pEGFP-c-fos在T24细胞的表达及在贝毒检测中的应用
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摘要
目的：构建含c-fos启动子的pEGFP-c-fos重组质粒载体。将重组质粒载体转入膀胱移行细胞瘤T24细胞中，筛选获得稳定表达株。加入贝毒后，利用细胞中绿色荧光蛋白表达水平的变化进行贝毒素的检测，建立一种以细胞为基础的受体水平的贝毒检测方法。
方法：用PCR扩增c-fos启动子片断，进行VspI、EcoRI双酶切，获得含粘性末端的片断。将该片段与同样双酶切的pEGFP-N1质粒进行连接，构建pEGFP-c-fos重组质粒载体。用PCR、双酶切鉴定，最后测序鉴定。利用脂质体法将重组质粒pEGFP-c-fos转入人膀胱移行细胞瘤T24细胞中，通过G418筛选得到稳定表达株。加入梯度神经性贝毒(neurotoxic shellfish poisoning，NSP)，或加入梯度麻痹性贝毒(Paralytic shellfish poison，PSP)后在经10ng／ml的NSP处理，利用激光共聚焦荧光显微镜观察细胞中绿色荧光蛋白表达的变化，并拍摄不同贝毒水平下绿色荧光蛋白的表达图像。利用美国Image-pro Plus专业图像分析软件对图像进行荧光强度定量分析，绘出绿色荧光表达强度与不同浓度NSP、PSP毒素的关系曲线。
结论：构建pEGFP-c-fos重组质粒载体并使之在T24表达。筛选得到的稳定表达株在经过NSP诱导下，发出较强的荧光，表明重组质粒pEGFP-c-fos在T24细胞中成功表达。建立了绿色荧光表达强度与不同浓度NSP毒素或PSP毒素的关系曲线，为构建以细胞为基础的受体水平的NSP和PSP贝毒检测方法奠定了基础。
Objective: A recombinant plasmid pEGFP-c-fos with c-fos promoter and EGFP was constructed, and then transfected into T24 cell. The positive transfectants were obtained by screening. Based on the changes of the expression of EGFP in the T24 cell which induced by the HABs toxins, a new method to detect HABs toxins was founded in receptor level.
Methods: The c-fos with cohesive terminus was obtained after amplified by PCR and then digested by VspI and EcoRI, and then ligated with the plasmid pEGFP digested by the same restriction enzymes. The recombinant plasmid pEGFP-c-fos, which was identified by PCR, digesting, and sequencing, was transfected into T24 cell through LipofectAMINE2000. The positive transfectants were obtained by G418. The changes of the expression of EGFP in T24 cell with addition of increasing NSP or PSP were detected by Laser scanning confocal microscope, and quantitative analysis was carried out by Image-pro Plus software.
Conclusions: A recombinant plasmid pEGFP-c-fos was constructed and a new pEGFP-c-fos-T24 cell strain was established successfully. The dose-effect relationship between the intensity of green fluorescence and the level of NSP or PSP was detected. A rapid cell-based assay for detection HABs toxin was established
primarily.
方法：用PCR扩增c-fos启动子片断，进行VspI、EcoRI双酶切，获得含粘性末端的片断。将该片段与同样双酶切的pEGFP-N1质粒进行连接，构建pEGFP-c-fos重组质粒载体。用PCR、双酶切鉴定，最后测序鉴定。利用脂质体法将重组质粒pEGFP-c-fos转入人膀胱移行细胞瘤T24细胞中，通过G418筛选得到稳定表达株。加入梯度神经性贝毒(neurotoxic shellfish poisoning，NSP)，或加入梯度麻痹性贝毒(Paralytic shellfish poison，PSP)后在经10ng／ml的NSP处理，利用激光共聚焦荧光显微镜观察细胞中绿色荧光蛋白表达的变化，并拍摄不同贝毒水平下绿色荧光蛋白的表达图像。利用美国Image-pro Plus专业图像分析软件对图像进行荧光强度定量分析，绘出绿色荧光表达强度与不同浓度NSP、PSP毒素的关系曲线。
结论：构建pEGFP-c-fos重组质粒载体并使之在T24表达。筛选得到的稳定表达株在经过NSP诱导下，发出较强的荧光，表明重组质粒pEGFP-c-fos在T24细胞中成功表达。建立了绿色荧光表达强度与不同浓度NSP毒素或PSP毒素的关系曲线，为构建以细胞为基础的受体水平的NSP和PSP贝毒检测方法奠定了基础。
Objective: A recombinant plasmid pEGFP-c-fos with c-fos promoter and EGFP was constructed, and then transfected into T24 cell. The positive transfectants were obtained by screening. Based on the changes of the expression of EGFP in the T24 cell which induced by the HABs toxins, a new method to detect HABs toxins was founded in receptor level.
Methods: The c-fos with cohesive terminus was obtained after amplified by PCR and then digested by VspI and EcoRI, and then ligated with the plasmid pEGFP digested by the same restriction enzymes. The recombinant plasmid pEGFP-c-fos, which was identified by PCR, digesting, and sequencing, was transfected into T24 cell through LipofectAMINE2000. The positive transfectants were obtained by G418. The changes of the expression of EGFP in T24 cell with addition of increasing NSP or PSP were detected by Laser scanning confocal microscope, and quantitative analysis was carried out by Image-pro Plus software.
Conclusions: A recombinant plasmid pEGFP-c-fos was constructed and a new pEGFP-c-fos-T24 cell strain was established successfully. The dose-effect relationship between the intensity of green fluorescence and the level of NSP or PSP was detected. A rapid cell-based assay for detection HABs toxin was established
primarily.
引文
[1] 周名江，利钧，于仁诚等．赤潮藻毒素研究进展．中国海洋药物，1999，3：33-39
[2] 丘建文 麻痹性贝毒研究概况海洋环境科学，1991，10(2)：65-68
[3] 于仁诚，周名江．麻痹性贝毒研究进展．海洋与湖沼，1998，29(3)：330-338．
[4] Baden D G. Marine roxins. Handbook of Clinical Neurology, 1995, 21(65): 141.
[5] 曹际娟，赵昕，郭皓．神经性贝类毒素．检验检疫科学，1999，9(6)：56-58．
[6] 刘洪英，彭双清，沈勇等．刺尾鱼毒素细胞毒性的作用．卫生毒理学杂志，1999，13(3)：200
[7] V. Morales-Tlalpan, U Vaca. Modulation of the maitotoxin response by intracellular and extracellular cations. Toxicon, 2002, 40: 493-500
[8] Kim M. McGinnis, Margaret E. Gnegy, Nicole Falk, et al. Cytochrome c translocation does not lead to caspase activation in maitotoxin-treated SH-SYSY neuroblastoma cells. Neurochemistry International 2003, 42: 517-523
[9] Li D, Sun L, Chen Z, et al. Survey of the distribution of red tide toxins (okadaic acid and dinophytoxin-1) in the Dalian Bay sea area of China by micellar electrokinetic capillary chromatography. Electrophoresis, 2001. 22(16): 3583-8
[10] Vale P, de M Sampayo M A. Determination of paralytic shellfish toxins in Portuguese shellfish by automated pre-column oxidation. Toxicon, 2001, 39(4): 561-71
[11] Leao J M, Gago A, Rodriguez-Vazquez J A. Solid-phase Extraction and high-performance liquid chromatography procedures for the analysis of paralytic shellfish toxins. J Chromatogr A, 1998, 6: 798(1-2): 131-6
[12] 郭皓，免疫方法在藻毒素及贝毒素检测中的应用，卫生研究，1999，28(2)：122-144
[13] Buzy A, Thibault P, Layeock M V. Development of a capillary electrophoresis method for the characterization of enzymatic products arising from the carbamoylase digestion of paralytic shellfish poisoning toxins. J Chromatogr A, 1994, 668(1-2): 301-316
[14] Ravn H. Toxicological and chemical aspects of paralytic shellfish poisoning (PSP). Vigo: Intergovemmental Oceanographic commission of UNESCO, 1995, 558
[15] Bouaicha N, HENNION M C, Sandra P. Toxcion[J ]. 1997, 35: 276-281.
[16] Buzy A, Thibault P, Laycock M V. Development of a capillary electrophoresis method for the characterization of enzymatic products arising from the carbamoylase digestion of paralytic shellfish poisoning toxins. J Chromatogr A, 1994, 668(1-2): 301-316
[17] Park D L, Adams W N, Graham S L, et al. Variability of the mouse bioassay for determination of Parallytic Shellfish Poisoning Toxins. J AOAC Int, 1986, 69: 547-550
[18] Sommer H, Meyer K F. Paralytic shellfish poisoning. Arch Pathol, 24: 560-598.
[19] Association of official agricultural Chemists.Official methods of analysos 16th ed. 1995, Washington DC
[20] Manger R L, Leja L S, Lee S Y, et al. Detection of sodium channel toxins: directed cytotoxicity assays of purified cigyatoxins, brevetoxins, saxitoxins, and seafood extracts. J AOAC Int, 1995, 78: 521-527.
[21] Manger R L, Leja L S, Lee S Y, et al. Tetrazolium-based cell bioassay for neurotoxins active on voltage-sensitive sodium channels: semiautomated assay for saxitoxins, brevetoxins, ciguatoxins. Anal Biochem, 1993, 214: 190-194.
[22] Elizabeth R, Fairey J, Stewart G, et al. A Cell-Based Assay for Brevetoxins Saxitoxins and Ciguatoxins Using a Stably Expressed e-fos-Luciferase Reporter Gene. Analytical Biochemistry, 1997, 251: 129-132.
[23] Powell C L, Doueette G J. A receptor binding assay for paralytic shellfish poisoning toxins: recent advances and applications. Nat Toxins, 1999, 7(6): 393-400
[24] Vieytes M R, Cabado A G, Alfonso A, et al. Solid-phase radioreeeptor assay for paralytic shellfish toxins. Anal Biochem. 1993, 211(1): 87-93.
[25] Sheng M, Greenberg M E. The regulation and function of c-fos other immediate early genes in the nervous system. Meuron, 1990, 4: 477-482.
[26] Viviani R. Eutrophication, marine biotoxins, human health. Science of the Total Environment, 1992, suppl: 631.
[27] Cullinan W E. Fos expression in forebrain afferents to the hypothalanmic paraventricular mucleus following swim stress. Comp-Neurol, 1996, 368(1): 88-99.
[28] Helmreich D L. The effect of adrenalectomy on stress-induced c-los mRNA expresion in the rat brain. Brain Res, 1996, 706(1): 137-144.
[29] Shimomura O, Johson FH, Saiga Y et al. J Cell. Comp. Physio 1, 1962; 59: 223
[30] Prasher D, Eckenrode V, Ward W, et al. Primary Structure of the Aequores victaria Green-fluorescent Protein. Gene, 1992, 111: 229-233.
[31] Chalfiee M, Tu Y, Euskirchen G, et al. Green Fluorescent Protein as a Marker For Gene Expression. Science, 1994, 263: 802-805.
[32] Ward W W, Cody C W, Prasher D C, et al. Photochem, Photobiol, 1989; 49: 623
[33] Cody C W, Prasher D C, Westter, et al. Biochem isty, 1993; 32: 1212
[34] Heim R, Cubitt A, Tsien R. Nature, 1995; 373: 663
[35] Delagrave S, Hantin RE, Silv CM, et al. Bio/Technology, 1995; 13: 151
[36] Toshio. I,. Nobuo S, Keishi. H,. et. al.. Prostaglandin A1 enhances c-los expression and actibating protein-1 activity. Molecular and Cellular Endocrinology, 2000, 164: 77-85
[37] Chalfiee M, Tu Y, Euskirchen G, et al. Green Fluorescent Protein as a Marker For Gene Expression. Science, 1994, 263: 802-805.
[38] Velez P, Sierralta J, Alcayaga C. A functional assy for paralytic shellfish toxins that use a recombinant sodium channels. Toxicon, 2001, 39(7): 929-935
[39] Fairey E R, Bottein CM, Sheets M F, et al. Modification of the cell based assay for brevetoxins using human cardiac voltage dependent sodium channels expressed in HEK-293 cells. Biosens Bioelectron, 2001, 16(7-8): 519-586
[40] Kerr D S, Bdggs D M, Saba H I, A neurophysiological method of rapid detection and analysis marine algal toxins. Toxicon, 1999, 37(12): 1803-1825.
[41] Second international conference on harmful algae management and mitigation. QingDao. 2001, 12-14
[42] Flanagan A F, Callanan K R, Donlon J. A cytotoxicity assay for the detection and differentiation of two families ofshellfish toxins. Toxicon, 2001, 39: 1021-1027
[43] 金冬雁，黎孟枫，等译．分子克隆实验指南，科学技术出版社，第二版．1992，786-787．
[44] 刘洁生，杨维东，车军，等．重组质粒HF443-EGFP的构建及其在赤潮毒素检测中的应用初探
[45] William W, Richard D. Sequence of the 5'-flanking region of the rat c-fos proto-oncogene. Gene, 1994, 143: 261-264.
[46] 卢圣栋．现代分子生物学实验技术，中国协和医科大学出版社，第二版．1999，392-397．
[47] Hawley T S, Telford W G, Ramezani A, et al. Four-color flow cytometfic detection of retrovirally expressed red, yellow, green and cyan fluorescent proteins. Biotechniques, 2001, 30: 1028-1034.
[48] Alexander E R, Masaki H, Martin W, et al, Identification of the morphology of a thin film by flurescence laser-scanning confocal microscopy, Polymer, 1998, 39(26): 7149-7151
[49] Peng Z, Kirk, T, Xu Z L, The development of three-dimensional imaging techniques of wear particle analysis, Wear, 1997, 203-204: 418-424
[50] Made-Yasmin, Dechraoui, Jerome N, et al. Ciguatoxins and brevetoxins, neurotoxic polyether compounds active on sodium channels, Toxicon, 1999, 37: 125-143
[51] Laurence S D, Steven M P, Kathleen R E, et al, A rapid assay for the brevetoxin group of sodium channel activators based on fluorescence monitoring of synaptoneurosomal membrane potential, Toxicon, 2003, 41: 191-198
[52] 方晓明，唐毅锋，刘俊平．高效液相色谱／四极杆-飞行时间质谱测定神经性贝毒．色谱，2004，22(1)：20-23
[53] 曹际娟，卫锋，马惠蕊等．贝类毒素检测技术及研究进展．检验建议科学2004，14(1)：53-56
[54] 宋泽，关于我国水产品质量安全管理体系建设的探讨．中国渔业经济，2003，5：37-39
[55] 蔡友琼，乔庆林，徐捷．我国贝类卫生现状及贝类净化概况．渔业现代化，2000．6：7-9
[56] 于维军．近年我国水产品出口遭遇国外技术性贸易壁垒概括．中国水产，2003，12：33-35
[57] 赵洪根．日本水产品食品卫生要求介绍．中国水产，2000，2：44-47
[2] 丘建文 麻痹性贝毒研究概况海洋环境科学，1991，10(2)：65-68
[3] 于仁诚，周名江．麻痹性贝毒研究进展．海洋与湖沼，1998，29(3)：330-338．
[4] Baden D G. Marine roxins. Handbook of Clinical Neurology, 1995, 21(65): 141.
[5] 曹际娟，赵昕，郭皓．神经性贝类毒素．检验检疫科学，1999，9(6)：56-58．
[6] 刘洪英，彭双清，沈勇等．刺尾鱼毒素细胞毒性的作用．卫生毒理学杂志，1999，13(3)：200
[7] V. Morales-Tlalpan, U Vaca. Modulation of the maitotoxin response by intracellular and extracellular cations. Toxicon, 2002, 40: 493-500
[8] Kim M. McGinnis, Margaret E. Gnegy, Nicole Falk, et al. Cytochrome c translocation does not lead to caspase activation in maitotoxin-treated SH-SYSY neuroblastoma cells. Neurochemistry International 2003, 42: 517-523
[9] Li D, Sun L, Chen Z, et al. Survey of the distribution of red tide toxins (okadaic acid and dinophytoxin-1) in the Dalian Bay sea area of China by micellar electrokinetic capillary chromatography. Electrophoresis, 2001. 22(16): 3583-8
[10] Vale P, de M Sampayo M A. Determination of paralytic shellfish toxins in Portuguese shellfish by automated pre-column oxidation. Toxicon, 2001, 39(4): 561-71
[11] Leao J M, Gago A, Rodriguez-Vazquez J A. Solid-phase Extraction and high-performance liquid chromatography procedures for the analysis of paralytic shellfish toxins. J Chromatogr A, 1998, 6: 798(1-2): 131-6
[12] 郭皓，免疫方法在藻毒素及贝毒素检测中的应用，卫生研究，1999，28(2)：122-144
[13] Buzy A, Thibault P, Layeock M V. Development of a capillary electrophoresis method for the characterization of enzymatic products arising from the carbamoylase digestion of paralytic shellfish poisoning toxins. J Chromatogr A, 1994, 668(1-2): 301-316
[14] Ravn H. Toxicological and chemical aspects of paralytic shellfish poisoning (PSP). Vigo: Intergovemmental Oceanographic commission of UNESCO, 1995, 558
[15] Bouaicha N, HENNION M C, Sandra P. Toxcion[J ]. 1997, 35: 276-281.
[16] Buzy A, Thibault P, Laycock M V. Development of a capillary electrophoresis method for the characterization of enzymatic products arising from the carbamoylase digestion of paralytic shellfish poisoning toxins. J Chromatogr A, 1994, 668(1-2): 301-316
[17] Park D L, Adams W N, Graham S L, et al. Variability of the mouse bioassay for determination of Parallytic Shellfish Poisoning Toxins. J AOAC Int, 1986, 69: 547-550
[18] Sommer H, Meyer K F. Paralytic shellfish poisoning. Arch Pathol, 24: 560-598.
[19] Association of official agricultural Chemists.Official methods of analysos 16th ed. 1995, Washington DC
[20] Manger R L, Leja L S, Lee S Y, et al. Detection of sodium channel toxins: directed cytotoxicity assays of purified cigyatoxins, brevetoxins, saxitoxins, and seafood extracts. J AOAC Int, 1995, 78: 521-527.
[21] Manger R L, Leja L S, Lee S Y, et al. Tetrazolium-based cell bioassay for neurotoxins active on voltage-sensitive sodium channels: semiautomated assay for saxitoxins, brevetoxins, ciguatoxins. Anal Biochem, 1993, 214: 190-194.
[22] Elizabeth R, Fairey J, Stewart G, et al. A Cell-Based Assay for Brevetoxins Saxitoxins and Ciguatoxins Using a Stably Expressed e-fos-Luciferase Reporter Gene. Analytical Biochemistry, 1997, 251: 129-132.
[23] Powell C L, Doueette G J. A receptor binding assay for paralytic shellfish poisoning toxins: recent advances and applications. Nat Toxins, 1999, 7(6): 393-400
[24] Vieytes M R, Cabado A G, Alfonso A, et al. Solid-phase radioreeeptor assay for paralytic shellfish toxins. Anal Biochem. 1993, 211(1): 87-93.
[25] Sheng M, Greenberg M E. The regulation and function of c-fos other immediate early genes in the nervous system. Meuron, 1990, 4: 477-482.
[26] Viviani R. Eutrophication, marine biotoxins, human health. Science of the Total Environment, 1992, suppl: 631.
[27] Cullinan W E. Fos expression in forebrain afferents to the hypothalanmic paraventricular mucleus following swim stress. Comp-Neurol, 1996, 368(1): 88-99.
[28] Helmreich D L. The effect of adrenalectomy on stress-induced c-los mRNA expresion in the rat brain. Brain Res, 1996, 706(1): 137-144.
[29] Shimomura O, Johson FH, Saiga Y et al. J Cell. Comp. Physio 1, 1962; 59: 223
[30] Prasher D, Eckenrode V, Ward W, et al. Primary Structure of the Aequores victaria Green-fluorescent Protein. Gene, 1992, 111: 229-233.
[31] Chalfiee M, Tu Y, Euskirchen G, et al. Green Fluorescent Protein as a Marker For Gene Expression. Science, 1994, 263: 802-805.
[32] Ward W W, Cody C W, Prasher D C, et al. Photochem, Photobiol, 1989; 49: 623
[33] Cody C W, Prasher D C, Westter, et al. Biochem isty, 1993; 32: 1212
[34] Heim R, Cubitt A, Tsien R. Nature, 1995; 373: 663
[35] Delagrave S, Hantin RE, Silv CM, et al. Bio/Technology, 1995; 13: 151
[36] Toshio. I,. Nobuo S, Keishi. H,. et. al.. Prostaglandin A1 enhances c-los expression and actibating protein-1 activity. Molecular and Cellular Endocrinology, 2000, 164: 77-85
[37] Chalfiee M, Tu Y, Euskirchen G, et al. Green Fluorescent Protein as a Marker For Gene Expression. Science, 1994, 263: 802-805.
[38] Velez P, Sierralta J, Alcayaga C. A functional assy for paralytic shellfish toxins that use a recombinant sodium channels. Toxicon, 2001, 39(7): 929-935
[39] Fairey E R, Bottein CM, Sheets M F, et al. Modification of the cell based assay for brevetoxins using human cardiac voltage dependent sodium channels expressed in HEK-293 cells. Biosens Bioelectron, 2001, 16(7-8): 519-586
[40] Kerr D S, Bdggs D M, Saba H I, A neurophysiological method of rapid detection and analysis marine algal toxins. Toxicon, 1999, 37(12): 1803-1825.
[41] Second international conference on harmful algae management and mitigation. QingDao. 2001, 12-14
[42] Flanagan A F, Callanan K R, Donlon J. A cytotoxicity assay for the detection and differentiation of two families ofshellfish toxins. Toxicon, 2001, 39: 1021-1027
[43] 金冬雁，黎孟枫，等译．分子克隆实验指南，科学技术出版社，第二版．1992，786-787．
[44] 刘洁生，杨维东，车军，等．重组质粒HF443-EGFP的构建及其在赤潮毒素检测中的应用初探
[45] William W, Richard D. Sequence of the 5'-flanking region of the rat c-fos proto-oncogene. Gene, 1994, 143: 261-264.
[46] 卢圣栋．现代分子生物学实验技术，中国协和医科大学出版社，第二版．1999，392-397．
[47] Hawley T S, Telford W G, Ramezani A, et al. Four-color flow cytometfic detection of retrovirally expressed red, yellow, green and cyan fluorescent proteins. Biotechniques, 2001, 30: 1028-1034.
[48] Alexander E R, Masaki H, Martin W, et al, Identification of the morphology of a thin film by flurescence laser-scanning confocal microscopy, Polymer, 1998, 39(26): 7149-7151
[49] Peng Z, Kirk, T, Xu Z L, The development of three-dimensional imaging techniques of wear particle analysis, Wear, 1997, 203-204: 418-424
[50] Made-Yasmin, Dechraoui, Jerome N, et al. Ciguatoxins and brevetoxins, neurotoxic polyether compounds active on sodium channels, Toxicon, 1999, 37: 125-143
[51] Laurence S D, Steven M P, Kathleen R E, et al, A rapid assay for the brevetoxin group of sodium channel activators based on fluorescence monitoring of synaptoneurosomal membrane potential, Toxicon, 2003, 41: 191-198
[52] 方晓明，唐毅锋，刘俊平．高效液相色谱／四极杆-飞行时间质谱测定神经性贝毒．色谱，2004，22(1)：20-23
[53] 曹际娟，卫锋，马惠蕊等．贝类毒素检测技术及研究进展．检验建议科学2004，14(1)：53-56
[54] 宋泽，关于我国水产品质量安全管理体系建设的探讨．中国渔业经济，2003，5：37-39
[55] 蔡友琼，乔庆林，徐捷．我国贝类卫生现状及贝类净化概况．渔业现代化，2000．6：7-9
[56] 于维军．近年我国水产品出口遭遇国外技术性贸易壁垒概括．中国水产，2003，12：33-35
[57] 赵洪根．日本水产品食品卫生要求介绍．中国水产，2000，2：44-47