CN106480003B - The combination of helicobacter pylori dominant antigen and screening technique based on CD4+T cellular immunity - Google Patents

The combination of helicobacter pylori dominant antigen and screening technique based on CD4+T cellular immunity Download PDF

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CN106480003B
CN106480003B CN201610931700.1A CN201610931700A CN106480003B CN 106480003 B CN106480003 B CN 106480003B CN 201610931700 A CN201610931700 A CN 201610931700A CN 106480003 B CN106480003 B CN 106480003B
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helicobacter pylori
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吴超
邹全明
孙合强
袁寒梅
赵�卓
谭燃景
李滨
郭刚
章金勇
敬海明
秦溢
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Third Military Medical University TMMU
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Abstract

The present invention relates to a kind of combination of helicobacter pylori dominant antigen and preparation method based on CD4+T cellular immunity, dominant antigen combination includes the following three types and its homologous protein: carnine acidohydrogenase, II type citrate synthase and urease B subunit；Its amino acid sequence is respectively as shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3.The CD4+T cellular immunity dominant antigen combination that the present invention is screened has obvious immanoprotection action, and protecting effect is better than H.pylori holoprotein antigen, has stronger helicobacter pylori Scavenging activity, while causing lighter pathology damage.Three kinds of Immunodominant Antigenics provided by the present invention can induce body and generate strong immune response for antigen.Therefore response is generated for immune protective dominant antigen by induction body; or body directly is immunized with protective antigens will helicobacter pylori infections be played with effective immanoprotection action, it can be used for that further helicobacter pylori is preventative and the research of therapeutic multivalent vaccine.

Description

The combination of helicobacter pylori dominant antigen and screening technique based on CD4+T cellular immunity
Technical field
The invention belongs to technical field of pharmaceutical biotechnology, are related to a kind of helicobacter pylori dominant antigen combination, and in particular to one Helicobacter pylori dominant antigen combination and screening technique of the kind based on CD4+T cellular immunity.
Background technique
Helicobacter pylori (Helicobacter pylori, H.pylori) is colonized in gastric mucosa, is I class that gastric cancer occurs Virulence factor is the master that the gastrointestinal diseases such as chronic gastritis, gastroduodenal ulcer, gastric mucosa associated lymphoid tissue lymthoma occur Want pathogenic factor.Global infection rate is more than 50%, therefore there is an urgent need to a kind of effective helicobacter pylori vaccines.
The screening of candidate antigens is the core of vaccine development.Different from the virus with Limiting antigen number, bacterium is due to containing There are hundreds of a large amount of antigens, be difficult to carry out systemic screening to it, so that it is determined that the protective antigens of its immunodominance.This The research and development of anti-bacterial vaccine are hampered always.
Research has shown that stomach lining part th1 and th17 cell have played important function in anti-H.pylori infection.It utilizes CD4+T cell Dominant Epitopes H.pyloriaA (88-100), it was demonstrated that in the restricted crowd of HLA-DRB1*1501, The CD4+T response of H.pyloriaA (88-100) epitope specificity and stomach trouble severity are closely related.Hitzler, I. couple H.pylori immune mouse B cell and CD4+T cell response is analyzed, and data show th1 and th17 cell wherein Play main protective effect, rather than the humoral immunity that B cell mediates.Likewise, the research of DeLyria, E.S. are also supported Protective effect of the th17 in H.pylori is immune.
But the Immunodominant Antigenic of th1 and th17 is also never by screening system mistake, based on this novel helicobacter pylori Vaccine is not also developed.
Summary of the invention
In view of this, the purpose of the present invention is to provide a kind of helicobacter pylori advantages based on CD4+T cellular immunity to resist Original combination.
The present invention also provides the screening techniques of above-mentioned dominant antigen combination.
In order to achieve the above objectives, the invention provides the following technical scheme:
Helicobacter pylori dominant antigen combination based on CD4+T cellular immunity, includes the following three types and its homologous protein: secondary Xanthosine acidohydrogenase (inosine 5'-monophosphate dehydrogenase, IMPDH), II type citric acid close Enzyme (type II citrate synthase, CS II) and urease B subunit (urease subunit beta, UreB)； Its amino acid sequence is respectively as shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3.
Preferably, the mass ratio of three kinds of antigen is 1:1:1, and the total antigenic content of vaccine is 100 μ g.
Application of the above-mentioned dominant antigen combination in the vaccine of preparation prevention or treatment helicobacter pylori infections.
Preferably, the vaccine is albumen or nucleic acid vaccine.
Preferably, the vaccine also includes medically acceptable immunologic adjuvant.
It is further preferred that the immunologic adjuvant is in Freund's adjuvant, aluminium adjuvant, CPG ODN 1826 or AddaVax It is any one or several.
The screening technique of above-mentioned dominant antigen combination, comprising the following steps:
1) helicobacter pylori thallus is cracked, helicobacter pylori holoantigen lysate is obtained, then according to each molecular weight of albumen Size be sieved into 30 components with molecule；
2) the spleen CD4+T from helicobacter pylori infections and immune mouse is stimulated respectively using 30 components of step 1) Cell is used3H-TdR incorporation methods detect CD4+T cell proliferative conditions, and are mapped with proliferation index, obtain stimulation CD4+T cell and increase Grow ability strongest advantage component PC05 and subdominant component PC17；
3) mouse is immunized respectively and carries out attacking poison by the advantage component PC05 and subdominant component PC17 filtered out using step 2) Protection is compareed with the full bacterial immunity of H.pylori and PBS, determines that advantage component PC05 has stronger immune clearance energy Power, while causing lighter inflammation damnification；
4) use the method for high performance liquid chromatography-mass spectrometry LC-MS/MS by institute in the resulting advantage component PC05 of step 3) The protein component contained is defined, and determines that PC05 contains 11 kinds of albumen；
5) utilize genetic engineering method synthesis step 4) determine 11 kinds of albumen；
6) stimulate PC05 specific C D4+T cell respectively with 11 kinds of albumen that step 5) synthesizes, screening and identification go out Th1 and The strongest H.pylori antigen of Th17 response, totally three kinds.
Preferably, 8M Urea Lysis helicobacter pylori thallus is used in step 1).
The beneficial effects of the present invention are:
After H.pylori infects gastric mucosa, H.pylori antigen can be huge by Dendritic Cells (dendritic cells, DC) The antigen presenting cells such as phagocyte (antigen-presenting cells, APC) identify and process submission.APC then swashs Original CD4+T cell livingThe Th1 cell response of secretion inducing IFN-γ is with secretion IL-17A's Th17 cell response.IL-17 can promote to express gastric epithelial cell, interstitial cell, endothelial cell, the mucous membrane of IL-17 receptor Lamina propria monocyte etc. secretes the cell factors such as IL-1, IL-6, IL-8 and TNF-α, and raises neutrophil leucocyte removing H.pylori。
Therefore applicant attempts the Immunodominant Antigenic that the systematicness from H.pylori full bacterium filters out Th1 and Th17, and base Novel helicobacter pylori vaccine is developed in this.In the process, each antigen of the full bacterium of H.pylori, which is included into synchronize, comments It surveys, until Immunodominant Antigenic is screened out.Meanwhile the immune protective rate of each antigen is verified in mouse model and inflammation is commented Point etc. indexs.As a result, successfully filtering out three dominant antigens of Th1 and Th17: IMPDH, CS II and UreB.Immunoprotection is real Verifying bright its has stronger H.pylori Scavenging activity and lighter immunopathogenesis is caused to damage.This is the following novel helicobacter pylorus Designing and developing for bacteria vaccine provides new candidate antigens.
The CD4+T cellular immunity dominant antigen combination screened as the result is shown has obvious immanoprotection action, protection Effect is better than H.pylori holoprotein antigen, has stronger helicobacter pylori Scavenging activity, while lighter pathology being caused to damage Wound.Three kinds of Immunodominant Antigenics provided by the present invention can induce body and generate strong immune response for antigen. Therefore response is generated for immune protective dominant antigen by induction body, or body directly is immunized with protective antigens will be right Helicobacter pylori infections play effective immanoprotection action, can be used for the preventative and therapeutic multivalence of further helicobacter pylori The research of vaccine.
Detailed description of the invention
In order to keep the purpose of the present invention, technical scheme and beneficial effects clearer, the present invention provides following attached drawing and carries out Illustrate:
Figure 1A~1D is that H.pylori holoantigen has immanoprotection action, and excites IFN-γ and IL-17A in gastric mucosa Response, wherein Figure 1A is bacteria planting amount, and Figure 1B -1 and Figure 1B -2 are stomach single cell suspension Flow cytometry, and Fig. 1 C is CD3+T, CD4+T cell quantity, Fig. 1 D are Mouse Gastric Mucous Membrane IFN-γ and IL-17A mRNA expression.
Fig. 2 is that H.pylori holoantigen is divided into 30 components according to molecular size range with molecular sieve chromatography.
Fig. 3 A and Fig. 3 B are advantage component screening, and wherein Fig. 3 A is that/infected group CD4+T lymphocyte is immunized in each component stimulation Proliferative conditions, Fig. 3 B are that each component stimulation be immunized/is uninfected by a group CD4+T lymphopoiesis situation.
Fig. 4 A~4C is H.pylori holoantigen, the evaluation of PC05, PC17 immunoprotection, and wherein Fig. 4 A is fixed for gastric mucosa bacterium Plant amount, Fig. 4 B are gastric tissue pathological score, and Fig. 4 C is gastric tissue pathological section.
Fig. 5 A~5C is that advantage component PC05 excites stronger Th1 and Th17 response, and wherein Fig. 5 A is that H.pylori is complete After antigen, PC05, PC17 Immunization, Mouse Gastric Mucous Membrane IFN-γ and IL-17AmRNA expression, Fig. 5 B are antigen-specific Property CD4+T cell Flow cytometry, Fig. 5 C be PC05 excite specificity T h1 and Th17 response level be better than PC17 and The full bacterium antigen of H.pylori.
Fig. 6 A~6D is that P5 (IMPDH), P10 (CS II) and P11 (UreB) excite stronger specificity T h1 and Th17 Response.
Fig. 7 is that Immunodominant Antigenic IMPDH, CS II and UreB has better immunity protection function.
Specific embodiment
Below in conjunction with attached drawing, a preferred embodiment of the present invention will be described in detail.
Helicobacter pylori Strains B0 of the present invention is to tame to obtain in BALB/c mouse body.
Materials and methods
(1) strain and mouse
Helicobacter pylori Strains B0: this bacterial strain is the domestication strain that helicobacter pylori is tamed in BALB/c mouse body, Its main feature is that being easier to be colonized in BALB/c mouse stomach, and lead to pathological reaction.From helicobacter pylori carrier's gastric tissue point From culture Helicobacter pylori Strains, the brain heart infusion agar culture containing 10% (volume ratio) rabbit blood is inoculated in by " trilinear method " Base.The single bacterium colony of picking is cultivated, and is accredited as helicobacter pylori by gene sequencing, is named as Helicobacter pylori Strains M. Then BALB/c mouse is infected with Helicobacter pylori Strains M.Then repeat it is above-mentioned isolate and purify scheme, from BALB/c mouse stomach group Knit separation helicobacter pylori.The thus obtained Helicobacter pylori Strains for being easier to be colonized in BALB/c mouse, are named as pylorus Screw rod bacteria strain B0.
For Helicobacter pylori Strains B0 in the brain heart infusion agar culture medium containing 10% (volume ratio) rabbit blood, 37 DEG C micro- aerobic Under the conditions of cultivate.Two days later, Helicobacter pylori Strains are transferred to 10% (volume ratio) fetal calf serum (FBS) Bu Shi meat from plate Soup culture medium expands culture.Take the helicobacter pylori of exponential phase of growth for infection and experiment in vitro.Six to eight week old without spy Determine cause of disease (SPF) female BAl BIc/c mouse, is purchased from Third Military Medical University's Experimental Animal Center.
(2) main agents and source
Specifically it is shown in Table 1.
The main agents of the invention of table 1. and source
The screening technique of dominant antigen combination of the invention, comprising the following steps:
1) 8M Urea Lysis helicobacter pylori thallus is used, obtains helicobacter pylori holoantigen Urea Lysis liquid, then root 30 components are sieved into molecule according to the size of each molecular weight of albumen；
2) the spleen CD4+T from helicobacter pylori infections and immune mouse is stimulated respectively using 30 components of step 1) Cell is used3H-TdR incorporation methods detect CD4+T cell proliferative conditions, and are mapped with proliferation index, obtain stimulation CD4+T cell and increase Grow ability strongest advantage component PC05 and subdominant component PC17；
3) mouse is immunized respectively and carries out attacking poison by the advantage component PC05 and subdominant component PC17 filtered out using step 2) Protection is compareed with the full bacterial immunity of H.pylori and PBS, determines that advantage component PC05 has stronger immune clearance energy Power, while causing lighter inflammation damnification；
4) use the method for high performance liquid chromatography-mass spectrometry LC-MS/MS by institute in the resulting advantage component PC05 of step 3) The protein component contained is defined, and determines that PC05 contains 11 kinds of albumen；
5) utilize genetic engineering method synthesis step 4) determine 11 kinds of albumen；
6) stimulate PC05 specific C D4+T cell respectively with 11 kinds of albumen that step 5) synthesizes, screening and identification go out Th1 and The strongest H.pylori antigen of Th17 response, totally three kinds；
7) mouse is immunized respectively and carries out attacking malicious protection for Th1 the and Th17 cellular immunity dominant antigen filtered out using step 6) Experiment is compareed so that PC05 and PBS is immune.Determine that three kinds of Immunodominant Antigenics all have apparent immune clearance ability, simultaneously Cause lighter inflammation damnification.
Embodiment 1:
The full bacterial immunity mouse excitation CD4+T cell response evaluation of H.pylori
1. animal immune and challenge viral dosage scheme
Experimental animal: 6~8 week old of BALB/c mouse female.
Antigen:
(1) it be immunized/attacks malicious group of (I/C): H.pylori and inactivates full bacterium.100 μ g/ are only.
(2) it be immunized/does not attack malicious group (U/C): phosphate buffer (PBS).
Adjuvant: Freund's adjuvant.100 μ l/ are only.
Immunization ways: subcutaneous injection.
Immune volume: 200 μ l/ are only.
Immunization protocol: subcutaneous inoculation 3 times (the 0,2,4th week).Complete Freund's adjuvant is used for the first time, is cannotd be used up for the second time entirely not Family name's adjuvant, is not added adjuvant for the third time.
Final immunization latter week, 1.0 × 109CFU helicobacter pylori stomach-filling, once a day, continuous 4 days.It attacks after poison the 4th week Mouse is put to death, helicobacter pylori definite value amount, CD4+T cell response in mouse gastric tissue is detected, analyzes its immune protective effect.
2. Mouse Gastric Mucous Membrane helicobacter pylori definite value amount detects.
Real-time quantitative PCR detects helicobacter pylori field planting amount in stomach.Bacterium is extracted with bacterial genomes extracts kit DNA detects its field planting amount according to helicobacter pylori 16SrDNA.The result is shown in Figure 1 A.
The sequence of helicobacter pylori 16SrDNA is as follows:
Upstream primer: 5 '-TTTGTTAGAGAAGATAATGACGGTATCTAAC-3 ', as shown in SEQ ID NO.4；
Downstream primer: 5 '-CATAGGATTTCACACCTGACTGACTATC-3 ', as shown in SEQ ID NO.5.
The sequence of fluorescence probe is as follows:
FAM-CGTGCCAGCAGCCGCGGT-TAMRA, as shown in SEQ ID NO.6.
3. Mouse Gastric Mucous Membrane CD4+T cell response detects
Mouse gastric tissue is dissected along greater curvature and lesser curvature, is softly washed 2 times with sterile PBS, to remove swill.And After be put into 10ml Hank's balanced salt solution (HBSS, be free of Ca, My), dithiothreitol (DTT) containing 1mM (DTT), 1mM ethylenediamine Tetraacethyl (EDTA) and 2% fetal calf serum (FCS), 37 DEG C of incubation 45min.Then, by gained mixture by sterile steel mesh with It removes indigested tissue and obtains single cell suspension.Postdigestive single cell suspension is washed twice with sterile PBS.It is then glimmering with being immunized The antibody (FITC-CD3, APC-CD4) of signal dyes and uses flow cytomery.With reference to Figure 1B -1, Figure 1B -2 and Fig. 1 C.
Real-time quantitative PCR detects Mouse Gastric Mucous Membrane part mRNA level in-site CD4+T cellullar immunologic response.Trizol method extracts stomach Mucous membrane total serum IgE, reverse transcription detect IFN-γ and IL-17A by real-time quantitative PCR at cDNA, and with SYBR Green incorporation methods Expression.The result is shown in Figure 1 D.
The sequence of IFN-γ is as follows:
Upstream primer: 5 '-GATCCTTTGGACCCTCTGACTT-3 ', as shown in SEQ ID NO.7；
Downstream primer: 5 '-TGACTGTGCCGTGGCAGTAA-3 ', as shown in SEQ ID NO.8.
The sequence of IL-17A is as follows:
Upstream primer: 5 '-CTCCAGAAGGCCCTCAGACTAC-3 ', as shown in SEQ ID NO.9；
Downstream primer: 5 '-GGGTCTTCATTGCGGTGG-3 ', as shown in SEQ ID NO.10.
Embodiment 2:
The full bacterium antigen of H.pylori is divided into different component according to molecular size range by sieve chromatography
Firstly, Helicobacter pylori Strains B0 is dissolved in 8mol/L urea, dithiothreitol (DTT) (DTT) 1g/ containing 10mmol/L 6ml is gently mixed 18 hours under the conditions of 4 DEG C.Secondly, 12000g is centrifuged, supernatant is collected, is filtered with 0.2 μM of filter.Again, By sieve chromatography, the protein of different molecular weight is divided into 30 groups, and mixed protein component is named as PC01~PC30 (figure 2).Molecular sieve selects 200 chromatographic column of 10/300GL Superdex, and applied sample amount 1ml, flow set 0.5mL/min are collected 1ml/ pipe.Sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) electrophoretic analysis of gained protein component, and it is pungent with two It can each protein component concentration of peaceful sour (BCA) detection kit measurement.As a result see Fig. 2.
Embodiment 3:
3H-TdR incorporation methods, which detect different component, stimulates CD4+T degree of cell proliferation
Take BALB/c mouse peritoneal macrophage is used as antigen presenting cell (APC).Every hole adds in 96 hole round bottom plates Enter 1 × 105APC and the complete RPMI1640 culture medium of 200 μ L.By itself and H.pylori bacterial strain B0 holoantigen or each protein component PC01~PC30 is in 37 DEG C of 5%CO2It is co-cultured in incubator, the ultimate density of each antigen is 50 μ g/mL, and every group sets 3 multiple holes.10 After hour, it is immunized/infects from H.pylori respectively and H.pylori be immunized/is uninfected by rear 4 weeks mouse, with magnetic bead sorting spleen Dirty CD4+T lymphocyte.1×105Spleen CD4+T lymphocyte and above-mentioned APC are co-cultured 96 hours.It was added at last 18 hours31 hole μ Ci/ H-TdR.Later, every hole CPM value is detected with liquid scintillation counter, and is indicated with stimulus index (SI), SI definition For experimental group CPM/ negative control CPM.As a result see Fig. 3 A and Fig. 3 B.
Embodiment 4:
The full bacterium of H.pylori, the evaluation of PC05, PC17 immunity protection function
1. animal immune and challenge viral dosage scheme
Experimental animal: 6~8 week old of BALB/c mouse female.
Antigen: H.pylori inactivates full bacterium, PC05, PC17, and 100 μ g/ are only.Equivalent PBS control.
Adjuvant: Freund's adjuvant.100 μ l/ are only.
Immunization ways: subcutaneous injection.
Immune volume: 200 μ l/ are only.
Immunization protocol: subcutaneous inoculation 3 times (the 0,2,4th week).Complete Freund's adjuvant is used for the first time, is cannotd be used up for the second time entirely not Family name's adjuvant, is not added adjuvant for the third time.
Final immunization latter week, 1.0 × 109CFU helicobacter pylori stomach-filling, once a day, continuous 4 days.It attacks after poison the 4th week Mouse is put to death, helicobacter pylori definite value amount, pathology damage, CD4+T cell response in mouse gastric tissue are detected, analyzes its immune guarantor Protect effect.
2. Mouse Gastric Mucous Membrane helicobacter pylori definite value amount detects.
Real-time quantitative PCR detects helicobacter pylori field planting amount in stomach.Bacterium is extracted with bacterial genomes extracts kit DNA detects its field planting amount according to helicobacter pylori 16SrDNA.As a result see Fig. 4 A.
The sequence of helicobacter pylori 16SrDNA is as follows:
Upstream primer: 5 '-TTTGTTAGAGAAGATAATGACGGTATCTAAC-3 ', as shown in SEQ ID NO.4；
Downstream primer: 5 '-CATAGGATTTCACACCTGACTGACTATC-3 ', as shown in SEQ ID NO.5.
The sequence of fluorescence probe is as follows:
FAM-CGTGCCAGCAGCCGCGGT-TAMRA, as shown in SEQ ID NO.6.
3. Mouse Gastric Mucous Membrane inflammatory score
A small amount of gastric tissue is longitudinally taken along greater curvature, is fixed with formalin, paraffin embedding, 5 μm of slices, and with hematoxylin- Eosin stains.Then histopathological scores are carried out under microscope.As a result see Fig. 4 B and Fig. 4 C.
Standards of grading are as follows: 0, without conspicuousness lesion；0.5, there is slight exception, such as small inflammatory infiltration stove or extensive nothing The mucous membrane metaplasia of inflammation；1.0, a kind of slight inflammatory cell infiltration, usually invades body of gland basal part；1.5, mild infiltration, then In addition slight epithelial proliferation or extensive mucilage cell metaplasia；2.0, inflammatory cell invades body of gland and/or submucosa；2.5, Inflammatory cell invades body of gland, while submucosa has mucilage cell metaplasia and/or slight epithelial hyperplasia；3.0, large stretch of inflammation Body of gland and submucosa are invaded, is often accompanied by moderate mucilage cell metaplasia and slightly to moderate epithelial proliferation；3.5,3.0 or more inflammation Disease is with obvious epithelial hyperplasia；4.0, the strong inflammatory infiltration of mucous layer is invaded, body of gland normal configuration is destroyed, and usual companion There are apparent epithelial proliferation and extensive mucilage cell metaplasia；4.5, serious inflammation is with mucous membrane focal ulcer；5.0, wide The general inflammation invaded under mucous membrane and mucous membrane, with gland structure destruction and ulcer.
4. Mouse Gastric Mucous Membrane CD4+T cell response detects
Mouse gastric tissue is dissected along greater curvature and lesser curvature, is softly washed 2 times with sterile PBS, to remove swill.And After be put into 10ml Hank's balanced salt solution (HBSS, be free of Ca, My), dithiothreitol (DTT) containing 1mM (DTT), 1mM ethylenediamine Tetraacethyl (EDTA) and 2% fetal calf serum (FCS), 37 DEG C of incubation 45min.Then, by gained mixture by sterile steel mesh with It removes indigested tissue and obtains single cell suspension.Postdigestive single cell suspension is washed twice with sterile PBS.Then use Trizol Method extracts its total serum IgE, and reverse transcription detects IFN-γ and IL- by real-time quantitative PCR at cDNA, and with SYBR Green incorporation methods The expression of 17A.As a result see Fig. 5 A.
The sequence of IFN-γ is as follows:
Upstream primer: 5 '-GATCCTTTGGACCCTCTGACTT-3 ', as shown in SEQ ID NO.7；
Downstream primer: 5 '-TGACTGTGCCGTGGCAGTAA-3 ', as shown in SEQ ID NO.8.
The sequence of IL-17A is as follows:
Upstream primer: 5 '-CTCCAGAAGGCCCTCAGACTAC-3 ', as shown in SEQ ID NO.9；
Downstream primer: 5 '-GGGTCTTCATTGCGGTGG-3 ', as shown in SEQ ID NO.10.
5. mouse boosting cell antigentic specificity CD4+T cell response detects.
Polishing separates H.pylori infecting mouse splenic lymphocytes.Then 1 × 107Lymphocyte and 100 μ g H.pylori bacterial strain B0 holoantigen co-cultures, and adds 5U/ml recombined small-mouse interleukin 2 (rmIL-2), the hole 3ml/ is complete RPMI1640 culture medium, 12 orifice plates.37 DEG C, 5%CO2After incubator culture 5 days, dead cell is removed using Ficoll gradient centrifugation. Living cells is passed on, 20U/ml rmIL-2, complete RPMI1640 culture are added, until obtaining within the 12nd day the full bacterium specificity of H.pylori Lymphocyte.In the meantime, half amount is carried out when needing changes liquid.By the specific lymphocyte 1 × 10 of preparation5It is corresponding to submission The APC cell 1 × 10 of antigen5It is co-cultured 5 hours in 96 hole round bottom plates, every hole adds 200 μ L golgistop containing BD complete RPMI1640 culture medium.Then fluorescent labeled antibody FITC-CD3, APC-CD4, PE-IFN- γ, PerCP-Cy5.5-IL- are used 17A is dyed and is used flow cytometry analysis.As a result see Fig. 5 B and Fig. 5 C.
Embodiment 5:
High performance liquid chromatography-mass spectrometry parses protein component
PC05 protein component is parsed with tablets by HPLC-MS (LC-MS/MS Analysis).By PC05 egg Informal voucher band in PAGE gel from being transferred in EP pipe and with protease cracking, and pyrolysis product is by Maxis 4G UHR Q-TOF Mass spectrograph is detected.The data of all records Proteome DiscovererTM1.4Software software is analyzed (match Silent winged generation that science and technology, the U.S.) and be compared with ncbi.fasta database (ftp: //ftp.ncbi.nih.gov/), thus Determine the specific protein component of protein groups PC05.
PC05 protein component and amino acid sequence
PC05 includes 11 protein components (table 2).
11 protein components of table 2.PC05
P5inosine 5'-monophosphate dehydrogenase (IMPDH) carnine acidohydrogenase
MRILQRALTFEDVLMVPRKSSVLPKDVSLKSRLTKNISLNIPFISAAMDTVTEHKTAIAMARLGGIGIV HKNMDIQTQVKEITKVKKSESGVINDPIFIHAHRTLADAKVITDNYKISGVPVVDDKGLLIGILTNRDVRFETDLSK KVGDVMTKMPLVTARVGISLEEARDLMHKHKIEKLPIVDKDNVLKGLITIKDIQKRIEYPEANKDDFGRLRVGAAIG VGQLDRAEMLVKAGVDALVLDSAHGHSANILHTLEEIKKSLVVDVIVGNVVTKEATSDLISAGADAIKVGIGPGSIC TTRIVAGVGMPQVSAIDNCVEVASKFDIPVIADGGIRYSGDVAKALALGASSVMIGSLLAGTEESPGDFMIYQGRQY KSYRGMGSIGAMTKGSSDRYFQEGVASEKLVPEGIEGRVPYRGKVSDMIFQLVGGVRSSMGYQGAKNILELYQNAEF VEITSAGLKESHVHGVDITKEAPNYYG
P10type II citrate synthase (CS II) II type citrate synthase
MSVTLVNNENNERYEFETIESTRGPKAVDFSKLFETTGFFSYDPGYSSTAGCQSKISYVNGKKGELYYR GHRIEDLVAKYKYVDVCKLLLTGELPKNQDESLEFELELRHRSFVHESLLNMFSAFPSNAHPMAKLSSGVSILSTLY STHQNMHTEEDYQTMARRIVAKIPTLAAICYRNEVGAPIIYPDIARSYVENILFMLRGYPYSRLKHTTQGEVEITPL EVEAFDKILTLHADHSQNASSTTVRNVASTGVHPYAAISAGISALWGHLHGGANEKVLLQLEEIGDVKNVDKYIARV KDKNDNFKLMGFGHRVYKSYDPRAKILKGLKDELHQKGVKMDERLSEIAAKVEEIALKDEYFIERNLYPNVDFYSGT ILRALKIPVRFFTPVFVIGRTVGWCAQLLEHVKSPQARITRPRQVYVGD
P11urease subunit beta (UreB) urease B subunit
MKKISRKEYASMYGPTTGDKVRLGDTDLIAEVEHDYTIYGEELKFGGGKTLREGMSQSNNPSKEELDLI ITNALIVDYTGIYKADIGIKDGKIAGIGKGGNKDMQDGVKNNLSVGPATEALAGEGLIVTAGGIDTHIHFISPQQIP TAFASGVTTMIGGGTGPADGTNATTITPGRRNLKFMLRAAEEYSMNIGFLAKGNASNDASLADQIEAGAIGLKIHED WGTTPSAINHALDVADKYDVQVAIHTDTLNEAGCVEDTMAAIAGRTMHTYHTEGAGGGHAPDIIKVAGEHNILPAST NPTIPFTVNTEAEHMDMLMVCHHLDKSIKEDVQFADSRIRPQTIAAEDTLHDMGIFSITSSDSQAMGRVGEVITRTW QTADKNKKEFGRLKEEKGDNDNFRIKRYLSKYTINPAIAHGISEYVGSVEVGKVADLVLWSPAFFGVKPNMIIKGGF IALSQMGDANASIPTPQPVYYREMFAHHGKAKYDANITFVSQAAYDKGIKEELGLERQVLPVKNCRNITKKDMQFND TTAHIEVNSETYHVFVDGKEVTSKPANKVSLAQLFSIF
Embodiment 6:
The clonal expression of 11 kinds of albumen in PC05 component
H.pylori DNA is extracted with bacterial genomes extracts kit.Gene order design according to 11 kinds of albumen is up and down Primer is swum, PCR amplification obtains target gene.It selects pGEX-6P-1 as vector plasmid, induces table in engineering bacteria E.Coli It reaches.The mesh containing GST label is separated with GST label protein purification filler (Glutathione Sepharose 4Fast Flow) Albumen.GST label then is cut off with Prescission Protease, obtains destination protein.It is stand-by that BCA method measures concentration.
Embodiment 7:
CD4+T lymphocyte dominant antigen is identified in PC05 component
Polishing separates PC05 component and mouse spleen lymphocyte is immunized.Then 1 × 107Lymphocyte and 100 μ g PC05 groups Point antigen co-cultures, and adds 5U/ml recombined small-mouse interleukin 2 (rmIL-2), the complete RPMI1640 culture medium in the hole 3ml/, and 12 Orifice plate.37 DEG C, 5%CO2After incubator culture 5 days, dead cell is removed using Ficoll gradient centrifugation.Living cells is passed on, is added 20U/ml rmIL-2, complete RPMI1640 culture, until obtaining the full bacterium specificity of H.pylori or single proteantigen spy on the 12nd day Anisotropic lymphocyte.In the meantime, half amount is carried out when needing changes liquid.By the specific lymphocyte 1 × 10 of preparation5With pass respectively It has been in the APC cell 1 × 10 of 11 kinds of antigen in PC055It is co-cultured 5 hours in 96 hole round bottom plates, every hole adds 200 μ L containing BD The complete RPMI1640 culture medium of golgistop.Then with fluorescent labeled antibody FITC-CD3, APC-CD4, PE-IFN- γ, PerCP-Cy5.5-IL-17A is dyed and is used flow cytometry analysis.As a result see Fig. 6 A and Fig. 6 B.
Embodiment 8:
Dominant antigen IMPDH, CS II and the evaluation of UreB immunity protection function
1. animal immune and challenge viral dosage scheme
Experimental animal: 6~8 week old of BALB/c mouse female.
Antigen: IMPDH, CS II and UreB, 100 μ g/ are only.Equivalent PBS control.
Adjuvant: Freund's adjuvant.100 μ l/ are only.
Immunization ways: subcutaneous injection.
Immune volume: 200 μ l/ are only.
Immunization protocol: subcutaneous inoculation 3 times (the 0,2,4th week).Complete Freund's adjuvant is used for the first time, is cannotd be used up for the second time entirely not Family name's adjuvant, is not added adjuvant for the third time.
Final immunization latter week, 1.0 × 109CFU helicobacter pylori stomach-filling, once a day, continuous 4 days.It attacks after poison the 4th week Mouse is put to death, helicobacter pylori definite value amount, pathology damage, CD4+T cell response in mouse gastric tissue are detected, analyzes its immune guarantor Protect effect.
2. Mouse Gastric Mucous Membrane helicobacter pylori definite value amount detects.
Real-time quantitative PCR detects helicobacter pylori field planting amount in stomach.Bacterium is extracted with bacterial genomes extracts kit DNA detects its field planting amount according to helicobacter pylori 16SrDNA.As a result see Fig. 6 C.
The sequence of helicobacter pylori 16SrDNA is as follows:
Upstream primer: 5 '-TTTGTTAGAGAAGATAATGACGGTATCTAAC-3 ', as shown in SEQ ID NO.4；
Downstream primer: 5 '-CATAGGATTTCACACCTGACTGACTATC-3 ', as shown in SEQ ID NO.5.
The sequence of fluorescence probe is as follows:
FAM-CGTGCCAGCAGCCGCGGT-TAMRA, as shown in SEQ ID NO.6.
3. Mouse Gastric Mucous Membrane inflammatory score
A small amount of gastric tissue is longitudinally taken along greater curvature, is fixed with formalin, paraffin embedding, 5 μm of slices, and with hematoxylin- Eosin stains.Then histopathological scores are carried out under microscope.As a result see Fig. 6 D.
Standards of grading are as follows: 0, without conspicuousness lesion；0.5, there is slight exception, such as small inflammatory infiltration stove or extensive nothing The mucous membrane metaplasia of inflammation；1.0, a kind of slight inflammatory cell infiltration, usually invades body of gland basal part；1.5, mild infiltration, then In addition slight epithelial proliferation or extensive mucilage cell metaplasia；2.0, inflammatory cell invades body of gland and/or submucosa；2.5, Inflammatory cell invades body of gland, while submucosa has mucilage cell metaplasia and/or slight epithelial hyperplasia；3.0, large stretch of inflammation Body of gland and submucosa are invaded, is often accompanied by moderate mucilage cell metaplasia and slightly to moderate epithelial proliferation；3.5,3.0 or more inflammation Disease is with obvious epithelial hyperplasia；4.0, the strong inflammatory infiltration of mucous layer is invaded, body of gland normal configuration is destroyed, and usual companion There are apparent epithelial proliferation and extensive mucilage cell metaplasia；4.5, serious inflammation is with mucous membrane focal ulcer；5.0, wide The general inflammation invaded under mucous membrane and mucous membrane, with gland structure destruction and ulcer.
4. Mouse Gastric Mucous Membrane CD4+T cell response detects
Mouse gastric tissue is dissected along greater curvature and lesser curvature, is softly washed 2 times with sterile PBS, to remove swill.And After be put into 10ml Hank's balanced salt solution (HBSS, be free of Ca, My), dithiothreitol (DTT) containing 1mM (DTT), 1mM ethylenediamine Tetraacethyl (EDTA) and 2% fetal calf serum (FCS), 37 DEG C of incubation 45min.Then, by gained mixture by sterile steel mesh with It removes indigested tissue and obtains single cell suspension.Postdigestive single cell suspension is washed twice with sterile PBS.Then use Trizol Method extracts its total serum IgE, and reverse transcription detects IFN-γ and IL-17A at cDNA, and with SYBR Green incorporation methods real-time quantitative PCR Expression.As a result see Fig. 7.
The sequence of IFN-γ is as follows:
Upstream primer: 5 '-GATCCTTTGGACCCTCTGACTT-3 ', as shown in SEQ ID NO.7；
Downstream primer: 5 '-TGACTGTGCCGTGGCAGTAA-3 ', as shown in SEQ ID NO.8.
The sequence of IL-17A is as follows:
Upstream primer: 5 '-CTCCAGAAGGCCCTCAGACTAC-3 ', as shown in SEQ ID NO.9；
Downstream primer: 5 '-GGGTCTTCATTGCGGTGG-3 ', as shown in SEQ ID NO.10.
Finally, it is stated that preferred embodiment above is only used to illustrate the technical scheme of the present invention and not to limit it, although logical It crosses above preferred embodiment the present invention is described in detail, however, those skilled in the art should understand that, can be Various changes are made to it in form and in details, without departing from claims of the present invention limited range.
SEQUENCE LISTING
<110>Military Medical Univ No.3, P.L.A
<120>the helicobacter pylori dominant antigen combination based on CD4+T cellular immunity and screening technique
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Claims (6)

1. the helicobacter pylori dominant antigen based on CD4+T cellular immunity combines, which is characterized in that include the following three types: secondary Huang Purine nucleosides acidohydrogenase, II type citrate synthase and urease B subunit；Its amino acid sequence respectively as SEQ ID NO.1, Shown in SEQ ID NO.2 and SEQ ID NO.3.
2. dominant antigen combination according to claim 1, which is characterized in that the mass ratio of three kinds of antigen is 1:1:1, vaccine Total antigenic content is 100 μ g.
3. dominant antigen combination of any of claims 1 or 2 is in the vaccine of preparation prevention or treatment helicobacter pylori infections Using.
4. application according to claim 3, which is characterized in that the vaccine is albumen or nucleic acid vaccine.
5. application according to claim 3, which is characterized in that the vaccine also includes medically acceptable immune assistant Agent.
6. application according to claim 5, which is characterized in that the immunologic adjuvant is Freund's adjuvant, aluminium adjuvant, CPG Any one or more of ODN 1826 or AddaVax.
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