猪源干扰素刺激基因PPBP荧光定量PCR检测方法的建立及初步应用
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摘要
根据猪源干扰素刺激基因PPBP(pro-platelet basic protein)基因全长序列,使用引物设计软件Primer Premier5.0设计合成特异性引物,经过PCR扩增,将基因扩增的片段连接到相应的载体上,成功构建重组质粒p3XFLAG-CMV-7.1-PPBP。重组质粒经过筛选、鉴定以及纯化后,经10倍系列稀释作为质控样品,用于实时荧光定量PCR中PPBP标准曲线的构建,并检测重复性、灵敏性和特异性。结果显示标准曲线线性关系R2>0.99;特异性试验中也能检测到PPBP扩增曲线;组间和组内变异系数均小于5%。使用建立的SYBR GreenⅠ荧光定量RT-PCR检测PRRSV感染组织和未感染组织中PPBP的表达情况,能够检测出组织中PPBP的含量存在差异。本研究初步建立了检测猪PPBP基因的SYBR GreenⅠ荧光定量RT-PCR的方法,为后续猪传染性疾病和猪PPBP之间相互关系的研究提供一个特异灵敏的检测和手段。
The study was to develop a real-time assay for detecting swine pro-platelet basic protein. According to PPBP gene sequence, specific primer was designed by primer design software Primer Premier5.0 The resulting fragments were cloned into the vector to construct recombinant plasmids of p3 XFLAG-CMV-7.1-PPBP. The recombinant plasmid was used as standard products to establish standard curve of porcine PPBP and detect repeatability, sensitivity and specificity. The results show a precise linear relationship with a correlation coefficient R2 >0.99. The amplification efficiency is 102%. Dissolve curve appears one peak. The variation coefficient is less than 0.5% within and between assays. The SYBR GreenⅠ real-time PCR assay was used to detect porcine PPBP from mock pigs and pigs infected with PRRSV. PPBP was found in this samples and was significant different between the infected and mock group.This SYBR Green Ⅰ real-time PCR assay developed in this study has highly sensitivity, specificity, stability and repeatability, which could be a tool for detecting relationship between pig infectious diseases and porcine PPBP.
The study was to develop a real-time assay for detecting swine pro-platelet basic protein. According to PPBP gene sequence, specific primer was designed by primer design software Primer Premier5.0 The resulting fragments were cloned into the vector to construct recombinant plasmids of p3 XFLAG-CMV-7.1-PPBP. The recombinant plasmid was used as standard products to establish standard curve of porcine PPBP and detect repeatability, sensitivity and specificity. The results show a precise linear relationship with a correlation coefficient R2 >0.99. The amplification efficiency is 102%. Dissolve curve appears one peak. The variation coefficient is less than 0.5% within and between assays. The SYBR GreenⅠ real-time PCR assay was used to detect porcine PPBP from mock pigs and pigs infected with PRRSV. PPBP was found in this samples and was significant different between the infected and mock group.This SYBR Green Ⅰ real-time PCR assay developed in this study has highly sensitivity, specificity, stability and repeatability, which could be a tool for detecting relationship between pig infectious diseases and porcine PPBP.
引文
[1]罗乐,赵碧,郑敏,等.鸭源PKC基因荧光定量PCR检测方法的建立[J].西北农业学报,2016,25(3):342-346.
[2]Unver N,Esendagli G,Yilmaz G,et al.CXCL7-induced macrophage infiltration in lung tumor is independent of CXCR2 expression CXCL7-induced macrophage chemotaxis in LLC tumors[J].Cytokine,2015,75(2):330-337.
[3]Wang Y S,Liao K W,Chen M F,et al.Canine CXCL7and its functional expression in dendritic cell undergoing maturation[J].Vet Immunol Immunopathol,2010,135(1):128-136.
[4]González-Cortés C,Diez-Tascón C,Guerra-Laso J M,et al.Non-chemotactic influence of CXCL7 on human phagocytes.Modulation of antimicrobial activity against L.pneumophila[J].Immunobiology,2012,217(4):394-401.
[5]袁继红.实时荧光定量PCR技术的实验研究[J].现代农业科技,2010(13):20-22.
[6]陈旭,齐风坤,康立功,等.实时荧光定量PCR技术研究进展及其应用[J].东北农业大学学报,2010,41(8):148-155.
[7]朱捷,杨成君,王军.荧光定量PCR技术及其在科研中的应用[J].生物技术通报,2009(2):73-76.
[8]刘小荣,张笠,王勇平.实时荧光定量PCR技术的理论研究及其医学应用[J].中国组织工程研究,2010,14(2):329-332.
[9]马改妮,刘珂,郇贝丽,等.猪谷氧还蛋白Ⅰ(GLRXⅠ)分子实时荧光定量PCR检测方法的建立[J].中国动物传染病学报,2016,24(3):78-82.
[10]郇贝丽,刘珂,马改妮,等.猪血清淀粉样蛋白3分子SYBR GreenⅠ实时荧光定量PCR检测方法的建立及初步研究[J].中国动物传染病学报,2017,25(3):80-86.
[11]Romeiro M F,Souza W M,Tolardo A L,et al.Evaluation and optimization of SYBR Green real-time reverse transcription polymerase chain reaction as a tool for diagnosis of the Flavivirus genus in Brazil[J].Rev Soc Bras Med Trop,2016,49(3):279-285.
[12]Machado A M,de Souza W M,de Pádua M,et al.Development of a one-step SYBR Green I real-time RT-PCR assay for the detection and quantita,tion of araraquara and rio mamore hantavirus[J].Viruses,2013,5(9):2272-2281.
[13]Tian H,Wu J,Shang Y,et al.The development of a rapid SYBR one step real-time RT-PCR for detection of porcine reproductive and respiratory syndrome virus[J].Virol J,2010,7:90.
[14]Brown A J,Sepuru K M,Rajarathnam K.Structural basis of native CXCL7 monomer binding to CXCR2 receptor N-domain and glycosaminoglycan heparin[J].Int J Mol Sci,2017,18(3).pii:E508.
[15]Guo Q,Jian Z,Jia B,et al.CXCL7 promotes proliferation and invasion of cholangiocarcinoma cells[J].Oncol Rep,2017,37(2):1114-1122.
[2]Unver N,Esendagli G,Yilmaz G,et al.CXCL7-induced macrophage infiltration in lung tumor is independent of CXCR2 expression CXCL7-induced macrophage chemotaxis in LLC tumors[J].Cytokine,2015,75(2):330-337.
[3]Wang Y S,Liao K W,Chen M F,et al.Canine CXCL7and its functional expression in dendritic cell undergoing maturation[J].Vet Immunol Immunopathol,2010,135(1):128-136.
[4]González-Cortés C,Diez-Tascón C,Guerra-Laso J M,et al.Non-chemotactic influence of CXCL7 on human phagocytes.Modulation of antimicrobial activity against L.pneumophila[J].Immunobiology,2012,217(4):394-401.
[5]袁继红.实时荧光定量PCR技术的实验研究[J].现代农业科技,2010(13):20-22.
[6]陈旭,齐风坤,康立功,等.实时荧光定量PCR技术研究进展及其应用[J].东北农业大学学报,2010,41(8):148-155.
[7]朱捷,杨成君,王军.荧光定量PCR技术及其在科研中的应用[J].生物技术通报,2009(2):73-76.
[8]刘小荣,张笠,王勇平.实时荧光定量PCR技术的理论研究及其医学应用[J].中国组织工程研究,2010,14(2):329-332.
[9]马改妮,刘珂,郇贝丽,等.猪谷氧还蛋白Ⅰ(GLRXⅠ)分子实时荧光定量PCR检测方法的建立[J].中国动物传染病学报,2016,24(3):78-82.
[10]郇贝丽,刘珂,马改妮,等.猪血清淀粉样蛋白3分子SYBR GreenⅠ实时荧光定量PCR检测方法的建立及初步研究[J].中国动物传染病学报,2017,25(3):80-86.
[11]Romeiro M F,Souza W M,Tolardo A L,et al.Evaluation and optimization of SYBR Green real-time reverse transcription polymerase chain reaction as a tool for diagnosis of the Flavivirus genus in Brazil[J].Rev Soc Bras Med Trop,2016,49(3):279-285.
[12]Machado A M,de Souza W M,de Pádua M,et al.Development of a one-step SYBR Green I real-time RT-PCR assay for the detection and quantita,tion of araraquara and rio mamore hantavirus[J].Viruses,2013,5(9):2272-2281.
[13]Tian H,Wu J,Shang Y,et al.The development of a rapid SYBR one step real-time RT-PCR for detection of porcine reproductive and respiratory syndrome virus[J].Virol J,2010,7:90.
[14]Brown A J,Sepuru K M,Rajarathnam K.Structural basis of native CXCL7 monomer binding to CXCR2 receptor N-domain and glycosaminoglycan heparin[J].Int J Mol Sci,2017,18(3).pii:E508.
[15]Guo Q,Jian Z,Jia B,et al.CXCL7 promotes proliferation and invasion of cholangiocarcinoma cells[J].Oncol Rep,2017,37(2):1114-1122.