在大肠杆菌 BL21(DE3)中表达和纯化破伤风毒素片段 C。

Expression and Purification of Tetanus Toxin Fragment C in Escherichia coli BL21(DE3).

机构信息

School of Life Science and Engineering, Lanzhou University of Technology, Lanzhou 730050, China

Key Laboratory of Drug Screening and Deep Processing for Traditional Chinese and Tibetan Medicine of Gansu Province, Lanzhou 730050, China

出版信息

Protein Pept Lett. 2020;27(11):1132-1140. doi: 10.2174/0929866527666200528113327.

DOI:10.2174/0929866527666200528113327

PMID:32484098

Abstract

BACKGROUND

Tetanus is an infectious disease caused by Clostridium secreting tetanus toxin in anaerobic environment. The fragment C of Tetanus toxin (TTc) has been widely studied as a candidate vaccine to replace the existing tetanus toxoid vaccine.

OBJECTIVE

In this study, we established a simple method to purify recombinant protein TTc with ion-exchange chromatography from Escherichia coli expression systems.

METHODS

The TTc gene sequence was cloned into pET26b (+) vector and transferred to E. coli BL21 (DE3) for expression. The fermentation conditions (IPTG concentration, Induction temperature, Induction time) were optimized to obtain more soluble proteins. The soluble proteins were purified by Anion exchange chromatography and Cation exchange chromatography. The sequence of columns in the purification process was discussed. Finally, the stability of purified TTc protein were determined, the secondary structure of the purified TTc protein was determined by circular dichroism. The molecular weight of the purified TTc protein was determined by liquid chromatograph- mass spectrometer. Furthermore, we verified the immunogenicity of the purified protein in mice.

RESULTS

The purity of TTc improved from 34% to 88% after the first anion exchange column, and the final yield of recombinant TTc (purity > 95%) can reach 84.79% after the following cation exchange chromatography. The recombinant TTc had a molecular weight of 51.737 KDa, was stable at 4 °C and weak alkaline environment, was a β-sheet secondary structure, and had strong immunogenicity.

CONCLUSION

The purification method we developed might be an efficient method for the industrial production of tetanus recombinant TTc vaccine.

摘要

背景

破伤风是一种由在厌氧环境下分泌破伤风毒素的梭菌引起的传染病。破伤风毒素的 C 片段（TTc）已被广泛研究作为替代现有破伤风类毒素疫苗的候选疫苗。

目的

本研究旨在建立一种从大肠杆菌表达系统中用离子交换层析法纯化重组蛋白 TTc 的简单方法。

方法

将 TTc 基因序列克隆到 pET26b（+）载体中，并转移到大肠杆菌 BL21（DE3）中进行表达。优化发酵条件（IPTG 浓度、诱导温度、诱导时间）以获得更多可溶性蛋白。用阴离子交换层析和阳离子交换层析纯化可溶性蛋白。讨论了纯化过程中柱子的顺序。最后，测定了纯化 TTc 蛋白的稳定性，用圆二色性测定了纯化 TTc 蛋白的二级结构。用液相色谱-质谱法测定了纯化 TTc 蛋白的分子量。此外，我们在小鼠中验证了纯化蛋白的免疫原性。