A Mycobacterium tuberculosis protein polypeptide and its application

Abstract

This invention relates to an anti-Mycobacterium tuberculosis (Mtb) protein polypeptide and its applications, belonging to the field of biomedicine. Specifically, it discloses a Mycobacterium tuberculosis protein polypeptide Mce3A-P6 and its applications. This invention proposes that the Mce3A-P6 polypeptide and antibodies prepared using the Mce3A-P6 polypeptide as an antigen can effectively block the interaction between Mce3A and the host cell receptor GPR108, thereby significantly preventing Mtb invasion of host cells and ultimately inhibiting the Mtb infection process. Therefore, it has clinical application value in the treatment or prevention of tuberculosis.

Description

Technical Field

[0001] This invention relates to an anti-tuberculosis mycobacterium protein polypeptide and its application, belonging to the field of biomedicine. Background Technology

[0002] Mycobacterium tuberculosis ( Mycobacterium tuberculosis Tuberculosis (TB) caused by *M. tuberculosis bacterium* (Mtb) infection is a serious infectious disease threatening human health. As a facultative intracellular pathogen, Mtb requires invasion of host cells to establish infection. Studies have shown that some effector proteins located on the surface of Mtb cells contain polypeptide sequences that bind with high specificity to alveolar epithelial cells (A549) and monocyte-macrophage (U937) cell lines, potentially promoting Mtb invasion of host cells. Therefore, antibodies generated based on immunization using these polypeptide sequences have the potential to block Mtb invasion (Ocampo M). et al ., Specific interaction between Mycobacterium tuberculosislipoprotein-derived peptides and target cells inhibits mycobacterial entry invitro, Chem Biol Drug Des, 2014,84(6):626-641; Ocampo M et al ., Functional, biochemical and 3D studies of Mycobacterium tuberculosis protein peptides for an effective anti-tuberculosis vaccine, Crit Rev Microbiol, 2014, 40(2):117-145; Rodríguez DC et al ., Mce4F Mycobacterium tuberculosis protein peptidescan inhibit invasion of human cell lines, Pathog Dis, 2015, 73(3):ftu020). The mammalian cell entry (Mce) family is a class of key effector proteins located on the surface of Mycobacterium tuberculosis (Mtb) cells and that may mediate Mtb invasion of host cells.

[0003] Studies have shown that Mce3A and Mce3E can promote the invasion of inert latex microbeads into HeLa cells (El-Shazly SARS-CoV-2 cells). et al (Internalization by HeLa cells of latex beads coated with mammalian cellentry (Mce) proteins encoded by the mce3 operon of Mycobacterium tuberculosis, J Med Microbiol, 2007, 56(Pt 9):1145-1151), but their mechanism of action in the infection process has not been reported; moreover, the molecular mechanism by which Mce family proteins promote pathogen invasion has not been elucidated. Summary of the Invention

[0004] Our research revealed that Mce3A interacts with the cell surface receptor GPR108 to promote Mtb invasion of host cells, a process dependent on the C-terminal domain of Mce3A (amino acid positions 317-425). We then designed overlapping polypeptide sequences based on this interacting domain and immunized mice to obtain specific antibodies. Further screening revealed polypeptide antibodies that significantly inhibited Mtb invasion. The results showed that the antibody produced by immunizing mice with Mce3A-P6 effectively blocked the Mtb invasion process. Therefore, this polypeptide is an important target for the development of novel tuberculosis drugs and vaccines.

[0005] This invention identifies the C-terminal domain (amino acids 317-425) of the surface protein Mce3A, associated with Mtb invasion of host cells, as promoting Mycobacterium tuberculosis infection by recognizing and binding to the G protein-coupled receptor 108 (GPR108) on the host cell surface. Further prediction of potential B-cell epitopes within this domain revealed amino acids 327-422 in the key domain of Mce3A for host cell invasion as potential B-cell epitopes. We hypothesize that antibodies targeting this epitope may block the interaction between Mce3A and the host cell surface receptor, thereby preventing Mtb invasion. Based on the B-cell epitope sequence of Mce3A (positions 327-422), we designed overlapping peptides and prepared peptide antibodies. Final screening revealed that antibodies induced by peptide P6 effectively blocked the interaction between Mce3A and GPR108, thus preventing Mtb invasion of host macrophages and inhibiting the Mtb infection process.

[0006] The present invention provides a Mycobacterium tuberculosis protein polypeptide, characterized in that its amino acid sequence is the C-terminal domain of Mce3A, specifically at amino acid positions 317-425, more specifically at amino acid positions 327-422, and more preferably at amino acid positions WANYFPVTRAVPEPPSIRQCIPGPA.

[0007] In addition, it is a polypeptide containing an amino acid sequence including the amino acid sequence shown in (a), (b), or (c) below:

[0008] (a) The amino acid sequence shown in SEQ ID NO:1;

[0009] (b) A polypeptide or analogue derived from the amino acid sequence in (a) by substitution, deletion or addition of one or more amino acids;

[0010] (c) A polypeptide or analogue derived from (a) with an overall amino acid sequence similarity of more than 85%, more than 88%, and more than 96% with respect to (a) and (b).

[0011] This invention provides the use of molecules that interact with the aforementioned Mycobacterium tuberculosis protein polypeptide in the preparation of medicaments for the treatment or prevention of diseases caused by mycobacteria.

[0012] Specifically, the molecules are small molecule compounds, polypeptides, proteins, and antibodies.

[0013] Preferably, the mycobacterium is selected from Mycobacterium tuberculosis.

[0014] The present invention also provides the use of the aforementioned Mycobacterium tuberculosis protein polypeptide in the preparation of drugs or vaccines for diseases caused by mycobacterial infection.

[0015] Specifically, the Mycobacterium tuberculosis protein polypeptide is used as a target to prepare molecules that can bind to or interact with it.

[0016] Furthermore, the molecules are small molecule compounds, polypeptides, proteins, or antibodies.

[0017] Preferably, the Mycobacterium tuberculosis protein polypeptide is used as a vaccine to prevent or treat diseases caused by mycobacterial infection.

[0018] More preferably, the mycobacterium is selected from Mycobacterium tuberculosis, and the disease is selected from tuberculosis.

[0019] Beneficial effects: The Mce3A-P6 polypeptide provided by this invention can serve as a new target for the development of anti-tuberculosis infection drugs; it can also be used directly as a drug and vaccine to induce antibodies in vivo to prevent mycobacterial invasion and infection. Attached Figure Description

[0020] Figure 1 Identify the key structural domains of Mce3A that mediate Mtb invasion of host cells.

[0021] Figure 2 To investigate the effect of host cell receptor GPR108 on Mce3A-mediated Mtb invasion of the host.

[0022] Figure 3 Detection of the titer of polyclonal antibodies prepared from peptides with overlapping amino acid domains at positions 327-422 of Mce3A.

[0023] Figure 4 Detection of the efficacy of polyclonal antibodies prepared from Mce3A-based peptides with overlapping amino acid domains at positions 327-422 in blocking Mtb infection.

[0024] Figure 5 Mce3A-P6 peptide and its antibody can directly block the interaction between the pathogen invasion protein Mce3A and the host cell receptor GPR108. Detailed Implementation

[0025] The present invention will be further illustrated below with specific embodiments in order to better understand the present invention, but these embodiments do not constitute a limitation thereof.

[0026] Example 1: Detection of the effects of Mce3A and its truncated form on Mce3A-mediated Mtb invasion of host cells

[0027] The main operating steps are as follows:

[0028] 1. Design primers and construct GST-Mce3A and its truncated forms (including truncated forms at positions 1-317 and 317-425). The primer sequences are as follows.

[0029] pGEX6P-1-Mce3A-F: CGGGATCCATGAGACGCGGGCCGGGTCGA

[0030] pGEX6P-1-Mce3A-R: CGGAATTCTCATGGCTGCTCCCCCGC

[0031] pGEX6P-1-Mce3A (1-317)-F: CGGGATCCATGAGACGCGGGCCGGGTCGA

[0032] pGEX6P-1-Mce3A (1-317)-R: CGGAATTCTCA CGTGCGCCCGTCGGCCCC

[0033] pGEX6P-1-Mce3A (317-425)-F: CGGGATCCCTACAACTCGATTGTGGCGCTA

[0034] pGEX6P-1-Mce3A (317-425)-R: CGGAATTCTCATGGCTGTCCCCGC

[0035] 2. The constructed plasmid was transformed into [a specific material] using the thermal shock method. E. coli (BL21) In Escherichia coli, the expression of GST fusion protein was induced by adding IPTG to Escherichia coli liquid culture medium.

[0036] 3. Collect E. coli cells by centrifuging at 8000 rpm for 15 minutes after induction at 16℃ for 18 hours. After the cells are disrupted by ultrasonic disruption, the supernatant is collected by high-speed centrifugation. GST affinity resin is added to the supernatant and incubated at 4℃ for 4 hours. Then, elution buffer is added and the mixture is concentrated in a concentration tube to obtain the GST fusion protein.

[0037] 4. One day in advance, seed RAW264.7 cells into 6-well plates and add GST, GST-Mce3A, GST-Mce3A (1-317) and GST-Mce3A (317-425) proteins at a concentration of 20 µg / mL one hour before infection.

[0038] 5. Using wild-type MtbH37Rv and Mtb∆ respectively Mce3A 、Mtb∆ Mce3A : Mce3A The strain infected RAW264.7 cells pretreated with GST fusion protein. Two hours after infection, the cells were washed three times with PBS and collected. The cells were then lysed with 0.02% SDS, multiplied in 7H9 liquid medium, and then plated on 7H10 solid medium.

[0039] 6. Invert the 7H10 solid culture medium into a 37℃ constant temperature bacterial incubator, and count the bacterial colony count after 2-3 weeks of incubation.

[0040] The results are as follows Figure 1 As shown, compared to the GST and GST-Mce3A (1-317) treatment groups, the GST-Mce3A and GST-Mce3A (317-425) treatment groups achieved a blocking effect of over 90% against Mce3A-mediated Mtb invasion. This suggests that the key functional domain of Mce3A-mediated pathogen invasion is positions 317-425.

[0041] Example 2: Detection of the effects of the Mce3A (317-425) domain and the GPR108 receptor in host cells on Mce3A-mediated Mycobacterium tuberculosis invasion.

[0042] The specific operating steps are as follows:

[0043] 1. Design online (http: / / crispr.mit.edu / ) sgRNA sequences targeting the murine GPR108 protein: F: 5'- CACCGATGACCGCGAGGCCTTCGAT-3'; R: 5'- AAACATCGAAGGCCTCGCGGTCATC-3'.

[0044] 2. After annealing the double-stranded GPR108 sgRNA, it was ligated into the pX458 vector and transfected into RAW264.7 cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA).

[0045] 3. Flow cytometry was used to sort the cells 24 hours after transfection. The green fluorescently labeled RAW264.7 cells obtained by flow cytometry were cultured in 96-well plates. After the RAW264.7 cells grew into monoclonal cells and their knockout efficiency was identified, the cells were cryopreserved for Mtb infection experiments.

[0046] 4. Wild-type and GPR108 knockout RAW264.7 cells were seeded in 6-well plates and treated with Mtb and Mtb ∆, respectively. Mce3A Mtb ∆ Mce3A : Mce3A Mtb ∆ Mce3A : Mce3A RAW264.7 cells were infected with strain (∆317-425). Two hours after infection, the cells were washed three times with PBS and collected. The cells were then lysed with 0.02% SDS and spread onto 7H10 solid medium after being multiplied by 7H9 liquid medium. The 7H10 solid medium was inverted in a 37°C constant temperature bacterial incubator, and the bacterial colony count was calculated after 2-3 weeks of culture.

[0047] The results are as follows Figure 2 As shown, compared with the wild-type RAW264.7 cell infection group, GPR108 knockout can block Mce3A-mediated Mtb invasion by 90%, suggesting that GPR108 is a key receptor for Mce3A to invade host cells.

[0048] Example 3: Prediction of B-cell epitopes of key domains of Mtb Mce3A invading host cells

[0049] ABCpred was used to predict potential B-cell epitope peptides in the key structural domains (amino acid positions 317-425) of Mce3A invading host cells. The prediction results are shown in Table 1.

[0050] Table 1: Prediction results of B-cell epitope peptides of Mtb Mce3A

[0051] polypeptide number amino acid sites peptide length polypeptide sequence 1 5-12 8 PGRHRLHD 2 35-39 5 TGSLR 3 52-57 6 GLVMDS 4 75-82 8 GRIEWAQN 5 95-98 4 IRYI 6 111-112 2 AF 7 122-162 41 PQNPSRARLSAGAVLHSKNVSTEINTVFENVVDLLNMIDPL 8 176-184 9 RGQGERIGQ 9 201 1 D 10 205 1 G 11 207-208 2 WR 12 211-223 13 KNFTDTYDAAAQD 13 242-243 2 ST 14 257 1 N 15 261 1 N 16 264 1 G 17 268 1 D 18 286-290 5 KYNPE 19 300 1 W 20 303 1 D 21 307 1 Y 22 310-313 4 WGGA 23 327-422 96 GNDPYVYPDNLPVVAAKGPGPGRPGCGPLPDATHNFPVRQLVTNTGWGTGLDIRPNPGIGHPCWANYFPVTRAVPEPPSIRQCIPGPAIGPNPAAG

[0052] Example 4: Design of Mce3A protein overlapping peptides

[0053] Amino acids 327-422 in the key domain of Mce3A for invading host cells are potential B-cell epitopes. We hypothesize that antibodies targeting this epitope may block the interaction between Mce3A and host cell surface receptors, thereby preventing Mtb from invading host cells. Based on the 327-425 domain of Mce3A, we designed overlapping peptides, progressively extracting peptides of 25 amino acids in length from the N-terminus to the C-terminus of the target protein, moving 12-13 amino acids at a time (with 12-13 amino acid overlaps between adjacent peptide sequences), ultimately forming an overlapping peptide library.

[0054] Table 2: Mtb Mce3A overlapping polypeptide sequences

[0055] polypeptide name amino acid sequence Serial Number Mce3A-P1 GNDPYVYPDNLPVVAAKGGPGGRPG SEQ ID NO:1 Mce3A-P2 VAAKGGPGGRPGCGPLPDATHNFPV SEQ ID NO:2 Mce3A-P3 CGPLPDATHNFPVRQLVTNTGWGTG SEQ ID NO:3 Mce3A-P4 RQLVTNTGWGTGLDIRPNPGIGHPC SEQ ID NO:4 Mce3A-P5 LDIRPNPGIGHPCWANYFPVTRAVP SEQ ID NO:5 Mce3A-P6 WANYFPVTRAVPEPPSIRQCIPGPA SEQ ID NO:6 Mce3A-P7 EPPSIRQCIPGPAIGPNPAA SEQ ID NO:7

[0056] Example 5: Preparation of polyclonal antibodies by immunizing mice with peptides

[0057] 1. Peptide-carrier protein coupling modification, the specific operation steps are as follows:

[0058] Because synthesized peptides typically have small molecular weights and weak immunogenicity, they are difficult to adequately induce an immune response to produce antibodies. Therefore, it is common practice to conjugate peptides with carrier proteins carrying numerous antigenic epitopes to fully activate the immune system and prepare sufficient quantities of antibodies. In this embodiment, we selected the most commonly used carrier protein, mcKLH (mariculture keyhole limpet hemocyanin), also known as hemocyanin (cat# 77605, Thermoscientific).

[0059] (1) Dissolve the synthesized polypeptide (2 mg / mL) in 1×PBS.

[0060] (2) Prepare mcKLH protein to 10 mg / mL using ddH2O.

[0061] (3) Mix the peptide and mcKLH protein in equal volumes and incubate at room temperature for 2 hours.

[0062] (4) ThermoZeba was adopted. TM The spin desalting column (cat# 89889, Thermoscientific) was desalted.

[0063] 2. The specific steps for vaccine preparation and mouse immunization are as follows:

[0064] (1) Vaccine preparation: The single-dose vaccine consists of 50 μg (peptide-carrier protein) plus 50 μL of MF59 adjuvant (cat#vac-adx-10), for a total of 100 μL. Use a pipette to blow and swirl 50 times, then seal with sealing film and shake to mix for 10 minutes to ensure complete emulsification.

[0065] (2) Immunization of mice: 6-8 week old female BALB / c mice (purchased from Beijing Vital River Company) were immunized by applying 100 μL to the thigh muscles of the mice. The inner thigh was thoroughly disinfected with cotton balls before exposing the muscles for immunization. The left and right legs were immunized alternately, once every two weeks, for a total of 3 immunizations.

[0066] 3. The specific steps for obtaining immune serum are as follows:

[0067] (1) The mice were anesthetized with 200 μL of tribromoethanol.

[0068] (2) Obtain as much mouse blood as possible by collecting blood from the heart.

[0069] (3) After standing at room temperature for 2 hours, centrifuge at 4000 rpm for 10 minutes and take serum for ELISA detection.

[0070] The results are as follows Figure 3 As shown: ELISA results show that we obtained specific antibodies against 7 peptides.

[0071] Example 6: Polypeptide Antibody Purification

[0072] The specific operating steps are as follows:

[0073] 1. Column packing: Fill an empty PD-10 column with 2 mL of binding / washing buffer (NaCl, 0.5 M; Na2HPO4, 20 mM, pH 8.0), and then fill the column with 5 mL of well-mixed Protein A / G gel resin.

[0074] 2. Column washing: Drain the liquid from the column and wash the column again with 10 times the volume of binding / washing buffer.

[0075] 3. Sample loading: Add the prepared serum containing peptide antibodies into the column in batches, depending on the column's capacity.

[0076] 4. Elute contaminating proteins: Rinse the column with binding / washing buffer until the buffer is free of proteins.

[0077] 5. Antibody Collection: Add elution buffer (7.507 g glycine dissolved in double-distilled water, pH adjusted to 3.0 with hydrochloric acid, and finally brought to a final volume of 500 mL) to the column, and collect the eluent (approximately 0.3–0.4 mL / tube) until the eluent is free of protein. Measure the protein content in each collection tube and combine the protein tubes. (Note: Approximately 150 μL of 1 M Tris-HCl, pH 9.0 buffer should be added to the collection tubes beforehand to prevent antibody inactivation in an overly acidic environment).

[0078] 6. The eluted antibody was collected by PBS dialysis. The obtained antibody protein was quantified and used to process cells for functional studies.

[0079] Example 7: Determination of the protective effect of different peptides on antibody production at the cellular level

[0080] The specific operating steps are as follows:

[0081] 1. Take freshly cultured wild-type Mtb H37Rv strain, disperse it using ultrasonication (using a BACspreader bacterial ultrasonic dispersion counter, Guangdong Tibikang), and then measure the OD. 600 nm Count (OD) 600 0.6 = 1 × 10 8 ).

[0082] 2. Mtb was added to Raw264.7 cells at MOI=1, along with antibodies against different peptides (0.5 μg / mL). Mouse IgG (0.5 μg / mL) was added to the control group, and the cells were co-cultured at 37°C.

[0083] 3. After incubating cells with Mtb for 1 hour, wash three times with PBS to remove bacteria that have not entered the cells, add 500 μL of 0.05% SDS to lyse the cells, and plate the cells to detect the amount of bacteria that have entered the cells.

[0084] The results are as follows Figure 4 As shown, compared with the IgG-treated control group, the bacterial count in macrophages treated with the Mce3A-P6 peptide-specific antibody decreased by about 50%-70%, indicating that the Mce3A-P6 peptide antibody has a significant inhibitory effect on Mtb invasion of host cells in the early stage of Mtb infection.

[0085] Example 8: Detection of the blocking effect of Mce3A-P6 peptide and its antibody on the interaction between pathogen invasion protein Mce3A and host cell receptor GPR108.

[0086] The specific operating steps are as follows:

[0087] 1. Transfect pEGFP-N1-GPR108 plasmid into 293T cells to overexpress the GFP-GPR108 fusion protein.

[0088] 2. After 36 hours of cell transfection, the cells were digested with trypsin and evenly aliquoted into five 1.5 mL centrifuge tubes.

[0089] 3. Incubation between peptides and proteins:

[0090] (1) Mouse IgG (negative control) at a final concentration of 5 μg / mL and mouse Mce3A-P6 monoclonal antibody at a final concentration of 5 μg / mL were incubated with GST-Mce3A fusion protein labeled with 594 fluorescence (AAT Bioquest, 1230) at room temperature for 10 minutes.

[0091] (2) Incubate 5 μg / mL bovine serum albumin (BSA) and 5 μg / mL Mce3A-P6 peptide with the two tubes of 293T cells overexpressing GFP-GPR108 fusion protein from step 2 at room temperature for 10 minutes.

[0092] 4. The two tubes of samples incubated in step 3 (1) were added to the two tubes of 293T cells overexpressing the GFP-GPR108 fusion protein prepared in step 2. The samples incubated in step 3 (2) were added with 5 μg / mL of GST-Mce3A fusion protein labeled with 594 fluorescence (AAT Bioquest, 1230). The remaining tube of cells in step 2 was added with an equal volume of PBS as a control group.

[0093] 5. All samples were incubated at room temperature for 1 hour, and then the cells were washed three times with PBS.

[0094] 6. Flow cytometry was used to detect the 594 fluorescence intensity on the surface of each sample cell, which represents the amount of Mce3A protein bound to the surface of GFP-GPR108 overexpressing cells.

[0095] like Figure 5 As shown, both the antibody and the Mce3A-P6 peptide can block the binding of Mce3A protein to the cell surface to varying degrees. Compared with the IgG and BSA treatment groups used as negative controls, the amount of Mce3A protein bound to the cell surface decreased by approximately 40%-60% in both the Mce3A-P6 peptide antibody treatment group and the Mce3A-P6 peptide treatment group.

Claims

1. A Mycobacterium tuberculosis protein polypeptide, characterized in that, Its amino acid sequence is WANYFPVTRAVPEPPSIRQCIPGPA.

2. The use of the Mycobacterium tuberculosis protein polypeptide according to claim 1 in the preparation of a drug for preventing diseases caused by Mycobacterium tuberculosis infection.

3. The application according to claim 2, characterized in that, The drug in question is a vaccine.

4. The application according to claim 3, characterized in that, The Mycobacterium tuberculosis protein polypeptide according to claim 1 is used as a vaccine to prevent tuberculosis caused by Mycobacterium tuberculosis infection.

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