Egg yolk antibody for cat parvovirus and preparation method thereof

Abstract

According to the present invention, the FPV-BJ strain (2 # seed virus) is adopted as the immunogen, and is inactivated to prepare the egg yolk antibody, the research results show that the egg yolk antibody with the FPV-HI titer of 1: 2048 can be obtained under the condition of the immunogen concentration of 105.0 TCID50 / ml, the obtained egg yolk antibody is higher than the existing reported level, and the egg yolk antibody can be further prepared into the feeding egg yolk antibody egg yolk powder for pet food, according to the present invention, the egg yolk antibody freeze-drying preparation or the egg yolk antibody injection preparation can be used as the drug, and the egg yolk antibody freeze-drying preparation is optimized, such that the treatment period and the treatment effect of the affected cat using the egg yolk antibody freeze-drying preparation or the egg yolk antibody injection preparation can be significantly improved; the symptom recovery time and the recovery time are shorter than those of sick cats without using the egg yolk antibody, so that the egg yolk antibody injection preparation prepared by the invention can effectively relieve clinical symptoms and greatly shorten the curing time.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an egg yolk antibody against feline parvovirus and its preparation method. Background Technology

[0002] Feline parvovirus (FPV) is a common pathogen infecting felines, belonging to the Parvoviridae family. The disease it causes is commonly known as feline panleukopenia, feline infectious enteritis, or feline distemper, and is an acute, highly contagious disease. The virus is a single-stranded DNA virus, non-enveloped, approximately 20–24 nm in diameter, with an icosahedral symmetry structure. Its genome contains two open reading frames, encoding non-structural proteins (NS1 and NS2) and capsid proteins (VP1 and VP2), respectively. VP2 protein is the main component of the viral particle, accounting for 90% of the viral particle, and can stimulate the body to produce neutralizing antibodies. After infection with feline parvovirus, symptoms typically include high fever, vomiting, diarrhea, and a severely reduced white blood cell count. It is particularly harmful to kittens around 6 months of age, with extremely high morbidity and mortality rates. Since its first discovery in 1928, feline panleukopenia virus (FPV) has become widespread in various feline species worldwide. In 1984, the first strain of FPV was isolated from a natural case in my country. Subsequently, the virus has been found in many regions, posing a serious threat to the health of cats.

[0003] In recent years, with the rapid development of the pet economy, especially the cat economy, the number of pet cats in households has been increasing, and they move frequently between regions. However, the vaccination coverage rate is low, and feline panleukopenia (FPA) remains prevalent in many areas, causing huge economic losses to the cat breeding industry. Prevention of FPA mainly relies on vaccination, which stimulates the body to produce antibodies for protection. However, active immunization takes a long time and cannot meet the needs of emergency prevention. Furthermore, the FPA virus is constantly evolving, with multiple mutant strains emerging. The only approved trivalent inactivated vaccine in China has significantly reduced protective efficacy against these mutant strains, leading to frequent immunization failures. In clinical treatment, due to the lack of specific drugs for viral diseases, the treatment of affected cats mainly relies on symptomatic therapy or antibiotics to prevent complications, but the efficacy is not ideal. Search results show that many domestic companies are currently conducting research on drugs for the treatment of feline panleukopenia. For example, Shanghai Peger Hospital Management Co., Ltd. (CN202311081616.1) has developed a feline panleukopenia egg yolk antibody with a high neutralizing titer. It has a good preventive and adjunctive therapeutic effect on feline panleukopenia, with a neutralizing titer of up to 1:1024. The antibody level can still be maintained above 1:512 after being stored at room temperature for more than six months. Changchun Xinuo Biotechnology Co., Ltd. has developed a bivalent lyophilized egg yolk antibody for feline infectious rhinoconjunctivitis and feline panleukopenia (CN201910521212.7). The neutralizing antibody titer of feline infectious rhinoconjunctivitis virus in this bivalent lyophilized egg yolk antibody is not less than 1:128, and the titer of feline panleukopenia virus HI antibody is not less than 1:512. Beijing Huachi Qiansheng Biotechnology Co., Ltd. has developed a feline triple egg yolk antibody (CN202210407205.6), which contains feline parvovirus egg yolk antibodies, feline herpesvirus egg yolk antibodies, and feline calicivirus egg yolk antibodies. All three antibodies exhibit high antibody titers and a synergistic effect, significantly improving the therapeutic efficacy of the triple egg yolk antibody against feline panleukopenia, feline herpesvirus, and feline calicivirus infections. Tianjin Heleant Biotechnology Co., Ltd. (CN202010001198.0) has developed a protective solution containing feline panleukopenia egg yolk antibodies. The natural plant active ingredients and lactic acid bacteria in this solution promote the excretion of toxins and waste from the cat's body, while also having a calming and soothing effect, enhancing immunity, and preventing stress responses from antibody-induced immune responses. Therefore, developing a highly specific and effective preventative and therapeutic agent against feline parvovirus is of significant practical importance. Summary of the Invention

[0004] To enrich the relevant prevention and treatment methods for feline parvovirus infection, this invention aims to provide a feline parvovirus egg yolk antibody, its preparation method, and its application.

[0005] To solve the above technical problems, the present invention adopts the following technical means: A feline parvovirus yolk antibody, characterized in that the feline parvovirus yolk antibody is obtained by immunizing laying hens with inactivated feline parvovirus FPV-BJ strain, and then extracting and purifying it from the egg yolk. The feline parvovirus FPV-BJ strain has the following accession number: CGMCC No. 46199; classification name: feline parvovirus; accession date: September 22, 2024; and depositary institution: China General Microbiological Culture Collection Center.

[0006] The method for preparing feline parvovirus yolk antibodies includes the following steps: (1) Preparation of inactivated antigen of feline parvovirus FPV-BJ strain; (2) Immunization of laying hens, wherein the laying hens are Hy-Line white laying hens; (3) Isolation and purification of egg yolk antibodies.

[0007] The specific method for immunizing laying hens includes: selecting healthy Hy-Line White laying hens aged 24-26 weeks, isolating and observing them for 7 days, then selecting healthy laying hens with normal diet and water intake for immunogen inoculation with an immunogen concentration of 10. 5.0 TCID 50 The specific immunization procedure for the FPV-BJ strain inactivated vaccine ( / ml) is as follows: First immunization: subcutaneous injection in the neck, dose 1.0 mL / bird; Second immunization: 14 days after the first immunization, subcutaneous injection in the neck, dose 1.5 mL / bird; Third immunization: 14 days after the second immunization, subcutaneous injection in the neck, dose 1.5 mL / bird; After the third immunization, the FPV-HI titer of the yolk fluid is measured. When the FPV-HI titer is ≥1:512, hyperimmune eggs are collected and stored at 10-12℃; During the production process after the third immunization, a booster immunization is performed every 30 days. The booster immunization is injected into the pectoral muscle, and the dose is 1.5 mL each time. The specific steps for the isolation and purification of the egg yolk antibodies include: (1) After sterilization, the egg white and yolk of the high-immunity eggs were separated by a separator. The yolk was diluted with 10 times the volume of pH 5.0 acetate-sodium acetate buffer, stirred until the yolk was completely dissolved, and allowed to stand at 2-8℃ for 12 hours to precipitate. Then, the supernatant was collected by centrifugation at 4500r / min for 15 minutes. (2) Take the supernatant and dilute it twice with 0.1M acetate buffer at pH 5.2. Stir for 30 min, add octanoic acid to a final concentration of 1%, let it stand to separate into layers, take the aqueous phase and centrifuge, collect the supernatant, filter it with a 0.45μm filter membrane, and collect the filtrate, which is the purified FPV egg yolk antibody; take the purified egg yolk antibody to perform a hemagglutination inhibition test, and the hemagglutination inhibition titer of the prepared egg yolk antibody is ≥1:1024.

[0008] The present invention also claims protection for the use of the described feline parvovirus yolk antibody in the preparation of a medicament for the prevention or treatment of feline parvovirus infection.

[0009] Preferably, the drug is a lyophilized preparation of egg yolk antibody or an injectable preparation of egg yolk antibody.

[0010] Preferably, the lyophilized egg yolk antibody formulation is prepared by the following method: (1) The FPV egg yolk antibody solution was placed into a 30kDa dialysis bag for dialysis to remove salt until the submucosa fluid and 2% barium chloride no longer produced a white precipitate. Then it was concentrated until the hemagglutination inhibition titer of the antibody was 1:1024. It was then filtered and sterilized using a 0.22μm filter to obtain the egg yolk antibody stock solution, which was then stored aseptically at 2-4℃. (2) Add freeze-drying protectant to the egg yolk antibody stock solution, mix evenly, filter sterilize using a 0.22μm filter, aseptically dispense quantitatively, and then freeze-dry under vacuum to make the moisture content ≤3.0%. After freeze-drying, seal to obtain FPV freeze-dried egg yolk antibody. The freeze-drying protectant is made of sucrose, gelatin, sodium butyrate, Solomon's seal polysaccharide, and skim milk powder. The preparation method is as follows: Weigh 25g of sucrose, 5g of gelatin, 1.5g of Solomon's seal polysaccharide, 0.5g of sodium butyrate, and 25g of skim milk powder. First, add the gelatin to 100mL of PBS buffer, heat to 75℃ and stir to dissolve it, then cool to 45℃, add the weighed other ingredients, stir and mix, filter to remove bacteria, and keep warm at 45℃ for later use. The method for preparing the freeze-dried egg yolk antibody is as follows: weigh the egg yolk antibody stock solution and mix it with the above-mentioned freeze-drying protectant, and then freeze-dry it. The freeze-drying procedure includes: setting the temperature to -5~-2℃, pre-freezing for 1 hour, then cooling to -45~-55℃ and maintaining for 2 hours, evacuating the freeze dryer to 0.1~0.2 mBar, raising the temperature to -30~-25℃ and maintaining for 24 hours; raising the temperature to -15~-10℃ and maintaining for 15 hours; raising the temperature to 0-10℃ and maintaining for 15 hours, thereby preparing the freeze-dried egg yolk antibody with a moisture content ≤4%.

[0011] The present invention also claims protection for the use of the feline parvovirus yolk antibody in the preparation of a feed-grade yolk antibody powder that can be used in the processing of pet food.

[0012] Based on the above technical solutions, the present invention has achieved the following beneficial effects: This invention isolated three FPV strains, of which strain #2 was the most virulent, with a 10 4.0 TCID 50At a rate of 1 mL / mL, FPV can induce typical symptoms such as vomiting and diarrhea in all cats, with a mortality rate reaching up to 100%. The disease is characterized by rapid onset, short course, and severe symptoms, and existing vaccines do not provide 100% protection against the #2 strain. Therefore, to meet the needs of FPV vaccine and therapeutic drug development, the isolated #2 strain was preserved and further tested. This invention uses the FPV-BJ strain (#2 strain) as an immunogen, which, after inactivation, is used for the preparation of yolk antibodies. Studies have shown that an immunogen concentration of 10... 5.0 TCID 50 At a concentration of 1 / ml, an egg yolk antibody with an FPV-HI titer of 1:2048 can be obtained, which is higher than the level reported in the prior art. Furthermore, it was formulated into feed-grade egg yolk antibody powder for pet food, and lyophilized or injectable egg yolk antibody preparations for pharmaceutical use. The lyophilized egg yolk antibody preparation was optimized. In terms of treatment cycle and efficacy, cats using the lyophilized or injectable egg yolk antibody preparations showed faster symptom recovery and overall recovery time than cats not using the egg yolk antibody. This demonstrates that the egg yolk antibody injectable preparation prepared in this invention can effectively alleviate clinical symptoms and significantly shorten the cure time. Attached Figure Description

[0013] Figure 1 CPE caused by FPV virus proliferation.

[0014] Figure 2 Results of indirect immunofluorescence assay.

[0015] Figure 3 : FPV virus PCR identification results, in which lanes 1-3 are virus strains 2#, 3#, and 7# respectively.

[0016] Figure 4 TCID testing was performed on the P1, P5, P10, and P15 generations of viruses after propagation of strains #2, #3, and #7. 50 Measurement. Detailed Implementation

[0017] The following embodiments are intended to make the technical concept and advantages of the present invention more intuitive, rather than to limit its scope. Any technical solution that does not depart from the spirit of the present invention and is reasonably deduced or optimized by those skilled in the art is considered to fall within the protection scope of the present invention. Example 1: Isolation of FPV-BJ strain

[0018] 1.1 Sample Source: Pathological samples were collected and provided by Shandong Zhongke Kangyuan Biotechnology Co., Ltd. and Beijing Shihua Kangyuan Biotechnology Co., Ltd. Eight cats diagnosed with FPV infection and deceased were collected from multiple cooperating pet hospitals in Linyi City, Shandong Province and Changping District, Beijing between April and September 2021. Post-mortem examination revealed obvious enterotoxemia. The intestines and intestinal contents were frozen at -80°C for later use. Clinical sample information is as follows: Table 1. Clinical Sample Information 1.2 Sample processing

[0019] Grind 0.5g each of the collected intestinal tract and intestinal contents in a mortar with liquid nitrogen. Dilute with PBS buffer containing 2% penicillin and antibiotics at a ratio of 1:5 (w / v), centrifuge at 12000 rpm for 10 min, collect the supernatant, filter it first through a 0.45 μm microporous membrane, and then filter it through a 0.22 μm microporous membrane for sterilization. Store the filtrate at -80℃ for later use. 1.3 Isolation and Culture of Viruses

[0020] F81 cells were cultured in T25 cell culture flasks until the cell density reached about 60%. After thawing the filtrate prepared in (2), it was seeded into F81 cells at a ratio of 1:10 (v / v). The flasks were placed in a 37°C incubator and allowed to absorb for 2 hours. Then, the filtrate was replaced with DMEM maintenance medium containing 2% serum and cultured in a 37°C, 5% CO2 incubator for 48-96 hours. Every 12 hours, the lesions were observed.

[0021] If no pathogenesis is observed in the first generation, the first-generation culture is subjected to three freeze-thaw cycles between -80°C and room temperature, followed by centrifugation at 12,000 rpm for 10 minutes at 4°C. The supernatant is collected and inoculated onto F81 cells at a density of approximately 60% at a ratio of 1:10 (v / v). After adsorption for 2 hours, the medium is replaced, and the cells are cultured for 3-5 days. Pathogenesis is then observed. This blind passage process is repeated for 3-5 generations until pathogenesis occurs. Figure 1 As shown.

[0022] Cultures exhibiting pathological passages were subjected to three freeze-thaw cycles between -80°C and room temperature. After centrifugation at 12,000 rpm for 10 minutes at 4°C, the supernatant was collected and aliquoted into sterile centrifuge tubes. The supernatant was then stored at -80°C for later use. This was the original seed virus, designated as P0 progeny.

[0023] After the above separation operations, seed viruses were obtained from samples 2, 3 and 7, and named seed viruses 2#, 3# and 7# respectively. 1.4 Indirect Immunofluorescence Assay (IFA)

[0024] F81 cells were cultured in 6-well cell culture plates until the cell mass reached about 85%. Cytotoxic agents 2#, 3#, and 7#, which had been passaged to the 5th generation, were diluted with culture medium and seeded into the cells at a rate of 2 mL per well. A blank control was also set up. After culturing for 2 hours, the supernatant was discarded, and the cells were washed three times with PBS. Pre-cooled formaldehyde fixative was added and incubated at 4°C for 25 minutes. After discarding the fixative, the cells were washed three times with PBS. 100 μL of 5% skim milk powder solution was added to each well, and the cells were blocked at 37°C for 1 hour. The blocking solution was discarded, and the cells were washed three times with PBS. 100 μL of FPV-positive cat serum (primary antibody) was added to each well, and the cells were incubated at 37°C for 1 hour. The positive cat serum was discarded, and the cells were washed three times with PBST. 100 μL of FITC-labeled rabbit anti-cat IgG was added to each well, and the cells were incubated at 37°C for 1 hour. The secondary antibody was discarded, and the cells were washed three times with PBST. 400 μL of 1:2000 diluted DAPI (4',6-diamidino-2-phenylindole) was added to each well, and the cells were stained for 15 minutes. The cells were washed three times with PBST, and a small amount of PBS was added for observation under an inverted biofluorescence microscope.

[0025] The results are as follows Figure 2 As shown, specific green fluorescent signals appeared in F81 cells inoculated with cytotoxic virus, indicating that FPV virus was successfully isolated. Among them, virus #2 showed the strongest fluorescence, while virus #7 showed the weakest. In the epidemiological investigation, the disease course (time from onset to death) of cat #2 was the shortest, while that of cat #7 was longer. 1.5 PCR Identification

[0026] Based on the FPV genome sequence (FPV-Cu4 strain, GenBank: M38246.1) in GenBank, PCR primers for FPV identification were designed. The upstream primer sequence was 5'-ATGAGTGATGGAGCAG-3', and the downstream primer sequence was 5'-TGCCAATCTCCTGGATT-3'. The expected amplified DNA fragment size was approximately 379 bp. Genomic DNA was extracted from infected F81 cell cultures using a viral DNA extraction kit for PCR identification. Specific results are shown below. Figure 3 As shown, all three strains yielded specific bands upon PCR amplification. Sequencing and sequence alignment revealed that all three strains isolated in this application were FPV.

[0027] 1.6 FPV's TCID 50 Measurement 1) Select a 25cm section of F81 cells that are confluent in a monolayer. 2For cell culture flasks, add 1 mL of trypsin for 60 seconds, discard the solution, add 1 mL of DMEM culture medium containing 10% serum to stop the digestion, disperse the cell clumps, add 9 mL of DMEM culture medium containing 10% serum and mix well. Add 100 μL of cell suspension to each well of a 96-well plate, seal the plate with sealing film, and place it in a 37°C, 5% CO2 cell culture incubator for 24 hours. Discard the solution.

[0028] 2) Take 10 1.5 mL centrifuge tubes, add 900 µL of DMEM culture medium to each tube, add 100 µL of the virus isolate to the first centrifuge tube and mix well. Draw 100 µL from the first centrifuge tube and add it to the second centrifuge tube, then dilute 10-fold to a final volume of 1×10⁻⁶. -2 ~1×10 -12 The virus solution was then added to a 96-well plate, with 8 replicates for each dilution. 100 μL of virus solution was inoculated into each well, and 100 μL of DMEM maintenance medium containing 2% serum was added to each well. Another column was used as a normal cell control, with 200 μL of DMEM maintenance medium containing 2% serum added to each well of the control cells.

[0029] 3) Observe and record the wells showing cytopathic effects every morning and afternoon for 4 days, and calculate the TCID of the virus using the Reed-Muench method. 50 value.

[0030] TCID tests were performed on P1, P5, P10, and P15 generations of viruses from strains #2, #3, and #7, respectively. 50 The measurement results are as follows: Figure 4 As shown in the figure. The results indicate that among the isolated viruses, virus strain #2 isolated from Changping, Beijing, has the highest relative TCID. 50 Its P1 generation reached 10 8.2 TCID 50 / mL, after being passaged to the 10th generation, the toxicity remained basically stable at 10. 10.0 TCID 50 Approximately / mL; After passage, the toxicity of seed #3 stabilizes after the 10th generation, at approximately 10. 8.5 TCID 50 / mL; the virulence of the 7# seed virus showed a decreasing trend after passage. In the epidemiological survey, the 2# seed virus had the shortest disease course (time from onset to death) and the most severe symptoms, while the 7# seed virus had a longer disease course. 1.7 Pathogenicity test of FPV 1.7.1 Virus Challenge Experiments on Susceptible Cats

[0031] Sixteen healthy, susceptible cats aged 3-4 months (approximately 12-16 weeks) were selected and randomly divided into four groups. Groups 1, 2, and 3 were orally administered 1 mL (10 mL) of P12 generation virus culture suspension containing viruses #2, #3, and #7, respectively. 4.0 TCID 50 The control group received an equal volume of PBS orally. The experimental cats in each group were isolated and fed separately. The onset of illness in the experimental cats was observed daily. The time from which typical symptoms such as fever, persistent depression, vomiting, diarrhea, or death appeared was defined as the onset time. The average onset time and 14-day survival rate of the affected cats were recorded. Details are shown in Table 2 below. Table 2 Results of FPV Pathogenicity Tests

[0032] Therefore, it can be seen that when using a lower dose of the seed virus for challenge, group 1 (seed virus #2) showed a shorter average time to onset of illness (one cat developed symptoms within 24 hours of challenge, recorded as day 1; two cats developed symptoms between 24 and 48 hours, recorded as day 2; and one cat developed symptoms between 48 and 72 hours, recorded as day 3; the sum of these days is 8 days, therefore the average time to onset of illness is 2.0 days). Furthermore, all animals died from viral infection during the experiment, with a survival rate of 0 / 4. Infected cats exhibited severe vomiting, diarrhea, and a prone, arched-back posture due to abdominal pain. All experimental animals died on day 6 after challenge. Relatively speaking, among the three strains, seed virus #2 was the most virulent. The following experiments were further conducted based on seed virus #2. 1.7.2 Determination of the toxicity effect of FPV 2# seed virus

[0033] Nine adult cats that had not received vaccines such as FPV-preventing vaccines (e.g., Feline vaccine) were selected and randomly divided into three groups of three. The cats were treated with different concentrations of 10... 4.0 TCID 50 / mL, 10 5.0 TCID 50 / mL, 10 6.0 TCID 50 Adult cats were challenged with 1 mL of a viral culture suspension ( / mL) orally. Post-infection disease progression and survival were observed and recorded. No drug treatment was administered during the experiment. Results showed that 10 4.0 TCID 50 In the / mL challenge group, all patients exhibited symptoms of depression and vomiting on day 3 post-challenge, and all died on day 12 post-challenge; 10 5.0 TCID 50 / mL and 10 6.0 TCID 50In the / mL challenge group, all patients exhibited symptoms of depression and vomiting on day 2 post-challenge, and all died on day 10 post-challenge. This demonstrates that FPV 2# seed virus possesses strong toxicity and lethality. 1.7.3 Challenge test of FPV 2# virus against cats vaccinated with FamiPure

[0034] Six healthy adult cats were selected and randomly divided into two groups of three. Each group received the full course of the Fever Plus vaccine (Zototen) according to the immunization schedule. Fourteen days after the completion of the vaccination program, the cats were challenged with a 10% concentration of the vaccine. 4.0 TCID 50 / mL, 10 5.0 TCID 50 1 mL of viral culture suspension per mL was used for challenge, administered orally. Post-infection disease progression was observed, and survival rates were recorded. 10 4.0 TCID 50 On day 5 after challenge with / mL, all patients exhibited symptoms of depression and vomiting. They survived by day 14, but were emaciated and occasionally experienced diarrhea. 10 5.0 TCID 50 On day 3 after challenge with FPV 2#, all animals exhibited symptoms of lethargy and vomiting. On day 14, one animal died, and the remaining two continued to show symptoms, specifically occasional vomiting and diarrhea. This demonstrates that existing commercially available vaccines do not provide effective protection against FPV 2# seed virus.

[0035] Based on the above experiments, it can be seen that this invention isolated three FPV strains, among which strain #2 has the strongest virulence, with a 10 4.0 TCID 50 At a rate of 1 mL / mL, the virus can cause all cats to exhibit typical symptoms such as vomiting and diarrhea, with a mortality rate as high as 100%. The disease is characterized by rapid onset, short course, and severe symptoms, and existing vaccines do not provide 100% protection against the #2 virus. Therefore, to meet the needs of FPV vaccine development, the isolated #2 virus was preserved and further tested.

[0036] Microbial preservation: The feline parvovirus strain 2# isolated in this invention was named FPV-BJ strain and submitted to the China Center for Type Culture Collection for preservation, with accession number: CGMCC No. 46199; classification name: feline parvovirus; preservation date: September 22, 2024. 1.8 Amplification of the VP2 fragment of the FPV-BJ strain

[0037] Total DNA was extracted from FPV cell cultures, and the FPV VP2 gene was amplified by PCR using FPV VP2-specific primers and high-fidelity DNA polymerase. PCR conditions were: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 45 s, 58℃ annealing for 30 s, 72℃ extension for 1 min, for a total of 32 cycles; followed by a final extension at 72℃ for 10 min. A template-free control was also included. The PCR product was purified and sequenced in a cloning vector, and the determined nucleotide sequence was converted to an amino acid sequence, as shown in SEQ ID No: 1. By comparing the VP2 protein sequence of this invention with the sequences of previously reported FPV strains, it was found that the FPV-BJ strain of this invention has two important mutations at key sites of VP2 compared with the vaccine strain from Pfizer and other strains isolated in Beijing. Specifically, the mutation is V at position 232 and E at position 323. It can be seen that the changes at these sites have led to significant changes in viral replication capacity and virulence. Compared with previously reported strains, it can adapt to F81 cells more quickly, and has a faster replication rate, higher viral titer, and higher lethality.

[0038] Table 3. Comparison of key amino acid sites in VP2 of different FPV strains

[0039] Example 2: Preparation of Immunogen 2.1 Propagation of seed viruses

[0040] Adherent F81 cells were diluted to 1.0 × 10⁶ cells using 10% fresh bovine serum DMEM cell culture medium. 6 - 2.0×10 6 At a density of cells / mL, 5 mL was seeded into T25 cell culture flasks and cultured at 37°C in a 5% CO2 incubator for 24-48 hours. After the cells reached a confluent monolayer, they were digested with 0.25% trypsin solution and transferred to T75 cell culture flasks. Next, the cells were resuspended in 15 mL of 2% new bovine serum DMEM cell maintenance medium and seeded with feline parvovirus FPV-BJ strain at 0.01 MOI. The cells were then cultured at 37°C in a 5% CO2 incubator for 96-120 hours. The viral load was harvested, and cytopathic effects were observed. Viral content was determined, and purity was tested using PCR. After passing the purity test, the cells were quantitatively aliquoted and stored below -70°C. 2.2 Preparation of Viral Fluid

[0041] Discard the culture medium from the F81 cells that have grown into a monolayer in the T75 cell culture flask, digest them with 0.25% trypsin solution, collect the cells, expand the culture to T175 cell culture flasks, resuspend them in 50 mL of 2% new bovine serum DMEM cell maintenance medium, inoculate with feline parvovirus FPV-BJ strain at 0.01 MOI, and continue to culture at 37℃ and 5% CO2 for 96-120 hours. Harvest the virus solution, observe cytopathic effects, and calculate the virus content. 2.3 Vaccine Preparation

[0042] (1) Inactivation: Add 0.1% volume of BEI to the harvested virus stock solution or dilution solution, mix well, inactivate at 4℃ for 24 hours, and then hydrolyze at 37℃ for 2 hours.

[0043] (2) Sterility test: The test shall be conducted in accordance with the appendix of the Chinese Veterinary Pharmacopoeia to ensure sterile growth.

[0044] (3) Inactivation test: The inactivated antigen was sterile and the inactivation solution was seeded into adherent F81 cells at 0.01 MOI. The cells were cultured at 37℃ and 5% CO2 for 6 days and then blindly passaged for 2 generations. The success of inactivation was judged by observing the cytopathic effect (CPE).

[0045] (4) Vaccine preparation: Dilute the antigen solution with known antigen content to 2×10 using sterile PBS. 4.0 TCID 50 / mL, 2×10 5.0 TCID 50 / mL and 2×10 6.0 TCID 50 Mix the sample with adjuvant (ISA 206 adjuvant) at a volume ratio of 1:1, stir at 2000 rpm for 10 minutes, dispense into 1 mL portions, and store at 4°C for later use. Example 3

[0046] 3.1 Selection of laying hens This invention uses two types of laying hens, Hy-Line White and Hy-Line Brown, for testing. The hens were divided into two groups of 20 birds each, based on their breed. The inactivated antigen prepared in Example 2 was used to immunize both groups of hens, and the immunization program is as follows: First vaccination: Subcutaneous injection in the neck, dose 1.0 mL / bird; Second vaccination: 14 days after the first vaccination, administer a subcutaneous injection in the neck at a dose of 1.5 mL / bird; Third vaccination: 14 days after the second vaccination, administer a subcutaneous injection in the neck at a dose of 1.5 mL / bird.

[0047] Start collecting eggs 14 days after the three immunizations, at fixed times in the morning, afternoon, and evening every day, by fixed personnel, to avoid a decrease in egg production rate due to stress or other reasons. Mark the collected eggs and store them in refrigeration at 4°C for preservation.

[0048] Subsequently, randomly select 40 eggs from each group of eggs for separation of egg white and egg yolk. Add 10 times the volume of acetic acid-sodium acetate buffer to the egg yolk liquid for dilution. After standing overnight, centrifuge at 4000 rpm for 15 minutes, take out the supernatant, and adjust the pH value to 7.0 - 7.2. Then, use the micro hemagglutination - hemagglutination inhibition test to measure the HI titer of feline parvovirus. After measurement, the FPV - HI titer of the Hy - line White laying hens group is 1:512, while the FPV - HI titer of the Hy - line Brown laying hens group is 1:128. It can be seen that the antibody level of the Hy - line White laying hens after immunization is higher than that of the Hy - line Brown laying hens. Therefore, the present invention selects Hy - line White laying hens as the laying hens for production.

[0049] 3.2 Determination of the optimal antigen content To determine the optimal antigen content, the present invention selects 30 Hy - line laying hens at 23 weeks of age, and immunizes them with inactivated vaccines with antigen contents of 10 4.0 TCID 50 / ml, 10 5.0 TCID 50 / ml, and 10 6.0 TCID 50 / ml respectively. The specific immunization process is as follows: Primary immunization: Subcutaneous injection in the neck, with a dose of 1.0 mL / feather; Secondary immunization: 14 days after the primary immunization, subcutaneous injection in the neck, with a dose of 1.5 mL / feather; Tertiary immunization: 14 days after the secondary immunization, subcutaneous injection in the neck, with a dose of 1.5 mL / feather.

[0050] Start collecting eggs after the tertiary immunization, at fixed times in the morning, afternoon, and evening every day, by fixed personnel, to avoid a decrease in egg production rate due to stress or other reasons. Mark the collected eggs and store them in refrigeration at 4°C for preservation. Monitor the antibody titer in the egg yolk liquid. An FPV - HI titer ≥ 1:128 indicates qualified eggs. When the titer is lower than 1:128, stop collecting, and count the average egg production rate within 100 days after the tertiary immunization. The specific situation is shown in Table 4 below: Table 4 Determination of the optimal antigen content [[ID=三十五]

[0051] Based on the above experiments, it can be known that both the medium - dose group and the high - dose group can obtain the highest antibody titer of 1:2048. However, the egg production rate of the high - dose group is slightly lower than that of the medium - dose group. Considering the comprehensive production cost, determine the immunogen concentration to be 10 5.0 TCID50 The medium-dose group ( / ml) represents the optimal immunogenic concentration for preparing egg yolk antibodies.

[0052] 3.3 Production and Collection of High Immunity Eggs Select healthy Hy-Line White laying hens aged 24-26 weeks, isolate and observe them for 7 days, then select healthy hens with normal diet and water intake to be vaccinated with an immunogen at a concentration of 10. 5.0 TCID 50 The specific immunization procedure for the FPV-BJ strain inactivated vaccine ( / ml) is as follows: First vaccination: Subcutaneous injection in the neck, dose 1.0 mL / bird; Second vaccination: 14 days after the first vaccination, administer a subcutaneous injection in the neck at a dose of 1.5 mL / bird; Third vaccination: 14 days after the second vaccination, administer a subcutaneous injection in the neck at a dose of 1.5 mL / bird.

[0053] After the third immunization, the FPV-HI titer of the egg yolk fluid was measured. When the FPV-HI titer was ≥1:512, the hyperimmune eggs were collected and stored at 10-12℃. During the production process after the third immunization, a booster immunization was performed every 30 days. The booster immunization was injected into the pectoral muscle, and the dose was 1.5 mL each time.

[0054] After the third immunization, eggs were collected. After separating the egg yolk and egg white, the FPV-HI titer of the egg yolk antibody was measured. When the FPV-HI titer of the immunized eggs was ≥1:512, a large number of immunized eggs were collected.

[0055] After rinsing and soaking the collected high-immunity eggs in tap water for 10 minutes, transfer them to a 1% benzalkonium chloride solution and soak for another 20 minutes. After drying, wipe them with 75% ethanol for disinfection and use them for later use.

[0056] 3.4 Isolation and purification of egg yolk antibodies (1) After sterilization, the egg white and yolk of the high-immunity eggs were separated by a separator. The yolk was diluted with 10 times the volume of pH 5.0 acetate-sodium acetate buffer, stirred until the yolk was completely dissolved, and allowed to stand at 2-8℃ for 12 hours to precipitate. Then, the supernatant was collected by centrifugation at 4500r / min for 15 minutes.

[0057] (2) Take the supernatant and dilute it twice with 0.1M acetate buffer at pH 5.2. Stir for 30 min, add octanoic acid to a final concentration of 1%, let it stand to separate into layers, take the aqueous phase and centrifuge, collect the supernatant, filter it with a 0.45μm filter membrane, and collect the filtrate, which is the purified FPV egg yolk antibody.

[0058] The purified egg yolk antibody was subjected to a hemagglutination inhibition test, and the hemagglutination inhibition titer of the prepared egg yolk antibody was ≥1:1024. Example 4 Preparation of egg yolk antibody preparation

[0059] 4.1 Preparation of Egg Yolk Antibody Powder for Feed The hyperimmune eggs collected in Example 3 were surface sterilized, the yolks were aseptically separated, homogenized, pasteurized and inactivated, and then 15g of sucrose, 10g of gelatin and 25g of skim milk powder (all raw materials were sterilized) were added to every 1000g of egg yolk liquid. After being thoroughly mixed, the mixture was freeze-dried at low temperature to obtain feed-grade egg yolk antibody powder, which can be used in the processing of pet food.

[0060] 4.2 Preparation of Lyophilized Egg Yolk Antibody Formulation (1) The FPV egg yolk antibody solution prepared in 3.4 of Example 3 was placed into a 30kDa dialysis bag for dialysis to remove salt until the submucosa fluid and 2% barium chloride no longer produced a white precipitate. Then it was concentrated until the hemagglutination inhibition titer of the antibody was 1:1024. It was then filtered and sterilized using a 0.22μm filter to obtain the egg yolk antibody stock solution, which was then sterilely stored at 2-4℃.

[0061] (2) Add freeze-drying protectant to the egg yolk antibody stock solution, mix evenly, filter sterilize using a 0.22μm filter, aseptically dispense quantitatively, and then freeze-dry under vacuum to make the moisture content ≤3.0%. After freeze-drying, seal to obtain FPV freeze-dried egg yolk antibody.

[0062] The freeze-drying protectant is made of sucrose, gelatin, sodium butyrate, Solomon's seal polysaccharide, and skim milk powder. The preparation method is as follows: Weigh 25g of sucrose, 5g of gelatin, 1.5g of Solomon's seal polysaccharide, 0.5g of sodium butyrate, and 25g of skim milk powder. First, add the gelatin to 100mL of PBS buffer, heat to 75℃ and stir to dissolve it, then cool to 45℃, add the weighed other ingredients, stir and mix, filter to remove bacteria, and keep warm at 45℃ for later use.

[0063] The method for preparing the freeze-dried egg yolk antibody is as follows: weigh the egg yolk antibody stock solution and mix it with the above-mentioned freeze-drying protectant, and then freeze-dry it. The freeze-drying procedure includes: setting the temperature to -5~-2℃, pre-freezing for 1 hour, then cooling to -45~-55℃ and maintaining for 2 hours, evacuating the freeze dryer to 0.1~0.2 mBar, raising the temperature to -30~-25℃ and maintaining for 24 hours; raising the temperature to -15~-10℃ and maintaining for 15 hours; raising the temperature to 0-10℃ and maintaining for 15 hours, thereby preparing the freeze-dried egg yolk antibody with a moisture content ≤4%.

[0064] 4.3 Preparation of Egg Yolk Antibody Injection Formulation (1) Separation of egg yolk: The hyperimmune eggs collected in Example 3 were surface sterilized, the egg yolks were aseptically separated, and the mixture was stirred evenly to obtain egg yolk liquid; (2) Dilution: Mix the egg yolk solution with sterile PBS buffer at pH=7.4 at a volume ratio of 1:2, and place the mixture in a centrifuge tube after mixing. (3) First precipitation: Add PEG-6000 at a concentration of 3.5% (w / v), stir thoroughly, heat in a water bath at 37℃ for 15-30 min, remove after complete dissolution, and let stand at room temperature for 1 h; (4) Centrifugation: Centrifuge at 12000 rpm for 30 min. The layers are yellow fat layer, clear layer and semi-solid layer from top to bottom. (5) Filtration: Pour out the supernatant, filter with a fast filter paper, collect the filtrate and record the volume of the filtrate, and discard the precipitate at the same time; (6) Secondary precipitation: According to the volume of filtrate, add PEG-6000 with a concentration of 8.5% (w / v), shake well, and heat in a water bath at 37°C for 15-30 minutes to fully dissolve; (7) Centrifugation: Centrifuge at 12,000 rpm for 30 min; (8) Dissolve: Discard the supernatant, add sterile PBS buffer with pH=7.4 to the precipitate and mix well to ensure that the precipitate is completely suspended, dispersed and dissolved. The amount of PBS buffer added is the same as the volume of egg yolk solution in step (1). (9) Third precipitation: Add PEG-6000 at a concentration of 12% (w / v), shake well, and heat in a water bath at 37°C for 15-30 min to fully dissolve; (10) Centrifugation: Centrifuge at 12,000 rpm for 30 min; (11) Sterilization and dispensing: Discard the supernatant, add sterile physiological saline to the precipitate for solvent and dilution, dilute to an FPV-HI titer of 1:256, then filter sterilize with a sterile 0.22μm filter, dispense into appropriate ampoules, and freeze for storage. Example 5: Clinical trial of the preventive effect of egg yolk antibody-containing egg yolk powder

[0065] The clinical preventive effect of the egg yolk antibody powder prepared in Example 4 was evaluated. Twenty weaned kittens aged 12-16 weeks were selected and tested negative for both FPV antigen and antibody. They were randomly divided into four groups of five kittens each. The specific experimental protocol is as follows: Group 1, Experimental Group 1: The egg yolk antibody powder prepared in Example 4.1 was administered orally to each cat at a dose of 1 g / kg body weight / day for 7 consecutive days; Group 2, Experimental Group 2: The egg yolk antibody powder prepared in Example 4.1 was administered orally to each cat at a dose of 0.5 g / kg body weight / day for 7 consecutive days; Group 3, Experimental Group 3: The egg yolk antibody powder prepared in Example 4.1 was administered orally to each cat at a dose of 0.25 g / kg body weight / day for 7 consecutive days; Group 4, the blank control group, was given 2g of egg yolk powder prepared from eggs produced by ordinary Hy-Line White chickens according to method 4.1 orally for 7 consecutive days.

[0066] On day 4, weaned kittens from groups 1-4 were challenged with the virus using FPV-BJ strain culture medium (10... 4.0 TCID 50 Animals were challenged orally at a dose of 1 mL per animal. All four groups of animals were isolated and fed in isolation. The cats' condition was observed and recorded daily for 14 consecutive days. The criteria for diagnosis of disease were: depression, decreased appetite, fever, vomiting, diarrhea, and a positive fecal FPV test. The experimental results are shown in Table 5 below.

[0067] Table 5. Application of egg yolk antibody and egg yolk powder in the prevention of FPV infection.

[0068] Based on the above experimental results, the morbidity and mortality rates in the blank control group were both 100%. Compared with the blank control group, the egg yolk antibody powder of the present invention showed excellent preventive effects at oral doses of 1 g / kg body weight / day and 0.5 g / kg body weight / day. None of the individuals showed clinical symptoms of FPV infection, and all remained healthy. However, when the oral dose was reduced to 0.25 g / animal, one weaned kitten developed diarrhea, and FPV was detected in its feces, thus indicating the onset of the disease. However, its symptoms quickly improved, and no experimental animals died. Therefore, the egg yolk antibody powder of the present invention has excellent preventive effects against FPV infection, exhibiting a dose-dependent effect. The optimal oral dose was determined to be ≥0.5 g / kg body weight. Example 6: Emergency Prevention Trial of FPV Infection

[0069] The clinical preventive effect of the egg yolk antibody powder prepared in Example 4.2 was evaluated. Fifteen weaned kittens aged 12-16 weeks were selected, and both FPV antigen and antibody tests were negative. The selected kittens were fed according to standard methods for 7 days, and then randomly divided into 3 groups. FPV-BJ strain virus culture medium (10...) was used. 7.0 TCID 50 Animals were challenged orally with a dose of 1 mL per rotavirus (v / mL). Emergency prophylaxis was administered 24 hours post-challenge using a lyophilized yolk antibody formulation. The specific experimental protocol is as follows: Group 1: The lyophilized egg yolk antibody preparation prepared in Example 4.2 was administered orally to each cat at a dose of 1 g / kg body weight / day, continuously until the symptoms disappeared; Group 2: Cats were given the same lyophilized egg yolk antibody preparation as Control Group 1 orally, at a dose of 1 g / kg body weight / day, continuously until symptoms disappeared. The lyophilization protectant for Control Group 1 was made from sucrose, gelatin, sodium butyrate, and skim milk powder. The preparation method was as follows: Weigh 26.5 g sucrose, 5 g gelatin, 0.5 g sodium butyrate, and 25 g skim milk powder. First, add the gelatin to 100 mL of PBS buffer, heat to 75°C and stir to dissolve. Then cool to 45°C, add the other weighed ingredients, stir and mix, filter to sterilize, and incubate at 45°C for later use. Other procedures were the same as in 4.2.

[0070] Group 3: Cats were given the same lyophilized egg yolk antibody preparation as Control Group 2 orally, at a dose of 1 g / kg body weight / day, continuously until symptoms disappeared. The lyophilization protectant for Control Group 2 was made from sucrose, gelatin, Polygonatum odoratum polysaccharide, and skim milk powder. The preparation method was as follows: Weigh 25 g of sucrose, 5 g of gelatin, 2 g of Polygonatum odoratum polysaccharide, and 25 g of skim milk powder. First, add the gelatin to 100 mL of PBS buffer, heat to 75°C and stir to dissolve. Then cool to 45°C, add the other weighed ingredients, stir and mix, filter to sterilize, and incubate at 45°C for later use. Other procedures were the same as in 4.2.

[0071] The three groups of experimental animals were kept in isolation, and the cats' condition was observed daily for 30 consecutive days. Fecal samples were collected and tested for FPV virus using test strips. The criteria for determining disease in cats were a positive test strip result and the presence of typical FPV infection symptoms.

[0072] Kittens in groups 1-3 all showed obvious FPV infection symptoms 24 hours after challenge, including lethargy, diarrhea, decreased appetite, increased salivation, nasal and ocular discharge, and FPV virus was detected in their feces using test strips. However: In Group 1, the animals' mental state began to recover on days 2-3 after oral medication, and their appetite gradually returned to normal. From days 4-6, symptoms such as diarrhea, drooling, nasal discharge, and eye discharge largely disappeared, and the animals recovered by days 8-9 after oral medication. PCR testing showed negative results for FPV virus. Therefore, the recovery period for this treatment regimen was 8-9 days. In Groups 2 and 3, recovery of mental state was observed around days 5-6 after oral medication, and all animals were observed to have recovered by around day 12 after oral medication. This shows that under high-intensity viral challenge, kittens quickly developed infection symptoms, while the recovery period for treatment with the lyophilized egg yolk antibody formulation of this invention was approximately 8-9 days. The control group showed delayed observation of drug efficacy and a longer recovery period. This indicates that the addition of sodium butyrate and Polygonatum odoratum polysaccharide to the lyophilized formulation contributes to the effectiveness of the egg yolk antibody drug. Example 7: Therapeutic effect of FPV egg yolk antibody injection preparation

[0073] Twenty cats with positive FPV tests admitted to pet hospitals in Linyi, Shandong Province were selected. After communicating with their owners, they agreed to participate in the experiment. They were divided into two groups according to age, weight, and disease condition, and were treated as subjects. The first group was the experimental group, and the second group was the control group.

[0074] The treatment regimen for the experimental group was as follows: subcutaneous injection of serotonin-clavulanate potassium (20 mg / kg body weight, once daily), recombinant feline ω-interferon (30 IU / kg body weight, once daily), prednisolone (0.5 mg / kg body weight, once daily), malopistan (0.1 mg / kg body weight, once daily), and feline calicivirus egg yolk antibody injection preparation prepared in Example 4 (0.5 mL / kg body weight, once daily). The injection site for the egg yolk antibody was fixed daily.

[0075] The treatment regimen for the control group was: subcutaneous injection of amoxicillin-clavulanate potassium (20 mg / kg body weight, once daily), recombinant feline ω-interferon (30 IU / kg body weight, once daily), prednisolone (0.5 mg / kg body weight, once daily), and maropistan (0.1 mg / kg body weight, once daily).

[0076] During the treatment, no local abnormal reactions such as redness, swelling, nodules, hair loss, or ulceration were observed at the injection site of the egg yolk antibody in the 10 cats in the experimental group, nor were any systemic adverse reactions observed.

[0077] In the experimental group, the cats' mental state began to recover on the 2nd-3rd day of treatment, and their appetite gradually returned to normal. Starting on the 3rd day, pain symptoms were relieved, and increased diarrhea, drooling, nasal discharge, and eye discharge were controlled. They recovered completely after 5-6 days of treatment, and PCR testing showed negative results for FPV virus. Therefore, the recovery period for this treatment regimen is 5-6 days.

[0078] In the control group, the cats' mental state and appetite gradually recovered on days 3-4 of treatment; from day 5, the increased salivation, nasal discharge, and eye discharge were controlled; on days 8-9, diarrhea and pain symptoms were relieved; and on days 12-14, diarrhea was essentially gone, and their mental state and appetite returned to normal. PCR testing showed negative results for FPV virus. Therefore, using conventional treatment methods, the recovery period is 12-14 days.

[0079] In terms of treatment cycle and treatment effect, cats that used FPV egg yolk antibody injection preparation had faster symptom recovery time and recovery time than cats that did not use egg yolk antibodies. This shows that the egg yolk antibody injection preparation prepared by this invention can effectively alleviate clinical symptoms and significantly shorten the cure time.

[0080] The above embodiments are merely illustrative, and the present invention is not limited thereto. Those skilled in the art can make equivalent substitutions, combinations, or improvements to the details, steps, or parameters without departing from the spirit of the present invention, and all such substitutions, combinations, or improvements shall fall within the protection scope of the present invention.

Claims

1. An egg yolk antibody of feline parvovirus, characterized by, The yolk antibody of the feline parvovirus is obtained by immunizing laying hens with inactivated feline parvovirus FPV-BJ strain, and then extracted and purified from the egg yolk, the preservation number of the feline parvovirus FPV-BJ strain is CGMCC No. 46199; the classification and naming is feline parvovirus; the preservation time is September 22, 2024.

2. The method of claim 1, wherein the feline parvovirus yolk antibody is prepared by, Comprise the following steps: (1) Preparation of inactivated antigen of feline parvovirus FPV-BJ strain; (2) Immunization of laying hens, the laying hens are Hyline white laying hens; (3) Isolation and purification of yolk antibody.

3. The method of claim 2, wherein the feline parvovirus yolk antibody is prepared by, The specific method for immunizing the laying hens includes: selecting healthy Hyline White laying hens at the age of 24-26 weeks, isolating and feeding for 7 days, and then selecting healthy laying hens with normal diet and water for inoculation of immunogen, wherein the content of the inoculated immunogen is 10 5.0 TCID 50 / ml of FPV-BJ strain inactivated vaccine, and the specific immunization process is as follows: first immunization: subcutaneous injection at the neck with a dose of 1.0 mL / hen; second immunization: subcutaneous injection at the neck 14 days after the first immunization with a dose of 1.5 mL / hen; third immunization: subcutaneous injection at the neck 14 days after the second immunization with a dose of 1.5 mL / hen; the FPV-HI titer of yolk fluid is determined after the third immunization, and when the FPV-HI titer is greater than or equal to 1:512, high-immune eggs are collected and stored in an environment at 10-12℃; during the production process after the third immunization, a booster immunization is performed every 30 days, and the injection site of the booster immunization is the chest muscle, and the injection dose is 1.5 mL each time.

4. The method of claim 2 or 3, wherein the feline parvovirus yolk antibody is prepared by, The specific steps of isolation and purification of the yolk antibody comprise: (1) Separate the egg white and yolk of the high-immune egg after disinfection with a separator, dilute the yolk with 10 times the volume of pH=5.0 acetic acid-sodium acetate buffer, stir until the yolk is completely dissolved, then stand still at 2-8 DEG C for 12 hours, and then collect the supernatant by centrifugation at 4500 r / min for 15 minutes; (2) Take the supernatant, dilute it 2 times with pH=5.2, 0.1M acetate buffer, stir for 30 minutes, add caprylic acid with a final concentration of 1%, stand still to separate the layers, then take the water phase and centrifuge, collect the supernatant, filter it with a 0.45 μm filter membrane, and collect the filtrate, which is the purified FPV yolk antibody; take the purified yolk antibody and perform a hemagglutination inhibition test, and the prepared yolk antibody has a hemagglutination inhibition titer of ≥1:1024.

5. The yolk antibody of the feline parvovirus according to claim 1 for use in the preparation of a medicine for preventing or treating infection of the feline parvovirus.

6. Use according to claim 5, characterized in that, The medicine is a yolk antibody freeze-dried preparation or a yolk antibody injection preparation.

7. Use according to claim 6, characterized in that, The yolk antibody freeze-dried preparation is prepared by the following method: (1) Put the FPV yolk antibody solution into a 30 kDa dialysis bag for desalting by dialysis until the membrane underflow does not produce white precipitate with 2% barium chloride, and then concentrate to a hemagglutination inhibition titer of 1:1024, filter with a 0.22 μm filter to sterilize, and then store the yolk antibody stock solution at 2-4 DEG C under sterile conditions; (2) Add a freeze-drying protective agent to the yolk antibody stock solution, mix uniformly, filter with a 0.22 μm filter to sterilize, and then perform sterile quantitative packaging, vacuum freeze-drying, so that the water content is ≤3.0%, and then seal after freeze-drying, to obtain the FPV freeze-dried yolk antibody; The freeze-drying protective agent is made of sucrose, gelatin, sodium butyrate, polygonatum multiflorum polysaccharide and skimmed milk powder, and the preparation method comprises the following steps: weigh 25 g of sucrose, 5 g of gelatin, 1.5 g of polygonatum multiflorum polysaccharide, 0.5 g of sodium butyrate and 25 g of skimmed milk powder, first add the gelatin to 100 mL of PBS buffer, heat to 75 DEG C and stir to dissolve, then cool to 45 DEG C, add the other ingredients, stir and mix, filter to sterilize, and then store at 45 DEG C. The preparation method of the freeze-dried egg yolk antibody is as follows: weighing the egg yolk antibody stock solution and mixing with the freeze-drying protectant, and then performing freeze-drying, the freeze-drying procedure comprises: setting the temperature to-5~-2℃, pre-freezing for 1h, and then reducing the temperature to-45~-55℃ for 2h, vacuumizing the freeze-drying machine to 0.1~0.2mBar, increasing the temperature to-30~-25℃, and maintaining for 24h; increasing the temperature to-15~-10℃, and maintaining for 15h; increasing the temperature to 0-10℃, and maintaining for 15h, thereby obtaining the freeze-dried egg yolk antibody, and the water content is ≤4%.

8. Use of the egg yolk antibody of feline parvovirus according to claim 1 in the preparation of egg yolk antibody egg yolk powder for feed, which can be used in the processing of pet food.

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