实时定量PCR测定治疗性HBV DNA疫苗工程菌的质粒拷贝数  

Determination of plasmid copy number of therapeutic HBV DNA vaccine engineered strain by real-time quantitative PCR

作　　者：陈孟璋 何星垚 林汉森 CHEN Mengzhang;HE Xingyao;LIN Hansen(Guangzhou Guangyao Yigan Biological Products Co.,Ltd.,Guangzhou 511495,China)

机构地区：[1]广州广药益甘生物制品股份有限公司,广东广州511495

出　　处：《广东药科大学学报》2023年第5期87-92,共6页Journal of Guangdong Pharmaceutical University

基　　金：广东省重大科技专项(2012A080204009)。

摘　　要：目的 比较不同的定量方法和总DNA制备方法对实时定量PCR(real-time quantitative PCR, qPCR)测定治疗性HBV DNA疫苗工程菌的质粒拷贝数(plasmid copy number, PCN)的影响,为建立快速简便的治疗性HBV DNA疫苗工程菌的PCN测定方法提供参考。方法 通过试剂盒提取法、Triton X-100煮沸法和培养基煮沸法分别制备总DNA样品用于qPCR检测,使用绝对定量方法和相对定量方法计算不同制备方法样品的PCN,并对测定结果进行统计学分析。结果 试剂盒提取法制备的样品经绝对定量和相对定量计算PCN,结果分别为43.10±4.02和42.92±3.98,显著低于Triton X-100煮沸法的结果(69.32±6.51和68.58±6.42)以及培养基煮沸法的结果(75.73±7.46和76.15±7.50)。任一样品制备方法,比较其绝对定量和相对定量结果,差异均无统计学意义。结论 qPCR的绝对定量和相对定量计算方法都适用于治疗性HBV DNA疫苗工程菌PCN测定,Triton X-100煮沸法更适合于总DNA模板制备。Objective To compare the effects of different quantitative methods and total DNA preparation methods on the plasmid copy number(PCN)determination of therapeutic HBV DNA vaccine engineered strain by real-time quantitative PCR(qPCR),so as to provide reference for the establishment of a rapid and simple method for the PCN determination of therapeutic HBV DNA vaccine engineered strain.Methods Total DNA samples were prepared for qPCR by kit extraction method,Triton X-100 boiling method and medium boiling method,respectively.The PCN of samples prepared by different methods was calculated by absolute and relative quantification methods,and the results were statistically analyzed.Results The absolute and relative quantification results of PCN of samples prepared by kit extraction method were 43.10±4.02 and 42.92±3.98,respectively,which were significantly lower than those obtained by Triton X-100 boiling method(69.32±6.51)and medium boiling method(75.73±7.46).There was no significant difference between the absolute and relative quantitative results of any sample preparation method.Conclusion Both the absolute and relative quantitative methods of qPCR are suitable for the determination of PCN of therapeutic HBV DNA vaccine engineered strain,and the Triton X-100 boiling method is more suitable for the preparation of total DNA template.

关 键 词：HBV疫苗 DNA疫苗 实时定量PCR 质粒拷贝数 

分 类 号：R392.1[医药卫生—免疫学]