不同层析介质纯化H5N1型流感病毒的研究

Purification of H5N1 influenza virus by different chromatography media

目的分别用新型复合层析介质Capto Core700和传统分子筛介质Sepharose 4FF纯化MDCK细胞制备的H5N1型流感病毒浓缩液,并比较分离纯化效果。方法用Capto Core700与Sepharose 4FF纯化灭活H5N1型流感病毒浓缩液,透射电镜分析不同样品中病毒颗粒的形态;分别用单向免疫扩散法(single radial immunodiffusion,SRID)、福林酚法(Lowry法)、双抗体夹心ELISA、qPCR检测不同层析介质纯化前后病毒血凝素含量、总蛋白质含量、宿主细胞蛋白质(host cell protein,HCP)含量和宿主细胞DNA(host cell DNA,HCD)含量,计算病毒抗原回收率和杂质去除率;用动物实验检测不同纯化病毒的免疫原性,并用t检验分析两种层析介质纯化效果差异。结果Capto Core700与Sepharose 4FF纯化的H5N1型流感病毒在透射电镜下均呈典型的流感病毒形态;两种层析介质纯化后病毒血凝素回收率差异无统计学意义(P>0.05),但前者的杂质(总蛋白质、HCP、HCD)去除率均高于后者,且差异有统计学意义(P<0.05);动物实验表明两种层析介质纯化的病毒样品均具有良好的免疫原性。结论相对于Sepharose 4FF层析介质,Capto Core700在保证病毒抗原蛋白回收率的基础上可以更有效地去除HCP、HCD和总蛋白质等工艺相关杂质。本研究为MDCK细胞生产H5N1型流感病毒疫苗纯化工艺开发提供了参考。

Objective To purify H5N1 influenza virus concentrate prepared by MDCK cells with a new mixed-mode chromatography medium Capto Core700 and the traditional medium Sepharose 4FF,and to compare the separation and purification efficacy of the two media.Methods Capto Core700 and Sepharose 4FF were used to purify inactivated H5N1 influenza virus concentrate.The morphology of virus particles in different samples was then observed under a transmission electron microscope.Single radial immunodiffusion(SRID),Folin-Phenol(Lowry)method,double-antibody sandwich ELISA and qPCR were used to detect hemagglutinin,total protein,host cell protein(HCP)and host cell DNA(HCD)before and after purification.The recovery rate of virus antigen and the removal rate of impurities were calculated.The immunogenicity of the viruses purified with different media was analyzed using animal experiments.Difference in the purification efficacy of the two chromatography media was analyzed by t-test.Results H5N1 influenza viruses purified by Capto Core700 or Sepharose 4FF showed the typical influenza virus morphology under transmission electron microscope.There was no significant difference in the recovery rate of hemagglutinin between the two chromatography media(P>0.05),but compared with Sepharose 4FF,Capto Core700 had a higher removal rate of impurities(total protein,HCP,HCD)and the difference was statistically significant(P<0.05).Animal experiments showed that the viruses purified by the two chromatography media had good immunogenicity.Conclusions Compared with Sepharose 4FF chromatography medium,Capto Core700 could more effectively remove process-related impurities such as HCP,HCD and total protein without affecting the recovery rate of viral antigen.This study provided reference for the development of purification technology in the production of H5N1 influenza virus vaccine in MDCK cells.