CN1299767C - Production of fowl leukocyte interferon - Google Patents
Production of fowl leukocyte interferon Download PDFInfo
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- CN1299767C CN1299767C CNB200510038332XA CN200510038332A CN1299767C CN 1299767 C CN1299767 C CN 1299767C CN B200510038332X A CNB200510038332X A CN B200510038332XA CN 200510038332 A CN200510038332 A CN 200510038332A CN 1299767 C CN1299767 C CN 1299767C
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Abstract
The present invention discloses a production method of fowl leukocyte interferon. In the method, healthy fowl leukocytes are induced by newcastle disease viruses(NDV) as an inducing agent and then processed by cultivation, virus inactivation, degerming, separation, purification, etc. so that a fowl leukocyte interferon freeze-drying powder injection is prepared. In the present invention, fowl fresh whole blood is successfully used as a raw material, and the present invention has the advantages of simple and convenient preparation process, easy transportation and preservation after freeze-drying, and reliable quality of products. After being injected into fowl bodies, the injection has the characteristics of good curative effect, safety, reliability, no toxic or side effect, etc.; the injection can be used for treating vital diseases, such as avian influenza viruses, new castle diseases, fowl infectious bronchitis, gosling plague, duck plague, etc.
Description
Technical field
The present invention relates to a kind of biological preparation, specifically is to be used for sick fowl leukocyte interferons such as urgent prevention and treatment gal virus sexually transmitted disease such as bird flu, newcastle, infectious bronchitis of fowl, gosling plague, duck pestilence.
Background technology
To the viral infectious control of poultry, routine is used vaccine, chemical drugs and antibiotic at present.Yet the appearance of fastbacteria in chemicals and antibiotic extensive use, the especially food chain has caused the worry of people to environment and human health.Cytokine such as interferon with respect to traditional therapeutic modality, provide infusive choice as the natural medium antivirus action of immune response.Interferon is under specific inducer effect, by one group of immunoreactive protein with multiple function that cell produces, mainly is glycoprotein, has antiviral, antitumor and immunomodulating three big functions.The research of interferon is relatively more active in modern veterinary and a modern biology frontier.
So far not seeing on the market has a kind of reasonable price, fowl leukocyte interferon preparation evident in efficacy.
Summary of the invention
The objective of the invention is to have preparation human alpha type LeIF (interferon in this chamber, IFN) on successful theory and the practical basis, the fowl leukocyte interferon goods of development and exploitation are for birds antiviral property disease is injected new treatment articles with biological product market.
Technical solution of the present invention is as follows:
The preparation method of fowl leukocyte interferon is characterized in that may further comprise the steps:
(1), the fresh anticoagulation of aseptic collection fowl, separated and collected leukocyte suspension;
(2), add startup in the fowl leukocyte suspension and start the superinduction cultivation in short-term with thick pure system fowl leukocyte interferon;
(3), adding derivant---newcastle disease virus (NDV) and the nutritional solution that contains fowl serum or blood plasma induce to be cultivated 18-24 hour;
(4), collect supernatant, deactivation newcastle disease virus etc., Entkeimung;
(5) divide the fowl leukocyte interferon semifinished product that installs, freezing preservation.
The preparation method of above-mentioned fowl leukocyte interferon is characterized in that:
(1), fresh anticoagulation bottle 30min is housed, collect blood sallow layer, obtain being rich in fowl whole blood leukocyte suspension, concentration of cell suspension is diluted to 1 * 10 with warm in advance culture fluid so that 800rpm～2500rpm is centrifugal
7/ ml cell suspension;
(2), in diluting good cell suspension, add a small amount of thick pure system fowl leukocyte interferon, making its ultimate density is 100 units/ml, places 37 ℃ of shaking bath stir culture 2 hours;
(3), add NDV in the fowl leukocyte suspension after startup, making its ultimate density is 100～150 HAUs/ml, and the while adds RPMI 1640 nutritional solutions that contain 10% fowl serum or fowl blood plasma of 1～1.5 times of volume of former whole blood in leukocyte suspension, adding final concentration is the kanamycin of 10 μ g/ml, transfer the suspension acid-base value to pH7.4,37 ℃ of shaking table stir culture 18～24 hours;
(4), through the centrifugal 30min of 2000rpm～3000rpm, get supernatant, add 6N HCl and transfer to pH 2.0, put 4 ℃ 4～5 days, jolting every day 2～3 times.Reuse 6N NaOH transfers to pH7.2, promptly obtains the fowl leukocyte interferon semifinished product.
Among the present invention: " pre-temperature culture fluid " is meant: put and be heated in room temperature or the water-bath near 37 ℃ being kept at 4 ℃ of RPMI, 1640 grades; " spend the night " in the present technique field, to be often referred to and placed 12 hours～18 hours at 4 ℃ or 37 ℃; PB contains 0.5M KCNS solution; Alleged " fowl " of the present invention comprises chicken, goose, duck etc.
The fowl leukocyte interferon of the present invention's preparation after separation and purification, is made lyophilized injectable powder, and specification is: interferon is tired 〉=20,000 unit/bottles; 2～8 ℃ or room temperature≤25 ℃ preservation.During use, add the 2ml aseptic double-distilled water after, can be dissolved as even suspension rapidly, can carry out intramuscular injection after shaking up gently.Poult 500～1000 units/only/day; Adult fowl 10000～20000 units/only/day.Inject course of treatment 3～5 times every day 1 time.
The present invention has successfully solved problems such as source, fowl fresh blood source and pollution, confirms not have visible untoward reaction through strict field experiment and regional experiment.After fowl leukocyte interferon injects animal body, viral diseases such as treatment bird flu, newcastle, infectious bronchitis of fowl, gosling plague, duck pestilence, have good effect, safe and reliable, have no side effect and characteristics such as free from environmental pollution.
When fowl has been fallen ill in treatment, should in time isolate the fowl that do not fall ill, and give the injection of equivalent chemoprophylaxis.When using the fowl leukocyte interferon of the present invention's preparation, should implement the comprehensive prevention ways such as sterilization of environment simultaneously, prevent to infect again or the recurrence of eqpidemic disease.
The specific embodiment
Fowl leukocyte interferon preparation technology
One, preparation anticoagulant (heparin sodium of every 12500 unit adds Hank ' s liquid 10ml, and every milliliter is 1250 units) and antibiotic (kanamycin, final concentration is 100 μ g/ml during use).
Two, in being added with anticoagulant and antibiotic sterile chamber in advance, aseptic collection fowl whole blood shakes up, and places standby at 4 ℃ of refrigerators.
Three, it tires preparation newcastle disease virus (F system), and titration.
Four, press product description regulation dissolving RPMI 1640 powder,, place standby at 4 ℃ of refrigerators after the aseptic detection through seitz filter pressure filtration.
Five, semifinished product interferon preparation
(1), fresh anticoagulation bottle 30min is housed, collect the sallow layer, obtain being rich in leukocytic suspension, be diluted to 1 * 10 with warm in advance culture fluid so that 800rpm～2500rpm is centrifugal
7/ ml cell suspension;
(2), in diluting good cell suspension, add a small amount of thick pure fowl leukocyte interferon, making its ultimate density is 100 units/ml, places 37 ℃ of shaking bath stir culture 2 hours;
(3), add NDV in the fowl leukocyte suspension after startup, making its ultimate density is 100～150 HAUs/ml, and the while adds RPMI 1640 nutritional solutions that contain 10% fowl serum or fowl blood plasma of 1～1.5 times of volume of former whole blood in leukocyte suspension, adding final concentration is the kanamycin of 10 μ g/ml, adjust this suspension acid-base value to pH7.4,37 shaking table stir culture 18～24 hours;
(4), through the centrifugal 30min of 2000rpm～3000rpm, get supernatant, add 6N HCl and transfer to pH and placed jolting every day 2～3 times 4～5 days for 2.0,4 ℃.Reuse 6N NaOH transfers to pH7.2, promptly obtains the fowl leukocyte interferon semifinished product.Leave and take sample and carry out steriling test, residual poison check etc.
With above-mentioned rough LeIF under 4 ℃ through the centrifugal 30min of 2000rpm～3000rpm; get supernatant seitz filter pressure filtration; aseptic collection filtrate; resampling carry out steriling test, residual poison check, safety verification, exogenous virus check and titration etc. all qualified after; add final concentration contain 5% germfree defatted milk, 5%GS and 10% fowl serum (or blood plasma) as the freeze drying protectant mixing after, insert freeze dryer and preserve.
The interferon semifinished product is according to the following step refining (potassium thiocyanate saltout purification):
(1), in rough interferon, add the 5M KCNS of 4 ℃ of pre-coolings of 1/10 amount, add stirring slowly, add the back and spend the night with 4 ℃ of pre-cooling 2N HCl accent pH to 3.5 (adding 3ml/min slowly) 4 ℃.
(2), supernatant discarded, get precipitation, 4 ℃ of 2000rpm 20min.
(3), abandon supernatant, add in the precipitation 1/15 the amount-20 ℃ of pre-cooling 95% ethanol.Add a small amount of ethanol earlier, the fast shelves of Potter-Elvehjem Tissue Grinders grind 3min, add to capacity ethanol again, transfer to pH4.0 with 2N HCl.
(4), 4 ℃ of 2500rpm 20min, abandon precipitation.
(5), supernatant transfers to pH5.0～5.5 with 1N NaOH.
(6), 4 ℃ of 2000rpm 20min, abandon precipitation.
(7), supernatant transfers to pH5.6 with 0.1N NaOH.
(8), 4 ℃ of 2000rpm 20min are centrifugal, abandon precipitation.
(9), supernatant transfers to pH8.0 with 1N NaOH.
(10), 4 ℃ of 2000rpm 20min are centrifugal, abandon supernatant.
(11), add original volume 1/10～1/15 amount pH8.0 0.1M PB (containing 0.5MKCNS) in the precipitate.
(12), electromagnetic agitation spends the night, and makes it abundant dissolving.
(13), 4 ℃ of 2000rpm 20min are centrifugal, abandon precipitation.
(14), supernatant transfers to pH3.0 with 2N HCl.
(15), 4 ℃ of 2000rpm 20min are centrifugal, abandon supernatant.
(16), add 1/100 amount pH8.0 0.1M PB dissolving in the precipitate with the pH7.6PB dialysis, remove CNS.
(17), use HNO
3, 0.1N AgNO
3Detect CNS
-, till dialysis eliminates.
(18), the centrifugal 1h of 30000 * g, abandon precipitation.
(19), measure interferon and tire and specific activity specific activity 〉=1 * 10
5Iu/mg albumen is qualified.
(20), aseptic subpackaged ,-70 ℃ of preservations.This is fowl leukocyte interferon laboratory reference standard product.
Claims (2)
1, the preparation method of fowl leukocyte interferon is characterized in that:
(1), with the centrifugal fresh anticoagulation bottle of the fowl 30min that is equipped with of 800rpm～2500rpm, collect blood sallow layer, obtain being rich in fowl whole blood leukocyte suspension, with warm in advance culture fluid concentration of cell suspension is diluted to 1 * 10
7/ ml cell suspension;
(2), in diluting good cell suspension, add a small amount of thick pure system fowl leukocyte interferon, making its ultimate density is 100 units/ml, places 37 ℃ of shaking bath stir culture 2 hours;
(3), add NDV in the fowl leukocyte suspension after startup, making its ultimate density is 100～150 HAUs/ml, and the while adds RPMI 1640 nutritional solutions that contain 10% fowl serum or fowl blood plasma of 1～1.5 times of volume of former whole blood in leukocyte suspension, adding final concentration is the kanamycin of 10 μ g/ml, transfer the suspension acid-base value to pH7.4,37 ℃ of shaking table stir culture 18～24 hours;
(4), through the centrifugal 30min of 2000rpm～3000rpm, get supernatant, add 6N HCl and transfer to pH 2.0, put 4 ℃ 4～5 days, jolting every day 2～3 times, reuse 6N NaOH transfers to pH7.2, promptly obtains the fowl leukocyte interferon semifinished product.
2, the preparation method of fowl leukocyte interferon according to claim 1 is characterized in that: refining according to the following step to the fowl leukocyte interferon semifinished product:
(1), in rough interferon, add the 5M KCNS of 4 ℃ of pre-coolings of 1/10 amount, add stirring slowly, add the back with 4 ℃ of pre-cooling 2N HCl accent pH to 3.5,4 ℃ are spent the night;
(2), supernatant discarded, get precipitation, 4 ℃ of 2000rpm 20min;
(3), abandon supernatant, add-20 ℃ of pre-cooling 95% ethanol of 1/15 amount in the precipitation, add a small amount of ethanol earlier, the fast shelves of Potter-Elvehjem Tissue Grinders grind 3min, add to capacity ethanol again, transfer to pH4.0 with 2N HCl;
(4), 4 ℃ of 2500rpm 20min, abandon precipitation;
(5), supernatant transfers to pH5.0～5.5 with 1N NaOH;
(6), 4 ℃ of 2000rpm 20min, abandon precipitation;
(7), supernatant transfers to pH5.6 with 0.1N NaOH;
(8), 4 ℃ of 2000rpm 20min are centrifugal, abandon precipitation;
(9), supernatant transfers to pH8.0 with 1N NaOH;
(10), 4 ℃ of 2000rpm 20min are centrifugal, abandon supernatant;
(11), add original volume 1/10～1/15 amount pH8.00.1M PB in the precipitate, contain 0.5MKCNS among the PB;
(12), electromagnetic agitation spends the night, and makes it abundant dissolving;
(13), 4 ℃ of 2000rpm 20min are centrifugal, abandon precipitation;
(14), supernatant transfers to pH3.0 with 2N HCl;
(15), 4 ℃ of 2000rpm 20min are centrifugal, abandon supernatant;
(16), add 1/100 amount pH8.0 0.1M PB dissolving in the precipitate with pH7.6 PB dialysis, remove CNS
-
(17), use HNO
3, 0.1N AgNO
3Detect CNS
-, till dialysis eliminates;
(18), the centrifugal 1h of 30,000 g, abandon precipitation;
(19), measure interferon and tire and specific activity specific activity 〉=1 * 10
5Iu/mg albumen is qualified;
(20), aseptic subpackaged ,-70 ℃ of preservations, this is fowl leukocyte interferon laboratory reference standard product.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB200510038332XA CN1299767C (en) | 2005-02-05 | 2005-02-05 | Production of fowl leukocyte interferon |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB200510038332XA CN1299767C (en) | 2005-02-05 | 2005-02-05 | Production of fowl leukocyte interferon |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1712065A CN1712065A (en) | 2005-12-28 |
| CN1299767C true CN1299767C (en) | 2007-02-14 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB200510038332XA Expired - Fee Related CN1299767C (en) | 2005-02-05 | 2005-02-05 | Production of fowl leukocyte interferon |
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Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105496972A (en) * | 2015-12-07 | 2016-04-20 | 宋宏婷 | Preparation method of chicken interferon lyophilized preparation |
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2005
- 2005-02-05 CN CNB200510038332XA patent/CN1299767C/en not_active Expired - Fee Related
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| CN1712065A (en) | 2005-12-28 |
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