03基因工程載體.ppt

第三章 基因工程载体;基因克隆载体;载体的功能及特征;载体的功能;载体应具备的条件;质粒;第一节 质粒克隆载体;一、 质粒的一般生物学特性;一、质粒的一般生物学特性;质粒DNA的复制;质粒的自主复制性：拷贝数的控制机制 – 质粒DNA复制启动控制;质粒的不亲和性;质粒转移;携带特殊的遗传标记;Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile 5.2.0.0. Copyright 2004-2011 Aspose Pty Ltd.;二、构建质粒克隆载体的基本策略;三、质粒克隆载体的构建;四、常用质粒载体类型;1、克隆质粒载体;pBR322质粒;CAP protein binding site - 591-554 (compl. strand); mRNA (LacZ) starts at nt position 507 (compl. strand); lac repressor binding site - 507-487 (compl. strand). ;pBR322插入失活效应;筛选重组子的示意图; BamHI片段(Tcr) ;pUC系列的质粒载体;Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile 5.2.0.0. Copyright 2004-2011 Aspose Pty Ltd.;Multiple Cloning Sites pUC18;Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile 5.2.0.0. Copyright 2004-2011 Aspose Pty Ltd.;pUC系列质粒载体的优点 ;pUC18 / 19：正选择标记 lacZ’ 的显色原理;利用菌落颜色筛选重组子;2、表达质粒载体;表达载体与克隆载体的区别;表达型载体（expression vector）;表达型载体（expression vector）;三种表达型载体结构图;表达型载体（expression vector）;3、多功能质粒载体;pGEM系列载体 ;pGEM3Z载体;Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile 5.2.0.0. Copyright 2004-2011 Aspose Pty Ltd.;2743 bp;Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile 5.2.0.0. Copyright 2004-2011 Aspose Pty Ltd.;1、克隆载体，全长3.19kb，具有Amp抗性基因和复制起始点 2、MCS两侧有SP6、T7RNA聚合酶启动子---可进行体外转录；为插入片段的测序提供特异引物位点 3、MCS为于lacZ’基因内部（插入失活）—蓝白斑筛选 4、引入丝状噬菌体f1(+) 复制起始点-----可以产生单链DNA，并分泌至上清中，;Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile 5.2.0.0. Copyright 2004-2011 Aspose Pty Ltd.;Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile 5.2.0.0. Copyright 2004-2011 Aspose Pty Ltd.;分泌型融合表达载体，有两个启动子（Plac,Pspa），启动子下游装有SPA的信号肽序列和两个结构基因ZZ。产生与SPA蛋白ZZ段融合的蛋白，并可将融合蛋白分泌出胞 4.59kb，含Amp抗性基因和原核复制起始点 有f1复制起始点，可产生单链DNA;4、穿梭质粒载体;Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile 5.2.0.0. Copyright 2004-2011 Aspose Pty Ltd.;穿梭质粒;第二节 λ噬菌体载体;一、 λ噬菌体的生物学特性;1、λ噬菌体的生活周期;Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile 5.2.0.0. Cop