CN105367659A - Preparation method and application of urease activity inhibition yolk antibody - Google Patents
Preparation method and application of urease activity inhibition yolk antibody Download PDFInfo
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- CN105367659A CN105367659A CN201510795911.2A CN201510795911A CN105367659A CN 105367659 A CN105367659 A CN 105367659A CN 201510795911 A CN201510795911 A CN 201510795911A CN 105367659 A CN105367659 A CN 105367659A
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Abstract
The present invention belongs to the technical field of biology, and specifically discloses a preparation method and application of a urease activity inhibition yolk antibody. By use of gene recombination technology, helicobacter pylori urease (Urease) B subunit DNA fragment is cloned, and after base mutation, constructed and cloned on to a prokaryotic expression plasmid vector, genetic engineering strain BL21 (DE3) is transformed, IPTG is used for inducing expression of recombinant antigen protein urease B subunit, and a soluble form of expression can be successfully implemented; lysis of cells is performed, and urease B subunit recombinant protein can be isolated and purified; the purified recombinant protein urease B subunit as an antigen and an adjuvant are prepared into a microsphere vaccine for laying hen immunization, the yolk antibody in egg yolk can be extracted by pot group-type countercurrent extraction, and the yolk antibody titer and specificity can be detected by ELISA and other techniques. The obtained yolk antibody is added into milk for direct drinking for curing and prevention of acute and chronic gastritis, peptic ulcer and other stomach diseases caused by helicobacter pylori infection.
Description
Technical field
The invention belongs to biological technical field, be suitable for the disease of stomach such as acute or chronic gastritis, peptide ulceration that prevention and therapy helicobacter pylori infection causes, be specially a kind of preparation method and the application thereof that suppress urease activity yolk antibody.
Background technology
The generation of the disease of stomach such as helicobacter pylori (Helicobacterpylori, Hp) and acute or chronic gastritis, peptide ulceration, cancer of the stomach and gastric MALT lymphoma is closely related.Within 1994, the World Health Organization is classified as a class carcinogen.Current prosperity
countryhelicobacter Pylori Infection Rate reaches 30%, in development
countrythe infection rate of the Hp of crowd is up to more than 50%.Can not only gastric ulcer healing be accelerated after eradicating HP, and can significantly reduce gastritis recurrence rate.But it is still very difficult thoroughly will to eradicate HP at present, the treatment also not having a kind of single microbiotic to infect HP is effective, must adopt Multiple Classes of Antibiotics combination therapy, as conbined usage such as omeprazole, metronidazole, amoxycilline Trihydrate bp, clarithromycin and bismuth preparations.But combined with antibiotic is more, side effect is larger, resistance also becomes more complicated, patient costly, compliance is poor, side reaction is many, Hp resistance significantly strengthens, and easily recurs after drug withdrawal.Hp vaccine is one of the most promising method being considered to prevention crowd Hp infection; but because Hp infects at stomach mucous membrane; the humoral immunization that conventional vaccine immunity produces and cellular immunization are difficult to reach this field; its provide protection is limited; also be that helicobacter pylori and subunit vaccine prophylactic treatment helicobacter pylori cause the major cause of stomach trouble difficulty up to now, up to the present do not have vaccine listing truly.Hp vaccine its be mainly prevention, but whether have effect for the treatment of infection population or patient, research report not relevant at present.
Urase is distributed in Hp surface, wide expression and high conservative, and larger relative molecular mass and grainy texture are also conducive to stimulating body to produce immune response, can be used as the candidate antigens of Hp vaccine.Hp urase is made up of, in six aggressiveness A, B Liang Ge subunit.Wherein B subunit relative molecular mass about 64000, antigenicity is strong, and body can be made to produce more effective provide protection.This patent adopts transgenation Optimizing Reconstruction Hp urase B subunit (UreaseBsubunit, UreB) DNA sequence dna, and be building up to prokaryotic expression carrier, realize its solution expression with high efficiency in intestinal bacteria, avoid the formation of non-activity inclusion body.Yolk immunoglobulin (IgY) is originated cheap due to it, output is high, cost is low, easy preparation, has the advantage of the features such as thermostability, acidproof and resistant protease and animal germline genetis method distance, and by as oral food, the infection of prevention and corntrol enteron aisle, oral cavity bacterium, these research and apply are the spy preparing anti-Hp, and different in nature IgY antibody provides possibility, and for prevention and therapy Hp infect stomach trouble open a new route.
Found by existing literature search, deliver as old Zhe etc. and be entitled as " structure of helicobacter pylori clinical strains urease B subunit prokaryotic expression system and the qualification of recombinant protein immunity thereof " (" journal of Zhejiang university: medicine ", 2003,1st phase (1): 4-8).Known from literary composition, urase prokaryotic expression has more existing inclusion body and expresses, and inclusion body is expressed and often caused protein denaturation to lose original natural structure, and protein renaturation length consuming time and technology is more complicated, significantly increase protein expression and purification cost.Liu Xiaofengs etc. are entitled as " expressing the preparation of the attenuated Salmonella typhimurium oral vaccine of helicobacter Pylori urease B subunit " (" China's digestion magazine ", 2001, 09 phase (9): 522-525), to prokaryotic expression and the vaccine research of urase B subunit, but because Hp infects at stomach mucous membrane, the humoral immunization that conventional vaccine immunity produces and cellular immunization are difficult to reach this field, its provide protection is limited, also be that helicobacter pylori and subunit vaccine prophylactic treatment helicobacter pylori cause the major cause of stomach trouble difficulty up to now, up to the present vaccine listing truly is not had.Hp vaccine its be mainly prevention, but whether have effect for the treatment of infection population or patient, research report not relevant at present.
Through finding existing patented technology retrieval, beautiful justice victory waits people in " helicobacter pylori being settled down to the glycoprotein with inhibit activities " literary composition, filter out specifically in conjunction with the glycoprotein of helicobacter pylori urase from milk and egg, that suppresses bacterium determines life, but content is low in the feed to the analysis found that this glycoprotein, the glycoprotein of preparation yields poorly, cost is high.Helicobacter pylori urease gene is inserted attenuation salmonella cell by the people such as Meyer in " helicobacter pylori live vaccine ", makes vaccine, although can produce immune response, attenuation salmonella exists anti-poison and loses the risk of urease gene.According to the regulation of U.S. food and drug regulatory agency (FDA), can not Resistance plasmid be there is in living vaccine, in humans and animals body, also usually cannot maintain the stability immune response of recombinant plasmid with antibiosis.The people such as Nie Zuoming are with using with the Heliobacter pylori antigen of ultrasonic grinding pylorus and the extract of the HP urea urase mixture immunity bird inlay as mixing (Urease) antigen, prepare the IgY antibody of anti-Hp, but this antigenic component is complicated, cause the specificity of its immunoglobulin (Ig) and, specific aim and antibody titers be strong high, often due to loss or the thalline sudden change of somatic surface antigen, be difficult to reach the yolk antibody suppressing the object of growth of H. pylori to produce; In addition there is people to be separated urase from helicobacter pylori, obtain antibody as immunogen, but this antibody can not suppress the field planting of helicobacter pylori.In sum, need
novelthe screening exploitation of Antybody therapy technology, realizes the integrated control to helicobacter pylori with this.
Summary of the invention
The present invention, just for the deficiencies in the prior art and defect, provides a kind of preparation method suppressing urease activity yolk antibody that a kind of safety, cost are low, have efficient prevention and therapy helicobacter pylori.
Another object of the present invention is to provide the application of above-described suppression urease activity yolk antibody.
Concrete technical scheme of the present invention is as follows:
A kind of preparation method suppressing urease urease activity yolk antibody, the method comprises the following steps: first helicobacter pylori urease subunit B gene 17 10bp is carried out transgenation, be subcloned into prokaryotic expression carrier again, and formed fusion expression plasmid pET32a-ureB; Genetic engineering bacterium (the bacterial strain of this genetic engineering bacterium will be formed in plasmid pET32a-ureB transformation of E. coli
preservationat Chinese microorganism strain
preservationmanagement
the councilcommon micro-organisms center, address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica,
preservationdate is on October 15th, 2015,
preservationbe numbered CGMCCNO:11509, Classification And Nomenclature is colon bacillus Escherichiacoli), genetic engineering bacterium is carried out abstraction and purification, obtains helicobacter pylori urease subunit B soluble proteins; Again helicobacter pylori urease subunit B soluble proteins is mixed with adjuvant, make recombinant helicobacterpylori urease subunit B microspheres vaccine, by 2-5 point intramuscular immunisation 3-4 laying hen; Collect egg, adopt yolk antibody in pot group type countercurrent extraction yolk, by titre and the specificity of ELISA and Westernblotting technology for detection yolk antibody.
In described e. coli expression product, the restructuring urase B albumen of 80-95% is helicobacter pylori urease subunit B soluble proteins, by using the recombinant helicobacterpylori urease subunit B soluble proteins after Ni-NTA resin purification, after purity of protein SDS-PAGE electrophoresis, its purity of gray scale scanning is at 85-98%.
Containing Muramyl dipeptide 5-50ug/g, lipopolysaccharides 5-40ug/g in described adjuvant.
The preparation of helicobacter pylori B subunit fragments microspheres vaccine: described restructuring urease subunit B microspheres vaccine contains whiteruss and span80, be 5-9:1 by whiteruss and span80a by its volume ratio, and 400-600rpm mechanical stirring is about 8-12min, mixes and prepares oil phase.Purified protein antigens and Muramyl dipeptide and lipopolysaccharides are dissolved in the sodium alginate soln of 20mg/ml, the final concentration of purified protein antigens, Muramyl dipeptide and lipopolysaccharides is respectively 150-550ug/ml, 5-50ug/ml and 5-40ug/ml, mixing, by the aperture spray-dryer ejection of diameter 0.03-0.05mm, stir in the oil phase of aforesaid liquid paraffin and span80a, emulsification 3-6h, both obtained microspheres vaccine.
In described egg, its antiurease yolk antibody titre reaches more than 1:10000.
The pot group type countercurrent extraction of described yolk antibody, in less than 30 DEG C condition multiple tank countercurrent extraction 6-8h, extraction rate reached is to 90-98%.
Yolk antibody in described pot group type countercurrent extraction yolk, for the first time, gets egg yolk liquid and UP water mixes by 1:2-5 volume ratio, slowly stirs 1-2h, the centrifugal 10-15min of 12000rpm below 30 DEG C, collects supernatant A.For the second time, get the resuspended first time centrifugation of UP water of first time centrifugation 8-20 times of weight, mix below 30 DEG C, stir the centrifugal 10-15min of 1-2h, 12000rpm, collect supernatant liquor.This supernatant liquor and fresh egg yolk liquid are mixed in 1:2-5 ratio below 30 DEG C, supplies supernatant liquor with UP water, stir the centrifugal 10-15min of 1-2h, 12000rpm, collect supernatant liquor B.Get the precipitation of the centrifugal acquisition of the resuspended second time of UP water of the Sediment weight of the centrifugal acquisition of 8-20 times of second time for the third time, mix below 30 DEG C, stir 1-2h, the centrifugal 10-15min of 12000rpm, collects supernatant liquor, and precipitation is managed elsewhere, the supernatant liquor this time obtained and first time extract centrifugal after be deposited in less than 30 DEG C and mix, stir the centrifugal 10-15min of 1-2h, 12000rpm, collect supernatant liquor.The supernatant liquor this time obtained and fresh egg yolk liquid are pressed 1:2-5 volume ratio and are mixed below 30 DEG C, supply supernatant liquor with UP water, stir the centrifugal 10-15min of 1-2h, 12000rpm, collect supernatant C.Merge supernatant A, supernatant liquor B and supernatant C.Regulate pH to 5-5.5 with HCI, stirring and evenly mixing, adds solid sodium chloride and reaches 8-9.5%, and 4 DEG C of centrifugal 10-15min of standing 6-10h, 12000rpm, collecting precipitation, purity reaches more than 80%, and antibody titers reaches 1:10
4-1:10
6.Preserve after pellet frozen drying is formed dry powder, preserve 1 year its vigor for-20 DEG C and remain on about 90%.
Immunization laying hen: by the restructuring urease subunit B microspheres vaccine of preparation at chest with 2-5 point intramuscular injection immunity SPF level laying hen, just exempting from dosage is 200-400ug/; Once, immunizing dose is only adjusted to 100-200ug/ to interval 1-2 week booster immunization, within one week, starts to collect egg after second time booster immunization; Detect antibody titers, after this when yolk antibody titre is lower than 10
4time can carry out reinforced immunological, immunizing dose be 200-600ug/ only.After the purity, the specificity that detect antibody by the method such as SDS-PAGE, Westernblot and ELISA and titre of tiring, collect and meet the requirements of egg.
Positively effect of the present invention is embodied in:
(1) preparing in vaccination process, genetic engineering technique is adopted to achieve restructuring urease subunit B solubility expression a large amount of in vitro, overcome the situation that inclusion body appears in conventional urease subunit B prokaryotic expression, keep the native conformation of helicobacter pylori urease subunit B, expression level is high, and subsequent purification is easy.Adopt pot group type countercurrent extraction technology, energy-saving and emission-reduction, save the time, reduce cost.The yolk antibody of the anti-helicobacter pylori urease subunit B of preparation, in vivo to animal model and the patient of helicobacter pylori infection, embodies good protected effect.
(2) yolk antibody is mixed with milk or yogurt milk, 94% or more is reached to the curative ratio of mice with helicobacter pylori infection; 95% or more is reached to the stomach trouble patient clarified a diagnosis because of helicobacter pylori infection is efficient.
Embodiment:
In order to make object of the present invention, technical scheme and advantage clearly understand, below in conjunction with embodiment, the present invention is described in further detail, but this should be interpreted as that the scope of the above-mentioned theme of the present invention is only limitted to following embodiment.
Embodiment 1:
A kind of preparation method suppressing urease urease activity yolk antibody, the method comprises the following steps: helicobacter pylori urease subunit B gene 17 10bpDNA fragment is carried out transgenation (mutant nucleotide sequence is shown in annex), after being suddenlyd change, sequence is subcloned into prokaryotic expression carrier, and forms fusion expression plasmid pET32a-ureB; Genetic engineering bacterium (the bacterial strain of this genetic engineering bacterium will be formed in plasmid pET32a-ureB transformation of E. coli
preservationat Chinese microorganism strain
preservationmanagement
the councilcommon micro-organisms center, address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica,
preservationdate is on October 15th, 2015,
preservationbe numbered CGMCCNO:11509, Classification And Nomenclature is colon bacillus Escherichiacoli), application Ni-NTA0.1 ~ 1.0mMIPTG expresses at 20 DEG C ~ 30 DEG C inducible proteins, through genetic engineering bacterium is carried out abstraction and purification, obtain the recombinant helicobacterpylori urease subunit B soluble proteins of soluble form; Again helicobacter pylori urease subunit B soluble proteins is mixed with adjuvant, make recombinant helicobacterpylori urease subunit B microspheres vaccine, by 2-5 point intramuscular immunisation 3-4 laying hen; Collect egg, adopt yolk antibody in pot group type countercurrent extraction yolk, by titre and the specificity of ELISA and Westernblotting technology for detection yolk antibody.
The purifying of the gene clone of helicobacter pylori urease subunit B fragment and soluble form restructuring urease subunit B albumen:
According to the helicobacter pylori urease subunit B sequence of synthetic, design a pair pcr amplification primer, upstream primer: F:5 ' CCGGAATTCATGAAAAAAATCTCTCGTAAA-3 ' and downstream primer R:5 ' CCGCTCGAGCTAGAAAAT-GCTAAAGAGTTG-3 ', helicobacter pylori urease subunit B gene 17 10bp fragment sequence is shown in sequence table:
Using the helicobacter pylori urease subunit B full-length gene optimized as template, annealing temperature is set to 57 DEG C and carries out pcr amplification.Reclaim PCR primer to cut back to close through EcoRI and XhoI enzyme, carry out connecting, transforming with same pET32a (+) prokaryotic expression carrier through double digestion process.According to a conventional method recombinant plasmid is identified, recombinant plasmid called after pET32a-UreB.By recombinant plasmid pET32a-UreB Transformed E .coliBL21 (DE3) correct for qualification.Next day, picking list colony inoculation contained the LB liquid nutrient medium of kantlex (50ug/ml) in 3ml, in 37 DEG C, and 200rpm shaking culture; After incubated overnight, access with the inoculative proportion of 0.5-5%, before induction, fermentation condition temperature is 37 DEG C, mixing speed 800rpm, air flow 1vvm, pH6.0-8.0, fermented liquid OD value 0.6-0.8 when induction, temperature is 20 DEG C ~ 30 DEG C, IPTG concentration 0.1 ~ 1.0mM, mixing speed 800rpm, air flow 1vvm, pH6.0-8.0, induction 8h, collected by centrifugation thalline.Sonicated cells.4 DEG C of centrifugal 10min of 12000rpm, collect supernatant liquor.The purifying of recombinant protein is carried out with Invitrogen company Ni-NTA resin specification sheets; And apply the urase B albumen of Bradford method to purifying and carry out quantitative analysis.
Whiteruss and span80a are 5-9:1 by its volume ratio by the preparation of helicobacter pylori urease subunit B split vaccines, and 400-600rpm mechanical stirring is about 8-12min, mixes and prepares oil phase.Purified protein antigens and Muramyl dipeptide and lipopolysaccharides are dissolved in the sodium alginate soln of 20mg/ml, the final concentration of purified protein antigens and Muramyl dipeptide and lipopolysaccharides is respectively 150-550ug/ml, 5-50ug/ml and 5-40ug/ml, mixing, by the aperture spray-dryer ejection of diameter 0.03-0.05mm, stir in above-mentioned oil phase, emulsification 3-6h, both obtained microspheres vaccine.
The preparation of the yolk antibody of anti-helicobacter pylori
(1) to the immunity of laying hen
By the vaccine of preparation to chest muscle 4 injecting immune SPF level laying hens, just exempting from dosage is 400ug/; Once, immunizing dose is only adjusted to 200ug/ to the 2 weeks booster immunizations in interval, within one week, starts to collect egg after third time immunity; Detect antibody titers, after this when yolk antibody titre is lower than 10
4time can carry out reinforced immunological, immunizing dose be 200ug/ only.After the purity, the specificity that detect antibody by the method such as SDS-PAGE, Westernblot and ELISA and titre of tiring, collect and meet the requirements of egg.
(2) antibody separation and purification first time, below 30 DEG C, get 5L egg yolk liquid and 15LUP water mixes, slowly stir 1h, the centrifugal 15min of 12000rpm, collection supernatant liquor (A).For the second time, the UP water getting first time centrifugation 10 times of weight is resuspended, mixes below 30 DEG C, stirs the centrifugal 15min of 1h, 12000rpm, collects supernatant liquor.This supernatant liquor and fresh egg yolk liquid are mixed in 1:1 ratio below 30 DEG C, stirs the centrifugal 15min of 1h, 12000rpm, collect supernatant liquor (B).Get the precipitation of the centrifugal acquisition of the resuspended second time of UP water of 10 times of weight for the third time, mix below 30 DEG C, stir the centrifugal 15min of 1h, 12000rpm, collect supernatant liquor, precipitation is managed elsewhere.The supernatant liquor this time obtained and second time extract centrifugal after be deposited in 30 DEG C below and mix, the centrifugal 10min of stirring 1h, 12000rpm, collection supernatant liquor.The supernatant liquor this time obtained and fresh egg yolk liquid mix below 30 DEG C, stir the centrifugal 15min of 1h, 12000rpm, collect supernatant liquor (C).Collect A, B, C.Regulate pH to 5-5.5 with HCI, stirring and evenly mixing, adds solid sodium chloride and reaches 8-9.5%, and 4 DEG C of centrifugal 15min of standing 6h, 12000rpm, collecting precipitation, purity reaches more than 80%, and antibody titers reaches 1:10
4-1:10
6.Preserve after pellet frozen drying is formed dry powder, preserve 1 year its vigor for-20 DEG C and remain on about 90%.
The yolk antibody purity of anti-helicobacter pylori and Potency Analysis
By above-mentioned collecting precipitation, dissolve with PBS, and be further purified and concentrate in super filter tube (molecular weight cut-off is 100KD), collect yolk antibody solution (D), carry out protein quantification by Bradford method, carry out Sterility testing simultaneously.
(1) yolk antibody purity check gets above-mentioned C, solution D, add the SDS-PAGE electrophoresis sample-loading buffer of reductive agent beta-mercaptoethanol respectively (see the Molecular Cloning: A Laboratory guide third edition in 2002, Pehanorm Bruce work, the U.S.), the purity of the yolk antibody of the anti-human A group colyliform obtained in embodiment 1 is detected by 12% separation gel; Constant current 15mA, gel coomassie brilliant blue staining.Gel is applied the purity of gray scale scanning sample: object band is in 66KD and 25KD position, and other are foreign protein.
(2) concentration of the yolk antibody for anti Helicobacter Pylori obtained in yolk antibody concentration determination Bradford determination of protein concentration embodiment 2, and by every ml yolk calculating antibody output.
(3) Activity determination
The urase B recombinant protein coating buffer of purifying is diluted to 10ug/mL by the experiment of A:ELISA indirect enzyme-linked immunosorbent, and elisa plate every hole bag is by 100 μ L, and 4 DEG C are spent the night; PBST adds 100 μ L final concentrations after washing plate be the BSA of 5%, and room temperature closes 2h; PBST adds the purifying IgY (1: 100 ~ 1: 10000) of different extension rate after washing plate, incubated at room 2h; PBST washes plate, the anti-chicken IgY of donkey (100 μ L hole) that the horseradish peroxidase (HRP) adding 1:5000 dilution marks, and 37 DEG C of wet boxes hatch 1h; PBST washes plate and adds TMB (100 μ L/ hole) colour developing, 37 DEG C of lucifuge effect 10min; Add 2mol/LH
2sO
4stop buffer (50 μ L/ hole) termination reaction; Enzyme mark automatic analyser 450nm wavelength measures its absorbance value (OD
450nm).Set incoherent band His label protein as contrast in experiment in test, absorbancy OD value more than 2 times is positive criteria, measures antibody titers.
B: urase B recombinant protein is carried out the SDS-PAGE gel electrophoresis of 12% by protein immunoblot experiment, utilize albumen electrotransfer system that albumen is gone to NC film, 5%BSA room temperature shaker closes 2h, and the IgY antibody adding above-mentioned purifying makes primary antibodie (1: 1000), room temperature effect 2h or 4 DEG C spends the night, PBST washs 3 times, each 10min, adds two anti-(the goat-anti chicken IgY of HRP mark) of 1: 5000 dilution, room temperature effect 1h, TBST washs 3 times, each 10min, DAB colour developing.Set incoherent band His label protein as contrast in test.Result is shown as single band, shows the yolk antibody energy specific immunity identification urase B antigen protein prepared.
Result shows, the yolk antibody of anti-helicobacter pylori prepared by the present invention, and antibody titers can reach more than 1:10000, and can specific recognition urase B albumen.
(4) yolk antibody interior therapeutic
The interior therapeutic of yolk antibody: yolk antibody is mixed with milk, final concentration reaches 2mg/mL; To feed helicobacter pylori infection C57/B6 mouse, set up stomach trouble animal model; Contain yolk antibody milk 100 μ L by oral administration gavage mouse every day, investigate germ amount after 2 weeks, result obviously reduces mouse germ amount, and gastric mucosal is good, and curative ratio reaches more than 94%.Further, it orally to be clarified a diagnosis because of stomach trouble patient 20 example of helicobacter pylori infection, the sense of discomfort of 24 hours patient gasteremphraxis etc. disappears, and crowd is efficient reaches 95%.
Carry out the helicobacter pylori urease subunit B gene 17 10bp fragment sequence after transgenation as follows:
1
ATGAAAAAAATCTCTCGTAAAGAATACGTTTCTATGTACGGTCCGACCACCGGTGACAAA
61
GTTCGTCTGGGTGACACCGACCTGATCGCTGAAGTTGAACACGACTACACCATCTACGGT
121
GAAGAACTGAAATTCGGTGGTGGTAAAACCCTGCGTGAAGGTATGTCTCAGTCTAACAAC
181
CCGTCTAAAGAAGAACTGGACCTGATCATCACCAACGCTCTGATCGTTGACTACACCGGT
241
ATCTACAAAGCTGACATCGGTATCAAAGACGGTAAAATCGCTGGTATCGGTAAAGGTGGT
301
AACAAAGACATGCAGGACGGTGTTAAAAACAACCTGTCTGTTGGTCCGGCTACCGAAGCT
361
CTGGCTGGTGAAGGTCTGATCGTTACCGCTGGTGGTATCGACACCCACATCCACTTCATC
421
TCACCCCAACAAATCCCTACAGCTTTTGCAAGCGGTGTAACAACCATGATTGGTGGCGGA
481
ACTGGTCCTGCTGATGGCACTAATGCGACTACTATCACTCCAGGCAGAAGAAATTTAAAA
541
TGGATGCTCAGAGCGGCTGAAGAATATTCTATGAACTTAGGTTTCTTGGCTAAAGGTAAC
601
GCTTCTAACGACGCGAGCTTAGCCGATCAAATTGAAGCTGGTGCGATTGGCTTTAAAATC
661
CACGAAGACTGGGGCACCACTCCTTCTGCAATCAATCATGCGTTAGATGTTGCAGACAAA
721
TACGATGTGCAAGTCGCTATCCACACAGACACTTTGAATGAAGCCGGTTGCGTGGAAGAC
781
ACTATGGCAGCTATTGCCGGACGCACTATGCACACTTTCCACACTGAAGGTGCTGGCGGC
841
GGACACGCTCCTGATATTATTAAAGTAGCTGGTGAACACAACATTCTTCCCGCTTCCACT
901
AACCCCACTATCCCTTTCACTGTGAATACAGAAGCAGAACACATGGACATGCTTATGGTG
961
TGCCACCACTTGGATAAAAGCATTAAAGAAGATGTTCAGTTCGCTGATTCAAGGATCCGC
1021
CCTCAAACCATTGCGGCTGAAGACACTTTGCATGACATGGGGATTTTCTCAATCACCAGC
1081
TCTGACTCTCAAGCTATGGGTCGTGTGGGTGAAGTTATCACTAGAACTTGGCAAACAGCT
1141
GACAAAAACAAAAAAGAATTTGGCCGCTTGAAAGAAGAAAAAGGCGATAACGACAACTTC
1201
AGGATCAAACGCTACTTGTCTAAATACACCATTAACCCAGCGATCGCTCATGGGATTAGC
1261
GAGTATGTAGGTTCTGTAGAAGTGGGCAAAGTGGCTGACTTGGTATTGTGGAGTCCCGCA
1321
TTCTTTGGCGTAAAACCCAACATGATCATCAAAGGCGGGTTCATTGCGTTGAGTCAAATG
1381
GGTGACGCGAACGCTTCTATCCCTACCCCACAACCAGTTTATTACAGAGAAATGTTCGCT
1441
CATCATGGTAAAGCCAAATACGATGCAAACATCACTTTTGTGTCTCAAGCGGCTTATGAC
1501
AAAGGCATTAAAGAAGAATTAGGGCTTGAAAGACAAGTGTTGCCGGTAAAAAATTGCAGA
1561
AACATCACTAAAAAAGACATGCAATTCAACGACACTACCGCTCACATTGAAGTCAATCCT
1621
GAAACTTACCATGTGTTCGTGGATGGCAAAGAAGTAACTTCTAAACCAGCCAATAAAGTG
1681
AGCTTGGCGCAACTCTTTAGCATTTTCTAG。
Claims (8)
1. one kind is suppressed the preparation method of urease activity yolk antibody, it is characterized in that: first clone helicobacter pylori urease subunit B gene fragment, carry out base mutation and form special urease B gene fragment, be subcloned on prokaryotic expression plasmid vector, change into genetic engineering bacterium, then expressed by 0.1 ~ 1.0mMIPTG inducible protein under the condition of 25 DEG C ~ 30 DEG C, the restructuring urease subunit B of final 80-95% is with soluble form protein expression; Through affinity protein purification purifying urease subunit B recombinant protein, it is mixed with adjuvant, be prepared into recombinant helicobacterpylori urease subunit B microspheres vaccine, by 2-5 point intramuscular immunisation 3-4 laying hen; Collect egg, adopt yolk antibody in pot group type countercurrent extraction yolk, by titre and the specificity of ELISA and Westernblotting technology for detection yolk antibody.
2. the preparation method of suppression urease activity yolk antibody according to claim 1, is characterized in that: the preserving number of described genetically engineered bacteria strain is CGMCCNO:11509.
3. the preparation method of suppression urease activity yolk antibody according to claim 1, it is characterized in that: described carries out purifying to restructuring urease subunit B albumen, it is by using Ni-NTA affine resin purification helicobacter pylori urease subunit B soluble proteins, and after SDS-PAGE electrophoresis, gray scale scanning shows that purity of protein reaches 85%-98%.
4. the preparation method of suppression urease activity yolk antibody according to claim 1, is characterized in that: containing Muramyl dipeptide 5-50ug/g, lipopolysaccharides 5-40ug/g in described adjuvant.
5. the preparation method of suppression urease activity yolk antibody according to claim 1, it is characterized in that: described restructuring urease subunit B microspheres vaccine contains whiteruss and span80, be 5-9:1 by whiteruss and span80a by its volume ratio, and 400-600rpm mechanical stirring is about 8-12min, mix and prepare oil phase, purified protein antigens and Muramyl dipeptide and lipopolysaccharides are dissolved in the sodium alginate soln of 20mg/ml, purified protein antigens, the final concentration of Muramyl dipeptide and lipopolysaccharides is respectively 150-550ug/ml, 5-50ug/ml and 5-40ug/ml, mixing, by the aperture spray-dryer ejection of diameter 0.03-0.05mm, stir in the oil phase of aforesaid liquid paraffin and span80a, emulsification 3-6h, both microspheres vaccine was obtained.
6. the preparation method of suppression urease activity yolk antibody according to claim 1, is characterized in that: in described egg, and its antiurease yolk antibody titre reaches more than 1:10000.
7. the preparation method of suppression urease activity yolk antibody according to claim 1, is characterized in that: the pot group type countercurrent extraction of described yolk antibody, and in lower than the condition multiple tank countercurrent extraction 6-8h of 30 DEG C, extraction rate reached is to 90-98%.
8. the preparation method of suppression urease activity yolk antibody according to claim 7, it is characterized in that: yolk antibody in described pot group type countercurrent extraction yolk, for the first time, egg yolk liquid is got and UP water mixes by 1:2-5 volume ratio below 30 DEG C, slow stirring 1-2h, the centrifugal 10-15min of 12000rpm, collects supernatant A; For the second time, get the resuspended first time centrifugation of UP water of first time centrifugation 8-20 times of weight, mix below 30 DEG C, stir the centrifugal 10-15min of 1-2h, 12000rpm, collect supernatant liquor, this supernatant liquor and fresh egg yolk liquid are mixed in 1:2-5 ratio below 30 DEG C, supplies supernatant liquor with UP water, stir 1-2h, the centrifugal 10-15min of 12000rpm, collects supernatant liquor B; Get the precipitation of the centrifugal acquisition of the resuspended second time of UP water of the Sediment weight of the centrifugal acquisition of 8-20 times of second time for the third time, mix below 30 DEG C, stir 1-2h, the centrifugal 10-15min of 12000rpm, collects supernatant liquor, and precipitation is managed elsewhere, the supernatant liquor this time obtained and first time extract centrifugal after be deposited in less than 30 DEG C and mix, stir the centrifugal 10-15min of 1-2h, 12000rpm, collect supernatant liquor; The supernatant liquor this time obtained and fresh egg yolk liquid are pressed 1:2-5 volume ratio and are mixed below 30 DEG C, supply supernatant liquor with UP water, stir the centrifugal 10-15min of 1-2h, 12000rpm, collect supernatant C; Merge supernatant A, supernatant liquor B and supernatant C.
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| CN115074288A (en) * | 2022-07-04 | 2022-09-20 | 福建益昕葆生物制药有限公司 | Anti-diarrhea egg yolk antibody coated lactobacillus preparation, preparation method and application thereof |
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