猪CR1-like单克隆抗体的制备及CR1-like在猪红细胞膜表面分布状态的研究

发布时间：2018-04-26 02:28

本文选题：猪红细胞CR1-like + 单克隆抗体　；参考：《山西农业大学》2015年硕士论文

【摘要】：目的：为进一步研究动物红细胞CR1天然分布状态及其功能调控机制,本试验以猪为研究对象,制备猪CR1-like单克隆抗体,应用该抗体对CR1-like分子在猪红细胞膜表面的分布进行深入研究,以期为课题组进一步研究动物红细胞免疫功能的分子机制提供理论数据和技术手段。方法：运用Gene ious软件分析猪CR1-like基因cDNA序列的生物信息学特征,合成免疫抗原肽段；采用单克隆抗体制备技术建立分泌猪CR1-like单克隆抗体的小鼠杂交瘤细胞,以亲和层析、中压蛋白纯化的技术纯化所分泌的单克隆抗体；通过SDS-PAGE方法对所产单抗的蛋白特性及纯度进行鉴定,并应用间接免疫荧光技术研究了该单抗的免疫活性。利用该抗体,应用激光共聚焦显微镜观察天然猪红细胞CR1-like膜分布状态；人工制备免疫复合物C3b-beads为报告因子,以流式细胞仪检测膜流动性变化对猪红细胞CR1-like发挥免疫粘附功能的影响。结果：获得了分泌猪CR1-like单克隆抗体的小鼠杂交瘤细胞株,并成功生产、纯化出小鼠抗猪CR1-like单克隆抗体；应用所产单抗间接免疫荧光染色猪红细胞,于荧光显微镜下观察,猪红细胞表面可见绿色荧光；激光共聚焦显微镜观察猪CR1-like分布状态,经膜预固定处理的猪红细胞表面其绿色荧光分布成片状；相比之下,未经膜固定处理的猪红细胞表面其荧光点呈颗粒状分布,较膜固定组分布明显成簇化。流式细胞仪检测发现,膜固定组平均荧光强度值为302.35,未固定组为638.44,阴性对照组为4.7,统计得：膜固定组、未固定组均明显高于阴性对照组,差异极显著(P0.01),同时未固定组明显高于膜固定组,差异极显著(P0.01)。结论：本研究成功获得可稳定传代生产猪CR1-like单克隆抗体的杂交瘤细胞株(型号：CRT-2 CL-5):生产、纯化出具有生物活性的猪CR1-like单克隆抗体；天然状态下猪红细胞表面的CR1-like分布呈分散状态,发挥免疫功能时,CR1-like表现出多价高效的结合特性,其分布状态表现为颗粒样成簇分布；红细胞膜流动性是影响猪红细胞CR1-like发挥免疫粘附功能的重要生理基础。
[Abstract]:Objective: to further study the natural distribution of CR1 in animal erythrocytes and its functional regulation mechanism, porcine monoclonal antibodies against CR1-like were prepared in this study. The distribution of CR1-like molecules on porcine erythrocyte membrane was studied by using this antibody in order to provide theoretical data and technical means for further study on the molecular mechanism of animal erythrocyte immune function. Methods: Gene ious software was used to analyze the bioinformatics characteristics of porcine CR1-like gene cDNA sequence, to synthesize the immune antigen peptide, and to establish murine hybridoma cells secreting porcine CR1-like monoclonal antibody by affinity chromatography. The monoclonal antibody was purified by the technique of medium pressure protein purification, the protein characteristics and purity of the monoclonal antibody were identified by SDS-PAGE method, and the immune activity of the monoclonal antibody was studied by indirect immunofluorescence technique. The distribution of CR1-like membrane in natural porcine red blood cells was observed by laser confocal microscopy, and the immune complex C3b-beads was prepared as a reporter factor. The effect of membrane fluidity on immune adherence of porcine erythrocyte CR1-like was detected by flow cytometry. Results: murine hybridoma cell lines secreting monoclonal antibodies to porcine CR1-like were obtained, and mouse monoclonal antibodies against porcine CR1-like were successfully produced and purified. Green fluorescence was observed on the surface of porcine red blood cells. The distribution of porcine CR1-like was observed by laser confocal microscopy. The fluorescence points on the surface of porcine red blood cells treated without membrane fixation showed granular distribution, which was more obvious than that of membrane fixation group. Flow cytometry showed that the average fluorescence intensity of membrane fixation group was 302.35, that of unfixed group was 638.44, and that of negative control group was 4.7. The statistical results showed that the average fluorescence intensity of membrane fixation group was significantly higher than that of negative control group. The difference was very significant (P 0.01), and the difference was significantly higher in the group without fixation than in the group of membrane fixation, and the difference was very significant (P 0.01). Conclusion: the hybridoma cell line (type 1: CRT-2 CL-5), which can be used to produce monoclonal antibody against porcine CR1-like, was successfully obtained in this study. The monoclonal antibody against porcine CR1-like with biological activity was purified from the hybridoma cell line (type 1: CRT-2 CL-5). The distribution of CR1-like on the surface of porcine red blood cells was dispersed in natural state, and the distribution of CR1-like showed multivalent and efficient binding characteristics, and the distribution of CR1-like was granulocylike cluster distribution. Erythrocyte membrane fluidity is an important physiological basis to affect the immune adherence function of porcine erythrocyte CR1-like.
【学位授予单位】：山西农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.4

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