Isolation, Identification And The Genome Cloning Analysis Of Feline Parvovirus And Expression Of VP2 Gene In E.coli

Feline panleukopenia is a highly contagious viral disease of kittens with a high mortality caused by feline panleukopenia virus (FPV), a member of parvoviridae. Not only can FPV infect all members of the cat family , but also other wild animals are susceptible to infection, such as raccoons, coatimundis, and ringtails etal, which has pose a serious threat to the economic animals and wildlife. In this study, one Feline panleukopenia virus (FPV)isolate named CC-1 was isolated and identified; The genome sequence of isolate CC-1 was obtained by the molecular biology techonology DNA sequencing. Then the molecular characteristics and phylogenetic of the FPV CC-1 isolate were analyzed by bioinformatics, his study is assisting to the study of molecular epidemiology of the Feline panleukopenia virus isolated from the nature and might offer an effective way to Feline Parvovirus infection. The recombinant plasmid pGEX-4T-VP2 was transformed into E.coli BL21 and the expression product-recombinant nucleocapsid protein of FPV was obtained under the optimized condition of host cell cultivation and IPTG induction.FPV is a small virus with a single-stranded positive-sense DNA genome and is a member of the family Parvovirus. The ssDNA genome is about 5200bp nucleotides in length, and has terminal palindromes of about 150 bases at the 3′and 5′ends. Promoters at genomic maps units 4 and 40 give rise to messages (R1~R3) for the non-structural proteins (NS) and capsid proteins (VP), respectively. NS1 and NS2, and VP1 and VP2 are formed by alternative splicing from the same mRNA.Isolation and identification of the FPV CC-1 isolate. The spleen of cats collected form Changchun region were FPV positive by PCR. Inoculate the tissue lysate on monolayer of F81 cell, 48h post-inoculate the obvious cytopathogenic effects were observed. The FPV -like particles about 20nm were observed in the infected cell culture lysate by electron microscopy.It was demonstrated to be a Feline Parvovirus by a series of systematic identification such as morphology test, physicochemically test, serological neutralization test, artificial Feline's infection test, and molecular biology test.According to the reported complete nucleotide sequence of FPV (CU-4) in GenBank, Three pairs of primers were designed and synthesized. VP1,VP2 and NS1 gene of isolate was amplified.The products of PCR were cloned into pGM-T vector,pGM-T vector and PMD18-T. the positive clone was identified by enzyme cutting,PCR and sequencing.As a results, The whole sequence of VP1gene was analyzed using DNAStar software.The results indicated that the VP1 gene with 2306bp long had a single opening read frame 2184bp in length and coded a polypeptide of 727 amino acids.The sequence analysis demonstrated that represent reference strains loaded down from GenBank,the result of ORF sequence analysis indicated that CC-1 strain exhibited 99.3% homology of the nucleotides and amino acid with that of the XJ-1strain in our county; The whole sequence of VP2gene with 1806bp long had a single opening read frame 1775bp in length and coded a polypeptide of 584 amino acids.The sequence analysis demonstrated that represent reference strains loaded down from GenBank,the result of ORF sequence analysis indicated that CC-1 strain exhibited 97.3%～99.8% homology of amino acid ; The whole sequence of NS1gene with 2.35kb long had a single opening read frame 2007bp in length and coded a polypeptide of 668 amino acids. Share 99.01%,98.91%,98.86%,98.76%,98.66%,99.4%identity of nucleotides and 99.2%,99.13%,99.07%,99.13%,99.07%,99.4% identity of amino acids with that of the 193/70 strain,PLI-IV strain,TU-2 strain,CU-4 strain,94-1 strain,XJ-1 strain respectively.The distances of NS1 gene of all strain analyzed by phylogenetic tree,we can conclude that CC-1 strain is near to MEM,CPV strain and far to PPV strain. This study clarifies the molecular characteristics of genetic variation of domestic strains of FPV. And have a significance in clinic and epidemiological study.The FPV VP2 gene was cloned into pGEX-4T-1 and transferred into E.coil BL21. an 64.6Ku expressed fusion protein was induced under 1.0mmol/L IPTG 5h and 37oC, which was identified by SDS-PAGE .The FPV VP2 protein could be expressed by pGEX-4T-VP2 in BL21 at high level, up to 21% of the total protein of the induced bacteria. The recombinant protein was confirmed by Western-blot that there was a positive reaction with standard positive blood serum. The result indicated that the protein had favourable immunogenicity. The provided favourable antigen for establishment of indirect ELISA which could detect FPV antibody.