CN120005006A

CN120005006A - A preparation method and application of cat Chapahama virus antibody

Info

Publication number: CN120005006A
Application number: CN202510055552.0A
Authority: CN
Inventors: 李海玲; 冯亚莉
Original assignee: Shenyang Chongye Biotechnology Co ltd
Current assignee: Shenyang Chongye Biotechnology Co ltd
Priority date: 2025-01-14
Filing date: 2025-01-14
Publication date: 2025-05-16

Abstract

The invention discloses a preparation method and application of a cat ChapaHama virus antibody, belonging to the field of biotechnology, comprising the following steps of S1, mixing FECHPV VLPS protein with an adjuvant and emulsifying to obtain FECHPV VLPS vaccine; S2, taking an immune auxiliary agent, wherein the immune auxiliary agent contains allium macrostemon extract and eucommia ulmoides leaf extract, the solvent is water for injection, the allium macrostemon extract and the eucommia ulmoides leaf extract are obtained by special enzymolysis extraction, S3, selecting healthy egg laying hens, inoculating FECHPV VLPS vaccine and the immune auxiliary agent for immunization, S4, sterilizing the collected immune eggs, and collecting yolk liquid to prepare FeChPV yolk antibody liquid, namely the cat Chapahar virus antibody. The feline Cha Paha equine virus antibody or the pharmaceutical preparation containing the antibody prepared by the method is used for preparing the medicine for preventing, relieving, assisting in treating or treating animal diseases caused by feline Cha Paha equine virus infection.

Description

Preparation method and application of cat Chapahama virus antibody

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a preparation method and application of a cat ChapaHama virus antibody.

Background

Cat Cha Paha Ma Bingdu (Feline chaphamaparvovirus, feChPV) is a novel feline parvovirus that infects domestic and wild cats and causes fatal infectious diseases. FeChPV was first found in cat stool samples from one animal holding system in Canada in 2019 and was thought to be associated with outbreaks of diarrhea and vomiting in cats, followed by sequential findings in Italy and Turkey. FECHPV DNA was then detected in cats with or without symptoms of gastroenteritis and cats with or without upper respiratory disease. Clinically, it will also be referred to as "novel cat plague".

Etiology results showed that the nucleic acid positive rate of FeChPV was 81.08% (30/37) in cats with all upper respiratory disease symptoms in the domestic housing. Common infection rate of FeChPV positive cats and other common viruses is as high as 80% (24/30) under normal conditions. FeChPV is also speculated to replicate in the immune organs of cats and cause upper respiratory symptoms, encephalitis and lymphadenitis. FeChPV can infect dogs and cats and potentially cross-species transmission, the potential health effects of FeChPV on cats and their potential as enteropathogens deserves further investigation.

At present, no specific therapeutic drug aiming at FeChPV is available, symptomatic treatment, antibacterial, anti-inflammatory and other methods are usually adopted clinically, and serious cases can be treated by being matched with interferon and antiviral drugs at the same time, but the specific treatment cannot be realized. Therefore, in order to effectively prevent FeChPV infection, development of safe and effective specific medicines is needed.

Disclosure of Invention

The invention aims to provide a preparation method and application of a cat ChapaHama virus antibody, so as to efficiently prepare a yolk antibody with high titer and good prevention and auxiliary treatment effects on FeChPV.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

in a first aspect, the invention provides a method for preparing FeChPV egg yolk antibodies, comprising the steps of:

S1, mixing FECHPVVLPS protein with an adjuvant and performing emulsification treatment to obtain FECHPV VLPS vaccine, wherein the VLPs content in FECHPV VLPS vaccine is 50-100 mug;

S2, taking an immune auxiliary agent, wherein the immune auxiliary agent contains 2-4wt% of allium macrostemon extract and 2-4wt% of eucommia ulmoides leaf extract, and the solvent is water for injection;

S3, selecting healthy egg laying hens, and inoculating FECHPVVLPS vaccine and an immune auxiliary agent for immunization after isolated feeding observation, wherein the immunization program comprises the following steps:

S3.1, basic immunization, namely carrying out injection inoculation FECHPVVLPS on neck subcutaneous or chest muscle of each healthy egg-laying hen for 1-2mL of vaccine per chicken, and then injecting 1-2mL of immune auxiliary agent per chicken;

s3.2, secondary immunization, namely, performing secondary injection inoculation at intervals of 10-15 days after basic immunization, performing injection inoculation on neck subcutaneous or chest muscles for FECHPV VLPS vaccine 1-2 mL/chicken, and then injecting 1-2 mL/chicken with an immune auxiliary agent;

S3.3, performing three times of immunization, namely performing three times of injection inoculation at intervals of 10-15 days after the second immunization, performing injection inoculation on neck subcutaneous or chest muscle for FECHPV VLPS vaccine 1-2 mL/chicken, and then injecting 1-2 mL/chicken with an immune auxiliary agent;

s3.4, after three times of immunization, collecting immunized eggs when the FeChPV antibody titer in egg yolk liquid is higher than 1:4000;

S4, sterilizing immune eggs, collecting egg yolk liquid, preparing FeChPV egg yolk antibody liquid, preparing FeChPV egg yolk antibody liquid, mixing egg yolk liquid with Tris-HCl buffer solution with the volume ratio of 1:1-1:5 of pH7.3-7.5, adding polyethylene glycol PEG6000 with the total volume of 2-5% (W/V) after shaking and mixing for 20-30 seconds, fully shaking and mixing, centrifuging for 20-30 minutes at 4 ℃ by using a low-temperature refrigerated centrifuge under the conditions of 10000-12000r/min, filtering by using polypropylene 750B filter cloth, collecting filtrate, filtering by using a filter column filter until liquid is clarified, obtaining clarified liquid, adding polyethylene glycol PEG6000 with the total volume of 5-10% (W/V) into the clarified liquid, standing for 30-40 minutes after shaking and mixing at room temperature, centrifuging for 20-30 minutes by using a low-temperature refrigerated centrifuge at 4 ℃ under the conditions of 10000-12000r/min to obtain precipitate A, centrifuging for 20-30 minutes by using a sterile pH7.3-7.5 (PBS with the total volume of 10-10 mL), preparing sterile sediment solution with the PBS with the total volume of 10-10 mL (PBS with the total volume of 10 ℃ 7.5) after shaking and mixing for 20-40 minutes by using the sterile PBS with the pH 7.3-120 r/min, dissolving by using the sterile PBS with the total volume of 10mL of 7B with the pH7 to prepare sterile sediment solution after shaking and shaking for 1-10.5 min, feChPV egg yolk antibody liquid, namely the cat Chapahama virus antibody, is obtained.

Further, in step S1, the preparation method of FECHPV VLPS proteins includes the following steps:

n1, synthesizing and cloning a nucleotide sequence of FeChPV capsid protein VP1 into a pET-28a prokaryotic expression vector after optimizing the sequence through escherichia coli codon bias analysis;

n2, E.coli induced expression to obtain soluble FeChPV VP1;

n3, expressing FeChPV VP in colibacillus to form icosahedron structure after purification by transmission electron microscope, and obtaining FECHPV VLPS protein by FeChPV VP1 automatically folding and assembling Virus-Like Particles (VLPs) according to the reported picture of (Novel parvovirus in an outbreak offatal enteritis in European hedgehogs(Erinaceus europaeus),Italy,2022)ChPV Particles.

Further, in the step S2, the preparation method of the allium macrostemon extract comprises the steps of taking allium macrostemon for smashing, adding water for injection, of which the mass is 8-12 times that of the allium macrostemon, adjusting the pH value to 4.5-5.0, adding cellulase of which the mass is 0.5-2% of the allium macrostemon and pectase of which the mass is 0.5-2% of the allium macrostemon, carrying out enzymolysis for 2-3 hours at 50-55 ℃, adjusting the pH value to 6.5-7, adding ficin of which the mass is 0.5-1.5% of the allium macrostemon, carrying out enzymolysis for 1-2 hours at 60-65 ℃, carrying out boiling water bath enzyme deactivation for 15-20 minutes, adding Serratin enzyme of which the mass is 0.5-1.5% of the allium macrostemon after cooling to room temperature, carrying out enzymolysis for 1-1.5 hours at 45-50 ℃, carrying out centrifugation for 10-15 minutes at 5000-6000r/min, taking supernatant, and carrying out ultrafiltration with a 3kDa ultrafiltration membrane to obtain the freeze-dried allium macrostemon extract.

Further, in the step S2, the preparation method of the eucommia ulmoides leaf extract comprises the steps of taking eucommia ulmoides leaves, crushing, adding 8-12 times of water for injection, adjusting the pH value to 4.5-5.5, adding 0.5-2% of cellulase in the eucommia ulmoides leaves, performing enzymolysis for 2-3h at 50-60 ℃, adjusting the pH value to 6.5-7.0, adding 0.5-1.5% of Serratinase in the eucommia ulmoides leaves, performing enzymolysis for 1-2h at 45-50 ℃, performing enzyme deactivation for 15-20min in a boiling water bath, performing enzymolysis for 1-1.5 min at 50-60 ℃, performing enzyme deactivation for 15-20min in a boiling water bath, adding 0.5-1.5% of nattokinase in the eucommia ulmoides leaves, performing enzymolysis for 50-80min at 50-55 ℃, performing enzyme deactivation for 15-20min in a boiling water bath, performing enzymolysis for 1-2h at 45-50 ℃ and 15-20min in a temperature, performing enzymolysis for 5000-6000 r/10 kDa, performing ultrafiltration on the supernatant to obtain an ultra-dried extract, and performing ultrafiltration to obtain a freeze-dried supernatant, and performing ultrafiltration to obtain the supernatant.

In the above steps, the reagents used for adjusting the pH include Phosphate Buffer (PBS), hydrochloric acid (HCl), citric acid, sodium hydroxide (NaOH) and sodium bicarbonate.

The invention also provides a cat ChapaHama virus antibody, which is prepared by adopting the preparation method.

The invention also provides a pharmaceutical preparation which comprises the cat Cha Paha Ma Bingdu antibody and pharmaceutically acceptable auxiliary components, wherein the administration route of the pharmaceutical preparation is oral administration or injection administration.

The invention also provides an application of the cat ChapaHama virus antibody or the pharmaceutical preparation in preparing medicines for preventing, relieving, assisting in treating or treating animal diseases caused by cat Cha Paha horse virus infection.

The invention has the beneficial effects that:

1. FECHPV VLPS the vaccine does not contain viral genetic material, has high safety, mimics the viral structure but does not contain infectious genetic material, and reduces the risk of infection after vaccination.

2. FECHPV VLPS can be expressed in a variety of cellular and biological expression systems, which provides a variety of options for vaccine production, helping to optimize the production process.

3. By inoculating FECHPV VLPS vaccine in hen and collecting yolk antibody, antibody specific to FeChPV can be obtained, which is helpful for specific diagnosis and treatment.

4. The use of egg yolk antibodies as an immunization strategy is more economical to produce than traditional monoclonal antibodies, especially for large-scale applications.

5. Through three immunization procedures, high efficiency of the yolk antibody, i.e. titers higher than 1:4000, is ensured, which means that the antibody has higher neutralizing capacity and affinity, and can more effectively fight FeChPV.

6. Egg yolk antibodies are derived from egg yolk, are natural and safe biological products and generally have lower toxic and side effects and anaphylactic reaction risks.

7. The preparation method of FeChPV egg yolk antibody not only can provide a safe and effective immune response, but also can play an important role in a plurality of fields such as vaccine development, disease treatment, drug delivery and the like.

8. The immune adjuvant contains Bulbus Allii Macrostemi extract and folium Eucommiae extract. During immunization, these extracts enhance the immune response in a number of ways. They act as immunomodulating substances on the hen's immune system, stimulating the activity of immune cells (e.g., lymphocytes, etc.), causing the organism to mount a stronger immune response to FECHPV VLPS vaccine, promoting better uptake and processing of antigen (FECHPV VLPS protein) in the vaccine by antigen presenting cells (e.g., dendritic cells), and then more effectively transferring antigen information to T lymphocytes, which in turn activate B lymphocytes to generate more antibodies against FeChPV, thereby increasing the FeChPV antibody titer in immunized eggs.

9. In the preparation process of the allium macrostemon extract, the effective components in the allium macrostemon can be released through a plurality of enzymolysis steps. Firstly, regulating pH value, adding cellulase and pectase, and making enzymolysis at a certain temperature to decompose cell wall component of allium macrostemon so as to make effective component in cell be more easily dissolved out. And then adding enzymes such as ficin, serratia peptidase and the like for further enzymolysis, and decomposing macromolecular substances in the allium macrostemon into high-efficiency micromolecular active ingredients. The small molecule components have better immunity enhancing effect and good bioavailability, are easily absorbed and utilized by organisms, thereby better exerting the regulating effect on immune systems, such as stimulating proliferation and differentiation of immune cells, enhancing the immune response of the organisms to antigens and finally being helpful for enhancing the immune titer.

10. The preparation of the eucommia ulmoides leaf extract also adopts a multi-step enzymolysis process. Firstly, the cell wall structure of eucommia ulmoides leaves is destroyed by enzymolysis of cellulase, so that intracellular substances are released. And then, regulating the pH value and adding Serratia peptidase, bromelain, nattokinase and the like for enzymolysis, so that macromolecular substances in the eucommia ulmoides leaves can be decomposed into effective micromolecular active ingredients. The small molecule components contain substances capable of regulating an immune system, and can activate an immune signal path of an organism, enhance the functions of immune cells and promote the activation and proliferation of antibody-producing cells (B lymphocytes), so that the immune response intensity of the organism to vaccine antigens is improved, and the antibody titer in immune eggs is further improved.

11. The allium macrostemon extract and the eucommia ulmoides leaf extract play a synergistic role through different immunoregulatory pathways. The small molecular active ingredient in Bulbus Allii Macrostemi extract mainly acts on some links of immune system to promote antigen presentation or regulate T lymphocyte function, and the small molecular ingredient in folium Eucommiae extract can activate other immune cells (such as B lymphocyte) or enhance immune signal transduction. When the two are combined, the two are matched with each other, so that the immune response of the organism is enhanced from multiple angles, more antibodies are generated, and the immune titer is synergistically improved.

Drawings

FIG. 1 is a diagram FeChPV Westernblot of example 1 of the present invention.

FIG. 2 is a graph showing the results of ELISA detection of the titer of the lyophilized powder for antibody of example 1 of the present invention.

FIG. 3 is a graph showing the effect of temperature on potency of an antibody lyophilized powder according to example 1 of the present invention.

FIG. 4 is a graph showing the effect of pH on the potency of an antibody lyophilized powder according to example 1 of the present invention.

FIG. 5 is a graph showing the effect of storage time on potency of an antibody lyophilized powder according to example 1 of the present invention.

Detailed Description

Further, the present invention is described in detail below by way of specific embodiments and examples with reference to the accompanying drawings. The detailed description and examples are set forth herein only for the purposes of illustration and are not intended to limit the invention.

The specific experimental procedures or conditions are not noted in the examples and may be followed by the operations or conditions of conventional experimental procedures described in the literature in this field. The reagents and instruments used are not noted by manufacturers and are conventional reagent products commercially available.

The biological material sources involved in the embodiment of the invention are cat with FeChPV positive PCR identification in pet hospitals, and egg-laying hen is commercial sea-blue brown chicken.

Example 1

(One) preparation FECHPV VLPS of protein:

(1) The FeChPV (GenBank accession No. NC_ 076446.1) sequence was downloaded from NCBI. The nucleotide sequence encoding FeChPV capsid protein VP1 is obtained. VP1 full length 1527bp, VP1 nucleotide sequence encoding FeChPV of 509 amino acids. The FeChPV VP gene nucleotide sequence is fused with small ubiquitin-like modified protein (SUMO) after optimization treatment by an escherichia coli codon matching tool (JCAT, brunswager http:// www.jcat.de, university of Technology, germany), the delegated optimization and fusion treatment unit is manufactured by a biological (Shanghai) stock company, so as to increase the solubility and stability of the protein, and the fused SUMO-VP1 sequence is cloned into a pET-28a prokaryotic expression vector to construct a pET-28a-SUMO-VP1 recombinant plasmid. And transforming the pET-28a-SUMO-VP1 recombinant plasmid into BL21 competence, and screening out clones containing the recombinant plasmid to obtain BL21-pET-28a-SUMO-VP1 competent cells. The fused SUMO-VP1 sequence is shown as SEQ ID NO. 1.

(2) Transferring pTF16 plasmid into BL21-pET-28a-SUMO-VP1 competent cells, screening positive escherichia coli colonies in a kanamycin-containing resistance plate, picking single escherichia coli colony for shaking, identifying single escherichia coli colonies for induced expression by PCR, identifying the correct identified strain as BL21-pET-28a-SUMO-VP1-pTf16, adding 50% (v/v) glycerol, uniformly mixing, and preserving at-80 ℃ to obtain BL21-pET-28a-SUMO-VP1-pTF16 glycerol bacteria.

PCR primer sequence:

T7-F:TAATACGACTCACTATAGG;

T7 TER-R:GCTAGTTATTGCTCAGCGG。

(3) Taking the preserved BL21-pET-28a-SUMO-VP1-pTF16 glycerol bacteria, culturing the glycerol bacteria in a LB culture medium containing kanamycin at the temperature of 16 ℃ and the speed of 200r/min until OD ₆₀₀ = 0.6, respectively sampling 1mL, adding 0.1mmol/L, 0.3mmol/L, 0.5mmol/L, 0.7mmol/L and 1.0mmol/L isopropyl-beta-D-thiogalactoside (IPTG) to induce the expression of the target protein VP1, and setting the uninduced expression bacteria as a negative control. Inducing target protein to express at the condition of 16 ℃ with the final concentration of IPTG of 0.5mmol/L protein expression quantity being maximum, selecting the concentration as the concentration of the induced expression, centrifuging at 12000r/min for 30min after 14h of the induced expression, collecting thalli, crushing the thalli by using a high-pressure low-temperature cell disruption instrument, centrifuging at 12000r/min for 20min, and collecting supernatant to obtain the target protein liquid for the induced expression.

(4) Westernblot identification of BL21-pET-28a-SUMO-VP1-pTF16 protein:

Mixing the target protein solution induced to be expressed with SDS loading buffer solution (SDS-PAGE SAMPLE Loading Buffer) at a volume ratio of 1:5, boiling at 100 ℃ for 10min to denature the protein to obtain denatured protein sample solution, preparing SDS-PAGE gel by using a 10% SDS-PAGE rapid gel kit, performing electrophoresis with a voltage of 60V, adjusting the voltage to 120V after 30min, and continuing to run the gel to the bottom of the gel. Transferring film with film transferring instrument, cross-flow of 300mA, transferring film transferring time of 45min, and transferring protein in gel onto NC film. Taking out NC membrane, cleaning, soaking in 5% skimmed milk powder sealing solution, sealing at 37deg.C for 2 hr, and washing off residual sealing solution with phosphate Tween buffer (PBST). His-tagged murine primary antibody diluted 1:5000 was incubated overnight at 4℃to allow specific binding of the primary antibody to the His tag on the protein of interest and membranes were washed using PBST. Goat anti-mouse secondary antibody labeled with HRP diluted 1:1000 was incubated at 37℃for 2h, the secondary antibody specifically bound to the primary antibody, and the HRP label was brought to the target protein band, and the membrane was washed with PBST. And developing the NC film by using a protein imager and photographing. The HRP-labeled secondary antibody generates a chromogenic reaction under the action of a chromogenic substrate, so that the target protein band is displayed. The presence and molecular weight of the target protein can be intuitively observed by photographing and recording the color development result.

(5) Purification of BL21-pET-28a-SUMO-VP1-pTF16 protein:

VP1 expressed in E.coli was purified using Ni-NTA affinity column. Since VP1 protein carries His tag, it can be specifically combined with nickel ion on Ni-NTA column. According to SDS-PAGE results, after protein liquid is fully combined with a chromatographic column, a single obvious band is visible after the elution by imidazole with the concentration of 200mM, and a FECHPV WESTERN blot diagram is shown in figure 1. The purified VP1 was dialyzed overnight to complete self-assembly of VLPs, giving FECHPV VLPS samples.

(6) Transmission electron microscopy observation FECHPV VLPS protein:

The FECHPV VLPS protein samples were adsorbed on copper grids at room temperature for 5min and the excess samples were blotted off with filter paper. The grid was stained with 3wt% phosphotungstic acid for 5min, excess stain was gently blotted with filter paper and virus-like particles were observed under a transmission microscope. Under TEM, the morphology and size of VLPs, as well as their distribution on the grid, can be observed. After purification, FECHPV VLPS samples were observed by transmission electron microscopy and expressed in E.coli to form icosahedral structures, consistent with the reported pictures of (Novel parvovirus in an outbreak of fatal enteritis in European hedgehogs(Erinaceus europaeus),Italy,2022)ChPV particles.

(II) preparation FECHPV VLPS of vaccine:

slowly adding FECHPV VLPS protein obtained above into commercial vaccine adjuvant at room temperature, emulsifying for 10min at 14000r/min to ensure complete emulsification, and preparing into FECHPV VLPS vaccine.

Among the commercial vaccine adjuvants employed are Sigma-Aldrich Freund's complete and Freund's incomplete adjuvants. Adding FECHPV VLPS protein into Freund's complete adjuvant at room temperature for emulsification to obtain FECHPV VLPS vaccine as basic injection of first needle, wherein the VLPs content in FECHPV VLPS vaccine is 100 μg, adding FECHPV VLPS protein into Freund's incomplete adjuvant at room temperature to obtain FECHPV VLPS vaccine as secondary injection and tertiary injection, wherein the VLPs content in FECHPV VLPS vaccine of secondary immunization is 80 μg, and the VLPs content in FECHPV VLPS vaccine of tertiary immunization is 50 μg.

(III) preparing an immune auxiliary agent:

adding Bulbus Allii Macrostemi extract 3wt% and folium Eucommiae extract 3wt% into injectable water, and mixing to obtain immune adjuvant.

The preparation method of the allium macrostemon extract comprises pulverizing allium macrostemon, adding 10 times of water for injection, adjusting pH to 4.8, adding cellulase with 1.2% of allium macrostemon mass and pectase with 1.2% of allium macrostemon mass, performing enzymolysis at 53 ℃ for 2.5h, adjusting pH to 6.8, adding ficin with 1.0% of allium macrostemon mass, performing enzymolysis at 62 ℃ for 1.5h, inactivating enzyme in boiling water bath for 18min, cooling to room temperature, adding Serratin with 1.0% of allium macrostemon mass, performing enzymolysis at 48 ℃ for 1h, inactivating enzyme in boiling water bath for 18min, cooling to room temperature, centrifuging at 5500r/min for 12min, collecting supernatant, performing ultrafiltration with 3kDa ultrafiltration membrane to obtain component smaller than 3kDa, and lyophilizing to constant weight to obtain allium macrostemon extract.

The preparation method of the eucommia ulmoides leaf extract comprises the steps of taking eucommia ulmoides leaves, crushing the eucommia ulmoides leaves, adding water for injection, adjusting the pH value to be 5.0, adding cellulase with the mass of 1.2% of the eucommia ulmoides leaves, carrying out enzymolysis for 2.5 hours at 55 ℃, adjusting the pH value to be 6.8, adding Serratia peptidase with the mass of 1.0% of the eucommia ulmoides leaves, carrying out enzymolysis for 1.5 hours at 48 ℃, inactivating enzyme for 18 minutes in a boiling water bath, cooling to room temperature, adding bromelain with the mass of 1.0% of the eucommia ulmoides leaves, carrying out enzymolysis for 1 hour at 55 ℃, carrying out enzymolysis for 18 minutes in a boiling water bath, carrying out enzymolysis for 60 minutes at 52 ℃, carrying out enzymolysis for 18 minutes in a boiling water bath, carrying out centrifugation for 12 minutes at 5500r/min, taking supernatant, carrying out ultrafiltration by adopting a 5kDa ultrafiltration membrane to obtain a component smaller than 5kDa, and carrying out freeze-drying to constant weight, thus obtaining the eucommia ulmoides leaf extract.

(IV) immunogenicity evaluation:

Selecting a Chinese cat with clinical manifestation health at 5 weeks of age, injecting FECHPV VLPS vaccine 1mL (first needle basic immunity injection) subcutaneously, injecting 1mL of immunity auxiliary agent subcutaneously, performing second booster immunization after 21 days, injecting 1mL of secondary immunity injection subcutaneously, injecting 1mL of immunity auxiliary agent subcutaneously, performing third booster immunization after 21 days, injecting 1mL of three immunity injections subcutaneously, and injecting 1mL of immunity auxiliary agent subcutaneously. Serum was collected on days 0, 21, 28, 42, and 63 after the first vaccine injection. Enzyme-linked immunosorbent assay (ELISA) to detect antibody titers in cat serum:

The reagent comprises a coated antigen (FECHPV VLPS protein), an enzyme-labeled secondary antibody (anti-cat IgG-HRP labeled antibody), a substrate solution (TMB substrate), a stop solution (sulfuric acid solution), a washing solution (PBS buffer containing 0.05% Tween-20), a dilution solution (PBS buffer with pH7.4 and 0.01M), and a blocking solution (PBS buffer containing 1wt% BSA; PBS buffer with pH7.4 and 0.01M).

The method comprises diluting the coated antigen to a proper concentration of 5 μg/mL with a coating buffer (carbonate buffer, pH 9.6), adding 150 μl per well, and placing in a 4 ℃ refrigerator overnight to allow the antigen to be firmly adsorbed on the walls of the wells of the ELISA plate. Pouring out the liquid in the holes of the ELISA plate, filling each hole with the washing liquid, standing for 30 seconds, pouring out, repeating the washing for 3 times to remove unbound antigen, and finally beating on absorbent paper. 200 μl of blocking solution was added to each well and incubated at 37deg.C for 2 hours to block the sites on the ELISA plate not occupied by antigen, preventing nonspecific adsorption in the subsequent step. Cat serum samples were diluted with dilutions at different fold volume ratios of 1:10, 1:20, 1:40, 1:100, 1:300, 1:600, 1:900, 1:1800, 1:4000, 1:4800, 1:6000, 1:6800, 1:7000, 3 wells per dilution fold, 200 μl of diluted serum sample was added per well, and incubated at 37 ℃ for 2 hours to allow sufficient binding of antibodies in serum to the coating antigen. Pouring out the liquid in the wells of the ELISA plate, filling each well with the washing liquid, standing for 30 seconds, pouring out, and repeating the washing for 3 times to remove unbound serum components. The secondary enzyme-labeled antibody was diluted to working concentration (1:5000 volume fold) with 200 μl/well incubated at 37 ℃ for 1 hour to bind the secondary enzyme-labeled antibody to the cat antibody already bound to the antigen. Pouring out the liquid in the wells of the ELISA plate, filling each well with the washing liquid, standing for 30 seconds, pouring out, and repeating the washing for 3 times to remove the unbound ELISA secondary antibodies. 200 μl of substrate solution (TMB substrate) was added to each well, developed for 30min at room temperature in the dark, and the reaction was stopped when the positive control wells (wells containing samples of antibodies to the coated antigen) showed a significant color change, while the negative control wells (wells containing only diluent) had little color change. 100. Mu.L of stop solution (sulfuric acid solution) was added to each well to terminate the chromogenic reaction. At this time, the color of the solution changes, and the TMB substrate changes from blue to yellow after color development. Absorbance (OD) values were read per well using a microplate reader at the appropriate wavelength of 450 nm. The threshold value is determined based on the OD values of the positive and negative controls. The OD value of the sample is greater than or equal to the critical value, namely that antibodies exist in serum, and the sample is negative when the OD value is smaller than the critical value. Serum samples were subjected to a series of dilutions at different ratios, and the above steps were repeated for testing. The highest dilution factor that can produce a positive result is the antibody titer. The detection results were 1:40 on day 0, 1:900 on day 21, 1:4000 on day 28, 1:6000 on day 42 and 1:6800 on day 63.

Comparative example for immunogenicity evaluation:

Selecting a cat with the clinical manifestation of health at the age of 5 years, subcutaneously injecting FECHPV VLPS vaccine 1mL (first needle basic immunity injection) without immune auxiliary agent, performing secondary booster immunization after 21 days, subcutaneously injecting 1mL of secondary immunity injection without immune auxiliary agent, performing tertiary booster immunization after 21 days, subcutaneously injecting 1mL of tertiary immunity injection without immune auxiliary agent, and performing primary booster immunization after 21 days. Serum was collected on days 0, 21, 28, 42, and 63 after the first vaccine injection. Enzyme-linked immunosorbent assay (ELISA) for detecting antibody titers in cat serum was as above. The results of the assay were 1:20 on day 0, 1:600 on day 21, 1:1800 on day 28, 1:4000 on day 42, and 1:4800 on day 63. The immune auxiliary agent has good effect of improving the antibody titer.

And (V) evaluating safety:

Selecting 5 healthy cats with the clinical manifestation of 4-8 weeks, injecting basic immunity injection (1 mL/each day) and immunity auxiliary agent (1 mL/each day) in a single dose, selecting 5 healthy cats with the clinical manifestation of 4-8 weeks, injecting basic immunity injection (4.5 mL/each day) and immunity auxiliary agent (4.5 mL/each day) in a single multiple dose, selecting 5 healthy cats with the clinical manifestation of 4-8 weeks, injecting basic immunity injection (twice daily, 1mL each time, continuously injecting for 5 days) and immunity auxiliary agent (twice daily, 1mL each time, continuously injecting for 5 days) in multiple times, observing the health state of the cats, and determining FECHPV VLPS vaccine and immunity auxiliary agent to be safe and reliable without obvious toxic and side effects.

Preparing immune eggs, feChPV yolk antibody liquid and antibody freeze-dried powder:

(1) Healthy egg laying hens are selected, after isolated feeding and observation for 7 days, FECHPV VLPS vaccines and immune auxiliaries are inoculated for immunization, and the immunization procedure comprises:

(1.1) basic immunization, namely, carrying out subcutaneous injection inoculation FECHPV VLPS vaccine 1.5 mL/chicken on the neck of each healthy egg-laying hen, and then injecting 1.5 mL/chicken of an immune auxiliary agent;

(1.2) secondary immunization, namely, carrying out secondary injection inoculation at intervals of 14 days after basic immunization, carrying out injection inoculation FECHPV VLPS vaccine 1.5 mL/chicken under the skin of the neck, and then injecting 1.5 mL/chicken of an immune auxiliary agent;

(1.3) three immunizations, namely, performing a third injection inoculation at intervals of 14 days after the second immunization, performing injection inoculation FECHPV VLPS vaccine 1.5 mL/chicken under the skin of the neck, and then injecting 1.5 mL/chicken of an immune auxiliary agent;

(1.4) after three immunizations, after day 14, the egg yolk liquid was assayed for FeChPV antibody titers at 1:8000 and the immunized eggs were collected.

(2) The preparation method of the FeChPV yolk antibody liquid comprises the steps of mixing the yolk solution with Tris-HCl buffer solution with the volume ratio of 1:3 at the pH of 7.4, adding polyethylene glycol PEG6000 with the total volume of 3% (W/V) after shaking and mixing for 25 seconds, fully shaking and mixing, centrifuging at the temperature of 4 ℃ for 25 minutes by using a low-temperature refrigerated centrifuge under the condition of 11000r/min, collecting supernatant, filtering by using polypropylene 750B filter cloth, collecting filtrate, filtering by using a filter column filter until liquid is clarified to obtain clarified liquid, adding polyethylene glycol PEG6000 with the total volume of 8% (W/V) into the clarified liquid, shaking and mixing, standing at room temperature for 35 minutes, centrifuging at the temperature of 4 ℃ by using a low-temperature refrigerated centrifuge under the condition of 11000r/min, removing the supernatant to obtain precipitate A, dissolving the precipitate A with the sterile PBS buffer solution with the total volume of 10mL of 7.4, centrifuging at the temperature of 53 mL of 10mL, dissolving the polyethylene glycol PEG6000 with the pH of 10mL of PBS, centrifuging at the temperature of 53 ℃ for 4, centrifuging at the temperature of 53 mL, centrifuging at the temperature of 50 ℃ for 35min, and centrifuging at the temperature of 53 mL, dissolving the pH of 10mL of the PBS with the pH of 50 for 4, fully centrifuging at the temperature of 35 ℃ until the temperature of 35 ℃ is completely dissolving the supernatant with the PBS with the pH of 50 to obtain the sterile solution with the PBS with the pH of 50 at the temperature of 35 ℃ of 50.

(3) Adding 5g of egg yolk antibody liquid into 1mL of freeze-drying protective liquid, adding the egg yolk antibody liquid into the freeze-drying protective agent, uniformly mixing, filtering and sterilizing by using a microporous filter membrane with the diameter of 0.22 mu m, taking filtrate for sterile split charging, and pre-freezing at the temperature of-40 ℃ to obtain the refined cat Cha Paha equine virus antibody.

The freeze-drying protective agent comprises 20g of trehalose, 6g of dextran and 8g of mannitol in every 100mL of freeze-drying protective agent, and is prepared by sterilizing the freeze-drying protective agent under high pressure with sterile water to reach 100 mL.

(4) The refined cat Cha Paha Ma Bingdu antibody is subjected to vacuum freeze drying, wherein the set temperature is-40 ℃ for pre-freezing for 0.5h, the vacuum degree is 0.1mBar, the vacuum degree is-40 ℃ for 8h, the vacuum degree is-20 ℃ for 16h, the vacuum degree is 5 ℃ for 10h, and the vacuum degree is 10 ℃ for 4h, so that the refined cat Cha Paha Ma Bingdu antibody freeze-dried powder, short for antibody freeze-dried powder, is obtained.

The titer detection of the antibody freeze-dried powder comprises the steps of firstly diluting FECHPV VLPS protein to 2 mu g/mL by using a PBS buffer solution, coating a plate, standing overnight at 4 ℃, washing the plate 3 times by using TBST, blocking by using 5% skimmed milk, reacting for 2 hours at 37 ℃, diluting the antibody freeze-dried powder by using the PBS buffer solution according to the mass ratio of the PBS buffer solution to the antibody freeze-dried powder=50:1, adopting an unimmunized egg yolk antibody as a negative control, reacting for 1 hour at 100 mu L per hole at 37 ℃, adding 100 mu L of the HRP-labeled rabbit anti-chicken IgY secondary antibody after dilution after washing and drying, reacting for 1 hour at 37 ℃, and reading OD value at 450nm after washing, drying and developing and stopping reaction. As the results in fig. 2 show, the ELISA titers of the 4 parallel-like antibody lyophilized powders were 1:128000.

(Eight) clinical treatment effect test:

The PCR identification of 25 cats positive in a pet hospital is selected, wherein 25 cats with single FeChPV positive symptoms (symptoms of illness cat have listlessness, inappetence, diarrhea, vomiting and fever) are randomly divided into 5 groups (5 groups of A, B, C, D and E), wherein the group of A is only orally taken with antibody freeze-dried powder, 1.5 g/time per 5 kg of body weight, and is continuously taken for 10 days, the group of B is orally taken with antibody freeze-dried powder (same as the group A) every day, and is matched with auxiliary treatment (cefquinome is orally taken at 0.5 g/time per 5 kg of body weight, once per day), methoxam is orally taken at 0.5 g/time per 5 kg of body weight, and the group of C is not taken with antibody freeze-dried powder, and is only subjected to auxiliary treatment (cefquinome is orally taken at 0.5 g/time per 5 kg of body weight, once per day), methoxam is taken at 0.5 g/time per day, and D is subcutaneously injected with injection containing antibody freeze-dried powder every 5 kg of body weight, and the injection containing antibody freeze-dried powder is taken at 0.5 kg of body weight, and the injection containing 5 mg/time of 5 mg/day of 5m is taken after oral liquid is sterilized, and the injection containing the antibody is taken at 0.5 kg of 5 kg of body freeze-dried powder is taken at once per day, and the injection containing 5 kg of antibody freeze-dried powder is taken orally after oral liquid is taken at 0 kg of 5 kg of body weight.

The symptoms of the diseased cat have the symptoms of listlessness, inappetence, diarrhea, vomiting and fever, and the cure condition is observed and detected:

The cure rate of group A is 80% (4/5) within 5 days, and the cure rate is 80% (4/5) within 10 days. The cure rate of group B is 80 (4/5) within 5 days, and the cure rate is 100% (5/5) within 10 days. The cure rate of group C is 20% (1/5) within 5 days, and the cure rate of group C is 40% (2/5) within 10 days. The cure rate of group D is 80% (4/5) within 5 days, the cure rate of group E is 100% (5/5) within 10 days, and the cure rate of group E is 100% (5/5) within 5 days. The specific cases are shown in table 1 below.

TABLE 1 progress of symptomatic rehabilitation

The antibody of the embodiment is used in the disease period, so that the treatment effect can be well achieved, and the recovery of the disease course of cats can be remarkably accelerated by matching with an auxiliary treatment means.

(Nine) antibody stability detection:

1. influence of temperature on potency of antibody lyophilized powder:

After treating the antibody lyophilized powder for 15min at 37 ℃, 50 ℃, 65 ℃, 75 ℃, 85 ℃ and 95 ℃ in 6 different temperature gradients, the ELISA titers of the antibodies were determined according to the method of experiment (seven) above, 3 replicates were performed for each temperature gradient. As shown in the results of FIG. 3, the titer of the antibody freeze-dried powder is not changed basically below 65 ℃ and is stable, the titer of the antibody freeze-dried powder is reduced rapidly under the treatment condition of 75-85 ℃ but can be maintained above 1:16000, and the titer of the antibody freeze-dried powder is inactivated under the treatment condition of 95 ℃. Therefore, the freeze-dried powder of the antibody is basically not affected by temperature below 75 ℃, and has good stability, but the antibody titer is rapidly reduced when the temperature exceeds 85 ℃, and the influence of the temperature on the product is considered in the application process.

2. Effect of pH on potency of antibody lyophilized powder:

The ELISA titers of the antibody lyophilized powders were determined by the method of experiment (seven) above by subjecting the antibody lyophilized powders to the following different pH conditions of 1.5, 2.5, 3.5, 4.5, 5.5, 6.5, 7.5, 8.5, 9.5, 10.5 and 11.5 for 1 hour at 37 ℃.3 replicates were run for each experiment. As shown in the results of FIG. 4, the antibody lyophilized powder was reduced in titer by 4 dilutions at pH values below 2.5, no change in antibody lyophilized powder titer was observed in the pH range of 3.5-9.5, and reduced in titer by 3 dilutions at pH values above 10.5. The strong acid and alkali can influence the activity of part of the freeze-dried powder of the antibody, and attention is paid to the storage and use so as not to influence the use effect.

3. Antibody lyophilized powder storage conditions and time test:

3 different temperature gradients of 25 ℃, 45 ℃ and 65 ℃ are selected, the antibody freeze-dried powder is filled in capsules, the capsules are respectively placed under the above 3 temperature conditions, sampling is carried out every month, and whether ELISA titer is changed is determined according to the method of the experiment (seventh). As shown in the results of FIG. 5, the antibody lyophilized powder was stored for 15 months at 25℃with no change in titer for 18 months, the antibody lyophilized powder was reduced by one dilution, the antibody lyophilized powder was stored for 12 months at 45℃with one dilution, the antibody lyophilized powder was stored for 18 months with 2 dilutions, the antibody lyophilized powder was stored for 9 months at 65℃with 1 dilution and 18 months with 3 dilutions of ELISA titer, but was still maintained at 1:16000 or more. The freeze-dried powder of the antibody has good storage stability.

Example 2

This example uses FECHPV VLPS vaccine prepared in example 1.

(II) preparing an immune auxiliary agent:

Adding Bulbus Allii Macrostemi extract 2wt% and folium Eucommiae extract 2wt% into injectable water, and mixing to obtain immune adjuvant.

The preparation method of Bulbus Allii Macrostemi extract comprises pulverizing Bulbus Allii Macrostemi, adding 8 times of water, adjusting pH to 4.5, adding cellulase and pectase at 0.5% of Bulbus Allii Macrostemi, performing enzymolysis at 50deg.C for 2 hr, adjusting pH to 6.5, adding ficin at 0.5% of Bulbus Allii Macrostemi, performing enzymolysis at 60deg.C for 1 hr, inactivating enzyme in boiling water bath for 15min, cooling to room temperature, adding Serratin at 0.5% of Bulbus Allii Macrostemi, performing enzymolysis at 45deg.C for 1 hr, inactivating enzyme in boiling water bath for 15min, cooling to room temperature, centrifuging at 5000r/min for 10min, collecting supernatant, ultrafiltering with 3kDa ultrafiltration membrane to obtain component smaller than 3kDa, and lyophilizing to constant weight to obtain Bulbus Allii Macrostemi extract.

The preparation method of folium Eucommiae extract comprises pulverizing folium Eucommiae, adding 8 times of water, adjusting pH to 4.5, adding cellulase with 0.5% of folium Eucommiae mass, performing enzymolysis at 50deg.C for 2 hr, adjusting pH to 6.5, adding Serratia peptidase with 0.5% of folium Eucommiae mass, performing enzymolysis at 45deg.C for 1 hr, inactivating enzyme in boiling water bath for 15min, cooling to room temperature, adding bromelain with 0.5% of folium Eucommiae mass, performing enzymolysis at 50deg.C for 1 hr, inactivating enzyme in boiling water bath for 15min, cooling to room temperature, adding nattokinase with 0.5% of folium Eucommiae mass, performing enzymolysis at 50deg.C for 50min, inactivating enzyme in boiling water bath for 15min, cooling to room temperature, centrifuging at 5000r/min for 10min, collecting supernatant, ultrafiltering with 5kDa ultrafiltration membrane to obtain component less than 5kDa, and lyophilizing to constant weight to obtain folium Eucommiae extract.

(III) preparing immune eggs, feChPV yolk antibody liquid and antibody freeze-dried powder:

(1.1) basic immunization, namely carrying out injection inoculation on chest muscles of each healthy egg-laying hen for FECHPV VLPS vaccine 1 mL/chicken, and then injecting an immune auxiliary agent for 1 mL/chicken;

(1.2) secondary immunization, namely, carrying out secondary injection inoculation at intervals of 10 days after basic immunization, carrying out injection inoculation on chest muscles by FECHPV VLPS vaccine 1 mL/chicken, and then injecting 1 mL/chicken of immune auxiliary agent;

(1.3) three immunizations, namely, performing a third injection inoculation at intervals of 10 days after the second immunization, performing injection inoculation on chest muscles with FECHPV VLPS vaccine 1 mL/chicken, and then injecting 1 mL/chicken of an immune auxiliary agent;

(1.4) after three immunizations, after 22 days, the immunized eggs were collected after detecting FeChPV antibody titers in the egg yolk liquid of 1:4800.

(2) The preparation method of the FeChPV yolk antibody liquid comprises the steps of mixing the yolk solution with Tris-HCl buffer solution with the volume ratio of 1:1 at the temperature of 7.3, adding polyethylene glycol PEG6000 with the total volume of 2% (W/V), fully shaking and uniformly mixing, centrifuging at the temperature of 4 ℃ for 20min by using a low-temperature refrigerated centrifuge at the temperature of 10000r/min, taking supernatant, filtering by using polypropylene 750B filter cloth, collecting filtrate, filtering by using a filter column filter until liquid is clarified to obtain clarified liquid, adding polyethylene glycol PEG6000 with the total volume of 5% (W/V) into the clarified liquid, shaking and uniformly mixing, standing at room temperature for 30min, centrifuging at the temperature of 4 ℃ by using a low-temperature refrigerated centrifuge at the temperature of 10000r/min, discarding supernatant to obtain precipitate A, dissolving precipitate A with PBS buffer solution with the pH of 10mL of sterile pH7.3 (the precipitate A is dissolved by using PBS buffer solution with the pH of 10mL of sterile pH 7.3), adding PBS with the pH of 10% (W/V) to obtain supernatant, centrifuging at the temperature of 10mL of 10 ℃ for 4 min, centrifuging at the temperature of 50 ℃ for 4 min, and fully dissolving the egg yolk antibody with the pH of 50B with the pH of 10.4 ℃ for 4 min.

The titer of the antibody lyophilized powder in this example was measured by using ELISA antibody assay method, firstly diluting FECHPV VLPS protein to 2. Mu.g/mL with PBS buffer, coating the plate overnight at 4 ℃, washing the plate 3 times with TBST, blocking with 5% skim milk, and allowing the plate to act for 2 hours at 37 ℃, diluting the antibody lyophilized powder with PBS buffer at a mass ratio of PBS buffer to antibody lyophilized powder=50:1, using the non-immunized egg yolk antibody as negative control, allowing each well to act for 1 hour at 100. Mu.L at 37 ℃, adding 100. Mu.L of HRP-labeled rabbit anti-chicken IgY secondary antibody after dilution after washing and drying, allowing each well to act for 1 hour at 37 ℃, and reading OD value at 450nm after washing, drying, color development and termination reaction. ELISA titers were 1:84000.

Example 3

This example uses FECHPV VLPS vaccine prepared in example 1.

(II) preparing an immune auxiliary agent:

Adding 4wt% of Bulbus Allii Macrostemi extract and 4wt% of folium Eucommiae extract into injectable water, and mixing to obtain immune adjuvant.

The preparation method of Bulbus Allii Macrostemi extract comprises pulverizing Bulbus Allii Macrostemi, adding 12 times of water, adjusting pH to 5.0, adding 2% cellulase and 2% pectase, performing enzymolysis at 55deg.C for 3 hr, adjusting pH to 7, adding 1.5% ficin, performing enzymolysis at 65deg.C for 2 hr, inactivating enzyme in boiling water bath for 20min, cooling to room temperature, adding 1.5% Serratase, performing enzymolysis at 50deg.C for 1.5 hr, inactivating enzyme in boiling water bath for 20min, cooling to room temperature, centrifuging at 6000r/min for 15min, collecting supernatant, ultrafiltering with 3kDa ultrafiltration membrane to obtain component smaller than 3kDa, and lyophilizing to constant weight to obtain Bulbus Allii Macrostemi extract.

The preparation method of folium Eucommiae extract comprises pulverizing folium Eucommiae, adding water 12 times of folium Eucommiae, adjusting pH to 5.5, adding cellulase 2% of folium Eucommiae, performing enzymolysis at 60deg.C for 3h, adjusting pH to 7.0, adding Serratia peptidase 1.5% of folium Eucommiae, performing enzymolysis at 50deg.C for 2h, inactivating enzyme in boiling water bath for 20min, cooling to room temperature, adding bromelain 1.5% of folium Eucommiae, performing enzymolysis at 60deg.C for 1.5h, inactivating enzyme in boiling water bath for 20min, cooling to room temperature, adding nattokinase 1.5% of folium Eucommiae, performing enzymolysis at 55deg.C for 80min, inactivating enzyme in boiling water bath for 20min, cooling to room temperature, centrifuging at 6000r/min for 15min, collecting supernatant, ultrafiltering with 5kDa ultrafiltration membrane to obtain component less than 5kDa, and lyophilizing to constant weight to obtain folium Eucommiae extract.

(1.1) basic immunization, namely, carrying out subcutaneous injection inoculation on the neck of each healthy egg-laying hen with FECHPV VLPS mL of vaccine per chicken, and then injecting 2mL of immune auxiliary agent per chicken;

(1.2) secondary immunization, namely, carrying out secondary injection inoculation at 15-day intervals after basic immunization, carrying out injection inoculation FECHPV VLPS vaccine 2 mL/chicken under the skin of the neck, and then injecting immune auxiliary agent 2 mL/chicken;

(1.3) three immunizations, namely, performing a third injection inoculation at 15-day intervals after the second immunization, performing injection inoculation FECHPV VLPS vaccine 2 mL/chicken under the skin of the neck, and then injecting an immune auxiliary agent 2 mL/chicken;

(1.4) after three immunizations, after 15 days, the egg yolk liquid was assayed for FeChPV antibody titers at 1:10000, and immunized eggs were collected.

(2) The preparation method of the FeChPV yolk antibody liquid comprises the steps of mixing the yolk solution with Tris-HCl buffer solution with the volume ratio of 1:5 at the pH of 7.5, adding polyethylene glycol PEG6000 with the total volume of 5% (W/V) after shaking and mixing for 30 seconds, fully shaking and mixing, centrifuging at the temperature of 4 ℃ for 30 minutes by using a low-temperature refrigerated centrifuge under the condition of 12000r/min, collecting supernatant, filtering by using polypropylene 750B filter cloth, collecting filtrate, filtering by using a filter column filter until liquid is clarified to obtain clarified liquid, adding polyethylene glycol PEG6000 with the total volume of 10% (W/V) into the clarified liquid, shaking and mixing, standing for 40 minutes at room temperature, centrifuging at the temperature of 4 ℃ by using a low-temperature refrigerated centrifuge under the condition of 12000r/min, removing the supernatant to obtain precipitate A, dissolving the precipitate A with the sterile PBS buffer solution with the total volume of 5 (10 mL of the precipitate A is dissolved by using the sterile PBS buffer solution with the pH of 10 mL), adding the sterile PBS with the pH of 15% (W/V) to obtain the supernatant, centrifuging at the temperature of 120 ℃ for 5 ℃ after shaking and standing for 40 minutes at the temperature of 120 ℃ under the temperature of 120 ℃ for 4 min, centrifuging to obtain the supernatant with the sterile PBS with the pH of 50 for 5, centrifuging at the temperature of 120 ℃ for 5, and standing for 4 ℃ for 4 min.

The titer of the antibody lyophilized powder in this example was measured by using ELISA antibody assay method, firstly diluting FECHPV VLPS protein to 2. Mu.g/mL with PBS buffer, coating the plate overnight at 4 ℃, washing the plate 3 times with TBST, blocking with 5% skim milk, and allowing the plate to act for 2 hours at 37 ℃, diluting the antibody lyophilized powder with PBS buffer at a mass ratio of PBS buffer to antibody lyophilized powder=50:1, using the non-immunized egg yolk antibody as negative control, allowing each well to act for 1 hour at 100. Mu.L at 37 ℃, adding 100. Mu.L of HRP-labeled rabbit anti-chicken IgY secondary antibody after dilution after washing and drying, allowing each well to act for 1 hour at 37 ℃, and reading OD value at 450nm after washing, drying, color development and termination reaction. ELISA titers were 1:182000.

Comparative example 1

(One) this comparative example employed FECHPV VLPS vaccine prepared in example 1.

(II) preparing immune eggs, feChPV yolk antibody liquid and antibody freeze-dried powder:

(1) Healthy egg laying hens are selected, after isolated feeding and observation for 7 days, FECHPV VLPS vaccine is inoculated for immunization, and the immunization procedure comprises:

(1.1) basic immunization, namely, carrying out subcutaneous injection inoculation FECHPV VLPS vaccine 1.5 mL/chicken on the neck of each healthy egg-laying hen without injection of an immune auxiliary agent;

(1.2) secondary immunization, namely, carrying out secondary injection inoculation at intervals of 14 days after basic immunization, carrying out injection inoculation FECHPV VLPS vaccine 1.5 mL/chicken subcutaneously on the neck, and not injecting an immune auxiliary agent;

(1.3) three immunizations, namely, performing a third injection inoculation at intervals of 14 days after the second immunization, performing injection inoculation FECHPV VLPS vaccine 1.5 mL/chicken subcutaneously on the neck, and not injecting an immune auxiliary agent;

(1.4) after three immunizations, after 20 days, the egg yolk liquid was assayed for FeChPV antibody titers at 1:4000, and immunized eggs were collected.

And (III) potency detection of the antibody freeze-dried powder, namely, diluting FECHPV VLPS protein to 2 mu g/mL by using a PBS buffer solution, coating a plate, standing overnight at 4 ℃, washing the plate 3 times by using TBST, blocking by using 5% skimmed milk, and reacting for 2 hours at 37 ℃, diluting the antibody freeze-dried powder by using the PBS buffer solution according to the mass ratio of PBS buffer solution to antibody freeze-dried powder=50:1, adopting an unimmunized egg yolk antibody as a negative control, reacting for 1 hour at 100 mu L per hole at 37 ℃, adding 100 mu L of the HRP-labeled rabbit anti-chicken IgY secondary antibody after dilution after washing and drying, reacting for 1 hour at 37 ℃, and reading OD value at 450nm after washing, drying and developing and stopping reaction. ELISA titers were 1:60000.

(IV) clinical treatment effect test:

the cure rate of group A is 40% (2/5) within 5 days, and the cure rate is 40% (2/5) within 10 days. The cure rate of group B is 40 (2/5) within 5 days, and 60% (3/5) within 10 days. The cure rate of group C is 20% (1/5) within 5 days, and the cure rate of group C is 40% (2/5) within 10 days. The cure rate of group D is 40% (2/5) in 5 days, 60% (3/5) in 10 days, 60% (3/5) in 5 days, and 60% (3/5) in 5 days. The specific cases are shown in table 2 below.

TABLE 2 progress of symptomatic rehabilitation

The result of comparative example 1 shows that the non-injection of the immune auxiliary agent can affect the reduction of the potency of the antibody freeze-dried powder and the treatment effect is poor.

Comparative example 2

(II) preparing an immune auxiliary agent:

The preparation method of Bulbus Allii Macrostemi extract comprises pulverizing Bulbus Allii Macrostemi, adding 10 times of water, adjusting pH to 4.8, adding cellulase 1.2% of Bulbus Allii Macrostemi and pectase 1.2% of Bulbus Allii Macrostemi, performing enzymolysis at 53deg.C for 2.5 hr, cooling to room temperature, adjusting pH to 6.8, adding Serratinidase 1.0% of Bulbus Allii Macrostemi, performing enzymolysis at 48deg.C for 1 hr, inactivating enzyme in boiling water bath for 18min, cooling to room temperature, centrifuging at 5500r/min for 12min, collecting supernatant, ultrafiltering with 3kDa ultrafiltration membrane to obtain component smaller than 3kDa, and lyophilizing to constant weight to obtain Bulbus Allii Macrostemi extract.

The preparation method of folium Eucommiae extract comprises pulverizing folium Eucommiae, adding water 10 times of folium Eucommiae, adjusting pH to 5.0, adding cellulase 1.2% of folium Eucommiae, performing enzymolysis at 55deg.C for 2.5 hr, adjusting pH to 6.8, adding bromelain 1.0% of folium Eucommiae, performing enzymolysis at 55deg.C for 1 hr, inactivating enzyme in boiling water bath for 18min, cooling to room temperature, centrifuging at 5500r/min for 12min, collecting supernatant, ultrafiltering with 5kDa ultrafiltration membrane to obtain component smaller than 5kDa, and lyophilizing to constant weight to obtain folium Eucommiae extract.

(1.4) after three immunizations, after 18 days, the egg yolk liquid was assayed for FeChPV antibody titers at 1:4400 and the immunized eggs were collected.

And (III) potency detection of the antibody freeze-dried powder, namely, diluting FECHPV VLPS protein to 2 mu g/mL by using a PBS buffer solution, coating a plate, standing overnight at 4 ℃, washing the plate 3 times by using TBST, blocking by using 5% skimmed milk, and reacting for 2 hours at 37 ℃, diluting the antibody freeze-dried powder by using the PBS buffer solution according to the mass ratio of PBS buffer solution to antibody freeze-dried powder=50:1, adopting an unimmunized egg yolk antibody as a negative control, reacting for 1 hour at 100 mu L per hole at 37 ℃, adding 100 mu L of the HRP-labeled rabbit anti-chicken IgY secondary antibody after dilution after washing and drying, reacting for 1 hour at 37 ℃, and reading OD value at 450nm after washing, drying and developing and stopping reaction. ELISA titers were 1:78000.

(IV) clinical treatment effect test:

The cure rate of group A is 40% (2/5) within 5 days, and the cure rate is 60% (3/5) within 10 days. The cure rate of group B is 40 (2/5) within 5 days, and 60% (3/5) within 10 days. The cure rate of group C is 20% (1/5) within 5 days, and the cure rate of group C is 40% (2/5) within 10 days. The cure rate of group D is 60% (3/5) in 5 days, the cure rate of group E is 60% (3/5) in 10 days, and the cure rate of group E is 60% (3/5) in 5 days. The specific cases are shown in table 3 below.

TABLE 3 progress of symptomatic rehabilitation

The result of comparative example 2 shows that the modification of the preparation method of the immune auxiliary agent can affect the reduction of the potency of the antibody freeze-dried powder and the deterioration of the treatment effect.

In the above examples, the enzyme source is cellulase from Shandong Jinda Biotechnology Co., ltd, enzyme activity is 10 ten thousand U/g, pectase from Shandong Yu XingBio technology Co., ltd, enzyme activity is 3 ten thousand U/g, ficin from Nanjing flourishing biological technology Co., ltd, enzyme activity is 10 ten thousand U/g, serratinidase from Jin Huashi KWUFAT Biotechnology Co., enzyme activity is 10 ten thousand U/g, bromelain from Shanxi Shang Nuo reaches biological technology Co., ltd, enzyme activity is 4 ten thousand U/g, nattokinase from Shanxi Sanhe pharmaceutical Co., ltd, and enzyme activity is 4 ten thousand U/g.

Claims

1. The preparation method of the cat Chapahama virus antibody is characterized by comprising the following steps of:

S1, mixing FECHPV VLPS protein and an adjuvant, and performing emulsification treatment to obtain FECHPV VLPS vaccine;

S3, selecting healthy egg laying hens, and inoculating FECHPV VLPS vaccine and immune auxiliary agent for immunization after isolated feeding observation;

s4, sterilizing the collected immune eggs, collecting yolk liquid, preparing FeChPV yolk antibody liquid, preparing FeChPV yolk antibody liquid, mixing the yolk liquid with Tris-HCl buffer solution with the volume ratio of 7.3-7.5 at the speed of 1:1-1:5, adding polyethylene glycol PEG6000 with the total volume of 2-5% (W/V) after shaking and mixing for 20-30 seconds, fully shaking and mixing, centrifuging for 20-30 minutes at 4 ℃ by using a low-temperature refrigerated centrifuge under the conditions of 10000-12000r/min, collecting supernatant, filtering by using polypropylene 750B filter cloth, collecting filtrate, filtering by using a filter column type filter until liquid is clarified, obtaining clarified liquid, adding polyethylene glycol PEG6000 with the total volume of 5-10% (W/V) into the clarified liquid, shaking and mixing, standing at room temperature for 30-40min, centrifuging at 4 ℃ by using a low-temperature refrigerated centrifuge for 20-30min, obtaining precipitate A, centrifuging for obtaining a buffer solution A with the total volume of 2-5% (W/V) at 4 ℃ by using a low-12000 r/min, centrifuging for 20-30min, dissolving in PBS with the buffer solution with the total volume of 5% (PBS) at the temperature of 10000-120 r/min, centrifuging for 20.3-20 min, and standing for 4 ℃ for 20 min, fully dissolving the supernatant by using the buffer solution with the sterile pH of 20-20.5% (PBS) at the temperature of 10000W/V) at the temperature of 20-120 r/min, and shaking for 20 min, and fully centrifuging for obtaining the supernatant.

2. The method for preparing a cat sham virus antibody according to claim 1, wherein in the step S1, the method for preparing FECHPV VLPS protein comprises the following steps:

n2, E.coli induced expression to obtain soluble FeChPV VP1;

N3, after purification, the product is observed by a transmission electron microscope to FeChPV VP to express in escherichia coli to form an icosahedron structure, and FeChPV VP1 is automatically folded and assembled into virus-like particles to obtain FECHPV VLPS protein.

3. The method of claim 1, wherein the amount of VLPs in the FECHPV VLPS vaccine in step S1 is 50-100 μg.

4. The preparation method of the cat-clash hamburger virus antibody according to claim 1, wherein in the step S2, the preparation method of the allium macrostemon extract comprises the following steps of taking allium macrostemon, crushing, adding water for injection, adjusting the pH value to 4.5-5.0, adding cellulase with the weight of 0.5-2% of the allium macrostemon and pectase with the weight of 0.5-2% of the allium macrostemon, performing enzymolysis for 2-3 hours at 50-55 ℃, adjusting the pH value to 6.5-7, adding ficin with the weight of 0.5-1.5% of the allium macrostemon, performing enzymolysis for 1-2 hours at 60-65 ℃, inactivating enzyme for 15-20 minutes in a boiling water bath, adding Serratin enzyme with the weight of 0.5-1.5% of the allium macrostemon after cooling to room temperature, performing enzymolysis for 1-1.5 hours at 45-50 ℃, inactivating enzyme for 15-20 minutes in a boiling water bath, performing centrifugation for 10-15 minutes at 5000-6000r/min after cooling to room temperature, taking 3kDa supernatant fluid, performing ultrafiltration to obtain a freeze-dried extract with the same weight of the allium macrostemon the extract;

The preparation method of the eucommia ulmoides leaf extract comprises the following steps of taking eucommia ulmoides leaves, crushing, adding water for injection, wherein the mass of the water is 8-12 times that of the eucommia ulmoides leaves, adjusting the pH value to 4.5-5.5, adding cellulase, the mass of which is 0.5-2%, carrying out enzymolysis for 2-3 hours at 50-60 ℃, adjusting the pH value to 6.5-7.0, adding Serratinase, the mass of which is 0.5-1.5%, carrying out enzymolysis for 1-2 hours at 45-50 ℃, carrying out boiling water bath enzyme deactivation for 15-20 minutes, cooling to room temperature, adding bromelain, the mass of which is 0.5-1.5%, carrying out enzymolysis for 1.5 hours at 50-60 ℃, carrying out boiling water bath enzyme deactivation for 15-20 minutes, adding nattokinase, the mass of which is 0.5-1.5% of the temperature is 0.5-1.5%, carrying out enzymolysis for 50-80 minutes at 50-55 ℃, carrying out boiling water bath enzyme deactivation for 15-20 minutes, cooling to room temperature, carrying out centrifugation for 10-15 minutes at 5000-6000r/min, carrying out ultrafiltration membrane, and carrying out freeze-drying to obtain an ultra-dried extract, wherein the ultra-dried extract is obtained by adopting the ultra-dried extract of the eucommia ulmoides leaves, and the ultra-dried extract, and the extract is obtained after the freeze-dried extract is subjected to 5 kDa.

5. The method of claim 1, wherein in step S3, the immunization procedure comprises:

s3.1, basic immunization, namely carrying out injection inoculation FECHPV VL PS on neck subcutaneous or chest muscle of each healthy egg-laying hen for 1-2mL of vaccine per chicken, and then injecting 1-2mL of immune auxiliary agent per chicken;

s3.4 after three times of immunization, when the FeChPV antibody titer in the egg yolk liquid is higher than 1:4000, collecting immunized eggs.

6. A feline panhama virus antibody produced by the method of any one of claims 1-5.

7. A pharmaceutical formulation comprising the cat Cha Paha Ma Bingdu antibody of claim 6 and a pharmaceutically acceptable adjunct ingredient.

8. The pharmaceutical formulation of claim 7, wherein the pharmaceutical formulation is administered orally or by injection.

9. Use of the feline Cha Paha Ma Bingdu antibody of claim 6 in the manufacture of a medicament for preventing, ameliorating, adjunctively treating, or treating an animal disorder caused by equine viral infection in a cat Cha Paha.

10. Use of a pharmaceutical formulation according to claim 7 or 8 for the manufacture of a medicament for the prevention, alleviation, co-treatment or treatment of an animal disease caused by equine viral infection in cat Cha Paha.