WO2007068154A1

WO2007068154A1 - A method for preparing specific igy against mutant avian influenza virus and the preparation tηereof

Info

Publication number: WO2007068154A1
Application number: PCT/CN2006/000212
Authority: WO
Inventors: Shing Paau; Rongjian Yang; Chang'an Wang; Tingying Cai; Haiwei Bao
Original assignee: Jason Medical Group (Far East) Ltd
Priority date: 2005-10-31
Filing date: 2006-02-13
Publication date: 2007-06-21
Also published as: CN1958608A; CN1958608B

Abstract

The present invention provides a method for preparing specific IgY against mutant avian influenza virus and the preparation thereof. To solve the problems in prior art, such as that the existing products can not resist mutant avian influenza virus, the finished product, i.e. specific IgY against mutant avian influenza virus, can be obtained by preparing a vaccine against mutant avian, influenza virus, innoculating avians capable of laying eggs with the vaccine, obtaining immunized eggs against avian influenza virus, extracting raw IgY from the yolks of those eggs, purifing the raw IgY, and filtering off bacteriums and viruses through, special equipments. Specifically, it may be specific IgY against mutant avian influenza virus, sepcific IgY against polypeptide HA2 of avian influenza virus, or specific IgY against the receptor of avian influenza virus. Specific complex IgY is obtained by mixing specific IgY against mutant avian influenza virus and specific IgY against secondary invader in appropriate proportion. And various preparations capable of preventing and curing avian influenza can be obtained by mixing specific IgY against mutant avian influenza virus or specific complex IgY against mutant avian influenza with, 99.99-80.0% adjuvants.

Description

Preparation method and preparation of anti-variant avian influenza virus specific Y

The invention relates to an antibody and a novel preparation thereof, in particular to an anti-variant avian influenza virus specific IgY and a novel preparation thereof, and belongs to the technical field of medical and health. Background technique

Avian influenza is the abbreviation for avian influenza (English for avian influenza, AI for short), which is avian infection caused by influenza A virus, mainly in chicken, turkey, pearl and other Birds, especially migratory waterfowl such as wild ducks and swans. In recent years, bird flu has become more and more frequent in the world, which has not only brought heavy blows to the poultry breeding industry in these countries, but also seriously threatened human health and life. The World Health Organization has classified avian influenza as a Class A animal disease.

In view of the serious harm of avian flu, scientists have been exploring how to prevent and treat bird flu, with a particular focus on vaccine research, and others have tried to control the general anti-avian flu virus IgY using common conventional methods. However, due to the avian influenza virus and human influenza virus, its antigenicity will continue to drift and mutate, and the rate of development of avian influenza vaccine cannot keep up with the speed of virus mutation, so the actual effect is not ideal; Resistance to certain avian influenza virus IgY, due to the constant mutation of the avian influenza virus, has no practical value. At present, the drugs such as “Tamimin” and “Leeqing” that are sold by countries all over the world have certain therapeutic effects on influenza A. In the absence of specific drugs for avian flu, people should not treat it as a treatment. The expedient choice of avian flu. However, these drugs have obvious side effects on the central nervous system and are prone to the spread of drug-resistant toxic beads. Recently, avian influenza virus strain resistant to [Tamiflu] has appeared in Indonesia; thus limiting its clinical Applications. Moreover, these drugs are not likely to be used to prevent avian flu. Technical content

In view of the above defects of the prior art, the present invention solves the problem of the avian influenza virus variation and the existing anti-avian influenza products have obvious side effects on the central nervous system, are prone to drug resistance, and the prevention effect is not obvious, so as to provide a Anti-variant avian influenza virus-specific IgY and its preparation.

In order to solve the above technical problems, the present invention provides a method for preparing an anti-variant avian influenza virus-specific IgY, which comprises the following steps: (51) Preparing an anti-mutation vaccine against mutant avian influenza virus by method;

(52) using the anti-variation bird flu vaccine, intensive injection immunization of the laying poultry, and extracting the anti-variable avian influenza immune egg produced by the immunized poultry;

(53) taking the egg yolk of the anti-variant avian influenza-immunized egg to prepare a crude extract of the specific avian influenza-resistant IgY;

(54) purifying the crude avian influenza-specific IgY extract to obtain a strain-resistant IgY-specific pure product;

(55) The anti-variant avian influenza-specific IgY pure product is filtered to filter out various bacteria and viruses to obtain a variant AgY-specific finished product.

In the present invention, the anti-variant avian influenza specific IgY may be an anti-variant avian influenza virus-specific IgY. At this time, in the step (S1), the anti-variant avian influenza vaccine may be prepared as follows:

Selected representative and most frequently occurring avian influenza virus strains, including Avian influenza A (H5N1) strain, H5N2 strain, H7N7 and H9N2 strains;

The H5N1, H5N2, H7N7 and H9N2 strains were cultured in the chicken embryo allantoic sac by the conventional chicken embryo allantoic sac, and the virus-containing allantoic fluid was collected, and the chicken red blood cell method was used for crude extraction, and then the sucrose density gradient was used for speeding. Purification by centrifugation or gel column chromatography to obtain purified four avian influenza viruses;

The purified four avian influenza viruses were separately added to 20% sodium dodecyl sulfate (SDS) at a final concentration of 2.0%, and lysed for 30 minutes to prepare four avian influenza viruses including H5N1, H5N2, H7N7 and H9N2. a lysate of a polypeptide in a conserved region;

Taking at least one of the four avian influenza virus lysates, adding Freund's adjuvant in a ratio of 1-10:1-10, and placing it in a high-speed homogenizer to homogenize at a high speed of 8,000-30,000 rpm to form a water-in-oil liquid, that is, a vaccine containing a plurality of avian influenza virus lysing polypeptide components

To prepare a variant avian influenza virus-specific IgY, in the step (SI) of the present invention, an anti-variant avian influenza vaccine can also be prepared as follows:

Inactivate or deactivate the avian influenza virus vaccine provided by the local quarantine machine, add 20% sodium dodecyl sulphate (SDS), the final concentration is 2.0%, and lyse for 30 minutes to obtain avian influenza virus lysate; The 1:1:1-10 ratio was added to the Freund's adjuvant, placed in a high-speed homogenizer, and homogenized at 8,000-30,000 rpm to form a water-in-oil emulsion, thereby preparing a novel vaccine against variant avian influenza.

In the present invention, the anti-variant avian influenza specific IgY may also be an anti-avian influenza virus polypeptide HA ₂ specific IgY. In this case, in the step (S1), the avian influenza virus polypeptide HA ₂ may be prepared as follows: antigen:

The hemagglutinin (HA ₂ ) gene was cloned from the Avian influenza A RNA by RT-PCR, and the signal peptide and the fragment of the transmembrane region were removed;

The pGEM-T vector was inserted first, and the sequence of the obtained gene was confirmed by sequencing. Digested with EcoR I and Not I, the target fragment was electrophoresed and linked to the yeast expression vector PPIC9K digested with the same double enzyme;

Transformation of E. coli, picking positive clones, extracting the plasmid, and after correcting the restriction enzyme, electroporating Pichia pastoris KM71 and GS115;

Positive clones were screened on histidine-free medium, and then high-copy transformants were selected on medium containing different concentrations of G418; single colonies were picked and inoculated into the medium and cultured overnight at 28 ° shaker;

After the dilution, the culture is continued. When the absorbance of the bacterial concentration reaches OD600 is about 0.8, the medium is replaced with a medium containing methanol, and the culture is continued for 24-48 hours;

The samples were sampled at different times in the culture, and the expression level of HA in the supernatant was determined by ELISA. The time of the highest expression was harvested, and the cell pellet was removed by centrifugation, and the supernatant contained a large amount of expression product;

The dialysis bag with a molecular weight cut off of 10 kd was precipitated with 50% ammonium sulfate, and dialyzed against distilled water for 24 hours, and separated by SepHAcryl S-200 and SepHAcryl S-100 column to obtain purified avian influenza HA ₂ antigen;

The avian influenza virus polypeptide HA ₂ antigen is added to Freund's adjuvant in a ratio of 1-10:1-10, and placed in a high-speed homogenizer to homogenize at a high speed of 8,000-30,000 rpm to form a water-in-oil emulsion, which is obtained. Avian influenza HA ₂ expressing protein vaccine.

In the present invention, the anti-variant avian influenza specific IgY may also be an anti-avian influenza virus receptor-specific IgY. At this time, in the step (S1), the avian influenza virus receptor vaccine may be prepared as follows: Take the influenza virus receptor, and mix the glycoprotein j and "9-0-acetoacetylneuraminic acid" expressed protein in a ratio of 1:1 to prepare a solution of 200 μg/mL, then 1-10:1. The virus-containing vaccine was prepared by adding Freund's adjuvant in a ratio of -10, placing it in a high-speed pulper, and homogenizing at a high speed of 8,000-30,000 rpm to form a water-in-oil emulsion. Wherein, the influenza virus receptor can be obtained by the following steps: cloning from influenza virus receptors, namely "glycoprotein" and "9-0-acetyl-N-acetylneuraminic acid" RNA by RT-PCR The peptide antigen gene is deleted from the signal peptide and the fragment of the transmembrane region; the pGEM-T vector is inserted first, and the obtained gene sequence is confirmed by sequencing, and then digested with the restriction enzymes EcoR I and Not l, and the target fragment is electrophoresed and recovered. It is ligated with the yeast expression vector PPIC9K digested with the same double enzyme; transformed into Escherichia coli, picked positive clones, and extracted plasmids. After digestion and identification, the electroporated Pichia pastoris KM71 and GS115; The positive clones were screened on the acid medium, and then the high-copy transformants were selected on the medium containing different concentrations of G418, and a single colony was picked and inoculated into the medium, and cultured overnight at 28 ° shaker; the culture was continued after dilution. When the absorbance of the bacteria concentration reaches OD600 is about 0.8, the medium is changed to the medium containing methanol, and the culture is continued for 24-48 hours; the samples are sampled at different times of the culture, and the supernatant is determined by ELISA. Protein expression, the highest expression amount of time selected from the harvested cell pellet was removed by centrifugation, the supernatant containing a large amount i.e. the expression product; 50% sulfur by Ammonium acid precipitation, dialysis bag with molecular weight cutoff of 10kd was dialyzed against distilled water for 24 hours, and SepHAcryl S-200 and SepHAcryl S-100 column chromatography were used to obtain purified influenza virus receptors - "glycoprotein" and "9" respectively. The antigenic component of -0-acetyl-N-acetylneuraminic acid.

The present invention also provides a method for preparing an anti-secondary pathogen-specific IgY, which comprises the following steps -

(521) Prepare secondary infection pathogen antigens as follows: Group A cluster B hemolytic streptococcus, Streptococcus pneumoniae, Haemophilus influenzae, MRSA golden Staphylococcus aureus, and tuberculosis bacteria 1-10:1 -10:1-10:1-10:1-10 ratio mixing, to obtain a mixture of pathogenic bacteria, and then add the pathogenic bacteria mixture to Freund's adjuvant in a ratio of 1-10:1-10, with high speed The slurry is treated at 8,000-30,000 rpm to form a water-in-oil emulsion, that is, a secondary antigen-infecting complex antigen is prepared;

(522) using the secondary infection pathogenic antigen, injecting and immunizing the laying poultry, and extracting the immune egg against the secondary infection pathogenic bacteria produced by the immunological poultry;

(523) taking the egg yolk against the secondary infection-infecting pathogen, and preparing the crude IgY extract against the secondary pathogenic bacteria;

(524) purifying the crude IgY-specific anti-secondary pathogen-specific pathogen-specific IgY product;

(525) The anti-secondary infection-infecting pathogen-specific IgY pure product is filtered to filter out various bacteria and viruses to obtain a finished IgY-resistant product against secondary pathogenic bacteria.

The present invention also provides a method for preparing an anti-avian influenza specific complex IgY, wherein the variant avian influenza specific IgY obtained by the method according to any one of claims 1 to 6 is obtained according to claim 8. The anti-secondary infection pathogenic bacteria specific I _g Y prepared by the method is mixed in a ratio of 1 - 10: 1 - 10 to prepare a specific complex IgY against the avian influenza.

The invention also provides an anti-variant avian influenza preparation, which comprises:

An anti-variant avian influenza specific IgY prepared according to the method of any one of claims 1 to 6 in an amount of 0.01 to 20.0%, or a content of 0.01 to 20.0%, which is obtained by the method according to claim 9. Anti-variant avian influenza specific composite IgY;

And accessories with a content of 99.99 - 80.0%;

The preparation is an atomizing agent, a nasal spray, a nasal drop, an eye drop, a throat spray, a mouth spray, a buccal tablet, an oral solution, a hand lotion, a spray disinfectant, a capsule or an injection.

The invention utilizes anti-variant avian influenza immunoglobulin (IgY) to protect local mucosa (nasal cavity, upper respiratory tract) cells from avian influenza virus (including avian influenza virus after mutation), which is the key to blocking infection. The adsorption of avian influenza virus directly acts as a blocking effect and is one of the mechanisms for preventing the specific avian influenza IgY. The anti-variant avian influenza-specific IgY can also neutralize the newly released virus of avian influenza patients, and lose the ability to spread and spread again, thus achieving the dual purpose of both treatment and prevention of avian influenza transmission. detailed description

IgY (Immunoglobulin of yolk) is an IgG-like immunoglobulin with a neutralizing antibody that specifically binds to the corresponding antigen, thereby changing or inhibiting the state or activity of the antigen (such as a virus). Prevents the antigen (such as virus) from being adsorbed to susceptible cells; in addition, IgY binds to its corresponding virus to form an immune complex, which is easily swallowed by macrophages.

As mentioned above, the antigens of avian flu are constantly drifting and mutating. In addition to seriously affecting the actual effects of the relevant vaccines, it also brings certain difficulties to the development of anti-avian influenza-specific IgY, and it is not possible to follow the conventional methods.

Studies have revealed that, like human influenza viruses, there are two peptide antigens on the surface of avian influenza viruses, namely hemagglutinin (HA) and neuraminidase (NA). These two antigenic structures are subject to change, ie drift or variation. Among them, HA is composed of a heavy chain (HA0 and a light chain (HA ₂ ) linked by a disulfide chain, and its antibody inhibits blood coagulation and neutralizes the virus, and is the most important protective antibody. The carboxyl group of HA ₂ polypeptide The end is located in the viral envelope, and the amino terminus (ie, the fusion) of the HA ₂ polypeptide is hidden inside the three-dimensional structure of the HA protein. When the avian influenza virus contacts the susceptible cells, the disulfide chain of HA is broken and cleavage into HAi and HA. ₂ , at this time, the amino terminal of HA ₂ , ie, the fusion, will be exposed, causing the viral envelope to fuse with the susceptible cell membrane, and then the viral scorpion scorpion enters the cytoplasm and proliferate. HA ₂ is conserved among various types and subtypes. The protein has a relatively stable structure and small variation, and is a protein component that mediates fusion of the avian influenza virus envelope with the susceptible cell membrane; therefore, the present invention provides a novel anti-HA ₂ against the variability of avian influenza virus. IgY antibodies, with this specific anti-HA ₂ polypeptide specific antibodies prevent fusion of the avian influenza virus envelope and the cell membrane susceptible to the avian influenza virus can not enter the cells, thereby achieving the prevention and treatment of avian influenza of.

In addition, a large number of experimental studies have found that avian influenza virus, like human influenza virus, requires the virus to bind to specific receptors on target cells when infecting human cells. The specific receptors for influenza viruses A and B are "sugars." Protein" and "9-0-acetoacetyl neuraine". According to this feature, the present invention provides a specific IgY which is resistant to a cold virus receptor, and the specific antibody and the influenza virus-specific receptor on the surface of the cell membrane in vivo are bound by spraying or oral administration or injection. If the flu virus loses its receptor, it cannot bind to the target cells, and naturally it will not infect the human body.

1. Because IgY is a polyclonal antibody, it can interact with a variety of pathogens, which is one of its advantages; therefore, it is possible to produce an anti-avian influenza-specific IgY against multiple subtypes, rather than the current vaccine. It works against a certain subtype of avian influenza virus, which can achieve better practical prevention and treatment effects.

2. In the present invention, according to the screening analysis of human influenza avian influenza subtypes confirmed by Asian countries and European countries in recent years, the most representative avian influenza viruses are selected as follows: Type A avian influenza Virus H5N1 strain, H5N2 strain, H7N7 and H9N2 strain.

3. Cultivation of representative avian influenza virus and purification: In the present invention, the H5N1, H5N2, H7N7 and H9N2 strains are cultured in the chicken embryo allantoic sac by conventional chicken embryo allantoic sac, and then the urine containing the virus is collected. The cyst fluid is then crudely extracted by chicken red blood cell method, and then the virus is purified by sucrose density gradient ultracentrifugation or gel column chromatography.

4. Preparation of vaccine components: In the present invention, four methods are used to prepare anti-mutation vaccine components.

4.1, virus lysis method for the production of avian influenza virus polypeptide antigen

The above-mentioned various avian influenza viruses were separately added to 20% sodium dodecyl sulfate (SDS) at a final concentration of 2.0%, and lysed for 30 minutes to obtain the above various avian influenza virus lysates. SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) test analysis, concentrated gel 4%, separation gel 7%, 240V, electrophoresis for 30 minutes, and then stained with Coomassie blue, observe the protein band; Detection to determine hemagglutinin heavy chain (HA0, hemagglutinin light chain (HA ₂ ), neuraminidase (NA:), nuclear protein (NP:), P protein (IV P ₂ , P ₃ ), matrix protein (1⁄4, M ₂ ) and non-structural proteins (NS, NS ₂ ). Consistent with the results of experiments conducted by Enami M et al.

4.2. Genetic engineering recombinant method for the production of avian influenza virus polypeptide HA ₂ antigen

The hemagglutinin (HA ₂ ) gene was cloned from the Avian influenza virus RNA by RT-PCR, and the signal peptide and the fragment of the transmembrane region were removed. The pGEM-T vector was inserted first. After sequencing, the obtained gene sequence was confirmed to be correct, and digested with restriction enzymes EcoR I and Not I, and the target fragment was electrophoresed and linked, and ligated with the yeast expression vector PPIC9K digested with the same double enzyme. Transform E. coli. Positive clones were picked, plasmids were extracted, and after digestion and identification, Pichia pastoris KM71 and GS115 were electrotransformed. Positive clones were screened on histidine-free medium and then screened for high copy transformants on media containing different concentrations of G418. A single colony was picked and inoculated into the medium and incubated overnight at 28 degrees on a shaker. Continue to culture after dilution. When the absorbance of the bacterial concentration reaches OD600 is about 0.8, the medium is changed to a medium containing methanol, and the culture is continued for 24-48 hours. The samples were sampled at different times in the culture, and the expression level of HA in the supernatant was determined by ELISA. Harvest the time with the highest expression. The cell pellet was removed by centrifugation. The supernatant contains a large amount of expressed product. The dialysis bag with a molecular weight cut off of 10 kd was precipitated with 50% ammonium sulfate, and dialyzed against distilled water for 24 hours, and separated by SepHAcryl S-200 and SepHAcryl S-100 column to obtain a vaccine component of the purified avian influenza virus HA ₂ antigen.

4.3. Genetic engineering recombinant method for making influenza virus receptor antigen

The signal peptide and the transmembrane region fragment were removed from the influenza virus receptors, namely the "glycoprotein" and "9-0-acetoacetylneuraminic acid" RNA clone polypeptide antigen genes by RT-PCR. The pGEM-T vector was inserted first. After sequencing, the obtained gene sequence was confirmed to be correct, digested with restriction enzymes EcoR I and Not I, and the target fragment was electrophoresed and linked, and ligated with the yeast expression vector pPIC9K digested with the same double enzyme. Transform E. coli. Pick positive clones, extract plasmids, and identify them by enzyme digestion. Transformation of Pichi _a p _aS toris KM71 and GS115. Positive clones were screened on histidine-free medium and then screened for high copy transformants on media containing different concentrations of G418. A single colony was picked and inoculated into the medium and incubated overnight at 28 degrees on a shaker. Continue to culture after dilution. When the absorbance of the bacterial concentration reaches OD600 is about 0.8, the medium is replaced with a medium containing methanol, and the culture is continued for 24-48 hours. The samples were sampled at different times in the culture, and the expression level of the receptor protein in the supernatant was determined by ELISA. Harvest the time with the highest expression. The cell pellet was removed by centrifugation. The supernatant contains a large amount of expressed product. After dialysis by 50% ammonium sulfate, the dialysis bag with a molecular weight cut off of 10kd was dialyzed against distilled water for 24 hours, and after SepHAcryl S-200 and SepHAcryl S-100 column chromatography, respectively, the purified influenza virus receptor-"glycoprotein" was obtained. And a vaccine component of the antigen of "9-0-acetoacetyl neuraminic acid".

4.4. Direct application of the ready-to-use avian influenza vaccine after cleavage and processing as an anti-mutation vaccine component containing avian influenza virus polypeptide antigen: This vaccine already contains the H5N1 avian influenza virus antigen component, which is lysed by the above-mentioned virus lysis method as an anti-mutation The material of the vaccine.

5, making anti-mutation vaccine

5.1, virus lysis peptide component vaccine

The four avian influenza virus lysates, such as H5N1, H5N2, H7N7 and H9N2 prepared by the above cleavage method, are uniformly mixed in a ratio of 1-10:1-10:1-10: 1-10, generally 1:1:1. :1, a mixed lysate; or two or three of H5N1, H5N2, H7N7, and H9N2 may be mixed into a virus lysate, or only one of them may be selected as a single virus lysate. Then add one of the three virus lysates to the Freund's adjuvant in a ratio of 1-10:1-10 (usually 1:1), place it in a high-speed homogenizer, and mix it at 8,000-30,000 rpm. The formation of a water-in-oil liquid, that is, a vaccine for a viral polypeptide component containing a plurality of avian influenza virus lysing components.

5.2. Avian influenza virus peptide HA ₂ vaccine

Purified avian influenza virus HA ₂ expressing protein (200 μg/mL) prepared according to the method of genetic engineering recombinant avian influenza virus polypeptide HA ₂ in 4.2 in a ratio of 1-10:1-10 (generally 1 :1 ratio) Adding Freund's adjuvant, placing it in a high-speed homogenizer, and homogenizing it at a high speed of 8,000-30,000 rpm to form a water-in-oil emulsion, thereby preparing a vaccine containing the avian influenza virus HA ₂ expressing protein.

5.3. Making avian influenza virus polypeptide component vaccine using ready-made avian influenza vaccine

Purchase of avian influenza vaccine from the Hong Kong Department of Health, which has the effect of preventing the H5N1 avian influenza virus. The vaccine is mixed and spliced by the virus lysis method as described above, and then the lysate is added in a ratio of 1-10:1-10, generally in a ratio of 1:1, and the Freund's adjuvant is added. Into a high-speed pulper, homogenized at a high speed of 8,000-30 ₅ 000 rpm to form a water-in-oil emulsion, that is, a "vaccine-cleaving polypeptide component vaccine" is prepared.

5.4, influenza virus receptor component vaccine 5.4.1 Purchasing the venom receptors “glycoprotein” and “9-0-acetyl-N-acetylneuraminic acid” from the American Virus Center (ATCC) first mixed in a 1:1 ratio to a concentration of 200 μg/mL Solution; then, in a ratio of 1-10:1-10, generally in a ratio of 1:1, adding Freund's adjuvant, and then placed in a high-speed homogenizer, homogenized at 8,000-30,000 rpm to form water-in-oil The emulsion, that is, a vaccine for which one of the influenza virus receptor components is prepared.

5.4.2 Expression of purified influenza virus receptors "glycoprotein" and "9-0-acetyl-N-acetylneuraminic acid" prepared by the method described in 4.3 Genetic engineering recombinant influenza virus receptor-expressing protein described above. The protein is mixed in a ratio of 1:1 to prepare a solution having a concentration of 200 μg/mL, and then added to a high-speed homogenizer at a ratio of 1-10:1-10, generally at a ratio of 1:1, and then placed in a high-speed homogenizer. The water-in-water emulsion is formed by high-speed homogenization at 8,000-30,000 rpm to prepare another influenza virus receptor component vaccine.

6, preparation of secondary infection pathogen complex antigen

Group A cluster B hemolytic streptococcus (2 X 109/mL), Streptococcus pneumoniae (2X 109/mL), Haemophilus influenzae (2X 109/mL) and MRSA Staphylococcus aureus (2X 109/mL) and Tuberculosis bacteria (2X 109 / mL) in a ratio of 1-10:1-10:1-10:1-10:1-10, usually in an equal proportion, mixed to prepare a mixture of pathogenic bacteria, and then the disease The bacterial mixture is added in an amount of 1-10:1-10 (generally in a ratio of 1:1) to an equal amount of Freund's adjuvant, and treated with a high-speed homogenizer at 8,000-30,000 rpm to form a water-in-oil emulsion. Infected with pathogenic bacteria complex antigen.

7. Preparation of anti-avian flu immune eggs

Four different anti-mutation vaccines prepared by the above four methods, namely, avian influenza virus lysing polypeptide component vaccine, avian influenza virus polypeptide HA ₂ vaccine, avian influenza vaccine lysing polypeptide component vaccine, and influenza virus receptor component vaccine, The laying hens were immunized separately, and once again intensively injected every two weeks, three times of immunization; after 20 days of the first immunization, the immunized eggs produced by the immunized hens were seized and coded.

8, preparation of anti-secondary infection bacterial immune eggs

The flu prepared by the above method is infected with the bacterial complex antigen, and the laying hen is immunized, and the injection is once again intensively every two weeks, and the immunization is performed three times. After the first immunization for 20 days, the immunized hen is taken. The immunized egg produced. The extracted immune eggs are coded and labeled.

The above immunization methods and frequency of injection can be appropriately adjusted and changed according to the immune response of the hen, and the same immunological techniques as described above can be applied, and the different antigens mentioned above are used for the laying mother duck or the laying geese or laying turkey or Different egg and poultry such as laying ostriches are immunized, and various different immune eggs are obtained.

9. Anti-variant avian influenza specific IgY and anti-secondary infection specificity IgY crude extract preparation

The immune eggs are first classified and labeled according to the different immunized birds and the vaccine or antigen used for immunization. Wash the immunized egg with running water, scrub with alcohol, and beat the egg with eggbeater, egg yolk Sift the sieve to remove the egg white, leave the egg yolk, stir evenly; add distilled water 4-6 times the volume of the egg yolk solution, dilute and mix, and adjust the pH to 5.5-6.0 with 1.0N HCI solution.

The pH-adjusted dilution was further stirred well, then cooled to 2-6 C, and allowed to stand for 12-24 hours; the dilution was centrifuged at 10,000 rpm for 20 minutes; the supernatant supernatant obtained by separation was taken. Perform ultrafiltration concentration 10-20 times; then add 2.0% sodium alginate solution to a final concentration of 0.1%, stir until turbidity occurs; then add 2.0% CaCl ₂ solution to a final concentration of 0.1%, stir evenly, 4 °C was allowed to stand for 8-12 hours; centrifuged at 8,000 rpm for 20 minutes, and the supernatant was taken; 0.45 μηι membrane was serially sterilized by 0.22 μιη membrane filtration; Ultipor VFTM DV50 virus removal filter was used to remove virus;

Through the above steps, four kinds of crude avian influenza-specific IgY extracts can be prepared, which are two kinds of virus-cleaving components and vaccine lysing components, respectively, and anti-variable avian influenza virus-specific IgY crude extract and anti-avian influenza. The viral polypeptide HA ₂ specific IgY crude extract, the anti-influenza virus receptor-specific IgY crude extract; in addition, a specific IgY crude extract against secondary infected bacteria can also be prepared. Finally, the prepared specific IgY crude extract corresponding to different vaccines or antigens is encoded and labeled.

10. Purification of specific IgY against variant avian influenza-specific IgY and anti-secondary infection bacteria The above five crude extracts were dissolved in pH 7.0, 0.01 M PB (phosphate buffer) solution, respectively. Through ion exchange column and gel chromatography column chromatography, four kinds of anti-variant avian influenza-specific IgY pure products and one specific IgY pure product against secondary infected bacteria were prepared.

11. The bacteria virus and virus removal device of the virus filtration system manufactured by Pall Ultrafine Filtration Company of the United States is used to thoroughly filter out various bacteria and viruses to ensure that the prepared IgY is free of any viruses and bacteria. Among them, the first bacterial filtering device removes bacteria such as Salmonella by using a 0.22 μπ membrane sterilizing filter; the second mycoplasma filtering device removes mycoplasma by using a Ο.ΐιη membrane in addition to a mycoplasma filter; The virus filtering device removes a variety of viruses including avian influenza virus and enterovirus using the Ultipor VFTM DV50 virus removal filter.

Finally, four kinds of anti-variant avian influenza-specific IgY can be obtained, which are two kinds of virus lysing components and vaccine lysing components respectively. Anti-variant avian influenza virus-specific IgY, anti-avian influenza virus polypeptide HA ₂ specific IgY, anti- Influenza virus receptor-specific IgY; a specific IgY against secondary infected bacteria can also be produced. By using the above specific anti-variant avian influenza-specific IgY and anti-secondary infection-specific IgY, other chemical components or traditional Chinese medicine ingredients can be added to prepare various compound medicines.

The above various anti-variant avian influenza-specific IgY, or specific IgY against secondary infected bacteria, or a combination of these IgY and chemicals, Chinese medicine, etc., are formulated with distilled water to a concentration of 0.01 - 20.0%. Solvent; can be made into a variety of new aerosols, mouth sprays, nasal sprays, nasal drops, eye drops, throat sprays, hand sanitizers, spray disinfectants, capsules and injections and other dosage forms. Due to IgY It can resist the destruction of pepsin and intestinal trypsin and chymotrypsin; therefore, it can be made into oral tablets, oral liquids or capsules for oral administration, and can also achieve the effects of prevention and treatment.

It is also possible to mix four different anti-avian influenza-specific IgY and specific IgY against secondary infected bacteria in a ratio of 1 - 10: 1 - 10 to prepare a specific compound against avian influenza. IgY, formulated with distilled water to form a compound solvent with a concentration of 0.01 - 20.0%, to form a new atomizing agent.

Anti-variant avian influenza-specific complex IgY with 0.01 - 20.0% anti-variant avian influenza-specific IgY, or anti-variant avian influenza-specific IgY and specific IgY against secondary infected bacteria, and this series of IgY Adding a combination of chemical and traditional Chinese medicines with 99.99 - 80.0% of excipients, it can be made into various clinically acceptable dosage forms, such as nebulizers, nasal sprays, nasal drops, eye drops, and throat sprays. Dosage forms, oral sprays, buccal tablets, oral liquids, hand sanitizers, spray disinfectants, capsules, and injections are used to prevent and treat avian influenza and secondary infections.

Among them, the anti-influenza virus receptor-specific IgY has a high specific antibody binding potency against human influenza virus in addition to specific inhibition of various types of avian influenza viruses; this is because of the avian influenza virus and The specific receptors for both human influenza viruses are the same. Therefore, the IgY can be used or mixed with the specific IgY against the secondary infection-infecting bacteria to prepare an anti-human influenza-specific complex IgY, or a combination of a chemical or a Chinese medicine; and it is provided with 99.99 - 80.0% Excipients, made into a variety of clinically acceptable dosage forms, such as aerosols, nasal sprays, nasal drops, eye drops, throat sprays, mouth sprays, hand lotions, buccal tablets, spray disinfectants, capsules And injections and other dosage forms for the prevention and treatment of human influenza and secondary infections.

The invention utilizes anti-variant avian influenza virus immunoglobulin (IgY) to protect local mucosa (nasal cavity, upper respiratory tract) cells from avian influenza virus, which is the key to blocking the infection of avian influenza virus (including after mutation) The adsorption of avian influenza virus directly acts as a blocking effect and is one of the mechanisms for preventing the specific avian influenza IgY. Anti-Variative Avian Influenza-specific IgY can also neutralize the newly released virus from avian influenza patients, and lose the ability to spread and spread again, thus achieving the dual purpose of both treatment and prevention of avian influenza transmission. This is the uniqueness of its innovation.

Since HA ₂ is a protein component that mediates fusion of an influenza virus membrane with an intracellular small body membrane, the present invention produces an I _g Y against the avian influenza virus polypeptide HA ₂ , and the anti-HA ₂ antibody can prevent the avian influenza virus. The fusion of the envelope with the intracellular small body membrane, and even affect the fusion of the avian influenza virus and the cell membrane, so that the viral nucleocapsid can not enter the cell, thereby effectively preventing the avian influenza virus. Thus, even if the variable region of the avian influenza virus (heavy chain - ΗΑ^ is mutated, as described above, the conserved region (light chain - ΗΑ ₂ ) is unchanged, the anti-avian prepared by the method of the present invention The specific IgY of each polypeptide antigen (including the conserved region) of influenza virus or the specific IgY of the anti-HA ₂ expressing protein still exerts an effective repression effect on it. In addition, the anti-influenza virus receptor-specific Y produced by the present invention can bind to a specific influenza virus receptor inherent in the surface of a cell membrane in a human body, so that even if the avian influenza virus invades the human body, there is no receptor, these viruses It can't be copied, it is bound to die. Because different subtypes of avian influenza virus and human influenza virus have the same specific receptors; therefore, this special anti-influenza virus receptor IgY is against a variety of different subtypes or variants of avian influenza viruses and humans. The inhibitory effect of the flu virus is the same, which solves the problem that the bird flu virus and the human flu virus type are many and easily mutated and difficult to deal with by another clever new method.

These features of the series of anti-variant avian influenza-specific IgY of the present invention overcome the ineffectiveness of the avian influenza vaccine against the variant avian influenza virus and the fact that the currently widely used antiviral drugs have little effect on the actual killing of the avian influenza virus. There are still a lot of toxic side effects on the human body. In particular, this IgY has the advantage of not inducing a mutation in the virus, and does not cause resistance to the avian influenza virus; therefore, it can overcome the existing resistance of the existing [Tenmin] and [Leeqing]. Fatal shortcomings of sex.

The invention designs four different methods for preparing avian influenza vaccine for poultry, and can select one of them according to different situations, and immunely lay eggs and poultry (chicken, duck, goose, ostrich, etc.), so that a stronger immune response occurs, resulting in A composite antibody with more types and stronger binding capacity. The various specific IgYs against various subtypes of avian influenza viruses have significantly prevented the prevention of various common avian influenza viruses, especially the avian influenza viruses, which are more common than the general avian influenza vaccines. This is because different subtypes of avian influenza viruses have the same 1^ ₂ conserved region, and the anti-HA ₂ specific IgY of the present invention can specifically target the HA ₂ conserved region; in addition, no matter which type of avian influenza virus Both must rely on specific receptors to bind to target cells, and the specific anti-influenza virus IgY of the present invention effectively inhibits this specific receptor; therefore, these two distinctive special IgY pairs of different subtypes The variant avian influenza virus will have a suppressive effect. At the same time, after the human body is infected with the avian influenza virus, the pathogenic bacteria will take advantage of it, which will inevitably cause secondary infection. The anti-multiple pathogen-specific complex IgY prepared by the invention has a very common pathogenic bacteria causing secondary infection. Good immune conditioning, especially the resistant bacteria MRSA and tuberculosis, which can effectively kill common bactericidal drugs, which are not possible with avian flu vaccine and general antiviral drugs. Therefore, this specific complex IgY can not only fundamentally prevent and treat avian influenza caused by various subtypes of avian influenza virus in different countries; moreover, even when a certain avian influenza virus mutates again The new avian influenza caused by the variant avian influenza virus, the anti-variant avian influenza specific IgY produced by the invention can also effectively deal with it. It has become a powerful weapon for preventing avian flu on a large scale, which is more convenient, safer and more effective than the general avian flu vaccine.

Experimental example

Experimental Example 1 : Anti-variant avian influenza virus specificity prepared by virus lysis method IgY

H5N1, H5N2, H7N7, H9N2 avian influenza virus strains were provided by the National University of Singapore Microbiology Laboratory in the form of academic cooperation research, respectively, cultured in conventional chicken embryo allantoic sac, and then purified separately. 20 mg of H5N1 viral protein, 10 mg of H5N2 viral protein, 10 mg of H7N7 viral protein, and 10 mg of H9N2 viral protein.

1. The four avian influenza viruses purified above were separately added with 20% sodium dodecyl sulfate (SDS), and the final concentration was 2.0%, and the cells were lysed for 30 minutes to obtain the above various influenza virus lysates. After SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) test analysis, the gel was concentrated 4%, and the gel was separated by 7 ° /. , 240V, electrophoresis for 30 minutes. Cooma's bright blue staining, observe the protein band. Detection confirmed that hemagglutinin heavy chain (HA0, hemagglutinin light chain (HA ₂ ), neuraminidase (NA), nuclear protein (NP), P protein (Pl, P2 P3), matrix protein (Ml, M2) And non-structural proteins (NS, NS2). Consistent with the experimental results of Enami M, etc. The lysate of the above four viruses can be prepared.

2. Mix the above four virus lysates in a ratio of 1:0.5:0.5:0.5, and then add the Freund's adjuvant to the high-speed homogenization in a ratio of 1:1 to prepare avian influenza virus lysate anti-mutation vaccine; finally, The virus-producing hens were immunized with the virus lysing component vaccine, and once again intensively injected every two weeks, three times of immunization; after 20 days of the first immunization, the immunized eggs produced by the immunized hens were seized and coded. And extract as follows:

2.1, first wash the immune eggs with running water, alcohol scrubbing disinfection, eggbeater to break the immune eggs, egg yolk sieve to filter the egg whites, leave the egg yolk, stir evenly; add distilled water 4-6 times the volume of the egg yolk liquid, carry out Dilute and mix well, adjust the pH to 5.5-6.0 with 1.0 N HCI solution.

2.2. The pH-adjusted dilution solution is further stirred well, then cooled to 2-60 C, and allowed to stand for 12-24 hours; the dilution is centrifuged at 10,000 rpm for 20 minutes; the supernatant obtained by separation is subjected to ultrafiltration. Ultrafiltration concentration 10-20 times; then add 2.0% sodium alginate solution to a final concentration of 0.1%, stir until turbidity; add 2.0% CaC12 solution to a final concentration of 0.1%, stir evenly, 40C The cells were allowed to stand for 8-12 hours; centrifuged at 8,000 rpm for 20 minutes, and the supernatant was taken; the 0.45 μm film was sterilized by filtration through a 0.22 μηι membrane; Ultipor VFTM DV50 virus removal filter was used to remove the virus; That is, a dry powder of crude IgY specific for anti-variant avian influenza virus is prepared.

3. The crude extract dry powder was dissolved in pH 7.0, 0.01 M PB (phosphate buffer) solution, and then separated by ion exchange column and gel chromatography column. That is, a pure anti-variant avian influenza virus-specific IgY product is prepared.

4. In order to ensure that the prepared IgY is free of any viruses and bacteria; no matter the prepared IgY crude product or IgY pure product, it must be filtered and sterilized by the following virus removal process:

4.1. The bacterial virus filtering device of the virus filtration system manufactured by Pall Ultrafme Filtration Company of the United States is used to thoroughly filter out various bacteria and viruses.

4.2. The first bacterial filtration device removes Salmonella by using a 0.22μιη membrane sterilization filter.

(Salmonella) and other bacteria; the second mycoplasma filtration device removes the mycoplasma with a 0.1 μηι membrane in addition to the mycoplasma filter; the third virus filtering device removes the package with the Ultipor VFTM DV50 virus removal filter. A variety of viruses including avian influenza virus and enterovirus.

Experimental Example 2: Activity detection of anti-HA ₂ specific chicken IgY by genetic engineering recombinant HA ₂ expression protein

1. The signal peptide and the fragment of the transmembrane region were removed from the avian influenza virus RA by cloning the hemagglutinin (HA ₂ ) gene by RT-PCR. The pGEM-T vector was inserted first. After sequencing, the obtained gene sequence was confirmed to be correct, and the target fragment was digested with restriction enzymes EcoR I and Notl, and the target fragment was ligated with the yeast expression vector PPIC9K digested with the same double enzyme. Transform E. coli. Positive clones were picked, plasmids were extracted, and after digestion and identification, Pichia pastoris KM71 and GS115 were electroporated. Positive clones were screened on histidine-free medium and then screened for high copy transformants on media containing different concentrations of G418. A single colony was picked and inoculated into the medium and incubated overnight at 28 degrees on a shaker. Continue to culture after dilution.

2. When the absorbance of the bacteria concentration reaches OD600 is about 0.8, the medium is changed to a medium containing methanol, and the culture is continued for 24-48 hours. The samples were sampled at different times in the culture, and the expression level of HA in the supernatant was determined by ELISA. Harvest the time with the highest expression. The cell pellet was removed by centrifugation. The supernatant contains a large amount of expressed product. The dialysis bag with a molecular weight cut off of 10 kd was precipitated with 50% ammonium sulfate, and dialyzed against distilled water for 24 hours, and subjected to SepHAcryl S-200 and SepHAcryl S-100 column chromatography to obtain a purified avian influenza virus HA ₂ antigen component.

3. The purified avian influenza virus HA ₂ expression protein (200 μg/mL) prepared is added to Freund's adjuvant in a ratio of 1-10:1-10 (generally in a ratio of 1:1), and placed at high speed. The slurry is homogenized at a high speed of 8,000 to 30,000 rpm to form a water-in-oil emulsion, that is, a vaccine containing the avian influenza virus HA ₂ expressing protein is prepared.

4. The vaccine for the avian influenza virus HA ₂ expression protein was then immunized in the same manner as in Test Example 1 and the anti-avian influenza virus polypeptide HA ₂ specific IgY was prepared in the same manner.

Experimental Example 3: Preparation of anti-variant avian influenza virus specific duck IgY using "Avian influenza virus vaccine lysing polypeptide component vaccine"

1. Purchase 50 avian influenza vaccines from the Hong Kong Department of Health. Mix the avian influenza vaccines and prepare the lysate by the above-mentioned virus lysis method. Then add the lysate to the Freund's adjuvant in a ratio of 1:1 and put it into the high speed. The homogenizer is high-speeded to prepare a vaccine containing the lysing components of four influenza virus polypeptides of H5N1, H5N2, H7N7 and H9N2.

2. Using the "Avian Influenza Vaccine Peptide Cleavage Ingredients Vaccine" to immunize the laying ducks, the anti-variant avian influenza virus-specific duck IgY was prepared by the method described above. H5N1, H5N2 and H7N7, H9N2 avian influenza viruses were used as detection antigens respectively, and the antibody binding titer of the anti-variant avian influenza virus-specific duck IgY to these four different antigens was detected by ELISA.

The result is as follows: Antigen-antibody binding titer

H5N1 avian influenza virus 1:2,048

Anti-variant avian influenza virus duck IgY H5N2 avian influenza virus 1:2,048

(Immune with "vaccine lysing component") H7N7 avian influenza virus 1:2,048

H9N2 avian influenza virus 1:2,048

Experimental Example 4: Preparation of anti-avian influenza virus by genetic engineering recombinant influenza virus receptor-expressing protein vaccine Receptor-specific IgY

1. Purchase of ready-made influenza virus receptors "glycoprotein" and "9-0-acetyl-N-acetylneuraminic acid" from the American Virus Center (ATCC), mixed in a 1:1 ratio, and mixed to make 200 micrograms. /mL concentration solution, then in a ratio of 1-10:1-10, generally in a ratio of 1:1, adding Freund's adjuvant, and then placed in a high-speed homogenizer, homogenized at 8,000-30,000 rpm, forming The water-in-oil emulsion is a vaccine for the preparation of a viral receptor.

2. Recombinant influenza virus receptor-expressing protein by genetic engineering. The specific operation methods are as follows.

2.1. The signal peptide and the fragment of the transmembrane region were removed by RT-PCR from the influenza virus receptor--glycoprotein and the "9-0-acetyl-N-acetylneuraminic acid" RNA clone polypeptide antigen gene. The pGEM-T vector was inserted first. After sequencing, the obtained gene sequence was confirmed to be correctly digested with restriction enzymes EcoR I and Not I, and the target fragment was electrophoresed and linked to the yeast expression vector pPIC9K digested with the same double enzyme. Transform E. coli. Positive clones were picked, plasmids were extracted, and after digestion and identification, Pichia pastoris KM71 and GS115 were electroporated. Positive clones were screened on histidine-free medium and then screened for high copy transformants on media containing different concentrations of G418. A single colony was picked and inoculated into the medium and cultured overnight on a 28-degree shaker. Continue to culture after dilution. When the concentration of the bacteria reaches OD600 and the absorbance is about 0.8, the medium is changed to a medium containing methanol and cultured for 24-48 hours. Samples were taken at different times in the culture, and the expression level of the receptor protein in the supernatant was determined by ELISA. Harvest the time with the highest expression. The cell pellet was removed by centrifugation. The supernatant contains a large amount of expressed product. After dialysis by 50% ammonium sulfate, the dialysis bag with a molecular weight cut off of 10kd was dialyzed against distilled water for 24 hours, and after SepHAcryl S-200 and SepHAcryl S-100 column chromatography, respectively, the purified influenza virus receptor-"glycoprotein" was obtained. And "9-0-acetyl-N-acetylneuraminic acid" antigen component.

2.2. The purified two influenza virus receptors, a "glycoprotein" and a "9-0-acetoacetylneuraminic acid", were first mixed in a ratio of 1:1 to prepare a solution of 200 μg/mL. In a ratio of 1-10:1-10, generally in a ratio of 1:1, adding Freund's adjuvant, and then placed in a high-speed homogenizer, homogenized at 8,000-30,000 rpm to form a water-in-oil emulsion, which is obtained. Viral receptor component vaccine.

2.3. The vaccine using the influenza virus receptor to express the protein was then tested in the same manner as in Test Example 1. The egg laying hens were immunized and prepared against the influenza virus receptor-specific IgY in the same manner.

Experimental Example 5: Anti-influenza secondary infection specific chicken IgY

1. A cluster of B-type hemolytic streptococcus, Streptococcus pneumoniae, Haemophilus influenzae, MRSA Staphylococcus aureus, and tuberculosis were cultured by the National University of Singapore Microbiology Laboratory. Mix the above five pathogenic bacteria in a ratio of 1:1:1:0.5:0.5, make 10mL of bacterial solution and then add 10mL of Freund's adjuvant and then place them in a high-speed homogenizer for high-speed homogenization to obtain secondary infection bacteria. Compound antigen 20 mL.

2. The hens were immunized with the complex antigen containing whole bacteria by the same method as described in the present invention, and the specific IgY against influenza secondary infection was prepared in the same manner. Then, it was mixed with the anti-variant avian influenza virus-specific IgY prepared in the above Experimental Example 1 at a ratio of 1 to 10:10 -1 to obtain a specific complex IgY against mutant avian influenza and secondary infection.

3. Finally, using H5N1, H5N2 and H7N7, H9N2 avian influenza virus and A cluster B hemolytic streptococcus, Streptococcus pneumoniae, Haemophilus influenzae, MRSA Staphylococcus aureus, and tuberculosis as detection antigens, by ELISA The antibody titer of this complex IgY against these antigens was examined.

The results are as follows _:

From the above test results, the anti-variant avian influenza and secondary infection-specific complex IgY prepared according to the method described herein, the four subtypes of avian influenza virus, and several common secondary infection pathogens are There is a higher antibody binding potency.

Experimental Example 6: Anti-variant avian influenza specific IgY and anti-secondary infection specificity IgY containing

"SDS-PAGE (sodium dodecyl sulfate-polypropylene gel) electrophoresis method for different anti-variant avian influenza-specific IgY and specific IgY against secondary infection bacteria prepared according to the above process The crude extract was tested and found to contain 45-52% IgY. The crude IgY extract was passed through the column to obtain pure IgY. The purity was PAGE pure by SDS-PAGE analysis, as shown in the following table: IgY pure pure Y content

Anti-variant avian influenza specific IgY 99.99%

Specificity against bacteria secondary to infection IgY 99.99%

Experimental Example 7: Specific anti-variant avian influenza specific IgY and anti-secondary infection specificity Compound IgY activity assay

1. The specific IgY against variant avian influenza and the specific IgY against secondary infected bacteria prepared by the special immunization initiation method and the improved process technology invented by the present inventors adopt "ELISA method" (enzyme-linked immunosorbent assay) ) Perform activity detection.

2. The test results showed that the various anti-variant avian influenza-specific IgYs were produced, and the type I "anti-variant avian influenza virus-specific IgY" prepared by immunizing with the split virus antigen was representative of the above four kinds. The antibody binding potency of H5N1, H5N2 and H7N7 and H9N2 avian influenza viruses reached above 1:2048. The antibody binding titer of type II "anti-HA ₂ specific ¥" obtained by immunization with HA ₂ expressing protein antigen to H5N1, H5N2, H7N and H9N2 avian influenza viruses also reached 1:1,024 or more; Avian influenza vaccine lysing component vaccine" The cockroach type "anti-variant avian influenza virus-specific IgY" obtained by immunization The antibody binding valence of four avian influenza viruses such as X-inch H5N1, H5N2, H7N and H9N2 also reached 1:1024 or more; The type IV anti-influenza virus receptor-specific IgY produced by immunization with the "influenza virus receptor vaccine" has an antibody binding titer of more than 1:1024 for four avian influenza viruses such as H5N1, H5N2, H7N and H9N2. In addition, as mentioned above, the specific receptors for both avian influenza virus and human influenza virus are the same; therefore, anti-influenza virus receptor-specific sputum prepared by immunization with influenza virus receptor vaccine against human influenza The antibody binding potency of the virus is also high, and the test results also reach 1:1024. Secondly, the specific IgY against secondary infection bacteria has a slightly lower antibody titer for representative pneumococci, A cluster B type hemolytic streptococcus, MRSA S. aureus, tuberculosis, and Haemophilus influenzae. , but both are above 1:512. See the table below for details: Four anti-variant avian influenza specific IgY titer test results

H3N2 1:1,024

H2N2 1:1,024

Anti-variant avian influenza virus specificity IgY (III)

H7 7 1:1,024

(Immune vaccine production by "Avian influenza vaccine lysis")

H9N2 1:1,024

H5N1 1:1,024

H5N2 1:1,024

Anti-influenza virus receptor specific IgY(IV)

H7N7 1:1,024

(Immune vaccine production with "influenza virus receptor")

H9N2 1:1,024

Human influenza virus H3N2 1:1,024 Specificity against secondary infection bacteria IgY titer test results

(Note: The sample concentration for detection is lmg/mL)

Example 1 : Preparation of anti-variant influenza virus specific chicken by virus lysis method IgY

1. The following four avian influenza viruses were selected: H5N1 strain, H5N2 strain, H7N7 strain, and H9N2 strain.

2. The above four influenza virus strains of H5N1, H5N2, H7N7 and H9N2 were respectively cultured in the chicken embryo allantoic sac according to the conventional chicken embryo allantoic sac method described below; the allantoic fluid containing the virus was collected, and then described below. The chicken red blood cell method was crudely extracted; the virus was further purified by the sucrose density gradient ultracentrifugation method described below to obtain purified influenza virus suspensions of H5N1, H5N2, H7N7 and H9N2, respectively.

3. The chicken embryo allantoic method is operated as follows: disinfect the egg shell in the upper part of the chicken embryo egg chamber with 70% alcohol, and punch a 1.0 cm long hole on it; add a drop of sterile liquid paraffin on the shell above the embryo body On the membrane; 0.1-0.2 mL of the virus specimen was injected into the allantoic cavity with a lmL tuberculin syringe, and then the opening was sealed with a sterile tape; placed in a 33-35 ° C incubator for 2-4 days; placed in 4 °C 6-18h _; disinfect the chamber egg shell with 75% alcohol, enlarge the opening on the eggshell, and use a syringe with a pure 18-gauge needle to extract the virus-containing allantoic fluid into a 50 mL centrifuge tube.

4. The method for crude extraction of chicken red blood cells is as follows: First, the above-mentioned collected allantoic fluid is centrifuged at 4,000 rpm for 30 minutes to remove debris and precipitated; formaldehyde-formed red blood cells are added per liter of allantoic fluid to a final degree of 2.5. -3.5%, mix well, place at 4 ° C for about 10 hours; centrifuge at 2,000 rpm 10 Minutes, go to the supernatant; wash the red blood cells twice in 0 °C saline (in an ice bath), every half minute; centrifuge at 2,000 rpm for 5 minutes at 4 ° C, remove the supernatant; add the original in the precipitated red blood cells One-fifth of the volume of allantoic fluid was pH 7.6, 0.01 M phosphate buffered saline, and the mixture was stirred, placed in a 37-inch water bath for 3 hours, and centrifuged at 2,000 rpm for 10 minutes to obtain a crude influenza virus suspension.

5. Purification of influenza virus by sucrose density gradient ultracentrifugation, the specific method is as follows: Take the crude influenza virus suspension l.OrnL, gently add to the 40% and 60% discontinuous density gradient of sucrose solution, after 100,000 g centrifuge for 2 hours, take the middle layer of the sucrose layer with liquid, add pH 7.2, 0.01 M phosphate buffered saline 1.0 mL, mix, dialysis and sugar removal, that is, a pure influenza virus suspension.

6. Purify each of the four influenza virus solutions H5N1, H5N2, H7N7 and H9N2, 1. OrnL (concentration: 40,000 HAU, equivalent to 200 micrograms of viral protein), and add 20% sodium dodecyl sulfate (SDS), respectively. The final concentration was 2.0%, and the solution was lysed at 37 ° C for 30 minutes to obtain the above four influenza virus lysates, respectively.

7. Mix the four influenza virus lysates in a ratio of 1.0:0.5:0.5:1.0 to make a mixed lysate, and then add the Freund's adjuvant to the mixed lysate according to a ratio of 1:1. In a high-speed homogenizer, homogenization was carried out at a high speed of 8,000 rpm to form a water-in-oil liquid, that is, a vaccine containing a plurality of influenza virus lysing components was prepared, and 150 mL was prepared.

8. 50 healthy laying hens were immunized with this anti-mutation vaccine, and each intramuscular injection of lmL was injected every two weeks for a total of three times.

9. The immune eggs will be collected on the 20th day after the first injection. By the next month, the 7,500 immunized eggs will be washed with running water, disinfected with 75% ethanol, and dried to break the eggshell. Egg yolk sieve to remove egg white, add egg yolk and 5 times distilled water, mix well, adjust pH to 5.5-6.0 with l.ON HCI, stir evenly, stand at 2-6 °C overnight, centrifuge at 10,000 rpm for 20 minutes, take supernatant Perform ultrafiltration concentration 10-20 times; then add 2.0% sodium alginate solution to a final concentration of 0.1%, stir until turbidity occurs; then add 2.0% CaCI2 solution to a final concentration of 0.1%, stir evenly, 4 °C 10 hours overnight; centrifuge at 8,000 rpm for 20 minutes, remove the supernatant; 0.45 μπι membrane in series with 0.22 μιη membrane filtration sterilization, Ultipor VFTM DV50 virus removal filter to remove virus; freeze-drying; that is, to produce anti-variant avian influenza virus specificity IgY crude extract 750 grams.

10. The above IgY crude extract was passed through an ion exchange resin column and a gel chromatography column to obtain 150 g of the specific IgY specific anti-variant avian influenza virus.

Example 2: Preparation of anti-variant avian influenza virus specific goose by virus lysis method IgY

1. First, the following four avian influenza viruses were selected: H5N1 strain, H5N2 strain, H7N7 strain, and H9N2 strain.

2. H5N1, H5N2, H7N7 and H9N2 four avian influenza virus strains were cultured in the chicken embryo allantoic sac respectively by conventional chicken embryo allantoic sac; the virus-containing allantoic fluid was collected and then crudely extracted by chicken red blood cell method; The virus was purified by gel column chromatography to obtain purified H5N1, H5N2, and H7N7, respectively. And H9N2 four influenza virus suspensions, the specific operation is as follows.

3. The above four avian influenza viruses were cultured by the chicken embryo allantoic sac method. The steps are as follows: 70% alcohol disinfects the egg shell in the upper part of the chicken embryo egg chamber, and punches a 1.0 cm long hole on it; add a drop of sterility The liquid paraffin is placed on the shell membrane above the embryo body; 0.1-0.2 mL of the virus specimen is injected into the allantoic cavity with a lmL tuberculin syringe, and then the opening is sealed with a sterile adhesive tape; 33-35 ° C incubator culture 2-4 days; placed at 4 °C for 6-18h _; disinfect the chamber egg shell with 75% alcohol, enlarge the opening on the eggshell, and extract the virus-containing allantoic fluid with a syringe equipped with a pure 18-gauge needle. Place in a 50 mL centrifuge tube.

4. The above four avian influenza viruses are crudely extracted with chicken red blood cells. The steps are as follows: First, the collected allantoic fluid is centrifuged at 4,000 rpm for 30 minutes to remove debris and precipitated; for each liter of allantoic fluid, formaldehyde red blood cells are added to The final degree is 2.5-3.5%, the mixture is evenly mixed, placed at 4 ° C for about 10 hours; centrifuged at 2,000 rpm for 10 minutes, the supernatant is removed; the red blood cells are washed twice in 0 ° C physiological saline (in an ice bath), each half Minutes; centrifuge at 2,000 rpm for 5 minutes at 4 ° C, remove the supernatant; add one-fifth of the volume of the original allantoic fluid to the precipitated red blood cells, pH 7.6, 0.01 M phosphate buffered saline, stir well, and place at 37 ° C water bath for 3 hours, centrifuged at 2,000 rpm for 10 minutes to obtain a crude avian influenza virus suspension.

5. Do not purify the above four avian influenza viruses by column chromatography. The procedure is as follows: 3-5 mL of the above suspension is applied to a SepHAdex G200 gel column (3 X 50 cm); 0.05 M, pH 7.4, PBS solution elution; flow rate 15-20mL / hour; collection of 5.0mL per tube; determination of elution curve at 280nm, and determination of influenza virus elution peak by hemagglutination titer; collection of virus eluate, which is purified avian influenza virus liquid.

6. Take 10 mL of purified H5N1, H5N2, H7N7 and H9N2 four avian influenza virus solutions (concentration: 40,000 HAU, equivalent to 2,000 micrograms of viral protein), and add 20% sodium dodecyl sulfate (SDS), respectively. At 2.0%, the above four avian influenza virus lysates were obtained by cleavage at 37 ° C for 30 minutes.

7. Mix the four avian influenza virus lysates at a ratio of 1.0:0.5:0.5·丄0 to prepare a mixed lysate, and then add the Freund's adjuvant to the mixed lysate at a ratio of 1:1. In a high-speed homogenizer, it is homogenized at a high speed of 8,000 rpm to form a water-in-oil liquid, that is, a vaccine containing a plurality of avian influenza virus lysing components is prepared, and 1500 mL is prepared.

8. Select 100 female gooses with high immune response ability. Use the above vaccine containing multiple influenza virus lysing components, and 1500mL to immunize the goose, each injection of 5mL antigen, after the first injection, each Intensive injections were given once every two weeks for a total of three times. In the seventh month after the first injection, the eggs produced by these geese were labeled and IgY was extracted according to the conventional method. ELISA (enzyme-linked immunosorbent assay) Detecting the titer of the prepared IgY, selecting a geese with a particularly strong immune response capable of producing an IgY titer of 256, and then using the eggs produced by the geese to hatch the fine goose species, until they grow up to 2 - 3 months, 50 of them were selected as the preferred high-immunity responsive special goose.

9. Immune to the preferred special goose by intensive immunization with subcutaneous injection, each The mother goose injects 5 mL of antigen each time, and intensively injects once every two weeks, and immunizes three times in one month. After 20 days of immunization, the immunized eggs produced by the goose are terminated until the 7th month. 7,500 immunized goose eggs.

10. Prepare the prepared immunized eggs with running water, sterilize with 75% ethanol, dry them, crush the eggshells, remove the egg whites with egg yolk sieve, remove the egg yolk and add 5 times of distilled water, mix well and adjust the pH with 1.0N HCI solution. 5.5-6.0, Stir well, stand at 2-6 °C overnight, centrifuge at 10,000 rpm for 20 minutes, remove the supernatant for 10-20 times by ultrafiltration; then add 2.0% sodium alginate solution to a final concentration of 0.1% Stir until turbidity occurs; add 2.0% CaCI2 solution to a final concentration of 0.1%, stir evenly, overnight at 4 °C for 10 hours; centrifuge at 8,000 rpm for 20 minutes, remove the supernatant; 0.45 μιη membrane in series with 0.22 μπι membrane filtration The strain, Ultipor VFTMDV50 removes the virus except the virus filter; freeze-drying, 2, 200 grams of crude goose IgY extract specific for the variant avian influenza virus.

11. The above crude IgY extract was subjected to ion exchange resin column and gel chromatography column chromatography to obtain 400 g of pure goose IgY specific anti-variant avian influenza virus.

Example 3: Preparation of anti-avian influenza virus polypeptide HA ₂ specific ostrich IgY by genetic engineering

1. The signal peptide and the fragment of the transmembrane region were removed by cloning the hemagglutinin (HA ₂ ) gene from the influenza A virus RNA by RT-PCR. The pGEM-T vector was inserted first. After sequencing, the obtained gene sequence was confirmed to be correct, and the target fragment was digested with restriction enzymes EcoR I and Notl, and the target fragment was ligated with the yeast expression vector PPIC9K digested with the same double enzyme. Transform E. coli. Positive clones were picked, plasmids were extracted, and after digestion and identification, Pichia pastoris KM71 and GS115 were electrotransformed. Positive clones were screened on histidine-free medium and then screened for high copy transformants on media containing different concentrations of G418. A single colony was picked and inoculated into the medium and incubated overnight at 28 degrees on a shaker. Continue to culture after dilution. When the absorbance of the bacterial concentration reaches OD600 is about 0.8, the medium is changed to a medium containing methanol, and the culture is continued for 24-48 hours. The samples were sampled at different times in the culture, and the expression level of HA in the supernatant was determined by ELISA. Harvest the time with the highest expression. The cell pellet was removed by centrifugation. The supernatant contains a large amount of expressed product. After dialysis by 50% ammonium sulfate, the dialysis bag with a molecular weight cut off of 10 kd was dialyzed against distilled water for 24 hours, and after SepHAcryl S-200 and SepHAcryl S-100 column chromatography, 1500 mL of the purified influenza virus HA ₂ vaccine was obtained.

2. Select 50 female ostriches with high immune response ability, and immunize the female ostrich with 1500 mL of the above-mentioned purified influenza virus HA ₂ vaccine, each injection of 10 mL vaccine, after the first injection, every two weeks. Intensive injections were made three times. In the seventh month after the first injection, the eggs produced by these ostriches were labeled and IgY was extracted according to the conventional method. The ELISA method (enzyme-linked immunosorbent assay) was used to detect the results. The IgY potency, select the ostrich that has a particularly strong immune response with an IgY titer of 256, and then use the eggs produced by the ostrich to hatch the excellent ostrich species until they grow up to 2-3 months. , 10 of them were selected as the preferred high-immunity responsive egg laying ostrich. 3. Using the above-mentioned method, 300 mL of HA ₂ expressing protein vaccine, using the booster immunization method of subcutaneous injection, respectively, to immunize the preferred special egg laying ostrich, each ostrich dose of 8-10 mL per vaccine, every two Zhou re-intensified the injection once, and immunized three times in one month; after 20 days of the first immunization, 100 eggs were collected from the ostrich.

4. Wash 100 eggs of the immunized ostrich eggs with running water, disinfect with 75% ethanol, dry them, crush the eggshells and filter the egg whites with egg yolk. Leave the egg yolk with 5 times distilled water and mix well with 1.0N. HCI solution was adjusted to pH 5.5-6.0, stirred evenly, left at 2-6 ° C overnight, centrifuged at 10,000 rpm for 20 minutes, and the supernatant was concentrated by ultrafiltration for 10-20 times; then 2.0% sodium alginate solution was added. At the final concentration of 0.1%, stir until turbidity occurs; then add 2.0% CaCI2 solution to a final concentration of 0.1%, stir and hook, 4V overnight; centrifuge at 8,000 rpm for 20 minutes, take the supernatant; 0.45μιη film in series with 0.22 The membrane was sterilized by μιη membrane filtration, and the virus was removed by Ultipor VFTM DV50 virus removal filter; lyophilized, 1,000 g of crude extract of anti-HA ₂ specific ostrich IgY was prepared.

5. The above IgY crude extracts were successively passed through an ion exchange resin column and a gel column chromatography to obtain 200 g of an anti-HA ₂ specific ostrich IgY pure product.

Example 4: Preparation of anti-variant avian influenza virus specificity by avian influenza vaccine component lysing polypeptide vaccine turkey IgY

1. Take 50 avian influenza vaccines (300 g/1.5 mL each) and mix them evenly. Add 20% sodium dodecyl sulfate (SDS) to a final concentration of 2.0% and cleave for 30 minutes at 37 ° C. Avian influenza virus lysate was obtained.

2. The lysate is added to Freund's adjuvant in a ratio of 1:1, placed in a high-speed homogenizer, and homogenized at 8,000 rpm to form a water-in-oil liquid, which is a vaccine component lysing polypeptide vaccine. 150 mL. Then, 50 healthy hamsters were immunized with this vaccine, and each intramuscular injection of 1 mL was injected every two weeks for a total of three times.

3. The immune eggs were collected on the 20th day after the first injection. On the 50th day, a total of 1,200 immune eggs were collected. They were washed with running water, disinfected with 75% ethanol, and dried to break the eggshell. The egg white was removed by egg yolk sieve, and the egg yolk was added with 5 times distilled water. The pH was adjusted to 5.5-6.0 with 1.0 N HCI. Stir well, stand at 2-6 ° C overnight, centrifuge at 10,000 rpm for 20 minutes, and take the supernatant. Perform ultrafiltration concentration 10-20 times; then add 2.0% sodium alginate solution to a final concentration of 0.1%, stir until turbidity occurs; then add 2.0% CaCI2 solution to a final concentration of 0.1%, stir evenly, 4 °C Overnight; centrifuge at 8,000 rpm for 20 minutes, remove the supernatant; 0.45 μπι membrane in series with 0.22 μπι membrane filtration sterilization, Ultipor VFTM DV50 virus removal filter to remove virus; freeze-drying; that is, to obtain anti-variant avian influenza virus-specific IgY rough 120 grams.

4. The above IgY crude extract is passed through an ion exchange resin column and a gel chromatography column to obtain an anti-variant avian influenza virus-specific IgY pure product which is immunized with avian influenza vaccine component lysed polypeptide vaccine. . Example 5: Preparation of specific IgY against secondary infected bacteria

1. Preparation of secondary infection pathogen complex antigen: group A cluster B type hemolytic streptococcus (2 X 109/mL), Streptococcus pneumoniae (2 X 109/mL), Haemophilus influenzae (2 X 109/mL) And mixed with S. aureus (2 X 109 / inL) in equal proportions to prepare a mixture of pathogenic bacteria, and then add the mixture of pathogenic bacteria in a 1:1 ratio to the same amount of Freund's adjuvant, using a high-speed homogenizer At 8,000 rpm, the water-in-water mixed bacterial antigen is secondary to infection with the pathogenic bacteria complex antigen.

2. Preparation of anti-secondary infection bacterial immune eggs: Apply the prepared influenza secondary infection bacterial composite antigen, and immunize 5 laying hens; each muscle is injected with lmL. The injection was intensively every two weeks, and the immunization was performed three times. After the first immunization for 20 days, the immunized eggs produced by the immunized hens were seized, and by the seventh month, a total of 7,500 immunized eggs were collected.

3. Preparation of specific anti-secondary infection bacteria IgY crude extract

Wash the seized immune eggs with running water, scrub with alcohol, then use the eggbeater to break the immune eggs, filter the egg whites with egg yolk sieve, leave the egg yolk, stir evenly; press 4-6 according to the volume of egg yolk Distilled water was added thereto, diluted and uniformly mixed, and the pH was adjusted to 5.5-6.0 with a 1.0 N HCI solution.

The pH-adjusted dilution is further stirred well, then cooled to 2-6 ° C and allowed to stand for 12-24 hours; the dilution is centrifuged at 10,000 rpm for 20 minutes; the supernatant obtained by separation is subjected to ultrafiltration. Ultrafiltration concentration 10-20 times; then add 2.0% sodium alginate solution to a final concentration of 0.1%, stir until turbidity; add 2.0% CaC12 solution to a final concentration of 0.1%, stir 4 °C, let stand for 8-12 hours; centrifuge at 8,000 rpm for 20 minutes, take the supernatant; 0.45 μπι membrane in series with 0.22μηι membrane filtration sterilization; Ultipor VFTM DV50 virus removal filter to remove virus; freeze-drying, that is, anti-drug A specific IgY crude extract of secondary bacterial infection, 700 grams.

4. Purification of specific IgY against secondary infection bacteria; the prepared specific IgY crude extract against secondary infected bacteria is dissolved in pH 7.0, 0.01 M PB (phosphate buffer) solution, and then After ion exchange column and gel chromatography column chromatography, 150 g of specific IgY pure product against secondary infected bacteria was prepared.

Example 6: Any of the four anti-variant avian influenza-specific IgY prepared by the method detailed in Experimental Examples 1-4 and the specific IgY against secondary infected bacteria were mixed in a ratio of 2:1. Avian influenza specific complex IgY 200g (anti-variant avian influenza specific IgY 133.33g, anti-secondary specific IgY 66.66g) was used as a main raw material to prepare an anti-avian influenza and secondary infection atmospheric spray. Anti-variant avian influenza specific compound IgY 200g

Glycerin 4,000g

PEG400 5,000g

Menthol 200g

Flavor 80g Ethanol 2,000mL

Distilled water added to 200 liters

The anti-variant avian influenza specific compound IgY is dissolved in 160 liters of distilled water, and glycerin is added to mix. Thin lotus brain, flavor plus sweet and sour steamed into ethanol to dissolve, add PEG400 and mix, add to the above solution under stirring, stir well, recharge the essential oil alcohol

Filter, add distilled water to the full amount, adjust the pH to 7.0 with a 0.1 mol, NaOH solution. The drug solution is poured into a normal pressure spray bottle, each 20 mL, and the package is 10,000 anti-variant avian influenza specific IgY atmospheric pressure sprays.

Example 7: An anti-avian influenza virus polypeptide HA ₂ protein-specific ostrich IgY 100g prepared by the method detailed in Example 3 was used as a main raw material to prepare a genetically engineered anti-variant avian influenza atmospheric pressure spray. . Anti-HA2 specific ostrich IgY 100g

2,000g

Right 2,500g

Brain 100g

30g

l,000mL

Add to 100 liters

The anti-HA ₂ protein-specific ostrich IgY was dissolved in 80 liters of distilled water, and glycerin was added to mix. Menthol and flavor are dissolved in ethanol, mixed with PEG400, added to the above solution under stirring, stirred and filtered, and distilled water is added to the whole amount. The pH is adjusted to 7.0 with 0.1 mol of NaOH solution and filled. The drug solution was poured into a normal pressure spray bottle, each of which was 20 mL. After the whole test, the package obtained 10,000 anti-variant avian influenza IgY atmospheric pressure sprays.

Example 8: 1.0 g of anti-variant avian influenza virus-specific IgY was prepared by the method detailed in Example 1, and the specific IgY against the secondary infected bacteria was prepared by the method detailed in Example 5, and the ratio was 1:1. 1 Proportionally mixed into 2.0 g of anti-variant avian influenza specific composite IgY, and this composite IgY was used as the main raw material to prepare a buccal tablet. Anti-variant avian influenza specific compound IgY 2.0g

Carboxymethylcellulose 5.0g

Magnesium stearate 0.6g

Aspartame 1.2g

Mint Flavor 0.8g

Orange flavor l.Og

Ethanol (30% concentration) l,000mL

Sorbitol is added to dissolve carboxymethyl cellulose into 1% ethanol solution to make 1,000 tablets The preparation process is as follows:

1. The sorbitol and the aspartame are well mixed and passed through a 60 mesh sieve for two times;

2. The carboxymethyl cellulose was dispersed in 30% ethanol to make a 1% ethanol solution.

3. One item should be granulated with a proper amount of 2 pieces of soft material, 14 mesh screen, ventilated and dried at 60 °C, and sieved with 18 mesh. Use a 40 mesh sieve to sift out the appropriate amount of fine powder and mix well with IgY and spray into the mint and orange flavor. Stir well, then mix with magnesium stearate, mix well with the whole batch of granules, and seal for more than 4 hours for each tablet. 0.6g. After passing the inspection, it will be packaged and fully tested.

Example 9: An anti-variant avian influenza virus-specific IgY 5.0 g was prepared by the method detailed in Example 1, and a novel injection for preventing and treating influenza was prepared. Anti-variant avian influenza virus specific pure IgY 5g

Pluronic F68 10g

Polyvinylpyrrolidone (PVP) 10g

PEG400 HOg

Polysorbate -80 10g

Water for injection added to l,000mL

1. Water for injection, add: Pluronic and PVP, heat to dissolve; take polysorbate-80, add PEG400 to mix; add the above mixture under stirring; add 0.1% needle with activated carbon, stir at 60 °C for 15min ; decarbonization filtration; the filtrate was sealed and heated at 100 ° C for 30 min, and cooled to room temperature for use.

2. Take activated water treated with activated carbon, add anti-influenza virus-specific pure IgY lyophilized powder to the sterilization container, continue to stir, add to the above mixture in a fine stream, and use O.lmol NaOH. The solution was adjusted to a pH of 6.0 to 7.5, and the whole amount was made up with decarbonized sterilized water for injection.

3. Sterilized 0.45μπι series 0.22μηι microporous membrane for sterilization filtration. The container is filled in a sterile container in a sterile potting machine. Each 2mL contains pure anti-variant avian influenza virus-specific pure IgY lyophilized powder 5mg. 500 IgY injections are available for light inspection, leak detection, printing and packaging. Specifications 5mg/2mL.

Example 10: Production of anti-variant avian influenza IgY hand-picked quantitative pressure spray can

Formulated with 200 liters of anti-variant avian influenza specific IgY combination solution according to the following formula: Anti-variant avian influenza specific IgY 300g

Aspartame 240g

Orange Flavor 600g

Peach Flavor 200g

Mint flavor l,400g

Distilled water added to 200 liters

1. Take 180 liters of distilled water, then add aspartame and flavor respectively, stir well, then Slowly add IgY while stirring; then add distilled water to 200 liters, stir well, measure the pH value of the solution, adjust the pH to 6.5-7.5 with 1mol NaOH solution according to the measured pH value;

2. After the solution is prepared, it is necessary to produce 20,000 manual quantitative pressure spray cans in the pressure tank. After filling 10ml of each spray can, it must be filled with pressure nitrogen or Freon as a booster (propellant). Install a metering valve, seal, and get.

Example 11: Preparation of specific anti-secondary infection bacteria IgY atmospheric spray

The specific IgY 50g against secondary infection bacteria was used as a main raw material to prepare an anti-secondary infection atmospheric pressure spray. Specificity against secondary infection bacteria IgY 50g

Glycerin l,000g

PEG400 l, 250g

Menthol 50g

Flavor 15g

Ethanol 5000mL

Distilled water added to 50 liters

The specific IgY against secondary infected bacteria was dissolved in distilled water, and glycerin was added to mix. Menthol and flavor are dissolved in ethanol, mixed with PEG400, added to the above solution under stirring, stirred, filtered, and distilled water is added to the whole amount, and the pH is adjusted to 6.5 with 0.1 mol of NaOH solution. The drug solution is poured into an atmospheric pressure spray bottle, each of which is 20 ml. After the whole test is passed, 2,500 anti-secondary infections of IgY atmospheric pressure spray are obtained. 500 IgY injections were obtained for inspection, leak detection, printing and packaging. Specifications 5mg/2ml.

Claims

A method for preparing an anti-variant avian influenza specific IgY, comprising the steps of:

(51) preparing a vaccine against avian influenza;

(52) using the anti-variation bird flu vaccine to intensify immunization of laying poultry and extracting anti-avian flu immune right eggs produced by immunized poultry;

(53) taking the egg yolk of the anti-avian influenza-immunized egg to prepare a crude extract of the specific avian influenza specific IgY;

(55) The anti-variant avian influenza-specific IgY pure product is filtered to filter out various bacteria and viruses to obtain a variant AgY-specific finished product. Book

The method for producing a variant avian influenza-specific IgY according to claim 1, wherein the anti-variant avian influenza-specific IgY is an anti-variant avian influenza virus-specific IgY, in the step (S1) To prepare avian influenza virus lysing component vaccine according to the following procedure:

The purified four avian influenza viruses were separately added to 20% sodium dodecyl sulfate (SDS) at a final concentration of 2.0%, and lysed for 30 minutes to prepare four kinds of birds, H5N1, Leg 2, H7N7 and H9N2. Influenza virus lysate;

Taking at least one of the four avian influenza virus lysates, adding Freund's adjuvant in a ratio of 1-10: 1-10, and placing it in a high-speed homogenizer at a high speed of 8,000-30, OOO rpm The formation of a water-in-oil liquid, that is, an anti-variation vaccine containing a plurality of avian influenza virus lysing components.

The method for producing a variant avian influenza-specific IgY according to claim 1, wherein the anti-variant avian influenza-specific IgY is an anti-variant avian influenza virus-specific IgY, in the step (S1) In the following steps, prepare avian influenza vaccine:

The avian influenza virus lysate containing inactivated or reduced avian influenza virus was added to 20% sodium dodecyl sulfate (SDS) to a final concentration of 2.0%, and lysed for 30 minutes to prepare an avian influenza virus lysate; Adding Freund's adjuvant in a ratio of 1-10: 1-10, placing it in a high-speed homogenizer, and homogenizing it at a high speed of 8,000- 30, OOO rpm to form a water-in-oil emulsion, which produces an anti-mutation containing viral components. Avian flu vaccine.

The method for producing a variant avian influenza-specific IgY according to claim 1, wherein the anti-variant avian influenza-specific IgY is an anti-avian influenza virus polypeptide HA ₂ specific IgY, in the step ( In S1), prepare the vaccine containing the avian influenza virus polypeptide ΗΑ _{2 ή} 3ε _{# as} follows:

The hemagglutinin (ΗΑ ₂ ) gene was cloned from the Avian influenza A RNA by RT-PCR, and the signal peptide and the fragment of the transmembrane region were removed;

The pGEM-Τ vector was inserted first, and the obtained gene sequence was confirmed by sequencing. After digestion with the restriction enzymes EcoR I and Not I, the target fragment was electrophoresed and linked with the yeast expression vector PPIC9K digested with the same double enzyme;

Transformation of E. coli, picking positive clones, extracting plasmids, and then correcting the correct digestion, electroporation of Pichia pastor is KM71 and GS115;

After the dilution, the culture is continued. When the concentration of the bacteria reaches OD600, the absorbance is about 0.8, and the medium is replaced with the medium containing methanol, and the cultivation is continued for 24-48 hours;

After dialysis by 50% ammonium sulfate, the dialysis bag with a molecular weight cutoff of 10 kd was dialyzed against distilled water for 24 hours, and after separation with SepHAcryl S-200 and SepHAcryl S-100 column, the purified avian influenza virus polypeptide HA ₂ antigen component was obtained;

The purified avian influenza virus polypeptide HA ₂ antigen component is added to Freund's adjuvant in a ratio of 1-10: 1-10, and placed in a high-speed homogenizer to homogenize at a high speed of 8,000-30 rpm to form a water-in-oil emulsion. , that is, an anti-variation vaccine containing avian influenza HA ₂ expressing protein was prepared.

The method for producing a variant avian influenza-specific IgY according to claim 1, wherein the anti-variant avian influenza-specific IgY is an anti-influenza virus receptor-specific IgY, in the step (S1) In the following procedure, prepare a vaccine containing influenza virus receptors:

Take the influenza virus receptor, and mix the "glycoprotein" and "9-0-acetyl-N-acetylneuraminic acid" expressed protein in a ratio of 1:1 to prepare a solution with a concentration of 200 μg/mL, then 1- 10: The ratio of 1-10 was added to Freund's adjuvant, and then placed in a high-speed homogenizer, and homogenized at a high speed of 8,000--30, OOO rpm to form a water-in-oil emulsion, thereby preparing a vaccine containing an influenza virus receptor. 6. The method for producing a variant avian influenza-specific IgY according to claim 5, wherein the influenza virus receptor is produced by the following steps:

RT-PCR was used to remove the signal peptide and the transmembrane region fragments from the influenza virus receptors, namely the "glycoprotein" and "9-0-acetyl-N-acetylneuraminic acid" RNA clone polypeptide antigen genes;

The pGEM-T vector was inserted first, and the obtained gene sequence was confirmed by sequencing. After digestion with the restriction enzymes EcoR I and Not I, the target fragment was electrophoresed and linked with the yeast expression vector PPIC9K digested with the same double enzyme;

Transformation of Escherichia coli, picking positive clones, extracting plasmids, and digesting the correct ones, electroporating Pichia pastoris KM71 and GS115;

Positive clones were screened on histidine-free medium, and then high-copy transformants were selected on medium containing different concentrations of G418, and single colonies were picked and inoculated into the medium, and cultured overnight at 28 ° shaker;

After the dilution, the culture is continued. When the absorbance of the bacteria concentration reaches 0D600 is about 0.8, the medium is replaced with the medium containing methanol, and the cultivation is continued for 24-48 hours;

At different times of culture, the expression level of the receptor protein in the supernatant was determined by ELISA, and the time was selected at the highest dosage, and the cell pellet was removed by centrifugation, and the supernatant contained a large amount of expression product;

After dialysis by 50% ammonium sulfate, the dialysis bag with a molecular weight cut off of 10kd was dialyzed against distilled water for 24 hours, and after SepHAcryl S-200 and SepHAcryl S-100 column chromatography, respectively, the purified influenza virus receptor-glycoprotein was obtained. And the antigenic component of "9-0-acetyl-N-acetylneuraminic acid".

The method for producing a variant avian influenza-specific IgY according to any one of claims 1 to 6, wherein

In the step (S3), preparing a crude avian influenza-specific IgY extract according to the following steps; taking the egg yolk of the anti-avian influenza-immunized egg, stirring uniformly; adding distilled water according to the volume of the egg yolk solution 4-6 times Dilute and mix well, adjust the pH to 5. 5-6. 0 with 1. ON HCI solution; further stir evenly, then cool it to 2-6 ° C, let stand for 12-24 hours; The mixture is stirred at 10,000 rpm for 20 minutes, and the resulting supernatant is placed in an ultrafilter for ultrafiltration to be concentrated 10-20 times; a solution of 2.0% sodium alginate is added to a final concentration of 0.1%, stirred until appears. turbidity; was added 2. 0% CaCl ₂ solution to a final concentration of 0.1%, stirring homogeneously, 4 ° C was allowed to stand for 8-12 hours; centrifuged at 8,000 rpm for 20 minutes, the supernatant, then by 0. 45Mm The membrane was sterilized by membrane filtration of 0. 22μιη, and the virus was removed by Ultipor VFTM DV50 virus removal filter, and then freeze-dried to prepare a crude extract of specific IgY against mutant avian influenza virus;

In the step (S4), the crude avian influenza-specific IgY extract is purified by the following steps: 0. 01M PB (phosphorus In the acid salt buffer), the ion-exchange column and gel chromatography column chromatography are respectively carried out to obtain a pure anti-variant avian influenza IgY product;

In the step (S5), the anti-variant avian influenza-specific IgY pure product is subjected to filtration treatment by the following steps: removing the bacteria such as Salmonella by using a 0.22 Mm membrane sterilization filter; The Mym membrane removes the mycoplasma from the mycoplasma filter; the virus is removed by the Ultipor VFTM DV50 virus removal filter to remove various viruses including avian influenza and enterovirus.

A method for preparing a specific IgY against a secondary pathogen-infected pathogen, which comprises the steps of:

(521) Prepare secondary infection pathogen antigens as follows: Group A cluster B hemolytic streptococcus, Streptococcus pneumoniae, Haemophilus influenzae, MRSA Staphylococcus aureus, and tuberculosis 1-10: 1- 10: 1-10: 1-10: 1-10 Mix in proportion to prepare a mixture of pathogenic bacteria, and then add the pathogenic bacteria mixture to Freund's adjuvant in a ratio of 1-10: 1-10, and homogenize with high speed. The device is treated with 8,000-30,000 rpni to form a water-in-oil emulsion, which is a composite antigen for secondary infection pathogenic bacteria;

(524) purifying the crude IgY-expressing anti-secondary pathogen-specific pathogen-specific IgY product;

(525) filtering the pure anti-secondary pathogen-specific IgY product to filter out various bacteria and viruses to obtain a finished IgY-resistant product against secondary pathogenic bacteria.

A method for preparing a specific avian influenza-specific complex IgY, which comprises the variant avian influenza-specific IgY obtained by the method according to any one of claims 1 to 6, and the method of claim 8 The anti-secondary infection pathogen-specific IgY prepared by the method is mixed in a ratio of 1 - 10: 1 - 10 to prepare a specific complex IgY against the avian influenza.

10. An anti-avian influenza preparation, characterized in that it comprises:

The content of the anti-variant avian influenza specific IgY prepared by the method according to any one of claims 1 to 6, or the content is 0. 01 - 20. 0% by weight The anti-variant avian influenza specific composite IgY prepared by the method of claim 9;

And an auxiliary material having a content of 99.99-80. 0%;