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
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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。
Enterovirus 71 (EV71) is one of the important etiologic agent for young children, which can cause various kind of diseases. The most common disease caused dy EV71 is HFMD(Hand, Foot, and Mouth Disease) bisease brain stem encephalitis and other nervous system infection to death. In rescent years, EV71 distrubutes throughout the world.
Enterovirus is a single-stranded, positive-sense RNA virus whose genome (≈7500 nucleotides) consists of one open reading frame (ORF), and express as large polyprotein that can be divided into 3 regions:P1, P2 and P3. The four structural proteins, VP1,VP2, VP3 and VP4, are encoded within P1 region while the non-structural proteins, such as proteases 3CD.The proteins VP1,VP2 and VP3 are exposed on the surface of virus particles while VP4 locat inside the virus capsid connect with the genomic RNA closely. The VP1, VP2, VP3 and VP4 constitute the area of antigen of EV71 together.Research date showed that mainly antigens decision of the virus located on VP1, but VP2,VP3 and VP4 have combined capabilities of the antigens with antibodies. Existing experimental results showed that a separate VP1 or VP3 just had limited immunogenicity, if we can obtain VP1-VP4, the immunogenicity will be improved obviously.
In view of this,to the EV71 virus isolated from Fuyang first we identify it by serological and molecular biology methods, and then cloned the P1 and 3CD genes using RT-PCR method, constructed bicistronic shuttle vector pShuttle-CMV-P1-3CD and single gene shuttle plasmid pShuttle-CMV-P1 and pShuttle-CMV-3CD by means of vector pcDNA3.0BA. By the way homologous recombination in E. coli, the recombinant adenovirus vector rAd-P1-3CD, rAd-P1, rAd-3CD are gained, and transfect them into 293 cells to obtain the corresponding recombinant adenovirus. According to RT-PCR analysis results, all of the recombinant exogenous genes of the three kinds of obtained bicistronic recombinant adenovirus rAd-P1-3CD, rAd-P1 and rAd-3CD have been transcripted mRNA. Immunofluorescence and immunehist-ochemistry results showed that just the bicistronic recombinant adenovirus rAd-P1-3CD could be detected in specific target protein, and the rAd-P1, rAd-3CD failed to detect the expression of specific product. The results indicate that all of three kinds of adenovirus can express EV71 P1 and 3CD genes effectively, the expression products of the recombinant adenovirus containing just P1or 3CD could not be recognized by specific antibodies, but the co-expression products of bicistronic recombinant adenovirus containing P1 gene and 3CD protease gene can be certified having EV71-specific antigen. Promote that P1 protein should be cleaved by 3CD protease and then to show antigenicity. Through the way of the intranasal, intragastric and subcutaneous injection to immune BALB/C mice, the results showed by ELISA assay is that all of the experimental groups serum produced anti-EV71IgG antibody. And the antibody level arose by rAd-P1-3CD is higher than immuned together with rAd-P1/rAd-3CD. Three different immunity means results showed that intragastric is the best inmunity way, intranasal second and subcutaneous injection produce the lowest antibody level. By the limited times of neutralization tests, it is failed detecting EV71 specific protection antibodies of the serum, and the concrete results are being further experimental demonstration.
This study cloned containing all of EV71 antigenic role P1 gene and has cleaving effect protease 3CD gene successfully. Results showed the interaction between the proteins of EV71 virus, laid a good foundation for studying the relationship of EV71 structural proteins and non-structural proteins; It is the exploitative experiment for finding the best immunity way in the aspect of interrelated recombinant adenovirus vaccine, and set the stage for genetic engineering vaccine of EV71 research.
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。
Enterovirus 71 (EV71) is one of the important etiologic agent for young children, which can cause various kind of diseases. The most common disease caused dy EV71 is HFMD(Hand, Foot, and Mouth Disease) bisease brain stem encephalitis and other nervous system infection to death. In rescent years, EV71 distrubutes throughout the world.
Enterovirus is a single-stranded, positive-sense RNA virus whose genome (≈7500 nucleotides) consists of one open reading frame (ORF), and express as large polyprotein that can be divided into 3 regions:P1, P2 and P3. The four structural proteins, VP1,VP2, VP3 and VP4, are encoded within P1 region while the non-structural proteins, such as proteases 3CD.The proteins VP1,VP2 and VP3 are exposed on the surface of virus particles while VP4 locat inside the virus capsid connect with the genomic RNA closely. The VP1, VP2, VP3 and VP4 constitute the area of antigen of EV71 together.Research date showed that mainly antigens decision of the virus located on VP1, but VP2,VP3 and VP4 have combined capabilities of the antigens with antibodies. Existing experimental results showed that a separate VP1 or VP3 just had limited immunogenicity, if we can obtain VP1-VP4, the immunogenicity will be improved obviously.
In view of this,to the EV71 virus isolated from Fuyang first we identify it by serological and molecular biology methods, and then cloned the P1 and 3CD genes using RT-PCR method, constructed bicistronic shuttle vector pShuttle-CMV-P1-3CD and single gene shuttle plasmid pShuttle-CMV-P1 and pShuttle-CMV-3CD by means of vector pcDNA3.0BA. By the way homologous recombination in E. coli, the recombinant adenovirus vector rAd-P1-3CD, rAd-P1, rAd-3CD are gained, and transfect them into 293 cells to obtain the corresponding recombinant adenovirus. According to RT-PCR analysis results, all of the recombinant exogenous genes of the three kinds of obtained bicistronic recombinant adenovirus rAd-P1-3CD, rAd-P1 and rAd-3CD have been transcripted mRNA. Immunofluorescence and immunehist-ochemistry results showed that just the bicistronic recombinant adenovirus rAd-P1-3CD could be detected in specific target protein, and the rAd-P1, rAd-3CD failed to detect the expression of specific product. The results indicate that all of three kinds of adenovirus can express EV71 P1 and 3CD genes effectively, the expression products of the recombinant adenovirus containing just P1or 3CD could not be recognized by specific antibodies, but the co-expression products of bicistronic recombinant adenovirus containing P1 gene and 3CD protease gene can be certified having EV71-specific antigen. Promote that P1 protein should be cleaved by 3CD protease and then to show antigenicity. Through the way of the intranasal, intragastric and subcutaneous injection to immune BALB/C mice, the results showed by ELISA assay is that all of the experimental groups serum produced anti-EV71IgG antibody. And the antibody level arose by rAd-P1-3CD is higher than immuned together with rAd-P1/rAd-3CD. Three different immunity means results showed that intragastric is the best inmunity way, intranasal second and subcutaneous injection produce the lowest antibody level. By the limited times of neutralization tests, it is failed detecting EV71 specific protection antibodies of the serum, and the concrete results are being further experimental demonstration.
This study cloned containing all of EV71 antigenic role P1 gene and has cleaving effect protease 3CD gene successfully. Results showed the interaction between the proteins of EV71 virus, laid a good foundation for studying the relationship of EV71 structural proteins and non-structural proteins; It is the exploitative experiment for finding the best immunity way in the aspect of interrelated recombinant adenovirus vaccine, and set the stage for genetic engineering vaccine of EV71 research.
引文
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[53陈全木.第71型肠道病毒外壳蛋白VP1-VP4基因转殖动物建立与评估口服疫苗可开发性的研究.国立中兴大学生物医学研究所硕博士论文2009年11月27日http://ir.lib.nchu.edu.tw/handle/309270000/15917.
[54]Sunil K. Lal, Purnima Kumar, Wee M. Yeo, et al. The VP1 Protein of Human Enterovirus 71 Self-Associates via an Interaction Domain Spanning Amino Acids 66-297[J]. Journal of Medical Virology.Volume 78 Issue 5, Pages 582-590, Published Online:22 Mar 2006.
[55]Damian Guang Wei Foo, Sylvie Alonso, et al. Passive protection against lethal enterovirus 71 infection in newborn mice by neutralizing antibodies elicited by a synthetic peptide[J]. Microbes and Infection. Volume 9, Issue 11, September 2007, Pages 1299-1306.
[56]Yu-Chen Hu, John Tsu-An Hsu, Jen-Huang Huang, et al. Formation of enterovirus-like particle aggregates by recombinant baculoviruses co-expressing P1 and 3CD in insect cells[J]. Biotechnology Letters. Volume 25:919-925, Number 12,2003.
[57]图片来源：www.cmesn.com.
[58]廖国阳,毕胜利,杨建勇等.双顺反子载体构建及其在基因联合免疫中的应用[J].中华实验和临床病毒学杂志,Chinese J Exp Clin Virol June 2006.Vol 20.No.2.
[59]图片来源：AdEasyTM Adenoviral Vector System.
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[57]图片来源：www.cmesn.com.
[58]廖国阳,毕胜利,杨建勇等.双顺反子载体构建及其在基因联合免疫中的应用[J].中华实验和临床病毒学杂志,Chinese J Exp Clin Virol June 2006.Vol 20.No.2.
[59]图片来源：AdEasyTM Adenoviral Vector System.
[1]McMinn P, Lindsay K, Perera D, et al. Phylogenetic analysis of enterovirus 71 strains isolated during linked epidemics in Malaysia, Singapore, and Western Australia[J]. J Virol,2001,75(16):7732-8.
[2]Shih SR, Ho MS, L in KH, et al. Genetic analysis of enterovirus 71 isolated from fatal and nonfatal cases of hand, foot and mouth disease during an epidemic in Taiwan, 1998. Virus Res,2000,68:127-136.
[3]Melnick JL. Enterovirus type 71 infections:a varied clinical pattern sometimes mimicking paralytic poliomyelitis [J].Rev Infect Dis,1984,6 Suppl 2:S387-390.
[4]Chan LG, Parashar UD, Lye MS, et al. Deaths of children during an outbreak of hand, foot, and mouth disease in Sarawak, Malaysia [J].Clin Infect Dis,2000, 31(3):678～683.
[5]Cardosa MJ,Perera D,Brown BA,et al. Molecular epidemiology of human enterovirus 71 strains and recent outbreaks in the Asia Pacific region:comparative analysis of t he VP1 and VP1 genes[J]. Emerg Infect Dis,2003,9 (4):461-468.
[6]Liu CC. An outbreak of enterovirns 71 infection in Taiwan,1998:epidemiolngical and clinical manifestations[J]. J Clin Virol,2000,17(2):23-30.
[7]Ho M, Chen ER, Hsu KH, et al. An epidemic of enterovirus 71 infection in Taiwan. Taiwan Enterovirus Epidemic Working Group [J]. N Engl J Med,1999,341 (13):929-935.
[8]Lin TY, Twu SJ, Ho MS, et al. Enterovirus 71 outbreaks, Taiwan:occurrence and recognition [J]. Emerg Infect Dis,2003,9(3):291-293.
[9]McMinn P, Stratov I, Nagarajan, et al. Neurological manifestation of enterovirus 71 infection in children during an outbreak of hand,foot,and mouth disease in western Australia [J]. Clin Infect Dis,2001,32(2):236～242.)
[10]Fujimoto T, Chikahira M,Yoshida S, et al. Outbreak of central nervous system disease associated with hand, foot, and mouth disease in Japan during the summer of 2000:detection and molecular epidemiology of enterovirus 71 [J]. Microbiol Immunol,2002,46(9):621～627.
[11]Mizuta K, Abiko C, Murata T, et al. Frequent importation of enterovirus 71 from surrounding countries into the local community of Yamagata, Japan, between 1998 and 2003[J]. J Clin Microbiol.2005; 43(12):6171-5.
[12]Tu PV, Thao NT, Perera D, et al. Epidemiologic and virologic investigation of hand, foot, and mouth disease, southern Vietnam,2005[J]. Emerg Infect Dis.2007; 13(11):1733-41.
[13]Cardosa MJ, Perera D, Brown BA, et al. Molecular epidemiology of human enterovirus 71 strains and recent outbreaks in the Asia-Pacific region:comparative analysis of the VP1 and VP4 genes[J]. Emerg Infect Dis.2003;9(4):461-8.
[14]Yong Zhang, Xiao-Juan Tana, Hai-YanWang, et al. An outbreak of hand, foot, and mouth disease associated with subgenotype C4 of hu man enterovirus 71 in Shandong, China[J] Journal of Clinical Virology 44 (2009) 262-267
[15]Jon M. Bible, Panagiotis Pantelidis, Paul K. S. Chan, et al. Genetic evolution of enterovirus 71:epidemiological and pathological implications Rev [J]. Med. Virol.2007; 17:371-379.
[16]Shih-Chen Huang, Yun-Wei Hsu Hsuan-Chen Wang, et al.Appearance of intratypic recombination of enterovirus 71 in Taiwan from 2002 to 2005[J]. Virus Research 131 (2008) 250-259.
[17]董晓楠,应剑,陈应华.1970-2004年全球肠道病毒71型分离株的分子流行病学分析[J].科学通报,2007,52(9)：1021-1027.
[18]Chang L Y, Huang LM, Gau SS, et al. Neurodevelopment and cognition in children after enterovirus 71 infection [J]. N Engl J Med,2007,356 (12):1226-1234.
[19]Susan Shur-Fen Gau, MD, PhDa,et al. Attention-Deficit/Hyperactivity-Related Symptoms Among Children With Enterovirus 71 Infection of the Central Nervous System[J]. Pediatrics.2008 Aug; 122(2):e452-8.
[20]Luan-Yin Chang, MD, PhDa, I-Shou Chang, et al. HLA-A33 Is Associated With Susceptibility to Enterovirus 71 Infection[J]. Pediatrics.2008 Dec; 122(6):1271-6.
[21]Wang SM, Lei HY, Huang KJ et al. Pathogenesis of enterovirus71 brainstem encephalitis in pediatric patients:the roles of cytokines and cellular immune activation in patients with pulmonary edema[J]. J Infect Dis 2003; 188:564-570.
[22]S.-M. Wang, H.-Y. Lei, Let al. Cerebrospinal fluid cytokines in enterovirus 71 brain stem encephalitis and echovirus meningitis infections of varying severity[J]. Clinical Microbiology and Infection:Volume 13 Number 7, July 2007.
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[24]杨绍基.肠道病毒71型感染[J].新医学：2008年6月第39卷第6期.
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