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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2017, Vol. 37 Issue (1): 1-6    DOI: 10.13523/j.cb.20170101
研究报告     
EV71病毒中和表位和诺如病毒P结构域嵌合蛋白的原核表达
李超, 刘波, 陶玉芬, 李昕潼, 刘建生, 刘红旗
中国医学科学院&北京协和医学院医学生物学研究所感染和免疫实验室 昆明 650118
Expression of Chimeric Protein of Norovirus P Domain and Neutralizing Epitopes of EV71 in Prokaryotic Escherichia coli
LI Chao, LIU Bo, TAO Yu-fen, LI Xin-tong, LIU Jian-sheng, LIU Hong-qi
Infection and Immunity Laboratory, Institute of Medical Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Kunming 650118, China
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摘要：

目的：构建肠道病毒71型（Enterovirus71，EV71）的线性中和抗原表位与诺如病毒P结构域融合基因的重组质粒，在大肠杆菌中表达诺如病毒P结构域与EV71中和抗原表位的嵌合蛋白。方法：根据已报道的3个EV71线性中和抗原表位的氨基酸序列，按大肠杆菌密码子表达使用的偏好性优化和设计各线性中和抗原表位的核苷酸序列，将这些表位以单个或不同的组合克隆至含诺如病毒P结构域和GST标签的质粒中，经测序确认后，分别转化到E. coli BL21（DE3）感受态细胞中，通过IPTG诱导融合蛋白表达。用GST融合蛋白纯化磁珠对融合蛋白进行纯化，最后通过免疫印迹法确认融合蛋白的表达及嵌合蛋白的抗原性。结果：测序结果表明，成功地构建了含EV71病毒3个单表位和4个串联中和抗原表位的诺如病毒P结构域重组质粒，而且这7个含线性中和抗原表位的嵌合蛋白在大肠杆菌中都以可溶形式得到了表达。免疫印迹分析表达蛋白的抗原性结果表明，表达的嵌合蛋白都能与抗诺如病毒P结构域抗血清反应。除了含单表位的SP55和SP28嵌合蛋白外，其它的嵌合蛋白均能与抗EV71病毒的抗血清反应。结论：成功地在大肠杆菌中表达了诺如病毒P结构域和EV71病毒中和抗原表位的嵌合蛋白，且具有抗原性，这为诺如病毒和EV71病毒的二价疫苗及检测方法的研发奠定了基础。
关键词： 肠道病毒71型;  中和抗原表位;  诺如病毒;  亚单位疫苗    
Abstract:

Objective:To construct recombinant plasmids and express chimeric proteins of neutralizing epitopes of Human Enterovirus 71 and Norovirus P domain. Methods:Nucleotide sequences of the neutralizing epitopes of EV71 were designed and optimized according to their reported sequences of amino acids and the codon bias usage of E. coli for expression. These nucleotide fragments were cloned into the plasmid vector containing norovirus P domain and GST tag via the loop2 of P domain, which was confirmed by sequencing. The constructs were transformed into the bacteria BL21(DE3) and induced with IPTG for protein expression. The GST-fused proteins were purified via GST affinity beads. Immunoblot analysis was used to detect the GST tag and evaluate the immunogenicity of chimeric proteins. Results:Seven recombinant plasmids were constructed successfully as confirmed by sequencing. The expressed 7 chimeric proteins were expressed and soluble in E. coli, which was determined by SDS-PAGE and Commassie blue staining. Immunoblot analysis via the anti-GST antibody revealed the expression of GST-fused proteins. Further analysis of immunogenicity showed that all 7 chimeric proteins could react with anti-NoV P domain antibody. Five out of 7 chimeric proteins could recognize anti-EV71 polyclonal antibody except two epitopes of SP55 and SP28 chimeric proteins. Conclusion:The chimeric proteins of NoV P domain and EV71 neutralizing epitopes were successfully expressed and antigenic, which lays a solid foundation for developing the bivalent subunit vaccine and the detection kit for NoV and EV71 viruses.
Key words: Enterovirus 71    Norovirus    Neutralizing epitopes    Subunit vaccine
收稿日期: 2016-08-04 出版日期: 2017-01-25
ZTFLH:  Q939.99  
基金资助:

国家自然科学基金（81571549），云南省重点新产品开发专项项目（2016BC004），中国医学科学院医学与健康科技创新工程协同创新团队项目（2016-12M-3-026）资助项目
通讯作者: 刘红旗     E-mail: lhq@Imbcams.com.cn
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引用本文:

李超, 刘波, 陶玉芬, 李昕潼, 刘建生, 刘红旗. EV71病毒中和表位和诺如病毒P结构域嵌合蛋白的原核表达[J]. 中国生物工程杂志, 2017, 37(1): 1-6.

LI Chao, LIU Bo, TAO Yu-fen, LI Xin-tong, LIU Jian-sheng, LIU Hong-qi. Expression of Chimeric Protein of Norovirus P Domain and Neutralizing Epitopes of EV71 in Prokaryotic Escherichia coli. China Biotechnology, 2017, 37(1): 1-6.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20170101    或    https://manu60.magtech.com.cn/biotech/CN/Y2017/V37/I1/1

[1] Kapikian A Z, Wyatt R G, Dolin R, et al. Visualization by immune electron microscopy of a 27-nm particle associated with acute infections nonbacterial gastroenteritis. Journal of Virology, 1972, 10(5):1075-1081.
[2] Dolin R. Noroviruses-challenges to control. N Engl J Med, 2007, 357(11):1072-1073.
[3] Yang F, Ren L L, Xiong Z H, et al. Enterovirus 71 outbreak in the People's Republic of China in 2008. Journal of Clinical Microbiology, 2009, 47(7):2351-2352.
[4] Chen S C, Chang H L, Yan T R, et al. An eight-year study of epidemiologic features of Enterovirus 71 infection in Taiwan. Am J Trop Med Hyg, 2007, 77(1):188-191.
[5] Prasad B V V, Hardy M E, Dokland T, et al. X-ray crystallographic structure of the Norwalk virus capsid. Science, 1999, 286(5438):287-290.
[6] Cao S, Lou Z Y, Tan M, et al. Structural basis for the recognition of blood group trisaccharides by norovirus. Journal of Virology, 2007, 81(11):5949-5957.
[7] Tan M, Fang P A, Chachiyo T, et al. Noroviral P particle:structure, function and applications in virus-host interaction. Virology, 2008, 382(1):115-123.
[8] Tan M, Jiang X. The P domain of norovirus capsid protein forms a subviral particle that binds to histo-blood group antigen receptors. Journal of Virology, 2005, 79(22):14017-14030.
[9] Fang H, Tan M, Xia M, et al. Norovirus P particle efficiently elicits innate, humoral and cellular immunity. PLoS One, 2013, 8(4):e63269.
[10] Tan M, Huang P W, Xia M, et al. Norovirus P particle, a novel platform for vaccine development and antibody production. Journal of Virology, 2011, 85(2):753-764.
[11] Liu C C, Chou A H, Lien S P, et al. Identification and characterization of a cross-neutralization epitope of Enterovirus 71. Vaccine, 2011, 29(26):4362-4372.
[12] Foo D G W, Alonso S, Phoon M C, et al. Identification of neutralizing linear epitopes from the VP1 capsid protein of Enterovirus 71 using synthetic peptides. Virus Research, 2007, 125(1):61-68.
[13] Li Y X, Zhao H, Cao R Y, et al. Recombinant tandem multi-linear neutralizing epitopes of human Enterovirus 71 elicited protective immunity in mice. Virology Journal, 2014, 11(1):79-86.
[14] 高文超, 国泰, 王玉琳. EV71抗原表位的研究方法及研究进展. 中国生物制品学杂志, 2014, 27(6):852-855. Gao W C, Guo T, Wang Y L. Method for and advance in research on neutralizing epitope of EV71. Chin J Biologicals, 2014, 27(6):852-855.
[15] Tan M, Jiang X. Norovirus P particle, a subviral nanoparticle for vaccine development against norovirus, rotavirus and influenza virus. Nanomedicine, 2012, 7(6):889-897.
[16] Xia M, Tan M, Wei C, et al. A candidate dual vaccine against influenza and noroviruses. Vaccine, 2011, 29(44):7670-7677.
[17] Wang L Y, Cao D J, Wei C, et al. A dual vaccine candidate against norovirus and hepatitis E virus. Vaccine, 2014, 32(4):445-452.
[18] Li P, Zheng Q S, Wang Q, et al. Immune responses of recombinant adenoviruses expressing immunodominant epitopes against Japanese encephalitis virus. Vaccine, 2008, 26(46):5802-5807.
[19] Su C X, Duan X G, Wang X Q, et al. Heterologous expression of FMDV immunodominant epitopes and HSP70 in P. pastoris and the subsequent immune response in mice. Veterinary Microbiology, 2007, 124(3-4):256-263.
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