Fusion protein pp19128r and its use in preventing or treating mycobacterium tuberculosis infection

Abstract

This invention discloses the fusion protein PP19128R and its application in the prevention or treatment of Mycobacterium tuberculosis infection. Specifically, it discloses a fusion protein comprising tandem HTL, CTL, and B-cell epitopes, PorB, RS-09, and PADRE. This invention screened 39 epitopes targeting Mycobacterium tuberculosis, which exhibit excellent immunogenicity and antigenicity, are non-toxic and non-sensitizing, and have high population coverage. The helper peptide PADRE is further added to enhance the immunogenicity of the vaccine, and PorB and RS-09 are added to endow the vaccine with targeted delivery function. In vitro experiments demonstrate that PP19128R can stimulate an immune response in human peripheral blood mononuclear cells, making it a superior protective antigen. PP19128R 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 fusion protein PP19128R and its application in the prevention or treatment of Mycobacterium tuberculosis infection. Specifically, it relates to the PP19128R 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 primarily transmitted through the respiratory tract, caused by Mycobacterium tuberculosis (MTB). MTB 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 TB cases 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 a 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), below the WHO's 50% protective efficacy threshold. Therefore, researching and developing novel, effective, and safe tuberculosis vaccines is crucial to achieving the goal of "ending the tuberculosis epidemic."

[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 PP19128R. 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 105-119, 125-141, 147-162, 168-185, 191-207, 213-225, 231-246, 252-267, 273-285, 291-304, 310-327, 333-347, 353-370, 376-391, 397-410, 416-432, 438-455, 461-478, and 484-501 of SEQ ID No. 1.

[0007] The tandem polypeptide 2 may include a polypeptide whose amino acid sequence is shown in positions 505-513, 517-525, 529-538, 542-551, 555-563, 567-575, 579-587, 591-599, 603-612, 616-624, 628-636, and 640-648 of SEQ ID No. 1.

[0008] The tandem polypeptide 3 may include a polypeptide whose amino acid sequence is shown at positions 651-683, 686-707, 710-741, 744-768, 771-809, 812-848, 851-878, and 881-912 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 19 HTL epitopes (amino acid sequences of positions 105-119, 125-141, 147-162, 168-185, 191-207, 213-225, 231-246, 252-267, 273-285, 291-304, 31...). The 19 HTL epitopes are obtained by tandemly connecting positions 0-327, 333-347, 353-370, 376-391, 397-410, 416-432, 438-455, 461-478, and 484-501. Specifically, the 19 HTL epitopes can be tandemly connected using amino acid linkers (such as GGPPG), and the amino acid sequence of the tandem polypeptide 1 can specifically be positions 105-501 of SEQ ID No. 1.

[0011] The tandem polypeptide 2 may be a tandem CTL epitope, consisting of 12 CTL epitopes (amino acid sequences of positions 505-513, 517-525, 529-538, 542-551, 555-563, 567-575, 579-587, 591-599, 603-612, 616-624, 628-636, and 640-648 of SEQ ID No. 1). Specifically, the 12 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 505-648 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 651-683, 686-707, 710-741, 744-768, 771-809, 812-848, 851-878 and 881-912 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 651-912 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, GGPPG, tandem polypeptide 2, AAY, 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 1-75; adjuvant peptide 2 whose amino acid sequence is SEQ ID No. 1, which is positions 918-924; and / or accessory peptide whose amino acid sequence is SEQ ID No. 1, which is positions 81-99.

[0018] The adjuvant peptide may be a TLR-2 agonist porin B (PorB) or a TLR-4 agonist RS-09, and the accessory peptide may be PADRE.

[0019] Specifically, the adjuvant peptide 1 may be PorB, and the adjuvant peptide 2 may be RS-09.

[0020] The amino acid sequence of adjuvant peptide 1 (PorB) may be positions 1-75 of SEQ ID No. 1, the amino acid sequence of adjuvant peptide 2 (RS-09) may be positions 918-924 of SEQ ID No. 1, and the amino acid sequence of accessory peptide (PADRE) may be positions 81-99 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 without changing the function of the fusion protein 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 PorB, the helper peptide PADRE, 19 HTL epitopes, 12 CTL epitopes, 8 B-cell epitopes, RS-09, and a 6×His tag.

[0027] Furthermore, the fusion protein PP19128R may be any of the following:

[0028] A1) The amino acid sequence is the protein of positions 105-912 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 105-912 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 1-924 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 A4) obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in positions 1-924 of SEQ ID No. 1.

[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] The A1) may be an antigenic fusion protein (positions 105-912 of SEQ ID No. 1) formed by linking tandem polypeptide 1 (including 19 tandem HTL epitopes), tandem polypeptide 2 (including 12 tandem CTL epitopes), and tandem polypeptide 3 (including 8 tandem B cell epitopes) through an amino acid linker.

[0035] A4) may be a fusion protein (positions 1-924 of SEQ ID No. 1) obtained by fusing adjuvant peptide 1 (PorB) and accessory peptide (PADRE) at the N-terminus of A1) and adjuvant peptide 2 (RS-09) at the C-terminus to enhance immunogenicity.

[0036] Furthermore, the fusion protein described in A6) can be a fusion protein with the same function obtained by attaching a His tag to the C-terminus of A4).

[0037] Further, A6) The fusion protein includes 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 PP19128R 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 whose coding sequence is SEQ ID No. 2, positions 313-2736 of SEQ ID No. 2, or positions 1-2772 of SEQ ID No. 2;

[0050] B2) The nucleotide sequence is the DNA molecule of SEQ ID No. 2, positions 313-2736 of SEQ ID No. 2, or positions 1-2772 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 PP19128R shown in SEQ ID No. 1.

[0053] The DNA molecule shown in positions 313-2736 of SEQ ID No. 2 may be a DNA molecule encoding the fusion protein PP19128R, whose amino acid sequence is shown in positions 105-912 of SEQ ID No. 1.

[0054] The DNA molecule shown in positions 1-2772 of SEQ ID No.2 may be a DNA molecule encoding the fusion protein PP19128R whose amino acid sequence is shown in positions 1-924 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 313-2736 of SEQ ID No. 2, or positions 1-2772 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(+)-PP19128R.

[0061] The recombinant vector pET-28a(+)-PP19128R 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(+)-PP19128R expresses the fusion protein PP19128R with the amino acid sequence shown in SEQ ID No. 1.

[0062] D4) The recombinant microorganism may be BL21 / pET-28a(+)-PP19128R. BL21 / pET-28a(+)-PP19128R is a recombinant microorganism obtained by introducing the recombinant vector pET-28a(+)-PP19128R 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 for any of the fusion proteins described herein, or any of the following applications of the biomaterials described herein:

[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 including 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 313-2736 of SEQ ID No. 2, or positions 1-2772 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 against 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 TLR2 agonist PorB and the TLR4 agonist RS-09 to endow the vaccine molecule with targeted delivery function and enhance its immunogenicity. Furthermore, immunoinformatics tools were used to predict and analyze the antigenicity, immunogenicity, physicochemical parameters, secondary structure, tertiary structure, and immunostimulation of the vaccine. The results show that the polypeptide fusion protein PP19128R provided by this invention has an antigenicity of 0.8067, an immunogenicity of 9.29811, and a solubility index of 0.900675. Its global population coverage reached 82.24% and 93.71% in Class I and Class II, respectively. Secondary structure prediction showed that PP19128R has an α-helix composition of 39.46%, a β-sheet composition of 11.61%, and a helix composition of 48.92%. Tertiary structure analysis showed that the Z-score and favorite region of PP19128R were -6.28 and 87.2%, respectively. The binding energies of PP19128R with TLR2 and TLR4 were -1324.77 kcal / mol and -1278 kcal / mol, respectively.

[0091] This invention further prepared the fusion protein PP19128R. 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 PP19128R can induce innate and adaptive immune responses characterized by significantly elevated levels of cytokines such as IFN-γ, TNF-α, IL-4, IL-6, IL-10, and IL-17A. Simultaneously, in vitro experiments demonstrated that the Mycobacterium tuberculosis peptide fusion protein PP19128R 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 PP19128R of this invention can be prepared through genetic engineering. Using PP19128R 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 diagram illustrates the construction of the PP19128R vaccine and shows the predicted secondary structure. Figure 4 B represents the amino acid sequence of the fusion protein HP19128R.

[0097] Figure 5 Z-score, Ramachandran plot and 3D model of PP19128R vaccine.

[0098] Figure 6 This is a schematic diagram of the interaction between the PP19128R vaccine and the toll-like receptor 2 (TLR2).

[0099] Figure 7 This is a schematic diagram of the interaction between the PP19128R vaccine and the toll-like receptor 4 (TLR4).

[0100] Figure 8 The results of C-ImmSim Server predictions for natural killer cells (NK), macrophages (MA), dendritic cells (DC), epithelial cells, and B cells induced by the PP19128R vaccine.

[0101] Figure 9 The results of C-ImmSim Server prediction of helper T (TH) cells, cytotoxic T (TC) cells, B cells and antibodies induced by PP19128R vaccine are presented.

[0102] Figure 10 The cytokine levels induced by the PP19128R vaccine on C-ImmSim Server are shown. Three injections of the PP19128R 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 cytokine induced by the PP19128R vaccine were analyzed. Cytokine concentrations are expressed in ng / ml.

[0103] Figure 11A schematic diagram of the construction of the recombinant vector pET-28a(+)-PP19128R and the SDS-PAGE purification of the vaccine molecule after in vitro cloning and expression.

[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 the PP19128R vaccine. IFN-γ was detected using the 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 13 The levels of cytokines produced by human peripheral blood mononuclear cells (PBMCs) induced by the PP19128R vaccine were measured. The levels of interleukin-2 (IL-2, A), tumor necrosis factor-α (TNF-α, B), interferon-γ (IFN-γ, C), IL-10 (D), IL-4 (E), IL-6 (F), and IL-17A (G) were detected using a human Th1 / Th2 / Th17 cytokine assay kit. PBMCs from healthy controls (HC), latent tuberculosis-infected individuals (LTBI), and active tuberculosis patients (ATB) were stimulated in vitro with the PP19128R 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 presented as mean + SEM. p < 0.05 was considered statistically significant. SEM, standard error of the mean.

[0106] Figure 14 Principal component analysis and correlation heat analysis were performed on seven cytokines (IL-2, TNF-α, IFN-γ, IL-10, IL-4, IL-6 and IL-17A) induced by the PP19128R vaccine in three population groups (HC, ATB, LTBI).

[0107] Figure 15 Simple linear regression analysis was performed to determine the significant correlation of seven cytokines (IL-2, TNF-α, IFN-γ, IL-10, IL-4, IL-6, and IL-17A) induced by the PP19128R vaccine in the LTBI population.

[0108] Figure 16Simple linear regression analysis was performed to determine the significant correlation of seven cytokines (IL-2, TNF-α, IFN-γ, IL-10, IL-4, IL-6, and IL-17A) induced by the PP19128R vaccine in the ATB population. Detailed Implementation

[0109] 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.

[0110] 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.

[0111] The Escherichia coli BL21(DE3) competent cells in the following examples were purchased from Shanghai Jingnuo Biotechnology Co., Ltd.

[0112] The carrier pET-28a(+) used in the following examples was purchased from Novagen.

[0113] The preparation methods of the main reagents in the following examples are as follows:

[0114] 1. Preparation of LB liquid culture medium (1000ml):

[0115]

[0116] Add deionized water to a final volume of 1000 ml, then autoclave at 121°C for 15 minutes.

[0117] 2. Preparation of LB solid culture medium (1000ml):

[0118]

[0119] Add deionized water to a final volume of 1000 ml, then autoclave at 121°C for 15 minutes.

[0120] 3. Preparation of buffer solution for purification of the soluble expression form of the target protein:

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

[0122]

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

[0124] (2) Preparation of buffer solution required for purification of the target protein expressed in inclusion bodies:

[0125] ①Inclusion body protein lysis buffer, pH 8.0 (1000ml):

[0126]

[0127] Dissolve in deionized water and bring the volume to 1000ml. Adjust the pH to 8.0 with NaOH.

[0128] ②Inclusion body protein washing buffer, pH 6.3 (1000ml):

[0129]

[0130] Dissolve in deionized water and bring the volume to 1000ml. Adjust the pH to 6.3 with NaOH.

[0131] ③Inclusion body protein elution buffer, pH 4.5 (1000ml):

[0132]

[0133] Dissolve in deionized water and bring the volume to 1000ml. Adjust the pH to 4.5 with NaOH.

[0134] The nucleotide sequence (SEQ ID No. 2) of the PP19128R gene involved in the following examples is shown below:

[0135]

[0136] Example 1: Prediction, screening, and identification of dominant immune epitopes

[0137] 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. Epitope vaccines utilize genetic engineering to express or artificially synthesize antigenic epitopes of pathogenic microorganisms in vitro, and then use them as vaccines. The key to epitope vaccine design is epitope screening. In this embodiment, 18 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.

[0138] 1. Selection of antigens

[0139] 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. Furthermore, 10 antigens have been used in protective studies in animal models. The remaining 19 antigens also induced strong immune responses. Therefore, we selected 18 antigens for epitope prediction and screening. The 18 candidate antigens are Rv1736c, Rv1737c, Rv1980c, Rv1981c, Rv2659c, Rv3429, Rv3873, Rv3879, Rv2031c, Rv2626c, Rv2656c, Rv2659c, Rv1511, Rv3872, Rv3425, Rv3878, Rv2660c, and Rv2653c.

[0140] 2. HTL epitope prediction and screening

[0141] 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, 19 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 AllerTOPv.2.0* and Aller FP* (i.e., Allergen FP v.1.0*) are used to predict sensitization. 1 indicates sensitization, and 2 indicates no sensitization.

[0142] 2. CTL epitope prediction and screening

[0143] 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, 12 CTL immunogenicity 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 Aller FP* (i.e., Allergen FP v.1.0*) are used to predict sensitization. 1 indicates sensitization, and 2 indicates no sensitization.

[0144] 3. Prediction and screening of B-cell epitopes

[0145] 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 .

[0146] Finally, 19 HTL epitopes, 12 CTL epitopes, and 8 B-cell epitopes were identified for constructing the active ingredient (peptide fusion protein) of the vaccine molecule, totaling 39 epitopes. The amino acid sequences of the 39 epitopes are shown in Table 1.

[0147] Table 1. Amino acid sequences of the 39 immunodominant epitopes finally identified through screening.

[0148] Epitope Sequence (5'-3') Serial Number HTL tabletop GAAFSWYTYSPTRVR SEQ ID No. 1, bits 105-119 HTL tabletop DFLPVVLEFAATVDPEA Positions 125-141 of SEQ ID No. 1 HTL tabletop LYYQIHVLIGLALFAL SEQ ID No. 1, bits 147-162 HTL tabletop LYESRLLRIASPMFHFGI SEQ ID No. 1, bits 168-185 HTL tabletop MMGELFWTVVPYVTMTI SEQ ID No. 1, bits 191-207 HTL tabletop AVTLASILPVLAV SEQ ID No. 1, bits 213-225 HTL tabletop ATMGSYALLVFFGLFL SEQ ID No. 1, bits 231-246 HTL tabletop PAYNINISLPSYYPDQ SEQ ID No. 1, bits 252-267 HTL tabletop FLFYSGFYLPMYW SEQ ID No. 1, bits 273-285 HTL tabletop SFLFYSGFYLPMYW Positions 291-304 of SEQ ID No. 1 HTL tabletop DLRVHDLRHSGAVLAAST SEQ ID No. 1, bits 310-327 HTL tabletop QSTARFILAYLPRWQ SEQ ID No. 1, bits 333-347 HTL tabletop AAAEQLRLMYNSANMTAK SEQ ID No. 1, bits 353-370 HTL tabletop VAPSVMPAAAAGSSAT Positions 376-391 of SEQ ID No. 1 HTL tabletop GLSAAAAKLAGLVF SEQ ID No. 1, bits 397-410 HTL tabletop DTGAGARPAASPLAAPV SEQ ID No. 1, bits 416-432 HTL tabletop TGREAAHLRAFRAYAAHS Positions 438-455 of SEQ ID No. 1 HTL tabletop VTPAAASGVPGARAAAAA SEQ ID No. 1, bits 461-478 HTL tabletop YPVLAVQAWAAFHDMTLR Positions 484-501 of SEQ ID No. 1 CTL tabletop ELFWTVVPY Positions 505-513 of SEQ ID No. 1 CTL tabletop GVAGTIFAV Positions 517-525 of SEQ ID No. 1 CTL tabletop EMKEGRYEVR Positions 529-538 of SEQ ID No. 1 CTL tabletop NVMEEHQVRR Positions 542-551 of SEQ ID No. 1 CTL tabletop RRDAYIRRV Positions 555-563 of SEQ ID No. 1 CTL tabletop ASYTGPDGR Bits 567-575 of SEQ ID No. 1 CTL tabletop LRPTEVDSL Positions 579-587 of SEQ ID No. 1 CTL tabletop GGTHPTTTY Positions 591-599 of SEQ ID No. 1 CTL tabletop EVSAQAATAF Bits 603-612 of SEQ ID No. 1 CTL tabletop EIAANREER Bits 616-624 of SEQ ID No. 1 CTL tabletop ETAAELAPR Bits 628-636 of SEQ ID No.1 CTL tabletop ATHGANVSL Bits 640-648 of SEQ ID No. 1 B-cell epitopes LIRRASTFNTSRIDHLYVDPHQPGARLFLHYGD Positions 651-683 of SEQ ID No. 1 B-cell epitopes LRDAPYFRPNADPVLPRLKAAA Bits 686-707 of SEQ ID No. 1 B-cell epitopes TLCSTKQIDDAFDWSEQNPYLQRKAQIIVDYY Positions 710-741 of SEQ ID No. 1 B-cell epitopes LEDEMKEGRYEVRAELPGVDPDKDV SEQ ID No. 1, bits 744-768 B-cell epitopes GVGTEQRNLSVVAPSQFTFSSRSPDFVDETAGQSWCAIL SEQ ID No. 1, bits 771-809 B-cell epitopes AGLNAPRRNRVGRQHGWPADVPSAEQRRAQRQRDLEA bits 812-848 of SEQ ID No. 1 B-cell epitopes GLANAYNDTRRKVVPPEEIAANREERRR Positions 851-878 of SEQ ID No. 1 B-cell epitopes TDQRLLDLLPPAPVDVNPPGDERHMLWFELMK Positions 881-912 of SEQ ID No. 1

[0149] Example 2: Construction, physicochemical properties, and structural analysis of peptide fusion proteins

[0150] 1. Population coverage and construction of peptide fusion proteins

[0151] Based on the HTL, CTL, and B-cell epitopes predicted and screened using the aforementioned bioinformatics tools, the following 39 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 exhibiting no toxicity or sensitization; and the B-cell epitope with the highest predicted score (in total). These epitopes were then used to construct a polypeptide fusion protein (e.g., PP19128R). 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 across 16 geographic regions. The analysis results showed that the CTL epitope (Class I) coverage rates of the PP19128R vaccine (a vaccine with the polypeptide fusion protein PP19128R as the active ingredient) in populations in Central Africa, East Africa, East Asia, Europe, North Africa, North America, Northeast Asia, Oceania, South America, South Asia, Southeast Asia, Southwest Asia, West Africa, and globally were 57.38%, 61.10%, 85.63%, 82.00%, 65.52%, 82.17%, 92.53%, 76.33%, 65.56%, 70.59%, 91.15%, 64.29%, 59.42%, and 82.24%, respectively. Similarly, the HTL epitope (Class II) coverage rates of the PP19128R vaccine in populations in Central Africa, East Africa, East Asia, Europe, North Africa, North America, Northeast Asia, Oceania, South America, South Asia, Southeast Asia, Southwest Asia, West Africa, and globally were 82.75%, 89.22%, 73.85%, 99.06%, 80.01%, 99.93%, 97.97%, 97.55%, 98.12%, 97.75%, 88.86%, 83.80%, 88.35%, and 93.71%, respectively.

[0152] The novel tuberculosis polypeptide fusion protein constructed in this invention comprises four parts (PorB, PADRE, multi-epitope fusion protein, and RS-09), named PP19128R. Figure 4 ).

[0153] First, the selected 39 epitopes were linked using amino acid linkers (GPGPG, AAY, KK). Specifically, in this embodiment, 19 HTL epitopes were linked together using GGPPG amino acid linkers to obtain tandem HTL epitopes (amino acid sequences of SEQ ID No. 1, positions 105-501); 12 CTL epitopes were linked together using AAY amino acid linkers to obtain tandem CTL epitopes (amino acid sequences of SEQ ID No. 1, positions 505-648); and 8 B-cell epitopes were linked together using KK amino acid linkers to obtain tandem B-cell epitopes (amino acid sequences of SEQ ID No. 1, positions 651-912). The tandem HTL, CTL, and B-cell epitopes were then linked together 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:

[0154] Multiepitope fusion protein: tandem HTL epitope - AAY - tandem CTL epitope - KK - tandem B cell epitope.

[0155] Then, the TLR2 agonist PorB (IALTLAALPVAAMADVTLYGTIKAGVETSRSVAHNGAQAASVETGTGIVDLGSKIGFKGQEDLGNGLKAIWQVEQ, positions 1-75 of SEQ ID No. 1) and the TLR4 agonist RS-09 (APPHALS, positions 918-924 of SEQ ID No. 1) were added to the initiation (amino terminus) and termination (carboxyl terminus of SEQ ID No. 1), respectively, as adjuvants to endow the vaccine molecule with targeted delivery capabilities and enhance its immunogenicity. Furthermore, the PADRE helper peptide (AGLFQRHGEGTKATVGEPV, positions 81-99 of SEQ ID No. 1) was added after the N-terminal adjuvant PorB to further enhance the immunogenicity of the epitope vaccine molecule.

[0156] Finally, to facilitate protein purification, a His tag (6×His) was added to the C-terminus of the above multi-epitope fusion protein.

[0157] The final constructed peptide fusion protein was named PP19128R. Figure 4 The amino acid sequence of the polypeptide fusion protein PP19128R is shown in SEQ ID No. 1. Figure 4 The gene encoding PP19128R is shown in SEQ ID No. 2.

[0158] Based on this, the antigenicity, allergenicity, immunogenicity, and toxicity prediction of the constructed polypeptide fusion protein PP19128R were analyzed using VaxiJen v2.0, ANTIGENpro, allergtop v.2.0, Allergen FPv.1.0, IEDB immunogenicity server, and Toxin Pred server. The results showed that the PP19128R vaccine (i.e., the polypeptide fusion protein PP19128R) consists of 930 amino acids. Analysis using the Expasy Protparam server revealed its relative molecular mass to be 98557.86 Da, its theoretical pI to be 9.20, and its estimated half-life to be 20 hours (mammalian reticulocytes, in vitro), 30 minutes (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, with an antigenicity of 0.8067 and an immunogenicity of 9.29811. Additionally, the solubility of the PP19128R vaccine predicted by the Protein-Sol server was 0.900675, higher than the average threshold of 0.45, indicating that the PP19128R vaccine has good solubility.

[0159] 2. Secondary and tertiary spatial structure analysis of peptide fusion proteins

[0160] 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), disorder 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 prediction is based on a critical value of 0.25. Secondary structure predictions show that PP19128R has 39.46% α-helices, 11.61% β-sheets, and 48.92% helices. Tertiary structure analysis shows that PP19128R has a Z-score of -6.28 and a favorite region of 87.2%.

[0161] 3. 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. Furthermore, 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, the 3D model of the PP19128R vaccine was validated using the ProSA-web server and UCLA-DOE LAB SAVES v6.0 software. The z-score of the PP19128R vaccine predicted by the ProSA-web server was -5.59 before optimization. Figure 5 (A), after optimization, is -6.28 ( Figure 5 (B). Furthermore, the Ramachandran plot generated by UCLA-DOE LAB SAVES v6.0 suggests that the candidate vaccine models comprise 70.8% core, 22.8% allow, 4.4% generic, and 2.0% disall. Figure 5 (C) Interestingly, after optimization, these figures changed to 87.2% cores, 9.0% permissions, 1.6% builds, and 2.2% disables. Figure 5 (D). Furthermore, the maximum deviation rate of amino acid residues in the PP19128R vaccine decreased from 23.2% to 18.8%.

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

[0165] 1. Molecular docking and molecular dynamics simulation of peptide fusion proteins with Toll-like receptors 2 (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. The TLR2 protein database (PDB) structure file (PDB ID: 6NIG) 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 based on the 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. The results showed that molecular docking of the PP19128R vaccine with TLRs using the ClusPro 2.0 server generated 30 model complexes. Analysis of the binding energies of these model complexes revealed that the optimal PP19128R-TLR2 complex, with the lowest binding energy, was -1324.77 kcal / mol. Figure 6 (A). Furthermore, we explored potential binding sites between PP19128R and TLR2, discovering 17 hydrogen-bonded binding sites between the PP19128R vaccine and TLR2. Figure 6 (B) Similarly, we selected the PP19128R-TLR4 complex model with the lowest binding energy for further analysis. The binding energy of the PP19128R-TLR2 complex is -1278 kcal / mol ( Figure 7 The PP19128R vaccine has 10 hydrogen-bonded binding sites with TLR2 (A). Figure 7 (B)

[0167] 2. Immunosimulation

[0168] Both specific and non-specific immune responses play crucial roles in the host's clearance and eradication of Mycobacterium tuberculosis. Therefore, this invention simulates and analyzes the immune response induced by the PP19128R vaccine molecule. The immune simulation was 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.

[0169] This invention has found that the PP19128R vaccine can activate NK cells and maintain their number at 310-380 cells / mm². 3 between( Figure 8 (A). Interestingly, the PP19128R vaccine can activate the proliferation and differentiation of macrophages and dendritic cells; all three simulated immunizations can induce presenting-2 type macrophages (A). Figure 8 The middle B cells and dendritic cells form three proliferation peaks ( Figure 8 (C). Unlike dendritic cells, the number of resting and active macrophages remained stable at 90 cells / mm² within 90 days after the first simulated immunization with the PP19128R vaccine. 3 However, after day 90, the number of resting macrophages increased sharply while the number of active macrophages decreased rapidly, eventually stabilizing at 15 cells / mm. 3 ( Figure 8 (B). The PP19128R vaccine can induce significantly high levels of epithelial cells (B). Figure 8 (Middle D). Similar to macrophages and dendritic cells, the PP19128R vaccine can stimulate B lymphocyte differentiation and proliferation, resulting in a higher number of presenting-1 and active B lymphocytes (600 cells / mm²) on day 40 after the first mimic immunization. 3 ), Day 70 (710 cells / mm) 3 ) and day 110 (470 cells / mm 3 Three peaks appeared respectively. Figure 8 (E). Furthermore, we observed a similar trend in immunoglobulins and immune complexes (E). Figure 8 (F).

[0170] In addition, we analyzed the immunomodulatory effects of the PP19128R vaccine on specific immune cells. The results showed that PP19128R vaccine-simulated immunization induced three peaks in the number of memory helper T lymphocytes, with the peak reaching 12,000 cells / mm² after the third immunization. 3 ( Figure 9 (A). Furthermore, we found that PP19128R mimic injection stimulated three peaks in the number of active helper T lymphocytes on days 20, 40, and 75 after the initial immunization. Figure 9 (B) Unlike helper T lymphocytes, PP19128R has a weaker ability to stimulate the immune system to form memory cytotoxic T lymphocytes. Figure 9 In the middle C), the number of active cytotoxic T lymphocytes peaked at 900 cells / mm² on day 50 after the first immunization. 3 ), and then gradually decreased; while resting cytotoxic T lymphocytes showed the completely opposite trend. Figure 9 Excitingly, we found that PP19128R can induce T lymphocytes to differentiate into Th1 lymphocytes, mediating a robust Th1 immune response. Figure 9 (Middle E). Furthermore, we observed that PP19128R could induce a rapid peak in regulatory T cells (155 cells / mm²) after initial immunization. 3 ), and then gradually decrease ( Figure 9 (F). Finally, we also analyzed the ability of PP19128R to induce IFN-γ production by immune cells. The results showed that three immunizations with PP19128R could induce three peaks in cytokines IFN-γ (410,000 ng / ml, 400,000 ng / ml and 380,000 ng / ml) and IL-2 (200,000 ng / ml, 690,000 ng / ml and 480,000 ng / ml). Figure 10 ).

[0171] Example 4: Construction of recombinant plasmids for polypeptide fusion proteins and their in vitro expression

[0172] 1. The PorB, PADRE, 19 HTL epitopes, 12 CTL epitopes, 8 B-cell epitopes, RS-09, 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 PP19128R (amino acid sequence as shown in SEQ ID No. 1). Figure 4The gene sequences corresponding to each part shown in Figure A are joined together by the gene sequences corresponding to the linkers EAAAK, GGPPG, AAY, and KK to form a complete gene, namely the PP19128R gene (nucleotide sequence as shown in SEQ ID No. 2). BamHI and XhoI recognition sites are added to both ends of the PP19128R gene (SEQ ID No. 2) to obtain DNA fragment 1 (GGATCC+SEQ ID No. 2+CTCGAG), which is then sent to Shanghai Sangon Biotech for the synthesis of the target gene.

[0173] 2. The artificially synthesized DNA fragment 1 from step 1 was digested with restriction endonucleases BamHI and XhoI, and the digestion products were recovered.

[0174] 3. The vector pET-28a(+) was digested with restriction endonucleases BamHI and XhoI, and the vector backbone was recovered.

[0175] 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(+)-PP19128R( Figure 11 (A)

[0176] 5. The structure of the recombinant vector is described as follows:

[0177] The recombinant vector pET-28a(+)-PP19128R 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(+)-PP19128R expresses the fusion protein PP19128R with the amino acid sequence shown in SEQ ID No. 1.

[0178] 6. The recombinant vector pET-28a(+)-PP19128R was introduced into *Escherichia coli* BL21(DE3) to obtain the recombinant bacterium BL21 / pET-28a(+)-PP19128R. 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(+)-PP19128R, it is the target recombinant bacterium.

[0179] 7. Expression of peptide fusion proteins

[0180] The recombinant strain BL21 / pET-28a(+)-PP19128R 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.

[0181] 8. Purification of peptide fusion proteins

[0182] (1) Take 100ml of the fermentation broth from step 7, centrifuge at 5000rpm for 10min, and collect the cell precipitate.

[0183] (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.

[0184] (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).

[0185] 9. Identification of polypeptide fusion proteins

[0186] 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 98.57 kDa, consistent with expectations.

[0187] Example 5: In vitro experimental verification of the cellular immune response induced by PP19128R vaccine

[0188] 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.

[0189] 1. PP19128R vaccine ELISPOT trial

[0190] Peripheral blood (5 ml) was collected from each of healthy controls (HC, n=21), patients with latent tuberculosis infection (LTBI, n=25), and patients with active tuberculosis (ATB, n=19), 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 of PP19128R (100 μg / ml) and 50 μl of auto-induction medium (AIM) 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. Results showed that PP19128R vaccine-induced IFN-γ levels were high in PBMCs from HC, LTBI-infected individuals, and ATB patients. + The number of T cells was higher than that induced by AIM. Figure 12 This indicates that PP19128R can stimulate IFN-γ in all three population groups. + T cell proliferation has good immunogenicity.

[0191] 2. Detection of Th1 / Th2 / Th17 cytokines induced by PP19128R vaccine

[0192] 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 PP19128R (100 μg / ml) and incubated in a 37°C CO2 incubator for 48 h. Simultaneously, PBMCs stimulated with AIM medium were used as a negative control. The PBMC cell culture mixture was transferred to a new tube, centrifuged at 500g 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).

[0193] To assess the consistency of the immune response induced by the PP19128R vaccine in computer simulations and in vitro, we performed cytokine assays on PBMCs collected from HC, LTBI, and ATB patients. The results showed no significant difference in IL-2 levels among HC, ATB, LTBI, and negative controls, regardless of whether PP19128R or AIM stimulation was used. Figure 13 (A). The PP19128R vaccine induced significantly higher levels of TNF-α in HCs and ATB patients than AIM culture medium induced levels in HCs. Figure 13 (Middle B). The IFN-γ level induced by PP19128R vaccine in HCs was significantly higher than that induced by AIM medium in HCs. Figure 13 (C). IL-10 induced by PP19128R vaccine in ATB, HCs and LTBI ( Figure 13 D), IL-4 ( Figure 13 (E) and IL-6 ( Figure 13 The levels of cytokines induced by the PP19128R vaccine in ATB and LTBI patients were significantly higher than those induced by AIM culture medium in HCs. Figure 13 (G).

[0194] 3. Correlation analysis and simple linear regression analysis of PP19128R vaccine-induced cytokines

[0195] To further explore the characteristics of cellular immune responses induced by the PP19128R vaccine, we also explored the major cytokines and their correlations based on principal component analysis and correlation analysis. Principal component analysis results showed that (1) in healthy individuals, PP19128R-induced cytokines such as IL-6, IL-10, IL-17A, and TNF-α played more important roles, but no significant correlations were found among the cytokines. Figure 14 (A) ;(2) In the ATB population, PP19128R-induced cytokines such as IL-2, IL-6 and TNF-α play a more important role, and there is a correlation between TNF-α and IFN-γ or IL-6, a correlation between IFN-γ and IL-6, and a correlation between IL-10 and IL-17A. Figure 14 (3) In the LTBI population, PP19128R-induced cytokines such as IL-2, IL-6, IL-10 and TNF-α are crucial. Furthermore, correlations were found between IL-2 and TNF-α or IL-4 or IL-17A, between TNF-α and IL-4 or IL-17A, and between IL-10 and IL-6. Figure 14 (C)

[0196] Further analysis of the aforementioned correlated cytokines using simple linear regression revealed that: (1) in LTBI subjects, PP19128R induced TNF-α and IL-2 (R2=0.965, P<0.0001, Y=0.006311*X-0.1866, Figure 15 IL-4 and IL-2 (R) 2 =0.7855, P=0.0036, Y=0.7980*X-1.781, Figure 15 IL-4 and TNF-α (R) 2 =0.7065, P=0.0090, Y=117.8*X-224.6, Figure 15 Middle C), IL-6 and IL-10 (R2=0.6343, P=0.0180, Y=0.01476*X-172.9, Figure 15 IL-17A and IL-2 (R) 2 =0.6574, P=0.0146, Y=0.2586*X-0.5414, Figure 15 (E) and IL-17A and TNF-α (R) 2 =0.6302, P=0.0186, Y=39.42*X-48.09, Figure 15There is a significant positive correlation between cytokines such as IFN-γ and TNF-α in patients with ATB; (2) In patients with ATB, PP19128R-induced IFN-γ and TNF-α (R) are significantly positively correlated. 2 =0.6708, P=0.0242, Y=107.7*X-523.7, Figure 16 IL-6 and TNF-α (R) 2 =0.7989, P=0.0067, Y=0.1831*X-2862, Figure 16 IL-6 and IFN-γ (R) 2 =0.7218, P=0.0155, Y=0.001323*X-14.71, Figure 16 (C) and IL-17A and IL-10(R) 2 =0.6224, P=0.0350, Y=7.554*X-7.930, Figure 16 Significant positive correlations exist between cytokines such as D and α.

[0197] 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 1-924 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. 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 2772nd position of SEQ ID No.

2.

4. 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.

5. The preparation method according to claim 4, characterized in that, The host cell is a microorganism.

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