柯萨奇病毒A组16型不同纯化方法的比较研究  

Comparison methods for the purification of coxsakievirus vaccine

作　　者：杨婷[1] 李华[1] 谢天宏[1] 杨水芝[1] 洪超[1] 岳磊[1] 朱凡丽 张也[1] 宋霞[1] 龙润乡[1] 杨蓉[1] 罗芳宇 谢忠平[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,昆明650118

出　　处：《中国病毒病杂志》2015年第4期293-299,共7页Chinese Journal of Viral Diseases

基　　金：云南省科技计划项目(2013FZ134;2012BC006)

摘　　要：目的评价不同方法纯化柯萨奇病毒A组16型(Coxsakievirus A 16,CA16)的可行性和有效性。方法将CA16病毒收获液经切向流超滤浓缩后,分别进行离心、氯仿抽提、分子筛和蔗糖垫层/梯度离心,按《中国药典》(三部)(2010版)的检定要求检测不同纯化方法得到的样品,检测指标包括:感染性滴度、抗原含量、蛋白含量、比活性、HPLC纯度、残余牛血清白蛋白、残余抗生素、免疫原性。结果从比活性来看,分子筛纯化法是其他方法的2~21倍,明显优于其他工艺;经HPLC检测,分子筛纯化样品纯度>98%,而其他工艺均在50%以下;分子筛法所得纯化样品中牛血清白蛋白残留量符合《中国药典》要求(<50ng/剂),去除率达99.97%;分子筛法和蔗糖垫层/梯度离心法所得纯化样品中抗生素残留量符合《中国药典》要求(<50ng/剂),分子筛法去除率在80%以上;从免疫小鼠后的抗体阳转率来看,离心和氯仿抽提样品组阳转率为100%,分子筛组为80%,蔗糖离心组为50%;从抗体几何平均滴度(geometric mean titer,GMT)来看,氯仿抽提组最高,为1∶912。结论以Sepharose 6Fast Flow作为层析介质的分子筛法可得到高纯度的CA16病毒,综合分析。Objective To evaluate different methods for the purification of Coxsakievirus A16(CA16)vaccine. Methods Harvested CA16 was concentrated by tangential flow filtration and then purified through centrifugation,chloroform extraction,size exclusion chromatography,sucrose cushion centrifuge or sucrose discontinuous density gradient centrifugation.Purified viruses from different procedures are analyzed according to the regulations of the Chinese Pharmacopoeia(Volume Ⅲ 2010Edition).The infection titer,antigen content,protein content,specific activity,purity,residue of bovine serum albumin and antibiotics were detected. Results The specific activity of purified viruses by size exclusion chromatography was 2-21 folds higher than that from other procedures;its purity measured by HPLC was over 98%,yet under 50% with other purification methods.The residue of bovine serum albumin and antibiotics in the samples from size exclusion chromatography was in accordance with the regulation of the Chinese Pharmacopoeia,and the proportions of removal by size exclusion chromatography were 99.97%.The positive rates of neutralization antibody in immunized mice with samples purified by centrifugation and chloroform extraction were 100%,and the positive rates in the size exclusion chromatography and centrifugation in sucrose groups were 80% and 50%.The highest Genometric Mean Titer(GMT)was seen in the chloroform extraction group(1∶912). Conclusions High-purity CA16 viruses were obtained by size exclusion chromatography using Sepharose 6Fast Flow.Comprehensively,the size exclusion Chromatography is preferable for purification for purification of CA16.

关 键 词：柯萨奇病毒A组16型 纯化方法 质量控制 

分 类 号：R373.23[医药卫生—病原生物学]