Gene cloning, expression and homology modeling of hemolysin gene from Aeromonas hydrophila.
Hemolysin is a significant toxin secreted by Aeromonas hydrophila, which contributes pathogenicity of fish to humans. The complete ORF of hemolysin gene (1886 bp) was amplified using PCR. It was cloned in TA and sub-cloned in pET28a vector then transformed into Escherichia coli BL21(DE3) codon plus RP cells expressed by the induction with 1.0 mM of IPTG. The expected size of expressed protein was 68.0 kDa estimated by migration in 12% SDS-PAGE. Anti-His monoclonal antibodies were used to substantiate the recombinant protein by Western blotting. The percent similarity between hemolysin of A. hydrophila with other hemolytic toxins revealed that the hemolysin/aerolysin/cytotoxin sequence varied from 99.35 to 50.40%. Homology modeling was used to construct 3-D structure of hemolysin of A. hydrophila with the known crystal 3-D structure (PDB: 1XEZ). This protein can be used for immunoassays and it is suitable for vaccine candidate against A. hydrophila infection.
Singh
Vijai
V
National Bureau of Fish Genetic Resources, Canal ring road, P.O. Dilkusha, Lucknow 226002, India. vijaisingh15@gmail.com
Somvanshi
Pallavi
P
Rathore
Gaurav
G
Kapoor
D
D
Mishra
B N
BN
D016980:Aeromonas hydrophila / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005393:Fish Diseases; D005399:Fishes; D015870:Gene Expression; D006460:Hemolysin Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D006801:Humans; D008958:Models, Molecular; D016366:Open Reading Frames / Q000235:genetics; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D040681:Structural Homology, Protein
D001426:Bacterial Proteins; D006460:Hemolysin Proteins; D011994:Recombinant Proteins