CN105496972A - Preparation method of chicken interferon lyophilized preparation - Google Patents
Preparation method of chicken interferon lyophilized preparation Download PDFInfo
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- CN105496972A CN105496972A CN201510886044.3A CN201510886044A CN105496972A CN 105496972 A CN105496972 A CN 105496972A CN 201510886044 A CN201510886044 A CN 201510886044A CN 105496972 A CN105496972 A CN 105496972A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/19—Cytokines; Lymphokines; Interferons
- A61K38/21—Interferons [IFN]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/555—Interferons [IFN]
Abstract
The invention discloses a preparation method of chicken interferon lyophilized preparation and belongs to the field of animal vaccines. The preparation method of the chicken interferon lyophilized preparation comprises six steps as follows: healthy chicken blood is collected sterilely and is subjected to centrifugation at the temperature of 10 DEG C for 20 min, stratum intermedium leukocytes are sucked, washed twice with NH4Cl with the mass percentage of 0.85% and then subjected to centrifugation, precipitated leukocytes are collected, NH4Cl is removed, an Eagle's MEM mixed culture solution containing 5% of chicken serum is added in a volume ratio of the leukocytes to the culture solution being 1:15 for suspension, the chicken leukocytes are diluted to be in a range from 10*10<7>/ml to 25*10<7>/ml, and a chicken leukocyte suspension is prepared. The preparation method of the chicken interferon lyophilized preparation has the characteristics that the long-acting chicken virus interferon preparation is prepared, chicken are injected with the preparation every 10 days, and the preparation has good effect on chicken virus diseases.
Description
Technical field
The present invention relates to a kind of preparation method of lyophilized preparation, particularly a kind of preparation method of chicken interferon lyophilized preparation.
Background technology
Chicken interferon studies one of animal interferon the earliest, and just there is the relevant report of chicken interferon the eighties in 20th century.The chicken interferon found comprises INF-α, β, ω, δ and INF-γ, has completed gene clone, order-checking and location now.In recent years, there is further research to the inductive condition of chicken interferon and physics and chemistry activity, a large amount of clinical trials has been carried out to chicken LeIF simultaneously.Research shows, it to many viral infectious of chicken as epidemic diarrhea, fowl plague, transmissible gastroenteritis etc., and to bovine viral diarrhoea, gosling plague, lamb diarrhoea etc., all there is significant curative effect, test confirms to there is cross-reactive between the animal such as chicken interferon and cattle and sheep.Not yet there is a kind of interferon formulation being used for the treatment of chicken virus efficiently at present.Existing chicken interferon repeated drug taking necessary every day, expensive, therapeutic effect is not good enough.Development trend from now on finds a kind of efficiently cheap long-acting interferon preparation, can be used in clinical, and for people, be mainly used as immunostimulant and have no side effect, animal is mainly used in the treatment of virosis.The long-acting interferon of chicken is not yet had to put on market at present.We intend setting up chicken long-acting interferon by genetic modification, and a kind of effective preparation of development, 3-5 puts on market.
Interferon (IFN) is the protein that a class has extensive biologic activity, there is the multiple effects such as conditioner body immunity function, antiviral, antitumor, be the important component part of body defending system, chicken interferon is the choice drug for the treatment of chicken viral diseases.Chicken is the main project of modern agricultural development, and it is the large problem involved the interests of the state and the people that development poultry is produced.The subject matter of puzzlement poultry husbandry development is disease problems, and the infectious disease of chicken is the maximum harm to chicken, and the economic loss that China causes because of the infectious disease of chicken every year reaches billions of unit, the serious development hindering poultry husbandry.In the infectious disease of chicken, viral disease is occupied an leading position, and accounts for 85%.Viral infectious (as fowl plague, foot and mouth disease, chicken reproductive and respiratory syndrome etc.) for chicken mainly prevents by vaccine at present.The chicken viral diseases overwhelming majority has available vaccine to prevent.
In recent years, along with the continuous appearance of the new strain of fowl disease poison, variant, the control of China's chicken virus is faced with formidable challenges, current prevention and therapy measure can not control the development of epidemic disease effectively, its harm is day by day serious, therefore in the urgent need to a kind of effectively preventing measure and efficient medicine.To treatment only reliable hyper-immune serum or the Viral interference preparation of the viral infectious of animal.Due to the impact by price and Animal adaptability, there is no the hyper-immune serum that can the supply treatment for chicken viral diseases at present.Although worked out the interferon formulation that may be used for treating viral disease, as polyinosini, chicken interferon etc., there is major defect in these products, and this quasi drugs half-life is short, weak curative effect, price are high.To infected animal necessary repeated drug taking every day, add the cost of medication, often fall flat, make the application of this kind of medicine be subject to serious restriction.Up to the present, also do not have a kind of effectively and the medicine of practicality for the treatment of chicken viral diseases, so develop the important means that efficient antiviral drugs is treatment chicken viral diseases, it is necessary to be that institute is produced in control chicken viral infectious disease development poultry.
Summary of the invention
Goal of the invention of the present invention is: for above-mentioned Problems existing, and provide a kind of and prepare long-acting fowl disease poison interferon formulation, injection in 10 days once, has the preparation method of the chicken interferon lyophilized preparation of good result to chicken viral diseases.
The technical solution used in the present invention is as follows:
The preparation method of a kind of chicken interferon lyophilized preparation of the present invention, comprises the following steps,
Step one: aseptic collection healthy chicken blood, centrifugal 20min under the condition of 10 DEG C, drawing intermediate layer leukocyte, is 0.85%NH with mass fraction
4cl washes twice rear centrifugal, collecting precipitation leukocyte, removing NH
4cl, according to leukocyte: the Eagle`sMEM mixed-culture medium that culture fluid volume ratio 1:15 adds containing 5% chicken serum suspends, and chicken leukocyte count is diluted in 10 × 10
7individual/ml ~ 25 × 10
7between individual/ml, obtained chicken leukocyte suspension;
Step 2: get a chicken leukocyte suspension, adds the chicken LeIF containing 100 active units, is placed in 37 DEG C of shaking bath stir culture 36h;
Step 3: according to the addition of every milliliter of chicken leukocyte suspension 50HANDV II, a certain amount of Avian pneumo-encephalitis virus NDV II is added in cell suspension, pH is regulated to be 7.2, at water-bath constant temperature oscillator 37 DEG C of rotating and culturing 24h, after having cultivated, with 6MHCl, the pH of culture fluid is adjusted to 3.5, acidification 4d at 4 DEG C, then by the NaOH solution of mass body volume concentrations 6%, pH is adjusted to 7.2, centrifugal segregation precipitates, get supernatant, the obtained rough liquid of chicken LeIF;
Step 4: get the rough liquid of a chicken LeIF, by NaOH solution adjust ph to 8.5, under condition of ice bath, according to the rough liquid of interferon: macrogol ester mol ratio 1:5 adds macrogol ester in solution, macrogol ester addition is five parts, be warming up to 4 DEG C, stirring reaction 2h, five parts of macrogol esters are added in solution, be warming up to 12 DEG C, stirring reaction 2h, five parts of macrogol esters are added in solution, be warming up to 25 DEG C, stirring reaction 2h, five parts of macrogol esters are added to solution, be warming up to 36 DEG C, stirring reaction 2h, after complete reaction, add the half Guang glycine cessation reaction of 0.75 Μ, obtain the interferon that macrogol ester is modified,
Owing to have employed technique scheme, adopt macrogol ester to modify interferon, the molecular size of interferon molecule can be expanded, interferon is not easily excreted through renal metabolism, thus extend the life period of interferon in chicken body; The stability of interferon can be improved, prolong half-life simultaneously, reduce immunogenicity, improve the water solublity of interferon.
Step 5: get the interferon that a macrogol ester is modified, get appropriate amount of deionized water as solvent, interferon according to macrogol ester is modified: dialkyl succinylsuccinate ester sodium sulfonate mol ratio 1:1 adds dialkyl succinylsuccinate ester sodium sulfonate in solution, be 2.6 with salt acid for adjusting pH, according to dialkyl succinylsuccinate ester sodium sulfonate: alpha-cyanoacrylate butyl ester mol ratio 1:2.5 adds alpha-cyanoacrylate butyl ester in solution, stir 10min, the pH of solution is regulated to be 5.8 with NaOH again, continue stirring reaction, large nanosphere is removed with 0.8 μm of microporous filter membrane, obtain nanosphere milky colloidal solution,
Owing to have employed technique scheme, by interferon with alpha-cyanoacrylate butyl ester for carrier, preparation becomes nanosphere particle diameter, can agglomerating target administration; Have simultaneously and there is good dispersibility, can peptizaiton after arrival target spot, improve the drug action power of interferon.
Step 6: get a nanosphere milky colloidal solution, be fully immersed in the saline of 0.85% ~ 0.9%, holding temperature is under the condition of 3.2 ~ 3.9 DEG C, according to nanosphere liliquoid: permeability protection liquid volume ratio 1:1.5 adds permeability protection liquid under infrasonic wave oscillating condition, continue to vibrate 30 ~ 45s under infrasonic wave condition, under solution being placed in the environment of vacuum 0.98, set temperature is-12 DEG C, under solution being placed in after freezing 24h the environment of vacuum 0.98, drop in liquid nitrogen, after freezing 3h, solution is taken out under being placed in the environment of vacuum 0.98, set temperature is-20 DEG C, air pressure is recovered to normal pressure according to the speed of 120Pa/min, obtain chicken interferon lyophilized preparation.
Owing to have employed technique scheme, DMSO under the condition of 3.9 DEG C is minimum to cytotoxicity for temperature, but temperature is too low, may form little ice crystal, thus cause mechanical damage to cell in cryopreserving liquid, therefore controls temperature at 3.2 ~ 3.9 DEG C.
Under infrasonic wave oscillating condition, cryoprotective agent can be spread apart rapidly, infiltrate through the inside of liliquoid, interferon molecule is placed in cryoprotective agent completely.
Under vacuum conditions, drop in liquid nitrogen after frozen in advance, intercept air heat exchange, improve freezing efficiency, can lower the temperature rapidly, improve the efficiency of frozen dehydration, thus avoid in refrigerating process, occur little ice crystal, make interferon directly form vitreous body, improve frozen survival rate, realize active harmless lyophilizing.
The preparation method of a kind of chicken interferon lyophilized preparation of the present invention; described permeability protection liquid by 3% ~ 3.8% DMSO; the gelatin of 4.8% ~ 5.6%; the monosaccharide of 0.6% ~ 1.0%, the half Guang glycine of 2.8% ~ 3.2%, the human serum albumin of 1.8% ~ 2.4%; the tween 80 0 of 1.4% ~ 2.0%; the NaCl of 0.5% ~ 0.7%, the manna of 4.9% ~ 5.1%, the ethyl acetate of 20% ~ 34% and the deionized water composition of 44.2% ~ 58.2%.
Owing to have employed technique scheme; human serum albumin is a kind of good lyophilized preparation protection liquid; but adopt human albumin as protection liquid high cost, therefore prepare other components of permeability protection liquid and replace most people's serum albumin greatly to reduce costs.
Adopt the DMSO of 3% ~ 3.8% preparation to become permeability protection liquid, while osmotic protection cytoactive, protectant cytotoxicity is reduced to minimum, reduces the cytotoxic damage that protective agent causes frozen interferon in refrigerating process.
Monosaccharide not only can provide certain nutrition for cellular metabolism, as impermeability protective agent, can ensure to reduce permeability protective agent content, can not impact frozen effect simultaneously.
Normal saline reduces protectant osmotic pressure, reduces and causes protectant permeability to damage in frozen process.
The preparation method of a kind of chicken interferon lyophilized preparation of the present invention; described permeability protection liquid by 3.4% DMSO; the gelatin of 5.2%; the monosaccharide of 0.8%, the half Guang glycine of 3%, the human serum albumin of 2.1%; the tween 80 0 of 1.7%; the NaCl of 0.6%, the manna of 5%, the ethyl acetate of 27% and the deionized water composition of 51.2%.
Owing to have employed technique scheme, this ratio is optimum.
The preparation method of a kind of chicken interferon lyophilized preparation of the present invention, described monosaccharide comprises the glucose of 0.3% ~ 0.5% and the fructose of 0.5% ~ 0.7%.
The preparation method of a kind of chicken interferon lyophilized preparation of the present invention, described infrasonic frequency is 8 ~ 13Hz.
Owing to have employed technique scheme, this frequency and internal organs vibration frequency are close, thus make interferon better adapt to cryopreserving liquid environment, improve protective agent osmotic effect.
The preparation method of a kind of chicken interferon lyophilized preparation of the present invention, the molecular weight of described macrogol ester is 42KDa.
Owing to have employed technique scheme, this molecular weight is optimum.
The preparation method of a kind of chicken interferon lyophilized preparation of the present invention, the concentration of described DMSO is 0.34 ~ 0.37g/mL.
In sum, owing to have employed technique scheme, the invention has the beneficial effects as follows:
1, prepare long-acting fowl disease poison interferon formulation, injection in 10 days once, has good result to chicken viral diseases, improves the stability of interferon, prolong half-life, reduces immunogenicity, improves the water solublity of interferon.
2, by interferon with alpha-cyanoacrylate butyl ester for carrier, preparation becomes nanosphere particle diameter, can agglomerating target administration, has simultaneously and has good dispersibility, can peptizaiton after arrival target spot, improves the drug action power of interferon.
3, preparing other components of permeability protection liquid replaces most people's serum albumin greatly to reduce costs; improve the efficiency of frozen dehydration; thus avoid in refrigerating process, occur little ice crystal; interferon is made directly to form vitreous body; improve frozen survival rate, realize active harmless lyophilizing.
Detailed description of the invention
The present invention is described in detail below.
In order to make the object of invention, technical scheme and advantage clearly understand, below in conjunction with embodiment, the present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.
Embodiment 1
A preparation method for chicken interferon lyophilized preparation, comprises the following steps,
Step one: aseptic collection healthy chicken blood, centrifugal 20min under the condition of 10 DEG C, drawing intermediate layer leukocyte, is 0.85%NH with mass fraction
4cl washes twice rear centrifugal, collecting precipitation leukocyte, removing NH
4cl, according to leukocyte: the Eagle`sMEM mixed-culture medium that culture fluid volume ratio 1:15 adds containing 5% chicken serum suspends, and chicken leukocyte count is diluted in 10 × 10
7individual/ml, obtained chicken leukocyte suspension;
Step 2: get a chicken leukocyte suspension, adds the chicken LeIF containing 100 active units, is placed in 37 DEG C of shaking bath stir culture 36h;
Step 3: according to the addition of every milliliter of chicken leukocyte suspension 50HANDV II, a certain amount of Avian pneumo-encephalitis virus NDV II is added in cell suspension, pH is regulated to be 7.2, at water-bath constant temperature oscillator 37 DEG C of rotating and culturing 24h, after having cultivated, with 6MHCl, the pH of culture fluid is adjusted to 3.5, acidification 4d at 4 DEG C, then by the NaOH solution of mass body volume concentrations 6%, pH is adjusted to 7.2, centrifugal segregation precipitates, get supernatant, the obtained rough liquid of chicken LeIF;
Step 4: get the rough liquid of a chicken LeIF, by NaOH solution adjust ph to 8.5, under condition of ice bath, according to the rough liquid of interferon: macrogol ester mol ratio 1:5 adds macrogol ester in solution, the molecular weight of macrogol ester is for being 42KDa, macrogol ester addition is five parts, be warming up to 4 DEG C, stirring reaction 2h, five parts of macrogol esters are added in solution, be warming up to 12 DEG C, stirring reaction 2h, five parts of macrogol esters are added in solution, be warming up to 25 DEG C, stirring reaction 2h, five parts of macrogol esters are added to solution, be warming up to 36 DEG C, stirring reaction 2h, after complete reaction, add the half Guang glycine cessation reaction of 0.75 Μ, obtain the interferon that macrogol ester is modified,
Step 5: get the interferon that a macrogol ester is modified, get appropriate amount of deionized water as solvent, interferon according to macrogol ester is modified: dialkyl succinylsuccinate ester sodium sulfonate mol ratio 1:1 adds dialkyl succinylsuccinate ester sodium sulfonate in solution, be 2.6 with salt acid for adjusting pH, according to dialkyl succinylsuccinate ester sodium sulfonate: alpha-cyanoacrylate butyl ester mol ratio 1:2.5 adds alpha-cyanoacrylate butyl ester in solution, stir 10min, the pH of solution is regulated to be 5.8 with NaOH again, continue stirring reaction, large nanosphere is removed with 0.8 μm of microporous filter membrane, obtain nanosphere milky colloidal solution,
Step 6: get a nanosphere milky colloidal solution, be fully immersed in the saline of 0.85%, holding temperature is under the condition of 3.2 DEG C, according to nanosphere liliquoid: permeability protection liquid volume ratio 1:1.5 adds permeability protection liquid under infrasonic wave oscillating condition, infrasonic frequency is 8Hz, permeability protection liquid comprises the DMSO of 3%, the concentration of DMSO is 0.34g/mL, the gelatin of 5.6%, the monosaccharide of 0.6%, the half Guang glycine of 3.2%, the human serum albumin of 1.8%, the tween 80 0 of 2.0%, the NaCl of 0.5%, the manna of 5.1%, the ethyl acetate of 20% and the deionized water of 58.2%, wherein monosaccharide comprises the glucose of 0.3% and the fructose of 0.7%, continue to vibrate 30s under infrasonic wave condition, under solution being placed in the environment of vacuum 0.98, set temperature is-12 DEG C, under solution being placed in after freezing 24h the environment of vacuum 0.98, drop in liquid nitrogen, after freezing 3h, solution is taken out under being placed in the environment of vacuum 0.98, set temperature is-20 DEG C, recovers air pressure to normal pressure, obtain chicken interferon lyophilized preparation according to the speed of 120Pa/min.
Embodiment 2
A preparation method for chicken interferon lyophilized preparation, comprises the following steps,
Step one: aseptic collection healthy chicken blood, centrifugal 20min under the condition of 10 DEG C, drawing intermediate layer leukocyte, is 0.85%NH with mass fraction
4cl washes twice rear centrifugal, collecting precipitation leukocyte, removing NH
4cl, according to leukocyte: the Eagle`sMEM mixed-culture medium that culture fluid volume ratio 1:15 adds containing 5% chicken serum suspends, and chicken leukocyte count is diluted in 25 × 10
7individual/ml, obtained chicken leukocyte suspension;
Step 2: get a chicken leukocyte suspension, adds the chicken LeIF containing 100 active units, is placed in 37 DEG C of shaking bath stir culture 36h;
Step 3: according to the addition of every milliliter of chicken leukocyte suspension 50HANDV II, a certain amount of Avian pneumo-encephalitis virus NDV II is added in cell suspension, pH is regulated to be 7.2, at water-bath constant temperature oscillator 37 DEG C of rotating and culturing 24h, after having cultivated, with 6MHCl, the pH of culture fluid is adjusted to 3.5, acidification 4d at 4 DEG C, then by the NaOH solution of mass body volume concentrations 6%, pH is adjusted to 7.2, centrifugal segregation precipitates, get supernatant, the obtained rough liquid of chicken LeIF;
Step 4: get the rough liquid of a chicken LeIF, by NaOH solution adjust ph to 8.5, under condition of ice bath, according to the rough liquid of interferon: macrogol ester mol ratio 1:5 adds macrogol ester in solution, the molecular weight of macrogol ester is for being 42KDa, macrogol ester addition is five parts, be warming up to 4 DEG C, stirring reaction 2h, five parts of macrogol esters are added in solution, be warming up to 12 DEG C, stirring reaction 2h, five parts of macrogol esters are added in solution, be warming up to 25 DEG C, stirring reaction 2h, five parts of macrogol esters are added to solution, be warming up to 36 DEG C, stirring reaction 2h, after complete reaction, add the half Guang glycine cessation reaction of 0.75 Μ, obtain the interferon that macrogol ester is modified,
Step 5: get the interferon that a macrogol ester is modified, get appropriate amount of deionized water as solvent, interferon according to macrogol ester is modified: dialkyl succinylsuccinate ester sodium sulfonate mol ratio 1:1 adds dialkyl succinylsuccinate ester sodium sulfonate in solution, be 2.6 with salt acid for adjusting pH, according to dialkyl succinylsuccinate ester sodium sulfonate: alpha-cyanoacrylate butyl ester mol ratio 1:2.5 adds alpha-cyanoacrylate butyl ester in solution, stir 10min, the pH of solution is regulated to be 5.8 with NaOH again, continue stirring reaction, large nanosphere is removed with 0.8 μm of microporous filter membrane, obtain nanosphere milky colloidal solution,
Step 6: get a nanosphere milky colloidal solution, be fully immersed in the saline of 0.9%, holding temperature is under the condition of 3.9 DEG C, according to nanosphere liliquoid: permeability protection liquid volume ratio 1:1.5 adds permeability protection liquid under infrasonic wave oscillating condition, infrasonic frequency is 13Hz, permeability protection liquid comprises the DMSO of 3.8%, the concentration of DMSO is 0.37g/mL, the gelatin of 4.8%, the monosaccharide of 1.0%, the half Guang glycine of 2.8%, the human serum albumin of 2.4%, the tween 80 0 of 1.4%, the NaCl of 0.7%, the manna of 4.9%, the ethyl acetate of 34% and the deionized water of 44.2%, wherein monosaccharide comprises the glucose of 0.5% and the fructose of 0.5%, continue to vibrate 45s under infrasonic wave condition, under solution being placed in the environment of vacuum 0.98, set temperature is-12 DEG C, under solution being placed in after freezing 24h the environment of vacuum 0.98, drop in liquid nitrogen, after freezing 3h, solution is taken out under being placed in the environment of vacuum 0.98, set temperature is-20 DEG C, recovers air pressure to normal pressure, obtain chicken interferon lyophilized preparation according to the speed of 120Pa/min.
Embodiment 3
A preparation method for chicken interferon lyophilized preparation, comprises the following steps,
Step one: aseptic collection healthy chicken blood, centrifugal 20min under the condition of 10 DEG C, drawing intermediate layer leukocyte, is 0.85%NH with mass fraction
4cl washes twice rear centrifugal, collecting precipitation leukocyte, removing NH
4cl, according to leukocyte: the Eagle`sMEM mixed-culture medium that culture fluid volume ratio 1:15 adds containing 5% chicken serum suspends, and chicken leukocyte count is diluted in 20 × 10
7between individual/ml, obtained chicken leukocyte suspension;
Step 2: get a chicken leukocyte suspension, adds the chicken LeIF containing 100 active units, is placed in 37 DEG C of shaking bath stir culture 36h;
Step 3: according to the addition of every milliliter of chicken leukocyte suspension 50HANDV II, a certain amount of Avian pneumo-encephalitis virus NDV II is added in cell suspension, pH is regulated to be 7.2, at water-bath constant temperature oscillator 37 DEG C of rotating and culturing 24h, after having cultivated, with 6MHCl, the pH of culture fluid is adjusted to 3.5, acidification 4d at 4 DEG C, then by the NaOH solution of mass body volume concentrations 6%, pH is adjusted to 7.2, centrifugal segregation precipitates, get supernatant, the obtained rough liquid of chicken LeIF;
Step 4: get the rough liquid of a chicken LeIF, by NaOH solution adjust ph to 8.5, under condition of ice bath, according to the rough liquid of interferon: macrogol ester mol ratio 1:5 adds macrogol ester in solution, the molecular weight of macrogol ester is for being 42KDa, macrogol ester addition is five parts, be warming up to 4 DEG C, stirring reaction 2h, five parts of macrogol esters are added in solution, be warming up to 12 DEG C, stirring reaction 2h, five parts of macrogol esters are added in solution, be warming up to 25 DEG C, stirring reaction 2h, five parts of macrogol esters are added to solution, be warming up to 36 DEG C, stirring reaction 2h, after complete reaction, add the half Guang glycine cessation reaction of 0.75 Μ, obtain the interferon that macrogol ester is modified,
Step 5: get the interferon that a macrogol ester is modified, get appropriate amount of deionized water as solvent, interferon according to macrogol ester is modified: dialkyl succinylsuccinate ester sodium sulfonate mol ratio 1:1 adds dialkyl succinylsuccinate ester sodium sulfonate in solution, be 2.6 with salt acid for adjusting pH, according to dialkyl succinylsuccinate ester sodium sulfonate: alpha-cyanoacrylate butyl ester mol ratio 1:2.5 adds alpha-cyanoacrylate butyl ester in solution, stir 10min, the pH of solution is regulated to be 5.8 with NaOH again, continue stirring reaction, large nanosphere is removed with 0.8 μm of microporous filter membrane, obtain nanosphere milky colloidal solution,
Step 6: get a nanosphere milky colloidal solution, be fully immersed in the saline of 0.87%, holding temperature is under the condition of 3.7 DEG C, according to nanosphere liliquoid: permeability protection liquid volume ratio 1:1.5 adds permeability protection liquid under infrasonic wave oscillating condition, infrasonic frequency is 11Hz, permeability protection liquid comprises the DMSO of 3.4%, the concentration of DMSO is 0.35g/mL, the gelatin of 5.2%, the monosaccharide of 0.8%, the half Guang glycine of 3%, the human serum albumin of 2.1%, the tween 80 0 of 1.7%, the NaCl of 0.6%, the manna of 5%, the ethyl acetate of 27% and the deionized water of 51.2%, wherein monosaccharide comprises the glucose of 0.4% and the fructose of 0.6%, continue to vibrate 40s under infrasonic wave condition, under solution being placed in the environment of vacuum 0.98, set temperature is-12 DEG C, under solution being placed in after freezing 24h the environment of vacuum 0.98, drop in liquid nitrogen, after freezing 3h, solution is taken out under being placed in the environment of vacuum 0.98, set temperature is-20 DEG C, recovers air pressure to normal pressure, obtain chicken interferon lyophilized preparation according to the speed of 120Pa/min.
Embodiment 4
The present embodiment carries out Performance Detection to the freeze-dried mixed powder of embodiment 1 ~ 3 gained, and result is as shown in table 1.
Room temperature bioactivity test method: freeze-dried mixed dose of embodiment 1 ~ 3 gained is kept in 37 DEG C of incubators, preserve 24 months, respectively 1 week, 2 weeks, January, February, April, August, December, after 16 months, 20 months, 24 months, with agar diffusion method, the interferon measuring each time point institute sample thief is tired change.
Table 1 embodiment 1 ~ 3 obtained freeze-drying agent room temperature bioactivity experimental result
Embodiment 5
The effect that the lyophilized preparation of this experimental example to embodiment 3 prevents and treats fowl plague carries out clinical experiment.
Experimental technique: choose wean chick 90, wherein 10 as the isolated rearing of counteracting toxic substances group, 10 as the isolated rearing of blank group, 60 are divided into 6 groups as a control group, often organize 10, isolated rearing, uses the preparation for treating of comparative example 1 ~ 6 respectively, other 10 as test group, with the preparation for treating of embodiment 1.Feeding manner is free choice feeding normal diet, freely drinks water, and after 24h, get the fowl plague virus liquid after the chicken intramuscular injection inoculation dilution of test group, matched group and counteracting toxic substances group, inoculum concentration is 2ml/; Start immunotherapy targeted autoantibody after infection, intramuscular injection, injection dosage is 0.2ml/kg body weight; Death condition after record 120h, result is as shown in table 2.
The agent of table 2 embodiment 1 obtained freeze-drying is to the prevention effect experimental result of fowl plague
Wherein, the reagent combination of comparative example 1 ~ 6 is as shown in table 4.
The reagent combination of table 4 comparative example 1 ~ 6
As can be seen from Table 4, the agent of embodiment 1 obtained freeze-drying effectively can prevent and treat fowl plague, and effect obviously will to be better than in anti-fowl plague yolk antibody, anti-fowl plague transfer factor, alpha-interferon any one is used alone or wherein two kinds of situations about combinationally using.
Embodiment 6
Field controls therapeutic test and therapeutic test is promoted in field, the results are shown in Table 5,6
Table 5 field controls therapeutic test statistics
Therapeutic test statistics is promoted in table 6 field
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any amendments done within the spirit and principles in the present invention, equivalent replacement and improvement etc., all should be included within protection scope of the present invention.
Claims (7)
1. a preparation method for chicken interferon lyophilized preparation, is characterized in that: comprise the following steps,
Step one: aseptic collection healthy chicken blood, centrifugal 20min under the condition of 10 DEG C, drawing intermediate layer leukocyte, is 0.85%NH with mass fraction
4cl washes twice rear centrifugal, collecting precipitation leukocyte, removing NH
4cl, according to leukocyte: the Eagle`sMEM mixed-culture medium that culture fluid volume ratio 1:15 adds containing 5% chicken serum suspends, and chicken leukocyte count is diluted in 10 × 10
7individual/ml ~ 25 × 10
7between individual/ml, obtained chicken leukocyte suspension;
Step 2: get a chicken leukocyte suspension, adds the chicken LeIF containing 100 active units, is placed in 37 DEG C of shaking bath stir culture 36h;
Step 3: according to the addition of every milliliter of chicken leukocyte suspension 50HANDV II, a certain amount of Avian pneumo-encephalitis virus NDV II is added in cell suspension, pH is regulated to be 7.2, at water-bath constant temperature oscillator 37 DEG C of rotating and culturing 24h, after having cultivated, with 6MHCl, the pH of culture fluid is adjusted to 3.5, acidification 4d at 4 DEG C, then by the NaOH solution of mass body volume concentrations 6%, pH is adjusted to 7.2, centrifugal segregation precipitates, get supernatant, the obtained rough liquid of chicken LeIF;
Step 4: get the rough liquid of a chicken LeIF, by NaOH solution adjust ph to 8.5, under condition of ice bath, according to the rough liquid of interferon: macrogol ester mol ratio 1:5 adds macrogol ester in solution, macrogol ester addition is five parts, be warming up to 4 DEG C, stirring reaction 2h, five parts of macrogol esters are added in solution, be warming up to 12 DEG C, stirring reaction 2h, five parts of macrogol esters are added in solution, be warming up to 25 DEG C, stirring reaction 2h, five parts of macrogol esters are added to solution, be warming up to 36 DEG C, stirring reaction 2h, after complete reaction, add the half Guang glycine cessation reaction of 0.75 Μ, obtain the interferon that macrogol ester is modified,
Step 5: get the interferon that a macrogol ester is modified, get appropriate amount of deionized water as solvent, interferon according to macrogol ester is modified: dialkyl succinylsuccinate ester sodium sulfonate mol ratio 1:1 adds dialkyl succinylsuccinate ester sodium sulfonate in solution, be 2.6 with salt acid for adjusting pH, according to dialkyl succinylsuccinate ester sodium sulfonate: alpha-cyanoacrylate butyl ester mol ratio 1:2.5 adds alpha-cyanoacrylate butyl ester in solution, stir 10min, the pH of solution is regulated to be 5.8 with NaOH again, continue stirring reaction, large nanosphere is removed with 0.8 μm of microporous filter membrane, obtain nanosphere milky colloidal solution,
Step 6: get a nanosphere milky colloidal solution, be fully immersed in the saline of 0.85% ~ 0.9%, holding temperature is under the condition of 3.2 ~ 3.9 DEG C, according to nanosphere liliquoid: permeability protection liquid volume ratio 1:1.5 adds permeability protection liquid under infrasonic wave oscillating condition, continue to vibrate 30 ~ 45s under infrasonic wave condition, under solution being placed in the environment of vacuum 0.98, set temperature is-12 DEG C, under solution being placed in after freezing 24h the environment of vacuum 0.98, drop in liquid nitrogen, after freezing 3h, solution is taken out under being placed in the environment of vacuum 0.98, set temperature is-20 DEG C, air pressure is recovered to normal pressure according to the speed of 120Pa/min, obtain chicken interferon lyophilized preparation.
2. the preparation method of a kind of chicken interferon lyophilized preparation as claimed in claim 1; it is characterized in that: described permeability protection liquid by 3% ~ 3.8% DMSO; the gelatin of 4.8% ~ 5.6%; the monosaccharide of 0.6% ~ 1.0%, the half Guang glycine of 2.8% ~ 3.2%, the human serum albumin of 1.8% ~ 2.4%; the tween 80 0 of 1.4% ~ 2.0%; the NaCl of 0.5% ~ 0.7%, the manna of 4.9% ~ 5.1%, the ethyl acetate of 20% ~ 34% and the deionized water composition of 44.2% ~ 58.2%.
3. the preparation method of a kind of chicken interferon lyophilized preparation as claimed in claim 1 or 2; it is characterized in that: described permeability protection liquid by 3.4% DMSO; the gelatin of 5.2%; the monosaccharide of 0.8%, the half Guang glycine of 3%, the human serum albumin of 2.1%; the tween 80 0 of 1.7%; the NaCl of 0.6%, the manna of 5%, the ethyl acetate of 27% and the deionized water composition of 51.2%.
4. the preparation method of a kind of chicken interferon lyophilized preparation as claimed in claim 3, is characterized in that: described monosaccharide comprises the glucose of 0.3% ~ 0.5% and the fructose of 0.5% ~ 0.7%.
5. the preparation method of a kind of chicken interferon lyophilized preparation as described in claim 1 or 4, is characterized in that: described infrasonic frequency is 8 ~ 13Hz.
6. the preparation method of a kind of chicken interferon lyophilized preparation as claimed in claim 5, is characterized in that: the molecular weight of described macrogol ester is for being 42KDa.
7. the preparation method of a kind of chicken interferon lyophilized preparation as claimed in claim 6, is characterized in that: the concentration of described DMSO is 0.34 ~ 0.37g/mL.
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| CN109125713A (en) * | 2017-06-19 | 2019-01-04 | 杭州俊丰生物工程有限公司 | A kind of chicken interferon-α Pharmaceutical composition |
| CN112075632A (en) * | 2020-09-08 | 2020-12-15 | 深圳市先康达生物科技有限公司 | Stem cell promoter and application thereof |
| CN113827705A (en) * | 2021-09-28 | 2021-12-24 | 河南省健达动保有限公司 | Animal placenta lipopolysaccharide and animal blood interferon compound immunopotentiator |
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Application publication date: 20160420 |