Study On The Key Preparation Technology And The Immune Response Mechanismof The Infectious Spleen And Kidney Necrosis Virus Rhabdovirus Combined Inactivated Vaccine

Siniperca chuatsi is an important economic edible fish in China,commonly known as"Osmanthus fish",which belong to Perciformes,Siniperca Gill.In recent years,the breeding industry of Siniperca chuatsi is subjected to heavy losses because of the increasing of breeding scales and the outbreak of infectious diseases.How to effectively prevent and treat infectious diseases has become the primary problem of the sustainable development of Siniperca chuatsi breeding.To immunize vaccines is one of the most effective ways to prevent fish infectious diseases.Inactivated vaccines cultured with cell are widely used in the production of fishery vaccines due to their simple operation and low cost.The epidemic etiology of Siniperca chuatsi is complex and diverse,so the single antigen vaccine can no longer meet the market demand.In order to develop the dual inactivated vaccines of infectious spleen and kidney necrosis virus?ISKNV?and Siniperca chuatsi rhabdovirus?SCRV?,this study screened the culture cells these are sensitive to virus,confirmed the inactivation conditions of the inactivated agents,and established the detection method of the double antibody sandwich antigen of rhabdovirus.In addition,the change of the immune factors expression was detected by fluorescence quantitative method after immuning Siniperca chuatsi,and the specific immune response pathway caused by the dual inactivated vaccines of ISKNV&SCRV was preliminarily explored,which laid the foundation for clarifying the immune response mechanism of Siniperca chuatsi that induced by the dual inactivated vaccines.The specific experimental results are as follows:?1?In order to cultivate a high-valent virus that is suitable to vaccine production,in this study,two single-shaped cells named as CPB-E1and CPB-F1were isolated and purified from the multi-formed carp brain tissue cells?CPB?by paper separation method.The growth curves of the two cells were measured by viable cell counting method.The results indicated that CPB-E1entered the decline phase on the 7th day,CPB-F1 entered the decline phase on the 5th day,and CPB-E1 grew longer,which is more suitable for virus proliferation.The ISKNV and SCRV viruses were inoculated into the two cells respectively to compare the sensitivity of the two cells to the virus.The virus titer results showed that the ISKNV virus was inoculated with CPB-E1:10^7.63TCID₅₀/mL;CPB-F1:10^6.92TCID₅₀/mL;CPB:10^6.5TCID₅₀/mL.The SCRV virus was inoculated with CPB-E1:10^12.67TCID₅₀/mL;CPB-F1:10^12.33TCID₅₀/mL;CPB:10^11.39TCID₅₀/mL.The virus titer of CPB-E1 cells was higher than CPB-F1 and CPB.In summary,CPB-E1 cells were selected for subsequent experiments because they are more suitable for virus growth and more sensitive.?2?In this study,formaldehyde and BPL,which are currently used in inactivated viruses,were used to inactivate ISKNV and SCRV viruses to determine the optimal inactivation conditions for the dual inactivated vaccines.The SCRV virus was respectively inactivated for 4,6,12,24,48,and 72 hours with BPL solution at a final concentration of 0.5‰,1‰,2‰at4?and formaldehyde solution at a final concentration of 2‰,1‰at 28?,80r/min.The ISKNV virus was respectively inactivated for 4,6,12,24,48,and 72 hours with BPL solution at a final concentration of 0.5‰,1‰at 4?and formaldehyde solution at a final concentration of 2‰,1‰at 28?,80r/min.The inactivated virus was blindly passaged for three generations to observe the cytopathic effect?CPE?,and the fish body safety test was conducted.The results revealed that the SCRV virus can be completely inactivated with the BPL solution at a final concentration of 2‰at 4?for 72h and formaldehyde solution at a final concentration of 2‰or1‰at 28?for respectively 6 h or 12 h.The ISKNV virus can be completely inactivated with1‰BPL solution at 4?for 6h and 2‰formaldehyde inactivation at 28?for 72 h.?3?In order to detect the antigen content of inactivated vaccine,the mice was immunized with purified SCRV virus and recombinant G protein respectively.9 monoclonal hybridoma cells were obtained after fusing,cloning and screening.After biological characteristics identification such as antibody subtype and epitope screening,it was confirmed that 4E12 1F3 and 4H8 1F11 can recognize SCRV virus better.4H8 1F11 was used as a coating antibody,and 4E12 1F3 was biotinylated as a detection antibody,and a double antibody sandwich ELISA assay method was established.The standard curve of rational function measured by this method is y=?a+bx?/?1+cx+dx^2??R=0.999?,and the minimum detection limit is 15.6 ng/mL.The diluted virus solution was detected by this method.The result found that the antigen content is positively correlated with the virus titer and the antigen content can be sensitive to the change of the virus titer,which indicated that the method is reliable and can be used to detect the antigen content of the SCRV virus of the vaccine.?4?The healthy Siniperca chuatsi were immunized with the prepared ISKNV&SCRV dual inactivated vaccines.At 7 days and 14 days after immunization,the two tissues of the head kidney and spleen were took and the fluorescence quantitative detection was performed to check the expression levels of specific immune factors MHCI,MHCII,CD4,CD8,IgM in order to explore the specific immunity mechanism of Siniperca chuatsi caused by the dual inactivated vaccines.The expression levels of these factors at 14d were generally higher than these at 7d.The MHCI factor was significantly up-regulated 4.9-fold?P<0.05?in the head kidney and3.3-fold?P<0.01?in the spleen at 14d.The CD8 factor expression was the highest in the head kidney at 14d and significantly up-regulated 4.0-fold?P<0.05?,and it was inhibited in the spleen.MHCII was up-regulated in the head kidney,and significantly up-regulated 1.5-fold?P<0.05?at7d and up-regulated 1.6-fold at 14d.It was down-regulated in the spleen at 7d and slightly up-regulated at 14d.CD4 was highly significantly up-regulated 4.8-fold and 9.5-fold?P<0.01?in the head kidney at 7d and 14d respectively,and the expression level in the spleen was down-regulated at 7d,and significantly down-regulated?P<0.05?at 14d.The expression levels of IgM were up-regulated in the head kidney and it was significantly up-regulated 1.2-fold?P<0.05?at 7d and 2.0-fold?P<0.05?at 14d.The above results indicated that the ISKNV&SCRV dual inactivated vaccines successfully induced cellular and humoral immunity in the fish body,and stimulated the MHC-type complex-mediated immune pathway.