本文關(guān)鍵詞：腸道病毒71型VP1基因及2A基因和蛋白質(zhì)特征　出處：《鄭州大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 腸道病毒71型 VP1基因 2A基因 同源性 系統(tǒng)發(fā)生樹 蛋白結(jié)構(gòu)模擬圖

【摘要】：腸道病毒71型(Enterovirus71, EV71)屬于小RNA病毒科腸道病毒屬,感染后可以導(dǎo)致手足口病、皰疹性咽峽炎、無(wú)菌性腦膜炎、腦炎和脊髓灰質(zhì)炎樣麻痹性疾病、神經(jīng)源性肺水腫等多種與神經(jīng)系統(tǒng)相關(guān)的疾病。近幾年在亞太地區(qū)的大流行中,出現(xiàn)嚴(yán)重中樞神經(jīng)系統(tǒng)感染病例,甚至死亡病例,因此有關(guān)EV71病毒的致病性、致病機(jī)理、診斷及疫苗方面的研究日益受到重視。國(guó)家衛(wèi)生部已于2008年5月2日決定,將手足口病納入傳染病防治法規(guī)定的丙類傳染病進(jìn)行管理。EV71為什么會(huì)導(dǎo)致少數(shù)重癥甚至死亡病例的出現(xiàn),毒力決定因子有哪些9 EV71的VP1基因編碼的蛋白質(zhì)是病毒重要的中和抗原決定簇,VP1基因序列除了是腸道病毒屬內(nèi)不同血清型分類的依據(jù),還可以作為小RNA病毒科內(nèi)不同屬的分類參考。研究表明EV71的11個(gè)病毒蛋白中,對(duì)酵母菌產(chǎn)生毒性的只有2A蛋白,而2A蛋白是水解酶,在正常的生理意義下,哺乳細(xì)胞被腸病毒感染后,宿主的生長(zhǎng)會(huì)受到抑制,其中一個(gè)機(jī)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),因而抑制宿主蛋白質(zhì)合成。也有報(bào)道指出,小兒麻痹病毒的2A蛋白會(huì)抑制釀酒酵母(Saccharomyces cerevisiae)的細(xì)胞生長(zhǎng),可能的原因是2A切割了一些用來(lái)進(jìn)行轉(zhuǎn)錄的蛋白質(zhì)因子。短暫的表達(dá)EV71 2A蛋白酶引起細(xì)胞凋亡,感染表達(dá)的EV71 2A會(huì)導(dǎo)致eIF4G I的裂解,而eIF4GI是宿主蛋白合成的關(guān)鍵因素。 目的 本課題旨在研究腸道病毒71型輕癥和重癥病例毒株VP1基因、2A基因的核苷酸序列及預(yù)測(cè)的2A蛋白質(zhì)結(jié)構(gòu)特征及是否有差別。方法 通過(guò)RD細(xì)胞分離病毒,然后RT-PCR鑒別診斷EV71,分別擴(kuò)增VPl、2A基因并進(jìn)行核苷酸序列測(cè)定,用DNAMAN軟件進(jìn)行同源性比較并構(gòu)建系統(tǒng)發(fā)生樹,通過(guò)同源建模用chimera軟件將2A氨基酸序列預(yù)測(cè)出蛋白空間結(jié)構(gòu)模擬圖,探討二級(jí)、三級(jí)結(jié)構(gòu)是否有差異�？寺�2A基因并構(gòu)建EV71 2A基因的重組表達(dá)質(zhì)粒pIRES2-EGFP-2A。 結(jié)果 1.RD細(xì)胞的培養(yǎng)及病毒分離：成功培養(yǎng)RD細(xì)胞,無(wú)污染,接種后腸道病毒的致細(xì)胞病變(CPE)表現(xiàn)為：細(xì)胞圓縮、胞漿顆�；�、核固縮以及最后細(xì)胞破裂。 2. RT-PCR鑒別診斷EV71：50份臨床手足口病標(biāo)本中有30份檢測(cè)是EV71,其中輕型(手足口)13份,重型(手足口并發(fā)腦炎或心肌炎)17份。 3.VPl、2A基因的擴(kuò)增：5株輕癥和5株重癥的標(biāo)本經(jīng)EV71 VP1、2A基因特異性引物擴(kuò)增,產(chǎn)物分別為1082 bp、539 bpo 4.EV71病毒VP1、2A基因核苷酸序列測(cè)定與同源性比較、構(gòu)建系統(tǒng)發(fā)生樹：對(duì)擴(kuò)增正確的10份PCR產(chǎn)物進(jìn)行VP1基因和2A基因測(cè)序和遺傳學(xué)分析,通過(guò)同源性比較和構(gòu)建系統(tǒng)發(fā)生樹發(fā)現(xiàn),此10株EV71病毒和中國(guó)大陸已發(fā)表的5株EV71病毒(fuyangEU703814.1、xi anHM003207.1 shandongEU753418.1、shenzhenFJ607337.1、henanGU366191.1)全部屬于C基因型,且核苷酸同源性較高,VP1基因和2A基因的同源性分別在94.7%-99.4%和93.6%-99.3%范圍內(nèi)。本次分離的10株EV71病毒與A、B基因型代表株比較,核苷酸同源性分別為81.0%-84.6%和78.4%-82.2%,差異較大。與已知的C1、C2、C3亞型代表株比較,核苷酸同源性在87.8%-90.2%,差異≥10%,與已知的C4亞型代表株比較,核苷酸同源性在96.8%-99.6%,因此認(rèn)為可將這10株病毒劃分為C4亞型。在系統(tǒng)發(fā)生樹上,這10株病毒形成一個(gè)較獨(dú)立的分支。 5.2A蛋白空間結(jié)構(gòu)模擬圖：從重癥57號(hào)和輕癥20號(hào)的2A蛋白結(jié)構(gòu)模擬圖可以看出,57號(hào)有三個(gè)蛋白結(jié)構(gòu)域與20號(hào)不同；其它4個(gè)重癥(36、37、43、55號(hào))和4個(gè)輕癥(30、31、40、52號(hào))患者病毒的2A蛋白結(jié)構(gòu)也有一些不同,但沒有發(fā)現(xiàn)輕、重癥各自之間一致性的結(jié)構(gòu)特征。 6.2A基因克�。航�(jīng)過(guò)PCR擴(kuò)增和核酸測(cè)序證實(shí),已成功獲得2A基因。將空的T載體轉(zhuǎn)入DH5a感受態(tài)細(xì)胞獲得DH5a(T)。 7.重組表達(dá)質(zhì)粒pIRES2-EGFP-2A的構(gòu)建：將輕、重型病例各一例分離的EV71的2A基因DNA插入pIRES2-EGFP,成功構(gòu)建重組質(zhì)粒pIRES2-EGFP-2A,通過(guò)PCR擴(kuò)增、雙酶切和核酸測(cè)序已證實(shí)。 結(jié)論 1.本次分離毒株均屬于EV71 C基因亞型,且與中國(guó)大陸其他省區(qū)分離的毒株遺傳關(guān)系緊密。 2. EV71 VP1、2A基因分別在經(jīng)測(cè)序的10個(gè)毒株之間遺傳關(guān)系緊密且輕癥和重癥病例毒株之間均不存在差異。 3.輕癥、重癥2A蛋白質(zhì)二級(jí)、三級(jí)結(jié)構(gòu)存在差異但未找到各自一致性的蛋白質(zhì)結(jié)構(gòu)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.

【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R373

【參考文獻(xiàn)】

相關(guān)期刊論文 前6條

1 崔愛利,許文波,李秀珠,胡家瑜,凌華,唐偉,楊智宏,張燕,陳立,Hiroyuki Shimizu;腸道病毒71型的RT-PCR診斷及基因特征[J];病毒學(xué)報(bào);2004年02期

2 何家鑫,沈曉娜;手足口病流行特點(diǎn)及其防治[J];海峽預(yù)防醫(yī)學(xué)雜志;2001年03期

3 ;Molecular epidemiology and evolution of worldwide enterovirus 71 strains isolated from 1970 to 2004[J];Chinese Science Bulletin;2007年11期

4 張愛香,李燕婷,張家琪,吳寰宇,李秀珠,黃惠敏,顧寶柯,蔣杰辰;一起由腸道病毒71型引起手足口病爆發(fā)的調(diào)查[J];上海預(yù)防醫(yī)學(xué)雜志;2001年12期

5 黃學(xué)勇;陳豪敏;馬宏;于賀娟;晁靈;許汴利;;2009年河南腸道病毒71型VP1基因特征分析[J];中國(guó)預(yù)防醫(yī)學(xué)雜志;2010年07期

6 何雅青,楊帆,李良成,楊洪,陳淑霞,金奇;我國(guó)深圳地區(qū)手足口病患者腸道病毒71型的分離與鑒定[J];中華實(shí)驗(yàn)和臨床病毒學(xué)雜志;1999年01期

相關(guān)碩士學(xué)位論文 前1條

1 唐啟慧;腸道病毒71型VP1基因抗原的表達(dá)及其腺病毒載體穿梭質(zhì)粒的構(gòu)建與鑒定[D];中國(guó)科學(xué)院研究生院（南海海洋研究所）;2008年

，

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