Quadruple egg yolk antibody for cats as well as preparation method and application of quadruple egg yolk antibody

Abstract

The invention relates to the technical field of antibodies, in particular to a quadruple egg yolk antibody for cats as well as a preparation method and application of the quadruple egg yolk antibody. The quadruple egg yolk antibody for cats, provided by the invention, comprises egg yolk antibodies aiming at four viruses, namely cat parvovirus, cat herpes virus type I, cat calicivirus and cat coronavirus, can specifically neutralize the viruses, has relatively high neutralizing antibody titer, and can be used for preventing and treating infection of the viruses and diseases caused by the infection. The quadruple egg yolk antibody for cats provided by the invention has the advantages that the preparation method is simple and efficient, the egg yolk antibody is high in yield and purity, the prepared antibody is high in titer and low in cost, large-scale production is easy to realize and the like, and the quadruple egg yolk antibody has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of antibodies, and in particular to a cat-use quadruple yolk antibody and a preparation method and application thereof. Background Art

[0002] Feline parvovirus (FPV) causes feline distemper, also known as feline panleukopenia, feline infectious enteritis, etc. The main clinical features are sudden high fever, vomiting, diarrhea and severe leukopenia. It is the most common acute, highly contagious and fatal infectious disease in domestic cats. FPV is a single-stranded DNA virus that can infect a variety of animals such as cats, raccoons and mustelids under natural conditions. Among them, small-sized cats and minks are the most susceptible. FPV can be transmitted in many ways, such as direct or indirect contact infection. Infected animals excrete toxins through excretions and secretions such as feces, urine, vomitus, and saliva, contaminating the environment, utensils, feed, etc. FPV infection is often endemic, with a mortality rate of 60% to 70%, and up to more than 90%, which is extremely harmful to young felines.

[0003] Feline rhinotracheitis, also known as infectious rhinotracheitis or viral rhinotracheitis, is caused by Felineherpesvirus type 1 (FHV 1) and is generally transmitted through the respiratory and digestive tracts. FHV 1 is a double-stranded DNA virus that is highly contagious, has an acute onset, and is highly contagious through contact.

[0004] Feline calicivirus infection is a multiple oral and respiratory disease of cats caused by feline calicivirus (FCV). The main symptoms include oral ulcers, fever and sneezing. It has a high mortality rate and seriously endangers the health of infected animals.

[0005] Feline coronavirus (FCoV) is a virus that is widely prevalent in cats. There are two biotypes: feline infectious peritonitis virus (FIPV) and feline enteric coronavirus (FECV). FECV usually only causes mild enteritis with mild symptoms; while FIPV is a fatal disease of domestic and wild cats, with a high mortality rate, a wide range of prevalence and high contagiousness. There is currently no vaccine or specific medicine for FIPV, which seriously affects the health of pet cats.

[0006] In pet clinical medicine, cats are often cross-infected with the above four infectious diseases, and usually have secondary bacterial infections with complex symptoms. However, the prior art lacks specific antiviral drugs for the above four viruses. Once infected, symptomatic treatment or antibiotics are often used for treatment, but the treatment effect and specificity are poor. If passive immunization is used, the cost of antiserum and monoclonal antibodies is high and the sources are limited, which currently cannot meet clinical needs. Compared with antiserum and monoclonal antibodies, the preparation of yolk antibodies (IgY) against the above four viruses by immunizing laying hens has obvious advantages in the prevention and treatment of these viral infections and their related diseases. Summary of the invention

[0007] The invention provides a quadruple yolk antibody for cats and a preparation method and application thereof.

[0008] As for egg yolk antibodies for cats, currently, inactivated virus liquid or inactivated vaccine prepared therefrom is used as immunogen to immunize laying hens and harvest egg yolk antibodies. However, the cultivation and preparation of virus liquid have high requirements on conditions and qualifications, and it is difficult to achieve large-scale production. In addition, using virus liquid as an immunogen will cause egg yolk antibodies to have certain safety hazards. The present invention uses viral antigen proteins as immunogens to develop egg yolk antibodies for cats. By screening a large number of antigen proteins of feline parvovirus, feline herpesvirus type I, feline calicivirus and feline coronavirus, the optimal antigen protein sequence is determined. Using these antigen proteins as immunogens can significantly improve the specific neutralizing antibody titer of the obtained egg yolk antibodies.

[0009] Specifically, the present invention provides the technical solution described below.

[0010] In a first aspect, the present invention provides a method for preparing a quadruple yolk antibody for cats, the method comprising: immunizing laying hens with feline parvovirus capsid protein VP2, feline herpesvirus type I gD protein, feline calicivirus capsid protein VP1, and feline coronavirus spike protein S truncated form as immunogens, and separating yolk or yolk antibodies; Among them, the amino acid sequence of the feline parvovirus capsid protein VP2 is shown in SEQ ID NO.1; the amino acid sequence of the feline herpesvirus type I gD protein is shown in SEQ ID NO.4; the amino acid sequence of the feline calicivirus capsid protein VP1 is shown in SEQ ID NO.5; and the amino acid sequence of the feline coronavirus spike protein S truncate is shown in SEQ ID NO.8.

[0011] For the antigenic proteins of feline parvovirus, feline herpesvirus type I, feline calicivirus and feline coronavirus, the present invention screens and immunogenicity verifies different types of antigenic proteins and different antigenic protein regions and truncated proteins of different lengths, and finally selects four proteins, namely, feline parvovirus capsid protein VP2, feline herpesvirus type I gD protein, feline calicivirus capsid protein VP1 and feline coronavirus spike protein S truncated body, as antigenic proteins. By comparison, it is found that the antigenic proteins using the above amino acid sequences can more efficiently induce laying hens to produce specific neutralizing antibodies against the above viruses, significantly improve the titer of specific neutralizing antibodies, and the obtained yolk antibodies have good preventive and therapeutic effects on feline parvovirus, feline herpesvirus type I, feline calicivirus and feline coronavirus.

[0012] Preferably, the above four antigenic proteins are used separately as immunogens to immunize laying hens to obtain egg yolk antibodies against the four viruses, and then the egg yolk antibodies against the four viruses are mixed to prepare quadruple yolk antibodies.

[0013] Specifically, feline parvovirus capsid protein VP2, feline herpesvirus type I gD protein, feline calicivirus capsid protein VP1 and feline coronavirus spike protein S truncation were used as immunogens to immunize laying hens respectively, and after separating the four yolk antibodies obtained by immunizing laying hens with the four immunogens respectively, the four yolk antibodies were mixed.

[0014] Alternatively, laying hens are immunized with feline parvovirus capsid protein VP2, feline herpesvirus type I gD protein, feline calicivirus capsid protein VP1 and feline coronavirus spike protein S truncation as immunogens, respectively, and egg yolks containing yolk antibodies against feline parvovirus capsid protein VP2, feline herpesvirus type I gD protein, feline calicivirus capsid protein VP1 and feline coronavirus spike protein S truncation are isolated, and the four types of egg yolks are mixed.

[0015] The present invention finds that, compared with mixing four antigen proteins as immunogens to immunize laying hens, immunizing the four antigen proteins separately to prepare egg yolk antibodies and then mixing them is more conducive to improving the titer of the produced egg yolk antibodies.

[0016] Preferably, the mass ratio of the egg yolk mixture containing egg yolk antibodies against feline parvovirus capsid protein VP2, feline herpesvirus type I gD protein, feline calicivirus capsid protein VP1 and feline coronavirus spike protein S truncation is (0.8-1.2):(1.8-2.2):(3.8-4.2):(2.8-3.2).

[0017] Preferably, the four types of egg yolks are respectively mixed with freeze-drying protective agents and then freeze-dried to obtain egg yolk powders, and then the four types of egg yolk powders are mixed.

[0018] In the above method, the preparation method of the immunogen includes: connecting the coding genes of feline parvovirus capsid protein VP2, feline herpesvirus type I gD protein, feline calicivirus capsid protein VP1 and feline coronavirus spike protein S truncated body to expression vectors respectively, constructing expression vectors of four immunogens, introducing the expression vectors into Escherichia coli, obtaining recombinant Escherichia coli expressing the four immunogens respectively, culturing the recombinant Escherichia coli, and isolating feline parvovirus capsid protein VP2, feline herpesvirus type I gD protein, feline calicivirus capsid protein VP1 and feline coronavirus spike protein S truncated body from the culture.

[0019] Preferably, the gene encoding the feline parvovirus capsid protein VP2 is shown as SEQ ID NO.9; the gene encoding the feline herpesvirus type I gD protein is shown as SEQ ID NO.12; the gene encoding the feline calicivirus capsid protein VP1 is shown as SEQ ID NO.13; the gene encoding the feline coronavirus spike protein S truncation is shown as SEQ ID NO.16.

[0020] The above coding gene sequence is a sequence obtained by codon optimization, which is beneficial to increase the expression amount of each antigen protein in Escherichia coli and improve the preparation efficiency of the immunogen.

[0021] Preferably, the expression vector is pET28a. The Escherichia coli is BL21 (DE3) strain.

[0022] The above antigen protein is expressed in the form of inclusion bodies, and therefore, the method for isolating the antigen protein from the culture includes the steps of inclusion body renaturation and antigen protein purification. Preferably, the antigen protein purification is carried out using an affinity chromatography column, and the inclusion body is first renatured before purification by the affinity chromatography column; after purification by affinity chromatography, the purified antigen protein, i.e., the immunogen, is obtained by desalting. Preferably, urea is used for inclusion body renaturation, and preferably 8M, 4M and 2M urea are used to treat the inclusion body in sequence. After inclusion body renaturation, affinity chromatography purification is preferably performed using a Ni column.

[0023] In the above method, the immunization method includes: emulsifying feline parvovirus capsid protein VP2, feline herpesvirus type I gD protein, feline calicivirus capsid protein VP1 and feline coronavirus spike protein S truncated form with adjuvants respectively, and immunizing laying hens respectively (i.e., primary immunization), performing a first booster immunization 12-15 days after the primary immunization, and performing a second booster immunization 12-15 days later.

[0024] Preferably, the concentration of the immunogen used in the immunization is 1-3 mg / mL, and the volume ratio of the immunogen to the adjuvant emulsification is 1:1~3:7; wherein, the dose of the primary immunization is 400-600 μg of immunogen / laying hen, and the adjuvant used is Freund's complete adjuvant; the immunization dose of the first booster immunization and the second booster immunization is 200-400 μg of immunogen / laying hen, and the adjuvant used is Freund's incomplete adjuvant.

[0025] Preferably, the laying hens are 6-8 months old.

[0026] Preferably, the immunization injection method is 6-10 point injection in the muscle.

[0027] In the above method, the method for separating yolk antibodies comprises: firstly extracting yolk antibodies from immune chicken eggs by water dilution method, and then performing ethanol precipitation treatment.

[0028] The water dilution method for extracting yolk antibodies comprises: separating yolk from collected immune chicken eggs, diluting with deionized water (preferably in an amount of 8-10 times the volume), adjusting the pH value to 5.0-5.4, standing at 3-5°C for 1.5-2.5h, centrifuging to remove precipitation, and retaining the supernatant.

[0029] The ethanol precipitation treatment comprises: mixing the supernatant with ice ethanol to make the final concentration of ethanol 55-65%, slowly stirring at 3-5°C for 15-40 minutes, centrifuging and discarding the supernatant, dissolving the precipitate with saline, adding ice ethanol again to make the final concentration 20-30%, stirring at 3-5°C for 3-30 minutes, centrifuging and collecting the precipitate, dissolving the precipitate with saline or water, and then obtaining a higher purity egg yolk antibody through DEAE ion exchange.

[0030] In a second aspect, the present invention provides a tetravalent egg yolk antibody for cats prepared by the method described above.

[0031] In the present invention, the quadruple yolk antibody for cats includes yolk antibody of feline parvovirus, yolk antibody of feline herpesvirus type I, yolk antibody of feline calicivirus and yolk antibody of feline coronavirus.

[0032] Preferably, the cat quadruple yolk antibody is a cat quadruple yolk antibody yolk powder, wherein the mass ratio of the yolk containing specific feline parvovirus capsid protein VP2 yolk antibody, the yolk containing specific feline herpesvirus type I gD protein yolk antibody, the yolk containing specific feline calicivirus capsid protein VP1 yolk antibody and the yolk containing specific feline coronavirus yolk antibody is (0.8-1.2): (1.8-2.2): (3.8-4.2): (2.8-3.2).

[0033] Preferably, egg yolk containing specific feline parvovirus capsid protein VP2 yolk antibody, egg yolk containing specific feline herpesvirus type I gD protein yolk antibody, egg yolk containing specific feline calicivirus capsid protein VP1 yolk antibody and egg yolk containing specific feline coronavirus yolk antibody are mixed with a lyophilization protectant respectively, and then freeze-dried to obtain egg yolk powder, and then the four kinds of egg yolk powder are mixed in a ratio of (0.8-1.2): (1.8-2.2): (3.8-4.2): (2.8-3.2). The lyophilization protectant includes sucrose, trehalose and gelatin, and the mass ratio thereof is (8-12): (28-32): (8-12). The mass ratio of the egg yolk to the lyophilization protectant is 1000: (45-55).

[0034] In a third aspect, the present invention provides the use of the above-mentioned quadruple yolk antibody for cats in the preparation of a product for preventing and / or treating a cat viral infection or a disease caused by the viral infection; The virus is one or more selected from feline parvovirus, feline herpesvirus type I, feline calicivirus and feline coronavirus.

[0035] Preferably, the product is a medicine.

[0036] In a fourth aspect, the present invention provides a pharmaceutical composition comprising the above-mentioned tetravalent egg yolk antibody for cats.

[0037] In a fifth aspect, the present invention provides a method for preventing and / or treating a feline viral infection or a disease caused by the viral infection, the method comprising: administering an effective dose of the above-mentioned cat tetravalent egg yolk antibody to a test cat; The virus is one or more selected from feline parvovirus, feline herpesvirus type I, feline calicivirus and feline coronavirus.

[0038] The beneficial effects of the present invention include at least: the cat-use quadruple yolk antibody provided by the present invention includes antibodies against four viruses, namely, feline parvovirus, feline herpesvirus type I, feline calicivirus and feline coronavirus, which can specifically neutralize the above viruses, have a high neutralizing antibody titer, and can be used to prevent and treat the infection of the above viruses and the diseases caused by the infection, and have a high protection efficiency. The cat-use quadruple yolk antibody provided by the present invention has the advantages of simple and efficient preparation method, and can prepare four kinds of yolk antibodies with high antibody titers in one preparation process, high yield of yolk antibodies, high purity, high antibody titer, low cost, easy to achieve large-scale production, etc., has a wide range of application prospects, is conducive to meeting animal welfare and increasing the resistance requirements of sick cats, and is of great significance for overcoming the problems of poor therapeutic effect and treatment specificity, drug resistance and toxic side effects in existing treatment methods for feline plague, feline rhinotracheitis, feline calicivirus and feline coronavirus infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0040] Figure 1 It is a colony PCR verification diagram of the engineering strain in Example 1 of the present invention; wherein, M: 1kb DNA marker; the remaining lanes are agarose gel electrophoresis results of PCR verification using SEQ ID NO.33 and SEQ ID NO.34, SEQ ID NO.35 and SEQ ID NO.36, SEQ ID NO.37 and SEQ ID NO.38, SEQ ID NO.39 and SEQ ID NO.40, SEQ ID NO.41 and SEQ ID NO.42, SEQ ID NO.43 and SEQ ID NO.44, SEQ ID NO.45 and SEQ ID NO.46, SEQ ID NO.47 and SEQ ID NO.48 as primers, respectively.

[0041] Figure 2 It is the SDS-PAGE verification diagram of VP2, NS1, gB, gD, VP1, VP1 truncation, S and S truncation antigen proteins in Example 3 of the present invention; M: protein Marker, lane A: eluate after VP2 affinity chromatography; lane B: eluate after NS1 affinity chromatography; lane C: eluate after gB affinity chromatography; lane D: eluate after gD affinity chromatography; lane E: eluate after VP1 affinity chromatography; lane F: eluate after tVP1 affinity chromatography; lane G: eluate after S affinity chromatography; lane H: eluate after tS affinity chromatography.

[0042] Figure 3 It is a diagram of DEAE ion exchange and SDS-PAGE analysis in IgY purification in Example 5 of the present invention; wherein, A: Peak 1: NaCl concentration is 100mM, Peak 2: NaCl concentration is 200mM; B: M: protein Marker, Lane 1: loading solution, Lane 2: Peak 1 eluate, Lane 3: Peak 2 eluate. DETAILED DESCRIPTION

[0043] The present invention provides a method for preparing a quadruple yolk antibody for cats, the method comprising: firstly, heterologously expressing feline parvovirus capsid protein VP2 (SEQ ID NO.1), feline herpesvirus type I gD protein (SEQ ID NO.4), feline calicivirus capsid protein VP1 (SEQ ID NO.5), and feline coronavirus spike protein S truncated body (SEQ ID NO.8) by using a prokaryotic expression system. Then, the purified antigen proteins are emulsified with adjuvants, and immunized with 6-8 month old laying hens respectively; a total of 3 immunizations are performed, and the first booster immunization is performed two weeks after the initial immunization, and the second booster immunization is performed two weeks after the initial immunization.

[0044] One week after the second booster immunization, take one collected egg, crack the shell, remove the egg white and yolk membrane, and collect the yolk liquid; dilute the yolk liquid 8-10 times with sterile water, adjust the pH to 5.0-5.4 with HCl, and obtain the yolk water-soluble substance after centrifugation, and then obtain the yolk antibody with a purity greater than 95% after ethanol precipitation and DEAE ion exchange.

[0045] In some specific embodiments of the present invention, expression plasmids containing gene sequences encoding VP2 of FPV, gD of FHV 1, VP1 of FCV, and S protein truncations of FCoV are constructed by genetic engineering methods, expressed in Escherichia coli, and then the expression products are separated and purified as immunogens, mixed and emulsified with adjuvants, and healthy laying hens are immunized, eggs are collected, and yolk antibodies in the egg yolks are extracted. The antibodies are natural immunoglobulins and are easy to use, highly effective, and non-toxic.

[0046] In some specific embodiments of the present invention, the method for constructing a recombinant antigen protein expression vector, transforming and inducing the expression of a target protein comprises the following steps: (1) inserting the gene fragments of VP2 of FPV, gD of FHV 1, VP1 of FCV and S protein truncated form of FCoV into the multiple cloning site of the expression vector pET28a, respectively, to construct recombinant expression vector plasmids pET28a-VP2, pET28a-gD, pET28a-VP1 and pET28a-tS; (2) transferring the recombinant expression vector plasmid constructed in (1) into the host cell E. coli BL21 (DE3) to obtain a recombinant strain; (3) inducing the recombinant strain to express the four antigen proteins, namely, VP2 protein, gD protein, VP1 protein and S truncated form protein. (4) purifying the four antigen proteins and emulsifying them with adjuvants in a certain proportion, and immunizing 6-8 month old laying hens respectively after emulsification; a total of three immunizations are performed, with the first booster immunization being performed two weeks after the first immunization, and the second booster immunization being performed two weeks after the first immunization. (5) Isolate and purify IgY antibodies, and measure the neutralizing titer of IgY against FPV, FHV1, FCV, and FCoV, respectively.

[0047] In a specific embodiment of the present invention, the optimal antigen protein is finally determined by screening 8 antigen proteins including VP2 protein, NS1 protein, gB protein, gD protein, VP1 protein, VP1 truncated protein, S protein and S truncated protein.

[0048] In some specific embodiments of the present invention, the eight antigenic proteins, namely, VP2 protein, NS1 protein, gB protein, gD protein, VP1 protein, VP1 truncated protein, S protein and S truncated protein, are the proteins described in GenBank: AAC37929.1, GenBank: WQF67429.1, GenBank: QQG63349.1, GenBank: AAB30980.1, GenBank: CAA77636.1 and GenBank: ACS44218.1, respectively, or are truncated proteins of these proteins.

[0049] In some specific embodiments of the present invention, the amino acid sequences of the eight antigenic proteins, namely, VP2 protein, NS1 protein, gB protein, gD protein, VP1 protein, VP1 truncated protein, S protein and S truncated protein, are shown as SEQ ID NO.1-8, respectively.

[0050] In some specific embodiments of the present invention, the nucleotide sequences of the genes encoding VP2, NS1, gB, gD, VP1, VP1 truncation, S and S truncation proteins are shown as SEQ ID NOs. 9-16, respectively.

[0051] In some specific embodiments of the present invention, the method of transforming the expression plasmid containing VP2, NS1, gB, gD, VP1, VP1 truncation, S and S truncation antigen protein gene sequences into Escherichia coli BL21 (DE3) includes: taking out the competent state from a -80°C refrigerator and placing it in an ice bath to thaw it, and after thawing, adding the above 50ng of plasmid to the competent state; heat shock in a 42°C water bath for 90s, and then placing on ice for 2min; adding 1mL LB to culture, and shaking and culturing at 37°C and 220rpm for 60min; taking 100μL of bacterial solution and applying it to an agarose culture plate containing ampicillin resistance.

[0052] In some specific embodiments of the present invention, the inducible expression method of VP2, NS1, gB, gD, VP1, VP1 truncated, S and S truncated antigen proteins comprises: picking up the constructed BL21-VP2, BL21-NS1, BL21-gB, BL21-gD, BL21-VP1, BL21-tVP1, BL21-S and BL21-tS engineering bacteria single colonies respectively into 5 mL LB medium and culturing at 37°C overnight, transferring to fresh TB medium at a 2% inoculum amount, and culturing at 37°C until OD 600 After the pH value is 0.6-0.8, the expression is induced at 16°C for 18-22 hours. After the induction, the cells are collected by centrifugation at 8000 rpm for 20 minutes.

[0053] In some specific embodiments of the present invention, the purification of the above eight antigenic proteins comprises the following steps: (1) collecting the bacteria and disrupting the cells after inducing expression, and obtaining a cell disrupted liquid precipitate by high-speed centrifugation; (2) using an inclusion body purification method combined with affinity column chromatography purification, and performing desalting after purification to obtain recombinant VP2, NS1, gB, gD, VP1, VP1 truncation, S and S truncation protein eluates, respectively.

[0054] In some specific embodiments of the present invention, the collected bacteria are ultrasonically disrupted, and then the precipitate is collected by centrifugation. The purified protein solution is treated with a desalting column according to the inclusion body purification method and combined with Ni-NTA resin purification. The obtained protein solution is concentrated by ultrafiltration to obtain purified VP2, NS1, gB, gD, VP1, VP1 truncation, S and S truncation antigen proteins, which are the immunogens.

[0055] In some specific embodiments of the present invention, the immunization includes: (1) primary immunization: the purified antigen protein (concentration 1.5-2.5 mg / mL) is emulsified with Freund's complete adjuvant at a volume ratio of 1:1, and the injection amount for each laying hen is 500 μg of antigen protein; (2) the first booster immunization is performed two weeks after the primary immunization, and the second booster immunization is performed two weeks after the primary immunization: the desalted antigen protein (concentration 1.5-2.5 mg / mL) is emulsified with Freund's incomplete adjuvant at a volume ratio of 1:1, and the injection amount for each laying hen is 300 μg of antigen protein.

[0056] In some specific embodiments of the present invention, healthy laying hens aged 6-8 months are selected for isolation and rearing and the immunogen is injected into 6-10 points of the muscle.

[0057] In some specific embodiments of the present invention, the purification of yolk antibodies (IgY) comprises the following steps: disinfecting eggs with 75% ethanol, breaking the eggshell with sterilized tweezers, collecting the yolk, and dissolving the yolk with 9 volumes of sterile ddH2O at pH 5.2, standing at 4°C for 2 hours, centrifuging and discarding the precipitate, precipitating IgY in the supernatant with two steps of ethanol, resuspending the obtained IgY precipitate with 0.028M NaCl solution, and further purifying IgY with DEAE again, so that the purity of the final IgY is greater than 95%.

[0058] In some specific embodiments of the present invention, the two-step ethanol precipitation method of IgY includes: mixing the supernatant and ethanol in a volume ratio of 4:6, slowly stirring at 4°C for 15-40 minutes, centrifuging and discarding the supernatant, dissolving the precipitate with saline, adding ethanol again to a final concentration of 20-30%, slowly stirring at 4°C for 3-30 minutes, centrifuging and discarding the supernatant, dissolving the precipitate with saline or water, and then obtaining a higher purity egg yolk antibody through DEAE ion exchange.

[0059] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0060] The homologous recombination enzymes used in the following examples were purchased from Beijing Quanshijin Biotechnology Co., Ltd. + Resins were purchased from Sigma, and other reagents not specifically indicated were purchased from Shanghai Sangon Biotechnology Co., Ltd. Gene and primer synthesis were from Beijing Bioengineering Biotechnology Co., Ltd. Experimental procedures that were not described in detail were performed according to laboratory manuals such as Molecular Cloning.

[0061] The nucleotide sequences of the primers used in the following examples are shown in Table 1.

[0062] Table 1

[0063] Example 1 Construction of expression plasmids and engineering strains of 8 antigenic proteins 1. First, according to the amino acid sequences of the antigenic proteins screened and determined by the present invention: feline parvovirus capsid protein VP2 (SEQ ID NO.1), feline parvovirus functional protein NS1 (SEQ ID NO.2), feline herpesvirus type I gB protein (SEQ ID NO.3), feline herpesvirus type I gD protein (SEQ ID NO.4), feline calicivirus capsid protein VP1 (SEQ ID NO.5), feline calicivirus capsid protein VP1 truncated form (tVP1, SEQ ID NO.6), feline coronavirus spike protein S (SEQ ID NO.7) and feline coronavirus spike protein S truncated form (tS, SEQ ID NO.8), the codon preference of Escherichia coli expression was optimized, and the VP2, NS1, gB, gD, VP1, tVP1, S and tS gene fragments were synthesized by a gene company, and the sequences were SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.21, SEQ ID NO.22, SEQ ID NO.23, SEQ ID NO.24, SEQ ID NO.25, SEQ ID NO.26, SEQ ID NO.27, SEQ ID NO.28, SEQ ID NO.29, SEQ ID NO.30, SEQ ID NO.31, SEQ ID NO.32, SEQ ID NO.33, SEQ ID NO.34, SEQ ID NO.35 As shown in ID NO.16, the restriction sites at both ends of the above-mentioned target gene fragment are NdeI and XhoI respectively.

[0064] 2. Using the plasmid of the synthetic gene fragment as a template, the upstream and downstream primers corresponding to each gene fragment were NdeI-VP2-F (SEQ ID NO.17) and XhoI-VP2-R (SEQ ID NO.18), NdeI-NS1-F (SEQ ID NO.19) and XhoI-NS1-R (SEQ ID NO.20), NdeI-gB-F (SEQ ID NO.21) and XhoI-gB-R (SEQ ID NO.22), NdeI-gD-F (SEQ ID NO.23) and XhoI-gD-R (SEQ ID NO.24), NdeI-VP1-F (SEQ ID NO.25) and XhoI-VP1-R (SEQ ID NO.26), NdeI-tVP1-F (SEQ ID NO.27) and XhoI-tVP1-R (SEQ ID NO.28), NdeI-SF (SEQ ID NO.29) and XhoI-SR (SEQ ID NO.30). NO.30), NdeI-tS-F (SEQ ID NO.31) and XhoI-tS-R (SEQ ID NO.32). VP2, NS1, gB, gD, VP1, VP1 truncation, S and S truncation gene fragments were amplified, and the PCR products were recovered after agarose gel electrophoresis.

[0065] Use NdeI and XhoI to double-digest the pET28a expression vector plasmid and the gel recovery products VP2, NS1, gB, gD, VP1, VP1 truncated, S and S truncated gene fragments, respectively. After gel recovery, use Solution I ligase to connect the vector and the target gene fragment. After reacting at 16℃ for 4-6h, add the ligation product to E. coli DH5α competent cells, ice bath for 30min, react at 42℃ for 1min 30s, ice bath for 2min, add 1mL LB liquid culture medium, incubate at 250rpm, and incubate at 37℃ for 1h. Collect the bacteria by centrifugation at 4000rpm, remove 800μL of supernatant and resuspend the bacteria, apply ampicillin-resistant LB culture medium plates, and culture at 37℃ overnight. Pick a single colony into 5mL LB liquid culture medium and culture at 250rpm and 37℃ overnight.

[0066] For the above single colonies, the primers shown in the sequences of SEQ ID NO.33 and SEQ ID NO.34, SEQ ID NO.35 and SEQ ID NO.36, SEQ ID NO.37 and SEQ ID NO.38, SEQ ID NO.39 and SEQ ID NO.40, SEQ ID NO.41 and SEQ ID NO.42, SEQ ID NO.43 and SEQ ID NO.44, SEQ ID NO.45 and SEQ ID NO.46, SEQ ID NO.47 and SEQ ID NO.48 were used for colony PCR identification (the length of the identified gene fragments was 250 bp). After the positive clones were sequenced and analyzed, the results were as follows: Figure 1 The positive transformant plasmids with correct sequencing were extracted and used, and were named pET28a-VP2, pET28a-NS1, pET28a-gB, pET28a-gD, pET28a-VP1, pET28a-tVP1, pET28a-S and pET28a-tS, respectively.

[0067] 3. Obtaining engineered strains for expressing VP2, NS1, gB, gD, VP1, VP1 truncation, S and S truncation antigen proteins.

[0068] The correctly sequenced pET28a-VP2, pET28a-NS1, pET28a-gB, pET28a-gD, pET28a-VP1, pET28a-tVP1, pET28a-S and pET28a-tS expression vector plasmids were transformed into the Escherichia coli expression host BL21 (DE3), and the transformants were verified by colony PCR. Finally, the expression of feline parvovirus capsid protein VP2 (SEQ ID NO.1), feline parvovirus functional protein NS1 (SEQ ID NO.2), feline herpesvirus type I gB protein (SEQ ID NO.3), feline herpesvirus type I gD protein (SEQ ID NO.4), feline calicivirus capsid protein VP1 (SEQ ID NO.5), feline calicivirus capsid protein VP1 truncated form (SEQ ID NO.6), feline coronavirus spike protein S (SEQ ID NO.7) and feline coronavirus spike protein S truncated form (SEQ ID NO.8) were obtained. NO.8) engineered strains BL21-VP2, BL21-NS1, BL21-gB, BL21-gD, BL21-VP1, BL21-tVP1, BL21-S and BL21-tS.

[0069] Example 2 Induced expression and purification of 8 antigenic proteins 1. Inducible expression of VP2, NS1, gB, gD, VP1, VP1 truncated form, S and S truncated form antigenic proteins Pick out the constructed single colonies of BL21-VP2, BL21-NS1, BL21-gB, BL21-gD, BL21-VP1, BL21-tVP1, BL21-S and BL21-tS engineering strains respectively and culture them in 5 mL LB medium at 37°C overnight, transfer them to fresh TB medium at a 2% inoculum volume, and culture them at 37°C until OD 600 After the concentration of 0.6-0.8, the expression was induced at 16°C for 18-22 hours. Finally, the cells were collected by centrifugation at 8000 rpm for 20 minutes.

[0070] 2. Purification of VP2, NS1, gB, gD, VP1, VP1 truncated form, S and S truncated form antigenic proteins (1) Resuspend the collected cells in 10 mM PB (pH 7.4) (add 20 mL PB to 1 g of wet cells), then disrupt the cells by ultrasonication at 35% power for 30 min in an ice bath.

[0071] (2) After ultrasonic disruption, centrifuge at 12000 rpm for 30 min, collect the precipitate and supernatant, and take 40 μL of each sample for SDS-PAGE analysis.

[0072] (3) Resuspend the pellet in Buffer A (10 mM PB, 8 M urea), place in an ice bath, add a rotor, and incubate at 220 rpm for 60 min.

[0073] (4) After incubation for 60 min, slowly add an equal volume of PB solution to dilute the original 8 M urea to 4 M and stir for 10 min; use the same method to dilute the urea concentration to 2 M.

[0074] (5) Centrifuge the diluted solution at 12,000 rpm for 30 min and collect the supernatant by filtering it through a 0.22 μM sterile membrane.

[0075] (6) Column equilibration: Load 3 mL of filler into the NTA-Ni column and rinse with 10 column volumes of water; then equilibrate the column with 5 column volumes of Buffer B (10 mM PB, 2 M urea).

[0076] (7) Loading: Slowly add the supernatant after passing through the membrane into the purification column.

[0077] (8) Wash the column with Buffer C (10 mM PB, 10 mM imidazole) for 10 column volumes.

[0078] (9) For the first elution, use Buffer D (10 mM PB, 10 mM imidazole) to dissolve the target protein and take 40 μL of sample for SDS-PAGE analysis.

[0079] (10) Elution for the second time: Use Buffer E (10 mM PB, 500 mM imidazole) to dissolve the target protein and take 40 μL sample for SDS-PAGE analysis.

[0080] (11) After use, wash the column with 10 times the column volume of water and then store the column in 20% ethanol.

[0081] 3. Desalting of recombinant proteins The desalting column was balanced with 4 column volumes of PBS (pH 7.4) buffer; the protein eluate prepared in step 2 was loaded on the desalting column at 30% of the column volume, and then eluted with PBS and collected by an AKT automatic sample collector. After desalting, the desalting column was treated with 0.5M NaOH solution, and finally the alkaline solution in the desalting column was washed clean with PBS and stored.

[0082] Example 3 Identification of VP2, NS1, gB, gD, VP1, VP1 truncated form, S and S truncated form antigenic proteins The purity and molecular weight of the VP2, NS1, gB, gD, VP1, VP1 truncated form, S and S truncated form antigen proteins prepared in step 3 of Example 2 were determined by 10% SDS-PAGE electrophoresis. The results showed that ( Figure 2 ), at 50 kDa ( Figure 2 Lane A), 80 kDa ( Figure 2 Lane B), 100 kDa ( Figure 2 Lane C), 45 kDa ( Figure 2 Lane D), 80 kDa ( Figure 2 Lane E), 20 kDa ( Figure 2 Lane F), 175 kDa ( Figure 2 G lane) and 35 kDa ( Figure 2 There is an obvious band near the H lane, which is consistent with the molecular weight of the target protein. The protein concentration was measured by Nanodrop, and it was finally calculated that 1L of bacterial solution could produce about 78mg, 37mg, 18mg, 65mg, 86mg, 30mg, 12mg and 82mg of protein.

[0083] Example 4 Antigen emulsification and immunization Select healthy 180-day-old laying hens for isolation and rearing. Primary immunization: Use a 30kDa ultrafiltration tube to concentrate the desalted VP2, NS1, gB, gD, VP1, VP1 truncated, S and S truncated antigen proteins in Example 2 to a concentration of 2 mg / mL, use a medical tee, and emulsify them with Freund's complete adjuvant in a volume ratio of 1:1. Push and pull the two syringes repeatedly, push and pull for 10 minutes every 2 hours, and push and pull 5 times in total to completely emulsify the antigen. Select 6-10 sites for injection in the muscle of the chicken wing, and the injection amount for each laying hen is 500 μg of antigen protein.

[0084] The first booster immunization and the second booster immunization: Use a 30kDa ultrafiltration tube to concentrate the VP2, NS1, gB, gD, VP1, VP1 truncated, S and S truncated antigen proteins after desalting in Example 2 to a concentration of 1.6 mg / mL, use a medical tee, and emulsify with Freund's incomplete adjuvant at a volume ratio of 1:1, push and pull the two syringes repeatedly, push and pull for 10 minutes every 2 hours, and push and pull 5 times in total to completely emulsify the antigen. Select 6-10 sites to be injected in the muscle of the chicken wing, and the injection amount for each laying hen is 400 μg of antigen protein; the first booster immunization is carried out 2 weeks after the initial immunization, and the second booster immunization is carried out 2 weeks after the initial immunization.

[0085] Example 5 Purification of yolk antibodies 1. Take the yolk of the immune laying hen of Example 4, add 9 times the volume of water to dilute and stir for 10 min, adjust the pH to 5.2 with HCl, and let it stand at 4 ° C for 2 h, centrifuge at 4 ° C, 12000 r / min for 25 min, remove the precipitate, take 2 mL of the supernatant, add ice ethanol (-20 ° C) to make its final concentration 60% (v / v), stir at 4 ° C for 20 min and then centrifuge (12000r / min, 4 ° C, 25min), collect the precipitate and add 0.028 mol / L NaCl solution, filter at 4 ° C (0.22μM filter membrane) to remove the lipoprotein precipitate, add ice ethanol (-20 ° C) to the collected filtrate to make its final concentration 25% (v / v), stir at 4 ° C for 20 min and then centrifuge (12000r / min, 25min, 4 ° C), and dissolve the precipitate with purified water (10mL).

[0086] 2. DEAE ion exchange (1) Use PB (pH 7.4, 10 mM) to equilibrate the DEAE ion exchange column.

[0087] (2) The solution obtained in step 1 was replaced with PB (pH 7.4, 10 mM) by ultrafiltration, and then the sample was loaded at 2 mL / min. After loading, the column was equilibrated again with PB (pH 7.4, 10 mM).

[0088] (3) Elute the impurities using a mixture of PB (pH 7.4, 10 mM) and 100 mM NaCl.

[0089] (4) Finally, the target protein IgY was eluted using a mixture of PB (pH 7.4, 10 mM) and 200 mM NaCl. After elution, the protein was desalted through a desalting column and stored at -80°C.

[0090] The results show that ( Figure 3 ), in DEAE ion exchange, two peaks appeared ( Figure 3 A), namely 100mM NaCl elution and 200mM NaCl elution, after SDS-PAGE ( Figure 3 B) The results showed that the purity of IgY was greater than 95%.

[0091] Example 6 Detection of the titer of egg yolk antibodies The titer determination adopted the neutralizing antibody detection method, and the neutralizing antibody titers of VP2, NS1, gB, gD, VP1, VP1 truncated, S and S truncated antigen proteins were determined to be 1:2187, 1:408, 1:387, 1:1024, 1:512, 1:128, 1:32 and 1:729 respectively. The results showed that VP2, gD, VP1 and S truncated as antigen proteins can obtain significantly higher yolk antibody titers. Therefore, VP2, gD, VP1 and S truncated with higher antibody titers were selected as antigen proteins to obtain the corresponding yolk antibodies.

[0092] The above results show that the egg yolk antibody prepared by the present invention has a high neutralizing antibody titer and can effectively neutralize feline parvovirus, feline herpesvirus type I, feline calicivirus and feline coronavirus.

[0093] Example 7 Preparation method of quadruple yolk antibody yolk powder for cats The four different types of immune eggs collected in Example 4, namely, eggs containing specific feline parvovirus capsid protein VP2 yolk antibodies, eggs containing specific feline herpesvirus type I gD protein yolk antibodies, eggs containing specific feline calicivirus capsid protein VP1 yolk antibodies, and eggs containing specific feline coronavirus spike protein S truncated yolk antibodies, were surface disinfected, the yolks were aseptically separated, and the slurry was homogenized. After pasteurization and virus inactivation, 10 g of sucrose, 30 g of trehalose and 10 g of gelatin were added per 1000 g of egg yolk, the mixture was fully mixed and then freeze-dried at low temperature. Then, the four different egg yolk powders were fully mixed in a mass ratio of 1:2:4:3 to obtain cat quadruple yolk antibody egg yolk powder.

[0094] Example 8 Clinical preventive effect test The clinical preventive effect of the quadruple yolk antibody egg yolk powder for cats (hereinafter referred to as quadruple yolk powder) prepared in Example 7 was evaluated, and the specific method and results are as follows.

[0095] 1. Application of cat quadruple yolk antibody in FPV prevention Fifteen weaned kittens aged 10 to 12 weeks were randomly divided into three groups, with 5 in each group, namely Group 1 (Group 1 taking quadruple yolk powder orally), Group 2 (Group 2 taking quadruple yolk powder orally), and Group 3 (Control group taking ordinary egg yolk powder of the same breed as the experiment but not immunized, hereinafter referred to as ordinary egg yolk powder). The specific experimental plan is: Group 1: Each individual was orally administered 2 g of quadruple egg yolk powder daily for 10 consecutive days.

[0096] Group 2: Each individual was orally administered 0.5 g of quadruple egg yolk powder and 1.5 g of ordinary egg yolk powder daily for 10 consecutive days.

[0097] Group 3: Each individual was orally administered 2 g of ordinary egg yolk powder daily for 10 consecutive days.

[0098] On the 6th day, groups 1 to 3 were challenged orally with FPV-CZY cell culture virus attenuated strain, with a FPV virus titer of 10 5 TCID 50 / 0.1mL, the dose is 200μL / cat. The 3 groups were completely isolated for feeding. The cats' status was observed and recorded daily for two consecutive weeks. The criteria for disease diagnosis were: depression, loss of appetite, fever, vomiting, diarrhea, weight loss, positive FPV in feces, etc.

[0099] Test results: The results showed (Table 2) that the morbidity rate of Group 3 (ordinary egg yolk powder control group) was 100%. Compared with the control group, Group 1 (oral 2g quadruple egg yolk powder group) had a good preventive effect, and all 5 individuals did not show clinical symptoms of FPV infection. In Group 2 (oral 0.5g quadruple egg yolk powder and 1.5g ordinary egg yolk powder group), two individuals showed vomiting and diarrhea symptoms on the 6th and 7th days. During the observation period, the clinical symptoms of Group 2 were significantly milder than those of the control group (Group 3), and the average duration of symptoms was also shorter.

[0100] The above results show that the quadruple yolk antibody of the present invention has a good protective clinical effect against FPV infection, and the protective effect is dose-dependent.

[0101] Table 2 Results of the quadruple yolk antibody experiment in preventing FPV infection

[0102] 2. Application of Feline Quadruple Yolk Antibody in FHV Prevention Fifteen weaned kittens aged 10 to 12 weeks were randomly divided into three groups, with 5 in each group, namely Group 1 (Group 1 taking quadruple egg yolk powder orally), Group 2 (Group 2 taking quadruple egg yolk powder orally), and Group 3 (Group 3 taking ordinary egg yolk powder orally). The specific experimental plan is: Group 1: Each individual was orally administered 2 g of quadruple egg yolk powder daily for 10 consecutive days.

[0103] Group 2: Each individual was orally administered 0.5 g of quadruple egg yolk powder and 1.5 g of ordinary egg yolk powder daily for 10 consecutive days.

[0104] Group 3: Each individual was orally administered 2 g of ordinary egg yolk powder daily for 10 consecutive days.

[0105] On the 6th day, groups 1 to 3 were challenged with FHV cell culture virus orally, with a FHV virus titer of 10 5 TCID 50 / 0.1mL, the dose is 200μL / cat. The 3 groups were completely isolated for feeding. The cats' status was observed and recorded daily for two consecutive weeks. The criteria for disease onset were: depression, loss of appetite, sneezing, increased eye and nasal secretions, coughing, nasal congestion, conjunctival edema, and positive FHV in eye and nasal secretions.

[0106] Test results: The results showed (Table 3) that the morbidity rate of Group 3 (ordinary egg yolk powder control group) was 100%. Compared with the control group, Group 1 (oral 2g quadruple egg yolk powder group) had a good preventive effect, and all 5 individuals did not show clinical symptoms of FHV infection. In Group 2 (oral 0.5g quadruple egg yolk powder and 1.5g ordinary egg yolk powder group), one individual showed symptoms of sneezing and increased eye and nasal secretions from the 6th to the 9th day. During the observation period, the clinical symptoms of Group 2 were significantly milder than those of the control group (Group 3), and the average duration of symptoms was also shorter.

[0107] The above results show that the quadruple egg yolk antibody of the present invention has a good protective clinical effect on FHV infection, and the protective effect is dose-dependent.

[0108] Table 3 Results of the quadruple yolk antibody experiment in preventing FHV infection

[0109] 3. Application of cat quadruple yolk antibody in FCV prevention Fifteen weaned kittens aged 10 to 12 weeks were randomly divided into three groups, with 5 in each group, namely Group 1 (Group 1 taking quadruple egg yolk powder orally), Group 2 (Group 2 taking quadruple egg yolk powder orally), and Group 3 (Group 3 taking ordinary egg yolk powder orally). The specific experimental plan is: Group 1: Each individual was orally administered 2 g of quadruple egg yolk powder daily for 10 consecutive days.

[0110] Group 2: Each individual was orally administered 0.5 g of quadruple egg yolk powder and 1.5 g of ordinary egg yolk powder daily for 10 consecutive days.

[0111] Group 3: Each individual was orally administered 2 g of ordinary egg yolk powder daily for 10 consecutive days.

[0112] On the 6th day, groups 1 to 3 were challenged with FCV cell culture virus orally, with a FCV virus titer of 2×10 5 TCID 50 / 0.1mL, the dose is 150μL / cat. The 3 groups were completely isolated for feeding. The cats' condition was observed and recorded daily for two consecutive weeks. The criteria for disease diagnosis were: fever, depression, sneezing, thick secretions from the eyes and nose, conjunctivitis, drooling, oral ulcers, tracheitis and bronchitis, and positive FCV in eye and nose secretions.

[0113] Test results: The results showed (Table 4) that the incidence rate of Group 3 (ordinary egg yolk powder control group) was 100%. Compared with the control group, Group 1 (oral 2g quadruple egg yolk powder group) had a good preventive effect, and all 5 individuals did not show clinical symptoms of FCV infection. In Group 2 (oral 0.5g quadruple egg yolk powder and 1.5g ordinary egg yolk powder group), 2 individuals showed symptoms of sneezing, drooling, and increased eye and nasal secretions from the 7th to the 9th day. During the observation period, the clinical symptoms of Group 2 were significantly milder than those of the control group (Group 3), and the average duration of symptoms was also shorter.

[0114] The above results show that the quadruple egg yolk antibody of the present invention has a good protective clinical effect against FCV infection, and the protective effect is dose-dependent.

[0115] Table 4 Results of the quadruple yolk antibody experiment in preventing FHV infection

[0116] 4. Application of cat quadruple yolk antibody in the prevention of FCoV Fifteen weaned kittens aged 10 to 12 weeks were randomly divided into three groups, with 5 in each group, namely Group 1 (Group 1 taking quadruple egg yolk powder orally), Group 2 (Group 2 taking quadruple egg yolk powder orally), and Group 3 (Group 3 taking ordinary egg yolk powder orally). The specific experimental plan is: Group 1: Each individual was orally administered 2 g of quadruple egg yolk powder daily for 10 consecutive days.

[0117] Group 2: Each individual was orally administered 0.5 g of quadruple egg yolk powder and 1.5 g of ordinary egg yolk powder daily for 10 consecutive days.

[0118] Group 3: Each individual was orally administered 2 g of ordinary egg yolk powder daily for 10 consecutive days.

[0119] On the 6th day, groups 1 to 3 were challenged with FCoV orally, and the FCoV virus titer was 1.5×10 5 TCID 50 / 0.1mL, the dose is 200μL / cat. The 3 groups were completely isolated for feeding. The cats' status was observed and recorded daily for two consecutive weeks. The criteria for disease onset were: depression, loss of appetite, vomiting, diarrhea, weight loss, and positive detection of FCoV in feces.

[0120] Test results: The results showed (Table 5) that the morbidity rate of Group 3 (ordinary egg yolk powder control group) was 100%. Compared with the control group, Group 1 (oral 2g quadruple egg yolk powder group) had a good preventive effect, with only one individual showing clinical symptoms of FCoV infection, which appeared on the 8th to 11th day, and the symptoms were mainly mild diarrhea. In Group 2 (oral 0.5g quadruple egg yolk powder and 1.5g ordinary egg yolk powder group), 3 individuals showed vomiting and diarrhea symptoms on the 6th and 12th days. During the observation period, the clinical symptoms of Groups 1 and 2 were significantly milder than those of the control group (Group 3), and the average duration of symptoms was also shorter.

[0121] The above results show that the quadruple egg yolk antibody of the present invention has a good protective clinical effect against FCoV infection, and the protective effect is dose-dependent.

[0122] Table 5 Results of the quadruple yolk antibody experiment in preventing FCoV infection

[0123] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a quadruple yolk antibody for cats, characterized in that: The method comprises: immunizing laying hens with feline parvovirus capsid protein VP2, feline herpesvirus type I gD protein, feline calicivirus capsid protein VP1 and feline coronavirus spike protein S truncated body as immunogens, and separating egg yolk or egg yolk antibody; Among them, the amino acid sequence of the feline parvovirus capsid protein VP2 is shown in SEQ ID NO.1; the amino acid sequence of the feline herpesvirus type I gD protein is shown in SEQ ID NO.4; the amino acid sequence of the feline calicivirus capsid protein VP1 is shown in SEQ ID NO.5; and the amino acid sequence of the feline coronavirus spike protein S truncate is shown in SEQ ID NO.

8.

2. The method according to claim 1, characterized in that The method comprises: using the feline parvovirus capsid protein VP2, the feline herpesvirus type I gD protein, the feline calicivirus capsid protein VP1 and the feline coronavirus spike protein S truncated form as immunogens to immunize laying hens respectively, separating four kinds of egg yolk antibodies obtained by immunizing laying hens with the four kinds of immunogens respectively, and then mixing the four kinds of egg yolk antibodies.

3. The method according to claim 1, characterized in that The method comprises: using the feline parvovirus capsid protein VP2, the feline herpesvirus type I gD protein, the feline calicivirus capsid protein VP1 and the feline coronavirus spike protein S truncate as immunogens to immunize laying hens respectively, isolating egg yolk containing egg yolk antibodies against the feline parvovirus capsid protein VP2, the feline herpesvirus type I gD protein, the feline calicivirus capsid protein VP1 and the feline coronavirus spike protein S truncate, and mixing the four types of egg yolks.

4. The method according to any one of claims 1 to 3, characterized in that The preparation method of the immunogen comprises: connecting the coding genes of the feline parvovirus capsid protein VP2, the feline herpesvirus type I gD protein, the feline calicivirus capsid protein VP1 and the feline coronavirus spike protein S truncated body to expression vectors respectively to construct expression vectors of four immunogens, introducing the expression vectors into Escherichia coli to obtain recombinant Escherichia coli expressing the four immunogens respectively, culturing the recombinant Escherichia coli, and isolating the feline parvovirus capsid protein VP2, the feline herpesvirus type I gD protein, the feline calicivirus capsid protein VP1 and the feline coronavirus spike protein S truncated body from the culture.

5. The method according to claim 4, characterized in that The coding gene of the feline parvovirus capsid protein VP2 is shown in SEQ ID NO.9; the coding gene of the feline herpesvirus type I gD protein is shown in SEQ ID NO.12; the coding gene of the feline calicivirus capsid protein VP1 is shown in SEQ ID NO.13; the coding gene of the feline coronavirus spike protein S truncation is shown in SEQ ID NO.

16.

6. The method according to any one of claims 1 to 3 and 5, characterized in that: The immunization method comprises: emulsifying the feline parvovirus capsid protein VP2, the feline herpesvirus type I gD protein, the feline calicivirus capsid protein VP1 and the feline coronavirus spike protein S truncated body with adjuvants respectively, and then immunizing laying hens respectively, performing a first booster immunization at an interval of 12-15 days after the initial immunization, and performing a second booster immunization at an interval of 12-15 days.

7. The method according to claim 6, characterized in that The concentration of the immunogen used in the immunization is 1-3 mg / mL, and the volume ratio of the immunogen to the adjuvant emulsification is 1:1-3:7; The dose of the first immunization is 400-600 μg of immunogen / laying hen, and the adjuvant used is Freund's complete adjuvant; the dose of the first booster immunization and the second booster immunization is 200-400 μg of immunogen / laying hen, and the adjuvant used is Freund's incomplete adjuvant; And / or, the laying hens are 6-8 months old.

8. The cat tetravalent egg yolk antibody prepared by the method according to any one of claims 1 to 7.

9. Use of the tetravalent egg yolk antibody for cats according to claim 8 in the preparation of a product for preventing and / or treating viral infection in cats or diseases caused by the viral infection; The virus is one or more selected from feline parvovirus, feline herpesvirus type I, feline calicivirus and feline coronavirus.

10. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the tetravalent egg yolk antibody for cats according to claim 8.

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