CN1462635A - Microzyme vaccine and bacterin for SARS epidemical tissue and its manufacturing method - Google Patents
Microzyme vaccine and bacterin for SARS epidemical tissue and its manufacturing method Download PDFInfo
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- CN1462635A CN1462635A CN03126573A CN03126573A CN1462635A CN 1462635 A CN1462635 A CN 1462635A CN 03126573 A CN03126573 A CN 03126573A CN 03126573 A CN03126573 A CN 03126573A CN 1462635 A CN1462635 A CN 1462635A
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Abstract
An apidemic lung tissue yeast SARS vaccine in the form of aerosol, tablet or injection for specifically preventing SARS is prepared from the epidemic lung tissue yeast of SARS through amplification, deactivating and detoxicating. A tissue yeast mycelium for diagnosing SARS or suspected SARS is prepared from said epidemic lung tissue yeast of SARS through washing, ultrasonic pulverizing and repeated freezing-thawing.
Description
One, technical field:
The popular lung tissue yeast vaccine of SARS and rhzomorph and manufacture method thereof: belong to biological technical field.
Two, background technology:
The popular lung tissue yeast vaccine of SARS and rhzomorph and manufacture method thereof: the present invention to 2003 lasts a wheat harvesting period at GuangZhou, China 18, on April, successfully identify and confirm that SARS, SARS pathogen are " popular lung tissue yeast " by surplus state China, primary disease is popular lung tissue blastomycosis, and to draft exploitation this pathogen relevant scientific research and produce series of products (Fig. 1～5) Fig. 1 be the being seen pathogen of animal model lung tissue section.Fig. 2 is the pathogen of pulmonary vascular smooth muscle, Fig. 3 is the pathogen in the lung tissue alveolar, and Fig. 4 is a large amount of pathogen in the lung tissue columnar epithelium, and Fig. 5 is pathogen granule and sphaerocyst daughter in the cerebral tissue, Fig. 6 is the pathogen pure culture, also is the raw material of making SARS vaccine and rhzomorph.
The invention provides a kind of by the popular lung tissue yeast of the popular lung tissue yeast (hereinafter to be referred as organizing yeast) " SARS " of SARS vaccine "; (hereinafter to be referred as organizing born of the same parents' Yeast vaccine) makes " the popular lung tissue saccharomycetin of SARS " (hereinafter to be referred as organizing saccharomycetin) with the above-mentioned same yeast of organizing
To whole data of being retrieved, all do not find the product and the production method thereof of or correlation technique content similar with the present invention.
Three, summary of the invention:
The popular lung tissue yeast of SARS and rhzomorph and manufacture method thereof: manufacturing organize the yeast vaccine, be used for pre-preventing tissue blastomycosis and infect, make histoplasmin, be used for specific diagnosis and organize blastomycosis.
The popular lung tissue yeast of the present invention can be used as scientific research, SARS, SARS test and commercial use.Remove above-mentioned manufacturing vaccine and skin test diagnostic reagent, after also available additive method is handled, make specific diagnosis reagent, and use this pathogen and organize the yeast test cdna to arrange to draw gene mapping or other scientific researches, drug study.
1, key problem in technology
Organize the yeast vaccine, mainly organizing yeast (dead bacterium) to make by bacterial strain amplification, deactivation, detoxification forms, its ablation method be make organize yeast in water-bath or in the suitable environment of heating with 100 ℃ 5-50 minute, or 60-95 ℃, deactivation in 1-10 hour, adopt 1%-3% formalin-inactivated and detoxification simultaneously, reach the deactivation purpose, and keep organizing the yeast vaccine antigen good.
2, technical scheme
Organizing the yeast vaccine is to organize the yeast vaccine with the method manufacturing of deactivation and formaldehyde detoxification.Also can will organize the yeast nutritional control simultaneously, constantly go down to posterity repeatedly (in animal or culture medium) or make and organize the yeast virulence to descend, make the live vaccine of organizing the yeast attenuation, be used for the above-mentioned prevention of organizing blastomycosis to infect with other artificial methods.
Organize the clinical consumption unit of yeast vaccine to calculate by containing the bacterium number, supplying the vaccine each (bottle) of subcutaneous or intramuscular injection is 10 * 10
8/ ml/ props up, and is used for collunarium and spray larynx aerosol, and its dosage is 5 * 10
9/ 5ml.And make the dosage form series organize the yeast vaccine, aerosol, collunarium drop (liquid) and be used for tablet, the subcutaneous or intramuscular injection injection of confession that the Sublingual contains, and lyophilized injectable powder etc.It is 2～20 times of inactivated vaccine decrement that the dosage of yeast attenuated live vaccine is organized in employing.
3, technique effect
The popular lung tissue blastomycosis of SARS is considered to a kind of deadly infectious disease now, and the yeast vaccine is organized in the present invention's development, thoroughly solves the optimal path that propagation is stretched and the susceptible person prevents of primary disease.Universally acknowledged, vaccine is the most effective, most convenient, the most complete and the most most economical method that keeps off infection, and organizes succeeding in developing of yeast vaccine, will make contributions for the whole society and to the whole mankind's health.
Organize saccharomycetin: its manufacture method is the yeast of organizing that adopts above-mentioned deactivation, through the repeated multiple times centrifuge washing, add an amount of aseptic solvent, with ultrasonic grinding and freeze repeatedly molten after, with appropriate solvent with 1: 100-10000 dilution, organize the blastomycosis skin test to use for specific diagnosis.
Make a definite diagnosis disconnected for the etiologic card of acute epidemic diseases, be to eliminate the epiphytotics source of infection and to patient's the timely treatment and the key of prevention, organize saccharomycetin to succeed in developing, a kind of specific diagnosis articles for use are provided, to organizing blastomycosis or doubtful primary disease, make a definite diagnosis fast and can make these patient's symptomatic treatments, in time given treatment to, early the health clothes.
For realizing that above-mentioned target the present invention adopts technical scheme as follows:
Organize yeast vaccine and the fabrication schedule of organizing saccharomycetin
Yeast vaccine seed system is organized in manufacturing
Production is adopted the seed lot system with strain.Criticize by means of generation from primordial seed, amplification back lyophilizing saves as main seed lot.From main seed lot preparation work seed lot.Main seed lot is consistent with primordial seed with the various characteristics of work seed lot.The work seed lot is used for producing.
The calibrating of seed lot strain
Form and cultural character
Bacterial classification inoculation to be checked in fat, the husky Borrow's culture medium of improvement or other appropriate medias, is histoplasma capsulatum, and on blood agar culture-medium, it is spherical that bacterium colony just is, and brain shape, pink are to brown.
Strain goes down to posterity and preserves
The strain lyophilizing is stored in 2-8 ℃, and main seed lot must not go down to posterity behind breakdown above 5 generations.
The stock solution preparation
The production seed
To work behind the seed lot strain breakdown, and be inoculated in the husky Borrow of improvement or other appropriate medias, and put 37 ℃ of cultivations and go down to posterity, and generally be no more than 24 hours, the production labor for preparing suitable number is thus made seed.
The production culture medium
With his culture medium of the base of the Nutrient agar of pH7.0-7.4 and nutrient broth or the approval of national drug administrative authority.Contain harmful in the culture medium or other anaphylactogen materials.
Bacterial classification inoculation and cultivation
Adopt large size vial closed to cultivate kind of a method, inoculation was cultivated 22-24 hour for rearmounted 37 ℃, and in culture sterilization sampling, smear staining microscopy, as finding assorted bacterium should be discarded.
Collect and sterilization
After cultivating end, collect bacterium liquid, direct heating sterilization or adding final concentration are the formalin of 1.0-%-1.2%, put 37 ℃, 7 days, sampling was planted in husky Borrow's culture medium of improvement and brave red agar and is done sterility test, behind 4 ℃ of nor-aldehyde of centrifuge washing, add aseptic PBS, put 2-8 ℃ of preservation to commercial weight.
The semi-finished product preparation
The stock solution of single batch or many batches of assay approvals merges, and with PBS dilution (phosphate buffer), mixes with stock solution, makes every 1ml contain 1-5 * 10
9
Form and purposes
This strain through heat inactivation, detoxification treatment, is made every bottle and is contained bacterium 1-5 * 10 after cultivating with histoplasma capsulatum
8Ml does not contain antiseptic, is used for the biological preparation of pre-preventing tissue yeast infection.
That gets not packing organizes yeast stock solution, with ultrasonic filter pulverize and freeze repeatedly molten after, with 1: the dilution of 100-10000 creates " tissue saccharomycetin " for organizing the yeast infection patient to make the usefulness of skin test." tissue yeast vaccine " and the concrete manufacture method of " tissue saccharomycetin " are as follows;
Organize the yeast vaccine and organize the saccharomycetin manufacture method
1. definition, composition and purposes
This strain is with after organizing yeast to cultivate, through formalin deactivation, detoxification treatment, make every bottle and contain bacterium 1-5 * 10
9/ ml does not contain antiseptic and makes.Be used for the prevention of the popular lung tissue blastomycosis of SARS.
2. make
2.1 basic demand
2.1.1 facility and quality of production management
Implement by China's " Good Manufacturing Practice and Quality Control of Drug " requirement.China Association of Chemistry Pharmaceutical Industry, chemical industry is published new, calendar year 2001 P14-37:96-105.
2.1.2 raw material and adjuvant
Meet the requirement of existing " the main raw and auxiliary material quality control standard of Chinese biological goods ".Do not include the chemical reagent of above-mentioned standard in, be not less than chemical pure.Chinese biological standard of articles committee, chemical industry is published new 2000 version P49-62.
2.1.3 industrial water
Industrial water meets national drinking water standard, existing " Chinese pharmacopoeia version standard in 2000 that purified water and water for injection meet." the main raw and auxiliary material quality control standard of Chinese biological goods " version in 2000, Chinese biological standard of articles committee compiles, P1-2.
Use utensil 2.1.4 produce
Be directly used in utensils such as the metal of production or glass, strict clean and handle or sterilization treatment in the source of reducing phlegm and internal heat.
2.15 produce and examine and determine and use animal
Rabbit, mice, Cavia porcellus meet the cleaning level.
2.2 strain
2.2.1 strain name and source
Manufacturing separates acquisition and lyophilizing preservation or keeping of national specified unit and distribution with strain by the direct sample in locality.
2.2.2 the foundation of seed lot
Production is adopted the seed lot system with strain.Criticize from primordial seed and to go down to posterity, amplification back lyophilizing saves as main seed lot.From main seed lot preparation work seed lot.Main seed lot is criticized consistent with the various characteristics of work seed lot with primordial seed.The work seed lot is used for producing.
2.2.3 seed lot strain calibrating
2.2.3.1 form and cultural character
In the husky Borrow's agar of improvement or other appropriate medias, for organizing yeast, bacterium colony just is spherical, brain shape, pink to brown on blood agar culture-medium with bacterial classification inoculation to be checked.
2.2.3.2 biochemical reaction
Used strain is all used antibacterial biochemical identification system, is accredited as and organizes yeast.
2.2.3.3 serum agglutination test
With 37 ℃ of cultures of cultivating 20-24 hour,,, make it contain bacterium 10 * 10 with the dilution of sterile physiological sodium chloride solution with 1% formalin-inactivated
8Ml and organizes yeast immunologic diagnosis serum, after fully mixing, puts 37 ℃ and spends the night, and the high dilution of serum of seeing coagulation with naked eyes is an agglutination titer.Agglutination titer should be not less than former serum titer partly.
2.2.3.4 virulence test
Take to organize yeast respectively with 37 ℃ of husky Borrow's liquid cultures of improvement of cultivating 20-24 hour, be diluted to the sterile physiological sodium chloride solution and contain bacterium 1.2 * 10
9/ ml, 8 * 10
8Ml, 4 * 10
8/ ml, 2 * 10
8/ ml isoconcentration bacterium liquid, lumbar injection body weight 14-16g mice, the same sex or male and female half and half, each concentration bacterium liquid uses at least 5 mices, and every 0.5ml observed 3-7 days, calculated LD
50
2.2.3.5 toxicity test
Adopt 37 ℃ of husky Borrow's liquid cultures of improvement of cultivating 20-24 hour to be suspended in the physiological sodium chloride solution 70 ℃ of heat 2 hours (or additive method) sterilization, not adding preservative agents respectively.After bactericidal assay is qualified, mix by a certain percentage, be diluted to every 1ml and contain bacterium 4.0 * 10
9, 2.0 * 10
9And 1.0 * 10
9Totally 3 concentration, 5 of each concentration bacteria suspension 0.5ml lumbar injection body weight 16-18g mices were observed 3 days, injection 1.0 * 10
9To 2.0 * 10
9The mice of individual dead thalline should all survive.
2.2.3.6 antigen test
Selecting at least 3 of rabbit about body weight 2kg,, inject 8 times with bacterium liquid injection rabbit with above-mentioned immunity test, takes a blood sample and does quantitative agglutination test in last injection back 7-10 days, and mensuration is tired.2/3 rabbit anteserum agglutination titer should be not less than 1: 2560.
2.2.3.7 immunity test
Be no more than 1.1% ± 0.1% formalin sterilization with final concentration,, make it contain bacterium 2.0 * 10 with the not bacterium liquid dilution of adding preservative agent
9Ml.Select at least 30 of body weight 14-16g mices, each subcutaneous injection bacterium liquid 0.5ml, injection in 5 days is 1 time at interval; inject altogether 3 times, after the last immunity 7-10 days, with 2 MLD toadstools; observed 3 days, the control group mice death toll should be not less than 80%, and immune group mice protective rate should be not less than 70%.
2.2.4 strain goes down to posterity and preserves
2.2.4.1 the strain lyophilizing is stored in 2-8 ℃, main seed lot must not go down to posterity behind breakdown above 5 generations.
2.2.4.2 check strain properties before producing, be used for producing after qualified.
2.3 stock solution preparation
Use seed 2.3.1 produce
To work behind the seed lot strain breakdown, and be inoculated in the husky Borrow's culture medium of improvement or other appropriate medias, and put 37 ℃ of cultivations and go down to posterity, and generally be no more than 24 hours, the production labor for preparing suitable number is thus made seed.
Use culture medium 2.3.2 produce
Other culture medium with husky Borrow's culture medium of the improvement of pH7.2-7.4 or the approval of national drug administrative authority.Do not contain harmful in the culture medium or other anaphylactogen materials.
2.3.3 bacterial classification inoculation and cultivation
Adopt sealing conical flask kind method, the rearmounted 37 ℃ of shaking tables of inoculation were cultivated 22-24 hour, and pure bacterium test is done in sampling before the sterilization in incubation, and smear staining microscopy, as finding assorted bacterium should be discarded.
2.3.4 collect and sterilization
After cultivating end, heat 100 ℃ immediately, sterilization in 10-30 minute, add the formalin that final concentration is 1.0%-1.2% then, put 37 ℃, 7 days, sampling is planted in the husky Borrow's culture medium of improvement and is done sterility test, behind 4 ℃ of nor-aldehyde of centrifuge washing, add aseptic PBS and wash secondary PBS and mend, put 2-8 ℃ of preservation to commercial weight.
2.4 stock solution calibrating
Undertaken by 3.1.
2.5 semi-finished product preparation
The stock solution of single batch or many batches of assay approvals merges, and with the PBS dilution, mixes with stock solution, makes every 1ml contain histoplasma capsulatum 1 * 10
9
2.6 semi-finished product calibrating
Undertaken by 3.2.
2.7 in batches
By this edition rules general rule＜biological product rules in batches〉carry out." Chinese biological goods rules " version in 2000, biological product standards committee compiles, P13
2.8 packing or lyophilizing.
By this edition rules general rule＜biological product packing rules〉carry out." Chinese biological goods rules " version in 2000, biological product standards committee compiles, P14
2.9 specification
This product specification is every bottle 1-5 * 10
9/ ml includes an amount of protective agent.
2.10 packing
By this edition rules general rule＜biological product packing rules〉carry out." Chinese biological goods rules " version in 2000, biological product standards committee compiles, P16
3 calibratings
3.1 stock solution calibrating
3.1.1 there is not assorted bacterium.
3.1.2 sterility test
By this edition rules general rule＜biological product sterility test rules〉the A item carries out." Chinese biological goods rules " version in 2000, biological product standards committee compiles, P29-34
3.1.3 concentration determination
Carry out concentration determination with reference to " Chinese bacterial turbidity standard ", quite per 1 milliliter contains bacterium and should be 1 * 10
9Calculate by this concentration during dilution, error is no more than 10%.
3.1.4 stock solution is preserved
Stock solution leaves standstill and is stored in 2-8 ℃.
Stock solution is gathered storage life, is no more than 4 months from the stock solution merging.Storage life is no more than 6 months from the collection sterilization.
3.2 semi-finished product calibrating
3.2.1 sterility test
By this edition rules general rule＜biological product sterility test rules〉the A item carries out." Chinese biological goods rules " Chinese biological standard of articles committee compiles, P29-34
3.2.2 formulation concentrations
Every 1ml contains histoplasma capsulatum 1 * 10
9
3.3 finished product calibrating
3.3.1 discrimination test
Carry out slide agglutination test with the specific immunity serum of this bacterium, should be obvious agglutination.
3.3.2 outward appearance
Shake grumeleuse or the foreign body that does not loose for should be the milky suspension after white suspension or white lyophilized injectable powder, the dissolving, should not containing.
3.3.3 chemical assay
By this edition rules appendix＜biological product chemistry and other calibration methods〉carry out." Chinese biological goods rules " Chinese biological standard of articles committee compiles, P669-696.
3.3.3.1PH value
Be 6-7.
3.3.3.2 free formaldehyde content: should not be higher than 0.1g/L.
3.3.3.3 lactose
" Chinese pharmacopoeia version in 2000, Chinese biological standard of articles committee compiles P600-603 and carries out by existing.Contain lactose.
3.3.4 sterility test
By this edition rules general rule＜biological product sterility test rules〉the A item carries out." Chinese biological goods rules " version in 2000, Chinese biological standard of articles committee compiles, P29-34.
3.3.5 abnormal toxicity test
By this edition rules general rule＜biological product abnormal toxicity test rules〉carry out.Injected in mice 0.5ml, Cavia porcellus injection 5ml." Chinese biological goods rules " version in 2000, Chinese biological standard of articles committee compiles, p40
3.3.6 concentration determination
Carry out concentration determination with " Chinese bacterial turbidity standard ", every bottle contains organizes yeast 1-5 * 10
9/ ml.
3.3.7 quality standard
Indication
Can improve the body specific immunity after the inoculation, be used for pre-preventing tissue blastomycosis and infect.
Organize the manufacture method of saccharomycetin
Finish on (3,1,4) the fabrication schedule basis of organizing the yeast vaccine, get and organize yeast stock solution centrifuge washing three times after the above-mentioned merging, fully pulverize with ultrasound wave, and freezing repeatedly below-100 ℃ molten three times simultaneously, take out, 4 ℃ 12000 time/minute centrifugal one hour, abandon precipitation, get supernatant, with water for injection 1: 100～10000 dilutions, pass through sterility test, abnormal toxicity test is transferred pH6-7, for the popular lung tissue blastomycosis of SARS or seemingly organize the usefulness of blastomycosis patient skin test with fixed attention, each 0.1ml, intradermal injection.
Claims (5)
1. the present invention relates to the popular lung tissue yeast of a kind of SARS vaccine, rhzomorph and manufacture method thereof is characterized in that: adopt the popular lung tissue yeast of SARS to make the specificity vaccine through strain amplification, deactivation, detoxification, or carry out attenuation through manual method and make attenuation prevention live vaccine, for the usefulness of pre-preventing tissue yeast infection.With above-mentioned homology strain through centrifuge washing, Ultrasonic Pulverization, freeze molten manufacturing repeatedly and organize saccharomycetin, or handle the back with additive method and produce specific diagnosis reagent, or make scientific research, gene order test and commercial use with, medicament screening experiment.
2. the ablation method of organizing the yeast vaccine according to claim 1 and 2 is characterized in that: adopt 100 ℃ 1-60 minute, or 60-95 ℃, 1-10 hour, organize the yeast inactivated vaccine with 1-3% formalin-inactivated and poison-removing method manufacturing.
3. the use content of histoplasma capsulatum as claimed in claim 1 or 2 vaccine, but packing 1-5ml is 10 * 10 with containing the bacterium number
8/ ml, adopt the dosage organize the yeast attenuated live vaccine be the inactivated vaccine decrement 2-20 doubly.
The yeast viable bacteria of organizing with artificial attenuation is made histoplasma capsulatum's attenuated live vaccine with reference to this manufacture method or other suitable methods.
4. according to the yeast vaccine of organizing as claimed in claim 1, can be made into the injection of drop that the spray larynx uses with aerosol, collunarium, tablet that the Sublingual contains, subcutaneous or intramuscular injection and lyophilized injectable powder etc.
5. according to the saccharomycetin of organizing as claimed in claim 1, it is characterized in that: adopt the yeast of organizing of deactivation, through repeatedly washing, ultrasonic grinding and freeze molten repeatedly, use The suitable solvent, with 1: the 100-10000 dilution, for the usefulness of organizing the blastomycosis specific diagnosis.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN03126573A CN1462635A (en) | 2003-05-16 | 2003-05-16 | Microzyme vaccine and bacterin for SARS epidemical tissue and its manufacturing method |
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|---|---|---|---|
| CN03126573A CN1462635A (en) | 2003-05-16 | 2003-05-16 | Microzyme vaccine and bacterin for SARS epidemical tissue and its manufacturing method |
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| CN1462635A true CN1462635A (en) | 2003-12-24 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100413537C (en) * | 2004-01-29 | 2008-08-27 | 国家人类基因组南方研究中心 | SARS coronary virus full virus vaccine |
-
2003
- 2003-05-16 CN CN03126573A patent/CN1462635A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100413537C (en) * | 2004-01-29 | 2008-08-27 | 国家人类基因组南方研究中心 | SARS coronary virus full virus vaccine |
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