论文总字数：18609字

摘 要

衣原体外膜蛋白抗原决定簇rabOmp蛋白序列长为216个氨基酸，分子量23.8kDa，抗原决定簇大多存在于抗原物质的表面，有些存在于抗原物质的内部，须经酶或其他方式处理后才暴露出来，在大肠杆菌中通常以包涵体的形式表达，要通过复杂的复性和纯化才能获得。为避免复杂的复性过程，将rabOmp融合本研究开发的新型増溶性融合标签，期望最大程度的获得可溶性表达的融合蛋白，以pET-ff53bff为模板对目的基因rabOmp进行PCR扩增，PCR产物进行回收后，对实验室所构建的带有截短体Ffase209，Ffase217，Ffase243，Ffase350的融合标签的pET-28a载体分别进行Nde1、Xco1双酶切，得到重组表达载体。然后与目的基因相连接，得到重组质粒，然后转化至E.coli BL21感受态细胞，挑选得到的重组子使用相应的引物进行菌落PCR鉴定。产物进行电泳分析，结果证明目的基因和重组载体成功连接，而且带有Ffase209的融合标签在一定程度上可以最好地提高目的蛋白表达量。

关键词：抗原决定簇 克隆表达 融合标签 重组蛋白

Cloning and expression of the outer membrane protein antigen determinant RabOmp of Chlamydia pneumoniae in Escherichia coli

Abstract

The sequence of the Chlamydia outer membrane protein antigen determinant was 216 amino acids, and the molecular weight was 23.8kDa,Antigenic determinants are on the surface of antigen material, some inside an antigenic substance, must be treated by enzymes or other way after exposed, in Escherichia coli, usually in the form of inclusion body expression to through complex refolding and purification to obtain.In order to avoid the complex process, rabOmp fusion the research and development of new increasing water solubility of fusion tags,to pet-ff53bff as template of target gene rabOmp PCR amplification, PCR products were recovered after that of laboratory construction with truncated Ffase209, Ffase217, Ffase243, pET28a vector Ffase350 fusion tags respectively for NDE 1 and Xho 1 double enzyme digestion. Then with the objective gene connected to obtain the recombinant plasmid, and then transformed into E.coli BL21 competent cells, the selection of recombinants using corresponding primers identified by colony PCR. Finally the optimal recombinant bacteria IPTG was added to induce the expression of protein.The results of electrophoresis analysis showed that the target gene was successfully amplified and the recombinant vector was successfully connected, and the fusion tag with Ffase209 could be improved to a certain extent.

Key Words: Antigenic determinant；Cloning and expression；Fusion tag；Recombinant protein

目 录

摘要 Ⅰ

ABSTRACT Ⅱ

第一章 文献综述 1

1.1抗原决定簇 1

1.1.1抗原决定簇简介 1

1.1.2抗原决定簇分类及应用 1

1.1.3衣原体外膜蛋白抗原决定簇 2

1.2 克隆表达 2

1.2.1融合标签概述 2

1.2.2融合标签在研究中的应用 3

1.3本课题研究意义和研究内容 4

第二章 衣原体外膜蛋白抗原决定簇在大肠杆菌中的克隆表达 5

2.1实验材料 5

2.1.1 菌株与质粒 5

2.1.2 实验试剂 5

2.1.3 主要设备及仪器 6

2.1.4培养基 7

2.2 实验方法 8

2.2.1 质粒的提取 8

2.2.2目的基因的PCR扩增 9

2.2.3 目的基因和载体的连接 9

2.2.4大肠杆菌感受态的制备及转化感受态 10

2.2.5 菌落PCR鉴定 11

2.2.6 重组蛋白在大肠杆菌中的表达 11

2.2.7 SDS-PAGE 聚丙烯酰胺凝胶电泳 11

2.3结果与讨论 13

2.3.1 目的基因扩增结果 13

2.3.2 质粒载体的构建 13

2.3.3 菌落PCR鉴定 14

2.3.4 SDS-PAGE分析 15

第三章 结论与展望 16

3.1 结论 16

3.2 展望 16

参考文献 17

致谢 20

1. 文献综述

1.1抗原决定簇

1.1.1抗原决定簇简介

请支付后下载全文，论文总字数：18609字