Abstract: Foot-and-mouth disease (FMD) is an acute and highly contagious animal disease that is caused by FMD virus (FMDV), and it brings huge economical damage to the livestock industry once breaking out. Vaccination with inactivated FMDV is widely used to prevent FMD prevalence, but there are several limitations to the conventional inactivated vaccine such as virus escape and high production costs. To avoid these deficiencies, recombinant protein subunit vaccines are being developed for the prevention and control of FMD. The structural protein VP1 of FMDV contains most of the pivotal immune epitopes, therefore numerous researchers focus on VP1 for diagnosis and vaccines development for FMDV. Compared with the eukaryotic expression system, the prokaryotic expression system has the advantages of fast growth, easy scaling up and low cost, making it more suitable for animal vaccine production. However, VP1 tends to form insoluble and inactive inclusion bodies when expressed in the prokaryotic system. In this study, a strategy for soluble expression of FMDV VP1 (serotype O/MYA/98) in the prokaryotic system was firstly designed by fusion with two tags of small ubiquitin-related modifier (SUMO) and RNA-interacting domain (RID). Through the PCR (polymerase chain reaction) overlap technique, the gene of RID-SUMO-VP1 was constructed and cloned into pET28a(+) vector. Then the recombinant plasmid was transformed into E. coli BL21(DE3). After induction with 1 mmol/L IPTG for 4 h at 18℃, the fusion protein of RID-SUMO-VP1 was efficiently expressed up to 79% insoluble form, while VP1 fused with one tag of SUMO or RID was still expressed as inclusion bodies. After cell disruption, a simple method of two-step ammonium sulfate precipitation was developed, which could separate RID-SUMO-VP1 from the lysate with a purity of 93%. ELISA (enzyme linked immunosorbent assay) showed that RID-SUMO-VP1 could react with O-type FMDV positive mice serum. These results demonstrated that soluble and immunogenic FMDV VP1 can be expressed in E. coli by fusion with a double-tag of RID and SUMO. Our study lays the foundation for the development of immunodiagnostic and novel subunit vaccines of FMD.

Key words: Foot-and-mouth disease virus of serotype O, structural protein VP1, small ubiquitin-related modifier (SUMO), RNA-interacting domain (RID), soluble expression, immune response

摘要： 口蹄疫(Foot-and-mouth Disease, FMD)是由口蹄疫病毒(Foot-and-mouth Disease Virus, FMDV)引起的急性、高度传染性疾病，一旦大规模爆发会给畜牧业造成巨大的经济损失。接种灭活病毒疫苗是目前防控口蹄疫的最有效手段，但传统灭活疫苗存在病毒逃逸、生产成本高等诸多局限。重组亚单位蛋白疫苗免疫原性好且安全性高，是发展新型口蹄疫疫苗的重要方向。口蹄疫病毒结构蛋白VP1因含主要抗原表位，而成为口蹄疫亚单位疫苗及诊断试剂的开发热点。与真核系统相比，原核表达系统生产周期短、易于放大生产、成本低廉，更适于动物疫苗的开发，但VP1采用原核系统表达会形成无活性的不溶包涵体。本工作中首次同时融合小分子泛素相关修饰物(Small Ubiquitin-related Modifier, SUMO)和RNA作用结构域(RNA-interacting Domain, RID)两种助溶标签来实现FMDV VP1 (O型/MYA/98)在原核系统中的高效可溶表达。首先采用PCR (聚合酶链反应)重叠技术，以pET-28a(+)为载体构建了RID-SUMO-VP1重组质粒，将重组质粒转化至E. coli BL21(DE3)，在18℃下通过1 mmol/L IPTG (异丙基-β-D-硫代半乳糖苷)诱导4 h后，融合蛋白RID-SUMO-VP1高效表达，可溶表达比例达79%，而融合SUMO或RID单一标签的VP1仍主要表达为不溶的包涵体。然后采用硫酸铵两步沉淀法对工程菌的破碎上清进行纯化，得到纯度为93%的RID-SUMO-VP1，酶联免疫吸附测定(ELISA)实验表明其能够与O型FMDV阳性小鼠血清反应。研究结果表明，采用RID及SUMO双标签融合策略能极大提高VP1在原核系统中的可溶表达效率，且表达的VP1具备良好的抗原性，这为开发新型FMDV免疫诊断试剂和安全高效的亚单位疫苗奠定了良好基础。

关键词: O型口蹄疫病毒, 结构蛋白VP1, 小分子泛素相关修饰物（SUMO）, RNA作用结构域 （RID）, 可溶表达, 免疫反应