山羊环鸟苷酸-腺苷酸合成酶的原核表达及其酶活性分析  

Prokaryotic expression and enzymatic activity of goat cyclic GMP-AMP synthase

作　　者：韦雨松 张艳 田慧慧 翟军军 陈国华[1] 房永祥[1] 景志忠[1] 屈雷[2] 米晓云[3] 何小兵[1] WEI Yusong;ZHANG Yan;TIAN Huihui;ZHAI Junjun;CHEN Guohua;FANG Yongxiang;JING Zhizhong;QU Lei;MI Xiaoyun;HE Xiaobing(State Key Laboratory for Animal Disease Control and Prevention/Key Laboratory of Veterinary Public Health,Ministry of Agriculture and Rural Affairs/Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730046,China;School of Life Science,Yulin University,Yulin 719000,China;Xinjiang Key Laboratory of Animal Infectious Diseases/lnstitute of Veterinary Medicine,Xinjiang Academy of Animal Science,Urumqi 830013,China)

机构地区：[1]中国农业科学院,兰州兽医研究所,动物疫病防控全国重点实验室,农业农村部兽医公共卫生重点实验室,甘肃兰州730046 [2]榆林学院生命科学学院,陕西榆林719000 [3]新疆畜牧科学院兽医研究所新疆动物疫病研究重点实验室,新疆乌鲁木齐830013

出　　处：《中国兽医科学》2024年第8期1096-1102,共7页Chinese Veterinary Science

基　　金：新疆动物疫病研究重点实验室开放基金项目(2023KLB003);国家自然科学基金项目(32060810,31960710);甘肃省自然科学基金项目(20JR10RA018,17JR5RA325)。

摘　　要：为了研究山羊环鸟苷酸-腺苷酸合成酶(gcGAS)的生物学功能,通过构建含gcGAS基因的pET28a-gcGAS原核表达载体,在Rosetta(DE3)表达菌中进行表达,经纯化获得了纯度较高的重组gcGAS蛋白,并测定其酶促活性。结果显示,在优化表达条件下,实现了重组gcGAS蛋白的可溶性表达,其能以ATP和GTP为底物合成了第二信使分子2′3′-c GAMP。结论:原核表达的可溶性重组gcGAS蛋白具有生物酶催化活性,这为后续gcGAS介导的抗病毒天然免疫机制研究及其应用开发奠定了一定的基础。To investigate the biological functions of the goat cyclic GMP-AMP synthase(gcGAS)in innate immune response,the prokaryotic expression vector pET28a-SUMO-gcGAS was constructed and was transformed into Rosetta(DE3),and soluble expression was achieved through optimized condition.The recombinant gcGAS protein was purified and its enzymatic activity was tested in vitro.The results showed that the soluble recombinant gcGAS protein was expressed and it catalyzed the synthesis of 2′3′-c GAMP using ATP and GTP.In conclusion,the recombinant gcGAS protein had enzymatic activity,which laid a foundation of the molecular mechanism of the gcGAS in antiviral innate immunity and development of the c GAMP.

关 键 词：环鸟苷酸-腺苷酸合成酶 环鸟苷酸-腺苷酸 原核表达 蛋白纯化 酶活性 

分 类 号：S852.2[农业科学—基础兽医学]