188宝金博页面版

华中农业大学 08 届硕士学位论文 2.4.2 单抗的特异性和结合活性........................................................................43 2.4.3 单抗对溶血活性的抑制............................................................................44 第三章 溶血素单抗 1A11 识别表位的研究 ...................................................................45 3.1 材料......................................................................................................................45 3.1.1 材料............................................................................................................45 3.1.2 主要试剂....................................................................................................45 3.1.3 主要试剂配制............................................................................................45 3.2 方法......................................................................................................................47 3.2.1 单克隆抗体腹水的纯化............................................................................47 3.2.2 抗体浓度的测定........................................................................................47 3.2.3 噬菌体肽库的亲和筛选............................................................................47 3.2.4 阳性噬菌体外源DNA序列分析 ...............................................................48 3.3 结果......................................................................................................................49 3.3.1 单抗 1A11 的提纯及检测 .........................................................................49 3.3.2 噬菌体肽库淘筛过程中的富集................................................................50 3.3.3 噬菌体结合活性分析................................................................................51 3.3.4 阳性噬菌体的测序和分析........................................................................51 3.4 讨论......................................................................................................................53 第四章 溶血素抗原表位氨基酸的缺失研究..................................................................55 4.1 材料......................................................................................................................55 4.1.1 质粒及菌株................................................................................................55 4.1.2 材料............................................................................................................55 4.2 缺失溶血素抗原表位氨基酸..............................................................................55 4.2.1 缺失基因引物序列....................................................................................55 4.2.2 PCR反应体系 .............................................................................................56 4.2.3 反应程序....................................................................................................57 4.2.4 缺失突变蛋白的表达................................................................................57 4.2.5 缺失突变蛋白溶血活性的鉴定................................................................57 4.2.6 缺失突变蛋白免疫原性检测....................................................................57 4.2.7 缺失突变蛋白与 1A11 单抗结合活性的检测 .........................................57 4.3 结果......................................................................................................................58 4.3.1 缺失SLY抗原表位氨基酸的克隆 ............................................................58 4.3.2 缺失突变表达产物的SDS-PAGE分析.....................................................59 4.3.3 缺失突变蛋白的溶血活性检测................................................................60 4.3.4 缺失突变蛋白Western blot分析 ...............................................................61 4.4 讨论......................................................................................................................62 本研究的主要结果和创新点............................................................................................64 参考文献............................................................................................................................65 致谢....................................................................................................................................72 2