CN110680917A - Preparation method and application of anti-propionibacterium acnes and staphylococcus composite IgY antigen - Google Patents
Preparation method and application of anti-propionibacterium acnes and staphylococcus composite IgY antigen Download PDFInfo
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/05—Actinobacteria, e.g. Actinomyces, Streptomyces, Nocardia, Bifidobacterium, Gardnerella, Corynebacterium; Propionibacterium
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
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- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0014—Skin, i.e. galenical aspects of topical compositions
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/10—Anti-acne agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
- C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
- C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
- C07K16/1271—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Micrococcaceae (F), e.g. Staphylococcus
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/70—Multivalent vaccine
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
- C07K2317/11—Immunoglobulins specific features characterized by their source of isolation or production isolated from eggs
Abstract
The invention discloses a preparation method and application of a composite IgY antigen for resisting propionibacterium acnes and staphylococcus, belonging to the technical field of resisting propionibacterium acnes and staphylococcus and comprising the following steps: the method comprises the following steps: preparing immunogen, mixing propionibacterium acnes and thallus fragments with golden and white staphylococcus and thallus fragments, mixing with Freund's complete adjuvant and Freund's incomplete adjuvant respectively, stirring and emulsifying to obtain water-in-oil Freund's complete adjuvant, Freund's incomplete adjuvant propionibacterium acnes and staphylococcus immunogen; step two: and (4) immunizing, and injecting the Freund's complete adjuvant immunogen into the laying hens. The specific anti-acne propionibacterium and staphylococcus composite IgY antibody prepared by the invention is locally applied to the face and the skin of the whole body, has no limitation and side effect of the medicine for the whole body, has quick effect, small dosage and good curative effect, and does not damage the normal micro-ecological environment on the surface of the mucous membrane skin.
Description
Technical Field
The invention relates to preparation and application of anti-acne IgY, in particular to a preparation method and application of a composite IgY antigen for resisting propionibacterium acnes and staphylococcus, belonging to the technical field of resisting propionibacterium acnes and staphylococcus.
Background
Propionibacterium acnes, also known as Propionibacterium acnes and Propionibacterium acnes, belong to the kingdom of bacteria, the phylum Actinomyces, class, subclass, order, the suborder Propionibacterium, the family, and the genus Propionibacterium, are typically gram-positive anaerobic bacteria that grow relatively slowly, are short rods, are intracellular parasites, belong to the normal flora of the skin, and generally reside in the hair follicles and sebaceous glands of the skin. The genes of propionibacterium acnes control the production of enzymes that break down skin and immunogenic proteins. Propionibacterium acnes live on fatty acids in pores, when the pores are blocked, the Propionibacterium acnes grow fiercely, saturated fatty acids are decomposed, a large amount of free fatty acids are generated, the fatty acids permeate into the skin through the pores to cause skin stress reaction, acne, red swelling and the like are generated, bacteria such as staphylococcus and the like adhere to the skin through effused pus, and skin tissues are damaged.
Acne is a chronic inflammatory dermatosis of the pilosebaceous unit caused by propionibacterium acnes, which is better developed in young people, and is characterized by polymorphous skin lesions such as acne, pimples, pustules, nodules and the like which are better developed on the face, the upper chest and the back in clinical manifestation. Acne occurs all year round and can occur repeatedly, and can be naturally relieved or healed after puberty. However, patients often leave permanent damage to the facial connective tissue hyperplasia lumps and depressions, which seriously affect the appearance and cause psychological trauma, and are clinically classified into 3-degree and 4-degree according to the nature and severity of acne lesions: level 1: acne alone; and 2, stage: in addition to acne, there are also some inflammatory papules; and 3, level: in addition to acne, there are also more inflammatory papules or pustules; 4, level: in addition to acne, inflammatory papules and pustules, there are nodules, cysts or scars. Non-inflammatory lesions of acne manifest as open and closed comedones. A typical skin lesion of closed comedones is a skin-colored pimple of about 1 mm in size, with no obvious hair follicle opening. Open comedones present as dome-shaped papules with a markedly dilated follicular opening. The further progression of the acne can evolve into various inflammatory lesions, manifested as inflammatory papules, pustules, nodules, and cysts. The inflammatory papules are red and have different diameters of 1-5 mm; the pustules are consistent in size and are filled with white pus; the diameter of the nodule is more than 5 mm, and the nodule can feel hard and painful when touched; the cyst is located deeper and filled with a mixture of pus and blood. These lesions may also fuse to form large inflammatory plaques and sinus tracts, etc. After the inflammatory lesions subside, pigmentation, persistent erythema, depressed or hypertrophic scars often remain.
Acne occurs mainly due to hyperseborrhea, Propionibacterium acnes infection after the blockage of the pilosebaceous canal, and secondary staphylococcal infection and inflammatory reaction. In adolescence, the level of androgen, particularly testosterone, in a human body is rapidly increased, the sebaceous gland development is promoted, a large amount of sebum is produced, meanwhile, abnormal keratinization of a pilosebaceous canal causes the canal to be blocked, sebum discharge is obstructed, and a keratotic plug, namely microacne, is formed. A plurality of microorganisms in hair follicles, especially Propionibacterium acnes, grow and multiply due to the fact that sebum contains more fatty acids and other components. The lipase produced by propionibacterium acnes breaks down sebum to produce free fatty acids, while chemotactic inflammatory cells and mediators, ultimately inducing and exacerbating the inflammatory response.
The daily care for acne is to wash face with warm water 1-2 times daily, and avoid using oils, powder cosmetics and ointment and cream containing glucocorticoid, and avoid squeezing with hand or scratching skin. Common methods for acne treatment are topical: tretinoin, benzoyl peroxide, antibiotics, azelaic acid, sulfur lotion, etc.; oral antibiotics: tetracyclines, and secondly macrolides, with a course of treatment of 6-12 weeks. Severe acne is treated with isotretinoin orally, with the treatment course targeting a minimum cumulative dose of 60 mg/kg. Anti-androgen therapy: such as: the oral contraceptive compound cyproterone acetate tablet is suitable for female moderate and severe acne patients, and is accompanied with androgen over-high expression or polycystic ovary syndrome. Oral contraceptives are also contemplated for use in female patients with delayed-type acne and significantly exacerbated premenstrual acne. Oral glucocorticoids: mainly used for fulminant or polymeric acne, and follows the principle of short-term, small dose and combined application with other methods. Other treatments include physical treatments such as photodynamic therapy, tartaric acid therapy, laser therapy, etc. for patients who are intolerant or unwilling to receive medication. Fractional treatment of acne includes: level 1: topical treatment is adopted, preferably topical retinoic acid preparation. And 2, stage: combining with topical retinoic acid and benzoyl peroxide or antibiotic, and optionally oral antibiotic. And 3, level: combination therapy is often required, with oral antibiotics being preferred in combination with topical benzoyl peroxide and/or retinoic acid drugs. Indicated female patients may also be considered anti-androgen therapy. 4, level: oral isotretinoin is the most effective treatment and can be used as first line therapy. For inflammatory papules and pustules, antibiotics can be systematically applied together with externally applied benzoyl peroxide, and oral isotretinoin sequence treatment is carried out after skin lesions are obviously improved. The maintenance treatment of acne is: after the skin lesion is obviously subsided, the maintenance therapy is continued, preferably the external vitamin A acid medicament is selected, the maintenance therapy is carried out for 6-12 months, and the external vitamin A acid medicament can be combined with benzoyl peroxide if necessary.
Secondary staphylococcal infection usually occurs in more than 2 grades, and has inflammatory papules and pustules, inflammatory plaques and sinuses in long-term inflammatory reaction, nodules, cysts, scars and the like accompanying the hyperplasia of the tissues of the hooves. Nodules, cysts and scars form leaving permanent inflammatory lesions. Secondary staphylococcal infections are therefore the main cause of permanent skin damage, and long-term antibiotic use also leads to dysbacteriosis and bacterial resistance. Therefore, a prevention and treatment means and a method which solve the problems of the quantity of propionibacterium acnes and staphylococcus existing on the surface of the skin and do not destroy the micro-ecological environment of the organism are urgently needed.
Staphylococci are a large family with many different species and types, and in addition to their individual specific antigens, there are a number of common antigens inherited from families of different species and types. According to the principle, the specific anti-propionibacterium acnes and staphylococcus composite IgY antibody generated by the hen immunized by the propionibacterium acnes and the staphylococcus in a mixed mode can generate specific immune reaction with the immunized propionibacterium acnes and the immunized staphylococcus and can also generate specific immune reaction with the propionibacterium acnes and the immunized staphylococcus which have the same antigenic determinant. When the IgY antibody and the bacterial surface antigen have immunoreaction, Propionibacterium acnes can be prevented from entering cells and staphylococci can be adhered to inflammatory parts, phagocytic cells are promoted to eliminate the Propionibacterium acnes and the staphylococci, and therefore infection is reduced, and the effect of preventing and treating diseases is achieved. When the IgY antibody has an immune reaction with a bacterial surface antigen, the IgY antibody changes the bacterial surface structure, inhibits some functional activities of bacteria, inhibits bacterial growth, reduces and inhibits adhesion of bacteria to cells, and promotes phagocytosis and elimination of bacteria by phagocytes such as lobular cells and macrophages. In fact, the specific IgY antibody is clinically used for preventing and treating decayed teeth and infant rotavirus diarrhea, preventing the infection of pseudomonas aeruginosa of a cholecystic fibrosis patient and reducing the gastritis caused by helicobacter pylori.
The anti-acne propionibacterium and staphylococcus compound IgY antibody has different action principles with the current antibiotics, and has specific combination with corresponding antigens on the surface of bacteria, thereby inhibiting the activity of the bacteria, preventing the bacteria from being adhered to tissue cells, rejecting the existence of the bacteria at inflammation parts, changing the surface structure and electric field of the bacteria, promoting the phagocytosis and elimination of the bacteria by phagocytes, simultaneously completely not inducing the generation of drug-resistant strains, not destroying the balance of local and overall micro-ecology, and simultaneously avoiding the abuse of the antibiotics and the occurrence of wide drug-resistant strains, thereby showing the importance and feasibility of the specific anti-acne propionibacterium and staphylococcus compound IgY antibody preparation. According to the action principle of the specific IgY antibody, the specific anti-Propionibacterium acnes and staphylococcus composite IgY antibody preparation can effectively treat all acnes and skin infectious inflammations caused by Propionibacterium acnes and staphylococcus bacteria and bacteria with common antigenic determinants with the bacteria, such as: acne, acute and chronic skin inflammation, suppuration, furuncle and subcutaneous carbuncle and swelling caused by acne infection and skin damage.
Disclosure of Invention
The main purpose of the invention is to solve the problems of simple acne and suppurative acne caused by propionibacterium acnes, staphylococcus and bacteria with common antigenic determinants; the skin furuncle and subcutaneous carbuncle swelling caused by the antigen and the preparation method and the application of the antigen are provided, and the antigen can prevent the formation of facial pimples and can resist propionibacterium acnes and staphylococcus composite IgY.
The purpose of the invention can be achieved by adopting the following technical scheme:
a method for preparing an anti-propionibacterium acnes and staphylococcus composite IgY antigen comprises the following steps:
the method comprises the following steps: preparing immunogen, mixing propionibacterium acnes and thallus fragments with golden and white staphylococcus and thallus fragments, mixing with Freund's complete adjuvant and Freund's incomplete adjuvant respectively, stirring and emulsifying to obtain water-in-oil Freund's complete adjuvant, Freund's incomplete adjuvant propionibacterium acnes and staphylococcus immunogen;
step two: immunizing, namely injecting the Freund's complete adjuvant immunogen into the laying hens;
step three: the preparation method of the anti-propionibacterium acnes and staphylococcus composite IgY antibody extract comprises the steps of collecting eggs of immunized hens, mixing egg yolk liquid with distilled water, stirring, adjusting acid and alkali, and taking supernatant to obtain the anti-propionibacterium acnes and staphylococcus composite IgY antibody extract.
Preferably, the immunogen preparation in step one comprises:
(1) taking 40-90 parts of pathogenic propionibacterium acnes and propionibacterium acnes bacterial fragment fragments, and 10-60 parts of golden and white staphylococcus and golden and white staphylococcus bacterial fragment fragments to mix into bacterial liquid;
(2) mixing the bacterial liquid with the same volume with the Freund's complete adjuvant, stirring and emulsifying to obtain water-in-oil Freund's complete adjuvant Propionibacterium acnes, golden yellow and white staphylococcus immunogen;
(3) mixing the bacterial liquid with the same volume with the Freund's incomplete adjuvant, stirring and emulsifying to obtain water-in-oil Freund's incomplete adjuvant Propionibacterium acnes, golden yellow and white staphylococcus immunogen;
(4) each milliliter of Freund's complete adjuvant immunogen and Freund's incomplete adjuvant immunogen contains 20 hundred million to 40 hundred million bacteria.
3. Preferably, the immunization in step two comprises:
(1) injecting the Freund's complete adjuvant immunogen into the laying hens at 30-100 points of the muscle under the skin, and injecting 0.7-1.5 ml of immunogen into each laying hen;
(2) injecting 1.0 ml-1.5 ml of Freund's incomplete adjuvant immunogen into the laying hens at 50-80 points of muscle every other week;
(3) after 3-4 times of boosting immunization by using Freund's incomplete adjuvant immunogen, 1.0-1.5 ml of Freund's incomplete adjuvant immunogen is injected to the laying hens at 50-80 points of subcutaneous and muscle for 5 times, and the boosting immunization is carried out for 9-10 times in total.
Preferably, the preparation of the anti-propionibacterium acnes and staphylococcus complex IgY antibody extract in step three comprises:
(1) collecting eggs of hens immunized for 30 days, cleaning the surface of the eggs with boiled water, sterilizing with alcohol, collecting yolk, removing yolk membrane, and collecting yolk liquid;
(2) adding distilled water into the yolk liquid and the distilled water according to the volume ratio of 1: 6-9, fully stirring, adjusting the pH value to 4.9-5.2 by using 1N HCl, and standing for 10 hours or overnight at the temperature of 4 ℃;
(3) centrifuging the yolk diluent at a high speed for 30 minutes, taking a supernatant, and placing the yolk diluent supernatant into a hollow fiber ultrafilter with the molecular weight cutoff of 10kDa for ultrafiltration concentration by 8-15 times;
(4) adding 0.02-5.0% chlorhexidine gluconate, fully stirring, adjusting the pH value to 7.0-7.5 by using 1N NaOH, and standing for 10 hours or overnight at the temperature of 4 ℃;
(5) centrifuging at high speed for 15-30 minutes, taking the supernatant, and filtering and sterilizing the supernatant by a 0.45-0.22 mu disinfection microporous filter membrane to obtain the extract of the abandoned lipid and lipoprotein of the anti-propionibacterium acnes and staphylococcus composite IgY antibody;
(6) adding ammonium sulfate into the extract of the anti-propionibacterium acnes and staphylococcus composite IgY antibody without lipid substances to 55-60% of saturation degree, fully stirring, and standing for 3-11 hours at room temperature;
(7) centrifuging at a high speed for 10 minutes, taking the precipitate, diluting the precipitate to the original volume by using distilled water, adding ammonium sulfate to reach the saturation degree of 33-40%, fully stirring, and standing at room temperature for 3-11 hours;
(8) centrifuging at high speed for 10 min, collecting precipitate, dissolving with distilled water, filling into dialysis bag, dialyzing with distilled water to remove salt to obtain pure product of anti-Propionibacterium acnes and Staphylococcus composite IgY antibody.
Preferably, the egg-laying hens in the immunization include domestic local chickens raised in a normal environment, domestic local chickens raised in a clean environment, domestic local chickens raised in an SPF environment, white-black-bone chickens, harlequin broilers, Lehangian chickens, and Ming-star chickens.
Preferably, eggs of the immunized hens are collected at 28 th day after the first immunization, and the eggs of the immunized hens from 28 th day to 320 th day after the first immunization have high titer of the anti-propionibacterium acnes and staphylococcus complex IgY antibodies.
Preferably, the purity of the extracted crude antibody extract of the anti-propionibacterium acnes and staphylococcus complex IgY is determined by reducing SDS-PAGE.
Preferably, in the step one, the thallus fragments are inactivated by carbolic acid, and simultaneously, the bacterial thallus fragments are crushed by ultrasonic by an ultrasonic crusher.
A compound IgY antigen for resisting Propionibacterium acnes and staphylococci is prepared from the medicine acceptable additive through preparing spray, aerosol, liniment, ointment and adhesive plaster.
Preferably, the prepared specific anti-acne propionibacterium and staphylococcus composite IgY antibody is locally applied to the skin for preventing and treating different clinical and pathological manifestations of acne and secondary acne infectious inflammation.
The invention has the beneficial technical effects that: according to the preparation method and the application of the anti-propionibacterium acnes and staphylococcus composite IgY antigen, the prepared specific anti-propionibacterium acnes and staphylococcus composite IgY antibody is locally applied to the skin of the face and the whole body, so that the preparation method has the advantages of no limitation and side effect of systemic medication, quick response, small dosage, good curative effect, no absorption, no damage to the micro-ecological environment of normal flora of the face and the skin of the whole body, no imbalance of local flora and the like, no toxic or side effect, no problem that the local use of antibiotics is easy to generate drug resistance and the like; the IgY antibody does not activate a complement system, does not produce nonspecific binding with rheumatoid factors, does not fix staphylococcus aureus protein A, does not bind with protein G and human cell Fc receptors, and is not easy to generate allergic reaction. The anti-acne propionibacterium and staphylococcus compound antibody extract does not produce acute toxic reaction and chronic toxic reaction and belongs to the actual non-toxic level; the anti-acne propionibacterium and staphylococcus compound IgY antibody extract has negative three-cause test, belongs to a product without teratogenicity, carcinogenesis and mutagenesis, and has no irritation to conjunctiva, mucosa and skin.
Detailed Description
In order to make the technical solutions of the present invention more clear and definite for those skilled in the art, the present invention is further described in detail with reference to the following examples, but the embodiments of the present invention are not limited thereto.
The preparation method and application of the anti-propionibacterium acnes and staphylococcus composite IgY antigen provided by the embodiment comprise the following steps:
the method comprises the following steps: preparing immunogen, mixing propionibacterium acnes and thallus fragments with golden and white staphylococcus and thallus fragments, mixing with Freund's complete adjuvant and Freund's incomplete adjuvant respectively, stirring and emulsifying to obtain water-in-oil Freund's complete adjuvant, Freund's incomplete adjuvant propionibacterium acnes and staphylococcus immunogen;
step two: immunizing, namely injecting the Freund's complete adjuvant immunogen into the laying hens;
step three: the preparation method of the anti-propionibacterium acnes and staphylococcus composite IgY antibody extract comprises the steps of collecting eggs of immunized hens, mixing egg yolk liquid with distilled water, stirring, adjusting acid and alkali, and taking supernatant to obtain the anti-propionibacterium acnes and staphylococcus composite IgY antibody extract.
In step one, the immunogen preparation comprises: (1) taking 40-90 parts of pathogenic propionibacterium acnes and propionibacterium acnes bacterial fragment fragments, and 10-60 parts of golden and white staphylococcus and golden and white staphylococcus bacterial fragment fragments to mix into bacterial liquid; (2) mixing the bacterial liquid with the same volume with the Freund's complete adjuvant, stirring and emulsifying to obtain water-in-oil Freund's complete adjuvant Propionibacterium acnes, golden yellow and white staphylococcus immunogen; (3) mixing the bacterial liquid with the same volume with the Freund's incomplete adjuvant, stirring and emulsifying to obtain water-in-oil Freund's incomplete adjuvant Propionibacterium acnes, golden yellow and white staphylococcus immunogen; (4) each milliliter of Freund's complete adjuvant immunogen and Freund's incomplete adjuvant immunogen contains 20 hundred million to 40 hundred million bacteria.
In step two, the immunization comprises: (1) injecting the Freund's complete adjuvant immunogen into the laying hens at 30-100 points of the muscle under the skin, and injecting 0.7-1.5 ml of immunogen into each laying hen; (2) injecting 1.0 ml-1.5 ml of Freund's incomplete adjuvant immunogen into the laying hens at 50-80 points of muscle every other week; (3) after 3-4 times of boosting immunization by using Freund's incomplete adjuvant immunogen, 1.0-1.5 ml of Freund's incomplete adjuvant immunogen is injected to the laying hens at 50-80 points of subcutaneous and muscle for 5 times, and the boosting immunization is carried out for 9-10 times in total.
The preparation of the anti-acne propionibacterium and staphylococcus composite IgY antibody extract in the third step comprises the following steps: (1) collecting eggs of hens immunized for 30 days, cleaning the surface of the eggs with boiled water, sterilizing with alcohol, collecting yolk, removing yolk membrane, and collecting yolk liquid; (2) adding distilled water into the yolk liquid and the distilled water according to the volume ratio of 1: 6-9, fully stirring, adjusting the pH value to 4.9-5.2 by using 1N HCl, and standing for 10 hours or overnight at the temperature of 4 ℃; (3) centrifuging the yolk diluent at a high speed for 30 minutes, taking a supernatant, and placing the yolk diluent supernatant into a hollow fiber ultrafilter with the molecular weight cutoff of 10kDa for ultrafiltration concentration by 8-15 times; (4) adding 0.02-5.0% chlorhexidine gluconate, fully stirring, adjusting the pH value to 7.0-7.5 by using 1N NaOH, and standing for 10 hours or overnight at the temperature of 4 ℃; (5) centrifuging at high speed for 15-30 minutes, taking the supernatant, and filtering and sterilizing the supernatant by a 0.45-0.22 mu disinfection microporous filter membrane to obtain the extract of the abandoned lipid and lipoprotein of the anti-propionibacterium acnes and staphylococcus composite IgY antibody; (6) adding ammonium sulfate into the extract of the anti-propionibacterium acnes and staphylococcus composite IgY antibody without lipid substances to 55-60% of saturation degree, fully stirring, and standing for 3-11 hours at room temperature; (7) centrifuging at a high speed for 10 minutes, taking the precipitate, diluting the precipitate to the original volume by using distilled water, adding ammonium sulfate to reach the saturation degree of 33-40%, fully stirring, and standing at room temperature for 3-11 hours; (8) centrifuging at high speed for 10 min, collecting precipitate, dissolving with distilled water, filling into dialysis bag, dialyzing with distilled water to remove salt to obtain pure product of anti-Propionibacterium acnes and Staphylococcus composite IgY antibody.
Laying hens in immunization comprise domestic local chickens, white-feather black-bone chickens, helan brown chickens, Lehang chickens, Roman chickens and star chickens which are bred in a common environment, a clean environment and an SPF environment, eggs of the immunized hens are collected on the 28 th day after the first immunization, the eggs of the immunized hens from the 28 th day to the 320 th day after the first immunization all have high-titer anti-propionibacterium acnes and staphylococcus composite IgY antibodies, the purity of the extracted crude extract of the anti-propionibacterium acnes and staphylococcus composite IgY antibodies is determined by adopting reducing SDS-PAGE, thallus fragments are inactivated by adopting carbolic acid in the first step, and meanwhile, the bacteria thallus fragments are ultrasonically crushed by an ultrasonic crusher.
The specific anti-acne propionibacterium and staphylococcus compound IgY antibody is locally applied to the skin to prevent and treat different clinical and pathological manifestations of acne and secondary acne infectious inflammation.
Example 1:
the production process flow for preparing 600 g of crude extract of the anti-propionibacterium acnes and staphylococcus composite IgY antibody comprises the following steps:
the method comprises the following steps: preparation of immunogens
(1) Propionibacterium acnes and Staphylococcus aureus isolated from patients with suppurative acne were identified as Propionibacterium acnes and Staphylococcus aureus by culturing Propionibacterium acnes in a meat culture medium and culturing Staphylococcus aureus in a 10% fresh sheep blood tryptic broth culture medium;
(2) inactivating the thallus fragments by carbolic acid, and ultrasonically crushing bacteria for 30 minutes by using an ultrasonic crusher to crush and crack the bacteria thallus fragments into fine and/or molecular fragments;
(3) mixing 65:25:10 parts of propionibacterium acnes, staphylococcus aureus and staphylococcus albus thallus fragments of the propionibacterium acnes;
(4) wherein the proportion of the bacteria to the thallus fragment fragments is 75 parts of the bacteria and 25 parts of the thallus fragment fragments;
(5) mixing 400ml of thallus fragments and bacterial liquid of the thallus fragments with 400ml of Freund's complete adjuvant, and stirring and emulsifying by using a high-speed stirrer to prepare water-in-oil Freund's complete adjuvant immunogen;
(5) the Freund's incomplete adjuvant immunogen is prepared by the following steps: mixing 400ml of thallus fragments and bacterial liquid of the thallus fragments with 400ml of Freund's incomplete adjuvant, stirring and emulsifying by using a high-speed stirrer to prepare water-in-oil Freund's incomplete adjuvant immunogen, wherein each milliliter of immunogen contains 30 hundred million bacteria;
step two: immune egg laying hen
(1) Injecting the Freund's complete adjuvant immunogen into a 30-week-old Holland brown laying hen subcutaneously and at 80-point muscle using a syringe, each with 1.5ml of immunogen;
(2) injecting 1.5ml of Freund's incomplete adjuvant immunogen into egg laying hens subcutaneously and at 80-point muscle every other week for 4 times;
(3) injecting 1.5ml of Freund's incomplete adjuvant immunogen subcutaneously and 80-point intramuscularly to the laying hens for 5 times every 4 weeks, wherein the immunization is performed for 10 times, and the total number of the immunized hens is 400;
step three: preparation of crude extract of anti-acne propionibacterium and staphylococcus composite IgY antibody
(1) Collecting eggs of immunized hens which are immunized 50-90 days after the first immunization, soaking the eggs in 70% alcohol for surface disinfection, taking egg yolks, removing egg yolk membranes and taking egg yolk liquid;
(2) adding distilled water into yolk liquid and distilled water at a volume ratio of 1:8, stirring, adjusting pH to 5.0 with 1N HCl, and standing in a freezer at 4 deg.C for 10 hr;
(3) centrifuging the yolk diluent at a high speed of 10000rpm for 30 minutes, taking the supernatant, placing the supernatant into a hollow fiber ultrafilter with the molecular weight cutoff of 100kDa for ultrafiltration and concentration by 10 times, adding 2.0 percent chlorhexidine gluconate, fully stirring, adjusting the pH value to be 7.2 by using 1N NaOH, and placing for 10 hours at 4 ℃;
(4) centrifuging at high speed of 10000rpm for 20 min, collecting supernatant, and filtering and sterilizing the supernatant with 0.45 μ sterilized microporous filter membrane to obtain crude extract of anti-Propionibacterium acnes and Staphylococcus composite IgY antibody;
(5) the titer of the specific IgY antibody in each mg of protein in the extract is 1:1000/mg of protein by an ELISA method, or the specific activity of the specific IgY antibody in each mg of protein in the crude extract is 1:1000, and the purity of the IgY antibody in the crude extract is 31.3 percent by non-reducing SDS-PAGE.
Example 2:
the production process flow for preparing 600 g of crude extract of the anti-propionibacterium acnes and staphylococcus composite IgY antibody comprises the following steps:
the method comprises the following steps: preparation of immunogens
(1) Propionibacterium acnes and Staphylococcus aureus isolated from patients with suppurative acne were identified as Propionibacterium acnes and Staphylococcus aureus by culturing Propionibacterium acnes in a meat culture medium and culturing Staphylococcus aureus in a 10% fresh sheep blood tryptic broth culture medium;
(2) and (3) inactivating the thallus fragments by carbolic acid. Further, the bacterial cell fragments are crushed and cracked into fine and/or molecular fragments by ultrasonic crushing for 30 minutes by an ultrasonic crusher;
(3) mixing the propionibacterium acnes and propionibacterium acnes thallus fragments of golden yellow and white staphylococcus and golden yellow and white staphylococcus thallus fragments 65:25:10 parts. Wherein the proportion of the bacteria to the thallus fragment fragments is 75 parts of the bacteria and 25 parts of the thallus fragment fragments;
(4) mixing 400ml of thallus fragments and bacterial liquid of the thallus fragments with 400ml of Freund's complete adjuvant, and stirring and emulsifying by using a high-speed stirrer to prepare water-in-oil Freund's complete adjuvant immunogen;
(5) the Freund's incomplete adjuvant immunogen is prepared by the following steps: mixing 400ml of thallus fragments and bacterial liquid of the thallus fragments with 400ml of Freund's incomplete adjuvant, stirring and emulsifying by using a high-speed stirrer to prepare water-in-oil Freund's incomplete adjuvant immunogen, wherein each milliliter of immunogen contains 30 hundred million bacteria;
step two: immune egg laying hen
(1) Injecting the Freund's complete adjuvant immunogen into a 30-week-old Holland brown laying hen subcutaneously and at 80-point muscle using a syringe, each with 1.5ml of immunogen;
(2) injecting 1.5ml of Freund's incomplete adjuvant immunogen into egg laying hens subcutaneously and at 80-point muscle every other week for 4 times;
(3) injecting 1.5ml of Freund's incomplete adjuvant immunogen subcutaneously and 80-point intramuscularly to the laying hens for 5 times every 4 weeks, wherein the immunization is performed for 10 times, and the total number of the immunized hens is 400;
step three: preparation of crude extract of anti-acne propionibacterium and staphylococcus composite IgY antibody
(1) Collecting eggs of immunized hens which are immunized 50-90 days after the first immunization, soaking the eggs in 70% alcohol for surface disinfection, taking egg yolks, removing egg yolk membranes and taking egg yolk liquid;
(2) adding distilled water into yolk liquid and distilled water at a volume ratio of 1:8, stirring, adjusting pH to 5.0 with 1N HCl, and standing in a freezer at 4 deg.C for 10 hr;
(3) centrifuging the yolk diluent at a high speed of 10000rpm for 30 minutes, taking the supernatant, placing the supernatant into a hollow fiber ultrafilter with the molecular weight cutoff of 100kDa for ultrafiltration and concentration by 10 times, adding 2.0 percent chlorhexidine, fully stirring, adjusting the pH value to be pH7.2 by using 1NNaOH, and placing for 10 hours at 4 ℃;
(4) centrifuging at high speed of 10000rpm for 20 min, collecting supernatant, and filtering and sterilizing the supernatant with 0.45 μ sterilized microporous filter membrane to obtain crude extract of anti-Propionibacterium acnes and Staphylococcus composite IgY antibody;
(5) adding ammonium sulfate into crude extract of anti-Propionibacterium acnes and staphylococcus composite IgY antibody to 55% of saturation, fully stirring, standing at room temperature for 4 hours, centrifuging at high speed at 10000rpm for 15 minutes, taking precipitate, diluting the precipitate to the original volume by using distilled water, adding ammonium sulfate to 33% of saturation, fully stirring, and standing at room temperature for 4 hours;
(6) centrifuging at a high speed of 10000rpm for 15 minutes, taking the precipitate, diluting and dissolving the precipitate with distilled water, filling the precipitate into a dialysis bag, and dialyzing the precipitate with distilled water to remove salt to obtain a pure product of the anti-propionibacterium acnes and staphylococcus composite IgY antibody; the titer of the specific IgY antibody per mg of protein in the anti-propionibacterium acnes and staphylococcus compound IgY antibody pure product is 1:4000/mg of protein, or the specific activity of the specific chicken IgY antibody per mg of protein in the anti-propionibacterium acnes and staphylococcus compound IgY antibody pure product is 1:4000, which is determined by an ELISA method. Among the purified IgY antibodies, the purity of the IgY antibodies was 86.4% as determined by non-reducing SDS-PAGE.
Experimental example 3:
the method is used for immunizing a helan brown laying hen with the propionibacterium acnes and staphylococcus compound immunogen, taking an egg of the immunized hen 40-60 days after the first immunization, preparing an extract of the anti-propionibacterium acnes and staphylococcus compound IgY antibody according to the method, and measuring the biological potency of the anti-propionibacterium acnes and staphylococcus compound IgY antibody combined with different antigens by using an ELISA method.
TABLE 1 titer of anti-Propionibacterium acnes and Staphylococcus Complex IgY antibody extracts
From the above experimental results it is clear that: the propionibacterium acnes and staphylococcus thallus fragment plus thallus fragment immunogen immunizes laying hens, and can generate anti-propionibacterium acnes and staphylococcus IgY antibodies respectively; according to different dosages used by each bacterium when the laying hens are immunized, the titer of the generated specific IgY antibody is different, the titer of the IgY antibody generated by the bacterium with large immunization dosage is high, and the titer is low otherwise; the propionibacterium acnes and staphylococcus composite IgY antibody can react with the propionibacterium acnes and staphylococcus mixed antigen, the titer of the total specific IgY antibody is also increased, and the titer has a certain superposition effect; the experiments show that the anti-acne propionibacterium and staphylococcus composite IgY antibody can treat acne, acute and chronic acne infectious inflammation and the like caused by the bacteria; simultaneously, an anti-propionibacterium acnes and staphylococcus composite IgY antibody can be used for treating acute and chronic acne infectious inflammation caused by any one of the bacteria; therefore, the invention can be expanded to acne caused by bacteria having common antigenic determinants with the bacteria, acute and chronic acne infectious inflammation and the like, and can be used for treating the acne caused by the anti-propionibacterium acnes and staphylococcus composite IgY antibody.
Experimental example 4:
the method is used for immunizing a helan brown laying hen with the propionibacterium acnes and staphylococcus compound immunogen, eggs of the immunized hens which are 40-60 days after the first immunization are taken, purified products of the anti-propionibacterium acnes and staphylococcus compound IgY antibodies are prepared according to the method, and the biological potency of the anti-propionibacterium acnes and staphylococcus compound IgY antibodies in combination with different antigens is determined by an ELISA method.
TABLE 2 titer of purified product of anti-Propionibacterium acnes and Staphylococcus complex IgY antibody
From the above experimental results it is clear that: the purified product of the anti-propionibacterium acnes and staphylococcus composite IgY antibody prepared by the method of the invention has higher biological value of the specific IgY antibody combined with propionibacterium acnes and staphylococcus composite antigen and the respective antigens of propionibacterium acnes, golden yellow and white staphylococcus bacteria compared with the specific IgY antibody of the extract of the anti-propionibacterium acnes and staphylococcus composite IgY antibody prepared by the method of the invention in the experimental example 1, and is basically increased in proportion; the propionibacterium acnes and staphylococcus compound immunogen are used for immunizing laying hens and can generate specific IgY antibodies for resisting the three bacteria respectively; according to different dosages used by each bacterium when the laying hens are immunized, the titer of the generated specific IgY antibody is different, the titer of the IgY antibody generated by the bacterium with large immunization dosage is high, and the titer is low otherwise; the anti-propionibacterium acnes and staphylococcus composite IgY antibody can react with propionibacterium acnes and staphylococcus composite antigen, the titer of the total specific IgY antibody is increased, and the titer has a certain superposition effect; the experiment shows that the anti-propionibacterium acnes and staphylococcus composite IgY antibody can treat acute and chronic acne, secondary acne infectious pustule and the like caused by the three bacteria; simultaneously, an anti-propionibacterium acnes and staphylococcus composite IgY antibody can be used for treating acute and chronic acne, secondary acne infectious pustule and the like caused by any one of the three bacteria or fungi; therefore, the invention can be expanded to the treatment of acute and chronic acne, secondary acne infectious pustule, rash and the like caused by bacteria with common antigenic determinants with the three bacteria, and the anti-propionibacterium acnes and staphylococcus composite IgY antibody can be used for treating the rash and the like.
Experimental example 5:
comparing the immune response of different chicken species and the ability to produce complex IgY antibodies against propionibacterium acnes and staphylococcus: the method for preparing the crude extract of the specific anti-propionibacterium acnes and staphylococcus complex IgY antibody comprises the steps of immunizing large-scale cage-rearing helvetia brown laying hens, star laying hens and lihang laying hens respectively, and stocking the laying hens, adopting eggs of the immunized hens 40-100 days after the first immunization, extracting the crude extract of the anti-propionibacterium acnes and staphylococcus complex IgY antibody according to the method of claim 1, and determining the biological potency of the anti-propionibacterium acnes and staphylococcus complex IgY antibody combined antigen by using an ELISA method.
TABLE 3 immune response of egg-laying hens of different breeds to the same antigen and the specific IgY antibody titer
From the above experimental results it is clear that: under the same immune condition, the crude extracts of the anti-propionibacterium acnes and staphylococcus compound IgY antibodies of different chicken species prepared by the method have basically consistent biological potency of the specific anti-propionibacterium acnes and staphylococcus compound IgY antibodies, wherein the biological potency of the specific anti-propionibacterium acnes and staphylococcus compound IgY antibodies is slightly changed but the difference is not obvious only in the crude extracts of the anti-propionibacterium acnes and staphylococcus compound IgY antibodies prepared by different batches of local chickens.
Experimental example 6:
the two immunogens of propionibacterium acnes, staphylococcus thallus fragments, propionibacterium acnes, staphylococcus thallus fragments and staphylococcus thallus fragment fragments, and the like, prepared by the method of the invention, immunize the helveticus nilotica broilers and laying hens, respectively prepare crude antibody extracts for resisting propionibacterium acnes and staphylococcus and crude antibody extracts for resisting propionibacterium acnes and staphylococcus thallus fragments and staphylococcus fragments and IgY antibody, and determine the combined biological potency of the propionibacterium acnes and staphylococcus antigens by applying an ELISA method.
TABLE 4 biological potency of different immunogen induced Hailan Brown mother specific IgY binding to three bacteria
From the above experimental results it is clear that: under the same immunization condition, two different immunogens such as propionibacterium acnes, staphylococcus thallus fragments and other fragments are prepared according to the method, and two IgY antibodies such as an anti-propionibacterium acnes and staphylococcus thallus fragments IgY antibody crude extract and an anti-propionibacterium acnes and staphylococcus thallus fragment IgY antibody crude extract of eggs of hens immunized 40-100 days after the first immunization are prepared. The two IgY antibodies respectively react with the antigen of the propionibacterium acnes and the staphylococcus thallus fragment, and as a result, the biological binding titer of the two IgY antibodies has certain difference, wherein the biological binding titer of the IgY antibodies resisting the propionibacterium acnes, the staphylococcus thallus fragment and the thallus fragment is higher and more stable, and the biological binding titer of the IgY antibodies resisting the propionibacterium acnes and the staphylococcus thallus fragment is different from batch to batch, namely 1:600/mg protein-1: 1200/mg protein. These results indicate that the immunogenic effect of the propionibacterium acnes and staphylococcus thallus fragments plus thallus fragment immunogen is better than that of the propionibacterium acnes and staphylococcus thallus fragment immunogen alone.
Experimental example 7:
the immune effect of the propionibacterium acnes, staphylococcus thallus fragments and thallus fragment immunogen 60-point intramuscular and subcutaneous injections is compared with the immune effect of single 3-point intramuscular injection or single several-point subcutaneous injection.
TABLE 5 comparison of the biological potency of specific IgY antibodies produced by Hailan-Brown hens induced by different immunization modalities
From the above experimental results it is clear that: the same immunogen prepared by the method of the invention immunizes the same helan brown laying hen in different immunization modes, and the crude extract of the anti-propionibacterium acnes and staphylococcus compound IgY antibody of the egg of the hen immunized 40-100 days after the first immunization is prepared by the method of the invention, and the biological potency of the anti-propionibacterium acnes and staphylococcus compound IgY antibody is the highest in the 60-point intramuscular and subcutaneous injection immunization mode of the invention, so the immunization method of the invention is the best.
Experimental example 8:
and (3) comparing the time length of the high biological titer specific IgY antibody generated by the propionibacterium acnes, staphylococcus thallus fragments and thallus fragment immunogen 60-point intramuscular and subcutaneous injection immunization with the time length of the high biological titer specific IgY antibody generated by single 3-point intramuscular injection or single 3-point subcutaneous injection immunization.
TABLE 6 comparison of the time periods for the production of high titers of specific IgY antibodies by Hailan brown hens induced by different immunization regimes
From the above experimental results it is clear that: the same immunogen prepared by the method of the invention is used for immunizing the same Hailan brown laying hen in different immunization modes, and the extract of the anti-Propionibacterium acnes and staphylococcus composite IgY antibody of the egg of the hen immunized 50-280 days after the first immunization is prepared by the method of the invention. The titer change of the anti-propionibacterium acnes and staphylococcus composite IgY antibody for 2-9 months is measured by an ELISA method, and the result shows that the titer of the specific anti-propionibacterium acnes and staphylococcus composite IgY antibody is basically unchanged by a 60-point intramuscular injection and subcutaneous injection immunization method after 2-9 months, while the capacity of producing the specific anti-propionibacterium acnes and staphylococcus composite IgY antibody by hens which are immunized by single-point intramuscular injection or single-point subcutaneous injection is obviously reduced, and the titer of the specific IgY antibody is also obviously reduced. This further illustrates that the 60-point intramuscular and subcutaneous injection immunization of the invention has significant advantages in terms of the duration of the production of high titer specific anti-Propionibacterium acnes and staphylococcal complex IgY antibodies, with effects significantly better than those of the other two methods.
Experimental example 9:
the method of the invention for 10 times of immunization injection is compared with the effect of the traditional method for three times of immunization injection.
TABLE 7 comparison of the ability of Hailan-Brown hens induced by different immunization times to produce high titers of specific IgY antibodies
From the above experimental results it is clear that: the production capacity of specific anti-propionibacterium acnes and staphylococcus composite IgY antibodies of the hylobacterium acnes and staphylococcus is compared with that of the hylobacterium acnes and staphylococcus composite IgY antibodies of the hylobacterium nilgholicus brown laying hens immunized by the immunization method for 10 times of the traditional immunization method for 3 times, and the crude extract of the anti-propionibacterium acnes and staphylococcus composite IgY antibodies after 150-200 days of the first immunization and the extract of the anti-propionibacterium acnes and staphylococcus composite IgY antibodies after 280-300 days of the first immunization are prepared by the method. The biological valence of the specific IgY antibody induced by the two immunization methods at different time is determined by an ELISA method, and the result shows that the biological valence of the specific IgY antibody generated by the two immunization methods in the time of 150-200 days after the first immunization is not changed greatly, so that the 10-time immunization method is better than the traditional three-time immunization method; after the time of 280-300 days, the 10 times of immunization method is much better than the traditional three times of immunization method, and the biological potency of the specific anti-propionibacterium acnes and staphylococcus composite IgY antibody is very obviously different from that of the specific anti-propionibacterium acnes and staphylococcus composite IgY antibody. The hen immunized by the 10 times of immunization method of the invention can generate specific anti-acne propionibacterium and staphylococcus composite IgY antibody with high titer for a longer time.
Experimental example 10:
the different composition proportions of the propionibacterium acnes and the staphylococcus aureus and the staphylococcus albus have influence on the generation of the specific anti-propionibacterium acnes and staphylococcus composite IgY antibody.
TABLE 8 comparison of the specific IgY antibody production induced by the immunogen of three bacteria in different composition ratios
From the above experimental results it is clear that: although the same immunogen prepared by the method has different composition proportions of propionibacterium acnes, golden yellow and white staphylococcus, the extract of the anti-propionibacterium acnes and staphylococcus composite IgY antibody of the eggs of the immunized hens which are immunized 40-60 days after the first immunization is prepared by the method, and the biological titers of the anti-propionibacterium acnes and staphylococcus composite IgY antibody are basically consistent. This indicates that the biological potency of the total anti-propionibacterium acnes and staphylococcus complex IgY antibodies was unchanged, despite the different compositional proportions of propionibacterium acnes, staphylococcus aureus and staphylococcus albus.
Experimental example 11:
the IgY antibodies generated by the composite antigens with different composition ratios of the propionibacterium acnes, the golden yellow staphylococcus and the white staphylococcus respectively resist the specific combination influence of the propionibacterium acnes, the golden yellow staphylococcus and the white staphylococcus.
TABLE 9 Effect of IgY antibodies produced by different composition ratios of the three bacteria on the specific binding of the three different bacteria
From the above experimental results it is clear that: the method is characterized in that an immunogen is prepared from three bacteria such as propionibacterium acnes, staphylococcus aureus and staphylococcus albus according to different composition proportions, the immunogen is used for immunizing egg-laying Hailan brown hens, and an anti-propionibacterium acnes and staphylococcus composite IgY antibody extract for immunizing eggs of the hens 40-60 days after the first immunization is prepared according to the method, wherein the anti-propionibacterium acnes antibody titer, the anti-staphylococcus aureus antibody titer and the anti-staphylococcus albus antibody titer are increased and decreased basically the same as the increase and decrease of the immunization doses of the different bacteria respectively. The change of the antibody titer is increased or decreased within the effective IgY antibody activity range, which shows that although the antibody titer fluctuates to a certain extent according to different composition ratios of three bacteria, such as propionibacterium acnes, golden yellow and staphylococcus albus, the biological titer of the IgY antibodies for inducing the chickens to generate the anti-propionibacterium acnes, the golden yellow and the staphylococcus albus is still within the effective range.
Experimental example 12:
effect of anti-propionibacterium acnes and staphylococcus complex IgY antibodies on neutrophil phagocytosis of staphylococcus aureus: the anti-acne propionibacterium acnes and staphylococcus composite IgY antibody extract of the eggs of the hens which are immunized 30-120 days after the first immunization, live staphylococcus aureus and human earlobe blood or venous blood are prepared by the method, and are incubated for 30 minutes in an incubator with 37 ℃, 5% CO2 and saturated humidity, so that the live staphylococcus aureus can be promoted to be phagocytosed by human earlobe blood and venous blood neutrophils. The phagocytosis rate of neutrophils increases with the increase of the IgY concentration.
TABLE 10 Effect of specific IgY antibodies on the phagocytosis of viable Staphylococcus aureus by the neutrophils in the earlobe blood
Note: phagocytosis ratio (%): group 1, group 3 p < 0.01; group 1, group 2 p < 0.05; 2 groups, 3 groups p < 0.01.
TABLE 11 Effect of specific IgY antibodies on the phagocytosis of viable Staphylococcus aureus by neutrophils in venous blood
Note: phagocytosis ratio (%): group 1, group 3 p < 0.01; group 1, group 2 p < 0.05; 2 groups, 3 groups p < 0.01.
From the above experimental results it is clear that: the anti-acne propionibacterium and staphylococcus compound antibody extract of the eggs of the hens immunized 30-120 days after the first immunization is prepared by the method, and can promote human neutrophils to enhance phagocytosis of live staphylococcus aureus. This opsonophagocytosis promotion may be associated with altered bacterial surface structure and electric field effects. This illustrates the mechanism of action of the anti-Propionibacterium acnes and staphylococcal composite IgY antibody extract prepared by the method of the present invention in treating sexual inflammation caused by Propionibacterium acnes, golden yellow and Staphylococcus albus.
Example 13: spray agent
Filtering with 0.22 μ sterilized microporous membrane, sterilizing, and aseptically packaging into sterile spray and aerosol cans.
Example 14: special for female
| Component (A) | Weight ratio of |
| Anti-acne propionibacterium and staphylococcus composite IgY antibody extract | 0.15 |
| Retinoic acid | 0.05 |
| Cyproterone acetate | 0.1 |
| Water (W) | 90.28 |
| Glycerol | 5 |
| Sodium guaiazulene sulfonate | 0.30 |
| EDTA disodium salt | 0.1 |
| Aloe extract | 0.5 |
| Propolis (propolis) | 2 |
| Solubilizer | 0.5 |
| EGF | 0.5 |
| Small cucumber | 0.02 |
| Preservative | 0.5 |
Filtering with 0.22 μ sterilized microporous membrane, sterilizing, and aseptically packaging into sterile spray and aerosol cans.
Example 15:
filling into sterilized aluminum foil and plastic hose.
In summary, in this embodiment, according to the preparation method and the application of the anti-propionibacterium acnes and staphylococcus complex IgY antigen of this embodiment, the purity of the crude extract of the anti-propionibacterium acnes and staphylococcus complex IgY antibody extracted by the method of the present invention is determined by non-reducing SDS-PAGE, and the purity of the chicken IgY antibody reaches more than 31%; the purity of the anti-propionibacterium acnes and staphylococcus composite IgY antibody purified product extracted by the method is determined by non-reducing SDS-PAGE, and the purity of the IgY antibody reaches more than 82 percent; the biological value of the extracted crude extract of the anti-propionibacterium acnes and staphylococcus compound IgY antibody is determined by an ELISA method, the biological value of the specific anti-propionibacterium acnes and staphylococcus compound IgY antibody combined with propionibacterium acnes and staphylococcus compound antigen reaches the value of more than or equal to 1: 400-600 of the specific anti-propionibacterium acnes and staphylococcus compound IgY antibody in each mg of protein, namely the value is more than or equal to 1: 400-600/mg of protein, or the specific activity of the specific anti-propionibacterium acnes and staphylococcus compound IgY antibody in each mg of protein in the crude extract is more than or equal to 1: 400-600; the biological activity of the pure anti-propionibacterium acnes and staphylococcus composite IgY antibody extracted by the method of the invention is as follows: the titer is determined by an ELISA method, and the biological titer of the specific anti-propionibacterium acnes and staphylococcus compound IgY antibody combined with propionibacterium acnes and staphylococcus compound antigen reaches or is more than or equal to 1: 1600-2400 per milligram of protein, namely the titer is more than or equal to 1:1600/mg of protein, or the specific activity of the specific anti-propionibacterium acnes and staphylococcus compound IgY antibody in each milligram of protein in a pure product is more than or equal to 1: 1600; the biological value of the extracted anti-propionibacterium acnes and staphylococcus composite IgY antibody water extract is measured by an ELISA method, and the biological value of the specific anti-propionibacterium acnes antigen reaches the specific anti-propionibacterium acnes antigen combination value of 1: 200-400/mg protein in each mg protein; the biological value of the specific anti-staphylococcus mixed antigen reaches that the specific combined staphylococcus mixed antigen in each milligram of protein is more than or equal to 1: 200-400/mg of protein, and the biological activity of the anti-propionibacterium acnes IgY antibody pure product extracted by the method is as follows: the titer is determined by an ELISA method, and the biological titer of the specific anti-acne propionibacterium antigen reaches the specific anti-acne propionibacterium antigen combination titer of 1: 800-1600 mg protein in each mg protein; the biological potency of the specific anti-staphylococcus mixed antigen reaches 1: 800-1600 mg of specific combined staphylococcus mixed antigen in each mg of protein; the prepared specific anti-acne propionibacterium and staphylococcus compound IgY antibody is locally applied to the face and the skin of the whole body, has no limitation and side effect of the whole body application, has the effects of quick response of the local application, small dosage, good curative effect, no absorption, no damage to the micro-ecological environment of normal flora of the face and the skin of the whole body, no cause of local flora imbalance and the like, no toxic or side effect, no problem of easy generation of drug resistance of the local application of antibiotics and the like; the IgY antibody does not activate a complement system, does not produce nonspecific binding with rheumatoid factors, does not fix staphylococcus aureus protein A, does not bind with protein G and human cell Fc receptors, and is not easy to generate allergic reaction. The anti-acne propionibacterium and staphylococcus compound antibody extract does not produce acute toxic reaction and chronic toxic reaction and belongs to the actual non-toxic level; the anti-acne propionibacterium and staphylococcus compound IgY antibody extract has negative three-cause test, belongs to a product without teratogenicity, carcinogenesis and mutagenesis, and has no irritation to conjunctiva, mucosa and skin.
Claims (10)
1. A preparation method of an anti-Propionibacterium acnes and staphylococcus composite IgY antigen is characterized by comprising the following steps:
the method comprises the following steps: preparing immunogen, mixing propionibacterium acnes and thallus fragments with golden and white staphylococcus and thallus fragments, mixing with Freund's complete adjuvant and Freund's incomplete adjuvant respectively, stirring and emulsifying to obtain water-in-oil Freund's complete adjuvant, Freund's incomplete adjuvant propionibacterium acnes and staphylococcus immunogen;
step two: immunizing, namely injecting the Freund's complete adjuvant immunogen into the laying hens;
step three: the preparation method of the anti-propionibacterium acnes and staphylococcus composite IgY antibody extract comprises the steps of collecting eggs of immunized hens, mixing egg yolk liquid with distilled water, stirring, adjusting acid and alkali, and taking supernatant to obtain the anti-propionibacterium acnes and staphylococcus composite IgY antibody extract.
2. The method for preparing the anti-propionibacterium acnes and staphylococcus complex IgY antigen according to claim 1, wherein the preparation of the immunogen in the first step comprises:
(1) taking 40-90 parts of pathogenic propionibacterium acnes and propionibacterium acnes bacterial fragment fragments, and 10-60 parts of golden and white staphylococcus and golden and white staphylococcus bacterial fragment fragments to mix into bacterial liquid;
(2) mixing the bacterial liquid with the same volume with the Freund's complete adjuvant, stirring and emulsifying to obtain water-in-oil Freund's complete adjuvant Propionibacterium acnes, golden yellow and white staphylococcus immunogen;
(3) mixing the bacterial liquid with the same volume with the Freund's incomplete adjuvant, stirring and emulsifying to obtain water-in-oil Freund's incomplete adjuvant Propionibacterium acnes, golden yellow and white staphylococcus immunogen;
(4) each milliliter of Freund's complete adjuvant immunogen and Freund's incomplete adjuvant immunogen contains 20 hundred million to 40 hundred million bacteria.
3. The method for preparing the anti-propionibacterium acnes and staphylococcus composite IgY antigen according to claim 1, wherein the immunization in the second step comprises:
(1) injecting the Freund's complete adjuvant immunogen into the laying hens at 30-100 points of the muscle under the skin, and injecting 0.7-1.5 ml of immunogen into each laying hen;
(2) injecting 1.0 ml-1.5 ml of Freund's incomplete adjuvant immunogen into the laying hens at 50-80 points of muscle every other week;
(3) after 3-4 times of boosting immunization by using Freund's incomplete adjuvant immunogen, 1.0-1.5 ml of Freund's incomplete adjuvant immunogen is injected to the laying hens at 50-80 points of subcutaneous and muscle for 5 times, and the boosting immunization is carried out for 9-10 times in total.
4. The method for preparing the anti-propionibacterium acnes and staphylococcus complex IgY antigen according to claim 1, wherein the preparation of the anti-propionibacterium acnes and staphylococcus complex IgY antibody extract in the third step comprises:
(1) collecting eggs of hens immunized for 30 days, cleaning the surface of the eggs with boiled water, sterilizing with alcohol, collecting yolk, removing yolk membrane, and collecting yolk liquid;
(2) adding distilled water into the yolk liquid and the distilled water according to the volume ratio of 1: 6-9, fully stirring, adjusting the pH value to 4.9-5.2 by using 1N HCl, and standing for 10 hours or overnight at the temperature of 4 ℃;
(3) centrifuging the yolk diluent at a high speed for 30 minutes, taking a supernatant, and placing the yolk diluent supernatant into a hollow fiber ultrafilter with the molecular weight cutoff of 10kDa for ultrafiltration concentration by 8-15 times;
(4) adding 0.02-5.0% chlorhexidine gluconate, fully stirring, adjusting the pH value to 7.0-7.5 by using 1N NaOH, and standing for 10 hours or overnight at the temperature of 4 ℃;
(5) centrifuging at high speed for 15-30 minutes, taking the supernatant, and filtering and sterilizing the supernatant by a 0.45-0.22 mu disinfection microporous filter membrane to obtain the extract of the abandoned lipid and lipoprotein of the anti-propionibacterium acnes and staphylococcus composite IgY antibody;
(6) adding ammonium sulfate into the extract of the anti-propionibacterium acnes and staphylococcus composite IgY antibody without lipid substances to 55-60% of saturation degree, fully stirring, and standing for 3-11 hours at room temperature;
(7) centrifuging at a high speed for 10 minutes, taking the precipitate, diluting the precipitate to the original volume by using distilled water, adding ammonium sulfate to reach the saturation degree of 33-40%, fully stirring, and standing at room temperature for 3-11 hours;
(8) centrifuging at high speed for 10 min, collecting precipitate, dissolving with distilled water, filling into dialysis bag, dialyzing with distilled water to remove salt to obtain pure product of anti-Propionibacterium acnes and Staphylococcus composite IgY antibody.
5. The method of claim 1, wherein the immunized laying hens comprise domestic local chickens, white-black-bone chickens, helan-brown chickens, lesho chickens, roman chickens and star chickens raised in a normal environment, a clean environment and an SPF environment.
6. The method of claim 1, wherein the eggs of the immunized hens are collected 28 days after the first immunization, and the eggs of the immunized hens from 28 days to 320 days after the first immunization have the anti-Propionibacterium acnes and staphylococcal IgY complex antibodies with high titer.
7. The method for preparing the anti-propionibacterium acnes and staphylococcus complex IgY antigen according to claim 1, wherein the purity of the extracted crude anti-propionibacterium acnes and staphylococcus complex IgY antibody is determined by reducing SDS-PAGE.
8. The method for preparing the anti-propionibacterium acnes and staphylococcus composite IgY antigen as claimed in claim 1, wherein in the step one, the thallus fragments are inactivated by carbolic acid, and the bacteria thallus fragments are crushed by ultrasonic crusher.
9. The application of the preparation method of the anti-propionibacterium acnes and staphylococcus compound IgY antigen is characterized in that pharmaceutically acceptable additives are added to prepare a spray, an aerosol, a liniment lotion, an ointment and an adhesive plaster containing the anti-propionibacterium acnes and staphylococcus compound IgY antibody.
10. The use of the antigen of claim 9 for the preparation of a specific anti-acne Propionibacterium, Staphylococcus complex IgY antibody for topical application to the skin for the prevention and treatment of acne of various clinical and pathological manifestations, as well as secondary acne infectious inflammation.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114558130A (en) * | 2022-03-19 | 2022-05-31 | 广西博生生物科技有限公司 | Preparation method of collagen and anti-propionibacterium acnes gamma protein combined preparation |
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| CN1283663C (en) * | 2005-01-12 | 2006-11-08 | 江西3L医用制品集团有限公司 | Antithemolytic streptococcus, staphylococcus and candida albicans lgG antibody and preparation thereof |
| CN103890173A (en) * | 2011-08-19 | 2014-06-25 | 鸵鸟制药株式会社 | Antibody and antibody-containing composition |
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| KR20020057785A (en) * | 2001-11-13 | 2002-07-12 | 이남형 | THE PRODUCTION METHOD OF EGG CONTAINING MIXING IgY ABOUT Propionibacterium acnes, Staphylococcus epidermidis, E.coli CAUSING ACNES AND THEREOF PREVENTING COSMETICS FOR ACNES SKIN CONTAINING MIXING IgY |
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| CN114558130B (en) * | 2022-03-19 | 2023-11-14 | 广西博生生物科技有限公司 | Preparation method of collagen and anti-acne propionibacterium gamma protein combined preparation |
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