本文關(guān)鍵詞：河南省2008年手足口病病原體（EV71）分離株全基因組序列　出處：《鄭州大學(xué)》2010年碩士論文　論文類(lèi)型：學(xué)位論文

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【摘要】： 目的 了解河南省2008年手足口病病原體流行特征,獲得一株EV71分離株的全基因組核苷酸序列并對(duì)其進(jìn)行同源性分析,構(gòu)建系統(tǒng)發(fā)生樹(shù),獲得該EV71毒株的基因型。 材料與方法 收集2008年河南省18個(gè)地市在門(mén)診就診和住院的手足口臨床診斷病例的血清和/或糞便(咽拭子、皰疹液、腦脊液)標(biāo)本,運(yùn)用國(guó)家CDC推薦的分子生物學(xué)方法對(duì)其進(jìn)行鑒定；同時(shí)選取部分標(biāo)本接種人橫紋肌肉瘤細(xì)胞(RD細(xì)胞)進(jìn)行病毒分離。提取其中一株EV71分離毒株的RNA,設(shè)計(jì)八對(duì)引物,用反轉(zhuǎn)錄聚合酶鏈?zhǔn)椒磻?yīng)(reverse transcription PCR即RT-PCR)擴(kuò)增其全基因組序列,產(chǎn)物純化后另外設(shè)計(jì)八對(duì)引物對(duì)其進(jìn)行雙向測(cè)序；用Clustal (1.8) X、GeneDoc、DNAStar、MEGA3等軟件進(jìn)行核苷酸整理、拼接組裝、校對(duì)、同源性比較、氨基酸序列分析、基因進(jìn)化樹(shù)構(gòu)建等分子變異研究；與GenBank中EV71毒株的各基因亞型代表株的VP1段進(jìn)行同源性分析并對(duì)該株毒株定型。 結(jié)果 1樣本檢測(cè)結(jié)果 共檢測(cè)435份手足口病臨床診斷病例的不同標(biāo)本,EV71檢出率為16.78%,Cox.A16檢出率為1.38%,經(jīng)檢驗(yàn)差異具有統(tǒng)計(jì)學(xué)意義(P0.01)。檢出陽(yáng)性病例中,男、女性別比為2.3:1(54/23),但差異無(wú)統(tǒng)計(jì)學(xué)意義。對(duì)69份標(biāo)本進(jìn)行了細(xì)胞培養(yǎng)和病毒分離,7份標(biāo)本出現(xiàn)明顯致細(xì)胞病變效應(yīng)(CPE),其中EV71占85.7%(6/7),Cox.A16占14.3%(1/7)。對(duì)分離株再次用RT-PCR檢測(cè),陽(yáng)性率達(dá)100%。 2一株EV71分離株的全基因組序列分析 EV71 HENAN08分離株全基因組序列長(zhǎng)度為7405bp(未包括多聚腺苷酸尾),其中腺嘌呤核苷酸(A)占27.01%,鳥(niǎo)嘌呤核苷酸(G)占24.00%,胸腺嘧啶核苷酸(T)占24.92%,胞嘧啶核苷酸(C)占24.07%。A和T豐富。從基因組的5'末端開(kāi)始有742個(gè)堿基的5'非編碼區(qū)(5'UTR),與SHZH98株、BrCr株、Zhejiang08株和TW2086株(743個(gè)堿基)不同。5'UTR之后為6579bp的編碼區(qū),編碼含2193個(gè)氨基酸的多聚蛋白,其后為84個(gè)堿基的3′非編碼區(qū)(3′UTR)。與其它EV71毒株相比,HENAN08株在編碼區(qū)沒(méi)有核苷酸的缺失和插入,但在5′UTR和3′UTR區(qū)存在個(gè)別核苷酸的缺失和插入。 核苷酸同源性比較：在整個(gè)基因組,包括5′UTR區(qū)、編碼區(qū)(P1、P2和P3)和3′UTR區(qū),和AnhuiFY08株以及Zhejiang08株的同源性均高于95%；在P1區(qū),HENAN08與AnhuiFY08、SHZH98、Zhejiang08、SHZH03株的同源性均90%,與BrCr和TW2086的同源性均80%,而與Cox.A16的同源性最低,70%；在3′UTR區(qū),與標(biāo)準(zhǔn)株BrCr以及TW2086的同源性最低。 氨基酸同源性比較：在整個(gè)編碼區(qū),EV71 HENAN08株與其它國(guó)內(nèi)、外EV71株的同源性均較高,與Cox.A16同源性較低；在P2和P3區(qū),HENAN08與其它國(guó)內(nèi)EV71株的同源性均較高,而與標(biāo)準(zhǔn)株BrCr以及TW2086的同源性低于Cox.A16;結(jié)構(gòu)蛋白VP1區(qū),EV71 HENAN08株與AnhuiFY08、SHZH98、標(biāo)準(zhǔn)株BrCr、Zhejiang08、SHZH03、TW2086株的同源性均95%,與Cox.A16的同源性最低,為72.7%。結(jié)構(gòu)蛋白VP4區(qū)的同源性比較都為100%。 結(jié)論 1 EV71是引起2008年河南省手足口病的優(yōu)勢(shì)病原體。 2河南省首次成功提取EV71病毒株的全部基因片段,基因序列號(hào)為GU366191。 3本次分析的EV71分離株同安徽省分離的毒株在基因親緣關(guān)系和流行時(shí)間關(guān)系上最接近,同屬于一種基因型——C基因型C4亞型。
[Abstract]:objective
To understand the epidemiological characteristics of HFMD pathogens in Henan Province in 2008, we obtained a complete nucleotide sequence of a EV71 strain and analyzed its homology. The phylogenetic tree was constructed to obtain the genotype of the EV71 strain.
Materials and methods
18 cities in Henan province in 2008 were collected and serum in outpatient and hospitalized HFMD cases and / or feces (swab, herpes fluid, cerebrospinal fluid specimens), using molecular biology method recommended by national CDC to identify them at the same time; selected specimens were inoculated human rhabdomyosarcoma cells (RD cells) virus the extraction separation. One strain EV71 isolated strains of RNA, eight pairs of primers were designed by reverse transcription polymerase chain reaction (reverse transcription PCR RT-PCR) to amplify the whole genome sequence of the purified product, also designed eight pairs of primers were sequenced to Clustal; (1.8) X, GeneDoc, DNAStar, MEGA3 and other software nucleotide finishing, assembly, proofreading, homology, amino acid sequence analysis, gene molecular phylogenetic tree constructed with GenBank mutation; EV71 strain subtypes strains VP1 The segment was analyzed by homology and the strain of the strain was stereotyped.
Result
1 sample test results
A total of 435 test samples of different specimens of clinically diagnosed cases of HFMD, the detection rate of EV71 was 16.78%, Cox.A16 positive rate was 1.38%, the difference was statistically significant (P0.01). The positive cases, male and female sex ratio was 2.3:1 (54/23), but the difference was not statistically significant. The 69 specimens were cell culture and virus isolation, 7 specimens appeared obvious cytopathic effect (CPE), which accounted for 85.7% of EV71 (6/7), Cox.A16 (1/7) accounted for 14.3%. Of the isolates again with RT-PCR detection, the positive rate was 100%.
Whole genome sequence analysis of 2 EV71 isolates
EV71 HENAN08 isolates complete genome sequence of length 7405bp (not including Polya tail), which accounted for 27.01% of adenine nucleotides (A), guanine nucleotide (G) accounted for 24%, accounted for 24.92% of thymine nucleotide (T), cytosine nucleotide (C) for 24.07%.A and T rich. From the 5'end of the genome has 742 base 5' (5'UTR), and the encoding region of SHZH98 strain, BrCr strain, Zhejiang08 strain and TW2086 strain (743 BP) in different.5'UTR 6579bp encoding region, encoding 2193 amino acids of the polyprotein, followed by a 84 BP 3 'non encoding region (3' UTR). Compared with other EV71 strains, HENAN08 strains in the encoding region without nucleotide deletion and insertion, but in the 5 'UTR and 3' UTR region deletion and insertion of individual nucleotides.
The nucleotide homology in the whole genome, including the 5 'UTR region, encoding region (P1, P2 and P3) and 3' UTR region, and AnhuiFY08 strain and Zhejiang08 strain homology were higher than 95%; in P1, HENAN08 and AnhuiFY08, SHZH98, Zhejiang08, SHZH03 strain homology was 90%, and the BrCr and TW2086 homology 80% with Cox.A16, and the lowest homology, 70%; in 3 'UTR region, and the standard strain BrCr and TW2086 the lowest homology.
The homology of amino acid encoding: in the whole area, EV71 HENAN08 and other domestic and foreign strains, EV71 strains are of high homology, the homology with Cox.A16 is low; in P2 and P3, HENAN08 and other domestic EV71 strains are of high homology, and standard strains BrCr and TW2086 homology is lower than Cox.A16; VP1 structural protein HENAN08 strain and AnhuiFY08, EV71, SHZH98, Zhejiang08, SHZH03 standard strain BrCr, strain TW2086, the homology was 95% Cox.A16, and the lowest homology, VP4 72.7%. structural protein homology is 100%.
conclusion
1 EV71 is the dominant pathogen of hand foot and mouth disease in Henan Province in 2008.
2 the whole gene fragment of EV71 virus strain was successfully extracted in Henan province for the first time. The sequence number of the gene was GU366191.
3 the EV71 isolates from this analysis were closest to the Anhui isolates, and they belonged to a genotype C genotype C4 subtype.

【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】：2010
【分類(lèi)號(hào)】：R373.2

【引證文獻(xiàn)】

相關(guān)博士學(xué)位論文 前1條

1 田曉靈;內(nèi)蒙古自治區(qū)手足口病主要病原分子生物學(xué)特征和人腸道病毒71型致病病理學(xué)研究[D];內(nèi)蒙古農(nóng)業(yè)大學(xué);2013年

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本文編號(hào)：1430477