人端粒酶催化亚基(hTERT)实时荧光定量PCR标准品的构建  

Construction of a standard plasmid for detecting hTERT with real-time PCR

作　　者：王有为[1] 韩之波[2] 康健[1] 孟磊[3] 韩忠朝[1,2,3] 

机构地区：[1]细胞产品国家工程研究中心天津昂赛细胞基因工程有限公司,天津300457 [2]中国医学科学院北京协和医学院血液学研究所实验血液学国家重点实验室,天津300020 [3]中国医学科学院血液学研究所泰达生命科学技术研究中心,天津300457

出　　处：《中国卫生检验杂志》2010年第8期1853-1854,1857,共3页Chinese Journal of Health Laboratory Technology

摘　　要：目的:构建用于检测人端粒酶催化亚基(hTERT)实时荧光定量PCR标准品。方法:通过逆转录PCR扩增得到目的片段后连接至pMD18-T载体,转化DH5α感受态细胞。分别经PCR和测序验证重组质粒的正确性。分光光度计测量重组质粒的吸光值,换算成拷贝数浓度后作梯度稀释制得质粒标准品。10倍梯度稀释质粒标准品后进行实时荧光定量PCR分析。结果:人端粒酶催化亚基(hTERT)基因片段成功克隆至pMD18-T载体之中,重组质粒序列完全正确。实时荧光定量PCR分析10倍梯度稀释的质粒标准品所得标准曲线良好。结论:成功构建了人端粒酶催化亚基(hTERT)实时荧光定量PCR标准品。Objective：To construct a recombinant plasmid as the standards for hTERT detection by real-time PCR.Methods：Total RNA was extracted from Hela.A fragment of hTERT was amplified by reverse transcription PCR.The PCR production was ligated with pMD18-T vector and transformed to DH5α.Recombinant plasmid was identified by PCR and sequencing.The concentration of recombinant plasmid was analyzed through its absorption at 260 nm.Standard curve was constructed by real-time PCR with ten-fold serially dilution of recombinant plasmid.Results：A fragment of hTERT was successfully cloned into pMD18-T.Dictated by real-time PCR,standard curve could be received from the concentration of 108 to 102 copys/μl.Conclusion：The standard plasmid for detecting hTERT with real-time PCR has been constructed successfully.

关 键 词：端粒酶逆转录酶 逆转录多聚酶链式反应 多聚酶链式反应 

分 类 号：R331[医药卫生—人体生理学]