CN106421768A - Helicobacter pylori multi-subunit vaccine based on CD4+T cellular immunity and preparing method - Google Patents

Abstract

The invention relates to a helicobacter pylori multi-subunit vaccine based on a CD4+T cellular response and a preparing method. The helicobacter pylori multi-subunit vaccine comprises three antigens, and the three antigens are inosine 5'-monophosphate dehydrogenase, type II citrat esynthase and urease subunit beta; the antigens also comprise homologous proteins. According to the helicobacter pylori multi-subunit vaccine based on the CD4+T cellular immunity, the CD4+T cellular response has the main anti-H.pylori infection effect on the gastric mucosa local position, more-extensive downstream related immune cell responses can be motivated through ways such as cell factor secreting, and the more-extensive and effective immune protection effect is developed at the gastric mucosa local position. Meanwhile, the helicobacter pylori multi-subunit vaccine has multiple subunits, and the immune effect is more stable and effective.

Description

Helicobacter pylori multi-subunit vaccine and preparation method based on CD4+ T cell immunity

technical field

The invention belongs to the technical field of medicine and biology, and relates to a multi-subunit vaccine, in particular to a Helicobacter pylori multi-subunit vaccine based on CD4+T cell immunity and a preparation method thereof.

Background technique

Helicobacter pylori (H. pylori) is a spiral gram-negative bacterium that colonizes the gastric mucosa and is closely related to the occurrence of chronic gastritis, gastric ulcer, gastric mucosa-associated lymphoid tissue lymphoma, and gastric cancer. With a global infection rate of over 50%, there is an urgent need for an effective H. pylori vaccine.

The oral recombinant Helicobacter pylori vaccine developed by the China National Immunization Engineering Technology Research Center has made a major breakthrough in solving this problem. The single protein component of the vaccine, the urease B subunit (UreB), was selected based on the humoral response. However, not every protein of H. pylori was screened during the development process. Phase III clinical trials showed that the protection rate of the vaccine was 71.8% (95% CI 48.2-85.6) in the first year, and dropped sharply to 55.0% (95% CI 0.9-81.0) in the second year. Therefore, its effectiveness and stability still need to be further improved.

The development of existing H. pylori vaccines is based on antibody responses and is a single subunit vaccine. Humoral immunity plays a certain role in anti-Helicobacter pylori infection, but humoral immunity alone is not enough to completely eradicate Helicobacter pylori. The protective effect of vaccines based on this design is not ideal.

Contents of the invention

In view of this, the object of the present invention is to provide a Helicobacter pylori multi-subunit vaccine based on CD4+T cell immunity.

The invention also provides the preparation method and application of the multi-subunit vaccine.

In addition, the present invention also provides a corresponding primer set based on CD4+ T cell immunity and its application.

To achieve the above object, the present invention provides the following technical solutions:

The Helicobacter pylori multi-subunit vaccine based on CD4+ T cell immunity contains three antigens, namely inosine 5'-monophosphate dehydrogenase (IMPDH), type II citrate synthase (type II citrat esynthase, CSⅡ) and urease B subunit (urease subunit beta, UreB); the antigen also includes its homologous protein.

Preferably, the mass ratio of the three antigens is 1:1:1, and the total antigen content of the vaccine is 100 μg.

The primer sequence of the antigen is as follows:

Inosine nucleotide dehydrogenase:

Upstream primer: 5'-CCGGAATTCATGAGAATTTTACAAAGGGCT-3', as shown in SEQ ID NO.1;

Downstream primer: 5'-CCGCTCGAGTTACCCATAATAATTAGGGGC-3', as shown in SEQ ID NO.2.

Type II citrate synthase:

Upstream primer: 5'-CGCGGATCCATGTCTGTTACTTTA-3', as shown in SEQ ID NO.3;

Downstream primer: 5'-CCGGAATTCTTAATCCCCTACATAG-3', as shown in SEQ ID NO.4.

Urease B subunit:

Upstream primer: 5'-CCGGAATTC ATGAAAAAGATTAGCAGAAAAG-3', as shown in SEQ ID NO.5;

Downstream primer: 5'-CCGCTCGAGCTTTTCTGCTAATCTTTTTCAT-3', as shown in SEQ ID NO.6.

Preferably, the vaccine further comprises a medically acceptable immune adjuvant.

Further preferably, the immune adjuvant is any one or more of Freund's adjuvant, aluminum adjuvant, CPG ODN 1826 or AddaVax.

The preparation method of the above-mentioned Helicobacter pylori multi-subunit vaccine based on CD4+T cell immunity comprises the following steps:

1) Extract Helicobacter pylori DNA, use the primer sequences of hypoxanthine nucleotide dehydrogenase, type II citrate synthase and urease B subunit, and extract from the Helicobacter pylori DNA by polymerase chain reaction (PCR). Amplified separately to obtain the corresponding three gene sequences;

2) Constructing expression vectors and engineering bacteria, respectively inducing and expressing corresponding proteins, and purifying to obtain three target proteins;

3) Use the three target proteins obtained in step 2) to form a three-subunit vaccine with a mass ratio of 1:1:1, add a medically acceptable adjuvant for emulsification, and obtain the vaccine.

Preferably, the Helicobacter pylori DNA in step 1) is extracted using a bacterial genome DNA extraction kit.

Preferably, the conditions of polymerase chain reaction in step 1) are as follows:

1a) 94°C, 5 minutes;

1b) 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 1 minute; the number of cycles is 30;

1c) 72°C, 10 minutes.

Preferably, the concrete method of constructing expression vector and engineered bacterium in step 2) is: reclaim target gene sequence with OMEGA gel recovery kit, digest target gene and pGEX-6P-1 plasmid, construct with the Ligation Mix kit of Takara company Expression vector, and then according to transfer into BL21 competent cells, construct engineering bacteria E.Coli.

Preferably, the induction expression condition in step 2) is to use 1 mmol/L isopropyl-β-D-thiogalactopyranoside (IPTG) aqueous solution, the induction temperature is 37° C., and the induction time is 3 hours.

Preferably, the purification method in step 2) comprises steps:

2a) collecting the cultured bacteria and lysing to obtain the supernatant;

2b) Add 1‰ GST filler with the mass of the original bacterial solution, and shake overnight at 4°C to fully combine;

2c) Centrifuge, discard the supernatant, and wash 2 to 3 times with 10 mMol/L PBS of 2 to 3 times the volume of the filler;

2d) Under the condition of 4°C, the volume ratio of PBS and filler is 1:1, and the digestion with Prescission Protease is carried out overnight;

2e) Collect the supernatant, which is the target protein.

Application of the above-mentioned Helicobacter pylori multi-subunit vaccine based on CD4+T cell immunity in the preparation of preparations for preventing or treating Helicobacter pylori infection.

Preferably, the preparation is a vaccine, more preferably a protein vaccine or a nucleic acid vaccine.

A primer set based on CD4+ T cell immunity, including three pairs of primers:

(A) primers for inosine nucleotide dehydrogenase, including forward primer and reverse primer, the sequence is as follows:

Upstream primer: 5'-CCGGAATTCATGAGAATTTTACAAAGGGCT-3', as shown in SEQ ID NO.1;

Downstream primer: 5'-CCGCTCGAGTTACCCATAATAATTAGGGGC-3', as shown in SEQ ID NO.2.

(B) Type II citrate synthase primers, including forward primers and reverse primers, the sequences are as follows:

Upstream primer: 5'-CGCGGATCCATGTCTGTTACTTTA-3', as shown in SEQ ID NO.3;

Downstream primer: 5'-CCGGAATTCTTAATCCCCTACATAG-3', as shown in SEQ ID NO.4.

(C) Primers for the urease B subunit, including a forward primer and a reverse primer, the sequences are as follows:

Upstream primer: 5'-CCGGAATTC ATGAAAAAGATTAGCAGAAAAG-3', as shown in SEQ ID NO.5;

Downstream primer: 5'-CCGCTCGAGCTTTTCTGCTAATCTTTTTCAT-3', as shown in SEQ ID NO.6.

The application of the above primer set in the preparation of Helicobacter pylori multi-subunit vaccine.

Preferably, the above primer set is used in the preparation of preparations for preventing or treating Helicobacter pylori infection.

The beneficial effects of the present invention are:

The Helicobacter pylori vaccine of the present invention is based on CD4+ T cell immunization, and the CD4+ T cell response plays a major role in anti-H. pylori infection in the local gastric mucosa, which can stimulate a wider range of downstream related Immune cell response plays a more extensive and effective immune protection role in the local gastric mucosa. At the same time, the present invention is multi-subunit, and the immune effect is more stable and effective.

Based on th1 and th17 cell responses, the applicant successfully screened 3 protective CD4+ T cell immunodominant antigens, IMPDH, CSⅡ and UreB. These three antigens can elicit a dominant CD4+ T cell response. The immune challenge protection experiment also confirmed that immunization with these three antigens respectively exerted obvious immune protection effect.

The trivalent subunit vaccine of the present invention has been subjected to preclinical research in mouse models. At the same time, the protective function of CD4+ T cells in the trivalent subunit vaccine was confirmed through the adoptive experiment of specific CD4+ T cells of the trivalent subunit vaccine, and the protective mechanism was clarified. This provides a theoretical and experimental basis for the later optimization and clinical research of the new trivalent subunit vaccine.

Description of drawings

In order to make the purpose, technical scheme and beneficial effect of the present invention clearer, the present invention provides the following drawings for illustration:

Figure 1 shows three kinds of Helicobacter pylori (H. pylori) protective CD4+ T cell immunodominant antigens, IMPDH, CSⅡ and UreB, target gene amplification and expression vector construction;

Figure 2 shows the purification of three proteins: IMPDH, CSⅡ and UreB;

Figure 3a, Figure 3b, Figure 3c and Figure 3d are multi-subunit vaccine challenge protection experiments, gastric mucosal Helicobacter pylori colonization and inflammation scores;

Figure 4a, Figure 4b and Figure 4c are the immune response detection in the multi-subunit vaccine challenge protection experiment;

Figure 5a, Figure 5b and Figure 5c are multi-subunit vaccine-specific CD4+ T cell adoptive experiments;

In the accompanying drawings, *P<0.05, **P<0.01, ***P<0.001, NS has no statistical difference (P>0.05).

detailed description

The preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

The Helicobacter pylori strain B0 involved in the present invention is domesticated in BALB/c mice.

Materials and Methods

(1) Strains and mice

Helicobacter pylori strain B0: This strain is a domesticated strain of Helicobacter pylori domesticated in BALB/c mice, and its characteristic is that it is easier to colonize the stomach of BALB/c mice and cause pathological reactions. The Helicobacter pylori strain was isolated and cultured from the stomach tissue of the Helicobacter pylori carrier, and was inoculated on the brain-heart infusion agar medium containing 10% (volume ratio) rabbit blood by the "three-line method". A single colony was picked and cultured, and identified as Helicobacter pylori by gene sequencing, named as Helicobacter pylori strain M. BALB/c mice were then infected with H. pylori strain M. Then repeat the above isolation and purification scheme, and isolate Helicobacter pylori from the gastric tissue of BALB/c mice. The H. pylori strain thus obtained that is easier to colonize BALB/c mice was named H. pylori strain B0.

Helicobacter pylori strain B0 was cultured in brain heart infusion agar medium containing 10% (volume ratio) rabbit blood under microaerobic conditions at 37°C. Two days later, the Helicobacter pylori strain was transferred from the plate to 10% (volume ratio) fetal bovine serum (FBS) Brucella broth medium for expansion. Helicobacter pylori in exponential growth phase was used for infection and in vitro experiments. Six to eight-week-old specific pathogen-free (SPF) female BALB/c mice were purchased from the Experimental Animal Center of Third Military Medical University.

(2) Main reagents and sources

See Table 1 for details.

Table 1. Main reagents and sources of the present invention

Example 1:

Figure 1 and Figure 2 show the amplification of the three target genes of IMPDH, CS II and UreB and the construction of expression vectors, as well as the induced expression and purification process.

Expression and purification of IMPDH, CS II and UreB proteins

1. Amplification of three protein target genes of IMPDH, CS II and UreB

Primer sequence:

IMPDH

Upstream primer: 5'-CCGGAATTCATGAGAATTTTACAAAGGGCT-3', as shown in SEQ ID NO.1;

Downstream primer: 5'-CCGCTCGAGTTACCCATAATAATTAGGGGC-3', as shown in SEQ ID NO.2;

CSⅡ

Upstream primer: 5'-CGCGGATCCATGTCTGTTACTTTA-3', as shown in SEQ ID NO.3;

Downstream primer: 5'-CCGGAATTCTTAATCCCCTACATAG-3', as shown in SEQ ID NO.4;

UreB

Upstream primer: 5'-CCGGAATTC ATGAAAAAGATTAGCAGAAAAG-3', as shown in SEQ ID NO.5;

Downstream primer: 5'-CCGCTCGAGCTTTTCTGCTAATCTTTTTCAT-3', as shown in SEQ ID NO.6.

The PCR reaction conditions are shown in Table 2.

Table 2. PCR reaction conditions

2. Expression vector construction

The target gene sequence was recovered with OMEGA gel extraction kit. Digest the target gene and pGEX-6P-1 plasmid. The expression vector was constructed with the Ligation Mix kit of Takara Company. Then, according to the transfer of BL21 competent cells, the engineering bacteria were constructed.

3. Induced expression

Induction conditions: IPTG 1mmol/L, 37°C, 3 hours.

4. Protein Purification Protocol

2a) collecting the cultured bacteria and lysing to obtain the supernatant;

2b) Add 1‰ GST filler with the mass of the original bacterial solution, and shake overnight at 4°C to fully combine;

2c) Centrifuge, discard the supernatant, and wash 2 to 3 times with 10 mMol/L PBS of 2 to 3 times the volume of the filler;

2d) Under the condition of 4°C, the volume ratio of PBS and filler is 1:1, and the digestion with Prescission Protease is carried out overnight;

2e) Collect the supernatant, which is the target protein.

Table 3 shows the number of amino acids and bases of these three proteins.

Table 3. The number of amino acids and bases of the three proteins

protein name Relative molecular weight (kD) amino acid number base number IMPDH 51.80 481 1443 CSⅡ 48.32 426 1278 UreB 61.55 569 1707

Example 2: Evaluation of immune protection function of multi-subunit vaccine

1. Experimental protocol for animal immunization and challenge

Experimental animals: BALB/c female mice aged 6-8 weeks.

Antigen: multi-subunit vaccine, H. pylori inactivated whole bacteria, 100μg/piece. Equal amount of PBS control.

Adjuvant: Freund's adjuvant. 100μl/piece.

Immunization method: subcutaneous injection.

Immunization volume: 200μl/monkey.

Immunization scheme: subcutaneous immunization 3 times (week 0, 2, 4). Complete Freund's adjuvant was used for the first time, incomplete Freund's adjuvant was used for the second time, and no adjuvant was added for the third time.

One week after the last immunization, 1.0×10 ⁹ CFU of Helicobacter pylori was administered orally, once a day, for 4 consecutive days. Mice were sacrificed at 4 weeks after challenge, and the fixed amount of Helicobacter pylori, pathological damage, and CD4+ T cell response in the gastric tissue of the mice were detected, and the immune protection effect was analyzed.

2. Determination of the fixed amount of Helicobacter pylori in mouse gastric mucosa

Real-time quantitative PCR was used to detect the amount of Helicobacter pylori colonization in the stomach. Bacterial DNA was extracted with a bacterial genome extraction kit, and the amount of colonization was detected based on Helicobacter pylori 16SrDNA. The results are shown in Figure 3a.

The sequence of Helicobacter pylori 16SrDNA is as follows:

Upstream primer: 5'-TTTGTTAGAGAAGATAATGACGGTATCTAAC-3', as shown in SEQ ID NO.7;

Downstream primer: 5'-CATAGGATTTCACACCTGACTGACTATC-3', as shown in SEQ ID NO.8.

The sequences of the fluorescent probes are as follows:

FAM-CGTGCCAGCAGCCGCGGT-TAMRA, as shown in SEQ ID NO.9.

3. Mouse Gastric Mucosa Inflammation Scoring

A small amount of gastric tissue was taken longitudinally along the greater curvature of the stomach, fixed in formalin, embedded in paraffin, sectioned at 5 μm, and stained with hematoxylin-eosin. Histopathological scoring was then performed under a microscope. The scoring criteria are: 0, no significant lesions; 0.5, mild abnormalities, such as small inflammatory infiltrates or extensive non-inflammatory mucosal metaplasia; 1.0, a mild inflammatory cell infiltration, usually invading Gland base; 1.5, mild infiltration, plus mild epithelial hyperplasia or extensive mucinous cell metaplasia; 2.0, inflammatory cell invasion into gland and/or submucosa; 2.5, inflammatory cell invasion into gland, At the same time, there is mucus cell metaplasia and/or mild epithelial cell hyperplasia in the submucosa; 3.0, extensive inflammation invades glands and submucosa, often accompanied by moderate mucus cell metaplasia and mild to moderate epithelial cell hyperplasia; 3.5, 3.0 The above inflammation is accompanied by obvious epithelial cell hyperplasia; 4.0, strong inflammatory infiltration that invades the mucosal layer, the normal structure of the gland is destroyed, and is usually accompanied by obvious epithelial hyperplasia and extensive mucus cell metaplasia; 4.5, severe inflammation with Mucosal focal ulceration; 5.0, extensive invasion of mucosal and submucosal inflammation with destruction of glandular structures and ulceration. The results are shown in Figure 3b.

4. Detection of mouse gastric mucosal CD4+ T cell response

Mouse stomach tissues were dissected along the greater and lesser curvatures of the stomach, and gently washed twice with sterile PBS to remove food residues. Then put into 10ml Hank's balanced salt solution (HBSS, without Ca, My), containing 1mM dithiothreitol (DTT), 1mM ethylenediaminetetraacetic acid (EDTA), and 2% fetal calf serum (FCS), Incubate at 37°C for 45min. Then, the resulting mixture was passed through a sterile steel mesh to remove undigested tissue to obtain a single cell suspension. The digested single-cell suspension was washed twice with sterile PBS. Then the total RNA was extracted by Trizol method, reverse transcribed into cDNA, and the expression of IFN-γ and IL-17A was detected by real-time quantitative PCR by SYBR Green incorporation method (Fig. 4a).

The sequence of IFN-γ is as follows:

Upstream primer: 5'-GATCCTTTGGACCCTCTGACTT-3', as shown in SEQ ID NO.10;

Downstream primer: 5'-TGACTGTGCCGTGGCAGTAA-3', as shown in SEQ ID NO.11.

The sequence of IL-17A is as follows:

Upstream primer: 5'-CTCCAGAAGGCCCTCAGACTAC-3', as shown in SEQ ID NO.12;

Downstream primer: 5'-GGGTCTTCATTGCGGTGG-3', as shown in SEQ ID NO.13.

5. Antigen-specific CD4+ T cell response detection of mouse splenocytes

Separation of H. pylori-infected mouse spleen lymphocytes by grinding. Then 1×10 ⁷ lymphocytes were co-cultured with 100 μg trivalent vaccine antigen, 5 U/ml recombinant mouse interleukin-2 (rmIL-2) was added, 3 ml/well complete RPMI1640 medium, 12-well plate. After culturing in a 5% CO2 incubator at 37°C for 5 days, dead cells were removed by Ficoll gradient centrifugation. Live cells were subcultured, added with 20U/mlrmIL-2, cultured in complete RPMI1640, and H. pylori specific lymphocytes were obtained on the twelfth day. During this period, half-volume fluid changes were performed as needed. Co-culture 1×10 ⁵ prepared specific lymphocytes with 1×10 ⁵ APC cells presenting IMPDH, CS II and UreB antigens respectively in 96-well round bottom plate for 5 hours, add 200 μL BDgolgistop complete RPMI1640 to each well Medium. Then stained with fluorescently labeled antibodies FITC-CD3, APC-CD4, PE-IFN-γ, PerCP-Cy5.5-IL-17A and analyzed by flow cytometry. The results are shown in Figures 4b and 4c.

Example 3:

Functional evaluation of adoptive protection of multi-subunit vaccine-specific CD4+ T cells

1. Mouse splenocyte antigen-specific CD4+ T cell culture and magnetic bead sorting

Separation of H. pylori-infected mouse spleen lymphocytes by grinding. Then 1×10 ⁷ lymphocytes were co-cultured with 100 μg trivalent vaccine antigen or inactivated Helicobacter pylori, plus 5 U/ml recombinant mouse interleukin-2 (rmIL-2), 3 ml/well complete RPMI1640 medium, 12 orifice plate. After culturing in a 37°C, 5% CO ₂ incubator for 5 days, dead cells were removed by Ficoll gradient centrifugation. The live cells were subcultured, added with 20U/ml rmIL-2, cultured in complete RPMI1640, and H. pylori specific lymphocytes were obtained on the twelfth day. CD4+ T cells were sorted with Miltenyi Immunomagnetic Beads Separation Kit, and the purity of the sorting was detected by flow cytometry.

2. Mouse splenocyte antigen-specific CD4+T adoptive and Helicobacter pylori challenge

Experimental animals: BALB/c female mice aged 6-8 weeks.

Adoptive method: tail vein injection.

Amount of adoptive cells: 2×10 ⁶ /only.

One week after the cells were adopted, 1.0×10 ⁹ CFU of Helicobacter pylori was administered orally, once a day, for 4 consecutive days. Mice were sacrificed at 4 weeks after challenge, and the fixed amount of Helicobacter pylori, pathological damage, and CD4+ T cell response in the gastric tissue of the mice were detected, and the immune protection effect was analyzed.

3. Determination of the fixed amount of Helicobacter pylori in mouse gastric mucosa

Real-time quantitative PCR was used to detect the amount of Helicobacter pylori colonization in the stomach. Bacterial DNA was extracted with a bacterial genome extraction kit, and the amount of colonization was detected based on Helicobacter pylori 16SrDNA. The results are shown in Figure 5a.

The sequence of Helicobacter pylori 16SrDNA is as follows:

Upstream primer: 5'-TTTGTTAGAGAAGATAATGACGGTATCTAAC-3', as shown in SEQ ID NO.7;

Downstream primer: 5'-CATAGGATTTCACACCTGACTGACTATC-3', as shown in SEQ ID NO.8.

The sequences of the fluorescent probes are as follows:

FAM-CGTGCCAGCAGCCGCGGT-TAMRA, as shown in SEQ ID NO.9.

4. Mouse Gastric Mucosa Inflammation Scoring

A small amount of gastric tissue was taken longitudinally along the greater curvature of the stomach, fixed in formalin, embedded in paraffin, sectioned at 5 μm, and stained with hematoxylin-eosin. Histopathological scoring was then performed under a microscope. The scoring criteria are: 0, no significant lesion; 0.5, mild abnormalities, such as small inflammatory infiltrates or extensive non-inflammatory mucosal metaplasia; 1.0, a mild inflammatory cell infiltration, usually invading Gland base; 1.5, mild infiltration, coupled with mild epithelial hyperplasia or extensive mucinous cell metaplasia; 2.0, inflammatory cell invasion into gland and/or submucosa; 2.5, inflammatory cell invasion into gland, At the same time, there is mucus cell metaplasia and/or mild epithelial cell hyperplasia in the submucosa; 3.0, extensive inflammation invades glands and submucosa, often accompanied by moderate mucus cell metaplasia and mild to moderate epithelial cell hyperplasia; 3.5, 3.0 The above inflammation is accompanied by obvious epithelial cell hyperplasia; 4.0, strong inflammatory infiltration that invades the mucosal layer, the normal structure of the gland is destroyed, and is usually accompanied by obvious epithelial hyperplasia and extensive mucus cell metaplasia; 4.5, severe inflammation with Focal mucosal ulceration; 5.0, extensive invasion of mucosal and submucosal inflammation, accompanied by glandular structure destruction and ulceration. The results are shown in Figure 5b.

5. Monitoring of mouse splenocyte antigen-specific CD4+ T cell response.

Separation of mouse splenic lymphocytes by grinding. Then 1×10 ⁵ lymphocytes and 1×10 ⁵ APC cells presented with trivalent vaccine antigen or inactivated Helicobacter pylori were co-cultured in 96-well round bottom plate for 5 hours, and 200 μL of complete RPMI1640 medium containing BDgolgistop was added to each well . Then stained with fluorescently labeled antibodies FITC-CD3, APC-CD4, PE-IFN-γ, PerCP-Cy5.5-IL-17A and analyzed by flow cytometry. The results are shown in Figure 5c

Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that it can be described in terms of form and Various changes may be made in the details without departing from the scope of the invention defined by the claims.

SEQUENCE LISTING

<110> The Third Military Medical University of the Chinese People's Liberation Army

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gggtcttcat tgcggtgg 18

Claims (10)

1. The Helicobacter pylori multi-subunit vaccine based on CD4+ T cell immunity is characterized in that it contains three kinds of antigens, namely inosine nucleotide dehydrogenase, type II citrate synthase and urease B subunit ; The antigen also includes its homologous protein. 2. The Helicobacter pylori multi-subunit vaccine according to claim 1, wherein the mass ratio of the three antigens is 1:1:1, and the total antigen content of the vaccine is 100 μg. 3. the Helicobacter pylori multi-subunit vaccine according to claim 1, is characterized in that, the primer sequence of described antigen is as follows: Inosine nucleotide dehydrogenase: Upstream primer: 5'-CCGGAATTCATGAGAATTTTACAAAGGGCT-3', as shown in SEQ ID NO.1; Downstream primer: 5'-CCGCTCGAGTTACCCATAATAATTAGGGGC-3', as shown in SEQ ID NO.2. Type II citrate synthase: Upstream primer: 5'-CGCGGATCCATGTCTGTTACTTTA-3', as shown in SEQ ID NO.3; Downstream primer: 5'-CCGGAATTCTTAATCCCCTACATAG-3', as shown in SEQ ID NO.4. Urease B subunit: Upstream primer: 5'-CCGGAATTC ATGAAAAAGATTAGCAGAAAAG-3', as shown in SEQ ID NO.5; Downstream primer: 5'-CCGCTCGAGCTTTTCTGCTAATCTTTTTCAT-3', as shown in SEQ ID NO.6. 4. The preparation method of the Helicobacter pylori multi-subunit vaccine according to any one of claims 1 to 3, characterized in that it comprises the following steps: 1) Extract Helicobacter pylori DNA, use the primer sequences of hypoxanthine nucleotide dehydrogenase, type II citrate synthase and urease B subunit, and amplify from the Helicobacter pylori DNA by polymerase chain reaction , get the corresponding three gene sequences; 2) Constructing expression vectors and engineering bacteria, respectively inducing and expressing corresponding proteins, and purifying to obtain three target proteins; 3) Use the three target proteins obtained in step 2) to form a three-subunit vaccine with a mass ratio of 1:1:1, add a medically acceptable adjuvant for emulsification, and obtain the vaccine. 5. preparation method according to claim 4, it is characterized in that, in step 2), the concrete method of constructing expression vector and engineering bacterium is: reclaim target gene sequence with OMEGA glue recovery kit, restriction endonuclease target gene and pGEX-6P -1 plasmid, the Ligation Mix kit of Takara Company was used to construct the expression vector, and then according to transfer into BL21 competent cells, the engineering bacterium E.Coli was constructed. 6. The preparation method according to claim 4, characterized in that, the induction expression condition in step 2) is that 1 mmol/L isopropyl-β-D-thiogalactoside aqueous solution is used, and the induction temperature is 37°C , induction time 3 hours. 7. preparation method according to claim 4, is characterized in that, the purification method in step 2) comprises steps: 2a) collecting the cultured bacteria and lysing to obtain the supernatant; 2b) Add 1‰ GST filler with the mass of the original bacterial solution, and shake overnight at 4°C to fully combine; 2c) Centrifuge, discard the supernatant, and wash 2 to 3 times with PBS of 2 to 3 times the volume of the filler; 2d) Under the condition of 4°C, the volume ratio of PBS and filler is 1:1, and the digestion with Prescission Protease is carried out overnight; 2e) Collect the supernatant, which is the target protein. 8. Use of the Helicobacter pylori multi-subunit vaccine according to any one of claims 1 to 3 in the preparation of preparations for preventing or treating Helicobacter pylori infection. 9. A primer set based on CD4+ T cell immunity, including three pairs of primers: (A) primers for inosine nucleotide dehydrogenase, including forward primer and reverse primer, the sequence is as follows: Upstream primer: 5'-CCGGAATTCATGAGAATTTTACAAAGGGCT-3', as shown in SEQ ID NO.1; Downstream primer: 5'-CCGCTCGAGTTACCCATAATAATTAGGGGC-3', as shown in SEQ ID NO.2. (B) Type II citrate synthase primers, including forward primers and reverse primers, the sequences are as follows: Upstream primer: 5'-CGCGGATCCATGTCTGTTACTTTA-3', as shown in SEQ ID NO.3; Downstream primer: 5'-CCGGAATTCTTAATCCCCTACATAG-3', as shown in SEQ ID NO.4. (C) Primers for the urease B subunit, including a forward primer and a reverse primer, the sequences are as follows: Upstream primer: 5'-CCGGAATTC ATGAAAAAGATTAGCAGAAAAG-3', as shown in SEQ ID NO.5; Downstream primer: 5'-CCGCTCGAGCTTTTCTGCTAATCTTTTTCAT-3', as shown in SEQ ID NO.6. 10. The use of the primer set according to claim 9 in the preparation of Helicobacter pylori multi-subunit vaccines.