A polypeptide with activity against mycobacterium tuberculosis, preparation method and application thereof

Abstract

The purpose of this invention is to provide a polypeptide with anti-tuberculosis activity, its preparation method, and its application, belonging to the field of biotechnology. The preparation method of the polypeptide of this invention includes the following steps: (1) preparing a linear polypeptide, the sequence of which is shown in SEQ ID NO:1; (2) subjecting the thiol groups of two cysteine ​​residues in the linear polypeptide to a dehydration condensation reaction with 1,3-dihydroxymethylurea to obtain the polypeptide. The polypeptide of this invention has low cytotoxicity and good anti-tuberculosis effect.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a polypeptide with anti-tuberculosis activity, its preparation method, and its application. Background Technology

[0002] Antimicrobial peptides exhibit diverse structures and are found in mammalian species. They are natural host defensins secreted by the body's innate immune system, exhibiting significant inhibitory and bactericidal effects against microorganisms (bacteria, viruses, etc.) and even tumor cells. BMAP-27 is a well-known peptide derived from bovine peptides, possessing an amphiphilic α-helix terminal formed by the cation NH2. It has been shown to have potent cytotoxic activity against bacteria, fungi, viruses, and parasites. However, BMAP-27 possesses certain cytotoxicity, thus limiting its application as a drug. BMAP-18 is a truncated form of BMAP-27; while reducing toxicity to mammalian and insect cells, its toxicity and antibacterial efficacy remain unsatisfactory.

[0003] Tuberculosis (TB) is a globally prevalent zoonotic disease that continues to threaten human health and life. Globally, TB remains one of the top 10 causes of death. The number of TB deaths worldwide rose from 1.4 million in 2019 to 1.5 million in 2020, and further increased to 1.6 million in 2021. The number of new cases of multidrug-resistant / rifampicin-resistant TB was 437,000 in 2020, increasing to 450,000 in 2021. Because commonly used TB treatments such as ethambutol, isoniazid, and rifampicin all have certain drug toxicity, easily causing hepatotoxicity, readily developing drug resistance, and exhibiting unstable treatment effects, TB continues to pose a significant threat and cause considerable damage to human safety and livestock development.

[0004] Tuberculosis pathogens include Mycobacterium tuberculosis. Currently, there is a lack of effective anti-tuberculosis drugs with few side effects. Summary of the Invention

[0005] The purpose of this invention is to provide a polypeptide that is low in cytotoxicity and highly effective against Mycobacterium tuberculosis.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] A polypeptide with anti-tuberculosis activity has the following structural formula:

[0008]

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

[0010] (1) Prepare a linear polypeptide, the sequence of which 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-dihydroxymethylurea to obtain the polypeptide.

[0012] In this invention, the molar ratio of the linear polypeptide to 1,3-dihydroxymethylurea is 1:8-12.

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

[0014] In this invention, the solvent for the linear polypeptide is one of a mixed solution of trifluoroacetic acid and guanidine hydrochloride, an aqueous solution of urea, acetonitrile, or PBS buffer; the solvent for 1,3-dihydroxymethylurea is one of water, acetonitrile, methanol, or DMF.

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

[0016] In this invention, the concentration of the linear polypeptide in the solvent is 1 mmol / L-8 mmol / L, and the concentration of 1,3-dihydroxymethylurea in the solvent is 18 mmol / L-80 mmol / L.

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

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

[0019] The present invention also provides the use of the polypeptide in the preparation of a drug for treating Mycobacterium tuberculosis.

[0020] The peptide SAH1 of this invention has anti-Mycobacterium tuberculosis activity and has the following advantages compared with the original peptide BMAP-18: (1) Safety and fewer side effects: SAH1 has significantly lower cytotoxicity to mouse macrophages than BMAP-18. The cytotoxic IC50 value of SAH1 is 300 μM, while that of BMAP-18 is 150 μM, thus significantly improving safety. (2) Better anti-Mycobacterium tuberculosis effect: In the minimum inhibitory concentration test, SAH1 showed extremely strong anti-BCG bacterial infection activity at a concentration of 12.5 μM, while under the same conditions, the minimum inhibitory concentration of BMAP-18 was 300 μM, and its antibacterial ability was only one-twenty-fourth that of SAH1. Scanning electron microscopy also directly showed that the antibacterial ability of SAH1 was significantly improved compared with BMAP-18. (3) Stronger resistance to protease hydrolysis: When trypsin at a concentration of 5 μg / mL was added to BMAP-18, all the 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 is an antimicrobial peptide that, in the post-antibiotic era, can achieve therapeutic effects while avoiding drug resistance side effects due to its unique mechanism of action. Therefore, it has very broad application prospects and is of great research value. Attached Figure Description

[0021] Figure 1 The reaction principle for preparing polypeptide SAH1.

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

[0023] Figure 3 It is a polypeptide GRFC 4 RFRC 8 Mass spectrum of KFKKLFKKLS-OH.

[0024] Figure 4 This is a chromatogram of the preparation of a polypeptide modified with 1,3-dihydroxymethylurea.

[0025] Figure 5 This is the mass spectrum of a polypeptide modified with 1,3-dihydroxymethylurea.

[0026] Figure 6 The cell viability of RAW264.7 cells treated with different concentrations of SAH1 and BMAP-18 is shown on the x-axis, where concentration is in μM and cell viability is in %.

[0027] Figure 7This is the fitting result of the IC50 values ​​of SAH1 and BMAP-18 on RAW264.7 cells.

[0028] Figure 8 The effects of SAH1 and BMAP-18 on the cell membrane integrity of BCG were observed using scanning electron microscopy. (A) is the negative control, (B) is BCG treated with BMAP-18, and (C) is BCG treated with SAH1.

[0029] Figure 9 The effects of SAH1 on the BCG cell membrane and internal structure were observed using transmission electron microscopy.

[0030] Figure 10 These are chromatograms obtained by RP-HPLC detection of the remaining peptide 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 These are chromatograms obtained by RP-HPLC detection of the remaining peptide of SAH1 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 Implementation

[0032] The BCG bacteria used in this application are attenuated bovine tuberculosis bacteria, ATCC number bio-77983, purchased from the China Institute of Veterinary Drug Control.

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

[0034] Example 1: Screening and preparation of peptide SAH1

[0035] 1. Screening of peptide SAH1

[0036] The BMAP-18 sequence (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). By modifying BMAP-18 through point mutations at any amino acid site or random point mutations at two amino acid sites, compound SAH1 was found to exhibit highly effective anti-tuberculosis effects while reducing cytotoxicity. Figure 1The specific modification method is as follows: The amino acids at positions 4 and 8 of BMAP-18 are mutated to cysteine ​​to obtain GRFCRCKFKKLFKKLS-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-dihydroxymethylurea to form a cyclic peptide, with the following structure:

[0037]

[0038] 2. Preparation of peptide SAH1

[0039] (1) Linear-chain polypeptide GRFC 4 RFRC 8 Preparation of KFKKLFKKLS-OH

[0040] Polypeptide synthesis using Fmoc solid-phase synthesis: GRFC 4 RFRC 8 KFKKLFKKLS-OH (from N-terminus to C-terminus).

[0041] Coupling the first amino acid: Weigh 0.2 mmol of Wang resin and add it to a solid-phase reaction column containing quinolone. Wash twice with DMF, then swell the resin with DMF for 30 min. Weigh 383.4 mg (1 mmol) of Fmoc-Ser(tBu)-OH and 142.11 mg (1 mmol) of Oxyma (ethyl 2-oxime cyanoacetate), dissolve them in DMF, then add 0.25 g (2 mmol) of DIC (N,N'-diisopropylcarbodiimide), activate for 3 min, and then add it to the DMF-swollen resin. Initiate the coupling reaction at room temperature for 1 h. Detect the reaction endpoint with ninhydrin. If the resin is colorless and transparent, terminate the reaction; if the resin is colored, extend the reaction by 1 h. After the reaction is complete, remove the reaction solution and wash the resin three times with DMF to remove the Fmoc protecting group: add a 20% piperidine aqueous solution to submerge the resin, stir with a glass rod, react for 3 minutes, remove the piperidine solution, wash the resin three times with DMF, add another 20% piperidine aqueous solution to submerge the resin, react for 4 minutes, remove the reaction solution, and wash the resin 3-5 times with DMF.

[0042] The second protective amino acid was coupled according to the method of coupling Fmoc-Ser(tBu)-OH.

[0043] Using the method described above, the remaining amino acids were sequentially coupled from the C-terminus to the N-terminus. The final peptide GRFC was obtained. 4 RFRC 8 KFKKLFKKLS-OH resin.

[0044] peptide GRFC 4 RFRC 8 KFKKLFKKLS-OH resin was washed three times with DMF and twice with DCM (dichloromethane). After vacuum drying, the cleavage reaction was performed as follows: lysis buffer was added, and the reaction was carried out at room temperature for 1 hour. The resin was filtered, and the filtrate was collected. The cleavage was repeated once with a small amount of lysis buffer, and the filtrates from both reactions were combined. The filtrate was slowly added to ice-cold ether, precipitated, centrifuged, and the supernatant was discarded. The crude linear polypeptide GRFC was obtained by drying under reduced pressure. 4 RFRC 8 KFKKLFKKLS-OH. The lysis buffer is a mixed solution of TFA (trifluoroacetic acid), TIS (triisopropylsilane), and H2O in a volume ratio of 95:2.5:2.5.

[0045] crude linear peptide GRFC 4 RFRC 8 KFKKLFKKLS-OH was lyophilized using the following method: it was frozen into a solid state in liquid nitrogen, then suspended in a lyophilizer for further lyophilization to obtain a solid powder. The linear polypeptide GRFC was identified using LC-MS. 4 RFRC 8 KFKKLFKKLS-OH, chromatogram as follows Figure 2 Mass spectrum as shown Figure 3 The mass spectrometry data are as follows:

[0046] M = 2292.3, found: 1147[M + 2H + ] 2+ 765.25[M+3H + ] 3+ 574.33[M+4H + ] 4+ 459.75[M+5H + ] 5+ .

[0047] linear peptide GRFC 4 RFRC 8 The structural formula of KFKKLFKKLS-OH is as follows:

[0048]

[0049] The sequence of the linear polypeptide (SEQ ID NO:1): GRFCRFRCKFKKLFKKLS.

[0050] (2) Two cysteine ​​residues in a linear polypeptide were modified and bound using 1,3-dihydroxymethylurea.

[0051] crude linear peptide GRFC4 RFRC 8 KFKKLFKKLS-OH reacts directly with 1,3-dihydroxymethylurea under acidic conditions to yield a bound cyclic peptide, reducing operational steps, improving production efficiency, and minimizing the use of organic solvents. The specific steps are as follows:

[0052] Step 1: Take crude linear polypeptide GRFC 4 RFRC 8 KFKKLFKKLS-OH was dissolved in a 1:1 mixture of TFA (trifluoroacetic acid) and guanidine hydrochloride to obtain a 2 mmol / L polypeptide solution; 1,3-dihydroxymethylurea was dissolved in water to obtain a 20 mmol / L aqueous solution of 1,3-dihydroxymethylurea.

[0053] Step 2: At room temperature, anhydrous trifluoroacetic acid was added to a 2 mmol / L peptide solution, followed by a 20 mmol / L aqueous solution of 1,3-dihydroxymethylurea. The reaction was carried out at room temperature for 1 minute to obtain crude 1,3-dihydroxymethylurea-modified peptide. The volume ratio of the peptide solution, anhydrous trifluoroacetic acid, and 1,3-dihydroxymethylurea aqueous solution was 1:1:1.

[0054] Step 3: The crude 1,3-dihydroxymethylurea-modified peptide was purified by semi-preparative high-performance liquid chromatography (HPLC) under the following conditions: Mobile phase: A: H₂O containing 0.1% (v / v) TFA; B: acetonitrile containing 0.1% (v / v) TFA; Elution program: 0-30 min; mobile phase B 0-60%, increment 2% B per minute; Column: Waters C18; Flow rate: 15 mL / min; Detection wavelength: 214 nm. Figure 4 It is evident that the prepared 1,3-dihydroxymethylurea-modified peptide has high purity.

[0055] The purified 1,3-dihydroxymethylurea-modified peptide was lyophilized. The lyophilization method was as follows: it was frozen into a solid state in liquid nitrogen, then suspended in a lyophilizer for lyophilization to obtain a solid powder. The purified 1,3-dihydroxymethylurea-modified peptide was identified by mass spectrometry. 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+ .

[0056] The structural formula of the 1,3-bis(hydroxymethyl)urea-modified polypeptide is as follows:

[0057]

[0058] The polypeptide modified with 1,3-dihydroxymethylurea is denoted as polypeptide SAH1.

[0059] Example 2: Detection of cell viability of RAW264.7 cells treated with SAH1 and BMAP-18 using the CCK-8 assay.

[0060] The toxicity of SAH1 and BMAP-18 to RAW264.7 cells was determined using the CCK-8 assay, also known as the WST (chemical name: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-dithiophenyl)-2H-tetrazole monosodium salt) colorimetric assay. The specific method was as follows: In a 96-well plate, 1 × 10⁶ cells were seeded per well. 5 RAW264.7 cells were cultured, and 100 μL of serially diluted SAH1 peptide solution (3.125 μM to 200 μM) was added to each well. The cells were incubated at 37°C and 5% CO2 for 24 hours. Positive control wells were prepared using DMEM complete medium instead of the SAH1 peptide solution, with all other conditions the same as the experimental wells. Blank wells were prepared without cell seeding, containing only DMEM complete medium. After incubation, 10 μL of CCK-8 reaction solution (Soluble Biotech, Beijing) was added to each well, and the cells were incubated at 37°C for another 2 hours. Finally, the absorbance at 450 nm was measured using a microplate reader (Synersymx, Biotek) to assess cell viability. Cell viability was calculated relative to the positive control wells using the formula: Cell viability = (Experimental well absorbance - Blank well absorbance) / (Positive control well absorbance - Blank well absorbance) * 100%.

[0061] In addition, the cell viability was determined using the same method after treatment with serially diluted BMAP-18 aqueous solutions at concentrations ranging from 3.125 μM to 200 μM.

[0062] Depend on Figure 6 It can be seen that at all concentration gradients, the cytotoxicity of SAH1 is significantly lower than that of BMAP-18. SAH1 at concentrations below 50 μM does not have significant cytotoxicity on RAW264.7 cells (cell survival rate > 90%), and BMAP-18 at concentrations below 25 μM does not have significant cytotoxicity on RAW264.7 cells (cell survival rate > 90%).

[0063] Example 3: Fitting results of IC50 values ​​of SAH1 and BMAP-18 on RAW264.7 cells

[0064] In a 96-well plate, 1×10⁻⁶ ppm is laid in each well. 5RAW264.7 cells were used. 100 μL of serially diluted SAH1 peptide aqueous solution (concentrations ranging from 3.125 μM to 200 μM) was added to each well, and the cells were incubated at 37°C and 5% CO2 for 24 hours. Positive control wells were prepared using DMEM complete medium instead of the SAH1 peptide aqueous solution, with the same conditions as the experimental wells. After incubation, 10 μL of CCK-8 reaction solution (Beijing Solarbio) was added to each well, and the cells were incubated at 37°C for another 2 hours. Finally, the absorbance at 450 nm was measured using a microplate reader (Synersymx, Biotek). The OD value of the positive control wells was B0, and the OD value of the wells with added antimicrobial peptide was B. The ratio B / B0 is called the inhibition rate. The concentration of antimicrobial peptide corresponding to an inhibition rate of 50% is called the IC50. The smaller the IC50 value, the stronger the cytotoxicity of the antimicrobial peptide. A line graph was constructed with antimicrobial peptide concentration on the x-axis and inhibition rate on the y-axis, and the IC50 was obtained by fitting the graph.

[0065] Using the same method, the IC50 of BMAP-18 on RAW264.7 cells was obtained.

[0066] Depend on Figure 7 As can be seen, the IC50 values ​​of SAH1 and BMAP-18 against RAW264.7 cells were 300 μM and 150 μM, respectively, indicating that SAH1 was less cytotoxic than BMAP-18.

[0067] Example 4: MIC test to determine the ability of SAH1 to inhibit the growth of BCG bacteria

[0068] The antibacterial effects of different concentrations of SAH1 on BCG bacteria were investigated. SAH1 was serially diluted in the range of 6.25–3200 μM using 7H9 medium as the solvent to obtain SAH1 solutions of various concentrations. BMAP-18 was serially diluted in the range of 6.25–3200 μM using 7H9 medium as the solvent to obtain BMAP-18 solutions of various concentrations. Rifampin, isoniazid, and ethambutol were serially diluted in the range of 25–12800 μM using 7H9 medium as the solvent to obtain rifampin, isoniazid, and ethambutol solutions of various concentrations, respectively. All drug solutions were filtered through a 0.22 μm filter to remove bacteria.

[0069] Set up an SAH1 experimental group. Add 0.1 mL of SAH1 solution of various concentrations to each centrifuge tube, then add 1.5 × 10⁻⁶ ppm. 70.1 mL of BCG bacteria (cfu / mL) was used as a positive control, replacing the SAH1 solution with 7H9 medium, otherwise the same as the SAH1 test group. A negative control was also set up, replacing the BCG bacteria with the same volume of ultrapure water, otherwise the same as the SAH1 test group. Additionally, BMAP-18, rifampin, isoniazid, and ethambutol test groups were set up, identical to the SAH1 test group, except that BMAP-18, rifampin, isoniazid, or ethambutol were used instead of the SAH1 solution. After incubating each centrifuge tube at 37°C for 7 days, resazurin indicator was added and allowed to act for 2 days, observing the color changes. Two days later, if the medium was blue, it indicated no bacteria in the centrifuge tube, suggesting that the drug concentration had an antibacterial effect; if the medium was purple and red, it indicated the presence of surviving bacteria in the centrifuge tube, suggesting that the drug concentration had no antibacterial effect.

[0070] 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, rifampin, isoniazid and ethambutol, indicating that the antibacterial ability of SAH1 was significantly improved compared with that of BMAP-18.

[0071] Table 1 Minimum inhibitory concentrations of each drug

[0072] drug Minimum inhibitory concentration SAH1 12.5μM BMAP-18 300μM Rifampicin 400μM Isoniazid 800μM ethambutol 800μM

[0073] Example 5: Scanning electron microscopy observation of the effects of SAH1 and BMAP-18 on BCG cell membrane integrity.

[0074] Collect BCG bacteria grown to the logarithmic phase, suspend them in fresh 7H9 medium, and adjust the OD. 600nm =0.5, SAH1 to a final concentration of 12.5 μM and BMAP-18 to a final concentration of 300 μM were added, respectively. The negative control used sterile ddH2O instead of the antimicrobial peptide. The mixtures were incubated at 37℃ for 1 h. After incubation, the bacterial cells were centrifuged, washed three times with PBS (0.1 M, pH 7.0), and the bacterial pellet was resuspended in 2.5% glutaraldehyde. After mixing, the pellet was incubated overnight at 4℃ to fix the bacteria. The bacterial pellet was washed three times with PBS (0.1 M, pH 7.0), and 1% OsO4 aqueous solution was added. The mixture was incubated for 1 h, and then washed three times with PBS (0.1 M, pH 7.0) by centrifugation. The pellet was then dehydrated sequentially with different concentrations of ethanol (30%, 50%, 70%, 80%, 90%, 100%), each dehydration lasting 15–20 min. Then, the sample was reacted in a mixture of ethanol and isoamyl acetate (v:v = 1:1) for 30 minutes, and finally transferred to pure isoamyl acetate for about 1 hour. It was then dehydrated with liquid carbon dioxide in a critical point desiccator, followed by gold coating at 45 mA for 2 minutes. The treated sample was then observed using a scanning electron microscope.

[0075] Depend on Figure 8 It is evident that, at the minimum inhibitory concentration, SAH1 causes significantly greater damage to the BCG cell membrane and cells than BMAP-18. After the mycobacterial cell membrane is damaged, the contents leak out, leading to bacterial shrinkage and eventual death.

[0076] Example 6: Observation of the effects of SAH1 on the cell membrane and intracellular structure of BCG cells using transmission electron microscopy.

[0077] Following the method described in Example 5, logarithmic-phase BCG was pre-dehydrated with ethanol at different concentration gradients and then transferred to anhydrous acetone for 20 min. Next, it was incubated with a mixture of anhydrous acetone and EPON812 resin (V / V = 1:1) at room temperature for 1 h, followed by incubation with a mixture of anhydrous acetone and EPON812 resin (V / V = 1:3) at room temperature for 3 h. Then, the bacterial sample was soaked in EPON812 resin overnight, and this process was repeated once. The treated sample was placed in EP tubes containing EPON812 resin and incubated at 70°C for at least 9 h. Finally, sections were prepared using an ultramicrotome and stained sequentially with uranyl acetate and basic lead citrate for 5–10 min. The bacterial samples were then observed under a transmission electron microscope. A negative control was also included, treated using the same method as the sample, except that no antibacterial substances were used to treat the logarithmic-phase BCG.

[0078] Depend on Figure 9 It is evident that BCG cells treated with SAH1 suffered significant loss of cell contents, resulting in cavities and ultimately bacterial death.

[0079] Example 7: Trypsin test to determine the resistance of SAH1 and BMAP-18 to protease hydrolysis.

[0080] A 4 mg / mL SAH1 solution and a 10 μg / mL trypsin solution were prepared using PBS (pH 7.4) buffer as the solvent. 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 time points after the reaction, and the decomposition of SAH1 was detected by RP-HPLC. The RP-HPLC chromatographic conditions were as follows: a C18 reversed-phase Bio-Rad column (250 × 4 mm, 30 nm pore size, 7 μm particle size) was used; linear gradient elution with 10–90% acetonitrile aqueous solution (both acetonitrile and water containing 0.1% trifluoroacetic acid (w / v)) was performed from 0 to 40 minutes; the flow rate was 1.2 mL / min.

[0081] In addition, using the same method described above, the hydrolysis of BMAP-18 by trypsin was detected by replacing the SAH1 aqueous solution with a 4 mg / ml BMAP-18 aqueous solution.

[0082] The amount of remaining peptide was determined by RP-HPLC after SAH1 reacted with trypsin at different time points. (See Table 2.) Figure 10 and Figure 11 As can be seen, after SAH1 was treated with trypsin for 15 min, 30 min, and 60 min, the residual peptide percentages were 67.59%, 45.89%, and 30.21%, respectively. In contrast, after BMAP-18 was treated with trypsin for 15 min, only 17.76% of the peptide remained, and after 30 min, the peptide was almost completely hydrolyzed. Therefore, SAH1 exhibits significantly enhanced resistance to protease hydrolysis compared to BMAP-18.

[0083] Table 2. Residual polypeptides of SAH1 and BMAP-18 after trypsin administration.

[0084]

[0085] Note: The calculation method for residual peptides in Table 2 is as follows: with the HPLC peak area of ​​the peptide at the initial reaction time of 0 min as 100%, the residual peptides are calculated using the following formula: RP-HPLC peak area of ​​residual peptide / RP-HPLC peak area of ​​initial peptide * 100%.

Claims

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

2. The method for preparing the polypeptide according to claim 1, characterized in that... Includes the following steps: (1) Prepare a linear polypeptide, the sequence of which 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-dihydroxymethylurea 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-dihydroxymethylurea is 1:8-12.

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

5. The preparation method according to claim 4, characterized in that... The solvent for the linear polypeptide is one of the following: a mixed solution of trifluoroacetic acid and guanidine hydrochloride, an aqueous solution of urea, acetonitrile, or PBS buffer; the solvent for 1,3-dihydroxymethylurea is one of the following: water, acetonitrile, methanol, or 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 1 mmol / L-8 mmol / L, and the concentration of 1,3-dihydroxymethylurea in the solvent is 18 mmol / L-80 mmol / 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; 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℃ and the reaction time is 0-10min.

10. The use of the polypeptide of claim 1 in the preparation of a drug for treating Mycobacterium tuberculosis.

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