肠道病毒71型VP1基因和5’非编码区核酸序列分析

发布时间：2018-04-12 23:23

本文选题：手足口病 + 肠道病毒71型　；参考：《郑州大学》2011年硕士论文

【摘要】：肠道病毒71型(Enterovirus71, EV71)是引起儿童手足口病(Hand, foot and mouth disease, HFMD)暴发流行的主要病原体之一,它还能够引起无菌性脑膜炎、神经源性肺水肿等与神经系统相关的疾病,致残及病死率较高。2008年春夏之际,我国部分省区(市)出现了EV71引起的手足口病的严重疫情,重症和死亡病例不断出现。2008年5月卫生部将手足口病纳入丙类传染病进行管理。EV71是小RNA病毒科(Picornaviridae)肠道病毒属(Entero virus)的成员,VP1是其主要的中和决定因子,能够直接决定其抗原性,具有与肠道病毒血清型完全对应的遗传多样性。EV71的5’非编码区(Untranslated region, UTR)含有一个Ⅰ型的内部核糖体进入位点(Internal ribosome entry site, IRES)和其它结构,在病毒复制和翻译起始过程中发挥重要的作用,对脊髓灰质炎病毒的研究发现5'UTR涉及病毒毒力。对不同临床背景的EV71毒株的VP1和5'UTR核酸序列的测定和分析,有助于阐明EV71的VPl和5'UTR与致病性的关系；确定EV71毒株的型别对于EV71的亚型的监测和防控策略的制定有较大的意义；目前有关EV71的研究中,IRES在EV71的5'UTR区的定位和长度仍不明确,确定IRES在EV71的5'UTR区中的准确位置,可以为研究IRES的功能提供更加精确的位点。目的 1.比较不同临床结局EV71毒株VP1和5'UTR的核酸序列。 2.确定研究中EV71毒株的型别。 3.比较不同临床结局EV71毒株5'UTR的RNA二级结构。 4.确定EV71内部核糖体进入位点(IRES) 5'及3’端分界。方法 1.标本来源：粪便标本采集自2010年3月至5月间在郑州市第六人民医院住院的手足口病患儿。 2.病毒核酸提取：提取标本处理液中的病毒RNA。 3.EV71的检测：用EV71特异性引物对提取的病毒RNA进行RT-PCR检测。 4.VP1和5'UTR的逆转录PCR：挑选不同临床结局的EV71毒株的RNA,分别用VP1和5'UTR特异性引物对VP1和5'UTR进行扩增。 5.5'UTR的克隆与鉴定：经过凝胶提取和纯化,将所得的5'UTR片段与载体进行连接,转化入感受态细胞中,筛选阳性克隆,增菌培养后提取质粒,以5'UTR特异性引物对提取的质粒进行PCR检测。 6.分析比较VP1和5'UTR序列,构建系统进化树：将不同临床结局EV71毒株VP1、5'UTR测序后用MEGA5进行核酸序列的分析。用MEGA5构建系统进化树。 7.5'UTR的RNA二级结构预测：用RNA draw 1.1来预测5'UTR的RNA二级结构。结果 1.获得了EV71 VP1和5'UTR完整的核苷酸序列。 2.研究中11个毒株的VP1核苷酸序列(891bp)间的一致率都在97.6%以上,重症组和轻症组毒株间VP1序列的一致率在97.8%-100%之间。11个毒株VP1氨基酸序列(297个氨基酸)间的一致率为100%。9个毒株的5'UTR核苷酸序列(743bp)间的一致率均在96.9%以上,重症组和轻症组毒株间5'UTR序列的一致率在96.9%-99.0%之间。 3.2010年郑州EV71毒株的VP1核酸序列与C4a亚型毒株的平均一致率为97.4%,属于C4a亚型。根据5'UTR核苷酸序列的系统进化分析结果与根据VP1核苷酸序列的结果一致。 4.重症以及死亡毒株的5'UTR RNA二级结构比轻症毒株的二级结构复杂和紧凑。 5.确定EV715'UTR的IRES定位于第101～592核苷酸之间的区域。结论 1.研究中EV71毒株VP1和5'UTR核苷酸序列同源性较高,VP1氨基酸序列完全一致,未发现重症和死亡毒株特异性的差异位点。 2.研究中的11个EV71毒株均属于C4a亚型。 3.根据EV715'UTR分型的结果与根据VP1核酸序列分型的结果一致。 4.EV715'UTR的内部核糖体进入位点定位于第101～592核苷酸之间的区域。
[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 .

【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R373

【参考文献】