CN109053880A - 4 type Yolk antibody of aviadenovirus and preparation method thereof - Google Patents
4 type Yolk antibody of aviadenovirus and preparation method thereof Download PDFInfo
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- CN109053880A CN109053880A CN201810977191.5A CN201810977191A CN109053880A CN 109053880 A CN109053880 A CN 109053880A CN 201810977191 A CN201810977191 A CN 201810977191A CN 109053880 A CN109053880 A CN 109053880A
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- aviadenovirus
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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
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
- C07K2317/11—Immunoglobulins specific features characterized by their source of isolation or production isolated from eggs
Abstract
The invention belongs to veterinary biologics technical fields, and in particular to the preparation method of 4 type Yolk antibody of aviadenovirus, including A) 4 type inactivated vaccine Immune Laying Hens of aviadenovirus are used, adaptive immune egg further obtains yolk liquid；B above-mentioned yolk liquid is mixed with Tris-HCl buffer salt solution), phenol solution is added, is mixed, is stood, low-speed centrifugal, supernatant is collected；C the Solutol HS15 of final concentration of 0.2~1.0 (m/v)) is added into supernatant, is stirred, stands certain time, supernatant is collected in centrifugation；D) that supernatant is purified, concentration, aseptic procedures to get.4 type Yolk antibody preparation method of aviadenovirus provided by the invention is not because be related to the operation such as subsequent gel chromatography, it is at low cost, it is easy to operate, while passing through the improvement to extraction step and purification step, so that the rate of recovery of antibody, degreasing rate are significantly higher than existing method, significant progress is achieved.
Description
Technical field
The invention belongs to veterinary biologics technical fields, and in particular to 4 type Yolk antibody of aviadenovirus and its preparation side
Method.
Background technique
Yolk antibody (immunoglobulin of egg yolk, IgY) is immunoglobulin present in bird egg Huang, is
The antibody to filial generation with protective effect generated in yolk is transferred to by the IgG in fowl blood.The molecular mass of IgY is about
180ku is made of, wherein light chain molecule quality about 22~30ku 2 light chains and 2 heavy chains, heavy chain molecule quality be 67~
70ku, isoelectric point is close to 5.2.Compared with IgG, IgY has the advantage of animal germline genetis method distance, is more suitable for production specificity
Antibody.Since IgY has unique physicochemical property and biological characteristics, the IgY of anti-specific antigen is being generated as fowl host just
Gradually by the concern of scholar.
In yolk about containing 30% lipid (moisture accounts for about 48%, 18%) protein accounts for about, wherein most fat with
Protein exists in the form of lipoprotein.Yolk includes water-solubility protein (α-livitin, β-livitin, γ-yolk
Globulin (IgY)).And the IgY of high quality, it must be with high purity, meanwhile, antibody recovery rate wants high.
And to extract IgY and first have to obtain water soluble ingredient (water soluble fraction, WSF), it is domestic at present
Outer scholar has expanded extensive research, as poloxamer is added into egg yolk liquid by inventor in prior art CN104072609A
Extract WSF；As in CN101717445B inventor with polyethylene glycol obtain water-solubility protein WSF；V.Ntakarutimana etc. will
TBS buffer salt solution containing chloroform be added in egg yolk liquid extract WSF [NTAKARUTIMANA V, DEMEDTS P,
VanSANDE M, et al.A simple and economical strategy for downstream processing
of specific antibodie s to human transferrin from egg yolk[J].J Immunol
Methods, 1992,153 (1/2): 133-140.].But the rate of recovery of Yolk antibody not more than 80% in the above method.
Another committed step for preparing of high quality Yolk antibody is purification step.Current more common purification process
Salting out method, gel-filtration chromatography, ion exchange chromatography, hydrophobic chromatography etc..Wherein, salting out method is simple, economical, but mentions
The IgY purity taken is low (being no more than 70%), and fatty residual quantity is high；And although chromatography can obtain the IgY (90%) of high-purity,
It is its low yield, and costly, is not suitable for large-scale application, is only applicable to scientific research.
Summary of the invention
The present invention is intended to provide 4 type Yolk antibody of aviadenovirus and preparation method thereof, this method is suitable for actual production ovum
Yellow antibody, because its is at low cost, and the Yolk antibody rate of recovery reaches as high as 96.6%, and prior fat residual quantity is low, is lower than
1%.
In order to achieve the above object, the invention adopts the following technical scheme: the preparation side of 4 type Yolk antibody of aviadenovirus
Method, comprising the following steps:
A 4 type inactivated vaccine Immune Laying Hens of aviadenovirus) are used, adaptive immune egg, after sterilized, protein isolate collects ovum
Huang further obtains yolk liquid；
B) above-mentioned yolk liquid is mixed with Tris-HCl buffer salt solution, be added phenol solution, mix, stand, low speed from
The heart collects supernatant；
C the Solutol HS15 of final concentration of 0.2~1.0 (m/v)) is added into supernatant, stirring is mixed
It closes, stands, supernatant is collected in centrifugation；
D the supernatant for) obtaining step C) is purified, is concentrated, aseptic procedures, obtains Yolk antibody.
Preferably, the step B) in yolk liquid with Tris-HCl buffer salt by 1:(3~6) volume ratio mix.
Preferably, the step B) in yolk liquid with phenol solution by 1:(1~3) volume ratio mix.
Preferably, the purification step specifically:
Into above-mentioned supernatant by 1:1 mass ratio be added phosphate buffer, stirring and dissolving, be added final concentration 0.01~
The CMC value of 1% (m/v) is the detergent of 0.1~8.0mM, is mixed, and is stood, and centrifugation collects precipitating, by precipitating sterile physiological
Salt water dissolution after, through macroporous resin adsorption remove detergent to get.
Preferably, the CMC value that final concentration of 0.06~1% (m/v) is added in the step S2 is going for 0.1~8.0mM
Dirty agent.The substance that goes of CMC value within the above range includes but is not limited to SDS, sodium taurocholate, CHAPS, NaTDC.More preferably
Ground, the CMC value of the detergent are 0.1~3.0mM.Because detergent of the CMC value within the scope of this 0.1~3.0mM is for pure
Change, the purity highest of Yolk antibody obtained, fatty residual quantity is minimum.More selection of land, the CMC value of the detergent are 1.33, when
Preferred stain release agent is SDS when the CMC value of detergent is 1.33.
" CMC value " described herein refers to critical concentration when monomer detergent starts to be gathered into micelle, referred to as critical micro-
Group concentration (ctitical micelle concentration, CMC).
Correspondingly, the present invention also provides a kind of 4 type Yolk antibodies of the aviadenovirus that above-mentioned preparation method is prepared.
Preferably, lipid residual quantity is lower than 1% in the Yolk antibody.
The one of innovative point of the present invention is in WSF extraction process, is improved to traditional phenol extraction method,
It is extracted jointly using phenol+Solutol HS15, it is found that antibody recovery rate is single use phenol extraction
1.68 again.
But also containing more lipid in extract at this time, another innovative point of the invention is, using CMC value
It is purified for the detergent of 0.1~8.0mM, specifically, when the CMC value of detergent is 0.1~3.0mM, ovum obtained
The purity highest of yellow antibody, fatty residual quantity is minimum, and only 0.65~1.0%, and when CMC value is 1.33, degreasing rate highest,
Lipid residual quantity is 0.65%.And the mechanism for obtaining said effect, it is not yet clear.
The invention has the following advantages that
4 type Yolk antibody preparation method of aviadenovirus provided by the invention is not because be related to the behaviour such as subsequent gel chromatography
Make, it is at low cost, it is easy to operate, while by the improvement to extraction step and purification step, so that the rate of recovery of antibody, degreasing rate
It is significantly higher than existing method, achieves significant progress.
Specific embodiment
The specific embodiment of form by the following examples makees further specifically above content of the invention
It is bright.But the range that this should not be interpreted as to the above-mentioned theme of the present invention is only limitted to following embodiment.
4 type inactivated vaccine of aviadenovirus herein is purchased from Guangdong Yu Yue Bioisystech Co., Ltd, wherein every 0.1ml fowl
4 type virus liquid hold-up >=10 of gland8.5TCID50。
Embodiment 1, the preparation of 4 type Yolk antibody of aviadenovirus
A) 100 age in days laying hens are immunized in subcutaneous injection 4 type inactivated vaccine of 2ml aviadenovirus, are immunized 3 times altogether, when Immunity at intervals
Between be 21d, 5d collects egg after last time is immune, obtains height and exempts from egg；
B height) is exempted from egg to be cleaned with sterile saline, is dried, then with 95% alcohol spray disinfectant, dries, uses ovum
Yellow separator protein isolate collects yolk, abandons vitellinae membrana, obtains yolk liquid；
C) above-mentioned yolk liquid is mixed with the Tris-HCl buffer salt solution of pH7.2 by the volume ratio of 1:5, by yolk liquid:
Phenol solution is added in phenol=1:2 volume ratio, is sufficiently stirred, and mixes, and stands 30min, and low-speed centrifugal collects supernatant；
D the Solutol HS15 of final concentration of 0.6 (m/v)) is added into supernatant, is sufficiently stirred mixed
Even, 4 DEG C of standings 6h, 5000r/min are centrifuged 10min, collect supernatant；
E phosphate buffer) is added by the mass ratio of 1:1 toward above-mentioned supernatant, final concentration 0.06% is added in stirring and dissolving
(m/v) SDS is mixed, and 4 DEG C of standings 12h, 5000r/min are centrifuged 20min, collect precipitating, and precipitating sterile saline is molten
Xie Hou removes detergent through macroporous resin adsorption, obtains concentrate, filtration sterilization, packing to get.
Embodiment 2~3, the preparation of 4 type Yolk antibody of aviadenovirus
Embodiment 4~5 is the difference from embodiment 1 is that step D) in the Solutol HS15 that is added it is whole
Concentration is respectively 0.2% (m/v), 1.0% (m/v), remaining parameter and operation are same as Example 1.
Embodiment 4~5, the preparation of 4 type Yolk antibody of aviadenovirus
Embodiment 4~5 is the difference from embodiment 1 is that step E) in the final concentration of SDS that is added be respectively 0.01% (m/
V), 1% (m/v), remaining parameter and operation are same as Example 1.
Embodiment 6, the preparation of 4 type Yolk antibody of aviadenovirus
Embodiment 6 the difference from embodiment 1 is that, detergent is the Triton X-100 that CMC value is 0.24, remaining parameter
It is same as Example 1 with operating.
Embodiment 7, the preparation of 4 type Yolk antibody of aviadenovirus
Embodiment 7 the difference from embodiment 1 is that, detergent is the NP-40 that CMC value is 0.34, remaining parameter and operation
It is same as Example 1.
Embodiment 8, the preparation of 4 type Yolk antibody of aviadenovirus
Embodiment 8 the difference from embodiment 1 is that, detergent is the CHAPS that CMC value is 8.0, remaining parameter and operation with
Embodiment 1 is identical.
Comparative example 1, the preparation of 4 type Yolk antibody of aviadenovirus
Comparative example 1 the difference from embodiment 1 is that, detergent is the Tween 80 that CMC value is 0.012, remaining parameter with
It operates same as Example 1.
Comparative example 2, the preparation of 4 type Yolk antibody of aviadenovirus
Comparative example 2 the difference from embodiment 1 is that, omit step D), remaining parameter with operation it is same as Example 1.
Comparative example 3, the preparation of 4 type Yolk antibody of aviadenovirus
Comparative example 3 the difference from embodiment 1 is that, omit step C), remaining parameter with operation it is same as Example 1.
Test example one, safety detection
10 age in days health laying hens are chosen, is divided into 12 groups, every group 5, Examples 1 to 8 and comparative example 1~3 is respectively adopted
The Yolk antibody solution branch chicken of preparation injects (every 5ml), and remaining one group is used as blank control group.Injection front and back temperature detection,
Sooner or later 1 time, and observe injection portion have it is without exception.
The results show that each group laying hen injection front and back body temperature difference is ± 0.1 DEG C, and each group compared with blank control group
Spirit, appetite and the drink of laying hen are intended to normally, and injection site is without exception, show Examples 1 to 8 and ovum prepared by comparative example 1~3
Yellow antibody is highly-safe.
Test example two, antibody recovery rate, degreasing rate comparative test
Examples 1 to 8 and 1~3 step B of comparative example are detected using enzyme linked immunosorbent assay) egg yolk liquid and step E)
The content of the content of antibody in gained concentrate and fat, as the following formula 1, the 2 calculating antibody rate of recovery and degreasing rate；Testing result
As shown in table 1 below.
Formula 1:
Formula 2:
1 antibody recovery rate of table, degreasing rate comparison result
By upper table 1 it is found that the Yolk antibody good quality that the embodiment of the present invention 1~8 is prepared, antibody recovery rate exist
On 90.0%, degreasing rate is 98% or more.Especially embodiment 1, antibody recovery rate are up to 96.6%, and degreasing rate reaches
99.35%.Comparative example 1 and embodiment 6~8, the result of comparative example 1 it is found that CMC value within the scope of 0.1~3.0mM most
It is good, when especially CMC is 1.33, and it can lead to degreasing rate sharp fall, and antibody recovery rate when exceeding this range
There is reduction by a small margin.
Comparative example 1 and embodiment 2,3 test results are it is found that when only with phenol or polyethylene glycol 15- hydroxy stearate
When acid esters extracts water soluble ingredient, antibody recovery rate is no more than 60%, and polyethylene glycol 15- is added on the basis of phenol
Hydroxy stearic acid ester, so that antibody recovery rate improves 68%%.
Test example three, Yolk antibody bioactivity
Yolk antibody potency made from Examples 1 to 8 and comparative example 1~3, detection are detected using AGP test experimental method
As a result as shown in table 2 below.
2 Yolk antibody potency comparison result of table
By upper table 2 it is found that the potency of the Yolk antibody prepared through the embodiment of the present invention 1~8 is in 1:130 or more.And it is right
Ratio 1~3 is decreased significantly compared with Example 1.
The above-described embodiments merely illustrate the principles and effects of the present invention, and is not intended to limit the present invention.It is any ripe
The personage for knowing this technology all without departing from the spirit and scope of the present invention, carries out modifications and changes to above-described embodiment.Cause
This, institute is complete without departing from the spirit and technical ideas disclosed in the present invention by those of ordinary skill in the art such as
At all equivalent modifications or change, should be covered by the claims of the present invention.
Claims (10)
1. the preparation method of 4 type Yolk antibody of aviadenovirus, which comprises the following steps:
A 4 type inactivated vaccine Immune Laying Hens of aviadenovirus) are used, adaptive immune egg, after sterilized, protein isolate collects yolk,
Further obtain yolk liquid；
B above-mentioned yolk liquid is mixed with Tris-HCl buffer salt solution), phenol solution is added, is mixed, is stood, low-speed centrifugal, is received
Collect supernatant；
C the Solutol HS15 of final concentration of 0.2~1.0 (m/v)) is added into supernatant, is stirred,
It stands, supernatant is collected in centrifugation；
D the supernatant for) obtaining step C) is purified, is concentrated, aseptic procedures, obtains Yolk antibody.
2. preparation method as described in claim 1, which is characterized in that the step B) in yolk liquid and Tris-HCl buffer salt
By 1:(3~6) volume ratio mixing.
3. preparation method as claimed in claim 1 or 2, which is characterized in that the step B) in yolk liquid pressed with phenol solution
1:(1~3) volume ratio mixing.
4. preparation method as described in claim 1, which is characterized in that the purification step specifically:
Phosphate buffer is added by the mass ratio of 1:1 into above-mentioned supernatant, final concentration 0.01~1% is added in stirring and dissolving
(m/v) CMC value is the detergent of 0.1~8.0mM, is mixed, and is stood, and centrifugation collects precipitating, by precipitating sterile saline
After dissolution, through macroporous resin adsorption remove detergent to get.
5. preparation method as claimed in claim 4, which is characterized in that be added final concentration of 0.06~1% in the step S2
(m/v) CMC value is the detergent of 0.1~8.0mM.
6. preparation method as described in claim 4 or 5, which is characterized in that the CMC value of the detergent is 0.1~3.0mM.
7. preparation method as claimed in claim 6, which is characterized in that the CMC value of the detergent is 1.33.
8. preparation method as claimed in claim 7, which is characterized in that the detergent is SDS.
9. a kind of 4 type Yolk antibody of the aviadenovirus that the preparation method as described in claim 1~8 is any is prepared.
10. 4 type Yolk antibody of aviadenovirus as claimed in claim 9, which is characterized in that lipid remains in the Yolk antibody
Amount is lower than 1%.
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| CN201810977191.5A CN109053880A (en) | 2018-08-26 | 2018-08-26 | 4 type Yolk antibody of aviadenovirus and preparation method thereof |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1202496A (en) * | 1998-05-21 | 1998-12-23 | 山东农业大学 | Process for preparing refined vitelline antibody |
| CN105175536A (en) * | 2015-09-30 | 2015-12-23 | 大连理工大学 | Egg yolk antibody extraction method suitable for large-scale industrial production |
| CN106928349A (en) * | 2017-03-08 | 2017-07-07 | 广州博恒生物科技有限公司 | A kind of type Yolk antibody preparation method of aviadenovirus 4 |
| CN108373501A (en) * | 2018-04-08 | 2018-08-07 | 倪同艳 | A kind of composite yolk antibody of resisting fowl bacterial epidemic disease |
-
2018
- 2018-08-26 CN CN201810977191.5A patent/CN109053880A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1202496A (en) * | 1998-05-21 | 1998-12-23 | 山东农业大学 | Process for preparing refined vitelline antibody |
| CN105175536A (en) * | 2015-09-30 | 2015-12-23 | 大连理工大学 | Egg yolk antibody extraction method suitable for large-scale industrial production |
| CN106928349A (en) * | 2017-03-08 | 2017-07-07 | 广州博恒生物科技有限公司 | A kind of type Yolk antibody preparation method of aviadenovirus 4 |
| CN108373501A (en) * | 2018-04-08 | 2018-08-07 | 倪同艳 | A kind of composite yolk antibody of resisting fowl bacterial epidemic disease |
Non-Patent Citations (1)
| Title |
|---|
| SRIRAM V: "Improved Recovery of Immunoglobulin Fraction from Egg Yolk of Chicken Immunized with AsialoGM1", 《RUSS J IMMUNOL》 * |
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