CN113527437A

CN113527437A - Peptide KC-19 with antibacterial activity

Info

Publication number: CN113527437A
Application number: CN202110981649.6A
Authority: CN
Inventors: 吴刘记; 陈雪艳; 贾兴锰
Original assignee: Henan Agricultural University
Current assignee: Henan Agricultural University
Priority date: 2021-08-25
Filing date: 2021-08-25
Publication date: 2021-10-22

Abstract

The invention discloses a peptide KC-19 with antibacterial activity, and the amino acid sequence of the peptide KC-19 is KCHLHPLCKGEALELFSVG. The broad-spectrum antifungal activity experiment of the peptide KC-19 showed that the peptide KC-19 mainly achieved the inhibitory effect on fungi through the aggregation of hyphae. The highest inhibitory rates of KC‑19 against Helicobacter umbilis, Curvus crescentus and Fusarium graminearum were 47.0%, 47.8% and 25.1%, respectively.

Description

Peptide KC-19 with antibacterial activity

Technical Field

The invention relates to a peptide KC-19 with antibacterial activity.

Background

Fungal plant pathogens are an important group of microorganisms that cause significant economic losses to agricultural production around the world. For decades, chemical pesticides have been commonly used to control the growth and spread of fungal pathogens. However, the use of pesticides both pollutes the environment and harms human health, and the pesticide has a limited range of action. Therefore, a new green, environment-friendly and safe bactericide is urgently needed to be found to replace the chemical pesticide currently used.

Antimicrobial peptides are a class of polypeptides that are widely found throughout the biological world. The antimicrobial peptides can be used as part of the immune system of plants, insects or mammals to combat the invasion of pathogenic microorganisms. The antibacterial peptide has the characteristics of wide antibacterial spectrum, small molecular weight, good thermal stability and water solubility, difficult generation of drug resistance and the like. In the case of many pathogenic bacteria with obvious resistance to the existing antibiotics, the discovery and research of antibacterial peptides provide possibility for developing new antibacterial drugs.

Research over the past decades has focused primarily on traditional peptides derived from traditional ORFs. In recent years, a new class of polypeptides, non-traditional peptides, derived from previously unannotated non-coding regions has received much attention as important functional endogenous peptides in plants. Studies have shown that non-traditional peptides play important roles in various biological processes, such as translational regulation, plant development and stress response.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a peptide with antibacterial activity.

The technical scheme of the invention is as follows: the amino acid sequence of the peptide KC-19 with antibacterial activity is KCHLHPLCKGEALELFSVG.

The peptide KC-19 is derived from an intergenic region, has the molecular weight of 2194.11Da and the isoelectric point of 6.9.

The peptide KC-19 is applied to inhibiting the activity of plant pathogenic fungi.

The plant pathogenic fungi are Helminthosporium zeae, curvularia lunata or fusarium graminearum.

The invention has the beneficial effects that: the broad-spectrum antifungal activity experiment of the peptide KC-19 shows that the peptide KC-19 mainly achieves the effect of inhibiting fungi through hypha aggregation. The KC-19 has the highest inhibition rates of 47.0%, 47.8% and 25.1% on Helminthosporium zeae, curvularia lunata and Fusarium graminearum respectively.

Drawings

FIG. 1 is a schematic diagram of a method of hyphal growth rate measurement;

FIG. 2 is a mass spectrum of the synthesized peptide KC-19;

FIG. 3 is a high performance liquid chromatogram of the synthesized peptide KC-19;

FIG. 4 shows the phenotypic identification of peptide KC-19 on Helminthosporium zeae, one of which is selected as a representative in three replicates per treatment, with a scale of 1 cm;

FIG. 5 shows the growth inhibition rate of peptide KC-19 on Helminthosporium zeae. Growth inhibition rate calculation formula: i% ([ (C-d) - (T-d) ]/(C-d) × 100%;

FIG. 6 shows the effect of peptide KC-19 on the hyphal morphology of Helminthosporium zeae at 36hpi, with a scale of 20 μm;

FIG. 7 shows the phenotypic identification of Curvularia lunata by peptide KC-19, with one of the three replicates per treatment selected as a representative, at a scale of 1 cm;

FIG. 8 shows the growth inhibition rate of Curvularia lunata by peptide KC-19. Growth inhibition rate calculation formula: i% ([ (C-d) - (T-d) ]/(C-d) × 100%;

FIG. 9 shows the effect of peptide KC-19 on Curvularia lunata hypha morphology at 36hpi, with a scale of 20 μm;

FIG. 10 phenotypic identification of peptide KC-19 on Fusarium graminearum, one of which was selected as a representative at each three replicates per treatment, at 1cm scale;

FIG. 11 shows the growth inhibition rate of peptide KC-19 on Fusarium graminearum. Growth inhibition rate calculation formula: i% ([ (C-d) - (T-d) ]/(C-d) × 100%;

FIG. 12 shows the effect of peptide KC-19 on the morphology of Fusarium graminearum hyphae at 36hpi, with a scale of 20 μm.

Detailed Description

1 materials and methods

1.1 Experimental materials

1.1.1 pathogenic bacteria

Helminthosporium zeae, curvularia lunata and fusarium graminearum are all stored in the laboratory.

1.1.2 culture Medium

The culture media used in this study were all Potato Dextrose Agar (PDA) media, configured as follows:

(1) fresh potato (200 g) is peeled, cut into pieces and put into 1000ml of distilled water.

(2) Decocting in boiling water for 20min, and filtering with gauze.

(3) Adding 20g of sucrose and 20g of agar powder into the filtrate, and adding distilled water to reach the volume of 1000 ml.

(4) Sterilizing at 121 deg.C under high pressure for 21min, and pouring into flat plate.

1.2 Experimental methods

1.2.1 Synthesis of peptides

The synthesis is carried out by a Fmoc (9-fluorenylmethoxycarbonyl) polypeptide solid-phase synthesis method, and the sequence is from C terminal (carboxyl terminal) to N terminal (amino terminal). Firstly, an amino acid with Fmoc protection is connected on a resin, and the Fmoc protection group in the resin is removed by 20 percent piperidine/DMF solution to expose active NH₂And the second Fmoc-protected amino acid is connected by the HBTU method until the whole peptide chain is connected. The peptide chain was eluted from the resin with DMF solvent to give the crude product. The specific experimental method is as follows:

(1) resin synthesis

Fmoc-AA-King resin

Removal of 1 molecule H by carboxyl group on Fmoc-AA-OH and-OH on Queen resin₂And O is obtained through reaction. The reaction is as follows:

the specific synthesis steps are as follows:

adding weighed queen resin into a round-bottom flask, adding a proper amount of dry DMF for swelling, stirring by using a magneton, adding 1.5eq of Fmoc-AA-OH, adding 6eq of pyridine, slowly adding 3eq of 2, 6-dichlorobenzoyl chloride by using a dropper after the Fmoc-AA-OH is dissolved, tightly covering by using a rubber plug after the addition is finished, and carrying out shake reaction for more than 3 hours. After removing the solution by suction filtration through a frit funnel, the resin was washed with DMF (3 times), MeOH (1 time), DCM (3 times) and MeOH (2 times). The tubes were then transferred to a collection tube and dried overnight in a vacuum desiccator.

Synthesis of Fmoc-AA-2-Cl Trt resin

The Fmoc-AA-2-Cl Trt Resin is obtained by reacting carboxyl on Fmoc-AA-OH with-Cl on 2-Cl Trt Resin to remove 1 molecule HCl, and is mainly used for synthesizing three amino acids of Pro, Cys and His at the C end. The reaction is as follows:

the specific synthesis steps are as follows:

the weighed 2-Cl Trt resin and 2eq of Fmoc-AA-OH were added to a round bottom flask, the appropriate amount of dried DCM swollen resin and dissolved amino acids were added, and 6eq of DIPEA were added. The reaction mixture was capped tightly with a stopper and stirred at room temperature for 2-3 h. HPLC grade MeOH (1g resin plus 10ml) was added and stirred for 30 min. After removing the solution by suction filtration through a frit funnel, the resin was washed with DMF (3 times), MeOH (1 time), DCM (3 times) and MeOH (2 times). The tubes were then transferred to a collection tube and dried overnight in a vacuum desiccator.

c.rink Amide MBHA resin Synthesis

The modified MBHA resin is obtained by connecting and reacting a Linker Amide group with MBHA resin. The method comprises the following specific steps:

MBHA resin is added into a reactor, 2eq HB is added to activate Rink Amide, and 6 times amount of NMM is added. And reacting for 3-4 h. End-capping, transfer the resin to the sand core funnel and wash 3 times with DMF, 1 time with MeOH, 3 times with DCM, and 3 times with MeOH, respectively. The resin was transferred to vacuum and dried.

(2) Weighing the amount of resin and swelling

The resin amount required is calculated from the number of moles of the peptide to be synthesized and the substitution degree of the resin (the resin amount is the number of moles of the peptide to be synthesized/substitution degree of the resin), and then the resin is weighed out and placed in a reactor to which the corresponding number is attached. Placing the reactor with the resin on a frame, adding 3-5 times of DMF of resin bed volume, and soaking for 30 min.

(3) Deprotection and washing thereof

Adding 20% piperidine/DMF solution about 3 times of the volume of the resin bed layer, introducing nitrogen to react for 5min, pumping out, adding 20% piperidine/DMF solution about 3 times of the volume of the resin bed layer, and introducing nitrogen to react for 15 min.

(4) Ninhydrin detection after deprotection

A small amount of resin is taken out and put into a small detection test tube, ninhydrin is added, reaction is carried out for 3min at the temperature of 110-120 ℃, and then the color development is observed. Different amino acids will appear differently colored, most amino acid resins will appear dark blue or blue, while Pro, Asn, Asp, Gln, Glu, His, and Cys will appear mostly red or reddish brown. However, since the peptide sequence differs during synthesis, the color exhibited by an amino acid does not completely match the above description, and the amino acid may exhibit another color under the influence of the above amino acid. However, when the ninhydrin detection reaction carried out after deprotection clearly distinguishes the color exhibited by the resin from the color exhibited by the resin upon connection, it is indicated that the deprotection reaction has proceeded completely.

(5) Ligation reaction

Solvent:

DMF (N, N-dimethylformamide), DCM (dichloromethane)

The raw material ratio is as follows:

AA: HBTU: NMM 3: 2.85: 6 (molar ratio)

Adding amino acid into a reactor, adding a minimum amount of DMF capable of dissolving the amino acid, adding HBTU, adding NMM, and reacting for 60 min.

Note: firstly, nitrogen is used before adding DMF to dissolve so as to prevent amino acid from leaking.

② adding HBTU after the amino acid is completely dissolved.

③ the amount of DMF solvent added during the reaction is based on the total immersion of the resin.

And fourthly, properly stirring the airflow by the nitrogen.

(6) Post ligation NT detection

A small amount of resin is taken out by a dropper into a detection tube, and the resin is washed once by DMF and MeOH respectively, and the subsequent steps are the same as deprotection detection. After the completion of the ligation, the resin was generally colorless and transparent, and in a few cases, it was pale yellow. When the color is obviously different from the color displayed by the protection before the connection is complete, the connection is complete.

(7) End capping

Proportioning: 5% -10% acetic anhydride/DMF + NMM (acetic anhydride: NMM ═ 1: 1)

Adding a blocking solution with the volume 3 times that of the resin layer, introducing nitrogen, stirring for 30min, then pumping off the solution, washing the opening of the reactor with DCM, washing with DMF for 5 times, and cleaning the blocking solution.

(8) Washing after connection

The washing after ligation is the same as the washing after deprotection, but three times of washing after ligation.

Repeating the previous steps until one peptide chain is completed

(9) Resin transfer

The deprotected and washed resin was transferred to a sand core funnel with DMF solvent, washed once with methanol, three times with DCM solvent and twice with methanol. And (3) gathering the resin in the center of the sand core funnel as much as possible by using methanol for the last washing, draining, transferring the resin into a small collecting pipe, sticking a label, and placing the small collecting pipe in a vacuum drier for draining for preparing cutting.

(10) Cutting of

a. Preparation of cutting fluid

Solution A: TFA: thioanisole: phenol: EDT (electro-thermal transfer coating): water 87.5: 5: 2.5: 2.5: 2.5

And B, liquid B: TFA: and (3) TIS: water 95: 3: 2

b. Selection and dosage of cutting fluid

Generally, the peptide chain containing Cys, Met and Trp with unprotected side chain is selected from solution A, and the other peptides are all selected from solution B. 10-15ml of cutting fluid is generally added to 1g of resin.

c. Cutting time

The addition of the cutting solution to the resin was started and the filtrate was precipitated with ether. For short peptides (such as penta-and hexa-peptides) cutting for 2h, more than 10 cutting for 2.5h, and for more than 30 peptides, the time is prolonged appropriately.

Adding appropriate amount of cutting fluid, reacting on shaking table, vacuum filtering, collecting filtrate, and washing the filtrate with diethyl ether in a centrifuge tube. Centrifuging in a centrifuge (rotation speed of about 4000 rad/min), pouring off the supernatant, mashing the precipitate with a glass rod, washing with diethyl ether, and centrifuging the precipitate again. The washing was repeated three times as above.

And (3) putting the crude product into a vacuum drier for pumping, then purifying by using HPLC (high performance liquid chromatography) to obtain a pure product with the purity of more than 90%, and performing mass spectrum identification.

1.2.2 preparation of peptide solutions

The synthesized peptide was dissolved in sterile water, and then prepared into a mother solution of 1mM peptide concentration with sterile water, and the prepared peptide solution was dispensed into 2ml EP tubes and stored at-20 ℃. The mother liquor is diluted to 100 μm with PDA culture medium for later use.

1.2.3 preparation of the test strains

(1) The strains stored at 4 ℃ were patted on the medium using a punch.

(2) The bacterial cake was inoculated on new PDA medium.

(3) The cells were cultured at 28 ℃ for 4 days in the dark for activation.

(4) The activated strain is used for beating a bacterial cake on a culture medium by a puncher.

(5) The beaten cake was inoculated on another fresh PDA medium.

(6) Cultured at 28 ℃ for 4 days in the dark for subsequent experiments.

1.2.4 growth inhibition test

(1) PDA culture medium is sterilized at 121 deg.C for 21min, and cooled to 60 deg.C.

(2) Then mixing with peptide solution to obtain mixture with final peptide concentration of 100 μm, and using sterile water instead of peptide solution as control.

(3) The mixture was mixed well and poured into a sterile petri dish with a diameter of 30 mm.

(4) After the culture medium solidified, a 5mm cake was taken from the outer edge of the four-day-old fungus colony, and the center of the culture medium was inoculated with the hypha facing downward.

(5) The medium was cultured in the dark at 28 ℃.

(6) The colony diameter was recorded photographically and measured every 3h or 6h (FIG. 1).

1.2.5 calculation of growth inhibition Rate

(3) After mixing well, the mixture was poured into a sterile 30mm petri dish.

(5) The medium was cultured in the dark at 28 ℃.

(6) The colony diameter was recorded and measured every 3h or 6 h.

(7) The growth inhibition rate was calculated from the measurement results of the diameter. The growth inhibition rate calculation formula is as follows: i% ([ (C-d) - (T-d) ]/(C-d) × 100%, where C is the diameter of the control colony. D is the cake diameter (5mm) and T is the colony diameter for non-traditional peptide treatment.

1.2.6 study of hyphal morphology

(3) After mixing well, the mixture was poured into a sterile 30mm petri dish.

(5) The medium was cultured in the dark at 28 ℃.

(6) Hyphal morphology was observed 36h after inoculation with a conventional light microscope.

2 analysis of results

2.1 Synthesis and Mass Spectrometry identification of peptides

The structure and molecular weight of the peptide KC-19 are consistent with the expected values through mass spectrum analysis (figure 2), and the purity of the synthesized peptide is more than 90 percent through high performance liquid chromatography (figure 3), so that the purity of the synthesized peptide is met, and the peptide KC-19 can be used for subsequent experiments.

2.2 broad-spectrum antifungal Activity of peptide KC-19

The peptide KC-19 has antibacterial activity on Helminthosporium zeae, curvularia lunata and Fusarium graminearum.

2.2.1 resistance of peptide KC-19 to Helminthosporium zeae

Growth of C.hirsutum was performed on the peptide KC-19-containing medium and the control medium (no peptide), respectively, and colonies were observed at 0hpi, 6hpi, 9hpi, 12hpi, 15hpi, 18hpi, 21hpi, 24hpi, 27hpi, 30hpi, 33hpi, 36hpi, and it was found that the growth rate was significantly lower on the peptide KC-19 medium than the control (FIG. 4) although the colonies all grew extensively over time.

Further observation of the growth inhibition rate revealed that the inhibition rate was between 6.9% and 47.0%, and the highest inhibition rate was achieved at 33hpi (fig. 5). These results indicate that the peptide KC-19 has antibacterial activity against Helminthosporium zeae.

Finally, in order to study the mode of action of the antibacterial activity of the peptide KC-19, after the peptide KC-19 is treated for 36 hours, the mycelium of the Helminthosporium zeae is observed by using a common optical microscope, and the control mycelium is sparse, and the mycelium of the Helminthosporium zeae can be aggregated after the peptide KC-19 is treated, so that the peptide KC-19 inhibits the growth and outward expansion of the mycelium by causing the aggregation of the mycelium (figure 6).

2.2.2 resistance of peptide KC-19 to Curvularia lunata

Curvularia lunata grew on the peptide KC-19-containing medium and the control medium (containing no peptide), respectively, and colonies were observed at 0hpi, 9hpi, 12hpi, 15hpi, 18hpi, 21hpi, 24hpi, 27hpi, 30hpi, 33hpi, and 36hpi, and it was found that the growth rate on the peptide KC-19 medium was significantly lower than the control although the colonies were all in extended growth with the passage of time (FIG. 7).

Further observation of the growth inhibition rate revealed that the inhibition rate was between 23.8% and 47.8%, and the highest inhibition rate was reached at 9hpi (fig. 8). These results indicate that the peptide KC-19 has antibacterial activity against Helminthosporium zeae.

Finally, in order to study the mode of action of the antibacterial activity of the peptide KC-19, after the peptide KC-19 is treated for 36 hours, Curvularia lunata hyphae are observed by a common optical microscope, and the control hyphae are found to be sparse, and the accumulation of the Curvularia lunata hyphae can be caused after the peptide KC-19 is treated, which shows that the peptide KC-19 inhibits the growth and outward expansion of the hyphae by causing the accumulation of the hyphae (figure 9).

2.2.3 resistance of peptide KC-19 to Fusarium graminearum

Fusarium graminearum was grown on the peptide KC-19-containing medium and the control medium (no peptide), respectively, and colonies were observed at 0hpi, 6hpi, 12hpi, 18hpi, 24hpi, 30hpi, 36hpi, 42hpi, 48hpi, 54hpi, 60hpi, and it was found that over time, although colonies were all growing in extension, the growth rate on the peptide KC-19 medium was significantly less than the control (FIG. 10).

Further observation of the growth inhibition rate revealed that the inhibition rate was between 8.8% and 25.1%, and the highest inhibition rate was reached at 24hpi (FIG. 11). These results indicate that peptide KC-19 has antibacterial activity against Fusarium graminearum.

Finally, in order to study the mode of action of the antibacterial activity of the peptide KC-19, after the peptide KC-19 is treated for 36 hours, fusarium graminearum hyphae is observed by using a common optical microscope, and the contrast hyphae are found to be sparse, and the fusarium graminearum hyphae can be gathered after the peptide KC-19 is treated, so that the peptide KC-19 inhibits the growth and outward expansion of the hyphae by causing the hyphae to gather (figure 12).

The broad-spectrum antifungal activity experiment of the peptide KC-19 shows that the peptide KC-19 mainly achieves the effect of inhibiting fungi through hypha aggregation.

KC-19 is dissolved in water according to the concentration of KC-19 in-vitro antibacterial experiment to prepare a peptide solution with the molar concentration of 100 mu M. Culturing Curvularia lunata with PDA culture medium, washing spore with water to obtain a concentration of 1 × 10⁶The corn leaves were sprayed with the spore suspension, and 24 hours later, the leaves were sprayed with the peptide solution, while the same spray was applied with an equal amount of sterile water as a control. The disease incidence of the corn leaves is observed every day, and the disease spot area of the corn leaves after the peptide treatment is found to be obviously smaller than that of the control leaves.

Claims

1. A peptide KC-19 with antibacterial activity, wherein the amino acid sequence of the peptide KC-19 is KCHLHPLCKGEALELFSVG.

2 . The peptide KC-19 according to claim 1 , wherein the peptide KC-19 is derived from the intergenic region, has a molecular weight of 2194.11 Da and an isoelectric point of 6.9. 3 .

3. The peptide KC-19 according to claim 1 is used for inhibiting the activity of phytopathogenic fungi.

4. The application of KC-19 as claimed in claim 3, wherein the phytopathogenic fungi are Helmenospora umbilifera, Curvus crescentus or Fusarium graminearum.