Benzonase酶去除流感疫苗中Vero细胞DNA条件的优化  

Optimization of condition for removal of Vero cell DNA in influenza H5N1 vaccine with Benzonase nuclease

作　　者：徐国峰[1] 耿兴良 宋绍辉[1] 马磊[1] 廖国阳[1] 李卫东[1] 

机构地区：[1]中国医学科学院北京协和医学院医学生物学研究所云南省重大传染病疫苗研发重点实验室,云南昆明650118

出　　处：《中国生物制品学杂志》2017年第5期540-545,共6页Chinese Journal of Biologicals

基　　金：国家科技部重大专项(2013ZX10004003-003-002);中国医学科学院医学科技创新团队(2016-I2M-3-026);云南省创新团队(2015HC027)

摘　　要：目的优化非限制性内切酶Benzonase去除Vero细胞制备的H5N1流感疫苗中残余细胞DNA的条件,并结合两步柱层析法使疫苗中DNA残余达到质量要求。方法向H5N1流感病毒浓缩液中添加不同终浓度的Benzonase酶(10、20、30、40、50、60 U/ml),置于37℃酶解不同时间(0.5、1、1.5、2、2.5、3 h),经100 k D超滤离心管,用不同p H(7.10、7.30、7.50、7.70、7.90)的PBS进行洗滤浓缩。将Benzonase酶处理的病毒液经蔗糖密度梯度离心纯化,去除部分杂蛋白,电镜下观察病毒的形态。结合两步柱层析法进一步去除Vero细胞DNA。结果 H5N1流感病毒液经终浓度为10 U/ml的Benzonase酶,37℃处理2.5 h后,用p H为7.50的PBS溶液进行洗滤,其DNA去除率可达99%以上。Benzonase酶处理前后病毒形态一致,结构完整,轮廓清晰。在用酶处理去除原液中大部分Vero细胞残余DNA的基础上,结合两步柱层析法基本可使疫苗残余外源DNA达到100 pg/剂的限定标准。结论非限制性核酸内切酶Benzonase可高效降解H5N1流感病毒液中的DNA,此方法降低流感疫苗中Vero细胞DNA简单可行,大大降低了后续工艺去除DNA的压力,同时结合两步柱层析法可使疫苗中DNA残余达到限定标准,为以Vero细胞为基质大流行流感疫苗的研发奠定了基础。Objective To optimize the conditions for removal of Vero cell DNA in influenza H5N1 vaccine with Benzonase nuclease,and make the residual DNA content meet the requirement for quality control by two step column chromatography.Methods Concentrated influenza H5N1 virus liquid was treated with Benzonase nuclease at various concentrations （10,20,30,40,50 and 60 U/ml） at 37 ℃ for various （0.5,1,1.5,2,2.5 and 3） hours,and filtered by 100 kD ultrafiltration centrifugal tubes,then concentrated with PBS at various pH values （7.10,7.30,7.50,7.70 and 7.90）.The virus liquid after treatment with Benzonase nuclease was purified by sucrose density gradient centrifugation to remove partial foreign proteins,and observed for morphology by microscopy.Vero cell DNA was further removed by two step column chromatography.Results After the harvested influenza H5N1 virus liquid was treated with 10 U/ml Benzonase nuclease at 37 ℃ for 2.5 h,and filtered by 100 kD ultrafiltration centrifugal tube with PBS buffer （pH 7.50）,more than 99% of DNA was removed.The morphologies of virus before and after treatment with Benzonase nuclease were in agreement,while the virus structures were intact and the outlines were clear.After the majority of Vero cell DNA in bulk was removed by treatment with Benzonase nuclease,the residual foreign DNA met the limited standard of 100 pg/dose after two step column chromatography.Conclusion Benzonase nuclease efficiently degraded the DNA in influenza H5N1 virus liquid,which was simple and feasible to reduce Vero cell DNA in influenza vaccine.It greatly reduced the pressure of the subsequent process to remove DNA and,in combination with two step column chromatography,made the DNA residue in the vaccine meet the limit standard,which laid a foundation of development of Vero cell culture-derived pandemic influenza vaccines.

关 键 词：Benzonase酶 H5N1流感病毒 VERO细胞 DNA 超滤 柱层析 

分 类 号：R373.13[医药卫生—病原生物学] R392-33[医药卫生—基础医学]