H3亚型流感重组腺病毒的纯化工艺研究  

Purification of a recombinant adenovirus containing an epitope of the hemagglutinin antigen of the influenza virus

作　　者：张萍[1,2] 郭海宁[1,2] 解长占 韩继成[2] 孙文超[2] 崔卓栋 南福龙 马海彬[2] 温树波[2] 肖朋朋[2] 庄忻雨 张赫[2] 张金勇[1,2] 赵冠宇[2,3] 鲁会军[2] 金宁一[2] 

机构地区：[1]吉林农业大学动物科学技术学院,吉林长春130118 [2]军事医学科学院军事兽医研究所,吉林长春130122 [3]吉林大学动物医学学院,吉林长春130062

出　　处：《中国病原生物学杂志》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)

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.

关 键 词：H3亚型流感 重组腺病毒疫苗 纯化 

分 类 号：R373.13[医药卫生—病原生物学]