【摘要】：家禽黏膜免疫作為動(dòng)物機(jī)體抵抗病原感染的第一道免疫屏障有著重要的意義。分泌型免疫球蛋白A(SIgA)做為黏膜免疫的效應(yīng)因子,對(duì)病原菌在黏膜表面黏附、增殖,啟動(dòng)免疫學(xué)生物活性,清除病原及保護(hù)機(jī)體抗感染等方面發(fā)揮重要作用。 雜交瘤技術(shù),是20世紀(jì)70年代到80年代誕生的生物高新技術(shù),應(yīng)用免疫學(xué)的抗體產(chǎn)生原理和細(xì)胞雜交技術(shù),獲得單一抗原決定簇的高度均一的相應(yīng)抗體,該種抗體特異性好,敏感性高,來(lái)源穩(wěn)定,容易標(biāo)準(zhǔn)化,與多克隆抗體相比,具有顯著的特點(diǎn)。 本研究首次以新鮮的雞膽汁為材料,采用硫酸銨鹽析法從雞膽汁中粗提純雞SIgA,應(yīng)用SDS-聚丙烯酰胺凝膠(SDS-PAGE)電泳鑒定和純化SIgA重鏈,再用純化的雞SIgA重鏈蛋白與等體積的弗氏完全佐劑充分混合頸背部皮下多點(diǎn)注射8周齡雌性Balb/c小鼠,SIgA 50μg/只,隔2周后用弗氏不完全佐劑與之混合,共免疫2次,超強(qiáng)免疫純抗原以100μg/只尾靜脈和腹腔注射。3d后取小鼠脾細(xì)胞與NS0骨髓瘤細(xì)胞,在聚乙二醇(PEG-1500)作用下進(jìn)行細(xì)胞融合,當(dāng)融合細(xì)胞長(zhǎng)滿細(xì)胞培養(yǎng)板時(shí),用間接ELISA進(jìn)行陽(yáng)性雜交瘤的篩選,通過(guò)有限稀釋法3次克隆后,篩選出1株能夠穩(wěn)定分泌抗雞SIgA單克隆抗體的雜交瘤細(xì)胞株5H9。經(jīng)間接ELISA檢測(cè)驗(yàn)證,其細(xì)胞培養(yǎng)上清和腹水效價(jià)為1:400-1:800,1:16000,表明所獲得的單克隆抗體具有和SIgA高度親和力和特異性。6個(gè)月內(nèi)連續(xù)凍存和復(fù)蘇3次,并連續(xù)傳代10次后,抗雞SIgA雜交瘤細(xì)胞株仍能穩(wěn)定分泌抗體,并制備出抗雞SIgA單克隆抗體,為進(jìn)一步研究家禽黏膜免疫機(jī)制和雞傳染性疾病的早期診斷與防治提供了重要的技術(shù)基礎(chǔ)。
[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.
【學(xué)位授予單位】：河南科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R392

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