A polypeptide against mycobacterium tuberculosis, a preparation method and application thereof

Abstract

The application provides an anti-mycobacterium tuberculosis polypeptide, a preparation method and application thereof, and belongs to the technical field of biotechnology. The preparation method of the anti-mycobacterium tuberculosis polypeptide comprises the following steps: (1) preparing a straight-chain polypeptide, wherein the sequence of the straight-chain polypeptide is as shown in SEQ ID NO:1; and (2) performing a dehydration condensation reaction on the mercapto groups of two cysteine residues in the straight-chain polypeptide and N,N'-bis(2-hydroxyethyl) terephthalamide to obtain the polypeptide. The polypeptide has small cytotoxicity and strong anti-mycobacterium tuberculosis activity.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a polypeptide against Mycobacterium tuberculosis, a preparation method and application thereof. BACKGROUND

[0002] Disease caused by Mycobacterium tuberculosis infection is still a major global infectious disease problem. This bacterium mainly infects macrophages and other immune cells in the lungs due to inhalation of aerosolized infectious microdroplets, and rapidly spreads to adjacent lymph nodes and other tissues. Current estimates indicate that at least 100 million people are infected with latent Mycobacterium tuberculosis. Mycobacterium tuberculosis can persist in tissues as a viable organism without causing overt disease in the host for their lifetime, and can eventually reactivate in a small fraction of individuals, leading to active tuberculosis and transmission to new hosts. Although developed public health infrastructure combined with effective chemotherapy regimens, tuberculosis has been effectively controlled and largely eliminated in most developed countries, but the disease continues to ravage underdeveloped and resource-poor countries.

[0003] Antibacterial peptides are determined as potential alternative therapies against antibacterial diseases (such as tuberculosis) due to different modes of action, which are 20-60 amino acid biomolecules that play a crucial role in innate immunity, and can kill pathogenic bacteria together with cytokines and other host immunomodulatory molecules by separate administration or in combination with other drugs for treating tuberculosis. Dozens of antibacterial peptides have been shown to be effective in killing Mycobacterium tuberculosis and other tuberculosis pathogens in vitro. Antibacterial peptides are cationic, amphipathic, and have bactericidal activity, which makes them the most effective therapeutic agents against tuberculosis. BMAP-27 is a 27-residue alpha-helical interrupted glycopeptide with a natural antibacterial peptide with an amidated C-terminal, however, BMAP-27 shows certain cytotoxic activity to mammalian cells (human red blood cells and neutrophils), while it will be degraded in 10 minutes in a mouse bronchoalveolar lavage fluid environment. BMAP-18 is modified from BMAP-27, which reduces toxicity without weakening its bactericidal capacity.

[0004] Tuberculosis pathogens include Mycobacterium tuberculosis and the like. There is a lack of drugs against Mycobacterium tuberculosis with small side effects and good effects in the prior art. SUMMARY

[0005] The purpose of the present application is to provide a polypeptide with small cytotoxicity and strong anti-Mycobacterium tuberculosis.

[0006] In view of the problem of the prevalence of tuberculosis pathogens, the application provides an application of a drug against infection of tuberculosis pathogens.

[0007] The purpose of the present application can be achieved by the following technical solutions:

[0008] A polypeptide against Mycobacterium tuberculosis, the structural formula of which is as follows:

[0009]

[0010] The application further provides a preparation method of the polypeptide, comprising the following steps:

[0011] (1) preparing a straight-chain polypeptide, the sequence of which is as shown in SEQ ID NO: 1;

[0012] (2) performing a dehydration condensation reaction on the mercapto groups of the two cysteine residues in the straight-chain polypeptide and N,N'-bis(2-hydroxyethyl)terephthalamide to obtain the polypeptide.

[0013] In the application, the molar ratio of the straight-chain polypeptide to N,N'-bis(2-hydroxyethyl)terephthalamide is 1:8-12.

[0014] In the application, the straight-chain polypeptide and N,N'-bis(2-hydroxyethyl)terephthalamide in step (2) are dissolved in solvents respectively, and then mixed to perform the dehydration condensation reaction.

[0015] In the application, the solvent of the straight-chain polypeptide is one of a mixed solution of trifluoroacetic acid and guanidine hydrochloride, an aqueous urea solution, acetonitrile and a PBS buffer; and the solvent of N,N'-bis(2-hydroxyethyl)terephthalamide is one of water, acetonitrile, methanol and DMF.

[0016] In the application, the concentration of the aqueous urea solution is 4-8 mol / L.

[0017] In the application, the concentration of the straight-chain polypeptide in the solvent is 1 mmol / L-8 mmol / L, and the concentration of N,N'-bis(2-hydroxyethyl)terephthalamide in the solvent is 15 mmol / L-40 mmol / L.

[0018] In the application, an acid reaction regulator is added in the reaction in step (2); the acid reaction regulator is trifluoroacetic acid; and the volume ratio between the straight-chain polypeptide solution and the acid reaction regulator is 1:1-1:3.

[0019] In the application, the reaction temperature in step (2) is 0-40℃, and the reaction time is 0-10 min.

[0020] The application further provides application of the polypeptide in preparation of a medicine against Mycobacterium tuberculosis.

[0021] The polypeptide SAH8 has activity against Mycobacterium tuberculosis, and has the following advantages compared with the polypeptide BMAP-18 before modification: (1) safety and less side effects: the cytotoxicity of SAH8 on mouse macrophages is significantly reduced compared with BMAP-18, and the IC50 value of cytotoxicity of SAH8 is 335 μM, and the IC50 value of cytotoxicity of BMAP-18 is 150 μM, so the safety is significantly improved; (2) better anti-Mycobacterium tuberculosis effect: in the minimum bacteriostatic test, SAH8 can exhibit very strong anti-BCG infection effect at a concentration of 25 μM, while under the same conditions, the minimum bacteriostatic concentration of BMAP-18 is 300 μM, and the bacteriostatic capacity is only one twelfth of that of SAH8. It is also directly observed by scanning electron microscope that the bacteriostatic capacity of SAH8 is significantly improved compared with BMAP-18; (3) stronger anti-protease hydrolysis capacity: after adding trypsin at a concentration of 5 μg / mL in BMAP-18, only 17.76% remains after 15 min, while in the same environment, 66.08% of SAH8 is not hydrolyzed after 15 min, and 39.14% of SAH8 remains after 30 min. The polypeptide SAH8 is an antibacterial peptide, and in the post-antibiotic era, it can achieve the effect of treating diseases while avoiding the side effects of drug resistance, so it has very wide application prospect and great research value. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 Reaction principle for preparing the polypeptide SAH8.

[0023] Figure 2 is a crude straight-chain polypeptide GRFKRFRKKFC 11 KLFC 15 Chromatogram of KLS-OH.

[0024] Figure 3 is a polypeptide GRFKRFRKKFC 11 KLFC 15 Mass spectrum of KLS-OH.

[0025] Figure 4 is a chromatogram for preparing the polypeptide modified by N,N'-bis(2-hydroxyethyl)terephthalamide.

[0026] Figure 5 is a mass spectrum of the polypeptide modified by N,N'-bis(2-hydroxyethyl)terephthalamide.

[0027] Figure 6 The cell survival rate of RAW264.7 cells treated with SAH8 and BMAP-18 at different concentrations is shown, the abscissa is the concentration, the unit is μM, and the ordinate is the cell survival rate, the unit is %.

[0028] Figure 7is the fitting result of IC50 values of SAH8 and BMAP-18 on RAW264.7 cells.

[0029] Figure 8 is the chromatogram of RP-HPLC for detecting the polypeptide remaining of BMAP-18 in the environment of trypsin (5 μg / mL) at different times, the reaction time of Figure A is 0 min, the reaction time of Figure B is 15 min, and the reaction time of Figure C is 30 min.

[0030] Figure 9 is the chromatogram of RP-HPLC for detecting the polypeptide remaining of SAH8 in the environment of trypsin (5 μg / mL) at different times, the reaction time of Figure A is 0 min, the reaction time of Figure B is 15 min, the reaction time of Figure C is 30 min, and the reaction time of Figure D is 60 min. DETAILED DESCRIPTION

[0031] The BCG bacteria in the present application is attenuated Mycobacterium bovis, and the ATCC number is bio-77983, which is purchased from China Institute for Control of Veterinary Drug.

[0032] The technical solutions of the present application are described in detail below with reference to the accompanying drawings, but the protection scope of the present application is not limited to the following examples.

[0033] Example 1 Screening and preparation of polypeptide SAH8

[0034] 1. Screening of polypeptide SAH8

[0035] The sequence of BMAP-18 (from N-terminal to C-terminal) is: G-R-F-K-R-F-R-K-K-F-K-K-L-F-K-K-L-S-OH (Gly-Arg-Phe-Lys-Arg-Phe-Arg-Lys-Lys-Phe-Lys-Lys-Leu-Phe-Lys-Lys-Leu-Ser-OH). By means of random two-site amino acid point mutation and the like, BMAP-18 is modified, and finally it is found that the compound SAH8 has high anti-Mycobacterium tuberculosis effect while reducing the cytotoxicity. For example, Figure 1 The specific modification method is as follows: the amino acids at positions 11 and 15 of BMAP-18 are mutated into cysteine to obtain G-R-F-K-R-F-R-K-K-F-C-K-L-F-C-K-L-S-OH (Gly-Arg-Phe-Lys-Arg-Phe-Arg-Lys-Lys-Phe-Cys-Lys-Leu-Phe-Cys-Lys-Leu-Ser-OH), and then dehydrated condensation reaction is carried out with N,N'-bis(2-hydroxyethyl)terephthalamide to form a cyclic peptide, and the structure is as follows:

[0036]

[0037] 2. Preparation of polypeptide SAH8

[0038] (1) Linear polypeptide GRFKRFRKKFC 11 KLFC 15 Preparation of KLS-OH

[0039] Fmoc solid phase synthesis of polypeptide: GRFKRFRKKFC 11 KLFC 15 KLS-OH.

[0040] Coupling of the first amino acid: 0.2 mmol Wang resin was weighed and added to the solid phase reaction column with sand. After washing with DMF twice, the resin was swelled with DMF for 30 min. 383.4 mg (1 mmol) of Fmoc-Ser(tBu)-OH and 142.11 mg (1 mmol) of Oxyma (2-hydroximoyl acetic acid ethyl ester) were dissolved in DMF, and then 0.25 g (2 mmol) of DIC (N,N'-diisopropylcarbodiimide) was added. After activation for 3 min, it was added to the resin swelled with DMF, and the coupling reaction was carried out at room temperature for 1 h. The reaction end point was detected by ninhydrin. If the resin was colorless and transparent, the reaction was terminated. If the resin was colored, the reaction was extended for 1 h. After the reaction was completed, the reaction solution was removed, and the resin was washed with DMF three times. The Fmoc protecting group was removed: 20% piperidine aqueous solution was added to the resin in a concentration sufficient to cover the resin, and a glass rod was stirred for 3 min. The piperidine solution was removed, and the resin was washed with DMF three times. 20% piperidine aqueous solution was added to the resin in a concentration sufficient to cover the resin again, and it was reacted for 4 min. The reaction solution was removed, and the resin was washed with DMF 3-5 times.

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

[0042] The remaining amino acids were sequentially coupled from the C-terminal to the N-terminal using the above method. Finally, the polypeptide GRFKRFRKKFC 11 KLFC 15 KLS-OH resin was obtained.

[0043] The polypeptide GRFKRFRKKFC 11 KLFC 15 KLS-OH resin was washed with DMF three times and DCM (dichloromethane) twice. After vacuum drying, the cleavage reaction was carried out as follows: the cleavage solution was added, and it was reacted at room temperature for 1 h. The resin was filtered, and the filtrate was collected. The cleavage was repeated once more with a small amount of cleavage solution, and the two filtrates were combined. The filtrate was slowly added to ice ethyl ether, and precipitation and centrifugation were performed. The supernatant was discarded, and the crude linear polypeptide GRFKRFRKKFC11 KLFC 15 KLS-OH. Wherein, the cleavage solution is a mixed solution of TFA (trifluoroacetic acid), TIS (triisopropylsilane) and H2O in a volume ratio of 95:2.5:2.5.

[0044] The crude linear polypeptide GRFKRFRKKFC 11 KLFC 15 KLS-OH was freeze-dried, and the freeze-drying method was as follows: frozen into a solid in liquid nitrogen, and then hung in a freeze dryer for freeze-drying to obtain a solid powder. The linear polypeptide GRFKRFRKKFC 11 KLFC 15 KLS-OH, the chromatogram is as follows Figure 2 , the mass spectrum is as follows Figure 3 , and the mass spectrum data are as follows:

[0045] M = 2263.89, found: 1132.83 [M + 2H + ] 2+ , 755.67 [M + 3H + ] 3+ , 567.17 [M + 4H + ] 4+ , 454.00 [M + 5H + ] 5+ .

[0046] The linear polypeptide GRFKRFRKKFC 11 KLFC 15 The structural formula of KLS-OH is as follows:

[0047]

[0048] The sequence of the linear polypeptide (SEQ ID NO: 1) is GRFKRFRKKFCKLFCKLS.

[0049] (2) The two cysteine residues in the linear polypeptide are modified and bound by using N,N'-bis(2-hydroxyethyl)terephthalamide

[0050] The crude linear polypeptide GRFKRFRKKFC 11 KLFC 15 KLS-OH is directly reacted with N,N'-bis(2-hydroxyethyl)terephthalamide under acidic conditions to obtain a bound cyclic peptide, reduce the operation process, improve the production efficiency, and at the same time reduce the use of organic solvents. The specific steps are as follows:

[0051] Step one: take the crude linear polypeptide GRFKRFRKKFC 11 KLFC 15KLS-OH was dissolved in a mixture of TFA (trifluoroacetic acid) and guanidine hydrochloride with a volume ratio of 1:1 to obtain a polypeptide solution of 2 mmol / L; N,N'-bis(2-hydroxyethyl)terephthalamide was dissolved in water to obtain an aqueous solution of N,N'-bis(2-hydroxyethyl)terephthalamide of 20 mmol / L.

[0052] Step two: anhydrous trifluoroacetic acid was added to the polypeptide solution of 2 mmol / L, followed by the addition of the aqueous solution of N,N'-bis(2-hydroxyethyl)terephthalamide of 20 mmol / L, and the reaction was carried out at room temperature (25°C) for 1 minute to obtain a crude product of N,N'-bis(2-hydroxyethyl)terephthalamide-modified polypeptide. The volume ratio of the polypeptide solution, anhydrous trifluoroacetic acid, and the aqueous solution of N,N'-bis(2-hydroxyethyl)terephthalamide was 1:1:1.

[0053] Step three: the crude product of N,N'-bis(2-hydroxyethyl)terephthalamide-modified polypeptide was purified by semi-preparative high-performance liquid chromatography under the following conditions: mobile phase: A: H2O containing 0.1% (volume percent) TFA, B: acetonitrile containing 0.1% (volume percent) TFA, elution program: 0-30 min, mobile phase B 0 to 60%, 2% B increment per minute; column: Waters C18, flow rate: 15 mL / min, detection wavelength: 214 nm. The purified product was obtained by lyophilization. Figure 4 It can be seen that the prepared N,N'-bis(2-hydroxyethyl)terephthalamide-modified polypeptide has high purity.

[0054] The purified N,N'-bis(2-hydroxyethyl)terephthalamide-modified polypeptide was lyophilized by the following method: frozen into a solid in liquid nitrogen, and then hung in a lyophilizer for lyophilization to obtain a solid powder. The purified N,N'-bis(2-hydroxyethyl)terephthalamide-modified polypeptide was identified by mass spectrometry, as shown in Figure 5 , and the mass spectrometry data are as follows:

[0055] M = 2479.36, found: 1240.42 [M+2H + ] 2+ , 826.75 [M+3H + ] 3+ , 620.08 [M+4H + ] 4+ , 496.33 [M+5H + ] 5+ .

[0056] The structural formula of the N,N'-bis(2-hydroxyethyl)terephthalamide-modified polypeptide is as follows:

[0057]

[0058] The polypeptide modified by N,N'-bis(2-hydroxyethyl)terephthalamide is denoted as polypeptide SAH8.

[0059] Example 2 CCK-8 method for detecting cell survival rate of RAW264.7 cells treated by SAH8 and BMAP-18

[0060] The CCK-8 method, i.e. WST (chemical name: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-dithiophenyl)-2H-tetrazolium monosodium salt) colorimetric test was used to determine the toxicity of SAH8 and BMAP-18 to RAW264.7 cells. The specific method is as follows: 1 x 10 5 RAW264.7 cells were plated in each well of a 96-well plate, and 100 μL of a serially diluted polypeptide SAH8 aqueous solution with a concentration of 3.125 μM to 200 μM was added to each experimental well, and incubated at 37°C in a 5% CO2condition for 24 hours. A positive control well was set, in which DMEM complete medium was used to replace the polypeptide SAH8 aqueous solution, and other conditions were the same as the experimental well. A blank well was also set, in which no cells were plated and only DMEM complete medium was added. After incubation, 10 μL of CCK-8 reaction solution (Beijing Solabio) was added to each well, and incubated at 37°C for another 2 hours. Finally, the absorbance at 450 nm was detected by an enzyme marker (Synersymx, Biotek) to detect the cell viability. According to the formula cell survival rate = (absorbance of experimental well - absorbance of blank well) / (absorbance of positive control well - absorbance of blank well) * 100%, the cell survival rate relative to the positive control well was calculated.

[0061] In addition, the same method was used to detect the survival rate of cells treated by a serially diluted BMAP-18 aqueous solution with a concentration of 3.125 μM to 200 μM.

[0062] From Figure 6 It can be seen that at each concentration gradient, the cytotoxicity of SAH8 was significantly lower than that of BMAP-18, and when the concentration of SAH8 was within 30 μM, it had no significant cytotoxicity to RAW264.7 cells (cell survival rate > 90%), and when the concentration of BMAP-18 was within 25 μM, it had no significant cytotoxicity to RAW264.7 cells (cell survival rate > 90%).

[0063] Example 3 Fitting results of IC50 values of SAH8 and BMAP-18 on RAW264.7 cells

[0064] In a 96-well plate, 1 x 10 5RAW264.7 cells were used. 100 μL of serially diluted SAH8 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 SAH8 peptide aqueous solution, with the same procedures 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 plotted with antimicrobial peptide concentration on the x-axis and inhibition rate on the y-axis. 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 SAH8 and BMAP-18 against RAW264.7 cells were 335 μM and 150 μM, respectively, indicating that SAH8 was significantly less cytotoxic than BMAP-18.

[0067] Example 4: MIC test to determine the ability of SAH8 to inhibit the growth of BCG bacteria

[0068] The antibacterial effects of different concentrations of SAH8 against BCG bacteria were investigated. SAH8 was serially diluted in the range of 6.25–3200 μM using 7H9 medium as the solvent to obtain SAH8 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 the SAH8 experimental group. Add 0.1 mL of SAH8 solution of various concentrations to each centrifuge tube, and then add 1.5 × 10⁻⁶ ppm. 70.1 mL of BCG bacteria at 1.0 x 107cfu / mL; a positive control was set up by replacing the SAH8 solution with 7H9 medium, and the other conditions were the same as those in the SAH8 test group; a negative control was set up by replacing the BCG bacteria with the same volume of ultrapure water, and the other conditions were the same as those in the SAH8 test group. In addition, a BMAP-18 test group, a rifampicin test group, an isoniazid test group, and an ethambutol test group were set up, and the conditions were the same as those in the SAH8 test group, except that the SAH8 solution was replaced with BMAP-18, rifampicin, isoniazid, or ethambutol. After the centrifuge tubes were incubated at 37°C for 7 days, resazurin indicator was added and allowed to act for 2 days, and the color change was observed. After two days, if the culture medium was blue, it indicated that there were no bacteria in the centrifuge tube, and it was indicated that the drug at this concentration had a bacteriostatic effect; if the culture medium was purple and red, it indicated that there were surviving bacteria in the centrifuge tube, and it was indicated that the drug at this concentration had no bacteriostatic effect.

[0070] The results are shown in Table 1. The minimum bacteriostatic concentration of SAH8 was 25 μM, which was significantly lower than that of BMAP-18, rifampicin, isoniazid, and ethambutol, indicating that the bacteriostatic ability of SAH8 was significantly improved compared with BMAP-18.

[0071] Table 1 Minimum bacteriostatic concentrations of various drugs

[0072] Drug Minimum inhibitory concentration SAH8 25 μM BMAP-18 300 μM Rifampicin 400 μM Isoniazid 800 μM Ethambutol 800 μM

[0073] Example 5 Trpsin test for determining the ability of SAH8 and BMAP-18 to resist protease hydrolysis

[0074] A 4 mg / ml SAH8 solution and a 10 μg / ml trypsin solution were prepared using PBS (pH 7.4) buffer as a solvent, 250 μl of the trypsin solution was added to 250 μl of the SAH8 solution, and the reaction was performed in an ice bath. Samples were taken at different times after the reaction, and the decomposition of SAH8 was detected by RP-HPLC. The RP-HPLC chromatographic conditions were as follows: a C18 reverse-phase Bio-Rad analysis column, 250 x 4 mm, 30 nm pore size, 7 μm particle size was used; a linear gradient elution of 10-90% acetonitrile aqueous solution was used for 0-40 minutes, and both the acetonitrile and the water contained 0.1% trifluoroacetic acid (w / v), and the flow rate was 1.2 mL / min.

[0075] In addition, the same method was used to replace the SAH8 aqueous solution with a 4 mg / ml BMAP-18 aqueous solution to detect the hydrolysis of BMAP-18 by trypsin.

[0076] The amount of remaining polypeptide was determined by RP-HPLC after SAH8 and trypsin were allowed to react for different periods of time. As shown in Table 2, Figure 8 and Figure 9As can be seen, after trypsin treatment for 15 min, 30 min and 60 min, the residual polypeptides of SAH8 accounted for 66.08%, 39.14% and 9.82% respectively, while after trypsin treatment for 15 min, the residual polypeptides of BMAP-18 only accounted for 17.76%, and after trypsin treatment for 30 min, the polypeptides were almost completely hydrolyzed. It can be seen that the anti-protease hydrolysis ability of SAH8 is significantly enhanced compared with BMAP-18.

[0077] Table 2 Residual polypeptides of SAH8 and BMAP-18 under the action of trypsin

[0078]

[0079] Note: In Table 2, the calculation method of residual polypeptides is as follows: taking the initial reaction Omin polypeptide HPLC peak area as 100%, the residual polypeptides are calculated by the following formula: residual polypeptide RP-HPLC peak area / initial polypeptide RP-HPLC peak area*100%.

Claims

1. A polypeptide against Mycobacterium tuberculosis, having the following structure:

2. A method of producing the polypeptide of claim 1, wherein comprising the following steps: (1) preparing a straight-chain polypeptide, the sequence of which is as shown in SEQ ID NO: 1; (2) subjecting the mercapto groups of two cysteine residues in the straight-chain polypeptide to a dehydration condensation reaction with N, N'-bis (2-hydroxyethyl) terephthalamide to obtain the polypeptide.

3. The method of claim 2, wherein The molar ratio of the straight-chain polypeptide to N, N'-bis (2-hydroxyethyl) terephthalamide is 1: 8-12.

4. The method of claim 3, wherein In step (2), the straight-chain polypeptide and N, N'-bis (2-hydroxyethyl) terephthalamide are respectively dissolved in a solvent, and then mixed to perform the dehydration condensation reaction.

5. The method of claim 4, wherein The solvent for the straight-chain polypeptide is one of a mixed solution of trifluoroacetic acid and guanidine hydrochloride, an aqueous urea solution, acetonitrile, and a PBS buffer; and the solvent for N, N'-bis (2-hydroxyethyl) terephthalamide is one of water, acetonitrile, methanol, and DMF.

6. The method of claim 5, wherein The concentration of the aqueous urea solution is 4-8 mol / L.

7. The method of claim 6, wherein The concentration of the straight-chain polypeptide in the solvent is 1 mmol / L-8 mmol / L, and the concentration of N, N'-bis (2-hydroxyethyl) terephthalamide in the solvent is 15 mmol / L-40 mmol / L.

8. The method of claim 7, wherein An acid reaction regulator is added in the reaction in step (2); the acid reaction regulator is trifluoroacetic acid; and the volume ratio between the straight-chain polypeptide solution and the acid reaction regulator is 1: 1-1:

3.

9. The method of claim 8, wherein The reaction temperature in step (2) is 0-40℃, and the reaction time is 0-10 min. 10.Use of the polypeptide in claim 1 in the preparation of a drug against Mycobacterium tuberculosis.

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