CN101293915A - Method for preparing anti-duck plague transfer factor - Google Patents
Method for preparing anti-duck plague transfer factor Download PDFInfo
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- CN101293915A CN101293915A CNA2008100535449A CN200810053544A CN101293915A CN 101293915 A CN101293915 A CN 101293915A CN A2008100535449 A CNA2008100535449 A CN A2008100535449A CN 200810053544 A CN200810053544 A CN 200810053544A CN 101293915 A CN101293915 A CN 101293915A
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Abstract
The invention discloses a preparation method of a natural high-effective biological active substance anti-duck plague virus transfer factor. The preparation method comprises the following steps of: inoculating duck plague virus into an allantoic fluid obtained from a 10-day-aged chick embryo, and preparing duck plague virus antigen; extracting the duck plague virus antigen; immunizing a pig with the extracted duck plague virus antigen; and extracting the anti-duck plague virus transfer factor from the immunized pig. The inventive anti-duck plague virus transfer factor can prevent duck plague and protect normal body cells from being infected by the duck plague virus, so as to reduce incidence rate.
Description
Technical field
The present invention relates to medicine and formulation art thereof, more particularly, relate to a kind of preparation method of anti-duck plague transfer factor.
Background technology
Duck plague is a kind of transmissible disease that is caused by duck plague virus, and is mainly in the duck after 20 ages in days.Cardinal symptom is: fervescence, have difficulty in breathing, shed tears, draw green loose stool, sick duck head and neck swelling, so the title of " epidemic infection with swollen head " is arranged.Duck plague sickness rate and lethality rate are all very high
Although the medicine or the preparation of some anti-duck plagues have been arranged at present: because the specific aim of existing medicine preparation is not strong, curative effect is limited, and mostly therapeutic preparation is after morbidity application, unable to get up prophylactic effect.For the susceptible duck group of duck plague disease, annual is the effective ways of prevention duck plague to duck inoculation duck plague vaccine regularly.Secondly should strengthen nutrition or drug intervention, improve the tired power of self exempting from.Improving body by medicine also is a kind of effective ways that prevent duck plague to Hang Li or adjusting immunizing power, and transfer factor can play this effect.
Transfer factor (TF, Transfer Factor) is a kind of low molecular weight polypeptide-nucleotide complex that can shift sensitization information that the T lymphocyte discharges, it can be transferred to certain specific cellular immune function of donor the normal lymphocyte of acceptor specifically, participate in the immune response of body, improve the acceptor cell immune function of human body, non-bacterial infections such as opposing fungi, virus; Promote the antibody-secreting effect of bone-marrow-derived lymphocyte simultaneously, can discharge Interferon, rabbit and interleukin by inducing cell, thereby strengthen the immunologic function of acceptor.
The extracting method of the transfer factor of having reported at present is the preparation method about non-specific transfer factor mostly.The content of the patent application of having reported is that the tissue after the fragmentation is directly filtered extraction behind frozen centrifugation, have the incomplete defective of cytoclasis equally, and its technology is loaded down with trivial details, the time is longer, loss of activity to product is also bigger, so yield is difficult to improve.
Pig spleen and thymus gland are immunologically competent cell accumulative places, and material source is very extensive, therefore extract specific transfer factor from this class cell, important significance for theories and realistic meaning are arranged, but have not yet to see report.
Summary of the invention
Technical problem to be solved by this invention is, overcomes the deficiencies in the prior art, and the preparation method of the anti-duck plague transfer factor of a kind of efficiency natural pure bioactive material is provided.
The preparation method of the anti-duck plague transfer factor of the present invention, carry out according to following steps:
(1) preparation virus antigen: duck plague is inoculated in duck embryo allantoic liquid in 10 day age, 33 ℃ following 48-96 hour, get the duck embryo allantoic liquid, in the dress heavy wall ampoule, ultrasonic smashing, centrifugal 20 minutes of 5000rpm discards sediment and stays supernatant;
(2) extract virus antigen: get centrifugal supernatant, discontinuous gradient density centrifugation purifying extracts virus antigen then; With virus antigen centrifugal after, get supernatant liquor and pack into the discontinuous gradient density centrifugation pipe of 15%, 30%, 45% and 60% sucrose preparation, ultracentrifugation, 165000 rev/mins 3 hours, determine centrifuge tube position, duck plague place according to the molecular weight of duck plague, sucking-off is standby;
(3) with the virus antigen immune swine of said extracted: select healthy immune swine, get the virus antigen of preparation, with the subcutaneous multi-point injection immune swine of said extracted virus antigen, immune 3-4 time altogether, preceding twice respectively with complete freund adjuvant and incomplete freund adjuvant hybrid antigen, beat even with high-speed homogenizer, in animal skin or subcutaneous multi-point injection immunity, after the 3rd time and the 4th in muscle or the intravenous injection of animal, each immunity is a week at interval, get animal venous blood after last immune 7-10 days, detect the specific antibody titres of above-mentioned virus with immunofluorescence method or ELISA method, the virus-specific immunizing potency appears and after, slaughter animal, get spleen and thymus gland on ice, be transferred to-20 ℃ of preservations, standby;
(4) from immune swine, extract anti-duck plague transfer factor:
1. preparing raw material and pre-treatment: with the spleen and the lymph node tissue of above-mentioned virus inoculation immune swine, clean the pig spleen with cold tri-distilled water, remove tissues such as its surperficial manadesma, fat, sterile saline washes repeatedly again;
2. broken: the pig spleen of wash clean is shredded, add the cold saline of 2 times of volumes, smash to pieces 3 times with 1000r/min with high-speed tissue mashing machine under freezing condition, each 3min makes homogenate;
3. freeze thawing: homogenate is placed-80 ℃ of quick freezing, melts in 30 ℃ of the water-baths, alternate freezing and thawing 4-6 time, behind each the thawing all with tissue refiner's high-speed homogenization pulverizing;
4. extract: add the cold saline of 3 times of volumes, place refrigerated centrifuge in 5 ℃ with 4000r/min centrifugal 30 minutes, get the blood red liquid in upper strata, filter with G2 sand core funnel again, get filtrate;
5. ultrafiltration: is 6000 daltonian hollow fiber ultrafiltration membrane pressurization ultrafiltration with above-mentioned filtrate with shut off value, and filtrate is that molecular weight is less than 6000 daltonian transfer factor;
6. degerming: the filtrate that ultrafiltration is obtained is the filter membrane pressure filtration degerming of 0.22um with the aperture;
7. packing: 4 ℃ of sealings are preserved, and promptly get described anti-duck plague transfer factor extracting solution.
The preparation method of a kind of anti-duck plague transfer factor of the present invention has following sorrow point:
(1) the starting material source is easy, pollution-free, inexpensive.Pig spleen and thymus gland belong to the tankage of butchering the back pig.
(2) behind the broken tissue, add the step of low temperature freeze thawing treatment, particularly, do not make the small-molecule substance loss of activity, and the duck plague transfer factor is extracted fully 30 ℃ of dissolvings down.
(3) process using more advanced hollow fiber ultrafiltration membrane ultra-filtration method, thereby shortened preparation time, guarantee that activity is not destroyed in the separating substances process.
(4) extract is a kind of low molecular weight polypeptide-nucleotide complex through biochemical measurement and some composition analyses, be a kind of egg that do not contain from matter, soluble small-molecule substance, molecular weight is below 6000 dalton.Through proofs such as inside and outside biological activity test and pharmacology, toxicity tests, have the purity height, immunocompetence is strong, rapid-action and characteristics such as it is long to hold time, easily absorption, no any untoward reaction, its determined curative effect, safe and reliable, not only can treat but also can strengthen resistance against diseases.This extract can be made formulations such as oral liquid, is convenient to use.
Anti-duck plague transfer factor of the present invention can suppress duck plague comprehensively or partly, can protect the normal body cell to avoid virus infection.Therefore use transfer factor of the present invention can play the prevention duck plague and infect, can protect the normal body cell to avoid the effect of duck plague infringement, thereby reduce sickness rate.Use transfer factor to treat for the duck of suffering the duck plague infection simultaneously, can effectively improve clinical symptom, improve curative ratio, reduce mortality ratio.
Embodiment
Below in conjunction with embodiment the present invention is described further.
Embodiment 1:
The preparation of anti-duck plague transfer factor
1. preparation virus antigen: duck plague is inoculated in duck embryo allantoic liquid in 10 day age, 33 ℃ following 48-96 hour, get the duck embryo allantoic liquid, in the dress heavy wall ampoule, ultrasonic smashing, centrifugal 20 minutes of 5000rpm discards sediment and stays supernatant;
2. get centrifugal supernatant, discontinuous gradient density centrifugation purifying extracts virus antigen then; With virus antigen centrifugal after, get supernatant liquor and pack into the discontinuous gradient density centrifugation pipe of 15%, 30%, 45% and 60% sucrose preparation, ultracentrifugation, 165000 rev/mins 3 hours, determine this centrifuge tube position, virus place according to the molecular weight of different virus, sucking-off is standby;
3. use the virus antigen immune swine of said extracted: with the subcutaneous multi-point injection immune swine of said extracted virus antigen, immunity is 4 times altogether, preceding twice respectively with complete freund adjuvant and incomplete freund adjuvant hybrid antigen, beat even with high-speed homogenizer, in animal skin or subcutaneous multi-point injection immunity, after the 3rd time and the 4th in muscle or the intravenous injection of animal, each immunity is a week at interval, get animal venous blood after last immune 7-10 days, detect the specific antibody titres of above-mentioned virus with immunofluorescence method or ELISA method, the virus-specific immunizing potency appears and after, slaughter animal, get spleen and thymus gland on ice, be transferred to-20 ℃ of preservations, standby;
4. from immune swine, extract anti-duck plague transfer factor:
(1) preparing raw material and pre-treatment: with spleen and the lymph node tissue of virus immunity pig, clean the pig spleen with cold tri-distilled water, remove tissues such as its surperficial manadesma, fat, sterile saline washes repeatedly again;
(2) fragmentation: the pig spleen of wash clean is shredded, add the cold saline of 2 times of volumes, smash to pieces 3 times with 1000r/min with high-speed tissue mashing machine under freezing condition, each 3min makes homogenate;
(3) freeze thawing: homogenate is placed-80 ℃ of quick freezing, melts in 30 ℃ of the water-baths, alternate freezing and thawing 4-6 time, behind each the thawing all with tissue refiner's high-speed homogenization pulverizing;
(4) extract: add the cold saline of 3 times of volumes, place refrigerated centrifuge in 5 ℃ with 4000r/min centrifugal 30 minutes, get the blood red liquid in upper strata, filter with G2 sand core funnel again, get filtrate;
(5) ultrafiltration: is 6000 daltonian hollow fiber ultrafiltration membrane pressurization ultrafiltration with above-mentioned filtrate with shut off value, and filtrate is that molecular weight is less than 6000 daltonian transfer factor;
(6) degerming: the filtrate that ultrafiltration is obtained is the filter membrane pressure filtration degerming of 0.22um with the aperture;
(7) packing: by the requirement of oral liquid, add 15% syrup, add standard sanitas (stupid sodium formiate 0.25%) back packing, the 10ml/ bottle;
(8) packing, 4 ℃ of sealings are preserved, and promptly get described anti-duck plague transfer factor extracting solution.
Embodiment 2:
The preparation of anti-duck plague transfer factor
(1) preparing raw material: healthy the no medical history of choosing 6 the monthly age 10 of pigs, usefulness duck plague vaccine subcutaneous injection immunity, after 15 days, again immunity once, the method agent is the same.Slaughtered in 15-20 days behind the second immunisation, get spleen and thymus gland, shift and put-20 ℃ of preservations on ice, standby;
(2) pre-treatment: behind the weighing pig spleen, clean the pig spleen, cut off its manadesma and fatty tissue, use cold tri-distilled water washes clean again with scissors with cold tri-distilled water;
(3) fragmentation: the pig spleen of above wash clean is shredded, add the cold saline of 2 times of volumes, smash to pieces 3 times with high-speed tissue mashing machine (1000r/min) under freezing condition, each 3min makes homogenate;
(4) freeze thawing: homogenate (adorning with vessel) as for Ultralow Temperature Freezer (80 ℃) lining quick freezing, is melted alternate freezing and thawing 10 times in 30 ℃ of the water-baths;
(5) extract: add the cold saline of 3 times of volumes, place the centrifugal 30min of refrigerated centrifuge (4000r/min, 5 ℃), get supernatant liquid (blood red), filter with G2 sand core funnel again, get filtrate;
(6) ultrafiltration: is 6000 daltonian hollow fiber ultrafiltration membrane pressurization ultrafiltration with above-mentioned filtrate with shut off value, and filtrate is that molecular weight is less than 6000 daltonian transfer factor;
(7) degerming: use sterile filters (the filter membrane aperture is 0.22um) pressure filtration respectively;
(8) packing:, add standard sanitas (Sodium Benzoate 0.25%) back packing, the 2ml/ bottle by the requirement of injection liquid;
(9) preserve: 4 ℃ of sealings are stored.
Embodiment 3:
Detection to the anti-duck plague transfer factor of prepared of the present invention
Anti-duck plague transfer factor (calling " this product " in the following text) to embodiment 1 described prepared is carried out following detection:
(1) ultraviolet spectrophotometry: this product has a high absorption peak at the 250.0-252.0nm place, and ABS260/ABS280＞2.0.
(2) reference liquid is faint yellow, and the pH value is between 6.0-6.5.
(3) 20% sulphosalicylic acids detect: this product does not have muddiness and deposited phenomenon, illustrates that albumen test is all negative, and it does not contain macro-molecular protein.
(4) determining content of peptides: it is 1.130mg/ml that this product is measured content of peptides through biuret method.
(5) nucleic acid content is measured: it is 602.78ug/ml that this product is measured nucleic acid content through orcin method.
(6) bacteriological detection: no aerobic, anaerobism, saprophytic microorganism and fungi exist in this product.
(7) E-rosette rate of formation relatively: there is the mouse erythrocyte receptor on duck peripheral blood lymphocyte surface, and can be with it in conjunction with forming the E-rosette.And transfer factor is combined with certain promoter action to lymphocyte and rat are erythrocytic.The E-rosette rate of formation average out to 31.9% of this product is than the increase by 25.9% (P≤0.01) of control group.
(8) get 5 of healthy mices, oral this product concentrated solution is equivalent to 50 times of normal oral liquid dosage, the viability of observing small white mouse changes the result: do not have any toxic reaction, also do not have the phenomena of mortality and occur, illustrate that this product is safe, does not have any toxicity and side effect.
(9) do the experiment of rabbit skin test with PPD, find that this product has the active function of transfer immunity.
(10) do skin test with duck plague antigen and detect, faint positive reaction is arranged, illustrate that this product has good transfer activity and specificity.
Except that above-mentioned syrup formulation, anti-duck plague transfer factor of the present invention also can be made into other oral liquids.
In addition, further can obtain the powder of anti-duck plague transfer factor,, can be made into capsule, tablets and other formulations in conjunction with conventional auxiliary material by conventional freeze-dry process.
Claims (1)
1. the preparation method of an anti-duck plague transfer factor is characterized in that, carries out according to following steps:
(1) preparation virus antigen: duck plague is inoculated in duck embryo allantoic liquid in 10 day age, 33 ℃ following 48-96 hour, get the duck embryo allantoic liquid, in the dress heavy wall ampoule, ultrasonic smashing, centrifugal 20 minutes of 5000rpm discards sediment and stays supernatant;
(2) extract virus antigen: get centrifugal supernatant, discontinuous gradient density centrifugation purifying extracts virus antigen then; With virus antigen centrifugal after, get supernatant liquor and pack into the discontinuous gradient density centrifugation pipe of 15%, 30%, 45% and 60% sucrose preparation, ultracentrifugation, 165000 rev/mins 3 hours, determine centrifuge tube position, duck plague place according to the molecular weight of duck plague, sucking-off is standby;
(3) with the virus antigen immune swine of said extracted: select healthy immune swine, get the virus antigen of preparation, with the subcutaneous multi-point injection immune swine of said extracted virus antigen, immune 3-4 time altogether, preceding twice respectively with complete freund adjuvant and incomplete freund adjuvant hybrid antigen, beat even with high-speed homogenizer, in animal skin or subcutaneous multi-point injection immunity, after the 3rd time and the 4th in muscle or the intravenous injection of animal, each immunity is a week at interval, get animal venous blood after last immune 7-10 days, detect the specific antibody titres of above-mentioned virus with immunofluorescence method or ELISA method, the virus-specific immunizing potency appears and after, slaughter animal, get spleen and thymus gland on ice, be transferred to-20 ℃ of preservations, standby;
(4) from immune swine, extract anti-duck plague transfer factor:
1. preparing raw material and pre-treatment: with the spleen and the lymph node tissue of above-mentioned virus inoculation immune swine, clean the pig spleen with cold tri-distilled water, remove tissues such as its surperficial manadesma, fat, sterile saline washes repeatedly again;
2. broken: the pig spleen of wash clean is shredded, add the cold saline of 2 times of volumes, smash to pieces 3 times with 1000r/min with high-speed tissue mashing machine under freezing condition, each 3min makes homogenate;
3. freeze thawing: homogenate is placed-80 ℃ of quick freezing, melts in 30 ℃ of the water-baths, alternate freezing and thawing 4-6 time, behind each the thawing all with tissue refiner's high-speed homogenization pulverizing;
4. extract: add the cold saline of 3 times of volumes, place refrigerated centrifuge in 5 ℃ with 4000r/min centrifugal 30 minutes, get the blood red liquid in upper strata, filter with G2 sand core funnel again, get filtrate;
5. ultrafiltration: is 6000 daltonian hollow fiber ultrafiltration membrane pressurization ultrafiltration with above-mentioned filtrate with shut off value, and filtrate is that molecular weight is less than 6000 daltonian transfer factor;
6. degerming: the filtrate that ultrafiltration is obtained is the filter membrane pressure filtration degerming of 0.22um with the aperture;
7. packing: 4 ℃ of sealings are preserved, and promptly get described anti-duck plague transfer factor extracting solution.
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| CNA2008100535449A CN101293915A (en) | 2008-06-17 | 2008-06-17 | Method for preparing anti-duck plague transfer factor |
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| CN104162156A (en) * | 2013-07-24 | 2014-11-26 | 河南联合英伟饲料有限公司 | Composition for resisting duck plague, freeze-dried powder, and preparation method and use of freeze-dried powder |
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| CN104208678A (en) * | 2013-09-30 | 2014-12-17 | 郑州后羿制药有限公司 | Veterinary antivirus composition, freeze-dried powder, preparation method and applications of the composition |
| CN104208683A (en) * | 2013-09-30 | 2014-12-17 | 郑州后羿制药有限公司 | Pharmaceutical composition for preventing duck viral diseases, freeze-dried powder, preparation method and applications of the composition |
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| 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 |
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2008
- 2008-06-17 CN CNA2008100535449A patent/CN101293915A/en active Pending
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| CN104162156A (en) * | 2013-07-24 | 2014-11-26 | 河南联合英伟饲料有限公司 | Composition for resisting duck plague, freeze-dried powder, and preparation method and use of freeze-dried powder |
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| CN104208676A (en) * | 2013-09-30 | 2014-12-17 | 郑州后羿制药有限公司 | Composition for preventing swine fever, porcine pseudorabies, and porcine circovirus disease, mixed freeze-dried powder, and preparation method of the composition |
| CN104208681A (en) * | 2013-09-30 | 2014-12-17 | 郑州后羿制药有限公司 | Mixed freeze-dried powder for preventing duck viral diseases and preparation method thereof |
| CN104208678A (en) * | 2013-09-30 | 2014-12-17 | 郑州后羿制药有限公司 | Veterinary antivirus composition, freeze-dried powder, preparation method and applications of the composition |
| CN104208683A (en) * | 2013-09-30 | 2014-12-17 | 郑州后羿制药有限公司 | Pharmaceutical composition for preventing duck viral diseases, freeze-dried powder, preparation method and applications of the composition |
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| CN104208678B (en) * | 2013-09-30 | 2016-04-20 | 郑州后羿制药有限公司 | A kind of antivirus veterinary composition, lyophilized powder, preparation method and application |
| CN104208683B (en) * | 2013-09-30 | 2016-06-01 | 郑州后羿制药有限公司 | The pharmaceutical composition of a kind of anti-duck viral disease, lyophilized powder, preparation method and application |
| CN105497867A (en) * | 2014-09-26 | 2016-04-20 | 天津嘉瑞生物科技有限公司 | Preparation method for transfer factor capable of resisting avian infectious laryngotracheitis virus |
| 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 |
| CN105560282A (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 |
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Open date: 20081029 |