CN1488645A - Specific antibody IgY for anti SARS virus, and preparing method thereof and composition containing same and its use - Google Patents
Specific antibody IgY for anti SARS virus, and preparing method thereof and composition containing same and its use Download PDFInfo
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- CN1488645A CN1488645A CNA031397158A CN03139715A CN1488645A CN 1488645 A CN1488645 A CN 1488645A CN A031397158 A CNA031397158 A CN A031397158A CN 03139715 A CN03139715 A CN 03139715A CN 1488645 A CN1488645 A CN 1488645A
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Abstract
The invention specific antibody IgY which has SARS resistant antigen and the pressurized spray, oral peroral medicine, and diagnosing medicine. The IgY is produced by using SARS pathogen coronavirus or gene project vaccine as antigen, using lipidosome as adjuvant, immune fowl. The invention provides methods such as water diluting method, salting out method, CM-sephadex C-50 positive ion chromatography, PS6FF dewatering layer chromatography for producing IgY. The cost is low and the efficiency of the antibody is good.
Description
Technical field
The present invention relates to specific antibody IgY preparation and application, particularly anti-SARS virus specific antibody IgY and preparation method and contain the anti-SARS composition and the purposes of this antibody.
Background technology
Severe acute respiratory syndrome (claiming SARS (Severe Acute Respiratory Syndrome) again, Severe Acute RespiratorySyndrome, the abbreviation SARS) outburst of a plurality of countries and regions and diffusion in the whole world at the beginning of 2003 causes the national governments and the people's extensive concern.On April 16th, 2003, The World Health Organization (WHO) announces that a kind of human past is the pathogenic agent of SARS from undiscovered novel coronavirus.Show that through preliminary study this sars coronavirus mainly is through respiratory infectious.At present to this new strong infective virus except that with external routine disinfection sterilization, defend the measure such as isolation body is not also had the specificity prevention method.
Summary of the invention
The object of the present invention is to provide a kind of anti-SARS virus specific IgY and preparation method thereof, the production cost of this antibody is reduced, be produced on a large scale and the cycle is short, antibody titer is high.Simultaneously, the composition as the anti-SARS virus of effective ingredient preparation with IgY of the present invention is provided, and, comprises sprays, oral preparation and diagnostic preparation etc. any formulation that said composition is applied to make prevention, diagnoses and treat SARS.
The specific antibody IgY of anti-SARS virus of the present invention, it is with laying hen under the SARS virus antigen immune, the specific antibody IgY that separation and purification makes from egg yolk.
The preparation method of the specific antibody IgY of a kind of anti-SARS virus of the present invention, comprise the extraction of SARS virus antigen prepd, immune hen, IgY etc., as antigen, dilute with water method, salting-out process, CM-Sephadex C-50 positively charged ion chromatography column chromatography, PS6FF hydrophobic chromatography column chromatography extract IgY with deactivation of SARS virus or its recombinant vaccine.
The preparation method of anti-SARS virus specific IgY of the present invention is as described below:
The preparation of virus antigen suspension: with sars coronavirus, be inoculated in the Vero monolayer cell bottle, put in 37 ℃ of incubators and cultivate, treat that cytopathy is obviously when (++ ++), gather in the crops viral liquid, after taking out sterility test and virus titer sample, add formaldehyde solution, its ultimate density must not surpass 0.05%, puts certain hour inactivation of viruses under the proper temperature.Filter the back and add the Thiomersalate sanitas, its ultimate density must not surpass 0.01%.
Virus titer: with EMEM the viral suspension work being increased progressively dilution for 10 times is 10
4～10
-8, the diluent with each sars coronavirus is inoculated in the little culture plate of putting of Vero monolayer cell respectively then.Every kind of extent of dilution inoculated 4 porocytes, 200 μ l/ holes.Do not add the cell contrast of virus simultaneously.Put into 37 ℃, 5%CO
2Cultivate in the incubator.Every day is the observation of cell pathology under inverted microscope, and makes detail record.The last result of determination to the 7th day.Press the TCID that the Reed-Muench accumulative is calculated SARS virus
50(the special infective dose of half tissue culture/0.2ml).
Inactivation test: the inactivation treatment viral suspension is placed 4 ℃, behind the dialysis 24h, inoculation Vero cell, it is 3m that every ml sample inoculation is no less than
2The cell sheet is put under the temperature of cultivating virus and was cultivated 14 days, must not have cytopathy to occur.
The present invention has used novel positive charge liposome-DC cholesterol (2.5mg/ml) and has been immunological adjuvant, before using its viral suspension is mixed, and DC-cholesterol final concentration reaches 1.6mg/ml.
SARS virus antigen, with the inactivation of viruses suspension, 5～10 times of dilutions mix with liposome or freund adjuvant, make the virus antigen of immune usefulness.
Get above-mentioned this virus antigen of deactivation 1.0ml at every turn and contain adjuvant, hen is carried out subcutaneous or intramuscular injection, respectively at 0,14 and 21 day injecting immune; Also desirable this virus antigen of deactivation 2.5～5ml, to every chicken wings intravenous injection, can be respectively at 0,7, carried out immunity in 21 days.
Collect immune egg, take out yolk, stir, the distilled water diluting that adds 6～7 times of volumes is regulated pH value to 5.0～5.3, under 3～5 ℃ of temperature, stirring is spent the night, high speed centrifugation, 30 minutes, get its supernatant liquor, satisfy with 50% again and close ammonium sulfate precipitation once, use 33% saturated ammonium sulphate more once, detect antibody activity with ELISA, adopt the SDS-PAGE method to measure purity, the Lowry method is measured protein concentration.IgY purity of the present invention reaches 97%, output 18.0mg/ml.
To carry out CM-Sephadex C-50 positively charged ion chromatography column chromatography again through the anti-SARS virus IgY that the rare method of water, salting-out process extract.With pH5.5,10～50mM PBS balance, with contain 0.5M NaCl, the pH5.5 phosphoric acid buffer carries out wash-out, flow velocity 10～500cm/h, elutriant is collected automatically by every pipe 5.0ml, collect 0.03～0.1M NaCl albumen elution peak, and detect each proteic antibody activity with ELISA.Antibody activity albumen elutriant will be arranged further through Phenyl Sepharose 6 Fast Flow (PS6FF) hydrophobic chromatography column chromatographies.With containing 1M ammonium sulfate pH5.0,25mM PBS solution equilibria, flow velocity 10～500cm/h, elutriant is collected automatically by every pipe 5.0ml, with 10～50mM, pH5.5, PBS buffer solution elution, collect the white elution peak of 80%～100%B liquid eggs, and detect each proteic antibody activity with ELISA.The albumen elutriant of antibody activity is left and taken in collection, 0.22 μ membrane filtration degerming, and lyophilize promptly gets the pure product of anti-SARS virus IgY.
Among the IgY and experiment: purification specificity IgY is done 2 times with EMEM increase progressively after the dilution, respectively with sars coronavirus (TCID to be infected
100) 37 ℃ of incubation 2h.Before test 1～2 day, the Vero passage cell strain is inoculated in 96 porocyte culture plates, put 37 ℃, 5%CO
2Incubator is cultivated, and makes it grow into the monolayer cell of even compact.Take out during test, remove cell culture fluid, wash once with Hanks liquid.Get above-mentioned IgY with micro sample adding appliance and resist-viral mixed solution, add respectively in the cell cultures plate hole, every kind of antibody virus mixture adds 2 holes respectively, every hole 0.2ml.Do positive antibody (IgY) contrast (1: 2) simultaneously, cell contrast and sars coronavirus contrast.Tissue Culture Plate is put into 37 ℃, 5%CO
2Cultivate in the incubator.Every day is observations under inverted microscope, observes altogether 3～5 days.To each porocyte pathology of virus control obviously (++～++ ++), positive antibody control wells and cell control well are normal, get final product result of determination.Still can suppress cytopathy with the high dilution of specific IgY antibody to be checked and occur, be NAT.IgY NAT of the present invention 〉=1: 128.
IgY technology of preparing production cost of the present invention is low, the cycle is short, antibody titer is high, stable, have stronger special neutralization active to SARS virus, and effect is rapid.
The composition of a kind of anti-SARS of the present invention, the specific antibody IgY that contains described anti-SARS virus is as effective constituent.
The preparation that the purposes of the composition of a kind of anti-SARS of the present invention, the composition of this anti-SARS are applied to make prevention, diagnosis and treat any formulation of SARS.
The composition of described anti-SARS of the present invention is a liquid, in the small-sized portable type spray tank of packing into, can carry out respiratory tract, mouth spray use.
Multiple composition be can prepare with anti-SARS virus specific IgY of the present invention as effective special composition, prevention, diagnosis, treatment SARS disease are applied to.As with anti-SARS virus specific IgY of the present invention as major ingredient and a certain proportion of distilled water, the composition of Natural menthol, sweeting agent, food flavour and Sodium Benzoate preparation, the spraying of it can being packed in the small-sized portable type spray tank is used, have safe in utilization, convenient, characteristics such as that IgY plays a role is special, quick, preventive effect is good can effectively be prevented the generation of SARS virus through respiratory tract infection.
The purposes of the composition of a kind of anti-SARS of the present invention, the composition of described anti-SARS are applied to prepare food, beverage and the healthcare products that prevent SARS.
Studies show that human body alimentary canal also is one of route of infection of SARS virus.The preparation of compositions of anti-SARS of the present invention is become food, beverage and healthcare products, can reach the effect of prevention, minimizing digestive tract infection rate.
Embodiment
The present invention will be further described by following examples:
One, the preparation of anti-SARS-IgY
Embodiment 1
Under the P3 condition, sars coronavirus kind and Vero cell count are inoculated by a certain percentage, put 37 ℃, CO
2Incubator is cultivated, and after 7 days, collects viral liquid, carries out virus titer and measures, and filters purifying, and formalin-inactivated behind the deactivation safety experiment, obtains deactivation of SARS virus suspension (TCID
50Be 10
-6.67).Mix with a certain amount of finger plastid adjuvant then, make deactivation of SARS virus antigen.
In the injection of the subcutaneous immunity of laying hen, every some 0.25ml, every laying hen 1ml, the immunity time is 0 day, 14 days, 21 days, three immunity.Begin to collect egg in immunity for the first time after 20 days.In molten device, put into yolk, after stirring, add the distilled water diluting of 6～7 times of volumes, regulate 5.0～5.3,4 ℃ of stirrings of pH value spend the night (12 hours).Centrifugal 30 minutes of 8000rpm gets supernatant liquor, obtains the IgY of roughing out.Through 50% saturated ammonium sulphate once, use 33% saturated ammonium sulphate more once, obtain the IgY of purifying, further extract as the available positively charged ion chromatography of needs column chromatography, hydrophobic chromatography column chromatography, and detect each proteic antibody titer with the ELISA test kit.With 0.22 μ membrane filtration degerming, lyophilize.
Embodiment 2
The SARS virus recombinant vaccine that will have good prophylactic effect, in the immunization of laying hen muscle, every laying hen 1ml, the immunity time is 0,14,21 days, totally three immunity.Begin to collect egg in immunity for the first time after 20 days, stir, add the distilled water diluting of 6～7 times of volumes, regulate 5.0～5.3,4 ℃ of stirrings of pH value and spend the night.Centrifugal 30 minutes of 8000rpm gets supernatant liquor, obtains the IgY of roughing out, through 50% saturated ammonium sulphate once, use 33% saturated ammonium sulphate more once, obtain the IgY of purifying, and detect each proteic antibody activity with the ELISA test kit, with 0.22 μ membrane filtration degerming, lyophilize.
Two, the preparation of anti-SARS composition product
Embodiment 3
Contain the preparation of anti-SARS specific IgY sprays: get IgY2.0 of the present invention, Natural menthol 0.10g, Sodium Benzoate 0.10g, sweeting agent 0.5g, food flavour 0.4ml.Natural peppermint is added in the distilled water of 100ml, 60 ℃ dissolve; Other solid composition is dissolved in 450ml distilled water not, and is then that various dissolved liquid mixing are even, is settled to 1000ml with distilled water.Be sub-packed in the small-sized portable type spray tank, can effectively prevent the generation of SARS virus through respiratory tract infection.
Embodiment 4
Diagnose with the preparation and the method that detect the SARS pathogeny: the main preparation that detects the SARS pathogeny with specific IgY Antibody Preparation of the present invention consists of: have the microwell plate of anti-SARS virus specific IgY antibody sandwich, enzyme labelling anti-SARS virus specific antibody HRP-anti-SARS-IgY.The former working concentration is 1: 300～3000; Latter's working concentration is 1: 300～1500.Its method may further comprise the steps: will be with specific IgY antibody sandwich of the present invention on solid phase carrier, behind washing, the shrouding, add sample to be checked (as serum, saliva), add the anti-SARS-IgY of enzyme labelling specific antibody HRP-then, the washing back adds the colour developing of substrate colour developing liquid, add the stop buffer termination reaction, the colorimetric result of determination.It is special, simple to operate, quick, practical that this preparation has detection, is fit to each different medical unit and uses.
Embodiment 5
Contain yogurt and the ice-cream production of anti-SARS IgY: will separate according to the egg yolk of embodiment 1 or the production of 2 schemes, ratio in 5% joins in yogurt and the ice-cream prescription, make the food that contains anti-SARSIgY, prevent, reduce the digestive tract infection rate by eating.
Embodiment 6
Contain the production of the milk of anti-SARS IgY: will separate according to the egg yolk of embodiment 1 or the production of 2 schemes, the ratio in 5% joins in the milk, makes the beverage that contains anti-SARS IgY, prevents, reduces the digestive tract infection rate by drinking.
Embodiment 7
Contain anti-SARS virus IgY yolk powder capsule: the egg yolk that the foregoing description 1 or 2 schemes are produced is separated, collected centrifugally, carry out lyophilize, make yolk powder, incapsulate,, prevent, reduce the digestive tract infection rate by taking as oral healthcare products.
Claims (10)
1, the specific antibody IgY of anti-SARS virus is characterized in that: it is with laying hen under the SARS virus antigen immune, the specific antibody IgY that separation and purification makes from egg yolk.
2, the preparation method of a kind of specific antibody IgY of anti-SARS virus, comprise the extraction of SARS virus antigen prepd, immune hen, IgY etc., it is characterized in that: as antigen, dilute with water method, salting-out process, CM-Sephadex C-50 positively charged ion chromatography column chromatography, PS6FF hydrophobic chromatography column chromatography extract IgY with deactivation of SARS virus or its recombinant vaccine.
3, preparation method according to claim 2 is characterized in that: when described water dilution method extracts the IgY crude product, with 6～7 times of volume distilled water agitation and dilution yolk, regulate pH value to 5.0～5.3, under 4 ℃ of temperature, stirred conventional centrifugal 20～30 minutes 12～24 hours.
4, preparation method according to claim 2 is characterized in that: described salting-out process is selected 50% saturated ammonium sulphate for use once, uses 33% saturated ammonium sulphate more once.
5, preparation method according to claim 2 is characterized in that: the elutriant of described CM-Sephadex C-50 positively charged ion chromatography column chromatography is the phosphoric acid buffer of 0.5M NaCl, pH5.5.
6, preparation method according to claim 2 is characterized in that: the elutriant of described PS6FF hydrophobic chromatography column chromatography is the PBS buffer solution elution of 10～50mM, pH5.5.
7, the composition of a kind of anti-SARS is characterized in that: the specific antibody IgY that contains anti-SARS virus as claimed in claim 1 is as effective constituent.
8, the purposes of the composition of a kind of anti-SARS is characterized in that: the preparation that the composition of the described anti-SARS of claim 7 is applied to prepare prevention, diagnosis and treats any formulation of SARS.
9, the purposes of the composition of anti-SARS according to claim 8 is characterized in that: the composition of described anti-SARS is a liquid, in the small-sized portable type spray tank of packing into, can carry out respiratory tract, mouth spray use.
10, the purposes of the composition of a kind of anti-SARS is characterized in that: the composition of the described anti-SARS of claim 7 is applied to prepare food, beverage and the healthcare products that prevent SARS.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA031397158A CN1488645A (en) | 2003-07-08 | 2003-07-08 | Specific antibody IgY for anti SARS virus, and preparing method thereof and composition containing same and its use |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA031397158A CN1488645A (en) | 2003-07-08 | 2003-07-08 | Specific antibody IgY for anti SARS virus, and preparing method thereof and composition containing same and its use |
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|---|---|
| CN1488645A true CN1488645A (en) | 2004-04-14 |
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ID=34155073
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA031397158A Pending CN1488645A (en) | 2003-07-08 | 2003-07-08 | Specific antibody IgY for anti SARS virus, and preparing method thereof and composition containing same and its use |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1903878B (en) * | 2005-07-26 | 2011-09-07 | 复旦大学 | Anti SARS virus human source nature antibody IgG Fab section |
| WO2021212755A1 (en) * | 2020-04-24 | 2021-10-28 | 成都钰康生物科技有限公司 | Anti-novel coronavirus antibody and preparation method therefor and use thereof |
-
2003
- 2003-07-08 CN CNA031397158A patent/CN1488645A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1903878B (en) * | 2005-07-26 | 2011-09-07 | 复旦大学 | Anti SARS virus human source nature antibody IgG Fab section |
| WO2021212755A1 (en) * | 2020-04-24 | 2021-10-28 | 成都钰康生物科技有限公司 | Anti-novel coronavirus antibody and preparation method therefor and use thereof |
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