摘要OmpF是大肠杆菌最重要的膜孔蛋白之一，也是目前研究的最清楚的蛋白质之一。它横跨脂质双分子层而形成一个离子通道，这个通道是大肠杆菌与外界进行物质交换的重要渠道，小分子量的水溶性物质也能通过该离子通道穿越外膜。OmpF膜孔蛋白与大肠杆菌许多生理活动密切相关，因此，研究OmpF膜孔蛋白的结构可以对后续膜孔蛋白与抗生素的结合机理提供理论基础。

本文主要采用基因重组技术，在大肠杆菌基因组中通过PCR技术扩增到目的基因片段，对PCR产物进行纯化后，先用TA克隆得到含有目的序列的质粒，再通过酶切酶连技术构建OmpF表达载体，并转化到大肠杆菌表达菌株BL21（DE3）中获得OmpF的表达株。IPTG诱导小量培养后利用SDS-PAGE检测OmpF的表达情况，优化条件进行大量表达分离纯化OmpF 蛋白，为后续的质谱分析奠定基础。79537

毕业论文关键词  OmpF  载体构建  蛋白表达与纯化  质谱分析

毕业设计说明书外文摘要

Title        Expression and mass spectrometry analysis of  membrane pore protein（OmpF）           

Abstract OmpF is one of the most important membrane pore proteins in Escherichia coli and one of proteins that we know the structure clearly。 OmpF protein crosses the lipid bilayer and forms ion channels。 The channel is so important that Escherichia coli exchange materials with the extracellular membranes and small molecular weight of water-soluble substances can pass through the ion channels from the outer membrane。 Many physiological activities are closely related with OmpF membrane protein in Escherichia coli。 Therefore, the study of OmpF protein structure can provide a theoretical basis of the mechanism of membrane protein binding with antibiotic。

Gene recombination technique were used in this paper to extract OmpF gene fragments from the genome in Escherichia coli and finally got OmpF protein。 We used PCR technique to amplify gene fragment。 The PCR products were purified, followed with TA connection processing to form a kind of plasmid containing OmpF target gene fragment。 The OmpF expression vector was constructed by enzyme digestion and enzyme linking technique。 Small quantity of expression was done to clarify the gene expression by SDS-PAGE。 OmpF protein was expressed and purified used for mass spectrometry analysis。

Keywords  OmpF,  Vector construction,  Protein expression and purification,  Mass spectrometry 

目   次

1  引言 1

1。1  生物膜介绍 1

1。2  膜蛋白研究现状 1

1。3  膜蛋白OmpF表达载体的构建 1

1。4  OmpF表达纯化与质谱分析 2

2 仪器和材料 3

2。1 仪器 3

2。2 材料 3

3 实验过程 7

3。1 引物设计 7

3。2 基因组提取 8

3。3 质粒小提 8

3。4 PCR 10

3。5 酶切 11

3。6 凝胶回收 12

3。7 酶连 14

3。8 TA克隆 15

结  论 21

致  谢