免疫重組DNA技术.pptVIP

Recombinant DNA Technology; Identification of recombinant clones ;相关帮助; 1. Screening ;1. Insertion inactivation;pBR322;（1）Tetracyclin (四环素)：;如果在Tetr上插入外源DNA，导致四环素抗性基因失活，可用四环素加环丝氨酸平板培养基选择重组克隆。;;（2）氨苄青霉素抗性插入失活选择过程;2. alpha-complementation;?-半乳糖苷酶;Procudure:;;利用插入的外源基因的表达产物特性进行直接选择。（只在特定条件下）。 ;小鼠的二轻叶酸还原酶（DHFR）对三甲氧苄二氨嘧啶有抗性。;4. Hybridization;Hybridization to identify the interested DNA or its RNA product Radiolabeled probes which is complementary to a region of the interested gene Probes: An oligonucleotide derived from the sequence of a protein product of the gene A DNA fragment/oligo from a related gene of another species Blotting the DNA or RNA on a membrane Hybridize the labeled probe with DNA membrane (Southern) or RNA (Northern) membrane;;;; A Southern blot result;Very easy to identify the positive clone;;4.3 Northern blot;2. Electrophoresis analysis enzymatic digestion PCR reaction;Electrophoresis analysis is based on the principal that the molecular weight of recombinant vector is larger than the wild one.;2.2 Analysis after digestion;Before digestion;;;Example of an electrophoresis gel after digestion;筛选过程;2.3 PCR analysis; The polymerase chain reaction(PCR) is to used to amplify a sequence of DNA using a pair of primers each complementary to one end of the the DNA target sequence. ;The PCR cycle;Template;Template;Primers;Enzymes and PCR Optimization;Selection by immunochemistry technology ;利用抗体作为“探针”来检测转入受体菌并且表达出相应的蛋白质的外源基因。;待测基因 产物蛋白;2. 放射性抗体检测法过程;二、免疫沉淀检测法;;三、酶联免疫吸附测定（ELISA）;待测基因 产物蛋白;2. ELISA检测的一般步骤;加入一抗，反应后冲洗掉未结合的抗体。;加入二抗，与一抗结合后再将未结合的二抗冲洗掉。二抗只识别一抗。;加入无色的底物，被二抗上所带的酶催化反应转变成有色物质（或发光）。;;在特殊的分光光度仪（酶标仪）上比色，打印出结果。;3.ELISA的局限性;四、免疫印迹（western blot）法;② 聚丙烯酰胺凝胶电泳（PAGE）：;点样;③蛋白质电泳的分子量标准;;④凝胶中的蛋白???染色：;直接染色电泳结果;（2）Western blotting;Western装置;② Blot;;1）先用一抗（待测蛋白的单克隆抗体）与 膜上的蛋白结合。;④结果;4. DNA sequencing;DNA sequencing;;32pGpCpTpGpCpTpApGpGpTpGpCpCpGpApGpC;Sanger sequencing;This figure is a representation of an acrylamide sequencing gel. Notice that the sequence