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

发布时间：2019-03-16 18:19

【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。
[Abstract]:Aim to express enterovirus 71 (enterovirus 71, EV 71) like virus granules (virus-like particle,VLP) in Hanson yeast and analyze its immunogenicity. Methods the recombinant strain pMV-P1-3CD/AU.30 L, which stably integrated P1 and 3CD genes, was obtained by transforming the recombinant plasmid p MV-P1-3CD into yeast AU0501,. The recombinant strain with stable integration of P1 and 3CD genes was cultured in fermentor. After purification, the fermentation products were detected by SDS-PAGE,Western blot,HPLC, electron microscopy and dynamic light scattering analysis. EV71 vaccines were prepared by different methods and immunized mice and rabbits respectively. The geometric mean titers of neutralizing antibodies in serum of mice and rabbits were detected to evaluate the immunogenicity of the vaccines and the effects of different adjuvant on the immunogenicity of the vaccines. Results SDS-PAGE analysis of the recombinant strain p-MV-P1-3CD/AU expression product and vaccine solution showed that the VP3,VP1 and VP0 protein bands were found at 26 000, 33 000 and 35 000 relative molecular weight. The protein with relative molecular weight of 33 000 could specifically bind to EV71-VP1 monoclonal antibody. The purity of the original vaccine solution was over 99%, and the VLP, particles of 30 nm were found to be good at one degree under electron microscope. The highest serum GMT in mice was 1536, and the highest GMT in rabbits was 586. Both aluminum phosphate adjuvant and aluminum hydroxide adjuvant could significantly increase the serum GMT. in mice. Conclusion the co-expression of P1 and 3CD genes of EV71 in Hanson yeast can form EV71 VLP, and have good immunogenicity, which provides experimental basis for the development of EV71 VLP vaccine in the future.
【作者单位】：北京民海生物科技有限公司研发中心;
【分类号】：R392

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