基因工程-6-重组DNA构建与筛选复习课程.ppt

（2）DEAE-葡萄糖转染技术 （3）电穿孔转染技术 （4）脂质体载体法 （5）细胞核的显微注射法 三、重组DNA导入动物细胞的方法 （1）磷酸钙转染技术 （6）活体电穿孔技术 活体电穿孔法(in vivo electroporation) 是将外源基因通过电场作用，导入动物目标组织或器官。 活体电穿孔法的特点： 靶器官的选择面广 对导入的外源基因片段的大小没有限制,从几KB 或十几KB 的表达载体, 到100～200KB 的YAC、BAC 基因组,都有成功导入并获得表达的报道。 活体电穿孔法操作简单快速,电穿孔的时间只有几秒钟,而且DNA片段不需要特殊的纯化操作。 研究实例一 杀蚊毒素在大肠杆菌中的异源表达 The toxin is composed of two proteins, BinA and BinB, with approximate molecular weights of 42 and 51 kDa, respectively. The two proteins require each other for full activity, and maximum activity is obtained when both components are present in equimolar ratio. 1.背景介绍 2.外源基因的获得 PCR引物： BinA BS51F; 5’-CGA AGC TTG TGC GAT TCA AAA GAC AAT-3’ BS51R; 5’-CCT CGA GAA TGA ATA TTT GAA TTT AAA GAG-3’ BinB BS42F; 5’-GAA GCT TGA GAA ATT TGG ATT TTA TTG ATT-3’ BS42R; 5’-GGC GGT CGA CTT AGT TTT GAT CAT CTG TAA TA-3’ The restriction sites added at the 5’ end of these primers (HindIII in BS42F and BS51F, SalI in BS42R, and XhoI in BS51R) are underlined. Genes encoding BinA and BinB were amplified from plasmid pET-42f and pET-51f Fig Schematic diagram of pGEX-BinA and pGEX-BinB. Polymerase chain reaction (PCR) products of binA and binB genes were digested with HindIII at the 5’ end and made blunt ended using Klenow polymerase. The 3’ end of binA was digested with SalI, whereas XhoI was used for binB. The digested fragments then were ligated to pGEX-4T-2 between SmaI and XhoI sites. 2.重组DNA构建 3.重组子筛选与表达 Escherichia coli JM109 containing pGEX-BinA or pGEXBinB was grown in Luria-Bertani (LB) broth containing 100 lg of ampicillin/ml until OD600 reached 0.4. To induce production of the toxin, 1 mmol/l isopropyl-b-D-thiogalactopyranoside (IPTG) was added, and the culture was further grown for 5 h. The induced culture then was collected and analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS). 4.表达产物活性测试 第四节 重组DNA分子的筛选 外源基因与载体连接后转化大肠杆菌可能的三种情况： （1） （2） （3） 转化 E.coli 一、重组体的筛选 遗传检测方法 抗药性标记插入失活 β-半乳糖苷酶显色反应 插入序列表型特征 免疫化学方法 免疫沉淀法 放射性抗体检测 核酸杂交方法 转译筛选法 （