中国药物评价
中國藥物評價
중국약물평개
Chinese Journal of Drug Evaluation
2014年
5期
257-261
,共5页
邱少辉%方鑫%赵冉%邹烨宁%何鹏%梁争论%胡忠玉
邱少輝%方鑫%趙冉%鄒燁寧%何鵬%樑爭論%鬍忠玉
구소휘%방흠%조염%추엽저%하붕%량쟁론%호충옥
乙型肝炎疫苗%荧光染色法%残留 DNA
乙型肝炎疫苗%熒光染色法%殘留 DNA
을형간염역묘%형광염색법%잔류 DNA
Hepatitis B vaccine%Fluorescence method%Residual DNA
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。
目的：對熒光染色法測定重組酵母乙肝疫苗原液中殘留 DNA 的影響因素進行探索分析，以瞭解該方法對本疫苗殘留DNA 檢定的適用性。方法：參照探針雜交法對乙肝疫苗原液殘留 DNA 的測定結果，對乙肝疫苗原液中可能存在 Tween-20、PEG、蛋白等物質對熒光染色法測定殘留 DNA 含量的影響進行分析，對該方法的線性範圍、用于乙肝疫苗原液殘留 DNA 檢定的準確性和重複性進行瞭研究，併對不同來源 DNA 標準存在的差異進行比較。結果：熒光法測定酵母乙肝疫苗殘留 DNA 線性範圍為2．5～80 ng? mL-1；髮現 Tween-20、PEG 等對殘留 DNA 檢測影響較小，加標迴收率均在80％～120％之間；而蛋白質對檢測影響較大，經酚-三氯甲烷抽提後可有效去除蛋白質榦擾，迴收率達到90％左右，CV 小于10％；同時髮現不同來源的 DNA 標準品存在熒光標記效率的差異。結論：乙肝疫苗原液經處理去除蛋白榦擾後可採用熒光染色法進行殘留 DNA 含量的測定，但應註意使用與疫苗錶達繫統相同宿主來源的 DNA 標準品。
목적：대형광염색법측정중조효모을간역묘원액중잔류 DNA 적영향인소진행탐색분석，이료해해방법대본역묘잔류DNA 검정적괄용성。방법：삼조탐침잡교법대을간역묘원액잔류 DNA 적측정결과，대을간역묘원액중가능존재 Tween-20、PEG、단백등물질대형광염색법측정잔류 DNA 함량적영향진행분석，대해방법적선성범위、용우을간역묘원액잔류 DNA 검정적준학성화중복성진행료연구，병대불동래원 DNA 표준존재적차이진행비교。결과：형광법측정효모을간역묘잔류 DNA 선성범위위2．5～80 ng? mL-1；발현 Tween-20、PEG 등대잔류 DNA 검측영향교소，가표회수솔균재80％～120％지간；이단백질대검측영향교대，경분-삼록갑완추제후가유효거제단백질간우，회수솔체도90％좌우，CV 소우10％；동시발현불동래원적 DNA 표준품존재형광표기효솔적차이。결론：을간역묘원액경처리거제단백간우후가채용형광염색법진행잔류 DNA 함량적측정，단응주의사용여역묘표체계통상동숙주래원적 DNA 표준품。
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.
