| Vibrio harveyi is Gram-negative bacterial pathogens which infect many aquacultureanimals. It is a facultative anaerobe that inhabits predominately in aquatic and marineenvironments and also a severe pathogen to crustaceans and induces luminous vibriosis infarmed penaeid shrimp. In addition to crustaceans, V. harveyi is an important fish pathogenand infect a wide range of species, causing economic losses throughout the world.Currently, the control of V. harveyi in aquaculture relies mainly on antibiotics. Since theuse of antibiotic compounds is associated with various side effects, such asantibiotic-resistant mutant bacterial strains, contamination of environment, and potentialfood safety hazards. Alternative means of disease control, such as vaccine, have beeninvestigated in recent years.Reactive oxygen species (ROS) are products of the normal metabolic activities of livingorganism and are produced in response to various stimuli. There is a normal balancebetween the production of ROS and their destruction while disrupting the balance betweengeneration and depletion of ROS, Oxidative stress occurs and leads to cell damages. Amicro antioxidant system composed by superoxide dismutase (SOD), glutathione reductase(GR), glutathione peroxidase (GPx) and glutathione S-transferase (GST) plays an importantrole in maintaining the activity of ROS balance which can effectively protect the cells fromoxidative damage.In this study, GST and GPx genes were cloned by PCR amplification from thechromosomal DNA of Vibrio harveyi ZJ0603. The nucleotide sequences were determined.The ORF of the GST gene consists of615bp, encoding a polypeptide of202amino acids.Sequence analysis showed that homologies of the amino acid sequence with VibrioOwensii, V. Retiferianus and V. Cambellii are respectively99%,99%and97%. The ORF ofthe GPx gene （Without Signal peptde）consists of477bp, encoding a polypeptide of158amino acids. Sequence analysis showed that homologies of the amino acid sequence withVibrio Owensii, V. cholerae and V. alginolyticus are respectively99%，99%and97%.The GST and GPx genes were subcloned into pET-28a (+).The prokaryotic expressionplasmids pET-GST and pET-GPx were transformed into BL21(DE3). After a4h inductionwith0.08mmol/L IPTG at37degrees of GST and6h induction with1.0mmol/L IPTG at 37degrees of GPx, the expressed proteins possessed an N-terminal His-tag sequence waspurified by Ni(+)-affinity chromatography and identified with SDS-PAGE. The singleproteins brand were detected by SDS-PAGE, and molecular weight of the purified proteinswere28.2kDa and22.5kDa, respectively.Orange-spotted grouper (Epinephelus coioides) were immunized with purified GST orGPx proteins by intramuscular injection. The immunized fish were then challenged with0.1mL of V. harveyi (1×108CFU/mL) five weeks later. Challenge result demonstrated thatthe protection of the immunized group (GST and GPx) were72.50%and77.50%, theresult of ELISA showed that specific antibody titers against different vaccines weredetected in experimental groups, and reached to the top level at fifth week after vaccinated,which suggest purified GST and GPx proteins act as the antigen offer immune protectionsto Orange-spotted grouper.The GST and GPx genes were subcloned into pcDNA3.1(+).The eukaryotic expressionplasmids pcDNA-GST and pcDNA-GPx. Epinephelus coioides was intramuscular injectedwith pcDNA-GST or pcDNA-GPx. The detection of DNA and mRNA levels of twoplasmid expression and distribution in fish tissue showed that these recombinant plasmidDNA is distributed in liver, injected muscle, head kidney and spleen7days and28daysafter injection. The ELISA and Western-blot results indicated that the target proteins wereexpressed to activate antibody response in vivo. Challenging experiment with live bacteria,the RPS of recombinant plasmid pcDNA-GST and pcDNA-GPx are80%and82.5%,respectively. |
PDF Full Text Request