A mussel byssus polypeptide with antibacterial properties and its applications

Abstract

The present invention discloses a mussel foot thread polypeptide with antibacterial properties and its application. This mussel foot thread polypeptide has obvious inhibitory effects on both Gram-positive bacteria and Gram-negative bacteria, can inhibit the formation of their biofilms, and at the same time shows different antibacterial activities against multi-drug resistant bacteria. It has low cytotoxicity and good biocompatibility, and exhibits good adhesion ability. Its peptide chain is short and its molecular weight is small. It is a small molecule polypeptide with great value and shows broad application potential in multiple fields, and has important value in the fields of drug preparation, medical devices and daily chemical products.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to a mussel foot thread polypeptide with antibacterial properties and its application. Background Art

[0002] Infectious diseases caused by pathogenic bacteria are one of the major diseases threatening human life and health. The most common and effective method for preventing and treating bacterial infections is antibiotic treatment. However, many traditional antibiotics have weakened or even almost lost their antibacterial activity against diseases caused by many bacteria. In addition, methicillin-resistant Staphylococcus aureus (MRSA) is the main risk pathogen inducing the development of drug resistance. Therefore, there is an urgent need for new antibacterial agents to combat multi-drug resistant bacteria. In addition, biofilms formed by bacterial colonization and development are another serious clinical problem. Since biofilms can survive in harsh environments, higher doses of antibiotics are required to completely eradicate pathogens. Therefore, there is an urgent need for alternative treatment strategies for treating bacterial infections by combating pathogens and their biofilms through various different actions.

[0003] Mussels are important species in marine aquaculture. Marine mussel adhesion proteins have high strength, high toughness and water resistance, as well as extremely strong functions of adhering to substrates, which are related to their special molecular structure, DOPA (dopamine)-mediated intermolecular cross-linking and the interaction mode with substrates. Moreover, they also have good biocompatibility and degradability, and are a class of bioadhesives with great advantages and potential. Mussel foot thread protein type 5 is considered to be the adhesion protein molecule with the most DOPA content in the mussel attachment plaque and plays a direct adhesion role in interfacial adhesion. Direct extraction of mussel foot thread protein has limitations and is a high-difficulty, uneconomical and low-yield way of obtaining. Preparing natural mussel foot thread protein Mfps by genetic engineering recombination is an effective green biological manufacturing approach to improve the protein yield and purity. Currently, there are few studies on mining the core region in mussel foot thread protein type 5.

[0004] Antimicrobial peptides (AMPs) have gradually attracted wide attention due to their broad antibacterial activity, unique mechanism of action and low drug resistance. However, the clinical application of natural AMPs is hindered by their high sensitivity to proteolytic degradation and non-specific toxicity to mammalian cells. At the same time, the existing antibacterial preparations have poor inhibitory effects on bacterial biofilms. The high lysine content in Mfp-5 reminds people of the high occurrence rate of this residue in antimicrobial peptides, and antimicrobial peptides exert their effects by disrupting the bacterial cell membrane. Based on relevant research, it shows that peptides rich in lysine and DOPA derived from Mfp-5 can indeed exhibit antibacterial activity. Based on this discovery, the most unique region in mussel foot thread type 5 is further found, in order to serve as an alternative to similar antimicrobial peptides to solve the problems existing in the existing antimicrobial peptides. Summary of the Invention

[0005] The object of the present invention is to overcome the deficiencies of the prior art and provide a mussel foot thread polypeptide with antibacterial properties and its applications.

[0006] To achieve the above object of the invention, the present invention adopts the following technical solutions:

[0007] A mussel foot thread polypeptide, the amino acid sequence of which is shown in SEQ ID NO.1.

[0008] The application of the mussel foot thread polypeptide in the preparation of antibacterial drugs; the antibacterial refers to antibacterial against Gram-positive bacteria, Gram-negative bacteria or multi-drug resistant bacteria; the Gram-positive bacteria are Staphylococcus aureus; the Gram-negative bacteria are Escherichia coli or Pseudomonas aeruginosa.

[0009] The multi-drug resistant bacteria are methicillin-resistant Staphylococcus or multi-drug resistant Escherichia coli.

[0010] The mussel foot thread protein has good biocompatibility and degradability and has a variety of applications in the field of medical devices. It can be used as a bridge between implantable metal stents and bioactive substances, and as a microscopic nano-level protective film to effectively protect wounds or skin mucosae. It can be used as a medical adhesive for the treatment of surgical incisions, burns, persistent ulcers and skin diseases, and has the effects of relieving pain, inhibiting bacteria and promoting healing.

[0011] The mussel foot thread polypeptide has rich multi-basal groups and lysine components, can promote cell adhesion and crawling, accelerate wound healing, and at the same time has anti-inflammatory and moisturizing effects, and can be used for the development of anti-aging and moisturizing skin care products.

[0012] The mussel foot thread polypeptide has broad-spectrum adhesiveness and anti-inflammatory properties, and can be used in sensitive skin care products to help relieve skin inflammation and enhance skin barrier function.

[0013] The interfacial adhesion and bonding ability of the mussel foot thread polypeptide give it unique advantages in drug preparation, and its characteristics of promoting cell adhesion and migration have important application prospects in the preparation of drugs that promote cell adhesion or migration.

[0014] By means of bioinformatics and genetic engineering, the present invention fully analyzes the sequence characteristics of mussel byssus polypeptide, analyzes the structural characteristics through structural modeling, and on the basis of correlation analysis, discovers and extracts the key adhesion regions of mussel byssus polypeptide. The excavated mussel byssus polypeptide solves the problems of low yield and poor purity in direct extraction by genetic engineering means, retains its high bonding strength, and at the same time has obvious inhibitory effects on both Gram-positive bacteria and Gram-negative bacteria, can inhibit the formation of their biofilms, has broad-spectrum antibacterial activity and medical value, low cytotoxicity and good biocompatibility. It is used to solve the unoptimistic treatment situation caused by the abuse of existing antibiotics and the formation of bacterial biofilms, and at the same time solve the defects of strong cytotoxicity and low biocompatibility of natural antibacterial peptides.

[0015] I. Excavation of Mussel Byssus Polypeptide

[0016] Based on the comprehensive analysis of the sequence characteristics, structural characteristics and molecular evolution characteristics of type 5 mussel byssus protein according to the core functional region of mussel adhesive protein mentioned in Patent 1 (Guangzhou Jinan University Research and Development Center of Medical Biotechnology Co., Ltd. A novel self-assembled mussel adhesive protein and its application: China, CN117986340B. 2024.07.30), through multi-dimensional information mining, it is found that the conserved domain Domain3 of type 5 mussel byssus protein is its key structural region. This region is extracted from the full-length type 5 mussel byssus protein, and the key core functional region cMfp of mussel adhesive protein is screened out.

[0017] II. Construction of Escherichia coli Vector of Mussel Byssus Polypeptide and Its Expression in Escherichia coli

[0018] The mussel byssus polypeptide involved in the present invention has significant innovation, and its key feature is that it can achieve efficient biosynthesis and expression optimization by genetic engineering means. Specifically, the synthesis process of this polypeptide can be optimized by the codon preference of different expression host systems and combined with a suitable recombinant expression vector. In the present invention, the selectable expression systems include but are not limited to Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris, insect Sf9 host cells and mammalian HEK293 cells. After cloning the target gene into the recombinant expression plasmid and transforming it into the host cell, the host cell is activated by an inducer to achieve overexpression of the target protein. In the preferred embodiment of the present invention, pET20b(+) is recommended as the recombinant expression vector, and Escherichia coli BL21(DE3) is used as the expression host. This combination can significantly improve the expression efficiency of the target protein, while ensuring its biological activity and stability, providing a solid foundation for subsequent industrial applications and research.

[0019] III. Isolation and Purification of Mussel Foot Protein Polypeptide Expression Product

[0020] The fermentation broth was homogenized by a high-pressure homogenizer and centrifuged at 12,000 rpm for 30 min using a BeckMan (JXN-26) high-speed centrifuge in the United States. The supernatant was loaded onto a HiprepTM 16 / 10 CM FF cation exchange column for separation at a loading flow rate of 1 ml / min. The mobile phase was a three-phase eluent: phase A was 0.02 M sodium chloride and 20 mM PB, phase B was 0.5 M sodium chloride and 20 mM PB, and phase C was 2 M sodium chloride and 20 mM PB. The elution flow rate was 2 ml / min. The purification of the expression product was detected by SDS-PAGE electrophoresis.

[0021] IV. Determination of the Minimum Inhibitory Concentration of Mussel Foot Protein Polypeptide

[0022] First, Gram-negative bacteria - Escherichia coli and Gram-positive bacteria Staphylococcus aureus were inoculated into LB medium and TSA medium and cultured for 12 hours. After obtaining the bacterial solution, it was diluted to a concentration of 10 6 CFU / ml, inoculated into a 96-well plate, and 50 μl of cell suspension and 50 μl of diluted protein solution were added to each well. After co-culturing for 24 h, the OD600 absorbance value was measured to confirm the MIC, and the cultured suspension was diluted to a certain multiple and plated for solid plate counting.

[0023] V. Detection of Different Antibacterial Activities of Mussel Foot Protein Polypeptide against Drug-Resistant Bacteria and Non-Drug-Resistant Bacteria

[0024] Bacterial suspensions of overnight-activated Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Escherichia coli, and multidrug-resistant Escherichia coli were centrifuged to discard the supernatant, resuspended with PBS, and diluted to approximately 10 6 CFU / mL. 300 μL of the diluted bacterial solution and 300 μL of protein solutions with different concentrations were mixed in a 2 mL EP tube and cultured at 37°C and 220 rpm. Photos were taken at 2 h and 6 h to observe the turbidity changes, and samples were taken for plating.

[0025] VI. Detection of the Ability of Mussel Foot Protein Polypeptide to Inhibit Biofilm

[0026] Bacterial suspensions of overnight-activated Staphylococcus aureus and Escherichia coli were diluted with medium to approximately 10 6CFU / mL. Inoculate 500 μL of the bacterial suspension and 500 μL of the protein dilution solution into a 24-well microplate. After culturing for 24 and 48 hours, remove the supernatant medium and wash the underlying biofilm 3 times with phosphate-buffered saline (PBS). Then, fix the biofilm in methanol for 15 minutes and stain it with 0.1% (w / v) CV for 10 minutes. Wash the wells with sterile water and dry them at 37°C for 30 minutes. Capture images using an optical microscope. Then dissolve the crystal violet by shaking (80 r / min) with 500 μL of 33% glacial acetic acid at 37°C for 30 minutes. Measure the OD 595 absorbance value at

[0027] VI. Detection of the cytotoxicity of mussel byssus polypeptide

[0028] Determine the cytotoxicity of mussel byssus polypeptide by MTT cell proliferation assay. Use the coating method to air-dry and modify the bottom of the wells of a 96-well plate with solutions of different protein concentrations. After resuscitating and activating L929 cells, inoculate them into the 96-well plate, 1x10 4个 cells / well and culture them in a constant temperature incubator at 37°C with 5% CO2 for 24 hours (for the blank control, use PBS instead of the protein solution). At the end of the culture, remove the medium, add MTT solution with a final concentration of 0.5 mg / mL to each well, and continue culturing for 4 h. At the end of the culture, carefully remove the medium, add 200 μL of formazan solution to each well, and slowly shake using a shaker for decolorization to ensure complete dissolution. After 10 minutes, measure the absorbance of the solute at 570 nm using an enzyme-labeling instrument.

[0029] VII. Test for the in vitro adhesion ability of mussel byssus polypeptide

[0030] Drop 30 ng of the protein solution above a plastic culture dish, bond pipette tips of different sizes to the dish, and dry them in a humid environment at 25°C for 12 h, then observe the bonding situation.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] The mussel byssus polypeptide of the present invention has obvious inhibitory effects on both Gram-positive bacteria and Gram-negative bacteria, can inhibit the formation of their biofilms, and at the same time shows different antibacterial activities against multi-drug resistant bacteria, has low cytotoxicity and good biocompatibility, and shows good adhesion ability. Its peptide chain is short and its molecular weight is small. It is a small molecule polypeptide with great value and has broad application prospects.

[0033] 1. As a new type of antibiotic for the treatment or prevention of bacterial infections, it provides new ideas and treatment plans for the clinical treatment of multi-drug resistant bacterial infections.

[0034] 2. Solve the defects of strong cytotoxicity and low biocompatibility of natural antimicrobial peptides.

[0035] 3. As the most unique region in mussel foot thread protein type 5, it is expected to be used as an adhesion domain for protein design. Brief Description of the Drawings

[0036] To more clearly illustrate the embodiments of the present invention, the drawings related to the embodiments will be briefly introduced below.

[0037] Figure 1 It is the mass spectrometry detection chart of mussel foot thread polypeptide;

[0038] Figure 2 It is the predicted alph-helical wheel chart of mussel foot thread polypeptide;

[0039] Figure 3 It is the plasmid chart of the expression vector of mussel foot thread polypeptide;

[0040] Figure 4 It is the electrophoresis chart for screening the expression bacteria of mussel foot thread polypeptide;

[0041] Figure 5 It is the purification effect chart of mussel foot thread polypeptide by cation exchange chromatography;

[0042] Figure 6 It is the antibacterial effect chart of mussel foot thread polypeptide against non-resistant bacteria;

[0043] Figure 7 It is the antibacterial effect chart of mussel foot thread polypeptide against drug-resistant bacteria;

[0044] Figure 8 It is the crystal violet quantification chart of mussel foot thread polypeptide inhibiting biofilm formation;

[0045] Figure 9 It is the detection result chart of the cytotoxicity of mussel foot thread polypeptide;

[0046] Figure 10 It is the effect chart of the in vitro adhesion ability of mussel foot thread polypeptide. Detailed Embodiments

[0047] The present invention will be further described below in conjunction with specific embodiments. It should be understood that the cited embodiments are only used to illustrate the present invention and not to limit the scope of the present invention.

[0048] Example 1: Mining of Mussel Foot Protein Polypeptide cMfp

[0049] A total of 405 relevant protein information entries were retrieved from the NCBI database. Combining the results of blastp alignment and manual verification, 4 target entries with complete nucleic acid and protein sequences were selected: Mytilus galloprovincialis, Mytilus edulis, Mytilus coruscus, and Mytilus californianus. The amino acid composition of the byssus proteins of these four mussels was analyzed and found to be mainly composed of glycine (Gly), lysine (Lys), tyrosine (Try), and serine (Ser). Based on the obtained basic sequence information, multiple sequence alignment was used to further analyze the characteristics and distribution of specific sequence sites. The threshold was set at a Max score of over 100 to ensure that the obtained sequences had high similarity and possible homology. The results showed that the 4 type 5 mussel byssus proteins were significantly divided into three relatively conserved regions. MEME was used to mine and predict the protein conserved motifs to identify possible functional domains or binding site protein motifs. The mining results were consistent with the multiple sequence alignment results. At the same time, it could be intuitively found from the visualized motif map that the domain3 region had the highest enrichment of tyrosine and lysine in the type 5 mussel byssus protein. The ProtParam tool was used to analyze the physicochemical properties of the sequence, including protein molecular weight, theoretical isoelectric point, amino acid composition, instability coefficient, and lipophilicity index, etc. The fasta file of the protein sequence was uploaded to the SignalP 6.0 platform to predict whether there were potential signal peptide cleavage sites and their positions in the given amino acid sequence. The AbinitioRelax application was used for Robetta de novo prediction to predict the initial structure of the protein. At the same time, the Rosetta all-atom force field was used for all-atom refinement to obtain a more accurate protein structure model. The structure models of 4 type 5 mussel byssus proteins were constructed de novo using the Alphfold2 algorithm. PSI-PRED was used as the secondary structure predictor, and I-TASSER was used for homology modeling to predict the tertiary structure of the protein. By correlating the primary sequence features, three-dimensional structure features, and the distribution of molecular evolution sites, the key mussel mucin core functional region cMfp was screened out. The mass spectrometry detection of the obtained protein is as Figure 1 shown. The results of the alph-helical wheel diagram showed that lysine and tyrosine in the cMfp region had the potential to form alternating helical loops. Further spatial structure simulation found that a helical-like conformation could indeed be formed, as Figure 2 shown.

[0050] Example 2: Construction and Expression Screening of Expression Vector of Mussel Foot Protein Polypeptide cMfp

[0051] The codons of the core polypeptide of mussel foot protein type 5 were optimized for codon preference according to that of Escherichia coli. The full gene of the core polypeptide fragment of mussel foot protein type 5 was synthesized by a gene synthesis company. The obtained cMfp gene fragment was inserted between NdeI and EcoRⅠ of the pET20b(+) vector to form the pET20b(+)-cMfp vector. The plasmid map of the vector is as Figure 3 shown.

[0052] According to the instructions of the TIANgel Midi Purification kit provided by Takara, the obtained pET20b(+) - cMfp vector ligation product was introduced into TOP10 competent cells. Subsequently, the transformed cells were placed on a resistance plate containing ampicillin (Amp+), and cultured at 37°C. Single clone colonies were picked from the plate, and positive clones were identified by PCR. The colonies identified as positive were inoculated into 5 mL of ordinary LB medium containing 100 μg / mL ampicillin and cultured overnight at 37°C. After the culture was completed, the plasmid was extracted and sent for sequencing detection to confirm the accuracy of the sequence.

[0053] The pET20b(+)-cMfp plasmid was transformed into Escherichia coli BL21(DE3)pLysS competent cells. Randomly select 10 single clone colonies, and screen out positive clones by PCR. The positive colonies were inoculated into 20 mL test tubes, added with 5 mL of LB medium containing 100 μg / mL ampicillin, and cultured overnight at 37°C. Subsequently, according to the inoculation amount of 1:100, 50 μL of the overnight cultured bacterial solution was inoculated into 5 mL of LB medium containing 50 μg / mL ampicillin, and cultured at 37°C for about 3 hours until the OD600 value reached 0.6 - 0.8. When the OD600 value reached the target range, 200 μL of the bacterial solution was taken, centrifuged at 12000 rpm for 1 minute, and the bacteria before induction expression were collected. Then, 1 mM IPTG was added to the remaining medium, and the culture was continued at 37°C for 3 hours to induce protein expression. After the induction was completed, 200 μL of the bacterial solution was taken again, centrifuged at 10000 rpm for 1 minute, and the bacteria after induction expression were collected. 80 μL of 5% SDS solution was added to the bacteria before and after induction expression, and boiled at 100°C for 10 minutes until the solution became clear. Then 20 μL of 5xloading buffer was added, and boiled at 100°C for 10 minutes again to complete the sample preparation. Finally, the samples before and after induction expression were identified by SDS-PAGE electrophoresis, and the results are as Figure 4 shown.

[0054] Example 3: Purification of Mussel Foot Protein Polypeptide cMfp

[0055] First, accurately weigh 100 g of wet weight of BL21(DE3) / pET20b(+)-cMfp cells. Then, use 1000 ml of 20 mM phosphate buffer (PB) and 0.15 M sodium chloride solution (pH 7.4) as the resuspension buffer to fully resuspend the cells. After resuspension, place the cell suspension in a low-temperature environment at 4°C and perform 3 crushing treatments using a high-pressure homogenizer, with the pressure set at 800 Pascals for each crushing. After crushing, centrifuge the sample at 12,000 rpm for 30 minutes to separate cell debris and unbroken cells. Finally, collect the supernatant for subsequent experimental operations. Select a cation exchange column according to the characteristics of the cMfp protein. Before sample loading, pre-treat the column by washing away ethanol with 3 - 5 column volumes of distilled water and equilibrating the column with 5 column volumes of binding buffer. The recommended flow rate is 1 ml / min. After equilibration, load the sample and then equilibrate with the binding buffer for at least 5 column volumes until the absorption peak reaches a stable baseline. Wash away unbound and non-specific proteins with the washing buffer (20 mM PB 0.5 M NaCl), at least 3 - 5 column volumes. Finally, elute with the elution buffer (20 mM PB 2 M NaCl), usually 5 - 10 column volumes, and collect the fractions from the rising to the falling of the absorption peak until it returns to a stable baseline to obtain the purified novel self-assembled mussel adhesive protein cMfp. Detect the purification status by SDS-PAGE electrophoresis, and the results are as Figure 5 shown. The obtained mussel foot thread polypeptide has an amino acid sequence as shown in SEQ ID NO.1.

[0056] Example 4: Determination of Minimum Inhibitory Concentration of Mussel Foot Protein Polypeptide cMfp against Gram-Positive and Gram-Negative Bacteria

[0057] Determine the minimum inhibitory concentration (MIC) of the polypeptide using the microbroth dilution method. Inoculate Gram-negative bacteria - Escherichia coli and Gram-positive bacteria Staphylococcus aureus into LB medium and TSA medium and culture at 37°C for 12 hours. After obtaining the bacterial solution, dilute it to a concentration of 10 6 CFU / ml. Prepare a 2 mg / ml stock solution of the polypeptide for standby. Use the two-fold dilution method to sequentially prepare a series of gradient polypeptide solutions. Inoculate 50 μl of the cell suspension and 50 μl of the diluted protein solution into a 96-well plate, and add 100 μl of medium to each well. Set positive controls (containing bacterial solution but no polypeptide) and negative controls (containing neither bacterial solution nor polypeptide) respectively. After culturing for 12 - 16 h, use an enzyme-linked immunosorbent assay (ELISA) reader to detect the OD 600 absorbance value to confirm the MIC, and dilute the cultured suspension to a certain multiple and plate it for solid plate counting method. The results are as Figure 6 shown.

[0058] Example 5: Detection of Bacteriostatic Activity of Mussel Foot Protein Polypeptide cMfp against Drug-Resistant and Non-Drug-Resistant Bacteria

[0059] Gram-negative drug-resistant bacteria - MDR-E. coli (multidrug-resistant Escherichia coli) and non-drug-resistant Escherichia coli, as well as gram-positive drug-resistant bacteria - MRSA (methicillin-resistant Staphylococcus aureus) and non-drug-resistant Staphylococcus aureus were inoculated into TSB medium and cultured at 37°C for 12 hours. Take 200 μL of the overnight-activated MRSA and MDR-E. coli bacterial suspensions, centrifuge at 8000 rpm for 1 minute to discard the medium, resuspend with 200 μL of PBS and dilute to approximately 10 6 CFU / mL. Take 200 μL of the diluted bacterial solution and 200 μL of protein solutions at different concentrations, mix well in a 2 mL EP tube, culture at 37°C and 220 rpm, take pictures at 2 h and 6 h to observe the turbidity change and sample for plating. The results are as Figure 7 shown.

[0060] Example 6: Detection of the Ability of Mussel Foot Protein Polypeptide cMfp to Inhibit Biofilm Formation

[0061] Gram-negative bacteria - Escherichia coli and gram-positive bacteria Staphylococcus aureus were inoculated into LB medium and TSA medium and cultured at 37°C for 12 hours. The overnight-activated Escherichia coli and Staphylococcus aureus bacterial suspensions were diluted with the medium to approximately 10 6 CFU / mL. 500 μL of the bacterial suspension and 500 μl of the protein dilution were inoculated into a 24-well microplate. After culturing for 24 and 48 hours, the supernatant medium was removed, and the underlying biofilm was washed 3 times with phosphate-buffered saline (PBS). Then, the biofilm was fixed in methanol for 15 minutes and stained with 0.1% (w / v) CV for 10 minutes. The wells were washed with sterile water and dried at 37°C for 30 minutes. Images were captured using an optical microscope. Then, 500 μl of 33% glacial acetic acid was used to dissolve crystal violet by shaking (80 r / min) at 37°C for 30 minutes. The OD 595 absorbance was measured with a microplate reader. The results are as Figure 8 shown.

[0062] Example 7: Cytotoxicity Detection of Mussel Foot Protein Polypeptide cMfp

[0063] The purified and filter-sterilized cMfp protein solution was diluted with PBS to concentrations of 0.8, 0.4, 0.2, and 0.05 mg / ml. Take 10 μL of protein solutions at different concentrations and add them to the wells of a 96-well plate respectively to coat the bottom of the plate. After the protein solution was dried, a protein film layer was formed, washed 3 times with PBS, and air-dried. The wells without any treatment were used as the control group. After completing the modification of the bottom of the plate, the 96-well plate was irradiated under an ultraviolet lamp for 1 hour. Subsequently, well-grown L929 cells were digested with trypsin and seeded at 1×10 per well 4The cells were inoculated into a protein-modified 96-well plate and an unmodified 96-well plate at a density of [number of cells]. The cells were cultured in a cell incubator at 37 °C for 24 hours. After culturing to the designated time point, 10 μL of MTT solution was added to each well and cultured for another 4 hours. Carefully aspirate all the supernatant in the wells, then add 100 μL of formazan solubilization solution to each well. After the formazan is completely dissolved, use a microplate reader to measure the absorbance at 570 nm and plot the absorbance curve. The experimental results are as Figure 9 shown.

[0064] Example 8: In Vitro Adhesion Ability Test of Mussel Foot Protein Polypeptide cMfp

[0065] Take an appropriate amount of sample solution and pipette 10 μL of it onto the surface of a culture dish, which is equivalent to a sample amount of approximately 30 ng. Subsequently, place pipette tips of different specifications (including 1 mL, 200 μL, and 10 μL) above the sample solution to ensure that the pipette tips are in full contact with the sample. After the solution dries naturally, carefully invert the lid of the culture dish and evaluate the bonding effect by observing whether the pipette tips are firmly adhered to the lid of the culture dish. The specific observation results are as Figure 10 shown. The pipette tips of the three specifications of 1 mL, 200 μL, and 10 μL all showed good bonding performance and could firmly adhere to the lid of the culture dish, indicating that these pipette tips have high stability and reliability in micro-sample handling.

Claims

1. A mussel byssus polypeptide, the amino acid sequence of which is shown in SEQ ID NO.

1.

2. Use of the mussel byssus polypeptide according to claim 1 in the preparation of an antibacterial drug; the antibacterial means against Staphylococcus aureus or Escherichia coli.

3. Use of the mussel byssus polypeptide according to claim 1 in the preparation of an antibacterial drug; the antibacterial means against methicillin-resistant Staphylococcus aureus or multidrug-resistant Escherichia coli.

4. Use of the mussel byssus polypeptide according to claim 1 in the preparation of a bioadhesive for protecting wounds or skin and mucous membranes.

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