CN116063387A

CN116063387A - Proline protection type enzymolysis-resistant antibacterial peptide and preparation method and application thereof

Info

Publication number: CN116063387A
Application number: CN202210818325.5A
Authority: CN
Inventors: 王家俊; 徐浩然; 吴万鹏; 单安山
Original assignee: Northeast Agricultural University
Current assignee: Northeast Agricultural University
Priority date: 2022-07-12
Filing date: 2022-07-12
Publication date: 2023-05-05
Anticipated expiration: 2042-07-12
Also published as: CN116063387B

Abstract

The invention discloses a proline protection type anti-enzymolysis antibacterial peptide, a preparation method and application thereof, belonging to the technical field of biology, wherein the sequence of the proline protection type anti-enzymolysis antibacterial peptide is shown as SEQ ID No.1, and a simplification principle is combined with a polypeptide design strategy for reasonably arranging amino acids to avoid protease cleavage sites, so that the anti-enzymolysis peptide with high antibacterial activity, low toxicity and high stability is designed. Through tests, the enzymolysis-resistant antibacterial peptide WPRP5 has obvious inhibition effect on most gram-negative bacteria and gram-positive bacteria tested, and the average antibacterial activity reaches 2.83 mu M. Meanwhile, the hemolytic activity and cytotoxicity are low, and the cell selectivity index is as high as 70.9; in addition, the anti-enzymolysis peptide WPRP5 still maintains higher antibacterial activity in the artificial simulated intestinal juice, the artificial simulated gastric juice and the 10mg/mL chymotrypsin solution; therefore, the anti-enzymolysis antibacterial peptide WPRP5 provided by the invention is an anti-enzymolysis antibacterial peptide with higher clinical application value.

Description

Proline protection type enzymolysis-resistant antibacterial peptide and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a proline protection type enzymolysis-resistant antibacterial peptide, and a preparation method and application thereof.

Background

The problem of resistance to antibiotics abuse has become one of the greatest crisis worldwide. Different from human administration, the feeding antibiotics have the defects of low supervision and general abuse phenomenon, serious exceeding of the residual antibiotics in animals, lowered animal productivity and the like, and restrict the development of animal husbandry. Therefore, the development of novel and green feed antibiotic substitution technology has no doubt great practical significance. The antibacterial peptide is a kind of small molecular polypeptide for resisting the invasion of external microorganisms and eliminating in-vivo mutant cells of animal and plant organisms, and has the advantages of wide antibacterial spectrum, multiple varieties, difficult generation of resistance mutation and the like. It is therefore considered the most powerful alternative to antibiotics. However, natural antimicrobial peptides still have disadvantages such as insufficient potency, high cytotoxicity to mammals, poor stability in salt ion and protease environments, and the like. And as a feed additive, the protease has poor stability and high production cost, which are certainly the biggest barriers preventing the protease from being further popularized and applied. Therefore, the novel synthetic peptide is developed through drug design, and is the best way for solving the application bottleneck of the antibacterial peptide.

In order to solve these problems, covalent bond modification strategies such as cyclization, peptoid and D-type amino acid intercalation are used for improving protease tolerance of artificially synthesized peptides, however, the strategies also greatly increase the synthesis difficulty while improving the protease resistance of the polypeptides, and the peptides cannot be produced in low-cost industrialization through a prokaryotic or eukaryotic expression system. Different from a chemical synthesis strategy for avoiding protease cleavage sites by covalent bond modification, the purpose of improving the resistance of polypeptide protease can be achieved by reasonably arranging natural amino acids to avoid the protease cleavage sites. The cleavage sites of proteases are not identical, but these specific cleavage sites are affected by the amino acid arrangement. For example, trypsin cleaves Arg and Lys preferentially at the P1 position and has a higher specificity for Arg. Cleavage of Arg and Lys by trypsin is hindered when Pro is in the P1 'position, but cleavage of Lys in the P1 position by trypsin is not hindered by Pro in the P1' position when Lys is in the P1 position and Trp is in the P2 position. Similarly, pro at P1', arg at P1 and Met at P2 will not inhibit trypsin cleavage. In addition to the above, the cleavage activity of trypsin on Lys at the P1 position can be blocked in the following cases: asp is at both the P2 and P1' positions; cys at position P2 and Asp at position P1'; his is at the P1' position; cys is at position P2 and Tyr is at position P1'. For Arg at position P1, the amino acid positions that can reduce the cleavage efficiency of trypsin are arranged as follows: arg is at position P2 and His is at position P1'; cys at position P2 and Lys at position P1'; arg is at both the P2 and P1' positions. Chymotrypsin has a high degree of specificity for aromatic amino acids. But Met or Pro in the P1' position prevents chymotrypsin hydrolysis of Trp in the P1 position. In addition, pro at the P1' position also blocks cleavage of Tyr and Phe at the P1 position by chymotrypsin. These rules can be applied reasonably in the modification process of the natural peptide, and can well enhance the protease resistance of the natural peptide. However, according to previous studies, the effectiveness of this strategy is highly dependent on the precise positioning of the amino acids in the sequence, and increasing the resistance of the polypeptide protease while probably destroying the balance of structural parameters of the propeptide, reducing the activity of the propeptide and increasing its cytotoxicity, severely limiting its applicability.

Disclosure of Invention

Based on the problems of the background technology, the invention aims to provide the proline protection type anti-enzymolysis antibacterial peptide WPRP5, which combines a simplification principle with a polypeptide design strategy for reasonably arranging amino acids to avoid protease cleavage sites, so as to design the anti-enzymolysis peptide with high antibacterial activity, low toxicity and high stability, wherein the amino acid sequence of the anti-enzymolysis peptide is shown as SEQ ID No.1, and the molecular formula of the anti-enzymolysis peptide is shown as formula (I):

the invention also aims to provide a preparation method of the proline protection type enzymolysis-resistant antibacterial peptide WPRP5, which comprises the following steps:

(1) The method is characterized by selecting pepsin, trypsin and chymotrypsin which are common in the gastrointestinal tract of pigs as resistance targets, respectively selecting Trp and Arg as hydrophobic and charged cores according to a simplification principle and a protease cleavage site theory, and protecting the Trp and Arg by utilizing Pro to respectively avoid the cleavage effect of the protease chymotrypsin and pepsin on tryptophan and the cleavage effect of the trypsin on arginine, wherein a molecular skeleton model is (WPRP) _n When n=5, the sequence is shown as SEQ ID No. 1;

(2) Obtaining peptide resin by a solid-phase chemical synthesis method through a polypeptide synthesizer, and cutting the obtained peptide resin by TFA to obtain polypeptide;

(3) After reversed-phase high performance liquid chromatography purification and mass spectrum identification, the preparation of the polypeptide is completed, and then the detection of in-vitro antibacterial activity, hemolytic activity, cytotoxicity, enzymolysis resistance and action mechanism is carried out, so that the polypeptide is named as enzymolysis resistance antibacterial peptide WPRP5.

The invention also aims to provide an application of the proline-protected enzymolysis-resistant antibacterial peptide WPRP5 in preparing a medicament for treating gram-negative bacteria or/and gram-positive bacteria infectious diseases.

The invention has the beneficial effects and advantages that: the enzymolysis-resistant antibacterial peptide WPRP5 has obvious inhibition effect on gram-negative bacteria and gram-positive bacteria such as escherichia coli, salmonella typhimurium, salmonella pullorum, staphylococcus aureus, bacillus subtilis, pseudomonas aeruginosa and the like, and the average antibacterial activity reaches 2.83 mu M. Meanwhile, the hemolytic activity and cytotoxicity are low, and the cell selectivity index is as high as 70.9; in addition, WPRP5 still maintains high antimicrobial activity in artificial simulated intestinal fluid, artificial simulated gastric fluid and 10mg/mL chymotrypsin solution. Therefore, the anti-enzymolysis antibacterial peptide WPRP5 is an anti-enzymolysis antibacterial peptide with higher clinical application value.

Drawings

FIG. 1 is a mass spectrum of an anti-enzymolysis antimicrobial peptide WPRP 5;

FIG. 2 is a chromatogram of the anti-enzymolysis antimicrobial peptide WPRP 5;

FIG. 3 shows the haemolytic activity of the anti-enzymolysis antimicrobial peptide WPRP 5;

FIG. 4 is a cytotoxicity assay of the anti-enzymolysis antimicrobial peptide WPRP 5;

FIG. 5 is a Tricine-SDS-PAGE of the protease treated anti-enzymolysis antimicrobial peptide WPRP 5;

fig. 6 is a TEM image of e.coli 25922 treated with the anti-enzymolysis antimicrobial peptide WPRP5.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but embodiments of the present invention are not limited thereto.

Example 1

Design of anti-enzymolysis antibacterial peptide WPRP 5: trp and Arg are selected as hydrophobic and charged cores respectively according to a simplification principle and a protease cleavage site theory, pro is utilized to protect Trp and Arg, cleavage of tryptophan by chymotrypsin and pepsin and cleavage of arginine by trypsin are avoided respectively, and a molecular skeleton model of the anti-enzymolysis peptide is (WPRP) _n (n=1, 2, 3 … …). When the number of the repeated units n is 5, the polypeptide is named as anti-enzymolysis antibacterial peptide WPRP5, and the amino acid sequence is shown in table 1:

TABLE 1 amino acid sequence and major structural parameters of the anti-Enzymolytic antibacterial peptide WPRP5

Molecular structural formula of enzymolysis-resistant antibacterial peptide WPRP5

Example 2

The method for synthesizing the anti-enzymolysis antibacterial peptide WPRP5 by the solid phase chemical synthesis method comprises the following specific steps:

1. the preparation of the polypeptides is carried out one by one from the C end to the N end, and is completed by a polypeptide synthesizer. Fmoc-X (X is the first amino acid at the C-terminal of each antibacterial peptide) is firstly accessed into Wang resin, and then Fmoc groups are removed to obtain X-Wang resin; fmoc-Y-Trt-OH (9-fluorenylmethoxycarbonyl-trimethyl-Y, Y being the second amino acid at the C-terminus of each antimicrobial peptide); the synthesis is sequentially carried out from the C end to the N end according to the procedure until the synthesis is completed, and the side chain protected resin without Fmoc groups is obtained.

2. Adding a cutting reagent into the obtained peptide resin, reacting for 2 hours at 20 ℃ in a dark place, and filtering; washing precipitated TFA (trifluoroacetic acid), mixing the washing solution with the filtrate, concentrating by a rotary evaporator, adding pre-cooled anhydrous diethyl ether with volume about 10 times, precipitating at-20 ℃ for 3 hours, separating out white powder, centrifuging at 2500g for 10min, collecting precipitate, washing the precipitate with anhydrous diethyl ether, and vacuum drying to obtain polypeptide, wherein the cutting reagent is prepared by mixing TFA, water and TIS (triisopropylchlorosilane) according to a mass ratio of 95:2.5:2.5.

3. Performing column balancing with 0.2mol/L sodium sulfate (pH 7.5 is adjusted) for 30min, dissolving polypeptide with 90% acetonitrile water solution, filtering, performing C18 reverse phase normal pressure column, performing gradient elution (eluent is methanol and sodium sulfate water solution mixed according to volume ratio of 30:70-70:30), flowing at 1mL/min, detecting wave at 220nm, collecting main peak, and lyophilizing; further purification using a reverse phase C18 column, eluent a was 0.1% tfa/water; the eluent B is 0.1 percent TFA/acetonitrile solution, the elution concentration is 25 percent B-40 percent B, the elution time is 12min, the flow rate is 1mL/min, and then the main peak is collected and freeze-dried.

4. Identification of antibacterial peptides: the obtained antibacterial peptide is analyzed by electrospray mass spectrometry, and the molecular weight shown in the mass spectrum is basically consistent with the theoretical molecular weight shown in table 1 (see figure 1), and the purity of the antibacterial peptide is more than 95% (see figure 2).

Example 3: and detecting the antibacterial activity, the hemolytic activity, the cytotoxicity, the enzymolysis resistance and the action mechanism of the prepared enzymolysis resistant antibacterial peptide WPRP5 in vitro:

1. determination of antibacterial Activity: the peptides are formulated as a stock solution for use. Minimum inhibitory concentration of anti-enzymolysis peptide by micro broth dilution methodDegree. Single colonies were inoculated into MHB, cultured at 220rpm at 37℃to logarithmic growth phase, and their concentration was adjusted to OD with MHB _600nm =0.1, finally diluted 1000-fold further with MHB to 0.5-1×10 ⁵ CFU/mL; to line A in a 96-well plate, 95. Mu.L of 0.2% BSA diluent was added, and the remaining wells were added with 50. Mu.L of 0.2% BSA diluent. Add 5. Mu.L of 1.28mM peptide to row A well, mix well, then aspirate 50. Mu.L to row B and push this, dilute the fold to row G, mix well and aspirate 50. Mu.L to discard. Three replicates were set for each assay peptide; 50 mu L of bacterial liquid is added into the row A-G holes of the 96-well plate, 50 mu L of bacterial liquid is also added into the row 1-6 holes of the H-well plate to serve as positive control, 50 mu L of fresh MHB culture medium is added into the row 7-12 holes to serve as negative control, and the mixture is evenly mixed and then is cultured for 24 hours at 37 ℃. The absorbance was measured at 492nm using an enzyme-labeled instrument to determine the minimum inhibitory concentration. The detection results are shown in Table 2.

TABLE 2 antibacterial Activity of anti-Enzymolytic antibacterial peptide WPRP5

As can be seen from Table 2, the anti-enzymolysis antimicrobial peptide WPRP5 has high-efficiency antibacterial activity on most gram-negative bacteria and gram-positive bacteria tested,

2. determination of haemolytic Activity: collecting 1mL of fresh blood of a human, dissolving the fresh blood into 2mL of PBS solution after anticoagulation of heparin, and collecting red blood cells after centrifugation of 1000g for 5min; after 3 washes with PBS, the suspension was resuspended in 10mL PBS; uniformly mixing 50 mu L of erythrocyte suspension and 50 mu L of anti-enzymolysis antibacterial peptide WPRP5 solution with different concentrations, incubating for 1h at constant temperature in a 37 ℃ incubator, taking out, and centrifuging at 4 ℃ for 5min at 1000 g; taking supernatant, and measuring absorbance value at 570nm by using an enzyme-labeling instrument; each group was averaged and analyzed by comparison. Wherein 50. Mu.L of erythrocytes were added with 50. Mu.L of PBS as a negative control; 50. Mu.L of erythrocytes plus 50. Mu.L of 0.1% Triton x-100 served as positive control. The minimum hemolysis concentration is the concentration of the antimicrobial peptide at which the antimicrobial peptide causes a 10% hemolysis rate. The detection results are shown in FIG. 3 and Table 3.

TABLE 3 calculation of the Selectivity index of the peptide WPRP5

* The selectivity index was calculated with 256. Mu.M at a minimum hemolysis concentration > 128. Mu.M

As can be seen from FIG. 3 and Table 3, the anti-enzymolysis antimicrobial peptide WPRP5 has no obvious hemolytic activity in the detection range.

For analysis of the potential for clinical application of the anti-enzymatic antimicrobial peptide WPRP5, it can be assessed synthetically by the selectivity index (ratio of haemolytic to bacteriostatic concentration). From table 3, it can be seen that the anti-enzymolysis antibacterial peptide WPRP5 has a higher selectivity index, which indicates that the designed anti-enzymolysis antibacterial peptide WPRP5 has the potential of clinically replacing antibiotics.

3. Cytotoxicity assay: resuscitating cells frozen in liquid nitrogen, inoculating into culture medium containing 10% foetal calf serum and 1% double antibody, and inoculating into culture medium containing 5% CO at 37deg.C ₂ Subculturing under the condition. The cultured cells were digested with 0.25% pancreatin and adjusted to 2-4X 10 with medium ⁵ cells/mL. 50. Mu.L of the cell suspension was mixed with 50. Mu.L of the polypeptide at various concentrations in 96-well plates at 37℃in 5% CO ₂ Incubation was carried out for 24h, followed by addition of 25. Mu.L MTT (5 mg/mL) per well and incubation was continued for 4h. After the incubation, the supernatant was discarded, the bottom crystals were dissolved in 100. Mu.L of DMSO, and the absorbance value per well was measured at 570nm using an ELISA reader. Medium wells served as blank. The detection results are shown in FIG. 4. As can be seen from fig. 4, the anti-enzymolysis antimicrobial peptide WPRP5 has a certain toxicity to RAW 264.7, and has a low toxicity to the other three test cells, and the cell survival rates of PK-15, HEK 293T and IPEC-J2 are all over 50% after the treatment with the high concentration of the anti-enzymolysis antimicrobial peptide WPRP5.

4. Protease resistance: taking a proper volume of anti-enzymolysis antibacterial peptide WPRP5 mother solution (2.56 mM), vacuum drying, adding an equal volume of simulated human gastrointestinal fluid or 10mg/mL chymotrypsin solution, incubating for 6 hours at 37 ℃, performing ultrasonic vibration, taking out a proper volume of sample, and directly detecting the change of MIC value of the strain to be detected. Heating the rest sample in water bath at 100deg.C for 30min, centrifuging at 13000 Xg for 30min, collecting supernatant, and detecting retention of anti-enzymolysis antibacterial peptide WPRP5 treated by simulated human gastrointestinal fluid and 10mg/mL chymotrypsin solution by SDS-PAGE protein gel. The test results are shown in Table 4 and FIG. 5

TABLE 4 minimum inhibitory concentration of protease-treated peptide WPRP5 against E.coli 25922

^a Manual simulation intestinal juice formula: taking 6.8g of monopotassium phosphate, adding 500mL of water to dissolve the monopotassium phosphate, and adjusting the pH value to 6.8 by using 0.1mol/L sodium hydroxide solution; another 10g of pancreatin is taken, dissolved by adding a proper amount of water, the two solutions are mixed, and the mixture is diluted to 1000mL by adding water.

^b 10mg/mL

^c Manual simulated gastric juice formula: 16.4mL of diluted hydrochloric acid was taken, about 800mL of water and 10g of pepsin were added, and after shaking, the mixture was diluted to 1000mL with water.

As can be seen from Table 4, the antibacterial activity of artificial simulated intestinal juice and 10mg/mL chymotrypsin solution against the enzymolysis antimicrobial peptide WPRP5 is not obviously affected; as can be seen from fig. 5, the protease-treated anti-enzymolysis antimicrobial peptide WPRP5 has a similar protein band to the control group, which demonstrates that the anti-enzymolysis antimicrobial peptide WPRP5 can resist the cleavage of artificial simulated intestinal juice and 10mg/mL chymotrypsin solution. The results show that the anti-enzymolysis antibacterial peptide WPRP5 with brand new design has excellent capability of resisting the hydrolysis of high-concentration protease.

4. Determination of antibacterial action mechanism: and observing the structural change of the thalli after the enzymolysis-resistant antibacterial peptide WPRP5 is treated by a transmission electron microscope. Coli e.coli 25922 single colonies were picked and grown overnight in MHB medium and transferred to new MHB for growth to mid-log phase. The bacterial solution is then centrifuged and resuspended in phosphate buffer to a final concentration of OD _600nm =0.2. Adding anti-enzymolysis antibacterial peptide WPRP5 parent peptide to make its final concentration beMIC values. After incubation at room temperature for 2 hours, the supernatant was removed by centrifugation, and the bacterial pellet was fixed with 1% osmium acid fixative for 2 hours. And centrifuging the immobilized thalli, collecting the precipitate, washing the thalli three times by PBS, removing the immobilized liquid, dehydrating the thalli by using 50%, 70%, 90% and 100% ethanol solutions according to a gradient for 8min each time, dehydrating the thalli by using 100% ethanol for 10min each time, replacing the thalli by using a mixed solution of ethanol and acetone (1:1, v/v) for 10min, and replacing the thalli by using pure acetone for 10min. After the replacement, the bacterial precipitate was collected by centrifugation, and then embedded with an embedding solution of acetone and resin (1:1, v/v) for 30min, and then replaced with 100% pure resin overnight. After the sample is formed into blocks, the blocks are sliced by an ultrathin microtome, and the blocks are observed by the microtome after being subjected to double-dyeing by uranium acetate-lead citrate, and the result is shown in figure 6. As shown in fig. 6, compared with the control group, the cell membrane of the escherichia coli treated by the anti-enzymolysis antibacterial peptide WPRP5 is folded, a part of the region forms holes, the cytoplasm flows out, and the cytoplasm wrapped by the cell membrane is scattered around, which indicates that the anti-enzymolysis antibacterial peptide WPRP5 damages the bacterial cell membrane through the action mode of a carpet model and induces bacterial death.

5. The results show that the design strategy of the anti-enzymolysis antibacterial peptide combining the simplification principle and the design strategy of the polypeptide of the protease cleavage site is reasonably arranged and avoided, the designed anti-enzymolysis antibacterial peptide WPRP5 has excellent antibacterial activity and high cell selectivity, and also has extremely strong resistance to artificial simulated intestinal juice and high-concentration chymotrypsin, so that the anti-enzymolysis antibacterial peptide WPRP5 has higher clinical application potential; meanwhile, the membrane-breaking antibacterial mechanism of the anti-enzymolysis antibacterial peptide WPRP5 is similar to a carpet model, so that the possibility of drug resistance of the anti-enzymolysis antibacterial peptide WPRP5 can be effectively reduced, and the anti-enzymolysis antibacterial peptide WPRP5 has higher potential for replacing antibiotics.

Claims

1. The proline protection type enzymolysis resistant antibacterial peptide WPRP5 is characterized in that the amino acid sequence is shown in SEQ NO.1, and the molecular formula is shown in formula (I):

2. the preparation method of the proline-protected enzymolysis-resistant antibacterial peptide WPRP5, which is characterized by comprising the following steps:

(3) After reversed-phase high performance liquid chromatography purification and mass spectrum identification, the preparation of the polypeptide is completed, then the detection of in-vitro antibacterial activity, hemolytic activity, cytotoxicity, enzymolysis resistance and action mechanism is carried out, and finally the polypeptide is named as enzymolysis resistance antibacterial peptide WPRP5.

3. The use of a proline-protected anti-enzymolysis antimicrobial peptide WPRP5 in accordance with claim 1 in the manufacture of a medicament for the treatment of gram negative bacterial or/and gram positive bacterial infectious diseases.