A targeted antimicrobial peptide against Salmonella and preparation method thereof

Abstract

The present invention discloses a targeted antimicrobial peptide for Salmonella and a preparation method thereof. The targeted antimicrobial peptide for Salmonella is prepared by selecting a short peptide sequence without positive charge from a plurality of peptide segments with affinity for Salmonella obtained by screening a phage peptide library. The sequence only has the function of binding to Salmonella but does not have bactericidal activity. Based on the affinity sequence, positive charge and hydrophobic amino acids are added to the sequence in combination with the structure-function relationship study. The targeted antimicrobial peptide for Salmonella of the present invention can specifically kill Salmonella, ensuring that other bacteria are not affected while killing Salmonella, allowing beneficial bacteria to survive and maintaining the balance of microbial structure.

Description

Technical Field

[0001] The present invention relates to the technical field of antimicrobial peptides, and in particular to an antimicrobial peptide targeted at Salmonella and a preparation method thereof. Background Art

[0002] Salmonella is a pathogen that can cause salmonellosis in humans and animals. The disease is mainly manifested by enteritis and sepsis, and can be fatal in severe cases. Salmonella is often a chronic or latent infection in adult livestock and poultry, making infected livestock and poultry a mobile source of infection, increasing the risk of Salmonella outbreaks in young livestock, poultry, and humans, not only causing serious economic losses to the breeding industry, but also seriously threatening human health. In developed countries, Salmonella is the number one cause of human diarrhea. With the development of my country's economy, the ranking of Salmonella in the causes of human diarrhea has been rising, and it is already in the top three in first-tier cities.

[0003] At present, the means of controlling chronic or latent infections in adult livestock and poultry and preventing salmonellosis outbreaks are mainly based on the large-scale use of broad-spectrum antibiotics. However, the large-scale use of broad-spectrum antibiotics has the following three disadvantages: 1. Inducing the production of drug-resistant bacteria: After bacteria acquire multidrug resistance, they often become "incurable", which seriously threatens human life safety; 2. Broad-spectrum antibiotics will kill beneficial bacteria while killing Salmonella, and the lack of beneficial bacteria is not conducive to the health and resistance of livestock and poultry; 3. Food safety issues: Antibiotics remaining in livestock and poultry products enter the human body, continue to induce the production of drug-resistant bacteria and kill beneficial bacteria in the human intestine. 4. Ecological environmental pollution: Antibiotics entering the environment will destroy the bacterial composition in the environment and cause ecological imbalance. Therefore, the development of antibiotic substitutes is imminent.

[0004] Antimicrobial peptides are an alternative to antibiotics. Antimicrobial peptides have the advantages of fast sterilization rate, no residue, no pollution, etc., and the mechanism of action of antimicrobial peptides is diverse. They can not only reach the surface of bacterial cell membrane through electrostatic interaction, and cause loss of cell membrane integrity and outflow of contents through physical destruction, but also kill cells intracellularly by inducing cell apoptosis, autophagy or necrosis, reducing the possibility of pathogenic microorganisms developing resistance to them.

[0005] However, most of the existing antimicrobial peptides have a broad-spectrum antibacterial effect, killing pathogenic bacteria while indiscriminately killing other beneficial bacteria. The lack of beneficial bacteria is not conducive to the health and resistance of livestock and poultry. Summary of the invention

[0006] In order to overcome the shortcomings of the prior art, the purpose of the present invention is to provide a targeted antimicrobial peptide for Salmonella and a preparation method thereof. Based on the structural and functional relationship of broad-spectrum antimicrobial peptides, a targeted antimicrobial peptide for Salmonella is designed, which can exert a targeted killing effect on Salmonella without affecting other bacteria.

[0007] The purpose of the present invention is achieved by the following technical solutions:

[0008] A targeted antimicrobial peptide against Salmonella, wherein positively charged and hydrophobic amino acids are added to the amino acid sequence shown in SEQ ID NO.1.

[0009] The targeted antimicrobial peptide for Salmonella of the present invention selects a short peptide sequence VTPPTQHQ without positive charge from a plurality of peptide segments with Salmonella affinity obtained by screening a phage peptide library. The sequence only has a function of binding to Salmonella but does not have bactericidal activity. On the basis of the affinity sequence, positive charges are added to the sequence in combination with structure-function relationship research to enhance its mutual attraction with negatively charged bacterial cell membranes and hydrophobic amino acids are added to enhance its penetration into bacterial cell membranes.

[0010] Furthermore, the number of positive charges is 4, the positively charged amino acid is arginine, and the hydrophobic amino acid is tryptophan. The symmetrical distribution of amino acids in antimicrobial peptides helps to improve their antibacterial activity, so four arginines are connected in pairs and placed on both sides of the tryptophan center.

[0011] The method for preparing a targeted antimicrobial peptide against Salmonella comprises the following steps:

[0012] 1) The preparation is carried out one by one from the C-terminus to the N-terminus of the polypeptide, and is completed by a peptide synthesizer; first, 9-fluorenylmethoxycarboxyl-X1 is connected to Wang resin, and then the 9-fluorenylmethoxycarboxyl group is removed to obtain X1-Wang resin; 9-fluorenylmethoxycarboxyl-X2-trimethyl is connected to another Wang resin, and then the 9-fluorenylmethoxycarboxyl group is removed to obtain X2-trimethyl-Wang resin; that is, X 2n-1 (n>0) is through 9-fluorenylmethoxycarboxyl-X 2n-1 Connect to Wang resin, and then remove the 9-fluorenylmethoxycarboxyl group to obtain X 2n-1 -Wang resin; X 2n (n>0) is through 9-fluorenylmethoxycarboxy-trimethyl-X 2n Connect to Wang resin, and then remove the 9-fluorenylmethoxycarboxyl group to obtain X 2n -trimethyl-Wang resin, until the peptide is synthesized from the C-terminus to the N-terminus to obtain a peptide resin with the 9-fluorenylmethoxycarboxyl group side chain protection removed; wherein X n(n>0) is the nth amino acid from the C-terminus of each polypeptide;

[0013] 2) adding a cleavage reagent to the peptide resin obtained in step 1), reacting at 20° C. in the dark, filtering to obtain a filtrate and a precipitate; then washing the precipitate, mixing the washing liquid of the precipitate with the filtrate, concentrating, adding 10 times the volume of pre-cooled anhydrous ether, precipitating at -20° C., centrifuging after white powder precipitates, collecting the precipitate, washing the precipitate with anhydrous ether, and vacuum drying to obtain a polypeptide;

[0014] 3) Using sodium sulfate at pH 7.5 and concentration of 0.2 mol / L for column balance, dissolving the polypeptide obtained in step 2) with 90% acetonitrile aqueous solution, filtering, using C18 reverse phase atmospheric pressure column, using eluent for gradient elution, with detection wave of 220 nm, collecting the main peak, and freeze-drying;

[0015] 4) The polypeptide lyophilized in step 3) is further purified by using a C18 reverse phase atmospheric pressure column and an eluent, and then the main peak is collected and lyophilized to obtain a targeted antimicrobial peptide against Salmonella;

[0016] Further, in step 2), the washing liquid for washing the precipitate is trichloroacetic acid.

[0017] Furthermore, in step 2), the cleavage reagent is trichloroacetic acid, water and triisopropylsilyl chloride in a mass ratio of 95:2.5:2.5.

[0018] Furthermore, in step 3), phosphoric acid is used to adjust the pH to 7.5.

[0019] Furthermore, in step 3), the eluent is methanol and sodium sulfate aqueous solution in a volume ratio of 30:70 to 70:30.

[0020] Further, in step 4), the eluents are eluent A and eluent B, eluent A is an aqueous solution with a concentration of 0.1% TFA; eluent B is an acetonitrile solution with a concentration of 0.1% TFA, and the elution concentration is 25% to 40% of eluent B; wherein, during elution, eluent A and eluent B are added together for elution.

[0021] Furthermore, in steps 3) and 4), the elution flow rate of the C18 reverse phase atmospheric pressure column is 1 mL / min.

[0022] Furthermore, in step 4), the purity of the targeted antimicrobial peptide against Salmonella is analyzed by electrospray ionization mass spectrometry, and the purity of the antimicrobial peptide of the present invention is greater than 95%.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] Based on the structure-function relationship of broad-spectrum antimicrobial peptides, the present invention designs targeted antimicrobial peptides for Salmonella, which can specifically kill Salmonella, ensuring that while killing Salmonella, other bacteria are not affected, allowing beneficial bacteria to survive and maintaining the balance of the microbial structure. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is a conformation diagram of the spiral wheel of Example 1;

[0026] Figure 2 The figure is a comparison chart of the cytotoxicity of Example 1 and Comparative Example 1. DETAILED DESCRIPTION

[0027] The present invention is further described below in conjunction with the accompanying drawings and specific implementation methods. It should be noted that, under the premise of no conflict, the various embodiments or technical features described below can be arbitrarily combined to form a new embodiment.

[0028] Example 1

[0029] A method for preparing a targeted antimicrobial peptide against Salmonella, comprising the following steps:

[0030] 1) The preparation is carried out one by one from the C-terminus to the N-terminus of the polypeptide, and is completed by a peptide synthesizer; first, 9-fluorenylmethoxycarboxyl-X1 is connected to Wang resin, and then the 9-fluorenylmethoxycarboxyl group is removed to obtain X1-Wang resin; 9-fluorenylmethoxycarboxyl-X2-trimethyl is connected to another Wang resin, and then the 9-fluorenylmethoxycarboxyl group is removed to obtain X2-trimethyl-Wang resin; that is, X 2n-1 (n>0) is through 9-fluorenylmethoxycarboxyl-X 2n-1 Connect to Wang resin, and then remove the 9-fluorenylmethoxycarboxyl group to obtain X 2n-1 -Wang resin; X 2n (n>0) is through 9-fluorenylmethoxycarboxy-trimethyl-X 2n Connect to Wang resin, and then remove the 9-fluorenylmethoxycarboxyl group to obtain X 2n -trimethyl-Wang resin, until the peptide is synthesized from the C-terminus to the N-terminus to obtain a peptide resin with the 9-fluorenylmethoxycarboxyl group side chain protection removed; wherein X n (n>0) is the nth amino acid from the C-terminus of each polypeptide;

[0031] 2) Adding a cleavage agent to the peptide resin obtained in step 1), reacting at 20° C. in the dark for 2 h, filtering to obtain a filtrate and a precipitate; then washing the precipitate with TFA (trifluoroacetic acid), mixing the washing liquid of the precipitate with the filtrate, concentrating using a rotary evaporator, adding 10 times the volume of pre-cooled anhydrous ether, and precipitating at -20° C. for 3 h. After a white powder is precipitated, centrifuging at a speed of 2500 r / min for 10 min, collecting the precipitate, and then washing the precipitate with anhydrous ether, and vacuum drying to obtain a polypeptide; wherein the cleavage agent is trichloroacetic acid, water and triisopropylsilyl chloride in a mass ratio of 95:2.5:2.5;

[0032] 3) Using (phosphoric acid adjusted) sodium sulfate with a pH of 7.5 and a concentration of 0.2 mol / L to balance the column for 30 minutes, dissolving the polypeptide obtained in step 2) with a 90% acetonitrile aqueous solution, filtering, using a C18 reverse phase atmospheric pressure column, using an eluent for gradient elution, with a flow rate of 1 mL / min and a detection wave of 220 nm, collecting the main peak, and lyophilizing; wherein the eluent is methanol and sodium sulfate aqueous solution in a volume ratio of 30:70 to 70:30.

[0033] 4) The polypeptide lyophilized in step 3) was further purified using a C18 reverse phase atmospheric pressure column with an eluent at a flow rate of 1 mL / min, and the main peak was collected and lyophilized to obtain Figure 1 The amino acid helical projection is a targeted antimicrobial peptide for Salmonella; wherein the eluents are eluent A and eluent B (eluent A and eluent B are added together), eluent A is an aqueous solution with a concentration of 0.1% TFA; eluent B is an acetonitrile solution with a concentration of 0.1% TFA, and the elution concentration is 25% to 40% eluent B;

[0034] 5) The purity of the targeted antimicrobial peptide against Salmonella was analyzed by electrospray ionization mass spectrometry. The purity of the antimicrobial peptide in this example was greater than 95%.

[0035] Comparative Example 1

[0036] The positively charged and hydrophobic amino acids added in Comparative Example 1 are the same as those in Example 1, and Comparative Example 1 is a broad-spectrum antimicrobial peptide.

[0037] The amino acid sequences of the amino acids in Example 1 and Comparative Example 1 are compared in Table 1:

[0038] Table 1 Amino acid sequences of Example 1 and Comparative Example 1

[0039] Amino acid sequence Peptide chain length Charge Example 1 <![CDATA[VTPPTQHQRRWWWRR-NH2]]> 15 +4 Comparative Example 1 <![CDATA[RRWWWRR-NH2]]> 7 +4

[0040] Wherein, -NH2 in Example 1 and Comparative Example 1 are the same hydrophobic amino acid (tryptophan),

[0041] The positive charges used were all arginine.

[0042] Performance Testing

[0043] 1. Antibacterial activity of Example 1 and Comparative Example 1

[0044] The antimicrobial peptides of Example 1 and Comparative Example 1 were prepared into storage solutions for use. The minimum inhibitory concentrations of Example 1 and Comparative Example 1 were determined by the microbroth dilution method. 0.01% acetic acid (containing 0.2% BSA) was used as the diluent, and a series of gradient antimicrobial peptide solutions were prepared in sequence by the two-fold dilution method. 100 μL of the above solution was placed in a 96-well cell culture plate, and then an equal volume of the test bacterial solution (~10 μL) was added. 5 / mL) in each well. Positive control (containing bacterial solution but not antimicrobial peptide) and negative control (containing neither bacterial solution nor peptide) were set up respectively. Culture at 37°C for 20 hours, and the minimum inhibitory concentration was the concentration at which no turbidity was observed at the bottom of the well with the naked eye. Example 1 and Comparative Example 2 Killing effect of Salmonella typhimurium, Salmonella choleraesuis, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis and Lactobacillus rhamnosus.

[0045] Table 2 Antibacterial activity of Example 1 and Comparative Example 1

[0046]

[0047] According to Table 2, Comparative Example 1 has a strong killing effect on Salmonella typhimurium, Salmonella choleraesuis, Pseudomonas aeruginosa, Staphylococcus, Staphylococcus epidermidis and Lactobacillus rhamnosus. The targeted antimicrobial peptide of Example 1 has a strong antibacterial activity against Salmonella, but a weak antibacterial activity against Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis and Lactobacillus detected simultaneously, and has a more obvious selective bactericidal effect, and has the ability to maintain the balance of microbial structure while exerting antibacterial activity.

[0048] II. Cytotoxicity of the antimicrobial peptides of Example 1 and Comparative Example 1 on mouse macrophages

[0049] The cells that had reached more than 80% plating and were cultured in a medium containing 10% fetal bovine serum and 1% double antibody were washed three times with PBS and digested with 0.25% trypsin. The digestion was terminated with a medium without serum and double antibody and the cell concentration was adjusted to 1×10 5cells / mL; in a 1.5ml EP tube, dilute 2.56mM antimicrobial peptide in 0.2% BSA solution containing 0.1% acetic acid to 256μM to 4μM; take 50μL of cell suspension and mix with different concentrations of peptides in a 96-well plate, set up three parallel groups for each short peptide, and incubate the 96-well plate at 37°C and 5% CO2 overnight, then add 50μL MTT (5mg / mL) to each well, continue incubation for 3 hours, gently aspirate the supernatant and add 100μL DMSO to each well to dissolve the bottom crystals. Use an enzyme marker to measure the absorbance value of the 96-well plate at 570nm, and use the formula survival rate = [(A-A0) / (A t -A0)]×100% (A is the absorbance after treatment with synthetic narrow-spectrum antimicrobial peptides, A0 and A t The absorbance values ​​of the negative control wells without cells and antimicrobial peptides and the positive control wells without antimicrobial peptide treatment were calculated to determine the cell survival rate after antimicrobial peptide treatment. Figure 2 shown.

[0050] like Figure 2 As shown, the cell survival rate of the antimicrobial peptide of Example 1 at the maximum measured concentration of 64 μM is still maintained at 100% and above (there is a systematic error), indicating that the targeted antimicrobial peptide against Salmonella of the present invention has lower cytotoxicity and has the potential to be developed into a drug.

[0051] The above-mentioned embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and substitutions made by technicians in this field on the basis of the present invention shall fall within the scope of protection required by the present invention. Sequence Listing <110>Weike Health Technology (Guangzhou) Co., Ltd. <120>An antimicrobial peptide targeting Salmonella and its preparation method <160> 1 <210>1 <211>15 <212> PRT <213>Artificial sequence <400>1 Val Thr Pro Pro Thr Gln His Gln Arg Arg Trp Trp Trp Arg Arg-NH2 1 5 10 15

Claims

1. A targeted antimicrobial peptide against Salmonella, characterized in that: The amino acid sequence of the targeted antimicrobial peptide against Salmonella is shown in SEQ ID NO.

1.

2. The method for preparing a targeted antimicrobial peptide against Salmonella according to claim 1, characterized in that: The following steps are involved: 1) The preparation is carried out one by one from the C-terminus to the N-terminus of the polypeptide, and is completed by a peptide synthesizer; first, 9-fluorenylmethoxycarboxyl-X1 is connected to Wang resin, and then the 9-fluorenylmethoxycarboxyl group is removed to obtain X1-Wang resin; 9-fluorenylmethoxycarboxyl-X2-trimethyl is connected to another Wang resin, and then the 9-fluorenylmethoxycarboxyl group is removed to obtain X2-trimethyl-Wang resin; that is, X 2n-1 is through 9-fluorenylmethoxycarboxyl-X 2n-1 Connect to Wang resin, and then remove the 9-fluorenylmethoxycarboxyl group to obtain X 2n-1 -Wang resin; X 2n It is through 9-fluorenylmethoxycarboxy-trimethyl-X 2n Connect to Wang resin, and then remove the 9-fluorenylmethoxycarboxyl group to obtain X 2n -trimethyl-Wang resin, until the peptide is synthesized from the C-terminus to the N-terminus to obtain a peptide resin with the 9-fluorenylmethoxycarboxyl group side chain protection removed; wherein X n The nth amino acid from the C-terminus of each polypeptide; Where n>0; 2) adding a cleavage reagent to the peptide resin obtained in step 1), reacting at 20° C. in the dark, filtering to obtain a filtrate and a precipitate; then washing the precipitate, mixing the washing liquid of the precipitate with the filtrate, concentrating, adding 10 times the volume of pre-cooled anhydrous ether, precipitating at -20° C., centrifuging after white powder precipitates, collecting the precipitate, washing the precipitate with anhydrous ether, and vacuum drying to obtain a polypeptide; 3) Using sodium sulfate at pH 7.5 and concentration of 0.2 mol / L for column balance, dissolving the polypeptide obtained in step 2) with 90% acetonitrile aqueous solution, filtering, using C18 reverse phase atmospheric pressure column, using eluent for gradient elution, with detection wave of 220 nm, collecting the main peak, and freeze-drying; 4) The polypeptide lyophilized in step 3) is further purified using a C18 reverse phase atmospheric pressure column and an eluent, and then the main peak is collected and lyophilized to obtain a targeted antimicrobial peptide against Salmonella.

3. The method for preparing a targeted antimicrobial peptide against Salmonella according to claim 2, characterized in that: In step 2), the washing liquid for washing the precipitate is trichloroacetic acid.

4. The method for preparing a targeted antimicrobial peptide against Salmonella according to claim 2, characterized in that: In step 2), the cleavage reagent is trichloroacetic acid, water and triisopropylsilyl chloride in a mass ratio of 95:2.5:2.

5.

5. The method for preparing a targeted antimicrobial peptide against Salmonella according to claim 2, characterized in that: In step 3), phosphoric acid is used to adjust the pH to 7.

5.

6. The method for preparing a targeted antimicrobial peptide against Salmonella according to claim 2, characterized in that: In step 3), the eluent is methanol and sodium sulfate aqueous solution in a volume ratio of 30:70 to 70:

30.

7. The method for preparing a targeted antimicrobial peptide against Salmonella according to claim 2, characterized in that: In step 4), the eluents are eluent A and eluent B, eluent A is an aqueous solution with a concentration of 0.1% TFA; eluent B is an acetonitrile solution with a concentration of 0.1% TFA, and the elution concentration is 25% to 40% of eluent B; during elution, eluent A and eluent B are eluted together.

8. The method for preparing a targeted antimicrobial peptide against Salmonella according to claim 2, characterized in that: In steps 3) and 4), the elution flow rate of the C18 reverse phase atmospheric pressure column is 1 mL / min.

9. The method for preparing a targeted antimicrobial peptide against Salmonella according to claim 2, characterized in that: In step 4), the purity of the targeted antimicrobial peptide against Salmonella is analyzed by electrospray ionization mass spectrometry.

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