CN105169381B - A kind of helicobacter pylori multivalence epitope vaccine and preparation method thereof - Google Patents
A kind of helicobacter pylori multivalence epitope vaccine and preparation method thereof Download PDFInfo
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- CN105169381B CN105169381B CN201510336891.2A CN201510336891A CN105169381B CN 105169381 B CN105169381 B CN 105169381B CN 201510336891 A CN201510336891 A CN 201510336891A CN 105169381 B CN105169381 B CN 105169381B
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Abstract
The present invention provides a kind of helicobacter pylori polyepitope vaccines, its activity is a polypeptide, is made of urease A and B subunit, adhesin HpaA, the advantage Th of heat shock protein HSP60 and B cell antigen epi-position or section and neutrophil activating protein NAP and b subunit of cholera toxin.The present invention synthesizes an artificial gene by gene synthesis technology, it includes urease A and B subunit, adhesin HpaA, the advantage Th of heat shock protein HSP60 and B cell antigen epi-position or the gene orders of section and neutrophil activating protein NAP, the artificial gene and b subunit of cholera toxin gene are mutually coupled, form a fusion.By the Bacillus coli expression fusion, after protein purification, multivalence epitope vaccine is obtained.The vaccine can excitating organism generate for urease, adhesin HpaA, heat shock protein HSP60 and the T cell immune response of neutrophil activating protein NAP and antibody humoral immune response, it is diseases related available for prevention Helicobacter pylori infection.
Description
Technical field
The invention belongs to biomedicine fields, and in particular to a kind of novel helicobacter pylori multiepitope vaccine and its system
Preparation Method and application.
Background technology
Helicobacter pylori (Helicobacter pylori, Hp) is that chronic gastritis, peptic ulcer and duodenum are burst
The important virulence factor of ulcer, closely related with gastric cancer, Hp is classified as I class carcinogens by the World Health Organization (WHO).Pylorus spiral shell
The infection rate of swing arm bacterium is very high, and global population Hp infection rates are more than 50%, and population of China Hp infection rates are 58.07%, and house is presented
Front yard aggregation, situation are more acute.The method for the treatment of Hp infection at present, is based primarily upon antibiotic " three " medicinal treatment, deposits
It is costly, patient compliance is poor, drug-fast bacteria increases and the shortcomings of intestinal bacilli illness, should not be extensive in Hp infection populations
It promotes the use of.Experimental study shows that immunity inoculation can prevent even to treat Hp infection, and crowd is made to obtain persistent immunity.
It is a kind of effective method for controlling Hp infectious diseases to develop effective preventative and therapeutic Hp vaccines, is increasingly becoming
The hot spot that domestic and foreign scholars competitively study.
Helicobacter pylori contains abundant urease (Ure), accounts for about the 5%~10% of whole cell albumen, inside Hp
With surface wide expression.General bacterium is difficult to survive in the high acid environment of stomach, but Ure can hydrolyze urea and generate ammonia to neutralize
Hydrochloric acid in gastric juice, and pathologic damage can be caused to stomach lining.Therefore, urease is the important colonization factor and virulence factor of Hp.Urea
Enzyme is made of A and B Liang Ge subunits (UreA and UreB), and wherein urease B subunit is urease activity subunit, in each Hp bacterium
In strain there is good conservative, be acknowledged as the ideal candidates antigen of Hp vaccines.Newest experimental study shows urease A
Subunit equally has stronger antigenicity, and good protective effect can be equally obtained for the Hp vaccines of urease A subunits；It is deep and remote
Door pylori adhesin HpaA is Hp flagellum sheath memebrane proteins, is almost present in all Hp bacterial strains surfaces being clinically separated, is Hp
One of main adhesion factor, and amino acid sequence is highly conserved, zoopery is proved the necessary factor that HpaA is field planting, is
A kind of ideal protective antigens of Hp vaccine developments.Helicobacter pylori heat shock protein (HSP) positioned at Hp surfaces, have compared with
Strong immunogenicity can induce mouse and generate the anti-Hp antibody of specificity.Hp heat shock proteins HSP60 is one kind of Hp ureases
Molecular chaperones, mediation Hp and Gastric Mucosal Cells are sticked；Helicobacter pylori neutrophil activating protein (NAP) is the master of Hp
One of virulence factor is wanted, has chemotaxis to neutrophil leucocyte, monocyte, leads to neutrophil infiltration stomach lining, and lure
Neutrophil NADPH oxidase activation is led, reactive oxygen intermediates (ROI) is generated, causes mucosal inflammation and tissue damage.
NAP antigenicities are strong, and most of Hp infected patients generate NAP antibody.Secondly, NAP is a kind of good Th1 types polarization cellular immunity
Adjuvant.
The thinking of the present invention is as follows：According to body to the immune protective mechanism of Hp, it is bis- sub- to reasonably select urease A and B
Base, adhesin HpaA, the advantage Th of heat shock protein HSP60 and B cell antigen epi-position or section and intramolecular mucosal adjuvant
CTB and Th1 type cellular immunity adjuvant NAP pass through the order of connection of the bioinformatics software to epitope or section, interval sequence
Row and multiple copies number, are analyzed and are determined, design scientific and reasonable, structure novel a Hp multivalence epitope vaccine
CWAE.Using the panimmunities such as Western Blot, ELISA method detection Hp multivalence epitope vaccines CWAE immunogenicity and
Immunologic specificity, research shows that Hp multivalence epitope vaccines CWAE can excite BALB/c mouse to generate for the bis- Asias of urease A and B
Base, adhesin HpaA, heat shock protein HSP60 and the T cell immune response of neutrophil activating protein NAP and high titre table
Position specific antibody, has good immunogenicity and immunologic specificity.
Invention content
The first aspect of the invention is there is provided one kind for Hp keys adhesion factor and virulence factor urease A and B
Double subunits, adhesin HpaA, heat shock protein HSP60 and neutrophil activating protein NAP Hp multivalence epitope vaccines.
There is provided the preparation methods of Hp multivalence epitope vaccines for the second aspect of the invention.
The third aspect of the invention is to disclose the purposes of Hp multivalence epitope vaccines.
The first aspect of the invention is there is provided one kind for the bis- subunits of urease A and B, adhesin HpaA, heat shock
The Hp multivalence epitope vaccines CWAE of albumen HSP60 and neutrophil activating protein NAP.The Hp multivalence epitopes vaccine is mainly by more
Epitope peptide WAE and b subunit of cholera toxin are formed, and the amino acid sequence of multi-epitope peptide WAE is such as shown in (sequence 3), Hp multivalence epitopes
The whole amino acid sequence of vaccine CWAE is such as shown in (sequence 1).
The novel Hp multivalence epitopes vaccine CWAE of the present invention has the following advantages：(1) Hp multivalence epitopes vaccine CWAE will urinate
The bis- subunits of plain enzyme A and B, adhesin HpaA, the dominant antigen epitope of heat shock protein HSP60 or section and neutrophil activation
Albumen NAP and Mucosal Adjuvants CTB are scientifically and rationally combined, can excite for the bis- subunits of Hp ureases A and B,
The T cell immune response and specificity humoral of adhesin HpaA, heat shock protein HSP60 and neutrophil activating protein NAP
Immune response.(2) the bis- subunits of Hp ureases A and B, adhesin HpaA heat shock protein HSP60 there are biology toxicity, Hp
Multivalence epitope vaccine CWAE contains only the advantage Th of this 4 kinds of Hp antigen proteins and B cell antigen epi-position or section, avoids Hp
The biology toxicity of toxin protein, and the epitope-specific antibodies for this 4 kinds of Hp antigen proteins can be excited, have higher
Immunologic specificity, the immunological disease rationality injury for preventing cross reaction from generating.(3) antiurease of natural Hp ureases excitation resists
Body, it is impossible to which urease inhibiting effective enzymatic activity, Hp multivalence epitope vaccines CWAE of the invention, which can be excited, effectively inhibits Hp urea enzyme activity
The neutralizing antibody of property.(4) the molecular weight very little of Hp virulence factors and adhesion factor epitope peptide, immunogenicity is very low, this hair
Bright Hp multivalence epitope vaccines CWAE takes " immunologic adjuvant in coupling molecule " and " epitope multiple copies " two kinds of designs to think
Road enhances the immunogenicity of Hp virulence factors and adhesion factor epitope peptide, has well solved epiposition vaccine immunogenicity
The defects of universal relatively low, can induce the epitope-specific antibodies of high titre.
The second aspect of the invention is to provide the preparation method of Hp multivalence epitope vaccines, and details are as follows for technology path：
(1) structure design of novel Hp multivalence epitopes vaccine antigen molecule
According to body to the immune protective mechanism of helicobacter pylori, Hp urine is rationally screened by bioinformatics software
The advantage Th and B cell antigen epi-position of the bis- subunits of plain enzyme A and B, adhesin HpaA and heat shock protein HSP60, and to antigen table
The order of connection, intervening sequence and the multiple copies number of position, are analyzed and are determined, design a scientific and reasonable, structure novel
Hp multivalence epitope vaccines CWAE.
(2) structure of recombinant expression plasmid pETCWAE (containing fusion CWAE)
A recombinant expression carrier pETC for containing b subunit of cholera toxin (CTB) gene is built first；Then pass through gene
Synthetic technology synthesizes multi-epitope peptide WAE (cell antigen epitope and Th1 containing the more a adhesion factors of Hp and virulence factor
Type cellular immunity adjuvant NAP) nucleotide sequence, and be inserted into recombinant expression carrier pETC, build recombinant expression carrier
pETCWAE。
(3) prokaryotic expression of fusion protein CWAE and purifying
Recombinant expression carrier pETCWAE is transformed into e. coli bl21 (DE3), builds recombination engineered strain
BL21(DE3)/pETCWAE.Using IPTG induced expressions, and pass through Ni-NTP nickel ion affinity chromatographs and can obtain high-purity and melt
Hop protein CWAE, as Hp multivalence epitopes vaccine.
There is provided the purposes of Hp multivalence epitope vaccines for the third aspect of the invention.Hp multivalence epitope vaccines CWAE can be used
It is diseases related in prevention and treatment Helicobacter pylori infection.
Description of the drawings
Fig. 1：The Molecular Design feature of novel Hp multivalence epitopes vaccine CWAE.
Fig. 2：The double digestion identification of recombinant expression carrier pETCWAE.
Swimming lane 1：DNA Marker；Swimming lane 2：pETCWAE/Nco I+Xho I
Fig. 3：The prokaryotic expression of Hp multivalence epitope peptide fusion proteins CWAE.
Swimming lane 1：Protein Marker；Swimming lane 2：BL21 (DE3)/pET22b whole bacterial proteins (being induced through IPTG)；Swimming lane 3：
BL21 (DE3)/pETCWAE inclusion body proteins (being induced through IPTG)；Swimming lane 4：BL21 (DE3)/pETCWAE soluble proteins (warps
IPTG is induced)；Swimming lane 5：BL21 (DE3)/pETCWAE whole bacterial proteins (being induced through IPTG)；Swimming lane 6：BL21(DE3)/pETCWAE
Whole bacterial protein (induces) without IPTG
Fig. 4：The Ni-NTA affinitive layer purifications of fusion protein CWAE.
Swimming lane 1：Protein Marker；Swimming lane 2：CWAE inclusion body proteins (loading albumen)；Swimming lane 3,4：Foreign protein；Swimming lane
5,6：CWAE protein samples after purification
Fig. 5：Hp multivalence epitope vaccines CWAE induces the detection of anti-Hp urease antibodies.
Hp multivalence epitopes vaccine CWAE, Hp urease and urease B subunit can be induced compared with high titre antiurease antibody.
Fig. 6：Hp multivalence epitope vaccines CWAE induces the detection of anti-UreA antibody.
Hp multivalence epitope vaccine CWAE and Hp ureases can induce the anti-UreA antibody compared with high titre.
Fig. 7：Hp multivalence epitope vaccines CWAE induces the detection of anti-UreB antibody.
Hp multivalence epitope vaccines CWAE, urease and urease B subunit can induce the anti-UreB antibody compared with high titre.
Fig. 8：Hp multivalence epitope vaccines CWAE induces the detection of heat resistanceheat resistant shock protein HSP60 antibody.
Only Hp multivalence epitopes vaccine CWAE could induce the heat resistanceheat resistant shock protein HSP60 antibody compared with high titre.
Fig. 9：Hp multivalence epitope vaccines CWAE induces the detection of anti-neutrophil activating protein NAP antibody.
Only Hp multivalence epitopes vaccine CWAE could induce the anti-neutrophil activating protein NAP antibody compared with high titre.
Figure 10：Hp multivalence epitope vaccines CWAE induces the detection of anti-adhesin HpaA antibody.
Only Hp multivalence epitopes vaccine CWAE could induce the anti-adhesin HpaA antibody compared with high titre.
Figure 11：Hp multivalence epitope vaccines CWAE induces UreA183-203The detection of epitope-specific antibodies.
Only Hp multivalence epitopes vaccine CWAE can generate the UreA compared with high titre183-203Epitope-specific antibodies.
Figure 12：Hp multivalence epitope vaccines CWAE induces UreB327-334The detection of epitope-specific antibodies.
Only Hp multivalence epitopes vaccine CWAE can generate the UreB compared with high titre327-334Epitope-specific antibodies.
Figure 13：Hp multivalence epitope vaccines CWAE induces HpaA132-141The detection of epitope-specific antibodies.
Only Hp multivalence epitopes vaccine CWAE can excite the HpaA compared with high titre132-141Epitope-specific antibodies.
Figure 14：Hp multivalence epitope vaccines CWAE induces HSP60189-203The detection of epitope-specific antibodies.
Only Hp multivalence epitopes vaccine CWAE can excite the HSP60 compared with high titre189-203Epitope-specific antibodies.
Figure 15：Breeder reaction of the Hp multivalence epitope vaccine CWAE sensitized mices spleen lymphocytes to antigenic stimulus.
Specific embodiment
Material
1.IPTG solution：It weighs 1.2g IPTG to be placed in 50ml centrifuge tubes, adds in 40ml sterile waters, abundant mixing dissolving
Afterwards, it is settled to 50ml.With 0.22 μm of filter filtration sterilization, aliquot packing, -20 DEG C of preservations.
Ampicillin 2. (Amp) reservoir (100mg/mL)：Weighing 100mg ampicillins (Amp), to be dissolved in 1mL sterile
The storage liquid of a concentration of 100mg/mL is made in water, and by 0.22 μm of bacterial filter filtration sterilization, solution packing is stored in -20 DEG C
In refrigerator.
3. culture medium：(1) LB fluid nutrient mediums：10g tryptones, 5g yeast extracts and 5g NaCl are weighed, adds distillation
Water adjusts pH to 7.4, autoclaving to 1000ml.(2) LB solid mediums：1.5g agar powders/100ml LB cultures
Liquid, after high pressure sterilization, pour plate.
4.DNA electrophoretic buffers (50x TAE)：Weigh 242g Tris, 37.2g Na2EDTA·2H2O and 57.1ml ice second
Acid, adds water to 1000ml, and when use dilutes 50 times.
5.SDS-PAGE electrophoretic buffers (5X)：Weigh Tris powder 15.1g, glycine 94g, SDS 5.0g；It adds in about
The deionized water of 800ml, stirring and dissolving；Deionized water is added to be settled to 1L, room temperature preservation；Pay attention to：Water should be allowed along wall by adding during water
It slowly flows down, to avoid many foams are generated due to SDS.
6. Coomassie brilliant blue protein staining reagent：(1) Coomassie brilliant G-250 dye liquor (quantification of protein use)：Coomassie
Brilliant blue G-250 100mg are dissolved in 95% ethyl alcohol of 50ml, are then added in 86% phosphatase 11 00ml, are diluted to distilled water
1000ml.(2) coomassie brilliant blue R_250 dye liquor：0.29g coomassie brilliant blue R_250s are dissolved in 250ml destainers.(3) it is fixed
Liquid：500ml ethyl alcohol, 100ml glacial acetic acid are diluted to 1000ml with distilled water.(4) destainer：250ml ethyl alcohol, 80ml glacial acetic acid are used
Distilled water is diluted to 1000ml.(5) liquid is preserved：87% glycerine of 25ml is dissolved in 225ml destainers.
7.RNase solution As (10mg/ml)：10mg RNase A are dissolved in 987 μ l water, add in 10 μ l 1M Tris-
After HCl (pH7.5) and 3 μ l 5M NaCl, boiling water bath 15min, be slowly cooled to room temperature, be divided into aliquot be stored in -20 DEG C it is spare.
8.Lysozyme solution (10mg/ml)：10mg Lysozyme are dissolved in 1ml 10mM Tris-HCl (pH8.0).
9. experimental animal：BALB/c mouse is SPF grades, male, 8~10 week old, purchased from Ningxia Medical University experimental animal
Center.
10.ELISA reagents：(1) coating buffer：1.6g Na2CO3, 2.9g NaHCO3, 0.2g NaN3, add distilled water to 1L,
PH value is adjusted to 9.6.(2) cleaning solution：0.2g KH are weighed respectively2PO4, 2.9g Na2HPO4·12H2O, 8.0g NaCl, 0.2g
KCl, 0.5ml Tween-20 add in ddH2O and are settled to 1000ml (PBST).(3) confining liquid：3.0g BSA are weighed to be dissolved in
In 100ml washing buffers, 4 DEG C of preservations after filtration sterilization.(4) substrate solution：Soluble one-component tmb substrate solution.(5) it terminates
Liquid：Distilled water 178.3ml is measured, concentrated sulfuric acid 21.7ml (1M H are added dropwise2SO4)。
Lymphocyte proliferation assay main agents 11. (1) mouse spleen lymphocyte separating liquid (has up to section for biotechnology
Limit company) (2) RPMI-1640 complete culture solutions：It is green that 10% fetal calf serum, 100U/ml are added in RPMI-1640 basic culture solutions
Mycin, 100 μ g/ml streptomysins.(3) the endless full nutrient solutions of RPMI-1640：Weigh 10.4g RPMI-1640 dry powder, 2.4g
HEPES、0.75g NaHCO3, add deionized water to 1000ml, pH 7.4, heat sterilization, packing.
13.BHI blood plates：3.5g BHI dry powder is weighed, adds distilled water 93ml, 1.5g agar powder, 121 DEG C of sterilizing 13min,
It is cooled to 60 DEG C and takes off fiber sheep blood, polymyxin B (5 μ g/ml of final concentration), vancomycin (10 μ of final concentration hereinafter, adding in 7ml
G/ml) and methoxybenzyl aminopyrimidine (5 μ g/ml of final concentration), dispense to culture dish, it is spare after cooling.
14. urea meat soup：Glucose 1g, peptone 1g, potassium dihydrogen phosphate 2g, sodium chloride 5g are weighed, adds distilled water
1000ml corrects PH to 7.2, adds 0.4% phenol red solutions of 2ml, high pressure sterilization (68.95kPa) 20 minutes is cooled to 60 DEG C
50% urea liquids of 1ml filtered with bacteria filter are added in during left and right, it is spare after mixing.
Example 1：The Molecular Design of helicobacter pylori multivalence epitope vaccine CWAE
According to " body is to the immune protective mechanism of Hp " and " the bis- subunits of Hp ureases A and B, HSP60 and HpaA etc. are crucial
The immunological properties of virulence factor and adhesion factor epitope " screen UreA by bioinformatics27-53、UreA183-203、
HpaA132-141、HSP60189-203、UreB185-225、UreB327-385Wait epitopes or section and intramolecular Th1 type cellular immunities
Adjuvant NAP and Mucosal Adjuvants CTB is used for the structure of novel Hp multivalence epitopes vaccine CWAE.Then, pass through epiposition vaccine
The theoretical analysis with bioinformatics of structure, to the order of connection of selected epitope or section, intervening sequence and antigen
Epitope copy number is analyzed and is determined that final design provides the Hp polyvaccines CWAE of scientific and reasonable structure.Through
The bioinformatics softwares such as DNAstar and MOE are analyzed, and CWAE has preferable isoelectric point, antigenicity and higher structure.From design
Theoretically, CWAE can excitating organism generate more strongly immunogenic and immunologic specificity, for the bis- subunits of Hp ureases A and B,
Adhesin HpaA, heat shock protein HSP60 and neutrophil activating protein NAP T cell immune response and antibody (IgA,
IgG, sIgA) humoral immune response, being effectively prevented and treated for Hp infectious diseases can be reached.
As a result：The Molecular Design feature and thinking of helicobacter pylori multivalence epitope vaccine CWAE is such as shown in (Fig. 1).
Example 2：The structure of recombinant expression carrier pETCWAE (containing fusion CWAE)
(1) gene chemical synthesis of multi-epitope peptide WAE nucleotide sequences
By the amino acid sequence of the multi-epitope peptide WAE of design early period, converted according to e. coli codon preferences principle
Into corresponding nucleotide sequence, the auspicious biotechnology company of commission Nanjing spun gold carries out gene chemical synthesis.
(3) connection of multi-epitope peptide WAE genes and pETC expression vectors
Recombinant plasmid pUCWAE and pETC (laboratory is built early period) are extracted by small amount plasmid extraction kit (Tiangeng),
Double digestion is carried out respectively with Kpn I/Xho I, and reaction system is as follows：
37 DEG C of endonuclease reaction 2h then with 1% agarose gel electrophoresis, observe electrophoresis result.Agarose is utilized respectively to coagulate
Glue DNA recovery purifyings kit recycles multi-epitope peptide WAE genes and pETC double digestion products.By the pETC linearized vectors of recycling
With WAE genes by complementary cohesive end, (4 DEG C overnight) connects into the DNA of closed hoop under the action of T4DNA ligases
Molecule forms recombinant expression plasmid pETCWAE.T4DNA ligase linked systems are as follows：
As a result：Utilize Nco I/Xho I double digestions recombinant plasmid pETCWAE to be checked, 37 DEG C of reaction 2h, with 1% agar
Sugared detected through gel electrophoresis, the DNA fragmentation for finding double digestion are 1797bp, in the same size with the theory of fusion CWAE.Again will
PETCWAE delivers Nanjing Jin Sirui biotechnologies company, and gene sequencing, card are carried out using T7 promoters and terminator universal primer
The nucleotide sequence of fusion CWAE is completely correct in bright pETCWAE, and without frameshift mutation.Recombinant expression carrier pETCWAE
Double digestion collection of illustrative plates is such as shown in (Fig. 2).
Example 3：The prokaryotic expression of multivalence epitope peptide fusion protein CWAE
It will verify that correct recombinant expression plasmid pETCWAE is transformed into E.coli BL21 (DE3) bacterial strain.It is made in advance
On the LB tablets containing 50 μ g/mL Amp got ready, oese scribing line engineering strain pETCWAE/BL21 (DE3) is inverted in
37 DEG C of incubators, after being incubated overnight 12~16h, picking single bacterium colony is inoculated in the LB culture mediums containing 50 μ g/mL Amp, 37
DEG C, 180rpm cultivates 12~16h.Recombinant bacterium is inoculated in containing 50 μ g/mL Amp LB culture mediums with 2% inoculum concentration respectively, 37
DEG C, which is inoculated in the LB Liquid Cultures containing 50 μ g/mL Amp by 180rpm shaken overnights, morning next day with 1% inoculum concentration again
In base, 37 DEG C, after 180rpm shaking flasks 3h, adding in IPTG makes final concentration reach 1mmol/L, 37 DEG C, 180rpm induced expressions, with sky
Carrier bacterium pET22b/BL21 (DE3) is as negative control.
As a result：It is 7, a concentration of 1mmol/L of IPTG, induction that genetic engineering is recombinated pETCWAE/BL21 (DE3) in pH value
37 DEG C of temperature, induced expression 6h.It is compared with control strain, genetic engineering recombination strain pETCWAE/BL21 (DE3) is in about 70KD
There is destination protein band in place, is consistent (Fig. 3) with the theoretical size of fusion protein CWAE.Fusion protein CWAE is mainly to forgive
The form of body protein exists.
Example 4：The purifying of multivalence epitope peptide fusion protein CWAE
It will be added in chromatographic column after the abundant mixing of Ni-NTA fillers, solution can slowly be flowed out by gravity, be treated completely
After addition, upper sieve plate is kept flat in column；It is added in into the column filled after suitable deionized water rinses ethyl alcohol well,
The Binding buffer balances of 8 times of column volumes are added in, it can loading after balance；After collecting thalline, add per 100mg thalline
Enter 1-5ml Binding Buffer, ultrasound cracking thalline；12000rpm is centrifuged, and abandons supernatant, precipitation is resuspended in and is added in advance
In the Binding Buffer of 1-5mM PMSF, ultrasonication is carried out, until inclusion body is cleaned up (in cleaner milky white
Color shape)；Precipitation is resuspended in the Binding Buffer of the urea containing 8M, ice bath 1h makes solubilization of inclusion bodies；By cellular lysate liquid
Upper prop is loaded, flow velocity is 10 times of column volume/hours；Pillar is rinsed using the Wash buffer of 15 times of column volumes, collection flows through
Peak；It is eluted using the Elution Buffer of 5 times of column volumes, collects eluting peak；Successively using the Binding of 3 times of column volumes
It after the deionized water washing of Buffer and 5 times of column volume, is balanced with 20% ethyl alcohol of 3 times of column volumes, 4 DEG C of preservations.
As a result：After Ni-NTA affinitive layer purifications, high-purity fusion protein CWAE can be obtained, purity is 96.8% (figure
4)。
Embodiment 5：The immunogenicity of Hp multivalence epitope vaccines CWAE and immunologic specificity research
(1) BALB/c mouse is immune
Experiment packet：SPF grades of BALB/c mouses are randomly divided into 5 groups, respectively Hp ureases (Ure) immune group, Hp urine
Plain enzyme B subunits (UreB) immune group, Hp multivalence epitopes vaccine (CWAE) immune group, recombinant cholera toxin b subunit (rCTB) are immune
Group and PBS immune groups.Every group of 6 BALB/c mouses, are grouped as follows shown totally in detail by 30：
Table 1：SPF grades of BALB/c mouse groupings and immunization protocol
Immunization ways：It is sterilized with 75% chronic ethanol treated mice abdomen, then fusion protein and Freund is subcutaneously injected in abdomen multiple spot
The blended emulsifier of Freund's complete adjuvant；Booster immunization is primary week about, the 2nd and 3 week plus incomplete Freund's adjuvant, and the 4th week directly
Inject fusion protein solution booster immunization.
Sero-fast acquisition：Tail vein is taken to take a blood sample before being immunized every time, detect the change of serum specific antibody level
Change；The 5th day after final immunization, eyeball of mouse blood sampling is plucked, collects blood, placement treats that serum is kept completely separate, 3000rpm centrifugations 5
Minute packing serum, -70 DEG C freeze it is spare.
(2) ELISA of specific antibody is detected in antiserum
By antigen (natural Hp ureases, UreA, rUreB, HSP60, HpaA, NAP, UreA183-203、UreB327-334、
HpaA132-141、HSP60189-203) with coating buffer it is diluted to 5 μ g/ml or 10 μ g/ml, 100 μ l/ holes coating elisa plate, 4 DEG C of mistakes
Night.After washing 4 times with cleaning solution, 300 μ l confining liquids, 37 DEG C of closing 2h are added in per hole.After washing 4 times with cleaning solution, by antiserum (mouse
The anti-UreB antiserums of anti-Ure antiserums, mouse, the anti-CWAE antiserums of mouse and the anti-CTB antiserums of mouse) and mouse negative serum multiple proportions it is dilute
Elisa plate is added in after releasing, 100 μ l/ holes are incubated 60min at 37 DEG C.After washing 4 times with cleaning solution, the goat-anti of HRP labels is added in
Mouse IgG (1: 10000), 100 μ l/ holes, 1h is incubated at 37 DEG C.After washing 4 times with cleaning solution, tmb substrate developing solution, room temperature are added in
15min is protected from light, 50 μ l terminate liquids is added to terminate reaction.Each hole OD is measured with microplate reader450Value.
As a result：A concentration of 5 μ g/ml of coating of natural Hp ureases, are detected, Hp multivalence epitope vaccines CWAE by ELISA
The antiurease antibody with urease (Ure) and urease B subunit (UreB) similar level can be generated, and cholera toxin B is sub-
Base (CTB) cannot generate anti-native urease antibody (Fig. 5)；A concentration of 5 μ g/ml of coating of urease A subunits (UreA), pass through
ELISA detections, Hp multivalence epitope vaccine CWAE and urease (Ure) immune group can generate the anti-UreA antibody of higher level,
And urease B subunit (UreB) and b subunit of cholera toxin (CTB) cannot generate anti-UreA specific antibodies (Fig. 6)；Urease B
A concentration of 5 μ g/ml of coating of subunit (UreB), are detected by ELISA, Hp multivalence epitope vaccines CWAE, urease (Ure) and urine
Plain enzyme B subunits (UreB) can generate the anti-UreB antibody of higher level, and b subunit of cholera toxin (CTB) cannot generate it is anti-
UreB antibody (Fig. 7)；A concentration of 5 μ g/ml of coating of heat shock protein HSP60, are detected, only Hp multivalence epitopes by ELISA
Vaccine CWAE can generate the anti-HSP60 antibody (Fig. 8) of higher level；The coating a concentration of 5 of neutrophil activating protein NAP
μ g/ml, are detected by ELISA, and only Hp multivalence epitopes vaccine CWAE immune groups can generate the anti-NAP antibody of higher level
(Fig. 9)；A concentration of 5 μ g/ml of coating of adhesin HpaA, are detected, only Hp multivalence epitopes vaccine CWAE immune groups by ELISA
The anti-HpaA antibody (Figure 10) of higher level can be generated；Urease epitope UreA183-203And UreB327-334Coating it is dense
It spends for 10 μ g/ml, is detected by ELISA, only Hp multivalence epitopes vaccine CWAE immune groups can generate the urea of higher level
Enzyme epitope (UreA183-203And UreB327-334) specific antibody (Figure 11 and Figure 12)；Adhesin epitope HpaA132-141And heat
Shock protein HSP60 epitopes HSP60189-203A concentration of 10 μ g/ml of coating, detected by ELISA, only Hp multivalence table
Position vaccine CWAE immune groups can generate the anti-HpaA of higher level132-141And HSP60189-203Specific antibody (Figure 13 and figure
14)。
The above results illustrate Hp multivalence epitope vaccines CWAE can stimulate body generate for urease, urease A subunits,
Urease B subunit, heat shock protein HSP60, adhesin HpaA, neutrophil activating protein NAP, epitope UreA183-203、
UreB327-334、HpaA132-141、HSP60189-203High titre specific antibody, have good immunogenicity.
(3) mouse spleen lymphocyte proliferation experiment
The mouse being immunized through antigen is dislocated and is put to death, after steeping 75% alcohol 5min, moves into superclean bench.It is careful with scissors
The abdomen crust of mouse is cut off, then cuts off the abdominal cavity of mouse, mouse spleen (kermesinus) is taken out with tweezers.In the sterile small of 20ml
4~5ml EZ-Sep are put into beakerTMMouse 1X lymphocyte separation mediums；Nylon wire is fixed with tweezers, then uses syringe
Piston is lightly ground mouse spleen so that the unicellular of dispersion is entered through nylon wire in lymphocyte separation medium；Have outstanding
The separating liquid of spleen cell is immediately transferred in centrifuge tube, covers 1640 culture mediums of about 200 μ l before centrifugation again；800g from
Heart 30min.Lymphocyte can float up after centrifugation, assemble below 1640 coatings.With containing 10% calf serum
RPMI-1640 culture mediums are prepared into certain density cell suspension (5x 106/ml).In 96 hole flat-bottomed plates, add per hole
Enter 100 μ l cells, while add in CWAE, urease, urease A subunits, urease B subunit, neutrophil activating protein (20
μ g/ml), helicobacter pylori lysate and each 100 μ l of physiological saline, final volume is 200 μ l/ holes, and every group is done 3 parallel holes.
The 96 hole flat-bottomed plates added are placed into 37 DEG C of 5%CO2After cultivating 60h in incubator, MTT solution is added in into each hole
(5mg/ml) 30 μ l, continue after cultivating 4h, supernatant are gently sucked out, and are read after 100 μ l of DMSO oscillation mixings are added in per hole with microplate reader
Take OD570Value.Stimulus index calculation formula is as follows：
As a result：The mouse spleen lymphocyte that Hp multivalence epitope vaccine CWAE sensitization is crossed is sub- through Hp ureases, urease A
It is thin that apparent lymph can occur for base, urease B subunit, neutrophil activating protein NAP, Hp lysate and CWAE stimulations
Born of the same parents' breeder reaction, and the mouse spleen lymphocyte of PBS immune groups receives that lymphocyte increasing does not occur during above-mentioned antigenic stimulus
Reaction is grown, illustrates that it is immune to maintain it for the Th epitopes or section of Hp toxin proteins and adhesion factor in multivalence epitope vaccine CWAE
Characteristic is learned, body can be stimulated to generate specificity cellular immunity response (Figure 15).
Claims (7)
1. a kind of helicobacter pylori multivalence epitope vaccine, active constituent is a polypeptide, mainly by b subunit of cholera toxin
(CTB) it is formed with multi-epitope peptide WAE, amino acid sequence such as SEQ ID NO:Shown in 1.
2. helicobacter pylori multivalence epitope vaccine as described in claim 1, coding nucleotide sequence such as SEQ ID NO:
Shown in 2.
3. helicobacter pylori multivalence epitope vaccine as described in claim 1, multi-epitope peptide WAE is mainly bis- by urease A and B
Subunit, adhesin HpaA, the advantage Th of heat shock protein HSP60 and B cell antigen epi-position or section and neutrophil leucocyte swash
Living protein NAP is formed, amino acid sequence such as SEQ ID NO:Shown in 3.
4. helicobacter pylori multivalence epitope vaccine as claimed in claim 3, the wherein nucleotides sequence of encoding polyepitope peptide WAE
Row such as SEQ ID NO:Shown in 4.
5. include the expression vector of the nucleotide sequence described in claim 2, transgenic cell line and host strain.
6. helicobacter pylori multivalence epitope vaccine as described in claim 1, b subunit of cholera toxin (CTB) and multi-epitope peptide
Intervening sequence between WAE is DPRVPSS.
7. the preparation method of helicobacter pylori multivalence epitope vaccine as claimed in claim 4, is closed by gene synthesis technology
Into the nucleotide sequence of multi-epitope peptide WAE, merged in the downstream of b subunit of cholera toxin (CTB) gene, structure contains fusion
The recombinant expression carrier pETCWAE of gene C WAE and its recombination engineering bacteria, after the fermentation of recombination engineered strain, through Ni-
NTA nickel ion displacement chromatographies obtain the fusion protein CWAE of the vaccine.
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| CN112048008B (en) * | 2020-08-12 | 2021-06-15 | 河北医科大学第四医院（河北省肿瘤医院） | Helicobacter pylori B cell tolerance epitope and antibody prepared from same |
| CN112190704B (en) * | 2020-10-13 | 2022-11-15 | 宁夏医科大学 | M cell targeting recombinant lactobacillus vaccine, preparation method and application |
| CN112107682B (en) * | 2020-10-13 | 2023-05-05 | 宁夏医科大学 | M cell targeting recombinant lactobacillus vaccine, preparation method and application |
| CN114907491B (en) * | 2022-06-21 | 2023-06-16 | 中国科学院西北生态环境资源研究院 | Multi-epitope peptide, helicobacter pylori octavalent multi-epitope vaccine and preparation method |
| CN116068170B (en) * | 2022-09-28 | 2023-09-15 | 北京金沃夫生物工程科技有限公司 | Test paper and kit for detecting Helicobacter pylori |
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