A self-adjuvanted heat-stable nano multivalent tuberculosis vaccine and its preparation method and application

Abstract

The present invention belongs to the field of nano vaccine technology, and specifically relates to a self-adjuvanted heat-stable nano multivalent tuberculosis vaccine and its preparation method and application. The vaccine uses a self-adjuvanted heat-stable nano self-assembly scaffold protein as a carrier, and based on its ability to carry multi-component antigens on its surface, a heat-resistant multivalent tuberculosis vaccine is prepared, which can significantly improve the antigen presentation and immunogenicity of the vaccine, and also has excellent immune broad spectrum and stability, and good safety. Compared with conventional recombinant subunit vaccines, it has outstanding advantages and is very suitable for the current prevention and treatment of tuberculosis.

Description

Technical Field

[0001] The invention belongs to the technical field of nano vaccines, and specifically relates to a self-adjuvant type heat-stable nano multivalent tuberculosis vaccine and a preparation method and application thereof. Background Art

[0002] Although many studies have been conducted on new tuberculosis vaccines at home and abroad, there are many defects in this type of vaccine. For example, the number of antigens contained in recombinant subunit vaccines is limited, the effect of single immunization is poor, and the requirements for combined use of adjuvants are high; the side effects of viral scaffold vaccines are high, and the anti-viral scaffold antibodies produced in the body after a single injection of the patient make it impossible to vaccinate multiple times, and the versatility is not strong; the attenuated live vaccine has poor safety and there is a risk of pathogen recovery; the inactivated vaccine has poor stability and high manufacturing costs, and it is easy to spread the risk of pathogens during production and use. Therefore, actively exploring vaccines that can simultaneously improve antigen presentation, immunogenicity, immune broad spectrum, stability and safety is an effective way to effectively solve the current tuberculosis vaccine problems. Summary of the invention

[0003] In view of the problems existing in the tuberculosis vaccine in the prior art, the present invention provides a self-adjuvanted heat-stable nano multivalent tuberculosis vaccine. The vaccine uses a self-adjuvanted heat-stable self-assembling scaffold protein as a carrier, and based on its ability to carry multi-component antigens on its surface, a heat-resistant multivalent tuberculosis vaccine is prepared. The antigen presentation and immunogenicity of the vaccine can be significantly improved, while also having excellent immune broad spectrum and stability, and good safety. Compared with conventional recombinant subunit vaccines, the vaccine has outstanding advantages and is very suitable for the current prevention and treatment of tuberculosis.

[0004] The first aspect of the present invention provides a method for preparing a self-adjuvanted heat-stable nano multivalent tuberculosis vaccine, the specific steps comprising:

[0005] The N-terminus or C-terminus exposed on the outer surface of the thermostable self-assembling scaffold protein is fused with a membrane-penetrating peptide, and the adjuvant molecule is loaded inside the thermostable self-assembling scaffold protein to form a self-adjuvant thermostable self-assembling scaffold protein with membrane-penetrating function;

[0006] At least two immunodominant antigens of Mycobacterium tuberculosis are selected, and the self-adjuvanted heat-stable self-assembling scaffold protein is conjugated with the Mtb immunodominant antigen to obtain a self-adjuvanted heat-stable nano multivalent tuberculosis vaccine.

[0007] The main reasons why the present invention uses a heat-stable self-assembling scaffold protein to encapsulate adjuvants and use it as an antigen delivery carrier are: (1) The scaffold protein is in the form of a virus-like particle, which can effectively recognize the pattern recognition receptors on the surface of antigen-presenting cells and enhance the antigen presentation ability; (2) The outer surface of the scaffold protein can carry a variety of characteristic antigens for the preparation of multivalent vaccines; (3) The scaffold protein has ultra-high stability, which can significantly improve the thermal stability of the carried antigens, effectively avoid vaccine degradation, and overcome the cold chain dependence in vaccine preparation, storage and transportation; (4) The scaffold protein is essentially a nano self-assembling protein, and the outer surface is easy to transform and modify, and can carry polypeptide elements with different functions; (5) The interior of the scaffold protein is a hollow structure, which is convenient for encapsulating exogenous adjuvant molecules, so that it has a specific self-adjuvant effect.

[0008] Furthermore, the cell-penetrating peptide includes one or more of R9, R8, TAT (49-57), ARF (1-22), pVEC, MPG, CADY, Penetratin, Pep-1, MAP, trasportan, P28, VT5, Bac7, Pep-7, and C105Y.

[0009] Furthermore, the adjuvant molecule includes CPG ODN, dsRNA, IL-12 DNA, interleukin-2 / immunoglobulin (IL-2 / Ig) DNA or Poly-ICLC.

[0010] Furthermore, the loading process of the adjuvant molecule includes: first placing the thermostable self-assembling scaffold protein in a buffer solution, depolymerizing it at a temperature of 100-120°C or an acidic condition of pH=2, then lowering the temperature from 100-120°C to 3-4°C or restoring the pH from 2 to neutral, immediately adding the adjuvant molecule and incubating, so that the depolymerized thermostable self-assembling scaffold protein loads the adjuvant molecule during the self-assembly process, and obtaining a self-adjuvant thermostable self-assembling scaffold protein. Among them, the thermostable self-assembling scaffold protein has the performance of disassembly and reassembly, and can be automatically disassembled under high temperature or high acid conditions. The adjuvant molecule is added to restore the original condition of the thermostable self-assembling scaffold protein, so that the thermostable self-assembling scaffold protein re-self-assembles, and then the adjuvant molecule is effectively encapsulated inside the scaffold.

[0011] More specifically, the thermostable self-assembling scaffold protein is incubated in a neutral Tris solution at 100-120°C for 1-2h, or the neutral Tris solution containing the thermostable self-assembling scaffold protein is adjusted to pH=2 and placed at room temperature for not less than 6h for depolymerization; then the temperature is lowered from 100-120°C to 3-4°C or the pH is restored from 2 to neutral, and an adjuvant molecule is immediately added and incubated, so that the depolymerized thermostable self-assembling scaffold protein is loaded with the adjuvant molecule during the self-assembly process, thereby obtaining a self-adjuvant-type thermostable self-assembling scaffold protein.

[0012] Furthermore, the self-adjuvant type heat-stable self-assembling scaffold protein is conjugated with the Mtb immunodominant antigen, specifically comprising the following steps:

[0013] S1: Fusion of the LPXTGX motif gene to the N-terminus or C-terminus of at least two immunodominant antigens of Mycobacterium tuberculosis to obtain the LPXTGX-antigen gene sequence;

[0014] S2: fusing the (G)n oligoglycine motif gene to the N-terminus or C-terminus exposed on the outer surface of the self-adjuvant type thermostable self-assembling scaffold protein to obtain the (G)n-scaffold gene sequence;

[0015] S3: cloning the constructed LPXTGX-antigen gene sequence and (G)n-scaffold gene sequence into a prokaryotic expression plasmid to obtain a recombinant plasmid;

[0016] S4: transforming, inducing expression and purifying the recombinant plasmid to collect LPXTGX-antigen and (G)n-scaffold protein;

[0017] S5: LPXTGX-antigen and (G)n-scaffold protein were conjugated to obtain a self-adjuvanted thermostable nano multivalent tuberculosis vaccine.

[0018] Furthermore, the at least two immunodominant antigens of Mycobacterium tuberculosis are immunodominant antigens at different stages of Mycobacterium tuberculosis infection, including at least two of the four types of antigens: antigens associated with active bacterial replication, antigens associated with bacterial virulence, antigens associated with the localization or secretion of different driver proteins, and antigens associated with pathogen dormancy. The combined use of immunodominant antigens at different stages of Mtb infection can achieve better immune and protective effects.

[0019] Furthermore, antigens associated with active bacterial replication include, but are not limited to, Ag85, ESAT6, and CFP10;

[0020] Antigens associated with bacterial virulence, including but not limited to EsxV, EsxW, and TB10.4;

[0021] Antigens associated with the localization or secretion of different kinesins, including but not limited to PPE18 and PPE42;

[0022] Antigens associated with pathogen dormancy, including but not limited to Rv1813 and Rv2660.

[0023] Furthermore, the Mycobacterium tuberculosis immunodominant antigens are Ag85, ESAT6, CFP10, TB10.4, PPE18 and Rv1813.

[0024] Furthermore, the LPXTGX motif and the (G)n oligoglycine motif are a group of motif combinations that can form covalent bonds; the LPXTGX motif and the (G)n oligoglycine motif can be replaced by other motif combinations, including SpyTag / SpyCatcher, SnoopTag / SnoopCatcher, SdTag / SdCatcher or DogTag / DogCatcher.

[0025] Furthermore, in step S2, the self-adjuvant type thermostable self-assembling scaffold protein is genetically fused with a (G)n oligoglycine motif gene at a position different from that of the cell-penetrating peptide.

[0026] Furthermore, in step S4, the target protein is purified by size exclusion chromatography, and the effluent fractions are collected, concentrated, and the protein concentration is determined, and then stored at -20°C.

[0027] Furthermore, in step S5, LPXTGX-antigen and (G)n-scaffold protein M78 are mixed in a molar ratio of 1.5-3:1, and Sortase is added to conjugate the two to prepare a self-adjuvanted thermostable nano multivalent tuberculosis vaccine.

[0028] The second aspect of the present invention provides a self-adjuvanted heat-stable nano multivalent tuberculosis vaccine obtained by the preparation method of the above self-adjuvanted heat-stable nano multivalent tuberculosis vaccine.

[0029] The third aspect of the present invention provides an application of the above-mentioned self-adjuvanted heat-stable nano multivalent tuberculosis vaccine in the field of tuberculosis prevention and treatment.

[0030] The beneficial effects achieved by one or more technical solutions of the present invention are as follows:

[0031] Mtb is an intracellular bacterium. The host needs to present antigens to activate T cells first to exert an effective immune response. There are abundant pattern recognition receptors on the surface of antigen presenting cells. It is a better strategy to develop vaccine types that can bind to these receptors. Moreover, due to the complexity of the Mtb genome itself, it presents different infection states after entering the human body, and a single type of TB vaccine is difficult to eliminate tuberculosis in all populations. Therefore, the present invention uses a self-adjuvant type heat-stable self-assembly scaffold protein as a carrier for antigen delivery. The carrier itself has good stability and can safely and efficiently present antigens to cells. Moreover, the heat-stable scaffold protein encapsulates the adjuvant molecules in the structure, and can also avoid the degradation of the adjuvant molecules, effectively exert the adjuvant effect, and then enhance the immune effect. At the same time, the present invention uses multi-component immunodominant antigens at different stages of Mycobacterium tuberculosis infection to couple to the scaffold protein, which can achieve the effect of comprehensive immune prevention and treatment of tuberculosis. Compared with conventional recombinant subunit vaccines, the vaccine not only has excellent antigen presentation and immunogenicity, but also has outstanding immune broad spectrum and stability, and has good safety, which is very suitable for the prevention and treatment of current tuberculosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a schematic diagram of the modification of the M scaffold protein transmembrane peptide described in Example 1 of the present invention;

[0033] Figure 2 This is a schematic diagram of the loading of adjuvant molecules inside the M scaffold protein according to Example 1 of the present invention;

[0034] Figure 3 This is a schematic diagram of the construction of the LPXTGX-antigen gene sequence and the (G)n-M78 gene sequence in Example 1 of the present invention;

[0035] Figure 4 The electrophoresis diagrams of the six LPXTGX-antigens and (G)n-M78 purification;

[0036] Figure 5 This is a negative staining transmission electron microscopy image in Example 1 of the present invention;

[0037] Figure 6 This is a graph showing the antigen activity detected by ELISA in Example 1 of the present invention;

[0038] Figure 7 This is a graph showing antigen activity before and after high temperature treatment by ELISA in Example 1 of the present invention;

[0039] Figure 8 This is a graph showing changes in antigen activity during long-term high-temperature storage detected by ELISA in Example 1 of the present invention;

[0040] Fig. 9This is a graph showing changes in cell fluorescence intensity detected by flow cytometry in Example 1 of the present invention;

[0041] Fig.10 The percentage of activated T cells detected by flow cytometry in Example 1 of the present invention;

[0042] Fig.11 The percentage of memory T cells detected by flow cytometry in Example 1 of the present invention;

[0043] Fig.12 This is a graph for detecting the secretion level of IFN-γ in Example 1 of the present invention;

[0044] Fig.13 The serum antigen-specific antibody level of Ag85 detected by ELISA in Example 1 of the present invention;

[0045] Fig.14 The serum antigen-specific antibody level of PPE18 detected by ELISA in Example 1 of the present invention;

[0046] Fig.15 The serum antigen-specific antibody level of TB10.4 detected by ELISA in Example 1 of the present invention;

[0047] Fig.16 The serum antigen-specific antibody level of Rv1813 detected by ELISA in Example 1 of the present invention;

[0048] Fig.17 This is a graph showing the detection of the aspartate aminotransferase (GOT) index in the serum of mice reflecting the liver function in Example 1 of the present invention;

[0049] Fig.18 This is a graph showing the detection of alanine aminotransferase (GPT) in the serum of mice reflecting liver function in Example 1 of the present invention;

[0050] Fig.19 This is a blood urea nitrogen (BUN) index detection chart reflecting kidney function in the serum of mice in Example 1 of the present invention;

[0051] Fig. 20 This is a graph showing the detection of blood uric acid (UA) in the serum of mice reflecting kidney function in Example 1 of the present invention. DETAILED DESCRIPTION

[0052] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in combination with specific embodiments and comparative examples.

[0053] Example 1

[0054] 1. Engineering of self-adjuvant thermostable nanoscaffold proteins

[0055] (1) External surface modification

[0056] The M scaffold protein derived from a hyperthermophilic bacterium was selected, and its amino acid sequence (SEQ ID NO.1) is: MRKYEGMLTARGVKVGIVASRFNHAIVERLIEGAIDCFLRHEGKEEDLTLVRVPGSWEIPMAVKELLLKEDVEGVVALGVLIRGATPHFDYIANELSKGLAMLSLEHRKPVSFGVITADTLEQAIERAGTKHGNKGWDAMLSVIEMINLFKDIR

[0057] The 3D spatial structure of the M scaffold protein was predicted by relevant software. It is a 20-sided spherical structure self-assembled by 60 identical monomers, similar to virus-like particles. According to software analysis, the N-terminus and C-terminus of the monomer are exposed on the outer surface of the scaffold. The C-terminus was fused with the membrane-penetrating peptide pVEC to obtain a thermostable self-assembling scaffold protein with membrane-penetrating function. The schematic diagram is shown in the figure. Figure 1 shown.

[0058] (2) Adjuvant internal encapsulation

[0059] CPG ODN, which is composed of oligodeoxynucleotides, is easily degraded and difficult to be taken up by cells, so it needs a stable carrier for delivery. The thermostable self-assembling scaffold protein has a hollow structure inside, which can effectively load the adjuvant molecule CPG ODN inside the scaffold protein, and then effectively deliver the adjuvant molecule into the cell through the cell-penetrating peptide. The thermostable scaffold can avoid the degradation of adjuvant molecules and effectively exert the adjuvant effect. The specific schematic diagram is as follows Figure 2 shown.

[0060] The steps of internal encapsulation of the adjuvant are as follows: the thermostable self-assembling scaffold protein fused with the cell-penetrating peptide is incubated in a neutral Tris solution at 110°C for 2 h for depolymerization, and then the temperature of the neutral Tris solution is lowered to 4°C, and the adjuvant molecule is immediately added and incubated, so that the depolymerized thermostable self-assembling scaffold protein loads the adjuvant molecule during the self-assembly process, and finally a self-adjuvant type thermostable self-assembling scaffold protein carrying the cell-penetrating peptide on the surface and encapsulating the sensitive adjuvant molecule CPG ODN inside is obtained, which is named M78.

[0061] 2. Screening of dominant antigens at different stages of tuberculosis infection

[0062] Multi-component immunodominant antigens at different stages of Mtb infection were screened, and Ag85, ESAT6 and CFP10 related to active bacterial replication, TB10.4 related to bacterial virulence, PPE18 related to the localization or secretion of different driver proteins, and Rv1813 related to pathogen dormancy were obtained, namely, six Mtb immunodominant antigens Ag85, ESAT6, CFP10, TB10.4, PPE18 and Rv1813.

[0063] 3. Construction and preliminary characterization of a self-adjuvanted thermostable nano multivalent tuberculosis vaccine

[0064] The vaccine is constructed and assembled by gene fusion and covalent binding. The Sortase system is used. Sortase is an enzyme from Gram-positive bacteria that can covalently link proteins with C-terminal LPXTGX motifs to other proteins with N-terminal oligoglycine motifs. Specific method: The LPXTGX motif is fused to the C-terminus of the antigen, and the (G)n oligoglycine motif is fused to the N-terminus of the scaffold protein surface, and the purified antigen and scaffold protein are expressed separately. The scaffold protein is mixed with the antigen, and Sortase is added. After the reaction, the scaffold protein with surface-directed antigen coupling is obtained.

[0065] (1) Gene construction and protein expression

[0066] The LPXTGX motif gene was fused to the C-terminus of the six Mtb immunodominant antigens to obtain the LPXTGX-antigen gene sequence; the (G)n oligoglycine motif gene was fused to the N-terminus exposed on the outer surface of M78 to obtain the (G)n-M78 gene sequence. Figure 3 As shown, the constructed LPXTGX-antigen gene sequence and (G)n-M78 gene sequence were cloned into a prokaryotic expression plasmid to obtain a recombinant plasmid, which was then transformed into Escherichia coli to induce protein expression. The target protein was purified by size exclusion chromatography (SEC), and the effluent fractions were collected to obtain 6 LPXTGX-antigens and (G)n-M78, which were concentrated and the protein concentration was determined, and then stored at -20°C. Figure 4 The electrophoresis diagrams of the six LPXTGX-antigens and (G)n-M78 after purification are shown, wherein TB1-TB6 are the six antigens Ag85, ESAT6, CFP10, TB10.4, PPE18, and Rv1813 fused with LPXTGX, respectively.

[0067] (2) Vaccine component assembly

[0068] The collected 6 LPXTGX-antigens and (G)n-M78 were mixed at a molar ratio of 1.5:1, and Sortase was added to allow the two to react fully. The free antigens not coupled to the scaffold were removed by SEC, and the effluent fractions were collected, concentrated and the protein concentration was determined, and stored at -20°C to prepare a self-adjuvanted thermostable nano multivalent tuberculosis vaccine, namely TB 6 -M78.

[0069] (3) Preliminary characterization of the vaccine

[0070] A. Negative stain transmission electron microscopy

[0071] Figure 5 Negative staining transmission electron microscopy results are shown, from which it can be seen that the purified TB 6 -M78 presents a uniform and consistent nanoparticle spherical morphology, indicating that the directional loading of antigens on the surface of the scaffold protein does not significantly affect its original morphology.

[0072] B. ELISA Identification

[0073] Figure 6 The results of ELISA showed that the purified TB 6 -M78 can bind to the specific antibodies of the corresponding antigens respectively, indicating that six TB dominant antigens were successfully coupled to the surface of the scaffold protein.

[0074] 4. Performance evaluation of self-adjuvanted thermostable nano multivalent tuberculosis vaccine

[0075] (1) Stability assessment

[0076] Since the constructed self-adjuvant thermostable self-assembling scaffold protein M78 is derived from hyperthermophilic bacteria, the scaffold protein itself is thermostable. Therefore, the focus is on evaluating whether the artificially modified self-adjuvant thermostable scaffold can improve the thermostability and long-term storage stability of multivalent Mtb immunodominant antigens.

[0077] A. Thermal Stability Evaluation

[0078] TB 6 -M78 and 6 free antigen mixtures (i.e., Ag85, ESAT6, CFP10, TB10.4, PPE18, Rv1813 mixture, named TB 6 ) were placed at 37°C and 65°C for a certain period of time, and the changes in the affinity of the two for specific antibodies were identified by ELISA to evaluate the effect of high temperature on antigen activity. The results are shown in Figure 7 .

[0079] ELISA results showed that compared with the untreated TB 6-M78 binding to specific antibodies did not change significantly, while TB 6 The binding capacity with specific antibodies was significantly reduced, indicating that TB 6 -M78 can withstand high temperature treatment and maintain antigen activity.

[0080] B. Long-term storage stability assessment

[0081] TB 6 -M78 and 6 free antigen mixtures (i.e., Ag85, ESAT6, CFP10, TB10.4, PPE18, Rv1813 mixture, named TB 6 ) were placed at 37°C for 4 weeks, and the affinity changes of the two for specific antibodies were identified by ELISA. The affinity change curve over time was drawn to evaluate the effect of long-term high temperature storage on antigen activity. The results are shown in Figure 8 .

[0082] ELISA results showed that the TB 6 -M78 showed a slight decrease in binding affinity with specific antibodies, while TB 6 The binding capacity with specific antibodies decreased significantly after one week of high temperature treatment, indicating that TB 6 -M78 can withstand long-term high temperature treatment and maintain high antigen activity.

[0083] (2) Antigen presentation assessment

[0084] For intuitive observation, the fluorescent dye FITC was artificially added to the other end of LPXTGX-antigen that was not fused to LPXTGX to construct a fluorescently labeled TB 6 -M78 (named FITC-TB 6 -M78). FITC-TB 6 -M78 and FITC-TB 6 Dendritic cells DC2.4 were treated separately, and the changes in fluorescence intensity in the cells were detected by flow cytometry to analyze the effect of M78 on antigen presentation ability. The results are shown in Fig. 9 .

[0085] Flow cytometry results showed that the free mixed antigen FITC-TB 6 Compared with FITC-TB 6 -M78-treated cells showed stronger fluorescence intensity, indicating that FITC-TB 6 -M78 can be effectively taken up by antigen-presenting cells and has stronger antigen-presenting ability.

[0086] (3) Cellular immunity assessment

[0087] Establish a mouse immune model to evaluate the cellular immunity level of the vaccine in animals. BALB / c mice were divided into three groups: negative control group, mixed antigen TB 6 Vaccine group, multivalent antigen TB 6 -M78 vaccine group. Mice were injected intramuscularly with TB vaccine containing the same molar amount of antigen 6 and TB 6 -M78, and the negative control group was injected with the same volume of sterile PBS. After 10 and 56 days of immunization, spleen tissues of mice were collected, single cell suspensions were prepared, and the levels of cellular immune indicators were detected.

[0088] A. T cell activation

[0089] like Fig.10 The results of flow cytometry showed that after 10 days of immunization, compared with the mixed antigen TB 6 Vaccine group, multivalent antigen TB 6 -M78 vaccine group can rapidly induce CD4 + and CD8 + T cell activation, indicating that it can rapidly activate cellular immune responses.

[0090] B. Percentage of memory T cells

[0091] like Fig.11 The results of flow cytometry showed that after 56 days of immunization, compared with the mixed antigen TB 6 Vaccine group, multivalent antigen TB 6 -M78 vaccine group can induce a higher proportion of central memory T cells (TCM) and effector memory T cells (TEM) in mice, indicating that after one injection, TB 6 -M78 can maintain longer-lasting cellular immunity levels by activating memory T cells.

[0092] C. IFN-γ secretion

[0093] Ten days after immunization, spleen cells isolated from spleen were used for TB 6 -M78 and TB 6 The secretion level of IFN-γ was detected. Fig.12 As shown, compared with the mixture antigen TB 6 Vaccine group, multivalent antigen TB 6 -The spleen cells of mice treated with the M78 vaccine group could secrete higher levels of IFN-γ, which is essential for controlling Mtb infection.

[0094] (4) Humoral immunity assessment

[0095] The mouse immune model was established to evaluate the humoral immunity level of the vaccine in animals. BALB / c mice were divided into three groups: negative control group, mixed antigen TB 6 Vaccine group, multivalent antigen TB 6 -M78 vaccine group. Mice were injected intramuscularly with TB vaccine containing the same molar amount of antigen 6 and TB 6 -M78, and the negative control group was injected with the same volume of sterile PBS. The immunization nodes were 0 and 2 weeks, and the peripheral blood of mice was collected every other week, the serum was separated, and the mice were killed at the 8th week. The indirect ELISA method was used to detect the specific antibody levels against 6 dominant antigens in the mouse serum, and the results only showed the serum specific antibody levels against Ag85, TB10.4, PPE18, and Rv1813.

[0096] Figure 13-16 The ELISA test results showed that compared with the mixture antigen TB 6 Vaccine group, TB 6 -M78 induced high titers of specific antibodies against multiple antigens in mice after one dose, and maintained high titer antibody levels until week 8 after the second dose, indicating that multivalent nanovaccines can rapidly induce humoral immune responses and maintain high persistence.

[0097] (5) Safety assessment

[0098] The levels of liver and kidney function indicators in the serum of mice were detected after immunization, including aspartate aminotransferase (GOT) and alanine aminotransferase (GPT) reflecting liver function, and blood urea nitrogen (BUN) and blood uric acid (UA) reflecting kidney function.

[0099] Figure 17-20 The test results showed that there was no significant change in the liver and kidney function indicators in the serum of mice in each immunized group, indicating that vaccine immunization had no effect on the liver and kidney function of mice, had no toxic side effects, and was safe.

[0100] In summary, this embodiment provides a self-adjuvanted heat-stable nano multivalent tuberculosis vaccine, which loads 6 dominant antigens at different stages of Mtb infection on the surface of an engineered self-adjuvanted heat-stable self-assembling scaffold protein to prepare a new multivalent tuberculosis vaccine containing multiple dominant antigens. Compared with simply mixing free antigens, this multivalent vaccine can significantly improve the antigen presentation and immunogenicity of the vaccine, while having excellent immune broad spectrum and stability, and good safety. This self-adjuvanted heat-stable nano multivalent tuberculosis vaccine has a high market development and application value.

Claims

1. A method for preparing a self-adjuvanted heat-stable nano multivalent tuberculosis vaccine, characterized in that: The N-terminus or C-terminus of the protein with the amino acid sequence as shown in SEQ ID NO.1 is fused with a membrane-penetrating peptide, and the adjuvant molecule is loaded inside the protein to form a self-adjuvant type thermostable self-assembling scaffold protein with membrane-penetrating function; Six immunodominant antigens of Mycobacterium tuberculosis were selected, and the self-adjuvanted heat-stable self-assembling scaffold protein was conjugated with the Mtb immunodominant antigens to obtain a self-adjuvanted heat-stable nano multivalent tuberculosis vaccine; The Mycobacterium tuberculosis immunodominant antigens are Ag85, ESAT6, CFP10, TB10.4, PPE18 and Rv1813; The adjuvant molecule is CPG ODN.

2. The method for preparing the self-adjuvanted heat-stable nano multivalent tuberculosis vaccine according to claim 1, characterized in that: The loading process of the adjuvant molecule includes: first placing the protein with an amino acid sequence as shown in SEQ ID NO.1 in a buffer solution, depolymerizing it at a temperature of 100-120°C or under acidic conditions of pH=2, then lowering the temperature from 100-120°C to 3-4°C or restoring the pH from 2 to neutral, immediately adding the adjuvant molecule and incubating, to obtain a self-adjuvant type thermally stable self-assembling scaffold protein.

3. The method for preparing the self-adjuvanted heat-stable nano multivalent tuberculosis vaccine according to claim 1, characterized in that: The self-adjuvant type heat-stable self-assembling scaffold protein is conjugated with the Mtb immunodominant antigen, comprising the following steps: S1: The LPXTGX motif gene was fused to the N-terminus or C-terminus of six Mycobacterium tuberculosis immunodominant antigens to obtain the LPXTGX-antigen gene sequence; S2: The (G)n oligoglycine motif gene is fused to the N-terminus or C-terminus exposed on the outer surface of the self-adjuvant type thermostable self-assembling scaffold protein to obtain the (G)n-scaffold gene sequence; S3: cloning the constructed LPXTGX-antigen gene sequence and (G)n-scaffold gene sequence into a prokaryotic expression plasmid to obtain a recombinant plasmid; S4: transforming, inducing expression and purifying the recombinant plasmid to collect LPXTGX-antigen and (G)n-scaffold protein; S5: LPXTGX-antigen and (G)n-scaffold protein were conjugated to obtain a self-adjuvanted thermostable nano multivalent tuberculosis vaccine.

4. The method for preparing the self-adjuvanted heat-stable nano multivalent tuberculosis vaccine according to claim 3, characterized in that: In step S2, the self-adjuvant type thermostable self-assembling scaffold protein is genetically fused with a (G)n oligoglycine motif gene at a position different from that of the cell-penetrating peptide.

5. The method for preparing the self-adjuvanted heat-stable nano multivalent tuberculosis vaccine according to claim 3, characterized in that: In step S4, the target protein is purified by size exclusion chromatography, and the effluent fractions are collected, concentrated, and the protein concentration is determined, and then stored at -20°C.

6. The self-adjuvanted heat-stable nano multivalent tuberculosis vaccine obtained by the method for preparing the self-adjuvanted heat-stable nano multivalent tuberculosis vaccine according to any one of claims 1 to 5.

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