發(fā)布時(shí)間：2018-06-19 10:13

  本文選題：腸道病毒71型 + 手足口病　； 參考：《山東大學(xué)》2013年碩士論文

【摘要】：腸道病毒71型(enterovirus71, EV71)為小RNA病毒科腸道病毒屬的成員,足引起嬰幼兒手足口病(hand, foot and mouth disease, HFMD)的主要病原體之一。輕癥患者只引起發(fā)熱、皰疹等癥狀,重癥患者會(huì)引起嚴(yán)重的中樞神經(jīng)系統(tǒng)(central nervous system, CNS)癥狀如無菌性腦膜炎、腦干腦炎、脊髓灰質(zhì)炎樣麻痹等,病死率較高。目前EV71的致病機(jī)制尚不明確,不同臨床癥狀的產(chǎn)生可能是由于病毒毒力不同,也可能是個(gè)體易感性的不同。 本研究首先采集HFMD患兒的糞便標(biāo)本,運(yùn)用RD細(xì)胞分離腸道病毒并對(duì)其進(jìn)行EV71的鑒定：其次對(duì)EV71的全基因組序列進(jìn)行分析,尋找基因組序列的差異；最后分別在5'UTR、VP2、3D區(qū)的基礎(chǔ)上對(duì)EV71的致病機(jī)制進(jìn)行研究,以尋找EV71致神經(jīng)系統(tǒng)病變的原因,為闡明EV71的致病機(jī)制提供重要基礎(chǔ)。 目的 1.從HFMD患者的糞便標(biāo)本中分離EV71,為進(jìn)一步的序列研究提供基礎(chǔ)。 2.了解山東省臨沂市分離的EV71病毒株的全基因組序列特征,尋找不同臨床癥狀病毒株基因組序列上的差異,為進(jìn)一步研究提供毒力候選位點(diǎn) 3.對(duì)FV715'UTR、VP2、3D區(qū)的毒力候選位點(diǎn)進(jìn)行研究,探索EV71的致病機(jī)制。 方法 1.將采集自200%2010年山東省臨沂市HFMD患者的糞便標(biāo)本進(jìn)行處理,接科,于RD細(xì)胞進(jìn)行初步分離,運(yùn)用腸道病毒通用引物及EV71特異性引物對(duì)分離到的病毒RNA進(jìn)行RT-PCR擴(kuò)增,篩選出EV71。 2.根據(jù)GenBank中EV71的基因組序列設(shè)計(jì)引物,對(duì)SDLY11病毒株進(jìn)行全基因組序列擴(kuò)增并測(cè)序,運(yùn)用DNAstar和MEGA4軟件對(duì)EV71的全基因組序列進(jìn)行分析。 3.根據(jù)SDLY01、SDLY11、SDLY48、SDLY96、SDLY107、SDLY153病毒株的全基因組序列設(shè)計(jì)引物,對(duì)8株EV71的5UTR區(qū)進(jìn)行RT-PCR擴(kuò)增并序列分析。 4.參考SDLY11、SDLY107全序列測(cè)序結(jié)果設(shè)計(jì)VP2、3D區(qū)擴(kuò)增引物,以構(gòu)建表達(dá)載體VP2-pBluescriptSK(+)、3D-Flag-pcDNA3.1,通過體外轉(zhuǎn)染細(xì)胞來表達(dá)蛋白,并檢測(cè)蛋白的致細(xì)胞病變作用。乳酸脫氫酶實(shí)驗(yàn)(LDH)檢測(cè)不同臨床癥狀病毒株的VP2蛋白、3D蛋白對(duì)細(xì)胞損傷的影響,Annexin-V與PI的聯(lián)合使用可檢測(cè)3D蛋白引起的細(xì)胞凋亡情況,通過MTT實(shí)驗(yàn)來檢測(cè)3D蛋白對(duì)細(xì)胞增殖的影響。 結(jié)果 1.本研究分離了2009～2010年的24份標(biāo)本,經(jīng)初步鑒定20份標(biāo)本為EV71,4份標(biāo)本為非EV71的腸道病毒,其中采集自2009年的22份樣本中有18例為EV71(81.8%)。 2. SDLY11病毒株基因組全長(zhǎng)7405nt,5'UTR包含742nt,3'UTR包含84nt,中間為6579nt的ORF,編碼2193個(gè)氨基酸的多聚蛋白。經(jīng)序列分析顯示SDLY11屬于V71的C4亞型。通過輕重癥重癥患者病毒株進(jìn)行比對(duì)分析發(fā)現(xiàn)5UTR中的2處核苷酸突變(T40C、C575T)及VP2區(qū)的氨基酸突變(T144S)可能與病毒引起不同臨床癥狀有關(guān) 3.8株EV71分離株的5UR區(qū)核苷酸序列為741nt～745nt,與C4亞型的同源性最高,同臨床癥狀病毒株的5UTR進(jìn)行序列比對(duì)發(fā)現(xiàn)有2處枋苷酸位點(diǎn)突變(G265A、(703T)。 4. SDLY11、SDLY107兩個(gè)病毒株的VP2蛋白均可在Vero細(xì)胞中表達(dá),LDH實(shí)驗(yàn)對(duì)VP2蛋白引起的細(xì)胞損傷程度進(jìn)行分析,結(jié)果顯示兩組未出現(xiàn)明顯不同。 5. SDLY11、SDLY107的3D蛋白可在RD細(xì)胞中表達(dá),LDH實(shí)驗(yàn)結(jié)果顯示SDLY11株3D蛋白轉(zhuǎn)染組造成的細(xì)胞損傷程度大于SDLY107株3D蛋白轉(zhuǎn)染組,細(xì)胞凋亡結(jié)果未有明顯不同,而進(jìn)一步的MTT實(shí)驗(yàn)結(jié)果顯示,SDLY107株3D蛋白轉(zhuǎn)染組的細(xì)胞增殖能力強(qiáng)于SDLY11株3D蛋白轉(zhuǎn)染組。 結(jié)論 1.初步判定2009年山東省臨沂市流行的HFMD以EV71為主,SDLY11為C4亞型,為近年來中國(guó)大陸地區(qū)流行的EV71亞型。 2.5'UTR區(qū)的4處突變(T40C、G265A、C575T、G703T)可能與病毒產(chǎn)生的神經(jīng)系統(tǒng)癥狀相關(guān),這可為進(jìn)一步的研究提供候選位點(diǎn)。 3.不同臨床癥狀病毒株的VP2蛋白可能對(duì)細(xì)胞損傷的影響不大,但3D蛋白會(huì)引起細(xì)胞損傷情況、細(xì)胞增殖能力的不同,這為EV71致病機(jī)制的研究提供了重要基礎(chǔ)。
[Abstract]:Enterovirus 71 ( EV71 ) is one of the main pathogens of small RNA virus family Enterovirus , which is one of the main pathogens of hand , foot and mouth disease ( HFMD ) .

In this study , the fecal samples of HFMD children were collected , and the EV71 was isolated by RD cells . The whole genome sequence of EV71 was analyzed to find out the difference of genomic sequence .
Finally , the pathogenic mechanism of EV71 was studied on the basis of 5 ' untranslated region , VP2 and 3D region respectively , in order to find out the cause of EV71 induced nervous system disease and provide an important basis for the pathogenesis of EV71 .

Purpose

1 . EV71 was isolated from stool specimens from HFMD patients , providing the basis for further sequence research .

2 . To understand the whole genome sequence characteristics of EV71 virus isolated from Linyi City , Shandong Province , and to find out the difference in the genome sequence of different clinical symptom virus strains , and provide the virulence candidate sites for further research .

3 . To study the virulence candidate sites of FV715 ' , VP2 and 3D regions , and explore the pathogenesis of EV71 .

method

1 . The stool specimens collected from HFMD patients in Linyi City of Shandong Province in 2010 were treated and isolated . The isolated virus RNA was amplified by RT - PCR using the general primers of Enterovirus and EV71 specific primers , and EV71 was screened out .

2 . According to the genomic sequence of EV71 in GenBank , the whole genome sequence of the strain SDLY11 was amplified and sequenced , and the whole genome sequence of EV71 was analyzed by DNAstar and MEGA4 software .

3 . RT - PCR amplification and sequence analysis were carried out on 8 strains of EV71 based on the whole genome sequence of SDLY01 , SDLY11 , SDLY48 , SDLY96 , SDLY107 and SDLY153 .

4 . The expression vector VP2 - pBluescriptSK ( + ) , 3D - Flag - pcDNA3.1 was designed by reference to SDLY11 and SDLY107 sequencing results .

Results

1 . This study has isolated 24 specimens from 2009 to 2010 , initially identified as EV71 and 4 specimens as non - EV71 enteroviruses , of which 18 of 22 samples collected from 2009 were EV71 ( 81.8 % ) .

2 . The genome of SDLY11 strain has a full length of 7405nt . The 5 ' untranslated region consists of the 742nt , the 3 ' untranslated region contains 84nt , the middle is 6579nt , and the polyprotein of 2193 amino acids is encoded . The sequence analysis shows that SDLY11 belongs to the C4 subtype of V71 . By comparison , it is found that amino acid mutation ( T40C , C575T ) and amino acid mutation ( T144S ) in VP2 region may be related to different clinical symptoms .

3 . The nucleotide sequence of the 5UR region of EV71 isolates was 741nt ~ 745nt , and the highest homology with the C4 isoforms was observed .

4 . VP2 protein of two strains of SDLY11 and SDLY107 could be expressed in Vero cells , and the degree of cell injury induced by VP2 protein in LDH experiment was analyzed .

5 . The 3D protein of SDLY11 and SDLY107 could be expressed in RD cells . The results of LDH experiment showed that the degree of cell damage caused by the 3D protein transfection group of SDLY11 strain was greater than that of SDLY107 strain . The results showed that the cell proliferation ability of SDLY107 strain was stronger than that of SDLY11 strain .

Conclusion

1 . It is preliminarily determined that the HFMD , which is popular in Linyi City in Shandong Province in 2009 , is EV71 , and SDLY11 is a C4 subtype , which is an EV71 subtype that has been popular in mainland China in recent years .

At 4 mutations ( T40C , G265A , C575T , G703T ) in the 2.5 ' - region may be associated with the neurological symptoms of the virus , which may provide candidate sites for further studies .

3 . VP2 protein of different clinical symptom virus strains may have little effect on cell injury , but 3D protein may cause cell injury and cell proliferation ability , which provides an important basis for the study of EV71 pathogenic mechanism .
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R373

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