Abstract:Rainbow trout is an important cold-water fish farmed worldwide, and it is cultured on a large scale in Gansu, Qinghai, and Xinjiang Province in China. With the development of rainbow trout farming and increase in production recently, diseases have gradually become an important limiting factor for rainbow trout development, causing huge economic losses, threatening the health of rainbow trout, and sustainable aquaculture industry development in China. The major rainbow trout pathogens are infectious hematopoietic necrosis virus (IHNV) and Aeromonas salmonicida. IHNV is the causative agent of IHN and causes necrosis of the kidneys, spleens, and hematopoietic tissues of fish, with a mortality greater than 90%, causing great economic losses to the rainbow trout farming industry worldwide. The IHNV genome encodes five structural and one non-structural proteins. Among them, the glycoprotein, also known as the G protein, is the only surface protein and main antigen of the virus. G protein stimulates neutralizing antibodies in the host, inducing cellular immunity, and playing an important role in viral pathogenicity and immune responses. Currently, most studies have focused on DNA vaccines targeting the IHNV G protein. A. salmonicida causes furunculosis and ulcers in various fish species, including rainbow trout. Currently, internationally commercialized vaccines against A. salmonicida are widely used, however China still mainly depends on antibiotics for disease control. Therefore, in China, developing a vaccine against A. salmonicida is necessary. Herein, the IHNV-G protein gene was amplified by PCR and ligated into the pGEX-4T-1 plasmid to obtain 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 obtain the A. salmonicida vaccine carrier SC18032201-G, which expressed the G protein of IHNV as a polyvalent vaccine. The SC18032201 with pGEX-4T-1 plasmid (SC18032201-pGEX) and wild-type strain SC18032201 were used as negative controls. Isopropyl-β-D-thiogalactopyranoside (IPTG) was used to induce G protein in SC18032201-G cells. 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 demonstrated that after IPTG induction, specific reaction bands were detected by the recombinant vaccine carrier SC18032201-G carrying the pGEX-4T-1-G plasmid, but not by SC18032201-pGEX and wild-type A. salmonicida SC18032201, which indicated that the G protein was expressed in A. salmonicida SC18032201-G. The expression of G protein was optimized by adjusting the induction time, IPTG concentration, and culture temperature. Western blotting showed that the best induction condition for recombinant G protein expression by SC18032201-G was 0.2 mmol/L IPTG at 28 ℃ for 8 h. The optimized conditions were used for the incubation and induction of SC18032201-G, and the bacteria were inactivated with formaldehyde. Then the inactivated bacteria were emulsified with Montanide™ ISA 763A VG as adjuvant to prepare an oil-based vaccine. A PBS control was prepared using the same method. Rainbow trout were immunized by intraperitoneal injection with a vaccine or phosphate buffered saline (PBS) control. Forty-five days post-vaccination, 10 rainbow trout from each group were randomly selected for blood sampling from the caudal vertebrae. Blood was stored at 4 °C overnight and centrifuged to obtain the serum for antibody detection. The serum was diluted two-fold (1:2−1:256) and incubated at a 1:1 ratio with IHNV viral culture medium [100× TCID50(50% tissue culture infective dose)]. The mixture was added to a monolayer of carp epithelial tumor cells (EPC) with eight replicates per gradient. The cells were incubated at 15 ℃ and observed for 7 d. The cytopathic effect (CPE) of the culture was recorded, and the highest serum dilution that inhibited 50% of the CPE was recorded as the neutralizing antibody level. 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 titers of the immunized and control groups was highly significant (P<0.01). Forty-five days after immunization, rainbow trout were infected with 1×106 CFU/mL A. salmonicida SC18032201 by immersion and observed continuously for 30 d. The mortality of rainbow trout was recorded for 30 d, and the relative survival percentage was calculated. The results showed that the relative survival percentage of rainbow trout vaccinated against A. salmonicida was 100% after 45 d of immunization, which was significantly different from that of the control group. In conclusion, we constructed an A. salmonicida vaccine carrier that expresses the G protein of IHNV, which provides effective protection against A. salmonicida and induces the specific neutralizing antibody of IHNV in rainbow trout. The vaccine carrier can be used as a polyvalent vaccine for major rainbow trout pathogens and as an effective route for disease control in rainbow trout farming in the future.
