Research On The Key Techniques Of Vaccine Testing For Infectious Spleen And Kidney Necrosis Virus Of Siniperca Chuatsi

Infectious spleen and kidney necrosis virus(ISKNV)is mainly viral agents causing high mortality and significant economic losses to mandarin fish industry.Vaccination is the most effective strategy against this disease.In the process of vaccine preparation,safety and efficacy are important to evaluate the quality of vaccines.Rapid inactivation test and determination of effective antigen are the key technologies to evaluate the safety and efficacy of inactivated vaccines.Therefore,in present study,inactivation test and determination of effective antigen of inactivated vaccine for ISKNV were developed,and these methods were used to optimize inactivation parameter and duration for antigen storage.The main results were as follows:1.An early gene of ISKNV with highest transcript level was confirmed by qRT-PCR from transcriptomic profiles of Mandarin fish brain cells(CPB)after ISKNV infection,and then a fast inactivation test was developed for infectious spleen and kidney necrosis virus(ISKNV).First,we drew standard curves concerning relationship between Ct value and copy numbers of plasmid pMD099,and the linear equation is:Ct=-3.42lgX+39.455(R2=0.998),the minimum detection limit is 3 copies/μL.Results of repeated trials showed variation of coefficients in intra-and-inter groups were less than2%,indicating the method has high sensitivity and repeatability.Results of sensitivity test showed that ISKNV transcripts were detected from cells inoculated with one copy of ISKNV virus at 7 d after inoculation.Results of cell blind passage trial and fish safety test showed that 0.1% final concentration of formaldehyde inactivated ISKNV inoculated cells passaging three generations did not appear CPE,and did not cause clinical symptoms and death,indicating this rapid virus inactivation test has higher sensitivity than cell blind passage trial and the fish safety test.2.It is confirmed by RT-PCR that ORF003 L has an 80 bp intron named IN-3 based on sequences blast between ISKNV Unigenes obtained from CPB cells after ISKNV inoculation with ISKNV genome.A fast detection method of ISKNV inactivation based on mRNA detection by using nested PCR primer spanning IN-3 to amplify cDNA ofCPB cell after ISKNV inoculation.Results showed that two electrophoretic bands were detected from cells inoculated with one copy ISKNV virus at 7 days after inoculation,one is 209 bp,the other is 289 bp,can eliminate the influence of viral DNA residue in samples.Results indicated that ISKNV ORF003 L mRNA was detected from cells inoculated with 0.1% formaldehyde inactivated ISKNV at 7 days after inoculation by nested RT-PCR.However,results of cell blind passage trial and fish safety test showed that 0.1% final concentration of formaldehyde inactivated ISKNV inoculated cells passaging three generations did not appear CPE,and did not cause clinical symptoms and death,indicating this rapid virus inactivation test has higher sensitivity than cell blind passage trial and the fish safety test.3.The particles of ISKNV were purified by sucrose density gradient centrifugation.The monoclonal antibodies were prepared by conventional hybridoma technique.Two positive hybridomas named 1C8 1B9 and 3B12 6B3 were acquired,each of which was used for ascites production.The antibody titer of ascites reached 1:106~1:103by indirect ELISA determination.The monoclonal antibody was purified by Protein G-Sepharose affinity chromatography column from collected ascites.A double antibody sandwich ELISA detection method for quantification of the effective antigen of ISKNV inactivated vaccine was established using monoclonal antibody 1C8 1B9 as coating antibody and biotin-labeling monoclonal antibody 3B12 6B3 as detection antibody.The linear range of standard curve was 4.69~300 ng/mL and the minimum detection limit is 0.9 ng/mL.Results showed that the effective antigen of ISKNV inactivated vaccine were positively correlated with the viral titer of pre-inactivation.4.Inactivation condition of ISKNV was optimized,and duration for antigen storage were determinedusing the proposed fast inactivation test and ELISA detection method.ISKNV was inactivated by Formalin,BPL and BEI in different conditions.Results showed that ISKNV was completely inactivated by 0.2% Formalin treated for 72 h,0.1%BPL treated for 6 h,4% BEI treated for 72 h.Effective antigen of ISKNV inactivated by BPL was higher than Formalin and BEI.ISKNV inactivated by BPL at 4 ℃ for 6h was optimal.Inactivated ISKNV was preserved at 4 ℃ and-80 ℃ for 1,3,6,12 month.ELISA results showed that effective antigen of inactivated ISKNV was higher at-80 ℃than 4 ℃ and remained stable in 180 d.It is confirmed that duration for antigen storage was in 180 d at-80 ℃.