CN109400705A - A kind of anti-helicobacter pylori duck Yolk antibody and preparation method thereof - Google Patents
A kind of anti-helicobacter pylori duck Yolk antibody and preparation method thereof Download PDFInfo
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- CN109400705A CN109400705A CN201811475967.XA CN201811475967A CN109400705A CN 109400705 A CN109400705 A CN 109400705A CN 201811475967 A CN201811475967 A CN 201811475967A CN 109400705 A CN109400705 A CN 109400705A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
- C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
- C07K16/121—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Helicobacter (Campylobacter) (G)
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/02—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from eggs
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
- C07K2317/11—Immunoglobulins specific features characterized by their source of isolation or production isolated from eggs
Abstract
The invention discloses anti-helicobacter pylori duck Yolk antibodies and preparation method thereof.It is that laying duck is immunized in antigen that the anti-helicobacter pylori duck Yolk antibody, which is with helicobacter pylori, collects the duck's egg that laying duck is immunized, obtains after isolating and purifying to duck's egg yolk.Anti-helicobacter pylori duck Yolk antibody high specificity of the invention, purity is high, and compared with the anti-helicobacter pylori IgY of egg compared to having stronger inhibiting effect to helicobacter pylori, Antibody preparation is at low cost, convenient simple simultaneously, suitable for large-scale production, it can be used for by the prevention and treatment of the microbial disease of helicobacter pylorus, and exploitation helicobacter pylori detection kit immunological response and diagnosis.
Description
Technical field
The invention belongs to biomedicine fields, and in particular to anti-helicobacter pylori duck Yolk antibody and preparation method thereof.
Background technique
Helicobacter pylori (Helicobacter pylori, Hp) is Gram-negative bacteria, wherein lipopolysaccharides, flagellum and urine
Plain acid (urease) is most important to the existence of Hp, and Hp penetrates thick-layer mucus by flagellum, by ingredients such as lipopolysaccharides and pili
It is adhered on the epithelial cell of gastric mucosa layer, the memebrane protein of Hp has also assisted in and the adherency of epithelial cell, this is the pathogenic weight of Hp
Want factor.Hp is survived in stomach and duodenal each region, the chronic inflammation that stomach lining can be caused slight, even lead to stomach
And duodenal ulcer and gastric cancer.Since thallus can secrete strong urea acid (urease), urea is promptly resolved into two
Carbonoxide and ammonia are to neutralize gastric acid, therefore thallus can survive in acid stomach.
Hp is a kind of allelic heterogeneity microorganism, can secrete a variety of virulence factors.Including urease, adhesion factor, phosphatide
Enzyme A, hemolysin, CagA+ Hp (Cag A), vacuolate cytotoxin (Vac A) etc..Cag A and Vac A are two
Important pathogenic virulence factor, wherein Cag A can destroy the close connection between cell and cell, destroy barrier cell, make
CagA is injected host cell by more bacteriums, destroys stomach lining immediately, is flowed into gastric acid in the duodenum of alkaline environment and is made
At duodenal ulcer.Vac A is present in all bacterial strains, may act on cell, mitochondria, causes releasing for Cytochrome P450
It puts, can be discharged with mast cell zygotic induction inflammatory factor, aggravate tissue damage, cause inflammatory reaction, it can also be in epithelial cell
Film forms chosen anion channel, promotes HCO3Outflow reduces gastric acid secretion.
The whole world is more than that 50% population infection has a Hp, the height and social and economic level of disease incidence, densely populated degree,
Public health condition and water source supply have compared with close relationship.Its main circulation way has " excrement-mouth " to propagate (fecal-
Oral) and " mouth-mouth " propagate (oral-oral), in " excrement-mouth " route of transmission, Hp with the update of gastric epithelial and
It falls off, successively passes through Stomach duodenum, small intestine and large intestine, be finally discharged from excrement, eaten the food of faecal contamination,
Then Hp in may be infected.In " mouth-mouth " route of transmission, Hp is fallen in gastric juice, by gastroesophageal reflux, enters oral cavity
In, to be attached to oral cavity, especially on plaque, propagated finally by saliva.Since the special eating habit of China makes Hp
Infection be particularly acute in China, and have apparent Season Distributing Regularity, using 7~August part as peak.In Asia, in
The infection rate difference 60%, 50%, 40%, 70% of the juvenile helicobacter pylori such as domestic ground, Hong-Kong, Vietnam, India.
Common anti-Hp drug has clarithromycin, tetracycline, fortimicin, furazolidone, organic colloidal both at home and abroad at present
Bismuth agent, stomach obtain happy (LE Wei Tab) etc., and the course for the treatment of is generally fortnight, and this therapeutic modality makes the anti-medicine of patient Chang Yin helicobacter pylori
The problem of property, can not eradicate helicobacter pylori and recurrent exerbation completely.
Yolk antibody (IgY) is to use the birds that lay eggs as bioreactor to prepare antibody, low in cost, than with rabbit,
The animals such as sheep, horse, ox prepare antibody with apparent superiority.The immunoglobulin (Ig) of birds and the immune ball of mammal
Albumen is also very different on immunological characteristic.The immune system of birds can identify more immune response sites and more
Antigenic determinant, therefore antibody caused by albumen or biomolecule of the birds immune system to mammal has higher potency
And affinity.
Yolk antibody is a kind of antibody resource with very big potentiality to be exploited, but is concentrated mainly on chicken yolk antibody at home
Production and application, the production cost of duck is cheaper compared with chicken.Some researches show that one can produce for adult duck 1 year
250 eggs, each egg can isolate the antibody of 75~120mg.But at present with laying duck immune body production duck Yolk antibody
Research is also seldom, does not report by the duck Yolk antibody of antigen and preparation method thereof correlative study of helicobacter pylori more.
Using Antybody therapy helicobacter pylori infections compared to traditional treatment method with safe and efficient, specificity is strong
The characteristics of.Duck Yolk antibody can by intervening the sticking of bacterium, virulence, locomitivity block the cell-ECM of helicobacter pylori
Between propagate and its attachment on epithelial cell, achieve the purpose that eradicate helicobacter pylori.
The present invention is directed to be specifically bound by the anti-helicobacter pylori duck Yolk antibody of specificity and helicobacter pylori, suppression
The adhesive ability of helicobacter pylori processed blocks its intercellular propagation, prevents the effects of discharging virulence factor downstream, finally reaches
To the purpose for the treatment of helicobacter pylori infections.
Summary of the invention
The purpose of the present invention is to provide anti-helicobacter pylori duck Yolk antibodies and preparation method thereof.Anti- pylorus of the invention
Helicobacter duck Yolk antibody potency height, high specificity, purity is high, and have stronger inhibiting effect to helicobacter pylori, resist simultaneously
Body prepares at low cost, convenience simply, is suitable for large-scale production, can be used for the prevention by the microbial disease of helicobacter pylorus and control
It treats, and exploitation helicobacter pylori detection kit immunological response and diagnosis.
The first aspect of the present invention provides a kind of anti-helicobacter pylori duck Yolk antibody, which derives from
It is that laying duck is immunized in antigen that duck's egg yolk, which is with Helicobacter pylori Strains, collects the duck's egg yolk institute that laying duck is immunized
Obtained IgY includes the deficiency IgY molecule of defect Fc heavy chain, and duck egg yolk IgY is different from chicken with yolk IgY, and duck egg yolk IgY is wrapped
The deficiency IgY molecular proportion chicken with yolk IgY molecule contained is small, it is easier to adhere to and enter gastric mucosa, the antibody titer of duck egg yolk IgY
For 1:640000~1:1280000,2 times or more higher than the antibody titer of chicken with yolk IgY, duck yolk antibody IgY can replace chicken
Yolk antibody IgY.
A kind of anti-helicobacter pylori duck yolk antibody IgY is provided as a preferred technical solution, the anti-helicobacter pylori duck
It is that laying duck is immunized in antigen that yolk antibody IgY, which is with Helicobacter pylori Strains, the duck's egg that laying duck is immunized is collected, to duck
Egg yolk carries out resulting IgY after purification.The antibody titer of the anti-helicobacter pylori duck yolk antibody IgY be 1:640000~
1:1280000, can reach 1:1280000, and the chicken yolk antibody being prepared under identical immunizing dose and immunization method
The antibody titer of IgY is up to 1:640000.
In the above-mentioned technical solutions, the laying duck is that the health of 20 week old is laid eggs duck.
In the above-mentioned technical solutions, the Helicobacter pylori Strains are by helicobacter pylori ATCC11637, helicobacter pylorus
Any 2 kinds or 3 kinds in the helicobacter pylori of bacterium ATCC26695 and CagA gene knockout are mixed to get.Mixed proportion be by
According to 1~2:1 of bacterium number~2:1~2, preferably 1:1:1.The helicobacter pylori of the CagA gene knockout can be normal according to this field
The method of the gene knockout of rule is prepared, and the helicobacter pylori for gene knockout can be that can be bought by commercial sources
One of obtained helicobacter pylori.When above-mentioned helicobacter pylori is used in combination, the helicobacter pylori of CagA gene knockout is excellent
Select the helicobacter pylori other than helicobacter pylori ATCC 11637, helicobacter pylori ATCC 26695.
In the above-mentioned technical solutions, after Helicobacter pylori Strains ultrasonic treatment forms cell fragment, as anti-
Laying duck is immunized in original.
The second aspect of the present invention provides a kind of preparation method of above-mentioned yolk antibody for anti Helicobacter Pylori, including as follows
Step:
(1) preparation of antigen: helicobacter pylori is seeded on blood agar plate, after culture 3~4 days, collects thallus, dilute
Being interpreted into helicobacter pylorus bacteria concentration is 2 × 109The bacterium solution of cfu/mL, ultrasonic treatment, obtains helicobacter pylori bacterium solution；
(2) laying duck is immunized: the helicobacter pylori bacterium solution of step (1) is pressed with Freund's complete adjuvant or incomplete Freund's adjuvant
The volume ratio of 1:1 mixes, emulsification, Freund's complete adjuvant emulsification vaccine and incomplete Freund's adjuvant emulsification vaccine is made, to laying duck
It is immunized, is immunized altogether five times, one exempts to emulsify vaccine using Freund's complete adjuvant, and immunizing dose is 0.8-1.2mL/, in duck
Dorsal sc injection；One exempt from after 21d, 35d, 56d be changed to incomplete Freund's adjuvant emulsification vaccine carry out two exempting from respectively, three exempt from and
Four exempt from, and immunizing dose is 0.3-0.7mL/, in duck dorsal sc injection；After one exempts from, the helicobacter pylorus of step (1) 77d
Bacterium bacterium solution carries out five and exempts from, and immunizing dose is 0.3-0.7mL/, in the root intramuscular injection of duck leg；
(3) three exempt from the daily collection duck's egg of beginning in latter 1 week, collect duck yolk, and the purified anti-helicobacter pylori duck yolk that obtains resists
Body IgY.
In the above-mentioned technical solutions, in step (1), the helicobacter pylori bacterium solution can be prepared into following methods
Arrive: by helicobacter pylori, that is, helicobacter pylori ATCC11637, helicobacter pylori ATCC26695 and CagA gene knockout it is deep and remote
Any 2 kinds or 3 kinds in Helicobacter pylori are seeded on blood agar plate respectively, after culture 3~4 days, respectively collect thallus, respectively
Being diluted to helicobacter pylorus bacteria concentration is 2 × 109The bacterium solution of cfu/mL, then by each bacterium solution according to bacterium number ratio 1~2:1~2 (2
When kind) or 1~2:1~2:1~2 (at 3 kinds) mixing, obtain helicobacter pylori bacterium solution.
In the above-mentioned technical solutions, in step (1), the step of the described ultrasonic treatment are as follows: helicobacter pylorus will be diluted to
Bacteria concentration is 2 × 109The bacterium solution of cfu/mL is placed in ice-water bath, ultrasound 20s, the interval 20s at power 200W, repeats 15~25
It is secondary, preferably 20 times.
In the above-mentioned technical solutions, in step (3), the purification step are as follows: in the duck yolk of collection, be added 2~3
After the oscillation of times volume PBS buffer solution, supernatant is collected in centrifugation, and PEG6000 is added to final concentration 8.5%, is stirred at room temperature, is centrifuged
Liquid is discarded supernatant, after PBS dissolution is added in precipitating, PEG6000 is added to final concentration 12%, is stirred at room temperature, centrifugation discards
Clear liquid after dissolution is resuspended with PBS in precipitating, is added saturated ammonium sulfate ammonium sulfate into solution final concentration of 33~35%, will precipitate
It is dissolved in the PBS buffer solution isometric with former duck yolk amount, dialyses 18~48 hours, the anti-pylorus spiral shell that must be purified through membrane filtration
Bacillus duck yolk antibody IgY.
In the above-mentioned technical solutions, in step (3), three exempt to start within latter 1 week daily collection duck's egg, collect duck yolk, add
After entering 2~3 times of volume PBS buffer solution oscillations, the supernatant through being collected by centrifugation detects antibody using enzyme linked immunosorbent assay
Potency chooses antibody titer in the duck's egg of 1:160000 or more and obtains yolk supernatant, then further purified, and obtains efficiently
The yolk antibody for anti Helicobacter Pylori of valence, antibody titer reach 1:1280000 or more.
The immune system of duck and its duck IgY of generation are different from chicken, and duck IgY includes the unexistent deficiency IgY of chicken IgY points
Son, is the deficiency IgY molecule of no Fc heavy chain, and molecular weight is small, it is easier to adhere to and enter gastric mucosa, moreover, duck IgY is to institute
Immune antigen reactivity is much stronger than chicken IgY.Under identical immunizing dose and immunization method, duck is than the antibody in the yolk of chicken
Potency is higher, the antibody titer of duck egg yolk IgY 2 times or more higher than the antibody titer of chicken with yolk IgY.Experimental result of the present invention is shown
The difference of duck IgY and chicken IgY, it was demonstrated that duck IgY to antigen reactivity be immunized far by force with chicken IgY.Furthermore Ovum Anas domestica yolk more than
Egg, IgY yield are much higher than egg yolk.Market price of the market price of duck also below chicken.Therefore, duck IgY product can replace chicken
The product of IgY, a kind of antibody resource with very big potentiality to be exploited.
The third aspect of the present invention provides above-mentioned yolk antibody for anti Helicobacter Pylori in preparation and prevents and treats helicobacter pylorus
Bacterium causes the application in the preparation of disease.Anti-helicobacter pylori duck Yolk antibody and helicobacter pylori specifically bind, and inhibit deep and remote
The adhesive ability of Helicobacter pylori blocks its intercellular propagation, prevents the effects of discharging virulence factor downstream, is finally reached and controls
Treat the purpose of helicobacter pylori infections.The preparation is drug, health care product and food additives.Specifically: by above-mentioned method
With ratio of adjuvant, different preparations, including liquid system is made by a certain percentage in the yolk antibody for anti Helicobacter Pylori being prepared
Agent, granular preparation, paste, tablet or capsule preparations etc. can be used for preparing the disease for preventing and treating Helicobacter pylori Infection
Drug, health care product, and the food additives of disease caused by anti-helicobacter pylori can be prevented and treated.The auxiliary material and ratio are refused
It is particularly limited to, this field customary adjuvant and ratio can be used.
The utility model has the advantages that
(1) yolk antibody for anti Helicobacter Pylori of the present invention is laid eggs duck by pylorospirobacillus immune and is obtained, and has spy
Anisotropic feature strong, potency is high has good effect for preventing and treating the microbial disease of helicobacter pylorus and immunology detection.
(2) the anti-helicobacter pylori duck Yolk antibody that high-titer can be obtained using method of the invention, identical immune
Under dosage and immunization method, the potency of obtained anti-helicobacter pylori duck IgY is up to 1:1280000, and chicken under the same conditions
The highest potency of anti-helicobacter pylori IgY is only 1:640000.
(3) there is safe and efficient, specificity compared to traditional treatment method using Antybody therapy helicobacter pylori infections
Strong feature.Duck Yolk antibody can by intervening the sticking of bacterium, virulence, locomitivity block the cell-of helicobacter pylori thin
Intercellular spread and its attachment on epithelial cell achieve the purpose that eradicate helicobacter pylori.
Detailed description of the invention
Fig. 1 shows chicken and the duck vitelligenous IgY potency comparison results of institute after pylorospirobacillus immune respectively.
Fig. 2 indicates the SDS-PAGE map of yolk antibody for anti Helicobacter Pylori after purification.Wherein swimming lane 1-2 is after purification
Yolk antibody, loading total protein concentration are respectively 20 μ g；Swimming lane 3 is albumen Marker.
Fig. 3 indicates 1*109Anti-helicobacter pylori duck IgY is added or be not added in the helicobacter pylori of CFU, is cultivated 24 hours
Culture solution clarity afterwards compares photo.
Fig. 4 indicates 1*109Anti-helicobacter pylori duck IgY is added and be not added in the helicobacter pylori of CFU, is cultivated 24 hours
Microscope photo afterwards.
Specific embodiment
Present invention will be further explained below with reference to specific examples.It should be understood that these examples be merely to illustrate the present invention and
It is not used in and limits application range of the invention.The test method of specific experiment condition is not specified in following example, usually according to
Condition described in normal condition or molecular cloning, or according to condition provided on product description.Here is used in example
Material, reagent etc. be commercially available unless otherwise specified.
The preparation of 1 yolk antibody for anti Helicobacter Pylori of embodiment
(1) preparation of Heliobacter pylori antigen
Respectively by 3 kinds of helicobacter pyloris (helicobacter pylori of ATCC 11637, ATCC 26695 and CagA gene knockout)
It is seeded on blood agar plate, is placed in 37 DEG C, cultivates under micro- aerobic environment, after cultivating 3 days, wash lower bacterium with sterilizing PBS respectively
It falls and receives bacterium, measure A600, determine cell concentration, bacterium solution it is dense to be obtained respectively with sterilizing PBS dilution by cell according to its cell concentration
Degree is 2 × 109The helicobacter pylori of the ATCC 11637 of cfu/mL, ATCC 26695 and CagA gene knockout, according to bacterium number 1:
1:1 mixing, then by power 200W ultrasound 20s, interval 20s be repeated 20 times, cell fragment must be contained by carrying out fracturing cell walls
Bacterium solution, that is, helicobacter pylori bacterium solution, as antigen；
(2) pylorospirobacillus immune duck
The Heliobacter pylori antigen of step (1) and Freund's complete adjuvant or incomplete Freund's adjuvant are pressed to the volume ratio of 1:1
Mixing and emulsifying, the Freund's complete adjuvant emulsification vaccine and incomplete Freund's adjuvant for obtaining Heliobacter pylori antigen respectively emulsify vaccine,
20 week old of immunity inoculation is laid eggs duck, and specific immunization method is as follows: one exempts to emulsify vaccine, immunizing dose using Freund's complete adjuvant
Only for 1ml/, in duck dorsal sc injection；One exempt from after 21d, 35d, 56d be changed to incomplete Freund's adjuvant emulsification vaccine respectively into
Row two is exempted from, three exempt to exempt from four, and immunizing dose is 0.5ml/, in duck dorsal sc injection；After one exempts from, 77d is changed to step (1)
Obtained helicobacter pylori bacterium solution carries out five and exempts from, and immunizing dose is 0.5ml/, in the root intramuscular injection of duck leg.
(3) duck Yolk antibody purifies
With reference to the method that Diana Pauly purifies egg yolk IgY in the PEG6000 that JOVE in 2011 is delivered, this method is being passed through
Allusion quotation PEG6000 has been purified on the basis of IgY and has been adjusted, and the working concentration of PEG6000 is by original in second step precipitation process
12% is reduced to 8.5%, effectively increases the purity of IgY.Consider that PEG6000 dialysis-effect may not be very well, with reference to Wang Jia
PEG can be removed by ammonium sulfate precipitation in political affairs version " DNA techniques handbook " page 70, then improve Diana Pauly's
Method increases 33% saturated ammonium sulphate IgY.
Specific purification step is as follows: breaking Ovum Anas domestica putamina, with Yolk separator careful separation duck yolk, guarantees duck vitellinae membrana not
Rupture.Duck yolk is rolled for several times in ordinary filter paper, albumen is absorbed to clean, duck yolk rupture of membranes collection duck yolk as far as possible.With 2
The 10mM PBS pH7.4 (similarly hereinafter) of times duck yolk volume dilutes duck yolk, is stirred at room temperature；It is heavy by final concentration 3.5%PEG6000
The duck yolk to form sediment after diluting is centrifuged 10min (similarly hereinafter) in the case where 4 DEG C of 10000rpm.Supernatant is collected after centrifugation, through ordinary filter paper mistake
It filters off and removes insoluble matter；PEG6000 is added to its final concentration 8.5%, 20min is stirred at room temperature, centrifugation discards supernatant, precipitating duck ovum
The PBS dissolution of corpus luteum product (~12ml) is added PEG6000 to its final concentration 12%, precipitates duck Yolk antibody, be stirred at room temperature
20min；Supernatant is abandoned in centrifugation, and precipitating is resuspended with 6~8ml PBS to be dissolved.33% saturated ammonium sulfate is added under the conditions of 4 DEG C, stirs
1h, centrifugation abandon supernatant, 1ml PBS are added in precipitating, dissolution is resuspended.Dissolved precipitating dialyses to PBS, and 20 hours or more, every 6
Replacement dialyzate is primary within~8 hours.The duck Yolk antibody by dialysis is collected, 20~100 times is diluted with PBS, measures wavelength
Absorbance under 280nm calculates IgY content (concentration=1.45 IgY × A280-0.74×A260, unit: mg/ml).
The anti-helicobacter pylori duck IgY of purified acquisition identifies purity through reproducibility SDS-PAGE, as a result such as Fig. 2.Fig. 2's
The results show that the anti-helicobacter pylori duck IgY purity that purifying obtains should be 90% or more.
(4) Elisa method detects IgY potency
In above-mentioned steps (2), third time is immunized (three exempt from) and starts within 1 week daily collection duck's egg, ELISA indirect Determination duck afterwards
Helicobacter pylorus bacteria specific antibodies IgY potency in egg yolk liquid, potency collect duck's egg up to 1:160000 or more is rear.If five exempt from rear potency
Decline lower than 1:160000 or titre, carries out booster immunization, booster immunization method and dosage with the 5th time immune (five exempt from), one week
After collect duck's egg.
The duck's egg of collection is carried out after purification using the method for above-mentioned (3), potency is detected with ELISA indirect method, for monitoring
Immunization schedule and the potency for determining antibody obtained.
Specific step is as follows for ELISA method detection IgY potency:
4.1 coated elisa plate
Helicobacter pylori proteantigen, the every hole the every 100ng of the package amount of antigen, every hole are diluted with carbonate buffer solution
100 μ L, 4 DEG C overnight.
4.2 washing
ELISA Plate is rinsed with PBST, is patted dry on blotting paper, cleaning solution is filled it up in hole and stands 1min, is patted dry on blotting paper,
It washs 3 times repeatedly.
4.3 addition measuring samples
Doubling dilution is carried out after sample is diluted 10,000 times to 640,000 times, by the sample after each doubling dilution, draws 100
The hole μ L/ is to ELISA Plate, 37 DEG C of incubation 2h.
4.4 washing
ELISA Plate is rinsed with PBST, is patted dry on blotting paper, cleaning solution is filled it up in hole and stands 1min, is patted dry on blotting paper,
It washs 5 times repeatedly.
4.5 secondary antibody
Goat-anti duck (or anti-chicken) enzyme labelled antibody is diluted to working concentration, every hole 100 μ L, 37 DEG C of incubation 1h with PBST.
4.6 washing
ELISA Plate is rinsed with PBST, is patted dry on blotting paper, cleaning solution is filled it up in hole and stands 1min, is patted dry on blotting paper,
It washs 5 times repeatedly.
4.7 colour developing
Take 4.86mL 0.1M citric acid solution, 5.14mL 0.2M Na2HPO4Solution, 0.5mL TMB solution, 32 μ L
0.75%H2O2Solution mixing, the every hole of ELISA Plate adds 100 μ L mixed liquors, is protected from light 10-15min.
4.8 terminating
2moL/L sulfuric acid solution is added in every hole, and 100/ hole terminates reaction.
4.9 result judgement
The OD value (wavelength 450nm) of microplate reader test sample, when sample OD value is 2.1 times or more of negative control OD value
Dilute the potency that multiple proportions is duck Yolk antibody.
In addition, laying hen (20 week old) is immunized according to the method for above-mentioned (1)~(3), anti-helicobacter pylori ovum gallinaceum is obtained
Yellow antibody carries out bioactivity, as the comparative example with duck Yolk antibody.
Fig. 1 be duck and chicken respectively after the above method is immunized compared with Yolk antibody potency obtained as a result, duck IgY potency
1:1280000 is reached as high as, chicken IgY potency reaches as high as 1:640000, in the entire immune period, in addition to six exempt from, duck IgY
There is higher potency than chicken IgY.In Fig. 1 six exempt from plus refer to March the 6th exempt from after time point after 3 months again.
The antibacterial experiment in vitro of 2 duck Yolk antibody of embodiment
By Hp strain inoculated in fluid nutrient medium (brucella broth), Hp bacterium solution is obtained in 37 DEG C of shake cultures, bacterium solution is dense
It spends to 2*109CFU/mL.Take anti-helicobacter pylori duck IgY (referred to as IgY) solution (11.5mg/mL) after purification through 0.22 μM
Membrane filtration is spare.Hp bacterium solution, the final concentration of 1*10 of bacterium solution is added by 1:1 volume ratio in IgY9CFU/mL, setting are negative
It compares (brucella broth) and is not added with IgY processing group, after culture 24 hours, observe liquid clarity (Fig. 3) and simultaneously removed from office respectively
Blue Albert'stain Albert is observed thalli morphology (Fig. 4).Fig. 3's the results show that add IgY compared with the processing group for being not added with IgY antibody
The processing group of antibody is more clarified, and illustrates that IgY of the invention has very strong anti-helicobacter pylori activity.Fig. 4's the results show that
Compared with the processing group for being not added with IgY, it is added to helicobacter pylori in the processing group of IgY antibody and ball-type change has occurred, illustrate IgY
The activity of helicobacter pylori is neutralized.
It is prepared by the freeze-dried powder of 3 duck yolk of embodiment
It collects 1 method of embodiment such as and obtained immune duck's egg is immunized, break Ovum Anas domestica putamina, with Yolk separator careful separation duck
Yolk guarantees that duck vitellinae membrana does not rupture.Duck yolk is rolled for several times in ordinary filter paper, albumen is absorbed to clean, duck ovum as far as possible
Yellow rupture of membranes collects duck yolk.Duck yolk liquid is laid in sterile tinfoil disk, so that its thickness is no more than 0.5cm, be put into -80 DEG C
Refrigerator freezing takes out tinfoil disk afterwards for 24 hours, is put into freeze dryer and is lyophilized, and 48h is lyophilized in 0.150mbar, and taking-up is ground into freeze-dried powder.
4 helicobacter pylori infections model therapeutic test of embodiment
By the duck yolk freeze-dried powder that must be fallen of embodiment 3, it is put in 4 DEG C of refrigerator cold-storages and saves, take a packet duck yolk daily
Freeze-dried powder (7g/ packet), takes 30 days as a treatment course altogether, takes within two hours after supper, takes rear fasting, cold water or normal-temperature water clothes
With alcohol prohibition during taking.C-13 or C-14 breath test result (respectively DOB value or DMP value) display, after treatment one month,
Patient shows negative (table 1) to helicobacter pylori.Table 1. is before and after treatment to the testing result of helicobacter pylori
Claims (8)
1. a kind of anti-helicobacter pylori duck yolk antibody IgY, which is characterized in that the duck yolk antibody IgY derives from duck's egg yolk,
Be with Helicobacter pylori Strains it is that laying duck is immunized in antigen, collects the obtained IgY of duck's egg yolk that laying duck is immunized
Deficiency IgY molecule comprising defect Fc heavy chain, duck egg yolk IgY are different from chicken with yolk IgY, the defect that duck egg yolk IgY is included
Type IgY molecular proportion chicken with yolk IgY molecule is small, it is easier to adhere to and enter gastric mucosa, the antibody titer of duck egg yolk IgY is 1:
640000~1:1280000,2 times or more higher than the antibody titer of chicken with yolk IgY, duck yolk antibody IgY can replace chicken with yolk
Antibody IgY.
2. a kind of anti-helicobacter pylori duck yolk antibody IgY, which is characterized in that the anti-helicobacter pylori duck yolk antibody IgY is
It is that laying duck is immunized in antigen with Helicobacter pylori Strains, collects the duck's egg that laying duck is immunized, duck's egg yolk is carried out pure
Resulting IgY after change.
3. anti-helicobacter pylori duck yolk antibody IgY according to claim 1 or 2, which is characterized in that the pylorus spiral shell
Bacillus strain is by the helicobacter pylorus of helicobacter pylori ATCC 11637, helicobacter pylori ATCC 26695 and CagA gene knockout
Bacterium is mixed to get according to 1~2:1 of bacterium number~2:1~2.
4. anti-helicobacter pylori duck yolk antibody IgY according to claim 1 or 2, which is characterized in that the pylorus spiral shell
After bacillus strain ultrasonic treatment forms cell fragment, laying duck is immunized as antigen.
5. anti-helicobacter pylori duck yolk antibody IgY according to claim 1 or 2, which is characterized in that the laying duck is
The health of 20 week old is laid eggs duck.
6. the preparation method of anti-helicobacter pylori duck yolk antibody IgY as claimed in claim 1 or 2, includes the following steps:
(1) preparation of antigen: helicobacter pylori is seeded on blood agar plate, after culture 3~4 days, is collected thallus, is diluted to
Helicobacter pylorus bacteria concentration is 2 × 109The bacterium solution of cfu/mL, ultrasonic treatment, obtains helicobacter pylori bacterium solution；
(2) laying duck is immunized: the helicobacter pylori bacterium solution of step (1) and Freund's complete adjuvant or incomplete Freund's adjuvant are pressed into 1:1
Volume ratio mixing, emulsification, be made Freund's complete adjuvant emulsification vaccine and incomplete Freund's adjuvant emulsification vaccine, to laying duck carry out
It is immune, it is immunized altogether five times, one exempts to emulsify vaccine using Freund's complete adjuvant, and immunizing dose is 0.8-1.2mL/, in duck back
Subcutaneous injection；One exempt from after 21d, 35d, 56d are changed to that incomplete Freund's adjuvant emulsification vaccine carries out two exempting from respectively, three exempt from and four
Exempt from, immunizing dose is 0.3-0.7mL/, in duck dorsal sc injection；After one exempts from, the helicobacter pylori of step (1) 77d
Bacterium solution carries out five and exempts from, and immunizing dose is 0.3-0.7mL/, in the root intramuscular injection of duck leg；
(3) three exempt to start within latter 1 week daily collection duck's egg, collect duck yolk, purified to obtain anti-helicobacter pylori duck Yolk antibody
IgY。
7. the preparation method of anti-helicobacter pylori duck yolk antibody IgY according to claim 6, which is characterized in that in step
Suddenly in (1), the step of the described ultrasonic treatment are as follows: will be diluted to helicobacter pylorus bacteria concentration is 2 × 109The bacterium solution of cfu/mL is placed in
In ice-water bath, ultrasound 20s, the interval 20s at power 200W is repeated 15~25 times.
8. the preparation method of anti-helicobacter pylori duck yolk antibody IgY according to claim 6, which is characterized in that in step
Suddenly in (3), the purification step are as follows: in the duck yolk of collection, after 2~3 times of volume PBS buffer solution oscillations are added, are centrifuged, receive
Collect supernatant, PEG6000 is added to final concentration 8.5%, is stirred at room temperature, centrifugation discards supernatant liquid, and PBS dissolution is added in precipitating
Afterwards, PEG6000 is added to final concentration 12%, is stirred at room temperature, centrifugation discards supernatant liquid, after dissolution is resuspended with PBS in precipitating, is added
Precipitating is dissolved in isometric with former duck yolk amount by saturated ammonium sulfate ammonium sulfate into solution final concentration of 33~35%, stirring
In PBS buffer solution, dialyse 18~48 hours, the anti-helicobacter pylori duck yolk antibody IgY that must be purified through membrane filtration.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111205367A (en) * | 2020-02-23 | 2020-05-29 | 珠海华敏医药科技有限公司 | Helicobacter pylori yolk antibody freeze-dried powder, preparation method and application preparation |
| 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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| CN1250056A (en) * | 1999-09-08 | 2000-04-12 | 中德联合研究院 | Pyloric bacillary spirillum resisting yolk immunoglobulin product and its application |
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| CN108794585A (en) * | 2018-05-28 | 2018-11-13 | 广东工业大学 | A kind of Yolk antibody preparation method and application of anti-helicobacter pylori |
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Cited By (3)
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| CN111205367A (en) * | 2020-02-23 | 2020-05-29 | 珠海华敏医药科技有限公司 | Helicobacter pylori yolk antibody freeze-dried powder, preparation method and application preparation |
| 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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