Sequence Analysis Of Canine Pavovirus LM Strain And Eukaryotic Expression Of VP2 Protein

Canine parvovirus(CPV), a member of parvoviridae, is the cause of highly severe disease of dog industry. CPV can cause haemorrhagic gastroenteritis and myocarditis in dogs and make a significant reduction in leukocyte, which result in high morbidity and mortality in puppies.In this study, CPV-LM strain were serially passaged and were identified on its morphology, physical chemistry and biology. The virus can propagate in F81 cells and show cytopathic effect, such as swellen, rounded, broken off, etc. Electron microscope observation showed that the virus was non-enveloped and had a 20 nm capsid of cubic symmetry. CPV can agglutinated RBC of swine and viruses produced a HA titer of 210 after four passages in cells, the hemagglutination activity can be inhibited by VP2 specific Monoclonal antibody (McAb). Immunoenzyme staining test (IEST) showed that the CPV infected F81 cells and can react to specific McAb.The genome of CPV-LM strain was divided into five segments and was amplified using polymerase chain reaction (PCR). Genome about 4974 bp in length containing the 3' end palindrome structure and the open reading frames was obtained after gene sequenced and spliced. CPV-LM strain was characterized as CPV type-2 variants according to the classification of CPV. Sequence analysis of NS gene showed that there were relatively more nucleotide substitutions throughout the region, but most were silent mutations, with a similarity of 99% with reference strains. Compared the VP2 coding region with that of the reference strains indicated four amino acid substitutions, including A-347→T,V-562→L,S-564→N and G-568→A. Furthermore, CPV-LM strain belongs the same branch of Vac1 strain, CPV-Cv strain, 388/05-3 strain and CPVint strains, with a homology level of 99% compared to CPV-2 type strains and 97.9% compared to other antigenic types.The recombinant expression plasmid pEGFP-VP2 was constructed by insertion of VP2 gene into the eukaryotic expression vector pEGFP-C1. The plasmid pEGFP-VP2 and pEGFP-C1 were transfected 293T cells, respectively. The granular green fluorescence in transfected cells were observed at 72 hours post transfection under the fluorescencent microscope, The results proved the transient expression of fugenic VP2 gene with correct reading frame.PCR amplified VP2 gene fragments containg BamHⅠ/EcoRⅠor BamHⅠ/XhoⅠrestriction enzyeme sites with two pairs of specific primers were inserted into baculovirus expression vector pFastBac 1 and pFastBac HT, respectively. The four kinds of recombinant transfer vector were obtained, designated as pFastBac 1-VP2 (BamHⅠ/EcoRⅠ), pFastBac 1-VP2 (BamHⅠ/XhoⅠ), pFastBac-HT-VP2 (BamHⅠ/ EcoRⅠ) and pFastBac-HT-VP2 (BamHⅠ/EcoRⅠ). The four recombinant transfer vectors were transfered into DH10bac, four corresponding recombinant bacmids were screened by PCR. And then they were transfected into insect cells Sf9, obtained recombinant baculovirus, named rBV-VP2(B/E),rBV-VP2(B/X),rBV HT-VP2(B/E) and rBVHT-VP2(B/X). IEST, SDS-PAGE and Western-blot analysis showed that the four recombinant baculovirus were successfully expressed in baculovirus-expression system with native protein reactive activity.In this study, CPV-LM strain was biologically identified and genome about 4974 bp in length containing the 3' end palindrome structure and the reading frames was determined. The genome sequencing of this isolate will enrich the genomic information of CPV and laid foundation for reverse genetics of CPV. The analysis of the major capsid protein VP2 gene sequence laid the foundation to explore the CPV antigenic drift. The expression of VP2 protein by the baculovirus-expression system makes it possible for the screening of the CPV monoclonal antibody and evaluation of genetic engineering vaccine.