CN108794585A - A kind of Yolk antibody preparation method and application of anti-helicobacter pylori - Google Patents
A kind of Yolk antibody preparation method and application of anti-helicobacter pylori Download PDFInfo
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- CN108794585A CN108794585A CN201810525606.5A CN201810525606A CN108794585A CN 108794585 A CN108794585 A CN 108794585A CN 201810525606 A CN201810525606 A CN 201810525606A CN 108794585 A CN108794585 A CN 108794585A
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- helicobacter pylori
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- yolk antibody
- asn
- antibody
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Abstract
The invention discloses applications of the helicobacter pylori HopQ in preparing yolk antibody for anti Helicobacter Pylori.The sequence SEQ ID NO of the helicobacter pylori HopQ:Shown in 1.The present invention uses helicobacter pylori HopQ as antigen, prepare the Yolk antibody of specific anti-helicobacter pylori, confrontation HopQ-CEACAM1 interactions and combination that can be specific, helicobacter pylori is prevented to be adsorbed onto on the epithelial cell of gastric mucosa, it reduces it to invade the natural immune system of body and induce corresponding infection, so as to effectively prevent the relevant disease of helicobacter pylori infections initiation.Meanwhile the Yolk antibody of the anti-helicobacter pylori can also be added in various drugs, health products and food, the infection for preventing helicobacter pylori；The detection kit as helicobacter pylori can also be prepared, for quickly, accurately detect helicobacter pylori.
Description
Technical field
The invention belongs to biotechnologies, and in particular to the prevention and treatment of helicobacter pylori infections more specifically relate to
And a kind of preparation method and applications of the Yolk antibody of anti-helicobacter pylori.
Background technology
Helicobacter pylori（Helicobacter pylori）It is scientist Barry Marshall and Robin Warren hairs
Existing, which can infect stomach tissue and cause ulcer, and this two researchers also obtain because being found that helicobacter pylori
Obtained Nobel's physiology and Medicine in 2005.
Helicobacter pylori is the virulence factor of gastric ulcer and gastric cancer.It is estimated that the world population of half infects pylorus spiral shell
Bacillus.Many the infecteds generate gastritis symptom, and in every 5 the infecteds, just have 1 people that may greatly suffer from gastric ulcer.?
Under many situations, this is a kind of quick fatal disease of disease progression.Although gastritis and gastric ulcer are subject to using antibiotic
Treatment, but usually require to use two to three kinds of different types of antibiotic, and be not all effective to all patients.If not
It is effectively treated, long-term helicobacter pylori inflammation eventually causes the generation of gastric cancer.And gastric cancer be a kind of height lethal,
It is difficult to the cancer cured.In the whole world, gastric cancer affects 1,000,000 people every year, has more than 700,000 people every year and dies of gastric cancer.Currently,
The main reason for gastric cancer generates is considered as helicobacter pylori（Helicobacter pylori）Infection, but lack effectively treatment
The method goods drug of gastric cancer；On the other hand, due to the continuous diffusion and enhancing of antibiotic resistance, the anti-main deformation of helicobacter pylori
It obtains more complicated.
Attachment to stomach lining is to establish the first step of bacteria planting, and being adhered to for helicobacter pylori surface is glutinous with people's stomach
Key effect is played in the combination of film.A few days ago, a publication is in the research report on international magazine Nature Microbiology
In, the researcher from mechanisms such as Technical University at Munich is found that a kind of new method to inhibit or treat pylorus by research
The infection or complication that helicobacter causes.In order to enduringly survive in mankind's stomach, helicobacter pylori must be adsorbed onto gastric mucosa
Epithelial cell on, in article researcher detect helicobacter pylori in epithelial cell adsorption capacity specificity obviously change
Become, i.e. the adhesion factor HopQ on helicobacter pylori surface can be attached to the relevant cell adherence of carcinomebryonic antigen point of stomach tissue
Son（CEACAMs）On；Researcher Bernhard B. Singer think, compare stick produced by the bacterium reported in the past because
For son, the structure that the combination found is not rely on carbohydrate is studied here, this seems to indicate that, helicobacter pylori exists
It can keep specifically stabilization, CEACAMs that can't occur in the stomach tissue of health in the acidic environment of stomach, but
When helicobacter pylori causes gastric inflammation, CEACAMs just will appear.HopQ-CEACAM1 interactions keep cytotoxin related
Gene A（CagA）Transposition become host cell, enhance the release of pro-inflammatory mediator, such as interleukin-8.It finds under study for action,
If helicobacter pylori lacks adhesion factor HopQ, helicobacter pylori cannot be made to be colonized in gastric epithelial cell, to further
Induce inflammatory reaction.This also just illustrates that adhesion factor HopQ is considerable to helicobacter pylori field planting gastric epithelial cell.If
Missing HopQ or HopQ can not bring into normal play effect, then can inhibit the activation for the Cag virulence accesses that HopQ mediates, such as hereditary or anti-
The HopQ functions that body mediates are shown, also can suppress a series of inflammation infection after following helicobacter pylori is infected.As it can be seen that
HopQ-CEACAM1 interactions are a kind of potential promising new therapy targets, for treating pyloric disease relevant disease.
Invention content
The purpose of the present invention is to provide applications of the helicobacter pylori HopQ in preparing yolk antibody for anti Helicobacter Pylori.
The helicobacter pylori HopQ genes play an important role during helicobacter pylori adheres to Gastric Mucosal Cells, this hair
The bright anti-helicobacter pylori yolk prepared as antigen using helicobacter pylori HopQ resists, and can be very good that helicobacter pylori is prevented to adhere to
In Gastric Mucosal Cells, you can reduce the infection of helicobacter pylori, reduce the probability that helicobacter pylori causes disease.
Another object of the present invention is to provide a kind of preparation methods of the Yolk antibody of anti-helicobacter pylori.
It is still another object of the present invention to provide a kind of Yolk antibodies of anti-helicobacter pylori.
It is still another object of the present invention to provide the applications of the Yolk antibody of the anti-helicobacter pylori.
It is still another object of the present invention to provide a kind of detection of Yolk antibody including anti-helicobacter pylori examinations
Agent box.
The above-mentioned purpose of the present invention is achieved by following scheme：
Applications of the helicobacter pylori HopQ (37-453) in preparing yolk antibody for anti Helicobacter Pylori, the helicobacter pylori
The sequence of HopQ such as SEQ ID NO:Shown in 1.
Whether helicobacter pylori HopQ can successfully be adhered on gastric mucosal cell with important helicobacter pylori
Influence, when helicobacter pylori can not be adhered to by HopQ on gastric mucosal cell, then helicobacter pylori cause infection it is several
Rate is decreased obviously, and the probability that helicobacter pylori infections cause relevant disease will be also substantially reduced.
The helicobacter pylori HopQ structures combined with CEACAMs are the beta hairpin knots in the extracellular 3+4 spirals binding domain of HopQ
Structure, gene order are 180-218.The present invention using 37-463 in HopQ sequence.It is used as by helicobacter pylori HopQ
The Cag virulence accesses that with the antibody of specific recognition HopQ, then can competitively inhibit HopQ mediations are prepared in antigen
Activation inhibits next a series of inflammation infection.
A kind of preparation method of the Yolk antibody of anti-helicobacter pylori, the method includes following processes：
S1. helicobacter pylori HopQ genetic fragments are first cloned, inducible protein on expression vector is then recombinated and expressed, and
Obtained albumen is detached and purified, HopQ recombinant proteins are obtained；The amino acid sequence such as SEQ of the HopQ recombinant proteins
ID NO:Shown in 2；
S2. laying poultry is immunized with HopQ recombinant proteins, then collects the produced egg of poultry after being immunized；
S3. it extracted from the yolk of the egg of collection, purify the antibody of anti-helicobacter pylori, and freezed, dry, obtain anti-pylorus
The Yolk antibody dry powder of helicobacter.
Laying poultry is immunized using helicobacter pylori HopQ, makes to generate antibody in laying poultry body, and produce yolk and include
There is the egg of the antibody of anti-helicobacter pylori.Laying poultry after absorbing helicobacter pylori HopQ antigens repeatedly, laying poultry body
Interior generation antibody there is the antibody of anti-helicobacter pylori HopQ in the yolk for the egg given birth to.The antibody can inhibit HopQ-
CEACAM1 is interacted and is combined, and helicobacter pylori is prevented to be adsorbed onto on the epithelial cell of gastric mucosa, is reduced it and is invaded body day
The right corresponding infection of immune system and induction, has preventive and therapeutic action for gastric ulcer and gastric cancer.
Preferably, the sequence of the helicobacter pylori HopQ such as SEQ ID NO:Shown in 1.
Preferably, when laying poultry being immunized in step S2, HopQ recombinant proteins, which can be equipped with adjuvant and inject, exempts from
Epidemic disease.
Preferably, when carrying out injecting immune to poultry, the dosage of per injection is 300~500ug/；Frequency injection is 3
~5 times；Per injection interval time is 2 weeks.
Preferably, when in the egg of collection helicobacter pylori antibody titre be less than 104When, laying poultry can be strengthened
It is immune；The laying poultry is laying hen.
The present invention also protects the Yolk antibody for the anti-helicobacter pylori that above-mentioned preparation method obtains, the helicobacter pylorus simultaneously
The sequence of bacterium HopQ such as SEQ ID NO:Shown in 1.
The Yolk antibody of the anti-helicobacter pylori is preparing the application in preventing helicobacter pylori infections vaccine also at this
In the protection domain of invention.
The Yolk antibody of the anti-helicobacter pylori as prevent helicobacter pylori infections additive application also this
In the protection domain of invention.
Preferably, the additive can add in drug, health and/functional food drug or food.
The present invention also protects a kind of kit of detection helicobacter pylori, and the kit includes the anti-helicobacter pylorus
The Yolk antibody of bacterium.
Compared with prior art, the invention has the advantages that：
（1）The present invention uses helicobacter pylori HopQ as antigen, prepares the Yolk antibody of specific anti-helicobacter pylori, can
It with the confrontation HopQ-CEACAM1 interactions of specificity and combines, helicobacter pylori is prevented to be adsorbed onto the epithelial cell of gastric mucosa
On, it reduces it and invades the natural immune system of body and induce corresponding infection.
（2）In order to solve the problems, such as the antigenic source needed for Yolk antibody large-scale production, genetic engineering and albumen are utilized
The technology of recombination prepares Heliobacter pylori antigen, easy to operate, at low cost.
（3）Yolk antibody for anti Helicobacter Pylori of the present invention can be added in various drugs, health products or food, be used for
Prevent the infection of helicobacter pylori；The detection kit as helicobacter pylori can also be prepared, for quickly, accurately examining
Survey helicobacter pylori.
Specific implementation mode
The present invention is made with reference to specific embodiment and further being elaborated, the embodiment is served only for explaining this
Invention, is not intended to limit the scope of the present invention.Test method used in following embodiments is normal unless otherwise specified
Rule method；Used material, reagent etc., unless otherwise specified, for the reagent and material commercially obtained.
Embodiment 1
1, the preparation of helicobacter pylori HopQ antigens
First using the 37-453bp genes of helicobacter pylori HopQ as template, annealing temperature is set as 50 DEG C of progress PCR amplifications.Recycling
PCR product is recycled through Kpn I and III digestions of Hind, is connected with the XL1-Blue expression vectors for also passing through double digestion processing
It connects, convert.Recombinant plasmid is identified according to a conventional method, recombinant plasmid is named as pQE-HopQ (37-453).It will identify just
True recombinant plasmid pQE-HopQ (37-453) conversions XL1-Blue expresses bacterium.Next day picking single bacterium colony is inoculated in 3mL and contains AMP
The LB liquid medium of (100ug/ml), in 37 DEG C, 200rpm shaken cultivations；After being incubated overnight, with the inoculative proportion of 0.5-5%
Access, fermentation condition temperature is 37 DEG C, speed of agitator 200rpm before induction, and ventilatory capacity 1vvm, Ph 6.0-8.0 induces it
When zymotic fluid OD value 0.6-0.8, temperature is 20 DEG C~30 DEG C, IPTG 0.1~1.0mM of concentration, speed of agitator 200rpm, ventilatory capacity
1vvm, pH 6.0-8.0 induce 5h, and thalline were collected by centrifugation.Sonicated cells.4 DEG C, 12000rpm centrifugation 10min, are collected
Supernatant.The purifying of recombinant protein is carried out with GE Healthcare company Ni-NTA resins specifications；And apply Bradford methods
Quantitative analysis is carried out to helicobacter pylori HopQ (37-453) albumen of purifying.
2, the preparation of anti-helicobacter pylori HopQ specific IgYs
（1）Laying hen is immunized
The helicobacter pylori HopQ antigens of above-mentioned preparation and isometric Freund's complete adjuvant or incomplete Freund's adjuvant are carried out
2-4h is emulsified to get microspheres vaccine.SPF grades of laying hens are immunized to multi-point injection under bilateral wing in the vaccine of preparation, just exempt from dosage
Only for 500ug/；It is primary to be spaced 2 weeks booster immunizations, immunizing dose is adjusted to 400ug/ only, and third time is immune to be started to receive in latter week
Collect egg, is immunized 3~5 times altogether；Yolk antibody titre is detected, hereafter when Yolk antibody titre is less than 104When can carry out strengthen exempt from
Epidemic disease, immunizing dose are 200ug/.By the methods of SDS-PAGE, Western blot and ELISA detect antibody purity,
After specificity and potency titre, the egg for reaching requirement is collected.
（2）The isolation and purification of antibody
Egg surface dirt to be cleaned with clear water, eggshell is wiped with 75% alcohol swab, broken shell detaches yolk with yolk separator,
Yolk is placed on neutral filter paper gently to roll until surface egg white thoroughly removes；Vitellinae membrana is punctured, its yolk liquid is taken, measures its body
Product；Distilled water is measured, PH=5.0-5.2 is adjusted, is proportionally added into distilled water 1:10（Yolk：Distilled water）；It is sufficiently stirred, 4 DEG C of mistakes
Night（16h）；Taken after the filtering of neutral filter paper filtrate centrifugation 1000g, 1h, 4 DEG C, above reset and add saturated ammonium sulfate and incubated for 4 DEG C to 50% saturation degree
1h is educated, 6000g centrifuges 30min, and precipitation is resuspended to original volume with distilled water and 19% sodium sulphate is added（190g/L）It is incubated under room temperature
20min.6000g centrifuges 20min, and precipitation is resuspended to original volume with distilled water and 14% sodium sulphate is added（140g/L）It is incubated under room temperature
20min, then centrifuge, pellet frozen is dried to form dry powder to get to the Yolk antibody dry powder of anti-helicobacter pylori, -20 DEG C are protected
It deposits spare.
The bacterial enzyme mark of 2 yolk antibody for anti Helicobacter Pylori of embodiment dyes
Take helicobacter pylori J99（ATCC700824）Make the dyeing of enzyme mark, smear and heat fixation.By anti-pylorus made from embodiment 1
The Yolk antibody of pylori helicobacter pylori is distributed into 3 concentration with the PBS that pH is 7.4,0.01mol/L（1:10,1:100,
1:1000）（mg：mL）It is added dropwise and is fully washed after 37 DEG C of effect 30min on slide.Secondary antibody is added dropwise（HRP enzyme mark rabbit-anti chickens IgY）
（Promega Corporation）DAB developing solutions and micro- sem observation is added dropwise, the IgY of not immune chicken is used in combination（Egg ovum before immune
Huang purifying）As negative control.The result shows that negative control is not colored, 3 concentration（1:10,1:100,1:1000）'s
Staining power is respectively +++, +++ ,+, illustrate anti-helicobacter pylori Yolk antibody can and helicobacter pylori combine, embodiment 1
The Yolk antibody of the anti-helicobacter pylori of preparation can be with specific recognition helicobacter pylori.
The titration of 3 yolk antibody for anti Helicobacter Pylori of embodiment
By the Yolk antibody of anti-helicobacter pylori made from embodiment 1 from 1：10 doubling dilutions are to 1：51200 detect its potency.
ELISA indirect enzyme-linked immunosorbents are tested, by the coatings of recombinant helicobacterpylori HopQ (37-453) made from embodiment 1
Liquid is diluted to 10 μ g/mL, and elisa plate is coated with 100 μ L per hole, covers 96 hole elisa Plates with sealer, then 37 DEG C of incubation 2h are dried
Liquid in hole is washed 1 time with PBST, and blotting paper pats dry；100 μ L 30mg/mL skimmed milk powers are added per hole to be closed, set 37
DEG C incubate 1h, then dry hole in liquid, washed 3 times with PBST（4 min of indwelling each time after washing）, patted dry with blotting paper；
Yolk antibody doubling dilution liquid is added after PBST board-washings（1:160~1:10240）, 100 holes μ L/ be added enzyme reaction plate in, most
PBS is added as blank control in a line afterwards, and 37 DEG C of incubation 1h are dried, washed 3 times with PBST（Indwelling 4min each time after washing）,
Blotting paper pats dry；L is added per hole:3000 rabbit-anti chicken IgY-HRP antibody（Promega Corporation）100 μ L, set 37
DEG C incubate 1h.Drying, is washed 3 times with PBST（Indwelling 4min each time after washing）, then patted dry with blotting paper；It is added per hole fresh
The substrate solution of preparation（10 3,3 ', 5,5 '-tetramethyl benzidines of mg（TMB）5mL absolute ethyl alcohols are dissolved in, it is dilute with 4.5mL distilled water
It releases, then adds 5mL phosphate citrate buffers, 10 μ L hydrogen peroxide are added before sample-adding）100 μ L set 37 DEG C of incubation 15min, then
The H of a concentration of 2 mol/L of 50 μ L is added per hole2SO4Terminate reaction.ELISA Plate is set into microplate reader 490nm strong points and surveys OD values.Extinction
It is positive criteria to spend OD values in control 2 times or more, measures antibody titer.The result shows that anti-helicobacter pylori prepared by the present invention
Yolk antibody, antibody titer is up to 1:10000 or more, show that HopQ has good antigenicity, be expected to become one it is new deep and remote
The Vaccine molecules of Helicobacter pylori；Show that yolk antibody for anti Helicobacter Pylori prepared by embodiment 1 has helicobacter pylori simultaneously
Good specificity.
4 anti-helicobacter pylori helicobacter pylori Yolk antibody pathogenic microorganisms of embodiment detects
According to《Chinese Pharmacopoeia》2015 editions " non-sterile product limit test of microbe of annex 1106：The inspection of Control bacteria examination method "
Method carries out Control bacteria examination to the Yolk antibody of anti-helicobacter pylori made from embodiment 1, bile tolerance Grain-negative is not detected
Bacterium, escherichia coli, detection of Salmonella, pseudomonas aeruginosa, staphylococcus aureus, clostridium and Candida albicans.
As a result prove that the Yolk antibody of anti-helicobacter pylori made from embodiment 1 is safe, does not carry other pathogens.
5 yolk antibody for anti Helicobacter Pylori avian flu virus detection of embodiment
The Yolk antibody of anti-helicobacter pylori made from embodiment 1 is configured to 1mg/mL solution with purified water, uses avian flu
Malicious gold-immunochromatographyreagent reagent for assay box is detected, and test result shows that avian influenza virus is negative.
The result shows that the Yolk antibody of anti-helicobacter pylori made from embodiment 1 is safe, vaccine can be prepared as
Or food additives, medicated premix are used.
A kind of Yoghourt containing the Yolk antibody for inhibiting helicobacter pylori of embodiment 6
（1）Prepare raw material：Primary raw material includes milk powder, xylitol and the Yolk antibody of helicobacter pylori；
（2）Prepare lotion：Milk powder 125g is weighed, 75 DEG C are added in milk powder, 1000mL warm waters are configured to lotion；
（3）Homogeneous：By step（2）In lotion on homogenizer homogeneous 2min, the pressure of homogenizer be maintained at 10MPa；
（4）Sterilizing：By step（3）Lotion after middle homogeneous keeps the temperature 10min in 90 DEG C of water-bath and carries out sterilization processing, then
40 DEG C are cooled to, feed liquid is obtained；
（5）Inoculation：Aseptically, the Yolk antibody 10mg that sterile inhibition helicobacter pylori is added is pressed separately by leavening
It is inoculated into feed liquid according to the ratio of 1g/1000mL, and is stirred for uniformly, obtaining inoculation feed liquid；
（6）Fermentation：Aseptically, inoculation feed liquid equivalent filling in the vial of sterilizing and is sealed, it then will sealing
Good vial is positioned in 42 DEG C of constant incubators the 5h that ferments；
（7）After ferment：By step（6）Vial after middle fermentation is put into 1 DEG C~4 DEG C of refrigerator to ferment after progress, rear to ferment
It at least keeps obtaining the Yoghourt containing the Yolk antibody for inhibiting helicobacter pylori for 24 hours.
Embodiment 7
The IgY of IgY yolk powder or 1g purifying containing anti-helicobacter pylori of the 10g containing anti-helicobacter pylori is added in per 100g milk powder
Freeze-dried powder, products obtained therefrom can be used for infant and adult prevents HP infection, or infect the auxiliary treatment of HP patient.
Embodiment 8
Be added purifying IgY freeze-dried powders of the 1g containing anti-helicobacter pylori in per 100ml milk, products obtained therefrom can be used for infant and at
People prevents HP infection, or has infected the auxiliary treatment of HP patient.
Embodiment 9
The purifying IgY freeze-dried powders of anti-helicobacter pylori press 1 with hericium erinaceum polysaccharide:5 ratios mix, and filling capsule can be used for protecting
The health food of stomach lining.
Embodiment 10
The purifying IgY freeze-dried powders of anti-helicobacter pylori add pharmaceutical adjunct, and oral solution, granule tablet or capsule is made, can use
In treatment diseases such as chronic gastritis, duodenal ulcer caused by HP infects；It can also be used for preventing the infection of HP.
The purifying IgY of 1 preparation-obtained anti-helicobacter pylori of embodiment is also used as additive in other drugs, guarantor
Application in strong sex pill and various other foods, to prevent the relevant disease of helicobacter pylori infections initiation.In addition also
The Yolk antibody of 1 preparation-obtained anti-helicobacter pylori of embodiment can be used to prepare to the detection kit of helicobacter pylori
In, for quickly detecting helicobacter pylori.
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than is protected to the present invention
The limitation of shield range can also be made on the basis of above description and thinking for those of ordinary skill in the art
Other various forms of variations or variation, there is no necessity and possibility to exhaust all the enbodiments.It is all the present invention
All any modification, equivalent and improvement etc., should be included in the protection of the claims in the present invention made by within spirit and principle
Within the scope of.
Sequence table
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<120>A kind of Yolk antibody preparation method and application of anti-helicobacter pylori
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aacaacctga acgagcgggc caagaccctg gccggcggca ccaccaactc ccccgcctac 300
caggccaccc tgctggccct gcggtccgtg ctgggcctgt ggaactccat gggctacgcc 360
gtgatctgcg gcggctacac caagtccccc ggcgagaaca accagaagga cttccactac 420
accgacgaga acggcaacgg caccaccatc aactgcggcg gctccaccaa ctccaacggc 480
acccactcct acaacggcac caacaccctg aaggccgaca agaacgtgtc cctgtccatc 540
gagcagtacg agaagatcca cgaggcctac cagatcctgt ccaaggccct gaagcaggcc 600
ggcctggccc ccctgaactc caagggcgag aagctggagg cccacgtgac cacctccaag 660
taccagcagg acaaccagac caagaccacc acctccgtga tcgacaccac caacgacgcc 720
cagaacctgc tgacccaggc ccagaccatc gtgaacaccc tgaaggacta ctgccccatc 780
ctgatcgcca agtcctcctc ctccaacggc ggcaccaaca acgccaacac cccctcctgg 840
cagaccgccg gcggcggcaa gaactcctgc gccaccttcg gcgccgagtt ctccgccgcc 900
tccgacatga tcaacaacgc ccagaagatc gtgcaggaga cccagcagct gtccgccaac 960
cagcccaaga acatcaccca gccccacaac ctgaacctga actccccctc ctccctgacc 1020
gccctggccc agaagatgct gaagaacgcc cagtcccagg ccgagatcct gaagctggcc 1080
aaccaggtgg agtccgactt caacaagctg tcctccggcc acctgaagga ctacatcggc 1140
aagtgcgacg cctccgccat ctcctccgcc aacatgacca tgcagaacca gaagaacaac 1200
tggggcaacg gctgcgccgg cgtggaggag acccagtccc tgctgaagac ctccgccgcc 1260
gacttcaaca accagacccc ccagatcaac caggcccaga acctggccaa caccctgatc 1320
caggagctgg gcaacaaccc cttccggaac atgggcatga tcgcctcctc caccaccaac 1380
aacggcgccc tgaacggcct gggcgtgcag gtgggctaca agcagttctt cggcgagaag 1440
aagcggtggg gcctgcggta ctacggcttc ttcgactaca accacgccta catcaagtcc 1500
aacttcttca actccgcctc cgacgtgtgg acctacggcg tgggctccga cctgctgttc 1560
aacttcatca acgacaagaa caccaacttc ctgggcaaga acaacaagat ctccttcggc 1620
ctgttcggcg gcatcgccct ggccggcacc tcctggctga actcccagtt cgtgaacctg 1680
aagaccatct ccaacgtgta ctccgccaag gtgaacaccg ccaacttcca gttcctgttc 1740
aacctgggcc tgcggaccaa cctggcccgg cccaagaaga aggactccca ccacgccgcc 1800
cagcacggca tggagctggg cgtgaagatc cccaccatca acaccaacta ctactccttc 1860
ctggacacca agctggagta ccggcggctg tactccgtgt acctgaacta cgtgttcgcc 1920
tac 1923
<210> 2
<211> 641
<212> PRT
<213>Helicobacter pylori (Helicobacter pylori)
<400> 2
Met Lys Lys Thr Lys Lys Thr Ile Leu Leu Ser Leu Thr Leu Ala Ala
1 5 10 15
Ser Leu Leu His Ala Glu Asp Asn Gly Val Phe Leu Ser Val Gly Tyr
20 25 30
Gln Ile Gly Glu Ala Val Gln Lys Val Lys Asn Ala Asp Lys Val Gln
35 40 45
Lys Leu Ser Asp Thr Tyr Glu Gln Leu Ser Arg Leu Leu Thr Asn Asp
50 55 60
Asn Gly Thr Asn Ser Lys Thr Ser Ala Gln Ala Ile Asn Gln Ala Val
65 70 75 80
Asn Asn Leu Asn Glu Arg Ala Lys Thr Leu Ala Gly Gly Thr Thr Asn
85 90 95
Ser Pro Ala Tyr Gln Ala Thr Leu Leu Ala Leu Arg Ser Val Leu Gly
100 105 110
Leu Trp Asn Ser Met Gly Tyr Ala Val Ile Cys Gly Gly Tyr Thr Lys
115 120 125
Ser Pro Gly Glu Asn Asn Gln Lys Asp Phe His Tyr Thr Asp Glu Asn
130 135 140
Gly Asn Gly Thr Thr Ile Asn Cys Gly Gly Ser Thr Asn Ser Asn Gly
145 150 155 160
Thr His Ser Tyr Asn Gly Thr Asn Thr Leu Lys Ala Asp Lys Asn Val
165 170 175
Ser Leu Ser Ile Glu Gln Tyr Glu Lys Ile His Glu Ala Tyr Gln Ile
180 185 190
Leu Ser Lys Ala Leu Lys Gln Ala Gly Leu Ala Pro Leu Asn Ser Lys
195 200 205
Gly Glu Lys Leu Glu Ala His Val Thr Thr Ser Lys Tyr Gln Gln Asp
210 215 220
Asn Gln Thr Lys Thr Thr Thr Ser Val Ile Asp Thr Thr Asn Asp Ala
225 230 235 240
Gln Asn Leu Leu Thr Gln Ala Gln Thr Ile Val Asn Thr Leu Lys Asp
245 250 255
Tyr Cys Pro Ile Leu Ile Ala Lys Ser Ser Ser Ser Asn Gly Gly Thr
260 265 270
Asn Asn Ala Asn Thr Pro Ser Trp Gln Thr Ala Gly Gly Gly Lys Asn
275 280 285
Ser Cys Ala Thr Phe Gly Ala Glu Phe Ser Ala Ala Ser Asp Met Ile
290 295 300
Asn Asn Ala Gln Lys Ile Val Gln Glu Thr Gln Gln Leu Ser Ala Asn
305 310 315 320
Gln Pro Lys Asn Ile Thr Gln Pro His Asn Leu Asn Leu Asn Ser Pro
325 330 335
Ser Ser Leu Thr Ala Leu Ala Gln Lys Met Leu Lys Asn Ala Gln Ser
340 345 350
Gln Ala Glu Ile Leu Lys Leu Ala Asn Gln Val Glu Ser Asp Phe Asn
355 360 365
Lys Leu Ser Ser Gly His Leu Lys Asp Tyr Ile Gly Lys Cys Asp Ala
370 375 380
Ser Ala Ile Ser Ser Ala Asn Met Thr Met Gln Asn Gln Lys Asn Asn
385 390 395 400
Trp Gly Asn Gly Cys Ala Gly Val Glu Glu Thr Gln Ser Leu Leu Lys
405 410 415
Thr Ser Ala Ala Asp Phe Asn Asn Gln Thr Pro Gln Ile Asn Gln Ala
420 425 430
Gln Asn Leu Ala Asn Thr Leu Ile Gln Glu Leu Gly Asn Asn Pro Phe
435 440 445
Arg Asn Met Gly Met Ile Ala Ser Ser Thr Thr Asn Asn Gly Ala Leu
450 455 460
Asn Gly Leu Gly Val Gln Val Gly Tyr Lys Gln Phe Phe Gly Glu Lys
465 470 475 480
Lys Arg Trp Gly Leu Arg Tyr Tyr Gly Phe Phe Asp Tyr Asn His Ala
485 490 495
Tyr Ile Lys Ser Asn Phe Phe Asn Ser Ala Ser Asp Val Trp Thr Tyr
500 505 510
Gly Val Gly Ser Asp Leu Leu Phe Asn Phe Ile Asn Asp Lys Asn Thr
515 520 525
Asn Phe Leu Gly Lys Asn Asn Lys Ile Ser Phe Gly Leu Phe Gly Gly
530 535 540
Ile Ala Leu Ala Gly Thr Ser Trp Leu Asn Ser Gln Phe Val Asn Leu
545 550 555 560
Lys Thr Ile Ser Asn Val Tyr Ser Ala Lys Val Asn Thr Ala Asn Phe
565 570 575
Gln Phe Leu Phe Asn Leu Gly Leu Arg Thr Asn Leu Ala Arg Pro Lys
580 585 590
Lys Lys Asp Ser His His Ala Ala Gln His Gly Met Glu Leu Gly Val
595 600 605
Lys Ile Pro Thr Ile Asn Thr Asn Tyr Tyr Ser Phe Leu Asp Thr Lys
610 615 620
Leu Glu Tyr Arg Arg Leu Tyr Ser Val Tyr Leu Asn Tyr Val Phe Ala
625 630 635 640
Tyr
Claims (10)
1. applications of the helicobacter pylori HopQ in preparing yolk antibody for anti Helicobacter Pylori, the helicobacter pylori HopQ's
Nucleotide sequence such as SEQ ID NO:Shown in 1.
2. a kind of preparation method of the Yolk antibody of anti-helicobacter pylori, which is characterized in that the method includes following processes：
S1. helicobacter pylori HopQ genes inducible protein is connected on recombinant expression carrier to express, and by obtained albumen into
Row separation and purifying, obtain HopQ recombinant proteins；The amino acid sequence of the HopQ recombinant proteins such as SEQ ID NO:Shown in 2；
S2. laying poultry is immunized with HopQ recombinant proteins, then collects the produced egg of poultry after being immunized；
S3. it extracted from the yolk of the egg of collection, purify the antibody of anti-helicobacter pylori, and freezed, dry, obtain anti-pylorus
The Yolk antibody dry powder of helicobacter.
3. the preparation method of the Yolk antibody of anti-helicobacter pylori according to claim 2, which is characterized in that the pylorus
The sequence of helicobacter HopQ such as SEQ ID NO:Shown in 1.
4. the preparation method of the Yolk antibody of anti-helicobacter pylori according to claim 2, which is characterized in that right in step S2
When laying poultry carries out immune, HopQ recombinant proteins can be equipped with adjuvant and carry out injecting immune；When carrying out injecting immune to poultry, often
The dosage of secondary injection recombinant protein is 300~500ug/；Frequency injection is 3~5 times；Per injection interval time is 2 weeks.
5. the preparation method of the Yolk antibody of anti-helicobacter pylori according to claim 2, which is characterized in that when the egg of collection
Middle helicobacter pylori antibody titre is less than 104When, reinforced immunological can be carried out to laying poultry；The laying poultry is to lay eggs
Chicken.
6. a kind of Yolk antibody for the anti-helicobacter pylori that any preparation method of claim 3~5 obtains.
7. the Yolk antibody of anti-helicobacter pylori described in claim 6 is preparing answering in preventing helicobacter pylori infections vaccine
With.
8. the Yolk antibody of anti-helicobacter pylori described in claim 6 is answered as the additive for preventing helicobacter pylori infections
With.
9. applying according to claim 8, which is characterized in that the additive can be applied in drug, health products or food.
10. a kind of kit of detection helicobacter pylori, which is characterized in that the kit includes to resist described in claim 6
The Yolk antibody of helicobacter pylori.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109400705A (en) * | 2018-12-04 | 2019-03-01 | 大连先锋生物科技有限公司 | A kind of anti-helicobacter pylori duck Yolk antibody and preparation method thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103360491A (en) * | 2013-07-10 | 2013-10-23 | 深圳市宝舜泰生物医药股份有限公司 | Anti-helicobacter pylori FlaA protein antibody IgY, preparation method and application thereof |
| WO2018015468A1 (en) * | 2016-07-20 | 2018-01-25 | Technische Universität München | Agents and methods for the prevention or treatment of h. pylori infections |
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2018
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103360491A (en) * | 2013-07-10 | 2013-10-23 | 深圳市宝舜泰生物医药股份有限公司 | Anti-helicobacter pylori FlaA protein antibody IgY, preparation method and application thereof |
| WO2018015468A1 (en) * | 2016-07-20 | 2018-01-25 | Technische Universität München | Agents and methods for the prevention or treatment of h. pylori infections |
Non-Patent Citations (3)
| Title |
|---|
| DANIEL A BONSOR: "The Helicobacter pylori adhesin protein HopQ exploits the dimer interface of human CEACAMs to facilitate translocation of the oncoprotein CagA", 《THE EMBO JOURNAL》 * |
| GENBANK: "outer membrane beta-barrel protein [Helicobacter pylori]", 《GENBANK DATABASE》 * |
| VERENA KÖNIGER: "Helicobacter pylori exploits human CEACAMs via HopQ for adherence and translocation of CagA", 《NATURE MICROBIOLOGY》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109400705A (en) * | 2018-12-04 | 2019-03-01 | 大连先锋生物科技有限公司 | A kind of anti-helicobacter pylori duck Yolk antibody and preparation method thereof |
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