KR100812637B1 - Recombinant canine rotavirus vp4 protein comprising epitope and antibody - Google Patents

Abstract

A recombinant canine rotavirus VP4 protein and an antibody thereof are provided to treat or prevent canine rotavirus infection symptoms more efficiently. A recombinant canine rotavirus VP4 protein consists of an amino acid sequence selected from the group consisting of sequences described in SEQ ID : NO. 3, 6, 9, 12, 15, 18 and 21. A gene codes the recombinant canine rotavirus VP4 protein. A recombinant expression vector includes the gene operably linked to a control sequence of an expression vector such as pMAL-c2X. A method for preparing the recombinant canine rotavirus fiber protein comprises the steps of: (a) preparing the recombinant expression vector; (b) transforming a host cell using the recombinant expression vector; (c) culturing the transformed host cell to express the gene; and (d) isolating a recombinant canine rotavirus VP4 protein from a culture material obtained from the step(c). An antibody against the recombinant canine rotavirus fiber protein is characterized in that it is isolated from yolk of eggs laid by immunized egg layers. A vaccine composition for treating or preventing canine rotavirus infection symptoms comprises the recombinant canine rotavirus VP4 protein as an effective ingredient.

Description

Recombinant Canine Rotavirus VP4 Protein Comprising Epitope and Antibody

1A is an electrophoretic photograph of a gene encoding the full length VP4 protein of canine rotavirus.

Figure 1b is an electrophoresis picture of the gene encoding the recombinant canine rotavirus VP4 protein of the present invention.

Figure 1c shows a cloning vector comprising the gene encoding the recombinant canine rotavirus VP4 protein of the present invention.

Figure 2a shows a recombinant expression vector comprising a gene encoding the recombinant canine rotavirus VP4 protein of the present invention.

Figure 2b is an electrophoresis picture showing that the gene of the recombinant canine rotavirus VP4 protein of the present invention is inserted into the recombinant expression vector of Figure 2a.

Figure 2c is a Western blot showing that the recombinant canine rotavirus VP4 protein of the present invention can be expressed through genetic recombination techniques.

3 is a graph showing that the recombinant canine rotavirus VP4 protein of the present invention exerts immunogenicity.

Figure 4 is a graph showing the change in the antibody over time egg yolk of the laying hens receiving the recombinant canine rotavirus VP4 protein of the present invention.

Figure 5a is a graph showing that the dog rotavirus-induced infection is reduced in the dog receiving the yolk antibody to the recombinant dog rotavirus VP4 protein of the present invention.

5B is a graph showing that dog rotavirus infection is prevented and treated in a dog receiving yolk antibody against the recombinant canine rotavirus VP4 protein of the present invention.

The present invention relates to a recombinant dog rotavirus VP4 protein comprising an antigenic determinant and an antibody thereto, and more particularly, to a recombinant dog rotavirus VP4 protein comprising an antigenic determinant of a dog rotavirus VP4 protein, a gene encoding the protein. , A recombinant expression vector comprising the gene, a host cell transformed with the recombinant expression vector, a vaccine composition containing the recombinant canine rotavirus VP4 protein, an antibody against the recombinant canine rotavirus VP4 protein, and the antibody containing the antibody. A pharmaceutical composition for treating or preventing canine rotavirus infection.

Pathogenic viruses that cause serious digestive problems in dogs and cause economic and emotional harm include canine parvovirus type 2 (CPV-2), canine adenovirus (CAV), and canine coronavirus (canine coronavirus); CCV), canine rotavirus (CRV), and the like (Mochizuki et al. 2001 J. Vet. Med. Science. Vol . 63, pp. 573-575).

The economic losses caused by the above mentioned pathogenic viruses should take into account the morbidity caused by each virus, but there are no statistics on the digestive disease disease caused by such viruses in Korea. However, in Japan, where the situation is similar, the prevalence of each virus is canine parvovirus (CPV-2); 23.4%, canine adenovirus (CAV); 2.2%, canine coronavirus (CCV); 55.4%, canine rotavirus (CRV); 2.1% was reported (Mochizuki et al. 2001). Based on these statistics, inferring the situation of the entire domestic dog can be said that the economic losses caused by the viruses are very huge.

In the absence of defense ability due to vaccine break, a problem caused by the use of vaccines to prevent infectious diarrhea in dogs, intestinal infection of pathogens can be prevented by neutralizing pathogenic viruses in the intestine by orally administering yolk antibodies to each pathogen.

In the past, virus culture was used as an antigen for egg yolk antibody production. To this end, the host cells must be cultured, followed by the inoculation of the virus, followed by culturing the host cells infected by the virus. The antigen production process is complicated, the antigen production period is long, and the yield is low, and the production cost is very high and egg yolk. The titer of the antibody is also low.

 On the other hand, "dog rotavirus" is a virus that infects puppies and causes diarrhea, and excretes green and viscous diarrhea, but blood incorporation usually does not occur, and the lesion is confined to the small intestine, but causes congestion throughout. Histologically, the small intestinal villi atrophy and the villi columnar epithelial cells are destroyed, so that epithelial cells in the impression or cuboid can be observed and the absorption function is reduced. However, an efficient vaccine has not been put to practical use yet.

The inventors analyzed the VP4 protein of canine rotavirus and prepared the recombinant canine rotavirus VP4 protein and its antibody to include the antigenic determinant and then administered it to the dog, thereby preventing and treating the dog rotavirus infection. It was confirmed that the present invention was completed.

In one aspect, the present invention provides a recombinant canine rotavirus VP4 protein comprising an antigenic determinant of canine rotavirus VP4 protein.

In another aspect, a gene encoding the recombinant canine rotavirus VP4 protein is provided.

In another aspect, there is provided a recombinant expression vector that is included such that the gene encoding the recombinant canine rotavirus VP4 protein is operably linked to a regulatory sequence of the expression vector.

In another aspect, a host cell transformed with the recombinant expression vector is provided.

As another aspect, (1) producing a recombinant expression vector by operably linking the gene encoding the recombinant dog rotavirus VP4 protein to the regulatory sequence of the expression vector; (2) transforming the host cell with the recombinant expression vector; (3) culturing the transformed host cell to express the gene; (4) It provides a method for producing a recombinant canine rotavirus VP4 protein comprising the step of separating the recombinant canine rotavirus VP4 protein from the culture.

In another aspect, there is provided an antibody against the recombinant canine rotavirus VP4 protein, in particular an egg yolk antibody.

As another aspect, (1) immunizing a laying hen with said recombinant canine rotavirus VP4 protein; (2) providing a method for producing an egg yolk antibody against a recombinant dog rotavirus VP4 protein, comprising the step of separating the egg yolk antibody against the recombinant dog rotavirus VP4 protein from the egg yolk from the egg laid.

In another aspect, a vaccine composition for treating or preventing canine rotavirus infection containing the recombinant canine rotavirus VP4 protein as an active ingredient is provided.

In another aspect, the present invention provides a pharmaceutical composition for the treatment or prevention of canine rotavirus infection containing an antibody against the recombinant canine rotavirus VP4 protein as an active ingredient.

In still another aspect, there is provided a feed composition for treating or preventing canine rotavirus infection containing an antibody against the recombinant canine rotavirus VP4 protein as an active ingredient.

In another aspect, the present invention provides a method for treating or preventing a dog rotavirus infection by administering the vaccine composition to a mammal other than a human.

In another aspect, the present invention provides a method for treating or preventing canine rotavirus infection by administering the pharmaceutical composition to a mammal other than a human.

The present invention relates to a VP4 protein of a canine rotavirus (hereinafter simply referred to as "CRV") recombined to include an epitope of a VP4 protein derived from canine rotavirus.

Specifically, the recombinant CRV VP4 protein of the present invention is fragmented to include a site to which an antibody binds, ie, an antigenic determinant, by analyzing the VP4 protein, which is a protein constituting the outer capsid of a dog rotavirus. , SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 18 and SEQ ID NO: 21, consisting of the amino acid sequences set forth in SEQ ID NO: each of which is CRV Va, CRV Vb, CRV Vc, CRV Vd, CRV Ve, CRV Vf, CRV Vg.

In the present invention, the recombinant CRV VP4 protein includes a glycoprotein including the protein and all derivatives derived therefrom, including the protein consisting of the amino acid sequence described in SEQ ID NO.

The recombinant CRV VP4 protein of the present invention comprises the steps of: (1) preparing a recombinant expression vector by operably linking a gene encoding the recombinant CRV VP4 protein to a regulatory sequence of the expression vector; (2) transforming the host cell with the recombinant expression vector; (3) culturing the transformed host cell to express the gene; (4) can be prepared by the step of separating the recombinant CRV VP4 protein from the culture.

Genes encoding the proteins are required to produce the recombinant CRV VP4 proteins of the present invention by the above-described genetic recombination techniques. The gene encoding the recombinant CRV VP4 protein according to the present invention may be artificially synthesized using a nucleic acid synthesizer or the like by referring to the nucleotide sequence of the gene, or encoding a recombinant CRV VP4 protein of interest as a template for genomic DNA of a dog rotavirus. It can be prepared by PCR using oligonucleotide having a sequence complementary to both ends of the gene as a primer. On the other hand, due to the degeneracy of the codon, the gene encoding the recombinant CRV VP4 protein of the present invention may exist in various base sequences, all of which are included in the scope of the present invention.

The gene encoding the recombinant CRV VP4 protein of the present invention is operably linked to the regulatory sequence of the expression vector according to step (1) to produce a recombinant expression vector. Here, "expression vector" refers to a DNA molecule capable of introducing and expressing a foreign gene into a host cell, which may be a plasmid, phage particle or simply a potential genomic insert. Various expression vectors for effectively inducing the expression of foreign genes in specific host cells are commercially available. For example, pUC8, pBR322, pET / Rb, pGEX, pET28a, etc. can be used in the present invention, and pMAL-c2x may be used. It is preferable. A DNA molecule obtained by operably linking a gene encoding a recombinant CRV VP4 protein of the present invention to a regulatory sequence of such expression vector is referred to as a "recombinant expression vector".

The recombinant expression vector thus prepared is transformed into host cells according to step (2) above. The host cell of the invention can be a prokaryotic or eukaryotic cell. A host having a high DNA introduction efficiency and a high expression efficiency of the introduced DNA is usually used. Known eukaryotic and prokaryotic hosts such as E. coli, Pseudomonas, Bacillus, Streptomyces, fungi, yeast can be used, with E. coli being preferred.

Methods for transforming into host cells include conventional transformation methods such as cationic lipid-mediated transfection, electroporation, DEAE-dextran mediated transfection. Calcium phosphate transfection can be used. With reference to what is known in the art, another optimal transformation method may be introduced depending on the type of host cell, expression vector, and the like.

The transformed host cells can be cultured in a nutrient medium suitable for the production of the recombinant CRV VP4 protein according to the invention according to step (3) above. For example, host cells may be cultured by small or large scale fermentation, shake flask culture in a laboratory or industrial fermentor conducted under conditions that facilitate expression and / or isolation of recombinant CRV VP4 protein with a suitable medium. Cultivation is carried out in a suitable nutrient medium containing carbon, nitrogen sources and inorganic salts using known techniques. Such suitable media may be obtained commercially or as known in the literature (eg, catalog of the American Type Culture Collection).

Recombinant CRV VP4 protein produced in transformed host cells can be isolated using conventional protein separation methods according to step (4) above. For example, the recombinant CRV VP4 protein can be isolated from the culture according to methods including, but not limited to, centrifugation, filtration, extraction, spray drying, evaporation or precipitation. Furthermore, recombinant CRV VP4 proteins may be purified via known methods, including chromatography (eg, ion exchange, affinity, hydrophobicity and size exclusion), electrophoresis, fractional solubility (eg, ammonium sulfate precipitation), SDS-PAGE or extraction. It can be purified.

On the other hand, antibodies to the recombinant CRV VP4 protein of the invention, in particular egg yolk antibodies, also form an aspect of the invention.

Monoclonal and polyclonal antibodies specific for canine rotavirus can be prepared using the recombinant CRV VP4 protein of the present invention as an antigen. Polyclonal antibodies are generally immunized with one or more collections of a mammal with an appropriate amount of antigen and, when the antibody titer reaches a certain value, recovered from the antiserum of the mammal and purified using known procedures if desired. And store in frozen buffered solution until use. On the other hand, the monoclonal antibody can isolate the B lymphocytes generated by injecting the antigen into the mammal and fusion with myeloma cells to make hybridoma cells and culture the antibody. Details of this process are well known in the art.

In the case of egg yolk antibodies, (1) immunizing the laying hen with the recombinant CRV VP4 protein; (2) can be prepared by separating the egg yolk antibody against the recombinant CRV VP4 protein from the egg yolk of the eggs laid from the laying hen.

The scattering system used in the present invention is not particularly limited, and it is preferable to use, for example, a white leghorn system, a Rhode island system, a high-line brown system, etc. having a high scattering rate. Routes for administering antigens to laying hens include intravaginal, intramuscular, intraocular or subcutaneous injection, and the like. Antigens of the invention, ie recombinant CRV VP4 protein, can be increased in immunity by using an adjuvant such as Freund's complete adjuvant or incomplete adjuvant.

Additional antigen boosters can be performed until sufficient antibodies are obtained. Preferably, about 3 times immunization at about 2 weeks interval, and 6 to 10 weeks interval if necessary. The eggs are collected from the immunized laying hens, the desired antibodies are isolated therefrom, the amount of antibody derived is measured, and when the increase reaches a steady state, the eggs are finally recovered.

The amount of the antibody derived can be measured according to conventional methods in the art. Preferably, it is measured by enzyme immunosorbent method or microtiter method. More preferably, after reacting the antigen of the present invention (recombinant CRV VP4 protein) attached to the microplate with the antibody isolated from egg yolk of the immunized egg according to the present invention, the secondary antibody is reacted and the substrate solution is added thereto. Enzyme Linked Immunosorbent Assay (ELISA) is used to quantify antibodies induced by enzymatic color reaction and measurement at specific wavelengths.

Egg yolk antibody of the present invention is to separate the egg yolk from the recovered eggs, dilute it with distilled water, adjust the pH of the dilution to 4.0 to 6.0, freeze, centrifugation by thawing the frozen body, and filter the formed supernatant Can be separated. Preferably, egg yolk is separated from the recovered eggs and diluted to 5 to 15 times by adding distilled water, and then the pH of the diluted solution is adjusted to about 5.0 and then frozen at -10 ° C to -30 ° C for at least 10 to 20 hours, and frozen. The sieve is thawed at room temperature and centrifuged for 20 to 40 minutes at 5,000 to 15,000 × g, 1 ° C to 10 ° C, and the supernatant formed is separated by filtration with filter paper.

In the above, both the recombinant CRV VP 4 protein and the antibodies thereof of the present invention can be used to treat or prevent canine rotavirus infection. Recombinant CRV VP4 protein can be treated or prevented through active immunity, and antibodies against recombinant CRV VP4 protein can be treated or prevented through canine rotavirus infection. Active immunity means that the living body itself is immunized when the pathogen invades itself, and passive immunity refers to immunity obtained by receiving an immune body produced in another living body in its body.

Recombinant CRV VP4 protein and antibody thereof which can treat or prevent canine rotavirus infection by this mechanism can be used as an active ingredient of vaccines and pharmaceutical compositions. In this case, the active ingredient is suitably used in the form of incorporation into a pharmaceutically acceptable carrier, rather than being used alone.

Here, pharmaceutically acceptable carriers include carriers, excipients and diluents commonly used in the pharmaceutical art. Pharmaceutically acceptable carriers for use in the vaccine compositions and pharmaceutical compositions of the invention include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, Alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The vaccine composition and the pharmaceutical composition of the present invention are each formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, or the like, external preparations, suppositories, or sterile injectable solutions according to conventional methods. Can be used.

When formulated, it may be prepared using conventional diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, and the like. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations contain at least one excipient in the active ingredient, for example starch, calcium carbonate, sucrose, lactose, gelatin It can be prepared by mixing. In addition to simple excipients, lubricants such as magnesium stearate, talc can also be used. Liquid preparations for oral use include suspensions, solvents, emulsions, and syrups, and various excipients such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used diluents such as water and liquid paraffin. have. Formulations for parenteral administration include sterile aqueous solutions, water-insoluble solvents, suspensions, emulsions, lyophilized formulations and suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

In particular, in the case of a liquid formulation, it is preferable to mix and sterilize a bactericide by filtration through a bacterial capture filter or the like. The so sterilized composition may be solidified, for example, by lyophilization, which, in use, is dissolved in sterile water or sterile diluent.

The vaccine composition and the pharmaceutical composition according to the present invention can be administered to mammals such as mice, mice, livestock, dogs, humans, etc. by various routes. All modes of administration can be expected, for example by oral, intravenous, intramuscular, subcutaneous, intraperitoneal injection.

The dosage of the vaccine composition and the pharmaceutical composition according to the present invention is selected in consideration of the age, weight, sex, physical condition, etc. of the animal. The amount necessary to induce an immunoprotective response in the animal without any adverse side effects may vary depending on the presence of any of the recombinant CRV VP4 proteins and excipients used as immunogens. Generally each dose contains 0.1 to 1000 μg protein, preferably 0.1 to 100 μg per sterile solution of the immunogenic amount of the recombinant CRV VP4 protein or antibody of the invention. In the case of vaccine compositions, an initial dose may optionally be followed by optionally repeated antigen stimulation.

Meanwhile, the antibody against the recombinant CRV VP4 protein of the present invention may be mixed with a conventional feed to prepare a feed composition for treating or preventing the infection of dog adenoviruses. Feeds include by-products such as pork, beef, and chicken, as well as corn, rice, rice straw, grasses, grasses, ansiridge, hay, and wild grasses, but are not limited thereto. Do. The method of adding and blending the antibody against the recombinant CRV VP4 protein of the present invention to such a feed includes a method such as mechanical mixing, adsorption, occlusion, but is not limited thereto.

Hereinafter, the examples are only for illustrating the present invention in more detail, and the scope of the present invention is not limited by these examples in accordance with the gist of the present invention, those skilled in the art. Will be self-evident.

Example 1 Acquisition and Gene Recombination of Canine Rotavirus Strains

1-1: Securing Canine Rotavirus Strain

37 ° C., to monolayer MA104 cells in Dulbecco's Modified Eagle medium [D-MEM (Gibco Co. USA)] with 10% FBS (Gibco Co. USA) and 1% penicillin-streptomycin (Gibco Co. USA). Incubated in a 5% CO ₂ incubator. Cultured MA104 cells were infected with canine rotavirus (ATCC-VR964). Approximately 70% of MA104 cells infected with CRV were treated in a 37 ° C., 5% CO ₂ incubator until cytopathic effect (CPE) occurred. 3 days Incubated.

1-2: RNA Extraction and cDNA Synthesis of Canine Rotavirus

CRV genomic RNA was extracted using Viral gene-spin ^TM viral DNA / RNA Extraction kit (iNtRON Biotechnology Inc. Korea). The extracted CRV genomic RNA was treated with DNase I (TaKaRa Co. Japan) and M-MLV reverse transcriptase (iNtRON Biotechnology Inc. Korea) was used to synthesize the first strand cDNA according to the manufacturer's manual.

1-3: Recombination of the gene encoding VP4 protein of dog rotavirus

The gene encoding the VP4 protein of the dog rotavirus was prepared by PCR using a primer specific for the gene of the CRV VP4 protein. The primer specific for the gene of the CRV VP4 protein was based on GenBank data (NCBI D13401). Was prepared to amplify the full-length gene (2.2Kb, SEQ ID NO: 22) of the CRV VP4 protein (CVR Va-F and CVR Vg-R primers in Table 1).

On the other hand, the gene encoding the recombinant CRV VP4 protein according to the present invention was amplified by PCR using the oligonucleotide shown in Table 1 as a template as a template and prepared as a primer. Namely, 1.0 μl of DNA template, 4 μl of 25 mM MgCl ₂ , 10 × EX Taq. Distilled water was added to 5 µl of buffer solution, 4 µl of 2.5 mM dNTP, 1 µl of forward primer for each fragment, 1 µl of reverse primer, and 0.5 µl of EX Taq DNA polymerase (5U / µl) so that the total reaction volume was 50 µl. Initiate denaturation at 94 ° C. for 10 minutes, denaturation at 94 ° C. for 1 minute, annealing at 55 ° C. for 1 minute, and repeating for 30 minutes at 72 ° C. for 30 minutes, and further extending at 72 ° C. for 10 minutes. Was carried out. The PCR product thus amplified was electrophoresed at 100V for 30 minutes on a 1% agarose gel, and then stained with ethidium bromide to identify a band.

As shown in Figure 1a, the product obtained through the PCR was confirmed that the gene encoding the 2.3kb CRV VP4 protein (lane 1: 1kb DNA ladder, lane 2: PCR product, lane 3: restriction enzyme of PCR product) Degradation products).

Dark text indicates restriction enzyme recognition sites.

On the other hand, the gene encoding the recombinant CRV VP4 protein according to the present invention was amplified by PCR using the oligonucleotide shown in Table 1 as a template as a template and prepared as a primer. Namely, 1.0 μl of DNA template, 4 μl of 25 mM MgCl ₂ , 10 × EX Taq. Distilled water was added to 5 µl of buffer solution, 4 µl of 2.5 mM dNTP, 1 µl of forward primer for each fragment, 1 µl of reverse primer, and 0.5 µl of EX Taq DNA polymerase (5U / µl) so that the total reaction volume was 50 µl. And 10 cycles of total denaturation at 94 ° C for 10 minutes, 1 minute at 94 ° C for 1 minute, annealing at 57 ° C for 1 minute, and extended for 2 minutes at 72 ° C for 30 minutes, and further extended at 72 ° C for 10 minutes. Was carried out. The PCR product thus amplified was electrophoresed at 100V for 30 minutes on a 1.2% agarose gel, and then stained with ethidium bromide to identify a band.

As a result of the electrophoresis analysis, the size of 344, 362, 314, 331, 353, 353, 317bp (CRV Va , CRV Vb , CRV Vc , CRV Vd , CRV Ve , CRV Vf , CRV Vg ) Seven genes encoding the recombinant CRV VP4 protein having were identified (lane 1: 100 bp DNA ladder, lane 2: CRV Va, lane 3: CRV Vb, lane 4: CRV Vc, lane 5: CRV Vd, lane 6: CRV Ve). Lane 7: CRV Vf, lane 8: CRV Vg).

The obtained PCR product was cloned into pGEM T easy vector (Promega Co, USA) as shown in FIG. 1C to confirm the base sequence on the basis of GenBank's database, and the homology of the analyzed base sequence to the US National Center for NCBI Comparison was made with the BLAST program provided by Biotechnology Information. As a result, it showed 99% similarity with the gene of CRV VP4 protein.

Example 2: Recombinant Expression Vector Construction and Protein Expression and Identification

2-1: Recombinant Expression Vector Construction

344, 362, 314, 331, 353, 353, and 317 bp (CRV Va ,) obtained by selecting positive clones for the genes of the seven recombinant CRV VP4 proteins previously obtained and cleaving each plasmid with restriction enzymes BamH I and Sal I The expression vector pMAL-c2x prepared by cleavage with the same restriction enzyme as the DNA fragment of CRV Vb , CRV Vc , CRV Vd , CRV Ve , CRV Vf , CRV Vg ) was ligated (see FIG. 2A). This was transformed into E. coli competent JM109 to select positive clones.

The recombinant expression vector was separated therefrom, digested with restriction enzymes BamH I and Sal I, and subjected to electrophoresis using agarose gel. As shown in FIG. 2B, seven genes encoding the recombinant CRV VP4 protein were identified (lane 1: λ DNA HindIII size marker, lane 2: pDMCRVva, lane 3: pDMCRVvb, lane 4: pDMCRVvc, lane 5: pDMCRVvd, lane 6: pDMCRVvf, lane 7: pDMCRVvg, lane 8: 100 bp ladder marker).

2-2: Expression and Isolation of Recombinant CRV VP4 Protein Using Escherichia Coli

The positive clones obtained above were taken and inoculated into 5 ml of LB broth (Merck co.) To which 100 μg ampicillin was added, followed by shaking culture at 37 ° C. and 200 rpm for 12 to 16 hours. Inoculated with 100 ml of LB broth added and incubated at 37 ° C. and 200 rpm until the absorbance (OD ₆₀₀ ) became 0.5-0.6. When the absorbance (OD ₆₀₀ ) was 0.5-0.6, 0.3 mM IPTG (isopropyl-β-D-thio-galactopyranoside, DUCHEFA Co. Netherland) was added, and then incubated for 3 hours 30 minutes-4 hours. The culture was collected by centrifugation at 6,000 rpm for 10 minutes at 4 ° C. to recover the cells, and then resuspended in cell lysis buffer (100 mM NaH ₂ PO ₄ , 300 mM NaCl, 10 mM imidazole, pH 8.0) and lysozyme 1 mg / It was added to the ml and left for 15 minutes on ice. The lysed cells were crushed using an ultrasonic grinder (BRANSON SONIFIER 450, Branson Ultrasonic. USA), and centrifuged at 4 ° C. and 10,000 × g for 20 minutes to collect supernatant and pellets separately to collect recombinant CRV VP4 protein. Obtained.

2-3: Analysis of Recombinant CRV VP4 Protein

Whether the protein obtained previously was a CRV VP4 truncated protein was confirmed by Western blot analysis. At this time, two sheets of SDS-polyacrylamide gel were simultaneously made and used, and the proteins were also loaded into two SDS-polyacrylamide gels in half and each was subjected to electrophoresis. After electrophoresis, one sheet was stained with Kusami Blue R-250 (SIGMA Co. USA) to visually see the protein, and the other sheet was blotted onto a nylon membrane (Amersham Pharmacia Biosciences, UK) and then 3% BSA. Blocked at 4 ° C. for 16 hours with / PBS. The membrane was then washed once with TBST (100 mM Tris-Cl pH7.5, 0.9% NaCl, 0.1% Tween 20), and the anti-MBP antibody (NEB Co. USA) was diluted 20,000 times with the primary antibody for 1 hour at room temperature. The reaction was carried out and washed three times with TBST. Here, a 10,000-fold dilution of alkaline phosphatase conjugated anti-rabbit IgG (Sigma Co. USA) with a secondary antibody was added to the membrane and allowed to react for 1 hour at room temperature. The membranes were then washed three times with TBST for 5 minutes each, once with TNM (100 mM Tris-Cl pH9.5, 100 mM NaCl, 5 mM MgCl ₂ ) and then BCIP (5-Bromo-4-Chloro-3-Indolyl). Phosphate, Amersham Pharmacia Bioscience, UK) and NBT (Nitro blue tetrazolium, Amersham Pharmacia Bioscience, UK) induced color development.

As shown in Figure 2c it was confirmed that the size of about 50-60kDa protein is detected in lanes 3-8 (lane 1: protein molecular weight marker (BIO-RAD, USA); lanes 2-8: recombinant CRV Va ~ Vg protein ). This is a result of the protein expressed in E. coli specifically reacted with anti-MBP antiserum, thereby confirming that the protein expressed in E. coli is a recombinant CVR Va ~ Vg protein.

2-4: Purification of recombinant CRV VP4 protein and analysis of protein concentration

Recombinant CRV VP4 protein was purified using pMAL ^™ protein fusion and purification system (NEB Co. USA). After incubating the bacteria in 1 L LB broth and centrifuging with reference to Example 2-2, the cells were secured. The recovered cells were crushed with an ultrasonic grinder, and only the supernatant was collected and mixed with the resin contained in the kit. The reaction was carried out for 3 hours with stirring at ℃. Thereafter, glass wool was loaded to load the aqueous protein solution reacted with the propylene glycol tube. After this, all procedures were performed according to the manufacturer's manual. In addition, the purified recombinant CRV VP4 protein concentration was obtained using the BCA (Bicinochoninic acid) Kit (Pierce Co.).

Example 3: Immunogenicity Study of Recombinant CRV VP4 Protein

3-1: Antiserum Preparation for Recombinant CRV VP4 Protein

In order to carry out an immunogenicity test for the recombinant CRV VP4 protein obtained in Example 2, immunization was performed to 8-week-old BALB / C mice (Samtako BioKorea. Korea). Eight recombinant CRV VP4 proteins and the inactivated virus were emulsified by mixing in equal amounts with the Frauns Complete Adjuvant (Sigma Chemical Co.). 0.5 ml of the emulsion was injected intramuscularly, and further inoculation was emulsified with a Freund's incomplete adjuvant (Sigma Chemical Co.) at two week intervals after the first inoculation, and performed twice in the same manner as the first inoculation. Blood was collected from mice before each inoculation, and serum was separated, and the antibody titer was confirmed by immunoassay for immunogenicity by ELISA.

As shown in FIG. 3 and Table 2, the antibody having the highest CRV Vd (16,000) was shown. In addition, the immunity of the CRV virus itself was 21,000, and the antibody titers of the remaining proteins were 2,500, 2,700 and 2,800 (CRV Vb, CRV Vc, CRV Vg).

3-2: Neutralization test using antiserum against recombinant CRV VP4 protein

In order to determine whether the antiserum of each of the above-produced CRV VP4 protein has a neutralizing effect on CRV, hemagglutinin inhibition test was performed to conduct a neutralization test. Each antiserum was diluted from 10-fold to 2-fold, and dispensed into 96 spheres (NUNC,). An equal amount of 4HA CRV virus was reacted at 37 ° C. for 1 hour, and 50 μl of 1% red blood cells (RBC) were mixed and mixed well. After standing at 25 ° C. for 40 minutes, the hemagglutination reaction was observed. This hemagglutination reaction was performed three times for each antiserum.

As shown in Table 2, it showed the best neutralization score in anti-CRV Vd.

Neutralization Results of Recombinant CRV VP4 Protein According to the Invention serum Antibodies Neutralization test Anti-CRV 21000 80 Anti-CRV Vb 2500 80 Anti-CRV Vc 2700 80 Anti-CRV Vd 16000 1400 Anti-CRV Vg 2800 1200

Example 4 Egg Yolk Antibody Production and Identification Using Recombinant CRV VP4 Protein

4-1: Production of Egg Yolk Antibody Against Recombinant CRV VP4 Protein

Immunization was performed on 28-week-old ISA-Brown laying hens for antibody production against the recombinant CRV VP4 protein obtained in this experiment. Breeding and feeding control of the laying hens was carried out in H farm, Gyeonggi-do, and emulsified by mixing the same amount of purified recombinant CRV VP4 protein and Frowns complete adjuvant (Sigma Chemical Co.). 1 ml of the emulsion was intramuscularly injected into four places of 0.25 ml in the hen's pectoral muscle, and the additional inoculation was emulsified with a Freund's incomplete adjuvant (Gibco Co. USA) every two weeks after the first inoculation in the same manner as the first inoculation. Three times in total. Before each inoculation, blood was collected from the subarcacular vein of the laying hens and serum was separated and stored at -20 ° C. Eggs were collected daily and stored in a low temperature room at 8 ° C for use in the experiment.

4-2: Confirmation of Egg Yolk Antibody from Egg Yolk of Laying Hens

ELISA was performed to analyze the antibody titer of the recombinant CRV VP4 protein from the antiserum and egg yolk isolated. Dilute the recombinant CRV VP4 protein to 5 μg / ml with carbonate-bicarbonate buffer (pH 9.6) and dispense 100 μl into each sphere of the Microtest III flexible assay plate (Falcon 3911, BD Biosciences, USA). Coating overnight at 4 ° C. The coated plate was washed three times with a washing buffer (PBST, 0.02M NaH ₂ PO ₄ , 0.13M NaCl, 0.05% Tween 20, pH 7.2), followed by 3% BSA (bovine serum albumin, pH 7.3, Sigma Co). USA) / PBS was dispensed into each sphere at 175 μL and incubated at 37 ° C. for 2 hours. After washing 5 times with PBST and diluting buffer solution containing 3% BSA / PBS and washing buffer solution (PBST, phosphate buffered saline-Tween 20) in the same amount, the antiserum and egg yolk obtained were diluted 2,000-fold each, three times in succession. After diluting with, the recombinant CRV VP4 protein was coated on the spheres and reacted at 37 ° C. for 2 hours. As a secondary antibody, alkaline phosphatase conjugated rabbit anti-chicken IgG (Jackson ImmunoResearch Laboratories Inc. USA) was diluted 5,000-fold and reacted by dispensing the spheres of the plate at 37 ° C for 2 hours. The substrate buffer was reacted at 37 ° C. for 20 minutes using a solution in which a phosphate substrate tablet (ρ-nitrophenyl phosphate, PIERCE Co. USA) was dissolved in 10% diethanolamine (pH 9.8) containing 0.5 mM MgCl ₂ . . After the reaction was blocked using 5M NaOH as a stop solution, the reaction was investigated by measuring absorbance at 405 nm using a microplate reader (EL-800, BIO-TEK Instrument Inc. USA).

As shown in FIG. 4, the antibody titer steadily increased from 3 weeks after immunization, and showed the highest antibody titer at 6 weeks (antibody is about 500,000). From then on, antibody titers tended to remain constant until the measurement expiration date. This phenomenon is presumed to be caused by the accumulation of antibodies formed in the blood from the immediate post-immune 2 weeks to transfer to yolk from 3 weeks.

4-3: Isolation of Egg Yolk Antibodies

Eggs obtained from the inoculated egg hens were collected and egg yolk proteins were separated by Kim et al. (1998, MS Thesis, Dankook University). First, only egg yolk was separated from the collected eggs and diluted 10-fold with distilled water. The diluted egg yolk solution was adjusted to pH 5.0 and frozen overnight at -20 ° C. The frozen egg yolk was thawed at room temperature and centrifuged at 10,000 x g at 4 ° C for 30 minutes to collect the supernatant. The supernatant was filtered to separate the aqueous fraction (WSF) and freeze-dried and stored. Ion-exchange chromatography using DEAE was carried out to purify egg yolk antibodies. That is, equilibrate with 0.025M phosphate buffer in a column containing DEAE, dispense 0.025M phosphate buffer solution containing egg yolk antibody in the column, wash the column, and elute with 0.25M phosphate buffer (pH 8.0). . At this time, the eluted protein was fractionated by 3 ml per tube and purified by measuring the absorbance at 280 nm using a spectrophotometer.

Example 5 Clinical Trials Using Egg Yolk Antibodies

5-1: Test animal and test design

Clinical trials using egg yolk antibody were performed for 24 days with 14 dogs of weaning and weaning age of 3.38 ± 0.7 (SD). The test design was as follows.

(1) control; Normal lyophilized yolk 1g / day administration group

(2) Low concentration treatment (LAB): 1g / day administration group of egg yolk antibody containing egg yolk antibody against CRV Vd

(3) High concentration treatment group (HAB): 3g / day egg yolk antibody containing yolk antibody against CRV Vd

5-2: Administration of specification control and yolk antibody, CRV

All dogs were bred separately in each metabolic cage. During the test period, the diet was given for the general weaning diet (for 2 ~ 5kg), and the feeding amount was provided twice in the morning and afternoon divided by 3% of the body's recommended daily daily salary. The yolk antibody and normal yolk powder were orally administered from the start of the test to the end of the test. CRV (6 × 10 ⁵ pfu / ml) was orally administered on the 3rd and 4th days of the test, and the visual loss, loss of appetite, diarrhea, Eye and nasal discharge, vomiting and mortality were checked.

As shown in Fig. 5a and Table 2, the mortality rate of all treatments was 0% during the 14-day test period, and the secretion of eyes and nose was 71% in the control group, which was higher in comparison with LAB 43% and HAB 33%. In contrast, control loss and loss of appetite were higher in the control than in the treatments.

Infectious disease alleviation by egg yolk antibody against recombinant CRV VP4 protein of the present invention Treatment Clinical symptoms Loss of energy Loss of appetite Discharge Our company Control 1/7 2/7 5/7 0/7 LAB 0/7 1/7 3/7 0/7 HAB 0/6 0/6 2/6 0/6

5-3: Analysis Items

Serum was isolated from blood from the veins of the dogs for the diagnosis of canine rotavirus antibody at the start of the test, 3, 5, 7, 10, and 14 days. ELISA was performed to analyze IgG titers in serum from test subjects. The supernatant was harvested by freezing and thawing CRV virus cultures three times and centrifuging at 2500 rpm for 10 minutes. 7% polyethylene glycol (PEG MW6000) and 2.3 g of NaCl were added per 100 ml of the collected supernatant, followed by stirring at 4 ° C. for 16 hours, followed by centrifugation at 1000 g for 30 minutes, to obtain a precipitate. The precipitate was re-dissolved by TEN (Tris 10 mM, EDTA 1 mM, NaCl 100 mM, pH 7.4) buffer at an initial concentration of 1/10 and used as an antigen for ELISA. Dilute 220 μl of CRV antigen with 11 ml of carbonate-bicarbonate buffer (pH 9.6) and dispense 100 μl into each sphere of Microtest III flexible assay plate (Falcon 3911, BD Biosciences, USA) at 4 ° C. Coating overnight. The coated plate was washed three times with washing buffer (PBST, 0.02M NaH ₂ PO ₄ , 0.13M NaCl, 0.05% Tween 20, pH 7.2), and then 3% BSA (bovine serum albumin, pH 7.3, Sigma Co. USA) / PBS was dispensed in each sphere at 175 μL and then incubated at 37 ° C. for 2 hours, followed by 5 washes with PBST. After diluting serum serially by three-fold dilution using a dilution buffer solution in which 3% BSA / PBS and phosphate buffered saline-Tween 20 (PBST) were mixed in the same amount, the CRV antigen-coated spheres The reaction was performed at 37 ° C. for 2 hours. As a secondary antibody, an alkaline phosphatase conjugated dog IgG antibody (Kirkegaard and Perry Laboratories Inc. Gaithersburg, MD) was diluted 2,500 times and reacted by dispensing the spheres of the plate at 37 ° C. for 2 hours. The substrate buffer was reacted at 37 ° C. for 20 minutes using a solution of phosphate substrate tablet (ρ-nitrophenyl phosphate, PIERCE Co. USA) dissolved in 10% diethanolamine (pH 9.8) containing 0.5 mM MgCl ₂ . . After blocking the reaction using 5M NaOH as a stop solution, the reaction was investigated by measuring the absorbance at 405 nm using a microplate reader (EL-800, BIO-TEK Instrument Inc. USA).

As shown in FIG. 5B, IgG antibody titers were measured in serum obtained from the test subjects. On the 14th day of the test end date, the control group had about 20 times higher IgG antibody titer than the test start date. At about the end of the experiment, IgG antibody titers were similar to the start of the test. In addition, HAB maintained a constant level of antibody titer until day 7, and IgG antibody titer increased approximately 4 times at the end of the test. Four days after the virus administration, the control group had 7 and 4 times higher IgG antibody titer than the yolk antibody treated groups (LAB and HAB), respectively. There was a significant difference between control and egg yolk antibody treatments (P <0.1), but no significant difference between LAB and HAB. It was observed that runny nose emerged from the control during the experiment. These secretions may have caused re-infection of the condensation, resulting in increased IgG antibody titer.

The recombinant canine rotavirus VP4 protein and antibodies thereto according to the present invention can be useful for more effectively treating or preventing infection with canine rotavirus.

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Claims (16)

A recombinant dog consisting of an amino acid sequence represented by SEQ ID NO: selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, and SEQ ID NO: 21 Rotavirus VP4 Protein. delete A gene encoding a recombinant canine rotavirus VP4 protein according to claim 1. A recombinant expression vector comprising a gene encoding a recombinant canine rotavirus VP4 protein according to claim 3 operably linked to a regulatory sequence of the expression vector. The method of claim 4, wherein The recombinant expression vector, characterized in that the expression vector is pMAL-c2X. A host cell transformed with the recombinant expression vector according to claim 4. The method of claim 6, A host cell, characterized in that E. coli. (1) preparing a recombinant expression vector by operably linking a gene encoding the recombinant canine rotavirus VP4 protein according to claim 1 to a regulatory sequence of the expression vector; (2) transforming the host cell with the recombinant expression vector; (3) culturing the transformed host cell to express the gene; (4) A method for producing a recombinant canine rotavirus VP4 protein, comprising separating the recombinant canine rotavirus VP4 protein from the culture. An antibody against the recombinant canine rotavirus VP4 protein according to claim 1. The method of claim 9, An antibody, characterized in that it is an egg yolk antibody against the VP4 protein of the recombinant canine rotavirus isolated from the egg yolk laid from the laying hens immunized with the recombinant canine rotavirus VP4 protein. (1) immunizing the laying hen with the recombinant canine rotavirus VP4 protein according to claim 1; (2) a method for producing an egg yolk antibody against a recombinant dog rotavirus VP4 protein, comprising separating the egg yolk antibody against the recombinant dog rotavirus VP4 protein from the egg yolk that is laid from the laying hen. A vaccine composition for the treatment or prevention of canine rotavirus infection comprising the recombinant canine rotavirus VP4 protein according to claim 1 as an active ingredient. A pharmaceutical composition for treating or preventing canine rotavirus infection comprising the antibody according to claim 9 as an active ingredient. Feed composition for the treatment or prevention of canine rotavirus infection containing the antibody according to claim 9. A method for treating or preventing canine rotavirus infection by administering the vaccine composition according to claim 12 to a mammal, except for a human. A method for treating or preventing a dog rotavirus infection by administering the pharmaceutical composition according to claim 13 to a mammal, except for a human.

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