泛素基因TaqMan实时荧光定量PCR检测方法的建立及应用
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摘要
为建立一种泛素基因TaqMan实时荧光定量PCR检测方法,以期为泛素基因的动态检测提供有效的试验手段。根据GenBank中公布的泛素基因序列设计1对荧光定量PCR引物及1条探针,建立一种快速检测泛素基因的实时荧光定量PCR方法,并对建立的方法进行条件优化与评价。结果显示,建立的实时荧光定量PCR方法的线性关系好,以质粒标准品构建的标准曲线的相关系数r~2为0.999;灵敏性、特异性及重复性均较高;应用建立的方法对多种种属来源细胞进行检测,能检测出不同类型细胞中泛素的含量。结果表明,成功建立了一种快速、准确检测泛素TaqMan实时荧光定量PCR方法。
To develop a real-time TaqMan reverse transcription-PCR(RT-PCR) assay for effective detection of ubiquitin gene,based on the ubiquitin gene sequence published in GenBank,a pair of PCR primers and a probe were designed.Then a real-time TaqMan RT-PCR assay for accurate quantification of ubiquitin gene was developed and optimized.Results showed that the real-time TaqMan RT-PCR assay had a good linear relationship.The standard curve correlation coefficient r~2 was 0.999.It also had a high sensitivity and reproducibility as well as specificity.The method was used to detect the contents of ubiquitin in different types of cells.In conclusion,a rapid and accurate real-time Taq Man reverse transcription-PCR for quantification of ubiquitin gene was successfully developed.
To develop a real-time TaqMan reverse transcription-PCR(RT-PCR) assay for effective detection of ubiquitin gene,based on the ubiquitin gene sequence published in GenBank,a pair of PCR primers and a probe were designed.Then a real-time TaqMan RT-PCR assay for accurate quantification of ubiquitin gene was developed and optimized.Results showed that the real-time TaqMan RT-PCR assay had a good linear relationship.The standard curve correlation coefficient r~2 was 0.999.It also had a high sensitivity and reproducibility as well as specificity.The method was used to detect the contents of ubiquitin in different types of cells.In conclusion,a rapid and accurate real-time Taq Man reverse transcription-PCR for quantification of ubiquitin gene was successfully developed.
引文
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[15]TAI H C,SCHUMAN E M.Ubiquitin,the proteasome and protein degradation in neuronal function and dysfunction[J].Nature Reviews Neuroscience,2008,9(11):826-838.
[16]STIEREN E S,EI AYADI A,XIAO Y,et al.Ubiquilin-1is a molecular chaperone for the amyloid precursor protein[J].Journal of Biological Chemistry,2011,286(41):35689-35698.
[17]段强,席学礼,樊继军,等.泛素羧基末端水解酶-1与神经退行性疾病[J].现代预防医学,2010,37(17):3374-3375.DUAN Qiang,XI Xue-li,FAN Ji-jun,et al.Ubiquitin carboxyl-terminal hydrolase-1 and neurodegenerative diseases[J].Modern Preventive Medicine,2010,37(17):3374-3375.(in Chinese)
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[2]陈季武,郑丽娜,王帮正,等.泛素化介导的非蛋白质降解功能[J].生物化学与生物物理进展,2012,39(7):613-621.CHEN Ji-wu,ZHENG Li-na,WANG Bang-zheng,et al.The nonproteolytic functions mediated by ubiquitylation[J].Progress in Biochemistry and Biophysics,2012,39(7):613-621.(in Chinese)
[3]倪晓光,赵平.泛素-蛋白酶体途径的组成和功能[J].生理科学进展,2006(3):255-258.NI Xiao-guang,ZHAO Ping.The composition of ubiquitin-proteasome pathway and its function[J].Progress in Physiological Sciences,2006(3):255-258.(in Chinese)
[4]陈科,程汉华,周荣家.自噬与泛素化蛋白降解途径的分子机制及其功能[J].遗传,2012,34(1):7-20.CHEN Ke,CHENG Han-hua,ZHOU Rong-jia.Molecular mechanisms and functions of autophagy and the ubiquitin-proteasome pathway[J].Hereditas,2012,34(1):7-20.(in Chinese)
[5]杨玖霞,张浩,王志龙,等.E3泛素连接酶调控植物抗病分子机理研究进展[J].植物保护,2015,41(4):1-8,28.YANG Jiu-xia,ZHANG Hao,WANG Zhi-long,et al.Recent progresses in the regulation mechanism of E3ligases in plant disease resistance[J].Plant Protection,2015,41(4):1-8,28.(in Chinese)
[6]杨娜,侯巧明,南洁,等.泛素连接酶的结构与功能研究进展[J].生物化学与生物物理进展,2008,35(1):14-20.YANG Na,HOU Qiao-ming,NAN Jie,et al.Progress in structural and functional study of ubiquitin ligases[J].Progress in Biochemistry and Biophysics,2008,35(1):14-20.(in Chinese)
[7]MANZANILLO P S,AYRES J S,WATSON R O,et al.The ubiquitin ligase parkin mediates resistance to intracellularpathogens[J].Nature,2013,501(7468):512-516.
[8]王佳悦,赵博.泛素化在肿瘤治疗方面的应用及展望[J].生命的化学,2017,37(6):879-887.WANG Jia-yue,ZHAO Bo.Applications and prospects of ubiquitin in oncotherapy[J].Chemistry of Life,2017,37(6):879-887.(in Chinese)
[9]王希明,张晔,刘云鹏,等.MAPK/ERK通路及泛素连接酶c-Cbl调控埃克替尼抑制肺癌HCC827细胞增殖和诱导细胞凋亡的研究[J].现代肿瘤医学,2014,22(5):1024-1027.WANG Xi-ming,ZHANG Ye,LIU Yun-peng,et al.Icotinib inhibits proliferation and induces apoptosis via inactivation of the c-Cbl dependent MAPK/E[J].Journal of Modern Oncology,2014,22(5):1024-1027.(in Chinese)
[10]WANG X,FU Y,BAN L,et al.Selection of reliable reference genes for quantitative real-time RT-PCRin alfalfa[J].Genes Genet Syst,2015,90(3):175-180.
[11]WAGGONER J J,ROJAS A,MOHAMED-HADLEY A,et al.Real-time RT-PCR for Mayaro virus detection in plasma and urine[J].Journal of Clinical Virology,2018,98:1-4.
[12]兰雨,杨平地.泛素-蛋白酶体通路在凋亡调控中的作用[J].国外医学.输血及血液学分册,2001(3):204-206.LAN Yu,YANG Ping-di.Role of ubiquitin proteasome pathway in apoptosis regulation[J].International Journal of Blood Transfusion and Hematology,2001(3):204-206.(in Chinese)
[13]BUDHIDARMO R,DAY C L.The ubiquitin-associated domain of cellular inhibitor of apoptosis proteins facilitates ubiquitylation[J].Journal of Biological Chemistry,2014,289(37):25721-25736.
[14]何珊,张令强.线性泛素化修饰研究进展[J].遗传,2015,37(9):911-917.HE Shan,ZHANG Ling-qiang.Research progress in linear ubiquitin modification[J].Hereditas,2015,37(9):911-917.(in Chinese)
[15]TAI H C,SCHUMAN E M.Ubiquitin,the proteasome and protein degradation in neuronal function and dysfunction[J].Nature Reviews Neuroscience,2008,9(11):826-838.
[16]STIEREN E S,EI AYADI A,XIAO Y,et al.Ubiquilin-1is a molecular chaperone for the amyloid precursor protein[J].Journal of Biological Chemistry,2011,286(41):35689-35698.
[17]段强,席学礼,樊继军,等.泛素羧基末端水解酶-1与神经退行性疾病[J].现代预防医学,2010,37(17):3374-3375.DUAN Qiang,XI Xue-li,FAN Ji-jun,et al.Ubiquitin carboxyl-terminal hydrolase-1 and neurodegenerative diseases[J].Modern Preventive Medicine,2010,37(17):3374-3375.(in Chinese)
[18]TAKALO M,HAAPASALO A,NATUNEN T,et al.Targeting ubiquilin-1 in Alzheimer’s disease[J].Expert Opinion on Therapeutic Targets,2013,17(7):795-810.
[19]HUBER C,DIAS-SANTAGATA D,GLASER A,et al.Identification of mutations in CUL7 in 3-M syndrome[J].Nature Genetics,2005,37(10):1119-1124.