| 斑马鱼血管新生相关因子PTPRB的原核表达及其多克隆抗体制备 |
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| 引用本文： | 冯鹏霏,肖蕊,张永德,林勇,宋漫玲,潘传燕,余艳玲,罗辉,罗洪林.斑马鱼血管新生相关因子PTPRB的原核表达及其多克隆抗体制备[J].南方农业学报,2021,52(4):1090-1097. |
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| 作者姓名： | 冯鹏霏 肖蕊 张永德 林勇 宋漫玲 潘传燕 余艳玲 罗辉 罗洪林 |
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| 作者单位： | 1. 广西水产科学研究院/广西水产遗传育种与健康养殖重点实验室, 南宁 530021;2. 西南大学动物科学学院, 重庆 402460 |
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| 基金项目： | 广西创新驱动发展专项（桂科AA17204080-3，桂科AA18242031-2）；广西水产遗传育种与康养殖重点实验室自主研发项目（17-A-01-02，19-A-01-05） |
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| 摘 要： | 【目的】原核表达斑马鱼（Danio rerio）蛋白酪氨酸磷酸酶受体B（PTPRB）并制备多克隆抗体，为研究斑马鱼PTPRB基因功能及血管发育相关信号传导通路打下基础。【方法】采用无缝克隆技术将斑马鱼PTPRB基因插入原核表达载体pET-B2m构建重组表达质粒，转化大肠杆菌B21感受态细胞后采用IPTG进行诱导表达，然后以诱导表达的融合蛋白免疫大耳兔制备多克隆抗体，并采用Western blotting和ELISA检测多克隆抗体的特异性及免疫效价。【结果】斑马鱼PTPRB蛋白亲水性均值为-0.490，属于亲水性蛋白，且具有较丰富的潜在抗原表位位点，分布均匀，无典型的跨膜区。将斑马鱼PTPRB基因插入原核表达载体pET-B2m成功构建获得重组表达质粒pET-B2-PTPRB，转化B21感受态细胞后经IPTG诱导表达，即获得35.0 kD的融合蛋白。融合蛋白PTPRB主要以包涵体形式存在；以纯化融合蛋白PTPRB免疫大耳兔，其血清抗体效价为1∶2048000，说明采用融合蛋白PTPRB可有效刺激大耳兔产生较强的免疫反应，获得较高效价的PTPRB多克隆抗体。Western blotting检测结果显示，PTPRB多克隆抗体具良好抗原特异性。采用Protein A/G亲和层析柱对制备获得的PTPRB多克隆抗体进行亲和层析纯化，可获得高纯度的多克隆抗体，纯化后的PTPRB多克隆抗体浓度在10 mg/mL以上。【结论】构建的斑马鱼PTPRB基因原核表达载体能高效表达具备良好免疫原性的融合蛋白PTPRB，以融合蛋白PTPRB免疫大耳兔可获得高效价、高特异性的PTPRB多克隆抗体，为研究斑马鱼PTPRB蛋白功能提供有利工具，也为揭示PTPRB在鱼类血管发育中的作用机制提供技术支持。
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| 关 键 词： | 斑马鱼 PTPRB 原核表达 多克隆抗体 |
| 收稿时间： | 2020-05-25 |
Prokaryotic expression and polyclonal antibody preparation of Danio rerio angiogenesis related factor PTPRB |
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| FENG Peng-fei,XIAO Rui,ZHANG Yong-de,LIN Yong,SONG Man-ling,PAN Chuan-yan,YU Yan-ling,LUO Hui,LUO Hong-lin.Prokaryotic expression and polyclonal antibody preparation of Danio rerio angiogenesis related factor PTPRB[J].Journal of Southern Agriculture,2021,52(4):1090-1097. |
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| Authors: | FENG Peng-fei XIAO Rui ZHANG Yong-de LIN Yong SONG Man-ling PAN Chuan-yan YU Yan-ling LUO Hui LUO Hong-lin |
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| Affiliation: | 1. Guangxi Academy of Fishery Sciences/Guangxi Key Laboratory of Genetic Breeding and Healthy Aquaculture, Nanning 530021, China;2. School of Animal Science, Southwest University, Chongqing 402460, China |
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| Abstract: | 【Objective】Prokaryotic expression of zebrafish(Danio rerio) protein tyrosine phosphatase receptor B(PTPRB) and preparation of polyclonal antibodies were conducted to lay the foundation for studying the function of zebrafish PTPRB gene and signal transduction pathways related to vascular development.【Method】The zebrafish PTPRB gene was inserted into the prokaryotic expression vector pET-B2m to construct the recombinant expression plasmid by seamless cloning technology. The recombinant plasmid was transformed into Escherichia coli B21 competent cells, and induced to express by IPTG. Then the polyclonal antibody was prepared by immunizing rabbits with the induced fusion protein. The specificity and immune titer of the polyclonal antibody were detected by Western blotting and ELISA.【Result】The average hydrophilicity of zebrafish PTPRB protein was-0.490, which belonged to hydrophilic protein. It had abundant potential epitopes, distributed evenly and had no typical transmembrane region. The zebrafish PTPRB gene was inserted into the prokaryotic expression vector pET-B2m, and the recombinant expression plasmid pET-B2-PTPRB was successfully constructed. The recombinant expression plasmid was transformed into B21 competent cells and induced by IPTG to express a 35.0 kD fusion protein. The fusion protein PTPRB mainly existed in the form of inclusion bodies;the purified fusion protein PTPRB was used to immunize big-eared rabbits, and the serum antibody titer was 1:2048000, indicating that the fusion protein PTPRB could effectively stimulate the big-eared rabbits to produce a stronger immune response and obtain PTPRB polyclonal antibody with high titer. Western blotting results showed that PTPRB polyclonal antibody has good antigen specificity. High purity polyclonal antibody against PTPRB was obtained by using protein A/G affinity chromatography column. The concentration of the purified PTPRB polyclonal antibody was above 10 mg/mL.【Conclusion】The constructed zebrafish PTPRB gene prokaryotic expression vector can efficiently express the fusion protein PTPRB with good immunogenicity. High titer and high specificity polyclonal antibody against PTPRB can be obtained by immunizing big-eared rabbits with the fusion protein PTPRB, which provides a tool for studying the function of zebrafish PTPRB protein and a way to reveal the mechanism of PTPRB in fish vascular development. |
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