条纹斑竹鲨单域抗体IgNAR抗体水平检测方法的建立与应用  

The creation and utilization of a technique to measure the quantities of single-domain IgNAR antibodies in Chiloscyllium plagiosum

作　　者：龙杨颖 姜啸风[1] 陈剑清[2] 吕正兵[1] LONG Yangying;JIANG Xiaofeng;CHEN Jianqing;LYU Zhengbing(College of Life Sciences and Medicine,Zhejiang Sci-Tech University,Hangzhou 310018,China;Shaoxing Biomedical Research Institute,Zhejiang Sci-Tech University,Shaoxing 312000,China)

机构地区：[1]浙江理工大学生命科学与医药学院,杭州310018 [2]浙江理工大学绍兴生物医药研究院,绍兴312000

出　　处：《生命的化学》2024年第12期2342-2352,共11页Chemistry of Life

摘　　要：依据条纹斑竹鲨免疫球蛋白新抗原受体(immunoglobulin new antigen receptor,IgNAR)的同源保守序列,设计并合成多肽序列,并将合成多肽免疫新西兰大白兔,制备抗条纹斑竹鲨IgNAR的特异性多克隆抗体。通过在NCBI等数据库获得条纹斑竹鲨的单域抗体IgNAR的序列,利用Clustal X和Genedoc软件进行序列比对分析,找出其中高度保守的片段,按照抗原表位标准对氨基酸数量、亲水性、二级结构等进行评估,筛选出满足要求的片段作为免疫原序列,合成后免疫新西兰大白兔,制备抗条纹斑竹鲨IgNAR的特异性多克隆抗体,得到的抗条纹斑竹鲨IgNAR多克隆抗体通过酶联免疫吸附测定(enzyme-linked immunosorbent assay,ELISA)、免疫印迹(Western blot,WB)、免疫荧光等实验评价其与条纹斑竹鲨单域抗体的特异性识别、结合能力。获得了具有较高效价和较好特异性的抗条纹斑竹鲨IgNAR多克隆抗体,并将其应用于检测免疫后鲨鱼IgNAR的效价水平,免疫后的血清中富集了较高水平IgNAR,同时在细胞水平上检测免疫原与VNAR的结合位点。通过筛选得到条纹斑竹鲨的保守序列,合成抗原表位并用于免疫新西兰大白兔,获得了抗条纹斑竹鲨IgNAR的特异性多克隆抗体,可用于靶向检测条纹斑竹鲨IgNAR水平,为鲨鱼IgNAR的开发应用提供了可靠的检测工具,助力于鲨鱼IgNAR在临床诊断、治疗上的进一步发展。Using the conserved sequence of immunoglobulin new antigen receptor(IgNAR)the striped bamboo shark as a reference,a peptide sequence was designed and synthesized to generate a specific polyclonal antibody against the striped bamboo shark IgNAR.New Zealand white rabbits were vaccinated with the synthesized peptide.Sequence alignment analysis was conducted to identify highly conserved regions using the single-domain antibody IgNAR against the striped bamboo shark sequence,which was obtained from databases such as NCBI,Clustal X,and GeneDoc software.Amino acid quantity,hydrophilicity,secondary structure,etc.were evaluated based on antigenic epitope standards,and fragments that met the criteria were selected as immunogenic sequences.After synthesis,New Zealand white rabbits were immunized to prepare specific polyclonal antibodies against striped bamboo shark IgNAR.The monoclonal antibodies developed against striped bamboo shark IgNAR were assessed using enzyme-linked immunosorbent assay(ELISA),Western blot(WB),immunofluorescence,and other experiments to test specific recognition and binding ability with single-domain antibodies of striped bamboo shark.Higher potency and better specificity anti-striped bamboo shark IgNAR polyclonal antibody was obtained and used to identify the binding sites of immunogens and VNAR at the cellular level.The study also assessed the potency level of IgNAR in immunized sharks,with higher levels of IgNAR enriched in immunized sera.A specific polyclonal antibody against striped bamboo shark IgNAR was obtained by screening for the shark’s conserved sequence,synthesizing the antigenic epitope,and immunizing New Zealand white rabbits with it.This allows for targeted detection of the shark’s IgNAR levels and serves as a reliable detection tool for the development of shark IgNAR applications,and will facilitate the advancement of shark IgNAR in clinical diagnosis and treatment.

关 键 词：多克隆抗体 单域抗体 抗体 IgNAR VNAR 

分 类 号：R392[医药卫生—免疫学]