(chǔ)。為了探究chZAP是否具有抗ALV-J復制作用,分別通過慢病毒重組載體轉(zhuǎn)染和原核表達獲得chZAP作用于ALV-J感染的細胞系。通過構(gòu)建chZAP慢病毒重組載體將chZAP基因插入DF-1細胞基因組內(nèi),免疫熒光和Western blot檢測證實chZAP在DF-1中超表達后,接種ALV-J,隨后進行熒光定量PCR檢測。檢測結(jié)果表明超表達的chZAP能夠顯著抑制ALV-J的復制,但其抑制效果呈現(xiàn)表達水平依賴性。通過原核表達的chZAP作用于ALV-J感染細胞,觀察chZAP是否可以抑制ALV-J的復制,結(jié)果表明原核表達的chZAP能夠顯著抑制ALV-J的復制。因此,證明chZAP能夠有效抑制ALV-J的復制�；谝陨辖Y(jié)果,推測chZAP在組織中的表達可能會影響ALV-J的組織嗜性。通過免疫組織化學及絕對定量PCR,證明chZAP在各組織中廣泛表達,但是表達水平呈現(xiàn)差異性;表達蛋白定位于細胞質(zhì)和細胞核中;在大部分組織中,chZAP自然表達水平較高時,病毒負載量較低,而在個別組織中如脾,chZAP自然表達水平不是很高,但是ALV-J感染后表達呈現(xiàn)顯著上升趨勢,病毒負載量相對其他組織亦較低,所以chZAP對組織的保護程度有可能不僅取決于其自然表達水平,亦取決于其可誘導程度。然而,SPSS分析顯示chZAP的表達與ALV-J感染程度不呈明顯相關(guān)。目前有研究表明,ALV-J的組織嗜性主要與env、LTR基因和ALV-J受體chNHE1有關(guān)。所以,盡管chZAP作為宿主防御因子在過表達時能夠顯著抑制ALV-J的復制,但是,chZAP只是影響ALV-J的組織嗜性而不是ALV-J組織嗜性的決定因子。綜上所述,本研究表明chZAP是限制ALV-J復制的一個宿主抗病毒因子;chZAP影響ALV-J的組織嗜性,但不是ALV-J組織嗜性的一個決定性因素。此發(fā)現(xiàn)為深入研究宿主防御蛋白體系和病毒-宿主的相互作用機制研究奠定科學基礎(chǔ);亦為ALV-J的防治工作提供了新的思路與方法。
[Abstract]:The CCCH type zinc finger antiviral protein (ZAP) is one of the host defense factors, with mRNA binding characteristics. When RNA virus infection, it will pass through the action of the virus mRNA and make it lose its infectivity. It can antagonize the mutant virus.J subgroup of avian leukemic virus (ALV-J) as a tumor causing RNA virus, due to its own hyper mutagenicity and the complexity of the disease. No effective vaccines or drugs have been developed now, which seriously endangers the poultry industry in China. Therefore, CCCH ZAP is expected to be the next generation of anti ALV-J drugs. However, the genetic characteristics, tissue distribution and antiviral effects of avian CCCH ZAP have not yet been studied. This laboratory has proved that the chicken bile exudate (exosome) has a inhibition of AL in the early stage of the laboratory. The function of V-J replication is that the new protein in chicken bile exosome, that is, CCCH type zinc finger antiviral protein (ZAP), is found in chicken bile. Therefore, it is presumed that CCCH type ZAP has the effect of inhibiting ALV-J replication. Based on the above findings, we first analyzed the conservatism of chZAP gene sequence in chicken. The liver, spleen, kidney and DF-1 cells of normal SPF chicken obtained the CCCH type zinc finger antiviral protein (chZAP) gene in chicken. Sequence alignment analysis found that the chZAP gene sequences of different tissues and DF-1 cells of chicken were identical, and the zinc finger antiviral proteins of different species (avian, rat and human) had species difference, but the four CCCH type zinc The sequence alignment results show that the CCCH type zinc finger body is completely preserved in the process of animal evolution, which lays a theoretical foundation for the study of its antiviral properties. In order to explore whether chZAP has the anti ALV-J replication effect, the transfection of the lentivirus recombinant vector and the prokaryotic expression to obtain chZAP respectively. The chZAP gene was inserted into the genome of the DF-1 cell by constructing the chZAP lentivirus recombinant vector. The immunofluorescence and Western blot detection confirmed that after the overexpression of chZAP in DF-1, the chZAP was inoculated ALV-J, and then the fluorescent quantitative PCR was detected. The results showed that the chZAP of the overexpression could significantly inhibit the replication of the ALV-J. The inhibitory effect showed expression level dependence. Through the effect of prokaryotic expression of chZAP on ALV-J infected cells, it was observed that chZAP could inhibit the replication of ALV-J. The results showed that the chZAP of the prokaryotic expression could inhibit the replication of ALV-J significantly. Therefore, it was proved that chZAP could effectively inhibit the replication of ALV-J. Based on the above results, we speculate that chZAP is in the tissue. Expression may affect the tissue eosinophilia of ALV-J. Through immunohistochemistry and absolute quantitative PCR, it is proved that chZAP is widely expressed in various tissues, but the expression level is different; the expression protein is located in the cytoplasm and nucleus; in most tissues, when the level of chZAP is high, the load of the virus is low and in a few groups. The natural expression level of chZAP was not very high in the fabric like spleen, but the expression of ALV-J showed a significant upward trend after infection, and the load of the virus was lower than that of other tissues. So the protection degree of chZAP on the tissue may not only depend on the level of its natural expression, but also on the degree of its inducement. However, the SPSS analysis shows that the expression of chZAP and ALV- There is no obvious correlation between the degree of J infection. Studies have shown that the tissue basophilia of ALV-J is mainly related to the env, LTR and ALV-J receptor chNHE1. Therefore, although chZAP can significantly inhibit the replication of ALV-J, as the host defense factor is overexpressed, chZAP is only a determinant of the tissue basophilia of ALV-J, not the ALV-J tissue eosinophilia. To sum up, this study shows that chZAP is a host antiviral factor restricting ALV-J replication; chZAP affects the tissue basophilia of ALV-J, but is not a decisive factor in ALV-J tissue eosinophilia. This discovery provides a scientific basis for the in-depth study of the host defense protein system and the interaction mechanism of the virus host; and the prevention and control of ALV-J. The work provides new ideas and methods.

【學位授予單位】：山東農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S852.65

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