本文選題：腸道病毒71型 + 核苷酸序列�。� 參考：《大連醫(yī)科大學(xué)》2012年碩士論文

【摘要】：目的：了解2009～2011年大連地區(qū)EV71流行情況；對(duì)分離出的EV71毒株進(jìn)行部分基因片段的核苷酸和氨基酸序列測(cè)定，構(gòu)建系統(tǒng)發(fā)生樹(shù)和同源性分析，以確定其基因型別；對(duì)與毒株毒力相關(guān)基因序列進(jìn)行比較分析，探尋毒株毒力與基因序列之間的關(guān)系，為今后EV71的防治及防控提供科學(xué)依據(jù)。 方法：收集大連市手足口病監(jiān)測(cè)哨點(diǎn)醫(yī)院門(mén)診就診病例和手足口樣病例，將來(lái)自監(jiān)測(cè)哨點(diǎn)醫(yī)院的咽拭子和糞便標(biāo)本用熒光RT-PCR方法檢測(cè)為EV71的陽(yáng)性標(biāo)本接種于RD細(xì)胞，經(jīng)傳代培養(yǎng)后分離EV71病毒，，提取病毒RNA，利用RT-PCR方法分別擴(kuò)增出VP1、VP4、5’UTR基因片斷，對(duì)擴(kuò)增產(chǎn)物進(jìn)行核苷酸序列測(cè)定，用DNAStar軟件與EV71各基因亞型代表株進(jìn)行序列比較，分析其核苷酸序列和氨基酸序列的同源性，以及不同臨床癥狀的毒株與核苷酸和氨基酸序列之間的關(guān)系。 結(jié)果： 1、2009～2011年期間共檢測(cè)533份樣品，其中EV71陽(yáng)性333份，陽(yáng)性率為62.48%。533份病例中，男317例，女216例，男女比例為1.47:1。其中男性患者EV71陽(yáng)性率為61.51%，女型患者EV71陽(yáng)性率為63.89%。0～5歲為手足口病高發(fā)年齡，以托幼兒童為主。 2、VP1區(qū)序列分析表明：選取9株RD細(xì)胞分離培養(yǎng)的EV71毒株進(jìn)行VP1序列擴(kuò)增并測(cè)序，其結(jié)果用DNAStar軟件分析，這9株EV71毒株均屬于C4a亞型，其核苷酸之間的同源性為88.7%～99.9%；氨基酸之間的同源性為96.7%～100%。與BRCR-CA-70的核苷酸同源性為79.4%～81.5%；與B型核苷酸同源性為：81.8%～84.7%；與C亞型核苷酸同源性為85.2%～99.2%,而與C4同源性高達(dá)90.0%～99.2%，其中與C4a的同源性最高為93.7%～99.2%，與C4b的同源性為90.0%～94.1%。對(duì)重癥來(lái)源的毒株，在第227位核苷酸發(fā)生了T→C的堿基置換，而輕癥來(lái)源的毒株均未發(fā)生改變。對(duì)于氨基酸序列比較上未發(fā)現(xiàn)一致性改變。 3、VP4區(qū)序列分析表明：9株毒株的VP4氨基酸序列高度一致，只是在第52位dalian1-09株發(fā)生了K→R的改變，第62位氨基酸dalian4-10株發(fā)生了T→S的改變，其余各株氨基酸序列均一致。沒(méi)有發(fā)現(xiàn)重癥來(lái)源的毒株和輕癥來(lái)源的毒株在氨基酸或核苷酸序列上相應(yīng)固定的變異。 4、5’UTR區(qū)序列分析表明：9株毒株在第208位核苷酸,重癥來(lái)源的樣品發(fā)生了堿基置換（G→A）；在第254位核苷酸，重癥來(lái)源的樣品發(fā)生了堿基置換（A→G）。氨基酸序列未發(fā)現(xiàn)一致性改變。 結(jié)論： 1、2009年-2011年大連地區(qū)手足口病的主要病原體為EV71，其基因型別為C4a亞型，這與中國(guó)大陸的大部分地區(qū)流行株相一致。 2、基于VP4的基因分型與VP1的基因分型相一致，均為C4a亞型，且VP4序列高度保守。 3、在毒株的毒力分析上，重癥來(lái)源的毒株，在VP1的第227位核苷酸發(fā)生了T→C的堿基置換，而輕癥來(lái)源的毒株均未發(fā)生改變。VP4的核苷酸序列和氨基酸序列均未發(fā)現(xiàn)與臨床癥狀相對(duì)應(yīng)的改變，而在5’UTR序列上發(fā)現(xiàn)了第208位核苷酸的堿基置換（G→A），這一結(jié)果還有待于進(jìn)行下一步的研究。
[Abstract]:Objective: to investigate the prevalence of EV71 in Dalian from 2009 to 2011, to determine the nucleotide and amino acid sequences of partial gene fragments of isolated EV71 strains, and to construct phylogenetic tree and homology analysis to determine their genotypes. In order to find out the relationship between virulence and gene sequence of virulence of virus Methods: the outpatient and hand foot mouth like cases of surveillance sentinel hospital of hand, foot and mouth in Dalian were collected. Throat swabs and stool samples from sentinel hospital were inoculated into Rd cells by fluorescence RT-PCR method. After passage culture, EV71 virus was isolated, virus RNAs were extracted, and VP1- VP4- 5 UTR gene fragments were amplified by RT-PCR method. The amplified products were sequenced by nucleotide sequencing and compared with the representative strains of EV71 gene subtypes by DNAStar software. The homology of nucleotide sequence and amino acid sequence, and the relationship between nucleotide sequence and amino acid sequence of virus strains with different clinical symptoms were analyzed. Results: From 2009 to 2011, a total of 533 samples were detected, of which 333 were positive for EV71, the positive rate was 62.48.533 cases, 317 males and 216 females, the ratio of male to female was 1.47: 1. The positive rate of EV71 was 61.51% in male patients and 63.89% in female patients. 2 sequence analysis of VP1 region showed that 9 EV71 strains isolated from Rd cells were amplified by VP1 sequence and sequenced. The results showed that the 9 EV71 strains belonged to C4a subtype by DNAStar software. The homology between nucleotides and amino acids was 88.7 and 99.9, respectively. The nucleotide homology with BRCR-CA-70 was 79.4%; with B type was 84.8%; with C subtype was 99.2; with C subtype was 99.2, and with C4 was 90.099.2. The highest homology with C4a was 93.799.2and 90.0799.2 with C4a, 90.0799.2with C4b and 90.094.1 with C4b. The base replacement of T-C was observed in the 227 nucleotides of the strains from the severe disease, while none of the strains from the mild disease changed. For amino acid sequence comparison, no change of consistency was found. The sequence analysis of VP4 region showed that the amino acid sequence of VP4 was highly consistent among the 9 strains, except for the change of K-R in the 52nd dalian1-09 strain, the change of T-S in the 62nd amino acid dalian4-10 strain, and the identical amino acid sequence of the other strains. No variations in amino acid or nucleotide sequences were found in severe and mild source strains. The sequence analysis of the UTR region showed that at the 208 nucleotides, the nucleotide substitution of G ~ (+) was found in the samples from severe source, and at the 254 nucleotides, the nucleotide substitutions of A ~ (1 +) were found in the samples from the severe source. The amino acid sequence showed no change in consistency. Conclusion: The main pathogen of HFMD in Dalian from 2009 to 2011 is EV71.The genotype of EV71is C4a subtype, which is consistent with the epidemic strains in most areas of mainland China. 2. The genotyping based on VP4 was consistent with that of VP1, all of them were C4a subtypes, and the VP4 sequence was highly conserved. 3. In the virulence analysis of the virulence of the virus strain, the base substitution of T-C was observed in the 227th nucleotide of VP1. The nucleotide sequence and amino acid sequence of VP4 did not change with clinical symptoms. On the 5'UTR sequence, the sequence of the 208th nucleotide was found, and the result is still to be studied in the next step.
【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R373.2

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