盐穗木病程相关蛋白HcPR10抗血清的制备及鉴定

Preparation and identification of antiserum against HcPR10 from Halostachys caspica

关键词:  盐穗木病程相关蛋白HcPR10, 原核表达, 蛋白纯化, 抗体制备及鉴定

Key words:  pathogenesis-related protein 10 from Halostachys caspica(HcPR10), prokaryotic expression, protein purification, antibody preparation and identification

摘要:

病程相关蛋白(PRs)在植物抗病抗逆过程中发挥重要作用。盐穗木病程相关蛋白基因HcPR10(GenBank:KF673356)来自盐穗木(Halostachys capsica)在600 mmol·L^-1 NaCl胁迫下的盐抑制差减文库。为探究盐穗木病程相关蛋白HcPR10发挥生物学功能的机制,该研究通过体外表达和纯化HcPR10重组蛋白制备特异性的HcPR10多克隆抗体。并采用双酶切构建原核重组表达载体pET28a-HcPR10,转化至大肠杆菌(Escherichia coli)BL21诱导表达,通过正交分析优化重组蛋白可溶性诱导表达的条件,利用Ni-NTA亲和层析柱纯化融合蛋白,免疫BALB/c小鼠制备多克隆抗体,基于纯化获得的His-HcPR10重组蛋白和转HcPR10拟南芥总蛋白,分别利用ELISA和Western Blotting检测抗血清效价和特异性。结果表明:成功构建重组表达载体pET28a-HcPR10; 正交结果显示诱导温度27 ℃,诱导转速200 r·min^-1,IPTG浓度0.7 mmol·L^-1,诱导时间6 h条件下可诱导表达大量可溶性目的蛋白; ELISA检测抗HcPR10血清效价达1:243 000,Western Blotting印迹结果显示制备的抗血清可以与重组蛋白和转基因拟南芥(Arabidopsis thaliana)中异源表达的HcPR10蛋白特异性结合。该研究获得了效价高、特异性强的盐穗木病程相关蛋白HcPR10抗血清,为进一步研究HcPR10的亚细胞定位及生物学功能奠定了基础。

Abstract:

Pathogenesis-related proteins(PRs)play important roles in plants in response to pathogen attack and diverse environmental stresses. HcPR10(GenBank: KF673356)was isolated from the Suppression Subtractive Hybridization cDNA libraries of Halostachys caspica under the stress of 600 mmol·L^-1 NaCl. In order to investigate the biological function of HcPR10, the specific polyclonal antibody of HcPR10 was prepared by expression and purification of recombinant HcPR10 protein in vitro. In this study, recombinant prokaryotic expression vector pET28a-HcPR10 was constructed by double digestion and then was transformed into Escherichia coli strain BL21 to induce HcPR10 expression. We explored the optimal expression condition for soluble recombinant protein in BL21 by orthogonal analysis. Fusion proteins which were purified by the Ni-NTA affinity chromatography column were injected to BALB/c mice for preparing the HcPR10 polyclonal antibody. The titer and specificity of HcPR10 antiserum were detected respectively by ELISA and Western Blotting using recombinant protein His-HcPR10 and total protein of transgenic HcPR10 Arabidopsis thaliana. The results were as follows: The recombinant expression vector pET28a-HcPR10 was successfully constructed; The maximum amount of soluble fusion protein was obtained under the 27 ℃, 200 r·min^-1 and 0.7 mmol·L^-1 IPTG for induction 6 h; The antiserum for HcPR10 possessing 1:243 000 titer could bind specifically to recombinant protein His-HcPR10 and the heterologous protein from transgenic HcPR10 Arabidopsis thaliana. The high titer and specific antiserum for HcPR10 has been prepared successfully, the results of this study provide the foundation for further investigating the subcellular localization and biological function of HcPR10.