發(fā)布時(shí)間：2018-04-22 15:08

  本文選題：手足口病 + 病原學(xué)　； 參考：《寧波大學(xué)》2012年碩士論文

【摘要】：目的： 1、通過對(duì)臨床表現(xiàn)為手足口病(Hand-foot-and-mouth disease，HFMD)的生物樣本進(jìn)行檢測(cè)，鑒定寧波地區(qū)引起HFMD病原種類，分析HFMD的病原學(xué)流行特征，為HFMD的綜合防治提供科學(xué)依據(jù)。 2、在全基因組測(cè)序的基礎(chǔ)上，了解寧波市EV71分離株的全基因特征，分區(qū)段分析腸道病毒71型(Enterovirus71，EV71)寧波株與其他地區(qū)代表株的同源性和親緣進(jìn)化關(guān)系，全面反映進(jìn)化動(dòng)態(tài)；分析VP1區(qū)潛在的B細(xì)胞抗原位點(diǎn)，以及計(jì)算毒株是否承受正向選擇壓力。 方法： 1、依托寧波市疾病預(yù)防控制中心病毒研究室，收集2009~2010年寧波地區(qū)臨床表現(xiàn)為手足口病的樣本共446份，通過熒光定量RT-PCR和RT-PCR方法確定腸道病毒型別，序列拼接和比對(duì)使用DNAMAN和DNAStar軟件，使用SPSS17.0軟件進(jìn)行數(shù)據(jù)統(tǒng)計(jì)，分析HFMD的病原學(xué)流行特征。 2、選擇2010年的EV71陽性樣本進(jìn)行病毒分離和核酸提取，通過逆轉(zhuǎn)錄聚合酶鏈反應(yīng)對(duì)EV71核酸進(jìn)行全基因組序列擴(kuò)增，同時(shí)測(cè)定其他30株EV71的VP1區(qū)序列，序列測(cè)定后使用相關(guān)生物信息軟件進(jìn)行基因片段的拼接、比對(duì)和分析，并將寧波株與GeneBank上不同時(shí)間、不同地點(diǎn)的EV71代表株序列進(jìn)行同源性和親緣進(jìn)化分析，分析潛在抗原為點(diǎn)，以及計(jì)算毒株是否承受正向選擇壓力。 結(jié)果： 1、2009~2010年共檢測(cè)寧波市HFMD病例各類樣本446份，其中有191份EV71(55.20%)；121份CoxA16(34.97%)；13份CoxA6(3.76%)；7份CoxA10；4份CoxA12；4份�？刹《�9型(Echovirus9，ECHO9)；2份CoxA2；CoxA4、CoxB1、CoxB4和其它腸道病毒各1份。在檢測(cè)到的346份總腸道病毒陽性標(biāo)本中，男性232份，女性114份，男女比例為2.04:1，發(fā)病人群主要集中在1~5歲年齡組，全年份均有發(fā)病，有較明顯的季節(jié)性，發(fā)病主要集中在4~7月，12月有不明顯小高峰，2010年檢測(cè)人數(shù)較2009年有下降趨勢(shì)，峰值也有所偏移，2009和2010年的發(fā)病高峰分別在4月和6月。 2、通過全基因組測(cè)序得到寧波4株EV71基因組序列，全長(zhǎng)均為7406bp�；蚪M5'端和3'端各有長(zhǎng)743bp和81bp的非編碼區(qū)，中間為長(zhǎng)6582bp的編碼區(qū)序列，編碼2193個(gè)氨基酸的多聚蛋白。與參考序列相比，寧波株編碼區(qū)沒有核苷酸的插入和缺失。同源性和親緣進(jìn)化分析推測(cè)寧波株EV71與中國大陸正在流行的C4a進(jìn)化分支最為接近。分析VP1蛋白的親水性，柔韌性，表面可能性及蛋白質(zhì)二級(jí)結(jié)構(gòu)，預(yù)測(cè)VP1蛋白以第39~40、159~167、212~220、287~290位氨基酸成為抗原表位的可能性較大。寧波株EV71編碼區(qū)沒有受到明顯正向進(jìn)化壓力的作用，處于凈化選擇狀態(tài)。 小結(jié)： 1、病原學(xué)分型：2009~2010年共檢測(cè)寧波市HFMD病例各類樣本446份，其中有191份EV71(55.20%)；121份CoxA16(34.97%)；13份CoxA6(3.76%)；7份CoxA10；4份CoxA12；4份�？刹《�9型(Echovirus9，ECHO9)；2份CoxA2；CoxA4、CoxB1、CoxB4和其它腸道病毒各1份。 2、檢測(cè)到總腸道病毒核酸陽性346份，男性232份，女性114份，男女比例為2.04:1，發(fā)病人群主要集中在1~5歲年齡組，全年份均有發(fā)病，有較明顯的季節(jié)性，發(fā)病主要集中在4~7月，12月有不明顯小高峰，2010年檢測(cè)人數(shù)較2009年有下降趨勢(shì)，峰值也有所偏移，2009和2010年的發(fā)病高峰分別在4月和6月。 3、寧波4株EV71全基因組核苷酸序列全長(zhǎng)均為7406bp，GeneBank登錄號(hào)為JF830007、JN864018~JN864020。 4、同源性分析：寧波株EV71與中國大陸流行的C4a基因亞型的同源性最高。 5、親緣進(jìn)化分析：從各區(qū)段進(jìn)化樹上看出，寧波株EV71與中國大陸流行的C4基因亞型的同源性最高，屬于C4a進(jìn)化分支。 6、正向選擇壓力：寧波株EV71編碼區(qū)沒有受到明顯正向進(jìn)化壓力的作用，處于凈化選擇狀態(tài)。 7、預(yù)測(cè)B細(xì)胞抗原位點(diǎn)：結(jié)合VP1蛋白的親水性，，柔韌性，表面可能性的重合區(qū)域，并分析VP1的蛋白質(zhì)二級(jí)結(jié)構(gòu)，預(yù)測(cè)VP1蛋白以第39~40、159~167、212~220、287~290位氨基酸成為抗原表位的可能性較大。
[Abstract]:Purpose :

1 . By using the biological samples of Hand - foot - and - mouth disease ( HFMD ) , the pathogenic types of HFMD were identified and the epidemiological characteristics of HFMD were analyzed and the scientific basis was provided for the comprehensive prevention and treatment of HFMD .

2 . On the basis of whole genome sequencing , we know the whole gene characteristics of EV71 isolate in Ningbo City , and analyze the homology and genetic evolution relationship between the strains of Enterovirus 71 ( EV71 ) and other regions , and comprehensively reflect the evolution dynamics .
Potential B - cell antigen sites in VP1 region were analyzed , and whether the virulent strain was subjected to positive selection pressure was calculated .

Method :

1 . Based on the research room of the disease prevention and control center of Ningbo City , we collected 446 samples of hand - foot - mouth disease in Ningbo area from 2009 to 2010 . By means of quantitative RT - PCR and RT - PCR , DNA MAN and DNAStar software were used to analyze the epidemic characteristics of HFMD .

2 , selecting EV71 positive samples in 2010 to carry out virus isolation and nucleic acid extraction , carrying out complete genome sequence amplification on EV71 nucleic acid through reverse transcription polymerase chain reaction , simultaneously measuring VP1 region sequences of other 30 EV71 , carrying out homology and genetic evolution analysis on EV71 representative strain sequences at different times and different sites on the Ningbo strain and GeneBank , analyzing the potential antigen as a point , and calculating whether the virulent strain is subjected to positive selection pressure .

Results :

1 . 446 samples of HFMD cases were tested in Ningbo City from 2009 to 2010 , 191 of them were EV71 ( 55.20 % ) ;
121 parts CoxA16 ( 34.97 % ) ;
13 parts CoxA6 ( 3.76 % ) ;
7 parts CoxA10 ;
4 parts CoxA12 ;
4 copies of Ethyvirus 9 ( Echovirus 9 , ECHO9 ) ;
2 parts CoxA2 ;
CoxA4 , CoxB1 , CoxB4 and other enteroviruses were detected in 1 part . Among the 346 samples tested , 232 male and 114 female , and the male and female ratio were 2.04 : 1 .

Compared with the reference sequence , there were no nucleotide insertions and deletions in the coding region of Ningbo strain .

Summary :

1 . Pathogenic typing : 446 samples of HFMD cases in Ningbo were detected in 2009 - 2010 , 191 of them were EV71 ( 55.20 % ) ;
121 parts CoxA16 ( 34.97 % ) ;
13 parts CoxA6 ( 3.76 % ) ;
7 parts CoxA10 ;
4 parts CoxA12 ;
4 copies of Ethyvirus 9 ( Echovirus 9 , ECHO9 ) ;
2 parts CoxA2 ;
CoxA4 , CoxB1 , CoxB4 , and other Enterovirus 1 part .

2 . There were 346 positive nucleic acid positive in total enteroviruses , 232 male and 114 female , and the male and female ratio was 2.04 : 1 . The incidence of the disease was mainly in the age group of 1 - 5 years .

3 . The whole length of the whole genome of EV71 was 7406bp , and GeneBank accession number was JF8007 , JF84018 - JF84018 . The nucleotide sequence of EV71 was 7406bp .

4 . Homology analysis : The homology of EV71 between Ningbo strain EV71 and C4a gene in mainland China is the highest .

5 . Genetic evolution analysis : From the evolution tree of each section , the homology of EV71 between Ningbo strain EV71 and Chinese mainland is the highest , belonging to the evolutionary branch of C4a .

6 . Positive selection pressure : the EV71 coding region of Ningbo strain is not affected by obvious positive evolution pressure , and is in a clean selection state .

7 . Prediction of B cell antigen site : The overlapping region of the hydrophilicity , flexibility and surface possibility of VP1 protein was predicted , and the secondary structure of VP1 protein was analyzed .

【學(xué)位授予單位】：寧波大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R725.1

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