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

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2．5～80 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。%Objective:To analyze the influencing factors of fluorescence method in determination of residual DNA in recombinant hep-atitis 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 ~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.