RECOMBINANT PORCINE PARVOVIRUS ANTIGENIC PROTEIN AND USE THEREOF

Abstract

The present invention provides: a recombinant expression vector comprising a gene encoding a porcine parvovirus VP2 protein; a recombinant plant or a recombinant insect cell transformed with the vector; and a vaccine composition for a porcine parvovirus and a composition for diagnosing porcine parvovirus, both of which contain a porcine parvovirus VP2 protein obtained from the recombinant plant or the recombinant insect cell. When the recombinant plant or recombinant insect cell of the present invention is used, the porcine parvovirus antigenic protein can be produced with high efficiency, and the porcine parvovirus antigenic protein production method using the recombinant plant or recombinant insect cell has excellent safety and stability compared with other antigen production methods.

Claims

1. A recombinant expression vector comprising a gene encoding a porcine parvovirus (PPV) VP2 protein, the gene being expressed by the nucleotide sequence of SEQ ID NO: 3 or 4.

2. A recombinant plant expressing a porcine parvovirus VP2 protein, transformed with the recombinant expression vector of claim 1.

3.-9. (canceled)

10. The recombinant plant of claim 2, wherein the plant is Nicotiana sp. plant.

11. The recombinant plant of claim 10, wherein the Nicotiana sp. plant is at least one selected from the group consisting of Nicotiana acuminata, Nicotiana africana, Nicotiana alata, Nicotiana attenuata, Nicotiana benthamiana, Nicotiana clevelandii, Nicotiana exigua, Nicotiana glauca, Nicotiana glutinosa L., Nicotiana langsdorffii, Nicotiana longiflora, Nicotiana occidentalis, Nicotiana obtusifolia, Nicotiana otophora, Nicotiana plumbaginifolia, Nicotiana quadrivalvis, Nicotiana rustica L., Nicotiana suaveolens Lehm., Nicotiana sylvestris, Nicotiana tabacum L. and Nicotiana tomentosiformis Goodsp.

12. The recombinant plant of claim 11, wherein the Nicotiana sp. plant is Nicotiana benthamiana.

13. A recombinant insect cell expressing a porcine parvovirus VP2 protein, transformed with the recombinant expression vector of claim 1.

14. The recombinant insect cell of claim 13, wherein the insect cell is Sf-9 cell.

15. The recombinant plant of claim 14, wherein the Sf-9 cell derived from Spodoptera frugiperda.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0046] FIG. 1 shows the sequence of VP2 protein for each porcine parvovirus strain.

[0047] FIG. 2A is a schematic diagram of a recombinant expression vector structure comprising a gene encoding PPV 82-opt VP2 protein.

[0048] FIG. 2B is a schematic diagram of a recombinant expression vector structure comprising a gene encoding PPV 07-opt VP2 protein.

[0049] FIG. 3A shows the Western blotting results of recombinant PPV 82-opt VP2 proteins expressed in plants.

[0050] Rbc:6His:VP2 represents the expression of PPV VP2 in plants transformed with an expression vector comprising RuBisCo transit peptide, 6×His, and codon-optimized PPV VP2 in sequence; and Rbc:VP2:6His represents the expression of PPV VP2 in plants transformed with an expression vector comprising RuBisCo transit peptide, codon-optimized PPV VP2, and 6×His in sequence.

[0051] FIG. 3B shows the Western blotting results of recombinant PPV 07-opt VP2 protein expressed in plants.

[0052] FIG. 3C shows the Western blotting results of PPV VP2 expression in plants transformed with expression vectors comprising non-codon-optimized PPV VP2.

[0053] 6His:VP2 represents the expression of PPV VP2 in plants transformed with an expression vector comprising 6×His and non-codon-optimized PPV VP2; and VP2:6His represents the expression of PPV VP2 in plants transformed with an expression vector comprising non-codon-optimized PPV V2 and 6×His.

[0054] FIG. 4 shows the Western blotting results of recombinant PPV 07-opt VP2 protein expressed in insect cells.

[0055] FIG. 5A shows the results of evaluating, in pigs, the immunogenicity of recombinant PPV 82-opt VP2 protein expressed in plants.

[0056] FIG. 5B shows the 96-well plate used in the evaluation of the immunogenicity of recombinant PPV 82-opt VP2 protein expressed in plants.

[0057] FIG. 6A shows the results of evaluating, in guinea pigs, the immunogenicity of recombinant PPV 07-opt VP2 protein expressed in plants and insect cells.

[0058] FIG. 6B shows the 96-well plate used in the evaluation of the immunogenicity of recombinant PPV 07-opt VP2 protein expressed in plants and insect cells.

[0059] FIG. 7A shows the results of verifying, with respect to guinea pig red blood cells, the hemagglutination ability of recombinant PPV 82-opt VP2 protein expressed in plants.

[0060] FIG. 7B shows the results of verifying, with the respect to guinea pig red blood cells, the hemagglutination ability of recombinant PPV 07-opt VP2 protein expressed in plants.

DETAILED DESCRIPTION

[0061] Hereinafter, the present disclosure will be described in more detail with reference to examples. These examples are provided only for the purpose of illustrating the present disclosure in more detail, and therefore, according to the purpose of the present disclosure, it would be apparent to a person skilled in the art that these examples are not construed to limit the scope of the present disclosure.

Examples

Example 1: Construction of Recombinant Porcine Parvovirus (PPV) VP2 (PPV 82-Opt, PPV 07-Opt) Antigen Plant Expression Vectors

[0062] To target VP2 having the highest antigenicity, the PPV VP2 DNA sequence obtained from specimens of the aborted and stillborn fetuses was used, and the sequence was optimized for Nicotiana benthamiana through the Genscript's codon optimization program, and synthesized by Bioneer company. The nucleotide sequences of the optimized PPV 82-opt VP2 and PPV 07-opt VP2 are composed of 1740 bp and 1737 bp, respectively.

[0063] 1-1. Recombinant PPV 82-Opt VP2 Expression Vector

[0064] For plant expression of PPV VP2 antigens, the polynucleotide (SEQ ID NO: 1) encoding the RuBisCo transit peptide, the polynucleotide (SEQ ID NO: 2) encoding six consecutive histidine resides, and the polynucleotide (SEQ ID NO: 3) encoding PPV 82 VP2 protein were sequentially linked between the CaMV 35S promoter gene and the NOS terminator of the pCAMBIA1300 vector, thereby constructing a recombinant PPV 82-opt VP2 plant expression vector (FIG. 2A).

[0065] 1-2. Recombinant PPV 07-Opt VP2 Expression Vector

[0066] For plant expression of PPV VP2 antigens, the polynucleotide (SEQ ID NO: 1) encoding the RuBisCo transit peptide, the polynucleotide (SEQ ID NO: 2) encoding six consecutive histidine resides, and the polynucleotide (SEQ ID NO: 4) encoding PPV 07 VP2 protein were sequentially linked between the CaMV 35S promoter gene and the NOS terminator of the pCAMBIA1300 vector, thereby constructing a recombinant PPV 07-opt VP2 plant expression vector (FIG. 2B).

Example 2: Expression of Recombinant PPV VP2 (PPV 82-Opt, PPV 07-Opt) Protein in Plants

[0067] To express recombinant PPV 82-opt VP2 or PPV 07-opt VP2 protein in plants, the recombinant expression vectors prepared in Example 1 were transformed into the agrobacterium strain GV3101 through electroporation using Gene Pulser XCell (Bio-Rad, USA) according to the manufacturer's instructions. The transformed GV3101 cells were grown in YEP medium (10 g of yeast extract, 10 g of peptone, 5 g of NaCl, 50 mg/L kanamycin, and 25 mg/L rifampicin) comprising an appropriate antibiotic until the cells reach a stationary phase. The cultures were centrifuged, and precipitates were re-suspended in an infiltration media (10 mM MES, 10 mM MgCl.sub.2, and 100 mM acetosyringone) to an OD.sub.600 of 0.5, and then incubated at room temperature for 1 hour. Agro-infiltration was performed by infiltrating the Agrobacterium suspension into Nicotiana benthamiana leaves grown to the age of 4-6 weeks under the condition of 25° C. through a syringe or by vacuum infiltration.

Example 3: Verification of Expression of Recombinant Porcine PPV VP2 (PPV 82-Opt, PPV 07-Opt) Protein in Plants

[0068] The leaf tissue with a size of about 3 cm.sup.2 was ground using 100 μl of a grinding 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]), and the insoluble debris was centrifuged at 20,000×g for 10 minutes at 4° C. The sample was subjected to separation on 12% SDS-PAGE gels, and transferred onto PVDF membranes (Millipore Merck KGaA of Darmstadt, Germany). Then, the sample was incubated with monoclonal His antibody (1:1,000 dilution, Invitrogen) and detected by the chemiluminescent substrate.

[0069] The use of the plant expression vector constructed by linking, in sequence, the polynucleotide encoding RubisCo transit peptide, the polynucleotides encoding six consecutive histidine residues, and the polynucleotides encoding porcine parvovirus VP2 protein (PPV 82-opt VP2 protein or PPV 07-opt VP2 protein) resulted in favorable expressions as confirmed in FIG. 3A (left panel) and FIG. 3B, but the use of the plant expression vector, constructed by linking histidines to the rear end of the porcine parvovirus VP2 protein, resulted in unfavorable protein expression (FIG. 3A, right panel).

[0070] The porcine parvovirus VP2 was not expressed in the plants transformed with the expression vectors comprising non-codon-optimized porcine parvovirus VP2 (FIG. 3C).

Example 4: Construction of Recombinant PPV 07-Opt VP2 Antigen Insect Cell Expression Vector

[0071] The PPV 07-opt VP2 sequence was amplified, cloned into a pDrive vector, and then sequenced. For Baculovirus recombinant expression, cloning was performed using pFastBac™ HT B vector (Invitrogen Company).

Example 5: Expression and Verification of Recombinant PPV 07-Opt VP2 Protein in Insect Cells

[0072] In order to express the recombinant PPV 07-opt VP2 protein in an insect, the recombinant expression vector was transformed into DH10Bac through heat shock at 42° C. according to the manufacturer's instructions. The transformed DH10Bac was incubated in S.O.C media for 4 hours at 37° C. The culture was diluted to 1/10, and incubated in LB media supplemented with gentamycin (7 ug/ml) antibiotic at 37° C. for 48 hours. After 10 colonies were transferred to 10 plates, the recombinant PPV 07-opt VP2 was sequenced using PCR with pUC/M13 primer. Sf-9 cells were transfected using Cellfectin II reagent (Invitrogen Company).

[0073] Transfection was performed at 27° C., and after five days, the cells were harvested, and protein extraction was performed at 4° C. for 30 minutes. Since the histidines were bound to the N-terminus of the recombinant PPV VP2 protein, the protein was separated using NI-NTA resin, for purification of the VP2 protein.

[0074] The purified protein was transferred onto a PVDF membrane, and then the membrane was blocked with 5% BSA. The membrane was incubated with the primary antibody mouse-His (1:1,000) at room temperature for 1 hour, followed by washing with TBST five times, and then incubated with the second antibody mouse-HRP (1:5,000) at room temperature for 1 hour, followed by washing with TBST five times, and then protein expression was visualized by the enhanced chemiluminescence (ECL) reaction (FIG. 4).

Example 6: Evaluation of Immunogenicity of Recombinant PPV VP2 Proteins

[0075] 6-1. Evaluation of Immunogenicity of Recombinant PPV 82-Opt VP2 Protein

[0076] The recombinant PPV 82-opt VP2 protein expressed in plants was mixed with an aluminum hydroxide gel adjuvant at 1:1, and 100-110 days old pigs were subjected to primary injection with 25,000 HA unit and, after two weeks, secondary injection. After two weeks, the blood was collected to measure the HI titer. As a result, a mean antibody titer of 2.sup.7 was confirmed (FIGS. 5A and 5B).

[0077] 6-2. Evaluation of Immunogenicity of Recombinant PPV 07-OP VP2 Proteins

[0078] Guinea pigs were subjected to first injection with the recombinant PPV 07-opt VP2 protein expressed in the insect cells and the recombinant PPV 07-opt VP2 protein expressed using the plant expression system, in 500 HA unit each and, after two weeks, second injection. After two weeks, the blood was collected to measure the HI titer. As a result, the recombinant PPV 07-opt VP2 protein expressed in plants showed a mean antibody titer of 2.sup.10, and the recombinant PPV 07-opt VP2 protein expressed in the insect cells showed a mean antibody titer of 2.sup.11 (FIGS. 6A and 6B).

Example 7: Hemagglutination Assay (HA)

[0079] 7-1. Hemagglutination Ability of Recombinant PPV 82-Opt VP2 Protein

[0080] PPV is characterized by agglutination with guinea pig red blood cells, and hemagglutination reaction was conducted referring to literature by Senda et al. (1986). The PPV 82-opt VP2 protein expressed in Nicotiana benthamiana was diluted to 2-fold in the 96-well U plate, mixed with 0.6% guinea pig red blood cells, and incubated at 37° C. for 1 hour, and then the results were analyzed.

[0081] The recombinant PPV 82-opt VP2 protein showed 2.sup.11 HA unit (8 g/plant) in terms of hemagglutination ability with guinea pig red blood cells, which verified antigenicity of the recombinant PPV 82-opt VP2 protein expressed in plants (Table 1 and FIG. 7A).

TABLE-US-00001 TABLE 1 Hemagglutination ability with guinea pig red blood cells Classification (Hemagglutination assay, HA) Nicotiana benthamiana (negative 0 control) Recombinant PPV 82-opt VP2 2.sup.11(2048) protein extracted from plants

[0082] 7-2. Hemagglutination Ability of Recombinant PPV 07-Opt VP2 Protein

[0083] PPV is characterized by agglutination with guinea pig red blood cells, and hemagglutination reaction was conducted referring to literature by Senda et al. (1986). The PPV 07-opt VP2 protein expressed in Nicotiana benthamiana was diluted to 2-fold in the 96-well U plate, mixed with 0.6% guinea pig red blood cells, and incubated at 37° C. for 1 hour, and then the results were analyzed.

[0084] The recombinant PPV 07-opt VP2 protein showed 2.sup.5 HA unit (8 g/plant) in terms of hemagglutination ability with guinea pig red blood cells, which verified antigenicity of the recombinant PPV 07-opt VP2 protein expressed in plants (Table 2 and FIG. 7B).

TABLE-US-00002 TABLE 2 Hemagglutination ability with guinea pig red blood cells Classification (Hemagglutination assay, HA) Nicotiana benthamiana (negative 0 control) Recombinant PPV 07-opt VP2 2.sup.5(32) protein extracted from plants