非洲猪瘟病毒D1133L蛋白腺苷三磷酸酶活性的测定  

Determination of ATPase activity of African swine fever virus D1133L protein

作　　者：崔卉梅 张婷 杨博 杨金柯 赵登率 袁兴国 陈学辉 郝雨 闫文倩 张克山 刘霞[1] 郑海学[2] 刘湘涛[2] CUI Hui-mei;ZHANG Ting;YANG Bo;YANG Jin-ke;ZHAO Deng-shuai;YUAN Xing-guo;CHEN Xue-hui;HAO Yu;YAN Wen-qian;ZHANG Ke-shan;LIU Xia;ZHENG Hai-xue;LIU Xiang-tao(College of Life Science and Technology,Gansu Agricultural University,Lanzhou 730070,China;State Key Laboratory of Veterinary Etiological Biology/Key Laboratory of Animal Virology,Ministry of Agriculture and Rural Affairs/National Footand-Mouth Disease Reference Laboratory/Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730046,China)

机构地区：[1]甘肃农业大学生命科学技术学院,甘肃兰州730070 [2]中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室农业农村部畜禽病毒学重点开放实验室国家口蹄疫参考实验室,甘肃兰州730046

出　　处：《中国兽医科学》2022年第5期572-578,共7页Chinese Veterinary Science

基　　金：甘肃省科技重大专项(20ZD7NA006);中国农业科学院基本科研业务费项目(Y2020XK09);国家自然科学基金联合项目(31941002)。

摘　　要：为测定非洲猪瘟病毒(ASFV)编码的D1133L蛋白的腺苷三磷酸酶活性,首先原核表达和纯化了D1133L蛋白。然后,利用Kinase-Glo Plus Luminescent Kinase Assay Platform试剂盒优化了反应条件,建立了D1133L蛋白腺苷三磷酸酶活性的测定方法,测定了D1133L蛋白腺苷三磷酸酶活性。结果显示,在25μL的反应体系中,当蛋白浓度为1.8μmol/L,Cu^(2+)浓度为10 mmol/L,37℃反应70 min时D1133L蛋白具有最佳腺苷三磷酸酶活性,可水解体系中40.04%的腺苷三磷酸。本结果为进一步明确D1133L功能,揭示其解旋机制和靶向D1133L解旋活性的高通量化学药物筛选奠定了基础。In order to determine the adenosine triphosphatase(ATPase)activity of D1133L protein encoded by African swine fever virus(ASFV),D1133L protein was expressed and purified using prokaryotic expression system and affinity chromatography system separately firstly.Then,the reaction conditions were optimized by using the Kinase-Glo Plus Luminescent Kinase Assay Platform kit,the determination method of D1133L protein ATPase activity was established,and the D1133L protein ATPase activity was determined.The results showed that D1133L protein had the best ATPase activity and could hydrolyze 40.04%adenosine triphosphate in the 25μL reaction system when the protein concentration was 1.8μmol/L,Cu^(2+)concentration was 10 mmol/L and the reaction time was 70 min at 37℃.The results laid the foundation for further confirming function of D1133L,revealing its unwind mechanisms and screening high-throughput chemical drugs target activity of D1133L.

关 键 词：非洲猪瘟病毒 D1133L 腺苷三磷酸酶 活性测定 

分 类 号：S852.659.1[农业科学—基础兽医学]