铁蛋白与口蹄疫病毒VP1在大肠杆菌中融合表达及纳米颗粒自组装

doi:10.13560/j.cnki.biotech.bull.1985.2020-0758

摘要/Abstract

摘要：

口蹄疫是由口蹄疫病毒引起的世界上最重要的畜牧疾病之一,严重影响世界畜牧业的发展,而疫苗免疫仍然是对疫情预防和控制的最有效手段。铁蛋白具有自组装和生物修饰的特性,在纳米疫苗等领域具有广阔的应用前景。选用O型口蹄疫病毒的vp1基因和幽门螺杆菌铁蛋白基因,通过融合PCR将vp1基因构建到铁蛋白亚基基因前端,在大肠杆菌中表达后通过His标签进行镍柱亲和层析纯化。将纯化好的重组蛋白进行Western blotting检测、质谱分析和透射电镜观察,发现重组蛋白VP1-Ferritin在大肠杆菌中获得表达,并可自组装成纳米颗粒。

关键词: 口蹄疫病毒, 铁蛋白, 纳米颗粒, 自组装

Abstract:

Foot-and-mouth disease is one of the most important livestock diseases in the world caused by foot-and-mouth disease virus,which seriously affects the development of animal husbandry in the world,and vaccine immunization is still the most effective means for the prevention and control of the epidemic. Ferritin has the characteristics of self-assembly and biological modification,and has broad application prospects in the field of nano vaccines. In this study,the vp1 gene of type O foot-and-mouth disease virus and the Helicobacter pylori ferritin gene were selected for nanoparticles assembly. The vp1 gene was fused to ferritin subunit gene by overlap PCR. After being expressed in Escherichia coli,it was purified by nickel column affinity chromatography by His Tag. The purified recombinant protein was then subjected to Western blotting detection,mass spectrometry analysis and transmission electron microscopy observations. It was found that the recombinant protein VP1-Ferritin was expressed in E. coli and self-assembled into nanoparticles.

Key words: foot-and-mouth disease virus, ferritin, nanoparticles, self-assemble

张伟业, 宋浩志, 刘兴健, 李轶女, 张志芳. 铁蛋白与口蹄疫病毒VP1在大肠杆菌中融合表达及纳米颗粒自组装[J]. 生物技术通报, 2021, 37(2): 96-102.

ZHANG Wei-ye, SONG Hao-zhi, LIU Xing-jian, LI Yi-nü, ZHANG Zhi-fang. Fusion Expression of Ferritin and Foot-and-Mouth Disease Virus VP1 in Escherichia coli and Self-assembly of Nanoparticles[J]. Biotechnology Bulletin, 2021, 37(2): 96-102.

图/表 9

表1 融合PCR扩增VP1-Ferritin用引物

引物名称	引物序列（5'-3'）
F1	CGGGATCCAACATGACCACCTCCACAGGTGAGTC
R1	GCGTCGACGAATTCTTAGCTCTTGCGGGACTTGGCGATG
F2	CCTGTGAAACAGTTGTTG TCCGGTGGCGACATCATCAAG
R2	CTTGATGATGTCGCCACCGGACAACAACTGTTTCACAGG

图1 FMDV VP1与Ferritin的PCR扩增结果（A）M：DNA分子质量标准;1、2：FMDV VP1的 PCR扩增产物;（B）M：DNA分子质量标准;1、2：Ferritin PCR扩增产物

图2 FMDV VP1-Ferritin的PCR扩增 M：DNA分子质量标准;1、2：FMDV VP1-Ferritin 融合PCR扩增产物

图3 重组质粒酶切验证结果 M：DNA分子质量标准;1：BamHI和EcoRI双酶切的FMDV VP1-Ferritin

图4 pET-28a VP1-Ferritin表达产物SDS-PAGE电泳检测结果= M：蛋白质分子量标准;1-7：IPTG诱导的pET-28a VP1-Ferritin表达样品;8：IPTG诱导的pET-28a表达样品;9：未诱导的pET-28a VP1-Ferritin表达样品

图5 重组蛋白VP1-Ferritin纯化后洗脱结果 M：蛋白质分子质量标准;1-12分别为：穿透液（1,2）;NTA0（3）;NTA25（4,5）;NTA50（6,7）;NTA100（8,9）;NTA250（10,11）;NTA500（12）洗脱液收集样品

图6 重组蛋白His-VP1-Ferritin的Western blotting分析结果 1：蛋白质分子质量标准;2-5：纯化重组蛋白His-VP1-Ferritin（稀释10,20,50和100倍）;6：pET-28a空载;7：未纯化重组蛋白His-VP1-Ferritin（稀释10倍）

表2 重组蛋白His-VP1-Ferritin的质谱鉴定结果

Master Protein Accession	Description	Coverage/%	# Peptides	# PSMs	# Unique Peptides	# AAs	MW/kD	calc. pI	Score Sequest HT：Sequest HT	Abundances	# Protein Groups
pET-28a	VP1-Ferritin	78	42	193	30	415	46.3	7.28	438.62	65020355405	1

图7 原核表达His-VP1-Ferritin和空白对照蛋白样品电镜观察图（scale bars=50 nm）

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