An antimicrobial lipopeptide CFH resistant to protease hydrolysis and its preparation method and application

Abstract

The present invention provides an antimicrobial lipopeptide CFH resistant to protease hydrolysis and a preparation method and application thereof, belonging to the field of bioengineering. 14 ‑(FHHFHHH) 3 ‑(Dab) 6 The present invention provides a self-assembly driving force for the antimicrobial lipopeptide through myristic acid, the template polypeptide provides the antimicrobial lipopeptide with hydrophobic force to kill pathogens, and Dab provides the antimicrobial lipopeptide with positive charges for adsorbing pathogen membranes and avoiding recognition by trypsin, thereby producing a highly stable antimicrobial lipopeptide CFH. The antimicrobial lipopeptide CFH of the present invention has a highly effective bactericidal activity against swine paratyphoid Salmonella in the presence of trypsin, chymotrypsin, artificial gastric juice and artificial intestinal juice, and has no toxicity to human red blood cells, making it have the potential to become a substitute for feed antibiotics.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to an antibacterial lipopeptide CFH resistant to protease hydrolysis, and a preparation method and application thereof. Background Art

[0002] Antimicrobial lipopeptide is a non-natural peptide with antimicrobial activity, which is a fatty acid coupled to a polypeptide through dehydration condensation. The fatty acid part is usually composed of C 12 , C 14 or C 16 The polypeptide part is usually composed of 10-20 amino acid residues and has broad-spectrum antibacterial activity against Gram-negative bacteria, Gram-positive bacteria and some fungi. Similar to antimicrobial peptides, antimicrobial lipopeptides kill pathogenic microorganisms by physically destroying bacterial membranes. Therefore, compared with common antibiotics, antimicrobial lipopeptides are less likely to develop drug resistance and are a highly potential alternative to feed antibiotics.

[0003] In recent years, antimicrobial lipopeptides have been shown to have high antimicrobial activity in vitro, but they have poor oral stability and are easily hydrolyzed into small peptide fragments by proteases in the gastrointestinal tract, making them difficult to feed directly as feed additives in animal husbandry. Therefore, on the basis of retaining the high antimicrobial activity of antimicrobial lipopeptides, further overcoming the technical bottleneck of poor enzyme stability is the most critical link in solving the application of antimicrobial lipopeptides as a substitute for feed antibiotics in animal husbandry. Summary of the invention

[0004] Based on the above shortcomings, the purpose of the present invention is to provide an antimicrobial lipopeptide CFH resistant to protease hydrolysis, which aims to resist hydrolysis by multiple proteases and inhibit the growth of swine Salmonella paratyphi, so that it has the potential to become a substitute for feed antibiotics.

[0005] The object of the present invention is achieved by: an antimicrobial lipopeptide CFH resistant to protease hydrolysis, the amino acid sequence of which is C 14 -(FHHFHHH)3-(Dab)6, where C 14 It is myristic acid containing fourteen carbon atoms, and Dab is diaminobutyric acid.

[0006] Furthermore, the molecular formula of the antimicrobial lipopeptide CFH as described above is shown in formula (I):

[0007]

[0008] Another object of the present invention is to provide a method for preparing an antimicrobial lipopeptide CFH resistant to protease hydrolysis, the method steps are as follows:

[0009] S1: Design a polypeptide including 3 structural fragments, fragment 1 is myristic acid containing 14 carbon atoms; fragment 2 is a template polypeptide, and its template sequence is (FHHFHHH)3; fragment 3 is diaminobutyric acid; wherein the myristic acid provides a self-assembly driving force for the antimicrobial lipopeptide, the template polypeptide provides the antimicrobial lipopeptide with hydrophobic force to kill pathogens, and the diaminobutyric acid provides the antimicrobial lipopeptide with a positive charge to adsorb pathogen membranes and avoid being recognized by trypsin, and its sequence is C 14 -(FHHFHHH)3-(Dab)6;

[0010] S2: A peptide resin is obtained by solid phase chemical synthesis, and the obtained peptide resin is cut by TFA to obtain a polypeptide, and after reverse phase high performance liquid chromatography purification and mass spectrometry identification, the preparation of the polypeptide is completed;

[0011] S3: After undergoing antibacterial activity tests containing multiple proteases and hemolytic activity tests, it was finally named antimicrobial lipopeptide CF H.

[0012] Another object of the present invention is to provide an antibacterial lipopeptide CFH resistant to protease hydrolysis for use in the preparation of a drug for treating swine paratyphoid Salmonella infectious diseases.

[0013] The beneficial effects and advantages of the present invention are as follows: the present invention uses myristic acid as a fatty acid chain to provide the antibacterial lipopeptide with the necessary hydrophobic force for self-assembly, and the antibacterial lipopeptide can hide the enzyme cleavage site inside the nanostructure after self-assembly and avoid being recognized by proteases; at the same time, the arginine that is easily recognized by trypsin is replaced by Dab, and is free from the recognition of trypsin, and an antibacterial lipopeptide CFH that can resist hydrolysis by multiple proteases is successfully created, and biological activity detection is performed on it, and it is found that it has high bactericidal activity against swine paratyphoid Salmonella in the presence of trypsin, chymotrypsin, artificial gastric juice and artificial intestinal juice, and has low hemolytic toxicity. The anti-enzymatic lipopeptide CFH only causes 6.05% hemolysis of red blood cells at a concentration of 128 μM, and it has the development potential to become a substitute for feed antibiotics. Therefore, the present invention is a newly designed protease-resistant antibacterial lipopeptide, which provides a theoretical basis and technical support for the research and development of feed-type antibiotic substitutes. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 It is the high performance liquid chromatogram of the antimicrobial lipopeptide CFH of the present invention.

[0015] Figure 2 It is a matrix-assisted laser desorption / ionization time-of-flight mass spectrum of the antibacterial lipopeptide CFH of the present invention.

[0016] Figure 3 It is a graph showing the hemolytic activity of the antimicrobial lipopeptide CFH and melittin ME of the present invention.

[0017] Figure 4 This is the self-assembly image of the antimicrobial lipopeptide CFH of the present invention. DETAILED DESCRIPTION

[0018] The present invention will be further described below with reference to the accompanying drawings:

[0019] Example 1

[0020] Design of antimicrobial lipopeptide CFH

[0021] The sequence is: C 14 -(FHHFHHH)3-(Dab)6; the sequence is shown in Table 1.

[0022] Table 1 Amino acid sequence of the antimicrobial lipopeptide CFH.

[0023]

[0024] The molecular formula is shown in formula (I):

[0025]

[0026] The positive charge number of the antimicrobial lipopeptide CFH is +6. The antimicrobial lipopeptide CFH is not only resistant to hydrolysis by a variety of proteases, but also has no toxicity to human red blood cells, and has the potential to be developed as a substitute for feed antibiotics.

[0027] Example 2

[0028] Synthesis of Antimicrobial Lipopeptide CFH by Solid Phase Chemical Synthesis

[0029] 1. The preparation is carried out one by one from the C-terminus to the N-terminus, and is completed by a peptide synthesizer. First, Fmoc-X (X is the first amino acid at the C-terminus of the antimicrobial lipopeptide) is connected to the Wang resin, and then the Fmoc group is removed to obtain the X-Wang resin; then Fmoc-Y-Trt-OH (9-fluorenylmethoxycarboxyl-trimethyl-Y, Y is the second amino acid at the C-terminus of each antimicrobial peptide); according to this procedure, it is synthesized from the C-terminus to the N-terminus in sequence until the synthesis is completed, and the side chain protection resin with the Fmoc group removed is obtained;

[0030] 2. Add a cleavage agent to the peptide resin obtained above, react at 20°C in the dark for 2 hours, and filter; wash with precipitated TFA (trifluoroacetic acid), mix the washing liquid with the above filtrate, concentrate on a rotary evaporator, add about 10 times the volume of pre-cooled anhydrous ether, precipitate at -20°C for 3 hours, precipitate a white powder, centrifuge at 2500g for 10 minutes, collect the precipitate, wash the precipitate with anhydrous ether, and vacuum dry to obtain a polypeptide, wherein the cleavage agent is a mixture of TFA, water and TIS (triisopropylsilyl chloride) in a mass ratio of 95:2.5:2.5;

[0031] 3. Use 0.2M sodium sulfate (phosphoric acid adjusted to pH 7.5) to balance the column for 30 minutes, dissolve the polypeptide with 90% acetonitrile aqueous solution, filter, C18 reverse phase atmospheric pressure column, use gradient elution (eluent is methanol and sodium sulfate aqueous solution mixed in a volume ratio of 30:70 to 70:30), flow rate is 1mL / min, detection wave is 220nm, collect the main peak, and freeze-dry; further purify using reverse phase C18 column, eluent A is 0.1% TFA / water solution; eluent B is 0.1% TFA / acetonitrile solution, elution concentration is 25%B to 40%B, elution time is 12min, flow rate is 1mL / min, collect the main peak as above, and freeze-dry;

[0032] 4. Identification of antimicrobial lipopeptides: The antimicrobial lipopeptides obtained above were analyzed by electrospray ionization mass spectrometry. The molecular weight (e.g. Figure 1 , 2 The molecular weight of the antimicrobial lipopeptide is basically consistent with the theoretical molecular weight in Table 1, and the purity of the antimicrobial lipopeptide is greater than 95%.

[0033] Example 3

[0034] 1. Determination of bactericidal activity: The minimum bactericidal concentration of the antimicrobial lipopeptide CFH in the presence of trypsin, chymotrypsin, artificial gastric juice and artificial intestinal juice was determined by the microbroth dilution method. 2 mg / ml BSA (containing 0.01% acetic acid) was used as the diluent, and then the pH value of the BSA dilution was adjusted to 6.0 using PBS solution as the buffer solution. A series of gradient antimicrobial lipopeptide solutions were prepared in sequence using the two-fold dilution method. 100 μL of the above solution was placed in a 96-well cell culture plate, and then an equal volume of the test bacterial solution (~10 5 / mL) in each well. Set up a positive control (containing bacterial solution but not antimicrobial lipopeptide) and a negative control (containing neither bacterial solution nor antimicrobial lipopeptide). Culture at 37℃ for 14-18h, then take 10μl of bacterial solution from each well and dilute it to an appropriate multiple and evenly spread it on the MHA plate, culture at 37℃ for 14-18h, and the concentration that kills more than 99.9% is the minimum bactericidal concentration. Protease treatment group: Antimicrobial lipopeptide was mixed with trypsin, chymotrypsin, artificial gastric juice and artificial intestinal juice in a volume ratio of 1:1, and the final concentration of protease was 8mg / ml. The mixed solution was incubated at 37℃ for 8h before the minimum bactericidal concentration of antimicrobial lipopeptide was determined. The test results are shown in Table 2.

[0035] Table 2 Antibacterial activity of antimicrobial lipopeptide CFH against Swine paratyphoid 21493.

[0036] Processing Groups Minimum bactericidal concentration (μM) Control group 16 Trypsin 16 Chymotrypsin 16 Artificial gastric juice 16 Artificial intestinal fluid 16

[0037] It can be seen from Table 2 that the antimicrobial lipopeptide CFH exhibits high bactericidal activity against swine paratyphoid 21493, and still maintains high bactericidal activity in the presence of trypsin, chymotrypsin, artificial gastric juice and artificial intestinal juice.

[0038] 2. Determination of hemolytic activity: Collect 1 mL of fresh human blood, dissolve it in 2 mL of PBS solution after anticoagulation with heparin, centrifuge at 3000 rpm for 10 minutes, collect red blood cells; wash with PBS solution for 3 times, and resuspend with 10 mL of PBS solution; take 50 μL of red blood cell suspension and 50 μL of antimicrobial lipopeptide solution of different concentrations, mix them evenly, and incubate them in a constant temperature incubator at 37°C for 1 hour; then centrifuge at 4°C and 3000 rpm for 10 minutes; take out the supernatant and measure the light absorption value at 570 nm with an enzyme marker. 50 μL of red blood cells plus 50 μL of PBS solution is used as a negative control, and 50 μL of red blood cells plus 50 μL of 0.1% Tritonx-100 is used as a positive control. The minimum hemolytic concentration is the antimicrobial lipopeptide concentration when the antimicrobial lipopeptide causes a hemolysis rate of 10%. The test results are shown in Figure 3 .pass Figure 3 It can be seen that the antimicrobial lipopeptide CFH did not show hemolytic activity within the detection range. At a concentration of 128 μM, it caused 6.05% hemolysis of red blood cells and failed to cause 10% hemolysis of red blood cells, which was significantly different from the control group bee venom. This shows that the antimicrobial lipopeptide CFH has the potential to be developed into a feed-type antimicrobial peptide.

[0039] 3. Determination of nanostructure: The nanostructure of antimicrobial lipopeptide was prepared by heat shock-cold shock method. The antimicrobial lipopeptide was diluted to 16 μM with deionized water, then steamed at high temperature for 15 min, then rapidly cooled on ice for 30 min, and incubated at room temperature for 12 h. 10 μl of the incubated antimicrobial lipopeptide CFH was added dropwise to the copper mesh and adsorbed for 2 min, then stained with 0.2% phosphotungstic acid for 2 min. After the copper mesh was dried, the nanostructure of the antimicrobial lipopeptide CFH was observed using a transmission electron microscope. The results are shown in Figure 2. Figure 4 .pass Figure 4 It can be seen that the antimicrobial lipopeptide CFH forms regular spherical micelles, which hide the amino acid residues inside the micelles to prevent the protease from contacting the amino acids, thereby hindering the protease from recognizing the antimicrobial lipopeptide and degrading it.

Claims

1. An antimicrobial lipopeptide CFH resistant to protease hydrolysis, characterized in that: Its amino acid sequence is C 14 -(FHHFHHH)3-(Dab)6, whose molecular formula is shown in formula (I), wherein C 14 is myristic acid containing fourteen carbon atoms, Dab is diaminobutyric acid, 2. The method for preparing an antimicrobial lipopeptide CFH resistant to protease hydrolysis according to claim 1, characterized in that: The steps are as follows: S1: Design a polypeptide including 3 structural fragments, fragment 1 is myristic acid containing 14 carbon atoms; fragment 2 is a template polypeptide, and its template sequence is (FHHFHHH)3; fragment 3 is diaminobutyric acid; wherein the myristic acid provides a self-assembly driving force for the antimicrobial lipopeptide, the template polypeptide provides the antimicrobial lipopeptide with hydrophobic force to kill pathogens, and the diaminobutyric acid provides the antimicrobial lipopeptide with a positive charge to adsorb pathogen membranes and avoid being recognized by trypsin, and its sequence is C 14 -(FHH FHHH)3-(Dab)6; S2: A peptide resin is obtained by solid phase chemical synthesis, and the obtained peptide resin is cut by TFA to obtain a polypeptide, and after reverse phase high performance liquid chromatography purification and mass spectrometry identification, the preparation of the polypeptide is completed; S3: After undergoing antibacterial activity tests containing multiple proteases and hemolytic activity tests, it was finally named antimicrobial lipopeptide CFH.

3. Use of the antimicrobial lipopeptide CFH resistant to protease hydrolysis according to claim 1 in the preparation of a drug for treating swine paratyphoid Salmonella infectious diseases.

Images