CN101906157A - Duck leukocyte interferon for treating duck viral disease - Google Patents
Duck leukocyte interferon for treating duck viral disease Download PDFInfo
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- CN101906157A CN101906157A CN 200910069135 CN200910069135A CN101906157A CN 101906157 A CN101906157 A CN 101906157A CN 200910069135 CN200910069135 CN 200910069135 CN 200910069135 A CN200910069135 A CN 200910069135A CN 101906157 A CN101906157 A CN 101906157A
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Abstract
The invention discloses a preparation and activity detection method for duck leukocyte interferon for treating duck viral disease. Duck source newcastle disease virus is used as an inducer for inducing healthy duck leukocyte. The method comprises the processes of raw material preparation, mixing, water bath stirring culture, centrifugation and harvesting, virus inactivation, sterilization, anti-virus activity detection and the like. The fresh complete blood of a duck is successfully used as a raw material, the preparation process is simple and convenient, and the product quality is reliable. After the duck leukocyte interferon is used for a duck body, the duck leukocyte interferon has the characteristics of good treatment effect, safety, reliability, no toxic or side effect and the like. The duck leukocyte interferon for broad spectrum virus resistance has the function of treating the viral diseases such as duck plague, duck viral hepatitis, duck source newcastle disease, duck source egg drop syndrome and the like. The duck leukocyte interferon is used for the animal body, and has the advantages of good treatment effect, safety, reliability, low using amount, no toxic or side effect, low cost and the like.
Description
Technical field
The invention belongs to the veterinary drug technical field, relate to a kind of preparation method of duck leukocyte interferon for the treatment of duck viral disease and the mensuration of antiviral activity thereof.
Background technology
Along with the develop rapidly of national economy and the science adjustment of the structure of agricultural production, foster duck has already become a very fast industry of animal husbandry development speed.Under the big good background that foster duck already develops rapidly, the flowability of duck increases greatly, and the intensification production degree of duck further improves, and the duck disease is produced the influence that is caused to duck and become more and more outstanding.How effectively to prevent and treat the duck disease, reduce financial loss, become one of problem that everybody is concerned about very much.At present, mainly be to be the generation that means are controlled duck viral disease with the epidemic prevention.Each side discovers in recent years, (interference effect of virus is caused by a kind of material Interferon, rabbit, this material is referred to as Interferon, rabbit) have outside the antivirus action, also has antitumor action, to a series of physiologically actives such as immune regulating effect and anti-cells, but in veterinary field, animal interferon fails to be widely used, major cause is that the animal interferon security that is obtained at present is not high, the manufacturing process complexity, cost height, fetch long price, effect is not remarkable, and popularization is restricted.
The object of the invention is intended to overcome above-mentioned shortcoming, and a kind of duck leukocyte interferon for the treatment of duck viral disease is provided.This duck leukocyte interferon is used for the treatment of diseases such as duck plague, duck viral hepatitis, duck source property newcastle disease, duck source property egg-decreasing syndrome as a kind of broad-spectrum antiviral medicament.
Interferon anti-reflecting virus has following characteristics:
1, interferon anti-reflecting virus be not kill but suppress virus, in case remove Interferon, rabbit, virus often again the breeding.Have only the Interferon, rabbit with enough concentration, life-time service just might be utterly destroyed it.Therefore Interferon, rabbit needs the life-time service treatment.
2, Interferon, rabbit has species specificity, and human interferon generally only is used for the treatment of people's disease.Therefore will resist the duck virus disease will prepare the duck Interferon, rabbit.
3, different cell category and different animal, different virus infection type are to the susceptibility difference of Interferon, rabbit.Therefore, the Interferon, rabbit clinical effectiveness that type is different is also different.
Research report about Interferon, rabbit is a lot, and its preparation method is day by day ripe, but because Interferon, rabbit has species specificity, at different species will usually result of treatment be best with disturbing accordingly.Simultaneously different cell categories and different animals, different virus infection type are to the susceptibility difference of Interferon, rabbit.Therefore, the Interferon, rabbit clinical effectiveness that type is different is also different.Resist duck viral disease and will prepare the duck Interferon, rabbit.Yet there are no about utilizing duck blood to prepare the report of duck leukocyte interferon.
Therefore, providing the preparation method of duck leukocyte interferon of a kind of effective prevention and treatment duck viral disease, 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 improve on the basis of existing technology, a kind of preparation method of duck leukocyte interferon is provided.
Implementation of the present invention is as follows for achieving the above object:
A kind of preparation of duck leukocyte interferon and the measuring method of antiviral activity thereof is characterized in that concrete implementation step is as follows:
(1) preparation duck serum: aseptic collection duck blood, 4 ℃ of refrigerators are placed, and separate out naturally, use 0.2%CaCl
2Handle blood plasma, remove and defibrinate, 0.22 μ m filtration sterilization, it is standby to put-20 ℃ of preservations;
(2) preparation white corpuscle: the healthy duck blood of aseptic collection, add the two anti-and an amount of Citric Acid antithrombotics of 100 μ g/ml, 4 ℃ of refrigerators are placed; With the centrifugal 30min of 4000rpm, collect the middle level white corpuscle, the sallow layer, cleaning recentrifuge with PBS separates, the collecting precipitation white corpuscle dilutes the white corpuscle throw out with having added the two DMEM nutrient solutions anti-and above-mentioned duck serum of 100 μ g/ml, and making the white corpuscle concentration dilution is 1 * 10
6/ ml;
(3) add the high human leukocyte interferon of tiring in the leukocyte suspension as starting Interferon, rabbit, make to contain antiviral activity of interferon 100～500 units/ml in the nutrient solution, cultivate 3～4h in 37 ℃ of stirring in water bath;
(4) in the leukocyte suspension that starts, add an amount of inducer duck source property Avian pneumo-encephalitis virus (with preceding in 9 age in days duck embryos amplification and to measure hemagglutinative titer be 1: 1280), making its ultimate density is 320 HAUs/ml, 37 ℃ of shaking baths cultivation 18～24h;
When (5) treating that Interferon, rabbit reaches the climax, centrifugal collection supernatant adds hydrochloric acid and transfers to pH2.0～3.0,4 ℃, places 3～5 days, and inactivation of viruses adds NaHCO again
3Transfer to pH7.0～8.0, aseptic 4 ℃ of centrifugal 4000rpm30min collect the supernatant Interferon, rabbit, remove precipitation and impurity, promptly get rough duck leukocyte interferon;
(6) the duck leukocyte interferon antiviral activity detects, key step is: 10 age in days SPF duck embryos remove four limbs, head and internal organ, add 2 times of volume trysinizations, about 37 ℃ of digestion 10min, wait into and outwell pancreatin when cotton-shaped, add the DMEM substratum and blow and beat repeatedly, preparation DEF, blow and beat even after-filtration, get a filtrate and check cell density, with 1 * 10 at cell numeration plate
4The empty density of cells/ is laid on 96 orifice plates with DEF, every hole 100 μ l, treat that cell grows up to individual layer after, add the duck leukocyte interferon of 10 times of gradient dilutions, 37 ℃, CO
2, about effect 24h, meet VSV virus 1000TCID
50, 37 ℃, CO
2, to cultivate about 24h, there is 75% pathology in microscopy positive control hole, and adding the duck leukocyte interferon hole has and reaches and less than 50% o'clock the record result.
The invention has the beneficial effects as follows:, prepared duck leukocyte interferon treatment duck viral disease according to the species specificity of interferon therapy.Though genetic engineering interferon research at present is awfully hot, it has the preparation cost height, yields poorly, and is restricted in clinical expansion.The present invention utilizes duck blood to prepare the method for duck leukocyte interferon: it has lot of advantages: preparation cost is low, and using dosage is low, good effect.Under the prerequisite of ensuring the quality of products, utilize duck blood to prepare the cost that duck leukocyte interferon can reduce medicine.Therefore this product has great market prospect and good economic benefits.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
Embodiment 1: a kind of preparation method of duck leukocyte interferon, and concrete steps are as follows:
(1) preparation duck serum: aseptic collection duck blood, 4 ℃ of refrigerators are placed, and separate out naturally, use 0.2%CaCl
2Handle blood plasma, remove and defibrinate, 0.22 μ m filtration sterilization, it is standby to put-20 ℃ of preservations, and consumption is 4～8%;
(2) preparation white corpuscle: the healthy duck blood of aseptic collection, add the two anti-and an amount of Citric Acid antithrombotics of 100 μ g/ml, 4 ℃ of refrigerators are placed; Centrifugal with 4000rpm, 30min collects the middle level white corpuscle, the sallow layer cleans recentrifuge with PBS and separates the collecting precipitation white corpuscle, dilute the white corpuscle throw out with having added the two DMEM nutrient solutions anti-and above-mentioned duck serum of 100 μ g/ml, making the white corpuscle concentration dilution is 1 * 10
6/ ml;
(3) add the high human leukocyte interferon of tiring in the leukocyte suspension as starting Interferon, rabbit, make to contain antiviral activity of interferon 400 units/ml in the nutrient solution, cultivate 4h in 37 ℃ of stirring in water bath;
(4) add an amount of inducer duck source property Avian pneumo-encephalitis virus in the leukocyte suspension that starts, making its ultimate density is 512 HAUs/ml, and 37 ℃ of shaking baths are cultivated 18h;
When (6) treating that Interferon, rabbit reaches the climax, 4 ℃ of 4000rpm are centrifugal, collect supernatant, and add hydrochloric acid and transfer to pH3.0,4 ℃, to place 3 days, inactivation of viruses adds NaHCO again
3Transfer to the centrifugal 30min of pH7.0～8.0,4 ℃ 4000rpm, collect the supernatant Interferon, rabbit, remove precipitation and impurity, filtration sterilization promptly gets rough duck leukocyte interferon.
Embodiment 2: the method that a kind of duck leukocyte interferon antiviral activity detects, and concrete steps are as follows:
(1) 10 age in days SPF duck embryos are removed four limbs, head and internal organ, with being added with the two anti-PBS flushings of 100 μ g/ml 2 times, flush away blood shreds, and adds 2 times of volume trysinizations, about 37 ℃ of 10min;
(2) etc. shred to be organized into and outwell pancreatin when cotton-shaped, add the DMEM nutrient solution, liquid-transfering gun is blown and beaten repeatedly;
(3) filtered through gauze after the piping and druming is evenly removed indigested bulk and impurity, is prepared into DEF;
(4) get a filtrate and check cell density, with 1 * 10 at cell numeration plate
4Cells/ hole density is laid on 96 orifice plates with DEF, every hole 100 μ l;
(5) treat that cell grows up to individual layer after, add the duck leukocyte interferon of 10 times of gradient dilutions of 100 μ l, 37 ℃, CO
2, about effect 24h;
(6) meet VSV virus 1000 TCID
50, 37 ℃, CO
2, cultivate about 24h;
(7) there is 75% pathology in microscopy positive control hole, and adding the duck leukocyte interferon hole has and reach and less than 50% o'clock the record result.The results are shown in Table 1:
Table 1 testing data
Data show in the table 1, half protective number (PD
50) between 10
-4With 10
-5Between, press the Reed-Muench method and calculate.Formula is:
By formula: be lower than the logarithm of the logarithm+distance proportion * dilution factor of 50% percentile duck leukocyte interferon=-4-0.79=-4.79, check numerical table.
Embodiment 3: this duck leukocyte interferon is to the application on duck
To the duck leukocyte interferon of embodiment 1 described prepared, the veterinary biologics clinical experimental study data requirement of announcing No. 442 issue according to the Ministry of Agriculture carries out following research:
1, experimental animal: 80 of the healthy ducks of 20 ages in days, usual manner is fed.
2, attack poison: attack poison a little less than the equivalent duck plague to 60, all cause morbidity.
3, test method: treat that symptom occurs, carry out the clinical treatment test immediately.Be divided into 4 groups at random, 20 every group.Low dosage duck leukocyte interferon solution dosage is 20IU/kg; Middle dosage duck leukocyte interferon solution dosage is 60IU/kg; High dosage duck leukocyte interferon solution dosage is 100IU/kg, and blank is organized not administration, the injection, homogeneous day once, continuous 7 days.
4, test-results: low dose group sx, spirit, appetite etc. all take a turn for the better; Middle dosage group is all got well with high dose group, and it is normal that spirit, appetite etc. are recovered.
By the foregoing description, we find that the duck leukocyte transfer factor extracted plays therapeutic action for the virus disease of duck.Therefore the present invention can produce popularization on a large scale.
The present invention can make various formulations, and oral dosage form, syrup formulation etc. also can further can obtain the powder of duck leukocyte interferon by conventional freeze-dry process, in conjunction with conventional auxiliary material, can be made into capsule, tablets and other formulations.
Claims (3)
1. duck leukocyte interferon that is used for the treatment of duck viral disease, it is characterized in that: raw material is preferably duck blood.
2. duck leukocyte interferon according to claim 1 is characterized in that: inducer can be duck plague virus, duck viral hepatitis virus, duck source property Avian pneumo-encephalitis virus, duck source property egg-decreasing syndrome virus, wherein preferred duck source property Avian pneumo-encephalitis virus.
3. duck leukocyte interferon according to claim 1 is characterized in that: active detection can be selected DEF, chick embryo fibroblast, Wish cell etc., wherein preferred DEF.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200910069135 CN101906157A (en) | 2009-06-04 | 2009-06-04 | Duck leukocyte interferon for treating duck viral disease |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200910069135 CN101906157A (en) | 2009-06-04 | 2009-06-04 | Duck leukocyte interferon for treating duck viral disease |
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| CN101906157A true CN101906157A (en) | 2010-12-08 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102397531A (en) * | 2011-11-15 | 2012-04-04 | 青岛绿曼生物工程有限公司 | Compound propolis composition for treating duck plague and preparation method thereof |
-
2009
- 2009-06-04 CN CN 200910069135 patent/CN101906157A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102397531A (en) * | 2011-11-15 | 2012-04-04 | 青岛绿曼生物工程有限公司 | Compound propolis composition for treating duck plague and preparation method thereof |
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Application publication date: 20101208 |