• 中国核心期刊(遴选)数据库收录期刊
  • 中文科技期刊数据库收录期刊
  • 中国期刊全文数据库收录期刊
  • 中国学术期刊综合评价数据库统计源期刊等

• 评价技术与方法 •    下一篇

荧光染色法测定重组酵母乙型肝炎疫苗原液中残留DNA的影响因素分析

邱少辉,方鑫,赵冉,邹烨宁,何鹏,梁争论,胡忠玉   

  1. 中国食品药品检定研究院,中国食品药品检定研究院,北京科兴生物制品有限公司,北京科兴生物制品有限公司,中国食品药品检定研究院,中国食品药品检定研究院,中国食品药品检定研究院
  • 收稿日期:2014-08-18 修回日期:2014-08-18 出版日期:2014-10-25 发布日期:2014-10-25
  • 基金资助:
    “艾滋病和病毒性肝炎等重大传染病防治”国家科技重大专项

Analysis of influencing factors of determination of residual DNA in recombinant hepatitis B vaccine bulk by fluorescence method

  1. National Institutes for Food and Drug Control,National Institutes for Food and Drug Control,Sinovac Biotech Ltd Beijing,Sinovac Biotech Ltd Beijing,National Institute for Food and Drug Control,National Institutes for Food and Drug Control,
  • Received:2014-08-18 Revised:2014-08-18 Online:2014-10-25 Published:2014-10-25
  • Supported by:
    National Science and Technology Key Projects on “Major Infectious Diseases such as HIV/AIDS, Viral Hepatitis Prevention and Treatment”

摘要: 目的: 对荧光染色法测定重组酵母乙肝疫苗原液中残留DNA的影响因素进行探索分析,以了解该方法对本疫苗残留DNA检定的适用性。方法: 参照探针杂交法对乙肝疫苗原液残留DNA的测定结果,对乙肝疫苗原液中可能存在Tween-20、PEG、蛋白等物质对荧光染色法测定残留DNA含量的影响进行分析,对该方法的线性范围、用于乙肝疫苗原液残留DNA检定的准确性和重复性进行了研究,并对不同来源DNA标准存在的差异进行比较。结果: 荧光法测定酵母乙肝疫苗残留DNA线性范围为2.5 ng.mL-1~80 ng.mL-1;发现Tween-20、PEG等对残留DNA检测影响较小,加标回收率均在80%-120%之间;而蛋白质对检测影响较大,经酚-三氯甲烷抽提后可有效去除蛋白质干扰,回收率达到90%左右,CV小于10%;同时发现不同来源的DNA标准品存在荧光标记效率的差异。 结论: 乙肝疫苗原液经处理去除蛋白干扰后可采用荧光染色法进行残留DNA含量的测定,但应注意使用与疫苗表达系统相同宿主来源的DNA标准品。

Abstract: Objective: To analyze the influencing factors of fluorescence method in determination of residual DNA in recombinant hepatitis B vaccine bulk, and to evaluate the applicability of this method in hepatitis B vaccine control. Methods: According to the result of residual DNA in the bulk of hepatitis B vaccine determined by Digoxin-labeled DNA hybridization method, interference factors such as Tween-20, PEG and protein were explored. The linear range of the method, as well as accuracy and precision were evaluated for residual DNA determination in hepatitis B vaccine bulk. DNA standards from different sources were also investigated for their variance in labeling efficiency. Results: The linear range of fluorescence method was between 2.5 ng.mL-1~80 ng.mL-1, which could be eliminated by phenol-chloroform extraction. Interference of protein was observed in determination of residual DNA by fluorescence method, with the recovery rate of about 90% and the coefficient of variation below 10%. Meanwhile, variance in fluorescence labeling efficiency of DNA standards from different sources was found. Conclusion: The fluorescence method is applicable in residual DNA detection in the bulk of hepatitis B vaccine pretreated with phenol-chloroform. However, DNA standard from the same source with the hepatitis B vaccine expression system should be used in the detection.