CN106480003A - Helicobacter pylori dominant antigen combination based on CD4+T cellular immunization and screening technique - Google Patents

Helicobacter pylori dominant antigen combination based on CD4+T cellular immunization and screening technique Download PDF

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CN106480003A
CN106480003A CN201610931700.1A CN201610931700A CN106480003A CN 106480003 A CN106480003 A CN 106480003A CN 201610931700 A CN201610931700 A CN 201610931700A CN 106480003 A CN106480003 A CN 106480003A
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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 helicobacter pylori dominant antigen combination based on CD4+T cellular immunization and preparation method, the combination inclusion of this dominant antigen following three kinds and its homologous protein：Carnine acidohydrogenase, II type citrate synthase and UreaB；Its aminoacid sequence is respectively as shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3.The CD4+T cellular immunization dominant antigen combination that the present invention is screened has obvious immanoprotection action, and its protected effect is better than H.pylori whole protein antigen, has higher helicobacter pylori Scavenging activity, causes lighter pathology damage simultaneously.The all inducible body of three kinds of Immunodominant Antigenic provided by the present invention is directed to antigen and produces strong immune response.Therefore pass through to induce body to be directed to the generation response of immune protective dominant antigen; or directly with protective antigen immunity body, helicobacter pylori infections will be played with effective immanoprotection action, can be used for the preventative research with therapeutic multivalent vaccine of further helicobacter pylori.

Description

Helicobacter pylori dominant antigen combination based on CD4+T cellular immunization and screening technique
Technical field
The invention belongs to technical field of pharmaceutical biotechnology, it is related to a kind of helicobacter pylori dominant antigen combination and in particular to one Plant the combination of helicobacter pylori dominant antigen and the screening technique based on CD4+T cellular immunization.
Background technology
Helicobacter pylori (Helicobacter pylori, H.pylori) is colonized in gastric mucosa, is I class of stomach carcinogenesis Virulence factor, is the master that the gastrointestinal disease such as chronic gastritiss, gastroduodenal ulcer, gastric mucosa associated lymphoid tissue lymphoma occur Want paathogenic factor.Global infection rate more than 50%, therefore in the urgent need to a kind of effective helicobacter pylori vaccine.
The screening of candidate antigens is the core of vaccine development.Different from the virus with Limiting antigen number, antibacterial is due to containing There is hundreds of a large amount of antigen, be difficult to it is carried out with systematicness screening, so that it is determined that the protective antigen of its immunodominance.This Hamper the research and development of anti-bacterial vaccine always.
Research has shown that, gastric mucosa local th1 and th17 cell have played important function in anti-H.pylori infects.Using 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 is closely related with the gastropathy order of severity.Hitzler, I. couple The mouse B cell of H.pylori immunity and CD4+T cell response are analyzed, and data display th1 and th17 cell are wherein Serve main protective effect, rather than the humoral immunization of B cell mediation.Likewise, DeLyria, the research of E.S. is also supported Protective effect in H.pylori immunity for the th17.
But, the Immunodominant Antigenic of th1 and th17 also never by screening system mistake, based on this new helicobacter pylori Vaccine is not also developed.
Content of the invention
In view of this, it is an object of the invention to provide a kind of helicobacter pylori advantage based on CD4+T cellular immunization resists Former combination.
Present invention also offers the screening technique of above-mentioned dominant antigen combination.
For reaching above-mentioned purpose, the present invention provides following technical scheme：
Helicobacter pylori dominant antigen combination based on CD4+T cellular immunization, including following three kinds 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 UreaB (urease subunit beta, UreB)； Its aminoacid 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 antigens is 1：1：1, the total antigenic content of vaccine is 100 μ g.
Above-mentioned dominant antigen group is combined in the application in preparation prevention or the vaccine for the treatment of helicobacter pylori infections.
Preferably, described vaccine is albumen or nucleic acid vaccine.
Preferably, described vaccine also comprises medically acceptable immunological adjuvant.
It is further preferred that described immunological adjuvant be Freund adjuvant, aluminium adjuvant, in CPG ODN 1826 or AddaVax Any one or several.
The screening technique of above-mentioned dominant antigen combination, comprises the following steps：
1) crack helicobacter pylori thalline, obtain helicobacter pylori holoantigen lysate, then according to each molecular weight of albumen Size molecule be sieved into 30 components；
2) utilize step 1) 30 components stimulate the spleen CD4+T coming from helicobacter pylori infections and immune mouse respectively Cell, uses3H-TdR incorporation methods detect CD4+T cell proliferative conditions, and with proliferation index mapping, obtain stimulating CD4+T cell to increase Ability of growing advantage component PC05 the strongest and subdominant component PC17；
3) utilizing step 2) immune mouse carries out counteracting toxic substances respectively for advantage component PC05 that filters out and subdominant component PC17 Protection, is compareed with the full bacterial immunity of H.pylori and PBS, determines that advantage component PC05 has higher immune clearance energy Power, causes lighter inflammation damnification simultaneously；
4) use the method for HPLC-MS LC-MS/MS by step 3) institute in advantage component PC05 of gained The protein component containing carries out clearly, determining that PC05 contains 11 kinds of albumen；
5) utilize engineered method synthesis step 4) 11 kinds of albumen determining；
6) with step 5) 11 kinds of albumen synthesizing stimulate PC05 specific C D4+T cell respectively, Screening and Identification go out Th1 and Th17 response H.pylori antigen the strongest, totally three kinds.
Preferably, step 1) in use 8M Urea Lysis helicobacter pylori thalline.
The beneficial effects of the present invention is：
After H.pylori infection gastric mucosa, H.pylori antigen can be huge by dendritic cell (dendritic cells, DC) The antigen presenting cells such as phagocyte (antigen-presenting cells, APC) are identified and are processed submission.APC swashs then Original CD4+T cell aliveThe Th1 cell response of secretion inducing IFN-γ and the Th17 of secretion IL-17A Cell response.It is intrinsic that IL-17 can promote to express the gastric epithelial cell of IL-17 receptor, Interstitial cell, endotheliocyte, mucosa The secretion cytokine such as IL-1, IL-6, IL-8 and TNF-α such as layer mononuclear cell, and raise neutrophilic granulocyte removing H.pylori.
Therefore applicant attempts the Immunodominant Antigenic that systematicness from H.pylori full bacterium filters out Th1 and Th17, and base Develop new helicobacter pylori vaccine in this.In the process, each antigen of the full bacterium of H.pylori is all included into synchronous commenting Survey, until Immunodominant Antigenic is screened out.Meanwhile, verify that the immune protective rate of each antigen and inflammation are commented in mouse model Grade index.As a result, three dominant antigens of Th1 and Th17 are successfully filtered out：IMPDH, CS II and UreB.Immunoprotection is real Verify that bright its has higher H.pylori Scavenging activity and cause lighter immunopathogenesis to damage.This is following new helicobacter pylorus Designing and developing of bacteria vaccine provides new candidate antigens.
Result shows that screened CD4+T cellular immunization dominant antigen combination has obvious immanoprotection action, its protection Effect is better than H.pylori whole protein antigen, has higher helicobacter pylori Scavenging activity, causes lighter pathology to damage simultaneously Wound.The all inducible body of three kinds of Immunodominant Antigenic provided by the present invention is directed to antigen and produces strong immune response. Therefore pass through to induce body to be directed to the generation response of immune protective dominant antigen, or directly will be right with protective antigen immunity body Helicobacter pylori infections play effective immanoprotection action, can be used for that further helicobacter pylori is preventative and therapeutic multivalence The research of vaccine.
Brief description
In order that the purpose of the present invention, technical scheme and beneficial effect are clearer, the present invention provides drawings described below to carry out Explanation：
Figure 1A～1D has immanoprotection action for H.pylori holoantigen, 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 is stomach single cell suspension Flow cytometry, and Fig. 1 C is CD3+T, CD4+T cell quantity, Fig. 1 D is Mouse Gastric Mucous Membrane IFN-γ and IL-17A mRNA expression.
Fig. 2 is, with molecular sieve chromatography, according to molecular size range, H.pylori holoantigen is divided into 30 components.
Fig. 3 A and Fig. 3 B is advantage component screening, and wherein Fig. 3 A swashs immunity/infected group CD4+T lymphocyte for each group intermuscular needling Proliferative conditions, Fig. 3 B for each group intermuscular needling swash immunity/be uninfected by organize CD4+T lymphopoiesis situation.
Fig. 4 A～4C is H.pylori holoantigen, PC05, PC17 immunoprotection is evaluated, and wherein Fig. 4 A is fixed for gastric mucosa antibacterial Plant amount, Fig. 4 B is gastric tissue pathological score, and Fig. 4 C is gastric tissue pathological section.
Fig. 5 A～5C excites higher Th1 and Th17 response for advantage component PC05, 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 is antigen-specific Property CD4+T cell Flow cytometry, Fig. 5 C excites specificity T h1 for PC05 and Th17 response level be better than PC17 and H.pylori full bacterium antigen.
Fig. 6 A～6D excites higher specificity T h1 and Th17 for P5 (IMPDH), P10 (CS II) and P11 (UreB) Response.
Fig. 7 has more preferable immunity protection function for Immunodominant Antigenic IMPDH, CS II and UreB.
Specific embodiment
Below in conjunction with accompanying drawing, the preferred embodiments of the present invention are described in detail.
Helicobacter pylori Strains B0 according to the present invention is to tame to obtain in BALB/c mouse body.
Materials and methods
(1) strain and mice
Helicobacter pylori Strains B0：This bacterial strain tames, for helicobacter pylori, the domestication strain obtaining in BALB/c mouse body, It is characterized in being easier to being colonized in BALB/c mouse stomach, and lead to pathological reaction.Divide from helicobacter pylori carrier gastric tissue From culture Helicobacter pylori Strains, it is inoculated in the brain heart infusion agar culture containing 10% (volume ratio) Sanguis Leporis seu oryctolagi 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. Helicobacter pylori Strains M is then used to infect BALB/c mouse.Then repeat above-mentioned to isolate and purify scheme, from BALB/c mouse stomach group Knit and separate helicobacter pylori.The thus obtained Helicobacter pylori Strains being easier to be colonized in BALB/c mouse, are named as pylorus Screw rod bacteria strain B0.
, in the brain heart infusion agar culture medium containing 10% (volume ratio) Sanguis Leporis seu oryctolagi, 37 DEG C micro- aerobic for Helicobacter pylori Strains B0 Under the conditions of cultivate.Two days later, Helicobacter pylori Strains are transferred to 10% (volume ratio) hyclone (FBS) Bu Shi meat from flat board Soup culture medium amplification culture.The helicobacter pylori taking exponential phase of growth is used for infection and experiment in vitro.The no spy of six to eight week old Determine cause of disease (SPF) female BAl BIc/c mice, be 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 table 1. present invention and source
The screening technique of the dominant antigen combination of the present invention, comprises the following steps：
1) use 8M Urea Lysis helicobacter pylori thalline, obtain helicobacter pylori holoantigen Urea Lysis liquid, Ran Hougen It is sieved into 30 components according to the size molecule of each molecular weight of albumen；
2) utilize step 1) 30 components stimulate the spleen CD4+T coming from helicobacter pylori infections and immune mouse respectively Cell, uses3H-TdR incorporation methods detect CD4+T cell proliferative conditions, and with proliferation index mapping, obtain stimulating CD4+T cell to increase Ability of growing advantage component PC05 the strongest and subdominant component PC17；
3) utilizing step 2) immune mouse carries out counteracting toxic substances respectively for advantage component PC05 that filters out and subdominant component PC17 Protection, is compareed with the full bacterial immunity of H.pylori and PBS, determines that advantage component PC05 has higher immune clearance energy Power, causes lighter inflammation damnification simultaneously；
4) use the method for HPLC-MS LC-MS/MS by step 3) institute in advantage component PC05 of gained The protein component containing carries out clearly, determining that PC05 contains 11 kinds of albumen；
5) utilize engineered method synthesis step 4) 11 kinds of albumen determining；
6) with step 5) 11 kinds of albumen synthesizing stimulate PC05 specific C D4+T cell respectively, Screening and Identification go out Th1 and Th17 response H.pylori antigen the strongest, totally three kinds；
7) utilizing step 6) immune mouse carries out counteracting toxic substances protection respectively for Th1 the and Th17 cellular immunization dominant antigen that filters out Experiment, is compareed with PC05 and PBS immunity.Determine that three kinds of Immunodominant Antigenic are respectively provided with obvious immune clearance ability, simultaneously Cause lighter inflammation damnification.
Embodiment 1：
H.pylori full bacterial immunity mice excites CD4+T cell response evaluation
1. animal immune and challenge viral dosage scheme
Laboratory animal：BALB/c mouse female 6～8 week old.
Antigen：
(1) immunity/counteracting toxic substances group (I/C)：H.pylori inactivates full bacterium.100 μ g/ are only.
(2) non-immunity/counteracting toxic substances group (U/C)：Phosphate buffer (PBS).
Adjuvant：Freund adjuvant.100 μ l/ are only.
Immunization wayses：Subcutaneous injection.
Immune volume：200 μ l/ are only.
Immunization protocol：Subcutaneous inoculation 3 times (the 0,2,4th week).First time complete Freund's adjuvant, cannots be used up entirely not for the second time Family name's adjuvant, is not added with adjuvant for the third time.
Final immunization one week after, 1.0 × 109CFU helicobacter pylori gavage, once a day, continuous 4 days.The 4th week after counteracting toxic substances Put to death mice, helicobacter pylori definite value amount, CD4+T cell response in detection mice gastric tissue, analyze its immune protective effect.
2. Mouse Gastric Mucous Membrane helicobacter pylori definite value amount detection.
Real-time quantitative PCR detects gastric helicobacter pylori field planting amount.Extract antibacterial with bacterial genomes extracts kit DNA, detects its field planting amount according to helicobacter pylori 16SrDNA.Result is shown in Figure 1A.
The sequence of helicobacter pylori 16SrDNA is as follows：
Forward 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 fluorescent probe is as follows：
FAM-CGTGCCAGCAGCCGCGGT-TAMRA, as shown in SEQ ID NO.6.
3. Mouse Gastric Mucous Membrane CD4+T cell response detection
Dissect mice gastric tissue along greater gastric curvature and lesser gastric curvature, with the soft washing of aseptic PBS 2 times, to remove food debriss.And After put in 10ml Hank's balanced salt solution (HBSS, without Ca, My), dithiothreitol, DTT containing 1mM (DTT), 1mM ethylenediamine Tetraacethyl (EDTA), and 2% hyclone (FCS), 37 DEG C of incubation 45min.Then, by gained mixture pass through aseptic steel mesh with Remove indigested tissue and obtain single cell suspension.Postdigestive single cell suspension is washed twice with aseptic PBS.Then use immunity glimmering 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 detection Mouse Gastric Mucous Membrane local mRNA level in-site CD4+T cellullar immunologic response.Trizol method extracts stomach Mucosa total serum IgE, reverse transcription becomes cDNA, and detects IFN-γ and IL-17A with SYBR Green incorporation methods by real-time quantitative PCR Expression.Result is shown in Fig. 1 D.
The sequence of IFN-γ is as follows：
Forward 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：
Forward 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：
Complete for H.pylori bacterium antigen is divided into different component according to molecular size range by sieve chromatography
First, Helicobacter pylori Strains B0 is dissolved in 8mol/L carbamide, dithiothreitol, DTT (DTT) 1g/ containing 10mmol/L 6ml, is gently mixed 18 hours under the conditions of 4 DEG C.Secondly, 12000g is centrifuged, and collects supernatant, 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).10/300GL Superdex 200 chromatographic column selected by molecular sieve, and applied sample amount is 1ml, and flow set is 0.5mL/min, collects 1ml/ manages.Sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) electrophoretic analysiss of gained protein component, and pungent with two Thujic acid (BCA) detection kit can measure each protein component concentration.Result is shown in Fig. 2.
Embodiment 3：
3H-TdR incorporation methods detection different component stimulates CD4+T degree of cell proliferation
Take BALB/c mouse peritoneal macrophage as antigen presenting cell (APC).In 96 hole round bottom plates, every hole adds 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%CO2Co-culture in incubator, the ultimate density of each antigen is 50 μ g/mL, and every group sets 3 multiple holes.10 After hour, respectively the mice of 4 weeks after H.pylori immunity/infection and H.pylori immunity/be uninfected by, use magnetic bead sorting spleen Dirty CD4+T lymphocyte.1×105Spleen CD4+T lymphocyte is co-cultured 96 hours with above-mentioned APC.Added at last 18 hours3H-TdR 1 μ Ci/ hole.Afterwards, detect every hole CPM value with liquid scintillation counter, and represented with stimulation index (SI), SI defines For experimental group CPM/ negative control CPM.Result is shown in Fig. 3 A and Fig. 3 B.
Embodiment 4：
The full bacterium of H.pylori, PC05, PC17 immunity protection function are evaluated
1. animal immune and challenge viral dosage scheme
Laboratory animal：BALB/c mouse female 6～8 week old.
Antigen：H.pylori inactivates full bacterium, PC05, PC17, and 100 μ g/ are only.Equivalent PBS control.
Adjuvant：Freund adjuvant.100 μ l/ are only.
Immunization wayses：Subcutaneous injection.
Immune volume：200 μ l/ are only.
Immunization protocol：Subcutaneous inoculation 3 times (the 0,2,4th week).First time complete Freund's adjuvant, cannots be used up entirely not for the second time Family name's adjuvant, is not added with adjuvant for the third time.
Final immunization one week after, 1.0 × 109CFU helicobacter pylori gavage, once a day, continuous 4 days.The 4th week after counteracting toxic substances Put to death mice, helicobacter pylori definite value amount, pathology damage, CD4+T cell response in detection mice gastric tissue, analyze its immunity and protect Shield effect.
2. Mouse Gastric Mucous Membrane helicobacter pylori definite value amount detection.
Real-time quantitative PCR detects gastric helicobacter pylori field planting amount.Extract antibacterial with bacterial genomes extracts kit DNA, detects its field planting amount according to helicobacter pylori 16SrDNA.Result is shown in Fig. 4 A.
The sequence of helicobacter pylori 16SrDNA is as follows：
Forward 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 fluorescent probe is as follows：
FAM-CGTGCCAGCAGCCGCGGT-TAMRA, as shown in SEQ ID NO.6.
3. Mouse Gastric Mucous Membrane inflammatory score
Longitudinally take a small amount of gastric tissue along greater gastric curvature, with formalin fix, paraffin embedding, 5 μm of sections, and with hematoxylin- Eosin stains.Then histopathological scores are carried out under microscope.Result is shown in Fig. 4 B and Fig. 4 C.
Standards of grading are：0th, no significance pathological changes；0.5th, there is slight exception, such as little inflammatory infiltration stove or widely nothing The mucosa metaplasia of inflammation；1.0th, a kind of slight inflammatory cell infiltration, generally invades and body of gland basilar parts；1.5th, mild infiltration, then Plus slight epithelial proliferation or extensive myxocyte metaplasia；2.0th, inflammatory cell is invaded and body of gland and/or tela submucosa；2.5、 Inflammatory cell is invaded and body of gland, and tela submucosa has myxocyte metaplasia and/or slight epithelial hyperplasia simultaneously；3.0th, large stretch of inflammation Invade and body of gland and Submucosa, be often accompanied by moderate myxocyte metaplasia and slightly to moderate epithelial proliferation；3.5th, more than 3.0 inflammation Disease is with obvious epithelial hyperplasia；4.0th, invade and mucous layer strong inflammatory infiltration, body of gland normal configuration is destroyed, and usual companion There are obvious epithelial proliferation and extensive myxocyte metaplasia；4.5th, serious inflammation is with mucosa focal ulcer；5.0th, wide General invade and mucosa and mucosa under inflammation, with gland structure destroy and ulcer.
4. Mouse Gastric Mucous Membrane CD4+T cell response detection
Dissect mice gastric tissue along greater gastric curvature and lesser gastric curvature, with the soft washing of aseptic PBS 2 times, to remove food debriss.And After put in 10ml Hank's balanced salt solution (HBSS, without Ca, My), dithiothreitol, DTT containing 1mM (DTT), 1mM ethylenediamine Tetraacethyl (EDTA), and 2% hyclone (FCS), 37 DEG C of incubation 45min.Then, by gained mixture pass through aseptic steel mesh with Remove indigested tissue and obtain single cell suspension.Postdigestive single cell suspension is washed twice with aseptic PBS.Then use Trizol Method extracts its total serum IgE, and reverse transcription becomes cDNA, and detects IFN-γ and IL- with SYBR Green incorporation methods by real-time quantitative PCR The expression of 17A.Result is shown in Fig. 5 A.
The sequence of IFN-γ is as follows：
Forward 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：
Forward 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 antigenic specificity CD4+T cell response detection.
Polishing separates H.pylori infecting mouse splenocyte.Then 1 × 107Lymphocyte and 100 μ g H.pylori bacterial strain B0 holoantigen co-cultures, plus 5U/ml recombined small-mouse interleukin II (rmIL-2), and 3ml/ hole is complete RPMI1640 culture medium, 12 orifice plates.37 DEG C, 5%CO2After incubator is cultivated 5 days, dead cell is removed using Ficoll gradient centrifugation. Living cells are passed on, plus 20U/ml rmIL-2, complete RPMI1640 culture, obtained H.pylori full bacterium specificity to the 12nd day Lymphocyte.In the meantime, carry out half amount when needing and change liquid.Specific lymphocyte 1 × 10 by preparation5Corresponding to submission The APC cell 1 × 10 of antigen596 hole round bottom plates co-culture 5 hours, it is complete that every hole adds 200 μ L golgistop containing BD RPMI1640 culture medium.Then fluorescent-labeled antibody FITC-CD3, APC-CD4, PE-IFN- γ, PerCP-Cy5.5-IL- are used 17A is dyeed and is used flow cytometry analysis.Result is shown in Fig. 5 B and Fig. 5 C.
Embodiment 5：
HPLC-MS parses protein component
Parse PC05 protein component with tablets by HPLC-MS (LC-MS/MS Analysis).By PC05 egg Informal voucher band is transferred in EP pipe and uses protease cracking from PAGE gel, 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 fly your science and technology, U.S. of generation) and with ncbi.fasta data base (ftp://ftp.ncbi.nih.gov/) compare, thus Determine the concrete protein component of protein groups PC05.
PC05 protein component and aminoacid 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) UreaB
MKKISRKEYASMYGPTTGDKVRLGDTDLIAEVEHDYTIYGEELKFGGGKTLREGMSQSNNPSKEELDLI ITNALIVDYTGIYKADIGIKDGKIAGIGKGGNKDMQDGVKNNLSVGPATEALAGEGLIVTAGGIDTHIHFISPQQIP TAFASGVTTMIGGGTGPADGTNATTITPGRRNLKFMLRAAEEYSMNIGFLAKGNASNDASLADQIEAGAIGLKIHED WGTTPSAINHALDVADKYDVQVAIHTDTLNEAGCVEDTMAAIAGRTMHTYHTEGAGGGHAPDIIKVAGEHNILPAST NPTIPFTVNTEAEHMDMLMVCHHLDKSIKEDVQFADSRIRPQTIAAEDTLHDMGIFSITSSDSQAMGRVGEVITRTW QTADKNKKEFGRLKEEKGDNDNFRIKRYLSKYTINPAIAHGISEYVGSVEVGKVADLVLWSPAFFGVKPNMIIKGGF IALSQMGDANASIPTPQPVYYREMFAHHGKAKYDANITFVSQAAYDKGIKEELGLERQVLPVKNCRNITKKDMQFND TTAHIEVNSETYHVFVDGKEVTSKPANKVSLAQLFSIF
Embodiment 6：
The clonal expression of 11 kinds of albumen in PC05 component
Extract H.pylori DNA with bacterial genomes extracts kit.Gene order according to 11 kinds of albumen designs up and down Trip primer, PCR amplification obtains genes of interest.From pGEX-6P-1 as vector plasmid, engineering bacteria E.Coli induces table Reach.Separate the mesh containing GST label with GST label protein purification filler (Glutathione Sepharose 4Fast Flow) Albumen.Then use Prescission Protease to excise GST label, obtain destination protein.It is stand-by that BCA method measures concentration.
Embodiment 7：
CD4+T lymphocyte dominant antigen identification in PC05 component
Polishing separates PC05 component immune mouse splenocyte.Then 1 × 107Lymphocyte and 100 μ g PC05 groups Antigen is divided to co-culture, plus 5U/ml recombined small-mouse interleukin II (rmIL-2), 3ml/ hole complete RPMI1640 culture medium, 12 Orifice plate.37 DEG C, 5%CO2After incubator is cultivated 5 days, dead cell is removed using Ficoll gradient centrifugation.Living cells are passed on, plus 20U/ml rmIL-2, complete RPMI1640 culture, obtained H.pylori full bacterium specificity to the 12nd day or single proteantigen is special Different in nature lymphocyte.In the meantime, carry out half amount when needing and change liquid.Specific lymphocyte 1 × 10 by preparation5With pass respectively It has been in the APC cell 1 × 10 of 11 kinds of antigens in PC05596 hole round bottom plates co-culture 5 hours, every hole adds 200 μ L and contains BD Golgistop complete RPMI1640 culture medium.Then use fluorescent-labeled antibody FITC-CD3, APC-CD4, PE-IFN- γ, PerCP-Cy5.5-IL-17A is dyeed and is used flow cytometry analysis.Result is shown in Fig. 6 A and Fig. 6 B.
Embodiment 8：
Dominant antigen IMPDH, CS II and UreB immunity protection function are evaluated
1. animal immune and challenge viral dosage scheme
Laboratory animal：BALB/c mouse female 6～8 week old.
Antigen：IMPDH, CS II and UreB, 100 μ g/ are only.Equivalent PBS control.
Adjuvant：Freund adjuvant.100 μ l/ are only.
Immunization wayses：Subcutaneous injection.
Immune volume：200 μ l/ are only.
Immunization protocol：Subcutaneous inoculation 3 times (the 0,2,4th week).First time complete Freund's adjuvant, cannots be used up entirely not for the second time Family name's adjuvant, is not added with adjuvant for the third time.
Final immunization one week after, 1.0 × 109CFU helicobacter pylori gavage, once a day, continuous 4 days.The 4th week after counteracting toxic substances Put to death mice, helicobacter pylori definite value amount, pathology damage, CD4+T cell response in detection mice gastric tissue, analyze its immunity and protect Shield effect.
2. Mouse Gastric Mucous Membrane helicobacter pylori definite value amount detection.
Real-time quantitative PCR detects gastric helicobacter pylori field planting amount.Extract antibacterial with bacterial genomes extracts kit DNA, detects its field planting amount according to helicobacter pylori 16SrDNA.Result is shown in Fig. 6 C.
The sequence of helicobacter pylori 16SrDNA is as follows：
Forward 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 fluorescent probe is as follows：
FAM-CGTGCCAGCAGCCGCGGT-TAMRA, as shown in SEQ ID NO.6.
3. Mouse Gastric Mucous Membrane inflammatory score
Longitudinally take a small amount of gastric tissue along greater gastric curvature, with formalin fix, paraffin embedding, 5 μm of sections, and with hematoxylin- Eosin stains.Then histopathological scores are carried out under microscope.Result is shown in Fig. 6 D.
Standards of grading are：0th, no significance pathological changes；0.5th, there is slight exception, such as little inflammatory infiltration stove or widely nothing The mucosa metaplasia of inflammation；1.0th, a kind of slight inflammatory cell infiltration, generally invades and body of gland basilar parts；1.5th, mild infiltration, then Plus slight epithelial proliferation or extensive myxocyte metaplasia；2.0th, inflammatory cell is invaded and body of gland and/or tela submucosa；2.5、 Inflammatory cell is invaded and body of gland, and tela submucosa has myxocyte metaplasia and/or slight epithelial hyperplasia simultaneously；3.0th, large stretch of inflammation Invade and body of gland and Submucosa, be often accompanied by moderate myxocyte metaplasia and slightly to moderate epithelial proliferation；3.5th, more than 3.0 inflammation Disease is with obvious epithelial hyperplasia；4.0th, invade and mucous layer strong inflammatory infiltration, body of gland normal configuration is destroyed, and usual companion There are obvious epithelial proliferation and extensive myxocyte metaplasia；4.5th, serious inflammation is with mucosa focal ulcer；5.0th, wide General invade and mucosa and mucosa under inflammation, with gland structure destroy and ulcer.
4. Mouse Gastric Mucous Membrane CD4+T cell response detection
Dissect mice gastric tissue along greater gastric curvature and lesser gastric curvature, with the soft washing of aseptic PBS 2 times, to remove food debriss.And After put in 10ml Hank's balanced salt solution (HBSS, without Ca, My), dithiothreitol, DTT containing 1mM (DTT), 1mM ethylenediamine Tetraacethyl (EDTA), and 2% hyclone (FCS), 37 DEG C of incubation 45min.Then, by gained mixture pass through aseptic steel mesh with Remove indigested tissue and obtain single cell suspension.Postdigestive single cell suspension is washed twice with aseptic PBS.Then use Trizol Method extracts its total serum IgE, and reverse transcription becomes cDNA, and detects IFN-γ and IL-17A with SYBR Green incorporation methods real-time quantitative PCR Expression.Result is shown in Fig. 7.
The sequence of IFN-γ is as follows：
Forward 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：
Forward primer：5 '-CTCCAGAAGGCCCTCAGACTAC-3 ', as shown in SEQ ID NO.9；
Downstream primer：5 '-GGGTCTTCATTGCGGTGG-3 ', as shown in SEQ ID NO.10.
Finally illustrate, preferred embodiment above only in order to technical scheme to be described and unrestricted, although logical Cross above preferred embodiment the present invention to be described in detail, it is to be understood by those skilled in the art that can be In form and various changes are made to it, without departing from claims of the present invention limited range in details.
SEQUENCE LISTING
<110>Military Medical Univ No.3, P.L.A
<120>Helicobacter pylori dominant antigen combination based on CD4+T cellular immunization and screening technique
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<170> PatentIn version 3.3
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Gly Ile Gly Ile Val His Lys Asn Met Asp Ile Gln Thr Gln Val Lys
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<223>Helicobacter pylori 16SrDNA forward primer
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<213>Artificial sequence
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<223>Helicobacter pylori 16SrDNA downstream primer
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<223>IFN-γ forward primer
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tgactgtgcc gtggcagtaa 20
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<213>Artificial sequence
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<223>IL-17A forward primer
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ctccagaagg ccctcagact ac 22
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gggtcttcat tgcggtgg 18

Claims (8)

1. the helicobacter pylori dominant antigen based on CD4+T cellular immunization combine it is characterised in that include following three kinds and its with Source protein：Carnine acidohydrogenase, II type citrate synthase and UreaB；Its aminoacid sequence is respectively such as Shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3.
2. dominant antigen combination according to claim 1 is it is characterised in that the mass ratio of three kinds of antigens is 1：1：1, vaccine Total antigenic content is 100 μ g.
3. the dominant antigen group described in claim 1 or 2 is combined in preparation prevention or the vaccine for the treatment of helicobacter pylori infections Application.
4. application according to claim 3 is it is characterised in that described vaccine is albumen or nucleic acid vaccine.
5. application according to claim 3 is it is characterised in that described vaccine also comprises medically acceptable immunity assistant Agent.
6. application according to claim 5 is it is characterised in that described immunological adjuvant is Freund adjuvant, aluminium adjuvant, CPG Any one or several in ODN 1826 or AddaVax.
7. the screening technique of the dominant antigen combination described in claim 1 or 2 is it is characterised in that comprise the following steps：
1) crack helicobacter pylori thalline, obtain helicobacter pylori holoantigen lysate, then big according to each molecular weight of albumen Little molecule is sieved into 30 components；
2) utilize step 1) 30 components stimulate the spleen CD4+T coming from helicobacter pylori infections and immune mouse thin respectively Born of the same parents, use3H-TdR incorporation methods detect CD4+T cell proliferative conditions, and with proliferation index mapping, obtain stimulating CD4+T cell proliferation Ability advantage component PC05 the strongest and subdominant component PC17；
3) utilizing step 2) immune mouse carries out counteracting toxic substances protection respectively for advantage component PC05 that filters out and subdominant component PC17 Experiment, is compareed with the full bacterial immunity of H.pylori and PBS, determines that advantage component PC05 has higher immune clearance ability, with The lighter inflammation damnification of Shi Zaocheng；
4) use the method for HPLC-MS LC-MS/MS by step 3) contained in advantage component PC05 of gained Protein component carry out clearly, determining that PC05 contains 11 kinds of albumen；
5) utilize engineered method synthesis step 4) 11 kinds of albumen determining；
6) with step 5) 11 kinds of albumen synthesizing stimulate PC05 specific C D4+T cell respectively, and Screening and Identification goes out Th1 and Th17 should Answer H.pylori antigen the strongest, totally three kinds.
8. preparation method according to claim 7 is it is characterised in that step 1) in use 8M Urea Lysis helicobacter pylori Thalline.
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