Use of periplaneta americana polypeptide hfdt1 as an immunoadjuvant for preparing brucellosis vaccine

Abstract

The present application belongs to the field of biomedical technology, and more particularly relates to the application of Periplaneta americana polypeptide HFDT1 as an immune adjuvant in the preparation of a brucellosis vaccine. The present application uses Brucella OMP10, BP26 and L7 / L12 proteins in combination with HFDT1 to induce mice to produce higher titers of anti-OMP10, BP26 and L7 / L12 protein specific IgG antibodies, and the titers increase with the increase in the number of immunizations, while promoting the body to produce mixed Th1 / Th2 type cellular immune responses and stimulating the proliferation and differentiation of mouse spleen T and B cells, and also facilitating mass production. The Periplaneta americana polypeptide HFDT1 described in the present application is obtained by convenient mass synthesis, and can be used as a novel small molecule immune adjuvant to enhance specific immune responses of the body.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and more specifically, relates to the application of American cockroach polypeptide HFDT1 as an immune adjuvant in the preparation of brucellosis vaccines. Background Technology

[0002] Natural products are widely distributed in nature, are non-toxic or have very low toxicity, and generally possess various biological activities such as anti-oxidation, antibacterial, anti-inflammatory, anti-tumor, and immunomodulatory effects. These natural products with potential pharmacological and beneficial effects are increasingly considered by the public to be safer than synthetic compounds. Currently, many naturally sourced products are widely used in modern medicine for anti-inflammatory, analgesic, and anti-tumor purposes. Further research has revealed that natural products have great potential in the field of vaccine adjuvants. Active substances such as soapberry, medicinal mushrooms, and propolis have been proven to have certain anti-inflammatory, antioxidant, anti-tumor, and immunomodulatory effects, and have been shown to have certain adjuvant effects.

[0003] With the continuous and rapid development of immunology and genetic engineering technologies, research on novel vaccines such as DNA vaccines and subunit vaccines has also reached new heights. However, these vaccines generally suffer from poor immunogenicity, requiring suitable adjuvants to enhance their immune response, improve the body's protective efficacy, reduce vaccine dosage, and lower production costs. Currently, although hundreds of candidate vaccine adjuvants are under development, only seven have been approved for marketing by the U.S. Food and Drug Administration: aluminum salt adjuvants, MF59, virus-like particles, AS04, AS03, AS01, and CpG1018. Immunological adjuvants are tools that enable vaccines to fully exert their immunogenicity. Currently, animal vaccine adjuvants are mainly aluminum salt adjuvants and oil emulsion adjuvants. However, aluminum adjuvants and oil emulsion adjuvants have significant drawbacks. While effective in enhancing serum antibodies, they can cause injection site reactions and have a weak ability to induce cellular immunity, thus limiting their application to some extent. Summary of the Invention

[0004] The purpose of this invention is to provide the application of American cockroach polypeptide HFDT1 as an immune adjuvant in the preparation of brucellosis vaccines.

[0005] This invention provides the application of American cockroach polypeptide HFDT1 as an immune adjuvant in the preparation of brucellosis vaccine, wherein the amino acid sequence of American cockroach polypeptide HFDT1 is shown in SEQ ID NO.1.

[0006] The immunoadjuvant provided by this invention is the American cockroach polypeptide HFDT1. When used in combination with Brucella OMP10, BP26, and L7 / L12 proteins, it can induce mice to produce higher titers of specific IgG antibodies against OBL proteins, with the titer increasing with the number of immunizations. Simultaneously, it promotes a mixed Th1 / Th2 cellular immune response and stimulates the proliferation and differentiation of T and B cells in the mouse spleen. Furthermore, this polypeptide can be easily mass-produced. It can serve as a novel small-molecule immunoadjuvant to enhance the body's specific immune response, thereby enabling the preparation of vaccines with higher potency.

[0007] Furthermore, the vaccine is prepared by mixing the American cockroach polypeptide HFDT1 with the membrane protein and ribosomal protein of Brucella, wherein the membrane protein is OMP10 protein and BP26 protein, and the ribosomal protein is L7 / L12 protein.

[0008] Furthermore, in the vaccine, the mass ratio of OMP10 protein, BP26 protein, L7 / L12 protein and American cockroach polypeptide HFDT1 is 0.5~1.5:0.5~1.5:0.5~1.5:1~2.

[0009] Furthermore, the amino acid sequence of the OMP10 protein is shown in SEQ ID NO.2.

[0010] Furthermore, the amino acid sequence of the BP26 protein is shown in SEQ ID NO.3.

[0011] Furthermore, the amino acid sequence of the L7 / L12 protein is shown in SEQ ID NO.4.

[0012] Furthermore, the vaccine also includes pharmaceutically acceptable excipients.

[0013] Furthermore, the vaccine is in the form of a solution, which is an oral liquid preparation or an injection.

[0014] The beneficial effects of this invention are as follows: The American cockroach polypeptide HFDT1 described in this invention can be used as an immune adjuvant. The amino acid sequence of the American cockroach polypeptide HFDT1 is shown in SEQ ID NO.1. This invention utilizes OBL protein in combination with HFDT1 to induce mice to produce higher titers of anti-OBL protein-specific IgG antibodies, and the titer increases with the number of immunizations. This not only enhances immunity but also facilitates large-scale production.

[0015] This invention induces purified influenza virus OBL protein and uses HFDT1 as an adjuvant in animal experiments. During the experiment, all mice showed no significant changes in physical signs, and their appetite, coat color, and mental state were good, indicating that the cockroach peptide HFDT1 has no toxic effects on mice. The OBL protein used in conjunction with HFDT1 induced mice to produce higher titers of anti-OBL protein-specific IgG antibodies. In the OBL+HFDT1 group of this invention, after three immunizations, the titers of IgG antibodies specifically binding to OBL protein were higher than those in the OBL group and the S2 commercial vaccine group. Simultaneously, the OBL+HFDT1 group promoted the proliferation and differentiation of splenic T and B lymphocytes in mice, and the CD3+ of the spleen in the OBL+HFDT1 group increased. ＋ CD8 ＋ CD3 ＋ CD4 ＋ Both T cell and germinal center B cell counts were higher in the OBL group. The OBL+HFDT1 group also stimulated the body to produce a mixed Th1 / Th2 immune response, enhancing the body's Th1-biased immune response and strengthening the Th2-biased immune response to a certain extent. It has a better effect on enhancing the immune response than the S2 vaccine positive control group. The American cockroach polypeptide HFDT1 of this invention can be easily synthesized in large quantities and is preliminarily judged to be a novel small molecule immune adjuvant to enhance the body's specific immune response. Attached Figure Description

[0016] Figure 1 The image shows the purification results of each protein.

[0017] Figure 2 The graph shows the results of the P / N ratio measurement of specific IgG antibodies in the serum of mice after immunization.

[0018] Figure 3 The image shows the results of the determination of specific IgG antibody titer in the serum of mice after immunization.

[0019] Figure 4 For flow cytometry determination of mouse CD3 ＋ CD4 ＋ Image showing the results of cell proliferation and differentiation.

[0020] Figure 5 For flow cytometry determination of mouse CD3 ＋ CD8 ＋ Image showing the results of cell proliferation and differentiation.

[0021] Figure 6 For flow cytometry determination of mouse CD45 ＋ CD95 ＋ GL7 ＋ Image showing the results of cell proliferation and differentiation.

[0022] Figure 7This is a statistical graph showing the results of flow cytometry analysis of T and B lymphocyte proliferation and differentiation in mouse spleen cells. In this graph, A represents CD3+. ＋ CD4 ＋ Statistical graph of positive cells, B represents CD3. ＋ CD8 ＋ Statistical graph of positive cells, C represents CD45 ＋ CD95 ＋ GL7 ＋ Statistical graph of positive cells.

[0023] Figure 8 To measure CD3 in mouse spleen cells using immunofluorescence ＋ CD4 ＋ Figure showing the results of T lymphocyte proliferation and differentiation.

[0024] Figure 9 Immunofluorescence assay of mouse spleen cell B220 ＋ Ki67 ＋ Image showing the results of B lymphocyte proliferation and differentiation.

[0025] Figure 10 The images show the results of IFN-γ, IL-2, and IL-4 cytokine assays in mouse spleen cell supernatants. In the images, A represents the IFN-γ cytokine assay, B represents the IL-2 cytokine assay, and C represents the IL-4 cytokine assay. Detailed Implementation

[0026] The present invention will now be described in detail with reference to specific embodiments, but these should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments can be obtained commercially unless otherwise specified.

[0027] The inventive concept of this invention is as follows: Current technology shows that only seven adjuvants are currently approved for marketing by the U.S. Food and Drug Administration: aluminum salt adjuvants, MF59, virus-like particles, AS04, AS03, AS01, and CpG1018. Immunoadjuvants are tools for fully realizing the immunogenicity of vaccines. Currently, animal vaccine adjuvants are mainly aluminum salt adjuvants and oil emulsion adjuvants. However, aluminum adjuvants and oil emulsion adjuvants have significant drawbacks; while effective in enhancing serum antibodies, they can cause injection site reactions and have a weak ability to induce cellular immunity.

[0028] Based on this, the present invention provides an application of the American cockroach polypeptide HFDT1 as an immune adjuvant in brucellosis vaccines. HFDT1, as a small molecule polypeptide, has advantages such as low cost of artificial synthesis, rapid energy provision, high digestibility and absorption, promotion of metabolism, low toxicity, and low immunogenicity, making it a promising novel immune adjuvant. The combination of OBL protein and HFDT1 can exert a good immune effect and shows promising application prospects in brucellosis prevention and control.

[0029] In the following examples, the PBS used was prepared as follows: 0.2 g / L KCl, 8 g / L NaCl, 0.27 g / L KH2PO4, 1.42 g / L Na2HPO4, with water as the solvent and pH 7.4.

[0030] Refolding solution I: NaCl 0.5mM, imidazole 5mM, Tris 20mM, solvent ddH2O. After adjusting the pH to 8.0, filter through a 0.45μm filter membrane and store at 4℃ for later use.

[0031] Refolding solution II: NaCl 0.5mM, imidazole 5mM, Tris 20mM, urea 2M, solvent ddH2O. After adjusting the pH to 8.0, filter through a 0.45μm filter membrane and store at 4℃ for later use.

[0032] Refolding solution III: NaCl 0.5mM, imidazole 5mM, Tris 20mM, urea 4M, solvent ddH2O, adjust pH to 8.0, filter through 0.45μm filter membrane, store at 4℃ for later use.

[0033] Refolding solution IV: NaCl 0.5mM, imidazole 5mM, Tris 20mM, urea 6M, solvent ddH2O. After adjusting the pH to 8.0, filter through a 0.45μm filter membrane and store at 4℃ for later use.

[0034] FBS-PBS buffer: Weigh 1g of NaN3 and dilute to 10mL with ddH2O to obtain 10% NaN3. Add 40mL of FBS and 4mL of 10% NaN3 to 1956mL of PBS, mix with a magnetic stirrer, and store at 4℃. The amino acid sequence of the American cockroach HFDT1 polypeptide provided by this invention is shown in SEQ ID NO.1, SEQ ID NO.1: RRNLLAASLLND. This polypeptide was synthesized by Shanghai Sangon Biotech.

[0035] The prokaryotic expression recombinant plasmids pET-28a-OMP10, pET-28a-BP26, and pET-28a-L7 / L12 were synthesized and constructed by Beijing Qingke Biotechnology Co., Ltd.

[0036] The amino acid sequence of OMP10 is shown in SEQ ID NO.2, the amino acid sequence of BP26 is shown in SEQ ID NO.3, and the amino acid sequence of L7 / L12 is shown in SEQ ID NO.4.

[0037] SEQ ID NO. 2: MHHHHHHCETTGPGSGNAPIIAHTPAGIEGSWVDPNGIASSFNGGIFETRTTDTNEKLAEGNYLYLSPQLVEINMRSIVRGTTSKVNCALVSPTQLNCTSSAGSRFSLTRRNAG.

[0038] SEQ ID NO.3: MHHHHHHQENQMTTQPARIAVTGEGMMTASPDMAILNLSVLRQAKTAREAMTANNEAMTKVLDAMKKAGIEDRDLQTGGINIQPIYVYPDDKNNLKEPTITGYSVSTSLTVR VRELANVGKILDESVTLGVNQGGDLNLVNDNPSAVINEARKRAVANAIAKAKTLADAAGVGLGRVVEISELSRPPMPMPIARGQFRTMLAAAPDNSVPIAAGENSYNVSVNVVFEIK.

[0039] SEQ ID NO.4:MHHHHHHADLAKIVEDLSALTVLEAAELSKLLEEKWGVSAAAPVAVAAAGGAAPAAAAEEKTEFDVVLADGGANKINVIKEVRALTGLGLKEAKDLVEGAPKAVKEGASKDEAEKIKAQLEAAGAKVELK.

[0040] Example 1 I. Protein preparation.

[0041] 1. Experimental Methods: The prokaryotic expression recombinant plasmids pET-28a-OMP10, pET-28a-BP26, and pET-28a-L7 / L12 were transformed into competent expression strain BL-21. Expression was induced using LB medium containing kanamycin (50 μg / ml) to obtain the corresponding bacterial suspensions. Specific steps are as follows: The competent strain BL-21 was removed at -80℃ and slowly thawed on ice. 1 μg of each recombinant plasmid was slowly added to BL-21 via rotation. The mixture was incubated on ice for 30 min, then incubated in a preheated 42℃ metal bath for 2 min. After incubation on ice for 2 min, 900 µL of antibiotic-free LB liquid medium was slowly added along the tube wall. The mixture was activated at 37℃ for 1 hour. After centrifugation at 5000 rpm for 1 min, the supernatant was discarded. The remaining liquid in the tube was gently pipetted to resuspend the broth, and then spread onto a plate containing kanamycin. The mixture was incubated overnight at 37℃. Single colonies grown on the plate were picked and cultured in LB liquid medium containing kanamycin resistance at 200 rpm for 12 hours to activate the expression of the corresponding protein.

[0042] The above-mentioned bacterial culture was added to LB liquid medium containing kanamycin resistance at a volume ratio of 1:100 for large-scale induction expression. After incubation at 37°C with shaking at 200 rpm for 4 hours, the OD of the bacterial culture was adjusted. 600 When the pH reached 0.6, the cells were induced with 0.2 M isopropyl-β-D-thiogalactopyranoside (IPTG) at 22°C for 18 h to obtain the induced protein. The cells were collected by centrifugation at 10,000 rpm for 5 min. The cells were washed twice with PBS, discarding the supernatant as much as possible on the last wash and retaining the precipitate. The cells were resuspended in 40 mL of PBS and then sonicated at low temperature for 45 min (5 s on, 8 s off). Afterward, the cells were centrifuged at 10,000 rpm for 10 min at 4°C to collect inclusion bodies. The washing process was repeated once. The inclusion bodies were dissolved in LE Buffer and then sonicated at low temperature for 15 min (3 s on, 5 s off). Afterward, the cells were centrifuged at 10,000 rpm for 30 min at 4°C. The supernatant was collected and used for Ni-NTA affinity chromatography.

[0043] The column loading, refolding, and purification steps are as follows: Step 1: Take 2 mL of Ni-NTA affinity chromatography medium nickel column, add it to the chromatography column, discard the original preservation solution, add 5 column volumes of LE Buffer to equilibrate for 5 min, and then circulate the collected supernatant through the column at a constant flow rate of 1 mL / min for 8 hours.

[0044] Step 2: Use a urea-containing refolding solution to slowly circulate through the column. The urea concentrations are from high to low: refolding solution I, refolding solution II, refolding solution III, and refolding solution IV.

[0045] Step 3: Wash the nickel column with 5 column volumes of washing buffer containing a low concentration of imidazole.

[0046] Step 4: Elute the protein with 5 column volumes of elution buffer containing 250 mM imidazole.

[0047] Step 5: Samples from each stage were validated by SDS-PAGE gel electrophoresis.

[0048] 2. Experimental Results: The results are as follows Figure 1 As shown, Figure 1 The results of SDS-PAGE analysis of purified OMP10, BP26, and L7 / L12 proteins showed that after extensive induction of protein expression, the target bands appeared at the corresponding positions: BP26, L7 / L12, and OMP10. The target bands were relatively uniform and of high purity.

[0049] II. Immunological and serological tests in mice.

[0050] 1. Experimental Setup: OBL protein was obtained by mixing equal masses of OMP10, BP26, and L7 / L12 proteins. The OBL protein was then combined with HFDT1 for animal experiments. Eight 6-week-old female BALB / c mice were used in each group, and all were immunized via intradermal multi-point injection. Negative controls were the PBS group and the HFDT1 group; experimental groups were divided into the OBL group (100 µg OBL) and the OBL+HFDT1 group (100 µg OBL + 50 µg HFDT1). The positive control was the S2 vaccine group, which was purchased from the Yunnan Provincial Institute for Endemic Disease Control. Mouse Serum Collection: Whole blood was collected from mice via the orbital vein on day 10 after each immunization. The collected whole blood was allowed to stand at room temperature for 2 hours, followed by centrifugation at 5000 rpm for 10 minutes. The supernatant serum was then gently aspirated and stored at -20°C for later use. The immunization cycle was week 0, week 2, and week 4, for a total of three immunizations, with a total volume of 200 µL each time. Blood was drawn from the orbital venous plexus 10 days after each immunization. The immunization grouping and procedure are shown in Table 1 below.

[0051] Table 1: Mouse Immunization Grouping and Flowchart 2. Experimental Data Analysis: GraphadPrism 9.5 was used for statistical analysis. The unpaired Mann-Whitoey test was used to compare the means of two groups. One-way ANOVA was used to compare the means of multiple groups. A p-value ≤ 0.05 was considered statistically significant. * indicates the following in the figures / graphs: P ≤0.05; "**": P ≤0.01; "***": P ≤0.001; "****": P≤0.0001. The mean of the ELISA-measured specific IgG antibody titers was calculated using the geometric mean method. Error bars represent the within-group standard deviation.

[0052] 3. Antibody Titer Detection: Antigen Coating: Dilute OBL with coating buffer to 3 µg / mL and coat with 100 µL / well, incubate overnight at 4°C. Blocking: Discard the coating buffer, wash three times with 1% PBST, 200 µL / well, 5 min each time. Block with 5% skim milk prepared with 1% PBST, 200 µL / well, incubate at 37°C for 2 hours. Primary Antibody Incubation: Discard the blocking buffer, wash three times with 1% PBST, 200 µL / well, 5 min each time. Add mouse serum diluted with 1% PBST, 50 µL / well, incubate at 37°C for 2 hours. Secondary Antibody Incubation: Discard the primary antibody, wash three times with PBST, 200 µL / well, 5 min each time. Add goat anti-mouse IgG-HRP antibody (Solepro) diluted with PBST at a volume ratio of 1:3000, 50 µL / well, incubate at 37°C for 2 hours. Color development: Remove secondary antibody, wash three times with PBST (200 µL / well, 5 min each time). Add 100 µL / well of TMB substrate and incubate at room temperature in the dark for 20 min. Stop reading: Add 100 µL / well of 10% sulfuric acid to stop color development, and measure absorbance at OD 450 nm. Use unincubated primary antibody wells as blanks. The cutoff value is defined as 2.1 times the OD value of the blank well. The highest serum dilution exceeding the cutoff value is determined as its titer.

[0053] Figure 2 The P / N ratio of specific IgG antibodies in mouse immune serum was determined for the purposes of this invention. Results showed that the level of specific IgG antibodies in mouse serum gradually increased with the number of immunizations. In the experimental groups, compared with the OBL group, the level of specific IgG antibodies in the OBL+HFDT1 group was increased, showing a trend consistent with the positive control group S2, and was higher than that of the positive control group.

[0054] Figure 3 The titer of specific IgG antibodies in mouse immune serum was measured. The results showed that after three immunizations, the titers of OBL protein-specific IgG antibodies in the OBL group, OBL+HFDT1 group, and S2 vaccine group reached 1:25600, 1:51200, and 1:25600, respectively. Among them, the OBL+HFDT1 group had the highest titer of OBL protein-specific IgG antibodies, which was even higher than that in the positive control group.

[0055] 4. Detection of T / B cell proliferation and differentiation in mouse spleen: To investigate the effect of HFDT1 on lymphocyte transformation, spleen cells were collected from mice in each group 10 days after the second booster immunization, and flow cytometry and immunofluorescence were performed on spleen sections.

[0056] The flow cytometry experimental procedure is as follows: (1) After cervical dislocation, the spleen of the mouse is taken in a clean bench and placed in a culture dish. It is weighed and photographed. FBS-PBS is added to keep it moist. (2) 3 mL of FBS-PBS is added to the culture dish. A 200-mesh grinding screen is placed on the dish. The spleen is ground thoroughly and the suspension is collected in a 10 mL centrifuge tube. The culture dish is then rinsed with 2 mL of FBS-PBS and the suspension is collected. The cells are centrifuged at 1200 rpm for 5 min and the supernatant is discarded. (3) 2 mL of RBC lysis buffer is added and mixed with a pipette tip and lysed for 5 min (note that the lysis is thorough). (4) 5 mL of FBS-PBS is added and the cells are centrifuged at 1200 rpm for 5 min and the supernatant is discarded. (5) 5 mL of FBS-PBS is added and the cells are washed once at 1200 rpm for 5 min. The cells are resuspended in 1 mL of FRS-PBS. (6) 250 μL of cell suspension is added to each flow cytometry tube and mixed with the flow cytometry antibodies FITC, APC, Percp, and PE (BD). (7) Add 2 ml of FBS-PBS, centrifuge at 1200 rpm for 5 min and wash once, discarding the supernatant; (8) Resuspend in 800 μL of FBS-PBS and run on the instrument. Immunofluorescence of spleen sections was performed by Wuhan Sewell Technology Co., Ltd.

[0057] Figure 4 , Figure 5 , Figure 6 CD3 ＋ CD4 ＋ T cells, CD3 ＋ CD8 ＋ T cells and GCB (CD45) ＋ GL7 ＋ CD95 ＋ Cell flow cytometry results, Figure 7 Statistical graphs of various immune cells, Figure 8 , Figure 9 Spleen slices B220 ＋ Ki67 ＋ CD3 ＋ CD4 ＋ Immunofluorescence results collectively reflect the proliferation and differentiation levels of mouse spleen lymphocytes. Flow cytometry results showed that the OBL+HFDT1 group, the OBL group, and the S2 vaccine group all promoted T / B cell activation and upregulated CD3. ＋ CD4 ＋ T cells, CD3 ＋ CD8 ＋ T cells and GCB cells enhance cellular and humoral immunity. In the OBL+HFDT1 group, CD4+ and CD8+ cells were particularly effective. ＋The proportions of T and GCB cells were higher in the PBS group than in the OBL group, indicating that HFDT1 may have a certain adjuvant effect, promoting the proliferation and differentiation of T / B cells. Similarly, immunofluorescence results showed that in the PBS and HFDT1 groups without antigen stimulation, only naive T cells remained in the spleen, with a low distribution; in the OBL group, CD3... ＋ CD4 ＋ Cell density was significantly higher in the OBL+HFDT1 group than in the PBS group, forming local clusters; CD3 in the OBL+HFDT1 group was significantly higher. ＋ CD4 ＋ Cell density formed numerous clusters, and dense follicle-like structures appeared in the white pulp region, suggesting that HFDT1 can enhance antigen presentation and promote CD3. ＋ CD4 ＋ T cell proliferation and differentiation. Next is B220. ＋ Ki67 ＋ Double-positive cells = activated and proliferating B cell population, mainly enriched in the germinal center, which is the core effector cell of humoral immune response. The germinal center is also a key site for the production of high-affinity antibodies and long-lasting memory B cells. In the PBS and HFDT1 groups, there was no antigen stimulation, and the spleen was in an immune quiescent state. ＋ Ki67 ＋ Fewer cells; S2 vaccine group, OBL group, OBL+HFDT1 group B220 ＋ Ki67 ＋ The number of cells was significantly higher than that in the PBS and HFDT1 groups, which could induce B cell proliferation and germinal center response to a certain extent; compared with the OBL group, the OBL+HFDT1 group formed a large number of clusters of B220 cells. ＋ Ki67 ＋ Cells suggest that HFDT1 can promote CD4 by enhancing TB cell interactions. ＋ Differentiation towards Tfh facilitates the differentiation of B cells into effector cells in the germinal center, significantly expanding the germinal center response. The spatial distribution results of immunofluorescence sections and the quantitative data from flow cytometry provide multidimensional corroboration, jointly confirming the synergistic effect of HFDT1 on humoral immunity.

[0058] 5. Th1 / Th2 Mixed Immune Response Detection: To investigate the immune type and response induced by HFDT1, spleen cells were collected from mice 10 days after the second booster immunization. Using OBL protein as a specific stimulus, the expression levels of IFN-γ, IL-2, and IL-4 cytokines were measured by ELISA. The experimental steps are as follows:

[0059] Step 1: Obtaining mouse spleen cells: Isolation of mouse spleen cells: 10 days after immunization, mice were euthanized by cervical dislocation, immersed in 75% ethanol for 10 minutes, and the spleen was dissected and isolated under aseptic conditions to obtain spleen cells. The specific operation steps are as follows: Spleen grinding: The extracted mouse spleen was washed twice with PBS, placed in a 35 mm cell culture dish, and 1 mL of 1640 cell culture medium was added. The spleen was cut into small pieces with sterile surgical scissors, and the shredded tissue was ground in a 200-mesh cell filter until no obvious tissue pieces were found. The spleen cells remaining on the nylon mesh were washed off with 1640 cell culture medium, and the spleen cells were transferred to a 50 mL centrifuge tube.

[0060] Cell washing: After centrifuging spleen cells at 1500 rpm for 5 min at 4℃, discard the supernatant, add 5 mL of 1640 cell culture medium, gently pipette to resuspend, and wash spleen cells twice.

[0061] Red blood cell lysis: Resuspend the washed and centrifuged spleen cells in 1 mL of 1640 cell culture medium, add 4 mL of red blood cell lysis buffer, lyse on ice for 4 min, and add an equal volume of 1640 complete culture medium containing 10% fetal bovine serum to terminate the lysis. If the lysis is incomplete, the lysis can be repeated once.

[0062] Cell culture: After lysis, centrifuge the cells at 1500 rpm for 5 min at 4°C, discard the supernatant, add 10 mL of 1640 complete culture medium, gently pipette to resuspend the cells, and wash twice. Resuspend the cells in 5 mL of 1640 complete culture medium and transfer to a T25 culture flask for culture until use.

[0063] Step 2: Spleen cell stimulation: The obtained spleen cells were resuspended in 1640 cell culture medium and plated to a cell concentration of 5 × 10⁻⁶ cells / mL. 6 Cells / mL were seeded in 2 mL six-well plates and stimulated with a final OBL protein concentration of 10 μg / mL. The plates were then incubated at 37°C in a 5% CO2 incubator for 24 hours.

[0064] Step 3: Indirect ELISA assay of cytokines: After stimulation, cells were collected, and the cell suspension was centrifuged at 1500 rpm for 5 min. The supernatant was collected and the secretion of specific INF-γ, IL-2 and IL-4 cytokines of spleen lymphocytes was detected according to the instructions of the cytokine assay kit of Shanghai Enzyme-Linked Chemical Co., Ltd., to evaluate the type and level of immune response induced by HFDT1 enhancement.

[0065] Figure 10The results show the assay results for each cytokine. The results indicated that after stimulation with OBL protein, the expression levels of IFN-γ and IL-2 in spleen cells of mice immunized in the OBL+HFDT1 group were significantly higher than those in the OBL group, and the differences were statistically significant. This suggests that HFDT1 can enhance the body's Th1-biased immune response. Figure 10 A and C. Figure 10 B represents the level of IL-4 cytokine expression in spleen cells of mice in each group after stimulation with OBL protein. The results showed that the OBL+HFDT1 group had a higher level of IL-4 cytokine expression than the OBL group, and the difference was statistically significant, indicating that HFDT1 can enhance the body's Th2-biased immune response to some extent.

[0066] This invention preliminarily demonstrates that the American cockroach polypeptide HFDT1 can enhance the body's production of anti-OBL antibodies, with the immune effect increasing with increasing dosage. It also promotes the proliferation and differentiation of T and B lymphocytes in the spleen, suggesting its adjuvant activity. HFDT1, used in combination with OBL protein, can promote the proliferation and differentiation of CD4⁺ (HTL) and CD8⁺ / CTL. HTL activates the bactericidal function of macrophages by secreting interferon-γ (IFN-γ), inhibiting the survival and replication of intracellular Brucella. CTL directly exerts cytotoxic effects, directly killing Brucella-infected target cells by releasing perforin and granzyme, or inducing apoptosis of infected cells through the Fas / FasL death receptor pathway, thereby blocking pathogen spread. Simultaneously, it can stimulate the formation of humoral immune germinal center B cells, producing long-lasting memory B cells and high-affinity antibodies to clear pathogens.

[0067] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0068] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0069] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. The application of American cockroach polypeptide HFDT1 as an immune adjuvant in the preparation of brucellosis vaccine, characterized in that, The amino acid sequence of the American cockroach polypeptide HFDT1 is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, The vaccine is prepared by mixing the American cockroach polypeptide HFDT1 with the membrane protein and ribosomal protein of Brucella, wherein the membrane protein is OMP10 protein and BP26 protein, and the ribosomal protein is L7 / L12 protein.

3. The application according to claim 2, characterized in that, In the vaccine, the mass ratio of OMP10 protein, BP26 protein, L7 / L12 protein and American cockroach polypeptide HFDT1 is 0.5~1.5:0.5~1.5:0.5~1.5:1~2.

4. The application according to claim 2, characterized in that, The amino acid sequence of the OMP10 protein is shown in SEQ ID NO.

2.

5. The application according to claim 2, characterized in that, The amino acid sequence of the BP26 protein is shown in SEQ ID NO.

3.

6. The application according to claim 2, characterized in that, The amino acid sequence of the L7 / L12 protein is shown in SEQ ID NO.

4.

7. The application according to claim 1, characterized in that, The vaccine also includes pharmaceutically acceptable excipients.

8. The application according to claim 1, characterized in that, The vaccine is in the form of a solution, which can be an oral liquid preparation or an injection.

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