Establishment And Application Of Molecular Biological And Immunological Methods For Tilapia Lake Virus

The frequent occurrence of tilapia diseases in recent years has caused huge economic losses to the global tilapia breeding industry and severely restricted the sustainable development of the tilapia industry.Since 2009,a new fish disease outbreak in many countries-Tilapia Lake Virus(Tilapia Lake Virus,TiLVD)caused by Tilapia Lake Virus(TiLVD)has been reported to the whole country.The tilapia farming industry in the world brings huge threats and serious economic losses.At present,there are no effective prevention and control measures for TiLVD.Early diagnosis and vaccine immunization are the main methods for the prevention and control of viral diseases.In order to meet the detection needs of TiLV in different occasions and at different levels,the main research content of this paper includes two aspects: one is to design specific primers and probes based on the gene sequence of the conserved regions of the TiLV genome,and establish real-time quantitative PCR(real-time RPA,qPCR)and real-time RPA(real-time RPA)two molecular biological methods to meet the needs of detecting TiLV nucleic acid under different conditions;the second is to successfully develop two monoclonal antibodies(MAb)based on the protein encoded by the TiLV S10 gene.The MAb was used to establish three TiLV immune methods: indirect immunofluorescence(IFA),western-blot and immunohistochemistry,laying the foundation for subsequent TiLVD vaccine development and S10 protein function research.The research content and results are as follows:1.Establishment and application of TiLV molecular biological methodsIn this study,one pair of qPCR-specific probe primers and 12 pairs of real-time RPA primers and probes were designed based on the gene sequence of the conserved regions of the TiLV genome.Through the screening of primer probes and optimization of reaction conditions,the Taq Man probe was successfully established.Two nucleic acid detection methods,qPCR and real-time RPA,and their sensitivity,specificity and reproducibility were evaluated.The results showed that the optimal reaction conditions for qPCR were 95℃ for 30 s,95℃ for 5 s,60℃ for 30 s,and 40 cycles;the lowest detection limit was 62 copies.The best reaction conditions for Real-time RPA are 39℃ for 30 minutes,and the lowest detection limit is 620 copies.Through the treatment of grass carp reovirus,carp spring viremia virus,koi herpes virus,carp edema virus,frog iridescent virus,largemouth bass iridescent virus,mandarin fish rhabdovirus,infectious spleen and kidney necrosis,infectious hematopoietic organs The detection of pathogens such as necrotic virus,Streptococcus agalactiae,Aeromonas hydrophila,Aeromonas velvetii,etc.,has not been observed to have cross-reactions with other common fish pathogens,confirming that both methods have good effects on TiLV Specificity.The established qPCR and real-time RPA methods were used to detect 35 clinical tissue samples and 60 artificially infected tissue samples.Compared with the results of virus isolation,the diagnostic sensitivity of real-time RPA and qPCR were93.33% and 100%,respectively,and the specificity was 100 % for both.The sensitivity of qPCR is 10 times higher than that of real-time RPA.qPCR is more suitable for the quantitative analysis of TiLV in the laboratory and the detection of samples with lower virus content,and real-time RPA is more suitable for clinical diagnosis of TiLVD and preliminary screening of TiLV infection in basic units and farms with poor equipment.2.Establishment and application of TiLV immunological methodsDue to the lack of specific antibodies against TiLV,there is currently no immunological detection method to detect TiLV.Preliminary research and analysis have shown that the protein encoded by the TiLV genome segment 10(S10)gene has strong immunogenicity and is a good candidate antigen protein for preparing antibodies.This study first designed specific primers based on the S10 gene sequence,amplified the target fragment by PCR,and constructed the recombinant expression vector p ET32a-S10.The recombinant vector was transformed into BL21(DE3)competent cells to express the target protein,and a recombinant protein with a molecular weight of 38 k Da was obtained by IPTG induction.After immunizing6～8 week-old female BALB/c mice with the purified recombinant protein several times,two hybridoma cell lines that can stably secrete MAb against the S10 protein were screened and named 2C3 and 2E3,respectively.The subtype test results showed that the 2C3 antibody was of the Ig G1/к type,and the 2E3 antibody was of the Ig G2a/к type.The results of antibody titer determination showed that the titers of the two MAbs were 1:12800 and 1:51200,respectively.The two purified MAbs were used to successfully establish three immune methods for TiLV: IFA,western-blot and immunohistochemistry.IFA results showed that 2C3 and 2E3 only reacted positively with TiLV-infected TiB cells;Western blot results showed that both 2C3 and 2E3 could recognize S10 recombinant protein and TiLV;immunohistochemical results showed that both antibodies were infected with TiLV.Strong positive signals were detected in non-fish liver and swim bladder,indicating that both tissues have higher virus content.Therefore,the anti-TiLV-S10 protein MAbs prepared in this study have strong specificity and high potency,and provide important materials and support for the subsequent development of TiLV vaccines,immunotherapy,establishment of detection methods,and S10 protein function research.