• 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 基因沉默抑制子3（suppressor of gene silencing 3, SGS3）是植物特有的 RNA 结合蛋白，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。截至目前尚缺乏针对该蛋白的特异性检测试剂，本研究拟制备多克隆抗体，以期为进一步探究番茄斑萎病毒（TSWV）侵染过程中SGS3蛋白的生物学功能提供核心检测工具。
    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 抗血清稀释浓度为1∶1500时目的条带清晰，NbSGS3b抗血清检测效果最佳。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 Suppressor of gene silencing 3 (SGS3) is a plant-specific RNA-binding protein that plays an important role in host antiviral defense. Tomato spotted will orthotospovirus (TSWV) is a destructive plant pathogen whose infection exhibits complex interactions with the host gene-silencing regulatory network. Currently, specific detection reagents for SGS3 are still lacking. Therefore,this study aimed to prepare polyclonal antibodies against NbSGS3 to provide a reliable detection tool for further exploring the biological functions of SGS3 during TSWV infection.
    Methods The full-length coding sequence of NbSGS3 from Nicotiana benthamiana as well as its N-terminal truncated (622−1908 bp) and C-terminal truncated (1−1365 bp) mutants were cloned by RT-PCR. Prokaryotic expression vectors pET28a-NbSGS3 and its truncated derivatives were constructed. The recombinant proteins were induced and purified then used to immunize New Zealand white rabbits to obtain antisera. Antibody specificity was verified. Western blot and qRT-PCR were further performed to examine the accumulation of NbSGS3 protein and the transcript level of NbSGS3 upon TSWV infection.
    Results Clear target bands were observed at an antiserum dilution ratio of 1:1500 and the NbSGS3 antiserum exhibited the best detection performamce. 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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