Development And Immune Effect Evaluation Of Largemouth Bass Ranavirus Inactivated Vaccine

Largemouth bass is one of the fastest-growing species of freshwater aquaculture fish in my country.It has the advantages of fast growth,delicious meat and no intermuscular spines,and is welcomed by consumers.With the continuous expansion of the breeding scale,density and seed circulation,a variety of diseases frequently occur in the breeding of largemouth bass,covering viral diseases,bacterial diseases and parasitic diseases.Among them,Largemouth bass virus is one of the most important viral disease pathogens.The typical symptoms of largemouth bass infected with LMBV are local ulcers on the body surface,hepatosplenomegaly,and the liver turns white and yellow.At present,there is no effective drug for the diseases caused by LMBV,and the current stage is mainly to prevent or cut off the transmission route of the pathogen.In this thesis,by developing a safe,high-efficiency and low-cost inactivated vaccine for largemouth bass frog iridovirus disease,the vaccine is immunized by intraperitoneal injection,which can obtain a better immune protection effect within a certain dose range,and effectively reduce LMBV to largemouth bass.The fatality rate lays the foundation for the subsequent development and application of vaccines.The full text is mainly divided into the following four parts:1.Based on the major capsid protein（MCP）gene of LMBV,specific primers and Taq Man probes were designed,and a Taq Man q PCR detection method for LMBV was established by optimizing the reaction conditions.The results showed that the method used the p MD-LMBV-MCP recombinant plasmid as the standard,and the established standard curve had a good linear relationship in the concentration range of 4.6×10²copies/μL～4.6×10⁸ copies/μL,and the correlation coefficient（R²）was 1.The amplification efficiency（E）is 99.2%.Using this method to detect 30 clinical samples,the results show that the positive rate of LMBV is 40%,which is significantly higher than 13.3%of conventional PCR.The established Taq Man q PCR detection method has strong specificity,high sensitivity and good repeatability,and can be used for The detection and epidemiological investigation of clinical LMBV provide reliable detection methods for the early diagnosis of LMBV.2.A continuous cell line was established from mandarin fish spinal cord tissue and used to study the susceptibility to largemouth bass Rana iridovirus.The cell line,named SCC,has been subcultured for 40 passages.The best growth of SCC cells can be observed in L-15 medium supplemented with 20%fetal bovine serum at 28℃.Karyotype analysis showed that the number of normal diploid chromosomes in the 36th generation SCC cells was 48.Partial amplification and sequencing of 16S r RNA and COI genes confirmed that the SCC cell line was derived from mandarin fish.Typical lesions appeared after SCC cells were inoculated with LMBV.The viral titer of the supernatant of the lesioned cells on the7th day of infection was 10¹⁰ TCID₅₀/m L,and LMBV could be stably passaged on SCC cells.Through electron microscope observation of diseased cells,a large number of virus particles with a diameter of 132nm～174nm can be seen.3.Through the LMBV sensitivity experiment,it was screened that EPC is more sensitive to LMBV,and has advantages over the other three cells in terms of lesion time and viral load,and can be used for the expansion of LMBV culture.Using EPC,the optimal cell culture method for LMBV was optimized,and the inoculum dose,incubation time and incubation temperature were tested.The results showed that the virus inoculated cells with a dilution ratio of 1:10 had the best lesion effect and high viral load;after 50min incubation,the lesion effect was the best,and the viral load was the highest;LMBV cultured at 24℃had the best lesion effect and viral load Highest.In order to prepare a vaccine with better immune effect,the inactivation conditions,vaccine adjuvant and immunization dose were optimized,and the optimal inactivation conditions were finally selected.The time is 48h;the optimal immunization dose is 1312 adjuvant 10⁹ TCID₅₀/ml.4.To evaluate the inactivated vaccine against largemouth bass frog iridovirus cells by detecting the expression of immune-related genes,the changes of immune enzyme activity and antibody level,and the immune protection rate.The changes in the expression levels of CD8-α,MHCI-α,Ig M,TNF-α,IL-8,and IL-1βwere found through the detection results.At different times after immunization,the six immune genes all showed a first increase and then a decrease to varying degrees.trend.The enzymatic activity results of T-AOC,SOD,ACP and AKP showed that there was basically no significant change in the enzymatic activity of the experimental group of T-AOC compared with the control group.At different times,the enzyme activities of SOD,ACP and AKP in the immunized group basically kept increasing at first and then decreased.ELISA results showed that,compared with the control group,different doses of the vaccine significantly increased antibody levels at different times,and the higher the immunization dose,the higher the antibody level.The largemouth bass frog iridovirus disease inactivated vaccine was used to challenge the sea bass.The results showed that the mortality rate of the immunized sea bass was significantly lower than that of the control group,indicating that the developed inactivated vaccine has a better immune effect.This study is based on the isolated largemouth bass iridovirus,an inactivated vaccine prepared by inactivation method.After immunizing sea bass,different immune indicators were used to evaluate their immune effects,and it was found that they had better immune protection,and the highest immune protection rate was 88.9%,indicating that this vaccine has development value and application prospects.