山羊IL-17单克隆抗体的制备与鉴定
发布时间：2018-07-27 20:33
【摘要】：IL-17是由IL-17+T细胞产生的细胞因子,它不仅在人类过敏反应、自身免疫疾病、肿瘤发生发展中起到一定的炎性作用,在机体抵抗细菌、真菌、病毒、寄生虫感染中也发挥着关键作用。有关IL-17在山羊机体抗感染免疫、山羊炎症反应中的作用还很不清楚,主要与缺乏山羊IL-17单克隆抗体有关。本研究用纯化的山羊r IL-17免疫BABL/c小鼠,采集小鼠血清,建立山羊IL-17抗体的间接ELISA检测方法;取小鼠脾细胞与SP2/0骨髓瘤细胞进行细胞融合,筛选能稳定分泌特异性IL-17单克隆抗体的杂交瘤细胞。研究取得如下结果:1.IL-17包被浓度为8μg/m L,小鼠免疫血清孵育60 min,酶标二抗的稀释比例为1:5000,作用60 min的条件下,ELISA检测结果最佳;2.获得2株能稳定分泌特异性r IL-17抗体的杂交瘤细胞株,命名为B6、H8;3.B6、H8细胞株的染色体数目分别为为104条、84条;4.B6和H8分泌的单克隆抗体亚型为IgG1,抗体轻链为κ型;5.B6、H8细胞培养上清效价分别为1:28、1:29,小鼠腹水效价为1:105、1:106,纯化单克隆抗体效价均为1:105。6.B6细胞株能特异性识别r IL-17和IL-17+293T细胞培养上清,不与pET32a标签融合蛋白反应,H8细胞株只特异性识别rIL-17。Western blot结果显示,B6与重组rIL-17、IL-17+293T细胞培养上清有特异性免疫结合。本研究成功建立了山羊IL-17抗体的间接ELISA检测方法,筛选到一株能稳定分泌特异性IL-17单克隆抗体的杂交瘤细胞-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.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.4
本文编号：2149061
[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.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.4
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