Canine parvovirus (CPV) belongs to the genus Parvovirus of Parvoviridae. CPV is thought to have emerged from feline panleukopenia parvovirus (FPLV) or a closely-related virus (10). Two antigenic variants, CPV-2a and CPV-2b, are now distributed worldwide. A third variant, CPV-2c, was detected in Italy in 2000 and is now circulating in Italy, Portugal, Germany and Vietnam together with types 2a and 2b (3, 6, 14).
CPV2 has a linear single-stranded DNA genome of 5323nt in length with hairpin structures at both ends (18). This virus has two major open reading frames (ORFs); one encodes two nonstructural proteins (NS1 and NS2) and the other encodes the capsid proteins VP1 and VP2. VP1 and VP2 are localized within the same DNA sequence and are translated from different start sites. Translation of VP2 begins at the residue 165 of VP1 (16, 18). VP3 is a truncated form of VP2 with approximately the first 20 amino acid residues lysised by proteinase(15). The virus particle consists of 60 subunits with no more than 10 VP1, the remainder comprising VP2 and VP3 (22). Langeveld et al. mapped the antigen epitopes of CPV2 using a complete set of overlapping nonpeptides (740 nonpeptides) of the capsid proteins VP1 and VP2. They found 10 antigenic sites six of which localized on the surface of the virus: three in the first 21 amino acid residues, one in loop1 (located in residue 100) and two in loop3 (residue 300) (11).
CPV2 can infect dogs at different ages with high infectivity, especially to young puppies. The best way for prophylaxis was vaccine inoculation. Traditional vaccines consist of either attenuated or inactivated microorganisms delivered by injection. Despite the low-virulence of the strain used, attenuated vaccines can cause severe diseases in immunodeficient individuals, and inactivated vaccines can still harbor toxins that compromise their safety. These problems can be circumvented by subunit vaccines. Research on synthetic peptide vaccines has shown that amino acid residues 2-21 of CPV VP2 are preferable for inducing neutralizing antibody (5). However, production of recombinant vaccine proteins in conventional expression systems is expensive, requiring large-scale fermentation equipments and stringent purification processes. These fermentation processes for vaccine production may be a prohibitively expensive technology for developing countries or for application in animal health. A plant expression system is an attractive alternative to conventional production systems to obtain recombinant proteins in large quantities at low cost, and is also free of human or animal pathogens and toxins (12). Fusion proteins of CPV amino acid residues 2-22 of VP2 (2L21) with either GUS (9) or CTB (13) were expressed in tobacco, respectively. Neutralizing antibodies were induced when animals were inoculated with these chimeric antigens (9, 13).
In this study, a strain of CPV2a was isolated and identified. Also, the N-terminal 414 amino acid residues of the vp2 gene that included the whole 6 exposed epitopes of CPV2 was cloned into a binary vector and used for tobacco transformation. Transgenic tobacco plants were obtained after selection on kanamycin-containing medium. The integration and expression of vp2 gene in transgenic tobacco was confirmed by PCR and Western blotting.
Isolation and Identification of Canine Parvovirus Serotype 2a and Its VP2 Protein Expression in Transgenic Tobacco
- Received Date: 13 December 2007
- Accepted Date: 10 April 2008
Abstract: A strain of canine parvovirus（CPV）was isolated from feces of an ill puppy in an animal hospital in Wuhan, China. It was designated as CPV/WH02/06. This isolate was identified as serotype CPV-2a by the hemagglutination test, CPV Ag detection strip, electron microscopy, and PCR. The vp2 gene was cloned and sequenced and assigned GenBank accession number EU377537. A 1242 bp segment of the 5’ region of the vp2 gene was cloned and inserted into the binary vector pBI121 and used for Agrobacterium-mediated tobacco transformation. Transgenic tobacco plants were selected on MS medium supplemented with 100 μg/mL kanamycin and 100 μg/mL timentin. Integration of the vp2 gene into the tobacco genome was confirmed by PCR using T1 progeny plants, and the expression of the VP2 protein was confirmed by Western blotting.