CN1250056A - Pyloric bacillary spirillum resisting yolk immunoglobulin product and its application - Google Patents
Pyloric bacillary spirillum resisting yolk immunoglobulin product and its application Download PDFInfo
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- CN1250056A CN1250056A CN 99117588 CN99117588A CN1250056A CN 1250056 A CN1250056 A CN 1250056A CN 99117588 CN99117588 CN 99117588 CN 99117588 A CN99117588 A CN 99117588A CN 1250056 A CN1250056 A CN 1250056A
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Abstract
Pyloric helicobacterium vaccine is used to immunize laying hen and the laid egg is used to extract anti-HPIgY. Anti-HpIgY is produced into health food or other biological preparation for the prevention and treatment of diseases caused by pyloric helicobacterium. Purified anti-HPIgY may be used in the clinical detection and diagnosis of pyloric helicobacterium. The product has excellent activity and specificity.
Description
The present invention relates to a kind of Yolk immunoglobulin product, and related to its application, particularly a kind of antibody product of anti-helicobacter pylori and application with antibody activity.
Recent study shows that helicobacter pylori (HP) is close with being related of chronic gastritis, peptide ulceration.HP is classified as the risk factor that the first kind causes cancer of the stomach by The World Health Organization (WHO) at present, and the HP propagation has worldwide, and the HP infection rate (grownup) among China various places nature crowd is all more than 50%.Develop into single antibiotic therapy → two kind of pharmacological agent → triple treatment → anti-secretory drugs of bismuth agent and the antibiotic of the methods of treatment of HP are united radical cure HP.But single antibiotic or two kinds of pharmacological agent curative effects are relatively poor, and the triple treatment side effects of bismuth agent are big, and compliance is poor.Anti-secretory drugs and antibiotic are united radical cure and are had the problem of tolerance difference in recent years, and do not have enough clinical practices.And, the restriction in China because of animal model, HP vaccine progress is slow.Therefore adopt the passive immunization protection to infect the new approach that provides for control HP.
The object of the present invention is to provide a kind of anti-helicobacter pylori Yolk immunoglobulin with antibody activity (anti--HPIgY) product, and products obtained therefrom is mixed with healthcare products, biotechnological formulation or clinical detection reagent, be used for the control of the relative disease that causes because of helicobacter pylori infection and clinical diagnosis or the detection of helicobacter pylori.
The object of the present invention is achieved like this:
Anti-helicobacter pylori Yolk immunoglobulin with antibody activity is (anti--HPIgY) product.This product can obtain from following steps: preparation helicobacter pylori vaccine, with helicobacter pylori vaccine immunization hen, collect its ovum gallinaceum that produces then, and therefrom extract Yolk immunoglobulin.
Can adopt following step for effectively preparing the helicobacter pylorus vaccine: at first separate obtaining HP from HP the infected's lesions position, then coat on the blood agar plate, 37 ℃ of anaerobism were cultivated 72 hours, plate streaking cultured continuously with the HP test positive, collect thalline and get HP bacterium liquid, ultrasonic grinding adds freund adjuvant, and emulsification gets the HP vaccine.
From yolk, extract Yolk immunoglobulin, adopt water dilution method or polyoxyethylene glycol method more superior.Its extracting efficiency height.Cost is low.It is a kind of free of contamination extracting method.Wherein the water dilution method step is: from immune ovum gallinaceum, separate yolk, with distilled water diluting egg liquid, adjust suitable pH value, standing over night, the centrifuging and taking supernatant liquor concentrates, lyophilize is resisted-HPIgY.
Polyoxyethylene glycol two-step precipitation method step is: from immune ovum gallinaceum, separate yolk, with 2 times of PBS dilution yolk, add 3.5% polyethylene glycol 6000, stirring was left standstill 1-4 hour, and the centrifuging and taking supernatant adds 8.5% polyoxyethylene glycol, stirring was left standstill 1-4 hour, centrifugal must the precipitation will be precipitated lyophilize, be resisted-HPIgY.
Provided by the present invention resisting-HPIgY product is laid eggs hen by the helicobacter pylori vaccine immunization and is got.Have very strong specificity, the relative disease that the control helicobacter pylori infection is caused has good effect, and the immunoglobulin (Ig) based article can strengthen the human immunologic function.It is carrier that the present invention adopts ovum gallinaceum that hen produces, safety non-toxic.Hen is easily raised and management, is convenient to industrialization.
Describe the present invention in detail below in conjunction with embodiment.
Embodiment 1: the helicobacter pylori Immunoglobulin of Yolk with antibody activity is (anti--HPIgY) product, can obtain by following steps: preparation helicobacter pylori (HP) vaccine, with HP vaccine immunization bird inlay, collect its continuous output ovum gallinaceum, therefrom extract anti--HPIgY again.
The preparation of embodiment 2:HP vaccine is got gastritis or stomach ulcer patient's mucosa tissue piece and is coated on the blood agar plate, and 37 ℃ of anaerobism were cultivated 72 hours, HP is detected male plate streaking cultured continuously, ultrasonic grinding adds freund adjuvant, and emulsification gets the HP vaccine.
Embodiment 3: collection gained immunity egg separation and Extraction in the following way resists-HPIgY: separate yolk with 4-10 times of distilled water diluting yolk, adjust suitable substance P H value, and 4 ℃ of standing over night, the centrifuging and taking supernatant liquor, concentrated, lyophilize must resist-HPIgY.
Embodiment 4: with 2 times of PBS dilution yolk, add 3.5% polyethylene glycol 6000 (PEG6000), stir and left standstill 1-4 hour, the centrifuging and taking supernatant adds 8.5%PEG6000, stirs and leaves standstill 1-4 hour, and centrifugal must the precipitation will be precipitated lyophilize, must resist-HPIgY.
Embodiment 5: get among the embodiment 1-4 that any product carries out indirect hemagglutination or its activity is surveyed in the indirect ELISA experiment.
Indirect hemagglutination test: will resist-the HPIgY diluent coagulates in the plate aperture between adding, and adds one in tanning of HP sensitization blood cell again, and put plate jolting five minutes on the blood clotting micro oscillator then, 37 ℃ of insulations 30 minutes are bead-like in the visible hole and precipitate, be i.e. the strong positive result.
The indirect ELISA test: HP is antigen coated in each hole of polyethylene micro-reaction plate, and 4 ℃ are spent the night, and add PBS-T and wash plate, add the BSA sealing, 37 ℃ of incubations washed plate in 30 minutes and will resist-HPIgY dilution drop in reacting hole, 37 ℃ of incubations 2 hours, wash plate and add anti--IgYHRP, 37 ℃ of incubations 1 hour are washed plate the OPD substrate are added reacting hole, add 30% hydrogen peroxide again, developed the color 5-10 minute, add the reaction of 2.5N hydrochloric acid color development stopping, microplate reader detects, and the OD value is big more to be that antibody activity is good more.
Embodiment 6: will slightly carry anti--HPIgY and cross DEAE-Sephadex A50 gel chromatographic columns, after the SephadexG200 gel chromatographic columns, lyophilize get final product pure anti--HPIgY.
Embodiment 7: get 30 of healthy mices, and establish control group, fasting be can't help water 12 hours before the administration, anti--HPIgY solution of 15% is irritated stomach by each 0.4 milliliter/10 grams, gave a medicine every 8 hours, twenty four hours is given anti--HPIgY3 time altogether, and the animal maximum tolerated dose is 18 gram/kg/day.Be equivalent to 3750 times of clinical dosages.Above result shows, and anti--HPIgY preparation safety is nontoxic.
Embodiment 8: gather anti--50 on HPIgY immunity egg, separate obtaining 800 milliliters of yolk, thin up to 5000 milliliter, centrifugal 4000 milliliters of the clear liquids that obtain, the employing Hollow Fiber Ultrafiltration extracts, be concentrated at last 400 milliliters stand-by, wherein contain and slightly carry anti--HPIgY12 gram.To add the 5ml water dissolution among the sucrose 10g, and sterilize 30 minutes, auxiliary material and 50 milliliters of ultrafiltrateds of handling are mixed for 100 ℃, freezing dried bath, aseptic pulverizing is packaged into bag, and every pouch includes 0.5 gram, is used for the treatment of the chronic gastritis and the peptide ulceration that cause because of the HP infection.
Embodiment 9: gather 100 on the immune egg of anti--HPIgY, obtain 1600 milliliters in yolk, add 2 times of PBS dilution egg liquid, and add 3.5% PEG6000 and stir and to leave standstill 2 hours, centrifugal 3500 milliliters of the clear liquids that obtain, add again PEG6000 to final concentration be 12%, left standstill centrifugal collecting precipitation 2 hours.With 100ml dissolved in distilled water IgY precipitation, by biofilter degerming, lyophilize, slightly carry anti--HPIgY with each conventional capsule dress 200mg and carry out can, products obtained therefrom is used to prevent helicobacter pylori infection, human body instructions of taking every day 2-3 time, each 3 capsules.
Claims (8)
1, a kind of pyloric bacillary spirillum resisting yolk immunoglobulin is (anti--HPIgY) product.This product can obtain from following steps: preparation helicobacter pylori (HP) vaccine, and with helicobacter pylori (HP) vaccine immunization healthy hens, collect it then and lay eggs, from laying eggs, it extracts anti--HPIgY again.
2, according to claim 1 resisting-the HPIgY product, it is characterized in that in order effectively to prepare helicobacter pylori (HP) vaccine, can adopt following step: at first separate obtaining HP from HP the infected's lesions position, then coat on the blood agar plate, 37 ℃ of anaerobism were cultivated 72 hours, with the plate streaking cultured continuously of HP test positive, collect thalline and get HP bacterium liquid, ultrasonic grinding adds freund adjuvant, and emulsification gets the HP vaccine.
3, according to claim 1 resisting-the HPIgY product, it is characterized in that: adopt water dilution method to extract Yolk immunoglobulin, its step: from immune ovum gallinaceum, separate yolk, with distilled water diluting egg liquid, adjust suitable pH value, standing over night, the centrifuging and taking supernatant liquor, concentrate, lyophilize is resisted-HPIgY.
4, according to claim 1 resisting-the HPIgY product, it is characterized in that: adopt polyoxyethylene glycol two-step precipitation method to extract Yolk immunoglobulin, its step: from immune ovum gallinaceum, separate yolk, with 2 times of PBS dilution yolk, add 3.5% polyethylene glycol 6000, stirring was left standstill 1-4 hour, and the centrifuging and taking supernatant adds 8.5% polyoxyethylene glycol, stirring was left standstill 1-4 hour, centrifugal must the precipitation will be precipitated lyophilize, be resisted-HPIgY.
5, the healthcare products or the biotechnological formulation of forming by any one described resisting-HPIgY product in the claim 1-4 item that are used to prevent and treat swollen chronic gastritis, stomach ulcer, duodenal ulcer disease and the cancer of the stomach that causes.
6, application rights requires the healthcare products or the biotechnological formulation of any one described resisting in the 1-4 item-chronic gastritis, stomach ulcer, duodenal ulcer disease and cancer of the stomach that HPIgY product preparation control HP causes.
7, contain in the claim 1-4 item any one described anti--HPIgY product is used to prevent and treat the microbial chronic gastritis of helicobacter pylorus, stomach ulcer, the healthcare products of duodenal ulcer disease and cancer of the stomach or biotechnological formulation.
8, be used as the detection diagnostic reagent of helicobacter pylori by any one described resisting-HPIgY product in the claim 1-3 item.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB991175883A CN100360566C (en) | 1999-09-08 | 1999-09-08 | Pyloric bacillary spirillum resisting yolk immunoglobulin product and its application |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB991175883A CN100360566C (en) | 1999-09-08 | 1999-09-08 | Pyloric bacillary spirillum resisting yolk immunoglobulin product and its application |
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| Publication Number | Publication Date |
|---|---|
| CN1250056A true CN1250056A (en) | 2000-04-12 |
| CN100360566C CN100360566C (en) | 2008-01-09 |
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| Application Number | Title | Priority Date | Filing Date |
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| CNB991175883A Expired - Fee Related CN100360566C (en) | 1999-09-08 | 1999-09-08 | Pyloric bacillary spirillum resisting yolk immunoglobulin product and its application |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004037862A1 (en) * | 2002-10-25 | 2004-05-06 | Jason Medical Group (Far East) Limited | Special igy of resist allergic and its preparation |
| CN101411362B (en) * | 2008-10-14 | 2012-05-30 | 大连韩伟企业集团有限公司 | Yoghourt containing vitellus immunoglobulin with helicobacter pylori resistance |
| CN106075436A (en) * | 2016-07-12 | 2016-11-09 | 闫小君 | A kind of anti-helicobacter pylori oral formulations without antibiotic |
| CN106632672A (en) * | 2016-12-05 | 2017-05-10 | 徐州安替生物科技有限公司 | Preparation method of egg yolk antibody containing helicobacter pylori resisting IgY and application |
| CN109400705A (en) * | 2018-12-04 | 2019-03-01 | 大连先锋生物科技有限公司 | A kind of anti-helicobacter pylori duck Yolk antibody and preparation method thereof |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1290580C (en) * | 1998-04-01 | 2006-12-20 | 中德联合研究院 | Chick lecithal immune globulin products against rolavirus and its use and its testing method of activity |
| CN1101404C (en) * | 1998-07-29 | 2003-02-12 | 中德联合研究院 | Anti Bacillus pyocyaneu Flugge vitelline immunoglobulin products and use thereof |
-
1999
- 1999-09-08 CN CNB991175883A patent/CN100360566C/en not_active Expired - Fee Related
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004037862A1 (en) * | 2002-10-25 | 2004-05-06 | Jason Medical Group (Far East) Limited | Special igy of resist allergic and its preparation |
| CN101411362B (en) * | 2008-10-14 | 2012-05-30 | 大连韩伟企业集团有限公司 | Yoghourt containing vitellus immunoglobulin with helicobacter pylori resistance |
| CN106075436A (en) * | 2016-07-12 | 2016-11-09 | 闫小君 | A kind of anti-helicobacter pylori oral formulations without antibiotic |
| CN106075436B (en) * | 2016-07-12 | 2020-01-03 | 闫小君 | An oral preparation containing no antibiotic for resisting helicobacter pylori |
| CN106632672A (en) * | 2016-12-05 | 2017-05-10 | 徐州安替生物科技有限公司 | Preparation method of egg yolk antibody containing helicobacter pylori resisting IgY and application |
| CN109400705A (en) * | 2018-12-04 | 2019-03-01 | 大连先锋生物科技有限公司 | A kind of anti-helicobacter pylori duck Yolk antibody and preparation method thereof |
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| Publication number | Publication date |
|---|---|
| CN100360566C (en) | 2008-01-09 |
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