CN1546526A - Method for preparing IgY for SARS - Google Patents
Method for preparing IgY for SARS Download PDFInfo
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- CN1546526A CN1546526A CNA2003101077318A CN200310107731A CN1546526A CN 1546526 A CN1546526 A CN 1546526A CN A2003101077318 A CNA2003101077318 A CN A2003101077318A CN 200310107731 A CN200310107731 A CN 200310107731A CN 1546526 A CN1546526 A CN 1546526A
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Abstract
The invention discloses a method for preparing IgY for SARS, which belongs to a process for manufacturing anti-SARS biological pharmaceuticals. The process comprises the steps of making vaccinogen, preparing antibody, homogenating vitelline, diluting, low-temperature stewing, filtering, purifying, and pulverizing. The said antibody preparation process is disclosed in the invention. The process according to the invention can be applied in preparing anti-SARS medicament.
Description
Technical field:
The present invention relates to a kind of making method of anti-sars type pneumonia yolk antibody, is a kind of making method of biologics.
Background technology:
From on November 16th, 2002 Chinese Foshan find the first routine atypical pneumonia (SevereAcute Respiratory Syndrome, SARS) since the case, SARS at first spreads in Guangdong Province, and rapid spread is to all over the world, according to the recent statistics of The World Health Organization (WHO),, there have been 28 countries and regions to become the morbidity area of SARS in the world by the end of April 26, SARS case 4836 examples take place in the whole world altogether, dead 293 examples.The domestic patient in China's Mainland has reached 2753 examples, dead 122 examples.China Guangdong Province becomes initial morbidity ground and the popular area of big area of this SARS.From that time that SARS takes place, people have just carried out a large amount of tracing to its morbific real arch-criminal-pathogenic agent, and have been separated to coronavirus in the SARS patient body.The scientist of Holland ERASMUS laboratory in colleges and universities completes successfully the animal model of coronavirus experiment, and the SARS illness that conclusive evidence causes the whole world to be wreaked havoc is caused by a kind of novel coronavirus.This result of study has confirmed the main flow viewpoint of global scientist based on recent research, and international viral educational circles confirms that " Coase hypothesis " (KOCH ' POSTULATES) four conditions of cause of disease are all satisfied.April 16, WHO announces that in Geneva the SARS cause of disease finds, and definite designation is a sars coronavirus.The scientist of various countries passes through work day and night simultaneously, has successfully decoded sars coronavirus full gene group sequence.According to the complete genome sequence analytical results of SARS virus, the structural similitude of SARS virus genome structure and other coronavirus.The main protein that prediction obtains has RNA polymerase albumen (polysaccharase 1a, 1b), spike protein (Spike protein, S), little membranin (small membrane protein, E), membranin (Membrane protein, M) and nucleocapsid protein (Nucleocapsid protein, N) etc.According to nucleotide sequence and deduced amino acid comparative result, find that sars coronavirus does not belong to any a group among three groups of known coronavirus, all different with the coronavirus of known people and animal, be a kind of new coronavirus.
At American National bioinformation center (NCBI), with the SARS virus is that search sequence is searched for, the sequence similarity of finding following several virus and SARS virus is higher: people's coronavirus (Humancoronavirus 229E), the hepatitis virus of mouse (Murine hepatitis virus, MHV), the infectious bronchitis virus of fowl (Avian infectious bronchitis virus, IBV), the coronavirus of ox (Bovine coronavirus, BCV), Porcine epidemic diarrhea virus (Porcineepidemic diarrhea virus, PEDV), transmissible gastro-enteritis virus (Transmissiblegastroenteritis virus, TGEV).On the whole, the genome structure of several viruses is closely similar.By the comparison between the viral genome, find in the position of 14k-16k it is all comparatively conservative zones of all coronavirus.The RNA polymerase that this regional code RNA relies on (RNA-dependent RNA polymerase, RDRP).The result of study of WHO announcement simultaneously proves that (Feline InfectiousPeritonitis virus, hyper-immune serum FIPV) can suppress the propagation of SARS virus with TGEV, HCoV 229E, MHV and cat peritonitis virus.From height exempt from the serum of animal (as rabbit, chicken, ox, sheep etc.) or just the Ruzhong separate IgG, in passive immunotherapy, have very important significance and be worth.But the IgG that these sources provide yields poorly, and the cost height can only be confined to breadboard immunology diagnosis and fundamental research usually.
Summary of the invention:
The making method that the purpose of this invention is to provide a kind of anti-sars type pneumonia yolk antibody to reduce cost, improves the stability of antibody, prevents multiple disease, improves the performance of biologics.
Above-mentioned purpose realizes by following technical scheme:
The making method of anti-sars type pneumonia yolk antibody, described method comprises: make vaccinogen, Antibody Preparation, yolk homogenate, dilution, low temperature spends the night, filter purifying, powder process, the preparation method of wherein said anti-SARS fowl yolk antibody is: select 100-140 age in days SPF fowl, first immunisation 1.5-3.5ml, 2-4 position of chest muscle injection, 2-4 week repeats booster immunization at interval, and antibody titer is monitored in blood sampling weekly, and two exempt from 1-2 collects after week through the qualified fowl ovum of antibody test, blood sampling detects antibody weekly, it is qualified that NAT is not less than 1:256, and three exempt from the decision of basis of time antibody detection situation, and injection volume and two is exempted from identical.
This technical scheme has following beneficial effect:
1. in recent decades, Chinese scholars has been done many work with regard to the research and development of yolk antibody, has proved that the egg of immune chicken provides the most convenient of specific IgG, the most cheap source.Ovum Gallus domesticus Flavus IgG is a kind of 7S immunoglobulin, and is slightly different with Mammals IgG.This proteic molecular weight is about 180kDa, contains two subunits, and promptly the light chain of the heavy chain of 67-70kDa and 22-30kDa claims IgY (Yolk Immunoglobulin) again.
2. the content of IgG is as many as even will be higher than IgG in the female bird serum in the yolk.Female bird is produced egg a middle of the month and is prepared yolk antibody and be equivalent to the 500ml serum antibody.Female bird is transferred to birds, beasts and eggs by two kinds of approach with its whole homotype antibody: at first be that blood plasma in the blood circulation is transferred to antibody in the sophisticated ovarian follicle and (just entered into yolk), the a-sphaeroprotein that this selectivity is transferred to ovary follicle mainly is IgG, and this antibody by the decision of IgG acceptor shifts and determining the whole IgG subgroup selectivity that exist in the source of parents blood to shift to ovary follicle.Second approach of the antibody transfer process of carrying out in uterine tube only relates to IgM and IgA, and the position of transfer is at egg white, and this prepares for us and utilizes yolk IgG that the condition that makes things convenient for is provided.
3.IgY biological characteristics determined its wide application field.Aspect immunodiagnosis, fowl IgY has not activation of human complement, immunological characteristic such as do not combine, with several no cross reactions of IgG, so obtained widespread use at medical field for a long time with albumin A, Protein G, Mammals Fc acceptor, complement and Rheumatoid factors, polyclonal.Aspect immunotherapy, heat-resisting, the acidproof and stable performance because of IgY, 180 days antibody titers of 120 days ,-20 ℃ preservations of 4 ℃ of preservations of water solvent are constant, if freeze-drying or spraying drying then preservation period can prolong greatly, can be through oral route treatment young animal or human infectious intestinal disease, as baby's diseases prevention food, the prevention of dental caries, and the control of infant's rotavirus, human influenza, toxoplasmosis or the like.Abroad, the report of using anti-pig pathogenic colon bacillus yolk antibody and anti-calf Salmonellas yolk antibody treatment pig pathogenic colon bacillus and calf Salmonella infection has been arranged, domestic also have similar research.Domestic widely used have infectious bursal disease, newcastle disease, gosling plague, duck plague, chicken egg-decreasing syndrome, duck hepatitis, chicken viral sacroiliitis, infectious laryngotracheitis of chicken yolk antibody or the like.Yolk antibody facts have proved the treatment of chicken, duck, goose, pig, ox, and is without any side effects to body.
4. yolk has many advantages as the source of specific antibody IgY.As: female bird is easy to raise, and expense is not high; Collect egg instant; Need not extract animal blood, not damaged meets modern protection of animal rule; It is little to produce the required antigen amount of effective antibody response; At home, fowl is the laboratory animal that takes the lead in realizing SPFization and industrialization, helps large-scale production; Bird helps disease control with far away with the Mammals sibship of artificial representative, and the immunoglobulin (Ig) with fowl production human can not be incorporated into the pathogenic micro-organism that causes human diseases in patient's the body, can solve the trouble and worry in the treatment.Poultry is as an effective antibody producing person, after with single adjuvant antigen immune, but long term maintenance antibody titer, the level of 1 all serum antibodies promptly is reacted in the yolk after immunity, and IgG only is secreted in the yolk, fast, special, longer duration, the characteristics of factor strong resistance make yolk antibody extremely adapt to pressing in the current SARS epidemic prevention to external world, can play a significant role for SARS treatment and control.
Description of drawings: accompanying drawing 1 is technological line figure of the present invention.
The specific embodiment of the present invention:
Embodiment 1:
The making method of anti-sars type pneumonia yolk antibody, described method comprises: make vaccinogen, Antibody Preparation, yolk homogenate, dilution, low temperature spend the night, filter purifying, powder process.
The present invention's 1 concrete operational path that provides with reference to the accompanying drawings finishes, and detailed process is explained as follows in detail:
1. immunogenic preparation.Select the SARS virus culture through the deactivation of β-third lactones, polyoxyethylene glycol-6000 concentrates 8-15 and doubly does water.MAUTAMIDEISA-70 is an oil phase, and by 15-20: the 50-80 emulsify at a high speed becomes the water-in-oil-type immunogen.
2. the preparation of anti-SARS fowl yolk antibody.Select the SPF fowl of 100-140 age in days, 120 age in days SPF fowl for example, first immunisation 1.5-3.5ml, 2-4 position of chest muscle injection.2-4 week repeats booster immunization (two exempt from) at interval.Antibody titer is monitored in blood sampling weekly, and two exempt from 1-2 collects after week through the qualified fowl ovum of antibody test.Blood sampling detects antibody weekly, and NAT is not less than 1: 256 for qualified.Three exempt from the decision of basis of time antibody detection situation, and injection volume and two is exempted from identical.
3. the purification of anti-SARS fowl yolk antibody.
The qualified egg of anti-SARS fowl yolk antibody, with 1: the golden iodine aqueous solution of 1000-1500 dilution is clean with the fowl ovum, airing, separates yolk in Bechtop.Isolating yolk and distilled water are by 1: the 2-4 mixed, put into rotating speed that stamp mill divides with 8000-20000/ stir 2-4 minute standby.
(1) the yolk liquid and the solvent (acetone 1-2 part, sherwood oil 4-6 part) of above-mentioned preparation are pressed 1: (8000-20000 rev/min of the mixed of 0.5-1.5 and high-speed stirring, 1-4 minute) back centrifugal 10000 the gram 10-15 minute, so repeat 2-4 time, keep water and abandon oil phase and precipitation.
(2) to make its final concentration be 0.14-0.2% and to transfer PH be 6.0-7.0 with adding sodium alginate in the yolk liquid of above-mentioned preparation, high-speed stirring (8000-20000 rev/min, 2-4 minute), and centrifugal 10000 grams 10-15 minute, 140-150 order copper mesh filters, abandons precipitation.Be mixed and made into the ammoniumsulphate soln of 50-60% and transfer PH to 7.0-8.0 with saturated ammonium sulphate solution and filtrate, (8000-20000 rev/min of high-speed stirring, 2 minutes) centrifugal 10000 grams in back abandoned supernatant in 10-15 minute, precipitation is with the dissolving of 20-40% ammonium sulfate and transfer (8000-20000 rev/min of PH7.0-8.0 high-speed stirring, 2 minutes) centrifugal 10000 grams in back 10-20 minute, so repeat after the secondary with PBS dissolution precipitation and dialysis.
4.SARS the preparation technology of powdery yolk.
The NAT of yolk and serum is qualified egg more than 256 times after measured, and birds, beasts and eggs are clean with the golden iodine aqueous solution of dilution in 1: 1000, airing separate yolk in Bechtop.Isolating yolk and distilled water are by 1: the 2-4 mixed, and put into stamp mill and stirred 2-4 minute with the rotating speed that 8000-20000/ divides, it is standby to be put in refrigerator-freezer below-10 ℃.With 1: 4 ℃ of the yolk liquid of 2-4 dilution proportion 10000 grams were abandoned precipitation in centrifugal 30-40 minute, and 140-150 order copper mesh filters, and filtrate concentrates 2-4 doubly.120-160 ℃ of spraying drying temperature in, temperature out 55-85 ℃.The sealing of spraying drying yolk powder is stored in the 2-8 ℃ of freezer, and validity period 1 year was preserved validity period 1 month for 10-20 ℃.
5. virus neutralization tests.
The fowl embryo median infective dose (ELD of virus
50) measure: SARS virus is pressed concentration gradient 10 with physiological saline
-7, 10
-8, 10
-9, 10
-10, 10
-11, 10
-12, 10
-13Dilute, inoculate 10-20 age in days SPF fowl embryo respectively, one group of each extent of dilution, every group of 4-8 fowl embryo, inoculum size 0.1-0.5ml/ embryo.Discard inoculation dead embryo before back 48 hours, write down and infected number in 48-120 hour and do not infect number, press Reed-Muech method calculating chicken embryo median infective dose.
Virus neutralization tests: virus is diluted 10 respectively
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6, 10
-7, 10
-8, 10
-9, mix with the antibody stoste of equivalent respectively, fully behind the mixing, put 4 ℃ of reactions 2-4 hour.The SPF fowl embryo of 10-20 age in days is divided into three winding kind viruses: first group is the normal serum control group, the inoculation normal serum; Second group is antibody toxicity control group, only inoculates antibody; The mixed solution of the 3rd winding kind virus and antibody, dosage of inoculation 0.1-05ml/ embryo.After the inoculation, 37 ℃ of cultivations, the chicken embryo of adding up 24 hours postoperative infections and not infecting until 130-150 hour, calculates the viral neutralization index of high-immunity yolk purification antibody, and 1: 256 is above qualified.
Claims (8)
1. the making method of an anti-sars type pneumonia yolk antibody, it is characterized in that: described method comprises: make vaccinogen, Antibody Preparation, yolk homogenate, dilution, low temperature spends the night, filter purifying, powder process, the preparation method of wherein said anti-SARS fowl yolk antibody is: select 100-140 age in days SPF fowl, first immunisation 1.5-3.5ml, 2-4 position of chest muscle injection, 2-4 week repeats booster immunization at interval, antibody titer is monitored in blood sampling weekly, and two exempt from 1-2 collects after week through the qualified fowl ovum of antibody test, and blood sampling detects antibody weekly, NAT is not less than 1: 256 for qualified, and three exempt from injection volume and two exempts from identical.
2. the making method of anti-sars type pneumonia yolk antibody according to claim 1 is characterized in that: described immunogenic preparation is: select the SARS virus culture through the deactivation of β-third lactones, polyoxyethylene glycol-6000 concentrates 8-15 and doubly does water.MAUTAMIDEISA-70 is an oil phase, and by 15-20: the 50-80 emulsify at a high speed becomes the water-in-oil-type immunogen.
3. the making method of anti-sars type pneumonia yolk antibody according to claim 1 and 2, it is characterized in that: described homogenate mode is: with 1: the golden iodine aqueous solution of 1000-1500 dilution is clean with the fowl ovum, airing, separate yolk in Bechtop, isolating yolk and distilled water are by 1: the 2-4 mixed, and put into stamp mill and stirred 2-4 minute with the rotating speed that 8000-20000/ divides.
4. the making method of anti-sars type pneumonia yolk antibody according to claim 1 and 2, it is characterized in that: described filtration and purification process comprises:
(1) the yolk liquid and the solvent (acetone 1-2 part, sherwood oil 4-6 part) of above-mentioned preparation are pressed 1: (8000-20000 rev/min of the mixed of 0.5-1.5 and high-speed stirring, 1-4 minute) back centrifugal 10000 the gram 10-15 minute, so repeat 2-4 time, keep water and abandon oil phase and precipitation;
(2) to make its final concentration be 0.14-0.2% and to transfer PH be 6.0-7.0 with adding sodium alginate in the yolk liquid of above-mentioned preparation, high-speed stirring (8000-20000 rev/min, 2-4 minute), and centrifugal 10000 grams 10-15 minute, 140-150 order copper mesh filters, abandons precipitation.Be mixed and made into the ammoniumsulphate soln of 50-60% and transfer PH to 7.0-8.0 with saturated ammonium sulphate solution and filtrate, (8000-20000 rev/min of high-speed stirring, 2 minutes) centrifugal 10000 grams in back abandoned supernatant in 10-15 minute, precipitation is with the dissolving of 20-40% ammonium sulfate and transfer (8000-20000 rev/min of PH7.0-8.0 high-speed stirring, 2 minutes) centrifugal 10000 grams in back 10-20 minute, so repeat after the secondary with PBS dissolution precipitation and dialysis.
5. the making method of anti-sars type pneumonia yolk antibody according to claim 3, it is characterized in that: described filtration and purification process comprises:
(1) the yolk liquid and the solvent (acetone 1-2 part, sherwood oil 4-6 part) of above-mentioned preparation are pressed 1: (8000-20000 rev/min of the mixed of 0.5-1.5 and high-speed stirring, 1-4 minute) back centrifugal 10000 the gram 10-15 minute, so repeat 2-4 time, keep water and abandon oil phase and precipitation;
(2) to make its final concentration be 0.14-0.2% and to transfer PH be 6.0-7.0 with adding sodium alginate in the yolk liquid of above-mentioned preparation, high-speed stirring (8000-20000 rev/min, 2-4 minute), and centrifugal 10000 grams 10-15 minute, 140-150 order copper mesh filters, abandons precipitation.Be mixed and made into the ammoniumsulphate soln of 50-60% and transfer PH to 7.0-8.0 with saturated ammonium sulphate solution and filtrate, (8000-20000 rev/min of high-speed stirring, 2 minutes) centrifugal 10000 grams in back abandoned supernatant in 10-15 minute, precipitation is with the dissolving of 20-40% ammonium sulfate and transfer (8000-20000 rev/min of PH7.0-8.0 high-speed stirring, 2 minutes) centrifugal 10000 grams in back 10-20 minute, so repeat after the secondary with PBS dissolution precipitation and dialysis.
6. according to the making method of claim 1 or 2 or 5 described anti-sars type pneumonia yolk antibodies, it is characterized in that: the preparation process of described SARS powdery yolk comprises: the NAT of yolk and serum is qualified egg more than 256 times after measured, golden iodine aqueous solution with dilution in 1: 1000 birds, beasts and eggs are clean, airing separates yolk in Bechtop.Isolating yolk and distilled water are by 1: the 2-4 mixed, and put into stamp mill and stirred 2-4 minute with the rotating speed that 8000-20000/ divides, it is standby to be put in refrigerator-freezer below-10 ℃.With 1: 4 ℃ of the yolk liquid of 2-4 dilution proportion 10000 grams were abandoned precipitation in centrifugal 30-40 minute, and 140-150 order copper mesh filters, and filtrate concentrates 2-4 doubly.120-160 ℃ of spraying drying temperature in, temperature out 55-85 ℃.
7. the making method of anti-sars type pneumonia yolk antibody according to claim 3, it is characterized in that: the preparation process of described SARS powdery yolk comprises: the NAT of yolk and serum is qualified egg more than 256 times after measured, golden iodine aqueous solution with dilution in 1: 1000 birds, beasts and eggs are clean, airing separates yolk in Bechtop.Isolating yolk and distilled water are by 1: the 2-4 mixed, and put into stamp mill and stirred 2-4 minute with the rotating speed that 8000-20000/ divides, it is standby to be put in refrigerator-freezer below-10 ℃.With 1: 4 ℃ of the yolk liquid of 2-4 dilution proportion 10000 grams were abandoned precipitation in centrifugal 30-40 minute, and 140-150 order copper mesh filters, and filtrate concentrates 2-4 doubly.120-160 ℃ of spraying drying temperature in, temperature out 55-85 ℃.
8. the making method of anti-sars type pneumonia yolk antibody according to claim 4, it is characterized in that: the preparation process of described SARS powdery yolk comprises: the NAT of yolk and serum is qualified egg more than 256 times after measured, golden iodine aqueous solution with dilution in 1: 1000 birds, beasts and eggs are clean, airing separates yolk in Bechtop.Isolating yolk and distilled water are by 1: the 2-4 mixed, and put into stamp mill and stirred 2-4 minute with the rotating speed that 8000-20000/ divides, it is standby to be put in refrigerator-freezer below-10 ℃.With 1: 4 ℃ of the yolk liquid of 2-4 dilution proportion 10000 grams were abandoned precipitation in centrifugal 30-40 minute, and 140-150 order copper mesh filters, and filtrate concentrates 2-4 doubly.120-160 ℃ of spraying drying temperature in, temperature out 55-85 ℃.
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| CNA2003101077318A CN1546526A (en) | 2003-12-02 | 2003-12-02 | Method for preparing IgY for SARS |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111474344A (en) * | 2020-03-20 | 2020-07-31 | 西安国联质量检测技术股份有限公司 | Preparation method of novel coronavirus IgY antibody immunodetection reagent |
| CN111505282A (en) * | 2020-04-21 | 2020-08-07 | 西安国联质量检测技术股份有限公司 | Preparation and use methods of novel coronavirus immunofluorescence detection reagent based on IgY antibody and kit containing novel coronavirus immunofluorescence detection reagent |
| CN111574622A (en) * | 2020-04-07 | 2020-08-25 | 刘会芳 | Antibody for resisting novel human coronavirus pneumonia and preparation method thereof |
| WO2021174806A1 (en) * | 2020-03-05 | 2021-09-10 | 广州万孚生物技术股份有限公司 | 2019-ncov novel coronavirus rapid detection immunochromatographic test strip |
| 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 |
| CN114835804A (en) * | 2022-05-19 | 2022-08-02 | 安徽中起生物科技有限公司 | Feline infectious peritonitis egg yolk antibody composition and preparation method and application thereof |
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2003
- 2003-12-02 CN CNA2003101077318A patent/CN1546526A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021174806A1 (en) * | 2020-03-05 | 2021-09-10 | 广州万孚生物技术股份有限公司 | 2019-ncov novel coronavirus rapid detection immunochromatographic test strip |
| CN111474344A (en) * | 2020-03-20 | 2020-07-31 | 西安国联质量检测技术股份有限公司 | Preparation method of novel coronavirus IgY antibody immunodetection reagent |
| CN111574622A (en) * | 2020-04-07 | 2020-08-25 | 刘会芳 | Antibody for resisting novel human coronavirus pneumonia and preparation method thereof |
| CN111505282A (en) * | 2020-04-21 | 2020-08-07 | 西安国联质量检测技术股份有限公司 | Preparation and use methods of novel coronavirus immunofluorescence detection reagent based on IgY antibody and kit containing novel coronavirus immunofluorescence detection reagent |
| 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 |
| CN114835804A (en) * | 2022-05-19 | 2022-08-02 | 安徽中起生物科技有限公司 | Feline infectious peritonitis egg yolk antibody composition and preparation method and application thereof |
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