CN1089608C - Process for producing transfer factors resisting virus of respiratory tract - Google Patents
Process for producing transfer factors resisting virus of respiratory tract Download PDFInfo
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- CN1089608C CN1089608C CN99115750A CN99115750A CN1089608C CN 1089608 C CN1089608 C CN 1089608C CN 99115750 A CN99115750 A CN 99115750A CN 99115750 A CN99115750 A CN 99115750A CN 1089608 C CN1089608 C CN 1089608C
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- transfer factors
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Abstract
The present invention relates to a preparation technology of transfer factors for resisting respiratory tract viruses, which comprises the following steps: firstly, preparing respiratory tract virus antigens; secondly, centrifugally purifying the respiratory tract virus antigens according to discontinuous gradient density; then immunizing animals with the extracted respiratory tract virus antigens; finally, extracting the transfer factors for resisting respiratory tract viruses from the immunized animals. The transfer factors can be prepared into injections or nose drop medicines, have the characteristics of effective prevention and treatment in both the superficiality and origin.
Description
The present invention relates to a kind of drug manufacture technology that is used to prevent and treat common respiratory virus infection, particularly a kind of transfer factors resisting virus of respiratory tract production method for preparing by the specific antigen immunity mammal of respiratory syncytial virus, adenovirus, parainfluenza virus, rhinovirus, influenza virus and herpesvirus extraction.
Flu is the upper respiratory tract infection that is caused by virus, is human modal disease, and normal because this disease causes multiple severe complications, is considered to " first of all kinds of diseases and ailments ".The whole world is used to prevent and to treat the expense of flu surprising, is example with Britain, though more than 5,000 ten thousand populations are only arranged, the annual expense that is used to prevent and treats flu and complication surpasses tens billion of dollars.Domestic and international existing anti-cold medicine mainly is to alleviate cold symptoms, and complication prevention takes place, and can not cure upper respiratory tract infection from the etiology angle.
The objective of the invention is to overcome the shortcoming of above-mentioned prior art, propose a kind of transfer factors resisting virus of respiratory tract drug manufacture technology, both had and to prevent, can treat the characteristics for the treatment of both the principal and secondary aspects of a disease again.
Little according to the transfer factor molecular weight, non-immunogenicity, and the immunology principle of no species specificity between mammal, the specific antigen that the present invention adopts common Respirovirus such as respiratory syncytial virus, adenovirus, parainfluenza virus, rhinovirus, influenza virus and herpesvirus to extract, the immunity mammal, get its spleen, lymph node tissue, extract active ingredient, develop anti-multiple virus specific transfer factor with molecular sieve filtration and dialysis process.Because the human body antiviral mainly is by the specific cell of cellular immunization and humoral immunization and antibody recognition, cause specific immunoreation, thereby kill and remove the body inner virus, transfer factor can pass to the human immunocyte to the immunologic information that has at virus-specific by the specific antigen immunity, thereby cause the special and immunoreation fast of human body, kill and remove virus.In addition, Respirovirus infringement human body must at first find suitable virus receptor at upper respiratory tract, causes epithelial cell to infect, and invades human body then.Immunologic cytotoxicity cell-specific killing ability and the adjusting mucosal immunity mechanism of transfer factors resisting virus of respiratory tract of the present invention by strengthening upper respiratory tract plays the effect that prevents and treat above-mentioned viral infection.
Production technology of the present invention is as follows:
At first, preparation Respirovirus antigen is with respiratory syncytial virus, adenovirus, parainfluenza virus, rhinovirus and herpes simplex virus, be inoculated in the Hep-2 cell or the bhk cell that have grown up to monolayer respectively, hatch for 37 ℃, every day observation of cell pathological changes situation, when CPE occurs +++or ++ ++ the time, collecting cell and supernatant are put the refrigerator cryogenic freezing and are preserved.
Influenza virus is inoculated in 10 days instar chicken embryo allantoic fluids and amniotic cavity, 33 ℃ 48～72 hours, collect allantoic fluid and amniotic fluid, with chicken or the susceptible malicious HA of Cavia porcellus RBC flow measurement.
Secondly, with discontinuous gradient density centrifugation purified virus antigen
The infection cell CPE+++ of respiratory syncytial virus, adenovirus, parainfluenza virus, rhinovirus and herpes simplex virus or ++ ++ after, scrape cell with a little culture fluid, in the dress heavy wall ampoule, ultrasonic smashing, centrifugal 20 minutes of 5000rpm discards sediment and stays supernatant.Pack into the discontinuous gradient density centrifugation pipe of 15%, 30%, 45% and 60% sucrose preparation, ultracentrifugation, 165000 * g 3 hours determines this centrifuge tube position, virus place according to the molecular weight of different virus, sucking-off is standby.Chick embryo allantoic liquid is got in influenza virus inoculation back 48～72 hours, and Ultrasonic Pulverization is got centrifugal supernatant, and the discontinuous gradient density centrifugation is extracted virus antigen, and extracting method is with above-mentioned other virus.
Then, use the virus antigen immune animal of being extracted
Select healthy mammal (30～50 kilograms of sheep body weight, 60～90 kilograms of pig body weight), get the virus antigen of above-mentioned preparation, immune animal, immunity is 5～6 times altogether.Preceding twice respectively with complete freund adjuvant and incomplete freund adjuvant hybrid antigen, beat with high-speed homogenizer even, in animal skins or subcutaneous multi-point injection immunity.In muscle or the intravenous injection of animal, each immunity is a week at interval after the 3rd time.Last immunity was got animal venous blood after 7 days, detected 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 lymph tuberosity tissue, the cryogenic refrigerator stored frozen is standby.
At last, extract the animal spleen and the lymph node tissue of transfer factors resisting virus of respiratory tract virus immunity, remove tissues such as its surperficial fascia, fat, sterile saline washes repeatedly, shred, through the tissue refiner high speed pulverization, multigelation 4～6 times, all pulverize behind each the thawing, extract with the method for ultrafiltration or dialysis then with the tissue refiner high-speed homogenization.When extracting with dialysis process, dialysis solution is 0.9% normal saline, puts the dialysis of 4 ℃ of refrigerators, places after 24 hours the collection dialysis solution and with filtering with microporous membrane, degerming less than 0.2 μ m, and it is standby to put the refrigerator stored frozen.
The safety test of transfer factors resisting virus of respiratory tract
1. pyrogen testing: get 5 of healthy adult rabbits, auricular vein injection transfer factors resisting virus of respiratory tract 2ml, respectively at before the injection, injection back 30 minutes, 1 hour, 2 hours, anus is surveyed body temperature, the average intensification is no more than 0.5 ℃.
2. acute toxicity testing: get 5 tail vein injection transfer factors resisting virus of respiratory tract of healthy mice 0.5ml, do not have death in 72 hours.
3. hypersensitive test: get 5 of body weight 250～350g healthy guinea pigs, injection this product 0.5ml after 2 weeks, again from intravenous injection this product 1ml, injects in back 15 minutes next day of continuous 3 times, observes animal and does not have phenomenons such as obviously perpendicular hair, dyspnea, spasm, collapse and death.
4. sterility test: with transfer factors resisting virus of respiratory tract, be inoculated in respectively in aerobe, anaerobe and the mycete culture tube, cultivating in 5 days for 30～35 ° does not have bacterial growth.
5. determination of activity: adopt active lymphocyte E-Flos Rosae Rugosae test, the lymphocyte bow structure percentage control tube of this product is high more than 30%.
The transfer factors resisting virus of respiratory tract that extracts by the inventive method has following physicochemical property:
1. the present invention is colourless or is little yellow liquid the optical test outward appearance because of containing a small amount of hypoxanthine.
2.PH value is 7.0～7.2.
3. ultra-violet absorption spectrum is a key property of this product, cause contains nucleotide, polypeptide micromolecular material composition, and purine that wherein contains and pyrimidine base base all have conjugated double bond structure, the ultraviolet light of energy strong absorption 250～255nm wavelength, its maximum absorption wavelength is at 252 ± 2nm.
4. the present invention is not because of containing the high molecular weight protein composition, with ultra-violet absorption spectrum OD value 260nm/280nm 〉=2.0 purity indexs as preparation.
5. determining content of peptides carries out (Chinese biological goods specification, nineteen ninety version first one 301 pages), the every ml of content of peptides 〉=500 μ g by the Lowry method.
6. ribose assay (by first one 321 pages of Chinese biological goods specification nineteen ninety versions), the every ml of ribose content 〉=50 μ g.
Transfer factors resisting virus of respiratory tract not only has preventive effect to upper respiratory tract infection, also has therapeutic efficiency.Experimental results show that nontoxic, side effect through pharmacology, toxicology etc., and be applicable to the acute upper respiratory tract viral infection, recurrent respiratory tract infection, acute pharyngitis, laryngopharynx, bronchitis and viral conjunctivitis, but and preventing cold through clinical verification.Especially to child, old people and human body patient lower for the resistance, can play the effect of raise immunity preventing cold.
Embodiment
1. transfer factors resisting virus of respiratory tract atomizing nasal drop
But the every ml of Zhi Bei transfer factors resisting virus of respiratory tract adds the broad ectrum antibiotic that 8000 units celebrate big syphilis or other topical application as stated above.
Respiratory virus infection at first is the viral infection airway epithelial cell and produces in part breeding or its metabolism and to diffuse into blood and cause General Symptoms, destroys the local mucous membrane function simultaneously, causes local dysbacteriosis and pathogenic bacterium to invade, and causes secondary infection.The contained transfer factors resisting virus of respiratory tract of preventing respiratory transfer factor atomizing nasal drop can play specificity and strengthen T lymphocytes in human body identification and kill virus ability, stop virus further to spread at human body, contained antibiotic composition can suppress or kill the breeding of local pathogenic bacterium simultaneously, prevent secondary infection, alleviate or eliminate clinical symptoms.Usage: collunarium: be grown up each 4～5, each 2～3 of children's, every day 4～5 times.
Eye drip: each 1～2, every day 3～4 times.
Atomizing: each 2ml, every day 1～2 time (can with medicine and the antibiotic coupling of reducing phlegm).Storage and effect duration: store in 4 ℃ of refrigerators or freezing (not multigelation), 18 months effect duration.
2. transfer factors resisting virus of respiratory tract injection
The transfer factors resisting virus of respiratory tract of method for preparing is after aseptic filtration, and in the aseptic subpackaged ampoule, every 2ml, fire fire and seal.
The transfer factors resisting virus of respiratory tract injection can directly inject heavy dose of reactive antiviral transfer factor in the human body, can rapidly and efficiently transfer the human immune system, strengthens anti-virus ability.Usage: intramuscular injection, the each 1～2ml of child, each 2～4ml that is grown up, every day or the next day annotate
Penetrate once.Storage and expiration date: 4 ℃ of refrigerators or 18 months effect duration of stored frozen (not multigelation).
Claims (1)
1, the production technology of transfer factors resisting virus of respiratory tract, at first prepare Respirovirus antigen,, use the virus antigen immune animal of being extracted then secondly with discontinuous gradient density centrifugation purified virus antigen, from immune animal, extract transfer factors resisting virus of respiratory tract at last, it is characterized in that:
With respiratory syncytial virus, adenovirus, parainfluenza virus, rhinovirus and herpes simplex virus, be inoculated in the Hep-2 cell or the bhk cell that have grown up to monolayer respectively, hatch for 37 ℃, every day observation of cell pathological changes situation, when CPE occurs +++or ++ +++after, scrape cell with a little culture fluid, in the dress heavy wall ampoule, ultrasonic smashing, centrifugal 20 minutes of 5000rpm, discard sediment and stay supernatant, pack into 15%, 30%, the discontinuous gradient density centrifugation pipe of 45% and 60% sucrose preparation, ultracentrifugation, 165000 * g3 hour, determine this centrifuge tube position, virus place according to the molecular weight of different virus, sucking-off is standby;
Influenza virus is inoculated in 10 days instar chicken embryo allantoic fluids and amniotic cavity, 33 ℃ following 48-72 hour, get chick embryo allantoic liquid, Ultrasonic Pulverization is got centrifugal supernatant, the discontinuous gradient density centrifugation is extracted virus antigen then;
Select healthy mammal and get the virus antigen of preparation, immune animal, immune 5-6 time altogether, preceding twice respectively with complete freund adjuvant and incomplete freund adjuvant hybrid antigen, beat even with high-speed homogenizer, in animal skins or subcutaneous multi-point injection immunity, the 3rd later muscle or intravenous injection animal, each immunity is a week at interval, last immunity was got animal venous blood after 7 days, detected 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 lymph tuberosity tissue;
Animal spleen and lymph node tissue with virus immunity, remove tissues such as its surperficial fascia, fat, sterile saline washes repeatedly, shred, through the tissue refiner high speed pulverization, multigelation 4-6 time is all pulverized with the tissue refiner high-speed homogenization behind each the thawing, extracts with the method for ultrafiltration or dialysis then;
When extracting with dialysis process, dialysis solution is 0.9% normal saline, puts the dialysis of 4 ℃ of refrigerators, places after 24 hours the collection dialysis solution and gets final product with filtering with microporous membrane, degerming less than 0.2 μ m.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN99115750A CN1089608C (en) | 1999-04-12 | 1999-04-12 | Process for producing transfer factors resisting virus of respiratory tract |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN99115750A CN1089608C (en) | 1999-04-12 | 1999-04-12 | Process for producing transfer factors resisting virus of respiratory tract |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1234276A CN1234276A (en) | 1999-11-10 |
| CN1089608C true CN1089608C (en) | 2002-08-28 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN99115750A Expired - Fee Related CN1089608C (en) | 1999-04-12 | 1999-04-12 | Process for producing transfer factors resisting virus of respiratory tract |
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| CN (1) | CN1089608C (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0101200A2 (en) * | 1982-07-20 | 1984-02-22 | BIO-COM Inc. | Transfer factor for use in treating viral infections |
| EP0143445A2 (en) * | 1983-11-25 | 1985-06-05 | Amtron, Inc. | Process for obtaining transfer factor from colostrum, transfer factor so obtained and use thereof |
-
1999
- 1999-04-12 CN CN99115750A patent/CN1089608C/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0101200A2 (en) * | 1982-07-20 | 1984-02-22 | BIO-COM Inc. | Transfer factor for use in treating viral infections |
| EP0143445A2 (en) * | 1983-11-25 | 1985-06-05 | Amtron, Inc. | Process for obtaining transfer factor from colostrum, transfer factor so obtained and use thereof |
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| Publication number | Publication date |
|---|---|
| CN1234276A (en) | 1999-11-10 |
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