CN103393721B - Compound specific transfer factor preparation technology and medical usage - Google Patents
Compound specific transfer factor preparation technology and medical usage Download PDFInfo
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Abstract
The present invention discloses a kind of compound specific transfer factor preparation technology and medical usage, adopt nanotechnology and chemical technology method, by pathogenic virus, antibacterial, parasite, various tumor, make cracking or the subunit vaccine of various prevention people, animal infectious disease by biological product requirement, then do adjuvant with fat milk and become animal specific immunity antigen; Inject various animal with immunizing antigen, period measures the antibody horizontal that in immune animal, related antigen produces, after meeting the requirements, and immune animal; Rubbed by immune organ and tissue meat grinder, add normal saline, suction filter pump is filtered, and nanotechnology treatments, multiple specific transfer factor is made in ultrafiltration.Have activity stabilized, output is high, scale is large, and anti-infectious disease spectrum is very wide, and technological means is advanced, constant product quality; The utilization of resources is abundant, significantly reduces production cost; Different user needs can be met; Life cycle of the product is short, can increase work efficiency greatly; Medicine group can prepare polytype preparation, easy to use.
Description
Technical field
The present invention discloses a kind of compound specific transfer factor preparation technology, additionally provides the medical usage of compound specific transfer factor simultaneously, belongs to biomedicine technical field.
Background technology
Immune system is an important anti-epidemic system in man and animal body, refers in man and animal body and participates in the former immunne response of enantiopathy, perform the general name of a series of organs of immunologic function, cell and molecule.This system is made up of immune organ, immunocyte or immune molecule, is the material base producing immunne response.Immune system is made up of central immune organ and peripheral immune organ.Maincenter comprises: bone marrow, thymus and fabricius bursa; Periphery comprises: lymph node, spleen, Ha Deershi gland, mucomembranous immune system (intestinal mucosa, lactiferous ducts mucosa, uterine mucosa etc.).Vertebrate immune system is equipped with to be used for identifying and to invade the pathogenic organism of body, as antibacterial, virus, fungus, parasite, tumor protect health to exempt from its infringement.Immune system generally includes cellular component and non-cell components.
Immune cellular component comprises usually said " lymphocyte " or leukocyte, and it exists several types.The cellular component of maturation immunity system causes the first nonspecific response to invasion pathogen usually, only and participate in replying the secondary specificity of pathogen.
In the initial response first to pathogenic infection, be called that cytophagous leukocyte is located and attacks invasion pathogen.Usual phagocyte is by internalization, or " eating up " pathogen, then disease for digest substance.In addition, leukocyte is produced and is secreted chemical substance in response pathogenic infection, expects that this chemical substance is attacked pathogen or contributes to instructing the attack to pathogen.
Only have when invading pathogenic infection and continuing to escape primary immune response, just need the specificity secondary immune response to pathogen.Because this secondary immune response normally postpones, therefore also referred to as " delayed hypersensitivity ".Mammal is by himself, usual until about 14 secondary immune responses of talent's initiation to pathogen after pathogenic infection.Secondary immune response is also referred to as specified pathogen acquired immunity originally.Pathogen has one or more characteristic proteins and is called " antigen ".Secondary immune response is called that the leukocyte " study " of bone-marrow-derived lymphocyte or " B-cell " and T lymphocyte or " T-cell " identifies one or more antigens of pathogen.B-cell and T-cell one work and produce thing and be called the protein of " antibody ".It is specific for one or more specific antigens in pathogen.
T-cell mainly causes secondary immune response to pathogen or antigenic substance or delayed hypersensitivity.There is the T-cell of three types: T-accessory cell, blocking statutes T-cell and antigen specific T-cell, the latter is also called cytotoxicity (being meant to " cell killing ") T-lymphocyte (CTL) or T-killer cell or NK cell (NK) cell.T-accessory cell and inhibition T-cell, although be not specific to some antigen, perform regulatory function (such as, the inflammation of usual concomitant infections), and this regulatory function helps from infection host removing pathogen or antigenic substance.Antibody only forms a part for immune system non-cell components, and identification specificity antigen is " antigenic specificity ".Then the antibody produced mainly helps leukocyte to locate and eliminates the pathogen in health.Usually, once leukocyte generation is the antibody for pathogen, leukocyte and all progenitor cell spy continuous generation antibody thereof.Eliminate after infecting, a small amount of T-cell corresponding with identified antigen and B-cell are held in " dormancy " state.When corresponding morbid substance or antigenic agents again infect host, " dormancy " T-cell and B-cell-stimulating also induced immunne response fast in about 48 hours.By response so, the secondary immune response of immune system mounts substance, thinks that immune system has " memory " to this pathogen.
The immune system of animal produces less albumen and is called " transfer factor ", as a part for the secondary immune response to pathogen infection.Transfer factor is the another kind of non-cell components of animal immune system.It is generally acknowledged in antigenic specificity transfer factor structure with antibody class seemingly, but molecule is much smaller.Antigenic specificity transfer factor and antibody all comprise former specific position.In addition, transfer factor and antibody all comprise the interactional high conservative region of the upper acceptor site of effector lymphocyte respective with it.Transfer factor and antibody molecule are the 3rd regions " linker ", connect antigen-specific sites and high conservative region.
Transfer factor is from having immunocompetent lymphocyte a kind of material discharged.The transfer factor that immunocompetent lymphocytes discharges under antigenic stimulus can make again unsensitized lymphocyte transformation for having immunocompetent lymphocyte.Therefore, some of donor lymphocyte specific or unspecific cellular immune function can be transferred to nonimmune receptor by transfer factor, make receptor have the cell immunocompetent of donor, to improve and to bring out the immune defence ability of receptor, improve recipient immune state.
The transfer factor of extracting from normal healthy animal immune organ claims common transfer factor (being also non-single transfer factor); Claim specificity vaccine (antigen) with the vaccine that specific pathogen organism is made, immune various animal, the extract getting animal immune organ claims specific transfer factor.
For preparing transfer factor both at home and abroad at present, antigen makes nonstandard, much do not meet biological product code requirement, and be mostly a kind of animal of a kind of antigen immune, gather one or both immune organ and tissues, extracting method uses multigelation smudge cells, by centrifugal, dialysis, the product made like this, how unstable activity is, yield poorly, small scale and anti-infectious disease spectrum very narrow, means are backwardness relatively.
Summary of the invention
The invention discloses a kind of compound specific transfer factor preparation technology, solve tradition preparation transfer factor exist active unstable, yield poorly, small scale and the very narrow shortcoming of anti-infectious disease spectrum.
Invention further provides the medical usage of compound specific transfer factor preparation technology.
the preparation technology of a kind of compound specific transfer factor provided by the invention, is characterized in that comprising the following steps:
1) by the vaccine of multiple for people and animals Pathogenic organisms or pathogen stock solution with hydrostatic pressing 200-300Mpa, 20-30 minute deactivation;
2) add 0.5%-0.7% by pathogen stock solution total amount and remove ketone nitrogen 101, shaking table mixing 1-2 hour, process with under nanometer ram machine 200-300 pressure, 10-40 nanometer condition, ketone nitrogen 101 is cleared out with gel filtration, add 20%-30% fat milk as immunological adjuvant, obtain cracking Seedling antigen or subunit seedling antigen;
3) by step 2) antigen immune injection 2-3 kinds of dissimilar animals, gather animal immune system organ-tissue, by meat mincer disrupting tissue, add 2-3 times of normal saline, make homogenate with homogenizer, filter pump filters, filtrate uses nanometer ram machine process, adjust passage to determine 40-60 nano-pore, finally use 5000-10000 membrane ultrafiltration, obtain compound specific transfer factor;
Described Pathogenic organisms comprises virus, antibacterial, parasite;
Described animal immune system organ-tissue comprises tonsil, lymph node, thymus, spleen.
Compound specific transfer factor prepared by described preparation technology is preparing the purposes in immunopotentiating agent and non-specific immunotherapy.
Modern biotechnology and biochemical technology are combined, adopt nanotechnology and chemical technology method, by pathogenic virus, antibacterial, parasite, various tumor, by biological product requirement make various prevention people, animal infectious disease cracking or subunit vaccine (antigen), then do adjuvant with fat milk and become animal specific immunity antigen; Various animal is injected with immunizing antigen, period measures the antibody horizontal that in immune animal, related antigen produces, after meeting the requirements, take animal tonsil, lymph node, thymus, spleen, blood leucocyte, the defatted milk of immune milk, sheep milk, Immune Laying Hens removes Ovum Gallus domesticus album yolk Ye Deng immuning tissue organ; Its extraction process is rubbed by immune organ and tissue meat grinder, adds normal saline, and suction filter pump is filtered, and nanotechnology treatments, multiple specific transfer factor is made in ultrafiltration.This transfer factor is a peptide species, polynucleotide and other become the compound substance that is grouped into, relative molecular weight is approximately 2000-5000.Transfer factor itself does not have antigenicity, relies on antigenic activation, non-primed lymphocyte can be excited to become primed lymphocyte, plays cell immunoreceptor.Transfer factor is without species specificity, mutually can transmit between different genera, therefore, various animal can be used, as the immune organs such as horse, cattle, sheep, pig, chicken (tonsil, lymph node, thymus, spleen, blood leucocyte and Placenta Hominis etc.), prepare and meet people, animal infectious disease treatment specific transfer factor.
good effect of the present invention is:antigen production standard, simultaneously immune two or more animal, and gather multiple immune organ and tissues of animal, adopt homogenizer to make homogenate, filter pump filtration, nanometer ram machine process, ultrafiltration obtains compound specific transfer factor; Have activity stabilized, output is high, scale is large, and anti-infectious disease spectrum is very wide, and technological means is advanced, constant product quality; The utilization of resources is abundant, significantly reduces production cost; Different user needs can be met; Life cycle of the product is short, can increase work efficiency greatly; Medicine group can prepare polytype preparation, easy to use.
detailed description of the invention:
The present invention is further described by following examples, do not limit the present invention in any way, under the prerequisite not deviating from the technology of the present invention solution, professional and technical personnel in the field made for the present invention easily realize any change or change falling within the claims in the present invention scope.
embodiment 1
the preparation of compound Aids specific transfer factor:
1) change after equine infectious anemia virus donkey leukocyte strain being drawn nutritional solution with the fresh medium containing 1-2% seed liquor, 37 degree are continued to cultivate, after connecing poison, every day there is pathological changes in the cell of observing after 5-7 days about 80%, with receive during compared with control cells significant difference poison every milliliter of viral level >=105TCD50, equivalent is introduced AIDS virus respectively and is cultivated stock solution and bacillus calmette-guerin vaccine, hydrostatic pressing deactivation (300Mpa, 30min);
2) add 0.6% by virion stock solution total amount and remove ketone nitrogen 101, shaking table mixes 1.5 hours, processes, clears out ketone nitrogen 101 with gel filtration, add 30% fat milk as immunological adjuvant, obtain cracking Seedling antigen with under nanometer ram machine 200-300 pressure, 20 nanometer conditions;
3) by step 2) the healthy horse of antigen immune, donkey each 3 and laying hen 100, milch cow 5 (milk goats) is by fundamental immunity and reinforced immunological, when meeting the requirements, horse is taked after butchering, donkey immune system tissues organ (comprises tonsil, lymph node, thymus, spleen, blood leucocyte), except sarolemma and fat meat grinder rub, add two times of normal saline, homogenate is made with homogenizer, suction filter pump is filtered, with filtrate and collection immune egg, remove Ovum Gallus domesticus album, get yolk liquid and immune milk, degrease, get albumen, three kinds of solution are fully mixed, with nanometer ram machine, adjust 40 nano-pore process twice, with 10,000 ultrafilter membrane ultrafiltration, filtrate is concentrated makes compound Aids specific transfer factor.
By the compound Aids specific transfer factor of preparation by complement fixation test and agar gel diffusion test result, tire >=3.
Their " biological sibship are close, and in its body, contained chemical composition is also close " of HIV (human immunodeficiency virus) and equine infectious anemia virus, reach the object of " treating different diseases with the same therapeutic principle " with " way for the treatment of the poisonous disease with poisonous drugs produces antitoxic immunity ".
Equine infectious anemia (ELAV) and acquired immune deficiency syndrome (AIDS) (HIV) belong to Retroviridae, lentivirus member together.Both morphosis, antigenic characteristic, gene structure become and virus and host continuous action etc. in very similar, and ELAV virus has unique quick Development process and the boundary of the obvious course of disease, becomes the ideal animals model of the mutual relation between the genovariation of research HIV and clinical symptoms.ELAV vaccination Ma Tihou; animal is not caused to fall ill; and can lasting immunoprotection be obtained; may be there is low-level viral nucleic acid to copy; this is most important to the feasibility of HIV vaccine; in the recent period to the quantitative tracking and monitoring of ELAV vaccine nucleic acid level in animal body, from a side reflection in order to replace HIV vaccine to be have practicality.
embodiment 2
dog makes dog coupling compound specific transfer factor with pentavaccine
Dog comprises rabies, canine distemper, dog parainfluenza, hepatitis infectiosa canis virus and Canine Parvovirus five kinds of weak poison with pentavaccine, by China Veterinary Drugs Supervisory Inst.'s keeping, qualification and supply.Five kinds of seeds culture of viruses used include original seed culture of viruses, basic seed culture of viruses and production seed culture of viruses three grades, and three grades of seeds culture of viruses are the cell culture that seed culture of viruses adapts to, and wherein 1-5 is on behalf of original seed culture of viruses; 6-10 is on behalf of basic seed culture of viruses; 11-15 is on behalf of production seed culture of viruses.Three class seeds culture of viruses can, best with original seed culture of viruses cell culture stock solution.
Vaccine is 1 batch with 10,000 agent and mixes, namely measure containing each 15 the minimum immunity of mad dog, canine distemper and dog parainfluenza Seedling by every agent, the consumption measured containing Canine Parvovirus and each 10 the minimum immunity of hepatitis infectiosa canis virus to take out from each Seedling semi-finished product mixed liquor bottle be up to the standards, aseptic mixing, be this vaccine to wet Seedling, put-30 DEG C for subsequent use.Every agent 1.4ml.I.e. Rb/E3 Seedling 0.1ml; CDV/R-20/8 Seedling 0.5ml; CPIV/A-20/8 Seedling 0.5ml; YCA18 Seedling 0.1ml; CR86106 Seedling 0.2ml.Protective agent is 30% fat milk, is 1:3, fully mixes, subpackage, lyophilizing with the ratio of wet Seedling.
1) by dog with pentavaccine with hydrostatic pressing 200Mpa, deactivation in 30 minutes;
2) add 0.5%% by virion stock solution total amount and remove ketone nitrogen 101, shaking table mixes 2 hours, processes, clears out ketone nitrogen 101 with gel filtration, add 20% fat milk as immunological adjuvant, obtain subunit seedling antigen with under nanometer ram machine 200 pressure, 10 nanometer conditions;
3) select horse 5, pig 10, Canis familiaris L. 50 injection subunit seedling antigen, fundamental immunity consumption is 5 times of body zooprophylazis amount, and every 7 days once, continues one month; Second month is reinforced immunological, and consumption is 15 times of body zooprophylazis amount, also continues one month, and once, period detects immune animal associated antibodies, butchers when peaking in immunity in every 15 days.Get that above-mentioned animal immune system organ-tissue (comprising tonsil, lymph node, thymus, spleen, blood leucocyte) removes muscle, fat meat grinder rubs, add 2 times of normal saline, homogenate is made with homogenizer, suction filter pump is filtered, 40 nano-pore process twice is adjusted with nanometer ram machine, finally use 10,000 ultrafilter membrane ultrafiltration, filtrate is compound dog coupling specific transfer factor.
By the compound dog coupling specific transfer factor of preparation by antibody test result, minimum antibody is tired and is respectively:
Rabies >=1.10; Canine distemper >=1.50; Dog parainfluenza >=1.40; Hepatitis infectiosa canis virus >=1.10; Canine Parvovirus >=1.16.
embodiment 3
the preparation of compound coupling bacterium specific transfer factor
1) adopt equal portions ratio to coordinate Lantigen B, b type influenza " haemophilus Conjugate vaccines ", then add 30% fat milk mixing, with hydrostatic pressing 200Mpa, deactivation in 20 minutes in the ratio of above-mentioned total amount 1:1;
2) add 0.5% by virion stock solution total amount and remove ketone nitrogen 101, shaking table mixes 1 hour, processes, clear out ketone nitrogen 101 with gel filtration with under nanometer ram machine 250 pressure, 30 nanometer conditions, add 20% fat milk as immunological adjuvant, obtain cracking compound coupling bacterium specific transfer factor antigen;
3) 4 dry goods are selected, 10 pigs carry out above-mentioned antigen immune injection, gather animal immune system organ-tissue, by meat mincer disrupting tissue, add 2 times of normal saline, homogenate is made with homogenizer, filter pump filters, and filtrate uses nanometer ram machine process, adjusts fixed 40 nano-pores of passage, finally use 10000 membrane ultrafiltration, obtain compound coupling bacterium specific transfer factor.
Compound coupling bacterium specific transfer factor is detected by circulating antibody, and minimum antibody is tired and is respectively:
Lantigen B >=1.10; B type influenza " haemophilus Conjugate vaccines " >=1.50; For compound coupling bacterium specific transfer factor, this transfer factor.Manufacture method is easy, safely without side reaction, therapeutic domain is wide, not only has therapeutical effect to above-mentioned pathogen, can also improve non-specific immunity, and antianaphylaxis disease effects might as well.
embodiment 4:
the preparation of compound coupling parasite specific transfer factor:
the making of parasite antigen:the parasite vaccine that can go on the market from current state approval is introduced or is introduced concentrated parasite stock solution from parasite R&D institution.Schistosomicide 1000 milliliters, toxoplasma 1000 milliliters, coccidiosis 1000 milliliters, cysticercosis 1000 milliliters, malaria worm 1000 milliliters, adds bacillus calmette-guerin vaccine 1000 milliliters, fully mixes, homogenate is made with homogenizer, with nanometer ram machine, select 40 nano-pores, twice repeatedly, add 1:130% fat milk again, be parasite immunity antigen.
1) by parasite immunity antigen with hydrostatic pressing 200Mpa, deactivation in 20 minutes;
2) add 0.6% by virion stock solution total amount and remove ketone nitrogen 101, shaking table mixes 1.5 hours, processes, clears out ketone nitrogen 101 with gel filtration, add 25% fat milk as immunological adjuvant, obtain cracking parasite immunity antigen with under nanometer ram machine 200 pressure, 30 nanometer conditions;
3) horse 10 is selected, pig 50, fundamental immunity is carried out by body zooprophylazis consumption 10 times injection parasite immunity antigen, every 10 days once, be used in conjunction 3 times, reinforced immunological is done again after one month, by body zooprophylazis consumption 20 times, every first quarter moon 1 time, be used in conjunction 3 times, detect immune circular correlation antibody during this period, after composite demand, butcher horse, pig, collect immune system organs's tissue, by meat mincer disrupting tissue, add 3 times of normal saline, homogenate is made with homogenizer, filter pump filters, filtrate uses nanometer ram machine process, adjust fixed 30 nano-pores of passage, finally use 10000 membrane ultrafiltration, obtain compound parasite specific transfer factor.
Compound parasite specific transfer factor is by immunology detection, and recycling-guard antibody is the positive.
embodiment 5
the preparation of the compound coupling tumor specific immune factor:
Prepared by lung cancer vaccine (antigen)
Choose autologous squamous cell lung carcinoma and lung carcinoma glanular cell or this type of cancer cell tissue culture 1000 milliliters as specific immunity antigen, at several antibacterial Seedling of cooperation as nonspecific immunity vaccine (antigen), bacillus calmette-guerin vaccine 1000 milliliters, Corynebacterium parvum vaccine 1000 milliliters, in cooperation cytokine as immunological adjuvant, add interleukin-2, each 500 milliliters of interleukin-10, fully mix, with nanometer ram machine, 40 nano-pore process 3 times, are compound tumor specific transfer factor antigen.
1) by lung cancer vaccine with hydrostatic pressing 200Mpa, deactivation in 25 minutes;
2) add 0.5% by virion stock solution total amount and remove ketone nitrogen 101, shaking table mixes 1 hour, processes, clears out ketone nitrogen 101 with gel filtration, add 20% fat milk as immunological adjuvant, obtain cracking lung cancer vaccine antigen with under nanometer ram machine 200 pressure, 30 nanometer conditions;
3) horse 3, pig 10, chicken 50 cracking lung cancer vaccine antigen immune is selected, gather animal immune system organ-tissue, by meat mincer disrupting tissue, add 3 times of normal saline, make homogenate with homogenizer, filter pump filters, filtrate uses nanometer ram machine process, adjust fixed 50 nano-pores of passage, finally use 510000 membrane ultrafiltration, obtain compound pulmonary carcinoma specific transfer factor.
Compound pulmonary carcinoma specific transfer factor through immunoassay, CD8
+and CD4
+t cell is the positive.
embodiment 6
Various different pharmaceutical preparation can be made with different in nature transfer factor prepared by embodiment 1 ~ 5:
1, directly vein or intramuscular dose are made in embedding;
2, add 2.5% mannitol and make lyophilized formulations;
3, powder is made in the drying of stock solution concentrated spray;
4, powder is added customary adjuvant and make tablet, capsule, ointment transdermal agent and dry powder agent;
5, stock solution injects and can be made into liquid spray with fat milk.The milch cow that specific immunity is good, milk sheep or laying hen, collect milk, sheep milk defat, egg goes Ovum Gallus domesticus album to get yolk liquid, by nanotechnology treatments, also can be made into out various pharmaceutical dosage form;
6, compound transspecific sex factor can in 1:1 ratio and related immune promoter, one or more compositions, implement combined pharmaceutical formulation (as lentinan, B glucosan, cordycepin, astragalus polysaccharides, ginsenoside, compound vitamin, mineral and trace element, aminoacids complex, nanometer colostrum, immunity skimmed milk and Ovum Gallus domesticus album derivant and relevant interferon, the cytokines such as interleukin neoplasm necrosis) further to improve immune effect, meet the needs of more multi-resistance infectious disease.
Claims (2)
1. a preparation technology for compound specific transfer factor, is characterized in that comprising the following steps:
1) by the vaccine of multiple for people and animals Pathogenic organisms or pathogen stock solution with hydrostatic pressing 200-300Mpa, 20-30 minute deactivation;
2) add 0.5%-0.7% by pathogen stock solution total amount and remove ketone nitrogen 101, shaking table mixing 1-2 hour, process with under nanometer ram machine 200-300 pressure, 10-40 nanometer condition, ketone nitrogen 101 is cleared out with gel filtration, add 20%-30% fat milk as immunological adjuvant, obtain cracking Seedling antigen or subunit seedling antigen;
3) by step 2) antigen or subunit seedling antigen immune injection 2-3 kinds of dissimilar animals, gather animal immune system organ-tissue, by meat mincer disrupting tissue, add 2-3 times of normal saline, make homogenate with homogenizer, filter pump filters, filtrate uses nanometer ram machine process, adjust passage to determine 40-60 nano-pore, finally use 5000-10000 membrane ultrafiltration, obtain compound specific transfer factor;
Described Pathogenic organisms comprises virus, antibacterial, parasite;
Described animal immune system organ-tissue comprises tonsil, lymph node, thymus, spleen.
2. the compound specific transfer factor that according to claim 1 prepared by preparation technology is preparing the purposes in immunopotentiating agent and non-specific immunotherapy.
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| CN101057864A (en) * | 2006-04-17 | 2007-10-24 | 肇庆学院 | Technology for preparing anti influenza virus transfer factors |
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| CN103316045A (en) * | 2013-06-26 | 2013-09-25 | 西安斯凯达生物制品有限公司 | Combined specific transfer factor for livestock and poultry and preparation method of combined specific transfer factor |
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| CN1098289A (en) * | 1993-08-04 | 1995-02-08 | 姜光斗 | The immune ribonucleic acid of resisting polytypic hepatitis and transfer factor and technology |
| CN101057864A (en) * | 2006-04-17 | 2007-10-24 | 肇庆学院 | Technology for preparing anti influenza virus transfer factors |
| CN101684140A (en) * | 2008-09-25 | 2010-03-31 | 天津生机集团股份有限公司 | Preparation method of anti-avian influenza virus, newcastle disease virus, chicken bursa virus specific transfer and antimicrobial peptide |
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