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作物学报 ›› 2022, Vol. 48 ›› Issue (1): 259-264.doi: 10.3724/SP.J.1006.2022.03010

• 研究简报 • 上一篇    

玉米SSIIb蛋白多克隆抗体的制备及其应用

余国武(), 青芸, 何珊, 黄玉碧*()   

  1. 四川农业大学农学院作物科学国家级实验教学示范中心, 四川成都 611130
  • 收稿日期:2020-02-11 接受日期:2021-06-08 出版日期:2022-01-12 网络出版日期:2021-06-17
  • 通讯作者: 黄玉碧
  • 作者简介:E-mail: 13862@sicau.edu.cn, Tel: 028-86290868第一联系人：**同等贡献
  • 基金资助:
    国家转基因生物新品种培育重大专项(2016ZX08003-001);国家自然科学基金项目(31501322);四川省博士后科研特别资助项目(03130104);留学回国人员科技活动择优资助项目资助(00124300)

Preparation and application of polyclonal antibody against SSIIb protein from maize

YU Guo-Wu(), QING Yun, HE Shan, HUANG Yu-Bi*()   

  1. National Demonstration Center for Experimental Crop Science Education, College of Agronomy, Sichuan Agricultural University, Chengdu 611130, Sichuan, China
  • Received:2020-02-11 Accepted:2021-06-08 Published:2022-01-12 Published online:2021-06-17
  • Contact: HUANG Yu-Bi
  • About author:First author contact:** Contributed equally to this work
  • Supported by:
    National Major Project for Transgenic Organism Breeding(2016ZX08003-001);National Natural Science Foundation of China(31501322);Special Postdotoral Research Project of Sichuan Province(03130104);Scientific Research Foundation for Returned Overseas Chinese Scholars(00124300)

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摘要：

抗体是蛋白质功能研究的重要试剂。玉米蛋白的功能研究因缺乏相应的抗体而难以开展。玉米淀粉合酶IIb与淀粉合酶IIa均属于淀粉合酶, 其主要负责支链淀粉的延伸。但对淀粉合酶IIb的报道较少, 本研究以原核表达的淀粉合酶IIb的C端(455~704 aa) GST融合蛋白为抗原, 免疫新西兰大白兔, 分离血清制备SSIIb多克隆抗体。Western blot试验发现, SSIIb多克隆抗体不但能识别SSIIb-C (455~704 aa)抗原, 而且能识别玉米不同发育阶段胚乳中SSIIb蛋白。利用该多克隆抗体的Western blot试验表明, 玉米SSIIb蛋白的表达在授粉后10 d到30 d的胚乳中表达模式是先增后降, 授粉后15 d表达量最高, 而半定量RT-PCR的结果表明授粉后20 d SSIIb的转录水平最高。这些结果表明成功制备了特异的玉米SSIIb的多克隆抗体, 并能在Western blot中特异识别玉米内源SSIIb蛋白, 为进一步深入研究SSIIb蛋白功能奠定基础。

关键词: SSIIb, 多克隆抗体, 原核表达, Western blot

Abstract:

Antibody is an important reagent for protein function research. It is difficult to carry out protein function research due to the lack of corresponding antibody. Maize starch synthase IIa and starch synthase IIb (SSIIb) belong to starch synthase, which are mainly responsible for the extension of amylopectin. However, the studies of starch synthase IIb were still in lack. In this study, we immunized New Zealand white rabbits with recombinant GST fusion protein (455-704 aa) at the C-terminal of starch synthase IIb as antigen and prepared polyclonal antibody against SSIIb protein. Western blot showed that the polyclonal antibody of SSIIb could recognize not only SSIIb-C (455-704 aa) antigen, but also SSIIb protein in endosperm of maize at different developmental stages. Western blot analysis using the polyclonal antibody revealed that the expression pattern of SSIIb protein in maize endosperm increased first and then decreased from 10 to 30 days after pollination, and the highest expression level was found at 15 days after pollination. Semi-quantitative RT-PCR results indicated that the transcription level of SSIIb was the highest at 20 days after pollination. These results indicated that the specific polyclonal antibody against SSIIb was successfully prepared, and it could specifically recognize the endogenous SSIIb protein of maize by Western blot, which laid a foundation for further study of the function of SSIIb protein.

Key words: SSIIb, polyclonal antibody, prokaryotic expression, Western blot

图1

SSIIb-C (455~704 aa)基因片段的RT-PCR扩增及pGEX-6p-1-SSIIb-C (455~704 aa)载体的酶切验证 A: 玉米SSIIb蛋白结构域图; B: pGEX-6p-1-SSIIb-C (455~704 aa)载体构建图示; C: SSIIb-C (455~704 aa)基因片段PCR扩增图; M: marker DL2000; 1: 以水为模板的阴性对照PCR; 2: 以玉米20 d胚乳cDNA为模板PCR扩增出ZmSSIIb-C (455~704 aa); D: 重组质粒pGEX-6p-1-SSIIb的BamH I和EcoR I双酶切鉴定; M: marker DL5000; 1: pGEX-6p-1-SSIIb-C (455~704 aa)载体BamH I和EcoR I双酶切; 2: pGEX-6p-1-SSIIb-C (455~704 aa)重组质粒对照。"

图1

SSIIb-C (455~704 aa)基因片段的RT-PCR扩增及pGEX-6p-1-SSIIb-C (455~704 aa)载体的酶切验证 A: 玉米SSIIb蛋白结构域图; B: pGEX-6p-1-SSIIb-C (455~704 aa)载体构建图示; C: SSIIb-C (455~704 aa)基因片段PCR扩增图; M: marker DL2000; 1: 以水为模板的阴性对照PCR; 2: 以玉米20 d胚乳cDNA为模板PCR扩增出ZmSSIIb-C (455~704 aa); D: 重组质粒pGEX-6p-1-SSIIb的BamH I和EcoR I双酶切鉴定; M: marker DL5000; 1: pGEX-6p-1-SSIIb-C (455~704 aa)载体BamH I和EcoR I双酶切; 2: pGEX-6p-1-SSIIb-C (455~704 aa)重组质粒对照。"

图2

GST-SSIIb-C (455~704 aa)融合蛋白的诱导表达与纯化 A: GST-SSIIb-C (455~704 aa)融合蛋白诱导表达的考马斯亮蓝染色图; B: GST-SSIIb-C (455~704 aa)融合蛋白纯化的考马斯亮蓝检测图。1~4: 纯化的GST-SSIIb-C (455~704 aa)融合蛋白; BSA作为上样量对照。"

图2

GST-SSIIb-C (455~704 aa)融合蛋白的诱导表达与纯化 A: GST-SSIIb-C (455~704 aa)融合蛋白诱导表达的考马斯亮蓝染色图; B: GST-SSIIb-C (455~704 aa)融合蛋白纯化的考马斯亮蓝检测图。1~4: 纯化的GST-SSIIb-C (455~704 aa)融合蛋白; BSA作为上样量对照。"

图3

ZmSSIIb多克隆抗体的特异性和灵敏度分析 泳道1~6: 纯化的GST-SSIIb-C (455~704 aa)蛋白作为抗原, 每孔道对其的上样量依次为400、200、100、50、25、12.5 ng, 抗体稀释比例为1:2000。"

图3

ZmSSIIb多克隆抗体的特异性和灵敏度分析 泳道1~6: 纯化的GST-SSIIb-C (455~704 aa)蛋白作为抗原, 每孔道对其的上样量依次为400、200、100、50、25、12.5 ng, 抗体稀释比例为1:2000。"

图4

玉米ZmSSIIb抗体在胚乳不同发育阶段SSIIb蛋白的表达分析中的应用及ZmSSIIb基因表达半定量RT-PCR分析 A: 玉米胚乳中的ZmSSIIb蛋白在授粉后不同时期的表达检测, β-actin用于上样量对照; SSIIb抗体稀释比为1:500; β-actin抗体稀释比例为1:2000; B: 玉米胚乳不同发育阶段ZmSSIIb基因半定量表达分析; ZmTXN作为内参基因。"

图4

玉米ZmSSIIb抗体在胚乳不同发育阶段SSIIb蛋白的表达分析中的应用及ZmSSIIb基因表达半定量RT-PCR分析 A: 玉米胚乳中的ZmSSIIb蛋白在授粉后不同时期的表达检测, β-actin用于上样量对照; SSIIb抗体稀释比为1:500; β-actin抗体稀释比例为1:2000; B: 玉米胚乳不同发育阶段ZmSSIIb基因半定量表达分析; ZmTXN作为内参基因。"

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