新型冠状病毒非结构蛋白14的表达及酶活性分析  

Expression and enzymatic activity analysis of non-structure protein 14 of SARS-CoV-2

作　　者：陈双 徐婧雯[2] 丁开云 刘建生[2] 彭小忠[2,3] 刘红旗 CHEN Shuang;XU Jingwen;DING Kaiyun;LIU Jiansheng;PENG Xiaozhong;LIU Hongqi(Department of Immunology,School of Basic Medicine of Kunming Medical University,Kunming 650500;Institute of Medical Biology,CAMS&PUMC,Kunming 650118;Department of Biochemistry and Molecular Biology,Institute of Basic Medical Sciences CAMS,School of Basic Medicine PUMC,Beijing 100005,China)

机构地区：[1]昆明医科大学、基础医学院、免疫学系,云南昆明650500 [2]中国医学科学院、北京协和医学院、医学生物学研究所,云南昆明650118 [3]中国医学科学院基础医学研究所、北京协和医学院基础学院、生物化学与分子生物学系,北京100005

出　　处：《基础医学与临床》2023年第1期51-58,共8页Basic and Clinical Medicine

基　　金：国家自然科学基金(32170166);昆明市科技计划项目[昆科计字(2020-1-N-034)号]。

摘　　要：目的获得纯度较高和有酶活性的新型冠状(新冠)病毒(SARS-CoV-2)非结构蛋白14(Nsp14)。方法根据大肠埃希氏菌(E.coli)的密码子偏好性,分析新冠病毒原始株基因nsp14的稀有密码子,并对其进行优化。将nsp14克隆至4个不同的表达载体并进行表达;通过对表达量和可溶性的比较分析,选择一个表达效果最好的重组表达质粒并优化表达条件;目的蛋白经谷胱甘肽亲和柱纯化后,用半胱氨酸家族蛋白酶(3C)切除融合标签,再分别利用谷胱甘肽亲和柱和分子筛柱纯化蛋白质。最后通过尿素聚丙烯酰胺凝胶电泳对其活性进行鉴定。结果Nsp14的基因在大肠杆菌中表达存在较多的稀有密码子,其中部分稀有密码子呈现近距离分布和串联分布。pGEX6P1-GST-OPTI-Nsp14重组质粒在大肠杆菌中表达的可溶性效果最好,其最佳表达温度为30℃。经纯化后获得了较纯的不含标签的Nsp14,该蛋白具有核酸酶活性。结论本研究成功地表达和纯化出具有核酸酶活性的新冠病毒Nsp14,为进一步推进对新冠病毒Nsp14的结构和功能研究奠定了基础,为筛选靶向Nsp14的抗病毒治疗药物提供了有利的条件。Objective To obtain the non-structure protein 14(Nsp14)of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)with higher purity and enzymatic activity.Methods This study firstly analyzed the rare codons in the gene of nsp14 according to the codon usage bias of E.coli,followed by codon optimization.The optimized nucleotide fragment of nsp14 was cloned into four kinds of expressing vectors respectively.Comparative analysis of yield and solubility was performed among these expressed four fusion proteins.The best one was chosen for further optimization of expressing conditions.After the fusion protein was purified by glutathione affinity column,the fusion tag was removed by 3C protease,and then the protein was purified by glutathione affinity column and molecular sieve column for further analysis of enzymatic activity through urea polyacrylamide gel electrophoresis.Results Many rare codons were found in expression of SARS-CoV-2 nsp14 in E.coli,among which some rare codons were distributed in close range and tandem.The best recombinant plasmid for expressing the fusion protein was pGEX6P1-GST-OPTI-Nsp14,which gave an eptimal expression in 30℃with high yield and solubility.After purification,a higher purity of Nsp14 with nuclease activity was obtained.Conclusions This study shows that the SARS-CoV-2 Nsp14 protein with nuclease activity is successfully prepared,which lays a foundation for the further research on the structure and function of SARS-CoV-2 Nsp14,and provides favorable conditions for screening antiviral drugs targeting at Nsp14 of SARS-CoV-2.

关 键 词：新型冠状病毒(SARS-CoV-2) 非结构蛋白14(Nsp14) 原核表达 蛋白质纯化 核酸酶活性 

分 类 号：R34[医药卫生—基础医学]