CN101757027A - Method for extracorporeally preparing transfer factor against specificity of infectious bronchitis virus of chickens - Google Patents
Method for extracorporeally preparing transfer factor against specificity of infectious bronchitis virus of chickens Download PDFInfo
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- CN101757027A CN101757027A CN200810152416A CN200810152416A CN101757027A CN 101757027 A CN101757027 A CN 101757027A CN 200810152416 A CN200810152416 A CN 200810152416A CN 200810152416 A CN200810152416 A CN 200810152416A CN 101757027 A CN101757027 A CN 101757027A
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Abstract
The invention relates to a method for extracorporeally preparing a transfer factor against the specificity of infectious bronchitis viruses of chickens. In the invention, splenic organs or peripheral blood of chickens are used as raw materials to make a lymphocyte monolayer, and then phytohemagglutinin and infectious bronchitis viruses of chickens are used to culture lymphocytes through induction so as to extracorporeally produce a large quantity of transfer factors against the specificity of the infectious bronchitis viruses of chickens. The anti-specificity transfer factor prepared by using the method of the invention is used for treating infectious bronchitis of chickens and can raise the immunity of poultry.
Description
Technical field
The present invention relates to the preparation method of the anti-specificity of infectious bronchitis virus of chickens transfer factor of external preparation.
Background technology
Transfer factor is a kind of low molecular polypeptide and the nucleic acid complexes that extracts from lymphocyte, energy specificity or non-specific ground human body immunity improving function, and premunition information is described as the cellular immunization activator.Its no species specificity can transmit between kind, and animal origin can be used for the mankind.
The preparation method of transfer factor routine is: fresh or refrigerated spleen of animal and lymph node are removed fat and connective tissue about 25 ℃, clean with purified water then; The tissue of cleaning is cut into small pieces, adds suitable quantity of water, use tissue mashing machine's smudge cells, make homogenate, add an amount of purified water, mixing is put-20 ℃ of multigelations 5～8 times, and melt temperature must not be above 37 ℃; With the homogenate of freeze thawing centrifugal (4 ℃ of centrifuging temperatures), go precipitation, stay supernatant standby; Supernatant dress bag filter was dialysed the semi-finished product filtration sterilization 24-48 hour; The configuration of finished product and check.
Transfer factor is through inducing, by the defensive peptide class active substance that resists exogenous pathogen that the animal immune system of defense produces, molecular weight is little, activity is strong, has antibacterium, fungus, virus and protozoon effect, even cancerous cell also had lethal effect, use very extensive.Transfer factor is as having specificity and nonspecific immunologically competent cell regulatory factor, is widely used in treating any zoosis toxicity disease, fungal infection, cellular immunization weakens or the auxiliary treatment of defective disease and malignant tumor, and the curative effect of getting well is arranged.But low, the no specificity of technology very complicated, the response rate of preparation transfer factor.The present invention is by the anti-specificity of infectious bronchitis virus of chickens transfer factor of external preparation high specificity, output capacity height.
Summary of the invention
The objective of the invention is to overcome the transfer factor that reaches for preparing in the prior art and do not have specificity at disease, shortcomings such as therapeutic effect instability provide that a kind of external immunization is produced high specificity, cost is low, output capacity is high, the preparation method of the anti-specificity of infectious bronchitis virus of chickens transfer factor of better effects if.
Technical solution of the present invention is summarized as follows:
The preparation method of the anti-specificity of infectious bronchitis virus of chickens transfer factor of a kind of external preparation, form by following steps:
(1) aseptic experimental chicken spleen or the anticoagulation taked prepares the lymphocyte monolayer with spleen or peripheral blood;
(2) to the cultivation of going down to posterity of lymphocyte monolayer;
(3) after the lymph passage cell grows up to monolayer,, promptly obtain the anti-specificity of infectious bronchitis virus of chickens transfer factor of external preparation with phytohaemagglutinin and avian infectious bronchitis virus inducing culture.
Stimulating the mass concentration of chicken spleen cell and the lymphopoietic phytohaemagglutinin agent of Sanguis Gallus domesticus is 50-100mg/L.
Advantage of the present invention:
The present invention is a raw material with less chicken spleen or peripheral blood, make the lymphocyte monolayer, at In vitro culture, can produced in vitro go out the mixture of a large amount of anti-specificity of infectious bronchitis virus of chickens transfer factors with phytohaemagglutinin and avian infectious bronchitis virus inducing culture induction of lymphocyte monolayer then, the anti-specificity of infectious bronchitis virus of chickens transfer factor of method preparation of the present invention need not further purification, directly mixture is used for birds, can play anti-avian infectious bronchitis virus and antimicrobial effect, improve the immunity of birds simultaneously.
The specific embodiment
The present invention is further illustrated below in conjunction with specific embodiment.
Embodiment 1:
The preparation method of the anti-specificity of infectious bronchitis virus of chickens transfer factor of a kind of external preparation, step:
(1) the aseptic anticoagulation of taking prepares the lymphocyte monolayer with peripheral blood, and step is:
Aseptic vein is taked 25 milliliters of animal's whole bloods, add 0.8% heparin solution 0.3ml anticoagulant, left standstill 45 minutes, with all blood plasma more than the band glueballs suction pipe absorption erythrocyte layer, the leukocytic cream that particularly is right after on the erythrocyte layer will be drawn as far as possible, be sub-packed in the sterilization centrifuge tube, with PBS (pH is 7.2) liquid cyclic washing 2～3 times, 800～1200 rev/mins of centrifugal speeds, carry out leukocyte and cultivate with containing 40 milliliters of 30% calf serum lactoalbumin hydrolysates then, being sub-packed in 100 milliliters of capacity behind the uniform mixing cultivates in square vase or the brick bottle, the jam-pack bottle stopper was put in 37 ℃ of calorstats or in the brick bottle machine and is cultivated, through 2～3 days, leukocyte is evenly adherent, promptly makes the lymphocyte monolayer.
(3) to the cultivation of going down to posterity of lymphocyte monolayer;
Before doing the passage cell cultivation, at first culture bottle is placed microscopically to observe, to determine that cell has grown up to monolayer, can carry out the cultivation of going down to posterity of cell.
(4) after the lymph passage cell grows up to monolayer, (final concentration is the inducing culture of 1280 HAUs/ml) with phytohaemagglutinin (final concentration is 100mg/L) and avian infectious bronchitis virus.
(5) through cultivating 2～3 days, the centrifuge cell culture fluid is collected supernatant, and supernatant stirs dialysis at 4 ℃ of lower magnetic forces, and heat sterilization is promptly made the Idiotype transfer factor.
The inspection item of the mensuration of transfer factor concentration, the method for bacteriostatic experiment, semi-finished product and finished product are all with embodiment 1.This method produce transfer factor be yellow clear liquid, content of peptides 0.43g/L, ribose content 0.35g/L.
Embodiment 2
The preparation method of the anti-specificity of infectious bronchitis virus of chickens transfer factor of a kind of external preparation, step:
(1) the aseptic experimental chicken spleen of taking prepares the lymphocyte monolayer with spleen, and step is:
A, processing chicken spleen
Get fresh chicken spleen and place aseptic glass container, adding PBS (pH is 7.2) solution soaking half an hour or being dipped in mass percentage concentration is in 1% the bromo geramine solution, taking-up is with alcohol disinfecting spleen surface, remove spleen fat and peplos with dissecting scissors and tweezers, wash once with PBS (pH is 7.2) liquid, spleen being cut into segment moves in the plate again, with eye scissors spleen is cut into the 1mm3 fritter at last, reuse PBS (pH is 7.2) liquid washing 2-3 time is with removal hemocyte, coloring matter and shred the cell of mechanical damage in the process;
B, digestion and dispersion tissue's piece
PBS (pH7.2) liquid that the last step was cleaned sops up, the piece of tissue that shreds is moved in the conical flask, by 5 times of amount adding 0.25% trypsin solutions (pH is 7.6～7.8) of piece of tissue volume, puts in 37 ℃ of water-baths and digests 20～40 minutes.Shook one time conical flask every 10 minutes, so that piece of tissue is scattered, in order to continuing digestion, up to tissue become loose, surperficial when scared till, at this moment from water-bath, take out conical flask, inhale and remove trypsin solution, reuse PBS (pH is 7.2) liquid washing 2～3 times, wash with 0.5% lactoalbumin hydrolysate-Hanks liquid,, filter with four layers of gauze with the big slightly tubule piping and druming several of bore;
C, cell counting
Adopt blood counting chamber to count, method of counting is identical with numeration of leukocyte;
The packing of D, cell suspension and cultivation
Press the cell counting result, cell suspension is adjusted to 600,000/ml cells suspension with the DMEM nutritional solution.With cell suspension branch pack into Tissue Culture Flask (100ml) and cell rolling bottle, dividing loading amount is 1/5 of this bottle capacity, closes the lid, and carries out sign, Tissue Culture Flask and rolling bottle is placed on respectively on CO2 gas incubator and the Rotary Machine cultivates then;
E, observation
Place 37 ℃ of cultured cells, need observe day by day.If cell is grown, then want the morphological characteristic of observation of cell and judge that its residing growth stage is until forming the lymphocyte monolayer.
(3) to the cultivation of going down to posterity of lymphocyte monolayer
Before doing the passage cell cultivation, at first place microscopically now to examine culture bottle, to determine that cell has grown up to monolayer, can carry out the cultivation of going down to posterity of cell.
(4) after the lymph passage cell grows up to monolayer, with phytohaemagglutinin and avian infectious bronchitis virus inducing culture
After spleen lymph passage cell grows up to monolayer, change and to keep liquid and add phytohaemagglutinin (final concentration is 75mg/L) and avian infectious bronchitis virus simultaneously (final concentration is the inducing culture of 640 HAUs/ml).
(5) inducing culture was taken a sample cell culture after 24 hours after ultrasonication, and detected the concentration of transfer factor.
The mensuration of transfer factor concentration: quantitatively many quantitative to transfer factor:, be a transfer factor unit with content of peptides 1mg with Folin-phenol method or spectrophotometry content of peptides with content of peptides.This method produce transfer factor be yellow clear liquid, content of peptides 0.38g/L, ribose content 0.28g/L.
The check of semi-finished product and finished product:
The semi-finished product calibrating: semi-finished product carry out a detection such as bacterial endotoxin, sterility test, pH value.
Finished product calibrating: finished product do respectively transfer factor and a check such as discrimination test, outward appearance detection, pH value, content of peptides, SAE, proteins react, sterility test, bacterial endotoxin, undue toxicity.
Embodiment 3
The preparation method of the anti-specificity of infectious bronchitis virus of chickens transfer factor of a kind of external preparation, step:
(1) gets fresh chicken spleen and place aseptic glass container, adding PBS (pH is 7.2) solution soaking half an hour or being dipped in mass percentage concentration is in 1% the bromo geramine solution, taking-up is with alcohol disinfecting spleen surface, remove spleen fat and peplos with dissecting scissors and tweezers, wash once with PBS (pH is 7.2) liquid, again spleen is cut into segment and moves in the plate, with eye scissors spleen is cut into the 1mm3 fritter at last, reuse PBS (pH is 7.2) liquid washing 2-3 time;
(2) will go up PBS (pH7.2) liquid that cleaned of step and sop up, the piece of tissue that shreds is moved in the conical flask, measure adding 0.25% trypsin solutions (pH is 7.6～7.8), and put in 37 ℃ of water-baths and digested 20～40 minutes by 5 times of piece of tissue volume.Shook one time conical flask every 10 minutes, so that piece of tissue is scattered, in order to continuing digestion, up to tissue become loose, surperficial when scared till, at this moment from water-bath, take out conical flask, inhale and remove trypsin solution, reuse PBS (pH is 7.2) liquid washing 2～3 times, wash with 0.5% lactoalbumin hydrolysate-Hanks liquid,, filter with four layers of gauze with the big slightly tubule piping and druming several of bore;
(3) cell suspension is adjusted to 600,000/ml cells suspension with the DMEM nutritional solution, divides pack into Tissue Culture Flask and cell rolling bottle then, be placed on respectively on CO2 gas incubator and the Rotary Machine and cultivate;
(4) through 2～3 days, leukocyte is evenly adherent, promptly makes the lymphocyte monolayer, can carry out the cultivation of going down to posterity of cell.After growing up to monolayer, change and to keep liquid and use phytohaemagglutinin (final concentration is 70mg/L) and avian infectious bronchitis virus simultaneously (final concentration is the inducing culture of 640 HAUs/ml).
(5) through cultivating 1～2 day, the centrifuge cell culture fluid is collected supernatant, the supernatant dialysis, and heat sterilization, supernatant are the Idiotype transfer factor.
Application example:
100 15 age in days broiler (aseptic through physical examination) are divided into totally 5 groups of A, B, C, D, E, test behind B, C, the strong malicious counteracting toxic substances of D, E group process artificial challenge's 0.5ml avian infectious bronchitis virus.A group is healthy group, infective virus not, not administration; B organizes negative control group, infective virus, not administration; The C group is medicine low dose group of the present invention, by fowl 0.5ml/ chicken/sky intramuscular injection; The D group is medicine high dose group of the present invention, feeds by fowl 1.0ml/ chicken/sky injection; The E group is for astragalus polysaccharides group (the biological company limited of Tianjin Shengji Group's share), by fowl 0.5g/ chicken/sky injection injection.The result shows, adopts the avian infectious infectious bronchitis virus disease of Drug therapy of the present invention that tangible curative effect is arranged.
Specific transfer factor experimental result of the present invention is as follows:
This result of the test shows, this medicine is low, high-dose therapy group and matched group difference highly significant (P＜0.01), illustrates that medicine of the present invention has significant therapeutical effect to the avian infectious bronchitis virus disease that virus causes.And with " astragalus polysaccharides " group therapeutic equivalence (P＞0.01).
Above-mentionedly resist the detailed description that the preparation method of specificity of infectious bronchitis virus of chickens transfer factor is 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 (2)
1. the preparation method of the anti-specificity of infectious bronchitis virus of chickens transfer factor of external preparation is characterized in that being made up of following steps:
(1) aseptic chicken spleen or the Sanguis Gallus domesticus taked prepares the lymphocyte monolayer with spleen or peripheral blood;
(2) to the cultivation of going down to posterity of lymphocyte monolayer;
(3) after the lymph passage cell grows up to monolayer,, promptly obtain the anti-specificity of infectious bronchitis virus of chickens transfer factor of external preparation with phytohaemagglutinin and avian infectious bronchitis virus inducing culture.
2. according to the preparation method of the anti-specificity of infectious bronchitis virus of chickens transfer factor of external preparation in claims 1, it is characterized in that: stimulating the mass concentration of chicken spleen cell and the lymphopoietic phytohaemagglutinin agent of Sanguis Gallus domesticus is 50-100mg/L.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102697809A (en) * | 2012-05-04 | 2012-10-03 | 郑州后羿制药有限公司 | Preparation method of in-vitro anti-gosling-plague transfer factors |
| CN104523751A (en) * | 2014-12-09 | 2015-04-22 | 郑州后羿制药有限公司 | Method for in vitro preparation of anti-Pestedes petits ruminant virus transfer factor |
| CN105497065A (en) * | 2014-09-26 | 2016-04-20 | 天津嘉瑞生物科技有限公司 | Preparation method of avian infectious laryngotracheitis virus resisting specific transfer factor |
| CN105561287A (en) * | 2014-10-14 | 2016-05-11 | 天津嘉瑞生物科技有限公司 | Preparation method of anti-duck hepatitis virus transfer factor |
| CN105663162A (en) * | 2014-11-19 | 2016-06-15 | 天津嘉瑞生物科技有限公司 | In vitro preparation method of duck plague virus resisting specific transfer factor |
| CN105663161A (en) * | 2014-11-19 | 2016-06-15 | 天津嘉瑞生物科技有限公司 | In vitro preparation method of avian infectious laryngotracheitis virus resisting specific transfer factor |
-
2008
- 2008-10-21 CN CN200810152416A patent/CN101757027A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102697809A (en) * | 2012-05-04 | 2012-10-03 | 郑州后羿制药有限公司 | Preparation method of in-vitro anti-gosling-plague transfer factors |
| CN105497065A (en) * | 2014-09-26 | 2016-04-20 | 天津嘉瑞生物科技有限公司 | Preparation method of avian infectious laryngotracheitis virus resisting specific transfer factor |
| CN105561287A (en) * | 2014-10-14 | 2016-05-11 | 天津嘉瑞生物科技有限公司 | Preparation method of anti-duck hepatitis virus transfer factor |
| CN105663162A (en) * | 2014-11-19 | 2016-06-15 | 天津嘉瑞生物科技有限公司 | In vitro preparation method of duck plague virus resisting specific transfer factor |
| CN105663161A (en) * | 2014-11-19 | 2016-06-15 | 天津嘉瑞生物科技有限公司 | In vitro preparation method of avian infectious laryngotracheitis virus resisting specific transfer factor |
| CN104523751A (en) * | 2014-12-09 | 2015-04-22 | 郑州后羿制药有限公司 | Method for in vitro preparation of anti-Pestedes petits ruminant virus transfer factor |
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Open date: 20100630 |