兔抗条纹斑竹鲨IgNAR多克隆抗体的制备和应用  

Preparation and application of rabbit polyclonal antibody against Immunoglobulin new antigen receptor of whitespotted bamboo shark

作　　者：肖国凯 郝珮羽 冯世涛 柳晓春 范益华 吕建欣 隋建新[2] 于文功[1] 顾玉超[1] XIAO Guo-kai;HAO Pei-yu;FENG Shi-tao;LIU Xiao-chun;FAN Yi-hua;LV Jian-xin;SUI Jian-xin;YU Wen-gong;GU Yu-chao(Key Laboratory of Marine Drugs,Ministry of Education,Shandong Provincial Key Laboratory of Glycoscience and Glycoengineering,School of Medicine and Pharmacy,Ocean University of China,Qingdao 266003,China;College of Food Science and Engineering,Ocean University of China,Qingdao 266003,China)

机构地区：[1]中国海洋大学海洋药物教育部重点实验室,山东省糖科学与糖工程重点实验室,医药学院,山东青岛266003 [2]中国海洋大学食品科学与工程学院,山东青岛266003

出　　处：《中国海洋药物》2022年第2期37-42,共6页Chinese Journal of Marine Drugs

基　　金：中央高校基本科研业务费(202042011,202064005)资助。

摘　　要：目的 制备条纹斑竹鲨(Chiloscyllium plagiosum)IgNAR的多克隆抗体,为条纹斑竹鲨单域抗体的开发提供工具。方法 合成条纹斑竹鲨IgNAR恒定区的一段多肽作为抗原,免疫新西兰大白兔,制备多克隆抗体血清,采用间接ELISA法测定抗体的效价,并通过Western Blot检测多克隆抗体的特异性。此外,利用制备的多克隆抗体对条纹斑竹鲨外周血淋巴细胞进行免疫荧光和流式分析。结果 制备的抗血清效价高达1 000万,该多克隆抗体不仅能识别条纹斑竹鲨的IgNAR,还能识别护士鲨(Ginglymostoma cirratum)的IgNAR,但对条纹斑竹鲨的灵敏度更高。通过免疫荧光和流式细胞术确定了该多克隆抗体还可以识别条纹斑竹鲨外周血淋巴细胞跨膜型IgNAR。结论 成功制备了条纹斑竹鲨IgNAR的多克隆抗体,该抗体可用于Western Blot、免疫荧光和流式细胞术等实验,为条纹斑竹鲨单域抗体的开发提供了有力工具。Objective To prepare a rabbit polyclonal antibody against the Immunoglobulin new antigen receptor(IgNAR) of whitespotted bamboo shark(Chiloscyllium plagiosum), and provide a tool for the development of single domain antibody of whitespotted bamboo shark. Methods We synthesized a peptide with the sequence from whitespotted bamboo sharks IgNAR constant regions and immunized New Zealand white rabbits. The polyclonal antibody was prepared, and the titer of the antibody was determined by indirect ELISA. The specificity of the polyclonal antibody to IgNAR was determined by Western Blot. In addition, the polyclonal antibody was used to detect the transmembrane IgNAR of whitespotted bamboo shark peripheral blood lymphocytes by immunofluorescence and flow cytometry. Results The titer of the antiserum was 10 000 000. The polyclonal antibody could detect IgNAR from both whitespotted bamboo shark and nurse shark(Ginglymostoma cirratum) through Western Blot, and had a higher sensitivity to the IgNAR of whitespotted bamboo shark. The polyclonal antibody could recognize transmembrane IgNAR of whitespotted bamboo shark peripheral blood lymphocytes by immunofluorescence and flow cytometry. Conclusion The polyclonal antibody against IgNAR of whitespotted bamboo shark was successfully produced. It could be used in Western Blot, immunofluorescence and flow cytometry, which would be a useful tool for the development of single domain antibody from whitespotted bamboo shark.

关 键 词：条纹斑竹鲨 IgNAR 单域抗体 多克隆抗体 

分 类 号：R965[医药卫生—药理学]