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

作　　者：李攀 刘术敏 刘晓雅 程英杰 吴常伟 黄恩启 LI Pan;LIU Shumin;LIU Xiaoya;CHENG Yingjie;WU Changwei;HUANG Enqi(Anhui Zhifei Longcom Biopharmaceutical Co.,Ltd.,Hefei 230088,Anhui Province,China)

机构地区：[1]安徽智飞龙科马生物制药有限公司,安徽合肥230088

出　　处：《中国生物制品学杂志》2024年第8期952-956,共5页Chinese Journal of Biologicals

基　　金：重大新药创制科技重大专项(2018ZX09739002).

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NV P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,SECHPLC)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.2 P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NV P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。Objective To obtain the soluble expression of P protein of norovirus(NV)GⅡ.2[P16]and analyze its antigenic characteristics.Methods The recombinant plasmid G I.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 DH5αcompetent 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 antigenantibody 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 GⅡ.2[P16]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 GⅡ.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 

分 类 号：Q786[生物学—分子生物学]