Fusion protein, mRNA (messenger Ribonucleic Acid) for coding fusion protein and application of fusion protein in inhibiting mycobacterium tuberculosis

Abstract

The invention relates to the technical field of biology, in particular to a fusion protein, mRNA for coding the fusion protein and application of the fusion protein to inhibition of mycobacterium tuberculosis. The invention provides a fusion protein based on an antibacterial peptide combination and bacteriophage pigtail fiber protein or lysin and mRNA for coding the fusion protein, the mRNA can effectively express the antibacterial peptide and the bacteriophage pigtail fiber protein / lysin in macrophages, and after being infected with mycobacterium tuberculosis, the mRNA can effectively express the antibacterial peptide and the bacteriophage pigtail fiber protein / lysin. The antibacterial peptide combination carried by the phage pigtail fiber protein / lysin expressed in the cells specifically targets and kills the mycobacterium tuberculosis, so that the aim of removing the mycobacterium tuberculosis in the cells is fulfilled. Compared with the existing antituberculous drugs, the mRNA preparation disclosed by the invention can be massively expressed in cells, the expression duration is long, and mycobacterium tuberculosis of the cells can be effectively eliminated.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to fusion proteins and their encoding mRNAs, and their application in inhibiting Mycobacterium tuberculosis. Background Technology

[0002] Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb). Macrophages are the primary host cells for Mtb during infection. Mtb's complex immune escape mechanisms within macrophages allow it to evade the host's immune system after entering the body. Currently, most existing drugs are only effective at killing extracellular pathogens; there is no effective treatment strategy to disrupt Mtb's immune escape and clear pathogens from within macrophages, leading to recurrent TB. Therefore, developing new and effective treatments to clear intracellular bacteria is a crucial strategy for TB control, especially for drug-resistant TB.

[0003] mRNA (messenger RNA) is a single-stranded RNA molecule transcribed from DNA, carrying genetic information and serving as a template for protein synthesis. As a form of gene therapy, mRNA technology boasts high translation efficiency, high safety, broad target range, ease of manufacturing, and novel mechanisms. With the development of COVID-19 mRNA vaccines and continuous technological advancements, the expression level and stability of mRNA have been significantly improved, ensuring efficient and sustained protein synthesis. These improvements enable mRNA to be applied to the treatment of multiple diseases, including personalized medicine, vaccine development, and chronic diseases. However, mRNA drug formulations are still primarily focused on vaccine development and anti-tumor research, with limited research on mRNA drugs for treating Mycobacterium tuberculosis. Therefore, providing an mRNA drug formulation capable of eliminating Mycobacterium tuberculosis intracellularly has significant clinical value, but still faces numerous challenges. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide a fusion protein and its encoding mRNA and its application in inhibiting Mycobacterium tuberculosis. The present invention provides a method for delivering a fusion mRNA of an antimicrobial peptide combination (LL-37, UB2, E50-52) with the tail fibrin GP6 or lysin GP10 of mycobacterial phage L5 into human monocyte (THP1) induced macrophages. By utilizing the cell's autonomous expression of the antimicrobial peptide and GP6 or GP10 fusion protein, after infection with Mycobacterium tuberculosis, the antimicrobial peptide combination carried by the intracellularly expressed GP6 or GP10 specifically targets and eliminates Mycobacterium tuberculosis, thereby achieving the purpose of eliminating intracellular Mycobacterium tuberculosis.

[0005] The present invention provides fusion proteins comprising at least two of the following: antimicrobial peptide LL-37, antimicrobial peptide UB2, and antimicrobial peptide E50-52.

[0006] In some embodiments, it also includes tail fibrin GP6 and / or lysozyme GP10.

[0007] In some embodiments, the fusion protein includes:

[0008] (1) The antimicrobial peptide LL-37 has the amino acid sequence shown in SEQ ID No. 10;

[0009] The antimicrobial peptide UB2 has the amino acid sequence shown in SEQ ID No. 11;

[0010] The antimicrobial peptide E50-52 has the amino acid sequence shown in SEQ ID No. 12;

[0011] The tail fibrin GP6 has the amino acid sequence shown in SEQ ID No. 13;

[0012] The solubilin GP10 has the amino acid sequence shown in SEQ ID No. 14;

[0013] or

[0014] (2) A sequence based on the amino acid sequence shown in (1) by substitution, deletion and / or addition of one or more amino acids, and an amino acid sequence that has the same or similar function as the amino acid sequence shown in (1).

[0015] or

[0016] (3) An amino acid sequence that has at least 80% sequence identity with the amino acid sequence shown in (1) and has the same or similar function as the amino acid sequence shown in (1).

[0017] In some embodiments, the fusion protein comprises sequentially linked components from the N-terminus to the C-terminus:

[0018] Antimicrobial peptides LL-37, UB2, and E50-52; or

[0019] Tail fibroin GP6, antimicrobial peptide LL-37, antimicrobial peptide UB2, and antimicrobial peptide E50-52; or

[0020] Dissolving agent GP10, antimicrobial peptide LL-37, antimicrobial peptide UB2 and antimicrobial peptide E50-52.

[0021] In some embodiments, the sequential linking includes linker linking, wherein the linker is independently selected from any of the amino acid sequences shown in SEQ ID No. 19-21.

[0022] In some embodiments, the fusion protein has any one of the amino acid sequences shown in SEQ ID No. 15-17.

[0023] This invention provides biomaterials comprising at least one of the following (4) to (6):

[0024] (4) The nucleic acid molecule encoding the fusion protein;

[0025] (5) An expression vector comprising the aforementioned nucleic acid molecules;

[0026] (6) Transform or transfect the host cells of the recombinant vector.

[0027] In some embodiments, the nucleic acid molecule includes DNA and / or mRNA, and the nucleic acid molecule has:

[0028] (7) A nucleotide sequence as shown in any of SEQ ID No. 1-8; or

[0029] (8) A nucleotide sequence that encodes the same protein as (7), but differs from the nucleotide sequence shown in (7) due to the degeneracy of the genetic code; or

[0030] (9) A nucleotide sequence obtained by substituting, deleting, or adding one or more nucleotide sequences to the nucleotide sequence shown in (7) or (8), and which has the same or similar function to the nucleotide sequence shown in (7) or (8); or

[0031] (10) A nucleotide sequence that is more than 95% identical to the nucleotide sequence described in (7), (8) or (9).

[0032] In some embodiments, the nucleic acid molecule is mRNA, the 5' end of which has 5'-CAP and 5'-UTR modifications, and the 3' end has 3'-UTR and Poly(A) modifications.

[0033] In some embodiments, the backbone vector of the expression vector is a pRSF vector.

[0034] The present invention provides the following (11) and / or (12) uses in the preparation of medicaments for the prevention and / or treatment of Mycobacterium tuberculosis infection or products for inhibiting Mycobacterium tuberculosis:

[0035] (11) The fusion protein;

[0036] (12) The aforementioned biomaterials.

[0037] This invention provides medicaments or products for preventing and / or treating Mycobacterium tuberculosis infection, including:

[0038] (13) The fusion protein; and / or

[0039] (14) The aforementioned biological material.

[0040] This invention provides a fusion protein based on an antimicrobial peptide combination and bacteriophage caustic fibrin or lysin, and the mRNA encoding it. The invention delivers a fusion mRNA expressing antimicrobial peptides LL-37, UB2, and E50-52 with the caustic fibrin GP6 or lysin GP10 of mycobacterial phage L5 into human monocyte (THP1)-induced macrophages. Utilizing the cells' autonomous expression of antimicrobial peptides and caustic fibrin, after infection with Mycobacterium tuberculosis, the antimicrobial peptide combination carried by the intracellularly expressed bacteriophage caustic fibrin specifically targets and eliminates Mycobacterium tuberculosis, achieving complete eradication of the bacteriophage. The drug formulation provided by this invention offers a rapid and simple method with high and prolonged expression levels, quickly stimulating THP1 cells to produce antimicrobial peptides that target Mycobacterium tuberculosis, enhancing the intracellular clearance capacity of macrophages for Mycobacterium tuberculosis. Attached Figure Description

[0041] Figure 1 A schematic diagram of the mRNA structure is shown, where AMP is the antimicrobial peptide.

[0042] Figure 2 This invention demonstrates the inhibitory effect of optimized single polypeptides encoding LL-37, UB2, and E50-52, and unoptimized artificially synthesized mRNA formulations, on Mycobacterium tuberculosis in macrophages.

[0043] Figure 3 The mRNA formulations encoding single polypeptides LL-37, UB2, and E50-52, as well as the combined polypeptides LL-37~UB2~E50-52, were shown to inhibit the growth of Mycobacterium tuberculosis in macrophages.

[0044] Figure 4 Immunofluorescence images showing GFP-labeled GP6 and GP10 proteins co-cultured with H37Rv;

[0045] Figure 5This study demonstrates the antibacterial activity of mRNA formulations encoding the LL-37~UB2~E50-52 combination peptide, the LL-37~UB2~E50-52 antimicrobial peptide combination fused with phage tail fibrin GP6, and the LL-37~UB2~E50-52 antimicrobial peptide combination fused with lysin GP10 against Mycobacterium tuberculosis within macrophages. Detailed Implementation

[0046] This invention provides a fusion protein and its encoding mRNA, and its application in inhibiting Mycobacterium tuberculosis. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0047] This invention provides a fusion protein based on an antimicrobial peptide combination and phage caustic fibrin or lysin, and its encoding mRNA. The mRNA can effectively express the antimicrobial peptides and phage caustic fibrin / lysin within macrophages. After infection with Mycobacterium tuberculosis, the antimicrobial peptide combination carried by the intracellularly expressed phage caustic fibrin / lysin specifically targets and kills Mycobacterium tuberculosis, thereby achieving the goal of clearing Mycobacterium tuberculosis from cells. Compared to current anti-tuberculosis drugs, the mRNA formulation of this invention can be expressed in large quantities within cells for a long duration, effectively clearing Mycobacterium tuberculosis from cells.

[0048] Antimicrobial peptides (AMPs), also known as host defense peptides, are a class of short peptides that are usually positively charged and possess antimicrobial activity. As components of the innate immune response, AMPs play a crucial role in controlling infection and maintaining the latent state of tuberculosis. Studies have shown that antimicrobial peptides such as human neutrophil peptides (HNPs), NZX, LL37, ubiquitin-derived peptides (Ub2), PR-39, E6, and hepcidin possess direct antimicrobial activity against Mtb. However, high production costs and susceptibility to degradation limit the clinical therapeutic potential of antimicrobial peptides.

[0049] Bacteriophages are viruses that invade bacteria, specifically recognizing the cell envelope of their bacterial host by producing proteins. Mycobacterial phage L5 is a phage that infects mycobacteria, especially Mycobacterium tuberculosis. Its tail protein GP6 and lysin GP10 are tools used to rapidly capture mycobacteria. Mycobacterial phage proteins can be used as pathogen capture platforms to improve the effectiveness of existing diagnostic methods.

[0050] This invention provides codon-optimized antimicrobial peptides LL-37, UB2, E50-52, GP6 (the tail fibrin of Mycobacterium phage L5), and GP10 (the lysosome), as well as antimicrobial peptide combinations LL-37~UB2~E50-52, GP6~LL-37~UB2~E50-52, and GP10~LL-37~UB2~E50-52 obtained by linking via a flexible protein linker. The specific amino acid and nucleotide sequences are shown in Table 1 below. The linker's amino acid sequences include SSGGGSRSGGGS (SEQ ID NO:19), SGGSGGSS (SEQ ID NO:20), and RGGSSGGSGGS (SEQ ID NO:21).

[0051] Table 1. Amino acid and nucleic acid sequences involved in this invention.

[0052]

[0053]

[0054]

[0055]

[0056]

[0057] The test materials used in this invention are all commercially available products. The invention will be further illustrated below with reference to specific embodiments.

[0058] Example 1 Construction and amplification of recombinant plasmids

[0059] 1. Synthesize the cDNA sequences of the mRNAs in SEQ ID No. 1~9.

[0060] 2. The DNA sequence encoding mRNA was ligated to the pRSF vector plasmid via SfiI restriction enzyme digestion and ligation. First, the vector and fragment were digested with the SfiI enzyme, and the digestion products were recovered using a gel electrophoresis apparatus. Then, the concentration of the recovered products was determined using NanoDrop. Finally, the vector and fragment were added to the ligation reaction system in the appropriate ratio according to their concentrations and incubated at room temperature for 1 hour.

[0061] 3. Transform the ligated plasmid into E. coli and inoculate it into a solid culture medium for overnight culture.

[0062] 4. Pick a single colony from the culture medium and perform colony PCR. Perform electrophoresis on the PCR products, and select colonies with the correct electrophoretic band size for first-generation sequencing.

[0063] 5. For E. coli colonies with correct nucleic acid sequences after sequencing, perform shake culture amplification, extract endotoxin-free plasmids according to the TIANGEN kit instructions, measure the concentration, and store for later use.

[0064] Example 2: In Vitro Transcription (IVT) of the Target mRNA Fragment

[0065] 1. Target fragment PCR: After obtaining a large amount of purified plasmid, the DNA sequence encoding the target mRNA, including the T7 sequence, was amplified by PCR using high-fidelity Taq polymerase. The amplification products were electrophoresed on a 1% agarose gel.

[0066] 2. Purification of the target PCR fragment: Place the electrophoresis product under UV light, cut off the correctly sized fragment, and add GSB solution to the gel at 55℃. Recover the gel solution using a recovery column, wash, and finally add ddH2O to recover the target PCR fragment.

[0067] 3. In vitro synthesis of target mRNA: In vitro transcription was performed using the T7 transcription kit (Novizan). The reaction system is as follows:

[0068] Table 2

[0069]

[0070] 4. Gently mix the components in Table 2 with a pipette, collect by short-term centrifugation, and incubate at 37°C for 2-5 hours.

[0071] 5. Mix RNA, 7.5M lithium chloride, and water in a 1:1:1 ratio and allow to settle at -20℃ for 2 hours. Collect the precipitate, centrifuge at 12,000 rpm for 15 minutes, and discard the supernatant. Then wash 2-3 times with pre-cooled 70% ethanol. After the last centrifugation, discard the supernatant, air dry, and resuspend in an appropriate amount of RNase-free water.

[0072] 6. To determine the concentration of the above RNA solution, take 10 μg of total RNA, dilute it to 15 μL, and incubate the RNA solution at 65°C for 5 minutes, then place it on ice for 5 minutes. Perform the capping operation. The reaction system is as follows:

[0073] Table 3

[0074]

[0075] 7. Gently mix the components in Table 3 using a pipette, briefly centrifuge to collect, and incubate at 37°C for 30 min. The reaction product is the target mRNA, with 5'-CAP and 5'-UTR sequentially linked to the 5' end, and 3'-UTR and poly A sequentially linked to the 3' end, as shown in the attached diagram. Figure 1 As shown.

[0076] Example 3: Detection of the antibacterial effect of a single antimicrobial peptide expressed in macrophages against Mycobacterium tuberculosis in macrophages.

[0077] 1. Target mRNAs encoding the following peptides were prepared and purified according to the methods described in Examples 1 and 2: LL-37 peptide, UB2 peptide, E50-52 peptide, and sGFP, and labeled as LL-37, UB2, E50-52, and GFP, respectively.

[0078] 2. Human mononuclear cell line THP-1 cells were seeded into 24-well plates one day in advance and cultured in 1640 medium containing 10% FBS. Phlorizate (PMA) was added to a final concentration of 100 ng / mL and cultured for 24 h to induce the mononuclear cells to adhere to the plate and become macrophages.

[0079] 3. The mRNA prepared above was transfected into macrophages using Lipofectamine 3000 (Thermo). At the same time, three groups of cells were cultured with artificially synthesized LL-37 peptide (amino acid sequence as shown in SEQ ID NO:10), UB2 peptide (amino acid sequence as shown in SEQ ID NO:11), and E50-52 peptide (amino acid sequence as shown in SEQ ID NO:12), respectively.

[0080] 4. After 6 hours of culture, GFP green fluorescence was observed under a fluorescence microscope, indicating successful mRNA expression.

[0081] 5. Replace the cell culture medium with 1640 medium containing Mycobacterium tuberculosis H37Rv (MOI=2.5) and 10% FBS, incubate for 2 hours, then replace the cell culture medium with ordinary 1640 medium containing 10% FBS and continue incubation for 5 days.

[0082] 6. After 5 days of culture, collect the supernatant. Add 500 μL of PBS lysis buffer containing 0.5% Triton X-100 to each well, and mix by pipetting 20–30 times. Mix the lysis buffer into the supernatant of the corresponding well. Transfer 100 μL of the above Mycobacterium tuberculosis mixed solution to a 1.5 ml centrifuge tube containing 900 μL of PBS. Perform three concentration gradients sequentially to obtain 10⁻⁶ concentrations of the Mycobacterium tuberculosis mixed solution. -1 10 -2 10 -3 Dilution. Take 100 μL of 10... -2 and 10 -3 The diluted solution was inoculated onto 7H10 medium, spread evenly, and incubated upside down in a 37 ℃ incubator for 3–4 weeks. Colonies were then counted.

[0083] 7. See the appendix for detailed results. Figure 2 The results showed that, compared with the control group expressing GFP, the expression of a single antimicrobial peptide in macrophages could reduce the number of Mycobacterium tuberculosis, while the three groups of artificially synthesized peptide control groups had no significant inhibitory effect on Mycobacterium tuberculosis.

[0084] Example 4: Detection of the antibacterial effect of macrophage-expressed combined antimicrobial peptides against Mycobacterium tuberculosis in macrophages

[0085] 1. Prepare and purify target mRNAs encoding the following peptides according to the methods described in Examples 1, 2 and 3: LL-37~UB2~E50-52, LL-37 peptide, UB2 peptide, E50-52 peptide and sGFP, and label them as L~U~E, LL-37, UB2, E50-52 and GFP, respectively.

[0086] 2. Human mononuclear cell line THP-1 cells were induced to adhere to the culture vessel and become macrophages. The mRNA prepared above was transfected into macrophages using Lipofectamine 3000 transfection reagent. After culturing for 6 hours, GFP green fluorescence was observed under a fluorescence microscope, indicating that the mRNA was successfully expressed.

[0087] 3. Replace the cell culture medium with 1640 medium containing H37Rv (MOI=2.5) and 10% FBS, incubate for 2 hours, then replace the cell culture medium with ordinary 1640 medium containing 10% FBS and continue incubation for 5 days.

[0088] 4. After culturing for 5 days, collect the supernatant and Mycobacterium tuberculosis in the cells, inoculate different concentrations of dilution onto 7H10 medium, spread evenly, invert and incubate at 37 ℃ for 3-4 weeks, and count the colonies.

[0089] 5. See the attached document for detailed results. Figure 3The results showed that, compared with the control group expressing GFP, the expression of the combination of antimicrobial peptides in macrophages could effectively inhibit the activity of Mycobacterium tuberculosis, and the antibacterial effect was better than that of the antimicrobial peptide group alone.

[0090] Example 5: Detection of the targeted antibacterial effect of the fusion mRNA of the combined antimicrobial peptides LL-37~UB2~E50-52 and phage fibrin GP6 or lysin GP10 expressed in macrophages against Mycobacterium tuberculosis.

[0091] 1. GFP-labeled GP6 and GP10 proteins were co-cultured with H37Rv, respectively. See the attached table for details. Figure 4 The results showed that both GP6 and GP10 could bind to H37Rv, indicating that GP6 and GP10 can serve as target-binding proteins for Mycobacterium tuberculosis.

[0092] 2. Prepare and purify the target mRNAs encoding the following polypeptides according to the methods described in Examples 1, 2 and 3: LL-37~UB2~E50-52, GP6~LL-37~UB2~E50-52, GP10~LL-37~UB2~E50-52 and sGFP, and label them as L~U~E, GP6~L~U~E, GP10~L~U~E and GFP, respectively.

[0093] 3. Human mononuclear cell line THP-1 cells were induced to adhere to the wall and become macrophages. The mRNA prepared above was transfected into macrophages using Lipofectamine 3000 transfection reagent. After culturing for 6 hours, GFP green fluorescence was observed under a fluorescence microscope, indicating that the mRNA was successfully expressed.

[0094] 4. Replace the cell culture medium with 1640 medium containing H37Rv (MOI=2.5) and 10% FBS, incubate for 2 hours, then replace the cell culture medium with ordinary 1640 medium containing 10% FBS and continue incubation for 5 days.

[0095] 5. After culturing for 5 days, collect the supernatant and Mycobacterium tuberculosis in the cells, inoculate different concentrations of dilution onto 7H10 medium, spread evenly, invert and incubate at 37 ℃ for 3-4 weeks, and count the colonies.

[0096] 6. See the attached document for detailed results. Figure 5 The results showed that the tandem combination of GP6 and GP10 with the combined antimicrobial peptides enhanced the antibacterial activity of macrophages, and the antibacterial effect of the GP6 combination was more significant.

[0097] As can be seen from the above embodiments, the mRNA preparation based on the fusion of antimicrobial peptides and bacteriophage fibrin / lysin provided by the present invention can be expressed in large quantities in cells for a long duration, and can effectively clear Mycobacterium tuberculosis from cells.

[0098] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A fusion protein, characterized in that, It includes at least two of the following: antimicrobial peptide LL-37, antimicrobial peptide UB2, and antimicrobial peptide E50-52.

2. The fusion protein according to claim 1, characterized in that, It also includes tail fibrin GP6 and / or lysin GP10.

3. The fusion protein according to claim 1 or 2, characterized in that, (1) The antimicrobial peptide LL-37 has the amino acid sequence shown in SEQ ID No. 10; The antimicrobial peptide UB2 has the amino acid sequence shown in SEQ ID No. 11; The antimicrobial peptide E50-52 has the amino acid sequence shown in SEQ ID No. 12; The tail fibrin GP6 has the amino acid sequence shown in SEQ ID No. 13; The solubilin GP10 has the amino acid sequence shown in SEQ ID No. 14; or (2) A sequence based on the amino acid sequence shown in (1) by substitution, deletion and / or addition of one or more amino acids, and an amino acid sequence that has the same or similar function as the amino acid sequence shown in (1). or (3) An amino acid sequence that has at least 80% sequence identity with the amino acid sequence shown in (1) and has the same or similar function as the amino acid sequence shown in (1).

4. The fusion protein according to any one of claims 1 to 3, characterized in that, The fusion protein comprises sequentially linked components from the N-terminus to the C-terminus: Antimicrobial peptides LL-37, UB2, and E50-52; or Tail fibroin GP6, antimicrobial peptide LL-37, antimicrobial peptide UB2, and antimicrobial peptide E50-52; or Dissolving agent GP10, antimicrobial peptide LL-37, antimicrobial peptide UB2 and antimicrobial peptide E50-52.

5. The fusion protein according to claim 4, characterized in that, The sequential linking includes linker linking, wherein the linker is independently selected from any of the amino acid sequences shown in SEQ ID No. 19-21.

6. The fusion protein according to any one of claims 1 to 5, characterized in that, The fusion protein has any one of the amino acid sequences shown in SEQ ID No. 15-17.

7. A biomaterial, characterized in that, Includes at least one of the following (4) to (6): (4) A nucleic acid molecule encoding the fusion protein according to any one of claims 1 to 6; (5) An expression vector comprising the aforementioned nucleic acid molecules; (6) Transform or transfect the host cells of the recombinant vector.

8. The biomaterial according to claim 7, characterized in that, The nucleic acid molecule includes DNA and / or mRNA, and the nucleic acid molecule has: (7) A nucleotide sequence as shown in any of SEQ ID No. 1-8; or (8) A nucleotide sequence that encodes the same protein as (7), but differs from the nucleotide sequence shown in (7) due to the degeneracy of the genetic code; or (9) A nucleotide sequence obtained by substituting, deleting, or adding one or more nucleotide sequences to the nucleotide sequence shown in (7) or (8), and which has the same or similar function to the nucleotide sequence shown in (7) or (8); or (10) A nucleotide sequence that is more than 95% identical to the nucleotide sequence described in (7), (8) or (9).

9. The following (11) and / or (12) in the preparation of medicaments for the prevention and / or treatment of Mycobacterium tuberculosis infection or products for inhibiting Mycobacterium tuberculosis: (11) The fusion protein according to any one of claims 1 to 6; (12) The biomaterial as described in claim 7 or 8.

10. A drug for the prevention and / or treatment of Mycobacterium tuberculosis infection or a product for inhibiting Mycobacterium tuberculosis, characterized in that, include: (13) The fusion protein according to any one of claims 1 to 6; and / or (14) The biomaterial as described in claim 7 or 8.

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