CN102973939A - IgY antibody preparation having mixed specificity for resistance to streptococcus mutans and streptococcus sobrinus, and preparation method and IgY total toothpaste - Google Patents
IgY antibody preparation having mixed specificity for resistance to streptococcus mutans and streptococcus sobrinus, and preparation method and IgY total toothpaste Download PDFInfo
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- CN102973939A CN102973939A CN2012103986901A CN201210398690A CN102973939A CN 102973939 A CN102973939 A CN 102973939A CN 2012103986901 A CN2012103986901 A CN 2012103986901A CN 201210398690 A CN201210398690 A CN 201210398690A CN 102973939 A CN102973939 A CN 102973939A
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Abstract
The invention discloses an IgY antibody preparation having mixed specificity for resistance to streptococcus mutans and streptococcus sobrinus. The IgY antibody preparation is prepared by mixing 10-70 parts by weight of streptococcus mutans bacteria and bacteria pieces and 30-90 parts by weight of streptococcus sobrinus bacteria and bacteria pieces into an antigen via laying hens. The invention also discloses a preparation method of the IgY antibody preparation having the mixed specificity for resistance to the streptococcus mutans and the streptococcus sobrinus, and a toothpaste which contains the IgY antibody preparation having the mixed specificity for resistance to the streptococcus mutans and the streptococcus sobrinus and the IgY antibody preparation having the mixed specificity for resistance to the anti-porphyromonas gingivalis and the fusobacterium nucleatum and is used for preventing saprodontia, gingivitis and halitosis thereof. The toothpaste used for preventing saprodontia, gingivitis, periodontitis and halitosis halitosis thereof has obvious effects for preventing saprodontia, gingivitis, periodontitis and halitosis bacteria thereof.
Description
Technical field
The present invention relates to the oral-cavity article technical field, particularly a kind of toothpaste that is used for caries prevention and gingivitis and halitosis thereof that contains Streptococcus mutans and Streptococcus sobrinus mixing specific IgY antibody preparation and preparation method thereof and contain Streptococcus mutans and Streptococcus sobrinus mixing specific IgY antibody preparation, Fusobacterium nucleatum and porphyromonas gingivalis mixing specific IgY antibody preparation.
Background technology
3 ~ 6 years old child's caries prevalence rate of China is 25.49% ~ 92.72%, and adult's caries prevalence rate is 40.37%.Wherein Streptococcus mutans and Streptococcus sobrinus are main cariogenic bacterias.The generation of bacterial infection disease at first is that adhesin by the bacterium surface combination is present in corresponding receptor generation specific binding in epithelial cell, endotheliocyte, leukocyte or the extracellular matrix and is settled in certain organs in the body with it.The infection of Streptococcus mutans and Streptococcus sobrinus utilize just the synthetic a large amount of extracellular polysaccharide of the sucrose in the food consist of dental plaque between matter, help antibacterial to facing adhere to and antibacterial between mutually adhere to, form bacterial plaque.People expect to reach by the toxicity that suppresses Streptococcus mutans and Streptococcus sobrinus the purpose of preventing decayed tooth always.Streptococcus mutans and Streptococcus sobrinus IgY were subject to extensive concern as a kind of novel biological auti-cariosity material in recent years.The toothpaste that is used at present anticaries is generally toothpaste with fluoride, has also destroyed the probiotics of keeping microecological balance in the oral cavity when removing cariogenic bacteria,
In addition, China's gingiva, periodontal sickness rate account for more than 70% of population, porphyromonas gingivalis (Porphyromonas gingivalis) is the main suspicious pathogenic bacterium of the periodontal disease such as marginal gingivitis, chronic periodontitis, aggressive periodontitis, its pili can be combined with the glyceraldehyde-3-phosphate dehydrogenase on mouth epithelial cells surface, invade gingival epithelium cell and Gingival Fibroblasts, escape from immune is removed.Porphyromonas gingivalis produces dipeptides acyl aminopeptidase degrade connective tissues, promotes the active increase of MMP-2 and MMP-1.The arginine of porphyromonas gingivalis and lysine specific cysteine protease (RgpA, RgpB, Kgp) can be hydrolyzed L-12 and cause INN γ to produce minimizing.Part porphyromonas unicellular hair can be induced Expression of Macrophages L-1 α, L-1 β, L-6, TNF
αCause alveolar bone to destroy.In the generation of patients with periodontal disease simultaneous halitosis, its halitosis mainly contains Fusobacterium nucleatum and causes.At present do not have effectively to treat the method for halitosis, use Antibiotics Drug therapy halitosis that a lot of drawbacks are arranged.
Setting about from main cause of disease bacterium porphyromonas gingivalis and Fusobacterium nucleatum for prevention and treatment periodontal disease and halitosis is key point.
The toothpaste that is used at present prevention and treatment periodontitis is generally the toothpaste that contains medicinal herb components.Although the patent of this class has a lot, all without clear and definite curative effect.
Existing bibliographical information shows and uses clinically specific IgY antibody caries prevention and treatment infant rotavirus diarrhoea, prevention pulmonary fibrosis patients charrin's disease, and it is remarkable to alleviate the microbial stomachache effect of helicobacter pylorus, and has no side effect and addiction.
Find that in the IgY of the numerous species that we study the periodontal disease that has the experiment proved that trend and the curative effect that does not have or do not have significantly to suppress periodontal disease and halitosis because of the IgY antibody that the bacterial immunity hen produces.Briefly, not every IgY has obvious therapeutical effect.
Because the suspicious cause of disease bacterium of periodontal disease and halitosis has 11 kinds more than, but main suspicious cause of disease bacterium comprises porphyromonas gingivalis, Fusobacterium nucleatum, companion actinomycetes Actinobacillus, the treponema dentium of dwelling, Fu Shi bacteroid, capnocytophaga gingivalis etc.When the IgY that explores the preparation correspondence was used for the treatment of periodontal disease and halitosis, we inferred that porphyromonas gingivalis, Fusobacterium nucleatum, companion actinomycetes Actinobacillus, the treponema dentium of dwelling, Fu Shi bacteroid, capnocytophaga gingivalis all might cause periodontal disease and halitosis; Because our prior unpredictable which bacterium in the middle of this is the principal element that causes periodontal disease and halitosis, which bacterium is to cause that periodontal disease and halitosis thereof are secondary causes.
We think must have the test of suitable anti-periodontal disease and halitosis to determine which bacterium is the principal element that causes periodontal disease and halitosis.Thereby medicine or the health product of the standby anti-periodontal disease of guidance system and halitosis.
Present prior art does not have relevant prompting guidance technology personnel to find that resisting porphyromonas gingivalis, Fusobacterium nucleatum mixed IgY are the keys that solves treatment periodontal disease and halitosis thereof.
The technical staff can't in advance under the prerequisite of the experiment of not doing anti-periodontal disease and halitosis, learn that in advance porphyromonas gingivalis, Fusobacterium nucleatum are the principal elements that causes periodontal disease and halitosis thereof.
The technical staff can't be in advance under the prerequisite of the experiment of not doing anti-periodontal disease and halitosis, learns that in advance resisting porphyromonas gingivalis, Fusobacterium nucleatum mixed IgY are than the better effects if of independent resisting porphyromonas gingivalis IgY, independent anti-Fusobacterium nucleatum IgY.
Summary of the invention
One of technical problem to be solved by this invention is to provide a kind of Streptococcus mutans and Streptococcus sobrinus mixing specific IgY antibody preparation.
Two of technical problem to be solved by this invention is to provide a kind of preparation method that contains Streptococcus mutans and Streptococcus sobrinus mixing specific IgY antibody preparation.
Three of technical problem to be solved by this invention is to provide a kind of toothpaste that is used for caries prevention and gingivitis and halitosis thereof that contains Streptococcus mutans and Streptococcus sobrinus mixing specific IgY antibody preparation, resisting porphyromonas gingivalis and Fusobacterium nucleatum mixing specific IgY antibody preparation.
Streptococcus mutans and Streptococcus sobrinus mixing specific IgY antibody preparation as first aspect present invention, it is characterized in that, be mixed into the antigen immune bird inlay and prepare by the Streptococcus sobrinus thalline of the Streptococcus mutans thalline of 10～70 weight portions and bacterial chip and 30～90 weight portions and bacterial chip.
Preparation method as Streptococcus mutans and the Streptococcus sobrinus mixing specific IgY antibody preparation of second aspect present invention is characterized in that, comprises the steps:
A. Streptococcus mutans and Streptococcus sobrinus incubation step
A.1 bacterial species: Streptococcus mutans (S.mutans) and Streptococcus sobrinus (S.sobrinus);
A.2 get Streptococcus mutans (S.mutans) MT8148 bacterial strain and Streptococcus sobrinus (S.sobrinus) 6715 bacterial strains, respectively through Streptococcus mutans, the Streptococcus sobrinus of BHI-S culture medium separation and Culture and amplification cultivation, and confirm as Streptococcus mutans, Streptococcus sobrinus through evaluation and obtain Streptococcus mutans thalline and bacterial chip, Streptococcus sobrinus thalline and bacterial chip;
B. immunogen preparation process:
B.1 get Streptococcus mutans thalline and bacterial chip 10～70 weight portions, Streptococcus sobrinus thalline and bacterial chip 30～90 weight portions are mixed into bacterium liquid antigen;
B.2 isopyknic bacterium liquid antigen is mixed with isopyknic Fu Shi Freund's complete adjuvant, use the high speed agitator stirring and emulsifying, it is former finally to make Water-In-Oil Fu Shi Freund's complete adjuvant Streptococcus mutans and Streptococcus sobrinus mixed immunity; Every milliliter of Fu Shi Freund's complete adjuvant mixed immunity is former to contain antibacterial 1,000,000,000-8,000,000,000;
B.3 isopyknic mixed bacteria liquid is mixed with isopyknic freund 's incomplete adjuvant, use the high speed agitator stirring and emulsifying, finally make Water-In-Oil freund 's incomplete adjuvant Streptococcus mutans and the Streptococcus sobrinus mixed immunity is former, contain antibacterial 1,000,000,000-8,000,000,000 during every milliliter of freund 's incomplete adjuvant mixed immunity is former;
C. immune step:
Use syringe with the former subcutaneous and intramuscular injection of Fu Shi Freund's complete adjuvant mixed immunity to bird inlay, then every bird inlay injection 0.5ml-3.0ml immunogen uses the subcutaneous and intramuscular injection of the former 0.5ml-3.0ml of freund 's incomplete adjuvant mixed immunity to bird inlay week about.Behind the booster immunization secondary, after this every re-using the subcutaneous and intramuscular injection of the former 0.5ml-3.0ml of freund 's incomplete adjuvant mixed immunity ten weeks to bird inlay booster immunization five times, altogether immunity eight times of front and back;
D. the preparation of Streptococcus mutans and Streptococcus sobrinus mixing specific IgY extract:
Collect for the first time rear the 28th day immune hen egg of immunity of immunity, the ethanol of use 75% carries out the egg surface sterilization, open eggshell and get egg yolk, remove membrane of yolk and get egg yolk liquid, with egg yolk liquid and the distilled water ratio adding distil water of 1:10 by volume, fully stir, and use glacial acetic acid to regulate acid-base value to pH5.30-5.50, then-20 ℃ of cold storage of placement were taken out and are thawed in 3 days afterwards, get supernatant, with concentrated 20 times of egg yolk liquid, regulate PH to 7.0-8.0 with 1NNaOH with the gel filtration system, put-20 ℃ of preservations.
The preparation method of above-mentioned Streptococcus mutans and Streptococcus sobrinus mixing specific IgY antibody preparation, behind described step D, increase by an IgY purification step, the saturation that specifically Streptococcus mutans and Streptococcus sobrinus mixing specific IgY extract is added sulphuric acid amine to 55%, fully stir, room temperature left standstill 2 hours, then high speed centrifugation is 10 minutes, get precipitation, to precipitate and use distilled water diluting to the substance accumulated amount, the saturation that adds again sulphuric acid amine to 33%, fully stir, room temperature left standstill 2 hours, and then high speed centrifugation is 10 minutes, get precipitation, with the bag filter of packing into after the distilled water diluting dissolving, through the hollow fiber filter desalination, namely get Streptococcus mutans and Streptococcus sobrinus mixing specific IgY antibody preparation sterling with distilled water.
In a preferred embodiment of the invention, described step D replaces with following methods: collect for the first time rear the 28th day immune hen egg of immunity of immunity, with 75% alcohol disinfecting egg shell, smash eggshell, leave and take egg yolk.Behind 10 times of dilutions of deionized water egg yolk, regulate PH to 5.30-5.50. with glacial acetic acid and anhydrous sodium acetate, get supernatant after 4 ℃ of sedimentations, add 2% chitosan and sodium alginate, 2% calcium chloride and sodium polyphosphate flocculating sedimentation are got supernatant again and are removed unnecessary deionized water with the ultrafiltration of gel filtration system, with concentrated about 20 times of egg yolk liquid, regulate PH to 7.0-8.0 with the 1N sodium hydroxide, put-20 ℃ of preservations.
As containing of third aspect present invention above-mentioned Streptococcus mutans and Streptococcus sobrinus mixing specific IgY antibody preparation, resisting porphyromonas gingivalis and Fusobacterium nucleatum mix the toothpaste that is used for caries prevention, gingivitis and periodontitis and halitosis thereof of specific IgY antibody preparation, include by Streptococcus mutans and Streptococcus sobrinus mixing specific IgY antibody preparation, resisting porphyromonas gingivalis and Fusobacterium nucleatum and mix the anti-pathogenic bacterium preparation that the specific IgY antibody preparation consists of, described anti-pathogenic bacterium preparation is comprised of the materials in percentage by mass that accounts for the toothpaste gross mass:
Resisting porphyromonas gingivalis, Fusobacterium nucleatum mix specific IgY antibody and Streptococcus mutans, Streptococcus sobrinus mixing specific IgY antibody are pressed the mixed preparation 0.1-3.0% of 1:1 mass ratio, hibitane 0.01-2.0%, glycerol 1.0-30.0%, polyvinylpyrrolidone 0.1-2.0%, and wherein hibitane, glycerol, polyvinylpyrrolidone consist of the protective agent of resisting porphyromonas gingivalis and Fusobacterium nucleatum mixing specific IgY antibody preparation, Streptococcus mutans and Streptococcus sobrinus mixing specific IgY antibody preparation.
In a preferred embodiment of toothpaste of the present invention, also comprise the toothpaste unguentum, described toothpaste unguentum is comprised of the materials in percentage by mass that accounts for the toothpaste gross mass: Ka Bopuer 0.1～0.5%, sodium carboxymethyl cellulose 0.5～1.3%, carrageenan 0.2～0.5%, xanthan gum 0.3～0.7%, glycerol 5.0～20.0%, sorbitol 5.0～70.0%, aspartame 0.1～0.5%; Saccharin sodium 0.1～0.3%, PEG-6000.5～5.0%, PEG-12000.5～5.0%, cocamido propyl betaine 0.1-2.0%, essence 0.8～2.0%, SiO
21.0～30%, the deionized water surplus.
The toothpaste that the present invention is used for caries prevention, gingivitis and periodontitis and halitosis thereof has a significant effect to caries prevention, gingivitis and periodontitis and halitosis pathogenic bacteria thereof.
The IgY toothpaste of caries prevention of the present invention, gingivitis and periodontitis and halitosis thereof is different from the advantage of toothpaste with fluoride and has namely also protected the probiotics of keeping microecological balance in the oral cavity when removing cariogenic bacteria, has remedied the weak point of traditional anticaries method.
The IgY toothpaste of caries prevention of the present invention, gingivitis and periodontitis and halitosis thereof is used for distortion preventing streptococcus, Streptococcus sobrinus and the microbial dental caries of anaerobism of common antigenic determinant, gingivitis is arranged and by porphyromonas gingivalis and Fusobacterium nucleatum and the microbial periodontitis of anaerobism of common antigenic determinant is arranged and halitosis etc. with it with it.
The specific embodiment
Describe implementation of the present invention in detail below in conjunction with embodiment.The related strain of following embodiment is all provided by Medical College, Shanghai Communication Univ..
Embodiment 1:
The preparation of Streptococcus mutans and Streptococcus sobrinus mixing specific IgY antibody preparation:
A. Streptococcus mutans and Streptococcus sobrinus incubation step
A.1 bacterial species bacterial species: Streptococcus mutans (S.mutans) and Streptococcus sobrinus (S.sobrinus);
A.2 get Streptococcus mutans (S.mutans) MT8148 bacterial strain and Streptococcus sobrinus (S. sobrinus) 6715 bacterial strains, respectively through Streptococcus mutans, the Streptococcus sobrinus of BHI-S culture medium separation and Culture and amplification cultivation, and confirm as Streptococcus mutans, Streptococcus sobrinus through evaluation and obtain Streptococcus mutans thalline and bacterial chip, Streptococcus sobrinus thalline and bacterial chip;
B. immunogen preparation process:
B.1 get Streptococcus mutans thalline and bacterial chip 10～70 weight portions, Streptococcus sobrinus thalline and bacterial chip 30～90 weight portions are mixed into bacterium liquid antigen;
B.2 isopyknic bacterium liquid antigen is mixed with isopyknic Fu Shi Freund's complete adjuvant, use the high speed agitator stirring and emulsifying, it is former finally to make Water-In-Oil Fu Shi Freund's complete adjuvant Streptococcus mutans and Streptococcus sobrinus mixed immunity; Every milliliter of Fu Shi Freund's complete adjuvant mixed immunity is former to contain antibacterial 1,000,000,000-8,000,000,000;
B.3 isopyknic mixed bacteria liquid is mixed with isopyknic freund 's incomplete adjuvant, use the high speed agitator stirring and emulsifying, finally make Water-In-Oil freund 's incomplete adjuvant Streptococcus mutans and the Streptococcus sobrinus mixed immunity is former, contain antibacterial 1,000,000,000-8,000,000,000 during every milliliter of freund 's incomplete adjuvant mixed immunity is former;
C. immune step:
Use syringe with the former subcutaneous and intramuscular injection of Fu Shi Freund's complete adjuvant mixed immunity to bird inlay, then every bird inlay injection 0.5ml-3.0ml immunogen uses the subcutaneous and intramuscular injection of the former 0.5ml-3.0ml of freund 's incomplete adjuvant mixed immunity to bird inlay week about.Behind the booster immunization secondary, after this every re-using the subcutaneous and intramuscular injection of the former 0.5ml-3.0ml of freund 's incomplete adjuvant mixed immunity ten weeks to bird inlay booster immunization five times, altogether immunity eight times of front and back;
D. the preparation of Streptococcus mutans and Streptococcus sobrinus mixing specific IgY extract:
Collect for the first time rear the 28th day immune hen egg of immunity of immunity, the ethanol of use 75% carries out the egg surface sterilization, open eggshell and get egg yolk, remove membrane of yolk and get egg yolk liquid, with egg yolk liquid and the distilled water ratio adding distil water of 1:10 by volume, fully stir, and use glacial acetic acid to regulate acid-base value to pH5.30-5.50, then-20 ℃ of cold storage of placement were taken out and are thawed in 3 days afterwards, get supernatant, with concentrated 20 times of egg yolk liquid, regulate PH to 7.0-8.0 with 1NNaOH with the gel filtration system, put-20 ℃ of preservations.
The E.IgY purification step:
The saturation that Streptococcus mutans and Streptococcus sobrinus mixing specific IgY extract is added sulphuric acid amine to 55%, fully stir, room temperature left standstill 2 hours, then high speed centrifugation is 10 minutes, get precipitation, to precipitate and use distilled water diluting to the substance accumulated amount, the saturation that adds again sulphuric acid amine to 33%, fully stir, room temperature left standstill 2 hours, and then high speed centrifugation is 10 minutes, get precipitation, with the bag filter of packing into after the distilled water diluting dissolving, through the hollow fiber filter desalination, namely get Streptococcus mutans and the direct sterling of Streptococcus sobrinus mixing specific IgY antibody with distilled water.
Streptococcus mutans, Streptococcus sobrinus mixing specific IgY sterling are measured through the ELISA method, tiring of specific IgY antibody in Streptococcus mutans, the Streptococcus sobrinus mixing specific IgY sterling in every milligram of albumen is 1:1280000/mg albumen, perhaps claim resisting porphyromonas gingivalis and Fusobacterium nucleatum to mix in the specific IgY sterling, the specific activity of the specific IgY antibody in every milligram of albumen is 1:1280000/mg albumen.In IgY antibody sterling, the purity of IgY antibody is determined as 89.3% through irreducibility SDS-PAGE.
Embodiment 2
The preparation of Streptococcus mutans and Streptococcus sobrinus mixing specific IgY antibody preparation:
A. Streptococcus mutans and Streptococcus sobrinus incubation step
A.1 bacterial species bacterial species: Streptococcus mutans (S.mutans) and Streptococcus sobrinus (S.sobrinus);
A.2 get Streptococcus mutans (S.mutans) MT8148 bacterial strain and Streptococcus sobrinus (S.sobrinus) 6715 bacterial strains, respectively through Streptococcus mutans, the Streptococcus sobrinus of BHI-S culture medium separation and Culture and amplification cultivation, and confirm as Streptococcus mutans, Streptococcus sobrinus through evaluation and obtain Streptococcus mutans thalline and bacterial chip, Streptococcus sobrinus thalline and bacterial chip;
B. immunogen preparation process:
B.1 get Streptococcus mutans thalline and bacterial chip 10～70 weight portions, Streptococcus sobrinus thalline and bacterial chip 30～90 weight portions are mixed into bacterium liquid antigen;
B.2 isopyknic bacterium liquid antigen is mixed with isopyknic Fu Shi Freund's complete adjuvant, use the high speed agitator stirring and emulsifying, it is former finally to make Water-In-Oil Fu Shi Freund's complete adjuvant Streptococcus mutans and Streptococcus sobrinus mixed immunity; Every milliliter of Fu Shi Freund's complete adjuvant mixed immunity is former to contain antibacterial 1,000,000,000-8,000,000,000;
B.3 isopyknic mixed bacteria liquid is mixed with isopyknic freund 's incomplete adjuvant, use the high speed agitator stirring and emulsifying, finally make Water-In-Oil freund 's incomplete adjuvant Streptococcus mutans and the Streptococcus sobrinus mixed immunity is former, contain antibacterial 1,000,000,000-8,000,000,000 during every milliliter of freund 's incomplete adjuvant mixed immunity is former;
C. immune step:
Use syringe with the former subcutaneous and intramuscular injection of Fu Shi Freund's complete adjuvant mixed immunity to bird inlay, then every bird inlay injection 0.5ml-3.0ml immunogen uses the subcutaneous and intramuscular injection of the former 0.5ml-3.0ml of freund 's incomplete adjuvant mixed immunity to bird inlay week about.Behind the booster immunization secondary, after this every re-using the subcutaneous and intramuscular injection of the former 0.5ml-3.0ml of freund 's incomplete adjuvant mixed immunity ten weeks to bird inlay booster immunization five times, altogether immunity eight times of front and back;
D. the preparation of Streptococcus mutans and Streptococcus sobrinus mixing specific IgY extract:
Collect for the first time rear the 28th day immune hen egg of immunity of immunity, with 75% alcohol disinfecting egg shell, smash eggshell, leave and take egg yolk.Behind 10 times of dilutions of deionized water egg yolk, regulate PH to 5.30-5.50. with glacial acetic acid and anhydrous sodium acetate, get supernatant after 4 ℃ of sedimentations, add 2% chitosan and sodium alginate, 2% calcium chloride and sodium polyphosphate flocculating sedimentation are got supernatant again and are removed unnecessary deionized water with the ultrafiltration of gel filtration system, with concentrated about 20 times of egg yolk liquid, regulate PH to 7.0-8.0 with the 1N sodium hydroxide, put-20 ℃ of preservations.
The E.IgY purification step:
The saturation that Streptococcus mutans and Streptococcus sobrinus mixing specific IgY extract is added sulphuric acid amine to 55%, fully stir, room temperature left standstill 2 hours, then high speed centrifugation is 10 minutes, get precipitation, to precipitate and use distilled water diluting to the substance accumulated amount, the saturation that adds again sulphuric acid amine to 33%, fully stir, room temperature left standstill 2 hours, and then high speed centrifugation is 10 minutes, get precipitation, with the bag filter of packing into after the distilled water diluting dissolving, through the hollow fiber filter desalination, namely get Streptococcus mutans and the direct sterling of Streptococcus sobrinus mixing specific IgY antibody with distilled water.
Streptococcus mutans, Streptococcus sobrinus mixing specific IgY sterling are measured through the ELISA method, tiring of specific IgY antibody in Streptococcus mutans, the Streptococcus sobrinus mixing specific IgY sterling in every milligram of albumen is 1:1280000/mg albumen, perhaps claim resisting porphyromonas gingivalis and Fusobacterium nucleatum to mix in the specific IgY sterling, the specific activity of the specific IgY antibody in every milligram of albumen is 1:1280000/mg albumen.In IgY antibody sterling, the purity of IgY antibody is determined as 89.3% through irreducibility SDS-PAGE.
Embodiment 3:
Resisting porphyromonas gingivalis and Fusobacterium nucleatum mix the preparation of specific IgY antibody preparation:
A. porphyromonas gingivalis and Fusobacterium nucleatum incubation step
A.1 bacterial species: porphyromonas gingivalis (Porphyromonas gingivalis) and Fusobacterium nucleatum (Fusobacterium nucleatum);
A.2 get porphyromonas gingivalis (P.g) ATCC33277 bacterial strain and Fusobacterium nucleatum (Fn) ATCC10953 bacterial strain, respectively through the unicellular bacterium of the porphyromonas of BHI-S culture medium separation and Culture and amplification cultivation, Fusobacterium nucleatum, and confirm as porphyromonas gingivalis, Fusobacterium nucleatum through evaluation and obtain porphyromonas gingivalis thalline and bacterial chip, Fusobacterium nucleatum thalline and bacterial chip;
B. immunogen preparation process:
B.1 get porphyromonas gingivalis thalline and bacterial chip 10～70 weight portions, Fusobacterium nucleatum thalline and bacterial chip 30～90 weight portions are mixed into bacterium liquid antigen;
B.2 isopyknic bacterium liquid antigen is mixed with isopyknic Fu Shi Freund's complete adjuvant, use the high speed agitator stirring and emulsifying, it is former finally to make Water-In-Oil Fu Shi Freund's complete adjuvant porphyromonas gingivalis and Fusobacterium nucleatum mixed immunity; Every milliliter of Fu Shi Freund's complete adjuvant mixed immunity is former to contain antibacterial 1,000,000,000-8,000,000,000;
B.3 isopyknic mixed bacteria liquid is mixed with isopyknic freund 's incomplete adjuvant, use the high speed agitator stirring and emulsifying, finally make Water-In-Oil freund 's incomplete adjuvant porphyromonas gingivalis and the Fusobacterium nucleatum mixed immunity is former, contain antibacterial 1,000,000,000-8,000,000,000 during every milliliter of freund 's incomplete adjuvant mixed immunity is former;
C. immune step:
Use syringe with the former subcutaneous and intramuscular injection of Fu Shi Freund's complete adjuvant mixed immunity to bird inlay, then every bird inlay injection 0.5ml-3.0ml immunogen uses the subcutaneous and intramuscular injection of the former 0.5ml-3.0ml of freund 's incomplete adjuvant mixed immunity to bird inlay week about.Behind the booster immunization secondary, after this every re-using the subcutaneous and intramuscular injection of the former 0.5ml-3.0ml of freund 's incomplete adjuvant mixed immunity ten weeks to bird inlay booster immunization five times, altogether immunity eight times of front and back;
D. resisting porphyromonas gingivalis and Fusobacterium nucleatum mix the preparation of specific IgY extract:
Collect for the first time rear the 28th day immune hen egg of immunity of immunity, the ethanol of use 75% carries out the egg surface sterilization, open eggshell and get egg yolk, remove membrane of yolk and get egg yolk liquid, with egg yolk liquid and the distilled water ratio adding distil water of 1:10 by volume, fully stir, and use glacial acetic acid to regulate acid-base value to pH5.30-5.50, then-20 ℃ of cold storage of placement were taken out and are thawed in 3 days afterwards, get supernatant, with concentrated 20 times of egg yolk liquid, regulate PH to 7.0-8.0 with 1N NaOH with the gel filtration system, put-20 ℃ of preservations.
The E.IgY purification step:
Resisting porphyromonas gingivalis and Fusobacterium nucleatum are mixed the saturation that the specific IgY extract adds sulphuric acid amine to 55%, fully stir, room temperature left standstill 2 hours, then high speed centrifugation is 10 minutes, get precipitation, to precipitate and use distilled water diluting to the substance accumulated amount, the saturation that adds again sulphuric acid amine to 33%, fully stir, room temperature left standstill 2 hours, and then high speed centrifugation is 10 minutes, get precipitation, with the bag filter of packing into after the distilled water diluting dissolving,, namely get resisting porphyromonas gingivalis and Fusobacterium nucleatum and mix the direct sterling of specific IgY antibody through the hollow fiber filter desalination with distilled water.
Resisting porphyromonas gingivalis and Fusobacterium nucleatum mix specific IgY antibody preparation sterling and measure through the ELISA method, the tiring of specific IgY antibody that resisting porphyromonas gingivalis and Fusobacterium nucleatum mix in the specific IgY sterling in every milligram of albumen is 1:32000/mg albumen, perhaps claim resisting porphyromonas gingivalis and Fusobacterium nucleatum to mix in the specific IgY sterling, the specific activity of the specific IgY antibody in every milligram of albumen is 1:32000.In IgY antibody sterling, the purity of IgY antibody is determined as 89.3% through irreducibility SDS-PAGE.
Embodiment 4:
Resisting porphyromonas gingivalis and Fusobacterium nucleatum mix the preparation of specific IgY antibody preparation:
A. porphyromonas gingivalis and Fusobacterium nucleatum incubation step
A.1 bacterial species: porphyromonas gingivalis (Porphyromonas gingivalis) and Fusobacterium nucleatum (Fusobacterium nucleatum);
A.2 get porphyromonas gingivalis (P.g) ATCC33277 bacterial strain and Fusobacterium nucleatum (Fn) ATCC10953 bacterial strain, respectively through the unicellular bacterium of the porphyromonas of BHI-S culture medium separation and Culture and amplification cultivation, Fusobacterium nucleatum, and confirm as porphyromonas gingivalis, Fusobacterium nucleatum through evaluation and obtain porphyromonas gingivalis thalline and bacterial chip, Fusobacterium nucleatum thalline and bacterial chip;
B. immunogen preparation process:
B.1 get porphyromonas gingivalis thalline and bacterial chip 10～70 weight portions, Fusobacterium nucleatum thalline and bacterial chip 30～90 weight portions are mixed into bacterium liquid antigen;
B.2 isopyknic bacterium liquid antigen is mixed with isopyknic Fu Shi Freund's complete adjuvant, use the high speed agitator stirring and emulsifying, it is former finally to make Water-In-Oil Fu Shi Freund's complete adjuvant porphyromonas gingivalis and Fusobacterium nucleatum mixed immunity; Every milliliter of Fu Shi Freund's complete adjuvant mixed immunity is former to contain antibacterial 1,000,000,000-8,000,000,000;
B.3 isopyknic mixed bacteria liquid is mixed with isopyknic freund 's incomplete adjuvant, use the high speed agitator stirring and emulsifying, finally make Water-In-Oil freund 's incomplete adjuvant porphyromonas gingivalis and the Fusobacterium nucleatum mixed immunity is former, contain antibacterial 1,000,000,000-8,000,000,000 during every milliliter of freund 's incomplete adjuvant mixed immunity is former;
C. immune step:
Use syringe with the former subcutaneous and intramuscular injection of Fu Shi Freund's complete adjuvant mixed immunity to bird inlay, then every bird inlay injection 0.5ml-3.0ml immunogen uses the subcutaneous and intramuscular injection of the former 0.5ml-3.0ml of freund 's incomplete adjuvant mixed immunity to bird inlay week about.Behind the booster immunization secondary, after this every re-using the subcutaneous and intramuscular injection of the former 0.5ml-3.0ml of freund 's incomplete adjuvant mixed immunity ten weeks to bird inlay booster immunization five times, altogether immunity eight times of front and back;
D. resisting porphyromonas gingivalis and Fusobacterium nucleatum mix the preparation of specific IgY extract:
Collect for the first time rear the 28th day immune hen egg of immunity of immunity, with 75% alcohol disinfecting egg shell, smash eggshell, leave and take egg yolk.Behind 10 times of dilutions of deionized water egg yolk, regulate PH to 5.30-5.50. with glacial acetic acid and anhydrous sodium acetate, get supernatant after 4 ℃ of sedimentations, add 2% chitosan and sodium alginate, 2% calcium chloride and sodium polyphosphate flocculating sedimentation are got supernatant again and are removed unnecessary deionized water with the ultrafiltration of gel filtration system, with concentrated about 20 times of egg yolk liquid, regulate PH to 7.0-8.0 with the 1N sodium hydroxide, put-20 ℃ of preservations.
The E.IgY purification step:
Resisting porphyromonas gingivalis and Fusobacterium nucleatum are mixed the saturation that the specific IgY extract adds sulphuric acid amine to 55%, fully stir, room temperature left standstill 2 hours, then high speed centrifugation is 10 minutes, get precipitation, to precipitate and use distilled water diluting to the substance accumulated amount, the saturation that adds again sulphuric acid amine to 33%, fully stir, room temperature left standstill 2 hours, and then high speed centrifugation is 10 minutes, get precipitation, with the bag filter of packing into after the distilled water diluting dissolving,, namely get resisting porphyromonas gingivalis and Fusobacterium nucleatum and mix the direct sterling of specific IgY antibody through the hollow fiber filter desalination with distilled water.
Resisting porphyromonas gingivalis and Fusobacterium nucleatum mix specific IgY antibody preparation sterling and measure through the ELISA method, the tiring of specific IgY antibody that resisting porphyromonas gingivalis and Fusobacterium nucleatum mix in the specific IgY sterling in every milligram of albumen is 1:32000/mg albumen, perhaps claim resisting porphyromonas gingivalis and Fusobacterium nucleatum to mix in the specific IgY sterling, the specific activity of the specific IgY antibody in every milligram of albumen is 1:32000.In IgY antibody sterling, the purity of IgY antibody is determined as 89.3% through irreducibility SDS-PAGE.
Experimental example 1:
Streptococcus mutans, Streptococcus sobrinus mixed immunity are former; Porphyromonas gingivalis, the Fusobacterium nucleatum mixed immunity is former in method immunity bird inlay of the present invention, get for the first time rear 30 days immune egg of immunity, and prepare the extract that the extract of Streptococcus mutans and Streptococcus sobrinus mixing specific IgY antibody and resisting porphyromonas gingivalis and Fusobacterium nucleatum mix specific IgY antibody by method of the present invention, use the ELISA method and measure Streptococcus mutans and Streptococcus sobrinus mixing specific IgY antibody, resisting porphyromonas gingivalis and Fusobacterium nucleatum mix the not biological value of synantigen (seeing Table 1) of specific IgY antibodies:
Tiring of the extract of the extract of table 1 Streptococcus mutans and Streptococcus sobrinus mixing specific IgY antibody, resisting porphyromonas gingivalis and Fusobacterium nucleatum mixing specific IgY antibody
Can be clear that according to above-mentioned experimental result:
⑴. Streptococcus mutans, two kinds of bacterium thalline+bacterial chips of Streptococcus sobrinus, porphyromonas gingivalis, two kinds of bacterium thalline of Fusobacterium nucleatum+bacterial chip immunogen immune bird inlay can produce the respectively specific IgY antibody of anti-these several antibacterials;
. generation to the specific IgY antibody of various antibacterials tire not with immune bird inlay the time every kind of antibacterial dosage of using change, that is: be not that tiring of the large bacteriogenic IgY antibody of immunizing dose is also high, this is relevant with the immunogenicity of antigen;
⑶. the mixing specific IgY antibody and can both react its superposition of having tired of Streptococcus mutans and Streptococcus sobrinus mixing specific IgY antibody and Streptococcus mutans and Streptococcus sobrinus;
⑷. by above-mentioned experiment as seen, a kind of Streptococcus mutans, Streptococcus sobrinus mixing specific IgY antibody can prevent by above-mentioned bacterial dental caries, gingivitis, periodontal disease and halitosis thereof etc. mix the mixing of specific IgY antibody with resisting porphyromonas gingivalis and Fusobacterium nucleatum after;
⑸. can also expand to thus by with above-mentioned antibacterial the bacterial dental caries, gingivitis, periodontal disease, halitosis etc. of common antigenic determinant being arranged, can use Streptococcus mutans of the present invention and Streptococcus sobrinus mixing specific IgY antibody and resisting porphyromonas gingivalis and Fusobacterium nucleatum to mix the specific IgY Antybody therapy.
Experimental example 2:
Streptococcus mutans, Streptococcus sobrinus mixed immunity are former; Porphyromonas gingivalis, the Fusobacterium nucleatum mixed immunity is former in method immunity bird inlay of the present invention, get the egg of the rear 60 days immune hen of for the first time immunity, and prepare the sterling of Streptococcus mutans and Streptococcus sobrinus mixing specific IgY antibody by method of the present invention, resisting porphyromonas gingivalis and Fusobacterium nucleatum mix the sterling of specific IgY antibody, use the sterling that the ELISA method is measured Streptococcus mutans and Streptococcus sobrinus mixing specific IgY antibody, the sterling combination of resisting porphyromonas gingivalis and Fusobacterium nucleatum mixing specific IgY antibody is the biological value of synantigen (seeing Table 2) not:
Tiring of the sterling of the sterling of table 2 Streptococcus mutans and Streptococcus sobrinus mixing specific IgY antibody, resisting porphyromonas gingivalis and anti-Fusobacterium nucleatum IgY antibody
Can be clear that according to above-mentioned experimental result:
⑴. the Streptococcus mutans of preparing by method of the present invention and the sterling of Streptococcus sobrinus mixing specific IgY antibody are than preparing specific binding Streptococcus mutans, the Streptococcus sobrinus hybrid antigen of the extract of Streptococcus mutans, Streptococcus sobrinus IgY antibody by method of the present invention in the experimental example 1, and the antibacterials such as Streptococcus mutans, Streptococcus sobrinus separately the biological value of antigen all want high, be to increase in proportion substantially;
⑵. the sterling that the resisting porphyromonas gingivalis of preparing by method of the present invention and Fusobacterium nucleatum mix specific IgY antibody is than preparing specific binding porphyromonas gingivalis and the Fusobacterium nucleatum hybrid antigen that resisting porphyromonas gingivalis and Fusobacterium nucleatum mix the extract of specific IgY by method of the present invention in the experimental example 1, and the antibacterials such as porphyromonas gingivalis, Fusobacterium nucleatum separately the biological value of antigen all want high, be to increase in proportion substantially;
⑶. Streptococcus mutans, the former immune bird inlay of Streptococcus sobrinus mixed immunity can produce the respectively specific IgY antibody of anti-these two kinds of antibacterials; The former immune bird inlay of porphyromonas gingivalis and Fusobacterium nucleatum mixed immunity also can produce the respectively specific IgY antibody of anti-these two kinds of antibacterials;
Produce to the specific IgY antibody of various antibacterials tire not with immune bird inlay the time every kind of antibacterial dosage of using change, that is: it is also high not to be that the large antibacterial of immunizing dose produces tiring of IgY antibody, this is relevant with the immunogenicity of antigen;
⑸. the hybrid antigen of Streptococcus mutans and Streptococcus sobrinus mixing specific IgY antibody and Streptococcus mutans and Streptococcus sobrinus can both react, its superposition of having tired; The hybrid antigen of two kinds of bacterium such as resisting porphyromonas gingivalis and Fusobacterium nucleatum mixing specific IgY and porphyromonas gingivalis, Fusobacterium nucleatum can both react, and tiring of total specific IgY antibody also increased its superposition of having tired;
⑹. by above-mentioned experiment as seen, mix specific IgY with resisting porphyromonas gingivalis and Fusobacterium nucleatum with Streptococcus mutans and Streptococcus sobrinus mixing specific IgY antibody and can prevent by above-mentioned bacterial dental caries, gingivitis, periodontal disease and halitosis thereof etc.;
⑺. can also expand to thus by with above-mentioned antibacterial bacterial dental caries, gingivitis periodontal disease and the halitosis thereof etc. of common antigenic determinant being arranged, can use Streptococcus mutans of the present invention and Streptococcus sobrinus mixing specific IgY, resisting porphyromonas gingivalis and Fusobacterium nucleatum to mix specific IgY prevention and treatment.
Experimental example 3:
The activity of dental caries, gingivitis and periodontitis and the full effective toothpaste of halitosis IgY thereof is tired
Anti-dental caries, gingivitis and periodontitis and halitosis IgY thereof are made tire result such as table 3 behind the tooth paste product by this patent prescription:
Annotate: preparation and the toothpaste unguentum of the Streptococcus mutans that is provided by this patent with the full effective toothpaste of IgY of the caries prevention after the protective agent, gingivitis and periodontitis and halitosis thereof and preparation, resisting porphyromonas gingivalis and the Fusobacterium nucleatum mixing specific IgY of Streptococcus sobrinus mixings specific IgY are formulated
It is formulated by preparation, resisting porphyromonas gingivalis and Fusobacterium nucleatum mixing specific IgY and the toothpaste unguentum of Streptococcus mutans and Streptococcus sobrinus mixing specific IgY merely not add protectant dental caries, gingivitis and periodontitis and the full effective toothpaste of halitosis IgY thereof.
The full effective toothpaste of IgY with the caries prevention gingivitis after the protective agent and periodontitis and halitosis thereof that obvious this patent provides can effectively be protected the activity of IgY in toothpaste of dental caries, gingivitis and periodontitis and halitosis thereof.
Experimental example 4:
The anti-pathogenic bacterium preparation that is made of Streptococcus mutans and Streptococcus sobrinus mixing specific IgY antibody preparation, resisting porphyromonas gingivalis and Fusobacterium nucleatum mixing specific IgY antibody preparation detects the effect that the anti-sucrose dependency of Streptococcus mutans adheres to
Method:
In containing the BHI culture medium of 1% sucrose, add by Streptococcus mutans and Streptococcus sobrinus mixing specific IgY antibody preparation, resisting porphyromonas gingivalis and Fusobacterium nucleatum and mix the anti-pathogenic bacterium preparation that the specific IgY antibody preparation consists of, 0.22 μ m filter opening membrane filtration degerming.
Negative control group: non-specific IgY, do not contain the similar toothpaste supernatant of IgY;
Blank group: the BHI that does not contain the anti-pathogenic bacterium preparation that is consisted of by Streptococcus mutans and Streptococcus sobrinus mixing specific IgY antibody preparation, resisting porphyromonas gingivalis and Fusobacterium nucleatum mixing specific IgY antibody preparation.
Add 50 μ l in all test tubes and derive from S.mutans8148, the S.sobrinus6715 that same test tube is cultivated 18h, 30 ° of overturning angles are cultivated 18h(, and this is first test tube: the A test tube).
Take out first test tube (A test tube), the test tube 3 times of slightly overturning is poured bacterium liquid into second batch and is cleaned test tube (B test tube), and contained antibacterial is non-adhesion antibacterial.The centrifugal supernatant of abandoning of bacterium liquid in the B test tube with PBS buffer washing bacterial cell twice, is cleaned culture medium, adds the 3mlPBS buffer, vortex concussion, fully suspendible antibacterial again.
Adhere to test tube wall side adding 3ml PBS from the nothing of first test tube (A test tube), behind the vortex concussion 5min, will contain germy liquid rotating and move in the 3rd batch of clean tube (C test tube).These antibacterials are the antibacterial of non-tight adhesion.The centrifugal supernatant of abandoning of bacterium liquid in the C test tube adds 3ml PBS buffer again, vortex concussion, fully suspendible antibacterial.
In first test tube (A test tube), add the ultrasonic concussion of PBS again, make the antibacterial of tight adhesion on the test tube wall all shake lower.The centrifugal supernatant of abandoning of test tube adds 3ml PBS buffer again, and vibration evenly.
Under spectrophotometer, measure the OD550nm value of antibacterial in all test tubes, calculate the percentage adhesion rate of antibacterial, the antibacterial percentage adhesion rate of tight adhesion=A/(A+B+C) * 100%.
The result
By Streptococcus mutans and Streptococcus sobrinus mixing specific IgY antibody preparation, resisting porphyromonas gingivalis and Fusobacterium nucleatum mix anti-pathogenic bacterium preparation that the specific IgY antibody preparation consists of to have (IgY) raw material of dental caries that the more commercially available egg of (IgY) raw material of dental caries that anti-sucrose dependency adhesive attraction immunity extracts and gingivitis and halitosis thereof extracts and gingivitis and halitosis thereof to suppress S. mutans sucrose dependency adhesive attraction to Streptococcus mutans more remarkable, and (IgY) raw material that adds dental caries that the immunity of 0.2% concentration extracts and gingivitis and halitosis thereof can adhere to the S. mutans sucrose dependency and drop to 15.43% from contrasting 70.68%; (IgY) raw material that adds the dental caries of commercially available extraction of 0.2% concentration and gingivitis and halitosis thereof can adhere to the S. mutans sucrose dependency from contrasting 70.68% and drop to the concrete experimental data of 45.83%(and see Table 4)
Table 4. mixes the specific IgY antibody preparation to the antibacterial result of Streptococcus mutans and Streptococcus sobrinus In Vitro Anti adhesive attraction by Streptococcus mutans, Streptococcus sobrinus mixing specific IgY antibody and resisting porphyromonas gingivalis, Fusobacterium nucleatum
Annotate: mix the anti-pathogenic bacterium preparation lyophilized powder lot number that the specific IgY antibody preparation consists of by Streptococcus mutans and Streptococcus sobrinus mixing specific IgY antibody preparation, resisting porphyromonas gingivalis and Fusobacterium nucleatum: 20091012, contain adjuvant, tire: 1:64-128 ten thousand, commercially available chicken IgY lyophilized powder lot number: 20090916, contain adjuvant, tire: 1:32 ten thousand.
Experimental example 5:
Bacteriostatic test
The porphyromonas gingivalis anaerobism is cultivated 48h, after identifying several colonies typicals is suspended in the BHI culture medium, and adjusting bacterial concentration is (2 * 10
8) CPU/L～(1 * 10
9) CPU/L, with aseptic BHI culture medium IgY is diluted, adjust the unicellular bacterium IgY of resisting porphyromonas final concentration and be 5.0,1.0,0.1gL.
Get the unicellular bacterium 100 μ l of porphyromonas and place 96 well culture plates, add again the unicellular bacterium IgY100 of each concentration group resisting porphyromonas μ l, every group of 3 holes, the blank hole does not add bacterium liquid and only adds the BHI culture medium.96 well culture plates are cultivated 24h, 72h in 37 ℃, anaerobism.
The A pH-value determination pH is got anaerobism cultivation 24h, 72h and is got 96 well culture plates, surveys A with microplate reader
490, read absorbance (A).
Statistical procedures is used the t check.
The result
Resisting porphyromonas gingivalis IgY extracts
The purity of the resisting porphyromonas gingivalis IgY of the different batches that the application water dilution method extracts reaches 31.4～34.6%, reaches 1:800 in conjunction with tiring of porphyromonas gingivalis.
The unicellular bacterium IgY of resisting porphyromonas purification, the IgY purity of 55% saturated ammonium sulphate of different batches reaches 58.3%～61.2%, the 33% saturated ammonium sulfate further IgY purity of precipitation reaches 87.6%～89.1%, 33% saturated ammonium sulfate purification reach 1:3200 in conjunction with tiring of the unicellular bacterium of porphyromonas.
The unicellular bacterium IgY of resisting porphyromonas bacteriostasis rate sees Table 5.
The resisting porphyromonas gingivalis IgY of table 5. purification is to the suppression ratio of porphyromonas gingivalis
As can be seen from Table 5, resisting porphyromonas gingivalis IgY has remarkable effect to suppressing the unicellular bacteria growing rate of porphyromonas.
The anti-Fusobacterium nucleatum IgY of purification is to the suppression ratio of Fusobacterium nucleatum
Tool nucleic acid Fusobacterium anaerobism is cultivated 48h, after identifying several colonies typicals is suspended in the BHI culture medium, and adjusting bacterial concentration is (2 * 10
8) CPU/L～(1 * 10
9) CPU/L, with aseptic BHI culture medium IgY is diluted, adjust anti-tool nucleic acid Fusobacterium IgY final concentration and be 2.0,1.0,0.1g/L.
Get tool nucleic acid Fusobacterium 100 μ l and place 96 well culture plates, add again the anti-tool nucleic acid of each concentration group Fusobacterium IgY100 μ l, every group of 3 holes, the blank hole does not add bacterium liquid and only adds the BHI culture medium.96 well culture plates are cultivated 24h, 72h in 37 ℃, anaerobism.
The A pH-value determination pH is got anaerobism cultivation 24h, 72h and is got 96 well culture plates, surveys A with microplate reader
490, read absorbance (A).
Statistical procedures is used the t check.
The result
Anti-Fusobacterium nucleatum IgY extracts
The purity of the anti-tool nucleic acid Fusobacterium IgY of the different batches that the application water dilution method extracts reaches 31.4～34.6%, reaches 1:25600 in conjunction with tiring of Fusobacterium nucleatum.
The unicellular bacterium IgY of resisting porphyromonas purification, the IgY purity of 55% saturated ammonium sulphate of different batches reaches 58.3%～61.2%, the 33% saturated ammonium sulfate further IgY purity of precipitation reaches 87.6%～89.1%, 33% saturated ammonium sulfate purification reach 1:51200 in conjunction with tiring of tool nucleic acid Fusobacterium.The results are shown in Table 6
Table 6
As can be seen from Table 6, anti-Fusobacterium nucleatum IgY has remarkable effect to suppressing the Fusobacterium nucleatum rate of growth.
Claims (6)
1. Streptococcus mutans and Streptococcus sobrinus mixing specific IgY antibody preparation, it is characterized in that, be mixed into the antigen immune bird inlay and prepare by the Streptococcus sobrinus thalline of the Streptococcus mutans thalline of 10～70 weight portions and bacterial chip and 30～90 weight portions and bacterial chip.
2. the preparation method of Streptococcus mutans claimed in claim 1 and Streptococcus sobrinus mixing specific IgY antibody preparation is characterized in that, comprises the steps:
A. Streptococcus mutans and Streptococcus sobrinus incubation step
A.1 bacterial species: Streptococcus mutans (S.mutans) and Streptococcus sobrinus (S.sobrinus);
A.2 get Streptococcus mutans (S.mutans) MT 8148 bacterial strains and Streptococcus sobrinus (S.sobrinus) 6715 bacterial strains, respectively through Streptococcus mutans, the Streptococcus sobrinus of BHI-S culture medium separation and Culture and amplification cultivation, and confirm as Streptococcus mutans, Streptococcus sobrinus through evaluation and obtain Streptococcus mutans thalline and bacterial chip, Streptococcus sobrinus thalline and bacterial chip;
B. immunogen preparation process:
B.1 get Streptococcus mutans thalline and bacterial chip 10～70 weight portions, Streptococcus sobrinus thalline and bacterial chip 30～90 weight portions are mixed into bacterium liquid antigen;
B.2 isopyknic bacterium liquid antigen is mixed with isopyknic Fu Shi Freund's complete adjuvant, use the high speed agitator stirring and emulsifying, it is former finally to make Water-In-Oil Fu Shi Freund's complete adjuvant Streptococcus mutans and Streptococcus sobrinus mixed immunity; Every milliliter of Fu Shi Freund's complete adjuvant mixed immunity is former to contain antibacterial 1,000,000,000-8,000,000,000;
B.3 isopyknic mixed bacteria liquid is mixed with isopyknic freund 's incomplete adjuvant, use the high speed agitator stirring and emulsifying, finally make Water-In-Oil freund 's incomplete adjuvant Streptococcus mutans and the Streptococcus sobrinus mixed immunity is former, contain antibacterial 1,000,000,000-8,000,000,000 during every milliliter of freund 's incomplete adjuvant mixed immunity is former;
C. immune step:
Use syringe with the former subcutaneous and intramuscular injection of Fu Shi Freund's complete adjuvant mixed immunity to bird inlay, then every bird inlay injection 0.5ml-3.0ml immunogen uses the subcutaneous and intramuscular injection of the former 0.5ml-3.0ml of freund 's incomplete adjuvant mixed immunity to bird inlay week about.Behind the booster immunization secondary, after this every re-using the subcutaneous and intramuscular injection of the former 0.5ml-3.0ml of freund 's incomplete adjuvant mixed immunity ten weeks to bird inlay booster immunization five times, altogether immunity eight times of front and back;
D. the preparation of Streptococcus mutans and Streptococcus sobrinus mixing specific IgY extract:
Collect for the first time rear the 28th day immune hen egg of immunity of immunity, the ethanol of use 75% carries out the egg surface sterilization, open eggshell and get egg yolk, remove membrane of yolk and get egg yolk liquid, with egg yolk liquid and the distilled water ratio adding distil water of 1:10 by volume, fully stir, and use glacial acetic acid to regulate acid-base value to pH5.30-5.50, then-20 ℃ of cold storage of placement were taken out and are thawed in 3 days afterwards, get supernatant, with concentrated 20 times of egg yolk liquid, regulate PH to 7.0-8.0 with 1N NaOH with the gel filtration system, put-20 ℃ of preservations.
3. preparation method as claimed in claim 2, it is characterized in that, behind described step D, increase by an IgY purification step, the saturation that specifically Streptococcus mutans and Streptococcus sobrinus mixing specific IgY extract is added sulphuric acid amine to 55%, fully stir, room temperature left standstill 2 hours, then high speed centrifugation is 10 minutes, get precipitation, to precipitate and use distilled water diluting to the substance accumulated amount, add again the saturation of sulphuric acid amine to 33%, fully stir, room temperature left standstill 2 hours, then high speed centrifugation is 10 minutes, gets precipitation, with the bag filter of packing into after the distilled water diluting dissolving, through the hollow fiber filter desalination, namely get Streptococcus mutans and Streptococcus sobrinus mixing specific IgY antibody preparation sterling with distilled water.
4. preparation method as claimed in claim 2 is characterized in that, described step D replaces with following methods: collect for the first time rear the 28th day immune hen egg of immunity of immunity, with 75% alcohol disinfecting egg shell, smash eggshell, leave and take egg yolk.Behind 10 times of dilutions of deionized water egg yolk, regulate PH to 5.30-5.50. with glacial acetic acid and anhydrous sodium acetate, get supernatant after 4 ℃ of sedimentations, add 2% chitosan and sodium alginate, 2% calcium chloride and sodium polyphosphate flocculating sedimentation are got supernatant again and are removed unnecessary deionized water with the ultrafiltration of gel filtration system, with concentrated about 20 times of egg yolk liquid, regulate PH to 7.0-8.0 with the 1N sodium hydroxide, put-20 ℃ of preservations.
5. contain the toothpaste that is used for caries prevention and gingivitis and halitosis thereof that Streptococcus mutans and Streptococcus sobrinus mixing specific IgY antibody preparation, resisting porphyromonas gingivalis and Fusobacterium nucleatum mix the specific IgY antibody preparation, include by Streptococcus mutans and Streptococcus sobrinus mixing specific IgY antibody preparation, resisting porphyromonas gingivalis and Fusobacterium nucleatum and mix the anti-pathogenic bacterium preparation that the specific IgY antibody preparation consists of, described anti-pathogenic bacterium preparation is comprised of the materials in percentage by mass that accounts for the toothpaste gross mass:
Resisting porphyromonas gingivalis, Fusobacterium nucleatum mix specific IgY antibody and Streptococcus mutans, Streptococcus sobrinus mixing specific IgY antibody are pressed the mixed preparation 0.1-3.0% of 1:1 mass ratio, hibitane 0.01-2.0%, glycerol 1.0-30.0%, polyvinylpyrrolidone 0.1-2.0%, and wherein hibitane, glycerol, polyvinylpyrrolidone consist of the protective agent of resisting porphyromonas gingivalis and Fusobacterium nucleatum mixing specific IgY antibody preparation, Streptococcus mutans and Streptococcus sobrinus mixing specific IgY antibody preparation.
6. toothpaste as claimed in claim 5, it is characterized in that, also comprise the toothpaste unguentum, described toothpaste unguentum is comprised of the materials in percentage by mass that accounts for the toothpaste gross mass: Ka Bopuer 0.1～0.5%, sodium carboxymethyl cellulose 0.5～1.3%, carrageenan 0.2～0.5%, xanthan gum 0.3～0.7%, glycerol 5.0～20.0%, sorbitol 5.0～70.0%, aspartame 0.1～0.5%; Saccharin sodium 0.1～0.3%, PEG-6000.5～5.0%, PEG-12000.5～5.0%, cocamido propyl betaine 0.1-2.0%, essence 0.8～2.0%, SiO
21.0～30%, the deionized water surplus.
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| 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 |
| CN110478267A (en) * | 2019-09-27 | 2019-11-22 | 中合泰克(南京)生物科技有限公司 | A kind of efficient anti-caries children's toothpaste and preparation method thereof |
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