CN106928349A - A kind of type Yolk antibody preparation method of aviadenovirus 4 - Google Patents
A kind of type Yolk antibody preparation method of aviadenovirus 4 Download PDFInfo
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- CN106928349A CN106928349A CN201710135189.9A CN201710135189A CN106928349A CN 106928349 A CN106928349 A CN 106928349A CN 201710135189 A CN201710135189 A CN 201710135189A CN 106928349 A CN106928349 A CN 106928349A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/02—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from eggs
Abstract
The present invention relates to a kind of type refined vitelline antibody preparation method of aviadenovirus 4, it is comprised the following steps：1) using the immune chicken of the type inactivated vaccine of aviadenovirus 4, obtain height and exempt from egg；2) yolk is collected, is crushed using colloid mill after removal vitellinae membrana and frenulum, obtain egg yolk liquid；3) by Tris HCL cushioning liquid that the PH of egg yolk liquid and 0.1mol/L is 7.2~7.4 with 1:6~8 volume ratio mixing, is stirring evenly and then adding into the cyclodextrin of final concentration of 1.0~2.0% (m/v), is sufficiently stirred for, 4 DEG C of 1~2h of standing, and fully at 4 DEG C after balance, 4000r/min is centrifuged 15~20min, collects supernatant；4) supernatant of collection purified, concentrated, aseptic process is obtained final product Yolk antibody.The type Yolk antibody of aviadenovirus 4 as obtained in the inventive method is stable in properties, potency is active strong, is adapted to the extensive preparation of the type Yolk antibody of aviadenovirus 4.
Description
Technical field
The invention belongs to veterinary biologicses technical field, and in particular to a kind of type Yolk antibody preparation side of aviadenovirus 4
Method.
Background technology
The type of aviadenovirus 4 (FAV-4) in fowl I groups of adenovirus is currently study hotspot in the world, has strong cause to chicken
Characteristic of disease, major lesions show as inclusion body hepatitis and hydropericardium-hepatitis syndrome, and the disease can directly trigger chicken farm 20~40
There is more than 30% death rate in its age chicken, in addition its clinically also usually with IBV, exhale the lonely disease of intestines
The accompanying infections such as poison, H9 subtype avian influenza virus, cause kind of laying hen an obvious respiratory diseases occur, arthritis, and serious lay eggs
The problems such as decline and lopsided egg.Therefore the disease has become one of great epidemic disease for having a strong impact on chicken farm production performance at present.
Yolk antibody refers to the antibody for specific antigen extracted from immune eggs, due to only having IgG classes in yolk
Antibody, therefore it is called Yolk immunoglobulin IgG (yolk antibody), referred to as IgY.When body is subject to exotic antigen
After stimulation, the B cell differentiation in the bursa of farbricius turns into thick liquid cell, and secreting specificity antibody enters blood circulation, when blood flows through ovum
During nest, specific antibody (mainly IgG) is gradually accumulated in egg cell, forms Yolk antibody, and IgG divides a word with a hyphen at the end of a line into egg cell is
The result of receptor acting, thus IgG can largely accumulate in egg cell, higher than the IgG in blood, single yolk is (about for concentration
Yolk antibody content is up to 200mg or so in 15ml).Antibody is extracted from yolk in addition compared with extracting antibody from animal blood serum
Simplicity, therefore Yolk antibody has obvious technical advantage.
Additionally, Yolk antibody has preferable stability in various environment.Under the conditions of less than 75 DEG C, Yolk antibody has
Good heat endurance, after 90 DEG C for the treatment of 15min, most of Yolk antibody loses binding activity, in PH<When 4, only a small amount of ovum
Yellow antibody loses activity.In the range of pH4~12, the activity of Yolk antibody is barely affected, in pH>When 12, Yolk antibody
Lose binding activity rapidly.Experiment shows that Yolk antibody has the characteristic of tolerance multigelation, and even across 5 freeze thawing, it resists
Former binding activity is barely affected.Can preserve at room temperature 6 months, 4 DEG C preserve up to more than 5 years, and it is left that activity only declines 5%
It is right.
Due to possessing above-mentioned superior biology, physicochemical property, thus Yolk antibody preparation and application receive it is more next
More concerns.Yet with the Yolk antibody production technology existing defects therefore strong influence product product of prior art
Not only antibody titer is difficult to ensure that for matter, such as some rough antibody products, and would generally pollute Avianreovirus, fowl lymph
The exogenous pathogenic original such as leukemia virus, RE outgrowth factor, Escherichia coli, salmonella, mycoplasma.In injection of antibodies
When prevention or treatment disease, it is most likely that cause vertical transmission cause of disease or some other bacterium, the diffusion even disease of virus
Outburst, bring serious economic loss.Further, since the producer of these rough antibody is often attempted by adding heavy dose
The measure such as formaldehyde, penicillin and streptomycin extend shelf-life of its product, increase so-called use " security ", but result is often
Be cause it is bigger stress be with serious medicament residue.And divide greatly containing substantial amounts of lipoprotein and lecithin etc. in rough antibody
Sub- material, these materials do not have any therapeutic action and volume big, and viscosity is high, injections difficult and are difficult to absorb, right after injection
Body stress be big, easily cause allergic reaction, be EP.Easily caused after injection the bleeding of injection site local organization,
Swelling, necrosis, the tissue of necrosis can cause organism fever, and heating can cause the functions such as body nerve modulation, Humoral immunity
Disorder, so as to cause morbidity colony feed intake to reduce, resistance declines, secondary other diseases.Still further aspect, in Yolk antibody
The very easy oxidation deterioration of unrighted acid and phospholipid of presence, so that the storage life of Yolk antibody is greatly shortened, because
This, set up it is a kind of can largely produce, the extensive use of product purity and activity method of purification high to Yolk antibody will have and weigh
Want meaning.
The separating-purifying to Yolk antibody mainly uses the hydrophily and hydrophobicity of antibody protein to separate at present.It is main
Wanting method has salting out method, ultrafiltration concentration method, chromatography, ion exchange chromatography, caprylic acid precipitation etc..Such as Publication No.：CN
The Chinese patent application " Yolk antibody of chicken inclusion body hepatitis and preparation method thereof " of 106065030A is with aviadenovirus I groups of serum
4 type viral inactivation vaccine immunization laying hens, obtain high-immunity egg, collect yolk, are made after extracting inactivation with the method for acidifying water-octanoic acid
The standby Yolk antibody into chicken inclusion body hepatitis.
The content of the invention
To solve technical problem present in prior art, it is an object of the invention to provide a kind of type ovum of aviadenovirus 4
Yellow preparation method for antibody.
The technical scheme that the present invention is provided is as follows：
The present invention provides a kind of type Yolk antibody preparation method of aviadenovirus 4, and it is comprised the following steps：
(1) using the hy-line brown hen of immune 100 ages in days of the type inactivated vaccine of aviadenovirus 4, obtain height and exempt from egg；
(2) collect height and exempt from egg yolk, crushed using colloid mill after removal vitellinae membrana and frenulum, obtain egg yolk liquid；
(3) PH of the egg yolk liquid for obtaining step (2) and 0.1mol/L be 7.2~7.4 Tris-HCL cushioning liquid with
1:6~8 volume ratio mixing, is stirring evenly and then adding into the cyclodextrin of final concentration of 1.0~2.0% (m/v), is sufficiently stirred for, 4 DEG C
1~2h is stood, fully at 4 DEG C after balance, 4000r/min is centrifuged 15~20min, collects supernatant；
(4) supernatant that step (3) is collected purified, concentrated, aseptic process is obtained final product Yolk antibody.
Preferably, final concentration of the 1.2% of above-mentioned steps (3) cyclodextrin.
Through the egg yolk liquid after homogenate water solubility can be largely being dissolved close to neutral Tris-HCL cushioning liquid
Protein, and by adding a certain amount of cyclodextrin, by centrifugation, using density contrast, water insoluble active ingredient is removed, so that fully
Water-solubility protein liquid (WSF) is isolated on ground from egg yolk, improves the rate of recovery of Yolk antibody.The present inventor is in process of the test
It was found that, when the large usage quantity of cyclodextrin, Yolk antibody can be caused to lose, only in certain concentration range, fully remove water
The rate of recovery of Yolk antibody higher can be kept while insoluble composition, it is preferable that end of the described cyclodextrin in system
Concentration be 1.0~2.0% (m/v), it is further preferred that described cyclodextrin in system final concentration of 1.2%.
Further, supernatant is purified, concentrated specially in above-mentioned steps (4)：
(1) toward adding saturated ammonium sulfate solution in supernatant, until ammonium sulfate in supernatant final concentration of 80%, mix
Even, 4 DEG C of standing 12h, 6000rpm centrifugation 15min, abandoning supernatant takes precipitation；
(2) in mass ratio it is 1:Add sterilized water in the precipitation that 1 ratio is obtained toward step (1), stirring and dissolving, then with
1:The ratio of (2~3) (v/v) adds the acetate buffer that pH is 4.8~5.2, is sufficiently stirred for 20~30min, adds dense eventually
It is the Solutol HS15 of 0.6~1.2% (m/v) to spend, and continues to stir 30~60min, 4 DEG C of standing 12h,
4000rpm is centrifuged 20min, collects supernatant；
(3) saturated ammonium sulfate solution is added in the supernatant collected toward step (2), until end of the ammonium sulfate in supernatant
Concentration is 60%, and 4 DEG C of standing 12h, 4000rpm centrifugation 20min, abandoning supernatant takes precipitation, then uses precipitation
SephadexG25 column chromatographies carry out desalination, obtain final product concentrate.
Preferably, in the step (2) Solutol HS15 final concentration of 0.8%.
Further, acetate buffer is prepared by following methods in the step (2)：Per 1000mL purified waters
Middle dissolving 0.886g sodium acetates and 2.82ml glacial acetic acids, then using the NaOH regulations pH to 4.8~5.2 of 1N.
Further, aseptic process is specially in the step (4)：The concentrate obtained after purified, concentration is used
Filtering accuracy is 0.45 μm of cylindrical filter cartridge refined filtration, then with the cylindrical filter cartridge filtration sterilization that filtering accuracy is 0.2 μm, sterile working
Packing, obtains final product Yolk antibody.
Further, step (1) height exempts from being specially for egg：To the hy-line brown hen hypodermic injection of 100 ages in days
The type inactivated vaccine of 2mL aviadenovirus 4 is immunized, and is immunized 3 times altogether, is 21d per adjacent time interval immune twice, in last time
5d collects egg after immune, obtains final product height and exempts from egg.
Further, step (2) egg yolk liquid is specially：Height exempted from into egg carry out surface with 75% alcohol to disappear
Poison, isolates yolk, crosses 60 mesh nylon mesh removal vitellinae membrana and frenulum, is subsequently placed in 4~6min of grinding in colloid mill, obtains final product egg
Yellow liquor；
Further, the type inactivated vaccine of aviadenovirus of the present invention 4 is the fowl produced with continuous cell line LMH cells
Adenovirus inactivated vaccine.
Above-mentioned Solutol HS15 is obtained by oxirane and hydroxy stearate acid reaction, is a kind of
Nontoxic, harmless medical auxiliary materials, the present inventor adds a certain amount of by substantial amounts of experiment discovery in acetate buffer environment
Solutol HS15, can largely remove the unrighted acid and fat egg in water-solubility protein liquid
It is white to wait composition, improve the purity and stability of Yolk antibody, it is preferable that the Solutol HS15 is in system
In final concentration of 0.6~1.2% (m/v), it is further preferred that the Solutol HS15 is in system
Final concentration of 0.8%.
The present invention type Yolk antibody of aviadenovirus 4 that also protection any of the above methods described is prepared.
Compared with prior art, advantage of the invention is that：
(1) the type Yolk antibody preparation method of aviadenovirus 4 that the present invention is provided has easy, quick, efficient advantage, makes
The type Yolk antibody of aviadenovirus 4 is stable in properties, potency is active strong, be adapted to the extensive system of the type Yolk antibody of aviadenovirus 4
It is standby.
(2) it is high using the type Yolk antibody purity of aviadenovirus 4 obtained in the preparation method for providing of the invention, can effectively remove
The composition such as unrighted acid and lipoprotein, while obtained antibody liquid exists without bacterium, without mycoplasma, without exogenous virus,
It is safe；The malicious animal protection test result of attacking of the type of aviadenovirus 4 virus shows the type Yolk antibody of aviadenovirus 4 to Qingyuan City fiber crops
Yellow chick safety, protective rate is high.
Brief description of the drawings
The change of the neutralizing antibody average level of chicken serum after the injection of Fig. 1 Yolk antibodies.
Specific embodiment
The present invention is further described below by way of specific embodiment, but the present invention is not limited only to following examples.
It is prepared by the type inactivated vaccine of 1 aviadenovirus of embodiment 4
(1) preparation of continuous cell line LMH cells：LMH cells are digested into dispersion passage with 0.025%EDTA- pancreatin, is used
Containing 5~10% NBCSs and appropriate dual anti-DMEM nutrient solutions in 37 DEG C, 5%CO2Cultivated in incubator；
(2) poison breeding is planted：Final volume 1 is pressed with the type seed culture of viruses of aviadenovirus 4:200 are inoculated into the LMH cells of step 1 preparation,
And inhaled after 30 minutes in 37 DEG C of absorption and abandon virus liquid, with containing 1~2% NBCS, appropriate dual anti-and 2mM glutamine
DMEM nutrient solutions are in 37 DEG C, 5%CO2Culture was to 58 ± 2 hours；
(3) collection virus, concentration and purifying：When 80% cytopathy occur in LMH cells, harvesting venom is in -20 DEG C
Twice, 5000rpm collects supernatant, i.e. virus stock solution used to multigelation after 4 DEG C of centrifugation 10min, and potency is carried out to viral supernatant liquid
Determine, its potency may be up to 107.0/0.1mL；Virus stock solution used is concentrated 10 times by the hollow fiber column ultrafilter of 50K, is as made
Seedling diseases venom；
(4) viral level is determined：Seedling virus liquid is done into 10 times with DMEM nutrient solutions to be serially diluted, 10 are taken-5、10-6、10-7、10-8、10-95 dilution factors are inoculated with 48 hole confluent monolayers LMH Tissue Culture Plates, and each dilution factor repeats 5 holes, while setting up the moon
Property compared with control cells hole；Per hole 0.1mL, after 37 DEG C of absorption 30min, add containing 1~2% NBCS, appropriate dual anti-and 2mM paddy
The DMEM nutrient solutions 0.3mL of glutamine is in 37 DEG C, 5%CO2Culture 120 hours, observation of cell lesion (CPE) calculates TCID50,
Per 0.1mL viral levels 108.0TCID50More than, can be used to prepare vaccine；
(5) inactivation of virus：Using 0.1% Formalin inactivation seedling virus liquid, as vaccine antigen；
(6) preparation of vaccine finished product：
1. prepared by oil phase：94 parts of high-quality injection white oil is taken, 2 parts of aluminum stearate, 4 parts of Span-80 is first slow by white oil
Heating, adds 4% Span-80 and 2% aluminum stearate, is heated when stirring, and transparent is until aluminum stearate is fully dissolved to
Only, autoclaving is standby；
2. water is mutually prepared：96 parts of 4 parts of Tween-80s added after sterilizing of vaccine antigen are taken, starts stirring, make Tween-80 complete
Untill CL, water phase is made；
3. emulsify：Emulsified using IKA emulsifying agents, 16000rpm, emulsified 5 minutes；
4. dispense：The vaccine of preparation is dispensed according to every bottle of 250mL.
It is prepared by the type Yolk antibody of 2 aviadenovirus of embodiment 4
(1) using the hy-line brown hen of immune 100 ages in days of the type inactivated vaccine of aviadenovirus 4, obtain height and exempt from egg, specially：
The type inactivated vaccine of hy-line brown hen hypodermic injection 2mL aviadenovirus 4 to 100 ages in days is immunized, and is immunized 3 times altogether, exempts from twice per adjacent
The time interval of epidemic disease is 21d, and 5d collects egg after last time is immune, obtains final product height and exempts from egg；
(2) height is exempted from into egg carries out surface sterilization with 75% alcohol, isolates yolk, crosses 60 mesh nylon mesh removal vitellinae membrana
And frenulum, it is subsequently placed in colloid mill and grinds 5min, obtain final product egg yolk liquid；
(3) PH of the egg yolk liquid for obtaining step (2) and 0.1mol/L be 7.2 Tris-HCL cushioning liquid with 1:8
Volume ratio mixes, and is stirring evenly and then adding into the cyclodextrin of final concentration of 1.2% (m/v), is sufficiently stirred for, 4 DEG C of 1~2h of standing, fills
At 4 DEG C after balance-dividing, 4000r/min centrifugation 20min collect supernatant；
(4) supernatant that step (3) is collected purified, concentrated, specially：
1. toward adding saturated ammonium sulfate solution in supernatant, until ammonium sulfate in supernatant final concentration of 80%, mix
Even, 4 DEG C of standing 12h, 6000rpm centrifugation 15min, abandoning supernatant takes precipitation；
2. in mass ratio it is 1:Sterilized water, stirring and dissolving, then with 1 are added in the precipitation that 1. 1 ratio obtains toward step:
The ratio of (2~3) (v/v) adds the acetate buffer that pH is 4.8, is sufficiently stirred for 25min, adds final concentration of 0.8% (m/
V) Solutol HS15, continues to stir 40min, 4 DEG C of standing 12h, 4000rpm centrifugation 20min, in collection
Clear liquid, wherein acetate buffer are prepared as：0.886g sodium acetates and 2.82ml glacial acetic acids are dissolved in per 1000mL purified waters, so
Afterwards using the NaOH regulations pH to 4.8 of 1N；
3. saturated ammonium sulfate solution is added in the supernatant 2. collected toward step, until end of the ammonium sulfate in supernatant is dense
It is 60% to spend, and 4 DEG C of standing 12h, 4000rpm centrifugation 20min, abandoning supernatant takes precipitation, then uses precipitation
SephadexG25 column chromatographies carry out desalination, obtain final product concentrate.
(5) concentrate of step (4) is purified, concentration carries out aseptic process, specially：After by purified, concentration
To concentrate using the cylindrical filter cartridge refined filtration that filtering accuracy is 0.45 μm, then with the cylindrical filter cartridge mistake that filtering accuracy is 0.20 μm
Bacterium is filtered, sterile working packing obtains final product Yolk antibody.
It is prepared by the type Yolk antibody of 3 aviadenovirus of embodiment 4
(1) using the hy-line brown hen of immune 100 ages in days of the type inactivated vaccine of aviadenovirus 4, obtain height and exempt from egg, specially：
The type inactivated vaccine of hy-line brown hen hypodermic injection 2mL aviadenovirus 4 to 100 ages in days is immunized, and is immunized 3 times altogether, exempts from twice per adjacent
The time interval of epidemic disease is 21d, and 5d collects egg after last time is immune, obtains final product height and exempts from egg；
(2) height is exempted from into egg carries out surface sterilization with 75% alcohol, isolates yolk, crosses 60 mesh nylon mesh removal vitellinae membrana
And frenulum, it is subsequently placed in colloid mill and grinds 6min, obtain final product egg yolk liquid；
(3) PH of the egg yolk liquid for obtaining step (2) and 0.1mol/L be 7.4 Tris-HCL cushioning liquid with 1:6
Volume ratio mixes, and is stirring evenly and then adding into the cyclodextrin of final concentration of 2% (m/v), is sufficiently stirred for, 4 DEG C of standing 1h, fully balance
Afterwards at 4 DEG C, 4000r/min centrifugation 20min collect supernatant；
(4) supernatant that step (3) is collected purified, concentrated, specially：
1. toward adding saturated ammonium sulfate solution in supernatant, until ammonium sulfate in supernatant final concentration of 80%, mix
Even, 4 DEG C of standing 12h, 6000rpm centrifugation 15min, abandoning supernatant takes precipitation；
2. in mass ratio it is 1:Sterilized water, stirring and dissolving, then with 1 are added in the precipitation that 1. 1 ratio obtains toward step:
The ratio of (2~3) (v/v) adds the acetate buffer that pH is 5.0, is sufficiently stirred for 25min, adds final concentration of 1.2% (m/
V) Solutol HS15, continues to stir 50min, 4 DEG C of standing 12h, 4000rpm centrifugation 20min, in collection
Clear liquid, wherein acetate buffer are prepared as：0.886g sodium acetates and 2.82ml glacial acetic acids are dissolved in per 1000mL purified waters, so
Afterwards using the NaOH regulations pH to 5.0 of 1N；
3. saturated ammonium sulfate solution is added in the supernatant 2. collected toward step, until end of the ammonium sulfate in supernatant is dense
It is 60% to spend, and 4 DEG C of standing 12h, 4000rpm centrifugation 20min, abandoning supernatant takes precipitation, then uses precipitation
SephadexG25 column chromatographies carry out desalination, obtain final product concentrate.
(5) concentrate of step (4) is purified, concentration carries out aseptic process, specially：After by purified, concentration
To concentrate using the cylindrical filter cartridge refined filtration that filtering accuracy is 0.45 μm, then with the cylindrical filter cartridge mistake that filtering accuracy is 0.20 μm
Bacterium is filtered, sterile working packing obtains final product Yolk antibody.
It is prepared by the type Yolk antibody of embodiment 4-6 aviadenovirus 4
The preparation process of the type Yolk antibody of 4,5,6 aviadenovirus of embodiment 4 is substantially the same manner as Example 2, and difference is, institute
The final concentration for stating addition cyclodextrin in step (3) is respectively 0.5%, 2.5%, 3.0% (m/v).
It is prepared by the type Yolk antibody of 1 aviadenovirus of comparative example 4
The preparation process of the type Yolk antibody of 1 aviadenovirus of comparative example 4 is substantially the same manner as Example 2, and difference is, the step
Suddenly (3) are added without cyclodextrin.
It is prepared by the type Yolk antibody of 2 aviadenovirus of comparative example 4
The preparation process of the type Yolk antibody of 2 aviadenovirus of comparative example 4 is substantially the same manner as Example 2, and difference is, the step
Suddenly (4) are added without Solutol HS15.
It is prepared by the type Yolk antibody of 3 aviadenovirus of comparative example 4
The preparation process of the type Yolk antibody of 3 aviadenovirus of comparative example 4 is substantially the same manner as Example 2, and difference is, the step
Suddenly pH is that 4.8 acetate buffer replaces with purified water in (4).
Test example one, the type Yolk antibody quality control of aviadenovirus 4
According to《People's Republic of China's regulations》Embodiment of the present invention 2-6 and comparative example 1-3 is obtained
The type Yolk antibody of aviadenovirus 4 carry out steriling test, mycoplasma inspection, exogenous virus detection, as a result do not detect.
Test example two, the type Yolk antibody safety testing of aviadenovirus 4
Choose by SPF hatchings, and raise to 10 day healthy chicken in age and do safety test in isolation room.It is classified as 9
Group, every group 5, the Yolk antibody solution branch intramuscular injection chicken with embodiment 2-6 and obtained in comparative example 1-3, injects respectively
Amount is respectively 5mL, wherein there is one group not inject as a control group.Test chicken is raised in isolation room.Temperature detection before injection, after injection
Temperature detection 2d, it is sooner or later each 1 time, and observe injection site whether there is abnormal conditions appearance.
Result shows, compares with control group, before embodiment of the present invention 2-6 and Yolk antibody injection obtained in comparative example 1-3
± 0.1 DEG C is with the body temperature difference after injection, and the spirit of each group experiment chicken, appetite and drink are intended to normally, injection site
Skin and muscle situation without exception occur, and show that the Yolk antibody of present invention offer is safe.
Influence of the test example three, cyclodextrin to protein recovery
The egg yolk liquid that step (2) in embodiment 2-6 obtains and the supernatant that step (3) is obtained are determined using BCA methods respectively
The concentration of middle protein, calculates protein recovery, to evaluate the influence for adding various concentrations cyclodextrin to the Yolk antibody rate of recovery,
Result see the table below 1.
Influence of the cyclodextrin of the various concentrations of table 1 to protein recovery
| Group | Cyclodextrin final concentration (m/v) | Protein recovery (%) |
| Embodiment 2 | 1.2% | 96.4 |
| Embodiment 3 | 2.0% | 95.8 |
| Embodiment 4 | 0.5% | 90.5 |
| Embodiment 5 | 2.5% | 84.2 |
| Embodiment 6 | 3.0% | 80.4 |
During Yolk antibody is prepared, can in the Tris-HCL cushioning liquid close to neutrality through the egg yolk liquid after homogenate
Largely to dissolve water soluble protein, adding a certain amount of cyclodextrin can effectively remove by centrifugation, using density contrast,
Water insoluble active ingredient is removed, so as to fully isolate water-solubility protein liquid (WSF) from egg yolk.As seen from the above table, ring is worked as
The consumption of dextrin is smaller, and protein recovery is relatively low；When its large usage quantity, Yolk antibody can be caused to lose, hence it is evident that to reduce albumen and return
Yield, when its in system final concentration of 1.2% and 2.0% when, can be kept while fully removing water insoluble active ingredient
The rate of recovery of Yolk antibody higher.
Test example four, the agar gel diffusion test method detection type Yolk antibody potency of aviadenovirus 4
1% agar is prepared with deionized water, after after agar, about 2mm thick flat board, in agar after solidification
The plum blossom hole of a diameter of 3mm is beaten on plate.The type viral antigen liquid of aviadenovirus 4, other holes are added in the medium pore of plum blossom hole
Sequentially add the Yolk antibody liquid of different extension rates.Situation about being occurred according to precipitation line judges antibody titer, knot
Fruit see the table below 2.
The potency of the Yolk antibody liquid of table 2
| Group | Embodiment 2 | Embodiment 3 | Embodiment 4 | Embodiment 5 | Embodiment 6 | Comparative example 1 | Comparative example 2 | Comparative example 3 |
| Potency | 1:132 | 1:130 | 1:120 | 1:108 | 1:100 | 1:94 | 1:80 | 1:98 |
From embodiment 4-6 and comparative example 1, the concentration of cyclodextrin too low (or without) or too high can cause to be obtained
The potency of Yolk antibody liquid significantly reduce, from comparative example 2, Solutol HS15 resists to improving yolk
The potency of body fluid plays an important roll, and from comparative example 3, acetate buffer solution has to the potency for improving Yolk antibody liquid
Certain effect, at the same explanation, only and when in acetate buffer solution environment, Solutol HS15 ability
Play one's part to the full.
Test example five, SDS-PAGE detects the type Yolk antibody purity of aviadenovirus 4
Detect embodiment 2, Yolk antibody solution purity obtained in comparative example 1-3 respectively using SDS-PAGE, as a result show,
Embodiment 2 is the visible obvious and single albumen of 67~70kD (heavy chain) and 22~30kD (light chain) place in relative molecular weight
Band, and illustrate its type Yolk antibody purity of aviadenovirus 4 extracted almost without foreign protein at other relative molecular weights
Height, and comparative example 1-3 has more obvious multiple bands, illustrate the type Yolk antibody impurity of aviadenovirus 4 that it is extracted it is many,
Purity is low.
Test example six, the type Yolk antibody neutralization test method of aviadenovirus 4
By existing《Republic of China Veterinary Pharmacopoeia》Neutralization test method detect Yolk antibody obtained in embodiment 2 respectively
Potency, as a result see the table below shown in 3 and Fig. 1.
The situation of change of the neutralizing antibody of chicken serum after the injection of the Yolk antibody of table 3
Result shows, the NAT produced after the obtained type Yolk antibody of aviadenovirus 4 of the present invention is immune it is high and
Duration is long.
Test example seven, the type Yolk antibody liquid of aviadenovirus 4 attack malicious animal protection test
10 Japanese instar chickling 50 is taken, is divided into 5 groups, every group 10, respectively embodiment 2, comparative example 1-3 and control group, respectively
The type Strain 1mL (10 of group difference intramuscular injection aviadenovirus 44ECL50/ mL), attacking poison 48 hours, intramuscular injection respectively is implemented
Example 2, Yolk antibody 1mL/ obtained in comparative example 1-3, control group injecting normal saline.Continuous Observation 15 days, counts each group ovum
The protective rate of yellow antibody, and the state of chick is observed, as a result see the table below shown in 4.
The Yolk antibody liquid of table 4 attacks malicious animal protection test result
Result shows, Yolk antibody obtained in the embodiment of the present invention 2 the type virus of aviadenovirus 4 is attacked poison chick have compared with
Good protective effect, protective rate is up to 100%, and the protective rate of comparative example 1 and 2 is only 40% and 30%, shows cyclodextrin and gathers
Ethylene glycol 15- hydroxy stearic acid esters can effectively improve the Yolk antibody rate of recovery, purity, improve potency activity.Can by comparative example 3
Know, the acetate buffer that pH is 4.8 is replaced with purified water, obtained Yolk antibody protective rate can be caused to reduce, only 70%,
Show that acetate buffer has certain effect to the purity tool for improving Yolk antibody, while show, only and when being 4.8 in pH
Under acetate buffer environment, Solutol HS15 can just play one's part to the full.
Test example eight, stability test
The embodiment of the present invention 2 and Yolk antibody liquid obtained in comparative example 1-3 are placed in 37 DEG C of environment, respectively preserve 1 week,
2 weeks, 3 weeks, January, June, December, using agar gel diffusion test method detect the type Yolk antibody potency of aviadenovirus 4, the results are shown in Table 5.
The potency change of the different holding time each group Yolk antibodies of table 5
| Group | 1 week | 2 weeks | 3 weeks | January | June | December |
| Embodiment 2 | 1:132 | 1:132 | 1:132 | 1:132 | 1:130 | 1:128 |
| Comparative example 1 | 1:94 | 1:90 | 1:86 | 1:70 | 1:32 | - |
| Comparative example 2 | 1:80 | 1:75 | 1:70 | 1:64 | 1:26 | - |
| Comparative example 3 | 1:98 | 1:94 | 1:90 | 1:85 | 1:56 | 1:22 |
Result shows that the type Yolk antibody of aviadenovirus 4 that the embodiment of the present invention 2 is refining to obtain preserves 12 in 37 DEG C of environment
Moon potency change is little, has good stability.
The above is only the preferred embodiment of the present invention, it is noted that it is right that above-mentioned preferred embodiment is not construed as
Limitation of the invention, protection scope of the present invention should be defined by claim limited range.For the art
For those of ordinary skill, without departing from the spirit and scope of the present invention, some improvements and modifications can also be made, these change
Enter and retouch and also should be regarded as protection scope of the present invention.
Claims (9)
1. the type Yolk antibody preparation method of a kind of aviadenovirus 4, it is characterised in that comprise the following steps：
(1) using the hy-line brown hen of immune 100 ages in days of the type inactivated vaccine of aviadenovirus 4, obtain height and exempt from egg；
(2) collect height and exempt from egg yolk, crushed using colloid mill after removal vitellinae membrana and frenulum, obtain egg yolk liquid；
(3) PH of the egg yolk liquid for obtaining step (2) and 0.1mol/L be 7.2~7.4 Tris-HCL cushioning liquid with 1:6~
8 volume ratio mixing, is stirring evenly and then adding into the cyclodextrin of final concentration of 1.0~2.0% (m/v), is sufficiently stirred for, and 4 DEG C stand 1
At 4 DEG C after~2h, fully balance, 4000r/min is centrifuged 15~20min, collects supernatant；
(4) supernatant that step (3) is collected purified, concentrated, aseptic process is obtained final product Yolk antibody.
2. the type Yolk antibody preparation method of aviadenovirus according to claim 14, it is characterised in that in the step (4)
Supernatant is purified, concentration step is specially：
(1) toward adding saturated ammonium sulfate solution in supernatant, until ammonium sulfate in supernatant final concentration of 80%, mix, 4
DEG C 12h, 6000rpm centrifugation 15min are stood, abandoning supernatant takes precipitation；
(2) in mass ratio it is 1:Sterilized water, stirring and dissolving, then with 1 are added in the precipitation that 1 ratio is obtained toward step (1):(2
~3) ratio of (v/v) add the acetate buffer that pH is 4.8~5.2, be sufficiently stirred for 20~30min, add final concentration of
The Solutol HS15 of 0.6~1.2% (m/v), continues to stir 30~60min, and 4 DEG C stand 12h, 4000rpm
Centrifugation 20min, collects supernatant；
(3) saturated ammonium sulfate solution is added in the supernatant collected toward step (2), until final concentration of the ammonium sulfate in supernatant
It is 60%, 4 DEG C of standing 12h, 4000rpm centrifugation 20min, abandoning supernatant takes precipitation, then uses precipitation
SephadexG25 column chromatographies carry out desalination, obtain final product concentrate.
3. the type Yolk antibody preparation method of aviadenovirus according to claim 14, it is characterised in that in the step (3)
Final concentration of the 1.2% of cyclodextrin.
4. the type Yolk antibody preparation method of aviadenovirus according to claim 24, it is characterised in that in the step (2)
Final concentration of the 0.8% of Solutol HS15.
5. the type Yolk antibody preparation method of aviadenovirus according to claim 24, it is characterised in that in the step (2)
Acetate buffer is prepared by following methods：0.886g sodium acetates and 2.82ml ice second are dissolved in per 1000mL purified waters
Acid, then using the NaOH regulations pH to 4.8~5.2 of 1N.
6. the type Yolk antibody preparation method of aviadenovirus according to claim 14, it is characterised in that in the step (4)
Aseptic process is specially：Will after purified, concentration the concentrate that obtains using the cylindrical filter cartridge refined filtration that filtering accuracy is 0.45 μm,
Again with the cylindrical filter cartridge filtration sterilization that filtering accuracy is 0.20 μm, sterile working packing obtains final product Yolk antibody.
7. the type Yolk antibody preparation method of aviadenovirus according to claim 14, it is characterised in that the step (1) is high
Exempt from being specially for egg：The type inactivated vaccine of hy-line brown hen hypodermic injection 2mL aviadenovirus 4 to 100 ages in days is immunized, and exempts from altogether
Epidemic disease 3 times, is 21d per adjacent time interval immune twice, and 5d collects egg after last time is immune, obtains final product height and exempts from egg.
8. the type Yolk antibody preparation method of aviadenovirus according to claim 14, it is characterised in that step (2) egg
Yellow liquor is specially：Height is exempted from into egg carries out surface sterilization with 75% alcohol, isolates yolk, crosses the removal of 60 mesh nylon mesh
Vitellinae membrana and frenulum, are subsequently placed in 4~6min of grinding in colloid mill, obtain final product egg yolk liquid.
9. the type Yolk antibody of aviadenovirus 4 for being prepared according to any methods describeds of claim 1-8.
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| CN109053880A (en) * | 2018-08-26 | 2018-12-21 | 广州渔跃生物技术有限公司 | 4 type Yolk antibody of aviadenovirus and preparation method thereof |
| CN111320689A (en) * | 2020-01-09 | 2020-06-23 | 广州渔跃生物技术有限公司 | Preparation method of duck reovirus, duck parvovirus and duck astrovirus trivalent egg yolk antibody |
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| CN111320689A (en) * | 2020-01-09 | 2020-06-23 | 广州渔跃生物技术有限公司 | Preparation method of duck reovirus, duck parvovirus and duck astrovirus trivalent egg yolk antibody |
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Application publication date: 20170707 |