Molecular Characteristics And Primary Application Of Expressed VP2 Of Feline Panleukopenia Virus In Some Areas Of Zhejiang Province From 2017 To 2020

Feline panleukopenia(FP)is an acute highly contagious disease caused by feline panleukopenia virus(FPV)in cats and felines,also known as feline distemper,feline infectious enteritis and feline parvovirus infection.FPV infects a wide range of hosts with high mortality,especially in cats,minks and raccoons.Vaccination is one of the main ways to prevent feline panleukopenia.Understanding the genetic variation of feline panleukopenia virus is of great significance for the diagnosis and prediction,as well as the study of new vaccines for the prevention and treatment of the disease.From 2017 to 2020,23 clinical samples of the cases suspected of feline panleukopenia virus infection were collected from Zhejiang animal hospital.The virus were obtained by inoculating in the FK81 monolayers for three times.DNA was extracted and identified by PCR.A total of 20 samples were positive for FPV.One of the FPV isolates,ZJFPV2,was selected for electron microscope observation,whole genome sequence identification and analysis.VP2 gene sequence alignment results showed that the nucleotide homology between 20 FPV isolates was 98.7%～100.0%,and that between the isolates and 37 reference strains was 97.3%～100.0%.The homology between the isolate and the reference strain was very high,and the variation was not significant.The 20 FPV isolates belonged to group G2 and G3 respectively,and their genotypes had obvious Asian characteristics.The isolation,identification and molecular epidemiological analysis of FPV can provide a basis for the subsequent development of an effective vaccine against the epidemic strain of FPV.The VP2 gene of ZJFPV2 isolate was amplified by PCR and inserted into prokaryotic expression vector.The recombinant plasmids pET32a(+)-VP2 and pET28a(+)-VP2 were constructed and transformed into E.coli BL21(DE3)for induction by IPTG.The results showed that the recombinant plasmids pET32a(+)-VP2 was constructed successfully and could be expressed in E.coli BL21(DE3).Western blot analysis showed that pET32a(+)-VP2 and pET28a(+)-VP2 successfully fused with His-tag protein.After pET32a(+)-VP2 was highly expressed in BL21(DE3),the recombinant VP2 protein was purified by his tag protein purification column and used as detection antigen.The optimal conditions of the reaction were determined as follows:The concentration of antigen coating was 5.64μg/mL,the concentration of negative and positive serum was 1:40,the coating conditions were 4℃overnight,the blocking time was 1 h,and the color development time was 15 min.The method had high repeatability and specificity.These results laid a foundation for the detection of FPV VP2 antibody.The VP2 gene of ZJFPV2 isolate was amplified by PCR and inserted into different eukaryotic expression vectors.The recombinant plasmids pcDNA3.1(+)-VP2 and pCAGGS-VP2 were constructed.pcDNA3.1(+)-VP2 and pCAGGS-VP2 were transfected into FK81 cells.The results of indirect immunofluorescence showed that VP2 gene was expressed in pcDNA3.1(+)and pCAGGS eukaryotic expression plasmids.The recombinant plasmids pCAGGS-VP2 and pcDNA3.1(+)-VP2 were used as DNA vaccine to immunize mice respectively,and the empty vector was set as the negative control.Two weeks after the third immunization,the serum was obtained and the antibody level was detected by indirect ELISA.The results showed that the specific antibody could be detected in the serum of mice,but not in the control group.These results indicate that the eukaryotic expression plasmids pCAGGS-VP2 and pcDNA3.1(+)-VP2 constructed in this study can induce mice to produce specific antibodies,which provides a basis for further research on FPV nucleic acid vaccine.