Cloning and Expression of Recombinant Chondroitin AC Lyase Protein of Flavobacterium columnare in a Prokaryotic Expression System

Background: Aquaculture, the fastest-growing food production sector worldwide, faces significant challenges due to disease outbreaks, particularly from bacterial pathogens such as Flavobacterium columnare. These pathogens cause severe economic losses, necessitating the development of effective diagnostic tools and vaccines. Methods: This study aimed to express the chondroitin AC lyase gene from F. columnare, a key virulence factor in prokaryotic expression vector. The gene was PCR-amplified, cloned into the pET-32a vector and expressed in Escherichia coli BL21-D3 cells. Positive clones were confirmed through PCR and restriction enzyme digestion. The expressed protein was analyzed by SDS-PAGE and Western blot, with protein expression tested under varying incubation temperatures. In silico analysis was performed to predict secondary and tertiary structures, including identification of key motifs and antigenic sites. Antigenicity was confirmed through an Agar Gel Immunodiffusion (AGID) assay. Result: The SDS-PAGE and Western blot analysis revealed a prominent band at ~77 kDa, indicating successful expression of the recombinant protein. Expression levels varied with incubation temperature. In silico analysis identified significant antigenic sites and the AGID assay showed strong immune reactions, confirming the protein’s potential as a vaccine component.