CN1354187A - Hen egg yolk antibody for resisting pyloric helicobacterium cytotoxin, its preparation and application - Google Patents
Hen egg yolk antibody for resisting pyloric helicobacterium cytotoxin, its preparation and application Download PDFInfo
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- CN1354187A CN1354187A CN 01129140 CN01129140A CN1354187A CN 1354187 A CN1354187 A CN 1354187A CN 01129140 CN01129140 CN 01129140 CN 01129140 A CN01129140 A CN 01129140A CN 1354187 A CN1354187 A CN 1354187A
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Abstract
The present invention relates to a chicken vitellus antibody (anti-HPTxIgY) with antibody activity for resisting pyloric helicobacterium cytotoxin, its preparation method and application in the preparation of medicine and health-care product for curing related diseases resulted from pyloric helicobacterium infection and preparation for detecting these related diseases. The above-mentioned antibody is undergone the process of IgY purity checking treatment. PAGE electrophoressi can be used for detecting purified vitallus antibody purity. Only one electrophoretic band is produced in the electrophoresis, and an UV spectrophotometer is used for determination so as to calculate the albumen concentration (mg/ml) which is (1.45XOD 280nm-0.74XOD 260nm)X dilute multiple. The above-mentioned method is as follows: preparing pyloric helicobacterium cytotoxin antigen, immunizing health hen with said antigen, collecting egg and extracting anti-HPTxIgY from the egg.
Description
(1), technical field
The present invention relates to a kind of yolk antibody product-hen egg yolk antibody for resisting pyloric helicobacterium cytotoxin, and related to its preparatory technology and in pharmaceutically application with antibody activity.
(2), technical background
(Helicobacter pylori HP) is the closely-related pathogenic bacterium of the generation with chronic gastritis, peptide ulceration and non-ucler dyspepsia that recent two decades is found to helicobacter pylori.WHO classifies it as I class carcinogen, HP can produce multiple protein molecular with cytotoxic activity, it discharges VacA (vacuolating cytotoxin), CagA toxin such as (cytotoxin sample albumen) and makes the downright bad and reaction that causes inflammation of gastric epithelial after gastric mucosa adheres to field planting.So cytotoxic molecule plays an important role in the HP pathogenic course.At present, because of the relative disease of HP infection, for example: chronic gastritis, peptide ulceration, cancer of the stomach etc.
The treatment measure of HP is the field that people pay close attention to always, at present, prevent and treat the method for helicobacter pylori infection clinically, the general combination medicine scheme that adopts more, commonly used as bismuth agent+nitro glyoxaline microbiotic+tsiklomitsin, proton pump retarding agent+new macrolide antibiotic, life-time service can produce antibiotics resistance bacterial strain and variant and cause difficulty and antibiotic life-time service can give patient for further treatment to bring other side effect.And, the restriction in China because of animal model, HP vaccine progress is slow.Therefore adopting the passive immunization protection is the new way that control HP infects.
Not seeing in the prior art has by adopting helicobacter pylori cytotoxin antigen, and the immune health hen is collected the ovum that it produces, and extracts the report of anti--HPTxIgY method again from ovum.
(3), Fa Ming content
One of technical problem to be solved by this invention is: at the prior art above shortcomings, provide a kind of hen egg yolk antibody for resisting pyloric helicobacterium cytotoxin with antibody activity (anti--HPTxIgY).
Two of technical problem to be solved by this invention is: the preparation method that above-mentioned pyloric bacillary spirillum resisting yolk antibody is provided.
Three of technical problem to be solved by this invention is: provide above-mentioned anti-helicobacter pylori cytotoxin yolk antibody to be used for because of infecting the microbial relative disease of helicobacter pylorus in preparation, pharmaceutical preparation or healthcare products as the prevention of chronic gastritis, peptide ulceration and diseases such as non-ucler dyspepsia and cancer of the stomach or treatment are used, and the application that is used for the detection reagent of the clinical diagnosis of helicobacter pylori or detection in preparation.
I.e. anti--the HPTxIgY of the anti-helicobacter pylori cytotoxin yolk antibody that provided one of is provided in the present invention, it is characterized in that: described anti--HPTxIg press YSDS-PAGE electrophoresis (7.5% separation gel) detection, collection of illustrative plates presents single district band; Be blue through electrophoresis Coomassie brilliant blue R250 dyeing; High molecular band place clear single specific enzymes occurred and dyes band during immunity marking Western-blotting detected; Carrying out dual wavelength mensuration with ultraviolet spectrophotometer to calculate protein concentration (mg/ml) is: (1.45 * OD
80nm-0.74 * OD
60nm) * extension rate.
What the present invention solved the problems of the technologies described above two is by adopting such technical scheme to realize that promptly a kind of method for preparing anti-helicobacter pylori toxin yolk antibody is characterized in that adopting following step to finish:
1, preparation HP cytotoxin antigen
(1) extraction method: separate to obtain toxogenic HP bacterial strain, coat on the brucella agar, 37 ℃ of little aerobic cultivations 72h, must pure strain after, enlarged culturing T2h in the brucella broth substratum.Collect culture supernatant, obtain toxin by ultrafiltration and chromatographic separation, added complete freund adjuvant by weight 1: 1, emulsification gets HP toxin antigen.
Or (2) synthesis method: make up the expression plasmid that contains HP cytotoxin structure gene VacA or CagA, in the intestinal bacteria system, express, expression product is through separation and purification, preparation HP vacuolating cytotoxin (VacA) and cell toxicant toxin (CagA), mixed as immunizing antigen by weight 1: 1, add isopyknic freund adjuvant, the emulsification mixing gets HP toxin antigen.
2, HP toxin antigen is prepared antibody by following step:
(1). to the immunity of the hen that lays eggs:
Select the hen that lays eggs (health) isolated rearing for use.Carry out multi-point injection under the cock skin, as 5 points, the toxin protein amount is equivalent to 10
10The CFU/ml bacterium produces.Per two all booster immunizations once amount to immunity 8 times later on; Began to collect egg after 7 days, 4 ℃ store for future use.
(2). the extraction of yolk antibody
1.. adopt water-soluble method to extract yolk antibody.From immune ovum gallinaceum, separate yolk, with distilled water diluting, adjust suitable pH value and leave standstill 8h for 4 ℃, the centrifuging and taking supernatant concentrates, lyophilize, is resisted-HPTxIgY.
Or 2. adopt membrane filter method to extract yolk antibody.Separate yolk, with distilled water diluting, 4 ℃ leave standstill 12h after, be that the film of 200KD and 150KD filters respectively with the molecular weight, collect the proteic substance of 150KD-200KD, lyophilize is resisted-HPTxIgY.
Above-mentioned resisting provided by the present invention-HPTxIgY product is laid eggs hen by the helicobacter pylori cytotoxin immunity and is got, and has specificity, and the relative disease that the control helicobacter pylori infection is caused has good effect.It is carrier that the present invention adopts ovum gallinaceum that hen produces, and safety non-toxic is easy to industrialization.
(4), the description of the drawings
Accompanying drawing has provided preparation method's of the present invention process flow sheet.
(5), embodiment
Below in conjunction with accompanying drawing embodiments of the invention are further described this:
Embodiment 1: the product with helicobacter pylori cytotoxin yolk antibody of antibody activity, can obtain by following steps: preparation helicobacter pylori (HP) cytotoxin antigen, with HP toxin-antitoxin antigen immune healthy hens, collect it then and lay eggs, from it is laid eggs, extract anti--HPTxIgY again.
The antigenic preparation of embodiment 2:HP cytotoxin separates to obtain toxogenic HP bacterial strain, coat on the brucella agar, 37 ℃ of little aerobic cultivations 72h, must pure strain after, enlarged culturing T2h in the brucella broth substratum.Collect culture supernatant, obtain toxin by ultrafiltration and chromatographic separation, added complete freund adjuvant by weight 1: 1, emulsification gets HP toxin antigen.
Embodiment 3: make up the expression plasmid that contains HP cytotoxin structure gene VacA or CagA, in the intestinal bacteria system, express, expression product is through separation and purification, preparation HP vacuolating cytotoxin (VacA) and cell toxicant toxin (CagA), mixed as immunizing antigen by weight 1: 1, add isopyknic freund adjuvant, the emulsification mixing gets HP toxin antigen.
Embodiment 4: in the following way separation and Extraction anti--HPTxIgY: from immune ovum gallinaceum, separate yolk, with distilled water diluting, adjust suitable pH value and leave standstill 8h for 4 ℃, the centrifuging and taking supernatant concentrates, lyophilize, is resisted-HPTxIgY.
Embodiment 5: separate yolk, with distilled water diluting, 4 ℃ leave standstill 12h after, be that the film of 200KD and 150KD filters respectively with the molecular weight, collect the proteic substance of 150KD-200KD, lyophilize is resisted-HPTxIgY.
Embodiment 6: the product of getting among the embodiment 1,4,5 carries out the IgY purity test: use the purity of the yolk antibody of PAGE electrophoresis detection purifying, only occur an electrophoresis band in the electrophoresis, be the yolk antibody of purification.
Embodiment 7: the product of getting among the embodiment 1,4,5 carries out the inspection of IgY concentration: that will extract does 5-10 dilution doubly with PBS, measures to calculate protein concentration with ultraviolet spectrophotometer then.
Protein concentration (mg/ml)=(1.45 * OD
280nm-0.74 * OD
260nm) * extension rate
Embodiment 8: get that any product carries out the IgY titration among the embodiment 1,4,5: adopt Ovshinsky double diffusion method to carry out the IgY titration, it is 1 that IgY tires: 640-1: 25600.
Embodiment 9: gather 50 on the immune egg of anti--HPTxIgY, separate and obtain 800ml yolk, thin up is to 5000ml, the centrifugal clear liquid 4000ml that obtains, the employing Hollow Fiber Ultrafiltration extracts, it is stand-by to be concentrated into 400ml at last, wherein contains essence and carries, and (refers to that promptly purity resists at the IgY more than 99.5%-HPTxIgY) 8 restrains.Isomaltose 10 grams are added the 5ml water dissolution, sterilized 30 minutes, auxiliary material and the 50ml ultrafiltrated of handling mixed for 100 ℃, lyophilize, 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 10: gather 100 on the immune egg of anti--HPTxIgY, obtain 1600ml yolk, add 2 times of PBS diluents, and add 3.5%PEG6000 and stir and left standstill 2 hours, the centrifugal clear liquid 3500ml that obtains, add again PEG6000 to final concentration be 12%, left standstill centrifugal collecting precipitation 2 hours.With 100ml dissolved in distilled water IgY precipitation, by the biofilter degerming, lyophilize is slightly carried anti--HPTxIgY with each conventional capsule dress 200mg and is carried out can, and products obtained therefrom is used to prevent HP to infect, human body instructions of taking every day 2-3 time, each 3 capsules.
Embodiment 11: anti--the external antagonism toxin of HPTxIgY biological activity test: anti--HPTxIgY ((30～90ug/ml) mix under aseptic condition at 1: 1 for 10ug～200ug/ml) and HP toxin.Behind 37 ℃ of 2h, get the 0.1ml supernatant and add in the Hela cell cultures, discovery IgY concentration can be blocked the toxic action of HP toxin to the Hela cell fully at 100～200ug/ml, and the activity of cell LDH (serum lactic dehydrogenase) does not increase; Thereafter serial dilution, its protection activity also lowers thereupon, and the microscope morphological observation also obtains identical result: normal Hela cell is adherent polymorphism growth, no particle in the cell, and refractive power is good.Behind the HP detoxifying function, cell is spherical to be become, and occurs many particles in the endochylema, and when concentration＞50ug/ml, the Hela cell is many with cracking, the obvious shrinkage of residual cells volume.Behind the antagonism toxin, the form of cell changes and alleviates.
Embodiment 12: anti--HPTxIgY observes in the body biologic activity: set up the model that rodent HP infects with reference to pertinent literature, test be divided into the treatment group (low dosage (10mg/Kg), middle dosage (25mg/Kg), high dosage (50mg/Kg) be anti--HPTxIgY), control group; Treat after 10,20,30 days, observe the cardinal principle situation of animal, pathology of gastric mucosa is checked, indexs such as rapid urease test and content of toxins.Found that: middle and high dosage group is after treatment 10 days, and the stomach mucous membrane content of toxins obviously descends, and the pathology of gastric mucosa of animal checks that index has clear improvement after 20 days, and content of toxins is negative.
Embodiment 13: anti--HPTxIgY observes with microbiotic combined utilization biologic activity: set up the model that rodent HP infects with reference to pertinent literature, test be divided into simple anti--HPTxIgY treatment group, merely microbiotic (Bismuth Potassium Citrate, clarithromycin, tinidazole) treatment group, unite group.Treat after 5,10,15 days, observe the gastric mucosa of animal pathological examination, indexs such as rapid urease test and content of toxins.Found that: unite group than two simple groups after treatment 5 days, the pathology of gastric mucosa of animal improvement, stomach mucous membrane content of toxins and HP quantity all have obvious decline; The stomach mucous membrane content of toxins of simple resisting-HPTxIgY treatment group is less than simple antibiotic therapy group.
Claims (4)
1, (resist-HPTxIgY), it is characterized in that: state anti--HPTxIg and press YSDS-PAGE electrophoresis (7.5% separation gel) detection, collection of illustrative plates presents single district band to a kind of anti-HP toxin chicken yolk antibody; Be blue through electrophoresis Coomassie brilliant blue R250 dyeing; High molecular band place clear single specific enzymes occurred and dyes band during immunity marking Western-blotting detected; Carrying out dual wavelength mensuration with ultraviolet spectrophotometer to calculate protein concentration (mg/ml) is: (1.45 * OD
280nm-0.74 * OD
260nm) * extension rate.
2, a kind of preparation hen egg yolk antibody for resisting pyloric helicobacterium cytotoxin, the method for promptly anti--HPTxIgY is characterized in that: adopt following processing step:
(1). preparation HP cytotoxin antigen:
1.. extraction method: separate to obtain toxogenic HP bacterial strain, coat on the brucella agar, 37 ℃ of little aerobic cultivations 72h, must pure strain after, enlarged culturing T2h in the brucella broth substratum; Collect culture supernatant, obtain toxin by ultrafiltration and chromatographic separation, added complete freund adjuvant by weight 1: 1, emulsification gets HP toxin antigen;
Or 2.. synthesis method: make up the expression plasmid that contains HP cytotoxin structure gene VacA or CagA, in the intestinal bacteria system, express, expression product is through separation and purification, preparation HP vacuolating cytotoxin (VacA) and cell toxicant toxin (CagA), mixed as immunizing antigen by weight 1: 1, add isopyknic freund adjuvant, the emulsification mixing gets HP toxin antigen;
(2). the HP toxin antigen with gained prepares antibody by following step again:
1.. to the immunity of the hen that lays eggs:
Select the hen that lays eggs (health) isolated rearing for use, carry out injection under the cock skin, make the toxin protein amount be equivalent to 10
10The CFU/ml bacterium produces, and later per two all booster immunizations once amount to immunity 8 times, and 4 ℃ store for future use;
2.. the extraction of yolk antibody
A. adopt water-soluble method to extract yolk antibody; From immune ovum gallinaceum, separate yolk, with distilled water diluting, adjust suitable pH value and leave standstill 8h for 4 ℃, the centrifuging and taking supernatant concentrates, lyophilize, is resisted-HPTxIgY;
Or b. adopts membrane filter method to extract yolk antibody: separate yolk, with distilled water diluting, 4 ℃ leave standstill 12h after, be that the film of 200KD and 150KD filters respectively with the molecular weight, collect the proteic substance of 150KD-200KD, lyophilize is resisted-HPTxIgY.
3, hen egg yolk antibody for resisting pyloric helicobacterium cytotoxin, promptly anti--HPTxIgY is used for the treatment of the relative disease medicine that causes because of helicobacter pylori infection or the application in the healthcare products in preparation.
4, hen egg yolk antibody for resisting pyloric helicobacterium cytotoxin, promptly anti--HPTxIgY are used for detecting the application of the preparation of the relative disease that causes because of helicobacter pylori infection in preparation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 01129140 CN1354187A (en) | 2001-12-02 | 2001-12-02 | Hen egg yolk antibody for resisting pyloric helicobacterium cytotoxin, its preparation and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 01129140 CN1354187A (en) | 2001-12-02 | 2001-12-02 | Hen egg yolk antibody for resisting pyloric helicobacterium cytotoxin, its preparation and application |
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Cited By (7)
| 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 |
| CN101838322A (en) * | 2009-03-18 | 2010-09-22 | 中国医学科学院基础医学研究所 | Malaria recombinant antigen, IgY immune body and malaria detection kit |
| CN109251247A (en) * | 2018-08-31 | 2019-01-22 | 菏泽睿智科技开发有限公司 | A kind of extracting method of helicobacter pylori Yolk antibody |
| CN110467673A (en) * | 2018-05-10 | 2019-11-19 | 北京万华生物工程有限公司 | It is a kind of remove gastrointestinal tract helicobacter pylori substance and application |
| CN111228481A (en) * | 2018-11-28 | 2020-06-05 | 万华普曼生物工程有限公司 | Chicken IgY bifunctional antibody for treating helicobacter pylori |
| CN113166234A (en) * | 2019-11-18 | 2021-07-23 | （株）Ad生物技术 | Preparation method of anti-Helicobacter pylori egg yolk antibody |
| CN116854814A (en) * | 2023-07-05 | 2023-10-10 | 江苏润洁生物科技有限公司 | Preparation method and application of helicobacter pylori egg yolk antibody |
-
2001
- 2001-12-02 CN CN 01129140 patent/CN1354187A/en active Pending
Cited By (8)
| 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 |
| CN101838322A (en) * | 2009-03-18 | 2010-09-22 | 中国医学科学院基础医学研究所 | Malaria recombinant antigen, IgY immune body and malaria detection kit |
| CN110467673A (en) * | 2018-05-10 | 2019-11-19 | 北京万华生物工程有限公司 | It is a kind of remove gastrointestinal tract helicobacter pylori substance and application |
| CN109251247A (en) * | 2018-08-31 | 2019-01-22 | 菏泽睿智科技开发有限公司 | A kind of extracting method of helicobacter pylori Yolk antibody |
| CN111228481A (en) * | 2018-11-28 | 2020-06-05 | 万华普曼生物工程有限公司 | Chicken IgY bifunctional antibody for treating helicobacter pylori |
| CN113166234A (en) * | 2019-11-18 | 2021-07-23 | （株）Ad生物技术 | Preparation method of anti-Helicobacter pylori egg yolk antibody |
| CN116854814A (en) * | 2023-07-05 | 2023-10-10 | 江苏润洁生物科技有限公司 | Preparation method and application of helicobacter pylori egg yolk antibody |
| CN116854814B (en) * | 2023-07-05 | 2024-03-26 | 江苏润洁生物科技有限公司 | Preparation method and application of helicobacter pylori egg yolk antibody |
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