Establishment Of An Indirect ELISA Method For Tiger-originated Feline Panleukopenia Virus And Its Effect On Apoptotic Pathway

Feline pandeukopenia is an acute,highly lethal infection caused by Feline panleucopenia virus(FPV),also known as feline fever virus,feline infectious enteritis virus,and cat parvovirus.Under natural conditions,it can infect bear,tiger,mink,leopard and other carnivorous animals.It has the characteristics of acute onset,short duration,frequent morbidity and high case-fatality rate.The disease causes a serious threat to wildlife and special economic animals,so controlling the prevalence,diagnosis and prevention of FPV in China is of great significance.In this study,tiger feces of infectious hemorrhagic enteritis in a tiger forest garden in Heilongjiang Province were taken as the research object.Through the methods of virus isolation and identification,animal regression test and molecular biological detection,it is proved that the pathogen of Panthera tigris altaica is the tiger parvovirus,named FPV-HER-NEFU.The parvovirus NS1 protein plays an important role in the replication and transcription of the virus.The research of NS1 gene has important guiding role in the research on the biological characteristics and diagnosis and prevention of parvovirus.In this study,the full-length NS1 protein gene of Amur tiger parvovirus was cloned and its coding protein was analyzed by bioinformatics and molecular genetic evolution tree was constructed.The 2007 bp target gene was successfully amplified and ligated into the expression vector pGEX-6P-1 to construct the recombinant expression vector pGEX-FPV-NS1.The positive recombinant plasmid was transformed into E.coli competent Rosetta and induced by IPTG.The soluble protein of interest is 102.6 kDa and has good immunogenicity.We used this recombinant protein as a diagnostic antigen to establish an indirect ELISA assay for tiger-type parvovirus.The optimal conditions were determined by matrix method,including antigen concentration of 250 ng/well,serum dilution of 1:100,and closed liquid bottom.The substance was 5%skim milk,the concentration of the secondary antibody was 1:8000,and the substrate action time was 25 minutes.The experimental results show that the established indirect ELISA method has higher specificity and sensitivity.The experimental results show that the established indirect ELISA method has higher specificity and sensitivity.Finally,TdT-mediated dUTP Nick-End Labeling(Tunel)fluorescence staining,DNA fragmentation analysis,real-time fluorescence quantification,Western Blot and other methods were used to investigate the effect of FPV on apoptosis of F81 cells.The results showed that the apoptosis rate of F81 cells increased with the prolongation of viral infection time.F81 cells infected with FPV significantly activated intracellular Caspase-9 and Caspase-3,while Caspase-8,Fas and FasL were no significant change in expression.FPV may induce apoptosis of F81 cells through Caspase-9/-3 cascade activation,and FPV infection can up-regulate the expression of Bax in the cell and down-regulate the inhibitory molecule Bcl-2 expression.In summary,this study successfully isolated a parvovirus FPV-HER-NEFU strain from the feces of Siberian tiger,obtained its major ORF sequence and bioinformatics analysis by cloning and sequencing;and expressed pGEX-FPV The-NS1 protein is an antigen,and an indirect ELISA assay for feline serum antibodies has been established.This study will help to analyze the molecular prevalence and genetics of Chinese tiger-derived parvovirus,and lay a foundation for the screening of the tiger-type parvovirus vaccine strain and the diagnosis of the disease.The preliminary study on the effect of FPV on the apoptosis of F81 cells provides a theoretical basis for further study of the pathogenesis of FPV infection on host cells,and also provides new ideas for the study of other viruses.