Objective A polyclonal antiserum against Mab21 domain of duck cyclic guanosine monophosphate-adenosine monophosphate synthase (du-cGAS) was prepared with immunological properties characterized.

Methods Based on the cGAS genomic sequence of Pekin ducks available in GenBank, the Mab21 domain was identified through bioinformatics analysis. Specific primers targeting the domain were designed. Coding sequence of the domain was amplified using cDNA from Muscovy duck embryo fibroblast (MDEF) cells as the template and cloned into prokaryotic expression vector pET-28a(+). The recombinant plasmid was then transformed into Escherichia coli BL21 (DE3) competent cells for prokaryotic expression. Purified du-cGAS-Mab21 recombinant protein was used to immunize BALB/c mice for serum specimen to evaluate the antibody specificity by western blot.

Results The coding sequence of the Mab21 domain was successfully amplified, and the recombinant expression plasmid pET-28a-du-Mab21 constructed. After IPTG induction and Ni-column affinity purification, a du-Mab21 recombinant protein with a relative molecular mass of approximately 43kDa that could be eluted with 150 mmol·L⁻¹ imidazole was obtained. The prepared anti-du-Mab21 polyclonal antiserum recognized the recombinant protein as well as the endogenous du-cGAS of molecular weight of approximately 55 kDa in MDEF cells but not the pre-immune mouse serum, nor in LMH cells.

Conclusion A polyclonal serum specific to the Mab21 domain of du-cGAS with high immunological reactivity and specificity was successfully obtained to provide reliable materials for studies on the cGAS-STING pathway and natural antiviral immune mechanism of waterfowl.