【摘要】：IL-17是由IL-17+T細(xì)胞產(chǎn)生的細(xì)胞因子,它不僅在人類過(guò)敏反應(yīng)、自身免疫疾病、腫瘤發(fā)生發(fā)展中起到一定的炎性作用,在機(jī)體抵抗細(xì)菌、真菌、病毒、寄生蟲(chóng)感染中也發(fā)揮著關(guān)鍵作用。有關(guān)IL-17在山羊機(jī)體抗感染免疫、山羊炎癥反應(yīng)中的作用還很不清楚,主要與缺乏山羊IL-17單克隆抗體有關(guān)。本研究用純化的山羊r IL-17免疫BABL/c小鼠,采集小鼠血清,建立山羊IL-17抗體的間接ELISA檢測(cè)方法;取小鼠脾細(xì)胞與SP2/0骨髓瘤細(xì)胞進(jìn)行細(xì)胞融合,篩選能穩(wěn)定分泌特異性IL-17單克隆抗體的雜交瘤細(xì)胞。研究取得如下結(jié)果:1.IL-17包被濃度為8μg/m L,小鼠免疫血清孵育60 min,酶標(biāo)二抗的稀釋比例為1:5000,作用60 min的條件下,ELISA檢測(cè)結(jié)果最佳;2.獲得2株能穩(wěn)定分泌特異性r IL-17抗體的雜交瘤細(xì)胞株,命名為B6、H8;3.B6、H8細(xì)胞株的染色體數(shù)目分別為為104條、84條;4.B6和H8分泌的單克隆抗體亞型為IgG1,抗體輕鏈為κ型;5.B6、H8細(xì)胞培養(yǎng)上清效價(jià)分別為1:28、1:29,小鼠腹水效價(jià)為1:105、1:106,純化單克隆抗體效價(jià)均為1:105。6.B6細(xì)胞株能特異性識(shí)別r IL-17和IL-17+293T細(xì)胞培養(yǎng)上清,不與pET32a標(biāo)簽融合蛋白反應(yīng),H8細(xì)胞株只特異性識(shí)別rIL-17。Western blot結(jié)果顯示,B6與重組rIL-17、IL-17+293T細(xì)胞培養(yǎng)上清有特異性免疫結(jié)合。本研究成功建立了山羊IL-17抗體的間接ELISA檢測(cè)方法,篩選到一株能穩(wěn)定分泌特異性IL-17單克隆抗體的雜交瘤細(xì)胞-B6。
[Abstract]:IL-17 is a cytokine produced by IL-17 T cells. It not only plays an inflammatory role in human allergic reaction, autoimmune disease, tumorigenesis and development, but also resists bacteria, fungi and viruses. Parasites also play a key role in infection. The role of IL-17 in goat anti-infection immunity and goat inflammation is still unclear, which is mainly related to the lack of monoclonal antibody against goat IL-17. In this study, we immunized BABL/c mice with purified goat r IL-17, collected mouse serum, established indirect ELISA detection method for goat IL-17 antibody, took mouse spleen cells and SP2/0 myeloma cells for cell fusion, To screen hybridoma cells which can secrete monoclonal antibody to specific IL-17 stably. The results were as follows: 1. When the concentration of IL-17 was 8 渭 g / mL, the mice immune serum was incubated for 60 minutes, the dilution ratio of the second antibody was 1: 5 000, and the best Elisa results were obtained under the condition of 60 min. Two hybridoma cell lines with stable secretion of specific r IL-17 antibody were obtained. The number of chromosomes named B6H83B6H8 cell lines were 104, 84, respectively. The monoclonal antibody subtypes secreted by B6 and H8 were IgG1, the antibody light chain was kappa 5.B6H8 cell culture, the titer of monoclonal antibody was 1 28: 1: 29, the titer of mouse ascites was 1 10 5: 1 10 6, and the purified monoclonal antibody was 1 10 5: 10 6. The antibody light chain was kappa type 5. B6 H8 cell line, the titer of monoclonal antibody was 1 28% 1: 29, and the mouse ascites titer was 1 10 5: 1 10 6. The results showed that 1:105.6.B6 cell lines could specifically recognize the supernatant of r IL-17 and IL-17 293T cells. The H8 cell line which did not react with pET32a tag fusion protein could specifically recognize rIL-17.Western blot. The results showed that B6 could bind to the supernatant of recombinant rIL-17 IL-17 293T cells. In this study, an indirect ELISA detection method for goat IL-17 antibody was successfully established, and a hybridoma cell line -B6, which could stably secrete specific IL-17 monoclonal antibody, was screened.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S852.4

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