Marine microalgae-derived antibacterial oligopeptide and application thereof

Abstract

The invention relates to the technical field of biology, in particular to a marine microalgae-derived antibacterial oligopeptide and application thereof. The antibacterial oligopeptide is Qz5707, Qz16317, Qz1933 or Qz13244 which is derived from marine microalgae, and the antibacterial oligopeptide is prepared from the marine microalgae; and modifying any one or more amino acids in the oligopeptide to obtain a sequence. The antibacterial oligopeptide disclosed by the invention has relatively strong antibacterial activity and wide antibacterial spectrum, can be applied to aquatic feed additives and food preservatives, and can also be developed into antibacterial agents, antibacterial drugs and the like, so that the antibacterial oligopeptide has a wide application prospect.

Description

Technical Field

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

[0002] With the widespread use of antibiotics, bacterial resistance is becoming increasingly serious and has become a major challenge in the field of global public health. The development speed of traditional antibiotics is difficult to keep up with the speed of bacterial resistance, so the development of new antibacterial drugs is imminent.

[0003] Antimicrobial peptides (AMPs) are a class of natural immune molecules widely present in organisms. They have the characteristics of broad-spectrum antimicrobial activity and are not easy to develop drug resistance. They are considered to be an ideal alternative to traditional antibiotics. In recent years, the discovery of new antimicrobial peptides from marine organisms has become a research hotspot. Marine microalgae, as important primary producers in marine ecosystems, are diverse and rich in metabolites, and are a potential treasure trove of antimicrobial 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 species. Their types are closely related to the survival mode, living environment and distribution tissues of marine organisms. At present, compared with the development of terrestrial resources, the discovery of marine AMPs has just started. The development and utilization of new marine AMPs resources will provide ideal molecular design skeletons and templates for the development of new peptide drugs, laying an important foundation for the development of new peptide drugs. Summary of the invention

[0004] The present invention aims to provide an antibacterial short peptide derived from marine microalgae and application thereof.

[0005] To achieve the above purpose, the present invention adopts the following technical solution: An antibacterial short peptide derived from marine microalgae, wherein the antibacterial short peptide is Qz5707, Qz13244, Qz16317 or Qz1933 derived from marine microalgae; and a sequence obtained by modifying any one or several amino acids in the short peptide; Among them, the antimicrobial 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 antimicrobial short peptide Qz16317 is 19 amino acids in length, has a net charge of +7, a molecular weight of 2354.85 Da, and an isoelectric point of 12.01; The antimicrobial 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.

[0006] The amino acid sequence of the antibacterial short peptide Qz5707 is KHIHRVIKNLLKATRR.

[0007] The amino acid sequence of the antibacterial short peptide Qz16317 is GLADVVRRRLKRTRRKMKD.

[0008] The amino acid sequence of the antibacterial short peptide Qz1933 is LKKALKRYVMYLQR.

[0009] The amino acid sequence of the antibacterial short peptide Qz13244 is AKIVPRITTMLRRIRKSLQKKQ.

[0010] An application of the antimicrobial short peptides derived from marine microalgae: application of one or more of the antimicrobial short peptides in the preparation of antimicrobial agents and / or antibacterial drugs.

[0011] The bacteria are Gram-negative bacteria, Gram-positive bacteria or fungi.

[0012] The bacteria is Escherichia coli Escherichia coli , Staphylococcus aureus Staphylococcus aureus , Micrococcus luteus Micrococcus luteus Pichia pastoris Shepherd's pie One or more of the .

[0013] An application of the antimicrobial short peptides derived from marine microalgae, and an application of one or more of the antimicrobial short peptides derived from marine microalgae in the preparation of aquatic feed additives and food preservatives.

[0014] The advantages of the present invention are: The antibacterial short peptide of the present invention has a broad-spectrum antibacterial activity, has strong antibacterial activity against Gram-negative bacteria and fungi, has certain antibacterial activity against Gram-positive bacteria, has high stability and good solubility, can be used as an aquatic feed additive, and can also be developed into an antibacterial agent and antibacterial drug, etc., and therefore has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The inhibitory effect of 4 antibacterial short peptides and ampicillin sodium provided by the present invention on Escherichia coli E. coli Growth activity. Among them, A: Ampicillin sodium 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.

[0016] Figure 2 Inhibition of Staphylococcus aureus by 4 kinds of antibacterial short peptides and ampicillin sodium provided by the present invention S. golden Growth activity. Among them, A: Ampicillin sodium 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 .

[0017] Figure 3 Four kinds of antibacterial short peptides and ampicillin sodium provided by the present invention inhibit Micrococcus luteus Yellow muscle Growth activity. Among them, A: Ampicillin sodium inhibits Micrococcus luteus Yellow muscle ; B: Qz13244 inhibits Micrococcus luteus M. yellow ; C: Qz16317 inhibits Micrococcus luteus Yellow muscle ; D: Qz5707 inhibits Micrococcus luteus Yellow muscle ; E: Qz1933 inhibits Micrococcus luteus Yellow M. .

[0018] Figure 4 Four kinds of antibacterial short peptides provided by the present invention inhibit Pichia pastoris P. to the shepherd Growth activity. Among them, A: Qz13244 inhibits Pichia pastoris P. to the shepherd ; B: Qz16317 inhibits Pichia pastoris P. to the shepherd ; C: Qz1933 inhibits Pichia pastoris P. to the shepherd ; D: Qz5707 inhibits Pichia pastoris P. to the shepherd .

[0019] The various microbial curves were used as positive controls, showing the normal growth of microorganisms; PBS alone was used as a negative control, indicating that the experimental system was not contaminated. DETAILED DESCRIPTION

[0020] In order to better understand the purpose and product function of the present invention, the following is a further detailed description of the four marine microalgae-derived antibacterial short peptides and their applications in combination with specific examples and drawings.

[0021] Example 1: Acquisition of antimicrobial short peptides Extraction of golden spherical algae Aureococcus anophagefferenRNA was used to construct a sequencing library. After removing the adapter sequence, poly-N sequence and low-quality reads, the obtained sequence was assembled de novo using the Trinity program, contigs were clustered into unigenes using TGICL, and then open reading frames were predicted using CodAn. The Blast program (e value was 1e-5) was used to predict potential AMPs sequences from protein sequences, and those with matching AMPs lengths less than 80% were filtered out. The AMPs prediction software AMPml (CTDD and PAAC models), CAMP and DBAASP were used to analyze the characteristics of the obtained protein sequences. New short peptides with hydrophobicity and amphipathicity greater than 1 were selected, namely Qz5707, Qz13244, Qz16317 and Qz1933.

[0022] The above short peptides were further purified by solid phase synthesis to obtain crude polypeptides (biosynthesized, purity>90%); that is, short peptide Qz16317, whose amino acid sequence is GLADVVRRRLKRTRRKMKD (sequence 1); short peptide Qz1933, whose amino acid sequence is LKKALKRYVMYLQR (sequence 2); short peptide Qz13244, whose amino acid sequence is AKIVPRITTMLRRIRKSLQKKQ (sequence 3); short peptide Qz5707, whose amino acid sequence is KHIHRVIKNLLKATRR (sequence 4). Specifically, the 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 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 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 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.

[0023] Example 2: Antibacterial activity detection of short peptides The short peptides Qz13244, Qz16317, Qz1933 and Qz5707 obtained above were dissolved in sterile PBS at a concentration of 10 mg / ml, filtered and sterilized, and then frozen in small portions; and then diluted with PBS to the concentration required for the experiment.

[0024] The strains involved in this embodiment include: Escherichia coli Escherichia coli , Staphylococcus aureus Staphylococcus aureus , Micrococcus luteus Micrococcus luteus Pichia pastoris Shepherd's pie .

[0025] The specific method of antibacterial test is as follows: First, collect the fresh bacterial liquid in the logarithmic growth phase, wash it three times with phosphate buffered saline (PBS), and adjust the bacterial concentration to about 10 4 cfu / mL. 50μL of the peptides of different concentrations required for the experiment were mixed with 50μL of different bacterial suspensions and incubated at 37°C for 2h. PBS was used as a blank control. Finally, 20μL of the mixture was distributed into a 96-well plate, each well containing 200μL of culture medium, and each peptide concentration had three parallel wells. The plate was placed in a microplate reader (Biotek, Winooski, VT, USA) and incubated at 37°C (Pichia at 28°C). The OD600 value was measured every 1 hour to generate a growth curve (see Figure 1-Figure 4 ). The results showed that the MIC of the four short peptides against Staphylococcus aureus was 0.25-4.32μM, and the bactericidal activity was much higher than that of ampicillin sodium (MIC was 13.46μM). Among them, the short peptide Qz5707 had the strongest activity in killing Staphylococcus aureus, and its MIC level was only 1 / 50 of that of ampicillin sodium. The MIC of the four short peptides against Escherichia coli was 2.93-26.54μM, of which the strongest one was Qz13244, whose MIC was 4.6 times lower than that of ampicillin sodium (13.46μM), and Qz13244 had the most significant effect in killing yeast growth. The MIC of the four short peptides against Micrococcus luteus was 3.94-6.63μM, which could completely inhibit its growth. In terms of inhibiting fungal growth, Qz16317 could only inhibit the growth of yeast at the experimental concentration but could not kill it, but the MIC of the other three peptides against Pichia pastoris was 1.47-7.88μM. In summary, the four short peptides derived from microalgae exhibit bactericidal ability and broad-spectrum bactericidal / fungicidal properties comparable to commonly used antibiotics. They can be added to aquatic feed as additives and food preservatives, and will have better application prospects.

Claims

1. An antibacterial short peptide derived from marine microalgae, characterized in that: The antibacterial short peptide is Qz5707, Qz13244, Qz16317 or Qz1933 derived from marine microalgae; and a sequence obtained by modifying any one or several amino acids in the short peptide; Among them, the antimicrobial 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 antimicrobial short peptide Qz16317 is 19 amino acids in length, has a net charge of +7, a molecular weight of 2354.85 Da, and an isoelectric point of 12.01; The antimicrobial 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 is 22 amino acids in length, has a net charge of +8, a molecular weight of 2665.33 Da, and an isoelectric point of 12.

48.

2. The antimicrobial short peptide derived from marine microalgae according to claim 1, characterized in that: The amino acid sequence of the antibacterial short peptide Qz5707 is KHIHRVIKNLLKATRR.

3. The antimicrobial short peptide derived from marine microalgae according to claim 1, characterized in that: The amino acid sequence of the antibacterial short peptide Qz16317 is GLADVVRRRLKRTRRKMKD.

4. The antimicrobial short peptide derived from marine microalgae according to claim 1, characterized in that: The amino acid sequence of the antibacterial short peptide Qz1933 is LKKALKRYVMYLQR.

5. The antimicrobial short peptide derived from marine microalgae according to claim 1, characterized in that: The amino acid sequence of the antibacterial short peptide Qz13244 is AKIVPRITTMLRRIRKSLQKKQ.

6. A use of the antimicrobial short peptide derived from marine microalgae according to claim 1, characterized in that: The use of one or more of the antibacterial short peptides in the preparation of antibacterial agents and / or antibacterial drugs.

7. The use of the antimicrobial short peptide derived from marine microalgae according to claim 6, characterized in that: The bacteria are Gram-negative bacteria, Gram-positive bacteria or fungi.

8. The use of the antibacterial short peptide derived from marine microalgae according to claim 6, characterized in that: The bacteria is Escherichia coli Escherichia coli , Staphylococcus aureus Staphylococcus aureus , Micrococcus luteus Micrococcus luteus Pichia pastoris Pichia pastoris One or more of the .

9. A use of the antimicrobial short peptide derived from marine microalgae according to claim 1, characterized in that: Application of one or more antimicrobial short peptides derived from marine microalgae in the preparation of aquatic feed additives or food preservatives.

Images