The Preparation And Immunization Research For Oral Vaccine Of Main Pathogen Inclusion Bodies Of Grouper

Aquaculture has become the fastest growing food industry around the world in recent years.With the intensive development of aquaculture,aquatic diseases occur increasingly.Infectious diseases caused by bacteria,viruses,fungi and parasites restrict the development of aquaculture and cause serious economic losses.Antibiotics were used more and more extensively,leading to the increase of antibiotic-resistant microorganisms.The abuse of antibiotics coulde make the food safety problems more serious,and damage human health and the development of environmentally-friendly aquaculture.Vaccination strategies are very effective in protecting fish from disease and infection,which receives more and more attention from various countries.However,simple and effective vaccines are still under development to meet the market demand.For fish breeding,the onset often occurs in the juvenile stage,which is difficult to prevent and controland could result in high mortality and huge economic losses.Therefore,the immunization pathway becomes a bottleneck for the development of fish immunization strategies,and only when an effective oral vaccine is developed will it have industrial application value.In order to control the diseases caused by the most common bacterial pathogen,vibrio sp.and the most fetal virus,nervous necrosis virus,recombinant key vaccine antigen of the pathogens were prepared.The two antigens were the grouper nervous necrosis virus major capsid protein(gNNVmcp)and vibrio parahaemolyticusouter membrane protein K(vpOmpK),respectively.The prokaryotic expression vectors pET-SUMO-gNNVmcp and pET-28a-vpOmpK were constructed by molecular cloning.The recombinant gNNVmcp and vpOmpK were successfully expressed in the form of inclusion bodies.Inclusion body antigens were produced in large quantity using the high-density fermentation,and were prepared easily by centrifugation with higher purity.The formalin inactivated vaccines of Vibrio parahaemolyticus and Vibrio harveyi were also prepared.Feeds for groupers including inactivated Vibrio vaccine feed(Vph)or inclusion body vaccine feed(IB)were made.Healthy juvenile fish of hybrid grouper(Epinephelus fuscogutatus? × Epinephelus lanceolatus?)was used to test the effectiveness of vaccines.In the immunization trial,six experimental groups were compared: the control,vibrio experimental group(Vph),Vibrio inactivated + low dose inclusion body group(Vph + IB-l),Vibrio inactivated + high dose inclusion body group(Vph + IB-h),low dose inclusion body group(IB-l)and high dose inclusion body group(IB-h).The expression of immune-related genes in different immune organs of grouper was checked after oral immunization of fish.As a result,the expression of igm in Vph+IB-h vaccine group was significantly up-regulated in the hindgut.Interestingly,the expression of igm in Vph vaccine group and igd in Vph+IB-h vaccine group showed a significantly up-regulation in blood cells.The il-1? expression in Vph-immunized group in head kidney was significantly up-regulated compared with other groups.The tnf-? expression showed a significant up-regulation in IB-h group.The inclusion bodies were incubated with serum to extract proteins that could bind to inclusion bodies in serum.In the proteins which bound to antigen inclusion bodies,more immunoglobulins were identified by mass spectrometry in the Vph+IB-h experimental group and the IB-l experimental group.All the experimental results suggested that the inclusion body oral vaccine had a certain regulatory effect on fish immunoglobulin genes and immune-related genes,and it was expected to be a new generation of fish immune vaccine.