| 抗鸡源EphA2蛋白小鼠多克隆抗体的制备及其特性研究 |
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| 引用本文： | 姚晓慧,李拓凡,谢泉,万志敏,邵红霞,秦爱建,叶建强.抗鸡源EphA2蛋白小鼠多克隆抗体的制备及其特性研究[J].中国家禽,2020(3):29-34. |
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| 作者姓名： | 姚晓慧 李拓凡 谢泉 万志敏 邵红霞 秦爱建 叶建强 |
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| 作者单位： | 扬州大学兽医学院;江苏高校动物重要疫病与人兽共患病防控协同创新中心;扬州大学农业科技发展研究院（国际联合实验室）;教育部农业与农产品安全国际合作联合实验室 |
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| 基金项目： | 国家自然科学基金面上项目(31472171、31972661);江苏高校优势学科建设工程资助项目。 |
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| 摘 要： | 为深入探究鸡源EphA2生物学功能,试验首先对鸡源EphA2基因进行了原核分段克隆以及真核表达载体构建。重组原核表达载体分别命名为pGEX-6p-1-EphA2-A(1-534aa)和pGEX-6p-1-EphA2-B(585-972aa),真核表达载体命名为pcDNA3.1-EphA2。重组原核表达载体转化BL21后经IPTG诱导以及SDS-PAGE分析发现,融合蛋白GST-EphA2-A和GST-EphA2-B均获得有效表达。以纯化的GST-EphA2-A和GST-EphA2-B蛋白免疫小鼠制备了抗鸡EphA2多克隆抗体。间接免疫荧光试验以及Western blot试验表明,所制备的抗鸡EphA2多克隆抗体不仅能识别转染pcDNA3.1-EphA2的DF-1及LMH细胞中表达的外源性EphA2,而且还能有效识别内源性鸡源EphA2蛋白。本研究中鸡EphA2的成功表达及其多克隆抗体研制为后续研究鸡EphA2的功能提供了分子生物学工具。
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| 关 键 词： | 鸡源EphA2 原核分段克隆表达 真核表达 多克隆抗体 反应性 |
Preparation of Polyclonal Antibody against Chicken EphA2 and Its Characteristics |
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| YAO Xiaohui,LI Tuofan,XIE Quan,WAN Zhimin,SHAO Hongxia,QIN Aijian,YE Jianqiang.Preparation of Polyclonal Antibody against Chicken EphA2 and Its Characteristics[J].China Poultry,2020(3):29-34. |
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| Authors: | YAO Xiaohui LI Tuofan XIE Quan WAN Zhimin SHAO Hongxia QIN Aijian YE Jianqiang |
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| Institution: | (Ministry of Education Key Laboratory for Avian Preventive Medicine,Key Laboratory of Jiangsu Preventive Veterinary Medicine,Yangzhou University,Yangzhou,Jiangsu 225009;Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses,Yangzhou University,Yangzhou,Jiangsu 225009;Institutes of Agricultural Science and Technology Development,Yangzhou University,Yangzhou,Jiangsu 225009;Joint International Research Laboratory of Agriculture and Agri-Product Safety,the Ministry of Education of China,Yangzhou University,Yangzhou,Jiangsu 225009) |
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| Abstract: | In this study,the chicken EphA2 gene was cloned into prokaryotic expression vector separately in two parts and the eukaryotic expression vector was constructed.The recombinant prokaryotic expression vectors were named pGEX-6p-1-EphA2-A(1-534aa)and pGEX-6p-1-EphA2-B(585-972aa),respectively,while the eukaryotic expression vector was named as pcDNA3.1-EphA2.After the recombinant prokaryotic expression vectors were transformed into BL21 and induced by IPTG,SDS-PAGE analysis showed that the fusion proteins GST-EphA2-A and GST-EphA2-B could be efficiently expressed.Polyclonal antibodies against chicken EphA2 were prepared by immunizing mice with the purified GST-EphA2-A and GST-EphA2-B proteins respectively.Indirect immunofluorescent assay and Western blot showed that the generated polyclonal antibodies against the chicken EphA2 could not only recognize the exogenous EphA2 expressed in DF-1 and LMH cells,but also react with the endogenous chicken EphA2 protein effectively.In this study,the successful expression of chicken EphA2 and the preparation of polyclonal antibodies against EphA2 provide the molecular biology tools for the further research on the function of chicken EphA2. |
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| Keywords: | chicken EphA2 clone and expression of prokaryotic vector separately eukaryotic expression polyclonal antibody reactivity |
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