Polypeptide having Anti-mycobacterium tuberculosis activity, preparation method therefor and use thereof

Abstract

A polypeptide having anti-mycobacterium tuberculosis activity, a preparation method therefor and the use thereof, belonging to the technical field of biology. The preparation method for the polypeptide comprises the following steps: (1) preparing a straight-chain polypeptide, the sequence of the straight-chain polypeptide being shown as SEQ ID NO: 1; and (2) carrying out a dehydration condensation reaction on sulfydryl of two cysteine residues in the straight-chain polypeptide and 1,3-bis(hydroxymethyl)urea to obtain the polypeptide. The polypeptide has small cytotoxicity and good anti-mycobacterium tuberculosis effect.

Description

A polypeptide with anti-mycobacterium tuberculosis activity, preparation method and application thereof Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a polypeptide with anti-mycobacterium tuberculosis activity, a preparation method and an application thereof. Background Art

[0002] Antimicrobial peptides are structurally diverse and found in mammalian species. They are natural host defense proteins secreted by the innate immune system and have significant inhibitory and antimicrobial effects on microorganisms (bacteria, viruses, etc.) and even tumor cells. BMAP-27 is a well-known peptide derived from cattle with an amphipathic α-helical terminus formed by the cationic NH2. It has been shown to have potent antibacterial activity against bacteria, fungi, viruses, and parasites. However, BMAP-27 exhibits certain cytotoxicity, making it unsuitable for pharmaceutical use. BMAP-18 is a truncated form of BMAP-27. Although its toxicity to mammalian and insect cells is reduced, its toxicity and antibacterial effects remain unsatisfactory.

[0003] Tuberculosis (TB) is a globally prevalent zoonosis that continues to endanger human health and life. Worldwide, TB remains one of the top 10 causes of death in humans. The global number of tuberculosis deaths increased from 1.4 million in 2019 to 1.5 million in 2020, and then to 1.6 million in 2021. The number of patients with multidrug-resistant / rifampicin-resistant TB was 437,000 in 2020, and increased to 450,000 in 2021. Because commonly used drugs for the treatment of tuberculosis, such as ethambutol, isoniazid, and rifampicin, have certain drug toxicity, are prone to hepatotoxicity, are prone to drug resistance, and have unstable treatment effects, tuberculosis still poses a huge threat and damage to human safety and animal husbandry development.

[0004] The pathogens of tuberculosis include Mycobacterium tuberculosis, etc. The existing technology lacks drugs that have little side effects and good effects against Mycobacterium tuberculosis.

[0005] Summary of the Invention

[0006] The purpose of the present invention is to provide a polypeptide which has low cytotoxicity and good effect against Mycobacterium tuberculosis.

[0007] The purpose of the present invention is achieved by adopting the following technical solutions:

[0008] A polypeptide with anti-Mycobacterium tuberculosis activity, the structural formula is as follows:

[0009] The present invention also provides a method for preparing the polypeptide, comprising the following steps:

[0010] (1) preparing a linear polypeptide, wherein the sequence of the linear polypeptide is as shown in SEQ ID NO: 1;

[0011] (2) The thiol groups of the two cysteine ​​residues in the linear polypeptide undergo a dehydration condensation reaction with 1,3-bis(hydroxymethyl)urea to obtain the polypeptide.

[0012] In the present invention, the molar ratio of the linear polypeptide to 1,3-bis(hydroxymethyl)urea is 1:8-12.

[0013] In the present invention, the linear polypeptide and 1,3-bis(hydroxymethyl)urea in step (2) are dissolved in solvents respectively, and then mixed to carry out a dehydration condensation reaction.

[0014] In the present invention, the solvent for the linear polypeptide is one of a mixed solution of trifluoroacetic acid and guanidine hydrochloride, urea aqueous solution, acetonitrile, and PBS buffer; the solvent for 1,3-bis(hydroxymethyl)urea is one of water, acetonitrile, methanol, and DMF.

[0015] In the present invention, the concentration of the urea aqueous solution is 4-8 mol / L.

[0016] In the present invention, the concentration of the linear polypeptide in the solvent is 1 mmol / L-8 mmol / L, and the concentration of 1,3-bis(hydroxymethyl)urea in the solvent is 18 mmol / L-80 mmol / L.

[0017] In the present invention, an acid reaction regulator is added to the reaction in step (2); the acid reaction regulator is trifluoroacetic acid; and the volume ratio between the linear polypeptide solution and the acid reaction regulator is 1:1 to 1:3.

[0018] In the present invention, the reaction temperature in step (2) is 0-40° C., and the reaction time is 0-10 min.

[0019] The present invention also provides use of the polypeptide in preparing drugs for resisting Mycobacterium tuberculosis.

[0020] The polypeptide SAH1 of the present invention has anti-Mycobacterium tuberculosis activity and has the following advantages over the unmodified polypeptide BMAP-18: (1) Safety and fewer side effects: SAH1 has significantly lower cytotoxicity against mouse macrophages than BMAP-18, with the cytotoxicity IC50 value of SAH1 being 300 μM and that of BMAP-18 being 150 μM, thus significantly improving safety. (2) Better anti-Mycobacterium tuberculosis effect: In the minimum inhibitory test, SAH1 exhibited a strong anti-BCG bacterial infection effect at a concentration of 12.5 μM, while under the same conditions, the minimum inhibitory concentration of BMAP-18 was 300 μM, and the antibacterial activity was only one-twenty-fourth of that of SAH1. Scanning electron microscopy also visually observed that SAH1 had a significantly improved antibacterial activity compared to BMAP-18. (3) Stronger resistance to protease hydrolysis: When 5 μg / mL of trypsin was added to BMAP-18, all peptides were hydrolyzed after 30 minutes. However, in the same environment, 45.89% of SAH1 remained unhydrolyzed after 30 minutes, and 30.21% remained after 1 hour. SAH1, an antimicrobial peptide, has a unique mode of action in the post-antibiotic era, which can achieve therapeutic effects while avoiding the side effects of drug resistance. Therefore, it has a very broad application prospect and is of great research value. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Reaction principle for preparing peptide SAH1.

[0022] Figure 2 is the crude linear polypeptide GRFC 4 RFRC 8 Chromatogram of KFKKLFKKLS-OH.

[0023] Figure 3 is the peptide GRFC 4 RFRC 8 Mass spectrum of KFKKLFKKLS-OH.

[0024] FIG4 is a preparative chromatogram of a 1,3-bis(hydroxymethyl)urea-modified peptide.

[0025] FIG5 is a mass spectrum of a peptide modified with 1,3-bis(hydroxymethyl)urea.

[0026] FIG6 shows the cell viability of RAW264.7 cells treated with SAH1 and BMAP-18 at various concentrations, wherein the horizontal axis represents the concentration in μM and the vertical axis represents the cell viability in %.

[0027] FIG7 is the fitting results of the IC50 values ​​of SAH1 and BMAP-18 in RAW264.7 cells.

[0028] FIG8 is a scanning electron microscopy observation showing the effects of SAH1 and BMAP-18 on BCG cell membrane integrity, wherein (A) is a negative control, (B) is BCG treated with BMAP-18, and (C) is BCG treated with SAH1.

[0029] FIG9 shows the effects of SAH1 on the BCG cell membrane and internal cell structure observed by transmission electron microscopy.

[0030] Figure 10 is a chromatogram of the RP-HPLC detection of the remaining polypeptide of BMAP-18 in a trypsin (5 μg / mL) environment at different times. The reaction time in Figure A is 0 min, the reaction time in Figure B is 15 min, and the reaction time in Figure C is 30 min.

[0031] Figure 11 is a chromatogram of the residual polypeptide of SAH1 detected by RP-HPLC at different times under the action of trypsin (5 μg / mL). The reaction time in Figure A is 0 min, the reaction time in Figure B is 15 min, the reaction time in Figure C is 30 min, and the reaction time in Figure D is 60 min. DETAILED DESCRIPTION

[0032] The BCG bacteria in this application is attenuated bovine tuberculosis Mycobacterium, ATCC number is bio-77983, purchased from China Veterinary Drug Administration.

[0033] The technical solutions of the present invention will be described in detail below with reference to the accompanying drawings, but the protection scope of the present invention is not limited to the following embodiments.

[0034] Example 1 Screening and preparation of polypeptide SAH1

[0035] 1. Screening of the SAH1 peptide

[0036] The sequence of BMAP-18 (from N-terminus to C-terminus) is: GRFKRFRKKFKKLFKKLS-OH (Gly-Arg-Phe-Lys-Arg-Phe-Arg-Lys-Lys-Phe-Lys-Lys-Leu-Phe-Lys-Lys-Leu-Ser-OH). BMAP-18 was modified by introducing point mutations at any or two random amino acid sites, ultimately leading to the discovery that the compound SAH1 exhibits highly effective anti-Mycobacterium tuberculosis activity while reducing cytotoxicity. As shown in Figure 1, the specific modification method is as follows: the amino acids at positions 4 and 8 of BMAP-18 are mutated to cysteine ​​to obtain GRFCRFRCKFKKLFKKLS-OH (Gly-Arg-Phe-Cys-Arg-Phe-Arg-Cys-Lys-Phe-Lys-Lys-Leu-Phe-Lys-Lys-Leu-Ser-OH), which is then subjected to a dehydration condensation reaction with 1,3-bis(hydroxymethyl)urea to form a cyclic peptide with the following structure:

[0037] 2. Preparation of SAH1 Peptide

[0038] (1) Linear polypeptide GRFC 4 RFRC 8 Preparation of KFKKLFKKLS-OH

[0039] Fmoc solid phase peptide synthesis: GRFC 4 RFRC 8 KFKKLFKKLS-OH (from N-terminus to C-terminus).

[0040] To couple the first amino acid: Weigh 0.2 mmol of Wang resin onto a solid-phase reaction column loaded with oxazolidinone. Wash the column twice with DMF, then swell the resin with DMF for 30 minutes. Dissolve 383.4 mg (1 mmol) of Fmoc-Ser(tBu)-OH and 142.11 mg (1 mmol) of Oxyma (ethyl 2-oximecyanoacetate) in DMF. Add 0.25 g (2 mmol) of DIC (N,N'-diisopropylcarbodiimide), activate the column for 3 minutes, and then add the column to the DMF-swollen resin. Incubate the column at room temperature for 1 hour. Monitor the reaction endpoint with ninhydrin. If the resin becomes colorless and transparent, terminate the reaction. If the resin develops color, continue the reaction for 1 hour. After the reaction is completed, the reaction solution is removed, and the resin is washed three times with DMF and then the Fmoc protecting group is removed: a 20% piperidine aqueous solution is added to submerge the resin, stirred with a glass rod, and reacted for 3 minutes before removing the piperidine solution. The resin is washed three times with DMF, and a 20% piperidine aqueous solution is added again to submerge the resin. After reacting for 4 minutes, the reaction solution is removed and the resin is washed 3-5 times with DMF.

[0041] The second protected amino acid was coupled according to the method of coupling Fmoc-Ser(tBu)-OH.

[0042] Using the above method, the remaining amino acids were coupled sequentially from the C-terminus to the N-terminus. Finally, the peptide GRFC was obtained. 4 RFRC 8 KFKKLFKKLS-OH resin.

[0043] Peptide GRFC 4 RFRC 8 The KFKKLFKKLS-OH resin was washed 3 times with DMF and 2 times with DCM (dichloromethane). After vacuum drying, the cleavage reaction was carried out. The specific method is as follows: add cleavage solution, react at room temperature for 1 hour, filter the resin, collect the filtrate, repeat the cleavage once with a small amount of cleavage solution, and combine the two filtrates. The filtrate was slowly added to ice ether, precipitated, centrifuged, the supernatant was discarded, and the crude linear polypeptide GRFC was obtained by vacuum drying. 4 RFRC 8 KFKKLFKKLS-OH. The lysis solution is a mixed solution of TFA (trifluoroacetic acid), TIS (triisopropylsilane) and H2O in a volume ratio of 95:2.5:2.5.

[0044] Crude linear polypeptide GRFC 4 RFRC 8 KFKKLFKKLS-OH was freeze-dried. The freeze-drying method was as follows: freeze into solid form with liquid nitrogen, then hang in a freeze dryer and freeze-dry to obtain solid powder. LC-MS was used to identify the linear polypeptide GRFC. 4 RFRC 8KFKKLFKKLS-OH, the chromatogram is shown in Figure 2, the mass spectrum is shown in Figure 3, and the mass spectrum data are as follows:

[0045] M=2292.3,found:1147[M+2H + ] 2+ ,765.25[M+3H + ] 3+ ,574.33[M+4H + ] 4+ ,459.75[M+5H + ] 5+ .

[0046] linear polypeptide GRFC 4 RFRC 8 The structural formula of KFKKLFKKLS-OH is as follows:

[0047] Sequence of the linear polypeptide (SEQ ID NO: 1): GRFCRFRCKFKKLFKKLS.

[0048] (2) Using 1,3-bis(hydroxymethyl)urea to modify and bind two cysteine ​​residues in a linear polypeptide

[0049] Crude linear polypeptide GRFC 4 RFRC 8 KFKKLFKKLS-OH reacts directly with 1,3-bis(hydroxymethyl)urea under acidic conditions to obtain bound cyclic peptides, which reduces the operation process, improves production efficiency, and reduces the use of organic solvents. The specific steps are as follows:

[0050] Step 1: Take crude linear polypeptide GRFC 4 RFRC 8 KFKKLFKKLS-OH was dissolved in a mixed solution of TFA (trifluoroacetic acid) and guanidine hydrochloride in a volume ratio of 1:1 to obtain a 2 mmol / L polypeptide solution; 1,3-bis(hydroxymethyl)urea was dissolved in water to obtain a 20 mmol / L 1,3-bis(hydroxymethyl)urea aqueous solution.

[0051] Step 2: At room temperature, add anhydrous trifluoroacetic acid to a 2 mmol / L peptide solution, followed by a 20 mmol / L 1,3-bis(hydroxymethyl)urea aqueous solution. Incubate at room temperature for 1 minute to obtain a crude 1,3-bis(hydroxymethyl)urea-modified peptide. The volume ratio of the peptide solution, anhydrous trifluoroacetic acid, and 1,3-bis(hydroxymethyl)urea aqueous solution is 1:1:1.

[0052] Step 3: The crude 1,3-bis(hydroxymethyl)urea-modified peptide was purified by semi-preparative HPLC using the following conditions: Mobile phases: A: H₂O containing 0.1% (volume percentage) TFA, B: acetonitrile containing 0.1% (volume percentage) TFA; elution time: 0-30 min, mobile phase B from 0 to 60% in 2% increments per minute; column: Waters C₁ₒ column; flow rate: 15 mL / min; detection wavelength: 214 nm. As shown in Figure 4, the prepared 1,3-bis(hydroxymethyl)urea-modified peptide was of high purity.

[0053] The purified 1,3-bis(hydroxymethyl)urea-modified peptide was lyophilized. The lyophilization method was as follows: freezing into a solid in liquid nitrogen, then hanging in a lyophilizer for lyophilization, and lyophilization to obtain a solid powder. The purified 1,3-bis(hydroxymethyl)urea-modified peptide was identified by mass spectrometry, as shown in Figure 5. The mass spectrometry data are as follows: M = 2376.9, found: 1188.92 [M+2H + ] 2+ ,792.92[M+3H + ] 3+ ,595.00[M+4H + ] 4+ ,476.25[M+5H + ] 5+ .

[0054] The structural formula of the 1,3-bis(hydroxymethyl)urea-modified peptide is as follows:

[0055] The 1,3-bis(hydroxymethyl)urea-modified polypeptide was designated as polypeptide SAH1.

[0056] Example 2 Detection of cell viability of RAW264.7 cells by CCK-8 assay using SAH1 and BMAP-18

[0057] The cytotoxicity of SAH1 and BMAP-18 on RAW 264.7 cells was determined by CCK-8 assay, namely WST (chemical name: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-dithiophenyl)-2H-tetrazolium monosodium salt) colorimetric assay. The specific method is as follows: 1×10 5RAW 264.7 cells were plated in each experimental well. 100 μL of a serially diluted SAH1 peptide solution at concentrations ranging from 3.125 μM to 200 μM was added to each experimental well and incubated at 37°C, 5% CO₂ for 24 hours. Positive control wells were prepared with DMEM complete medium instead of the SAH1 peptide solution, while the rest of the wells were treated the same way. Blank wells were also prepared without cells and filled with DMEM complete medium. After incubation, 10 μL of CCK-8 reaction solution (Beijing Solebo) was added to each well and incubated at 37°C for an additional 2 hours. Finally, cell viability was assessed by measuring absorbance at 450 nm using a microplate reader (Synersymx, Biotek). Cell viability relative to the positive control wells was calculated using the formula: cell viability = (absorbance of the experimental well - absorbance of the blank well) / (absorbance of the positive control well - absorbance of the blank well) * 100%.

[0058] In addition, the same method was used to detect the survival rate of cells after treatment with serially diluted BMAP-18 aqueous solutions with concentrations ranging from 3.125 μM to 200 μM.

[0059] As shown in Figure 6, the cytotoxicity of SAH1 was significantly lower than that of BMAP-18 at each concentration gradient. When the SAH1 concentration was within 50 μM, it had no obvious cytotoxicity to RAW264.7 cells (cell survival rate > 90%), and when the BMAP-18 concentration was within 25 μM, it had no obvious cytotoxicity to RAW264.7 cells (cell survival rate > 90%).

[0060] Example 3 Fitting results of IC50 values ​​of SAH1 and BMAP-18 on RAW264.7 cells

[0061] In a 96-well plate, 1 × 10 5 RAW 264.7 cells were added to each experimental well. 100 μL of a serially diluted SAH1 peptide solution at concentrations ranging from 3.125 μM to 200 μM was added to each experimental well and incubated at 37°C, 5% CO₂ for 24 hours. A positive control well was set up, with DMEM complete medium replacing the SAH1 peptide solution. The other wells were treated the same as the experimental wells. After incubation, 10 μL of CCK-8 reaction solution (Beijing Solebao) was added to each well and incubated at 37°C for an additional 2 hours. Finally, the absorbance at 450 nm was measured using a microplate reader (Synersymx, Biotek). The OD value of the positive control well was designated as B0, while the OD value of the experimental well with antimicrobial peptide added was designated as B. The ratio B / B0 was defined as the inhibition rate. The concentration of antimicrobial peptide that resulted in a 50% inhibition rate was designated as the IC₅₀. Lower IC₅₀ values ​​indicate greater cytotoxicity. A line graph was constructed with antimicrobial peptide concentration as the abscissa and inhibition rate as the ordinate, and the IC₅₀ was calculated by fitting.

[0062] The same method was used to obtain the IC50 of BMAP-18 on RAW 264.7 cells.

[0063] As shown in FIG7 , the IC50 values ​​of SAH1 and BMAP-18 against RAW 264.7 cells were 300 μM and 150 μM, respectively. The results showed that the cytotoxicity of SAH1 was weaker than that of BMAP-18.

[0064] Example 4 MIC test to determine the ability of SAH1 to inhibit the growth of BCG bacteria

[0065] The antibacterial effect of various concentrations of SAH1 against BCG bacteria was investigated. SAH1 was serially diluted in 7H9 medium over a range of 6.25-3200 μM to obtain SAH1 solutions of various concentrations. BMAP-18 was serially diluted in 7H9 medium over a range of 6.25-3200 μM to obtain BMAP-18 solutions of various concentrations. Rifampicin, isoniazid, and ethambutol were serially diluted in 7H9 medium over a range of 25-12800 μM to obtain rifampicin solutions, isoniazid solutions, and ethambutol solutions of various concentrations, respectively. Each drug solution was filtered through a 0.22 μm filter to remove bacteria.

[0066] The SAH1 test group was set up. 0.1 mL of SAH1 solution of each concentration was added to each centrifuge tube, and then 1.5×10 7 A 0.1 mL volume of BCG bacteria (containing 0.1 cfu / mL) was added. A positive control was performed with 7H9 medium replacing the SAH1 solution, with all other parameters being the same as the SAH1 group. A negative control was performed with the same volume of ultrapure water replacing the BCG bacteria, with all other parameters being the same as the SAH1 group. Additionally, BMAP-18, rifampicin, isoniazid, and ethambutol groups were performed, identical to the SAH1 group, differing only in that BMAP-18, rifampicin, isoniazid, or ethambutol were used in place of the SAH1 solution. After incubation at 37°C for 7 days, resazurin indicator was added and incubated for 2 days, with color changes observed. After two days, if the culture medium was blue, the tube was free of bacteria, indicating that the drug at that concentration had an antibacterial effect. If the culture medium was purple or red, viable bacteria were present in the tube, indicating that the drug at that concentration had no antibacterial effect.

[0067] The results are shown in Table 1. The minimum inhibitory concentration of SAH1 was 12.5 μM, which was significantly lower than that of BMAP-18, rifampicin, isoniazid, and ethambutol, indicating that the antibacterial ability of SAH1 was significantly improved compared with BMAP-18.

[0068] Table 1 Minimum inhibitory concentration of each drug

[0069] Example 5 Scanning electron microscopy observation of the effects of SAH1 and BMAP-18 on BCG cell membrane integrity

[0070] Collect BCG bacteria that have grown to the logarithmic phase, suspend them in fresh 7H9 medium, and adjust the OD 600nm = 0.5, SAH1 at a final concentration of 12.5 μM and BMAP-18 at a final concentration of 300 μM were added, respectively. Sterile ddH2O was used instead of the antimicrobial peptide as a negative control, and each was incubated at 37°C for 1 hour. After incubation, the cells were centrifuged and washed three times with PBS (0.1 M, pH 7.0). The pellet was resuspended in 2.5% glutaraldehyde, mixed, and incubated at 4°C overnight to fix the bacteria. The pellet was washed three times with PBS (0.1 M, pH 7.0), and a 1% aqueous solution of OsO4 was added for 1 hour. The pellet was then washed three times with PBS (0.1 M, pH 7.0) by centrifugation. The cells were then dehydrated using various concentrations of ethanol (30%, 50%, 70%, 80%, 90%, and 100%) for 15-20 minutes each time. The samples were then treated in a mixture of ethanol and isoamyl acetate (v:v = 1:1) for 30 minutes, then transferred to pure isoamyl acetate for approximately 1 hour. Dehydrated with liquid carbon dioxide in a critical point dryer, the samples were then coated with gold at a current of 45 mA for 2 minutes. The treated samples were then observed using a scanning electron microscope.

[0071] As shown in Figure 8, at the minimum inhibitory concentration, the degree of damage to the BCG cell membrane and cells by SAH1 is significantly greater than that by BMAP-18. After the mycobacterial cell membrane is damaged, the contents leak out, causing the bacteria to shrink and eventually die.

[0072] Example 6 Transmission electron microscopy observation of the effect of SAH1 on BCG cell membrane and internal cell structure

[0073] Logarithmic-phase BCG cells were treated according to the method described in Example 5. After pre-dehydration with gradient ethanol concentrations, the cells were transferred to anhydrous acetone for 20 minutes. Next, the cells were treated with a mixture of anhydrous acetone and EPON812 resin (V / V = 1:1) at room temperature for 1 hour, followed by a mixture of anhydrous acetone and EPON812 resin (V / V = 1:3) at room temperature for 3 hours. The bacterial samples were then soaked in EPON812 resin overnight, and this process was repeated once. The treated samples were placed in an EP tube containing EPON812 resin and incubated at 70°C for at least 9 hours. Finally, the cells were sectioned using an ultrathin microtome and stained with uranyl acetate and alkaline lead citrate for 5-10 minutes, followed by observation under a transmission electron microscope. A negative control was also performed, using the same treatment method as the sample, except that no antimicrobial agent was used to treat the logarithmic-phase BCG cells.

[0074] As shown in Figure 9 , after SAH1 treatment, the contents of BCG cells were largely lost, resulting in cavities and leading to bacterial death.

[0075] Example 7 Trypsin test to determine the ability of SAH1 and BMAP-18 to resist protease hydrolysis

[0076] A 4 mg / ml SAH1 solution and a 10 μg / ml trypsin solution were prepared in PBS (pH 7.4) buffer. 250 μl of the trypsin solution was added to 250 μl of the SAH1 solution, and the reaction was carried out in an ice bath. Samples were taken at different times after the reaction, and the decomposition of SAH1 was analyzed by reversed-phase HPLC. The RP-HPLC chromatographic conditions were as follows: a C18 reverse-phase Bio-Rad analytical column, 250 × 4 mm, 30 nm pore size, 7 μm particle size; elution was performed using a linear gradient of 10-90% acetonitrile in water (0-40 minutes), with both acetonitrile and water containing 0.1% trifluoroacetic acid (w / v), at a flow rate of 1.2 mL / min.

[0077] In addition, the same method as above was used to replace the SAH1 aqueous solution with a 4 mg / ml BMAP-18 aqueous solution to detect the hydrolysis of BMAP-18 by trypsin.

[0078] SAH1 was treated with trypsin for different time periods, and the amount of remaining peptides was determined by RP-HPLC. As shown in Table 2, Figures 10, and 11, after 15, 30, and 60 minutes of trypsin treatment, the remaining peptides in SAH1 were 67.59%, 45.89%, and 30.21%, respectively. In contrast, after 15 minutes of trypsin treatment of BMAP-18, only 17.76% of the peptides remained, and after 30 minutes of treatment, the peptides were nearly completely hydrolyzed. This demonstrates that SAH1 has significantly enhanced resistance to proteolysis compared to BMAP-18.

[0079] Table 2 Residual peptides of SAH1 and BMAP-18 after trypsin treatment

[0080] Note: The calculation method of the residual peptide in Table 2 is: the HPLC peak area of ​​the peptide at the initial reaction time of 0 min is taken as 100%, and the residual peptide is calculated using the following formula: RP-HPLC peak area of ​​the residual peptide / RP-HPLC peak area of ​​the initial peptide*100%.

Claims

1. A polypeptide having anti-mycobacterium tuberculosis activity, the structural formula of which is as follows:

2. The method for preparing the polypeptide according to claim 1, characterized in that The steps include: (1) preparing a linear polypeptide, wherein the sequence of the linear polypeptide is as shown in SEQ ID NO: 1; (2) The thiol groups of the two cysteine ​​residues in the linear polypeptide undergo a dehydration condensation reaction with 1,3-bis(hydroxymethyl)urea to obtain the polypeptide.

3. The preparation method according to claim 2, characterized in that The molar ratio of the linear polypeptide to 1,3-bis(hydroxymethyl)urea is 1:8-12.

4. The preparation method according to claim 3, characterized in that In step (2), the linear polypeptide and 1,3-bis(hydroxymethyl)urea are dissolved in solvents respectively, and then mixed to carry out a dehydration condensation reaction.

5. The preparation method according to claim 4, characterized in that The solvent of the linear polypeptide is one of a mixed solution of trifluoroacetic acid and guanidine hydrochloride, a urea aqueous solution, acetonitrile, and a PBS buffer solution; the solvent of 1,3-bis(hydroxymethyl)urea is one of water, acetonitrile, methanol, and DMF.

6. The preparation method according to claim 5, characterized in that The concentration of the urea aqueous solution is 4-8 mol / L.

7. The preparation method according to claim 6, characterized in that The concentration of the linear polypeptide in the solvent is 1mmol / L-8mmol / L, and the concentration of 1,3-bishydroxymethylurea in the solvent is 18mmol / L-80mmol / L.

8. The preparation method according to claim 7, characterized in that In step (2), an acid reaction regulator is added to the reaction; the acid reaction regulator is trifluoroacetic acid; and the volume ratio between the linear polypeptide solution and the acid reaction regulator is 1:1 to 1:

3.

9. The preparation method according to claim 8, characterized in that In step (2), the reaction temperature is 0-40° C. and the reaction time is 0-10 min.

10. Use of the polypeptide according to claim 1 in the preparation of drugs against Mycobacterium tuberculosis.

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