| 水稻小热休克蛋白OsHSP20抗体特性鉴定及应用 |
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| 引用本文： | 何婧,刘寒,郭留明,李静 张恒木.水稻小热休克蛋白OsHSP20抗体特性鉴定及应用[J].中国水稻科学,2019,33(3):235-240. |
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| 作者姓名： | 何婧 刘寒 郭留明 李静 张恒木 |
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| 作者单位： | 1.浙江师范大学 化学与生命科学学院,浙江 金华 3210042 浙江省农业科学院 病毒学与生物技术研究所/省部共建植物有害生物国家重点实验室培育基地, 杭州 310021; |
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| 基金项目： | 国家自然科学基金资助项目(31601603)；浙江省重点研发计划资助项目(2019C02018)。 |
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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。
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| 关 键 词： | 小热休克蛋白 多克隆抗体 蛋白质印迹法 |
| 收稿时间： | 2019-01-21 |
| 修稿时间： | 2019-02-17 |
Characterization and Application of Antibodies Against a Small Heat Shock Protein OsHSP20 from Oryza sativa |
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| HE Jing,LIU Han,GUO Liuming,LI Jing,ZHANG Hengmu.Characterization and Application of Antibodies Against a Small Heat Shock Protein OsHSP20 from Oryza sativa[J].Chinese Journal of Rice Science,2019,33(3):235-240. |
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| Authors: | HE Jing LIU Han GUO Liuming LI Jing ZHANG Hengmu |
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| Institution: | 1.College of Chemistry and Life Science, Zhejiang Normal University, Jinhua 321004, China2 State Key Laboratory Breeding Base for Zhejiang Sustainable Pest and Disease Control/Institute of Virology and Biotechnology, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China; |
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| Abstract: | 【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. |
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| Keywords: | small heat shock protein polyclonal antibodies Western blot |
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