Determination of the optimal conditions of cloning Aerolysin gene from the common carp pathogen Aeromonas hydrophila in Escherichia coli BL۲۱

Aeromonas hydrophila is a gram-negative bacterium which associated with gastrointestinal diseases and septicaemia. This pathogenic bacterium has several virulence factors ranging from pili to the excreted protein which called (Aerolysin) with minor and major effects, respectively. Additionally, Aeromonas hydrophila is a widely distributed bacterium that commonly causes ulcers in cyprinid fish such as carps and secondary diseases in humans as well. In the present study, characteristics and haemolytic activities of the recombinant Aerolysin protein and optimal conditions for cloning are determined using the synthesized cloning/expression Aerolysin gene, assembled into the Escherichia coli BL۲۱ (DE۳) through pGEX-۶P۱ vector, using SDS-PAGE and western blotting techniques. The results declared that, the Aerolysin gene (۱۴۸۲ bp) was cloned by transforming the recombinant pGEX-۶P۱ vector into Escherichia coli BL۲۱ (DE۳) as a prokaryotic expression host. The SDS-PAGE results indicated that the estimated protein size was ۵۴ KDa. Recombinant Aerolysin protein synthesis at both selected temperatures, ۲۵°C and ۳۷°C, indicated that ۱ mM of isopropyl-β-D-thiogalactopyranoside (IPTG) was the optimum concentration for induction. However, the recombinant protein was unable to synthesize in the absence of IPTG inducer. Western blot analysis indicated the efficient sensitivity and specificity of the recombinant Aerolysin protein. In conclusion, the recombinant protein showed potential advantages for immunoassay approaches in order to decrease the economic losses caused by disease in the aquaculture industry.

Keywords:

Aeromonas hydrophila , Escherichia coli common Carp , Fish disease , Haemolytic activity , Recombinant protein , Aerolysin