CN102977208B - Preparation method, application and medicine composition and preparation of specific egg yolk immunoglobulin (IgY) and acinetobacter baumannii, as well as preparation and kit - Google Patents
Preparation method, application and medicine composition and preparation of specific egg yolk immunoglobulin (IgY) and acinetobacter baumannii, as well as preparation and kit Download PDFInfo
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Abstract
The invention provides a preparation method, application and medicine composition and preparation of specific egg yolk immunoglobulin (IgY) and acinetobacter baumannii, as well as a preparation and a kit. The preparation method is characterized by comprising the following steps of: preparing antigen liquor, immunizing hens, and separating and purifying the IgY. In-vivo and in-vitro experiments prove that the prepared specificity IgY has specific binding activity to the acinetobacter baumannii, can be used for effectively inhibiting the growth of the acinetobacter baumannii and alleviating a toxic effect generated by the acinetobacter baumannii, can be applied to prevention, treatment and diagnostic analysis on infectious diseases and complications of the acinetobacter baumannii, and has wide application prospect.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of preparation method of Acinetobacter bauamnnii special yolk immunoglobulin (Ig), application and pharmaceutical composition, preparation and test kit.
Background technology
Acinetobacter bauamnnii (Acinetobacter baumannii) is non-fermentative gram-negative bacilli, it is the flora causing human infection in acinetobacter calcoaceticus, mainly cause respiratory tract infection, microbemia, urinary system infection, wound infection, meningitis etc., also peritonitis, endocarditis, endophthalmitis etc. can be caused, modal infection site is lung, is the important pathogenic former of Nosocomial Pneumonia.
Along with a large amount of uses of clinical antibacterials, the resistance of Acinetobacter bauamnnii is also more and more stronger, multi-drug resistant is occurred to Common Antibiotics such as cephalosporins, aminoglycoside, quinolones, even comprise imipenum to the mould class medicine of the blue or green alkene of carbon or Meropenem all occurs resistance, this brings extreme difficulties to clinical treatment.
Yolk immunoglobulin (Egg yolk immunoglobulin, IgY) to be bird bone-marrow-derived lymphocyte under antigenic substance stimulates produce and be deposited in yolk can with a kind of immunoglobulin (Ig) of antigen generation specific reaction, be the material for improving resistibility that bird is formed in long-term evolution process.IgY, except having the advantages such as high specificity, cheap and easy to get, stable in properties, has no side effect, not easily produces resistance compared with microbiotic, thus gain great popularity compared with chemicals.Special yolk antibody has been considered as the primary drug candidate of substitute antibiotics by U.S. FDA, and Japanese government drops into huge fund and helps " fowl source antibody industry ".
At present, multiple specific IgY obtains application in human and animal's prevention and treatment of diseases.The application of specific IgY in human diseases control comprises infants with rotavirus diarrhoea, helicobacter pylori infection, carious tooth etc., but the application in the control of Acinetobacter bauamnnii infectious diseases yet there are no report.
Summary of the invention
The pharmaceutical composition, preparation and the test kit that the object of this invention is to provide a kind of preparation method of Acinetobacter bauamnnii special yolk immunoglobulin (Ig) (IgY) and application and prepared by this IgY.
Specifically, the preparation method of described Acinetobacter bauamnnii special yolk immunoglobulin (Ig) of the present invention comprises the steps:
(1) antigen liquid is modulated
By Acinetobacter bauamnnii strain inoculation in TSB liquid nutrient medium, after 37 DEG C of cultivation 18-24h, collected by centrifugation thalline, thalline is with normal saline dilution to 10
8-10
10cfu/mL, adds the formaldehyde that bacteria liquid amasss 0.5% in bacterium liquid, and 37 DEG C of deactivation 24h, shake frequently, after steriling test is qualified, add isopyknic Freund's complete adjuvant or Freund's incomplete adjuvant respectively, makes full bacterium inactivated vaccine after fully emulsified, 4 DEG C of preservations;
(2) immune hen
Select the bird inlay of 140-160 age in days, inject 2 points in every thoracic muscle, immunity is carried out in neck subcutaneous injection 1, immunity three times altogether, the vaccine of first immunisation is the antigen liquid adding Freund's complete adjuvant, the vaccine of booster immunization is the antigen liquid adding Freund's incomplete adjuvant, and three times immunizing dose increases progressively, and is respectively every chicken 1.0mL, 1.5mL and 2.0mL, each immunization interval 14 days, after collecting first immunisation, the egg of 1-160 days is preserved in 4 DEG C of classification;
(3) separation, purifying Yolk immunoglobulin
Get immune egg, clean sterilization, shell breaking, albumen is removed with Yolk separator, then draw albumen further with aseptic filter paper, puncture membrane of yolk and collect yolk, yolk liquid adds the deionized water dilution of 6-10 times of volume, pH to 5.0 is adjusted with hydrochloric acid, stir evenly rear 4 DEG C of placements to spend the night, at 4 DEG C, the centrifugal 25min of 10000rpm, gets supernatant liquor, after saltouing, solution of saltouing obtains lyophilized powder through ultrafiltration, lyophilize.
Wherein, described Acinetobacter bauamnnii bacterial classification can use commercially available Acinetobacter bauamnnii standard bacteria, and hospital distributing also can be used from the multidrug resistance Acinetobacter bauamnnii obtained.
Described application of the present invention comprises the application of Acinetobacter bauamnnii special yolk immunoglobulin (Ig) in the medicine for the preparation of control Acinetobacter bauamnnii infectious diseases of above-mentioned preparation and the application in the medicine for the preparation of analyzing and diagnosing Acinetobacter bauamnnii infectious diseases.
In above-mentioned application, described Acinetobacter bauamnnii infectious diseases generally refer in the infective respiratory tract infection of Acinetobacter bauamnnii, microbemia, urinary system infection, wound infection, meningitis, peritonitis, endocarditis, endophthalmitis one or more, refer in particular to Acinetobacter bauamnnii infectious pneumonia or microbemia.
The present invention also provides a kind of pharmaceutical composition and preparation thereof.Described pharmaceutical composition comprises the Acinetobacter bauamnnii special yolk immunoglobulin (Ig) of above-mentioned preparation and pharmaceutically acceptable auxiliary material.Described preparation is the preparation prepared by aforementioned pharmaceutical compositions, is oral preparations, injection formulations or external preparation, comprises tablet, capsule, microcapsule, injection, transfusion, ointment etc.
As tablet, according to therapeutic dose and can tire, appropriate above-mentioned Acinetobacter bauamnnii special yolk Sandoglobulin is mixed with weighting agent (as amylum pregelatinisatum), disintegrating agent (as Microcrystalline Cellulose), lubricant (as micropowder silica gel, talcum powder), compressing tablet or direct compression after dry granulation, then carry out dressing as required.As capsule, according to therapeutic dose and can tire, appropriate above-mentioned Acinetobacter bauamnnii special yolk Sandoglobulin be mixed with weighting agent (as dextrin), inserts capsule.As microcapsule, according to therapeutic dose and can tire, by the solution of appropriate above-mentioned Acinetobacter bauamnnii special yolk immunoglobulin (Ig) and capsule material (as Tisuacryl monomer) according to emulsion polymerization two-step approach, take PLURONICS F87 as stablizer, make nanocapsule or micro-capsule, then make other formulation further.As injection, the solution of certain density above-mentioned Acinetobacter bauamnnii special yolk immunoglobulin (Ig) can be mixed with isotonic regulator (as glucose), stablizer (as halfcystine), 0.1 μm of Entkeimung, according to therapeutic dose and packing of tiring.As ointment, according to therapeutic dose and can tire, appropriate above-mentioned Acinetobacter bauamnnii special yolk immunoglobulin (Ig) be mixed with blank ointment base (mixture as being made up of Macrogol 4000 and poly(oxyethylene glycol) 400) and sanitas (as ethyl p-hydroxybenzoate) and is prepared.
In addition, Acinetobacter bauamnnii special yolk immunoglobulin (Ig) prepared by the present invention can also the form of test kit provide, described test kit comprises the Acinetobacter bauamnnii special yolk immunoglobulin (Ig) of above-mentioned preparation, can also comprise enzyme substrates and other various additives such as stablizer, buffer reagent.
Vivo and vitro experiment shows, the specific IgY that the present invention prepares and Acinetobacter bauamnnii have specific binding activity, effectively can suppress the growth of Acinetobacter bauamnnii and the toxic effect of generation thereof, can be used for control and the diagnositc analysis of Acinetobacter bauamnnii infectious diseases and complication thereof, have broad application prospects.
Accompanying drawing explanation
Fig. 1 is the electrophorogram of the specific IgY after each step purifying of M group and Q group.Band in figure: the ultrafiltration component of 1.M group-specific IgY; The secondary of 2.M group-specific IgY is saltoutd component; 3.IgY standard substance; The component of once saltouing of 4.M group-specific IgY; The water soluble ingredient of 5.M group-specific IgY; The ultrafiltration component of 6.Q group-specific IgY; The secondary of 7.Q group-specific IgY is saltoutd component; The component of once saltouing of 8.Q group-specific IgY; The water soluble ingredient of 9.Q group-specific IgY.
Fig. 2 represents the antibody titer with different time yolk water soluble ingredient after M group-specific IgY immunity.
Fig. 3 represents the antibody titer with different time yolk water soluble ingredient after Q group-specific IgY immunity.
Fig. 4 represents the growth-inhibiting effect of M group-specific IgY to M bacterium.
Fig. 5 represents the growth-inhibiting effect of Q group-specific IgY to M bacterium.
Fig. 6 represents the growth-inhibiting effect of M group-specific IgY to Q bacterium.
Fig. 7 represents the growth-inhibiting effect of Q group-specific IgY to Q bacterium.
Embodiment
One, the preparation of IgY
Adopt the Acinetobacter bauamnnii of two kinds of different sourcess as antigen immune hen respectively in the following examples 1,2, prepare two kinds of Acinetobacter bauamnnii specific IgYs, respectively called after M group and Q group.
Embodiment 1(M group-specific IgY)
(1) antigen liquid is modulated
By Acinetobacter bauamnnii standard bacteria (ATCC-BAA1605, purchased from American standard biological product collecting center (American type culture collection), available from Dalian Chen Yu biochemical reagents company) (be called for short M bacterium) be inoculated in TSB liquid nutrient medium, after 37 DEG C of cultivation 20h, collect thalline in the centrifugal 15min of 6000rpm, thalline is with normal saline dilution to 10
9cfu/ml, blood counting chamber counts.Then in bacterium liquid, add the formaldehyde that bacteria liquid amasss 0.5%, 37 DEG C of deactivation 24h, shake frequently.After steriling test is qualified, adds isopyknic Freund's complete adjuvant or Freund's incomplete adjuvant respectively, after fully emulsified, makes full bacterium inactivated vaccine, 4 DEG C of preservations.Before injection as there is demixing phenomenon in vaccine, then use after needing fully mixing.
(2) immune hen
Select the bird inlay of 140-160 age in days (sea blue laying hen, purchased from Dalian flood livestock company limited), inject 2 points in every thoracic muscle, immunity is carried out in neck subcutaneous injection 1, immunity three times altogether, the vaccine of first immunisation is the antigen liquid adding Freund's complete adjuvant, the vaccine of booster immunization is the antigen liquid adding Freund's incomplete adjuvant, three times immunizing dose increases progressively, be respectively every chicken 1.0mL, 1.5mL and 2.0mL, each immunization interval 14 days, after collecting first immunisation, the egg of 1-160 days is preserved in 4 DEG C of classification.
(3) separation, purifying Yolk immunoglobulin
Get sorted immune egg, clean sterilization, shell breaking, removes albumen with Yolk separator, then draws albumen further with aseptic filter paper, punctures membrane of yolk and collects yolk.Yolk liquid adds the deionized water dilution of 6 times of volumes, adjusts pH to 5.0, stir evenly rear 4 DEG C of placements and spend the night, the centrifugal 25min of 10000rpm at 4 DEG C, get supernatant liquor and water soluble ingredient (WSF) with hydrochloric acid.Saltout through the ammoniumsulphate soln of 50% saturation ratio, 14wt% metabisulfite solution after elder generation, solution of saltouing obtains lyophilized powder through ultrafiltration (Vivaflow50 ultrafiltration system, molecular weight cut-off is 100KD), lyophilize again.
Embodiment 2(Q group-specific IgY)
Hospital distributing is processed from the multidrug resistance Acinetobacter bauamnnii obtained (being called for short Q bacterium) by same method, modulation antigen liquid.Other steps similarly to Example 1, prepare Yolk immunoglobulin lyophilized powder.
The non-specific IgY of embodiment 3()
Except replacing with except physiological saline by bacterium liquid, by method similarly to Example 1, prepare Yolk immunoglobulin lyophilized powder.
Two, purity detecting
Adopt SDS-PAGE(damping fluid to adopt Tris-glycine system, resolving gel concentration is 7.5%, and concentrated gum concentration is 4%), under non reducing conditions, detect the specific IgY purity (as shown in Figure 1) after each step purifying.
As can be seen from Figure 1, be separated through water dilution method and obtain yolk antibody, then saltout for twice through ammonium sulfate and sodium sulfate, the purge process of last ultrafiltration desalination, antibody purity progressively improves, and ultrafiltration and secondary saltout the purity of component more than IgY standard substance (purchased from Promega company).
Three, bioactivity
Adopt indirect elisa method to measure antibody titer after immunity in different time yolk water soluble ingredient (WSF), concrete steps are as follows: being buffered liquid (0.05M carbonate buffer solution, pH9.6) with bag, to dilute antigen bacteria (M bacterium or Q bacterium) to concentration be 10
9cfu/mL, coated elisa plate (every hole 100 μ L), 37 DEG C of incubation 2h, washings (PBS solution of 0.05%Tween20) washes plate three times; Confining liquid (PBS solution of 1%BSA) closes (every hole 100 μ L), 37 DEG C of incubation 1h, and washings washes plate three times; Add the WSF(every hole 100 μ L with the specific IgY of diluent (PBS solution of 0.1%BSA) doubling dilution or non-specific IgY respectively), with the WSF of non-immune group egg be negative control, with three immune serum (extracting vein bloods on the same day, serum in the centrifugal 10min of 5000rpm obtains) be blank (negative control, positive control and blank are respectively every hole 100 μ L) for positive control, with diluent, 37 DEG C of incubation 2h, washings washes plate three times; Add HRP and mark Tu Kangji IgG(available from Sigma) (every hole 100 μ L), 37 DEG C of incubation 1h, washings washes plate three times; Add TMB working fluid (purchased from Huamei Bio-Engrg Co.) (every hole 100 μ L), shady place reaction 20min; Add 2M H
2sO
4carry out stopping (every hole 50 μ L), microplate reader detects OD value (determined wavelength 450nm).The maximum dilution multiple of OD value corresponding to 2.1 times of negative control value is as its valence value (as shown in Figure 2, the result of Q group-specific IgY as shown in Figure 3 for the result of M group-specific IgY).
As can be seen from Figure 2, M group all raises from tiring in 10 after first immunisation day, within 25 days, tires and reaches 12800, within 30 ~ 55 days, reach peak value and be 25600, and high-titer (more than 6400) may persist to latter 120 days of immunity.As seen from Figure 3, Q group also raises from tiring in 10 after first immunisation day, within 25 days, tires and reaches 12800, within 40 ~ 60 days, reach peak value and be 25600, and high-titer (more than 6400) may persist to latter 120 days of immunity.And physiological saline immune group IgY(and non-specific IgY) to tire be 200 always, illustrate and do not have specific IgY to produce.
Four, bacteriostatic experiment
5,10,20mg/mL specific IgY lyophilized powder (M group or Q group) to be dissolved in TSB liquid nutrient medium to desired concn:, in addition, non-specific IgY lyophilized powder is dissolved in TSB liquid nutrient medium to 10mg/mL(negative control 1), cefoperzone sodium and sulbactam sodium is dissolved in TSB liquid nutrient medium to 0.2mg/mL(positive control), get TSB liquid nutrient medium (negative control 2) in addition, respectively that these solution are degerming through 0.22 μm of filtering with microporous membrane.M bacterium or Q bacterium are joined in above-mentioned solution respectively, makes final bacterial concentration be 10
7cfu/mL, in 37 DEG C, hatch under 80rpm concussion speed, sample 100 μ L every 2h, with the blank of TSB liquid nutrient medium for measuring, adopt microplate reader to measure light absorption value (determined wavelength 600nm), result is as shown in figs. 4-7.
Fig. 4 represents the growth-inhibiting effect of M group-specific IgY to M bacterium, and Fig. 5 represents the growth-inhibiting effect of Q group-specific IgY to M bacterium.As can be seen from the specific IgY of Fig. 4,5, M groups and Q group concentration be 5,10 and 20mg/mL time all can suppress the growth of M bacterium completely, substantially identical with positive control medicine bacteriostatic action, but not the growth of specific IgY to M bacterium does not have restraining effect.
Fig. 6 represents the growth-inhibiting effect of M group-specific IgY to Q bacterium, and Fig. 7 represents the growth-inhibiting effect of Q group-specific IgY to Q bacterium.As can be seen from Fig. 6,7, M group-specific IgY concentration be 5,10 and 20mg/mL time be dose-dependently to the growth-inhibiting effect of Q bacterium; Q group-specific IgY concentration be 5,10 and 20mg/mL time to the growth-inhibiting effect of Q bacterium also in dose-dependently, the growth of Q bacterium can be suppressed completely when concentration is 20mg/mL.
Five, to the effect of mouse pneumonia
Get Kunming mouse 50 (Dalian Medical Univ's Experimental Animal Center, credit number SCXK(the Liao Dynasty) 2008-0002), be divided into 5 groups at random, often organize 10.4 groups of mouse intranasals instillation Acinetobacter bauamnnii (Q bacterium) (2 × 10
11cfu/mL, 50 μ L/10g body weight) set up murine pneumonia model; 1 group, as blank group, is not infected Acinetobacter bauamnnii.
Cefoperzone sodium and sulbactam sodium, specific IgY (M group or Q group) respectively with physiological saline solution to desired concn (cefoperzone sodium and sulbactam sodium 20mg/mL, specific IgY 20mg/mL), degerming rear for subsequent use with 0.22 μm of filtering with microporous membrane.
Each group of mouse is respectively at infecting front 24h intraperitoneal injection once, every 10g body weight 150 μ L; And after infection every 24h intraperitoneal injection once, continuous five days, every 10g body weight 250 μ L.The each group of medicine given is as shown in table 1.
Observe the clinical manifestation of each group of mouse, body weight, Temperature changing, the change of white corpuscle (WBC) number, mortality ratio, lung tissue microbial culture, lung tissue outward appearance and lung pathology section.After Acinetobacter bauamnnii infects, except blank group, respectively organizing WBC number in Mouse Weight, body temperature, peripheral blood all has decline in various degree, and the decline of specific IgY (M group and Q group) treatment group is starkly lower than negative control group, close with positive controls; Except blank group, all the other are respectively organized mouse lung tissue microbial culture and are all positive, and specific IgY (M group and Q group) treatment group and positive controls bacterial count are lower than negative control group; Except blank group, all the other respectively organize mouse lung tissue outward appearance damage in various degree, and lung pathology section display alveolar structure destroys, pulmonary fibrosis, but specific IgY (M group and Q group) treatment group and positive controls degree of injury are lower than negative control group; The each group of mouse mortality ratio of four days is respectively: blank group is 0, and negative control group is 90%, and positive controls and specific IgY treatment group are 20%.
Table 1
| Group | Mouse | The medicine given |
| Blank group | Non-pneumonia infection | Physiological saline |
| Negative control group | Pulmonary inflammation model | Physiological saline |
| Positive controls | Pulmonary inflammation model | Cefoperzone sodium and sulbactam sodium |
| Specific IgY treatment group 1 | Pulmonary inflammation model | M group-specific IgY |
| Specific IgY treatment group 2 | Pulmonary inflammation model | Q group-specific IgY |
Six, formulation examples
1. tablet: mixed with amylum pregelatinisatum 375g, Microcrystalline Cellulose 100g, micropowder silica gel 15g by specific IgY (M group or Q group) lyophilized powder 10g, compressing tablet after dry granulation, makes 1000 altogether.
2. capsule: mixed with dextrin 490g by specific IgY (M group or Q group) lyophilized powder 10g, insert capsule, makes 1000 altogether.
3. microcapsule: by specific IgY (M group or Q group) solution 40mL(25mg/mL) with Tisuacryl monomer 10g according to emulsion polymerization two-step approach, be stablizer with PLURONICS F87, make micro-capsule.
4. ointment: by 25g Macrogol 4000 and 64.9g poly(oxyethylene glycol) 400 heating and melting, adding 0.1g ethyl p-hydroxybenzoate makes it dissolve, then be under agitation cooled to rapidly about 35 DEG C, add 10ml specific IgY (M group or Q group) solution (100mg/ml), stir and get final product.
5. injection: lactose 1g, glucose 4.5g, halfcystine 0.05g are dissolved in a small amount of water, add specific IgY (M group or Q group) solution 40ml(25mg/ml), add water and be settled to 100ml, ampoule is sub-packed in after 0.1 μm of filtering with microporous membrane, every bottle of 1ml, seals after lyophilize and hunts leak.
Claims (4)
1. the application of Acinetobacter bauamnnii special yolk immunoglobulin (Ig) in the medicine for the preparation of control or analyzing and diagnosing Acinetobacter bauamnnii infectious diseases;
Described Acinetobacter bauamnnii infectious diseases is Acinetobacter bauamnnii infectious pneumonia or microbemia;
Described Acinetobacter bauamnnii bacterial classification is that commercially available Acinetobacter bauamnnii standard bacteria or hospital distributing are from the multidrug resistance Acinetobacter bauamnnii obtained;
The preparation method of described Acinetobacter bauamnnii special yolk immunoglobulin (Ig), comprises the steps:
(1) antigen liquid is modulated
By Acinetobacter bauamnnii strain inoculation in TSB liquid nutrient medium, after 37 DEG C of cultivation 18-24h, collected by centrifugation thalline, thalline is with normal saline dilution to 10
8-10
10cfu/mL, adds the formaldehyde that bacteria liquid amasss 0.5% in bacterium liquid, and 37 DEG C of deactivation 24h, shake frequently, after steriling test is qualified, add isopyknic Freund's complete adjuvant or Freund's incomplete adjuvant respectively, makes full bacterium inactivated vaccine after fully emulsified, 4 DEG C of preservations;
(2) immune hen
Select the bird inlay of 140-160 age in days, inject 2 points in every thoracic muscle, immunity is carried out in neck subcutaneous injection 1, immunity three times altogether, the vaccine of first immunisation is the antigen liquid adding Freund's complete adjuvant, the vaccine of booster immunization is the antigen liquid adding Freund's incomplete adjuvant, and three times immunizing dose increases progressively, and is respectively every chicken 1.0mL, 1.5mL and 2.0mL, each immunization interval 14 days, after collecting first immunisation, the egg of 1-160 days is preserved in 4 DEG C of classification;
(3) separation, purifying Yolk immunoglobulin
Get immune egg, clean sterilization, shell breaking, albumen is removed with Yolk separator, then draw albumen further with aseptic filter paper, puncture membrane of yolk and collect yolk, yolk liquid adds the deionized water dilution of 6-10 times of volume, pH to 5.0 is adjusted with hydrochloric acid, stir evenly rear 4 DEG C of placements to spend the night, at 4 DEG C, the centrifugal 25min of 10000rpm, gets supernatant liquor, after saltouing, solution of saltouing obtains lyophilized powder through ultrafiltration, lyophilize.
2. a pharmaceutical composition, is characterized in that, described pharmaceutical composition comprises Acinetobacter bauamnnii special yolk immunoglobulin (Ig) and pharmaceutically acceptable auxiliary material;
The preparation method of described Acinetobacter bauamnnii special yolk immunoglobulin (Ig), comprises the steps:
(1) antigen liquid is modulated
By Acinetobacter bauamnnii strain inoculation in TSB liquid nutrient medium, after 37 DEG C of cultivation 18-24h, collected by centrifugation thalline, thalline is with normal saline dilution to 10
8-10
10cfu/mL, adds the formaldehyde that bacteria liquid amasss 0.5% in bacterium liquid, and 37 DEG C of deactivation 24h, shake frequently, after steriling test is qualified, add isopyknic Freund's complete adjuvant or Freund's incomplete adjuvant respectively, makes full bacterium inactivated vaccine after fully emulsified, 4 DEG C of preservations;
(2) immune hen
Select the bird inlay of 140-160 age in days, inject 2 points in every thoracic muscle, immunity is carried out in neck subcutaneous injection 1, immunity three times altogether, the vaccine of first immunisation is the antigen liquid adding Freund's complete adjuvant, the vaccine of booster immunization is the antigen liquid adding Freund's incomplete adjuvant, and three times immunizing dose increases progressively, and is respectively every chicken 1.0mL, 1.5mL and 2.0mL, each immunization interval 14 days, after collecting first immunisation, the egg of 1-160 days is preserved in 4 DEG C of classification;
(3) separation, purifying Yolk immunoglobulin
Get immune egg, clean sterilization, shell breaking, albumen is removed with Yolk separator, then draw albumen further with aseptic filter paper, puncture membrane of yolk and collect yolk, yolk liquid adds the deionized water dilution of 6-10 times of volume, pH to 5.0 is adjusted with hydrochloric acid, stir evenly rear 4 DEG C of placements to spend the night, at 4 DEG C, the centrifugal 25min of 10000rpm, gets supernatant liquor, after saltouing, solution of saltouing obtains lyophilized powder through ultrafiltration, lyophilize;
Described Acinetobacter bauamnnii bacterial classification is that commercially available Acinetobacter bauamnnii standard bacteria or hospital distributing are from the multidrug resistance Acinetobacter bauamnnii obtained.
3. the preparation prepared by pharmaceutical composition according to claim 2, is characterized in that, described preparation is oral preparations, injection formulations or external preparation.
4. a test kit, is characterized in that, described test kit comprises Acinetobacter bauamnnii special yolk immunoglobulin (Ig);
The preparation method of described Acinetobacter bauamnnii special yolk immunoglobulin (Ig), comprises the steps:
(1) antigen liquid is modulated
By Acinetobacter bauamnnii strain inoculation in TSB liquid nutrient medium, after 37 DEG C of cultivation 18-24h, collected by centrifugation thalline, thalline is with normal saline dilution to 10
8-10
10cfu/mL, adds the formaldehyde that bacteria liquid amasss 0.5% in bacterium liquid, and 37 DEG C of deactivation 24h, shake frequently, after steriling test is qualified, add isopyknic Freund's complete adjuvant or Freund's incomplete adjuvant respectively, makes full bacterium inactivated vaccine after fully emulsified, 4 DEG C of preservations;
(2) immune hen
Select the bird inlay of 140-160 age in days, inject 2 points in every thoracic muscle, immunity is carried out in neck subcutaneous injection 1, immunity three times altogether, the vaccine of first immunisation is the antigen liquid adding Freund's complete adjuvant, the vaccine of booster immunization is the antigen liquid adding Freund's incomplete adjuvant, and three times immunizing dose increases progressively, and is respectively every chicken 1.0mL, 1.5mL and 2.0mL, each immunization interval 14 days, after collecting first immunisation, the egg of 1-160 days is preserved in 4 DEG C of classification;
(3) separation, purifying Yolk immunoglobulin
Get immune egg, clean sterilization, shell breaking, albumen is removed with Yolk separator, then draw albumen further with aseptic filter paper, puncture membrane of yolk and collect yolk, yolk liquid adds the deionized water dilution of 6-10 times of volume, pH to 5.0 is adjusted with hydrochloric acid, stir evenly rear 4 DEG C of placements to spend the night, at 4 DEG C, the centrifugal 25min of 10000rpm, gets supernatant liquor, after saltouing, solution of saltouing obtains lyophilized powder through ultrafiltration, lyophilize;
Described Acinetobacter bauamnnii bacterial classification is that commercially available Acinetobacter bauamnnii standard bacteria or hospital distributing are from the multidrug resistance Acinetobacter bauamnnii obtained.
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| CN108992669B (en) * | 2018-08-27 | 2022-03-22 | 广州汇高生物科技有限公司 | Composite yolk antibody composition for preventing and treating respiratory tract infection, aerosol inhalation solution, preparation process and application |
| CN109010824A (en) * | 2018-08-27 | 2018-12-18 | 广州汇高生物科技有限公司 | A kind of special yolk immune globulin composite and its preparation |
| CN111060684A (en) * | 2019-12-31 | 2020-04-24 | 锐纳生物技术(杭州)有限公司 | In-vitro diagnostic kit preservative with dual effects |
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| CN102188449A (en) * | 2011-04-15 | 2011-09-21 | 广西壮族自治区兽医研究所 | Preparation method of high-immune whole-egg powder for resisting piglet diarrhea |
| CN102617731A (en) * | 2012-04-19 | 2012-08-01 | 华中农业大学 | Porcine circovirus-resistant type 2 egg yolk antibody and preparation method and application thereof |
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