Bioinformatics analysis and polyclonal antibody preparation of Mesocricetus auratus DHX30

DHX30, as members of the DExD/H family, plays a crucial regulatory role in RNA metabolism and antiviral innate immune response. To facilitate comprehensive investigation of DHX30's multifunctional involvement in viral life cycles, a highly specific anti-DHX30 antibody is needed. In this paper, we used bioinformatic analyses combined with the Immune Epitope Database (IEDB) to identify immunodominant B-cell epitopes in the Mesocricetus auratus DHX30 protein. A hydrophilic peptide sequence demonstrating strong antigenic potential was selected for recombinant antigen production. The corresponding DHX30 truncated gene fragment was cloned into the pET-28a vector to generate the pET-28a-DHX30-N expression construct. Following transformation into E. coli BL21(DE3) competent cells, recombinant protein expression was induced and subsequently purified via Ni-NTA affinity chromatography. Six-week-old female KM mice were immunized intraperitoneally with the purified antigen. The antibody titer was determined by indirect ELISA, demonstrating high serum titers. Specificity validation through immunoblotting and immunocytochemical analyses confirmed the antibody's exceptional target recognition capability. These antibodies are expected to develop detection methods for further research.