An oral recombinant protein vaccine against Helicobacter pylori and a method for preparing its antigen

Abstract

The present invention relates to the field of biomedicine, and particularly to a Helicobacter pylori oral recombinant protein vaccine and a method for preparing an antigen thereof. There are technical problems in the prior art such as low tolerance of oral recombinant protein vaccines and dilution, degradation or even denaturation and inactivation of antigens. The present invention provides a Helicobacter pylori oral recombinant protein vaccine and a method for preparing an antigen thereof. Through the preparation method of the present invention, the protein purity reaches more than 90% after purification, and the yield of protein per liter of fermentation broth can obtain 96 mg of protein. Moreover, animal experiments prove that it can effectively stimulate the body to produce an immune response and has a good immune protection effect. After the immune protection experiment, its protection rate reaches 80%.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to an oral recombinant protein vaccine for Helicobacter pylori and a method for preparing its antigen. Background Art

[0002] Helicobacter pylori (Hp) is a Gram-negative, spiral-shaped, microaerophilic bacterium that can persist in the stomach, mucosa and duodenal epithelium. In 1982, Australian scholars Marshall and Warren first reported the isolation of Helicobacter pylori from the gastric mucosa of patients with chronic gastritis and gastric ulcers. It is the only known microbial species that can survive in the human stomach. A large number of studies have confirmed that Hp is a common pathogen of gastrointestinal diseases and the main cause of chronic type B gastritis, gastric and duodenal ulcers. Hp is also a key cause of gastrointestinal cancer and gastric mucosal lymphoid tissue lymphoma. It causes genetic instability of gastric epithelial cells by regulating intracellular signals, affects the DNA damage repair system, and promotes the tumor transformation of gastric epithelial cells. [i] In 1994, the International Agency for Research on Cancer listed it as the primary carcinogen for gastric cancer. ii The list of carcinogens published by the World Health Organization's International Agency for Research on Cancer in 2017 shows that Helicobacter pylori is a Class I carcinogen and can induce tumors such as gastric cancer and lymphoproliferative gastric lymphoma.

[0003] H. pylori infection is prevalent worldwide, exceeding 50% globally. This prevalence is primarily concentrated in developing countries such as Africa and Brazil, while infection rates are relatively low in developed regions such as Europe, Japan, and South Korea. In China, H. pylori infection exceeds 600 million people, and gastric cancer contributes to an estimated 200,000 deaths annually. While current antibiotic treatments for H. pylori infection have demonstrated some positive clinical benefits, they still face significant limitations: 1) toxic side effects; 2) the development of drug-resistant strains; 3) high cost; 4) long treatment courses and poor patient compliance; and 5) inconsistent efficacy. Vaccines are the most cost-effective and effective means of controlling infectious diseases. Therefore, H. pylori vaccines, designed to stimulate a specific immune response against Helicobacter pylori, can prevent or treat H. pylori infection. However, the difficulty of large-scale H. pylori cultivation and the presence of potentially carcinogenic components in crude antigens have significantly hampered the development of whole-bacterium vaccines. Genetically engineered vaccines, characterized by safety, effectiveness, affordability, and ease of deployment and application, are a key area of ​​focus for H. pylori vaccines. Despite intense research efforts both domestically and internationally, success has been limited.

[0004] The ability of Hp to colonize the stomach is a prerequisite for its pathogenicity, and adhesion is key to colonization. Hp colonizes exclusively in the gastric epithelium and the epithelial regions of the esophagus and duodenum, suggesting that this adhesion is mediated by specific adhesin receptors. N-acetylneuraminyllactose-binding fibrillary hemagglutinin (NLBH, HpaA) is known to be one of the main adhesins of Hp. It binds to multiple surface receptors on gastric epithelial cells, including ganglioside GM3, sulfatide cerebroside (SLC), and the sialyllactose component of laminin, enabling Hp to adhere tightly to gastric epithelial cells. Blocking Hp adhesion clearly can prevent and treat Hp infection.

[0005] The human mucosal immune system (HMIS) is mainly composed of mucosal and submucosal lymphocytes diffusely distributed in the respiratory tract, genitourinary tract, and digestive tract. It is the largest organ in the human body, with a surface area larger than that of the skin. Among them, the intestinal mucosa is the most important mucosal part of the human body. The human intestine has an area of ​​about 400 cm 2 surface area. In the intestinal mucosal system, there are unique lymphoid tissues and a large number of other lymphocytes, which recognize and resist foreign dangerous pathogens through their innate immunity and acquired immunity, thereby maintaining the body's immune homeostasis. In the intestinal mucosa, due to the presence of abundant immune cells, antigens can induce systemic humoral immunity and cellular immune responses through the uptake and presentation of immune cells, such as the production of antibodies (mainly sIgA) and corresponding cytotoxic T cells (CTL) against the antigen. At the same time, since the activated antigen-specific lymphocytes home to remote mucosal effector sites, local mucosal immune responses can also be induced. Therefore, intestinal mucosal immunity has great potential for achieving systemic or local immunotherapy. Intestinal mucosal vaccination is also currently considered to be the only way to defend against intestinal pathogens.

[0006] Bacterial DNA is a natural ligand for Toll-like receptor 9 (TLR9). Synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG sequences can mimic bacterial DNA structure and elicit similar activity. CpG ODNs can trigger TLR9-expressing immune cells (including human plasmacytoid dendritic cells and B cells), generating innate immune responses characterized by Th1 immunity and its associated cytokines. When used as vaccine adjuvants, CpG ODNs can enhance the function of professional antigen-presenting cells and promote antigen-specific humoral and cellular immune responses. Based on their structure and biological function, CpG-containing sequences can be divided into three major categories: CpG-A, CpG-B, and CpG-C. CpG-B molecules are the most commonly used in clinical vaccine research. The structure and function of CpG-ODNs are closely related. In fact, the higher-order structure of the molecule determines whether the CpG-ODN is localized intracellularly to early or late lysosomes, which are associated with different signaling pathways. Multimeric CpG-A ODNs are primarily localized to early lysosomes, where they lead to a strong induction of IFN-α in plasmacytoid dendritic cells. Monomeric CpG-B ODNs are concentrated in late endosomal compartments and can promote cell maturation of plasmacytoid dendritic cells and B cells. CpG-C ODNs are localized to both compartments, inducing IFN-α production and cell maturation. Other structural modifications that affect the biological effects of CpG-containing sequences include linking two or more short phosphorothioate backbone CpG ODNs via non-nucleoside chemical linkers to produce linear chimeric immunomodulatory compounds and / or formulations of CpG-containing nanoparticle compounds. 1018 is a synthetic CpG-B class oligonucleotide with a phosphorothioate backbone and sequence The first CpG motif (underlined) is a sequence active against mouse TLR-9, while the second CpG motif (double-underlined) is active against human and non-human primate TLR-9. CpG 1018 has been used as an adjuvant in Heplisav-B, an improved HBV vaccine for adults (age >18 years). Although other HBV vaccines are widely used, they are typically administered in a three-dose regimen, while Heplisav-B offers a two-dose regimen. Summary of the Invention

[0007] The purpose of the present invention is to provide an oral recombinant protein vaccine for Helicobacter pylori and a method for preparing its antigen, so as to solve the technical problems in the prior art of low tolerance of oral recombinant protein vaccines and antigen dilution, degradation and even denaturation and inactivation.

[0008] To achieve the above objectives, the present invention provides the following technical solutions:

[0009] The present invention provides an oral recombinant protein vaccine for Helicobacter pylori, which comprises an antigen and an adjuvant, wherein the antigen is HpaA, the adjuvant is CpG1018, and the amino acid sequence of HpaA is shown in SEQ ID NO: 1.

[0010] The present invention also provides a method for preparing an antigen of an oral recombinant protein vaccine for Helicobacter pylori, comprising the following steps:

[0011] S1. Plasmid construction and protein expression: The HpaA gene with the nucleotide sequence shown in SEQ ID NO: 2 is linked to an expression vector, and the constructed expression vector is transferred into recombinant engineering bacteria for induced expression;

[0012] S2, fermentation;

[0013] S3, purification;

[0014] S31, resuspend the bacteria; dilute the bacteria and solution A at a mass ratio of 1:15 and resuspend them in solution A until the bacteria are evenly suspended in solution A;

[0015] S32, breaking the bacterial cells and collecting the supernatant by centrifugation;

[0016] S33, SP FF chromatography: equilibrate for 2-3 CV; load sample; re-equilibrate for 3-5 CV; elute with Solution B for 10 CV, collect the eluted peak, dilute the eluted peak with Solution A, Cond < 6.0 ms / cm, and collect the filtrate by filtration;

[0017] S34, SP HP chromatography: equilibrate for 2-3 CV; load sample; re-equilibrate for 2-3 CV; elute with Solution B for 10 CV and collect the elution peak, dilute the elution peak, mix with Solution C, and filter the supernatant to collect the filtrate;

[0018] S35, ButyL HP chromatography: Equilibrate with solution D for 2-3 CV; load sample; re-equilibrate for 2-3 CV; elute with solution A for 10 CV and collect the elution peak;

[0019] S36, concentrate, replace the liquid, sterilize and filter to obtain the HpaA stock solution.

[0020] Furthermore, the expression vector in S1 is pET-29b(+), and the recombinant engineering bacteria is pET-HpaA / BL21.

[0021] Furthermore, the step S2 includes the following sub-steps:

[0022] S21. Initiation of the strain: The fourth generation of pET29-HpaA / BL21 seed bacteria was inoculated into a flask containing LB medium at a ratio of 1:600 ​​v / v, and cultured at 36.0-38.0°C, 200-240 rpm, for 4-8 hours to obtain the fifth generation production strain;

[0023] S22, seed tank culture: take the fifth generation production bacteria at a ratio of 0.5-2% and inoculate it into a seed tank, fill the tank with LB medium, and culture it for 3.0-8.0 hours with deep aeration: 10.0-50.0 L / min, 36.0-38.0°C, 50-300 rpm, pH 6.70-7.30 to obtain the sixth generation production bacteria;

[0024] S23, fermenter culture: The 6th generation production strain is inoculated into the fermenter at an inoculum size of 8%-12%, and cultured at 36.0-38.0°C, deep aeration 50.0-350.0 L / min, 100-500 rpm, pH 6.70-7.30 for 3.0-7.0 hours, completing the strain amplification;

[0025] S24, induction culture: temperature: 29.0-31.0°C; deep aeration: 50.0-350.0 L / min; rotation speed: 100-500 rpm; pH: 6.70-7.30; induction time: 4.0 hours;

[0026] S25. Collect the bacterial precipitate by centrifugation.

[0027] Furthermore, after the completion of S21, S22, and S24, bacterial liquid samples were taken for pure bacterial examination and microscopic examination, and the bacteria were Gram-negative rods. After the completion of S21, S22, S23, and S25, the OD600 values ​​of the bacterial liquid were greater than 1.50, greater than 1.50, 20.00, and not more than 1.00, respectively; glycerol was added at 0.015 L / min starting at 10.00 OD600 of the S23 bacterial liquid until the end of induction; concentrated culture medium was added at 0.02 L / min at the beginning of induction in S24.

[0028] Furthermore, the S32 is specifically as follows: shearing and homogenizing the resuspended bacteria: homogenizing at 2-8°C, 700-900 Bar, 3 times; centrifuging the homogenized liquid at 12000g centrifugal force for 30 minutes; collecting the supernatant, clarifying and filtering using a 0.6-0.8μm deep filter plate, and then clarifying and filtering using a 0.65μm filter element.

[0029] Furthermore, in the S3, liquid A is 20mM PB, pH 6.0; liquid B is 20mM PB+1M NaCl, pH 6.0; liquid C is 20mM PB+3M (NH4)2SO4, pH 6.0; and liquid D is 20mM PB+1.5M (NH4)2SO4, pH 6.0.

[0030] Furthermore, the S36 is a three-step chromatography sample solution treated by ultrafiltration and concentration using a 10KDAA membrane package, concentrated to 1 / 5-1 of the original volume, and an equal volume of E solution is added, and diafiltration is continuously performed for 5-10 volumes. After dialysis, the sample is concentrated to 1 / 2-1 of the volume of the three-step chromatography sample solution; the E solution is 20mM PB+5mM L-Cys·HCl+5% mannitol, pH 8.0.

[0031] Based on the above technical solution, the embodiments of the present invention can produce at least the following technical effects:

[0032] (1) The protein purity of the purified protein provided by the present invention reaches more than 90%, and the yield per liter of fermentation liquid can obtain 96 mg of protein. Animal experiments have shown that it effectively stimulates the body to produce an immune response and has a good immune protection effect.

[0033] (2) The oral recombinant protein vaccine for Helicobacter pylori provided by the present invention has high tolerance, and EDTA chelates and consumes extracellular Ca 2+ The ability to protect antigens.

[0034] (3) The Helicobacter pylori oral recombinant protein vaccine provided by the present invention has been tested for immune protection, and its protection rate has reached 80%. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 is a schematic diagram of the plasmid pET29 of an embodiment of the present invention;

[0036] Figure 2 This is the result of enzyme digestion identification of the recombinant plasmid pET29-HpaA in the embodiment of the present invention, M: DNA molecular weight standard; 1: recombinant plasmid pET29-HpaA enzyme digestion sample;

[0037] Figure 3 This is a graph showing the protein expression results of the recombinant engineered bacteria pET29-HpaA / BL21 according to an embodiment of the present invention;

[0038] Figure 4 This is the statistical result of the serum-specific antibody IgG titer of the embodiment of the present invention. DETAILED DESCRIPTION

[0039] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work shall fall within the scope of protection of the present invention.

[0040] SEQ ID NO:1:MSPHIIETNE VALKLNYHPA SEKVQALDEK ILLLRPAFQY SDNIAKEYENKFKNQTALKV EQILQNQGYK VINVDSSDKD DLSFSQKKEG YLAVAMNGEI VLRPDPKRTIQKKSEPGLLFSTGLDKMEGV LIPAGFIKVT ILEPMSGESL DSFTMDLSEL DIQEKFLKTT HSSHSGGLVS TMVKGTDNSNDAIKSALNKI FANIMQEIDK KLTQKNLESY QKDAKELKNK RNR

[0041] SEQ ID NO:2: catatgagcc cgcacattat cgaaaccaac gaagtggcgc tgaaactgaactaccacccg gcgagcgaaa aagtgcaggc gctggacgaa aaaattctgc tgctgcgccc ggcattccagtactctgata acatcgcgaa agaatacgaa aacaaattca aaaaccagac cgcgctgaaa gttgaacagatcctgcagaa ccagggctac aaagtgatca acgtggactc tagcgacaaa gatgacctgt ccttcagccagaaaaaagaa ggctacctgg cagttgcgat gaacggcgaa atcgttctgc gcccggaccc gaaacgtaccatccagaaga aaagcgaacc gggtctgctg ttttccaccg gcctggataa aatggaaggc gtgctgatcccggcgggttt catcaaagtt accatcctgg aaccgatgag cggtgaatcc ctggactctt tcaccatggatctgtctgaa ctggatatcc aggaaaaatt cctgaaaacc acccacagca gccacagcgg tggtctggttagcacgatgg ttaaaggcac cgataacagc aacgacgcga tcaaatccgc gctgaacaaa atcttcgcgaacatcatgca ggaaattgat aaaaaactga cccagaaaaa cctggaaagc taccagaaag atgctaaagaactgaaaaac aaacgtaacc gttaaggatc

[0042] Example 1

[0043] S1. Plasmid construction and protein expression (such as Figure 1Using D1801434-WP_187900158.1 as the HpaA sequence, the HpaA DNA sequence was codon-optimized in Escherichia coli to obtain a gene sequence as shown in SEQ ID NO: 2. The gene as shown in SEQ ID NO: 2 was linked to the expression vector plasmid pET29, and the constructed plasmid was transferred into the recombinant engineering bacteria pET-HpaA / BL21 for induced expression.

[0044] The target gene was synthesized by Shanghai Shenggong Bioengineering Co., Ltd. Figure 2 As shown, the results of enzyme digestion of the recombinant plasmid pET29-HpaA showed that a 5.3 kb vector fragment and a 711 bp target fragment were obtained after enzyme digestion, which was consistent with the expected results and the recombinant plasmid was constructed correctly.

[0045] The engineering strain was BL21(DE3), with the genotype F-ompT hsdSB(rB-mB-)gal dcm(DE3), which was obtained from Shanghai Sangon Biotechnology Co., Ltd. Shanghai Sangon Biotechnology Co., Ltd. was commissioned to transform the recombinant expression vector pET29b(+)-HpaA into the host strain BL21(DE3).

[0046] Determination of target protein expression in recombinant engineering bacteria pET-HpaA / BL21:

[0047] Inoculate 10 ml of LB medium with pET29-HpaA / BL21 seed bacteria and incubate at 37°C on a shaker at 220 rpm for 12-16 hours. Add 400 μl of the culture to 20 ml of kanamycin-resistant LB liquid medium and incubate at 37°C on a shaker at 220 rpm until the OD600 reaches 0.6-0.8. Take 1 ml of the culture, centrifuge at 10,000 rpm for 10 minutes, remove the supernatant, and store at -20°C. This will be used as the uninduced sample for testing. Add IPTG to a final concentration of 0.5 mmol / L to the remaining culture, and induce at 30°C on a shaker at 220 rpm for 4 hours. Take 1 ml of the induced culture, centrifuge at 10,000 rpm for 10 minutes, remove the supernatant, and store at -20°C. Resuspend the sample in 1 ml of PB and use it as the test solution. Take 40μl of the solution to be tested, add it to 10μl of 5× protein loading buffer, heat it in a metal bath at 100℃ for 5-10 minutes, cool it to room temperature, and centrifuge it for 10 seconds. Take 10μl of the supernatant and perform 12% SDS-PAGE electrophoresis. The results are as follows: Figure 3 The results of SDS-PAGE electrophoresis showed that the molecular weight of the HpaA protein expressed by the recombinant engineering bacteria pET-HpaA / BL21 was approximately 26 kDa, which was consistent with the theoretical molecular weight.

[0048] S2, fermentation;

[0049] S21. Initiation of bacterial strains: The fourth generation of pET29-HpaA / BL21 seed bacteria was inoculated into a 2000 ml Erlenmeyer flask at a ratio of 1:600 ​​v / v. The flask contained 600 ml of LB medium and cultured at 36.0-38.0°C, 200-240 rpm, for 4-8 h to obtain the fifth generation production strain. A bacterial liquid sample was taken for pure bacterial test and microscopic examination. The pure bacterial test result should be free of foreign bacteria, and the microscopic examination result should be Gram-negative bacilli, with a bacterial liquid OD600 value greater than 1.50.

[0050] S22. Seed tank culture: Take the 5th generation production bacteria and inoculate them into the seed tank at a ratio of 0.5-2%. Fill the tank with 30 L of LB medium and culture for 3.0-8.0 hours under deep ventilation: 10.0-50.0 L / min, 36.0-38.0°C, 50-300 rpm, pH 6.70-7.30 to obtain the 6th generation production bacteria; and take a bacterial liquid sample for pure bacteria inspection and microscopic examination. The pure bacteria inspection result should contain no foreign bacteria, and the microscopic examination result should be Gram-negative bacilli, and the bacterial liquid OD600 value should be greater than 1.50.

[0051] S23, fermenter culture: The 6th generation production strain was inoculated into the fermenter at an inoculum size of 8%-12%, and cultured at 36.0-38.0°C, deep aeration 50.0-350.0 L / min, 100-500 rpm, and pH 6.70-7.30 for 3.0-7.0 hours. When the OD600 of the bacterial solution was around 10.00, glycerol was added at a rate of 0.015 L / min until the induction was completed. When the OD600 was around 20.00 (this was the 7th generation), the strain amplification was completed and the induction stage was ready.

[0052] S24. Induction culture: When the OD600 of the bacterial solution is around 20.00 and the fermenter temperature is controlled at 29.0-31.0°C (set to 30.0°C), add the sterile-filtered inducer (isopropyl-β-D-thiogalactoside) at a final concentration of 0.5 mmol to begin induction. Induction parameters: temperature: 29.0-31.0°C (set to 30.0°C); deep aeration: 50.0-350.0 L / min; rotation speed: 100-500 rpm; pH: 6.70-7.30; induction time: 4.0 hours. At the beginning of induction, add concentrated culture medium (yeast extract, tryptone) at a feed rate of 0.02 L / min until the induction is completed. At the end of the culture, take a sample of the bacterial solution for pure bacteria test and microscopic examination. The pure bacteria test result should be free of foreign bacteria, and the microscopic examination result should be Gram-negative rods.

[0053] S25. Collect the bacterial precipitate by centrifugation and transfer the fermentation broth to a bacterial broth storage tank. Collect the bacterial cells using a continuous flow centrifugation (≥12,000 rpm) at a rate of 0.8 to 2.0 L / min. The OD600 value of the centrifuged supernatant should not exceed 1.00. After centrifugation, collect the precipitate (bacteria). Store at -20.0 to -30.0°C.

[0054] S3, purification;

[0055] S31. Resuspend the cells; Solution A is 20 mM PB, pH 6.0; dilute the cells and solution A at a mass ratio of 1:15, add solution A, and resuspend until the cells are evenly suspended in solution A;

[0056] S32, breaking the bacterial cells and collecting the supernatant by centrifugation;

[0057] S33, SP FF chromatography: Solution B is 20 mM PB + 1 M NaCl, pH 6.0; equilibrate for 2-3 CV; load sample; re-equilibrate for 3-5 CV; elute with Solution B for 10 CV, collect the eluted peak, dilute the eluted peak with Solution A, Cond < 6.0 ms / cm, and collect the filtrate by filtration;

[0058] S34, SP HP chromatography: Solution C is 20mM PB + 3M (NH4)2SO4, pH 6.0; equilibrate for 2-3CV; load sample; re-equilibrate for 2-3CV; elute with Solution B for 10CV, collect the elution peak, dilute the elution peak, mix with Solution C, and collect the supernatant by filtration;

[0059] S35, ButyL HP chromatography: Solution D is 20mM PB + 1.5M (NH4)2SO4, pH 6.0; Equilibrate with Solution D for 2-3CV; Load the sample; Re-equilibrate for 2-3CV; Elute with Solution A for 10CV and collect the elution peak;

[0060] S36, concentrate, replace the solution, sterilize and filter to obtain HpaA stock solution:

[0061] Solution E is 20 mM PB + 5 mM L-Cys·HCl + 5% mannitol, pH 8.0;

[0062] The three-step chromatography sample solution was treated by ultrafiltration and concentration using a 10KDAA membrane package, concentrated to 1 / 5-1 of the original volume, and an equal volume of E solution was added. The solution was dialyzed continuously for 5-10 volumes. After dialysis, the sample was concentrated to 1 / 2-1 of the volume of the three-step chromatography sample solution. Under sterile conditions, the HpaA stock solution was filtered using a 0.22μm filter to obtain the HpaA stock solution.

[0063] Example 2

[0064] (1) Experimental grouping and immunization procedure Female BALB / c mice were randomly divided into 3 groups. The normal saline (blank) group had 5 mice. The vaccine group was gavaged with recombinant Helicobacter pylori vaccine (Escherichia coli). Each mouse was given the following vaccine (the volume was adjusted to 400 μL), as shown in the table below. The challenge control group was immunized with 400 μL of normal saline.

[0065] Immunization was performed by gavage, with the patient fasting one day in advance and without water the next morning.

[0066]

[0067] (2) Resuscitation and rejuvenation of Hp strains

[0068] Three days before the infection, Hp strains were revived and rejuvenated, and the concentration of the bacterial solution was adjusted to 1×10 7 CFU / ml.

[0069] (3) Attack the virus

[0070] Ten days after the fourth immunization, the mice were challenged with Hp virus, with 0.4 ml injected into the stomach. Two weeks after the challenge, the mice were killed and the stomach tissues were taken to detect Hp colonization in the stomach (qPCR detection of colonization amount), and the protection rate was calculated.

[0071] Immune protection test results

[0072]

[0073] Example 3 3.1

[0075]

[0076] 3.2 Purchase several female BALB / c mice and immunize them. Deprive them of food one day before gavage immunization and water the next morning.

[0077] Pre-immunization treatment: After administering the gastric acid neutralizing solution, administer the vaccine immediately (the entire immunization process should be completed within 30 minutes), continue fasting for 2 hours, and then resume feeding and drinking water to the mice.

[0078] 3.3 Sampling

[0079] 14 days after the last immunization, tail blood was collected from each group to detect IgG, and stomach and intestine were sampled to detect sIgA.

[0080] 3.4 Sample testing

[0081] Serum IgG and intestinal tissue supernatant sIgA were detected by ELISA. UreB / HpaA coating was determined for each group according to the immune antigen [UreB (5μg / ml) and HpaA (5μg / ml)].

[0082] The serum was titrated into eight gradients of 1:50, 1:200, 1:800, 1:3200, 1:12800, 1:51200, 1:204800 and 1:819200; the intestinal tissue supernatant was titrated into eight titers of 1:10, 1:20, 1:40, 1:80, 1:160, 1:320, 1:640 and 1:1280; the gastric tissue supernatant was titrated into eight titers of 1:10, 1:20, 1:40, 1:80, 1:160, 1:320, 1:640 and 1:1280; the vaginal washing fluid was titrated into eight titers of 1:5, 1:10, 1:20, 1:40, 1:80, 1:160, 1:320 and 1:640.

[0083] ELISA was used to detect serum IgG and intestinal tissue supernatant sIgA. The coating of each group was determined according to the immune antigen. HpaA (5 μg / ml) and serum IgG were as follows. Figure 4 As shown, the positive conversion rate was calculated by diluting the intestinal supernatant at a ratio of 1:20, as shown in the table below.

[0084]

[0085] ELIZA assay

[0086] (1) Experimental instruments

[0087] Water purifier (model ZYMICRO-II-20T Sichuan Excellence Water Treatment Equipment Co., Ltd.), ELISA plate (model), ELISA reader (model A51119600 Thermo Fisher Scientific), microplate washer (model 1575 Bio-Rad Laboratories), electric constant temperature incubator (model DHP-9052 Shanghai Yiheng Scientific Instrument Co., Ltd.), pipettes (model 10uL, 100uL, 200uL, 300uL, 1000uL, 5mL, 10mL Eppendorf Company)

[0088] Experimental reagents

[0089] Purified water (resistivity not less than 18.25 MΩ·cm), sodium carbonate (Na2CO3), sodium bicarbonate (NaHCO3), 12-hydrated sodium hydrogen phosphate (Na2HPO4·12H2O), 2-hydrated sodium dihydrogen phosphate (NaH2PO4·2H2O), sucrose, casein, BSA, Proclin 300, aminopyrine, calf blood, polysorbate 20 (Tween 20), sodium chloride (NaCl), potassium chloride (KCl), potassium dihydrogen phosphate (KH2PO4), sodium thimerosal, TMB color development solution, sulfuric acid, and HRP-labeled goat anti-mouse IgG secondary antibody.

[0090] 3) Preparation of experimental reagents

[0091] Coating solution: Weigh 14.345 g of Na2CO3 and 30.68 g of NaHCO3 on an electronic balance, add 500 mL of distilled water, dissolve and mix, and then adjust the volume to 1000 mL. Label it with 10xCBS. Dilute it 10-fold with UP water to 1xCBS during coating.

[0092] Antibody diluent (enzyme dilution): Weigh 5.80 g of Na2HPO4·12H2O, 0.592 g of NaH2PO4·2H2O, 5.00 g of casein, and 0.50 g of aminopyrine on an electronic balance, add 500 mL of distilled water, dissolve and mix thoroughly, add 0.5 mL of Tween 20 with a pipette, make the volume up to 1000 mL, add 1000 mL of calf serum and 1 mL of Proclin 300, and mix thoroughly. Washing solution: Dissolve 23.48 g / bag of PBS in 2000 mL of pure water, add 1 mL of Tween 20, and mix thoroughly.

[0093] Blocking solution: Weigh 5.80 g of Na2HPO4·12H2O, 0.59 g of NaH2PO4·2H2O, 100.00 g of sucrose, 1.00 g of casein, and 10.00 g of BSA on an electronic balance, add 500 mL of distilled water, dissolve and mix, and make up to 1000 mL. Add 300 1 mL of Proclin and mix thoroughly.

[0094] Stop solution (2 mol / L sulfuric acid): Measure 443.9 mL of ultrapure water into a reagent bottle, then slowly add 56.1 mL of concentrated sulfuric acid using a pipette and mix thoroughly.

[0095] Experimental methods

[0096] (1) Coating: Dilute the antigen to the required concentration (Ureb coating concentration 5 μg / mL, HpaA coating concentration 5 μg / mL, SS1 whole bacterial protein 50 μg / mL) with coating solution, coat the ELISA plate with 100 μL / well, shake well and spread evenly, and place in a refrigerator at 4°C overnight or at 37°C for 2 hours.

[0097] (2) Blocking: Wash the plate 3 times with washing solution, adding 300 μL of solution each time, shaking for 30 seconds, and aspirating for 2.5 seconds. Block the enzyme-linked strip with 200 μL of blocking solution per well and place in a refrigerator at 4°C overnight or at 37°C for 2 hours.

[0098] (3) Adding primary antibody: Wash the plate three times with washing solution, adding 300 μL of solution each time, shaking for 30 seconds, and aspirating for 2.5 seconds. Dilute the serum of vaccine-immunized animals to 1:12800 with antibody diluent. Simultaneously, use the serum obtained from animals immunized with physiological saline as a negative control, dilute according to the lowest dilution of the serum in the corresponding experimental group, shake well, and incubate at 37°C in an incubator for 2 hours.

[0099] (4) Adding secondary antibody: Wash the plate three times with washing buffer, adding 300 μL of solution each time, shaking for 30 seconds, and aspirating for 2.5 seconds. Dilute the HRP-labeled secondary IgG antibody 1:10,000 with antibody diluent, add 100 μL / well, shake well, and incubate at 37°C for 1 hour.

[0100] (5) Color development: Wash the plate five times with washing solution, adding 300 μL of solution each time, shaking for 30 seconds, and aspirating for 2.5 seconds. Add 100 μL / well of TMB color development solution and develop the color in a dark incubator at 37°C for 15 minutes.

[0101] (6) Termination of the reaction: When color development is complete, add 2 mol / L H2SO4 (50 μL / well) to terminate the reaction. Measure the OD value of each well at 450 nm using a microplate reader.

[0102] (7) Statistical method: A sample / A negative value > 2.1 was used as the positive standard.

Claims

1. A method for preparing an antigen for an oral recombinant protein vaccine for Helicobacter pylori, characterized in that: The following steps are involved: S1. Plasmid construction and protein expression: The nucleotide sequence of HpaA gene as shown in SEQ ID NO: 2, which is modified according to the codon preference of Escherichia coli, is connected to the expression vector plasmid pET29, and the constructed expression vector is transferred into the recombinant engineering bacteria for induced expression; S2, fermentation; S3, purification; S31, resuspend the bacteria; add the bacteria to solution A at a mass ratio of 1:15 to dilute and resuspend until the bacteria are evenly resuspended in solution A; S32, breaking the bacteria, centrifuging and filtering to collect the supernatant; the S32 specifically comprises: shearing and homogenizing the resuspended bacteria: homogenizing at 2-8°C, 700-900 Bar, 3 times; centrifuging the homogenized liquid at 12000g centrifugal force for 30 minutes; collecting the supernatant, clarifying and filtering using a 0.6-0.8μm deep filter plate, and then clarifying and filtering using a 0.65μm filter element; S33, SP FF chromatography: balance for 2-3CV; load sample; re-balance for 3-5CV; solution B, 10CV elution, collect elution peak, dilute the elution peak with solution A, Cond<6.0 ms / cm, filter and collect filtrate; S34, SP HP chromatography: balance for 2-3CV; load sample; re-balance for 2-3CV; solution B, 10CV elution, collect elution peak, dilute elution peak, mix with solution C, filter supernatant and collect filtrate; S35, ButyL HP chromatography: D solution, equilibrate for 2-3CV; load sample; re-equilibrate for 2-3CV; A solution, elute for 10CV and collect elution peak; S36, concentration, liquid replacement, sterilization and filtration to obtain HpaA stock solution; the S36 is a three-step chromatography sample solution subjected to ultrafiltration and concentration using a 10KDAA membrane package, concentrated to 1 / 5-1 of the original volume, and an equal volume of E solution is added, and dialysis is performed continuously for 5-10 volumes. After dialysis, the sample is concentrated to 1 / 2-1 of the volume of the three-step chromatography sample solution; the A solution is 20mM PB, pH 6.0; the B solution is 20mM PB+1MNaCl, pH 6.0; the C solution is 20mM PB+3M (NH4)2SO4, pH 6.0; the D solution is 20mM PB+1.5M (NH4)2SO4, pH 6.0; the E solution is 20mM PB+ 5mM L-Cys·Hcl + 5% mannitol, pH 8.

0.

2. The method for preparing the antigen of the Helicobacter pylori oral recombinant protein vaccine according to claim 1, characterized in that: The expression vector in S1 is pET-29b (+), and the recombinant engineering bacteria is pET-HpaA / BL21.

3. The method for preparing the antigen of the oral recombinant protein vaccine of Helicobacter pylori according to claim 1, characterized in that: The S2 comprises the following sub-steps: S21, strain initiation: the fourth generation of pET29-HpaA / BL21 seed bacteria is inoculated in a triangular flask at a ratio of 1:600 ​​v / v, wherein the triangular flask contains LB medium, and cultured at 36.0-38.0°C, 200-240 rpm, for 4-8 hours to obtain the fifth generation production strain; S22, seed tank culture: take the fifth generation production bacteria and inoculate them into the seed tank at a ratio of 0.5-2%, the tank is filled with LB medium, and cultured for 3.0-8.0 hours in deep aeration: 10.0-50.0 L / min, 36.0-38.0℃, 50-300 rpm, pH 6.70-7.30 to obtain the sixth generation production strain; S23, fermentation tank culture: the 6th generation production strain is inoculated into the fermentation tank at an inoculation rate of 8%-12%, and cultured at 36.0-38.0°C, deep aeration 50.0-350.0 L / min, 100-500 rpm, pH 6.70-7.30 for 3.0-7.0 hours, and the strain amplification is completed; S24, induction culture: temperature: 29.0-31.0°C; deep ventilation: 50.0-350.0 L / min; Speed: 100~500rpm; pH: 6.70~7.30; induction time 4.0 hours; S25. Collect the bacterial precipitate by centrifugation.

4. The method for preparing the antigen of the oral recombinant protein vaccine of Helicobacter pylori according to claim 3, characterized in that: After the completion of S21, S22, S23, S24, and S25, bacterial liquid samples were taken for pure bacteria inspection and microscopic examination. The bacteria were Gram-negative bacilli. The OD600 value of the bacterial liquid was greater than 1.50 after the completion of S21, the OD600 value of the bacterial liquid was greater than 1.50 after the completion of S22, the OD600 value of the bacterial liquid was about 20.00 after the completion of S23, and the OD600 value of the bacterial liquid did not exceed 1.00 after the completion of S25. Glycerol was added at 0.015 L / min starting at 10.00 OD600 of the S23 bacterial liquid until the induction was completed; concentrated culture medium was added at 0.02 L / min at the beginning of induction of S24.

5. An oral recombinant protein vaccine for Helicobacter pylori, characterized in that: The vaccine comprises an antigen and an adjuvant, wherein the antigen is HpaA, the adjuvant is CpG1018, the amino acid sequence of HpaA is shown in SEQ ID NO: 1, and the antigen is prepared by the method for preparing the antigen of the Helicobacter pylori oral recombinant protein vaccine according to claim 1.

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