本文選題：手足口病 + 腸道病毒71型　； 參考：《中國協(xié)和醫(yī)科大學(xué)》2010年碩士論文

【摘要】： 腸道病毒71型(Enterovirus 71, EV71)是導(dǎo)致嬰幼兒感染的重要病原之一,可引起多種疾病,具有較廣的疾病譜。其中由EV71引起的兒童手足口病(Hand, Foot, and Mouth Disease, HFMD)最為常見,而且在嬰幼兒容易造成腦干腦炎等神經(jīng)系統(tǒng)感染導(dǎo)致死亡。EV71近年有多地區(qū)流行的趨勢。 EV71在分類上屬于小RNA病毒科腸道病毒屬,基因組為正鏈單股RNA�；蚪M全長7500bp,編碼區(qū)僅有一個開放閱讀框(ORF),編碼約2200個氨基酸的多聚蛋白(Polyprotein),該多聚蛋白可進一步被水解成P1、P2、P3三個前體蛋白,P1蛋白在3CD蛋白酶的切割作用下生成VP1、VP2、VP3與VP4四種結(jié)構(gòu)蛋白。其中VP1、VP2和VP3裸露于病毒顆粒的表面,VP4位于病毒顆粒衣殼內(nèi)側(cè)與基因組RNA緊密連接,共同構(gòu)成EV71的抗原區(qū)。研究資料表明,雖然該病毒主要抗原決定區(qū)位于VP1,但是VP2、VP3以及VP4上均有抗原抗體結(jié)合功能；已有的實驗結(jié)果顯示單獨的VP1和VP3免疫原性有限,若能獲得VP1～VP4,免疫原性將會得到明顯提高。 鑒于此,我們首先對從流行區(qū)分離的EV71病毒進行了血清學(xué)和分子生物學(xué)方面的鑒定,采用RT-PCR的方法克隆了P1和3CD基因,借助于載體pcDNA3.0BA勾建雙順反子穿梭質(zhì)粒pShuttle-CMV-P1-3CD和單基因的穿梭質(zhì)粒pShuttle-CMV-P1與pShuttle-CMV-3CD.經(jīng)同源重組獲得了重組腺病毒質(zhì)粒rAd-P1-3CD, rAd-P1、rAd-3CD,分別轉(zhuǎn)染293細(xì)胞進行包裝,獲得相應(yīng)的重組腺病毒rAd-P1-3CD、rAd-P1和-Ad-3CD,經(jīng)RT-PCR檢測表明,三個重組腺病毒均已轉(zhuǎn)錄目的基因nRNA;免疫熒光和免疫組化檢測結(jié)果表明僅雙順反子重組腺病毒rAd-P1-3CD中可檢測到特異性目的蛋白,而rAd-P1、rAd-3CD中未能檢測到特異性的表達產(chǎn)物,結(jié)果表明重組腺病毒可有效表達EV71 P1和3CD基因,僅含P1或3CD基因的重組腺病毒表達產(chǎn)物未能被特異性抗體所識別,而含P1及3CD蛋白酶基因的雙順反子重組腺病毒表達產(chǎn)物具有EV71特異抗原性,提示P1蛋白只有在3CD蛋白酶的切割作用下才具有抗原性；重組腺病毒通過滴鼻、灌胃和皮下注射的方式分別免疫BALB/C小鼠,ELISA去檢測結(jié)果表明：實驗組小鼠血清中均檢測到抗EV71特異性IgG的抗體,且由rAd-P1-3CD免疫后產(chǎn)生的抗體水平明顯高于rAd-P1和rAd-3CD共免疫產(chǎn)生的抗體水平；三種不同的免疫方式結(jié)果證明,灌胃免疫方式最佳,滴鼻次之,皮下注射產(chǎn)生的抗體水平最低。目前有限次數(shù)的中和試驗還未能在實驗組血清中檢測到EV71特異性保護作用,其原因還有待進一步研究。 本實驗成功克隆了包含EV71全部結(jié)構(gòu)蛋白區(qū)基因P1和具有切割作用的蛋白酶3CD,初步探索了EV71病毒蛋白之間的相互作用,為研究EV71結(jié)構(gòu)蛋白與非結(jié)構(gòu)蛋白之間的關(guān)系打下了基礎(chǔ)；為找尋最合理的相關(guān)重組腺病毒疫苗的免疫方式做了初步的探索實驗,為EV71基因工程疫苗的研究做了前期的基礎(chǔ)工作。
[Abstract]:Enterovirus 71 (EV71) is one of the most important pathogens leading to infantile infection, which can cause a variety of diseases and has a wide spectrum of diseases. Hand, foot and mouth disease (Handhand, foot and mouth disease, Foot, and Mouth Disease, HFMD) caused by EV71 is the most common disease in children. In recent years, death caused by nervous system infection such as brainstem encephalitis in infants and young children has a trend of epidemic in many areas. EV71 belongs to the genus of enterovirus of small RNA virus family, and its genome is positive chain single stranded RNA. The genome is 7500 BP in length, with only one open reading frame (ORF) encoding about 2200 amino acids. The polyprotein can be further hydrolyzed into three precursor proteins, P1P2P3-3, to produce VP1VP2VP3 by cleavage of 3CD protease. And four structural proteins of VP4. VP1VP2 and VP3 were exposed to the surface of virus particles. VP4 was located inside the capsid of virus particles and tightly connected with genomic RNA, forming the antigenic region of EV71. The results showed that although the main antigen-determining region of the virus was located in VP1, VP2VP3 and VP4 had antigen-antibody binding function, and the previous results showed that the immunogenicity of VP1 and VP3 alone was limited. The immunogenicity of VP1 + VP4 will be improved obviously if VP1 + VP4 can be obtained. In view of this, we first carried out serological and molecular biological identification of EV71 virus isolated from endemic areas, and cloned P1 and 3CD genes by RT-PCR method. The double cisonic shuttle plasmid pShuttle-CMV-P1-3CD and the single gene shuttle plasmid pShuttle-CMV-P1 and pShuttle-CMV-3 CD1 were constructed with the aid of the vector pcDNA3.0BA. Recombinant adenovirus plasmids rAd-P1-3CD1, rAd-P1rAd-3CD3 were obtained by homologous recombination. The recombinant adenovirus rAd-P1-3CD-P1 and -Ad-3CD1 were transfected into 293 cells for packaging, respectively. The results showed that the recombinant adenovirus rAd-P1-3CD-P1 and -Ad-3CDwere obtained by RT-PCR assay. The results of immunofluorescence and immunohistochemistry showed that only the specific target protein could be detected in the recombinant adenovirus rAd-P1-3CD, while the specific expression product could not be detected in the rAd-P1rAd-3CD. The results showed that the recombinant adenovirus could effectively express EV71 P1 and 3CD genes, and only the recombinant adenovirus expression products containing P1 or 3CD gene could not be recognized by specific antibodies. The recombinant adenovirus expressing products containing P1 and 3CD protease genes have the specific antigenicity of EV71, suggesting that P1 protein is antigenicity only under the action of 3CD protease, and the recombinant adenovirus is expressed by nasal drip. BALB/C mice were immunized with Elisa by intragastric and subcutaneous injection respectively. The results showed that the antibody against EV71 specific IgG was detected in the serum of the experimental group. The level of antibody produced by rAd-P1-3CD was significantly higher than that produced by co-immunization of rAd-P1 and rAd-3CD. The results of three different immunization methods showed that the best immunization method was intragastric administration, followed by nasal drip, and the lowest antibody level was produced by subcutaneous injection. At present, the limited number of neutralization tests have not detected the specific protective effect of EV71 in the serum of the experimental group, and the reason remains to be further studied. In this study, the gene P1 containing the entire structural protein region of EV71 and the cleavage protease 3 CD3 were cloned successfully, and the interaction between the proteins of EV71 virus was preliminarily explored, which laid a foundation for the study of the relationship between EV71 structural proteins and non-structural proteins. In order to find the most reasonable immunization method of recombinant adenovirus vaccine, the preliminary experiment was carried out, and the preliminary basic work was done for the study of EV71 gene engineering vaccine.
【學(xué)位授予單位】：中國協(xié)和醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R373

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