Expression of recombinant proteins and preparation of monoclonal antibodies for IFNa and IFNd in grass carp (Ctenopharyngodon idella)

To understand the synthesis, secretion and immune functions of type I interferons (IFN) in Ctenopharyngodon idella, CiIFNa and CiIFNd were expressed in Escherichia coli (E. coli) cells and purified. The mature peptides of CiIFNa and CiIFNd were cloned into pET-21d and pEHISTEVb expression vectors, respectively, and transformed in E. coli Rosetta cells. The recombinant proteins were expressed as inclusion bodies after IPTG induction. Following denaturation with guanidine hydrochloride, renaturation and concentration, the recombinant proteins were purified by size exclusion chromatography and used for immunization of mice. Hybridoma cells were obtained using the PEG method and injected into the abdominal cavity of mice to generate ascites. Four monoclonal antibodies of CiIFNa and CiIFNd (2 antibodies each) were purified, and characterized by SDS-PAGE, ELISA, Western blot and immunofluorescence assay. It has been shown that the monoclonal antibodies produced had good specificity and high titers against the antigens and could specifically recognize recombinant proteins expressed in E. coli and eukaryotic cells. The CiIFNa antibodies did not react with CiIFNd and vice versa. Taken together, the CiIFNa and CiIFNd monoclonal antibodies prepared in this study may provide the basis for further in-depth study of the cellular sources and protein expression profiles of IFNs in grass carp.