CN101690812B - Concentrated freeze-dried yolk antibody composite preparation for Newcastle disease-infectious bursal disease and preparation process thereof - Google Patents
Concentrated freeze-dried yolk antibody composite preparation for Newcastle disease-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 Newcastle disease-infectious bursal disease, which contains the following components in part by weight: 10.0 to 99.5 parts of Newcastle disease-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 indigowoad root, 50 to 100 parts of fresh rehmannia, 50 to 80 parts of danshen root and 30 to 50 parts of liquorice. The preparation is a freeze-dried preparation prepared by combining the components such as the Newcastle disease-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 Newcastle disease-infectious bursal disease, has the effect of treating the Newcastle disease-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 newcastle disease-infectious bursal disease concentrated freeze-dried yolk antibody composite preparation, and preparation method thereof.
Background technology
Newcastle disease-infectious bursal disease is the formidable enemy of poultry husbandry.The infection medicine that is applied to prevent and treat newcastle disease-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 type 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 newcastle disease-infectious bursal disease.But their medicines are residual big, can pass through food source contact scar, cause human diseases.
(2) chemosynthesis medicine
This type 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 auxiliary treatment newcastle disease-infectious bursal disease, but it is big to have toxic and side effects; Be prone to residue in the body, cause liver, kidney etc. to organize irreversible infringement (seeing Chinese Pharmacopoeia); 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 newcastle disease-infectious bursal disease.
More than two types of 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 newcastle disease-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 newcastle disease, 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 type anti-infectives is from special chicken yolk antibody.Their speed of actions are fast, and curative effect is accurate, high specificity, but because only to newcastle disease-infectious bursal disease virus, invalid to mixed infection and secondary infection, have to sometimes with above-mentioned three types of medicine auxiliary treatment; Secondly, present most yolk antibodies are that direct goods are that original high-immunity yolk goods claim that also rough yolk antibody (presses Jiang Yanfen such as breeding enterprise oneself, Wang Yaping; Pan Rui etc.: the development and the application of anti-avian influenza, newcastle, fabricius bursa three high immunity yolk antibodies, Gansu Agriculture University,'s journal, 2004; 39 < 1 >, p57, method preparation); 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 the extraction freeze-dry process at present, have been refined into yolk antibody extraction freeze-dried products and (pressed Wang Li, Zhou Yunlu, Hu Xiaomiao, Zhou Xueli etc. such as breeding enterprise oneself: infectious bursal disease; The Preliminary development and the effect of newcastle duplex yolk antibody lyophilized formulations are pacified little agricultural sciences, 1999; 27 < 5 >: p509, method preparation), 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., owing to cost an arm and a leg, be difficult to accepted extensively, as for more difficult carrying out in newcastle disease-infectious bursal disease by clinical.
Based on above-mentioned technical background, the present invention is directed to newcastle disease-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 newcastle disease-infectious bursal disease yolk antibody composite preparation; Overcome the shortcoming in the prior art; Reduce cost with 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 method for preparing of said compound formulation is provided, further reduces production costs, enhance productivity.
Above-mentioned purpose of the present invention realizes through following technical scheme:
A kind of newcastle disease-infectious bursal disease concentrated freeze-dried yolk antibody composite preparation is provided, and it contains the component of following parts by weight:
(1) yolk antibody of Newcastle disease-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 Isatidis 10-50, Radix Rehmanniae 50-100, Radix Salviae Miltiorrhizae 50-80, Radix Glycyrrhizae 30-50.
Said 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 albumin 1.0-6.0 or the sucrose 1.0-3.0.
Described yolk antibody of Newcastle disease-infectious bursal disease virus can be in the prior art through the newcastle disease-ibdv vaccine height produced of the laying hen of reinforced immunological exempt to purify in 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 Newcastle disease-infectious bursal disease virus of the present invention preferably makes according to following method:
(1) produce anti-newcastle disease-infectious bursal disease 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 newcastle disease-ibdv vaccine at every turn, injection volume is respectively 0.5ml, 1.5ml and 2.0ml, each week age at interval;
(1.3) accomplish the 3rd immunity after 21 days; Collect egg; Yolk antibody in the yolk is carried out immunology detection; The yolk antibody blood clotting of newcastle disease suppresses to tire and reaches 1: 512 yolk antibody agar diffusion above, infectious bursal disease virus simultaneously and tire that to reach 1: 32 above egg be that newcastle disease, infectious bursal disease virus height are exempted from egg, and collection storage is subsequent use 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 Newcastle disease-infectious bursal disease virus
The height of the anti-newcastle disease-infectious bursal disease virus that (2.1) 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 through siphon; After again with precipitate with 8 times of sterile deionized water dilutions, and adjustment pH value to 6.0 stirred 5 minutes then, after under 4 ℃ condition, leaving standstill 24 hours, extracted the secondary supernatant through siphon; Merge supernatant twice, remove endotoxin through adsorption filtration, it is subsequent use to obtain to remove malicious supernatant; Wherein, the adjustment of said pH value can use 0.1N HCl or 0.1N NaOH to accomplish; Said adsorption filtration can use active carbon to accomplish, and also can use known any method to accomplish;
(2.3) the malicious supernatant that removes that (2.2) step is obtained is removed the impurity such as yolk microgranule in the supernatant; Again filtered solution is removed low molecular impurity, and, be yolk antibody of Newcastle disease-infectious bursal disease virus trapped substance simmer down to magma; Wherein, the said impurity of removing in the supernatant such as yolk microgranule can pass through through the 1000KD membrane filtration or through 10000 rev/mins of completion in centrifugal 10 minutes; Said filtered solution is removed low molecular impurity can be through accomplishing through 30～100KD membrane filtration.
The method for distilling of described polysaccharide can be existing arbitrarily method, preferably extracts according to following method 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 through the hot reflux concentration technology to obtain concentrated medicament;
B. with the concentrated medicament of step a preparation through 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%, and hold over night discards the ethanol supernatant; Precipitate is removed ethanol; Be dissolved in water then and through the 100KD membrane filtration, remove macromole impurity such as foreigh protein removing, endotoxin, collect filtered solution and filter through 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, hold 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 method for preparing of described compound formulation, may further comprise the steps:
1) produces anti-newcastle disease-infectious bursal disease virus height according to conventional method and exempt from egg;
The height of the anti-newcastle disease-infectious bursal disease virus that 2) 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 5 minutes then; After under 4 ℃ condition, leaving standstill 48 hours, extract a supernatant through siphon; After again with precipitate with 8 times of sterile deionized water dilutions, and adjustment pH value to 6.0 stirred 5 minutes then, after under 4 ℃ condition, leaving standstill 24 hours, extracted the secondary supernatant through 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 through ultrafiltration and concentration, obtain yolk antibody of Newcastle disease-infectious bursal disease virus trapped substance;
3) with step 2) yolk antibody of Newcastle disease-infectious bursal disease virus that obtains, extract herbal polysaccharide from said Chinese medicine and mix according to described weight portion ratio and be dissolved in the sterile deionized water; Make the yolk antibody blood clotting of newcastle disease in every ml soln suppress to tire >=1: 2048, the yolk antibody agar diffusion of infectious bursal disease virus tires >=and 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 newcastle disease of the present invention-infectious bursal disease concentrated freeze-dried yolk antibody composite preparation.
Wherein, step 2) adjustment of described pH value preferably uses 0.1N HCl or 0.1N NaOH to accomplish.
Step 2) described adsorption filtration preferably uses active carbon to accomplish.
Step 2) described impurity of removing in the supernatant such as yolk microgranule were preferably accomplished through the 1000KD membrane filtration or through 10000 rev/mins in centrifugal 10 minutes; Said filtered solution is removed low molecular impurity and is preferably accomplished through 30～100KD membrane filtration.
The described lyophilization of step 4) preferably is sub-packed in cillin bottle with solution and is placed in the vacuum freeze drier; Pre-cooling is to-40 ℃; At-40 ℃ of following lyophilization 2-8 hours, then per hour heat up about 3 ℃ then, the 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 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, needs to lean on gravity natural sedimentation yolk microgranule to reach and reclaim 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.The present invention is from producing reality; 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 method for preparing 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 property of yolk antibody of Newcastle disease-infectious bursal disease virus and polysaccharide and they are combined the compound freeze-dried formulation of processing 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 newcastle disease-infectious bursal disease 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 effectively, prevention effect more rapidly, concrete prevention effect can be referring to following clinical trial:
Test the clinical application effect experiment of 1. newcastle disease of the present invention-infectious bursal disease concentrated freeze-dried yolk antibody composite preparations
[being tried disease chicken situation]
Tried the disease chicken and be to make a definite diagnosis suffer from newcastle-chickling of the 14-84 age in days that infectious bursal disease virus infects with breed chicken, do not have the kind restriction.
[medical diagnosis on disease standard]
With sick chicken glandular stomach thelorrhagia, muscular stomach cutin membrane bleed bottom, small intestinal have island shape hemorrhagic necrosis and fabricius bursa enlargement, hemorrhage be principal character, hold concurrently following any two or multinomial symptom:
1, the purplish red blackout of sick chicken cockscomb, fervescence, dyspnea gets rid of a rhinorrhea, lassitude, anorexia is trembled, dehydration, highly collapse;
2, wryneck is nodded, the sagging lower limb paralysis of wing, and intermittent diarrhea is drawn watery white feces or is drawn the yellow green loose stool;
3, the whole body serous coat is hemorrhage, and subcutaneous and chest muscle and lower limb flesh are hemorrhage, and lymphonodi caecales is hemorrhage, hemorrhage of rectum, renal tubules urate deposition.
[laboratory observation method]
11825 chickens that brood of making a definite diagnosis newcastle disease and infectious bursal disease mixed 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; The antibody preparation method for preparing is referring to " Wang Li, Zhou Yunlu, Hu Xiaomiao, Zhou Xueli etc.: infectious bursal disease; the Preliminary development and the effect of newcastle duplex yolk antibody lyophilized formulations; pacify little agricultural sciences, 1999,27 < 5 >: p509 ";
C group---rough yolk antibody treatment group; The antibody preparation method for preparing is referring to " Jiang Yanfen, Wang Yaping, Pan Rui etc.: the development and the application of anti-avian influenza, newcastle, fabricius bursa three high immunity yolk antibodies, Gansu Agriculture University,'s journal, 2004,39 < 1 >, p57 ";
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 blood clotting of the newcastle disease of every plumage part (ND) suppresses (HI) tire 1: 512, the yolk antibody agar diffusion (AGP) of infectious bursal disease virus (IBDV) and tired 1: 16.
[healing evaluation criterion]
1, the hemorrhage pathological changes of fabricius bursa enlargement of morbidity chickling disappears, is white powder;
Hemorrhage pathological changes such as 2, glandular stomach thelorrhagia, muscular stomach cutin membrane bleed bottom, small intestinal have island shape hemorrhagic necrosis, the whole body serous coat is hemorrhage, lymphonodi caecales is hemorrhage, hemorrhage of rectum disappear;
3, subcutaneous and chest muscle and lower limb flesh are hemorrhage, and renal tubules urate deposition pathological changes disappears;
4, cockscomb gloss glow, the difficulty that breathes no more with get rid of symptom such as a rhinorrhea, the nervous symptoms that wryneck is nodded with the sagging lower limb paralysis of wing disappears, lassitude, anorexia, intermittent diarrhea, tremble, dewatering symptom disappears.
[laboratory observation result]
Therapeutic outcome shows, uses that the cure rate to sick chicken is 94.3% behind the preparation of the present invention, and the yolk antibody extraction freeze-dried products that uses prior art is 86.6% to the cure rate of sick chicken, and the rough yolk antibody of prior art is to the cure rate of sick chicken only 80.9%.Respectively three experimental group cure rates are carried out t check in twos through the applying biological statistics; Show that fully all there are utmost point significant difference (u＞2.58 each other in they; P＜0.01), therefore proved that the therapeutic effect of compound formulation of the present invention is superior to existing other preparation.Experimental result is seen table 1.
Table 1. compound formulation of the present invention clinical application effect compared with prior art
Annotate: " * * " all represent and organizes data with other of index and relatively have highly significant property difference in the table.
2) antibody is effectively protected, and promotes the efficacy stability performance
On the basis that molecular biology, modern molecular immunology and Chinese medicine combine in modern times; Utilize the intermolecular hydrogen bonding structural theory; After mixing compositions such as yolk antibody of Newcastle disease-infectious bursal disease virus and polysaccharide 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 Newcastle disease-infectious bursal disease virus is peripheral, thereby be developed into a kind of newcastle disease-infectious bursal disease concentrated freeze-dried yolk antibody composite preparation through hydrogen bond.Compositions such as polysaccharide are the one type of 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 Newcastle disease-infectious bursal disease virus surface.Its existence has improved yolk antibody of Newcastle disease-infectious bursal disease virus opposing high temperature, ultraviolet etc. and has induced degeneration; Surface structure, conformation and the function of yolk antibody of Newcastle disease-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 experiment of 2. newcastle disease of the present invention-infectious bursal disease concentrated freeze-dried yolk antibody composite preparations
We have done this experiment for the heat-resistant quality of identifying preparation of the present invention.
The preparation (A) and the yolk antibody of the prior art of the embodiment of the invention 1 are extracted freeze-dried products (B), rough yolk antibody (C); Place 37 ℃ of environment; Preserve 1 week, 2 weeks, 3 weeks, January, December respectively, after 24 months, 36 months, 48 months; With ND-HI and IBDV-AGP algoscopy, the yolk antibody blood clotting inhibition (HI) of measuring newcastle disease (ND) is respectively tired, the yolk antibody agar diffusion (AGP) of infectious bursal disease virus (IBDV) is tired, and the result sees table 2,3.
The yolk antibody blood clotting of the newcastle disease of the different preparations of the every plumage part of table 2. suppresses the variation that (HI) tires
The variation that the yolk antibody agar diffusion (AGP) of the infectious bursal disease virus of the different preparations of the every plumage part of table 3. is tired
Learn by last two tables; Preparation of the present invention has very strong heat resistanceheat resistant performance; To suppress (HI) to tire still be 1: 512, the yolk antibody agar diffusion (AGP) of infectious bursal disease virus (IBDV) is tired still be 1: 16 to the yolk antibody blood clotting of the newcastle disease (ND) of the every plumage part of this series products when in 37 ℃ environment, preserving 36 months; Be qualified products; And the yolk antibody extraction freeze-dried products of the prior art that uses in the experiment can only be stored 12 months in this environment, and rough yolk antibody also can only store for 3 weeks.So, the advantage that preparation of the present invention has heat resistanceheat resistant storage, convenient transportation and uses, overcome present other like product storage, transportation and use aspect deficiency.
3) do not produce drug resistance, can not pollute, use cost is low
Owing to 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 to the people yet; 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) method for preparing of the present invention can further reduce production costs, enhance productivity under the prerequisite of the therapeutic effect that guarantees compound formulation.
In the method for preparing 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 with 0.1N HCl or 0.1N NaOH solution adjustment pH value to 5.2, under 4 ℃ condition, leaves standstill 6 hours; And the inventive method is diluted 11 times with sterile deionized water with high-immunity yolk liquid, and with 0.1N HCl or 0.1N NaOH solution adjustment pH value to 6.00, under 4 ℃ condition, leaves standstill 48 hours.Facts have proved that in production in enormous quantities, lean under the gravity natural sedimentation situation, the former lacks 25-35% than the latter at the antibody yield in the same environment.Therefore, method for preparing of the present invention can further reduce production costs, enhance productivity under the prerequisite of the therapeutic effect that guarantees compound formulation.
The specific embodiment
Further specify content of the present invention through specific embodiment below.
Embodiment 1. kinds of traditional Chinese medicines glycan newcastle disease-infectious bursal disease concentrated freeze-dried yolk antibody composite preparations
1. composition:
99.5 kilograms of yolk antibody of Newcastle disease-infectious bursal disease virus;
0.5 kilogram of herbal polysaccharide;
Said herbal polysaccharide extracts from following parts by weight of Chinese traditional medicine material compositions: Radix Isatidis 10, Radix Rehmanniae 50, Radix Salviae Miltiorrhizae 50, Radix Glycyrrhizae 30.
2. preparation:
(1), the height of preparation anti-newcastle disease-infectious bursal disease virus 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 newcastle disease-ibdv vaccine at every turn, injection volume is respectively 0.5ml, 1.5ml and 2.0ml, each week age at interval;
(1.3) accomplish the 3rd immunity after 21 days; Collect egg; Yolk antibody in the yolk is carried out immunology detection; The yolk antibody blood clotting of newcastle disease suppresses to tire and reaches 1: 512 yolk antibody agar diffusion above, infectious bursal disease virus simultaneously and tire that to reach 1: 32 above egg be that newcastle disease, infectious bursal disease virus height are exempted from egg, and collection storage is subsequent use 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 Newcastle disease-infectious bursal disease virus
The height of the anti-newcastle disease-infectious bursal disease virus that (2.1) 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 through siphon; After again with precipitate with 8 times of sterile deionized water dilutions, and use 0.1N HCl or 0.1N NaOH adjustment pH value to 6.0, stirred then 5 minutes, after under 4 ℃ condition, leaving standstill 24 hours, extract the secondary supernatant through siphon; Merge supernatant twice, remove by filter endotoxin through activated carbon adsorption, it is subsequent use to obtain to remove malicious supernatant;
(2.3) with (2.2) step obtain remove 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 Newcastle disease-infectious bursal disease virus trapped substance simmer down to magma;
(3) preparation polysaccharide
(3.1) with Radix Isatidis, Radix Rehmanniae, Radix Salviae Miltiorrhizae and Radix Glycyrrhizae according to 10 parts of Radix Isatidis, 50 parts of Radix Rehmanniae: the weight portion of 50 parts of Radix Salviae Miltiorrhizaes, 30 parts in Radix Glycyrrhizae gets material, chopping, clean the back mixes; 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 through 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%, and hold over night discards the ethanol supernatant; Precipitate is removed ethanol; Be dissolved in water then and through the 100KD membrane filtration, remove macromole impurity such as foreigh protein removing, endotoxin, collect filtered solution and filter through 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, hold 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 newcastle disease-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 of 0.5 kilogram of step (3) preparation are mixed; Be dissolved in the sterile deionized water; Making the yolk antibody blood clotting of newcastle disease in every ml soln (ND) suppress (HI) tires and is >=1: 2048; The yolk antibody agar diffusion (AGP) of infectious bursal disease virus (IBDV) is tired and is >=1: 512, is sub-packed in cillin bottle then, places vacuum freeze drier dry.The vacuum freezing condition: 1, pre-cooling is to-40 ℃; 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] these article are faint yellow, are 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] advances by following method
Press the dated plumage part of label 1.IBDV neutralization index is measured, be diluted to 1 plumage part/ml with normal saline, measure antibody titer by " Chinese veterinary drug allusion quotation " note neutralization test sessile antibody virus dilution method, neutralization index should be 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 is 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, passive immunotherapy is carried out in every intramuscular injection originally article 1 plumage part; 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.
4. get 30 of the healthy susceptible chickens in age in 3-6 week, be divided into 3 groups at random, 10 every group.The 1st group is the normal healthy controls group, does not inject any medicine, isolated rearing separately.2nd, 3 groups of every chicken difference intramuscular injection ND Beijing virulent strain 10
5ELD
50After 24 hours, passive immunotherapy is carried out in the 2nd group of every intramuscular injection originally article 1 plumage part; The 3rd group of injecting normal saline.Observe every group of morbidity, death condition to the 14 days.
The 1st group of duration of test chicken of result of determination should all be good for and be lived.The 3rd group is the counteracting toxic substances matched group, should be behind counteracting toxic substances morbidity in 24-48 hour, after 48 hours beginning dead, all morbidities in 72 hours, all dead in 14 days.The 2nd group is early infection treatment group, injects behind this preparation to begin to recover normal in 24-48 hour, and observation finishes to survive 9 at least, and it is qualified to be judged to.
5.ND-HI the yolk antibody blood clotting of measuring newcastle disease (ND) with IBDV-AGP suppress (HI) tire should be >=1: 512, the yolk antibody agar diffusion (AGP) of infectious bursal disease virus (IBDV) tires and should be >=1: 16.
[effect and purposes] is used for early stage urgent prevention and treats newcastle disease, infectious bursal disease.
[usage and consumption]
1, every 1 plumage part below prevention consumption 25 ages in days, 25-35 age in days 1.5 plumage parts, 35-45 age in days 2 plumage parts, the above 2.5-4 plumage of 45 ages in days part.But duplicate injection in case of necessity 2-3 time.
2, every 2.0 plumage part below therapeutic dose 35 ages in days, every 2.5-3.0 plumage part more than 35 ages in days; When being in a bad way, multiplicable consumption also can 1 time on the 1st, logotype 2-3 day.
[points for attention]
1. with behind these article passive immunity, must not carry out the immunity of newcastle disease attenuated live vaccines and infectious bursal disease live-vaccine in 5 days.
2. these article are sure not heat and are used use immediately after diluting with normal saline.
3. these article are oral invalid.
These article 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 newcastle disease-infectious bursal disease concentrated freeze-dried yolk antibody composite preparations
1. composition:
65 kilograms of yolk antibody of Newcastle disease-infectious bursal disease virus;
6 kilograms of herbal polysaccharides;
10 kilograms of sucrose;
2. preparation:
The method for preparing 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 Isatidis, 20 parts of Radix Rehmanniae, 10 parts in Radix Glycyrrhizae;
The building-up process of compound formulation and method be with embodiment 1, just becomes 10 kilograms of 65 kilograms of yolk antibody of Newcastle disease-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 newcastle disease-infectious bursal disease concentrated freeze-dried yolk antibody composite preparation
1. composition:
40 kilograms of yolk antibody of Newcastle disease-infectious bursal disease virus;
20 kilograms of herbal polysaccharides;
6 kilograms of albumin;
2. preparation:
The method for preparing 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 Radix Glycyrrhizae, 30 parts of Radix Salviae Miltiorrhizaes;
The building-up process of compound formulation and method be with embodiment 1, just becomes 6 kilograms of 40 kilograms of yolk antibody of Newcastle disease-infectious bursal disease virus, 20 kilograms of polysaccharide and albumin at synthesis material.
3. quality standard:
With embodiment 1.
Embodiment 4. single medicinal material glycan newcastle disease-infectious bursal disease concentrated freeze-dried yolk antibody composite preparations
1. composition:
10 kilograms of yolk antibody of Newcastle disease-infectious bursal disease virus;
5 kilograms of herbal polysaccharides;
1 kilogram of albumin;
1 kilogram of sucrose;
2. preparation:
The method for preparing of yolk antibody and polysaccharide is with embodiment 1, and just the extraction of polysaccharide source becomes Radix Rehmanniae;
The building-up process of compound formulation and method be with embodiment 1, just becomes 1 kilogram of 10 kilograms of yolk antibody of Newcastle disease-infectious bursal disease virus, 5 kilograms of polysaccharide, 1 kilogram of albumin and sucrose at synthesis material.
3. quality standard:
With embodiment 1.
Claims (10)
1. newcastle disease-infectious bursal disease concentrated freeze-dried yolk antibody composite preparation is characterized in that it contains the component of following parts by weight:
(1) yolk antibody of Newcastle disease-infectious bursal disease virus 10.0-99.5;
(2) herbal polysaccharide 0.5-20.0;
Described herbal polysaccharide extracts from following parts by weight of Chinese traditional medicine compositions: Radix Isatidis 10-50, Radix Rehmanniae 50-100, Radix Salviae Miltiorrhizae 50-80 and Radix Glycyrrhizae 30-50.
2. the described compound formulation of claim 1 is characterized in that: further comprise 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 albumin 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 Newcastle disease-infectious bursal disease virus makes according to following method:
(1) produces anti-newcastle disease-infectious bursal disease virus height according to conventional method and exempt from egg;
(2) purification of yolk antibody of Newcastle disease-infectious bursal disease virus
The height of the anti-newcastle disease-infectious bursal disease virus that (2.1) 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 through siphon; After again with precipitate with 8 times of sterile deionized water dilutions, and adjustment pH value to 6.0 stirred 5 minutes then, after under 4 ℃ condition, leaving standstill 24 hours, extracted the secondary supernatant through siphon; Merge supernatant twice, remove endotoxin through adsorption filtration, it is subsequent use to obtain to remove malicious supernatant;
(2.3) the malicious supernatant that removes that (2.2) step is obtained is removed the impurity such as yolk microgranule in the supernatant; Again filtered solution is removed low molecular impurity, and be magma through ultrafiltration and concentration, be yolk antibody of Newcastle disease-infectious bursal disease virus trapped substance.
5. the described compound formulation of claim 1 is characterized in that, described polysaccharide extracts according to following method 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 through the hot reflux concentration technology to obtain concentrated medicament;
B. with the concentrated medicament of step a preparation through 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 95% ethanol of 4-8 times of volume, and hold over night discards the ethanol supernatant; Precipitate is removed ethanol; Be dissolved in water then and through the 100KD membrane filtration, remove macromole impurity such as foreigh protein removing, endotoxin, collect filtered solution and filter through 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 amount 99% ethanol precipitation of 5 times of volumes, stir, hold 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 method for preparing of the described compound formulation of claim 1 may further comprise the steps:
1) produces anti-newcastle disease-infectious bursal disease virus height according to conventional method and exempt from egg;
The height of the anti-newcastle disease-infectious bursal disease virus that 2) 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 5 minutes then; After under 4 ℃ condition, leaving standstill 48 hours, extract a supernatant through siphon; After again with precipitate with 8 times of sterile deionized water dilutions, and adjustment pH value to 6.0 stirred 5 minutes then, after under 4 ℃ condition, leaving standstill 24 hours, extracted the secondary supernatant through 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 through ultrafiltration and concentration, obtain yolk antibody of Newcastle disease-infectious bursal disease virus trapped substance;
3) with step 2) yolk antibody of Newcastle disease-infectious bursal disease virus that obtains, extract herbal polysaccharide from said Chinese medicine and mix according to described weight portion ratio and be dissolved in the sterile deionized water; Make the yolk antibody blood clotting of newcastle disease in every ml soln suppress to tire >=1: 2048, the yolk antibody agar diffusion of infectious bursal disease virus tires >=and 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 newcastle disease of the present invention-infectious bursal disease concentrated freeze-dried yolk antibody composite preparation.
7. the described method for preparing of claim 6 is characterized in that: step 2) adjustment of described pH value uses 0.1N HCl or 0.1N NaOH to accomplish.
8. the described method for preparing of claim 6 is characterized in that: step 2) completion of described adsorption filtration use active carbon.
9. the described method for preparing of claim 6 is characterized in that: step 2) the described impurity of removing in the supernatant such as yolk microgranule are to accomplish in centrifugal 10 minutes through the 1000KD membrane filtration or through 10000 rev/mins; It is to accomplish through 30～100KD membrane filtration that said filtered solution is removed low molecular impurity.
10. the described method for preparing 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, and pre-cooling is to-40 ℃, 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 in 25 ℃ of environment, keeps 3-5 hour.
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| CN104208678B (en) * | 2013-09-30 | 2016-04-20 | 郑州后羿制药有限公司 | A kind of antivirus veterinary composition, lyophilized powder, preparation method and application |
| CN105505888A (en) * | 2015-12-29 | 2016-04-20 | 浙江美保龙生物技术有限公司 | Infectious bursa disease virus diluent and preparation method thereof |
| CN106310253A (en) * | 2016-08-29 | 2017-01-11 | 青岛润达生物科技有限公司 | Composite product of ganoderma lucidum polysaccharides and egg yolk antibody |
| CN109010823B (en) * | 2018-08-27 | 2022-03-18 | 广州汇高生物科技有限公司 | Composition for resisting various pathogenic bacteria, preparation process and application thereof, spray and preparation process thereof |
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| CN1380098A (en) * | 2002-05-20 | 2002-11-20 | 张勇飞 | Anti-infection compound preparation and its preparation method |
| CN101057971A (en) * | 2006-04-20 | 2007-10-24 | 河南农业大学 | Medicine for controlling livestock-poultry virus infectious disease |
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| CN1380098A (en) * | 2002-05-20 | 2002-11-20 | 张勇飞 | Anti-infection compound preparation and its preparation method |
| CN101057971A (en) * | 2006-04-20 | 2007-10-24 | 河南农业大学 | Medicine for controlling livestock-poultry virus infectious disease |
| CN101214378A (en) * | 2008-01-14 | 2008-07-09 | 中国农业大学 | Yam polysaccharide complex immunity globulin anti-infectious preparations and preparation thereof |
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