Characterization and Application of Antibodies Against a Small Heat Shock Protein OsHSP20 from Oryza sativa

doi:10.16819/j.1001-7216.2019.9009

Abstract

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.

Key words: small heat shock protein, polyclonal antibodies, Western blot

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(37 kD)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Western blot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。

关键词: 小热休克蛋白, 多克隆抗体, 蛋白质印迹法

CLC Number:

Q785
S511.01

Jing HE, Han LIU, Liuming GUO, Jing LI, Hengmu ZHANG. 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.

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.

Figures/Tables 4

Fig. 1. Analysis of recombinant OsHSP20 (A) and specificity of L and S antibodies (B and C) by SDS-PAGE and Western blot. M, Protein marker; Lanes 1, 7, and 9, E. coli BL21(DE3) transformed with plasmid pET-32a(+); Lane 2, E. coli BL21 (DE3) harboring recombinant plasmid pET-OsHSP20 without IPTG; Lanes 3-5, E. coli BL21 (DE3) harboring recombinant plasmid pET-OsHSP20 induced by IPTG for 1, 2, and 3 h, respectively; Lanes 8 and 10, E. coli BL21 (DE3) harboring recombinant plasmid pET-OsHSP20 induced by IPTG for 1 h.

Fig. 2. Titer examination of L (A) and L (B) antibodies by ACP-ELISA.

Fig. 3. Sensitivity analysis of L (A) and S (B) antibodies by dot-ELISA. Sixteen groups of dot-ELISA (three repeats per group).

Fig. 4. Detection of HSP20s with Western blot in different plant samples. M, Protein marker; HS, Heat shock treatment; Os, Oryza sativa; Nb, Nicotiana benthamiana; Zm, Zea mays; Ca, Capsicum annuum; So, Spinacia oleracea; Ec, E. coli expressing recombinant OsHSP20 proteins; Bc, Brassica chinensis Linn.; Rs, Raphanus sativus; Bp, Brassica pekinensis.

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