水稻小热休克蛋白OsHSP20抗体特性鉴定及应用
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摘要
【目的】在前期克隆了一个水稻小热休克蛋白(OsHSP20)的基础上,进一步分析该蛋白多克隆抗体的免疫反应特性和应用范围,为深入鉴定该类基因的功能奠定基础。【方法】利用合成多肽和其原核表达产物分别免疫家兔制备了相应的多克隆抗体;ACP(Antigen-coated plate)-ELISA和dot-ELISA用于分析比较多克隆抗体效价和灵敏度;Western blot用于鉴定多克隆抗体的特异性和应用范围。【结果】Western blot分析显示,利用重组OsHSP20蛋白及其合成多肽所制备的多克隆抗体均可以在原核表达OsSHSP样品中特异性检测到1条大小与预期一致(37kD)的条带,表明两种方法获得的多克隆抗体都具有高度的特异性;ACP(Antigen-coated plate)-ELISA和dot-ELISA分析表明,利用重组蛋白制备的多克隆抗体效价和灵敏度均高于利用多肽制备的抗体。进一步的Westernblot分析显示该抗体可用于多种植物小热休克蛋白(SHSP)的检测。【结论】制备了OsHSP20蛋白质抗体,且具有高度的特异性和较高的效价和灵敏度,可广泛地用于多种单、双子叶植物HSP20蛋白的表达分析,有助于OsHSP20及其他植物同源蛋白的功能鉴定。
【Objective】In previous studies, OsHSP20, a gene encoding small heat shock protein, was cloned from Oryza sativa. Here its polyclonal antibodies were prepared and analyzed for their properties of immunological reactions and range of application. 【Method】 Synthetic peptide and recombinant protein of OsHSP20 were used to immunize the rabbits for preparing the polyclonal antibodies against the protein, respectively. Antigen-coated plate(ACP)-and dot-enzyme linked immunosorbent assays(ELISA) were used for comparing the titer and sensitivity of antibodies.Western blotting was used to determine the antibody specificity and the range of application. 【Result】In Western blotting assays, both antibodies were strongly reacted with a band of protein with an expected size of 37 kD, suggesting that they were specific against the OsHSP20. ACP-and dot-ELISA assays showed that the polyclonal antibody prepared from the recombinant protein had a higher titer and sensitivity than that of peptide. Further assays showed that the polyclonal antibody could be used for detection of the SHSP homologs of plants. 【Conclusion】The polyclonal antibodies were successfully prepared with high specificity, titer, and sensitivity and could be widely used to analyze the expression of HSP20 protein in a variety of monocotylous and dicotyledonous plants, which could contribute to the functional characterization of OsHSP20 and its homologs in different plants.
【Objective】In previous studies, OsHSP20, a gene encoding small heat shock protein, was cloned from Oryza sativa. Here its polyclonal antibodies were prepared and analyzed for their properties of immunological reactions and range of application. 【Method】 Synthetic peptide and recombinant protein of OsHSP20 were used to immunize the rabbits for preparing the polyclonal antibodies against the protein, respectively. Antigen-coated plate(ACP)-and dot-enzyme linked immunosorbent assays(ELISA) were used for comparing the titer and sensitivity of antibodies.Western blotting was used to determine the antibody specificity and the range of application. 【Result】In Western blotting assays, both antibodies were strongly reacted with a band of protein with an expected size of 37 kD, suggesting that they were specific against the OsHSP20. ACP-and dot-ELISA assays showed that the polyclonal antibody prepared from the recombinant protein had a higher titer and sensitivity than that of peptide. Further assays showed that the polyclonal antibody could be used for detection of the SHSP homologs of plants. 【Conclusion】The polyclonal antibodies were successfully prepared with high specificity, titer, and sensitivity and could be widely used to analyze the expression of HSP20 protein in a variety of monocotylous and dicotyledonous plants, which could contribute to the functional characterization of OsHSP20 and its homologs in different plants.
引文
[1]Vierling E.The roles of heat shock proteins in plants.Annu Rev Plant Physiol Plant Mol Biol,2003,42(4):579-620.
[2]Haslbeck M,Franzmann T,Weinfurtner D,Buchner J.Some like it hot:The structure and function of small heat shock proteins.Nat Struct Mol Biol,2005,12(10):842-846.
[3]Arrigo AP.Small stress proteins:Chaperones that act as regulators of intracellular redox state and programmed cell death.Biol Chem,1998,379(1):19-26.
[4]Kappe G,Leunissen J A,De Jong W W.Evolution and diversity of prokaryotic small heat shock proteins.Prog Mol Subcell Biol,2002,28:12-17.
[5]Zou J,Liu A,Chen X,Zhou X,Gao G,Wang W.Expression analysis of nine rice heat shock protein genes under abiotic stresses and ABA treatment.Plant Physiol,2009,166(8):851-861
[6]Scharf K D,Siddique M,Vierling E.The expanding family of Arabidopsis thaliana small heat stress proteins and a new family of proteins containing alpha-crystallin domains(Acd proteins).Cell Stress Chap,2001,6(3):225-237.
[7]Li J,Xiang C Y,Yang J,Chen J P,Zhang H M.Interaction of HSP20 with a viral RdRp changes its sub-cellular localization and distribution pattern in plants.Sci Rep,2015,5:14016
[8]MacRae T H.Structure and function of small heat shock/alpha-crystallin proteins:Established concepts and emerging ideas.Cell Mol Life Sci,2000,57(6):899-913.
[9]van Montfort R,Slingsby C,Vierling E.Structure and function of the small heat shock protein/alpha-crystallin family of molecular chaperones.Adv Protein Chem,2002,59:105-156.
[10]项聪英,蔡年俊,李静,羊健,陈剑平,张恒木.一个水稻小热休克蛋白基因的克隆和鉴定.中国水稻科学,2016,30(6):587-592.Xiang C Y,Cai N J,Li J,Yang J,Chen J P,Zhang H M.Cloning and characterization of a small heat shock protein(SHSP)gene in rice plant.Chin J Rice Sci,2016,30(6):587-592.(in Chinese with English abstract)
[11]蔡年俊,郭留明,李静,项聪英,羊健,陈剑平,张恒木.一个水稻小热休克蛋白的异源表达及寡聚特性分析.中国水稻科学,2017,31(5):483-488.Cai N J,Guo L M,Li J,Xiang C Y,Yang J,Chen J P,Zhang H M.Heterologous expression and oligomeric identification of a small heat shock protein(SHSP)from Oryza sativa.Chin J Rice Sci,2017,31(5):483-488.(in Chinese with English abstract)
[12]Saha S,Raghava G P.Prediction of continuous B-cell epitopes in an antigen using recurrent neural network.Prote-struct Funct&Bioinform,2010,65(1):40-48.
[13]徐宜为.免疫检测技术.2版.北京:科学出版社,1991.Xu Y W.Immunoassay Technology.2nd ed.Beijing:Science Press,1991.(in Chinese)
[14]Shang H L,Xie Y,Zhou X P,Qian Y J,Wu J X.Monoclonal antibody-based serological methods for detection of Cucumber green mottle mosaic virus.Virol J,2011,8:228-236.
[15]梁国栋.最新分子生物学实验技术.北京:科学出版社,2001.Liang G D.The Newest Experimental Techniques of Molecular Biology.Beijing:Science Press,2001.(in Chinese)
[16]沈关心,周汝麟.现代免疫学实验技术.武汉:湖北科学技术出版社,1998.Shen G X,Zhou R L.Modern Immunology Experimental Technology.Wuhan:Hubei Science and Technology Press,1998.(in Chinese)
[17]邵健忠,项黎新.应用Dot-ELISA技术检测草鱼出血病病毒的研究.水产学报,1996,20(1):6-12.Shao J Z,Xiang L X.Study on detection of hemorrhagic disease of grass carp by Dot-ELISA.J Fish China,1996,20(1):6-12.
[18]Salonen E M,Vaheri A.Immobilization of viral and mycoplasma antigens and of immunoglobulins on polystyrene surface for immunoassays.J Immun Meth,1979,30(3):209-218.
[2]Haslbeck M,Franzmann T,Weinfurtner D,Buchner J.Some like it hot:The structure and function of small heat shock proteins.Nat Struct Mol Biol,2005,12(10):842-846.
[3]Arrigo AP.Small stress proteins:Chaperones that act as regulators of intracellular redox state and programmed cell death.Biol Chem,1998,379(1):19-26.
[4]Kappe G,Leunissen J A,De Jong W W.Evolution and diversity of prokaryotic small heat shock proteins.Prog Mol Subcell Biol,2002,28:12-17.
[5]Zou J,Liu A,Chen X,Zhou X,Gao G,Wang W.Expression analysis of nine rice heat shock protein genes under abiotic stresses and ABA treatment.Plant Physiol,2009,166(8):851-861
[6]Scharf K D,Siddique M,Vierling E.The expanding family of Arabidopsis thaliana small heat stress proteins and a new family of proteins containing alpha-crystallin domains(Acd proteins).Cell Stress Chap,2001,6(3):225-237.
[7]Li J,Xiang C Y,Yang J,Chen J P,Zhang H M.Interaction of HSP20 with a viral RdRp changes its sub-cellular localization and distribution pattern in plants.Sci Rep,2015,5:14016
[8]MacRae T H.Structure and function of small heat shock/alpha-crystallin proteins:Established concepts and emerging ideas.Cell Mol Life Sci,2000,57(6):899-913.
[9]van Montfort R,Slingsby C,Vierling E.Structure and function of the small heat shock protein/alpha-crystallin family of molecular chaperones.Adv Protein Chem,2002,59:105-156.
[10]项聪英,蔡年俊,李静,羊健,陈剑平,张恒木.一个水稻小热休克蛋白基因的克隆和鉴定.中国水稻科学,2016,30(6):587-592.Xiang C Y,Cai N J,Li J,Yang J,Chen J P,Zhang H M.Cloning and characterization of a small heat shock protein(SHSP)gene in rice plant.Chin J Rice Sci,2016,30(6):587-592.(in Chinese with English abstract)
[11]蔡年俊,郭留明,李静,项聪英,羊健,陈剑平,张恒木.一个水稻小热休克蛋白的异源表达及寡聚特性分析.中国水稻科学,2017,31(5):483-488.Cai N J,Guo L M,Li J,Xiang C Y,Yang J,Chen J P,Zhang H M.Heterologous expression and oligomeric identification of a small heat shock protein(SHSP)from Oryza sativa.Chin J Rice Sci,2017,31(5):483-488.(in Chinese with English abstract)
[12]Saha S,Raghava G P.Prediction of continuous B-cell epitopes in an antigen using recurrent neural network.Prote-struct Funct&Bioinform,2010,65(1):40-48.
[13]徐宜为.免疫检测技术.2版.北京:科学出版社,1991.Xu Y W.Immunoassay Technology.2nd ed.Beijing:Science Press,1991.(in Chinese)
[14]Shang H L,Xie Y,Zhou X P,Qian Y J,Wu J X.Monoclonal antibody-based serological methods for detection of Cucumber green mottle mosaic virus.Virol J,2011,8:228-236.
[15]梁国栋.最新分子生物学实验技术.北京:科学出版社,2001.Liang G D.The Newest Experimental Techniques of Molecular Biology.Beijing:Science Press,2001.(in Chinese)
[16]沈关心,周汝麟.现代免疫学实验技术.武汉:湖北科学技术出版社,1998.Shen G X,Zhou R L.Modern Immunology Experimental Technology.Wuhan:Hubei Science and Technology Press,1998.(in Chinese)
[17]邵健忠,项黎新.应用Dot-ELISA技术检测草鱼出血病病毒的研究.水产学报,1996,20(1):6-12.Shao J Z,Xiang L X.Study on detection of hemorrhagic disease of grass carp by Dot-ELISA.J Fish China,1996,20(1):6-12.
[18]Salonen E M,Vaheri A.Immobilization of viral and mycoplasma antigens and of immunoglobulins on polystyrene surface for immunoassays.J Immun Meth,1979,30(3):209-218.