Antibacterial Short Peptides Derived from Marine Microalgae and Their Applications

Abstract

The present invention relates to the field of biotechnology, and specifically relates to an antibacterial short peptide derived from marine microalgae and its applications. The antibacterial short peptides are Qz5707, Qz16317, Qz1933 or Qz13244 derived from marine microalgae; and sequences obtained by modifying any one or several amino acids in the short peptides. The antibacterial short peptides of the present invention have strong antibacterial activity and a broad antibacterial spectrum, and can be applied to aquaculture feed additives and food preservatives, and can also be developed into antibacterial agents and antibacterial drugs, etc., so they have broad application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to an antibacterial short peptide derived from marine microalgae and its application. Background Art

[0002] With the widespread use of antibiotics, the problem of bacterial drug resistance has become increasingly serious and has become a major challenge in the field of global public health. The research and development speed of traditional antibiotics is difficult to keep up with the speed of the emergence of bacterial drug resistance. Therefore, it is urgent to develop new antibacterial drugs.

[0003] Antimicrobial peptides (AMPs) are a class of natural immune molecules widely present in organisms, with characteristics such as broad-spectrum antibacterial activity and difficulty in generating drug resistance, and are considered ideal substitutes for traditional antibiotics. In recent years, exploring new AMPs from marine organisms has become a research hotspot. As an important primary producer in the marine ecosystem, marine microalgae are diverse in species and rich in metabolites, and are potential treasure houses of antibacterial peptides. Among them, different types of antimicrobial peptide molecules are widely present in different classes of marine organisms, participating in the construction of an efficient immune defense system that conforms to the survival characteristics of the species. Their types are closely related to the survival mode, living environment and tissues of marine organisms. At present, compared with the development of land resources, the exploration of marine AMPs has just started. Developing and utilizing new marine AMPs resources will provide an ideal molecular design skeleton and template for the development of new peptide drugs, and lay an important foundation for the development of new peptide drugs. Summary of the Invention

[0004] The purpose of the present invention is to provide an antibacterial short peptide derived from marine microalgae and its application.

[0005] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0006] An antibacterial short peptide derived from marine microalgae, the antibacterial short peptide is Qz5707, Qz13244, Qz16317 or Qz1933 derived from marine microalgae; and sequences obtained by modifying any one or several amino acids in the short peptide;

[0007] Among them, the antibacterial short peptide Qz5707 has a length of 16 amino acids, a net charge of +6, a molecular weight of 1983.44 Da, and an isoelectric point of 12.31;

[0008] The antibacterial short peptide Qz16317 has a length of 19 amino acids, a net charge of +7, a molecular weight of 2354.85 Da, and an isoelectric point of 12.01;

[0009] The antibacterial short peptide Qz1933 has a length of 14 amino acids, a net charge of +5, a molecular weight of 1810.28 Da, and an isoelectric point of 10.56;

[0010] The antimicrobial short peptide Qz13244 is 22 amino acids in length, with a net charge of +8, a molecular weight of 2665.33 Da, and an isoelectric point of 12.48.

[0011] The amino acid sequence of the antimicrobial short peptide Qz5707 is KHIHRVIKNLLKATRR.

[0012] The amino acid sequence of the antimicrobial short peptide Qz16317 is GLADVVRRRLKRTRRKMKD.

[0013] The amino acid sequence of the antimicrobial short peptide Qz1933 is LKKALKRYVMYLQR.

[0014] The amino acid sequence of the antimicrobial short peptide Qz13244 is AKIVPRITTMLRRIRKSLQKKQ.

[0015] Application of an antimicrobial short peptide derived from marine microalgae as described above: Application of one or more of the antimicrobial short peptides in the preparation of antimicrobial agents and / or antibacterial drugs.

[0016] The bacteria are Gram-negative bacteria, Gram-positive bacteria or fungi.

[0017] The bacteria are Escherichia coli Escherichia coli , Staphylococcus aureus Staphylococcus aureus , Micrococcus luteus Micrococcus luteus and Pichia pastoris Pichia pastoris One or more of them.

[0018] Application of an antimicrobial short peptide derived from marine microalgae as described above, Application of one or more of the antimicrobial short peptides derived from marine microalgae in the preparation of aquaculture feed additives and food preservatives.

[0019] Advantages of the present invention:

[0020] The antimicrobial short peptides of the present invention have broad-spectrum antibacterial activity, strong antibacterial activity against Gram-negative bacteria and fungi, and certain antibacterial activity against Gram-positive bacteria. The short peptides have high stability and good solubility, and can be applied to aquaculture feed additives, and can also be developed into antimicrobial agents and antibacterial drugs, etc. Therefore, they have broad application prospects. Description of the Drawings

[0021] Figure 1 Growth inhibitory activity of 4 antimicrobial short peptides and sodium ampicillin provided in the examples of the present invention against Escherichia coli E. coli Among them, A: Sodium ampicillin inhibits Escherichia coli E. coli ; B: Qz5707 inhibits Escherichia coli E. coli ; C: Qz13244 inhibits Escherichia coli E. coli; D: Qz1933 inhibits Escherichia coli E. coli ; E: Qz16317 inhibits Escherichia coli E. coli。

[0022] Figure 2 The anti - Staphylococcus aureus growth activities of the four antibacterial short peptides and sodium ampicillin provided by the examples of the present invention S. aureus Among them, A: Sodium ampicillin inhibits Staphylococcus aureus S. aureus ; B: Qz13244 inhibits Staphylococcus aureus S. aureus ; C: Qz16317 inhibits Staphylococcus aureus S. aureus ; D: Qz1933 inhibits Staphylococcus aureus S. aureus ; E: Qz5707 inhibits Staphylococcus aureus S. aureus .

[0023] Figure 3 The anti - Micrococcus luteus growth activities of the four antibacterial short peptides and sodium ampicillin provided by the examples of the present invention M. luteus Among them, A: Sodium ampicillin inhibits Micrococcus luteus M. luteus ; B: Qz13244 inhibits Micrococcus luteus M. luteus ; C: Qz16317 inhibits Micrococcus luteus M. luteus ; D: Qz5707 inhibits Micrococcus luteus M. luteus ; E: Qz1933 inhibits Micrococcus luteus M. luteus .

[0024] Figure 4 The anti - Pichia pastoris growth activities of the four antibacterial short peptides provided by the examples of the present invention P. pastori Among them, A: Qz13244 inhibits Pichia pastoris P. pastori ; B: Qz16317 inhibits Pichia pastoris P. pastori ; C: Qz1933 inhibits Pichia pastoris P. pastori ; D: Qz5707 inhibits Pichia pastoris P. pastori .

[0025] The curves of various microorganisms are used as positive controls to show the normal growth of microorganisms; separate PBS is used as a negative control to illustrate that the experimental system is not contaminated. Detailed implementation manners

[0026] To better understand the purpose and product functions of the present invention, the following further describes in detail the four antibacterial short peptides derived from marine microalgae of the present invention and their applications in combination with specific examples and drawings.

[0027] Example 1: Obtaining of antibacterial short peptides

[0028] Extraction of *Aureococcus anophagefferens* Aureococcus anophagefferen RNA was extracted to construct a sequencing library. After removing adapter sequences, poly-N sequences, and low-quality reads from the obtained sequences, de novo assembly was performed using the Trinity program, contigs were clustered into unigenes using TGICL, and then open reading frames were predicted using CodAn. Potential AMPs sequences were predicted from the protein sequences using the Blast program (e value of 1e-5), and those with a match length less than 80% of AMPs were filtered out. The obtained protein sequences were characterized using AMP prediction software AMPml (CTDD and PAAC models), CAMP, and DBAASP. New short peptides with hydrophobicity and amphiphilicity both greater than 1 were selected, namely Qz5707, Qz13244, Qz16317, and Qz1933.

[0029] Furthermore, the above short peptides were purified by solid-phase synthesis to obtain crude polypeptides (synthesized by Sangon Biotech, purity > 90%); namely, short peptide Qz16317 with the amino acid sequence GLADVVRRRLKRTRRKMKD (Sequence 1); short peptide Qz1933 with the amino acid sequence LKKALKRYVMYLQR (Sequence 2); short peptide Qz13244 with the amino acid sequence AKIVPRITTMLRRIRKSLQKKQ (Sequence 3); short peptide Qz5707 with the amino acid sequence KHIHRVIKNLLKATRR (Sequence 4). Specifically, short peptide Qz16317 has a length of 19 amino acids, a net charge of +7, a molecular weight of 2354.85 Da, and an isoelectric point of 12.01; short peptide Qz1933 has a length of 14 amino acids, a net charge of +5, a molecular weight of 1810.28 Da, and an isoelectric point of 10.56; short peptide Qz13244 has a length of 22 amino acids, a net charge of +8, a molecular weight of 2665.33 Da, and an isoelectric point of 12.48; short peptide Qz5707 has a length of 16 amino acids, a net charge of +6, a molecular weight of 1983.44 Da, and an isoelectric point of 12.31.

[0030] Example 2: Detection of the antibacterial activity of short peptides

[0031] The obtained short peptides Qz13244, Qz16317, Qz1933, and Qz5707 were respectively dissolved in sterile PBS at a concentration of 10 mg / ml, filtered through a 0.22 μm filter to remove bacteria, and then divided into small aliquots and stored frozen; then they were diluted with PBS to the required experimental concentration.

[0032] The strains involved in this example include: *Escherichia coli* Escherichia coli , *Staphylococcus aureus* Staphylococcus aureus , *Micrococcus luteus* Micrococcus luteus and *Pichia pastoris* Pichia pastoris .

[0033] The specific method of the antibacterial test is as follows: First, collect the fresh bacterial suspensions of each strain in the logarithmic growth phase mentioned above, wash them 3 times with phosphate buffer solution (PBS), and adjust the bacterial concentration to approximately 10 4 cfu / mL. Mix 50 μL of different concentrations of polypeptides required for the experiment with 50 μL of different bacterial suspensions, incubate at 37 °C for 2 h, and use PBS as a blank control. Finally, dispense 20 μL of the mixture into a 96-well plate, with each well containing 200 μL of medium, and there are three parallel wells for each peptide concentration. The plate is placed in a microplate reader (Biotek, Winooski, VT, USA), incubated at 37 °C (Pichia pastoris at 28 °C), and the OD600 value is measured every 1 hour to generate a growth curve (see Figures 1 - 4 ). The results showed that the MIC of the 4 short peptides against Staphylococcus aureus was 0.25 - 4.32 μM, and the bactericidal activity was much higher than that of sodium ampicillin (MIC was 13.46 μM). Among them, the short peptide Qz5707 had the strongest activity against Staphylococcus aureus, and its MIC level was only 1 / 50 of that of sodium ampicillin. The MIC of the 4 short peptides against Escherichia coli was 2.93 - 26.54 μM, and the strongest one was Qz13244, whose MIC was 4.6 times lower than that of sodium ampicillin (13.46 μM), and Qz13244 also had the most significant effect on killing the growth of yeast. The MIC of the 4 short peptides against Micrococcus luteus was 3.94 - 6.63 μM, which could completely inhibit its growth. In terms of inhibiting the growth of fungi, Qz16317 could only inhibit the growth of yeast but not kill it at the experimental concentration, while the MIC of the other three polypeptides against Pichia pastoris was 1.47 - 7.88 μM. In summary, the 4 short peptides derived from microalgae demonstrated bactericidal ability comparable to that of common antibiotics and broad-spectrum antibacterial / fungal characteristics. Therefore, adding them to aquatic feed as additives and food preservatives will have better application prospects.

Claims

1. An antibacterial short peptide derived from marine microalgae, characterized in that: The antibacterial short peptides are Qz5707, Qz13244, Qz16317 or Qz1933 derived from marine microalgae; Among them, the antibacterial short peptide Qz5707 has a length of 16 amino acids, a net charge of +6, a molecular weight of 1983.44 Da, and an isoelectric point of 12.31; The antibacterial short peptide Qz16317 has a length of 19 amino acids, a net charge of +7, a molecular weight of 2354.85 Da, and an isoelectric point of 12.01; The antibacterial short peptide Qz1933 has a length of 14 amino acids, a net charge of +5, a molecular weight of 1810.28 Da, and an isoelectric point of 10.56; The antibacterial short peptide Qz13244 has a length of 22 amino acids, a net charge of +8, a molecular weight of 2665.33 Da, and an isoelectric point of 12.48; The amino acid sequence of the antibacterial short peptide Qz5707 is KHIHRVIKNLLKATRR; The amino acid sequence of the antibacterial short peptide Qz16317 is GLADVVRRRLKRTRRKMKD; The amino acid sequence of the antibacterial short peptide Qz1933 is LKKALKRYVMYLQR; The amino acid sequence of the antibacterial short peptide Qz13244 is AKIVPRITTMLRRIRKSLQKKQ.

2. Use of the antibacterial short peptide derived from marine microalgae according to claim 1, characterized in that: Use of one or more of the antibacterial short peptides Qz5707, Qz13244 or Qz1933 in the preparation of antibacterial agents; The bacterium is Escherichia coli Escherichia coli , Staphylococcus aureus Staphylococcus aureus , Micrococcus luteus Micrococcus luteus and Pichia pastoris Pichia pastoris or one or more of them.

3. Use of the antibacterial short peptide derived from marine microalgae according to claim 1, characterized in that: Use of the antibacterial short peptide Qz16317 in the preparation of antibacterial agents; The bacterium is Escherichia coli Escherichia coli , Staphylococcus aureus Staphylococcus aureus , Micrococcus luteus Micrococcus luteus or one or more of them.

4. Use of the antibacterial short peptide derived from marine microalgae according to claim 1, characterized in that: Use of one or more of the antibacterial short peptides derived from marine microalgae in the preparation of aquaculture feed additives or food preservatives.

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