KR101886286B1 - Method of manufacturing dual-antigenic combined antigen protein of Porcine Parvovirus and Classical Swine Fever Virus - Google Patents

Abstract

The present invention relates to a method for producing a virus-like particle having multiple antigens, which comprises virus-like particles obtained by fusing the VP2 protein, an important antigen of porcine parvovirus, and the epitope region of E2 protein, an important antigen of swine fever virus, ≪ / RTI >
When the fusion protein of the present invention as described above is used, virus particles having the simultaneous antigenicity of two viruses can be produced, and thus it can be effectively used for the prevention and treatment of infectious diseases caused by multiple viruses.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a method for preparing a dual antigenic complex antigen protein for porcine parvovirus and swine fever virus,

The present invention relates to a method for preparing a co-antigenic complex antigen protein against porcine parvovirus and swine fever virus, and more particularly, to a method for producing an epitope of E2 protein, which is an important antigen of VP2 protein and swine fever virus, And a method for producing virus-like particles having the simultaneous antigenicity of two viruses by fusion expression of the top region.

Pig parvovirus infection is a communicable disease of pigs caused by infection with Porcine Parvovirus (PPV). When a pregnant pig is infected, it does not show any special clinical symptoms in general sows and piglets. However, , It is a disease in which symptoms of reproductive disorder such as miscarriage, a stillbirth from death, and mummy degeneration caused by discoloration of black are exhibited.

Swine fever is a serious economic loss to the pigs because the Classical Swine Fever Virus (CSFV) attacks the fetus through the placenta and causes fetal absorption, malformation, miscarriage and premature birth, It is an epidemic that causes.

In order to prevent pig parvovirus infection, pig parvovirus vaccine, which has been used worldwide to date, has a virus vaccine in which virus is inactivated using formalin after tissue culture of porcine parvovirus. However, It is extremely difficult to do. As a result, the price of vaccine production is about 1 thousand won per vaccine, making it the most expensive vaccine for swine disease. In addition, since it is difficult to completely inactivate the virus, there is a problem in that the use of the Sadox vaccine causes an infection caused by the vaccine strain, and the vaccination for prevention causes the infectious disease. There is no effective treatment method for swine fever virus. Currently, inactive vaccine or inactivated vaccine is used as a preventive measure, but there is a problem about the efficacy of domestic vaccine.

VP2 protein is one of the major structural antigens for pig parvovirus, and it is possible to form virus-like protein (VLP) only by VP2 expression. Various antigen production methods have been reported.

As a prior art, a method of producing VP2 protein using cells of the insect cell line Sf family has been disclosed (Korean Patent No. 014220). However, in the method of producing a protein using a cell line, a large amount of cell culture conditions are required, a large amount of cell culture medium is required, and the cost of the culture medium is high, which is not economical. Recently, effective production of VLP using silkworm larva has been reported.

On the other hand, one silkworm larva can replace 100 ~ 200 ml of insect cell culture, so mass production of protein is easy. Insect cell line is a single cell, but when the insect bioreactor having various cells is used, Can be increased. In addition, the price of one silkworm is about 100 won, which is advantageous in comparison with a cell culture medium of 500 ㎖ to 50,000 won.

Korean Patent Laid-Open No. 10-2012-0125627 (November 16, 2012) Korean Patent No. 10-0142210 (Mar. 27, 1998) Korean Patent No. 10-0320165 (December 26, 2001) Korean Patent No. 10-1313906 (2013.09.23)

Je et al., Biotechnol. Lett., 23: 1809-1817, 2001 Guo C. et al, Arch Virol. May 2014 May 159 (5): 963-70

In order to solve the inconvenience of producing vaccines and diagnostic antigens for the prevention, diagnosis and treatment of infectious diseases of porcine parvovirus and swine fever virus, the present inventors have developed a vaccine against pandemic fever virus We have tried to develop a virus - like particle which has concurrent antigenicity against two viruses by fusing and expressing important antigen epitope of E2 protein.

As a result, the inventors have devised a method for producing a virus-like particle having two antigenicities simultaneously using a silkworm larva (Bombyx mori Nucleopolyrovirus) and silkworm larvae instead of producing a single antigen protein for each virus, Thereby completing the present invention.

Accordingly, an object of the present invention is to provide a virus-like particle having simultaneous antigenicity against porcine parvovirus and swine fever virus, and a method for producing the same.

According to one aspect of the present invention, the present invention provides a method of producing a porcine parvovirus VP2 (viral protein 2) protein and (b) a swine fever virus (classical swine fever virus) Parvovirus and swine fever virus simultaneously antigenic complex antigen protein.

The VP2 is the major structural protein of porcine parvovirus and thus is involved in inducing the production of neutralizing antibodies in the immunized host (Guo C. et al, Arch Virol. 2014 May; 159 (5) : 963-70). The amino acid sequence of VP2 is represented by SEQ ID No. 1, and the nucleic acid sequence encoding the VP2 is represented by SEQ ID No. 2.

E2 is one of the four structural proteins of the swine fever virus, Capsid, E ^rns , E1 and E2. It is known as a major defense antigen involved in the production of antibodies that neutralize virus by constituting the envelope. The amino acid sequence corresponding to the epitope of E2 is the CKEDYRY site represented by SEQ ID NO: 3, and the nucleotide sequence encoding the same is represented by SEQ ID NO: 4.

The term "fusion" in the specification of the present invention means that it is bonded to the amino acid at the N-terminus or C-terminus through a peptide bond.

The term "co-antigenic complex antigen protein" in the specification of the present invention means a complex antigen protein in which two or more viruses or antigens from bacteria are fused to simultaneously exhibit the characteristics of each antigen. The complex antigen protein may comprise an antigen from each virus or an epitope thereof.

In one embodiment of the present invention, the epitope of the E2 protein may be fused to the N-terminus or the C-terminus of the VP2 protein. Preferably, the epitope of the E2 protein is fused to the N-terminus of the VP2 protein.

According to another aspect of the present invention, there is provided a composition for a porcine parvovirus and a porcine febrile virus concurrent antigenic vaccine comprising the complex antigen protein as an active ingredient.

According to another aspect of the present invention, the present invention provides a recombinant protein comprising (a) a nucleic acid sequence encoding a complex antigen protein according to any one of claims 1 to 3 and (b) a promoter operably linked to the nucleic acid sequence Lt; RTI ID = 0.0 > expression < / RTI >

In one embodiment of the present invention, the promoter is a promoter operable in an insect cell and is selected from the group consisting of a polyhedrin promoter derived from baculovirus, an IE-1 promoter, an IE-2 promoter, a p35 promoter, p10 ≪ / RTI > promoter and the gp64 promoter. In certain embodiments, the promoter may be a polyhedrin promoter.

The term "polyhedrin promoter " in the context of the present invention serves to form polyhedra, which are large structures for protecting virus particles from the external environment.

In one embodiment of the present invention, the construct may further comprise a transcription termination sequence.

According to another aspect of the present invention, there is provided a co-transfected host cell for constructing said complex antigen protein.

The term "transfection" in the specification of the present invention refers to a phenomenon in which a nucleic acid is introduced into a cell and a normal viral particle having an infectivity is formed by separating and purifying only the nucleic acid from the virus and putting it in the host cell of the virus.

The transfection can be carried out using a nucleic acid of Baculovirus. Since baculovirus forms polymorphisms, the presence or absence of polyhedrosis protein in the host can be determined.

In one embodiment of the invention, the host cell may be an insect cell.

Since the host cell may be a cell line isolated from living insects or a cell contained in the body of a living insect, the term "culture" herein used to describe the concrete steps of the method for producing a complex antigen protein of the present invention In vitro culture of cell lines isolated from live insects and breeding of live insects with said host cells. The insect may be a silkworm.

According to another aspect of the present invention, the present invention provides a method for preparing porcine parvovirus and porcine swine fever virus co-antigen complex antigen protein comprising the steps of: (a) culturing a construct for the expression of said complex antigen protein Culturing an infected host cell; And (b) isolating the porcine parvovirus and the porcine fever virus simultaneously antigenic complex antigen protein from the host cells cultured in the step (a).

The features and advantages of the present invention are summarized as follows:

The present invention provides a virus-like particle in which a VP2 protein, an important antigen of a porcine parvovirus, and an epitope region of an E2 protein, an important antigen of a swine fever virus, are fused and a method for producing the virus-like particle.

The use of the fusion protein of the present invention enables the production of viral particles having the simultaneous antigenicity of two viruses, which can be effectively used for the prevention and treatment of infectious diseases caused by a plurality of viruses.

FIG. 1A shows a process of cloning a gene (VP2-E2) obtained by fusing a major epi- top of swine fever virus antigen protein E2 into a porcine parvovirus VP2 gene into a baculovirus transfer vector pBmKSK3 and a process of producing a recombinant silkworm nuclear polyhedrosis virus It is an overview.
Fig. 1B shows pBmKSK3-N-VP2 cloned into the baculovirus transfer vector pBmKSK3 by gene (VP2-E2) obtained by fusing the main epitope of the antigen protein E2 of swine fever virus to the N-terminus of the VP2 gene of porcine parvovirus FIG.
2A shows the results of analysis of VP2-E2 protein expressed in Bm5 cell line by protein electrophoresis analysis.
FIG. 2B is a photograph showing the results of Western blot analysis of a VP2-E2 protein expressed in a Bm5 cell line using a porcine parvovirus antibody and a swine fever virus E2 epitope antibody.
FIG. 3 is a photograph showing the results of analysis of VP2-E2 protein expressed in hemolymph and fat body of silkworm larva by protein electrophoresis analysis and Western blot analysis.
FIG. 4 shows the result of VLP isolation from hemolymphs and fat bodies of the cell line and silkworm larvae, followed by transmission electron microscopy.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Example 1. Production of Recombinant Silkworm Nuclear Polyhedrosis Virus

Polymerase chain reaction (PCR) was performed using the genomic DNA of porcine parvovirus as a template and primers represented by SEQ ID No. 5 and SEQ ID No. 6, A DNA fragment coding for the CKEDYRY amino acid, which is the major epitope of the E2 antigen, was obtained.

Also, a DNA fragment encoding the CKEDYRY epitope of E2 together with the C-terminus of the VP2 protein was obtained using the same template as Sequence Listing No. 7 and Sequence Listing No. 8.

Each DNA fragment was cloned into a T & A cloning vector (RBC Bioscience), digested with Sac II and Xba I, inserted into pBmKSK3 (Korean Patent No. 10-0320165) obtained by digesting with Sac II and Xba I using this as an insert Transformation vectors pBmKSK3-N-VP2 and pBmKSK3-VP2-C were prepared (Fig. 1A). In this vector the VP2 protein encoding DNA fragment is located downstream of the polyhedrin promoter.

The information of the 5th to 8th sequences of the Sequence Listing is as summarized in Table 1.

Sequence List division order Fifth sequence CSFV E2-CKEDYRY-PPV VP2-F primer 5'-CCGCGGCCACCATGTGCAAGGAAGATTACAGGTACAGTGAAAATG-3 ' 6th sequence PPV-VP2-R primer 5'-TCTAGACTAGTATAATTTTCTTGGTATAAG-3 Seventh sequence PPV-VP2-F primer 5'-CCGCGGCCACCATGAGTGAAAATGTGGAAC-3 ' Eighth sequence PPV VP2-CSFV-E2-CKEDYRY-R 5'-TCTAGACTAGTACCTGTAATCTTCCTTGCA GTATAATTTTCTTGG-3 '

Genomic DNA of the prepared transcription vector and the recombinant virus bBpGOZA (Je et al., Biotechnol. Lett., 23: 1809-1817, 2001) was mixed with 100 μl of TC-100 medium (WelGene), and cell pectin II II, Invitrogen) were mixed in 100 μl of TC-100 medium and each was left at 27 ° C for 15 minutes. The mixture was then mixed and left at 27 ° C for 15 minutes.

The resulting mixture was divided into a silkworm cell line Bm5 (Bombyx mori 5) at a concentration of 2.0 x 10 ⁶ cells per 25 mm 2 culture flask, inoculated into the culture medium, and further cultured to obtain recombinant silkworm nuclear polyhedrosis virus rBp-N-VP2 and rBp-VP2 Respectively.

The recombinant viruses prepared in this manner were purified by a plain virus isolation method (Plaque assay). To prepare a large amount of virus, pure viruses were transferred to a T-25 flask (SPL Inc.) The virus was able to be obtained in large quantities by re-inoculation and proliferation. The obtained viruses were quantitatively used by the dilution ending method (King and Possee, 1992, The Baculovirus Expression System, A Laboratory Guide) (see FIG.

Example 2. Protein electrophoresis analysis of VLPs against swine parvovirus and swine fever virus in silkworm cell line

The recombinant silkworm nuclear polyhedrosis virus prepared in Example 1 was divided into Bm5 cells, which are insect cell lines, in an amount of 1.0 x 10 ⁶ per 60 x 15 mm culture plate so that there is one virus particle per cell, Respectively. At the end of 3 days after inoculation, the cells were observed under an inverted microscope, and it was judged that they were infected when polyhedrin proteins appeared in the infected cells. As a control, wild type baculovirus BmNPV-K1 (domestic isolate, KCTC 0491BP) was used as a control.

These recombinant viruses were collected from the plate and the culture was centrifuged at 1,000xg for 5 minutes. Cell lysis solution (20 mM Tris-HCl pH 7.5, 50 mM NaCl, 5% glycerol, 1% NP-40) was added to the precipitate and left on ice for 30 minutes. To the reaction solution was added 5X sample buffer (0.6 ml of 1 M Tris-HCl (pH 6.8), 5 ml of 50% glycerol, 2 ml of 10% SDS, 0.5 ml of 2-mercaptoethanol, 1 ml of 1% , 0.9 ml H ₂ O) was added and mixed, heated at 100 ° C for 10 minutes with hot water, left on ice for a while, and then centrifuged at 13,000 rpm for 10 minutes.

Protein electrophoresis was performed using a 10% protein electrophoresis gel as a supernatant. After electrophoresis, the gel was stained with coomassie brillant blue to confirm the presence of the protein at about 64 kDa, the predicted size of porcine parvovirus VP2 and porcine fever virus episode fusion protein (Fig. 2a).

For accurate identification of the target protein, peptides composed of the sera produced by inoculating porcine parvovirus into the abdominal cavity of rabbits and the epitope CKEDYRY amino acid of the swine fever virus E2 were synthesized and inoculated into the abdominal cavity of rabbits to obtain Western blot analysis Respectively.

After protein electrophoresis with the protein samples prepared as described above for Western blot analysis, the proteins present in the gel were electro-transferred to a nitrocellulose membrane (Pall Corp.). The electrophoretically transferred nitrocellulose membrane was treated for 1 hour in skim milk in TBS-T buffer (20 mM Trs, 150 mM NaCl, 0.5% Tween 20, pH 7.4) dissolved in 5% Serum of porcine parvovirus and serum of swine fever virus major antigen epithelium were treated as primary antibody for 4 hours, and then washed three times for 10 minutes with TBS-T buffer solution. The secondary antibody against the rabbit serum diluted in TBS-T buffer was then applied to the membrane and washed three times for 10 minutes each. Finally, the treated membrane was treated with a Western HRP substrate (Millipore, USA) and developed on an X-ray film to observe specific bands.

As a result, at 64 kDa position observed in the protein electrophoresis analysis, specific bands were observed for the porcine parvovirus sera and the swine fever virus sera, respectively, and a 64 kDa protein was identified as a specific protein for these two viruses (Fig. 2B). As a result of comparing the results obtained by fusion of N-terminal and C-terminal of porcine parvovirus VP2 with that of swine fever virus epitope, higher expression could be confirmed by fusion expression at the N-terminus.

Example 3. Production of VP2 protein from silkworm larva

1 x 10 ⁵ recombinant silkworm nuclear polyhedrosis virus particles prepared in Example 1 were inoculated into a silkworm larva of the fifth day of the first day using a 1 ml syringe. At this time, wild type baculovirus BmNPV-K1 was used as a control. Blood lymphedema and fat bodies were collected from infected larvae while bred for 5 days after inoculation and observed visually. Hemolymph was collected by cutting the leg end of the silkworm larvae into a cooled tube on ice, and the fat body was incised to dissect the silkworm larvae, and only the white fat body was collected in an ice-cooled tube.

Cell lysis solution (20 mM Tris-HCl pH 7.5, 50 mM NaCl, 5% glycerol, 1% NP-40) was added to 1 μl of hemolymph and 50 mg of lipid collected by the above method, Respectively. To the reaction solution was added 5X sample buffer (0.6 ml of 1 M Tris-HCl (pH 6.8), 5 ml of 50% glycerol, 2 ml of 10% SDS, 0.5 ml of 2-mercaptoethanol, 1 ml of 1% , 0.9 ml H ₂ O) were added and mixed, heated at 100 ° C for 10 minutes with hot water, left on ice for a while, and then centrifuged at 13,000 rpm for 10 minutes.

The supernatant was then subjected to protein electrophoresis using a 10% protein electrophoresis gel and western blot analysis of Example 2, respectively. After electrophoresis, the gel was stained with a coomassie brillant blue. As a result, a specific protein at about 64 kDa, which is a predicted position of the fusion protein, was identified only when the E2 epitope was fused to the N-terminus of VP2 It was possible. Western blot analysis showed the same result (Fig. 3).

Example 4 Purification of VLP Using Ammonium Sulfate

Using the Bm5 cell line infected with the recombinant silkworm nuclear polyhedrosis virus in Examples 2 and 3 and the samples collected from hemolymphs and lipids isolated from the silkworm larvae, pigs parvovirus VP2 protein and pig fever virus The VLPs formed by E2 fusion expression were purified.

Specifically, the following purification method was used according to the hemolymph and lipid body of Bm5 cell line and silkworm larvae.

Example 4-1. Method for purifying VLP from Bm5 cell line

The recombinant silkworm nuclear polyhedrosis virus prepared in Example 1 was infected into the insect cell line Bm5 cells at a density of 2.0 x 10 ⁶ in a T-25 flask so as to become one virus particle per cell. Three days after inoculation, the cells were observed under an inverted microscope and examined for the presence or absence of polyhedrosis protein in the infected cells. At this time, wild type baculovirus BmNPV-K1 was used as a control.

To collect the recombinant viruses thus produced in a flask, the culture was centrifuged at 1,000 g for 5 minutes. Washed with 1 ml of PBS wash buffer, and treated with 800 [mu] l of 25 mM sodium bicarbonate to give an osmotic shock. Thereafter, the mixture was allowed to stand for 10 minutes on ice, followed by vortexing for 1 minute, followed by centrifugation at 10000 rpm and 4 ° C for 7 minutes.

The centrifuged supernatant was transferred to a new tube, treated with 20% of 200% ammonium sulfate (100%), and placed in a 4 ° C refrigerator for 20 minutes. Then, the virus particle precipitate was obtained by centrifugation at 10000 g and 4 ° C for 30 minutes.

Example 4-2. VLP purification method from platycodon of silkworm larva

On the 1st day of silkworm larvae, 1 x 10 ⁵ viral particles were inoculated into silkworm larvae using a 1 ml syringe. Wild-type baculovirus BmNPV-K1 was used as a control in Example 3. Blood lymphs were collected from the infected larvae while being raised for 5 days after the inoculation and observed with naked eyes. The blood limb was cut in the tip of the silkworm larva and collected in a tube cooled in ice.

100 [mu] l of the collected hemolymph was diluted in 700 [mu] l of PBS washing solution and centrifuged at 10000 rpm at 4 [deg.] C for 7 minutes. After centrifugation, the supernatant was transferred to a new tube, treated with 20% ammonium sulfate 100%, and placed in a refrigerator at 4 ° C for 20 minutes. Then, the virus particle precipitate was obtained by centrifugation at 10000 g and 4 ° C for 30 minutes.

Example 4-3. Method of purifying VLP from fat body of silkworm larva

On the 1st day of silkworm larvae, 1 x 10 ⁵ viral particles were inoculated into silkworm larvae using a 1 ml syringe. Wild-type baculovirus BmNPV-K1 was used as a control in Example 3. Five days after the inoculation, the infected larvae were cut and the white fat bodies were collected by observing with naked eyes. This fat body was immersed in a cooled tube in an amount of 50 ㎍, and then treated with 720 의 of sodium bicarbonate for osmotic shock.

Thereafter, the mixture was allowed to stand for 10 minutes on ice, followed by vortexing for 1 minute, followed by centrifugation at 10000 rpm and 4 ° C for 7 minutes. The centrifuged supernatant was transferred to a new tube, treated with 20% of 200% ammonium sulfate (100%), and placed in a 4 ° C refrigerator for 20 minutes. Then, the virus particle precipitate was obtained by centrifugation at 10000 g and 4 ° C for 30 minutes.

Example 5. Confirmation of VLP by energy transmission electron microscope

In Example 4, a sample of VLP purified from Bm5 cell line and larvae isolated from silkworm larvae and lipids was adsorbed on a carbon-coated grid, and stained with phosphotungstic acid for 5 minutes , Dried at room temperature for 40 minutes, and then photographed using an energy transmission electron microscope (Libra 120, Carl Zeiss, Germany). Analysis of the photographs revealed that the size of porcine parvovirus-like particles was 20 nm, which was similar to the original viral particle size including nucleic acids (FIG. 4).

Having described specific portions of the present invention in detail, those skilled in the art will appreciate that these specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

<110> Chungbuk National University Industry Academic Cooperation Foundation <120> Method of manufacturing dual-antigenic combined antigen protein          of Porcine Parvovirus and Classical Swine Fever Virus <130> PN160381 <160> 8 <170> Kopatentin 2.0 <210> 1 <211> 567 <212> PRT <213> Porcine Parvovirus <400> 1 Met Ser Glu Asn Val Glu Gln His Asn Pro Ile Asn Ala Gly Thr Glu   1 5 10 15 Leu Ser Ala Thr Gly Asn Glu Ser Gly Gly Gly Val Gly Val Ser Thr Gly              20 25 30 Ser Phe Asn Asn Gln Thr Glu Phe Gln Tyr Leu Gly Glu Gly Leu Val          35 40 45 Arg Ile Thr Ala His Ala Ser Arg Leu Ile His Leu Asn Met Pro Glu      50 55 60 His Glu Thr Tyr Lys Arg Ile His Val Leu Asn Ser Glu Ser Gly Val  65 70 75 80 Ala Gly Gln Met Val Gln Asp Asp Ala His Thr Gln Met Val Thr Pro                  85 90 95 Trp Ser Leu Ile Asp Ala Asn Ala Trp Gly Val Trp Phe Asn Pro Ala             100 105 110 Asp Trp Gln Leu Ile Ser Asn Asn Met Thr Glu Ile Asn Leu Val Ser         115 120 125 Phe Glu Gln Glu Ile Phe Asn Val Val Leu Lys Thr Ile Thr Glu Ser     130 135 140 Ala Thr Ser Pro Pro Thr Lys Ile Tyr Asn Asn Asp Leu Thr Ala Ser 145 150 155 160 Leu Met Val Ala Leu Asp Thr Asn Asn Thr Leu Pro Tyr Thr Pro Ala                 165 170 175 Ala Pro Arg Ser Glu Thr Leu Gly Phe Tyr Pro Trp Leu Pro Thr Lys             180 185 190 Pro Thr Gln Tyr Arg Tyr Tyr Leu Ser Cys Thr Arg Asn Leu Asn Pro         195 200 205 Pro Thr Tyr Thr Gln Gln Ser Gln Gln Ile Thr Asp Thr Ile Gln Thr     210 215 220 Gly Leu His Ser Asp Ile Met Phe Tyr Thr Ile Glu Asn Ala Val Pro 225 230 235 240 Ile His Leu Leu Arg Thr Gly Asp Glu Phe Ser Thr Gly Ile Tyr His                 245 250 255 Phe Asp Thr Lys Pro Leu Lys Leu Thr His Ser Trp Gln Thr Asn Arg             260 265 270 Ser Leu Gly Leu Pro Pro Lys Leu Leu Thr Glu Pro Thr Thr Glu Gly         275 280 285 Asp Gln His Pro Gly Thr Leu Pro Ala Ala Asn Thr Arg Lys Gly Tyr     290 295 300 His Gln Thr Met Asn Asn Ser Tyr Thr Glu Ala Thr Ala Ile Arg Pro 305 310 315 320 Ala Gln Val Gly Tyr Asn Thr Pro Tyr Met Asn Phe Glu Tyr Ser Asn                 325 330 335 Gly Gly Pro Phe Leu Thr Pro Ile Val Pro Thr Ala Asp Thr Gln Tyr             340 345 350 Asn Asp Asp Glu Pro Asn Gly Ala Ile Arg Phe Thr Met Gly Tyr Gln         355 360 365 His Gly Gln Leu Thr Thr Ser Ser Gln Glu Leu Glu Arg Tyr Thr Phe     370 375 380 Asn Pro Gln Ser Lys Cys Gly Arg Ala Pro Lys Gln Gln Phe Asn Gln 385 390 395 400 Gln Ala Pro Leu Asn Leu Glu Asn Thr Asn Asn Gly Thr Leu Leu Pro                 405 410 415 Ser Asp Pro Ile Gly Gly Lys Pro Asn Met His Phe Met Asn Thr Leu             420 425 430 Asn Thr Tyr Gly Pro Leu Thr Ala Leu Asn Asn Thr Ala Pro Val Phe         435 440 445 Pro Asn Gly Gln Ile Trp Asp Lys Glu Leu Asp Thr Asp Leu Lys Pro     450 455 460 Arg Leu His Val Thr Ala Pro Phe Val Cys Lys Asn Asn Pro Pro Gly 465 470 475 480 Gln Leu Phe Val Lys Ile Ala Pro Asn Leu Thr Asp Asp Tyr Asn Ala                 485 490 495 Asp Ser Pro Gln Gln Pro Arg Ile Ile Thr Tyr Ser Asn Phe Trp Trp             500 505 510 Lys Gly Thr Leu Thr Phe Thr Ala Lys Met Arg Ser Ser Asn Met Trp         515 520 525 Asn Pro Ile Gln Gln His Thr Thr Thr Ala Glu Asn Ile Gly Lys Tyr     530 535 540 Ile Pro Thr Asn Ile Gly Gly Met Lys Met Phe Pro Glu Tyr Ser Gln 545 550 555 560 Leu Ile Pro Arg Lys Leu Tyr                 565 <210> 2 <211> 1704 <212> DNA <213> Porcine Parvovirus <400> 2 atgagtgaaa atgtggaaca acacaaccct attaatgcag gcactgaatt gtctgcaaca 60 ggaaatgaat ctgggggggt tggtgtgtct acaggtagtt tcaataatca aacagaattt 120 caatacttgg gggagggctt ggttagaatc actgcacacg catcaagact catacatcta 180 aatatgccag aacatgaaac atacaaaaga atacatgtac taaattcaga atcaggggtg 240 gcgggacaaa tggtacaaga cgatgcacac acacaaatgg taacaccttg gtcactaata 300 gatgctaacg catggggggt gtggttcaat ccagcagact ggcagttaat atccaacaac 360 atgacagaaa taaacttagt tagttttgaa caagaaatat tcaatgtagt acttaaaaca 420 attacagaat cagcaacctc accaccaacc aaaatatata ataatgatct aactgcaagc 480 ttaatggtag cactagacac caataacaca cttccataca caccagcagc acctagaagt 540 gaaacacttg gtttttatcc atggttacct acaaaaccaa ctcaatacag atattaccta 600 tcatgcacca gaaacctaaa tccaccaaca tacactggac aatcacaaca aataacagac 660 acaatacaaa caggactaca cagtgacatt atgttctaca caatagaaaa tgcagtacca 720 attcatcttc taagaacagg agatgaattc tccacaggaa tatatcactt tgacacaaaa 780 ccactaaaat taactcactc atggcaaaca aacagatctc taggactgcc tccaaaacta 840 ctaactgaac ctaccacaga aggagaccaa cacccaggaa cactaccagc agctaacaca 900 agaaaaggtt atcaccaaac aatgaataat agctacacag aagcaacagc aattaggcca 960 gctcaggtag gatataatac accatacatg aattttgaat actccaatgg tggaccattt 1020 ctaactccta tagtaccaac agcagacaca caatataatg atgatgaacc aaatggtgct 1080 ataagattta caatgggtta ccaacatgga caattaacca catcttcaca agagctagaa 1140 agatacacat tcaatccaca aagtaaatgt ggaagagctc caaagcaaca atttaatcaa 1200 caggcaccac taaacctaga aaatacaaat aatggaacac ttttaccttc agatccaata 1260 ggagggaaac ctaacatgca tttcatgaat acactcaata catatggacc attaacagca 1320 ctaaacaata ctgcacctgt atttccaaat ggtcaaatat gggataaaga acttgataca 1380 gatctaaaac ctagactaca tgttacagct ccatttgttt gtaaaaacaa tccaccagga 1440 caactatttg taaaaatagc accaaaccta acagatgatt acaatgctga ctctcctcaa 1500 caacctagaa taataactta ttcaaacttt tggtggaaag gaacactaac attcacagca 1560 aaaatgagat ccagtaatat gtggaaccct attcaacaac acacaacaac agcagaaaac 1620 attggtaaat atattcctac aaatattggt ggcatgaaaa tgtttccaga atattcacaa 1680 cttataccaa gaaaattata ctag 1704 <210> 3 <211> 7 <212> PRT <213> Classical Swine Fever Virus <400> 3 Cys Lys Glu Asp Tyr Arg Tyr   1 5 <210> 4 <211> 21 <212> DNA <213> Classical Swine Fever Virus <400> 4 tgcaaggaag attacaggta c 21 <210> 5 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> artificial sequences <400> 5 ccgcggccac catgtgcaag gaagattaca ggtacagtga aaatg 45 <210> 6 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> artificial sequences <400> 6 tctagactag tataattttc ttggtataag 30 <210> 7 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> artificial sequences <400> 7 ccgcggccac catgagtgaa aatgtggaac 30 <210> 8 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> artificial sequences <400> 8 tctagactag tacctgtaat cttccttgca gtataatttt cttgg 45

Claims (12)

(a) porcine parvovirus VP2 (viral protein 2) protein and (b) a porcine parvovirus fused with epitope of the classical swine fever virus E2 protein represented by SEQ ID NO: 3, Virus-like particles.
The virus-like particle according to claim 1, wherein the epitope of the E2 protein is fused to the N-terminus or the C-terminus of the VP2 protein.
3. The virus-like particle according to claim 2, wherein the epitope of the E2 protein is fused to the N-terminus of the VP2 protein.
A composition for swine parvovirus and swine fever virus concurrent antigenic vaccine comprising the virus-like particle according to any one of claims 1 to 3 as an active ingredient.
1. A construct for expressing a virus-like particle comprising (a) a nucleic acid sequence encoding a virus-like particle according to any one of claims 1 to 3 and (b) a promoter operably linked to the nucleic acid sequence.
6. The construct of claim 5, wherein the promoter is a promoter operable in insect cells.
7. The virus-like particle expressing kit according to claim 6, wherein the promoter is selected from the group consisting of polyhedrin promoter, IE-1 promoter, IE-2 promoter, p35 promoter, p10 promoter and gp64 promoter Truck.
6. The construct of claim 5, wherein the construct further comprises a transcription termination sequence.
A co-transfected host cell for expression of a virus-like particle according to claim 5.
10. The host cell according to claim 9, wherein the host cell is an insect cell.
11. The host cell according to claim 10, wherein the insect is a silkworm.
A method for producing virus-like particles of swine parvovirus and swine fever virus simultaneously comprising the steps of:
(a) culturing the host cell according to claim 9; And
(b) isolating and isolating porcine parvovirus and porcine fowl virus simultaneously antigenic virus-like particles from the host cells cultured in said step (a).

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