CN109503710A - Helicobacter pylori resistant Yolk antibody and the preparation method and application thereof - Google Patents
Helicobacter pylori resistant Yolk antibody and the preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a kind of helicobacter pylori resistant Yolk antibodies, it is by tri- kinds of albumen enriched Mixtures of Ure, VacA, CagA for separating from Hp lysate as immunogene, injecting immune is carried out to bird inlay, water dilution method, NaCl salting-out method and thiophilic affinity chromatography extraction purification antibody are then used from immunity eggs yolk.The Yolk antibody purity is high as obtained by method preparation has stronger neutralization helicobacter pylori activity, can be applied to enterogastric diseases drug caused by preparation prevention and treatment Helicobacter pylori infection.
Description
Technical field
The present invention relates to biological medicine technology, more particularly to a species specificity helicobacter pylori resistant Yolk antibody and its
Preparation method and application.
Background technique
Helicobacter pylori (Hp) is a kind of spiral shape, micro- anaerobism, the gram-negative very harsh to growth conditions requirement
Property bacillus.Nineteen eighty-three separates successfully from the Mucosa Biopsy tissue of chronic active gastritis patient for the first time, is currently known energy
Enough only microbe species survived in people's stomach.Helicobacter pylori disease includes being burst by the caused gastritis of Hp infection, alimentary canal
Ulcer, lymphoproliferative gastric lymphoma etc..The poor prognosis of helicobacter pylori disease is gastric cancer.Hp is colonized in mucosa tissue,
The gastric ulcer of 67%-80% and 95% duodenal ulcer are as caused by Hp.
Hp has infection in the crowd of the different races in the world, different regions, it may be said that is widest slow in adult
Property bacterial infection.Total trend is: Hp infection rate increases with the age and is risen, and developing country is about 80%, developed country
About 40%, male is slightly above women.The infection age in China, infection rate was within -40 years old 20 years old earlier than developed country 20 years or so
45.4%-63.6%, 70 years old or more up to 78.9%.In addition, the infection rate of the northern area of China is higher than southern area.
How Hp destroys gastric cells so that finally causing gastric cancer, generally believes 3 hatching eggs mainly expressed in vivo with it now
Bai Youguan is urease (Ure), vacuolating cytotoxin (VacA) and cytotoxin-associated gene A (CagA) respectively.Ure is spiral shell
A kind of enzyme that Bacillus is rich in, it can change the local strong acidic environment that Hp is grown in stomach, to the Pseudomonas in stomach
Parasitic to play an important role with pathogenic, the urea enzyme amount of Hp synthesis is very big, accounts for about the 5% of bacterial protein, almost all of Hp is thin
Bacterium contains urease.VacA full length gene 5961bp, open reading frame 2888bp, are present in the gene of all Hp bacterial strains
In group, expression product is the toxalbumin that epithelial cell can be made to generate vacuolar degeneration, and also referred to as cavitating toxin, VacA toxin is
The important virulence factor that Hp is generated.CagA overall length is that 3444~5925bp is differed, and molecular weight is Hp between 128kD-140kD
One of virulence mark, it is pathogenic and clinically relevant disease severity is related with Hp.
Currently, the method for clinical treatment Hp infection mainly has standard triple therapy or bismuth agent tetrad containing clarithromycin to treat
Method or lavo-ofloxacin triple therapy, but these conjoint therapies on the one hand it is costly bring biggish financial burden to patient,
Another aspect long-term administration can generate serious side effect to human body, and be easy to produce drug resistance, and patient is difficult to receive.Cause
This, exploitation can reduce expense and Small side effects have the product of good result again, seems very for preventing and treating Hp infection
It is important.As passive immunity method is gradually known, so that the Yolk antibody of specific helicobacter pylori resistant prevents and treats Hp
Infection becomes a kind of effective method.
Chicken yolk antibody (IgY), i.e. chicken generate corresponding specific antibody, and transport, store after specific antigen is immune
In yolk.The property of IgY and the IgG of mammal are similar, and molecular weight is about 180KD, by two light chains and two heavy chain groups
At isoelectric point is about 5.2.But compared with general mammal IgG, IgY have stronger heat-resisting, acidproof, anti-ionic strength and
Certain resistance to enzymic degradation ability.IgY is more stable in PH4.0-11.0, activity decline rapidly when PH3.0-3.5, and when PH12 is living
Property is also declined.IgY has higher resistance to pepsin, but very sensitive to trypsase.The ovum of specific pathogen bacterium
Yellow antibody can directly attach on the cell wall of pathogen, change the integrality of pathogenic cell, directly inhibit the growth of pathogen；
Yolk antibody can attach on the pili of bacterium, be allowed to that intestinal mucosa epithelial cell cannot be attached to；A part of Yolk antibody exists
Under the action of enteron aisle digestive ferment, it is degraded in combination with segment, these segments variable small peptide (Fab) part antibody-containing, these
Small peptide is easy to by intestinal absorption, into blood after pathogen can be prevented from sticking in conjunction with specific pathogen adhesion factor
Permissive cell and lose pathogenic, and the stable region (Fc) of IgY is partially left at enteral.
It therefore, can using the Yolk antibody of three kinds of protein immunization bird inlays preparation helicobacter pylori resistant of above-mentioned Hp
To prevent Hp from attaching to gastric mucosa and its toxic protein destruction parietal cell and grow it can not in stomach acidity environment, from
And it can prevent and treat and caused enterogastric diseases are infected by Hp.
In prior art implementation, certain main for passing through the bacterial strain total protein that cracking Hp is extracted or pronuclear recombination expression
Bird inlay is immunized according to certain immune programme in a peculiar albumen of Hp such as FlaA albumen, then collects egg, method is diluted with water
The Yolk antibody of the extraction purification helicobacter pylori resistant from egg with ammonium sulfate precipitation method or the PEG precipitation method, or directly pass through
Powdery yolk is made for food additives or functional health product in freeze-drying.
Problems and disadvantages of the existing technology are mainly that the helicobacter pylori resistant Yolk antibody of its preparation neutralizes Hp
Activity is weaker to reduce its effect for preventing and treating enterogastric diseases caused by Hp infects.The reason of causing this main problem master
It embodies both ways, the pathogenic Ingredients Active of Helicobacter pylori is weaker in the immunogene being on the one hand immunized for laying hen
Or abundance is lower smaller so as to cause the anti-Hp Yolk antibody affinity of immune generation, the on the other hand extraction purification ovum from egg
Purity is inadequate when yellow antibody, and be used to prevent and treat Hp infection the requirement of helicobacter pylori resistant Yolk antibody its with higher degree.
Summary of the invention
Goal of the invention: in view of the above-mentioned problems existing in the prior art, the application is living by improving the Hp specific pathogenetic factor
Property and abundance enhancing immune effect and improve purification process improve Yolk antibody purity method, obtain the ovum of anti-Hp a kind of
Yellow antibody, which, which has, relatively neutralizes by force Hp activity, can efficiently prevent and treat enterogastric diseases caused by Hp infects, and provide its preparation
Method and application.
Technical solution: a kind of preparation method of helicobacter pylori resistant Yolk antibody of the present invention, including following step
It is rapid:
(1) immunity eggs are collected: using the immune laying hen of laying eggs of Helicobacter pylori antigen, after being immunized
Collect egg, fresh-keeping preservation；
(2) water dilution method extracts Yolk antibody: the immunity eggs for taking step (1) to collect, and removal albumen is received under aseptic condition
Collect yolk, ultrapure water magnetic agitation is added, adjust PH to 5.0~5.2, continue magnetic agitation, is placed in -20 DEG C~-80 DEG C overnight
It solidifies, after the completion first 4 DEG C of 1~4h of defrosting, then room temperature is thawed to complete, supernatant, then the filtering of sterilized filter paper, receipts is collected by centrifugation
Collect filter liquor；
(3) NaCl salting-out method purifies: solid sodium chloride is added in the filter liquor collected under stirring to step (2),
PH to 4.0, magnetic agitation are adjusted after mixing evenly, and centrifugation is collected precipitating and dried；
(4) thiophilic affinitive layer purification: taking the precipitating buffer solution of step (3) collection, and loading elutes after filter membrane,
Eluent centrifugation is collected, membrane filtration is used after desalination and concentration, packing freeze-drying obtains the Yolk antibody of helicobacter pylori resistant.
In step (1), the Helicobacter pylori antigen be enrichment Hp specific antigen, mainly include Ure,
The helicobacter pylori of three kinds of albumen such as VacA, CagA cracks isolate.It is preferably BioReagents, article No. purchased from brand
For the Helicobacter pylori antigen of A144.
Under normal circumstances, immune to lay eggs laying hen 3~5 times in step (1), each immunization interval time is 14~30
It, each immunizing dose is 50~200 μ g, and immune end started to collect egg after 7 days.
, it is preferable to use Helicobacter pylori antigen is immune is laying eggs laying hen 3 times in the application step (1), the
When primary immunization, ultrasonic emulsification after Helicobacter pylori antigen is mixed with isometric Freund's complete adjuvant；Second
When immune, ultrasonic emulsification after Helicobacter pylori antigen is mixed with isometric incomplete Freund's adjuvant；Third time is exempted from
When epidemic disease, adjuvant is not used.
Further, it in step (1), is immunized the subcutaneous two o'clock of the nape of the neck and pectoralis major two o'clock is taken to infuse for the first time and for the second time
It penetrates, third time is immune to take chicken wings tail vein injection Helicobacter pylori antigen.
It further, is for the first time every 50~200 μ g immunogene of laying hen with second of immunizing dose in step (1),
0.2~0.5ml of volume injected；Third time immunizing dose be every 50~200 μ g immunogene of laying hen, volume injected 0.2~
0.5ml。
4 DEG C of the egg preservations that step (1) is collected.
In step (2), yolk: ultrapure water volume ratio is 1:7~10, and magnetic agitation 10min after ultrapure water is added.
In step (2), yolk solution PH to 5.0~5.2 is adjusted with HCl, sets uniform stirring 2h~8h on magnetic stirring apparatus,
Stirring rate is 100~200rpm/min.
In step (2), solidification is thawed again, is to reduce interference of the lipid to subsequent salt precipitation for abundant grease removal, improve
Yolk antibody purity.Centrifugal condition after defrosting: 13500g/min, 10 DEG C, 15min.
In step (3), the additional amount of sodium chloride is the 8.2%~9.4% of total volume (w/v), is then sufficiently stirred
10min。
In step (4), the precipitating buffer solution for taking step (3) to collect, loading is eluted after crossing 0.45 μm of filter membrane, is collected
Eluent is centrifuged, with 0.22 μm of membrane filtration after desalination and concentration.
In step (4), the buffer is specifically 50mM NaH2PO4, 0.5M Na2SO4, PH8.0.
In step (4), elution requirement is 50mM NaH2PO4, PH8.0, flow velocity is 2~5ml/min.
Above method preparation gained helicobacter pylori resistant Yolk antibody is also within the scope of the present invention.
Stomach and intestine caused by above-mentioned helicobacter pylori resistant Yolk antibody prevents and treats Helicobacter pylori infection in preparation
Application in tract disease drug is also within the scope of the present invention.
Further, anti-Hp Yolk antibody provided by the invention can neutralize the cell toxicant of Hp toxic protein induction in vitro
Property effect, Hp can also be neutralized in vivo to inhibit its growth, the antibody, which can be further processed, is processed into food addition
Agent or functional health product are applied to prevent and treat enterogastric diseases caused by Hp infects.
The utility model has the advantages that being compared to the prior art, the present invention uses tri- kinds of Ure, VacA, CagA separated from Hp lysate
Protein enrichment mixture carries out injecting immune as immunogene, to bird inlay, then dilute with water from immunity eggs yolk
Interpretation of the law, NaCl salting-out method and thiophilic affinity chromatography extraction purification antibody.The Yolk antibody purity is high as obtained by method preparation,
With stronger neutralization helicobacter pylori activity, for the subsequent stomach caused by preparation prevention and treatment Helicobacter pylori infection
Intestines problem drug research lays the foundation.
Detailed description of the invention
Fig. 1 is the anti-Hp Yolk antibody preparation method flow chart of the application；
Fig. 2 is second of laying hen immune rear 10th day antiserum titre in embodiment 1；
Fig. 3 is the anti-Hp Yolk antibody 10%SDS-PAGE electrophoretogram of embodiment 3；
Fig. 4 is the anti-Hp Yolk antibody potency that embodiment 4 purifies；
Fig. 5 is the cytotoxicity result that in the anti-Hp Yolk antibody of the purifying of embodiment 5 and Hp toxic protein induces；
Fig. 6 is anti-Hp IgG potency result in mice serum after the anti-Hp Yolk antibody of embodiment 6 treatment Hp infecting mouse.
Specific embodiment
The application is explained in detail combined with specific embodiments below.
Material source:
Immunogene obtains: by consulting dependent merchandise product and comparative analysis, finally having selected brand is
BioReagents, the helicobacter pylori that article No. is A144 crack isolate.The product is using with independent intellectual property rights pure
Change method separation and concentration Hp specific antigen from H. pylori bacterial lysate, mainly three kinds including Ure, VacA, CagA etc.
Albumen.From domestic agency Nanjing Han Rui Biotechnology Co., Ltd buy this immunogene of 1mg, concentration 2.5mg/ml,
And dispensed, it is stored in -20 DEG C.
H.Pylori for stomach Helicobacter pylori infecting mouse model foundation is derived from duodenal ulcer patients body.
1 Immune Laying Hens of embodiment and antiserum titre measurement
3 healthy laying hen sub-cage rearings laid eggs are chosen, respectively number #1, #2, #3.By immunogene when first immunisation
It being sufficiently mixed with isometric Freund's complete adjuvant, ultrasonic emulsification is complete, and take the subcutaneous two o'clock of the nape of the neck and pectoralis major two o'clock to inject,
Immunizing dose is every 100 μ g immunogene of laying hen, volume injected 0.5ml.It is spaced and carries out within 30 days being immunized for second, it is this time immune
It is for Freund's complete adjuvant to be changed to incomplete Freund's adjuvant with first immunisation difference, immunizing dose and volume injected are the same as the first time
It is immune.It is immune that it is spaced the third time of progress in 30 days after immune for the second time, is this time immunized and does not use adjuvant, chicken wings tail vein is taken to infuse
It penetrates, immunizing dose is every 50 μ g immunogene of laying hen, volume injected 0.5ml.This time immune end started to collect chicken after 7 days
Egg writes affiliated laying hen number exactly in egg shell when collecting egg every time and raw egg date, egg is stored in 4 DEG C.
Second it is immune after the 10th day, every chicken is by chicken wings tail vein blood about 0.5ml, 4 DEG C of placement half an hour, so
10000rpm afterwards, 4 DEG C of centrifugation 10min, separation serum detect antiserum titre.
Indirect ELISA method is used when detecting antiserum titre, wherein indirect ELISA method is as follows: with immune primordial covering
ELISA Plate, 2 μ g/ml, 50 holes μ l/, 4 DEG C overnight.Next day inclines coating buffer, after the artificial board-washing of 0.05%PBST 3 times, is added
0.5%BSA closing, 200 holes μ l/, 37 DEG C, 1h.It discards confining liquid, after the artificial board-washing of 0.05%PBST 3 times, is added from 1:1000
Start above-mentioned antiserum totally 7 dilutions of doubling dilution, blank control PBS, 50 holes μ l/, 37 DEG C, 1h.0.05%PBST
After artificial board-washing 3 times, the rabbit-anti chicken IgY secondary antibody of the addition diluted horseradish peroxidase-labeled of 1:2000,50 holes μ l/, 37 DEG C,
1h.After the artificial board-washing of 0.05%PBST 5 times, chromogenic substrate TMB, 50 holes μ l/, room temperature, 10min is added.
OD450nm is read in microplate reader, as a result as shown in Fig. 2, wherein abscissa is antiserum extension rate, ordinate
It is OD450nm, baseline is 2.1 times of blank control wells OD value, and the above are the positives to be worth for baseline, and the following are feminine genders to be worth for baseline.As a result
The antiserum titre of display #1 and #2 laying hen reaches 1:4000 or more, and the antiserum titre of #3 laying hen reaches 1:16000 or more,
Illustrate that the application immunogen immune laying hen can generate the anti-Hp special yolk antibody of higher affinity.
The anti-Hp Yolk antibody of 2 extraction purification of embodiment
In conjunction with attached drawing 1 Yolk antibody preparation flow figure and embodiment 1 in immunity eggs collect, further using water it is dilute
Interpretation of the law, NaCl salting-out method and thiophilic affinity chromatography extraction purification antibody.
Preparation: experiment with all utensils (2,250ml beaker, 250ml glass reagent bottle 2,1,250ml graduated cylinder,
Yolk separator 1, glass bar 1, filter paper 2 opens, magnetic stir bar 1) and reagent (ultrapure water, PBS) mention the previous day high temperature
High pressure sterilization, for use.
Water dilution method extracts Yolk antibody: it is soaked in bromogeramine after washing with water egg surface, immersion 1~
3min, after taking-up is dried with gauze, then with 75% wipes of alcohol one time, naturally dry is ensured of aseptically removes as far as possible
Albumen.Yolk and albumen are separated using Yolk separator, yolk is placed in rolling in sterilizing filter paper and sops up residual albumen, punctures ovum
Yellow membrane makes yolk liquid stream enter to sterilize in beaker, measures (yolk volume about 15ml collected by a general egg).According to yolk:
Ultrapure water volume ratio is respectively that ultrapure water (generally about 105ml, 120ml, 135ml) dilution is added in 1:7,1:8,1:9, sets magnetic force and stirs
It mixes and 10min is sufficiently stirred on device.Yolk solution PH to 5.0 or 5.2 is adjusted with 1M HCl, sets uniform stirring on magnetic stirring apparatus
2h,4h,8h.Yolk solution is transferred in sterile 50ml centrifuge tube, -20 DEG C or -80 DEG C solidifications overnight are placed.It shifts within second day
To 4 DEG C of defrostings 1h or 4h, transfers to room temperature and wait for thawing completely.It is centrifuged (13500g/min, 10 DEG C, 15min), supernatant is turned
It moves to filtering in sterilizing filter paper and discards precipitating, filter liquor is spare into sterilizing glass reagent bottle.
NaCl salting-out method purifying: measuring above-mentioned filtrate volume with graduated cylinder and pour into sterilizing beaker, past while stirring
Solid sodium chloride is slowly added in filter liquor, additional amount is the 8.8% or 9.4% of total volume (w/v), and 10min is sufficiently stirred.With
1M HCl adjusts yolk solution PH to 4.0, sets uniform stirring 2h on magnetic stirring apparatus.Mixed liquor is transferred to 50ml centrifuge tube
In, it is centrifuged (3700g, 10 DEG C, 20min), discards supernatant, centrifuge tube back-off is dried.
Thiophilic affinitive layer purification: above-mentioned all precipitating 50ml 50mM NaH are taken2PO4, 0.5M Na2SO4, PH8.0 is dilute
Dissolution is released, then with 0.45 μm of membrane filtration into sterilizing glass reagent bottle.According to thiophilic affinity chromatography prepacked column specification into
The thiophilic affinitive layer purification of row Yolk antibody.Eluent is collected into 15ml 100KD ultra-filtration centrifuge tube, centrifugation (4000rpm,
10 DEG C, 15min), during which changed clothes 2 times with 5ml PBS, make its desalination and concentration to about 2ml volume.With 0.22 μm of membrane filtration, divide
Dress freeze-drying, obtains the Yolk antibody of helicobacter pylori resistant.
Embodiment 3
The anti-Hp Yolk antibody concentration mensuration of 2 gained of embodiment and purity detecting
Anti- Hp Yolk antibody detects its A280 and A260 after purification, with ultramicrospectrophotometer, with the inspection of PBS blank
It surveys.As A280/A260 < 1.5, with Lowry-Kalokar formula: sample protein content (mg/ml)=1.45A280-
0.74A260 calculates Yolk antibody concentration, and a general egg can be with the above ovum of extraction purification 80mg according to method in embodiment 2
Yellow antibody.
The purity of above-mentioned antibody purification is detected using SDS-PAGE and dying method with coomassie brilliant blue, as a result such as Fig. 3
Shown, wherein M is Protein Marker, when swimming lane 1,2 is respectively from #3 laying hen in 2018/8/18 and 201,8/8,/11 two
Between put the Yolk antibody of extraction purification in the egg given birth to denaturation reduction electrophoretic band, heavy chain about 70KD, light chain about 25KD, swimming
Road 3,4 is to be denaturalized non-reduced electrophoretic band.Show to can achieve according to the Yolk antibody purity of method extraction purification in embodiment 2
95% or more, fully meet the purity requirement of the helicobacter pylori resistant Yolk antibody for preventing and treating Hp infection.
The anti-Hp Yolk antibody titration of embodiment 4
Anti- Hp Yolk antibody measures its potency using indirect ELISA method after purifying is quantitative.Indirect ELISA method is such as
Under: immunogene coated elisa plate is used, 2 μ g/ml, 50 holes μ l/, 4 DEG C are overnight.Next day inclines coating buffer, artificial with 0.05%PBST
After board-washing 3 times, 0.5%BSA is added and closes, 200 holes μ l/, 37 DEG C, 1h.Discard confining liquid, the artificial board-washing of 0.05%PBST 3 times
Afterwards, it is added the above-mentioned antibody purification of the doubling dilution since the 100 μ g/ml totally 7 concentration, blank control PBS, 50 holes μ l/, 37
DEG C, 1h.After the artificial board-washing of 0.05%PBST 3 times, the rabbit-anti chicken IgY bis- of the diluted horseradish peroxidase-labeled of 1:2000 is added
It is anti-, 50 holes μ l/, 37 DEG C, 1h.After the artificial board-washing of 0.05%PBST 5 times, addition chromogenic substrate TMB, 50 holes μ l/, room temperature,
10min。
OD450nm is read in microplate reader, as a result as shown in figure 4, from #3 laying hen in 2018/8/18 and 2018/8/11 liang
The potency of the anti-Hp Yolk antibody of extraction purification in the egg that a time point gives birth to, wherein abscissa is that the anti-Hp yolk of purifying is anti-
Bulk concentration, ordinate are OD450nm, and baseline is 2.1 times of blank control wells OD value, and the above are the positives to be worth for baseline, below baseline
For feminine gender value.Show that the potency of anti-Hp Yolk antibody can achieve 5 μ g/ml or more.
The cytotoxicity experiment induced in the anti-Hp Yolk antibody of embodiment 5 with Hp toxic protein
Experiments have shown that Hp toxic protein such as VacA, CagA can inhibit cell growth with inducing cytotoxic, it is anti-in order to verify
Whether Hp Yolk antibody has in vitro neutralizes the effect of these toxic proteins to make cell restore normal proliferative growth, according to following
Experimental method carries out.
(1) (10000 cells/wells are careful not to generate gas 100 μ l HEK293T cell suspensions of inoculation in 96 orifice plates
Bubble) for 24 hours, while blank group and control group is arranged in preculture, 2 multiple holes of every group of setting；
(2) the anti-Hp Yolk antibody (respectively 0,5,10,20mg/ml) of various concentration and the immunogene of 100 μ g/ml are isometric
Mixing, 37 DEG C of reaction 2h are separately added into 10 μ l reaction solutions to every hole, are incubated for 72h in the incubator；
(3) to every hole be added 10 μ l CCK-8 (suggest along cell wooden partition addition, after mix gently, not generate bubble),
2h is incubated in incubator；
(4) light absorption value at microplate reader measurement 450nm.
(5) suppression percentage=[(B-A)/(B-C)] × 100% is calculated
A: experimental group light absorption value (for containing culture medium, cell, drug to be measured and CCK-8 light absorption value)
B: control group light absorption value (for containing culture medium, cell, CCK-8 light absorption value)
C: blank group light absorption value (for the light absorption value containing culture medium, CCK-8)
As a result as shown in figure 5, being mentioned in the egg that 2018/8/18 and 201,8/8,/11 two time point gives birth to from #3 laying hen
It takes in the anti-Hp Yolk antibody of purifying and the cytotoxicity experiment of Hp toxic protein induction is as a result, wherein abscissa is anti-Hp yolk
Antibody concentration, ordinate are inhibitory rate of cell growth, and inhibiting rate is that negative can consider entirely without inhibiting effect.The result shows that
The Hp toxic protein of 50 μ g/ml, which can grow HEK293T cell, generates 60% inhibiting rate, and the anti-of above-mentioned three kinds of concentration is added
Just this inhibiting effect can be reversed after Hp Yolk antibody, and the anti-Hp Yolk antibody of 5mg/ml concentration can make Hp toxic protein
The cytotoxic effect of induction completely disappears.Therefore, anti-Hp Yolk antibody has very strong neutralization Hp activity in vitro.
Bacteriostatic experiment in the anti-Hp Yolk antibody body of embodiment 6
(1) prepared by H. pylori strain
The H.Pylori bacterial strain (CagA+, VacA+) separated in duodenal ulcer patients body is selected, using Liquid Culture
Method increases bacterium: freezing bacterial strain for -70 DEG C and recovers on campylobacter jejuni agar plate, transferred species is in brain-heart infusion medium, micro- oxygen
37 DEG C of 60~72h of culture of condition take a small amount of culture solution smear, and ne ar is observed after Grain stain and detects urease, peroxide
Change hydrogen enzyme and oxidizing ferment.It is that bacterial concentration is adjusted to 1x10 after the positive9Cfu/mL is inoculated with 6~8 week old female immediately
Balb/c mouse.
(2) Hp infecting mouse model is established
Stomach-filling is inoculated with the Hp of fresh cultured again after NaHCO3 the solution 0.5mL, 2h of filling feeding 0.1M after mouse 12h fasting for solids and liquids
Bacterium solution 0.5mL continues 4h fasting for solids and liquids.It is repeated 5 times after stomach-filling, midfeather 48h, wherein latter 3 times do not fill NaHCO3.Last sense
Dye took 5 mouse after l0 days at random, and solution takes stomach, and being detected in stomach with helicobacter pylori pH indicator method diagnostic kit has
Hp field planting, and substance on the inside of stomach lining is taken to be cultivated and passed on, it detects the Hp growth that can stablize passage, illustrates modeling
Success.Model mice is grouped at random and starts to be administered in next day.
(3) anti-Hp Yolk antibody drug treatment Hp infecting mouse
Experimental animal is grouped as described below: 1. blank control group 1 (normal mouse gives physiological saline)；2. empty
White control group 2 (normal mouse gives 100mg/ml anti-Hp IgY)；3. model control group (infecting mouse gives physiological saline)；
4. positive controls (infecting mouse gives clarithromycin)；5. negative control group (infecting mouse gives negative Yolk antibody)；
6. anti-Hp IgY treatment group (infecting mouse gives 100mg/ml anti-Hp IgY)；7. (infecting mouse is given for anti-Hp IgY treatment group
Give the anti-Hp IgY of 50mg/ml)；8. anti-Hp IgY treatment group (infecting mouse gives 10mg/ml anti-Hp IgY)；It is every group 5 small
Mouse, administration route are stomach-filling.Daily stomach-filling is primary, is spaced 1 day, totally 3 times, sodium bicarbonate is filled before each stomach-filling first with middle stomach function regulating
Acid prevents Yolk antibody from inactivating, and detects anti-Hp in every mice serum using indirect ELISA method after 4 weeks after taking Yolk antibody
IgG potency.
Indirect ELISA method is as follows: using immunogene coated elisa plate, 2 μ g/ml, 50 holes μ l/, 4 DEG C overnight.Next day inclines
Coating buffer is added 0.5%BSA and closes after the artificial board-washing of 0.05%PBST 3 times, 200 holes μ l/, and 37 DEG C, 1h.Discard closing
After the artificial board-washing of 0.05%PBST 3 times, 1000 times of diluted mouse resisting anteserums are added in liquid, 50 holes μ l/, and 37 DEG C, 1h.0.05%
After the artificial board-washing of PBST 3 times, the goat anti-mouse IgG secondary antibody of the diluted horseradish peroxidase-labeled of 1:20000,50 μ are added
The hole l/, 37 DEG C, 1h.After the artificial board-washing of 0.05%PBST 5 times, chromogenic substrate TMB, 50 holes μ l/, room temperature, 10min is added.
OD450nm is read in microplate reader, as a result as shown in fig. 6, wherein abscissa is each experiment group name, ordinate is
OD450nm, each group OD450nm are 5 mouse average values.The results show that anti-Hp IgY treatment group includes high, normal, basic three dosage
Anti- Hp IgG level is substantially reduced in the mice serum of group, is substantially on close level, is said with Clarithromycin in Treating positive controls
Bright anti-Hp Yolk antibody has very strong neutralization Hp activity in vivo to inhibit Hp to grow in gastrointestinal tract.
Therefore, it can be further processed into food additives or functional health product using the anti-Hp Yolk antibody, be used for
Prevent and treat enterogastric diseases caused by Hp infects.
Claims (10)
1. a kind of preparation method of helicobacter pylori resistant Yolk antibody, which comprises the following steps:
(1) immunity eggs are collected: using the immune laying hen of laying eggs of Helicobacter pylori antigen, being collected after immune
Egg, fresh-keeping preservation；
(2) water dilution method extracts Yolk antibody: the immunity eggs for taking step (1) to collect, and removal albumen collects ovum under aseptic condition
Ultrapure water magnetic agitation is added in Huang, adjusts PH to 5.0~5.2, continues magnetic agitation, is placed in -20 DEG C~-80 DEG C solidifications overnight,
First 4 DEG C of 1~4h of defrosting after the completion, then room temperature are thawed completely, and supernatant is collected by centrifugation, and filter liquor is collected in sterilized filter paper filtering；
(3) NaCl salting-out method purifies: solid sodium chloride is added in the filter liquor collected under stirring to step (2), stirs
PH to 4.0, magnetic agitation are adjusted after uniformly, centrifugation is collected precipitating and dried；
(4) thiophilic affinitive layer purification: taking the precipitating buffer solution of step (3) collection, and loading elutes after filter membrane, collects
Eluent is centrifuged, and membrane filtration is used after desalination and concentration, and packing freeze-drying obtains the Yolk antibody of helicobacter pylori resistant.
2. the preparation method of helicobacter pylori resistant Yolk antibody according to claim 1, which is characterized in that step (1)
In, the Helicobacter pylori antigen is enrichment Hp specific antigen, deep and remote including tri- kinds of albumen of Ure, VacA, CagA
Door pylori cracks isolate.
3. the preparation method of helicobacter pylori resistant Yolk antibody according to claim 1, which is characterized in that step (1)
In, it is laying eggs laying hen 3 times using Helicobacter pylori antigen is immune, when immune for the first time, by helicobacter pylori
Ultrasonic emulsification after specific antigen is mixed with isometric Freund's complete adjuvant；It is when immune for the second time, helicobacter pylori is special
Ultrasonic emulsification after property antigen is mixed with isometric incomplete Freund's adjuvant；When third time is immune, adjuvant is not used.
4. the preparation method of helicobacter pylori resistant Yolk antibody according to claim 3, which is characterized in that step (1)
In, it is immunized the subcutaneous two o'clock of the nape of the neck and pectoralis major two o'clock is taken to inject for the first time and for the second time, third time is immune to take chicken wings tail
It is injected intravenously Helicobacter pylori antigen.
5. the preparation method of helicobacter pylori resistant Yolk antibody according to claim 3, which is characterized in that step (1)
In, it is for the first time every 50~200 μ g immunogene of laying hen, 0.2~0.5ml of volume injected with second of immunizing dose；For the third time
Immunizing dose is every 50~200 μ g immunogene of laying hen, 0.2~0.5ml of volume injected.
6. the preparation method of helicobacter pylori resistant Yolk antibody according to claim 1, which is characterized in that step (3)
In, the additional amount of sodium chloride is the 8.2%~9.4% of total volume.
7. the preparation method of helicobacter pylori resistant Yolk antibody according to claim 1, which is characterized in that step (4)
In, the buffer is 50mM NaH2PO4, 0.5M Na2SO4, PH8.0.
8. the preparation method of helicobacter pylori resistant Yolk antibody according to claim 1, which is characterized in that step (4)
In, elution requirement are as follows: 50mM NaH2PO4, PH8.0, flow velocity is 2~5ml/min.
9. any preparation method preparation gained helicobacter pylori resistant Yolk antibody in claim 1-8.
Draw 10. helicobacter pylori resistant Yolk antibody described in claim 9 prevents and treats Helicobacter pylori infection in preparation
The application in enterogastric diseases drug risen.
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110025782A (en) * | 2019-04-22 | 2019-07-19 | 深圳市喆邦生物工程有限公司 | A kind of Chinese medicine composition of helicobacter pylori resistant and preparation method thereof |
| CN111345473A (en) * | 2020-03-13 | 2020-06-30 | 珠海华敏医药科技有限公司 | Probiotics composition containing yolk antibody IgY and application preparation |
| CN111793137A (en) * | 2019-12-12 | 2020-10-20 | 南京蛋球球生物医学技术合伙企业(有限合伙) | Hp tetravalent antigen and preparation method and application thereof |
| CN112079915A (en) * | 2020-09-14 | 2020-12-15 | 四川昕泰科技有限公司 | Polypeptide and preparation method and application thereof |
| CN113166234A (en) * | 2019-11-18 | 2021-07-23 | （株）Ad生物技术 | Preparation method of yolk antibody for resisting helicobacter pylori |
| CN113845590A (en) * | 2021-10-19 | 2021-12-28 | 大连先锋生物科技有限公司 | Block chain-based helicobacter pylori egg yolk antibody and preparation method thereof |
| CN113975387A (en) * | 2021-10-18 | 2022-01-28 | 广西康众洋生物技术有限公司 | Preparation method of helicobacter pylori-resistant egg yolk antibody embedded gel particles |
| CN114874317A (en) * | 2022-03-30 | 2022-08-09 | 广西博生生物科技有限公司 | Preparation method of anti-helicobacter pylori specific yolk antibody |
| WO2023029666A1 (en) * | 2021-09-06 | 2023-03-09 | 尤丽康（江苏）生物医药有限公司 | Helicobacter pylori composite antigen, antibody, preparation method and use |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110025782A (en) * | 2019-04-22 | 2019-07-19 | 深圳市喆邦生物工程有限公司 | A kind of Chinese medicine composition of helicobacter pylori resistant and preparation method thereof |
| CN113166234A (en) * | 2019-11-18 | 2021-07-23 | （株）Ad生物技术 | Preparation method of yolk antibody for resisting helicobacter pylori |
| CN111793137A (en) * | 2019-12-12 | 2020-10-20 | 南京蛋球球生物医学技术合伙企业(有限合伙) | Hp tetravalent antigen and preparation method and application thereof |
| CN111345473A (en) * | 2020-03-13 | 2020-06-30 | 珠海华敏医药科技有限公司 | Probiotics composition containing yolk antibody IgY and application preparation |
| CN112079915A (en) * | 2020-09-14 | 2020-12-15 | 四川昕泰科技有限公司 | Polypeptide and preparation method and application thereof |
| WO2023029666A1 (en) * | 2021-09-06 | 2023-03-09 | 尤丽康（江苏）生物医药有限公司 | Helicobacter pylori composite antigen, antibody, preparation method and use |
| CN113975387A (en) * | 2021-10-18 | 2022-01-28 | 广西康众洋生物技术有限公司 | Preparation method of helicobacter pylori-resistant egg yolk antibody embedded gel particles |
| CN113845590A (en) * | 2021-10-19 | 2021-12-28 | 大连先锋生物科技有限公司 | Block chain-based helicobacter pylori egg yolk antibody and preparation method thereof |
| CN114874317A (en) * | 2022-03-30 | 2022-08-09 | 广西博生生物科技有限公司 | Preparation method of anti-helicobacter pylori specific yolk antibody |
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