手足口病重要病毒序列分析及生物信息学表征

发布时间：2018-03-13 00:25

本文选题：手足口病　切入点：肠道病毒　出处：《泰山医学院》2012年硕士论文　论文类型：学位论文

【摘要】：目的手足口病是一种流行性传染病，多由肠道病毒引起，症状主要表现为手、足和口腔粘膜的疱疹，多数是良性、自限性疾病，也可出现心、肺和神经系统等严重并发症。我们利用对肠道病毒具有较高特异性的兼并引物和自行设计的分型引物，检测手足口病患儿咽拭子标本，同时，通过肠道病毒基因组VP1编码区序列的测定和分析，研究手足口病病原体检测和分型方法，掌握其遗传进化特征。人类肠道病毒属于小RNA病毒科肠道病毒属，该属包括脊髓灰质炎病毒、柯萨奇病毒、埃可病毒和肠道病毒，其中肠道病毒71型是引起手足口病最重要的病原体，由其引起的手足口病症状较重，常伴有无菌性脑膜炎、脑干脑炎、脊髓灰质炎样麻痹等并发症，严重者甚至会导致死亡。该病毒存在较明显的变异和重组现象，因此到目前为止仍没有很好的预防和治疗方法。本课题通过对肠道病毒71型VP1区序列及全基因组序列的分析，研究肠道病毒71型的变异、重组规律，为疫苗的研发、药物靶位的筛选及防控策略的完善等提供理论基础。除柯萨奇病毒A16（CVA16）和肠道病毒71型（EV71）以外，柯萨奇病毒A10（CVA10）、柯萨奇病毒A6（CVA6）也是手足口病比较常见的病原体。本课题通过对手足口病CVA10VPl蛋白的生物信息学研究，分析和预测该病毒VP1蛋白的理化特性、空间结构以及该蛋白的B细胞抗原表位。方法 1.标本采集标本采集自山东大学齐鲁儿童医院住院手足口病患儿的咽拭子标本。标本加入适当生理盐水，剧烈振荡洗下咽拭子上粘附的病毒及含有病毒的细胞等，自然沉淀后吸取上清液，加入适量抗生素，置4℃条件下，备用。 2. RNA提取采用北京天根公司病毒RNA提取试剂盒，按说明书进行。提取的RNA溶于60ulRnase-free ddH2O中，直接用于RT-PCR或置－20℃以下低温保存。 3.肠道病毒分子鉴定用肠道病毒通用实时荧光RT-PCR核酸检测试剂盒同时分别进行肠道病毒属、EV71和CVA16型进行分子鉴定。 4.肠道病毒VP1编码序列分析（1）引物设计合成对肠道病毒VP1区3＇端序列具有较高特异性的兼并引物040-011，同时通过检索GenBank中EV71和CVA10病毒基因型VP1序列，分别设计分型引物。（2）RT-PCR扩增通过RT-PCR方法扩增EV71、CVA16、CVA6和CVA10病毒基因型VP1区核苷酸序列。（3）DNA纯化琼脂糖凝胶电泳对扩增产物进行分离，利用北京天根公司琼脂糖凝胶DNA回收试剂盒对DNA进行回收、纯化。（4）序列测定和分析回收后的DNA进行序列测定。采用生物信息学软件DNAMAN5.2.2和BioEdit7.0.9.0进行序列比对并构建系统进化树。 5. EV71基因组重组分析使用基因重组分析软件RDP和Simplot_v3.5.1，对EV71基因组进行重组分析。 6. CVA10VP1蛋白生物信息学分析（1）CVA10VP1蛋白二级结构预测利用R4（Gamier-Osguthorpe-Robson方法）、HNN（Hierarchical人工神经网络预测法）、PHD（多重比对人工神经网络比对预测结构法）、Predator（单序列分析人工神经网络预测结构法）、SOPMA（改进型自我优化预测结构法）等方法预测VP1蛋白的二级结构（无规卷曲、β-片层、α-螺旋和β-转角）。（2）CVA10VP1蛋白三级结构预测利用Expasy（http://au.Expasy.org/tools/）中的SWISS-MODEL三级结构同源建模，预测VPl蛋白的空间构象并建模。（3）CVA10VP1蛋白亲水性、柔韧性、表面可能性和抗原表位预测和分析应用DNAstar软件的Protean，采用Kyte-Doolittle、Karplus-Schultz、Emini和Jameson-Wolf对氨基酸的亲水性、柔韧性、表面可能性及抗原指数进行单参数分析。（4）CVA10VP1蛋白抗原表位综合分析用ABCpred和DiscoTope服务器预测B细胞表位。将单参数预测结果汇总，结合二级结构中无规卷曲区域确定为B细胞优势表位。最后采用ABCpred和DiscoTope验证表位预测结果。结果 1.5个EV71济南分离株核苷酸同源性为95.7%-99.1%，氨基酸同源性为98.7%-100%。在进化关系上，它们与EV71C4a亚型的同源性较高，核苷酸同源性为90.8%-98.8%，故而属于C4a亚型。 2.2个CVA16济南分离株核苷酸和氨基酸同源性较高，分别为97.7%和96.8%。它们与CVA16B1b亚型的同源性较高，核苷酸和氨基酸的同源性分别为90.5%-95.5%和96.8%-100%。JN-C07和JN-B11与大部分毒株相比，无氨基酸变异位点出现。 3.39份肠道病毒通用型阳性非EV71、非CVA16标本，用040-011引物扩增后，有5例为CVA10，1例为CVA6。其余33份样本的肠道病毒血清型待定。 4.利用自行设计的CVA10分型引物P16、P11.2扩增上述33份血清型待定的标本，其中4份标本确定CVA10，核苷酸同源性为94.5%-99.0%，氨基酸同源性为97.2%-99.5%。 5.在线Blast程序比对，，JN-D04属于CVA6型，与2003年-2009年报告的12个CVA6毒株核苷酸同源性为75.1%-92.3%，同源性最高的是台湾2008年的EU908152，氨基酸同源性为82.0%-89.0%；氨基酸在767、773、776、777、792、806、812、824、827、841、843和845位发生变异。 6.2010年济南市2个病毒株在P1区与EV71C型具有较高相似度（Simplot图中显示相似度80%），在2B-3B区之间与B基因型相似度较高（75%）。3＇末端这两个病毒株与EV71各基因型代表株的相似度均不高（75%），而在3C到3＇UTR区与CVA16G-10相似较高（75%）。 7. JN-C10与CVA10D型基因组同源性最高，核苷酸和氨基酸的同源性分别为91.5%-97.6%和90.4%-98.6%。测序所得VPl基因长为732个核苷酸，其相对分子质量为60580，理论等电点为5.10。VPl蛋白二级结构中以无规卷曲为主，无跨膜区域，属于胞外蛋白。发现蛋白质数据库(PDB)一个已知结构的蛋白质（编号为3vbhA）与VP1具有65%的序列一致性，并以此为模板，得到VP1蛋白三级结构模型。最后，综合多种抗原表位分析方法得出，JN-C10VPl蛋白的B细胞抗原表位可能是12-23、100-110、115-125、169-180、195-215及220-2385个区段。结论 1.兼并引物040-011对HEVA类肠道病毒VP1区3＇端具有较高的特异性。 2.引起手足口病的肠道病毒，除EV71和CVA16外，CVA10和CVA6也是其常见病原体。 3. CVA10能引起重症手足口病和病毒性脑炎、癫痫等严重的临床表现。 4.2010年-2011年济南EV71分离株为C4a亚型，CVA16分离株为B1b亚型，CVA10分离株为D基因型，与近几年中国大陆各基因型优势株流行趋势基本一致。 5.2010年EV71济南分离株存在基因型内和型间双重组现象。 6. CVA10VP1基因编码蛋白存在多个抗原表位区域，可能是免疫诊断、药物作用和疫苗研制的靶位，将为CVA10诊断、治疗和预防提供参考依据。
[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.

【学位授予单位】：泰山医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R725.1

【参考文献】