CN1202860C - Anti-SARS virus transfer factor preparation method - Google Patents
Anti-SARS virus transfer factor preparation method Download PDFInfo
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- CN1202860C CN1202860C CNB031080065A CN03108006A CN1202860C CN 1202860 C CN1202860 C CN 1202860C CN B031080065 A CNB031080065 A CN B031080065A CN 03108006 A CN03108006 A CN 03108006A CN 1202860 C CN1202860 C CN 1202860C
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Abstract
The present invention discloses a preparation method for a severe acute respiratory syndrome (SARS) virus resistance transfer factor. Firstly, SARS viruses are cultured and proliferated by using the Hep-2 cells, human embryonic kidney cells, human embryonic lung cells or chick embryos, SARS virus antigens are prepared, protective antigens are prepared according to an SARS virus gene order by a gene engineering method in the mode of expression; secondly, the virus antigens are processed through centrifugal purification by discontinuous gradient density or processed through sedimentation concentration by using sucrose of 20%; thirdly, an animal is immunized by using the extracted virus antigens; finally, an SARS virus resistance transfer factor is extracted from the immunized animal. The SARS virus resistance transfer factor medicine of the present invention can perform the functions of preventing and treating virus infection through enhancing specific killing function of immunized killer cells and regulating a mucosal immunity mechanism, can be prepared into an injection medicine or a nose drop medicine. The present invention has the characteristics of prevention, treatment, and treatment of both manifestation and root cause of a disease.
Description
One, technical field
The invention belongs to field of virology, relate to and a kind ofly be used for prevention and treatment variation coronavirus (Coronavirus) or be called SARS (SARS) medicine for treating viral infections preparation method, particularly a kind of anti-sars virus transfer factor method that adopts the specific antigen immunity mammal of SARS virus and prepare.
Two, background technology
Atypical pneumonia (Severe Acute Respiratory Syndrome, SARS SARS) be to cause by the coronavirus that makes a variation, in November, 2002, the first the infected was made a definite diagnosis in China Guangdong Province, now in the whole world nearly more than 20 countries popular, according to the numeral that on April 22nd, 2003, World Health Organization (WHO) announced, existing 3947 examples in the whole world, dead 229 examples, mortality rate is up to 5.8%, and expansion trend is arranged.Cause global fear, seriously disturbed normal social activity, had a strong impact on global economic development, prevented and treated the top priority that SARS has become the whole mankind.So far, still the nonreactive coronavirus pharmaceutical effectively prevents and treats.
Three, summary of the invention
The objective of the invention is to, propose a kind of preparation method of anti-sars virus transfer factor medicine, both had and to prevent, can treat the characteristics for the treatment of both the principal and secondary aspects of a disease again.
The present invention is little according to the transfer factor molecular weight, non-immunogenicity, and between mammal, do not have kind of the immunology principle of the attribute opposite sex.The specific antigen that adopts SARS virus to extract, immune mammal is got its spleen, lymph node tissue, extracts active ingredient with molecular sieve filtration and dialysis process, develops the anti-sars virus specific transfer factor.Because the human body antiviral mainly is specific immunity cell and antibody recognition by cellular immunization and humoral immunization, causes 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 causes the special and immunoreation fast of human body, kills and remove virus.In addition, think that at present SARS virus mainly is by respiratory infectious, Respirovirus infringement human body generally is at first to find suitable virus receptor at upper respiratory tract, causes epithelial cell to infect, and invades human body then.
The preparation method of anti-sars virus transfer factor medicine of the present invention is as follows:
At first, preparation SARS virus antigen.From patient respiration road secretions, separating the SARS virus that obtains, be inoculated in the Hep-2 cell, HEKC, human embryonic lung cell or the Embryo Gallus domesticus that grow up to monolayer, hatch for 37 ℃, every day observation of cell pathological changes situation, when CPE occurs +++time or detect positive through virus antigen.Collecting cell and supernatant, ultrasonic smashing behind inactivation of virus put the refrigerator stored frozen.
Secondly, with 20% sucrose heavy layer concentrated antigen or with discontinuous gradient density centrifugation purified virus Proantigen.With the ultrasonic centrifugal 20min of antigen 5000rpm that smashes, discard sediment and stay supernatant, pack into the discontinuous gradient density centrifugation pipe of 15%, 30%, 45% and 60% sucrose preparation, ultracentrifugation, 165000 * g, 3h determines this centrifuge tube position, virus place according to the molecular weight of virus, and the sucking-off stored frozen is standby.
Then, use the virus antigen immune animal of being extracted.Select healthy mammal (sheep body weight 30kg～50kg, pig body weight 60kg～90kg), 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, the 3rd later muscle or intravenous injection animal, each immunity is a week at interval, and 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, after the virus-specific immunizing potency occurring, slaughter animal, get spleen and lymph tuberosity tissue, cryogenic freezing is preserved standby.
At last, extract the anti-sars virus transfer factor.The animal spleen and the lymph node tissue of virus immunity, go its surperficial fascia, fatty tissue, sterile saline washes repeatedly, shred, through the tissue refiner high speed pulverization, multigelation 4～6 times is all pulverized with the tissue refiner high-speed homogenization behind each the thawing again, extracts with the method for dialysis and ultrafiltration then.When extracting with dialysis and dialysis process, dialysis solution is 0.9% normal saline, puts 4 ℃ of refrigerators dialysis, collects dialysis solution behind the placement 24h, and with the filtering with microporous membrane degerming less than 0.2 μ m, and it is standby to put the refrigerator stored frozen.
Immunologic cytotoxicity cell-specific killing ability and the adjusting mucosal immunity mechanism of anti-sars virus transfer factor medicine of the present invention by strengthening respiratory tract can play the effect that prevents and treat above-mentioned viral infection.
Four, the specific embodiment
The present invention is described in further detail below in conjunction with embodiment that the inventor provides.
According to above-mentioned technical scheme, carry out the safety test of anti-sars virus transfer factor for the anti-sars virus transfer factor medicine that extracts.
The safety test of anti-sars virus transfer factor comprises:
1. pyrogen testing: get 5 of healthy adult rabbits, auricular vein injection anti-sars virus transfer factor 2ml, respectively at before the injection, injection back 30min, 1h, 2h, anus is surveyed body temperature, the average intensification is no more than 0.5 ℃.
2. acute toxicity testing: get 5 tail vein injection anti-sarses of healthy mice virus transfer factor 0.5ml, do not have death in the 72h.
3. hypersensitive test: get 5 of body weight 250g～350g healthy guinea pig, injection this product 0.5ml next day of continuous 3 times after 2 weeks, again from intravenous injection this product 1ml, in the 15min of injection back, observes animal and does not show phenomenons such as 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 anti-sars virus transfer factor 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, because of containing nucleotide, polypeptide micromolecular material composition wherein contains purine and phonetic purine alkali base all has conjugated double bond structure, the ultraviolet light of energy strong absorption 250nm～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 rules, 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 rules nineteen ninety versions), the every ml of ribose content 〉=50 μ g.
It below is the specific embodiment that the inventor provides.
Embodiment 1: anti-sars virus transfer factor atomizing nasal drop
But Zhi Bei the every ml of anti-sars virus transfer factor adds the broad ectrum antibiotic of 8000 unit gentamycins or other topical application as stated above.
Because of SARS virus mainly is to pass through respiratory infectious, respiratory virus infection at first is the viral infection airway epithelial cell and diffuses into blood at part breeding or its metabolite and cause General Symptoms, destroy the local mucous membrane function simultaneously, cause local dysbacteriosis pathogenic bacterium to invade, cause secondary infection.The contained anti-sars virus transfer factor of anti-sars virus 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: 30 months effect duration of stored frozen (not multigelation).At every turn with after put 4 ℃ of refrigerators.
Embodiment 2: anti-sars virus transfer factor injection
The anti-sars virus transfer factor of method for preparing is after aseptic filtration, and in the aseptic subpackaged ampoule, every 2ml, fire fire and seal.
Anti-sars virus transfer factor 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 the SARS anti-virus ability.
Usage: intramuscular injection: the each 1ml～2ml of child, each 2ml～4ml that is grown up, every day or next day injection are once.
Storage and expiration date: 30 months effect duration of stored frozen (not multigelation).
Claims (2)
1. anti-sars virus transfer factor preparation method is characterized in that, may further comprise the steps:
1) preparation SARS virus antigen
To from patient respiration road secretions, separate the SARS virus that obtains, be inoculated in the Hep-2 cell, HEKC, human embryonic lung cell or the Embryo Gallus domesticus that grow up to monolayer, hatch for 37 ℃, every day observation of cell pathological changes situation, when CPE occurs +++time or detect positive through virus antigen; Collecting cell and supernatant, ultrasonic smashing behind inactivation of virus put the refrigerator stored frozen;
2) concentrate with the heavy layer of sucrose, with discontinuous gradient density centrifugation purified virus antigen
With the ultrasonic centrifugal 20min of antigen 5000rpm that smashes, discard sediment and stay supernatant, pack into the discontinuous gradient density centrifugation pipe of 15%, 30%, 45% and 60% sucrose preparation, ultracentrifugation, 165000 * g, 3h, determine this centrifuge tube position, virus place according to the molecular weight of virus, the sucking-off stored frozen is standby;
3) use the virus antigen immune animal of being extracted
Select healthy mammal, get the virus antigen immune animal of above-mentioned preparation, immunity is 5～6 times 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, and 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, after the virus-specific immunizing potency occurring, slaughter animal, get spleen and lymph tuberosity tissue, cryogenic freezing is preserved standby;
4) from immune animal, extract the anti-sars virus transfer factor
The animal spleen and the lymph node tissue of virus immunity, go its surperficial fascia, fatty tissue, sterile saline washes repeatedly, shred, through the tissue refiner high speed pulverization, multigelation 4～6 times is all pulverized with the tissue refiner high-speed homogenization behind each the thawing again, extracts the anti-sars virus transfer factor with the method for dialysis and ultrafiltration then.
2. anti-sars virus transfer factor preparation method according to claim 1, it is characterized in that described when extracting with ultrafiltration and dialysis process, dialysis solution is 0.9% normal saline, put the dialysis of 4 ℃ of refrigerators, place and collect dialysis solution behind the 24h and get final product with filtering with microporous membrane degerming less than 0.2 μ m.
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| Application Number | Priority Date | Filing Date | Title |
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| CNB031080065A CN1202860C (en) | 2003-04-30 | 2003-04-30 | Anti-SARS virus transfer factor preparation method |
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| CNB031080065A CN1202860C (en) | 2003-04-30 | 2003-04-30 | Anti-SARS virus transfer factor preparation method |
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| CN1202860C true CN1202860C (en) | 2005-05-25 |
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Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN100432096C (en) * | 2003-11-28 | 2008-11-12 | 陆家海 | Transfer factor specific for SARS virus and method for preparing same |
| CN101314032B (en) * | 2007-05-30 | 2011-04-06 | 遵义医学院 | Preparation technique for specified transfer factor oral preparation for typhoid fever, paratyphoid fever |
| 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 |
| CN104523751A (en) * | 2014-12-09 | 2015-04-22 | 郑州后羿制药有限公司 | Method for in vitro preparation of anti-Pestedes petits ruminant virus transfer factor |
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