本文選題：手足口病　切入點(diǎn)：腸道病毒　出處：《泰山醫(yī)學(xué)院》2012年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：目的 手足口病是一種流行性傳染病，多由腸道病毒引起，癥狀主要表現(xiàn)為手、足和口腔粘膜的皰疹，多數(shù)是良性、自限性疾病，也可出現(xiàn)心、肺和神經(jīng)系統(tǒng)等嚴(yán)重并發(fā)癥。我們利用對(duì)腸道病毒具有較高特異性的兼并引物和自行設(shè)計(jì)的分型引物，檢測(cè)手足口病患兒咽拭子標(biāo)本，同時(shí)，通過(guò)腸道病毒基因組VP1編碼區(qū)序列的測(cè)定和分析，研究手足口病病原體檢測(cè)和分型方法，掌握其遺傳進(jìn)化特征。 人類(lèi)腸道病毒屬于小RNA病毒科腸道病毒屬，該屬包括脊髓灰質(zhì)炎病毒、柯薩奇病毒、�？刹《竞湍c道病毒，其中腸道病毒71型是引起手足口病最重要的病原體，由其引起的手足口病癥狀較重，常伴有無(wú)菌性腦膜炎、腦干腦炎、脊髓灰質(zhì)炎樣麻痹等并發(fā)癥，嚴(yán)重者甚至?xí)䦟?dǎo)致死亡。該病毒存在較明顯的變異和重組現(xiàn)象，因此到目前為止仍沒(méi)有很好的預(yù)防和治療方法。本課題通過(guò)對(duì)腸道病毒71型VP1區(qū)序列及全基因組序列的分析，研究腸道病毒71型的變異、重組規(guī)律，為疫苗的研發(fā)、藥物靶位的篩選及防控策略的完善等提供理論基礎(chǔ)。 除柯薩奇病毒A16（CVA16）和腸道病毒71型（EV71）以外，柯薩奇病毒A10（CVA10）、柯薩奇病毒A6（CVA6）也是手足口病比較常見(jiàn)的病原體。本課題通過(guò)對(duì)手足口病CVA10VPl蛋白的生物信息學(xué)研究，分析和預(yù)測(cè)該病毒VP1蛋白的理化特性、空間結(jié)構(gòu)以及該蛋白的B細(xì)胞抗原表位。 方法 1.標(biāo)本采集 標(biāo)本采集自山東大學(xué)齊魯兒童醫(yī)院住院手足口病患兒的咽拭子標(biāo)本。標(biāo)本加入適當(dāng)生理鹽水，劇烈振蕩洗下咽拭子上粘附的病毒及含有病毒的細(xì)胞等，自然沉淀后吸取上清液，加入適量抗生素，置4℃條件下，備用。 2. RNA提取 采用北京天根公司病毒RNA提取試劑盒，按說(shuō)明書(shū)進(jìn)行。提取的RNA溶于60ulRnase-free ddH2O中，直接用于RT-PCR或置－20℃以下低溫保存。 3.腸道病毒分子鑒定 用腸道病毒通用實(shí)時(shí)熒光RT-PCR核酸檢測(cè)試劑盒同時(shí)分別進(jìn)行腸道病毒屬、EV71和CVA16型進(jìn)行分子鑒定。 4.腸道病毒VP1編碼序列分析 （1）引物設(shè)計(jì) 合成對(duì)腸道病毒VP1區(qū)3＇端序列具有較高特異性的兼并引物040-011，同時(shí)通過(guò)檢索GenBank中EV71和CVA10病毒基因型VP1序列，分別設(shè)計(jì)分型引物。 （2）RT-PCR擴(kuò)增 通過(guò)RT-PCR方法擴(kuò)增EV71、CVA16、CVA6和CVA10病毒基因型VP1區(qū)核苷酸序列。 （3）DNA純化 瓊脂糖凝膠電泳對(duì)擴(kuò)增產(chǎn)物進(jìn)行分離，利用北京天根公司瓊脂糖凝膠DNA回收試劑盒對(duì)DNA進(jìn)行回收、純化。 （4）序列測(cè)定和分析 回收后的DNA進(jìn)行序列測(cè)定。采用生物信息學(xué)軟件DNAMAN5.2.2和BioEdit7.0.9.0進(jìn)行序列比對(duì)并構(gòu)建系統(tǒng)進(jìn)化樹(shù)。 5. EV71基因組重組分析 使用基因重組分析軟件RDP和Simplot_v3.5.1，對(duì)EV71基因組進(jìn)行重組分析。 6. CVA10VP1蛋白生物信息學(xué)分析 （1）CVA10VP1蛋白二級(jí)結(jié)構(gòu)預(yù)測(cè) 利用R4（Gamier-Osguthorpe-Robson方法）、HNN（Hierarchical人工神經(jīng)網(wǎng)絡(luò)預(yù)測(cè)法）、PHD（多重比對(duì)人工神經(jīng)網(wǎng)絡(luò)比對(duì)預(yù)測(cè)結(jié)構(gòu)法）、Predator（單序列分析人工神經(jīng)網(wǎng)絡(luò)預(yù)測(cè)結(jié)構(gòu)法）、SOPMA（改進(jìn)型自我優(yōu)化預(yù)測(cè)結(jié)構(gòu)法）等方法預(yù)測(cè)VP1蛋白的二級(jí)結(jié)構(gòu)（無(wú)規(guī)卷曲、β-片層、α-螺旋和β-轉(zhuǎn)角）。 （2）CVA10VP1蛋白三級(jí)結(jié)構(gòu)預(yù)測(cè) 利用Expasy（http://au.Expasy.org/tools/）中的SWISS-MODEL三級(jí)結(jié)構(gòu)同源建模，預(yù)測(cè)VPl蛋白的空間構(gòu)象并建模。 （3）CVA10VP1蛋白親水性、柔韌性、表面可能性和抗原表位預(yù)測(cè)和分析 應(yīng)用DNAstar軟件的Protean，采用Kyte-Doolittle、Karplus-Schultz、Emini和Jameson-Wolf對(duì)氨基酸的親水性、柔韌性、表面可能性及抗原指數(shù)進(jìn)行單參數(shù)分析。 （4）CVA10VP1蛋白抗原表位綜合分析 用ABCpred和DiscoTope服務(wù)器預(yù)測(cè)B細(xì)胞表位。將單參數(shù)預(yù)測(cè)結(jié)果匯總，結(jié)合二級(jí)結(jié)構(gòu)中無(wú)規(guī)卷曲區(qū)域確定為B細(xì)胞優(yōu)勢(shì)表位。最后采用ABCpred和DiscoTope驗(yàn)證表位預(yù)測(cè)結(jié)果。 結(jié)果 1.5個(gè)EV71濟(jì)南分離株核苷酸同源性為95.7%-99.1%，氨基酸同源性為98.7%-100%。在進(jìn)化關(guān)系上，它們與EV71C4a亞型的同源性較高，核苷酸同源性為90.8%-98.8%，故而屬于C4a亞型。 2.2個(gè)CVA16濟(jì)南分離株核苷酸和氨基酸同源性較高，分別為97.7%和96.8%。它們與CVA16B1b亞型的同源性較高，核苷酸和氨基酸的同源性分別為90.5%-95.5%和96.8%-100%。JN-C07和JN-B11與大部分毒株相比，無(wú)氨基酸變異位點(diǎn)出現(xiàn)。 3.39份腸道病毒通用型陽(yáng)性非EV71、非CVA16標(biāo)本，用040-011引物擴(kuò)增后，有5例為CVA10，1例為CVA6。其余33份樣本的腸道病毒血清型待定。 4.利用自行設(shè)計(jì)的CVA10分型引物P16、P11.2擴(kuò)增上述33份血清型待定的標(biāo)本，其中4份標(biāo)本確定CVA10，核苷酸同源性為94.5%-99.0%，氨基酸同源性為97.2%-99.5%。 5.在線Blast程序比對(duì)，，JN-D04屬于CVA6型，與2003年-2009年報(bào)告的12個(gè)CVA6毒株核苷酸同源性為75.1%-92.3%，同源性最高的是臺(tái)灣2008年的EU908152，氨基酸同源性為82.0%-89.0%；氨基酸在767、773、776、777、792、806、812、824、827、841、843和845位發(fā)生變異。 6.2010年濟(jì)南市2個(gè)病毒株在P1區(qū)與EV71C型具有較高相似度（Simplot圖中顯示相似度80%），在2B-3B區(qū)之間與B基因型相似度較高（75%）。3＇末端這兩個(gè)病毒株與EV71各基因型代表株的相似度均不高（75%），而在3C到3＇UTR區(qū)與CVA16G-10相似較高（75%）。 7. JN-C10與CVA10D型基因組同源性最高，核苷酸和氨基酸的同源性分別為91.5%-97.6%和90.4%-98.6%。測(cè)序所得VPl基因長(zhǎng)為732個(gè)核苷酸，其相對(duì)分子質(zhì)量為60580，理論等電點(diǎn)為5.10。VPl蛋白二級(jí)結(jié)構(gòu)中以無(wú)規(guī)卷曲為主，無(wú)跨膜區(qū)域，屬于胞外蛋白。發(fā)現(xiàn)蛋白質(zhì)數(shù)據(jù)庫(kù)(PDB)一個(gè)已知結(jié)構(gòu)的蛋白質(zhì)（編號(hào)為3vbhA）與VP1具有65%的序列一致性，并以此為模板，得到VP1蛋白三級(jí)結(jié)構(gòu)模型。最后，綜合多種抗原表位分析方法得出，JN-C10VPl蛋白的B細(xì)胞抗原表位可能是12-23、100-110、115-125、169-180、195-215及220-2385個(gè)區(qū)段。 結(jié)論 1.兼并引物040-011對(duì)HEVA類(lèi)腸道病毒VP1區(qū)3＇端具有較高的特異性。 2.引起手足口病的腸道病毒，除EV71和CVA16外，CVA10和CVA6也是其常見(jiàn)病原體。 3. CVA10能引起重癥手足口病和病毒性腦炎、癲癇等嚴(yán)重的臨床表現(xiàn)。 4.2010年-2011年濟(jì)南EV71分離株為C4a亞型，CVA16分離株為B1b亞型，CVA10分離株為D基因型，與近幾年中國(guó)大陸各基因型優(yōu)勢(shì)株流行趨勢(shì)基本一致。 5.2010年EV71濟(jì)南分離株存在基因型內(nèi)和型間雙重組現(xiàn)象。 6. CVA10VP1基因編碼蛋白存在多個(gè)抗原表位區(qū)域，可能是免疫診斷、藥物作用和疫苗研制的靶位，將為CVA10診斷、治療和預(yù)防提供參考依據(jù)。
[Abstract]:objective
HFMD is a kind of epidemic disease, caused by intestinal viruses, symptoms of hand, foot and mouth mucosa herpes, most are benign, self limiting disease, also can appear serious complications of heart, lung and nervous system. We use degenerate primers with high specificity for enterovirus the type of designed primers, HFMD detection swab specimens, at the same time, through the determination and sequence of VP1 encoding region of enterovirus genome analysis, research and classification of HFMD pathogen detection, the genetic evolution characteristics.
Human enterovirus RNA virus belongs to the small intestinal virus genus, the genus including polio virus, Coxsackie virus, ECHO virus and enterovirus, enterovirus 71 is the most important cause of HFMD pathogens caused by HFMD severe symptoms, often accompanied by aseptic meningitis, brain stem encephalitis, poliomyelitis like paralysis and other complications, and even lead to death. The mutation andrecombination are obvious, so far still no better prevention and treatment. This paper based on the analysis of enterovirus 71 VP1 sequence and genomic sequence, mutation, study of enterovirus type 71 recombinant rules for the vaccine research and development, and provide a theoretical basis for the screening of drug targets and control strategy of the perfect.
In addition to Coxsackie virus A16 (CVA16) and enterovirus 71 (EV71), coxsackievirus A10 (CVA10), coxsackievirus A6 (CVA6) is a common pathogen of HFMD. This paper studies the biological information of CVA10VPl protein of foot and mouth disease, physicochemical characteristics analysis and prediction of the virus VP1 protein, the spatial structure and the protein of B cell antigen epitope.
Method
1. specimen collection
Collected from Shandong University Qilu children's Hospital of HFMD from throat swabs specimens. Specimens with appropriate physiological saline, oscillation washing swab on the adhesion of virus and virus containing cells, natural precipitation to obtain supernatant, adding proper amount of antibiotics, the temperature of 4 DEG C, standby.
2. RNA extraction
Beijing Tian Gen virus RNA extraction kit was carried out according to the instructions. The extracted RNA was dissolved in 60ulRnase-free ddH2O, and was directly applied to RT-PCR or under 20 degrees Celsius for cryopreservation.
Molecular identification of 3. enterovirus
The general real-time fluorescent RT-PCR nucleic acid detection kit of enterovirus was used for the identification of enterovirus, EV71 and CVA16 respectively.
Analysis of the encoding sequence of 4. enterovirus VP1
(1) primer design
The primer 040-011 was synthesized with high specificity for the 3 'end sequence of enterovirus VP1 region. Meanwhile, the typing primers were designed by retrieving the EV71 and CVA10 virus genotype VP1 sequences in GenBank.
(2) RT-PCR amplification
The nucleotide sequences of EV71, CVA16, CVA6 and CVA10 virus genotype VP1 region were amplified by RT-PCR method.
(3) purification of DNA
The agarose gel electrophoresis was used to separate the amplified products, and the DNA was recovered and purified by the agarose gel DNA recovery kit of Beijing Tian Gen company.
(4) sequence determination and analysis
The reclaimed DNA was sequenced. The sequence alignment was compared with the bioinformatics software DNAMAN5.2.2 and BioEdit7.0.9.0, and the phylogenetic tree was constructed.
Analysis of 5. EV71 genome reorganization
The recombinant analysis software RDP and Simplot_v3.5.1 were used to reorganize the EV71 genome.
Bioinformatics analysis of 6. CVA10VP1 protein
(1) prediction of the two grade structure of CVA10VP1 protein
The use of R4 (Gamier-Osguthorpe-Robson), HNN (Hierarchical forecasting method of artificial neural network (PHD), multiple alignment of artificial neural network on prediction method, Predator (structure) prediction method of single sequence structure analysis of artificial neural network (SOPMA), improved self optimizing structure prediction method) methods two VP1 protein structure prediction (no random coil, beta sheet, alpha helix and beta angle).
(2) prediction of the three grade structure of CVA10VP1 protein
The spatial conformation and modeling of VPl protein are predicted by using the homologous modeling of the SWISS-MODEL three level structure in the Expasy (http://au.Expasy.org/tools/).
(3) the hydrophilicity, flexibility, surface possibility and epitope prediction and analysis of CVA10VP1 protein
Using DNAstar software Protean, Kyte-Doolittle, Karplus-Schultz, Emini and Jameson-Wolf were used for single parameter analysis of hydrophilicity, flexibility, surface potential and antigen index of amino acids.
(4) comprehensive analysis of epitopes of CVA10VP1 protein
ABCpred and DiscoTope servers were used to predict B cell epitopes. The single parameter prediction results were aggregated, combined with the random coil area in the two level structure to be the dominant epitope of B cells. Finally, ABCpred and DiscoTope were used to verify the prediction results.
Result
The nucleotide homology of the 1.5 EV71 Ji'nan isolates is 95.7%-99.1%, and the amino acid homology is 98.7%-100%.. On the evolutionary relationship, they have high homology with EV71C4a subtype, and the nucleotide homology is 90.8%-98.8%, so they belong to C4a subtype.
2.2 CVA16 Ji'nan isolate nucleotide and amino acid homology, respectively 97.7% and 96.8%. are highly homologous with the CVA16B1b subtype, nucleotide and amino acid homology were 90.5%-95.5% and 96.8%-100%.JN-C07 and JN-B11 compared with most of the strains, no amino acid mutation.
3.39 cases of enterovirus universal positive non EV71 and non CVA16 specimens were amplified by 040-011 primers. 5 cases were CVA10,1, CVA6. and the remaining 33 samples were enterovirus serotype.
4., the above 33 serotype pending specimens were amplified by the self designed CVA10 primer P16 and P11.2, 4 of which were identified as CVA10, the nucleotide homology was 94.5%-99.0%, and the amino acid homology was 97.2%-99.5%..
5. online Blast program on JN-D04, belongs to CVA6 type, and the 2003 -2009 report of 12 CVA6 strains nucleotide homology was 75.1%-92.3%, homology is the highest in Taiwan 2008 EU908152, the homology of amino acid was 82.0%-89.0%; amino acids in 767773776777792806812824827841843 and 845 variation.
6.2010 years of Ji'nan City, 2 strains in P1 and EV71C with high similarity (Figure Simplot show 80% similarity), and B genotype in high similarity between the 2B-3B region (75%) and the similarity of different genotypes of EV71 strains.3 'end of the two strains of the virus is not high (75%), and in the 3C to 3 UTR region and CVA16G-10 high similarity (75%).
7. JN-C10 and CVA10D genome nucleotide homology of nucleotide and amino acid homology were 91.5%-97.6% and 90.4%-98.6%. sequencing of the VPl gene is 732 nucleotides long, the relative molecular mass of 60580 and an isoelectric point of two secondary structure of 5.10.VPl protein mainly random coil, without transmembrane domain, which belongs to the extracellular protein protein database (PDB). It is found that a protein of known structure (3vbhA) sequence with 65% identity with VP1, and used as the template, VP1 three protein secondary structure model. Finally, multi epitope analysis, JN-C10VPl protein B cell epitope may be 12-23100-110115-125169-180195-215 and 220-2385 a section.
conclusion
1. annexed primer 040-011 has a high specificity on the 3 'end of the VP1 region of the HEVA enterovirus.
2. the enteroviruses that cause hand foot and mouth disease, except for EV71 and CVA16, are also the common pathogens of CVA10 and CVA6.
3. CVA10 can cause severe hand foot and mouth disease and viral encephalitis, epilepsy and other serious clinical manifestations.
In the 4.2010 years -2011, EV71 isolates from Ji'nan were C4a subtypes, CVA16 isolates were B1b subtypes, CVA10 isolates were D genotypes, which were basically consistent with the prevalent trend of dominant genotypes in mainland China in recent years.
In 5.2010 years, there was a double group of genotypes and genotypes in the EV71 isolates of EV71.
6., there are multiple epitope regions of CVA10VP1 gene encoded protein. It may be a target for immunodiagnosis, drug action and vaccine development. It will provide a reference for diagnosis, treatment and prevention of CVA10.

【學(xué)位授予單位】：泰山醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R725.1

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