CN103168924A - Method for preparing livestock and poultry feed additive (peptide YY egg yolk antibody powder) - Google Patents
Method for preparing livestock and poultry feed additive (peptide YY egg yolk antibody powder) Download PDFInfo
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- CN103168924A CN103168924A CN2013100517761A CN201310051776A CN103168924A CN 103168924 A CN103168924 A CN 103168924A CN 2013100517761 A CN2013100517761 A CN 2013100517761A CN 201310051776 A CN201310051776 A CN 201310051776A CN 103168924 A CN103168924 A CN 103168924A
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Abstract
The invention aims to provide a livestock and poultry feed additive, i.e., a peptide YY egg yolk antibody powder, and a preparation method thereof. The preparation method is simple in production process, convenient to use and obvious in effect of increasing the feed intake of animals. The preparation method mainly comprises the following steps: preparation of a peptide YY complete antigen, preparation of a peptide YY immune oilemulsion vaccine, specific immunization of peptide YY to laying hens, and preparation of egg yolk antibody powder. The peptide YY egg yolk antibody powder prepared by the method is high in titer, long in persistence time, long in storage time, safe, non-poisonous and pollution-free, and meets the demands of resource-saving and environmentally-friendly sustainable livestock industry development. Therefore, the prepared peptide YY egg yolk antibody powder as a safe and green feed additive can effectively promote appetite, increase feed intake and promote growth of animals.
Description
Technical field
The present invention relates to a kind of preparation method of biologically active peptide Yolk antibody powder, especially relate to a kind of preparation method of livestock and poultry phagostimulant junket junket peptide Yolk antibody powder.
Background technology
Junket junket peptide (PYY) is as the derivative hormone of class intestines and stomach, can not only make body produce full sense, reduce and ingest and body weight, but also affect alimentary canal physiology and function, mechanism of action is complicated, and coverage is extensive, becomes the novel appetite regulatory factor (Ou Rongdi that is concerned by people, Chen Yonghui, Shen Weijun etc. junket junket peptide physiological function and the application prospect [J] in the milking sow cultivation thereof. Animal nutrition journal, 2010,22 (5): 1142-1146.).PYY concentration in the rodent body of fasting and free choice feeding descends before ingesting and the phenomenon of increase explanation after ingesting, and PYY is a satiety signals that adjusting is ingested.The experiment of animal and human's body all confirms peripheral injection PYY
3-36Can produce anoretic effect, and ingest reduction and PYY
3-36Injection volume be dose-response relationship, long-term injection can make the weight of animals weightening finish reduce (Maria A., Papadimitriou, Alicja A., et al.Peptide YY
3-36-induced inhibition of food intake in female monkeys.Brain Research.2007,1175:60-65.).Adiposis patient fasting plasma PYY level is lower, and after taking in identical test meal, the PYY of fat object discharges lower than normal control, and the hyperphagia that prompting endogenous PYY deficiency causes is closely related with obesity, supplemented with exogenous PYY
3-36May become the fat effective ways for the treatment of.Studies show that, PYY and acceptor thereof have regulating action to ingesting, and can make body produce full sense, and minimizing is ingested.PYY can freely pass through blood-brain barrier, with receptors bind, hormone signal is converted to nerve signal, activate the premelanocyte cortin former/the neuronic expression of benzedrine regulating transcription thing (POMC/CART), promote the secretion of opium melanocortin, simultaneously can also suppress neuropeptide tyrosine/agouti chromoprotein GAP-associated protein GAP (NPY/AGRP) neuron activity, reduce the secretion of NPY and AGRP, thereby make body produce full sense, suppress to search for food (MartinN M, Small C J, Sajedi A, et al.Pre-obese and obese agouti mice are sensitive to the anorectic effects of peptide YY (3-36) but resistant to ghrelin.Int JObes Relat Metab Disord.2004, 28:886-893.).
In theory, inject during the antibody of the influential hormone that suppresses to ingest is in the blood and certain hormone in body by external source, namely adopt the immunological technique of antigen-antibody neutralization can reduce this hormone as the ingest ability of signal of inhibition, perhaps weaken hormone signal and pass to the ability of central nervous system from peripheral neverous system by adding certain hormone receptor antagonists, just can reduce the full sense effect of this hormone, promote feed intake.Part Study person just has realized that in the fifties in last century the antibody that utilizes hormone changes the possibility of breeding performonce fo animals.Antibody is as the important effect molecule of humoral immunity, is that the B cell accepts that Proliferation, Differentiation is the glycoprotein that thick liquid cell produces after antigenic stimulus, mainly be present in the body fluid such as serum, can with the specific combination of corresponding antigens, show immunologic function.Because antibody half life is longer, has avoided protein hormone easily to degrade in vivo and be difficult to inconvenience in practice.But because the material that is neutralized is normally endogenic, do not have in vivo immunogenicity, so must carry out crosslinked with a suitable immunity carrier (a kind of macromolecular endogenous protein), at present commonly used have bovine serum albumin (BSA), a human albumin (HSA) etc.When this material and carrier crosslinked after, will select adjuvant (keep immunogenicity Stable Release and can extend the immune response time) stimulate in and the immune response of recycled material, guarantee that immune product comprises part for the antibody with high specificity of target material, the biological action of target material in performance and in body.The immunoregulation technology utilizes the realization of immunology antigen-antibody neutralization reaction principle to the regulation and control of breeding performonce fo animals.In fact by giving exogenous antigen or the antibody of certain material of animal (affecting breeding performonce fo animals), immune neutralization reaction occurs in animal body, eliminate or weaken this kind of endogenous levels of substance, cause the normal physiological effect of this kind material disappear or weaken, thereby reach the purpose of regulation and control breeding performonce fo animals.
For solving in the animal production process, low and the growth retardation problem of the feed intake that causes under various adverse circumstance unfavorable conditions, the present invention utilizes the passive immunity technology, after antigen immune laying hen with the PYY preparation, utilize its egg that produces to be prepared into the Yolk antibody freeze-dried powder, realized both effectively utilizing the biology food calling effect of PYY antibody, also can partly replenish livetin, satisfy simultaneously the demand of the modern feed addictive of safe green, become a kind of biological technology products that haves a great attraction.
Summary of the invention
Technical problem to be solved by this invention:
Principle of the present invention is that comlete antigen take PYY and bovine serum albumin coupling preparation is as the basis, by formulating scientifically and rationally the laying hen immune programme for children, be prepared into freeze-dried powder extract yolk and freeze drying in producing egg after, working condition is simple, need not special expensive equipment.
So, the purpose of this invention is to provide a kind of low cost, high-titer, production technology simple, to the have clear improvement preparation method of feed addictive PYY Yolk antibody of effect of livestock and poultry feed intake.For achieving the above object, to adopt PYY, glutaraldehyde, bovine serum albumin, Fei Shi Freunds incomplete adjuvant etc. be raw material in the present invention.
Its feature comprises the following steps:
(1) preparation of PYY comlete antigen: the PYY that accurately takes certain mass is dissolved in the 100mL beaker that PBS solution is housed, and is called A liquid; The bovine serum albumin (BSA) that accurately takes 4 times of PYY quality is dissolved in equally PBS solution is housed, and is called in the 100mL beaker of B liquid.A, the B liquid PBS solution (10mL0.01M that all meets certain requirements, pH=7.4), then A liquid slowly is added drop-wise in B liquid, puts people's magnetic stirring apparatus rotor, dropwise adding 100 μ L concentration with pipette under stirring condition is 25% glutaraldehyde solution, sealed beaker, skin wraps up lucifuge with masking foil, and under room temperature, reaction after 24 hours, is all transferred to the conjugate reactant liquor in bag filter, under 4 ℃, with PBS solution dialysis 24 hours, liquid was changed twice in the centre.
(2) envelope antigen preparation: the envelope antigen preparation method is identical with the preparation method of PYY-BSA comlete antigen, take hemocyanin (KLH) as carrier protein, prepares envelope antigen PYY-KLH with glutaraldehyde as cross linker.
(3) immune programme for children: the healthy bird inlay of selecting 300 ages in days, mode according to chest muscle multi-point injection (general 4-6 point) is carried out immunity, the initial immunity antigen dose is 200 μ g/, after initial immunity every 15 days booster immunizations once, booster immunization is 3 times altogether, and the booster immunization antigen dose only is 100 μ g/.Add the Freund's complete adjuvant of equivalent during initial immunity in antigen, add the not formula Freunds incomplete adjuvant of equivalent during booster immunization.First and booster immunization ID is 0.5mL.
(4) anti-PYY Yolk antibody preparation
1. yolk sample pre-treatments: after egg is broken, remove the white of an egg and vitellinae membrana, get 0.3m L yolk liquid in centrifuge tube with syringe, according to yolk liquid: PBS: the ratio of chloroform=1: 1: 2 adds respectively PBS buffer solution and chloroform, after powerful vibration, the standing 1h of room temperature, the then centrifugal 15min of 3500r/min, get supernatant, to be measured in-20 ℃ of preservations.
2. powdery yolk preparation: collect weekly egg after initial immunity 1 time, antibody titer namely reach 1: 1600 the 4th week began to collect egg, tire to reach and week the 7th after 1: 4096 week finish to collect, with the aseptic separation yolk of the egg that gathers, utilize freeze drier to make powdery yolk.
3. antibody titer detects: measure antibody from yolk with indirect enzyme-linked immunosorbent absorption (ELISA) and tire, measure the OD value with the 450nm wavelength on ELIASA, thereby determine that the antibody titer of PYY in yolk is (surveying each hole OD value after blank hole zeroing, if positive greater than 2.1 times of the negative control OD value of stipulating.Being yolk to be measured under certain extension rate, absorbance/negative yolk absorbance 〉=2.1 under identical extension rate are decided to be the positive).
Table 1ELISA records mixed yolk powder antibody titer
Technique effect
1. the prepared PYY Yolk antibody production technology of the inventive method is simple, and the antigen raw materials is easy to get, and the laying hen immune time is few, can produce throughout the year, does not need the special expensive production equipment, and production cost is low, therefore is convenient to suitability for industrialized production, and rule of origin is secure;
2. prepare the PYY Yolk antibody with Yolk antibody, that antibody is held time is long, the high antibody time can reach for 4 weeks; The height of tiring, antibody titer reaches 1: 4096; Holding time is long, and lower 4 months of 4 ℃, aqua or-20 ℃ were preserved and tired constantly in 6 months, and the freeze-dried powder holding time is longer, is easy to promote the use of in herding is produced;
The PYY Yolk antibody of the present invention preparation have the height of tiring, safety non-toxic, have no drug resistance, excitant, easily absorb, a series of characteristics of instant effect, meet the development trend of Modern Animal Husbandry sustainable development spirit and feed addictive green non-pollution;
4. the PYY Yolk antibody of the present invention effect of searching for food except having obvious promotion, the powdery yolk at its place is nutritious, the Nutrients Digestion utilization rate is high, to promoting animal to overcome the feed intake that the adverse circumstance unfavorable conditions causes, good effect is arranged lowly.Zoopery confirms, compares with control group, and the average feed intake of PYY Yolk antibody powder interpolation group mouse improves within whole experimental period over 6%, and growth performance also is better than control group.
Description of drawings
1 part, accompanying drawing is a kind of technology of preparing route of additive for farm animal feed junket junket peptide Yolk antibody powder
Embodiment
According to above production procedure, below provide several specific embodiment.
Embodiment 1:
A. be 4mg (from the market buy, content 〉=99%) with PYY weight, ((10mL0.01M, pH=7.4) makes the coupling reaction product to PBS solution in glutaraldehyde solution, i.e. so-called PYY-BSA comlete antigen; Then take hemocyanin (KLH) as carrier protein, prepare envelope antigen PYY-KLH with glutaraldehyde as cross linker, preparation method is identical with the preparation method of PYY-BSA comlete antigen.
B. chest muscle multi-point injection (general 4-6 point, 0.5ml/ inferior) mode laying hen is carried out immunity, the initial immunity antigen dose be 200 μ g/ only, after initial immunity every 15 days booster immunizations once, booster immunization is 3 times altogether, and the booster immunization antigen dose only is 100 μ g/
C. the egg that produces is removed the white of an egg and vitellinae membrana, get 0.3mL yolk liquid in centrifuge tube with syringe, according to yolk liquid: PBS: the ratio of chloroform=1: 1: 2 adds respectively PBS buffer solution and chloroform, after powerful vibration, the standing 1h of room temperature, then the centrifugal 15min of 3500r/min, get supernatant, utilizes freeze drier to make powdery yolk.
Antibody titer is measured: measure antibody from yolk with indirect enzyme-linked immunosorbent absorption (ELISA) and tire, measure the OD value with the 450nm wavelength on ELIASA, thereby determine that the antibody titer of PYY in yolk is (surveying each hole OD value after blank hole zeroing, if positive greater than 2.1 times of the negative control OD value of stipulating.Being yolk to be measured under certain extension rate, absorbance/negative yolk absorbance 〉=2.1 under identical extension rate are decided to be the positive).
Embodiment 2: the animal feeding effect test
The junket junket peptide Yolk antibody powder of this method preparation is tired and can be reached 1: 4096.Add junket junket peptide Yolk antibody powder 40mg/kg in 21 age in days kunming mouse basal diets, tested 28 days by a definite date.As can be seen from Table II: test in the full phase, the test group mouse is compared average daily ingestion amount and all improves with control group mice, full phase feed intake increasing degree surpasses 6%, and wherein the 0-7 angel has improved 4.50%, improved in 8-17 days and improved 11.23% in 2.24%, 18-28 days; Test group is compared average daily gain with control group and has been improved 3.92% in the time of 0-7 days; Test group is compared serum junket junket peptide level and is all reduced with control group in test 0-17 days, wherein wherein reduces in 0-7 days in 0-7 days and has reduced by 4.32% in 6.63%, 8-17 days.
Table 2 daily ration adds junket junket peptide Yolk antibody powder to the impact of mouse growth performance and serum junket junket peptide level
Claims (3)
1. the preparation method of an additive for farm animal feed junket junket peptide (PYY) Yolk antibody powder, step comprises:
(1) preparation of PYY comlete antigen and evaluation: with PYY and bovine serum albumin coupling, make PYY-BSA coupled complex (PYY comlete antigen), detect the coupling situation through SDS-PAGE.
(2) the PYY-BSA coupled complex is to the immune programme for children of laying hen and Yolk antibody preparation thereof: PYY-BSA coupled complex and Freunds adjuvant mixing that (1) is made, fully emulsified, make oily newborn seedling, Immune Laying Hens, collect egg, and PYY Yolk antibody powder is made in the yolk liquid freeze drying that will contain the PYY specific antibody.
2. the preparation of the PYY-BSA coupled complex described in claim (1), its step comprises: accurately take 4mg PYY and be dissolved in the 100ml beaker that 10ml0.01M PBS (pH=7.4) solution is housed, be called A liquid; Accurately take 16mg bovine serum albumin (BSA) and be dissolved in the 100ml beaker that 10ml0.01M PBS (pH=7.4) solution is housed, be called B liquid.Then A liquid slowly is added drop-wise in B liquid, puts people's magnetic stirring apparatus rotor, dropwise adding 100ul concentration with pipette under stirring condition is 25% glutaraldehyde solution, sealed beaker, and skin wraps up lucifuge with masking foil, and under room temperature, reaction is 24 hours.The conjugate reactant liquor is all transferred in bag filter, dialysed 24 hours with PBS (pH value 7.4) under 4 ℃, liquid is changed twice in the centre.With the comlete antigen packing for preparing ,-20 ℃ save backup.Adopt the SDS-PAGE gel electrophoresis method to detect coupling degree: the lyophilized complex that takes a morsel, be dissolved in ph=7.40.005M PBS, on the concentrated glue that makes, once every sky adds the 5uL above-mentioned substance, beginning SDS-PAGE gel electrophoresis.
3. the newborn seedling of PYY oil that is mixed with of the PYY-BSA coupled complex described in claim (2) and Freunds adjuvant is to the immune programme for children of laying hen and utilize the institute's egg that produces extraction to prepare PYY Yolk antibody process, its step comprises: the healthy bird inlay of selecting 300 ages in days, mode according to chest muscle multi-point injection (general 4-6 point) is carried out immunity, the initial immunity antigen dose is 200ug/, after initial immunity every 15 days booster immunizations once, booster immunization is 3 times altogether, and the booster immunization antigen dose only is 100ug/.Add the Freund's complete adjuvant of equivalent during initial immunity in antigen, add the not formula Freunds incomplete adjuvant of equivalent during booster immunization.First and booster immunization ID is 0.5ml.Immunity detects Yolk antibody by ELISA and tires, and collects egg when being positive until stop collecting when being negative, and aseptic separation yolk utilizes freeze drier to make powdery yolk.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102016593A (en) * | 2008-04-07 | 2011-04-13 | 艾尼纳制药公司 | Methods of using a G protein-coupled receptor to identify peptide Y (PYY) secretagogues and compounds useful in the treatment of conditions modulated by PYY |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102016593A (en) * | 2008-04-07 | 2011-04-13 | 艾尼纳制药公司 | Methods of using a G protein-coupled receptor to identify peptide Y (PYY) secretagogues and compounds useful in the treatment of conditions modulated by PYY |
Non-Patent Citations (8)
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| 《中国优秀硕士学位论文全文数据库》 20071214 臧海军 "胆囊收缩速卵黄抗体粉再肉鸡饲粮中适宜添加量的研究" 1－3 , * |
| 《中国优秀硕士学位论文全文数据库》 20101118 梁哲 "酪酪肽完全抗原的制备及酪酪肽被动免疫对大鼠营养生理效应的影响" , * |
| 《动物营养学报》 20121130 李玉莲等 "免疫调控技术在畜禽采食量调控中的应用" 第2092-2095页 1－3 第24卷, 第11期 * |
| 《畜牧兽医》 20101230 侯志强等 "卵黄抗体研究进展" 第45-47页 1－3 , 第6期 * |
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| 李玉莲等: ""免疫调控技术在畜禽采食量调控中的应用"", 《动物营养学报》 * |
| 梁哲: ""酪酪肽完全抗原的制备及酪酪肽被动免疫对大鼠营养生理效应的影响"", 《中国优秀硕士学位论文全文数据库》 * |
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Application publication date: 20130626 |