CN101417122B - Recombination chicken alpha-interferon injection and preparation method thereof - Google Patents
Recombination chicken alpha-interferon injection and preparation method thereof Download PDFInfo
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- CN101417122B CN101417122B CN2008102379712A CN200810237971A CN101417122B CN 101417122 B CN101417122 B CN 101417122B CN 2008102379712 A CN2008102379712 A CN 2008102379712A CN 200810237971 A CN200810237971 A CN 200810237971A CN 101417122 B CN101417122 B CN 101417122B
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Abstract
The invention relates to a recombinant chicken Alpha-interferon injection and a preparation method, belonging to the technical field of animal medicine production. The recombinant chicken Alpha-interferon injection is characterized by consisting of the following components with the weight percentage: 1 to 5 percent of astragalus polyose, 0.01 to 0.05 percent of phenylformic acid, 5 to 20 percent of original liquid of chicken Alpha-interferon, 0.1 to 0.5 percent of albumin and the rest is injection water. The injection water is a solvent; and the size of a single interferon product is 10ml. The product can be used for saving the medicine cost during the culturing process by 10 to 35 percent; moreover, the product has very good effects on diarrhea and enteritis caused by chicken rotavirus; simultaneously the death rate caused by the descending of resistance caused by immunological suppression during the chickling period can be reduced.
Description
Technical field
The present invention relates to injection of a kind of recombination chicken alpha-interferon and preparation method thereof, belong to veterinary medicine thing production technical field.
Background technology
(Interferon IFN) is one of the important cytokine of vertebrate immune system to interferon, and it supports in multiple pathogenic microorganism, antitumor and the immunomodulating at body and plays an important role.The interferon of chicken no matter is natural or recombinant interferon all has stronger antiviral ability.Wherein the ability of chicken alpha-interferon resisiting influenza virus and vesicular stomatitis virus is the strongest, to Avian pneumo-encephalitis virus, and infectious bronchitis virus, Marek poison etc. all has stronger inhibition effect.
Publication number is the Chinese patent application of CN1594568, and the production method and the recombinant vector thereof that disclose a kind of recombination chicken alpha-interferon belong to the genetically engineered biological goods that obtain with molecular biology method.It is incorporated into ad-hoc location on the yeast with the gene order of chicken alpha-interferon by electric transform mode.Adopt the pichia yeast expression system expression alien gene, the purity of expressing foreign protein is higher than 70%, finds to suppress 100-1000TCID fully through 65636 times of dilutions
50The attack of vesicular stomatitis virus.Chicken alpha interferon to the transcribing of VSV and bird flu virus, that the translating phase is suppressed ability is especially obvious, Avian pneumo-encephalitis virus, infectious bursa of Fabricius virus etc. also all there is stronger inhibitory action, and also have powerful antitumor, antiproliferative effect, the Marek's disease virus of chicken is also had stronger inhibitory action.Though this patent document preferably resolves the preparation problem of recombined chicken alpha interferon, but in the use of concrete recombination chicken alpha-interferon, do not find suitable consumption, when use is excessive, can cause the body immune system disorder, when using deficiency, do not reach excellent prevention and therapeutic effect, to such an extent as to the outburst again of disease, and this patent can't be carried out normal large-scale production and application, can only be the laboratory product, have product limitation and the scale of application limitation.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, injection of a kind of recombination chicken alpha-interferon and preparation method thereof is provided.
A kind of injection of recombination chicken alpha-interferon is characterized in that component is as follows, all is weight percentage:
Astragalus polysaccharides 1%-5%, benzoic acid 0.1 ‰-0.5 ‰, chicken alpha-interferon stock solution 5%-20%, albumin 0.1%-0.5%, surplus is a water for injection.Water for injection is solvent, and single interferon product volume is 10ml.
The pH=5.0-6.0 of described recombination chicken alpha-interferon injection.
Described albumin is Sanguis Gallus domesticus albumin or Sanguis Bovis seu Bubali albumin.
It is the product that the disclosed preparation method of Chinese patent application of CN1594568 is prepared acquisition that described chicken alpha-interferon stock solution can be with reference to publication number.
A kind of preparation method of above-mentioned recombination chicken alpha-interferon injection is characterized in that, step is as follows:
(1) with astragalus polysaccharides, benzoic acid, albumin mixing, be dissolved in water for injection and autoclaving then and handle, get injection solvent;
(2) injection solvent that makes in the step (1) is mixed with chicken alpha-interferon stock solution and adds residue water for injection, injection stock solution;
(3) the injection stock solution that step (2) is made is regulated pH to 5.0-6.0, gets the injection of recombination chicken alpha-interferon.
(4) step (3) is made to such an extent that the injection of recombination chicken alpha-interferon is distributed into bottle under aseptic condition.
Described autoclaving is handled and can be adopted damp and hot autoclaving sterilization.
Regulating pH in the described step (3) adopts the 100mM phosphate buffered solution to regulate.
The vigor of every bottle of injection is 2500～8,000 ten thousand unit of activity after the middle packing of described step (4).Unit of activity (U) is meant can suppress 50% cytopathic interferon greatest dilution and is defined as an active unit.
Using method:
Prevention disease: inject each 1-5 ten thousand unit of activity/plumage, every day 1 time, 2-3 days
Treat common disease: inject each 2-8 ten thousand unit of activity/plumage, every day 1 time, 2-5 days continuously
Treatment serious symptom: inject each 4-12 ten thousand unit of activity/plumage, every day 1 time, 3-5 days continuously
Beneficial effect of the present invention is as follows:
The present invention is directed to newcastle disease, the infectious bronchitis disease has significant curative effect, the experiment proved that, newcastle disease, the sick cure rate of infectious bronchitis are reached 70%-90%, be higher than relevant similar fowl medicine in the past, and owing to adopt biological reagent, can effectively avoid chemosynthesis medicine to suffering from the pollution of Fowl meat matter, improve the birds edible quality.
This product can be saved breeding process medicine cost 10%-35% when using, and this product also has better curative effect to diarrhoea and the enteritis that the chicken rotavirus causes, can reduce chick stage simultaneously and cause the mortality rate that resistance descends and causes because of immunosuppressant.
Description of drawings
Fig. 1: the chicken alpha-interferon Yeast expression carrier makes up sketch map;
Fig. 2: chicken alpha-interferon mature protein gene PCR product electrophoretic analysis
1, dna molecular amount standard is followed successively by 2000bp from top to bottom, 750bp, 500bp, 100bp;
2, negative control;
3, PCR product, arrow are 530bp;
Fig. 3: the enzyme action of PCIZa-A-chIFN α positive colony carrier is identified
1, dna molecular amount standard is followed successively by 2000bp from top to bottom, 1000bp, 500bp, 250bp;
2, the 3rd, the recombinant expression carrier of ECOR I and Sal I enzyme action.Arrow is the band of 530bp;
Fig. 4: the PCR of transformed yeast identifies
1, negative control;
2, the reorganization on yeast, arrow is the band of 588bp;
3, dna molecular standard, arrow is followed successively by 1000bp from top to bottom, the band of 500bp;
4, positive recombination yeast, arrow indication are the band of 1100bp;
Fig. 5: the serious pathological changes of the vesicular stomatitis virus in positive control hole;
Fig. 6: the interferon diluted concentration is lower than 4
8The chick embryo fibroblast of handling;
Fig. 7: the chick embryo fibroblast in the negative control hole;
Fig. 8: chicken is identified in gel electrophoresis with alpha-interferon
1, is the protein standard, 2, be the yeast expression chicken with α-interferon result, 3, be the result of empty carrier expression of recombinant yeast;
The specific embodiment
The present invention is described in further details by following examples, but does not limit the scope of the invention.
Embodiment 1:
The barms (but gene structure method and sequence of referenced patent 200410041075.0) of screening high expressed
(1) extracts the total RNA of chicken Whole blood lymphocyte,, the PCR product is carried out agarose gel electrophoresis identify that the chicken alpha-interferon gene size of acquisition is 488bp by 30 circulations are set on the PCR instrument.The method of RT-PCR clones the gene of chicken alpha-interferon maturation protein.
(2) utilize chicken alpha-interferon mature protein gene (pMD18-T-chIFN α) that ECOR I and two restriction enzyme sites of Xba I are arranged by pcr amplification.The chicken alpha-interferon mature protein gene is downcut the back link to each other,, if can downcut and big or smallly then can be accredited as the positive for the band of 530bp again through ECOR I and XbaI enzyme cutting with the pPICZa-A yeast vector of same two kinds of digestion with restriction enzyme; The results are shown in Figure 3.
(3) preparation competence yeast, the Yeast expression carrier electricity that will contain the chicken alpha-interferon mature protein gene is transformed into yeast.After electric shock finishes, get 250 μ L conversion products and be coated with and contain on the antibiotic YPDS culture medium of the 100 μ g/ml Zeocin plate, put the positive transformant of bacterium colony that on the YPDS culture medium, to grow 28 ℃ of constant temperature culture 2-4 days.
(4) the high copy of the reorganization that screening is obtained bacterial strain carries out pcr amplification.After amplification finishes, get the 5ul reactant liquor do the zymic chromosome of PPICZa-A that agarose gel electrophoresis observes any genes of interest of end reorganization by primer amplification and positive expression of recombinant yeast bacterial strain by the fragment of above primer amplification, the results are shown in Figure 4.
(5) contain the screening of chicken alpha-interferon high expressed yeast strain
With the initial transformant that grows in the YPDS culture medium plate, photolithography inoculates successively that to contain Zeocin antibiotic concentration gradient be 250,500, and in the YPDS culture medium of 1000ug/ml, the high resistant strain of step-sizing Zeocin promptly contains the height copy bacterial strain of genes of interest.
(6) contain the abduction delivering of the yeast strain of chicken alpha-interferon mature protein gene
Distinguish the BMGY of picking two strains and 25ml from containing the high YPDS culture medium plate that copies the yeast strain of genes of interest, be cultured to OD through 28 ℃ of shaking tables
600During=2-6, the centrifugal 5min harvesting of room temperature 12000rpm, with the BMMY culture fluid of the 100ml 28 ℃ of abduction deliverings in the triangular flask with 1000ml that suspend, every 24h adds 0.5% methanol in culture medium, to keep inductive continuous expression with the cell of results.Finally get the supernatant of inducing 72h, be used to measure its antiviral activity after the dialysis.
(7) contain the preliminary purification of the expression product of chicken alpha-interferon mature protein gene high expressed yeast strain
The processing of culture supernatant:, after the collection supernatant keeps sample, contain the Tris-HCl buffer dissolving of 1mM PMSF with 50mM with the abduction delivering medium centrifugal.Impurity is removed in Tris-HCl buffer dialysis with 50mM, and reuse PEG20000 concentrates and obtains the chicken alpha interferon maturation protein.
Embodiment 2:
Fermentation technology
(1) preparation of seed liquor
With the streak inoculation of chicken alpha-interferon barms to YPDS+zeocin dull and stereotyped last 30 ℃ cultivate 96 hours after, choose single colony inoculation 30 ℃ of shaking tables in the YPD fluid medium and cultivated 16～18 hours, OD is surveyed in sampling under aseptic condition
630Between 0.6～0.8, pH value is between 5.5～6.0, and microscopy does not have assorted bacterium and has the strain that promptly can be used as the fermentation use.Fermentation uses seed liquor generally to be kept at 0～4 ℃, and the holding time must not be above 48 hours.
(2) fermentation condition
Fermentation medium is the BMGY/BMMY culture medium, and cultivation temperature is 28-30 ℃, and induction time 48-72 hour, it was 0.5-1% that 24-48 hour stream adds quantity of methyl alcohol, fermentation PH5.5-6.0, and inoculum concentration is 5-20%, speed of agitator is 450-500rpm/min.
Interferon sample " micro-cytopathic-effect inhibition assay " after the fermentation degerming is detected its biological activity.Observe visible positive control hole and occur 100% serious pathological changes (Fig. 5) fully, and the interferon of yeast expression carries out 10
7, 10
6, 10
5The cell hole that the dilution back is added has 90%, 50%, 0% pathological changes respectively, is lower than 10 in adding
5The cell hole of the recombination yeast chicken alpha-interferon of dilution, cell state good (Fig. 6).In the negative control hole normal chick embryo fibroblast (Fig. 7).The biologic activity that draws the recombination yeast chicken alpha interferon from The above results is 10
6U/ml.
Embodiment 3:
Back extraction process
Get the fermentation liquid that makes by embodiment 2 methods, extract as follows:
Adopt 10000-12000 rev/min of bactofugation earlier; Plate-and-frame filtration remove impurity and further degerming; Get clear liquid by the film bag ultrafiltration of 100,000 molecular weight again, remove macromolecular protein and impurity; Concentrated solution is collected in the film bag ultrafiltration of reuse 6,000 molecular weight, removes the impurity such as inorganic salt of some aldehyde, alcohols and small-molecular weight; Degerming again at last obtains rough interferon, and the qualification result in gel electrophoresis is seen Fig. 8.
Interferon biological activity after employing " micro-cytopathic-effect inhibition assay " Detection and Extraction, the result is as follows:
Get 60 liters of fermentation liquids, titration is 5.2 * 10
6U/ml, the parallel back extraction process of carrying out for three times is handled, and measurement volumes is also measured and is tired.
Embodiment 4:
1000 of chicken alpha-interferon 8,000 ten thousand unit of activity that preparation was administered once in a day, component is as follows:
Chicken alpha-interferon stock solution 1500ml (can be prepared) with reference to the method in the patent 200410041075.0
Benzoic acid 1-5g
Albumin 10-15g
(model of producing available from Chemical Reagent Co., Ltd., Sinopharm Group is that the model that 69003431 albumin or Bovin produce is the albumin of 10-B11)
Astragalus polysaccharides 100-500g
(pin of producing available from the prosperous plant material company limited in Sichuan sea with the astragalus polysaccharides of 70% content)
Water for injection add to cumulative volume be 10000ml
Preparation method: press above-mentioned amount with astragalus polysaccharides, benzoic acid, albumin mixing, be dissolved in the 500ml water for injection, after autoclaving is handled, 1000ml mixes with chicken alpha-interferon stock solution, add water for injection, regulate pH to 6.0-7.0 with the 100mM phosphate buffered solution simultaneously, it is long-pending to 10000ml to decide solution.Under aseptic condition, the interferon goods are packed as the finished product interferon of every bottle of 10ml, and roll cap seal and be stored in the 0-4 ℃ of refrigerator.Randomly drawing 2 bottles of finished product interferon, survey it with " micro-cytopathic-effect inhibition assay " and tire, is qualified to be not less than 8,000 ten thousand unit of activity.Unit of activity is defined as: be defined as an active unit can suppress 50% cytopathic interferon greatest dilution.
Embodiment 5:
Chicken alpha-interferon is to the zoopery of chicken viral diseases treatment
Case one chicken alpha-interferon is to the treatment of infectious bronchitis
Respectively 720 of 20 age in days SPF chickens, AA broiler, the brown laying hens of Luo Man, every kind all is divided into I, II, III three at random to be organized greatly, every group of 240 chickens, be divided into 24 groups more at random in every big group, 10 of every groups comprise 27 groups and 3 matched groups of 9 batches of parallel test groups of 3 lot numbers, are 3 * X
1-8Respectively at chicken alpha-interferon 0.5 ten thousand units, 10,000 units, 1.5 ten thousand units, 2.0 ten thousand units, 2.5 ten thousand units, 3.0 ten thousand units of three batches of intramuscular injection behind the counteracting toxic substances, X
8Be virus inoculation, non-treatment matched group injection 0.5ml normal saline.2 days begin treatments behind the counteracting toxic substances, the chicken alpha-interferon of the corresponding unit of intramuscular injection 0.5ml once a day, used three days continuously.Observe 14d continuously.Protective rate=(A * B-C)/A * B, A is the test group chicken, and B is the dead ratio of matched group, and C is the dead chicken number of test group.
Table 1 is respectively organized test chicken interferon injection volume (ten thousand units/only)
With the chicken alpha-interferon intramuscular injection for treating of 2.0 ten thousand a units/dosage, the SPF chicken can obtain the protective rate more than 70%, and the AA broiler can obtain the protective rate more than 65%; With 1.5 ten thousand units/only above dosage intramuscular injection for treating, the brown laying hen of Luo Man can obtain the protective rate more than 65%.Infect so chicken genetic engineering alpha-interferon can be used for the prophylactic treatment infectious bronchitis virus, present relative dose dependent during treatment.When using, intramuscular injection for treating is advisable with 20,000 units/plumage.
Case two: chicken alpha interferon is to the treatment of infectious laryngotracheitis
Get the brown commodity of Luo Man for 720 of laying hens, be divided into I, II, III three at random and organize greatly, every group of 240 chickens are divided into 24 groups in every big group more at random, comprise 21 groups and 3 matched groups of 9 batches of parallel test groups of 3 lot numbers, and 10 of every groups are 3 * X
1-8The strong malicious 0.05ml of LITV105EID50/0.1ml in every chicken trachea, 2 days begin treatments behind the counteracting toxic substances, the interferon of the corresponding unit of intramuscular injection 0.5ml of every chicken, X
1-7Respectively at chicken alpha-interferon 0.3 ten thousand units, 0.5 ten thousand units, 10,000 units, 1.5 ten thousand units, 2.0 ten thousand units, 2.5 ten thousand units, 3.0 ten thousand units of three batches of each lot numbers of intramuscular injection behind the counteracting toxic substances, X8 is the non-treatment matched group of virus inoculation.14d is observed in each group chicken isolated rearing respectively continuously.Protective rate=(A * B-C)/A * B, A is the test group chicken, and B is the dead ratio of matched group, and C is the dead chicken number of test group.
Table 2 is respectively organized test chicken interferon injection volume
With the chicken alpha-interferon intramuscular injection for treating of above dosage of 1.5 ten thousand units/only, the brown commodity of Luo Man can obtain protective rate more than 65% for laying hen.Chicken genetic engineering alpha-interferon can be used for the prophylactic treatment infectious laryngotracheitis virus to be infected, and presents relative dose dependent during treatment.When using, intramuscular injection for treating is advisable with 20,000 units/plumage.
Embodiment 6 chicken alpha-interferons are used the preventing/treating of virosis
Chicken alpha-interferon can be used for the prevention and the treatment of various virosiss.
When big meat chicken carries out prevention and control to commodity generations, finish the immunity of 21 ages in days intact after, since 23 ages in days,, used weekly 2 days with injection or water way, 2-6 ten thousand U/ plumages can effectively prevent the generation of virosis.
When big meat chicken is treated to commodity generations, with injection or water way, 2-6 ten thousand U/ plumages, logotype three days, and be equipped with the medicine of relief of symptoms or treatment secondary or mixed infection.When respiratory symptom occurring, be equipped with Macrocyclolactone lactone kind medicine; Be equipped with when renal swelling occurring and separate renal swelling class medicine; When the mixed infection of large intestine kind bacterium occurring, be equipped with cephalo-type or other class broad ectrum antibiotic.Adjust service time according to the state of an illness.
To laying hen or when planting chicken and carrying out prevention and control, can use 2 days according to every 1-2 week in season, consumption is 2-5 ten thousand U/ plumages.
To laying hen or plant the method that chicken is treated, with commodity big meat chicken of generation.
Claims (7)
1. the injection of a recombination chicken alpha-interferon is characterized in that, component is as follows, all is weight percentage:
Astragalus polysaccharides 1%-5%, benzoic acid 0.1 ‰-0.5 ‰, chicken alpha-interferon stock solution 5%-20%, albumin 0.1%-0.5%, surplus is a water for injection.
2. the injection of recombination chicken alpha-interferon according to claim 1 is characterized in that described albumin is Sanguis Gallus domesticus albumin or Sanguis Bovis seu Bubali albumin.
3. the preparation method of a recombination chicken alpha-interferon injection as claimed in claim 1 is characterized in that, step is as follows:
(1) with astragalus polysaccharides, benzoic acid, albumin mixing, be dissolved in water for injection and autoclaving then and handle, get injection solvent;
(2) injection solvent that makes in the step (1) is mixed with chicken alpha-interferon stock solution and adds residue water for injection, injection stock solution;
(3) the injection stock solution that step (2) is made is regulated pH to 5.0-6.0, gets the injection of recombination chicken alpha-interferon;
4. preparation method as claimed in claim 3 is characterized in that, described autoclaving is handled and adopted damp and hot autoclaving sterilization.
5. as the preparation method of recombination chicken alpha-interferon injection as described in the claim 3, it is characterized in that, regulate pH in the step (3) and adopt the 100mM phosphate buffered solution to regulate.
6. as the preparation method of recombination chicken alpha-interferon injection as described in the claim 3, it is characterized in that the injection of the recombination chicken alpha-interferon that described step (3) makes is distributed into bottle under aseptic condition.
7. as the preparation method of recombination chicken alpha-interferon injection as described in the claim 6, it is characterized in that the vigor of every bottle of injection is 2500～80,000,000 unit of activity after the packing.
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| CN102154418B (en) * | 2009-06-25 | 2012-11-21 | 天津艾森生物工程有限公司 | Preparation method of animal interferon |
| CN101785535B (en) * | 2010-04-08 | 2012-10-31 | 山西医科大学 | Feed additive containing chicken alpha interferon |
| CN104208662A (en) * | 2013-09-30 | 2014-12-17 | 郑州后羿制药有限公司 | Compound injection for treating poultry viral diseases and preparation method thereof |
| CN110627893A (en) * | 2019-11-01 | 2019-12-31 | 山东仙普爱瑞科技股份有限公司 | Post-extraction process of chicken alpha-interferon |
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| CN1594568A (en) * | 2004-06-24 | 2005-03-16 | 南京农业大学 | Method for preparing recombinant chicken alpha-interferon and its recombinant vector |
| CN1611604A (en) * | 2003-12-23 | 2005-05-04 | 青岛宝依特生物技术研究所 | Swine alpha-interferon gene synthesis, expression vector establishment and product preparing method |
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| CN1611604A (en) * | 2003-12-23 | 2005-05-04 | 青岛宝依特生物技术研究所 | Swine alpha-interferon gene synthesis, expression vector establishment and product preparing method |
| CN1594568A (en) * | 2004-06-24 | 2005-03-16 | 南京农业大学 | Method for preparing recombinant chicken alpha-interferon and its recombinant vector |
| CN1634569A (en) * | 2004-11-04 | 2005-07-06 | 山东华辰生物科技有限公司 | Animal interferon gene engineering biologically active preparation |
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Inventor after: Jiang Zhengjun Inventor after: Cui Liping Inventor after: Liu Shaojie Inventor after: Chen Buyan Inventor after: Liu Gang Inventor after: Kuang Baoxiao Inventor after: Wang Shaojuan Inventor after: Sun Lihong Inventor after: Su Xiaoming Inventor after: Liu Lulu Inventor after: Wang Maochao Inventor before: Jiang Zhengjun Inventor before: Chen Buyan Inventor before: Liu Gang Inventor before: Sun Lihong Inventor before: Su Xiaoming Inventor before: Liu Lulu Inventor before: Wang Maochao Inventor before: Cui Liping Inventor before: Liu Shaojie |
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Address after: 261061 Weifang high tech Zone, Shandong Chang Shun Street, No. 207 Patentee after: Shandong Huachen Pharmaceutical Co., Ltd. Address before: 262610 Weifang high tech Zone, Jin Road, No. 516, No. Patentee before: Shandong Sinostar Bioscience and Technology Co., Ltd. |
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