Fusion protein hp13138pb and its use in tuberculosis prevention

Abstract

The application discloses a fusion protein HP13138PB and application thereof in tuberculosis prevention. Specifically disclosed is a fusion protein comprising HTL, CTL and B cell epitopes in series, HBD-3, PSM alpha 4 and PADRE. The application screens 34 epitopes for mycobacterium tuberculosis, which have good immunogenicity and antigenicity and no toxicity and sensitization, and have the characteristics of high population coverage and the like. The immunogenicity of the vaccine is improved by further adding antibacterial peptide HBD-3 and auxiliary peptide PADRE, and PSM alpha 4 is added to endow the vaccine with targeted delivery function. In-vitro experiments prove that HP13138PB can stimulate human peripheral blood mononuclear cells to produce an immune response, and is an advantage protective antigen. The HP13138PB as a vaccine has the advantages of simple preparation method, low cost, high yield and higher safety and the like. The application has great value for the prevention and treatment of active tuberculosis and latent tuberculosis infection.

Description

Technical Field

[0001] This invention belongs to the field of immunology and relates to the fusion protein HP13138PB and its application in tuberculosis prevention, specifically involving a protein derived from Mycobacterium tuberculosis (…). Mycobacterium tuberculosis HP13138PB recombinant multiepitope antigen of MTB protein antigen and its application in the prevention of active and latent tuberculosis infection. Background Technology

[0002] Tuberculosis (TB) is caused by Mycobacterium tuberculosis (Mycobacterium tuberculosis). Mycobacteria tuberculosis Tuberculosis (TB) is an infectious disease caused by infection with Mycobacterium tuberculosis (MTB), transmitted through the respiratory tract. Mycobacterium tuberculosis infection is classified into two states: latent tuberculosis infection (LTBI) and active tuberculosis (ATB). MTB is an intracellular parasite that primarily causes long-term infection by attacking macrophages and inhibiting their apoptosis. A 2022 report from the World Health Organization (WHO) showed that there were 10.4 million new cases of TB and 1.4 million deaths globally in 2021. Since the 1990s, the WHO has developed a series of programs to stop TB and achieve the ambitious goal of ending TB. However, the number of newly diagnosed TB cases rebounded in 2021, reaching 6.4 million. These data suggest that TB is the second leading cause of death from a single pathogen.

[0003] Vaccination is the most effective way to prevent and control tuberculosis. Bacillus Calmette-Guerin (BCG) is the only approved tuberculosis vaccine, offering excellent protection against miliary tuberculosis and tuberculous meningitis in children. However, its efficacy against adult tuberculosis is poor (0%–80%), with protection lasting only 10–20 years. In immunocompromised patients, BCG vaccination may also lead to systemic dissemination of tuberculosis. Tuberculosis vaccine candidates evaluated in clinical trials can be divided into four categories: inactivated vaccines, live attenuated vaccines, subunit tuberculosis vaccines, and virus-vector-based tuberculosis vaccines. Currently, the highly anticipated subunit vaccine M72 / AS01E has completed phase II clinical trials. However, in 2019, the New England Journal of Medicine published final data from the M72 / AS01E vaccine's Phase 2b clinical trial (enrolling 3,500 adults aged 10–50 years). The data showed that at 36 months after a 3-year follow-up, the overall efficacy of the M72 / AS01E vaccine was 49.7% (95% CI 2.1–74.2), below the WHO's 50% protective efficacy threshold. Therefore, the development of new, effective, and safe tuberculosis vaccines is even more urgent.

[0004] With the rapid development of bioinformatics and immunoinformatics, peptide vaccines have become one of the most attractive vaccine development strategies. Through low-cost production technologies, peptides identified from MTB antigens can be accurately characterized as chemical entities (similar to classic drugs). Furthermore, peptides are chemically defined compounds with good stability. These excellent properties make peptide vaccines easy to transport and store. In addition, the lack of redundant elements overcomes some of the drawbacks of traditional vaccines, such as allergies and autoimmune reactions. As an interdisciplinary field based on informatics and modern immunology, immunoinformatics has led to a change in vaccine development models, accelerating research in the field of novel tuberculosis vaccines. Using bioinformatics tools, researchers can quickly and accurately process the large amounts of data generated during immunological research, significantly shortening vaccine development time. Summary of the Invention

[0005] The purpose of this invention is to provide a polypeptide fusion protein and its application in the prevention of tuberculosis. The technical problem to be solved is not limited to the described technical subject matter; other technical subjects not mentioned herein will be clearly understood by those skilled in the art through the following description.

[0006] To achieve the above objectives, the present invention first provides a fusion protein, which may be named HP13138PB. The fusion protein may include tandem polypeptide 1, tandem polypeptide 2, and tandem polypeptide 3. The tandem polypeptide 1 may include a polypeptide whose amino acid sequence is shown in positions 75-90, 96-109, 115-127, 133-146, 152-168, 174-189, 195-210, 216-233, 239-256, 262-279, 285-300, 306-319, and 325-340 of SEQ ID No. 1.

[0007] The tandem polypeptide 2 may include a polypeptide whose amino acid sequence is shown at positions 344-352, 356-365, 369-377, 381-389, 393-401, 405-414, 418-426, 430-438, 442-450, 454-462, 466-474, 478-486, and 490-498 of SEQ ID No. 1.

[0008] The tandem polypeptide 3 may include a polypeptide whose amino acid sequence is shown at positions 501-520, 523-542, 545-564, 567-586, 589-608, 611-630, 633-652, and 655-674 of SEQ ID No. 1.

[0009] Furthermore, the polypeptides may be linked together by amino acid linkers.

[0010] The tandem polypeptide 1 may be a tandem HTL epitope, consisting of 13 HTL epitopes (amino acid sequences of positions 75-90, 96-109, 115-127, 133-146, 152-168, 174-189, 195-210, 216-233, 239-256, 262-279, 285-300, 306-319, and 325-340 of SEQ ID No. 1). Specifically, the 13 HTL epitopes may be tandemly linked by an amino acid linker (such as GGPPG), and the amino acid sequence of the tandem polypeptide 1 may specifically be positions 75-340 of SEQ ID No. 1.

[0011] The tandem polypeptide 2 may be a tandem CTL epitope, consisting of 13 CTL epitopes (amino acid sequences of positions 344-352, 356-365, 369-377, 381-389, 393-401, 405-414, 418-426, 430-438, 442-450, 454-462, 466-474, 478-486, and 490-498 of SEQ ID No. 1). Specifically, the 13 CTL epitopes may be tandemly linked by an amino acid linker (such as AAY), and the amino acid sequence of the tandem polypeptide 2 may specifically be positions 344-498 of SEQ ID No. 1.

[0012] The tandem polypeptide 3 may be a tandem B-cell epitope, which is obtained by tandemly connecting eight B-cell epitopes (the amino acid sequences of which are positions 501-520, 523-542, 545-564, 567-586, 589-608, 611-630, 633-652 and 655-674 of SEQ ID No. 1, respectively). Specifically, the eight B-cell epitopes may be tandemly connected by an amino acid linker (such as KK), and the amino acid sequence of the tandem polypeptide 3 may be positions 501-674 of SEQ ID No. 1.

[0013] By linking tandem peptide 1 (tandem HTL epitope), tandem peptide 2 (tandem CTL epitope), and tandem peptide 3 (tandem B cell epitope) with amino acid linkers, a multi-epitope fusion protein is obtained, which can be used as an active ingredient to construct vaccine molecules.

[0014] Furthermore, the fusion protein can be, from the N-terminus to the C-terminus, the tandem polypeptide 1, the tandem polypeptide 2, and the tandem polypeptide 3.

[0015] Furthermore, the tandem polypeptides may be linked by amino acid linkers.

[0016] Further, the fusion protein, from N-terminus to C-terminus, may be the tandem polypeptide 1, amino acid linker, tandem polypeptide 2, amino acid linker, and tandem polypeptide 3 in sequence. Specifically, the fusion protein, from N-terminus to C-terminus, may be the tandem polypeptide 1, AAY, tandem polypeptide 2, KK, and tandem polypeptide 3 in sequence.

[0017] Furthermore, the fusion protein may further include adjuvant peptides and / or accessory peptides. Preferably, the fusion protein may further include adjuvant peptide 1 whose amino acid sequence is SEQ ID No. 1, which is positions 1-45; adjuvant peptide 2 whose amino acid sequence is SEQ ID No. 1, which is positions 680-699; and / or accessory peptide whose amino acid sequence is SEQ ID No. 1, which is positions 51-69.

[0018] The adjuvant peptide may be the antimicrobial peptide human beta-defensin-3 (HBD-3) or the TLR2 agonist phenol-soluble modulator α4 (PSMα4), and the accessory peptide may be PADRE.

[0019] Specifically, the adjuvant peptide 1 may be the antimicrobial peptide HBD-3, and the adjuvant peptide 2 may be PSMα4.

[0020] The amino acid sequence of adjuvant peptide 1 (HBD-3) may be positions 1-45 of SEQ ID No. 1, the amino acid sequence of adjuvant peptide 2 (PSMα4) may be positions 680-699 of SEQ ID No. 1, and the amino acid sequence of accessory peptide (PADRE) may be positions 51-69 of SEQ ID No. 1.

[0021] Furthermore, the fusion protein, from the N-terminus to the C-terminus, may be the adjuvant peptide 1, the helper peptide, the tandem polypeptide 1, the tandem polypeptide 2, the tandem polypeptide 3, and the adjuvant peptide 2.

[0022] Furthermore, the fusion protein, from N-terminus to C-terminus, may be, in sequence, the adjuvant peptide 1, the amino acid linker, the accessory peptide, the amino acid linker, the tandem polypeptide 1, the amino acid linker, the tandem polypeptide 2, the amino acid linker, the tandem polypeptide 3, the amino acid linker, and the adjuvant peptide 2.

[0023] Specifically, the fusion protein, from N-terminus to C-terminus, may be the adjuvant peptide 1, EAAAK, the helper peptide, GGPPG, the tandem polypeptide 1, AAY, the tandem polypeptide 2, KK, the tandem polypeptide 3, EAAAK, and the adjuvant peptide 2.

[0024] As is well known to those skilled in the art, amino acid linkers (also known as spacers or linkers) are short peptide sequences between polypeptides in a fusion protein. The purpose of using linkers to connect different epitopes is to prevent the formation of new epitopes at the junction of two epitopes and to protect the structure and function of the natural epitopes. Therefore, any linker that can achieve this purpose can be used to connect the epitopes described in this invention.

[0025] The amino acid linkers described in this article include, but are not limited to, EAAAK, GGPPG, AAY, KK, KKK, GGGSGGG, GGSSGG, GGSGSG, GGSGSG, GGGGS, and GSG.

[0026] In one embodiment of the present invention, the fusion protein includes the antimicrobial peptide HBD-3, the helper peptide PADRE, 13 HTL epitopes, 13 CTL epitopes, 8 B cell epitopes, the TLR2 agonist PSMα4, and a 6×His tag.

[0027] Furthermore, the fusion protein HP13138PB can be any of the following:

[0028] A1) The amino acid sequence is the protein consisting of positions 1-699 of SEQ ID No. 1;

[0029] A2) A protein that has more than 80% identity with and has the same function as the protein shown in A1) obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in positions 1-699 of SEQ ID No. 1.

[0030] A3) A fusion protein with the same function obtained by attaching a tag or signal peptide to the N-terminus and / or C-terminus of A1) or A2);

[0031] A4) The amino acid sequence is the protein consisting of positions 75-674 of SEQ ID No. 1;

[0032] A5) A protein that has more than 80% identity with and has the same function as the protein shown in A1) obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID No. 1 from positions 75 to 674.

[0033] A6) A fusion protein with the same function is obtained by attaching a tag or signal peptide to the N-terminus and / or C-terminus of A4) or A5).

[0034] Wherein, the fusion protein described in A3) can be a fusion protein with the same function obtained by attaching a His tag to the C-terminus of A1).

[0035] Further, the fusion protein described in A3) may be a protein whose amino acid sequence is SEQ ID No. 1, or a protein that has more than 80% identity with and has the same function as the protein shown in SEQ ID No. 1, obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID No. 1.

[0036] The substitutions described herein can be conservative substitutions (also known as conservative replacements) or non-conservative substitutions in non-core functional regions. As is known to those skilled in the art, conservative substitutions or non-conservative substitutions in non-core functional regions generally do not have a qualitative impact on protein function.

[0037] The tags mentioned in this article include, but are not limited to: GST (glutathione thiotransferase) tag protein, His tag protein (His-tag), MBP (maltose-binding protein) tag protein, Flag tag protein, SUMO tag protein, HA tag protein, Myc tag protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow-green fluorescent protein), mCherry (monomer red fluorescent protein), or AviTag tag protein.

[0038] In this article, identity refers to the similarity of amino acid or nucleotide sequences. The identity of amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the procedure, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, and setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, a search can be performed to calculate the identity of amino acid sequences, and then the identity value (%) can be obtained.

[0039] In this document, the 80% or more of identity can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0040] The present invention also provides a biomaterial, which may be any of the following:

[0041] D1) Nucleic acid molecules of any of the fusion proteins HP13138PB described in the encoded text;

[0042] D2) An expression cassette containing the nucleic acid molecules described in D1);

[0043] D3) A recombinant vector containing the nucleic acid molecule described in D1), or a recombinant vector containing the expression cassette described in D2);

[0044] D4) Recombinant microorganisms containing the nucleic acid molecules described in D1, or recombinant microorganisms containing the expression cassette described in D2), or recombinant microorganisms containing the recombinant vector described in D3);

[0045] D5) A recombinant host cell containing the nucleic acid molecule described in D1), or a recombinant host cell containing the expression cassette described in D2), or a recombinant host cell containing the recombinant vector described in D3).

[0046] In the above-mentioned biological materials, the nucleic acid molecule described in D1) can be any of the following:

[0047] B1) A DNA molecule whose coding sequence is SEQ ID No. 2, positions 1-2097 of SEQ ID No. 2, or positions 223-2022 of SEQ ID No. 2;

[0048] B2) The nucleotide sequence is a DNA molecule of SEQ ID No. 2, positions 1-2097 of SEQ ID No. 2, or positions 223-2022 of SEQ ID No. 2.

[0049] Furthermore, the expression cassette described in D2), the recombinant vector described in D3), the recombinant microorganism described in D4), and the recombinant host cell described in D5 can all express the nucleic acid molecule described in D1.

[0050] The DNA molecule shown in SEQ ID No. 2 may be a DNA molecule encoding the fusion protein HP13138PB shown in SEQ ID No. 1.

[0051] The DNA molecule shown in positions 1-2097 of SEQ ID No. 2 may be a DNA molecule encoding the fusion protein HP13138PB whose amino acid sequence is shown in positions 1-699 of SEQ ID No. 1.

[0052] The DNA molecule shown in positions 223-2022 of SEQ ID No. 2 may be a DNA molecule encoding the fusion protein HP13138PB whose amino acid sequence is shown in positions 75-674 of SEQ ID No. 1.

[0053] The nucleic acid molecule may also include a nucleic acid molecule obtained by codon preference modification based on the nucleotide sequence shown in SEQ ID No. 2, positions 1-2097 of SEQ ID No. 2, or positions 223-2022 of SEQ ID No. 2. Considering the degeneracy of codons and the codon preferences of different species, those skilled in the art can use codons suitable for the expression of a specific species as needed.

[0054] The vectors described herein refer to vectors capable of delivering exogenous DNA or target genes into host cells for amplification and expression. These vectors can be cloning vectors or expression vectors, including but not limited to: plasmids, bacteriophages (such as λ phage or M13 filamentous phage), granules (i.e., Cosmids), Ti plasmids, and viral vectors (such as retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, etc.). In one or more embodiments of this invention, the vector is pET-28a(+).

[0055] The microorganisms mentioned in this article may be bacteria, fungi, actinomycetes, protozoa, algae, or viruses. Among them, the bacteria may originate from the genus *Escherichia* (…). Escherichia sp. Erwinia ( ) Erwinia sp. ), Agrobacterium ( Agrobacterium sp. Flavobacterium ( Flavobacterium sp. Alcaligenes ( ) Alcaligenes sp. ), Pseudomonas spp. Pseudomonas sp. ), Bacillus spp. ( Bacillus sp. Examples of bacteria include, but are not limited to, Escherichia coli (E. coli). Escherichia coli Bacillus subtilis ( Bacillus subtilis ) or Bacillus pumilus ( Bacillus pumilus In one or more embodiments of the present invention, the microorganism is Escherichia coli BL21(DE3).

[0056] The host cell (also called the recipient cell) described herein may be a plant cell or an animal cell. The term "host cell" is understood to refer not only to a specific recipient cell but also to its offspring, which, due to natural, accidental, or intentional mutations and / or alterations, need not be identical to the original parent cell but are still included within the scope of the host cell. Suitable host cells are those known in the art.

[0057] The recombinant vectors described in this article refer to recombinant DNA molecules constructed by linking exogenous target genes with vectors in vitro. They can be constructed in any suitable manner, as long as the constructed recombinant vector can carry the exogenous target gene into the recipient cell and provide the exogenous target gene with the ability to replicate, integrate, amplify and / or express in the recipient cell.

[0058] D3) The recombinant vector may be pET-28a(+)-HP13138PB.

[0059] The recombinant vector pET-28a(+)-HP13138PB is obtained by replacing the fragment (small fragment) between the BamHI and XhoI recognition sites of the pET-28a(+) vector with the DNA fragment whose nucleotide sequence is the DNA fragment of SEQ ID No. 2 in the sequence listing, while keeping the other nucleotide sequences of the pET-28a(+) vector unchanged. The recombinant vector pET-28a(+)-HP13138PB expresses the fusion protein HP13138PB with the amino acid sequence shown in SEQ ID No. 1.

[0060] D4) The recombinant microorganism may be BL21 / pET-28a(+)-HP13138PB. BL21 / pET-28a(+)-HP13138PB is a recombinant microorganism obtained by introducing the recombinant vector pET-28a(+)-HP13138PB into Escherichia coli BL21(DE3).

[0061] The importation can be achieved through chemical transformation methods (such as Ca). 2+ The vector carrying the DNA molecule of the present invention can be transformed into host bacteria using any known transformation method, such as induced transformation, polyethylene glycol-mediated transformation, metal cation-mediated transformation, or electroporation transformation; alternatively, the DNA molecule of the present invention can be transduced into the host bacteria via bacteriophage transduction. The introduction can also be achieved by transfecting the host cell with the vector carrying the DNA molecule of the present invention using any known transfection method, such as calcium phosphate co-precipitation, liposome-mediated transformation, electroporation, or viral vector transfection.

[0062] This invention also provides any of the fusion proteins described herein, and / or any of the following applications of the biomaterial:

[0063] C1) Use in the preparation of products for the prevention and / or treatment of diseases caused by Mycobacterium tuberculosis infection;

[0064] C2) Application in the preparation of vaccines to prevent diseases caused by Mycobacterium tuberculosis infection;

[0065] C3) Application in the preparation of protective antigens against Mycobacterium tuberculosis;

[0066] C4) Application in screening and / or developing antibodies against Mycobacterium tuberculosis.

[0067] Furthermore, the product may be a reagent or a drug.

[0068] The protective antigen refers to the antigenic component of Mycobacterium tuberculosis that can stimulate the body to produce a protective immune response.

[0069] The Mycobacterium tuberculosis antibody may include, but is not limited to, full-length antibodies or antigen-binding fragments (such as Fab fragments, Fv fragments, Fab′ fragments, F(ab′)2 fragments, single-chain antibodies (ScFv), nanobodies (single-domain antibodies), bispecific antibodies or minimum recognition units (MRUs).

[0070] The present invention also provides products for the prevention and / or treatment of diseases caused by Mycobacterium tuberculosis infection, said products comprising any of the fusion proteins described herein.

[0071] Furthermore, the product may be a vaccine or a pharmaceutical composition.

[0072] The vaccine can be used to prevent Mycobacterium tuberculosis infection.

[0073] The active ingredient of the vaccine may include any of the fusion proteins described herein.

[0074] The vaccine may also include an adjuvant and / or a vaccine delivery system.

[0075] The adjuvant may be a substance that can stimulate the body to produce a stronger humoral and / or cellular immune response against the co-inoculated antigen. The adjuvants described herein may be those known to those skilled in the art, including but not limited to: plant adjuvants (such as alkylamines, phenolic compounds, quinine, saponins, sesquiterpenes, proteins, polypeptides, polysaccharides, glycolipids, phytohemagglutinins, etc.), bacterial adjuvants (such as cholera toxin, Escherichia coli heat-labile toxin, bacterial lipopolysaccharides, etc.), aluminum adjuvants and other inorganic adjuvants (such as calcium adjuvants), cytokine and nucleic acid adjuvants (such as monocyte clone stimulating factor, leukocyte cytokines IL-1, IL-2, IL-4, IL-5, IL-6, IFN-γ, CpG motifs, nucleic acid carriers, etc.), and emulsion adjuvants (such as Freund's adjuvant). The adjuvant may be pharmaceutically acceptable.

[0076] The vaccine delivery system described herein can be a substance capable of carrying antigens to the body's immune system, where they can be stored and exert their antigenic effects for an extended period. The vaccine delivery system described herein can be a salt gel adjuvant vaccine delivery system, an emulsion adjuvant vaccine delivery system, a liposome adjuvant vaccine delivery system, or a nano-adjuvant vaccine delivery system.

[0077] The active ingredient of the pharmaceutical composition may include any of the fusion proteins described herein.

[0078] The pharmaceutical composition may also include one or more pharmaceutically acceptable carriers. The pharmaceutically acceptable carrier may be a diluent, excipient, filler, binder, wetting agent, disintegrant, absorption enhancer, adsorbent, surfactant, or lubricant, but is not limited thereto.

[0079] Furthermore, the disease caused by Mycobacterium tuberculosis infection described in this article can be tuberculosis.

[0080] Furthermore, the tuberculosis may include active tuberculosis (ATB) and latent tuberculosis infection (LTBI).

[0081] The present invention also provides a method for preparing any of the fusion proteins described herein, the method comprising expressing a nucleic acid molecule encoding any of the fusion proteins described herein in a microorganism or a host cell to obtain the fusion protein.

[0082] Furthermore, the method may include the following steps:

[0083] G1) Construct a recombinant expression vector containing a nucleic acid molecule encoding any of the fusion proteins described herein;

[0084] G2) The recombinant expression vector is introduced into microorganisms to obtain recombinant microorganisms;

[0085] G3) The recombinant microorganism is cultured, and the fusion protein is obtained by isolation and / or purification;

[0086] Further, the nucleic acid molecule described in G1) may be a DNA molecule as shown in SEQ ID No. 2, positions 1-2097 of SEQ ID No. 2, or positions 223-2022 of SEQ ID No. 2.

[0087] Furthermore, the microorganism may be Escherichia coli BL21(DE3).

[0088] The inventors of this invention predicted and screened HTL, CTL, and B-cell epitopes targeting Mycobacterium tuberculosis using bioinformatics and immunoinformatics techniques. These epitopes exhibit excellent immunogenicity and antigenicity, are non-toxic and non-sensitizing, and have high population coverage. Based on this, the inventors added the antimicrobial peptide HBD-3 and the helper peptide PADRE to the epitope vaccine design to further enhance the immunogenicity of the epitope vaccine molecule, and added the TLR2 agonist PSMα4 to endow the vaccine molecule with targeted delivery capabilities. Furthermore, immunoinformatics tools were used to predict and analyze the antigenicity, immunogenicity, physicochemical parameters, secondary structure, tertiary structure, and immunostimulatory effects of the vaccine. The results showed that the polypeptide fusion protein HP13138PB provided by this invention has an antigenicity of 0.87, an immunogenicity of 2.79, and a solubility index of 0.55. Secondary structure prediction showed that HP13138PB has an α-helix composition of 31%, a β-strand composition of 11%, and a helix composition of 56%. Tertiary structure analysis showed that the Z-score and favorite region of HP13138PB were -4.47 and 88.22%, respectively. The binding energy of HP13138PB to TLR2 was -1224.7 kcal / mol.

[0089] This invention further prepared the fusion protein HP13138PB. The consistency between immunoinformatics and real-world experimental results was analyzed using enzyme-linked immunospot assays (ELISPOT) and Th1 / Th2 / Th17 cytokine detection experiments. Both immunoinformatics and real-world experimental results showed that the peptide fusion protein HP13138PB can induce innate and adaptive immune responses characterized by significantly elevated levels of cytokines such as IFN-γ, TNF-α, IL-4, and IL-10. Simultaneously, in vitro experiments demonstrated that the Mycobacterium tuberculosis peptide fusion protein HP13138PB can serve as an antigenic protein, stimulating an immune response in human peripheral blood mononuclear cells (PBMCs), making it a superior protective antigen. This invention can provide a new candidate vaccine for the development of tuberculosis vaccines.

[0090] The polypeptide fusion protein HP13138PB of this invention can be prepared through genetic engineering. Using HP13138PB as a vaccine offers advantages over bacterial protein vaccines, including simpler preparation methods, lower cost, higher yield, and greater safety. This invention is of great value for the prevention and treatment of active tuberculosis and latent tuberculosis infection. Attached Figure Description

[0091] Figure 1 This refers to the HTL epitope information of the final peptide fusion protein selected in Example 1 for construction.

[0092] Figure 2This refers to the CTL epitope information selected in Example 1 for the final construction of the polypeptide fusion protein.

[0093] Figure 3 This refers to the B-cell epitope information selected in Example 1 for the final construction of the polypeptide fusion protein.

[0094] Figure 4 This is a schematic diagram illustrating the construction of the HP13138PB vaccine.

[0095] Figure 5 The results show the predicted molecular solubility and secondary structure of the HP13138PB vaccine.

[0096] Figure 6 Three-dimensional model, Z-score, and Ramachandran plot of HP13138PB vaccine.

[0097] Figure 7 To predict the conformational B-cell epitopes of the HP13138PB vaccine using the ElliPro server.

[0098] Figure 8 This is a schematic diagram of the interaction between the HP13138PB vaccine and toll-like receptor 2 (TLR2).

[0099] Figure 9 The results of C-ImmSim Server's predictions for macrophages (MA) and dendritic cells (DC).

[0100] Figure 10 The results show the predictions of C-ImmSim Server for helper T (TH) cells, cytotoxic T (TC) cells, B cells, and antibodies.

[0101] Figure 11 A schematic diagram of the construction of the recombinant vector pET-28a(+)-HP13138PB.

[0102] Figure 12 Cytokine levels induced by the HP13138PB vaccine on C-ImmSim Server. Three injections of the HP13138PB vaccine were simulated in C-ImmSim Server, and the levels of IFN-γ, IL-4, IL-12, TGF-β, TNF-α, IL-10, IL-6, IFN-β, IL-18, IL-23, and IL-2 induced by the HP13138PB vaccine were analyzed. Cytokine concentrations are expressed in ng / ml.

[0103] Figure 13 For the detection of IFN-γ by enzyme-linked immunospot assay (ELISPOT) +T lymphocytes. Peripheral blood mononuclear cells (PBMCs) from healthy controls (HC), patients with latent tuberculosis infection (LTBI), and patients with active tuberculosis (ATB) were stimulated in vitro with HP13138PB vaccine. IFN-γ was detected using a human ELISPOT kit. + Spot-forming cells (SFCs) of T lymphocytes were analyzed. Data were assessed for normality using unpaired t-tests or Mann-Whitney tests. Data are presented as mean ± SEM. p < 0.05 was considered statistically significant. SEM values ​​are the standard error of the mean.

[0104] Figure 14 The levels of cytokines produced by human peripheral blood mononuclear cells (PBMCs) induced by HP13138PB vaccine were measured. The levels of interleukin-4 (IL-4, A), IL-6 (B), IL-10 (C), IL-17A (D), tumor necrosis factor-α (TNF-α, E), interferon-γ (IFN-γ, F), and IL-2 (G) cytokines were detected using a human Th1 / Th2 / Th17 cytokine assay kit. PBMCs from healthy controls (HC, n=21), latent tuberculosis-infected individuals (LTBI, n=24), and active tuberculosis patients (ATB, n=18) were stimulated in vitro with HP13138PB vaccine. PBMCs from HCs stimulated with AIM medium served as a negative control. One-way ANOVA or the Kruskal-Wallis test was used to compare differences based on data normality and homogeneity of variance. All data are presented as mean ± SEM. p < 0.05 was considered statistically significant. SEM, standard error of the mean. Detailed Implementation

[0105] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0106] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0107] The Escherichia coli BL21(DE3) competent cells in the following examples were purchased from Shanghai Jingnuo Biotechnology Co., Ltd.

[0108] The carrier pET-28a(+) used in the following examples was purchased from Novagen.

[0109] The preparation methods of the main reagents in the following examples are as follows:

[0110] 1. Preparation of LB liquid culture medium (1000 ml):

[0111]

[0112] Add deionized water to a final volume of 1000 ml, then autoclave at 121°C for 15 minutes.

[0113] 2. Preparation of LB solid culture medium (1000 ml):

[0114]

[0115] Add deionized water to a final volume of 1000 ml, then autoclave at 121°C for 15 minutes.

[0116] 3. Preparation of buffer solution for purification of the soluble expression form of the target protein:

[0117] (1) Soluble protein lysis buffer, pH 8.0 (1000 ml):

[0118]

[0119] Dissolve in deionized water and bring the volume to 1000 ml. Adjust the pH to 8.0 with NaOH.

[0120] (2) Preparation of buffer solution required for purification of the target protein expressed in inclusion bodies:

[0121] ① Inclusion body protein lysis buffer, pH 8.0 (1000 ml):

[0122]

[0123] Dissolve in deionized water and bring the volume to 1000 ml. Adjust the pH to 8.0 with NaOH.

[0124] ② Inclusion body protein washing buffer, pH 6.3 (1000 ml):

[0125]

[0126] Dissolve in deionized water and bring the volume to 1000 ml. Adjust the pH to 6.3 with NaOH.

[0127] ③ Inclusion body protein elution buffer, pH 4.5 (1000 ml):

[0128]

[0129] Dissolve in deionized water and bring the volume to 1000 ml. Adjust the pH to 4.5 with NaOH.

[0130] Example 1: Prediction, screening, and identification of immune dominant epitopes

[0131] When pathogenic microorganisms invade the body, they trigger an immune response. This immune response is not directed at the entire exogenous substance, but only at epitopes, usually a polypeptide. In this embodiment, 17 candidate antigens were predicted and screened for HTL epitopes, CTL epitopes, and B cell epitopes. The aim was to effectively obtain the optimal antigenic epitopes recognized by helper T lymphocytes (HTL), cytotoxic T cells (CTL), and B cells, respectively, for further use in vaccine preparation.

[0132] 1. Selection of antigens

[0133] Anat Zvi et al. screened 189 potential tuberculosis (TB) vaccine candidates from 3989 open reading frames across the entire Mycobacterium tuberculosis (MTB) genome using literature search and bioinformatics methods. In previous studies, 34 of these antigens had been identified as potential TB vaccine candidates. Of these 34 antigens, at least five have entered clinical trials, such as Ag85A (Rv3804c), Ag85B (Rv1886c), ESAT-6 (Rv3875), MTB72F (Rv0125), and Rv1196. In addition, 10 antigens have been used in protective studies in animal models. The remaining 19 antigens also induce strong immune responses. Therefore, we selected five antigens evaluated in clinical trials and 12 antigens used in preclinical studies for epitope prediction and screening. The 17 candidate antigens are Ag85A, Ag85B, ESAT6, EspA, Mpt63, MTB32A, PPE18, RpfB, TB10.4, CFP10, MPT51, MPT64, MTB8.4, PPE44, PPE68, RpfA, and RpfB.

[0134] 2. HTL epitope prediction and screening

[0135] HTL epitope prediction was performed using the Major Histocompatibility Complex (MHC) II server in IEDB (http: / / tools.iedb.org / mhcii / ). Parameter settings: IEDB recommended 2.22 was used as the prediction method; human was selected as the species; the MHC alleles were selected from the total reference set of human leukocyte antigens (HLA) (HLA-DR, HLA-DP, HLA-DQ); the epitope length was set to 15. Inclusion criteria: HTL epitope percentile ranking <0.5; peptide scores were obtained by comparison with 5 million 15-mers (peptides of 15 amino acids in length) in the SWISSPROT database (a lower score for epitopes binding to MHC II indicates higher affinity), and a percentile ranking <0.5 was obtained by comparison with 5 million 15-mers in the SWISSPROT database. VaxiJen v2.0 (http: / / www.ddg-pharmfac.net / vaxijen / VaxiJen / VaxiJen.html) was used to predict epitope antigenicity with a threshold of 0.4. Automatic cross-covariance (ACC) was used to transform the target selection and predict the probability of protection against a specific antigen. Finally, the IFN-γ epitope server (http: / / crdd.osdd.net / raghava / ifnepitope / index.php) was used to predict the IFN-γ inducibility of epitopes (negative / positive; a positive result indicating IFN-γ induction means the epitope can be further investigated). Through the above prediction and screening, 13 HTL immunodominant epitopes were ultimately identified as candidate epitopes for constructing vaccine molecules. For details on specific epitope sequences, please refer to [link to relevant documentation]. Figure 1 AllerTOP v.2.0 and Aller FP (i.e., Allergen FP v.1.0) () is used to predict sensitization. 1 indicates sensitization, and 2 indicates no sensitization.

[0136] 2. CTL epitope prediction and screening

[0137] The IEDB MHC I server (http: / / tools.iedb.org / mhci / ) was used to predict CTL epitopes. IEDB Recommendation 2020.09 (NetMHCpanEL 4.1) was the primary qualifier, and epitopes of all lengths of human HLA alleles were secondary qualifiers. Epitopes with a percentile <0.5 were eligible for the next step of analysis. Then, the Class I immunogenicity server (http: / / tools.iedb.org / immunogenicity / ) was used to analyze the immunogenicity of these CTL epitopes; epitopes with a percentile level <0.5 and an immune score >0 were selected for the next step. Finally, the VaxiJen v2.0 server was used to predict antigenicity with a threshold of 0.4. Through the above prediction and screening, 13 CTL immunodominant epitopes were ultimately identified as candidate epitopes for constructing vaccine molecules. For details on specific epitope sequences, please refer to [link to relevant documentation]. Figure 2 AllerTOP v.2.0 And Allergen FPv.1.0 Used to predict sensitization. 1 indicates sensitization, 2 indicates no sensitization.

[0138] 3. Prediction and screening of B-cell epitopes

[0139] B cells play a crucial role in the host's fight against various viruses. The ABCpred server (https: / / webs.iiitd.edu.in / raghava / abcpred / ABC_submission.html) was used to predict linear B-cell epitopes due to its high accuracy (65.93%). Epitope length was limited to 20, and the filtering threshold remained at the default 0.51 (a higher threshold implies higher specificity but lower sensitivity). Through the above prediction and screening, eight B-cell epitopes were ultimately identified as candidate epitopes for constructing vaccine molecules. For details regarding specific epitope sequences, please refer to [link to relevant documentation]. Figure 3 .

[0140] Ultimately, 13 HTL epitopes, 13 CTL epitopes, and 8 B-cell epitopes, totaling 34 epitopes, were identified for constructing the active ingredient (peptide fusion protein) of the vaccine molecule. The amino acid sequences of the 34 epitopes are shown in Table 1.

[0141]

[0142] Example 2: Construction, physicochemical properties, and structural analysis of peptide fusion proteins

[0143] 1. Population coverage and construction of peptide fusion proteins

[0144] Based on the HTL, CTL, and B-cell epitopes predicted and screened using the aforementioned bioinformatics tools, the following 34 epitopes were ultimately selected: HTL epitopes with the highest adjusted rank, antigenicity, and IFN-γ scores, and those exhibiting no toxicity or sensitization; CTL epitopes with the highest adjusted rank, immunogenicity, and antigenicity scores, and those exhibiting no toxicity or sensitization; and the B-cell epitope with the highest predicted score (for a total of 34 epitopes) to construct a polypeptide fusion protein (e.g., HP13138PB). Population coverage analysis of the selected immunodominant HTL and CTL epitopes was performed using the population coverage tool in the IEDB database (http: / / tools.iedb.org / population / ). The HLA allele genotype frequencies used in the IEDB database were obtained from the Allele Frequencies Database (http: / / www.allelefrequencies.net / ). This database provides allele frequencies for 115 countries and 21 ethnic groups divided into 16 geographic regions. The analysis results (Table 2) show that the coverage rates of the HP13138PB vaccine (a vaccine with the polypeptide fusion protein HP13138PB as the active ingredient) for HTL epitopes (Class I) in Central Africa, Central America, East Africa, East Asia, Europe, North Africa, North America, Northeast Asia, Oceania, South America, South Asia, Southeast Asia, Southwest Asia, West Africa, the West Indies, and globally were 68.55%, 2.78%, 78.26%, 82.31%, 96.91%, 86.09%, 92.37%, 83.48%, 68.69%, 80.54%, 85.36%, 70.17%, 83.21%, 81.57%, 90.04%, and 92.07%, respectively. Similarly, the HP13138PB vaccine coverage rates for HTL epitopes (Class II) in Central Africa, Central America, East Africa, East Asia, Europe, North Africa, North America, Northeast Asia, Oceania, South America, South Asia, Southeast Asia, Southwest Asia, West Africa, the West Indies, and globally were 75.27%, 98.55%, 83.73%, 67.46%, 69.62%, 73.75%, 86.59%, 90.66%, 94.22%, 91.72%, 66.72%, 83.45%, 68.85%, 86.21%, 72.98%, and 73.60%, respectively.

[0145]

[0146] a Projected population coverage.

[0147] bAverage number of epitope hits / HLA combinations recognized by the population.

[0148] c Minimum number of epitope hits / HLA combinations recognized by 90% of the population.

[0149] The novel tuberculosis polypeptide fusion protein constructed in this invention consists of four parts (HBD-3, PADRE, multi-epitope fusion protein, and PSMα4), named HP13138PB. Figure 4 ).

[0150] First, the selected 34 epitopes were linked using amino acid linkers (GPGPG, AAY, KK). Specifically, in this embodiment, 13 HTL epitopes were linked using GGPG amino acid linkers to obtain tandem HTL epitopes (amino acid sequences of SEQ ID No. 1, positions 75-340); 13 CTL epitopes were linked using AAY amino acid linkers to obtain tandem CTL epitopes (amino acid sequences of SEQ ID No. 1, positions 344-498); and 8 B-cell epitopes were linked using KK amino acid linkers to obtain tandem B-cell epitopes (amino acid sequences of SEQ ID No. 1, positions 501-674). The tandem HTL, CTL, and B-cell epitopes were then linked using amino acid linkers to obtain a multi-epitope fusion protein. This multi-epitope fusion protein can be used as an active ingredient in the construction of vaccine molecules. Specifically, in this embodiment, the linking method of the multi-epitope fusion protein is as follows:

[0151] Multiepitope fusion protein: tandem HTL epitope - AAY - tandem CTL epitope - KK - tandem B cell epitope.

[0152] Then, the antimicrobial peptide human β-defensin 3 (HBD-3, GIINTLQKYYCRVRGGRCAVLSCLPKEEQIGKCSTRGRKCCRRKK, positions 1-45 of SEQ ID No. 1) and the TLR2 agonist phenol-soluble modulator α4 (PSMα4, MAIVGTIIKIIKAIIDIFAK, positions 680-699 of SEQ ID No. 1) were added as adjuvants to enhance the immunogenicity of the vaccine. Furthermore, the PADRE helper peptide (AGLFQRHGEGTKATVGEPV, positions 51-69 of SEQ ID No. 1) was added after the adjuvant HBD-3 at the amino terminus to enhance the immunogenicity of the vaccine molecule.

[0153] Finally, a 6-His tag was added to the end of the amino acid sequence for protein purification.

[0154] The final constructed peptide fusion protein was named HP13138PB. Figure 4 The amino acid sequence of the polypeptide fusion protein HP13138PB is shown in SEQ ID No. 1, and its encoding gene is named HP13138PB gene. The nucleotide sequence of HP13138PB gene is shown in SEQ ID No. 2.

[0155] Based on this, VaxiJen v2.0, ANTIGENpro, allergtop v.2.0, Allergen FPv.1.0, IEDB immunogenicity server, and Toxin Pred server were used to perform antigenicity, allergenicity, immunogenicity, and toxicity prediction analysis on the constructed polypeptide fusion protein HP13138PB.

[0156] 2. Physicochemical properties and spatial structure analysis of peptide fusion proteins

[0157] The Expasy Protparam server (https: / / web.expasy.org / protparam / ) was used to predict the physicochemical parameters of peptide fusion proteins. It can predict vaccine physicochemical properties such as molecular weight, theoretical pI, amino acid composition, atomic composition, extinction coefficient, estimated half-life, instability index, aliphatic index, and gross hydrophilicity. The Protein-Sol server (https: / / protein-sol.manchester.ac.uk / ) was used to predict the solubility of peptide fusion proteins. Single amino acid sequences obtained from the Protein-Sol server were compared with data in the database. A solubility value greater than 0.45 indicates good solubility of the protein. The PSIPRED server (http: / / bioinf.cs.ucl.ac.uk / psipred / ) was used to construct the secondary structure of the peptide vaccine. It can effectively identify transmembrane topologies, transmembrane helices, folds, and domain recognition. RaptorX Property (http: / / raptorx.uchicago.edu / StructurePropertyPred / predict / ) predicts the secondary structure characteristics of peptide vaccines. This server uses an evolving machine learning model called Deep CNF to continuously calculate secondary structure (SS), disordered regions (DISO), and solvent accessibility (ACC). Secondary structures include α-helices, β-sheets, and random coils. Solvent accessibility is categorized into three states: hidden (below 10%), exposed (above 40%), and medial (between 10% and 40%). Order / disorder predictions are based on a critical value of 0.25.

[0158] The results showed that the HP13138PB vaccine (i.e., the polypeptide fusion protein HP13138PB) consists of 705 amino acids. Analysis using the Expasy Protparam server revealed its relative molecular mass to be 70245.98 Da, theoretical pI to be 9.41, and estimated half-lives to be 30 hours (mammalian reticulocytes, in vitro), 20 hours (yeast, in vivo), and 10 hours (E. coli, in vivo). Furthermore, we found that the instability index, aliphatic index, and grand average of hydropathicity (GRAVY) were 33.20, 79.32, and 0.04, respectively (Table 3). Additionally, the solubility of the HP13138PB vaccine predicted by the Protein-Sol server was 0.55, higher than the average threshold of 0.45. Figure 5 (A) indicates that the HP13138PB vaccine has good solubility.

[0159]

[0160] The secondary structure of HP13138PB is as follows: Figure 5 Studies have shown that α-helices and naturally unfolded protein regions are important types of "structural antigens" that facilitate naturally induced antibody recognition after infection. Results indicate that the HP13138PB vaccine contains 31% α-helices, 11% β-helices, and 56% helices. Furthermore, to support solvent accessibility, it is projected that 55% of amino acid residues will be exposed, 12% will be exposed to culture medium, and 31% will be buried.

[0161] 3. Prediction, optimization, and validation of the tertiary structure of peptide fusion proteins.

[0162] The I-TASSER server automatically searches for molecular structure prediction templates from protein databases using the multi-threaded LOMETS method. Therefore, the I-TASSER server (https: / / zhanggroup.org / / I-TASSER / ) was used to predict the three-dimensional structure of the vaccine. Then, the GalaxyRefine web server (https: / / galaxy.seoklab.org / cgi-bin / submit.cgi?type=REFINE) was used to optimize the tertiary structure of the peptide vaccine (i.e., the polypeptide fusion protein), optimizing the side chains and repackaging them based on previous research. The structure of the peptide vaccine was validated using the ProSA-web server (https: / / prosa.services.came.sbg.ac.at / prosa.php) and the ERRAT server (https: / / saves.mbi.ucla.edu / ) to identify potential errors. The ProSA-web server used a Z-score to indicate potential errors in the protein structure; a Z-score greater than 0 indicates an erroneous or unstable part found in the protein model. In addition, a Ramachandran diagram of the vaccine was generated using the SWISS-MODEL server (https: / / swissmodel.expasy.org / assess). A Ramachandran diagram is a method for visualizing energy-rich regions of amino acid residues in a protein structure relative to the dihedral angles of the backbone.

[0163] In this invention, we used the I-TASSER server to predict five three-dimensional models of peptide fusion proteins, with c-scores of respectively. 1.65 2.82 4.30 3.25 2.38. Generally, the C-score ranges from -2 to 5; the higher the value, the higher the model accuracy. Here, we selected a model with a C-score of -1.65, a TM score of 0.51 ± 0.15, and an expected root mean square deviation (RMSD) of 12.1 ± 4.4A. Figure 6 (A). Subsequently, we used the GalaxyRefine web server to optimize the loop and minimize the energy in the model to improve the consistency of protein modeling. GDT-HA represents the accuracy of building the backbone structure of the model. It represents the various interactions at the atomic level of the model; the smaller the value, the better the quality of the model structure. Therefore, a higher GDT-HA value and a lower MolProbity value indicate better model quality. Therefore, from the 5 optimized models (Table 4), model 4 was selected as the final 3D model of the HP13138PB vaccine. Figure 6 (B)

[0164]

[0165] 4. Conformation B cell epitope prediction

[0166] Conformational epitopes play a crucial role in stimulating immune responses. Constructed protein folding forms conformational B-cell epitopes, with over 90% of B-cell epitopes identified as discontinuous. ElliPro achieved the highest prediction accuracy compared to other structure-based epitope prediction methods, with a receiver operating characteristic (AUC) curve of 0.732, making it the best computational method for any protein. Therefore, we utilized the ElliPro server (http: / / tools.iedb.org / ellipro / ) for conformational B-cell epitope prediction. Our results showed a total of 267 residues distributed across three conformational B-cell epitopes (…). Figure 7 (and Table 5).

[0167]

[0168] Example 3: Molecular docking, dynamic simulation, and immune stimulation simulation analysis of peptide fusion proteins

[0169] 1. Molecular docking and molecular dynamics simulation of peptide fusion protein with Toll-like receptor 2 (TLR2)

[0170] Stable receptor-ligand complexes were obtained through computational molecular docking, and their binding affinity was predicted based on a scoring function. Therefore, the interaction between peptide vaccines and TLRs was evaluated. The protein database (PDB) structure file (PDB ID: 6NIG) for TLR2 was obtained from the NCBI Molecular Modeling Database (MMDB) (https: / / www.ncbi.nlm.nih.gov / structure / ). Molecular docking was then performed using the ClusPro 2.0 server (https: / / cluspro.bu.edu / home.php) to verify the interaction between TLRs and peptide-based vaccines. The server analyzed the molecular docking of peptide vaccines with TLRs using the following three steps: (1) sampling billions of conformations for rigid body docking; (2) clustering the 1000 lowest-energy structures using a root mean square standard deviation (RMSD) clustering method to find the largest cluster; and (3) removing spatial conflicts using energy minimization. Finally, hydrogen bonding and hydrophobic interactions were evaluated using the LigPlot+ program. Our results indicate that molecular docking of the HP13138PB vaccine with TLRs using the ClusPro 2.0 server yielded 30 model complexes. We selected the model with the lowest binding energy between HP13138PB and TLR2 (…). Figure 8 The total energy of HP13138PB vaccine (A) is -1224.7 kcal / mol. Subsequently, we used the LigPlot+ program to demonstrate the hydrophobic interactions between HP13138PB vaccine and TLRs. The results showed that there are 9 hydrogen bonds and 9 hydrophobic interactions between HP13138PB and TLR2 (A). Figure 8 (B)

[0171] Furthermore, molecular dynamics simulations are crucial for determining the stability between the receptor-ligand complex. Simulation predictions can enhance the understanding of the microstructure of the interaction between the vaccine and the TLR. Structural characterization and interaction analysis of the ligand (vaccine) and receptor (TLR2) were performed using Gromacs v5.1.515. All molecular dynamics simulations were conducted under the AMBER99 force field. Simultaneously, energy minimization was performed before simulation to ensure the correct geometry of the system, and the steepest descent algorithm was employed to avoid spatial conflicts. During the equilibrium phase (100 ps), the temperature was increased to 300 K and the pressure reached 1 bar. Our results show that energy minimization is performed in stages within the MD simulation protocol. At 300 Kelvin (constant number, volume, and temperature equilibrium (NVT)) and 1 bar (constant number, volume, and temperature equilibrium (NPT)), protein atoms and solvent molecules equilibrate around the protein molecules for 1 ns. Predictions show that the HP13138PB-TLR2 temperature profile fluctuates between 299 and 301 Kelvin with relatively small amplitudes. Figure 8 (C). The pressure chart results show that the fluctuation value of HP13138PB-TLR2 is 0.68 bar (C). Figure 8 (D).

[0172] 2. Immunosimulation

[0173] Immunological simulations were predicted using the C-ImmSim server (https: / / 150.146.2.1 / C-IMMSIM / index.php). This server can assess the immune response of B and T lymphocytes (including Th1 and Th2 lymphocytes) under simulated vaccine injection conditions. The C-ImmSim server parameters were set as follows: random seed = 12345, simulation volume = 10, simulation steps = 1000, and host alleles HLA-A0101, A0201, B0702, B0801, DRB10101, and DRB1501 were selected. Finally, the cellular immune response and cytokine levels induced by three vaccine injections were predicted.

[0174] This invention discovered that the HP13138PB vaccine activates macrophages to produce phagocytosis, maintaining a total macrophage count of 200 / mm². 3 about( Figure 9 (A). Furthermore, during the three immunization simulation injections, the number of viable macrophages remained at 150 cells / mm². 3 ( Figure 9 (A). Similarly, DC has the strongest antigen delegation capacity, with its total number maintained at 200 / mm. 3 ( Figure 9 (Middle B). T cell immune responses significantly clear MTB, including CD4. +T cells are crucial. Results showed that the total TH cell count significantly increased during the immunosimulation, peaking at the third injection, with peak counts of 12,000 memory and non-memory TH cells / mm². 3 and 95,000 pieces / mm 3 ( Figure 10 (A). Furthermore, at the third injection, the number of active TH cells in each state reached 8000 cells / mm². 3 ( Figure 10 (B) CD8 + T cells can kill MTBs by secreting cytotoxic substances. C-ImmSim server analysis showed that after immunization, the number of non-memory TC cells was 1150 / mm². 3 Reaching peak ( Figure 10 (C). Interestingly, when the number of active TC cells reached 600 / mm², 3 At its peak, the population size of resting TC cells in each state was at its lowest. Figure 10 (D). B cells primarily generate humoral immunity in the body, and the HP13138PB vaccine can activate B cells. We also found that after the third immunization simulation injection, the population per state was higher than 720 cells / mm². 3 ( Figure 10 (E). Furthermore, the H113132 vaccine can induce high levels of IgM and IgG antibodies, with the IgM + IgG antibody titer reaching 700,000 after the third dose. Among them, the IgM titer is higher than 350,000 (…). Figure 10 (Middle F).

[0175] Example 4: Construction of recombinant plasmids for polypeptide fusion proteins and their in vitro expression

[0176] 1. The HBD-3, PADRE, 13 HTL epitopes, 13 CTL epitopes, 8 B-cell epitopes, PSMα4, and 6×His tag described in Example 2 were linked together with linkers such as EAAAK, GGPPG, AAY, and KK to form a polypeptide fusion protein HP13138PB (amino acid sequence as shown in SEQ ID No. 1). Figure 1 The gene sequences corresponding to each part shown are joined together by the gene sequences corresponding to the linkers EAAAK, GGPPG, AAY, and KK to form a complete gene, namely the HP13138PB gene (nucleotide sequence shown in SEQ ID No. 2). BamHI and XhoI recognition sites were added to both ends of the HP13138PB gene (SEQ ID No. 2) to obtain DNA fragment 1 (GGATCC+SEQ ID No. 2+CTCGAG), which was then sent to Shanghai Sangon Biotech for the synthesis of the target gene.

[0177] 2. The artificially synthesized DNA fragment 1 from step 1 was digested with restriction endonucleases BamHI and XhoI, and the digestion products were recovered.

[0178] 3. The vector pET-28a(+) was digested with restriction endonucleases BamHI and XhoI, and the vector backbone was recovered.

[0179] 4. Ligate the enzyme digestion product obtained in step 2 with the vector backbone obtained in step 3 to obtain the recombinant plasmid (i.e., the recombinant vector). Name the recombinant vector pET-28a(+)-HP13138PB. Figure 11 (A)

[0180] 5. The structure of the recombinant vector is described as follows:

[0181] The recombinant vector pET-28a(+)-HP13138PB is obtained by replacing the fragment (small fragment) between the BamHI and XhoI recognition sites of the pET-28a(+) vector with the DNA fragment whose nucleotide sequence is the DNA fragment of SEQ ID No. 2 in the sequence listing, while keeping the other nucleotide sequences of the pET-28a(+) vector unchanged. The recombinant vector pET-28a(+)-HP13138PB expresses the fusion protein HP13138PB with the amino acid sequence shown in SEQ ID No. 1.

[0182] 6. The recombinant vector pET-28a(+)-HP13138PB was introduced into *Escherichia coli* BL21(DE3) to obtain the recombinant bacterium BL21 / pET-28a(+)-HP13138PB. Validation of the recombinant bacterium: The strain was inoculated onto LB solid medium plates (containing 100 ug / ml kanamycin), single colonies were picked and transferred to LB liquid medium, cultured at 37°C, and plasmids were extracted and sequenced. If the extracted plasmid is the recombinant plasmid pET-28a(+)-HP13138PB, it is the target recombinant bacterium.

[0183] 7. Expression of peptide fusion proteins

[0184] The recombinant strain BL21 / pET-28a(+)-HP13138PB was inoculated into LB liquid medium (containing 15 μg / ml kanamycin) and cultured overnight at 37°C and 220 r / min. The next day, it was transferred to LB liquid medium with the same antibiotic concentration at an inoculation rate of 1% (volume percentage) and cultured at 37°C and 220 r / min until OD. 600 When the value is approximately 0.6, IPTG inducer is added to a final concentration of 0.1 mM, and expression is induced overnight at 16℃ and 220 r / min to obtain the fermentation broth.

[0185] 8. Purification of peptide fusion proteins

[0186] (1) Take 100 ml of the fermentation broth from step 7, centrifuge at 5000 rpm for 10 min, and collect the cell precipitate.

[0187] (2) Resuspend the bacterial cells obtained in step (1) in 30 ml of soluble protein lysis buffer, mix by pipetting, and sonicate on ice. The sonication conditions are: 4.5 sec for operation, 9 sec for interval, for a total of 60 min, and the power is 125 W. Centrifuge the sonicated lysate at 12,000 × g for 20 min, discard the supernatant, add 10 ml of inclusion body protein lysis buffer to the precipitate, mix thoroughly by pipetting, and let stand overnight at room temperature.

[0188] (3) The next day, the overnight mixture obtained in step (2) was mixed with 2 ml of Ni-NTA and shaken at 200 rpm for 4 h at room temperature to ensure that the target protein (peptide fusion protein) was fully bound to Ni-NTA. The mixture was then transferred into a purification column and washed 3 times with inclusion body protein washing buffer, 10 ml each time (flow rate controlled at 3 ml / min). Then, it was eluted 5 times with inclusion body protein elution buffer, 500 μl each time (flow rate controlled at 3 ml / min). The collected eluents were combined and the protein concentration was measured to obtain the target protein solution (peptide fusion protein solution).

[0189] 9. Identification of polypeptide fusion proteins

[0190] The peptide fusion protein solution was subjected to 12% polyacrylamide gel electrophoresis, and the results are shown below. Figure 11 The B-type peptide fusion protein solution showed only one band of approximately 73.8 kDa, consistent with expectations.

[0191] Example 5: In vitro experimental verification of HP13138PB vaccine-induced cytokine levels

[0192] In this embodiment, the healthy controls (HC), latent tuberculosis-infected individuals (LTBI), and active tuberculosis patients (ATB) were obtained from the Department of Tuberculosis Medicine, Eighth Medical Center of the PLA General Hospital. The sample collection was approved by the Ethics Committee of the Eighth Medical Center of the PLA General Hospital, approval number: 309202204080808.

[0193] 1. HP13138PB vaccine ELISPOT trial

[0194] Peripheral blood (5 ml) was collected from each of healthy controls (HC, n=21), patients with latent tuberculosis infection (LTBI, n=24), and patients with active tuberculosis (ATB, n=18), and peripheral blood mononuclear cells (PBMCs) were isolated. A portion of the isolated PBMCs was added to 96-well ELISPOT plates (2.5 × 10⁻⁶). 5 Cells / well were stimulated with 50 μl HP13138PB (100 μg / ml), and 50 μl AIM medium was used as a negative control. The culture plates were incubated in a CO2 incubator at 37°C. After 24 h, Human IFN-γ ELISA was used. PRO This kit (MABTECH product, catalog number 3420-2HPT-2) detects positive interferon-gamma (IFN-γ) antibodies. + T cell spot count.

[0195] 2. Detection of Th1 / Th2 / Th17 cytokines induced by HP13138PB vaccine

[0196] Add the remaining PBMCs to a 96-well cell culture plate (2.5 × 10⁻⁶). 5 (Cells / well) (Mabtech AB, NackaStrand, Sweden). PBMCs were stimulated with 50 μl HP13138PB (100 μg / ml) and incubated in a 37°C CO2 incubator for 48 h. Simultaneously, PBMCs stimulated with AIM medium served as a negative control. The PBMC cell culture mixture was transferred to a new tube, centrifuged at 500 g for 10 min, and the supernatant was slowly transferred to another tube. The levels of interleukin-2 (IL-2), IL-4, IL-6, IL-10, IFN-γ, tumor necrosis factor-α (TNF-α), and IL-17A were detected using a human Th1 / Th2 / Th17 cytokine kit (BD Biosciences, catalog number 560484).

[0197] C-ImmSim server results showed that the HP13138PB vaccine significantly stimulated high levels of IFN-γ, TGF-β, IL-2, IL-10, and IL-12, forming three peaks. The highest peak values ​​for IFN-γ and IL-2 were 410,000 ng / ml and 550,000 ng / ml, respectively. Figure 12 To assess the consistency of the HP13138PB vaccine in inducing immune responses in computer simulations and in vitro, we performed ELISPOT and cytokine assays on PBMCs collected from HC, LTBI, and ATB patients. The results showed that HP13138PB vaccine induced IFN-γ levels in PBMCs from HC, LTBI, and ATB patients.+ The number of T cells was significantly higher than that induced by autoinduction medium (AIM). Figure 13 Simultaneously, the serum levels of IFN-γ, IL-2, IL-4, IL-6, IL-10, TNF-α, and IL-17A cytokines were measured in HCs, LTBI-infected individuals, and ATB patients. The results indicated that the HP13138PB vaccine induced IL-4 (…) in ATB patients, HCs, and LTBI-infected individuals. Figure 14 (A), IL-6 ( Figure 14 (B), IL-10 ( Figure 14 (C) and IL17A ( Figure 14 The levels of cytokines induced by HP13138PB vaccine in ATB patients and HCs were significantly higher than those induced by AIM medium in HCs. Figure 14 Furthermore, the HP13138PB vaccine induced significantly higher IFN-γ levels in ATB patients than the AIM medium induced in HC ( ). Figure 14 (Middle F). Regardless of whether HP13138PB stimulation or AIM stimulation was performed, there were no significant differences in IL-2 levels among HC, ATB, LTBI, and the negative control. Figure 14 (G).

[0198] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A fusion protein, characterized in that, The fusion protein is any one of the following: A1) The amino acid sequence is the protein consisting of positions 1-699 of SEQ ID No. 1; A2) A fusion protein with the same function obtained by attaching a tag or signal peptide to the N-terminus and / or C-terminus of A1); A3) The amino acid sequence of the protein is SEQ ID No.

1.

2. A biomaterial, characterized in that, The biomaterial is any one of the following: D1) A nucleic acid molecule encoding the fusion protein of claim 1; D2) An expression cassette containing the nucleic acid molecules described in D1); D3) A recombinant vector containing the nucleic acid molecule described in D1), or a recombinant vector containing the expression cassette described in D2); D4) Recombinant microorganisms containing the nucleic acid molecules described in D1, or recombinant microorganisms containing the expression cassette described in D2), or recombinant microorganisms containing the recombinant vector described in D3); D5) A recombinant host cell containing the nucleic acid molecule described in D1), or a recombinant host cell containing the expression cassette described in D2), or a recombinant host cell containing the recombinant vector described in D3).

3. The biomaterial according to claim 2, characterized in that, D1) The nucleic acid molecule is a DNA molecule whose coding sequence is SEQ ID No. 2 or the first 2097th position of SEQ ID No.

2.

4. The fusion protein of claim 1, and / or any of the following applications of the biomaterial of claim 2 or 3: C1) Its application in the preparation of vaccines to prevent diseases caused by Mycobacterium tuberculosis infection; C2) Application in the preparation of protective antigens against Mycobacterium tuberculosis; C3) Application in screening and / or developing antibodies against Mycobacterium tuberculosis.

5. The method for preparing the fusion protein according to claim 1, characterized in that, The preparation method includes expressing the nucleic acid molecule encoding the fusion protein of claim 1 in a microorganism or host cell to obtain the fusion protein.

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