A recombinant Helicobacter pylori antigen protein SecG and its preparation method and application

Abstract

The present invention discloses a recombinant antigen protein SecG of Helicobacter pylori and its preparation method and application. The amino acid sequence of the recombinant antigen protein SecG is shown in SEQ ID NO: 1, and the nucleotide sequence of the recombinant antigen protein SecG encoding gene is shown in SEQ ID NO: 2. The present invention obtains a high-purity antigen protein through the steps of plasmid construction, induced expression, affinity chromatography and exchange chromatography purification. The purification process is simple, low in cost, and highly operable. The antigen prepared after purification has high immunogenicity and immune titer and can be used as a genetically engineered subunit vaccine. As an important component protein in the membrane transport protein of Helicobacter pylori, the antigen protein SceG will become one of the most promising candidate antigens in the development of Helicobacter pylori genetically engineered vaccines.

Description

Technical Field

[0001] The present invention belongs to the technical field of biopharmaceuticals and relates to recombinant antigen proteins, in particular to a Helicobacter pylori recombinant antigen protein SecG and a preparation method and application thereof. Background Art

[0002] Helicobacter pylori (H. pylori) is a Gram-negative, opportunistic pathogen that parasitizes the human gastrointestinal tract. It is widely recognized to be closely linked to a variety of gastrointestinal diseases, particularly peptic ulcers, chronic gastritis, gastric cancer, and mucosa-associated lymphoid tissue lymphoma. H. pylori infection poses a serious health threat. Traditional clinical treatments for H. pylori infection, often using triple and quadruple therapies, can cure the infection to a certain extent, but they still pose challenges such as high antibiotic dosage, high costs, and high recurrence rates that require urgent attention.

[0003] Decades of scientific research have demonstrated that vaccination can effectively prevent and even treat a range of diseases caused by Helicobacter pylori infection. In 1993, Czinn et al. orally immunized mice with a lysate of Helicobacter pylori to induce high levels of secretory IgA (sIgA) in the mucosa. They also found that this immunization could prevent reinfection with Helicobacter pylori, confirming for the first time the protective effect of the vaccine. Subsequently, a growing number of researchers have begun researching vaccines targeting Helicobacter pylori. Therefore, vaccination is expected to become one of the most effective and promising means of preventing and treating Helicobacter pylori infection in the future.

[0004] Whole-cell Helicobacter pylori vaccines are complex in antigenic composition and face numerous challenges, including long production cycles, demanding culture conditions, low yields, and contamination. Consequently, the preparation of traditional vaccines using whole-cell Helicobacter pylori is challenging. With the continuous advancement of molecular biology, genetically engineered vaccines have become a research hotspot. Currently, several different Helicobacter pylori genomes and genes have been cloned for use in genetically engineered Helicobacter pylori vaccines, including the chemokines cheA and cheY, the flagellin genes flaA and flaB, the heat shock protein gene hspA, the urease genes UreA and UreB, and the vacuolating toxin gene vacA. While some of these antigens have demonstrated some protective immune responses, they currently suffer from low immunogenicity and inefficient recombinant expression, hindering large-scale protein production. Therefore, further research on Helicobacter pylori vaccines should focus not only on improving immunization methods and identifying highly effective and non-toxic adjuvants, but also on developing a diverse and more comprehensive set of immunizing antigens, ultimately aiming to create a highly effective human Helicobacter pylori vaccine through a combination of multivalent antigens. Summary of the Invention

[0005] In view of the problems that the existing Helicobacter pylori whole-cell vaccines have complex antigen components and Helicobacter pylori also has long production cycle, harsh culture conditions, low yield, and easy contamination, as well as the fact that in the development of Helicobacter pylori genetically engineered vaccines, there are few types of antigen proteins with high immune activity, and the recombinant antigens of existing known genes generally have low immune titers or are difficult to prepare in large quantities, the purpose of the present invention is to solve the above problems and provide a Helicobacter pylori recombinant antigen protein SecG and its preparation method and application. The present invention obtains a high-purity antigen protein through two-step purification by nickel ion affinity chromatography and cation exchange chromatography on the basis of recombinant expression. Animal experiments show that the recombinant antigen protein SecG has good immunogenicity, can induce an efficient mucosal immune response, and can be used as an effective vaccine candidate antigen.

[0006] Bioinformatics is currently a hot topic in biological research. This study leverages the structure of multiple Helicobacter pylori antigenic proteins in a database and, using the principles of "reverse immunology," screened for a novel recombinant antigen protein, SecG, targeting Helicobacter pylori. SecG is a subunit of the protein translocase on the H. pylori cell membrane. It stimulates protein translocation by undergoing cycles of topological inversion within the membrane, coordinating with the membrane insertion and deinsertion cycles of SecA. Previously, no studies have demonstrated that the H. pylori SecG protein can act as a specific H. pylori antigen and elicit an immune response. This study clones the selected antigen gene into a protein expression vector and recombinantly expresses it on a large scale within engineered bacteria. The purified antigen can then be used as a genetically engineered subunit vaccine, utilizing a simple, low-cost, and highly operational purification process. As a key component of the H. pylori membrane transporter, SecG is one of the most promising candidate antigens for the development of genetically engineered Helicobacter pylori vaccines.

[0007] To achieve the above object, the present invention provides a recombinant Helicobacter pylori antigen protein SecG, the amino acid sequence of the recombinant antigen protein SecG is shown in SEQ ID NO: 1.

[0008] SEQ ID NO:1 Amino acid sequence of SecG gene:

[0009] KEYGKSVLDETKTNKELSPLVPATGTLNPTLNPTLNPTLNPLEQAPTNPLMPQQTPKELPKEPLNVPSVESPKQNEKNEKNEKNEKNDAKENGIKGVEKTKENAKTPPTTHQKPKTHAQTNAHTNQKKDEK.

[0010] The nucleotide sequence of the gene encoding the recombinant antigen protein SecG is shown in SEQ ID NO: 2. The recombinant antigen protein SecG is prepared by selecting a peptide encoding gene with high specificity in the protein encoded by the Helicobacter pylori genome as a target gene fragment, and introducing the target gene fragment into a host cell via a prokaryotic expression vector for expression. The target gene fragment has the nucleotide sequence shown in SEQ ID NO: 2 (the underlined portion indicates the restriction enzyme cleavage site).

[0011] SEQ ID NO: 2 SecG gene nucleotide sequence:

[0012] CCATGG GCAAGGAATACGGCAAAAGCGTGCTGGATGAAACCAAAACCAATAAAAGAACTGAGCCCGCTGGTTCCGGCCACCGGTACCCTGAATCCGACCCTGAATCCTACCCTGAATCCAACCCTGAATCCCCTGGAACAGGCCCCGACCAATCCGCTGATGCCGCAGCAGACCCCGAAAGAACTGCCGAAAGAACCGCTGAAT GTGCCGAGCGTTGAAAGCCCGAAACAGAATGAAAAAAATGAAAAAAACGAGAAGAACGAGAAGAATGATGCCAAAGAAAATGGTATTAAGGGTGTGGAAAAAACCAAAGAAAATGCCAAAACCCCGCCGACCACCCATCAGAAACCGAAAACCCATGCCCAGACCAATGCACATACCAATCAGAAAAAAGATGAAAAAA CTCGAG .

[0013] The present invention also provides a method for preparing the recombinant Helicobacter pylori antigen protein SecG, comprising the following steps:

[0014] S1. Plasmid construction

[0015] The gene with the nucleotide sequence of SEQ ID NO: 2 is used as a target gene fragment, and the target gene fragment is connected to an expression vector plasmid to construct a recombinant expression plasmid containing the target gene fragment;

[0016] S2. Inducible expression

[0017] The recombinant expression plasmid is transformed into a host bacterium for expression of the recombinant protein for induction of expression, the induced expression bacteria are collected, the bacteria are crushed, centrifuged and the supernatant is collected;

[0018] S3. Product Purification

[0019] The supernatant was purified by nickel column affinity chromatography and cation exchange chromatography to obtain the Helicobacter pylori recombinant antigen protein SecG. The amino acid sequence of the recombinant antigen protein SecG is shown in SEQ ID NO: 1.

[0020] The preparation method of the above-mentioned Helicobacter pylori recombinant antigen protein SecG and the construction of the recombinant expression plasmid are routine operations in this field. The present invention adopts the principle of "reverse immunology" to design and screen to obtain the amino acid sequence encoding the potential Helicobacter pylori recombinant antigen protein SecG, and according to the amino acid sequence, performs codon optimization for Escherichia coli preference to obtain the target gene fragment with the nucleotide sequence as shown in SEQ ID NO: 2, and finally connects the target gene fragment to the expression vector plasmid by double enzyme digestion to obtain the recombinant expression plasmid. Those skilled in the art can obtain the recombinant expression plasmid by conventional means. In the present invention, step S1 specifically includes the following steps:

[0021] S11. Screening and obtaining the amino acid sequence encoding the recombinant antigen protein SecG of Helicobacter pylori, and performing codon optimization for Escherichia coli preference based on the amino acid sequence to obtain the target gene fragment, the nucleotide sequence of which is shown in SEQ ID NO: 2;

[0022] S12. Perform full gene synthesis on the target gene and connect it to the expression vector plasmid through the restriction sites of Xho I and Nco I to construct a recombinant expression plasmid.

[0023] The preparation method of the recombinant Helicobacter pylori antigen protein SecG and the induction expression are conventional operations in the art, and the present invention has no special limitations thereto. Those skilled in the art can induce the expression of the recombinant strain to produce the recombinant antigen protein according to conventional methods. In the present invention, step S2 specifically includes the following steps:

[0024] S21, transforming the recombinant expression plasmid into the expression host bacteria to obtain a recombinant strain that can express the recombinant antigen protein SecG, and culturing the recombinant strain to OD 600 0.6-0.8, and then induce expression with 0.3-0.5 mM isopropylthiogalactoside (IPTG) at 30-37 °C and 180-240 rpm for 4-8 h;

[0025] S22. Centrifuge the bacterial solution after induced expression and discard the supernatant to obtain precipitated bacteria. Add buffer A to the precipitated bacteria and suspend them evenly. After the bacteria are broken, centrifuge and discard the precipitate. Filter the supernatant through a 0.45 μm filter membrane through a suction filtration bottle for later use.

[0026] In the above-mentioned method for preparing the recombinant Helicobacter pylori antigen protein SecG, the expression plasmid vector is pET28a(+); and the host bacteria is Escherichia coli.

[0027] The method for preparing the recombinant Helicobacter pylori antigen protein SecG is described above. The present invention purifies the antigen protein by nickel column affinity chromatography and cation exchange chromatography. The specific operations of nickel column affinity chromatography and cation exchange chromatography can be conventional operations in the art. In the present invention, step S3 specifically includes the following steps:

[0028] S31, nickel column affinity chromatography

[0029] Equilibration: Equilibrate the nickel column with buffer A;

[0030] Loading: loading the supernatant from step S2;

[0031] Re-equilibration: Re-equilibrate the nickel column with buffer B;

[0032] Elution: Use buffer C to elute impurity proteins; then use buffer D to elute the target protein and collect the eluate;

[0033] S32, cation exchange chromatography

[0034] Equilibration: Equilibrate the cation column with buffer E;

[0035] Sample loading: dilute the eluate from step S31 with buffer E and then load the sample;

[0036] Re-equilibration: Re-equilibrate the cation column using buffer E;

[0037] Elution: Use buffer F to elute impurity proteins; then use buffer G to elute the target protein, collect the eluate, and obtain the Helicobacter pylori recombinant antigen protein SecG.

[0038] The preparation method of the above-mentioned Helicobacter pylori recombinant antigen protein SecG, preferably, the composition of the buffer A is: 20-50mM phosphate buffer at pH 6-7, 300-500mM NaCl, 0-30mM imidazole; the composition of the buffer B is: 20-50mM phosphate buffer at pH 6-7, 100-200mM NaCl, 0-30mM imidazole; the composition of the buffer C is: 20-50mM phosphate buffer at pH 6-7, 100-200mM NaCl, 40-60mM imidazole; the composition of the buffer D is: 20-50mM phosphate buffer at pH 6-7, 100-200mM NaCl, 90-120mM imidazole; the composition of the buffer E is: 20-50mM phosphate buffer at pH 6-7; the composition of the buffer F is: 20-50mM phosphate buffer at pH 6-7, 80-120mM NaCl; the composition of the buffer G is 20-50mM phosphate buffer at pH 6-7, 180-250mM NaCl.

[0039] The preparation method of the recombinant antigen protein SecG of Helicobacter pylori is not particularly limited to the method of bacterial cell disruption in the present invention. In step S2, a high-pressure homogenizer or an ultrasonic disruptor is preferably used to disrupt the bacteria. The high-pressure homogenizer disruption parameters are preferably: pressure 600-800 bar, flow rate 100-150 mL / min, repeated 4-8 times; the ultrasonic disruptor disruption parameters are preferably: ultrasonic 3-5s, stop 3-5s, and alternately disrupt the bacteria for 20-40min. Furthermore, the centrifugal operation parameters in step S2 can also adopt conventional operating parameters in the art. Preferably, the centrifugal speed is 10000-14000g and the centrifugal time is 20-40min.

[0040] In the above-mentioned method for preparing the recombinant Helicobacter pylori antigen protein SecG, the fillers in the nickel column and the cationic column can be conventional fillers in the art. Preferably, the filler in the nickel column is preferably Ni-agarose HP (Ni-Sepharose HP); the filler in the cationic column is preferably SP-agarose HP (SP-Sepharose HP).

[0041] The present invention also provides the use of the above-mentioned Helicobacter pylori recombinant antigen protein SecG in the preparation of Helicobacter pylori vaccines. Animal experiments have shown that the recombinant antigen protein SecG can effectively stimulate the body to produce a high level of immune response and has high immune activity. At the same time, immune protection evaluation experiments have confirmed that the SecG vaccine has a good protective effect, proving that the recombinant antigen protein SecG has vaccine application value. The present invention clones the selected antigen gene into a protein expression vector, recombines it in engineered bacteria for large-scale expression, and the purified antigen can be used as a genetically engineered subunit vaccine. As an important component protein of the Helicobacter pylori membrane transport protein, the recombinant antigen protein SecG will also be one of the most promising candidate antigens in the development of Helicobacter pylori genetically engineered vaccines.

[0042] The Helicobacter pylori recombinant antigen protein SecG provided by the present invention and its preparation method and application have the following beneficial effects:

[0043] (1) The Helicobacter pylori recombinant antigen protein SecG provided by the present invention has a high expression efficiency. Based on the principle of "reverse immunology", the present invention obtained the encoding amino acid sequence of the Helicobacter pylori recombinant antigen protein SecG as shown in SEQ ID NO: 1 through data analysis, and the sequence was codon-optimized for the expression host strain. The optimized nucleotide sequence is shown in SEQ ID NO: 2. The obtained nucleotide sequence was constructed into the expression vector plasmid pET28a(+) by full chemical synthesis and transformed into the recombinant protein expression host Escherichia coli BL21(DE3) for induced expression. The expression level of the expressed recombinant protein was higher than 20%.

[0044] (2) The preparation method of the recombinant Helicobacter pylori antigen protein SecG provided by the present invention has a simple purification process, a purity of the purified product exceeding 99%, good reproducibility, and a high recovery rate. This indicates that the recombinant antigen protein SecG is easy to prepare in large quantities and has a good application basis.

[0045] (3) The Helicobacter pylori recombinant antigen protein SecG provided by the present invention has high immune activity and immune protection effect. When the purified protein was co-injected with aluminum phosphate adjuvant to immunize BalB / c mice, the highest antibody titer produced by the immunized mice reached 1:128000, and the antibody positive rate after immunization reached 100%, proving that the recombinant antigen protein SecG can effectively stimulate the body to produce a high immune response and has high immune activity; when the recombinant antigen protein SecG was combined with LTs63K adjuvant and nasally immunized mice, the sIgA antibody titer produced by the recombinant antigen protein SecG immunized mice reached 1:128; the antibody positive rate after immunization reached 100%, indicating that the recombinant antigen protein SecG can stimulate mice to produce a high level of mucosal immune response. At the same time, the immune protection evaluation experiment confirmed that the SecG vaccine has a good protective effect, proving that the recombinant antigen protein SecG has vaccine application value and provides a new antigen selection for the development of recombinant Helicobacter pylori vaccine. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 The results of double enzyme digestion of pET28a(+) / SecG plasmid are shown. Lane 1 is the recombinant plasmid, and lane 2 is the result of double enzyme digestion. The separated fragments are approximately 5000 bp and 400 bp.

[0047] Figure 2 The results of induction identification of recombinant antigen protein SecG are shown. Lane 1 is the suspension of broken bacteria, lane 2 is the supernatant of broken bacteria, and lane 3 is the resuspension of broken bacteria precipitate.

[0048] Figure 3 The results of SDS-PAGE electrophoresis after nickel column affinity chromatography are shown in Figure 1. Lane 1 is the loading sample, lane 2 is the flow-through, lane 3 is the wash, and lane 4 is the elution of the target protein.

[0049] Figure 4 The results of SDS-PAGE electrophoresis after ion column chromatography, lanes 1-3 are the elution of the target protein;

[0050] Figure 5 The SDS-PAGE electrophoresis results of the prepared LTs63K are shown in Figure 1. Lane 1 is the loading sample, lane 2 is the flow-through, and lanes 3-5 are the elution of the target protein.

[0051] Figure 6 Shown are the results of specific antibody IgG detection.

[0052] Figure 7 Shown are the results of specific antibody sIgA detection. DETAILED DESCRIPTION

[0053] The technical solutions of the various embodiments of the present invention are clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments derived by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts belong to the present invention.

[0054] In the following examples, the E. coli expression strain E. coli BL21 (DE3) used was purchased from Chengdu Bio-Chenxi Biotechnology Co., Ltd. and preserved by the applicant; the Helicobacter pylori strain Helicobacter pylori ATCC700824 used was purchased from the American Type Culture Collection and preserved by the applicant; DNA markers, restriction endonucleases Nco I and Xho I, and nucleic acid markers were products of Takara, and protein markers were products of Thermo Fisher; the plasmid extraction kit was a product of Tiangen Biochemical Technology (Beijing) Co., Ltd. The nickel column affinity filler was Ni-Sepharose HP (cytiva), and the cationic column filler was SP-Sepharose HP (cytiva). The centrifuge model was Beckman Coulter, JXN-26.

[0055] Example 1

[0056] The recombinant Helicobacter pylori antigen protein SecG provided in this embodiment is prepared by the following steps:

[0057] S1. Plasmid construction

[0058] The gene with the nucleotide sequence of SEQ ID NO: 2 is used as the target gene fragment, and the target gene fragment is connected to the expression vector plasmid to construct a recombinant expression plasmid containing the target gene fragment. Step S1 specifically includes the following steps:

[0059] S11. Using the principle of "reverse immunology," a potential amino acid sequence encoding the Helicobacter pylori recombinant antigen protein SecG was designed and screened. Based on the amino acid sequence, codon optimization was performed for E. coli preference to obtain the target gene fragment, the nucleotide sequence of which is shown in SEQ ID NO: 2.

[0060] S12. The target gene was fully synthesized (completed by Wuhan Jinkairui Bioengineering Co., Ltd.) and connected to the expression vector pET28a(+) through the restriction sites of XhoI and NcoI to construct a recombinant expression plasmid. The plasmid sequencing results were exactly the same as the target gene alignment sequence. After analysis of the results, it was confirmed that there was no amino acid mutation. The recombinant plasmid was extracted from the recombinant SecG-pET28a(+)-E. coli Top 10 strain and double enzyme digestion was performed. The size of the enzyme digestion band was consistent with the theoretical result. The results are as follows Figure 1 As shown, lane 1 is the recombinant plasmid, and lane 2 is the result of enzyme digestion, with the separated fragments being approximately 5000 bp and 400 bp.

[0061] S2. Inducible expression

[0062] The recombinant expression plasmid is transformed into a host bacterium for recombinant protein expression, and the recombinant strain is induced to express, the induced expression bacteria are collected, crushed, centrifuged, and the supernatant is collected. Step S2 specifically includes the following steps:

[0063] S21. The recombinant plasmid was transformed into the expression strain E. coli BL21 (DE3) to obtain recombinant E. coli SecG-pET28a (+) -E. coli BL21 (DE3) that can express SecG. The recombinant strain was streaked and revived overnight. A single colony from the SecG-pET28a (+) -E. coli BL21 (DE3) plate was inoculated into 60 mL of kanamycin-resistant LB medium and cultured at 220 rpm and 37 ° C overnight. Then, 60 mL of the overnight cultured SecG-pET28a (+) -E. coli BL21 (DE3) was inoculated into 6 L of kanamycin-resistant TB medium and cultured at 220 rpm and 37 ° C for 2 h until the OD 600 When the pH value was 0.6-0.8, 3 mL of 1 M IPTG was added to make the final IPTG concentration 0.5 mM, and the expression was induced in a constant temperature shaker at 220 rpm and 37°C for 4 h.

[0064] S22, using a refrigerated floor-standing centrifuge, the bacterial solution after induced expression was centrifuged at 8000g for 20min to collect the bacteria. Take 10g of wet bacteria and add 300mL of buffer A (50mM PB, 500mM NaCl, 25mM imidazole, pH 6.5) to resuspend the bacteria. The bacterial solution was subjected to a high-pressure homogenizer to disrupt the cells. The cell disruption solution was centrifuged at 12000g for 30min to collect the supernatant. The supernatant was filtered through a 0.45μm filter membrane through a suction filtration bottle for standby use.

[0065] S3. Product Purification

[0066] The supernatant was purified by nickel column affinity chromatography and cation exchange chromatography to obtain the Helicobacter pylori recombinant antigen protein SecG, the amino acid sequence of which is shown in SEQ ID NO: 1. Step S3 specifically includes the following steps:

[0067] S31, nickel ion column affinity chromatography

[0068] Equilibration: Equilibrate the nickel column using buffer A (50 mM PB, 500 mM NaCl, 25 mM imidazole, pH 6.5) on the purifier.

[0069] Sampling: taking the supernatant of step S22 for sample loading;

[0070] Reequilibration: Reequilibrate the nickel column with buffer B (50 mM PB, 150 mM NaCl, 25 mM imidazole, pH 6.5);

[0071] Elution: Use buffer C (50mM PB, 150mM NaCl, 50mM imidazole, pH 6.5) to elute impurity proteins; then use buffer D (50mM PB, 150mM NaCl, 100mM imidazole, pH 6.5) to elute the target protein, collect the eluate, and purify the electrophoresis results as shown below. Figure 3 As shown, lane 1 is loading, lane 2 is flow-through, lane 3 is washing, and lane 4 is elution of the target protein.

[0072] S32, cation exchange chromatography

[0073] Equilibration: Equilibrate the cation column with buffer E (20 mM PB, pH 6.5);

[0074] Sample loading: The eluate from step S31 was diluted 10-fold with buffer E (20 mM PB, pH 6.5) and then loaded;

[0075] Reequilibration: Reequilibrate the cation column using buffer E (20 mM PB, pH 6.5);

[0076] Elution: Use buffer F (20mM PB, 100mM NaCl, pH 6.5) to elute impurity proteins; then use buffer G (20mM PB, 200mM NaCl, pH 6.5) to elute the target protein, collect the protein elution peak indicated by A280, and obtain the Helicobacter pylori recombinant antigen protein SecG. The amino acid sequence of the recombinant antigen protein SecG is shown in SEQ ID NO: 1. The protein purity was detected by 15% SDS-page grayscale analysis, and the concentration was determined by BCA method and then stored at -80°C for future use. The purification electrophoresis results are shown in Figure 4As shown, lanes 1-3 are the elution of the target protein. After 15% SDS-PAGE detection, the eluate showed a single target protein band with a molecular weight of approximately 15.8 kDa. The purity of the purified protein obtained was 99.4%.

[0077] Example 2

[0078] This example verifies and analyzes the expression efficiency of the recombinant strain, specifically including the following steps:

[0079] S1. Plasmid construction

[0080] The gene with the nucleotide sequence of SEQ ID NO: 2 is used as the target gene fragment, and the target gene fragment is connected to the expression vector plasmid to construct a recombinant expression plasmid containing the target gene fragment. Step S1 specifically includes the following steps:

[0081] S11. Using the principle of "reverse immunology," a potential amino acid sequence encoding the recombinant Helicobacter pylori antigen protein SecG was designed and screened. The amino acid sequence encoding the recombinant antigen protein SecG is shown in SEQ ID NO: 1. Based on the amino acid sequence, codon optimization was performed for E. coli tropism to obtain the target gene fragment. The optimized nucleotide sequence is shown in SEQ ID NO: 2 (the underlined portion indicates the restriction enzyme cleavage site).

[0082] S12. The target gene was fully synthesized and ligated to the expression vector pET28a(+) through the restriction sites of Xho I and Nco I to construct a recombinant expression plasmid. The plasmid sequencing results were identical to the target gene alignment sequence. The results were analyzed to confirm that there were no amino acid mutations. The recombinant plasmid was extracted from the recombinant SecG-pET28a(+)-E. coli Top 10 strain and double enzyme digestion was performed. The size of the enzyme digestion band was consistent with the theoretical result. The results are as follows Figure 1 shown.

[0083] S2. Inducible expression

[0084] The recombinant expression plasmid is transformed into a host bacterium for recombinant protein expression, and the recombinant bacterium is induced to express, and the induced expression bacteria are collected, broken, and then electrophoresis analysis and identification are performed. Step S2 specifically includes the following steps:

[0085] S21. Transform the recombinant plasmid into the expression strain E. coli BL21 (DE3) to obtain recombinant E. coli SecG-pET28a (+) -E. coli BL21 (DE3) that can express SecG. The recombinant strain SecG-pET28a (+) -E. coli BL21 (DE3) was streaked and revived overnight. A single colony from the SecG-pET28a (+) -E. coli BL21 (DE3) plate was inoculated into 10 mL of kanamycin-resistant LB medium and cultured overnight at 220 rpm and 37 ° C. 200 μL of the overnight culture solution was transferred to 20 mL of kanamycin-resistant LB medium and cultured at 220 rpm and 37 ° C for 2-3 hours, and secondary activation was performed until the OD 600 When the pH value was 0.6-0.8, 10 μL of 1M IPTG was added to make the final concentration of IPTG 0.5 mM, and then the mixture was placed in a constant temperature shaker at 220 rpm and 37°C to induce expression for 4 h.

[0086] S22. Use a refrigerated floor-standing centrifuge to centrifuge the induced bacterial solution at 8000g for 10 min, discard the supernatant, take 0.5g of precipitated bacteria, add 4mL buffer A (50mM PB, 500mM NaCl, 25mM imidazole, pH 6.5), and suspend evenly. Ultrasonicate the cells in an ice bath for 10min (power 100w, ultrasound 3s on, stop 3s), centrifuge at 4℃ and 10000g for 10min, and separate the supernatant and precipitate.

[0087] S23. Add 4 mL of buffer A to resuspend the pellet. Take 40 μL of the lysate, supernatant, and resuspended pellet, add 10 μL of 5× protein loading buffer (Sangon, catalog number: C508320-0010), and incubate at 100°C in a metal bath for 10 min.

[0088] S24, 10 μL of each of the treated lysate, centrifuged supernatant, and centrifuged pellet suspension was loaded as a sample and subjected to 15% SDS-PAGE electrophoresis. After staining with Coomassie Brilliant Blue, the gel was scanned and imaged using a gel scanning imaging system (BIO-RAD, ChemiDoc™ MP Imaging System). Figure 2 As shown, lane 1 is the suspension of broken bacteria, lane 2 is the supernatant of the broken bacteria liquid, and lane 3 is the resuspension of the broken bacteria precipitate. The grayscale analysis results show that the recombinant antigen protein SecG is expressed soluble in SecG-pET28a(+)-E.coli BL21(DE3), and the expression level accounts for 22% of the total protein.

[0089] Example 3

[0090] In this example, the recombinant antigen protein SecG prepared in Example 1 was used as an antigen to conduct an animal immunization experiment, as follows.

[0091] S1, recombinant antigen protein SecG and aluminum phosphate combined to immunize mice

[0092] The recombinant antigen protein SecG prepared in Example 1 was used as an antigen and combined with aluminum phosphate adjuvant to immunize mice by intramuscular injection. After the immunization, the specific antibodies in the mouse serum were detected to identify the immunogenicity of the recombinant protein.

[0093] Experimental animals: 40 BalB / c mice, 8-10 weeks old, were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. and grouped as shown in Table 1.

[0094] Table 1 Grouping of mice in the experimental study of combined immunization with recombinant antigen protein SecG and aluminum phosphate adjuvant

[0095]

[0096] 50 μg of recombinant antigen protein SecG or an equal volume of PBS mixed with aluminum phosphate adjuvant (1:1) was injected into the thigh muscles at 4°C for 30 min, 50 μL / side, for a total of 100 μL / mouse. Four immunizations were performed on days 0, 14, 21, and 28.

[0097] S2, recombinant antigen protein SecG and LTs63K adjuvant combined with intranasal immunization of mice

[0098] Experimental animals: 40 female BalB / c mice, 8-10 weeks old, purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd., LTs63K were prepared in-house (preparation method as in the literature: Feng Qiang. Construction, expression and characterization of recombinant Escherichia coli heat-labile enterotoxin and its mutants and its B subunit. [D]. Chongqing University. 2003). Preparation results are shown in the table below. Figure 5 As shown, lane 1 is loading, lane 2 is flow-through, and lanes 3-5 are target protein elution. The animal groups are shown in Table 2 below:

[0099] Table 2 Grouping of mice immunized with recombinant antigen protein SecG and LTs63K adjuvant

[0100]

[0101]

[0102] After mixing 50 μg of antigen and LTs63K adjuvant, gently mix them at 4°C for 30 minutes and place them in an ice box until ready for use. Pipette the prepared immunogen and slowly drip it into the mouse nasal cavity at 8 μL / side, for a total of 16 μL / mouse. Four immunizations were performed on days 0, 14, 21, and 28, using the same injection dose and immunization method as above.

[0103] S3. Mouse-specific antibody detection

[0104] Elisa detection of serum specific antibody IgG after mice were immunized with S31 and recombinant antigen protein SecG and aluminum phosphate adjuvant

[0105] Five days after the fourth immunization, orbital venous blood was collected from BalB / c mice, incubated at 4°C for 3 hours, and then centrifuged at 3000 rpm for 5 minutes to separate the serum. Elisa was used to detect changes in the level of SecG-specific IgG in the mouse serum. The specific steps are as follows:

[0106] a. Antigen coating: Dilute the purified SecG protein to 4 μg / mL in the coating solution, coat the ELISA plate with 100 μL / well, and incubate at 4°C overnight.

[0107] b. Blocking: 300 μL / well of blocking solution, incubate at 37°C for 1 hour, wash with PBST and store at 4°C until use;

[0108] c. Specimen dilution: Serum was serially diluted from 1:8000 to 1:128000;

[0109] d. Sample addition: Take the coated ELISA plate and add diluted serum in sequence, 100 μL / well, with duplicates for each sample, incubate at 37°C for 1 hour, and wash 4 times with PBST;

[0110] e. Add secondary antibody: dilute HRP-labeled goat anti-mouse IgG (Sangon, catalog number: D110087-0100) in antibody diluent 1:10,000, 100 μL / well, incubate at 37°C for 30 min, and wash four times with PBST;

[0111] f. Color development: Add 100 μL / well of substrate color development solution, incubate at 37°C for 10 min, then add 50 μL / well of stop solution, and measure the OD value at a wavelength of 450 nm on a microplate reader;

[0112] g. Result judgment: A 样品 / A 阴性 ≥2.1 is positive.

[0113] The coating solution in a is 0.05 mM carbonate / bicarbonate buffer, pH 9.6 (15 mM Na2CO3, 35 mM NaHCO3); the blocking solution in b is 10 mM PBS (pH 7.4) + 1% BSA; the PBST wash solution in d is 10 mM PBS (pH 7.4) + 0.05% Tween-20; the antibody dilution solution in e is 10 mM PBS (pH 7.4) + 0.05% Tween-20 + 0.5% BSA; the developing solution in f is TMB storage solution: substrate buffer: 3% hydrogen peroxide = 10:90:1, where the TMB storage solution is 1 mg / mL TMB dissolved in DMSO, and the substrate buffer is 0.53 mM citric acid (pH 5.0) and 100 mM Na2HPO4; the stop solution in f is 2 M H2SO4.

[0114] Test results such as Figure 6 As shown in Table 3, the highest antibody titer produced by mice immunized with the recombinant antigen protein SecG and aluminum phosphate adjuvant reached 1:128,000. The geometric mean titer of recombinant SecG in mice immunized with SecG was 1:59,888.86. As shown in Table 3, the antibody positivity rate after immunization reached 100%, indicating that the recombinant antigen protein SecG obtained by the present invention can induce an immune response in the immunized mice and produce specific antibodies, demonstrating that the recombinant antigen protein SecG obtained by the present invention has high immunogenicity and immune titer.

[0115] Table 3 Geometric mean titer of serum IgG in mice after SecG immunization

[0116]

[0117] Elisa detection of specific antibodies sIgA in vaginal lavage fluid of mice after intranasal immunization with S32, recombinant antigen protein SecG and LTs63K adjuvant

[0118] Five days after the fourth immunization, vaginal lavage fluid from BalB / c mice was collected four times using PBST (PBS containing 0.05% Tween 20), 75 μL per wash, and 300 μL per mouse. After collection, the fluid was vortexed for 1 minute and centrifuged at 12,000 g for 3 minutes. The supernatant was then analyzed by Elisa for changes in SecG-specific sIgA levels. The specific steps are as follows:

[0119] a. Antigen coating: Dilute the purified SecG protein to 4 μg / mL in the coating solution, coat the ELISA plate with 100 μL / well, and incubate at 4°C overnight.

[0120] b. Blocking: Add 300 μL of blocking solution per well, incubate at 37°C for 1 h, wash the plate with PBST, and store at 4°C until use.

[0121] c. Specimen dilution: Serially dilute the serum from 1:16 to 1:256.

[0122] d. Sample addition: Take the coated ELISA plate and add diluted serum in sequence, 100 μL / well, with duplicates for each sample, incubate at 37°C for 1 hour, and wash 4 times with PBST;

[0123] e. Add secondary antibody: dilute HRP-labeled goat anti-mouse IgA (Abcam, catalog number: Ab97235) in antibody diluent 1:10,000, 100 μL / well, incubate at 37°C for 30 min, and wash four times with PBST;

[0124] f. Color development: Add 100 μL / well of substrate color development solution, incubate at 37°C for 10 min, then add 50 μL / well of stop solution, and measure the OD value at a wavelength of 450 nm on a microplate reader;

[0125] g. Result judgment: A 样品 / A 阴性 ≥2.1 is positive.

[0126] The coating solution in a is 0.05 mM carbonate / bicarbonate buffer, pH 9.6 (15 mM Na2CO3, 35 mM NaHCO3); the blocking solution in b is 10 mM PBS (pH 7.4) + 1% BSA; the PBST wash solution in d is 10 mM PBS (pH 7.4) + 0.05% Tween-20; the antibody dilution solution in e is 10 mM PBS (pH 7.4) + 0.05% Tween-20 + 0.5% BSA; the developing solution in f is TMB storage solution: substrate buffer: 3% hydrogen peroxide = 10:90:1, where the TMB storage solution is 1 mg / mL TMB dissolved in DMSO, and the substrate buffer is 0.53 mM citric acid (pH 5.0) and 100 mM Na2HPO4; the stop solution in f is 2 M H2SO4.

[0127] Test results such as Figure 7 As shown in Table 4, the highest sIgA antibody titer produced by mice immunized with the recombinant antigen protein SecG reached 1:128; the geometric mean titer of recombinant SecG in mice immunized with SecG mucosa was 1:76.1. As shown in Table 4, the antibody positivity rate after immunization reached 100%, indicating that the recombinant antigen protein SecG obtained in the present invention can stimulate mice to produce a higher mucosal immune response and produce specific secretory antibodies.

[0128] Table 4 Geometric mean titer of sIgA in vaginal lavage fluid of mice after SecG immunization

[0129]

[0130] Example 4

[0131] This example will conduct experimental analysis and evaluation on the protective effect of the recombinant antigen protein SecG prepared in Example 1 after immunization, as follows.

[0132] S1. Oral gavage of mice: 10 days after the last intranasal immunization described in Example 3, mice were orally gavaged with live H. pylori ATCC700824 bacteria for a challenge experiment. The mice were fasted for 24 hours and deprived of water for 17 hours before gavage. The infection dose for each mouse was 2.0×10 7 CFU, and water and food were restored 2 h after gavage.

[0133] S2. Plate culture: One week after oral administration, mice were slaughtered, and the gastric tissue was minced and placed in PBS buffer, vortex-washed for 3 minutes. The washing stock solution and its 10-fold dilution were spread on Skirrow plates (15 g / L peptone (Haibo Bio), 2.5 g / L tryptone (Oxoid), 5 g / L yeast extract (Oxoid), 5 g / L sodium chloride (Cologne reagent), pH 7.4) containing 5% defibrinated sheep blood (Nanjing Maojie Biological) and 0.5% compound antibiotics (vancomycin 1.67 mg / mL, polymyxin 0.0694 mg / mL, trimethoprim 0.5 mg / mL, amphotericin B 0.2 mg / mL). The plates were incubated at 37°C microaerophilically (5% O2, 10% CO2, 85% N2) for 2-3 days and observed.

[0134] S3. Analysis and Identification: Based on the characteristics of H. pylori colonies, a rapid urease assay and microscopic examination were used to detect the presence of H. pylori on the plate to determine the infection rate of the mice. Vaccine protection rate = (control group infection rate - immunization group infection rate) / control group infection rate * 100%.

[0135] S4. Statistical Results: Table 5 shows the results of plate culture analysis of 20 mice in each of the control and experimental groups. All 20 mice in the control group tested positive for plate culture, with an infection rate of 100%. Eleven of the 20 mice in the experimental group tested positive for plate culture, with an infection rate of 55%. This indicates that the vaccine has a protective efficacy of 45%. This indicates that the recombinant antigen protein SecG obtained in the present invention can induce a strong immune response in immunized mice and inhibit Helicobacter pylori colonization in the mouse stomach, thereby providing a protective effect against Helicobacter pylori infection.

[0136] Table 5 Statistics of positive rates of Helicobacter pylori infection in mice after immunization

[0137] Mouse number control group Experimental group Mouse number control group Experimental group 1 + + 11 + - 2 + + 12 + + 3 + - 13 + + 4 + - 14 + - 5 + + 15 + + 6 + + 16 + - 7 + - 17 + - 8 + + 18 + - 9 + - 19 + + 10 + + 20 + +

[0138] Note: "+" indicates that both the rapid urease test and the microscopic examination are positive, "-" indicates that both the rapid urease test and the microscopic examination are negative

[0139] Those skilled in the art will appreciate that the embodiments herein are intended to help readers understand the principles of the present invention, and it should be understood that the scope of protection of the present invention is not limited to such specific descriptions and embodiments. Those skilled in the art may make various other specific variations and combinations based on the technical teachings disclosed herein without departing from the essence of the present invention, and such variations and combinations are still within the scope of protection of the present invention.

Claims

1. A use of a Helicobacter pylori recombinant antigen protein SecG in preparing a Helicobacter pylori mucosal immune vaccine, wherein the amino acid sequence of the Helicobacter pylori recombinant antigen protein SecG is shown in SEQ ID NO:

1.

2. The use according to claim 1, characterized in that: The nucleotide sequence of the recombinant antigen protein SecG encoding gene is shown in SEQ ID NO:

2.

3. The use according to claim 1, characterized in that: The Helicobacter pylori mucosal immune vaccine is a nasal drop vaccine comprising a recombinant antigen protein SecG and an adjuvant.

4. The use according to claim 3, characterized in that: The adjuvant is LTs63K adjuvant.

5. The use according to any one of claims 1 to 4, characterized in that: The preparation method of the Helicobacter pylori recombinant antigen protein SecG comprises the following steps: S1. Plasmid construction The gene with the nucleotide sequence of SEQ ID NO: 2 is used as a target gene fragment, and the target gene fragment is connected to an expression vector plasmid to construct a recombinant expression plasmid containing the target gene fragment; S2. Inducible expression The recombinant expression plasmid is transformed into a host bacterium for expression of the recombinant protein for induction of expression, the induced expression bacteria are collected, the bacteria are crushed, centrifuged and the supernatant is collected; S3. Product purification The supernatant was purified by nickel column affinity chromatography and cation exchange chromatography to obtain the Helicobacter pylori recombinant antigen protein SecG, the amino acid sequence of which is shown in SEQ ID NO: 1; The step S3 specifically includes the following steps: S31, nickel column affinity chromatography Equilibration: Equilibrate the nickel column with buffer A; Sampling: taking the supernatant of step S2 for sampling; Re-equilibration: Re-equilibrate the nickel column with buffer B; Elution: Use buffer C to elute impurity proteins; then use buffer D to elute the target protein and collect the eluate; S32, cation exchange chromatography Equilibration: Equilibrate the cation column with buffer E; Sample loading: dilute the eluate from step S31 with buffer E and then load the sample; Re-equilibration: Re-equilibrate the cation column using buffer E; Elution: Use buffer F to elute impurity proteins; then use buffer G to elute the target protein, collect the eluate, and obtain the Helicobacter pylori recombinant antigen protein SecG; The composition of the buffer A is: 20-50 mM phosphate buffer at pH 6-7, 300-500 mM NaCl, and 0-30 mM imidazole; the composition of the buffer B is: 20-50 mM phosphate buffer at pH 6-7, 100-200 mM NaCl, and 0-30 mM imidazole; the composition of the buffer C is: 20-50 mM phosphate buffer at pH 6-7, 100-200 mM NaCl, and 40-60 mM imidazole; the composition of the buffer D is: 20-50 mM phosphate buffer at pH 6-7, 100-200 mM NaCl, and 90-120 mM imidazole; the composition of the buffer E is: 20-50 mM phosphate buffer at pH 6-7; the composition of the buffer F is: 20-50 mM phosphate buffer at pH 6-7, 80-120 mM NaCl; buffer G consists of 20-50 mM phosphate buffer at pH 6-7, 180-250 mM NaCl.

6. The use according to claim 5, characterized in that: The step S1 comprises the following steps: S11. Screening and obtaining the amino acid sequence encoding the Helicobacter pylori antigen SecG, and performing Escherichia coli-biased codon optimization based on the amino acid sequence to obtain the target gene fragment, the nucleotide sequence of which is shown in SEQ ID NO: 2; S12. Perform full gene synthesis on the target gene and connect it to the expression vector plasmid through the restriction sites of Xho I and Nco I to construct a recombinant expression plasmid.

7. The use according to claim 5, characterized in that: The step S2 comprises the following steps: S21, transforming the recombinant expression plasmid into the expression host bacteria to obtain a recombinant strain that can express the recombinant antigen protein SecG, and culturing the recombinant strain to OD 600 0.6-0.8, then induce expression with 0.3-0.5 mM isopropylthiogalactoside at 30-37 °C and 180-240 rpm for 4-8 h; S22. Centrifuge the bacterial solution after induced expression and discard the supernatant to obtain precipitated bacteria. Add buffer A to the precipitated bacteria and suspend them evenly. After the bacteria are broken, centrifuge and discard the precipitate. Filter the supernatant through a 0.45 μm filter membrane through a suction filtration bottle for later use.

8. The use according to claim 7, characterized in that: In step S2, a high-pressure homogenizer or an ultrasonic disruptor is used to disrupt the bacteria. The high-pressure homogenizer disruption parameters are: pressure 600-800 bar, flow rate 100-150 mL / min, repeated 4-8 times; the ultrasonic disruptor disruption parameters are preferably: ultrasonication 3-5 seconds, stop 3-5 seconds, and alternating disruption for 20-40 minutes.

9. The use according to claim 5, characterized in that: The expression plasmid vector is pET28a(+); the host bacteria is Escherichia coli.

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