CN102697808B - In-vitro preparation method of transfer factors capable of resisting duck viral hepatitis - Google Patents
In-vitro preparation method of transfer factors capable of resisting duck viral hepatitis Download PDFInfo
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Abstract
The invention relates to an in-vitro preparation method of transfer factors capable of resisting duck viral hepatitis. In the method, the transfer factors capable of resisting the duck viral hepatitis are prepared through the induction of duck hepatitis virus by utilizing an in-vitro lymphocyte cell culture method; and chicken spleen or peripheral blood is used as a basic raw material to prepare single-layer lymphocyte cells, and then the single-layer lymphocyte cells are induced and cultured through phytohemagglutinin and the duck hepatitis virus, so that a large number of specific transfer factors capable of resisting the duck hepatitis virus are generated in vitro.. The transfer factors can effectively inhibit the virus of duck viral hepatitis, so that the normal bodies cannot be infected with the duck viral hepatitis, and the radical pre-prevention action can be realized. The method provided by the invention has the characteristics of being simple and easy to operate and the like.
Description
Technical field
The preparation method that the present invention relates to a kind of In Vitro Anti duck viral hepatitis transfer factor, belongs to field of immunology.
Background technology
Duck viral hepatitis is a kind of acute height lethal infectious disease of duckling being caused by DHV (DVH), the feature of this disease is that morbidity is anxious, the course of disease is short, infection is fast, incidence and mortality higher disease all, to aquaculture, brings heavy economic losses.
Transfer factor (TF, Transfer Factor) claim again transmission factor, can be used as cellular immunization promoter, it is a kind of low molecular weight polypeptide-nucleotide complex that can shift sensitization information that T lymphocyte discharges, this material is transferred to the normal lymphocyte of receptor specifically by certain specific cellular immune function of donor, participate in the immunoreation of body, improve the cellular immune function of receptor; Promote bone-marrow-derived lymphocyte secretory action simultaneously, can discharge interferon and interleukin by inducing immune cells, thereby strengthen the resistance of receptor, this transfer factor molecular weight is little, nontoxic, active strong, no antigen, do not play the advantages such as anaphylaxis, for treatment zoosis toxicity disease, cellular immunization weakens or when defect disease and malignant tumor, fungal disease, can play auxiliary therapeutic action.
Although had at present medicine and the preparation of some anti-duck viral hepatitiss, because existing medicine preparation specific aim is not strong, curative effect is limited, and mostly curative preparation be just to apply after morbidity, can not play basic therapeutical effect.
The extracting method of the transfer factor of having reported is at present mostly about the preparation method of non-single transfer factor, and is mostly the preparation method of transfer factor immunization in body.
Summary of the invention
The preparation method that the object of this invention is to provide a kind of In Vitro Anti duck viral hepatitis transfer factor.
The technical solution adopted in the present invention is as follows: a kind of preparation method of In Vitro Anti duck viral hepatitis transfer factor, take chicken spleen or peripheral blood as basic material, prepare monolayer lymphocyte, through phytohaemagglutinin (Phytohemagglutinin, PHA) and DHV inducing culture monolayer lymphocyte, thus promote a large amount of anti-DHV special transfer factors of external generation.
The concrete steps of preparation method of the present invention are as follows:
1) lymphocytic preparation: first select healthy chicken, take chicken spleen or anticoagulation under aseptic condition, make monolayer lymphocyte;
2) lymphocytic cultivation: the lymphocyte cultivation of going down to posterity, with form of single sheet, go down to posterity, standby;
3) inoculation of virus: DHV is inoculated in the allantoic cavity of 9-10 day instar chicken embryo, cultivate 72-120h virus of proliferation for 37 ℃, allantoic fluid is collected, then centrifugal after ultrasonic disruption is processed, stay precipitation, according to viral particle size, utilize density gradient high speed centrifugation, obtain comparatively pure virus;
4) preparation of transfer factor: through phytohaemagglutinin and DHV inducing culture monolayer lymphocyte, obtain anti-duck viral hepatitis transfer factor.
Described in step 1), in chicken spleen cell preparation process, pancreas enzyme concentration requires 0.22-0.25%, and pH is 7.3-7.6, and consumption is 5-7 times of organizer accumulated amount.
The lectins working concentration that promotes chicken spleen cell or blood lymphocyte differentiation and proliferation described in step 4) is 60-180mg/L.
The present invention can suppress duck viral hepatitis virus efficiently about the anti-duck viral hepatitis transfer factor of external preparation, and protection normal body cell is avoided the infection of duck viral hepatitis, reaches the fundamentally effect of prevention.Thisly by going down to posterity, cultivate and can produce a large amount of products, the rate of output is high, and cost is low, can save a large amount of human and material resources, can avoid product to carry foreign aid's virus and other sex pheromones.This preparation method has the features such as simple, easy to operate, production cost is low.
The specific embodiment
In conjunction with specific embodiments content of the present invention is further described.
Embodiment 1
A kind of preparation method of In Vitro Anti duck viral hepatitis transfer factor, the present embodiment is to take chicken peripheral blood as basic material, prepare monolayer lymphocyte, through phytohaemagglutinin and DHV inducing culture monolayer lymphocyte, thereby promote a large amount of anti-DHV special transfer factors of external generation, concrete steps are as follows:
1. anticoagulant collection: select healthy free from infection chicken, blood sampling site sterilization, with the asepsis injector of 50mL, heart gathers anticoagulation, and adding concentration is the heparin sodium 5mL of 1mg/mL;
2. separation of lymphocytes: anticoagulation and lymphocyte separation medium (are bought the biological company limited of Beijing Puli, in operation instruction operation) in the ratio of 1:1, add in the centrifuge tube after sterilization, lymphocyte separation medium is added to the bottom of centrifuge tube, the centrifugal 15min of 3000r/min, the white layer in the middle of collecting; Then with the Hanks liquid of pH7.0, repeatedly clean lymphocyte 4 times, 5000r/min, centrifugal 10min, collecting precipitation, with the calf serum DMEM(containing 30%, buy Invitrogen Corporation, by operation instruction operation) 80mL fully mixes, and is then sub-packed in the Tissue Culture Flask of 100mL, is placed in 5% CO
2, in 37 ℃ of calorstats, cultivate, the cultivation through 2 days, lymphocyte adherent growth forms monolayer;
3. lymphocytic cultivation: the cultivation of going down to posterity of monolayer lymphocyte, first will grow up to 37 ℃ of short time digestion of pancreatin of 0.22% for monolayer buffy coat, piping and druming disperses, add the Hanks liquid of pH7.0 to stop, wash again 3 times, get 1/3 and join clean culture bottle, 5% CO
2, 37 ℃ of constant temperature culture, observe once every day, till growing up to monolayer;
4. the inoculation of virus: DHV is inoculated in the allantoic cavity of instar chicken embryo on the 9th, cultivates 72h virus of proliferation, allantoic fluid is collected for 37 ℃, then centrifugal after ultrasonic disruption is processed, stay precipitation, according to viral particle size, utilize density gradient high speed centrifugation, obtain comparatively pure virus;
5. inducing culture: after monolayer lymphocyte grows up to, be replaced with maintenance medium (containing 5% calf serum DMEM), add concentration is the phytohaemagglutinin of 60mg/L simultaneously, and DHV (concentration is 1280 HAUs/mL) carries out inducing culture; Through the inducing culture of 3 days, then by cell bottle process micro oscillation, culture fluid dislocation is centrifugal in centrifuge tube, collect supernatant;
6. the preparation of transfer factor: supernatant utilizes ultrasonic cell disruption instrument (duty: broken 3s stops 3s, 400W, 10 min) after fragmentation, centrifuging and taking supernatant, through 0.22um membrane filtration, filtrate is 6000 daltonian doughnut membrane ultrafiltration through molecular cut off, utilize positive pressure ultrafiltration, collect filtrate filtration sterilization again, packing, obtains anti-duck viral hepatitis transfer factor.
Embodiment 2
A kind of preparation method of In Vitro Anti duck viral hepatitis transfer factor, the present embodiment is to take chicken peripheral blood as basic material, prepare monolayer lymphocyte, through phytohaemagglutinin and DHV inducing culture monolayer lymphocyte, thereby promote a large amount of anti-DHV special transfer factors of external generation, concrete steps are as follows:
1. anticoagulant collection: select healthy free from infection chicken, blood sampling site sterilization, with the asepsis injector of 50mL, heart gathers anticoagulation, and adding concentration is the heparin sodium 5mL of 1mg/mL;
2. separation of lymphocytes: anticoagulation and lymphocyte separation medium (are bought the biological company limited of Beijing Puli, in operation instruction operation) in the ratio of 1:1, add in the centrifuge tube after sterilization, lymphocyte separation medium is added to the bottom of centrifuge tube, the centrifugal 20min of 2500r/min, the white layer in the middle of collecting; Then with the Hanks liquid of pH7.2, repeatedly clean lymphocyte 4 times, 4000r/min, centrifugal 10min, collecting precipitation, with the calf serum DMEM(containing 30%, buy Invitrogen Corporation, by operation instruction operation) 80mL fully mixes, and is then sub-packed in the Tissue Culture Flask of 100mL, is placed in 5% CO
2, in 37 ℃ of Rotary Machines, cultivate, the cultivation through 3 days, lymphocyte adherent growth forms monolayer;
3. lymphocytic cultivation: the cultivation of going down to posterity of monolayer lymphocyte, first will grow up to 37 ℃ of digestion 2 min of pancreatin of 0.25% for monolayer buffy coat, piping and druming disperses, add the Hanks liquid of pH7.2 to stop, wash again 2 times, get 1/3 and join clean culture bottle, 5% CO
2, 37 ℃ of constant temperature culture, observe once every day, till growing up to monolayer;
4. the inoculation of virus: DHV is inoculated in the allantoic cavity of instar chicken embryo on the 10th, cultivates 96h virus of proliferation, allantoic fluid is collected for 37 ℃, then centrifugal after ultrasonic disruption is processed, stay precipitation, according to viral particle size, utilize density gradient high speed centrifugation, obtain comparatively pure virus;
5. inducing culture: after monolayer lymphocyte grows up to, be replaced with maintenance medium (containing 5% calf serum DMEM), add concentration is the phytohaemagglutinin of 180mg/L simultaneously, DHV (concentration is 1280 HAUs/mL) carries out inducing culture, inducing culture through 3 days, then by cell bottle process micro oscillation, culture fluid dislocation is centrifugal in centrifuge tube, collect supernatant;
6. the preparation of transfer factor: supernatant is through ultrasonic cell disruption instrument (duty: broken 3s stops 3s, 300W, 10 min) after fragmentation, centrifuging and taking supernatant, through 0.22um membrane filtration, filtrate is 6000 daltonian doughnut membrane ultrafiltration through molecular cut off, utilize positive pressure ultrafiltration, collect filtrate filtration sterilization again, packing, obtains anti-duck viral hepatitis transfer factor.
Embodiment 3
A preparation method for In Vitro Anti duck viral hepatitis transfer factor, the present embodiment mainly comprises that chicken spleen prepares monolayer lymphocyte, adds the steps such as phytohaemagglutinin and virus induction, concrete steps are as follows:
1. the collection of spleen: the chicken of choosing healthy free from infection, under aseptic condition, win spleen, then with sterile saline, rinse surperficial blood and visceral surface liquid, disinfect surface in alcohol, reject organ surface fatty tissue and fascia, with aseptic eye scissors and ophthalmic tweezers, from spleen inside, get portion of tissue, be placed in the beaker of 30mL, then shredded into the piece that diameter is less than 0.1cm, with the Hanks liquid of pH7.2, clean blood and the impurity in 3 broken tissues again, guarantee that as far as possible tissue is clean;
2. splenocyte preparation: the pH that adds 5 times of amounts in above-mentioned broken tissue is 7.3, the trypsin solution of concentration 0.22%, the water-bath of putting into 37 ℃ digests 20min, and every 5 min shake once, so that fully digestion of tissue, until tissue become transparent, till periphery is scared, illustrate that digestion is complete, can take out beaker, draw pancreatic juice, the Hanks liquid that adds preheating maintenance medium pH7.2, fully makes for 70 times tissue disperse with suction pipe piping and druming, then leaves and takes after filtration filtrate;
3. lymphocytic cultivation: adopt blood counting chamber to count, get filtrate 10uL, dilute 100 times, then micro-Microscopic observation, the same with cytometry rule, determines extension rate; To be sub-packed in Tissue Culture Flask with the filtrate after the dilution of DMEM liquid condition of culture: 5% CO
2, cultivate on 37 ℃ of calorstats, observe twice every day, examine under a microscope and whether form cell monolayer, determine the cultivation of going down to posterity;
4. the inoculation of virus: DHV is inoculated in the allantoic cavity of instar chicken embryo on the 9th, cultivates 120h virus of proliferation, allantoic fluid is collected for 37 ℃, then centrifugal after ultrasonic disruption is processed, stay precipitation, according to viral particle size, utilize density gradient high speed centrifugation, obtain comparatively pure virus;
5. inducing culture: go down to posterity and cultivate into after monolayer lymphocyte, replacing nutritional solution is maintenance medium, and add concentration is 100mg/L phytohaemagglutinin and DHV (concentration is 1280 HAUs/mL) simultaneously, carries out the inducing culture of 2 days;
6. the preparation of transfer factor: utilize ultrasonic cell disruption instrument (duty: broken 3s stops 3s, 400W, 10 min) after fragmentation, centrifuging and taking supernatant, filters, then is 6000 daltonian doughnut membrane ultrafiltration through molecular cut off, utilize positive pressure ultrafiltration, collect filtrate filtration sterilization again, packing, is anti-duck viral hepatitis transfer factor.
Embodiment 4
A preparation method for In Vitro Anti duck viral hepatitis transfer factor, the present embodiment mainly comprises that chicken spleen prepares monolayer lymphocyte, adds the steps such as phytohaemagglutinin and virus induction, concrete steps are as follows:
1. the collection of spleen: the chicken of choosing healthy free from infection, under aseptic condition, win spleen, then with sterile saline, rinse surperficial blood and visceral surface liquid, disinfect surface in alcohol, reject organ surface fatty tissue and fascia, with aseptic eye scissors and ophthalmic tweezers, from spleen inside, get portion of tissue, be placed in the beaker of 30mL, then shredded into the piece that diameter is less than 0.1cm, with the Hanks liquid of pH7.0, clean blood and the impurity in 3 broken tissues again, guarantee that as far as possible tissue is clean;
2. splenocyte preparation: the pH that adds 7 times of amounts in above-mentioned broken tissue is 7.6, the trypsin solution of concentration 0.25%, the water-bath of putting into 37 ℃ digests 30min, and every 5 min shake once, so that fully digestion of tissue, until tissue become transparent, till periphery is scared, illustrate that digestion is complete, can take out beaker, draw pancreatic juice, the Hanks liquid that adds preheating maintenance medium pH7.0, fully makes for 90 times tissue disperse with suction pipe piping and druming, then leaves and takes after filtration filtrate;
3. lymphocytic cultivation: adopt blood counting chamber to count, get filtrate 10uL, dilute 100 times, then micro-Microscopic observation, the same with cytometry rule, determines extension rate; To be sub-packed in cell spinner bottle with the filtrate after the dilution of DMEM liquid condition of culture: 5% CO
2, cultivate on 37 ℃ of calorstats, observe twice every day, examine under a microscope and whether form cell monolayer, determine the cultivation of going down to posterity;
4. the inoculation of virus: DHV is inoculated in the allantoic cavity of instar chicken embryo on the 10th, cultivates 120h virus of proliferation, allantoic fluid is collected for 37 ℃, then centrifugal after ultrasonic disruption is processed, stay precipitation, according to viral particle size, utilize density gradient high speed centrifugation, obtain comparatively pure virus;
5. inducing culture: go down to posterity and cultivate into after monolayer lymphocyte, replacing nutritional solution is maintenance medium, and add concentration is 140mg/L phytohaemagglutinin and DHV (concentration is 1280 HAUs/mL) simultaneously, carries out the inducing culture of 2 days;
6. the preparation of transfer factor: utilize ultrasonic cell disruption instrument (duty: broken 3s stops 3s, 400W, 10 min) after fragmentation, centrifuging and taking supernatant, filters, then is 6000 daltonian doughnut membrane ultrafiltration through molecular cut off, utilize positive pressure ultrafiltration, collect filtrate filtration sterilization again, packing, is anti-duck viral hepatitis transfer factor.
Experimental example 1: animal protection test:
Get 300 healthy ducklings of 12 ages in days, be divided at random contrast, test, blank three groups, 100 every group, in new environment, adapting to three days, free choice feeding, counteracting toxic substances dosage is 1.0 10
8only, after counteracting toxic substances, the 3rd day test group starts to inoculate anti-duck viral hepatitis transfer factor to CFU/, and every intramuscular injection 0.2ml/ only, once a day, matched group injection physiology salt, blank omnidistance normal saline, observes clinical symptoms and mortality rate as the clinical application effect of index reaction this product.Result shows: matched group sickness rate 100% and mortality rate 100%, blank group does not change, test group sickness rate 25%, and mortality rate 3%.
Experimental example 2: animal safety test
Get 150 of healthy mices, be divided at random blank, injection group, oral three groups, 50 every group, only, only, oral group only gavages this product 5 ml/, observes the variation of white mice injection group injection this product 5 ml/ blank oral and injection distilled water 2 ml/.Result shows that three groups all without ANOMALOUS VARIATIONS, and this product safety is described, has no adverse reaction.
Experimental example 3: irritation test
Get 30 TWOs, be divided into 2 groups of contrasts, subcutaneous injection, intradermal injection, adopt depilatory to be sloughed by hair rabbit spinal column both sides, guarantee the integrity of skin, then only inject this product 1 ml/, matched group injecting normal saline, is divided into the variation of single-dose, multiple dosing observation skin.Result demonstration, 10 of injecting normal saline is merely hit, and has 5 redness occurs, and detumescence in a day, respectively has 3 generations in test group, all detumescences in 12 hours.
Experimental example 4
This experimental example adopts the detection of various conventional methods to the anti-duck viral hepatitis transfer factor of invention:
1, the mensuration of content of peptides: with Folin-phenol method or spectrophotometry polypeptide, take duck peripheral blood as basic material content of peptides 0.46g/L, the transfer factor content of peptides 0.39g/L that the duck spleen of take is prepared as raw material.
2, protein measuring: adopt 20% sulfosalicylic acid method, muddiness and deposited phenomenon do not occur extracting solution, is negative, not containing protein.
3, aseptic detection: detect according to the 2011 editions > > of veterinary drug allusion quotation of the < < People's Republic of China (PRC), there is no bacterial growth.
4, Ribose concentration is measured: utilize spectrophotometry, take duck peripheral blood as basic material Ribose concentration 0.34g/L, the transfer factor Ribose concentration 0.43g/L that the duck spleen of take is prepared as raw material.
5, outward appearance and pH value are measured: yellow clear liquid, the value that acidity value is 7.0-7.3.
By above experimental example; we can obtain the anti-duck viral hepatitis transfer factor that the present invention extracts can not only suppress duck viral hepatitis virus safely and efficiently; can also protect normal body cell to avoid the infection of duck viral hepatitis, improve the immunity of body.The external preparation method of this product is feasible, and this inventive method is not only easy and simple to handle, reduces working strength, and production cost is low, should extensively promote clinically.
Claims (3)
1. the preparation method of an In Vitro Anti duck viral hepatitis transfer factor, it is characterized in that: take chicken spleen or peripheral blood as basic material, prepare monolayer lymphocyte, through phytohaemagglutinin and DHV inducing culture monolayer lymphocyte, thereby promote a large amount of anti-DHV special transfer factors of external generation, concrete steps are as follows:
1) lymphocytic preparation: first select healthy chicken, take chicken spleen or chicken anticoagulation under aseptic condition, make monolayer lymphocyte;
2) lymphocytic cultivation: the lymphocyte cultivation of going down to posterity, with form of single sheet, go down to posterity, standby;
3) inoculation of virus: DHV is inoculated in the allantoic cavity of 9-10 day instar chicken embryo, cultivate 72-120h virus of proliferation for 37 ℃, allantoic fluid is collected, then centrifugal after ultrasonic disruption is processed, stay precipitation, according to viral particle size, utilize density gradient high speed centrifugation, obtain comparatively pure virus;
4) preparation of transfer factor: through phytohaemagglutinin and DHV inducing culture monolayer lymphocyte, utilize after ultrasonic cell disruption instrument fragmentation, centrifuging and taking supernatant, through 0.22 μ m membrane filtration, filtrate is 6000 daltonian doughnut membrane ultrafiltration through molecular cut off, utilizes positive pressure ultrafiltration, collects filtrate filtration sterilization again, packing, obtains anti-duck viral hepatitis transfer factor; The duty of ultrasonic cell disruption instrument: broken 3s stops 3s, 400W, 10min.
2. the preparation method of a kind of In Vitro Anti duck viral hepatitis transfer factor according to claim 1, it is characterized in that: described in step 1), in chicken spleen cell preparation process, pancreas enzyme concentration requires 0.22-0.25%, pH is 7.3-7.6, and consumption is 5-7 times of organizer accumulated amount.
3. the preparation method of a kind of In Vitro Anti duck viral hepatitis transfer factor according to claim 1, is characterized in that: the lectins working concentration that promotes chicken spleen cell or blood lymphocyte differentiation and proliferation described in step 4) is 60-180mg/L.
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| 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 |
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| CN101759765A (en) * | 2008-10-21 | 2010-06-30 | 天津生机集团股份有限公司 | Method for extracorporeally preparing transfer factor against specificity of bursal disease virus of chickens |
| CN101953849A (en) * | 2010-10-19 | 2011-01-26 | 郑州后羿制药有限公司 | Method for preparing anti-duck viral hepatitis transfer factor |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101759765A (en) * | 2008-10-21 | 2010-06-30 | 天津生机集团股份有限公司 | Method for extracorporeally preparing transfer factor against specificity of bursal disease virus of chickens |
| CN101953849A (en) * | 2010-10-19 | 2011-01-26 | 郑州后羿制药有限公司 | Method for preparing anti-duck viral hepatitis transfer factor |
Non-Patent Citations (1)
| Title |
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| 傅先强等."第四章 鸡胚内病毒的培养".《养禽场禽病检验手册》.中国农业大学出版社,1997,(第1版),第87-90页. |
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