摘要
目的为了探索经济、高效的腺病毒的纯化工艺,采用阴离子交换层析和分子筛层析两步法对H3亚型流感重组腺病毒pacAd-H3-EHA-H1HA1new进行纯化,并评价纯化效果。方法利用7L生物反应器培养HEK-293细胞,用于对H3型流感重组腺病毒进行扩增;利用冻融的方法使病毒从细胞中释放,通过阴离子交换层析和分子筛层析法纯化病毒,用分光光度计检测纯化样品的纯度,并测定病毒的滴度和回收率。结果纯化后的样品经PCR鉴定正确,纯化前后的病毒滴度分别为5.6×10~9 TCID_(50)/ml和1.1×10^(10) TCID_(50)/ml,纯化后样品纯度(A260/A280值)分别为2.01和1.26,阴离子交换层析纯化的回收率为31.5%,分子筛层析纯化的回收率为81.4%,总回收率为25.6%。结论利用阴离子交换层析和分子筛层析两步法纯化H3亚型流感重组腺病毒的回收率和滴度高,该方法可用于重组腺病毒的纯化。
Objective To purify the recombinant adenovirus pacAd-H3-EHA-H1HA1 with the AKTA purifier-100 in order to establish an economical and efficient method for purification of adenovirus. Methods A recombinant adenovirus containing an epitope of the hemagglutinin antigen of influenza virus subtype H3 was cultured with HEK-293 cells in a 7L bioreactor.After freezing and thawing,the virus was purified using anion exchange chromatography and molecular sieve chromatography.The purified samples were tested with a spectrophotometer,and the titer and recovery rate of the virus were determined. Results The purified samples were identified as correct according to PCR,and the virus titer increased from 5.6×10^9 TCID50/ml to 1.1×10^10 TCID50/ml after purification.The purified samples had a purity of 2.01 and 1.26,respectively.The recovery rate of anion exchange chromatography was 31.5%,the recovery rate of purification by molecular sieve chromatography was 81.4%,and the total recovery rate was 25.6%. Conclusion In this study,a recombinant adenovirus containing an epitope of the hemagglutinin antigen of influenza virus subtype H3 was purified using two-step purification,and results indicated that the method of purification was feasible and effective at purification of the virus.Thus,this work has laid the foundation for vaccine production.
出处
《中国病原生物学杂志》
CSCD
北大核心
2016年第9期779-782,787,共5页
Journal of Pathogen Biology
基金
国家863项目(No.2012AA02A403)
国际合作交流项目(No.2014DFA30290)
吉林省自然科学基金项目(No.20130101093JC)
关键词
H3亚型流感
重组腺病毒疫苗
纯化
Influenza virus subtype H3
recombinant adenovirus
purification
