GⅡ.2[P16]型诺如病毒P蛋白的可溶表达、纯化及其抗原特性分析
Analysis of soluble expression,purification and antigenic characterization of P protein of GⅡ.2[P16]norovirus
李攀 1刘术敏 1刘晓雅 1程英杰 1吴常伟 1黄恩启1
作者信息
- 1. 安徽智飞龙科马生物制药有限公司,安徽合肥 230088
- 折叠
摘要
目的 获得可溶表达的GⅡ.2[P16]型诺如病毒(norovirus,NV)P蛋白,并分析其抗原特性.方法 通过优化合成GⅡ.2[P16]型NVP蛋白基因并克隆至pET-28a(+)载体上,构建重组质粒GⅡ.2-NV-pET-28a,转化DH5α感受态细胞,通过对表达菌种的优化,使P蛋白以可溶性形式表达.表达的P蛋白经Ni-NTA亲和层析纯化后,采用Western blot法检测纯化蛋白抗原性;体积排阻高效液相色谱(size exclusion high performance liquid chromatography,SEC-HPLC)法检测抗原纯度;ELISA法鉴定抗原抗体结合能力.结果 重组BL21 Star(DE3)pLySs工程菌目的蛋白以可溶形式高表达,经亲和层析纯化后,GⅡ.2P蛋白纯度为95.53％,且抗原性较好.结论 用原核表达菌种成功制备了可溶性表达的NVP蛋白,并明确了 P蛋白的抗原性,为GⅡ.2[P16]型NV检测试剂盒和重组亚单位疫苗的研制奠定了基础.
Abstract
Objective To obtain the soluble expression of P protein of norovirus(NV)G Ⅱ.2[P16]and analyze its antigenic characteristics.Methods The recombinant plasmid G Ⅱ.2-NV-pET-28a was constructed by optimizing the NV GⅡ.2[P16]P protein gene,synthesizing and cloning it into pET-28a(+)vector.The recombinant plasmid was transformed into DH5α competent cells,and the expression strain was optimized to make the P protein express in soluble form.The expressed P protein was purified by Ni-NTA affinity chromatography,of which the antigenicity was detected by Western blot,the purity was detected by size exclusion high performance liquid chromatography(SEC-HPLC),and the antigen-antibody binding ability was identified by ELISA.Results The target protein of recombinant BL21 Star(DE3)pLySs was highly expressed in soluble form.After purification by affinity chromatography,the purity of G Ⅱ.2 P protein was 95.53％,and the antigenicity was good.Conclusion Soluble NV P protein was successfully prepared by prokaryotic expression strain,and the antigenicity of P protein was confirmed,which laid a foundation of the development of NV G Ⅱ.2[P16]detection kits and the recombinant subunit vaccines.
关键词
诺如病毒/GⅡ.2[P16]/P蛋白/可溶性表达Key words
Norovirus(NV)/GⅡ.2[P16]/P protein/Soluble expression引用本文复制引用
基金项目
重大新药创制科技重大专项(2018ZX09739002)
出版年
2024
NETL
NSTL
万方数据