CN101757029A - Method for extracting transfer factors from cattle spleen - Google Patents
Method for extracting transfer factors from cattle spleen Download PDFInfo
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- CN101757029A CN101757029A CN200810154335A CN200810154335A CN101757029A CN 101757029 A CN101757029 A CN 101757029A CN 200810154335 A CN200810154335 A CN 200810154335A CN 200810154335 A CN200810154335 A CN 200810154335A CN 101757029 A CN101757029 A CN 101757029A
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Abstract
The invention provides a method for extracting transfer factors from cattle spleen, which comprises the following steps of grinding cells by a high-pressure homogenizer, precipitating by a flocculating agent, filtering by a micro-pore filtration membrane, ultra-filtering by a macro-pore filtration membrane, wherein the flocculating agent is added when in shell breaking, so that the protein in solution can be precipitated, and the solution can become transparent, thereby reducing the possibility of blocking a hollow-fiber ultra-filtration column in the ultra-filtration process. The method can greatly shorten the production cycle, save large amount of manpower and material resources and improve the production efficiency of the transfer factors, so as to provide a feasible path for meeting the industrial large-scale production.
Description
Technical field
The invention belongs to field of veterinary, particularly relate to a kind of method of from animal spleen, extracting transfer factor.What summary of the invention of the present invention related generally to is the method for extracting transfer factor from cattle spleen.
Background technology
(transfer factor TF) is a kind of material that can shift sensitization information that the T lymphocyte discharges, and it can give receptor with the cellular immunization information transfer of donor specifically, thereby strengthens the immunologic function of receptor in transfer factor.TF finds so far that from the fifties Chinese scholars is all carried out big quantity research to this, and is introduced in veterinary's immunity and the clinical treatment in 1979, and it has become a kind of widely used enhance immunity preparation abroad at present.
Because transfer factor do not have species specificity, therefore the source of transfer factor is comparatively extensive clinically, human common clinically have people's transfer factor, pig transfer factor, cattle transfer factor, sheep transfer factor, goose, monkey transfer factor etc.At present poultry go up common have cattle transfer factor, pig transfer factor, chicken transfer factor.Fowl poultry spleen is the refuse in the course of processing, therefore extremely wide application and development prospect is arranged, and has become the focus of transfer factor research.
Transfer factor be molecular weight less than 10000, the small-molecule mixture of the heterogencity of biologically active, its inclusions comprises free amino acid (16-18 kind), metallic elements such as nucleic acid, polypeptide and K, Na, Ca, Mg, Zn.Physicochemical property in the various TF, constituent difference is little, but content has than big-difference.
On immunology, TF can be divided into specific transfer factor and non-specific transfer factor two big classes.Extract again behind certain specific pathogen infection of specific transfer factor system employing or immune crowd, the animal and contain the active transfer factor of this antigen-specific, present clinical used hepatitis B virus special transfer factor, various tomour specific transfer factor, epidemic haemorrhagic fever virus transfer factor, the transfer factor of cloth Salmonella, herpesvirus transfer factor, encephalitis B transfer factor, tulase transfer factor etc.Non-specific transfer factor is meant with what nature crowd or animal leukocyte extracted to have an active transfer factor of panimmunity, immune function that can non-specific adjusting body, thus improve the resistance of body or the immune effect of vaccine.
Cellular immunization is mainly realized by the T lymphocyte, report transfer factors such as Wang Jianwen can strengthen the lymphocytic ratio of the young cock T of Luo Man, Ma Fenglong etc. discover that with chicken spleen transfer factor and newcastle I be that injection can significantly improve lymphocytic conversion ratio to Seedling to broiler simultaneously, proof chicken spleen TF has facilitation to the cellular immunization of chicken, can promote the lymphocytic propagation of T with ripe, the cellullar immunologic response ability of enhancing body.
Early stage classical transfer factor mechanism development test just finds that transfer factor has the activity of transmitting the specific cell immunologic information, and the performance of the stimulation of antigenic information and animal body immunologic function has directly and gets in touch.Existing numerous data confirm, but transfer factor activated lymphocyte E receptor, the formation number of increase E rosette.Therefore immunologic information is transferred to the receptor lymphocyte by TF, make cell obtain this immunologic information and produce the antigenic receptor of identification specificity, when this kind antigen exists, this specific recognition receptor will combine with specific antigen and trigger the recipient cell activation, causes its height differentiation and proliferation from strengthening lymphocytic activity.
After high winter jasmine was waited research report chicken injection TF, inoculation egg-decreasing syndrome inactivated vaccine can significantly improve the antibody titer of chicken body.Discovery chicken TF live vaccine such as Ma Fenglong and oil-emulsion inactivated vaccine all have synergism when head exempts from booster immunization, can significantly improve the humoral immunoresponse(HI) ability of immunity inoculation chicken, produce higher titer antibody, and prolong the antibody peak period, make it to keep the less time.Because the X lymphocyte is realizing that the humoral immunization function needs T lymphocytic auxiliary, generally believe that at present transfer factor mainly is an immune function of regulating body by the T lymphocyte to the influence of humoral immunization.
Liu Runzhen etc. prepare the transfer factor of specificity spleen after utilizing canine penton vaccine immunity pig, can significantly strengthen mice E rosette forming rate and peritoneal macrophage activate the phagocytic capacity.Chen Dekun etc. discover, to healthy mice injection and oral pig spleen transfer factor can strengthen the peripheral blood neutrophilic granulocyte to staphylococcic phagocytic rate and peritoneal macrophage to erythrocytic phagocytic rate.Therefore, transfer factor also has potentiation to the nonspecific immunity function of body.
Fan Zhenqing etc. discover, the heavy exponential decline of leukocyte count, lymphocyte percentage and spleen of the oral and injection TF mice that is caused by hydrocortisone capable of blocking.The immunosuppressant Rabbit Model that Miao Naifa adopts cyclophosphamide to set up is carried out the experiment of Lien Sus domestica transfer factor promoting leucocytes, and injection back 3d is that visible total white blood cells rises rapidly, and 5d can reach summit, and trend is normal behind the 8d.After inferring that thus TF enters body as immunoactivator, can make hemopoietic system produce a large amount of leukocyte at short notice, the while activated lymphocyte, increase the t helper cell generation and discharge more lymphokine, thereby promote T lymphocyte growth and division performance to resist the effect of radiation, anti-immunosuppressant, raise immunity.
The oral 0.5-1.0ML transfer factor of report chickling such as Wang Jianwen semifinished product is not compared with taking the transfer factor group behind the 7d, and weightening finish is obvious, and oral transfer factor group chicken essence is refreshing active, and feather is glossy.Jiang Qingyan etc. find that in test the transfer factor of injection spleen has obvious effects to the yellow chicken weightening finish in Guangdong.The normal growth of animal body is grown generally by T3, T4 and GH coordinated regulation and is finished.GH mainly promotes tissue growth, and T3, T4 mainly promote the differentiation of organ, tissue, and the growth promoting function of GH needs an amount of T3, the existence of T4.In this test, the TF chicken group of injection variable concentrations is blood T4 when 8 ages in week, and the GH level all is significantly higher than matched group, infers that thus the spleen transfer factor promotes that the inherent mechanism of the yellow chicken growth promoter in Guangdong is relevant with the rising of blood T4, GH level.
Along with improving constantly of scale, intensification degree cultured in modernization, viral disease has become puzzlement raiser's a great problem at present.Existing general solution is exactly immunity, yet for various reasons, can cause immuning failure unavoidably sometimes, breaks out atypical infectious disease, brings certain difficulty for diagnosis and treatment.Behind immuning failure, often rely on antibiotic therapy, caused the increase of Resistant strain and the generation of cross infection.Transfer factor is sent out as a kind of cellular immunization and is answered reinforcing agent, can play an important role aspect disease preventing and treating, improves the immune effect after the immunity, effectively improves the resistance against diseases of body.
Present existing transfer factor technology after normally the fat in the spleen of animal and trunk being picked out, is organized and is smashed to pieces, behind the multigelation, obtains the operating cost height by the semipermeable membrane dialysis.In addition, the conventional Laurence method that adopts promptly utilizes twice dialysis to reclaim transfer factor, and this method is simple to operate, but production cycle farm labourer preface loaded down with trivial details (production cycle needs 20 days approximately), length consuming time is wasted a large amount of manpower and materials.Therefore these methods all are difficult to carry out large-scale industrialization production.
Therefore, need a kind of quick, efficient and economic method of from animal spleen, obtaining transfer factor, to satisfy present suitability for industrialized production.
Summary of the invention
The object of the invention is to provide a kind of method of obtaining transfer factor from animal spleen, comprising:
1) with refrigerated animal spleen 1000g, be cut into small pieces after cleaning with distilled water, add the 1000ml distilled water, rub with tissue mashing machine;
2) high pressure homogenizer (60MPa) carries out cell breakage, adds flocculating agent 2.0g simultaneously;
3) under 4 ℃, with 7000 rev/mins, centrifugal 10 minutes, go precipitation, stay supernatant;
4) supernatant filters with microporous filter membrane, carries out ultrafiltration with ultrafilter membrane then, is the stock solution that contains transfer factor.
Contain the transfer factor of expection in the stock solution, remove the preparation of virus back according to the national drug management method then.
In a specific embodiments, described distilled water is the former distilled water that reduces phlegm and internal heat.
In another embodiment, described flocculating agent is a citric acid.
In another embodiment, the described microporous filter membrane preferred 0.22 μ m in footpath.
In another embodiment, the preferred molecular cut off of described ultrafilter membrane is 5000 daltonian ultrafilter membranes.
In another embodiment, described animal is domestic animal, preferred cattle.
Term:
" pyrogen " means the pyrogenicsubstance that can cause the rising of Homoiotherm abnormal body temperature by microorganisms.It comprises bacillary pyrogen, endogenous macromolecule pyrogen, the low molecule pyrogen of endogenous and chemical pyrogen etc.Here " pyrogen " of indication mainly is meant bacillary pyrogen, is metabolite, antibacterial corpse and the endotoxin of some antibacterial.What the pyrogenicity ability was the strongest is the product of gram negative bacilli, secondly is Grain-positive bacillus class, gram positive coccus then a little less than, mycete, yeast even virus also can produce pyrogen.Pyrogen is the many pure complex that form with protein bound of phospholipid normally.The many alcohol of phospholipid are active center of complex, and pyrogenic action is the strongest.Its chemical composition is because of the different differences to some extent of strain.Molecular weight is 5 * 104～5 * 105, and the big more pyrogenic action of molecular weight is also strong more.
The former method of reducing phlegm and internal heat comprises:
1. high temperature method: the heat resistance of pyrogen is good, and 60 ℃ of heating 1h destruction that is not decomposed do not degrade for 100 ℃, but 180 ℃ of 3～4h, 200 ℃ of 60min or 250 ℃ of 30～45min can make pyrogen thoroughly destroy.Therefore the syringe that uses when used container and other apparatus and injection in heat-resisting article such as glass, metallic article, the production process etc. all can adopt this method to destroy pyrogen.But under normally used injection pressure sterilizing condition, be not enough to destroy pyrogen.
2. absorption method: pyrogen can be by absorption such as active carbon, asbestos, kaolin in aqueous solution and is removed.Because activated carbon property is stable, adsorptivity has concurrently by force and helps filter and decolorization, thus be widely used in injection production, but should note adsorbing the loss of the principal agent that medicinal liquid causes.
3. ultrafiltration: the pyrogen molecular weight is 1 * 10
6About, volume is less, and about 1～5nm can pass through general filter and microporous filter membrane, but adopts ultrafiltration as being removed with 3.0～15nm ultrafilter membrane.
4. the way of distillation: pyrogen can be water-soluble but non-volatile, but can enter with the droplet of steam in the water for injection, when therefore preparing water for injection, pyrogen in the former water can be removed through distillation, but needs repeatedly distillation, and be added with every the foam device, single distills that often effect is undesirable.
5. acid-base method: pyrogen can be destroyed by strong acid, highly basic, strong oxidizer.Glass container and apparatus such as dosing destroy pyrogen with available potassium dichromate sulphuric acid cleaning solution or dilute sodium hydroxide processing such as glass drying oven, infusion bottles.
Other: comprise that ion exchange, gel filter method, hyperfiltration etc.
Beneficial effect
1. the present invention is in leaching process, and broken back uses high pressure homogenizer to carry out breaking cellular wall once more, makes tissue or cellular content fully be released;
2. added flocculating agent at breaking cellular wall simultaneously, made protein precipitation in the solution, the solution becomes clarification, in the process of ultrafiltration, reduced stop up the Hollow Fiber Ultrafiltration post may;
3. adopt production transfer factor of the present invention, the production cycle shortens greatly, makes to carry out the method that cell wall breaking, semipermeable membrane are dialysed by the method for traditional multigelation, and the production cycle shortens about 20 days; And adopt the transfer factor production cycle of the inventive method preparation only to need about 7 days, saved lot of manpower and material resources;
4. improved the production efficiency of transfer factor, the method of tradition multigelation is carried out the method that cell wall breaking, semipermeable membrane are dialysed, the transfer factor stock solution of producing, it is stock solution 2000ml about 2.0mg/mL that every 1000g spleen is produced content approximately, to produce content approximately be stock solution 1000ml about 7.0mg/mL and adopt the present invention to produce every 1000g spleen, and amounting to output increases by 175%.
The specific embodiment
The present invention will be further described below in conjunction with embodiment.
Embodiment 1: the preparation of transfer factor
1, cut and pluck, clean: will melt is that the animal spleen that partly freezes attitude is removed fat and blood vessel, and accurate weighing 1kg is with no thermal source distilled water water cleaning 3 times.
2, rub: the spleen after the grease removal takes by weighing in the meat grinder and just twists 3 times, does not have the thermal source distilled water with adding 1000ml in the spleen that rubs, and puts into and smashs cylinder to pieces, smart strand.
3, grind: the spleen slurry of smashing to pieces is put into colloid mill with its grinding, make homogenate.
4, cell breakage: with above-mentioned spleen homogenate, in high pressure homogenizer, pressure 60MPa carries out homogenizing, makes homogenate.
5, high pressure homogenize is good homogenate adds citric acid 2.0g, stirs, and puts in the refrigerated centrifuger, and 7000 rev/mins, centrifugal 15 minutes, 4 ℃ of Centrifugal Environment temperature discarded sediment, collects supernatant.
6, supernatant filters with the microporous filter membrane of 0.22 μ m, is that 5000 dalton's ultrafilter membranes carry out ultrafiltration with molecular cut off then, is stock solution.
Embodiment 2: to the detection of the transfer factor of prepared of the present invention
Transfer factor stock solution to embodiment 1 described prepared is carried out following detection according to National Drug Administration, national drug standards WS1-XG-036-2000:
1, appearance character is little yellow clear liquid, meets the appearance character of transfer factor solution.
2, the aminoacid identification is a positive reaction.
3, ultraviolet spectrophotometry: above-mentioned gained sample has an absworption peak at 261 ± 2nm place, and ABS
260/ ABS
280＞1.9.
4, pH value is between 6.0～7.5.
5,20% sulfosalicylic acid detects: above-mentioned gained sample does not have muddiness and deposited phenomenon, illustrates that albumino reaction all becomes negative, and it does not contain macro-molecular protein.
6, high molecular weight material detects, and does not have absworption peak before the insulin appearance time, and illustrating does not have the macromole that is higher than molecular weight 6000 in this product.
7, free amino acid detects: this product amino acid content 15mg among every 1ml.
7, transfer factor determination of activity: can make its function of receptors of thymus T cellular-restoring that takes off behind the E receptor according to transfer factor, thus the biological activity of reaction transfer factor.The percentage rate that above-mentioned gained sample forms the E rosette is respectively 32.3%, 30.2%, and comparison increases by 18.1%, 17.2% (P≤0.01) respectively according to group.
8, bacteriological analysis: no aerobic, anaerobism, saprophytic bacteria and fungus exist in the above-mentioned gained sample.
9, anaphylaxis, toxicity test: get 10 of healthy mices, male and female half and half, oral this product concentrated solution, be equivalent to 100 times of normal oral dose, observe the variation of the survival ability of white mice, the result: do not have any allergy, toxic reaction, also do not have any dead appearance, illustrate that this product is safe, does not have any toxicity and side effect.
10, nucleic acid content is measured: above-mentioned gained sample is measured nucleic acid content through orcin method and is respectively 631.29 μ g/mL, 603.98 μ g/mL.
11, determining content of peptides: above-mentioned gained sample is measured content of peptides through the Lowry method and is respectively: 7.38mg/mL, 7.05mg/mL.
By the foregoing description, we find the cattle spleen transfer factor content height that extracted, and production process is simple, the production efficiency height.Institute's production transfer factor stock solution meets the standard code of national code simultaneously.Therefore production method of the present invention can be produced popularization on a large scale.
Claims (6)
1. method of obtaining transfer factor from animal spleen comprises:
1) with refrigerated animal spleen 1000g, be cut into small pieces after cleaning with distilled water, add the 1000ml distilled water, rub with tissue mashing machine;
2) high pressure homogenizer (60MPa) carries out cell breakage, adds flocculating agent simultaneously and adds flocculating agent 2.0g;
3) under 4 ℃, with 7000 rev/mins, centrifugal 10 minutes, go precipitation, stay supernatant;
4) supernatant filters with microporous filter membrane, carries out ultrafiltration with ultrafilter membrane then, is the stock solution that contains transfer factor.
2. the process of claim 1 wherein that described distilled water is the former distilled water that reduces phlegm and internal heat.
3. claim 1 or 2 method, wherein said flocculating agent is a citric acid.
4. each method in the claim 1 to 3, the wherein said microporous filter membrane preferred 0.22 μ m in footpath.
5. each method in the claim 1 to 4, the preferred molecular cut off of wherein said ultrafilter membrane is 5000 daltonian ultrafilter membranes.
6. each method in the claim 1 to 5, wherein said animal is domestic animal, preferred cattle.
Priority Applications (1)
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| CN200810154335A CN101757029A (en) | 2008-12-23 | 2008-12-23 | Method for extracting transfer factors from cattle spleen |
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| CN200810154335A CN101757029A (en) | 2008-12-23 | 2008-12-23 | Method for extracting transfer factors from cattle spleen |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102357102A (en) * | 2011-09-30 | 2012-02-22 | 浙江华尔成生物药业股份有限公司 | Preparation method of transfer factor soluble powder for animals |
| CN105030827A (en) * | 2015-07-14 | 2015-11-11 | 天津瑞普生物技术股份有限公司 | Transfer factor and application thereof |
| CN107417765A (en) * | 2017-09-26 | 2017-12-01 | 珠海联邦制药股份有限公司 | The method that recombinant protein is isolated and purified in Escherichia coli self-dissolving expression system |
-
2008
- 2008-12-23 CN CN200810154335A patent/CN101757029A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102357102A (en) * | 2011-09-30 | 2012-02-22 | 浙江华尔成生物药业股份有限公司 | Preparation method of transfer factor soluble powder for animals |
| CN102357102B (en) * | 2011-09-30 | 2013-05-15 | 浙江华尔成生物药业股份有限公司 | Preparation method of transfer factor soluble powder for animals |
| CN105030827A (en) * | 2015-07-14 | 2015-11-11 | 天津瑞普生物技术股份有限公司 | Transfer factor and application thereof |
| CN107417765A (en) * | 2017-09-26 | 2017-12-01 | 珠海联邦制药股份有限公司 | The method that recombinant protein is isolated and purified in Escherichia coli self-dissolving expression system |
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Open date: 20100630 |