Helicobacter pylori vaccine recombinant protein antigen ureb-s and preparation method and application thereof

Abstract

The present application belongs to the field of biological pharmacy, and particularly relates to a Helicobacter pylori recombinant protein antigen UreB-s, a preparation method and application thereof. In the preparation of the existing genetic engineering vaccine, the selection of the antigen is very important, and there are problems in screening. Direct use of the original Helicobacter pylori protein often causes unsuccessful recombination expression, or difficult purification or low purity, and the problems of poor immune effect and poor protection, etc. In view of the above problems, the present application provides a Helicobacter pylori recombinant protein antigen UreB-s and a preparation method thereof, which is obtained by E. coli genetic engineering expression. The antigen UreB-s has the advantages of strong hydrophilicity, soluble expression, easy purification, high purity, simple preparation method, etc., has significant economic benefits, and animal experiments prove that it can effectively stimulate the body to produce immune response and has good immune protection effect, and can be used as a vaccine candidate component for preventing Helicobacter pylori infection.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biopharmaceuticals, and particularly relates to a Helicobacter pylori vaccine recombinant protein antigen UreB-s and a preparation method and application thereof. BACKGROUND

[0002] Helicobacter pylori (HP) is a gram-negative bacterium that exists in the human stomach and duodenal mucosa, and is one of the most common bacterial pathogens that endanger human health. The World Health Organization classified it as a class I carcinogen as early as 1994. This bacterium can cause many different gastrointestinal diseases, including gastritis, duodenitis, gastric ulcer, mucosa-associated lymphoid tissue lymphoma, gastric cancer, etc.

[0003] At present, the treatment of HP infection often adopts the combined treatment mode of proton pump inhibitors and antibiotics, but this treatment mode often has some defects, such as re-infection, increasing antibiotic resistance worldwide, side effects, patient compliance and cost, etc. Therefore, developing a vaccine against HP is a reliable method to control HP infection and block transmission.

[0004] The infection and pathogenesis of HP in organisms are generally related to various virulence factors, and urease is a main virulence factor for HP to survive in the stomach environment. A large amount of urease is produced during the growth of HP, accounting for 10%-15% of the weight of the entire produced protein, which is used to adjust the pH value of the surrounding environment, so that it can adapt to the acidic environment and resist damage caused by gastric acid. This urease is a multimer with a molecular weight of 550KDa, which is composed of A subunit (29.5KDa) and B subunit (66KDa). The B subunit contains the active site of the enzyme, participates in urease activity, induces Th17 cell response, induces the production of NF-κB and interleukin-8, etc. If the body can produce antibodies against it to destroy its function, the activity of urease can be blocked, and the colonization ability of HP can be greatly reduced.

[0005] The present application intends to prepare a genetically engineered recombinant protein by recombining part of the protein fragment of urease B subunit, to provide a new antigen for Helicobacter pylori vaccine. SUMMARY

[0006] The technical problem to be solved by the present application is that in the preparation of existing genetic engineering vaccines, the selection of antigens is very important. Screening problems or directly using Helicobacter pylori original proteins often cause problems such as unsuccessful recombination expression, difficulty in purification or low purity, poor immune effect, poor protection, etc. Therefore, it is urgent to develop a vaccine component with stable performance, high yield and good protection effect in the development of Helicobacter pylori vaccine.

[0007] The technical scheme for solving the above technical problems of the present application is to provide a Helicobacter pylori vaccine recombinant protein antigen UreB-s.The amino acid sequence of the Helicobacter pylori vaccine recombinant protein antigen UreB-s is shown in SEQ ID NO:1.

[0008] The amino acid sequence of the antigen UreB-s is shown in SEQ ID NO:1.

[0009] NPTIPFTVNTEAEHMDMLMVCHHLDKSIKEDVQFADSRIRPQTIAAEDTLHDMGIFSITSSDSQAMGRVGEVITRTWQTADKNKKEFGRLKEEKGDNDNF.

[0010] The nucleotide sequence encoding the antigen UreB-s is shown in SEQ ID NO:2.

[0011] The present application also provides a preparation method of the above-mentioned Helicobacter pylori vaccine recombinant protein antigen UreB-s, comprising the following steps:

[0012] a. plasmid construction and prokaryotic expression

[0013] The gene with the nucleotide sequence shown in SEQ ID NO:2 is linked in an expression vector plasmid, and the plasmid is constructed and transformed into a host bacterium for induced expression;

[0014] b. cell disruption and centrifugation

[0015] The bacterium after expression is resuspended and uniformly mixed with a cell disruption solution with a pH of 6.0-8.0, high-pressure homogenization is performed for cell disruption, and high-speed centrifugation is performed to collect the supernatant;

[0016] c. Ni column affinity purification

[0017] Ni affinity filler is used for preliminary purification, an A solution is used for equilibration chromatography column, and a B solution is used for gradient elution;

[0018] d. Q column anion exchange chromatography

[0019] The target protein purified in step c is used for equilibration Q chromatography column with a C solution, and a D solution is used for elution to obtain the Helicobacter pylori vaccine recombinant protein antigen UreB-s.

[0020] In the above-mentioned Helicobacter pylori vaccine recombinant protein antigen UreB-s, the SEQ ID NO:2 in step a is an optimized nucleotide sequence encoding the antigen UreB-s.

[0021] The nucleotide sequence encoding the antigen UreB-s is shown in SEQ ID NO:2.

[0022] ATGAACCCAACCATCCCATTTACCGTTAACACCGAAGCTGAACACATGGACATGCTGATGGTATGCCACCACCTGGACAAGAGCATCAAAGAAGATGTCCAGTTCGCAGATTCCCGTATTCGTCCACAAACCATTGCAGCAGAGGATACCCTGCATGACATGGGT ATCTTTAGCATCACCAGCTCCGACTCTCAGGCAATGGGCCGTGTTGGTGAAGTAATTACCCGTACTTGGCAGACCGCGGACAAAAACAAAAAAGAATTTGGTCGCCTGAAAGAAGAAAAAGGTGACAACGACAACTTTCTCGAGCACCACCACCACCACCACTGA.

[0023] In the aforementioned Helicobacter pylori vaccine recombinant protein antigen UreB-s, the expression vector described in step a is pET22b.

[0024] In the aforementioned Helicobacter pylori vaccine recombinant protein antigen UreB-s, the host bacterium mentioned in step a is E. coli BL21DE3.

[0025] In the aforementioned Helicobacter pylori vaccine recombinant protein antigen UreB-s, the induction expression conditions described in step a are a temperature of 16-37℃, a rotation speed of 180-220rpm, and the induction expression uses isopropyl thiogalactoside at a concentration of 0.1-0.5mM.

[0026] In the aforementioned Helicobacter pylori vaccine recombinant protein antigen UreB-s, the lysis solution described in step b is 20-50 mM PB at pH 6.0-8.0, 0.1-0.5 M NaCl, and 10-50 mM imidazole; the lysis conditions are: external circulation temperature -4-0℃, pressure 600-850 bar, power 20-30%, 4-6 cycles; and centrifugation conditions are: 12000-15000 rpm, 15-30 min.

[0027] In the aforementioned Helicobacter pylori vaccine recombinant protein antigen UreB-s, the Ni affinity filler mentioned in step c is NiSepharose High Performance (cytiva, catalog number: 17526802); the composition of solution A is: 20-50mM PB at pH 6.0-8.0, 0.1-0.5M NaCl and 10-50mM imidazole; the composition of solution B is: 20-50mM PB at pH 6.0-8.0, 0.1-0.5M NaCl and 0.5-1M imidazole.

[0028] In the Helicobacter pylori vaccine recombinant protein antigen UreB-s, the Q column anion exchange chromatography column filler in step d is Q Sepharose High performance (cytiva, item number: 17101401); the composition of the C solution is 10-30 mM PB at pH 6.0-8.0; and the composition of the D solution is 10-30 mM PB and 0.5-1 M NaCl at pH 6.0-8.0.

[0029] The application further provides a use of the Helicobacter pylori vaccine recombinant protein antigen UreB-s in a drug for preventing or treating Helicobacter pylori infection.

[0030] Further, the drug is a vaccine.

[0031] The application has the following beneficial effects:

[0032] The application screens part of protein structures with high specificity, good antigenicity and high hydrophilicity, named UreB-s protein, by spatial and structural positioning analysis of HP urease, combined with antigen epitope analysis prediction, and obtains the target gene fragment by E. coli partiality codon optimization according to the amino acid sequence of UreB-s, and obtains the E. coli expressed gene engineering recombinant Helicobacter pylori antigen protein after the gene fragment is introduced into a vector and recombinantly expressed.

[0033] Further, the application adopts a special protein purification method, and can purify the recombinant protein with a purity of more than 99.6%, and verifies that the recombinant protein can effectively stimulate humoral immune response, significantly improve serum IgG, effectively stimulate mucosal immune response, produce high sIgA, and has good protection effect through immune protection evaluation experiment, and can be used as an antigen component for preventing Helicobacter pylori infection, and is used for preparing a vaccine for preventing or treating Helicobacter pylori infection. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1The recombinant plasmid UreB-s double enzyme digestion identification results are shown; M: Takara DL5000 DNA Marker; Lane 1: plasmid UreB-s-pET28a; Lane 2: double enzyme digestion UreB-s-pET28a; the identification results show that the isolated fragments are about 5270bp and 303bp; 3: plasmid UreB-s-pET20b+; Lane 4: double enzyme digestion UreB-s-pET20b+; the identification results show that the isolated fragments are about 2970bp and 303bp; 5: plasmid UreB-s-pET22b; Lane 6: double enzyme digestion UreB-s-pET22b; the identification results show that the isolated fragments are about 5300bp and 303bp.

[0035] Figure 2 The UreB-s protein induction identification results are shown; Lane 1: UreB-s-pET20b+ whole bacteria liquid; Lane 2: UreB-s-pET20b+ broken bacteria supernatant; Lane 3: UreB-s-pET20b+ broken bacteria precipitate; Lane 4: UreB-s-pET22b whole bacteria liquid; Lane 5: UreB-s-pET22b broken bacteria supernatant; Lane 6: UreB-s-pET22b broken bacteria precipitate; M: Thermo Scientific Protein Ruler; 7: UreB-s-pET28a broken bacteria precipitate; Lane 8: UreB-s-pET28a broken bacteria supernatant; Lane 9: UreB-s-pET28a whole bacteria liquid; the identification results show that the UreB-s protein is about 12.57KD, and UreB-s-pET22b and UreB-s-pET28a are soluble expressed.

[0036] Figure 3 The UreB-s1 protein induction identification results are shown; Lane 1: UreB-s1-pET20b+ whole bacteria liquid; Lane 2: UreB-s-pET20b+ broken bacteria supernatant; Lane 3: UreB-s-pET20b+ broken bacteria precipitate; M: Thermo Scientific Protein Ruler; Lane 4: UreB-s-pET22b whole bacteria liquid; Lane 5: UreB-s-pET22b broken bacteria supernatant; Lane 6: UreB-s-pET22b broken bacteria precipitate; M: Thermo Scientific Protein Ruler; 7: UreB-s-pET28a whole bacteria liquid; Lane 8: UreB-s-pET28a broken bacteria supernatant; Lane 9: UreB-s-pET28a broken bacteria precipitate; M: Thermo Scientific Protein Ruler; the identification results show that the UreB-s1 protein is about 15KD, and UreB-s-pET20b+ and UreB-s-pET28a are inclusion body expressed.

[0037] Figure 4 The results are shown in the Ni affinity chromatography electrophoresis results; 1: Sample loading; 2: Flow-through; 3: 5% B; 4: 20% B; 5: 100% B; M: Thermo Scientific Protein Ruler;

[0038] Figure 5 The results of Q-column ion chromatography electrophoresis are shown below; 1: Sample loading; 2: Flow-through; M: Thermo Scientific Protein Ruler; 3: 5%-1D; 4: 5%-2D; 5: 20%D; 6: 40%-1D; 7: 40%-2D; 8: 100%D;

[0039] Figure 6 The results show the statistical results of serum specific antibody IgG titer.

[0040] Figure 7 The image shown is an electrophoresis image of LTs63K; 1: loading; 2: flow-through; 3: elution 1; 4: elution 2; M: ThermoScientificProtein Ruler.

[0041] Figure 8 The results show the statistical results of the specific antibody sIgA titer of vaginal irrigation fluid. Detailed Implementation

[0042] This invention, through structural and spatial localization analysis of urease and combined with "reverse vaccinology" technology, screened a subset of protein fragments located on the surface of the urease molecule that exhibit high specificity, high antigenicity, and high hydrophilicity, naming it the UreB-s protein, with its sequence shown in SEQ ID NO:1. Its encoding gene, SEQ ID NO:2, was cloned into an expression vector, transformed into engineered bacteria for large-scale expression, and purified to obtain the recombinant protein, which can be used to prepare genetically engineered subunit vaccines. This recombinant protein has advantages such as simple preparation process, low cost, and high operability, and is expected to become one of the candidate antigens for HP genetically engineered vaccines.

[0043] This invention successfully constructed a recombinant vector containing the UreB-s protein and achieved efficient expression. The expressed protein was correctly folded and stable. Furthermore, a high-purity target protein was obtained through purification using a combination of Ni and Q column chromatography. The purification process of this invention is simple, low-cost, and produces a stable purified protein that can elicit an immune response and provide immunoprotection in animal experiments, offering strong theoretical support for the development of Helicobacter pylori vaccines.

[0044] In the purification method for preparing recombinant proteins in this invention, a purity greater than 99% can be obtained from the engineered Escherichia coli expressing the recombinant subunit genetically engineered protein UreB-s. Based on the amino acid sequence, the molecular mass of the protein UreB-s constructed in this invention is predicted to be approximately 12.57 KD, and the isoelectric point is around 5.73.

[0045] The purification methods described in this invention mainly include Ni affinity purification and Q-column anion exchange chromatography. The protein purified by these methods, when detected by 15% SDS-PAGE, showed a single target protein band with a molecular weight of approximately 12.57 kDa. The protein purity was 99.6%. The purified UreB-s, co-injected with aluminum hydroxide adjuvant, into Balb / c mice for immunization. The results showed that the serum IgG level in the UreB-s plus adjuvant group was significantly higher than that in the negative control group (PBS group) (P < 0.01). Nasal immunization of Balb / c mice with UreB-s and LTs63K adjuvant significantly increased the antibody sIgA titer and induced a mucosal immune response. This demonstrates that the antigen UreB-s obtained using the purification method of this invention can effectively stimulate a high immune response, and the immunoprotective efficacy evaluation experiment confirmed its good protective effect.

[0046] The specific implementation of the present invention will be further explained and described below through examples, but this does not mean that the scope of protection of the present invention is limited to the scope described in the examples.

[0047] The Helicobacter pylori used in the examples was purchased from ATCC (J99 / Helicobacter pylori) in the United States. 700824); plasmid pET-22b was purchased from Thermo Fisher; Escherichia coli strain BL21(DE3) was purchased from Shanghai Chaoyan Biotechnology Co., Ltd., and is kept by the applicant; DNA Marker, restriction endonucleases Nco I and Xho I, T4 DNA ligase, and protein Marker are products of Thermo Fisher; plasmid extraction kit, gel extraction kit, bacterial genome extraction kit, and ultrathin extraction kit are products of Tiangen Biotech (Beijing) Co., Ltd.

[0048] The remaining reagents and equipment are ordinary commercially available products.

[0049] Example 1: Construction of a recombinant plasmid for the UreB-s gene

[0050] The specific steps are as follows:

[0051] (1) Firstly, the spatial and structural positioning analysis of HP urease was carried out by using "reverse vaccinology" through bioinformatics technology, and the partial protein structure with high specificity, good antigenicity and high hydrophilicity was screened out and named as UreB-s protein (amino acid sequence is SEQ ID NO: 1), and UreB-s1 and other proteins were also screened out, and the amino acid sequence of UreB-s1 is shown as SEQ ID NO: 3.

[0052] SEQ ID NO: 3 Amino acid sequence of UreB-s1

[0053] GPATEALAGEGLIVTAGGIDTHIHFISPQQIPTAFASGVTTMIGGGTGPADGTNATTITPGRRNLKWMLRAAEEYSMNLGFLAKGNTSNDASLADQIEAGAIGFKIHEDWGTTPSAINHALDVADKYDVQVAIHTDTLNEAGCVEDTMAAIA.

[0054] (2) According to the amino acid sequences of UreB-s and UreB-s1, the E. coli partial preference codon optimization was carried out to obtain the target gene fragment, and the sequences are SEQ ID NO: 2 and SEQ ID NO: 4.

[0055] SEQ ID NO: 4 Coding nucleotide sequence of UreB-s1

[0056] GGTCCGGCAACTGAAGCACTGGCTGGTGAGGGTCTGATCGTCACTGCGGGTGGCATCGATACCCACATTCACTTCATCTCTCCTCAGCAGATTCCGACCGCATTCGCTTCTGGCGTCACCACTATGATCGGTGGTGGTACCGGCCCTGCTGATGGTACCAACGCTACCACTATCACTCCGGT CGTCGTAATCTGAAATGGATGCTGCGTGCGGCAGAAGAATACAGCATGAACCTGGGCTTCCTGGCCAAAGGTAACGCATCTAACGACGCTTCCCTGGCGGACCAAATCGAAGCTGGCGCAATTGGCTTCAAAATCCATGAAGACTGGGGTACCACTCCTTCCGCGATTAACCACGCGCTGGATGTTGCAGATAAGTACGACGTGCAGGTCGCCATCCATACCGATACGCTGAACGAAGCGGGCTGCGTTGAGGACACGATGGCGGCAATTGCG.

[0057] (3) The target gene is synthesized and inserted into expression plasmid pET22b, pET20b+, pET28a, etc. expression vectors through Nde I and Xho I enzyme cutting sites (completed by Wuhan Jin Kai Rui Biological Engineering Co., Ltd.), and the plasmid sequencing result is compared with the submitted synthesized sequence information, and the nucleotide sequence is completely same.

[0058] (4) The synthesized plasmid is dissolved with 40 μl sterile water, 2 μl is taken to transform E. coli BL21 (DE3) competent cells, ice bath for 30 min, 42°C heat shock for 90 sec, and then quickly ice bath for 3 min. Add 1 ml of SOC medium, mix well, and place in a 37°C shaking bed at 220 rpm for 40 min.

[0059] (5) Take 100 μl of bacterial solution and spread on Amp resistant LB plate, and place in 37°C incubator for 16 h.

[0060] Example 2 Screening and identification of pET22b, pET20b+, pET28a / UreB-s / BL21 (DE3) positive recombinant plasmid

[0061] The specific operation steps are as follows:

[0062] (1) Pick single colonies well separated on the transformation plate, inoculate in the corresponding Amp and kana resistant LB medium, and incubate at 37°C with shaking overnight;

[0063] (2) Plasmid extraction: refer to the plasmid extraction kit instruction;

[0064] (3) Plasmid DNA was double digested by Nde I and Xho I; 37℃ for 2h;

[0065] (4) 1.0% agarose gel electrophoresis was used to detect the double digestion results, and the results are shown in Figure 1 , indicating that the recombinant plasmid construction was successful.

[0066] The double digestion reaction system is shown in Table 1:

[0067] Table 1 Double digestion reaction system

[0068] Reagents Volume μl CutSmart Buffer 2 Nde I 0.2 Nco I 0.2 Plasmid 6 Water, nuclease-free Up to 20

[0069] Example 3 Induction expression of recombinant protein UreB-s in prokaryotic expression system - Escherichia coli and identification of expression form

[0070] The specific operation steps are as follows:

[0071] (1) Take 100 μl of overnight culture of pET22b, pET20b+, pET28a / UreB-s / BL21(DE3) bacterial solution and add it to 10 mL Amp / kana resistant LB medium, and incubate at 220 rpm and 37℃ overnight. Take 400 μl of overnight culture and add it to 20 mL Amp / kana resistant LB medium, and incubate at 220 rpm and 37℃ for 2h. When the second activation reaches OD600 of 0.8, add 10 μl of IPTG (isopropyl-β-D-thiogalactoside) to make the final concentration 0.5 mM, and then place it in a shaker at 220 rpm and 37℃ for 4h of induction expression.

[0072] (2) Take out the bacterial solution after induction expression, centrifuge at 8000g for 15min, discard the supernatant, add 3ml of bacterial disruption solution (50mM PB, 0.5M NaCl, 20mM imidazole, pH 7.4) and mix well, ice bath ultrasonic lysis for 10min (ultrasonic 5s stop 6s), then 4℃ 12000g centrifuge for 30min, separate the supernatant and precipitate.

[0073] (3) Add 3ml of bacterial disruption solution to resuspend the precipitate, and take 40 μl of bacterial lysis solution, supernatant and resuspended precipitate respectively, add 10 μl of 5X protein loading buffer (Shengong, item number: C508320-0010), 100℃ for 10min, 12000g centrifuge for 3min.

[0074] (4) SDS-PAGE electrophoresis

[0075] The treated lysate, supernatant and precipitate were respectively sampled 10 μl, and the electrophoresis results are shown in Figure 2 The electrophoresis results show that UreB-s-pET20b+ is not expressed, UreB-s-pET22b and UreB-s-pET28a are normally expressed, and show single target protein bands with a molecular weight of about 12.57 KD, wherein UreB-s-pET22b is more soluble expressed, and has the best expression effect.

[0076] The identification results of UreB-s1 are shown in Figure 3 The identification results of UreB-s1 are shown in

[0077] Example 4 Preparation of UreB-s antigen

[0078] 1. Protein obtained by amplification culture

[0079] 30 mL of pET22b / UreB-s / BL21(DE3) bacterial solution cultured overnight was added into 3L Amp+ resistant TB culture medium, and cultured at 220 rpm and 37°C for 2-3 h. When the culture reached OD600 of 0.8-1, 1.5 ml of 1M IPTG was added to make the final concentration 0.5 mM, and the expression was induced at 220 rpm and 37°C for 4 h. The bacterial solution after induction was centrifuged at 8000 g for 15 min to collect the bacterial body, and then 200 ml of bacterial breaking solution (same as in Example 3) was added to resuspend the bacterial body. The bacterial solution was broken by high pressure homogenization: external cycle temperature -4°C, pressure 650 bar, power 25%, 6 cycles. The solution was centrifuged at 12000 g for 30 min, and the supernatant was taken.

[0080] 2. UreB-s purification

[0081] (1) Ni column affinity chromatography

[0082] The supernatant of the bacterial breaking solution was filtered through a 0.45 μm filter membrane for standby. The Ni column affinity chromatography column was equilibrated with A liquid (50 mM PB, 0.15 M NaCl, 20 mM imidazole, pH 7.4), and the filtered supernatant was sampled. Then 5% B liquid (50 mM PB, 0.15 M NaCl, 1 M imidazole, pH 7.4) + 95% A liquid was used to elute impurities, and 20% B liquid + 80% A liquid was used to elute the target protein. The electrophoresis results are shown in Figure 4

[0083] (2) Q column anion exchange chromatography

[0084] ​Take the eluted protein 14 ml in (1) and dilute to 140 ml with C liquid (20 mM PB, pH 7.4) for standby. Equilibrate the Q chromatography column with C liquid, load the sample, equilibrate with C liquid, and then elute with D liquid (20 mM PB, 0.5 M NaCl, pH 7.4) to collect the target protein for standby at 4°C. The electrophoresis results are shown in Figure 5 Figure 2, and the purity of the target protein is greater than 99%.

[0085] Example 5 Immunization of mice with UreB-s antigen combined with aluminum hydroxide adjuvant

[0086] Balb / C mice, female, 8-10 weeks old, purchased from Jiangsu Jizhuangkang Biotechnology Co., Ltd. Divide into immunization group (UreB-s antigen + aluminum hydroxide adjuvant), negative control group (PBS + aluminum hydroxide adjuvant) and blank control group (PBS), 20 in each group.

[0087] (1) First immunization, the immunization group is injected with 50 μg of UreB-s antigen and aluminum hydroxide adjuvant mixed at a volume ratio of 1:1, the negative control group is injected with 50 μg of PBS and aluminum hydroxide adjuvant mixed at a volume ratio of 1:1, and the blank control group is injected with PBS, both sides of the thigh muscle injection (100 μl / mouse).

[0088] (2) Second immunization, the second immunization is performed on the 14th day, and the injection amount and immunization method are the same as above;

[0089] (3) Third immunization, the third immunization is performed on the 21st day, and the injection amount and immunization method are the same as above;

[0090] (4) Fourth immunization, the third immunization is performed on the 28th day, and the injection amount and immunization method are the same as above.

[0091] Example 6 Elisa detection of serum specific antibody IgG after immunization of mice with antigen UreB-s combined with aluminum hydroxide adjuvant

[0092] Five days after the fourth immunization, the Balb / C mice were collected, and the orbital venous blood was collected and placed at 4°C for 3 h, then centrifuged at 3000 rpm for 5 min to separate the serum, and the UreB-s specific IgG level was detected by Elisa.

[0093] The specific operation steps are as follows:

[0094] (1) Antigen coating: take the coating liquid to dilute the UreB-s purified protein to 4 μg / mL, 100 μL / well coating the enzyme-labeled plate, 4°C overnight.

[0095] (2) Blocking: blocking solution 300 μL / well, 37°C incubation for 1 h, PBST plate washing, and 4°C storage for standby.

[0096] (3) Sample dilution: The serum was serially diluted by 1:4096 to 1:65536.

[0097] (4) Sample loading: The coated enzyme-labeled plate was taken and the diluted serum was added, 100 μL / well, double repeats for each sample, 37°C incubation for 1 h, PBST washing for 4 times;

[0098] (5) Secondary antibody loading: HRP-labeled goat anti-mouse IgG (Shanghai Yingay, item number: D110087-0100) was diluted by 1:10000, 100 μL / well, 37°C incubation for 30 min, PBST washing for 4 times;

[0099] (6) Color development: 100 μL / well of substrate color development solution was added, 37°C incubation for 10 min, then 50 μL / well of stop solution was added, and the OD value was determined on an enzyme-labeled instrument at 450 nm wavelength;

[0100] (7) Result judgment: A sample / A negative≥2.1 was positive.

[0101] Among them, the coating solution in (1) is 50 mM carbonate / bicarbonate buffer pH 9.6 (15 mM Na2CO3, 35 mM NaHCO3). The blocking solution in (2) is 10 mM PBS (pH 7.4) + 1% BSA. The PBST washing solution in (4) is 10 mM PBS (pH 7.4) + 0.05% Tween-20. The antibody diluent in (5) is 10 mM PBS (pH 7.4) + 0.05% Tween-20 + 0.5% BSA. The color developing solution in (6) is TMB storage solution: substrate buffer: 3% hydrogen peroxide = 10:90:1; TMB storage solution is 1 mg / mL TMB dissolved in DMSO; Substrate buffer (pH 5.0) is 0.1 M citric acid, 0.2 M Na2HPO4. The stop solution in (6) is 2 M H2SO4.

[0102] The results are shown in Table 2 and Figure 6 The results show that the highest titer of the antibody IgG produced by the UreB-s protein antigen immunized mice reaches 1:65536; the geometric mean titer of the recombinant UreB-s immunized mice to the recombinant UreB-s is 1:31651.8, the antibody positive rate after immunization reaches 100%, indicating that the recombinant UreB-s protein can make the immune mice produce specific antibodies in vivo.

[0103] Table 2 IgG geometric mean titer

[0104]

[0105] Example 7 Antigen UreB-s combined with LTs63K adjuvant for nasal immunization of mice

[0106] Balb / C mice, female, 8-10 weeks old, purchased from Jiangsu Jizhuangkang Biotechnology Co., Ltd., LTs63K self-made, the preparation method refers to "Feng Qiang. Construction, expression and property research of recombinant E. coli heat-labile enterotoxin and its mutant and B subunit. [D]. Chongqing University. 2003", the preparation result is shown as follows: Figure 7 The animals are grouped as shown in Table 3:

[0107] Table 3 UreB-s combined with LTs63K adjuvant immunization of mice

[0108]

[0109] (1) First immunization, the immunization group uses 50 μg of UreB-s antigen and 10 ug of LTs63K adjuvant mixed, then mixed with a homogenizer at 4°C for 30 min, and placed in an ice box for immunization standby. Take the prepared immunogen and slowly drop it into the mouse's nose: 10 μL / side, a total of 20 μL / mouse. The control group replaces the UreB-s antigen with an equal amount of PBS and operates the same way.

[0110] (2) Second immunization, the second immunization was performed on day 14, and the injection dose and immunization method were the same as above.

[0111] (3) Third immunization, the third immunization was performed on day 21, and the injection dose and immunization method were the same as above.

[0112] (4) Fourth immunization, the third immunization was performed on day 28, and the injection dose and immunization method were the same as above.

[0113] Example 8 Detection of specific antibody sIgA in vaginal lavage fluid of mice immunized with antigen UreB-s combined with LTs63K adjuvant by nasal instillation

[0114] Five days after the fourth immunization, the vaginal lavage fluid of Balb / C mice was collected with PBST (0.05% Tween 20 in PBS), 75 μL / time, lavaged 4 times, 300 μL / mouse. After collection, vortex for 1 min, then centrifuge at 12000g for 3 min to take the supernatant for Elisa detection of UreB-s specific sIgA level changes.

[0115] (1) Antigen coating: Take the coating solution to dilute the UreB-s purified protein to 4 μg / mL, 100 μL / well to coat the enzyme-labeled plate, and incubate at 4°C overnight.

[0116] (2) Blocking: Blocking solution 300 μL / well, 37°C incubation for 1 h, PBST plate washing, then 4°C storage standby.

[0117] (3) Sample dilution: The vaginal lavage fluid was serially diluted by 8 times from 1:8 to 1:128.

[0118] (4) Sample addition: The coated enzyme-labeled plate was taken, and the diluted serum was added, 100 μL / well, double repeats were made for each sample, 37°C incubation for 1 h, and PBST washing was performed for 4 times;

[0119] (5) Secondary antibody addition: HRP-labeled goat anti-mouse IgA (Abeam, item number: Ab97235) was diluted by 1:10000 with the antibody diluent, 100 μL / well, 37°C incubation for 30 min, and PBST washing was performed for 4 times;

[0120] (6) Color development: 100 μL / well of substrate color development liquid was added, 37°C incubation for 10 min, 50 μL / well of termination liquid was added, and the OD value was determined on the enzyme-labeled instrument at 450 nm wavelength;

[0121] (7) Result judgment: A sample / A negative≥2.1 was positive.

[0122] Among them, the coating liquid in (1) is 50 mM carbonate / bicarbonate buffer pH 9.6 (15 mM Na2CO3, 35 mM NaHCO3). The blocking liquid in (2) is 10 mM PBS (pH 7.4) + 1% BSA. The PBST washing liquid in (4) is 10 mM PBS (pH 7.4) + 0.05% Tween-20. The antibody diluent in (5) is 10 mM PBS (pH 7.4) + 0.05% Tween-20 + 0.5% BSA. The color development liquid in (6) is TMB storage liquid: substrate buffer: 3% hydrogen peroxide = 10:90:1; TMB storage liquid is 1 mg / mL TMB dissolved in DMSO; Substrate buffer (pH 5.0) is 0.1 M citric acid, 0.2 M Na2HPO4. The termination liquid in (6) is 2 M H2SO4.

[0123] The results are shown in Table 4 and Figure 8 The results show that the sIgA antibody titer of the UreB-s protein antigen immunized mice reaches 1:128; the geometric mean titer of the UreB-s immunized mice to the recombinant UreB-s is 1:68.6; the antibody positive rate after immunization reaches 100%, indicating that the UreB-s recombinant protein can stimulate the mucosal immune response of mice.

[0124] Table 4 Geometric mean titer of sIgA in vaginal lavage fluid of mice after UreB-s immunization

[0125]

[0126] Example 9 Evaluation of the protection against infection after UreB-s recombinant protein immunization

[0127] The specific steps are as follows:

[0128] (1) Gavage: Ten days after the last nasal immunization, mice were challenged by oral gavage with live Helicobacter pylori J99 bacteria. The mice were fasted for 24 hours and deprived of water for 17 hours before gavage. The infection dose per mouse was 2.0 × 10⁻⁶. 7 CFU was administered via gavage, and the patient was allowed to resume a liquid diet 2 hours later.

[0129] (2) Plate culture: One week after gavage, mice were slaughtered, and their stomach tissue was minced and placed in PBS buffer. The mixture was vortexed for 3 min, and then the washing solution and 10-fold dilution were spread on Skirrow plates containing 5% defibrinated sheep blood (Nanjing Maojie Biotechnology) 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) (containing peptone 15 g / L (Haibo Biotechnology), tryptone 2.5 g / L (Oxoid), yeast extract 5 g / L (Oxoid), sodium chloride 5 g / L (Kolomb reagent), pH 7.4). The plates were cultured at 37℃ under microaerophilic conditions (5% O2, 10% CO2, 85% N2) for 2-3 days and then observed.

[0130] (3) The presence of *HP* on the plate was detected by combining *HP* colony characteristics, rapid urease reagent, and microscopic examination to determine whether the mice were successfully infected with *HP*. The vaccine protection rate was calculated as follows: (Positive infection rate in the control group - Positive infection rate in the immunized group) / Positive infection rate in the control group * 100%.

[0131] The results are shown in Table 5 below. The plate culture results of 20 mice in the control group and 20 mice in the experimental group are statistically analyzed. 18 out of 20 mice in the control group tested positive for plate culture, with an infection rate of 90%. 11 out of 20 mice in the experimental group tested positive for plate culture, with an infection rate of 55%. The protective efficiency of the vaccine was 38.9%.

[0132] Table 5. Statistics on the positive rate of *Helicobacter pylori* infection after mouse immunization.

[0133] 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 + +

[0134] Note: "+" indicates that both the rapid urease test and microscopic examination are positive, and "-" indicates that both the rapid urease test and microscopic examination are negative.

[0135] It can be seen that the recombinant protein prepared by the application has good immunogenicity, can induce a stronger immune response in mice, and can effectively inhibit the colonization of H. pylori in the stomach of mice. The method for preparing the UreB-s recombinant protein provided by the application can quickly obtain the target protein with high purity, can stimulate the body to produce an immune response, and is expected to be used as a vaccine component for the prevention and treatment of H. pylori.

Claims

1. A method for preparing the recombinant protein antigen UreB-s for Helicobacter pylori vaccine, characterized in that, Includes the following steps: a. Plasmid construction and prokaryotic expression The gene with the nucleotide sequence shown in SEQ ID NO:2 was linked into an expression vector plasmid, the plasmid was constructed and transformed into a host bacterium for induced expression; the expression vector was pET22b. b. Sterilization and centrifugation After expression, the bacterial cells were collected, resuspended in a lysis buffer at pH 6.0-8.0, homogenized under high pressure, centrifuged at high speed, and the supernatant was collected. c. Ni column affinity purification Preliminary purification was performed using Ni-affinity packing material. The column was equilibrated with solution A, followed by gradient elution with solution B. The Ni-affinity packing material was Ni Sepharose High Performance. Solution A consisted of 20-50 mM PB, 0.1-0.5 M NaCl, and 10-50 mM imidazole at pH 6.0-8.

0. Solution B consisted of 20-50 mM PB, 0.1-0.5 M NaCl, and 0.5-1 M imidazole at pH 6.0-8.

0. d. Q-column anion exchange chromatography The target protein purified in step c was equilibrated with solution C and eluted with solution D to obtain the recombinant Helicobacter pylori vaccine protein antigen UreB-s. The Q column anion exchange chromatography packing material was Q Sepharose High performance. The composition of solution C was 10-30 mM PB at pH 6.0-8.

0. The composition of solution D was 10-30 mM PB at pH 6.0-8.0 and 0.5-1M NaCl.

2. The method for preparing the recombinant protein antigen UreB-s of Helicobacter pylori vaccine according to claim 1, characterized in that: The host bacterium mentioned in step a is E. coli BL21 DE3.

3. The method for preparing the recombinant protein antigen UreB-s of Helicobacter pylori vaccine according to claim 1, characterized in that: The induction conditions described in step a are a temperature of 16-37℃, a rotation speed of 180-220 rpm, and the induction expression uses isopropyl thiogalactoside at a concentration of 0.1-0.5 mM.

4. The method for preparing the recombinant protein antigen UreB-s of Helicobacter pylori vaccine according to claim 1, characterized in that: The lysis solution described in step b is 20-50 mM PB, 0.1-0.5 M NaCl, and 10-50 mM imidazole with a pH of 6.0-8.0; the lysis conditions are: external circulation temperature -4-0℃, pressure 600-850 bar, power 20-30%, 4-6 cycles; the centrifugation conditions are: 12000-15000 rpm, 15-30 min.

5. The use of the recombinant protein antigen UreB-s of Helicobacter pylori vaccine prepared by the method of claim 1 in the preparation of a vaccine to prevent Helicobacter pylori infection.

Images