Development And Properties Research Of Canine Distemper Virus N Protein Monoclonal Antibody And Analysis Of Canine Parvovirus VP2 Gene Mutation

Canine distemper is composed of Paramyxoviridae morbillivirus Canine distemper virus(CDV) caused an acute, febrile, highly contagious, viral infectious diseases. Canine parvovirus disease is caused by Canine parvovirus(CPV), is one of severe infectious diseases in dogs, with the main symptoms of enteritis and diarrhea. In order to control and prevent these diseases better, we analysis of CPV VP2 gene mutations and preparation N protein of CDV monoclonal antibody.The study used the CDV3 virus RNA as the template, constructing CDV N protein truncated(aa277-471), expression and purification in prokaryotic vector, and immuned BALB/c female mice to obtain the B lymphocyte. After CDV N protein truncated(aa277-471) ELISA screening, we obtained a hybridoma cell strain named 1N8, which subtype identification for Ig G1 a, kappa chain and immune fluorescence and WB detection positive.A series of synthesized peptides based on the amino acid sequence of the CDV N protein were screened one peptide 351NFGRSYFDP359 was difined as the minimal linear peptide epitope recognized by the mab 1N8. We propose that this motif is a linear B-cell epitope of the N protein. The alignment and comparison of the 1N8-defined epitope with other Morbillivirus sequences showed that the epitope is conserved among the Morbillivirus including Measles virus, Rindispest virus, Peste-des-petits-ruminants virus, and Rinderpest virus. The identified epitope may be useful for clinical applications, as well as utilized as a tool for further study regarding the structure and function of the CDV N protein.The variable regions of the heavy chain(VH) and light chain(VL) were amplified by RT-PCR from the hybridoma 1N8, which secretes the monoclonal antibody against CDV N protein(aa 277-471). The VL and VH CDSs were combined using SOE-PCR by a 12 amino acid flexible linker(SSGGGGSGGGGS), which produced the sc Fv gene(named sc Fv/1N8). After sequence analysis, the sc Fv/1N8 gene were cloned into the prokaryotic expression vector PET32 a with a His-tag. The recombinant sc Fv/1N8 protein was successfully expressed in recombinant Escherichia coli by IPTG induction. Moreover, the binding activity and specificity of the sc Fv was determined by indirect ELISA(His tag) and competitive ELISA. The recombinant sc Fv/1N8 protein reported here could provide the basis for further antiviral drug research on the sc Fv molecule.we study 351NFGRSYFDP359 epitope vaccine immune effect using expression vector of adenovirus. To construct the expression cassette containing 351NFGRSYFDP359 and CPV VP2 gene, The recombinant adenovirus packaging success by PCR and WB assay, at DNA and protein level. After immunization of mice, compared with commercial control vaccine, ELISA titer of recombinant CPV VP2 equivalent, but the relatively low ELISA titer of CDV.In the present study, sequence analysisi of several VP2 genes of CPV contributed to the predominant CPV strain as CPV-2a(Ser297Ala), with two CPV-2b(Ser297Ala). Sequence comparison revealed homologies of 99.3–99.9%, 99.9% and 99.3–99.7% within the CPV 2a isolates, within the CPV 2b isolates and between the CPV 2a and 2b isolates, respectively. In addition, several non-synonymous and synonymous mutations were also recorded. The phylogenetic tree revealed that most of the CPV strains from different areas in China were located in the formation of a large branch, which were grouped together along with the KU143-09 strain from Thailand and followed the same evolution. In this study, we provide an updated molecular characterization of CPV 2 circulation in China.