A tubercle bacillus ag85b-wfc fusion protein for nasal immunization and a preparation method and application thereof

Abstract

The application relates to the field of biological medicine, and particularly discloses a tubercle bacillus Ag85B-wFc fusion protein for nasal immunization, a preparation method and application thereof, an amino acid sequence of the Ag85B-wFc fusion protein is shown as SEQ ID NO. 1, and a nucleotide sequence for coding the protein is shown as SEQ ID NO. 2. The Ag85B-wFc fusion protein prepared by the application can induce stronger local mucosal immunity and systemic immune response than subcutaneous immunization after nasal immunization, and has a good protective effect on infection of tubercle bacillus.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a tuberculosis Ag85B-wFc fusion protein for nasal immunization, its preparation method, and its application. Background Technology

[0002] Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis (M. tb). It is one of the three major infectious diseases worldwide. In the past two decades, due to large-scale population migration, the incidence of TB has been increasing year by year in many places. Data from the "Global Tuberculosis Report 2022" shows that in 2021, there were 10.6 million new TB cases globally, with the incidence rate increasing by 3.6% compared to 2020. Therefore, the prevention and treatment of this disease is urgently needed.

[0003] BCG (Bacillus Calmette-Guérin) is currently the only vaccine clinically used to prevent tuberculosis and is also the most effective vaccine for preventing tuberculosis. Studies have shown that BCG has a good immune protection effect on newborns, but its effect on preventing adult pulmonary tuberculosis is very limited. Due to the continuous passage of BCG, the immune protection effect gradually weakens. Furthermore, during the production of BCG, some genes related to immune memory and protection are lost, shortening its protective period and correspondingly weakening the protective immune response. Currently, chemotherapy is the main treatment for tuberculosis, but it is characterized by long treatment courses, significant side effects, high costs, and a high likelihood of developing drug-resistant strains. Therefore, accelerating the development of safer and more effective new vaccines is urgently needed.

[0004] Currently, the most researched tuberculosis vaccines both domestically and internationally include viral vector vaccines, protein / adjuvant vaccines, mycobacterial whole-cell or extract vaccines, and mycobacterial live attenuated vaccines. These vaccines are primarily developed via subcutaneous or intramuscular administration. However, invasive (subcutaneous or intramuscular) administration requires the body to endure a certain degree of pain. Therefore, developing a non-invasive vaccine with good immunogenicity is of great significance. Summary of the Invention

[0005] To develop a Mycobacterium tuberculosis protein with good immunogenicity, this invention provides a Mycobacterium tuberculosis Ag85B-wFc fusion protein for nasal immunization, its preparation method, and its application. The Ag85B-wFc fusion protein prepared by this invention can induce stronger local mucosal and systemic immune effects than subcutaneous immunization after nasal immunization, with high immunogenicity and high antibody titer.

[0006] The present invention provides an Ag85B-wFc fusion protein, the amino acid sequence of which is shown in SEQ ID NO.1, and the nucleotide sequence encoding the protein is shown in SEQ ID NO.2.

[0007] The present invention also provides a pcDNA-Ag85B-wFc recombinant plasmid, wherein the pcDNA-Ag85B-wFc recombinant plasmid comprises the sequence of SEQ ID NO.2 of claim 1.

[0008] Further, the construction steps of the pcDNA-Ag85B-wFc recombinant plasmid are as follows: Hind of plasmid pcDNA3.1(+) is... The fragment between the XbaI restriction site and the DNA sequence shown in SEQ ID NO.2 of the sequence listing was replaced to obtain the recombinant plasmid pcDNA-Ag85B-wFc.

[0009] The present invention also provides a method for preparing the Ag85B-wFc fusion protein, comprising the following steps:

[0010] Construct the pcDNA-Ag85B-wFc recombinant plasmid;

[0011] The pcDNA-Ag85B-wFc recombinant plasmid was transformed into competent cells, which were then transfected into CHO cells. Monoclonal cells that stably expressed the Ag85B-mFc fusion protein were screened, and the fusion protein was then isolated, purified, and concentrated to obtain the Ag85B-wFc fusion protein.

[0012] Further, the construction steps of the pcDNA-Ag85B-wFc recombinant plasmid are as follows: Hind of plasmid pcDNA3.1(+) is... The fragment between the XbaI restriction site and the DNA sequence shown in SEQ ID NO.2 of the sequence listing was replaced to obtain the recombinant plasmid pcDNA-Ag85B-wFc.

[0013] The present invention also provides the use of the Ag85B-wFc fusion protein or the Ag85B-wFc recombinant plasmid in the preparation of drugs for the prevention and / or treatment of tuberculosis.

[0014] The present invention also provides the application of the Ag85B-wFc fusion protein in the preparation of an anti-tuberculosis vaccine.

[0015] Furthermore, the vaccine is a mucosal immune vaccine.

[0016] Furthermore, the mucosal immunity is nasal drop immunity.

[0017] Furthermore, the vaccine also includes mucosal adjuvants, preservatives, stabilizers, and emulsifiers.

[0018] Furthermore, the mucosal adjuvant is CpG ODN 1826.

[0019] Furthermore, the preservative is thimerosal.

[0020] Furthermore, the stabilizer includes gelatin, sorbitol, sugar molecules sucrose, and lactose.

[0021] Furthermore, the emulsifier includes lanolin, Span-80, Arlacel-80, and Tween-80.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0023] 1. The Ag85B-wFc fusion protein prepared in this invention induces stronger local mucosal and systemic immunity after nasal instillation compared to subcutaneous immunization. The nasal administration route reduces the transmission of pathogens associated with needle contamination. This provides a vaccine against Mycobacterium tuberculosis that can be administered via the nasal mucosa, which has significant clinical value for the prevention and control of Mycobacterium tuberculosis and important application value for the Mycobacterium tuberculosis vaccine industry.

[0024] 2. This invention, through immunization with Ag85B-wFc fusion protein and Ag85B-mFc and Ag85B via subcutaneous and nasal administration, found that the nasal administration of Ag85B-wFc fusion protein showed significantly higher efficacy than other groups. Details are as follows:

[0025] Cellular immunization efficacy: On day 42 post-immunization, the proportions of CD3+CD4+IFN-γ+ cells and CD3+CD8+IFN-γ+ cells in the lung and spleen tissues of the Ag85B-wFc nasal drop immunization group were significantly higher than those in the other five groups (p<0.05); the proportion of CD3+CD44+CD62L¯ memory T cells in the lung and spleen tissues of the Ag85B-wFc nasal drop immunization group was also significantly higher than that in the other five groups (p<0.05). On day 42 post-immunization, after stimulation of spleen cells with Ag85B antigen, the lymphocyte stimulation index of the Ag85B-wFc nasal drop immunization group was significantly higher than that of the other five groups (p<0.01); on day 42 post-immunization, the serum IFN-γ and IL-2 levels of the Ag85B-wFc nasal drop immunization group were higher than those of the other five groups (p<0.05).

[0026] Humoral immunity: The serum antibody titers of IgG, IgG1, and IgG2a in the Ag85B-wFc intranasal immunization group were significantly higher than those in the other 5 groups at 14, 28, and 42 days (p<0.05), with IgG2a being the predominant antibody type. On 42 days after the first immunization, the nasal mucosal rinsing fluid of mice in the Ag85B-wFc intranasal immunization group showed the highest specific IgG antibody levels, and the antibody titers were significantly higher than those in the other 5 groups (p<0.05). Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 The diagram shows the construction process of recombinant plasmids pcDNA-Ag85B and pcDNA-Ag85B-wFc; pcDNA-Ag85B-Fc in the diagram is pcDNA-Ag85B-wFc.

[0029] Figure 2 The identification results of pcDNA-Ag85B, pcDNA-Ag85B-wFc and pcDNA-Ag85B-mFc recombinant plasmids;

[0030] In the figure, A represents the double enzyme digestion identification result of pcDNA-Ag85B, M: DNA marker (DL10000), 1: pcDNA3.1+ empty plasmid, 2: pcDNA-Ag85B recombinant plasmid, and 3: Hind III / XbaI double enzyme digestion product of pcDNA-Ag85B recombinant plasmid.

[0031] B represents the results of double enzyme digestion identification of pcDNA-Ag85B-wFc; M: DNA marker (DL10000); 1: empty plasmid pcDNA3.1+; 2: recombinant plasmid pcDNA-Ag85B-wFc; 3: Hind III / XbaI double enzyme digestion product of pcDNA-Ag85B-wFc recombinant plasmid.

[0032] C represents the results of double enzyme digestion identification of pcDNA-Ag85B-mFc; M: DNA marker (DL10000); 1: empty pcDNA3.1+ plasmid; 2: pcDNA-Ag85B-mFc recombinant plasmid; 3: Hind III / XbaI double enzyme digestion product of pcDNA-Ag85B-mFc recombinant plasmid.

[0033] Figure 3 The following are the results of Western Blot analysis of the expression products (fusion proteins) after transfection with recombinant plasmids: 1: Expression product of CHO cells transfected with pcDNA-Ag85B-wFc recombinant plasmid; 2: Expression product of CHO cells transfected with pcDNA-Ag85B-mFc recombinant plasmid; 3: Expression product of CHO cells transfected with pcDNA-Ag85B recombinant plasmid; 4: CHO cells; M: Protein marker.

[0034] Figure 4 The results of SDS-PAGE and Western Blot analysis of the purified fusion protein are shown.

[0035] In the figure, A represents the SDS-PAGE detection result of the purified fusion protein, M represents the protein marker, 1 represents the purified product of Ag85B fusion protein, 2 represents the purified product of Ag85B-mFc fusion protein, and 3 represents the purified product of Ag85B-wFc fusion protein.

[0036] B represents the Western Blot results of the purified fusion protein; M: protein marker; 1: purified product of Ag85B-mFc fusion protein; 2: purified product of Ag85B-wFc fusion protein; 3: purified product of Ag85B fusion protein.

[0037] Figure 5 The diagram illustrates the gating strategies for cells. In the diagram, A represents the gating strategy for lymphocytes, and B represents the gating strategy for CD3+ T cells.

[0038] Figure 6 The effects of different immunization methods on FCM in lung tissue by Ag85B, Ag85B-wFc, and Ag85B-mFc are shown in the figure. In the figure, A is the flow cytometry of CD4+ and CD8+ T cells secreting IFN-γ in lung tissue; B is the statistical graph of the proportion of CD4+ T cells secreting IFN-γ in lung tissue; and C is the statistical graph of the proportion of CD8+ T cells secreting IFN-γ in lung tissue. * p<0.05, ** p<0.01, *** p<0.001.

[0039] Figure 7The effects of different immunization methods on splenic FCM of Ag85B, Ag85B-wFc, and Ag85B-mFc are shown in the figure. In the figure, A is the flow cytometry of CD4+ and CD8+ T cells secreting IFN-γ in the spleen; B is the statistical graph of the proportion of CD4+ T cells secreting IFN-γ in the spleen; and C is the statistical graph of the proportion of CD8+ T cells secreting IFN-γ in the spleen. * p<0.05, ** p<0.01, *** p<0.001.

[0040] Figure 8 The proportion of memory Tem cells in each immunogroup in lung tissue and spleen ( (n=5); In the figure, A is the flow cytometry plot of memory Tem cells in the lung tissue and spleen of each immune group; B is the statistical plot of the proportion of memory Tem cells in the lung tissue; C is the statistical plot of the proportion of memory Tem cells in the spleen. * p<0.05, ** p<0.01, *** p<0.001.

[0041] Figure 9 To investigate the effects of different immune groups on lymphocyte proliferation capacity, ** p<0.01, *** p<0.001.

[0042] Figure 10 The effects of different immunization groups on the levels of IFN-γ and IL-2 in mouse serum.

[0043] Figure 11 The figures show the antibody titers in the serum of mice in different immunization groups; in the figure, A is the IgG antibody titer in mouse serum; B is the IgG1 antibody titer in mouse serum; C is the IgG2a antibody titer in mouse serum; D is the ratio of antibody IgG2a / IgG1; and E is the IgG antibody titer in mouse nasal lavage fluid.

[0044] Figure 12 The results of the challenge protection experiment of Ag85B-wFc fusion protein are shown. In the figure, A represents the number of bacteria in the lungs of mice infected with Ag85B-wFc fusion protein; B represents the number of bacteria in the spleen of mice infected with Ag85B-wFc fusion protein.

[0045] Figure 13 The effect of Ag85-wFc fusion protein on H37Ra bacterial count (results of auramine O fluorescence staining). Detailed Implementation

[0046] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0047] The experimental materials used in this invention are as follows:

[0048] 1. Cell line: Chinese hamster ovary cells (CHO) were purchased from the Chinese Academy of Medical Sciences;

[0049] 2. Experimental animals: Several 6-8 week old female C57BL / 6J mice were purchased from the Experimental Animal Center of Ningxia Medical University, license number: SCXK (Ning) 2020-0001, and housed in an SPF-grade environment.

[0050] The fusion proteins of Ag85B, Ag85B-wFc, and Ag85B-mFc prepared in this invention and their related nucleotide sequences are shown below:

[0051] 1. The amino acid sequence of the Ag85B-wFc fusion protein is shown in SEQ ID NO.1, and the nucleotide sequence encoding the Ag85B-wFc fusion protein is shown in SEQ ID NO.2.

[0052] In SEQ ID NO.1, positions 1-25 are the signal peptide, positions 26-310 are Ag85B, positions 311-324 are the linker, and positions 325-557 are IgG2a Fc.

[0053] In SEQ ID NO.2, positions 1-75 are the signal peptide coding sequence, positions 76-930 are the Ag85B nucleic acid sequence, positions 931-972 are the linker coding sequence, and positions 973-1674 are the IgG2a Fc nucleic acid sequence.

[0054] 2. The amino acid sequence of the Ag85B-mFc fusion protein is shown in SEQ ID NO.3, and the nucleotide sequence encoding the Ag85B-mFc fusion protein is shown in SEQ ID NO.4.

[0055] 3. The amino acid sequence of the Ag85B fusion protein is shown in SEQ ID NO.5, and the nucleotide sequence encoding the Ag85B fusion protein is shown in SEQ ID NO.6.

[0056] Example 1: Ag85B-wFc fusion protein and its preparation method.

[0057] I. Construction of recombinant plasmids

[0058] 1. Construction of recombinant plasmids pcDNA-Ag85B-wFc, pcDNA-Ag85B, and pcDNA-Ag85B-mFc:

[0059] like Figure 1 As shown, the recombinant plasmid pcDNA-Ag85B-wFc: Hind of plasmid pcDNA3.1(+) and Xho The fragments between the restriction enzyme sites were replaced with the DNA molecule shown in SEQ ID NO.2, while keeping the other parts unchanged, to obtain the recombinant plasmid pcDNA-Ag85B-wFc.

[0060] Ag85B is the Ag85B gene carrying a signal peptide. wFc is the mouse IgG2a Fc fragment, in which the key amino acids E (glutamate) and K (lysine) on the Fc fragment that bind to complement Clq are both mutated to A (alanine). The amino acid residues His310 and His433 in the CH2 and CH3 regions of the IgG Fc fragment are essential amino acids for binding to FcRn. mFc is formed by mutating these two amino acids (His310 and His433) on wFc to alanine (Ala) in order to reduce the binding affinity of FcRn to IgG.

[0061] Recombinant plasmid pcDNA-Ag85B-mFc: Hindextrin from plasmid pcDNA3.1(+) and Xba The small fragments between the restriction enzyme sites were replaced with the DNA molecule shown in SEQ ID NO.4 of the sequence listing, while keeping the other parts unchanged, to obtain the recombinant plasmid pcDNA-Ag85B-mFc.

[0062] 2. Identification of recombinant plasmids pcDNA-Ag85B, pcDNA-Ag85B-wFc, and pcDNA-Ag85B-mFc

[0063] The prepared recombinant plasmids pcDNA-Ag85B, pcDNA-Ag85B-wFc, and pcDNA-Ag85B-mFc were transformed into competent DH5α cells, respectively. The plasmids were then plated on LB agar containing Amp and incubated at 37 °C for 30 min. After incubation, the plates were inverted and left to steep overnight. Single colonies with good growth were picked and inoculated into 200 mL LB liquid medium containing Amp and cultured at 37 °C with shaking at 200 rpm for 12–16 h. Recombinant plasmids were extracted using an endotoxin-free plasmid extraction kit (Beijing Tiangen Biotech Co., Ltd.).

[0064] The extracted recombinant plasmids were added according to the system in Table 1, mixed thoroughly, and then briefly centrifuged. The plasmids were then digested with enzymes at 37 °C for 1 h. The correctly digested recombinant plasmids were sent to Beijing Ruiboxingke Biotechnology Co., Ltd. for sequencing. Finally, agarose gel electrophoresis was used for detection.

[0065] Table 1 Enzyme digestion reaction system

[0066]

[0067] II. Establishment of stable cell lines

[0068] 1. Cell transfection

[0069] (1) Take CHO cells in good growth condition, digest them with trypsin, and then count the cells at a concentration of 1.0 × 10⁻⁶. 5 Cells were seeded at a density of 10 cells / well into 24-well culture plates and cultured in a cell culture incubator (37 °C, 5% CO2) until 70–90% confluence.

[0070] (2) Prepare transfection reagents for solutions A and B. Solution A: 25 μL Opti-MEM™ + 1.5 μL Lipofectamine™ 3000, mix gently. Solution B: 25 μL Opti-MEM™ + 0.5 μg recombinant plasmid + 1 μL LP3000™, mix gently.

[0071] Mix solution A and solution B and incubate at room temperature for 15 min. Discard the culture medium in the 24-well plate, wash twice with PBS, and add 1 mL of antibiotic-free culture medium. f. Add the mixture of solution A and solution B dropwise to the wells, gently shake the culture plate to mix, and incubate in a cell culture incubator for 24 h to obtain transfected cells.

[0072] 2. Screening of monoclonal cells

[0073] After digesting and counting the transfected cells, 1000, 2000, and 5000 cells were seeded into 10 cm culture dishes. Once the cells adhered, the medium was replaced with selection medium containing 600 μg / mL G418, and the emergence of monoclonal cells was observed. The entire monoclonal cell line was then transferred to a 96-well plate, and an appropriate amount of selection medium was added for further culture. After confluence, the cell supernatant was collected, and positive monoclonal cells expressing the target protein were selected by Western blotting. These cells were then passaged sequentially into 6-well plates and T25 culture flasks for expansion culture, with continuous Western blotting to identify the expression of the target protein. Expansion culture continued until T75 culture flasks were reached, during which time the medium was replaced with complete medium containing 300 μg / mL G418. Finally, CHO cell lines stably expressing Ag85B, Ag85B-wFc, and Ag85B-mFc were obtained through expansion culture.

[0074] 3. Expression and identification of fusion proteins

[0075] When the cells have grown to approximately 90% confluence with the culture dish, collect the cell culture medium, centrifuge at 500 g and 4 °C for 5 minutes, collect the cell supernatant and store it at -80 °C for later use. Then perform Western blotting for identification.

[0076] III. Isolation and purification of fusion proteins

[0077] 1. Isolation of fusion proteins

[0078] a. Screen for positive monoclonal cell lines that stably and highly express the fusion proteins Ag85B, Ag85B-wFc, and Ag85B-mFc, and expand their culture. When the cell confluence reaches approximately 90%, add sodium butyrate solution to a final concentration of 1 μM, and continue culturing in a 32°C, 5% CO2 incubator until most cells float. Collect the cell supernatant. Centrifuge at 2000 rpm for 5 min and filter through a 0.45 μm vacuum filter. Slowly add the filtered and pre-chilled cell culture supernatant to the pre-equilibrated packed column, and collect all the eluent. Then, wash and elute (elute the target protein with 5 column volumes of elution buffer, discard the first three drops, collect the eluent in an EP tube pre-filled with an appropriate amount of Tris, mixing continuously while adding). Determine the protein concentration using BCA, and take 5 μg for SDS-PAGE electrophoresis and Western blotting.

[0079] 2. Purification and concentration of fusion proteins

[0080] a. Equilibration: Take a new, sterile, dried ultrafiltration tube, add sterile deionized water until it completely passes through the membrane, incubate on ice for 10 minutes, and centrifuge at 4 °C and 2000 rpm for 5 minutes. b. Sample loading: Discard the liquid at the bottom of the tube, place the ultrafiltration tube on ice, add the separated target protein, ensuring it does not exceed the white line at the top of the tube, and centrifuge at 4 °C and 3000 rpm for 10 minutes. c. Replacement buffer system: When all the protein solution is concentrated to approximately 1 mL, slowly add an appropriate amount of sterile PBS solution along the tube wall, centrifuge at 4 °C and 3000 rpm for 10 minutes three times. d. After the final centrifugation, collect the concentrated protein solution in the tube, not exceeding 500 μL. e. Cleaning and storage of ultrafiltration tubes: Add an appropriate amount of deionized water, gently blow with a pipette tip, being careful not to touch the filter membrane, discard the liquid, and repeat 3 times; add 0.2 M NaOH solution, let stand at room temperature for 20 min, then centrifuge at 3000 rpm for 10 min, and pour out the liquid; immerse the tube core in a beaker filled with deionized water, and let stand for 2-4 h, changing the deionized water repeatedly to dilute the NaOH concentration; clean the 50 mL tube, fill it with 20% ethanol, remove the tube core so that it is completely immersed in the 50 mL centrifuge tube, cap it, and store it at 4 ℃. f. Detection: Determine the protein concentration of the collected concentrate using BCA, and take 5 μg for SDS-PAGE electrophoresis and Western blotting. Aliquot the collected eluent into small tubes and freeze at -80 ℃ for later use.

[0081] IV. Experimental Results

[0082] 1. Identification of recombinant plasmids

[0083] (1) The double digestion (Hind III and Xbal) product of the eukaryotic recombinant expression plasmid pcDNA-Ag85B was analyzed by 1% agarose gel electrophoresis, and a band of approximately 1000 bp was observed, which was consistent with the expected size (951 bp). Figure 2 The pcDNA-Ag85B recombinant plasmid, which was identified by double enzyme digestion, was sequenced. The sequencing results showed that the full-length Ag85B gene was 951 bp, and the homology with the Ag85B (NP_216402.1) gene sequence of M.tb H37Rv included in NCBI was 100%, indicating that the plasmid was constructed correctly.

[0084] (2) The double enzyme digestion (Hind III and Xbal) product of the eukaryotic recombinant expression plasmid pcDNA-Ag85B-wFc was analyzed by 1% agarose gel electrophoresis, and a band of approximately 1700 bp was observed, which was consistent with the expected size (1674 bp). Figure 2The pcDNA-Ag85B-wFc recombinant plasmid, which was correctly identified by double enzyme digestion, was sequenced. The sequencing results showed that the full-length Ag85B-wFc gene was 1674 bp. The signal peptide US6 gene showed 100% homology with the US6 gene sequence (AMB66244.1) of Envelope glycoprotein D [Humanal pHaherpesvirus 2] included in NCBI; the Ag85B gene showed 100% homology with the Ag85B gene sequence (NP_216402.1) of M.tb H37Rv included in NCBI; and the wFc gene (except for the mutation site) showed 100% homology with the heavy chain Fc sequence (P01863.1) of mouse IgG2a included in NCBI, indicating that the plasmid construction was correct.

[0085] (3) Sequencing identification of the eukaryotic recombinant expression plasmid pcDNA-Ag85B-mFc: The pcDNA-Ag85B-mFc recombinant plasmid that was correctly identified by double enzyme digestion was sequenced. The sequencing results showed that both the Ag85B and mFc genes were correct. (See...) Figure 2 C.

[0086] 2. Western blot analysis of the expression products (fusion proteins) after transfection with recombinant plasmids.

[0087] After transfection, monoclonal cells were transferred to 96-well plates and cultured further with selection medium until the cell density reached approximately 90%. Cells and cell supernatant were collected, and positive monoclonal cells expressing the protein were screened by Western blotting. A CHO cell control group was also included. Figure 3 .

[0088] 3. SDS-PAGE and Western Blot analysis of the purified fusion protein

[0089] SDS-PAGE analysis of the purified and concentrated Ag85B, Ag85B-mFc, and Ag85B-wFc fusion proteins revealed distinct bands at approximately 35 kDa and 70 kDa, respectively, consistent with the expected protein sizes. Figure 4 (A). Western blot analysis was performed on the concentrated fusion proteins of Ag85B, Ag85B-mFc, and Ag85B-wFc, and the results were consistent with the expected sizes. (See A). Figure 4 B.

[0090] Example 2: Study on the immunogenicity of the Ag85B-wFc fusion protein prepared above.

[0091] I. Experimental Methods

[0092] 1. Animal immunization program

[0093] This experiment used the prime-boost protocol: 6-8 week old C57BL / 6J mice (18-20 g) were randomly divided into 6 groups (group 1, group 2... group 6), and immunized according to the immunization methods and dosages in Table 2. Based on previous experiments, the protein immunization dose and the mucosal adjuvant CpG ODN1826 (abbreviated as CpG) dose were determined to be 20 μg / mouse. Mice in the mucosal immunization group were intraperitoneally anesthetized with 0.25-0.5 mg / g body weight of aphthylamine anesthetic. The same volume of nasal drops was used for both immunizations, ensuring identical doses and volumes. Specific immunization, blood collection, and tissue sampling times are shown in Table 3. The nasal drop immunization group is also known as the mucosal immunization group. The nasal immunization method is as follows: before immunization, the mouse is anesthetized (abdominal anesthesia with Avodin anesthetic, dosage is 0.2mL / 10g body weight). After complete anesthesia, the immunizing agent is dripped into the nasal cavity of the mouse one drop at a time, alternating between the left and right nasal cavities.

[0094] Table 2 Immunization Schedule

[0095]

[0096] Table 3 Immunization Time

[0097]

[0098] II. Experimental Results

[0099] (I) Flow cytometry (FCM) detection of IFN secreted after immunization in mice - γ CD4 + T, CD8 + Number of T cells and effector memory T cells

[0100] Forty-two days after the initial immunization, spleens were harvested from mice, and lymphocytes were obtained to prepare a lymphocyte suspension. Forty-two days after the initial immunization, lung tissue was harvested from mice, and a single-cell suspension was prepared. FCM was used to detect CD4+ secreting IFN-γ. + T cells (CD3) + CD4 + IFN-r + T cells and CD8 cells that secrete IFN-γ + T cells (CD3) + CD8 + IFN-r + T cells (including CD3+ cells) and memory T cells (CD3+ cells) + CD44 +CD62L - The number of memory T cells.

[0101] 1. Gating strategy for FCM detection

[0102] In flow cytometry, FSC is plotted on the x-axis and SSC on the y-axis, representing the CD4+ cells that secrete IFN-γ. + T cells, CD8 + T cells and memory T cells perform gating. Figure 4 shows the gating strategies for these two cell types, A and B respectively. In Figure 4, A uses FSC-A as the x-axis and SSC-A as the y-axis to circle the lymphocyte population; then, CD3 is circled using CD3 as the x-axis and SSC-A as the y-axis. + T cell population; finally, select CD3. + CD4 cells secrete IFN-γ in T cells + T cells (CD4) + IFN-γ + The number of CD8 cells secreting IFN-γ and the amount of IFN-γ secreted + T cells (CD8) + IFN-γ + The quantity of ). Figure 5 In B, with FSC-A as the x-axis and SSC-A as the y-axis, the lymphocyte population is first delineated; then, with CD3 as the x-axis and SSC-A as the y-axis, CD3 is delineated. + T cell population; finally, select CD3. + Effector memory T cells (CD44) + The number of CD62L¯ (the same gate strategy in spleen and lung tissue).

[0103] 2. Proportion of CD4+ T and CD8+ T cells secreting IFN-γ in lung tissue and spleen of each group.

[0104] like Figure 6 The results showed that the CD3 concentration in lung tissues from the Ag85B-wFc nasal immunization group, Ag85B-mFc nasal immunization group, Ag85B-wFc subcutaneous immunization group, Ag85B-mFc subcutaneous immunization group, and Ag85B and PBS nasal immunization groups were significantly increased. + CD4 + IFN-γ +The cell percentages were 1.63%±0.18%, 1.19%±0.11%, 0.81%±0.12%, 0.53%±0.02%, 0.89%±0.01%, and 0.33%±0.04%, respectively. The Ag85B-wFc nasal drop immunization group had a higher percentage than the other five groups, and the difference was statistically significant (p<0.05).

[0105] CD3 ions from lung tissues in the Ag85B-wFc nasal immunization group, Ag85B-mFc nasal immunization group, Ag85B-wFc subcutaneous immunization group, Ag85B-mFc subcutaneous immunization group, and Ag85B and PBS nasal immunization group + CD8 + IFN-γ + The cell percentages were 1.42%±0.23%, 0.79%±0.16%, 0.78%±0.13%, 0.50%±0.04%, 0.62%±0.16%, and 0.39%±0.04%, respectively. The Ag85B-wFc nasal drop immunization group had a higher percentage than the other five groups, and the difference was statistically significant (p<0.05).

[0106] like Figure 7 As shown, the CD3 levels in the spleen of the Ag85B-wFc nasal immunization group, the Ag85B-mFc nasal immunization group, the Ag85B-wFc subcutaneous immunization group, the Ag85B-mFc subcutaneous immunization group, and the Ag85B and PBS nasal immunization groups were... + CD4 + IFN-γ + The cell percentages were 4.75%±0.40%, 3.74%±0.16%, 3.03%±0.29%, 2.63%±0.06%, 2.21%±0.05%, and 1.78%±0.22%, respectively. The Ag85B-wFc nasal drop immunization group had a higher percentage than the other five groups, and the difference was statistically significant (p<0.05).

[0107] CD3 levels in the spleen of the Ag85B-wFc nasal immunization group, Ag85B-mFc nasal immunization group, Ag85B-wFc subcutaneous immunization group, Ag85B-mFc subcutaneous immunization group, and Ag85B and PBS nasal immunization group. + CD8 + IFN-γ + The proportions of cells were 4.52%±0.26%, 3.46%±0.08%, 2.33%±0.13%, 2.37%±0.28%, 3.60%±0.18%, and 1.82%±0.04%, respectively. The Ag85B-wFc nasal drop immunization group had a higher proportion than the other five groups, and the difference was statistically significant (p<0.05).

[0108] 3. Proportion of effector memory Tem cells in each immune group in lung tissue and spleen

[0109] The results are as follows Figure 8 As shown, the CD3 content of lung tissue in the Ag85B-wFc nasal immunization group, Ag85B-mFc nasal immunization group, Ag85B-wFc subcutaneous immunization group, Ag85B-mFc subcutaneous immunization group, and Ag85B and PBS nasal immunization group were analyzed. + CD44 + CD62L – The cell percentages were 29.50%±4.95%, 15.30%±1.83%, 16.20%±1.13%, 16.95%±2.48%, 15.45%±2.76%, and 11.80%±2.26%, respectively. The Ag85B-wFc nasal drop immunization group had significantly higher cell percentages than the other five groups (p<0.05).

[0110] The results are as follows Figure 9 As shown, the CD3 levels in the spleen of the Ag85B-wFc nasal immunization group, the Ag85B-mFc nasal immunization group, the Ag85B-wFc subcutaneous immunization group, the Ag85B-mFc subcutaneous immunization group, and the Ag85B and PBS nasal immunization groups were... + CD44 + CD62L – The cell percentages were 27.37%±4.23%, 18.27%±3.91%, 19.70%±2.60%, 19.20%±1.31%, 17.90%±2.60%, and 13.67%±1.51%, respectively. The Ag85B-wFc nasal drop immunization group had significantly higher cell percentages than the other five groups (p<0.05).

[0111] (II) Lymphocyte proliferation experiment

[0112] Mice in different immunization groups showed significant lymphocyte proliferation after their spleen lymphocytes were stimulated in vitro with Ag85B-specific antigen. The stimulation index (SI) of the Ag85B-wFc intranasal immunization group, Ag85B-wFc subcutaneous immunization group, and Ag85B-mFc subcutaneous immunization group were 2.55, 2.04, and 1.77, respectively. The SI of the Ag85B-wFc intranasal immunization group was significantly higher than that of the Ag85B-wFc and Ag85B-mFc subcutaneous immunization groups (p<0.01). The SI of the Ag85B-mFc, Ag85B, and PBS intranasal immunization groups were 1.74, 1.68, and 0.40, respectively, and their proliferation was significantly lower than that of the Ag85B-wFc (2.55) intranasal immunization group (p<0.001).

[0113] (III) Serum IFN-γ and IL-2 levels in mice

[0114] The results are as follows Figure 10 As shown, on day 42 after the first immunization, the serum IFN-γ levels in the Ag85B-wFc and Ag85B-mFc subcutaneous immunization groups were 150 and 138 pg / mL, respectively. The serum IFN-γ levels in the Ag85B-wFc, Ag85B-mFc, Ag85B, and PBS nasal drop immunization groups were 484, 260, 112, and 86 pg / mL, respectively. The Ag85B-wFc nasal drop immunization group had significantly higher levels than the other five groups, with statistically significant differences (P<0.001).

[0115] The serum IL-2 levels in the subcutaneous immunization groups of Ag85B-wFc and Ag85B-mFc were 56 and 52 pg / mL, respectively. The serum IL-2 levels in the nasal immunization groups of Ag85B-wFc, Ag85B-mFc, Ag85B, and PBS were 106, 53, 48, and 34 pg / mL, respectively. The nasal immunization group of Ag85B-wFc had significantly higher levels than the other five groups, and the difference was statistically significant (P<0.001).

[0116] (iv) Evaluation of humoral immune response

[0117] 1. IgG antibody titer in mouse serum

[0118] The results are as follows Figure 11 As shown in Figure A, on day 14 after the first immunization, the serum levels of specific IgG antibodies in mice in the Ag85B-wFc, Ag85B-mFc, Ag85B intranasal immunization group and the Ag85B-wFc, Ag85B-mFc subcutaneous immunization group began to increase. By day 28, the antibody level in the Ag85B-wFc intranasal immunization group reached its highest level, and then decreased by day 42. Moreover, the antibody levels in the Ag85B-wFc intranasal immunization group were significantly higher than those in the other five groups on days 14, 28, and 42, and the differences were statistically significant (P<0.01).

[0119] 2. IgG1 antibody titer in mouse serum

[0120] The results are as follows Figure 11As shown in Figure B, on day 14 after the first immunization, the serum levels of specific IgG1 antibodies in mice in the Ag85B-wFc, Ag85B-mFc, Ag85B intranasal immunization group and the Ag85B-wFc, Ag85B-mFc subcutaneous immunization group began to increase. By day 28, the antibody level in the Ag85B-wFc intranasal immunization group reached its highest level, and then decreased by day 42. Moreover, the antibody levels in the Ag85B-wFc intranasal immunization group were significantly higher than those in the other five groups on days 14, 28, and 42, and the differences were statistically significant (P<0.01).

[0121] 3. Antibody titer of IgG2a in mouse serum

[0122] The results are as follows Figure 11 As shown in Figure C, on day 14 after the first immunization, the serum levels of specific IgG2a antibodies in mice in the Ag85B-wFc, Ag85B-mFc, Ag85B intranasal immunization group and the Ag85B-wFc, Ag85B-mFc subcutaneous immunization group began to increase. By day 28, the antibody level in the Ag85B-wFc intranasal immunization group reached its highest level, and then decreased by day 42. Moreover, the antibody levels in the Ag85B-wFc intranasal immunization group were significantly higher than those in the other five groups on days 14, 28, and 42, and the differences were statistically significant (P<0.01).

[0123] 4. The ratio of antibody IgG2a to IgG1

[0124] Analysis of the ratio of specific antibodies IgG2a / IgG1 showed that the ratio in the Ag85B-wFc nasal drop immunization group was greater than 1 on days 14, 28, and 42, which was statistically significant compared with the PBS group. ** p<0.01 (VS PBS group). This indicates that nasal immunization with Ag85B-wFc drops mainly induces the body to produce IgG2a antibodies, and the immune response shifts towards the Th1 type.

[0125] 5. Antibody titer of IgG in mouse nasal lavage fluid

[0126] On day 42 after the first immunization, the highest level of specific IgG antibody was detected in the nasal mucosal rinsing fluid of mice in the Ag85B-wFc intranasal immunization group, which was significantly higher than that in the other five groups (P<0.05).

[0127] Example 3: Protection experiment of Ag85B-wFc fusion protein prepared above against viral challenge.

[0128] Six- to eight-week-old female C57BL / 6J mice were randomly divided into two groups of six each.

[0129] Group 1 (Ag85-wFc + H37Ra infection group): The first immunization was performed on day 1 of the experiment, and the second immunization was performed on day 15 of the experiment; both immunizations were administered via nasal drops; the immunizing agent for both immunizations was Ag85-wFc immunizing agent, with a single immunization volume of 40 μL (containing 20 μg of Ag85-wFc protein) per animal; on day 43 of the experiment, Mycobacterium tuberculosis H37Ra (10 μg) was injected via tail vein. 6 CFU (each)

[0130] Group 2 (PBS + H37Ra infection group): The first immunization was performed on day 1 of the experiment, and the second immunization was performed on day 15 of the experiment; both immunizations were administered via nasal drops; the immunizing agent used for both immunizations was the control immunizing agent, and the volume of a single immunization per animal was 40 μL; on day 43 of the experiment, Mycobacterium tuberculosis H37Ra (10) was injected via tail vein. 6 CFU (each)

[0131] Ag85-wFc immunizing agent: Prepared by mixing equal volumes of the Ag85-wFc fusion protein (adjusted for protein concentration with PBS) prepared in Example 1 and mucosal adjuvant. Control immunizing agent: Prepared by mixing equal volumes of PBS and mucosal adjuvant.

[0132] Twenty-eight days after injection of Mycobacterium tuberculosis H37Ra, mice were euthanized by cervical dislocation. The lungs and spleen of the mice were aseptically isolated.

[0133] I. Determination of Organ Bacterial Load by Plate Method

[0134] 1. Take half of the lung (and the whole spleen), place it in a cell sieve with a pore size of 40 μm, add 5 mL of PBS, grind, collect all cell suspension into a 15 mL centrifuge tube, mix well, and then perform a 10-fold serial dilution with PBS to obtain the diluted solution.

[0135] 2. Take 100 μL of the dilution, spread it on a 7H10 medium plate, seal it with sealing film, and incubate it upside down at 37°C for 21 days. Record the number of colonies on the plate. Set up 3 replicate plates for each dilution.

[0136] Organ bacterial load (CFU) = (N / dilution) × 40.

[0137] N represents the number of individual colonies on the plate.

[0138] See results Figure 12 Compared with the PBS+H37Ra-infected group, the number of bacteria in the lungs and spleen of the Ag85-wFc+H37Ra-infected group was significantly reduced (p<0.001, p<0.01, respectively).

[0139] II. Acid-fast and Auramine O staining

[0140] 1. Making paraffin slices

[0141] The remaining half of the lung was removed, fixed with 4% paraformaldehyde for 48 hours, embedded in paraffin, and then sectioned.

[0142] 2. Auramine O fluorescent staining of paraffin sections

[0143] The slides were sequentially immersed in two vats of xylene solution, each for 10 minutes to dewax. Then, the slides were immersed in two vats of anhydrous ethanol, each for 5 minutes. Next, the slides were sequentially immersed in 95% ethanol solution, 85% ethanol solution, and 75% ethanol solution, each for 5 minutes to dehydrate. Then, auramine O staining solution was added to the slides, and staining was performed in the dark for 15 minutes. The slides were rinsed with distilled water, then destained with acidic destaining solution for 2 minutes, rinsed with distilled water, and then stained with counterstaining solution for 2 minutes, and rinsed with distilled water. The slides were gently blotted dry, allowed to air dry, and examined under a fluorescence microscope.

[0144] See results Figure 13 Auramine O fluorescence staining results showed that M. tb was stained golden yellow: compared with the PBS+H37Ra infected group, the number of lung bacteria in the Ag85-wFc+H37Ra infected group was significantly reduced (p<0.001).

[0145] In summary, the Ag85B-wFc fusion protein prepared in this invention can induce stronger local mucosal and systemic immune responses than subcutaneous immunization in mice after intranasal immunization, and has a good protective effect against Mycobacterium tuberculosis infection.

[0146] It should also be noted that in this invention, wFc is a mouse IgG2a Fc fragment, and mFc represents the Fc fragment after the wFc gene is mutated. The mutation refers to the mutation of both amino acids E (glutamic acid) and K (lysine) at the key sites on the Fc fragment that bind to complement Clq to A (alanine).

[0147] 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 the preferred embodiments as well as all changes and modifications falling within the scope of the invention.

[0148] 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 an Ag85B-wFc fusion protein in the preparation of an anti-tuberculosis vaccine, characterized in that, The vaccine is a mucosal immunotherapy vaccine, the route of administration is nasal drop immunization, the vaccine also includes a mucosal adjuvant, the amino acid sequence of the Ag85B-wFc fusion protein is shown in SEQ ID NO.1, and the nucleotide sequence encoding the protein is shown in SEQ ID NO.

2.

2. An Ag85B-wFc fusion protein, characterized in that, The amino acid sequence of the Ag85B-wFc fusion protein is shown in SEQ ID NO.1, and the nucleotide sequence encoding the protein is shown in SEQ ID NO.

2.

3. A pcDNA-Ag85B-wFc recombinant plasmid, characterized in that, The pcDNA-Ag85B-wFc recombinant plasmid contains the sequence SEQ ID NO.2 of claim 2.

4. The pcDNA-Ag85B-wFc recombinant plasmid according to claim 3, characterized in that, The construction steps of the pcDNA-Ag85B-wFc recombinant plasmid are as follows: replace the fragment between the HindIII and XhoI restriction sites of plasmid pcDNA3.1(+) with the DNA sequence shown in SEQ ID NO.2 of the sequence listing to obtain the recombinant plasmid pcDNA-Ag85B-wFc.

5. A method for preparing the Ag85B-wFc fusion protein according to claim 2, characterized in that, Includes the following steps: Construct the pcDNA-Ag85B-wFc recombinant plasmid; The pcDNA-Ag85B-wFc recombinant plasmid was transformed into competent DH5α cells, plated on LB solid medium containing Amp, and cultured overnight. Single colonies were picked and inoculated into LB liquid medium containing Amp. After shaking culture, the recombinant plasmid was extracted and transfected into CHO cells. Positive monoclonal cells expressing the protein were screened out, and the cells were expanded to obtain a CHO cell line stably expressing the Ag85B-mFc fusion protein. Then, the fusion protein was isolated, purified, and concentrated to obtain the Ag85B-wFc fusion protein.

6. The method for preparing the Ag85B-wFc fusion protein according to claim 5, characterized in that, The construction steps of the Ag85B-wFc recombinant plasmid are as follows: replace the fragment between the HindIII and XhoI restriction sites of plasmid pcDNA3.1(+) with the DNA sequence shown in SEQ ID NO.2 of the sequence listing to obtain the recombinant plasmid pcDNA-Ag85B-wFc.

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