鸡CCCH型ZAP抑制J亚群禽白血病病毒复制及对其组织嗜性影响的研究

发布时间：2018-04-23 09:34

  本文选题：鸡锌指抗病毒蛋白 + J亚群禽白血病病毒　； 参考：《山东农业大学》2015年硕士论文

【摘要】：CCCH型锌指抗病毒蛋白(ZAP)为宿主防御因子之一,具mRNA结合特性,在RNA病毒感染时,会通过与病毒mRNA作用,而使之失去感染性,可对抗突变的病毒。J亚群禽白血病病毒(ALV-J)为致瘤RNA病毒,由于其自身的超突变性以及引起疾病的复杂性,至今未研制出有效的疫苗或药物,严重危害了我国的养禽业。因此,CCCH型ZAP有望成为下一代抗ALV-J的药物,然而对于禽CCCH型ZAP的基因特征、组织分布及抗病毒效果等问题,目前尚未有研究。本实验室前期证明,鸡胆汁外泌体(exosome)具有抑制ALV-J复制的功能,对其进行蛋白组学分析时发现鸡胆汁exosome内出现新型蛋白,即CCCH型锌指抗病毒蛋白(ZAP),因此推测可能是CCCH型ZAP具有抑制ALV-J复制的作用。基于上述发现,我们首先对鸡体内的chZAP基因序列的保守性进行了分析。分别从正常SPF鸡的肝脏、脾脏、肾脏及DF-1细胞中获得了鸡CCCH型锌指抗病毒蛋白(chZAP)基因。序列比对分析发现,鸡不同组织器官及DF-1细胞的chZAP基因序列完全一致;而不同物种(禽、鼠、人)之间的锌指抗病毒蛋白具有种属差异性,但其四个CCCH型锌指模体却具有保守性。序列比对结果表明CCCH型锌指模体在动物进化过程中被完整保留具有超保守性,为我们研究其抗病毒特性奠定了理论基础。为了探究chZAP是否具有抗ALV-J复制作用,分别通过慢病毒重组载体转染和原核表达获得chZAP作用于ALV-J感染的细胞系。通过构建chZAP慢病毒重组载体将chZAP基因插入DF-1细胞基因组内,免疫荧光和Western blot检测证实chZAP在DF-1中超表达后,接种ALV-J,随后进行荧光定量PCR检测。检测结果表明超表达的chZAP能够显著抑制ALV-J的复制,但其抑制效果呈现表达水平依赖性。通过原核表达的chZAP作用于ALV-J感染细胞,观察chZAP是否可以抑制ALV-J的复制,结果表明原核表达的chZAP能够显著抑制ALV-J的复制。因此,证明chZAP能够有效抑制ALV-J的复制。基于以上结果,推测chZAP在组织中的表达可能会影响ALV-J的组织嗜性。通过免疫组织化学及绝对定量PCR,证明chZAP在各组织中广泛表达,但是表达水平呈现差异性;表达蛋白定位于细胞质和细胞核中;在大部分组织中,chZAP自然表达水平较高时,病毒负载量较低,而在个别组织中如脾,chZAP自然表达水平不是很高,但是ALV-J感染后表达呈现显著上升趋势,病毒负载量相对其他组织亦较低,所以chZAP对组织的保护程度有可能不仅取决于其自然表达水平,亦取决于其可诱导程度。然而,SPSS分析显示chZAP的表达与ALV-J感染程度不呈明显相关。目前有研究表明,ALV-J的组织嗜性主要与env、LTR基因和ALV-J受体chNHE1有关。所以,尽管chZAP作为宿主防御因子在过表达时能够显著抑制ALV-J的复制,但是,chZAP只是影响ALV-J的组织嗜性而不是ALV-J组织嗜性的决定因子。综上所述,本研究表明chZAP是限制ALV-J复制的一个宿主抗病毒因子;chZAP影响ALV-J的组织嗜性,但不是ALV-J组织嗜性的一个决定性因素。此发现为深入研究宿主防御蛋白体系和病毒-宿主的相互作用机制研究奠定科学基础;亦为ALV-J的防治工作提供了新的思路与方法。
[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.

【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.65

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