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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 particle (VLP) of Enterovirus 71 (EV71) in Hansenula and analyze its immunogenicity. Methods The recombinant plasmid pMV-P1-3CD was transformed into Hansenula polymorpha AU0501, and the recombinant strain p MV-P1-3CD / AU stably integrated with P1 and 3CD genes was obtained. The recombinant strain was cultured in 30 L fermentor. The fermentation products were purified and analyzed by SDS-PAGE, Western blot, HPLC, electron microscopy and dynamic light scattering. The EV71 vaccine was prepared by different methods. The mice were immunized with rabbits and rabbits respectively. The geometric mean titers (GMTs) of neutralizing antibodies in serum were detected to evaluate the immunogenicity and the immunogenicity of different vaccines. Results SDS-PAGE analysis of the expressed product of p MV-P1-3CD / AU and the vaccine stock showed that the VP3, VP1 and VP0 protein bands were found at the relative molecular mass of 26 000, 33 000 and 35 000, and the relative molecular The protein with the mass of 33 000 could bind specifically with the EV71-VP1 monoclonal antibody. The purity of the vaccine stock was over 99%. The VLP of 30 nm was observed by electron microscopy and the particle homogeneity was good. The serum GMT of mouse serum was up to 1 536 , Rabbit serum GMT up to 586, aluminum phosphate adjuvant and aluminum hydroxide adjuvant can significantly increase serum GMT in mice. Conclusions The co-expression of P1 and 3CD genes in EV71 can be successfully established in Hansenula. EV71 VLP can be formed and has good immunogenicity, which provides an experimental basis for the future development of EV71 VLP vaccine.