GⅡ.17型诺如病毒样颗粒的制备及鉴定

Preparation and identification of Norovirus GⅡ.17 virus-like particles

[背景]诺如病毒(Norovirus,NoV)是引发全球人类急性胃肠炎的主要食源性致病原,具有广泛的遗传多样性,其中GⅡ.17型是亚洲地区的优势流行株,危害最为严重。研究表明,通过基因工程技术制备的诺如病毒样颗粒(virus-like particle,VLP)具有良好的免疫保护作用,是目前NoV疫苗研发的主要思路。[目的]利用大肠杆菌(Escherichia coli)原核表达系统制备GⅡ.17型诺如病毒的VLP,为GⅡ.17型NoV疫苗的研发奠定基础。[方法]合成GⅡ.17型NoV衣壳蛋白VP1的基因并克隆入pET28a-MsyB原核表达质粒中,将重组质粒转化大肠杆菌BL21(DE3)中进行表达,通过烟草蚀纹病毒(tobacco etch virus,TEV)蛋白酶去除融合标签,利用His-Tag镍柱纯化获得天然VP1蛋白,利用SDS-PAGE、Western Blot和透射电镜对纯化后的VP1蛋白反应原性及其形成的结构进行初步鉴定。[结果]MsyB-VP1融合蛋白能够以可溶形式大量表达,其最适表达条件为:IPTG浓度0.8 mmol/L,37℃诱导8 h;TEV蛋白酶酶切后的VP1纯化蛋白能够与鼠抗GⅡ.17型NoV VP1多克隆抗体特异性结合,并能够自组装形成直径约为40 nm的病毒样颗粒。[结论]利用原核表达系统成功制备了GⅡ.17型诺如病毒VLP,有望成为GⅡ.17型诺如病毒的候选疫苗。

[Background]Norovirus,featuring genetic diversity,is a major foodborne initiator of acute gastroenteritis in human worldwide.GⅡ.17,the most destructive genotype,is a novel variant emerging in Asia from 2014 through 2015 and has been predominant in Asia.Virus-like particles(VLPs)of Norovirus,prepared by genetic engineering,are safe with high immunogenicity,which are promising candidate vaccine of Norovirus.[Objective]This paper aims to prepare GⅡ.17 VLPs in Escherichia coli.[Methods]The gene encoding GⅡ.17 capsid protein VP1 was synthesized and cloned into pET28 a-MsyB vector.The recombinant plasmid was transformed into E.coli BL21(DE3)and expressed under the induction of IPTG.The fusion tags were removed by tobacco etch virus(TEV)protease,followed by purification with His-Tag nickel column to yield the natural VP1 protein.SDS-PAGE,Western Blot,and transmission electron microscope were employed to determine the reactogenicity and structure of VP1 protein.[Results]MsyB-VP1 can be expressed in soluble form,and the optimum conditions are as follows:IPTG at 0.8 mmol/L,and 37°C for 8 h.The purified VP1 protein digested by TEV protease can specifically bind to mouse anti-GⅡ.17 VP1 polyclonal antibody and assemble into VLPs(diameter:about 40 nm).[Conclusion]In this study,GⅡ.17 VLPs were prepared with E.coli,which can be used as the candidate GⅡ.17 vaccine.