Construction of pCDNA3.1-AopB gene construct as a DNA vaccine model against Aeromonas hydrophila in carp

Aeromonas hydrophila is one of the most important pathogenic bacteria in fish that causes hemorrhagic septicemia, ascites and mortality in different ages and in different fish species including carp. Immunization is one of the best ways to prevent this disease. The use of gene constructs containing bacterial-specific antigens has been developed in recent years to develop high-performance vaccines and to use them in easier methods such as oral administration in aquaculture. In this study, aopB protein of type III secretory system of A. hydrophila isolated from carp in Khuzestan province was used to design a gene construct for DNA vaccine which cloned into the expression eukaryotic plasmid pCDNA3.1. In this study, A. hydrophila isolated from carp with Aeromonas septicemia in carp farms of Khuzestan province which species confirmed by molecular and phenotypic methods. The aopB gene sequence was extracted from the NCBI gene bank and the gene encoding region was amplified using primers containing EcoRI and NheI enzyme cleavage regions. The PCR product was then inserted into the eukaryotic expression plasmid pCDNA3.1-HisA and was transfected to TOP10-F 'bacteria using heat shock. After screening for positive clones using colony PCR and sequencing, the pCDNA3.1-AopB gene construct was transferred to CHO cells using electroporation method and recombinant protein expression was assessed by Western blotting on cell lysis. PCR results and sequencing of gene constructs indicated the cloning of the aopB gene approximately 1000bp in plasmid pCDNA3.1-HisA. Western blot using anti-HisTag antibody showed the presence of a protein with a molecular weight of about 36kD. Conclusively, the pCDNA3.1-AopB gene construct containing the aopB gene was designed and validated. This gene construct can be used in clinical studies to evaluate the immunogenicity of carp in experimental pathogenicity with A. hydrophila.