Feline infectious rhinoconjunctivitis virus bivalent freeze-dried egg yolk antibody and its preparation method and application

Abstract

The present invention discloses a bivalent freeze-dried egg yolk antibody for feline infectious rhinoconjunctivitis virus, as well as a preparation method and application thereof. Screened feline infectious rhinoconjunctivitis virus CC3 strain and CC246 strain are made into inactivated vaccines to immunize laying hens to prepare highly immune eggs. The egg yolk is separated to extract antibodies to obtain a bivalent freeze-dried egg yolk antibody for feline infectious rhinoconjunctivitis virus, which can improve the protection against cats, and has high titer, good stability, rapid effect, and no drug resistance. The preparation method of the present invention adopts an acid dilution method to crudely extract antibodies from egg yolk, then adopts an ammonium sulfate precipitation method to purify the antibodies, and finally ultrafiltration concentration and desalination are performed, followed by freeze vacuum drying to prepare a bivalent freeze-dried egg yolk antibody. The egg yolk antibody preparation process is simple, has low consumption cost, high yield, stable properties, and is easy to store. It is safe, efficient, and pollution-free, and can be used for emergency prevention of feline infectious rhinoconjunctivitis virus to improve the protection rate.

Description

Technical Field

[0001] The present invention relates to the technical field of antibodies, in particular to a bivalent freeze-dried egg yolk antibody of feline infectious rhinoconjunctivitis virus, and a preparation method and application thereof. Background Art

[0002] Feline infectious rhinoconjunctivitis virus, also known as feline calicivirus (FCV), primarily causes upper respiratory tract symptoms, including coughing, rhinitis, conjunctivitis, and oral ulcers, with severe manifestations including pneumonia and gastroenteritis. The disease is highly contagious and primarily affects kittens under one year old. One of the main reasons for its high prevalence in cats is that cats often remain carriers of the virus. Many cats that have recovered clinically or are asymptomatic continue to shed the virus for years, becoming a significant source of infection. Their saliva, ocular and nasal secretions, and feces are the primary vectors of the virus. While FCV alone is not fatal, infected cats have a weakened immune system and are highly susceptible to co-infection with other feline upper respiratory viruses, which can increase mortality.

[0003] Currently, the disease is primarily prevented through vaccination. However, the vaccine consists of antigens, and the immunization method is active, requiring the body to produce antibodies for protection to take effect, which takes a long time. For unvaccinated cats living in a group, if one cat becomes ill, vaccination will not be enough to produce antibodies to protect the cats. Furthermore, vaccine penetration is very low, and once infected, there is no specific treatment available domestically or internationally.

[0004] Currently, cats are primarily treated with broad-spectrum antibiotics to prevent secondary infections and provide symptomatic treatment. However, antibiotics disrupt protein synthesis by damaging cell walls, leading to bacterial imbalance over time. Furthermore, they lack specificity and are prone to developing drug resistance, resulting in limited efficacy. Therefore, developing a specific drug for feline infectious rhinoconjunctivitis virus (FICV) to reduce morbidity and mortality is of great practical significance.

[0005] FCV comprises a large number of strains, with extensive antigenic variation between strains. This high level of antigenic variation makes it difficult to achieve broad protection against FCV in cats. Some studies have shown that different FCV strains differ by 20% to 40% in the capsid variant region. Due to the diverse FCV genome, FCV antigens also exhibit diverse antigenic diversity. This antigenic diversity means that no vaccine can neutralize all virus isolates. Studies have found that most vaccine strains, such as F9 or 255, offer broad cross-protection. Furthermore, due to the evolution of FCV and the occurrence of associated lethal diseases, many strains used in existing vaccines fail to achieve adequate efficacy, leading to frequent cases of immune failure. Although vaccination with homologous strains is highly effective, the level of cross-protection from one strain against another is quite limited, making it difficult to select antigens for vaccine development.

[0006] Currently, egg yolk antibodies have great application prospects due to their relatively simple preparation, low cost, and high yield. However, due to the high level of antigenic variability of FCV, a single egg yolk antibody is difficult to fully protect against prevalent feline infectious rhinoconjunctivitis virus (FICV), and no commercially available bivalent FICV egg yolk antibodies are available domestically or internationally. In light of this, the present invention demonstrated through serum neutralization tests that there is a lack of cross-protection between FICV CC3 and CC246 strains, and that homology is as low as 78.6%. Consequently, we developed a bivalent FICV egg yolk antibody. Summary of the Invention

[0007] In a first aspect, the present invention provides a bivalent freeze-dried egg yolk antibody against feline infectious rhinoconjunctivitis virus. The antibody is obtained by immunizing laying hens with inactivated vaccines prepared from FCV CC3 and FCV CC246 strains, separating the yolks of highly immune eggs, extracting antibodies, and mixing them. The neutralizing antibody titers of FCV CC246 and FCV CC3 strains are both ≥1:2048. The gene sequence of FCV CC3 strain is shown in SEQ ID No.1, and the gene sequence of FCV CC246 strain is shown in SEQ ID No.2.

[0008] Furthermore, the FCV CC3 strain was deposited in the General Microbiology Center of the China Culture Collection Administration on July 1, 2022. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC No. 45218, and the classification name is feline infectious rhinoconjunctivitis virus.

[0009] Furthermore, the FCV CC246 strain was deposited in the General Microbiology Center of the China Culture Collection Administration on July 1, 2022. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC No. 45217, and the classification name is feline infectious rhinoconjunctivitis virus.

[0010] Furthermore, the antibody is prepared by the following steps: taking FCV CC246 strain and FCV CC3 strain virus liquid, inactivating them by BEI, and mixing them with ISA 78 adjuvant to obtain corresponding vaccines; selecting 150-200 day-old Hy-Line white laying hens, and immunizing the laying hens of the FCV CC246 strain and FCV CC3 strain groups six times with the vaccine obtained in step S1; 14 days after the six immunizations, starting to detect the neutralizing titer of antibodies in egg yolks, and when the neutralizing antibody titer is ≥1:256, collecting the eggs for ammonium sulfate purification of IgY; ultrafiltration to remove salt, concentrating until the neutralizing antibody titer of FCV CC246 strain and FCV CC3 strain is not less than 1:2048, inactivating, and freeze-drying.

[0011] Furthermore, the immunization program for laying hens is: 6 immunizations, each immunization dose is 1.0 ml / hen, each immunization interval is 14 days, and the injection is performed into the leg or chest muscle.

[0012] In a second aspect, the present invention provides a method for preparing the above-mentioned bivalent freeze-dried egg yolk antibody to feline infectious rhinoconjunctivitis virus, comprising the following steps:

[0013] S1. Vaccine preparation:

[0014] The virus liquids of FCV CC246 and FCV CC3 were inactivated by BEI, mixed with ISA 78 adjuvant at a ratio of 1:1 (V:V) and emulsified.

[0015] S2. Immunity of laying hens:

[0016] 150-200-day-old Hy-Line white laying hens were selected, and the laying hens of the FCV CC246 strain and the FCV CC3 strain groups were immunized six times with the vaccine obtained in step S1. The laying hens were immunized six times, with a dose of 1.0 ml per chicken per immunization, and an interval of 14 days between each immunization, and the immunizations were injected into the leg or chest muscles.

[0017] S3. Collection of high-immunity eggs:

[0018] Fourteen days after the sixth immunization, the neutralization titer of the antibody in the egg yolk was tested separately. The egg yolk was diluted 10 times with acetic acid-sodium acetate buffer, centrifuged and the supernatant was collected. The titer of the neutralizing antibody against feline infectious rhinoconjunctivitis virus in the supernatant was tested. When the neutralizing antibody titer was ≥1:256, the eggs were collected separately.

[0019] S4. Purification of yolk antibodies:

[0020] Separate the yolks from the collected eggs, dilute them 7-10 times with acetic acid-sodium acetate buffer, stir until completely dissolved, and let stand at 2-8°C for 12-24 hours; 12000r / min-14000r / min Centrifuge for 10-15 minutes, remove the supernatant, and discard the precipitate; purify IgY with ammonium sulfate by adding solid ammonium sulfate to a final concentration of 33%-35% to the supernatant for precipitation and purification, stirring until the ammonium sulfate is completely dissolved, standing at 2-8°C for 2-24 hours, centrifuging at 12000-14000 rpm for 10-15 minutes, removing the precipitate, dissolving the precipitate in 1 / 4 volume of normal saline equal to the original volume, and adding solid ammonium sulfate to a final concentration of 33%-35% for precipitation and purification, repeating the precipitation twice; dissolving the third ammonium sulfate precipitate in the original volume of normal saline equal to the original volume of the third precipitate, stirring until the precipitate is completely dissolved, and ultrafiltration using a 30KD membrane to remove salts. When the volume of the concentrate is 1 / 10 of the original volume, replenish it with normal saline to the original volume, repeating at least 5 times until no white precipitate is produced in the submembrane fluid detected by 2% BaCl2 solution; concentrate the volume until the neutralizing antibody titer of FCV CC246 and FCV CC3 strains is not less than 1:2048;

[0021] S5. Inactivation:

[0022] The concentrated yolk antibody solution of feline infectious rhinoconjunctivitis virus (FCV) CC246 strain and the concentrated yolk antibody solution of feline infectious rhinoconjunctivitis virus (FCV) CC3 strain were sterilized by 0.22 μm filtration, and then β-propiolactone was added at a ratio of 1:4000 (V:V). The solution was inactivated at 2-8°C for 48-50 hours and then hydrolyzed at 37°C for 2 hours to obtain the yolk antibody stock solution of feline infectious rhinoconjunctivitis virus (FCV) CC246 strain and the yolk antibody stock solution of feline infectious rhinoconjunctivitis virus (FCV) CC3 strain.

[0023] S6. Freeze-drying:

[0024] Mix a stock solution of egg yolk antibodies against FCV CC246 and FCV CC3, and dilute with normal saline until the titer of neutralizing antibodies against FCV in each solution is no less than 1:2048. Add dextran 40 and mannitol proportionally to final concentrations of 4% and 2.5%, respectively. Mix thoroughly, sterile filter through a 0.22 μm filter, and dispense aseptically. Place a lyophilized butyl rubber stopper on the suspension, rapidly freeze-dry, and seal under vacuum. Store at 2-8°C.

[0025] In a third aspect, the present invention provides the use of the above-mentioned feline infectious rhinoconjunctivitis virus bivalent freeze-dried egg yolk antibody in an emergency preventive drug for feline infectious rhinoconjunctivitis.

[0026] In a fourth aspect, the present invention also provides a pharmaceutical composition for use against feline infectious rhinoconjunctivitis virus, comprising the above-mentioned bivalent freeze-dried egg yolk antibody against feline infectious rhinoconjunctivitis virus and a pharmaceutically acceptable carrier.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The present invention uses the screened feline infectious rhinoconjunctivitis virus CC3 strain and CC246 strain, which lack cross-protection and have low homology, to inoculate them into F81 cells to obtain high-content virus liquid. After the virus liquid is inactivated, it is mixed with an adjuvant, and high-immune eggs are prepared by immunizing laying hens with inactivated vaccines. The yolk is separated and antibodies are extracted to obtain bivalent freeze-dried yolk antibodies against feline infectious rhinoconjunctivitis virus, which can improve the protection against cats and has a high titer of up to 1:2048. It has good stability, rapid effect, and will not produce drug resistance. At the same time, when chickens are used to produce antibodies against mammalian tissue components, more antibodies can be produced with less antigen, and the IgY titer in the yolk of the immune laying hens can be maintained at a high level for a long time. In addition, the yolk antibodies are extracted from eggs, and antibodies can be obtained by collecting eggs laid by the immune laying hens, without damaging the laying hens themselves, and complying with animal welfare and modern animal protection regulations.

[0029] The invention discloses a method for preparing a bivalent freeze-dried egg yolk antibody against feline infectious rhinoconjunctivitis virus. The method comprises the following steps: crudely extracting antibodies from egg yolk using an acid dilution method, purifying the antibodies using an ammonium sulfate precipitation method, and finally, desalting by ultrafiltration and concentration, followed by freeze vacuum drying to prepare the bivalent freeze-dried egg yolk antibody. The egg yolk antibody preparation process is simple, has low consumption costs, high yield, stable properties, and is easy to store. The antibody is heat-resistant, acid-resistant, and does not inactivate at pasteurization temperatures. The antibody is safe, efficient, and pollution-free.

[0030] The feline infectious rhinoconjunctivitis virus bivalent freeze-dried egg yolk antibody of the present invention has good cross-protection effect, can provide effective and more extensive immune protection for cats infected with feline infectious rhinoconjunctivitis virus, can be used for emergency prevention of feline infectious rhinoconjunctivitis virus, and improves the protection rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments described in the present invention. For ordinary technicians in this field, other drawings can also be obtained based on these drawings.

[0032] Figure 1 These are the results of neutralization antibody detection of FCV CC246 and FCV CC3 yolk antibodies provided in Example 3 of the present invention, wherein A: F81 cells with pathological changes after FCV CC246 infection; B: F81 cells after virus neutralization by FCV CC246 yolk antibodies.

[0033] Figure 2 The results of neutralizing antibody detection of FCV CC3 strain yolk antibodies provided in Example 3 of the present invention, wherein A: FCVCC3-infected F81 cells with pathological changes; B: FCV CC246 strain yolk antibodies neutralizing the virus in F81 cells.

[0034] Figure 3 This is the FCV CC246 strain yolk antibody purity test result provided in Example 3 of the present invention.

[0035] Figure 4 This is the FCV CC3 strain yolk antibody purity test result provided in Example 3 of the present invention. DETAILED DESCRIPTION

[0036] To help those skilled in the art better understand the technical solutions of the present invention, the present invention will be further described in detail below with reference to the embodiments and accompanying drawings. It is apparent that the embodiments described are only a portion of the embodiments of the present invention, not all of them. All other embodiments derived by those skilled in the art based on the embodiments of the present invention without creative effort are also within the scope of protection of the present invention.

[0037] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified.

[0038] Example 1 Screening of strains

[0039] The present invention uses rabbit polyantisera prepared from 13 strains of feline infectious rhinoconjunctivitis virus (FCV CCSN-1, FCV CC3, FCV CC9, FCVCC10, FCV CC85, FCV CC246, FCV CC247, FCV CC256, FCV CC272, FCV CC295, FCV CC313, FCVCC316, and FCV CC320, which were isolated and identified by our company from diseased cat samples in Changchun, Shenyang, Harbin, Zhangjiakou, Beijing, Hangzhou, Yangzhou, Zhengzhou, etc., and rabbit polyantisera obtained after immunization with a triple inactivated vaccine (Miu Sanduo) for feline rhinotracheitis, calicivirus disease, and panleukopenia as a control, to conduct a cross-protection test with existing feline infectious rhinoconjunctivitis virus and determine the titer of feline infectious rhinoconjunctivitis virus neutralizing antibodies, thereby screening two strains with good cross-protection and high antibody titer from the 13 feline infectious rhinoconjunctivitis virus strains: FCV CC3 strain and CC246 strain. FCV antiserum can neutralize 6 different strains of feline infectious rhinoconjunctivitis virus (46.2%). FCV CC3 strain and CC246 strain antiserum can neutralize 12 different strains of feline infectious rhinoconjunctivitis virus (92.3%). FCV CC3 strain and FCVCC246 strain lack effective cross-protection between each other. The neutralizing antibody titers of FCV CC246 and FCV CC3 antiserum are not less than 1:2048. The results are shown in Tables 1 to 3. Sequence alignment analysis found that the sequence homology between FCV CC3 (gene sequence shown in SEQ ID No. 1) and FCV CC246 (gene sequence shown in SEQ ID No. 2) is low (78.6%).

[0040] Table 1 Strain screening results (1)

[0041]

[0042]

[0043] Table 2 Strain screening results (2)

[0044]

[0045] Table 3 Strain screening results (3)

[0046]

[0047]

[0048] The FCV CC246 strain was isolated and identified by our company and belongs to the Caliciviridae family, Calicivirus genus, and is a non-enveloped single-stranded positive-strand RNA virus. It was deposited in the General Microbiology Center of the China Culture Collection Administration Committee (CGMCC) on July 1, 2022, with the deposit number CGMCC No. 45217. The FCV CC3 strain was isolated and identified by our company and belongs to the Caliciviridae family, Calicivirus genus, and is a non-enveloped single-stranded positive-strand RNA virus. It was deposited in the General Microbiology Center of the China Culture Collection Administration Committee (CGMCC) on July 1, 2022, with the deposit number CGMCC No. 45218. Virus TCID 50 Not less than 10 9.5 / ml.

[0049] Example 2 Preparation of vaccine

[0050] FCV CC246 strain and FCV CC3 strain vaccines were prepared respectively.

[0051] The feline infectious rhinoconjunctivitis virus FCV CC246 strain and FCV CC3 strain screened in Example 1 were inoculated into F81 cells at an MOI of 0.01 to 0.1, cultured for 24 to 48 hours, and the pathological changes were observed daily. When the cells were completely detached, the cell culture was harvested, and the virus solution was harvested to obtain a virus solution with a content of not less than 10 9.5 TCID 50 / ml; the virus liquids were inactivated with BEI at a final concentration of 0.001 mol / L at 30°C for 20 hours, and then 10% of the BEI usage amount and 1 mol / L sodium thiosulfate were added to neutralize BEI. The two inactivated virus liquids were respectively mixed with ISA 78 adjuvant in a 1:1 (V:V) ratio and emulsified to obtain the corresponding vaccines.

[0052] The preparation method of BEI is as follows: weigh BEA and dissolve it in 0.2 mol / L NaOH, the weight volume ratio of BEA and 0.2 mol / L NaOH is 2.05g:100ml, and place it in a 37°C water bath for 1 hour to produce 0.1 mol / L BEI. This process is accompanied by a change in pH value, which drops from 12.5 to 8.5.

[0053] Example 3 Preparation of bivalent egg yolk antibodies

[0054] 1. Immunity of laying hens:

[0055] 150-200 day-old Hy-Line white laying hens were selected, and the laying hens of the FCV CC246 strain and FCV CC3 strain groups were immunized six times with the vaccine obtained in Example 2. In order to obtain stable and high-titer yolk antibodies, the laying hens were immunized six times with a dose of 1.0 ml per chicken each time, with an interval of 14 days between each immunization, and injected into the leg or chest muscle.

[0056] 2. Collection of high-immunity eggs:

[0057] Fourteen days after the sixth immunization, egg yolk neutralization titers were measured. Egg yolks were diluted 10-fold with acetic acid-sodium acetate buffer, centrifuged, and the supernatant (WSF) was assayed for FIRCV neutralizing antibody titers. Eggs were collected when neutralizing antibody titers were ≥1:256. As shown in Table 4, both antibody levels were at least 1:256.

[0058] Table 4 Antibody titer results after immunization of laying hens

[0059]

[0060] 3. Purification of yolk antibodies:

[0061] Separate the yolks from the collected eggs, dilute them 7-10 times with acetic acid-sodium acetate buffer, stir until completely dissolved, and let stand at 2-8°C for 12-24 hours; 12000r / min-14000r / min Centrifuge for 10 to 15 minutes, take the supernatant, and remove the precipitate; purify IgY with ammonium sulfate, add solid ammonium sulfate at a final concentration of 33% to 35% to the supernatant for precipitation and purification, stir until the ammonium sulfate is completely dissolved, let stand at 2 to 8°C for 2 to 24 hours, centrifuge at 12,000 r / min to 14,000 r / min for 10 to 15 minutes, take the precipitate, dissolve the precipitate in 1 / 4 volume of normal saline of the original volume, add solid ammonium sulfate at a final concentration of 33% to 35% for precipitation and purification, and repeat the precipitation twice; dissolve the third ammonium sulfate precipitate in the original volume of normal saline of the third precipitate, stir until the precipitate is completely dissolved, and remove salt by ultrafiltration using a 30KD membrane bag. When the volume of the concentrate is 1 / 10 of the original volume, add normal saline to the original volume, repeat at least 5 times, until no white precipitate is produced in the submembrane fluid detected by 2% BaCl2 solution; concentrate the volume to a titer of neutralizing antibodies against egg yolk antibodies of FCV CC246 strain and FCV CC3 strain of not less than 1:2048 (such as Figure 1 and 2 As shown), the purity is not less than 95.0% (as shown Figure 3 、 Figure 4 , as shown in Table 5 and Table 6).

[0062] Table 5 Purity and detection results of FCV (CC246) yolk antibodies

[0063]

[0064] Table 6 Purity and detection results of FCV (CC3) yolk antibodies

[0065]

[0066]

[0067] 4. Inactivation:

[0068] The concentrated yolk antibody solution of feline infectious rhinoconjunctivitis virus FCV CC246 strain and the concentrated yolk antibody solution of feline infectious rhinoconjunctivitis virus FCV CC3 strain were sterilized by 0.22 μm filtration, and β-propiolactone was added at a ratio of 1:4000 (V:V). The solution was inactivated at 2-8°C for 48-50 hours and hydrolyzed at 37°C for 2 hours to obtain the stock solution of feline infectious rhinoconjunctivitis virus FCV CC246 strain and the stock solution of feline infectious rhinoconjunctivitis virus FCV CC3 strain yolk antibody.

[0069] 5. Freeze-drying:

[0070] Mix a stock solution of egg yolk antibodies against FCV CC246 and FCV CC3, and dilute with normal saline until the titer of neutralizing antibodies against FCV in each solution is no less than 1:2048. Add dextran 40 and mannitol proportionally to final concentrations of 4% and 2.5%, respectively. Mix thoroughly, sterile filter through a 0.22 μm filter, and dispense aseptically. Place a lyophilized butyl rubber stopper on the suspension, rapidly freeze-dry, and seal under vacuum. Store at 2-8°C.

[0071] 6. Antibody quality standards

[0072] Appearance: White spongy loose mass.

[0073] The sterility test shall be carried out according to the sterility test or pure test method in the appendix of Part III of the current "Chinese Veterinary Pharmacopoeia", and no bacteria should grow.

[0074] The titer was determined using the neutralizing antibody detection method, and the feline infectious rhinoconjunctivitis virus neutralizing antibody titer was no less than 1:2048.

[0075] Bacterial endotoxins should be tested according to the bacterial endotoxin test method in Appendix I of the current "Chinese Veterinary Pharmacopoeia" and should be less than 10EU / ml.

[0076] The purity shall be tested according to the electrophoresis method in Appendix 1 of the current "Chinese Veterinary Pharmacopoeia" - cellulose acetate film electrophoresis method, and shall not be less than 95.0% of the total protein.

[0077] Example 4 Emergency Prevention Trial of Feline Infectious Rhinoconjunctivitis Virus

[0078] 1. Method

[0079] Twenty-five 2- to 3-month-old kittens (FCV neutralizing antibody titer no higher than 1:4) were randomly divided into seven groups, with five cats in each group: Group I (challenge control group 1), Group II (challenge control group 2), Group III (challenge control group 3), Group IV (egg yolk antibody group 1), Group V (egg yolk antibody group 2), Group VI (egg yolk antibody group 3), and Group VII (blank control group).

[0080] Cats in groups I, II, III, and VII were injected subcutaneously with normal saline at a dose of 1.5 ml / kg. Cats in groups IV, V, and VI were injected subcutaneously with a bivalent freeze-dried egg yolk antibody prepared for feline infectious rhinoconjunctivitis virus at a dose of 1.5 ml / kg. 24 hours later, cats in groups I and IV were infected with FCV (CC3 strain isolate, viral load 10 8.50 TCID 50 / ml), the dose was 0.25ml / nostril; Group II and Group V were infected with FCV virus (CC246 isolate, from Sino Biotechnology Co., Ltd.) by nasal drops, the dose was 0.25ml / nostril; Group III and Group VI were infected with FCV virus (CC295 isolate, from Sino Biotechnology Co., Ltd., because its sequence had low homology with FCV CC246 strain and FCV CC3 strain, which were 76.6% and 79.7% respectively. The virus content in the liquid after inoculation was as high as 10 8.70 TCID 50 Group VII was infected with MEM medium via nasal drops at a dose of 0.25 ml per nostril. The seven experimental cats in each group were housed separately, and their condition was observed and recorded daily for 14 consecutive days. Nasopharyngeal swabs were collected for PCR testing. Cats were identified as ill if their RT-PCR results were positive and they showed typical symptoms of FCV infection (high fever, oral ulcers, and respiratory symptoms).

[0081] 2. Results

[0082] 2.1 Clinical symptoms

[0083] Cats in Group I developed depression, eye and nasal discharge, ulcers, and elevated body temperature 2-3 days after challenge. Cats in Groups II and III developed depression, eye and nasal discharge, ulcers, and elevated body temperature 2 days after challenge. One cat in Groups IV and VI developed a small oral ulcer, sneezing, and a small amount of eye and nasal discharge. Cats in Group V and Group VII showed no obvious abnormal symptoms. Clinical symptoms in some cats improved 14 days after challenge.

[0084] 2.2 Body temperature changes

[0085] Cats in Group I experienced a temperature increase on day 3 after challenge, peaking at 40.8°C on day 6 and returning to normal 9 days after infection. In Group II, temperature increased on day 2, peaked on day 5, and reached a maximum temperature of 40.5°C. Temperatures returned to normal 8 days after infection. In Group III, three cats experienced a temperature increase on day 2, peaked on day 4, and reached a maximum temperature of 40.2°C. All cats returned to normal 9 days after infection. In Group IV, one cat experienced a temperature increase on days 5 and 6, which subsequently returned to normal. In Group VI, one cat experienced a temperature increase on day 3, which returned to normal on day 6. Temperature fluctuations in Groups V and VII were minimal. The scores for the control group and the egg yolk antibody group were significantly different (P < 0.01). The results are shown in Tables 7 and 8 below.

[0086] Table 7 Body temperature in the emergency prevention test of feline infectious rhinoconjunctivitis virus (Groups I to III)

[0087]

[0088] Table 8 Body temperature in the emergency prevention test of feline infectious rhinoconjunctivitis virus (Group IV to Group VII)

[0089]

[0090] 2.3 FCV PCR test results

[0091] Cats in Groups I, II, and III tested positive for FIRCV in eye and nasal swabs on days 2 and 3 after challenge, with virus shedding lasting for more than 7 days. Cats began to die on day 10 after challenge. Cats in Groups IV and VI tested positive in eye and nasal swabs on days 2 and 3, with virus shedding lasting for no more than 4 days. Cats in Groups V and VII all tested negative. The results are shown in Tables 9 and 10.

[0092] Table 9 Virus detection results of feline infectious rhinoconjunctivitis virus emergency prevention test (Group I to Group III)

[0093]

[0094] Note: "+" represents positive, "-" represents negative, and "*" represents the death of the test cat.

[0095] Table 10 Virus detection results of feline infectious rhinoconjunctivitis virus emergency prevention test (Group IV to Group VII)

[0096]

[0097]

[0098] Note: "+" represents positive, "-" represents negative, and "*" represents the death of the test cat.

[0099] 3 Conclusion

[0100] In an emergency prevention test, after being injected with the feline infectious rhinoconjunctivitis bivalent freeze-dried egg yolk of the present invention, the test cats can obtain protection of not less than 80% and there is no death.

[0101] The incidence rates of antibody groups I (challenge control group 1), II (challenge control group 2), and III (challenge control group 3) were all 100%, the protection rates were 0%, and the mortality rates were 60%, 40%, and 60%, respectively. The incidence rates of groups IV (egg yolk antibody group 1), V (egg yolk antibody group 2), and VI (egg yolk antibody group 3) were all 20%, 20%, and 0%, the protection rates were 80%, 100%, and 80%, and the mortality rates were all 0. Group VII (blank control group) did not develop any disease. The results are shown in Table 11.

[0102] Table 11 Results of morbidity, protection rate and mortality of test cats in each group

[0103]

[0104] The above experiments show that the feline infectious rhinoconjunctivitis bivalent freeze-dried egg yolk antibody of the present invention has a good cross-protection effect, can provide effective and broader immune protection for cats infected with feline infectious rhinoconjunctivitis virus, can be used for the emergency prevention of feline infectious rhinoconjunctivitis virus, and has broad application prospects.

[0105] The above description is merely illustrative of certain exemplary embodiments of the present invention. It goes without saying that those skilled in the art will be able to modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the above drawings and description are illustrative in nature and should not be construed as limiting the scope of protection of the claims.

Claims

1. A method for preparing bivalent freeze-dried egg yolk antibodies to feline infectious rhinoconjunctivitis virus, characterized in that: The following steps are involved: S1. Vaccine preparation: The virus liquids of FCV CC246 and FCV CC3 were inactivated by BEI, mixed with ISA 78 adjuvant at a ratio of 1:1 (V:V), and emulsified. The gene sequence of FCV CC3 is shown in SEQ ID No. 1, and the gene sequence of FCV CC246 is shown in SEQ ID No.

2. The FCV CC3 strain was deposited in the General Microbiology Center of the China National Center for Microbiological Culture Collection on July 1, 2022, with the deposit number CGMCC No. 45218 and the classification name "feline infectious rhinoconjunctivitis virus." The FCV CC246 strain was deposited in the General Microbiology Center of the China National Center for Microbiological Culture Collection on July 1, 2022, with the deposit number CGMCC No. 45217 and the classification name "feline infectious rhinoconjunctivitis virus." S2. Immunity of laying hens: 150-200 day-old Hy-Line White laying hens were selected and immunized with the vaccine obtained in step S1, respectively, in groups of FCV CC246 and FCV CC3. The immunization schedule for laying hens was as follows: each immunization dose was 1.0 ml per chicken, with an interval of 14 days between immunizations, for six immunizations, administered intramuscularly in the leg or chest. S3. Collection of high-immunity eggs: Fourteen days after the sixth immunization, the neutralization titer of the antibody in the egg yolk was tested separately. The egg yolk was diluted 10 times with acetic acid-sodium acetate buffer, centrifuged and the supernatant was collected. The titer of the neutralizing antibody against feline infectious rhinoconjunctivitis virus in the supernatant was tested. When the neutralizing antibody titer was ≥1:256, the eggs of each group were collected separately. S4. Purification of yolk antibodies: The yolks of the collected eggs were separated and diluted 7-10 times with acetic acid-sodium acetate buffer, stirred until completely dissolved, and allowed to stand at 2-8°C for 12-24 hours; centrifuged at 12000 r / min-14000 r / min for 10-15 minutes, and the supernatant was collected to remove the precipitate; IgY was purified by ammonium sulfate, and solid ammonium sulfate at a final concentration of 33%-35% was added to the supernatant for precipitation and purification, stirred until the ammonium sulfate was completely dissolved, and allowed to stand at 2-8°C for 2-24 hours, and centrifuged at 12000 r / min-14000 r / min for 1 minute. From 0 to 15 minutes, take the precipitate and dissolve it in 1 / 4 of the original volume of normal saline. Add solid ammonium sulfate at a final concentration of 33% to 35% for precipitation purification. Repeat the precipitation twice. Dissolve the third ammonium sulfate precipitate in the original volume of normal saline of the third precipitation and stir until the precipitate is completely dissolved. Use a 30KD membrane package to ultrafiltration to remove salt. When the volume of the concentrate is 1 / 10 of the original volume, add normal saline to the original volume. Repeat at least 5 times until no white precipitate is produced in the submembrane liquid detected by 2% BaCl2 solution. Concentrate the volume until the neutralizing antibody titer of FCV CC246 and FCV CC3 strains is not less than 1:2048. S5. Inactivation: The concentrated yolk antibody solution of FCV CC246 and FCV CC3 was sterilized by 0.22 μm filtration, and then β-propiolactone was added at a ratio of 1:4000 (V:V). The solution was inactivated at 2-8°C for 48-50 hours and then hydrolyzed at 37°C for 2 hours to obtain the FCV CC246 and FCV CC3 yolk antibody stock solutions. S6. Freeze-drying: The egg yolk antibody stock solution of feline infectious rhinoconjunctivitis virus (FCV) CC246 strain and the egg yolk antibody stock solution of feline infectious rhinoconjunctivitis virus (FCV) CC3 strain were mixed and diluted with normal saline so that the titer of feline infectious rhinoconjunctivitis virus neutralizing antibody in the mixture was not less than 1:2048; dextran 40 and mannitol were added in proportion to make the final concentrations of 4% and 2.5%, respectively, and the mixture was evenly mixed, sterilized and filtered through a 0.22 μm filter, aseptically quantitatively packaged, and covered with a lyophilized butyl rubber stopper. The mixture was quickly freeze-dried in a vacuum, sealed with a cap after lyophilization, and stored at 2-8°C.

2. Feline infectious rhinoconjunctivitis virus bivalent freeze-dried egg yolk antibody, characterized in that: The method as claimed in claim 1 is used for manufacturing.

3. Use of the bivalent freeze-dried egg yolk antibody to feline infectious rhinoconjunctivitis virus as claimed in claim 2 in the preparation of an emergency preventive drug for feline infectious rhinoconjunctivitis.

4. A pharmaceutical composition for resisting feline infectious rhinoconjunctivitis virus, characterized in that: The invention comprises the feline infectious rhinoconjunctivitis virus bivalent freeze-dried egg yolk antibody according to claim 2 and a pharmaceutically acceptable carrier.

Images