CN110237251B - Composite specificity egg yolk antibody oral spray and preparation method thereof - Google Patents

Composite specificity egg yolk antibody oral spray and preparation method thereof Download PDF

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CN110237251B
CN110237251B CN201910548855.0A CN201910548855A CN110237251B CN 110237251 B CN110237251 B CN 110237251B CN 201910548855 A CN201910548855 A CN 201910548855A CN 110237251 B CN110237251 B CN 110237251B
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yolk antibody
egg yolk
solution
actinobacillus
porphyromonas gingivalis
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邹节明
贾成刚
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Guilin Sanjin Pharmaceuticals Co Ltd
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Abstract

The invention discloses a compound specificity egg yolk antibody oral spray and a preparation method thereof. The oral spray disclosed by the invention has the advantages of simple and safe components, no toxic or side effect, no alcohol, mildness and no irritation, has a strong antibacterial effect on specific bacteria Porphyromonas gingivalis and actinobacillus concomitans, can effectively inhibit the bacteria from forming a biological film, reduces the generation and development of dental plaque, has a good prevention effect on gingivitis and periodontitis, and is simple and easy to prepare, low in production cost and good in product stability.

Description

Composite specificity egg yolk antibody oral spray and preparation method thereof
Technical Field
The invention belongs to the field of daily chemical disinfection products, and particularly relates to a composite specificity egg yolk antibody oral spray and a preparation method thereof, and more particularly relates to an anti-porphyromonas gingivalis and actinobacillus concomitatus specificity egg yolk antibody oral spray and a preparation method thereof.
Background
The oral cavity is an important component of the respiratory system and the digestive system of people, and oral health has a vital meaning on human health, but as the life pace of people increases, people have no time to clean and care the oral cavity, which directly results in that most people are plagued by oral diseases to a greater or lesser extent at present. Scientific researches show that oral diseases such as periodontitis, gingivitis, dental caries, halitosis and the like are caused by oral pathogenic bacteria hyperproliferation and oral flora imbalance. Therefore, in order to solve the problem of oral diseases, it is necessary to control the growth and reproduction of oral pathogenic bacteria from the source, reduce the adhesion of oral pathogenic bacteria to oral epithelial cells, and regulate the balance of oral flora.
Because the way of brushing teeth cleans the oral cavity is complicated and is not easy to use, products such as mouthwash, mouthspray modified by mouthwash and the like for cleaning the oral cavity appear, the action principle of the products is that a broad-spectrum antibacterial agent or antibiotics are used as main active ingredients, bacteria in the oral cavity are killed through the sterilization and bacteriostasis effects of the broad-spectrum antibacterial agent and the antibiotics, but various bacteria (including beneficial bacteria) in the oral cavity can be killed when the broad-spectrum antibacterial agent and the antibiotics have no specificity, and the oral cavity flora is disordered after long-term use, so that other oral problems appear. In addition, through researching the oral cavity spray on the market, we find that the spray nozzles of the oral cavity spray on the market are at the lower end, the spray amount is difficult to keep consistent, the atomization effect is poor, and the oral cavity spray contains alcohol and has irritation to the oral mucosa.
The present invention has been made in view of this.
Disclosure of Invention
The invention aims to solve the technical problems of overcoming the defects of the prior art and providing a composite specificity egg yolk antibody oral spray and a preparation method thereof, wherein the composite specificity egg yolk antibody can specifically inhibit actinobacillus actinomyces and porphyromonas gingivalis, and has the advantages of good specificity, high potency, safety and no toxic or side effect; the preparation method of the invention can prolong the time of maintaining the high titer of the yolk antibody, improve the yield and purity of the yolk antibody, ensure that the yolk antibody has higher biological activity, reduce the production cost of the yolk antibody and ensure that the yolk antibody technology can be better applied to industrialized production.
In order to solve the technical problems, the invention adopts the basic conception of the technical scheme that:
the first object of the invention is to provide a composite specificity egg yolk antibody oral spray, the components of the oral spray comprise:
Figure BDA0002104806520000021
pure water, the sum of the components is hundred percent.
Further, the compound specificity egg yolk antibody is added with a protective agent, and the protective agent comprises: 0.4-1% cetylpyridinium chloride.
In a further scheme, the compound specificity egg yolk antibody is a compound specificity egg yolk antibody for resisting porphyromonas gingivalis and actinobacillus. The specific yolk antibody for resisting porphyromonas gingivalis and actinobacillus actinomyces can specifically bind with the cell walls of actinobacillus gingivalis and actinobacillus gingivalis, inhibit the formation of bacterial biofilm, have the advantages of good specificity, high potency and no side effect, and have extremely strong pertinence to the porphyromonas gingivalis and the actinobacillus. The porphyromonas gingivalis is a main pathogenic bacterium capable of causing various oral diseases, and actinobacillus concomitans is a pathogenic bacterium causing invasive periodontitis, so that the compound specificity egg yolk antibody for resisting the porphyromonas gingivalis and actinobacillus concomitans has the effects of preventing the oral diseases such as periodontitis, gingivitis and the like, can be used as an effective component of medicines or living goods for resisting the periodontitis and the gingivitis, and is used in the production of medicines and living goods.
The second object of the invention is to provide a preparation method of the composite specificity egg yolk antibody oral spray, which comprises the following steps:
(1) Preparing a composite specificity egg yolk antibody mother solution A;
(2) Respectively weighing hydrogenated castor oil, menthol and borneol according to a proportion, placing the hydrogenated castor oil into a container, adding the menthol and the borneol under stirring, and dissolving the menthol and the borneol into the hydrogenated castor oil to prepare a solution B;
(3) Weighing sorbitol, sucralose and watermelon frost according to a proportion, and dissolving the sorbitol, the sucralose and the watermelon frost in proper amount of pure water to prepare a solution C;
(4) Weighing a compound specific egg yolk antibody mother solution A according to 1-10% of the total weight, uniformly mixing the compound specific egg yolk antibody mother solution A with a solution C, adding the solution B under stirring, fully uniformly mixing and emulsifying the solution B into an oil-in-water state, and supplementing pure water until the sum of all components is percentage;
(5) Filtering the solution with a filter membrane, and aseptically filling.
Further, the specific method of the step (1) comprises the following steps: weighing a compound specific egg yolk antibody, preparing the compound specific egg yolk antibody with pure water according to a certain antibody titer, adding 0.4-1% cetylpyridinium chloride as a protective agent, and fully and uniformly mixing to prepare a compound specific egg yolk antibody mother solution A;
preferably, the antibody titer is 1: 10240A standard was prepared with pure water as a complex specific egg yolk antibody mother liquor.
Further, the preparation method of the compound specific egg yolk antibody comprises the following steps:
(1) Culturing and extracting Porphyromonas gingivalis and actinobacillus;
(2) Preparation of immunogens: respectively taking porphyromonas gingivalis and actinomycetes thallus, carrying out ultrasonic crushing, mixing according to a proportion to obtain mixed bacterial suspension, and preparing an immune antigen by using the mixed bacterial suspension;
(3) Immunization of egg laying hens: selecting hen chicks which are not produced with eggs, starting to feed, and immunizing with an immune antigen 35-55 days before laying eggs;
(4) And (3) separating and purifying the anti-porphyromonas gingivalis and actinobacillus complex specificity egg yolk antibody.
The preparation method selects the hen chicks which are not produced with eggs to feed, and utilizes the immune antigen to immunize 35-55 days before laying eggs, so that the time for maintaining high titer of the yolk antibody can be prolonged, the yield and purity of the yolk antibody can be improved, the yolk antibody has higher bioactivity, the production cost of the yolk antibody can be reduced, and the yolk antibody technology can be better applied to industrialized production.
In a further scheme, in the step (2), after ultrasonic crushing, 10-30 parts of Porphyromonas gingivalis strain fragments and 70-90 parts of actinobacillus concomitantly strain fragments are mixed to obtain mixed bacterial suspension of the two bacteria.
The invention adopts 10-30 parts of Porphyromonas gingivalis thallus fragments and 70-90 parts of actinobacillus concomitantly to prepare mixed bacterial suspension in a compound way to prepare the immune antigen. Because the immunogenicity of porphyromonas gingivalis is relatively strong and the immunogenicity of actinobacillus accompaniment is relatively weak, the antigen compounded and configured in such a proportion can form a difference value on immune stimulation, so that the obtained yolk antibody can form better complementation on potency, and the newly obtained specific yolk antibody has stronger biological activity.
In a further scheme, in the step (4), the steps of separating and purifying the anti-porphyromonas gingivalis and actinobacillus complex specificity egg yolk antibody sequentially comprise: extracting egg yolk antibody by water extraction, extracting egg yolk antibody by affinity chromatography, ultrafiltering, concentrating, and freeze drying to obtain purified compound specific egg yolk antibody.
Further, the method for extracting the egg yolk antibody by water extraction method comprises the following steps:
(1) Collecting hen's eggs, sterilizing the egg shells with 75% alcohol, breaking the shells to collect yolk, measuring and recording the yolk liquid volume V 0 。
(2) Taking sorbitol aqueous solution with mass fraction of 5-10% and pH of 5.0-5.5, and mixing at 10-15V 0 Adding the mixture into the collected yolk liquid, stirring, standing, centrifuging to remove precipitate, and collecting water solution for later use.
The invention improves the extraction process of the egg yolk antibody, and sorbitol is added as a stabilizer on the basis of water extraction to reduce the potency loss of the egg yolk antibody in aqueous solution.
Further, the method for extracting the egg yolk antibody by affinity chromatography comprises the following steps:
(1) Filling the affinity chromatography column with an affinity filler coupled with 2-mercaptopyridine and agarose gel, and balancing the affinity column with 20mM sodium phosphate buffer solution containing 0.5M potassium sulfate and having pH of 7.5 after filling;
(2) Loading the water-soluble solution of the egg yolk antibody obtained by crude extraction, and balancing an affinity column by using 20mM sodium phosphate buffer solution with the pH of 7.5 and 0.5M potassium sulfate after loading;
(3) Eluting with 20mM sodium phosphate buffer solution with pH of 7.5 to obtain refined egg yolk antibody water solution, measuring and recording the volume V of the obtained water solution 1 。
The invention adopts the method of affinity chromatography to extract the compound specific egg yolk antibody, avoids the influence of adding high molecular polymer on the activity of the egg yolk antibody, and the water solution after affinity chromatography is the water solution containing the egg yolk antibody, wherein no high molecular polymer is combined, the subsequent treatment does not need salting out treatment, the process flow is reduced, and the influence of high concentration salt on the activity of the egg yolk antibody is avoided.
Further, the method for ultrafiltration concentration liquid exchange and freeze drying comprises the following steps:
(1) Ultrafiltering the water solution of egg yolk antibody with ultrafilter membrane bag to obtain 10V solution 1 After the replacement of the volume of PBS buffer solution is completed, concentrating the solution by using an ultrafiltration membrane bag;
(2) Subpackaging the concentrated yolk antibody solution into a disposable sterile culture dish, sequentially placing the sterile culture dish into a refrigerator at the temperature of-80 ℃ for freezing, and then performing freeze-drying in a vacuum freeze dryer, and taking out a freeze-dried sample to obtain a pure yolk antibody product;
preferably, the molecular retention of the ultrafiltration membrane package is 50kDa;
preferably, the mixture is frozen in a refrigerator at the temperature of minus 80 ℃ for 2 to 5 hours, and is frozen and dried in a vacuum freeze dryer for 12 to 15 hours.
The ultrafiltration membrane bag is adopted to replace dialysis for liquid exchange, the liquid exchange is more thorough, the ultrafiltration membrane bag can be used for concentrating the egg yolk antibody aqueous solution, the treatment capacity during freeze drying is reduced, and the treatment efficiency is improved.
In a further scheme, in the step (2), after the mixed bacterial suspension is obtained, the mixed bacterial suspension and Freund's complete adjuvant are compounded according to the volume ratio of 1:1, and stirred and emulsified to prepare the oil-in-oil Shui Fushi complete adjuvant antigen;
compounding the mixed bacterial suspension and Freund's incomplete adjuvant according to the volume ratio of 1:1, stirring and emulsifying to prepare the oil-in-Shui Fushi incomplete adjuvant antigen.
In a further aspect, the method of immunizing a hen that has not produced eggs in step (3) comprises:
the primary immunization uses water-in-oil Freund complete adjuvant antigen, the immunization dose is 0.8mL, and subcutaneous multipoint injection and intramuscular injection are adopted for immunization; the first boost was performed 10 days after the initial immunization, using water-in-oil Freund's incomplete adjuvant antigen at an immunization dose of 0.8mL, and then was performed once every 10 days, at least eight times, and the hen was collected after laying eggs.
According to the invention, an immunization scheme is improved, hen chicks which are not laid eggs are selected for raising, immunization is started 35-55 days before laying eggs, meanwhile, the immunization interval time is shortened, the immunization dosage is increased, the hen is continuously subjected to immune stimulation, the time for maintaining the high titer of the obtained egg yolk antibody is greatly increased after the immunization scheme is improved, the available high titer part of the egg yolk antibody obtained by the traditional preparation process can only select egg yolk antibody of 15 days, and the available high titer part of the egg yolk antibody obtained by the preparation process can be used for selecting egg yolk antibody of 60 days, so that the available yield is greatly increased.
Specifically, the preparation method of the compound specificity egg yolk antibody provided by the invention comprises the following steps:
A. Culturing and extracting Porphyromonas gingivalis and actinobacillus concomitatus:
porphyromonas gingivalis (P.g) ATCC33277 strain and actinobacillus concomitans (A.a) ATCC29523 strain from the Guangdong province microorganism strain collection were amplified and cultured in blood agar plate medium and identified by gram stain. Porphyromonas gingivalis (P.g) ATCC33277 strain was cultured for 3 days, actinobacillus (A.a) ATCC29523 strain was cultured for 1 day, and the bacteria were scraped with a loop in PBS buffer. And repeating centrifugal extraction for three times, and collecting bacterial precipitate to obtain the required bacterial thallus.
B. Preparation of immunogens:
B1. taking Porphyromonas gingivalis and actinobacillus actinomyces, respectively, and adjusting colony density to 3-5×10 with sterile PBS 9 Performing ultrasonic crushing on CFU/mL, and mixing the bacterial suspension according to the corresponding proportion to obtain mixed bacterial suspension of two bacteria;
B2. compounding the mixed bacterial suspension and Freund's complete adjuvant according to the volume ratio of 1:1, and stirring and emulsifying by using a high-speed stirrer to finally prepare the oil-in-Shui Fushi complete adjuvant antigen.
B3. Compounding the mixed bacterial suspension and Freund's incomplete adjuvant according to the volume ratio of 1:1, and stirring and emulsifying by using a high-speed stirrer to finally prepare the oil-in-Shui Fushi incomplete adjuvant antigen.
C. Immunization of egg laying hens:
selecting hen chicks which are not produced with eggs to feed, and starting immunization 35-55 days before laying eggs; primary immunization uses Freund's complete adjuvant antigen, the immune dose is 0.8mL, and subcutaneous multipoint injection and intramuscular injection are adopted for immunization; the first boost was performed 10 days after the initial immunization, using Freund's incomplete adjuvant antigen at an immunization dose of 0.8mL, and then was performed once every 10 days for eight times, and the hen was collected and purified in time after laying eggs.
D. Separating and purifying the anti-porphyromonas gingivalis and actinobacillus complex specificity egg yolk antibody:
d1: crude extraction of egg yolk antibody by modified water extraction:
(1) Collecting immunized hen eggs, sterilizing the egg shells with 75% alcohol, breaking the shells to collect yolk, measuring and recording the yolk liquid volume V 0 。
(2) Preparing 5-10% sorbitol aqueous solution with distilled water, adjusting pH to 5.0-5.5 with diluted hydrochloric acid, and then treating with 10-15V 0 Is added to the collected yolk liquid, and the mixture is stirred by a magnetic stirrer at a low levelStirring at a rotating speed for 30-60 minutes, and then standing at 4 ℃ overnight. After standing, centrifuging at 10000rpm for 20 min, discarding the precipitate, and collecting water-soluble solution.
D2: fine extraction of egg yolk antibody by affinity chromatography:
(1) The affinity column was packed with an affinity packing coupled with 2-mercaptopyridine and agarose gel, and after completion of the packing, the affinity column was equilibrated with 20mM sodium phosphate buffer (pH 7.5) containing 0.5M potassium sulfate.
(2) The water-soluble solution obtained in D1 was loaded at a flow rate of 5 mL/min, and after loading, the affinity column was equilibrated with 5 column volumes of 20mM sodium phosphate buffer (pH 7.5) containing 0.5M potassium sulfate to allow the egg yolk antibody to be fully affinity adsorbed to the affinity packing in the column.
(3) Eluting with 10 column volumes of 20mM sodium phosphate buffer (pH 7.5) to obtain solution containing high purity egg yolk antibody, measuring and recording the volume V 1 。
D3: ultrafiltration concentration liquid exchange and freeze drying
(1) The solution V obtained in D2 was packed with an ultrafiltration membrane having a molecular cut-off of 50kDa 1 Performing ultrafiltration to obtain a solution with 10V 1 After the change of the volume of PBS buffer solution is completed, the solution is slowly concentrated to 20-50mL by using an ultrafiltration membrane bag.
(2) Packaging the concentrated yolk antibody solution into disposable sterile culture dish, freezing at-80deg.C for 2-5 hr, freeze drying in vacuum freeze dryer for 12-15 hr, taking out the freeze dried sample to obtain pure yolk antibody, and recording as M Pure product 。
After the technical scheme is adopted, compared with the prior art, the invention has the following beneficial effects:
1. the added specific composite yolk antibody is prepared by taking porphyromonas gingivalis and actinobacillus accompaniment as composite antigens, and can be specifically combined to cell walls of the porphyromonas gingivalis and actinobacillus accompaniment to inhibit the two bacteria from forming a biological film in an oral cavity, so that the generation and development of dental plaque are reduced; according to the specific binding principle of antigen and antibody, the specific composite yolk antibody only aims at pathogenic bacteria selected as antigens, does not influence the growth and propagation of other beneficial bacteria, and can avoid causing further imbalance of oral flora. Experimental results show that the invention has stronger antibacterial effect on porphyromonas gingivalis and actinobacillus.
2. The egg yolk antibody is an immunoglobulin extracted from egg yolk, does not bind to rheumatoid factors, does not bind to protein A, protein G and mammal F receptors or complement, hardly has cross reaction with IgG, is extracted by adopting food and pharmaceutical grade raw materials, and is safe and has no side effect. Meanwhile, the cetylpyridinium chloride and the compound specificity egg yolk antibody are compounded, and then sorbitol is added, so that the cetylpyridinium chloride and the sorbitol form a protective agent system of the compound specificity egg yolk antibody together, the activity of the egg yolk antibody is ensured to be well maintained in an oral spray, and the stability of the egg yolk antibody is improved.
Therefore, the composite specificity egg yolk antibody oral spray disclosed by the invention has the advantages of simple and safe components, no toxic or side effect, no alcohol, mildness and no irritation, has a strong antibacterial effect on specific bacteria of Porphyromonas gingivalis and actinobacillus, can effectively inhibit the bacteria from forming a biological film, reduces the generation and development of dental plaque, and has a good prevention effect on gingivitis and periodontitis. The oral spray has the advantages of simple and easy preparation, convenient use, low production cost and good product stability.
The following describes the embodiments of the present invention in further detail with reference to the accompanying drawings.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention. It is evident that the drawings in the following description are only examples, from which other drawings can be obtained by a person skilled in the art without the inventive effort. In the drawings:
fig. 1 is a graph showing the stability trend of a composite specific egg yolk antibody, wherein sample a is a sample to which a protective agent is added, and sample b is a sample to which no protective agent is added.
FIG. 2 is an electrophoretogram of the complex-specific egg yolk antibodies against Porphyromonas gingivalis and Actinobacillus actinomycetes of example 1 and comparative example 1 of the present invention; in the figure, band 1.1 is the crude extract of the egg yolk antibody of example 1; strip 1.2 is the pure extract of the egg yolk antibody of example 1; strip 2.1 is crude egg yolk antibody extract of comparative example 1; strip 2.2 is the pure extract of the egg yolk antibody of comparative example 1; the strip M is a Marker;
FIG. 3 is a graph showing the titers of the compound specific egg yolk antibodies against Porphyromonas gingivalis and Actinobacillus actinomycetes according to example 1 and comparative example 1 of the present invention;
FIG. 4 is a graph showing the effect of the composite specific egg yolk antibody against Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans in inhibiting the formation of a biological membrane of Actinobacillus actinomycetemcomitans;
FIG. 5 is a graph showing the effect of the complex specific egg yolk antibody against Porphyromonas gingivalis and Actinobacillus actinomyces for inhibiting formation of Porphyromonas gingivalis biofilm;
FIG. 6 is a graph showing the effect of the composite specific egg yolk antibody against Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans on inhibiting the cell wall of Actinobacillus actinomycetemcomitans; in the figure, the larger black shadow is actinobacillus actinomycetemcomitans, and the tiny black small particles are egg yolk antibodies;
FIG. 7 is a graph showing the effect of the composite specific egg yolk antibody against Porphyromonas gingivalis and Actinobacillus actinomyces for inhibiting Porphyromonas gingivalis cell wall according to the present invention; in the figure, the larger black shadow is porphyromonas gingivalis, and the tiny black small particles are egg yolk antibodies.
It should be noted that these drawings and the written description are not intended to limit the scope of the inventive concept in any way, but to illustrate the inventive concept to those skilled in the art by referring to the specific embodiments.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions in the embodiments will be clearly and completely described with reference to the accompanying drawings in the embodiments of the present invention, and the following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the present invention.
Example 1
Taking 100g of egg yolk antibody oral spray as an example:
(1) Weighing 20mg of egg yolk antibody powder with titer of 5120 and 40mg of cetylpyridinium chloride powder, dissolving in a proper amount of pure water, adding pure water to 10g of the weight after full dissolution, and preparing a specific composite egg yolk antibody mother solution A;
(2) Weighing 0.5g of hydrogenated castor oil in a container, simultaneously weighing 0.01g of borneol and menthol for standby, placing the hydrogenated castor oil on a 1400-rpm stirrer for stirring, and slowly adding the weighed borneol and menthol until the borneol and menthol are completely dissolved in the hydrogenated castor oil to prepare a solution B;
(3) 1g of sorbitol, 0.005g of sucralose and 0.01g of watermelon frost are weighed and dissolved in a proper amount of pure water to prepare a solution C;
(4) Weighing 1g of specific composite egg yolk antibody mother liquor A, adding the solution C, and fully and uniformly mixing; then placing the mixed solution on a 1400-rpm stirrer for stirring, and slowly adding the solution B to fully emulsify the mixed solution into an oil-in-water aqueous solution; pure water was supplemented to a total weight of 100g;
(5) The sample was filtered through a 0.45 μm sterile filter and then dispensed into aerosol bottles at a dose of 20 mL/bottle under sterile conditions.
Example 2
Taking 200g of egg yolk antibody oral spray as an example:
(1) Weighing 40mg of egg yolk antibody powder with titer of 5120 and 200mg of cetylpyridinium chloride powder, dissolving in a proper amount of pure water, adding pure water to 20g of the weight after full dissolution, and preparing a specific composite egg yolk antibody mother solution A;
(2) Weighing 6g of hydrogenated castor oil in a container, simultaneously weighing 0.1g of borneol and menthol for standby, placing the hydrogenated castor oil on a stirrer with the speed of 1600 revolutions per minute for stirring, and slowly adding the weighed borneol and menthol until the borneol and menthol are completely dissolved in the hydrogenated castor oil to prepare a solution B;
(3) Weighing 20g of sorbitol, 0.1g of sucralose and 0.2g of watermelon frost, and dissolving in a proper amount of pure water to prepare a solution C;
(4) Weighing 20g of specific composite yolk antibody mother liquor A, adding the solution C, and fully and uniformly mixing; then placing the mixed solution on a stirrer with the speed of 1600 revolutions per minute for stirring, and slowly adding the solution B to fully emulsify the mixed solution into an aqueous solution in an oil-in-water state; pure water was supplemented to a total weight of 200g;
(5) The sample was filtered through a 0.45 μm sterile filter and then dispensed into aerosol bottles at a dose of 20 mL/bottle under sterile conditions.
Example 3
Preparation of a compound specificity egg yolk antibody for resisting porphyromonas gingivalis and actinobacillus:
A. culturing and extracting Porphyromonas gingivalis and actinobacillus concomitatus:
porphyromonas gingivalis (P.g) ATCC33277 strain and actinobacillus concomitans (A.a) ATCC29523 strain from the Guangdong province microorganism strain collection were amplified and cultured in blood agar plate medium and identified by gram stain. Porphyromonas gingivalis (P.g) ATCC33277 strain was cultured for 3 days, actinobacillus (A.a) ATCC29523 strain was cultured for 1 day, and the bacteria were scraped with a loop in PBS buffer. And repeating centrifugal extraction for three times, and collecting bacterial precipitate to obtain the required bacterial thallus.
B. Preparation of immunogens:
B1. taking Porphyromonas gingivalis and actinobacillus actinomyces, respectively, and adjusting colony density to 3-5×10 with sterile PBS 9 CFU/mL, then ultrasonic disruption is carried out on a ultrasonic disruption instrument for 25 minutes at 25Hz for 3s and at rest for 4s, and then the bacterial suspension is processed according to Porphyromonas gingivalis: mixing actinobacillus actinobacillus=30:70 to obtain mixed bacterial suspension of two bacteria;
B2. compounding the mixed bacterial suspension and Freund's complete adjuvant according to the volume ratio of 1:1, and stirring and emulsifying by using a high-speed stirrer to finally prepare the oil-in-Shui Fushi complete adjuvant antigen.
B3. Compounding the mixed bacterial suspension and Freund's incomplete adjuvant according to the volume ratio of 1:1.2, and stirring and emulsifying by using a high-speed stirrer to finally prepare the oil-in-Shui Fushi incomplete adjuvant antigen.
C. Immunization of egg laying hens:
selecting hen chicks which are not laid eggs to start feeding, and starting immunization 45 days before laying eggs; primary immunization uses Freund's complete adjuvant antigen, the immune dose is 0.8mL, and subcutaneous multipoint injection and intramuscular injection are adopted for immunization; the first boost was performed 10 days after the initial immunization, using Freund's incomplete adjuvant antigen at an immunization dose of 0.8mL, and then was performed once every 10 days for eight times, and the hen was collected and purified in time after laying eggs.
D. Separating and purifying the anti-porphyromonas gingivalis and actinobacillus complex specificity egg yolk antibody:
D1: crude extraction of egg yolk antibody by modified water extraction:
(1) Collecting immunized hen eggs, sterilizing the egg shells with 75% alcohol, breaking the shells to collect yolk, measuring and recording the yolk liquid volume V 0 。
(2) Preparing 8% sorbitol aqueous solution with distilled water, adjusting pH to 5.0-5.5 with diluted hydrochloric acid, and then treating with 12V 0 Is added to the collected yolk liquid, and the mixture is stirred on a magnetic stirrer at a low rotation speed for 40 minutes and then left to stand at 4 ℃ overnight. After standing, centrifuging at 10000rpm for 20 min, discarding the precipitate, and collecting water-soluble solution.
D2: fine extraction of egg yolk antibody by affinity chromatography:
(1) The affinity column was packed with an affinity packing coupled with 2-mercaptopyridine and agarose gel, and after completion of the packing, the affinity column was equilibrated with 20mM sodium phosphate buffer (pH 7.5) containing 0.5M potassium sulfate.
(2) The water-soluble solution obtained in D1 was loaded at a flow rate of 5 mL/min, and after loading, the affinity column was equilibrated with 5 column volumes of 20mM sodium phosphate buffer (pH 7.5) containing 0.5M potassium sulfate to allow the egg yolk antibody to be fully affinity adsorbed to the affinity packing in the column.
(3) Eluting with 10 column volumes of 20mM sodium phosphate buffer (pH 7.5) to obtain solution containing high purity egg yolk antibody, measuring and recording the volume V 1 。
D3: ultrafiltration concentration liquid exchange and freeze drying
(1) The solution V obtained in D2 was packed with an ultrafiltration membrane having a molecular cut-off of 50kDa 1 Performing ultrafiltration to obtain a solution with 10V 1 After the completion of the change of volume of PBS buffer, the solution was slowly concentrated to 50mL by ultrafiltration membrane.
(2) Packaging the concentrated yolk antibody solution into a disposable sterile culture dish, freezing at-80deg.C for 2 hr, freeze drying in vacuum freeze dryer for 12 hr, taking out the freeze dried sample to obtain pure yolk antibody, and recording as M Pure product 。
After the compound specificity egg yolk antibody is obtained, agarose gel electrophoresis experiments and potency detection experiments are respectively carried out on the compound specificity egg yolk antibody, the purity and potency of the compound specificity egg yolk antibody are checked, and the yield is counted.
Example 4
Preparation of a compound specificity egg yolk antibody for resisting porphyromonas gingivalis and actinobacillus:
A. culturing and extracting Porphyromonas gingivalis and actinobacillus concomitatus:
porphyromonas gingivalis (P.g) ATCC33277 strain and actinobacillus concomitans (A.a) ATCC29523 strain from the Guangdong province microorganism strain collection were amplified and cultured in blood agar plate medium and identified by gram stain. Porphyromonas gingivalis (P.g) ATCC33277 strain was cultured for 3 days, actinobacillus (A.a) ATCC29523 strain was cultured for 1 day, and the bacteria were scraped with a loop in PBS buffer. And repeating centrifugal extraction for three times, and collecting bacterial precipitate to obtain the required bacterial thallus.
B. Preparation of immunogens:
B1. taking out gingiva respectivelyPorphyromonas and actinomycetes with actinomycetes, colony density was adjusted to 3-5X 10 with sterile PBS 9 CFU/mL, then ultrasonic disruption is carried out on a ultrasonic disruption instrument for 25 minutes at 25Hz for 3s and at rest for 4s, and then the bacterial suspension is processed according to Porphyromonas gingivalis: mixing actinobacillus actinobacillus=10:90 to obtain mixed bacterial suspension of two bacteria;
B2. compounding the mixed bacterial suspension and Freund's complete adjuvant according to the volume ratio of 1:1, and stirring and emulsifying by using a high-speed stirrer to finally prepare the oil-in-Shui Fushi complete adjuvant antigen.
B3. Compounding the mixed bacterial suspension and Freund's incomplete adjuvant according to the volume ratio of 1:1, and stirring and emulsifying by using a high-speed stirrer to finally prepare the oil-in-Shui Fushi incomplete adjuvant antigen.
C. Immunization of egg laying hens:
selecting hen chicks which are not laid eggs to start feeding, and starting immunization 35 days before laying eggs; primary immunization uses Freund's complete adjuvant antigen, the immune dose is 0.8mL, and subcutaneous multipoint injection and intramuscular injection are adopted for immunization; the first boost was performed 10 days after the initial immunization, using Freund's incomplete adjuvant antigen at an immunization dose of 0.8mL, and then was performed once every 10 days for eight times, and the hen was collected and purified in time after laying eggs.
D. Separating and purifying the anti-porphyromonas gingivalis and actinobacillus complex specificity egg yolk antibody:
d1: crude extraction of egg yolk antibody by modified water extraction:
(1) Collecting immunized hen eggs, sterilizing the egg shells with 75% alcohol, breaking the shells to collect yolk, measuring and recording the yolk liquid volume V 0 。
(2) Preparing 8% sorbitol aqueous solution with distilled water, adjusting pH to 5.0-5.5 with diluted hydrochloric acid, and then treating with 10V 0 Is added to the collected yolk liquid, and the mixture is stirred on a magnetic stirrer at a low rotation speed for 30 minutes and then left to stand at 4 ℃ overnight. After standing, centrifuging at 10000rpm for 20 min, discarding the precipitate, and collecting water-soluble solution.
D2: fine extraction of egg yolk antibody by affinity chromatography:
(1) The affinity column was packed with an affinity packing coupled with 2-mercaptopyridine and agarose gel, and after completion of the packing, the affinity column was equilibrated with 20mM sodium phosphate buffer (pH 7.5) containing 0.5M potassium sulfate.
(2) The water-soluble solution obtained in D1 was loaded at a flow rate of 5 mL/min, and after loading, the affinity column was equilibrated with 5 column volumes of 20mM sodium phosphate buffer (pH 7.5) containing 0.5M potassium sulfate to allow the egg yolk antibody to be fully affinity adsorbed to the affinity packing in the column.
(3) Eluting with 10 column volumes of 20mM sodium phosphate buffer (pH 7.5) to obtain solution containing high purity egg yolk antibody, measuring and recording the volume V 1 。
D3: ultrafiltration concentration liquid exchange and freeze drying
(1) The solution V obtained in D2 was packed with an ultrafiltration membrane having a molecular cut-off of 50kDa 1 Performing ultrafiltration to obtain a solution with 10V 1 After the completion of the change of volume of PBS buffer, the solution was slowly concentrated to 35mL by ultrafiltration membrane.
(2) Packaging the concentrated yolk antibody solution into a disposable sterile culture dish, freezing at-80deg.C for 3 hr, freeze-drying in vacuum freeze dryer for 13 hr, taking out the freeze-dried sample to obtain pure yolk antibody, and recording as M Pure product 。
After the compound specificity egg yolk antibody is obtained, agarose gel electrophoresis experiments and potency detection experiments are respectively carried out on the egg yolk antibody, the purity and potency of the egg yolk antibody are checked, and the yield is counted.
Example 5
Preparation of a compound specificity egg yolk antibody for resisting porphyromonas gingivalis and actinobacillus:
A. culturing and extracting Porphyromonas gingivalis and actinobacillus concomitatus:
Porphyromonas gingivalis (P.g) ATCC33277 strain and actinobacillus concomitans (A.a) ATCC29523 strain from the Guangdong province microorganism strain collection were amplified and cultured in blood agar plate medium and identified by gram stain. Porphyromonas gingivalis (P.g) ATCC33277 strain was cultured for 3 days, actinobacillus (A.a) ATCC29523 strain was cultured for 1 day, and the bacteria were scraped with a loop in PBS buffer. And repeating centrifugal extraction for three times, and collecting bacterial precipitate to obtain the required bacterial thallus.
B. Preparation of immunogens:
B1. taking Porphyromonas gingivalis and actinobacillus actinomyces, respectively, and adjusting colony density to 3-5×10 with sterile PBS 9 CFU/mL, then ultrasonic disruption is carried out on a ultrasonic disruption instrument for 30min at 25Hz for 3s and at the stop of 4s, and then bacterial suspension is processed according to Porphyromonas gingivalis: mixing actinobacillus actinobacillus=25:75 to obtain mixed bacterial suspension of two bacteria;
B2. compounding the mixed bacterial suspension and Freund's complete adjuvant according to the volume ratio of 1:1, and stirring and emulsifying by using a high-speed stirrer to finally prepare the oil-in-Shui Fushi complete adjuvant antigen.
B3. Compounding the mixed bacterial suspension and Freund's incomplete adjuvant according to the volume ratio of 1:1.3, and stirring and emulsifying by using a high-speed stirrer to finally prepare the oil-in-Shui Fushi incomplete adjuvant antigen.
C. Immunization of egg laying hens:
selecting hen chicks which are not laid eggs to start feeding, and starting immunization 55 days before laying eggs; primary immunization uses Freund's complete adjuvant antigen, the immune dose is 0.8mL, and subcutaneous multipoint injection and intramuscular injection are adopted for immunization; the first boost was performed 10 days after the first boost using Freund's incomplete adjuvant antigen at an immunization dose of 0.8mL, and then the boost was performed once every 10 days for nine total boosts, and the hen was collected and purified immediately after laying eggs.
D. Separating and purifying the anti-porphyromonas gingivalis and actinobacillus complex specificity egg yolk antibody:
d1: crude extraction of egg yolk antibody by modified water extraction:
(1) Collecting hen eggs, sterilizing the egg shells with 75% alcohol, and breakingCollecting yolk from shell, measuring and recording yolk liquid volume V 0 。
(2) Preparing 8% sorbitol aqueous solution with distilled water, adjusting pH to 5.0-5.5 with diluted hydrochloric acid, and then treating with 15V 0 Is added to the collected yolk liquid, and the mixture is stirred on a magnetic stirrer at a low rotation speed for 60 minutes and then left to stand at 4 ℃ overnight. After standing, centrifuging at 10000rpm for 20 min, discarding the precipitate, and collecting water-soluble solution.
D2: fine extraction of egg yolk antibody by affinity chromatography:
(1) The affinity column was packed with an affinity packing coupled with 2-mercaptopyridine and agarose gel, and after completion of the packing, the affinity column was equilibrated with 20mM sodium phosphate buffer (pH 7.5) containing 0.5M potassium sulfate.
(2) The water-soluble solution obtained in D1 was loaded at a flow rate of 5 mL/min, and after loading, the affinity column was equilibrated with 5 column volumes of 20mM sodium phosphate buffer (pH 7.5) containing 0.5M potassium sulfate to allow the egg yolk antibody to be fully affinity adsorbed to the affinity packing in the column.
(3) Eluting with 10 column volumes of 20mM sodium phosphate buffer (pH 7.5) to obtain solution containing high purity egg yolk antibody, measuring and recording the volume V 1 。
D3: ultrafiltration concentration liquid exchange and freeze drying
(1) The solution V obtained in D2 was packed with an ultrafiltration membrane having a molecular cut-off of 50kDa 1 Performing ultrafiltration to obtain a solution with 10V 1 After the completion of the change of volume of PBS buffer, the solution was slowly concentrated to 20mL by ultrafiltration membrane.
(2) Packaging the concentrated yolk antibody solution into a disposable sterile culture dish, freezing at-80deg.C for 5 hr, freeze-drying in vacuum freeze dryer for 12 hr, taking out the freeze-dried sample to obtain pure yolk antibody, and recording as M Pure product 。
After the egg yolk antibody is obtained, agarose gel electrophoresis experiments and titer detection experiments are respectively carried out on the egg yolk antibody, the purity and the titer of the egg yolk antibody are checked, and the yield is counted.
Comparative example 1
In order to verify the effectiveness of the protective agent on the activity of the egg yolk antibody, the stability research is carried out, and the method comprises the following steps:
(1) Sample a is prepared according to example 2 of the present invention, sample b without cetylpyridinium chloride and sorbitol is prepared with reference to example 2 of the present invention, and the two samples are placed in a 37+ -1deg.C incubator for accelerated stability investigation, and potency changes of the two samples under accelerated conditions are investigated;
(2) And detecting the titer of the sample combined with actinobacillus actinomyces by ELISA method, sampling and detecting every two weeks, drawing a titer change trend graph, and comparing and analyzing.
As shown in fig. 1, fig. 1 is a graph showing the stability trend of the egg yolk antibody, wherein sample a is a sample with the protective agent system added, and sample b is a sample without the protective agent system added, and as can be seen from the graph, the initial titers of the two samples are the same and are 1024, the titer of sample a is stabilized at 1024 within 4 weeks, and then is reduced to 512 and is maintained at 512, which indicates that the cost performance is relatively stable and does not significantly decrease with time. The potency of sample b began to drop after 2 weeks, and dropped to 512 at week 4, with no significant drop in one month, but the potency continued to drop to 128 at week 6, after which the potency dropped rapidly, and had dropped to 32 at week 10, indicating a large loss of activity. By comparison, the activity of the compound specific egg yolk antibody is better maintained in the sample added with the protective agent.
Comparative example 2
The preparation method adopts the traditional preparation technology to prepare the compound specificity egg yolk antibody for resisting porphyromonas gingivalis and actinobacillus, and comprises the following steps:
A. culturing and extracting Porphyromonas gingivalis and actinobacillus concomitatus:
A1. bacterial species: porphyromonas gingivalis (Porphyromonas gingivalis, P.g) strain ATCC33277 and actinobacillus concomitans (Aggregatibacter actinomycetemcomitans, A.a) strain ATCC 29523;
A2. taking a porphyromonas gingivalis (P.g) ATCC33277 strain from the Guangdong province microorganism strain collection, and culturing and amplifying the strain in an anaerobic gas-producing bag at 37 ℃ for 3 days by using a blood agar plate culture medium; actinobacillus ATCC29523 strain from the microorganism strain collection of Guangdong province is taken and cultured in blood agar plate medium at 37 ℃ and 5% CO 2 Culturing and amplifying for 1 day under the condition; after culturing and amplifying, scraping bacteria from the culture medium, and centrifuging at the temperature of 4 ℃ and the speed of 8000rpm for 15min to obtain thalli.
B. Preparation of immunogens:
B1. taking porphyromonas gingivalis, and adjusting the concentration by using PBS solution to ensure that the porphyromonas gingivalis absorbs OD in an ultraviolet spectrophotometer 595 Reading at 1.1, and collecting actinobacillus companion, regulating concentration with PBS solution to make it have absorbance OD in ultraviolet spectrophotometer 595 The time reading is 1.2; performing ultrasonic crushing for 25min in a circulating ultrasonic mode for 3s at 25Hz on an ultrasonic crusher, and stopping for 4s, and mixing the seed bacterial suspension according to the volume ratio of 1:1 to obtain mixed bacterial suspension of two bacteria;
B2. compounding the mixed bacterial suspension and Freund's complete adjuvant according to the volume ratio of 1:1, and stirring and emulsifying by using a high-speed stirrer to finally prepare the oil-in-Shui Fushi complete adjuvant antigen;
B3. compounding the mixed bacterial suspension and Freund's incomplete adjuvant according to the volume ratio of 1:1, and stirring and emulsifying by using a high-speed stirrer to finally prepare the oil-in-Shui Fushi incomplete adjuvant antigen.
C. Immunization of egg laying hens:
selecting 20-week-old egg laying hens; primary immunization uses Freund's complete adjuvant antigen, and adopts subcutaneous multipoint injection immunization mode to inject 0.6mL into each hen; the first boost was performed 15 days after the first boost, 0.6mL was injected into each hen using Freund's incomplete adjuvant antigen, and then the boost was performed once every 15 days, five times total boost, and the immunized eggs were collected from the first boost and timely isolated.
D. Preparing a crude extract of the egg yolk antibody:
collecting hen after immunization Sterilizing egg shell with 75% alcohol, breaking shell, collecting yolk, and recording yolk liquid volume V 0 The method comprises the steps of carrying out a first treatment on the surface of the Adding 3V 0 Mixing evenly PBS buffer solution containing 3.0% (w/v) PEG6000, stirring for 40min, and standing for 12h at 2-4 ℃; centrifuging at 4deg.C and 10000rpm for 20 mm, collecting supernatant, and filtering to obtain filtrate V 1 The method comprises the steps of carrying out a first treatment on the surface of the Slowly adding PEG6000 into the filtrate to make the final concentration of PEG6000 be 12% (w/v), and stirring for 40min; centrifuging at 4deg.C and 10000rpm for 20 mm, and discarding supernatant to obtain precipitate, which is the crude yolk antibody M Crude product 。
E. The egg yolk antibody purification step:
specifically, V is used for crude extraction of anti-porphyromonas gingivalis and actinobacillus complex specific egg yolk antibody 0 Dissolving in PBS, adding V 0 The volume of saturated ammonium sulfate ensures that the saturation of the ammonium sulfate in the solution is 50 percent, and the solution is fully mixed and stirred for 40 minutes and is stood for 12 hours at the temperature of 4 ℃; centrifuging at 4deg.C and 10000rpm for 20 mm, and collecting precipitate; the precipitate was treated with 1/5V 0 Dissolving in PBS, and adding 1/10V 0 The volume of saturated ammonium sulfate ensures that the saturation of the ammonium sulfate in the solution is 33 percent, and the solution is fully and uniformly mixed and stirred for 40 minutes and is stood for 12 hours at the temperature of 4 ℃; centrifuging at 4deg.C and 10000rpm for 20 mm, collecting precipitate to obtain semi-pure yolk antibody M Semi-pure product The method comprises the steps of carrying out a first treatment on the surface of the By 1/5V 0 Dissolving the semi-pure yolk antibody product in a volume of PBS solution, dialyzing the sample in the PBS solution for 3 days by using a dialysis bag, timely replacing the PBS solution in the dialysis process, taking out the sample after the dialysis is completed, and sub-packaging the sample into a sterile plate; freezing the antibody solution at-80deg.C, and lyophilizing in a freeze dryer for 12 hr to obtain pure product M of egg yolk antibody with anti-Porphyromonas gingivalis and actinobacillus complex specificity Pure product 。
After the egg yolk antibody is obtained, agarose gel electrophoresis experiments and titer detection experiments are respectively carried out on the egg yolk antibody, the purity and the titer of the egg yolk antibody are checked, and the yield is counted.
Comparison of example 3 with comparative example 2
FIG. 2 is an electrophoresis chart of the anti-Porphyromonas gingivalis and actinobacillus concomitantly specific egg yolk antibodies obtained in example 3 and comparative example 2. As can be seen from the figure, the crude extract of the yolk antibody of example 3 has more protein bands, which indicates that the crude extract contains more impurity proteins; the purity of the antibody is greatly improved through a series of purification steps, and the purity of the egg yolk antibody in the pure product of the compound specificity egg yolk antibody is more than 95 percent through analysis and determination of reducing SDS-PAGE and gray value software.
The crude extract of the yolk antibody of comparative example 2 also contained a large amount of impurity protein, and the pure extract contained an impurity band of about 38kDa in addition to a heavy chain band of about 70kDa and a light chain band of about 20kDa, indicating that the pure extract of the yolk antibody obtained in comparative example 2 still contained a part of impurities, and the purity of the yolk antibody was about 75% as determined by reducing SDS-PAGE and gray-scale software analysis.
In conclusion, the improved preparation method of the egg yolk antibody greatly improves the purity of the egg yolk antibody.
FIG. 3 is a graph showing the potency of the anti-Porphyromonas gingivalis and actinobacillus concomitantly specific egg yolk antibodies obtained in example 3 and comparative example 2. As can be seen from the graph, the yolk antibody preparation obtained in comparative example 2 was tested by ELISA method, and the maximum specific activity of the yolk antibody preparation against Porphyromonas gingivalis per mg of yolk antibody preparation was 1:10240 for 15 days, the maximum specific activity against Actinobacillus actinomycetes was 1:5120 for 15 days, and the rising period and falling period of the titer of the yolk antibody obtained in comparative example 2 were long, and the maintenance period of the maximum specific activity was short. The yolk antibody preparation obtained in example 3 had a maximum specific activity against anti-Porphyromonas gingivalis of 1:20480 per mg of yolk antibody, a specific activity of 1:10240 or more for about 65 days, a maximum specific activity against Actinobacillus actinomycetes of 1:20480, a specific activity of 1:10240 or more for about 60 days, and the yolk antibody titer obtained in example 1 had a short rise period and a short fall period, and a long maintenance period of the larger specific activity. It is also seen from the figure that the titers of the yolk antibody obtained in example 3 against Porphyromonas gingivalis and against Actinobacillus actinomycetemcomitans are complementary in time. In summary, the improved antigen compounding scheme of the invention can make the titers of the egg yolk antibodies complementary in time, and increase the immune effect.
The results of the comprehensive comparison of the yolk antibody preparation method and effect of example 3 with those of comparative example 2 are shown in table 1.
Table 1 comparative table of the methods and effects of preparing yolk antibodies of example 3 and comparative example 2
Figure BDA0002104806520000171
The invention optimizes the immunization scheme, replaces the hen which has laid eggs with the hen which has not laid eggs to immunize, greatly shortens the rising period and the falling period of the titer of the yolk antibody, and obviously prolongs the maintenance period of the high titer of the yolk antibody. Meanwhile, the extraction method starts from each link of the preparation process, reduces the introduction of impurities as much as possible, reduces the titer loss of the egg yolk antibody in the extraction process, and has important significance for maintaining the high titer of the egg yolk antibody.
Test example 1
In order to verify the effect of the obtained pure product of the compound specificity egg yolk antibody against porphyromonas gingivalis and actinobacillus actinomyces on the formation of bacterial biomembrane, the effect of the compound specificity egg yolk antibody on the formation of the bacterial biomembrane of porphyromonas gingivalis (P.g) and actinobacillus (A.a) was examined by adopting a crystal violet staining method. The method comprises the following steps:
(1) Taking a porphyromonas gingivalis (P.g) ATCC33277 strain from the Guangdong province microorganism strain collection, and culturing and amplifying the strain in an anaerobic gas-producing bag at 37 ℃ for 3 days by using a blood agar plate culture medium; actinobacillus ATCC29523 strain from the microorganism strain collection of Guangdong province is taken and cultured in blood agar plate medium at 37 ℃ and 5% CO 2 Culturing and amplifying for 1 day under the condition; after the culture and amplification, the bacteria are scraped from the culture medium, and the bacteria are regulated to OD by using BHI liquid culture medium 595 The reading was 0.8 and then diluted 50-fold for use.
(2) The specific egg yolk antibody obtained in example 3 was used as an experimental group, and the initial concentration was diluted to 2.5mg/mL, and the 2-fold ratio was diluted to 3 concentration gradients; blank egg yolk antibody (non-specific egg yolk antibody) is used as negative control, and the initial concentration is diluted to 2.5mg/mL, and the 2-fold ratio is diluted to 2 concentration gradients; ampicillin at 0.1mg/mL was used as a positive control; blank medium was used as a blank control. Respectively taking 0.05mL of each of the bacterial strains and adding the bacterial strains into the 96-well ELISA plate, respectively taking 0.05mL of diluted bacteria and adding the diluted bacteria into the 96-well ELISA plate, fully and uniformly mixing the bacteria and culturing the bacteria under corresponding bacteria culturing conditions.
(3) After the bacteria culture was completed, the culture was aspirated, and the plate was washed twice with distilled water. Then, 0.05mL of 0.1% crystal violet solution was added to each 96-well ELISA plate, and the plate was stained at room temperature for 15min.
(4) The crystal violet was aspirated, the plate was washed 3 times with distilled water and air dried under natural conditions. Then 0.2mL of 95% ethanol solution is respectively added into the 96-well ELISA plate, and after fully mixing, an ELISA instrument is used for OD 590 And reading the plate and drawing the analysis result.
Results: FIGS. 4 and 5 are graphs showing the effect of the anti-Porphyromonas gingivalis and anti-Actinobacillus actinomycetemcomitans complex specific egg yolk antibodies on the inhibition of the formation of Actinobacillus actinobacillus and Porphyromonas gingivalis bacteria, respectively. As can be seen, 0.1mg/mL ampicillin as a positive control significantly inhibited the formation of bacterial biofilm; the inhibition effect of the specific egg yolk antibody on the bacterial biomembrane in the experimental group changes along with the concentration change, wherein the inhibition effect of the specific egg yolk antibody with the concentration of 1.25mg/mL on the bacterial biomembrane is basically the same as that of the positive control; bacteria normally grow in a blank medium serving as a blank control, and bacterial biofilm formation is not inhibited; the blank yolk antibody as a negative control was identical to the blank control, and the blank yolk antibody could not inhibit the formation of bacterial biofilm without difference in concentration gradient.
Test example 2
In order to verify the binding effect of the obtained pure product of the compound specificity yolk antibody against porphyromonas gingivalis and actinobacillus, the binding effect of the compound specificity yolk antibody against porphyromonas gingivalis (P.g) and actinobacillus (A.a) is tested by adopting a transmission electron microscopy method. The method comprises the following steps:
(1) Well-grown bacteria were collected from the blood plates using PBS, centrifuged, and washed twice with PBS solution. Regulating bacterial concentration to 10 7 CFU/mL, 1mL of bacterial solution was taken.
(2) Taking the compound specific egg yolk antibody obtained in the example 3 as an experimental group and taking blank egg yolk antibodies (non-specific egg yolk antibodies) as a control group, and respectively diluting to 6mg/mL; 1mL of the antibody solution of the experimental group and the control group were mixed with 1mL of bacteria, incubated at 37℃for 2 hours, and then left overnight at 4 ℃.
(3) The mixture was aspirated and washed twice with PBS-BSA wash; the colloidal gold-conjugated rabbit anti-chicken IgY antibodies were diluted 14-fold with PBS-BSA, and then 0.3mL of each was added to the samples of the experimental group and the control group, respectively, and the samples were incubated at 37℃for 2 hours.
(4) The cultures were aspirated, rinsed twice with PBS-BSA wash to remove unbound antibody; resuspended to the original volume with PBS solution. 5 μl of the sample solution was dropped on a 300 mesh copper mesh and adsorbed for 5min.
(5) Negative staining was performed with 2% phosphotungstic acid (ph=7.0, sodium or potassium phosphotungstate, pH adjusted with sodium or potassium hydroxide) for 5min, and after washing and drying, observation was performed under a hitachi HT7700 transmission electron microscope.
Results: FIGS. 6 and 7 are graphs showing the effect of the complex specific egg yolk antibodies on the cell walls of Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis, respectively. As shown in fig. 6, the compound specific egg yolk antibody in the experimental group is adsorbed on the cell wall of actinobacillus actinomycetemcomitans in a large amount, and the blank egg yolk antibody in the control group can only be dissociated around actinobacillus, but cannot be bound on the cell wall; similarly, as can be seen from FIG. 7, the complex specific egg yolk antibody in the experimental group was adsorbed on the cell wall of Porphyromonas gingivalis in a large amount, whereas the blank egg yolk antibody in the control group was free only around Porphyromonas gingivalis and could not be bound to the cell wall. The experiment proves that the obtained compound specificity egg yolk antibody can be specifically combined on the cell walls of actinomycetes and porphyromonas gingivalis, so that the growth and propagation of the actinomycetes and porphyromonas gingivalis are affected.
Test example 3
In order to verify the antibacterial effect of the oral spray provided by the invention on porphyromonas gingivalis and actinobacillus, an antibacterial experiment is carried out, and the method comprises the following steps:
(1) Porphyromonas gingivalis (strain ATCC 33277) from the Guangdong province microorganism collection was cultured in BHI liquid medium at 37℃under anaerobic conditions for 96 hours; actinobacillus concomitatus (ATCC 29523 strain) from Guangdong microorganism collection was cultured in BHI broth at 37℃with 5% CO 2 Culturing for 24 hours under the condition;
(2) Taking cultured bacterial strains, and diluting 100-1000 times as experimental bacterial suspension;
(3) Taking 1 sample to be tested prepared in the example 2, and sucking 5mL to 15mL sterile centrifuge tubes to be used as an experimental group; sucking 5mL of sterile physiological saline into a 15mL centrifuge tube to serve as a control group; respectively sucking 100 mu L of diluted experimental bacterial suspension, adding into an experimental group and a control group, fully mixing, and placing into a water bath kettle at 20+/-1 ℃ for incubation for 5min; taking experimental bacterial suspension, performing 5 times of 10-fold gradient dilution (respectively marked as 1×,2×,3×,4×and5×oforiginal bacterial liquid), and then taking 3×,4×and5×oforiginal bacterial liquid for flat plate coating; taking an experimental group and a control group which are subjected to incubation, respectively performing 2 gradient dilutions (the experimental group is marked as a sample 1×and2×; the control group is marked as a blank 1×and2×), and then respectively performing flat plate coating on an experimental group stock solution (marked as a sample 0×) and a control group stock solution (marked as a blank 0×) and dilutions thereof; after the completion of the plating, the plate was placed under culture conditions for the corresponding bacteria for a corresponding period of time.
(4) After the bacteria on the plate are cultured for a certain time, the plate is taken out for colony counting, and then the bacteriostasis rate is calculated according to the following formula: x= (a-B)/a×100%;
Wherein: x-antibacterial rate, A-average colony count of control group, B-average colony count of experimental group. The experimental results are as follows:
TABLE 2 antibacterial Properties of oral spray against Porphyromonas gingivalis and Actinobacillus actinomycetes
Figure BDA0002104806520000191
The foregoing description is only illustrative of the preferred embodiment of the present invention, and is not to be construed as limiting the invention, but is to be construed as limiting the invention to any and all simple modifications, equivalent variations and adaptations of the embodiments described above, which are within the scope of the invention, may be made by those skilled in the art without departing from the scope of the invention.

Claims (12)

1. The composite specificity egg yolk antibody oral spray is characterized by comprising the following components:
Figure FDA0004092918510000011
pure water, wherein the sum of all components is hundred percent;
the compound specificity egg yolk antibody is added with a protective agent, and the protective agent comprises: 0.4-1% cetylpyridinium chloride;
The preparation method of the compound specific egg yolk antibody comprises the following steps:
(1) Culturing and extracting Porphyromonas gingivalis and actinobacillus;
(2) Preparation of immunogens: respectively taking porphyromonas gingivalis and actinomycetes thallus, carrying out ultrasonic crushing, mixing according to a proportion to obtain mixed bacterial suspension, and preparing an immune antigen by using the mixed bacterial suspension;
(3) Immunization of egg laying hens: selecting hen chicks which are not produced with eggs, starting to feed, and immunizing with an immune antigen 35-55 days before laying eggs;
(4) Separating and purifying the compound specificity egg yolk antibody of the anti-porphyromonas gingivalis and the actinobacillus;
in the step (2), 10-30 parts of Porphyromonas gingivalis strain fragments and 70-90 parts of actinobacillus concomitantly strain fragments are mixed to obtain mixed bacterial suspension of the two bacteria.
2. A method for preparing the composite specific egg yolk antibody oral spray according to claim 1, which comprises the following steps:
(1) Preparing a composite specificity egg yolk antibody mother solution A;
(2) Respectively weighing hydrogenated castor oil, menthol and borneol according to a proportion, placing the hydrogenated castor oil into a container, adding the menthol and the borneol under stirring, and dissolving the menthol and the borneol into the hydrogenated castor oil to prepare a solution B;
(3) Weighing sorbitol, sucralose and watermelon frost according to a proportion, and dissolving the sorbitol, the sucralose and the watermelon frost in proper amount of pure water to prepare a solution C;
(4) Weighing a compound specific egg yolk antibody mother solution A according to 1-10% of the total weight, uniformly mixing the compound specific egg yolk antibody mother solution A with a solution C, adding the solution B under stirring, fully uniformly mixing and emulsifying the solution B into an oil-in-water state, and supplementing pure water until the sum of all components is percentage;
(5) Filtering the solution with a filter membrane, and aseptically filling.
3. The preparation method according to claim 2, wherein the specific method in the step (1) is as follows: weighing the compound specificity egg yolk antibody, preparing with pure water according to a certain antibody titer, adding 0.4-1% cetylpyridinium chloride as a protective agent, and fully and uniformly mixing to prepare a compound specificity egg yolk antibody mother solution A.
4. The method of claim 2, wherein the antibody titer is 1: 10240A standard was prepared with pure water as a complex specific egg yolk antibody mother liquor.
5. The method of any one of claims 2-4, wherein the method of preparing the composite specific egg yolk antibody comprises:
(1) Culturing and extracting Porphyromonas gingivalis and actinobacillus;
(2) Preparation of immunogens: respectively taking porphyromonas gingivalis and actinomycetes thallus, carrying out ultrasonic crushing, mixing in proportion to obtain mixed bacterial suspension of the two bacteria, and preparing immune antigens by using the mixed bacterial suspension;
(3) Immunization of egg laying hens: selecting hen chicks which are not produced with eggs, starting feeding, and immunizing by using the prepared immune antigen 35-55 days before laying eggs;
(4) And (3) separating and purifying the anti-porphyromonas gingivalis and actinobacillus complex specificity egg yolk antibody.
6. The method according to claim 5, wherein 10-30 parts of Porphyromonas gingivalis strain fragments and 70-90 parts of Actinobacillus actinomycetes strain fragments are mixed in the step (2) to obtain a mixed bacterial suspension of the two bacteria.
7. The method according to claim 5, wherein in the step (4), the step of separating and purifying the anti-Porphyromonas gingivalis and actinobacillus concomitantly specific egg yolk antibody comprises, in order: extracting egg yolk antibody by water extraction, extracting egg yolk antibody by affinity chromatography, ultrafiltering, concentrating, and freeze drying to obtain purified compound specific egg yolk antibody.
8. The method of claim 7, wherein the crude extraction of egg yolk antibody by aqueous extraction comprises:
(1) Collecting hen's eggs, sterilizing the egg shells with 75% alcohol, breaking the shells to collect yolk, measuring and recording the yolk liquid volume V 0 ；
(2) Taking sorbitol aqueous solution with mass fraction of 5-10% and pH of 5.0-5.5, and mixing at 10-15V 0 Adding the mixture into the collected yolk liquid, stirring, standing, centrifuging to remove precipitate, and collecting water solution for later use.
9. The method of claim 7, wherein the method of extracting egg yolk antibody by affinity chromatography comprises:
(1) Filling the affinity chromatography column with an affinity filler coupled with 2-mercaptopyridine and agarose gel, and balancing the affinity column with 20mM sodium phosphate buffer solution containing 0.5M potassium sulfate and having pH of 7.5 after filling;
(2) Loading the water-soluble solution of the egg yolk antibody obtained by crude extraction, and balancing an affinity column by using 20mM sodium phosphate buffer solution with the pH of 7.5 and 0.5M potassium sulfate after loading;
(3) Eluting with 20mM sodium phosphate buffer solution with pH of 7.5 to obtain refined egg yolk antibody water solution, measuring and recording the volume V of the obtained water solution 1 。
10. The method of claim 7, wherein the ultrafiltration concentrate exchange and freeze drying process comprises:
(1) Ultrafiltering the water solution of egg yolk antibody with ultrafilter membrane bag to obtain 10V solution 1 After the replacement of the volume of PBS buffer solution is completed, concentrating the solution by using an ultrafiltration membrane bag;
(2) And subpackaging the concentrated yolk antibody solution into a disposable sterile culture dish, sequentially putting into a refrigerator at the temperature of minus 80 ℃ for freezing, and then performing freeze drying in a vacuum freeze dryer, and taking out a freeze-dried sample to obtain a pure yolk antibody product.
11. The method according to claim 10, wherein the ultrafiltration membrane is used having a molecular cutoff of 50KDa.
12. The method of claim 10, wherein the frozen product is frozen in a freezer at-80 ℃ for 2-5 hours and in a vacuum freeze dryer for 12-15 hours.
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