A dimerized anti-lipolytic peptide and its preparation method and application

Abstract

The present invention discloses a dimerized anti-lipolysis peptide and a preparation method and application thereof. The amino acid sequence thereof is shown in SEQ ID No. 1, and the peptide comprises two groups of nonapeptide groups, the sequence of which is: KGGGRRRPC, the 5th position of which is D-arginine, the N-terminus of each group of nonapeptide groups is acetylated by decanoic acid, and the dimerization of the two groups of nonapeptide groups is achieved by relying on the intermolecular disulfide bond of the cysteine ​​located at the end of the sequence in each group of nonapeptide groups, and the peptide is named as a dimerized anti-lipolysis peptide (C-C 10 ) C‑C The dimeric anti-enzymatic lipolytic peptide is obtained by solid phase chemical synthesis. The present invention also discloses the use of the dimeric anti-enzymatic lipolytic peptide in the preparation of a drug for treating Gram-negative bacteria or / and Gram-positive bacteria infectious diseases. The dimeric anti-enzymatic lipolytic peptide of the present invention has extremely high broad-spectrum antibacterial activity, extremely low hemolytic activity and can resist degradation by multiple proteases, and has the potential to be used as an antibacterial drug for bacterial infection.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically designs a dimerization anti-lipolysis peptide and a preparation method and application thereof. Background Art

[0002] In the current situation where bacterial infections are increasing dramatically worldwide due to the overuse of antibiotics, it is imperative to find new non-antibiotic antimicrobial agents. Antimicrobial peptides (AMPs), as multifunctional small molecule polypeptides in the biological defense system, are considered to be potential substitutes for antibiotics in the livestock field because they are not easy to cause drug resistance. However, problems such as the easy degradation of natural antimicrobial peptides by proteases and insufficient antibacterial activity restrict their further application. Summary of the invention

[0003] Based on the above problems, the present invention provides a dimeric anti-lipolytic peptide, which has a broad-spectrum antibacterial activity and can resist degradation by multiple proteases, thereby solving the problem that natural antibacterial peptides are easily degraded by proteases and have insufficient antibacterial activity.

[0004] The technical scheme adopted by the present invention is as follows: a dimerized anti-lipolytic peptide, whose amino acid sequence is shown in SEQ ID No. 1, which includes two groups of nonapeptide groups, the sequence of the nonapeptide groups is: KGGGRRRPC, the 5th position of each group of the nonapeptide groups is D-arginine, the N-terminus of each group of the nonapeptide groups is acetylated with decanoic acid, and the dimerization of the two groups of the nonapeptide groups is achieved by relying on the intermolecular disulfide bonds of the cysteine ​​located at the terminal of the two groups of the nonapeptide groups.

[0005] Furthermore, the molecular formula of the dimerized anti-lipolytic peptide as described above is as shown in formula (I):

[0006]

[0007] Another object of the present invention is to provide a method for preparing the above-mentioned dimerized anti-lipolytic peptide, comprising the following steps:

[0008] S1: Design of arginine-rich anti-enzymatic unit "R D RRP" takes it as the positive charge center, where R D The amino acid sequence of the polypeptide is shown in SEQ ID No. 1.

[0009] S2: obtaining a peptide resin by solid phase chemical synthesis, cutting the peptide resin with TFA, and then purifying it with reverse phase high performance liquid chromatography and identifying it with mass spectrometry to obtain a polypeptide;

[0010] S3: The peptide was then subjected to minimum inhibitory concentration test, hemolytic activity test and protease stability test, and finally named as dimerization anti-lipolysis peptide (CC 10 ) C-C .

[0011] Another object of the present invention is to provide the use of the above-mentioned dimerized anti-lipolytic peptide in the preparation of a drug for treating Gram-negative bacteria and / or Gram-positive bacteria infectious diseases.

[0012] Furthermore, the Gram-positive bacteria are Staphylococcus aureus, Enterococcus faecalis or Staphylococcus epidermidis.

[0013] Furthermore, the Gram-negative bacteria are Escherichia coli, Salmonella typhimurium or Pseudomonas aeruginosa.

[0014] Another object of the present invention is to provide a drug suitable for treating and / or preventing Gram-positive bacteria and / or Gram-negative bacteria infection, wherein the drug contains the dimerized anti-lipolytic peptide as described above.

[0015] Beneficial effects and advantages of the present invention: The dimerized anti-lipolysis peptide (CC 10 ) C-C It has a broad spectrum of antibacterial activity, good biocompatibility, and excellent stability against a variety of proteases. In summary, the dimerized anti-lipolytic peptide (CC 10 ) C-C It has the potential to be developed as an antibacterial drug for bacterial infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Dimeric lipopeptide (CC 10 ) C-C Reverse phase high performance liquid chromatogram of

[0017] Figure 2 Dimeric lipopeptide (CC 10 ) C-C Mass spectrum of

[0018] Figure 3 Dimeric lipopeptide (CC 10 ) C-C Protease stability test diagram, including (a), trypsin, (b), chymotrypsin, and (c), pepsin. DETAILED DESCRIPTION

[0019] The present invention is further described below with reference to the accompanying drawings:

[0020] Example 1 Dimeric lipolytic peptide (CC 10 ) C-C Design Synthesis

[0021] Dimeric lipopeptide (CC 10 ) C-C The sequence and physicochemical parameters are as follows:

[0022] Design of arginine-rich anti-enzymatic unit "R D RRP" takes it as the positive charge center, where R D The D-arginine is used, one end of the anti-enzymatic unit is connected to lysine through a flexible connector "GGG", and the other end is connected to cysteine, and then the ε-amino group of lysine is connected to decanoic acid to provide hydrophobicity, thereby forming a nonapeptide group containing decanoic acid, and the two groups of nonapeptide groups are dimerized by the cysteine ​​located at the end of the two groups of nonapeptide groups relying on the intermolecular disulfide bond. Figure 1 Shown is the dimerized lipolytic peptide (CC 10 ) C-C The reversed-phase high performance liquid chromatogram is as follows: Figure 2 Shown is the dimerized lipolytic peptide (CC 10 ) C-C Mass spectrum of the dimerized lipopeptide (CC 10 ) C-C The sequences and physicochemical parameters are shown in Table 1.

[0023] Table 1 Dimeric lipolytic peptide (CC 10 ) C-C Sequence and physicochemical parameters

[0024]

[0025] Its molecular formula is shown in formula (I):

[0026]

[0027] Example 2

[0028] The minimum inhibitory concentration test method is as follows:

[0029] Dimeric lipopeptide (CC 10 ) C-C The dimeric lipopeptide (CC 10 ) C-CUsing 0.01% acetic acid (containing 0.2% BSA) as the diluent, a series of gradients of dimerized anti-lipolytic peptide (CC 10 ) C-C 50 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 CFU / mL) in each well. Positive controls (containing bacterial solution but not containing dimeric anti-lipolytic peptide (CC 10 ) C-C ) and negative control (containing neither bacterial solution nor dimeric anti-lipolytic peptide (CC 10 ) C-C ). The cells were incubated at 37°C for 18 h. The minimum inhibitory concentration was defined as the concentration at which no turbidity was observed at the bottom of the wells. The results are shown in Tables 2 and 3.

[0030] Table 2 Dimeric lipolytic peptide (CC 10 ) C-C Antibacterial activity against Gram-negative bacteria (μM)

[0031]

[0032] Table 3 Dimeric lipolytic peptide (CC 10 ) C-C Antibacterial activity against Gram-positive bacteria (μM)

[0033]

[0034] The results of Example 2 show that the dimerization anti-lipolysis peptide (CC 10 ) C-C It exhibits extremely strong broad-spectrum antibacterial activity.

[0035] Example 3

[0036] Dimeric lipopeptide (CC 10 ) C-C The hemolytic activity was detected as follows:

[0037] To evaluate the dimerization of lipopolysaccharide (CC 10 ) C-C The safety of the dimeric anti-lipolytic peptide (CC 10 ) C-CHemolytic behavior of human red blood cells (hRBC). Use sodium heparin anticoagulant tubes to collect 2mL of fresh blood from healthy volunteers, centrifuge at 1000×g for 5min, discard the supernatant, and collect the red blood cells. Wash the collected red blood cells three times with PBS buffer solution, and resuspend the blood cells. First, take a sterile 96-well culture plate, add PBS buffer solution to it, and then add the dimerized anti-lipolytic peptide (C-C10) CC to be tested, make a 2-fold gradient dilution, and then add an equal volume of blood cell solution, incubate at 37°C for 1h, use a 96-well culture plate centrifuge, centrifuge at 1000×g for 5min, and collect the supernatant. Use an enzyme reader to measure the absorbance value at 570nm. In this experiment, the 0.1% Triton X-100 group was used as the positive control for 100% hemolysis, and the group without the dimerized anti-lipolytic peptide (CC 10 ) C-C The group was used as a negative control, and the minimum hemolysis concentration was the minimum concentration of dimerization anti-lipolysis peptide (C-C10) CC when the dimeric anti-lipolysis peptide (C-C10) CC caused a 5% hemolysis rate. The results are shown in Table 4.

[0038] Table 4 Dimeric lipolytic peptide (CC 10 ) C-C Hemolytic activity

[0039]

[0040] a MHC is the lowest concentration of the dimerized lipolytic peptide (C-C10) CC that causes 10% hemolysis of human red blood cells (hRBCs), and when no detectable hemolytic activity was observed at 128 μM, 256 μM was used to calculate the therapeutic index;

[0041] As can be seen from Table 4, the dimerization of anti-lipolysis peptide (CC 10 ) C-C The peptide concentration was 128 μM and showed no hemolytic activity, indicating that the dimerization of lipolytic resistant peptide (CC 10 ) C-C Has good biocompatibility.

[0042] Example 4

[0043] Protease stability test:

[0044] Minimum inhibitory concentration assay was used to evaluate the activity of dimerized anti-lipolytic peptide (CC 10 ) C-C Protease stability. 10 ) C-CThe diluent (2.56 mM) was mixed with trypsin, chymotrypsin, and pepsin solutions of different concentrations (2, 4, 8, and 16 mg / ml) in equal volumes and incubated for 8 hours. Then, E. coli ATCC 25922 was used as the test strain, and the dimerization anti-lipolytic peptide (CC 10 ) C-C The antibacterial activity after protease treatment. Figure 3 shown.

[0045] Depend on Figure 3 It can be seen that the dimerization of lipolytic peptide (CC 10 ) C-C It has excellent protease stability.

[0046] Based on the results of Examples 1, 2, 3 and 4, the dimerization anti-lipolysis peptide (CC 10 ) C-C It has extremely high broad-spectrum antibacterial activity, extremely low hemolytic activity and can resist degradation by multiple proteases, indicating that it has the potential to be used as an antibacterial drug for bacterial infection. In summary, the dimerized anti-lipolytic peptide (CC 10 ) C-C It has great application potential.

Claims

1. A dimerized anti-lipolytic peptide, characterized in that: Its amino acid sequence is shown in SEQ ID No. 1, which includes two groups of nonapeptide groups, the sequence of the nonapeptide groups is: KGGGRRRPC, the 5th position of each group of nonapeptide groups is D-arginine, the N-terminus of each group of nonapeptide groups is acetylated by decanoic acid, and the dimerization of the two groups of nonapeptide groups is achieved by relying on the intermolecular disulfide bonds of the cysteine ​​located at the terminal of the two groups of nonapeptide groups, wherein the molecular formula of the dimerized anti-enzymatic lipolysis peptide is shown in formula (I):

2. The method for preparing a dimerized anti-lipolytic peptide according to claim 1, characterized in that: The steps are as follows: S1: Design of arginine-rich anti-enzymatic unit "R D RRP" takes it as the positive charge center, where R D The anti-enzymatic unit is D-arginine, one end of the anti-enzymatic unit is connected to lysine through a flexible connector "GGG", and the other end is connected to cysteine, and then the ε-amino group of lysine is connected to decanoic acid to provide hydrophobicity, thereby forming a nonapeptide group containing decanoic acid, and the two groups of nonapeptide groups are dimerized by the cysteine ​​located at the end of the two groups of nonapeptide groups relying on the intermolecular disulfide bond to obtain a polypeptide, the amino acid sequence of which is shown in SEQ ID No.1; S2: obtaining a peptide resin by solid phase chemical synthesis, cutting the peptide resin with TFA, and then purifying it with reverse phase high performance liquid chromatography and identifying it with mass spectrometry to obtain a polypeptide; S3: The peptide was then subjected to minimum inhibitory concentration test, hemolytic activity test and protease stability test, and finally named as dimerization anti-lipolysis peptide (CC 10 ) C-C .

3. Use of a dimeric anti-lipolytic peptide according to claim 1 in the preparation of a drug for treating Gram-negative bacteria and / or Gram-positive bacteria infectious diseases; the Gram-positive bacteria are Staphylococcus aureus, Enterococcus faecalis or Staphylococcus epidermidis; the Gram-negative bacteria are Escherichia coli, Salmonella typhimurium or Pseudomonas aeruginosa.

4. A drug suitable for treating and / or preventing Gram-positive and / or Gram-negative bacterial infections, characterized in that: The drug contains the dimeric anti-lipolytic peptide as claimed in claim 1.

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