KR100812636B1 - Recombinant canine adenovirus fiber protein comprising epitope and antibody - Google Patents

Abstract

A recombinant canine adenovirus fiber protein and an antibody thereof are provided to be useful for treating or preventing canine adenovirus infection symptoms efficiently. A recombinant canine adenovirus fiber 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 method for preparing the recombinant canine adenovirus fiber protein comprises the steps of: (a) linking a gene coding the recombinant canine adenovirus fiber protein operably to a control sequence of an expression vector to prepare a 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 adenovirus fiber protein from a culture material obtained from the step(c). An antibody against the recombinant canine adenovirus 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 adenovirus infection symptoms comprises the recombinant canine adenovirus fiber protein as an effective ingredient.

Description

Recombinant Canine Adenovirus Fiber Protein Comprising Epitope and Antibody

1A is an electrophoretic photograph of the gene encoding the full length fiber protein of canine adenovirus.

Figure 1b is an electrophoretic picture of the gene encoding the recombinant dog adenovirus fiber protein of the present invention.

Figure 1c shows a cloning vector comprising a gene encoding a recombinant canine adenovirus fiber protein of the invention.

Figure 2a shows a recombinant expression vector comprising a gene encoding a recombinant dog adenovirus fiber protein of the present invention.

Figure 2b is an electrophoresis picture showing that the gene of the recombinant dog adenovirus fiber 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 adenovirus fiber protein of the present invention can be expressed through genetic recombination techniques.

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

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

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

FIG. 5B is a graph showing the prevention and treatment of infection by canine adenovirus in a dog receiving yolk antibody against the recombinant canine adenovirus fiber protein of the present invention.

The present invention relates to a recombinant dog adenovirus fiber protein comprising an antigenic determinant and an antibody thereto, and more particularly, to a recombinant dog adenovirus fiber protein comprising an antigenic determinant of a dog adenovirus fiber 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 dog adenovirus fiber protein, an antibody against the recombinant dog adenovirus fiber protein, and the antibody containing the antibody. A pharmaceutical composition for treating or preventing canine adenovirus infections.

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, "canine adenovirus" is a virus that is transmitted from livestock and wild canines. There are no clinical symptoms in many cases of infection, but in young individuals, severe hepatitis, bladder edema, tonsillitis, and multiple vasculitis bleeding may be observed. have. In addition, unvaccinated individuals may exhibit severe systemic symptoms that cause cirrhosis of the liver. This canine adenovirus is a highly contagious virus and requires an effective defense against it.

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

In one aspect, the present invention provides a recombinant dog adenovirus fiber protein comprising an antigenic determinant of a dog adenovirus fiber protein.

In another aspect, a gene encoding the recombinant canine adenovirus fiber protein is provided.

In another aspect, there is provided a recombinant expression vector that is included such that the gene encoding the recombinant dog adenovirus fiber 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 a gene encoding a recombinant dog adenovirus fiber 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) It provides a method for producing a recombinant dog adenovirus fiber protein comprising the step of separating the recombinant dog adenovirus fiber protein from the culture.

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

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

In another aspect, the present invention provides a vaccine composition for the treatment or prevention of canine adenovirus infections containing the recombinant canine adenovirus fiber protein as an active ingredient.

In another aspect, the present invention provides a pharmaceutical composition for the treatment or prevention of canine adenovirus infections comprising an antibody against the recombinant canine adenovirus fiber protein as an active ingredient.

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

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

The present invention relates to a fibrous protein of a dog adenovirus (hereinafter simply referred to as "CAV") recombined to include an epitope of a canine rotavirus derived fiber protein.

Specifically, the recombinant CAV fiber protein of the present invention is a fragment obtained by analyzing the fibrous protein of the dog adenovirus to include a site to which the antibody binds, that is, an antigenic determinant, 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: selected from the group consisting of SEQ ID NO: 21, which consists of CAV Fa, CAV Fb, CAV Fc, CAV Fd, CAV, respectively It is briefly referred to as Fe, CAV Ff, CAV Fg.

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

The recombinant CAV fiber protein of the present invention comprises the steps of (1) preparing a recombinant expression vector by operably linking a gene encoding the recombinant CAV fiber 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 a step of separating the recombinant CAV fiber protein from the culture.

Genes encoding the proteins are required to produce the recombinant CAV fiber proteins of the present invention by the above-described genetic recombination techniques. The gene encoding the recombinant CAV fiber protein according to the present invention may be artificially synthesized using a nucleic acid synthesizer or the like with reference to the nucleotide sequence of the gene, or the genomic DNA of the dog adenovirus may be encoded as a template. 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 CAV fiber 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 CAV fiber protein of the present invention is operably linked to the regulatory sequences 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 that effectively induce the expression of foreign genes in a particular host cell are commercially available. In the present invention, for example, pUC8, pBR322, pET / Rb, pGEX, pET28a, etc. can be used, and using pMAL-c2x is possible. desirable. DNA molecules obtained by operably linking a gene encoding a recombinant CAV fiber protein of the present invention to a regulatory sequence of such an expression vector are referred to as "recombinant expression vectors".

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 CAV fiber protein according to the present invention according to step (3) above. For example, host cells can be cultured by small or large scale fermentation, shake flask culture in a laboratory or industrial fermentor conducted under conditions that facilitate the expression and / or isolation of recombinant CAV fiber 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 CAV fiber proteins produced in transformed host cells can be isolated using conventional protein separation methods according to step (4) above. For example, recombinant CAV fiber proteins can be isolated from the culture according to methods including, but not limited to, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation. Furthermore, recombinant CAV fiber proteins can be obtained 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 against recombinant CAV fiber proteins of the invention, in particular egg yolk antibodies, also form an aspect of the invention.

Monoclonal and polyclonal antibodies specific for canine adenovirus can be prepared using the recombinant CAV fiber protein of the present invention as an antigen. Polyclonal antibodies are generally immunized with at least one recovery of a mammal with an appropriate amount of antigen, and when the titer of the antibody reaches a certain level, it is recovered from the antiserum of the mammal and, if desired, using known procedures It can be purified and stored in frozen buffered solution until use. Meanwhile, the monoclonal antibody can isolate B lymphocytes generated by injecting an antigen into a mammal, fuse with myeloma cells to form hybridoma cells, and culture the same to prepare antibodies. Details of this process are well known in the art.

In the case of an egg yolk antibody, (1) immunizing a laying hen with said recombinant CAV fiber protein; (2) can be prepared by the step of separating the egg yolk antibody against the recombinant CAV fiber protein in the egg yolk of the egg 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 CAV fiber proteins, 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 CAV fiber 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, the recombinant CAV VP 4 protein of the present invention and antibodies thereto may be used to treat or prevent canine adenovirus infection. Recombinant CAV fiber proteins can be treated or prevented through active immunity, and antibodies against recombinant CAV fiber proteins can be passively immunized through passive immunity. 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 CAV fiber protein and antibody thereof which can treat or prevent canine adenovirus 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 an animal without any adverse side effects can vary depending on the presence of any of the recombinant CAV fiber proteins and excipients used as immunogens. Generally each dose contains 0.1 to 1000 μg of protein, preferably 0.1 to 100 μg, per ml of sterile solution of the immunogenic amount of the recombinant CAV fiber 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 CAV fiber 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 adenovirus. 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 CAV fiber 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 Adenovirus Strains

1-1: Securing Canine Adenovirus Strains

37 ° C. to allow CRFK cells to be monolayered on 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. The CRFK cells were infected with canine adenovirus (ATCC-VR800). In about 80% of the CRFK cells infected with CAV, the cells were cultured in 37 ° C., 5% CO ₂ incubator for 1 to 6 days until cytopathic effect (CPE) occurred.

1-2: DNA Extraction of Canine Adenoviruses

Using the Viral gene-spin ^TM viral DNA / RNA Extraction kit (iNtRON Biotechnology Inc. Korea), CAV genomic DNA was extracted according to the manufacturer's manual.

1-3: Recombination of genes encoding fiber proteins of dog adenoviruses

The gene encoding the fiber protein of the dog adenovirus was prepared by PCR using a primer specific for the gene of the CAV fiber protein. The primer specific for the gene of the CAV fiber protein was based on GenBank's data (NCBI Z37498). To amplify the entire CAV fiber protein gene (1.9kb). The PCR was performed with 1 μl of the genomic DNA template of the virus, 4 μl of 25 mM MgCl ₂ , 10 × EX Taq. 5 μL buffer, 4 μL 2.5 mM dNTP, forward primer (CAV Fa- ggA TCC ATg TTT gCT AAg ACg TCT ggg TC-SEQ ID NO: 1) 1 μl, reverse primer (CAV Fg- gTC gAC gAC CTA TTA TTg ATT TTC GCC TA-SEQ ID NO: 20) 1 μl, EX Taq DNA polymerase (5U / L) distilled water was added to 0.5 µl so that the total reaction volume was 50 µl, initiating denaturation at 94 ° C for 5 minutes, denaturing at 94 ° C for 1 minute, annealing at 54 ° C for 1 minute, and extending at 72 ° C for 2 minutes. Each cycle was repeated 30 times and the final extension was conducted at 72 ° C. for 10 minutes (MiniCycler ™ Thermocycler (MJ Research. Inc. USA)). The PCR product thus amplified was electrophoresed (RunOne Electrophoresis Cell, Embi Tec. USA) for 30 minutes at 100V on 1% agarose gel (SeaKem LE agarose, BMA.USA) and stained with ethidium bromide to confirm the band.

As shown in Figure 1a, the product obtained through the PCR was able to identify the gene (SEQ ID NO: 22) encoding a 1.9kb CAV fiber protein (lane 1: λ DNA Eco RI / Hin d III marker, lane 2 : PCR product, lane 3: restriction enzyme digest of PCR product, lane 4: 100 bp ladder marker).

* Bold typeface indicates restriction enzyme recognition site.

On the other hand, the seven fragments of the CAV fiber protein gene was amplified by PCR using a PCR amplified sample as a template and oligonucleotides shown in Table 1 as primers. 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 2.5mM 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. Process of denaturation at 95 ° C. for 5 minutes, denaturation at 95 ° C. for 1 minute, annealing at 54 ° C. for 1 minute, extension at 72 ° C. for 1 minute, and repeating extension at 72 ° C. for 10 minutes It was carried out through. 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, as shown in FIG. 1B, seven CAV fiber protein fragment genes having a size of 320, 314, 284, 335, 296, 362, and 323 bp were identified (lane 1: 100 bp DNA ladder, lane 2: CAV). Fa, lane 3: CAV Fb, lane 4: CAV Fc, lane 5: CAV Fd, lane 6: CAV Fe, lane 7: CAV Ff, lane 8: CAV Fg).

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 NCBI (National Center) compared to the BLAST program provided by for Biotechnology Information. The results showed 99% similarity to the dog adenovirus fiber protein gene.

Example 2 Recombinant Expression Vector Construction and Protein Expression and Identification

2-1: Construction of Recombinant Expression Vector

After selecting positive clones for the genes of the seven recombinant CAV fiber proteins obtained above, each plasmid was digested with restriction enzymes BamH I and Sal I to cut 320, 314, 284, 335, 296, 362, 323 bp (CAV Fa , DNA fragments of CAV Fb, CAV Fc, CAV Fd, CAV Fe, CAV Ff, CAV Fg) were obtained, and then the fragments and the expression vector pMAL-c2x prepared by cleavage with the restriction enzyme were ligated. This was transformed into E. coli competent cell JM109 to select positive clones (see FIG. 2A).

The recombinant expression vector was separated therefrom, cleaved with restriction enzymes BamH I and Sal I, and subjected to electrophoresis using an agarose gel. As a result, seven genes encoding the recombinant CAV fiber protein were identified (lane 1). : λ DNA HindIII size marker, lane 2: pDMCAVFa, lane 3: pDMCAVFb, lane 4: pDMCAVFc, lane 5: pDMCAVFd, lane 6: pDMCAVFf, lane 7: pDMCAVFg, lane 8: 100 bp ladder marker).

2-2: Expression and Isolation of Recombinant CAV Fiber Protein Using Escherichia Coli

The positive clones obtained above were inoculated into 5 ml 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. The cultured cells were inoculated in 100 ml LB broth to which 100 μg ampicillin was added and cultured at 37 ° C. and 200 rpm until the absorbance (OD ₆₀₀ ) was 0.5 to 0.6. When the absorbance (OD ₆₀₀ ) is 0.5 to 0.6, 0.3 mM IPTG (isopropyl-β-D-thio-galactopyranoside, DUCHEFA Co. Netherland) was added, and then cultured for 3 hours 30 minutes to 4 hours. The obtained culture was centrifuged 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). 1 mg / ml was added and left on ice for 15 minutes. Through this process, the lysed cells were crushed using an ultrasonicator (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 obtain recombinant CAV fiber protein. Obtained.

2-3: Analysis of Recombinant CAV Fiber Proteins

Whether the protein obtained previously was CAV fiber fragment protein was confirmed by Western blot analysis. Here, SDS-polyacrylamide gel was used to make two sheets at the same time, and proteins were also loaded into two SDS-polyacrylamide gels in half by dividing the engineered protein in the same 1.5 ml tube and electrophoresed respectively. 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. Blot 16 hours at 4 ° C. 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 primary antibody, anti-MBP antibody (NEB Co. USA) was diluted 20,000 times at room temperature. After the reaction for 3 hours, the mixture was washed three times with TBST. The secondary antibody, alkaline phosphatase conjugated gout anti-rabbit IgG (Sigma Co. USA), was diluted 10,000-fold and the dilution was added to the membrane and allowed to react at room temperature for 1 hour. Membrane TBST three times for 5 min each, washed 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) The color reaction was induced with Pharmacia Bioscience, UK) and NBT (Nitro blue tetrazolium, Amersham Pharmacia Bioscience, UK).

As shown in Figure 2c it was confirmed that 50 ~ 60kDa size protein is detected in lanes 2-8 (lane 1: protein molecular weight marker, lanes 2-8: recombinant CAV Fa ~ Fg protein). This is a result of the protein expressed in E. coli specifically reacted with anti-MBP antiserum, thereby confirming that the protein expressed by E. coli is a recombinant CAV Fa ~ Fg protein.

2-4: Purification of Recombinant CAV Fiber Protein and Analysis of Protein Concentration

Recombinant CAV fragment protein was purified using pMAL ^™ protein fusion and purification system (NEB Co. USA). After culturing the bacteria in 1 L LB broth with reference to Example 2-2 to secure the cells by centrifugation, and then pulverizing them with an ultrasonic grinder, the supernatant was collected and mixed with the resin contained in the kit at 4 ℃ The reaction was carried out for 3 hours with stirring. Subsequently, glass wool was loaded to load the aqueous protein solution reacted with the polypropylene tube. Then all procedures were performed according to the manufacturer's manual. In addition, the concentration of purified recombinant CAV fiber protein obtained above was confirmed using a BCA (Bicinochoninic acid) Kit (Pierce Co.).

Example 3: Immunogenicity Study of Recombinant CAV Fiber Proteins

3-1: Antiserum Preparation for Recombinant CAV Fiber Proteins

Immunization was performed to 8-week-old BALB / C mice (Samtako BioKorea. Korea) in order to perform immunogenicity and neutralization test for the recombinant CAV fiber protein obtained in Example 2. Seven recombinant CAV fiber proteins and the inactivated virus were emulsified by mixing in equal amounts with the Freund's Complete Adjuvant (Sigma Chemical Co.), respectively. 0.5 ml of the emulsion was injected intramuscularly, and further inoculation was emulsified with Frouze incomplete adjuvant (Sigma Chemical Co.) at two weeks intervals after the first inoculation and performed twice in the same manner as the first inoculation. Before each inoculation, blood was collected from the mice, and serum was isolated to confirm whether immunogenicity appeared by ELISA, and a portion of the separated serum was stored at −20 ° C. for neutralization test (to be described later).

As shown in FIG. 3 and Table 2, the antibody titer having the highest CAV Fg (15,000) was shown. In addition, the immunity of the CAV virus itself was 27,000, and the antibody values of the remaining proteins were 10,000, 11,000, and 13,000 (CAVFa, CAVFc, CAVFf).

3-2: Neutralization Test Using Antisera against Recombinant CAV Fiber Protein

In order to determine whether each antiserum on the CAV fibrous protein produced above had a neutralizing effect on CAV, hemagglutinin inhibition test was performed to conduct a neutralization test. Each antiserum was diluted from 10 to 2 fold and dispensed into 96 spheres (NUNC, GmbH & Co. KG). 4HA CAV virus was equally divided and reacted at 37 ° C. for 1 hour, and then 50 μl of 1% red blood cells (RBC) were mixed well, and left standing at 25 ° C. for 40 minutes. This hemagglutination reaction was performed three times for each antiserum.

As shown in Table 2, it showed the best neutralization score in anti-CAV Fc.

Neutralization Results of Recombinant CAV Fiber Proteins According to the Present Invention serum Antibodies Neutralization test Anti-CAV 27000 60 Anti-CAVFa 10000 80 Anti-CAVFc 11000 2400 Anti-CAVFf 13000 1400 Anti-CAVFg 15000 1200

Example 4 Production and Identification of Egg Yolk Antibodies Using Recombinant CAV Fiber Protein

4-1: Production of Egg Yolk Antibodies Against Recombinant CAV Fiber Proteins

Immunization was performed on 28-week-old ISA-Brown laying hens for antibody production against the recombinant CAV fiber cleaved protein obtained in this experiment. Breeding and feeding management of laying hens was carried out at H farm, Gyeonggi-do, and emulsified by mixing the same amount of purified recombinant CAV fiber protein and Fruze complete adjuvant (Sigma Chemical Co.). 1 ml of the emulsion was injected intramuscularly into four places of 0.25 ml in the hen's pectoral muscle, and the additional inoculation was emulsified with Frowns 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.

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

ELISA was performed to analyze the antibody titers of the recombinant CAV fiber protein from the separated antiserum and egg yolk. Dilute the recombinant CAV fiber protein to 5 μg / ml with carbonate-bicarbonate buffer (pH 9.6) and dispense 100 μl into each sphere of Microtest III flexible assay plate (Falcon 3911, BD Biosciences, USA). Coating overnight at 4 ° C. The coating film 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 in each sphere 175ul and incubated for 2 hours at 37 ℃. Subsequently, the antiserum and egg yolk obtained using a dilution buffer, which was washed five times with PBST and the same amount of 3% BSA / PBS and a washing buffer solution (PBST, phosphate buffered saline-Tween 20), were diluted 2,000-fold, respectively. After serial dilutions, the recombinant CAV fibrous protein-coated spheres were dispensed and reacted at 37 ° C. for 2 hours. On the other hand, 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 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. 4, the antibody titer steadily increased from 3 weeks after immunization, and showed the highest antibody titer at 6 weeks (antibody is about 450,000). Since then, antibody titers have tended to decrease. 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, and the diluted yolk solution was adjusted to pH 5.0 and frozen at -20 ° C overnight. The frozen egg yolk was thawed at room temperature and then centrifuged at 10,000 × 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. Subsequently, ion-exchange chromatography using DEAE was performed to purify egg yolk antibodies. That is, the column containing DEAE was equilibrated with 0.025M phosphate buffer, 0.025M phosphate buffer solution containing egg yolk antibody was dispensed on the column, the column was washed, and eluted with 0.25M phosphate buffer (pH 8.0). . At this time, the eluted protein was fractionated by 3 ml per tube, and the absorbance was measured at 280 nm using a spectrophotometer to confirm purification.

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 2.91 ± 0.7 (SD). The test design was as follows.

1) control group: normal lyophilized egg yolk 1 g / day

2) Low concentration treatment (LAB): yolk antibody 1 g / day administration group containing yolk antibody against CAV Fc

3) High concentration treatment group (HAB): 3g / day administration group of egg yolk antibody containing egg yolk antibody against CAV Fc

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

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, and CAV (9 × 10 ⁵ pfu / ml) was orally administered on the 3rd and 4th days after the start of the test. Eye, nose discharge and mortality were checked.

As shown in Fig. 5A and Table 3, there was no death in each treatment during the 14-day test period. The secretions of eyes and nose were 37.5% in control and 25% in LAB, which was higher than HAB 0%. Anorexia and loss of energy were observed in all treatments, but there was no significant difference.

Infectious disease alleviation by egg yolk antibody against recombinant CAV fiber protein of the present invention Treatment Clinical symptoms Loss of energy Loss of appetite diarrhea Discharge Our company Control 2/8 3/8 1/8 3/8 0/8 LAB 3/8 2/8 0/8 2/8 0/8 HAB 2/7 3/7 0/7 0/7 0/7

5-3: Analysis target

Serum was isolated by taking blood at the start of the test, 3, 5, 7, 9, and 14 days in the dog vein for the diagnosis of canine adenovirus antibodies. ELISA was performed to analyze IgG titers in serum from test subjects. The CAV virus culture was freeze-thawed three times, and then centrifuged at 2500 rpm for 10 minutes, and the supernatant was collected. After adding 7% polyethylene glycol (PEG MW6000) and 2.3 g of NaCl per 100 ml of the collected supernatant, the mixture was stirred at 4 ° C. for 16 hours, centrifuged at 1000 g for 30 minutes, and the precipitate was harvested. 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 CAV 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 three times using dilution buffer solution in which 3% BSA / PBS and phosphate buffered saline-Tween 20 (PBST) were mixed in the same amount, the CAV 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 the reaction was blocked 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 the serum obtained from the test subjects, and the control group was 1.3 times higher than LAB and HAB after 7 days of CAV administration. The IgG antibody titer of the control group increased 30-fold until the end of the test after CAV administration, and LAB increased 5-fold on day 5, but remained constant thereafter. HAB increased 10-fold on day 5 and began to decrease from day 7 and decreased 1.5-fold at the end of the trial. This resulted in the removal of the eyeballs caused by the secretions of the eyes during the test period, but the runny nose in the nose was difficult to remove because of the habit of wiping off the tongue when a foreign substance adhered to the nose. It is thought that re-infection through foreign body occurred in this process.

The recombinant canine adenovirus fiber protein and the antibody to the canine according to the present invention can be useful for more effectively treating or preventing infection with canine adenovirus.

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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 Adenovirus Fiber Protein. delete A gene encoding a recombinant canine adenovirus fiber protein according to claim 1. A recombinant expression vector comprising a gene encoding a recombinant canine adenovirus fiber 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 dog adenovirus fiber 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 dog adenovirus fiber protein, comprising separating the recombinant dog adenovirus fiber protein from the culture. An antibody against the recombinant canine adenovirus fiber protein according to claim 1. 10. The antibody according to claim 9, which is an egg yolk antibody against the fibrous protein of recombinant canine adenovirus isolated from the egg yolk of eggs laid from the laying hens immunized with the recombinant canine adenovirus fiber protein. (1) immunizing the laying hen with a recombinant dog adenovirus fiber protein according to claim 1; (2) a method for producing an egg yolk antibody against a recombinant dog adenovirus fiber protein, comprising separating the egg yolk antibody against the recombinant dog adenovirus fiber protein from the egg yolk laid from the laying hen. A vaccine composition for the treatment or prevention of canine adenovirus infections comprising the recombinant canine adenovirus fiber protein according to claim 1 as an active ingredient. A pharmaceutical composition for treating or preventing canine adenovirus infection, comprising the antibody according to claim 9 as an active ingredient. Feed composition for the treatment or prevention of dog adenovirus infection containing the antibody according to claim 9. A method of treating or preventing a canine adenovirus infection by administering the vaccine composition according to claim 12 to a mammal other than a human. A method of treating or preventing a canine adenovirus infection by administering the pharmaceutical composition according to claim 13 to a mammal other than a human.

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