Abstract：Bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) is a second messenger molecule widely present in bacteria and can participate in innate immune response through stimulator of interferon genes (STING) signaling pathway and is a potential vaccine adjuvant. In order to clarify its innate immune regulation mechanism and evaluate the effect of immunoadjuvant, the efficient synthesis of c-di-GMP needs to be solved urgently. In order to establish a relatively simple and efficient c-di-GMP biosynthesis method to prepare c-di-GMP in vitro, this study constructed Escherichia coli dinucleotide cyclase (DncV) recombinant expression vector, and the target protein DncV was obtained by IPTG (isopropy-β-D-thiogalactoside)-induced expression and His affinity purification; Detection of c-di-GMP from GTP catalyzed by DncV recombinant protein by ultra performance liquid chromatography (UPLC) in vitro. The results showed that the IPTG-inducible expression vector pET-28a-His-DncV was successfully constructed, and the purity of the recombinant protein obtained by affinity purification was 92%. The recombinant protein could generate c-di-GMP in one step by enzymatic reaction; Metal ions had a significant effect on the activity of the enzyme. Three kinds of metal ions (Mg2+, Mn2+, Co2+) could improve the catalytic efficiency, and the other 7 metal ions (Cu2+, Zn2+, Ni2+, Mo2+, Bo2+, Fe2+, Ca2+) inhibited the enzymatic reaction. This study established a method for the synthesis of c-di-GMP by E. coli DncV recombinant protein, which provides basic data for the large-scale preparation and functional study of c-di-GMP.
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