EGFP在大肠杆菌Ecoli中的表达与检测.pptxVIP

EGFP在E.coli中的表达与检测 3组contents一背景介绍二实验流程三实验步骤四预期结果 五参考文献第几章一、背景介绍1、绿色荧光蛋白（GFP）2、质粒3、大肠杆菌表达载体及表达系统4、SDS原理及蛋白分离与检测一绿色荧光蛋白1 -1.1发现下村修马丁-查尔菲钱学健发现GFP科研发光的遗传标签一绿色荧光蛋白（GFP）1 -1.1发现一绿色荧光蛋白（GFP）1 -1.2特性GFP 的分子质量为26 kDa，晶体结构如左图所示。蛋白质中央是一个圆柱形水桶样结构， 长420 nm，宽240 nm，由11 个围绕中心α螺旋的反平行β折叠组成，荧光基团的形成就是从该螺旋开始，桶的顶部由3 个短的垂直片段覆盖，底部由一个短的垂直片段覆盖，对荧光活性很重要的生色团则位于大空腔内。发色团是由其蛋白质内部第65～67位的“丝氨酸-酪氨酸-甘氨酸” 自身环化和氧化形成。一绿色荧光蛋白1 -1.2特性GFP在紫外光激发下发绿色荧光的最高吸收峰为395 nm，另一小吸收峰为470 nm，最大发射峰为505 nm。一绿色荧光蛋白1 -1.2特性优点：易于检测，荧光稳定，无毒害，通用性，易于构建载体，活细胞观察一绿色荧光蛋白1 -1.3应用一质粒2 -2.1pEGFP-N3质粒一质粒2 -2.1.1pEGFP-N3质粒描述pEGFP-N3 encodes a red-shifted variant of wild-type GFP (1–3) which has been optimized for brighter fluorescence and higher expression in mammalian cells. (Excitation maximum = 488 nm; emission maximum = 507 nm.) pEGFP-N3 encodes the GFPmut1 variant (4) which contains the double-amino-acid substitution of Phe-64 to Leu and Ser-65 to Thr. The coding sequence of the EGFP gene contains more than 190 silent base changes which correspond to human codon-usage preferences (5). Sequences flanking EGFP have been converted to a Kozak consensus translation initiation site (6) to further increase the translation efficiency in eukaryotic cells. The MCS in pEGFP-N3 is between the immediate early promoter of CMV (PCMV IE) and the EGFP coding sequences. Genes cloned into the MCS will be expressed as fusions to the N terminus of EGFP if they are in the same reading frame as EGFP and there are no intervening stop codons. SV40 polyadenylation signals downstream of the EGFP gene direct proper processing of the 3 end of the EGFP mRNA. The vector backbone also contains an SV40 origin for replication in mammalian cells expressing the SV40 T-antigen. A neomycin resistance cassette (Neor), consisting of the SV40 early promoter, the neomycin/kanamycin resistance gene of Tn5, and polyadenylation signals from the Herpes simplex virus thymidine kinase (HSV TK) gene, allows stably transfected eukaryotic cells to be selected using G418. A bacteri