CN110357955B - Composite specificity egg yolk antibody and preparation method and application thereof - Google Patents
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Abstract
The invention discloses a compound specificity egg yolk antibody and a preparation method and application thereof, wherein the preparation method of the compound specificity 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 of the invention prolongs the time for maintaining the high titer of the compound specificity egg yolk antibody, improves the yield, the purity and the bioactivity, reduces the production cost, and enables the egg yolk antibody technology to be better applied to industrialized production.
Description
Technical Field
The invention belongs to the technical field of immunology, and particularly relates to a compound specificity egg yolk antibody, a preparation method and application thereof, and more particularly relates to a specific egg yolk antibody for resisting porphyromonas gingivalis and actinobacillus concomitans, and a preparation method and application thereof.
Background
According to statistics, the prevalence rate of oral diseases such as gum and periodontal diseases in China is more than 70% of population. Oral diseases are closely related to pathogenic bacteria in the oral cavity, and Porphyromonas gingivalis (Porphyromonas gingivalis) and Actinobacillus actinomycetemcomitans (Aggregatibacter actinomycetemcomitans) are considered to be two bacteria that cause periodontitis. Porphyromonas gingivalis is the main suspected pathogenic bacteria of periodontal diseases such as chronic gingivitis, chronic periodontitis, invasive periodontitis and the like, and pili of the porphyromonas gingivalis can be combined with glyceraldehyde-3-phosphate dehydrogenase on the surface of oral epithelial cells, invade gingival epithelial cells and gingival fibroblasts, and evade immune clearance. Actinobacillus actinomycetemcomitans express a variety of virulence factors such as CagE protein, leukotoxin, lipopolysaccharide, and cytolethal swollenin, etc., which are involved in the pathogenesis of periodontal disease. Actinobacillus actinomycetemcomitans plays an important role in invasive periodontitis (juvenile periodontitis) and also causes complications such as endocarditis, meningitis, osteomyelitis and the like.
Preventing and treating oral diseases such as periodontitis, and effectively inhibiting pathogenic bacteria from oral pathogenic bacteria, is a relatively direct and feasible method. At present, some products aiming at periodontitis and gingivitis in the market mainly comprise Chinese herbal medicine components, antibiotic components or chemical antibacterial agent components, but the products have slow effects, are not pointed to pathogenic bacteria, can cause side effects such as drug resistance and the like, and have unsatisfactory action effects. Therefore, research and production of the product with the characteristics of strong pertinence, remarkable effect, no side effect and the like are of great significance for preventing and treating related oral diseases.
The egg yolk antibody is also called egg yolk immunoglobulin, is the only immunoglobulin in the egg yolk of poultry, is the egg yolk antibody which is used in many cases at present, and after the egg laying hen is immunized by antigen, the corresponding antibody can be produced in chicken serum, and the hen can effectively transfer the antibody in the serum into the egg yolk in an egg laying mode. Immunization with different antigens will produce specific egg yolk antibodies against the antigen. The yolk antibody does not need to collect blood, accords with the modern animal protection rule, has the advantages of large quantity of produced antibody, strong specificity, stable property and no toxic or side effect, and has the capability of combining with antigen and the growth inhibition effect on the growth of antigen bacteria.
The research and production of egg yolk antibody with the function of inhibiting pathogenic bacteria in oral cavity is one effective way of preventing and treating relevant oral diseases. The existing preparation process of the egg yolk antibody is not stable enough, and the obtained egg yolk antibody has uneven titer, low yield, low purity, poor biological activity and higher cost.
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 compound specificity egg yolk antibody, a preparation method and application thereof, wherein the compound 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 present invention is to provide a method for preparing a complex specific egg yolk antibody, comprising:
(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 into the collected yolk liquid, and the mixed liquid is placed on a magnetic stirrerStirring at a low rotation speed for 30-60 min, and then standing at 4deg.C 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 。
The second purpose of the invention is to provide the compound specificity egg yolk antibody prepared by the preparation method according to one scheme or the combination scheme.
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 third object of the invention is to provide an application of the specific egg yolk antibody in preparing medicines and/or living goods for preventing and/or treating oral diseases;
preferably, the specific egg yolk antibody is applied to the preparation of medicines and/or living goods for preventing and/or treating periodontitis and gingivitis.
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.
After the technical scheme is adopted, compared with the prior art, the invention has the following beneficial effects:
1. 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 antibacterial effect.
2. 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.
3. 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. The invention adopts the method of affinity chromatography to extract the yolk antibody, avoids the influence of adding high molecular polymer on the activity of the yolk antibody, and the water solution after affinity chromatography is the water solution containing the 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 yolk antibody is avoided. The ultrafiltration membrane bag is adopted to replace dialysis for liquid exchange, the liquid exchange is more thorough, the ultrafiltration membrane bag can concentrate the egg yolk antibody aqueous solution, the treatment capacity during freeze drying is reduced, and the treatment efficiency is improved.
4. The obtained egg yolk antibody is verified by a biological membrane inhibition experiment and a transmission electron microscope experiment. The transmission electron microscope experimental result shows that: the obtained compound specificity egg yolk antibody can be specifically combined to cell walls of porphyromonas gingivalis and actinobacillus, thereby affecting the growth and propagation of the two bacteria. The biological membrane inhibition experimental result shows that: the obtained compound specificity egg yolk antibody can inhibit the formation of biological films of porphyromonas gingivalis and actinobacillus actinomyces, and has remarkable effect, and the biological films are the precondition of forming dental plaque, so that the compound specificity egg yolk antibody can reduce the generation of dental plaque to a certain extent and inhibit the development of dental plaque. In conclusion, the composite specificity egg yolk antibody obtained by the invention has high biological activity and can be used as an effective component of medicines or articles for daily use for preventing periodontitis and periodontal disease.
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 diagram showing the electrophoresis of a complex-specific egg yolk antibody against Porphyromonas gingivalis and Actinobacillus actinomycetes in 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. 2 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. 3 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. 4 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. 5 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. 6 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
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 2
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=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 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 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 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 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, separating with centrifugal machine at 10000rpmThe pellet was discarded and the water-soluble solution was kept for further use after 20 minutes.
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
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 eggs after immunization, sterilizing the egg shells with 75% alcohol, breaking the shells to collect yolk and recording the 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 results of example 1 and comparative example 1
FIG. 1 is an electrophoresis chart of a composite specificity egg yolk antibody against Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans obtained in example 1 and comparative example 1. As can be seen from the figure, the crude extract of the egg yolk antibody of example 1 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 1 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 1 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. 2 is a graph showing the potency of the anti-Porphyromonas gingivalis and actinobacillus concomitantly specific egg yolk antibodies obtained in example 1 and comparative example 1. As can be seen from the graph, the yolk antibody preparation obtained in comparative example 1 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 1 were long, and the maintenance period of the maximum specific activity was short. The yolk antibody preparation obtained in example 1 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 1 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 1 with those of comparative example 1 are shown in table 1.
Table 1 comparative table of the methods for producing yolk antibodies of example 1 and comparative example 1 and the effects
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 culturing and amplifyingScraping the bacteria from the culture medium, and adjusting the bacteria to OD with 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 1 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. 3 and 4 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 1 as an experimental group and a blank egg yolk antibody (non-specific egg yolk antibody) 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. 5 and 6 are graphs showing the effect of the combination of a complex-specific egg yolk antibody on the cell walls of Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis, respectively. As shown in fig. 5, 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 the actinobacillus, but cannot be bound to the cell wall; similarly, as can be seen from FIG. 6, 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 only free 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.
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 (7)
1. A method for preparing a compound specific egg yolk antibody, comprising the steps of:
(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; after ultrasonic crushing, 10-30 parts of Porphyromonas gingivalis strain fragments and 70-90 parts of actinomycetes companion strain fragments are mixed to obtain mixed bacterial suspension of the two bacteria;
(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) The method for separating and purifying the anti-porphyromonas gingivalis and actinobacillus complex specificity egg yolk antibody sequentially comprises the following steps: crude extraction of the egg yolk antibody by a water extraction method, fine extraction of the egg yolk antibody by an affinity chromatography method, ultrafiltration concentration liquid exchange and freeze drying to obtain the purified compound specific egg yolk antibody;
the method for extracting the egg yolk antibody by the 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.
2. The method of claim 1, 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 。
3. The method of claim 1, 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.
4. The method according to claim 3, wherein the ultrafiltration membrane is used in a molecular cutoff of 50kDa.
5. The method of claim 1, 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.
6. The preparation method of any one of claims 1 to 5, wherein in the step (2), after the mixed bacterial suspension is obtained, the mixed bacterial suspension and Freund's complete adjuvant are compounded according to a 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.
7. The method of any one of claims 1 to 5, wherein the immunization of the hen without 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.
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