两种层析方法纯化轮状病毒效果的比较  

Purification of rotavirus by two chromatographic methods

作　　者：杨星[1] 吴晋元[1] 易山[1] 李鸿钧[1] 张光明[1] 郜岩[2] 贾琴妹[1] 赵晓南[1] 孙茂盛[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,昆明650118 [2]昆明医学院,昆明650031

出　　处：《中国生物制品学杂志》2012年第6期754-758,共5页Chinese Journal of Biologicals

基　　金：云南省社会发展科技计划国际合作项目(2008CA028);云南省应用基础研究计划面上项目(2009CD142)

摘　　要：目的比较两种不同的凝胶层析纯化方法对人轮状病毒(Rotavirus,RV)的纯化效果及其对病毒抗原性和免疫原性的影响。方法病毒收获液经超滤浓缩后,分别采用Q Sepharose Fast Flow(QFF)离子交换层析和Sepharose 4 Fast Flow(4FF)凝胶过滤层析纯化,采用A群轮状病毒诊断试剂盒(胶体金)检测纯化病毒的抗原性;电镜观察病毒的形态;荧光灶法测定病毒纯化前后的感染性滴度;双抗体夹心ELISA法测定抗原含量;Lowry法测定纯化前后样品的蛋白含量;SDS-PAGE和Western blot鉴定纯化的病毒蛋白;纯化病毒经灭活后,PAGE分析病毒的核酸带型,免疫小鼠,检测纯化病毒的免疫原性。结果QFF洗脱峰1和4FF洗脱峰1为RV抗原阳性,电镜观察可见完整的、大小约70 nm的RV颗粒;经QFF和4FF层析纯化后,病毒的感染性滴度分别为7.10和4.50 lgCCID50/ml,最终抗原回收率分别为65.3%和11.8%,病毒液总蛋白去除率分别为99.68%和99.52%;SDS-PAGE分析显示,经QFF和4FF纯化的样品未见明显杂蛋白条带,且特异性良好;纯化病毒灭活后,病毒基因组带型未发生改变,且保持了良好的抗原性和免疫原性。结论两种凝胶层析方法均能纯化人轮状病毒,综合比较,QFF离子交换层析纯化效果优于4FF凝胶过滤层析。Objective To compare the purification effects of two gel chromatographic methods on human rotavirus（RV） as well as their influence on antigenicity and immunogenicity of RV.Methods Virus harvest was concentrated by ultrafiltration,purified by Q Sepharose Fast Flow（QFF） ion exchange chromatography and Sepharose 4 Fast Flow（4FF） gel filtration chromatography,then observed for morphology by electron microscopy,and determined for antigenicity by diagnostic kit（colloidal gold） for group A RV,for infectious titers before and purification by fluorescent focus method,and for antigen content by double antibody sandwich ELISA.The purified viral protein was identified by SDS-PAGE and Western blot,analyzed for pattern of nucleic acid by PAGE,and used for immunization of mice to evaluate the immunogenicity.Results The elution peak 1 of QFF chromatography and elution peak 1 of 4FF chromatography were positive for RV antigen.Intact RV particles each at a diameter of about 70 nm were observed under electron microscope.After purification by QFF and 4FF chromatography,the infectious titers of RV were 7.10 and 4.50 lg CCID50 / ml,while the final recovery rates of antigen were 65.3% and 11.8%,and the removal rates of total protein were 99.68% and 99.52%,respectively.SDS-PAGE showed no obvious foreign protein bands in the samples purified by QFF and 4FF chromatography,indicating high specificity.The purified RV after inactivation showed no change in pattern of genome,of which high antigenicity and immunogenicity were maintained.Conclusion Both QFF and 4FF chromatography were suitable for purification of human RV,however,comprehensively,QFF ion exchange chromatography was superior to 4FF gel filtration chromatography.

关 键 词：轮状病毒 纯化 离子交换层析 凝胶过滤层析 

分 类 号：R392-33[医药卫生—免疫学] R373.2[医药卫生—基础医学]