• 中文核心期刊
  • CSCD来源期刊
  • 中国科技核心期刊
  • CA、CABI、ZR收录期刊

本氏烟NbSGS3蛋白的原核表达、多克隆抗体制备及应用

Prokaryotic Expression and Polyclonal Antibody Preparation/Application of NbSGS3 from Nicotiana benthamiana

    摘要
  • 摘要:
    目的 植物特有的RNA结合蛋白基因沉默抑制子3（Suppressor of gene silencing 3, SGS3），在寄主抵御病毒侵染中发挥重要作用。番茄斑萎病毒（Tomato spotted wilt orthotospovirus, TSWV）是危害严重的植物病原病毒，其侵染过程常与寄主基因沉默调控网络存在互作。鉴于现有研究缺少针对性的蛋白检测试剂，本研究制备多克隆抗体可为进一步探究番茄斑萎病毒（TSWV）侵染过程中SGS3蛋白发挥的生物学功能提供检测工具。
    方法 利用RT-PCR克隆本氏烟（Nicotiana benthamiana）SGS3基因（NbSGS3）编码区全长序列及其N端截短（622-1908 bp）和C端截短（1-1365 bp）突变体，构建原核表达载体pET28a-NbSGS3及其突变体载体，诱导产生重组蛋白，免疫新西兰白兔获得的抗血清并进行特异性检测。利用纯化的抗体经Western blot和qRT-PCR检测分析TSWV侵染本氏烟后NbSGS3蛋白和mRNA变化。
    结果 成功获得NbSGS3蛋白及截短突变体重组蛋白，制备的NbSGS3多克隆抗体能够特异性识别原核表达及本氏烟内源NbSGS3蛋白。Western blot结果表明，TSWV感染植株中可特异性检测到TSWV N蛋白，且NbSGS3蛋白表达量明显高于健康对照。qRT-PCR分析显示，TSWV侵染显著诱导NbSGS3基因上调（P＜0.01），与蛋白表达变化趋势一致。
    结论 本研究成功制备了NbSGS3多克隆抗体，可有效用于蛋白的免疫学检测。TSWV侵染本氏烟后激活NbSGS3 mRNA和蛋白上调表达，揭示NbSGS3受到TSWV侵染的调控。

     

    Abstract:
    Objective A polyclonal antibody of the plant viral protection-related RNA-binding protein suppressor of gene silencing 3 (SGS3) was prepared for detecting tomato spotted wilt virus (TSWV).
    Methods The full-length coding sequence of NbSGS3 from Nicotiana benthamiana as well as its N-terminal truncated (622-1908bp) and C-terminal truncated (1-1365bp) mutants were cloned by RT-PCR. Prokaryotic expression vectors pET28a-NbSGS3 and mutant vectors were constructed. The recombinant proteins were induced to immunize New Zealand white rabbits to obtain the antiserum and determine reaction specificity. Changes in NbSGS3 protein and mRNA after infecting N. benthamiana with TSWV were verified by western blot and qRT-PCR using purified antibodies.
    Results The recombinant proteins and truncated mutants of NbSGS3 were successfully secured. The prepared NbSGS3 polyclonal antibodies specifically recognized the prokaryotic expression and endogenous NbSGS3 in N. benthamiana. The western blot showed that the TSWV N protein could be specifically detected in the virus-infected plants, and the expression of NbSGS3 significantly higher than that in the healthy control. The qRT-PCR analysis indicated that the TSWV infection significantly up-regulated NbSGS3 (P＜0.01), which was consistent with the expression changes.
    Conclusion The successfully prepared NbSGS3 polyclonal antibodies could be adequately applied for immunological detection of the target proteins. TSWV-infected N. benthamiana displayed upregulated NbSGS3 mRNA and protein that positively confirmed the regulating function of the gene.

     

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