SPC-A1细胞应答呼吸道合胞病毒感染的敏感基因zlg10的克

发布时间：2018-12-31 11:04

【摘要】：呼吸道合胞病毒(respiratory syncytial virus,RSV)是引起婴幼儿严重下呼吸道感染的重要病毒性病原体。该病毒流行广泛,致病率高,多数儿童两岁前都感染过RSV,在老年人和免疫力低下的人群中也会暴发流行。RSV属副黏病毒科,肺炎病毒属,病毒粒子直径约150～300nm。RSV的基因组为单股负链RNA,长约15kb,编码11种蛋白质,包括3种核衣壳蛋白(核壳蛋白N、磷酸蛋白P、大聚合酶蛋白L);3种跨膜蛋白(融合蛋白F、附着蛋白G、小疏水蛋白SH);2种基质蛋白(M、M2)和2种非结构蛋白(NS1、NS2)。RSV感染的临床症状在不同年龄组往往不同,一般可致鼻炎、中耳炎、哮喘、支气管炎、细支气管炎和肺炎,尤其以后两种疾病最为严重,甚至可导致死亡。目前能有效治疗RSV感染的药物很少,且疗效不确定。世界卫生组织(WHO)已将研制RSV疫苗列为全球疫苗计划的优先发展项目之一。但迄今为止,尚无安全、有效的RSV疫苗获准上市。分子生物学研究已经证实当病毒感染宿主细胞时,宿主细胞会发生变化,其中有一些特异性的序列表达量有所改变。mRNA差异显示法(mRNA differential display,DDRT-PCR)是目前筛选差异表达基因最有效的方法之一。为了筛查到与病毒感染相关的宿主细胞敏感基因,本研究以呼吸道合胞病毒RSV感染的SPC-A1细胞与正常SPC-A1细胞为样本,通过mRNA差异显示技术获得了SPC-A1细胞应答RSV感染的mRNA差异表达谱,并且克隆到了50余个与呼吸道合胞病毒RSV致病相关的EST片段。随后选择在RSV感染后的SPC-A1细胞中高表达的EST片段g10-1为研究对象,经克隆测序g10-1长264bp,采用生物信息学方法对g10-1进行了鉴定后证实这是一个新基因片段,随后电子克隆获得的g10-1的延伸产物为761bp,含有一个编码54个氨基酸的读码框,命名为zlg10基因。zlg10编码的蛋白质与己知蛋白质均无显著同源性,亚细胞定位实验证实是一个新的核蛋白质。Northern Blot杂交分析验证后,设计了zlg10含完整阅读框的特异性扩增引物,通过RT—PCR方法克隆出了该基因。为了了解新基因zlg10在RSV感染细胞过程中如何被调节以及在RSV感染过
[Abstract]:Respiratory syncytial virus (respiratory syncytial virus,RSV) is an important viral pathogen causing severe lower respiratory tract infection in infants. The virus is widespread and has a high pathogenicity. Most children have been infected with RSV, before the age of two years. RSV belongs to paramyxovirus family, pneumonia virus family, and can also break out in the elderly and people with low immunity. The genome of virus particles about 150~300nm.RSV is about 15kb long, encoding 11 proteins, including three nucleocapsid proteins (core-shell protein N, phosphate protein P, large polymerase protein L);). Three transmembrane proteins (fusion protein F, attachment protein G, small hydrophobic protein SH);) The clinical symptoms of two kinds of matrix protein (MM-2) and two kinds of non-structural proteins (NS1,NS2). RSV infection) are often different in different age groups. They can cause rhinitis, otitis media, asthma, bronchitis, bronchiolitis and pneumonia. In particular, the last two diseases are the most serious and can even lead to death. At present, there are few drugs that can effectively treat RSV infection, and the effect is uncertain. The World Health Organization (WHO) has made the development of RSV vaccine one of the priorities of the global vaccine program. But so far, no safe, effective RSV vaccine has been approved to market. Molecular biology has shown that when the virus infects the host cell, the host cell changes, and some of the specific sequence expressions change. MRNA differential display (mRNA differential display, DDRT-PCR is one of the most effective methods for screening differentially expressed genes. In order to screen host cell-sensitive genes associated with viral infection, we used SPC-A1 cells infected with respiratory syncytial virus (RSV) RSV and normal SPC-A1 cells as samples. The differentially expressed mRNA profiles of SPC-A1 cells in response to RSV infection were obtained by mRNA differential display technique, and more than 50 EST fragments related to the pathogenesis of respiratory syncytial virus RSV were cloned. Subsequently, the highly expressed EST fragment g10-1 in SPC-A1 cells infected with RSV was selected as the study object. The g10-1 was cloned and sequenced to 264bp. the g10-1 gene fragment was identified by bioinformatics method and proved to be a new gene fragment. The extended product of g10-1 was 761 BP, which contained a reading frame encoding 54 amino acids and was named zlg10 gene. The protein encoded by zlg10 had no significant homology with known proteins. The subcellular localization experiment confirmed that it was a new nuclear protein. Northern Blot hybridization analysis and verified, then designed a specific amplification primer containing a complete reading frame for zlg10, and cloned the gene by RT-PCR method. To understand how the new gene zlg10 is regulated in RSV infected cells and how it is infected with RSV
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R373

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