Preparation of Polyclonal Antibody Containing CPN60-β for Controlling Rice Stripe Virus Disease
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摘要:
目的 制备针对水稻叶绿体蛋白Chaperonin-60-beta（CPN60-β）多克隆抗体，为进一步研究CPN60-β蛋白在由NSvc4蛋白所介导的水稻条纹病毒（Rice stripe virus, RSV）的运动和致病过程中的调控功能及作用机制提供技术支持和材料准备。 方法 通过RT-PCR从水稻幼嫩叶片基因组扩增CPN60-β基因的功能片段，与原核表达质粒pET-28a（+）构建重组质粒pET-CPN60-β，然后将重组质粒转化大肠杆菌BL21细胞进行诱导表达；将表达纯化的CPN60-β融合蛋白免疫雄性新西兰大白兔获得高效价高浓度的多克隆抗体。 结果 RT-PCR扩增获得的水稻叶绿体CPN60-β基因片段约为657 bp，构建的重组质粒pET-CPN60-β转化大肠杆菌BL21后在诱导剂IPTG浓度0.5 mmol·L−1、摇床温度37 ℃、转速220 r·min−1振荡培养3.5 h条件下成功表达CPN60-β融合蛋白。ELISA和SDS-PAGE检测表明，以纯化CPN60-β融合蛋白免疫雄性新西兰大白兔获得的多克隆抗体效价为1∶1 000 000，抗体质量浓度约300 μg·mL−1。 结论 明确CPN60-β基因原核表达条件；制备获得的水稻叶绿体CPN60-β蛋白的多克隆抗体质量浓度和效价较高，可用于后续研究CPN60-β蛋白在RSV病毒运动和致病过程中发挥的功能。 Abstract:Objective To elucidate the regulatory function and mechanism of the pathogenic processes mediated by the rice stripe virus (RSV)-encoding NSvc4 protein in rice, the high titer concentrated polyclonal antibody containing the chloroplast-related chaperonin-60-beta (CPN60-β) protein against the RSV disease was prepared. Method The CPN60-β fragment was amplified by RT-PCR from rice genome. The recombinant plasmid pET-CPN60-β was constructed with prokaryotic expression plasmid pET-28a (+) and further expressed in Escherichia coli BL21 cells. The CPN60-β fusion protein was purified and immunized into New Zealand rabbits to obtain polyclonal antibody. Titer and concentration of the polyclonal antibody against CPN60-β protein were detected by ELISA and SDS-PAGE for confirmation. Result The approximately 657bp chloroplast-related CPN60-β fragment was successfully amplified by RT-PCR. The reconstructed plasmid pET-CPN60-β positively expressed the CPN60-β fusion protein in E. coli BL21 cells under IPTG concentration of 0.5mmol·L−1 at 37 ℃ with a rotate speed of 220 r·min−1 and an induction time of 5 h. The purified polyclonal antibody had a high titer of 1:106 and a concentration of 300 μg·mL−1 as detected separately by ELISA and SDS-PAGE. Conclusion The conditions of CPN60-β prokaryotic expression were confirmed and the polyclonal antibody against chloroplast-related CPN60-β protein with high titer and concentration successfully prepared for further studies. -
Key words:
- rice /
- chloroplast /
- CPN60-β /
- polyclonal antibody /
- titer
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图 1 重组质粒pET-CPN60-β构建
注：A–CPN60-β基因RT-PCR产物琼脂糖凝胶电泳：M, DNA Marker；1, 阴性对照（水）；2, 水稻叶片样品。B– 重组质粒pET-CPN60-β PCR产物琼脂糖凝胶电泳：M, DNA Marker；1, 阴性对照（水）；2, 单克隆菌落。
Figure 1. Construction of recombinant plasmid pET-CPN60-β
Note: A–Agarose gel electrophoresis of CPN60-β RT-PCR products: M-DNA marker, 1-negative control (water), 2-rice leaf samples. B– Agarose gel electrophoresis of recombinant plasmid pET-CPN60-β PCR products: M-DNA marker, 1-negative control (water), 2-monoclonal colonies.
图 2 CPN60-β基因的表达与鉴定
注：A– SDS-PAGE检测CPN60-β基因的表达：M, 蛋白Marker；1, 不加IPTG的阴性对照；2, 加0.5 mmol·L−1 IPTG样品。B–Western blot鉴定CPN60-β基因的表达：M, 蛋白Marker；1, 加0.5 mmol·L−1 IPTG样品；2, 不加IPTG的阴性对照。
Figure 2. Expression and confirmation of CPN60-β gene
Note: A– Expression of CPN60-β detected by SDS-PAGE: M-protein marker, 1-negative control without IPTG, 2-0.5mM IPTG samples. B– Expression of CPN60-β protein confirmed by western blot: M-protein marker, 1-0.5 mmol·L−1 IPTG samples, 2-negative control without IPTG.
图 3 抗CPN60-β抗体的效价测定
注：A, 免疫过程中抗体效价测定。B, 免疫完成后抗体效价测定：1, 兔子编号1；2, 兔子编号2。
Figure 3. Titer of antibodies against CPN60-β
Note: A–Titer detection of antibodies before immunity. B–Titer detection of antibodies after immunity: 1-rabbit No. 1, 2-rabbit No. 2.
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