KR20110137743A - Composition for feeding containing high antigenic capsid protein of canine parvovirus - Google Patents

Abstract

PURPOSE: A high antigenic capsid protein for preventing and treating canine pavovirus and a feed composition containing the same are provided to induce immune response in an intestinal tract. CONSTITUTION: A canine pavovirus recombinant protein(VP2) has antigenic capsid site of sequence number 4. An expression vector contains a gene encoding the recombinant protein. The expression vector is pGAP Za. A method for preparing the recombinant protein comprises: a step of amplifying a gene encoding the antigenic capsid site of canine pavovirus using a primer of sequence numbers 1 and 2; a step of inserting the gene into the expression vector pGAP Za to prepare a recombinant expression vector; a step of transforming the recombinant expression vector into host cells to prepare a transformant; and a step of culturing the cells in a medium containing Zeocin and collecting the cultured product. The host cells are yeast(Pichia pastoris). A composition for preventing or treating canine pavovirus infection contains the recombinant protein as an active ingredient.

Description

Composition for feeding containing high antigenic capsid protein of canine parvovirus for preventing and treating canine parvovirus

The present invention relates to a parvovirus high antigenic capsid protein for canine parvovirus prevention and treatment and a feed composition comprising the same.

Canine parvovirus is one of the most common causes of canine diarrhea. It is the leading cause of canine infectious diseases in dogs with hemorrhagic enteritis and sometimes myocarditis, with an incidence rate of 42% and mortality rate of 20%.

This canine parvovirus infection is an infectious disease caused by canine parvovirus, the most serious damage is the intestinal tract, especially the small intestine. The normal small intestine has a small finger-shaped villi to increase its surface area and increase the absorption of moisture and nutrients, and the villi have many other microvilli, which are the main sites where canine parvoviruses attack.

The canine parvovirus vaccine currently in use is a venomous vaccine in which canine parvovirus is incubated in a tissue and then inactivated a strongly pathogenic virus using formalin, or attenuated by passage of pathogenic viruses for several decades in a laboratory. do.

Preventing canine parvovirus infection is the best way to prevent attenuated canine parvovirus vaccination, but should be repeated several times.

In addition, the antibody formation due to vaccination is mainly induced immunoglobulin G (IgG) corresponding to the systemic immune (humoral immunity), whereas the onset of the disease is induced in the intestinal system, so immunoglobulin A (intestinal mucosal immune antibody) ( Induction of IgA) is essential, but various antibiotics and antimicrobials that can improve canine parvovirus infection have been developed. However, there are no special treatments that can fundamentally solve canine parvovirus infection.

Therefore, the inventors of the present invention, while developing an effective treatment for canine parvovirus infection, delivers the antigen directly into the mucosa by the oral administration (form of feed), and directly induces mucosal immunity through the delivered antigen canine parvovirus infection To find a feed composition for the treatment or prevention of canine parvovirus infection that can fundamentally solve the present invention was completed.

An object of the present invention is to deliver a high antigenic capsid protein site which can be directly resolved canine parvovirus infection by delivering antigen directly into the mucosa by oral administration (form of feed), and directly induce mucosal immunity through the delivered antigen. Eggplant is to provide a canine parvovirus recombinant protein, a gene encoding the same, an expression vector comprising the gene.

Another object of the present invention is to provide a method for producing the recombinant protein and a feed composition for the treatment or prevention of canine parvovirus infections containing the protein.

The present invention provides a canine parvovirus recombinant protein having a high antigenic capsid protein site, a gene encoding the same, and an expression vector comprising the gene.

The present invention provides a method for producing the recombinant protein and a feed composition for the treatment or prevention of canine parvovirus infections containing the protein.

Hereinafter, the present invention will be described in detail.

The present invention provides a canine parvovirus recombinant protein having a high antigenic capsid protein site of SEQ ID NO: 4.

More specifically, the canine parvovirus recombinant protein of the present invention is infected with canine parvovirus to animal cells, and centrifuged. CPV-2a) gene was prepared, and then the canine parvovirus (CPV-2a) gene was cloned to analyze the entire sequence, and the analyzed canine parvovirus (CPV-2a) gene was used as a template for the PCR amplification method. Canine parvovirus (CPV-2a) recombinant proteins can be prepared by designing and expressing canine parvovirus (CPV-2a) fragment genes to include the original capsid protein sites.

The present invention provides a gene encoding the canine parvovirus recombinant protein.

More specifically, to prepare a canine parvovirus (CPV-2a) fragment gene to include a high antigenic capsid protein site, it can be prepared by PCR amplification using a specific primer (specific primer). The term “specific primer” refers to an artificial nucleotide sequence for producing a canine parvovirus (CPV-2a) fragment gene comprising a high antigenic capsid protein site by PCR amplification method. The specific primer can be prepared based on GenBank's database. In addition, a restriction enzyme recognition site is inserted into the specific primer, which is a conventional step for inserting the canine parvovirus (CPV-2a) fragment gene PCR amplified with the specific primer into the expression vector, which is commonly used in the art. It is a way. Specific primers prepared refer to SEQ ID NOs: 1 and 2.

The present invention provides an expression vector comprising a gene encoding the canine parvovirus recombinant protein.

In the present invention, the expression vector is characterized in that pGAP Za.

The "expression vector" refers to a preparation containing a nucleic acid sequence operatively linked to a suitable regulatory sequence capable of expressing a gene in a suitable host cell, said "operatively linked". The term “nucleic acid sequence binding” refers to a functional relationship.

The present invention

1) amplifying a gene encoding a high antigen capsid protein site of canine parvovirus with primers of SEQ ID NO: 1 and SEQ ID NO: 2;

2) preparing a recombinant expression vector by inserting the gene encoding the amplified high antigen capsid protein site into pGAP Za which is a vector for yeast expression;

3) obtaining a transformant by transforming the prepared recombinant expression vector into a host cell; And

4) culturing the obtained transformant in a medium containing Zeocin to express the gene according to claim 2 to obtain a culture, and recovering the protein from the culture;

It provides a method for producing a dog parvovirus recombinant protein comprising a.

In the present invention, the host cell is yeast ( Pichia) pastoris ), a host cell transformed with the expression vector of the present invention is characterized by using a yeast having high gene introduction efficiency and high expression efficiency of the introduced gene.

More specifically, the canine parvovirus recombinant protein is one or more expression regulators, as mentioned above, in which the canine parvovirus (CPV-2a) fragment gene is operatively linked, respectively, to regulate its expression. Inserted into a vector comprising an expression control sequence, transforming the host cell with a recombinant expression vector formed therefrom, and culturing the resulting transformant under medium and conditions appropriate for the gene to be expressed, and culturing It can be prepared by recovering substantially pure canine parvovirus (CPV-2a) recombinant protein from water.

The transformation method by introducing the expression vector into the host cell is calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, Cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection can be used. In this way, a transformant can be obtained.

The present invention provides a feed composition for the treatment or prevention of canine parvovirus infection, which contains a canine parvovirus recombinant protein as an active ingredient.

Feed composition of the present invention can be prepared by mixing the yolk antibody of the VP2 recombinant protein with a conventional feed. Feed used in animal breeding is extruded pellets (EP) made by mixing the general feed source in the form of pellets by applying heat and pressure, raw feed that is used as feed by making the pellets the material that can be used for animal feed. Moisture pellets (MPs), which are made of pellets by mixing raw feeds and powdered feeds (feeds mixed with a general feed source).

Canine parvovirus recombinant protein using yeast as a host cell according to the present invention directly expresses the capsid protein, which is a parvovirus high antigen site in yeast, and induces an immune response in the intestinal tract of the dog by administering it with a feed to immunoglobulin G in blood ( IgG) and immunoglobulin A (IgA), which is a mucosal immunity antibody, are directly induced into the intestinal mucosa, which is a cause of disease, and thus, parvovirus infection can be more efficiently and fundamentally treated and prevented.

1 is a photograph of the PCR amplified dog parvovirus capsid gene according to the present invention,
2 is a result confirming the presence or absence of the dog parvovirus capsid gene according to the restriction enzyme treatment of the vector inserted the dog parvovirus capsid gene according to the present invention,
(A: pGEM T-Easy cloning vector, B: pGAP Za yeast expression vector)
Figure 3 shows the nucleotide sequence (A) and amino acid sequence (B) of the canine parvovirus capsid gene according to the present invention,
Figure 4 shows the SDS-PAGE results for yeast transformed with an expression vector comprising a gene encoding a dog parvovirus recombinant protein according to the present invention,
(M: size mark, A: untreated yeast before transformation, B: yeast transformed with recombinant protein)
5 shows Western blot results for yeast transformed with an expression vector comprising a gene encoding a canine parvovirus recombinant protein of the present invention,
(M: size mark, A: untreated yeast before transformation, B: yeast transformed with recombinant protein)
Figure 6 shows the results of quantitative analysis of intestinal IgA derived from orally administered mice transformed with the recombinant protein of the present invention,
(1: untreated yeast treatment before transformation, 2: yeast treatment with a gene encoding a dog parvovirus recombinant protein transformed)
Figure 7 shows the results of quantitative analysis of IgG in the blood derived from the yeast-weighted dog transformed with the recombinant protein of the present invention,
Figure 8 shows the results of quantitative analysis of intestinal IgA derived from yeast-weighted dogs transformed with the recombinant protein of the present invention.

The present invention will be described in more detail with reference to the following examples. However, the following examples are provided to aid understanding of the present invention, and the scope of the present invention is not limited by these examples in any sense.

Hereinafter, the technical and scientific terms used herein will be understood by those skilled in the art without departing from the scope of the present invention. Descriptions of known functions and configurations that may be unnecessarily blurred are omitted.

Example 1 Cloning of Canine Parvovirus Capsid Gene

Gene cloning for capsid gene cloning of canine parvovirus (CPV-2a), which is currently in vogue, was synthesized based on GenBank # EU377537 as a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 2 to the following base sequences. At this time, in consideration of the yeast expression vector pGAP inside the multi-cloning positions (multiple cloning site) present in the Za of the EcoR I restriction enzyme has a forward portion and a reverse portion has been inserted into the Kpn I restriction enzyme any.

That is, the forward (SEQ ID NO: 1) primer was synthesized by GAATTC ATG AGT GAT GGA GCA GTT CA, and the reverse (SEQ ID NO: 2) primer by GGTACC TTA ATA TAA TTT TCT AGG TGC. Canine parvovirus (CPV-2a) was obtained from the National Veterinary Research and Quarantine Service and DNA was isolated using viral DNA extraction kit (Bioneer).

In order to amplify the canine parvovirus capsid gene using the isolated DNA as a template, PCR was performed using the forward primer of SEQ ID NO: 1 and the reverse primer of SEQ ID NO: 2.

As a result of PCR, 1.7 kb amplification products were obtained as shown in FIG. 1, and the amplified capsid gene was cloned into pGEM T-Easy (Promega), which is a cloning vector.

The cloned capsid gene was identified through sequencing and restriction enzyme ( EcoR I + Kpn I) treatment to determine the size and nucleotide sequence (SEQ ID NO: 3) and amino acid sequence (SEQ ID NO: 4) and amino acid sequence.

As a result, as shown in FIG. 2A, it was confirmed that the capsid gene was inserted in the 1.7 kb portion. After gel elution of the inserted capsid gene, the same restriction enzyme as the inserted capsid gene ( EcoR I + K p n I Ligation was carried out together with the fragment of pGAP Za, a yeast expression vector cut with). The ligation of the capsid gene and the pGAP Za vector is determined by E. coli colony selection, and the vector DNA is isolated and then cut back with the same restriction enzyme ( EcoR I + Kpn I) to insert the capsid gene in the yeast expression vector pGAP Za. It was confirmed.

As a result, it could be confirmed that the capsid gene in B and the yeast expression vector pGAP Za of FIG. 2 were inserted, and an expression vector including a gene encoding a canine parvovirus recombinant protein was prepared from the above result.

[ Example  2] In yeast  Confirmation of Protein Expression

The embodiment of yeast (for example Pichia one parvovirus expression containing the gene encoding the recombinant protein of the vector pastoris ), the cloned expression vector was cut with a restriction enzyme Avr II to make a linear, and then genes encoding recombinant canine parvovirus protein by electroporation (Gene Pulser, Bio-Rad). Was inserted into yeast and colony selection was performed on selection medium (YPD agar) containing Zeocin (Sigma).

After culturing the selected colonies for 3 days in YPD broth, the protein was obtained from the culture and the expression of the protein was confirmed by SDS-PAGE and Western blot.

As a result, as shown in Figure 4 it was confirmed that the protein of about 65 kDa size is expressed.

In addition, the electrophoretic gel was subjected to membrane transfer for western blot, and then, anti-CPV capsid HRP-conjugated as a primary antibody and DAB colorant (Serotec) for color development were used for expression of the protein. Confirmed. As a result, the expression of the recombinant protein was confirmed as shown in FIG. The recombinant protein was named VP2 protein.

[ Test Example  1] Confirmation of Intestinal Immunity in Mice

Prior to this experiment with dogs, it was confirmed that yeast preparations produced antibodies through oral administration using mice, which are experimental animals.

Ten 6-week-old ICR mice were prepared and divided into two groups of five animals each, group 1 using untreated yeast before transformation, and group 2 using transgenic yeast expressing the VP2 protein of Example 1 Oral administration of yeast per 1 × 10 ⁷ cells was administered three times at 2 week intervals.

After 7 days after the final administration three times, the mouse was sacrificed and the small intestine tissue was incised 5 cm, and then placed in 1 ml of PBS, the tissue was crushed using a tissue grinder, and the upper layer of the intestinal crushed fluid after centrifugation. The liquid was used as a sample.

ELISA was performed to confirm the presence of anti-CPV VP (capsid protein) immunoglobulin A (IgA) contained in the supernatant of the intestinal lysate. That is, the extracted capsid protein antigen was diluted with PBS, adjusted to 100 µg / ml, and coated on a 96-well plate (Maxisorp ™, Nunc, Denmark) at a concentration of 100 µl / well. The plate was blocked for 1 hour with PBS added with 1% skim milk, and then washed 3-4 times with PBST (PBS + 0.05% Tween-20, pH 7.4) buffer.

A total of 90 μl of the supernatant of the intestinal lysate was added per well, followed by washing at 37 ° C. for 1 hour and washing with PBST buffer three times, followed by goat anti-mouse IgA horseradish peroxidase diluted 1: 500. The reaction was carried out using HRP) -conjugated (Pierce) as the secondary antibody. Secondary antibodies were also washed three times with PBST buffer after 1 hour reaction at 37 ° C.

Detection of bound secondary antibody was carried out using a substrate solution [250 [mu] l of 100 mM citric acid buffer, pH 4.0, 10 [mu] l ABTS stock solution (ABTS 100 mg in 4.5 ml DW) 250 [mu] l H ₂ 0 _2. . The reaction with the substrate solution was carried out in the dark for 10 minutes, the OD value was measured by using an ELISA reader (Multiskan EX, Thermo Lab Systems, Beverly, MA) at an absorbance of 405 nm. The measured OD values were expressed as mean ± standard deviation (SD), and the values were tested for statistical significance by Student's t-test.

As a result, as can be seen in Figure 6, the immunoglobulin A (IgA) level in the transgenic yeast treatment group (No. 2) mice expressing the VP2 protein was added to the control group of the untreated yeast treatment group (No. 1) before the transformation Compared with the statistically significant (P <0.0001) high level, it was confirmed that immunoglobulin A was induced against the parvovirus capsid protein.

[ Test Example  2] Confirmation of intestinal immunity in dogs

Based on the test example 1, this experiment was conducted to determine the immune-induced response of the yeast agent to the dog parvovirus dog host animal.

(1) to be used in the experiment Self-sufficient  term- VP2  Maternal antibody IgG  Measure

First of all, the subjects checked the ELISA for the presence of anti-CPV-2a VP2 antibody, which may remain in the blood after blood collection. In other words, blood was collected from each dog, serum was isolated, recombinant VP2 was coated on the ELISA wells with antigen, and the dog's serum was used as a primary antibody (1: 100), and goat anti-canine IgG-HRP conjugated as a secondary antibody. ELISA was performed using (1: 1,000, Pierce).

As a result, as can be seen in Table 1 above, in the case of H335, the antibody value of blood was found to be a little high. As a result, it was confirmed by PCR that the dog was infected with Gapabovirus before the test dog. Although V583 did not have high antibody titer, V583 died of diarrhea and other canpabovirus infections.

In each of the other cases, it was confirmed that there were no significant high levels of antibody titers.

(2) IgG and IgA measurement of anti-VP2 antibodies after self-feeding of yeast expressing recombinant VP2

In this experiment, when feeding yeast expressing recombinant VP2, mucosa immunization against parvovirus was activated to determine whether antibody titer was performed. To this end, 12 dogs of 55 days of age, which are expected to be depleted of maternal antibodies, were prepared as shown in Table 2 below, followed by feeding yeast and measuring blood IgG and intestinal IgA for VP2. Feeding was carried out four times in total. Five days after each feeding, IgG was collected through blood collection, and diarrhea was induced, and IgA in feces was measured.

More specifically, Group 1 prepared six previously unvaccinated rats, three of them fed rVP-expressing yeast (CPV-SMD), and the other three fed rVP2 expressed in E. coli as antigen. This was done,

Group 2 is a group that feeds rVP-expressing yeast (CPV-SMD) using individuals who have been inoculated once with the commercialized parvo / corona vaccine (central vaccine),

Group 3 used a single dose of the commercialized parvo / corona vaccine (central vaccine) and two weeks later the DHPPL vaccine (Bay Korea).

Five days after the last dose, IgG was measured by blood collection. In other words, ELISA was performed in the same manner as the maternal antibody test. As a result of this experiment, changes in IgG in blood are shown in Table 3 below.

As can be seen in Table 3 and Figure 7, it was confirmed that the antibody titer in the IgG of blood in the dogs that received the recombinant VP in 9 dogs.

In order to detect immunoglobulin A (IgA) in the dog's feces, 1 g of fecal samples were diluted in 5 ml of PBS, centrifuged, and the supernatant was used as a sample. ELISA carried out after is the same as carried out in the mouse of Test Example 1. Only secondary antibody was used goat-anti canine IgA HRP-conjugated (Pierce).

As a result, as can be seen in Table 4 and FIG. 8, it was confirmed that the antibody value of the IgA in feces increased in seven dogs.

Canine parvovirus recombinant protein having a high antigenic capsid protein site expressed in the yeast of the present invention orally administered from the results of Test Examples 1 and 2 is immunoglobulin A, an antibody responsible for mucosal immunity through induction of an immune response in the intestine of a dog By inducing (IgA), it was found that it could be used as a preventive agent against parvovirus infection which may be a future infection, which would be more effective in treating or preventing parvovirus infection caused by infection before proper titer was induced. It is also the result of confirming that it can be done.

<110> INDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY <120> Composition for feeding containing high antigenic capsid protein          of canine parvovirus <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 1 gaattcatga gtgatggagc agttca 26 <210> 2 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 2 ggtaccttaa tataattttc taggtgc 27 <210> 3 <211> 1755 <212> DNA <213> canine parvovirus <400> 3 atgagtgatg gagcagttca accagacggt ggtcaacctg ctgtcagaaa tgaaagagct 60 acaggatctg ggaacgggtc tggaggcggg ggtggtggtg gttctggggg tgtggggatt 120 tctacgggta ctttcaataa tcagacagaa tttaaatttt tggaaaacgg atgggtggaa 180 atcacagcaa actcaagcag acttgtacat ttaaatatgc cagaaagtga aaattataga 240 agagtggttg taaataattt ggataaaact gcagttaacg gaaacatggc tttagatgat 300 actcatgcac aaattgtaac accttggtca ttggttgatg caaatgcttg gggagtttgg 360 tttaatccag gagattggca actaattgtt aatactatga gtgagttgca tttagttagt 420 tttgaacaag aaatttttaa 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 65 70 75 80 Arg Val Val Val Asn Asn Leu Asp Lys Thr Ala Val Asn Gly Asn Met                  85 90 95 Ala Leu Asp Asp Thr His Ala Gln Ile Val Thr Pro Trp Ser Leu Val             100 105 110 Asp Ala Asn Ala Trp Gly Val Trp Phe Asn Pro Gly Asp Trp Gln Leu         115 120 125 Ile Val Asn Thr Met Ser Glu Leu His Leu Val Ser Phe Glu Gln Glu     130 135 140 Ile Phe Asn Val Val Leu Lys Thr Val Ser Glu Ser Ala Thr Gln Pro 145 150 155 160 Pro Thr Lys Val Tyr Asn Asn Asp Leu Thr Ala Ser Leu Met Val Ala                 165 170 175 Leu Asp Ser Asn Asn Thr Met Pro Phe Thr Pro Ala Ala Met Arg Ser             180 185 190 Glu Thr Leu Gly Phe Tyr Pro Trp Lys Pro Thr Ile Pro Thr Pro Trp         195 200 205 Arg Tyr Tyr Phe Gln Trp Asp Arg Thr Leu Ile Pro Ser His Thr Gly     210 215 220 Thr Ser Gly Thr Pro Thr Asn Ile Tyr His Gly Thr Asp Pro Asp Asp 225 230 235 240 Val Gln Phe Tyr Thr Ile Glu Asn Ser Val Pro Val His Leu Leu Arg                 245 250 255 Thr Gly Asp Glu Phe Ala Thr Gly Thr Phe Tyr Phe Asp Cys Lys Pro             260 265 270 Cys Arg Leu Thr His Thr Trp Gln Thr Asn Arg Ala Leu Gly Leu Pro         275 280 285 Pro Phe Leu Asn Ser Leu Pro Gln Ala Glu Gly Gly Thr Asn Phe Gly     290 295 300 Tyr Ile Gly Val Gln Gln Asp Lys Arg Arg Gly Val Thr Gln Met Gly 305 310 315 320 Asn Thr Asn Ile Ile Thr Glu Ala Thr Ile Met Arg Pro Ala Glu Val                 325 330 335 Gly Tyr Ser Ala Pro Tyr Tyr Ser Phe Glu Ala Ser Thr Gln Gly Pro             340 345 350 Phe Lys Thr Pro Ile Ala Ala Gly Arg Gly Gly Ala Gln Thr Asp Glu         355 360 365 Asn Gln Ala Ala Asp Gly Asp Pro Arg Tyr Ala Phe Gly Arg Gln His     370 375 380 Gly Gln Lys Thr Thr Thr Thr Gly Glu Thr Pro Glu Arg Phe Thr Tyr 385 390 395 400 Ile Ala His Gln Asp Thr Gly Arg Tyr Pro Glu Gly Asp Trp Ile Gln                 405 410 415 Asn Ile Asn Phe Asn Leu Pro Val Thr Asn Asp Asn Val Leu Leu Pro             420 425 430 Thr Asp Pro Ile Gly Gly Lys Thr Gly Ile Asn Tyr Thr Asn Ile Phe         435 440 445 Asn Thr Tyr Gly Pro Leu Thr Ala Leu Asn Asn Val Pro Pro Val Tyr     450 455 460 Pro Asn Gly Gln Ile Trp Asp Lys Glu Phe Asp Thr Asp Leu Lys Pro 465 470 475 480 Arg Leu His Val Asn Ala Pro Phe Val Cys Gln Asn Asn Cys Pro Gly                 485 490 495 Gln Leu Phe Val Lys Val Ala Pro Asn Leu Thr Asn Glu Tyr Asp Pro             500 505 510 Asp Ala Ser Ala Asn Met Ser Arg Ile Val Thr Tyr Ser Asp Phe Trp         515 520 525 Trp Lys Gly Lys Leu Val Phe Lys Ala Lys Leu Arg Ala Ser His Thr     530 535 540 Trp Asn Pro Ile Gln Gln Met Ser Ile Asn Val Asp Asn Gln Phe Asn 545 550 555 560 Tyr Val Pro Ser Asn Ile Gly Gly Met Lys Ile Val Tyr Glu Lys Ser                 565 570 575 Gln Leu Ala Pro Arg Lys Leu Tyr             580

Claims (7)

Canine parvovirus recombinant protein (VP2) having a high antigenic capsid protein site of SEQ ID NO: 4. Gene encoding the canine parvovirus recombinant protein according to claim 1. An expression vector comprising a gene encoding a canine parvovirus recombinant protein according to claim 2. The method of claim 3, wherein
The expression vector is pGAP Za characterized in that the expression vector. 1) amplifying a gene encoding a high antigen capsid protein site of canine parvovirus with primers of SEQ ID NO: 1 and SEQ ID NO: 2;
2) preparing a recombinant expression vector by inserting the gene encoding the amplified high antigen capsid protein site into pGAP Za which is a vector for yeast expression;
3) obtaining a transformant by transforming the prepared recombinant expression vector into a host cell; And
4) culturing the obtained transformant in a medium containing Zeocin to express the gene according to claim 2 to obtain a culture, and recovering the protein from the culture;
Method for producing a dog parvovirus recombinant protein comprising a. 6. The method of claim 5,
The host cell is a yeast ( Pichia) pastoris ) method for producing a dog parvovirus recombinant protein, characterized in that. A feed composition for the treatment or prevention of canine parvovirus infection, comprising the canine parvovirus recombinant protein according to claim 1 as an active ingredient.

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