CN101260143A - Highly effective production method for canine leucocyte interferon - Google Patents
Highly effective production method for canine leucocyte interferon Download PDFInfo
- Publication number
- CN101260143A CN101260143A CNA2008100528981A CN200810052898A CN101260143A CN 101260143 A CN101260143 A CN 101260143A CN A2008100528981 A CNA2008100528981 A CN A2008100528981A CN 200810052898 A CN200810052898 A CN 200810052898A CN 101260143 A CN101260143 A CN 101260143A
- Authority
- CN
- China
- Prior art keywords
- interferon
- leukocyte
- canine
- leukocyte suspension
- rabbit
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a method for producing a canine leukocyte interferon in high efficiency. The particular steps of the method are as follows: (1) the dog blood is aseptically collected, and is added with antibiotics and anticoagulants, and then is put in a refrigerator at a temperature of 4.0 DEG C; the dog blood is centrifuged by the rotary speed of between 2000 and 4000rpm, and the gray yellow layer is collected, and a prepared culture solution is used to dilute cell precipitates until the leukocyte concentration is diluted to contain 1x10<5>-1x10<7> per ml; (2) the leukocyte suspension is added with interferon with the concentration of between 100 and 1000IU/ml and stirred and cultured in a water bath at a temperature of 37 DEG C for 2 to 3 hours; 3) the carboxymethyl Tuckahoe polysaccharide is added to the leukocyte suspension, which makes the final concentration of the leukocyte suspension reach between 25 and 100 mu g/ml, and the leukocyte suspension is stirred and cultured in a water bath at a temperature of 37 DEG C for 1 to 2 hours; 4) the starting leukocyte suspension is added with the proper deal of inducting agent newcastle disease viruses, which makes the final concentration of the leukocyte suspension reach between 128 and 1280 HA units/ml, and the leukocyte suspension is stirred and cultured in a shaking table at a temperature of 37 DEG C for 18 to 24 hours; 5) when the interferon reaches a high ceiling value, the supernatant is collected by centrifugation, and the leukocyte suspension has the pH value adjusted to 2.0 by adding hydrochloric acids and stands for 1 to 4 days for killing live viruses, so that coarse canine leukocyte interferon is obtained. The interferon produced by the method is high in content and obviously low in manufacturing cost.
Description
Technical field
The invention belongs to field of medicaments, particularly a kind of method of High-efficient Production canine leucocyte interferon.
Background technology
Canine viral disease is still the current serious restriction and supports the main transmissible disease that dog is already developed in a healthy way.And traditional transmissible disease treatment plan is difficult to gather effect clinically sometimes, and is especially few at the virus disease way.Canine viral disease does not still have effective medicine and methods of treatment so far.Once the someone carried out preventing and treating with Chinese herbal and crude drugs preparations the test of canine viral disease in recent years, but curative ratio is lower.Microbiotic and sulfa drugs are invalid substantially.At present, dog interferon is the active drug of treatment canine viral disease.
The problem that exists is: it is lower to use routine to induce canine leucocyte to produce Interferon, rabbit content with newcastle disease virus (NDV) in the production of canine leucocyte interferon.
Carboxymethylpachymaran is a kind of novel immunoregulation effect material that has, and the function that can stimulate the human immune system induces and urgees to give birth to Interferon, rabbit and produces, and also is a kind of leucoregulin.Carboxymethylpachymaran has strengthening immunity, antitumor, antiviral, anti-ly radiates, protects the liver, pharmacologically active widely such as hypnosis, can induce and urge to induce the human blood cell and produce Interferon, rabbit-a (IFN-a), interferon-r (IFN-r), interleukin II (IL-2), interleukin-6 (IL-6), tumour necrosis factor-a (TNF-a) is with grain one macrophage colony stimulating factor various kinds of cell active factores such as (GM-CSF).Carboxymethylpachymaran is a kind of novel biological response modifier (BRM).Have antiviral indirectly, antitumor action.
But about the method for utilizing newcastle disease virus F strain (NDV-F) and carboxymethylpachymaran to make compound interferon inducers efficient production Interferon, rabbit yet there are no report.
Therefore, providing the method for a kind of Interferon, rabbit content height, High-efficient Production canine leucocyte interferon that preparation cost is low, is one of this technical field scientific research personnel new problem of being badly in need of developing.
Summary of the invention
The objective of the invention is to overcome the weak point in the above-mentioned technology, a kind of method of High-efficient Production canine leucocyte interferon is provided.
Implementation of the present invention is as follows for achieving the above object:
A kind of method of High-efficient Production canine leucocyte interferon is characterized in that concrete implementation step is as follows:
(1) aseptic collection dog blood adds 10 μ g/ml kantlex and an amount of antithrombotics, and 4.0 ℃ of refrigerators are placed; With the centrifugal dog blood of 2000-4000rpm, collect the sallow layer, with the nutrient solution diluting cells throw out that configures, making the white corpuscle concentration dilution is that every ml contains 1 * 10
5-1 * 10
7
(2) add the small amount of interference element in the leukocyte suspension, making its ultimate density is 100-1000IU/ml, cultivates 0.5-2 hour in 37 ℃ of stirring in water bath;
(3) add carboxymethylpachymaran again, making its ultimate density is 25-100 μ g/ml, cultivates 1-2 hour in 37 ℃ of stirring in water bath;
(4) add an amount of inducer newcastle disease virus then again in above-mentioned leukocyte suspension, making its ultimate density is 128-1280 HAU/ml, 37 ℃ of shaking table stir culture 18-24 hour;
When (5) treating the Interferon, rabbit value of peaking, centrifugal collection supernatant adds hydrochloric acid and transfers to 2.0,4.0 ℃ of pH, places 1-4 days, and inactivation of viruses adds NaOH again and transfers to pH 7.0, promptly gets rough canine leucocyte interferon.
The invention has the beneficial effects as follows: the dog genetic engineering interferon has the preparation cost height, is subjected to arriving very much restriction in clinical expansion.The present invention utilizes dog blood to prepare the method for canine leucocyte interferon: it has lot of advantages: preparation cost is low, good effect.It is low that routine induces canine leucocyte to produce Interferon, rabbit content with NDV-F,
The present invention induces in the LeIF process in the NDV-F strain and adds carboxymethylpachymaran, significantly improves to utilize dog blood to prepare the content of canine leucocyte interferon, has significantly reduced the cost of medicine.After this product injects animal body, have good effect, safe and reliable, consumption is low, have no side effect and low cost and other advantages.
Embodiment
Below in conjunction with embodiment, to details are as follows according to embodiment provided by the invention:
Embodiment 1
A kind of method of High-efficient Production canine leucocyte interferon, concrete steps are as follows:
(1) white corpuscle preparation
Aseptic collection dog blood adds microbiotic and antithrombotics, and 4.0 ℃ of refrigerators are placed.With the centrifugal dog blood of 2000-4000rpm, collect the sallow layer, with the nutrient solution diluting cells throw out that configures, making the white corpuscle concentration dilution is that every ml contains 1 * 10
6Leukocytic separation should be carried out in back 48 hours in blood sampling.Viable count should reach more than 90%.
(2) induce virus
Adopt new castle disease virus (NDV) F strain, hemagglutinative titer reaches suitable titre and can go into operation.
(3) nutrient solution
Adopt cell culture medium RPMI-1640, include 5-10% dog serum and 10 μ g/ml kantlex or gentamicins, also available other suitable nutrient solution.
(4) production should meet drinking water standard with former water, and the water that is directly used in goods should meet the water for injection standard.
(5) preparation technology of rough Interferon, rabbit
(5.1) induce viral preparation
Adopt 9-10 age in days healthy chicken embryo, an amount of NDV-F of inoculation in the allantoic cavity of every embryo cultivated 48-72 hour for 37 ℃, and chick embryo development is good, virus is collected allantoic fluid, and is done sterility test after reaching suitable titre, merge after the assay was approved, titration is done in sampling, put-20 ℃ stand-by;
(5.2) leukocyte suspension
Use the centrifuging separated plasma, draw leukocytic cream, use the nutrient solution dilution of configuration in the step (3) then, contain 5 * 10 with every ml
6Be advisable;
(5.3) start and induce
Add a little LeIF in the leukocyte suspension, making its ultimate density is 600IU/ml, cultivates 0.5 hour in 37 ℃ of stirring in water bath, adds carboxymethylpachymaran again, and making its ultimate density is 100 μ g/ml, cultivates 1.5 hours in 37 ℃ of stirring in water bath;
(5.4) add 1024 HAUs/ml NDV-F strain again and induce, when treating the Interferon, rabbit value of peaking, collect supernatant,, be rough canine leucocyte interferon supernatant inactivation of viruses after acidifying and neutralisation;
When (5.5) treating the Interferon, rabbit value of peaking, centrifugal collection supernatant adds hydrochloric acid and transfers to pH2.0, and 4.0 ℃, to place 2 days, inactivation of viruses adds NaOH again and transfers to pH7.0, promptly gets rough canine leucocyte interferon;
(5.6) centrifugal collection supernatant promptly gets rough LeIF, puts-20 ℃ of preservations;
(5.7) keep sample and make test of sterility test, residual poison and safety verification respectively, and tire with micromethod titration Interferon, rabbit.The rough Interferon, rabbit of dog by the preparation of this method totally 1 time, 2 lot numbers have obtained the high yield Interferon, rabbit, on average tire to be 23800IU/ml.The routine that does not add carboxymethylpachymaran induces the group Interferon, rabbit and on average tires and be lower than 9450IU/ml.
The Interferon, rabbit titration: adopt CPE (cell cause a disease effect) to suppress the inhibition micromethod for the basis, the dilution inverse that still can protect half cell human amniotic cell Wish cell (50) to avoid the attack of bubble stomatitis virus with the high dilution of every ml Interferon, rabbit inspection product is decided to be Interferon, rabbit unit.Represent with international unit (IU), and proofread and correct the result with the national standard product.
Embodiment 2
A kind of method of High-efficient Production canine leucocyte interferon, concrete steps are as follows:
(1) aseptic collection dog blood adds 10 μ g/ml penicillin, Streptomycin sulphate and an amount of antithrombotics, and 4.0 ℃ of refrigerators are placed.With the centrifugal dog blood of 4000rpm, collect the sallow layer, with the 1640 substratum diluting cells throw outs that configure, making the white corpuscle concentration dilution is that every ml contains 1 * 10
7
(2) in leukocyte suspension, add 8% dog serum or blood plasma, mixing.
(3) add a little Interferon, rabbit in above-mentioned leukocyte suspension, making its ultimate density is 200-300IU/ml, cultivates 1 hour in 37 ℃ of stirring in water bath.
(4) add carboxymethylpachymaran again, making its ultimate density is 25 μ g/ml; Cultivated 2 hours in 37 ℃ of stirring in water bath.
(5) in the leukocyte suspension that starts, add 512 HAUs/ml NDV-F strain again and induce, 37 ℃ of shaking table stir culture 12-24 hour.
When (6) treating the Interferon, rabbit value of peaking, centrifugal with 3000rpm, centrifugal collection supernatant adds hydrochloric acid and transfers to pH2.0, and 4.0 ℃, to place 4 days, inactivation of viruses adds NaOH again and transfers to pH7.0, promptly gets rough canine cells Interferon, rabbit.Keep sample and measure Interferon, rabbit and tire, the rough Interferon, rabbit of dog by this method preparation totally 2 times, 4 lot numbers have obtained the high yield Interferon, rabbit, on average tire to be 25600IU/ml.The routine that does not add carboxymethylpachymaran induces the group Interferon, rabbit and on average tires and be 12560IU/ml.
Embodiment 3
A kind of method of High-efficient Production canine leucocyte interferon, concrete steps are as follows:
(1) aseptic collection dog blood adds 10 μ g/ml kantlex and an amount of antithrombotics, and 4.0 ℃ of refrigerators are placed.With the centrifugal dog blood of 4000rpm, collect the sallow layer, with the nutrient solution diluting cells throw out that configures, making the white corpuscle concentration dilution is that every ml contains 1 * 10
5
(2) add a little Interferon, rabbit in the leukocyte suspension, making its ultimate density is 800IU/ml, cultivates 2 hours in 37 ℃ of stirring in water bath.
(3) add carboxymethylpachymaran again, making its ultimate density is 50 μ g/ml, cultivates 2 hours in 37 ℃ of stirring in water bath.
(4) in the leukocyte suspension that starts, add 256-1280 HAU/mlNDV-F strain again and induce, in leukocyte suspension, add the DMEM substratum that contains 5% dog serum or blood plasma simultaneously, 37 ℃ of shaking table stir culture 12-24 hour.
When (5) treating the Interferon, rabbit value of peaking, centrifugal with 3000rpm, collect supernatant and promptly get rough canine leucocyte interferon.Keep sample and measure Interferon, rabbit and imitate and add, the rough Interferon, rabbit of dog by this method preparation totally 2 times, 3 lot numbers, on average tiring is 278900IU/ml.The routine that does not add carboxymethylpachymaran induces the group Interferon, rabbit and on average tires and be 11500IU/ml.
The Interferon, rabbit content height that uses this preparation method to produce has significantly reduced production cost.Concrete grammar is as follows: at first get the fresh anticoagulated whole blood of dog, the aseptic sallow layer of leaving and taking, use NDV-F and carboxymethylpachymaran to induce the healthy dogs LeIF as compound inducer, it includes leukocyte suspension, starts Interferon, rabbit, viral liquid and nutrient solution, make the canine leucocyte interferon of making through feedstock production → mixed → stirring in water bath, the inducing culture → centrifugal results → inactivation of viruses → degerming → packing of thawing → finished product packing operation.Keep sample and make sterility test, residual poison test and safety verification respectively.The rough Interferon, rabbit of dog by the preparation of this method totally 3 times, 4 lot numbers have obtained the high yield Interferon, rabbit, on average tire greater than 30720IU/ml, and sterility test, residual poison test are all negative, and all healthy survival in injection back has no adverse reaction in the animal body.
The dog interferon of the present invention's preparation has virus disease effects such as treatment canine viral disease, simultaneously bacteriosis is also had therapeutic action.Be used to prevent and treat the dog disease.As diseases such as canine parvovirus disease, canine distemper, infectious canine hepatitis.Usage and consumption: my son 20,000-100,000 IU/ head/sky, educate dog 30,000-500,000 IU/ head/sky.Inject every day 1 time, course of treatment 3 times, seriously ill consumption slightly.
Obtained the high yield Interferon, rabbit by the present invention, the rough Interferon, rabbit of dog by the preparation of this method totally 7 times, 6 lot numbers, wherein 4 lot numbers are tired and (be up to 40960IU/ml) more than 24200IU/ml; All the other are tired more than 19480IU/ml.On average tiring is about 24230IU/ml, and the Interferon, rabbit content height that uses this preparation method to produce has significantly reduced production cost.
Interferon, rabbit sterility test, the residual poison test that keeps sample is all negative, and all healthy survival in injection back has no adverse reaction in the animal body.Side effect has good result of treatment to the canine viral communicable disease.The present invention is a kind of broad-spectrum antiviral preparation, mainly treats canine viral disease, for example canine parvovirus disease, canine distemper, infectious canine hepatitis and dog rotavirus disease etc.By the injection said preparation, itself and peripheral cell are reacted, inducing cell produces antiviral protein, thereby suppresses the breeding of virus, plays antivirus action.
Clinical trial certificate:
The canine leucocyte interferon that utilizes the present invention to develop is treated totally 285 of dog diseases such as canine parvovirus disease, canine distemper, infectious canine hepatitis and dog rotavirus disease, cures 237, and curative ratio reaches 83.2%; Treat particularly wherein that canine parvovirus disease is efficient to reach 95.4%, curative ratio is up to 84.6%, the effect highly significant.Cure rate after viral diarrhea trouble dog is treated separately with Interferon, rabbit is apparently higher than the control group with interferon therapy.
Above-mentioned detailed description of the method for this High-efficient Production canine leucocyte interferon being carried out with reference to embodiment; be illustrative rather than determinate; can list several embodiment according to institute's limited range; therefore in the variation and the modification that do not break away under the general plotting of the present invention, should belong within protection scope of the present invention.
Claims (1)
1, a kind of method of High-efficient Production canine leucocyte interferon is characterized in that concrete implementation step is as follows:
(1) aseptic collection dog blood adds 10 μ g/ml kantlex and an amount of antithrombotics, and 4.0 ℃ of refrigerators are placed; With the centrifugal dog blood of 2000-4000rpm, collect the sallow layer, with the nutrient solution diluting cells throw out that configures, making the white corpuscle concentration dilution is that every ml contains 1 * 10
5-1 * 10
7
(2) add the small amount of interference element in the leukocyte suspension, making its ultimate density is 100-1000IU/ml, cultivates 0.5-2 hour in 37 ℃ of stirring in water bath;
(3) add carboxymethylpachymaran again, making its ultimate density is 25-100 μ g/ml, cultivates 1-2 hour in 37 ℃ of stirring in water bath;
(4) add an amount of inducer newcastle disease virus then again in above-mentioned leukocyte suspension, making its ultimate density is 128-1280 HAU/ml, 37 ℃ of shaking table stir culture 18-24 hour;
When (5) treating the Interferon, rabbit value of peaking, centrifugal collection supernatant adds hydrochloric acid and transfers to 2.0,4.0 ℃ of pH, places 1-4 days, and inactivation of viruses adds NaOH again and transfers to pH 7.0, promptly gets rough canine leucocyte interferon.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2008100528981A CN101260143A (en) | 2008-04-25 | 2008-04-25 | Highly effective production method for canine leucocyte interferon |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2008100528981A CN101260143A (en) | 2008-04-25 | 2008-04-25 | Highly effective production method for canine leucocyte interferon |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101260143A true CN101260143A (en) | 2008-09-10 |
Family
ID=39960866
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2008100528981A Pending CN101260143A (en) | 2008-04-25 | 2008-04-25 | Highly effective production method for canine leucocyte interferon |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101260143A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102838670A (en) * | 2012-09-25 | 2012-12-26 | 大连三仪动物药品有限公司 | Pretreating agent used for producing chicken leucocyte interferon and production method |
| CN106085954A (en) * | 2016-06-24 | 2016-11-09 | 安徽未名细胞治疗有限公司 | A kind of a large amount of human peripheral PBMC fast separating process |
-
2008
- 2008-04-25 CN CNA2008100528981A patent/CN101260143A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102838670A (en) * | 2012-09-25 | 2012-12-26 | 大连三仪动物药品有限公司 | Pretreating agent used for producing chicken leucocyte interferon and production method |
| CN106085954A (en) * | 2016-06-24 | 2016-11-09 | 安徽未名细胞治疗有限公司 | A kind of a large amount of human peripheral PBMC fast separating process |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104447978A (en) | Recombinant chicken interferon alpha and preparation method thereof | |
| CN1142187A (en) | Paramunity inducer based on combinations of poxvirus components, process to prepare it and its use as medicament | |
| CN104894065A (en) | NK (natural killer) cell culture medium and culture method of NK cell | |
| CN106139140B (en) | A kind of Muscovy duck parvovirus subunit vaccine | |
| CN101289666B (en) | Method for preparing recombination porcine alpha-type interferon | |
| CN103319608B (en) | Pig IFNalpha1-Fc fusion protein as well as encoding gene and expression method thereof | |
| CN106399266A (en) | Recombinant baculovirus for expressing dog serum albumin fused interferon gamma and application of recombinant baculovirus | |
| CN113201508A (en) | Recombinant Newcastle disease oncolytic virus and preparation method and application thereof | |
| CN101260143A (en) | Highly effective production method for canine leucocyte interferon | |
| CN112979796A (en) | Horse anti-A H5N1 tiger source influenza virus immunoglobulin and specific immunoglobulin and refining method thereof | |
| CN105671002A (en) | Construction and application of H9N2-subtype avian influenza virus cell high-yield vaccine strain | |
| CN101250224A (en) | Method for high-effective production of pig leucocyte interferon | |
| CN102154418B (en) | Preparation method of animal interferon | |
| CN112080478B (en) | Efficient propagation method and application of H5 subtype avian influenza virus | |
| CN108948163A (en) | Queensland nut plant alexin and its application | |
| CN104162160B (en) | Feed additive antiviral composition, lyophilized powder, preparation method and application | |
| CN101250223A (en) | Method for preparing bovine leukocyte interferon | |
| CN107019703A (en) | LSECtin is being used as the application in treating and/or preventing the target spot of Ebola virus | |
| CN112143714A (en) | Method for producing H7 subtype avian influenza virus inactivated vaccine by using low-immunity chick embryo | |
| CN101810638B (en) | Preparation method of endogenous interferon inducer injection and nanometer microencapsulation solution | |
| CN1887344A (en) | Animal interferon compound and its prepn and application | |
| CN110684768B (en) | Duck natural immunopotentiator based on pattern recognition receptor ligand | |
| CN104531691A (en) | Primers for obtaining genes of bovine interferon alpha and preparation method for recombinant bovine interferon alpha | |
| CN104450731A (en) | Production method of chicken gamma interferon protein | |
| CN103468769A (en) | Selenium-containing recombinant human albumin preparation method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Open date: 20080910 |