霍乱弧菌二核苷酸环化酶的可溶性原核表达及其表达产物的生物活性分析

Soluble prokaryotic expression of dinucleotide cyclase from Vibrio cholerae and bioactivity analysis of the expressed product

为获得具有生物活性的霍乱弧菌二核苷酸环化酶(DncV)蛋白与环二核苷酸c-di-GMP、c-di-AMP和3'3'-cGAMP,构建了DncV基因的原核表达载体pET-28a-SUMO-DncV,将其转化至大肠杆菌Rosetta(DE3),并通过优化诱导条件实现了可溶性表达;利用镍柱亲和层析纯化表达的重组蛋白产物,随后切除SUMO标签蛋白,再用亲和层析进一步纯化,获得了纯度较高的重组DncV蛋白;将获得的重组DncV蛋白通过体外酶促反应分别合成了c-di-AMP、c-di-GMP和3'3'-cGAMP,且合成产物能够诱导细胞产生Ⅰ型IFNs。结果表明,重组蛋白DncV及其产物c-di-GMP、c-di-AMP和3'3'-cGAMP均具有生物活性,这为后续抗病毒、抗肿瘤药物以及免疫增强剂和佐剂的开发奠定了基础。

To obtain bioactive dinucleotide cyclase in Vibrio cholerae(DncV)proteins and cyclic dinucleotides c-di-AMP,c-di-GMP or 3′3′-cGAMP,the prokaryotic expression vector pET-28a-SUMO-DncV was constructed and transformed intoEscherichia coli Rosetta(DE3),and the soluble recombinant protein DncV was expressed in medium by optimizing the conditions.Then,the recombinant protein DncV was purified via nickel column affinity chromatography,and the SUMO tag protein was removed using ULP protease by affinity chromatography to further obtain higher purity DncV protein.The recombinant DncV protein was used to synthesize c-di-AMP,c-di-GMP or 3′3′-cGAMP in vitro,which was confirmed to induce the production of typeⅠIFNs at the cellular level.The results indicated that recombinant protein DncV and CDNs have biological activity,which lay the foundation for the further development of anti-viral and anti-tumor drugs and immunopotentiators.