严重急性呼吸综合征冠状病毒2核衣壳蛋白的制备及其初步应用  

Preparation and preliminary application of recombinant severe acute respiratory syndrome coronavirus 2 nucleocapsid protein

作　　者：李光[1,2,3] 任仲 胡道奇 刘汨 李克强 张玉玲 周松辉 方晓兰 李卫平 滕淑静[1] LI Guang;REN Zhong;HU Dao-qi;LIU Mi;LI Ke-qiang;ZHANG Yu-ling;ZHOU Song-hui;FANG Xiao-lan;LI Wei-ping;TENG Shu-jing(Yueyang Vocational and Technical College,Yueyang 414000,Hunan Province,China)

机构地区：[1]岳阳职业技术学院,湖南岳阳414000 [2]湖南康润药业股份有限公司,湖南岳阳414000 [3]免疫诊断试剂湖南省工程研究中心,湖南岳阳414000

出　　处：《中国生物制品学杂志》2022年第2期184-188,共5页Chinese Journal of Biologicals

基　　金：湖南省自然科学基金(2020JJ7071);湖南省教育厅科研基金(20c1872);湖南省卫生健康委科研计划项目(202211005269).

摘　　要：目的在大肠埃希菌中表达严重急性呼吸综合征冠状病毒2(severe acute respiratory syndrome coronavirus 2,SARS-CoV-2)核衣壳蛋白(nucleocapsid protein,N蛋白),并进行纯化,用于制备SARS-CoV-2检测试剂。方法将含N蛋白基因的重组质粒pET28a转化感受态大肠埃希菌BL21(DE3),诱导表达N蛋白,考察Ni柱亲和层析与SP强阳离子交换层析和CM弱阳离子交换层析对重组N蛋白的纯化效率,制备胶体金免疫检测试剂并检测灭活血清。结果表达的重组N蛋白相对分子质量约49000,表达量约占包涵体总蛋白的30%。Ni^(2+)亲和层析对重组N蛋白的选择性较低,通过强阳离子交换与弱阳离子交换两步层析,实现了重组N蛋白在色谱柱上的复性和纯化,纯度大于90%。以此制备的免疫胶体金检测试剂可初步用于血清中相应抗体的检测。结论复性后的重组N蛋白,具有较好的生物活性,可用于诊断试剂开发,复性工艺对其他重组蛋白质的纯化具有借鉴意义。Objective To express the nucleocapsid(N)protein of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)in E.coli,purify the expressed product and use for preparation of detection reagent for SARS-CoV-2.Methods Recombinant plasmid pET28a containing the gene encoding N protein was transformed into competent E.coli BL21(DE3)and induced for expression.The purification efficiencies of N protein by nickel ion affinity chromatography,SP strong cation exchange chromatography and CM weak strong ion exchange chromatography were evaluated.Immune colloidal gold detection reagent was prepared and used for detection of inactivated serum.Results The expressed recombinant N protein,with a relative molecular mass of about 49000,contained about 30%of total somatic protein.Nickel ion affinity chromatography showed low selectivity to recombinant N protein.Through strong cation exchange and weak cation exchange chromatography,recombinant N protein was re-naturalized and purified on the column,of which the purity was more than 90%.The prepared immunocolloidal gold detection reagent could be preliminarily used for the detection of the corresponding antibody in sera.Conclusion The re-naturalized recombinant N protein has good biological activity and can be used for the development of diagnostic reagents.The re-naturalization process of recombinant N protein on column can be used as a reference for the purification of other recombinant proteins.

关 键 词：严重急性呼吸综合征冠状病毒2 核衣壳蛋白 包涵体 复性 纯化 免疫胶体金检测试剂 

分 类 号：R446.61[医药卫生—诊断学]