CN105560282A - Preparation method of anti-duck hepatitis virus transfer factor - Google Patents
Preparation method of anti-duck hepatitis virus transfer factor Download PDFInfo
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Abstract
The invention discloses a preparation method of an anti-duck hepatitis virus transfer factor. The method is carried out according to the steps of: inoculating a duck hepatitis virus to the allantoic fluid of a 9-11 days old chicken embryo or duck embryo to prepare a virus antigen; extracting a duck hepatitis virus antigen; immunizing healthy pigs with the extracted virus antigen; and extracting the anti-duck hepatitis virus transfer factor from the immunized pigs. Therefore, the transfer factor provided by the invention can play a role of preventing duck hepatitis virus infection and protecting normal body cells from the invasion of duck hepatitis virus, thereby reducing the morbidity. And the method has very good application prospect.
Description
Technical field
The present invention relates to field of veterinary, in particular, relate to a kind of preparation method of anti-DHV transfer factor.
Background technology
It is scorching that duck viral hepatitis is called for short duck liver, is to cause the one of duckling to propagate rapidly and height lethal infectious diseases by DHV, is the acute deadly infectious disease that a kind of liver presents hemorrhage inflammatory conditions.The principal character of this disease is liver enlargement is mottled hemorrhage, gollbladder dilation, and bile color is thin out, has hemorrhage speckle and nervous symptoms.In primary infection epidemic-stricken area, the mortality rate of this disease is very high, can reach more than 90%.
Duck viral hepatitis mainly betides 4 ~ 20 age in days ducklings, and middle one-tenth duck does not generally fall ill, and chicken and goose can not natural occurrences.The major source of infection of this disease is disease duck and the malicious duck of band, and mainly through digestive tract and respiratory tract infection, feeding and management is bad, and as being deficient in vitamin and mineral, duck shed is moist, crowded, and primary disease all can be impelled to occur.Primary disease betides duckling and hatches season, once outburst, propagate very fast, sickness rate can reach 100%, has carried out very large economic loss to cultivating industrial belt.
Transfer factor (Transferfactor, TF) be a kind of dialysed small-molecule substance-polypeptide nucleotide complex that can shift sensitization information that T lymphocyte discharges, the cellular immunization information of donor can be transferred to the normal lymphocyte of receptor by specifically, thus strengthen the immunologic function of receptor, be described as the triggering agent of T cell activity, the reinforcing agent of cellular immunization, cell immunomodulator and interferon and produce and start agent.There is premunition information, excite immunologic cellular activity, the effect such as immunity moderation function, enhancing body specificity and Nonspecific immunity function, the non-bacterial infection such as fungus, virus can be resisted.TF is containing Multiple components, and molecular weight is little, apyrogeneity, no antigen, has no side effect and without species variation, is a kind of novel and safe immune formulation.
The production of current transfer factor is mainly obtained by semipermeable membrane dialysis, and disposable volume of production is few, and operating cost is high, therefore needs a kind of efficient, quick, economic method to obtain transfer factor, and then realizes the large-scale industrial production of transfer factor.In addition, the place that pig spleen, thymus and lymph node tissue are assembled as immunologically competent cell, and be the leftover bits and pieces after live hog is slaughtered, the lower and material source of cost is widely, therefore from this kind of cell, extract specific transfer factor, have important theory significance and realistic meaning.
Summary of the invention
Technical problem to be solved by this invention overcomes the deficiencies in the prior art, provides the preparation method of the anti-DHV transfer factor of a kind of efficiency natural pure bioactive material.
The preparation method of the anti-DHV transfer factor of the present invention, carry out according to following steps:
(1) virus antigen is prepared: in Embryo Gallus domesticus DHV being inoculated in 9-11 age in days or duck embryo allantoic liquid, 33 DEG C of-37 DEG C of incubations, Embryo Gallus domesticus dead in 24h or duck embryo discard, the Embryo Gallus domesticus or the duck embryo allantocherion that have obvious pox speckle is collected after 120h, after phosphate buffer (PBS) washing, be placed in tissue refiner and make suspension, save backup in-20 DEG C of--40 DEG C of refrigerators;
(2) virus antigen is extracted: after obtaining virus antigen multigelation 3-5 time in step (1), in loading heavy wall ampoule, ultrasonic 15-20min, 4 DEG C, the centrifugal 20-30min of 5000rpm, discard precipitation, collect supernatant, by supernatant through 4 DEG C, the centrifugal 20-30min of 7000rpm, discard precipitation, collect supernatant, through 4 DEG C, the centrifugal 20-30min of 25000rpm, abandoning supernatant, get precipitation, dissolve by equal-volume PBS solution.Above-mentioned solution is loaded the discontinuous gradient density centrifugation pipe prepared with the sucrose of 15%, 30%, 45%, 60% and 80%, ultracentrifugation, 4 DEG C, the centrifugal 2-4h of 15000-20000rpm, according to the molecular weight determination DHV position of DHV, sucking-off being placed in-20 DEG C of--40 DEG C of refrigerators saves backup;
(3) with the virus antigen immune swine that step (2) gained extracts: select healthy immune swine, get the virus antigen of preparation, adopt subcutaneous multiple spot immunization method, injecting immune pig, immune 3-4 time altogether, front twice respectively with complete freund adjuvant and incomplete freund adjuvant hybrid antigen, beat with high-speed homogenizer, in animal skins or subcutaneous multi-point injection immunity, in the muscle of animal or intravenous injection after 3rd time and the 4th, each immunization interval one week, animals iv blood is got after last immune 7-10 days, the specific antibody titres of above-mentioned virus is detected with immunofluorescence method or ELISA method, after occurring that virus specific immunity is tired, slaughter animal, get spleen and thymus on ice, be transferred to-20 DEG C--40 DEG C preservations, for subsequent use,
(4) from immune swine, anti-DHV transfer factor is extracted:
1. raw-material preparation and pretreatment: get the spleen of the inoculation DHV immune swine of step (3) gained, thymus and lymph node tissue, the spleen of pig is cleaned with cold tri-distilled water, remove the tissues such as surperficial fascia, fat, then sterile saline rinses repeatedly;
2. broken: the pig spleen of wash clean to be shredded, adds the cold saline of 2 times of volumes, under freezing condition, smash 3 times with high-speed tissue mashing machine to pieces with 1000r/min, each 3min, obtained homogenate;
3. freeze thawing: homogenate is placed in-80 DEG C of quick freezing, melts in water-bath 30 DEG C, alternate freezing and thawing 4-8 time, all pulverizes with tissue refiner high-speed homogenization after each thawing;
4. extract: the cold saline adding 3 times of volumes, be placed in refrigerated centrifuge in 5 DEG C with the centrifugal 30min of 4000r/min, get the blood red liquid in upper strata, then with the filtration of G2 sand core funnel, get filtrate;
5. ultrafiltration: be 5000 daltonian hollow fiber ultrafiltration membrane pressurization ultrafiltration by above-mentioned filtrate shut off value, filtrate is that molecular weight is less than 5000 daltonian transfer factors;
6. degerming: filtrate aperture ultrafiltration obtained is that the filter membrane pressure filtration of 0.1-0.22um is degerming;
7. subpackage: 4 DEG C of sealings are preserved, and obtain anti-DHV transfer factor extracting solution finished product.
Advantage of the present invention and beneficial effect are:
1, raw material sources is abundant, pollution-free, cost is low.Pig spleen, thymus and lymph node tissue belong to the leftover bits and pieces of butchering rear pig.
2, after disrupting tissue, add the step of low temperature freeze thawing treatment, particularly dissolve below 37 DEG C, do not make small-molecule substance loss of activity, and DHV transfer factor can be made to be extracted fully.
3, technique have employed more advanced hollow fiber ultrafiltration membrane ultra-filtration method, thus shortens preparation time, ensures that in separating substances process, activity is not destroyed.
4, extract is through biochemical measurement and some component analyses, is a kind of low molecular weight polypeptide-nucleotide complex, be a kind of containing egg from matter, solvable small-molecule substance, mean molecule quantity is below 5000 dalton.Through inside and outside biological activity test and the proof such as pharmacology, toxicity test, have that purity is high, immunocompetence is strong, rapid-action and hold time long, easily absorb, without features such as any untoward reaction, its determined curative effect, safe and reliable, not only can treat but also can resistance against diseases be strengthened.This extract can make the dosage form such as oral liquid or injection, is convenient to application.
Anti-DHV transfer factor of the present invention effectively can suppress DHV, and normal body cell can be protected from viral infection.Therefore use the effect that transfer factor of the present invention can be played preventing duck hepatites virus infections, normal body cell can be protected to encroach on from DHV, thus reduce sickness rate.Simultaneously for suffering that the duck that DHV infects uses transfer factor to treat, effectively can improve clinical symptoms, improving cure rate, reduce mortality rate.
Detailed description of the invention
The present invention is described in further detail by following examples.It should be noted that: following embodiment is illustrative, is not determinate, can not limit protection scope of the present invention with following embodiment.
Embodiment 1
A preparation method for anti-DHV transfer factor, its step is as follows:
(1) virus antigen is prepared: be inoculated in by DHV in the chick embryo allantoic liquid of 10 ages in days, 37 DEG C of incubations, Embryo Gallus domesticus dead in 24h discards, the chick chorioallantoic membrane having obvious pox speckle is collected after 120h, after phosphate buffer (PBS) washing, be placed in tissue refiner and make suspension, save backup in-20 DEG C of refrigerators;
(2) extract virus antigen: after obtaining virus antigen multigelation 3 times in step (1), load in heavy wall ampoule, ultrasonic 20min, 4 DEG C, the centrifugal 30min of 5000rpm, discard precipitation, collect supernatant, by supernatant through 4 DEG C, the centrifugal 20min of 7000rpm, discard precipitation, collect supernatant, through 4 DEG C, the centrifugal 30min of 25000rpm, abandoning supernatant, get precipitation, dissolve by equal-volume PBS solution.Above-mentioned solution is loaded the discontinuous gradient density centrifugation pipe prepared with the sucrose of 15%, 30%, 45%, 60% and 80%, ultracentrifugation, 4 DEG C, the centrifugal 4h of 18000rpm, according to the molecular weight determination DHV position of DHV, sucking-off being placed in-20 DEG C of refrigerators saves backup;
(3) with the virus antigen immune swine that step (2) gained extracts: select healthy immune swine, get the virus antigen of preparation, adopt subcutaneous multiple spot immunization method, injecting immune pig, immunity 4 times altogether, front twice respectively with complete freund adjuvant and incomplete freund adjuvant hybrid antigen, beat with high-speed homogenizer, in animal skins or subcutaneous multi-point injection immunity, in the muscle of animal or intravenous injection after 3rd time and the 4th, each immunization interval one week, animals iv blood is got after last immune 8 days, the specific antibody titres of above-mentioned virus is detected with immunofluorescence method or ELISA method, after occurring that virus specific immunity is tired, slaughter animal, get spleen and thymus on ice, be transferred to-20 DEG C of preservations, for subsequent use,
(4) from immune swine, anti-DHV transfer factor is extracted:
1. raw-material preparation and pretreatment: get the spleen of the inoculation DHV immune swine of step (3) gained, thymus and lymph node tissue, the spleen of pig is cleaned with cold tri-distilled water, remove the tissues such as surperficial fascia, fat, then sterile saline rinses repeatedly;
2. broken: the pig spleen of wash clean to be shredded, adds the cold saline of 2 times of volumes, under freezing condition, smash 3 times with high-speed tissue mashing machine to pieces with 1000r/min, each 3min, obtained homogenate;
3. freeze thawing: homogenate is placed in-80 DEG C of quick freezing, melts in water-bath 30 DEG C, alternate freezing and thawing 6 times, all pulverizes with tissue refiner high-speed homogenization after each thawing;
4. extract: the cold saline adding 3 times of volumes, be placed in refrigerated centrifuge in 5 DEG C with the centrifugal 30min of 4000r/min, get the blood red liquid in upper strata, then with the filtration of G2 sand core funnel, get filtrate;
5. ultrafiltration: be 5000 daltonian hollow fiber ultrafiltration membrane pressurization ultrafiltration by above-mentioned filtrate shut off value, filtrate is that molecular weight is less than 5000 daltonian transfer factors;
6. degerming: filtrate aperture ultrafiltration obtained is that the filter membrane pressure filtration of 0.22 μm is degerming;
7. subpackage: 4 DEG C of sealings are preserved, and obtain anti-DHV transfer factor extracting solution finished product.
Embodiment 2
A preparation method for anti-DHV transfer factor, its step is as follows:
(1) virus antigen is prepared: be inoculated in by DHV in the duck embryo allantoic liquid of 10 ages in days, 33 DEG C of incubations, duck embryo dead in 24h discards, the duck embryo allantocherion having obvious pox speckle is collected after 120h, after phosphate buffer (PBS) washing, be placed in tissue refiner and make suspension, save backup in-20 DEG C of refrigerators;
(2) extract virus antigen: after obtaining virus antigen multigelation 3 times in step (1), load in heavy wall ampoule, ultrasonic 15min, 4 DEG C, the centrifugal 20min of 5000rpm, discard precipitation, collect supernatant, by supernatant through 4 DEG C, the centrifugal 20min of 7000rpm, discard precipitation, collect supernatant, through 4 DEG C, the centrifugal 20min of 25000rpm, abandoning supernatant, get precipitation, dissolve by equal-volume PBS solution.Above-mentioned solution is loaded the discontinuous gradient density centrifugation pipe prepared with the sucrose of 15%, 30%, 45%, 60% and 80%, ultracentrifugation, 4 DEG C, the centrifugal 2h of 15000rpm, according to the molecular weight determination DHV position of DHV, sucking-off being placed in-20 DEG C of DEG C of refrigerators saves backup;
(3) with the virus antigen immune swine that step (2) gained extracts: select healthy immune swine, get the virus antigen of preparation, adopt subcutaneous multiple spot immunization method, injecting immune pig, immunity 4 times altogether, front twice respectively with complete freund adjuvant and incomplete freund adjuvant hybrid antigen, beat with high-speed homogenizer, in animal skins or subcutaneous multi-point injection immunity, in the muscle of animal or intravenous injection after 3rd time and the 4th, each immunization interval one week, animals iv blood is got after last immune 10 days, the specific antibody titres of above-mentioned virus is detected with immunofluorescence method or ELISA method, after occurring that virus specific immunity is tired, slaughter animal, get spleen and thymus on ice, be transferred to-20 DEG C of DEG C of preservations, for subsequent use,
(4) from immune swine, anti-DHV transfer factor is extracted:
1. raw-material preparation and pretreatment: get the spleen of the inoculation DHV immune swine of step (3) gained, thymus and lymph node tissue, the spleen of pig is cleaned with cold tri-distilled water, remove the tissues such as surperficial fascia, fat, then sterile saline rinses repeatedly;
2. broken: the pig spleen of wash clean to be shredded, adds the cold saline of 2 times of volumes, under freezing condition, smash 3 times with high-speed tissue mashing machine to pieces with 1000r/min, each 3min, obtained homogenate;
3. freeze thawing: homogenate is placed in-80 DEG C of quick freezing, melts in water-bath 30 DEG C, alternate freezing and thawing 4 times, all pulverizes with tissue refiner high-speed homogenization after each thawing;
4. extract: the cold saline adding 3 times of volumes, be placed in refrigerated centrifuge in 5 DEG C with the centrifugal 30min of 4000r/min, get the blood red liquid in upper strata, then with the filtration of G2 sand core funnel, get filtrate;
5. ultrafiltration: be 5000 daltonian hollow fiber ultrafiltration membrane pressurization ultrafiltration by above-mentioned filtrate shut off value, filtrate is that molecular weight is less than 5000 daltonian transfer factors;
6. degerming: filtrate aperture ultrafiltration obtained is that the filter membrane pressure filtration of 0.22um is degerming;
7. subpackage: 4 DEG C of sealings are preserved, and obtain anti-DHV transfer factor extracting solution finished product.
Embodiment 3
A preparation method for anti-DHV transfer factor, its step is as follows:
(1) virus antigen is prepared: be inoculated in by DHV in the chick embryo allantoic liquid of 11 ages in days, 35 DEG C of incubations, Embryo Gallus domesticus dead in 24h discards, the chick chorioallantoic membrane having obvious pox speckle is collected after 120h, after phosphate buffer (PBS) washing, be placed in tissue refiner and make suspension, save backup in-40 DEG C of refrigerators;
(2) extract virus antigen: after obtaining virus antigen multigelation 5 times in step (1), load in heavy wall ampoule, ultrasonic 20min, 4 DEG C, the centrifugal 30min of 5000rpm, discard precipitation, collect supernatant, by supernatant through 4 DEG C, the centrifugal 30min of 7000rpm, discard precipitation, collect supernatant, through 4 DEG C, the centrifugal 30min of 25000rpm, abandoning supernatant, get precipitation, dissolve by equal-volume PBS solution.Above-mentioned solution is loaded the discontinuous gradient density centrifugation pipe prepared with the sucrose of 15%, 30%, 45%, 60% and 80%, ultracentrifugation, 4 DEG C, the centrifugal 4h of 20000rpm, according to the molecular weight determination DHV position of DHV, sucking-off being placed in-40 DEG C of refrigerators saves backup;
(3) with the virus antigen immune swine that step (2) gained extracts: select healthy immune swine, get the virus antigen of preparation, adopt subcutaneous multiple spot immunization method, injecting immune pig, immunity 4 times altogether, front twice respectively with complete freund adjuvant and incomplete freund adjuvant hybrid antigen, beat with high-speed homogenizer, in animal skins or subcutaneous multi-point injection immunity, in the muscle of animal or intravenous injection after 3rd time and the 4th, each immunization interval one week, animals iv blood is got after last immune 8 days, the specific antibody titres of above-mentioned virus is detected with immunofluorescence method or ELISA method, after occurring that virus specific immunity is tired, slaughter animal, get spleen and thymus on ice, be transferred to-40 DEG C of preservations, for subsequent use,
(4) from immune swine, anti-DHV transfer factor is extracted:
1. raw-material preparation and pretreatment: get the spleen of the inoculation DHV immune swine of step (3) gained, thymus and lymph node tissue, the spleen of pig is cleaned with cold tri-distilled water, remove the tissues such as surperficial fascia, fat, then sterile saline rinses repeatedly;
2. broken: the pig spleen of wash clean to be shredded, adds the cold saline of 2 times of volumes, under freezing condition, smash 3 times with high-speed tissue mashing machine to pieces with 1000r/min, each 3min, obtained homogenate;
3. freeze thawing: homogenate is placed in-80 DEG C of quick freezing, melts in water-bath 30 DEG C, alternate freezing and thawing 8 times, all pulverizes with tissue refiner high-speed homogenization after each thawing;
4. extract: the cold saline adding 3 times of volumes, be placed in refrigerated centrifuge in 5 DEG C with the centrifugal 30min of 4000r/min, get the blood red liquid in upper strata, then with the filtration of G2 sand core funnel, get filtrate;
5. ultrafiltration: be 5000 daltonian hollow fiber ultrafiltration membrane pressurization ultrafiltration by above-mentioned filtrate shut off value, filtrate is that molecular weight is less than 5000 daltonian transfer factors;
6. degerming: filtrate aperture ultrafiltration obtained is that the filter membrane pressure filtration of 0.22um is degerming;
7. subpackage: 4 DEG C of sealings are preserved, and obtain anti-DHV transfer factor extracting solution finished product.
Embodiment 4:
A preparation method for anti-DHV transfer factor, its step is as follows:
(1) virus antigen is prepared: be inoculated in by DHV in the duck embryo allantoic liquid of 9 ages in days, 35 DEG C of incubations, duck embryo dead in 24h discards, the chick chorioallantoic membrane having obvious pox speckle is collected after 120h, after phosphate buffer (PBS) washing, be placed in tissue refiner and make suspension, save backup in-20 DEG C of refrigerators;
(2) extract virus antigen: after obtaining virus antigen multigelation 4 times in step (1), load in heavy wall ampoule, ultrasonic 30min, 4 DEG C, the centrifugal 20min of 5000rpm, discard precipitation, collect supernatant, by supernatant through 4 DEG C, the centrifugal 30min of 7000rpm, discard precipitation, collect supernatant, through 4 DEG C, the centrifugal 30min of 25000rpm, abandoning supernatant, get precipitation, dissolve by equal-volume PBS solution.Above-mentioned solution is loaded the discontinuous gradient density centrifugation pipe prepared with the sucrose of 15%, 30%, 45%, 60% and 80%, ultracentrifugation, 4 DEG C, the centrifugal 4h of 18000rpm, according to the molecular weight determination DHV position of DHV, sucking-off being placed in-20 DEG C of refrigerators saves backup;
(3) with the virus antigen immune swine that step (2) gained extracts: select healthy immune swine, get the virus antigen of preparation, adopt subcutaneous multiple spot immunization method, injecting immune pig, immunity 4 times altogether, front twice respectively with complete freund adjuvant and incomplete freund adjuvant hybrid antigen, beat with high-speed homogenizer, in animal skins or subcutaneous multi-point injection immunity, in the muscle of animal or intravenous injection after 3rd time and the 4th, each immunization interval one week, animals iv blood is got after last immune 8 days, the specific antibody titres of above-mentioned virus is detected with immunofluorescence method or ELISA method, after occurring that virus specific immunity is tired, slaughter animal, get spleen and thymus on ice, be transferred to-20 DEG C of preservations, for subsequent use,
(4) from immune swine, anti-DHV transfer factor is extracted:
1. raw-material preparation and pretreatment: get the spleen of the inoculation DHV immune swine of step (3) gained, thymus and lymph node tissue, the spleen of pig is cleaned with cold tri-distilled water, remove the tissues such as surperficial fascia, fat, then sterile saline rinses repeatedly;
2. broken: the pig spleen of wash clean to be shredded, adds the cold saline of 2 times of volumes, under freezing condition, smash 3 times with high-speed tissue mashing machine to pieces with 1000r/min, each 3min, obtained homogenate;
3. freeze thawing: homogenate is placed in-80 DEG C of quick freezing, melts in water-bath 30 DEG C, alternate freezing and thawing 6 times, all pulverizes with tissue refiner high-speed homogenization after each thawing;
4. extract: the cold saline adding 3 times of volumes, be placed in refrigerated centrifuge in 5 DEG C with the centrifugal 30min of 4000r/min, get the blood red liquid in upper strata, then with the filtration of G2 sand core funnel, get filtrate;
5. ultrafiltration: be 5000 daltonian hollow fiber ultrafiltration membrane pressurization ultrafiltration by above-mentioned filtrate shut off value, filtrate is that molecular weight is less than 5000 daltonian transfer factors;
6. degerming: filtrate aperture ultrafiltration obtained is that the filter membrane pressure filtration of 0.22 μm is degerming;
7. subpackage: 4 DEG C of sealings are preserved, and obtain anti-DHV transfer factor extracting solution finished product.
Embodiment 5:
To the detection of anti-DHV transfer factor prepared by technique of the present invention
Following detection is carried out to anti-DHV transfer factor (calling in the following text " this product ") prepared by technique described in embodiment 1:
(1) Infrared spectrophotometry: this product has a high-selenium corn peak at 250.0-260.0nm place, and ABS260/ABS280>2.0.
(2) titer is faint yellow, and pH value is between 6.0-6.7.
(3) 20% sulfosalicylic acids detect: this product, without muddy and deposited phenomenon, illustrates that albumino reaction is feminine gender, and it is not containing macro-molecular protein.
(4) determining content of peptides: it is 1.784mg/ml that this product measures content of peptides through two vena contracta method.
(5) nucleic acid content measures: it is 666.34 μ g/ml that this product measures nucleic acid content through orcin method.
(6) bacteriological detection: exist without the need to oxygen, anaerobism, saprophytic bacteria and fungus in this product.
(7) E-red rose pigment rate compares: chicken periphery blood lymph cell surface has Mice red cell receptor, and can combine with it and form E-rosette, and transfer factor is combined with certain facilitation to lymphocyte and Rat Erythrocytes.The E-red rose pigment rate average out to 38.3% of this product, than the increase by 27.6% (P≤0.01) of matched group.
(8) get healthy mice 10, oral this product concentrated solution, be equivalent to 50 times of normal oral liquid dosage, observe the survival ability change of white mice, result: without any toxic reaction, also occurs without the phenomena of mortality, illustrate that this product is safe, without any Side effect.
(9) do the experiment of rabbit skin test with PPD, find that this product has the function of transfer immunity activity.
(10) do skin test with DHV antigen to detect, have faint positive reaction, illustrate that this product has good transfer activity and specificity.
Anti-DHV transfer factor of the present invention can be made into oral liquid or injection dosage form.If apply conventional freeze-dry process, the powder of anti-DHV transfer factor can be obtained further, in conjunction with customary adjuvant, can be made into capsule, tablets and other formulations.
Claims (1)
1. a preparation method for anti-DHV transfer factor, is characterized in that: the step of this preparation method is as follows:
(1) virus antigen is prepared: in Embryo Gallus domesticus DHV being inoculated in 9-11 age in days or duck embryo allantoic liquid, 33 DEG C of-37 DEG C of incubations, Embryo Gallus domesticus dead in 24h or duck embryo discard, the Embryo Gallus domesticus or the duck embryo allantocherion that have obvious pox speckle is collected after 120h, after phosphate buffer (PBS) washing, be placed in tissue refiner and make suspension, save backup in-20 DEG C of--40 DEG C of refrigerators;
(2) virus antigen is extracted: after obtaining virus antigen multigelation 3-5 time in step (1), in loading heavy wall ampoule, ultrasonic 15-20min, 4 DEG C, the centrifugal 20-30min of 5000rpm, discard precipitation, collect supernatant, by supernatant through 4 DEG C, the centrifugal 20-30min of 7000rpm, discard precipitation, collect supernatant, through 4 DEG C, the centrifugal 20-30min of 25000rpm, abandoning supernatant, get precipitation, dissolve by equal-volume PBS solution.Above-mentioned solution is loaded the discontinuous gradient density centrifugation pipe prepared with the sucrose of 15%, 30%, 45%, 60% and 80%, ultracentrifugation, 4 DEG C, the centrifugal 2-4h of 15000-20000rpm, according to the molecular weight determination DHV position of DHV, sucking-off being placed in-20 DEG C of--40 DEG C of refrigerators saves backup;
(3) with the virus antigen immune swine that step (2) gained extracts: select healthy immune swine, get the virus antigen of preparation, adopt subcutaneous multiple spot immunization method, injecting immune pig, immune 3-4 time altogether, front twice respectively with complete freund adjuvant and incomplete freund adjuvant hybrid antigen, beat with high-speed homogenizer, in animal skins or subcutaneous multi-point injection immunity, in the muscle of animal or intravenous injection after 3rd time and the 4th, each immunization interval one week, animals iv blood is got after last immune 7-10 days, the specific antibody titres of above-mentioned virus is detected with immunofluorescence method or ELISA method, after occurring that virus specific immunity is tired, slaughter animal, get spleen and thymus on ice, be transferred to-20 DEG C--40 DEG C preservations, for subsequent use,
(4) from immune swine, anti-DHV transfer factor is extracted:
1. raw-material preparation and pretreatment: get the spleen of the inoculation DHV immune swine of step (3) gained, thymus and lymph node tissue, the spleen of pig is cleaned with cold tri-distilled water, remove the tissues such as surperficial fascia, fat, then sterile saline rinses repeatedly;
2. broken: the pig spleen of wash clean to be shredded, adds the cold saline of 2 times of volumes, under freezing condition, smash 3 times with high-speed tissue mashing machine to pieces with 1000r/min, each 3min, obtained homogenate;
3. freeze thawing: homogenate is placed in-80 DEG C of quick freezing, melts in water-bath 30 DEG C, alternate freezing and thawing 4-8 time, all pulverizes with tissue refiner high-speed homogenization after each thawing;
4. extract: the cold saline adding 3 times of volumes, be placed in refrigerated centrifuge in 5 DEG C with the centrifugal 30min of 4000r/min, get the blood red liquid in upper strata, then with the filtration of G2 sand core funnel, get filtrate;
5. ultrafiltration: be 5000 daltonian hollow fiber ultrafiltration membrane pressurization ultrafiltration by above-mentioned filtrate shut off value, filtrate is that molecular weight is less than 5000 daltonian transfer factors;
6. degerming: filtrate aperture ultrafiltration obtained is that the filter membrane pressure filtration of 0.1-0.22um is degerming;
7. subpackage: 4 DEG C of sealings are preserved, and obtain anti-DHV transfer factor extracting solution finished product.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101293915A (en) * | 2008-06-17 | 2008-10-29 | 天津生机集团股份有限公司 | Method for preparing anti-duck plague transfer factor |
| 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 |
-
2014
- 2014-10-14 CN CN201410539958.8A patent/CN105560282A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101293915A (en) * | 2008-06-17 | 2008-10-29 | 天津生机集团股份有限公司 | Method for preparing anti-duck plague transfer factor |
| 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 (2)
| Title |
|---|
| 何庆雄: "鸭病毒性肝炎弱毒活疫苗临床免疫试验研究"", 《中国优秀硕士学位论文全文数据库农业科技辑》 * |
| 张桂枝主编: "《规模禽肠实验室工作手册》", 31 October 2013 * |
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