【作者】 叶建强；

【导师】 秦爱建；

【作者基本信息】 扬州大学 ， 预防兽医学， 2005， 博士

【摘要】 J亚群禽白血病病毒(ALV-J)是1989年被分离、鉴定出的一个新的禽白血病病毒亚群,与其它ALV亚群比较,该亚群病毒的一个显著特点是其env基因非常独特。ALV-J env基因与其它亚群的同源性很低,但是与正常存在于鸡体内的内源性EAV-HP序列有高达97%的同源性。该亚群病毒主要引起髓细胞瘤,而其它外源性ALV亚群主要引起淋巴瘤。ALV研究资料表明其致瘤性与env密切相关。为了进一步研究Env蛋白的功能和在致瘤中的作用,本研究首先构建了一个表达ALV-J env基因细胞系,并发现了该细胞具有类转化现象;进一步研究发现Env蛋白中存在与信号转导相关的基序;根据基序功能,首次提出将ALV-J Env蛋白分为三类,即抑制型、双功能型以及激活型Env蛋白;并提出了ALV-J Env蛋白致病致瘤理论;同时对双功能型env基因进行了克隆、表达,建立了检测ALV-J Env抗原/IgG免疫复合物的ELISA方法。 一、表达J亚群禽白血病病毒env基因细胞系的构建 将ALV-J 4817毒株env基因插入pcDNA3.1真核表达载体构建成转移载体pcDNA-env。用转移载体pcDNA-env转染鸡胚成纤维细胞系,通过抗Zeocin药物基因筛选获得转染阳性细胞。阳性细胞经传30代后,冻存,13个月后复苏,置不含药物的培养基中传代培养。连续传40代后,分别用PCR、Southern-blot、间接免疫荧光(IFA)及Western-blot对细胞中的env基因进行了检测,并进行了抗病毒感染分析。试验结果表明,通过Zeocin药物筛选获得了稳定表达ALV-J env基因的鸡胚成纤维细胞系,命名为pcDNA-env-DF1细胞;该细胞在体外抗ALV-J感染试验中,能抵抗10~4个TCID50病毒感染。这一细胞系的构建对进一步探索env基因功能具有重要意义。扬州大学博士学位论文二、J亚群禽白血病病毒Env蛋白功能与PI3K相关 稳定表达Al入l-J 4817毒株Env蛋白的peDNA.env一DFI细胞的生长形态特性研究表明,该细胞形态与正常细胞相比明显变圆、变大,提出其可能是一种类转化现象。利用3D矛SSM软件以及TMpred软件分别对4817毒株Env蛋白进行了分析,结果表明4817毒株Env蛋白具有信号转导功能,而且发现4817毒株Env蛋白胞浆区存在PI3K信号激活结构域Y尤月讨。用PI3K特异性抑制剂对peDNA·env-DFI细胞处理后发现,该细胞类转化形态能被逆转,证明peDNA-env一DFI细胞类转化现象直接与PI3K信号有关,提示4817毒株Env蛋白具有潜在的致瘤性,其机理可能通过胞浆区YxxM结构域激活PI3K,引起peDNA-env-DFI细胞的类转化,有关研究正在深入。三、J亚群禽白血病病毒Bnv蛋白分类及其特性 对不同ALV一J Env蛋白胞浆区氨基酸序列进一步比较发现,ALV一J毒株Env蛋白胞浆区除了YxxM结构域外,还存在ITIM和类ITAM结构域。根据Y双M、ITAM和mM作用和功能,作者提出将ALV一J Env蛋白分为抑制型、双功能型以及激活型三类。根据这一分类原则,在检索到的33个ALV一J Env蛋白中,有28个为双功能型Env蛋白,3个为抑制型Env蛋白,只有4817和UD3毒株2个为激活型Env蛋白;而检索到的所有EAV.HP均属于抑制型Env蛋白。 Env蛋白的抗原性分析发现,抑制型、双功能型以及激活型Env蛋白的胞浆区抗原性依次降低,但致病、致瘤能力越来越强,这种现象与其信号转导基序直接相关。分析ITIM基序对控制或抑制免疫细胞激活、增殖功能,提示抑制型、双功能型Env蛋白中ITIM基序与ALV一J免疫抑制相关,抑制型Env蛋白与EAV一HP可能是ITIM受体,EAV一HP可能为细胞内原癌基因,ALV一J env可能为病毒癌基因。四、J亚群禽白血病病毒双功能型即y墓因克隆及其表达 通过组织病理检查、组织切片特异性抗原检测、细胞培养技术以及间接免疫荧光试验佃A)从一疑似ALv一J感染的祖代肉用型种鸡群中分离、鉴定到一株ALV一J病毒,命名为Js.Nt毒株。随后对该毒株的env基因进行了PCR扩增、克隆、序列分析、转移表达质粒构建以及表达。结果表明,该毒株Env蛋白胞浆区存在Y双M和ITIM结构域,属于ALV一J双功能型Env蛋白;用该毒株env基因以及peDNA3.1载体成功构建了转移表达质粒PcDNA.NT.env;通过peDNA一T一e。更多还原

【Abstract】 Subgroup J avian leukosis virus (ALV-J) was first isolated in 1989 and identified as a new subgroup of avian leukosis virus. The env gene of ALV-J is very unique, has very low homology to other ALV subgroups, however, exhibits 97% identity to EAV-HP sequence, which is existed in the normal chicken’s genome. ALV-J most causes myeloid leukosis, while other exogenous ALVs mainly induce lymphoid leukosis. The previous research data showed that env gene is closely related to the ALV oncogenicity, however, the function of the envlope protein is still unclear. This paper is to further investigate the uniqueness of ALV-J env gene and to explore the function of env gene in the ALV-J pathogenicity and oncogenicity.1 Development of Chicken Embryo Fibroblast Cell Line Expressing env Gene of Subgroup J Avian Leukosis VirusThe recombinant of pcDNA-env was constructed by inserting the env gene of ALV-J 4817 strain into the vector pcDNA3.1. Chicken embryo fibroblast cell line transfected with the recombinant pcDNA-env was selected for the resistant cells to Zeocin. The resistant cells were passed for 30 generations and frozen. After 13 months, these cells were refreshed and cultured in the medium free of Zeocin. After another 40 passages, env gene and Env protein in the cells were detected by PCR, Southern-blot, IFA and Western-blot. The results showed that the cells transfected with the recombinant, designated as pcDNA-env-DF1 cells, could stably express the env gene of ALV-J. And pcDNA-env-DF1 cells had the characterization of resistance to ALV-J with a dosage of 104 TCID50. The pcDNA-env-DF1 cells developed in this paper had a vital significance for further exploring the function of ALV-J env gene.2 Function of Envelope Protein of Subgroup J Avian Leukosis Virus Associating with PI3KThe observation in pcDNA-env-DFl cells expressing Env protein of ALV-J 4817 strain showed that the cells appeared large size and became round in morphology comparing with normal cell. The cells look like transformed. Analysis results with 3D-PSSM software showed that there is signal transduction in Env protein of 4817. The further analysis for signal transduction motif released that there is a YxxM motif in cytoplasmic tail of the protein, which could specifically activate PI3K signal pathway. And the changed morphology of the cells could be reversed to normal by inhibitor LY294002 specific to PI3K. These results proved the phenomenon of the cells was directly related to the PI3K and indicated the envelope protein had potential oncogenicity and could activate PI3K through YxxM motif to result in the phenomenon. Some experiments associated with the phenomenon are being undertaken.3 Classification and Characterization of Envelope Protein of Subgroup J Avian Leukosis VirusFurther analysis showed that not only YxxM motif, but also ITIM and ITAM-like were existed in cytoplasmic tails of different ALV-J envelope proteins. Based on significance of YxxM and ITAM motifs in conducting active signal to induce cell proliferation and tumorogenisis, significance of ITIM in conducting inhibitory signal to control or inhibit cell proliferation and tumorogenisis and the distribution of these motifs in ALV-J envelope proteins, ALV-J envelope proteins were classified as three types, designated as inhibitive envelope protein, bi-functional envelope protein and active envelope protein. According to this classification, 28 of 33 ALV-J envelope proteins (Download from Genbank) were bi-functional envelope protein, 3 ones were inhibitive envelope protein and only 4817 and UD3 strain’s envelope proteins were active envelope proteins, while all EAV-HPs (Download from Genbank) were classified as inhibitive envelope protein. Based on mechanisms in signal transduction of these signal motifs, three models of signal transduction of the three types of envelope protein were figured out.Antigenic analysis of envelope suggests that the antigenicity of cytoplasmic tail of inhibitive, bi-functional and active envelope proteins be not only directly related to these motifs, but also decreas更多还原