CN117285618A - Preparation method and application of anti-human papilloma virus IgY antibody - Google Patents

Abstract

The invention discloses a preparation method and application of an anti-human papilloma virus IgY antibody. The method comprises the steps of preparing Cheng Fushi complete adjuvant immunogen and Freund's incomplete adjuvant immunogen by using fusion protein consisting of recombinant HPV 18 type virus L1 capsid protein and L2 capsid protein N-terminal 11-200 aa, immunizing an egg laying hen through subcutaneous and intramuscular 5-80 points, extracting IgY antibody from immunized hen egg yolk by using a water dilution method, purifying the IgY antibody by using a two-step ammonium sulfate salting-out method, and obtaining the anti-human papillomavirus IgY antibody. Preparations formulated using the specific anti-human papillomavirus IgY antibodies of the invention, such as: the spray, aerosol, gargle, washing liquid, suppository and the like can be used for preventing and treating human skin, mucous membrane warts, benign and malignant tumors and the like caused by human papilloma viruses, and provides a brand-new prevention and treatment method and means without toxic or side effect for the warts, benign and malignant diseases which are transmitted through sexually and are still not prevented and treated by the ideal method at present.

Description

Preparation method and application of anti-human papillomavirus IgY antibody

Technical field

The invention relates to a method for preparing a specific anti-human papillomavirus IgY antibody and its preventive application in preventing and treating multiple HPV serotype strain infections and tumors.

Background technique

Human papillomavirus (HPV) belongs to the genus Papillomavirus A of the family Papovaviridae. The viral genome consists of 7900 base pairs and is a circular spherical double-stranded closed-loop small DNA. HPV has a diameter of 45-55nm, icosahedral stereosymmetry, and contains 72 shell particles. It is wrapped by protein and has no capsule. Its molecular weight is 5×106 Daltons. HPV DNA has 8 open reading frames, and one mRNA molecule is transcribed and translated into 8 proteins, including capsid proteins (L1, L2) and functional proteins (E1, E2, E4, E5, E6, E7). Human skin keratinocytes and mucosal squamous epithelial cells are the natural hosts of HPV. HPV does not spread through the bloodstream and does not produce viremia. There are common antigens among various types of HPV, which are specific antigens and are present in the L1 protein; the L2 protein is a type-specific antigen, and the L2 constant region carries a surface-specific antigenic epitope. This epitope interacts with the host cell membrane and the virus enters cell. Each HPV type has varying degrees of antigenic cross-reactivity in the L1 and L2 capsid proteins. Antibodies against one type of HPV can interact with many other types of HPV viruses to achieve a broad spectrum of prevention and treatment, which will produce unexpected surprises. . For example, HPV16 and HPV18 share common antigens with other types of HPV. Anti-HPV16 or HPV18 antibodies can prevent or treat warts or tumors on the skin, mucous membranes, genitals, etc. caused by other types of HPV.

At present, more than 200 HPV genotypes have been discovered, including low-risk HPV types, which infect the skin and mucous membranes and produce warts and benign tumors; and high-risk HPV types that cause cancer (HPV5, 8-9, 14-25, 30-31, 33, 35, 39, 45, 51-53, 56, 58, 59, 68, 73, and 82), which cause cancer of the vulva, penis, anus, prostate, bladder, cervix, rectum, and oral cavity , tonsil cancer, etc. HPV is transmitted through direct or indirect contact with contaminated items, or through sexual transmission. Natural infection produces a poor response to anti-HPV antibodies. HPV has a unique infection mode. The damaged epithelial basement membrane is exposed. The HPV L1 protein binds to the heparin sulfate proteoglycans (HSPGs) of the granular layer epithelial basement membrane and changes its conformation, exposing the N-terminus of the L2 protein, which is e.g. Linase cleaves it, thereby rearranging the conformations of L1 and L2, allowing the virus to move to the granular layer keratinocyte zone, and its viral residues are exposed to epithelial cells (undifferentiated cells) endocytic groups that engulf the virus. The infected cells begin to differentiate and migrate to the upper layers of the epithelium and stratum corneum. Eventually, the epithelial cells die and fall off, and HPV is released into the lumen and infects new damaged and exposed basal layer cells. Escape of HPV natural immunity: ① The normal death of HPV-infected keratinocytes does not cause a significant inflammatory response, so it does not cause an immune response to clear the virus. ② HPV can down-regulate the expression and function of interferon (IFN) and interleukin-18 (IL-18), and reduce the phagocytic function of NK cells and macrophages. HPV escapes from acquired immunity: ①. The number of Langerhans cells (LC) in HPV-infected tissues decreases and the structure changes, resulting in obstacles to HPV antigen presentation. ②. The chance of LC coming into contact with viral antigens is small. ③.HPV reduces CD4+T cell response. ④. HPV induces tolerance of cytotoxic T lymphocyte (CTL) precursor cells.

Sites prone to HPV infection include the vulva, vagina, penis, cervix and around the anus, inside the anal canal, urethral opening, as well as in the armpits, umbilical fossa, between the toes, under the breasts, and oral mucosal surfaces. There are few specific treatments for HPV.

The L2 capsid protein contains a common epitope, and a neutralizing epitope was found between aa 11 and 200 at the N-terminus of L2. This epitope is highly conserved among different types of HPV. Varsani et al. generated chimeras inserted into HPV16 L2 aa 108-120 and then assembled into VLPs. HPV16 L1 immunization induced the production of L2-specific antibodies. Passive transfer of immune sera to mice cross-protects animals against vaginal mucosal challenge with all relevant pseudovirions (PsV) hrHPV (HPV16/18/45/31/33/52/58/35/39/51/59/68 /56/73/26/53/66/34) and lrHPV (HPV6/43/44) in vivo attack. .

Since the HPV infection route is direct infection of the virus from the damaged epithelium of the cavity, the blood antibody circulation produced by the vaccine has little or no effect on preventing and treating HPV infection. The local application of specific anti-HPV antibodies has positive effects. For example, clinical trials of IgY antibodies against multi-drug-resistant bacteria have been successful; specific anti-rotavirus IgY has significant efficacy in treating human rotavirus infections, reducing the number of diarrhea; egg yolk antibodies Can passively immunize to prevent and treat COVID-19 infection.

In view of the above reasons, an anti-human papillomavirus (HPV) IgY antibody is prepared, which can bind to various types of HPV with common antigens and passively immunize to prevent and treat skin and mucosal surface warts and tumors caused by various types of HPV. Eliminating HPV from the body has far-reaching social benefits and significant economic benefits.

Contents of the invention

The purpose of the present invention is to provide a method for preparing IgY antibodies specific against human papillomavirus (HPV), and the prepared IgY antibodies can bind to various types of HPV with common antigens. Using this IgY antibody preparations prevent and treat human skin and mucosal warts and benign and malignant tumors caused by various types of human papillomavirus (HPV).

The object of the present invention can be achieved by the following methods:

A specific anti-human papillomavirus (HPV) IgY antibody, which can bind to various types of HPV with a common antigen, is characterized by being obtained by the following method:

(1) Immunogen preparation:

Take 10~100 μg/ml of the fusion protein composed of the N-terminal 11-200 aa of the L1 capsid protein of the recombinant HPV 18 virus and the L2 capsid protein;

Mix an equal volume of HPV18 virus L1 and L2 capsid fusion protein liquid (for example: 100 ml of 10 to 100 μg/ml liquid) with an equal volume of Freund's complete adjuvant containing 5 to 10 mg/ml of live BCG (for example: 100 ml of liquid). ), mix with a high-speed homogenizer at 6000 rpm 5 times for 3 minutes each time, and emulsify until the water-in-oil Freund's complete adjuvant immunogen solution is obtained;

Mix an equal volume of HPV18 virus L1 and L2 capsid fusion protein liquid (for example: 100 ml of 10~100 μg/ml liquid) with an equal volume of Freund's incomplete adjuvant (for example: 100 ml), and pass it through a high-speed homogenizer. Stir 5 times at 6000rpm, 3 minutes each time, to emulsify into water-in-oil Freund's incomplete adjuvant immunogen solution;

Each milliliter of Freund's complete adjuvant immunogen and Freund's incomplete adjuvant immunogen contains 10-100 μg of the fusion capsid protein composed of the N-terminal 11-200 aa of HPV18 virus L1 and L2;

(2) Immunity:

Inject Freund's complete adjuvant immunogen subcutaneously and intramuscularly into laying hens at 5 to 80 o'clock. Each laying hen was injected with 1 ml of immunogen, and then 1 ml of Freund's incomplete adjuvant immunogen was administered every 1 to 2 weeks. The laying hens were injected subcutaneously and intramuscularly at 10-60 o'clock for a total of four times. After that, 1 ml of Freund's incomplete adjuvant immunogen was used subcutaneously and intramuscularly at 10-30 o'clock to strengthen the laying hens every 1 to 2 months. Immunity for one year;

(3) Preparation of anti-human papillomavirus (HPV) IgY antibody water dilution method extract:

Collect eggs from immunized hens 21 days after immunization. Wash the surface of the eggs with boiling water and soak them in 70% alcohol. Remove the eggshells to get the yolk. Remove the yolk membrane to get the yolk liquid. Mix the egg yolk liquid and distilled water at a volume ratio of 1:5 to 12 Add distilled water in the appropriate proportion and stir, adjust the pH to pH 4.9~5.3 with 1N HCl, and let it stand overnight at 4ºC;

Centrifuge the egg yolk dilution at high speed for 15 minutes at 4ºC to take the supernatant, then ultrafiltrate and concentrate it 10 to 20 times in a hollow fiber ultrafilter with a molecular weight cutoff of 10 to 50 kDa, and then add 0.02% to 5.0% chlorhexidine gluconate. , and use 1NNaOH to adjust the pH to pH 7.0~7.5, and let it stand at 4ºC overnight; then centrifuge at 4ºC for 10 minutes at high speed to take the supernatant, and filter and sterilize the supernatant through a 0.22μ~0.45μ sterilized microporous filter membrane. That is, the water extract of anti-human papillomavirus (HPV) IgY antibodies is obtained.

The fusion protein of HPV type 18 virus L1 capsid protein and L2 capsid protein N-terminal 11-200aa in the preparation of the immunogen is 10~100 μg.

The egg-laying hens in the immunization include Laihang chickens, Hyline brown chickens, Roman chickens, star chickens, and domestic native chickens raised in ordinary environments, clean environments, and SPF environments.

(4) Preparation of pure anti-human papillomavirus IgY antibodies:

The preparation step of pure anti-human papillomavirus IgY antibody is a continuation process after the preparation procedure of the anti-human papillomavirus IgY antibody water dilution method extract. The steps are: the extract prepared by the IgY antibody water dilution method is treated with glucose. Add ammonium sulfate to the chlorhexidine-treated anti-human papillomavirus IgY antibody extract to a saturation level of 45% to 60%. Add ammonium sulfate powder while stirring. Stir thoroughly to dissolve and mix, then let stand at room temperature for 2 to 12 seconds. hours, and then centrifuge in a high-speed centrifuge at 4ºC for 10 minutes. Discard the supernatant and collect the precipitate. Dilute the precipitate to the original volume with distilled water, and then slowly add ammonium sulfate powder to a saturation of 28%~35%. Add ammonium sulfate powder as you go. Stir, stir thoroughly, dissolve and mix, then let stand at room temperature for 2 to 12 hours, then centrifuge in a high-speed centrifuge at 4ºC for 10 minutes, discard the supernatant and remove the precipitate, dissolve the precipitate with distilled water, and use the hollow fiber ultrafiltration membrane to ultrafiltrate the IgY solution. salt to obtain pure anti-human papillomavirus IgY antibody.

The IgY purity of the anti-human papillomavirus IgY antibody water dilution extract prepared according to the method of the present invention was measured by SDS-PAGE electrophoresis, and the purity of the IgY antibody reached 30%-35%; The IgY purity of the pure anti-human papillomavirus IgY antibody prepared by the ammonium precipitation method was determined by SDS-PAGE electrophoresis, and the purity of the IgY antibody reached 85%-92%.

The IgY biological potency of the anti-human papillomavirus IgY antibody water dilution extract extracted according to the method of the present invention was determined by the ELISA (enzyme-linked immunosorbent assay) method. The biological efficacy of the specific anti-human papillomavirus IgY antibody The specific anti-human papillomavirus antigen IgY antibody binding biological titer per milligram of protein is 1:2048~4096/mg protein.

The biological titer of the pure IgY antibody prepared by the two-step ammonium sulfate precipitation method of the anti-human papillomavirus (HPV) IgY antibody extracted according to the method of the present invention was determined by the ELISA (enzyme-linked immunosorbent assay) method. The biological potency of human papillomavirus (HPV) antibodies reaches the specific anti-human papillomavirus (HPV) antigen IgY antibody binding biological potency per milligram of protein, which is 1:8192~16384 /mg protein.

The specific anti-human papillomavirus (HPV) IgY antibody prepared according to the method of the present invention also includes the following steps: adding pharmaceutically acceptable additives to prepare an antibody containing anti-human papillomavirus (HPV) IgY Antibody sprays, aerosols, gargles, lotions, and suppositories.

The specific anti-human papillomavirus (HPV) IgY antibody prepared according to the method of the present invention is locally applied to the mouth, throat, bronchus, vulva, vagina, as well as the skin and mucous membranes of the whole body. It has no systemic use. It has limitations and side effects. It also has the advantages of fast onset of effect, small dosage, good efficacy, non-absorption, and no damage to the normal microecological environment of the nasal cavity, oral cavity, throat, bronchus, vulva, vagina, as well as the skin and mucous membranes of the whole body. And has no toxic or side effects. The anti-human papillomavirus (HPV) IgY antibody extract does not produce acute toxic reactions or chronic toxic reactions and belongs to the actual non-toxic level; the anti-human papillomavirus (HPV) IgY antibody extract is negative in the triple test , a product that is non-teratogenic, carcinogenic, and mutagenic.

Detailed ways

The implementation of the present invention is described in detail below with reference to examples.

Example 1:

The production process flow of 1000 grams of anti-human papillomavirus (HPV) IgY antibody prepared by water dilution method:

Preparation of immunogen:

Weigh 5 mg of the fusion protein composed of the N-terminal 200 aa of the L1 capsid protein of the recombinant HPV 18 virus and the N-terminal 200 aa of the L2 capsid protein to prepare the Freund's complete adjuvant immunogen. Mix 50 ml of HPV18 virus L1 and L2 capsid fusion protein N-terminal 200 aa solution (5 mg fusion protein, concentration 100 μg/ml) with 50 ml of Freund's complete adjuvant (containing 1.0 g of live BCG vaccine, 20 mg/ml). Stir the high-speed homogenizer at 10,000 rpm for 3 minutes, stop for 5 minutes and stir again. Emulsify for a total of 5 times to finally prepare the water-in-oil Freund's complete adjuvant immunogen (the final immunogen contains fusion protein concentration of 50 μg/ml, live BCG vaccine 10 mg/ml ).

Weigh 5 mg of the fusion protein composed of the N-terminal 200 aa of the L1 capsid protein of the recombinant HPV 18 virus and the N-terminal 200 aa of the L2 capsid protein to prepare the Freund's incomplete adjuvant immunogen. Mix 50 ml of HPV18 virus L1 and L2 capsid fusion protein N-terminal 200 aa solution (5 mg fusion protein, concentration 100 μg/ml) with 50 ml of Freund's incomplete adjuvant, stir for 3 minutes with a high-speed homogenizer at 10000 rpm, and stop for 5 minutes. Then stir again and emulsify for a total of 5 times, and finally prepare the water-in-oil Freund's incomplete adjuvant immunogen (the final immunogen contains fusion protein concentration of 50 μg/ml).

(2) Immunization of laying hens: Use a 1ml sterile plastic syringe to extract 1ml of the water-in-oil complete Freund's adjuvant immunogen prepared above, divide it into 55 points and inject it into the wings of 20-week-old Hyline brown laying hens. , chest, thighs, back, etc., and injected into the thigh muscles at 5 points. After 2 weeks, use 1ml of the prepared water-in-oil Freund's incomplete adjuvant immunogen and inject it subcutaneously into the wings, chest, thighs, back, etc. of the laying hen at 37 points, and into the thigh at 3 points. intramuscular. The total number of vaccinated hens is 100;

(3) Preparation of anti-human papillomavirus (HPV) IgY antibody water dilution method extract:

Collect 2200 eggs from immunized hens on days 21 to 47 after the first immunization. Wash the surface of the eggs with boiling water and soak them in 70% alcohol. Remove the egg shells to obtain the egg yolk, remove the egg yolk membrane to obtain the egg yolk liquid, and mix the egg yolk liquid with Add distilled water at a volume ratio of 1:10, stir, adjust the pH to pH 5.1 with 1N HCl, and place in a 4°C freezer overnight;

Centrifuge the egg yolk dilution at 10,000 g at high speed for 15 minutes at 4ºC. Take the supernatant and concentrate it 11 times by ultrafiltration in a hollow fiber ultrafilter with a molecular weight cutoff of 50 kDa. Then add 1.0% chlorhexidine gluconate. Use 1N NaOH to adjust the pH to pH 7.1, let it stand at 4ºC overnight, and then centrifuge it at 10,000 g at 4ºC for 10 minutes. Take the supernatant and filter it through a 0.45μ sterilized microporous filter to sterilize. A water extract of anti-human papillomavirus (HPV) IgY antibody was obtained; the IgY water extract solution was measured by SDS-PAGE electrophoresis, and its IgY purity reached 34%; measured by ELISA (enzyme-linked immunosorbent assay), The titer of its specific IgY antibody is 1:4096/mg protein.

Example 2:

The production process for preparing 200 grams of pure anti-human papillomavirus (HPV) IgY antibody:

Preparation of immunogen:

Weigh 5 mg of the fusion protein composed of the N-terminal 200 aa of the L1 capsid protein of the recombinant HPV 18 virus and the N-terminal 200 aa of the L2 capsid protein to prepare the Freund's complete adjuvant immunogen. Mix 50 ml of HPV18 virus L1 and L2 capsid fusion protein N-terminal 200 aa solution (5 mg fusion protein, concentration 100 μg/ml) with 50 ml of Freund's complete adjuvant (containing 1.0 g of live BCG vaccine, 20 mg/ml). Stir the high-speed homogenizer at 10,000 rpm for 3 minutes, stop for 5 minutes and stir again. Emulsify for a total of 5 times to finally prepare the water-in-oil Freund's complete adjuvant immunogen (the final immunogen contains fusion protein concentration of 50 μg/ml, live BCG vaccine 10 mg/ml ).

Weigh 5 mg of the fusion protein composed of the N-terminal 200 aa of the L1 capsid protein of the recombinant HPV 18 virus and the N-terminal 200 aa of the L2 capsid protein to prepare the Freund's incomplete adjuvant immunogen. Mix 50 ml of HPV18 virus L1 and L2 capsid fusion protein N-terminal 200 aa solution (5 mg fusion protein, concentration 100 μg/ml) with 50 ml of Freund's incomplete adjuvant, stir for 3 minutes with a high-speed homogenizer at 10000 rpm, and stop for 5 minutes. Then stir again and emulsify for a total of 5 times, and finally prepare the water-in-oil Freund's incomplete adjuvant immunogen (the final immunogen contains fusion protein concentration of 50 μg/ml).

(2) Immunization of laying hens: Use a 1ml sterile plastic syringe to extract 1ml of the water-in-oil complete Freund's adjuvant immunogen prepared above, divide it into 55 points and inject it into the wings of 20-week-old Hyline brown laying hens. , chest, thighs, back, etc., and injected into the thigh muscles at 5 points. After 2 weeks, use 1ml of the prepared water-in-oil Freund's incomplete adjuvant immunogen and inject it subcutaneously into the wings, chest, thighs, back, etc. of the laying hen at 37 points, and into the thigh at 3 points. intramuscular. The total number of vaccinated hens is 100;

(3) Preparation of anti-human papillomavirus (HPV) IgY antibody water extract:

Collect 2,200 eggs from immunized hens on days 21 to 47 after the first immunization. Wash the surface of the eggs with boiling water and soak them in 70% alcohol. Remove the egg shells to get the yolks. Remove the yolk membrane to get the yolk liquid. Add distilled water at a volume ratio of 1:10, stir, adjust the pH to pH 5.1 with 1N HCl, and place in a 4°C freezer overnight;

Centrifuge the egg yolk dilution at 10,000 g at high speed for 15 minutes at 4ºC. Take the supernatant and concentrate it 11 times by ultrafiltration in a hollow fiber ultrafilter with a molecular weight cutoff of 50 kDa. Then add 1.0% chlorhexidine gluconate. And use 1N NaOH to adjust the pH to pH7.1, leave it at 4ºC overnight, and then centrifuge at 10000´g at 4ºC for 10 minutes, and take the supernatant;

(4) Preparation of pure anti-human papillomavirus (HPV) IgY antibodies:

Slowly add ammonium sulfate powder to the anti-human papillomavirus (HPV) IgY antibody supernatant treated with chlorhexidine gluconate (3) above, and stir while adding until the ammonium sulfate reaches 50% saturation, stir thoroughly After mixing, let stand at room temperature for 6 hours, then centrifuge the solution at 10,000 g for 10 minutes in a high-speed centrifuge at 4ºC. Discard the supernatant and collect the precipitate. Dilute the precipitate with distilled water to the original solution volume, and then slowly add amine sulfate powder. Add and stir until the ammonium sulfate reaches 32% saturation. Let it stand at room temperature for 6 hours. Then centrifuge the solution at 10,000 g for 10 minutes in a high-speed centrifuge at 4ºC. Discard the supernatant and collect the precipitate. Dissolve the precipitate with distilled water and use 10 kDa solution. The hollow fiber ultrafiltration membrane is used to remove salt by ultrafiltration to obtain pure anti-human papillomavirus (HPV) IgY antibody; the pure IgY antibody was measured by SDS-PAGE electrophoresis, and its purity was 85.8%; the pure IgY antibody was As measured by ELISA method, the IgY antibody titer was 1:16384/mg protein.

Example 3:

Anti-human papillomavirus (HPV) IgY antibody oral and throat spray and aerosol:

Water-extracted anti-human papillomavirus (HPV) IgY antibody 0.2g%

Orange flavor 0.6ml%

Peach flavor 0.35ml%

Aspartame 0.12g%

Distilled water to add to 100ml

Procedure for preparing 100ml anti-human papillomavirus (HPV) IgY antibody oral and throat spray or aerosol: Add 60ml distilled water into a beaker, then add the above ingredients in sequence, and finally add water to extract anti-human papillomavirus (human papillomavirus) papillomavirus, HPV) IgY antibody, then add distilled water to 100ml, mix thoroughly, then filter and sterilize with positive pressure through a 0.45μ sterilized microporous filter membrane, and aseptically fill into sterilized spray and aerosol bottles for packaging.

Example 4:

Anti-human papillomavirus (HPV) IgY antibody oral and throat spray and aerosol:

Pure anti-human papillomavirus (HPV) IgY antibody 0.05g%

Orange flavor 0.60ml%

Peach flavor 0.35ml%

Aspartame 0.12g%

Distilled water to add to 100ml

Procedure for preparing 100ml anti-human papillomavirus (HPV) IgY antibody oral and throat spray or aerosol: Add 60ml distilled water into a beaker, then add the above ingredients in sequence, and finally add purified anti-human papillomavirus (human papillomavirus) , HPV) IgY antibody, then add distilled water to 100ml, mix thoroughly, then filter and sterilize with positive pressure through a 0.45μ sterilized microporous filter membrane, and aseptically fill into sterilized spray and aerosol bottles for packaging.

Example 5:

Anti-human papillomavirus (HPV) IgY antibody rinse:

Water-extracted anti-human papillomavirus (HPV) IgY antibody 0.15g%

Orange flavor 0.60ml%

Peach flavor 0.45ml%

Aspartame 0.12g%

Distilled water to add to 100ml

Procedure for preparing 100ml anti-human papillomavirus (HPV) IgY antibody gargle: add 60ml distilled water into a beaker, then add the above ingredients in sequence, and finally add water to extract anti-human papillomavirus (HPV) IgY antibodies. Then add distilled water to 100 ml, mix thoroughly, filter and sterilize with positive pressure through a 0.45μ sterilized microporous filter membrane, and aseptically fill into sterilized plastic bottles for packaging.

Example 6:

Anti-human papillomavirus (HPV) IgY antibody rinse:

Purified anti-human papillomavirus (HPV) IgY antibody 0.05g%

Orange flavor 0.60ml%

Peach flavor 0.45ml%

Aspartame 0.12g%

Distilled water to add to 100ml

Procedure for preparing 100 ml of anti-human papillomavirus (HPV) IgY antibody gargle: add 60 ml of distilled water into a beaker, then add the above ingredients in sequence, and finally add purified anti-human papillomavirus (HPV) IgY antibody, and then Add distilled water to 100 ml, mix thoroughly, filter and sterilize with positive pressure through a 0.45μ sterilized microporous filter membrane, and aseptically fill into sterilized plastic bottles for packaging.

Example 7:

Preparation of specific anti-human papillomavirus (HPV) IgY antibodies and extraction of IgY antibodies by water dilution method:

(1) Preparation of immunogen:

Weigh 1 mg of the fusion protein consisting of the N-terminal 200 aa of the L1 capsid protein of the recombinant HPV 18 virus and the N-terminal 200 aa of the L2 capsid protein to prepare the Freund's complete adjuvant immunogen. Mix 10 ml of N-terminal 200 aa solution of HPV18 virus L1 and L2 capsid fusion protein (1 mg fusion protein, concentration 100 μg/ml) with 10 ml of Freund's complete adjuvant (containing 0.2 g of live BCG vaccine, 20 mg/ml). Stir with a high-speed mixer at 6000 rpm for 5 minutes, stop for 3 minutes, stir again, and emulsify for a total of 6 times to finally prepare the water-in-oil Freund's complete adjuvant immunogen (the immunogen contains fusion protein with a final concentration of 50 μg/ml, and live BCG vaccine 10 mg/ml).

Weigh 2 mg of the fusion protein composed of the N-terminal 200 aa of the L1 capsid protein of the recombinant HPV 18 virus and the N-terminal 200 aa of the L2 capsid protein to prepare the Freund's incomplete adjuvant immunogen. Mix 20 ml of HPV18 virus L1 and L2 capsid fusion protein N-terminal 200 aa solution (2 mg fusion protein, concentration 100 μg/ml) with 20 ml of Freund's incomplete adjuvant, stir with a high-speed mixer at 6000 rpm for 5 min, stop for 3 min and then repeat Stir for a total of 6 times to emulsify, and finally prepare the water-in-oil Freund's incomplete adjuvant immunogen (the immunogen contains the fusion protein at a final concentration of 50 μg/ml).

(2) Immunization of laying hens: Use a 1ml sterile plastic syringe to extract 1ml of the water-in-oil complete Freund's adjuvant immunogen prepared above, divide it into 50 points and inject it into the body of a 23-week-old Hyline brown laying hen. Subcutaneously in wings, chest, thighs, back, etc., and injected into thigh muscles at 6 points. After 2 weeks, use 1ml of the prepared water-in-oil Freund's incomplete adjuvant immunogen and inject it subcutaneously into the wings, chest, thighs, back, etc. of the laying hen at 40 points, and at 5 points. Within the thigh muscles. After 4 weeks, use 1 ml of the water-in-oil Freund's incomplete adjuvant immunogen prepared above and inject it subcutaneously into the wings, chest, thighs, back, etc. of the laying hen at 42 points, and at 3 points. Within the thigh muscles. The total number of vaccinated hens is 18.

(3) Preparation of anti-human papillomavirus (HPV) IgY antibody water dilution method extract:

Collect 60 eggs from immunized hens on the 50th to 53rd day after the first immunization. Wash the surface of the eggs with boiling water and soak them in 70% alcohol. Remove the eggshells to obtain the egg yolk. Remove the egg yolk membrane to obtain the egg yolk liquid. Mix the egg yolk liquid with Add distilled water at a volume ratio of 1:9, stir, adjust the pH to pH 5.0 with 1N HCl, and place in a 4°C freezer overnight;

Centrifuge the overnight egg yolk dilution at 10,000´g in a high-speed centrifuge at 4ºC for 15 minutes. Take the supernatant and concentrate it 10 times by ultrafiltration in a hollow fiber ultrafilter with a molecular weight cutoff of 50kDa. Then add 0.8% gluconic acid. Chlorhexidine, and use 1N NaOH to adjust the pH to pH 7.2, let it stand at 4ºC overnight, then centrifuge at 10000´g in a high-speed centrifuge at 4ºC for 10 minutes, take the supernatant, and pass the supernatant through a 0.45μ sterilized microporous filter. After membrane filtration and sterilization, a water extract of anti-human papillomavirus (HPV) IgY antibody was obtained; the IgY water extract solution was measured by SDS-PAGE electrophoresis, and its IgY purity reached 33%; by ELISA (enzyme-linked immunoassay) Adsorption experiment) determined that the specific anti-HPV 18 virus L1 capsid protein and L2 capsid protein N-terminal 200 aa fusion protein immunogen (antigen) IgY antibody titer is 1:4096/mg protein.

According to the above experimental results, it can be clearly seen that: ⑴. The anti-human papillomavirus (HPV) IgY antibody water dilution method extract prepared according to the method of the present invention has specific anti-HPV 18 virus L1 capsid protein IgY antibody with the N-terminal 200 aa fusion protein immunogen (antigen) of L2 capsid protein; ⑵. From the above experiments, it can be seen that the anti-human papillomavirus (HPV) IgY antibody titer can reach after the third immunization Peak (the first immunization was with Freund's complete adjuvant immunogen on day 0, the second immunization was with Freund's incomplete adjuvant immunogen on day 14, and the third immunization with Freund's incomplete adjuvant immunogen was on day 28 .); ⑶. It can be predicted from the above experiments that the anti-human papillomavirus (HPV) IgY antibody water dilution extract can prevent and treat human tissue lesions (warts or tumors) caused by HPV type 18 virus; ⑷. By This can also be extended to skin, mucosal warts or tumors caused by viruses that share common antigenic determinants with HPV type 18 virus, etc., which can be extracted using the anti-human papillomavirus (HPV) IgY antibody water dilution method of the present invention prevention and treatment.

Example 8:

Preparation and purification of specific anti-human papillomavirus (HPV) IgY antibodies:

(1) Preparation of immunogen:

Weigh 1 mg of the fusion protein consisting of the N-terminal 200 aa of the L1 capsid protein of the recombinant HPV 18 virus and the N-terminal 200 aa of the L2 capsid protein to prepare the Freund's complete adjuvant immunogen. Mix 10 ml of HPV18 virus L1 and L2 capsid fusion protein N-terminal 200aa solution (1 mg fusion protein, concentration 100 μg/ml) with 10 ml of Freund's complete adjuvant (containing 0.2 g of live BCG vaccine, 20 mg/ml). Stir the mixer at 6000rpm for 5 minutes, stop for 3 minutes and stir again. Emulsify 6 times in total, and finally prepare the water-in-oil Freund's complete adjuvant immunogen (the immunogen contains fusion protein with a final concentration of 50 μg/ml, and live BCG vaccine 10 mg/ml).

And prepare pure water extract of anti-human papillomavirus (HPV) IgY antibody according to the method of the present invention,

Weigh 2 mg of the fusion protein composed of the N-terminal 200 aa of the L1 capsid protein of the recombinant HPV 18 virus and the N-terminal 200 aa of the L2 capsid protein to prepare the Freund's incomplete adjuvant immunogen. Mix 20 ml of HPV18 virus L1 and L2 capsid fusion protein N-terminal 200 aa solution (2 mg fusion protein, concentration 100 μg/ml) with 20 ml of Freund's incomplete adjuvant, stir with a high-speed mixer at 6000 rpm for 5 min, stop for 3 min and then repeat Stir for a total of 6 times to emulsify, and finally prepare the water-in-oil Freund's incomplete adjuvant immunogen (the immunogen contains the fusion protein at a final concentration of 50 μg/ml).

(2) Immunization of laying hens: Use a 1ml sterile plastic syringe to extract 1ml of the water-in-oil complete Freund's adjuvant immunogen prepared above, divide it into 50 points and inject it into the body of a 23-week-old Hyline brown laying hen. Subcutaneously in wings, chest, thighs, back, etc., and injected into thigh muscles at 6 points. After 2 weeks, use 1ml of the prepared water-in-oil Freund's incomplete adjuvant immunogen and inject it subcutaneously into the wings, chest, thighs, back, etc. of the laying hen at 40 points, and at 5 points. Within the thigh muscles. After 4 weeks, use 1 ml of the water-in-oil Freund's incomplete adjuvant immunogen prepared above and inject it subcutaneously into the wings, chest, thighs, back, etc. of the laying hen at 42 points, and at 3 points. Within the thigh muscles. The total number of vaccinated hens is 18.

(3) Preparation of anti-human papillomavirus (HPV) IgY antibody water dilution method extract:

Collect 60 eggs from immunized hens on the 50th to 53rd day after the first immunization. Wash the surface of the eggs with boiling water and soak them in 70% alcohol. Remove the eggshells to obtain the egg yolk. Remove the egg yolk membrane to obtain the egg yolk liquid. Mix the egg yolk liquid with Add distilled water at a volume ratio of 1:9 and stir, adjust the pH to pH 5.0 with 1N HCl, and place it in a 4°C freezer overnight; centrifuge the overnight egg yolk dilution at 10,000´g in a high-speed centrifuge at 4°C for 15 minutes, and take the supernatant , ultrafiltrate and concentrate the supernatant 10 times through a hollow fiber ultrafilter with a molecular weight cutoff of 50kDa, then add 0.8% chlorhexidine gluconate, and use 1N NaOH to adjust the pH to pH 7.2, and leave it to stand at 4ºC overnight. Centrifuge at 10,000´g in a high-speed centrifuge at 4ºC for 10 minutes and take the supernatant.

(4) Preparation of pure anti-human papillomavirus (HPV) IgY antibodies:

Slowly add ammonium sulfate powder to the supernatant extracted from the chlorhexidine gluconate-treated anti-human papillomavirus (HPV) IgY antibody water dilution method in the above (3), and stir while adding until the ammonium sulfate reaches 50%. saturation, mix thoroughly and let stand at room temperature for 6 hours, then centrifuge the solution at 4ºC high-speed centrifuge at 10,000 g for 10 minutes, discard the supernatant and collect the precipitate, dilute the precipitate with distilled water to the original solution volume, and then slowly add Add ammonium sulfate powder while stirring until the ammonium sulfate reaches 33% saturation. Leave it at room temperature for 6 hours. Then centrifuge the solution at 10,000´g in a high-speed centrifuge at 4ºC for 10 minutes. Discard the supernatant and collect the precipitate, then dissolve it in distilled water. After precipitation, a 10kDa hollow fiber ultrafiltration membrane is used to remove salt by ultrafiltration to obtain pure anti-human papillomavirus (HPV) IgY antibody;

The pure IgY antibody was measured by SDS-PAGE electrophoresis, and its purity was 88%; the pure IgY antibody was measured by ELISA, and its specific IgY antibody binds to the N-terminal 200 aa fusion protein of HPV18 virus L1 capsid protein and L2 capsid protein. The biological potency of the immunogen (antigen) is 1:16384/mg protein.

According to the above experimental results, it can be clearly seen that: ⑴. The pure anti-human papillomavirus (HPV) IgY antibody prepared according to the method of the present invention has specific anti-HPV 18 virus L1 capsid protein and L2 capsid protein. IgY antibody of the N-terminal 200 aa fusion protein immunogen (antigen) of the shell protein; ⑵. From the above experiments, it can be seen that the anti-human papillomavirus (HPV) IgY antibody titer can reach the peak after the third immunization (0 The first immunization was with Freund's complete adjuvant immunogen on day 14, the second immunization was with Freund's incomplete adjuvant immunogen on day 14, and the third immunization with Freund's incomplete adjuvant immunogen was on day 28.); ⑶. It can be predicted from the above experiments that pure anti-human papillomavirus (HPV) IgY antibodies can prevent and treat human tissue lesions (warts or tumors) caused by HPV type 18 virus; ⑷. This can also be expanded to include Viruses that have common antigenic determinants with the above viruses, such as skin and mucous membrane warts or tumors caused by HPV16, as well as common warts, genital warts (condyloma acuminata), cervical cancer, and anal cancer induced by other viruses with common antigenic determinants , laryngeal cancer, etc. can be prevented and treated by using the pure anti-human papillomavirus (HPV) IgY antibody of the present invention.

Example 9:

Comparison of the induction of specific anti-human papillomavirus (HPV) IgY antibodies by different chicken species and the preparation of IgY antibodies extracted by water dilution:

(1) Preparation of immunogen:

Weigh 1 mg of the fusion protein consisting of the N-terminal 200 aa of the L1 capsid protein of the recombinant HPV 18 virus and the N-terminal 200 aa of the L2 capsid protein to prepare the Freund's complete adjuvant immunogen. Mix 10 ml of N-terminal 200 aa solution of HPV18 virus L1 and L2 capsid fusion protein (1 mg fusion protein, concentration 100 μg/ml) with 10 ml of Freund's complete adjuvant (containing 0.2 g of live BCG vaccine, 20 mg/ml). Stir with a high-speed mixer at 6000 rpm for 5 minutes, stop for 3 minutes, stir again, and emulsify for a total of 6 times to finally prepare the water-in-oil Freund's complete adjuvant immunogen (the immunogen contains fusion protein with a final concentration of 50 μg/ml, and live BCG vaccine 10 mg/ml).

Weigh 2 mg of the fusion protein composed of the N-terminal 200 aa of the L1 capsid protein of the recombinant HPV 18 virus and the N-terminal 200 aa of the L2 capsid protein to prepare the Freund's incomplete adjuvant immunogen. Mix 20 ml of HPV18 virus L1 and L2 capsid fusion protein N-terminal 200 aa solution (2 mg fusion protein, concentration 100 μg/ml) with 20 ml of Freund's incomplete adjuvant, stir with a high-speed mixer at 6000 rpm for 5 min, stop for 3 min and then repeat Stir for a total of 6 times to emulsify, and finally prepare the water-in-oil Freund's incomplete adjuvant immunogen (the immunogen contains the fusion protein at a final concentration of 50 μg/ml).

(2) Immune egg-laying hens: Select three types of chickens such as Hai-Line brown egg-laying hens, Laihang egg-laying hens, and star egg-laying hens. Use a 1ml sterile plastic syringe to extract the oil package prepared above. 1 ml of the immunogen in Waterford's complete adjuvant was injected subcutaneously into the wings, chest, thighs, back, etc. of the hens at 60 points for three types of 25-week-old egg-laying hens, and into the thigh muscles at 3 points. After 2 weeks, use 1 ml of the prepared water-in-oil Freund's incomplete adjuvant immunogen and inject it subcutaneously into the wings, chest, thighs, back, etc. of the laying hen at 45 points, and at 5 points. Within the thigh muscles. After 4 weeks, use 1ml of the prepared water-in-oil Freund's incomplete adjuvant immunogen and inject it subcutaneously into the wings, chest, thighs, back, etc. of the laying hen at 40 points, and inject it at 3 points Within the thigh muscles. The number of hens of each chicken breed to be immunized is 6.

(3) Preparation of anti-human papillomavirus (HPV) IgY antibody water dilution method extract:

Collect 20 eggs from the immunized hens on the 45th to 49th day after the first vaccination of each type of hen. Wash the surface of the eggs with boiling water and soak them in 70% alcohol. Remove the eggshells to obtain the yolks, and remove the yolk membrane to obtain the yolk liquid. , add distilled water to the egg yolk liquid and distilled water in a volume ratio of 1:9, stir, adjust the pH to pH 5.1 with 1N HCl, and place in a 4°C freezer overnight;

Centrifuge the overnight egg yolk dilution at 10,000´g in a high-speed centrifuge at 4ºC for 15 minutes. Take the supernatant and concentrate it 10 times by ultrafiltration in a hollow fiber ultrafilter with a molecular weight cutoff of 50kDa. Then add 0.8% gluconic acid. Chlorhexidine, and use 1N NaOH to adjust the pH to pH 7.2, let it stand at 4ºC overnight, then centrifuge at 10000´g in a high-speed centrifuge at 4ºC for 10 minutes, take the supernatant, and pass the supernatant through a 0.45μ sterilized microporous filter. After membrane filtration and sterilization, a water dilution extract of anti-human papillomavirus (HPV) IgY antibody was obtained; the IgY water extract solution was measured by SDS-PAGE electrophoresis, and its IgY purity reached 33.5%; by ELISA (enzyme The specific anti-HPV 18 virus L1 capsid protein and L2 capsid protein N-terminal 200 aa fusion protein immunogen (antigen) IgY antibody titer is 1:4096/mg protein, as determined by the immunosorbent assay), as shown in the table 1.

Table 1. Immune response capabilities of laying hens of different chicken breeds to the same immunogen and titers of specific IgY antibody aqueous extracts

According to the above experimental results, it can be clearly seen that under the same immunization conditions, the anti-human papillomavirus (HPV) IgY antibody water dilution extracts of different chicken species prepared according to the method of the present invention are The biological potency of specific anti-human papillomavirus (HPV) IgY antibodies is consistent.

Example 10:

Observe the persistence of the induced specific anti-human papillomavirus (HPV) IgY antibody production time—preparation of specific anti-human papillomavirus (HPV) IgY antibody and extraction of IgY antibody by water dilution method:

(1) Preparation of immunogen:

Weigh 2 mg of the fusion protein consisting of the N-terminal 200 aa of the L1 capsid protein of the recombinant HPV 18 virus and the N-terminal 200 aa of the L2 capsid protein to prepare the Freund's complete adjuvant immunogen. Mix 20 ml of N-terminal 200 aa solution of HPV18 virus L1 and L2 capsid fusion protein (2 mg fusion protein, concentration 100 μg/ml) with 20 ml of Freund's complete adjuvant (containing 0.4 g of live BCG vaccine, 20 mg/ml). Stir with a high-speed mixer at 6000 rpm for 5 minutes, stop for 3 minutes, stir again, and emulsify for a total of 6 times to finally prepare the water-in-oil Freund's complete adjuvant immunogen (the immunogen contains fusion protein with a final concentration of 50 μg/ml, and live BCG vaccine 10 mg/ml).

Weigh 10 mg of the fusion protein composed of the N-terminal 200 aa of the L1 capsid protein of the recombinant HPV 18 virus and the N-terminal 200 aa of the L2 capsid protein to prepare the Freund's incomplete adjuvant immunogen. Mix 100 ml of HPV18 virus L1 and L2 capsid fusion protein N-terminal 200 aa solution (10 mg fusion protein, concentration 100 μg/ml) with 100 ml of Freund's incomplete adjuvant, stir with a high-speed mixer at 6000 rpm for 5 min, stop for 3 min and then repeat Stir for a total of 6 times to emulsify, and finally prepare the water-in-oil Freund's incomplete adjuvant immunogen (the immunogen contains the fusion protein at a final concentration of 50 μg/ml).

(2) Immunization of laying hens: Use a 1ml sterile plastic syringe to extract 1ml of the water-in-oil complete Freund's adjuvant immunogen prepared above, divide it into 50 points and inject it into the body of a 23-week-old Hyline brown laying hen. A total of 32 egg-laying hens were immunized for the first time; 2 weeks later, the water-in-oil incomplete adjuvant prepared above was used. 1ml of immunogen was divided into 40 points and injected subcutaneously into the wings, chest, thighs, back, etc. of the laying hens, and 5 points were injected into the thigh muscles. A total of 32 laying hens were immunized for the second time; 4 After 2 weeks, use 1ml of the prepared water-in-oil Freund's incomplete adjuvant immunogen and inject it subcutaneously into the wings, chest, thighs, back, etc. of the laying hen at 42 points, and into the thigh at 3 points. Intramuscularly, a total of 32 laying hens were immunized for the third time; 6 weeks later, 1 ml of the water-in-oil Freund's incomplete adjuvant immunogen prepared above was used and injected into the wings of laying hens at 42 points. Subcutaneously on the chest, thighs, back, etc., and injected into the thigh muscles at three points, a total of 16 laying hens were immunized for the fourth time; 8 weeks later, the water-in-oil Freund's incomplete adjuvant prepared above was used for immunization The original 1ml was injected into the subcutaneous parts of the wings, chest, thighs, back, etc. of laying hens at 40 points, and injected into the thigh muscles at 3 points. A total of 16 laying hens were immunized in the fifth time; On the 130th day after immunization, use 1 ml of the water-in-oil Freund's incomplete adjuvant immunogen prepared by the above preparation method, and inject it into the subcutaneous parts of the laying hen's wings, chest, thighs, back, etc. at 25 points, and at 3 points Point injection into the thigh muscle, a total of 16 laying hens were immunized for the sixth time; on the 190th day after the first immunization, 1 ml of water-in-oil Freund's incomplete adjuvant immunogen prepared by the above preparation method was divided into 25 Injection into the subcutaneous parts of the wings, chest, thighs, back, etc. of laying hens, and injection into the thigh muscles at 3 points. A total of 16 laying hens were immunized for the seventh time; on the 250th day after the first immunization, Use 1ml of the water-in-oil Freund's incomplete adjuvant immunogen prepared by the above preparation method, and inject it into the subcutaneous parts of the wings, chest, thighs, back, etc. of the laying hen at 25 points, and into the thigh muscles at 3 points. , a total of 16 laying hens were immunized for the eighth time; on the 310th day after the first immunization, 1ml of the water-in-oil Freund's incomplete adjuvant immunogen prepared by the above preparation method was divided into 20 points and injected into the laying hens. The vaccine was injected subcutaneously into the wings, breasts, thighs, backs, etc. of the chickens, and injected into the thigh muscles at 5 points. A total of 16 egg-laying hens were immunized for the ninth time.

(3) Preparation of anti-human papillomavirus (HPV) IgY antibody water dilution method extract:

Collect 22 eggs from the immunized hen on the 47th day after the first immunization and 10 eggs from the immunized hen on the 323rd day. Wash the surface of the eggs with boiling water and soak them in 70% alcohol. Remove the eggshells and remove the yolks. Take the egg yolk liquid from the egg yolk film, add distilled water to the egg yolk liquid and distilled water in a volume ratio of 1:9, stir, adjust the pH to pH 5.1 with 1N HCl, and place it in a 4°C freezer overnight;

Centrifuge the overnight egg yolk dilution at 10,000´g in a high-speed centrifuge at 4ºC for 15 minutes. Take the supernatant and concentrate it 10 times by ultrafiltration in a hollow fiber ultrafilter with a molecular weight cutoff of 50kDa. Then add 0.8% gluconic acid. Chlorhexidine, and use 1N NaOH to adjust the pH to pH 7.2, let it stand at 4ºC overnight, then centrifuge at 10000´g in a high-speed centrifuge at 4ºC for 10 minutes, take the supernatant, and pass the supernatant through a 0.45μ sterilized microporous filter. Membrane filtration and sterilization were performed to obtain the water extract of anti-human papillomavirus (HPV) IgY antibodies; the IgY water extract solutions extracted by the egg water dilution method of three and nine times immunized laying hens were subjected to SDS-PAGE electrophoresis. It was determined that its IgY purity reached 33~34%; as determined by ELISA (enzyme-linked immunosorbent assay), its specific anti-HPV 18 virus L1 capsid protein and L2 capsid protein N-terminal 200 aa fusion protein immunogen (antigen) The titers of IgY antibodies are shown in Table 2.

Table 2. Comparison of the ability of Hy-Line brown hens to produce high-titer specific IgY antibodies after three immunizations and nine immunizations.

According to the above experimental results, it can be clearly seen that the specific anti-human papilloma of Hy-Line brown egg-laying hens immunized according to the nine-time immunization method of the present invention is compared with the Hy-Line brown egg-laying hens immunized by the traditional three-time immunization method. Virus (human papillomavirus, HPV) IgY antibody production ability is more durable. And according to the method of the present invention, the anti-human papillomavirus (HPV) IgY antibody water extract 47 days after the first immunization and the anti-human papillomavirus (HPV) IgY antibody water 323 days after the first immunization are prepared The biological potency of the extracts is the same. The nine-time immunization method of the present invention is better than the traditional three-time immunization method. The biological titer of its specific anti-human papillomavirus (HPV) IgY antibody is significantly different compared with the nine-time immunization method. Chickens take longer to produce high-titer specific anti-human papillomavirus (HPV) IgY antibodies. The immunization method of the present invention has no life-threatening or harmful effects on the immunized hens, and does not affect the hens' diet and egg production rate.

Example 11:

According to Example 7 of the present invention, the N-terminal 200 aa fusion protein of HPV type 18 virus L1 capsid protein and L2 capsid protein was used to immunize eggs produced by laying hens on the 47th day against human papillomavirus (HPV) The specific IgY antibodies in the IgY antibody water dilution method can produce cross-immune reactions with multiple types of HPV. The specific IgY antibody binding titers are shown in Table 3.

Table 3. Comparison of the cross-binding reactivity of anti-human papillomavirus (HPV) IgY antibodies to different types of HPV viruses

According to the above experimental results, it can be clearly seen that the anti-human papillomavirus (HPV) IgY antibody water dilution extract prepared according to the method of the present invention can interact with other types of HPV with common antigenic determinants. Cross-antigen antibody immune binding reaction, but the strength of the common antigenicity is also different, manifested in the different biological titers of IgY antibodies. This shows that the anti-human papillomavirus (HPV) IgY antibodies produced by the method of the present invention can produce antibodies against more antigenic determinants of HPV, which not only have a large number and types of antibodies, but also have high binding titers. The anti-human papillomavirus (HPV) IgY antibodies prepared by the method of the present invention can make up for or make up for the current inefficiency and inability of HPV prevention and treatment. The anti-human papillomavirus (HPV) IgY antibody prepared by the method of the present invention has broad spectrum and can effectively prevent and treat common warts, genital warts (genital warts), cervical cancer, anal cancer, and laryngeal cancer caused by many types of HPV. wait. The method of the present invention and the anti-human papillomavirus (HPV) IgY antibody make up for the defects of the HPV vaccine, truly block HPV from invading human epithelial cells, produce unexpected surprises, realize HPV can be prevented and cured, and achieve perfect results. Ideal for passive immunotherapy.

Claims (9)

1. An anti-human papillomavirus IgY antibody, characterized in that: it is obtained by the following preparation method: (1) Immunogen preparation: Take 10~100 μg/mg of the fusion protein composed of the N-terminal 11-200 aa of the L1 capsid protein of the recombinant HPV 18 virus and the L2 capsid protein; Mix an equal volume of HPV18 virus L1 and L2 capsid fusion protein solution with an equal volume of Freund's complete adjuvant containing 5~10 mg/ml live BCG vaccine, stir and emulsify, and prepare water-in-oil Freund's complete adjuvant. immunogen; Mix an equal volume of HPV18 virus L1 and L2 capsid fusion protein solution with an equal volume of Freund's incomplete adjuvant, stir and emulsify, and prepare a water-in-oil Freund's incomplete adjuvant immunogen; Each milliliter of Freund's complete adjuvant immunogen and Freund's incomplete adjuvant immunogen contains 10-100 μg of the fusion capsid protein composed of the N-terminal 11-200 aa of HPV18 virus L1 and L2; (2) Immunity: Inject Freund's complete adjuvant immunogen subcutaneously and intramuscularly into laying hens at 5 to 80 o'clock. Each laying hen was injected with 1 ml of immunogen, and then 1 ml of Freund's incomplete adjuvant immunogen was administered every 1 to 2 weeks. The laying hens were injected subcutaneously and intramuscularly at 10-60 o'clock for a total of four times. After that, 1 ml of Freund's incomplete adjuvant immunogen was used subcutaneously and intramuscularly at 10-30 o'clock to strengthen the laying hens every 1 to 2 months. Immunity for one year; (3) Preparation of anti-human papillomavirus IgY antibody aqueous extract: Collect eggs from immunized hens 21 days after immunization. Wash the surface of the eggs with boiling water and soak them in 70% alcohol. Remove the eggshells to get the yolk. Remove the yolk membrane to get the yolk liquid. Mix the egg yolk liquid and distilled water at a volume ratio of 1:5 to 12 Add distilled water in the appropriate proportion and stir, adjust the pH to pH 4.9~5.3 with 1N HCl, and let it stand overnight at 4ºC; Centrifuge the egg yolk dilution at high speed for 15 minutes at 4ºC to take the supernatant, then ultrafiltrate and concentrate it 10 to 20 times in a hollow fiber ultrafilter with a molecular weight cutoff of 10 to 50 kDa, and then add 0.02% to 5.0% chlorhexidine gluconate. , and use 1N NaOH to adjust the pH to pH 7.0 ~ 7.5, and let it stand at 4ºC overnight; then centrifuge at 4ºC for 10 minutes to take the supernatant, and filter the supernatant through a 0.22μ ~ 0.45μ sterilized microporous filter to sterilize , to obtain the aqueous extract of anti-human papillomavirus IgY antibodies. 2. The anti-human papillomavirus IgY antibody according to claim 1, characterized in that: the fusion protein of the HPV 18 virus L1 capsid protein and the N-terminal 11-200aa of the L2 capsid protein in the preparation of the immunogen . 3. The anti-human papillomavirus IgY antibody according to claim 1, characterized in that: in the preparation of the immunogen, the fusion of HPV 18 virus L1 capsid protein and L2 capsid protein N-terminal 11-200aa Protein 10~100μg/mg. 4. The anti-human papillomavirus IgY antibody according to claim 1, characterized in that: the egg-laying hens in the immunity include Laihang raised in a normal environment, a clean environment, and a SPF environment. Chickens, Highland brown chickens, Roman chickens, star chickens, and domestic native chickens. 5. A method for preparing pure anti-human papillomavirus IgY antibody, which is characterized by: including the following steps: (1) Immunogen preparation: Take 10~100 μg/ml of the fusion protein composed of the N-terminal 11-200 aa of the L1 capsid protein of the recombinant HPV 18 virus and the L2 capsid protein; Mix an equal volume of HPV18 virus L1 and L2 capsid fusion protein solution with an equal volume of Freund's complete adjuvant containing 5~10 mg/ml live BCG vaccine, stir and emulsify, and prepare water-in-oil Freund's complete adjuvant. immunogen; Mix an equal volume of HPV18 virus L1 and L2 capsid fusion protein solution with an equal volume of Freund's incomplete adjuvant, stir and emulsify, and prepare a water-in-oil Freund's incomplete adjuvant immunogen; Each milliliter of Freund's complete adjuvant immunogen and Freund's incomplete adjuvant immunogen contains 10-100 μg of the fusion capsid protein composed of the N-terminal 11-200 aa of HPV18 virus L1 and L2; (2) Immunity: Inject Freund's complete adjuvant immunogen subcutaneously and intramuscularly into laying hens at 5 to 80 o'clock. Each laying hen was injected with 1 ml of immunogen, and then 1 ml of Freund's incomplete adjuvant immunogen was administered every 1 to 2 weeks. The laying hens were injected subcutaneously and intramuscularly at 10-60 o'clock for a total of four times. After that, 1 ml of Freund's incomplete adjuvant immunogen was used subcutaneously and intramuscularly at 10-30 o'clock to strengthen the laying hens every 1 to 2 months. Immunity for one year; (3) Preparation of anti-human papillomavirus IgY antibody aqueous extract: Collect eggs from immunized hens 21 days after immunization. Wash the surface of the eggs with boiling water and soak them in 70% alcohol. Remove the eggshells to get the yolk. Remove the yolk membrane to get the yolk liquid. Mix the egg yolk liquid and distilled water at a volume ratio of 1:5 to 12 Add distilled water in the appropriate proportion and stir, adjust the pH to pH 4.9~5.3 with 1N HCl, and let it stand overnight at 4ºC; Centrifuge the egg yolk dilution at high speed for 15 minutes at 4ºC to take the supernatant, then ultrafiltrate and concentrate it 10 to 20 times in a hollow fiber ultrafilter with a molecular weight cutoff of 10 to 50 kDa, and then add 0.02% to 5.0% chlorhexidine gluconate. , and use 1N NaOH to adjust the pH to pH 7.0 ~ 7.5, and let it stand at 4ºC overnight; then centrifuge at 4ºC for 10 minutes to take the supernatant, and filter the supernatant through a 0.22μ ~ 0.45μ sterilized microporous filter to sterilize , to obtain the aqueous extract of anti-human papillomavirus IgY antibodies; (4) Preparation of pure anti-human papillomavirus IgY antibodies: Add amine sulfate to the chlorhexidine gluconate-treated anti-human papillomavirus IgY antibody aqueous extract to a saturation level of 45% to 60%, mix thoroughly, let stand at room temperature for 2 to 12 hours, and then centrifuge at 4ºC at high speed 10 minutes, take the precipitate, dilute the precipitate to the original volume with distilled water, add amine sulfate to a saturation of 28%~35%, stir thoroughly, let it stand at room temperature for 2~12 hours, then centrifuge at 4ºC for 10 minutes at high speed, and remove Precipitate, dissolve with distilled water and use hollow fiber ultrafiltration membrane to remove salt by ultrafiltration to obtain pure anti-human papillomavirus IgY antibody. 6. The preparation method of anti-human papillomavirus IgY antibodies according to claim 1 or 5, characterized in that: it also includes the following steps: adding pharmaceutically acceptable additives to prepare anti-human papillomavirus IgY antibodies. Sprays, aerosols, rinses, lotions, suppositories. 7. The anti-human papillomavirus IgY antibody prepared according to the method of the present invention is locally applied to the oral cavity, throat, and bronchial mucosa, as well as to the skin and mucosal surfaces of the vulva, vagina, hands, and feet to prevent and treat warts and tumors. 8. A kind of anti-human papillomavirus IgY antibody according to claim 1, characterized in that: it is obtained by the following preparation method: (1) Immunogen preparation: Take 10~100 μg/mg of the fusion protein composed of the N-terminal 11-200 aa of the L1 capsid protein of the recombinant HPV 18 virus and the L2 capsid protein; Mix an equal volume of HPV18 virus L1 and L2 capsid fusion protein solution with an equal volume of Freund's complete adjuvant containing 5~10 mg/ml live BCG vaccine, stir and emulsify, and prepare water-in-oil Freund's complete adjuvant. immunogen; Mix an equal volume of HPV18 virus L1 and L2 capsid fusion protein solution with an equal volume of Freund's incomplete adjuvant, stir and emulsify, and prepare a water-in-oil Freund's incomplete adjuvant immunogen; Each milliliter of Freund's complete adjuvant immunogen and Freund's incomplete adjuvant immunogen contains 10 μg of the fusion capsid protein composed of the N-terminal 11-200 aa of HPV18 virus L1 and L2; (2) Immunity: Inject Freund's complete adjuvant immunogen subcutaneously and intramuscularly into laying hens at 5 to 80 o'clock. Each laying hen was injected with 1 ml of immunogen, and then 1 ml of Freund's incomplete adjuvant immunogen was administered every 1 to 2 weeks. The laying hens were injected subcutaneously and intramuscularly at 10-60 o'clock for a total of four times. After that, 1 ml of Freund's incomplete adjuvant immunogen was used subcutaneously and intramuscularly at 10-30 o'clock to strengthen the immunity of the laying hens every one month. one year; (3) Preparation of anti-human papillomavirus IgY antibody aqueous extract: Collect eggs from immunized hens 21 days after immunization. Wash the surface of the eggs with boiling water and soak them in 70% alcohol. Remove the egg shells to get the yolk. Remove the yolk membrane to get the yolk liquid. Mix the egg yolk liquid and distilled water at a volume ratio of 1:5. Add distilled water and stir, adjust the pH to pH 4.9 with 1NHCl, and let it stand overnight at 4ºC; Centrifuge the egg yolk dilution at high speed for 15 minutes at 4ºC to take the supernatant, then ultrafiltrate and concentrate it 10 times in a hollow fiber ultrafilter with a molecular weight cutoff of 10~50kDa, then add 0.02% chlorhexidine gluconate, and use 1N NaOH Adjust the pH to pH 7.0 and let it stand at 4ºC overnight; then centrifuge at 4ºC for 10 minutes to take the supernatant, filter and sterilize the supernatant through a 0.22μ~0.45μ sterilized microporous filter membrane to obtain anti-human papilloma Aqueous extract of viral IgY antibodies. 9. A kind of anti-human papillomavirus IgY antibody according to claim 1, characterized in that: it is obtained by the following preparation method: (1) Immunogen preparation: Take 100 μg/mg of the fusion protein composed of the N-terminal 11-200 aa of the L1 capsid protein of the recombinant HPV 18 virus and the L2 capsid protein; Mix an equal volume of HPV18 virus L1 and L2 capsid fusion protein solution with an equal volume of Freund's complete adjuvant containing 10 mg/ml live BCG vaccine, stir and emulsify, and prepare a water-in-oil Freund's complete adjuvant immunogen. ; Mix an equal volume of HPV18 virus L1 and L2 capsid fusion protein solution with an equal volume of Freund's incomplete adjuvant, stir and emulsify, and prepare a water-in-oil Freund's incomplete adjuvant immunogen; Each milliliter of Freund's complete adjuvant immunogen and Freund's incomplete adjuvant immunogen contains 10-100 μg of the fusion capsid protein composed of the N-terminal 11-200 aa of HPV18 virus L1 and L2; (2) Immunity: Inject Freund's complete adjuvant immunogen subcutaneously and intramuscularly into laying hens at 5 to 80 o'clock. Each laying hen was injected with 1 ml of immunogen, and then 1 ml of Freund's incomplete adjuvant immunogen was administered every 1 to 2 weeks. The laying hens were injected subcutaneously and intramuscularly at 10-60 o'clock for a total of four times. After that, 1ml of Freund's incomplete adjuvant immunogen was used subcutaneously and intramuscularly at 10-30 o'clock to strengthen the immunity of the laying hens every 2 months. one year; (3) Preparation of anti-human papillomavirus IgY antibody aqueous extract: Collect eggs from immunized hens 21 days after immunization. Wash the surface of the eggs with boiling water and soak them in 70% alcohol. Remove the eggshells to get the yolk. Remove the yolk membrane to get the yolk liquid. Mix the egg yolk liquid and distilled water at a volume ratio of 1:12. Add distilled water and stir, adjust the pH to pH 5.3 with 1NHCl, and let it stand overnight at 4ºC; Centrifuge the egg yolk dilution at high speed for 15 minutes at 4ºC to take the supernatant, then ultrafiltrate and concentrate it 10-20 times in a hollow fiber ultrafilter with a molecular weight cutoff of 10-50kDa, then add 5.0% chlorhexidine gluconate, and use Adjust the pH to pH 7.5 with 1N NaOH, and let it stand at 4ºC overnight; then centrifuge at 4ºC for 10 minutes to take the supernatant, and filter and sterilize the supernatant through a 0.45μ sterilized microporous filter to obtain anti-human papillomavirus Aqueous extract of IgY antibodies.