CN1384119A - Composite yolk antibody for resisting fowl's viral blight and its prepn and application - Google Patents
Composite yolk antibody for resisting fowl's viral blight and its prepn and application Download PDFInfo
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Abstract
The present invention relates to a chicken's yolk antibody, anti-ANIgY, with antibody, activity to fowl influenza and new castle diseases, its preparation process and application in preparing medicine and health article for treating fowl's viral blight caused by fowl influenza and/or new castle disease. The bioactivity detection shows that anti-ANIgY has neutralizing and deactivating effect on AIV and NDV. IgY purity test shows that purified yolk antibody has only one electrophoresis zone. The preparation process of anti-ANIgY includes preparing AIN and NDV antigen, immunizing healthy hen with the antigen, collecting eggs and extracting anti-ANIgY from the eggs.
Description
Technical field
The present invention relates to a kind of composite yolk antibody material with antibody activity---and the resisting fowl's viral blight composite yolk antibody (anti--ANIgY) product, and relate to its preparation technology and application.
Technical background
Bird flu (AI), newcastle disease (ND) are the potent virus sexually transmitted diseases of serious harm aviculture.The cause of disease that causes bird flu (AI) is avian influenza virus (AIV), this Tobamovirus orthomyxoviridae family Influenza Virus.Newcastle disease (ND) is still pandemic main poultry diease so far all over the world, it is a kind of Vi transmissible disease, it causes enteron aisle hyperemia, serious expiratory dyspnea, nervous symptoms, egg drop reduction, mortality ratio are very high, (NDV) be the representative species of paramyxovirus, be named as paramyxovirus I type (PMVI).Bird flu and newcastle disease have a common feature, and promptly the virulence of its pathogenic factor (virus) all has great variation.Avian influenza virus and Avian pneumo-encephalitis virus all have can cause that infection does not but produce the strain of obvious clinical symptom, and they are to endanger two the most serious big virus diseases, and often merges generation.
The control of relevant chicken virus mixed infection, the raiser carries out vaccine inoculation with antibiotic medicines such as gold poison gram, viral economic benefits and social benefits, positive Thailand are glad usually, though lean on such medical treatment avian viral diseases that chicken group's surviving rate is had certain help, directly cause meat poultry food antibiotic remains, hormone residues, heavy-metal residual and pesticide residue to substantially exceed China from green non-pollution standard and the international export standard ordered; Other has the part breeding enterprise to adopt the Chinese medicine preparation mode chicken group to be carried out the control of such eqpidemic disease, but for various reasons, fowl is not satisfactory with the effect of Chinese medicine preparation, its major cause is that bird is bad to the absorption of Chinese drugs agentia, drug effect can not get good embodiment on the chicken body, thereby eqpidemic disease can't be fundamentally controlled at Ji Qunzhong.Below what time be the existing avian viral of China infect eqpidemic disease main controlling mode, drawback is all respectively arranged, the quality that is unfavorable for China's poultry and egg series products obtains definite effective and improves.Long-term edible such meat product will be to people's the healthy severe impairment of bringing.
Many in recent years countries comprise in use gradually that all biotechnological formulations such as live vaccine, deactivation vaccine, probiotic bacterium control viral infection bird eqpidemic disease, the effect of this biotechnological formulation control chicken virus infection in most cases is single, the level that is equivalent to chemoprophylaxis, disease to polyinfection is powerless, depend merely on vaccine prevention, immune effect is undesirable.Vaccine is basic to the prevention of healthy animal transmissible disease, but is a difficult problem so far to the treatment of certain proportion morbidity colony and polyinfection clinically.In the overall measure that improves bird productivity, significant is to utilize various biotechnological formulation prevention poultry dieases, except that vaccine, being applied in that vaccination produces before the immunizing power, urgent preventive vaccination during immuning failure, improving the poult resistance against diseases and promote that its production aspect is all significant of hyper-immune serum.
Chicken yolk antibody (IgY) is the IgG (immunoglobulin G) from chicken serum, and the structure of IgY is similar to IgG, has the space conformation of typical immunoglobulin (Ig).Studies show that IgY has the stability of height, to having good tolerance in heat, acid, the alkali environment, IgY also has the advantage that cost is low, be easy to form large-scale production simultaneously.
Do not see in the prior art that having by adopting associating newcastle disease multivalence strain (standard strain+local strain) and bird flu strain is antigen, the immune health hen is collected the ovum that it produces, the report of anti--ANIgY that extracts from ovum again and its production and application.
The content of invention
The present invention studies according to brutish this developing direction of medicine class biotechnological formulation, at viral blights such as bird flu, newcastle disease mainly by antibiotic medicine prevent and treat and the deficiency that exists, after adopting newcastle disease multivalence strain (standard strain+local strain) and bird flu strain to prepare complex antigen inoculation laying hen, induce its immunity system to produce high titre immunoglobulin.Carry the immunoglobulin (Ig) of (purity reaches more than 95%) by improveing water-soluble method essence.
The present invention can solve the brutish class impurity of the drug of biotechnological formulation too high levels in the past, immunoglobulin (Ig) effectiveness is not obvious, restricted condition is many, can't really carry out the difficult point that industrialization is produced, effectively improved the purity of immunoglobulin (Ig), make it directly act on the chicken body, its homology and utility all are higher than other biotechnological formulation; Solved the problem of antibiotic medicine simultaneously, for the quality that has improved this series products provides favourable assurance to poultry and egg series products residue.
The present invention is directed to above problem, develop a kind of convenience, the biological fowl medicine of non-antibiotic efficient, that have no side effect, be used for infecting the control that caused avian viral catches because of chicken AIV, NDV.Simpler because of its method for preparing immunoglobulin (Ig) wanting, fast, be easy to grasp, the extraction recovery ideal, economic benefit is considerable, is suitable for industrialization and makes.
The present invention is by being antigen with newcastle disease multivalence strain (standard strain+local strain) and bird flu strain, the immune health bird inlay, extract activeconstituents (anti--ANIgY).Provide new approaches and novel method for treatment fowl hybrid virus infects, and be that the immunity biological agent that further prepares anti-AI, ND polyinfection lays the foundation.Therefore:
One of technical problem to be solved by this invention is: at the prior art above shortcomings, provide a kind of composite yolk antibody with fowl's viral blights such as anti-AI, ND of antibody activity (anti--ANIgY).
Two of technical problem to be solved by this invention is: provide above-mentioned composite yolk antibody (anti--ANIgY) preparation method.
Three of technical problem to be solved by this invention is: provide above-mentioned composite yolk antibody (anti--ANIgY) preparation be used for bird because of AI, ND single and or the polyinfection checken pest, the prevention of avian influenza toxicity disease or bio-pharmaceutical preparation, fodder additives and the related products thereof of treatment that cause use.
The present invention one of solve the problems of the technologies described above the resisting fowl's viral blight that is provided composite yolk antibody (resist-ANIgY), it is characterized in that: described anti--ANIgY press YSDS-PAGE electrophoresis (7.5% separation gel) detection, collection of illustrative plates presents single district band; Be blue through electrophoresis Coomassie brilliant blue R250 dyeing; High molecular band place clear single specific enzymes occurred and dyes band during immunity marking Western-blotting detected; Through the active detection of the external antagonism viral organism of anti--ANIgY, show deactivation to AIV, NDV, AIV agar diffusion antibody titer is more than 1: 64 in the titration, and NDV hemagglutination inhibition test (HI test) antibody titer reaches 1: 320-1: 1084.
What the present invention solved the problems of the technologies described above two is by adopting such technical scheme to realize, the anti-AI of promptly a kind of preparation, ND composite yolk antibody (anti--ANIgY) method, it is characterized in that adopting following step to finish:
1. preparation complex antigen
(1), newcastle disease virus (NDV) strain is the cultivation of F48E8 and autonomous strain isolated HR98: a, employing tissue culture method, after Avian pneumo-encephalitis virus is inoculated in the chicken embryo culture, collect the amnion allantoic fluid, adding formalin or β-propionic acid lactones makes it to make antigen after the deactivation, add mineral oil or aluminium hydroxide gel again as adjuvant, oil emulsion inactivated antigen is made in emulsification; (2) bird flu strain H7 is inoculated in the chicken embryo culture after, collect the amnion allantoic fluid, add formalin and make inactivation antigen; Then with two kinds of antigens by mix at 1: 1 complex antigen, complex antigen mixes by 1: 1 with complete freund adjuvant before the immunity.
2. complex antigen is prepared antibody by following step:
(1). to the immunity of the hen that lays eggs: the A. fundamental immunity; Select the hen that lays eggs (health) isolated rearing for use.Carry out multi-point injection chicken complex antigen under the cock skin, 0.5ml/ only.B. reinforced immunological: fundamental immunity was carried out reinforced immunological after 7 days, only injected incomplete freund adjuvant complex antigen 0.5ml/.C. super-strengthening immunity: need super-strengthening immunity 1-3 time after 21 days.Injection volume 2.5ml/, to keep antibody titers.
Began to collect egg after 7 days, 4 ℃ store for future use.
(2). the extraction of yolk antibody
1.. adopt water-soluble method to extract yolk antibody.From immune ovum gallinaceum, separate yolk, with distilled water diluting, adjust suitable pH value and leave standstill 8h for 4 ℃, the centrifuging and taking supernatant concentrates, lyophilize, is resisted-ANIgY.Or 2. adopt membrane filter method to extract yolk antibody.Separate yolk, with distilled water diluting, 4 ℃ leave standstill 12h after, be that the film of 200KD and 150KD filters respectively with the molecular weight, collect the proteic substance of 150KD-200KD, lyophilize is resisted-ANIgY.
Provided by the present invention above-mentioned anti--the ANIgY product lays eggs hen by chicken AI, ND immunity and get, has specificity, to prevent and treat bird because of AI, ND single and or the infectious disease of the digestive tract that causes of polyinfection have good effect.It is carrier that the present invention adopts ovum gallinaceum that hen produces, and safety non-toxic is easy to industrialization.
The description of the drawings
Accompanying drawing has provided preparation method's of the present invention process flow sheet.
Embodiment
Below in conjunction with accompanying drawing embodiments of the invention are further described this:
Embodiment 1: have the AI of antibody activity, the product of ND composite yolk antibody, can obtain by following steps: preparation AI, ND antigen, and with the antigen immune healthy hens, collect it then and lay eggs, from laying eggs, it extracts anti--ANIgY again.
Embodiment 2: newcastle disease virus (NDV) strain is F48E8 and autonomous strain isolated HR98, the employing tissue culture method is cultivated, after the strong poison of newcastle disease is inoculated in the chicken embryo culture, collect the amnion allantoic fluid, adding formalin or β-propionic acid lactones makes it to make after the deactivation, add mineral oil or aluminium hydroxide gel again as adjuvant, oil emulsion inactivated antigen is made in emulsification; After bird flu strain H7 is inoculated in the chicken embryo culture, collect the amnion allantoic fluid, add formalin and make inactivation antigen; Two kinds of antigen by mix at 1: 1 complex antigen.
Embodiment 3: in the following way separation and Extraction anti--ANIgY: from immune ovum gallinaceum, separate yolk, with distilled water diluting, adjust suitable pH value and leave standstill 8h for 4 ℃, the centrifuging and taking supernatant concentrates, lyophilize, is resisted-ANIgY.
Embodiment 4: separate yolk, with distilled water diluting, 4 ℃ leave standstill 12h after, be that the film of 200KD and 150KD filters respectively with the molecular weight, collect the proteic substance of 150KD-200KD, carry out filtration sterilization with the malleation millipore filter.Filter membrane is a blend fiber ester filter membrane, and the aperture is 0.22 μ m, filter pressure＜1.0kg/cm
2Purification antibody with steriling test and safety verification after qualified is sub-packed in the 100mL vial, and lyophilize is resisted-ANIgY, put that 4 ℃ of refrigerators are preserved or-15 ℃ freeze to preserve standby.
Embodiment 5: the product of getting among the embodiment 1,3,4 carries out the IgY purity test: use the purity of the yolk antibody of PAGE electrophoresis detection purifying, only occur an electrophoresis band in the electrophoresis, be the yolk antibody of purification.
Embodiment 6: the product of getting among the embodiment 1,3,4 carries out the inspection of IgY concentration: the anti--ANIgY that will extract does 5-10 dilution doubly with 0.01mol/L PH7.2 PBS, then with adopting the biuret biochemical process to measure protein concentration.
Embodiment 7: with yolk antibody direct inoculation sulphur glycollate culture medium (T.G) tubule of purifying, each 2 on peptone from casein agar (G.A) inclined-plane, put 36 ± 1 ℃ for 1, put 25 ± 1 ℃ for 1, with 1 glucose peptone soup (G.P) tubule, inoculation 0.2mL puts 25 ± 1 ℃ in addition, all cultivate 5d, all asepsis growth.
Embodiment 8: safety verification is got 5 of the healthy chickens of 30 ages in days, and every subcutaneous injection 0.5mL observed for 1 week, all no abnormal reaction.
Embodiment 9: titration: get the yolk antibody liquid for preparing, fully shake up, measure AIV and NDV antibody with agar diffusion test and hemagglutination inhibition test (HI test) respectively.Wherein AIV agar diffusion antibody titer is more than 1: 64, and NDV hemagglutination inhibition test (HI test) antibody titer reaches 1: 320-1: 1084.
Embodiment 10: gather 50 on the immune egg of anti--ANIgY, separate and obtain 800ml yolk, thin up is to 5000ml, the centrifugal clear liquid 4000ml that obtains, the employing Hollow Fiber Ultrafiltration extracts, it is stand-by to be concentrated into 400ml at last, wherein contain essence carry (promptly refer to purity the IgY more than 95% anti--ANIgY) 12 grams.Maltose 10 grams are added the 50ml water dissolution, sterilized 30 minutes, auxiliary material and the 50ml ultrafiltrated of handling mixed for 100 ℃, lyophilize, aseptic pulverizing is packaged into bag, and every pouch includes 0.5 gram, is used for the treatment of single and or the avian viral transmissible disease that causes of polyinfection because of AI, ND.
Embodiment 11: gather 50 on the immune egg of anti--ANIgY, separate and obtain 800ml yolk, thin up is to 5000ml, the centrifugal clear liquid 4000ml that obtains, the employing Hollow Fiber Ultrafiltration extracts, it is stand-by to be concentrated into 400ml at last, wherein contain essence carry (promptly refer to purity the IgY more than 99.5% anti--ANIgY) 10 grams.Ultrafiltrated is pressed 5-10% and is added N.F,USP MANNITOL, mixes packing 0.1 gram/bottle, lyophilize.Be used for the treatment of single and or the deadly infectious disease that causes of polyinfection because of AIV, NDV.
Embodiment 12: experiment in vitro:
1.. molecular weight detection: SDS-PAGE electricity arteries and veins, separation gel length 8% concentrates gum concentration 5%, current stabilization 11mA electricity arteries and veins 5h, Marker adopts the low relative molecular mass standard protein, takes off gel and is used for conventional silver staining and electrophoretic blotting, and recording molecular weight is 180KD.
2.. purity check: immunoblotting (reference literature carries out), purity is 95%.
3.. the active detection: ELISA indirect test, working method is with reference to conventional ELISA indirect method, used antigen, anti--ANIgY, enzyme labelled antibody are all determined working concentration by the chessboard volumetry, virus antigen concentration is 100 μ g/ml, wraps by the every hole of plate application of sample 100 μ l tested resisting-the every hole of ANIgY application of sample 100 μ l, it is 1: 10000 that horseradish peroxidase is marked the anti-chicken IgG of anti-rabbit (Sigma company) working dilution, substrate is OPD, 2ml/ vitriol oil termination reaction, and microplate reader 492nm measures the OD value down.The result shows anti--ANIgY to the titre scope of two kinds of viruses 1: 64-1: between 1080.
Embodiment 13: anti--the external antagonism toxin of ANIgY biological activity assay:
Anti--ANIgY (10 μ g～200 μ g/ml) mixes under aseptic condition with ND strain (10～30 μ g/ml) at 1: 1.Behind 37 ℃ of 2h, get the 0.1ml supernatant and add in the cultivation of chicken respiratory epithelial cell, discovery IgY concentration can be blocked ND to the epithelial toxic action of chicken respiratory fully at 100～200 μ g/ml; Thereafter serial dilution, its protection activity also lowers thereupon, and the microscope morphological observation also obtains identical result: normal chicken respiratory epithelial cell is adherent polymorphism growth, no particle in the cell, and refractive power is good.After the ND effect, cell is spherical to be become, and occurs many particles in the endochylema, and when concentration＞50 μ g/ml, the IEC cell is many with cracking, the obvious shrinkage of residual cells volume; After the antagonism virus, the form of cell changes and alleviates.Illustrate anti--ANIgY at the external ND that can block to the epithelial toxicity of chicken respiratory, play the effect of protection cell.
Embodiment 14: chicken is in the body protection test: get 15 of 40 healthy age in days chickens; be divided into 3 groups at random; the 1st group every strong venom of subcutaneous injection newcastle disease virus and yolk purification antibody 1ml; (strong poison: after yolk antibody=mixing in 1: 4; put 37 ℃ of effect 1h). inject yolk extracting solution 1ml earlier for the 2nd group; the strong venom 0.5ml of injection NDV behind the 1h, control group subcutaneous injection 0.5ml NDV virulent strain.Observing 14d for the 1st group does not all have morbidity and death, and per 2 groups of observation 14d all do not have morbidity and dead, control group 4d sequela, and beginning death behind the 5d, whole dead behind the 7d.Illustrate that anti--ANIgY at physical efficiency antagonism ND, significantly improves the disease symptom of chicken, improve survival rate.
Embodiment 15: chicken is in the body protection test: get 15 of 40 healthy age in days chickens, be divided into 3 groups at random, the 1st group every subcutaneous injection chicken avian cholera venom and yolk purification antibody 1ml, (venom: after yolk antibody=1: 3 mixes, put 37 ℃ of effect 1h).Inject yolk extracting solution 1ml earlier for the 2nd group, the strong venom 0.5ml of injection AIV behind the 1h, the strong venom of control group subcutaneous injection 0.5ml AIV.Observing 14d for the 1st group does not all have morbidity and dead, observes 1 morbidity of 14d for per 2 groups, does not have dead.Control group 3d sequela, beginning death behind the 6d, all dead behind the 9d.Illustrate that anti--ANIgY at physical efficiency antagonism AI, significantly improves the disease symptom of chicken, improve survival rate.
Embodiment 16: oral resisting-ANIgY observes in the body biologic activity: the model of setting up the chicken infectation of bacteria with reference to pertinent literature, test is divided into treatment group " low dosage (10mg/kg), middle dosage (20mg/kg), high dosage (40mg/kg) resist-ANIgY ", control group; Behind the oral administration 3,5,7 days, indexs such as spirit of observation animal and respiratory symptom.Found that: spirit and the respiratory symptom of middle and high dosage group animal after taking 7 days have clear improvement.Illustrating that oral antibody (20mg/kg) can play improves clinical symptom, improves the effect of survival rate.
Claims (3)
1, (resist-ANIgY), it is characterized in that: described resisting-ANIgY presses YSDS-PAGE electrophoresis (7.5% separation gel) and detects a kind of composite yolk antibody of resisting fowl's viral blight, and collection of illustrative plates presents single district band; Be blue through electrophoresis Coomassie brilliant blue R250 dyeing; High molecular band place clear single specific enzymes occurred and dyes band during immunity marking Western-blotting detected; Through the active detection of the external antagonism viral organism of anti--ANIgY, show deactivation to AIV, NDV, AIV agar diffusion antibody titer is more than 1: 64 in the titration, and NDV hemagglutination inhibition test (HI test) antibody titer reaches 1: 320-1: 1084.
2, the anti-AI of a kind of preparation, ND chicken yolk antibody, the method for promptly anti--ANIgY is characterized in that: adopt following processing step:
1. preparation complex antigen
(1), newcastle disease virus (NDV) strain is the cultivation of F48E8 and autonomous strain isolated HR98: a, employing tissue culture method, after Avian pneumo-encephalitis virus is inoculated in the chicken embryo culture, collect the amnion allantoic fluid, adding formalin or β-propionic acid lactones makes it to make antigen after the deactivation, add mineral oil or aluminium hydroxide gel again as adjuvant, oil emulsion inactivated antigen is made in emulsification; (2) bird flu strain H7 is inoculated in the chicken embryo culture after, collect the amnion allantoic fluid, add formalin and make inactivation antigen; Then with two kinds of antigens by mix at 1: 1 complex antigen, complex antigen mixes by 1: 1 with complete freund adjuvant before the immunity;
(2) again gained antigen is prepared antibody by following step:
1.. to the immunity of the hen that lays eggs: the A. fundamental immunity; Select the hen that lays eggs (health) isolated rearing for use.Carry out multi-point injection chicken complex antigen under the cock skin, 0.5ml/ only; B. reinforced immunological: fundamental immunity was carried out reinforced immunological after 7 days, and injection complex antigen 0.5ml/ only; C. super-strengthening immunity: need super-strengthening immunity 1-3 time after 21 days.Injection volume 2.5ml/, to keep antibody titers;
Began to collect egg after 7 days, 4 ℃ store for future use.
2.. the extraction of yolk antibody
A. adopt water-soluble method to extract yolk antibody; From immune ovum gallinaceum, separate yolk, with distilled water diluting, adjust suitable pH value and leave standstill 8h for 4 ℃, the centrifuging and taking supernatant concentrates, lyophilize, is resisted-ANIgY;
Or B. adopts membrane filter method to extract yolk antibody: separate yolk, with distilled water diluting, 4 ℃ leave standstill 12h after, be that the film of 200KD and 150KD filters respectively with the molecular weight, collect the proteic substance of 150KD-200KD, lyophilize is resisted-ANIgY;
3, claim 1,2 described composite yolk antibodies are (anti--as ANIgY) to be used for the treatment of bird single and or the bird medicine of the relative disease that causes of polyinfection and application in the fodder additives because of AIV, NDV in preparation.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2007068154A1 (en) * | 2005-10-31 | 2007-06-21 | Jason Medical Group (Far East) Ltd | A method for preparing specific igy against mutant avian influenza virus and the preparation tηereof |
| CN102516389A (en) * | 2011-12-28 | 2012-06-27 | 黑龙江省农业科学院畜牧研究所 | Method for extracting avian influenza egg yolk antibodies |
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| CN102558347A (en) * | 2012-03-01 | 2012-07-11 | 广东紫金正天药业有限公司 | Yolk antibody for treating bird flu and infectious bronchitis and preparation method thereof |
| CN102894397A (en) * | 2011-07-27 | 2013-01-30 | 郑长义 | Production method of avian influenza immune egg |
| CN101575374B (en) * | 2009-05-18 | 2013-02-13 | 张训海 | Chicken Marek's virus antibody and preparation method thereof |
| CN113121679A (en) * | 2021-04-01 | 2021-07-16 | 仲恺农业工程学院 | High-immunity yolk antibody for pigeon Newcastle disease and preparation method and application thereof |
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2002
- 2002-05-17 CN CN02113744A patent/CN1384119A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007068154A1 (en) * | 2005-10-31 | 2007-06-21 | Jason Medical Group (Far East) Ltd | A method for preparing specific igy against mutant avian influenza virus and the preparation tηereof |
| CN101575374B (en) * | 2009-05-18 | 2013-02-13 | 张训海 | Chicken Marek's virus antibody and preparation method thereof |
| CN102894397A (en) * | 2011-07-27 | 2013-01-30 | 郑长义 | Production method of avian influenza immune egg |
| CN102516389A (en) * | 2011-12-28 | 2012-06-27 | 黑龙江省农业科学院畜牧研究所 | Method for extracting avian influenza egg yolk antibodies |
| CN102526727A (en) * | 2012-01-04 | 2012-07-04 | 河北科星药业有限公司 | Anti-bird-flue egg yolk antibody injection and application thereof |
| CN102526727B (en) * | 2012-01-04 | 2014-05-07 | 河北科星药业有限公司 | Anti-bird-flue egg yolk antibody injection and application thereof |
| CN102558347A (en) * | 2012-03-01 | 2012-07-11 | 广东紫金正天药业有限公司 | Yolk antibody for treating bird flu and infectious bronchitis and preparation method thereof |
| CN113121679A (en) * | 2021-04-01 | 2021-07-16 | 仲恺农业工程学院 | High-immunity yolk antibody for pigeon Newcastle disease and preparation method and application thereof |
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