天然免疫相关蛋白分子SPLUNC1的表达和抗体制备

发布时间：2018-06-18 18:08

本文选题：短的上腭、肺及鼻咽上皮克隆1(SPLUNC1) + 定点突变　；参考：《北京市结核病胸部肿瘤研究所》2010年硕士论文

【摘要】： 目的:真核表达SPLUNC1蛋白和制备鉴定SPLUNC1单克隆抗体内容:1.SPLUNC1真核表达载体的构建;2.m-SPLUNC1-Ig和h-SPLUNC1-Ig融合蛋白的表达及SPLUNC1蛋白的体外活性分析;3.SPLUNC1单克隆抗体的制备和鉴定分析。方法:从肺腺癌组织中扩增SPLUNC1基因,利用大引物法和巢式PCR实现基因定点突变,顺利构建PDH3-SPLUNC1的表达载体,同时构建PDM3-SPLUNC1的表达载体。利用Lipofectamine2000转染CHO/dhfr-细胞,利用二氢叶酸还原酶基因选择系统加用透析血清及MTX进行筛选分别获得持续表达m-SPLUNC1-Ig和h-SPLUNC1-Ig融合蛋白的细胞株。采用ELISA和免疫印迹实验分析h-SPLUNC1-Ig融合蛋白与LPS结合,了解h-SPLUNC1-Ig体外的生物活性。采用常规的免疫方法免疫Balb/c小鼠,取效价最高的Balb/c小鼠行下一步融合实验,利用有限稀释法获得稳定分泌SPLUNC1抗体的杂交瘤,分析其亲和力、亚类、表位以及与天然蛋白结合情况。结果:顺利完成了基因的定点突变,实现了PDH3-SPLUNC1和PDM3-SPLUNC1两种表达载体的构建,同时获得了一种无需切胶纯化的基因定点突变方法;获得了分别表达m-SPLUNC1-Ig和h-SPLUNC1-Ig两种融合蛋白的稳定表达株,能够持续稳定地表达融合蛋白,体外活性分析发现h-SPLUNC1-Ig融合蛋白具有体外活性,能够与革兰氏阴性菌细胞壁的脂多糖成分结合;单抗制备过程中获得了6株单克隆抗体,亚类均为IgG1,能够与天然SPLUNC1蛋白结合。结论:获得了具有体外活性的SPLUNC1融合蛋白和SPLUNC1单克隆抗体,能够与天然的SPLUNC1蛋白结合。
[Abstract]:Aim: to express SPLUNC1 protein and to prepare and identify SPLUNC1 monoclonal antibody. 1. Construction of Eukaryotic expression Vector of SPLUNC1. Expression of 2.m-SPLUNC1-Ig and h-SPLUNC1-Ig Fusion protein and activity Analysis of SPLUNC1 protein in Vitro. 3. Preparation and characterization of monoclonal antibody against SPLUNC1. Methods: SPL1 gene was amplified from lung adenocarcinoma tissue. The expression vector of PDH3-SPLUNC1 was successfully constructed by using large primer method and nested PCR to construct the expression vector of PDH3-SPL1, and the expression vector of PDM3-SPL1 was constructed at the same time. The cell lines expressing m-SPLUNC1-Ig and h-SPLUNC1-Ig fusion proteins were obtained by transfection of CHO / dhfr- cells with Lipofectamine2000, dihydrofolate reductase gene selection system and dialysis serum and MTX, respectively. The binding of h-SPL1-Ig fusion protein to LPS was analyzed by Elisa and Western blotting to study the bioactivity of h-SPL1-Ig in vitro. Balb / c mice were immunized with routine immune methods. Balb / c mice with the highest titer were selected for the next fusion experiment. Hybridomas secreting the antibody against SPLUNC1 were obtained by finite dilution method, and their affinity and subclasses were analyzed. Epitopes and their binding to natural proteins. Results: the site-directed mutation of the gene was successfully completed and the expression vectors PDH3-SPLUNC1 and PDM3-SPLUNC1 were constructed. At the same time, a method of site-directed mutation was obtained. The stable expression strains of m-SPLUNC1-Ig and h-SPLUNC1-Ig were obtained, which can express the fusion protein continuously and stably. The in vitro activity analysis of h-SPLUNC1-Ig fusion protein showed that h-SPLUNC1-Ig fusion protein had in vitro activity. It could bind to lipopolysaccharide from the cell wall of Gram-negative bacteria, and six monoclonal antibodies were obtained during the preparation of monoclonal antibodies, all of which were IgG1, which could bind to natural SPLUNC1 protein. Conclusion: the fusion protein of SPLUNC1 and the monoclonal antibody against SPLUNC1 have been obtained, which can bind to the natural SPLUNC1 protein.
【学位授予单位】：北京市结核病胸部肿瘤研究所
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392.1

【参考文献】