本文選題：EV71 + 全基因組��； 參考：《昆明醫(yī)科大學(xué)》2017年碩士論文

【摘要】：[目的]分析2016年昆明市手足口病(HFMD)患兒腸道病毒EV71分離株的全基因組序列特性(不包含多聚腺苷尾),以闡明當(dāng)年昆明流行EV71毒株的分子生物學(xué)特點(diǎn),通過(guò)與標(biāo)準(zhǔn)株比較,了解病毒的生物學(xué)性狀是否發(fā)生改變,進(jìn)一步了解其進(jìn)化的規(guī)律和發(fā)病特點(diǎn)。[方法]將昆明市婦幼保健院送檢的20個(gè)EV71咽拭子陽(yáng)性樣本提取液接種到非洲綠猴腎細(xì)胞(Vero-E6)進(jìn)行擴(kuò)增,從接種后生長(zhǎng)較好的細(xì)胞中隨機(jī)選取10株(KM01-KM10)的標(biāo)本用于后續(xù)實(shí)驗(yàn)。利用RT-PCR擴(kuò)增覆蓋基因組全長(zhǎng)的片段并測(cè)序,采用Over-lap extension PCR獲得10個(gè)分離株的基因組序列;從GenBank中選取來(lái)自不同地區(qū)、時(shí)期及不同類型的26個(gè)EV71毒株及1株CA16毒株的全基因組序列,與KM01-KM10進(jìn)行同源性、進(jìn)化、重組等分析。[結(jié)果]10株EV71昆明株基因組全部符合腸道病毒基因組的基本特征,獲得的基因組全長(zhǎng)均為7405nt,其開(kāi)放閱讀框?yàn)?582nt,編碼一個(gè)含2194個(gè)氨基酸的多聚蛋白,10株EV71全長(zhǎng)序列與挑選的標(biāo)準(zhǔn)菌株比較顯示,其編碼區(qū)內(nèi)未見(jiàn)有核苷酸的插入或缺失;10株EV71毒株的VP1、VP4、P1、P2、P3及全長(zhǎng)的核苷酸和氨基酸同源性分析顯示KM01-KM10與阜陽(yáng)EU703812菌株的相似度最高;VP1、P1、P2+P3與全長(zhǎng)區(qū)段的進(jìn)化分析顯示KM01-KM10處于C4亞型分支內(nèi)部的最末端,與阜陽(yáng)EU703812菌株處于同一簇,進(jìn)化關(guān)系很近。[結(jié)論]1.本研究獲得昆明10株EV71病毒(KM01-KM10)的全基因組序列。2.通過(guò)核苷酸及氨基酸序列的相似性比對(duì)和遺傳進(jìn)化分析顯示KM01-KM10病毒屬于C4亞型,它們與2008年分離的C4亞型EV71毒株親緣關(guān)系很近,可能起源于同一種系。3.昆明KM01-KM09等9株EV71病毒的全基因組序列沒(méi)有發(fā)生型內(nèi)重組現(xiàn)象,而KM10的可能發(fā)生重組。
[Abstract]:[objective] to analyze the whole genome sequence characteristics (excluding polyadenosine tail) of enterovirus EV71 isolated from children with hand, foot and mouth disease (HFMD) in Kunming in 2016, in order to elucidate the molecular biological characteristics of EV71 strain in Kunming, and compare it with the standard strain. To understand whether the biological characters of the virus have changed, to further understand the evolution of the law and the characteristics of the disease. [methods] Twenty EV71 pharyngeal swabs positive samples from Kunming Maternal and Child Health Hospital were inoculated into the kidney cells of African green monkey (Vero-E6). Ten samples (KM01-KM10) were randomly selected from the cells growing well after inoculation. The genomic sequences of 10 isolates were amplified by RT-PCR and sequenced by overlap extension, 26 EV71 strains from different regions, different periods and different types and 1 CA16 strain were selected from GenBank. Analysis of homology, evolution and recombination with KM01-KM10. [results] the genomes of 10 EV71 Kunming strains all accord with the basic characteristics of enterovirus genome. The total length of genome was 7405nt.The open reading frame was 6582nt.The full-length sequence of 10 EV71 strains encoding 2194 amino acids was compared with the selected standard strain. No nucleotide insertion or deletion was found in the coding region. Analysis of nucleotide and amino acid homology showed that KM01-KM10 had the highest similarity with Fuyang EU703812 strain. Evolution analysis of KM01-KM10 showed that KM01-KM10 had the highest similarity with the full-length region. Evolutionary analysis showed that KM01-KM10 had the highest similarity with the full-length region. The phylogenetic analysis of KM01-KM10 showed that KM01-KM10 had the highest similarity with that of Fuyang EU703812 strain. At the end of the subtype C4 branch, The strain EU703812 is in the same cluster as Fuyang strain, and the evolutionary relationship is very close. [conclusion] 1. In this study, the whole genome sequence of 10 strains of Kunming EV71 virus (KM01-KM10) was obtained. The similarity analysis of nucleotide and amino acid sequence and genetic evolution analysis showed that KM01-KM10 virus belongs to C4 subtype, which is closely related to the strain of C4 subtype EV71 isolated in 2008, and probably originated from the same strain. 3. The whole genome sequence of 9 strains of KM01-KM09 et al., Kunming KM01-KM09, etc., did not recombine in the whole genome, but the recombination of KM10 was possible.
【學(xué)位授予單位】：昆明醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R725.1

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