CN1318449C - Anti SARS specificity IgY and combination preparation thereof - Google Patents
Anti SARS specificity IgY and combination preparation thereof Download PDFInfo
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
- A61K39/215—Coronaviridae, e.g. avian infectious bronchitis virus
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1002—Coronaviridae
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- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/38011—Tombusviridae
- C12N2770/38034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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Abstract
The present invention discloses specific atypical pneumonia (SARS) resistant IgY, a preparation method and preparations thereof. The preparation method comprises the following steps: main causative viruses resulted in atypical pneumonia (SARS) are filtered out, single or compound causative virus antigen is respectively prepared on the basis of various causative virus filtration and differential combination, immunity eggs are prepared, a specific IgY crude extract of the various causative viruses used for resisting the atypical pneumonia (SARS) is prepared, and the specific IgY crude extract of the various causative viruses used for resisting the atypical pneumonia (SARS) is purified. A plurality of preparations of the specific atypical pneumonia (SARS) resistant IgY have the characteristics of specificity, safety and convenient operation for preventing and treating the atypical pneumonia (SARS).
Description
Technical field
The present invention relates to a kind of anti-sars type pneumonia (SARS) specific IgY and preparation method thereof, particularly relate to the composite IgY and preparation method thereof and the combination preparation that are used to prevent and treat the multiple combination of atypical pneumonia.
Background technology
Atypical pneumonia is called " SARS (Severe Acute Respiratory Syndrome) (Severe Acute Respiratory Syndrome; be called for short SARS); be the strong acute respiratory disease of a kind of infectivity, The World Health Organization (WHO) is the pathogenic agent that causes atypical pneumonia in the mutation of official confirmation coronavirus on the 16th April in 2003 again.This sick onset is anxious, is first symptom with heating, and cough (mostly being dry cough), few phlegm are arranged, and the severe patient appearance is breathed and quickened, shortness of breath, or obviously respiratory distress.
The method for the treatment of atypical pneumonia (SARS) at present in the world mainly is to use antiviral [as Ribavirin (ribavirin) etc.] and glucocorticosteroid and microbiotic or herbal medicine.At present, the whole world does not still have the chemicals that really virus is had definite curative effect, and the ubiquity severe side effect.Her Majesty the Queen in right of Canada as represented by the minister of Healt is according to the clinical experience of Canada with the centers for disease control and prevention of the united states expert, and Ribavirin (ribavirin) test invalid to diseases induced coronavirus, add many serious drug reaction reports, announced to stop using Ribavirin (ribavirin) on May 1st, 2003.Use glucocorticosteroid can make the compromised immune of human body, heavy dose of glucocorticosteroid that uses causes patient at last medicine to be had no reaction often, yields to no remedy and main causes of death at last.Herbal medicine has only opsonization to atypical pneumonia (SARS), has been difficult to the definite effective result of treatment.
IgY (Immunoglobulin of Yoik) belongs to the IgG immunoglobulin like protein, can combine with corresponding antigens generation specificity, thereby suppress or changed the state or the activity of this antigen (for some bacterium or virus), and, promote segmented cell or scavenger cell engulfing to bacterium or virus by opsonization; It can combine with viral, changes the conformation of virus surface, stops virus to be adsorbed in permissive cell, and simultaneously, the virus of formation-IgY immunocomplex can be by macrophage phagocytic.
Because IgY can specificity is combined with pathogenic agent and with its effective inhibition, just can overcome antiviral chemicals to the actual killing action of virus the little and fatal shortcoming fairly obvious to the human body toxic side effect, thereby reach with a definite target in view the purpose of the atypical pneumonia (SARS) that causes by this pathogenic agent of the coronavirus of variation from the prevention of morbidity root and treatment.
Technology contents
The purpose of this invention is to provide specific IgY of the main Causative virus of a kind of direct inhibition atypical pneumonia (SARS) and preparation method thereof; The present invention also aims to provide a kind of natural safety of making by the specific IgY that can directly suppress the main Causative virus of atypical pneumonia (SARS) to have novel combination preparation specific action, that prevent and treat atypical pneumonia (SARS) again.
The objective of the invention is to be achieved through the following technical solutions:
(1) preferably cause the main Causative virus of atypical pneumonia (SARS): according to the affirmation of The World Health Organization (WHO), the mutation of coronavirus is the pathogenic agent that causes atypical pneumonia (SARS).But the sample that contains coronavirus in extraction carries out finding in the genetic analysis that this virus variation speed is fast, and only the pathogeny of 4 kinds of atypical pneumonias (SARS) coronavirus has just been found in Hong Kong.The present invention analyzes the result of the gene ordering of country variant and geographic atypical pneumonia (SARS) coronavirus according to gene mapping, carry out sort merge by roughly similar principle, choosing has several atypical pneumonias of notable difference (SARS) coronavirus as representational main Causative virus.Totally 6 kinds of atypical pneumonias (SARS) coronavirus of propagating with Canada that propagate in 4 kinds of different atypical pneumonias (SARS) coronavirus in now preferred Hong Kong and Beijing is as representational atypical pneumonia (SARS) pathogenic agent.From now on, along with the constantly variation in the process of human-to-human transmission of this coronavirus, and further improving and deeply, can in time adjust and change the pathogenic agent combination of representational atypical pneumonia (SARS) this coronavirus gene ordering work in combination.In addition, also can choose different sars coronavirus combinations, as Hong Kong group (some type compositions), Beijing group (some type compositions), Canada's group (some type compositions) or the like according to country variant and area marshalling.
(2) preparation of the special complex antigen of atypical pneumonia (SARS) Causative virus: the present invention is an example with Hong Kong group.With the VeroE6 cell inoculation in 10%FCS DMEM nutrient solution, final concentration of cells 1 * 10
5/ ml.The above-mentioned training liquid of 15ml is placed the 100ml culturing bottle, this culturing bottle is placed on 37 ℃, 5%CO
2Cultivate into monolayer cell in the saturated incubator.Discard nutrient solution then, 4 kinds of different sars coronavirus strains in area, Hong Kong of collecting are diluted to 100 TCID 50/ml with 2%FCS and 1% mycillin DMEM nutrient solution.Get this diluent 10ml and add in the Tissue Culture Flask, and this Tissue Culture Flask is placed on 37 ℃, 5%CO
2Cultivate in the saturated humidity incubator after 24 hours, treat cytopathy reach++++ time results virus.With culturing bottle freeze thawing three times, collect culture supernatant during results virus, put into supercentrifuge,, collect supernatant with the centrifugation of 15000rpm rotating speed.The final concentration that adds formaldehyde to 0.1% again is under 37 ℃ of temperature, through the SARS virus of these 4 kinds of cultivations of difference deactivation in 24 hours.Cultured inactivation of viruses is 4 ℃ or-20 ℃ of preservations.Then, with the 4 kinds of different sars coronavirus in area, cultured Hong Kong in 1: 1: 1: 1 ratio or the mixing of other suitable proportions, add freund adjuvant in 1: 1 ratio or other suitable proportions again, insert in the high-speed homogenization machine with the homogenize of 10000～30000rpm rotating speed high speed pulverization, form water-in-oil solution by high-speed stirring, promptly make the special complex antigen that contains multiple veriform totivirus of atypical pneumonia (SARS) Causative virus.
(3) prepare immune egg
Use the patented technology (number of patent application: 00101270.3),, respectively bird inlay is carried out immunity that our company has applied for adopting atypical pneumonia (SARS) the Causative virus antigen of method for preparing; Strengthen injection more once every two weeks, planned immunization epidemic disease three times; Immunity for the first time is after 20 days, and the hen of searching after the immunity produces immune egg, and the different antigen of correspondence, and the immune egg of being searched is carried out coded markings.
Above immunization method and frequency of injection can suitably be adjusted and variation according to practical situation; Also can use above-mentioned same immunological technique, adopt above-mentioned antigen, respectively lay eggs duck or lay eggs female goose or the differences such as the turkey or the ostrich of laying eggs of the laying eggs bird of laying eggs be carried out immunity, obtain corresponding immune egg.
(4) preparation of anti-sars type pneumonia (SARS) Causative virus specific IgY crude extract: at first according to different and immune used antigen difference,, clean immune egg, use the wipes of alcohol wash disinfection again with flowing water with immune egg classification and label coding by the bird of immunity; Smash with the immune egg that beating machiae will be searched, remove egg white, stay yolk with the sieving of yolk sieve, stir, measure the volume of the yolk of gained, extraordinarily go into distilled water by the 4-6 of this volume, dilute and mix, adjust between the pH to 5.5-6.0 with NaOH solution;
The diluent of adjusting the pH value is further stirred, it is cooled to 2-6 ℃ then, left standstill 12 hours-24 hours; Diluent is added in the high-speed centrifuge centrifugation 20 minutes; Separating obtained supernatant added again carry out ultrafiltration in the ultra-fine filter and concentrate 10-20 doubly, the ratio in 0.1～0.3% adds sodium alginate in the slurry after concentrating, fully stirring; Add in the supercentrifuge centrifugally again, get supernatant, remove lipoprotein; Slurry after the centrifugation is added ultrafilter, cross the ultra micro film, carry out filtration sterilization; Product after the filtration sterilization is carried out lyophilize with freeze drier, promptly make anti-sars type pneumonia (SARS) Causative virus specific IgY crude extract dry powder finished product; At last, prepared corresponding different antigenic specific IgY crude extract is carried out coded markings.
(5) purifying of anti-sars type pneumonia (SARS) Causative virus specific IgY: adopt the disclosed patented technology (number of patent application 00101270.3 of our company, patent publication No. 1307061) crosses ion exchange column and gel exchange column respectively anti-sars type pneumonia (SARS) the IgY crude extract of producing by above technology is carried out purifying, promptly get pure IgY.
Use SDS-PAGE cataphoretic determination method, anti-sars type pneumonia (SARS) the Causative virus specific IgY crude extract of producing by above technology is detected, recording wherein pure IgY content is more than 50%.
(6) adopt the specifc immunity Activiation method of inventor's invention and the prepared anti-sars type of improved Technology pneumonia (SARS) Causative virus specific IgY to carry out the activity detection with reference to conventional " ELISA method " (enzyme linked immunosorbent assay).
Detected result shows that anti-sars type pneumonia (SARS) Causative virus specific IgY is tired to the antibodies of the 4 kinds of representative atypical pneumonias in Hong Kong (SARS) coronavirus and all reached more than 1: 512.
To be equipped with distilled water in order to anti-sars type pneumonia (SARS) the Causative virus specific IgY of top method preparation or physiological saline is deployed into the solvent of concentration more than 0.01%, make a kind of novel propellant, add in the various spraying guns, after atomizing, suck respiratory tract and lung's generation effect for the atypical pneumonia patient, just can effectively suppress to cause the Causative virus of atypical pneumonia, thereby play effective therapeutic action.
This anti-sars type of 0.01-80% pneumonia specific IgY is equipped with the auxiliary material of 99.99-20%, can be made into various clinical acceptable forms, as formulations such as propellant, nasal spray, nasal drop, throat spraying agent, mouth spraying agent, buccal tablets, be used for prevention and treatment atypical pneumonia (SARS).
Formulation as prevention usefulness, this anti-sars type pneumonia (SARS) Causative virus specific IgY can also 2: 1 or 3: 1 or other suitable proportions add with the disclosed patented technology (number of patent application 00101270.3 of our company, patent publication No. 1307061) Zhi Bei dental caries resistant bacteria IgY makes a kind of novel many effects specificity composite IgY.This many effects specificity composite IgY is equipped with auxiliary material makes sprays or buccal tablet, as long as often use this sprays spout chamber and throat, perhaps often contain this novel buccal tablet of clothes, just can be very convenient, both prevention of atypical pneumonia (SARS) like a cork, prevent decayed tooth again, eliminate oral inflammation and halitosis, reach the purpose of achieving many things at one stroke.
To the no specific medicament of atypical pneumonia (SARS), generally adopt symptomatic treatment and a large amount of glucocorticosteroid of throwing clothes microbiotic, broad-spectrum antiviral drug and immunosuppressor at present.As everyone knows, microbiotic does not have killing action to virus, and the antibiotic abuse of broad-spectrum antiviral has destroyed normal people's " colony balance " of human body on the contrary, causes " flora imbalance ".Clinical practice confirms that " flora imbalance " is not only the result that disease takes place, and also is the promoting factor of disease of viral infection, can aggravate disease even worsen conversely.A large amount of uses of immunosuppressor and hormone can directly cause patient's compromised immune, and it is poor that immunity function is more cured, secondary infectation of bacteria very easily, and finally more refractory is healed.
Because the maximum characteristics of the specific IgY of the present invention preparation are directly at the pathogenic agent generation specific inhibitory effect of atypical pneumonia (SARS), the present invention is designed to directly impose on the most concentrated lung of pathogenic agent and respiration channel with the formulation and the insecticide-applying way of practical application again, thus effect directly, target accurately, the disconnected source of effecting a permanent cure, effect be remarkable.Simultaneously, the present invention has fundamentally changed the situation that present treatment atypical pneumonia (SARS) generally needs a large amount of curative effects for oral administration and inapt pharmaceutical chemicals and hormone, avoid damaging patient's immunity function, also overcome conventional insecticide-applying way and will make a large amount of chemicalses enter after the human circulation just generation effect and can bring all dysgenic disadvantages to human body.
Prepared anti-sars type pneumonia (SARS) specific IgY of the present invention is again a kind of unique natural biotechnological formulation, use fool proof, without any side effects, because it only directly acts at the infective pathogen body of atypical pneumonia, probiotics all there is good provide protection; Simultaneously, it can not bring out the generation of drug resistance strain fully, also can be as conventional medicine, and probiotics is also killed with pathogenic agent and caused " flora imbalance "; Conversely; the specific IgY of anti-sars type pneumonia of the present invention (SARS) pathogenic agent can make up a kind of difficult by the normal microflora ecology of virus and pathogenic infection in pneumonia and respiratory tract; form the natural protection barrier of human body one, reach the dual purpose of not only treating but also preventing.In addition, the prepared specific IgY of the present invention is a kind of polyclonal antibody, and it can all produce restraining effect to several different pathogenic agent, and this point is meaningful especially for the coronavirus of pathogenic agent one variation of atypical pneumonia (SARS).Because this novel coronavirus makes a variation especially easily, has only found 4 kinds more than at present in Hong Kong; If develop vaccine, then need multiple vaccine, just can really reach the effect of effective control, this has just increased the difficulty of preventing this pernicious transmissible disease greatly.
And specificity Ig of the present invention not only produces effect to the variation coronavirus of the unsolicited atypical pneumonia (SARS) of directly making the As antigen immune; And, because distance bird far away has stronger immunogenicity on the mammalian proteins confrontation phylogenetics of high conservative; Therefore, as long as adopt the immune bird of immuno-stimulating patented technology (number of patent application 02121244.9) of my company's invention with certain antigen, prepared specific IgY has the pathogenic agent of " common antigenic determinant " all can have intensive to suppress active to every with this antigen.That is to say that both made coronavirus that certain variation has taken place, in fact its integral part can not become; Specific IgY of the present invention still can be at its integral part generation effect.Therefore, the coronavirus that new variation has taken place is suppressed and inactivation.Suppress ability just because of adopt anti-sars type pneumonia (SARS) specific IgY of method preparation of the present invention to have special wide spectrum; So, can solve the difficult problem that the development of giving relevant vaccine and new drug because of this novel coronavirus variation is fast especially brings quite big resistance.
Experimental example 1: will be encoded to Hong Kong 1 type, Hong Kong 2 types, Hong Kong 3 types, Hong Kong 4 types successively from 4 kinds of Hong Kong different atypical pneumonias (SARS) coronavirus.Hong Kong 1 type coronavirus is made antigen by the method for the 2nd of above-mentioned technology contents, uses this antigen immune hen again, and adopts the method for the 4th of the technology of the present invention content to prepare anti-Hong Kong 1 type coronavirus specific IgY.Then, serve as to detect antigen with Hong Kong 1 type coronavirus, detect prepared anti-sars type pneumonia (SARS) specific IgY antibodies with " ELISA method " (enzyme linked immunosorbent assay) and tire.Result such as following table
| IgY | Detection antigen | Antibodies is tired |
| Anti-Hong Kong 1 type coronavirus specific IgY | Hong Kong 1 type coronavirus | 1∶1024 |
Experimental example 2: serve as to detect to use antigen with Hong Kong 2 type coronavirus, Hong Kong 3 type coronavirus, Hong Kong 4 type coronavirus respectively, adopt " ELISA method " (enzyme linked immunosorbent assay) to detect anti-Hong Kong 1 type coronavirus specific IgY respectively these the three kinds antigenic antibodies of detection are tired.Result such as following table
| IgY | Detection antigen | Antibodies is tired |
| Anti-Hong Kong 1 type coronavirus specific IgY | Hong Kong 2 type coronavirus | 1∶256 |
| Hong Kong 3 type coronavirus | 1∶256 | |
| Hong Kong 4 type coronavirus | 1∶256 |
Find out from above detected result, adopt method of the present invention to produce Causative virus antigen, adopt again immuno-stimulating method of the present invention immunity lay eggs bird and the preparation corresponding IgY, prepared anti-Hong Kong 1 type coronavirus specific IgY has the ideal broad spectrum, not only as above the experimental example 1 indicated Hong Kong 1 type coronavirus antigen to direct immunization has very strong restraining effect, the antibodies of being surveyed tire quite high (up to 1: 1024); Other not other three kinds of atypical pneumonias (SARS) coronavirus of direct immunization that morphed are had certain restraining effect too, and detected antibodies is tired and is still belonged to ideal.
Experimental example 3: pressed 1: 1: 1 with Hong Kong 1 type coronavirus, Hong Kong 2 type coronavirus, Hong Kong 3 type coronavirus, Hong Kong 4 type coronavirus: 1 mixed is even, make complex antigen with the method steps of the technology of the present invention content (2) again, and adopt method immune hen of the present invention, prepare anti-sars type pneumonia (SARS) specific IgY.Use " ELISA method " (enzyme linked immunosorbent assay) to use antigen with the coronavirus that propagate in Hong Kong 1 type coronavirus, Hong Kong 2 type coronavirus, Hong Kong 3 type coronavirus, Hong Kong 4 type coronavirus and Beijing as detecting respectively then, detect prepared anti-sars type pneumonia (SARS) specific IgY these 5 kinds of antigenic antibodies of difference are tired.Detected result such as following table
| IgY | Detection antigen | Antibodies is tired |
| Anti-sars type pneumonia (SARS) specific IgY | Hong Kong 1 type coronavirus | 1∶512 |
| Anti-sars type pneumonia (SARS) specific IgY | Hong Kong 2 type coronavirus | 1∶512 |
| Anti-sars type pneumonia (SARS) specific IgY | Hong Kong 3 type coronavirus | 1∶512 |
| Anti-sars type pneumonia (SARS) specific IgY | Hong Kong 4 type coronavirus | 1∶512 |
| Anti-sars type pneumonia (SARS) specific IgY | The coronavirus that propagate in Beijing | 1∶256 |
Can see from above result, make the special complex antigen that contains multiple viral composition of multiple Causative virus with method of the present invention, use this special complex antigen again, use the immuno-stimulating method of the present invention immunity bird of laying eggs, prepared anti-sars type pneumonia (SARS) specific IgY, broad spectrum is good especially, except a few type atypical pneumonias (SARS) coronavirus as the antigen direct immunization being had quite high antibodies tires, as of the same race veriform atypical pneumonia (SARS) coronavirus of antigen direct immunization the good restraining effect is not arranged to having yet, reach suitable ideal effect.
Experimental example 4: make atypical pneumonia (SARS) Causative virus antigen according to the method for the technology of the present invention content (2) respectively, then respectively with this antigen immune bird inlay, the duck of laying eggs, the female goose of laying eggs, the lay eggs turkey and the ostrich of laying eggs.Adopt method of the present invention to prepare again respectively: (1) anti-sars type pneumonia (SARS) specificity chicken IgY, (2) anti-sars type pneumonia (SARS) specificity duck IgY, (3) anti-sars type pneumonia (SARS) specificity goose IgY, (4) anti-sars type pneumonia (SARS) specificity turkey IgY, (5) anti-sars type pneumonia (SARS) specificity ostrich IgY.Then, adopting " ELISA method " (enzyme linked immunosorbent assay) serves as to detect antigen with Hong Kong 1 type coronavirus, Hong Kong 2 type coronavirus, Hong Kong 3 type coronavirus, Hong Kong 4 type coronavirus respectively, detects the antibodies of this three kinds of anti-sars type pneumonia (SARS) specific IgY and tires.Result such as following table
| IgY | Detection antigen | Antibodies is tired |
| Anti-sars type pneumonia (SARS) specificity chicken IgY | Hong Kong 1 type coronavirus | 1∶1024 |
| Hong Kong 2 type coronavirus | 1∶1024 | |
| Hong Kong 3 type coronavirus | 1∶1024 | |
| Hong Kong 4 type coronavirus | 1∶1024 | |
| Hong Kong 1 type coronavirus | 1∶1024 |
| Anti-sars type pneumonia (SARS) specificity duck IgY | Hong Kong 2 type coronavirus | 1∶1024 |
| Hong Kong 3 type coronavirus | 1∶1024 | |
| Hong Kong 4 type coronavirus | 1∶1024 | |
| Anti-sars type pneumonia (SARS) specificity goose IgY | Hong Kong 1 type coronavirus | 1∶512 |
| Hong Kong 2 type coronavirus | 1∶512 | |
| Hong Kong 3 type coronavirus | 1∶512 | |
| Hong Kong 4 type coronavirus | 1∶512 | |
| Anti-sars type pneumonia (SARS) specificity turkey IgY | Hong Kong 1 type coronavirus | 1∶512 |
| Hong Kong 2 type coronavirus | 1∶512 | |
| Hong Kong 3 type coronavirus | 1∶256 | |
| Hong Kong 4 type coronavirus | 1∶256 | |
| Anti-sars type pneumonia (SARS) specificity ostrich IgY | Hong Kong 1 type coronavirus | 1∶256 |
| Hong Kong 2 type coronavirus | 1∶256 | |
| Hong Kong 3 type coronavirus | 1∶256 | |
| Hong Kong 4 type coronavirus | 1∶256 |
From above test-results as can be seen, adopt method of the present invention to make antigen, with immuno-stimulating method immune hen, duck, female goose, the lay eggs turkey or the ostrich of laying eggs, adopt method of the present invention to make anti-sars type pneumonia (SARS) specificity chicken IgY respectively again, anti-sars type pneumonia (SARS) specificity duck IgY, anti-sars type pneumonia (SARS) specificity goose IgY, anti-sars type pneumonia (SARS) specificity turkey IgY, five kinds of different IgY of anti-sars type pneumonia (SARS) specificity ostrich IgY, though the bird difference of immunity, prepared specific IgY all has the good restraining effect to several Causative virus that cause atypical pneumonia.Because duck IgY does not have the Fc section, if duck IgY is made injectable dosage forms, be injected in the human body, human body is difficult for producing rejection, and this is a duck IgY biggest advantage.Though immunity is laid eggs the ostrich and the prepared IgY antibodies of turkey of laying eggs tire from detect data seen lower a little, but, because ostrich and turkey egg individuality are big especially, it is all more much more than chicken immune egg and duck immunity egg and goose immunity egg that each immune egg can extract the IgY that obtains, this point also is very desirable for large-scale industrialization production.
Experimental example 5: atypical pneumonia (SARS) just morbidity patient is used with " anti-sars type pneumonia (SARS) specific IgY " of the present invention clinical observation as the combination propellant treatment of raw material making
(1) at the beginning of being admitted to hospital just that isolating for treatment diagnoses a disease really arbitrarily to select 50 among the people, sends out the patient;
(2) " anti-sars type pneumonia (SARS) IgY makes up propellant " of adopting the present invention to prepare after soup is opened, is poured in the PARI type compression atomizing air pump, sucks for the patient through atomizing.Under the prerequisite that continues the corresponding symptomatic treatment measure cooperation of nasal tube oxygen inhalation, sucked IgY combination propellant of the present invention once, and continued 5-10 minute at every turn in per two hours with other.
(3) every day the viewing test patient the symptom variation situation, make detail record;
(4) through smoking " anti-sars type pneumonia (SARS) IgY makes up propellant " continuously after three days, 32 patient Gao Re progressively go down, sx such as chilly, weak, general malaise.After smoking a week, it is lasting to have only 8 patients to generate heat, and dry cough, pectoralgia occur, and all the other patients are no longer heating all, and symptom obviously alleviates, and has 10 patient's symptoms to eliminate.
Experimental example 6: the clinical observation result that the combination propellant that atypical pneumonia (SARS) patient with severe symptoms uses " anti-sars type pneumonia (SARS) specificity composite IgY " of the present invention to make as raw material is treated:
(1) optional 50 isolate for treatment in hospital in, severe atypical pneumonia (SARS) patient;
(2) under the prerequisite that continues the air-breathing corresponding symptomatic treatment measure cooperation of nose conduit as usual, use the compression atomizing air pump to supply the patient to suck " anti-sars type pneumonia (SARS) specificity composite IgY combination propellant " of the present invention atomizing back with other.Sucked once in per two hours, and continued 5-10 minute at every turn.
(3) every day viewing test patient symptom situation, detail record.Result such as following table
| The treatment fate | Sxs such as heating and cough example number | Transference cures such as heating and cough example number | Do not have obviously and improve |
| 1-3 days | 18 | 0 | 32 |
| 4-7 days | 25 | 12 | 11 |
| 8-14 days | 5 | 27 | 12 |
| 15-21 days | 0 | 6 | 5 |
" anti-sars type pneumonia (SARS) specific IgY " of the present invention can directly play effective restraining effect at the Causative virus that causes atypical pneumonia with a definite target in view, is to deal with problems from the morbidity root; These pathogenic agent that neutralize on the one hand change the activity of these pathogenic agent, and promote that the human immune system removes these pathogenic agent.Particularly, specific IgY of the present invention has very strong wide spectrum and suppresses ability, even some variation has taken place Causative virus, still can produce certain restraining effect.Experimental result proves, " anti-sars type pneumonia (SARS) specific IgY " of the present invention combination preparation, is that " serotherapy " of " passive immunotherapy " is the same to the result of treatment of atypical pneumonia (SARS) equally with what adopted at present, and effect is more satisfactory; And, wide spectrum suppresses ability because specific IgY of the present invention has preferably, antibody activity is also much better than, can not produce side effects such as cross reaction again, purity height, source are extensively, price is much cheap again, therefore, and for prevention with treatment atypical pneumonia (SARS) this infection is fast, the big pernicious transmissible disease of harm has been opened up a brand-new approach.
Embodiment 1: preparation 100g anti-sars type pneumonia (SARS) specific IgY crude extract and pure IgY
(1) select the breeding duck of the high immunne response ability of tool:
Make antigen with pneumococcus, respectively immune 1000 ducks, per injection 1ml antigen after injection for the first time, is strengthened injection once every two weeks, totally three times again; In injecting the back 7th month for the first time, the IgY extracting respectively according to a conventional method behind the egg mark that these ducks produced wherein detects tiring of prepared IgY with ELISA method (enzyme-linked immunosorbent assay); Select and wherein can make IgY and tire 〉=200 of strong especially ducks of 256 immunne response ability, the egg that is produced with this batch duck is hatched good duck kind again, treats that it grows up to 2-3 month, as the extraordinary duck of preferred high immunne response ability, and selects wherein 40;
(2) preparation of atypical pneumonia (SARS) Causative virus complex antigen
The present invention is an example with Hong Kong group.With Vero E6 cell inoculation in 10%FCS DMEM nutrient solution, final concentration of cells 1 * 10
5/ ml.The above-mentioned nutrient solution of 15ml is placed the 100ml culturing bottle, this culturing bottle is placed on 37 ℃, 5% CO
2Cultivate into monolayer cell in the saturated incubator.Discard nutrient solution then, 4 kinds of different sars coronavirus strains in area, Hong Kong of collecting are diluted to 100 TCID 50/ml with 2%FCS and 1% mycillin DMEM nutrient solution.Get this diluent 10ml and add in the Tissue Culture Flask, and this Tissue Culture Flask is placed on 37 ℃, 5% CO
2Cultivate in the saturated humidity incubator after 24 hours, treat cytopathy reach++++ time results virus.With culturing bottle freeze thawing three times, collect culture supernatant during results virus, put into supercentrifuge,, collect supernatant with the centrifugation of 15000rpm rotating speed.The final concentration that adds formaldehyde to 0.1% again is under 37 ℃ of temperature, through the SARS virus of these 4 kinds of cultivations of difference deactivation in 24 hours.Cultured inactivation of viruses is 4 ℃ or-20 ℃ of preservations.Then, with the 4 kinds of different coronavirus in area, cultured Hong Kong in 1: 1: 1: 1 ratio or the mixing of other suitable proportions, add freund adjuvant in 1: 1 ratio or other suitable proportions again, insert in the high-speed homogenization machine with the homogenize of 10000～30000rpm rotating speed high speed pulverization, form water-in-oil solution by high-speed stirring, promptly make the special complex antigen that contains multiple veriform virus of atypical pneumonia (SARS) Causative virus.
(3) respectively preferred extraordinary duck is carried out reinforced immunological:
With (SARS) coronavirus complex antigen of being produced, the reinforced immunological method that adopts subcutaneous injection and wing intravenous injection to combine, respectively preferred extraordinary duck is carried out immunity, every duck per injection amount reaches 1-2ml antigen, strengthen injection more once every two weeks, be total to immunity three times in one month; Immunity was for the first time searched duck and is produced 1000 on immune egg after 20 days;
(4) preparation of anti-sars type pneumonia (SARS) specific IgY:
Smash with the immune egg that beating machiae will be searched, remove egg white, stay yolk, stir with the sieving of yolk sieve; Measure the volume of the yolk of gained, extraordinarily go into distilled water, dilute and mix by the 4-6 of this volume; Adjust between the pH to 5.5-6.0 with Na0H solution; The diluent of adjusting the pH value is further stirred, it is cooled to 2-6 ℃ then, left standstill 12 hours; Diluent is added in the high-speed centrifuge, separated 20 minutes with 8000-12000rpm rotating speed high speed centrifugation; Separating obtained supernatant reentered carry out ultrafiltration in the ultra-fine filter and concentrate 15 times, the ratio in 0.2% adds Lalgine in the slurry after concentrating, fully stirring; It is centrifugal to add supercentrifuge again, gets supernatant, removes lipoprotein; Slurry after the centrifugation is added ultrafilter, cross the ultra micro film, carry out filtration sterilization; Product after the filtration sterilization is carried out lyophilize with freeze drier, promptly make anti-sars type pneumonia (SARS) specific IgY crude extract dry powder finished product; Cross ion exchange column and gel exchange column antagonism atypical pneumonia (SARS) specific IgY crude extract and carry out purifying, promptly get pure IgY.
Embodiment 2: the preparation of anti-sars type pneumonia (SARS) Causative virus IgY propellant
Anti-sars type pneumonia (SARS) Causative virus specific IgY dry powder 300g adds in the pony mixer fully mixed;
By following prescription allotment 30 liters of anti-sars type pneumonia (SARS) Causative virus IgY combination solution
Anti-sars type pneumonia (SARS) Causative virus specific IgY 300g
Aspartame 36g
Fragrant citrus essence 90g
Honey peach essence 30g
Physiological saline adds to 30 liters
Modulation physiological saline is 25 liters earlier, adds aspartame and essence then, after stirring, slowly adds IgY more while stirring; Then, add physiological saline to 30 liter, stir; Measure the pH value of solution, regulate pH to 6.5-7.5 with 1.0N NaOH solution; Through the bacterium filtration sterilization, adopt aseptic bottle can anti-sars type pneumonia (SARS) Causative virus IgY sprays then, every dress 20ml.
Embodiment 3: preparation anti-sars type pneumonia (SARS) Causative virus IgY hand is pressed quantitative Incapacitating sprays
Anti-sars type pneumonia (SARS) Causative virus specific IgY dry powder 300g adds in the pony mixer fully mixed;
By following prescription allotment 200 liters of anti-sars type pneumonia (SARS) Causative virus IgY combination solution;
Anti-sars type pneumonia (SARS) Causative virus specific IgY 300g
Aspartame 240g
Fragrant citrus essence 600g
Honey peach essence 200g
Peppermint essence 1400g
Distilled water adds to 200 liters
Get 180 liters of distilled water earlier, add aspartame, essence then respectively, stir, slowly add IgY more while stirring; Then adding distil water to 200 liter stirs, and measures the pH value of solution value, according to the pH value of being surveyed with 1.0N NaOH solution adjusting pH to 6.5-7.5; After preparing solution is good, must be on the pressure-pot production line manual quantitative 20000 of the Incapacitating sprays of can, after every spray tank was irritated sufficient 10ml, quantitative valve was loaded onto as boosting agent (propellent) in charged pressure nitrogen or fluorine Lyons again, sealing, promptly.
Embodiment 4: the quantitative normal pressure spray jar of preparation anti-sars type pneumonia (SARS) Causative virus IgY
Pure IgY dry powder 300 weight parts of anti-sars type pneumonia (SARS) Causative virus specificity add in the pony mixer fully mixed;
Allocate 200 weight part anti-sars type pneumonia (SARS) Causative virus IgY combination solutions by following prescription;
Anti-sars type pneumonia (SARS) Causative virus specific IgY 300 weight parts
Aspartame 240 weight parts
Fragrant citrus essence 600 weight parts
Honey peach essence 200 weight parts
Peppermint essence 1400 weight parts
Distilled water adds to 200 parts by volume
Get distilled water 180 parts by volume earlier, add aspartame, essence then respectively, stir, slowly add composite IgY more while stirring; Then adding distil water to 200 parts by volume stirs, and measures the pH value of solution value, regulates between pH to 6.5-7.5 with 1.0N NaOH solution according to the pH value of being surveyed; Through the bacterium filtration sterilization, the can hand is pressed 20000 in quantitative normal pressure spray jar, every 10ml, every full 10ml of can.
Embodiment 5: preparation anti-sars type pneumonia (SARS) and the quantitative normal pressure spray jar of oral inflammation
Anti-sars type pneumonia (SARS) specificity composite IgY dry powder 150 weight parts, dental caries resistant bacteria IgY dry powder 150 weight parts, fully mixed in the adding pony mixer, make anti-sars type pneumonia (SARS) and oral inflammation composite IgY dry powder 300 weight parts;
Allocate 200 weight part anti-sars type pneumonia (SARS) and oral inflammation composite IgY combination solution by following prescription;
Anti-sars type pneumonia (SARS) and oral inflammation composite IgY 300 weight parts
Aspartame 240 weight parts
Fragrant citrus essence 600 weight parts
Honey peach essence 200 weight parts
Peppermint essence 1400 weight parts
Distilled water adds to 200 parts by volume
Get distilled water 180 parts by volume earlier, add aspartame, essence then respectively, stir, slowly add composite IgY more while stirring; Then adding distil water to 200 parts by volume stirs, and measures the pH value of solution value, regulates between pH to 6.5-7.5 with 1.0N NaOH solution according to the pH value of being surveyed; Through the bacterium filtration sterilization, the can hand is pressed 20000 in quantitative normal pressure spray jar, every 10ml, every full 10ml of can.
Embodiment 6:
The preparation method of anti-sars type pneumonia (SARS) specific IgY, the preparation method of this anti-sars type pneumonia (SARS) specific IgY comprises the following steps: with various single or compound atypical pneumonias (SARS) Causative virus antigen, respectively the bird of laying eggs is carried out immunity, prepare immune egg; The various Causative virus specific IgY of preparation anti-sars type pneumonia (SARS) crude extract; The purifying of anti-sars type pneumonia (SARS) Causative virus specific IgY crude extract; It is characterized in that:
With various single or compound atypical pneumonias (SARS) Causative virus antigen, respectively the bird of laying eggs is carried out immunity, the step for preparing immune egg is specially: with various single or compound atypical pneumonias (SARS) Causative virus antigen, respectively the bird of laying eggs is carried out immunity, strengthen injection more once every two weeks, the planned immunization epidemic disease was three times in one month, immunity is after 20 days for the first time, the bird of searching after the immunity produces immune egg, and to corresponding different antigen, the immune egg of being searched is carried out coded markings;
The step of preparation anti-sars type pneumonia (SARS) Causative virus specific IgY crude extract is specially: clean immune egg with flowing water, use the wipes of alcohol wash disinfection again, smash with the immune egg that beating machiae will be searched, remove egg white with the sieving of yolk sieve, stay yolk, stir, measure the volume of the yolk of gained, 4-6 by this volume extraordinarily goes into distilled water, dilute and mix, adjust between the pH to 5.5-6.0 with NaOH solution, the diluent of adjusting the pH value is further stirred, it is cooled to 2-6 ℃ then, left standstill 12 hours-24 hours, diluent is added in the high-speed centrifuge, centrifugation 20 minutes, separating obtained supernatant added again carry out ultrafiltration in the ultra-fine filter and concentrate 10-20 doubly, in the slurry after ratio in 0.1～0.3% concentrates the sodium alginate adding, fully stir, add in the supercentrifuge centrifugal again, get supernatant, remove lipoprotein, slurry after the centrifugation is added ultrafilter, cross the ultra micro film, carry out filtration sterilization, product after the filtration sterilization is carried out lyophilize with freeze drier, promptly make anti-sars type pneumonia (SARS) Causative virus specific IgY crude extract dry powder finished product, last, prepared corresponding different antigenic specific IgY crude extract is carried out coded markings;
The step of the purifying of anti-sars type pneumonia (SARS) Causative virus specific IgY crude extract is specially: anti-sars type pneumonia (SARS) the Causative virus specific IgY crude extract that above technology is produced was taken up in order of priority ion exchange column and gel chromatography column, promptly got pure IgY.
Claims (1)
1, the application of anti-sars type pneumonia (SARS) specific IgY in the preparation of preparation prevention and treatment atypical pneumonia.
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| CNB031361013A CN1318449C (en) | 2003-05-13 | 2003-05-13 | Anti SARS specificity IgY and combination preparation thereof |
| PCT/CN2003/000587 WO2004101621A1 (en) | 2003-05-13 | 2003-07-22 | A SPECIFIC IMMUNOGLOBULIN (Ig) Y AGAINST SEVERVE ACUTE RTESPIRATORY SYNDROME (SARS) AND A PREPARING METHOD THEREOF AND COMBINATION PREPARATIOS CONTAINING IT |
| AU2003255080A AU2003255080A1 (en) | 2003-05-13 | 2003-07-22 | A SPECIFIC IMMUNOGLOBULIN (Ig) Y AGAINST SEVERVE ACUTE RTESPIRATORY SYNDROME (SARS) AND A PREPARING METHOD THEREOF AND COMBINATION PREPARATIOS CONTAINING IT |
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|---|---|---|---|---|
| WO2005012360A2 (en) * | 2003-07-22 | 2005-02-10 | Crucell Holland B.V. | Binding molecules against sars-coronavirus and uses thereof |
| WO2006095180A2 (en) * | 2005-03-10 | 2006-09-14 | Ultra Biotech Limited | Humananized monoclonal antibodies against sars - associated coronavirus and treatment of patients with sars |
| CN113201068A (en) * | 2020-02-03 | 2021-08-03 | 深圳市雅臣智能生物工程有限公司 | Gene recombination anti 2019-nCoV and other coronavirus IgY and small molecule antibody and application thereof |
| US20210347858A1 (en) * | 2020-04-03 | 2021-11-11 | Firebreak, Inc. | Alimentary and systemic antiviral therapeutics |
| CN111548413B (en) * | 2020-04-24 | 2021-03-26 | 成都钰康生物科技有限公司 | Antibody for resisting novel coronavirus, preparation method and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1201693A (en) * | 1998-04-01 | 1998-12-16 | 中德联合研究所 | Chick lecithal immune globulin products against rolavirus and its use and its testing method of activity |
| CN1382736A (en) * | 2002-02-08 | 2002-12-04 | 北京市农林科学院畜牧兽医研究所 | Yolk antibody and antigen of pig's infective enterogastritis virus and its preparing process |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0657663B2 (en) * | 1988-08-12 | 1994-08-03 | 武彦 山本 | Method for producing multifunctional specific antibody |
| JP2002165567A (en) * | 2000-11-30 | 2002-06-11 | Society For Techno-Innovation Of Agriculture Forestry & Fisheries | Method for producing liquid food free from harmful bacterium |
| US6680376B2 (en) * | 2000-12-08 | 2004-01-20 | Good Biotech Corporation | Process for selectively isolating avian immunoglobulins |
| KR100381553B1 (en) * | 2001-02-23 | 2003-04-23 | 주식회사 중앙백신연구소 | Manufacturing method of special feed additives using the canine disease antigens |
-
2003
- 2003-05-13 CN CNB031361013A patent/CN1318449C/en not_active Expired - Fee Related
- 2003-07-22 AU AU2003255080A patent/AU2003255080A1/en not_active Abandoned
- 2003-07-22 WO PCT/CN2003/000587 patent/WO2004101621A1/en active Application Filing
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1201693A (en) * | 1998-04-01 | 1998-12-16 | 中德联合研究所 | Chick lecithal immune globulin products against rolavirus and its use and its testing method of activity |
| CN1382736A (en) * | 2002-02-08 | 2002-12-04 | 北京市农林科学院畜牧兽医研究所 | Yolk antibody and antigen of pig's infective enterogastritis virus and its preparing process |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1450086A (en) | 2003-10-22 |
| AU2003255080A1 (en) | 2004-12-03 |
| WO2004101621A1 (en) | 2004-11-25 |
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