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

Abstract

The present invention discloses a recombinant antigen protein TatB of Helicobacter pylori and a preparation method and application thereof. The amino acid sequence of the recombinant antigen protein TatB is shown in SEQ ID NO:1, and the nucleotide sequence of the recombinant antigen protein TatB 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, the cost is low, and the operability is strong. The purified antigen has high immunogenicity and immune titer and can be used as a genetic engineering subunit vaccine. As an important component protein of the Helicobacter pylori membrane transport protein, the recombinant antigen protein TatB will become one of the most promising candidate antigens in the development of Helicobacter pylori genetic engineering vaccines.

Description

Technical Field

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

[0002] Helicobacter pylori is a spiral or S-shaped Gram-negative microaerophilic bacterium. Helicobacter pylori infection is one of the most common infectious diseases in the world. In some developing countries, more than half of the population is infected with Helicobacter pylori. Epidemiological studies have shown that Helicobacter pylori infection is closely related to diseases such as chronic gastritis, peptic ulcer, gastric lymphoma and gastric cancer. In 1994, the World Health Organization classified Helicobacter pylori as a Class I biological carcinogen. Currently, the treatment of diseases caused by Helicobacter pylori infection mainly uses a "triple" or "quadruple" therapy combined with antibiotics to inhibit or eliminate Helicobacter pylori in the patient's body. Although the early treatment effect is significant, the treatment effect on patients who are infected again is significantly reduced, and recurrence is very likely to occur after stopping the drug.

[0003] As for the problems encountered in treatment after infection with Helicobacter pylori, people have to find new solutions in order to achieve the goal of not increasing bacterial resistance and improving the effect of clinical treatment. After decades of scientific research, a large number of experiments have proved that vaccination can effectively prevent and even treat a series of diseases caused by Helicobacter pylori infection. In 1993, Czinn et al. orally immunized mice with feline Helicobacter pylori lysate to induce mucosal production of high levels of secretory IgA (sIgA), and found that it could also prevent reinfection of feline Helicobacter pylori, confirming the protective effect of the vaccine for the first time. Subsequently, vaccines against Helicobacter pylori were studied by more and more scholars. 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] The antigen composition of Helicobacter pylori whole-cell vaccine is complex, and Helicobacter pylori also has many problems such as long production cycle, harsh culture conditions, low yield, and easy contamination. Therefore, there are many difficulties in preparing traditional vaccines with Helicobacter pylori whole-cell. With the continuous development of molecular biology, genetic engineering vaccines have become a hot research topic. At present, many different genomes or genes of Helicobacter pylori have been cloned for the study of Helicobacter pylori genetic engineering vaccines, including: chemokines cheA and cheY, flagellin genes flaA and flaB, heat shock protein gene hspA, urease genes UreA, UreB, vacuolating toxin gene vacA, etc. Although some of these antigenic proteins can produce certain immune protection effects, there are still problems such as low immune activity and low recombinant expression efficiency, which leads to difficulties in large-scale protein preparation. Therefore, in addition to improving the immunization method and screening efficient and non-toxic adjuvant types, further research on Helicobacter pylori vaccines should mainly develop a variety of more comprehensive and effective immune antigens, and form an efficient human Helicobacter pylori vaccine through the 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 also have long production cycles, harsh culture conditions, low yields, and easy contamination, as well as the fact that in the development of Helicobacter pylori genetic engineering 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 TatB and a preparation method and application thereof. The present invention obtains a high-purity antigen protein through two-step purification by nickel ion affinity chromatography and anion exchange chromatography on the basis of recombinant expression. Animal experiments show that the recombinant antigen protein TatB has good immunogenicity and high immune titer, and can induce efficient mucosal immune response, and can be used as an effective vaccine candidate antigen.

[0006] At present, bioinformatics is a hot topic in biological research. The present invention uses the study of the structures of various Helicobacter pylori antigen proteins in the database to screen out a new immune recombinant antigen protein TatB for Helicobacter pylori according to the principle of "reverse immunology". Tat is a system in Helicobacter pylori that transports folded proteins across the membrane, and its signal peptide contains a highly conserved twin arginine motif. The Tat system includes multiple proteins such as TatA and TatB, and their complexes form a transport channel on the plasma membrane. Previously, no studies have shown that the TatB protein of Helicobacter pylori can be used as a Helicobacter pylori-specific antigen to induce an immune response in the body. The present invention clones the selected TatB antigen gene into a protein expression vector, recombines it in an engineered bacterium for large-scale expression, and the antigen prepared after purification can be used as a genetic engineering subunit vaccine, and the purification process is simple, low cost, and strong operability. As an important component protein in the Helicobacter pylori membrane transport protein, the antigen protein TatB will also be one of the most promising candidate antigens in the development of Helicobacter pylori genetic engineering vaccines.

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

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

[0009] KFPQAVVDIKKETLEYQKLFENKVESLKGVKIEELEDAKVTAENEIKSIQDLMQDYKQSLENNAPPNHSNKEISNEEALNEEISNNESLKGAELLTNNPKEHDKEKEHVLE.

[0010] The nucleotide sequence of the recombinant antigen protein TatB encoding gene is shown in SEQ ID NO: 2. The recombinant antigen protein TatB is prepared by selecting a peptide encoding gene showing 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 for expression through a prokaryotic expression vector, and the target gene fragment has a nucleotide sequence as shown in SEQ ID NO: 2 (the underlined part indicates the restriction site).

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

[0012] CCATGGAGAAATTTCCGCAAGCAGTTGTTGATATCAAGAAAGAAACTTTGGAGTATCAGAAATTATTTGAGAACAAAGTTGAGTCCCTTAAGGGTGTGAAAATCGAGGAGTTAGAGGACGCTAAAGTCACAGCGGAGAATGAGATCAAAAGTATCCAAGACCTGATGCAA GATTATAAGCAAAGTCTGGAAAACAACGCCCCGCCGAATCATTCGAATAAAGAAATCAGCAATGAGGAGGCCCTGAATGAAGAAATTAGCAACAATGAAAGTCTGAAGGGTGCGGAGCTTCTGACAAACAACCCAAAGGAACACGACAAAGAGAAAGAGCATGTC CTCGAG .

[0013] The present invention also provides a method for preparing a recombinant Helicobacter pylori antigen protein TatB, 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 recombinant protein expression to induce expression, the induced expression bacteria are collected, the bacteria are broken and then centrifuged to collect the supernatant;

[0018] S3. Product purification

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

[0020] The preparation method of the above-mentioned Helicobacter pylori recombinant antigen protein TatB 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 TatB, and optimizes the codons preferred by Escherichia coli according to the amino acid sequence to obtain the target gene fragment with the nucleotide sequence as shown in SEQ ID NO: 2. Finally, the target gene fragment is connected to the expression vector plasmid by double enzyme digestion to obtain a recombinant expression plasmid. A skilled technician 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 TatB of Helicobacter pylori, and optimizing the codons preferred by Escherichia coli according to 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 antigen protein TatB of Helicobacter pylori, inducing expression is a conventional operation in the art, and the present invention has no special limitation thereto. Those skilled in the art can induce expression of the recombinant strain to produce the recombinant antigen protein in a conventional manner. 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 TatB, and culturing the recombinant strain until 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. After induced expression, the bacterial solution was centrifuged and the supernatant was discarded to obtain precipitated bacteria. Buffer A was added to the precipitated bacteria to be evenly suspended. After the bacteria were broken, the precipitate was discarded by centrifugation. The supernatant was filtered through a 0.45 μm filter membrane through a suction filtration bottle for later use.

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

[0027] The preparation method of the recombinant antigen protein TatB of Helicobacter pylori described above, the present invention purifies the antigen protein by nickel column affinity chromatography and anion column exchange chromatography, the specific operations of nickel column affinity chromatography and anion column exchange chromatography can adopt conventional operations in the art. In the present invention, the 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 of 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, anion exchange chromatography

[0034] Equilibration: Use buffer E to equilibrate the anion column;

[0035] Loading: diluting the eluate in step S31 with buffer E and then loading the sample;

[0036] Re-equilibration: Use buffer E to re-equilibrate the anion column;

[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 TatB.

[0038] The preparation method of the above-mentioned Helicobacter pylori recombinant antigen protein TatB, preferably, the composition of the buffer A is: 20-50mM phosphate buffer at pH 7-8, 300-500mM NaCl, 0-25mM imidazole; the composition of the buffer B is: 20-50mM phosphate buffer at pH 7-8, 100-200M NaCl, 0-25mM imidazole; the composition of the buffer C is: 20-50mM phosphate buffer at pH 7-8, 100-200mM NaCl, 30-40mM imidazole; the composition of the buffer D is: 20-50mM phosphate buffer at pH 7-8, 100-200mM NaCl, 80-120mM imidazole; the composition of the buffer E is: 20-50mM phosphate buffer at pH 7-8; the composition of the buffer F is: 20-50mM phosphate buffer at pH 7-8, 40-70mM NaCl; the composition of the buffer G is 20-50mM phosphate buffer at pH 7-8, 120-200mM NaCl.

[0039] The preparation method of the above-mentioned Helicobacter pylori recombinant antigen protein TatB, the present invention has no special restrictions on the method of bacterial (cell) disruption, in the step S2, preferably a high-pressure homogenizer or ultrasonic disruptor is used to disrupt the bacteria, and the high-pressure homogenizer bacteria disruption parameters are preferably: pressure 600-800 bar, flow rate 100-150 mL / min, repeated 4-8 times; ultrasonic disruptor bacteria disruption parameters are preferably: ultrasound 3-5s, stop 3-5s, alternate bacteria disruption for 20-40min. Further, the centrifugal operation parameters in step S2 can also adopt conventional operation parameters in the art, preferably, the centrifugal speed is 10000-14000g, and the centrifugal time is 20-40min.

[0040] In the preparation method of the recombinant Helicobacter pylori antigen protein TatB, the fillers in the nickel column and the anion column can be conventional fillers in the art. Preferably, the filler in the nickel column is Ni-agarose HP (Ni-Sepharose HP); the filler in the anion column is Q-agarose HP (Q-Sepharose HP).

[0041] The present invention also provides the use of the above-mentioned Helicobacter pylori recombinant antigen protein TatB in the preparation of Helicobacter pylori vaccine. Animal experiments have shown that the recombinant antigen protein TatB can effectively stimulate the body to produce a high level of immune response and has a high immune activity. At the same time, the immune protection evaluation experiment confirms that the TatB vaccine has a good protective effect, proving that the recombinant antigen protein TatB has vaccine application value. The present invention clones the selected antigen gene into a protein expression vector, recombines it in the engineered bacteria for large-scale expression, and the purified prepared 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 TatB 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 TatB provided by the present invention and its preparation method and application have the following beneficial effects:

[0043] (1) The Helicobacter pylori recombinant antigen protein TatB provided by the present invention has a high expression efficiency. According to the principle of "reverse immunology", the present invention obtains the encoding amino acid sequence of the Helicobacter pylori recombinant antigen protein TatB as shown in SEQ ID NO:1 through data analysis, and optimizes the codon of the sequence for the expression host strain. The optimized nucleotide sequence is shown in SEQ ID NO:2. The obtained nucleotide sequence is constructed into the expression vector plasmid pET28a(+) through full chemical synthesis and transformed into the recombinant protein expression host Escherichia coli BL21(DE3) for induced expression, and the expression amount of the expressed recombinant protein is higher than 37%.

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

[0045] (3) The recombinant antigen protein TatB of Helicobacter pylori provided by the present invention has high immune activity and immune protection effect. When the purified protein was co-injected with aluminum hydroxide adjuvant to immunize BalB / c mice, the IgG level in the serum of the TatB plus immune adjuvant group was significantly higher than that of the negative control group (PBS group) P<0.01, and the highest antibody titer produced by the immunized mice reached 1:64000, and the antibody positive rate after immunization reached 100%, proving that the recombinant antigen protein TatB can effectively stimulate the body to produce a higher immune response and has high immune activity; when the recombinant antigen protein TatB was combined with LTs63K adjuvant and nasally immunized mice, the sIgA antibody titer produced by the recombinant antigen protein TatB immunized mice reached 1:256; the antibody positive rate after immunization reached 100%, indicating that the recombinant antigen protein TatB can stimulate mice to produce a higher level of mucosal immune response. At the same time, the immune protection evaluation experiment confirmed that the TatB vaccine has a good protective effect, proving that the recombinant antigen protein TatB 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 restriction digestion of pET28a(+) / TatB plasmid are shown in Figure 1. Lane 1 is the recombinant plasmid, and lane 2 is the result of double restriction digestion. The separated fragments are approximately 5000 bp and 350 bp.

[0047] Figure 2 The results of TatB protein induction identification are shown in Figure 1. 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, lane 1 is the loading, lane 2 is the flow-through, and lanes 3-7 are the elution of the target protein;

[0049] Figure 4 The results of SDS-PAGE electrophoresis after ion column chromatography, lane 1 is the loading, lane 2 is the flow-through, and lanes 3-4 are the elution of the target protein;

[0050] Figure 5 The SDS-PAGE electrophoresis results of the prepared LTs63K, lane 1 is the loading, 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 embodiments of the present invention are clearly and completely described in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work 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 Marker, restriction endonuclease Nco I and Xho I, nucleic acid Marker are products of Takara, protein Marker is a product of Thermo Fisher; plasmid extraction kit is a product of Tiangen Biochemical Technology (Beijing) Co., Ltd. The nickel column affinity filler is Ni-Sepharose HP (cytiva), and the anion column filler is Q-Sepharose HP (cytiva). The model of the centrifuge is Beckman Coulter, JXN-26.

[0055] Example 1

[0056] The recombinant Helicobacter pylori antigen protein TatB 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. By using the principle of "reverse immunology", the amino acid sequence encoding the potential Helicobacter pylori recombinant antigen protein TatB was designed and screened, and the codon optimization for Escherichia coli preference was performed based on the amino acid sequence to obtain the target gene fragment, the nucleotide sequence of which is shown in SEQ ID NO: 2;

[0060] S12, the target gene was synthesized (completed by Wuhan Jinkairui Bioengineering Co., Ltd.), and the recombinant expression plasmid was constructed by connecting it to the expression vector pET28a(+) through the restriction sites of XhoI and Nco I. The plasmid sequencing results were exactly the same as the target gene alignment sequence, and the results were analyzed to confirm that there was no amino acid mutation. The recombinant plasmid was extracted from the recombinant TatB-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 identification after restriction digestion, and the separated fragments are approximately 5000 bp and 350 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, the bacteria are crushed and then centrifuged to collect the supernatant, and step S2 specifically includes the following steps:

[0063] S21. Transform the recombinant plasmid into the expression strain E. coli BL21 (DE3) to obtain recombinant E. coli TatB-pET28a (+)-E. coli BL21 (DE3) that can express TatB. The recombinant strain was streaked and revived overnight. A single colony from the TatB-pET28a (+)-E. coli BL21 (DE3) plate was inoculated with 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 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 to centrifuge the induced expression bacterial solution at 8000g for 20min to collect the bacteria. Take 10g of wet bacteria and add 300mL buffer A (50mM PB, 500mM NaCl, 25mM imidazole, pH 7.4) to resuspend the bacteria, and then use a high-pressure homogenizer to break the cells. The cell breaking solution is centrifuged at 12000g for 30min to collect the supernatant, and the supernatant is filtered through a 0.45μm filter membrane through a suction filtration bottle for standby use.

[0065] S3. Product purification

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

[0067] Equilibration: The nickel column was equilibrated with buffer A (50 mM PB, 500 mM NaCl, 25 mM imidazole, pH 7.4) on the purifier;

[0068] Sampling: taking the supernatant of step S22 for sampling;

[0069] Re-equilibration: Re-equilibrate the nickel column using buffer B (50 mM PB, 150 mM NaCl, 25 mM imidazole, pH 7.4);

[0070] Elution: Use buffer C (50mM PB, 150mM NaCl, 35mM imidazole, pH 7.4) to elute impurity proteins; then use buffer D (50mM PB, 150mM NaCl, 100mM imidazole, pH 7.4) to elute the target protein, collect the eluate, and purify the electrophoresis results as shown in Figure 3 As shown, lane 1 is loading, lane 2 is flow-through, and lanes 3-7 are elution of the target protein.

[0071] S32, anion exchange chromatography

[0072] Equilibration: The anion column was equilibrated with buffer E (20 mM PB, pH 7.4);

[0073] Sample loading: dilute the eluate from step S31 10 times with buffer E (20 mM PB, pH 7.4) and then load the sample;

[0074] Re-equilibration: Use buffer E (20 mM PB, pH 7.4) to re-equilibrate the anion column;

[0075] Elution: Use buffer F (20mM PB, 50mM NaCl, pH 7.4) to elute impurity proteins; then use buffer G (20mM PB, 150mM NaCl, pH 7.4) to elute the target protein, collect the protein elution peak indicated by A280, and obtain the Helicobacter pylori recombinant antigen protein TatB. The amino acid sequence of the recombinant antigen protein TatB 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 stored at -80°C for future use. The purification electrophoresis results are shown in Figure 4 As shown, lanes 3-4 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 16.8 kDa. The purity of the purified protein obtained was 99.2%.

[0076] Example 2

[0077] This example verifies and analyzes the expression efficiency of the recombinant strain, specifically including the following steps:

[0078] S1. Plasmid construction

[0079] 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:

[0080] S11. Through the principle of "reverse immunology", the amino acid sequence encoding the potential Helicobacter pylori recombinant antigen protein TatB was designed and screened, and the amino acid sequence encoding the recombinant antigen protein TatB is shown in SEQ ID NO: 1. According to the amino acid sequence, the codon optimization of Escherichia coli preference was performed to obtain the target gene fragment, and the optimized nucleotide sequence is shown in SEQ ID NO: 2 (the underlined part indicates the restriction site).

[0081] S12. The target gene was fully synthesized and connected 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 exactly the same as the target gene alignment sequence. The results were analyzed to confirm that there was no amino acid mutation. The recombinant plasmid was extracted from the recombinant TatB-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.

[0082] S2. Inducible expression

[0083] 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:

[0084] S21. Transform the recombinant plasmid into the expression strain E. coli BL21 (DE3) to obtain recombinant E. coli TatB-pET28a (+)-E. coli BL21 (DE3) that can express TatB. The recombinant strain was streaked and revived overnight. A single colony of the TatB-pET28a (+)-E. coli BL21 (DE3) plate was inoculated with 10 mL of kanamycin-resistant LB medium, and cultured overnight at 220 rpm and 37°C. 200 μL of the overnight cultured bacterial 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 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.

[0085] S22. Use a refrigerated floor-standing centrifuge to centrifuge the induced expression 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 7.4) and suspend evenly, disrupt the cells by ultrasonication on an ice bath for 10min (power 100w, ultrasonication for 3s on and off for 3s), centrifuge at 4℃ and 10000g for 10min, and separate the supernatant and precipitate.

[0086] S23. Add 4 mL of buffer A to resuspend the precipitate. Take 40 μL of the bacterial lysis solution, supernatant, and resuspended precipitate, add 10 μL of 5× protein loading buffer (Sinopharm, catalog number: C508320-0010), and place in a 100°C metal bath for 10 min.

[0087] S24, 10 μL of the treated lysed bacterial solution, centrifugal supernatant and centrifugal precipitate resuspension were taken as samples, and subjected to 12% SDS-PAGE electrophoresis. After staining with Coomassie brilliant blue, the samples were scanned and imaged by a gel scanning imaging system (BIO-RAD, ChemiDocTM MP Imaging System). Figure 2 As shown, lane 1 is a suspension of broken bacteria, lane 2 is a supernatant of broken bacteria, and lane 3 is a resuspension of broken bacteria precipitates. Grayscale analysis results show that the recombinant antigen protein TatB is expressed soluble in TatB-pET28a(+)-E.coli BL21(DE3), and the expression level accounts for 37% of the total protein.

[0088] Example 3

[0089] In this example, the recombinant antigen protein TatB prepared in Example 1 was used as an antigen to conduct an animal immunization experiment, as follows.

[0090] S1. Immunization of mice with recombinant antigen protein TatB and aluminum hydroxide

[0091] The recombinant antigen protein TatB prepared in Example 1 was used as an antigen and combined with aluminum hydroxide 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.

[0092] Experimental animals: BalB / c mice: 40, 8-10 weeks old, purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd., and grouped as shown in Table 1.

[0093] Table 1 Grouping of mice in the experiment of combined immunization with recombinant antigen protein TatB and aluminum hydroxide adjuvant

[0094]

[0095] 50 μg of recombinant antigen protein TatB or an equal volume of PBS and aluminum hydroxide adjuvant 1:1 was mixed, adsorbed at 4℃ for 30 minutes, and injected into the thigh muscles of both sides, 50 μL / side, a total of 100 μL / mouse. Four immunizations were performed on days 0, 14, 21, and 28, respectively.

[0096] S2, recombinant antigen protein TatB and LTs63K adjuvant combined with intranasal immunization of mice

[0097] Experimental animals: BalB / c mice, female, 8-10 weeks old, purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd., LTs63K self-made (preparation method as shown in the literature: Feng Qiang. Construction, expression and property research of recombinant Escherichia coli heat-labile enterotoxin and its mutants and its B subunit. [D]. Chongqing University. 2003), preparation results as shown Figure 5 As shown, lane 1 is loading, lane 2 is flow-through, and lanes 3-5 are target protein elution. Animal grouping is shown in Table 2 below:

[0098] Table 2 Grouping of mice immunized with recombinant antigen protein TatB and LTs63K adjuvant

[0099]

[0100] 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 for later use. Pipette the prepared immunogen and slowly drip it into the nasal cavity of mice: 9 μL / side, a total of 18 μL / mouse; four immunizations were performed on days 0, 14, 21, and 28, respectively, with the injection dose and immunization method being the same as above.

[0101] S3. Mouse-specific antibody detection

[0102] Elisa detection of serum specific antibody IgG after mice were immunized with S31, antigen TatB and aluminum hydroxide adjuvant

[0103] Five days after immunization, orbital venous blood was collected from mice, and after standing at 4°C for 3 h, the blood was centrifuged at 3000 rpm for 5 min to separate the serum. The changes in TatB-specific serum IgG levels were detected by Elisa.

[0104] a. Antigen coating: dilute the purified TatB protein to 4 μg / mL with coating solution, coat the ELISA plate with 100 μL / well at 4°C overnight.

[0105] b. Blocking: 300 μL / well of blocking solution, incubate at 37°C for 1 h, wash the plate with PBST and store at 4°C for later use.

[0106] c. Specimen dilution: Serum was serially diluted from 1:4000 to 1:128000.

[0107] d. Sample addition: Take the coated ELISA plate and add diluted serum in sequence, 100 μL / well, perform duplicates for each sample, incubate at 37°C for 1 hour, and wash 4 times with PBST;

[0108] e. Add secondary antibody: dilute HRP-labeled goat anti-mouse IgG (Sangon, catalog number: D110087-0100) with antibody diluent 1:10000, 100 μL / well, incubate at 37°C for 30 min, and wash 4 times with PBST;

[0109] 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 450 nm on a microplate reader;

[0110] g. Result judgment: A 样品 / A 阴性 ≥2.1 is positive.

[0111] Among them, the coating solution in a is 0.05mM carbonate / bicarbonate buffer pH9.6 (15mM Na2CO3, 35mMNaHCO3); the blocking solution in b is 10mM PBS (pH7.4) + 1% BSA; the PBST washing solution in d is 10mM PBS (pH7.4) + 0.05% Tween-20; the antibody dilution solution in e is 10mM PBS (pH7.4) + 0.05% Tween-20 + 0.5% BSA; the color development solution in f is TMB storage solution: substrate buffer: 3% hydrogen peroxide = 10﹕90﹕1, the TMB storage solution is 1mg / mL TMB dissolved in DMSO, the substrate buffer is 0.53mM citric acid (pH5.0), Na2HPO4 100mM; the stop solution in f is 2M H2SO4.

[0112] Test results such as Figure 6 As shown: the highest antibody titer produced by mice immunized with the recombinant TatB protein reached 1:64000, the geometric mean titer of TatB immunized mice to recombinant TatB was 1:46987.85, and the antibody positive rate after immunization reached 100%, indicating that the recombinant TatB protein obtained in the present invention can cause the immunized mice to produce an immune response and produce specific antibodies, proving that the TatB obtained in the present invention has high immunogenicity and immune titer.

[0113] Table 3 Geometric mean titer of serum IgG in mice after TatB immunization

[0114]

[0115]

[0116] Detection of specific antibody sIgAElisa in vaginal lavage fluid of mice immunized with S32, antigen TatB and LTs63K adjuvant by intranasal drops

[0117] Five days after the fourth immunization, vaginal lavage fluid of BalB / c mice was collected with PBST (PBS containing 0.05% Tween 20), 75 μL / time, lavage 4 times, and 300 μL / mouse was collected. After collection, vortex for 1 min, centrifuge at 12000g for 3 min, and the supernatant was used for Elisa to detect changes in TatB-specific sIgA levels. The specific steps are as follows:

[0118] a. Antigen coating: dilute the purified TatB protein to 4 μg / mL using the coating solution, coat the ELISA plate with 100 μL / well, and incubate at 4°C overnight.

[0119] b. Blocking: 300 μL / well of blocking solution, incubate at 37°C for 1 h, wash with PBST and store at 4°C for later use;

[0120] c. Specimen dilution: Serum was serially diluted from 1:16 to 1:256;

[0121] d. Sample addition: Take the coated ELISA plate and add diluted serum in sequence, 100 μL / well, perform duplicates for each sample, incubate at 37°C for 1 hour, and wash 4 times with PBST;

[0122] e. Add secondary antibody: dilute HRP-labeled goat anti-mouse IgA (Abcam, catalog number: Ab97235) with antibody diluent 1:10000, 100 μL / well, incubate at 37°C for 30 min, and wash 4 times with PBST;

[0123] 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 450 nm on a microplate reader;

[0124] g. Result judgment: A 样品 / A 阴性 ≥2.1 is positive.

[0125] Among them, the coating solution in a is 0.05mM carbonate / bicarbonate buffer pH9.6 (15mM Na2CO3, 35mMNaHCO3); the blocking solution in b is 10mM PBS (pH7.4) + 1% BSA; the PBST washing solution in d is 10mM PBS (pH7.4) + 0.05% Tween-20; the antibody dilution solution in e is 10mM PBS (pH7.4) + 0.05% Tween-20 + 0.5% BSA; the color development solution in f is TMB storage solution: substrate buffer: 3% hydrogen peroxide = 10﹕90﹕1, the TMB storage solution is 1mg / mL TMB dissolved in DMSO, the substrate buffer is 0.53mM citric acid (pH5.0), Na2HPO4 100mM; the stop solution in f is 2M H2SO.

[0126] Test results such as Figure 7 As shown in Table 4, the titer of sIgA antibodies produced by mice immunized with TatB protein antigen reached 1:256; the geometric mean titer of TatB immunized mice to recombinant TatB was 1:174.9; the antibody positive rate after immunization reached 100%, indicating that the recombinant antigen protein TatB obtained by the present invention can stimulate mice to produce mucosal immune response and produce specific secretory antibodies.

[0127] Table 4 Geometric mean titer of sIgA in vaginal lavage fluid of mice after TatB immunization

[0128]

[0129] Example 4

[0130] This example will conduct experimental analysis and evaluation on the protective effect of the recombinant antigen protein TatB prepared in Example 1 after immunization, as follows.

[0131] S1. Oral gavage of mice: 10 days after the last nasal immunization 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 water was cut off 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 oral gavage.

[0132] S2. Plate culture: One week after oral administration, the mice were slaughtered, the stomach tissue was cut into pieces and placed in PBS buffer, vortex washed for 3 minutes, and the washing stock solution and its 10-fold dilution were spread on Skirrow plates 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) (15 g / L peptone (Haibo Biological), 2.5 g / L tryptone (Oxoid), 5 g / L yeast extract powder (Oxoid), 5 g / L sodium chloride (Cologne reagent), pH 7.4), and cultured at 37°C microaerophilically (5% O2, 10% CO2, 85% N2) for 2-3 days and observed.

[0133] S3. Analysis and identification: Combined with the characteristics of Helicobacter pylori colonies, rapid urease reagent and microscopic examination were used to detect whether Helicobacter pylori grew on the plate, so as to determine the infection rate of mice. Among them, vaccine protection rate = (control group infection positive rate - immunization group infection positive rate) / control group infection positive rate * 100%.

[0134] S4. Result statistics: The plate culture statistics of 20 mice in the control group and the experimental group are shown in Table 5. The plate culture test of 20 mice in the control group was positive, and the infection rate was 100%. Among the 20 mice in the experimental group, 14 mice were positive in the plate culture, and the infection rate was 70%. It was concluded that the protective efficiency of the vaccine was 30%. It shows that the recombinant antigen protein TatB obtained by the present invention can induce mice to produce a strong immune response, and can inhibit the colonization of Helicobacter pylori in the stomach of mice, and produce a protective and preventive effect on Helicobacter pylori infection.

[0135] Table 5 Statistics of positive rates of Helicobacter pylori infection in mice after immunization

[0136]

[0137]

[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 should be understood that the protection scope of the present invention is not limited to such specific statements and embodiments. Those skilled in the art can make various other specific variations and combinations that do not deviate from the essence of the present invention based on the technical revelations disclosed by the present invention, and these variations and combinations are still within the protection scope of the present invention.

Claims

1. A recombinant Helicobacter pylori antigen protein TatB, characterized in that: The amino acid sequence of the recombinant antigen protein TatB is shown in SEQ ID NO:

1.

2. A method for preparing a recombinant Helicobacter pylori antigen protein TatB, characterized in that: The following steps are involved: S1. Plasmid construction Connecting the target gene fragment to the expression vector plasmid to construct a recombinant expression plasmid containing the target gene fragment; specifically comprising the following steps: S11, screening and obtaining the amino acid sequence encoding the Helicobacter pylori antigen TatB, and optimizing the codons preferred by Escherichia coli according to the amino acid sequence to obtain a target gene fragment containing a restriction site, the nucleotide sequence of which is shown in SEQ ID NO: 2; S12, synthesize the target gene, and connect it to the expression vector plasmid pET28a (+) through the restriction sites of Xho I and Nco I to construct a recombinant expression plasmid; S2. Inducible expression The recombinant expression plasmid is transformed into a host bacterium for recombinant protein expression to induce expression, the induced expression bacteria are collected, the bacteria are broken and then centrifuged to collect the supernatant; S3. Product purification The supernatant was purified by nickel column affinity chromatography and anion column exchange chromatography in sequence to obtain the Helicobacter pylori recombinant antigen protein TatB. The amino acid sequence of the recombinant antigen protein TatB is shown in SEQ ID NO:

1.

3. The method for preparing the recombinant Helicobacter pylori antigen protein TatB according to claim 2, 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 TatB, and culturing the recombinant strain until OD 600 0.6-0.8, and then induce expression with 0.3-0.5 mM isopropylthiogalactoside at 30-37°C and 180-240 rpm for 4-8 h; S22. After induced expression, the bacterial solution was centrifuged and the supernatant was discarded to obtain precipitated bacteria. Buffer A was added to the precipitated bacteria to be evenly suspended. After the bacteria were broken, the precipitate was discarded by centrifugation. The supernatant was filtered through a 0.45 μm filter membrane through a suction filtration bottle for later use.

4. The method for preparing the recombinant Helicobacter pylori antigen protein TatB according to claim 2, characterized in that: The step S3 specifically comprises 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, anion exchange chromatography Equilibration: Use buffer E to equilibrate the anion column; Loading: diluting the eluate in step S31 with buffer E and then loading the sample; Re-equilibration: Use buffer E to re-equilibrate the anion column; 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 TatB.

5. The method for preparing the recombinant Helicobacter pylori antigen protein TatB according to claim 4, characterized in that: The composition of the buffer A is: 20-50 mM phosphate buffer at pH 7-8, 300-500 mM NaCl, and 0-25 mM imidazole; the composition of the buffer B is: 20-50 mM phosphate buffer at pH 7-8, 100-200 M NaCl, and 0-25 mM imidazole; the composition of the buffer C is: 20-50 mM phosphate buffer at pH 7-8, 100-200 mM NaCl, and 30-40 mM imidazole; the composition of the buffer D is: 20-50 mM phosphate buffer at pH 7-8, 100-200 mM NaCl, and 80-120 mM imidazole; the composition of the buffer E is: 20-50 mM phosphate buffer at pH 7-8; the composition of the buffer F is: 20-50 mM phosphate buffer at pH 7-8, 40-70 mM NaCl; buffer G consists of 20~50mM phosphate buffer at pH 7~8, 120~200mM NaCl.

6. The method for preparing the recombinant Helicobacter pylori antigen protein TatB according to claim 2, characterized in that: In step S2, a high-pressure homogenizer or an ultrasonic disruptor is used to disrupt the bacteria. The parameters for the high-pressure homogenizer are: pressure 600-800 bar, flow rate 100-150 mL / min, repeated 4-8 times; the parameters for the ultrasonic disruptor are: ultrasonication for 3-5 seconds, stop for 3-5 seconds, and alternating the disruption for 20-40 minutes.

7. The method for preparing the recombinant Helicobacter pylori antigen protein TatB according to claim 2, characterized in that: The host bacteria is Escherichia coli.

8. Use of the recombinant Helicobacter pylori antigen protein TatB according to claim 1 in the preparation of Helicobacter pylori vaccine.

Images