CN103007278A - Anti-porphyromonas gingivalis and fusobacterium nucleatum compound specific IgY antibody, preparation method and toothpaste thereof - Google Patents
Anti-porphyromonas gingivalis and fusobacterium nucleatum compound specific IgY antibody, preparation method and toothpaste thereof Download PDFInfo
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- CN103007278A CN103007278A CN2012103965835A CN201210396583A CN103007278A CN 103007278 A CN103007278 A CN 103007278A CN 2012103965835 A CN2012103965835 A CN 2012103965835A CN 201210396583 A CN201210396583 A CN 201210396583A CN 103007278 A CN103007278 A CN 103007278A
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- fusobacterium nucleatum
- porphyromonas gingivalis
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- igy
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- Cosmetics (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention discloses an anti-porphyromonas gingivalis and fusobacterium nucleatum compound specific IgY preparation. According to the preparation, 10-70 parts by weight of porphyromonas gingivalis thalli and thalli fragments, and 30-90 parts by weight of fusobacterium nucleatum thalli and thalli fragments are compounded into an antigen to immune an egg laying hen. The invention also discloses the specific preparation method of the anti-porphyromonas gingivalis and fusobacterium nucleatum compound specific IgY preparation, and toothpaste containing the anti-porphyromonas gingivalis and fusobacterium nucleatum compound specific IgY preparation. By the IgY toothpaste which is used for treating and preventing gingivitis and ozostomia has obvious effects in preventing and treating the gingivitis and the ozostomia.
Description
Technical field
The present invention relates to the application of IgY antibody and preparation method thereof and this IgY preparation, be particularly related to compound specific IgY preparation of resisting porphyromonas gingivalis and Fusobacterium nucleatum and preparation method thereof, and with the prevention of this IgY antibody preparation toothpaste by these two kinds of microbial gingivitis and halitosis thereof.
Background technology
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 induce Expression of Macrophages L-1 α, L-1 β, L-6, TNF α to cause 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 thereof 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, and prevention pulmonary fibrosis patients charrin's disease alleviates the microbial stomachache effect of helicobacter pylorus remarkable, and has no side effect and addiction.
Find in the IgY of the numerous species that we study, some periodontal disease the experiment proved that trend and the curative effect that does not have or do not have significantly to suppress periodontal disease and halitosis thereof 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 thereof 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 thereof, 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 thereof; Because our prior unpredictable which bacterium in the middle of this is the principal element that causes periodontal disease and halitosis thereof, 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 thereof to determine which bacterium is the principal element that causes periodontal disease and halitosis thereof.Thereby medicine or the health product of the standby anti-periodontal disease of guidance system and halitosis thereof.
Present prior art does not have relevant prompting guidance technology personnel to find that resisting porphyromonas gingivalis, Fusobacterium nucleatum composite 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 thereof, 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 thereof, learns that in advance resisting porphyromonas gingivalis, Fusobacterium nucleatum composite 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 resisting porphyromonas gingivalis and the compound specific IgY preparation of Fusobacterium nucleatum.
Two of technical problem to be solved by this invention is to provide the preparation method of above-mentioned resisting porphyromonas gingivalis and the compound specific IgY preparation of Fusobacterium nucleatum.
Three of technical problem to be solved by this invention be to provide contain above-mentioned resisting porphyromonas gingivalis and the compound specific IgY preparation of Fusobacterium nucleatum be used for prevention by the toothpaste of these two kinds of microbial periodontal diseases and halitosis thereof.
Resisting porphyromonas gingivalis and the compound specific IgY preparation of Fusobacterium nucleatum as first aspect present invention, it is characterized in that, be combined into the antigen immune bird inlay and prepare by the Fusobacterium nucleatum thalline of the porphyromonas gingivalis thalline of 10～70 weight portions and bacterial chip and 30～90 weight portions and bacterial chip.
Preparation method as resisting porphyromonas gingivalis and the compound specific IgY preparation of Fusobacterium nucleatum of second aspect present invention is characterized in that, comprises the steps:
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 combined into bacterium liquid antigen;
B.2 isopyknic bacterium liquid antigen and isopyknic Fu Shi Freund's complete adjuvant is compound, use the high speed agitator stirring and emulsifying, finally make Water-In-Oil Fu Shi Freund's complete adjuvant porphyromonas gingivalis and Fusobacterium nucleatum complex immunogen; Every milliliter of Fu Shi Freund's complete adjuvant complex immunogen contains antibacterial 1,000,000,000-8,000,000,000;
B.3 isopyknic composite bacteria liquid and isopyknic freund 's incomplete adjuvant is compound, use the high speed agitator stirring and emulsifying, finally make Water-In-Oil freund 's incomplete adjuvant porphyromonas gingivalis and Fusobacterium nucleatum complex immunogen, contain antibacterial 1,000,000,000-8,000,000,000 in every milliliter of freund 's incomplete adjuvant complex immunogen;
C. immune step:
Use syringe with Fu Shi Freund's complete adjuvant complex immunogen subcutaneous and intramuscular injection to bird inlay, then every bird inlay injection 0.5ml-3.0ml immunogen uses the subcutaneous and intramuscular injection of freund 's incomplete adjuvant complex immunogen 0.5ml-3.0ml to bird inlay week about.Behind the booster immunization secondary, after this every re-using the subcutaneous and intramuscular injection of freund 's incomplete adjuvant complex immunogen 0.5ml-3.0ml ten weeks to bird inlay booster immunization five times, altogether immunity eight times of front and back;
D. the preparation of the compound specific IgY extract of resisting porphyromonas gingivalis and Fusobacterium nucleatum:
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 preparation method of the compound specific IgY preparation of above-mentioned resisting porphyromonas gingivalis and Fusobacterium nucleatum, behind described step D, increase by an IgY purification step, the saturation that specifically resisting porphyromonas gingivalis and the compound specific IgY extract of Fusobacterium nucleatum 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 the compound specific IgY preparation of resisting porphyromonas gingivalis and Fusobacterium nucleatum 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.
The toothpaste that is used for prevention of gingivitis and halitosis thereof as the above-mentioned resisting porphyromonas gingivalis of the usefulness of third aspect present invention and the compound specific IgY preparation preparation of Fusobacterium nucleatum, include anti-Periodontal Pathogens IgY preparation, described anti-Periodontal Pathogens IgY preparation is comprised of the materials in percentage by mass that accounts for the toothpaste gross mass:
The compound specific IgY preparation 0.01～0.5% of resisting porphyromonas gingivalis and Fusobacterium nucleatum, hibitane 0.01-2.0%, glycerol 1.0-30.0%, polyvinylpyrrolidone 0.1-2.0%, wherein hibitane, glycerol, polyvinylpyrrolidone consist of resisting porphyromonas gingivalis and the compound specific IgY preparation of Fusobacterium nucleatum protective agent.
A preferred embodiment at the toothpaste for prevention of gingivitis and halitosis thereof 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%, SiO21.0～30%, deionized water surplus.
The present invention is used for the treatment of with the toothpaste of prevention of periodontal disease and halitosis prevention and treatment periodontal disease and halitosis thereof is had a significant effect.
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 the compound specific IgY preparation of resisting porphyromonas gingivalis and Fusobacterium nucleatum:
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 combined into bacterium liquid antigen;
B.2 isopyknic bacterium liquid antigen and isopyknic Fu Shi Freund's complete adjuvant is compound, use the high speed agitator stirring and emulsifying, finally make Water-In-Oil Fu Shi Freund's complete adjuvant porphyromonas gingivalis and Fusobacterium nucleatum complex immunogen; Every milliliter of Fu Shi Freund's complete adjuvant complex immunogen contains antibacterial 1,000,000,000-8,000,000,000;
B.3 isopyknic composite bacteria liquid and isopyknic freund 's incomplete adjuvant is compound, use the high speed agitator stirring and emulsifying, finally make Water-In-Oil freund 's incomplete adjuvant porphyromonas gingivalis and Fusobacterium nucleatum complex immunogen, contain antibacterial 1,000,000,000-8,000,000,000 in every milliliter of freund 's incomplete adjuvant complex immunogen;
C. immune step:
Use syringe with Fu Shi Freund's complete adjuvant complex immunogen subcutaneous and intramuscular injection to bird inlay, then every bird inlay injection 0.5ml-3.0ml immunogen uses the subcutaneous and intramuscular injection of freund 's incomplete adjuvant complex immunogen 0.5ml-3.0ml to bird inlay week about.Behind the booster immunization secondary, after this every re-using the subcutaneous and intramuscular injection of freund 's incomplete adjuvant complex immunogen 0.5ml-3.0ml ten weeks to bird inlay booster immunization five times, altogether immunity eight times of front and back;
D. the preparation of the compound specific IgY extract of resisting porphyromonas gingivalis and Fusobacterium nucleatum:
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:
The saturation that resisting porphyromonas gingivalis and the compound specific IgY extract of Fusobacterium nucleatum 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 the direct sterling of the compound specific IgY antibody of resisting porphyromonas gingivalis and Fusobacterium nucleatum with distilled water.
The compound specific IgY preparation of resisting porphyromonas gingivalis and Fusobacterium nucleatum sterling is measured through the ELISA method, tiring of specific IgY antibody in the compound specific IgY sterling of resisting porphyromonas gingivalis and Fusobacterium nucleatum in every milligram of albumen is 1:32000/mg albumen, perhaps claim in the compound specific IgY sterling of resisting porphyromonas gingivalis and Fusobacterium nucleatum, 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 2:
The preparation of the compound specific IgY preparation of resisting porphyromonas gingivalis and Fusobacterium nucleatum:
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 combined into bacterium liquid antigen;
B.2 isopyknic bacterium liquid antigen and isopyknic Fu Shi Freund's complete adjuvant is compound, use the high speed agitator stirring and emulsifying, finally make Water-In-Oil Fu Shi Freund's complete adjuvant porphyromonas gingivalis and Fusobacterium nucleatum complex immunogen; Every milliliter of Fu Shi Freund's complete adjuvant complex immunogen contains antibacterial 1,000,000,000-8,000,000,000;
B.3 isopyknic composite bacteria liquid and isopyknic freund 's incomplete adjuvant is compound, use the high speed agitator stirring and emulsifying, finally make Water-In-Oil freund 's incomplete adjuvant porphyromonas gingivalis and Fusobacterium nucleatum complex immunogen, contain antibacterial 1,000,000,000-8,000,000,000 in every milliliter of freund 's incomplete adjuvant complex immunogen;
C. immune step:
Use syringe with Fu Shi Freund's complete adjuvant complex immunogen subcutaneous and intramuscular injection to bird inlay, then every bird inlay injection 0.5ml-3.0ml immunogen uses the subcutaneous and intramuscular injection of freund 's incomplete adjuvant complex immunogen 0.5ml-3.0ml to bird inlay week about.Behind the booster immunization secondary, after this every re-using the subcutaneous and intramuscular injection of freund 's incomplete adjuvant complex immunogen 0.5ml-3.0ml ten weeks to bird inlay booster immunization five times, altogether immunity eight times of front and back;
D. the preparation of the compound specific IgY extract of resisting porphyromonas gingivalis and Fusobacterium nucleatum:
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 resisting porphyromonas gingivalis and the compound specific IgY extract of Fusobacterium nucleatum 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 the direct sterling of the compound specific IgY antibody of resisting porphyromonas gingivalis and Fusobacterium nucleatum with distilled water.
The compound specific IgY preparation of resisting porphyromonas gingivalis and Fusobacterium nucleatum sterling is measured through the ELISA method, tiring of specific IgY antibody in the compound specific IgY sterling of resisting porphyromonas gingivalis and Fusobacterium nucleatum in every milligram of albumen is 1:32000/mg albumen, perhaps claim in the compound specific IgY sterling of resisting porphyromonas gingivalis and Fusobacterium nucleatum, 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 3:
Get separately porphyromonas gingivalis thalline and bacterial chip as bacterium liquid; Prepare separately resisting porphyromonas gingivalis specific IgY extract.All the other preparations are with reference to embodiment 1.
Embodiment 4:
Get separately the Fusobacterium nucleatum bacterial chip as bacterium liquid; Prepare separately anti-Fusobacterium nucleatum specific IgY extract.All the other preparations are with reference to embodiment 1.
Experimental example 1:
The immunogen of two kinds of bacteria combination such as porphyromonas gingivalis, Fusobacterium nucleatum is by method immunity bird inlay of the present invention, get for the first time rear 30 days immune egg of immunity, and prepare the extract of resisting porphyromonas gingivalis and the compound specific IgY of Fusobacterium nucleatum by method of the present invention, use the ELISA method and measure resisting porphyromonas gingivalis and the compound specific IgY of Fusobacterium nucleatum in conjunction with the biological value of synantigen (seeing Table 1) not:
Tiring of table 1 resisting porphyromonas gingivalis and anti-Fusobacterium nucleatum IgY antibody extract
Can be clear that according to above-mentioned experimental result:
⑴. 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-two kinds of 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 complex antigen of resisting porphyromonas gingivalis and Fusobacterium nucleatum composite IgY antibody and porphyromonas gingivalis, Fusobacterium nucleatum can both react, its superposition of having tired;
⑷. by above-mentioned experiment as seen, the compound specific IgY of a kind of resisting porphyromonas gingivalis and Fusobacterium nucleatum can be treated by above-mentioned bacterial periodontal disease, halitosis etc.;
⑸. also can treat by any bacterial periodontal disease in the above-mentioned antibacterial, halitosis etc. with a kind of resisting porphyromonas gingivalis and the compound specific IgY of Fusobacterium nucleatum simultaneously;
⑹. can also expand to thus by with above-mentioned antibacterial the bacterial periodontal disease, halitosis etc. of common antigenic determinant being arranged, can use the compound specific IgY treatment of resisting porphyromonas gingivalis of the present invention and Fusobacterium nucleatum.
Experimental example 2:
Porphyromonas gingivalis, Fusobacterium nucleatum complex immunogen are by 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 resisting porphyromonas gingivalis and the compound specific IgY preparation of Fusobacterium nucleatum sterling by method of the present invention, use the ELISA method and measure resisting porphyromonas gingivalis and the compound specific IgY of Fusobacterium nucleatum in conjunction with the biological value of synantigen (seeing Table 2) not:
Tiring of table 2 resisting porphyromonas gingivalis and anti-Fusobacterium nucleatum IgY antibody sterling
Can be clear that according to above-mentioned experimental result:
⑴. the sterling of preparing resisting porphyromonas gingivalis and the compound specific IgY of Fusobacterium nucleatum by method of the present invention is than preparing specific binding porphyromonas gingivalis and the Fusobacterium nucleatum complex antigen of the extract of resisting porphyromonas gingivalis and the compound specific IgY of Fusobacterium nucleatum 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;
⑵. porphyromonas gingivalis and Fusobacterium nucleatum complex immunogen immunity bird inlay can produce the respectively specific IgY antibody of anti-two kinds of 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 the large antibacterial of immunizing dose produces tiring of IgY antibody high yet, this is relevant with the immunogenicity of antigen;
⑷. the complex antigen of two kinds of bacterium such as the compound specific IgY of resisting porphyromonas gingivalis and Fusobacterium nucleatum and porphyromonas gingivalis, Fusobacterium nucleatum can both react, and tiring of total specific IgY antibody also increased its superposition of having tired;
⑸. by above-mentioned experiment as seen, the compound specific IgY of a kind of resisting porphyromonas gingivalis and Fusobacterium nucleatum can be treated by above-mentioned bacterial periodontal disease and halitosis thereof etc.;
⑹. also can treat by any bacterial periodontal disease in the above-mentioned antibacterial, halitosis etc. with a kind of resisting porphyromonas gingivalis and the compound specific IgY of Fusobacterium nucleatum simultaneously;
⑺. can also expand to thus by with above-mentioned antibacterial the bacterial periodontal disease, halitosis etc. of common antigenic determinant being arranged, can use the compound specific IgY prevention of resisting porphyromonas gingivalis of the present invention and Fusobacterium nucleatum and treatment.
Experimental example 3:
The activity of the IgY toothpaste of resist gingivitis and halitosis thereof is tired
With the IgY toothpaste of the resist gingivitis after the protective agent and halitosis thereof (instant RT) with do not add tire result such as the table 3 of the IgY toothpaste (immediately RT) of protectant resist gingivitis and halitosis thereof:
Table 3
The IgY toothpaste with the resist gingivitis after the protective agent and halitosis thereof that obvious this patent provides can effectively be protected the activity of IgY in toothpaste of resist gingivitis and halitosis thereof.
Experimental example 4:
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 5.0,1.0,0.1g/L.
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 A490 with microplate reader, reads 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 4.
The resisting porphyromonas gingivalis IgY of table 4. purification is to the suppression ratio of porphyromonas gingivalis
As can be seen from Table 4, 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 A490 with microplate reader, reads 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 anti-Fusobacterium nucleatum IgY of purification sees Table 5 to the suppression ratio of Fusobacterium nucleatum
Table 5
As can be seen from Table 5, anti-Fusobacterium nucleatum IgY has remarkable effect to suppressing the Fusobacterium nucleatum rate of growth.
Claims (6)
1. the compound specific IgY preparation of resisting porphyromonas gingivalis and Fusobacterium nucleatum, it is characterized in that, be combined into the antigen immune bird inlay and prepare by the Fusobacterium nucleatum thalline of the porphyromonas gingivalis thalline of 10～70 weight portions and bacterial chip and 30～90 weight portions and bacterial chip.
2. the preparation method of the compound specific IgY preparation of resisting porphyromonas gingivalis claimed in claim 1 and Fusobacterium nucleatum is characterized in that, comprises the steps:
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 combined into bacterium liquid antigen;
B.2 isopyknic bacterium liquid antigen and isopyknic Fu Shi Freund's complete adjuvant is compound, use the high speed agitator stirring and emulsifying, finally make Water-In-Oil Fu Shi Freund's complete adjuvant porphyromonas gingivalis and Fusobacterium nucleatum complex immunogen; Every milliliter of Fu Shi Freund's complete adjuvant complex immunogen contains antibacterial 1,000,000,000-8,000,000,000;
B.3 isopyknic composite bacteria liquid and isopyknic freund 's incomplete adjuvant is compound, use the high speed agitator stirring and emulsifying, finally make Water-In-Oil freund 's incomplete adjuvant porphyromonas gingivalis and Fusobacterium nucleatum complex immunogen, contain antibacterial 1,000,000,000-8,000,000,000 in every milliliter of freund 's incomplete adjuvant complex immunogen;
C. immune step:
Use syringe with Fu Shi Freund's complete adjuvant complex immunogen subcutaneous and intramuscular injection to bird inlay, then every bird inlay injection 0.5ml-3.0ml immunogen uses the subcutaneous and intramuscular injection of freund 's incomplete adjuvant complex immunogen 0.5ml-3.0ml to bird inlay week about.Behind the booster immunization secondary, after this every re-using the subcutaneous and intramuscular injection of freund 's incomplete adjuvant complex immunogen 0.5ml-3.0ml ten weeks to bird inlay booster immunization five times, altogether immunity eight times of front and back;
D. the preparation of the compound specific IgY extract of resisting porphyromonas gingivalis and Fusobacterium nucleatum:
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 resisting porphyromonas gingivalis and the compound specific IgY extract of Fusobacterium nucleatum 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 the compound specific IgY preparation of resisting porphyromonas gingivalis and Fusobacterium nucleatum sterling with distilled water.
4. preparation method as claimed in claim 2 or claim 3 is characterized in that, described step D replaces with following methods: collect after for the first time immunity of immunity the 28th day immune hen egg, 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. the toothpaste for preparing with resisting porphyromonas gingivalis claimed in claim 1 and the compound specific IgY preparation of Fusobacterium nucleatum, it is characterized in that, include anti-Periodontal Pathogens IgY preparation, described anti-Periodontal Pathogens IgY preparation is comprised of the materials in percentage by mass that accounts for the toothpaste gross mass:
The compound specific IgY preparation 0.01～0.5% of resisting porphyromonas gingivalis and Fusobacterium nucleatum, hibitane 0.01-2.0%, glycerol 1.0-30.0%, polyvinylpyrrolidone 0.1-2.0%, wherein hibitane, glycerol, polyvinylpyrrolidone consist of resisting porphyromonas gingivalis and the compound specific IgY preparation of Fusobacterium nucleatum protective agent.
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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| CN106380518A (en) * | 2016-08-30 | 2017-02-08 | 安徽安科生物工程（集团）股份有限公司 | Extraction technology of egg yolk immunoglobulins |
| CN106380518B (en) * | 2016-08-30 | 2019-07-12 | 安徽安科生物工程（集团）股份有限公司 | The extraction process of Yolk immunoglobulin |
| CN106467572A (en) * | 2016-08-31 | 2017-03-01 | 广东工业大学 | One species specificity composite yolk antibody and its preparation method and application |
| CN108728388A (en) * | 2018-07-24 | 2018-11-02 | 上海美加净日化有限公司 | Porphyromonas gingivalis, resisting porphyromonas gingivalis specific IgY preparation and Compound formula of toothpaste |
| CN110128541A (en) * | 2019-04-16 | 2019-08-16 | 广东工业大学 | A kind of preparation method and application of yolk antibody for preventing and treating chronic periodontitis |
| CN110237251A (en) * | 2019-06-24 | 2019-09-17 | 桂林三金药业股份有限公司 | A kind of compound special yolk antibody oral spray and preparation method thereof |
| CN110357955A (en) * | 2019-06-24 | 2019-10-22 | 桂林三金药业股份有限公司 | A kind of compound special yolk antibody and its preparation method and application |
| CN110237251B (en) * | 2019-06-24 | 2023-05-12 | 桂林三金药业股份有限公司 | Composite specificity egg yolk antibody oral spray and preparation method thereof |
| CN110357955B (en) * | 2019-06-24 | 2023-05-12 | 桂林三金药业股份有限公司 | Composite specificity egg yolk antibody and preparation method and application thereof |
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