Screening method and application of antibacterial peptide and antibacterial peptide

Abstract

The invention belongs to the technical field of biology, and discloses a screening method and application of an antibacterial peptide and the antibacterial peptide. According to the screening method of the antibacterial peptide provided by the invention, a to-be-selected antibacterial peptide expression vector constructed by taking a nucleotide fragment with a sequence as shown in SEQ ID NO: 2 as a ribosome binding site is adopted, and protein expression in a cell-free protein expression system can be realized under the condition of no other treatment (such as transformation, transfection and the like) or specific condition stimulation; according to the method, the to-be-selected antibacterial peptide fermentation liquor is used for preparing the antibacterial peptide rapidly, then the growth condition of pathogenic bacteria after the to-be-selected antibacterial peptide fermentation liquor and the pathogenic bacteria culture solution are mixed is observed, screening of the antibacterial activity of the antibacterial peptide can be achieved, and huge potential is shown in rapid and high-throughput screening of the antibacterial peptide.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a screening method and application of an antibacterial peptide and the antibacterial peptide. Background Art

[0002] Antibacterial peptides (AMPs) refer to small-molecule polypeptides with antibacterial biological activities that widely exist in organisms. They play a direct bactericidal role by destroying the integrity of bacteria and have abundant microbial targets. Compared with traditional antibiotics, AMPs have the advantages of a broad antibacterial spectrum, stable physicochemical properties, and low tendency to generate drug resistance, and have good application prospects in research fields such as medicine, veterinary medicine, and life sciences. Therefore, it is of great significance to develop an antibacterial agent that is not prone to generating drug resistance and is highly efficient and broad-spectrum.

[0003] Currently, the screening methods of antibacterial peptides mainly include microbial screening method, cytotoxicity screening method, high-throughput screening method, and surface plasmon resonance screening method. And the above methods all need to first prepare antibacterial peptides and then screen the obtained antibacterial peptides. Generally, the extraction method or genetic engineering technology is used to prepare antibacterial peptides. However, the extraction method has problems such as high cost and unsatisfactory biological activity of the extracted antibacterial peptides, while the genetic engineering technology has problems such as complex steps, high technical requirements, and the antibacterial peptide will inhibit the production strain, and it is impossible to well realize the preparation of antibacterial peptides, which seriously restricts the screening and research of antibacterial peptides and has great limitations. Summary of the Invention

[0004] Based on the problems existing in the preparation / extraction process of antibacterial peptide acquisition in the existing antibacterial peptide screening, such as complex process, high technical requirements, high cost, and unsatisfactory structure of the obtained antibacterial peptide, the inventors of the present invention have conducted extensive and in-depth research and a large number of experiments, and creatively found that: a candidate antibacterial peptide expression vector constructed with a nucleotide fragment shown in SEQ ID NO:2 as the ribosome binding site can achieve efficient protein expression in a cell-free protein expression system only by using a simple mixed culture method without other treatments (such as transformation, transfection, etc.) or specific condition stimulation, and obtain the corresponding antibacterial peptide, which greatly simplifies the operation steps, improves the experimental efficiency and reduces the cost, and shows great potential in realizing the high-throughput screening of antibacterial peptides and has very excellent application prospects. Based on this, the technical solution of the present invention is obtained.

[0005] In a first aspect, the present invention provides a method for screening antibacterial peptides. This screening method involves constructing a candidate antibacterial peptide expression vector containing a ribosome binding site with a nucleotide sequence as shown in SEQ ID NO:2. This candidate antibacterial peptide expression vector can achieve protein expression in a cell-free protein expression system under simple mixing conditions, thereby obtaining a fermentation broth containing the candidate antibacterial peptide. Finally, by using a conventional microbial screening method to detect the effect of co-culturing the candidate antibacterial peptide fermentation broth with a pathogenic bacteria culture solution on the cell concentration in the culture solution, the screening of active antibacterial peptides can be realized, which has the advantages of simple operation, high screening efficiency, and low cost.

[0006] Specifically, the method for screening antibacterial peptides provided by the present invention specifically includes: S1. Taking the candidate antibacterial peptide expression vector and performing protein expression in a cell-free protein expression system to obtain a candidate antibacterial peptide fermentation broth; S2. Taking the candidate antibacterial peptide fermentation broth and mixing it with a pathogenic bacteria culture solution for co-culturing, and detecting the cell concentration of the pathogenic bacteria in the culture solution to screen for active antibacterial peptides; wherein, the candidate antibacterial peptide expression vector sequentially includes, along the 5' to 3' direction: a promoter, a ribosome binding site, an AMPs coding gene, and a terminator, and the nucleotide sequence of the ribosome binding site is as shown in SEQ ID NO:2.

[0007] In the present invention, in step S1, in the candidate antibacterial peptide expression vector, the promoter is a sequence that indicates the recognition, binding, and start of transcription by RNA polymerase, which is a conventional technical means in existing genetic engineering technologies. Those skilled in the art can make adaptive selections according to actual needs, and the present invention does not make special limitations; the terminator is a sequence that indicates RNA polymerase to stop transcription and release RNA, which is a conventional technical means in existing genetic engineering technologies. Those skilled in the art can make adaptive selections according to actual needs, and the present invention does not make special limitations.

[0008] In some specific embodiments, in step S1, in the candidate antibacterial peptide expression vector, the nucleotide sequence of the promoter is preferably as shown in SEQ ID NO:1, and the nucleotide sequence of the terminator is preferably as shown in SEQ ID NO:3. At this time, the candidate antibacterial peptide expression vector can achieve better protein expression effects in a cell-free protein expression system.

[0009] In the present invention, in step S1, the design of the ribosome binding site in the candidate antibacterial peptide expression vector is the key to realizing "antibacterial peptide expression". The present invention does not make special limitations on other parts of the candidate antibacterial peptide expression vector. The candidate antibacterial peptide expression vectors designed by those skilled in the art based on what is disclosed in the present invention for other parts of the candidate antibacterial peptide expression vector are also covered by the protection scope of the present invention.

[0010] In the present invention, in step S1, the cell-free protein expression system is a technique for in vitro protein synthesis using cell extracts. This cell-free protein expression system does not rely on living cells and directly uses DNA or mRNA as a template to synthesize the target protein, which is a commonly used technical means in the art. Those skilled in the art can make adaptive selections according to actual needs, and the present invention does not make special limitations.

[0011] In the present invention, in step S1, the cell-free protein expression system is preferably prepared using common protein expression cells such as Escherichia coli and Saccharomyces cerevisiae as cell raw materials. Its preparation specifically includes: taking cells and performing ultrasonic disruption treatment to obtain the cell-free protein expression system. At this time, this cell-free protein expression system has the ability to highly express antibacterial polypeptides.

[0012] In some specific embodiments, the conditions of the ultrasonic disruption treatment include that the ultrasonic power is preferably 50 - 70 W, such as 50 W, 53 W, 55 W, 58 W, 60 W, 65 W, 68 W, 70 W or any value between them; the ultrasonic frequency is preferably 15 - 25 kHz, such as 15 kHz, 17.5 kHz, 19 kHz, 20 kHz, 23 kHz, 25 kHz or any value between them; the ultrasonic time is preferably 10 - 20 s, such as 10 s, 12 s, 15 s, 18 s, 20 s or any value between them; the interval time is preferably 5 - 15 s, such as 5 s, 8 s, 10 s, 12 s, 15 s or any value between them; the total time of the ultrasonic disruption treatment is preferably 40 - 50 s, such as 40 s, 42 s, 44 s, 45 s, 48 s, 50 s or any value between them.

[0013] In some specific embodiments, in step S1, based on the total volume of the solution in protein expression, the addition amount of the candidate antibacterial peptide expression vector is preferably 10 - 50 ng / μL, such as 10 ng / μL, 12.5 ng / μL, 15 ng / μL, 20 ng / μL, 25 ng / μL, 30 ng / μL, 40 ng / μL, 50 ng / μL or any value between them. At this time, the candidate antibacterial peptide expression vector can achieve better protein expression effects in the cell-free protein expression system.

[0014] In some specific embodiments, in step S1, the temperature of the protein expression is preferably 30 - 35 °C, such as 30 °C, 31 °C, 32 °C, 34 °C, 35 °C or any value between them; the time is preferably 1 - 24 h, such as 1 h, 2 h, 4, 6, 10, 15, 16, 20, 22, 24 or any value between them. At this time, the candidate antibacterial peptide expression vector can achieve better protein expression effects in the cell-free protein expression system.

[0015] In the present invention, in step S2, the pathogenic bacteria culture solution includes the pathogenic bacteria to be screened as the target. Those skilled in the art can make adaptive selections according to actual needs, and the present invention does not particularly limit it. More specifically, the pathogenic bacteria can be, but are not limited to, one or more of Vibrio harveyi, Vibrio alginolyticus, Vibrio parahaemolyticus, and Aeromonas hydrophila.

[0016] In some specific embodiments, in step S2, based on the total volume of the solution system in the co-culture, the addition concentration of the candidate antibacterial peptide fermentation broth is preferably 10-30% (v / v), such as 10% (v / v), 10.5% (v / v), 15% (v / v), 18% (v / v), 20% (v / v), 23% (v / v), 25% (v / v), 30% (v / v), or any value between them; the initial cell density of the pathogenic bacteria is preferably 10 3 ~10 5 cfu / mL, such as 1×10 3 cfu / mL, 5×10 3 cfu / mL, 9×10 3 cfu / mL, 1×10 4 cfu / mL, 5×10 4 cfu / mL, 1×10 5 cfu / mL, or any value between them.

[0017] In the invention, in step S2, those skilled in the art can make adaptive selections for the co-culture conditions according to the pathogenic bacteria to be screened as the target, and the present invention does not particularly limit it.

[0018] In some specific embodiments, in step S2, when the pathogenic bacteria are one or more of Vibrio harveyi, Vibrio alginolyticus, Vibrio parahaemolyticus, and Aeromonas hydrophila, the co-culture temperature is preferably 20-29°C, such as 20°C, 23°C, 25°C, 28°C, 29°C, or any value between them; the time is preferably 1-48 h, such as 1 h, 12 h, 24 h, 36 h, 48 h, or any value between them.

[0019] In the present invention, screening for the active antibacterial peptide by detecting the cell concentration of the culture broth specifically includes: continuously detecting the OD of the culture broth in the co-culture 600Values are used to plot the growth curve; and a culture solution of the pathogenic bacterium without adding the fermentation broth of the candidate antibacterial peptide under the same culture conditions is used as a blank control to compare the growth of the pathogenic bacterium to judge the antibacterial performance of the candidate antibacterial peptide, so as to achieve screening. Among them, detecting the cell concentration of the culture medium and judging the antibacterial performance of the candidate antibacterial peptide are conventional technical means in the microbial screening method of existing antibacterial peptides, and those skilled in the art can make adaptive selections according to actual needs, and the present invention does not particularly limit them.

[0020] Second, based on the great potential shown by the above antibacterial peptide screening method in realizing high-throughput screening of antibacterial peptides, the present invention also provides an application of the above antibacterial peptide screening method in the preparation of drugs for inhibiting pathogenic bacteria.

[0021] Third, the present invention also provides an antibacterial peptide, which comprises one or more of the polypeptides shown in the amino acid sequences such as SEQ ID NO: 6, SEQ ID NO: 10 or SEQ ID NO: 12.

[0022] In the present invention, the antibacterial peptide has a good inhibitory effect on one or more pathogenic bacteria among Vibrio harveyi, Vibrio alginolyticus, Vibrio parahaemolyticus and Aeromonas hydrophila.

[0023] Beneficial effects:

[0024] The present invention provides a screening method for antibacterial peptides. The screening method uses a candidate antibacterial peptide expression vector constructed with a nucleotide fragment shown in SEQ ID NO: 2 as a ribosome binding site, which can achieve protein expression in a cell-free protein expression system without other treatments (such as transformation, transfection, etc.) or specific condition stimulation, so as to rapidly prepare antibacterial peptides. Then, by observing the growth of pathogenic bacteria after mixing the fermentation broth of the candidate antibacterial peptide with the culture solution of pathogenic bacteria, the screening of the antibacterial activity of the antibacterial peptide can be achieved, showing great potential in realizing rapid and high-throughput screening of antibacterial peptides. Description of the drawings

[0025] Figure 1 It is a schematic structural diagram of the candidate antibacterial peptide expression vector provided in Example 1 of the present invention;

[0026] Figure 2 It is one of the experimental result diagrams of the screening of the candidate antibacterial peptide provided in Example 1 of the present invention (experimental group 1);

[0027] Figure 3 It is the second experimental result diagram of the screening of the candidate antibacterial peptide provided in Example 1 of the present invention (experimental group 2);

[0028] Figure 4It is the third experimental result diagram (experimental group 3) for the screening of candidate antimicrobial peptides provided in Example 1 of the present invention;

[0029] Figure 5 It is the fourth experimental result diagram (experimental group 4) for the screening of candidate antimicrobial peptides provided in Example 1 of the present invention;

[0030] Figure 6 It is the fifth experimental result diagram (control group) for the screening of candidate antimicrobial peptides provided in Example 1 of the present invention;

[0031] Figure 7 It is the sixth experimental result diagram (blank control group) for the screening of candidate antimicrobial peptides provided in Example 1 of the present invention;

[0032] Figure 8 It is the first experimental result diagram (experimental group 1) for the screening of candidate antimicrobial peptides provided in Example 2 of the present invention;

[0033] Figure 9 It is the second experimental result diagram (experimental group 2) for the screening of candidate antimicrobial peptides provided in Example 2 of the present invention;

[0034] Figure 10 It is the third experimental result diagram (experimental group 3) for the screening of candidate antimicrobial peptides provided in Example 2 of the present invention;

[0035] Figure 11 It is the fourth experimental result diagram (experimental group 4) for the screening of candidate antimicrobial peptides provided in Example 2 of the present invention;

[0036] Figure 12 It is the fifth experimental result diagram (control group) for the screening of candidate antimicrobial peptides provided in Example 2 of the present invention;

[0037] Figure 13 It is the sixth experimental result diagram (blank control group) for the screening of candidate antimicrobial peptides provided in Example 2 of the present invention;

[0038] Figure 14 It is the first experimental result diagram (experimental group 1) for the screening of candidate antimicrobial peptides provided in Example 3 of the present invention;

[0039] Figure 15 It is the second experimental result diagram (experimental group 2) for the screening of candidate antimicrobial peptides provided in Example 3 of the present invention;

[0040] Figure 16 It is the third experimental result diagram (experimental group 3) for the screening of candidate antimicrobial peptides provided in Example 3 of the present invention;

[0041] Figure 17 It is the fourth experimental result diagram (experimental group 4) for the screening of candidate antimicrobial peptides provided in Example 3 of the present invention;

[0042] Figure 18 It is the fifth (control group) of the experimental result diagrams for the screening of candidate antibacterial peptides provided in Example 3 of the present invention;

[0043] Figure 19 It is the sixth (blank control group) of the experimental result diagrams for the screening of candidate antibacterial peptides provided in Example 3 of the present invention;

[0044] Figure 20 It is the first (experimental group 1) of the experimental result diagrams for the screening of candidate antibacterial peptides provided in Example 4 of the present invention;

[0045] Figure 21 It is the second (experimental group 2) of the experimental result diagrams for the screening of candidate antibacterial peptides provided in Example 4 of the present invention;

[0046] Figure 22 It is the third (experimental group 3) of the experimental result diagrams for the screening of candidate antibacterial peptides provided in Example 4 of the present invention;

[0047] Figure 23 It is the fourth (experimental group 4) of the experimental result diagrams for the screening of candidate antibacterial peptides provided in Example 4 of the present invention;

[0048] Figure 24 It is the fifth (control group) of the experimental result diagrams for the screening of candidate antibacterial peptides provided in Example 4 of the present invention;

[0049] Figure 25 It is the sixth (blank control group) of the experimental result diagrams for the screening of candidate antibacterial peptides provided in Example 4 of the present invention. Detailed implementation mode

[0050] The embodiments of the present invention are described in detail below. The examples are intended to explain the present invention and should not be construed as limiting the present invention. For those without specific technical or conditions noted in the examples, the techniques or conditions described in the literature in the field or according to the product specifications are followed. The reagents or instruments without the manufacturer noted are all conventional products that can be obtained through commercial purchase.

[0051] The sequences involved in the present invention are specifically shown in Table 1.

[0052] Table 1.

[0053]

[0054]

[0055] Example 1

[0056] This example is used to illustrate the construction of candidate antimicrobial peptide expression vectors and the screening of candidate antimicrobial peptides with antibacterial activity against Vibrio harveyi, specifically including:

[0057] 1. Construction of candidate antimicrobial peptide expression vectors: (1) Referring to Figure 1 A plasmid vector and a control plasmid vector were constructed. The plasmid vector starts with the T7 promoter and sequentially includes a T7 promoter, a ribosome binding site, a T7 terminator, an ampicillin resistance gene, and a replicon along the 5' to 3' direction. The difference between the plasmid vector and the control plasmid vectors 1 and 2 lies in the different ribosome binding sites they contain, and the other fragments are the same. The specific nucleotide sequences of each fragment are shown in Table 2.

[0058] Table 2.

[0059]

[0060]

[0061] (2) According to Table 3, the synthesized AMPs coding genes were ligated onto the corresponding plasmid vectors or control plasmid vectors constructed above to obtain candidate antimicrobial peptide expression vectors, and these candidate antimicrobial peptide expression vectors were stored in ddH2O.

[0062] Table 3.

[0063]

[0064]

[0065] 2. Screening of candidate antimicrobial peptides: (1) A single colony of Escherichia coli Rosetta(DE3) (Beijing Na Biotechnology Co., Ltd., product number BNCC357922, the same below) was inoculated into a 2×YT plate medium (Haibo, product number HBDC003, the same below), cultured overnight at 37°C and 200 rpm for activation, and then a single colony was picked into 50 mL of 2×YT liquid medium and cultured at 37°C and 200 rpm for 16 h to obtain a seed solution.

[0066] (2) 300 μL of the seed solution was mixed with 2700 μL of 2×YT liquid medium, and the OD 600 value of the culture solution was adjusted to 0.1, and it was cultured at 37°C and 200 rpm. Starting from the 1st hour of culture, the OD 600 value was measured every half hour. When the OD 600 value reached 0.6, IPTG (final concentration 1 mM) was added to the culture solution according to the final concentration of 1 mM, and the culture was continued until the OD 600The value is 2.0. Centrifuge at 5000 g and 4 °C for 10 min, collect the precipitate and store it at -80 °C.

[0067] (3) After taking the precipitate and thawing it on ice for 30 min, add s30 buffer according to a volume ratio of 1:1. After vortexing and mixing evenly, under the conditions of 65 W and 20 kHz, perform ultrasonic disruption treatment for 45 s in the manner of ultrasonic treatment for 15 s and pause for 10 s. Immediately add 4.5 μL of DTT with a concentration of 1000 mM and invert and mix evenly. Centrifuge at 30000 g and 4 °C for 10 min to obtain a cell-free protein expression system.

[0068] Among them, the s30 buffer includes 14 mM potassium glutamate, 60 mM glutamic acid enzyme, 2 mM DTT and 10 mM Tris, and the pH value of the s30 buffer is adjusted to 8.2 with glacial acetic acid.

[0069] (4) Take 3.3 μL of the cell-free protein expression system, 4.9 μL of the supplement solution and 300 ng of the candidate antimicrobial peptide expression vector and mix them evenly, and make up to 10 μL with ddH2O. React at 30 °C and 180 rpm for 12 h to obtain the candidate antimicrobial peptide fermentation broth.

[0070] (5) Inoculate Vibrio harveyi (MCCC 1A20671) on a 2216E plate medium (Haibo, product number HB0132-1, the same below), culture it overnight at 30 °C and 200 rpm for activation. Pick a single colony and inoculate it into 50 mL of 2216E liquid medium, and culture it at 30 °C and 220 rpm until the OD 600 value is 1.0, and adjust the cell density of the culture solution to 10 4 cfu / mL with a new 2216E liquid medium to obtain a pathogenic bacteria culture solution.

[0071] (6) According to Table 4, respectively take each candidate antimicrobial peptide fermentation broth and add it to the pathogenic bacteria culture solution, and the addition amount of the candidate antimicrobial peptide fermentation broth is 10% (v / v). Culture at 30 °C and 220 rpm for 24 h, and perform OD 600 testing every 10 min during the culture process. The results are as Figures 2 - 7 shown.

[0072] Table 4.

[0073]

[0074] From Figures 2 - 7 the test results, it can be seen that the antimicrobial peptides AMPs-K5, AMPs-K58 and AMPs-K61 all have excellent inhibitory effects on Vibrio harveyi.

[0075] Example 2

[0076] This example is used to illustrate the screening of candidate antibacterial peptides with antibacterial activity against Vibrio alginolyticus, specifically including:

[0077] (1) Pick a single colony of Escherichia coli Rosetta(DE3)) and inoculate it into a 2×YT plate medium, and culture it overnight at 37°C and 200 rpm for activation. Pick a single colony into 50 mL of 2×YT liquid medium and culture it at 37°C and 200 rpm for 16 h to obtain a seed solution.

[0078] (2) Take 300 μL of the seed solution and mix it with 2700 μL of 2×YT liquid medium, and adjust the OD 600 value to 0.1, and culture it at 37°C and 200 rpm. Starting from the 1st h of culture, measure the OD 600 value every half hour. When the OD 600 value is 0.8, add IPTG (final concentration 1 mM) to the culture solution according to the final concentration of 1 mM, and continue to culture until the OD 600 value of the culture solution is 2.0, centrifuge at 5000 g and 4°C for 10 min, collect the precipitate and store it at -80°C.

[0079] (3) Take 300 μL of the seed solution and mix it with 2700 μL of 2×YT liquid medium, and adjust the OD 600 value to 0.1, and culture it at 37°C and 200 rpm. Starting from the 1st h of culture, measure the OD 600 value every half hour. When the OD 600 value is 0.8, add IPTG (final concentration 1 mM) to the culture solution according to the final concentration of 1 mM, and continue to culture until the OD 600 value of the culture solution is 2.0, centrifuge at 5000 g and 4°C for 10 min, collect the precipitate and store it at -80°C.

[0080] (4) Take 3.3 μL of the cell-free protein expression system, 4.9 μL of the supplement solution and 300 ng of the candidate antibacterial peptide expression vector, mix them evenly, and make up to 10 μL with ddH2O, and react at 30°C and 180 rpm for 12 h to obtain the candidate antibacterial peptide fermentation broth. Among them, the candidate antibacterial peptide expression vector is the candidate antibacterial peptide expression vector provided in Example 1 (specifically shown in Table 3).

[0081] (5) Inoculate Vibrio alginolyticus (MCCC 1K04633) on a 2216E plate medium and culture it overnight at 30 °C and 200 rpm for activation. Pick a single colony and inoculate it into 50 mL of 2216E liquid medium, and culture it at 30 °C and 220 rpm until the OD 600 value of the culture solution is 1.0, and use a new 2216E liquid medium to adjust the cell density of the culture solution to 10 4 cfu / mL to obtain a pathogenic bacteria culture solution.

[0082] (6) According to Table 5, respectively take each candidate antibacterial peptide fermentation broth and add it to the pathogenic bacteria culture solution, and the addition amount of the candidate antibacterial peptide fermentation broth is 10% (v / v). Culture it at 30 °C and 220 rpm for 24 h, and measure OD every 10 min during the culture process 600 and the results are as Figures 8 - 13 shown.

[0083] Table 5.

[0084]

[0085] From Figures 8 - 13 the test results, it can be seen that the antibacterial peptides AMPs-K5, AMPs-K58, and AMPs-K61 all have excellent inhibitory effects on Vibrio alginolyticus.

[0086] Example 3

[0087] This example is used to illustrate the screening of candidate antibacterial peptides with antibacterial activity against Vibrio parahaemolyticus, which specifically includes:

[0088] (1) Pick a single colony of Escherichia coli Rosetta (DE3) and inoculate it into a 2×YT plate medium, and culture it overnight at 37 °C and 200 rpm for activation. Pick a single colony into 50 mL of 2×YT liquid medium, and culture it at 37 °C and 200 rpm for 16 h to obtain a seed solution.

[0089] (2) Mix 300 μL of the seed solution with 2700 μL of 2×YT liquid medium, and adjust the OD 600 value of the culture solution to 0.1, and culture it at 37 °C and 200 rpm. Starting from the 1st h of culture, measure the OD 600 value every half hour. When the OD 600 value is 0.8, add IPTG (final concentration 1 mM) to the culture solution according to the final concentration of 1 mM, and continue to culture until the OD 600 value of the culture solution is 2.0, and centrifuge it at 5000 g and 4 °C for 10 min, collect the precipitate and store it at -80 °C.

[0090] (3) After the precipitate was thawed on ice for 30 min, S30 buffer was added in a volume ratio of 1:1. After vortex mixing, ultrasonic disruption was carried out at 65 W and 20 kHz for 45 s in the mode of ultrasonic treatment for 15 s and pause for 10 s. Immediately, 4.5 μL of DTT with a concentration of 1000 mM was added and mixed by inversion. Then, centrifugation was carried out at 30000 g and 4 °C for 10 min to obtain a cell-free protein expression system. Among them, the candidate antimicrobial peptide expression vector was the candidate antimicrobial peptide expression vector provided in Example 1 (specifically shown in Table 3).

[0091] (4) 3.3 μL of the cell-free protein expression system, 4.9 μL of the supplement solution and 300 ng of the candidate antimicrobial peptide expression vector were mixed evenly, and ddH2O was used to make up to 10 μL. The reaction was carried out at 30 °C and 180 rpm for 12 h to obtain the candidate antimicrobial peptide fermentation broth.

[0092] (5) Vibrio parahaemolyticus (MCCC 1H00056) was inoculated on a 2216E plate medium and cultured overnight at 30 °C and 200 rpm for activation. A single colony was picked and inoculated into 50 mL of 2216E liquid medium and cultured at 30 °C and 220 rpm until the OD 600 value was 1.0, and the cell density of the culture broth was adjusted to 10 4 cfu / mL with a new 2216E liquid medium to obtain a pathogenic bacteria culture solution.

[0093] (6) According to Table 6, each candidate antimicrobial peptide fermentation broth was added to the pathogenic bacteria culture solution, and the addition amount of the candidate antimicrobial peptide fermentation broth was 10% (v / v). The culture was carried out at 30 °C and 220 rpm for 24 h, and OD 600 was measured every 10 min during the culture process. The results are as Figures 14 - 19 shown.

[0094] Table 6.

[0095]

[0096] From Figures 14 - 19 the test results, it can be seen that the antimicrobial peptides AMPs-K5, AMPs-K58 and AMPs-K61 have excellent inhibitory effects on Vibrio parahaemolyticus.

[0097] Example 4

[0098] This example is used to illustrate the screening of candidate antimicrobial peptides with antibacterial activity against Aeromonas hydrophila, specifically including:

[0099] (1) Pick a single colony of Escherichia coli Rosetta(DE3) and inoculate it into a 2×YT plate medium. Culture it overnight at 37°C and 200 rpm for activation. Then pick a single colony into 50 mL of 2×YT liquid medium and culture it at 37°C and 200 rpm for 16 h to obtain a seed solution.

[0100] (2) Take 300 μL of the seed solution and mix it with 2700 μL of 2×YT liquid medium, and adjust the OD 600 value of the culture solution to 0.1. Culture it at 37°C and 200 rpm. Starting from the 1st h of culture, measure the OD 600 value every half hour. When the OD 600 value reaches 0.8, add IPTG (final concentration 1 mM) to the culture solution according to a final concentration of 1 mM, and continue to culture until the OD 600 value of the culture solution reaches 2.0. Centrifuge at 5000 g and 4°C for 10 min, collect the precipitate and store it at -80°C.

[0101] (3) Take 300 μL of the seed solution and mix it with 2700 μL of 2×YT liquid medium, and adjust the OD 600 value of the culture solution to 0.1. Culture it at 37°C and 200 rpm. Starting from the 1st h of culture, measure the OD 600 value every half hour. When the OD 600 value reaches 0.8, add IPTG (final concentration 1 mM) to the culture solution according to a final concentration of 1 mM, and continue to culture until the OD 600 value of the culture solution reaches 2.0. Centrifuge at 5000 g and 4°C for 10 min, collect the precipitate and store it at -80°C.

[0102] (4) Take 3.3 μL of the cell-free protein expression system, 4.9 μL of the supplement solution and 300 ng of the candidate antimicrobial peptide expression vector, mix them evenly, and make up to 10 μL with ddH2O. React at 30°C and 180 rpm for 12 h to obtain the candidate antimicrobial peptide fermentation broth. Among them, the candidate antimicrobial peptide expression vector is the candidate antimicrobial peptide expression vector provided in Example 1 (specifically shown in Table 3).

[0103] (5) Inoculate Aeromonas hydrophila (MCCC 1A00032) on a 2216E plate medium and culture it overnight at 30°C and 200 rpm for activation. Pick a single colony and inoculate it into 50 mL of 2216E liquid medium. Culture it at 30°C and 220 rpm until the OD 600 value of the culture solution reaches 1.0, and adjust the cell density of the culture solution to 10 4cfu / mL to obtain a pathogenic bacteria culture solution.

[0104] (6) Respectively take each candidate antimicrobial peptide fermentation broth according to Table 6 and add it to the pathogenic bacteria culture solution, and the addition amount of the candidate antimicrobial peptide fermentation broth is 10% (v / v). Cultivate at 30 °C and 220 rpm for 24 h, and perform OD 600 testing every 10 min during the cultivation process. The results are as Figures 20 - 25 shown.

[0105] Table 6.

[0106]

[0107] From Figures 20 - 25 the test results, it can be seen that the antimicrobial peptides AMPs-K5, AMPs-K58, and AMPs-K61 all have excellent inhibitory effects on Aeromonas hydrophila.

[0108] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention without departing from the principle and purpose of the present invention.

Claims

1. A method for screening antimicrobial peptides, characterized in that: The screening method comprises: S1, taking the antimicrobial peptide expression vector to be selected and expressing the protein in a cell-free protein expression system to obtain a fermentation broth of the antimicrobial peptide to be selected; S2, taking the fermentation broth of the antimicrobial peptide to be selected and mixing it with a pathogenic bacteria culture solution for co-culture, and detecting the concentration of pathogenic bacteria in the culture solution to screen for active antimicrobial peptides; The antimicrobial peptide expression vector to be selected includes, in order from 5' to 3', a promoter, a ribosome binding site, an AMPs encoding gene and a terminator, and the nucleotide sequence of the ribosome binding site is shown in SEQ ID NO:

2.

2. The method for screening antimicrobial peptides according to claim 1, characterized in that: In step S1, in the selected antimicrobial peptide expression vector, the nucleotide sequence of the promoter is shown as SEQ ID NO:1, and the nucleotide sequence of the terminator is shown as SEQ ID NO:

3.

3. The method for screening antimicrobial peptides according to claim 1, characterized in that: In step S1, the preparation of the cell-free protein expression system includes: taking cells and subjecting them to ultrasonic disruption to obtain the cell-free protein expression system; Optionally, the cell is Escherichia coli or Saccharomyces cerevisiae.

4. The method for screening antimicrobial peptides according to claim 1, characterized in that: In step S1, based on the total volume of the solution in the protein expression process, the amount of the selected antimicrobial peptide expression vector added is 10-50 ng / μL; Optionally, the protein is expressed at a temperature of 30 to 35° C. for 1 to 24 hours.

5. The method for screening antimicrobial peptides according to claim 1, characterized in that: In step S2, the pathogenic bacteria culture solution includes pathogenic bacteria, and the pathogenic bacteria include one or more of Harveyella, Vibrio alginolyticus, Vibrio parahaemolyticus and Aeromonas hydrophila.

6. The method for screening antimicrobial peptides according to claim 1, characterized in that: In step S2, based on the total volume of the solution system in the co-culture, the concentration of the selected antimicrobial peptide fermentation broth is 10-30% (v / v), and the initial cell density of the pathogenic bacteria is 10 3 ~10 5 cfu / mL.

7. The method for screening antimicrobial peptides according to claim 1, characterized in that: In step S2, the co-cultivation temperature is 20-29° C. and the time is 1-48 hours.

8. Use of the method for screening antimicrobial peptides according to claims 1 to 7 in the preparation of drugs for inhibiting pathogenic bacteria.

9. An antimicrobial peptide, characterized in that The antimicrobial peptide comprises one or more of the polypeptides with amino acid sequences as shown in SEQ ID NO:6, SEQ ID NO:10 or SEQ ID NO:

12.

10. The antimicrobial peptide according to claim 9, characterized in that The antimicrobial peptide has an inhibitory effect on one or more pathogenic bacteria selected from the group consisting of Harveyella, Vibrio alginolyticus, Vibrio parahaemolyticus and Aeromonas hydrophila.

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