Cloning and expression of an outer membrane protein ompTS of Aeromonas hydrophila and study of immunogenicity in fish

• Published in: Protein expression and purification 2007-02, Vol.51 (2), p.303-307
• Main Authors: Khushiramani, Rekha, Girisha, S.K., Karunasagar, Indrani, Karunasagar, Iddya
• Format: Article
• Language: English
• Subjects: Aeromonas hydrophila; Aeromonas hydrophila - genetics; Aeromonas hydrophila - pathogenicity; Animals; Bacterial Outer Membrane Proteins - biosynthesis; Bacterial Outer Membrane Proteins - genetics; Bacterial Outer Membrane Proteins - immunology; Carps; Cloning expression; Cloning, Molecular; Enzyme-Linked Immunosorbent Assay; Fish Diseases - immunology; Fish Diseases - prevention & control; Immunogenicity; Labeo rohita; Mice; Outer membrane protein; Polyclonal antibodies; Rabbits
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2006.07.021

The outer membrane proteins of the warm water fish pathogen, Aeromonas hydrophila have a role in the virulence of the organism and are potential candidates for vaccine development. In this study, the gene encoding an outer membrane protein designated ompTS was amplified by PCR excluding the region coding for signal peptide, cloned in pQE 30-UA Vector and expressed using induction with isopropyl thiogalactoside (IPTG). The size of the expressed protein was 37 kDa as estimated by migration in 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). Polyclonal antibodies were raised in mice and rabbit against the purified protein and the reaction of the antibody was confirmed by Western blotting using the purified protein and A. hydrophila cultures. The Indian major carp, Labeo rohita Hamilton was immunized using the purified protein and developed antibodies with mean titers of 1:4000 on day 14 and 1:12,000 on day 28 showing promise that the protein is highly immunogenic in fish.