Earthworm antimicrobial peptides and screening methods thereof and their application in the preparation of broad-spectrum antimicrobial products

Abstract

The present invention provides an earthworm antimicrobial peptide and a screening method thereof, as well as an application in the preparation of a broad-spectrum antimicrobial product. The amino acid sequence of the antimicrobial peptide is shown in SEQ ID NO.3, has 14 hydrophobic amino acid residues, and its isoelectric point is 11.61. The present invention mainly uses a bioinformatics method to find new earthworm antimicrobial peptides based on the whole genome or mitochondrial genome of earthworms. The obtained antimicrobial peptide has good antimicrobial activity against Gram-negative bacteria represented by Escherichia coli and Gram-positive bacteria represented by Staphylococcus aureus, and can be used to prepare antibacterial products, including antimicrobial drugs, additives and preservatives.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a novel earthworm antimicrobial peptide and a screening method thereof, as well as an application in the preparation of a broad-spectrum antimicrobial product. The novel earthworm antimicrobial peptide has antimicrobial activity against both Gram-negative and Gram-positive bacteria. Background Art

[0002] At present, bacterial resistance is a major problem faced by the whole world. The use of a large number of antibiotics in the fields of medicine and livestock and poultry farming has caused the emergence and widespread spread of bacterial resistance, which has brought serious challenges to the treatment of bacterial infectious diseases in medicine and veterinary clinics. Bacterial resistance has become an important factor that threatens human life, health and safety and cannot be ignored, as well as an important problem that the livestock and poultry farming industry needs to face. In order to overcome the increasingly serious phenomenon of bacterial resistance, it is necessary to continuously explore and find new antibacterial drugs.

[0003] Antimicrobial peptides are a class of peptides composed of different amino acids that are widely distributed in nature and exist in both prokaryotes and eukaryotes and have antibacterial or bactericidal effects. Antimicrobial peptides have good antibacterial effects, and bacteria are not easy to develop drug resistance after use, especially with antibiotics commonly used in current medical clinics and livestock and poultry breeding. Therefore, they are increasingly valued by researchers. Scientific research shows that earthworms contain a variety of antimicrobial peptides, and earthworm antimicrobial peptides have the potential to become new antibacterial drugs. The current method for finding earthworm antimicrobial peptides is mainly to first homogenize the earthworm body, and then use chemical reagents for separation, purification and extraction, such as patents CN00136778.1, CN200310115583.4, CN201010278565.8, CN201110317906.2, etc. However, these methods generally only obtain a mixture containing antimicrobial peptides, and it is not easy to separate and purify a single peptide chain with antibacterial effects, making it difficult to conduct later drug research and development. Summary of the invention

[0004] In view of the defects in the prior art, the purpose of the present invention is to provide a novel earthworm antimicrobial peptide and a screening method thereof, as well as an application in the preparation of a broad-spectrum antimicrobial product.

[0005] The object of the present invention is achieved through the following solutions:

[0006] The first aspect of the present invention provides an earthworm antimicrobial peptide, the amino acid sequence of which is shown in SEQ ID NO. 3, specifically MWRCVATRRRCAAVCRNCAAARR.

[0007] Furthermore, the earthworm antimicrobial peptide has 14 hydrophobic amino acid residues and its isoelectric point is 11.61.

[0008] The second aspect of the present invention provides the use of earthworm antimicrobial peptides in the preparation of broad-spectrum antimicrobial products.

[0009] Preferably, the earthworm antimicrobial peptide has antibacterial activity against Gram-negative bacteria and / or Gram-positive bacteria.

[0010] Preferably, the Gram-negative bacteria is Escherichia coli, and the Gram-positive bacteria is Staphylococcus aureus.

[0011] Preferably, the minimum inhibitory concentration of the earthworm antimicrobial peptide against Escherichia coli is 1024 μg / mL, and the minimum inhibitory concentration against Staphylococcus aureus is 512 μg / mL.

[0012] Preferably, the application includes developing and preparing derivative products such as antibacterial drugs, additives and preservatives using the earthworm antimicrobial peptide.

[0013] The third aspect of the present invention provides a method for screening earthworm antimicrobial peptides, which mainly uses bioinformatics methods to find new antimicrobial peptides based on the whole genome or mitochondrial genome of earthworms. Specifically, it includes the following steps:

[0014] (1) Screening potential earthworm antimicrobial peptides: First, download the whole genome DNA or mitochondrial genome DNA of earthworms from the NCBI database, then search for open reading frames (ORFs) in the whole genome DNA or mitochondrial genome DNA of earthworms, and use the antimicrobial peptide prediction server to search for peptides that may have antimicrobial activity, thereby obtaining potential earthworm antimicrobial peptide sequences;

[0015] (2) Synthesizing potential earthworm antimicrobial peptides: artificially synthesizing potential earthworm antimicrobial peptides according to the potential earthworm antimicrobial peptide sequences obtained in step (1);

[0016] (3) Antibacterial activity detection: The potential earthworm antimicrobial peptides synthesized in step (2) are subjected to antibacterial experiments to determine the minimum inhibitory concentration (MIC) and screen out earthworm antimicrobial peptides with antibacterial effects.

[0017] Preferably, in step (1), the method for finding the open reading frame is: input the corresponding sequence in the ORFfinder function of NCBI, if the sequence is whole genome DNA, select the standard gene encoding method, if it is mitochondrial genome DNA, select the gene encoding method of invertebrate mitochondria, search for open reading frame sequences of 30nt and above in the whole sequence, select "ATG" and other alternative start codons as the start site, calculate, and then choose to download all sequences in protein FASTA format.

[0018] Preferably, in step (1), the method of using the antimicrobial peptide prediction server to search for peptides that may have antimicrobial activity is: uploading the earthworm whole genome DNA or mitochondrial genome DNA to the PredictAntimicrobial Peptides function of the CAMPR3 server, selecting four methods of SVM, Random Forest, Artificial Neural Network and Discriminant Analysis for analysis, and selecting sequences that are judged as antimicrobial peptides by all four methods and whose antimicrobial peptide probabilities of SVM, RandomForest and Discriminant Analysis are greater than 0.90 as peptides that may have antimicrobial activity.

[0019] The earthworm antibacterial peptide of the present invention has 14 hydrophobic amino acid residues, is amphipathic, and easily forms an alpha helix. Its isoelectric point is 11.61 and has cationic properties. Therefore, it is possible to inhibit bacteria by destroying bacterial cell membranes or interacting with molecules in cells.

[0020] Compared with the prior art, the present invention has the following beneficial effects: the present invention obtains a pure single earthworm antimicrobial peptide, and the obtained peptide has antibacterial activity against Gram-negative bacteria represented by Escherichia coli and Gram-positive bacteria represented by Staphylococcus aureus, with a minimum inhibitory concentration of MIC 大肠杆菌 =1024μg / mL, MIC 金黄色葡萄球菌 =512 μg / mL. The antimicrobial peptide has good antibacterial effect and can be used to prepare antibacterial products, including antibacterial drugs, additives and preservatives. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings:

[0022] Figure 1 It is a HPLC purification report of the artificially synthesized antimicrobial peptide of the present invention, showing that the purity of the purified antimicrobial peptide is greater than 95%;

[0023] Figure 2 This is the MC report of the artificially synthesized antimicrobial peptide of the present invention. DETAILED DESCRIPTION

[0024] The present invention is described in detail below in conjunction with specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, several changes and improvements can also be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0025] At present, there have been relatively in-depth studies on the whole genome and mitochondrial genome of earthworms, and the genome sequences of some earthworm species have been determined. The present invention analyzes these newly determined gene sequences and uses bioinformatics methods to find single peptide chains with antibacterial effects, thereby providing a new source of antibacterial drugs.

[0026] Example 1: Screening of novel earthworm antimicrobial peptides

[0027] (1) Download the complete genome DNA sequence of earthworm GWHACBE00000000 from the National Center for Bioinformatics (CNCB-NGDC);

[0028] (2) Input the sequence of GWHACBE00000000 in the ORFfinder function of NCBI, select the complete sequence of invertebrate mitochondrial gene coding mode to search for ORFs sequences of 30 nt and above, select "ATG" and other alternative start codons for the start site, calculate, and then choose to download all sequences in protein FASTA format;

[0029] (3) Upload the sequence obtained in the previous step to CAMP R3 The Predict Antimicrobial Peptides function of the server selected four methods, namely SVM, Random Forest, Artificial Neural Network and Discriminant Analysis, for analysis, and selected three sequences (shown in Table 1) that were judged as antimicrobial peptides by all four methods, and whose antimicrobial peptide probabilities by SVM, Random Forest and Discriminant Analysis were greater than 0.90, and commissioned Gill Biochemical (Shanghai) Co., Ltd. to synthesize them artificially using the solid phase synthesis method, with a purity greater than 95%;

[0030] (5) The peptides synthesized in the previous step were diluted to 10 mg / L with sterile water, and antimicrobial peptides with better antibacterial effects were screened out through antibacterial tests. The specific steps are as follows:

[0031] Escherichia coli ATCC25922 (stored in our laboratory) and Staphylococcus aureus ATCC29213 (stored in our laboratory) cultured overnight were diluted 100 times with MH broth (purchased from Shanghai Titan Technology Co., Ltd.) and cultured to OD 600= 0.5, then dilute 1000 times with MH broth medium, then take 180 μL of bacterial solution, add 20 μL of 10 mg / L synthetic peptide, and culture for 18-24 hours, observe the bacterial solution, and judge that the bacterial solution is clear and has no precipitation as having antibacterial activity. Finally, the new antibacterial peptide SEQ ID NO.3: MWRCVATRRRCAAVCRNCAAARR was obtained, and its molecular weight is 2.681 kDa. Table 1 shows the synthetic peptide sequence and its antibacterial activity test results.

[0032] Table 1. Synthesized peptides and their antibacterial effects

[0033]

[0034] In the table, “-” represents sterile growth, and “+” represents bacterial growth.

[0035] Example 2: Determination of the Minimum Inhibitory Concentration of the New Earthworm Antimicrobial Peptide against Escherichia coli

[0036] 1. Reagents and Materials

[0037] The earthworm antimicrobial peptide MWRCVATRRRCAAVCRNCAAARR predicted by bioinformatics in Example 1 was commissioned to be synthesized by Jier Biochemical (Shanghai) Co., Ltd. with a purity greater than 95%. The purification results and MC report are as follows: Figure 1 and Figure 2 shown.

[0038] MH medium was purchased from Shanghai Titan Technology Co., Ltd.;

[0039] Escherichia coli ATCC25922 was stored in this laboratory;

[0040] 2. Determination of Minimum Inhibitory Concentration

[0041] (1) Streak culture of Escherichia coli ATCC25922 on MH agar plate and culture at 37°C overnight;

[0042] (2) Pick a single colony of Escherichia coli, inoculate it into MH broth, and culture it at 37°C and 180 rpm overnight;

[0043] (3) Take 50 μL of the overnight culture solution from step (2) and add it to 5 mL of MH broth medium. Cultivate at 37°C and 180 rpm until OD 600 = about 0.5;

[0044] (4) Add 10 μL of the bacterial solution obtained in step (3) into 10 mL of MH broth to obtain the bacterial solution required for the assay;

[0045] (5) The earthworm antimicrobial peptide (shown in SEQ ID NO. 3) of the present invention was diluted with sterile water to 10240, 5120, 2560, 1280, 640, 320, 160, 80, 40, 20, 10, and 5 μg / mL, respectively;

[0046] (6) Add 90 μL of the bacterial solution required for the assay obtained in step (4) to a sterile 96-well plate, and then add 10 μL of the earthworm antimicrobial peptide solution of various concentrations diluted in step (5) as the experimental group (three parallel experiments). The positive control group is: add 90 μL of the bacterial solution required for the assay obtained in step (4) and 10 μL of sterile water; the negative control group is: add 90 μL of sterile MH broth medium and 10 μL of sterile water; culture at 37°C for 18-24 hours;

[0047] (7) Observe the turbidity of the bacterial solution in each well and whether there is bacterial precipitation. The lowest concentration at which the bacterial solution is clear and has no precipitation is taken as the minimum inhibitory concentration. The experimental results are shown in Table 2:

[0048] Table 2. Determination of minimum inhibitory concentration of Escherichia coli

[0049]

[0050] In the table, “-” represents sterile growth, and “+” represents bacterial growth.

[0051] The test results show that the minimum inhibitory concentration of the earthworm antimicrobial peptide obtained in the present invention against Escherichia coli is MIC 大肠杆菌 =1024μg / mL.

[0052] Example 3: Determination of the Minimum Inhibitory Concentration of the Novel Earthworm Antimicrobial Peptide against Staphylococcus aureus

[0053] 1. Reagents and Materials

[0054] The earthworm antimicrobial peptide and MH culture medium described in the present invention are the same as those in Example 2.

[0055] Staphylococcus aureus ATCC29213 was stored in this laboratory;

[0056] 2. Determination of Minimum Inhibitory Concentration

[0057] (1) Staphylococcus aureus ATCC29213 was streaked on MH agar and cultured at 37°C overnight;

[0058] (2) Pick a single colony of Staphylococcus aureus, inoculate it into MH broth, and culture it at 37°C and 180 rpm overnight;

[0059] (3) Take 50 μL of the overnight culture solution from step (2) and add it to 5 mL of MH broth medium. Cultivate at 37°C and 180 rpm until OD600 = about 0.5;

[0060] (4) Add 10 μL of the bacterial solution obtained in step (3) into 10 mL of MH broth to obtain the bacterial solution required for the assay;

[0061] (5) The earthworm antimicrobial peptide of the present invention was diluted with sterile water to 10240, 5120, 2560, 1280, 640, 320, 160, 80, 40, 20, 10, and 5 μg / mL, respectively;

[0062] (6) Add 90 μL of the bacterial solution required for the assay obtained in step (4) to a sterile 96-well plate, and then add 10 μL of the earthworm antimicrobial peptide solution of various concentrations diluted in step (5) as the experimental group (three parallel experiments). The positive control group is: add 90 μL of the bacterial solution required for the assay obtained in step (4) and 10 μL of sterile water; the negative control group is: add 90 μL of sterile MH broth medium and 10 μL of sterile water; culture at 37°C for 18-24 hours;

[0063] (7) Observe the turbidity of the bacterial solution in each well and whether there is bacterial precipitation. The lowest concentration at which the bacterial solution is clear and has no precipitation is taken as the minimum inhibitory concentration. The experimental results are shown in Table 3:

[0064] Table 3. Minimum inhibitory concentration determination of Staphylococcus aureus

[0065]

[0066] In the table, “-” represents sterile growth, and “+” represents bacterial growth.

[0067] The test results show that the minimum inhibitory concentration of the earthworm antimicrobial peptide obtained in the present invention against Staphylococcus aureus is MIC 金黄色葡萄球菌 =512μg / mL. The peptide has good antibacterial activity against Escherichia coli and Staphylococcus aureus and can be used in the development of antibacterial drugs, additives and preservatives.

[0068] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art can make various changes or modifications within the scope of the claims, which does not affect the essence of the present invention. In the absence of conflict, the embodiments of the present application and the features in the embodiments can be combined with each other arbitrarily. Sequence Listing <110> Shanghai Jiao Tong University <120> Earthworm antimicrobial peptides and screening methods thereof and their application in the preparation of broad-spectrum antimicrobial products <130> KAG45922 <160> 3 <170> SIPO Sequence Listing 1.0 <210> 1 <211> 25 <212> PRT <213> Artificial Sequence <400> 1 Met Gly Tyr Cys Cys Cys Cys Arg Cys Ser Lys Gly Gly Ala Phe Cys 1 5 10 15 Gly Ala Leu Ala Ser Ala Pro Cys Ala 20 25 <210> 2 <211> 24 <212> PRT <213> Artificial Sequence <400> 2 Met Cys Phe Lys Arg Leu Asp Ala Gly Ala Lys His Cys Val Gly Cys 1 5 10 15 Arg Ala Ser Leu Cys Cys Cys Lys 20 <210> 3 <211> 23 <212> PRT <213> Artificial Sequence <400> 3 Met Trp Arg Cys Val Ala Thr Arg Arg Arg Cys Ala Ala Val Cys Arg 1 5 10 15 Asn Cys Ala Ala Ala Arg Arg 20

Claims

1. An earthworm antimicrobial peptide, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

3.

2. The earthworm antimicrobial peptide according to claim 1, characterized in that: The earthworm antimicrobial peptide has 14 hydrophobic amino acid residues and an isoelectric point of 11.

61.

3. Use of the earthworm antimicrobial peptide as claimed in claim 1 in the preparation of broad-spectrum antimicrobial products.

4. The use of the earthworm antimicrobial peptide according to claim 3 in the preparation of a broad-spectrum antimicrobial product, characterized in that: The earthworm antimicrobial peptide has antimicrobial activity against both Gram-negative bacteria and Gram-positive bacteria.

5. The use of the earthworm antimicrobial peptide according to claim 4 in the preparation of a broad-spectrum antimicrobial product, characterized in that: The Gram-negative bacteria is Escherichia coli, and the Gram-positive bacteria is Staphylococcus aureus.

6. The use of the earthworm antimicrobial peptide according to claim 5 in the preparation of a broad-spectrum antimicrobial product, characterized in that: The minimum inhibitory concentration of the earthworm antimicrobial peptide to Escherichia coli is 1024 μg / mL, and the minimum inhibitory concentration to Staphylococcus aureus is 512 μg / mL.

7. The use of the earthworm antimicrobial peptide according to claim 3 in the preparation of a broad-spectrum antimicrobial product, characterized in that: The application includes developing and preparing antibacterial drugs, additives and preservatives using the earthworm antibacterial peptide.

8. A method for screening earthworm antimicrobial peptides according to any one of claims 1 to 7, characterized in that: The steps include: (1) Screening potential earthworm antimicrobial peptides: first download the earthworm whole genome DNA or mitochondrial genome DNA, then search for open reading frames in the earthworm whole genome DNA or mitochondrial genome DNA, and use the antimicrobial peptide prediction server to search for peptides that may have antimicrobial activity, thereby obtaining potential earthworm antimicrobial peptide sequences; (2) Synthesizing potential earthworm antimicrobial peptides: artificially synthesizing potential earthworm antimicrobial peptides according to the potential earthworm antimicrobial peptide sequences obtained in step (1); (3) Antibacterial activity detection: The potential earthworm antimicrobial peptides synthesized in step (2) are subjected to antibacterial experiments to determine the minimum inhibitory concentration (MIC), and the earthworm antimicrobial peptides described in any one of claims 1 to 7 with antibacterial effects are screened out.

9. The method for screening earthworm antimicrobial peptides according to claim 8, characterized in that: In step (1), the method for finding the open reading frame is as follows: input the corresponding sequence into the ORFfinder function of NCBI, select the standard gene encoding method if the sequence is whole genome DNA, select the gene encoding method of invertebrate mitochondria if it is mitochondrial genome DNA, search for open reading frame sequences of 30nt and above in the whole sequence, select "ATG" and other alternative start codons for the start site, calculate, and then choose to download all sequences in protein FASTA format.

10. The method for screening earthworm antimicrobial peptides according to claim 8, characterized in that: In step (1), the method of using the antimicrobial peptide prediction server to search for peptides that may have antimicrobial activity is as follows: uploading the earthworm whole genome DNA or mitochondrial genome DNA to the Predict Antimicrobial Peptides function of the CAMPR3 server, selecting four methods, namely SVM, RandomForest, Artificial Neural Network and Discriminant Analysis, for analysis, and selecting sequences that are judged to be antimicrobial peptides by all four methods and whose antimicrobial peptide probabilities by SVM, Random Forest and Discriminant Analysis are greater than 0.90 as peptides that may have antimicrobial activity.

Images