Polypeptides having activity against mycobacterium tuberculosis, methods of making and uses thereof

Abstract

The application aims to provide a polypeptide with anti-mycobacterium tuberculosis activity, a preparation method and application thereof, and belongs to the technical field of biotechnology. The preparation method of the 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 with N,N'-bis(2-hydroxyethyl)terephthalamide to obtain the polypeptide. The polypeptide is small in cytotoxicity and high in anti-mycobacterium tuberculosis efficiency.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a polypeptide with anti-mycobacterium tuberculosis activity, a preparation method and application thereof. BACKGROUND

[0002] Antimicrobial peptides (AMPs) are small molecule polypeptides widely existing in nature, which have a unique antibacterial mechanism, including lysing pathogenic bacterial biofilm, binding to intracellular targets to induce death, etc. Due to the characteristics of wide antibacterial spectrum and difficulty in developing drug resistance, antimicrobial peptides are considered as potential antibiotic substitutes, and are expected to be used in livestock production, medical and health fields, etc. BMAP-27 is a well-known peptide derived from bovine, which has an amphiphilic alpha helix terminal formed by cation NH2. It has been proved to have strong killing activity on bacteria, fungi, viruses and parasites. However, BMAP-27 has certain cytotoxicity, and therefore cannot be used as a drug. BMAP-18 is modified from the bovine-derived natural antimicrobial peptide BMAP-27, which retains the N-terminal sequence with alpha helix structure while removes the C-terminal hydrophobic tail to reduce cytotoxicity, and can be used for treating bacterial infections of skin and mucosa

[0003] Since the treatment cycle of ethambutol and isoniazid, rifampicin and other commonly used drugs for treating tuberculosis is long, and has no therapeutic effect on pathogenic bacteria with drug resistance, tuberculosis still poses a great threat and damage to human safety and livestock development.

[0004] The pathogen of tuberculosis includes mycobacterium tuberculosis, etc. There is a lack of drugs with small side effects and good effects against mycobacterium tuberculosis in the prior art. SUMMARY

[0005] The purpose of the present application is to provide a polypeptide with small cytotoxicity and high efficiency against mycobacterium tuberculosis.

[0006] The purpose of the present application is achieved by adopting the following technical solutions:

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

[0008]

[0009] The present application also provides a preparation method of the polypeptide, comprising the following steps:

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

[0011] (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.

[0012] In the present application, the molar ratio of the straight-chain polypeptide to N,N'-bis(2-hydroxyethyl)terephthalamide is 1:7-15.

[0013] In the present application, the straight-chain polypeptide and N,N'-bis(2-hydroxyethyl)terephthalamide in step (2) are dissolved in corresponding solvents respectively, and then mixed to carry out the dehydration condensation reaction.

[0014] In the present 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 PBS buffer; and the solvent of N,N'-bis(2-hydroxyethyl)terephthalamide is one of water, acetonitrile, methanol and DMF.

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

[0016] In the present 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-80 mmol / L.

[0017] In the present application, an acid reaction regulator is added in the reaction of 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.

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

[0019] The present application further provides the use of the polypeptide in the preparation of a drug for resisting Mycobacterium tuberculosis.

[0020] The beneficial effects of polypeptide SAH4 compared with polypeptide BMAP-18 are as follows: the cytotoxicity of SAH4 to mouse macrophages is significantly lower than that of BMAP-18, the IC50 value of SAH4 is 400 μM, and that of BMAP-18 is 150 μM, thus the safety is significantly improved and the side effects are reduced; in the minimum bacteriostatic test, SAH4 has a strong anti-BCG bacterial 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 SAH4; when trypsin with a concentration of 5 μg / mL is added to BMAP-18, all the polypeptides are hydrolyzed after 30 min, while in the same environment, 66.68% of SAH4 is not hydrolyzed after 30 min, and 43.82% of SAH4 can still remain after 1 h. Polypeptide SAH4 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 by its unique mode of action, thus having a very wide application prospect and great research value. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 Reaction principle for preparing polypeptide SAH4.

[0022] Figure 2 is a crude straight-chain polypeptide GRFKRFC 7 KKFC 11 chromatogram of KLFKKLS-OH.

[0023] Figure 3 is polypeptide GRFKRFC 7 KKFC 11 mass spectrum of KLFKKLS-OH.

[0024] Figure 4 is the chromatogram for preparing the polypeptide modified by N,N'-bis(2-hydroxyethyl)terephthalamide.

[0025] Figure 5 is the mass spectrum of the polypeptide modified by N,N'-bis(2-hydroxyethyl)terephthalamide.

[0026] Figure 6 shows the cell survival rate of RAW264.7 cells treated with SAH4 and BMAP-18 at different concentrations, the abscissa is the concentration in μM, and the ordinate is the cell survival rate in %.

[0027] Figure 7 is the fitting result of IC50 values of SAH4 and BMAP-18 on RAW264.7 cells.

[0028] Figure 8Figure is the chromatogram of RP-HPLC for detecting the polypeptide residue of BMAP-18 in the environment of trypsin (5 μg / mL) at different time, 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.

[0029] Figure 9 Figure is the chromatogram of RP-HPLC for detecting the polypeptide residue of SAH4 in the environment of trypsin (5 μg / mL) at different time, 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

[0030] 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.

[0031] 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.

[0032] Example 1: Screening and preparation of polypeptide SAH4

[0033] 1. Screening of polypeptide SAH4

[0034] 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). BMAP-18 is modified by means of random point mutation of two amino acids to cysteine and cyclization, etc. Finally, it is found that polypeptide SAH4 has high anti-Mycobacterium tuberculosis effect while reducing the cytotoxicity. Figure 1 The specific modification method is as follows: the amino acids at positions 7 and 11 of BMAP-18 are mutated to cysteine to obtain G-R-F-K-R-F-C-K-K-F-C-K-L-F-K-K-L-S-OH (Gly-Arg-Phe-Lys-Arg-Phe-Cys-Lys-Lys-Phe-Cys-Lys-Leu-Phe-Lys-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:

[0035]

[0036] 2. Preparation of polypeptide SAH4

[0037] (1) Linear polypeptide GRFKRFC 7 KKFC 11 Preparation of KLFKKLS-OH

[0038] Fmoc solid phase synthesis of polypeptide: GRFKRFC 7 KKFC 11 KLFKKLS-OH.

[0039] Coupling of the first amino acid: 0.2 mmol Wang resin was weighed and added to the solid phase reaction column with sandstone. After washing with DMF for 2 times, 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-hydroxymethyl acetic acid ethyl ester) were dissolved with DMF, and then 0.25 g (2 mmol) of DIC (N,N'-diisopropyl carbodiimide) 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 prolonged for 1 h. After the reaction was completed, the reaction solution was removed, and the resin was washed with DMF for 3 times. The Fmoc protecting group was removed: 20% piperidine aqueous solution was added to the resin in a concentration of 20%, and the glass rod was stirred. After 3 min of reaction, the piperidine solution was removed, and the resin was washed with DMF for 3 times. 20% piperidine aqueous solution was added to the resin in a concentration of 20% again, and the reaction was carried out for 4 min. After the reaction solution was removed, the resin was washed with DMF for 3-5 times.

[0040] According to the method of coupling Fmoc-Ser(tBu)-OH, the second protective amino acid was coupled

[0041] According to the above method, the remaining amino acids were sequentially coupled from C-terminal to N-terminal. Finally, the polypeptide GRFKRFC 7 KKFC 11 KLFKKLS-OH resin.

[0042] The polypeptide GRFKRFC 7 KKFC 11 KLFKKLS-OH resin was washed with DMF for 3 times, and washed with DCM (dichloromethane) for 2 times. After vacuum drying, the polypeptide was cleaved from the resin, and the specific method was as follows: the cleavage solution was added, and the reaction was carried out at room temperature for 1 h. The resin was filtered, and the filtrate was collected. The polypeptide was cleaved again with a small amount of cleavage solution, and the two filtrates were combined. The filtrate was slowly added to ice ethyl ether, and the precipitate was centrifuged. The supernatant was discarded, and the crude linear polypeptide GRFKRFC 7 KKFC 11KLFKKLS-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.

[0043] The crude linear polypeptide GRFKRFC 7 KKFC 11 KLFKKLS-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 GRFKRFC 7 KKFC 11 KLFKKLS-OH was identified by LC-MS, and the chromatogram was as follows: Figure 2 , and the mass spectrum was as follows: Figure 3 , and the mass spectrum data were as follows:

[0044] M = 2263.89, found: 1132.83 [M+2H + ] 2+ , 755.67 [M+3H + ] 3+ , 567.17 [M+4H + ] 4+ , 454.00 [M+5H + ] 5+ The linear polypeptide GRFKRFC 7 KKFC 11 KLFKKLS-OH has the following structural formula:

[0045]

[0046] The sequence of the linear polypeptide (SEQ ID NO: 1) is GRFKRFCKKFCKLFKKLS.

[0047] (2) The two cysteine residues in the linear polypeptide were modified and bound by using N,N'-bis(2-hydroxyethyl)terephthalamide

[0048] The crude linear polypeptide GRFKRFC 7 KKFC 11 KLFKKLS-OH was 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 reduce the use of organic solvents. The specific steps are as follows:

[0049] Step one: Take the crude linear polypeptide GRFKRFC 7 KKFC 11KLFKKLS-OH was dissolved in a mixture of TFA (trifluoroacetic acid) and guanidine hydrochloride with a volume ratio of 1:1 to obtain a 2 mmol / L polypeptide solution; N,N'-bis(2-hydroxyethyl)terephthalamide was dissolved in water to obtain a 20 mmol / L N,N'-bis(2-hydroxyethyl)terephthalamide aqueous solution.

[0050] Step two: anhydrous trifluoroacetic acid was added to the 2 mmol / L polypeptide solution, followed by the addition of 20 mmol / L N,N'-bis(2-hydroxyethyl)terephthalamide aqueous solution at room temperature (25°C), and then the reaction was carried out at room temperature (25°C) for 1 minute to obtain the N,N'-bis(2-hydroxyethyl)terephthalamide-modified polypeptide crude product. The volume ratio of the polypeptide solution, anhydrous trifluoroacetic acid, and N,N'-bis(2-hydroxyethyl)terephthalamide aqueous solution was 1:1:1.

[0051] Step three: the N,N'-bis(2-hydroxyethyl)terephthalamide-modified polypeptide crude product 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 N,N'-bis(2-hydroxyethyl)terephthalamide-modified polypeptide was obtained by lyophilization. Figure 4 It can be seen that the prepared N,N'-bis(2-hydroxyethyl)terephthalamide-modified polypeptide has high purity.

[0052] 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 freeze dryer 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:

[0053] M = 2479.36, found: 1240.42 [M+2H + ] 2+ , 826.75 [M+3H + ] 3+ , 620.08 [M+4H + ] 4+ , 496.33 [M+5H + ] 5+ .

[0054] The structural formula of the N,N'-bis(2-hydroxyethyl)terephthalamide-modified polypeptide is as follows:

[0055]

[0056] The polypeptide modified by N,N'-bis(2-hydroxyethyl)terephthalamide is denoted as polypeptide SAH4.

[0057] Example 2 CCK-8 method for detecting cell survival rate of RAW264.7 cells treated by SAH4 and BMAP-18

[0058] 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 SAH4 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 SAH4 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 SAH4 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.

[0059] 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.

[0060] From Figure 6 It can be seen that at each concentration gradient, the cytotoxicity of SAH4 was significantly lower than that of BMAP-18, and when the concentration of SAH4 was within 50 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%).

[0061] Example 3 Fitting results of IC50 values of SAH4 and BMAP-18 on RAW264.7 cells

[0062] In a 96-well plate, 1 x 10 5RAW264.7 cells were used. 100 μL of serially diluted SAH4 peptide 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 SAH4 peptide solution, with all other conditions the same 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, Biotec). 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.

[0063] Using the same method, the IC50 of BMAP-18 on RAW264.7 cells was obtained.

[0064] Depend on Figure 7 As can be seen, the IC50 values ​​of SAH4 and BMAP-18 against RAW264.7 cells were 400 μM and 150 μM, respectively, indicating that SAH4 was significantly less cytotoxic than BMAP-18.

[0065] Example 4: MIC test to determine the ability of SAH4 to inhibit the growth of BCG bacteria

[0066] The antibacterial effects of different concentrations of SAH4 on BCG bacteria were investigated. SAH4 was serially diluted in the range of 6.25–3200 μM using 7H9 medium as the solvent to obtain SAH4 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.

[0067] Set up an SAH4 experimental group. Add 0.1 mL of SAH4 solution of various concentrations to each centrifuge tube, then add 1.5 × 10⁻⁶ ppm. 7BCG bacteria 0.1 mL; a positive control was set, using 7H9 medium instead of SAH4 solution, and the others were the same as the SAH4 test group; a negative control was set, using the same volume of ultrapure water instead of BCG bacteria, and the others were the same as the SAH4 test group. In addition, BMAP-18 test group, rifampicin test group, isoniazid test group, and ethambutol test group were set, which were the same as the SAH4 test group, except that BMAP-18, rifampicin, isoniazid, or ethambutol was used to replace the SAH4 solution. After the centrifuge tubes were incubated at 37°C for 7 days, resazurin indicator was added and reacted for 2 days, and the color change was observed. After two days, if the culture medium was blue, it indicated that there was no bacteria in the centrifuge tube, and it meant that the drug at this concentration had bacteriostatic effect; if the culture medium was purple and red, it indicated that there were surviving bacteria in the centrifuge tube, and it meant that the drug at this concentration had no bacteriostatic effect.

[0068] The results are shown in Table 1. The minimum bacteriostatic concentration of SAH4 was 25 μM, which was significantly lower than that of BMAP-18, rifampicin, isoniazid, and ethambutol, indicating that the bacteriostatic ability of SAH4 was significantly improved compared with BMAP-18.

[0069] Table 1 Minimum bacteriostatic concentration of each drug

[0070] Drug Minimum inhibitory concentration SAH4 25 μM BMAP-18 300 μM Rifampicin 400 μM Isoniazid 800 μM Ethambutol 800 μM

[0071] Example 5 Trpsin test for determining the ability of SAH4 and BMAP-18 to resist protease hydrolysis

[0072] SAH4 aqueous solution of 4 mg / ml and trypsin aqueous solution of 10 μg / ml were prepared using PBS (pH 7.4) as solvent, 250 μl of the trypsin solution was added to 250 μl of the SAH4 solution, and the reaction was carried out in an ice bath. Samples were taken at different times after the reaction, and the decomposition of SAH4 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 in 0-40 minutes, and both acetonitrile and water contained 0.1% trifluoroacetic acid (w / v), and the flow rate was 1.2 mL / min.

[0073] In addition, the same method was used to replace the SAH4 aqueous solution with BMAP-18 aqueous solution of 4 mg / ml to detect the hydrolysis of BMAP-18 by trypsin.

[0074] The amount of remaining polypeptide was determined by RP-HPLC at different time periods of SAH4 and trypsin reaction. As shown in Table 2, Figure 8 and Figure 9It can be seen that after SAH4 was treated by trypsin for 15 min, 30 min and 60 min, the residual polypeptides accounted for 84.84%, 66.68% and 43.82% respectively, while after BMAP-18 was treated by trypsin for 15 min, the residual polypeptides accounted for only 17.76%, and after 30 min, the polypeptides were almost completely hydrolyzed. It can be seen that the anti-protease hydrolysis ability of SAH4 is significantly enhanced compared with BMAP-18.

[0075] Table 2 Residual polypeptides of SAH4 and BMAP-18 under the action of trypsin

[0076]

[0077] Note: In Table 2, the calculation method of residual polypeptides is as follows: taking the polypeptide HPLC peak area of initial reaction 0 min 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 with anti-mycobacterium tuberculosis activity, 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 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: 7-15.

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 corresponding solvents, and then mixed to perform the dehydration condensation reaction.

5. The method of claim 4, wherein 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.

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-80 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 for resisting mycobacterium tuberculosis.

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