Fusion protein HP16118P and application thereof in differential diagnosis of mycobacterium tuberculosis latent infection

Abstract

The application discloses a fusion protein HP16118P and application thereof in differential diagnosis of latent infection of mycobacterium tuberculosis. Specifically disclosed is a fusion protein comprising HTL, CTL and B cell epitope in series, HBD-3, PADRE and PSM alpha 4. The application screens 35 epitopes for mycobacterium tuberculosis, which have good immunogenicity and antigenicity and no toxicity, no sensitization and other characteristics. Further, the auxiliary peptides HBD-3 and PADRE are added to improve the immunogenicity of the fusion protein, and the PSM alpha 4 is added to endow the fusion protein with a targeted delivery function. In vitro experiments prove that the HP16118P can stimulate human peripheral blood mononuclear cells to produce an immune response, and is a dominant protective antigen. The HP16118P has the advantages of simple preparation method, good specificity, high sensitivity, low cost and the like as a diagnostic molecule. The application has great value for differential diagnosis 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 HP16118P and its application in the differential diagnosis of latent Mycobacterium tuberculosis infection. Specifically, it relates to the HP16118P recombinant multiepitope antigen derived from Mycobacterium tuberculosis (MTB) protein antigen and its application in the differential diagnosis 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 belonging to the genus Mycobacterium of the family Mycobacteriaceae in the order Actinobacteriaceae. It primarily causes long-term infection by attacking macrophages and inhibiting their apoptosis. Since the 1990s, the World Health Organization has developed a series of programs to stop tuberculosis and achieve the ambitious goal of ending it. However, since the COVID-19 pandemic, the number of newly diagnosed TB cases in 2021 has rebounded, reaching 6.4 million. These data suggest that tuberculosis is the second leading cause of death from a single pathogen after COVID-19 infection.

[0003] Currently, detection methods for LTBI include TST and IFN-γ release assays (IGRAs), but neither of these methods can differentiate between LTBI and ATB. Therefore, researching efficient diagnostic molecules for LTBI and applying them to the differential diagnosis of LTBI, improving the sensitivity and specificity of LTBI diagnosis, and reducing the incidence of TB in infected individuals are of great significance for the prevention and control of TB. In the bactericidal mechanism of Mycobacterium tuberculosis, epitopes play an important role in triggering the immune response. Meanwhile, the development of bioinformatics and immunoinformatics has provided convenient conditions for the study of epitope molecules. Therefore, the prediction and screening of candidate antigens and immunodominant epitopes have become key factors in the diagnosis and prevention of LTBI. Multi-epitope molecules with multiple dominant epitopes can not only be recognized by more MHC molecules in the body and efficiently presented to T cells, but also more effectively compensate for problems such as immune escape of pathogens caused by mutations in a single dominant epitope.

[0004] With the rapid development of bioinformatics and immunoinformatics, peptide molecules have become one of the most attractive strategies for developing diagnostic molecules. 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 of peptides provide fusion proteins with the advantage of easy transport and preservation. Summary of the Invention

[0005] The purpose of this invention is to provide a polypeptide fusion protein and its application in the differential diagnosis of latent tuberculosis infection. 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 HP16118P. The fusion protein may include tandem polypeptide 1, tandem polypeptide 2, and tandem polypeptide 3. The tandem polypeptide 1 may include a polypeptide whose amino acid sequence is shown in positions 75-89, 95-111, 117-133, 139-151, 157-170, 176-193, 199-213, 219-233, 239-256, 262-276, 282-299, 305-320, 326-339, 345-360, 366-381, and 387-401 of SEQ ID No. 1.

[0007] The tandem polypeptide 2 may include a polypeptide whose amino acid sequence is shown at positions 405-413, 417-425, 429-438, 442-451, 455-464, 468-476, 480-488, 492-500, 504-512, 516-524, and 528-536 of SEQ ID No. 1.

[0008] The tandem polypeptide 3 may include a polypeptide whose amino acid sequence is shown at positions 539-591, 594-618, 621-658, 661-690, 693-714, 717-749, 752-779, and 782-844 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 16 HTL epitopes (amino acid sequences of positions 75-89, 95-111, 117-133, 139-151, 157-170, 176-193, 199-213, 219-233, 239-256, 262-276, 282-299, 305-320, 326-339, 345-360, 366-381, and 387-401, respectively) tandemly. Specifically, the 16 HTL epitopes may be tandemly linked by an amino acid linker (such as GGPPG), and the amino acid sequence of the tandem polypeptide 1 may specifically be positions 75-401 of SEQ ID No. 1.

[0011] The tandem polypeptide 2 may be a tandem CTL epitope, consisting of 11 CTL epitopes (amino acid sequences of positions 405-413, 417-425, 429-438, 442-451, 455-464, 468-476, 480-488, 492-500, 504-512, 516-524, and 528-536 of SEQ ID No. 1). Specifically, the 11 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 405-536 of SEQ ID No. 1.

[0012] The tandem polypeptide 3 may be a tandem B-cell epitope, which is obtained by tandemly connecting 8 B-cell epitopes (amino acid sequences of positions 539-591, 594-618, 621-658, 661-690, 693-714, 717-749, 752-779 and 782-844 of SEQ ID No. 1). Specifically, the 8 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 539-813 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 an amino acid linker, a multi-epitope fusion protein is obtained. This multi-epitope fusion protein can be used as an active ingredient to construct diagnostic 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 an adjuvant peptide and / or an accessory peptide. Preferably, the fusion protein may further include accessory peptide 1 whose amino acid sequence is from position 1 to 45 of SEQ ID No. 1, accessory peptide 2 whose amino acid sequence is from position 51 to 69 of SEQ ID No. 1, and / or an adjuvant peptide whose amino acid sequence is from position 819 to 838 of SEQ ID No. 1.

[0018] The accessory peptide may be HBD-3 and / or PADRE, and the adjuvant peptide may be the TLR-2 agonist PSMα4.

[0019] Specifically, the helper peptide 1 may be HBD-3, and the helper peptide 2 may be PADRE.

[0020] The amino acid sequence of the accessory peptide 1 (HBD-3) may be positions 1-45 of SEQ ID No. 1, the amino acid sequence of the accessory peptide 2 (PADRE) may be positions 51-69 of SEQ ID No. 1, and the amino acid sequence of the adjuvant peptide (PSMα4) may be positions 819-838 of SEQ ID No. 1.

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

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

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

[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 invention, the fusion protein includes helper peptides HBD-3 and PADRE, 16 HTL epitopes, 11 CTL epitopes, 8 B-cell epitopes, PSMα4, and a 6×His tag.

[0027] Furthermore, the fusion protein HP16118P may be any of the following:

[0028] A1) The amino acid sequence is the protein consisting of positions 1-838 of SEQ ID No. 1;

[0029] A2) A protein that has more than 80% identity with and has the same function as the protein shown in A1) obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in positions 1-838 of SEQ ID No. 1.

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

[0031] A4) The amino acid sequence is the protein consisting of positions 75-813 of SEQ ID No. 1;

[0032] A5) A protein that has more than 80% identity with and has the same function as the protein shown in A1) obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID No. 1 from positions 75 to 813.

[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 A4) may be an antigenic fusion protein (positions 75-813 of SEQ ID No. 1) formed by linking tandem polypeptide 1 (including 16 tandem HTL epitopes), tandem polypeptide 2 (including 11 tandem CTL epitopes), and tandem polypeptide 3 (including 8 tandem B cell epitopes) through an amino acid linker.

[0035] A1) may be a fusion protein (positions 1-838 of SEQ ID No. 1) obtained by fusing helper peptide 1 (HBD-3) and helper peptide 2 (PADRE) at the N-terminus of A4) and adjuvant peptide (PSMα4) at the C-terminus to enhance immunogenicity.

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

[0037] Further, A3) 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, GFP (green fluorescent protein), CFP (cyan fluorescent protein), YFP (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, by using blast as the procedure, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, and setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, a search can be performed to calculate the identity of amino acid sequences, and then the identity value (%) can be obtained.

[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) A nucleic acid molecule encoding any of the fusion proteins HP16118P described herein;

[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 1-2514 of SEQ ID No. 2, or positions 223-2439 of SEQ ID No. 2;

[0050] B2) The nucleotide sequence is a DNA molecule of SEQ ID No. 2, positions 1-2514 of SEQ ID No. 2, or positions 223-2439 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 HP16118P shown in SEQ ID No. 1.

[0053] The DNA molecule shown in positions 1-2514 of SEQ ID No. 2 may be a DNA molecule encoding the fusion protein HP16118P whose amino acid sequence is shown in positions 1-838 of SEQ ID No. 1.

[0054] The DNA molecule shown in positions 223-2439 of SEQ ID No. 2 may be a DNA molecule encoding the fusion protein HP16118P whose amino acid sequence is shown in positions 75-813 of SEQ ID No. 1.

[0055] The nucleic acid molecule may also include nucleic acid molecules obtained by codon preference modification based on the nucleotide sequence shown in SEQ ID No. 2, positions 1-2514 of SEQ ID No. 2, or positions 223-2439 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(+)-HP16118P.

[0061] The recombinant vector pET-28a(+)-HP16118P 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(+)-HP16118P expresses the fusion protein HP16118P with the amino acid sequence shown in SEQ ID No. 1.

[0062] D4) The recombinant microorganism may be BL21 / pET-28a(+)-HP16118P. BL21 / pET-28a(+)-HP16118P is a recombinant microorganism obtained by introducing the recombinant vector pET-28a(+)-HP16118P 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 identification and / or diagnosis of latent Mycobacterium tuberculosis infection;

[0066] C2) Application in the preparation of products for identifying and distinguishing between latent tuberculosis infected individuals and patients with active tuberculosis;

[0067] C3) Application in the preparation of products for identifying and distinguishing between latent tuberculosis infected individuals and healthy subjects;

[0068] C4) Application in the preparation of products for differentiating between patients with active tuberculosis and healthy subjects;

[0069] C5) Application in the prevention and control of diseases caused by Mycobacterium tuberculosis infection;

[0070] C6) Application in the preparation of diagnostic molecules for the differential diagnosis of latent Mycobacterium tuberculosis infection;

[0071] C7) Application in the preparation of protective antigens against Mycobacterium tuberculosis;

[0072] Application of C8 in screening and / or developing antibodies against Mycobacterium tuberculosis.

[0073] The protective antigen refers to the antigenic component of Mycobacterium tuberculosis that can stimulate the body to produce a protective immune response.

[0074] 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).

[0075] In the above applications, the disease caused by Mycobacterium tuberculosis infection can be tuberculosis.

[0076] Furthermore, the tuberculosis may include active tuberculosis (ATB) and latent tuberculosis infection (LTBI).

[0077] Furthermore, in the above applications, the application may include the following steps:

[0078] H1) The subject sample is co-cultured with a stimulus, which may be any of the fusion proteins described herein;

[0079] H2) detects the levels of secreted IL-5 and / or IL-17F in the sample.

[0080] Furthermore, the application also includes: identifying and / or diagnosing latent Mycobacterium tuberculosis infection based on the IL-5 level (i.e., identifying and / or diagnosing whether a subject is a latent tuberculosis infected person).

[0081] Furthermore, the application also includes: differentiating between latent tuberculosis infected individuals and active tuberculosis patients based on the IL-5 level, or differentiating between latent tuberculosis infected individuals and healthy subjects based on the IL-5 level.

[0082] Furthermore, the application also includes: differentiating between active tuberculosis patients and healthy subjects based on the IL-17F level, or differentiating between latent tuberculosis infected individuals and healthy subjects based on the IL-17F level.

[0083] The subject samples mentioned in this article may be blood samples or tissue samples.

[0084] The present invention also provides products for the identification and / or diagnosis of latent infection with Mycobacterium tuberculosis, said products may include any of the fusion proteins described herein.

[0085] Furthermore, the product may also include reagents for detecting IL-5 and / or IL-17F.

[0086] The products described in this article may be diagnostic molecules, pharmaceutical compositions, reagents, kits, chips, test strips, or test cards.

[0087] The diagnostic molecules, pharmaceutical compositions, reagents, kits, chips, test strips, or test cards mentioned above can be used to identify and / or diagnose latent Mycobacterium tuberculosis infection, or to differentiate between latent tuberculosis infected individuals and patients with active tuberculosis.

[0088] The active ingredients of the diagnostic molecules, pharmaceutical compositions, and reagents may include any of the fusion proteins described herein.

[0089] The diagnostic molecules may also include adjuvants.

[0090] 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.

[0091] 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.

[0092] 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.

[0093] Furthermore, the method may include the following steps:

[0094] G1) Construct a recombinant expression vector containing a nucleic acid molecule encoding any of the fusion proteins described herein;

[0095] G2) The recombinant expression vector is introduced into microorganisms to obtain recombinant microorganisms;

[0096] G3) The recombinant microorganisms are cultured, and the fusion protein is obtained by isolation and / or purification;

[0097] Further, the nucleic acid molecule described in G1) may be a DNA molecule as shown in SEQ ID No. 2, positions 1-2514 of SEQ ID No. 2, or positions 223-2439 of SEQ ID No. 2.

[0098] Furthermore, the microorganism may be Escherichia coli BL21(DE3).

[0099] The applications and methods described herein may be for disease diagnosis, disease prognosis and / or disease treatment purposes, or they may be for non-disease diagnosis, non-disease prognosis and non-disease treatment purposes; their direct purpose may be to obtain information on intermediate results of disease diagnosis, disease prognosis and / or disease treatment, or their direct purpose may be for non-disease diagnosis, non-disease prognosis and / or non-disease treatment purposes.

[0100] 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, and are non-toxic and non-sensitizing. Based on this, the inventors added the helper peptides HBD-3 and PADRE to the epitope molecule design to further enhance the immunogenicity of the epitope diagnostic molecule, and added the TLR2 agonist PSMα4 to endow the diagnostic molecule with targeted delivery capabilities and enhance its immunogenicity. Furthermore, immunoinformatics tools were used to predict and analyze the antigenicity, immunogenicity, physicochemical parameters, secondary structure, tertiary structure, and immune response of the diagnostic molecule. The results showed that the antigenicity of the polypeptide fusion protein HP16118P provided by this invention was 0.7381 and 0.60063, the immunogenicity was 6.43254, and the solubility index was 0.382. Secondary structure prediction showed that HP16118P has 41% α-helices, 7% β-sheets, and 50% random coils. The three-level structure analysis showed that the confidence score (C-score) of HP16118P was -1.98.

[0101] This invention further prepared the fusion protein HP16118P. The consistency between immunoinformatics and real-world experimental results was analyzed using ELISPOT and high-throughput liquid chromatography protein analysis. Both immunoinformatics and real-world experimental results showed that the peptide fusion protein HP16118P induced an increase in active B lymphocytes and produced high levels of IgG and IgM antibodies, induced an increase in the number of active cytotoxic T cells reaching a peak on day 50 post-stimulation, and also induced high levels of IFN-γ and IL-2. Simultaneously, in vitro experimental results demonstrated that the Mycobacterium tuberculosis peptide fusion protein HP16118P can serve as an antigenic protein, stimulating an immune response in human peripheral blood mononuclear cells (PBMCs), exhibiting immunogenicity, and inducing the production of IFN-γ. + This invention provides new candidate target molecules for the differential diagnosis of latent tuberculosis infection, including T lymphocytes and various cytokines.

[0102] Further diagnostic performance experiments showed that the beneficial effects of using IL-5 and / or IL-17F as biomarkers for differential diagnosis between ATB, LTBI and HC were: (1) IL-5 induced by the HP16118P diagnostic molecule could distinguish the LTBI population from the ATB (P=0.0372, AUC=0.8214, 95% CI [0.5843 to 1.000]) and HC (P=0.0026, AUC=0.9643, 95% CI [0.8770 to 1.000]) populations, with a sensitivity and specificity of 100% and 71.43% (ATB vs. LTBI), and 100% and 85.71% (HC vs. LTBI), respectively. (2) HP16118P diagnostic molecule-induced IL-17F can distinguish the ATB population from the HC population (P=0.0088, AUC=0.9184, 95% CI [0.7716 to 1.000]), with a sensitivity and specificity of 71.43% and 85.71%, respectively; (3) HP16118P diagnostic molecule-induced IL-17F can distinguish the LTBI population from the HC population (P=0.0038, AUC=0.9464, 95% CI [0.8299 to 1.000]), with a sensitivity and specificity of 87.50% and 85.71%, respectively.

[0103] The polypeptide fusion protein HP16118P of this invention can be prepared through genetic engineering. Using HP16118P as a diagnostic molecule, it exhibits strong antigenicity and immunogenicity, and its preparation method is simple, specific, sensitive, low-cost, and high-yield. This invention has significant value for the differential diagnosis of active tuberculosis and latent tuberculosis infection, as well as for the prevention and control of tuberculosis. Attached Figure Description

[0104] Figure 1 This refers to the HTL epitope information of the final peptide fusion protein selected in Example 1 for construction.

[0105] Figure 2 This refers to the CTL epitope information selected in Example 1 for the final construction of the polypeptide fusion protein.

[0106] Figure 3 This refers to the B-cell epitope information selected in Example 1 for the final construction of the polypeptide fusion protein.

[0107] Figure 4 This is a schematic diagram of the construction of HP16118P and the prediction results of its secondary structure.

[0108] Figure 5 This is a 3D model of the HP16118P.

[0109] Figure 6 The results of C-ImmSim Server predictions for HP16118P-induced natural killer (NK) cells, macrophages (MA), dendritic cells (DC), epithelial cells, and B cells, and the antibodies they produce.

[0110] Figure 7 The results show the predictions of C-ImmSim Server for HP16118P-induced helper T (TH) cells, cytotoxic T (TC) cells, and regulatory T (TR) cells.

[0111] Figure 8 The results show the predicted cytokine levels in HP16118P-induced cells using C-ImmSim Server.

[0112] Figure 9 A schematic diagram of the construction of the recombinant vector pET-28a(+)-HP16118P and the SDS-PAGE purification of the diagnostic molecule after in vitro cloning and expression.

[0113] Figure 10 For the detection of IFN-γ by enzyme-linked immunospot assay (ELISPOT) + T lymphocytes were used to stimulate peripheral blood mononuclear cells (PBMCs) from healthy controls (HC), patients with latent tuberculosis infection (LTBI), and patients with active tuberculosis (ATB) in vitro using HP16118P. IFN-γ levels were 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 expressed as mean ± SEM. p < 0.05 was considered statistically significant. SEM values ​​are the standard error of the mean.

[0114] Figure 11 This study aimed to determine the levels of cytokines produced by human peripheral blood mononuclear cells (PBMCs) induced by HP16118P. High-throughput liquid chromatography-protein analysis was used to detect the levels of 35 inflammatory cytokines induced by HP16118P (G-CSF, GM-CSF, HGF, IFN-α, IFN-γ, IL-1α, IL-1β, IL-10, IL-12p70, IL-13, IL-117F, 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-1α, MIP-1β, PD-1, SDF-1α, TIM-3, TIMP-1, TNF-α, VEGF-A, and VEGF-R2). All data are presented as mean ± SEM. SEM values ​​are the standard error of the mean. Cytokine concentrations are expressed in ng / ml.

[0115] Figure 12 Differences in cytokine production induced by HP16118P in human peripheral blood mononuclear cells (PBMCs) among the three groups were investigated. PBMCs from healthy controls (HC), latent tuberculosis-infected individuals (LTBI), and active tuberculosis patients (ATB) were stimulated in vitro with HP16118P. Differences were compared using one-way ANOVA or the Kruskal-Wallis test to ensure normality and homogeneity of variance. All data are presented as mean ± SEM. p < 0.05 was considered statistically significant. Detailed Implementation

[0116] 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.

[0117] 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.

[0118] The Escherichia coli BL21(DE3) competent cells in the following examples were purchased from Shanghai Jingnuo Biotechnology Co., Ltd.

[0119] The carrier pET-28a(+) used in the following examples was purchased from Novagen.

[0120] The preparation methods of the main reagents in the following examples are as follows:

[0121] 1. Preparation of LB liquid culture medium (1000ml):

[0122]

[0123] Add deionized water to a final volume of 1000 ml, then autoclave at 121°C for 15 minutes.

[0124] 2. Preparation of LB solid culture medium (1000ml):

[0125]

[0126] Add deionized water to a final volume of 1000 ml, then autoclave at 121°C for 15 minutes.

[0127] 3. Preparation of buffer solution for purification of the soluble expression form of the target protein:

[0128] (1) Soluble protein lysis buffer, pH 8.0 (1000ml):

[0129]

[0130] Dissolve in deionized water and bring the volume to 1000ml. Adjust the pH to 8.0 with NaOH.

[0131] (2) Preparation of buffer solution required for purification of the target protein expressed in inclusion bodies:

[0132] ①Inclusion body protein lysis buffer, pH 8.0 (1000ml):

[0133]

[0134] Dissolve in deionized water and bring the volume to 1000ml. Adjust the pH to 8.0 with NaOH.

[0135] ②Inclusion body protein washing buffer, pH 6.3 (1000ml):

[0136]

[0137] Dissolve in deionized water and bring the volume to 1000ml. Adjust the pH to 6.3 with NaOH.

[0138] ③Inclusion body protein elution buffer, pH 4.5 (1000ml):

[0139]

[0140] Dissolve in deionized water and bring the volume to 1000ml. Adjust the pH to 4.5 with NaOH.

[0141] The nucleotide sequence (SEQ ID No. 2) of the HP16118P gene involved in the following examples is shown below:

[0142]

[0143] Example 1: Prediction, screening, and identification of immune dominant epitopes

[0144] 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 diagnostic molecules utilize genetic engineering techniques to express or artificially synthesize antigenic epitopes of pathogenic microorganisms in vitro, and then use them as diagnostic molecules. The key to epitope diagnostic molecule design is epitope screening. In this embodiment, 15 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 the preparation of epitope diagnostic molecules.

[0145] 1. Selection of antigens

[0146] Anat Zvi et al. screened 189 potential tuberculosis (TB) candidate molecules 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 diagnostic candidate antigens. 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 15 antigens for epitope prediction and screening. The 15 candidate antigens are Rv1736c, Rv1737c, Rv2626c, Rv2656c, Rv2659c, Rv1511, Rv1980c, Rv1981c, Rv3873, Rv3878, Rv3879c, Rv3425, Rv1978, Rv2031c, and Rv3429.

[0147] 2. HTL epitope prediction and screening

[0148] 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, 16 HTL immunodominant epitopes were ultimately identified as candidate epitopes for constructing diagnostic 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*) were used to predict sensitization. 1 indicates sensitization, and 2 indicates no sensitization.

[0149] 2. CTL epitope prediction and screening

[0150] 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, 11 CTL immunodominant epitopes were ultimately identified as candidate epitopes for constructing diagnostic 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) were used to predict sensitization. 1 indicates sensitization, and 2 indicates no sensitization.

[0151] 3. Prediction and screening of B-cell epitopes

[0152] 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 diagnostic molecules. For details regarding specific epitope sequences, please refer to [link to relevant documentation]. Figure 3 .

[0153] Finally, 16 HTL epitopes, 11 CTL epitopes, and 8 B-cell epitopes were identified for constructing the diagnostic molecularly active component (peptide fusion protein), totaling 35 epitopes. The amino acid sequences of the 35 epitopes are shown in Table 1.

[0154] Table 1. Amino acid sequences of the 35 immunodominant epitopes finally identified through screening.

[0155]

[0156]

[0157] Example 2: Construction, physicochemical properties, and structural analysis of peptide fusion proteins

[0158] 1. Construction of peptide fusion proteins

[0159] Based on the HTL, CTL, and B-cell epitopes predicted and screened using the aforementioned bioinformatics tools, the HTL epitopes with the highest percentile rank, antigenicity, and IFN-γ scores, as well as those with no toxicity or sensitization, were selected. The CTL epitopes with the highest percentile rank, immunogenicity, and antigenicity scores, as well as those with no toxicity or sensitization, and the B-cell epitopes with the highest predicted scores (a total of 35 epitopes) were then used to construct a polypeptide fusion protein (such as HP16118P).

[0160] The novel tuberculosis polypeptide fusion protein constructed in this invention comprises four parts (HBD-3, PADRE, multi-epitope fusion protein, and PSMα4), and is named HP16118P. Figure 4 ).

[0161] First, the selected 35 epitopes were linked using amino acid linkers (GPGPG, AAY, KK). Specifically, in this embodiment, 16 HTL epitopes were linked using GGPG amino acid linkers to obtain tandem HTL epitopes (amino acid sequences of SEQ ID No. 1, positions 75-401); 11 CTL epitopes were linked using AAY amino acid linkers to obtain tandem CTL epitopes (amino acid sequences of SEQ ID No. 1, positions 405-536); 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 539-813). 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 diagnostic molecules. Specifically, in this embodiment, the linking method of the multi-epitope fusion protein is as follows:

[0162] Multiepitope fusion protein: tandem HTL epitope - AAY - tandem CTL epitope - KK - tandem B cell epitope.

[0163] Then, HBD-3 helper peptide (GIINTLQKYYCRVRGGRCAVLSCLPKEEQIGKCSTRGRKCCRRKK, positions 1-45 of SEQ ID No. 1) and PADRE helper peptide (AGLFQRHGEGTKATVGEPV, positions 51-69 of SEQ ID No. 1) were added to the initiation (amino terminus) of the multi-epitope fusion protein to further enhance the immunogenicity of the epitope diagnostic molecule. Furthermore, the TLR2 agonist PSMα4 (MAIVGTIIKIIKAIIDIFAK, positions 819-838 of SEQ ID No. 1) was added to the terminal (carboxyl terminus) of the multi-epitope fusion protein as an adjuvant to endow the diagnostic molecule with targeted delivery capabilities and enhance its immunogenicity.

[0164] Finally, to facilitate protein purification, a His tag (6×His) was added to the carboxyl terminus of the peptide fusion protein.

[0165] The final constructed peptide fusion protein was named HP16118P. Figure 4 The amino acid sequence of the polypeptide fusion protein HP16118P is shown in SEQ ID No. 1, and its encoding gene is named HP16118P gene. The nucleotide sequence of HP16118P gene is shown in SEQ ID No. 2.

[0166] Based on this, the antigenicity, allergenicity, immunogenicity, and toxicity prediction of the constructed peptide fusion protein HP16118P 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 HP16118P diagnostic molecule (i.e., the peptide fusion protein HP16118P) consists of 844 amino acids. Analysis using the Expasy Protparam server revealed its relative molecular mass to be 90265.44 Da, its theoretical pI to be 9.84, and its estimated half-life to be 30 hours (mammalian reticulocytes, in vitro), 20 minutes (yeast, in vivo), and 10 hours (E. coli, in vivo), indicating that HP16118P is a relatively stable protein molecule. Furthermore, we found that the instability index, aliphatic index, and grand average of hydropathicity (GRAVY) were 43.02, 75.09, and -2.7, respectively, with antigenicity of 0.7381 and 0.60063, and immunogenicity of 6.43254. Additionally, the solubility of HP16118P predicted by the Protein-Sol server was 0.382. These results indicate that HP16118P is a stable, hydrophilic, and moderately basic protein molecule with moderate solubility.

[0167] 2. Secondary and tertiary spatial structure analysis of peptide fusion proteins

[0168] The Expasy Protparam server (https: / / web.expasy.org / protparam / ) was used to predict the physicochemical parameters of peptide fusion proteins. It can predict the physicochemical properties of diagnostic molecules, 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 peptide fusion proteins. It can effectively identify transmembrane topologies, transmembrane helices, folds, and domain recognition. RaptorX Property (http: / / raptorx.uchicago.edu / StructurePropertyPred / predict / ) predicts the secondary structure features of peptide molecules. 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 prediction is based on a critical value of 0.25. Secondary structure prediction shows that HP16118P has 41% α-helices, 7% β-sheets, and 50% random coils. Tertiary structure analysis shows that HP16118P has a C-score of -1.98. Figure 5 ).

[0169] Example 3: Immunostimulation Simulation Analysis of Polypeptide Fusion Protein

[0170] 1. Immunomimetic modeling of peptide fusion proteins

[0171] Both specific and non-specific immune responses play important roles in the host's clearance and eradication of Mycobacterium tuberculosis. Therefore, this invention simulates and analyzes the immune response induced by the HP16118P diagnostic 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 epitope molecule 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 HP16118P-induced cellular immune response and cytokine levels were predicted.

[0172] This invention has discovered that the HP16118P diagnostic molecule can activate NK cells and maintain their number at 325-375 cells / mm². 3 between( Figure 6 (A). Interestingly, the HP16118P diagnostic molecule can activate the proliferation and differentiation of macrophages and dendritic cells, and can induce presenting-2 type macrophages (A). Figure 6 (B) and dendritic cells form a proliferation peak ( Figure 6 (C). Unlike dendritic cells, the number of resting and active macrophages remained stable at 90 cells / mm² on day 8 after HP16118P simulated immunization. 3 ( Figure 6 HP16118P can induce significantly high levels of epithelial cells (B). Figure 6 (Middle D). Similar to macrophages and dendritic cells, HP16118P can stimulate B lymphocyte differentiation and proliferation, resulting in a rapid peak in presenting-2 (520 cells / mm) after stimulation. 3 The number of active B lymphocytes on day 5 was 480 cells / mm². 3 Peak () Figure 6 (E). Subsequently, the induced active B lymphocytes produced high levels of IgG and IgM antibodies ( Figure 6 (F).

[0173] In addition, we analyzed the immunomodulatory effects of HP16118P on specific immune cells. The results showed that HP16118P-mimicking immunity could induce a peak number of memory helper T lymphocytes up to 5500 cells / mm². 3 ( Figure 7(A). Furthermore, we found that HP16118P simulated injection stimulated the number of active helper T lymphocytes to peak on day 10. Figure 7 Unlike helper T lymphocytes, HP16118P has a weaker ability to stimulate the immune system to form memory cytotoxic T lymphocytes. Figure 7 In the middle C), the number of active cytotoxic T lymphocytes peaked at 1000 cells / mm² on day 15 post-immunization. 3 ); while resting cytotoxic T lymphocytes show the completely opposite trend ( Figure 7 Excitingly, we found that HP16118P can induce T lymphocytes to differentiate into Th1 lymphocytes, mediating a robust Th1 immune response. Figure 7 (Middle E). Furthermore, we observed that HP16118P could induce a rapid increase in regulatory T cells on the second day after immunization, followed by a peak (140 cells / mm). 3 ()( Figure 7 Finally, we also analyzed the ability of HP16118P to induce IFN-γ production in immune cells. The results showed that HP16118P immunization could induce peak levels of cytokines IFN-γ (400,000 ng / ml) and IL-2 (100,000 ng / ml). Figure 8 ).

[0174] Example 4: Construction of recombinant plasmids for polypeptide fusion proteins and their in vitro expression

[0175] 1. The HBD-3, PADRE, 16 HTL epitopes, 11 CTL epitopes, 8 B-cell epitopes, PSMα4, and 6×His tag described in Example 2 were linked together with linkers such as EAAAK, GGPPG, AAY, and KK to form a polypeptide fusion protein HP16118P (amino acid sequence as shown in SEQ ID No. 1). Figure 4 The 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 HP16118P gene (nucleotide sequence shown in SEQ ID No. 2). BamHI and XhoI recognition sites are added to both ends of the HP16118P 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.

[0176] 2. The artificially synthesized DNA fragment 1 from step 1 was digested with restriction endonucleases BamHI and XhoI, and the digestion products were recovered.

[0177] 3. The vector pET-28a(+) was digested with restriction endonucleases BamHI and XhoI, and the vector backbone was recovered.

[0178] 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(+)-HP16118P( Figure 9 (A)

[0179] 5. The structure of the recombinant vector is described as follows:

[0180] The recombinant vector pET-28a(+)-HP16118P 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(+)-HP16118P expresses the fusion protein HP16118P with the amino acid sequence shown in SEQ ID No. 1.

[0181] 6. The recombinant vector pET-28a(+)-HP16118P was introduced into *Escherichia coli* BL21(DE3) to obtain the recombinant bacterium BL21 / pET-28a(+)-HP16118P. 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(+)-HP16118P, it is the target recombinant bacterium.

[0182] 7. Expression of polypeptide fusion proteins

[0183] The recombinant strain BL21 / pET-28a(+)-HP16118P 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.

[0184] 8. Purification of peptide fusion proteins

[0185] (1) Take 100ml of the fermentation broth from step 7, centrifuge at 5000rpm for 10min, and collect the cell precipitate.

[0186] (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.

[0187] (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).

[0188] 9. Identification of polypeptide fusion proteins

[0189] The peptide fusion protein solution was subjected to 12% polyacrylamide gel electrophoresis, and the results are shown below. Figure 9 The B-type peptide fusion protein solution showed only one band of approximately 90.26 kDa, consistent with expectations.

[0190] Example 5: In vitro experimental verification of the cellular immune response induced by the HP16118P diagnostic molecule.

[0191] 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.

[0192] 1. HP16118P diagnostic molecular ELISPOT assay

[0193] Peripheral blood (5 ml) was collected from each of healthy controls (HC, n=23), patients with latent tuberculosis infection (LTBI, n=24), 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). 5Cells / well were stimulated with 50 μl HP16118P (100 μg / ml), and 50 μl PBS 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 HP16118P-diagnostic molecules induced IFN-γ in PBMCs of HC, LTBI-infected individuals, and ATB patients. + The number of T cells was higher than that induced by PBS. Figure 10 This indicates that HP16118P can stimulate IFN-γ in all three population groups. + T cell proliferation has good immunogenicity.

[0194] 2. High-throughput liquid phase protein analysis experiment of HP16118P diagnostic molecule

[0195] 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 HP16118P (100 μg / ml) and incubated in a CO2 incubator at 37°C for 48 h. Simultaneously, HC-stimulated PBMCs were used as a negative control. The PBMCs cell culture medium mixture was transferred to a new tube and centrifuged at 500g for 10 min. Finally, the supernatant was slowly transferred to another tube, and the levels of 35 inflammatory cytokines (G-CSF, GM-CSF, HGF, IFN-α, IFN-γ, IL-1α, IL-1β, IL-10, IL-12p70, IL-13, IL-117F, 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-1α, MIP-1β, PD-1, SDF-1α, TIM-3, TIMP-1, TNF-α, VEGF-A, and VEGF-R2) induced by HP16118P were detected using high-throughput liquid chromatography protein analysis.

[0196] To assess the consistency of the HP16118P diagnostic molecule in computer-simulated and in vitro induced immune responses, we performed cytokine assays on PBMCs collected from HC, LTBI, and ATB patients. First, HP16118P induced PBMCs to secrete 35 cytokines, and the results showed ( Figure 11The concentrations of cytokines greater than 10,000 pg / ml included TIMP-1; greater than 1,000 pg / ml included GM-CSF, IL-6, IL-8, MCP-1, MIP-1β, and TNF-α; and greater than 100 pg / ml included IL-1α, IL-10, IL-23, TIM-3, and VEGF-A. The concentrations of cytokines less than 10 pg / ml included IFN-α, IL-12p70, IL-17F, and IL-31. These data indicate that HP16118P has strong immunogenicity and the ability to induce immune cells to produce various cytokines. Further analysis of the differences in HP16118P-induced cytokines among the three groups showed that... Figure 12 The levels of HP16118P-induced cytokines IL-1α (P = 0.0020), IL-1β (P = 0.0106), IL-17F (P = 0.0076), IL-2 (P = 0.0004), IL-5 (P = 0.0009), MIG (P = 0.0151), HGF (P = 0.0065), and TNF-α (P = 0.0117) were significantly lower in the LTBI group than in the HC group; the levels of HP16118P-induced cytokines IL-17F (P = 0.0171) and TIM-3 (P = 0.0224) were significantly lower in the ATB group than in the HC group; and the level of HP16118P-induced cytokine IL-5 (P = 0.0372) was significantly lower in the LTBI group than in the ATB group.

[0197] Example 6: Diagnostic performance of HP16118P diagnostic molecule

[0198] Based on Example 5 Figure 12As a result, IL-5 and IL-17F were further selected as biomarkers for differential diagnosis among ATB, LTBI, and HC. The results showed (Table 2): (1) IL-5 induced by the HP16118P diagnostic molecule could distinguish the LTBI population from the ATB (P=0.0372, AUC=0.8214, 95% CI [0.5843 to 1.000]) and HC (P=0.0026, AUC=0.9643, 95% CI [0.8770 to 1.000]) populations, with a sensitivity and specificity of 100% and 71.43% (ATB vs. LTBI), and 100% and 85.71% (HC vs. LTBI), respectively. (2) HP16118P diagnostic molecule-induced IL-17F can distinguish the ATB population from the HC population (P=0.0088, AUC=0.9184, 95% CI [0.7716 to 1.000]), with a sensitivity and specificity of 71.43% and 85.71%, respectively; (3) HP16118P diagnostic molecule-induced IL-17F can distinguish the LTBI population from the HC population (P=0.0038, AUC=0.9464, 95% CI [0.8299 to 1.000]), with a sensitivity and specificity of 87.50% and 85.71%, respectively.

[0199] Table 2. Sensitivity and specificity of HP16118P-induced IL-5 and IL-17F cytokines in diagnosing ATB and LTBI

[0200]

[0201] 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 1-838 of SEQ ID No. 1; A2) A fusion protein with the same function is obtained by attaching a His tag to the C-terminus of A1).

2. A biomaterial, characterized in that, The biomaterial is any one of the following: D1) The 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 nucleotide sequence of the nucleic acid molecule is SEQ ID No. 2 or positions 1-2514 of SEQ ID No.

2.

4. Any of the following applications of the fusion protein of claim 1 or the biomaterial of claim 2 or 3: C1) Use in the preparation of products for the identification and / or diagnosis of latent Mycobacterium tuberculosis infection; C2) Application in the preparation of products for identifying and distinguishing between latent tuberculosis infected individuals and patients with active tuberculosis; C3) Application in the preparation of products for identifying and distinguishing between latent tuberculosis infected individuals and healthy subjects; C4) Application in the preparation of products for differentiating between patients with active tuberculosis and healthy subjects; C5) Application in the preparation of protective antigens against Mycobacterium tuberculosis; The products are selected from reagents, kits, chips, and test strips.

5. A product for the identification and / or diagnosis of latent infection with Mycobacterium tuberculosis, characterized in that, The product includes the fusion protein of claim 1; the product is selected from reagents, kits, chips and test strips.

6. 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.

7. The preparation method according to claim 6, wherein the host cell is a microorganism.

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