Variance Analysis Of Canine Distemper Virus N Protein C Terminus And Development Of A Epitope ELISA Detection Method

Canine distemper（CD）is a viral disease that affects a wide variety of animal families,including domestic and wild species of dogs,ferrets,raccoons,and a variety of other species.The disease is highly contagious via inhalation and fatal 50%of the time.The distribution of CDV is worldwide and causes great economic loss to the animal husbandry.The nucleocapsid proteinis the major virion structural proteins of CDV-nucleocapsid and can induce an antigen-specific adaptive immune response of the body.It is an ideal target antigen for CDV diagnosis,with the advantage of highest expression in the virus and better immunogenicity.There is a high mutation region（aa408⁵19）at the C-terminus of the canine distemper N protein,with the features of high mutation rates,certain hydrophobic capacity and a large number of potential epitopes.Studies showed that C-side mutants could affect the viral virulence,and the difference between wild-type and attenuated strains of N protein was mainly embodied in the C-side hypervariable region.Therefore,the variation and functional analysis of the region will contribute to the further study.To further investigate the specific variation of C-terminal hypervariable region of N protein,102suspected canine distemper samples were collected for detection from Wuhan,Shanghai,Changchun and Dalian.The results showed that 19 samples were positive.Compared N gene of the positive samples with a dataset of several N gene sequences available from the NCBI database.The results indicated that there were 101 mutations in 336 nucleotide gene fragments.In the C-terminal hypervariable region of N-protein,36 sites mutated in the 112 amino acid deduced sequences,and the mutation rate was 32%,and significant differences in the C-terminal region of N protein were observed between the vaccine strains and the Chinese epidemic strains.In view of the above analysis,BALB/c mice were immunized with CDV truncated N protein（aa401-523）expressed by adenovirus vector.Immunized mouse spleen cells were fused with SP2/0myeloma cells,and 2 monoclonal antiboby strains were screened,and were named N1-C8 and N1-C41.The titer of cultural supernatant and mouse ascites of N1-C8 and N1-C41 were 1:1024,1:106 and 1:512,1:105,respectively,determined by indirect ELISA.Western blot results showed that there were differences between N1-C8 and N1-C41 MAbs in different virulence strains that identified may be related to the difference of N protein.The epitope of N1-C8 was linear epitope ⁴⁴⁰ENQGGDKYP IHFNDE⁴⁵⁴ by peptide scanning method,but the epitope of N1-C41 has not been screened,presumably due to its epitope was spatial conformation.The 440aa-454aa epitope ELISA assay was established with the screening the epitope as coating peptide.The ideal peptide coating concentration was set at 1μg/mL,the optimal blocking time was 4℃overnight,while 5%skimmed milk powder in PBST as blocking reagent showed best results,the temperature and time for reaction were 37℃and 2 h.The best dilution of primary antibody was 1:80;the secondary fold was 1:4000;the best incubation condition for serum and secondary antibody was37℃and 1 h.The critical value between positive and negative serum is 0.225.The sensitivity results showed that positive samples could be detected when the serum sample dilution factor was 1:320.The CV value of the intracranial repeatability test was 0.42%⁸.50%.The CV value of the variation coefficient between the plates was 0.36%⁸.92%,which was less than 10%.Specificity test revealed that the OD_450nm value of MEV positive serum was 0.207,and the OD_450nm value of ADV positive serum was 0.179,both were negative.The coincidence rates of 440aa-454aa epitope ELISA assay and commercial CDV ELISA kit were 88.2%,87.2%and 91.6%in the detection of positive serum,sample serum and negative serum,respectively,indicating that the selected epitope peptide showed high positive detection rate,with a certain diagnostic value.This study has ultimately opened up paths to the study of CDV ELISA assay,which lays a foundation for the further study of N protein epitope of CDV.