發(fā)布時(shí)間：2018-04-12 23:23

  本文選題：手足口病 + 腸道病毒71型　； 參考：《鄭州大學(xué)》2011年碩士論文

【摘要】：腸道病毒71型(Enterovirus71, EV71)是引起兒童手足口病(Hand, foot and mouth disease, HFMD)暴發(fā)流行的主要病原體之一,它還能夠引起無(wú)菌性腦膜炎、神經(jīng)源性肺水腫等與神經(jīng)系統(tǒng)相關(guān)的疾病,致殘及病死率較高。2008年春夏之際,我國(guó)部分省區(qū)(市)出現(xiàn)了EV71引起的手足口病的嚴(yán)重疫情,重癥和死亡病例不斷出現(xiàn)。2008年5月衛(wèi)生部將手足口病納入丙類傳染病進(jìn)行管理。EV71是小RNA病毒科(Picornaviridae)腸道病毒屬(Entero virus)的成員,VP1是其主要的中和決定因子,能夠直接決定其抗原性,具有與腸道病毒血清型完全對(duì)應(yīng)的遺傳多樣性。EV71的5’非編碼區(qū)(Untranslated region, UTR)含有一個(gè)Ⅰ型的內(nèi)部核糖體進(jìn)入位點(diǎn)(Internal ribosome entry site, IRES)和其它結(jié)構(gòu),在病毒復(fù)制和翻譯起始過(guò)程中發(fā)揮重要的作用,對(duì)脊髓灰質(zhì)炎病毒的研究發(fā)現(xiàn)5'UTR涉及病毒毒力。 對(duì)不同臨床背景的EV71毒株的VP1和5'UTR核酸序列的測(cè)定和分析,有助于闡明EV71的VPl和5'UTR與致病性的關(guān)系；確定EV71毒株的型別對(duì)于EV71的亞型的監(jiān)測(cè)和防控策略的制定有較大的意義；目前有關(guān)EV71的研究中,IRES在EV71的5'UTR區(qū)的定位和長(zhǎng)度仍不明確,確定IRES在EV71的5'UTR區(qū)中的準(zhǔn)確位置,可以為研究IRES的功能提供更加精確的位點(diǎn)。 目的 1.比較不同臨床結(jié)局EV71毒株VP1和5'UTR的核酸序列。 2.確定研究中EV71毒株的型別。 3.比較不同臨床結(jié)局EV71毒株5'UTR的RNA二級(jí)結(jié)構(gòu)。 4.確定EV71內(nèi)部核糖體進(jìn)入位點(diǎn)(IRES) 5'及3’端分界。 方法 1.標(biāo)本來(lái)源：糞便標(biāo)本采集自2010年3月至5月間在鄭州市第六人民醫(yī)院住院的手足口病患兒。 2.病毒核酸提�。禾崛�(biāo)本處理液中的病毒RNA。 3.EV71的檢測(cè)：用EV71特異性引物對(duì)提取的病毒RNA進(jìn)行RT-PCR檢測(cè)。 4.VP1和5'UTR的逆轉(zhuǎn)錄PCR：挑選不同臨床結(jié)局的EV71毒株的RNA,分別用VP1和5'UTR特異性引物對(duì)VP1和5'UTR進(jìn)行擴(kuò)增。 5.5'UTR的克隆與鑒定：經(jīng)過(guò)凝膠提取和純化,將所得的5'UTR片段與載體進(jìn)行連接,轉(zhuǎn)化入感受態(tài)細(xì)胞中,篩選陽(yáng)性克隆,增菌培養(yǎng)后提取質(zhì)粒,以5'UTR特異性引物對(duì)提取的質(zhì)粒進(jìn)行PCR檢測(cè)。 6.分析比較VP1和5'UTR序列,構(gòu)建系統(tǒng)進(jìn)化樹：將不同臨床結(jié)局EV71毒株VP1、5'UTR測(cè)序后用MEGA5進(jìn)行核酸序列的分析。用MEGA5構(gòu)建系統(tǒng)進(jìn)化樹。 7.5'UTR的RNA二級(jí)結(jié)構(gòu)預(yù)測(cè)：用RNA draw 1.1來(lái)預(yù)測(cè)5'UTR的RNA二級(jí)結(jié)構(gòu)。 結(jié)果 1.獲得了EV71 VP1和5'UTR完整的核苷酸序列。 2.研究中11個(gè)毒株的VP1核苷酸序列(891bp)間的一致率都在97.6%以上,重癥組和輕癥組毒株間VP1序列的一致率在97.8%-100%之間。11個(gè)毒株VP1氨基酸序列(297個(gè)氨基酸)間的一致率為100%。9個(gè)毒株的5'UTR核苷酸序列(743bp)間的一致率均在96.9%以上,重癥組和輕癥組毒株間5'UTR序列的一致率在96.9%-99.0%之間。 3.2010年鄭州EV71毒株的VP1核酸序列與C4a亞型毒株的平均一致率為97.4%,屬于C4a亞型。根據(jù)5'UTR核苷酸序列的系統(tǒng)進(jìn)化分析結(jié)果與根據(jù)VP1核苷酸序列的結(jié)果一致。 4.重癥以及死亡毒株的5'UTR RNA二級(jí)結(jié)構(gòu)比輕癥毒株的二級(jí)結(jié)構(gòu)復(fù)雜和緊湊。 5.確定EV715'UTR的IRES定位于第101～592核苷酸之間的區(qū)域。 結(jié)論 1.研究中EV71毒株VP1和5'UTR核苷酸序列同源性較高,VP1氨基酸序列完全一致,未發(fā)現(xiàn)重癥和死亡毒株特異性的差異位點(diǎn)。 2.研究中的11個(gè)EV71毒株均屬于C4a亞型。 3.根據(jù)EV715'UTR分型的結(jié)果與根據(jù)VP1核酸序列分型的結(jié)果一致。 4.EV715'UTR的內(nèi)部核糖體進(jìn)入位點(diǎn)定位于第101～592核苷酸之間的區(qū)域。
[Abstract]:Enterovirus 71 ( EV71 ) is one of the main pathogens causing the outbreak of hand , foot and mouth disease ( HFMD ) in children . It can also cause the disease , disability and mortality rate related to nervous system caused by aseptic meningitis , neurogenic pulmonary edema , etc .

The measurement and analysis of the VP1 and 5 ' flanking nucleic acid sequences of EV71 strain of different clinical backgrounds can help elucidate the relationship between the VPl and 5 ' UU' s of EV71 and the pathogenicity .
It is of great significance to determine the type of EV71 strain and to establish the monitoring and control strategy of EV71 subtype .
At present , in the study of EV71 , the location and length of the 5 ' untranslated region of the EV71 remains unclear , and it is determined that the exact location of the site in the 5 ' untranslated region of the EV71 can provide a more accurate site for the study of the function of the .

Purpose

1 . Comparison of the nucleic acid sequences of the EV71 strain VP1 and the 5 & # x2032 ; of different clinical outcomes .

2 . Determine the type of EV71 strain in the study .

3 . The RNA secondary structure of EV71 strain of EV71 strain with different clinical outcomes was compared .

4 . Identify the 5 ' and 3 ' end boundaries of the EV71 internal ribosome entry site .

method

1 . Specimen origin : feces samples were collected from children who were hospitalized in the Sixth People ' s Hospital of Zhengzhou from March to May 2010 .

2 . viral nucleic acid extraction : extracting viral RNA in the sample processing solution .

3 . Detection of EV71 : RT - PCR was performed on the extracted viral RNA with EV71 - specific primers .

4 . Reverse transcription - polymerase chain reaction ( RT - PCR ) : RNA of EV71 strain with different clinical outcomes was selected to amplify the VP1 and 5 ' untranslated regions with VP1 and 5 ' - specific primers , respectively .

5 . The cloning and identification of the 5 ' - region : After gel extraction and purification , the obtained 5 ' untranslated region was ligated with the vector , transformed into competent cells , positive clones were screened , and the plasmid was extracted after the enrichment culture , and the extracted plasmid was subjected to PCR detection with 5 ' untranslated primers .

6 . The sequences of VP1 and 5 ' were analyzed , and the phylogenetic trees were constructed . After sequencing the VP1 and 5 ' untranslated regions of EV71 strain of different clinical outcomes , the sequence of nucleic acid was analyzed with MEGA5 . The phylogenetic tree of the system was constructed by MEGA5 .

The RNA secondary structure prediction of the 7.5 ' untranslated region : RNA draw 1.1 was used to predict the secondary structure of the RNA secondary structure .

Results

1 . The complete nucleotide sequence of the EV71 VP1 and the 5 ' untranslated region was obtained .

2 . The coincidence rate between VP1 nucleotide sequence ( 891bp ) of 11 strains in the study was above 90.6 % . The coincidence rate of VP1 sequences between severe group and light disease group was 97.8 % -100 % . The coincidence rate among the VP1 amino acid sequences of 11 strains ( 297 amino acids ) was more than 96.9 % .

3 . The average coincidence rate of VP1 nucleic acid sequence and C4a subtype strain in Zhengzhou EV71 strain in 2010 was 97.4 % , belonging to the subtype C4a . The results of phylogenetic analysis according to the nucleotide sequence of the 5 ' USAF were consistent with the results according to the nucleotide sequence of VP1 .

4 . The secondary structure of the 5 ' UURNA secondary structure of the severe and dead strain is complicated and compact than the secondary structure of the light disease strain .

5 . The region between nucleotides 101 - 592 is determined to be located between nucleotides 101 - 592 .

Conclusion

1 . The amino acid sequence identity of EV71 strain was higher in the study , and the VP1 amino acid sequence was identical with that of EV71 strain , and no significant difference was found between severe and dead strain .

2 . The 11 EV71 strains in the study belong to C4a subtype .

3 . The results according to EV715 & # x2032 ; are consistent with the results according to the VP1 nucleic acid sequence typing .

4 . The internal ribosome entry site of the EV715 & # x2032 ; ' s is located in the region between nucleotides 101 - 592 .

【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】：2011
【分類號(hào)】：R373

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本文編號(hào)：1741905