新型冠状病毒S蛋白表达、纯化及应用  

Expression,purification and application of novel coronavirus S protein

作　　者：于彩君 李明明[1,2] 陈珂 盛军[1] 朱强强 YU Cai-jun;LI Ming-ming;CHEN Ke;SHENG Jun;ZHU Qiang-qiang(Key Laboratory of Pu-er Tea Science,Ministry of Education,Yunnan Agricultural University,Kunming 650201,China;College of Food Science and Technology,Yunnan Agricultural University,Kunming 650201,China)

机构地区：[1]普洱茶学教育部重点实验室,云南农业大学,云南昆明650201 [2]云南农业大学食品科学技术学院,云南昆明650201

出　　处：《生物技术》2022年第6期671-676,共6页Biotechnology

基　　金：昆明市科技计划项目(2020-1-H-039)。

摘　　要：[目的]克隆严重急性呼吸综合征冠状病毒2(Severe Acute Respiratory Syndrome Coronavirus 2,SARS-CoV-2)刺突糖蛋白(Spike,S蛋白),纯化重组蛋白为制备鼠多克隆抗体提供免疫原,并鉴定抗体活性。[方法]依据对S蛋白的氨基酸序列解析,设计并构建了S蛋白重组质粒S713 pcDNA3.1(+)-C-6His,经酶切和测序正确后,转入中国仓鼠卵巢细胞(CHO-K1)中进行诱导表达,SDS-PAGE鉴定融合蛋白表达情况。表达产物利用Ni柱和分子筛纯化,目的蛋白混合免疫佐剂AS03免疫ICR小鼠,制备S蛋白多克隆抗体并对通过中和实验进行鉴定。[结果]成功构建重组质粒S713 pcDNA3.1(+)-C-6His,在CHO-K1细胞中S蛋白以可溶性形式表达。可溶性目的蛋白纯化后,蛋白纯度为94.4%,作为免疫原制备鼠多克隆抗体,抗体效价为1∶400,中和实验显示该多抗具有保护作用。[结论]成功诱导表达、纯化重组S蛋白并制备其多克隆抗体,为深入研究新冠病毒感染、发病机制及新型肺炎的防治奠定基础。[Objective]To clone the Spike glycoprotein(S protein)of Severe Acute Respiratory Syndrome Coronavirus 2(SARS-COV-2)and prepare mouse polyclonal antibodies by purifying the recombinant protein as immunogen and identified the antibody activity.[Method]According to the amino acid sequence analysis of S protein,the recombinant S protein plasmid S713 pcDNA3.1(+)-C-6 His was designed and constructed.After correct digestion and sequencing,it was transferred into Chinese hamster ovary cells(CHO-K1)for induction expression.The expression of fusion protein was identified by SDS-PAGE.The expressed product was purified by Ni column and molecular sieve,and ICR mice were immunized with the target protein mixed with immune adjuvant AS03 to prepare S protein polyclonal antibody,which was identified by neutralization test.[Result]The recombinant plasmid S713 pcDNA3.1(+)-C-6 His was successfully constructed and S protein was expressed in a soluble form in CHO-K1 cells.The soluble target protein was purified with a purity of 94.4%and was used as an immunogen to prepare mouse polyclonal antibody with a titer of 1∶400.Neutralization experiments showed that the polyclonal antibody had protective effect.[Conclusion]The recombinant S protein was successfully induced,purified and polyclonal antibody was prepared,which laid a foundation for further study of novel coronavirus infection,pathogenesis and prevention and treatment of new pneumonia.

关 键 词：新冠病毒 刺突糖蛋白 蛋白纯化 中和实验 多克隆抗体 

分 类 号：S852.61[农业科学—基础兽医学]