肠道病毒71型类病毒颗粒在汉逊酵母中的表达及其免疫原性  

Expression of virus-like particles of enterovirus 71 in Hansenula polymorpha and their immunogenicity

作　　者：李国顺[1] 郭林[1] 方玉松[1] 陈磊[1] 张盼[1] 徐颖之 王凤伟[1] 顾美荣[1] 魏文进[1] 郑海发[1] 

机构地区：[1]北京民海生物科技有限公司研发中心,北京102600

出　　处：《中国生物制品学杂志》2015年第9期906-911,共6页Chinese Journal of Biologicals

摘　　要：目的于汉逊酵母中表达肠道病毒71型(enterovirus 71,EV71)类病毒颗粒(virus-like particle,VLP),并分析其免疫原性。方法将鉴定正确的重组质粒p MV-P1-3CD转化汉逊酵母AU0501,经筛选获得稳定整合P1和3CD基因的重组菌株p MV-P1-3CD/AU。30 L发酵罐培养重组菌株,发酵产物纯化后进行SDS-PAGE、Western blot、HPLC检测、电镜分析及动态光散射分析检测。采用不同方法制备EV71疫苗,分别免疫小鼠和家兔,检测动物血清中和抗体几何平均滴度(GMT),评价其免疫原性及不同佐剂对疫苗免疫原性的影响。结果重组菌株p MV-P1-3CD/AU表达产物及疫苗原液的SDS-PAGE分析显示,于相对分子质量26 000、33 000和35 000处均可见VP3、VP1和VP0蛋白条带,且相对分子质量33 000的蛋白可与EV71-VP1单克隆抗体发生特异性结合;疫苗原液的纯度达99%以上;电镜观察可见30 nm的VLP,颗粒均一度较好;小鼠血清GMT最高可达1 536,家兔血清GMT最高可达586,磷酸铝佐剂及氢氧化铝佐剂均可明显提高小鼠血清GMT。结论于汉逊酵母中成功实现EV71的P1和3CD基因的共表达,可形成EV71 VLP,且具有良好的免疫原性,为今后EV71 VLP疫苗的研制提供了实验依据。Objective To express the virus-like particles（VLPs）of enterovirus 71（EV71）in Hansenula polymorpha and analyze their immunogenicity. Methods Correctly constructed recombinant plasmid p MV-P1-3CD was transformed to H. polymorpha AU0501, based on which recombinant strain p MV-P1-3CD / AU stably integrated with P1 and 3CD genes was screened and cultured in a 30 L fermenter. The fermentation product was purified, and analyzed by SDS-PAGE,Western blot, HPLC, electron microscopy and dynamic light scattering（DLS）. EV71 vaccine was prepared by various methods and used for immunization of mice and rabbits. The GMTs of neutralizing antibody in sera of animals were determined to evaluate the immunogenicity and effects of various adjuvants. Results Both SDS-PAGE profiles of expressed product and bulk vaccine showed VP3, VP1 and VP0 protein bands with relative molecular masses of 26 000,33 000 and 35 000 respectively, of which the one with a relative molecular mass of 33 000 showed specific binding to monoclonal antibody against EV71-VP1. The bulk vaccine reached a purity of more than 99%. VLPs with good homogeneity, at a mean size of 30 nm, were observed by electron microscopy. The GMT of serum antibody of mice reached1 536 at most, while that of rabbits reached 586 at most. Aluminum phosphate adjuvant increased the GMT of serum antibody of mice significantly. Conclusion P1 and 3CD genes of EV71 were co-expressed in H. polymorpha, and the exoressed protein was self-assembled into VLPs with good immu-nogenicity, which provided an experimental basis for further preparation of EV71 VLP vaccine.

关 键 词：肠道病毒71型 类病毒颗粒 汉逊酵母 免疫原性 

分 类 号：R373.2[医药卫生—病原生物学] Q786[医药卫生—基础医学]