CN114634564B - Triple egg yolk antibody for cat, preparation method and application - Google Patents
Triple egg yolk antibody for cat, preparation method and application Download PDFInfo
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- CN114634564B CN114634564B CN202210407205.6A CN202210407205A CN114634564B CN 114634564 B CN114634564 B CN 114634564B CN 202210407205 A CN202210407205 A CN 202210407205A CN 114634564 B CN114634564 B CN 114634564B
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
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- A—HUMAN NECESSITIES
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/20—Antivirals for DNA viruses
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- A—HUMAN NECESSITIES
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- A61P31/12—Antivirals
- A61P31/20—Antivirals for DNA viruses
- A61P31/22—Antivirals for DNA viruses for herpes viruses
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Abstract
The invention relates to the technical field of biology, in particular to a triple egg yolk antibody for cats, a preparation method and application thereof. Wherein the triple egg yolk antibodies comprise a feline parvovirus egg yolk antibody, a feline herpesvirus egg yolk antibody and a feline calicivirus egg yolk antibody. The triple egg yolk antibody provided by the invention can be used for preventing and assisting in treating feline panleukopenia, feline rhinotracheitis and feline infectious rhinoconjunctivitis, and has high protection rate and good effect. Particularly has good treatment effect or adjuvant treatment effect on the feline panleukosis diarrhea, and can obviously shorten the treatment time of the feline panleukosis diarrhea. The invention expands the treatment way of the feline diarrhea and provides a new direction for the treatment of the feline diarrhea.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a triple egg yolk antibody for cats, a preparation method and application thereof.
Background
Feline distemper, also known as Feline panleukopenia and Feline infectious enteritis, is an acute high-contact infectious disease caused by Feline Parvovirus (FPV), and is mainly characterized by sudden hyperpyrexia, intractable vomiting, diarrhea, dehydration, circulatory disturbance and rapid decrease of leukocytes in clinical manifestations. The disease is highly contagious, and is more contagious in the incompletely vaccinated or unvaccinated cats, and more contagious in young cats of 3-5 months old. If the female cat is infected with the feline panleukopenia during pregnancy, the problems of dead fetus, abortion, neurological symptoms of the newborn kittens and the like can be caused. FPV can infect felines (e.g., tigers, leopards), ferrets (e.g., minks), and felines (e.g., raccoons) in addition to cats. The feline panleukosis has extremely high mortality, and under the normal condition, the mortality of cats under 1 year old is 50-60%, and the mortality of cats under 5 months old is 80-90%.
The Feline nasal ramus is an acute upper respiratory infectious disease of felidae, has the characteristics of strong infectivity, acute morbidity and high contact property, is also called infectious rhinotracheitis or viral rhinotracheitis, is caused by Feline herpesvirus type I (Feline herpesvirus type 1, FHV-1), and is generally infected through respiratory tract and digestive tract. The infection of nasal branches of cats is not seasonal, and cats at all ages are susceptible, especially young cats who are not immune or are immune-deficient or cats with weak constitution.
Feline calicivirus infection, also known as Feline infectious rhino-conjunctivitis, is a multiple oral and respiratory disease in felines caused by Feline Calicivirus (FCV), with the main symptoms of mouth ulcerations, fever, sneezing, conjunctivitis, chronic gastroenteritis, and the like. Some virulent strains appearing in recent years can cause acute systemic diseases, cause high mortality rate and seriously jeopardize the health of infected animals. The feline calicivirus is distributed in the world, not only infects cats, but also infects all felines, such as tigers, lions, leopards and the like, and poses certain threat to the health of wild animals.
Clinically, the three diseases of the cat are frequently cross-mixed and infected, and usually secondary bacterial infection is caused, so that the symptoms are more complicated. In the related technology, antiviral specific drugs aiming at the three viruses are lacked, and once infection occurs, symptomatic treatment or antibiotic application is usually adopted for treatment, but the treatment effect and the treatment specificity are poor, and drug resistance and toxic and side effects generally exist.
Disclosure of Invention
Therefore, the invention aims to solve the technical problems of poor treatment effect and treatment specificity, drug resistance and toxic and side effects in the existing methods for treating feline infectious diseases, feline rhinobronchitis and feline calicivirus infection, and provides the triple egg yolk antibody for the cats, the preparation method and the application.
Therefore, the invention provides a triple egg yolk antibody for cats, which comprises the following components: feline parvovirus egg yolk antibodies, feline herpesvirus egg yolk antibodies, and feline calicivirus egg yolk antibodies.
Optionally, in the triple egg yolk antibody, the neutralizing antibody titer of the feline parvovirus egg yolk antibody is not less than 1.
Preferably, in the triple egg yolk antibody, the neutralizing antibody titer of the feline parvovirus egg yolk antibody is not less than 1.
Optionally, the feline parvovirus egg yolk antibody, the feline herpesvirus egg yolk antibody and the feline calicivirus egg yolk antibody may be neutralizing antibodies. The antibody titer of each yolk antibody in the triple yolk antibody can be detected by methods commonly used in the art.
The invention also provides a pharmaceutical preparation which comprises the triple egg yolk antibody and pharmaceutically acceptable auxiliary components.
Alternatively, the pharmaceutical formulation may be a solid formulation, and the adjunct ingredients include at least one of vitamin C, sucrose, glucose, or sodium benzoate.
Optionally, in the solid preparation, the neutralizing antibody titer of the feline parvovirus egg yolk antibody is not less than 1.
Optionally, the pharmaceutical preparation is a liquid preparation, and the auxiliary ingredient comprises glucose and chlorobutanol, and/or sorbitol or mannitol.
Optionally, in the liquid formulation, the neutralizing antibody titer of the feline parvovirus egg yolk antibody is not less than 1.
The invention also provides application of the triple egg yolk antibody or the pharmaceutical preparation in preparation of a medicine for preventing, relieving, assisting in treating or treating animal diseases caused by at least one virus infection of feline parvovirus, feline herpesvirus and feline calicivirus.
Optionally, the prevention comprises emergency prevention; the adjuvant therapy comprises adjuvant therapy with at least one of antiinflammatory, antiemetic, antidiarrheal, nasal drop and eye drop.
Optionally, the animal disease caused by feline parvovirus infection includes feline distemper; the clinical symptoms of feline fever include depressed spirit, decreased appetite, elevated body temperature, vomiting, diarrhea, and/or positive FPV detected in feces; the diarrhea comprises watery or bloody diarrhea; the appetite decrease comprises a slight decrease in appetite, a decrease in appetite, or a loss of appetite.
Optionally, the animal diseases caused by feline herpesvirus infection include digestive tract diseases and respiratory tract diseases; the clinical symptoms of the respiratory tract diseases comprise mental depression, anorexia, eye and nose secretion increase, cough, sneeze, nasal obstruction, immobility, eye and conjunctival edema, conjunctivitis and/or eye and nose secretion detection of FHV positive: the ocular and nasal secretions are watery or viscous.
Optionally, the feline calicivirus-caused animal diseases include oral diseases and respiratory diseases; the clinical symptoms of the oral diseases comprise running water and oral ulcer; the clinical symptoms of the respiratory tract diseases include fever, mental depression, sneezing, mucous or thick secretions in the eyes or nose, conjunctivitis, increased secretions, tracheitis and/or bronchitis.
Optionally, the animal disease symptoms caused by feline parvovirus, feline herpesvirus, and feline calicivirus mixed infection include vomiting, diarrhea, bloody stool, watery or purulent discharge in the eyes and nose, or canker sores.
Optionally, the use includes use in the preparation of an oral medicament or an injectable medicament.
Optionally, the application refers to application in preparation of a medicine for preventing, relieving, adjunctively treating or treating feline panleukopenia diarrhea. The feline diarrhea can be diarrhea caused by feline parvovirus (feline parvovirus) infection, and the adjuvant therapy can be combined with other drugs for treating the feline diarrhea or combined with drugs for treating other symptoms of the feline parvovirus infection to treat the feline diarrhea.
The invention also provides a preparation method of the triple egg yolk antibody, which comprises the following steps:
(1) Preparing a triple inactivated vaccine, wherein the triple inactivated vaccine is an inactivated feline parvovirus serum-free concentrate, an inactivated feline herpesvirus serum-free concentrate, an inactivated feline calicivirus serum-free concentrate and an emulsion of Freund's adjuvant, wherein the ratio of the total volume of the inactivated feline parvovirus serum-free concentrate, the inactivated feline herpesvirus serum-free concentrate and the inactivated feline calicivirus serum-free concentrate to the volume of the Freund's adjuvant is 1; the inactivated feline parvovirus serum-free concentrated solution is obtained by concentrating and inactivating a feline parvovirus solution cultured in a serum-free manner, the concentration multiple is 2-3 times, and the content of the feline parvovirus in the feline parvovirus solution is not less than 10 6.0 TCID 50 0.1ml; the inactivated feline herpesvirus serum-free concentrated solution is obtained by concentrating and inactivating a serum-free cultured feline herpesvirus solution, wherein the concentration multiple is 2-3 times, and the content of the feline herpesvirus in the feline herpesvirus solution is not less than 10 6.5 TCID 50 0.1ml; the inactivated feline calicivirus serum-free concentrated solution is obtained by concentrating and inactivating serum-free cultured feline calicivirus solution, the concentration multiple is 2-3 times, and the content of feline calicivirus in the feline calicivirus solution is not less than 10 6.5 TCID 50 /0.1ml；
(2) Feeding laying hens of 25-26 weeks old by using a mixed feed, controlling the feeding environment temperature to be 13-25 ℃, the air humidity to be 55-65% and the illumination time to be not less than 15 hours each day, wherein the mixed feed contains 0.02-0.05 wt% of L-theanine, 0.02-0.05 wt% of Piper methysticum, 0.005-0.01 wt% of vitamin E, 0.004-0.012 wt% of zinc sulfate and 0.01-0.03 wt% of taurine based on the total weight of the mixed feed;
(3) Sequentially immunizing the laying hens for 3 times by using the triple inactivated vaccine prepared in the operation (1) in dark light 1-2 weeks after feeding is started in the operation (2), wherein the immunization dose of the first immunization is 1-1.5 ml per hen, and the immunization positions are neck subcutaneous and bilateral leg muscles; the immunization dose of the second immunization is 1.5-2 ml per mouse, and the immunization parts are neck subcutaneous and chest muscles; the immunization dose of the third immunization is 2-2.5 ml per mouse, and the immunization parts are neck subcutaneous part and chest subcutaneous part; the time interval between the first immunization and the second immunization is 10 to 15 days, and the time interval between the second immunization and the third immunization is 15 to 21 days;
(4) After 3 times of immunization, when the neutralizing antibody titer of the feline parvovirus egg yolk antibody in the egg yolk is not lower than 1;
(5) Sterilizing the immunized eggs, and aseptically separating the yolk.
Optionally, the preparation method further comprises the operation of performing antibody extraction treatment and purification treatment on the egg yolk.
Optionally, the concentration by 2 to 3 times means that the volume of the virus liquid after concentration is 1/2 to 1/3 of that before concentration, or the concentration of the virus in the virus liquid after concentration is 2 to 3 times of that before concentration.
In certain preferred embodiments, the inactivated virus serum-free concentrate can be prepared by the following method: respectively culturing the feline parvovirus, the feline herpesvirus and the feline calicivirus in F-81 cells (feline kidney cells) in a serum-free manner, and then concentrating and inactivating to respectively obtain an inactivated feline parvovirus serum-free concentrated solution, an inactivated feline herpesvirus serum-free concentrated solution and an inactivated feline calicivirus serum-free concentrated solution.
The invention also provides a preparation method of the liquid medicine preparation, which comprises the following steps: taking the yolk after aseptic separation in the preparation method, homogenizing, adding 3-5 volume times of phosphate buffer solution after disinfection, then adding n-octanoic acid with the final concentration of 0.1-1 wt%, standing, centrifuging, discarding the precipitate, taking the supernatant, and concentrating 6-10 times for later use; adding glucose with the final concentration of 1-3 wt%, chlorobutanol with the final concentration of 0.3-0.5 wt% and sorbitol with the final concentration of 0-0.5 wt% or mannitol with the final concentration of 0-5 wt% into the supernatant, mixing, adjusting the pH value to 6.0-6.5, and filtering the obtained mixture.
The invention also provides a preparation method of the solid pharmaceutical preparation, which comprises the following steps: taking the yolk after aseptic separation in the preparation method, homogenizing, sterilizing, adding pharmaceutically acceptable auxiliary components, mixing, and lyophilizing.
Optionally, the auxiliary components can be vitamin C, sucrose and sodium benzoate, and 10-15 g of vitamin C, 50-100 g of sucrose and 1-1.5 g of sodium benzoate are added into every 1000 g of egg yolk.
The technical scheme of the invention has the following advantages:
1. the triple egg yolk antibody for the cats, provided by the invention, simultaneously contains a feline parvovirus egg yolk antibody, a feline herpesvirus egg yolk antibody and a feline calicivirus egg yolk antibody, the three antibodies have higher antibody titer, and the three antibodies have a synergistic interaction effect, so that the treatment effect of the triple egg yolk antibody on feline plague, feline nasal bronchitis and feline calicivirus infection is remarkably improved.
2. The triple egg yolk antibody for the cat comprises a feline parvovirus egg yolk antibody, a feline herpesvirus egg yolk antibody and a feline calicivirus egg yolk antibody. According to the embodiment of the invention, the medicine containing the triple egg yolk antibody and the medicine containing the feline panleukopenia monoclonal antibody are respectively used for treating the cat suffering from the feline panleukopenia diarrhea, and the result shows that the treatment effect of the triple egg yolk antibody on the feline panemia diarrhea is obviously better than that of the feline panemia monoclonal antibody, and the treatment time is obviously shortened. The invention expands the treatment way of the feline diarrhea and provides a new direction for the treatment of the feline diarrhea.
3. The triple egg yolk antibody for the cats is obtained by directly immunizing laying hens with combined antigen containing feline parvovirus antigen, feline herpesvirus antigen and feline calicivirus antigen, so that the triple egg yolk antibody has the advantages of pure nature, low cost, basically no drug resistance and toxic or side effect and good specificity.
4. According to the preparation method of the triple egg yolk antibody for the cats, provided by the invention, the serum-free inactivated virus concentrated solution is used as the vaccine, so that on one hand, the laying hens can generate various types of antibodies aiming at one virus, on the other hand, the stress response of the laying hens can be relieved by the serum-free vaccine, the laying rate of the laying hens is prevented from being greatly reduced, on the other hand, the laying hens can be prevented from generating antiserum antibodies, and the purity and the level of target antibodies are increased;
the method is characterized in that immunization is carried out when the laying hens are 26-27 weeks old, and the laying hens are in the egg laying peak period, so that the phenomenon that the laying rate of the laying hens cannot reach a normal level due to the influence of immune stimulation can be avoided; in addition, the laying hens are specially fed one week before the start of immunization, so that the laying hens are calmed to further relieve immune stress reaction, meanwhile, the nutrition and the resistance of the laying hens are increased, and the specific immune reaction strength of the laying hens is enhanced;
the specific immunization locus is adopted, three times of immunization are carried out under dark light when the laying hens are in a sleep state, and meanwhile, the immunization dosage and the immunization interval time are optimized, so that the target antibody level and the target antibody maintenance time of the immunized laying hens are obviously enhanced.
5. The preparation method of the triple egg yolk antibody for the cats, provided by the invention, is simple to operate and low in preparation cost, and can be used for preparing three egg yolk antibodies with high antibody titer in one preparation process.
Detailed Description
The following examples are provided to further understand the present invention, not to limit the scope of the present invention, but to provide the best mode, not to limit the content and the protection scope of the present invention, and any product similar or similar to the present invention, which is obtained by combining the present invention with other prior art features, falls within the protection scope of the present invention.
The examples do not show the specific experimental steps or conditions, and can be performed according to the conventional experimental steps described in the literature in the field. The reagents or instruments used are not indicated by manufacturers, and are all conventional reagent products which can be obtained commercially.
The source or isolation method of the biological material involved in the examples of the present invention is as follows:
f-81 cells (source: ATCC); feline parvovirus (Liu Biluo, du Huo, zhang Xiao war, etc.. Isolation and identification of feline parvovirus [ J ]. Animal medicine Advances, 2013,34 (9): 124-127.); isolation and identification of feline herpesvirus type I (zhaoshuo, li lingling, wangbao et al.: isolation and identification of feline herpesvirus type I [ J ]. Experimental zoology.2012, 7 (2): 21-25.); the isolation of feline calicivirus (Gaoyiwei, xia Bing, hu Liang, etc. Leopard and Hu cat calicivirus and the comparative study of hyper-variable region genes [ J ]. Chinese preventive veterinary academy of sciences 2003,25 (3): 179-182.); laying hens (variety: nongda III, source: beinong Dai poultry Co., ltd., quzhou county).
The sources of reagents involved in the examples of the invention are as follows:
freund's adjuvant (source: sigma, lot: P191204); white oil adjuvant (source: zheng, aide good Biotechnology Ltd., batch No. 202104005S); vitamin C (source: shiyao group vitamin pharmaceutical (Shijiazhu) Co., ltd., lot number: 1211024527); sucrose (source: guangxi sugar industry group Daohi sugar Co., ltd., batch No. 20210220051204); sodium benzoate (from Yongda chemical reagents Co., ltd., tianjin, lot No. 20210514); n-octanoic acid (source: national chemical group, ltd., lot No. 20201210); glucose (origin: national chemical group, ltd., lot: 20160531); chlorobutanol (source: national chemical group, ltd., lot number: 20200918); feline panleukopenia monoclonal antibody (preparation of monoclonal antibody specific to tiger-derived feline panleukosis virus VP2 protein [ J ]. Proc. Economy animal bulletin, 2010,14 (2): 67-70.).
Example 1
The embodiment provides a preparation method of a triple egg yolk antibody for cats, which comprises the following steps:
(1) Preparation of triple inactivated vaccine
Respectively culturing Feline Parvovirus (FPV), feline Herpesvirus (FHV) and Feline Calicivirus (FCV) by using F-81 cells (the culture solution is DMEM culture medium), and culturingCulturing F-81 cells until above 80% CPE (cytopathic effect) occurs, repeatedly freezing and thawing the culture for 3 times, centrifuging at 3000r/min to collect each virus solution, and measuring TCID of virus in each virus solution 50 . The virus content of FPV in the cat parvovirus fluid is determined to be 1 multiplied by 10 6.5 TCID 50 0.1ml, FHV virus content of cat herpes virus liquid is 1 x 10 7.0 TCID 50 0.1ml, FCV virus content in feline calicivirus fluid 1X 10 6.5 TCID 50 /0.1ml。
After concentrating the collected three serum-free virus solutions by 2 times, respectively, carrying out inactivation treatment (the inactivation condition is formaldehyde with the final concentration of 0.055-0.2 wt% at 38 ℃ for 36 hours) to obtain three inactivated virus serum-free concentrated solutions, mixing the three inactivated virus serum-free concentrated solutions, and then mixing and emulsifying the mixed solutions with Freund's adjuvant according to the volume ratio of 1 to prepare the triple inactivated vaccine containing the inactivated FPV, the inactivated FHV and the inactivated FCV.
(2) Feeding of laying hens
Feeding 25-26 weeks old laying hens with a mixed feed, controlling the feeding environment temperature to be 13-25 ℃, the air humidity to be 55-65% and the illumination time to be not less than 15h every day, wherein the mixed feed contains 0.05wt% of L-theanine, 0.03wt% of Piper methysticum, 0.006wt% of vitamin E, 0.01wt% of zinc sulfate and 0.01wt% of taurine based on the total weight of the mixed feed;
(3) Immunization of egg-laying hens
1 week after feeding is started in the operation (2), injecting and immunizing laying hens in a healthy state and with good laying rate by using the triple inactivated vaccine obtained in the step (1) under dark light, wherein the immunization times are three times, the first immunization dose is 1.5 ml/egg, and the immunization parts are neck subcutaneous and bilateral leg muscles; the second immunization dose is 2 ml/mouse, and the immunization parts are neck subcutaneous and breast muscle; the third immunization dose is 2.5ml per mouse, and the immunization parts are neck subcutaneous part and chest subcutaneous part; the first two immunizations were separated by 14 days, and the third immunization was performed 21 days after the second immunization.
After the third immunization, collecting eggs, separating the eggs from egg white and yolk, and respectively measuring the neutralizing antibody titer of the FPV egg yolk antibody, the FHV egg yolk antibody and the FCV egg yolk antibody in the yolk, wherein when the neutralizing antibody titer of the FPV egg yolk antibody in the immunized eggs is not less than 1.
(3) And (3) disinfecting the immune eggs collected in the step (2), and performing aseptic separation on egg yolks to obtain the triple egg yolk antibody for the cats.
In this example, the immunized eggs from 10 th to 180 th days after the third immunization were collected, the titer of each yolk antibody in the eggs after 180 days was lower than the collection standard, and the collection was stopped, during which the average laying rate of the laying hens was 78.5%. Titer measurements were performed on the cats produced in this example using the triple egg yolk antibody, wherein the neutralizing antibody titer of the feline parvovirus egg yolk antibody was 1.
Example 2
A triple egg yolk antibody for cats was prepared according to the method of example 1, except that: in this example, the first immunization dose was 1 ml/mouse, the second immunization dose was 2 ml/mouse, and the third immunization dose was 2 ml/mouse.
In this example, the immunized eggs 12 to 150 days after the third immunization were collected, and after 150 days, the titer of each yolk antibody in the eggs was lower than the collection standard, and the collection was stopped, and during the collection period, the laying rate of the laying hens was 79.1% on average. Titer measurements were performed on the cats produced in this example using the triple egg yolk antibody, wherein the neutralizing antibody titer of the feline parvovirus egg yolk antibody was 1.
Example 3
A triple egg yolk antibody for cats was prepared according to the method of example 1, except that: in this example, the first immunization dose was 1.5 ml/mouse, the second immunization dose was 1.5 ml/mouse, and the third immunization dose was 2.5 ml/mouse.
In this example, the immunized eggs from 10 th to 150 th day after the third immunization were collected, and after 150 days, the titer of each yolk antibody in the eggs was lower than the collection standard, and the collection was stopped, and during the collection period, the laying rate of the laying hens was 79.2% on average. Titer measurements were performed on the cats produced in this example using the triple egg yolk antibody, wherein the neutralizing antibody titer of the feline parvovirus egg yolk antibody was 1.
Example 4
A triple egg yolk antibody for cats was prepared according to the method of example 1, except that: in this example, the first two immunizations were separated by 10 days, and the third immunization was performed 21 days after the second immunization.
In this example, the immunized eggs from day 15 to day 150 after the third immunization were collected, and after day 150, the titer of each yolk antibody in the eggs was lower than the collection standard, and the collection was stopped, and during the collection period, the average laying rate of the laying hens was 78.5%. Titer measurements were performed on the cats produced in this example using the triple egg yolk antibody, wherein the neutralizing antibody titer of the feline parvovirus egg yolk antibody was 1.
Example 5
A triple egg yolk antibody for cats was prepared according to the method of example 1, except that: in this example, the first two immunizations were separated by 15 days, and the third immunization was performed 15 days after the second immunization.
In this example, the immunized eggs from 15 th to 120 th day after the third immunization were collected, the titer of each yolk antibody in the eggs after 120 days was lower than the collection standard, and the collection was stopped, during which the laying rate of the laying hens was 78.9% on average. Titer measurements were performed on the cats produced in this example using the triple egg yolk antibody, wherein the neutralizing antibody titer of the feline parvovirus egg yolk antibody was 1.
Example 6
A triple egg yolk antibody for cats was prepared according to the method of example 1, except that: the concentration times of the three virus solutions in this example were 3 times.
In this example, the immunized eggs from 8 th to 180 th day after the third immunization were collected, the titer of each yolk antibody in the eggs after 180 days was lower than the collection standard, and the collection was stopped, during which the laying rate of the laying hens was 78.1% on average. Titer measurements were performed on the cats produced in this example using the triple egg yolk antibody, wherein the neutralizing antibody titer of the feline parvovirus egg yolk antibody was 1.
Example 7
The implementation provides a preparation method of triple egg yolk antibody yolk powder for cats, which comprises the following steps:
respectively carrying out surface sterilization on the immune eggs collected in the examples 1-6, carrying out aseptic separation on egg yolks, homogenizing, carrying out pasteurization and virus inactivation treatment, adding 10g of vitamin C, 50g of cane sugar and 1.5g of sodium benzoate into every 1000 g of egg yolk, fully mixing uniformly, and carrying out low-temperature freeze-drying to obtain the triple egg yolk antibody egg yolk powder for the cats.
Example 8
The embodiment provides a preparation method of a triple egg yolk antibody injection for cats, which comprises the following steps:
respectively carrying out surface sterilization on the immune eggs collected in the examples 1-6, carrying out aseptic separation on yolk, homogenizing, carrying out pasteurization and virus inactivation treatment, adding 3 times by volume of phosphate buffer solution with pH of 7.2, mixing uniformly, adding 0.1wt% of n-octanoic acid under stirring, standing overnight at 2-8 ℃ or standing at room temperature for 4-6 hours, centrifuging at the rotating speed of 8000r/min for 20 minutes, discarding precipitates, reserving supernatant, concentrating 6 times, adding 2wt% of glucose and 0.5wt% of trichloro-tert-butanol, mixing uniformly, adjusting the pH value to 6.0, carrying out suction filtration by using a 0.22 mu m filter membrane, sterilizing and removing impurities to obtain the yolk antibody injection.
Comparative example 1
A triple egg yolk antibody for cats was prepared according to the method of example 1, except that: in this comparative example, the Freund's adjuvant was replaced with an equal amount of white oil adjuvant. Since antibody levels were consistently below the requirements, hens were immunized a fourth time 21 days after triple immunization.
In the comparative example, the immunized eggs from 10 th to 60 th days after the fourth immunization were collected, the titer of each yolk antibody in the eggs after 60 days was lower than the collection standard, and the collection was stopped, during which time the laying rate of the laying hens was 76.9%. Titer determinations were performed on the triple egg yolk antibodies for cats prepared in this comparative example, wherein the neutralizing antibody titer of the feline parvovirus egg yolk antibody was 1.
Comparative example 2
A triple egg yolk antibody for cats was prepared according to the method of example 1, except that: the three virus liquids in the comparative example are not concentrated, are directly mixed after inactivation, and are mixed and emulsified with Freund adjuvant in equal volume to obtain the triple inactivated vaccine of the comparative example.
In the comparative example, the immunized eggs from 15 th to 120 th days after the third immunization were collected, the titer of each yolk antibody in the eggs after 120 days was lower than the collection standard, and the collection was stopped, during which time the laying rate of the laying hens was 82.6%. Titer determinations were performed on the triple egg yolk antibodies for cats prepared in this comparative example, wherein the neutralizing antibody titer of the feline parvovirus egg yolk antibody was 1.
Comparative example 3
A triple egg yolk antibody for cats was prepared according to the method of example 1, except that: the culture solution used in the preparation of the triple inactivated vaccine in the step (1) of the comparative example is a DMEM culture medium added with 2% by volume of fetal bovine serum.
In the comparative example, the immunized eggs from 10 th to 160 th days after the third immunization were collected, the titer of each yolk antibody in the eggs after 160 days was lower than the collection standard, and the collection was stopped, during which time the laying rate of the laying hens was 73.2%. Titer measurements were performed on the triple egg yolk antibodies for cats prepared in this comparative example, wherein the neutralizing antibody titer of the feline parvovirus egg yolk antibody was 1.
Comparative example 4
A triple egg yolk antibody for cats was prepared according to the method of example 1, except that: in the step (2) of this comparative example, a conventional complete feed (manufacturer: beijing Xingnong feed Co., ltd.) was used and the laying hens were fed under natural conditions.
In the comparative example, the immunized eggs from 10 th to 175 th day after the third immunization were collected, the titer of each yolk antibody in the eggs after 175 days was lower than the collection standard, and the collection was stopped, during which time the laying rate of the laying hens was 55.1%. Titer determinations were performed on the cats prepared in this comparative example using the triple egg yolk antibody, wherein the neutralizing antibody titer of the feline parvovirus egg yolk antibody was 1.
Comparative example 5
A triple egg yolk antibody for cats was prepared according to the method of example 1, except that: in step (2) of this comparative example, egg-laying hens of 30 to 31 weeks old were used.
In the comparative example, the immunized eggs from 10 th to 170 th day after the third immunization are collected, the titer of each yolk antibody in the eggs is lower than the collection standard after 170 days, the collection is stopped, and the laying rate of laying hens is 45.6% during the collection period. Titer determinations were performed on the triple egg yolk antibodies for cats prepared in this comparative example, wherein the neutralizing antibody titer of the feline parvovirus egg yolk antibody was 1.
Comparative example 6
A triple egg yolk antibody for cats was prepared according to the method of example 1, except that: in step (3) of this comparative example, the injection sites for the three immunizations were all bilateral leg muscles.
In the comparative example, the immunized eggs from 10 th to 130 th days after the third immunization were collected, the titer of each yolk antibody in the eggs after 130 days was lower than the collection standard, and the collection was stopped, during which time the laying rate of the laying hens was 79.1%. Titer measurements were performed on the triple egg yolk antibodies for cats prepared in this comparative example, wherein the neutralizing antibody titer of the feline parvovirus egg yolk antibody was 1.
Experimental example 1
This experimental example serves to illustrate the use of the triple egg yolk antibody for cats of the present invention in FPV, FHV and FCV infection. The immunized eggs collected in example 1 were prepared into triple yolk antibody yolk powder (hereinafter, referred to as "yolk powder" in this experimental example) or triple yolk antibody injection (hereinafter, referred to as "injection" in this experimental example) according to the methods of examples 7 and 8.
1.1 application of triple yolk antibody for cats in FPV infection
1.1.1 test design
25 weaned kittens aged 8-12 weeks were selected and randomly divided into 5 groups of 5, each group consisting of group 1 (oral yolk powder group, prevention), group 2 (injection group, prevention), group 3 (post-challenge treatment group), group 4 (challenge control group) and group 5 (blank control). The specific test scheme is as follows:
group 1: before counteracting toxic substance, 1 g/egg yolk powder is orally administered for 5 days.
Group 2: before toxin counteracting, subcutaneous injection is injected every day, 1 ml/injection, and continuous injection is carried out for 5 days.
Group 3: before counteracting toxic pathogen, normal diet.
Group 4: before counteracting toxic pathogen, normal diet.
Group 5: normal diet throughout the test period.
On day 6, FPV cell culture virus oral administration challenge was performed on groups 1-4, with FPV virus titer of 10 5 TCID 50 0.1ml, 300. Mu.l/cat. Group 5 was fed completely separately from groups 1 to 4. The status of the cats was observed and recorded daily for two weeks. The disease occurrence judgment standard is as follows: mental depression, anorexia, and increased body temperatureFPV positive is detected by vomit, diarrhea and feces.
1.1.2 test results
The results show that the attack control group (group 4) has 100% of morbidity and 100% of mortality, compared with the attack control group, the egg yolk powder oral group (group 1) and the injection group (group 2) have good emergency prevention effect, the morbidity is 40%, and the egg yolk powder oral group and the injection group both survive after the test. In the treatment group after challenge (group 3), the clinical symptoms of feline panleukopenia begin to appear from day 4 to day 5 after challenge, and the treatment is started at the moment, and the treatment mode is as follows: the yolk powder is orally taken at a dose of 1.5g/kg for 2 times/day, and adjuvant treatments such as fever reduction, inflammation diminishing, antiemetic, and diarrhea relieving are simultaneously carried out (amoxicillin potassium clavulanate, omeprazole and vitamin B complex, which are administered according to the prescribed dosage). On the 3 rd day from the beginning of treatment, diarrhea or bloody stool of 2 cats is obviously relieved, and the body temperature is gradually normal; on day 4, 3 cats had significantly reduced diarrhea symptoms, the diarrhea turned into soft stool, and the diet increased; on day 5, diarrhea symptoms of 5 cats are improved, the appetite is obviously increased, and on day 7, the mental state is completely recovered, clinical symptoms disappear, the appetite is recovered to before the test and even increased, and after day 7, the egg yolk powder is continuously eaten for 3 days for consolidation. The blank control (group 2) cats had no clinical symptoms of feline fever and the test was established.
The results show that the triple egg yolk antibody for cats has good emergency prevention effect and obvious treatment effect on FPV infection, and the cure rate is 100%.
TABLE 1 yolk antibody Emergency prevention test
Remarks are: grade 1 (relatively mild clinical symptoms): the body temperature is increased, the spirit is deep and depressed, the appetite is slightly reduced, occasionally vomit and slight diarrhea are caused, and FPV positive is detected in excrement; grade 2 (relatively severe clinical symptoms): rising body temperature, lethargy, depression, anorexia, emesis, watery sample or bloody diarrhea, and FPV positive detected in feces; grade 3 (severe clinical symptoms): body temperature rise, extreme depression, loss of appetite, vomiting, watery or bloody diarrhea, and positive FPV detected in feces.
1.2 application of triple yolk antibody for cats in FHV infection
1.2.1 test design
20 weaned kittens aged 8-12 weeks are selected and randomly divided into 4 groups, and each group comprises 5 cats, namely a group 1 (oral yolk powder group, prevention), a group 2 (injection group, prevention), a group 3 (after-treatment group after toxicity attack) and a group 4 (control group after toxicity attack). Blank group 5 blank "1.1 use of triple egg yolk antibody in FPV infection in cats" was used as reference. The specific test scheme is as follows:
group 1: before counteracting toxic materials, 1 g/egg yolk powder is orally taken every day for 5 days.
Group 2: before toxin counteracting, subcutaneous injection is injected every day, 1 ml/injection, and continuous injection is carried out for 5 days.
Group 3: before counteracting toxic pathogen, normal diet.
Group 4: before counteracting toxic pathogen, normal diet.
On day 6, FHV cell culture was performed on groups 1 to 4 to achieve nasal administration with a FHV virus titer of 10 5.5 TCID 50 0.1ml, 300. Mu.l/cat. The status of the cats was observed and recorded daily for two weeks. The disease occurrence judgment standard is as follows: depression, anorexia, sneezing, watery or sticky eye and nose secretion, cough, nasal obstruction, involuntary movement, edema of conjunctiva and eye, conjunctivitis, and positive FHV.
1.2.2 test results
The results show that the attack control group (group 4) has 100% of morbidity and 100% of mortality, compared with the attack control group, the egg yolk powder oral group (group 1) and the injection group (group 2) have good emergency prevention effects, the morbidity is 60% and 40%, and the egg yolk powder oral group and the injection group both survive after the test. In the treatment group after toxin attack (group 3), obvious clinical symptoms of nasal branches of cats appear from day 4 to day 5 after toxin attack, and treatment is started at the moment, wherein the treatment mode is as follows: the yolk powder is orally taken at a dose of 1.5g/kg for 2 times/day, and adjuvant treatment by eye drop and antiphlogistic treatment (amoxicillin potassium clavulanate and fibrate eye drop) is carried out. On the 3 rd day from the beginning of treatment, the clinical symptoms of the sick cats are obviously improved, the sick cats can drink water independently from no appetite, on the 4 th day, the symptoms are slightly relieved, on the 5 th day, the appetite is recovered and can eat independently, the health condition is obviously improved, on the 7 th day, the mental state is completely recovered, the clinical symptoms basically disappear, the appetite is recovered to the extent before the test and even increased, and after the 7 th day, the sick cats continue to eat the egg yolk powder for 3 days for consolidation.
The results show that the triple egg yolk antibody for cats has good emergency prevention effect and obvious treatment effect on FHV infection, and the cure rate is 100%.
1.3 application of triple yolk antibody for cats in FCV infection
1.3.1 test design
20 weaned kittens aged 8-12 weeks are selected and randomly divided into 4 groups, and each group comprises 5 cats, namely a group 1 (oral yolk powder group, prevention), a group 2 (injection group, prevention), a group 3 (after-treatment group after toxicity attack) and a group 4 (control group after toxicity attack). Blank group 5 blank "1.1 use of triple egg yolk antibody in FPV infection in cats" was used as reference. The specific test scheme is as follows:
group 1: before counteracting toxic substance, 1 g/egg yolk powder is orally administered for 5 days.
Group 2: before toxin challenge, injection is subcutaneously injected every day for 5 days, with 1ml per injection.
Group 3: before counteracting toxic pathogen, diet is normal.
Group 4: before counteracting toxic pathogen, normal diet.
On day 6, FCV cell culture virus of 2 × 10 titer was administered to groups 1-4 for nasal administration 5.0 TCID 50 0.1ml, and the dosage is 500. Mu.l/cat. The status of the cats was observed and recorded daily for two weeks. The disease occurrence judgment standard is as follows: fever, mental depression, sneeze, eye or nose mucous or thick secretion, conjunctivitis, watery mouth, oral ulcer, secretion increase, tracheitis, bronchitis, eye and nose secretion, FCV positive detection, etc.
1.3.2 test results
The results show that the attack control group (group 4) has 100% of morbidity, typical symptoms such as oral ulcer, eye and nose secretion increase, fever, sneezing, mental depression, diet reduction and the like and 40% of mortality, and compared with the attack control group, the egg yolk powder oral group (group 1) and the injection group (group 2) have good emergency prevention effect, the morbidity is 40% and 60%, and the test is finished and the egg yolk powder oral group and the injection group are alive. In the post-challenge treatment group (group 3), significant clinical symptoms began to appear on day 3 after challenge, mainly manifested as oral and lingual blisters, followed by ulceration, decreased appetite, poor spirit and increased eye and nose secretions. The disease cats in the post-challenge treatment group (group 3) were treated from the onset of typical symptoms by the following treatment regimen: yolk powder is orally taken every day at a dose of 1.5g/kg for 2 times/day, and is used for adjuvant treatment such as anti-inflammatory, antipyretic, antivirus and ulcer surface treatment (doxycycline, interferon, fibrate eye drop and vitamin B2, which are administered according to a prescribed dosage). On the 5 th day from the beginning of treatment, the clinical symptoms of the sick cats are obviously improved, the oral cavity blisters are reduced, the ulcers gradually begin to heal, the mental state is recovered and the clinical symptoms basically disappear by the 7 th day, and the triple egg yolk powder is continuously eaten for 3 days after the 7 th day for consolidation.
The results show that the triple egg yolk antibody for cats has good emergency prevention effect and obvious treatment effect on FCV infection, and the cure rate is 100%.
Experimental example 2
This experimental example is intended to illustrate the use of the triple yolk antibody for cats in the mixed infection of FPV, FHV and FCV according to the invention. The immune eggs collected in example 1 were prepared into triple yolk antibody yolk powder or triple yolk antibody injection according to the methods of examples 7 and 8.
Selecting 15 clinically infected kittens of 8-15 weeks old cats with feline distemper, feline herpes and feline calicivirus, detecting FPV, FHV and FCV nucleic acid positively, and showing symptoms of vomiting, diarrhea or bloody stool, watery or purulent secretion in eyes and noses and ulcer in oral cavity with different areas. The test cats were randomly divided into 3 groups of 5 cats each. Group 1 was treated with the triple egg yolk antibody yolk powder 2 times a day at 1.5 g/time, and at the same time, was treated with adjunctive therapies such as anti-inflammatory, antipyretic, antiemetic, antiviral, etc. (amoxicillin potassium clavulanate, omeprazole, cerinin, interferon, fibrate eye drops and vitamin B complex, dosed as prescribed). Group 2 was treated with triple yolk antibody injection 1 time per day at 1 ml/time, and at the same time, was treated with adjuvant treatments such as anti-inflammatory, antipyretic, antiemetic, antiviral, etc. (amoxicillin clavulanate potassium, omeprazole, cerinin, interferon, fibrate eye drops and vitamin B complex, administered at prescribed doses); group 3 was treated with feline panleukopenia mab by injection 1 time per day at a dose of 1 ml/time, and adjuvant treatments such as anti-inflammatory, antipyretic, antiemetic, and antiviral (amoxicillin clavulanate potassium, omeprazole, cerinine, interferon, fibrate eye drop, and vitamin B complex, administered at prescribed doses). The results show that the cure rates of the three modes are respectively 100%, 100% and 80%.
Group 1, on day 4 of treatment, diarrhea and bloody stool were significantly improved in 3 cats, and appetite was increased; on day 6, nasal secretion of 2 cats eyes is relieved, whole clinical symptoms are relieved, and diarrhea or bloody stool is obviously relieved; on the 8 th day, the nasal secretion of 4 cats are obviously improved, the clinical symptoms of 4 cats basically disappear, diarrhea and bloody stool become soft stool, the oral ulcer gradually heals, and the body temperature is normal; on day 10, clinical symptoms disappeared in all 5 cats.
Group 2, on day 4 of treatment, diarrhea and bloody stool were significantly improved in 2 cats, and appetite was increased; on day 6, 3 cat eye nasal secretions were significantly reduced; on day 8, 5 cat eye nasal secretions were significantly reduced, of which 3 had no diarrhea or bloody stools; on day 9, the clinical symptoms basically disappear, the oral ulcer is healed, the body temperature is normal, and on day 10 to day 11, the clinical symptoms of 5 cats disappear.
Group 3, treatment day 4, 2 cats had reduced diarrhea; on day 6, 3 cats had diarrhea or reduced bloody stools; on day 8, nasal secretion was improved in 2 cats, diarrhea and bloody stool were soft stool in 4 cats, and body temperature was normal; on the 10 th day, 3 cat eye nasal secretions are obviously reduced, 2 cat eye nasal secretions become sticky, and 4 cats gradually improve diarrhea or bloody stool; on day 12, 1 cat had continued to develop pneumonia due to diarrhea and fever; on day 13, cats with a shift to pneumonia died; on day 15, all clinical symptoms disappeared in the remaining 4 cats, the canker sore healed, and the body temperature was normal.
The results show that the triple egg yolk antibody yolk powder has a good adjuvant treatment effect on diarrhea caused by the feline panleukopenia virus, the treatment effect on the diarrhea is slightly superior to that of the egg yolk antibody injection, and the course of disease is shortened by about 1 day; compared with the feline panleukosis monoclonal antibody, the triple egg yolk antibody yolk powder shortens the whole treatment time by about 5 days. The treatment effect of the triple yolk antibody injection is also superior to that of the feline panleukopenia monoclonal antibody, and the course of disease is shortened by 4-5 days. The results show that the triple egg yolk antibody for the cats has obvious treatment effect.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications derived therefrom are intended to be within the scope of the invention.
Claims (8)
1. A preparation method of a triple egg yolk antibody is characterized by comprising the following steps:
(1) Preparing a triple inactivated vaccine, wherein the triple inactivated vaccine is an emulsion of inactivated feline parvovirus serum-free concentrate, inactivated feline herpesvirus serum-free concentrate, inactivated feline calicivirus serum-free concentrate and Freund's adjuvant, wherein the ratio of the total volume of the inactivated feline parvovirus serum-free concentrate, the inactivated feline herpesvirus serum-free concentrate and the inactivated feline calicivirus serum-free concentrate to the volume of the Freund's adjuvant is 1; the inactivated feline parvovirus serum-free concentrated solution is obtained by concentrating and inactivating serum-free cultured feline parvovirus solution, the concentration multiple is 2-3 times, and the content of the feline parvovirus in the feline parvovirus solution is not less than 10 6.0 TCID 50 0.1ml; the inactivated feline herpesvirus serum-free concentrated solution is obtained by concentrating and inactivating a serum-free cultured feline herpesvirus solution, wherein the concentration multiple is 2-3 times, and the content of the feline herpesvirus in the feline herpesvirus solution is not less than 10 6.5 TCID 50 0.1ml; the said extinguisherThe serum-free concentrated solution of the live feline calicivirus is obtained by concentrating and inactivating serum-free cultured feline calicivirus solution, the concentration multiple is 2 to 3 times, and the content of the feline calicivirus in the feline calicivirus solution is not less than 10 6.5 TCID 50 /0.1ml；
(2) Feeding laying hens of 25-26 weeks old by using a mixed feed, controlling the feeding environment temperature to be 13-25 ℃, the air humidity to be 55-65% and the illumination time to be not less than 15 hours each day, wherein the mixed feed contains 0.02-0.05 wt% of L-theanine, 0.02-0.05 wt% of Piper methysticum, 0.005-0.01 wt% of vitamin E, 0.004-0.012 wt% of zinc sulfate and 0.01-0.03 wt% of taurine based on the total weight of the mixed feed;
(3) Sequentially immunizing the laying hens for 3 times by using the triple inactivated vaccine prepared in the operation (1) in dark light 1-2 weeks after feeding is started in the operation (2), wherein the immunization dose of the first immunization is 1-1.5 ml per hen, and the immunization positions are neck subcutaneous and bilateral leg muscles; the immunization dose of the second immunization is 1.5-2 ml per mouse, and the immunization parts are neck subcutaneous and chest muscles; the immunization dose of the third immunization is 2-2.5 ml per mouse, and the immunization parts are neck subcutaneous part and chest subcutaneous part; the time interval between the first immunization and the second immunization is 10-15 days, and the time interval between the second immunization and the third immunization is 15-21 days;
(4) After 3 times of immunization, when the neutralizing antibody titer of the feline parvovirus egg yolk antibody in the egg yolk is not less than 1;
(5) Sterilizing the immunized eggs, and aseptically separating the yolk.
2. A triple egg yolk antibody for cats prepared by the preparation method of claim 1, comprising: feline parvovirus egg yolk antibodies, feline herpesvirus egg yolk antibodies, and feline calicivirus egg yolk antibodies.
3. The triple egg yolk antibody of claim 2, wherein in the triple egg yolk antibody, the feline parvovirus egg yolk antibody has a neutralizing antibody titer of not less than 1.
4. A pharmaceutical preparation comprising the triple egg yolk antibody of claim 2 or 3 and a pharmaceutically acceptable auxiliary ingredient.
5. The pharmaceutical formulation of claim 4, wherein the pharmaceutical formulation is a solid formulation, and the adjunct ingredients include at least one of vitamin C, sucrose, glucose, or sodium benzoate;
or the pharmaceutical preparation is a liquid preparation, and the auxiliary components comprise glucose and chlorobutanol and/or sorbitol or mannitol.
6. Use of the triple egg yolk antibody of claim 2 or 3 or the pharmaceutical formulation of claim 4 or 5 in the manufacture of a medicament for the prevention, alleviation, co-treatment or treatment of a disease in an animal caused by infection with at least one of feline parvovirus, feline herpesvirus and feline calicivirus.
7. A method of preparing a liquid pharmaceutical formulation, comprising:
taking the yolk after aseptic separation in claim 1, homogenizing, sterilizing, adding 3-5 times by volume of phosphate buffer solution, then adding n-octanoic acid with a final concentration of 0.1-1 wt%, standing, centrifuging, discarding the precipitate, taking the supernatant, concentrating 6-10 times for use;
adding glucose with the final concentration of 1-3 wt%, chlorobutanol with the final concentration of 0.3-0.5 wt% and sorbitol with the final concentration of 0-0.5 wt% or mannitol with the final concentration of 0-5 wt% into the supernatant, mixing, adjusting the pH value to 6.0-6.5, and filtering the obtained mixture.
8. A method of preparing a solid pharmaceutical formulation comprising:
homogenizing the aseptically separated egg yolk of claim 1, sterilizing, adding pharmaceutically acceptable adjuvants, mixing, and lyophilizing.
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