论文总字数：26905字

摘 要

大肠杆菌由于遗传背景清晰、发酵周期短，操作简单等原因而作为最常用的宿主菌株来进行外源蛋白的表达，但由于其本身存在一些缺陷如缺乏帮助蛋白质折叠的酶或辅助因子，因此导致蛋白错误折叠产生无活性的包涵体聚合物。尽管大量的方法相继产生用以弥补其缺陷，但在大肠杆菌中实现外源目的蛋白的可溶性表达仍存在问题。融合标签是蛋白质表达和纯化的常用工具，同时也是解决大肠杆菌自身缺陷的重要方法。

β-呋喃果糖苷酶（Ffase）具有高效分泌与折叠能力，故推测能将其开发成新型融合标签帮助蛋白折叠。基于本实验室前期研究，将开发的4种新型融合标签与单独在大肠杆菌中以包涵体形式表达的衣原体外膜蛋白抗原决定簇（rabOmp）融合，期望实现其可溶性表达。结果表明，衣原体外膜蛋白抗原决定簇在融合Ffase标签后，目的蛋白含量均增加，其中融合标签Ffase209，将其可溶性表达量提高至0.72g/L, 可溶性蛋白比例提高至81%，而单独表达rabOmp时可溶性蛋白比例不到25%。经镍柱纯化以及肠激酶酶切步骤得到的衣原体外膜蛋白用以药用研究。

关键词： 融合标签 衣原体外膜蛋白 可溶性表达 大肠杆菌

The efficient expression strategy of the Chlamydia pneumoniae out membrane protein

ABSTRACT

Escherichia coli as a simple and an inexpensive choice for recombinant protein production, is widely used, and has become the most popular expression platform. However, some bottlenecks exit when it was established for expressing soluble proteins from other organisms, like poor growth of the host, inclusion body (IB) formation, protein inactivity, and so on. Several efforts have been taken to conquer the limitations，including the use of fusion partner that improves proteins expression and solubility.

Highly efficient secretion was achieved in the expression of β-fructofuranosidase in E.coli, which make it possible to be developed as novel fusion tags. According to early research achievement, Truncated Ffase were highly soluble and secretory proteins, five truncated Ffase were fused to Chlamydia pneumoniae out membrane protein (rabOmp85) in order to promote its solubility in E.coli. The results shows that total expression of rabOmp85 was improved after fusing truncated Ffase fusion tags Ffase209, Ffase217, Ffase243, and Ffase350 to rabOmp, especially Ffase209, 50% of soluble fusion protein was achieved by fusion tag Ffase209 and Ffase217, while less than 25% of soluble protein were achieved without fusion tags. 81% of soluble protein and 0.72 g/L fusion protein were achieved at 30 ℃ after 6h induction by 0.2 g/L lactose, which is 2.5 times as the original condition. Compared to MBP and GST, the new fusion tag Ffase209 performed better to express rabOmp. RabOmp85 can be applied in pharmaceutical research after purification by nickel column and enterokinase digestion.

Key Words: fusion tag Chlamydia pneumoniae out membrane protein solubility Escherichia coli

目 录

摘 要 I

ABSTRACT II

第1章 文献综述 1

1.1 衣原体外膜蛋白简介 1

1.2 大肠杆菌中表达的问题 1

1.3 融合标签技术概述 2

1.3.1 增溶性融合标签 3

1.3.2 纯化型融合标签 5

1.4 本课题研究的意义与内容 7

第2章 Ffase截短体可溶性表达元件的构建 8

2.1 前言 8

2.2 材料与仪器 9

2.2.1 菌株与质粒 9

2.2.2 实验材料 10

2.2.3 主要仪器设备 11

2.2.4 培养基 12

2.3 实验方法 12

2.3.1 融合标签表达载体的构建 12

2.3.2 大肠杆菌感受态制备 14

2.3.3 重组质粒的转化 15

2.3.4 重组子的筛选与测序 15

2.3.5 重组菌的表达和细胞破碎 15

2.3.6 SDS-PAGE蛋白电泳 16

2.3.7 重组子BL21/pET-bff₂₀₉rabomp的诱导剂种类和浓度优化 16

2.3.8 融合标签的去除 16

2.3.9 重组蛋白纯化 16

2.4 结果与讨论 16

2.4.1 Ffase截短体的可溶性预测 16

2.4.2 含预测可溶性较高的Ffase截短体的载体构建 17

2.4.3 融合蛋白表达载体的构建 19

2.4.4 衣原体外膜蛋白抗原决定簇的融合表达 20

2.4.5 重组子BL21/pET-bff₂₀₉rabomp的诱导剂种类和浓度优化 21

2.4.6 重组蛋白bff₂₀₉rabomp的纯化 23

2.5 本章小结 23

第3章 结论与展望 25

3.1 结论 25

3.2 展望 25

参考文献 26

致 谢 28

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