氯仿抽提纯化CA 16病毒最优条件的筛选  

Optimization of condition for purification of Coxsackievirus A 16 virus by chloroform extraction

作　　者：张也[1] 李华[1] 杨婷[1] 岳磊[1] 谢天宏[1] 龙润乡[1] 罗芳宇 杨蓉[1] 何鑫 谢忠平[1] ZHANG Ye, LI Hua, YANG Ting, YUE Lei, XIE Tian-hong, LONG Run-xiang, LUO Fang-yu, YANG Rong, lie Xin, XIE Zhong-ping(Institute of Medical Biology, Chinese Academy of Medical Science and Peking Union Medical College, Yunnan Key Laboratory of Vaccine Research and Development on Severe Infectious Diseases, Kunming 650118, Yunnan Province, Chin)

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工程技术研究中心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》2018年第3期282-286,共5页Chinese Journal of Biologicals

基　　金：云南省重点新产品开发计划(2015BC008)

摘　　要：目的筛选氯仿抽提纯化柯萨奇病毒A组16型(Coxsakievirus A 16,CA16)的最优条件。方法根据《中国药典》三部(2015版)的相关要求,选择病毒感染性滴度、残余蛋白、残余牛血清白蛋白(bovine serum albumin,BSA)、残余卡那霉素及残余氯仿含量等指标,分析氯仿抽提的不同条件及超滤浓缩对纯化产物质量指标的影响。将CA16浓缩病毒液与氯仿按不同体积比进行一抽多洗纯化,分析不同比例氯仿对纯化效果的影响;将CA16超滤浓缩病毒液分别用不同浓度的PBS洗涤,以评价不同离子浓度的PBS洗涤对纯化病毒纯化液质量指标的影响。结果不同浓度的PBS洗涤液、病毒液与氯仿体积比对纯化液病毒感染性滴度均无明显影响,病毒回收率平均为81.7%;随着PBS洗涤次数的增多,洗液中的杂蛋白逐渐减少,氯仿比例及PBS离子浓度对蛋白去除率影响不明显,蛋白去除率均大于82%(82.69%~93.6%),平均去除率88.37%;不同氯仿比例、不同PBS浓度及不同洗涤次数对残余卡那霉素含量无明显影响,结果均低于30 ng/mL,经超滤浓缩后,其含量低于3.0 ng/mL;氯仿抽提纯化残余BSA的去除率可达68.87%,若结合超滤浓缩工艺,残余BSA的去除率可达94.74%;超滤浓缩后纯化病毒液残余氯仿去除率可达98%以上,最终纯化液残余氯仿含量小于6μg/mL。结论氯仿抽提方法结合超滤浓缩可得到符合要求的CA16病毒实验性疫苗。Objective To optimize the condition for purification of Coxsackievirus A 16（CA16）by chloroform extraction.Methods According to the requirements in Chinese Pharmacopoeia（Volume Ⅲ,2015 edition）,the effect of condition for chloroform extraction as well as concentration by ultrafiltration on quality of purified product was evaluated using infectious virus titer,residual protein,residual bovine serum albumin（BSA）,residual kanamycin and residual chloroform content as indicators. The concentrated CA16 virus was mixed with chloroform at various volume ratios to evaluate the influence of chloroform proportion on purification effect of CA16 virus by one time of extraction and several times of washing. The concentrated CA16 virus was mixed with PBS at various concentrations to evaluate the influence of PBS concentration on quality indexes of purified virus liquid. Results PBS concentration and the volume ratio of virus liquid to chloroform showed no significant effect on the infectious titer of purified virus liquid,and the mean recovery rate of virus was 81. 7%. With the increasing times of PBS washing,the foreign protein in washing solution decreased gradually.Chloroform proportion,PBS concentration and times of washing showed no significant influence on the residual kanamycin content. All the determination results of kanamycin content were less than 30 ng/mL,which were less than 3. 0 ng/mL after concentration by ultrafiltration. The removal rate of residual BSA was 68. 87% after chloroform extraction,which was 94. 74% after concentration by ultrafiltration. However,the removal rate of the residual chloroform was more than 98%after concentration by ultrafiltration,which was less than 6 μg/mL in final purification liquid. Conclusion Experimental CA16 vaccine was obtained by chloroform extraction combined with concentration by ultrafiltration.

关 键 词：柯萨奇病毒A组16型 氯仿抽提 疫苗 质量控制 

分 类 号：R373.23[医药卫生—病原生物学] R392-33[医药卫生—基础医学]