CN106380518B - The extraction process of Yolk immunoglobulin - Google Patents
The extraction process of Yolk immunoglobulin Download PDFInfo
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- CN106380518B CN106380518B CN201610781996.3A CN201610781996A CN106380518B CN 106380518 B CN106380518 B CN 106380518B CN 201610781996 A CN201610781996 A CN 201610781996A CN 106380518 B CN106380518 B CN 106380518B
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- yolk
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- 238000002347 injection Methods 0.000 claims description 16
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- RKCAIXNGYQCCAL-UHFFFAOYSA-N porphin Chemical compound N1C(C=C2N=C(C=C3NC(=C4)C=C3)C=C2)=CC=C1C=C1C=CC4=N1 RKCAIXNGYQCCAL-UHFFFAOYSA-N 0.000 claims 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
- C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
-
- 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
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Peptides Or Proteins (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention discloses a kind of extraction processes of Yolk immunoglobulin comprising following steps: a) prepared by antigen；B) laying hen is immune, collects egg；C) yolk of membrane removal clothing will be gone to stir evenly, is placed at -20 ± 3 DEG C and at least freezes 24-72h, quick-thawing, dilution is adjusted pH, stood overnight；D) centrifugal filtration is concentrated by ultrafiltration, is replaced with sodium chloride solution, ammonium sulfate precipitation, sodium chloride dissolution；E) dialysis, centrifugal filtration are to get Yolk immunoglobulin liquid.Compared with prior art, the present invention carries out the extraction purification of Yolk immunoglobulin using physical-chemical processes such as a series of centrifugation, filtering, precipitating, dialysis, it is not only easy to operate, and extraction cost is low, extraction process is easy to control, and extracted obtained Yolk immunoglobulin can be good at carrying out the treatment of periodontitis, effect is obvious, and extraction process disclosed by the invention can carry out large-scale industrial production.
Description
Technical field
The present invention relates to pharmaceutical technology fields, and in particular to a kind of extraction process of Yolk immunoglobulin.
Background technique
Three kinds of immunoglobulins, i.e. IgG, IgM and IgA are primarily present in carcass, wherein IgG is the principal immune ball egg of fowl
It is white, since there are many differences for the characteristic and the IgG characteristic of mammal of fowl IgG, thus fowl IgG is usually referred to as IgY.Work as fowl
Body is by can generate corresponding specific immunity antibody after the stimulation of specific antigen, and the IgY antibody that body generates can be from blood
In be largely transported in yolk, the antibody being stored in yolk in this way is known as Yolk immunoglobulin.Yolk immunoglobulin is
A kind of novel, efficient green preparation to diagnosis, prevents and treats disease caused by bacterium, virus with good effect.
Poultry egg (such as egg) can provide the source IgY, and yield is high, about containing 100~250mg's in one piece of egg
IgY, and how efficiently to extract a large amount of Yolk immunoglobulin, this is most important for the extensive use of IgY.
In the prior art, it is generally adopted for chicken yolk immune globulin and extraction method progress primary extraction is diluted with water, then in conjunction with salt
Analysis with ion exchange in conjunction with, hydrophobic chromatography the methods of in conjunction with gel filtration, however separating effect is bad, is unsuitable on a large scale
Production.
Periodontitis is common one of mouth disease, mainly includes at present that drug and operation are controlled for the treatment of periodontitis
It treats, wherein be mainly preservative and antimicrobial sustained-release agent for treating the drug of periodontitis, but such drug is easy to produce bacterium
Drug resistance and toxic side effect should not be used for a long time.Chinese patent discloses entitled " resisting porphyromonas gingivalis yolk immune globulin
The patent of invention of the preparation method (application number 2010101359598) of white vaccine ", the patent are pointed out using porphyromonas gingivalis
Immune Laying Hens, the processed manufactured preparation of the chicken yolk immune globulin extracted in this way can be used for the treatment of periodontitis, and effect
Significantly.But the patent be during extracting Yolk immunoglobulin use kit it is expensive, specifically, one
A kit is only capable of the yolk liquid of processing 200ml or so, therefore the production that can not carry out large-scale Yolk immunoglobulin is pure
Change.It can be seen that being directed to efficient, the inexpensive extraction of chicken yolk immune globulin, in technical aspect, there is also many at present
Bottleneck.In addition, carrying out the preparation of IgY in the patent using the laying hen without normal immunological, we have found in early stage research,
When large-scale breeding, the laying hen without normal immunological is susceptible to suffer from various poultry diseases, and the death rate is high, and the death rate after four months is high
Up to 90%, and the death rate of the immunized healthy laying hen after four months is only 1%.Therefore, method disclosed in the prior art is only
It can be confined to laboratory, cannot achieve large-scale production.
Summary of the invention
The object of the present invention is to provide the extraction works of a kind of yield and purity is high and Yolk immunoglobulin at low cost
Skill.
To achieve the above object, the technical solution adopted by the present invention is that: a kind of extraction process of Yolk immunoglobulin,
The following steps are included:
A) prepared by antigen: the unicellular strain of porphyromonas is collected, culture obtains porphyromonas gingivalis body, then inactivate,
3-5 DEG C of overnight, PBS buffer solution washing, is later 1.0 × 10 by concentration11The inactivation thallus of CFU/ml and isometric Freund are helped
Agent piping and druming is mixed into Water-In-Oil shape injection；
B) laying hen is immune: the healthy laying hen of 3-5 monthly age isolated rearing is selected, through pigeon breast portion subdermal muscle injecting immune 4 times, and prison
It surveys Specific antibody titre variation tendency in record yolk: starting largely to collect chicken when antibody titer has obvious ascendant trend
Egg；It is same to monitor Yolk antibody titre when finding that titre is begun to decline booster immunization 1 time, when Yolk antibody titre has obviously
Stop collecting egg when decline, by the In Shade storage of egg；
C) by egg cleaning, disinfection, broken shell removes egg white and membrane of yolk clothing, yolk is stirred evenly, is placed in -20 ± 3 DEG C
Under at least freeze 24-72h, 8-10 times of volume purified water dilution stirring 5-6h is added, extremely with hydrochloric acid solution tune pH in quick-thawing
5.0-5.1 is stood overnight at 20-23 DEG C；
D) by yolk dilution centrifugal filtration, obtained filtrate is concentrated by ultrafiltration, and is replaced with sodium chloride solution and liquid is concentrated by ultrafiltration
Twice, displacement gained concentrate ammonium sulfate precipitation, is redissolved using sodium chloride solution later；
E) by the dialysis of sodium chloride lysate, centrifugal filtration to get Yolk immunoglobulin liquid.
With the prior art using kit carry out Yolk immunoglobulin extraction compared with, the present invention using it is a series of from
The physical-chemical processes such as the heart, filtering, precipitating, dialysis carry out the extraction purification of Yolk immunoglobulin, not only easy to operate, Er Qieti
Take at low cost, extraction process is easy to control, and extracted obtained Yolk immunoglobulin can be good at carrying out periodontitis
Treatment, effect are obvious；
In addition, unlike the prior art, we prepare IgY using the laying hen after being immunized, it can be to avoid because of large-scale breeding
Caused by the high problem of the laying hen death rate；Meanwhile the present invention city Jun He in terms of titre, purity and yield IgY obtained
IgY no significant difference on field, therefore extraction process disclosed by the invention can carry out large-scale industrial production.
Specific scheme is the cultural method of step a) Porphyromonas gingivalis body are as follows:
1) aseptically, the unicellular strain of porphyromonas is drawn in Columbia Blood Agar plate with four zoning collimation methods
On, it is placed in 37 DEG C of anaerobic culture box and cultivates 3-5d to generating black colonies；
2) black colonies are inoculated in Anaerobic culturel 2-4d in 3.7% N of brain heart infusion fluid nutrient medium, tooth is collected by centrifugation
Gum Porphyromonas thallus.
Specifically, step a) Porphyromonas gingivalis body is with 1% formalin-inactivated, 4 DEG C overnight, sterile PBS buffer
It washes 3 times, later with concentration for 1.0 × 1011The inactivation thallus 1ml of CFU/ml is helped completely with equivalent incomplete Freund's adjuvant, Freund
Agent blow and beat respectively be mixed into two kinds in requisition for Water-In-Oil shape injection.
Scheme as a further preference, the method for Immune Laying Hens is as follows in step b):
Healthy laying hen is first immunized 4 times, every chicken per injection 1.0ml, and first immunisation is with Freund's complete adjuvant and inactivation
The injection that the injection that thallus mixes, rear 3 booster immunization incomplete Freund's adjuvants and inactivation thallus mix, it is immune every time
Interval time is 8-10d, Specific antibody titre variation tendency in monitoring record yolk: when antibody titer has obvious ascendant trend
When start largely to collect egg；When discovery titre begins to decline Shi Zaiyong incomplete Freund's adjuvant and inactivates the injection of thallus mixing
It is agent booster immunization 1 time, same to monitor Yolk antibody titre, stop collecting egg when Yolk antibody titre decreased significantly, it will
The laying hen of collection marks and stores in the cool.
Preferably, in step d) centrifugal filtration processing method are as follows: yolk dilution is centrifuged at 4 DEG C, is taken on clear liquid
To get filtrate, the filler in Filter column is made of gauze layer arranged up and down and medical absorbent cotton layer for Filter column filtering.Application
People, can be well by centrifugal clear liquid using the filler that gauze layer and medical absorbent cotton layer are constituted through a large amount of analysis and research discovery
The granular insoluble matter of the middle overwhelming majority and lipid material removal, thus greatly reduce the purifying difficulty in later period, while effectively
Ground improves the purity of Yolk immunoglobulin.
Further, step d) used molecular cut off that filtrate is concentrated by ultrafiltration 9-10 times for the ultrafiltration membrane of 100KD before this,
Then liquid is concentrated by ultrafiltration twice with 0.9% sodium chloride solution equivalent displacement, displacement gained concentrate is spare.It is to use because before
Purified water dilution dissolution yolk, and postorder needs to dissolve ammonium sulfate precipitation using sodium chloride, therefore chlorination is first used before precipitation
Liquid displacement will be concentrated by ultrafiltration in sodium solution, so the dissolution system of target protein can be replaced as sodium chloride solution by purified water, from
And facilitate precipitating, dissolution process, to remove impurity therein, realize the purpose of protein of interest.
Further, the concentrate that step d is obtained is by following precipitating and dissolution process: according to the ratio of 1ml:0.243g
Ammonium sulfate is added into displacement concentrate in example, and 1h is sufficiently stirred, is stored at room temperature 2h, is centrifuged at 4 DEG C, supernatant is abandoned, with 0.9%
Sodium chloride solution is completely dissolved precipitating；Preferred embodiment is displacement concentration liquid precipitate and dissolution process step cycle weight in step d
Again twice, being completely dissolved precipitating with sodium chloride solution in certain step is also the redissolution of sediment.
Specifically, the sodium chloride solution that step e) is by sodium chloride lysate with temperature is 2-8 DEG C, concentration is 0.9% is saturating
Then at 2-8 DEG C overnight analysis is centrifuged at 4 DEG C, gained clear liquid with 0.22 μm of membrane filtration to get Yolk immunoglobulin liquid,
Gained Yolk immunoglobulin liquid, which is placed at -20 DEG C, to be saved.
Preferably, step c) is that yolk after mixing evenly is placed at -20 ± 3 DEG C at least to freeze 48h, at this
Under part, the yield and extraction efficiency of target product can be effectively ensured.
Detailed description of the invention
Fig. 1 is the overnight layering result figure of different yolk liquid.
Specific embodiment
Disclosed technical solution in order to further illustrate the present invention, 1-10 is illustrated by the following examples,
Partial parameters in middle embodiment 5,7-10 are not within the scope of the present invention, it can be understood as are comparative examples:
Embodiment 1: the acquisition of egg
1) aseptically, the unicellular strain of porphyromonas (waiting supplementing in source) is drawn in brother's rival with four zoning collimation methods
On sub- blood agar plate, it is placed in 37 DEG C of anaerobic culture box and cultivates 4d to generating black colonies；
2) black colonies are inoculated in Anaerobic culturel 3d in 3.7% N of brain heart infusion fluid nutrient medium, gum is collected by centrifugation
Porphyromonas thallus；
3) by 1% formalin-inactivated of porphyromonas gingivalis body, 4 DEG C overnight, and sterile PBS buffer is washed 3 times, later with dense
Degree is 1.0 × 1011The inactivation thallus 1ml and equivalent incomplete Freund's adjuvant, Freund's complete adjuvant of CFU/ml blows and beats mixing respectively
At two kinds in requisition for Water-In-Oil shape injection.
4) healthy laying hen is first immunized 4 times, every chicken per injection 1.0ml, first immunisation be with Freund's complete adjuvant with go out
The injection that the injection that viable bacteria body mixes, rear 3 booster immunization incomplete Freund's adjuvants and inactivation thallus mix, exempts from every time
Epidemic disease interval time is 8-10d, Specific antibody titre variation tendency in monitoring record yolk: when antibody titer has obvious rise to become
Start largely to collect egg when gesture；When discovery titre begins to decline Shi Zaiyong incomplete Freund's adjuvant and inactivates the note of thallus mixing
It penetrates agent booster immunization 1 time, it is same to monitor Yolk antibody titre, stop collecting egg when Yolk antibody titre decreased significantly,
The laying hen of collection is marked and is stored in the cool.
Embodiment 2: the extraction of livitin
1) by egg cleaning, disinfection, broken shell removes egg white and membrane of yolk clothing, yolk is stirred evenly, is placed at -20 DEG C
For 24 hours, 9 times of volume purified waters dilution stirring 5h are added in quick-thawing for freezing, with hydrochloric acid solution tune pH to 5.0-5.1, in 20-23
It is stood overnight at DEG C；
2) yolk dilution is centrifuged at 4 DEG C, takes on clear liquid Filter column filtering to get filtrate, the filler in Filter column
It is to be made of gauze layer arranged up and down and medical absorbent cotton layer；
3) use molecular cut off be the ultrafiltration membrane of 100KD by filtrate be concentrated by ultrafiltration 9 times, then with 0.9% sodium chloride
Liquid is concentrated by ultrafiltration twice in solution even displacement, and ammonium sulfate is added into displacement gained concentrate according to the ratio of 1ml:0.243g,
1h is sufficiently stirred, is stored at room temperature 2h, is centrifuged at 4 DEG C, abandons supernatant, is completely dissolved precipitating with 0.9% sodium chloride solution, sets
It changes concentration liquid precipitate and dissolution process step cycle is repeated twice；
4) the sodium chloride solution dialysis that be 6 DEG C with temperature by sodium chloride lysate, concentration is 0.9%, is then stayed overnight for 4 DEG C,
It is centrifuged at 4 DEG C, gained clear liquid is with 0.22 μm of membrane filtration to get Yolk immunoglobulin liquid, gained Yolk immunoglobulin
Liquid is placed at -20 DEG C and saves.
Embodiment 3: the extraction of livitin
1) by egg cleaning, disinfection, broken shell removes egg white and membrane of yolk clothing, yolk is stirred evenly, is placed at -20 DEG C
48h is freezed, 9 times of volume purified waters dilution stirring 5h are added in quick-thawing, with hydrochloric acid solution tune pH to 5.0-5.1, in 20-23
It is stood overnight at DEG C；
2) yolk dilution is centrifuged at 4 DEG C, takes on clear liquid Filter column filtering to get filtrate, the filler in Filter column
It is to be made of gauze layer arranged up and down and medical absorbent cotton layer；
3) use molecular cut off be the ultrafiltration membrane of 100KD by filtrate be concentrated by ultrafiltration 9 times, then with 0.9% sodium chloride
Liquid is concentrated by ultrafiltration twice in solution even displacement, and ammonium sulfate is added into displacement gained concentrate according to the ratio of 1ml:0.243g,
1h is sufficiently stirred, is stored at room temperature 2h, is centrifuged at 4 DEG C, abandons supernatant, is completely dissolved precipitating with 0.9% sodium chloride solution, sets
It changes concentration liquid precipitate and dissolution process step cycle is repeated twice；
4) the sodium chloride solution dialysis that be 6 DEG C with temperature by sodium chloride lysate, concentration is 0.9%, is then stayed overnight for 4 DEG C,
It is centrifuged at 4 DEG C, gained clear liquid is with 0.22 μm of membrane filtration to get Yolk immunoglobulin liquid, gained Yolk immunoglobulin
Liquid is placed at -20 DEG C and saves.
Embodiment 4: the extraction of livitin
1) by egg cleaning, disinfection, broken shell removes egg white and membrane of yolk clothing, yolk is stirred evenly, is placed at -20 DEG C
72h is freezed, 9 times of volume purified waters dilution stirring 5h are added in quick-thawing, with hydrochloric acid solution tune pH to 5.0-5.1, in 20-23
It is stood overnight at DEG C；
2) yolk dilution is centrifuged at 4 DEG C, takes on clear liquid Filter column filtering to get filtrate, the filler in Filter column
It is to be made of gauze layer arranged up and down and medical absorbent cotton layer；
3) use molecular cut off be the ultrafiltration membrane of 100KD by filtrate be concentrated by ultrafiltration 9 times, then with 0.9% sodium chloride
Liquid is concentrated by ultrafiltration twice in solution even displacement, and ammonium sulfate is added into displacement gained concentrate according to the ratio of 1ml:0.243g,
1h is sufficiently stirred, is stored at room temperature 2h, is centrifuged at 4 DEG C, abandons supernatant, is completely dissolved precipitating with 0.9% sodium chloride solution, sets
It changes concentration liquid precipitate and dissolution process step cycle is repeated twice；
4) the sodium chloride solution dialysis that be 6 DEG C with temperature by sodium chloride lysate, concentration is 0.9%, is then stayed overnight for 4 DEG C,
It is centrifuged at 4 DEG C, gained clear liquid is with 0.22 μm of membrane filtration to get Yolk immunoglobulin liquid, gained Yolk immunoglobulin
Liquid is placed at -20 DEG C and saves.
Embodiment 5 (comparative example): the extraction purification of livitin
1) by egg cleaning, disinfection, broken shell removes egg white and membrane of yolk clothing, yolk is stirred evenly, is directly added into 9 times
Volume purified water dilution stirring 5h is stood overnight at 20-23 DEG C with hydrochloric acid solution tune pH to 5.0-5.1；
2) yolk dilution is centrifuged at 4 DEG C, takes on clear liquid Filter column filtering to get filtrate, the filler in Filter column
It is to be made of gauze layer arranged up and down and medical absorbent cotton layer；
3) use molecular cut off be the ultrafiltration membrane of 100KD by filtrate be concentrated by ultrafiltration 9 times, then with 0.9% sodium chloride
Liquid is concentrated by ultrafiltration twice in solution even displacement, and ammonium sulfate is added into displacement gained concentrate according to the ratio of 1ml:0.243g,
1h is sufficiently stirred, is stored at room temperature 2h, is centrifuged at 4 DEG C, abandons supernatant, is completely dissolved precipitating with 0.9% sodium chloride solution, sets
It changes concentration liquid precipitate and dissolution process step cycle is repeated twice；
4) the sodium chloride solution dialysis that be 2 DEG C with temperature by sodium chloride lysate, concentration is 0.9%, is then stayed overnight for 2 DEG C,
It is centrifuged at 4 DEG C, gained clear liquid is with 0.22 μm of membrane filtration to get Yolk immunoglobulin liquid, gained Yolk immunoglobulin
Liquid is placed at -20 DEG C and saves.
Embodiment 6: the extraction purification of livitin
1) by egg cleaning, disinfection, broken shell removes egg white and membrane of yolk clothing, yolk is stirred evenly, is placed at -20 DEG C
48h is freezed, 8 times of volume purified waters dilution stirring 5h are added in quick-thawing, with hydrochloric acid solution tune pH to 5.0-5.1, in 20-23
It is stood overnight at DEG C；
2) yolk dilution is centrifuged at 4 DEG C, takes on clear liquid Filter column filtering to get filtrate, the filler in Filter column
It is to be made of gauze layer arranged up and down and medical absorbent cotton layer；
3) use molecular cut off be the ultrafiltration membrane of 100KD by filtrate be concentrated by ultrafiltration 10 times, then with 0.9% sodium chloride
Liquid is concentrated by ultrafiltration twice in solution even displacement, and ammonium sulfate is added into displacement gained concentrate according to the ratio of 1ml:0.243g,
1h is sufficiently stirred, is stored at room temperature 2h, is centrifuged at 4 DEG C, abandons supernatant, is completely dissolved precipitating with 0.9% sodium chloride solution, sets
It changes concentration liquid precipitate and dissolution process step cycle is repeated twice；
4) the sodium chloride solution dialysis that be 6 DEG C with temperature by sodium chloride lysate, concentration is 0.9%, is then stayed overnight for 4 DEG C,
It is centrifuged at 4 DEG C, gained clear liquid is with 0.22 μm of membrane filtration to get Yolk immunoglobulin liquid, gained Yolk immunoglobulin
Liquid is placed at -20 DEG C and saves.
Embodiment 7 (comparative example): the extraction purification of livitin
1) by egg cleaning, disinfection, broken shell removes egg white and membrane of yolk clothing, yolk is stirred evenly, is placed at -20 DEG C
48h is freezed, 9 times of volume purified waters dilution stirring 5h are added in quick-thawing, with hydrochloric acid solution tune pH to 5.0-5.1, at 3-5 DEG C
Under stand overnight；
2) yolk dilution is centrifuged at 4 DEG C, takes on clear liquid Filter column filtering to get filtrate, the filler in Filter column
It is to be made of gauze layer arranged up and down and medical absorbent cotton layer；
3) use molecular cut off be the ultrafiltration membrane of 100KD by filtrate be concentrated by ultrafiltration 9 times, then with 0.9% sodium chloride
Liquid is concentrated by ultrafiltration twice in solution even displacement, and ammonium sulfate is added into displacement gained concentrate according to the ratio of 1ml:0.243g,
1h is sufficiently stirred, is stored at room temperature 2h, is centrifuged at 4 DEG C, abandons supernatant, is completely dissolved precipitating with 0.9% sodium chloride solution, sets
It changes concentration liquid precipitate and dissolution process step cycle is repeated twice；
4) the sodium chloride solution dialysis that be 6 DEG C with temperature by sodium chloride lysate, concentration is 0.9%, is then stayed overnight for 4 DEG C,
It is centrifuged at 4 DEG C, gained clear liquid is with 0.22 μm of membrane filtration to get Yolk immunoglobulin liquid, gained Yolk immunoglobulin
Liquid is placed at -20 DEG C and saves.
Embodiment 8 (comparative example): the extraction purification of livitin
1) by egg cleaning, disinfection, broken shell removes egg white and membrane of yolk clothing, yolk is stirred evenly, is placed at -20 DEG C
48h is freezed, 9 times of volume purified waters dilution stirring 5h are added in quick-thawing, with hydrochloric acid solution tune pH to 5.0-5.1, in 29-31
It is stood overnight at DEG C；
2) yolk dilution is centrifuged at 4 DEG C, takes on clear liquid Filter column filtering to get filtrate, the filler in Filter column
It is to be made of gauze layer arranged up and down and medical absorbent cotton layer；
3) use molecular cut off be the ultrafiltration membrane of 100KD by filtrate be concentrated by ultrafiltration 9 times, then with 0.9% sodium chloride
Liquid is concentrated by ultrafiltration twice in solution even displacement, and ammonium sulfate is added into displacement gained concentrate according to the ratio of 1ml:0.243g,
1h is sufficiently stirred, is stored at room temperature 2h, is centrifuged at 4 DEG C, abandons supernatant, is completely dissolved precipitating with 0.9% sodium chloride solution, sets
It changes concentration liquid precipitate and dissolution process step cycle is repeated twice；
4) the sodium chloride solution dialysis that be 6 DEG C with temperature by sodium chloride lysate, concentration is 0.9%, is then stayed overnight for 4 DEG C,
It is centrifuged at 4 DEG C, gained clear liquid is with 0.22 μm of membrane filtration to get Yolk immunoglobulin liquid, gained Yolk immunoglobulin
Liquid is placed at -20 DEG C and saves.
Embodiment 9 (comparative example): the extraction purification of livitin
1) by egg cleaning, disinfection, broken shell removes egg white and membrane of yolk clothing, yolk is stirred evenly, is placed at -20 DEG C
48h is freezed, 9 times of volume purified waters dilution stirring 5h are added in quick-thawing, with hydrochloric acid solution tune pH to 5.0-5.1, in 20-23
It is stood overnight at DEG C；
2) yolk dilution is centrifuged at 4 DEG C, takes on clear liquid Filter column filtering to get filtrate, the filler in Filter column
It is to be made of active carbon；
3) use molecular cut off be the ultrafiltration membrane of 100KD by filtrate be concentrated by ultrafiltration 9 times, then with 0.9% sodium chloride
Liquid is concentrated by ultrafiltration twice in solution even displacement, and ammonium sulfate is added into displacement gained concentrate according to the ratio of 1ml:0.243g,
1h is sufficiently stirred, is stored at room temperature 2h, is centrifuged at 4 DEG C, abandons supernatant, is completely dissolved precipitating with 0.9% sodium chloride solution, sets
It changes concentration liquid precipitate and dissolution process step cycle is repeated twice；
4) the sodium chloride solution dialysis that be 6 DEG C with temperature by sodium chloride lysate, concentration is 0.9%, is then stayed overnight for 4 DEG C,
It is centrifuged at 4 DEG C, gained clear liquid is with 0.22 μm of membrane filtration to get Yolk immunoglobulin liquid, gained Yolk immunoglobulin
Liquid is placed at -20 DEG C and saves.
Embodiment 10 (comparative example): the extraction purification of livitin
1) by egg cleaning, disinfection, broken shell removes egg white and membrane of yolk clothing, yolk is stirred evenly, is placed at -20 DEG C
48h is freezed, 9 times of volume purified waters dilution stirring 5h are added in quick-thawing, with hydrochloric acid solution tune pH to 5.0-5.1, in 20-23
It is stood overnight at DEG C；
2) yolk dilution is centrifuged at 4 DEG C, takes on clear liquid Filter column filtering to get filtrate, the filler in Filter column
It is to be made of diatomite；
3) use molecular cut off be the ultrafiltration membrane of 100KD by filtrate be concentrated by ultrafiltration 9 times, then with 0.9% sodium chloride
Liquid is concentrated by ultrafiltration twice in solution even displacement, and ammonium sulfate is added into displacement gained concentrate according to the ratio of 1ml:0.243g,
1h is sufficiently stirred, is stored at room temperature 2h, is centrifuged at 4 DEG C, abandons supernatant, is completely dissolved precipitating with 0.9% sodium chloride solution, sets
It changes concentration liquid precipitate and dissolution process step cycle is repeated twice；
4) the sodium chloride solution dialysis that be 6 DEG C with temperature by sodium chloride lysate, concentration is 0.9%, is then stayed overnight for 4 DEG C,
It is centrifuged at 4 DEG C, gained clear liquid is with 0.22 μm of membrane filtration to get Yolk immunoglobulin liquid, gained Yolk immunoglobulin
Liquid is placed at -20 DEG C and saves.
As a result it detects:
Protein content survey is carried out to the Yolk immunoglobulin liquid that embodiment 2-5,7-10 is prepared using Lorry method
Fixed, as the result is shown: 1, embodiment 2-5 testing result shows to prepare by the yolk of 200g striping clothing in terms of using embodiment 2-5
Obtained IgY total amount is respectively 497mg, 635mg, 651mg, 359mg, it can be seen that, before comprehensively considering optimal yolk liquid
Reason mode are as follows: be put into after being freezed 48 hours under -20 DEG C of environment and take out spare, so may insure the yield and extraction efficiency of IgY；
2, the result after yolk liquid is stood overnight after tune acid in embodiment 3,7,8 is as shown in Figure 1, it can be seen from the figure that after adjusting acid
The dwell temperature of yolk liquid influences very big, self sinking when yolk liquid dwell temperature is 20-23 DEG C on the layered effect of yolk liquid
Layered effect is dropped significantly better than 3-5 DEG C and 29-31 DEG C；Different filtering column packings is respectively adopted to target liquid in embodiment 3,9,10
It is filtered, the results show that the clarity of the filtrate obtained after different filler filterings has apparent difference, wherein with
Absorbent cotton and gauze are that the filtered filtrate clarity of Filter column of filler is best in embodiment 3, living with powdery in embodiment 9
Property charcoal be filler the filtered filtrate clarity of Filter column take second place, and in embodiment 10 diatomite be filler Filter column
Filtered filtrate clarity is worst, the supernatant obtained after yolk diluent centrifugation, by being filling with absorbent cotton and gauze
After the Filter column filtering of object, most granular insoluble matters and lipid material are successfully removed, and greatly reduce the later period
Difficulty is purified, so can effectively improve the purity of Yolk antibody.
Claims (10)
1. a kind of extraction process of Yolk immunoglobulin comprising following steps:
A) prepared by antigen: collecting the unicellular strain of porphyromonas, culture obtains porphyromonas gingivalis body, then inactivation, 3-5 DEG C
Overnight, concentration is later 1.0 × 10 by PBS buffer solution washing11The inactivation thallus and isometric Freund's adjuvant of CFU/ml is blown and beaten
It is mixed into Water-In-Oil shape injection；
B) laying hen is immune: the healthy laying hen of 3-5 monthly age isolated rearing is selected, through pigeon breast portion subdermal muscle injecting immune 4 times, and monitoring note
Specific antibody titre variation tendency in record yolk: start largely to collect egg when antibody titer has obvious ascendant trend；When
It was found that booster immunization 1 time when titre is begun to decline, same to monitor Yolk antibody titre, when Yolk antibody titre decreased significantly
Stop collecting egg, by the In Shade storage of egg；
C) by egg cleaning, disinfection, broken shell removes egg white and membrane of yolk clothing, yolk is stirred evenly, be placed in -20 ± 3 DEG C down toward
24-72h is freezed less, and 8-10 times of volume purified water dilution stirring 5-6h is added, with hydrochloric acid solution tune pH to 5.0- in quick-thawing
5.1, it is stood overnight at 20-23 DEG C；
D) by yolk dilution centrifugal filtration, obtained filtrate is concentrated by ultrafiltration, and is replaced with sodium chloride solution and liquid is concentrated by ultrafiltration twice,
Displacement gained concentrate ammonium sulfate precipitation, is redissolved using sodium chloride solution later；
E) sodium chloride solution of liquid after above-mentioned redissolution is dialysed, centrifugal filtration is to get Yolk immunoglobulin liquid.
2. the extraction process of Yolk immunoglobulin according to claim 1, it is characterised in that: porphyromonas in step a)
The cultural method of unit cell thallus are as follows:
1) aseptically, the unicellular strain of porphyromonas is drawn on Columbia Blood Agar plate with four zoning collimation methods, is set
3-5d is cultivated in 37 DEG C of anaerobic culture box to generating black colonies；
2) black colonies are inoculated in Anaerobic culturel 2-4d in 3.7% N of brain heart infusion fluid nutrient medium, gum porphin is collected by centrifugation
Quinoline unit cell thallus.
3. the extraction process of Yolk immunoglobulin according to claim 2, it is characterised in that: porphyromonas in step a)
Unit cell thallus is with 1% formalin-inactivated, and 4 DEG C overnight, and sterile PBS buffer is washed 3 times, later with concentration for 1.0 × 1011CFU/ml
Inactivation thallus 1ml and equivalent incomplete Freund's adjuvant, Freund's complete adjuvant blow and beat respectively be mixed into two kinds in requisition for oil
Wrap watery injection.
4. the extraction process of Yolk immunoglobulin according to claim 3, it is characterised in that: Immune Laying Hens in step b)
Method it is as follows:
Healthy laying hen is first immunized 4 times, every chicken per injection 1.0ml, and first immunisation is with Freund's complete adjuvant and inactivation thallus
The injection that the injection of mixing, rear 3 booster immunization incomplete Freund's adjuvants and inactivation thallus mix, each immunization interval
Time is 8-10d, Specific antibody titre variation tendency in monitoring record yolk: is opened when antibody titer has obvious ascendant trend
Begin a large amount of collection eggs；Add when discovery titre begins to decline Shi Zaiyong incomplete Freund's adjuvant with the injection that thallus mixes is inactivated
It is strong 1 time immune, it is same to monitor Yolk antibody titre, stop collecting egg when Yolk antibody titre decreased significantly, will collect
Laying hen mark and store in the cool.
5. the extraction process of Yolk immunoglobulin according to claim 4, it is characterised in that: centrifugal filtration in step d)
Processing method are as follows: yolk dilution is centrifuged at 4 DEG C, takes on clear liquid Filter column filtering to get filtrate, filling out in Filter column
Material is made of gauze layer arranged up and down and medical absorbent cotton layer.
6. the extraction process of Yolk immunoglobulin according to claim 5, it is characterised in that: step d) is used cut before this
It stays the ultrafiltration membrane that molecular weight is 100KD that filtrate is concentrated by ultrafiltration 9-10 times, is then replaced with 0.9% sodium chloride solution equivalent super
Twice, displacement gained concentrate is spare for filter concentration liquid.
7. the extraction process of Yolk immunoglobulin according to claim 6, it is characterised in that: the concentration that step d is obtained
Liquid is by following precipitating and dissolution process: ammonium sulfate is added into displacement concentrate according to the ratio of 1ml:0.243g, sufficiently stirs
1h is mixed, 2h is stored at room temperature, is centrifuged at 4 DEG C, supernatant is abandoned, is completely dissolved precipitating with 0.9% sodium chloride solution.
8. the extraction process of Yolk immunoglobulin according to claim 7, it is characterised in that: replace concentration in step d
Liquid precipitate and dissolution process step cycle are repeated twice.
9. the extraction process of Yolk immunoglobulin according to claim 6 or 7, it is characterised in that: step e) is by chlorine
Change the sodium chloride solution dialysis that sodium lysate with temperature is 2-8 DEG C, concentration is 0.9%, then at 2-8 DEG C overnight, at 4 DEG C from
The heart, for gained clear liquid with 0.22 μm of membrane filtration to get Yolk immunoglobulin liquid, gained Yolk immunoglobulin liquid is placed in -20
It is saved at DEG C.
10. the extraction process of Yolk immunoglobulin according to claim 9, it is characterised in that: step c) is will to stir
Yolk after uniformly, which is placed at -20 ± 3 DEG C, at least freezes 48h.
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| CN109613252A (en) * | 2018-10-22 | 2019-04-12 | 新疆医科大学第附属医院 | Echinococcus granulosus high immunity yolk antibody and test strips and preparation method and application |
| CN110128541A (en) * | 2019-04-16 | 2019-08-16 | 广东工业大学 | A kind of preparation method and application of Yolk antibody that preventing and treating chronic periodontitis |
| CN110240648A (en) * | 2019-06-17 | 2019-09-17 | 长春西诺生物科技有限公司 | Cat infectiousness nose conjunctivitis and feline panleukopenia bigeminy freeze-dried yolk antibody and preparation and application |
| CN112300275A (en) * | 2020-02-19 | 2021-02-02 | 南京蛋球球生物医学技术合伙企业(有限合伙) | Yolk antibody for inhibiting new coronavirus SARS-CoV-2 and its preparation method and application |
| CN111718410B (en) * | 2020-06-05 | 2022-10-11 | 江南大学 | Method for preparing yolk immunoglobulin |
| CN117164702B (en) * | 2023-09-13 | 2024-05-10 | 江苏润洁生物科技有限公司 | Preparation method and application of yolk globulin for clinical HPV infection |
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