A型口蹄疫病毒1D蛋白在大肠埃希菌中的可溶性表达、纯化及电子显微镜检测

doi:10.16098/j.issn.0529-1356.2017.06.016

摘要/Abstract

摘要：

目的本实验旨在表达出高可溶性的A型口蹄疫病毒1D蛋白，并通过电子显微镜检测，以期望形成纳米样颗粒。方法根据A型口蹄疫病毒核酸序列，得到FMDV A/GDMM/CHA/2013株的1D蛋白基因，并进行截短和优化，共132个氨基酸；同时，从结肠弯曲杆菌(Campylobacter coli）中分离得到135个氨基酸的铁蛋白(Fn)基因片段，将A型口蹄疫病毒1D蛋白与铁蛋白串联，设计并合成了口蹄疫病毒1D蛋白-铁蛋白片段，命名为CcFnt166AS。构建了CcFnt166AS融合Grifin、GST、MBP、Sumo、Thioredoxin、γ-crystallin、ArsC、PpiB、CeHSP17等9种不同可溶性标签的表达重组载体。分别转化至大肠埃希菌BL21(DE3)中，异丙基硫代半乳糖苷（IPTG）诱导，SDS-PAGE电泳对融合蛋白的可溶性表达进行检测，筛选出高可溶性表达的CcFnt166AS融合蛋白。重组蛋白通过Ni-NTA Agarose亲和纯化，进行电子显微镜检测。结果成功构建9种CcFnt166AS表达载体；9个标签中，MBP与CcFnt166AS蛋白相融合的可溶表达效果最好，并获得了高纯度的MBP-CcFnt166AS重组蛋白质；电子显微镜结果显示，MBP-CcFnt166AS形成了纳米样颗粒。结论本实验建立了稳定获得CcFnt166AS重组蛋白质的方法。

关键词: A型口蹄疫病毒, 1D蛋白, 铁蛋白, 融合标签, 可溶性, 纳米颗粒, 电子显微术, 大肠埃希菌

Abstract:

Objective To establish a method to express high soluble 1D protein from foot-and-mouth disease virus type A in Escherichia coli(E. coli). Methods Based on the nucleic acid sequences of the foot-and-mouth disease virus type A, the 1D protein genes of FMDV A/GDMM/CHA/2013 was obtained. Truncation and optimization were performed. The fusion tags were screened in order to obtain soluble expression of CcFnt166AS protein. The 135 aminoacid ferritin protein was isolated from Campylobacter coli and linked with the 1D protein, which was named as CcFnt166AS. We constructed prokaryotic expression vectors fused with nine different fusion tags (Grifin, GST, MBP, Sumo, Thioredoxin, γ-crystallin, ArsC, PpiB, CeHSP17)fragments, these recombinant plasmids were transformed into Escherichia coli BL21(DE3) and induced by isopropyl β-D-thiogalactoside(IPTG). SDS-PAGE analysis was used to identify the soluble expression of recombinant protein after ultrasonic decomposition. The fusion tag that could significantly promote soluble expression of CcFnt166AS protein was selected. Abundantly inducible expression recombinant protein was purified by Ni-NTA purification sysm and detected by electron microscopy. Results We successfully constructed the recombinant prokaryotic expression plasmid and obtained the high purity MBP-CcFnt166AS. The result of electron microscopy showed that MBP-CcFnt166AS formed nano-sized particles. Conclusion These result suggested that we established a method to generate CcFnt166AS recombinant protein, which may lay a foundation for the development and subsequent study of FMDV structure vaccine.

Key words: A-type foot-and-mouth disease virus, 1D protein, Ferritin, Fusion tag, solubility, Nanoparticle, Electron microscopy, Eschericha coli

郭玉堃明胜利郭婉莹杨国宇郭豫杰. A型口蹄疫病毒1D蛋白在大肠埃希菌中的可溶性表达、纯化及电子显微镜检测[J]. 解剖学报, 2017, 48(6): 726-731.

GUO Yu-kun MING Sheng-li GUO Wan-ying YANG Guo-yu GUO Yu-jie. Soluble expression, purification and electron microscopic detection of 1D protein from A-type foot-and-mouth disease virus in Escherichia coli[J]. Acta Anatomica Sinica, 2017, 48(6): 726-731.

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