铁蛋白Ferritin原核表达和纯化及纳米颗粒胞外自组装

doi:10.11843/j.issn.0366-6964.2018.01.009

摘要/Abstract

摘要：

旨在使用原核表达系统表达幽门螺杆菌铁蛋白Ferritin，获得具有天然纳米结构的铁蛋白纳米颗粒，从而通过表面修饰等方法应用于疾病治疗和抗原呈递等研究。将铁蛋白氨基酸序列按照大肠杆菌常用密码子进行优化，合成重组铁蛋白基因并克隆至原核表达载体。使用镍柱进行亲和层析纯化重组蛋白，并在透析复性后利用透射电子显微镜对体外自组装纳米颗粒进行鉴定。结果显示，本研究成功构建了pET-32a-Ferritin原核表达载体，并使用IPTG进行诱导表达获得His-Ferritin重组铁蛋白，且主要以包涵体形式存在。通过镍柱进行亲和层析纯化获得纯度为96%的重组蛋白，使用脱盐柱将重组蛋白梯度透析至2 mol·L^-1尿素缓冲液中进行复性后，使用电子显微镜观察胞外自组装重组蛋白为粒径在12~20 nm的均一的纳米结构，与天然铁蛋白纳米粒子类似。本研究成功建立了Ferritin蛋白原核表达体系，通过诱导表达、亲和层析纯化及复性获得了具有天然纳米结构的铁蛋白纳米颗粒，为其在生物医药领域的应用奠定基础。

关键词: 铁蛋白, 原核表达, 亲和层析, 胞外自组装

Abstract:

This study aimed to express Helicobacter Pylori nonheme iron-containing Ferritin protein in Escherichia coli, and obtain the Ferritin nano-particles with natural nanostructure, for the application in disease and antigen presentation study by surface modification. To improve the expression of Ferritin, Ferritin gene was synthesized according to the Escherichia coli biased codon and cloned to the pET-32a vector. The recombinant protein was purified by affinity chromatography using His-trap Ni²⁺ column, and refold by gradient dialysis. The refolding protein was detected by transmission electron microscope to identify the nano-structure of the self-assembling protein. The results showed that the prokaryotic expression vector pET-32a-Ferritin was successfully constructed, and the recombinant His-Ferritin protein was expressed after induced by IPTG. The recombinant Ferritin protein was mainly expressed in the form of inclusion body. By use of His-trap Ni²⁺ affinity chromatography column, we obtained the recombinant Ferritin protein with 96% purity. The purified protein was in gradient dialysis to 2 mol·L^-1 urea buffer with a desalting column to obtain the refold protein. The refold protein had a nanostructure with the diameter of 12-20 nm under the transmission electron microscope, which was similar to the natural nanoparticles. In this study, the prokaryotic expression system of Ferritin was successfully established and the recombinant protein with nanostructure was prepared by induction expression, affinity chromatography purification and refolding. This study lays the foundation for the application of Ferritin in biomedical field.

Key words: Ferritin, prokaryotic expression, affinity chromatography, self-assembling in vitro

中图分类号:

Q812

李志鹏, 刘福航, 崔奎青, 石德顺, 刘庆友. 铁蛋白Ferritin原核表达和纯化及纳米颗粒胞外自组装[J]. 畜牧兽医学报, 2018, 49(1): 75-82.

LI Zhi-peng, LIU Fu-hang, CUI Kui-qing, SHI De-shun, LIU Qing-you. Prokaryotic Expression and Purification of Ferritin and Nano-particles Self-assembling in vitro[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(1): 75-82.

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