Cloning and expression of Vibrio harveyi OmpK* and GAPDH* genes and their potential application as vaccines in large yellow croakers Pseudosciaena crocea.
Vibrio harveyi is a causative agent of vibriosis in the large yellow croaker Pseudosciaena crocea and causes severe losses to the aquaculture industry in China. The vaccines based on the outer membrane proteins (OMPs) of the pathogens are considered to be the optimum intervention for this disease. In this study, two V. harveyi OMP genes, OmpK* and glyceraldehyde-3-phosphate dehydrogenase (GAPDH*), were cloned, sequenced, and characterized. The recombinant proteins (r-OmpK and r-GAPDH) were expressed by the prokaryotic expression vector pET-30a(+) and purified with nickel-nitrilotriacetic acid affinity chromatography. Western blots showed that rabbit antisera against purified r-OmpK and r-GAPDH specifically reacted with the native OMP of V. harveyi. Large yellow croakers were immunized with r-OmpK and r-GAPDH. Specific antibody titer assessed by enzyme-linked immunosorbent and phagocytosis assays demonstrated that specific and innate immunity was stimulated in response to the OMPs of V. harveyi. Challenge results indicated that vaccination of large yellow croakers with r-OmpK and r-GAPDH increased relative survival (37.7% and 40.0%, respectively) against wild V. harveyi.
College of Biomedical Engineering and Instrument Science and Institute of Preventive Veterinary Medicine, Zhejiang University, Hangzhou 310027, China.
Chongwen
C
Zhang
Lian
L
Yu
Ronghua
R
Qian
D000818:Animals; D000907:Antibodies, Bacterial / Q000096:biosynthesis; D000917:Antibody Formation; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D001428:Bacterial Vaccines / Q000737:chemistry; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D005393:Fish Diseases / Q000276:immunology / Q000517:prevention & control*; D015870:Gene Expression; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D010473:Perciformes; D014611:Vaccination / Q000662:veterinary; D014733:Vibrio / Q000235:genetics* / Q000276:immunology; D014735:Vibrio Infections / Q000517:prevention & control / Q000662:veterinary*
D000907:Antibodies, Bacterial; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D001428:Bacterial Vaccines; D004269:DNA, Bacterial; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases