CN101791405B - Method for preparing yolk immunoglobulin vaccine for resisting porphyromonas gingivalis - Google Patents
Method for preparing yolk immunoglobulin vaccine for resisting porphyromonas gingivalis Download PDFInfo
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Abstract
The invention relates to a method for preparing a yolk immunoglobulin vaccine for resisting porphyromonas gingivalis, which comprises the following operating steps of: (1) culturing and separating an international standard strain of the porphyromonas gingivalis to obtain the porphyromonas gingivalis; (2) obtaining mycoprotein of the porphyromonas gingivalis; (3) mixing a whole bacterium soluble protein antigen of the porphyromonas gingivalis with a freunds incomplete adjuvant uniformly to prepare an intravenous injection preparation, performing an intravenous injection under a chicken wing of a laying hen, and collecting eggs laid by the laying hen; and (4) extracting yolk immunoglobulin for resisting the porphyromonas gingivalis in the eggs. The method has a low production cost and a high yield because 100 milligrams of the yolk immunoglobulin can be obtained from one egg; and the method is simple, convenient and quick to operate and has the advantages of heat resistance, acid resistance, good stability and the like. Under an acid condition of which the temperature is not over 75 DEG C and the pH is more than 4, the yolk immunoglobulin vaccine can still well maintain the biological activity, can be stored for about 3 months at the normal temperature, and can be stored for 6 to 12 months at 4 DEG C with non-decreased antibody activity.
Description
Technical field
The invention belongs to a kind of method for preparing of biological preparation, be specifically related to the method for preparing of yolk immunoglobulin vaccine for resisting porphyromonas gingivalis.
Background technology
Chronic periodontitis is one of the most common oral disease, is not only to cause one of human principal element of losing tooth, or the risk factor of some systemic diseases.There is report to claim that the probability of patients with periodontitis generation coronary heart disease and apoplexy is respectively 1.4 times and 2.1 times of the normal person of periodontal; Periodontitis can increase pathogenic organisms of respiratory tract in the pharyngeal field planting of patient, and the probability that the reduction of chronic pulmonary infection and pulmonary function takes place is 2 times of the good person of oral hygiene; The anemia of pregnant woman who suffers from the serious symptom periodontitis, the danger that premature labor and low birthweight infant are taken place is 7.5 times of periodontal normal pregnancies; The chronic periodontitis sickness rate obviously increases in the diabetics; Have 30% relevant among the acute or subacute infective endocarditis patient with odontogenic infection; Antibacterial produces a large amount of endotoxins and gets into blood, can bring out or increase the weight of atherosclerosis, causes valvulitis even ischemic heart desease.Tracing it to its cause, possibly be that chronic periodontitis involves a plurality of teeth, and the area of infection is big, and wound is open, and the toxin of antibacterial and generation gets into also field planting at other organs with blood circulation, the pathological changes that causes; Simultaneously because of suffering from odontoseisis, when chewing with tooth to the root tip compressing and microorganism and toxin are expressed in blood vessel and the lymphatic vessel, make the immunoreation of the antibacterial persistent infection stimulation of host in the periodontal tissue.Grain-negative anaerobe---porphyromonas gingivalis (Porphyromonas gingivalis; Pg) and other Grain-negative anaerobe are the main advantage pathogenic bacterium at chronic periodontitis diseased region and movable position; Be one of chronic periodontitis advantage pathogenic bacterium of generally acknowledging, increase the weight of closely related with the generation of periodontitis, the recurrence of treatment back or the state of an illness.
At present, the treatment of periodontitis mainly comprises Primary Care, Drug therapy and periodontal surgery etc.Conventional scaling, scrape and control with flap operation and can only temporarily control bacterial plaque, diminish inflammation, stop pathological changes to increase the weight of, effect is preferably arranged in the moderate periodontitis treatment.But severe periodontitis is owing to the complexity of its periodontal pocket structure causes the curettage instrument tool to get into difficulty, is difficult to thoroughly remove pathogenic bacterium under the gum.Subgingival curettage is had relatively high expectations for doctor's clinical manipulation, and the doctor must pass through and could effectively know after the systematic long-term training and diminish inflammation bacterial plaque.The difficult point that this still need solve in one quite long period basic hospital and the doctor of basic unit.In chronic periodontitis external curing medicine, mainly be antiseptic and antimicrobial sustained-release agent, like hibitane gargarism and minocycline hydrochloride ointment, but this type of medicine is easy to generate bacterial resistance and toxic and side effects, unsuitable life-time service.[1] such as Japan scholar Yokoyama with the plain immune hen of the gingiva behind the purification, the result finds that the plain Yolk immunoglobulin (IgY) of anti-gingiva can obviously suppress the degraded of gingiva element to human epithelial cell, stops cytostromatic destruction; Further clinical studies show Yolk immunoglobulin (IgY) ointment help clean scrape control with root planing after clinical symptoms improve and reduce the quantity [2] of porphyromonas gingivalis.Hamajima [3] uses the Yolk immunoglobulin of porphyromonas gingivalis 381 outer membrane protein of the anti-40kDa that recombinates can effectively suppress the copolymerization collection of Pg bacterium on oral cavity bacterium.But, adopt special yolk immunoglobulin biological preparation treatment chronic periodontitis not see correlational study at home as yet and report with relevant product.As far as penetrating in-house pathogenic bacterium, simple Primary Care is difficult to kill fully, often auxiliary antibiotic therapy.Yet the antibiotic frequent drug administration very easily causes bacterial resistance, and has toxic and side effects.
(immunoglobulin of yolk IgY) is polyclonal antibody in the egg yolk to Yolk immunoglobulin, has heat-resisting, acid and alkali-resistance, preparation cost is low, is easy to outstanding advantages such as commercialization.
Summary of the invention
For the approach of the treatment chronic periodontitis of seeking an effective and safe, the present invention provides a kind of method for preparing of yolk immunoglobulin vaccine for resisting porphyromonas gingivalis.
The present invention utilizes porphyromonas gingivalis to process antigen immune breeding chicken;, its institute prepares resisting porphyromonas gingivalis Yolk immunoglobulin (anti-Pg-IgY) from laying eggs; The research and development high effect nontoxic; Be easy to the Yolk immunoglobulin periodontal pocket biovaccine preparation of standardization preparation, rely on suitability for industrialized production, attempt to open up the new way of biological antibody control periodontitis.
The method for preparing of yolk immunoglobulin vaccine for resisting porphyromonas gingivalis of the present invention comprises following operating procedure:
(1) cultivation of porphyromonas gingivalis and evaluation
It is subsequent use to obtain porphyromonas gingivalis with the separation of porphyromonas gingivalis international standard strain culturing;
(2) obtain the porphyromonas gingivalis tropina
With isolated porphyromonas gingivalis in Medulla Bovis seu Bubali heart immersion (BHI) Sanguis caprae seu ovis fluid medium 37 ℃, 80% nitrogen (N
2), 10% carbon dioxide (CO
2), 10% hydrogen (H
2) increase bacterium anaerobism cultivation 48h, collect antibacterial, ultrasonic shatter broken fully to 90% cell, 4000r/min * 10min is centrifugal, collects supernatant, is the full bacterium soluble protein of porphyromonas gingivalis antigen, and 4 ℃ of preservations of temperature are subsequent use;
(3) laying hen immunity
Select the non-immune healthy Luo Man bird inlay at 5 monthly ages, isolated rearing; Be mixed into Water-In-Oil shape intravenous injection with the full bacterium soluble protein of 1mg/ml porphyromonas gingivalis antigen 1 ml and freund 's incomplete adjuvant 1ml piping and druming, to intravenous injection under the above-mentioned bird inlay chicken wing, every chicken injection volume is 0.5mg; Injecting immune once is total to injecting immune 4 times weekly; Simultaneously, collect the egg of giving birth to every day, 4 ℃ of preservations of temperature;
(4) purification resisting porphyromonas gingivalis Yolk immunoglobulin (anti-Pg-IgY)
With Yolk immunoglobulin purification system test kit (EGG stract
TMIgY Purification System, Promega company, the U.S.) to purify and give birth to the Yolk immunoglobulin of egg every day, said test kit comprises test kit A liquid and test kit B liquid, concrete operations are following:
A, with egg yolk and Ovum Gallus domesticus album separately collects egg yolk, slowly adds the test kit A liquid of 3 times of volumes of egg yolk, stirs 5min, and centrifugal 10000r/min * 15min under 4 ℃ of conditions of refrigerated centrifuger collects supernatant;
B, at the test kit B liquid of middle adding 1/3 volume of supernatant, stir 5min, centrifugal 10000r/min * 15min under 4 ℃ of conditions of refrigerated centrifuger abandons supernatant, the resuspended precipitation process of phosphate buffer (PBS) with aseptic pH value 7.2 obtains precipitate;
C, in precipitate, add the test kit B liquid of 1/3 volume; Stir 5min; Centrifugal 10000r/min * 15min under 4 ℃ of conditions of refrigerated centrifuger abandons supernatant, with the resuspended deposition of phosphate buffer (PBS) of aseptic pH value 7.2; Obtain highly purified resisting porphyromonas gingivalis Yolk immunoglobulin, 4 ℃ of preservations;
The relative molecular mass of resisting porphyromonas gingivalis Yolk immunoglobulin is about 180, and 2 contained subunits i.e. 65～70 heavy chain and 22～30 light chain.Yolk immunoglobulin is activating complement not, and planting system between mammal, that distance takes place is big, and the intersection serological reaction between immunoglobulin can not take place, thereby has avoided the false negative or the false positive results that produce in the immune detection process.Yolk immunoglobulin has the ability of heat-resisting, acidproof and certain antienzyme degraded.Stored 5 years or room temperature held 6 months for 4 ℃, its antibody activity is influenced hardly; It is dissolved in the normal saline and stored 6～7 years in 4 ℃, and antibody activity reduces and is no more than 5%; But after temperature surpassed 75 ℃, degeneration then appearred in Yolk immunoglobulin.When pH value greater than 3 the time, its antibody activity does not have obvious influence, is 5～7 o'clock quite stables at pH value especially; When pH value less than 3 the time, its antibody activity obviously descends.In addition, Yolk immunoglobulin also has the performance that anti-height oozes and the characteristic of anti-multigelation preferably.
With the resisting porphyromonas gingivalis Yolk immunoglobulin is main component, and preparation valid density is the gel of 1mg/ml.When clinical use, be made into the gel film of length x width x thickness=0.2cm * 0.1cm * 0.2mm by the doctor, be placed on the bag end of periodontal pocket and do not make this gel film skid off periodontal pocket, 1 time weekly, totally 4 weeks.
The yolk immunoglobulin vaccine for resisting porphyromonas gingivalis of the inventive method preparation is following than the advantage of currently available vaccines, existing vaccines: the research for the porphyromonas gingivalis immunization therapy concentrates on active immunity and passive immunity two aspects.The preparation major part that is used for the artificial active immunity is processed by pathogenic microorganism, is called as vaccine.The research type of prevention periodontitis vaccine comprises: deactivation killed vaccine, subunit vaccine, polypeptide vaccine, recombinant vaccine etc., these vaccines have been used for zoopery, and have obtained certain success.Have the scholar in zoopery, the full bacterial cell of periodontal pathogenic bacterium, outer film component or synthetic polypeptide to be used for active immunity, the result shows that the development of periodontitis can be blocked through active immunity.Klausen [4] etc. confirm that pili has the immunogenicity of height, and t cell responses increase significantly after the pili immunity also with partially purified porphyromonas gingivalis pili immunized mice.Make up ten minutes because of difficulty because the high-purity vaccine is purified, have many scholars to turn to and carry out the research of passive immunity prevention periodontitis.Artificial immunity has the fast advantage of generation effect, but because the non-autoimmune of this immunity system produces, is prone to be eliminated, so that immunization is held time is shorter.Okuda [5] etc. are with the hemagglutinin immunize rabbit of the porphyromonas gingivalis of purifying; Then the antiserum of the rabbit that obtains is inoculated in repeatedly the periodontal of the hamster that has infected porphyromonas gingivalis, the quantity of observing the porphyromonas gingivalis of finding immunized mice significantly reduces than matched group.
Through subcutaneous injection porphyromonas gingivalis (Pg) proteantigen, hen can produce corresponding specific antibody and can be delivered to the egg yolk from serum, is resisting porphyromonas gingivalis Yolk immunoglobulin (anti-Pg-IgY).It has following several big advantage:
1) immune protein transforms through hen, to people's toxicity reduction greatly.Yolk immunoglobulin is activating complement not, is a kind of very promising, safe antibody vaccine.
2) utilize hen as biomass generator, production cost is low, and output capacity is high; Environmental protection simultaneously, an egg can obtain 100 milligrams Yolk immunoglobulin, is higher than the antibody that other animals produce far away; Thereby the price of vaccine is reduced greatly, for the extensive use of vaccine provides maybe.
3) purify simple and efficiently, and have advantage such as heat-resisting, acidproof, good stability.In temperature is not higher than 75 ℃ and sour environment more than pH4, still can keep its BA well, can store under the room temperature about 3 months, can preserve 6-12 month and antibody activity does not descend for 4 ℃, be fit to long preservation and transportation.
4) operate administration by the doctor, the patient can have good compliance.Easy simple to operate is convenient to carry out in basic unit the preventing and controlling of chronic periodontitis.
Yolk immunoglobulin vaccine for resisting porphyromonas gingivalis of the present invention, high effect nontoxic has no side effect, and is aided with the biological preparation of protein antibody stabilizing agent, will have broad clinical application prospect and industrialization prospect.
Description of drawings
Fig. 1 is technology path figure of the present invention,
Fig. 2 is PAGE (SDS-PAGE) analysis chart before and after resisting porphyromonas gingivalis Yolk immunoglobulin (anti-Pg-IgY) purification,
Fig. 3 is a resisting porphyromonas gingivalis Yolk immunoglobulin absorbance change curve in time behind the antigen immune,
Fig. 4 is the clinical effect figure of resisting porphyromonas gingivalis Yolk immunoglobulin preparation after using for 4 weeks on the animal model,
Fig. 5 is the clinical effectiveness figure of sterile phosphate buffer after using for 4 weeks on the animal model,
Fig. 6 is that resisting porphyromonas gingivalis Yolk immunoglobulin preparation uses 4 week back tissue slice figure on animal model,
Fig. 7 is the tissue slice figure of sterile phosphate buffer after using for 4 weeks on the animal model.
The specific embodiment
Below in conjunction with accompanying drawing, the present invention is done explanation further through embodiment.
Embodiment:
The method for preparing of yolk immunoglobulin vaccine for resisting porphyromonas gingivalis is characterized in that comprising following operating procedure:
(1) cultivation of porphyromonas gingivalis and evaluation
With porphyromonas gingivalis international standard strains A TCC 33277 usefulness sterile phosphate buffer (PBS after PH7.2) 0.2ml fully dissolves, is inoculated in Medulla Bovis seu Bubali heart immersion (BHI) SBA solid medium, 37 ℃, 80% nitrogen (N
2), 10% carbon dioxide (CO
2), 10% hydrogen (H
2) anaerobism cultivated 7～10 days.Observing colonial morphology identifies.Porphyromonas gingivalis has black, convexity, glossy, ganoid colonies typical form, Gram-negative, and antibacterial is the coccobacillus or brevibacterium.It is subsequent use to preserve isolated porphyromonas gingivalis.
(2) obtain the porphyromonas gingivalis tropina
With isolated porphyromonas gingivalis in Medulla Bovis seu Bubali heart immersion (BHI) Sanguis caprae seu ovis fluid medium 37 ℃, 80% nitrogen (N
2), 10% carbon dioxide (CO
2), 10% hydrogen (H
2) increase bacterium anaerobism cultivation 48h, collect antibacterial, ultrasonic shatter broken fully to 90% cell, 4000r/min * 10min is centrifugal, collects supernatant, is the full bacterium soluble protein of porphyromonas gingivalis antigen, and 4 ℃ of preservations are subsequent use.
(3) laying hen immunity
Select totally 8 of the non-immune healthy Luo Man bird inlays at 5 monthly ages, isolated rearing.Be mixed into the Water-In-Oil shape with 1mg/ml porphyromonas gingivalis whole bacterial protein and the piping and druming of 1ml freund 's incomplete adjuvant, to intravenous injection under the hen chicken wing, 0.5mg/ml under every chicken wing.Immunity once more in the same manner weekly, immunity is 4 times altogether.Simultaneously, collect the egg of giving birth to every day, 4 ℃ of preservations.
(4) purification resisting porphyromonas gingivalis Yolk immunoglobulin (anti-Pg-IgY)
With Yolk immunoglobulin purification system test kit (EGG stract
TMIgY Purification System, Promega company, the U.S.) to purify and give birth to the Yolk immunoglobulin of egg every day, this test kit comprises test kit A liquid and test kit B liquid, operates as follows:
A, with egg yolk and Ovum Gallus domesticus album separately scratches the rotten clothing of egg yolk with needle point, collects egg yolk in beaker, claims the net weight (1g egg yolk is equal to the 1ml volume) of egg yolk.The test kit A liquid that slowly adds 3 times of volumes, behind the stirring 5min, centrifugal 10000r/min * 15min under 4 ℃ of conditions of refrigerated centrifuger collects supernatant.
B, measure the weight of supernatant, add the B liquid of 1/3 volume, stir 5min after, centrifugal 10000r/min * 15min under 4 ℃ of conditions of refrigerated centrifuger abandons supernatant, with sterile phosphate buffer (PBS, PH7.2) resuspended deposition.
C, measure the liquid weight after resuspended; The B liquid that adds 1/3 volume; After continuing to stir 5min, centrifugal 10000r/min * 15min under 4 ℃ of conditions of refrigerated centrifuger abandons supernatant; (PBS, PH7.2) resuspended deposition can obtain highly purified yolk immunoglobulin vaccine for resisting porphyromonas gingivalis with the sterile phosphate buffer.Add gentamycin sulfate and make its concentration reach 5 μ g/ml, be i.e. 4 ℃ of preservations to prevent germ contamination.
(5) PAGE of resisting porphyromonas gingivalis Yolk immunoglobulin (anti-Pg-IgY)
(SDS-PAGE) analyze
Preparation PAAG plate carries out the PAGE analysis with the special yolk antibody before and after the purification.Applied sample amount is 10 μ l, and gel is with Coomassie brilliant blue R-250 dyeing behind the electrophoresis, and (see figure 2) is observed in the decolouring back.Visible 2 clear bands on gel slab are respectively relative molecular mass and are 65000 heavy chain and 25000 light chain.Provable thus success from egg, extract specific resisting porphyromonas gingivalis Yolk immunoglobulin (IgY).
(6) indirect enzyme-linked immunosorbent absorbent measuring experiment (ELISA method) detects resisting porphyromonas gingivalis Yolk immunoglobulin (anti-Pg-IgY) titre
Encapsulate 96 orifice plates with porphyromonas gingivalis antigen with 10 μ g/ml, 4 ℃ are spent the night, 0.05% sterile phosphate cleaning mixture (PBS-Tween20) washing of inferior daily pH 7.4 5 times, and 5% calf serum sealing 12h, washing, button is done.With the sterile phosphate buffer with immunity after every Yolk immunoglobulin doubling dilution (beginning from 1:10, totally 8 concentration group) of purifying at a distance from 5 days afterwards add 100 μ l in every hole, hatch 1h for 37 ℃; (HRP-IgG, concentration was 1: 10 to the goat-anti chicken immune globulin of adding horseradish peroxidase-labeled after the washing button was done
5) 100 μ l, hatch 1h for 37 ℃.37 ℃ of lucifuge colour developings of o-phenylenediamine 15min, 2mol/L sulphuric acid cessation reaction reads the A450 value on ELIASA.(PBS PH7.2) is blank to the sterile phosphate buffer.Draw the absorbance change curve.Found behind the initial immunity to begin to produce specific antibody on the 5th day by the curve chart (see figure 3), As time goes on, antibody titer rises gradually, and 50-55d peaks to the immunity back, and tiring reaches 1: 10
5
(7) resisting porphyromonas gingivalis Yolk immunoglobulin (anti-Pg-IgY) bacteriostatic test
A, porphyromonas gingivalis in Medulla Bovis seu Bubali heart immersion (BHI) SBA culture medium after, several colonies typicals of picking are suspended in 37 ℃ of Medulla Bovis seu Bubali heart immersion (BHI) Sanguis caprae seu ovis fluid mediums, 80% nitrogen (N
2), 10% carbon dioxide (CO
2), 10% hydrogen (H
2) anaerobism cultivates 48-72h and increase;
B, (PBS PH7.2) dilutes the resisting porphyromonas gingivalis Yolk immunoglobulin, and its concentration is adjusted into 4.0mg/ml, 2.0mg/ml, 1.0mg/ml, 0.5mg/ml 0.0mg/ml with aseptic phosphate buffer.Get each concentration group resisting porphyromonas gingivalis Yolk immunoglobulin of 100 μ l and add in the 96 aseptic porocyte culture plates, each concentration group is done 3 multiple holes;
(PBS, PH7.2) as positive controls, negative control group and blank control group, allotment concentration is 4.0mg/ml, 2.0mg/ml, 1.0mg/ml, 0.5mg/ml 0.0mg/ml for c, minocycline hydrochloride, calf serum, sterile phosphate buffer.Every group of each concentration is got 100 μ l respectively and is added in the 96 aseptic porocyte culture plates, and every group of each concentration is done 3 multiple holes and compared experiment.
D, get porphyromonas gingivalis bacterium liquid 100 μ l and join in each hole.96 porocyte culture plates are put 37 ℃, 80% nitrogen (N
2), 10% carbon dioxide (CO
2), 10% hydrogen (H
2) anaerobism cultivation 48-72h.
E, taking-up 96 porocyte culture plates, ultraviolet spectrophotometer reads the A450 value, by the fungistatic effect in each hole of absorbance contrast.Experimental result show resisting porphyromonas gingivalis Yolk immunoglobulin (anti-Pg-IgY) to the fungistatic effect of porphyromonas gingivalis slightly inferior to minocycline hydrochloride; Than calf serum and sterile phosphate buffer tangible bacteriostasis is arranged, and have best Mlc at 1--2mg/ml.
F, zoopery: with sterile phosphate buffer (PH7.2) the resisting porphyromonas gingivalis Yolk immunoglobulin is mixed with the antibody medicinal liquid that concentration is 1mg/ml, supplies zoopery to use.
Select 10 of SD rats, artificial preparation chronic periodontitis model.The upper and lower jaw the 1st of inoculation+high-carbonhydrate diet processing rat bilateral is ground one's teeth in sleep in silk thread ligation method+porphyromonas gingivalis gingival sulcus, has waited to occur red swelling of gingiva, has been prone to begin experiment after hemorrhage and the manifestation of periodontitis in various degree such as tartar, periodontal pocket.The concrete grouping as follows:
Experimental group: use resisting porphyromonas gingivalis Yolk immunoglobulin preparation in modeling+periodontal pocket
Matched group: use sterile phosphate buffer (PH7.2) flushing in modeling+periodontal pocket
Model group: modeling
Respectively before treatment, observed and recorded result weekly after the treatment, 4 week the back put to death animals, index is following:
Gross examination of skeletal muscle: comprise the body weight of rat, the general state of rat
Clinical indices: GI, plaque index is visited and is examined bleeding index, loosening degree
Other inspections: imaging examination, histological examination
Through the observed and recorded in 4 weeks, can find that experimental group has tangible curative effect than matched group.The spy after using resisting porphyromonas gingivalis Yolk immunoglobulin preparation of experimental group rat is examined bleeding index and is obviously descended, and plaque index also descends than matched group.The effect of the also provable resisting porphyromonas gingivalis Yolk immunoglobulin of histological examination preparation in the chronic periodontitis treatment, (seeing Fig. 4-7).
List of references
1. La Heman must be congratulated in Hengshan Mountain.The active in vitro study of the plain Yolk immunoglobulin of resisting porphyromonas gingivalis gingiva.The oral microorganism immunity.2007，22(5)：352-355。(l.Yokoyama?K，Sugano?N，Rahman?AK，et?al.Activity?ofanti-Porphyromonas?gingivalis?egg?yolk?antibody?against?gingipains?in?vitro.Oral?MicrobiolImmunol.2007Oct；22(5)：352-355.)
2. the field, island must be congratulated in Hengshan Mountain.The plain Yolk immunoglobulin of resisting porphyromonas gingivalis gingiva is to the influence of periodontitis.China's stomatology, 2007,49 (3): 201-206.(2.Yokoyama?K，Sugano?N，Shimada?T，et?al.Effects?of?eggyolk?antibody?against?porphyromonas?gingivalis?gingipains?in?periodontitis?patients.J?OralSci，2007Sep；49(3)：201-6.)
3. Bin Dao, ball mountain, mud room, hada, Bick difficult to understand.It is active that resisting porphyromonas gingivalis 40-kDa Yolk immunoglobulin suppresses the outer membrane protein copolymerization.The Oral Biology archives; 2007; 52 (7): 697-704. (3.Hamajima S, MaruyamaM, Hijiya T; Hatta H, Abiko Y.Egg yolk-derived immunoglobulin (IgY) against Porphyromonasgingivalis 40-kDa outer membrane protein inhibits coaggregation activity.Arch Oral Biol.2007Jul; 52 (7): 697-704.)
The gram Lawson, Ai Wensi, Rameau this.Rat alveolar bone level after the field planting of gingiva bacteroid and the active research of tropina.The oral microorganism immunity; 1991,6:193-201. (4.Klausen B, Evans RT; Ramamurthy NS; Et al.Periodontal bone level and gingival proteincese activity in gnotobiotic rats immunized withBacteroides gingivalis.Oral Microbiol Immunol, 1991,6:193～201.)
5. field difficult to understand adds rattan, interior rattan.Active and the experiment of passive immunotherapy gingiva bacteroides infection hamster.Dentistry research magazine; 1988; 67:807-811. (5.Okuda K, Kato T, Naito Y.Protective efficacy of active and passiveimmunizations against experimental infection with Bacteroids gingivalis in ligated hamsters.J Dent Res; 1988,67:807～811.)
Claims (1)
1. the method for preparing of yolk immunoglobulin vaccine for resisting porphyromonas gingivalis is characterized in that comprising following operating procedure:
(1) cultivation of porphyromonas gingivalis and evaluation
It is subsequent use to obtain porphyromonas gingivalis with the separation of porphyromonas gingivalis international standard strain culturing;
(2) obtain the porphyromonas gingivalis tropina
With isolated porphyromonas gingivalis in Medulla Bovis seu Bubali heart immersion (BHI) Sanguis caprae seu ovis fluid medium 37 ℃, 80% nitrogen (N
2), 10% carbon dioxide (CO
2), 10% hydrogen (H
2) increase bacterium anaerobism cultivation 48h, collect antibacterial, ultrasonic shatter broken fully to 90% cell, 4000r/min * 10min is centrifugal, collects supernatant, is the full bacterium soluble protein of porphyromonas gingivalis antigen, and 4 ℃ of preservations of temperature are subsequent use;
(3) laying hen immunity
Select the non-immune healthy Luo Man bird inlay at 5 monthly ages, isolated rearing; Be mixed into Water-In-Oil shape intravenous injection with the full bacterium soluble protein of 1mg/ml porphyromonas gingivalis antigen 1 ml and freund 's incomplete adjuvant 1ml piping and druming, to intravenous injection under the above-mentioned bird inlay chicken wing, every chicken injection volume is 0.5mg; Injecting immune once is total to injecting immune 4 times weekly; Simultaneously, collect the egg of giving birth to every day, 4 ℃ of preservations of temperature;
(4) purification resisting porphyromonas gingivalis Yolk immunoglobulin (anti-Pg-IgY)
Purify with Yolk immunoglobulin purification system test kit and to give birth to the Yolk immunoglobulin of egg every day, said test kit comprises test kit A liquid and test kit B liquid, and concrete operations are following:
A, with egg yolk and Ovum Gallus domesticus album separately collects egg yolk, slowly adds the test kit A liquid of 3 times of volumes of egg yolk, stirs 5min, and centrifugal 10000r/min * 15min under 4 ℃ of conditions of refrigerated centrifuger collects supernatant;
B, at the test kit B liquid of middle adding 1/3 volume of supernatant, stir 5min, centrifugal 10000r/min * 15min under 4 ℃ of conditions of refrigerated centrifuger abandons supernatant, the resuspended precipitation process of phosphate buffer (PBS) with aseptic pH value 7.2 obtains precipitate;
C, in precipitate, add the test kit B liquid of 1/3 volume; Stir 5min; Centrifugal 10000r/min * 15min under 4 ℃ of conditions of refrigerated centrifuger abandons supernatant, with the resuspended deposition of phosphate buffer (PBS) of aseptic pH value 7.2; Obtain highly purified resisting porphyromonas gingivalis Yolk immunoglobulin, 4 ℃ of preservations; The relative molecular mass of resisting porphyromonas gingivalis Yolk immunoglobulin is about 180, and 2 contained subunits i.e. 65～70 heavy chain and 22～30 light chain.
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