CN104892756A - Antibacterial chicken egg yolk complex antibody as well as preparation method and application thereof - Google Patents
Antibacterial chicken egg yolk complex antibody as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses an antibacterial chicken egg yolk complex antibody as well as a preparation method and an application thereof. The preparation method specifically comprises steps as follows: 1) preparing immunizing antigens of oral pathogenic bacteria respectively; 2) immunizing each flock of healthy hens with one immunizing antigen of the oral pathogenic bacteria; 3) collecting egg yolk of immunized eggs, and performing extraction, purification and separation to obtain an antibody stock solution of each single bacterium; 4) mixing the antibody stock solutions of all the single bacteria, adding a stabilizer, and preparing the antibacterial chicken egg yolk complex antibody. The antibacterial chicken egg yolk complex antibody directly acts on human oropharynges, can be combined with specific pathogens of the oral pathogenic bacteria in a short time, can effectively kill specific oral pathogenic bacteria for a longer time, and is better in safety and wider in application range.
Description
Technical field
The present invention relates to biological antibody immunological technique field, specifically refer to a kind of antibacterial chicken yolk compound antibody and preparation method thereof and application.
Background technology
Also there is more shortcoming in current Broad spectrum antibiotics, not only can not realize specificity and kill, and likely destroy probiotic bacterium in the killing of oral cavity pathogen, and the long-time words used also may cause flora imbalance, produce resistant organism; Chemical process can destroy the structure of oral cavity pathogen, causes the death of pathogenic bacterium, but has hormesis to skin mucosa; Physical method has killing action to oral cavity pathogen, but has injury to human body skin mucous membrane.
Yolk antibody is the corresponding specific antibody that bird produces specific antigen.Yolk antibody is a kind of natural existence, noresidue, the immunoglobulin (Ig) that has no side effect, be antibiotic substitute.Current high immunity egg yolk is widely used in the urgent prevention and therapy of some diseases.
Now have no the antibacterial chicken yolk antibody killing oral cavity pathogen specially, and the existing method preparing chicken yolk antibody, then carry out immunity by hybrid antigen, thus the antibody obtained, but because hen body exists larger difference, it can the chicken yolk antibody of separation and Extraction cannot ensure monoxenic insect killing effect, is difficult to realize suitability for industrialized production.
Existingly prepare in the method for yolk antibody, selected immune chicken is the premunitive healthy bird inlay through selecting, checking and carried out some epidemic disease, still there is the bacterium of some chicken source property or/and viral mixing is stored in egg, and then the chicken yolk antibody affected in egg, as intestinal bacteria, Salmonellas, egg drop syndrome virus, chicken avian leukosis virus etc., be easy to the security having influence on antibody.At present, some researchs select band virose formaldehyde, diethyl enamine (BEI) and beta-propiolactone (BPL) to make inactivator, to chicken yolk antibody exogenous virus, there is inactivating efficacy, but have impact to the activity of chicken yolk antibody, be unfavorable for that very much chicken yolk antibody plays effect.
Summary of the invention
One is the object of the present invention is to provide to directly act on people's pars oralis pharyngis, can be combined with the specific pathogen of oral cavity pathogen in the short period of time, long period can effectively kill specificity oral cavity pathogen, and safety performance is better, uses crowd's antibacterial chicken yolk compound antibody more widely.
Another object of the present invention is the preparation method providing antibacterial chicken yolk compound antibody.
A further object of the invention is to provide the application of antibacterial chicken yolk compound antibody in anti-oral cavity pathogen.
The present invention is achieved through the following technical solutions: a kind of method preparing antibacterial chicken yolk compound antibody, comprises the following steps:
(1) prepare the immunizing antigen of oral cavity pathogen respectively, oral cavity pathogen comprises Hemolytic streptococcus, streptococcus aureus, Candida albicans, intestinal bacteria;
(2) healthy hens is hived off, respectively to the immunizing antigen of every group of a kind of oral cavity pathogens of healthy hens immunity;
(3) collect the immune egg that every group of immune hens produce respectively, and respectively the yolk of immune egg is extracted, purifying, separation, thus obtain each monoxenic antibody stoste;
(4) each monoxenic antibody stoste detects according to antibody titer and mixes, and makes antibacterial chicken yolk compound antibody with addition of stablizer.
Of the present invention prepare the deactivation of oral cavity pathogen bacterium after, dilute respectively, emulsification make each single bacterial immunity antigen; With this antigen injection immunity bird inlay, adaptive immune egg, is separated through sterilization, yolk protein, gets yolk, and water dilution method extraction, centrifugation obtain water soluble ingredient, obtain each single bacterium chicken yolk antibody crude extract with ammonium sulfate extraction; After crude extract buffer solution, ultrafiltration purification; By ultrafiltrated deactivation; According to univalent antibody bioactivity result, prepare multiple flora oral cavity yolk antibody, after Sterile Filtration, add appropriate stablizer make hemolytic hammer streptococcus aureus, Candida albicans intestinal bacteria yolk antibody finished product.This product every milliliter is tired containing corresponding special pathogenic autoantibody and is not less than 1:16.Be mainly used in the prevention of the corresponding pathogenic agent of population pharyngeal mucosa.
In order to better realize method of the present invention, further, in described step (1), the detailed process preparing the immunizing antigen of oral cavity pathogen is:
(1.1) obtain oral cavity pathogen from the throat swab of acute tonsillitis patient inflammation part, be inoculated in the blood agar of the fresh sheep blood of 10% or husky guarantor's substratum;
(1.2) judged by patient's inflammation part symptom, and confirm through gram's staining morphologic observation, Mei Liai assessing instrument and the order-checking of 16sRNA total length, the Main Pathogenic Bacteria in oral cavity comprises beta hemolytic streptococcus, streptococcus aureus, Candida albicans, intestinal bacteria;
(1.3) respectively above-mentioned four kinds of oral cavity pathogens of enlarged culturing, and to adjust bacterial concentration be 2 × 10
9;
(1.4) oral cavity pathogen of above-mentioned four kinds of enlarged culturing is divided into two parts, a by 0.5% formaldehyde solution deactivation 24 hours, another part adopts the corresponding bacterium of respective physical method cracking: 1. intestinal bacteria adopt sonioation method; 2. beta hemolytic streptococcus adopts Tissue Lysis instrument resonance cracking: add bacteria suspension and 50-200 μm of bead 50 μ g in 2mlEP pipe, vibrate 5 minutes, resonate 2-3 time with 25Hz; 3. streptococcus aureus adopts grinding broken; 4. Candida albicans adopts tissue homogenizer cracking;
(1.5) by the oral cavity pathogen of the identical type of deactivation and cracking in the mixing of 1:1 ratio, obtain the immunizing antigen of this kind of oral cavity pathogen;
(1.6) according to step (1.5), the immunizing antigen of four kinds of oral cavity pathogens is obtained respectively.
In order to realize method of the present invention better, further, in described step (2), the concrete immunologic process of hen is:
(2.1) in the immunizing antigen of oral cavity pathogen, equal-volume Freund's complete adjuvant is added, then be placed on Tissue Lysis instrument, with the frequency resonance 5 minutes of 25Hz, repeat twice, emulsification is carried out to the immunizing antigen of this oral cavity pathogen, through proof test, steriling test and physical behavior after the assay was approved, obtained first immunisation emulsion mixture;
(2.2) first immunisation is carried out to healthy hens, only get initial immunity emulsion mixture 1ml/, the immunizing antigen good to the position emulsification such as wing base portion deltoid muscle or chest muscle of healthy hens carries out muscle, subcutaneous multi-point injection, produces better immune response to make bird inlay;
(2.3) in the immunizing antigen of oral cavity pathogen, equal-volume Freund's incomplete adjuvant is added, by the described obtained immune emulsion mixture of step (2.1);
(2.4), during interval 2,4,6,10 weeks, only get immune emulsion mixture 1ml/, booster immunization is carried out to healthy hens.
In order to realize method of the present invention better, further, in described step (3), the yolk of immune egg is extracted, purifying, separation, the concrete steps obtaining single bacteria antibody stoste are as follows:
(3.1) egg that the hen of collecting adaptive immune gives birth to, Ovum Gallus domesticus album wash clean immunity collected afterwards, broken shell, be separated the yellow and white, removes egg white, collects yolk and take weight;
(3.2) by yolk in proportion 1:6 ~ 9 ratio add purified water dilution homogenate, with 1mol/L hydrochloric acid soln adjust pH4.8 ~ 5.5, under 2 ~ 8 DEG C of conditions leave standstill 1 ~ 24 hour;
(3.3) centrifugal with 4000 ~ 20000r/min after leaving standstill, remove throw out, get supernatant liquid, add 10% ~ 35% ammonium sulfate precipitation wherein, fully mix, overnight precipitation;
(3.4) after having precipitated, to throw out with after the centrifugal desalination of 4000 ~ 20000r/min, get the obtained yolk antibody crude extract of precipitation, ammonium sulfate supernatant liquor is removed;
(3.5) obtained precipitation 1/150 ~ 1/300 volume weak acidic buffer redissolves, and adjusts pH4.8 ~ 5.5, makes it fully dissolve, and keep envrionment temperature 2 ~ 8 DEG C to spend the night, get supernatant liquid;
(3.6) obtained supernatant liquor carries out ultrafiltration with retaining relative molecular weight 50 ~ 100kD ultra-filtration membrane, and carries out inactivation of virus;
(3.7) after deactivation, get its supernatant liquid, residue suspension liquid 4000 ~ 20000r/min is centrifugal goes precipitation, and merge supernatant liquid, obtain single bacteria antibody stoste, in the single bacteria antibody stoste obtained, chicken yolk antibody purity is not less than 80%.
In order to realize method of the present invention better, further, in described step (3.6), viral inactivation steps is, being placed on temperature by needing the solution of inactivation of virus is in the environment of 54 ~ 60 DEG C, keeps 8 ~ 20 hours.
In order to realize method of the present invention better, further, in described step (4), the method for acquisition single bacteria antibody stoste being carried out to bioactivity is ELISA method.
The antibacterial chicken yolk compound antibody prepared by aforesaid method, comprises anti-beta hemolytic streptococcus chicken yolk antibody, anti-candida albicans chicken yolk antibody, anti-Staphylococcus aureus chicken yolk antibody, Chinese People's Anti-Japanese Military and Political College's enterobacteria chicken yolk antibody.
In order to realize the present invention better, further, tire to antibacterial Ovum Gallus domesticus Flavus compound antibody and detect, wherein anti-beta hemolytic streptococcus chicken yolk antibody >=1:32, anti-candida albicans chicken yolk antibody tire >=1:16, Chinese People's Anti-Japanese Military and Political College's enterobacteria chicken yolk antibody of >=1:32, anti-Staphylococcus aureus chicken yolk antibody of tiring of tiring is tired >=1:16.
Antibacterial chicken yolk compound antibody is preparing oral cavity pathogen and is infecting the application in the preparation of the relative disease caused.
Antibacterial chicken yolk compound antibody preparation prevention and or treatment oral cavity pathogen infect application in the medicine of the relative disease caused, healthcare products, foodstuff additive and fodder additives.
The sterilized principle for germ of the antibacterial chicken yolk compound antibody of preparation of the present invention is that different oral cavity pathogens has different specific antigenss, and the proteantigen as streptococcus is cell wall constituent, has type specificity, has M, T, R and S etc., and M is relevant with pathogenic; The proteantigen of Candida albicans Pseudomonas is cell wall constituent, is Mannoproteins fragment and can distinguish with serological method the two kinds of Candida albicans serotypes come; The important antigen of Staphylococcus has staphylococcal protein A,SPA, teichoic acid and peptidoglycan; Escherichia coli has thalline O antigen, flagellum H antigen, pod membrane K antigen and envelope antigen etc.
Use several strain beta hemolytic streptococcus, Candida albicans, streptococcus aureus and intestinal bacteria immunity bird inlay, the anti-beta hemolytic streptococcus of specificity that this chicken produces, Candida albicans, streptococcus aureus and colibacillary chicken yolk antibody just not only can produce specific antigens-antibody response with the bacterial isolates of immunity, also can with not of the same race and type there is common antigen, namely the bacterial strain of common antigenic determinant produces antigen-antibody response.When chicken yolk antibody and bacterial surface antigen generation antigen-antibody response, just can change bacterial surface structures, some functionally activies of anti-bacteria, bacteria growing inhibiting, minimizing and anti-bacteria to the sticking of cell, promote the phagocytic cell such as segmented cell and scavenger cell engulfing and removing bacterium.
The present invention compared with prior art, has the following advantages and beneficial effect:
(1) the antibacterial chicken yolk compound antibody produced by the inventive method is mainly used in killing oral cavity pathogen, the reagent made only need at nasal cavity, oral cavity, pharynx, larynx and bronchial mucosa topical, it is rapid-action, consumption is few, both avoid limitation and the side effect of Formulations for systemic administration, also avoid the imbalance causing local flora;
(2) the antibacterial chicken yolk compound antibody emphasis described in the present invention is killed for beta hemolytic streptococcus, streptococcus aureus, Candida albicans, these 4 kinds of oral cavity pathogens of intestinal bacteria, this antibacterial chicken yolk compound antibody is in sterilization process, do not produce acute toxic reaction with bacterium, belong to actual nontoxic rank;
(3) the inventive method is by obtaining single bacterial classification antibody of oral cavity pathogen, but makes the compound antibody of configuration can kill the effect of pathogenic bacterium by independent, coordinates, not easily transformation reactions occurs without the need to complement or with other immunocyte;
(4) antibacterial chicken yolk compound antibody of the present invention is good to the killing effect of oral cavity pathogen, and life-time service can not produce Resistant strain, also can not cause flora imbalance;
(5) the antibacterial chicken yolk compound antibody described in the present invention is to the body surface of living body biological and the equal nonirritant of eyeball, and comparatively long resident, at spraying position, continues to carry out sterilization;
(6) tiring of the antibacterial chicken yolk compound antibody prepared by the present invention is higher, is generally greater than 1:16, therefore has good sterilization effect and more antigen binding site;
(7) the present invention adopts 54 ~ 60 DEG C, and 8 ~ 20 hours inactivation of viruses deactivation modes, had both maintained the biological activity of chicken yolk antibody, does not also have his malicious secondary material and gets involved;
(8) method described in the present invention, antagonist purified water dilute acid metallization processes, uses ammonium sulfate precipitation, applies the purification techniques such as centrifugal and ultrafiltration, makes yolk antibody purity be not less than 80%, makes the extraction efficiency that chicken yolk antibody keeps higher.
Accompanying drawing explanation
Fig. 1 is the schema of method in the present invention;
Fig. 2 is that in the present invention, antigen immune inject time and antibacterial chicken yolk antibody are tired trend map;
Fig. 3 is the SDS-PAGE analysis chart of purifying antibacterial chicken yolk antibody in the present invention.
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail, but embodiments of the present invention are not limited thereto, without departing from the idea case in the present invention described above, according to ordinary skill knowledge and customary means, make various replacement and change, all should comprise within the scope of the invention.
Embodiment 1:
The method preparing antibacterial chicken yolk compound antibody in the present embodiment, as shown in Figure 1, comprises the following steps:
(1) immunizing antigen of oral cavity pathogen is prepared respectively; The detailed process preparing the immunizing antigen of oral cavity pathogen is:
(1.1) obtain oral cavity pathogen from the throat swab of acute tonsillitis patient inflammation part, be inoculated in the blood agar of the fresh sheep blood of 10% or husky guarantor's substratum;
(1.2) judged by patient's inflammation part symptom, and confirm through gram's staining morphologic observation, Mei Liai assessing instrument and the order-checking of 16sRNA total length, the Main Pathogenic Bacteria in oral cavity comprises beta hemolytic streptococcus, streptococcus aureus, Candida albicans, intestinal bacteria;
(1.3) respectively above-mentioned four kinds of oral cavity pathogens of enlarged culturing, and to adjust bacterial concentration be 2 × 10
9;
(1.4) oral cavity pathogen of above-mentioned four kinds of enlarged culturing is divided into two parts, a by 0.5% formaldehyde solution deactivation 24 hours, another part adopts the corresponding bacterium of respective physical method cracking: 1. intestinal bacteria adopt sonioation method; 2. beta hemolytic streptococcus adopts Tissue Lysis instrument resonance cracking: add bacteria suspension and 50-200 μm of bead 50 μ g in 2mlEP pipe, vibrate 5 minutes, resonate 2-3 time with 25Hz; 3. streptococcus aureus adopts grinding broken; 4. Candida albicans adopts tissue homogenizer cracking;
(1.5) by the oral cavity pathogen of the identical type of deactivation and cracking in the mixing of 1:1 ratio, obtain the immunizing antigen of this kind of oral cavity pathogen;
(1.6) according to step (1.5), the immunizing antigen of four kinds of oral cavity pathogens is obtained respectively.
(2) healthy hens hived off, respectively to the immunizing antigen of a kind of oral cavity pathogen of every group of healthy hens immunity, the concrete immunologic process of hen is:
(2.1) in the immunizing antigen of oral cavity pathogen, equal-volume Freund's complete adjuvant is added, then be placed on Tissue Lysis instrument, with the frequency resonance 5 minutes of 25Hz, repeat twice, emulsification is carried out to the immunizing antigen of this oral cavity pathogen, through proof test, steriling test and physical behavior after the assay was approved, obtained first immunisation emulsion mixture;
(2.2) first immunisation is carried out to healthy hens, only get initial immunity emulsion mixture 1ml/, the immunizing antigen good to the position emulsification such as wing base portion deltoid muscle or chest muscle of healthy hens carries out muscle, subcutaneous multi-point injection, produces better immune response to make bird inlay;
(2.3) in the immunizing antigen of oral cavity pathogen, equal-volume Freund's incomplete adjuvant is added, by the described obtained immune emulsion mixture of step (2.1);
(2.4), during interval 2,4,6,10 weeks, only get immune emulsion mixture 1ml/, booster immunization is carried out to healthy hens.
As shown in Figure 2, along with antigen immune increases inject time, antibacterial chicken yolk compound antibody is tired and is presented rhythmicity, and immunization is tired and rised appreciably for one week.
(3) collect the immune egg that every group of immune hens produce respectively, and respectively the yolk of immune egg is extracted, purifying, separation, thus obtain each monoxenic antibody stoste; The yolk of immune egg is extracted, purifying, separation, the concrete steps obtaining single bacteria antibody stoste are as follows:
(3.1) egg that the hen of collecting adaptive immune gives birth to, Ovum Gallus domesticus album wash clean immunity collected afterwards, broken shell, be separated the yellow and white, removes egg white, collects yolk and take weight;
(3.2) by yolk in proportion 1:6 ~ 9 ratio add purified water dilution homogenate, with 1mol/L hydrochloric acid soln adjust pH4.8 ~ 5.5, under 2 ~ 8 DEG C of conditions leave standstill 1 ~ 24 hour;
(3.3) centrifugal with 4000 ~ 20000r/min after leaving standstill, remove throw out, get supernatant liquid, add 10% ~ 35% ammonium sulfate precipitation wherein, fully mix, overnight precipitation,
(3.4) after having precipitated, to throw out with after the centrifugal desalination of 4000 ~ 20000r/min, get the obtained yolk antibody crude extract of precipitation, ammonium sulfate supernatant liquor is removed;
(3.5) obtained precipitation 1/150 ~ 1/300 volume weak acidic buffer redissolves, and adjusts pH4.8 ~ 5.5, makes it fully dissolve, and keep envrionment temperature 2 ~ 8 DEG C to spend the night, get supernatant liquid;
(3.6) obtained supernatant liquor carries out ultrafiltration with retaining relative molecular weight 50 ~ 100kD ultra-filtration membrane, and carries out inactivation of virus, and viral inactivation steps is, being placed on temperature by needing the solution of inactivation of virus is in the environment of 54 ~ 60 DEG C, keeps 8 ~ 20 hours; (3.7) after deactivation, get its supernatant liquid, residue suspension liquid 4000 ~ 20000r/min is centrifugal goes precipitation, merges supernatant liquid, obtains single bacteria antibody stoste; The single bacteria antibody stoste purity extracted, as shown in Figure 3, the antibacterial chicken yolk antibody of 1 expression ammonium sulfate precipitation in Fig. 3,2 represent that the antibacterial chicken yolk antibody of precipitation is through super filter tube purifying, M represents protein markers, the antibacterial chicken yolk antibody concentration extracted by ammonium sulfate method is higher, and assorted band is few; Can be further purified after ultrafiltration, reduce foreign protein; Extract the main band of albumen molecular weight be about 70KD and about 25KD, gel imaging software analysis antibacterial chicken yolk antibody purity is respectively 85%, 94%.
(4) each monoxenic antibody stoste carries out antibody titer detection by ELISA method, finally tire as the 1:32 that tires for beta hemolytic streptococcus chicken yolk antibody, anti-candida albicans chicken yolk antibody 1:32, anti-Staphylococcus aureus chicken yolk antibody 1:16, the Chinese People's Anti-Japanese Military and Political College's enterobacteria chicken yolk antibody 1:16 that tires that tires that tires carries out four kinds of bacterium yolk antibodies mixing according to different antibodies, finally make the antibody mixed solution that total protein concentration is 1 ~ 2.5mg/ml, and make antibacterial chicken yolk compound antibody with addition of stablizer.
The antibacterial chicken yolk compound antibody prepared by above-mentioned method, tired by ELISA method antagonist and detect, wherein anti-beta hemolytic streptococcus chicken yolk antibody >=1:32, anti-candida albicans chicken yolk antibody tire >=1:16, Chinese People's Anti-Japanese Military and Political College's enterobacteria chicken yolk antibody of >=1:32, anti-Staphylococcus aureus chicken yolk antibody of tiring of tiring is tired >=1:16.
Embodiment 2:
The present embodiment is checking antibacterial chicken yolk compound antibody to the bacteriostasis property of beta hemolytic streptococcus, Candida albicans, streptococcus aureus and intestinal bacteria four kinds of oral cavity pathogens, has done following test:
Test organisms 24h slant culture PBS is washed down, make bacteria suspension (concentration of requirement is: with 100 μ L drip in contrast print on or in 5mL sample liquid, bacterial count recovered is 1 × 10
4~ 9 × 10
4cfu/ sheet or ml).
Get and managed by coupons (2.0cm × 3.0cm) or sample liquid (5ml) and contrast print or sample liquid (with sample homogeneous material, equal size, but not containing anti-biotic material, and through sterilising treatment) each 4 (being placed in sterilizing plate in) or 4.
Get above-mentioned bacteria suspension, respectively each by coupons or sample liquid and contrast print or sample liquid or interior dropping 100 μ L, even spread/mixing, start timing, effect 2, 5, 10, 20 minutes, respectively print or sample liquid (0.5mL) are dropped into containing 5mL PBS in vitro with aseptic tweezer, abundant mixing, do suitably dilution, then wherein 2 ~ 3 extent of dilution are got, draw 0.5mL respectively, be placed in two plates, pour into cool nutrient agar (bacterium) to 40 ~ 45 DEG C or sabouraud's agar (yeast) 15mL, rotate plate, make it full and uniform, after agar solidification, upset is dull and stereotyped, cultivate 48h(bacterium for 35 DEG C ± 2 DEG C) or 72h(yeast), to viable bacteria enumeration.
Test-results is as following table:
The antibacterial Ovum Gallus domesticus Flavus compound antibody of table one is to the fungistatic effect of four kinds of oral cavity pathogens
S. aureus-positive contrast bacterial concentration: 3.85 × 10
4cfu/ml; Candida albicans positive control bacterial concentration: 3.82 × 10
4cfu/ml; Intestinal bacteria positive control bacterial concentration: 1.89 × 10
4cfu/ml; Beta hemolytic streptococcus positive control bacterial concentration: 3.48 × 10
4cfu/ml; Negative control Sterile grows.
As seen from the above table, at the conditions of the experiments described above, antibacterial chicken yolk compound antibody stoste is to beta hemolytic streptococcus stoste, streptococcus aureus, intestinal bacteria and Candida albicans effect 2,5,10,20 minutes, and average bacteriostasis rate is 100%.
Embodiment 3:
The present embodiment is the security of antibacterial chicken yolk compound antibody on living organisms surface described in checking, has carried out repeatedly intact skin irritant test to the antibacterial chicken yolk compound antibody obtained.
Concrete process of the test is as follows:
Family's rabbit back backbone both sides are cut by hair by 24h before test, the left and right each about 3cm × 3cm of unhairing scope.Be applied on the 2.5cm × 2.5cm skin of side by antibacterial chicken yolk compound antibody stoste 0.5ml during test, opposite side skin is as blank.After smearing, 4h removes chicken yolk antibody stoste, smears every day once, smears 14d continuously.After smearing, 24h observations, as shown in the table at every turn,
Table two antibacterial chicken yolk compound antibody is to rabbit repeatedly intact skin irritant test result
By upper Biao Ke get, to rabbit repeatedly intact skin irritant test result be nonirritant.
The experiment sufficient proof of the present embodiment, the described skin surface of antibacterial chicken yolk compound antibody to living organisms does not have pungency, in actual use, spreads upon living organisms surface and has good security.
Embodiment 4:
The present embodiment, for the antibacterial chicken yolk compound antibody described in checking is in the security of living body biological eyeball surface, has carried out acute eye irritation test to the antibacterial chicken yolk compound antibody obtained.
Concrete process of the test is as follows:
Getting antibacterial chicken yolk compound antibody stoste 0.1ml instills in rabbit right eye conjunctival sac, after passive closed 4s, 30s with enough, flow velocity very fast but the normal saline flushing 30s that animal eye can not be caused to damage, left eye is blank.The local reaction of 1h, 24h, 48h, 72h, 7d, 14d and 21d after record eye drip.If there is not irritant reaction in 72h, or 7d, 14d, eye irritation reacts completely recovery, can premature termination test.Test-results is as shown in the table,
Table three antibacterial chicken yolk compound antibody is to rabbit eyes irritant test result
Average score means that 24h, 48h, 72h mark sum divided by hop count 3 observing time.
As seen from the above table, antibacterial chicken yolk compound antibody stoste is to Rabbits with Acute eye irritant test result nonirritant.
The described antibacterial chicken yolk compound antibody of the experiment sufficient proof of the present embodiment, when touching the eyeball of living organisms, to eyeball nonirritant, in actual use, do not worry because operating discomfort, cause this antibody-solutions to enter human eye, and cause the discomfort of human eye, embody this antibacterial chicken yolk compound antibody security in vivo applications further.
Embodiment 5:
The present embodiment for checking described in antibacterial chicken yolk compound antibody stability, to obtain antibacterial chicken yolk compound antibody carried out the stability test of microbial method.
Concrete process of the test is as follows:
The sample of antibacterial chicken yolk compound antibody stoste is placed in the environment of 37 DEG C after 90 days, then makes it to Candida albicans effect, as shown in the table,
Table four antibacterial chicken yolk compound antibody deposits the rear fungistatic effect to Candida albicans
Wherein positive control bacterial concentration: 3.56 × 10
4(2.57 × 10
4-4.30 × 10
4) cfu/ml, Negative control Sterile grows.
As seen from the above table, after this antibody places 90 days through 37 DEG C, stoste is to Candida albicans effect 2min, and average bacteriostasis rate is 100%.
Embodiment 6:
The present embodiment is long in the residence time of living body biological different sites for the antibacterial chicken yolk compound antibody described in checking, has carried out the experiment of drug residence time small animal imaging to the antibacterial chicken yolk compound antibody obtained.
Concrete process of the test is as follows:
Modulation pH to 8.5-10.0, joined by a small amount of fluorescence dye in antibacterial chicken yolk compound antibody stoste, temperature is 4 DEG C, and vertical mixing is spent the night.Then ultrafiltration is centrifugal removes unreacted fluorescence molecule, obtains the antibacterial chicken yolk compound antibody stoste containing fluorescence molecule.
Test method: mouth spray will be made containing fluorescence molecule antibacterial chicken yolk compound antibody stoste.Be divided into three groups of experimental group: a group: prohibit water 8 hours after spraying, non-fasting; B group: fasting 8 hours after spraying, can't help water; C group: prohibit water fasting 8 hours after spraying; Skin sprays: spray after rejecting part fur in back.
Spray site fluorescence intensity is gathered by small animal imaging, by fluorescence intensity weakened condition, judge the residence time of antibacterial chicken yolk compound antibody stoste at spray site, after mouth spray, the fluorescence of the antibacterial chicken yolk compound antibody stoste of different group weakens in time, after mouth spray, different tests group all can reach 8.5 hours in the residence time in oral cavity, and wherein fasting taboo water experimental group effectiveness comparison is good.Antibacterial chicken yolk compound antibody stoste containing fluorescence molecule is more than 70 hour in the skin place residence time.
The experiment of the present embodiment is to verify that this antibacterial chicken yolk compound antibody is sprayed in living animal surface or oral cavity; the time stopped all can reach more than 8 hours; there is longer protection period; in actual use, the spray time of this antibacterial chicken yolk compound antibody can be predicted accurately.Other parts of the present embodiment are same as the previously described embodiments, repeat no more here.
Although illustrate and describe embodiments of the invention, those having ordinary skill in the art will appreciate that: do not departing under principle of the present invention and aim and can carry out multiple change, amendment, replacement and modification to these embodiments, scope of the present invention is by claim and equivalents thereof.
Claims (10)
1. prepare a method for antibacterial chicken yolk compound antibody, it is characterized in that: comprise the following steps:
(1) immunizing antigen of oral cavity pathogen is prepared respectively;
(2) healthy hens is hived off, respectively to the immunizing antigen of every group of a kind of oral cavity pathogens of healthy hens immunity;
(3) collect the immune egg that every group of immune hens produce respectively, and respectively the yolk of immune egg is extracted, purifying, separation, thus obtain each monoxenic antibody stoste;
(4) each monoxenic antibody stoste detects according to antibody titer and mixes, and makes antibacterial chicken yolk compound antibody with addition of stablizer.
2. a kind of method preparing antibacterial chicken yolk compound antibody according to claim 1, it is characterized in that: in described step (1), the detailed process preparing the immunizing antigen of oral cavity pathogen is:
(1.1) obtain oral cavity pathogen from the throat swab of acute tonsillitis patient inflammation part, be inoculated in blood agar or substratum;
(1.2) judged by patient's inflammation part symptom, and after carrying out bacterium checking, determine that the Main Pathogenic Bacteria in oral cavity comprises beta hemolytic streptococcus, streptococcus aureus, Candida albicans, intestinal bacteria;
(1.3) the above-mentioned oral cavity pathogen of difference enlarged culturing, and adjust to suitable concentration;
(1.4) oral cavity pathogen of above-mentioned four kinds of enlarged culturing is divided into two parts, a by 0.5% formaldehyde solution deactivation 24 hours, another part makes oral cavity pathogen cracking;
(1.5) by the mixing of the oral cavity pathogen of the identical type of deactivation and cracking, the immunizing antigen of this kind of oral cavity pathogen is obtained;
(1.6) according to step (1.5), the immunizing antigen of four kinds of oral cavity pathogens is obtained respectively.
3. a kind of method preparing antibacterial chicken yolk compound antibody according to claim 2, it is characterized in that: in described step (2), the concrete immunologic process of hen is: in the immunizing antigen of oral cavity pathogen, add Freund's complete adjuvant, emulsification is carried out to immunizing antigen, through Over emulsfication and the immunizing antigen be up to the standards healthy hens is injected first, 2,4,6,10 weeks, interval, use the oral cavity pathogen immunizing antigen of Freund's incomplete adjuvant emulsification, and after the assay was approved, healthy hens is injected, booster immunization.
4. a kind of method preparing antibacterial chicken yolk compound antibody according to claim 1 or 2 or 3, is characterized in that: in described step (3), the yolk of immune egg is extracted, purifying, separation, the concrete steps obtaining single bacteria antibody stoste are as follows:
(3.1) egg that the healthy hens of collecting adaptive immune is given birth to, is separated the yellow and white, removes egg white, collects yolk and takes weight;
(3.2) yolk is added purified water dilution homogenate, be adjusted to weakly acidic condition, leave standstill 1 ~ 24 hour;
(3.3) centrifugal after leaving standstill, get supernatant liquid and saltout, wait for that it precipitates;
(3.4) after having precipitated, centrifugal, obtain the obtained yolk antibody crude extract of precipitation;
(3.5) add weak acidic buffer to redissolve, regulate pH to make it fully dissolve, leave standstill after 1 ~ 24 hour, get supernatant liquid;
(3.6) ultrafiltration, and carry out inactivation of virus;
(3.7) after deactivation, get its supernatant liquid, remaining suspension liquid carries out centrifugation, gets supernatant liquid and merges, and obtains single bacteria antibody stoste.
5. a kind of method preparing antibacterial chicken yolk compound antibody according to claim 4, it is characterized in that: in described step (3.6), viral inactivation steps is, being placed on temperature by needing the solution of inactivation of virus is in the environment of 54 ~ 60 DEG C, keeps 8 ~ 20 hours.
6. a kind of method preparing antibacterial chicken yolk compound antibody according to claim 1 or 2 or 3 or 5, is characterized in that: in described step (4), and the method for acquisition single bacteria antibody stoste being carried out to bioactivity is ELISA method.
7. a kind of antibacterial chicken yolk compound antibody that according to any one of claim 1 ~ 6 prepared by method, is characterized in that: comprise anti-beta hemolytic streptococcus chicken yolk antibody, anti-candida albicans chicken yolk antibody, anti-Staphylococcus aureus chicken yolk antibody, Chinese People's Anti-Japanese Military and Political College's enterobacteria chicken yolk antibody.
8. a kind of antibacterial chicken yolk compound antibody according to claim 7, it is characterized in that: tire to antibacterial Ovum Gallus domesticus Flavus compound antibody and detect, wherein anti-beta hemolytic streptococcus chicken yolk antibody >=1:32, anti-candida albicans chicken yolk antibody tire >=1:16, Chinese People's Anti-Japanese Military and Political College's enterobacteria chicken yolk antibody of >=1:32, anti-Staphylococcus aureus chicken yolk antibody of tiring of tiring is tired >=1:16.
9. antibacterial chicken yolk compound antibody described in any one of claim 1 ~ 8 is preparing oral cavity pathogen and is infecting the application in the preparation of the disease caused.
10. the antibacterial chicken yolk compound antibody described in any one of claim 1 ~ 8 prevents and/or treats oral cavity pathogen in preparation and infects application in the sterilizing agent of the relative disease caused, medicine, healthcare products, foodstuff additive and fodder additives.
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