猪谷氧还蛋白Ⅰ(GLRX1)分子实时荧光定量PCR检测方法的建立
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- 英文论文题名：DESIGN AND VALIDATION OF REAL TIME FLUORESENCE QUANTITATIVE RT-PCR ASSAY FOR DETECTION OF PORCINE GLUTAREDOXIN
- 论文作者：马改妮 ; 刘珂 ; 郇贝丽 ; 魏建超 ; 邵东华 ; 李蓓蓓 ; 邱亚峰 ; 石元元 ; 马志永
- 英文论文作者：Ma Gai-ni ; Liu Ke ; Huan Bei-li ; Shao Dong-hua ; Wei Jian-chao ; Li Bei-bei ; Qiu Ya-feng ; Shi Yuan-yuan ; Ma Zhiyong ; Shanghai Veterinary Research Institute ; CAAS
- 年：2016
- 作者机构：中国农业科学院上海兽医研究所;
- 论文关键词：猪谷氧还蛋白 ; SYBR GreenⅠ ; 实时荧光定量PCR
- 英文论文关键词：porcine GLRX1 ; SYBR Green real-time quantitative PCR assay
- 会议召开时间：2016-07-20
- 会议录名称：中国畜牧兽医学会兽医公共卫生学分会第五次学术研讨会论文集
- 语种：中文
- 分类号：S858.28
- 学会代码：SYGW
- 会议名称：中国畜牧兽医学会兽医公共卫生学分会第五次学术研讨会
- 会议地点：中国内蒙古通辽
- 主办单位：中国畜牧兽医学会兽医公共卫生学分会
- 学会名称：中国畜牧兽医学会兽医公共卫生学分会
- 页数：7
- 文件大小：694k
- 原文格式：D
- 会议级别：全国
摘要
本实验利用SYBR GreenⅠ荧光定量技术建立一种相对定量检测猪谷氧还蛋白1(GLRX1)的方法。针对猪GLRX1设计对特异性引物,将PCR扩增的片段分别连接到T-easy载体上构建重组质粒,经筛选、鉴定纯化后,倍比稀释作为质控样品,用于实时荧光定量PCR中GLRX1标准曲线的构建,并进行反应的灵敏性、特异性和重复性试验。结果显示标准曲线线性关系R2值均在0.99以上;特异性结果表明的只能检测到猪GLRX1扩增曲线;批内和批间重复性试验的变异系数均小于0.5%。利用本方法检测临床样品,各组织间GLRX1表达量有差异,。本研究初步建立了检测猪GLRX1基因的SYBR Green荧光定量RT-PCR的方法,为后续对猪传染性疾病与猪GLRX1之间相互关系的研究提供了一种特异、灵敏的检测方法。
The objective of the study is to establish a method for detecting porcine GLRX1 by SYBR GreenⅠrelative fluorescence quantitative RT-PCR.Special primers based on porcine GLRX1 were designed.Then these amplified fragments were cloned into T-easy.Using the recombinant plasmid as standard products,a real-time quantitative reverse transcription-polymerase chain reaction(RT-PCR) was performed to construct the standard curves of porcine GLRX1 and detect the sensitivity,specificity and repeatability.The results showed a precise linear relationship with a correlation coefficient of R2 >0.99.The amplification curve showing a single peak could only been detected for porcine GLRX1.The variation coefficient was less than 0.5% by within and between the group of repeatability tests.The clinical samples were detected 3 times by this method,and the expression of GLRX1 in different tissues was various.The developed real-time PCR assay was highly specific,sensitive,and reproducible and could be an available tool for monitoring the relationship between pig infectiousdiseases and porcine GLRX1.
The objective of the study is to establish a method for detecting porcine GLRX1 by SYBR GreenⅠrelative fluorescence quantitative RT-PCR.Special primers based on porcine GLRX1 were designed.Then these amplified fragments were cloned into T-easy.Using the recombinant plasmid as standard products,a real-time quantitative reverse transcription-polymerase chain reaction(RT-PCR) was performed to construct the standard curves of porcine GLRX1 and detect the sensitivity,specificity and repeatability.The results showed a precise linear relationship with a correlation coefficient of R2 >0.99.The amplification curve showing a single peak could only been detected for porcine GLRX1.The variation coefficient was less than 0.5% by within and between the group of repeatability tests.The clinical samples were detected 3 times by this method,and the expression of GLRX1 in different tissues was various.The developed real-time PCR assay was highly specific,sensitive,and reproducible and could be an available tool for monitoring the relationship between pig infectiousdiseases and porcine GLRX1.
引文
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[5]孟春春,段云兵.基因Ⅶ型新城疫病毒SYBR Green I实时荧光定量PCR检测方法的建立[J].中国动物传染病学报,2011,19(5):8-14.
[6]侯军委,周艳君.猪源TTV Taqman实时荧光定量PCR检测方法的建立和应用[J].中国动物传染病学报,2011,19(2):13-19.
[7]王蓉蓉,孙卫东.猪链球菌2型主要毒力因子三重荧光定量PCR检测方法的建立[J].中国动物传染病学报,2014,22(6):25-31.
[8]Stroher E,Millar A.The biological roles of glutaredoxins[J].Biochem J,2012,446(3):333-348.
[9]Kalinina EV,Chernov NN,Saprin AN.Involvement of thio-,peroxi-,and glutaredoxins in cellular redoxdependent processes[J].Biochemistry:Mosc,2008,73(13):1493-1510
[10]Cha M K,Kim I H.Preferential overexpression of glutaredoxin3 in human colon and lung carcinoma[J].Cancer Epidemiol.2009,33(3-4):281-287.
[11]Jurado J,Prieto-Alamo M J,Madrid-Rísquez J,Pueyo C.Absolute gene expression patterns of thioredoxin and glutaredoxin redox systems in mouse[J].J Biol Chem.2003,278(46):45546-45554.
[12]Lim J,Luderer U.Oxidative damage increases and antioxidant gene expression decreases with aging in the mouse ovary[J].Biol Reprod.2011,84(4):775-782.
[13]Liu X,Jann J,Xavier C,Wu H.Glutaredoxin 1(Grx1)Protects Human Retinal Pigment Epithelial Cells From Oxidative Damage by Preventing AKT Glutathionylation[J].Invest Ophthalmol Vis Sci.2015,56(5):2821-2832.
[14]Cater M A,Materia S,Xiao Z,et al.Glutaredoxin1 protects neuronal cells from copper-induced toxicity[J].Biometals.2014,27(4):661-672.
[15]Aesif SW,Kuipers I,van der Velden J,et al.Activation of the glutaredoxin-1 gene by nuclear factorκBenhances signaling.Free Radic Biol Med[J].2011,51(6):1249-1257.
[16]Morgan L,Estevez O,Mueller L,et al.The glutaredoxin GLRX-21 functions to prevent selenium-induced oxidative stress in Caenorhabditis elegans[J].Toxicol Sci.2010,118(2):530-543.
[2]Lillig C,Berndt C.Glutaredoxins in thiol/disulfide exchange[J].Antioxid Redox Signal,2013,18(13):1654-1655.
[3]HOLMGREN A.Antioxidant function of thioredoxin and glutaredoxin systems[J].Antioxid Redox Signal,2000,2(4):811-820.
[4]Sagemark J,Elgan TH,Burglin TR,et al.Redox properties and evolution of human glutaredoxins[J].Proteins,2007,68(4):879-892.
[5]孟春春,段云兵.基因Ⅶ型新城疫病毒SYBR Green I实时荧光定量PCR检测方法的建立[J].中国动物传染病学报,2011,19(5):8-14.
[6]侯军委,周艳君.猪源TTV Taqman实时荧光定量PCR检测方法的建立和应用[J].中国动物传染病学报,2011,19(2):13-19.
[7]王蓉蓉,孙卫东.猪链球菌2型主要毒力因子三重荧光定量PCR检测方法的建立[J].中国动物传染病学报,2014,22(6):25-31.
[8]Stroher E,Millar A.The biological roles of glutaredoxins[J].Biochem J,2012,446(3):333-348.
[9]Kalinina EV,Chernov NN,Saprin AN.Involvement of thio-,peroxi-,and glutaredoxins in cellular redoxdependent processes[J].Biochemistry:Mosc,2008,73(13):1493-1510
[10]Cha M K,Kim I H.Preferential overexpression of glutaredoxin3 in human colon and lung carcinoma[J].Cancer Epidemiol.2009,33(3-4):281-287.
[11]Jurado J,Prieto-Alamo M J,Madrid-Rísquez J,Pueyo C.Absolute gene expression patterns of thioredoxin and glutaredoxin redox systems in mouse[J].J Biol Chem.2003,278(46):45546-45554.
[12]Lim J,Luderer U.Oxidative damage increases and antioxidant gene expression decreases with aging in the mouse ovary[J].Biol Reprod.2011,84(4):775-782.
[13]Liu X,Jann J,Xavier C,Wu H.Glutaredoxin 1(Grx1)Protects Human Retinal Pigment Epithelial Cells From Oxidative Damage by Preventing AKT Glutathionylation[J].Invest Ophthalmol Vis Sci.2015,56(5):2821-2832.
[14]Cater M A,Materia S,Xiao Z,et al.Glutaredoxin1 protects neuronal cells from copper-induced toxicity[J].Biometals.2014,27(4):661-672.
[15]Aesif SW,Kuipers I,van der Velden J,et al.Activation of the glutaredoxin-1 gene by nuclear factorκBenhances signaling.Free Radic Biol Med[J].2011,51(6):1249-1257.
[16]Morgan L,Estevez O,Mueller L,et al.The glutaredoxin GLRX-21 functions to prevent selenium-induced oxidative stress in Caenorhabditis elegans[J].Toxicol Sci.2010,118(2):530-543.
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