CN108191975A - Mite allergen special yolk immune globulin antibody and its antiallergy preparation - Google Patents
Mite allergen special yolk immune globulin antibody and its antiallergy preparation Download PDFInfo
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- CN108191975A CN108191975A CN201810053167.2A CN201810053167A CN108191975A CN 108191975 A CN108191975 A CN 108191975A CN 201810053167 A CN201810053167 A CN 201810053167A CN 108191975 A CN108191975 A CN 108191975A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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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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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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Abstract
The present invention relates to a kind of mite allergen special yolk immune globulin antibody and its antiallergy preparation, mite allergen special yolk immune globulin antibody is mainly prepared by the following method：Mite allergen extracts, reinforced immunological is carried out to chicken, prepared by crude extract, essence carries, purifies.The anti-mite allergen special yolk immunoglobulin of the present invention and its antibody activity of preparation are high, have the defense reaction of specificity to sensitization local caused by mite, there is good deadening effect to sensitization local caused by other sensibiligens, non-immunogenicity, it has no adverse reaction to human skin mucous membrane, security performance is high.
Description
Technical field
The invention belongs to biomedicine fields, are related to a kind of Yolk immunoglobulin antibody and its antiallergy preparation, especially
It is related to a kind of mite allergen special yolk immune globulin antibody and its antiallergy preparation.
Background technology
Allergic rhinitis (allergic rhinitis, AR) are that body is exposed to the nose mainly mediated after allergen by IgE
The non-infectious chronic inflammatory disease of mucous membrane.A large amount of epidemiological survey both at home and abroad shows that global AR patient is more than 500,000,000.AR
It has become as main respiratory tract chronic inflammatory disease, brings and seriously affect to patients ' life quality and social economy.In face of such
The requirement that high illness rate and patient improves quality of life, clinically various treatment measures come into being, nasal cavity part sugar
Cortin, oral antihistamines and leukotriene receptor retarding agent are current most important therapies, but can only control symptom.
Desensitization treatment can make 70% or so patient generate tolerance to corresponding anaphylactogen, but the period of desensitizing, up to 3 years or more, expense is very
Height, and to be treated in the case where doctor observes, the compliance of patient is not high.
Mite is dermatophagoides pteronyssinus, dust mite etc. in the most important mite in subtropical zone and torrid areas.Dermatophagoides pteronyssinus using mankind's scurf as food,
And it mainly lives in mattress, bed bottom, pillow, carpet, furniture and push toy.In heat（20 DEG C or more）It is and moist（It is relatively wet
Degree is more than 80%）Environment in breed it is most fast.Dermatophagoides pteronyssinus allergen is included in its fecal pellets, when the fabric of contamination is touched
After dynamic, these particles are just exposed in air and can deposit again quickly, and mite resists as indoor main sensibiligen, mite
Original has direct stimulation to human airway epithelial cells and congenital immunity cell, easily causes allergic rhinoconjunctivitis and allergic asthma,
And contribute to the generation of atopic eczema and other anaphylaxis dermatosis.If being directed in local application has enzymatic activity allergen
Antibody, activity will be weakened, cannot be combined with IgE, and allergic reaction would not occur.
Yolk immunoglobulin IgY is one kind of antibody, is immunized using what biological high-tech was extracted from birds yolk
Globulin, combine potency and in and the ability of the antigens such as virus, bacterium, fungi and insect in terms of, IgY and human antibodies phase
Seemingly, IgY can specifically bind with the epiope on corresponding sex pheromone surface and inhibit anaphylactoid generation, these
Activity reduces after sex pheromone antigen is combined by IgY, and loses pathogenecity, is easy to by the IgY antigens combined by people
Para-immunity system identification, and pass through the degradations such as monocyte, because IgY will not change the inhereditary material and biology of sex pheromone
Characteristic is learned, so drug resistance will not be generated, this feature of IgY makes it be of great significance in topical application.Moreover, skin
Autopath can also apply, and beneficiaries are very extensive.Therefore, for mite allergen, there is an urgent need for exploitation exempting from including yolk newly
The antibody of epidemic disease globulin IgY.
Invention content
In view of the deficiencies of the prior art, the present invention proposes a kind of mite allergen special yolk immune globulin antibody and
Its antiallergy preparation, for treating the allergic rhinitis as caused by mite allergen, allergic rhinitis with asthma and allergic dermatitis
Etc. diseases.
In order to solve the above technical problems, the technical solution that the present invention takes is：
The present invention proposes a kind of preparation method of mite allergen specific IgY antibody, includes the following steps：
It is prepared by mite allergen：Purifying acarid body is chosen, using Coca ' s liquid extracting methods, mite allergen crude extract is made；
It is prepared by immune vaccine：The mite allergen crude extract is dissolved in abundant mixing in the PBS solution of 0.1mol/L, is obtained
Mite allergen solution, protein concentration 2mg/ml；1-1.5ml mite allergen solution is drawn, adds in 5.25-6ml physiological saline
Mixing obtains mite hybrid antigen solution I, by 1:Freund's complete adjuvant is added in mite hybrid antigen solution I and mixed by 1 ratio
Uniformly, emulsus mite complex antigen immunologic adjuvant I is made, as the immune seedlings of head；2-3ml mite allergen solution is drawn, adds in 5.25-
6ml physiological saline mixings obtain mite hybrid antigen solution II, by 1:Incomplete Freund's adjuvant is added in mite and mixed by 1 ratio to be resisted
It is uniformly mixed in original solution II, emulsus mite complex antigen immunologic adjuvant II is made, as reinforced immunological vaccine；
Reinforced immunological is carried out to chicken：Draw the head and exempt from 200 μ l of vaccine dose, respectively each two under the left and right wing of chicken at carry out skin
Lower injection carries out first immunisation, then primary using the reinforced immunological vaccine reinforced immunological every 10 days, altogether reinforced immunological four
It is secondary；It collects the produced egg of chicken by reinforced immunological respectively after third time and the 4th reinforced immunological, and is adopted under chicken wings
Blood, blood sampling volume 1-1.5ml, first immunisation and first time reinforced immunological are carried out in early period of laying eggs；
The extraction of IgY antibody：The egg of collection is carried out that yolk is taken to handle, 1 volume yolk adds the distilled water of 5-6 volumes, fully
After being uniformly mixed, pH to 5-6 is debugged, 4 DEG C is then placed it in and stands 8-10 hours, refrigerated centrifuge 15-30min takes
Clear liquid obtains crude extract；
Essence carries：It is primary that 60% sodium sulphate precipitation is added in the crude extract of acquisition, then with 14% metabisulfite solution precipitation twice, is taken
Clear liquid carries out heat sterilization, deionization obtains extract；
Purifying：Column chromatography purifying is carried out to the extract of acquisition by cellulose, obtains mite allergen specific IgY antibody.
Further, the mite allergen crude extract includes：Dermatophagoides pteronyssinus protein D er p1 and Der p2, dust mite albumen
Der f1 and Der f2.
Further, the purifying acarid body obtains by the following method：Acarid is seeded to flour culture medium, by described in
Flour culture medium merging temperature is 25 DEG C, is cultivated 4-6 weeks in the insulating box of relative humidity 75%, acarid group is formed, by the mite
Worm group, which is moved into together with part flour culture medium in the container equipped with fresh feed, continues culture 4-6 weeks, repeats aforesaid operations and obtains
Largely pure mite is obtained, a large amount of pure mites of acquisition are moved into whole mite culture mediums further breeds, and obtains purifying acarid body.
Further, the chicken of the progress reinforced immunological is one kind in plum forests chicken, the red chicken of hy-line brown hen and capital.
Further, the chicken age of the chicken for carrying out reinforced immunological is 120-150 days, and every chicken is 1.2-1.5kg.
Further, the produced chicken of chicken by reinforced immunological is collected respectively after third time and the 4th reinforced immunological
Egg, and 1 is extracted to every 6 of the chicken by reinforced immunological, it takes a blood sample under chicken wings, blood sampling volume 1-1.5ml.
Further, the speed of the refrigerated centrifuge is 12000r/min.
Further, the mite allergen specific IgY antibody is preparing treatment allergia nose as caused by mite allergen
Scorching and allergic rhinitis is with the purposes in the drug of asthma.
Further, the mite allergen specific IgY antibody is preparing treatment anaphylaxis skin as caused by mite allergen
Purposes in scorching drug, the allergic dermatitis include acne.
Simultaneously, it is proposed that a kind of antiallergy preparation according to made of foregoing mite allergen specific IgY antibody is pressed
Include following component according to weight：
Mite allergen specific IgY antibody or its crude extract 0.15-2%；
Hydroxyethyl cellulose 0.5-1%；
Xylitol 0.5-1.0%；
Glycerine 1.0-2.0%；
Polyoxyethylene sorbitan monoleate 0.3-0.5%；
Refrigerant alcohol 0.1-0.3%；
Chlorhexidine 0.01-0.02%；
Distilled water adds to 100%.
Beneficial effects of the present invention include at least：Mite allergen special yolk immune globulin antibody of the present invention and its anti-
Allergy preparations, can reach all standing for causing allergic rhinitis and the main sensitization source of allergic asthma, antibody activity obtained is high,
There is the defense reaction of specificity to sensitization local caused by mite, there is good deaden to imitate sensitization local caused by other sensibiligens
Should, non-immunogenicity has no adverse reaction to human skin mucous membrane, and security performance is high.
Description of the drawings
Fig. 1 is mite allergen crude extract electrophoretogram of the present invention.
Fig. 2 is mite allergen special yolk immune globulin IgY antibody electrophoretogram of the present invention.
Fig. 3 is mite allergen crude extract Western Blot of the present invention detection figures.
Fig. 4 is antiallergy preparation of the present invention film forming testing inspection figure.
Fig. 5 is antiallergy preparation animal surface skin of the present invention and cornea allergy and toxicity test detection figure.
Fig. 6 is that No. 1 example pharmaceuticals cell toxicity test of antiallergy preparation of the present invention inhibits matched curve figure.
Fig. 7 is that No. 2 example pharmaceuticals cell toxicity tests of antiallergy preparation of the present invention inhibit matched curve figure.
Fig. 8 is that No. 3 example pharmaceuticals cell toxicity tests of antiallergy preparation of the present invention inhibit matched curve figure.
Fig. 9 is antiallergy preparation bacterium blocking Test Drawing of the present invention.
Wherein：In Fig. 1, M is standard protein molecular weight, and 1 is 2,3 and 4 three kind of allergen mixing, prepared by 2 present invention
Mite allergen crude extract, 3 be A Luoge standard dermatophagoides pteronyssinus skin test antigens, and comprising Der p1 and Der p2 albumen, 4 be sieve's Ah case marker
Quasi- dust mite skin test antigen includes Der f1 and Der f2 albumen；
In Fig. 2, M is standard protein molecular weight, and A, B, C and D are respectively：The present invention repeats IgY antibody, the present invention that experiment obtains
The IgY antibody, anti-various bacteria antibody and blank control of acquisition are tested for the first time；A, b, c and d are respectively that A, B, C and D correspond to antibody
50 DEG C of water bath processings 30 minutes；
In Fig. 3,1 is 2,3 and 4 three kind of allergen mixing, and the 2 mite allergen crude extracts prepared for the present invention, 3 be A Luoge standards
Dermatophagoides pteronyssinus skin test antigen, 4 be A Luoge standard flour dust mite skin test antigens；
In Fig. 4, left side is respectively that No. 1 example pharmaceuticals, No. 2 example pharmaceuticals and No. 3 example pharmaceuticals, right side are blank control group；
In Fig. 5, D is：Upside is control group, and downside is No. 1 example pharmaceuticals experimental group；E is：Upside is control group, and downside is No. 2
Example pharmaceuticals experimental group；F is：Upside is control group, and downside is No. 3 example pharmaceuticals experimental groups；
In Fig. 9, G is blank control group, and H is No. 1 example pharmaceuticals, and I is No. 2 example pharmaceuticals, and J is No. 3 example pharmaceuticals.
Specific embodiment
In order to which those skilled in the art is made to more fully understand technical scheme of the present invention, with reference to specific embodiment to this
Invention is described in further detail.The embodiments described below is exemplary, and is only used for explaining the present invention, without being understood that
For limitation of the present invention.Particular technique or condition are not specified in embodiment, according to the described skill of document in the art
Art or condition are carried out according to product description.
Below in conjunction with specific Test Drawing to mite allergen special yolk immune globulin antibody of the present invention and
Its antiallergy preparation is specifically addressed.
The preparation method of mite allergen specific IgY antibody of the present invention, specifically includes following steps.
First, prepared by mite allergen
Mite allergen extraction raw material comes from purifying acarid body（Including>90% acarid body, a little excrement and different hairs
Educate stage mite）, from bedroom bedding, mattress, bed bottom, room angle collect dust after conventional treatment obtains mite, acarid is inoculated with
To flour culture medium, the flour culture medium is placed in temperature and is 25 DEG C, cultivated 4-6 weeks in the insulating box of relative humidity 75%, shape
Into acarid group, the acarid group is moved into together with part flour culture medium in the container equipped with fresh feed and continues to cultivate 4-
It 6 weeks, repeats aforesaid operations and obtains a large amount of pure mites, a large amount of pure mites of acquisition are moved into whole mite culture medium further breeds, and obtains
Acarid body is purified, then using Coca ' s liquid extracting methods, mite allergen crude extract is made.
According to some embodiments of the present invention, the concrete type of the container is unrestricted, preferably glass jar.
Coca ' s liquid extracting method of the present invention is the extracting method of the art routine, is mainly included：This is sent out
The purifying acarid body of bright acquisition is inactivated, removes lipoprotein, liquid nitrogen multigelation, mills successively, ultrasonication, coca ' s are molten
Liquid extracts, and obtains mite allergen crude extract.Fig. 1 is mite allergen crude extract electrophoretogram of the present invention, with reference to shown in Fig. 1, the present invention
The mite allergen crude extract shows protein band in molecular weight between 34-26kDa, near 17kDa and 25kDa, comprising point
Son amount is 27kDa, 15kDa dermatophagoides pteronyssinus major protein Der p1 and Der p2；And 25kDa, 14kDa dust mite major protein
Der f1 and Der f2.
Der p1 are dermatophagoides pteronyssinus albumen, and concentration is high in mite excrement, belongs to papain cysteine proteinase family
A kind of main allergen.Lead to the generation of IgE antibody in anaphylaxis atopic individuals after human body sucking Der p1, when sucking again
Der p1 are the I allergic reaction types and its homologous dust mite Der f1 occurred based on allergic rhinitis, since its antigen is determined
It is identical with Der p1 to determine cluster, the two has cross reaction.Der p2 are acarid body proteins, and severe asthma patient is more commonly right
Der p2 and Der f2 dust mite allergies, and children are more more sensitive than Der p1 to Der p2.Mite allergen of the present invention slightly carries
Object is simultaneously using tetra- kinds of Der p1, Der f1, Der p2 and Der f2 anaphylactogens, you can basically reaches allergic rhinitis and allergy
The all standing of the property main sensitization source of asthma.
2nd, mite allergen special yolk immune globulin IgY Antibody preparation
1. prepared by immune vaccine：The mite allergen crude extract being prepared is dissolved in the PBS solution of 0.1mol/L
Abundant mixing obtains mite allergen solution, protein concentration 2mg/ml；Draw 1-1.5ml mite allergen solution（It is as anti-
Original solution）, 5.25-6ml physiological saline mixings are added in, mite hybrid antigen solution I are obtained, by 1:1 ratio helps Freund completely
Agent is added in mite hybrid antigen solution I to push away to beat repeatedly and is uniformly mixed, and emulsus mite complex antigen immunologic adjuvant I is made, exempts from as head
Vaccine；
Draw 2-3ml mite allergen solution（As antigenic solution）, 5.25-6ml physiological saline mixings are added in, it is anti-to obtain mite mixing
Original solution II, by 1:Incomplete Freund's adjuvant is added in push away to beat repeatedly in mite hybrid antigen solution II and is uniformly mixed by 1 ratio,
Emulsus mite complex antigen immunologic adjuvant II is made, as reinforced immunological vaccine.
2. pair chicken carries out reinforced immunological：The present invention is preferably plum forests chicken, and chicken age is 120-150 days, and every plum forests chicken is
1.2-1.5kg collects the egg before being immunized as blank control group.It draws the head and exempts from 200 μ l of vaccine dose, points 4 points subcutaneous
Injection, respectively each two under the left and right wing of plum forests chicken at carry out being subcutaneously injected into carry out first immunisation, then every 10 days use institute
It is primary to state reinforced immunological vaccine reinforced immunological, altogether reinforced immunological four times；It is collected respectively after third time and the 4th reinforced immunological
1 is extracted by the produced egg of plum forests chicken of reinforced immunological, and to every 6 of the plum forests chicken by reinforced immunological, under chicken wings
Blood sampling, blood sampling volume 1-1.5ml, first immunisation and first time reinforced immunological are carried out in early period of laying eggs；The blank control group is same
When collect and egg and take a blood sample.
According to some embodiments of the present invention, the chicken for carrying out reinforced immunological can also be the red chicken of hy-line brown hen or capital
Deng.
3. the extraction of mite allergen special yolk immune globulin IgY antibody：The egg of collection is checked one by one, egg
Shell has the not bright-coloured rejecting of flaw, breakage or color, carries out that yolk is taken to handle, and 1 volume yolk adds the distilled water of 5-6 volumes, fills
Divide after being uniformly mixed, debug pH to 5-6, pH is preferably 5.5；Then 4 DEG C are placed it in and stands 8-10 hours, preferably 8
Hour；Refrigerated centrifuge 15-30min, preferably 15min, the speed of the refrigerated centrifuge is 12000r/min；Supernatant is taken, i.e.,
For crude extract.
4. essence carries：It is primary that 60% sodium sulphate precipitation is added in the crude extract of acquisition, then two are precipitated with 14% metabisulfite solution
It is secondary, supernatant is taken to carry out heat sterilization, deionization acquisition extract.
5. purifying：Column chromatography purifying is carried out to the extract of acquisition by cellulose, mite allergen specific IgY is obtained and resists
Body also obtains other albumen and includes transferrins, lysozyme etc..According to some embodiments of the present invention, the present invention may be used
DEAE celluosic resins are isolated and purified as ion-exchanger.
According to an embodiment of the invention, Fig. 2 is mite allergen special yolk immune globulin IgY antibody electrophoresis of the present invention
Figure, IgY is a kind of 7s immunoglobulins, is made of the heavy chain of 67-70kDa and the light chain of 22-30kDa, mite allergen is immunized
The chicken with yolk protein extract that chicken obtains（As crude extract or extract）Electrophoresis result is shown（As shown in Figure 2）：Purifying IgY exists
Molecular weight shows a main band between 55-72kDa, and molecular weight is the heavy chain of 68kDa, and one is shown between 26-34kDa
The smaller band of item, molecular weight are the light chain of 28kDa.50 DEG C of water bath processings property of protein stabilization after 30 minutes, shows the present invention
The mite allergen specific IgY of preparation has good thermal stability.
According to an embodiment of the invention, Fig. 3 is mite allergen crude extract Western Blot of the present invention detection figures, with reference to figure
Shown in 3, the present invention is tested through protein immunoblotting and is found, four kinds of antigens are main for display at 17-34kDa four in molecular weight
Band, and have hangover, show IgY antibody prepared by the present invention can allergen proteins specific bindings a variety of with mite, be that one kind is more
Clonal antibody.IgY antibody prepared by the present invention carries out ELISA detections, is common ELISA indirect methods, and experimental procedure is main
Including：1. coating：By antigen used coating dilution PBS1:100 dilution coatings；2. closing：PBS solution and bovine serum albumin
White 1:100 closings；3. add in detected sample（IgY antibody samples prepared by the present invention）：It is loaded 100 μ l；4. it adds in enzyme mark to resist
Body：PBS solution and horseradish peroxidase（1:10000）The secondary antibody donkey anti-chicken IgG of label；5. add 100 μ l of substrate solution；6. it terminates
Reaction：Add in terminate liquid H2SO4, 50 μ l；7. survey OD values.ELISA testing results show that it is 1 to repeat to test immune serum potency:
2048, it is 1 to test immune serum potency for the first time:1024, corresponding IgY antibody titers are 1:2048~4096 and 1:1024, blank
Control serum potency 1:128, albumen potency 1:64, it is known that IgY antibody titers prepared by the present invention have reached wanting for clinical practice
It asks.
According to some embodiments of the present invention, the mite allergen specific IgY antibody is preparing treatment by mite allergen
Caused allergic rhinitis and allergic rhinitis are with the purposes in the drug of asthma.
According to some embodiments of the present invention, the mite allergen specific IgY antibody is preparing treatment by mite allergen
Purposes in the drug of caused allergic dermatitis, the allergic dermatitis include acne.
Meanwhile the present invention proposes a kind of antiallergy according to made of foregoing mite allergen specific IgY antibody
Preparation includes following component according to weight：
Mite allergen specific IgY antibody or its crude extract 0.15-2%；
Hydroxyethyl cellulose 0.5-1%；
Xylitol 0.5-1.0%；
Glycerine 1.0-2.0%；
Polyoxyethylene sorbitan monoleate 0.3-0.5%；
Refrigerant alcohol 0.1-0.3%；
Chlorhexidine 0.01-0.02%；
Distilled water adds to 100%.
Cellulose is by being dehydrated glucopyranose units by the D- of β-(1 → 4)-glucosides key connection（AGU）The line of composition
Property syndiotactic homopolymer, hydroxyethyl cellulose using ethylene oxide due to being modified, instead of the part of hydroxyl on cellulose,
Good water solubility is made it have, aqueous solution has higher viscosity, can be used as hydrophilic gel and dispersion stabilizer etc..In this system
Act what is disperseed and stablize to IgY using the function of its repetitive unit in agent, simultaneously because its viscosity is higher, good salt is molten
Property, biocompatibility and keep the performances such as moisture, there is certain protective effect to mucous membrane, and IgY can be extended in the stagnant of part
Stay the time.The chlorhexidine is surfactant, and anti-corrosion is used in this preparation；The polyoxyethylene sorbitan monoleate is also that a kind of surface is lived
Property agent, serve in this preparation emulsification and solubilising, remaining ingredient form moisturizing, seasoning, stabilising system.It is 7-7.5 in pH
Under conditions of room temperature preservation half a year, preparation still clarifies, no precipitation.The present invention also uses hydroxypropyl methyl cellulose and carboxylic first respectively
Base cellulose replace hydroxyethyl cellulose carry out preparation preparation, although both high molecular materials can be used as excipient substance and
Dispersant, and its dispersibility is stronger than hydroxyethyl cellulose in theory, but the solution that the former prepares is slightly aobvious muddy, Hou Zheyou
Floccule precipitates to be formed（Adsorb polyoxyethylene sorbitan monoleate）, finally using dispersant of 0.5% hydroxyethyl cellulose as IgY preparations, use
In drug delivery.
Antiallergy preparation made of mite allergen specific IgY antibody of the present invention（Hereinafter referred to as：Anti- mite allergen system
Agent）Performance detection.
1. into film test：
It presses above-mentioned formula and prepares No. 1 example pharmaceuticals（Anti- mite allergen preparation of the present invention）, hydroxypropyl methyl is then used respectively
Cellulose and carboxymethyl cellulose prepare No. 2 and No. 3 example pharmaceuticals instead of hydroxyethyl cellulose, and No. 3 example pharmaceuticals have cotton-shaped
Object precipitates, centrifugation removal；1,2, No. 3 example pharmaceuticals 200ul is taken respectively, is sprayed on 6 well culture plates, it is micro- after drying at room temperature
Microscopic observation makees blank control with PBS buffer solution, and as a result only No. 1 example pharmaceuticals form the moderate film of coarse uniform thickness, and
2nd, No. 3 example pharmaceuticals are not uniform enough, thickness low LCL, and Fig. 4 is antiallergy preparation of the present invention film forming testing inspection figure, with reference to Fig. 4 institutes
Show.
2. animal surface skin and cornea allergy and toxicity test：
Experimental animal：Nude mice is the mouse of immune deficiency, easily infects, and skin and hair is rare, is easy to observe；
Experimental method：6 week old maturation nude mices are divided into tri- groups of D, E, F as experimental subjects, wherein, D is：Upside is control group, under
Side is No. 1 example pharmaceuticals experimental group；E is：Upside is control group, and downside is No. 2 example pharmaceuticals experimental groups；F is：Upside is control
Group, downside are No. 3 example pharmaceuticals experimental groups；The upside is not intervened for control group, and downside is experimental group, give 1 respectively, 2,
No. 3 example pharmaceuticals.Mouse is fixed with left hand, is sprayed on periorbit skin and cornea with experimental drug（It need to ensure fast when mouse opens eyes
Speed sprinkling）, sprayed per side one, every other day administration is administered 2 weeks, about 7-8 times in total.Observe the variation of nude mice skin and cornea.
Experimental result：Start to be administered in 2017-07-19,2017-08-02 last times are administered, and are administered 8 times in total.It does not see
Observing the skin of experimental group and control animals and cornea has the changes such as secretion, fash, ulcer；The skin of nude mice before and after experimental group
The color of skin and cornea, glossiness etc. have not observed apparent change, and Fig. 5 is antiallergy preparation animal surface skin of the present invention
Skin and cornea allergy and toxicity test detection figure, with reference to shown in Fig. 5, show invention formulation external application safety.
3. cell toxicity test：
Experiment packet：No. 1,2,3 example pharmaceuticals.Every group of drug sets 3 blocks of plates, observes the example pharmaceuticals of 0H, 24H and 48H
To the inhibiting effect of 239T cells.
Experimental method：Inhibiting effect of the drug to 293T cell lines is detected with mtt assay.Take the logarithm growth period 293T it is thin
Born of the same parents remove supernatant and are washed twice with PBS solution, and after being digested with pancreatin, digestion is terminated with full training.It is planted after cell count into 96 holes
Plate is adjusted to every 2000 cells in hole.5%CO2, after 37 DEG C are incubated 24 hours, 1,2,3 is separately added into according to concentration gradient setting
Number example pharmaceuticals and each 10ul of control group（Each concentration sets 3 multiple holes）.Immediately the Drug inhibition effect of each group 0H is surveyed.Respectively
MTT 10ul are added in per hole（2mg/ml）5%CO afterwards2, 37 DEG C are incubated 4 hours, remove supernatant, add in DMSO 150ul.Fully shake
After swinging absorbance is detected in OD 490nm with microplate reader.Absorbance after same method detection drug effect 24H, 48H.
The concentration gradient setting（Unit ul）It is as follows：
| 10 | 5 | 2 | 1 | 0.5 | 0.2 | 0.1 | 0 |
（The present invention 8 prescriptions of setting just calculate IC50）, time point detection is respectively：0H, 24H, 48H.
Experimental result：
Fig. 6-8 is respectively that 3 groups of example pharmaceuticals cell toxicity tests of antiallergy preparation of the present invention inhibit matched curve figure.With reference to figure
Shown in 6-8 matched curves, 0H results show example pharmaceuticals without obvious effect, and each hole cell quantity of 96 orifice plates is evenly distributed.3 groups
Each concentration gradients of example pharmaceuticals 24H are to cell without obvious inhibiting effect.Slightly inhibiting occur in the visible 3 groups of drugs of 48H results
Effect, shows that invention formulation low concentration application is safe.Calculate each group drug IC50 it is as follows：
No. 1 example pharmaceuticals IC50:1.6(1.561-1.646) ul
No. 2 example pharmaceuticals IC50:1.2(1.252-1.286) ul
No. 3 example pharmaceuticals IC50:1.6(1.456-1.739) ul.
4. microorganism barrier experiment：
Respectively with 1,2, No. 3 sample（It is filtered using 0.22um）It is intensive to be coated on Micro-Organism Culture Dish, respectively 100ul,
200ul, 300ul and blank control.Culture dish is exposed to 6h in air after air-drying, and cultivates, and calculates bacterium colony and is formed and taken pictures（Fig. 9
In, G is blank control group, and H is No. 1 example pharmaceuticals, and I is No. 2 example pharmaceuticals, and J is No. 3 example pharmaceuticals）.Bacterial clump calculates：
Blank group G：25；
No. 1 H groups 100ul：25； 200ul：18； 300ul：14；
No. 2 I groups 100ul：20； 200ul：19； 300ul：13；
No. 3 J groups 100ul：16； 200ul：6； 300ul：18；
The results show that 1, No. 2 example pharmaceuticals of the invention have bacterium deadening effect with concentration increase, have with blank control comparing difference
Conspicuousness（p<0.05), and No. 3 example pharmaceuticals effects are unstable.
Inventor has found that mite allergen special yolk immune globulin antibody and its antiallergy preparation of the present invention are reachable
To all standing of allergic rhinitis and the main sensitization source of allergic asthma, antibody activity obtained is high, to locally being caused caused by mite
The quick defense reaction for having specificity has good deadening effect to sensitization local caused by other sensibiligens, and non-immunogenicity is right
Human skin mucous membrane has no adverse reaction, and security performance is high.
The above embodiment is only the preferred embodiment of the present invention, it is impossible to the scope of protection of the invention is limited with this,
The variation and replacement for any unsubstantiality that those skilled in the art are done on the basis of the present invention belong to the present invention and want
Seek the range of protection.
Claims (10)
1. a kind of preparation method of mite allergen specific IgY antibody, which is characterized in that include the following steps：
It is prepared by mite allergen：Purifying acarid body is chosen, using Coca ' s liquid extracting methods, mite allergen crude extract is made；
It is prepared by immune vaccine：The mite allergen crude extract is dissolved in abundant mixing in the PBS solution of 0.1mol/L, is obtained
Mite allergen solution, protein concentration 2mg/ml；1-1.5ml mite allergen solution is drawn, adds in 5.25-6ml physiological saline
Mixing obtains mite hybrid antigen solution I, by 1:Freund's complete adjuvant is added in mite hybrid antigen solution I and mixed by 1 ratio
Uniformly, emulsus mite complex antigen immunologic adjuvant I is made, as the immune seedlings of head；2-3ml mite allergen solution is drawn, adds in 5.25-
6ml physiological saline mixings obtain mite hybrid antigen solution II, by 1:Incomplete Freund's adjuvant is added in mite and mixed by 1 ratio to be resisted
It is uniformly mixed in original solution II, emulsus mite complex antigen immunologic adjuvant II is made, as reinforced immunological vaccine；
Reinforced immunological is carried out to chicken：Draw the head and exempt from 200 μ l of vaccine dose, respectively each two under the left and right wing of chicken at carry out skin
Lower injection carries out first immunisation, then primary using the reinforced immunological vaccine reinforced immunological every 10 days, altogether reinforced immunological four
It is secondary；It collects the produced egg of chicken by reinforced immunological respectively after third time and the 4th reinforced immunological, and is adopted under chicken wings
Blood, blood sampling volume 1-1.5ml, first immunisation and first time reinforced immunological are carried out in early period of laying eggs；
The extraction of IgY antibody：The egg of collection is carried out that yolk is taken to handle, 1 volume yolk adds the distilled water of 5-6 volumes, fully
After being uniformly mixed, pH to 5-6 is debugged, 4 DEG C is then placed it in and stands 8-10 hours, refrigerated centrifuge 15-30min takes
Clear liquid obtains crude extract；
Essence carries：It is primary that 60% sodium sulphate precipitation is added in the crude extract of acquisition, then with 14% metabisulfite solution precipitation twice, is taken
Clear liquid carries out heat sterilization, deionization obtains extract；
Purifying：Column chromatography purifying is carried out to the extract of acquisition by cellulose, obtains mite allergen specific IgY antibody.
2. preparation method according to claim 1, which is characterized in that the mite allergen crude extract includes：Dermatophagoides pteronyssinus egg
White Der p1 and Der p2, dust mite protein D er f1 and Der f2.
3. preparation method according to claim 1, which is characterized in that the purifying acarid body obtains by the following method：
Acarid is seeded to flour culture medium, the flour culture medium is placed in temperature is 25 DEG C, is trained in the insulating box of relative humidity 75%
It supports 4-6 weeks, forms acarid group, the acarid group is moved into together with part flour culture medium in the container equipped with fresh feed
Continue culture 4-6 weeks, repeat aforesaid operations and obtain a large amount of pure mites, it is further that a large amount of pure mites of acquisition are moved into whole mite culture medium
Breeding obtains purifying acarid body.
4. preparation method according to claim 1, which is characterized in that the chicken for carrying out reinforced immunological is plum forests chicken, sea
One kind in blue brown chicken and the red chicken in capital.
5. preparation method according to claim 4, which is characterized in that the chicken age of the chicken for carrying out reinforced immunological is 120-
150 days, every chicken was 1.2-1.5kg.
6. preparation method according to claim 1, which is characterized in that received respectively after third time and the 4th reinforced immunological
Collection passes through the produced egg of chicken of reinforced immunological, and extracts 1 to every 6 of the chicken by reinforced immunological, takes a blood sample, adopts under chicken wings
Blood volume is 1-1.5ml.
7. preparation method according to claim 1, which is characterized in that the speed of the refrigerated centrifuge is 12000r/min.
8. preparation method according to claim 1, which is characterized in that prepared by the mite allergen specific IgY antibody
The allergic rhinitis as caused by mite allergen and allergic rhinitis are treated with the purposes in the drug of asthma.
9. preparation method according to claim 1, which is characterized in that prepared by the mite allergen specific IgY antibody
The purposes in the drug of the allergic dermatitis as caused by mite allergen is treated, the allergic dermatitis includes acne.
10. antiallergy preparation made of mite allergen specific IgY antibody according to claim 1, which is characterized in that press
Include following component according to weight：
Mite allergen specific IgY antibody or its crude extract 0.15-2%；
Hydroxyethyl cellulose 0.5-1%；
Xylitol 0.5-1.0%；
Glycerine 1.0-2.0%；
Polyoxyethylene sorbitan monoleate 0.3-0.5%；
Refrigerant alcohol 0.1-0.3%；
Chlorhexidine 0.01-0.02%；
Distilled water adds to 100%.
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| CN201810053167.2A CN108191975A (en) | 2018-01-19 | 2018-01-19 | Mite allergen special yolk immune globulin antibody and its antiallergy preparation |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1965665A (en) * | 2005-11-16 | 2007-05-23 | 雅臣药业集团（远东）有限公司 | Method for preparing immune milk or milk powder containing IgY antibody |
| CN101628119A (en) * | 2009-08-21 | 2010-01-20 | 北京新华联协和药业有限责任公司 | Dermatophagoides pteronyssinus (Der p) allergen diagnostic reagent and preparation method thereof |
| WO2016062832A2 (en) * | 2014-10-23 | 2016-04-28 | Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e. V. | Vaccine development against the poultry red mite dermanyssus gallinae |
-
2018
- 2018-01-19 CN CN201810053167.2A patent/CN108191975A/en not_active Withdrawn
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1965665A (en) * | 2005-11-16 | 2007-05-23 | 雅臣药业集团（远东）有限公司 | Method for preparing immune milk or milk powder containing IgY antibody |
| CN101628119A (en) * | 2009-08-21 | 2010-01-20 | 北京新华联协和药业有限责任公司 | Dermatophagoides pteronyssinus (Der p) allergen diagnostic reagent and preparation method thereof |
| WO2016062832A2 (en) * | 2014-10-23 | 2016-04-28 | Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e. V. | Vaccine development against the poultry red mite dermanyssus gallinae |
Non-Patent Citations (4)
| Title |
|---|
| WAYNE R. THOMAS: "House Dust Mite Allergens: New Discoveries and Relevance to the Allergic Patient", 《CURR ALLERGY ASTHMA REP》 * |
| 王伟青等: "《免疫学技术》", 30 June 2014, 重庆大学出版社 * |
| 石连等: "粉尘螨变应原Der f1 基因的原核表达及多克隆抗体的制备", 《皖南医学院学报》 * |
| 鲍尔: "《药物制剂包衣原理工艺及设备》", 30 April 2006, 中国医药科技出版社 * |
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