| Rainbow trout is an important cold-water farming fish in the world, and it is also cultured on a large scale in Gansu, Qinghai and Xinjiang in China. With the development of rainbow trout farming and the increasing of production in recent years, diseases has gradually become an important limiting factor for the development of the rainbow trout, causing huge economic losses and threatening the health of rainbow trout and sustainable development of aquaculture industry in China. Among the pathogen of rainbow trout, infectious hematopoietic necrosis virus (IHNV) and Aeromonas salmonicida are two major pathogens of rainbow trout. IHNV is the causative agent of infectious hematopoietic necrosis (IHN), and causes necrosis of kidneys, spleens, and hematopoietic tissues of fishes with a mortality more than 90%, which has caused great economic losses to the rainbow trout farming industry in the world. The genome of IHNV encodes five structural proteins and one non-structural protein. Among them, the glycoprotein, also known as the G-protein, is the only surface protein of the virus, which is also the main antigen of the virus. G protein can stimulate neutralizing antibodies in host, induce cellular immunity, and plays an important role in viral pathogenicity and immune response, etc. Currently, most of the studies focus on the DNA vaccines of IHNV G protein gene. Aeromonas salmonicida could cause furunculosis and ulcer in a variety of fish species, including rainbow trout. Currently, the international commercialized vaccines against Aeromonas salmonicida are widely used, while China still relies on antibiotics as the main control method. The development of vaccines against Aeromonas salmonicida has become particularly urgent in China. Therefore, in this study, the IHNV-G protein gene was amplified by PCR, and ligated into pGEX-4T-1 plasmid to get the G protein expression vector pGEX-4T-1-G.The recombinant plasmid pGEX-4T-1-G was transformed into A. salmonicida subsp. salmonicida SC18032201 by electronic transformation, to get the A. salmonicida vaccine carrier SC18032201-G, which could express G protein of IHNV as polyvalent vaccine. The SC18032201 with pGEX-4T-1 plasmid (SC18032201-pGEX) and the wild strain SC18032201 were set as negative controls. IPTG was used to induce the G protein in SC18032201-G. The G protein expressed by A. salmonicida SC18032201-G was detected by Western Blotting using mouse anti-His protein serum as the primary antibody and goat anti-mouse serum with HRP as the secondary antibody. The results of the Western Blotting showed that after IPTG induction, the specific reaction bands could be detected by the recombinant vaccine carrier SC18032201-G carrying the pGEX-4T-1-G plasmid, while not the SC18032201-pGEX and wild-type A. salmonicida SC18032201, which indicated that the G protein was expressed in A. salmonicida SC18032201-G.to the expression of G protein was optimized by induction time, IPTG concentration and culture temperature. The results of Western Blotting showed that the best induction conditions for recombinant G protein expressed by SC18032201-G was 0.2 mM IPTG at 28°C for 8 h. The optimized conditions were then used for the incubation and induction of SC18032201-G, and the bacteria was inactivated with formaldehyde. Then the inactivated bacteria was emulsified with Montanide? ISA 763A VG as adjuvant to prepare an oil-based d vaccine. PBS control was prepared according to the same method. The rainbow trout were immunized by intraperitoneal injection with vaccine or PBS control. At 45 days post vaccination, 10 rainbow trout in each group were randomly selected for blood sampling from the caudal vertebrae. The blood was stored at 4°C overnight and centrifugate to get the serum for the following antibody detection experiment. The serum was diluted in a 2-fold dilution gradient (1:2-1:256) and incubated at 1:1 with IHNV viral culture medium (100×TCID50). The mixture was added to the monolayer carp epithelial tumor cells (EPC) cells, with 8 replicates per gradient. The cells were incubated at 15℃ and observed for 7 d. The CPE of culture was recorded, and the highest dilution of serum that inhibited 50% of CPE was recorded as the level of neutralizing antibody. The results showed that the neutralizing antibody titer was 54.95±6.76 in the vaccinated group, and no neutralizing antibody potency was detected in the control group. The difference between the neutralizing antibody titer of the immunized group and that of the control group was highly significant (P<0.01). At the same time, after 45 days of immunization, the rainbow trout were infected with 1×106 CFU/mL of A. salmonicida SC18032201 by immersion, and observed continuously for 30 d. The death of rainbow trout recorded for 30 d, and the relative percentage survival was calculated. The results showed that the relative percentage survival of the vaccinated rainbow trout against A. salmonicida was 100% after 45 days of immunization, which was significantly different from that of the control group. In conclusion, we constructed an A. salmonicida vaccine carrier that express the G protein of IHNV in this study, which could not only provide effective protection against A. salmonicida, but also induce the specific neutralizing antibody of IHNV in rainbow trout. The vaccine carrier could be used as polyvalent vaccine for the major pathogens of rainbow trout, and could be used as an effective route of disease control in rainbow trout farming in the future. |
