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

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.