抗鸡SIgA单克隆抗体杂交瘤细胞株的建立

发布时间：2018-08-04 10:58

【摘要】：家禽黏膜免疫作为动物机体抵抗病原感染的第一道免疫屏障有着重要的意义。分泌型免疫球蛋白A(SIgA)做为黏膜免疫的效应因子,对病原菌在黏膜表面黏附、增殖,启动免疫学生物活性,清除病原及保护机体抗感染等方面发挥重要作用。杂交瘤技术,是20世纪70年代到80年代诞生的生物高新技术,应用免疫学的抗体产生原理和细胞杂交技术,获得单一抗原决定簇的高度均一的相应抗体,该种抗体特异性好,敏感性高,来源稳定,容易标准化,与多克隆抗体相比,具有显著的特点。本研究首次以新鲜的鸡胆汁为材料,采用硫酸铵盐析法从鸡胆汁中粗提纯鸡SIgA,应用SDS-聚丙烯酰胺凝胶(SDS-PAGE)电泳鉴定和纯化SIgA重链,再用纯化的鸡SIgA重链蛋白与等体积的弗氏完全佐剂充分混合颈背部皮下多点注射8周龄雌性Balb/c小鼠,SIgA 50μg/只,隔2周后用弗氏不完全佐剂与之混合,共免疫2次,超强免疫纯抗原以100μg/只尾静脉和腹腔注射。3d后取小鼠脾细胞与NS0骨髓瘤细胞,在聚乙二醇(PEG-1500)作用下进行细胞融合,当融合细胞长满细胞培养板时,用间接ELISA进行阳性杂交瘤的筛选,通过有限稀释法3次克隆后,筛选出1株能够稳定分泌抗鸡SIgA单克隆抗体的杂交瘤细胞株5H9。经间接ELISA检测验证,其细胞培养上清和腹水效价为1:400-1:800,1:16000,表明所获得的单克隆抗体具有和SIgA高度亲和力和特异性。6个月内连续冻存和复苏3次,并连续传代10次后,抗鸡SIgA杂交瘤细胞株仍能稳定分泌抗体,并制备出抗鸡SIgA单克隆抗体,为进一步研究家禽黏膜免疫机制和鸡传染性疾病的早期诊断与防治提供了重要的技术基础。
[Abstract]:Poultry mucosal immunity is of great significance as the first immune barrier against pathogenic infection in animals. Secretory immunoglobulin A (SIgA), as an effector of mucosal immunity, plays an important role in mucosal surface adhesion, proliferation, activation of immunological biological activity, elimination of pathogens and protection of organism against infection. Hybridoma technology, which was born in the 1970s and 1980s, uses immunological antibody production principles and cell hybridization techniques to obtain highly homogeneous corresponding antibodies of a single antigen determinant, which has good specificity. High sensitivity, stable source, easy standardization, compared with polyclonal antibody, has significant characteristics. In this study, fresh chicken bile was used for the first time to purify chicken Siga from chicken bile by ammonium sulfate salting-out method. SIgA heavy chain was identified and purified by SDS-polyacrylamide gel (SDS-PAGE) electrophoresis. Then the purified chicken SIgA heavy chain protein and the equal volume Freund's complete adjuvant were fully mixed into the neck and back subcutaneously and injected into 8 week-old female Balb/c mice with Siga 50 渭 g / mouse. After 2 weeks, they were mixed with Freund's incomplete adjuvant and immunized twice. The superimmune pure antigen was injected into the tail vein of 100 渭 g / g and intraperitoneally for 3 days. The spleen cells of mice were fused with NS0 myeloma cells in the presence of polyethylene glycol (PEG-1500). When the fusion cells were filled with cell culture plates, Indirect ELISA was used to screen the positive hybridoma. After three times of cloning by limited dilution method, a hybridoma cell line 5H9, which could stably secrete monoclonal antibody against chicken SIgA, was selected. The titer of supernatant and ascites was 1: 400-1: 800: 16000, which indicated that the monoclonal antibody obtained had high affinity and specificity with SIgA. Anti-chicken SIgA hybridoma cell lines can still secrete antibodies stably and produce monoclonal antibodies against chicken SIgA, which provides an important technical basis for further study on the mechanism of poultry mucosal immunity and the early diagnosis and control of chicken infectious diseases.
【学位授予单位】：河南科技大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

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