CN1569229A - IgY product for resisting mouth and upper respiratory tract infection and its application - Google Patents
IgY product for resisting mouth and upper respiratory tract infection and its application Download PDFInfo
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Abstract
The invention belongs to bioengineering technology field, it relates in particular to the preparation technology, the product prescriptions and specific application fields of multiple chicken yolk IgY with specific infection resisting function.
Description
Content of the present invention is about multiple IgY (chicken yolk immunoglobulin) product, technology of preparing and concrete application the thereof with specificity infection function.Particularly, mainly be meant anti-oral cavity and upper respiratory tract infection IgY product and application thereof.
Background technology
1, oral cavity and upper respiratory tract infection
Therefore oral cavity and upper respiratory tract (nose, pharynx, larynx) are except that skin, contact the most frequently in each system of human body with external environment condition, and the position of contact area maximum is infected by the pathogenic microorganism in the external environment condition very easily.Only the antibacterial that exists in the oral cavity promptly has 400 kinds more than, and comprising probiotics and some pathogenic bacterium, they can adhere to oral mucosa and dental surface, growth and breeding.Under normal condition, keeping a kind of useful dynamic equilibrium between the various types of microorganism in oral cavity and between they and the host.But under the influence of some inside and outside factors (cause as immunity degradation, antibiotic abuse that endogenous probiotics is killed, the external source pathogenic bacteria is attacked etc.), this balance is destroyed, and pathogenic microorganism is bred in a large number and caused infection
[1]
Streptococcus mutans, lactobacillus, actinomycetes etc. can cause dental caries, and further cause dental pulp disease and periapical disease; Infection such as porphyromonas gingivalis, spirillum, fusiform bacilarmature can cause gingivitis, periodontitis and the disease of cari oris mucosa (as necrotic gingivostomatitis)
[2]In addition, herpes simplex virus, candidiasis, staphylococcus aureus, Hemolytic streptococcus, Diplococcus pneumoniae etc. also can be separately or the mixed infection oral mucosa, cause different types of disease of cari oris mucosa.Pathogenic bacteria in the various chronic oral focuses (as gingivitis, periodontitis, dental pulp disease, necrotic gingivostomatitis etc.) (mainly is a streptococcus, Streptococcus viridans particularly) and the toxin that produces can be transported to other positions of body by blood circulation or lymphsystem, cause insecondary other diseases.Wherein relation is comparatively close bacterial endocarditis, rheumatic or a rheumatoid arthritis etc.
[3,4]Therefore, oral health and health are important component parts that guarantees animal economy health.
Multiple virus and pathogenic bacteria can cause acute upper respiratory tract infection, not only have stronger infectiousness, and can cause severe complication, should actively prevent and treat.Acute upper respiratory tract infection has 70%-80% to be caused by virus approximately.Mainly contain influenza virus (first, second, third), parainfluenza virus, respiratory syncytial virus, adenovirus, rhinovirus, echovirus, Coxsackie virus, Measles virus, rubella virus.Bacterial infection can directly or after viral infection take place, and for seeing, is hemophilus influenza, streptococcus pneumoniae and staphylococcus etc. with Hemolytic streptococcus secondly more.Accidental gram negative bacilli.Its infection mainly show as flu, pharyngolaryngitis or tonsillitis
[5]
The method of control oral cavity infection at present has oral cavity and cleaning of teeth (as brush teeth, ultrasonic toothwash, chewing gum etc.), whole body or locally gives fluorine, uses antibiotic or chemical drugs etc.Prevent and treat upper respiratory tract infection and mainly use antibiotic, antiviral drugs (can select medicine less), symptomatic treatment and vaccine methods such as (mainly being influenza vaccines).But that is that all right at present is ripe for the method for vaccine prevention, and price is expensive.Regular use chemical drugs and antibiotic cause the generation of drug resistance pathogenic bacterium easily, and because its effect non-specific, will destroy in the body colony balance normally, cause the generation of secondary infection and other diseases; Also there are certain direct harm (may cause fluorine tooth and other potential side effect as the long-term fluorine of giving) in chemical drugs and antibiotic to human body.
2, chicken IgY
The passive immunity method is in the direct using specific antibody and the toxin of pathogenic microorganism and generation thereof.Because its simple, specific effect, the particularly passive immunity that is undertaken by digestive tract and respiratory tract can also be avoided some side effect of active immunity (being vaccine), have higher safety and effectiveness, be subjected to people's attention just day by day.From the 1980's ends so far, existing both at home and abroad a large amount of research datas confirm the effectiveness of utilization polyclonal antibodies (abbreviation multi-resistance) and monoclonal antibody (abbreviation monoclonal antibody) passive immunity disease preventing and treating
[6,7,8,9]
In the passive immunity method there be the source of specific antibody: human immunoglobulin, cattle colostrums, chicken yolk immunoglobulin (IgY) and monoclonal antibody.Polyclonal antibody can be discerned the more epitope in viral pathogenic bacteria surface, make it to be difficult to escape by variation the neutralization of antibody, and preparation method is easy, cost is lower.
Under naturalness, IgY is transported to the egg yolk from hen blood, avoids various infectious disease to protect following chicken.With the laying hen after the specific antigen artificial immunity is the preferably source of large-scale production specific polyclonal antibody
[9]Compare with additive method, chicken IgY extraction process is simpler, and egg is people's daily bread, does not have toxic problem substantially.Can extract the IgY about 100 milligrams in each egg yolk, and a laying hen can produce 280 pieces of eggs every year, that is to say after the laying hen immunity inoculation that (antigen-specific antibodies wherein can reach 10% of total IgY to the annual antibody that can obtain about 30 grams, concrete ratio is different because of antigen type and other factors), be equivalent to the antibody production in 1 year of 12 rabbits.The production cost of IgY is lower more than 10 times than mammal IgG
[10,11]
Chicken IgY is similar to mammal IgG on structure, function, and molecular weight is 180kD, isoelectric point, IP 5.7-7.6.Stability to sour environment and temperature is poor slightly than mammal IgG, but also very stable.4 ℃ of preserve 5 years, room temperature following 6 months or 37 ℃ 1 month have no significant effect its antibody activity; The active maintenance more than 24 hours in the time of 65 ℃,, 70 ℃ are heated active remarkable decline after 90 minutes; Can be acidproof, alkaline-resisting and high pressure resistant, quite stable during pH value 4-11, active decline rapidly when PH reduces to 2-3, IgY is lower than 60 ℃ with temperature and handles 30 minutes still retaining part activity under the 400Mpa high pressure.Ma Peisong, Liu Yansong etc. be at 37 ℃, PH2.8, and the low concentration pepsin detected with enzyme-linked method after 1.5 hours, and the IgY activity only reduces by 25%
[10,11,12]
Domestic and international at present existing many chicken IgY are used to prevent and treat the research report of various infectious disease.Study secondary infection, pharyngolaryngitis and other upper respiratory tract infection etc. that more disease kind has children's's rotavirus diarrhea and some other digestive tract infection, helicobacter pylori infections relevant disease, dental caries and relevant oral disease, bacillus pyocyaneus to cause
[13,14,15,16]Wherein, about the existing patent application (Chinese patent publication number: CN1201693A, CN1250056A, CN1208732A, CN1307061A) of the research of chicken IgY anti-rotavirus, helicobacter pylori, bacillus pyocyaneus, dental caries Streptococcus mutans.But, on the preparation of pathogenic bacterium and antigenic selection thereof, specific IgY and purification technique and finished product formulation selection, still have big difference; On IgY output and concrete experimental result, also exist very big difference between the different research work.It should be noted that especially the pathogenic microorganism kind that oral cavity and upper respiratory tract infection relates to is more, the pathogenesis complexity, select at strain is this area focus and difficult point problem always.Relevant IgY research work in the past is quite scattered, and is more single on specific antigen is selected, and the concrete formula for a product of using of IgY is selected not attach great importance to.
Summary of the invention
The objective of the invention is, fully comparing and analyzing on the basis that correlational study makes progress both at home and abroad in recent years, propose selection of antigen and finished product prescription more safely and efficiently, the one-step optimization IgY that goes forward side by side preparation, purification technique are realized its extensive, low-cost and environment-friendly type production.Simultaneously, provide several chicken yolk immunoglobulin (IgY) products at main oral cavity and upper respiratory tract infection disease.
Below the IgY product preparation process that the present invention taked is narrated, about several main anti-oral cavities and the research situation of upper respiratory tract infection IgY product and prescription thereof, will in the specific embodiment of back, be described in detail one by one.
Preparation artificial immunity specific egg yolk immunoglobulin (IgY) is taked following steps:
1, preparation antigen: at certain sick best kind (or bacterium/strain) of selecting pathogenic bacterium or virus of planting; Various types of (or bacterium/strain) cultivates amplification respectively; Prepare particulate antigen respectively or extract antigen with appropriate method.
2, immune hen: various pathogenic bacterias/viral malicious antigen is immune hen respectively.Method with reference to Farrelly etc.
[17], initial immunity is got the antigenic solution of above preparation, adds fully mixing of equivalent Fu Shi Freund's complete adjuvant (Sigma), multiple spot muscle or subcutaneous injection Leghorn at reproduction age kind chicken (SPF level), 0.5 milliliter every (about 400 microgram antigens); 2 weeks of every interval again booster immunization once, 2-3 time altogether, booster immunization is used freund 's incomplete adjuvant instead.After antibody titer reaches the platform peak value, begin to collect hyperimmune egg.And detect antibody titer weekly, when antibody titer was tending towards descending, booster immunization was once again.The hyperimmune egg of collecting is pressed each bacterium/seed culture of viruses antigen type in 4 degrees centigrade of difference cold storing and fresh-keepings.
3, IgY preparation: various bacterium/seed culture of viruses IgY antibody extracts respectively.Get the good hyperimmune egg of classification, after the careful sassafras wash disinfection of 75% ethanol, shell breaking is removed albumen with the egg yolk separator as far as possible, egg yolk is rolled on aseptic filter paper blot afterwards, punctures membrane of yolk, collects egg yolk; In the deionized water dilution of 4 ℃ of pre-coolings of ratio adding in 1: 10, fully homogenize, clarification is spent the night; Get supernatant, the negative pressure leaching defat, 50% saturated ammonium sulphate, 4 ℃ of centrifugal 20min of 8000r/min, precipitation is resuspended after Sepharose S-200 gel filters purification with 0.1M PBS; Antibody purification solution ultrafiltration and concentration, packing is stored in 4 ℃ after the degerming after filtration, or preserves after the lyophilization.
4, IgY purity is identified and determining the protein quantity.Purity is identified with SDS-PAGE method (Laemmli system); The assay folin-phenol method.
5, the IgY antibody titer detects.Adopt the ELISA method to detect, by elisa plate, two resist the biotin labeled mouse-anti chicken IgY light chain monoclonal antibody of employing with each corresponding bacterium/seed culture of viruses antigenic solution bag.Detection is carried out according to routine.
6, antibody mixes: by different sick kinds, will be several and its characteristic bacterium/corresponding IgY of seed culture of viruses antigen with only mixed.
7, formula for a product: get the IgY mixtures of antibodies of above preparation, add adjuvant and other materials, make the IgY product of anti-oral cavity of each species specificity or respiratory tract infection by different formula for a product.
The present invention prepares the IgY antibody of anti-every kind of pathogenic bacterium/virus respectively, then according to the effect size of these pathogenic bacterium/viruses in certain disease takes place, selects best IgY antibody combination.This antibody combination both can guarantee the effective dose of major antigen specific antibody, can guarantee again at other antigenic wide spectrum effects, thereby improve curative effect most possibly.Particularly at the antibody optimum organization of several main oral cavities and upper respiratory tract infection disease.
Discover that the adhesion of pathogenic microorganism is the necessary condition that host infection takes place, and can effectively protect from infection to the pathogenic bacterium surface antigen that adhesion plays a major role by inhibition
[2]Therefore, among the present invention on selection of antigen cell wall and the adventitia fragment to contain the pathogenic bacterium surface antigen, both improved the content of effective IgY, kept the simplification of antigen preparation technology again.
The present invention takes ammonium sulfate precipitation method in the IgY preparation
[18], both improved the IgY yield, lower production cost, avoided preparation technology to use Organic substance environmental pollution problem again.
Behind 3 immunologic facilitations, the ELISA method detects, and the specific IgY antibody titer in the egg yolk reaches more than the 10-5; Detect through forint-phenol law, the egg yolk total protein response rate reaches more than 90%; Average every egg can obtain about 100 milligrams of IgY, and SDS-PAGE detects purity up to 95%, and fat content is lower than 1%; Whole process of preparation IgY antibody activity reduction ratio is lower than 5%.These qualification results have proved that fully the present invention adopts IgY preparation technology's effectiveness.
According to selected each oral cavity and the sick domestic and international study on prevention result who plants of upper respiratory tract infection, external pathogenic bacterium/virus that the present invention has carried out IgY and compound composition thereof suppresses experiment and preliminary zoopery, at different application, repetition test has also tentatively been determined the optimum formula that several IgY use.
Following examples will more specifically be described detailed content of the present invention, but content of the present invention is not limited only to this.
The IgY preparation of embodiment 1 caries prevention and products thereof prescription
Dental caries is a ubiquitous infectious disease among the crowd, and according to the national oral epidemiology survey data second time, the crowd of China more than 50% suffers from dental caries in various degree.And dental caries is the one of the main reasons that causes halitosis.Main at present whole body or the local method (mainly being fluoridize in the toothpaste) of fluorine of giving of adopting prevented and treated dental caries.But for a long time with the security hidden trouble of fluorine existence to human body.Number of research projects shows both at home and abroad, and using specificity chicken IgY passive immunity preventing decayed tooth is a kind of safe and effective potential alternative means
[2,19,20]
Streptococcus mutans is to enamel and the equal tool adhesive attraction of dentin, be the main pathogenic bacterium of dental caries, wherein (the total recall rate in the human dental bacterial plaque is 90% with serum c, e, f type, but have the serum cross reaction between the three) and the d/g type (also claim Streptococcus sobrinus, streptococcus sobrinus, recall rate in the human dental bacterial plaque reaches 9%) be main, but the latter's acid producing ability is stronger, so destructiveness may be bigger; Lactobacillus plays an important role in the process of dark dental caries (carious dentin) development at shallow dental caries (enamel caries) then to dentin tool adhesive attraction
[21]
Present embodiment prepares the two kinds of hypotypes (serum c type and d/g type) of Streptococcus mutans and the IgY antibody of lactobacillus respectively, and is mixed and made into product by a certain percentage.
1, antigen preparation:
Modification streptococcus reference culture Ingbritt (serum c type), Streptococcus sobrinus S.sobrinus 6715 (serum g type), lactobacillus reference culture ATCC4356 are inoculated in TS (pH7.0) culture fluid that contains sucrose 95%N respectively
2, 5%CO
2Anaerobism is cultivated, and cultivates 48h for 37 ℃, centrifugal separately collection thalline, and bacteria concentration to 2 * 109CFU/ml is regulated in PBS washing back.Ultrasonication, centrifugal 10 minutes of 1000g removes big bacterial debris and full cell; Centrifugal 20 minutes of 60000g removes supernatant, and collecting precipitation (cell wall and adventitia), PBS centrifuge washing once add PBS and make suspension, and regulating protein concentration is 1600 mcg/ml.
2, immune hen
Get the antigenic solution of above preparation, add fully mixing of equivalent Fu Shi Freund's complete adjuvant (Sigma), multiple spot muscle or subcutaneous injection Leghorn at reproduction age kind chicken (SPF level), 0.5 milliliter every (about 400 microgram antigens); 2 weeks of every interval again booster immunization once, 2-3 time altogether, booster immunization is used freund 's incomplete adjuvant instead.After antibody titer reaches the platform peak value, begin to collect hyperimmune egg.And detect antibody titer weekly, and when antibody titer is tending towards descending, booster immunization once (begin to collect hyperimmune egg after three months, every month booster immunization is once) again.The hyperimmune egg of collecting is in 4 degrees centigrade of classification cold storing and fresh-keepings.
3, IgY antibody extracts purification, detection, evaluation
IgY extracts the method that purification adopts water dilution, saturated ammonium sulphate to combine with Sepharose S-200 gel filtration.Purity is identified with determining the protein quantity and is adopted SDS-PAGE method (Laemmli system) and folin-phenol method respectively.The IgY antibody titer detects and adopts the ELISA method, and by elisa plate, two resist the biotin labeled mouse-anti chicken IgY light chain monoclonal antibody of employing with the antigenic solution bag.
4, antibody mixes: get the IgY antibody behind the purification, press c: g: the mixed of lactobacillus=3: 1: 1 is anti-tooth decay IgY solution, the lyophilizing preservation.
5, caries prevention IgY formula for a product: add antiseptic, wetting agent or other auxiliary elements.
Spray 1: anti-tooth decay IgY 4 grams of above preparation, Herba Menthae 1 gram, sodium benzoate 0.1 gram after the sterilized water dissolving, is settled to 1000 milliliters.
Spray 2: anti-tooth decay IgY 4 grams of above preparation, Herba Menthae 1 gram, sodium benzoate 0.1 gram, xylitol 50 grams after the sterilized water dissolving, are settled to 1000 milliliters.
Su mouth liquid: anti-tooth decay IgY 4 grams of above preparation, Herba Menthae 1 gram, sodium benzoate 0.1 gram, xylitol 50 grams after the sterilized water dissolving, add 100 glycerol, are settled to 1000 milliliters.
Chewing gum; 40 kilograms of gum base, 50 degrees centigrade softening after, add 30 kilograms of xylitol, 20 kilograms of Sorbitols, 10 kilograms of calcium lactate, 0.1 kilogram of Herba Menthae, fruit essence is an amount of, anti-tooth decay IgY 50 grams of potassium sorbate 20 grams and above preparation stir.Be total to double centner.
The IgY preparation of embodiment 2 treatment periodontal disease (gingivitis, periodontitis) and diseases of oral mucosa and products thereof prescription
Periodontal disease and diseases of oral mucosa all cause owing to multiple oral cavity pathogen mixed infection, the colony balance in the oral cavity to these two kinds of diseases play a part very important.
Periodontal disease be one group by antibacterial (porphyromonas gingivalis, actinobacillus actinomycetem comitans/companion's unwrapping wire haemophilus, borrelia buccale, fusiform bacilarmature etc.) infect cause, be the chronic disease of feature with the infringement and the breaking-up of periodontal tissue.It is to cause tooth to lack, influence the commonly encountered diseases of oral health in a large number.According to the position and the lesion nature of morbidity, periodontal disease is divided into gingivitis and periodontitis again.The long-term existence of periodontal focus also may cause general secondary infection (the particularly toxin of Streptococcus viridans and generation thereof) such as bacterial endocarditis.And the same with dental caries, periodontal disease is to cause another major reason of halitosis
[1]Diseases of oral mucosa is meant except that tumor, occurs in the disease of oral mucosa and soft tissue.The sick kind of diseases of oral mucosa is more, and prevalence is lower, and sick damage is varied, cause of disease complexity.Wherein the diseases of oral mucosa that causes owing to local infection has: borrelia buccale and fusiform bacilarmature cause the necrotic gingivostomatitis of (the Pu Liwo bacterium was increased in the middle of the report affected part was also arranged recently); Candidiasis infects thrush or the thrush (being mainly in infant) that causes; By caused membranous stomatitis of coccus infection such as staphylococcus aureus, Streptococcus viridans, Hemolytic streptococcus, Diplococcus pneumoniae etc.
[3]
Chicken IgY combination at the preferred antigens kind can effectively suppress above main pathogenic bacterium, thereby safeguards the benign balance of oral cavity flora, avoids the generation of oral disease.
1, antigen preparation
Cultivate amplification porphyromonas gingivalis, actinobacillus actinomycetem comitans/companion's unwrapping wire haemophilus, borrelia buccale, fusiform bacilarmature, middle Pu Liwo bacterium, Streptococcus viridans, candidiasis (adopting oral cavity reference culture or isolated strains) respectively.Regulate bacteria concentration to 2 * 10
9CFU/ml.Ultrasonication, centrifugal 10 minutes of 1000g removes big bacterial debris and full cell; Centrifugal 20 minutes of 60000g removes supernatant, and collecting precipitation (cell wall and adventitia), PBS centrifuge washing once add PBS and make suspension, and regulating protein concentration is 1600 mcg/ml.
2, immune hen
Get the antigenic solution of above preparation, add fully mixing of equivalent Fu Shi Freund's complete adjuvant (Sigma), multiple spot muscle or subcutaneous injection Leghorn at reproduction age kind chicken (SPF level), 0.5 milliliter every (about 400 microgram antigens); 2 weeks of every interval again booster immunization once, 2-3 time altogether, booster immunization is used freund 's incomplete adjuvant instead.After antibody titer reaches the platform peak value, begin to collect hyperimmune egg.And detect antibody titer weekly, and when antibody titer is tending towards descending, booster immunization once (begin to collect hyperimmune egg after three months, every month booster immunization is once) again.The hyperimmune egg of collecting is in 4 degrees centigrade of classification cold storing and fresh-keepings.
3, IgY antibody extracts purification, detection, evaluation
IgY extracts the method that purification adopts water dilution, saturated ammonium sulphate to combine with Sepharose S-200 gel filtration.Purity is identified with determining the protein quantity and is adopted SDS-PAGE method (Laemmli system) and folin-phenol method respectively.The IgY antibody titer detects and adopts the ELISA method, and by elisa plate, two resist the biotin labeled mouse-anti chicken IgY light chain monoclonal antibody of employing with the antigenic solution bag.
4, antibody mixes: get each the IgY antibody behind the purification, press the order of porphyromonas gingivalis, actinobacillus actinomycetem comitans/companion's unwrapping wire haemophilus, borrelia buccale, fusiform bacilarmature, middle Pu Liwo bacterium, Streptococcus viridans, candidiasis, with 2: 2: 1: 1: 1: 1: 1 mixed.
5, formula for a product:
Spray: the anti-oral cavity of above preparation and periodontal infection IgY 4 grams, Herba Menthae 1 gram, sodium benzoate 0.1 gram after the sterilized water dissolving, is settled to 1000 milliliters.
Su mouth liquid: the anti-oral cavity of above preparation and periodontal infection IgY 4 grams, Herba Menthae 1 gram, sodium benzoate 0.1 gram after the sterilized water dissolving, adds 100 milliliters of glycerol, is settled to 1000 milliliters.
The IgY preparation of embodiment 3 preventions, treatment influenza and complication thereof and products thereof prescription
Influenza is a kind of acute upper respiratory tract infection, has the recurrent in season, and can cause other respiratory tract complications, mainly cause by influenza infection, bacterial infection can directly or after viral infection take place, for seeing, is hemophilus influenza, streptococcus pneumoniae and staphylococcus etc. with Hemolytic streptococcus secondly more.Accidental gram negative bacilli.Its infection mainly show as flu, pharyngolaryngitis or tonsillitis.
1, antigen preparation
Popular influenza virus representative strains in 2002 according to the WHO announcement, select A1, A3, Type B influenza virus, the conventional Embryo Gallus domesticus method that goes down to posterity increases, and the blood clotting method is surveyed virus titer, after three kinds of type virus equal proportions were mixed, regulating concentration to hemagglutinative titer was 1: 640 and 1: 800.
Hemolytic streptococcus, staphylococcus aureus, Diplococcus pneumoniae are cultivated amplification separately, regulate bacteria concentration to 2 * 10
9CFU/ml.Ultrasonication, centrifugal 10 minutes of 1000g removes big bacterial debris and full cell; Centrifugal 20 minutes of 60000g removes supernatant, and collecting precipitation (cell wall and adventitia), PBS centrifuge washing once add PBS and make suspension, and regulating protein concentration is 1600 mcg/ml.
2, immune hen
The immunity respectively of influenza antigen and other pathogenic bacterium antigen, process is as follows:
Get influenza antigen, intravenous immune under Leghorn kind chicken at reproduction age (SPF level) wing, 5 milliliters of each every animals, it is 1: 800 that viral hemoagglutination is tired, weekly, continuous 5 weeks; Just exempt from 4 weeks of back once in chicken wing root place's intramuscular injection booster immunization, 0.5 milliliter of every side, totally 1 milliliter, it is 1: 640 that viral hemoagglutination is tired.Antibody titer reaches behind the platform peak value (about 5 * 10
-4), begin to collect hyperimmune egg.The hyperimmune egg of collecting is in 4 degrees centigrade of cold storing and fresh-keepings.
Get each pathogenic bacterium antigenic solution of above preparation, add fully mixing of equivalent Fu Shi Freund's complete adjuvant (Sigma), multiple spot muscle or subcutaneous injection Leghorn at reproduction age kind chicken (SPF level), 0.5 milliliter every (about 400 microgram antigens); 2 weeks of every interval again booster immunization once, 2-3 time altogether, booster immunization is used freund 's incomplete adjuvant instead.After antibody titer reaches the platform peak value, begin to collect hyperimmune egg.And detect antibody titer weekly, and when antibody titer is tending towards descending, booster immunization once (begin to collect hyperimmune egg after three months, every month booster immunization is once) again.The hyperimmune egg of collecting is in 4 degrees centigrade of classification cold storing and fresh-keepings.
3, IgY antibody extracts purification, detection, evaluation
IgY extracts the method that purification adopts water dilution, saturated ammonium sulphate to combine with Sepharose S-200 gel filtration.Purity is identified with determining the protein quantity and is adopted SDS-PAGE method (Laemmli system) and folin-phenol method respectively.The IgY antibody titer detects and adopts the ELISA method, and by elisa plate, two resist the biotin labeled mouse-anti chicken IgY light chain monoclonal antibody of employing with the antigenic solution bag.
4, antibody mixes: get the IgY antibody behind the purification, by the order of influenza virus, Hemolytic streptococcus, staphylococcus aureus, Diplococcus pneumoniae with 3: 2: 1: 1 mixed.
5, formula for a product
Spray: influenza IgY 4 grams of above preparation, Herba Menthae 1 gram, sodium benzoate 0.1 gram after the sterilized water dissolving, is settled to 1000 milliliters.
The IgY preparation of embodiment 4 treatment pharyngolaryngitis and products thereof prescription
Pharyngolaryngitis is the department of otorhinolaryngology commonly encountered diseases, how to be caused by the accompanying infection due to courses of infection or other upper respiratory diseases.Based on Hemolytic streptococcus, be streptococcus pneumoniae and staphylococcus etc. secondly.
1, antigen preparation
Hemolytic streptococcus, staphylococcus aureus, Diplococcus pneumoniae are cultivated amplification separately, regulate bacteria concentration to 2 * 10
9CFU/ml.Ultrasonication, centrifugal 10 minutes of 1000g removes big bacterial debris and full cell; Centrifugal 20 minutes of 60000g removes supernatant, and collecting precipitation (cell wall and adventitia), PBS centrifuge washing once add PBS and make suspension, and regulating protein concentration is 1600 mcg/ml.
2, immune hen
Get the antigenic solution of above preparation, add fully mixing of equivalent Fu Shi Freund's complete adjuvant (Sigma), multiple spot muscle or subcutaneous injection Leghorn at reproduction age kind chicken (SPF level), 0.5 milliliter every (about 400 microgram antigens); 2 weeks of every interval again booster immunization once, 2-3 time altogether, booster immunization is used freund 's incomplete adjuvant instead.After antibody titer reaches the platform peak value, begin to collect hyperimmune egg.And detect antibody titer weekly, and when antibody titer is tending towards descending, booster immunization once (begin to collect hyperimmune egg after three months, every month booster immunization is once) again.The hyperimmune egg of collecting is in 4 degrees centigrade of classification cold storing and fresh-keepings.
3, IgY antibody extracts purification, detection, evaluation
IgY extracts the method that purification adopts water dilution, saturated ammonium sulphate to combine with Sepharose S-200 gel filtration.Purity is identified with determining the protein quantity and is adopted SDS-PAGE method (Laemmli system) and folin-phenol method respectively.The IgY antibody titer detects and adopts the ELISA method, and by elisa plate, two resist the biotin labeled mouse-anti chicken IgY light chain monoclonal antibody of employing with the antigenic solution bag.
4, antibody mixes: get the IgY antibody behind the purification, the order of pressing Hemolytic streptococcus, staphylococcus aureus, Diplococcus pneumoniae was with 3: 1: 1 mixed.
5, formula for a product
Spray: anti-sphagitis IgY 4 grams of above preparation, Herba Menthae 1 gram, sodium benzoate 0.1 gram after the sterilized water dissolving, is settled to 1000 milliliters.
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20, Zhou Yi etc., resistance streptococcus Yolk immunoglobulin, tooth body dental pulp periodontal magazine, 2002,12 (9): 514-516
21, Gu Shuping etc., the dentin adhesive capacity experiment of lactobacillus and mutans streptococcus, tooth body dental pulp periodontal magazine, 1999,9 (1): 8
Claims (10)
1, the chicken yolk immunoglobulin (IgY) of artificial immunity preparation is used for: caries prevention, treatment periodontal disease and diseases of oral mucosa, prevention and treatment influenza, treatment pharyngolaryngitis.
2, a kind of artificial immunity chicken yolk immunoglobulin (IgY) preparation technology method, its process comprise that selection antigen bacteria/viral species, preparation antigen, IgY extract purification, IgY detects evaluation, antibody mixed.It is characterized in that, in specific aim that improves prepared IgY as far as possible and effectiveness, fully guaranteed the simple and easy, efficient and low-cost of whole preparation process, and be suitable for large-scale production.
3, preparation technology's method according to claim 2 is characterized in that:
Select best antigen bacteria/viral species according to the effect primary and secondary of pathogenic bacteria of all categories/virus in certain disease;
Cell wall and adventitia fragment to contain the pathogenic bacterium surface antigen on selection of antigen;
Prepare purification and the IgY antibody of preserving at every kind of pathogenic bacterium/virus respectively;
IgY extracts the method that purification adopts water dilution, saturated ammonium sulphate to combine with Sepharose S-200 gel filtration;
The evaluation of IgY purity, determining the protein quantity and IgY antibody titer detect SDS-PAGE method (Laemmli system), folin-phenol method and the ELISA method of adopting respectively;
According to the effect size of these pathogenic bacterium/viruses in certain disease takes place, select best combination of IgY antibody and ratio thereof.
4, a kind of anti-tooth decay IgY product.It is characterized in that the IgY antibody of resistance type streptococcus Ingbritt (serum c type), Streptococcus sobrinus S.sobrinus (serum g type) and lactobacillus was with 3: 1: 1 mixed.
5, a kind of IgY product for the treatment of periodontal disease and diseases of oral mucosa.It is characterized in that, the IgY antibody of resisting porphyromonas gingivalis, actinobacillus actinomycetem comitans/companion's unwrapping wire haemophilus, borrelia buccale, fusiform bacilarmature, middle Pu Liwo bacterium, Streptococcus viridans, candidiasis is with 2: 2: 1: 1: 1: 1: 1 mixed.
6, a kind of prevention, treatment influenza IgY product.It is characterized in that the IgY antibody of resisiting influenza virus, Hemolytic streptococcus, staphylococcus aureus, Diplococcus pneumoniae is with 3: 2: 1: 1 mixed.
7, a kind of IgY product for the treatment of pharyngolaryngitis.It is characterized in that the IgY antibody of anti-hemolysis streptococcus, staphylococcus aureus, Diplococcus pneumoniae was with 3: 1: 1 mixed.
8, the multiple prescription that is used for anti-oral cavity and upper respiratory tract infection IgY product is characterized in that, adds antiseptic, wetting agent, flavouring agent and/or other auxiliary elements in the IgY product.
9, prescription according to claim 8 is characterized in that, sodium benzoate or potassium sorbate are as antiseptic, and glycerol is as wetting agent and protein stabiliser, and Herba Menthae is as flavouring agent.
10, prescription according to claim 8 is characterized in that, adds xylitol in the anti-tooth decay IgY product as auxiliary antipathogenic composition.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 03149558 CN1569229A (en) | 2003-07-17 | 2003-07-17 | IgY product for resisting mouth and upper respiratory tract infection and its application |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 03149558 CN1569229A (en) | 2003-07-17 | 2003-07-17 | IgY product for resisting mouth and upper respiratory tract infection and its application |
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| Publication Number | Publication Date |
|---|---|
| CN1569229A true CN1569229A (en) | 2005-01-26 |
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| CN 03149558 Pending CN1569229A (en) | 2003-07-17 | 2003-07-17 | IgY product for resisting mouth and upper respiratory tract infection and its application |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1329091C (en) * | 2005-03-14 | 2007-08-01 | 刘国花 | Use of toothpaste containing anti-tooth decay IgY with dodecyl dimethyl lycine as blowing agent and toothpaste thereof |
| CN101455235A (en) * | 2009-01-07 | 2009-06-17 | 大连理工大学 | Special yolk antibody preparation for fruit fresh-keeping, preparation method and use thereof |
| CN109593129A (en) * | 2018-12-19 | 2019-04-09 | 广东工业大学 | A kind of Yolk antibody for pre- anti-caries and preparation method thereof and yolk antibody preparation |
| CN112957464A (en) * | 2021-02-24 | 2021-06-15 | 江秀坤 | Application of chicken egg yolk immunoglobulin in preparing throat-moistening health-care product |
-
2003
- 2003-07-17 CN CN 03149558 patent/CN1569229A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1329091C (en) * | 2005-03-14 | 2007-08-01 | 刘国花 | Use of toothpaste containing anti-tooth decay IgY with dodecyl dimethyl lycine as blowing agent and toothpaste thereof |
| CN101455235A (en) * | 2009-01-07 | 2009-06-17 | 大连理工大学 | Special yolk antibody preparation for fruit fresh-keeping, preparation method and use thereof |
| CN109593129A (en) * | 2018-12-19 | 2019-04-09 | 广东工业大学 | A kind of Yolk antibody for pre- anti-caries and preparation method thereof and yolk antibody preparation |
| CN109593129B (en) * | 2018-12-19 | 2022-09-06 | 广东工业大学 | Yolk antibody for preventing dental caries, preparation method thereof and yolk antibody preparation |
| CN112957464A (en) * | 2021-02-24 | 2021-06-15 | 江秀坤 | Application of chicken egg yolk immunoglobulin in preparing throat-moistening health-care product |
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