Canine parvovirus specific immune preparation, preparation method and application

Abstract

The present invention provides a canine parvovirus-specific antigen epitope fusion protein, a monoclonal antibody, an immunoglobulin preparation, a preparation method, and use thereof in preparing a drug or reagent for preventing or treating canine parvovirus disease. The mixed preparation of the nano antibody and the immunoglobulin preparation provided by the present invention can work synergistically, and clinical application results show that the canine parvovirus-specific immunoglobulin preparation prepared by the optimized method of the present invention is intravenously infused into sick dogs clinically diagnosed with canine parvovirus infection, and the nano antibody is used in combination, and the cure rate is as high as 97%, and the two have a good synergistic effect.

Description

Technical Field

[0001] The present invention relates to the biological field, in particular to canine parvovirus-specific immune preparations. The present invention also relates to the canine parvovirus-specific monoclonal antibodies, immunoglobulin preparations, preparation methods and use thereof in preparing drugs or reagents for preventing or treating canine parvovirus disease, belonging to the field of canine parvovirus prevention and control. Background Art

[0002] Canine parvovirus disease is an acute infectious disease caused by canine parvovirus (CPV) infecting dogs. It is characterized by severe vomiting, hemorrhagic enteritis, significant leukopenia and myocarditis. It has high morbidity, strong infectivity and high mortality. It is one of the most serious infectious diseases that harm dogs. At present, the clinical treatment drugs for canine parvovirus disease are mainly antiviral drugs and interferon injection, but these drugs are not effective. Canine parvovirus particles are 20-sided axially symmetrical and 25nm in size. Its capsid protein is composed of three structural proteins: VP1, VP2 and VP3. VP2 is composed of 8 antiparallel β-sheet motifs and 4 displayed polypeptide loops. The main B cell epitopes of CPV are concentrated on loop1 and loop3. However, since the above-mentioned antigenic epitopes are not all reflected on the surface of the virus particles, the effect of using canine parvovirus directly as an immunogen is not ideal and stable. Moreover, in the process of genetic evolution of parvovirus, with antigenic drift, new virus variants continue to appear and their host range continues to expand. In recent years, CPV2a, CPV2b, and CPV2c variants with different genotypes from CPV2 have been reported in different countries and regions. These variants can replicate, infect, and spread between dogs and cats. CPV2 has also been isolated from fur animals such as foxes and raccoon dogs.

[0003] Immunoglobulin is a type of globulin with antibody activity or chemical structure similar to that of antibodies. It is widely present in the blood, tissue fluid and exocrine fluid of animals, among which the content in serum and colostrum is the highest. Immunoglobulin is an important component of the body's immune system and plays an important role in anti-infection and immune regulation. After intravenous injection and absorption, the content of immunoglobulin in the recipient's blood can be rapidly increased, and through its specific neutralization and immune conditioning functions, it can prevent and treat various viral, bacterial, fungal, parasitic and other diseases. However, researchers in the prior art have tested the immune effect of CPV vaccines and found that only less than 20% of the vaccines can produce protective antibodies. Moreover, the commonly used antibodies on the market are mouse-derived IgG. If sick dogs use it repeatedly or for a long time, the mouse-derived components of the Fc fragment in IgG will cause dogs to produce specific antibodies after their immune response, which is not conducive to the treatment of canine parvovirus. In addition, macromolecular antibodies cannot effectively neutralize intracellular viruses, affecting the therapeutic effect. It can be seen that the therapeutic effects of these two methods are not ideal.

[0004] Therefore, developing a specific immune preparation with high purity, high concentration, high activity and high titer against canine parvovirus is an effective means for clinical treatment and emergency prevention of canine parvovirus disease. Summary of the invention

[0005] The present invention first provides a canine parvovirus-specific antigen epitope fusion protein, which contains most of the antigen epitopes of canine parvovirus and typical antigen epitopes of feline parvovirus, and fully exposes its antigen epitopes through bioinformatics analysis and optimization. The amino acid sequence of the fusion protein is shown in SEQ ID NO.1.

[0006] Furthermore, the present invention provides a nanobody against the above fusion protein, whose amino acid sequence is shown in SEQ ID NO.2.

[0007] Furthermore, the present invention provides an immunoglobulin preparation for the above-mentioned fusion protein and a preparation method thereof, the preparation method comprising the following steps: (1) preparing highly immune healthy dog ​​plasma or serum; (2) separating and purifying canine parvovirus-specific immunoglobulin in the plasma or serum prepared in step (1) by ion exchange chromatography; (3) ultrafiltering, concentrating, dialyzing, filtering and sterilizing the canine parvovirus-specific immunoglobulin purified in step (2), and packaging to obtain the immunoglobulin.

[0008] The content of canine parvovirus-specific immunoglobulin prepared by the method is greater than 50 mg / ml, the purity is greater than or equal to 98%, the endotoxin content is less than 9 IU, and the neutralizing antibody titer (TCID 50 / 200μl) reached above 1:800.

[0009] Preferably, the canine parvovirus-specific immunoglobulin preparation is a liquid or freeze-dried preparation with a pH of about 4.0, wherein the canine parvovirus-specific immunoglobulin mainly exists as IgG monomer. The canine parvovirus-specific immunoglobulin preparation of the present invention has a long shelf life and can be stored at 2-8°C for more than 2 years.

[0010] Furthermore, the present invention provides a use of the above-mentioned fusion antigen epitope, nanobody, and immunoglobulin preparation in the preparation of a drug or reagent for preventing or treating canine parvovirus disease.

[0011] Beneficial Effects

[0012] The antigen epitope fusion protein provided by the present invention has good immunogenicity, can induce the body to produce humoral immunity and cellular immunity, and can induce the body to produce neutralizing antibodies.

[0013] The nanoantibody provided by the present invention can specifically bind to CPV and has a high affinity. More importantly, the neutralizing activity of the expressed recombinant nanoantibody was detected by a neutralization test (VN) for CPV. The results showed that one of the nanoantibodies, CPVVHH2, had a neutralization titer of greater than 1:80 for CPV. It was preliminarily judged that the VHH had the ability to clear CPV, suggesting that this antibody can be used for the prevention and treatment of canine parvovirus.

[0014] The immunoglobulin preparation provided by the present invention has higher purity, higher protein content, lower endotoxin content and better antibody activity. The present invention adopts ion exchange chromatography to separate and purify immunoglobulins in plasma or serum, so that the purity of immunoglobulins in the product is greatly improved, and other impurities that cause allergic reactions are also greatly reduced. For example, two major allergic substances - anti-complement active substances (ACA) and antihypertensive substances (PKA) are significantly reduced.

[0015] The mixed preparation of the nano-antibody and the immunoglobulin preparation of the present invention can work synergistically. The nano-antibody can quickly increase the canine parvovirus antibody in the blood of sick dogs from zero or low concentration to a protective level, quickly neutralize and kill the parvovirus in the body of the sick dog, effectively prevent the continued infection and invasion of the body by the parvovirus, and play a role in quickly treating canine parvovirus disease. At the same time, the immunoglobulin itself has an immune conditioning effect on the body, activating the immune cells in the sick dog to phagocytize and remove invading pathogens, and quickly enhancing the immunity and resistance of the sick dog. The clinical application results show that the canine parvovirus-specific immunoglobulin preparation prepared by the optimized method of the present invention is intravenously infused into sick dogs clinically diagnosed with canine parvovirus infection, and the nano-antibody is used in combination, and the cure rate is as high as 97%, and the two have a good synergistic effect.

[0016] Definitions of terms used in this invention

[0017] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0018] The term "endotoxin" refers to a component in the cell wall of Gram-negative bacteria, called lipopolysaccharide. Lipopolysaccharide is toxic to the host. Endotoxin is only released when the bacteria die and dissolve or when the bacterial cells are destroyed by artificial methods, so it is called endotoxin. Its toxic component is mainly lipid A.

[0019] The terms "neutralizing antibodies and neutralizing antibody titer" refer to proteins that can specifically bind to antigens and have special disease resistance and are secreted by adaptive immune response cells in the body when pathogenic microorganisms invade the animal body. This protein is actually immunoglobulin. Neutralizing antibodies are soluble proteins that exist in blood, lymph and tissue fluids. When pathogenic microorganisms such as viruses invade the body, immune cells secrete neutralizing proteins into the blood. The latter bind to the particles of pathogenic microorganisms such as viruses in the blood, prevent viruses and other pathogenic microorganisms from infecting cells, and destroy the particles of pathogenic microorganisms such as viruses, thus "neutralizing" the viruses and other pathogenic microorganisms. The neutralizing antibody titer is an important indicator used to evaluate the activity and ability of neutralizing antibodies to neutralize pathogenic microorganisms. The cell neutralization test is generally used to determine the titer of neutralizing antibodies. The Reed-Muench method is used to calculate the highest dilution that can protect 50% of the cell wells to determine the titer of neutralizing antibodies. DETAILED DESCRIPTION

[0020] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, it should be understood that the embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications or replacements all fall within the protection scope of the present invention.

[0021] Example 1 Screening of canine parvovirus and feline parvovirus antigen epitope fusion proteins

[0022] With reference to the CPV and FPV gene coding sequences in GenBank, full-length amplification primers for the VP2 gene were designed using Primer Premier 5.0 software:

[0023] Upstream primer: GCAGACAGTAAAGCAGACAT (SEQ ID NO. 1);

[0024] Downstream primer: ATATTTAGGTCCTAGTGAGAT (SEQ ID NO.2) PCR amplification of VP2 gene was performed on the applicant and 42 suspected CPV-infected cat fecal cotton swabs and 35 suspected FPV-infected dog fecal cotton swabs collected from 2020 to 2023. The amplified fragment was connected to the pMD19-T vector and sent to Qingke Gene Company for sequencing. The Editseq program in DNAStar software was used to analyze the VP2 gene sequence obtained by sequencing, and the amino acid sequence encoded by it was deduced. The MegAlign program was used to compare the VP2 nucleotide and amino acid sequences of the CPV and FPV reference strains logged in GenBank and construct a phylogenetic tree. The Protean program was used to analyze the antigenic index and hydrophilicity of the VP2 protein. The results showed that the main antigenic determinants were mainly concentrated in loop2 (positions 200-250), while the amino acid sequences on loop1, loop3, and loop4 were relatively conservative. After comprehensive analysis, six regions with obvious hydrophilicity and high antigenicity index in VP2 were selected, including two sites that determine the host specificity and antigenicity of parvovirus. Bioinformatics was used to optimize and analyze them, and rigid or flexible linkers were selected to connect them. The amino acid sequence of the antigen epitope fusion protein finally obtained was:

[0025] MGGSGSGGVGISTGTHNNQGGSGGSLVHANMPGSYPWKPTITLPSHTGGGS

[0026] GGGSQGPFKTPIAAGRGGAQTDENQAADGDPRYAFGRQHGQKGETPERFTYIAGSGSPQDTGRYSEGDWIQNANF (SEQ ID NO. 3).

[0027] The above protein sequence was delivered to BGI for expression, purification and packaging before use.

[0028] Example 2 Screening of Parvovirus Fusion Protein Nanobodies

[0029] Vaccine preparation and immunization

[0030] The purified fusion protein was mixed with an equal volume of Freund's complete adjuvant / Freund's incomplete adjuvant at a final concentration of 300 μg / ml to prepare the vaccine. The vaccine prepared with Freund's complete adjuvant was used for the first immunization, and a 24-month-old healthy male alpaca was immunized by multiple intramuscular injections (4 points on both sides of the spine and under the armpits, 0.2 mL at each point). Multiple booster immunizations were performed on the 14th, 28th and 42nd days after the first immunization, respectively. The booster immunization used a vaccine prepared with Freund's incomplete adjuvant, and the immunization method and dosage were the same as the first immunization.

[0031] Antibody level monitoring

[0032] On the 14th day after the third and fourth immunizations, peripheral blood was collected from the immunized alpacas, and serum was separated to detect the CPV antibody titer by ELISA. The ELISA detection method is as follows:

[0033] (1) Coating: Dilute the purified CPV to 5 μg / mL with coating buffer and add to 96-well ELISA plate at 100 μL / well. Incubate at 4°C overnight. Discard the coating buffer and wash 5 times with PBST.

[0034] (2) Blocking: Add 300 μL PBSM (PBS containing 5% skim milk powder) to each well and block at 37°C for 2 hours. Discard the blocking solution, wash with PBST 5 times, spin dry and store at -20°C for later use.

[0035] (3) Dilution and incubation of test serum: Alpaca serum collected 14 days after the third and fourth immunizations was diluted to 1000-fold, 5000-fold, 25000-fold and 125000-fold, respectively, and added to the coated ELISA plate at 100 μL / well, 2 wells for each dilution. At the same time, alpaca serum that was not immunized with CPV was used as a negative control. After incubation at 37°C for 1 hour, the serum was discarded and washed 5 times with PBST.

[0036] (4) Secondary antibody incubation: Add 100 μL of 10,000-fold diluted Goat Anti-Alpaca IgG H&L (HRP) secondary antibody to each well, incubate at 37°C for 1 hour, then discard the secondary antibody and wash five times with PBST.

[0037] (5) Color development: Add 100 μL of TMB color development solution to each well and incubate in the dark for 15 min.

[0038] (6) Termination: Add 50 μL of 2 M sulfuric acid solution to terminate the color reaction.

[0039] (7) Result determination: OD450 value was measured. If the ratio of the average absorbance value of the sample serum to that of the negative serum was greater than 2.1, it was judged as positive. The highest dilution multiple at which the absorbance value reached the positive standard was taken as the antibody titer of the serum.

[0040] VHH gene amplification

[0041] On the 14th day after the fourth immunization, about 30 mL of peripheral blood was collected from the alpaca through the jugular vein and dispensed into anticoagulant tubes. The anticoagulant tubes were slowly inverted several times to prevent blood coagulation. Lymphocytes were separated within 2 hours according to the instructions of the lymphocyte separation solution. RNA was extracted using the Trizol method, and RNA with qualified quality was reverse transcribed according to the instructions of Takara's cDNA synthesis kit. PCR amplification of VHH genes and screening of phage libraries were performed according to the method of Yang Yanli et al. (Yang Yanli et al., 2019).

[0042] The selected single positive clone was amplified by shaking for antibody expression: the third round of natural single domain antibody library selected above was inoculated in 2×YT medium, and when OD600nm=0.5, 20MOI helper phage was added, and the mixture was allowed to stand for 30 min. After centrifugation, the precipitate was resuspended in 2×YT medium, and cultured for another 1 h. Then, it was spread on a 2×YT plate containing antibiotics and cultured overnight. The next day, 40 single colonies were selected and inoculated into 2×YT medium. When OD600=0.5, 20MOI helper phage was added, and the mixture was allowed to stand for 30 min. After centrifugation, the precipitate was resuspended in 2×YT medium, and cultured again. IPTG was added to induce expression for 8 h.

[0043] ELISA identification: 100 μL / well of the antigen epitope fusion protein was coated on the ELISA plate overnight, the coating solution was removed and the plate was washed three times, blocked with 200 μL / well BSA (3%) for 2 h, washed three times with PBST, and 100 μL / well of the single domain antibody library amplified in step (1) was added as the primary antibody (the library construction vector carried M13), incubated at 37°C for 2 h, washed three times with PBST, and M13-HRP was added as the secondary antibody. After termination, the OD450nm value was detected, and a value 3 times higher than the OD450nm value of the control group was considered positive.

[0044] According to the results of ELISA, monoclones with higher positive values ​​were selected and named CPVVHH1-3. The specificity of the three nanobodies to CPV was verified by indirect immunofluorescence experiments. Since the recombinant nanobodies carry 6×His tags, if they can specifically bind to CPV, the anti-His tag antibody with FITC tag is used as the secondary antibody, and the fluorescent signal can be seen. The results showed that all three nanobodies can specifically bind to CPV.

[0045] The neutralization activity of the expressed recombinant nanoantibodies was detected by the neutralization test (VN) of CPV. The results showed that one of the nanoantibodies, CPVVHH2, had a neutralization titer of more than 1:80 against CPV. It was preliminarily judged that the VHH had CPV neutralization activity, while the other antibodies did not have neutralization activity. This suggests that this antibody can be used for the prevention and treatment of canine parvovirus. PCR amplification was performed using universal primers of the pComb3x vector (upstream primer: AATACAGGTATCGCGTTCAAT (SEQ ID NO.4); downstream primer: CGCCGCCTTATTACCGTTTCGCGA (SEQ ID NO.5)). The PCR product was sent to the company for sequencing, and the sequencing results were analyzed using DNAMAN software. The final sequence obtained was:

[0046] QVQLVESGGGLVQPLSGGSLTAACASG FTLHYPAIGD FRQAPGKAGVSRECI

[0047] TY SGGQSYTDA ADSVKGRFSRTIDNVYLAKNTQMNKPSLEDTAVYYCAAEV NGLDALHAMCF QEGM D N W G Q T Q VTVSSAHYSEDP (SEQ ID NO. 6)

[0048] Example 3 Preparation of specific immunoglobulin preparation

[0049] Preparation of plasma or serum from highly immune healthy dogs

[0050] (1) A certain number of healthy meat dogs were selected and inoculated with the antigen epitope fusion protein (10 ug / ml) obtained in Example 1 on days 0 and 14, respectively, and the operation was performed according to the instructions;

[0051] (2) 10-14 days after the immunization, canine plasma or serum that meets the following requirements is selected as raw plasma or serum for the production of immunoglobulin: the antibody titer is measured by hemagglutination inhibition test (HI), and the antibody titer is not less than 1:1280;

[0052] (3) Blood was collected from the jugular vein, mixed, and stored at low temperature.

[0053] Isolation and purification of canine parvovirus-specific immunoglobulin by ion exchange chromatography

[0054] (1) thawing the plasma or serum prepared above, diluting it tenfold with 10 times the amount of 0.03 M acetate buffer with a pH value of 5.0, adjusting the pH value to 5.0 with 1.0 M acetic acid, and filtering to clarify;

[0055] (2) balancing the cation chromatography column with 0.03 M acetate buffer at a pH of 5.0 until the pH of the effluent is 5.0, and then loading the sample;

[0056] (3) After the protein peak appears, start collecting. After the sample is loaded, continue washing with 0.03M acetate buffer with a pH value of 5.0 until the protein peak disappears. This permeate (I) is the crude canine parvovirus-specific immunoglobulin;

[0057] (4) The anion chromatography column was equilibrated with 0.03 M acetate buffer at a pH of 5.0 until the pH of the effluent was 5.0, and the permeate (I) was loaded;

[0058] (5) After the sample is loaded, rinse with 0.03 M acetate buffer with a pH value of 5.0 until no peak appears, and elute the column with 0.2 M NaCl. Collect the protein from the time the peak appears until the peak disappears. This eluate (II) is the purified canine parvovirus-specific immunoglobulin;

[0059] Ultrafiltration concentrated dialysis immunoglobulin

[0060] The ultrafiltration concentration dialysis comprises the following steps: the collected protein solution after chromatography is dialyzed with 6 volumes of 4°C injection water until the ethanol content is less than 0.025% (0.025g of alcohol per 100ml) and the protein solution concentration is above 3% (3g of globulin per 100ml), 0.5mol / L hydrochloric acid solution is slowly added while stirring, the pH is adjusted to 4.0, and the solution is packaged or freeze-dried for storage.

[0061] Determination of protein concentration, purity and endotoxin content of canine parvovirus-specific immunoglobulin

[0062] The protein concentration, purity and endotoxin content of the prepared canine parvovirus-specific immunoglobulin were determined according to the methods specified in the Chinese Veterinary Pharmacopoeia and the Chinese Pharmacopoeia.

[0063] The prepared canine parvovirus-specific immunoglobulin has a protein concentration of 41 mg / ml, a protein purity of 98%, an endotoxin content of less than 9 IU, and a pH of 4.0.

[0064] Determination of neutralizing antibody titer

[0065] It is determined according to the cell neutralization test specified in the Veterinary Pharmacopoeia.

[0066] The canine parvovirus-specific immunoglobulin was diluted two-fold in a centrifuge tube using 2% serum culture medium according to Table 1.

[0067] Table 1 Sample dilution table

[0068]

[0069] Preparation of canine parvovirus virus solution

[0070] Take out from -80℃ refrigerator and measure TCID 50 For 10 -2.909 A canine parvovirus standard virus solution (provided by Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences) was dissolved and diluted to 200 TCID with 2% serum culture medium. 50 , prepare 15 ml for later use.

[0071] Neutralization test

[0072] Add 200 TCID to each canine parvovirus specific immunoglobulin tube for intravenous injection of different titers diluted according to Table 1 50 900 μl of virus solution was added to each tube. After mixing, the tube was placed in a 37°C incubator for 1 hour.

[0073] The cytopathic effect was determined as per the routine procedure: infected cells swelled, became round, and intercellular spaces widened; the cell monolayer was destroyed 48-72 hours after infection with the virus; no CPE was produced in the completely neutralized wells.

[0074] The final results verified that the neutralizing antibody titer of the above immunoglobulin was 1:1024 (TCID 50 / 200μl).

[0075] Experimental Example 4 Clinical application of a mixed preparation of nanoantibodies and canine parvovirus-specific immunoglobulin

[0076] 400 clinically ill dogs with clinical symptoms including depression, vomiting, hemorrhagic diarrhea, pale conjunctiva and nose, severe diarrhea in the form of jets, initial yellow with gray, mixed with a large amount of blood and mucous membranes, followed by tarry and fishy feces, clinical pathological significant decrease in white blood cell count, increased hematocrit value, increased gas and water in the digestive tract, and positive canine parvovirus antigen rapid diagnostic test strips were randomly divided into 4 groups. First, the nano-antibody preparation (50μg / dose / 0.5ml) was injected intraperitoneally at a dose of 0.5ml, twice on the 0th and 3rd days, and the above-mentioned canine parvovirus-specific immunoglobulin preparation was intravenously infused on the 5th day for 5 consecutive days, and the cure rate (%) was calculated.

[0077] The results showed (Table 2) that the cure rate of single infusion of canine parvovirus-specific immunoglobulin preparation was 75%, the cure rate of single infusion of nano-antibody was 60%, and the cure rate of the synergistic effect of the two was 97%, indicating that the two have obvious synergistic effect and can effectively treat or prevent canine parvovirus.

[0078] Table 2 Statistical results of cure rate

[0079]

[0080]

[0081] The above experiments show that the canine parvovirus-specific immunoglobulin preparation and nano-antibodies provided in this application have therapeutic and preventive effects on canine parvovirus, and there is a synergistic effect between the two. The combined vaccine can significantly increase the neutralizing antibodies against the real virus. In the future, the ratio and administration method of the two can be further optimized to develop a combined vaccine. After the combination, there is no mutual inhibition between the two antigen component vaccines, and they are well compatible. A higher neutralizing antibody titer can be produced against the virus, and the antibody titer level can be synergistically improved.

[0082] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0083] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0084] In addition, various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims

1. A canine parvovirus-specific antigen epitope fusion protein, characterized in that: The fusion protein contains most of the antigenic epitopes of canine parvovirus and typical antigenic epitopes of feline parvovirus, and its antigenic epitopes are fully exposed through bioinformatics analysis and optimization. The amino acid sequence of the fusion protein is shown in SEQ ID NO.

3.

2. A canine parvovirus-specific nanobody, characterized in that: The amino acid sequence of the nanobody is shown in SEQ ID NO.

6.

3. A method for preparing a canine parvovirus-specific immunoglobulin preparation, characterized in that: The preparation method comprises the following steps: (1) preparing highly immune healthy dog ​​plasma or serum; (2) separating and purifying canine parvovirus-specific immunoglobulin in the plasma or serum prepared in step (1) by ion exchange chromatography; (3) ultrafiltration, concentration, dialysis, filtration sterilization, and packaging of the canine parvovirus-specific immunoglobulin purified in step (2), wherein the immunogen in step (1) is the antigen epitope fusion protein described in claim 1.

4. Use of the antigen epitope fusion protein according to claim 1, the nanobody according to claim 2, and / or the immunoglobulin preparation prepared by the preparation method according to claim 3 in the preparation of a drug or reagent for preventing or treating canine parvovirus disease.

5. A composition, characterized in that The composition comprises the antigen epitope fusion protein according to claim 1, the nanobody according to claim 2, and / or the immunoglobulin preparation prepared by the preparation method according to claim 3.

6. A kit for preventing or treating canine parvovirus disease, characterized in that The kit comprises the antigen epitope fusion protein according to claim 1, the nanobody according to claim 2, and / or the immunoglobulin preparation prepared by the preparation method according to claim 3.

7. Use of the composition according to claim 5 and / or the kit according to claim 6 in the preparation of a drug or reagent for preventing or treating canine parvovirus disease.

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