A cell-free synthesis system, kit and application of antimicrobial peptides

Abstract

The present invention discloses a cell-free synthesis system, kit, and application of antimicrobial peptides, relating to the field of biotechnology. The system comprises a cell extract, a ribonucleotide mixture, an amino acid mixture, a buffer, salt ions, an energy source, an RNA polymerase, and a template; the template can be used to encode the antimicrobial peptide; and the salt ions include magnesium ions. The system offers advantages such as high synthesis efficiency, high safety, and low cost, and the synthesized antimicrobial peptide is stable and highly active.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a cell-free synthesis system, a kit and applications of an antimicrobial peptide. Background Art

[0002] Antimicrobial peptides are protein molecules with bactericidal and antiviral properties, and have broad application prospects in the production of antimicrobial agents, drugs and health products.

[0003] The synthesis of antimicrobial peptides mainly relies on chemical synthesis and natural product extraction. Chemical synthesis is suitable for the synthesis of smaller antimicrobial peptides, but there are still problems such as toxic intermediate byproducts, low synthesis efficiency, poor activity of antimicrobial peptides, and high synthesis costs. In related technologies, antimicrobial peptides (such as Nisin from Streptococcus lactis) are mostly produced through natural product extraction. This method has the advantages of high yield and low purification cost, but the antimicrobial peptides produced often have weak antimicrobial activity and single antimicrobial activity. In addition, long-chain antimicrobial peptides have the advantages of good broad spectrum and high antibacterial efficiency, but they are difficult to prepare in large quantities using traditional chemical and biological technologies, which limits their industrial application.

[0004] Therefore, it is necessary to provide a method for producing antimicrobial peptides with high synthesis efficiency, high safety and low cost, and the produced antimicrobial peptides have good stability and high activity. Summary of the Invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention provides a cell-free synthesis system for antimicrobial peptides, which has high synthesis efficiency, high safety, low cost, and good stability and high activity of the synthesized antimicrobial peptides.

[0006] The present invention also provides a kit.

[0007] The present invention also provides a method for producing the antimicrobial peptide.

[0008] The present invention also provides the use of the cell-free synthesis system or kit in the production of antimicrobial peptides.

[0009] According to the first embodiment of the present invention, a cell-free synthesis system of an antimicrobial peptide comprises: a cell extract, a ribonucleotide mixture, an amino acid mixture, a buffer, salt ions, an energy substance, an RNA polymerase, and a template;

[0010] The template is used to encode the antimicrobial peptide;

[0011] The salt ions include magnesium ions.

[0012] The cell-free synthesis system according to the embodiment of the present invention has at least the following beneficial effects:

[0013] The cell-free synthesis system of the embodiment has high synthesis efficiency, high safety, and low cost. Compared with chemical synthesis or natural product extraction methods, the antimicrobial peptides synthesized by this cell-free synthesis system can improve the antimicrobial activity by 30% to 60% and the stability by 80% to 160%. This cell-free synthesis system has great application prospects in the production of antimicrobial peptides, especially long-chain antimicrobial peptides.

[0014] According to some embodiments of the present invention, the cell extract includes at least one of an Escherichia coli extract, a Bacillus subtilis extract, and a yeast extract.

[0015] According to some embodiments of the present invention, in the cell-free synthesis system, the concentration of the cell extract is 30 w / v% to 70 w / v%, and can further be 40 w / v% to 60 w / v%. For example, it can be 40 w / v%, 42 w / v%, 44 w / v%, 46 w / v%, 48 w / v%, 50 w / v%, 52 w / v%, 54 w / v%, 56 w / v%, 58 w / v%, or 60 w / v%.

[0016] According to some embodiments of the invention, the ribonucleotide mixture is a substrate for synthesizing RNA.

[0017] According to some embodiments of the present invention, in the cell-free synthesis system, the concentration of the ribonucleotide mixture is 1 mM to 3 mM, for example, 1 mM, 1.3 mM, 1.5 mM, 1.7 mM, 2 mM, 2.2 mM, 2.5 mM, 2.7 mM or 3 mM.

[0018] According to some embodiments of the present invention, the amino acid mixture is a substrate for synthesizing the antimicrobial peptide, and the amino acid mixture includes at least one of natural amino acids and unnatural amino acids.

[0019] According to some embodiments of the present invention, the natural amino acids include at least one of alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, serine, tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, histidine, glycine and proline.

[0020] According to some embodiments of the present invention, in the cell-free synthesis system, the concentration of each amino acid in the amino acid mixture is 0.05 mM to 0.3 mM, and can further be 0.05 mM to 0.2 mM. For example, it can be 0.05 mM, 0.07 mM, 0.1 mM, 0.12 mM, 0.14 mM, 0.16 mM, 0.18 mM, or 0.2 mM.

[0021] According to some embodiments of the present invention, the buffer comprises at least one of 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), tris(hydroxymethylaminomethane) (Tris), and phosphate-buffered saline (PBS).

[0022] According to some embodiments of the present invention, the pH of the cell-free synthesis system is 7.0 to 8.5, for example, 7.0, 7.2, 7.5, 7.8, 8.0, 8.2 or 8.5.

[0023] According to some embodiments of the present invention, in the cell-free synthesis system, the concentration of the buffer is 10 mM to 50 mM, and can further be 10 mM to 30 mM. For example, it can be 10 mM, 12 mM, 14 mM, 16 mM, 18 mM, 20 mM, 22 mM, 24 mM, 26 mM, 28 mM, or 30 mM.

[0024] According to some embodiments of the present invention, the source of magnesium ions includes at least one of magnesium acetate, magnesium chloride, magnesium phosphate, magnesium sulfate, magnesium citrate, magnesium nitrate, and magnesium oxalate.

[0025] According to some embodiments of the present invention, in the cell-free synthesis system, the concentration of magnesium ions is 1 mM to 10 mM, and can further be 1 mM to 5 mM. For example, it can be 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, or 5 mM.

[0026] According to some embodiments of the present invention, the salt ions further include at least one of potassium ions, ammonium ions, and sodium ions.

[0027] According to some embodiments of the present invention, the source of potassium ions includes at least one of potassium chloride, potassium sulfate, potassium nitrate, potassium hydroxide, potassium carbonate, potassium bicarbonate, potassium acetate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate and potassium citrate.

[0028] According to some embodiments of the present invention, in the cell-free synthesis system, the concentration of potassium ions is 0 mM to 300 mM. Further, it can be 50 mM to 200 mM. Still further, it can be 50 mM to 150 mM. For example, it can be 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, or 150 mM.

[0029] According to some embodiments of the present invention, the source of ammonium ions includes at least one of ammonium chloride, ammonium sulfate, ammonium nitrate, ammonium phosphate, ammonium carbonate, ammonium bicarbonate, diammonium hydrogen phosphate, ammonium dihydrogen phosphate, and ammonium acetate.

[0030] According to some embodiments of the present invention, in the cell-free synthesis system, the concentration of ammonium ions is 0 mM to 50 mM, and can further be 10 mM to 30 mM, for example, 10 mM, 13 mM, 15 mM, 17 mM, 20 mM, 22 mM, 25 mM, 27 mM, or 30 mM.

[0031] According to some embodiments of the present invention, the source of sodium ions includes at least one of sodium chloride, sodium sulfate, sodium nitrate, sodium sulfite, sodium carbonate, sodium bicarbonate, sodium acetate, disodium hydrogen phosphate, sodium dihydrogen phosphate and sodium citrate.

[0032] According to some embodiments of the present invention, in the cell-free synthesis system, the concentration of sodium ions is 0 mM to 300 mM. Further, it can be 10 mM to 100 mM. Still further, it can be 30 mM to 70 mM. For example, it can be 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, or 70 mM.

[0033] According to some embodiments of the present invention, the energy substance includes a carbohydrate substance, and the carbohydrate substance includes at least one of glucose, fructose, glycerol, maltodextrin, and lactose.

[0034] According to some embodiments of the present invention, in the cell-free synthesis system, the concentration of the energy substance is 20mM to 200mM. Further, it can be 20mM to 100mM. Still further, it can be 30mM to 70mM. For example, it can be 30mM, 35mM, 40mM, 45mM, 50mM, 55mM, 60mM, 65mM, or 70mM.

[0035] According to some embodiments of the present invention, the RNA polymerase comprises T7 RNA polymerase, which can react with the template to transcribe the corresponding mRNA.

[0036] According to some embodiments of the present invention, in the cell-free synthesis system, the concentration of the RNA polymerase is 10 mg / L to 100 mg / L, and can further be 10 mg / L to 50 mg / L, for example, 10 mg / L, 20 mg / L, 30 mg / L, 40 mg / L, or 50 mg / L.

[0037] According to some embodiments of the present invention, the template is a DNA molecule capable of encoding the antimicrobial peptide. The template further comprises at least one of a promoter, a terminator, and an enhancer. For example, the template can be expressed by a T7 promoter.

[0038] According to some embodiments of the present invention, in the cell-free synthesis system, the concentration of the template is 0.1 μg / mL to 10 μg / mL, for example, 0.1 μg / mL, 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, 5 μg / mL, 6 μg / mL, 7 μg / mL, 8 μg / mL, 9 μg / mL, or 10 μg / mL.

[0039] According to some embodiments of the present invention, the cell-free synthesis system may further include a crowding agent.

[0040] According to some embodiments of the present invention, the crowding agent comprises at least one of polyethylene glycol (PEG), polyvinyl alcohol, and dextran.

[0041] According to some embodiments of the present invention, in the cell-free synthesis system, the concentration of the crowding agent is 0 w / v% to 5 w / v%, and can further be 1 w / v% to 3 w / v%. For example, it can be 1 w / v%, 1.2 w / v%, 1.4 w / v%, 1.6 w / v%, 1.8 w / v%, 2 w / v%, 2.2 w / v%, 2.4 w / v%, 2.6 w / v%, 2.8 w / v%, or 3 w / v%.

[0042] According to some embodiments of the present invention, the cell-free synthesis system may further include at least one of (1) to (3);

[0043] (1) Ethyl acetate;

[0044] (2) acylase;

[0045] (3) DNA molecules encoding acylase.

[0046] Acylation of antimicrobial peptides by acylase can effectively improve the stability and antibacterial activity of antimicrobial peptides.

[0047] According to some embodiments of the present invention, in the cell-free synthesis system, the concentration of ethyl acetate is 0 v / v% to 0.1 v / v%, and can further be 0.01 v / v% to 0.1 v / v%. For example, it can be 0.01 v / v%, 0.02 v / v%, 0.03 v / v%, 0.04 v / v%, 0.05 v / v%, 0.06 v / v%, 0.07 v / v%, 0.08 v / v%, 0.09 v / v%, or 0.1 v / v%.

[0048] According to some embodiments of the present invention, in the cell-free synthesis system, the concentration of the acylase is 10 mg / L to 100 mg / L, for example, 10 mg / L, 20 mg / L, 30 mg / L, 40 mg / L, 50 mg / L, 60 mg / L, 70 mg / L, 80 mg / L, 90 mg / L, or 100 mg / L.

[0049] According to some embodiments of the present invention, in the cell-free synthesis system, the concentration of the DNA molecule encoding the acylase is 0.1-1 mg / L, for example, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, or 1 mg / mL.

[0050] According to some embodiments of the present invention, the acylase may be Mycobacterium smegmatis acyltransferase (MsAcT). The DNA molecule encoding the acylase further comprises at least one of a promoter, a terminator, and an enhancer. For example, the acylase may be expressed by a T7 promoter.

[0051] According to the second embodiment of the present invention, an antimicrobial peptide production kit includes the above-mentioned cell-free synthesis system. Since the antimicrobial peptide production kit adopts all the technical solutions of the cell-free synthesis system of the above-mentioned embodiment, it has at least all the beneficial effects brought about by the technical solutions of the above-mentioned embodiment.

[0052] According to the third aspect of the present invention, a method for producing an antimicrobial peptide comprises the following steps:

[0053] The antimicrobial peptide is obtained by incubating the above cell-free synthesis system for reaction.

[0054] According to some embodiments of the present invention, the incubation temperature is 30°C to 40°C. For example, it can be 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C or 40°C.

[0055] According to some embodiments of the present invention, the incubation reaction time is 4 hours to 24 hours.

[0056] According to some embodiments of the present invention, the production method may further include isolating and / or detecting the antimicrobial peptide.

[0057] According to some embodiments of the present invention, the separation includes but is not limited to separation by ion exchange chromatography and / or dextran gel molecular chromatography.

[0058] According to some embodiments of the present invention, the detection includes but is not limited to detection by SDS-PAGE electrophoresis.

[0059] According to the fourth aspect of the present invention, the cell-free synthesis system or antimicrobial peptide production kit is used in the production of antimicrobial peptides.

[0060] Other features and advantages of the present invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments with reference to the accompanying drawings, in which:

[0062] Figure 1 The expression of the antimicrobial peptide synthesized by the cell-free synthesis system of Example 1 of the present invention;

[0063] Figure 2 The expression of the antimicrobial peptide synthesized by the cell-free synthesis system of Example 2 of the present invention;

[0064] Figure 3 The figures show the antibacterial effects of the antimicrobial peptides of Examples 1 and 2 of the present invention under different treatments; * indicates a significant difference (p<0.05), and *** indicates an extremely significant difference (p<0.001). DETAILED DESCRIPTION

[0065] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with the embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0066] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or those recommended by the manufacturer. All reagents or instruments used, if the manufacturer is not specified, are commercially available conventional products.

[0067] In the description of the present invention, the terms "comprises" and "having" and any variations thereof are intended to cover non-exclusive inclusions. For example, a process, method, system, product or apparatus that includes a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to the process, method, product or apparatus.

[0068] The terms "preferably" and "preferably" and the like in this disclosure refer to embodiments of the disclosure that may provide certain beneficial effects under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the description of one or more preferred embodiments does not imply that other embodiments are not useful, nor is it intended to exclude other embodiments from the scope of the present disclosure.

[0069] When a numerical range is disclosed herein, the range is considered continuous and includes the minimum and maximum values ​​of the range, as well as every value between such minimum and maximum values. Further, when a range refers to an integer, every integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe a feature or characteristic, the ranges can be combined. In other words, unless otherwise indicated, all ranges disclosed herein are understood to include any and all subranges subsumed therein.

[0070] In the embodiment of the present invention, the yeast extract used is derived from Pichia pastoris YeastStrain GS115 or SMD1168 (Thermo Fisher, C18100). The preparation method is as follows: Pichia pastoris (initial OD 600 The value was 0.05-0.5) and inoculated into YPD medium at 1 / 1000 (v / v), and cultured at 28°C with shaking until OD 600 The value is 5-6, centrifuged, washed, resuspended in PBS, and then crushed under high pressure, centrifuged, took the supernatant, and freeze-dried to obtain yeast extract.

[0071] In the embodiment of the present invention, the antimicrobial peptide DNA contains a T7 promoter, the amino acid sequence of the antimicrobial peptide is shown in SEQ ID NO.1, and the nucleotide sequence of the antimicrobial peptide DNA is shown in SEQ ID NO.2;

[0072] MSGRGKGGKGLGKGGAKRHRKVLRDNIQGITKPAIRRLARRGGVKRISGLIYEETRGVLKIFLENVIRDAVTYTEHARRKTVTAMDVVYALKRQGRTLYGFGG(SEQ ID NO.1);

[0073] TAATACGACTCACTATAGGGAGACCACAACGGTTTCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACAT ATGTCAGGTAGGGGGAAGGGCGGAAAAGGATTAGGTAAAGGTGGCGCGAAACGTCATCGTAAGG TGCTGCGTGACAATATTCAAGGTATTACCAAACCGGCGATTCGTCGTTTGGCGCGTCGCGGTGGCGTAAAACGCAT CAGCGGTCTGATCTACGAGGAAACCCGTGGCGTCTTGAAGATCTTCCTGGAAAACGTGATCCGCGACGCTGTGACG TACACTGAGCACGCCCGTAGAAAGACCGTTACCGCAATGGATGTTGTTTATGCGCTGAAGCGCCAGGGCCGTACCC TGTATGGTTTTGGTGGCTAAGTCGACCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGC (SEQ ID NO. 2); wherein, the underlined portion is the CDS coding region of the antimicrobial peptide.

[0074] Example 1

[0075] This example provides a cell-free synthesis system for antimicrobial peptides, the formula of which is shown in Table 1.

[0076] Table 1

[0077]

[0078]

[0079] The amino acid mixture consists of alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, serine, tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, histidine, glycine and proline, and the concentration of each amino acid is 0.1 mM.

[0080] A method for synthesizing an antimicrobial peptide comprises the following steps:

[0081] After the cell-free synthesis system shown in Table 1 was reacted at 37° C. overnight, the antimicrobial peptide was purified using ion exchange chromatography and dextran gel molecular chromatography to obtain the antimicrobial peptide.

[0082] The product obtained after overnight reaction at 37°C was designated as the DNA-added group; the sample obtained after purification of the DNA-added group was designated as the purified group; peptide synthesis was performed without the addition of antimicrobial peptide DNA, and the product obtained after overnight reaction at 37°C was designated as the no-DNA group. Each sample was subjected to SDS-PAGE electrophoresis to detect antimicrobial peptide expression.

[0083] Test results such as Figure 1 shown.

[0084] In the cell-free synthesis system of Example 1, the antimicrobial peptide was successfully expressed and purified.

[0085] Example 2

[0086] This example provides a cell-free synthesis system for antimicrobial peptides, the formula of which is shown in Table 2.

[0087] Table 2

[0088]

[0089]

[0090] The amino acid mixture consists of alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, serine, tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, histidine, glycine and proline, and the concentration of each amino acid is 0.1 mM;

[0091] The acylase DNA contains a T7 promoter. The amino acid sequence of the acylase is shown in SEQ ID NO. 3. The nucleotide sequence of the acylase DNA is shown in SEQ ID NO. 4.

[0092] MAKRILCFGDSLTWGWVPVEDGAPTERFAPDVRWTGVLAQQLGADFEVIEEGLSARTTNIDDPTDPRLNGASYLPSCLATHLPLDLVIIMLGTNDTKAYFRRTPLDIALGM SVLVTQVLTSAGGVGTTYPAPKVLVVSPPPLAPMPHPWFQLIFEGGEQKTTELARVYSALASFMKVPFFDAGSVISTDGVDGIHFTEANNRDLGVALAEQVRSLL(SEQID NO.3);

[0093] taatacgactcactataggcctctagaaataattttgtttaactttaagaaggagatatacat ATGGC TAAAAGGATACTATGTTTTGGAGATTCGTTGACCTGGGGTTGGGTTCCGGTTGAAGACGGCGCGCCAACCGAGCGT TTCGCACCAGATGTGCGCTGGACCGGTGTACTCGCGCAGCAGCTGGGTGCGGACTTCGAAGTTATCGAAGAAGGCC TGTCTGCGAGAACTACGAACATCGACGACCCGACCGATCCGCGTCTGAATGGTGCCAGCTACCTGCCGAGTTGCCT GGCGACCCATCTGCCTCTTGACCTCGTCATTATCATGCTGGGCACCAACGATACCAAGGCGTATTTCCGCCGTACC CCGCTGGACATCGCCCTGGGCATGTCCGTGTTGGTCACCCAAGTGTTGACGAGCGCTGGCGGTGTTGGCACGACGT ACCCGGCTCCGAAGGTGCTGGTCGTGAGCCCGCCTCCGCTGGCGCCGATGCCGCACCCGTGGTTTCAGCTGATTTT CGAGGGTGGTGAACAAAAAACCACTGAGCTGGCGCGTGTGTATAGCGCGTTGGCTTCTTTTATGAAAGTTCCGTTT TTCGATGCAGGGTCCGTTATCAGCACCGACGGTGTTGATGGTATTCACTTTACCGAGGCAAATAACCGCGATTTAG GCGTGGCCTTGGCTGAGCAAGTTCGTAGCCTGCTG TAAccaccgctgagcaataactagcataaccccttggggcctctaaacgggtcttgaggggttttttgctgaaa (SEQ ID NO. 4); wherein, the underlined portion is the CDS coding region of the acyl modification enzyme.

[0094] A method for synthesizing an antimicrobial peptide comprises the following steps:

[0095] After the cell-free synthesis system shown in Table 2 was reacted at 37° C. overnight, the antimicrobial peptide (acylated antimicrobial peptide) was purified using ion exchange chromatography and dextran gel molecular chromatography.

[0096] The product obtained after overnight reaction at 37°C was designated as the DNA-added group; the sample obtained after purification of the DNA-added group was designated as the purified group; peptide synthesis was performed without the addition of antimicrobial peptide DNA, and the product obtained after overnight reaction at 37°C was designated as the no-DNA group. Each sample was subjected to SDS-PAGE electrophoresis to detect antimicrobial peptide expression.

[0097] Test results such as Figure 2 shown.

[0098] In the cell-free synthesis system of Example 2, the antimicrobial peptide was successfully expressed and purified.

[0099] Test example

[0100] The antimicrobial peptides synthesized using the cell-free synthesis system of the present invention were used to treat Escherichia coli to test their antimicrobial properties. To verify the stability of the antimicrobial peptides (to test whether they would be degraded by bacterial lysate), the cells were divided into an untreated group and a group treated with bacterial lysate overnight for testing.

[0101] (1) Untreated group:

[0102] 1 μg of the antimicrobial peptide purified in Example 1 (unacylated antimicrobial peptide) or the antimicrobial peptide purified in Example 2 (acylated antimicrobial peptide) was added to 1 ml of LB medium, and the Escherichia coli suspension was inoculated at a dilution of 1 / 1000 (v / v) (initial OD 600 After culturing overnight at 37°C, the OD 600 value.

[0103] (2) Group co-treated with bacterial lysate overnight:

[0104] 10 μg of the antimicrobial peptide purified in Example 1 (unacylated antimicrobial peptide) or the antimicrobial peptide purified in Example 2 (acylated antimicrobial peptide) was added to 10 mL of bacterial lysate and treated at 37°C overnight to obtain a supernatant containing antimicrobial peptides. 1 mL of the supernatant containing antimicrobial peptides was added to 10 mL of LB medium and inoculated with Escherichia coli at a concentration of 1 / 1000 (v / v) (initial OD 600 The cells were cultured at 37°C overnight and the OD 600 value.

[0105] The preparation method of the bacterial lysate is as follows: 10 mL of 10 OD Escherichia coli is crushed by high pressure, and then sterilized by filtration using a 0.22 μm filter membrane to obtain the bacterial lysate.

[0106] Test results are shown in Figure 3 .

[0107] The antimicrobial peptides of Examples 1 and 2 both have good antimicrobial effects; and compared with the unacylated antimicrobial peptide of Example 1, the stability and activity of the acylated antimicrobial peptide of Example 2 are significantly improved.

[0108] The embodiments of the present invention are described in detail above in conjunction with the embodiments, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge scope of ordinary technicians in the relevant technical field without departing from the purpose of the present invention.

Claims

1. A cell-free synthesis system for antimicrobial peptides, characterized in that: The invention comprises at least one of an acylase, a DNA molecule encoding the acylase, a cell extract, a ribonucleotide mixture, an amino acid mixture, a buffer, salt ions, an energy substance, an RNA polymerase, a template, ethyl acetate, and a crowding agent; The amino acid sequence of the acylase is shown in SEQ ID NO: 3; The template can be used to encode the antimicrobial peptide; the amino acid sequence of the antimicrobial peptide is shown in SEQ ID NO: 1; The salt ions are composed of magnesium ions, potassium ions, ammonium ions and sodium ions; In the cell-free synthesis system, the concentration of the cell extract is 30 w / v% to 70 w / v%; the concentration of the ribonucleotide mixture is 1 mM to 3 mM; the concentration of each amino acid in the amino acid mixture is 0.05 mM to 0.3 mM; the concentration of the buffer is 10 mM to 50 mM; the concentration of the magnesium ion is 1 mM to 10 mM; the concentration of the energy substance is 20 mM to 200 mM; the concentration of the RNA polymerase is 10 mg / L to 100 mg / L; the concentration of the template is 0.1 μg / mL to 10 μg / mL; the concentration of the ethyl acetate is 0.01 v / v% to 0.1 v / v%; the concentration of the potassium ion is 50 mM to 200 mM; the concentration of the ammonium ion is 10 mM to 30 mM; the concentration of the sodium ion is 10 mM to 100 mM; and the concentration of the crowding agent is 1 w / v% to 3 w / v%; If present, the concentration of the acylase is 10 mg / L to 100 mg / L; If present, the concentration of the DNA molecule encoding the acylase is 0.1 mg / L to 1 mg / L.

2. The cell-free synthesis system according to claim 1, characterized in that In the cell-free synthesis system, the cell extract includes at least one of an Escherichia coli extract, a Bacillus subtilis extract, and a yeast extract; and / or The buffer comprises at least one of 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), tris(hydroxymethylaminomethane) (Tris), and phosphate-buffered saline (PBS); and / or The source of the magnesium ion includes at least one of magnesium acetate, magnesium chloride, magnesium phosphate, magnesium sulfate, magnesium citrate, magnesium nitrate, and magnesium oxalate; and / or The energy substances include carbohydrate substances.

3. The cell-free synthesis system according to claim 1, characterized in that The crowding agent includes at least one of polyethylene glycol, polyvinyl alcohol, and dextran.

4. A kit for producing antimicrobial peptides, characterized in that: The method comprises the cell-free synthesis system according to any one of claims 1 to 3.

5. A method for producing an antimicrobial peptide, characterized in that: The following steps are involved: The antimicrobial peptide is obtained by incubating the cell-free synthesis system according to any one of claims 1 to 3.

6. Use of the cell-free synthesis system according to any one of claims 1 to 3 or the antimicrobial peptide production kit according to claim 4 in producing antimicrobial peptides.

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