CN101690811A - Concentrated freeze-dried yolk antibody composite preparation for infectious bursal disease and preparation process thereof - Google Patents
Concentrated freeze-dried yolk antibody composite preparation for infectious bursal disease and preparation process thereof Download PDFInfo
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Abstract
The invention provides a concentrated freeze-dried yolk antibody composite preparation for infectious bursal disease, which contains the following components in part by weight: 10.0 to 99.5 parts of infectious bursal disease virus resistant yolk antibody and 0.5 to 20.0 parts of Chinese medicinal polysaccharide, wherein the Chinese medicinal polysaccharide is extracted from one or a composition of more than two of the following Chinese medicines in part by weight: 10 to 50 parts of Chinese angelica, 50 to 100 parts of szechwon tangshen root, 50 to 80 parts of membranous milkvetch root and 30 to 50 parts of Indian buead. The preparation is a freeze-dried preparation prepared by combining the components such as the infectious bursal disease virus resistant yolk antibody and the polysaccharide under the guidance of immunity engineering of Chinese medical science, is clinically used for the infectious bursal disease, has the effect of treating infectious bursal disease virus infection, and excites the immunity and disease resistance of organisms; and simultaneously, the polysaccharide component in the preparation has powerful protection effect on the yolk antibody.
Description
Technical field
The present invention relates to a kind of antibody composite preparation, be specifically related to a kind of infectious bursal disease concentrated freeze-dried yolk antibody composite preparation, and preparation method thereof.
Background technology
Infectious bursal disease is the formidable enemy of poultry husbandry.The infection medicine that is applied to prevent and treat infectious bursal disease at present has antibiotic, chemosynthesis medicine, Chinese medicine and preparation thereof, high immunity yolk antibody and other anti-infection bio preparations.
(1) antibiotic
This class anti-infectives mainly is the metabolite that is produced by Institute of Micro-biology such as antibacterial, fungus, actinomycetes, and artificial imitated synthetic or semisynthetic antibiotics.They are strong to the inhibition or the killing action of pathogenic microorganism, has a broad antifungal spectrum, and good effect mainly is the bacillary secondary infection of auxiliary treatment infectious bursal disease.But their medicines are residual big, can pass through food source contact scar, cause human diseases.
(2) chemosynthesis medicine
This class anti-infectives mainly comprises sulfonamides, quinolones, furans etc.Their has a broad antifungal spectrum, stable in properties, determined curative effect, easy to use, also be the bacillary secondary infection that is used for the auxiliary treatment infectious bursal disease, but it is big to have toxic and side effects, easily residue in the body, cause liver, kidney etc. to organize irreversible infringement (seeing Chinese Pharmacopoeia for details); Also have moroxydine, amantadine etc.,, caused food source property medicine residual, influenced the application of this type of medicine in the people doctor though can treat infectious bursal disease.
More than two class medicines except above-mentioned shortcoming, their common drawbacks also have the interference body normal flora, destroy the body microecological balance, cause microorganism species imbalance displacement, cause superinfection and other pathological changes; Moreover heavy dose is used repeatedly and can be caused drug resistance, reduces therapeutic effect, makes the responsive rate of its anti-infective drop to 10-15%, and induces out highly pathogenic bacterial strain.
(3) Chinese medicine and preparation thereof
Chinese medicine belongs to natural drug, has the characteristics of harmless advantage, and strengthening vital QI to eliminate pathogenic factors, integral body are adjusted and had no drug resistance, and are that antibiotics and chemosynthesis medicine are incomparable, can treat infectious bursal disease effectively.But, many anti-infective Chinese medicine preparation such as Radix Isatidis electuary, CHUANXINLIAN ZHUSHEYE etc. are arranged in the market.Though these medicine antibacterials spectrum is wide, have that drug effect is slow, effect is weak, consumption is big, difficult quality is stable so that use factor such as inconvenience.
(4) vaccine prevention
Though infectious bursal disease can carry out vaccine prevention, vaccine just seems of no avail under urgent situation about infecting.
(5) high immunity yolk antibody preparation
This class anti-infectives is from special chicken yolk antibody.Their speed of actions are fast, and curative effect is accurate, high specificity, but because only at chicken infectivity bursa of Fabricius virus, invalid to mixed infection and secondary infection, have to sometimes with above-mentioned three class medicine auxiliary treatment; Secondly, present most yolk antibody is that direct goods are that original high-immunity yolk goods also claim rough high immunity yolk antibody (to press Wang Zhijie such as breeding enterprise oneself, the development of infectious bursal disease yelk antibody liquid in high immunity, Xichang agricultural higher junior college journal, 2004,18 (2), the yolk antibody of the method preparation that p51 provides etc.), include many non-ingredients such as fat in the yolk and particulate protein, they influence the absorption of yolk antibody, and what have also has other disease propagation source; Moreover, even there are indivedual yolk antibody preparations to adopt extraction concentration technique to manufacture refining high immunity yolk antibody at present, major part also is that water preparation (is pressed Zhu Ruiliang such as breeding enterprise oneself, Qi Junzhi etc., the preparation method of anti-IBD high-immune yolk AI antibody and application, the Shandong animal and veterinary, 1994,60 (4), the yolk antibody of the method preparation that p35 provides etc.), again such as present market use by West China, Sichuan biological product Engineering Co., Ltd, the refining high immunity yolk antibody of the infectious bursal disease that enterprises such as Luoyang Pu Laike biological product Engineering Co., Ltd produce, they must cryopreservation, in storing and use, be easy to the degeneration inactivation, lose medical value.Therefore, these anti-infectious preparations are difficult to operate on drug market and have lost the meaning of commodity value.
(6) other anti-infective biotic factor preparations
Such as interferon, cytokine etc.,, be difficult to be accepted extensively, as for more difficult implementation in infectious bursal disease by clinical owing to cost an arm and a leg.
Based on above-mentioned technical background, the present invention is directed to infectious bursal disease and propose a kind of yolk antibody composite preparation, overcome many defectives that the infection medicine exists in the prior art.
Summary of the invention
The objective of the invention is to: propose a kind of infectious bursal disease yolk antibody composite preparation, overcome the shortcoming in the prior art, reduce cost and chemical sproof prerequisite under, effectively improved the prevention effect of eqpidemic disease, and can not produce food source contact scar and drug residue the people.
Another object of the present invention also is: a kind of preparation method of described compound formulation is provided, further reduces production costs, enhance productivity.
Above-mentioned purpose of the present invention is achieved through the following technical solutions:
A kind of infectious bursal disease concentrated freeze-dried yolk antibody composite preparation is provided, and it is formed by the feedstock production of following parts by weight:
(1) yolk antibody of infectious bursal disease virus 10.0-99.5;
(2) herbal polysaccharide 0.5-20.0;
Described herbal polysaccharide extracts one or more the compositions in following parts by weight of Chinese traditional medicine: Radix Angelicae Sinensis 10-50, Radix Codonopsis 50-100, Radix Astragali 50-80, Poria 30-50.
Described compound formulation can also further comprise conventional protein protective agent 1.0-10.0 weight portion.
Described protein protective agent, by weight, the mixture of one or both among preferred defatted milk powder 1.0-6.0 or the sucrose 1.0-3.0.
Described yolk antibody of infectious bursal disease virus can be in the prior art through the bursal disease vaccine height produced of the laying hen of reinforced immunological exempt to purify in the egg existing product of preparation repeatedly, purification preparation method can be the method for any purpose that realizes purifying in the prior art.
For the yield that further improves yolk antibody, reduce production costs, yolk antibody of infectious bursal disease virus of the present invention preferably makes in accordance with the following methods:
(1) produce anti-chicken infectivity bursa of Fabricius virus height according to conventional method and exempt from egg, can may further comprise the steps:
(1.1) the no-special pathogen laying hen is in the isolated rearing of clean plant;
(1.2) laying hen is carried out 3 immunity, give every chicken injection bursal disease vaccine at every turn, injection volume is respectively 0.5ml, 1.5ml and 2.0ml, each week age at interval;
(1.3) finish the 3rd immunity after 21 days, collect egg, yolk antibody in the yolk is carried out immunology detection, the yolk antibody agar diffusion of chicken infectivity bursa of Fabricius virus is tired and is reached 1: 32 above egg and exempt from egg for the chicken infectivity bursa of Fabricius virus height, and collection storage is standby under 16 ℃ of conditions; If it is not up to standard to detect These parameters, can carry out the 3rd immunity again, qualified until detecting.
(2) purification of yolk antibody of infectious bursal disease virus
(2.1) height of the anti-chicken infectivity bursa of Fabricius virus that step (1) is obtained is exempted from egg and is carried out disinfection, and smashes egg and isolates yolk;
(2.2) in isolated yolk of (2.1) step, add the sterile deionized water of 11 times of yolk weight, and adjust pH value to 6.00, stirred then 5 minutes, after under 4 ℃ condition, leaving standstill 48 hours, extract a supernatant by siphon; After again with precipitate with 8 times of sterile deionized water dilutions, and adjust pH value to 6.0, stirred then 5 minutes, after under 4 ℃ condition, leaving standstill 24 hours, extract the secondary supernatant by siphon; Merge supernatant twice, remove endotoxin through adsorption filtration, it is standby to obtain to remove malicious supernatant; Wherein, the adjustment of described pH value can use 0.1N HCl or 0.1N NaOH to finish; Described adsorption filtration can use active carbon to finish, and also can finish with known any method;
(2.3) the malicious supernatant that removes that (2.2) step is obtained is removed impurity such as yolk microgranule in the supernatant; Again filtered solution is removed low molecular impurity, and, be yolk antibody of infectious bursal disease virus trapped substance simmer down to magma; Wherein, the described impurity of removing in the supernatant such as yolk microgranule can be by finishing through the 1000KD membrane filtration or through 10000 rev/mins in centrifugal 10 minutes; Described filtered solution is removed low molecular impurity can be by finishing through 30～100KD membrane filtration.
The extracting method of described polysaccharide can be existing arbitrarily method, preferably extracts in accordance with the following methods to obtain:
A. with after described raw material of Chinese medicine chopping, cleaning, fully soak into cold water soak 2 hours to the medicine heart, the water that adds 12 times of raw material weights again is heated to the boiling back under 90 ℃ situation, decocts to concentrate simultaneously in 180 minutes by the hot reflux concentration technology to obtain concentrated medicament;
B. with the concentrated medicament of step a preparation by 10000 rev/mins centrifugal 20 minutes, discard precipitate, obtain supernatant and also concentrate through negative pressure, making it form dry is 25% concentrated medicament;
C. the concentrated medicament that step b is obtained adds the ethanol of 4-8 times of volume 95%, standing over night, discard the ethanol supernatant, precipitate is removed ethanol, be dissolved in water then and by the 100KD membrane filtration, remove macromole impurity such as foreigh protein removing, endotoxin, collect filtered solution and filter by the 1KD NF membrane again, remove organic molecule and impurity such as inorganic molecule and ion, the reverse current dialysis in material dilution back that is trapped 24 hours; Taking-up is concentrated into liquid, adds 5 times of volume 99% ethanol precipitations, stir, and standing over night, 3000-5000 rev/min is centrifugal 20 minutes; Precipitate is removed ethanol and through spray drying, is promptly obtained herbal polysaccharide used in the present invention.
The present invention also provides a kind of preparation method of described compound formulation, may further comprise the steps:
1) produces anti-chicken infectivity bursa of Fabricius virus height according to conventional method and exempt from egg;
2) height of the anti-chicken infectivity bursa of Fabricius virus that step 1) is obtained is exempted from egg and is carried out disinfection, smash egg and isolate yolk, the sterile deionized water that adds 11 times of yolk weight then therein, and adjustment pH value to 6.00, stirred then 5 minutes, after under 4 ℃ condition, leaving standstill 48 hours, extract a supernatant by siphon; After again with precipitate with 8 times of sterile deionized water dilutions, and adjust pH value to 6.0, stirred then 5 minutes, after under 4 ℃ condition, leaving standstill 24 hours, extract the secondary supernatant by siphon; Merge supernatant twice, remove endotoxin, obtain to remove malicious supernatant through adsorption filtration, remove by filter the impurity such as yolk microgranule in the supernatant, again filtered solution is removed low molecular impurity, and be magma by ultrafiltration and concentration, obtain yolk antibody of infectious bursal disease virus trapped substance;
3) with step 2) yolk antibody of infectious bursal disease virus that obtains, extract herbal polysaccharide from described Chinese medicine and mix according to described weight portion ratio and be dissolved in the sterile deionized water, the yolk antibody agar diffusion of chicken infectivity bursa of Fabricius virus in every ml soln is tired 〉=1: 512;
4) solution that step 3) is obtained is sub-packed in cillin bottle and carries out normal freeze-drying, and passes through Co
60The irradiation killing microorganisms promptly obtains infectious bursal disease concentrated freeze-dried yolk antibody composite preparation of the present invention.
Step 2 wherein) adjustment of described pH value preferably uses 0.1N HCl or 0.1N NaOH to finish.
Step 2) described adsorption filtration preferably uses active carbon to finish.
Step 2) described impurity of removing in the supernatant such as yolk microgranule were preferably finished through the 1000KD membrane filtration or through 10000 rev/mins in centrifugal 10 minutes; Described filtered solution is removed low molecular impurity and is preferably finished through 30～100KD membrane filtration.
The described lyophilization of step 4) preferably is sub-packed in solution cillin bottle and is placed in the vacuum freeze drier, be chilled to-40 ℃ in advance, then at-40 ℃ of following lyophilization 2-8 hours, then per hour heat up about 3 ℃, vacuum drying through 12-18 hour is warming up to 25 ℃, add a cover then, seal, in 25 ℃ of environment, kept 3-5 hour.
Prior art is divided into separation and purification, two steps of extraction purification of yolk antibody aspect the yolk antibody purification.In the separation and purification of first step yolk antibody, there are Polyethylene Glycol (PEG) method, dextran sulfate (DS) method, chloroform lifting manipulation, natural gum natural gum such as carrageenan (CAR) and xanthan gum (XAN) method, sad (CA) method, water dilution (WD) method, supercritical gas to extract (SFE) method etc.; The second step yolk antibody extracts on the purification, and ultrafiltration, ammonium sulfate precipitation method, the sodium sulfate sedimentation method, Polyethylene Glycol (PEG) sedimentation method, cold ethanol two-step precipitation method, co-precipitation method etc. are arranged.Comprehensive above-mentioned two steps, water dilution (WD) method combined with hyperfiltration method was a most worthy yolk antibody method of purification, because this integrated processes does not exist any external source to pollute as the medicine of producing pollution-free food.But, owing in production in enormous quantities, need to reach recovery antibody supernatant purpose, and the water of prior art dilution (WD) method can not get the antibody supernatant of capacity at short notice, has therefore influenced the response rate of antibody by gravity natural sedimentation yolk microgranule.The present invention is actual from producing, yolk antibody purification technique to existing water dilution (WD) method combined with hyperfiltration method has been done further improvement, improved the yolk antibody response rate, and then the preparation method of above-mentioned compound formulation has been proposed, on the basis that guarantees pharmaceutical effectiveness, reached the purpose of further boosting productivity, reducing cost.
Compared with prior art, compound formulation of the present invention has following beneficial effect:
1) has more comprehensive, effectively preventing effect
Under the guidance of traditional Chinese medical science immunoengineering, the present invention adopts the top physics and chemistry technology of modern immunological engineering technology and modern Chinese medicine preparation, the non-specific immunity of the specific immune of yolk antibody of infectious bursal disease virus and polysaccharide and they are combined the compound freeze-dried formulation of making to the opsonic action of body self resistance against diseases.Therefore, it is former that compound formulation of the present invention both can have been resisted protopathy---and chicken infectivity bursa of Fabricius virus infects, and can excite autoimmunity and whole disease-resistant function again; Both treating both the principal and secondary aspects of a disease was quick and durable again.Therefore compound formulation of the present invention simple antibody preparation of the prior art relatively and Chinese medicine preparation have more comprehensively, more effective, prevention effect that action time is longer.For example, 1 plumage part of infectious bursal disease concentrated freeze-dried yolk antibody composite preparation in 1 preparation of every chicken muscle injection embodiment of the invention, respectively can be after 1 hour from being detected yolk antibody of infectious bursal disease virus the injection chicken serum at infectious bursal disease virus (IBDV), rise gradually subsequently, peaked in 48 hours, be maintained to 72 hours, and descended thereupon, 144 hours basic disappearances.As seen compound formulation of the present invention more can press down effectively and kill chicken infectivity bursa of Fabricius virus than general antibiotic drug long action time in vivo, plays excellent curative.
Concrete therapeutic effect can be referring to following clinical trial: the clinical effectiveness experiment of testing 1. infectious bursal disease concentrated freeze-dried yolk antibody composite preparations of the present invention
[being tried disease chicken situation]
Being tried the disease chicken is to make a definite diagnosis the chickling of the 14-84 age in days of suffering from the chicken infectivity bursa of Fabricius virus infection and breed chicken, does not have the kind restriction.
[medical diagnosis on disease standard]
The morbidity chickling with the fabricius bursa enlargement, hemorrhagely be feature and have following one or both symptoms concurrently:
1, lassitude, anorexia, intermittent diarrhea draw watery white feces, tremble dehydration, highly collapse;
2, subcutaneous and chest muscle and lower limb flesh are hemorrhage, and is hemorrhage between glandular stomach and muscular stomach, the renal tubules urate deposition.
[experiment and observational technique]
12360 chickens that brood of making a definite diagnosis the infections chicken cloacal bursa virus infection are divided into four groups:
A group---compound formulation treatment group of the present invention, the method that provides by the embodiment of the invention 1 prepares antibody preparation;
B group---yolk antibody extracts freeze-dried products treatment group, and the method that provides by " Zhu Ruiliang, Qi Junzhi etc., the preparation method of anti-IBD high-immune yolk AI antibody and application, Shandong animal and veterinary, 1994,60 (4), p35 " prepares antibody preparation;
C group---rough yolk antibody treatment group, the method that provides by " Wang Zhijie, the development of infectious bursal disease yelk antibody liquid in high immunity, Xichang agricultural higher junior college journal, 2004,18 (2), p51 " prepares antibody preparation;
The D group---not treatment group is left intact.
Then, the A-C group uses corresponding preparations to treat respectively, and method is every sick chicken intramuscular injection every day 1 plumage part, continuous use 3 days.The yolk antibody neutralization index of the anti-chicken infectivity bursa of Fabricius virus of every plumage part (IBDV) answers 〉=105.0
[healing evaluation criterion]
1, the fabricius bursa enlargement of morbidity chickling disappears, is white powder;
2, lassitude, anorexia, intermittent diarrhea, tremble, dewatering symptom disappears;
3, subcutaneous and chest muscle and lower limb flesh are hemorrhage, and is hemorrhage between glandular stomach and muscular stomach, and renal tubules urate deposition pathological changes disappears.
[laboratory observation result]
Therapeutic outcome, preparation of the present invention is to the cure rate 93.8% of infectious bursal disease, and the refining high immunity yolk antibody product of prior art is to the cure rate 85.6% of infectious bursal disease, the informal at present cure rate 83.8% of producing the homemade rough high immunity yolk antibody of the enterprise of raising chickens of medicine number to infectious bursal disease.Respectively three experimental group cure rates are carried out t check in twos by the applying biological statistics, show that fully all there is utmost point significant difference (u＞2.58, p＜0.01) each other in they, therefore proved that the therapeutic effect of compound formulation of the present invention is better than other preparation.Experimental result sees Table 1.
Table 1. compound formulation of the present invention clinical application effect compared with prior art
Annotate: in the table "
*" all the expression with index other the group data relatively have highly significant difference.
In addition, through after the above-mentioned test, the fabricius bursa that two groups of the A in the experiment, D are tried the disease chicken is found the chicken bursa outward appearance pinkiness of A group healing, inner mucosa milky by analysing the method comparison of advancing; The fabricius bursa apparent size of the dead chicken of D group untreated differs, the enlargement that has, and the atrophy that has, what have is congested hemorrhage, the steatosis that has, what inner mucosa had is congested hemorrhage, the character shape that has lost mucosa that has.Simultaneously, by the fabricius bursa sample of A, two groups of chickens of D is done the histopathology sections observation, as can be seen: the chicken bursa folliculus cortilymph cell that the A group is cured is intensive, netted cell activation in the folliculus medullary substance, a small amount of lymphocyte is replaced by macrophage, and connective tissue proliferation becomes the reparation state between folliculus; And D group is not cured the chicken bursa lymph follicle and is almost played full disappearance, is replaced by connective tissue, presents typical infectious bursal disease pathological change (seeing accompanying drawing 1).
2) antibody is effectively protected, and promotes the efficacy stability performance
On the basis that modern molecular biology, modern molecular immunology and Chinese medicine combine, utilize the intermolecular hydrogen bonding structural theory, after compositions such as yolk antibody of infectious bursal disease virus and polysaccharide are mixed through vacuum lyophilization, make composition such as polysaccharide form the false hydration shell of multidimensional network space hydrogen bond structure in that yolk antibody of infectious bursal disease virus is peripheral, thereby be developed into a kind of infectious bursal disease concentrated freeze-dried yolk antibody composite preparation by hydrogen bond.Compositions such as polysaccharide are class materials that is rich in hydroxyl, and they can make up multidimensional network space hydrogen bond structure each other, form false hydration shell on the yolk antibody of infectious bursal disease virus surface.Its existence has improved yolk antibody of infectious bursal disease virus opposing high temperature, ultraviolet etc. and has induced degeneration; surface structure, conformation and the function of yolk antibody of infectious bursal disease virus molecule have directly been protected; therefore the yolk antibody in the compound formulation of the present invention is difficult for inactivation, can bring into play the infection effect more fully effectively.Concrete effect is referring to following experiment: test the heat-resisting storage effect of 2. infectious bursal disease concentrated freeze-dried yolk antibody composite preparations of the present invention
We have done this experiment for the heat-resistant quality of identifying preparation of the present invention.
Place 37 ℃ of environment with the preparation (A) of the embodiment of the invention 1, existing refining high immunity yolk antibody product (B) with by the existing homemade rough high immunity yolk antibody of method (C), preserve 1 week, 2 weeks, 3 weeks, January, June, December respectively, after 24 months, 36 months, 48 months, with yolk antibody of infectious bursal disease virus agar diffusion (AGP) method, measure the yolk antibody agar diffusion (AGP) of every plumage part and tire, the results are shown in Table 2.
The thermostability of the different anti-infectious bursal disease yolk antibody preparations of table 2.
As shown in Table 2, the preparation of the embodiment of the invention 1 has very strong heat resistanceheat resistant performance, preserves the yolk antibody agar diffusion (AGP) of 36 months every plumage parts and tire still at 1: 16 in 37 ℃ environment; And refining high immunity yolk antibody product of the prior art can only be stored 6 months in this environment, and rough high immunity yolk antibody also can only store for 3 weeks.So preparation of the present invention has the advantage of heat resistanceheat resistant storage, convenient transportation and use, overcome the defective of present other product.
3) other
Culture the field chicken, after country had issued antiviral agents such as forbidding moroxydine, amantadine, the virosis of aquaculture had become restriction to culture the bottleneck of development.But do not contain antibiotic or chemical synthetic drug in the compound formulation of the present invention, therefore can not make chicken produce drug resistance, can not produce the residual pollution of medicine yet the people; And with respect to preparations such as cytokine, interferon, compound formulation of the present invention has multiple advantages such as cost is low, easy to use.
4) preparation method of the present invention can further reduce production costs, enhance productivity under the prerequisite of the therapeutic effect that guarantees compound formulation.
In the preparation method of the present invention, adopt new yolk antibody purifying technique, promptly improved water dilution (WD) method combined with hyperfiltration method of the prior art.Prior art is diluted 7～10 times with sterile deionized water with high-immunity yolk liquid, and adjusts pH value to 5.2 with 0.1N HCl or 0.1N NaOH solution, leaves standstill under 4 ℃ condition 6 hours; And the inventive method is diluted 11 times with sterile deionized water with high-immunity yolk liquid, and adjusts pH value to 6.00 with 0.1N HCl or 0.1N NaOH solution, leaves standstill under 4 ℃ condition 48 hours.Facts have proved that by under the gravity natural sedimentation situation, the former lacks 25-35% than the latter at the antibody yield in the same environment in production in enormous quantities.Therefore, preparation method of the present invention can further reduce production costs, enhance productivity under the prerequisite of the therapeutic effect that guarantees compound formulation.
Description of drawings
Fig. 1 be in the experiment of embodiment 5 treatment group and not treatment group fabricius bursa to photograph and picture, wherein:
Fig. 1-A1 is the fabricius bursa mucosa macrostate of treatment group;
Fig. 1-A2 is the fabricius bursa mucosa macrostate of not treating group;
Fig. 1-B1 is the fabricius bursa tissue slice microscopically image (HE * 100) of treatment group;
Fig. 1-B2 is the fabricius bursa tissue slice microscopically image (HE * 100) of not treating group.
The specific embodiment
Further specify content of the present invention below by specific embodiment.
Embodiment 1. kinds of traditional Chinese medicines glycan infectious bursal disease concentrated freeze-dried yolk antibody composite preparations
1. composition:
99.5 kilograms of yolk antibody of infectious bursal disease virus
0.5 kilogram of herbal polysaccharide
Described herbal polysaccharide extracts the Chinese crude drug compositions from following weight ratio: Radix Angelicae Sinensis 10, Radix Codonopsis 50, the Radix Astragali 50, Poria 30.
2. preparation:
(1), the height of the anti-chicken infectivity bursa of Fabricius virus of preparation is exempted from egg.
(1.1) the no-special pathogen laying hen is in the isolated rearing of clean plant;
(1.2) laying hen is carried out 3 immunity, give every chicken injection bursal disease vaccine at every turn, injection volume is respectively 0.5ml, 1.5ml and 2.0ml, each week age at interval;
(1.3) finish the 3rd immunity after 21 days, collect egg, yolk antibody in the yolk is carried out immunology detection, the yolk antibody agar diffusion of chicken infectivity bursa of Fabricius virus is tired and is reached 1: 32 above egg and exempt from egg for the chicken infectivity bursa of Fabricius virus height, and collection storage is standby under 16 ℃ of conditions; If it is not up to standard to detect These parameters, can carry out the 3rd immunity again, qualified until detecting.
(2) purification of yolk antibody of infectious bursal disease virus
(2.1) height of the anti-chicken infectivity bursa of Fabricius virus that step (1) is obtained is exempted from egg and is carried out disinfection, and smashes egg and isolates yolk;
(2.2) go on foot the sterile deionized water that adds 11 times of yolk weight in the isolated yolk in (2.1), and use 0.1N HCl or 0.1N NaOH to adjust pH value to 6.00, stirred then 5 minutes, and after under 4 ℃ condition, leaving standstill 48 hours, extracted a supernatant by siphon; After again with precipitate with 8 times of sterile deionized water dilutions, and use 0.1N HCl or 0.1N NaOH to adjust pH value to 6.0, stirred then 5 minutes, after under 4 ℃ condition, leaving standstill 24 hours, extract the secondary supernatant by siphon; Merge supernatant twice, remove by filter endotoxin through activated carbon adsorption, it is standby to obtain to remove malicious supernatant;
What (2.3) (2.2) step is obtained removes malicious supernatant through the 1000KD membrane filtration or through 10000 rev/mins of impurity such as yolk microgranule of removing in the supernatant in centrifugal 10 minutes; Again filtered solution is removed low molecular impurity through 30～100KD membrane filtration, and, be yolk antibody of infectious bursal disease virus trapped substance simmer down to magma;
(3) preparation polysaccharide
(3.1) Radix Angelicae Sinensis, Radix Codonopsis, the Radix Astragali and Poria are got material, chopping, cleaned the back mixing according to the weight ratio of 10 parts of Radix Angelicae Sinensis, 50 parts of Radix Codonopsis, 50 parts of the Radixs Astragali, 30 parts in Poria, fully soak into cold water soak 2 hours to the medicine heart, 12 times the water that adds raw material weight again, be heated to the boiling back under 90 ℃ situation, under the hot reflux concentration technology, decoct 180 minutes concentrated simultaneously concentrated medicaments that obtain.
(3.2) with the concentrated medicament of step (3.1) preparation by 10000 rev/mins centrifugal 20 minutes, discard precipitate, obtain supernatant and also concentrate through negative pressure, making it form dry is 25% concentrated medicament;
(3.3) concentrated medicament that step (3.2) is obtained adds the ethanol of 4-8 times of volume 95%, standing over night, discard the ethanol supernatant, precipitate is removed ethanol, be dissolved in water then and by the 100KD membrane filtration, remove macromole impurity such as foreigh protein removing, endotoxin, collect filtered solution and filter by the 1KD NF membrane again, remove organic molecule and impurity such as inorganic molecule and ion, the reverse current dialysis in material dilution back that is trapped 24 hours; Take out concentrated solution, add 5 times of volume 99% ethanol precipitations, stir, standing over night, 3000-5000 rev/min is centrifugal 20 minutes; Precipitate is removed ethanol and through spray drying, is promptly obtained herbal polysaccharide used in the present invention.
(4) kinds of traditional Chinese medicines glycan infectious bursal disease concentrated freeze-dried yolk antibody composite preparation is synthetic
The yolk antibody of 99.5 kilograms of steps (2) preparation and the polysaccharide mixing of 0.5 kilogram of step (3) preparation are dissolved in the sterile deionized water, the yolk antibody agar diffusion (AGP) of chicken infectivity bursa of Fabricius virus in every ml soln (IBDV) is tired be 〉=1: 512, be sub-packed in cillin bottle then, place vacuum freeze drier dry.Vacuum freezing condition: 1, be chilled to-40 ℃ in advance; 2, at-40 ℃ of following lyophilization 2-8 hours, then per hour heat up about 3 ℃, through 12-18 hour vacuum drying to 25 ℃; 3, add a cover, sealing kept 3-5 hour in 25 ℃ of environment.After Co
60The irradiation killing microorganisms is finished product.3. quality standard:
[character] this product is faint yellow, is fine and close agglomerate.
[steriling test] undertaken by " Chinese veterinary drug allusion quotation ", answers asepsis growth.
[mycoplasma check] undertaken by " Chinese veterinary drug allusion quotation ", should not have the mycoplasma growth.
[exogenous virus check] undertaken by " Chinese veterinary drug allusion quotation ", should be up to specification.
With 5 of 14 age in days SPF chickens, each intramuscular injection 10 plumage part was observed 14 days [safety verification], should all be good for and live, and the injection site does not have inflammatory reaction.
[efficacy test] following method is appointed and is selected one
1. neutralization index is measured and is pressed the dated plumage part of label, is diluted to 1 plumage part/ml with normal saline, measures antibody titer by " Chinese veterinary drug allusion quotation " note neutralization test sessile antibody virus dilution method, and neutralization index answers 〉=10
5.0
2. the chickling protection is measured with 30 of 21 age in days SPF chickling, wherein 10, every intramuscular injection 1 plumage part is done contrast, isolated rearing for 20 in addition.24 hours; get whole passive immunity chickling together with 10 of contrast chickens; the strong malicious TL strain of every eye dripping inoculation IBD (dilution 30-60 doubly); the dead result of 96 hour records; counteracting toxic substances matched group chickling mortality rate should be more than 80%; passive immunity group chickling protective rate should be more than 90%, and 10 fabricius bursa of normal healthy controls group do not have any variation.
3. measure with 90 of 21 age in days SPF chickling infecting the chickling healing power, 30 give over to negative control, the strong malicious TL strain of all the other 60 every eye dripping inoculation IBD (dilution 30-60 doubly), infect after 12 hours, wherein 30, every intramuscular injection this product 1 plumage part is carried out passive immunotherapy, every day 1 time, continuous 3 days, in addition 30 as positive control, isolated rearing.Treated back 72 hours, and observed and write down and respectively organize dead result, counteracting toxic substances positive controls mortality rate should be more than 80%, and passive immunotherapy group healing power should be more than 90%, and 30 fabricius bursa of healthy negative control group do not have any variation.
[residual moisture mensuration] is undertaken by " Chinese veterinary drug allusion quotation ", should meet veterinary biologics general rule regulation.
[vacuum mensuration] is undertaken by " Chinese veterinary drug allusion quotation ", should be up to specification.
[effect and purposes] is used for the emergency treatment and the prevention of the early infection of infectious bursal disease.
[usage and consumption] intramuscular injection.
Chicken 1 plumage part of treatment consumption 14～35 ages in days, chicken 2 plumage parts that 35 ages in days are above; When being in a bad way, can adding 2～3 times of amounts and use, but also every day 1 time, logotype 2～3 days.
Chicken 1 plumage part of prevention consumption 14～35 ages in days, chicken 2 plumage parts that 35 ages in days are above; Urgent prevention, but duplicate injection 2～3 times.
[points for attention]
1. with behind this product passive immunity, must not carry out the immunity of attenuated living vaccine against infectious bursal disease in 5 days.
2. this product is sure not the high temperature heating and is used use immediately after diluting with normal saline.
3. this product is oral invalid.
This product can with antibiotic hybrid injection for animals.
[storing and effect duration] 2-8 ℃ or room temperature preservation; 36 months effect duration.
Embodiment 2. 3 flavor herbal polysaccharide type infectious bursal disease concentrated freeze-dried yolk antibody composite preparations
1. composition:
65 kilograms of yolk antibody of infectious bursal disease virus;
6 kilograms of herbal polysaccharides;
10 kilograms of sucrose;
2. preparation:
The preparation method of yolk antibody and polysaccharide is with embodiment 1, and just the extraction of polysaccharide source becomes following parts by weight of Chinese traditional medicine raw material: 10 parts of Radix Angelicae Sinensis, 20 parts of Radix Codonopsis, 10 parts in Poria;
The building-up process of compound formulation and method be with embodiment 1, just becomes 10 kilograms of 65 kilograms of yolk antibody of infectious bursal disease virus, 6 kilograms of polysaccharide and sucrose at synthesis material.
3. quality standard:
With embodiment 1.
3. liang of flavors of embodiment herbal polysaccharide type infectious bursal disease concentrated freeze-dried yolk antibody composite preparation
1. composition:
40 kilograms of yolk antibody of infectious bursal disease virus;
20 kilograms of polysaccharide;
6 kilograms of defatted milk powder;
2. preparation:
The preparation method of yolk antibody and polysaccharide is with embodiment 1, and just the extraction of polysaccharide source becomes following parts by weight of Chinese traditional medicine raw material: 10 parts in Poria, 30 parts of the Radixs Astragali;
The building-up process of compound formulation and method be with embodiment 1, just becomes 6 kilograms of 40 kilograms of yolk antibody of infectious bursal disease virus, 20 kilograms of polysaccharide and defatted milk powder at synthesis material.
3. quality standard:
With embodiment 1.
Embodiment 4. single medicinal material glycan infectious bursal disease concentrated freeze-dried yolk antibody composite preparations
1. composition:
10 kilograms of yolk antibody of infectious bursal disease virus;
5 kilograms of polysaccharide;
1 kilogram of defatted milk powder;
1 kilogram of sucrose;
2. preparation:
The preparation method of yolk antibody and polysaccharide is with embodiment 1, and just the extraction of polysaccharide source becomes Radix Codonopsis;
The building-up process of compound formulation and method be with embodiment 1, just becomes 1 kilogram of 10 kilograms of yolk antibody of infectious bursal disease virus, 5 kilograms of polysaccharide, 1 kilogram of defatted milk powder and sucrose at synthesis material.
3. quality standard:
With embodiment 1.
Claims (10)
1. an infectious bursal disease concentrated freeze-dried yolk antibody composite preparation is characterized in that, it contains the component of parts by weight down:
(1) yolk antibody of infectious bursal disease virus 10.0-99.5;
(2) herbal polysaccharide 0.5-20.0;
Described herbal polysaccharide extracts one or more the compositions in following parts by weight of Chinese traditional medicine: Radix Angelicae Sinensis 10-50, Radix Codonopsis 50-100, Radix Astragali 50-80, Poria 30-50.
2. the described compound formulation of claim 1 is characterized in that, further contains conventional protein protective agent 1.0-10.0 weight portion.
3. the described compound formulation of claim 2 is characterized in that: described protein protective agent by weight, is one or both the mixture among defatted milk powder 1.0-6.0 or the sucrose 1.0-3.0.
4. the described compound formulation of claim 1 is characterized in that, described yolk antibody of infectious bursal disease virus makes in accordance with the following methods:
(1) produces anti-chicken infectivity bursa of Fabricius virus height and exempt from egg;
(2) purification of yolk antibody of infectious bursal disease virus
(2.1) height of the anti-chicken infectivity bursa of Fabricius virus that step (1) is obtained is exempted from egg and is carried out disinfection, and smashes egg and isolates yolk;
(2.2) in isolated yolk of (2.1) step, add the sterile deionized water of 11 times of yolk weight, and adjust pH value to 6.00, stirred then 5 minutes, after under 4 ℃ condition, leaving standstill 48 hours, extract a supernatant by siphon; After again with precipitate with 8 times of sterile deionized water dilutions, and adjust pH value to 6.0, stirred then 5 minutes, after under 4 ℃ condition, leaving standstill 24 hours, extract the secondary supernatant by siphon; Merge supernatant twice, remove endotoxin through adsorption filtration, it is standby to obtain to remove malicious supernatant;
(2.3) the malicious supernatant that removes that (2.2) step is obtained is removed impurity such as yolk microgranule in the supernatant; Again filtered solution is removed low molecular impurity, and be magma by ultrafiltration and concentration, be yolk antibody of infectious bursal disease virus trapped substance.
5. the described compound formulation of claim 1 is characterized in that, described polysaccharide extracts in accordance with the following methods and obtains:
A. with after described raw material of Chinese medicine chopping, cleaning, fully soak into cold water soak 2 hours to the medicine heart, the water that adds 12 times of raw material weights again is heated to the boiling back under 90 ℃ situation, decocts to concentrate simultaneously in 180 minutes by the hot reflux concentration technology to obtain concentrated medicament;
B. with the concentrated medicament of step a preparation by 10000 rev/mins centrifugal 20 minutes, discard precipitate, obtain supernatant and also concentrate through negative pressure, making it form dry is 25% concentrated medicament;
C. the concentrated medicament that step b is obtained adds the ethanol of 4-8 times of volume 95%, standing over night, discard the ethanol supernatant, precipitate is removed ethanol, be dissolved in water then and by the 100KD membrane filtration, remove macromole impurity such as foreigh protein removing, endotoxin, collect filtered solution and filter by the 1KD NF membrane again, remove organic molecule and impurity such as inorganic molecule and ion, the reverse current dialysis in material dilution back that is trapped 24 hours; Take out concentrated solution, add 5 times of volume 99% ethanol precipitations, stir, standing over night, 3000-5000 rev/min is centrifugal 20 minutes; Precipitate is removed ethanol and through spray drying, is promptly obtained herbal polysaccharide used in the present invention.
6. the preparation method of the described compound formulation of claim 1 may further comprise the steps:
1) produces anti-chicken infectivity bursa of Fabricius virus height according to conventional method and exempt from egg;
2) height of the anti-chicken infectivity bursa of Fabricius virus that step 1) is obtained is exempted from egg and is carried out disinfection, smash egg and isolate yolk, the sterile deionized water that adds 11 times of yolk weight then therein, and adjustment pH value to 6.00, stirred then 5 minutes, after under 4 ℃ condition, leaving standstill 48 hours, extract a supernatant by siphon; After again with precipitate with 8 times of sterile deionized water dilutions, and adjust pH value to 6.0, stirred then 5 minutes, after under 4 ℃ condition, leaving standstill 24 hours, extract the secondary supernatant by siphon; Merge supernatant twice, remove endotoxin, obtain to remove malicious supernatant through adsorption filtration, remove by filter the impurity such as yolk microgranule in the supernatant, again filtered solution is removed low molecular impurity, and be magma by ultrafiltration and concentration, obtain yolk antibody of infectious bursal disease virus trapped substance;
3) with step 2) yolk antibody of infectious bursal disease virus that obtains, extract herbal polysaccharide from described Chinese medicine and mix according to described weight portion ratio and be dissolved in the sterile deionized water, the yolk antibody agar diffusion of chicken infectivity bursa of Fabricius virus in every ml soln is tired 〉=1: 512;
4) solution that step 3) is obtained is sub-packed in cillin bottle and carries out normal freeze-drying, and passes through Co
60The irradiation killing microorganisms promptly obtains infectious bursal disease concentrated freeze-dried yolk antibody composite preparation of the present invention.
7. the described preparation method of claim 6 is characterized in that: step 2) adjustment of described pH value uses 0.1N HCl or 0.1N NaOH to finish.
8. the described preparation method of claim 6 is characterized in that: step 2) described adsorption filtration uses active carbon to finish.
9. the described preparation method of claim 6 is characterized in that: step 2) the described impurity of removing in the supernatant such as yolk microgranule are to finish in centrifugal 10 minutes through the 1000KD membrane filtration or through 10000 rev/mins; It is to finish through 30～100KD membrane filtration that described filtered solution is removed low molecular impurity.
10. the described preparation method of claim 6, it is characterized in that: the described lyophilization of step 4) is solution to be sub-packed in cillin bottle be placed in the vacuum freeze drier, be chilled to-40 ℃ in advance, then at-40 ℃ of following lyophilization 2-8 hours, then per hour heat up about 3 ℃, vacuum drying through 12-18 hour is warming up to 25 ℃, adds a cover then, seals, and keeps 3-5 hour in 25 ℃ of environment.
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