新型冠状病毒S1蛋白的表达及活性鉴定  

Expression and Activity Identification of SARS-CoV-2 S1 Protein

作　　者：汪巧菊 胡雨萌 温亚亚 宋丽 孟闯 潘志明 焦新安 WANG Qiao-ju;HU Yu-meng;WEN Ya-ya;SONG Li;MENG Chuang;PAN Zhi-ming;JIAO Xin-an(Yangzhou University/Key Laboratory of Zoonoses of Jiangsu Province/Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses of Jiangsu Province/Key Laboratory of Prevention and Control of Ministry of Agriculture and Rural Affairs(Animal Origin)for Agrifood Safety and Quality/Joint Laboratory for International Cooperation in Agriculture and Agricultural Products Safety of the Ministry of Education,Yangzhou 225009)

机构地区：[1]扬州大学/江苏省人兽共患病学重点实验室/江苏省动物重要疫病与人兽共患病防控协同创新中心/农业农村部农产品质量安全生物性危害因子(动物源)控制重点实验室/教育部农业与农产品安全国际合作联合实验室,扬州225009

出　　处：《生物技术通报》2022年第3期157-163,共7页Biotechnology Bulletin

基　　金：中国博士后科学基金资助项目(2020M681741);江苏省高校优势学科建设工程资助项目(PAPD);江苏省重点研发计划(现代农业)重点项目(BE2021331)。

摘　　要：旨在获得具有免疫活性的SARS-CoV-2 S1蛋白,为COVID-19的免疫预防及临床诊断提供较好的候选抗原。该研究利用PCR技术扩增出刺突蛋白(Spike,S)的S1亚基序列,通过同源重组法将目的片段克隆至冷休克表达载体pCold I,转化至大肠杆菌BL21感受态细胞内诱导表达S1蛋白,经Ni^(2+)柱亲和层析纯化后,通过SDS-PAGE和Western blotting鉴定目的蛋白的表达情况。结果表明,已成功表达含His标签的S1蛋白,并且已获得浓度和纯度较高、能与S1多克隆抗体反应的目的蛋白。此外,蛋白刺激细胞的结果表明,原核表达的S1蛋白免疫原性良好,能诱导小鼠巨噬细胞上调表达多种细胞因子和趋化因子,促进RAW264.7细胞产生免疫反应。综上,该研究成功构建了pCold I-S1重组质粒,获得了SARS-CoV-2的良好候选抗原S1蛋白。This work aims to obtain SARS-CoV-2 S1 protein with high immunoactivity,and to provide a good candidate antigen for the immune prevention and clinical diagnosis of COVID-19.PCR was adapted to amplify the S1 subunit of S protein,and the homologous recombination to clone the target sequence into the cold shock expression vector pCold I,then the recombinant plasmid was transformed into competent cells Escherichia coli BL21 and induced to express S1 protein.After purification by Ni^(2+) column affinity chromatography,SDSPAGE and Western blotting were used to identify the expressions of the target protein.The results showed that the S1 protein containing His tag was expressed successfully,also,S1 protein with high concentration and purity was obtained,which reacted with S1 polyclonal antibody.In addition,the results of cell stimulation by protein demonstrated that S1 protein by prokaryotic expression presented promising immunogenicity,and it significantly increased the expression of cytokines and chemokines in mice macrophages,thus promoting the immune response of RAW264.7 cells.In sum,this study successfully constructed the recombinant plasmid pCold I-S1,and obtained S1 protein that could be a good candidate antigen for SARS-COV-2.

关 键 词：SARS-CoV-2 S1蛋白 原核表达 生物活性鉴定 

分 类 号：Q936[生物学—微生物学]