RECOMBINANT CANINE PARVOVIRUS 2A VP2 AND 2B VP2 ANTIGEN PROTEIN, AND USE THEREOF

Abstract

The present invention provides a recombinant expression vector comprising a gene encoding canine parvovirus 2a VP2 or 2b VP2 protein, a recombinant plant into which the vector is transformed, a vaccine composition against canine parvovirus, comprising canine parvovirus 2a VP2 or 2b VP2 protein obtained from the recombinant plant, and a composition for diagnosing canine parvovirus. When the recombinant plant of the present invention is used, canine parvovirus 2a VP2 or 2b VP2 antigen protein can be rapidly produced with high efficiency. Since the composition for diagnosing canine parvovirus according to the present invention uses a recombinant antigen protein, there is no possibility of contamination due to live virus handling, and thus the composition is safe, and the presence or absence of canine parvovirus infection can be rapidly diagnosed from a large amount of samples.

Claims

1. A recombinant expression vector comprising: i) a gene that is represented by the nucleotide sequence of SEQ ID NO: 3 and encodes a canine parvovirus 2a VP2 protein; or ii) a gene that is represented by the nucleotide sequence of SEQ ID NO: 4 and encodes for a canine parvovirus 2b VP2 protein.

2. A recombinant plant, wherein the recombinant plant is transformed with the recombinant expression vector of claim 1.

3. A vaccine composition against canine parvovirus, comprising i) the canine parvovirus 2a VP2 protein, or ii) the canine parvovirus 2b VP2 protein expressed in the plant of claim 2.

4.-10. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0059] FIG. 1 shows western blot results illustrating recombinant canine parvovirus 2a VP2 and 2b VP2 proteins expressed in Nicotiana benthamiana.

[0060] FIG. 2a shows hemagglutination assay results for recombinant canine parvovirus 2a VP2 protein expressed in Nicotiana benthamiana.

[0061] FIG. 2b shows hemagglutination assay results for recombinant canine parvovirus 2b VP2 protein expressed in Nicotiana benthamiana.

[0062] FIG. 3a shows hemagglutination inhibition (HI) titer test results in guinea pigs immunized with the recombinant canine parvovirus 2a VP2 protein expressed in Nicotiana benthamiana.

[0063] FIG. 3b shows hemagglutination inhibition (HI) titer test results in guinea pigs immunized with the recombinant canine parvovirus 2b VP2 protein expressed in Nicotiana benthamiana.

DETAILED DESCRIPTION

[0064] A better understanding of the present disclosure may be obtained via the following Examples which are set forth to illustrate, but are not to be construed as limiting the present disclosure.

EXAMPLES

Example 1. Production of Recombinant Canine Parvovirus 2a VP2 and 2b VP2 Proteins

[0065] 1-1. Preparation of Recombinant Plant Expression Vector Expressing Canine Parvovirus 2a VP2 and 2b VP2 Antigen

[0066] A canine parvovirus VP2 gene was synthesized in Bioneer Inc. after canine parvovirus 2a VP2 (SEQ ID NO: 5) and 2b VP2 (SEQ ID NO: 6) DNA sequences obtained from canine fecal samples were optimized to fit for Nicotiana benthamiana through GenScript's codon optimization program. The optimized polynucleotide sequences of 2a VP2 and 2b VP2 were 1752 bp and 1752 bp long, respectively.

[0067] For use in plant expression of canine parvovirus 2a VP2 and 2b VP2 antigens, a recombinant canine parvovirus 2a VP2 or 2b VP2 expression vector was constructed by sequentially linking a polynucleotide encoding RuBisCO transit peptide (SEQ ID NO: 1), a polynucleotide encoding 6 consecutive histidine residues (SEQ ID NO: 2), and a polynucleotide encoding canine parvovirus 2a VP2 protein (SEQ ID NO: 3) or 2b VP2 protein (SEQ ID NO: 4) between the CaMV 35S promoter gene and the NOS terminator in pCAMBIA1300 vector.

[0068] 1-2. Expression in Recombinant Canine Parvovirus 2a VP2 and 2b VP2 Protein in Plant

[0069] In order to express recombinant canine parvovirus 2a VP2 and 2b VP2 proteins in a plant, the recombinant expression vector constructed in Example 1 was transformed into Agrobacterium GV3101 by electroporation using Gene Pulser Xcell (Bio-Rad, USA) according to the manufacturer's instruction. The transformed GV3101 cells were grown in YEP broth containing suitable antibiotics (yeast extract 10 g, peptone 10 g, NaCl 5 g, kanamycin 50 mg/L, rifampicin 25 mg/L) until they reached a stationary phase. After centrifugation of the cell culture, the pellet was resuspended to 0.5 OD.sub.600 in an infiltration medium (10 mM Mes, 10 mM MgCl2, 100 mM acetosyringone). The bacterial suspension was incubated for 1 hour at room temperature. Agro-infiltration was carried out by applying the agrobacterial suspension to 4- to 6-week old Nicotiana benthamiana leaves at 25° C. through a syringe or vacuum-assisted infiltration.

[0070] 1-3. Expression

[0071] A leaf tissue with a size of about 3 cm.sup.2 was homogenized in 100 μl of a homogenization buffer (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 5 mM DTT and plant protease inhibitor cocktail [Sigma-Aldrich]), followed by centrifugation at 20,000×g, 4° C. for 10 min to remove insoluble debris. The sample was separated in 12% SDS-PAGE gel and transferred onto a PVDF membrane (Millipore Merck KGaA of Darmstadt, Germany). Subsequent to a reaction with a monoclonal anti-His antibody (1:1,000 dilution, Invitrogen), detection was made with a chemiluminescent substrate (FIG. 1).

[0072] As can be seen in FIG. 1, extensive expression was achieved with the plant expression vector in which a polynucleotide encoding RuBisCO transit peptide, a polynucleotide encoding 6 consecutive histidine residues, and a polynucleotide encoding canine parvovirus 2a VP2 protein or 2b VP2 protein were sequentially linked while poor expression was obtained with a plant expression vector in which the histidine residues were positioned behind the canine parvovirus 2a VP2 protein or 2b VP2 protein.

Example 2. Assay for Immunogenicity of Recombinant Canine Parvovirus 2a VP2 and 2b VP2 Proteins

[0073] 2-1. Hemagglutination Assay (HA)

[0074] For use HA tests, the recombinant canine parvovirus VP2 proteins were subjected to serial two-fold dilution in 0.6% porcine erythrocytes in Sorensen buffer (pH 6.8). HA titers were expressed as the highest dilution folds which allowed hemagglutination. HA tests were conducted using canine parvovirus 2a VP2 and 2b VP2 proteins expressed in Nicotiana benthamiana. As a test result, HA titers were measured to be 2.sup.15 HA units for canine parvovirus 2a VP2 protein and 2.sup.20 HA units for canine parvovirus 2b VP2 protein (FIGS. 2a and 2b).

[0075] 2-2. Immunogenicity Evaluation

[0076] Healthy female guinea pigs free of canine parvovirus-specific antibodies were divided into groups of three. The groups were immunized with the canine parvovirus 2a VP2 or 2b VP2 protein mixed with adjuvant A, a canine parvovirus inactivated vaccine, and PBS, respectively. As an immunogen, 100 μl of the recombinant canine parvovirus 2a VP2 or 2b VP2 protein (pertinent HA titer 1:2.sup.8) was intramuscularly injected into guinea pigs, followed by two rounds of boosting at intervals of two weeks. On day 28 after primary immunization, a blood sample was taken and coagulated at 37° C. for 2 hours. Sera were taken and inactivated at 56° C. for 30 min. HI was used for analyzing canine parvovirus-specific antibodies. After being mixed at 1:1 ratio with 25% kaolin solution, stirred for 1 hour, and mixed at 1:1 ratio with 50% pig blood corpuscle solution, the sera were separated to remove non-specific antibodies. The sera were subjected to serial two-fold dilution. The dilutions were reacted at 4° C. for 1 hour with a 1:1 dilution of the canine parvovirus 2a VP2 or 2b VP2 protein, expressed in Nicotiana benthamiana, having 8 HA units after back titration. Subsequently, 0.6% porcine blood corpuscle was 1:1 diluted and reacted at 4° C. for 2 hours before measurement.

[0077] As a result, a HI titer of 2.sup.6 was detected in two guinea pigs injected with the canine parvovirus 2a VP2 protein expressed in the plant (FIG. 3a).

[0078] Two guinea pigs injected with the canine parvovirus 2b VP2 protein expressed in the plant were measured to have a HI titer of 2.sup.8 (FIG. 3b).