Antimicrobial peptides from Polysiphonium lanuginosus and their applications

Abstract

The present application provides an antimicrobial peptide of Polysiphonium lanuginosa and its application in the preparation of an anti-infective drug, wherein the amino acid sequence of the antimicrobial peptide of Polysiphonium lanuginosa is SEQ ID NO.1 or SEQ ID NO.2. The antimicrobial peptide of Polysiphonium lanuginosa of the present application has a good antimicrobial effect on a variety of pathogens and drug-resistant pathogens including Staphylococcus aureus, Escherichia coli, Acinetobacter baumannii, and Pseudomonas aeruginosa, and has low cytotoxicity.

Description

Technical Field

[0001] The present application belongs to the field of proteins and the field of marine natural product development. Specifically, the present application provides an antimicrobial peptide from Polysiphon lindl. and an application thereof. Background Art

[0002] Under long-term, high-dose antibiotic pressure, bacteria can develop drug resistance through enzyme secretion, efflux system, sensitive structural gene mutation, etc. After years of use, antibiotic resistance has become a major problem in the prevention and treatment of infectious diseases. In addition to developing new antibiotics and controlling the abuse of antibiotics, the development of antimicrobial peptides is also a feasible way to solve this problem. This is because most antimicrobial peptides achieve their antibacterial effects by acting on bacterial cell membranes, which is different from the mechanism of most antibiotics acting on bacterial DNA replication or metabolism.

[0003] Polysiphonia is a common type of red algae, belonging to the Rhodophyta, Pinaceae, Polysiphonia genus. Polysiphonia mainly grows in rocks or rocky marshes in low tide zones. Common species along the coast of my country are Polysiphonia senticulosa Polysiphonia contains a large amount of phycobiliprotein components such as phycoerythrin, phycocyanin, allophycocyanin (generally believed to account for more than 30%) and other protein components.

[0004] It has been found that extracts from Polysiphonium have multiple functions such as antibacterial, antioxidant, and enhancing nonspecific immunity. Trying to develop antimicrobial peptides from Polysiphonium is a feasible way to combat drug-resistant bacteria. Polysiphonia Lanosa ) is a species of algae of the genus Polysiphonium that grows mainly on the Atlantic coast of Europe. Its growth environment, morphology, and phycobiliprotein composition are similar to those of the common Polysiphonium ( Polysiphonia senticulosa ) are different. Cotton Hair Polysiphon has been approved as a cosmetic by CFDA. Summary of the invention

[0005] In view of the above situation, the applicant tried to use Polysiphon odoratum as material to screen and identify antibacterial substances, especially antibacterial peptides.

[0006] In one aspect, the present application provides an antimicrobial peptide from Polysiphonium lanuginosa, wherein the amino acid sequence of the antimicrobial peptide from Polysiphonium lanuginosa is SEQ ID NO.1 or SEQ ID NO.2.

[0007] On the other hand, the present application provides the use of the above-mentioned Polysiphonium lanuginosa antimicrobial peptide in the preparation of anti-infection drugs.

[0008] Furthermore, the anti-infective drug is an anti-Staphylococcus aureus ( Staphylococcus aureus ), Escherichia coli ( Escherichia coli ), Acinetobacter baumannii ( Acinetobacter baumannii ) and Pseudomonas aeruginosa ( Pseudomonas aeruginosa) is an anti-infective drug that is used to treat one or more bacteria.

[0009] Furthermore, the anti-infection drug is an anti-infection drug for drug-resistant Acinetobacter baumannii or an anti-infection drug for drug-resistant Staphylococcus aureus.

[0010] On the other hand, the present application provides an anti-infective drug, which comprises the above-mentioned Polysiphonium lanuginosa antimicrobial peptide.

[0011] Furthermore, the anti-infective drug is in the form of an injection, an oral dosage form or an external dosage form.

[0012] Furthermore, the anti-infective drug also contains pharmaceutically acceptable excipients.

[0013] The antimicrobial peptide of Polysiphonium lanuginosa of the present application can be prepared by using a common solid phase synthesis method, or by using a genetic engineering method after designing a corresponding gene sequence, vector, and host.

[0014] The dosage forms applicable to the present application include, but are not limited to, coated or uncoated tablets, capsules, suspension granules, oral liquids, water injections, powder injections, sprays, creams, gels, etc. According to the dosage form, various pharmaceutically acceptable excipients can be selected in the preparation, including but not limited to coating materials, capsule shells, fillers, solvents, solubilizers, adhesives, stabilizers, antioxidants, pH adjusters, flavoring agents, lubricants, etc. These excipient components can be selected by those skilled in the art according to common knowledge of pharmacy.

[0015] The preparation of the present application does not exclude other anti-infective drugs, such as antibiotics.

[0016] The cotton wool polysiphon antimicrobial peptide of the present application is selected from red algae with cosmetic and edible precedents, and has low cytotoxicity. It has good antibacterial effects on a variety of pathogens including Staphylococcus aureus, Escherichia coli, Acinetobacter baumannii, and Pseudomonas aeruginosa, and in particular, still maintains a good MIC against drug-resistant bacteria. It provides a new type of choice for the development of new anti-infective drugs to combat clinical drug resistance of pathogens. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the absorption spectrum of water-soluble protein of Polysiphonia;

[0018] Figure 2 This is the peak shape diagram of papain hydrolysate passing through G75 column;

[0019] Figure 3 is the mass spectrum of PL-1;

[0020] Figure 4 is the mass spectrum of PL-2;

[0021] Figure 5is the MIC value of the antibacterial effect of PL-1 and PL-2;

[0022] Figure 6 The cytotoxicity test results of PL-1 and PL-2 are shown in Figure 2. DETAILED DESCRIPTION

[0023] Example 1: Main materials and equipment.

[0024] Seaweed:

[0025] Polysiphonia spp. Polysiphonia Lanosa ) Frozen products were purchased from Guangzhou Zhenghe Biotechnology Co., Ltd.;

[0026] Strains:

[0027] Non-obvious resistant strains:

[0028] Staphylococcus aureus ( Staphylococcus aureus )ATCC29213;

[0029] Escherichia coli ( Escherichia coli )ATCC25922;

[0030] Acinetobacter baumannii ( Acinetobacter baumannii )ATCC15151;

[0031] Pseudomonas aeruginosa ( Pseudomonas aeruginosa )ATCC 27853;

[0032] Resistant strains:

[0033] Staphylococcus aureus ( Staphylococcus aureus ) ATCC43300 (resistant to multiple penicillin antibiotics, etc.);

[0034] Acinetobacter baumannii ( Acinetobacter baumannii )ATCC19606 (resistant to multiple penicillins, chloramphenicol, erythromycin, norfloxacin, etc.).

[0035] The above strains were purchased from BNCC.

[0036] cell:

[0037] HUVEC cells were purchased from Sean Biotechnology.

[0038] Example 2: Extraction of water-soluble protein from Polysiphon lindl.

[0039] Since the commercially available Polysiphonium odoratum is frozen, for the sake of simplicity, the Polysiphonium odoratum protein was directly extracted by freeze-thaw crushing:

[0040] Place the frozen cotton wool Polysiphonium in PBS with pH 7.2 and 0.02M, which is 4-5 times the mass of the frozen cotton wool Polysiphonium, and freeze and thaw three times (thaw at room temperature - freeze at -20℃ - thaw at room temperature - freeze at -20℃ - thaw at room temperature), filter the obtained dark red liquid with gauze and centrifuge to remove algal fragments; add 5 times the volume of 90% v / v ethanol to the filtered liquid, let it stand overnight at -8℃ and then centrifuge; freeze and dry the obtained precipitate to obtain the water-soluble protein of Polysiphonium. The protein content in the product is about 60-65% as determined by the biuret method.

[0041] From the absorption spectrum of water-soluble protein from Polysiphonium Figure 1 ), absorption peaks at 280nm, 360nm, 546nm, 614nm, 498nm, 535nm, and 565nm can be observed, corresponding to phycobiliproteins such as phycoerythrin and phycocyanin. At the same time, there are also a large number of other protein components.

[0042] Example 3: Enzymatic hydrolysis of water-soluble protein from Polysiphon lanuginosa.

[0043] The water-soluble protein of Polysiphonium was prepared into a 20 mg / ml aqueous solution; after adding food-grade protease (purchased from Dongheng Huadao, food grade, prepared according to the marked activity) ingredients, enzymolysis was performed; the enzyme was inactivated at 90°C for 3 minutes; cooled, centrifuged, and optionally concentrated and freeze-dried. The specific enzymolysis conditions are shown in Table 1:

[0044] Table 1: Enzymatic hydrolysis conditions

[0045]

[0046] Example 4: Antimicrobial activity of the enzymatic hydrolysate of the water-soluble protein of Polysiphonium lanuginosa.

[0047] The strains listed in Example 1 were activated and then spread for culture. A single colony was picked and added to a test tube containing 5 ml of broth medium. The culture was shaken at 37° C. for 8 hours and the concentration of the bacterial solution was determined by McFarland turbidimetry.

[0048] Dilute the bacterial suspension to 1×10 6 CFU / ml; the water-soluble protein hydrolysate of Polysiphonium obtained in Example 3 was diluted twice using hydrolyzed casein medium (the initial concentration was calculated as 20 mg / ml); 100 μL of the diluted bacterial solution and 100 μL of the diluted enzymatic hydrolysate solution were added to each well of the plate (3 replicates); cultured at 37°C for 24 hours, the turbidity of the bacterial solution was observed, and the minimum enzymatic hydrolysate concentration for clarifying the bacterial solution was taken as the MIC of the enzymatic hydrolysate (the results of the 3 replicates were consistent, otherwise the experiment was repeated). The results are shown in Table 2:

[0049] Table 2: Antimicrobial MICs of the enzymatic hydrolysates of water-soluble proteins from Polysiphon lindl.

[0050]

[0051] The results showed that the antibacterial activity of the undigested water-soluble protein of Polysiphon odoratum and its trypsin hydrolysate was very weak, while that of the bromelain hydrolysate and the ficin hydrolysate was improved to a certain extent; compared with the undigested water-soluble protein of Polysiphon odoratum, the MIC of the papain hydrolysate was reduced by more than 10 times, and there was no obvious difference in the effect against resistant and non-resistant bacteria, so the antibacterial active ingredients can be further identified.

[0052] Example 5: Identification of antimicrobial peptides in papain hydrolysate.

[0053] The papain hydrolyzate of the water-soluble protein of Polysiphonia lanuginosa was prepared into a 20 mg / ml aqueous solution and passed through a G75 column (3 cm × 75 cm, 10 ml sample, flow rate 0.5 ml / min, 3 minutes / tube). A280 was used as an indicator and the peak shape was plotted according to the number of tubes. Figure 2 As shown, three peaks with a certain degree of distinction were observed, and the corresponding tubes of the three peaks were collected and tested for antibacterial activity against Staphylococcus aureus ATCC29213 and Escherichia coli ATCC25922. It was found that the antibacterial activity of peaks 1 and 2 was significantly lower than that of peak 3 with the smallest molecular weight. The molecular weight of the components therein by electrophoresis was basically lower than 2000.

[0054] Peak 3 from tubes 37-43 was selected for further desalting and then analyzed by LC-MS (ThermoFisher TSQ Altis). LC part: injection volume 3 μL; C18 column 75 μm×25 cm; flow rate 500 nL / min; column temperature 40°C; mobile phase A was 0.1% formic acid in water, mobile phase B was 0.1% formic acid in acetonitrile; linear elution for 1 hour. MS: data dependent acquisition mode; 120,000 resolution; 300-2000 M / z range scan; maximum injection time 100 ms. HCD-MS / MS: resolution: 30,000, dynamic exclusion time: 30 s, maximum injection time: 50 ms.

[0055] The mass spectra were analyzed by PEAKS Studio version 10.6. PEAKS DB searched the uniprot database and further predicted using antiBP2, obtaining a total of 5 antimicrobial peptides. On the ToxinPrep platform, toxicity predictions were performed based on the SVM algorithm and the synthesis difficulty predictions were performed, after which 3 peptides were excluded. The remaining hexapeptide PL-1 (SDVLKR, SEQ ID NO.1) and heptapeptide PL-2 (FLTDGAL, SEQ ID NO.2) were handed over to Nanjing GenScript Biotechnology Co., Ltd. for solid phase synthesis. The mass spectra are shown in Figure 2. Figure 3 ,4 As shown, the molecular weights around 717 and 735 in the figure are consistent with the expected molecular weights of PL-1 and PL-2, and the purity is relatively high.

[0056] Example 6: Antibacterial activity and toxicity of PL-1 and PL-2.

[0057] According to the method described in Example 3, the MIC values ​​of the synthesized PL-1 and PL-2 were detected. The results are shown in Table 3 and Figure 5 As shown:

[0058] Table 3: Antimicrobial MICs of PL-1 and PL-2

[0059]

[0060] The results showed that both PL-1 and PL-2 had significant antibacterial activity, with MIC values ​​much lower than those of the enzymatic hydrolysates, and there was almost no difference in antibacterial activity between resistant bacteria and non-resistant bacteria, indicating that PL-1 and PL-2 may be effective through the so-called membrane lysis matrix commonly found in antimicrobial peptides, so antibiotic-resistant bacteria have basically no resistance to them.

[0061] Cotton-haired Polysiphonium has been approved as a cosmetic by CFDA. There are many edible red algae of similar genera, and the enzymes used in this application are all food-grade enzymes specified in the food additive standards. The above situation endorses the safety of PL-1 and PL-2. On this basis, the applicant further used HUVEC cells to determine their cytotoxicity:

[0062] The purchased HUVEC cells were revived; after trypsin digestion, the cells were suspended by blowing, diluted and counted using a hemocytometer; 100 μL of serum-containing DMEM medium containing about 5000 HUVEC cells was added to each well of the plate, and cultured at 37°C, 5% CO2 for 24 hours; 100 μL of synthetic PL-1 and PL-2 solutions prepared with gradient concentrations using culture medium were added, and cultured at 37°C, 5% CO2 for 24 hours; resazurin staining was performed, and the survival rate was calculated using empty culture medium as a control.

[0063] Figure 6 The results showed that at a concentration of 200 μM, about 90% of HUVEC cells were still alive after 24 hours of culture, indicating that the toxicity of PL-1 and PL-2 to normal human cells was at a relatively low level among antimicrobial peptides, which was consistent with the software prediction results.

Claims

1. The antimicrobial peptide of Polysiphon lindl. is characterized in that: The amino acid sequence of the antimicrobial peptide from Polysiphonium lanuginosa is SEQ ID NO.1 or SEQ ID NO.

2.

2. The use of the antimicrobial peptide of Polysiphonium lanuginosa according to claim 1 in the preparation of an anti-infective drug, wherein the anti-infective drug is an anti-Staphylococcus aureus ( Staphylococcus aureus s), Escherichia coli ( Escherichia coli , Acinetobacter baumannii ( Acinetobacter baumannii ) and Pseudomonas aeruginosa ( Pseudomonas aeruginosa ) is an anti-infective drug that is used to treat one or more bacteria.

3. The use according to claim 2, wherein the anti-infective drug is an anti-infective drug for drug-resistant Acinetobacter baumannii or an anti-infective drug for drug-resistant Staphylococcus aureus.

4. An anti-infective drug, characterized in that: The anti-infective drug comprises the antimicrobial peptide of Polysiphon lindraceus according to claim 1; the anti-infective drug is an anti-Staphylococcus aureus ( Staphylococcus aureus s), Escherichia coli ( Escherichia coli , Acinetobacter baumannii ( Acinetobacter baumannii ) and Pseudomonas aeruginosa ( Pseudomonas aeruginosa ) is an anti-infective drug that is used to treat one or more bacteria.

5. The anti-infective drug according to claim 4, which is in the form of an injection, an oral dosage form or an external dosage form. The anti-infective drug according to claim 5 , further comprising a pharmaceutically acceptable excipient.

7. The method for preparing the antimicrobial peptide from Polysiphon lindracena according to claim 1, characterized in that: The method is a solid phase synthesis method or a genetic engineering method.

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