Preparation And Immune Effect Evaluation Of Yeast Oral Vaccine Against Largemouth Bass Ranavirus(LMBV)

Largemouth bass(Micropterus salmoides)is one of the five most successful exotic species introduced into aquaculture in China and plays an important role in freshwater fisheries.However,with the expansion of breeding scale and the increased stocking density,disease outbreaks are becoming more prominent.Among them,Largemouth bass ranavirus(LMBV)infects largemouth bass,leading to significant mortality and economic losses.Unfortunately,there are no safe and effective drugs against this disease.Vaccines can effectively prevent and control the occurrence of infectious diseases caused by viruses,and are in line with the national development direction of aquatic drug research and development.Oral vaccine can directly target the intestinal mucosal immune system,effectively activate systemic immunity and mucosal immunity.It is superior to injection vaccine or immersion vaccine in many aspects and has a good application prospect.Adjuvants can enhance the immune effect of vaccines and have a multiplier effect.Therefore,appropriate adjuvants are often added during vaccine development to enhance the protective effect of vaccines.Based on the yeast expression system,this study used the yeast a-lectin surface display technology to display the major capsid protein(MCP)of LMBV.The LMBV yeast oral vaccine EBY100-OMCP was prepared.Fusion expression of the B subunit of Escherichia coli heat-labile enterotoxin(LTB,mucosal immune adjuvant)and MCP protein to obtain yeast vaccine EBY100-LTB-OMCP.To evaluate the immune effect of the oral vaccines,we measured the activity of immune-related enzymes and detected the expression levels of immune-related genes.The serum neutralizing antibody titers and the RPS of the vaccine after LMBV challenge were evaluated.The specific research results are as follows:1.Preparation of yeast oral vaccines(1)The MCP gene sequence of LMBV(Gen Bank:MK836319.1)was codon-optimized for S.cerevisiae expression using the online software.The optimized MCP gene was named OMCP.The CAI of OMCP was 0.79(that of MCP was 0.65)and the GC content was 42.98%(that of MCP was 55.32%),which was easier for subsequent S.cerevisiae to express MCP protein.(2)OMCP and ltb were amplified in vitro and cloned into p YD1 vector(S.cerevisiae surface display vector)to obtain recombinant vectors p YD1-OMCP and p YD1-LTB-OMCP.The p YD1-OMCP and p YD1-LTB-OMCP were introduced into S.cerevisiae cells by electrotransformation.The recombinant yeast EBY100-OMCP and EBY100-LTB-OMCP were preliminarily screened by minimal dextrose plates(MDP),and the recombinant positive yeast could grow on MDP.Using the recombinant yeast EBY100-OMCP and EBY100-LTB-OMCP as templates respectively,the target fragments p YD1-OMCP(1391 bp)and p YD1-LTB-OMCP(1716 bp)could be amplified.The expression of EBY100-OMCP and EBY100-LTB-OMCP was induced by galactose in the dark for48 h.The expression of recombinant protein was detected by indirect immunofluorescence.EBY100-OMCP and EBY100-LTB-OMCP recombinant yeast cells could produce obvious specific red fluorescence.2.Evaluation of vaccine effect(1)Before the collective immunization,100μL 107CFU/m L recombinant yeasts were administered via oral gavage to Largemouth bass.Immunohistochemistry was used to analyze whether the yeast could deliver the antigen to the second intestine of the fish.The results showed that the presence of the antigen could be detected in the midgut of the fish.(2)The recombinant yeasts were induced to express the recombinant proteins in large quantities,then fully mixed yeast and feed(1×10~7 colony forming units(CFU)/g).Continuous oral immunization for 7 d.Healthy largemouth bass were randomly divided into four groups,namely the Control(ordinary feed),EBY100-p YD1(empty vector yeast feed),EBY100-OMCP(MCP vaccine)and EBY100-LTB-OMCP(LTB-MCP adjuvant vaccine).Non-specific and specific responses were measured at 1 d,7 d,14 d,21 d,28 d,35 d and 42 d after immunization.Compared with the control group,the activity of T-SOD in the immunized groups was significantly higher(P<0.05 or P<0.01).In the serum,the T-SOD activity of the EBY100-OMCP group was significantly increased at 7 dpi(258.5±24.4 U/m L).The T-SOD activity in the EBY100-LTB-OMCP group peaked at28 dpi(345.1±20.5 U/m L).The AKP activity in the serum of the immunized group increased significantly at 7 dpi and peaked at 21 dpi(EBY100-OMCP:31.3±1.95 U/100m L;BY100-LTB-OMCP:40.3±1.86 U/100 m L).The AKP activity in the intestinal mucus reached a peak at 14 dpi(EBY100-OMCP:338.6±17.44 U/gprot;BY100-LTB-OMCP:388.3±17.00 U/gprot).ACP activity in the serum and intestinal mucus began to decrease after 21 dpi in both vaccine groups.In the serum,the LZM activity of the two vaccine groups increased steadily,with highest values of 512.8±30 U/gprot(EBY100-OMCP group)and 696.6±17.00 U/gprot(EBY100-LTB-OMCP group).The LZM activity in the intestinal mucus of the two vaccine groups began to decrease after 28 dpi.(3)Throughout the immunization period,the immune enzyme activity of the EBY100-LTB-OMCP group was superior to that of the EBY100-OMCP group,especially at 21 dpi,28 dpi,and 35 dpi(P<0.05 or P<0.01).Compared with the control group,the expression levels of the four genes(TNF-α,IL-1β,Ig M,and Ig T)in the immunized groups showed a trend of increasing first and then decreasing.The expression of TNF-αand IL-1βin the head kidney was the highest at 21 dpi(EBY100-OMCP group:2.3-fold,2.2-fold;BY100-LTB-OMCP group:2.7-fold,2.3-fold),and the highest expression in the intestine was at 28 dpi(EBY100-OMCP group:4.8-fold,4.0-fold;BY100-LTB-OMCP group:5.6-fold,4.8-fold).The expression of Ig M in the head kidney(EBY100-OMCP group:3.8-fold;EBY100-LTB-OMCP group:4.3-fold)was higher than that in the intestine(EBY100-OMCP group:2.6-fold;BY100-LTB-OMCP group:3.2-fold),and the head kidney maintained a high level of Ig M for a long time(28–42 dpi).Ig T was more highly expressed in the intestine(EBY100-OMCP group:5.6-fold;BY100-LTB-OMCP group:6.7-fold)than in the head kidney(EBY100-OMCP group:3.2-fold;BY100-LTB-OMCP group:4.1-fold).(4)Serum neutralizing antibody titers of the two groups reached 1:85.Oral vaccination protected against LMBV infection.The relative percent survival was 52.1%(EBY100-OMCP)and 66.7%(EBY100-LTB-OMCP).In summary,oral LMBV yeast oral vaccine is safe and harmless,can significantly improve the specific and non-specific levels of largemouth bass,and can be used as a promising and effective candidate vaccines against LMBV infection.It is of great significance to the disease prevention and control of largemouth bass and has reference value for the development of other vaccines.