CN101298467A - Preparation of anti-newcastle disease virus transfer factor - Google Patents
Preparation of anti-newcastle disease virus transfer factor Download PDFInfo
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- CN101298467A CN101298467A CNA2008100535415A CN200810053541A CN101298467A CN 101298467 A CN101298467 A CN 101298467A CN A2008100535415 A CNA2008100535415 A CN A2008100535415A CN 200810053541 A CN200810053541 A CN 200810053541A CN 101298467 A CN101298467 A CN 101298467A
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Abstract
The invention discloses a preparation method for transferring gene of a natural effective pure biological active matter for resisting a newcastle disease virus. The invention is carried out according to the following steps: inoculating the newcastle disease virus into a chick embryo allantois liquid of ten days to prepare a virus antigen; extracting the virus antigen; using the extracted virus antigen for immunizing a pig; extracting the transferring gene for resisting the newcastle disease virus from the immunized pig; therefore, the transferring gene of the invention can be used for playing the role of preventing the infection of the newcastle disease virus and protecting a normal organism cell from being infected by the newcastle disease virus, thereby reducing the disease 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-newcastle disease virus transfer factor.
Background technology
Newcastle disease is the acute respiratory transmissible disease that is caused by newcastle disease virus.The maximum characteristics of newcastle disease virus are exactly to morph easily, and the anti-newcastle disease virus specific antibody that causes having occurred in the chicken body loses the effect of neutralization virus, thereby chicken is infected easily, even causes popular on a large scale.Sick chicken is subjected to newcastle disease virus to infect symptoms such as suffer from diarrhoea in the back, expiratory dyspnea, and weight person can cause death.
Although the medicine or the preparation of some anti-newcastle disease virus 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 chicken group of newcastle disease, annual regularly is the effective ways of prevention newcastle disease to the chicken inoculation newcastle disease vaccine.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 newcastle disease 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 all is the preparation method about non-specific transfer factor.For example, Chinese invention patent application " processing method---the complete filtering method of preparation transfer factor " (number of patent application: 200310118439.6) then disclose the preparation technology who adopts press filtration, ultrafiltration, the continuous filtration of nanofiltration several method.Another part Chinese invention patent application " processing method---the heating method of preparation transfer factor " (number of patent application: be the step that on the basis of preceding a application, adds heat treated by same applicant 200410039444.2), remain the preparation technology of the multiple filtration method of continuous employing.The content of above-mentioned patent application 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, and the time is longer, and is also bigger to the loss of activity of product, 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 a kind of preparation method of efficiency natural pure bioactive material anti-newcastle disease virus transfer factor is provided.
The preparation method of anti-newcastle disease virus transfer factor of the present invention, carry out according to following steps:
(1) preparation virus antigen: newcastle disease virus is inoculated in 10 days instar chicken embryo allantoic fluids, 33 ℃ following 48-72 hour, get chick 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, 110,000-165,000 rev/min 3 hours, according to centrifuge tube position, newcastle disease virus place, the sucking-off newcastle disease virus is standby;
(3) with the virus antigen immune swine of said extracted: select healthy pig, get the virus antigen of preparation, 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-newcastle disease virus transfer factor:
1. preparing raw material and pre-treatment: virus immunity is inoculated spleen and the lymph node tissue of pig, 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-newcastle disease virus transfer factor extracting solution.
The preparation method of a kind of anti-newcastle disease virus 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 newcastle disease virus 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-newcastle disease virus transfer factor of the present invention can suppress newcastle disease virus 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 newcastle disease virus and infect, can protect the normal body cell to avoid the effect of newcastle disease virus infringement, thereby reduce sickness rate.Use transfer factor to treat for the chicken of suffering the newcastle disease virus 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-newcastle disease virus transfer factor
1. preparation virus antigen: newcastle disease virus is inoculated in 10 days instar chicken embryo allantoic fluids, 33 ℃ following 48-72 hour, get chick 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, getting supernatant liquor packs into the discontinuous gradient density centrifugation pipe of 15%, 30%, 45% and 60% sucrose preparation, ultracentrifugation, 165000 rev/mins 3 hours, standby according to the centrifuge tube position sucking-off of newcastle disease virus place;
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 extract anti-newcastle disease virus transfer factor from immune swine:
(1) preparing raw material and pre-treatment: virus immunity is inoculated spleen and the lymph node tissue of 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-newcastle disease virus transfer factor extracting solution.
Embodiment 2:
The preparation of anti-newcastle disease virus transfer factor
(1) preparing raw material: healthy the no medical history of choosing 6 the monthly age 10 of pigs, usefulness newcastle disease virus 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-newcastle disease virus transfer factor of prepared of the present invention
Anti-newcastle disease virus transfer factor (calling " this product " in the following text) to embodiment 1 described prepared carries 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 1.260mg/ml that this product is measured content of peptides through biuret method.
(5) nucleic acid content is measured: it is 631.56ug/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 chicken 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 39.4% of this product is than the increase by 21.7% (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 newcastle disease virus 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-newcastle disease virus transfer factor of the present invention also can be made into other oral liquids.
In addition, further can obtain the powder of anti-newcastle disease virus 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-newcastle disease virus transfer factor is characterized in that, carries out according to following steps:
(1) preparation virus antigen: newcastle disease virus is inoculated in 10 days instar chicken embryo allantoic fluids, 33 ℃ following 48-72 hour, get chick 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, 110,000-165,000 rev/min 3 hours, according to centrifuge tube position, newcastle disease virus place, the sucking-off newcastle disease virus is standby;
(3) with the virus antigen immune swine of said extracted: select healthy pig, get the virus antigen of preparation, 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-newcastle disease virus transfer factor:
1. preparing raw material and pre-treatment: virus immunity is inoculated spleen and the lymph node tissue of pig, 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-newcastle disease virus transfer factor extracting solution.
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| CNA2008100535415A CN101298467A (en) | 2008-06-17 | 2008-06-17 | Preparation of anti-newcastle disease virus transfer factor |
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| CNA2008100535415A CN101298467A (en) | 2008-06-17 | 2008-06-17 | Preparation of anti-newcastle disease virus transfer factor |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101721427B (en) * | 2009-11-13 | 2012-07-11 | 烟台绿叶动物保健品有限公司 | Method for preparing gallinaceous Newcastle disease transspecific factor |
| CN103724419A (en) * | 2013-12-30 | 2014-04-16 | 天津瑞普生物技术股份有限公司 | Pig spleen transfer factor purification method |
| CN105194643A (en) * | 2015-10-16 | 2015-12-30 | 锦州景良动物药业有限公司 | Preparation method for chick specific immunopotentiator |
| CN105287634A (en) * | 2015-10-16 | 2016-02-03 | 天津瑞普生物技术股份有限公司 | Preparation method of anti-avian influenza virus transfer factor |
| 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 |
-
2008
- 2008-06-17 CN CNA2008100535415A patent/CN101298467A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101721427B (en) * | 2009-11-13 | 2012-07-11 | 烟台绿叶动物保健品有限公司 | Method for preparing gallinaceous Newcastle disease transspecific factor |
| CN103724419A (en) * | 2013-12-30 | 2014-04-16 | 天津瑞普生物技术股份有限公司 | Pig spleen transfer factor purification method |
| CN103724419B (en) * | 2013-12-30 | 2015-11-18 | 天津瑞普生物技术股份有限公司 | A kind of Pig spleen transfer factor purification method |
| 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 |
| CN105194643A (en) * | 2015-10-16 | 2015-12-30 | 锦州景良动物药业有限公司 | Preparation method for chick specific immunopotentiator |
| CN105287634A (en) * | 2015-10-16 | 2016-02-03 | 天津瑞普生物技术股份有限公司 | Preparation method of anti-avian influenza virus transfer factor |
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Open date: 20081105 |