WO2020087822A1 - Trypsin-resistant antimicrobial agent - Google Patents

Abstract

Provided is a trypsin-resistant antimicrobial agent, which is a mutant of MccC7, said mutant being R2A, R2H, R2L, R2T, R2Q, and R2K, wherein R2A, R2H, R2T and R2Q can resist to trypsin.

Description

Antibacterial peptide resistant to trypsin

This application claims the priority of the Chinese invention patent application with the application number 201811274568.7 and the name "a trypsin-resistant antibacterial peptide" filed on October 30, 2018.

Technical field

The invention belongs to the field of biological products, and specifically relates to a mutant new antibacterial peptide which adopts the Quickchange method to saturate and mutate the R residue 2 of MccC7, that is, an antibacterial peptide resistant to trypsin.

technical background

Antibacterial peptides have broad-spectrum antibacterial activity, and have a strong killing effect on bacteria, especially its killing effect on certain drug-resistant pathogenic bacteria, which has attracted people's attention; in addition, people have also found that The peptide has a killing effect on some viruses, fungi, protozoa and cancer cells, and can even improve immunity and accelerate the wound healing process; the wide biological activity of antibacterial peptides shows its good application prospects in medicine.

Bacteriocin is a class of protein antibiotics with a small molecular weight (<10kDa). The coding gene is usually located on the plasmid and is accompanied by the expression of immune genes to prevent the bacteriocin from poisoning the host bacteria. Some bacteriocins need to be processed and modified to mature, so plasmids expressing bacteriocins also carry processing genes. After translation, the modified bacteriocin may form a special structure to inhibit important life processes in the cell, such as replication, transcription, and translation.

MccC7 is encoded by the MccA gene, which is 21 bp long and is located on the pRYC7 plasmid of E. coli. At the same time, the processing and transportation modified immune gene (MccBCDEF) is also located on this plasmid and arranged in clusters to initiate transcription and translation when nutrition is scarce. The MccA gene contains 7 amino acids (MRTGNAN) after translation. The C-terminus of the MccB gene is covalently bonded to a molecule of AMP through the N-acyl phosphate bond. The MccDE gene adds a propylamine group to the phosphate group to form a mature MccC7.

Mature MccC7 is secreted out of the cell and is taken into the cell by the YejABEF transport complex of sensitive strains through the "Trojan horse" strategy. MccC7 removes the six amino acids at the N-terminus under the action of protease to release the toxic site (modified Aspartyl-adenylate), and then competitively bind to aspartyl tRNA synthetase, thereby effectively inhibiting the translation process, resulting in cell death.

The second residue R of wild Microcin C7 (MccC7) can be cleaved by trypsin, causing the inactivation of MccC7, which is very unfavorable for the application of antimicrobial peptides.

Therefore, an antimicrobial peptide resistant to trypsin is needed to solve the above-mentioned defects and lay a foundation for the application of new antimicrobial peptides.

Summary of the invention

The object of the present invention is to provide an antimicrobial peptide that is resistant to trypsin to enhance the application of antimicrobial peptides in immunoassays and complement binding experiments.

In order to solve the above technical problems, the present invention provides an antimicrobial peptide resistant to trypsin, which is secreted and expressed by Escherichia coli RZC29, which was deposited in China on September 4, 2018 The General Microbiology Center of the Microbial Culture Collection Management Committee (abbreviated as CGMCC, No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing), its deposit number is CGMCC NO.16421.

To solve the above technical problems, the present invention provides an antimicrobial peptide resistant to trypsin, which is secreted and expressed by Escherichia coli RZC23, which was deposited in China on September 4, 2018 The General Microbiology Center of the Microbial Culture Collection Management Committee (abbreviated as CGMCC, No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing), and its deposit number is CGMCC NO.16422.

In order to solve the above technical problems, the present invention provides an antimicrobial peptide resistant to trypsin, which is secreted and expressed by Escherichia coli RZC16, and the strain was deposited in China on September 4, 2018 The General Microbiology Center of the Microbial Culture Collection Management Committee (abbreviated as CGMCC, No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing), and its deposit number is CGMCC NO.16423.

In order to solve the above technical problems, the present invention provides an antimicrobial peptide resistant to trypsin, which is secreted and expressed by Escherichia coli RZC14, which was deposited in China on September 4, 2018 The General Microbiology Center of the Microbial Culture Collection Management Committee (abbreviated as CGMCC, No. 3, No.1 Hospital, Beichen West Road, Chaoyang District, Beijing), its deposit number is CGMCC NO.16424.

Preferably, the antibacterial peptides include mutants, and the mutants are R2A, R2H, R2L, R2T, R2Q, and R2K; wherein, the R2A, R2H, R2T, and R2Q have trypsin resistance; The PDZ88 plasmid is the original expression vector, and the Quickchange method was used to saturate and mutate residue 2 of MccC7.

Preferably, the extraction of the mutant includes the following steps:

S1, cultivate experimental strains;

S2, fixed-point mutation;

S3, screening mutant clones;

S4, isolation and purification of mutants;

S5, detect mutants have antibacterial activity;

S6, to detect the inhibitory effect of mutants on E. coli in vitro;

S7, to determine the stability in simulated gastrointestinal fluid.

Preferably, the cultivation of the experimental strain in step S1 is performed under the conditions of 37 ° C. and 220 rpm; the culture medium of the experimental strain includes 5 g yeast powder, 10 g peptone, 10 g sodium chloride, and 1 L deionized water.

Preferably, the culture medium of the experimental strain includes 30g yeast powder, 2g KH2PO4, 0.1g MgSO4 and 1L deionized water.

Preferably, the minimum inhibitory concentration range of the mutant in step S6 against E. coli is 0.03 μg / ml-0.5 μg / ml, and the minimum bactericidal concentration range is 0.25 μg / ml-2 μg / ml.

Preferably, the step S7 specifically includes:

a. Preparation of artificial gastric juice: take dilute hydrochloric acid 16.4ml, add about 800ml of water and shake with pepsin 10g, dilute to 1000ml with water;

b. Preparation of artificial intestinal juice: take 6.8g of potassium dihydrogen phosphate, add 500ml of water to dissolve, adjust the pH value to 6.8 with 0.1mol / L sodium hydroxide solution; take another 10g of pancreatin, add appropriate amount of water to dissolve, mix the two liquids, Dilute with water to 1000ml;

c. Preparation of the control solution: accurately weigh 50.00 mg of the sample, put it in a 25 ml volumetric flask, dissolve and dilute to the mark with water, shake well, and record it as CK88, CK14, CK16, CK23 and CK29, respectively, and set at 4 ℃ for use;

d. Simulate gastrointestinal digestion process.

The technical solution of the present invention has the following beneficial effects:

The present invention provides an antimicrobial peptide that is resistant to trypsin, uses the PDZ88 plasmid as the original expression vector, and uses the Quickchange method to saturate mutations in the R residue 2 of the encoding gene of MccC7. After cloning, transfer the verified vector into MC4100 host culture for 24 hours, use 96-well plate method to select active mutants, and then use the inhibition zone method to re-examine, and then isolate and purify the mutants with antibacterial effect. Identification. Mutants R2A, R2H, R2L, R2T, R2Q and R2K have antibacterial activity. Among them, R2A, R2H, R2T and R2Q can tolerate trypsin, laying a foundation for the application of new antibacterial peptides.

BRIEF DESCRIPTION

FIG. 1 is a comparison diagram of the bacteriostatic effect of the antimicrobial peptide of the present invention after mutation of the antimicrobial peptide at position 2;

2 is a graph showing the isolation and purification of R2A mutants in the antimicrobial peptide trypsin-resistant antimicrobial peptide of the present invention;

3 is a graph showing the results of isolation and purification of R2H mutants in the antimicrobial peptide trypsin-resistant antimicrobial peptide of the present invention;

4 is a diagram showing the results of isolation and purification of R2T mutants in the antimicrobial peptide trypsin-resistant antimicrobial peptide of the present invention;

Fig. 5 is a graph showing the results of isolation and purification of R2Q mutants in the antimicrobial peptide trypsin-resistant antimicrobial peptide of the present invention.

detailed description

In order to enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below in conjunction with specific embodiments.

In order to screen for new antimicrobial peptides resistant to trypsin, that is, secreted and expressed by Escherichia coli from the antimicrobial peptides, in this example, the Quickchange method was used to saturate and mutate the R residue 2 of MccC7. , R2T, R2Q and R2K have antibacterial activity, of which R2A, R2H, R2T and R2Q can tolerate trypsin, which lays the foundation for the application of new antibacterial peptides. The mutant uses PDZ88 plasmid as the original expression vector and uses the Quickchange method Saturation mutation was performed on residue 2 of MccC7.

The materials and methods for extracting mutants in this example are as follows

S1, culture experimental strain; DH5α (F endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG U80dlacZDM15 D (lacZYA-argF) U169, hsdR17

k) Purchased from TIANGEN BIOTECH. MC4100 (F-araD139 (argF-lac) U169 rspL150 relA1 flbB5301 fruA25 deoC1 ptsF25) is kept by our laboratory.

Culture conditions: 37 ° C, 220 rpm, final concentration of ampicillin 100 μg / ml. LB medium: 5g yeast powder, 10g peptone, 10g sodium chloride, dissolved in 1L deionized water; rich medium: 30g yeast powder, 2g KH2PO4, 0.1g MgSO4, dissolved in 1L deionized water.

S2, fixed-point mutation;

Quickchange mutation primers are shown in Table 2-1 and amplified with vector PDZ88 as template. The PCR system is as follows:

PCR program: (1) 98 ℃ 3min, (2) 98 ℃ 15s, (3) 55 ℃ 25s, (4) 72 ℃ n s, (5) 72 ℃ 5min. Repeat from (2) to (4) 15cycles, (The extension time n is calculated based on the length of the PCR product compared to the extension speed of 15-30s / kbp). The amplified product is recovered using WizardSVSV Gel and PCR Clean-UP system Kit (Promega). The recovered product is digested with Dpnl to remove residual template DNA. The digestion reaction system is as follows:

The above enzyme digestion system was recovered after reaction at 37 ° C for 1.5 to 3 hours, and directly transformed into DH5α competent cells, and cultured overnight at 37 ° C. Pick single clones and inoculate them in LB overnight at 37 ° C for plasmid identification.

Table 2-1: Quickchange mutation primers

Note: The red letters in the column of ^a amino acid sequence represent the residues after mutation, ^{b The lower} case letters in the primer sequence represent the codons of the mutated amino acid residues, ^c merger codons.

S3, screening for mutant clones; preparation of E. coli MC4100 chemically competent cells: 1. Pick a single colony (2-3 mm in diameter) from a plate incubated at 37 ° C for 16-20h and transfer to a medium containing 100ml of LB or SOB medium 1L flask. Incubate at 37 ° C with vigorous shaking for 3h. As a general rule of thumb, 1OD600 contains approximately E. coli DH5α109CFU / mL. 2. Transfer the bacteria to a sterile, single-use, 50ml polypropylene tube pre-chilled with ice, place on ice for 10min, and allow the culture to cool to 0 ° C. 3. Centrifuge the Sorvall GS3 special head (or its equivalent rotor) at 4100r / min for 10min at 4 ℃ to recover the cells. 4. Pour out the culture medium and invert the tube for 1 min to allow the last trace of culture medium to run out. 5. Resuspend each cell pellet with 30ml of pre-chilled 0.1mol / LCaCl2-MgCl2 solution (80mmol / L MgCl2, 20mmol / L CaCl2) per 50ml of initial culture solution. 6. Centrifuge the Sorvall GS3 rotor (or its equivalent rotor) at 4100r / min for 10min at 4 ℃ to recover the cells. 7. Pour out the culture fluid, and invert the tube for 1 min to allow the last trace of culture fluid to run out. 8. Resuspend each cell pellet with 2ml of 0.1mol / L CaCl2 (or TFB) pre-chilled with ice for every 50ml of initial culture. 9. At this time, you can use freshly prepared competent cells directly for transformation experiments, or you can freeze the cells at -70 ℃. The mutant plasmids sequenced correctly were transformed into MC4100 competent cells, cultured overnight, and selected for monoclonal identification.

A 96-well plate was used to detect the activity of antimicrobial peptides: 1. The engineered strain and MC4100 were streaked onto an ampicillin-resistant plate and incubated at 37 ° C for 20 h. 2. Pick the larger single clone into 4mL rich medium and shake it for 20-24h. 3. Centrifuge the bacterial solution and draw the supernatant into the EP tube. 4. Place the bacterial supernatant, 96-well plate and 2mL EP tube in a 65 ° C oven for 30 minutes. 5. Dilute the bacterial supernatant with an equal volume of LB medium and pipette 200 μL into a 96-well plate. 6. Connect 4-5μL of indicator bacteria to each well. MC4100 diluted supernatant is recorded as CK1, MC4100 (pDZ88) diluted supernatant is recorded as CK2, mutant strain diluted supernatant is recorded as pZCx (pZCx corresponds to the plasmid name in Table 2-1), MC4100 diluted supernatant without inoculation was recorded as CK3. 7. 37 ° C, shaking culture at 90 rpm for 3-4h. 8. Measure the absorbance at 600nm using a fully automatic quantitative plotting microplate reader (Synergy H1 Multi-Mode Reader, BioTek). 9. Data processing: The OD600 value of CK1 and CK3 is recorded as a, and the OD600 of CK2, pZCx and CK3 is recorded as b and bx. B≤bx≤a is determined to have antibacterial activity.

The Oxford Cup method was used to detect the antibacterial activity: the fermentation broth was centrifuged at 4 ° C and 10,000 rpm for 10 minutes, and the Oxford Cup diffusion method was used to determine the antibacterial activity (Chen Jiemei et al., 2014). The outer diameter of the Oxford cup was 8 mm and the inner diameter was 6 mm. Each Oxford cup was added with 100 μL Supernatant. When the LB medium is autoclaved and cooled to about 45 ° C, inoculate E. coli ATCC25922 fermentation broth to a final concentration of 104-105CFU / mL, mix well, and pour into a Petri dish with an Oxford cup. After the medium is solidified, take out the Oxford cup, add 200 μL of fermentation supernatant, pure product or 25 μg / ml chloramphenicol solution, and add an equal volume of LB medium to the negative control. Place the petri dish in the refrigerator at 4 ° C for 2-3h, and then incubate at 37 ° C for 12-18h.

S4, isolation and purification of mutants;

a. Transform the engineered bacteria to streak on the ampicillin LB plate and incubate overnight. The next day, pick a single clone and inoculate it in a 10ml test tube (4ml of rich medium filling solution, add ampicillin), incubate at 37 ℃, 220rpm for 12-16h, and inoculate 1% in a 1L Erlenmeyer flask (rich medium filling liquid volume 250ml, Ampicillin was added) and cultured at 37 ° C and 220 rpm for 24 hours.

b. The fermentation broth is centrifuged to remove bacteria to reach the standard of being clear and free of impurities. In order to prevent the centrifugal effect from reaching the standard, 0.45 microfiltration membrane is used to remove impurities after centrifuging two to three times.

c. Ammonium sulfate precipitation treatment. (1) The sample is subjected to ammonium sulfate precipitation treatment. After adding ammonium sulfate, it will be observed that the clear fermentation broth becomes turbid. Continue to add ammonium sulfate, stirring while adding. The specific amount added is converted by the solution of saturated ammonium sulfate in water. After stirring for 1h, the sample was centrifuged and HPLC was tested to see if there was any target substance in the supernatant. The target substance could not be detected as the standard, and then centrifuged to remove the supernatant to leave a precipitate. (2) The precipitated material collected is reconstituted. Reconstitute the collected precipitate with pure water. The amount of water used to achieve complete dissolution is the standard (except for some lipid substances that are insoluble in water in the precipitate). The reconstituted solution is filtered or centrifuged to remove insoluble materials and save for use ( (Do not need to be frozen at -20 degrees on the day).

d. Hydrophobic chromatography. BufferA: 1M / L sodium chloride, filtered for standby; BufferB: pure water, conductivity controlled under 2us / cm; sample: sodium chloride adjusted conductivity to 55ms / cm. Rinse the column with pure water until the baseline is equilibrated; A rinse the column 2CV; load the sample (determine the loading capacity according to the loading volume); A balance the column 3CV; B 0-100% linear gradient elution, observe the conductivity change and spectrum, collect the target substance Peak (B phase increases to about 10% and the target substance peak begins to appear); B3CV flushes the column until the baseline and conductivity drop to pure water conductivity, ready for the next sample.

e. Desalting G10 gel. The eluent is ethanol, eluting isocraticly.

S6, to detect the inhibitory effect of mutants on E. coli in vitro;

Follow the micro broth dilution method, the specific steps are as follows:

a. Preparation of liquid medicine: wild-type, mutant and colistin sulfate are made into 128μg / mL stock solution with sterilized ultrapure water, filtered and sterilized, ready to use.

b. Preparation of bacterial solution: the experimental strains were inoculated into agar plates, and after overnight cultivation at 37 ° C, 1 to 3 single colonies were picked and inoculated into MH broth culture solution, incubated at 37 ° C for 16 to 18 hours, and the growth turbidity reached 1 to 5 × 109CFU / mL. Dilute the original bacterial liquid 5-10 times with liquid medium to make the turbidity reach 5-7.5 × 108CFU / mL, and then dilute 1: 1000 as the test bacterial liquid.

c. Inoculation and culture: use a micropipette to take 50μl of the experimental drug stock solution into the first row of a 96-well plate, and then successively dilute it with MH broth medium to 64μg / ml, 32μg / ml, 16μg / ml, 8μg / ml, 4μg / ml, 2μg / ml, 1μg / ml, 0.5μg / ml, 0.25μg / ml, 0.125μg / ml┄┄, 50μl per well, add drug-free medium in wells 11 and 12 as controls . Use a micropipette to add the experimental bacterial solution to each row of the microplate in sequence, 50μl per well, so that the final concentration of the drug in each well is 32μg / ml, 16μg / ml, 8μg / ml, 4μg / ml, 2μg / ml, 1μg / ml, 0.5μg / ml, 0.25μg / ml, 0.125μg / ml, 0.0625μg / ml┄┄, add 50μl blank MH broth medium to the 12th well. Four replicates of each experimental strain were separately mixed, and the microplate was incubated at 37 ° C for 18-24 hours to observe the results.

d. MIC judgment: Observed under the light lined with a black bottom plate, the culture solution in the pores is dispersed and turbid when the bacteria grow or there is a round or wire mesh precipitate at the bottom of the U shape. The minimum drug concentration of the bacteria-free growth hole is The MIC of the drug.

S7, to determine the stability in simulated gastrointestinal fluid. ;

The test process is as follows:

Liquid medicine preparation:

a. Artificial gastric juice: take dilute hydrochloric acid 16.4ml, add about 800ml of water and shake with pepsin 10g, dilute to 1000ml with water.

b. Artificial intestinal juice: take 6.8g of potassium dihydrogen phosphate, add 500ml of water to dissolve, adjust the pH value to 6.8 with 0.1mol / L sodium hydroxide solution; take another 10g of pancreatin, add appropriate amount of water to dissolve, mix the two liquids, add water Dilute to 1000ml.

c. Preparation of control solution: accurately weigh 50.00mg of sample (content is calculated as 100%), put it in a 25ml volumetric flask, add water to dissolve and dilute to the mark, shake well, and record as CK88, CK14, CK16, CK23 and CK29 (correspondingly) The plasmids are pDZ88, pZC14, pZC14, pZC23 and pZC29), and set at 4 ℃ for use.

d. Simulate gastrointestinal digestion process:

Sample 1 (S1-x): Weigh 50.00mg of the sample precisely, put it in a 25ml volumetric flask, add 25ml of artificial gastric juice that has been in a constant temperature water bath at 37 ° C for 5min, and set the volume to S1-88, S1-14, S1-16 , S1-23 and S1-29 (corresponding plasmids are pDZ88, pZC14, pZC16, pZC23 and pZC29), quickly placed in a 37 ℃ water bath, accurately record the time, sampling at 1h.

Sample 2 (S2-x): Weigh another 50.00mg sample accurately, put it in a 50ml volumetric flask, add 25ml of artificial gastric juice that has been in a constant temperature water bath at 37 ℃ for 5min, quickly vortex to dissolve and quickly reset to 37 ℃ water bath During the treatment, the treatment was stopped at 1h, then the volume was adjusted to the mark with artificial intestinal fluid, the pH value was adjusted to 7.5, and it was reset in a 37 ° C water bath, and samples were taken at 1h. Samples are noted as S2-88, S2-14, S2-16, S2-23 and S2-29 (corresponding plasmids are pDZ88, pZC14, pZC16, pZC23 and pZC29).

Sample treatment: The samples taken from samples 1 and 2 were all terminated in a 100 ° C water bath for 10 minutes in a water bath. Then cool to room temperature, use HPLC method to detect the content, and compare with the content of the control solution.

The above method of this embodiment resulted in four strains, namely A, B, C and D,

The result data of the above examples are as follows: combined with Figure 1, Figure 2, Figure 3, Figure 4 and Figure 5, the experimental results of mutants with antibacterial activity: mutant clones were tested for antibacterial activity by 96-well plate method, the b , Bx value is calibrated with a, when bx / a ≤ b / a and b / a ≤ bx / a ≤ 1, it is determined that there is antibacterial activity, the calculation process is as described above. The active mutants in the 96-well plate were retested by the Oxford Cup method, and pZC15 and pZC18 were selected as negative controls. Figure 1 shows the comparison of antibacterial effects after mutation of antimicrobial peptide at position 2 of trypsin-resistant antimicrobial peptide: pZCx corresponds to The names of the plasmids in Table 2-1 are 200 μl of fermentation supernatant added to each well; CK is a positive control, 200 μl of 25 μg / ml chloramphenicol is added to each well; LB is a negative control, 200 μl of LB medium is added to each well. It can be seen from the results that the Oxford Cup method is consistent with the 96-well plate results. Mutants R2A, R2H, R2L, R2T, R2Q and R2K have antibacterial activity. Figures 2, 3, 4 and 5 are respectively pancreatic resistant Isolation and purification of R2A mutants in trypsin-resistant antimicrobial peptides of protease antimicrobial peptides, R2H mutant isolation and purification of trypsin-resistant antimicrobial peptides, antibacterial peptides of trypsin-resistant antimicrobial peptides Isolation and purification results of R2T mutant in peptide trypsin resistance, R2Q isolation and purification results of antimicrobial peptide in trypsin resistant antimicrobial peptide, corresponding strains are in the General Microbiology Center of China Microbial Culture Collection Management Committee The deposit numbers are CGMCC NO.16424, CGMCC NO.16423, CGMCC NO.16422 and CGMCC NO.16421; in Figures 2 to 5, the abscissa indicates the time of purification, the unit is min; the ordinate indicates The response value of the corresponding time is that the abscissa of the liquid chromatogram is time, and the unit of min is the response value, and there is no unit.

In vitro inhibition results of mutants against Escherichia coli in this example: the mutants have good inhibitory effects on E. coli in vitro, and the minimum inhibitory concentration (MIC) of E. coli ranges from 0.03μg / ml-0.5μg / ml, the minimum bactericidal concentration (MBC) range is 0.25μg / ml-2μg / ml. There is no obvious difference in the killing effect of E. coli resistant and non-resistant strains. It can efficiently inhibit colistin-resistant E. coli, see Table 3-2 for details.

Table 3-2 MccJ25's anti-killing test results on animal-derived E. coli

Note: E. coli ATCC25922 is a quality control strain, and the other strains are clinically isolated from animals. The upper right corner marked with * is a drug-resistant strain, and the resistance point of colistin sulfate resistance is> 2.

The stability results of the mutant in the simulated gastrointestinal fluid in this example: after the simulated gastric juice was treated for 1 hour, the content was reduced to about 90%, indicating that the wild type and the mutant were relatively stable in the gastric juice. Continuous treatment of simulated gastrointestinal fluid, that is, after treatment of simulated gastric juice for 1h, then add simulated intestinal fluid to continue treatment for 1h: the content of wild type drops to 0, indicating that wild type is very sensitive to the treatment of simulated gastrointestinal fluid; the content of mutant type is still more than 55%, It indicates that each mutant is tolerant to the treatment of simulated gastrointestinal fluid, as shown in Table 3-4.

Table 3-4 HPLC test results after simulated gastrointestinal fluid treatment

Note: The processing conditions are in accordance with the steps in the materials and methods.

In the present invention, the PDZ88 plasmid is used as the original expression vector, and the Quickchange method is used to saturate mutation of R residue 2 of the coding gene of MccC7. It is expected that a tryptic tolerant clone can be selected, and the correct vector is transferred to the MC4100 host culture 24 Hours, the active mutants were selected by 96-well plate method, and then rechecked by the bacteriostatic circle method, and then the mutants with bacteriostatic effect were separated, purified and identified. Mutants R2A, R2H, R2L, R2T, R2Q and R2K have antibacterial activity. Among them, R2A, R2H, R2T and R2Q can tolerate trypsin, laying a foundation for the application of new antibacterial peptides.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than limit them. Although the present invention has been described in detail with reference to the above embodiments, a person of ordinary skill in the art can still implement the specific embodiments of the present invention. Modifications or equivalent substitutions are made in a manner that does not deviate from the spirit and scope of the present invention, and all modifications or equivalent substitutions are within the protection scope of the claims to be approved.

Claims (10)

1. An antibacterial peptide resistant to trypsin, characterized in that the antibacterial peptide is secreted and expressed by Escherichia coli (Escherichia coli) RZC29. The Escherichia coli is an ordinary microorganism in the China Microbial Species Deposit Management Committee The deposit number of the center is CGMCC NO.16421.
2. An antibacterial peptide resistant to trypsin, characterized in that the antibacterial peptide is secreted and expressed by Escherichia coli (Escherichia coli) RZC23. The Escherichia coli is an ordinary microorganism in the China Microbial Species Deposit Management Committee The deposit number of the center is CGMCC NO.16422.
3. An antimicrobial peptide resistant to trypsin, characterized in that the antimicrobial peptide is secreted and expressed by Escherichia coli RZC16. The Escherichia coli is a general microorganism in the China Microbial Species Deposit Management Committee The deposit number of the center is CGMCC NO.16423.
4. An antimicrobial peptide resistant to trypsin, characterized in that the antimicrobial peptide is secreted and expressed by Escherichia coli RZC14. The Escherichia coli is a general microorganism in the China Microbial Species Deposit Management Committee The deposit number of the center is CGMCC NO.16424.
5. The antimicrobial peptide resistant to trypsin according to any one of claims 1 to 4, characterized in that the antimicrobial peptide includes mutants, and the mutants are R2A, R2H, R2L, R2T, R2Q and R2K; wherein, the R2A, R2H, R2T, and R2Q have trypsin resistance; the mutant uses the PDZ88 plasmid as the original expression vector, and uses the Quickchange method to saturate mutation of residue 2 of MccC7.
6. The antimicrobial peptide resistant to trypsin according to any one of claims 1 to 4, wherein the extraction of the mutant includes the following steps:

   S1, cultivate experimental strains;

   S2, fixed-point mutation;

   S3, screening mutant clones;

   S4, isolation and purification of mutants;

   S5, detect mutants have antibacterial activity;

   S6, to detect the inhibitory effect of mutants on E. coli in vitro;

   S7, to determine the stability in simulated gastrointestinal fluid.
7. The antimicrobial peptide resistant to trypsin according to claim 6, wherein the cultivation of the experimental strain in step S1 is carried out under the conditions of 37 ° C and 220 rpm; the culture medium of the experimental strain includes 5 g of yeast powder, 10g peptone, 10g sodium chloride and 1L deionized water.
8. The antimicrobial peptide resistant to trypsin according to claim 6, wherein the culture medium of the experimental strain comprises 30g yeast powder, 2g KH2PO4, 0.1g MgSO4 and 1L deionized water.
9. The antimicrobial peptide resistant to trypsin according to claim 6, wherein the minimum inhibitory concentration of the mutant in step S6 against E. coli is 0.03μg / ml-0.5μg / ml, and the minimum bactericidal concentration The range is 0.25μg / ml-2μg / ml.
10. The antimicrobial peptide resistant to trypsin according to claim 6, wherein the step S7 specifically comprises:

    a. Preparation of artificial gastric juice: take dilute hydrochloric acid 16.4ml, add about 800ml of water and shake with pepsin 10g, dilute to 1000ml with water;

    b. Preparation of artificial intestinal juice: take 6.8g of potassium dihydrogen phosphate, add 500ml of water to dissolve, adjust the pH value to 6.8 with 0.1mol / L sodium hydroxide solution; take another 10g of pancreatin, add appropriate amount of water to dissolve, mix the two liquids, Dilute with water to 1000ml;

    c. Preparation of the control solution: accurately weigh 50.00 mg of the sample, put it in a 25 ml volumetric flask, dissolve and dilute to the mark with water, shake well, and record it as CK88, CK14, CK16, CK23 and CK29, respectively, and set at 4 ℃ for use

    d. Simulate gastrointestinal digestion process.

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