Antibacterial peptide PPI45 / PPI47 as well as preparation method and application thereof

Abstract

The invention discloses an antibacterial peptide PPI45 / PPI47 as well as a preparation method and application of the antibacterial peptide PPI45 / PPI47. According to the invention, fungal defensin Plectasin is used as a template, and the self-assembled antibacterial peptide PPI45 / PPI47 is designed. The expression of the self-assembled antibacterial peptide PPI45 / PPI47 in pichia pastoris is realized by optimizing the gene sequence of the antibacterial peptide and constructing a specific expression vector, a perfect purification system is established, large-scale production can be realized, and the self-assembled antibacterial peptide PPI45 / PPI47 can be applied to the fields of antibacterial drug development, hydrogel wound treatment and the like, and has wide application value and market prospect.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering, and in particular to an antimicrobial peptide PPI45 / PPI47 and a preparation method and application thereof. Background Art

[0002] The increase in resistance to traditional antibiotics has promoted the development and application of antimicrobial peptides (AMPs). AMPs are widely present in animals, plants and microorganisms. They are one of the important components of the host's innate immune system and have inhibitory effects on microorganisms, viruses, parasites and even tumors (Liu et al., 2021). The multi-target mechanism of action of AMPs makes it difficult to develop drug resistance, but natural antimicrobial peptides have defects such as low expression, protease sensitivity, poor stability and short duration of action. Some antimicrobial peptides also show insufficient bactericidal activity. Therefore, how to improve their yield, activity and efficacy time has become the current difficulty and hope of antimicrobial peptide drug development (Jiang et al., 2021). Purposeful transformation of natural antimicrobial peptides to overcome deficiencies is of great significance in the development of antimicrobial peptide drugs. Therefore, the present invention designs and transforms antimicrobial peptides, maintains and improves antimicrobial activity, enables them to improve stability and targeting through specific regulation of self-assembly, and controls the release rate. These are the key technical problems to be solved by the present invention. At the same time, self-assembly into hydrogels also expands the clinical administration and application of antimicrobial peptides for the treatment of bacterial infections. Summary of the invention

[0003] The purpose of the present invention is to provide a self-assembling antimicrobial peptide PPI45 / PPI47 and a preparation method and application thereof.

[0004] To achieve the purpose of the present invention, in a first aspect, the present invention provides an antimicrobial peptide PPI45 / PPI47, wherein the amino acid sequence of the antimicrobial peptide PPI45 is shown in SEQ ID NO: 1, and the amino acid sequence of the antimicrobial peptide PPI47 is shown in SEQ ID NO: 2.

[0005] In a second aspect, the present invention provides a DNA sequence encoding the antimicrobial peptide PPI45 / PPI47.

[0006] In a third aspect, the present invention provides a nucleic acid construct, wherein the nucleic acid construct is a nucleic acid construct comprising a nucleic acid sequence encoding the antimicrobial peptide PPI45 / PPI47 and a nucleic acid sequence thereof. XOt I restriction site and Kex2 cleavage site, TAA and TAG terminator sequences are added at the 3' end and Not I restriction enzyme cutting site, and its nucleotide sequence is shown in SEQ ID NO:5 or SEQ ID NO:6.

[0007] In a fourth aspect, the present invention provides a biological material containing a DNA sequence encoding the antimicrobial peptide PPI45 / PPI47 or the nucleic acid construct.

[0008] The biological material includes, but is not limited to, recombinant DNA, expression cassette, transposon, plasmid vector, viral vector, engineered bacteria or transgenic cell line.

[0009] In a fifth aspect, the present invention provides an expression vector, wherein the starting vector is pPICZα A, carrying the DNA sequence encoding the antimicrobial peptide PPI45 / PPI47 or the nucleic acid construct.

[0010] Furthermore, the nucleic acid construct and the vector pPICZα A were respectively XOt I and Not I double enzyme digestion and ligation to obtain the recombinant yeast expression vector.

[0011] In a sixth aspect, the present invention provides a host cell containing the expression vector, preferably Pichia pastoris, more preferably Pichia pastoris X-33.

[0012] In a seventh aspect, the present invention provides a genetically engineered bacterium, wherein the genetically engineered bacterium is obtained by linearizing the expression vector and transforming it into Pichia pastoris X-33, and the resulting yeast transformant is the recombinant Pichia pastoris engineered bacterium.

[0013] In an eighth aspect, the present invention provides a method for culturing the genetically engineered bacteria, comprising the following steps: 1) Preparation of seed solution: Pick a single colony of yeast transformants from the YPD plate, inoculate it into 10 mL YPD liquid medium containing 100 μg / ml Zeocin, and culture it in a shaking incubator at 29°C, 250 rpm for 18-24 h. Inoculate it into 200 mL YPD liquid medium at a 1% v / v inoculum, and culture it in a shaking incubator at 29°C, 250 rpm for 16-18 h until the OD reaches 0. 600nm The value is 6, which means seed solution; 2) Fermentation culture: at 25℃~29℃, add the above seed solution to 2L basal salt medium at a 10% v / v inoculation rate, adjust the pH to 5.0, add 9.6ml PTM1, maintain the ventilation at 8vvm, the rotation speed at 600rpm, and the dissolved oxygen at more than 20%; 3) Feeding carbon source: observe the dissolved oxygen value slowly decreasing and then suddenly rising to more than 80%, start to flow 50% glucose solution containing 12‰ PTM1, the flow acceleration is 12-24mL / L / min, continue to flow for 6-8h, and increase the speed to 1000rpm; 4) Methanol induction: After adding glucose, starve the system for half an hour, and then start adding 100% methanol. The flow rate is gradually increased from 1 mL / L / min in the first hour to 6 mL / L / min in the sixth hour. The rotation speed is increased to 1100 rpm, the pH is increased to 5.5, and the dissolved oxygen is controlled above 20% until the end of fermentation.

[0014] The basic salt medium used in step 2) is as follows: 90 g glucose, 100 g NH 4 H 2 PO 4 , 40gK 2 SO 4 、30g MgSO 4 7H 2 O, 12g KH 2 PO4, 0.8 g CaSO 4 and 3g KOH, add water to make up to 2L.

[0015] In a ninth aspect, the present invention provides a method for expressing antimicrobial peptides PPI45 / PPI47 in recombinant Pichia pastoris, the method comprising: fermenting and culturing the genetically engineered bacteria, and isolating and purifying the antimicrobial peptides PPI45 / PPI47 from the fermentation product.

[0016] In a tenth aspect, the present invention provides any of the following uses of the antimicrobial peptide PPI45 / PPI47: (1) Used for the preparation of antibacterial drugs; (2) Used for preparing fungicides; (3) Used in the preparation of food or feed additives; (4) Used in the preparation of cosmetics.

[0017] The bacteria include Gram-positive bacteria and Gram-negative bacteria.

[0018] The Gram-positive bacteria include Staphylococcus aureus ( Staphylococcus aureus ), Staphylococcus epidermidis ( Staphylococcus epidermidis )、Streptococcus agalactiae( Streptococcus agalactiae ), Streptococcus dysgalactiae ( Streptococcus dysgalactiae ), Streptococcus suis ( Streptococcus suis )wait.

[0019] The Gram-negative bacteria include Escherichia coli ( Escherichia coli ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa )wait.

[0020] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects: The present invention uses the fungal defensin Plectasin as a template to design self-assembling antimicrobial peptides PPI45 / PPI47. By optimizing the antimicrobial peptide gene sequence and constructing a specific expression vector, the expression of the self-assembling antimicrobial peptides PPI45 / PPI47 in Pichia pastoris is achieved, and a complete purification system is established, which can achieve large-scale production and can be applied to the fields of antimicrobial drug development, hydrogel wound treatment, etc., with broad application value and market prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the result of linearized electrophoresis of the recombinant PPI45 / PPI47 vector in Example 3 of the present invention; wherein, M: Trans5KDNA marker; PPI45-1, PPI47-1: linearized recombinant vector PPI45 / PPI47; PPI45-2, PPI47-2: non-linearized recombinant vector PPI45 / PPI47.

[0022] Figure 2 This is the induction curve of the change of wet weight and total protein concentration of PPI45 / PPI47 expressed in the fermenter over time in Example 5 of the present invention.

[0023] Figure 3 These are the antibacterial activity test results of the fermentation supernatant at different induction times after the PPI45 / PPI47 recombinant yeast strain was induced to ferment for 120 hours in Example 5 of the present invention.

[0024] Figure 4 These are the results of Tricine-SDS-PAGE electrophoresis of the fermentation supernatant of the PPI45 / PPI47 recombinant yeast strain induced fermentation at different induction times in Example 5 of the present invention; wherein, M: ultra-low molecular weight protein marker; 1-6: represent the electrophoresis bands of the fermentation supernatant at 0h, 24h, 48h, 72h, 96h, and 120h of induction, respectively.

[0025] Figure 5 This is the Tricine-SDS-PAGE result of the antimicrobial peptide PPI45 / PPI47 after cation exchange chromatography purification in Example 6 of the present invention; wherein, PPI45 M: ultra-low molecular weight protein marker; 1: PPI45 fermentation supernatant; 2: penetration peak; 3: A solution; 4: 5% B peak; 5: 10% B peak; 6: 15% B peak; 7: 20% B peak.

[0026] PPI47 M: ultra-low molecular weight protein marker; 1: PPI47 fermentation supernatant; 2: A solution; 3: 10% B; 4: 10% B; 5: 15% B; 6: 20% B; 7: 20% B dialysis.

[0027] Figure 6This is the mass spectrometry identification result of the antimicrobial peptide PPI45 / PPI47 in Example 6 of the present invention.

[0028] Figure 7 The ANS fluorescence spectra and critical micelle concentrations of different concentrations of the antimicrobial peptide PPI45 / PPI47 of Example 7 of the present invention are shown.

[0029] Figure 8 The antimicrobial peptide PPI45 / PPI47 pair of Example 9 of the present invention Staphylococcus aureus Bactericidal time curve of ATCC 43300.

[0030] Fig. 9 The antimicrobial peptide PPI45 / PPI47 pair of Example 10 of the present invention Staphylococcus aureus Post-antibiotic effect (PAE) of ATCC 43300.

[0031] Fig.10 These are the hemolytic test results of the antimicrobial peptide PPI45 / PPI47 in Example 11 of the present invention.

[0032] Fig.11 These are the results of the cytotoxicity experiment of the antimicrobial peptide PPI45 / PPI47 in Example 12 of the present invention. DETAILED DESCRIPTION

[0033] The present invention aims to provide a self-assembling antimicrobial peptide PPI45 / PPI47 derived from a fungal defensin Plectasin and a preparation method and application thereof.

[0034] The present invention adopts the following technical solution: 1. Design of derivative peptide sequences: Based on bioinformatics, a variety of defensins homologous to Plectasin were compared and analyzed, and the amino acid sequences that changed during the evolution process were replaced and recombined to change the length, charge, isoelectric point and other physicochemical properties of the peptides, thereby obtaining a new fungal defensin Plectasin-derived self-assembling antimicrobial peptide PPI45 / PPI47, the sequences of which are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

[0035] 2. Codon optimization: The DNA sequence encoding the self-assembling antimicrobial peptide PPI45 / PPI47 was optimized according to the codon preference of Pichia pastoris, and the gene sequence encoding the Kex2 signal peptide cleavage site was inserted into the 5' end of the optimized gene sequence (SEQ ID NO:3, SEQ ID NO:4), and the termination codons (TAA and TAG) were connected to the ends to terminate the translation, and restriction endonucleases were added to both ends of the gene sequence. XOt I and Not The I restriction site has the nucleotide sequence shown in SEQ ID NO:5 and SEQ ID NO:6.

[0036] 3. Construction of expression vector: The DNA sequence shown in SEQ ID NO:5 or SEQ ID NO:6 and the vector pPICZαA were XOt I and Not I double enzyme digestion and ligation to obtain the recombinant yeast expression vector.

[0037] 4. Construction of genetically engineered bacteria: The recombinant expression vector is linearized and transformed into Pichia pastoris X-33 to screen for high-expression genetically engineered bacteria.

[0038] 5. The method for culturing the above-mentioned Pichia pastoris X-33 genetically engineered bacteria comprises the following steps: 1) Preparation of seed solution: Pick a single colony of yeast transformants from the YPD plate and inoculate it into 10 mL YPD liquid medium containing 100 μg / mL Zeocin. Incubate at 29°C, 250 rpm, shake for 18-24 h. Inoculate 1% inoculum into 200 mL YPD liquid medium. Incubate at 29°C, 250 rpm, shake for 16-18 h until OD reaches 0. 600nm The value is 6, and the seed solution is obtained; 2) Fermentation culture: at 25℃~29℃, add the above seed solution to 2L basal salt medium at a 10% inoculum volume, adjust the pH to 5.0, add 9.6mL PTM1, maintain the aeration at 8vvm, the rotation speed at 600rpm, and the dissolved oxygen at more than 20%; 3) Feeding carbon source: observe the dissolved oxygen value slowly decreasing and then suddenly rising to above 80%, start to flow 12‰ PTM1 50% glucose solution, the flow acceleration is 12-36mL / L / min, continue to flow for 6-8h, and increase the speed to 1000rpm; 4) Methanol induction: After glucose was added, the cells were starved for half an hour, and then 100% methanol was added. The flow rate was gradually increased from 1 mL / L / min in the first hour to 6 mL / L / min in the sixth hour. The speed was increased to 1100 rpm, the pH was increased to 5.5, and the dissolved oxygen was controlled above 20% until the end of the fermentation. The basic salt medium formula used in step 2) is: 100g NH 4 H 2 PO 4 , 40g K 2 SO 4 、30gMgSO 4 7H 2 O, 12g KH 2 PO4, 0.8 g CaSO 4 and 3 g KOH, add water to make up to 1.8 L, and mix with 200 mL 45% glucose.

[0039] 6. The present invention also provides a method for purifying the recombinant protein secreted by the genetically engineered bacteria Pichia pastoris X-33, which comprises the steps of dialysis desalination, ion exchange chromatography and freeze drying of the fermentation broth.

[0040] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0041] The culture medium and buffer formulations involved in the following examples are: LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl; solid LB medium was added with 2% agarose.

[0042] Low-salt LB medium: 10 g / L tryptone, 5 g / L yeast extract, 5 g / L NaCl; solid low-salt LB medium is added with 2% agar powder.

[0043] MHB medium: Weigh 2.4 g of powder and dissolve it in 90 mL of distilled water. Stir with a magnetic stirrer and adjust the volume to 100 mL after dissolution.

[0044] MHA medium: weigh 3.65 g of powder and dissolve it in 90 mL of distilled water. Stir with a magnetic stirrer and adjust the volume to 100 mL after dissolution.

[0045] YPD medium: 20 g / L peptone, 10 g / L yeast extract, 20 g / L glucose; solid YPD medium is added with 2% agar powder.

[0046] YPDS medium: peptone 20 g / L, yeast extract 10 g / L, sorbitol 182.2 g / L, glucose 20 g / L, agar powder 20 g / L.

[0047] BMGY medium (per liter): yeast extract 10 g, peptone 20 g, glycerol 10 mL, 13.4% amino acid-free yeast nitrogen base (YNB) 100 mL, 0.02% biotin 2 mL, 1 mol / L phosphate buffer, pH 6.0, 100 mL.

[0048] For the use of LB medium, low-salt LB, MH, YPD, YPDS and other culture media, refer to the Invitrogen Pichia pastoris operation manual.

[0049] 50 mM phosphate buffer (Solution A): 7.786 g Na 2 HPO4 , 4.407 g NaH 2 PO 4 , add deionized water to 950 mL, place on a magnetic stirrer until completely dissolved, adjust pH to 7.0, and make up to 1000 mL.

[0050] 1M NaCl 50 mM phosphate buffer (Solution B): 7.786 g Na 2 HPO 4 , 4.407 g NaH 2 PO 4 , 58.46 gNaCl, add deionized water to 950 mL, place on a magnetic stirrer until completely dissolved, adjust the pH to 7.0, and make up to 1000 mL.

[0051] The protein detection method involved in the following examples is Tricine-SDS-PAGE.

[0052] The protein concentration determination method involved in the following examples is the Bradford method.

[0053] The method for determining the protein molecular weight involved in the following examples is the MALDI-TOF MS method.

[0054] The protein purification method involved in the following examples is cationic chromatography.

[0055] The fermentation method involved in the following examples is a high-density fermentation method.

[0056] The strains and plasmids involved in the following examples are shown in Table 1: Table 1 Test strains and plasmids

[0057] Example 1 Design of antimicrobial peptide PPI45 / PPI47 Based on the sequence alignment of the fungal defensin plectasin, the strong antibacterial variant NZ2114, and the self-assembly variant PPI42, the 9th, 13th, and 14th amino acids at the differential sites were arranged and combined to design the derivative peptides PPI45 / PPI47, whose amino acid sequences are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

[0058] Example 2 Construction of yeast recombinant expression vector According to the preferred codon table of Pichia pastoris, the coding gene sequence was optimized. The gene sequence encoding the Kex2 signal peptide cleavage site was inserted at the 5' end, and the termination codons (TAA and TAG) were connected to the 3' end to terminate the translation, and restriction endonucleases were added at both ends of the gene sequence. XOt I and NotI restriction site. The nucleotide sequences of the obtained gene expression cassettes are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively. The designed gene sequence was synthesized by Sangon Biotechnology (Shanghai) Technology Service Co., Ltd. and ligated to the pPICZαA plasmid and stored in E. coli DH5α.

[0059] Example 3 Obtaining the gene fragment of antimicrobial peptide PPI45 / PPI47 1. Plasmid extraction The E. coli plasmid containing the recombinant expression vector was extracted according to the instructions of the plasmid extraction kit. Figure 1 shown.

[0060] 2. Linearization of the recombinant vector pPICZαA-PPI45 / PPI47 use Pme I. The constitutive recombinant expression vector pPICZαA-PPI45 / PPI47 was digested with enzymes. The enzyme digestion system and reaction conditions were as follows: After the above enzyme digestion system was added, it was placed in a 37°C water bath for 4 hours and detected by 1.5% agarose gel electrophoresis. The electrophoresis conditions were: 135-140 V, 35 min. Figure 1 ) shows that the pPICZαA-PPI45 / PPI47 recombinant vector is completely linearized.

[0061] 3. Purification and recovery of linearized recombinant plasmid vector The linearized recombinant plasmid was recovered using a common DNA product purification kit, and the specific operation steps were performed according to the kit instructions. The recovered product was detected by 1.5% agarose gel electrophoresis, lyophilized and concentrated, and stored at 4°C for later use.

[0062] Example 4 Construction of a recombinant yeast strain containing PPI45 / PPI47 genes 1. Preparation of competent cells of Pichia pastoris X-33 1) Pick a single X-33 colony from the YPD plate and inoculate it into 10 mL YPD liquid medium. Culture overnight at 29°C and 250 rpm. 2) Take the overnight culture of Pichia pastoris X-33 and inoculate it into 100 mL YPD liquid medium at 1% inoculation volume. Cultivate at 29°C and 250 rpm until OD 600nm The absorbance value is 1.1-1.3; 3) Centrifuge 50 mL of culture at 4000 rpm for 5 min at 4°C and resuspend in 50 mL of pre-cooled sterile water; 4) Centrifuge at 4000 rpm for 5 min at 4°C, remove the supernatant, and resuspend in 25 mL of pre-cooled sterile water; 5) Centrifuge at 4000 rpm for 5 min at 4°C, remove the supernatant, and resuspend in 2 mL of pre-cooled 1 M sorbitol; 6) Centrifuge at 4000 rpm for 5 min at 4°C, remove the supernatant, add 200 μL of pre-cooled 1M sorbitol and resuspend to obtain X-33 competent cells.

[0063] 2. Electrical conversion Take 100μL of yeast competent cells and add the linearized recombinant plasmid lyophilized powder, mix gently, transfer to an ice-precooled electroporation cup, place on ice for 5 minutes, and electroporate with parameters of 1.2KV, 25μF, and 400Ω. Immediately after electroporation, add 1mL of ice-precooled 1M sorbitol solution, mix well and transfer to a 2mL centrifuge tube, resuscitate at 29℃ for 2h, take 200μL of the resuscitated bacterial solution and spread it on a YPDS plate containing 100μg / mL Zeocin antibiotics, and invert and culture at 29℃ until a single colony grows.

[0064] 3. 48-well plate induction screening of positive transformants Add 500 μL of BMGY medium to each well of a 48-well plate, pick a single colony and place it in the 48-well plate. Set up blank control wells without bacteria, negative control wells with empty pPICZαA plasmid, and positive control wells that can be induced. After shaking and culturing at 29°C and 250 rpm for 24 hours, add 2.5 μL of methanol (final concentration of methanol is 0.5%) to each well, record it as 0h, add methanol every 24 hours, record it as 0h, 24h, 48h, 72h, respectively. After 96 hours of induction, collect the fermentation liquid in the 48-well plate in 1.5mL centrifuge tubes, centrifuge and take the supernatant for antibacterial activity detection.

[0065] Example 5 High-density fermentation of recombinant yeast strains A single transformant colony was picked from the YPD plate and inoculated into a 50 mL shake flask containing 10 mL YPD liquid medium (containing 100 μg / mL zeocin) and incubated at 29°C, 250 rpm for 18-24 h. A 1% v / v inoculum was inoculated into a 1 L shake flask containing 200 mL YPD seed medium and incubated at 29°C, 250 rpm for 16-18 h. The OD 600nm About 6, used as high-density fermentation seed liquid.

[0066] A 5L fermenter was used for high-density fermentation. The fermentation process was divided into three stages: (1) bacterial growth stage: 2L basal salt culture medium was added, sterilized at 121℃ for 20min, cooled to 29℃, pH adjusted to 5.0, 9.6mL PTM1 was added, and 200mL bacterial solution (1:10, volume ratio) was inoculated. The ventilation volume was maintained at 8vvm, the rotation speed was 600rpm, and the dissolved oxygen was maintained above 20%; (2) glucose addition growth stage: the dissolved oxygen value was observed to slowly decrease and then suddenly increase, and 50% glucose solution (12‰ PTM1) was started to be added, the flow acceleration was 24mL / L / min, and the flow was continued for 6h. The rotation speed was increased to 1000rpm, and other fermentation conditions remained unchanged; (3) methanol transition induction stage: fermentation conditions changed, after 6h of glucose addition, starvation for half an hour, 100% methanol was added, and the flow rate in the first hour was 1mL / L / min. Gradually increase to 6mL / L / min in the sixth hour, increase the rotation speed to 1100rpm, increase the pH to 5.5, control the dissolved oxygen above 20%, and keep other fermentation conditions unchanged until the end of fermentation.

[0067] Starting from the transition induction, samples were taken every 24 hours for protein expression analysis and antibacterial activity analysis. Figure 2 The induction curve of the wet weight and total protein concentration of PPI45 / PPI47 expressed in fermenter over time. Figure 3 The antibacterial effect of the supernatant of high-density fermentation of recombinant yeast strains. Figure 4 This is the electrophoresis diagram of fermentation supernatant protein.

[0068] Example 6 Purification of antimicrobial peptides PPI45 / PPI47 1. Cation exchange chromatography purification: The HiPrep SPFF cation exchange column (length 16 mm, inner diameter 10 mm, GE Healthcare) was equilibrated with solution A for 3-5 column volumes before loading. After the injection, the column was eluted with 50 mM phosphate elution buffer (solution A) at pH 7.0. After the penetration peak was eluted, the column was eluted with 50 mM phosphate elution buffer (solution B) at pH 7.0 containing 1 M NaCl. The elution peak was collected and the elution was monitored at UV280 nm. Figure 5 Tricine-SDS-PAGE images and Figure 6 Mass spectrometry detection for purified PPI45 / PPI47.

[0069] 2. 1kDa dialysis bag desalination The collected elution peak was dialyzed through a 1 kDa molecular weight cutoff dialysis bag at 4 ° C, and the water was changed every 2 hours for 6 times. The dialysate after dialysis was collected and freeze-dried in a low-temperature vacuum freeze dryer (-54 ° C, 0.0.016 mPa) to obtain the antimicrobial peptide PPI45 / PPI47 lyophilized powder product. The amino acid sequence of the antimicrobial peptide PPI45 is shown in SEQ ID NO: 1, and the amino acid sequence of the antimicrobial peptide PPI47 is shown in SEQ ID NO: 2.

[0070] Example 7 ANS fluorescence spectra and critical micelle concentration of antimicrobial peptides PPI45 / PPI47 A high concentration of 1,8-ANS solution was prepared using N,N-dimethylformamide (DMF) as solvent and ddH 2 O was diluted to 51.2µM, prepared and used immediately, and stored away from light. The peptide solutions of different concentrations incubated overnight at room temperature were mixed with equal volumes of 1,8-ANS solution (50µl) and added to a 96-well plate, incubated at room temperature for 30 minutes away from light; 0.01M PBS (pH7.4) was used as a negative control. The fluorescence spectrum was detected by a multifunctional microplate reader with an excitation wavelength of 360nm and an emission wavelength of 400-670nm. Critical micelle concentration: The inflection point concentration was calculated using the 490nm fluorescence value as the vertical axis and the concentration lg value as the horizontal axis. The results are shown in Figure 7 shown.

[0071] Example 8 Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of antimicrobial peptides PPI45 / PPI47 Antimicrobial peptide solutions with different concentration gradients (10, 20, 40, 80, 160, 320, 640, 1280 µg / mL) and diluted 10 5 CFU / mL bacterial suspension was added to a 96-well plate at 10µL:90µL and placed in a 37°C incubator for 12-18 h. White precipitate will appear at the bottom of the plate with bacterial growth. The concentration corresponding to the last well on the 96-well plate without bacterial growth is the MIC value of the antimicrobial peptide for the bacteria. The viable bacteria were counted for the dilution of microbial culture solution above the MIC concentration. The minimum concentration with a colony count lower than 99.9% of the initial inoculation is the minimum bactericidal concentration of the antimicrobial agent to kill the microorganism. The results are shown in Table 2.

[0072] Table 2 Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of antimicrobial peptides PPI45 / PPI47 against different bacterial species Example 9 Bactericidal curve of antimicrobial peptide PPI45 / PPI47 Add 10mL of 10 5Different concentrations of PPI45 / PPI47 antimicrobial peptide solutions were added to the CFU / mL bacterial culture solution to make the final concentrations 1×, 2×, and 4×MIC, and then cultured in a shaking incubator at 37°C and 250rpm. 100μl of bacterial solution samples were taken at 0, 0.5, 1, 2, 4, 6, 10, 22, and 24h, and then smeared and counted. The same volume of PBS (pH7.4) and antibiotics at a final concentration of 2×MIC were added as negative and positive controls, respectively. The results are shown in Figure 8 shown.

[0073] Example 10 Post-antibiotic effect (PAE) To 1mL, 10 8 Different concentrations of PPI45 / PPI47 antimicrobial peptide solution and ofloxacin were added to the CFU / mL bacterial culture medium to make the final concentrations 1×, 2×, and 4×MIC. The culture was incubated at 37°C, 250rpm shaking for 2h, 50µl was taken out, and 50ml of preheated MHB (37°C) liquid culture medium was added to dilute 1000 times to remove the peptide effect and rebuild the growth system. This time was defined as 0h. Samples were taken at 0, 0.5, 1, 2, 4, 6, 8, 10, 12, and 24h time points, and 10-fold gradient dilutions were plated and counted. The same volume of PBS (pH7.4) was added as a negative control test. Calculate the time required for the number of colonies in each group to be 10 times higher than the zero point after reconstruction, and compare the PAE by subtracting the time of the control group from that of each experimental group. The results are shown in Fig. 9 shown.

[0074] Example 11 Hemolytic activity test of antimicrobial peptides PPI45 / PPI47 The antimicrobial peptide PPI45 / PPI47 lyophilized powder was dissolved in sterile saline to prepare a stock solution with a concentration of 512 μg / mL, and the final concentration was 1 μg / mL after 2-fold dilution. Blood was collected from the eyeballs of 6-week-old SPF-grade ICR female mice using sodium heparin anticoagulant tubes. The collected blood was incubated at 4 o C, centrifuge at 1500rpm for 10 min, and wash the red blood cells three times with 10mM PBS (pH7.3) until the supernatant is colorless and transparent to make an 8% red blood cell suspension. Take 100μL of red blood cell suspension and antimicrobial peptide PPI45 / PPI47 solution, add them to a 96-well plate, incubate at 37℃ for 1h, centrifuge at 1500rpm for 5min, and aspirate the supernatant to an ELISA plate to detect the ultraviolet absorbance at 540nm. Physiological saline and 0.1% Triton X-100 were used as 0% and 100% hemolysis control experiments, respectively. The formula for calculating the degree of hemolysis is as follows (refer to Yang et al., 2017): Hemolysis (%) = [(Abs540nm antimicrobial peptide Ple-AB-Abs540nm saline) / (Abs540nm 0.1% Triton X-100-Abs540nm saline)] × 100% The results are as follows Fig.10 shown.

[0075] Example 12 Cytotoxicity experiment of antimicrobial peptide PPI45 / PPI47 The cell density was 2.5 × 10 5 HaCaT cells with 10 cells / mL were inoculated in a 96-well plate and cultured in a cell culture incubator for 24 h. Then, an equal volume of 2-256 µg / mL antimicrobial peptide solution diluted 2-fold with saline was added and continued for 24 h. The old culture medium was removed, and the cells were washed 3 times with saline. CCK-8 (10 µM, 100 µl / well) was added in the dark, incubated for 2 h, and the absorbance was measured at 450 nm. Saline was used as a negative control, and no cells were used as zero. Six replicates were set for each concentration.

[0076] Cell survival rate (%) = (As-Ab) / (Ac-Ab) × 100% As: absorbance value of the experimental group (culture medium containing cells, samples, CCK-8) Ac: absorbance value of control group (culture medium containing cells, no sample, CCK-8) Ab: absorbance value of zero adjustment group (culture medium without cells, no sample, CCK-8) The results are as follows Fig.11 shown.

[0077] The present invention successfully optimized the gene sequence encoding the antimicrobial peptide PPI45 / PPI47 and constructed the pPICZαA-PPI45 / PPI47 recombinant expression vector. PmeI was linearized and successfully transformed into Pichia pastoris X-33 to obtain a recombinant yeast strain. The self-assembly characteristics and antibacterial activity of the purified antimicrobial peptide PPI45 / PPI47 were tested. The results showed that the critical micelle concentrations of PPI45 / PPI47 for hydrogel formation in 0.01M PBS (pH7.4) were 973.2 µg / mL and 1016 µg / mL, respectively. PPI45 / PPI47 had good antibacterial activity against Gram-positive bacteria, with rapid bactericidal activity and long-lasting post-antibiotic effect. The hemolytic test results of PPI45 / PPI47 showed that PPI45 / PPI47 in the concentration range of 20-2560 μg / mL almost did not cause hemolysis of erythrocytes. The cytotoxicity test results of PPI45 / PPI47 showed that the cell survival rate of PPI45 / PPI47 was close to 100% at a concentration of 2-256 μg / mL. Experiments have shown that PPI45 / PPI47 has good antibacterial activity, high expression levels, low toxicity, and good self-assembly properties, which increases the possibility of further clinical application of antimicrobial peptides.

[0078] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. Antimicrobial peptides PPI45 / PPI47, among which: The amino acid sequence of the antimicrobial peptide PPI45 is shown in SEQ ID NO: 1, and the amino acid sequence of the antimicrobial peptide PPI47 is shown in SEQ ID NO:

2.

2. A DNA sequence encoding the antimicrobial peptide according to claim 1.

3. A nucleic acid construct, characterized in that The nucleic acid construct is a nucleic acid construct comprising a nucleic acid residue added to the 5' end of the DNA sequence of claim 2. XOt I restriction site and Kex2 cleavage site, TAA and TAG terminator sequences are added at the 3' end and Not I restriction enzyme cutting site, and its nucleotide sequence is shown in SEQ ID NO:5 or SEQ ID NO:

6.

4. A biological material containing the DNA sequence of claim 2 or the nucleic acid construct of claim 3; The biological material is recombinant DNA, expression cassette, transposon, plasmid vector, virus vector, engineering bacteria or transgenic cell line.

5. An expression vector, characterized in that The starting vector is pPICZα A, carrying the DNA sequence of claim 2 or the nucleic acid construct of claim 3.

6. A host cell containing the expression vector according to claim 5; Preferably, the host cell is Pichia pastoris.

7. A genetically engineered bacterium, characterized in that: The genetically engineered bacteria is obtained by linearizing the expression vector described in claim 5 and then transforming it into Pichia pastoris X-33, and the yeast transformant obtained is the recombinant Pichia pastoris engineered bacteria.

8. A method for expressing antimicrobial peptide PPI45 / PPI47 in recombinant Pichia pastoris, characterized in that: The method comprises: fermenting and culturing the genetically engineered bacteria according to claim 7, and separating and purifying the antimicrobial peptide PPI45 / PPI47 from the fermentation product.

9. Any of the following uses of the antimicrobial peptide according to claim 1: (1) Used for the preparation of antibacterial drugs; (2) Used for preparing fungicides; (3) Used in the preparation of food or feed additives; (4) Used in the preparation of cosmetics.

10. The use according to claim 9, characterized in that: The bacteria include Gram-positive bacteria and Gram-negative bacteria; The Gram-positive bacteria include Staphylococcus aureus ( Staphylococcus aureus ), Staphylococcus epidermidis ( Staphylococcus epidermidis )、Streptococcus agalactiae( Streptococcus agalactiae ), Streptococcus dysgalactiae ( Streptococcus dysgalactiae ), Streptococcus suis ( Streptococcus suis ); The Gram-negative bacteria include Escherichia coli ( Escherichia coli ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa ).

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