CN110357955A - A kind of compound special yolk antibody and its preparation method and application - Google Patents
A kind of compound special yolk antibody and its preparation method and application Download PDFInfo
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- CN110357955A CN110357955A CN201910548867.3A CN201910548867A CN110357955A CN 110357955 A CN110357955 A CN 110357955A CN 201910548867 A CN201910548867 A CN 201910548867A CN 110357955 A CN110357955 A CN 110357955A
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Abstract
The invention discloses a kind of compound special yolk antibody and its preparation method and application, the preparation method of compound special yolk antibody includes: (1) culture and extraction porphyromonas gingivalis and actinobacillus actinomycetem comitans；(2) it prepares immunogene: taking porphyromonas gingivalis and actinobacillus actinomycetem comitans thallus respectively, plastc ring is proportionally mixed to get after ultrasonication, prepare immunizing antigen using plastc ring；(3) bird inlay is immunized: the hen chick that selection did not produced egg starts to raise, and is immunized using immunizing antigen within 35-55 days before laying eggs；(4) resisting porphyromonas gingivalis and the compound special yolk antibody of actinobacillus actinomycetem comitans are isolated and purified.Preparation method of the invention extends the duration that compound special yolk antibody maintains high-titer, improves yield, purity and bioactivity, reduces production cost, and Yolk antibody technology is enable preferably to be applied to industrialization production.
Description
Technical field
The invention belongs to immunological technique fields, specifically, being related to a kind of compound special yolk antibody and its preparation
Methods and applications, more particularly to resisting porphyromonas gingivalis and actinobacillus actinomycetem comitans special yolk antibody and its system
Preparation Method and application.
Background technique
According to statistics, the mouth diseases illness rate such as China's gum, periodontal disease accounts for 70% of population or more.Mouth disease and mouth
Pathogenetic bacteria in chamber is closely related, porphyromonas gingivalis (Porphyromonas gingivalis) and with unwrapping wire unwrapping wire bar
Bacterium (Aggregatibacter actinomycetemcomitans) is regarded as causing two kinds of bacteriums of periodontitis.Gum
Detection of Porphyromonas is the main putative pathogen of the periodontosis such as marginal gingivitis, chronic periodontitis, aggressive periodontitis, pili
Human gingival epithelial cells and gum can be invaded into fiber in conjunction with the glyceraldehyde-3-phosphate dehydrogenase on mouth epithelial cells surface
Cell escapes immune clearance.Actinobacillus actinomycetem comitans express a variety of virulence factor such as CagE albumen, leucotoxin, lipopolysaccharides
Toxin etc. is expanded with cell-lethal, these virulence factors are related with the pathogenesis of periodontosis.Actinobacillus actinomycetem comitans are being invaded
It plays an important role in property periodontitis (juvenile periodontitis), can also cause the complication such as endocarditis, meningitis, osteomyelitis.
The mouth diseases such as periodontitis are prevented and treated, sets about from oral cavity pathogen, effectively inhibits these pathogenic bacteria, are a kind of
More direct and feasible method.Currently, on the market for periodontitis, gingivitis some products mainly contain Chinese herbal medicine at
Point, the product of antibiotic composition or chemical bacteriostatic agent ingredient, but the slow effect of this kind of product and often do not have for pathogenic bacteria
Specific aim and it will lead to the side effects such as drug resistance, function and effect are undesirable.Therefore, studying and produce has with strong points, work
It is of great significance with the product of the features such as significant, without side-effects for preventing and treating related oral disease.
Yolk antibody is also known as Yolk immunoglobulin, is unique immunoglobulin present in birds yolk, answers at present
With it is more be chicken yolk antibody, be that can produce corresponding antibody in chicken serum after bird inlay is immunized with antigen, hen energy again
The antibody in serum is effectively transferred in yolk in a manner of laying eggs.Different antigen is immune will to be generated for the antigen
Special yolk antibody.Yolk antibody meets modem animal safeguard rule without blood sampling, and the amount of antibody of generation is big, specificity
By force, property is stablized, and has no toxic side effect, and not only has and can also have growth to press down the growth of antigen bacteria with the ability of antigen binding
Production is used.
It is a kind of effective prevention and treatment that research and production, which have the Yolk antibody of inhibition oral cavity pathogen effect,
Related oral disease method.The preparation process of Yolk antibody is not sufficiently stable at present, and obtained Yolk antibody potency height is uneven, is produced
Measure low, purity is low, and bioactivity is poor, and cost is relatively high.
In view of this present invention is specifically proposed.
Summary of the invention
The technical problem to be solved in the present invention is that it is anti-to overcome the deficiencies of the prior art and provide a kind of compound special yolk
Body and its preparation method and application, the compound special yolk antibody can specificity inhibition actinobacillus actinomycetem comitans and gum porphins
Quinoline monad, specificity is good, potency is high, safe without toxic side effect；Preparation method of the invention can extend Yolk antibody maintenance
The duration of high-titer, while the yield and purity of Yolk antibody are improved, and make Yolk antibody bioactivity with higher, it drops
The production cost of low Yolk antibody enables Yolk antibody technology to be preferably applied to industrialization production.
In order to solve the above technical problems, the present invention is using the basic conception of technical solution:
The first object of the present invention is to provide a kind of preparation method of compound special yolk antibody, comprising:
(1) cultivate and extract porphyromonas gingivalis and actinobacillus actinomycetem comitans；
(2) prepare immunogene: take porphyromonas gingivalis and actinobacillus actinomycetem comitans thallus respectively, after ultrasonication according to
Ratio is mixed to get plastc ring, prepares immunizing antigen using plastc ring；
(3) bird inlay is immunized: the hen chick that selection did not produced egg starts to raise, and utilizes within 35-55 days before laying eggs
Immunizing antigen is immunized；
(4) resisting porphyromonas gingivalis and the compound special yolk antibody of actinobacillus actinomycetem comitans are isolated and purified.
The hen chick that preparation method selection of the invention did not produced egg starts to raise, and utilizes within 35-55 days before laying eggs
Immunizing antigen is immunized, and can be extended Yolk antibody and be maintained the duration of high-titer, while improving the yield of Yolk antibody and pure
Degree, and make Yolk antibody bioactivity with higher, the production cost of Yolk antibody is reduced, Yolk antibody technology is enable
Preferably it is applied to industrialization production.
Further embodiment, in step (2), after ultrasonication, by 10-30 parts of porphyromonas gingivalis bacterial strain fragment and companion
70-90 parts of bacterial strain fragment of the unwrapping wire Actinobacillus plastc rings for being mixed to get two kinds of bacterium.
The present invention is using 10-30 parts and actinobacillus actinomycetem comitans 70-90 parts of porphyromonas gingivalis monad bacterial chip
Complex configuration prepares immunizing antigen at plastc ring.Due to the immunogenicity of porphyromonas gingivalis is relatively strong and with putting
The immunogenicity of line Actinobacillus is relatively weak, therefore, can be in immune thorn with antigen made of such ratio complex configuration
Difference is formed on swashing, the Yolk antibody for obtaining it can form preferable complementation in potency, so that the specific ovum newly obtained
Yellow antibody has stronger bioactivity.
Further embodiment in step (4), isolates and purifies resisting porphyromonas gingivalis and the compound spy of actinobacillus actinomycetem comitans
The step of anisotropic Yolk antibody, successively includes: water extraction method coarse extraction Yolk antibody, affinity chromatography essence extraction Yolk antibody, surpasses
Liquid and freeze-drying are changed in filter concentration, obtain the compound special yolk antibody of purifying.
The method of further embodiment, water extraction method coarse extraction Yolk antibody includes:
(1) egg for collecting immune hen, with 75% alcohol disinfecting egg shell, broken shell collects yolk, measures and record
Egg yolk liquid volume V0。
(2) taking mass fraction is the aqueous sorbitol solution that 5-10%, pH are 5.0-5.5, with 10-15V0Volume be added
It into the egg yolk liquid of collection, stirs, stands, centrifugation goes to precipitate, and aqueous is spare.
Present invention improves over the extraction processes of Yolk antibody, and D-sorbite is added on the basis of water extraction as stabilization
Agent reduces its potency loss in aqueous solution.
Further embodiment, the method that affinity chromatography essence extracts Yolk antibody include:
(1) affinity column is loaded with the affine filler of 2- mercaptopyridine and agarose gel coupling, is used after the completion of filling
The 20mM of the potassium sulfate containing 0.5M, the sodium phosphate buffer of pH 7.5 balance affinity column；
(2) by the water-soluble solution loading of the Yolk antibody slightly raised, after completion of the sample, with the potassium sulfate containing 0.5M
The sodium phosphate buffer of 20mM, pH 7.5 balances affinity column；
(3) it is eluted with the sodium phosphate buffer of 20mM, pH 7.5, obtains the aqueous solution for the Yolk antibody that essence proposes, amount
The aqueous solution volume V for taking and recording1。
The present invention extracts compound special yolk antibody using the method essence of affinity chromatography, avoids addition high molecular polymerization
Object is to the activity influence of Yolk antibody, and the aqueous solution after affinity chromatography is the aqueous solution containing Yolk antibody, without height
Molecularly Imprinted Polymer combines, and subsequent processing does not need to carry out processing of saltouing, and reduces process flow, and avoid high concentration salinity pair
The active influence of Yolk antibody.
Further embodiment, ultrafiltration concentration changes liquid and the method for freeze-drying includes:
(1) ultrafiltration being carried out with aqueous solution of the ultrafiltration membrane packet to the Yolk antibody that essence raises and changing liquid, changing liquor is 10V1
The PBS buffer solution of volume, is changed after the completion of liquid, and solution is concentrated with ultrafiltration membrane packet；
(2) the Yolk antibody solution after concentration is dispensed into disposable sterilized culture dish, it is cold is sequentially placed into -80 DEG C of refrigerators
Freeze, be freeze-dried in vacuum freeze drier, the sample after taking out freeze-drying obtains Yolk antibody sterling；
Preferably, the molecule interception of the ultrafiltration membrane packet used is 50KDa；
Preferably, -80 DEG C of refrigerators are put into freeze 2-5 hours, it is small to be put into freeze-drying 12-15 in vacuum freeze drier
When.
The present invention carries out changing liquid using the substitution dialysis of ultrafiltration membrane packet, it is more thorough to change liquid, and ultrafiltration membrane packet can be anti-to yolk
Body aqueous solution is concentrated, and treating capacity when reducing freeze-drying improves treatment effeciency.
Further embodiment, in step (2), obtain after plastc ring by plastc ring and Freund's complete adjuvant according to
The volume ratio of 1:1 carries out compound, stirring and emulsifying, and Water-In-Oil Freund's complete adjuvant antigen is made；
Plastc ring and incomplete Freund's adjuvant are subjected to compound, stirring and emulsifying according to the volume ratio of 1:1, oily packet is made
Water incomplete Freund's adjuvant antigen.
Further embodiment, carrying out immune method to the hen for not producing egg in step (3) includes:
Initial immunity uses Water-In-Oil Freund's complete adjuvant antigen, immunizing dose 0.8mL, using subcutaneous multi-point injection and
The mode of intramuscular injection is immune；First time booster immunization is carried out after initial immunity 10 days, booster immunization uses Water-In-Oil Freund not
Freund's complete adjuvant antigen, immunizing dose 0.8mL is later primary every 10 days progress booster immunizations, at least booster immunization eight times, female
Chicken collects egg after laying eggs.
It present invention improves over immunization protocol, has chosen the hen chick that do not lay eggs and is raised, 35-55 days just before laying eggs
Start to be immunized, while shortening the immunization interval time, increase immunizing dose, immunostimulation persistently is carried out to hen, improves immune side
After case, obtained Yolk antibody maintains the duration of high-titer to greatly increase, and the Yolk antibody that conventional preparation techniques obtain is available
High-titer part can only choose 15 days Yolk antibodies, and the available high-titer portion of Yolk antibody that preparation process of the present invention obtains
It point can be used to choose 60 days Yolk antibodies, available yield greatly increases.
Specifically, the preparation method of compound special yolk antibody provided by the invention includes:
A. it cultivates and extracts porphyromonas gingivalis and actinobacillus actinomycetem comitans:
It fetches from porphyromonas gingivalis (P.g) the ATCC33277 bacterial strain of Guangdong Province's Culture Collection and companion
Unwrapping wire Actinobacillus (A.a) ATCC29523 bacterial strain reflects with blood agar plate culture medium amplification cultivation and by Gram's staining
It is fixed.Porphyromonas gingivalis (P.g) ATCC33277 strain culturing 3 days, the training of actinobacillus actinomycetem comitans (A.a) ATCC29523 bacterial strain
It supports 1 day, with oese scraping bacterium in PBS buffer solution.Repeated centrifugation is extracted three times, and it is required for collecting bacterial sediment
Microorganism.
B. immunogene is prepared:
B1. porphyromonas gingivalis and actinobacillus actinomycetem comitans thallus are taken respectively, adjust colony density to 3- with sterile PBS
5×109Then CFU/mL carries out ultrasonication, then bacteria suspension is hanged according to the Mixed Microbes that corresponding proportion is mixed to get two kinds of bacterium
Liquid；
B2. plastc ring and Freund's complete adjuvant is compound according to the volume ratio progress of 1:1, it is stirred using high speed agitator
Emulsification is mixed, Water-In-Oil Freund's complete adjuvant antigen is finally made.
B3. plastc ring and incomplete Freund's adjuvant is compound according to the volume ratio progress of 1:1, use high speed agitator
Stirring and emulsifying is finally made Water-In-Oil incomplete Freund's adjuvant antigen.
C. bird inlay is immunized:
The hen chick that selection did not produced egg starts to raise, and 35-55 days before laying eggs start to be immunized；Initial immunity
Using Freund's complete adjuvant antigen, immunizing dose 0.8mL is immunized by the way of subcutaneous multi-point injection and intramuscular injection；Just
First time booster immunization is carried out after secondary immune 10 days, booster immunization uses incomplete Freund's adjuvant antigen, and immunizing dose is
0.8mL, later primary every 10 days progress booster immunizations, booster immunization eight times altogether, hen collects egg simultaneously after laying eggs in time
Purification.
D. resisting porphyromonas gingivalis and the compound special yolk antibody of actinobacillus actinomycetem comitans are isolated and purified:
D1: improvement water extraction method coarse extraction Yolk antibody:
(1) immune hen egg is collected, with 75% alcohol disinfecting egg shell, broken shell collects yolk, measures and record egg
Yellow liquor volume V0。
(2) with the aqueous sorbitol solution of distilled water configuration 5-10%, its pH to 5.0-5.5 is adjusted with dilute hydrochloric acid, then
With 10-15V0Volume be added in the egg yolk liquid of collection, by mixed liquor on magnetic stirring apparatus with lower revolving speed stir 30-
It 60 minutes, is then placed in 4 DEG C of conditions and stands overnight.It stands later with centrifuge with 10000rpm revolving speed centrifugation 20 minutes, discards
Precipitating, water intaking soluble solution are spare.
D2: affinity chromatography essence extracts Yolk antibody:
(1) affinity column is loaded with the affine filler of 2- mercaptopyridine and agarose gel coupling, is used after the completion of filling
The 20mM sodium phosphate buffer (pH 7.5) of the potassium sulfate containing 0.5M balances affinity column.
(2) water-soluble solution obtained in D1 is subjected to loading with 5mL/ minutes flow velocitys, after completion of the sample, then with 5
The 20mM sodium phosphate buffer (pH 7.5) of the potassium sulfate containing 0.5M of column volume balances affinity column, keeps Yolk antibody sufficiently affine
It is adsorbed on the affine filler in pillar.
(3) it is eluted with the 20mM sodium phosphate buffer (pH 7.5) of 10 column volumes, makes to be adsorbed on affinity column
Yolk antibody is sufficiently eluted, and obtained solution is the aqueous solution of the higher Yolk antibody containing purity, is measured and is remembered
Record obtained liquor capacity V1。
D3: ultrafiltration concentration changes liquid and freeze-drying
(1) the ultrafiltration membrane packet for being 50KDa with molecule interception is to solution V obtained in D21It carries out ultrafiltration and changes liquid, it is molten to change liquid
Liquid is 10V1The PBS buffer solution of volume, is changed after the completion of liquid, continues that solution is slowly concentrated to 20-50mL with ultrafiltration membrane packet.
(2) the Yolk antibody solution after concentration is dispensed into disposable sterilized culture dish, is first put into -80 DEG C of refrigerator frosts
It 2-5 hours, is then placed in vacuum freeze drier and is freeze-dried 12-15 hours, the sample after taking out freeze-drying, as ovum
Yellow antibody sterling, is denoted as MSterling。
The second object of the present invention is to provide preparation method described in a kind of above-mentioned scheme or assembled scheme and is prepared
Compound special yolk antibody.
The special yolk antibody of resisting porphyromonas gingivalis and actinobacillus actinomycetem comitans provided by the invention can be special
Property combination actinobacillus actinomycetem comitans and porphyromonas gingivalis cell wall, inhibit the formation of bacterial biof iotalm, have specificity
Good, potency height, advantage without side-effects, have especially strong specific aim to porphyromonas gingivalis and actinobacillus actinomycetem comitans.
The third object of the present invention is to provide a kind of special yolk antibody as described above in preparation prevention and/or treatment
The drug of mouth disease and/or the application in daily necessity；
Preferably, special yolk antibody is in preparation prevention and/or treatment periodontitis, the drug of gingivitis and/or life
Application in articles.
Porphyromonas gingivalis is a kind of main pathogenic bacteria that can cause multiple oral disease, actinobacillus actinomycetem comitans
It is to cause a kind of pathogenic bacteria of aggressive periodontitis, therefore a kind of resisting porphyromonas gingivalis provided by the invention and put with unwrapping wire
The compound special yolk antibody of line bar bacterium can be used as periodontitis, tooth with the mouth diseases effect such as periodontitis, gingivitis is prevented
The drug of oulitis or the effective component of daily necessity are used in drug and daily necessity production.
After adopting the above technical scheme, compared with the prior art, the invention has the following beneficial effects:
1. the present invention is using 10-30 parts of porphyromonas gingivalis monad bacterial chip and actinobacillus actinomycetem comitans 70-90
Part complex configuration prepares immunizing antigen at plastc ring.Due to the immunogenicity of porphyromonas gingivalis is relatively strong and companion
The immunogenicity of unwrapping wire Actinobacillus is relatively weak, therefore, can be immune with antigen made of such ratio complex configuration
Difference is formed in stimulation, the Yolk antibody for obtaining it can form preferable complementation in potency, so that the specificity newly obtained
Yolk antibody has stronger bacteriostasis.
2. having chosen the hen chick that do not lay eggs present invention improves over immunization protocol and being raised, 35-55 days before laying eggs
It begins to be immunized, while shortening the immunization interval time, increase immunizing dose, immunostimulation persistently is carried out to hen, improve immune
After scheme, obtained Yolk antibody maintains the duration of high-titer to greatly increase, and the Yolk antibody that conventional preparation techniques obtain is available
High-titer part can only choose 15 days Yolk antibodies, and the available high-titer of Yolk antibody that preparation process of the present invention obtains
Part can be used to choose 60 days Yolk antibodies, and available yield greatly increases.
3. adding D-sorbite on the basis of water extraction as stabilization present invention improves over the extraction process of Yolk antibody
Agent reduces its potency loss in aqueous solution.The present invention extracts Yolk antibody using the method essence of affinity chromatography, avoids and adds
Enter high molecular polymer to the activity influence of Yolk antibody, the aqueous solution after affinity chromatography is to contain the water-soluble of Yolk antibody
Liquid is combined without high molecular polymer, and subsequent processing does not need to carry out processing of saltouing, and reduces process flow, and avoid
High concentration salinity is on the active influence of Yolk antibody.The present invention carries out changing liquid using the substitution dialysis of ultrafiltration membrane packet, and it is more thorough to change liquid
Bottom, and Yolk antibody aqueous solution can be concentrated in ultrafiltration membrane packet, and treating capacity when reducing freeze-drying improves treatment effeciency.
4. the present invention verifies the Yolk antibody of acquisition by biomembrane Inhibition test and transmission electron microscope.Transmission
Electron microscope experiment the result shows that: compound special yolk antibody obtained can be specific be integrated to porphyromonas gingivalis and
On the cell wall of actinobacillus actinomycetem comitans, to influence the growth and breeding of two kinds of bacteriums.Biomembrane inhibition assay result shows: institute
Obtain compound special yolk antibody can inhibit porphyromonas gingivalis and actinobacillus actinomycetem comitans biomembrane formation and
Significant effect, biomembrane is the premise to form plaque, therefore the compound special yolk antibody that the present invention obtains can be one
Determine the generation for reducing plaque in degree, inhibits the development of plaque.In conclusion the compound special yolk that the present invention obtains
Antibody has very high bioactivity, can be used as the effective component of pre- preventing parodontitis, the drug of periodontosis or daily necessity.
A specific embodiment of the invention is described in further detail with reference to the accompanying drawing.
Detailed description of the invention
Attached drawing is as a part of the invention, and for providing further understanding of the invention, of the invention is schematic
Examples and descriptions thereof are used to explain the present invention, but does not constitute an undue limitation on the present invention.Obviously, the accompanying drawings in the following description
Only some embodiments to those skilled in the art without creative efforts, can be with
Other accompanying drawings can also be obtained according to these attached drawings.In the accompanying drawings:
Fig. 1 is the resisting porphyromonas gingivalis of the embodiment of the present invention 1 and comparative example 1 and the compound spy of actinobacillus actinomycetem comitans
Anisotropic Yolk antibody electrophoretogram；Band 1.1 is the Yolk antibody crude extract of embodiment 1 in figure；Band 1.2 is embodiment 1
The pure extract of Yolk antibody；Band 2.1 is the Yolk antibody crude extract of comparative example 1；Band 2.2 is that the yolk of comparative example 1 is anti-
The pure extract of body；Band M is Marker；
Fig. 2 is the embodiment of the present invention 1 and the resisting porphyromonas gingivalis of comparative example 1 and the compound spy of actinobacillus actinomycetem comitans
Anisotropic Yolk antibody potency curve graph；
Fig. 3 is the compound special yolk antibody inhibition companion of resisting porphyromonas gingivalis of the present invention and actinobacillus actinomycetem comitans
The effect picture of unwrapping wire Actinobacillus biofilm formation；
Fig. 4 is the compound special yolk antibody inhibition tooth of resisting porphyromonas gingivalis of the present invention and actinobacillus actinomycetem comitans
The effect picture of gum Detection of Porphyromonas biofilm formation；
Fig. 5 is the compound special yolk antibody inhibition companion of resisting porphyromonas gingivalis of the present invention and actinobacillus actinomycetem comitans
The effect picture of unwrapping wire Actinobacillus cell wall；In figure biggish black shade be actinobacillus actinomycetem comitans, tiny black small
Grain is Yolk antibody；
Fig. 6 is the compound special yolk antibody inhibition tooth of resisting porphyromonas gingivalis of the present invention and actinobacillus actinomycetem comitans
The effect picture of gum Detection of Porphyromonas cell wall；In figure biggish black shade be porphyromonas gingivalis, tiny black small
Grain is Yolk antibody.
It should be noted that these attached drawings and verbal description are not intended to the design model limiting the invention in any way
It encloses, but illustrates idea of the invention by referring to specific embodiments for those skilled in the art.
Specific embodiment
In order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below in conjunction with the embodiment of the present invention
In attached drawing, the technical solution in embodiment is clearly and completely described, the following examples are intended to illustrate the invention, but
It is not intended to limit the scope of the invention.
Embodiment 1
The preparation of resisting porphyromonas gingivalis and the compound special yolk antibody of actinobacillus actinomycetem comitans:
A. it cultivates and extracts porphyromonas gingivalis and actinobacillus actinomycetem comitans:
It fetches from porphyromonas gingivalis (P.g) the ATCC33277 bacterial strain of Guangdong Province's Culture Collection and companion
Unwrapping wire Actinobacillus (A.a) ATCC29523 bacterial strain reflects with blood agar plate culture medium amplification cultivation and by Gram's staining
It is fixed.Porphyromonas gingivalis (P.g) ATCC33277 strain culturing 3 days, the training of actinobacillus actinomycetem comitans (A.a) ATCC29523 bacterial strain
It supports 1 day, with oese scraping bacterium in PBS buffer solution.Repeated centrifugation is extracted three times, and it is required for collecting bacterial sediment
Microorganism.
B. immunogene is prepared:
B1. porphyromonas gingivalis and actinobacillus actinomycetem comitans thallus are taken respectively, adjust colony density to 3- with sterile PBS
5×109CFU/mL stops 4s cyclic ultrasonic breaking 25min then on Ultrasonic Cell Disruptor with 25Hz, ultrasonic 3s, then by bacteria suspension
According to porphyromonas gingivalis: actinobacillus actinomycetem comitans=30:70 ratio is mixed to get the plastc ring of two kinds of bacterium；
B2. plastc ring and Freund's complete adjuvant is compound according to the volume ratio progress of 1:1, it is stirred using high speed agitator
Emulsification is mixed, Water-In-Oil Freund's complete adjuvant antigen is finally made.
B3. plastc ring and incomplete Freund's adjuvant is compound according to the volume ratio progress of 1:1.2, use high-speed stirred
Device stirring and emulsifying is finally made Water-In-Oil incomplete Freund's adjuvant antigen.
C. bird inlay is immunized:
It chooses the hen chick that do not lay eggs to start to raise, 45 days before laying eggs start to be immunized；Initial immunity is using not
Family name's Freund's complete adjuvant antigen, immunizing dose 0.8mL are immunized by the way of subcutaneous multi-point injection and intramuscular injection；Initial immunity
After 10 days carry out first time booster immunization, booster immunization use incomplete Freund's adjuvant antigen, immunizing dose 0.8mL, after
Primary every 10 days progress booster immunizations, booster immunization eight times altogether, hen collects egg in time and purifies after laying eggs.
D. resisting porphyromonas gingivalis and the compound special yolk antibody of actinobacillus actinomycetem comitans are isolated and purified:
D1: improvement water extraction method coarse extraction Yolk antibody:
(1) immune hen egg is collected, with 75% alcohol disinfecting egg shell, broken shell collects yolk, measures and record egg
Yellow liquor volume V0。
(2) with distilled water configuration 8% aqueous sorbitol solution, adjust its pH to 5.0-5.5 with dilute hydrochloric acid, then with
12V0Volume be added in the egg yolk liquid of collection, by mixed liquor on magnetic stirring apparatus with lower revolving speed stirring 40 minutes,
4 DEG C of conditions are then placed in stand overnight.It stands later with centrifuge with 10000rpm revolving speed centrifugation 20 minutes, discards precipitating, take
Water-soluble solution is spare.
D2: affinity chromatography essence extracts Yolk antibody:
(1) affinity column is loaded with the affine filler of 2- mercaptopyridine and agarose gel coupling, is used after the completion of filling
The 20mM sodium phosphate buffer (pH 7.5) of the potassium sulfate containing 0.5M balances affinity column.
(2) water-soluble solution obtained in D1 is subjected to loading with 5mL/ minutes flow velocitys, after completion of the sample, then with 5
The 20mM sodium phosphate buffer (pH 7.5) of the potassium sulfate containing 0.5M of column volume balances affinity column, keeps Yolk antibody sufficiently affine
It is adsorbed on the affine filler in pillar.
(3) it is eluted with the 20mM sodium phosphate buffer (pH 7.5) of 10 column volumes, makes to be adsorbed on affinity column
Yolk antibody is sufficiently eluted, and obtained solution is the aqueous solution of the higher Yolk antibody containing purity, is measured and is remembered
Record obtained liquor capacity V1。
D3: ultrafiltration concentration changes liquid and freeze-drying
(1) the ultrafiltration membrane packet for being 50KDa with molecule interception is to solution V obtained in D21It carries out ultrafiltration and changes liquid, it is molten to change liquid
Liquid is 10V1The PBS buffer solution of volume, is changed after the completion of liquid, continues that solution is slowly concentrated to 50mL with ultrafiltration membrane packet.
(2) the Yolk antibody solution after concentration is dispensed into disposable sterilized culture dish, is first put into -80 DEG C of refrigerator frosts
It 2 hours, is then placed in vacuum freeze drier and is freeze-dried 12 hours, the sample after taking out freeze-drying, as yolk are anti-
Body sterling, is denoted as MSterling。
After obtaining compound special yolk antibody, agarose gel electrophoresis experiment is carried out to it respectively and bioactivity is real
It tests, examines the purity and potency of compound special yolk antibody, and count its yield.
Embodiment 2
The preparation of resisting porphyromonas gingivalis and the compound special yolk antibody of actinobacillus actinomycetem comitans:
A. it cultivates and extracts porphyromonas gingivalis and actinobacillus actinomycetem comitans:
It fetches from porphyromonas gingivalis (P.g) the ATCC33277 bacterial strain of Guangdong Province's Culture Collection and companion
Unwrapping wire Actinobacillus (A.a) ATCC29523 bacterial strain reflects with blood agar plate culture medium amplification cultivation and by Gram's staining
It is fixed.Porphyromonas gingivalis (P.g) ATCC33277 strain culturing 3 days, the training of actinobacillus actinomycetem comitans (A.a) ATCC29523 bacterial strain
It supports 1 day, with oese scraping bacterium in PBS buffer solution.Repeated centrifugation is extracted three times, and it is required for collecting bacterial sediment
Microorganism.
B. immunogene is prepared:
B1. porphyromonas gingivalis and actinobacillus actinomycetem comitans thallus are taken respectively, adjust colony density to 3- with sterile PBS
5×109CFU/mL stops 4s cyclic ultrasonic breaking 25min then on Ultrasonic Cell Disruptor with 25Hz, ultrasonic 3s, then by bacteria suspension
According to porphyromonas gingivalis: actinobacillus actinomycetem comitans=10:90 ratio is mixed to get the plastc ring of two kinds of bacterium；
B2. plastc ring and Freund's complete adjuvant is compound according to the volume ratio progress of 1:1, it is stirred using high speed agitator
Emulsification is mixed, Water-In-Oil Freund's complete adjuvant antigen is finally made.
B3. plastc ring and incomplete Freund's adjuvant is compound according to the volume ratio progress of 1:1, use high speed agitator
Stirring and emulsifying is finally made Water-In-Oil incomplete Freund's adjuvant antigen.
C. bird inlay is immunized:
It chooses the hen chick that do not lay eggs to start to raise, 35 days before laying eggs start to be immunized；Initial immunity is using not
Family name's Freund's complete adjuvant antigen, immunizing dose 0.8mL are immunized by the way of subcutaneous multi-point injection and intramuscular injection；Initial immunity
After 10 days carry out first time booster immunization, booster immunization use incomplete Freund's adjuvant antigen, immunizing dose 0.8mL, after
Primary every 10 days progress booster immunizations, booster immunization eight times altogether, hen collects egg in time and purifies after laying eggs.
D. resisting porphyromonas gingivalis and the compound special yolk antibody of actinobacillus actinomycetem comitans are isolated and purified:
D1: improvement water extraction method coarse extraction Yolk antibody:
(1) immune hen egg is collected, with 75% alcohol disinfecting egg shell, broken shell collects yolk, measures and record egg
Yellow liquor volume V0。
(2) with distilled water configuration 8% aqueous sorbitol solution, adjust its pH to 5.0-5.5 with dilute hydrochloric acid, then with
10V0Volume be added in the egg yolk liquid of collection, by mixed liquor on magnetic stirring apparatus with lower revolving speed stirring 30 minutes,
4 DEG C of conditions are then placed in stand overnight.It stands later with centrifuge with 10000rpm revolving speed centrifugation 20 minutes, discards precipitating, take
Water-soluble solution is spare.
D2: affinity chromatography essence extracts Yolk antibody:
(1) affinity column is loaded with the affine filler of 2- mercaptopyridine and agarose gel coupling, is used after the completion of filling
The 20mM sodium phosphate buffer (pH 7.5) of the potassium sulfate containing 0.5M balances affinity column.
(2) water-soluble solution obtained in D1 is subjected to loading with 5mL/ minutes flow velocitys, after completion of the sample, then with 5
The 20mM sodium phosphate buffer (pH 7.5) of the potassium sulfate containing 0.5M of column volume balances affinity column, keeps Yolk antibody sufficiently affine
It is adsorbed on the affine filler in pillar.
(3) it is eluted with the 20mM sodium phosphate buffer (pH 7.5) of 10 column volumes, makes to be adsorbed on affinity column
Yolk antibody is sufficiently eluted, and obtained solution is the aqueous solution of the higher Yolk antibody containing purity, is measured and is remembered
Record obtained liquor capacity V1。
D3: ultrafiltration concentration changes liquid and freeze-drying
(1) the ultrafiltration membrane packet for being 50KDa with molecule interception is to solution V obtained in D21It carries out ultrafiltration and changes liquid, it is molten to change liquid
Liquid is 10V1The PBS buffer solution of volume, is changed after the completion of liquid, continues that solution is slowly concentrated to 35mL with ultrafiltration membrane packet.
(2) the Yolk antibody solution after concentration is dispensed into disposable sterilized culture dish, is first put into -80 DEG C of refrigerator frosts
It 3 hours, is then placed in vacuum freeze drier and is freeze-dried 13 hours, the sample after taking out freeze-drying, as yolk are anti-
Body sterling, is denoted as MSterling。
After obtaining compound special yolk antibody, agarose gel electrophoresis experiment is carried out to it respectively and bioactivity is real
It tests, examines the purity and potency of Yolk antibody, and count its yield.
Embodiment 3
The preparation of resisting porphyromonas gingivalis and the compound special yolk antibody of actinobacillus actinomycetem comitans:
A. it cultivates and extracts porphyromonas gingivalis and actinobacillus actinomycetem comitans:
It fetches from porphyromonas gingivalis (P.g) the ATCC33277 bacterial strain of Guangdong Province's Culture Collection and companion
Unwrapping wire Actinobacillus (A.a) ATCC29523 bacterial strain reflects with blood agar plate culture medium amplification cultivation and by Gram's staining
It is fixed.Porphyromonas gingivalis (P.g) ATCC33277 strain culturing 3 days, the training of actinobacillus actinomycetem comitans (A.a) ATCC29523 bacterial strain
It supports 1 day, with oese scraping bacterium in PBS buffer solution.Repeated centrifugation is extracted three times, and it is required for collecting bacterial sediment
Microorganism.
B. immunogene is prepared:
B1. porphyromonas gingivalis and actinobacillus actinomycetem comitans thallus are taken respectively, adjust colony density to 3- with sterile PBS
5×109CFU/mL stops 4s cyclic ultrasonic breaking 30min then on Ultrasonic Cell Disruptor with 25Hz, ultrasonic 3s, then by bacteria suspension
According to porphyromonas gingivalis: actinobacillus actinomycetem comitans=25:75 ratio is mixed to get the plastc ring of two kinds of bacterium；
B2. plastc ring and Freund's complete adjuvant is compound according to the volume ratio progress of 1:1, it is stirred using high speed agitator
Emulsification is mixed, Water-In-Oil Freund's complete adjuvant antigen is finally made.
B3. plastc ring and incomplete Freund's adjuvant is compound according to the volume ratio progress of 1:1.3, use high-speed stirred
Device stirring and emulsifying is finally made Water-In-Oil incomplete Freund's adjuvant antigen.
C. bird inlay is immunized:
It chooses the hen chick that do not lay eggs to start to raise, 55 days before laying eggs start to be immunized；Initial immunity is using not
Family name's Freund's complete adjuvant antigen, immunizing dose 0.8mL are immunized by the way of subcutaneous multi-point injection and intramuscular injection；Initial immunity
After 10 days carry out first time booster immunization, booster immunization use incomplete Freund's adjuvant antigen, immunizing dose 0.8mL, after
Primary every 10 days progress booster immunizations, booster immunization nine times altogether, hen collects egg in time and purifies after laying eggs.
D. resisting porphyromonas gingivalis and the compound special yolk antibody of actinobacillus actinomycetem comitans are isolated and purified:
D1: improvement water extraction method coarse extraction Yolk antibody:
(1) immune hen egg is collected, with 75% alcohol disinfecting egg shell, broken shell collects yolk, measures and record egg
Yellow liquor volume V0。
(2) with distilled water configuration 8% aqueous sorbitol solution, adjust its pH to 5.0-5.5 with dilute hydrochloric acid, then with
15V0Volume be added in the egg yolk liquid of collection, by mixed liquor on magnetic stirring apparatus with lower revolving speed stirring 60 minutes,
4 DEG C of conditions are then placed in stand overnight.It stands later with centrifuge with 10000rpm revolving speed centrifugation 20 minutes, discards precipitating, take
Water-soluble solution is spare.
D2: affinity chromatography essence extracts Yolk antibody:
(1) affinity column is loaded with the affine filler of 2- mercaptopyridine and agarose gel coupling, is used after the completion of filling
The 20mM sodium phosphate buffer (pH 7.5) of the potassium sulfate containing 0.5M balances affinity column.
(2) water-soluble solution obtained in D1 is subjected to loading with 5mL/ minutes flow velocitys, after completion of the sample, then with 5
The 20mM sodium phosphate buffer (pH 7.5) of the potassium sulfate containing 0.5M of column volume balances affinity column, keeps Yolk antibody sufficiently affine
It is adsorbed on the affine filler in pillar.
(3) it is eluted with the 20mM sodium phosphate buffer (pH 7.5) of 10 column volumes, makes to be adsorbed on affinity column
Yolk antibody is sufficiently eluted, and obtained solution is the aqueous solution of the higher Yolk antibody containing purity, is measured and is remembered
Record obtained liquor capacity V1。
D3: ultrafiltration concentration changes liquid and freeze-drying
(1) the ultrafiltration membrane packet for being 50KDa with molecule interception is to solution V obtained in D21It carries out ultrafiltration and changes liquid, it is molten to change liquid
Liquid is 10V1The PBS buffer solution of volume, is changed after the completion of liquid, continues that solution is slowly concentrated to 20mL with ultrafiltration membrane packet.
(2) the Yolk antibody solution after concentration is dispensed into disposable sterilized culture dish, is first put into -80 DEG C of refrigerator frosts
It 5 hours, is then placed in vacuum freeze drier and is freeze-dried 12 hours, the sample after taking out freeze-drying, as yolk are anti-
Body sterling, is denoted as MSterling。
After obtaining Yolk antibody, agarose gel electrophoresis experiment and bioactivity experiment are carried out to it respectively, examines yolk
The purity and potency of antibody, and count its yield.
Comparative example 1
Resisting porphyromonas gingivalis is prepared using conventional preparation techniques and the compound special yolk of actinobacillus actinomycetem comitans is anti-
Body, method and step are as follows:
A. it cultivates and extracts porphyromonas gingivalis and actinobacillus actinomycetem comitans:
A1. bacterial species: porphyromonas gingivalis (Porphyromonas gingivalis, P.g) ATCC33277 bacterial strain
With actinobacillus actinomycetem comitans (Aggregatibacter actinomycetemcomitans, A.a) ATCC29523 bacterial strain；
A2. porphyromonas gingivalis (P.g) ATCC33277 bacterial strain from Guangdong Province's Culture Collection is fetched,
Amplification 3 days is cultivated in 37 DEG C, anaerobic gas generation bag with blood agar plate culture medium；It fetches from Guangdong Province's Microbiological Culture Collection
The actinobacillus actinomycetem comitans ATCC29523 bacterial strain of the heart, with blood agar plate culture medium at 37 DEG C, 5%CO2Under the conditions of cultivate amplification
1 day；Bacterium is scraped down from culture medium after culture amplification, obtains thallus after 4 DEG C, 8000rpm pelleted by centrifugation 15min.
B. immunogene is prepared:
B1. taking porphyromonas gingivalis PBS solution to adjust concentration makes it in ultraviolet specrophotometer absorbance OD595When
Reading is 1.1, and taking actinobacillus actinomycetem comitans PBS solution to adjust concentration makes it in ultraviolet specrophotometer absorbance OD595When read
Number is 1.2；With 25Hz, ultrasonic 3s on Ultrasonic Cell Disruptor, stop 4s cyclic ultrasonic breaking 25min, then bacteria suspension will be planted according to body
Product carries out the plastc ring for being mixed to get two kinds of bacterium than 1:1；
B2. plastc ring and Freund's complete adjuvant is compound according to the volume ratio progress of 1:1, it is stirred using high speed agitator
Emulsification is mixed, Water-In-Oil Freund's complete adjuvant antigen is finally made；
B3. plastc ring and incomplete Freund's adjuvant is compound according to the volume ratio progress of 1:1, use high speed agitator
Stirring and emulsifying is finally made Water-In-Oil incomplete Freund's adjuvant antigen.
C. bird inlay is immune:
Choose 20 week old bird inlays；Initial immunity uses Freund's complete adjuvant antigen, using exempting from for subcutaneous multi-point injection
Epidemic disease mode injects 0.6mL to every hen；First time booster immunization is carried out after initial immunity 15 days, uses incomplete Freund's adjuvant
Antigen injects 0.6mL to every hen using the immunization ways of subcutaneous multi-point injection, later every 15 days progress booster immunizations one
Secondary, immunity eggs, and timely separation and Extraction are collected in booster immunization five times altogether since first time booster immunization.
D. prepared by Yolk antibody crude extract:
Immune rear hen egg is collected, with 75% alcohol disinfecting egg shell, broken shell collects yolk and simultaneously records egg yolk fluid
Product V0；3V is added0The PBS buffer solution of the PEG6000 containing 3.0% (w/v) of volume mixes, after stirring 40min, in 2~4 DEG C of items
12h is stood under part；At 4 DEG C, 10000rpm pelleted by centrifugation 20mim takes supernatant, and filtering obtains filtrate V1；Slowly add in filtrate
Enter PEG6000, make PEG6000 final concentration of 12% (w/v), stirs 40min；At 4 DEG C, 10000rpm pelleted by centrifugation 20mim, abandon
Supernatant must precipitate, as Yolk antibody crude product MCrude product。
E. Yolk antibody purification step:
Specifically by resisting porphyromonas gingivalis and the compound special yolk antibody coarse extraction V of actinobacillus actinomycetem comitans0
The PBS solution of volume dissolves, and adds V0The saturated ammonium sulfate of volume makes the saturation degree 50% of ammonium sulfate in the solution, mixes well
40min is stirred, stands 12h under the conditions of 4 DEG C；At 4 DEG C, 10000rpm pelleted by centrifugation 20mim takes precipitating；By precipitating 1/5V0
The PBS solution of volume dissolves, and adds 1/10V0The saturated ammonium sulfate of volume makes the saturation degree 33% of ammonium sulfate in the solution,
Stirring 40min is mixed well, stands 12h under the conditions of 4 DEG C；At 4 DEG C, 10000rpm pelleted by centrifugation 20mim takes precipitating, as
Half sterling M of Yolk antibodyHalf sterling；Use 1/5V0The PBS solution of volume dissolves half sterling of Yolk antibody, then with bag filter by sample
It is placed in PBS solution and dialyses 3 days, replace PBS solution in dialysis procedure in due course, take out sample after the completion of dialysis, be dispensed into sterile
In plate；Antibody-solutions are first put into -80 DEG C and are freezed, is then transferred into freeze drier and is freeze-dried 12h to get anti-tooth
Gum Detection of Porphyromonas and the compound special yolk antibody sterling M of actinobacillus actinomycetem comitansSterling。
After obtaining Yolk antibody, agarose gel electrophoresis experiment and bioactivity experiment are carried out to it respectively, examines yolk
The purity and potency of antibody, and count its yield.
The comparison result of embodiment 1 and comparative example 1
Fig. 1 is that embodiment 1 and the resisting porphyromonas gingivalis obtained of comparative example 1 and actinobacillus actinomycetem comitans are compound special
Property Yolk antibody electrophoretogram.As seen from the figure, the protein band of the Yolk antibody crude extract of embodiment 1 is more, illustrates slightly to mention
Object is taken to contain more impurity protein；The heavy chain band of the about 70KDa of only one in pure extract and the light chain of an about 20KDa
Band illustrates to substantially increase the purity of antibody by serial purification step, in the compound special yolk antibody sterling, ovum
The purity of yellow antibody is 95% or more through reproducibility SDS-PAGE and gray value software analysis measurement purity.
Yolk antibody crude extract in comparative example 1 also contains more impurity protein, in addition to one in pure extract
Outside the light chain bands of the heavy chain band of about 70KDa and an about 20KDa, there are also the impurity bands of a treaty 38KDa, illustrate to compare
The pure extract of Yolk antibody that example 1 obtains still contains partial impurities, the purity of the Yolk antibody through reproducibility SDS-PAGE and
Gray value software analysis measurement purity is 75% or so.
In conclusion the Yolk antibody preparation method improved in the present invention substantially increases the purity of Yolk antibody.
Fig. 2 is the resisting porphyromonas gingivalis and the compound specificity of actinobacillus actinomycetem comitans that embodiment 1 and comparative example 1 obtain
Yolk antibody potency curve graph.It can be seen that in figure, the yolk antibody preparation sterling obtained in comparative example 1 is measured through ELISA method,
In the Yolk antibody sterling, the maximum specific activity of every milligram of Yolk antibody sterling resisting porphyromonas gingivalis is 1:10240, is continued
15 days, the maximum specific activity of anti-actinobacillus actinomycetem comitans was 1:5120, continued 15 days, the Yolk antibody obtained in comparative example 1
Potency rising stage and decline phase are all very long, and the high specific active maintenance phase is very short.And the yolk antibody preparation obtained in embodiment 1
The maximum specific activity of the anti-resisting porphyromonas gingivalis of every milligram of Yolk antibody of sterling is 1:20480, and specific activity is in 1:10240 or more
Duration be about 65 days, the maximum specific activities of anti-actinobacillus actinomycetem comitans is 1:20480, and specific activity is in 1:10240 or more
Duration be about 60 days, Yolk antibody potency rising stage for obtaining in embodiment 1 and decline phase are all very short, larger specific activity
The maintenance phase it is very long.From on figure it is also seen that the Yolk antibody resisting porphyromonas gingivalis that obtains and anti-being put with unwrapping wire in embodiment 1
The potency of line bar bacterium forms complementation in time.In conclusion the antigen compound scheme that the present invention improves can make Yolk antibody
Potency form complementation in time, increase immune effect.
The result that the Yolk antibody preparation method and effect of embodiment 1 and comparative example 1 are comprehensively compared is as shown in table 1.
The comparison sheet of the Yolk antibody preparation method and effect of 1 embodiment 1 of table and comparative example 1
Present invention optimizes immunization protocols, are immunized with the hen that the hen substitution that do not lay eggs has been laid eggs, this contracts significantly
The potency rising stage and decline phase of short Yolk antibody, significantly extend the maintenance phase of Yolk antibody high-titer.Meanwhile the present invention
Extracting method start with from the links of preparation process, reduce impurity introducing to the greatest extent, reduce Yolk antibody in extraction process
Potency loss, this has important meaning to the high-titer for keeping Yolk antibody.
Test example 1
In order to verify obtained resisting porphyromonas gingivalis and the compound special yolk antibody sterling of actinobacillus actinomycetem comitans
To the influence that bacterial biof iotalm is formed, examine compound special yolk antibody to porphyromonas gingivalis using crystal violet staining assay
(P.g) and the influence of actinobacillus actinomycetem comitans (A.a) biofilm formation.Its method and step is as follows:
(1) porphyromonas gingivalis (P.g) ATCC33277 bacterial strain from Guangdong Province's Culture Collection is fetched,
Amplification 3 days is cultivated in 37 DEG C, anaerobic gas generation bag with blood agar plate culture medium；It fetches from Guangdong Province's Microbiological Culture Collection
The actinobacillus actinomycetem comitans ATCC29523 bacterial strain of the heart, with blood agar plate culture medium at 37 DEG C, 5%CO2Under the conditions of cultivate amplification
1 day；Bacterium is scraped down from culture medium after culture amplification, is adjusted bacterium to OD with BHI fluid nutrient medium595Place's reading
It is 0.8, then dilutes 50 times for use.
(2) make experimental group with the special yolk antibody that embodiment 1 obtains, initial concentration is diluted to 2.5mg/mL, and 2 times again
Than being diluted to 3 concentration gradients；Make negative control with blank Yolk antibody (non-specific Yolk antibody), initial concentration is diluted to
2.5mg/mL, 2 times of doubling dilutions are at 2 concentration gradients；Make positive control with the ampicillin of 0.1mg/mL；With blank culture
Base is as blank control.0.05mL is respectively taken to be added in 96 hole elisa Plates respectively, the bacterium difference for then taking 0.05mL to dilute
It is added in 96 hole elisa Plates, is sufficiently mixed uniformly, is cultivated under the conditions of corresponding Bacteria Culture.
(3) after Bacteria Culture, culture is sucked out, twice with distilled water board-washing.Then in 96 hole elisa Plates respectively
The crystal violet solution of 0.05mL 0.1% is added, dyes 15min at room temperature.
(4) crystal violet is sucked out to be air-dried under field conditions (factors) with distilled water board-washing 3 times.Then in 96 hole elisa Plates respectively
The ethanol solution of 0.2mL 95% is added, after mixing well, with microplate reader in OD590Locate read plate, plot analysis result.
As a result:
Fig. 3 and 4 is respectively that resisting porphyromonas gingivalis and the compound special yolk antibody of actinobacillus actinomycetem comitans inhibit companion
The effect picture that unwrapping wire Actinobacillus and porphyromonas gingivalis bacterium are formed.It can be seen that in figure, the 0.1mg/ as positive control
ML ampicillin can significantly inhibit the formation of bacterial biof iotalm；Special yolk antibody is to bacterial biof iotalm in experimental group
Inhibiting effect with its concentration change and change, wherein inhibition of the special yolk antibody of 1.25mg/mL to bacterial biof iotalm
It acts on essentially identical with the effect of positive control；As bacterium normal growth in the blank cultures of blank control, bacterium living beings
Film forms not suppressed；Blank Yolk antibody as negative control is identical as blank control, not the difference of concentration gradient,
Blank Yolk antibody cannot inhibit the formation of bacterial biof iotalm.
Test example 2
In order to verify obtained resisting porphyromonas gingivalis and the compound special yolk antibody sterling of actinobacillus actinomycetem comitans
To the combination effect of bacteria cell wall, examine compound special yolk antibody to porphyromonas gingivalis using transmission electron microscope method
(P.g) and the combination of actinobacillus actinomycetem comitans (A.a).Its method and step is as follows:
(1) well-grown bacterium is collected from blood plate using PBS, be centrifuged, washed with PBS solution secondary.It adjusts
Bacterial concentration is to 107CFU/mL takes 1mL bacterial solution therein.
(2) the compound special yolk antibody obtained using embodiment 1 is experimental group, blank Yolk antibody (non-specific ovum
Yellow antibody) it is control group, it is diluted to 6mg/mL respectively；Take 1mL experimental group and control group antibody-solutions and 1mL bacterium mixed respectively
It closes, is incubated for 2h under the conditions of 37 DEG C, is then stood overnight at 4 DEG C.
(3) mixture is sucked out, is washed twice with PBS-BSA washing lotion；By the rabbit-anti chicken IgY antibody PBS- of colloidal gold conjugate
BSA dilutes 14 times, then respectively 0.3mL is taken to be separately added into experimental group and control sample, and by sample incubation in 37 DEG C of conditions
Lower 2h.
(4) culture is sucked out, twice with the rinsing of PBS-BSA washing lotion, removes unbonded antibody；It is hanged again with PBS solution
Floating to original volume.It takes 5 μ L sample solution drop in the copper mesh of 300 mesh, adsorbs 5min.
(5) it is carried out with 2% phosphotungstic acid (pH=7.0, sodium phosphotungstate or potassium adjust pH with sodium hydroxide or potassium hydroxide)
Negative staining 5min, after cleaning-drying, using being observed under Hitachi's HT7700 transmission electron microscope.
As a result:
Figures 5 and 6 are respectively compound special yolk antibody combination actinobacillus actinomycetem comitans and porphyromonas gingivalis cell
The effect picture of wall.As seen from Figure 5, the compound special yolk antibody in experimental group is largely adsorbed on actinobacillus actinomycetem comitans
On cell wall, the blank Yolk antibody in control group can only be then free in around actinobacillus actinomycetem comitans, can not be integrated to it
On cell wall；Likewise, as seen from Figure 6, the compound special yolk antibody in experimental group is largely adsorbed on porphyromonas list
On the cell wall of born of the same parents bacterium, the blank Yolk antibody in control group can only be then free in around porphyromonas gingivalis, Wu Fajie
It closes on its cell wall.By this it is demonstrated experimentally that the present invention compound special yolk antibody obtained can be specifically bound
On the cell wall of actinobacillus actinomycetem comitans and porphyromonas gingivalis, to influence its growth and breeding.
The above is only presently preferred embodiments of the present invention, is not intended to limit the present invention in any form, though
So the present invention has been disclosed as a preferred embodiment, and however, it is not intended to limit the invention, any technology people for being familiar with this patent
Member without departing from the scope of the present invention, when the technology contents using above-mentioned prompt make it is a little change or be modified to
The equivalent embodiment of equivalent variations, but anything that does not depart from the technical scheme of the invention content, it is right according to the technical essence of the invention
Any simple modification, equivalent change and modification made by above embodiments, in the range of still falling within the present invention program.
Claims (10)
1. a kind of preparation method of compound special yolk antibody characterized by comprising
(1) cultivate and extract porphyromonas gingivalis and actinobacillus actinomycetem comitans；
(2) it prepares immunogene: taking porphyromonas gingivalis and actinobacillus actinomycetem comitans thallus respectively, after ultrasonication proportionally
It is mixed to get plastc ring, prepares immunizing antigen using plastc ring；
(3) bird inlay is immunized: the hen chick that selection did not produced egg starts to raise, and utilizes preparation within 35-55 days before laying eggs
Immunizing antigen be immunized；
(4) resisting porphyromonas gingivalis and the compound special yolk antibody of actinobacillus actinomycetem comitans are isolated and purified.
2. preparation method according to claim 1, which is characterized in that in step (2), after ultrasonication, by porphyromonas
10-30 parts and 70-90 parts of actinobacillus actinomycetem comitans bacterial strain fragment of the aeromonas strain fragment Mixed Microbes for being mixed to get two kinds of bacterium are outstanding
Liquid.
3. preparation method according to claim 1 or 2, which is characterized in that in step (4), isolate and purify resisting porphyromonas
The step of monad and actinobacillus actinomycetem comitans compound special yolk antibody successively includes: that water extraction method coarse extraction yolk is anti-
Body, affinity chromatography essence extract Yolk antibody, are concentrated by ultrafiltration and change liquid and freeze-drying, obtain the compound special yolk of purifying
Antibody.
4. preparation method according to claim 3, which is characterized in that the method packet of water extraction method coarse extraction Yolk antibody
It includes:
(1) egg for collecting immune hen, with 75% alcohol disinfecting egg shell, broken shell collects yolk, measures and record yolk
Liquid accumulates V0；
(2) taking mass fraction is the aqueous sorbitol solution that 5-10%, pH are 5.0-5.5, with 10-15V0Volume be added to receipts
It in the egg yolk liquid of collection, stirs, stands, centrifugation goes to precipitate, and aqueous is spare.
5. preparation method according to claim 3 or 4, which is characterized in that the side of affinity chromatography essence extraction Yolk antibody
Method includes:
(1) affinity column is loaded with the affine filler of 2- mercaptopyridine and agarose gel coupling, with containing after the completion of filling
The sodium phosphate buffer of 20mM, pH 7.5 of 0.5M potassium sulfate balances affinity column；
(2) by the water-soluble solution loading of the Yolk antibody slightly raised, after completion of the sample, with the 20mM of the potassium sulfate containing 0.5M,
The sodium phosphate buffer of pH 7.5 balances affinity column；
(3) it is eluted with the sodium phosphate buffer of 20mM, pH 7.5, obtains the aqueous solution for the Yolk antibody that essence mentions, measured simultaneously
Record obtained aqueous solution volume V1。
6. according to preparation method described in claim 3-5 any one, which is characterized in that ultrafiltration concentration changes liquid and freeze-drying
Method include:
(1) ultrafiltration being carried out with aqueous solution of the ultrafiltration membrane packet to the Yolk antibody that essence raises and changing liquid, changing liquor is 10V1Volume
PBS buffer solution is changed after the completion of liquid, and solution is concentrated with ultrafiltration membrane packet；
(2) the Yolk antibody solution after concentration is dispensed into disposable sterilized culture dish, be sequentially placed into -80 DEG C of refrigerator freezings,
It is freeze-dried in vacuum freeze drier, the sample after taking out freeze-drying obtains Yolk antibody sterling；
Preferably, the molecule interception of the ultrafiltration membrane packet used is 50KDa；
Preferably, -80 DEG C of refrigerators are put into freeze 2-5 hours, is put into vacuum freeze drier and is freeze-dried 12-15 hours.
7. preparation method described in -6 any one according to claim 1, which is characterized in that in step (2), it is outstanding to obtain Mixed Microbes
Plastc ring and Freund's complete adjuvant are subjected to compound, stirring and emulsifying according to the volume ratio of 1:1 after liquid, Water-In-Oil Freund is made
Freund's complete adjuvant antigen；
Plastc ring and incomplete Freund's adjuvant are subjected to compound, stirring and emulsifying according to the volume ratio of 1:1, Water-In-Oil is made not
Family name's Freund's incomplete adjuvant antigen.
8. preparation method described in -7 any one according to claim 1, which is characterized in that not producing egg in step (3)
Hen carry out immune method and include:
Initial immunity uses Water-In-Oil Freund's complete adjuvant antigen, immunizing dose 0.8mL, using subcutaneous multi-point injection and muscle
The mode of injection is immune；First time booster immunization is carried out after initial immunity 10 days, booster immunization is incomplete using Water-In-Oil Freund
Adjuvant antigen, immunizing dose 0.8mL is later primary every 10 days progress booster immunizations, at least booster immunization eight times, and hen produces
Egg is collected after egg.
9. a kind of compound special yolk antibody that the preparation method as described in claim 1-8 any one is prepared.
10. a kind of special yolk antibody as claimed in claim 9 is in preparation prevention and/or the drug for the treatment of mouth disease
And/or the application in daily necessity；
Preferably, special yolk antibody is in preparation prevention and/or the drug and/or daily necessity for the treatment of periodontitis, gingivitis
In application.
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