CN101759765A - Method for extracorporeally preparing transfer factor against specificity of bursal disease virus of chickens - Google Patents
Method for extracorporeally preparing transfer factor against specificity of bursal disease virus of chickens Download PDFInfo
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- CN101759765A CN101759765A CN200810152419A CN200810152419A CN101759765A CN 101759765 A CN101759765 A CN 101759765A CN 200810152419 A CN200810152419 A CN 200810152419A CN 200810152419 A CN200810152419 A CN 200810152419A CN 101759765 A CN101759765 A CN 101759765A
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Abstract
The invention relates to a method for extracorporeally preparing a transfer factor against the specificity of bursal disease viruses of chickens. In the invention, splenic organs or peripheral blood of chickens are used as raw materials to make a lymphocyte monolayer, lymphocytes are extracorporeally cultured, and then phytohemagglutinin and bursal disease viruses of chickens are used to culture the lymphocytes through induction so as to extracorporeally produce a large quantity of transfer factors against the specificity of the bursal disease viruses of chickens. The transfer factor against the specificity of bursal disease viruses of chickens extracorporeally prepared by using the method of the invention is used for treating infectious bursal diseases of chickens and can raise the immunity of poultry.
Description
Technical field
The present invention relates to the preparation method of external preparation specific transfer factor for resisting chicken bursa virus.
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 enhance immunity 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 and lymphoglandula are removed fat and reticular tissue about 25 ℃ with animal, 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 liquor standby; Supernatant liquor dress dialysis tubing was dialysed the work in-process 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 pathogenic agent that the animal immune system of defense produces, molecular weight is little, activity is strong, has antibacterium, fungi, virus and protozoon effect, even cancer cells 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 zoonosis toxicity disease, fungi infestation, cellular immunization weakens or the assisting therapy of defective disease and malignant tumour, and the curative effect of getting well is arranged.But low, the no specificity of technology very complicated, the rate of recovery of preparation transfer factor.The present invention is by external preparation specific transfer factor for resisting chicken bursa virus 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 result of treatment instability provide that a kind of external immunization is produced high specificity, cost is low, output capacity is high, the preparation method of the specific transfer factor for resisting chicken bursa virus of better effects if.
Technical solution of the present invention is summarized as follows:
A kind of preparation method of external preparation specific transfer factor for resisting chicken bursa virus, be made up of following steps:
(1) aseptic experimental chicken spleen or the anticoagulation taked prepares the lymphocyte individual layer with spleen or peripheral blood;
(2) to the cultivation of going down to posterity of lymphocyte individual layer;
(3) after the lymph passage cell grows up to individual layer,, promptly obtain external preparation specific transfer factor for resisting chicken bursa virus with phytohaemagglutinin and bursal disease virus of chickens inducing culture.
Stimulating the mass concentration of the phytohaemagglutinin agent of chicken splenocyte and chicken blood lymphocyte propagation is 50-150mg/L.
Advantage of the present invention:
The present invention is a raw material with less chicken spleen or peripheral blood, make the lymphocyte individual layer, in vitro culture, get final product the mixture that produced in vitro goes out a large amount of specific transfer factor for resisting chicken bursa virus with phytohaemagglutinin and bursal disease virus of chickens inducing culture induction of lymphocyte individual layer then, the specific transfer factor for resisting chicken bursa virus of method preparation of the present invention need not further purification, directly mixture is used for bird, can play resisting chicken bursa virus and antimicrobial effect, improve the immunizing power of bird simultaneously.
Embodiment
The present invention is further illustrated below in conjunction with specific embodiment.
Embodiment 1:
A kind of preparation method of external preparation specific transfer factor for resisting chicken bursa virus, step:
(1) the aseptic anticoagulation of taking prepares the lymphocyte individual layer with peripheral blood, and step is:
Aseptic vein is taked 25 milliliters of animal's whole bloods, add 0.8% heparin solution 0.3ml anti-freezing, left standstill 45 minutes, with all blood plasma more than the band glueballs suction pipe absorption red corpuscle layer, the leukocytic cream that particularly is right after on the red corpuscle layer will be drawn as far as possible, be sub-packed in the sterilization centrifuge tube, with PBS (pH is 7.2) liquid repetitive scrubbing 2～3 times, 800～1200 rev/mins of centrifugal speeds, carry out white corpuscle 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 thermostat containers or in the brick bottle machine and is cultivated, through 2～3 days, white corpuscle is evenly adherent, promptly makes the lymphocyte individual layer.
(3) to the cultivation of going down to posterity of lymphocyte individual layer;
Before doing the passage cell cultivation, at first culturing bottle is placed microscopically to observe, to determine that cell has grown up to individual layer, can carry out the cultivation of going down to posterity of cell.
(4) after the lymph passage cell grows up to individual layer, change and to keep liquid and add phytohaemagglutinin (final concentration is 120mg/L) and bursal disease virus of chickens simultaneously (final concentration is the inducing culture of 1280 HAUs/ml).
When lymphocyte go down to posterity grow up to individual layer after, phytohaemagglutinin and bursal disease virus of chickens inducing culture.
(5) through cultivating 2～3 days, the eccentric cell nutrient solution is collected supernatant, and supernatant liquor stirs dialysis at 4 ℃ of lower magnetic forces, and heat sterilization is promptly made the Idiotype transfer factor.
The Interventions Requested of the mensuration of transfer factor concentration, the method for bacteriostatic experiment, work in-process and finished product are all with embodiment 1.Present method produce transfer factor be yellow clear liquid, content of peptides 0.315g/L, ribose content 0.284g/L.
Embodiment 2
A kind of preparation method of external preparation specific transfer factor for resisting chicken bursa virus, step:
(1) the aseptic experimental chicken spleen of taking prepares the lymphocyte individual layer with spleen, and step is:
A, processing chicken spleen
Get fresh chicken spleen and place aseptic Glass Containers, adding PBS (pH is 7.2) solution soaking half an hour or being dipped in mass percentage concentration is in 1% the bromogeramine solution, taking-up is with alcohol disinfecting spleen surface, remove spleen fat and coating 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, use PBS (pH is 7.2) liquid washing again 2-3 time, with removal hemocyte, coloring matter and shred the cell of physical abuse in the process;
B, digestion and dispersion tissue's piece
PBS (pH7.2) liquid that the last step was cleaned sops up, the tissue block that shreds is moved in the Erlenmeyer flask, by 5 times of amount adding 0.25% trypsin solutions (pH is 7.6～7.8) of tissue block volume, puts in 37 ℃ of water-baths and digests 20～40 minutes.Shook one time Erlenmeyer flask every 10 minutes, so that tissue block is scattered, in order to continuing digestion, up to tissue become loose, surperficial when scared till, at this moment from water-bath, take out Erlenmeyer flask, inhale and remove trypsin solution, use PBS (pH is 7.2) liquid washing 2～3 times again, 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 white blood cell count(WBC);
The packing of D, cell suspension and cultivation
By the cell counting result, cell suspension is adjusted to 600,000/ml cells suspension with the DMEM nutritive medium.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 CO2gas 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 specificity of observation of cell and judge that its residing growth phase is until forming the lymphocyte individual layer.
(3) to the cultivation of going down to posterity of lymphocyte individual layer
Before doing the passage cell cultivation, at first place microscopically now to examine on culturing bottle, to determine that cell has grown up to individual layer, can carry out the cultivation of going down to posterity of cell.
(4) after the lymph passage cell grows up to individual layer, with phytohaemagglutinin and bursal disease virus of chickens inducing culture
After spleen lymph passage cell grows up to individual layer, change and to keep liquid and add phytohaemagglutinin (final concentration is 80mg/L) and bursal disease virus of chickens simultaneously (final concentration is the inducing culture of 640 HAUs/ml).
(5) inducing culture is 24 hours, with cell culture after ultrasonication, take a sample detect transfer factor and concentration.
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.Present method produce transfer factor be yellow clear liquid, content of peptides 0.493g/L, ribose content 0.372g/L.
The check of work in-process and finished product:
The work in-process calibrating: work in-process carry out a detection such as bacterial endotoxin, sterility test, pH value.
Finished product calibrating: finished product do respectively transfer factor and telling test, outward appearance detections, pH value, content of peptides, SAE, proteins react, sterility test, bacterial endotoxin, undue toxicity etc. check.
Embodiment 3
A kind of preparation method of external preparation specific transfer factor for resisting chicken bursa virus, step:
(1) gets fresh chicken spleen and place aseptic Glass Containers, adding PBS (pH is 7.2) solution soaking half an hour or being dipped in mass percentage concentration is in 1% the bromogeramine solution, taking-up is with alcohol disinfecting spleen surface, remove spleen fat and coating 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, use PBS (pH is 7.2) liquid washing 2-3 time again;
(2) will go up PBS (pH7.2) liquid that cleaned of step and sop up, the tissue block that shreds is moved in the Erlenmeyer 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 tissue block volume.Shook one time Erlenmeyer flask every 10 minutes, so that tissue block is scattered, in order to continuing digestion, up to tissue become loose, surperficial when scared till, at this moment from water-bath, take out Erlenmeyer flask, inhale and remove trypsin solution, use PBS (pH is 7.2) liquid washing 2～3 times again, 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 nutritive medium, divides pack into Tissue Culture Flask and cell rolling bottle then, be placed on respectively on CO2gas incubator and the Rotary Machine and cultivate;
(4) through 2～3 days, white corpuscle is evenly adherent, promptly makes the lymphocyte individual layer, can carry out the cultivation of going down to posterity of cell.After growing up to individual layer, change and to keep liquid and use phytohaemagglutinin (final concentration is 70mg/L) and bursal disease virus of chickens simultaneously (final concentration is the inducing culture of 1280 HAUs/ml).
(5) through cultivating 1～2 day, the eccentric cell nutrient solution is collected supernatant, the supernatant liquor dialysis, and heat sterilization, supernatant liquor are the Idiotype transfer factor.
Application example:
50 40 age in days fryer (aseptic through physical examination) are divided into totally 5 groups of A, B, C, D, E, and B, C, D, E group are attacked the poison back through the strong poison of artificial challenge's 0.5ml bursal disease virus of chickens and tested.A group is healthy group, infective virus not, not administration; B organizes negative control group, infective virus, not administration; The present invention of C group is the medicine low dose group, by fowl 0.5ml/ chicken/sky intramuscular injection; The D group is medicine high dose group of the present invention, 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.The result shows, adopts pharmacological agent infections chicken cloacal bursa virus disease of the present invention that tangible curative effect is arranged.
Specific transfer factor experimental result of the present invention is as follows:
This test-results shows, this medicine is low, high-dose therapy group and control group difference highly significant (P＜0.01), illustrates that medicine of the present invention has significant therapeutic action to the chicken bursal disease viral disease that virus causes.And with " astragalus polysaccharides " group therapeutic equivalence (P＞0.01).
Above-mentioned detailed description of the preparation method of specific transfer factor for resisting chicken bursa virus 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 (2)
1. the preparation method of an external preparation specific transfer factor for resisting chicken bursa virus is characterized in that being made up of following steps:
(1) aseptic chicken spleen or the chicken blood taked prepares the lymphocyte individual layer with spleen or peripheral blood;
(2) to the cultivation of going down to posterity of lymphocyte individual layer;
(3) after the lymph passage cell grows up to individual layer,, promptly obtain external preparation specific transfer factor for resisting chicken bursa virus with phytohaemagglutinin and bursal disease virus of chickens inducing culture.
2. according to the preparation method of external preparation specific transfer factor for resisting chicken bursa virus in claims 1, it is characterized in that: stimulating the mass concentration of the phytohaemagglutinin agent of chicken splenocyte and chicken blood lymphocyte propagation is 50-150mg/L.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102453088A (en) * | 2010-10-22 | 2012-05-16 | 曹吉祥 | Anti-chicken infectious bursal disease specific transfer factor and preparation method thereof |
| CN102697808A (en) * | 2012-05-04 | 2012-10-03 | 郑州后羿制药有限公司 | In-vitro preparation method of transfer factors capable of resisting duck viral hepatitis |
| 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 |
| 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 CN200810152419A patent/CN101759765A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102453088A (en) * | 2010-10-22 | 2012-05-16 | 曹吉祥 | Anti-chicken infectious bursal disease specific transfer factor and preparation method thereof |
| CN102697808A (en) * | 2012-05-04 | 2012-10-03 | 郑州后羿制药有限公司 | In-vitro preparation method of transfer factors capable of resisting duck viral hepatitis |
| CN102697808B (en) * | 2012-05-04 | 2014-02-05 | 郑州后羿制药有限公司 | In-vitro preparation method of transfer factors capable of resisting duck viral hepatitis |
| 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 |
| 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 |
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Open date: 20100630 |