牛G3BP1蛋白的原核表达及其对牛cGAS生物酶催化活性的影响  

Prokaryotic expression of bovine G3BP1 protein and its influence on enzymatic activity of the bovine cGAS

作　　者：田慧慧 何小兵[1] 陈国华[1] 高真贞 韦雨松 俞宗佑 房永祥[1] 米晓云[4] 景志忠[1] TIAN Hui-hui;HE Xiao-bing;CHEN Guo-hua;GAO Zhen-zhen;WEI Yu-song;YU Zong-you;FANG Yong-xiang;MI Xiao-yun;JING Zhi-zhong(State Key Laboratory for A nimal Disease Control and Prevention/Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730046,China;School of Life Science,Yulin University,Yulin 719000,China;China Agricultural Vet.Bio.Science and Technology Co.,Ltd.,Lanzhou 730046,China;Xinjiang Key Laboratory for 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,甘肃兰州730046 [2]榆林学院生命科学学院,陕西榆林719000 [3]中农威特生物科技股份有限公司,甘肃兰州730046 [4]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》2023年第12期1531-1536,共6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);甘肃省自然科学基金项目(20JR10RA018,17JR5RA325)。

摘　　要：为了探究牛G3BP1(bG3BP1)对DNA识别受体cGAS生物酶催化活性的影响,本研究提取牛血液淋巴细胞中总的RNA,经RT-PCR反转录成cDNA,克隆出bG3BP1基因全长;随后构建bG3BP1的原核表达载体pET-28a-SUMO-bG3BP1,将其转化至大肠杆菌中进行诱导表达,使用镍亲和层析柱对重组蛋白bG3BP1进行纯化,并切除His6-SUMO标签后再经镍柱纯化;最后将获得的bG3BP1蛋白用于牛cGAS(bcGAS)的体外酶促反应,采用ELISA方法检测酶促合成产率。结果显示,可溶性表达的bG3BP1蛋白促进了bcGAS的体外酶促反应,提高了第二信使分子2′3′-cGAMP的产率,这为2′3′-cGAMP的大规模生产及应用提供了研究思路和技术方法。To investigate the effect of bovine G3BP1(bG3BP1)on the enzymatic activity of DNA recog-nition receptor cGAS,the total RNA from bovine blood lymphocytes was extracted and reversely tran-scribed into cDNA by RT-PCR and the bG3BP1 gene were cloned.Subsequently,the prokaryotic expression vector pET-28a-SUMO-bG3BPl was constructed and transformed into competent E.coli cells.The recombi-nant protein bG3BPl was purified by nickel column affinity chromatography and purified further after removing SUMO label proteins to reach high purity.Finally,the bG3BP1 was used for the enzymatic reac-tion of bovine cGAS(bcGAS)in vitro,and the product of 2'3'-cGAMP was detected by ELISA.The results showed that soluble bG3BPl protein promoted the enzymatic reaction of bcGAS in vitro and increased the yield of the second messenger molecule 2'3'-cGAMP,which provided technical methods and research ideas for the large-scale production and application of 2'3'-cGAMP.

关 键 词：牛cGAS 牛G3BP1 原核表达 第二信使分子 酶促反应 

分 类 号：S858.23[农业科学—临床兽医学]