肠道病毒71型VP1-VP4在重组腺病毒系统中的表达及免疫学评价

发布时间：2018-05-23 22:30

本文选题：手足口病 + 肠道病毒71型　；参考：《中国协和医科大学》2010年硕士论文

【摘要】： 肠道病毒71型(Enterovirus 71, EV71)是导致婴幼儿感染的重要病原之一,可引起多种疾病,具有较广的疾病谱。其中由EV71引起的儿童手足口病(Hand, Foot, and Mouth Disease, HFMD)最为常见,而且在婴幼儿容易造成脑干脑炎等神经系统感染导致死亡。EV71近年有多地区流行的趋势。 EV71在分类上属于小RNA病毒科肠道病毒属,基因组为正链单股RNA。基因组全长7500bp,编码区仅有一个开放阅读框(ORF),编码约2200个氨基酸的多聚蛋白(Polyprotein),该多聚蛋白可进一步被水解成P1、P2、P3三个前体蛋白,P1蛋白在3CD蛋白酶的切割作用下生成VP1、VP2、VP3与VP4四种结构蛋白。其中VP1、VP2和VP3裸露于病毒颗粒的表面,VP4位于病毒颗粒衣壳内侧与基因组RNA紧密连接,共同构成EV71的抗原区。研究资料表明,虽然该病毒主要抗原决定区位于VP1,但是VP2、VP3以及VP4上均有抗原抗体结合功能；已有的实验结果显示单独的VP1和VP3免疫原性有限,若能获得VP1～VP4,免疫原性将会得到明显提高。鉴于此,我们首先对从流行区分离的EV71病毒进行了血清学和分子生物学方面的鉴定,采用RT-PCR的方法克隆了P1和3CD基因,借助于载体pcDNA3.0BA勾建双顺反子穿梭质粒pShuttle-CMV-P1-3CD和单基因的穿梭质粒pShuttle-CMV-P1与pShuttle-CMV-3CD.经同源重组获得了重组腺病毒质粒rAd-P1-3CD, rAd-P1、rAd-3CD,分别转染293细胞进行包装,获得相应的重组腺病毒rAd-P1-3CD、rAd-P1和-Ad-3CD,经RT-PCR检测表明,三个重组腺病毒均已转录目的基因nRNA;免疫荧光和免疫组化检测结果表明仅双顺反子重组腺病毒rAd-P1-3CD中可检测到特异性目的蛋白,而rAd-P1、rAd-3CD中未能检测到特异性的表达产物,结果表明重组腺病毒可有效表达EV71 P1和3CD基因,仅含P1或3CD基因的重组腺病毒表达产物未能被特异性抗体所识别,而含P1及3CD蛋白酶基因的双顺反子重组腺病毒表达产物具有EV71特异抗原性,提示P1蛋白只有在3CD蛋白酶的切割作用下才具有抗原性；重组腺病毒通过滴鼻、灌胃和皮下注射的方式分别免疫BALB/C小鼠,ELISA去检测结果表明：实验组小鼠血清中均检测到抗EV71特异性IgG的抗体,且由rAd-P1-3CD免疫后产生的抗体水平明显高于rAd-P1和rAd-3CD共免疫产生的抗体水平；三种不同的免疫方式结果证明,灌胃免疫方式最佳,滴鼻次之,皮下注射产生的抗体水平最低。目前有限次数的中和试验还未能在实验组血清中检测到EV71特异性保护作用,其原因还有待进一步研究。本实验成功克隆了包含EV71全部结构蛋白区基因P1和具有切割作用的蛋白酶3CD,初步探索了EV71病毒蛋白之间的相互作用,为研究EV71结构蛋白与非结构蛋白之间的关系打下了基础；为找寻最合理的相关重组腺病毒疫苗的免疫方式做了初步的探索实验,为EV71基因工程疫苗的研究做了前期的基础工作。
[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.
【学位授予单位】：中国协和医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R373

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