Polypeptide fusion protein cp13138p and its use in tuberculosis prevention

Abstract

This invention discloses the polypeptide fusion protein CP13138P and its application in tuberculosis prevention. Specifically, it discloses a fusion protein comprising tandem HTL, CTL, and B-cell epitopes, CTB, Pam2Cys, and PADRE. This invention screened 34 epitopes targeting Mycobacterium tuberculosis, which exhibit excellent immunogenicity and antigenicity, are non-toxic and non-sensitizing, and have high population coverage. In vitro experiments demonstrate that CP13138P can stimulate an immune response in human peripheral blood mononuclear cells (PBMCs), increasing IFN-γ in PBMCs. + The number of T lymphocytes is significantly increased, and they secrete significantly high levels of more than a dozen cytokines, including G-CSF, making them a dominant protective antigen. CP13138P, as a vaccine, has advantages such as simple preparation method, low cost, high yield, and enhanced safety. This invention has significant 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 polypeptide fusion protein CP13138P and its application in tuberculosis prevention. Specifically, it relates to the CP13138P recombinant multiepitope antigen derived from Mycobacterium tuberculosis (MTB) protein antigen and its application in the prevention of active and latent tuberculosis infection. Background Technology

[0002] Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis (MTB). It is transmitted through the respiratory tract and can affect various organs, with pulmonary tuberculosis being the most common. Mycobacterium tuberculosis infection is classified into two states: latent tuberculosis infection (LTBI) and active tuberculosis (ATB). MTB, commonly known as tuberclebacterium, belongs to the genus Mycobacterium of the family Mycobacteriaceae in the order Actinobacteriaceae. It is an intracellular parasite that primarily causes long-term infection by attacking macrophages and inhibiting their apoptosis.

[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 Phase 2b clinical trial of the M72 / AS01E vaccine (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), lower than the WHO's 50% protective efficacy threshold. Therefore, accelerating the research and development of new, effective, safe, and more widely applicable tuberculosis vaccines is of great significance for protecting public health and controlling the spread of tuberculosis.

[0004] With the rapid development of immunoinformatics and bioinformatics, peptide vaccines have become one of the most attractive vaccine development strategies. Through low-cost production technologies, peptides identified from Mycobacterium tuberculosis (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 the development time and reducing costs of peptide vaccines, bringing new hope to patients worldwide. 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 CP13138P. 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 134-151, 157-173, 179-191, 197-210, 216-233, 239-255, 261-276, 282-299, 305-320, 326-343, 349-364, 370-383, and 389-404 of SEQ ID No. 1.

[0007] The tandem polypeptide 2 may include a polypeptide whose amino acid sequence is shown in positions 408-416, 420-428, 432-440, 444-452, 456-464, 468-476, 480-488, 492-501, 505-513, 517-525, 529-538, 542-550, and 554-563 of SEQ ID No. 1.

[0008] The tandem polypeptide 3 may include a polypeptide whose amino acid sequence is shown at positions 566-585, 588-607, 610-629, 632-651, 654-673, 676-695, 698-717, and 720-739 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 134-151, 157-173, 179-191, 197-210, 216-233, 239-255, 261-276, 282-299, 305-320, 326-343, 349-364, 370-383, and 389-404 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 134-404 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 408-416, 420-428, 432-440, 444-452, 456-464, 468-476, 480-488, 492-501, 505-513, 517-525, 529-538, 542-550, and 554-563 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 408-563 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 566-585, 588-607, 610-629, 632-651, 654-673, 676-695, 698-717 and 720-739 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 566-739 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 (CTB) with amino acid sequence of positions 1-104 of SEQ ID No. 1, adjuvant peptide 2 (Pam2Cys) with amino acid sequence of positions 745-765 of SEQ ID No. 1, and / or accessory peptide (PADRE) with amino acid sequence of positions 110-128 of SEQ ID No. 1.

[0018] The adjuvant peptide may be a TLR4 agonist cholera toxin subunit B (CTB) or a TLR2 agonist Pam2Cys, and the accessory peptide may be PADRE.

[0019] Specifically, the adjuvant peptide 1 may be CTB, and the adjuvant peptide 2 may be Pam2Cys.

[0020] The amino acid sequence of adjuvant peptide 1 (CTB) may be positions 1-104 of SEQ ID No. 1, the amino acid sequence of adjuvant peptide 2 (Pam2Cys) may be positions 745-765 of SEQ ID No. 1, and the amino acid sequence of accessory peptide (PADRE) may be positions 110-128 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 TLR4 agonist CTB, the helper peptide PADRE, 13 HTL epitopes, 13 CTL epitopes, 8 B-cell epitopes, the TLR2 agonist Pam2Cys, and a 6×His tag.

[0027] Furthermore, the fusion protein CP13138P may be any of the following:

[0028] A1) The amino acid sequence is the protein consisting of positions 1-765 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) by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in positions 1-765 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 from positions 134 to 739 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 134 to 739.

[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] A1) The protein may be a fusion protein that does not include the His tag (i.e.: the adjuvant peptide 1-EAAAK-the accessory peptide-GPGPG-the tandem polypeptide 1-AAY-the tandem polypeptide 2-KK-the tandem polypeptide 3-EAAAK and the adjuvant peptide 2).

[0035] A4) The protein may be a multi-epitope fusion protein (i.e.: the tandem HTL epitope-AAY-the tandem CTL epitope-KK-the tandem B cell epitope).

[0036] The fusion protein described in A3) may be a fusion protein with the same function obtained by attaching a His tag to the C-terminus of A1).

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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, setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing a search to calculate the identity of amino acid sequences, then the identity value (%) can be obtained.

[0041] In this document, the 80% or more 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.

[0042] The present invention also provides a biomaterial, which may be any of the following:

[0043] D1) Nucleic acid molecules of any of the fusion proteins CP13138P described in the encoded text;

[0044] D2) An expression cassette containing the nucleic acid molecules described in D1);

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

[0046] 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);

[0047] 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).

[0048] In the above-mentioned biological materials, the nucleic acid molecule described in D1) can be any of the following:

[0049] B1) The coding sequence is a DNA molecule of SEQ ID No. 2, positions 1-2295 of SEQ ID No. 2, or positions 400-2217 of SEQ ID No. 2;

[0050] B2) The nucleotide sequence is a DNA molecule of SEQ ID No. 2, positions 1-2295 of SEQ ID No. 2, or positions 400-2217 of SEQ ID No. 2.

[0051] 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).

[0052] The DNA molecule shown in SEQ ID No. 2 may be a DNA molecule encoding the fusion protein CP13138P shown in SEQ ID No. 1.

[0053] The DNA molecule shown in positions 1-2295 of SEQ ID No. 2 may be a DNA molecule encoding the fusion protein CP13138P whose amino acid sequence is shown in positions 1-765 of SEQ ID No. 1.

[0054] The DNA molecule shown in positions 400-2217 of SEQ ID No. 2 may be a DNA molecule encoding the fusion protein CP13138P whose amino acid sequence is shown in positions 134-739 of SEQ ID No. 1.

[0055] 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 1-2295 of SEQ ID No. 2, or positions 400-2217 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.

[0056] 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(+).

[0057] The microorganisms described herein may be bacteria, fungi, actinomycetes, protozoa, algae, or viruses. Among them, the bacteria may originate from, but are not limited to, genera such as *Escherichia sp.*, *Erwinia sp.*, *Agrobacterium sp.*, *Flavobacterium sp.*, *Alcaligenes sp.*, *Pseudomonas sp.*, and *Bacillus sp.*, for example, *Escherichia coli*, *Bacillus subtilis*, or *Bacillus pumilus*. In one or more embodiments of the present invention, the microorganism is *Escherichia coli* BL21(DE3).

[0058] 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.

[0059] 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.

[0060] D3) The recombinant vector may be pET-28a(+)-CP13138P.

[0061] The recombinant vector pET-28a(+)-CP13138P 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(+)-CP13138P expresses the fusion protein CP13138P with the amino acid sequence shown in SEQ ID No. 1.

[0062] D4) The recombinant microorganism may be BL21 / pET-28a(+)-CP13138P. BL21 / pET-28a(+)-CP13138P is a recombinant microorganism obtained by introducing the recombinant vector pET-28a(+)-CP13138P into Escherichia coli BL21(DE3).

[0063] 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.

[0064] This invention also provides any of the fusion proteins described herein, and / or any of the following applications of the biomaterial:

[0065] C1) Use in the preparation of products for the prevention and / or treatment of diseases caused by Mycobacterium tuberculosis infection;

[0066] C2) Application in the preparation of vaccines to prevent diseases caused by Mycobacterium tuberculosis infection;

[0067] C3) Application in the preparation of protective antigens against Mycobacterium tuberculosis;

[0068] Application of C4 in screening and / or developing antibodies against Mycobacterium tuberculosis.

[0069] Furthermore, the product may be a reagent or a drug.

[0070] The protective antigen refers to the antigenic component of Mycobacterium tuberculosis that can stimulate the body to produce a protective immune response.

[0071] 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 minimal recognition units (MRUs).

[0072] 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.

[0073] Furthermore, the product may be a vaccine or a pharmaceutical composition.

[0074] The vaccine can be used to prevent Mycobacterium tuberculosis infection.

[0075] The active ingredient of the vaccine may include any of the fusion proteins described herein.

[0076] The vaccine may also include an adjuvant and / or a vaccine delivery system.

[0077] 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.

[0078] 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.

[0079] The active ingredient of the pharmaceutical composition may include any of the fusion proteins described herein.

[0080] 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.

[0081] Furthermore, the disease caused by Mycobacterium tuberculosis infection described in this article can be tuberculosis.

[0082] Furthermore, the tuberculosis may include active tuberculosis (ATB) and latent tuberculosis infection (LTBI).

[0083] 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.

[0084] Furthermore, the method may include the following steps:

[0085] G1) Construct a recombinant expression vector containing a nucleic acid molecule encoding any of the fusion proteins described herein;

[0086] G2) The recombinant expression vector is introduced into microorganisms to obtain recombinant microorganisms;

[0087] G3) The recombinant microorganisms are cultured, and the fusion protein is obtained by isolation and / or purification;

[0088] Further, the nucleic acid molecule described in G1) may be a DNA molecule as shown in SEQ ID No. 2, positions 1-2295 of SEQ ID No. 2, or positions 400-2217 of SEQ ID No. 2.

[0089] Furthermore, the microorganism may be Escherichia coli BL21(DE3).

[0090] 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 helper peptide PADRE to the epitope vaccine design to further enhance the immunogenicity of the epitope vaccine molecule, and added the TLR4 agonist CTB and the TLR2 agonist Pam2Cys 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 immune stimulation of the vaccine. The results show that the polypeptide fusion protein CP13138P provided by this invention is an epitope vaccine containing 771 amino acids, with a molecular weight of 77.5 kDa, a theoretical PI value of 9.78, an instability index of 31.03 (<40 indicates stability, >40 indicates instability) and an aliphatic amino acid index of 77.37 (which helps improve thermal stability), and a hydrophilicity GRAVY value of 0.048. Furthermore, this invention also found that the CP13138P epitope vaccine has a half-life exceeding 30 hours in mammalian cells, and half-lives exceeding 20 hours and 10 hours in yeast and Escherichia coli, respectively. The binding energies of CP13138P to TLR2 and TLR4 are -1313.3 kcal / mol and -1255.1 kcal / mol, respectively.

[0091] This invention further prepared the fusion protein CP13138P. The consistency between immunoinformatics and real-world experimental results was analyzed using ELISPOT and 35 cytokine detection experiments. Both immunoinformatics and real-world experimental results showed that the peptide fusion protein CP13138P can induce innate and adaptive immune responses characterized by significantly elevated levels of cytokines such as IFN-γ, IL-10, and IL-2. Simultaneously, in vitro experiments demonstrated that the Mycobacterium tuberculosis peptide fusion protein CP13138P 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.

[0092] The polypeptide fusion protein CP13138P of this invention can be prepared through genetic engineering. Using CP13138P as a vaccine offers advantages over bacterial protein vaccines, including simpler preparation methods, lower cost, higher yield, and greater safety. This invention has significant value for the prevention and treatment of active tuberculosis and latent tuberculosis infection. Attached Figure Description

[0093] Figure 1 This refers to the HTL epitope information of the final peptide fusion protein selected in Example 1 for construction.

[0094] Figure 2 This refers to the CTL epitope information selected in Example 1 for the final construction of the polypeptide fusion protein.

[0095] Figure 3 This refers to the B-cell epitope information selected in Example 1 for the final construction of the polypeptide fusion protein.

[0096] Figure 4 This is a schematic diagram illustrating the construction of the CP13138P vaccine.

[0097] Figure 5 The results show the predicted secondary structure of the CP13138P vaccine.

[0098] Figure 6 A schematic diagram of the interaction between the CP13138P vaccine and toll-like receptor 2 (TLR2).

[0099] Figure 7 This is a schematic diagram of the interaction between the CP13138P vaccine and the toll-like receptor 4 (TLR4).

[0100] Figure 8 The results show the predictions of C-ImmSim Server for cytotoxic T (TC) cells, macrophages (MA), dendritic cells (DC), and natural killer (NK) cells.

[0101] Figure 9 The results of C-ImmSim Server's prediction of helper T (TH) cells, PLB cells, B cells, and antibodies.

[0102] Figure 10 A schematic diagram of the construction of the recombinant vector pET-28a(+)-CP13138P.

[0103] Figure 11 The cytokine levels induced by the CP13138P vaccine on C-ImmSim Server are shown. Three injections of the CP13138P vaccine were simulated in C-ImmSim Server, and the levels of cytokines induced by the CP13138P vaccine, including IFN-γ, IL-4, IL-12, TGF-β, TNF-α, IL-10, IL-6, IFN-β, IL-18, IL-23, and IL-2, were analyzed. Cytokine concentrations are expressed in ng / ml.

[0104] Figure 12 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 CP13138P vaccine. IFN-γ was detected using a human ELISPOT kit. + Spot-forming cells (SFCs) of T lymphocytes were analyzed. Unpaired t-tests or Mann-Whitney tests were performed to assess normality. Data are presented as mean + SEM. p < 0.05 was considered statistically significant. SEM values ​​are the standard error of the mean.

[0105] Figure 13The levels of cytokines produced by human peripheral blood mononuclear cells (PBMCs) induced by the CP13138P vaccine were measured using a human 35-cytokine assay kit. The levels of G-CSF, GM-CSF, HGF, IFN-alpha, IFN gamma, IL-1alpha, IL-1beta, IL-10, IL-12p70, IL-13, IL-17F, IL-2, IL-21, IL-22, IL-23, IL-3, IL-31, IL-4, IL-5, IL-6, IL-8, IL-9, IP-10, MCP-1, MCP-3, MIG, MIP-1alpha, MIP-1beta, PD-1, SDF-1alpha, TIM-3, TIMP-1, TNF-α, VEGF-A, and VEGF-R2 cytokines were detected. PBMCs from healthy controls (HC, n=7), latent tuberculosis-infected individuals (LTBI, n=8), and active tuberculosis patients (ATB, n=7) were stimulated in vitro with CP13138P vaccine. PBMCs from HC were stimulated with AIM medium 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 shown as mean + SEM. p < 0.05 was considered statistically significant. SEM, standard error of the mean. Detailed Implementation

[0106] 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.

[0107] 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.

[0108] The Escherichia coli BL21(DE3) competent cells in the following examples were purchased from Shanghai Jingnuo Biotechnology Co., Ltd.

[0109] The carrier pET-28a(+) used in the following examples was purchased from Novagen.

[0110] The preparation methods of the main reagents in the following examples are as follows:

[0111] 1. Preparation of LB liquid culture medium (1000ml):

[0112]

[0113] Add deionized water to a final volume of 1000 ml, then autoclave at 121°C for 15 minutes.

[0114] 2. Preparation of LB solid culture medium (1000ml):

[0115]

[0116]

[0117] Add deionized water to a final volume of 1000 ml, then autoclave at 121°C for 15 minutes.

[0118] 3. Preparation of buffer solution for purification of the soluble expression form of the target protein:

[0119] (1) Soluble protein lysis buffer, pH 8.0 (1000ml):

[0120]

[0121] Dissolve in deionized water and bring the volume to 1000ml. Adjust the pH to 8.0 with NaOH.

[0122] (2) Preparation of buffer solution required for purification of the target protein expressed in inclusion bodies:

[0123] ①Inclusion body protein lysis buffer, pH 8.0 (1000ml):

[0124]

[0125] Dissolve in deionized water and bring the volume to 1000ml. Adjust the pH to 8.0 with NaOH.

[0126] ②Inclusion body protein washing buffer, pH 6.3 (1000ml):

[0127]

[0128] Dissolve in deionized water and bring the volume to 1000ml. Adjust the pH to 6.3 with NaOH.

[0129] ③Inclusion body protein elution buffer, pH 4.5 (1000ml):

[0130]

[0131] Dissolve in deionized water and bring the volume to 1000ml. Adjust the pH to 4.5 with NaOH.

[0132] Example 1: Prediction, screening, and identification of MTB immune dominant epitopes

[0133] 1. HTL epitope prediction and screening

[0134] 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 species were selected; 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 finally selected as candidate epitopes for constructing vaccine molecules. For details on specific epitope sequences, please refer to [link to relevant documentation]. Figure 1 AllerFP* (i.e., Allergen FP v.1.0) is used to predict sensitization. 1 indicates sensitization, and 2 indicates no sensitization. ToxinPred* represents toxicity prediction, with 1 indicating toxicity and 2 indicating no toxicity.

[0135] 2. CTL epitope prediction and screening

[0136] 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 finally selected as candidate epitopes for constructing vaccine molecules. For details on specific epitope sequences, please refer to [link to relevant documentation]. Figure 2 Aller FP* (i.e., AllergenFP v.1.0) is used to predict sensitization. 1 indicates sensitization, and 2 indicates no sensitization. Toxin Pred* represents toxicity prediction, with 1 indicating toxicity and 2 indicating no toxicity.

[0137] 3. Prediction and screening of B-cell epitopes

[0138] 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 selected as candidate epitopes for constructing vaccine molecules. For details regarding specific epitope sequences, please refer to [link to relevant documentation]. Figure 3 Aller FP* (i.e., Allergen FP v.1.0) is used to predict sensitization. 1 indicates sensitization, and 2 indicates no sensitization. Toxin Pred* represents toxicity prediction, with 1 indicating toxicity and 2 indicating no toxicity.

[0139] 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.

[0140] Table 1. The 34 immunodominant epitope sequences finally identified through screening.

[0141]

[0142]

[0143] Example 2: Construction, physicochemical properties, and structural analysis of peptide fusion proteins

[0144] 1. Population coverage and construction of peptide fusion proteins

[0145] 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. These epitopes were used to construct peptide fusion proteins (such as CP13138P; vaccines using CP13138P as the active ingredient are called CP13138P vaccines). 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 frequency database (http: / / www.allelefrequencies.net / ). This database provides allele frequencies for 115 countries and 21 ethnic groups across 16 geographic regions. The biggest difference between epitope vaccines and other traditional vaccines is their MHC restriction. Therefore, the population coverage of epitopes directly determines the global promotion and use of vaccines. The CP13138P vaccine contains 13 HTL epitopes and 13 CTL epitopes with global MHC class I and II allele coverage rates of 87.85% and 91.08%, respectively, and an overall population coverage rate of 98.92% (Table 2). Therefore, the CP13138P vaccine designed in this study has better population applicability.

[0146] Table 2. Population Coverage of Peptide Fusion Proteins

[0147]

[0148] a Projected population coverage.

[0149] bThe average number of epitope hits / HLA combinations recognized by the population.

[0150] c Minimum number of epitope hits / HLA combinations recognized by 90% of the population.

[0151] The novel tuberculosis polypeptide fusion protein constructed in this invention consists of four parts (CTB, PADRE, multi-epitope fusion protein, and Pam2Cys), named CP13138P. Figure 4 ).

[0152] 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 134-404); 13 CTL epitopes were linked using AAY amino acid linkers to obtain tandem CTL epitopes (amino acid sequences of SEQ ID No. 1, positions 408-563); 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 566-739). 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:

[0153] Multiepitope fusion protein: tandem HTL epitope - AAY - tandem CTL epitope - KK - tandem B cell epitope.

[0154] Then, the TLR4 agonist CTB (MTPQNITDLCAEYHNTQIYTLNDKIFSYTESLAGKREMAIITFKNGAIFQVEVPGSQHIDSQKKAIERMKDTLRIAYLTEAKVEKLCVWNNKTPHAIAAISMAN, positions 1-104 of SEQ ID No. 1) and the TLR2 agonist Pam2Cys (FNNFTVSFWLRVPKVSASHLE, positions 745-765 of SEQ ID No. 1) were added as adjuvants to enhance the immunogenicity of the vaccine. Furthermore, the PADRE helper peptide (AGLFQRHGEGTKATVGEPV, positions 110-128 of SEQ ID No. 1) was added after the adjuvant CTB at the amino terminus to enhance the immunogenicity of the vaccine molecule.

[0155] Finally, a 6-His tag was added to the end of the amino acid sequence for protein purification.

[0156] The final constructed peptide fusion protein was named CP13138P. Figure 4 The amino acid sequence of the polypeptide fusion protein CP13138P is shown in SEQ ID No. 1, and its encoding gene is named CP13138P gene. The nucleotide sequence of CP13138P gene is shown in SEQ ID No. 2.

[0157] 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 CP13138P.

[0158] 2. Physicochemical properties and secondary structure analysis of peptide fusion proteins

[0159] 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.

[0160] The results showed that the CP13138P vaccine (i.e., the polypeptide fusion protein CP13138P) consists of 771 amino acids. Analysis using the Expasy Protparam server revealed a relative molecular mass of 77.5 kDa, a theoretical PI value of 9.78, an instability index of 31.03 (<40 indicates stability, >40 indicates instability) and an aliphatic amino acid index of 77.37 (which contributes to improved thermal stability), and a hydrophilicity GRAVY value of 0.048. Furthermore, we found that the CP13138P epitope vaccine has a half-life exceeding 30 hours in mammalian cells, and half-lives exceeding 20 hours and 10 hours in yeast and E. coli, respectively (Table 3). These results indicate that the CP13138P vaccine is a thermostable vaccine molecule that can exist stably in vivo. In addition, the solubility of the CP13138P vaccine predicted by the Protein-Sol server was 0.495, higher than the average threshold of 0.45, indicating that the CP13138P vaccine has good solubility.

[0161] Table 3. Physicochemical properties of peptide fusion protein molecules predicted by Expasy Protparam

[0162]

[0163] The secondary structure of CP13138P 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 CP13138P vaccine contains 48.24% α-helices, 9.1% β-sheets, and 42.66% helices.

[0164] Example 3: Molecular docking, dynamic simulation, and immune stimulation simulation analysis of peptide fusion proteins

[0165] 1. Molecular docking and molecular dynamics simulation of peptide fusion proteins with TLR2 and TLR4

[0166] 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. Protein database (PDB) structure files for TLR2 and TLR4 were 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. To further evaluate the molecular docking of the CP13138P epitope vaccine with TLR2 and TLR4, we constructed 30 model complexes using the ClusPro 2.0 server. The binding energies of these model complexes were analyzed, and the lowest binding energy of the CP13138P-TLR2 complex was -1313.3 kcal / mol. Figure 6 The CP13138P vaccine and TLR2 have 21 hydrogen-bonded binding sites. Figure 6 (B); The lowest binding energy of the CP13138P-TLR4 complex was found to be -1255.1 kcal / mol. Figure 7 The CP13138P vaccine and TLR4 have 15 hydrogen-bonded binding sites. Figure 7 (B)

[0167] 2. Immunosimulation

[0168] Immunological simulations were predicted using the C-ImmSim server (https: / / 150.146.2.1 / C-IMMSIM / index.php). This server can assess the immune responses of innate and adaptive immune cells under simulated vaccine injection conditions. The C-ImmSim server parameters were set as follows: random seed = 20000, simulation volume = 10, simulation steps = 1000, and host alleles HLA-A*02:01, HLA-A*30:01, HLA-B*15:11, HLA-B*15:01, HLA-DRB1*09:01, and HLA-DRB1*15:01 were selected. Finally, the cellular immune responses and cytokine levels induced by three vaccine injections were predicted.

[0169] This invention reveals that the CP13138P vaccine can activate cytotoxic T cells (TCs) to differentiate into non-memory subsets and rapidly increase the number of active TC cells to a peak; it can also activate and maintain the total number of NK cells at 300-370 cells / mm². 3 Approximately three immune responses can induce rapid proliferation of active macrophages and cause three successively decreasing peaks in the number of type 2 macrophages and dendritic cells. Figure 8 Adaptive immune cells, especially CD4+ + T cells play a crucial role in clearing MTB. Results showed that three doses of the CP13138P vaccine induced significantly high levels of B lymphocytes and TH cells, with three progressively increasing peaks in both cell types and their subtypes. Figure 9 Interestingly, we found that the CP13138P vaccine could induce a significantly high level of Th1 cell response. Figure 9 ).

[0170] Example 4: Construction of recombinant plasmids for polypeptide fusion proteins and their in vitro expression

[0171] 1. The CTB, PADRE, 13 HTL epitopes, 13 CTL epitopes, 8 B-cell epitopes, Pam2Cys, 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 CP13138P (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 CP13138P gene (nucleotide sequence shown in SEQ ID No. 2). BamHI and XhoI recognition sites were added to both ends of the CP13138P 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.

[0172] 2. The artificially synthesized DNA fragment 1 from step 1 was digested with restriction endonucleases BamHI and XhoI, and the digestion products were recovered.

[0173] 3. The vector pET-28a(+) was digested with restriction endonucleases BamHI and XhoI, and the vector backbone was recovered.

[0174] 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 this recombinant vector pET-28a(+)-CP13138P( Figure 10 (A)

[0175] 5. The structure of the recombinant vector is described as follows:

[0176] The recombinant vector pET-28a(+)-CP13138P 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(+)-CP13138P expresses the fusion protein CP13138P with the amino acid sequence shown in SEQ ID No. 1.

[0177] 6. The recombinant vector pET-28a(+)-CP13138P was introduced into *Escherichia coli* BL21(DE3) to obtain the recombinant bacterium BL21 / pET-28a(+)-CP13138P. Validation of the recombinant bacterium: The strain was inoculated onto LB solid medium plates (containing 100 μg / 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(+)-CP13138P, it is the target recombinant bacterium.

[0178] 7. Expression of peptide fusion proteins

[0179] The recombinant strain BL21 / pET-28a(+)-CP13138P was inoculated into LB liquid medium (containing 15 μg / ml kanamycin) and cultured overnight at 37°C and 220 rpm. 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 rpm 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 °C and 220 r / min to obtain the fermentation broth.

[0180] 8. Purification of peptide fusion proteins

[0181] (1) Take 100ml of the fermentation broth from step 7, centrifuge at 5000rpm for 10min, and collect the cell precipitate.

[0182] (2) Resuspend the bacterial cells obtained in step (1) in 30 ml of soluble protein lysis buffer, mix well by pipetting, and then sonicate under ice bath conditions: 4.5 sec for operation, 9 sec for interval, for a total of 60 min, with a power of 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.

[0183] (3) The following day, the overnight mixture obtained in step (2) was mixed with 2 ml of Ni-NTA and vortexed 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 three times with inclusion body protein washing buffer, 10 ml each time (flow rate controlled at 3 ml / min). Then, it was eluted five 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).

[0184] 9. Identification of polypeptide fusion proteins

[0185] The peptide fusion protein solution was subjected to 12% polyacrylamide gel electrophoresis, and the results are shown below. Figure 10 The B-type peptide fusion protein solution showed only one band of approximately 77.5 kDa, consistent with expectations.

[0186] Example 5: In vitro experimental verification of cytokine levels induced by CP13138P vaccine

[0187] 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.

[0188] 1. CP13138P vaccine ELISPOT trial

[0189] Peripheral blood (5 ml) was collected from each of healthy controls (HC, n=25), patients with latent tuberculosis infection (LTBI, n=25), 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). 5Cells / well were stimulated with 50 μl CP13138P (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. + The number of T cell spots was measured. The results showed that, compared to AIM culture medium, the CP13138P vaccine molecule was able to stimulate LTBI (P<0.0001). Figure 11 (A) ATB (P = 0.0016) Figure 11 (B) and HC (P<0.0001, Figure 11 IFN-γ in PBMCs from three groups of individuals (C) + The number of T lymphocytes was significantly increased, indicating that the CP13138P epitope vaccine has broad immunogenicity and is the most promising candidate vaccine molecule.

[0190] 2. Detection of 35 cytokines induced by CP13138P vaccine

[0191] Add the remaining PBMCs to a 96-well cell culture plate (2.5 × 10⁻⁶). 5 (Cells / well) (Mabtech AB, Nacka Strand, Sweden). PBMCs were stimulated with 50 μl CP13138P (100 μg / ml) and incubated in a CO2 incubator at 37°C for 48 h. Simultaneously, PBMCs stimulated with AIM medium served as a negative control. Transfer the PBMCs cell culture medium mixture to a new tube, centrifuge at 500g for 10 min, and finally slowly transfer the supernatant to another tube. Use the Human 35 Cytokines Assay Kit (BDBiosciences, catalog number PPX-35) to detect the levels of G-CSF, GM-CSF, HGF, IFN-alpha, IFN gamma, IL-1alpha, IL-1beta, IL-10, IL-12p70, IL-13, IL-17F, IL-2, IL-21, IL-22, IL-23, IL-3, IL-31, IL-4, IL-5, IL-6, IL-8, IL-9, IP-10, MCP-1, MCP-3, MIG, MIP-1alpha, MIP-1beta, PD-1, SDF-1alpha, TIM-3, TIMP-1, TNF alpha, VEGF-A, and VEGF-R2 in the cell culture supernatant.

[0192] Results from the C-ImmSim server showed that the CP13138P vaccine significantly stimulated high levels of IFN-γ, TGF-β, IL-2, IL-10, and IL-18, forming three peaks. The highest peak values ​​for IFN-γ and IL-2 were 450,000 ng / ml and 750,000 ng / ml, respectively. Figure 12 To evaluate the consistency of the CP13138P vaccine in computer simulation and in vitro induced immune responses, we performed in vitro stimulation experiments on PBMCs collected from HCs, LTBI patients, and ATB patients and measured the levels of 35 cytokines in the culture supernatant. The results showed that, compared with the PBS negative control, the CP13138P vaccine stimulated PBMCs from ATB patients, HCs, and / or LTBI-infected individuals to secrete significantly higher levels of cytokines including G-CSF, IFN-γ, IL-1α, IL-1β, IL-10, IL-12p70, IL-13, IL-17F, IL-2, IL-22, IL-4, IL-5, IL-6, PD-1, TNF-α, and MIG. Figure 13 ).

[0193] The present invention has been described in detail above. Those skilled in the art will recognize that 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. While specific embodiments have been provided, 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.

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 1-765 of SEQ ID No. 1; A2) 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 A1).

2. 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 of claim 2, wherein, D1) The nucleic acid molecule is a DNA molecule whose coding sequence is SEQ ID No. 2 or the first 2295th positions 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. Use according to claim 4, characterized in that, The disease caused by Mycobacterium tuberculosis infection is tuberculosis.

6. A product for the prevention of disease caused by infection with Mycobacterium tuberculosis, characterised in that, The product includes the fusion protein of claim 1.

7. The product according to claim 6, characterized in that, The disease caused by Mycobacterium tuberculosis infection is tuberculosis.

8. 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 host cell to obtain the fusion protein.

9. The preparation method according to claim 8, characterized in that, The host cell is a microorganism.

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