CN1657541A - Process for preparating specificity complex IgY of anti coxsackie virus myocarditis and its compound preparation - Google Patents
Process for preparating specificity complex IgY of anti coxsackie virus myocarditis and its compound preparation Download PDFInfo
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- CN1657541A CN1657541A CN 200410005797 CN200410005797A CN1657541A CN 1657541 A CN1657541 A CN 1657541A CN 200410005797 CN200410005797 CN 200410005797 CN 200410005797 A CN200410005797 A CN 200410005797A CN 1657541 A CN1657541 A CN 1657541A
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Abstract
A process for preparing the coxsackie virus type myocarditis specific composite checken yolk immunoglobulin CVMD-IgY includes such steps as amplifying coxcackie virus in cells, purifying, deactivating, preparing its antigen, immunizing domestic fowls to obtain the immunized eggs, evtracting CVMD-IgY, and purifying. It can be used to prepare the composite medicines for preventing and treating viral myocarditis.
Description
Technical field
The present invention relates to be used to prevent and treat the medicine of the sick viral myocarditis of Sa Qi, more particularly, relate to a kind of preparation method and combination preparation thereof of specificity composite IgY (egg yolk immunoglobulin (Ig)) of anti-coxsackie myocarditis.
Background technology
1899, Fiedler (Fiedler) reported first because of heating, expiratory dyspnea, twitch, 3 routine viral myocarditis clinical cases that stupor is dead.Between after this more than 100 year,, researchs such as this sick cause of disease, pathogeny and immunity have been had very big breakthrough, but the treatment aspect still there is not big progress along with medical microbiology and relevant science and technology development.
After the seventies in 20th century, the viral myocarditis morbidity increases day by day, sickness rate height not only in children particularly, and consequence is serious, 1/3 patient can not recover normal heart function fully, 1/3 develops into chronic myocarditis and heart function disorder, and 1/3 final progress is for the death of chronic cardiac nonfunction or need heart transplantation
[1]According to epidemiology survey data both domestic and external, the morbidity of viral myocarditis is 2.3-5% among the crowd.The sickness rate of infection population reaches 12-33% during the viral prevalence, and sickness rate can reach 50% among the baby.The eruption and prevalence of viral myocarditis once took place in ground such as China Hubei, Yunnan, during local sickness rate reach 26.8-50%, case fatality rate is up to 23.6%
[2,3]Concerning medical circle, the diagnosis of viral myocarditis and treatment are its serious challenge that faces.
A lot of viruses can cause myocarditis, clear and definite so far plant virus surplus having ten, but enterovirus and adenovirus are the most common.Change of coxsackie b virus (hereinafter to be referred as CVB) in the special enterovirus has more than 50% to detect its IgM and neutralizing antibody in acute and chronic viral myocarditis patient approximately.In the biopsy and autopsy tissue of viral myocarditis, there is considerable sample can be checked through antigen and the nucleic acid of CVB.This external application CVB virus infected mice, the pathological change that can make its cardiac muscle produce similar Human virus's property myocarditis sample.Generally speaking, generally believe that at present CVB is the main virus that causes viral myocarditis
[4]CVB divides 6 types, and wherein the 1-5 type all can cause myocardium pathology.In addition, adenovirus, COxsackie A papova, echo, enteric cytopathogenic human orphan virus, simplexvirus etc. also can cause viral myocarditis.
By to the viral myocarditis The Animal Model Study, this sick pathogeny is clear and definite substantially.The direct effect of virus and the immune response of body are main mechanism.At acute and subacute stage, a large amount of viruses are duplicated in heart tissue, are bred and send out, and directly cause myocardial cell's dissolving and necrosis.Subsequently, scavenger cell, NK cell and T cell successively soak into cardiac muscle, the cytotoxicity of its mediation infects the myocardial cell at lytic virus, kill virus simultaneously, also damaged the myocardial cell, particularly under the situation that T cellular immunization is produced by inflammatory factor excessive activation such as IL-2 and cardiac response autoantibody, damage even more serious.In chronic phase, mainly show as virus and duplicate the low-level of myocardial cell's midium or long term, but coup injury myocardial structural and function, also sustainable activate immunity reaction and autoimmune response and cause myocardium indirect injury finally cause DCM (dilated cardiomyopathy).Therefore, the treatment of viral myocarditis acute phase based on antiviral, and acute later stage and chronic phase suppress the over-drastic inflammatory reaction and autoimmune response even more important
[5]
At present the treatment of viral myocarditis still there is not the specific method.Clinical in to alleviate symptomatic treatments such as heart burden, anti-arrhythmia and immunomodulatory.Report personnel selection immunoglobulin therapy is arranged in recent years by the mouse viral myocarditis that CVB and EMV (mouse brain myocarditis virus) cause, obtained better curative effect.No matter immunoglobulin (Ig) and virus are inoculated mouse simultaneously, still in the back use of 2 week of virus infection, myocardial cell's the necrosis and the infiltration of inflammatory cell all obviously reduce; The cytokine that inflammation is relevant descends; The survival time of mouse obviously prolongs.Particularly the former immunoglobulin (Ig) has almost completely suppressed the infringement of virus to cardiac muscle, does not have dead mouse in experimentation.And mechanism autoimmune disorder viral for immunoglobulin therapy is not fully aware of, thinks at present with following some is relevant: have certain neutralizing antibody in (1) immunoglobulin (Ig); (2) after lot of antibodies entered body by injection, it was saturated to make protection IgG avoid catabolic Fc acceptor, thereby promoted the decomposition of autoantibody; (3) immunoglobulin (Ig) resembles each complement component sewage lagoon in blood plasma, has reduced the complement in the blood, and reaction reduces inflammation; (4) suppress the release of the cytokine of inflammation-related, reduced the propagation and the infiltration of T cell
[6,7]
Prevent and treat virus disease with efficient immune serum of the mankind and immunoglobulin (Ig), using modern times always, particularly to the urgent prevention such as the HBV of some viruses, and the most effective present anti-HBV immunoglobulin (Ig) of people that height is tired that is still.In anti-SARS (atypical pneumonia) infected, total professor Jiang Suchun of institute of PLA had successfully treated self SARS infection with rehabilitation clients's serum, is recent noticeable example.But these methods of treatment exist all restrictions and potential danger factor.At first human efficient immune serum and immunoglobulin (Ig) source difficulty only just have enough serum available under the more situation of population infection.Secondly blood product safety is an open question always.And because the complement system of mammiferous IgG antibody energy activation of human, and can react with serum, Rheumatoid factors, polyclonal, disturb human body IgG, cause the cross immunity reaction, therefore aspect much, limited its application.
In view of above various reasons, the present patent application people has done initiative research in this respect, find that female bird has very good immunologic responsiveness to the various antigens of the mankind, can produce a large amount of persistence IgG with a small amount of antigen immune, reach yolk from serum again, can obtain a large amount of Yolk immunoglobulins (Immunoglobulin of Yolk is hereinafter to be referred as IgY) through the separation and Extraction purifying.
Because bird differs greatly with mammals on system takes place, therefore IgY can not combine with mammals generation serum cross reaction and Rheumatoid factors, polyclonal, can the human activin complement system yet, can not disturb IgG, avoid the toxic side effect of microbiotic and chemical synthetic drug and resistant organism to produce fully.And produce, extract, separate complete environmentally safe.Prevent and treat in the process, only pathogenic agent is damaged, human probiotics is not had any effect, thereby safeguarded local microecological balance.Specific IgY is at anti-dental caries, and the experimental study of opposing helicobacter pylori, salmonella and rotavirus infection has been obtained success [8,9].
IgY belongs to the IgG--immunoglobulin like protein, has the effect of neutralizing antibody, and it can combine with corresponding antigens generation specificity, thereby changes its corresponding antigen surface, as the configuration of virus surface, stops virus to be adsorbed in permissive cell; In addition, after its corresponding virus combination of IgY, can form immunocomplex, easily be engulfed by scavenger cell.
The applicant is having own exclusive patent core technology (JasonMedical Holdings Inc.USA) aspect the separation of IgY, extraction, the purifying, and first the collutory and the oral spray of specificity composite IgY preparation is tried out in human body disease preventing and treating and prevention and health care in the whole world
[10]
The applicant is engaged in the treatment of coxsackie myocarditis and the research work of prevention always.Dna vaccination and oral gene vaccine to Coxsackie virus are studied; find that these two kinds of vaccines all can stimulate body to produce certain immune response; isostructural virus infection there is the certain protection ability; but the antibody titers of humoral immunization is low; specific cell is also powerful inadequately; particularly Coxsackie virus and the enterovirus to other types do not have effective cross-protection, and these have limited its applying value.
Summary of the invention
The present invention will solve and be used to prevent and treat the more single and less-than-ideal problem of effect of myocarditic medicine in the prior art, with the new way of developing coxsackie myocarditis study on prevention.
For solving the problems of the technologies described above, the invention provides a kind of preparation method of specificity composite IgY (hereinafter to be referred as anti-CVMD-IgY) of anti-coxsackie myocarditis, comprising the following step:
(1) preparation causes the antigen of coxsackie myocarditis;
(2), and search the immune egg that described poultry produces with described antigen immune poultry;
(3) from described immune egg, extract anti-CVMD-IgY crude extract;
(4) described IgY crude extract is carried out purifying, obtain the pure product of anti-CVMD-IgY.
In (1) step of the method for the invention, can prepare CVB1-5 type antigen by following steps: the cultivation of virus, get the Hela cell and cultivate coxsackie B group 1-5 (CVB1-5) C-type virus C respectively; The purifying of virus adopts last diluted passage method eventually, and the strain of the pipe of CPE as purifying appears in the most last extent of dilution; Adopt formalin to carry out inactivation treatment; Mix with freund adjuvant at last, be prepared into CVB1-5 type antigen.
In (2) step of the method for the invention, can prepare immune egg: with described CVB1-5 type antigen the bird of laying eggs is carried out injecting immune, strengthen injection more once, amount to immunity three times every two weeks by following concrete steps; In immunity for the first time after 20 days, the bird of searching after the immunity produces immune egg.
In (2) step of the method for the invention, also comprise the lay eggs step of bird of the following breeding that preferably has high immunne response ability: with immune respectively many birds of laying eggs of described CVB1-5 type antigen, per injection 1ml antigen, after injection for the first time, strengthen injection more once every two weeks, totally three times; In injecting the back 7th month for the first time, behind these egg difference marks that bird produced of laying eggs, extract IgY wherein more according to a conventional method respectively; Detect tiring of prepared IgY respectively with enzyme-linked immunosorbent assay (ELISA) method, the IgY that the egg that bird produced if certain is only laid eggs is made tires greater than 256, then selects this bird of only laying eggs; Hatch good egg fowl veriety with the selected egg that bird produced of laying eggs again, treat that it grows up to 2-3 month, the special type that promptly the can be used as preferred high immunne response ability bird of laying eggs.Wherein, the described bird of laying eggs can be one or more in hen, duck, female goose, turkey or the ostrich.
In (2) step of the method for the invention, when the bird of laying eggs being carried out injecting immune with described CVB1-5 type antigen, the reinforced immunological method that adopts subcutaneous injection to combine with the wing intravenous injection: if the described bird of laying eggs is a hen, then every chicken per injection amount is 1-2ml; If the described bird of laying eggs is a duck, then per injection amount is 1-2ml; If the described bird of laying eggs is a female goose, then per injection amount is 5-10ml; If the described bird of laying eggs is female ostrich, then per injection amount is 10-20ml.
In (3) step of the method for the invention, extract anti-CVMD-IgY crude extract by following concrete steps: clean described immune egg, smash, elimination egg white stays yolk, stirs; 4-6 by the yolk volume extraordinarily goes into distilled water, dilutes and mixes; Transfer pH to 5.5-6.0 with 1.0N HCl solution; The diluent that mixes up the pH value is further stirred, it is cooled to 2-6 ℃ then, left standstill 12 hours-24 hours; With above-mentioned diluent under the 10000rpm condition, centrifugal 20 minutes; Getting separating obtained supernatant adds and carries out ultrafiltration in the ultra-fine filter and concentrate 10-20 doubly; Add 2.0% sodium alginate soln, to final concentration be 0.1%, be stirred to and turbidity and precipitation occurs, add 2.0% CaCl again
2Solution, to final concentration be 0.1%, stir, and under 4 ℃ of conditions, spend the night; Under the 8000rpm condition centrifugal 20 minutes, get supernatant, use 0.45 μ m membrane filtration degerming again; Carry out lyophilize again, make anti-CVMD-IgY crude extract.
In (4) step of the method for the invention, described anti-CVMD-IgY crude extract is dissolved in pH7.0,0.01M PB (phosphate buffered saline buffer), successively cross ion exchange column, gel chromatography column again, then adopt U.S. Pall film sterilizing filter and remove virus filter removal bacterium and virus, obtain the anti-CVMD-IgY finished product of purifying at last.Described ion exchange column is the DEAE-SephadexA50 ion exchange column, and described gel chromatography column is the SephadexG200 gel chromatography column.
In addition, the present invention also provides the made sprays with above-mentioned anti-CVMD-IgY, and wherein the concentration of anti-CVMD-IgY is 0.15%.In preferred version of the present invention, in the described anti-CVMD-IgY sprays, contain by weight proportion: anti-CVMD-IgY 0.15%, aspartame 0.15%, fragrant citrus essence 0.03%, honey peach essence 0.02%, peppermint essence 0.02%; Said components is dissolved in distilled water to 100ml.
When the described preparation of concrete preparation is sprays, get about 90ml distilled water earlier, add aspartame and described various essence then, after stirring, slowly add described anti-CVMD-IgY more while stirring, and stir; Adding distil water stirs to 100ml again; Transfer pH to 6.5-7.5 with 0.1N NaOH, 0.22 μ m membrane filtration degerming, the aseptic bottle can obtains bottled anti-CVMD-IgY sprays.
Described anti-CVMD-IgY preparation of the present invention can also be a colon colloidal sol capsule, contains in per 1000 burl enteric coated capsulees: anti-CVMD-IgY 5 grams, chitosan 148 grams, low-substituted hydroxypropyl cellulose (L-HPC) 15 grams, lactose 30 grams, and Magnesium Stearate is an amount of.
During the described colon colloidal sol capsule of concrete preparation, it is even to get chitosan, low substituted hydroxy-propyl fiber and lactose thorough mixing earlier, carries out drying under 80 ℃, be cooled to room temperature again, by equivalent progressively increase method and described anti-CVMD-IgY mixing, cross 80 mesh sieves then, add an amount of Magnesium Stearate mixing again; Last Autocapsulefillingmachine is filled No. 2 chitosan capsules, and every 0.2 gram contains 0.5 milligram of anti-CVMD-IgY; Again with the molten dressing sealing of HPMCP colon.
Described anti-CVMD-IgY preparation of the present invention can also be an intramuscular dose, wherein contains: anti-CVMD-IgY 3 grams, Pluronic 5 grams, polyvinylpyrrolidone (PVP) 5 grams, PEG400,66 grams, Polyoxyethylene Sorbitan Monooleate, 11 grams, and water for injection adds to 1000ml.
During the described intramuscular dose of concrete preparation, (1) gets water for injection 800ml earlier, adds Pluronic and polyvinylpyrrolidone, heating for dissolving; Other gets Polyoxyethylene Sorbitan Monooleate, adds the PEG400 mixing, adds aforementioned solution again, stirs; Add 0.1% needle-use activated carbon, 60 ℃ were stirred 15 minutes down, again decarbonization filtering; Airtight again heating, 100 ℃, 30 minutes, it was standby to be cooled to room temperature then; (2) water for injection that activated carbon treatment crosses of learning from else's experience in addition adds anti-CVMD-IgY in sterilising vessel, continue to stir down, is in the mixed solution that thread is added on above-mentioned (1) step, supplies content with taking off charcoal water for injection; (3) remove bacterium and virus with U.S. Pall film sterilizing filter with except that virus filter, embedding is in sterile chamber in aseptic filling and sealing machine, and every 2.0ml contains 6.0 milligrams of anti-CVMD-IgY.
In addition, also can be made into various clinical acceptable forms, formulations such as nasal spray, nasal drop, throat spraying agent, mouth spraying agent, buccal tablet, injection are used for prevention and treatment viral myocarditis.
Because the maximum characteristics of the specific IgY of the present invention's preparation are direct at the pathogenic agent generation specificity deactivation that causes viral myocarditis, thereby the disconnected source of can effecting a permanent cure, effect is remarkable.The prepared anti-CVMD-IgY of the present invention is a kind of unique natural biotechnological formulation, uses fool proof, without any side effects.
Description of drawings
No accompanying drawing.
Embodiment
Embodiment 1: anti-CVMD-IgY
1, preparation CVB 1-5 type antigen
Specifically may further comprise the steps:
The cultivation of virus is got the Hela cell and is cultivated coxsackie B group 1-5 (CVB1-5) C-type virus C respectively;
The purifying of virus adopts last diluted passage method eventually, and the strain of the pipe of CPE as purifying appears in the most last extent of dilution;
Adopt formalin to carry out inactivation treatment;
Mix with freund adjuvant at last, be prepared into CVB1-5 type antigen.
2, prepare immune egg
(1), the breeding duck that preferably has high immunne response ability
With described CVB1-5 type antigen immunity 100 ducks of laying eggs respectively, per injection 1ml antigen after injection for the first time, is strengthened injection once every two weeks, totally three times again; In injecting the back 7th month for the first time, behind these egg difference marks that duck produced of laying eggs, extract IgY wherein more according to a conventional method respectively; Detect tiring of prepared IgY respectively with enzyme-linked immunosorbent assay (ELISA) method, the tiring greater than 256 of the IgY that makes if certain duck is laid eggs then selected this duck; Hatch good duck kind with the egg that selected duck produced again; Treat that it grows up to 2-3 month, as the extraordinary duck of preferred high immunne response ability.
In addition, also available hen, female goose, turkey or ostrich replace the duck in the above-mentioned steps.
(2), respectively preferred extraordinary duck is carried out reinforced immunological
With prepared CVB1-5 type antigen, the reinforced immunological method that adopts subcutaneous injection and wing intravenous injection to combine is carried out immunity to preferred extraordinary duck respectively, and every duck per injection amount reaches 1-2ml antigen, strengthen injection more once every two weeks, interior immunity altogether three times; Immunity was for the first time searched duck and is produced immune egg after 20 days.In this step, if the bird of choosing of laying eggs is a hen, then per injection amount is 1-2ml; If the described bird of laying eggs is a female goose, then per injection amount is 5-10ml; If the described bird of laying eggs is female ostrich, then per injection amount is 10-20ml.
3, the crude extract of the anti-CVMD-IgY of preparation
Clean immune egg with flowing water, use the wipes of alcohol wash disinfection again; With beating machiae immune egg is smashed then, egg white is removed in the yolk sieving, stays yolk and stirs; Clean described immune egg, smash, elimination egg white stays yolk, stirs; 4-6 by the yolk volume extraordinarily goes into distilled water, dilutes and mixes; Transfer pH to 5.5-6.0 with 1.0N HCl solution; The diluent that mixes up the pH value is further stirred, it is cooled to 2-6 ℃ then, left standstill 12 hours-24 hours; With above-mentioned diluent under the 10000rpm condition, centrifugal 20 minutes; Getting separating obtained supernatant adds and carries out ultrafiltration in the ultra-fine filter and concentrate 15 times; Add 2.0% sodium alginate soln, to final concentration be 0.1%, be stirred to and turbidity and precipitation occurs, add 2.0%CaCl again
2Solution, to final concentration be 0.1%, stir, and under 4 ℃ of conditions, spend the night; Under the 8000rpm condition centrifugal 20 minutes, get supernatant, use 0.45 μ m membrane filtration degerming again; Carry out lyophilize again, promptly make anti-CVMD-IgY crude extract dry powder.
4, purify
Described anti-CVMD-IgY crude extract is dissolved in pH7.0,0.01M PB (phosphate buffered saline buffer), successively cross ion exchange column, gel chromatography column again, remove bacterium and virus with U.S. Pall film sterilizing filter with except that virus filter again, obtain the anti-CVMD-IgY finished product of purifying.
Anti-Coxsackie virus specificity composite IgY of the present invention belongs to r-sphaeroprotein class, and its molecular-weight average is 178Kda.When IgY of the present invention is used for enteron aisle, must avoid the acid solution in the stomach and the degraded of the enzyme in the intestinal fluid, make the activity that colon-specific drug delivery system (DDS) then can keep IgY.
The lung Foradil Aerolizer formoterol fumarate need with IgY and auxiliary material at low temperatures aseptic powder be broken into the particle of 1-6 μ m, by nitrogen pressure dry powder is sprayed into respiratory tract, from lung qi tube-surface capillary vessel suction blood, reach whole body therapeutic again.All suck auxiliary material N.F,USP MANNITOL, sorbyl alcohol, Xylitol, sucrose alcohol, sorbitol etc.
Lung's fluid inhalation needs to add the paucidisperse agent with sterile water for injection and makes IgY solution, as PVP series, and PLURONIC series, polysorbate series 20,40,60,80, glycerine, PEG, propylene glycol etc.
For IgY colon colloidal sol capsule, need not disintegration release in stomach, small intestine, and disintegration release in the colon of no digestive ferment, used auxiliary material has chitosan, dextran, the Pectin calcium beta-cyclodextrin, sodium alginate etc., molten special-purpose capsule of also available colon such as Japanese Aicello Chemical CO., the product of Ltd., or the colon colloidal sol capsule of Chaozhou, Guangdong capsule factory etc., what have then needs enteric coated outside sealing.
Also can be made into the conlon targeting release tablet, by time lag 5-6 hour and pH 7.0 left and right sides disintegration releases, design time lag type and pH responsive type colon DDS system.Used auxiliary material has poly-propionic acid water II resin, Eudragil S100, the dressing material of ethyl cellulose EC and EC and acrylic resin different ratios, control certain clothing film thickness (weightening finish), made tablet is not disintegration release in 3 hours under one's belt, release in 3 hours is no more than about 20%, 6 hour and arrives colon in small intestine, and basic release is complete more than 10 hours.
The IgY injection is IgY to be dissolved in contain in the aqueous solution of dispersion agent, isotonic regulator, tensio-active agent etc. after aseptic, becomes the clarification soup, and pH meets the human body requirement, and IgY is stable not to degrade.Auxiliary material has Poloxamer series, PVP series, PEG series, dehydration polysorbate class, pH stablizer, acetate buffer, PBS liquid, HCl and acetate salt buffer corner etc.
Embodiment 2 IgY lung Foradil Aerolizer formoterol fumarates
Prescription: IgY lyophilized powder 300 grams, injection sorbyl alcohol 3000 grams, high pure nitrogen is an amount of, makes 1000.
Preparation technology: with sorbyl alcohol in cleaning oven, 90 ℃ of dryings 4 hours, be chilled to after the room temperature and IgY lyophilized powder mixing in the clean area sterilising vessel, in the freezing crusher of sterilizing, be ground into the fine powder of 1-6 μ m again, use the can of powder filling machine topping up nitrogen again in the spray bottle, every 3.3 gram, 200 sprays.Be used for the treatment of viral myocarditis, each 2-4 spray, every day 3-4 time or follow the doctor's advice.
Embodiment 3 IgY liquid inhalant
Prescription: IgY lyophilized powder 1.5 grams, PVP
K-305 grams, Pluronic 3 grams, glycerine 20 grams, polysorbate 10 grams, high purity nitrogen is an amount of, and adds sterilized water for injection again to 1000ml.
Preparation technology:
1) gets PVP
K-30, Pluronic, glycerine, polysorbate is dissolved in 80% the water for injection, is heated to boiling, and it is standby to be chilled to room temperature again;
2) get 20% water for injection in addition, the aseptic room temperature that is chilled to adds IgY and makes its dissolving;
3) stir on one side, on one side with the 2nd) solution of item joins the 1st) in the solution of item, continue to stir 15 minutes, use then and spray bottle filling machine, fill the nitrogen can promptly.Every 20ml is used for lung and sucks, each 2-4 spray, every day 4-6 time or follow the doctor's advice.
Embodiment 4 IgY colon colloidal sol capsules
Prescription: IgY lyophilized powder 5 grams, chitosan 148 grams, low-substituted hydroxypropyl cellulose (L-HPC) 15 grams, lactose 30 grams, Magnesium Stearate is an amount of, makes 1000.
Preparation technology:
1) get chitosan, low-substituted hydroxypropyl cellulose and lactose thorough mixing are even, and 80 ℃ of dryings 2 hours are chilled to room temperature, by equivalent progressively increase method and IgY mixing, cross 80 mesh sieves, add the Magnesium Stearate mixing;
2) use Autocapsulefillingmachine, fill No. 2 chitosan capsules (Japanese Aicello Chemical company produce), every 0.2 gram contains 5 milligrams of IgY, uses HPMCP enteric coating liquid sealing again;
3) coating liquid prescription: HPMCP 50 grams are dissolved in acetone-ethanol (1: 1) 1000ml, and spray is wrapped and stated capsule then.Check it really to show, it does not discharge in 2 hours in simulated gastric fluid, and disintegration release in 3 hours is less than 20% in intestinal fluid, and release in 5-6 hour reaches 100% in colon.
Embodiment 5 IgY conlon targeting release capsules
Prescription: IgY lyophilized powder 10 grams, cane sugar powder 88 grams, Microcrystalline Cellulose (MCC) 58 grams, N.F,USP MANNITOL 29 grams, starch 14 grams, silicon-dioxide 3 grams, Magnesium Stearate 2 grams are made 1000.
Preparation technology:
1) get cane sugar powder, Microcrystalline Cellulose, N.F,USP MANNITOL, starch mixes, 80 ℃ of dryings 2 hours, by equivalent Superposition Method and IgY mixing, mistake 80 mesh sieves add silicon-dioxide and Magnesium Stearate mixing;
2) use Autocapsulefillingmachine, fill Chaozhou, Guangdong and produce molten No. 2 capsules of colon, every 0.2 gram contains 10 milligrams of IgY.
Embodiment 6 IgY conlon targeting release tablets
Prescription: IgY lyophilized powder 20 grams, Microcrystalline Cellulose (MCC) 200 grams, low-substituted hydroxypropyl cellulose 27 grams, lactose 50 grams, 5%PVP
K-3050% ethanol liquid is an amount of, and Magnesium Stearate 3 grams are made 1000.
Preparation technology:
1) get Microcrystalline Cellulose, the lactose mixing is crossed 80 mesh sieves, adds 5%PVP
K-3050% ethanol liquid is tackiness agent, makes suitable softwood.16 order nylon mesh are granulated, and temperature 50-60 ℃, air seasoning, control pellet moisture 3-5%; the whole grain of 14 order stainless steel meshs adds low-substituted hydroxypropyl cellulose, and IgY lyophilized powder mixing sifts out an amount of fine powder and adds Magnesium Stearate and mix evenly; mix with particle again, compressing tablet, every contains 20 milligrams of IgY.
2) the coating liquid preparation is got II polyacrylic acid resin (or Eudragil S100) and was made 6% ethanol-acetone (1: 1) coating liquid with ethyl cellulose by 7: 3, and coating liquid volume 10% adds the O diethyl phthalate, and stirs promptly.
3) get the label sieve and remove fine powder, put in the coating pan and rotate, make the sheet bed tempertaure at 39-41 ℃, adjusting rotating speed is 45 commentaries on classics per minutes, air pressure 196-392 kPa, heating power 40-50 ℃ temperature, open the gun spraying dressing, reach 10%, stop dressing until the label weightening finish, coating tablet was placed in moisture eliminator more than 4 hours, and packing promptly.
This coating tablet is disintegration release in 10 hours in simulated gastric fluid, and disintegration release in 3 hours is less than 20% in artificial intestinal fluid, and 6 hours release time lag time, time lag is prominent immediately later to be released, and this moment, just in colon, release reached more than 95% after 12 hours.
Embodiment 7 IgY injections
Prescription: IgY lyophilized powder 3 grams, Pluronic 5 grams, polyvinylpyrrolidone 5 grams, PEG400 66 grams, polysorbate-88 11 gram, water for injection adds to 1000ml.
Preparation technology:
1) gets water for injection 800ml and add Pluronic and polyvinylpyrrolidone heating for dissolving, other gets Polyoxyethylene Sorbitan Monooleate and adds the PEG400 mixing, under agitation add in the above-mentioned mixed solution, add 0.1% needle-use activated carbon again, 60 ℃ were stirred 15 minutes down, decarbonization filtering, again with filtrate heated sealed to 100 ℃, 30 minutes; It is standby to be cooled to room temperature then.
2) take needle-use activated carbon absorption in addition, take off charcoal, boil, and be chilled to the water for injection residual content of room temperature, in sterile chamber, add the dissolving of IgY lyophilized powder, continue to stir down, be thin notes and add the 1st) in the solution of item, supply full dose with taking off the charcoal sterile water for injection.
3) remove bacterium and virus with U.S. Pall film sterilizing filter with except that virus filter, can is in sterile chamber in aseptic filler, and every 2ml contains 6 milligrams of IgY, lettering, and packing is promptly.
Experimental example 1
In 1: 1: 1: 1: 1 ratio, get CVB1, the CVB2, CVB3, CCVB4, the CVB5 virus that cause viral myocarditis.Press the method for embodiment 1 again, make antigen,, make anti-CVMD-IgY purification thing at last with this antigen immune hen.Then, respectively with CVB1, CVB2, CVB3, CCVB4, CVB5 virus as detecting antigen, with the antibody titer of the prepared anti-CVMD-IgY of " ELISA " detection.The result is as follows
Table:
| ????IgY | Detection antigen | Antibody titer |
| Anti-CVMD-IgY | ????CVB1 | ????1∶1024 |
| ????CVB2 | ????1∶1024 | |
| ????CVB3 | ????1∶1024 | |
| ????CVB4 | ????1∶1024 | |
| ????CVB5 | ????1∶1024 |
Experimental example 2
In 1: 1: 1: 1: 1 ratio, get CVB1, the CVB2, CVB3, CCVB4, the CVB5 virus that cause viral myocarditis.Press the method for embodiment 1 again, make antigen,, make the anti-CVMD-IgY purification thing of chicken, duck, goose, turkey, ostrich at last respectively with this antigen difference immune hen, duck, female goose, turkey and ostrich.Then, respectively with CVB1, CVB2, CVB3, CCVB4, CVB5 virus as detecting antigen, with the antibody titer of the prepared anti-CVMD-IgY of " ELISA " detection.Result such as following table:
| ????IgY | Detection antigen | Antibody titer |
| Anti-CVMD-chicken IgY | ????CVB1 | ????1∶1024 |
| ????CVB2 | ????1∶1024 | |
| ????CVB3 | ????1∶1024 | |
| ????CVB4 | ????1∶1024 | |
| ????CVB5 | ????1∶1024 | |
| Anti-CVMD-duck IgY | ????CVB1 | ????1∶1024 |
| ????CVB2 | ????1∶1024 | |
| ????CVB3 | ????1∶1024 | |
| ????CVB4 | ????1∶1024 | |
| ????CVB5 | ????1∶1024 | |
| Anti-CVMD-goose IgY | ????CVB1 | ????1∶512 |
| ????CVB2 | ????1∶512 | |
| ????CVB3 | ????1∶512 | |
| ????CVB4 | ????1∶512 | |
| ????CVB5 | ????1∶512 | |
| Anti-CVMD-turkey IgY | ????CVB1 | ????1∶256 |
| ????CVB2 | ????1∶256 | |
| ????CVB3 | ????1∶256 | |
| ????CVB4 | ????1∶256 | |
| ????CVB5 | ????1∶256 | |
| Anti-CVMD-ostrich IgY | ????CVB1 | ????1∶256 |
| ????CVB2 | ????1∶256 | |
| ????CVB3 | ????1∶256 |
| ????CVB4 | ????1∶256 | |
| ????CVB5 | ????1∶256 |
From above test-results as can be seen, adopt method of the present invention to make antigen, with immuno-stimulating method immunity bird inlay, duck, female goose, turkey or ostrich, adopt method of the present invention to make anti-CVMD-chicken IgY, anti-CVMD-duck IgY, anti-CVMD-goose IgY, anti-CVMD-turkey IgY, five kinds of different IgY of anti-CVMD-ostrich IgY respectively again, though the bird difference of immunity, but prepared specific IgY all has higher antibody activity to the VCB1-5 type that causes viral myocarditis, and this shows that they all have higher inactivation virus function.Though immunity is laid eggs ostrich and lay eggs the prepared IgY antibody titer of turkey from detect data seen lower a little, but, because ostrich and turkey egg individuality are big especially, it is all more much more than chicken immune egg and duck immunity egg and goose immunity egg that each immune egg can extract the IgY that obtains, this point also is very desirable for large-scale industrialization production.
Experimental example 3: mouse test
Use the CVB infecting mouse, and allow the mouse spray eat with the sprays among the embodiment 3 simultaneously, mouse is injected with the injection among the embodiment 6.
Perhaps, mouse is injected with the injection among the embodiment 6 after 2 weeks, allowing the mouse spray eat with the sprays among the embodiment 3 with the CVB infecting mouse.
Observations shows, no matter be in virus infection, or use anti-CVMD-IgY of the present invention behind virus infection, the necrosis of mouse cardiac muscle cell and the infiltration of inflammatory cell all obviously reduce; The cytokine that inflammation is relevant descends; The survival time of mouse obviously prolongs.Particularly the former immunoglobulin (Ig) has almost completely suppressed the infringement of virus to cardiac muscle, does not have dead mouse in experimentation.
" anti-CVMD-IgY " of the present invention can directly play useful effect at the CVB1-5 type Causative virus that causes viral myocarditis with a definite target in view, is to deal with problems from the morbidity root; On the other hand, IgY is natural product, and it is wide to originate, and preparation is simple, and cost is low, and is without any side effects again, therefore, and for preventing and treating viral myocarditis and opened up a brand-new approach.
The reference that background technology part of the present invention is quoted:
[1] Wheeler DS, Kooy NW.A formidable challenge:the diagnosis and treatment of viral myocarditis inchildren.Crit Care Clin.2003 Jul; 19 (3): 365-91. (difficult challenge: the myocarditic Clinics and Practices of children virus)
[2] Bai Dengyun, Shi Huafang, Li Zhongheng waits an acute myocarditis to break out with nosetiology and tentatively inquires into.Yunnan medicine, 1995,6:109-111
[3] Shashi, Hubei health and epidemic prevention station, an acute myocarditis outbreak of epidemic.Chinese Medical Journal, 1980,203 (4): 203
[4]Hingorani?AD.Post?infectious?myocarditis.BMJ，1992，304：1676-1678
[5] Yang Yingzhen, " heart disease caused by viruses ", Science and Technology of Shanghai press, 2001,10:27-41
[6]Takada?H，Kishimoto?C?and?Hiraoka?Y.Therapy?with?immunoglobulin?suppresses?myocarditis?in?amurine?coxsackievirus?B3?model.Antiviral?and?anti-inflammatory?effects.Circulation.1995?92：1604-1611
[7]Kishimoto?C，Takada?H，Kawamata?H，et?al.Immunoglobulin?treatment?prevents?congestive?heartfailure?in?murine?encephalomyocarditis?viral?myocarditis?associated?with?reduction?of?inflammatory?cytokines.Jpharmacol?Exp?Ther.2001?Nov；299(2)：645-51.
[8]Lee?EN，Sunwoo?HH，Menninen?K，et?al.In?vitro?studies?of?chicken?egg?yolk?antibody(IgY)againstSalmonella?enteritidis?and?Salmonella?typhimurium.Poult?Sci?2002?May；81(5)：623-41
[9]Daniel?J，Smith，William?F，et?al.Passive?transfer?of?immunoglobulin?Y?antibody?to?Streotococcusmutans?glucan?binding?protein?B?can?confer?protection?against?experimental?dental?caries.Infection?andImmunity?2001，p：3135-3142
[10] Shi Junnan, Yang Rongjian, Wang Hanguo.The research 2001,11 (4) that the sick antibody development of dental caries is dynamic and Ao Liting key tooth reveals: 296-272
Claims (16)
1, a kind of preparation method of specificity composite IgY of anti-coxsackie myocarditis is characterized in that, comprises the following steps:
(1) preparation causes the antigen of coxsackie myocarditis;
(2), and search the immune egg that described poultry produces with described antigen immune poultry;
(3) the specificity composite IgY crude extract of the anti-coxsackie myocarditis of extraction from described immune egg;
(4) described IgY crude extract is carried out purifying, obtain the pure product of specificity composite IgY of anti-coxsackie myocarditis.
2, method according to claim 1 is characterized in that, in described (1) step, specifically may further comprise the steps:
The cultivation of virus is got the Hela cell and is cultivated coxsackie B group 1-5 (CVB1-5) C-type virus C respectively;
The purifying of virus adopts last diluted passage method eventually, and the strain of the pipe of CPE as purifying appears in the most last extent of dilution;
Adopt formalin to carry out inactivation treatment;
Mix with freund adjuvant at last, be prepared into CVB1-5 type antigen.
3, method according to claim 2 is characterized in that, in described (2) step, prepares immune egg by following concrete steps:
With described CVB1-5 type antigen the bird of laying eggs is carried out injecting immune, strengthen injection more once, amount to immunity three times every two weeks;
In immunity for the first time after 20 days, the bird of searching after the immunity produces immune egg.
4, method according to claim 3 is characterized in that, in described (2) step, also comprises the lay eggs step of bird of the following breeding that preferably has high immunne response ability:
With immune respectively many birds of laying eggs of described CVB1-5 type antigen, per injection 1ml antigen, after injection for the first time, strengthen injection more once every two weeks, totally three times;
In injecting the back 7th month for the first time, behind these egg difference marks that bird produced of laying eggs, extract IgY wherein more according to a conventional method respectively;
Detect tiring of prepared IgY respectively with enzyme-linked immunosorbent assay (ELISA) method, the IgY that the egg that bird produced if certain is only laid eggs is made tires greater than 256, then selects this bird of only laying eggs;
Hatch good egg fowl veriety with the selected egg that bird produced of laying eggs again, treat that it grows up to 2-3 month, the special type that promptly the can be used as preferred high immunne response ability bird of laying eggs.
5, method according to claim 4 is characterized in that, the described bird of laying eggs can be one or more in hen, duck, female goose, turkey or the ostrich.
6, method according to claim 5 is characterized in that, when the bird of laying eggs being carried out injecting immune with described CVB1-5 type antigen, and the reinforced immunological method that adopts subcutaneous injection to combine with the wing intravenous injection:
If the described bird of laying eggs is a hen, then every chicken per injection amount is 1-2ml;
If the described bird of laying eggs is a duck, then per injection amount is 1-2ml;
If the described bird of laying eggs is a female goose, then per injection amount is 5-10ml;
If the described bird of laying eggs is female ostrich, then per injection amount is 10-20ml.
7, according to each described method among the claim 1-6, it is characterized in that, in described (3) step, extract the specificity composite IgY crude extract of anti-coxsackie myocarditis by following concrete steps:
Clean described immune egg, smash, elimination egg white stays yolk, stirs;
4-6 by the yolk volume extraordinarily goes into distilled water, dilutes and mixes;
Transfer pH to 5.5-6.0 with 1.0N HCl solution;
The diluent that mixes up the pH value is further stirred, it is cooled to 2-6 ℃ then, left standstill 12 hours-24 hours;
With above-mentioned diluent under the 10000rpm condition, centrifugal 20 minutes;
Getting separating obtained supernatant adds and carries out ultrafiltration in the ultra-fine filter and concentrate 10-20 doubly;
Add 2.0% sodium alginate soln, to final concentration be 0.1%, be stirred to and turbidity and precipitation occurs, add 2.0%CaCl again
2Solution, to final concentration be 0.1%, stir, and under 4 ℃ of conditions, spend the night;
Under the 8000rpm condition centrifugal 20 minutes, get supernatant, use 0.45 μ m membrane filtration degerming again;
Carry out lyophilize again, make the specificity composite IgY crude extract of anti-coxsackie myocarditis.
8, method according to claim 7, it is characterized in that, in described (4) step, the specificity composite IgY crude extract of described anti-coxsackie myocarditis is dissolved in pH7.0,0.01M PB (phosphate buffered saline buffer), successively cross ion exchange column, gel chromatography column again, adopt U.S. Pall film sterilizing filter again and remove virus filter removal bacterium and virus, obtain the specificity composite IgY finished product of the anti-coxsackie myocarditis of purifying.
9, method according to claim 8 is characterized in that, described ion exchange column is the DEAE-SephadexA50 ion exchange column, and described gel chromatography column is the SephadexG200 gel chromatography column.
10, with the made preparation of specificity composite IgY of each anti-coxsackie myocarditis among the claim 1-9, it is characterized in that described preparation is a sprays, wherein the concentration of the specificity composite IgY of anti-coxsackie myocarditis is 0.15%.
11, the specificity composite IgY preparation of anti-coxsackie myocarditis according to claim 10 is characterized in that, in described IgY sprays, contains by weight proportion:
The specificity composite IgY 0.15% of anti-coxsackie myocarditis,
Aspartame 0.15%,
Fragrant citrus essence 0.03%,
Honey peach essence 0.02%,
Peppermint essence 0.02%;
Said components is dissolved in distilled water to 100ml.
12, the specificity composite IgY preparation of anti-coxsackie myocarditis according to claim 11 is characterized in that, when specifically preparing described preparation and being sprays,
Get about 90ml distilled water earlier, add aspartame and described various essence then, after stirring, slowly add the specificity composite IgY of described anti-coxsackie myocarditis more while stirring, and stir;
Adding distil water stirs to 100ml again;
Transfer pH to 6.5-7.5 with 0.1N NaOH, 0.22 μ m membrane filtration degerming, the aseptic bottle can obtains the specificity composite IgY sprays of bottled anti-coxsackie myocarditis.
13, with the made preparation of specificity composite IgY of each anti-coxsackie myocarditis among the claim 1-9, it is characterized in that described preparation is a colon colloidal sol capsule, contain in per 1000 burl enteric coated capsulees:
Specificity composite IgY 5 grams of anti-coxsackie myocarditis,
Chitosan 148 grams,
Low-substituted hydroxypropyl cellulose (L-HPC) 15 grams,
Lactose 30 grams,
And Magnesium Stearate is an amount of.
14, the specificity composite IgY preparation of anti-coxsackie myocarditis according to claim 13 is characterized in that, when specifically preparing described colon colloidal sol capsule,
It is even to get chitosan, low substituted hydroxy-propyl fiber and lactose thorough mixing earlier, under 80 ℃, carry out drying, be cooled to room temperature again, then by the progressively increase specificity composite IgY mixing of method and described anti-coxsackie myocarditis of equivalent, cross 80 mesh sieves, add an amount of Magnesium Stearate mixing again;
Last Autocapsulefillingmachine is filled No. 2 chitosan capsules, and every 0.2 gram contains 0.5 milligram of the specificity composite IgY of anti-coxsackie myocarditis; Again with the molten dressing sealing of HPMCP colon.
15, with the made preparation of specificity composite IgY of each anti-coxsackie myocarditis among the claim 1-9, it is characterized in that described preparation is an intramuscular dose, wherein contains:
Specificity composite IgY 3 grams of anti-coxsackie myocarditis,
The Pluronic5 gram,
Polyvinylpyrrolidone (PVP) 5 grams,
PEG400,66 grams,
Polyoxyethylene Sorbitan Monooleate, 11 grams,
And water for injection adds to 1000ml.
16, the specificity composite IgY preparation of anti-coxsackie myocarditis according to claim 15 is characterized in that, when specifically preparing described intramuscular dose,
(1) gets water for injection 800ml earlier, add Pluronic and polyvinylpyrrolidone, heating for dissolving; Other gets Polyoxyethylene Sorbitan Monooleate, adds the PEG400 mixing, adds aforementioned solution again, stirs; Add 0.1% needle-use activated carbon, 60 ℃ were stirred 15 minutes down, again decarbonization filtering; Airtight again heating, 100 ℃, 30 minutes, it was standby to be cooled to room temperature then;
(2) water for injection that activated carbon treatment crosses of learning from else's experience in addition adds the specificity composite IgY of anti-coxsackie myocarditis in sterilising vessel, continue to stir down, is in the mixed solution that thread is added on above-mentioned (1) step, supplies content with taking off charcoal water for injection;
(3) adopt U.S. Pall film sterilizing filter and remove virus filter and remove bacterium and virus, embedding is in sterile chamber in aseptic filling and sealing machine, and every 2.0ml contains 6.0 milligrams of the specificity composite IgYs of anti-coxsackie myocarditis.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102816240A (en) * | 2011-03-08 | 2012-12-12 | 中国人民解放军第三军医大学第二附属医院 | Fusion protein and fusion protein expression vector thereof |
| CN105597097A (en) * | 2015-03-17 | 2016-05-25 | 深圳市雅臣爱己生物工程有限公司 | Preparation method of compound IgY for resisting mycotoxin, bacterial exotoxin and pathogen as well as preparation thereof |
| CN107043415A (en) * | 2017-03-07 | 2017-08-15 | 尤丽康（江苏）生物医药有限公司 | A kind of extracting method of Yolk immunoglobulin |
| CN116769733A (en) * | 2023-08-02 | 2023-09-19 | 中国医学科学院医学生物学研究所 | Coxsackie virus B group 4 strain and application thereof |
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2004
- 2004-02-18 CN CN 200410005797 patent/CN1657541A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102816240A (en) * | 2011-03-08 | 2012-12-12 | 中国人民解放军第三军医大学第二附属医院 | Fusion protein and fusion protein expression vector thereof |
| CN105597097A (en) * | 2015-03-17 | 2016-05-25 | 深圳市雅臣爱己生物工程有限公司 | Preparation method of compound IgY for resisting mycotoxin, bacterial exotoxin and pathogen as well as preparation thereof |
| CN107043415A (en) * | 2017-03-07 | 2017-08-15 | 尤丽康（江苏）生物医药有限公司 | A kind of extracting method of Yolk immunoglobulin |
| CN116769733A (en) * | 2023-08-02 | 2023-09-19 | 中国医学科学院医学生物学研究所 | Coxsackie virus B group 4 strain and application thereof |
| CN116769733B (en) * | 2023-08-02 | 2023-11-03 | 中国医学科学院医学生物学研究所 | Coxsackie virus B group 4 strain and application thereof |
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