EV71多表位抗原亲和纯化及类病毒颗粒胞外自组装

doi:10.13523/j.cb.20160706

中国生物工程杂志

2016, Vol. 36

Issue (7): 34-40 DOI: 10.13523/j.cb.20160706

技术与方法

EV71多表位抗原亲和纯化及类病毒颗粒胞外自组装

薛玲^1,2, 刘江宁³, 张耀^1,2, 张纯¹, 王祺^1,2, 秦川³, 刘永东¹, 苏志国¹

1 中国科学院过程工程研究所生化工程国家重点实验室北京 100190;
2 中国科学院大学北京 100049;
3 中国医学科学院实验动物研究所北京 100021

Affinity Purification of Enterovirus 71 Fused Multi-Epitope Protein Antigen and Assembling It as Virus-like Particles in Vitro

XUE Ling^1,2, LIU Jiang-ning³, ZHANG Yao^1,2, ZHANG Chun¹, WANG Qi^1,2, QIN Chuan³, LIU Yong-dong¹, SU Zhi-guo¹

1 National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China;
2 University of Chinese Academy of Sciences, Beijing 100049, China;
3 Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences, Beijing 100021, China

全文: PDF(815 KB) HTML

摘要：

利用肠道病毒71型（EV71）衣壳蛋白优势表位肽段构建融合蛋白抗原能有效抑制病毒感染，有望成为继灭活病毒后更为安全有效的疫苗品种。该融合蛋白能通过原核体系有效表达但形成无序包涵体，采用常规层析介质难以实现目标蛋白与宿主杂质的有效分离，阻碍了对该抗原蛋白进行全面临床前活性及安全性评价。在原有融合蛋白抗原N端插入组氨酸标签，对形成的包涵体变性溶解后直接采用镍金属螯合亲和介质进行分离纯化，获得了纯度大于95%的抗原纯品，目标蛋白收率46.8%。采用透析方式脱除纯化样品中高浓度脲，发现直接透析至无脲的缓冲液中蛋白质大量沉淀，而先稀释至2mol/L脲的缓冲液中然后用G25脱盐柱完全脱除脲则无任何沉淀形成，获得近100%的蛋白质收率。透射电镜分析最终样品发现融合蛋白形成了10nm左右粒径均一的类病毒蛋白颗粒，且在pH 8.0的磷酸盐缓冲液中保持稳定。该研究结果为将EV71融合蛋白抗原发展为安全有效且低成本的手足口疫苗奠定了基础。

关键词： 手足口病疫苗; 亲和层析; 肠道病毒71型; 多表位融合抗原; 复性及自组装

Abstract:

Human enterovirus 71 (EV71) is one of the main causative pathogens for hand-foot-and-mouth disease (HFMD) which often infects infants and children under 5 years old. Although prophylactic vaccines of inactivated EV71 virus for HFMD have been clinically available, low productivity and safety concerns still prompt scientists to develop other vaccine substitutes. Recently, a kind of vaccine candidate VacA for EV71 has been designed and reported, which contained the main antigen peptides, was fused as a single protein and expressed through recombinant E. coli system. This candidate was demonstrated to have high neutralizing antibody titers as well as efficiently protect against virus infection. However, forming inclusion bodies in E. coli and failure of being separated from the host contaminants through route chromatographs hampered its path to go forward to whole pre-clinic and safety assessments.Another form of VacA as His-VacA was constructed by inserting a His tag at the N-terminal of VacA and explored to be separated through metal affinity chromatography. As still forming inclusion bodies, the fusion protein of His-VacA was directly purified at denatured state through a special mental affinity medium of cOmplete His-tag, which could tolerate low concentration of EDTA and DTT. Purity of about 95% for His-VacA was achieved after such one step chromatographic procedure, with a recovery of 46.8%. Dialysis was then adopted to remove urea in the purified protein, but protein almost completely precipitated when dialyzing against PB buffer without any additives. Nevertheless, in the case that the purified denatured protein was firstly diluted in a buffer containing 2mol/L urea and then exchanged to a buffer without urea through a desalting column of G25, no protein precipitate could be found, resulting in nearly 100% recovery. More inspirationally, His-VacA was found in the form of virus-like particles with a size of 10nm through transmission electron microscopy. Moreover, this protein particle is very stable in the final buffer and then could be used for the further pre-clinic assessments. All these results lay a foundation for developing His-VacA as a new kind of EV71 vaccine with the advantages of safer and cheaper.

Key words: Enterovirus 71 Affinity chromatography Hand-foot-and-mouse disease vaccine Refolding and aseembling Fused multi-epitope antigen

收稿日期: 2016-02-03 出版日期: 2016-03-02

ZTFLH:

Q81

基金资助:

国家自然科学基金（21576267），北京市自然科学基金（2162041），“重大新药创制”国家科技重大专项（2012ZX09101319004，2016ZX09101120-006）资助项目

通讯作者: 刘永东, 苏志国 E-mail: ydliu@ipe.ac.cn;zgsu@ipe.ac.cn

	服务
	把本文推荐给朋友
	加入引用管理器
	E-mail Alert
	RSS
	作者相关文章
	张纯
	王祺
	秦川
	刘永东
	苏志国
	薛玲
	刘江宁
	张耀

引用本文:

薛玲, 刘江宁, 张耀, 张纯, 王祺, 秦川, 刘永东, 苏志国. EV71多表位抗原亲和纯化及类病毒颗粒胞外自组装[J]. 中国生物工程杂志, 2016, 36(7): 34-40.

XUE Ling, LIU Jiang-ning, ZHANG Yao, ZHANG Chun, WANG Qi, QIN Chuan, LIU Yong-dong, SU Zhi-guo. Affinity Purification of Enterovirus 71 Fused Multi-Epitope Protein Antigen and Assembling It as Virus-like Particles in Vitro. China Biotechnology, 2016, 36(7): 34-40.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20160706 或 https://manu60.magtech.com.cn/biotech/CN/Y2016/V36/I7/34

[1] McMinn P C.An overview of the evolution of enterovirus 71 and its clinical and public health significance.Fems Microbiology Reviews,2002,26(1):91-107.
[2] Li R C,Liu L D,Mo Z J,et al.An inactivated enterovirus 71 vaccine in healthy children.New England Journal of Medicine,2014,370(9):829-837.
[3] Xing W J,Liao Q H,Viboud C,et al.Hand,foot,and mouth disease in China,2008-12:an epidemiological study.The Lancet Infectious Diseases,2014,14(4):308-318.
[4] Zhu F C,Xu W B,Xia J L,et al.Efficacy,safety,and immunogenicity of an enterovirus 71 vaccine in China.New England Journal of Medicine,2014,370(9):818-828.
[5] Zhang D M,Lu J Y,Lu J H.Enterovirus 71 vaccine:close but still far.International Journal of Infectious Diseases,2010,14(9):e739-743.
[6] 罗雯.肠道病毒71型疫苗研究进展.实用预防医学,2013,20(12):1535-1537.Luo W.Research progress on the development of enterovirus 71 vaccines.Practical Preventive Medicine,2013,20(12):1535-1537.
[7] Chung Y C,Huang J H,Lai C W,et al.Expression,purification and characterization of enterovirus-71 virus-like particles.World Journal of Gastroenterology,2006,12(6):921-927.
[8] Li H Y,Han J F,Qin C F,et al.Virus-like particles for enterovirus 71 produced from Saccharomyces cerevisiae potently elicits protective immune responses in mice.Vaccine,2013,31(32):3281-3287.
[9] Zhang C,Ku Z Q,Liu Q W,et al.High-yield production of recombinant virus-like particles of enterovirus 71 in Pichia pastoris and their protective efficacy against oral viral challenge in mice.Vaccine,2015,33(20):2335-2341.
[10] Zhao D,Sun B,Jiang H,et al.Enterovirus71 virus-like particles produced from insect cells and purified by multistep chromatography elicit strong humoral immune responses in mice.J Appl Microbiol,2015,119(4):1196-1205.
[11] Cifuente J O,Lee H,Yoder J D,et al.Structures of the procapsid and mature virion of enterovirus 71 strain 1095.Journal of Virology,2013,87(13):7637-7645.
[12] Ho M.Enterovirus 71:the virus,its infections and outbreaks.Journal of Microbiology,Immunology,and Infection,2000,33(4):205-216.
[13] Foo D G,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.
[14] Chiu C H,Chu C,He C C,et al.Protection of neonatal mice from lethal enterovirus 71 infection by maternal immunization with attenuated Salmonella enterica serovar Typhimurium expressing VP1 of enterovirus 71.Microbes and Infection,2006,8(7):1671-1678.
[15] Liu J N,Wang W,Duo J Y,et al.Combined peptides of human enterovirus 71 protect against virus infection in mice.Vaccine,2010,28(46):7444-7451.
[16] 郝翊,秦川.人肠道病毒71型亚单位疫苗的研制.中国比较医学杂志,2010,20(6):7-12.Hao Y,Qin C.Preparation of subunit vaccine for human enterovirus 71.Chinese Journal of Comparative Medicine,2010,20(6):7-12.
[17] Laemmli U K.Cleavage of structural proteins during assembly of head of bacteriophage-T4.Nature,1970,227(5259):680-685.
[18] Chen X J S,Casini G,Harrison S C,et al.Papillomavirus capsid protein expression in Escherichia coli:Purification and assembly of HPV11 and HPV16 L1.Journal of Molecular Biology,2001,307(1):173-182.
[19] Yang C Y,Pan H R,Wei M X,et al.Hepatitis E virus capsid protein assembles in 4M urea in the presence of salts.Protein Science,2013,22(3):314-326.
[20] Zhang Y,Song S X,Liu C Y,et al.Generation of chimeric HBc proteins with epitopes in E.coli:formation of virus-like particles and a potent inducer of antigen-specific cytotoxic immune response and anti-tumor effect in vivo.Cellular Immunology,2007,247(1):18-27.

[1]	张玲,曹小丹,杨海旭,李文蕾. 连续流层析技术在亲和层析中的应用及生产放大评估[J]. 中国生物工程杂志, 2021, 41(6): 38-44.
[2]	付大伟,孙莹莹,徐伟. 融合蛋白NusA-hRI的高效异源表达、纯化及活性分析[J]. 中国生物工程杂志, 2019, 39(3): 21-28.
[3]	王云龙, 赵二霞, 李玉林. Thymidine Kinase 1(TK1)重组蛋白的表达、纯化及鉴定[J]. 中国生物工程杂志, 2017, 37(9): 15-22.
[4]	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.
[5]	蒙国基, 邓义熹, 李乐, 罗豪晖, 于玉根. 变速上样在MabSelect填料上分离WLB303[J]. 中国生物工程杂志, 2016, 36(6): 65-75.
[6]	代云见, 张勇侠, 何勇智, 丛聪, 张涛, 王明蓉. 技术与方法Anti-IgE单链抗体纯化工艺研究及活性鉴定[J]. 中国生物工程杂志, 2015, 35(12): 51-57.
[7]	赵倩倩, 周晓今, 柳小庆, 杨文竹, 李素贞, 刘奇, 陈茹梅. 转基因玉米中植酸酶蛋白免疫亲和纯化体系的建立[J]. 中国生物工程杂志, 2013, 33(6): 125-130.
[8]	包海生, 高秀峰, 郑雪妮, 李永生. 壳聚糖锌离子固定化亲和层析填料的制备与性质[J]. 中国生物工程杂志, 2012, 32(05): 85-90.
[9]	程丽芳,沐万孟,张涛,江波. Bacillus Stearothermophilus IAM 11001 L-阿拉伯糖异构酶在大肠杆菌中的表达、纯化及活性研究[J]. 中国生物工程杂志, 2008, 28(9): 52-55.
[10]	窦佳,蔡河,姬芳玲,崔文举,王静云,包永明,安利佳. Gln49-磷脂酶A2基因工程定点突变及酶活性分析[J]. 中国生物工程杂志, 2007, 27(5): 21-27.
[11]	张青,张思河,苏明权,沈建军,韦晓明,郝晓柯. 人γ－精浆蛋白的亲和纯化及其糖基化分析[J]. 中国生物工程杂志, 2007, 27(2): 99-102.
[12]	余德荣,游力,赵跃然,王郡甫,许晓群,高春义. 重组hKGF2原核表达载体的构建及其蛋白质的纯化[J]. 中国生物工程杂志, 2007, 27(2): 9-13.
[13]	雷旭宇, 杨青, 包永明, 许建强, 栾雨时, 安利佳. 大连蛇岛蝮蛇类凝血酶在大肠杆菌中的表达与纯化[J]. 中国生物工程杂志, 2004, 24(9): 44-48.
[14]	金晓霞, 罗坚, 苏志国. 血浆蛋白分离纯化的进展——亲和技术的重要作用[J]. 中国生物工程杂志, 2004, 24(7): 74-81.
[15]	李义良, 宋海, 赵静, 张红锋, 李载平, 赵慕钧. LPTS抗体的制备和活性检测[J]. 中国生物工程杂志, 2004, 24(3): 44-47.

Viewed

Full text

Abstract

Cited

Shared

Discussed