肠道病毒71型VP1基因及2A基因和蛋白质特征

发布时间：2018-01-12 16:38

本文关键词：肠道病毒71型VP1基因及2A基因和蛋白质特征　出处：《郑州大学》2011年硕士论文　论文类型：学位论文

【摘要】：肠道病毒71型(Enterovirus71, EV71)属于小RNA病毒科肠道病毒属,感染后可以导致手足口病、疱疹性咽峡炎、无菌性脑膜炎、脑炎和脊髓灰质炎样麻痹性疾病、神经源性肺水肿等多种与神经系统相关的疾病。近几年在亚太地区的大流行中,出现严重中枢神经系统感染病例,甚至死亡病例,因此有关EV71病毒的致病性、致病机理、诊断及疫苗方面的研究日益受到重视。国家卫生部已于2008年5月2日决定,将手足口病纳入传染病防治法规定的丙类传染病进行管理。EV71为什么会导致少数重症甚至死亡病例的出现,毒力决定因子有哪些9 EV71的VP1基因编码的蛋白质是病毒重要的中和抗原决定簇,VP1基因序列除了是肠道病毒属内不同血清型分类的依据,还可以作为小RNA病毒科内不同属的分类参考。研究表明EV71的11个病毒蛋白中,对酵母菌产生毒性的只有2A蛋白,而2A蛋白是水解酶,在正常的生理意义下,哺乳细胞被肠病毒感染后,宿主的生长会受到抑制,其中一个机制就是：2A蛋白切割分解宿主的真核生物起始因子4G (eIF4G, eukaryotic translation initiation factor 4G),因而抑制宿主蛋白质合成。也有报道指出,小儿麻痹病毒的2A蛋白会抑制酿酒酵母(Saccharomyces cerevisiae)的细胞生长,可能的原因是2A切割了一些用来进行转录的蛋白质因子。短暂的表达EV71 2A蛋白酶引起细胞凋亡,感染表达的EV71 2A会导致eIF4G I的裂解,而eIF4GI是宿主蛋白合成的关键因素。目的本课题旨在研究肠道病毒71型轻症和重症病例毒株VP1基因、2A基因的核苷酸序列及预测的2A蛋白质结构特征及是否有差别。方法通过RD细胞分离病毒,然后RT-PCR鉴别诊断EV71,分别扩增VPl、2A基因并进行核苷酸序列测定,用DNAMAN软件进行同源性比较并构建系统发生树,通过同源建模用chimera软件将2A氨基酸序列预测出蛋白空间结构模拟图,探讨二级、三级结构是否有差异。克隆2A基因并构建EV71 2A基因的重组表达质粒pIRES2-EGFP-2A。结果 1.RD细胞的培养及病毒分离：成功培养RD细胞,无污染,接种后肠道病毒的致细胞病变(CPE)表现为：细胞圆缩、胞浆颗粒化、核固缩以及最后细胞破裂。 2. RT-PCR鉴别诊断EV71：50份临床手足口病标本中有30份检测是EV71,其中轻型(手足口)13份,重型(手足口并发脑炎或心肌炎)17份。 3.VPl、2A基因的扩增：5株轻症和5株重症的标本经EV71 VP1、2A基因特异性引物扩增,产物分别为1082 bp、539 bpo 4.EV71病毒VP1、2A基因核苷酸序列测定与同源性比较、构建系统发生树：对扩增正确的10份PCR产物进行VP1基因和2A基因测序和遗传学分析,通过同源性比较和构建系统发生树发现,此10株EV71病毒和中国大陆已发表的5株EV71病毒(fuyangEU703814.1、xi anHM003207.1 shandongEU753418.1、shenzhenFJ607337.1、henanGU366191.1)全部属于C基因型,且核苷酸同源性较高,VP1基因和2A基因的同源性分别在94.7%-99.4%和93.6%-99.3%范围内。本次分离的10株EV71病毒与A、B基因型代表株比较,核苷酸同源性分别为81.0%-84.6%和78.4%-82.2%,差异较大。与已知的C1、C2、C3亚型代表株比较,核苷酸同源性在87.8%-90.2%,差异≥10%,与已知的C4亚型代表株比较,核苷酸同源性在96.8%-99.6%,因此认为可将这10株病毒划分为C4亚型。在系统发生树上,这10株病毒形成一个较独立的分支。 5.2A蛋白空间结构模拟图：从重症57号和轻症20号的2A蛋白结构模拟图可以看出,57号有三个蛋白结构域与20号不同；其它4个重症(36、37、43、55号)和4个轻症(30、31、40、52号)患者病毒的2A蛋白结构也有一些不同,但没有发现轻、重症各自之间一致性的结构特征。 6.2A基因克隆：经过PCR扩增和核酸测序证实,已成功获得2A基因。将空的T载体转入DH5a感受态细胞获得DH5a(T)。 7.重组表达质粒pIRES2-EGFP-2A的构建：将轻、重型病例各一例分离的EV71的2A基因DNA插入pIRES2-EGFP,成功构建重组质粒pIRES2-EGFP-2A,通过PCR扩增、双酶切和核酸测序已证实。结论 1.本次分离毒株均属于EV71 C基因亚型,且与中国大陆其他省区分离的毒株遗传关系紧密。 2. EV71 VP1、2A基因分别在经测序的10个毒株之间遗传关系紧密且轻症和重症病例毒株之间均不存在差异。 3.轻症、重症2A蛋白质二级、三级结构存在差异但未找到各自一致性的蛋白质结构特征。
[Abstract]:Enterovirus 71 (Enterovirus71, EV71) belongs to a small RNA virus, enterovirus, infection can cause HFMD herpangina, aseptic meningitis, encephalitis and poliomyelitis like paralysis disease, neurogenic pulmonary edema and other diseases associated with the nervous system. In recent years in the Asia Pacific region in cases of severe pandemic, central nervous system infection and even death cases, so the pathogenicity of EV71 virus, the pathogenesis, diagnosis and vaccine research more and more attention. The Ministry of health has been decided in May 2, 2008, will be included HFMD infectious disease prevention law of the class C infectious disease management.EV71 why lead a few severe or death cases, what are the 9 virulence factors
The VP1 gene encoding EV71 protein is an important virus neutralizing antigen determinant, VP1 gene sequence in addition to enterovirus in different serotypes of the basis for classification, classification of reference can also be used as a small RNA virus Kone different genera. The research showed that 11 viral protein EV71, the toxicity on yeast only 2A protein. 2A protein is a hydrolase, physiological significance in mammalian cells was normal, intestinal virus infection, can inhibit the growth of host, is one such mechanism: 2A protein cutting host eukaryotic initiation factor 4G (eIF4G eukaryotic translation initiation factor 4G), and also inhibit host protein synthesis. The report pointed out that polio virus 2A protein inhibits yeast (Saccharomyces cerevisiae) on the cell growth, the possible reason is that 2A used to cut some transcription protein Cytokine. Transient expression of EV71 2A protease leads to apoptosis. The expression of EV71 2A will cause the cleavage of eIF4G I, and eIF4GI is the key factor of host protein synthesis.
objective
The aim of this study is to investigate the VP1 gene, 2A gene sequence and predicted 2A protein structure of enterovirus 71 type mild and severe cases.
Virus was isolated by RD cells, and differential diagnosis of RT-PCR EV71 were amplified by VPl and nucleotide sequencing of 2A gene and DNAMAN software, homology comparison and phylogenetic tree, the predicted amino acid sequence of 2A protein space structure model by homology modeling using chimera software to investigate the two level, three level structure there are differences. The cloned 2A gene and constructed EV71 2A recombinant expression plasmid pIRES2-EGFP-2A.
Result
1.RD cell culture and virus isolation: RD cells were successfully cultured without contamination. After inoculation, the cytopathic effect (CPE) of the enterovirus showed: cell shrinkage, cytoplasmic granulation, pyknosis and final cell disruption.
2. RT-PCR differential diagnosis of EV71:50 clinical HFMD specimens, 30 of the detection is EV71, of which 13 are light (HFMD), and 17 are severe (HFMD encephalitis or myocarditis).
3.VPl, 2A gene amplification: 5 strains of light and 5 severe specimens were amplified by EV71 VP1,2A specific primers, and the products were 1082 BP, 539 BPO, respectively.
Determination and comparison of homology of 4.EV71 virus VP1,2A gene nucleotide sequence and phylogenetic tree of VP1 gene and 2A gene sequencing and genetic analysis of 10 PCR amplified product of correct, by homology comparison and phylogenetic tree showed that 5 strains of the EV71 virus and 10 strains of EV71 virus (published in mainland China fuyangEU703814.1 Xi, anHM003207.1 shandongEU753418.1, shenzhenFJ607337.1, henanGU366191.1) all belong to the C genotype, and nucleotide homology, VP1 gene and 2A gene homology respectively in 94.7%-99.4% and 93.6%-99.3% range. The separation of the 10 strains of EV71 and A, B genotype strains, the nucleotide homology were 81.0%-84.6% and 78.4%-82.2% that is quite different. With the known C1, C2, C3 subtype strains, the homology of nucleotide sequence in 87.8%-90.2%, the difference is more than 10%, with known C4 subtype strains comparison, Nucleotide homology is in 96.8%-99.6%, so it is thought that these 10 viruses can be divided into C4 subtypes. In the phylogenetic tree, these 10 viruses form a more independent branch.
Figure 5.2A: Simulation of protein structure can be seen from the 2A protein structure of severe and mild No. 57 No. 20 No. 57 simulation, three protein domains and 20 different; the other 4 patients (36,37,43,55) and 4 mild (30,31,40,52) 2A protein structure in patients with virus also has some different, but did not find the light, the structure between the respective characteristics of severe consistency.
6.2A gene cloning: the 2A gene has been successfully obtained by PCR amplification and nucleic acid sequencing. The empty T vector is transferred into the DH5a receptive cell to obtain DH5a (T).
7. construction of recombinant expression plasmid pIRES2-EGFP-2A: insert 2A gene DNA of EV71 isolated from a case of light and severe cases into pIRES2-EGFP, construct recombinant plasmid pIRES2-EGFP-2A successfully, and have been confirmed by PCR amplification, double digestion and DNA sequencing.
conclusion
1. the isolates belonged to the subtype of EV71 C gene and closely related to the strains isolated from other provinces in mainland China.
The genetic relationship between the 2. EV71 VP1,2A genes in the sequenced 10 strains was close, and there was no difference between the light and the severe case strains.
3. light symptoms, grade two of severe 2A protein, and differences in the three level structure but not found to be consistent with the structure of protein.

【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R373

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