CN110669147A - A kind of antibacterial agent FAM-AMP containing gelatinase digestion sequence for detection of Saureus bacteria - Google Patents

Abstract

The invention belongs to the technical field of antibacterial agents, and specifically discloses an antibacterial agent FAM-AMP containing a gelatinase digestion sequence for detection of S. aureus bacteria. GKRWWKWWRR in the AMP antibacterial agent is an antimicrobial peptide, and PLGVRG is a gelatinase cleavage sequence, which can be recognized and cleaved by the gelatinase secreted by S. aureus, and the two are coupled by a peptide solid-phase synthesis method to form a gelatinase cleavage sequence. Site-based novel antibacterial agents. Then, the fluorescein FAM is labeled with the antibacterial agent AMP through the coupling agent, and the labeled FAM-AMP can further detect the concentration of S. aureus by capillary electrophoresis. The antibacterial agent FAM-AMP prepared by the invention can not only detect the concentration of S. aureus bacteria but also effectively sterilize, and has good biocompatibility, little interference to normal physiological activities of body cells, low toxicity and high safety.

Description

A kind of antibacterial agent FAM-containing gelatinase digestion sequence for detection of Saureus bacteria AMP

technical field

The invention belongs to the technical field of antibacterial agents, and specifically discloses an antibacterial agent FAM-AMP containing a gelatinase digestion sequence for detection of S. aureus bacteria.

Background technique

Antibiotics have made outstanding contributions to anti-infection. However, the widespread use and even abuse of antibiotics has led to the emergence of increasingly strong drug resistance in many important human pathogens. of new antibiotics. Antimicrobial peptides (AMPs) have incomparable advantages over traditional antibiotics. They have unique antibacterial mechanisms, rapid bactericidal effect and are not easy to induce bacterial resistance. They are a class of highly potential antibacterial drugs. Antibacterial peptides are considered to have broad application prospects in the pharmaceutical industry due to their high antibacterial activity, wide antibacterial spectrum, wide variety, and wide selection range, and the target strains are not easy to produce resistance mutations. However, there are still problems such as high toxicity, low stability and high production cost, which limit their development into drugs.

Recently, several techniques have been developed for bacterial detection, such as fluorescence imaging, surface-enhanced Raman spectroscopy, immunology and colorimetric assays. However, colorimetric assays, for example, are often susceptible to interference from environmental conditions (ie pH, ionic concentrations and solvents), thus compromising the specificity and accuracy of bacterial detection.

SUMMARY OF THE INVENTION

In order to solve the deficiencies in the prior art, the present invention combines luciferin FAM with AMP containing a gelatinase cleavage site to provide a novel antibacterial agent FAM-AMP. The antibacterial agent prepared by the present invention not only has excellent antibacterial properties It can be recognized and cut by the gelatinase secreted by S. aureus bacteria (Staphylococcus aureus), so a new method for detecting S. aureus bacteria is proposed. Through the capillary electrophoresis of fluorescence detection, each component of the sample is efficiently and rapidly separated according to the difference of its mobility and distribution coefficient, so as to realize the effective detection of S. aureus bacteria. Compared with the traditional method, the CE (capillary electrophoresis detection) adopted in the present invention has the advantages of high separation efficiency, high sensitivity, fast speed, low sample consumption, low cost and the like. The antibacterial peptide itself has no obvious antibacterial effect, but it will be cut by gelatinase in the bacterial liquid to release the antibacterial peptide sequence, so as to achieve the antibacterial purpose. It can be used for the detection of S. aureus bacteria and has broad application prospects.

The specific technical scheme of the present invention is as follows:

The antibacterial agent FAM-AMP prepared by the invention is composed of a fluorescent agent and an antibacterial peptide containing a gelatinase cleavage site.

The fluorescent agent in the prepared antibacterial agent FAM-AMP is 5-FAM, its maximum absorption wavelength is 492nm, and its fluorescence emission wavelength is 518nm.

The amino acid sequence of the antimicrobial peptide AMP containing a gelatinase cleavage site is GKRWWKWWRRPLGVRGC, wherein GKRWWKWWRR is an antimicrobial peptide, and PLGVRG is a gelatinase cleavage sequence.

AMP is synthesized by the conventional Fmoc solid-phase method, that is, the amino group is exposed after deprotection of the monomer amino acid protected by Fmoc on the solid-phase resin, and a peptide bond is formed with the carboxyl group of the amino acid in the solution through a condensation reaction, thereby connecting the amino acid to the resin. The peptide chain is extended from the C-terminus to the N-terminus until the desired peptide chain is synthesized.

The method of labeling AMP with fluorescent agent 5-FAM is as follows: Weigh 5-FAM, HOBT, and EDC equivalent to 3 times the molar amount of the resin with peptides, dissolve them in DMF, add DIEA to avoid light and react overnight, and finally use cleavage solution to label the AMP. The peptides were cleaved from the resin, purified by HPLC, and analyzed by mass spectrometry.

The antibacterial agent FAM-AMP prepared by the invention can further detect the concentration of S. aureus bacteria by fluorescence detection capillary electrophoresis (CE-FL).

The specific method for detecting the concentration of S. aureus by fluorescence capillary electrophoresis is as follows:

CE-FL adopts the testing system built by the laboratory. The electric field is provided by a high-voltage power supply (20kV), the inner diameter of 75μm, the effective length of 50cm (outer diameter of 365μm, total length of 75cm) polyamide gel-coated capillary is connected to the positive and negative electrophoresis tank, and fixed in the inverted fluorescence microscope detection window, 100W mercury lamp provides The light source is processed by excitation filter (BP 420±20nm) and dichroic mirror (DM 455). Finally, the optical fiber spectrometer Maya-2000pro converts the optical signal into electrical signal in the detection window to form an electrophoresis spectrum at the computer terminal. The capillaries were washed sequentially with 1M HCl, 1M NaOH and running buffer (25mM Na2B4O7, pH 9.3) for 30 min before use. Electrophoresis was performed with manual injection at the positive end.

Compared with the prior art, the present invention has the following beneficial effects:

The FAM-AMP antibacterial agent prepared by the present invention has no obvious antibacterial effect, but will be cut by gelatinase in S. aureus (Staphylococcus aureus) bacterial liquid to release antibacterial peptide sequences, thereby achieving antibacterial purpose.

The FAM-AMP antibacterial agent prepared by the invention can be recognized and cut off by the gelatinase secreted by S. aureus, thereby providing a new method for detecting S. aureus. Through the capillary electrophoresis of fluorescence detection, each component of the sample is efficiently and rapidly separated according to the difference of its mobility and distribution coefficient, so as to realize the effective detection of S. aureus bacteria.

The preparation process disclosed by the invention is simple, the raw material sources are wide, the reaction conditions are mild, the synthesis is easy, and the invention is suitable for popularization and use.

Description of drawings

Figure 1 is an HPLC chart of AMP.

Figure 2 is an HPLC chart of FAM-AMP.

Figure 3 is a mass spectrum of FAM-AMP.

Figure 4 is the UV absorption diagram of AMP and FAM-AMP.

Figure 5 is a graph of the fluorescence emission of FAM-AMP.

Figure 6 (A, B) is a graph showing the effect of the concentration of S. aureus on the digestion effect of FAM-AMP.

Figure 7 is a fitting curve diagram of the concentration of S. aureus bacteria on the effect of FAM-AMP enzyme cleavage.

Figure 8 is a graph showing the effect of FAM-AMP antibacterial agent on the growth curve of S. aureus.

Figure 9 shows the survival rate of S. aureus in different concentrations of FAM-AMP solution.

Figure 10 is a graph showing the effect of E. coli concentration on the digestion effect of FAM-AMP.

Figure 11 is a graph showing the effect of FAM-AMP antibacterial agent on the growth curve of E. coli.

Figure 12 shows the survival rate of S. aureus in different concentrations of AMP solution.

Detailed ways

The present invention will be described in detail below with reference to the embodiments, but the present invention is not limited to these embodiments.

Example 1

1. Synthesis of FAM-AMP

1) Synthesis of AMP

AMP is based on 2-Chlorotrityl Chloride resin with Fmoc protecting group, obtained by solid-phase peptide synthesis. Specifically: Weigh amino acids (chemically modified α-amino acids), HBTU, and HOBT equivalent to 5 times the molar amount of the resin, dissolve them in DMF, and add DIEA for coupling for 30 minutes. Then, 20% piperidine was added to react for 30 minutes to remove the Fmoc protecting group, and this step was repeated to reach the peptide chain required for synthesis. The synthesized peptide chain was purified by HPLC, and the purification effect was shown in FIG. 1 .

2) 5-FAM Fluorescein-labeled AMP

Weigh 30 mg of resin (resin with peptide chain synthesized above), dissolve 5-FAM, HOBT, and EDC in 3-fold molar amount of the resin in 1 mL of DMF, add 10 μL of DIEA to avoid light and react overnight, and finally use cleavage solution to remove 30 mg of resin. The labeled polypeptide was cleaved from the resin, purified by HPLC, and the molecular weight was determined by LC-MS.

2. Characterization of FAM-AMP antibacterial agent

1) Determination experiment of UV absorption of AMP, FAM-AMP antibacterial agent

After the synthesized AMP and FAM-AMP were filtered through a water system (0.22 μm) filter membrane, the dilution was doubled, and each sample was measured three times in parallel, and the average value was taken. Scan the UV absorption of AMP and FAM-AMP with a UV spectrophotometer. The range of 300nm ~ 800nm, UV absorption spectrum as shown in Figure 4. It can be seen from the figure that AMP alone has no obvious absorption at 300-800 nm, while FAM-AMP has an obvious absorption peak at 492 nm, indicating that 5-FAM is successfully labeled with AMP.

2) Determination experiment of fluorescence emission of FAM-AMP antibacterial agent

After the synthesized FAM-AMP antibacterial agent was filtered through a water system (0.22 μm) filter membrane, it was diluted twice, and each sample was measured three times in parallel, and the average value was taken. The fluorescence of FAM-AMP antibacterial agent was tested with a fluorescence spectrometer. As shown in FIG. 5 , it can be seen from the figure that the maximum emission wavelength of FAM-AMP is 518 nm.

3. The effect of S. aureus concentration on FAM-AMP digestion by CE-FL

In order to explore the effect of different concentrations of S. aureus on FAM-AMP digestion, different concentrations of S. aureus were incubated with FAM-AMP for 3 h, and the samples were analyzed by CE-FL to observe the change of fluorescence signal. The sample was excited at a wavelength of 480 nm, the injection time was 20 s, and the electrophoresis spectrum of the 518 nm channel (corresponding to the emission wavelength of FAM) was observed.

10 ⁶ CFU/mL to 10 ⁸ CFU/mL of S. aureus was mixed with FAM-AMP, incubated for 3 hours, and analyzed using CE-FL.

As shown in Figure 6 (A, B), the electrophoresis peak (P1) of FAM-AMP appeared at 240s, and with the increase of S. aureus concentration, a new electrophoresis peak (P2) appeared at 295s, and The fluorescence intensity of P2 gradually increased, while that of P1 gradually decreased. From this, we can infer that the peak at P2 is the electrophoresis peak of FAM-AMP enzymatic hydrolysis product. As the concentration of S. aureus increases, the effect of S. aureus on FAM-AMP enzymatic cleavage is better. When the concentration is 10 ⁸ At CFU/mL, the P1 peak almost disappeared, and FAM-AMP had been completely digested.

According to the relationship between the area of P1 and P2 and the concentration of S. aureus in Figure 6 (A, B), the linear fitting curve is shown in Figure 7, the higher the concentration of S. aureus, the higher the concentration of ln (S The smaller the value of _P1 /S _P2 ). This curve can be used to detect the concentration of S. aureus bacteria.

3. FAM-AMP antibacterial performance test

1) The effect of FAM-AMP antibacterial agent on the growth curve of S. aureus

In order to explore the effect of FAM-AMP antibacterial agent on the growth of S. aureus, the overnight cultured S. aureus was diluted to 10 ⁵ CFU/mL, 200 μL of bacterial solution was added to a 96-well plate, and 10 μL of 100 μM FAM- AMP antibacterial agent, three parallel samples for each group. The absorbance at 600 nm was detected by a microplate reader for 10 hours continuously, and then the growth of bacteria in the experimental group and the control group was observed according to the detected data (Figure 8).

It can be seen from the figure that after co-incubating for 4 hours, the OD _{600 value of the S. aureus control group has reached 1, while the OD 600 value} of the FAM-AMP antibacterial agent group is only 1/3 of the control group, which is about 0.3 (attached). Figure 9), showing a significant inhibitory effect, which may be caused by the action of AMP.

2) Determination of MIC ₉₀ value of FAM-AMP antibacterial agent

Prepare 15 μM, 30 μM, 45 μM, 60 μM, 75 μM, 90 μM, 105 μM of FAM-AMP with sterile water, take 10 ⁸ CFU/mL S. aureus bacterial solution and incubate with 250 μL of the sample for 1 hour, dilute 10 ³ , take 100 μL of the plate was coated and placed in a biochemical incubator for 15 hours. The number of colonies growing on the agar plate was counted, and each sample was measured in parallel three times (Figure 9).

As shown in the coating results of FIG. 9, the concentration of FAM-AMP antibacterial agent has a significant inhibitory effect on the survival rate of S. aureus, and the higher the concentration, the better the inhibitory effect. When the concentration of FAM-AMP antibacterial agent was 18 μM, the survival rate of S. aureus was only 3%, that is, the MIC ₉₀ was 18 μM.

Comparative Example 1

(1) The effect of CE-FL detection of E. coli concentration on the digestion of FAM-AMP

In order to explore the effect of E.coli on the digestion effect of FAM-AMP, 10 ⁸ CFU/mL E.coli was incubated with FAM-AMP for 3h, and the samples were analyzed by CE-FL to observe the changes of FRET. The sample was excited at a wavelength of 480 nm, the injection time was 20 s, and the electrophoresis spectrum of the 518 nm channel (corresponding to the emission wavelength of FAM) was observed.

As shown in Fig. 10, the electrophoretic peak (P1) of FAM-AMP appeared at 240s. 10 ⁸ CFU/mL E. coli was incubated with FAM-AMP for 3 hours, no new peak appeared at 295s, indicating that E. coli could not secrete gelatinase and could not recognize and cut AMP polypeptide sequence.

(2) The effect of FAM-AMP antibacterial agent on the growth curve of E. coli

In order to explore the effect of FAM-AMP antibacterial agent on the growth of E. coli bacteria, the overnight cultured E. coli bacteria were diluted to 10 ⁵ CFU/mL, 200 μL of bacterial solution was added to a 96-well plate, and 10 μL of 100 μM FAM- AMP antibacterial agent, three parallel samples for each group. The absorbance at 600nm was detected by a microplate reader for 10 hours continuously, and then the growth of bacteria in the experimental group and the control group was observed according to the measured data (Figure 11).

It can be seen from the figure that after 5 hours of co-incubation, the OD ₆₀₀ value of the E. coli control group has reached 1, and the FAM-AMP antibacterial agent group also showed an inhibitory effect (Fig. 11), but the effect was not as good as that of S. aureus. obvious (Fig. 9).

(3) Determination of MIC ₉₀ value of GKRWWKWWRR

Prepare 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM GKRWWKWWRR with sterile water, take 10 ⁸ CFU/mL S. aureus bacterial solution and incubate with 250 μL of the sample for 1 hour, dilute 10 ³ , take 100 μL of the plate was coated and placed in a biochemical incubator for 15 hours. The number of colonies growing on the agar plate was counted, and each sample was measured in parallel three times (Figure 12).

As shown in the results of FIG. 12 , the concentration of GKRWWKWWRR has a significant inhibitory effect on the survival rate of S. aureus, and the higher the concentration, the better the inhibitory effect. When the concentration was 10 μM, the survival rate of S. aureus was only 9%, that is, the MIC ₉₀ was 10 μM.

Comparative Example 2

1. Synthesis of FAM-AMP

1) Synthesis of AMP

AMP is based on 2-Chlorotrityl Chloride resin with Fmoc protecting group, obtained by solid-phase peptide synthesis. Specifically: Weigh amino acids (chemically modified α-amino acids), HBTU, and HOBT equivalent to 5 times the molar amount of the resin, dissolve them in DMF, and add DIEA for coupling for 30 minutes. Then 20% piperidine was added to react for 30 min to remove the Fmoc protecting group, and this step was repeated until the desired peptide chain (GKRWWKWWRR) was synthesized.

2) 5-FAM Fluorescein-labeled AMP

Weigh 30 mg of resin (resin with peptide chain synthesized above), dissolve 5-FAM, HOBT, and EDC in 3-fold molar amount of the resin in 1 mL of DMF, add 10 μL of DIEA to avoid light and react overnight, and finally use cleavage solution to remove 30 mg of resin. The labeled polypeptide was cleaved from the resin, purified by HPLC, and the molecular weight was determined by LC-MS.

This sequence is only an antibacterial sequence and has no enzyme cutting sequence, so it cannot specifically recognize the gelatinase secreted by S. aureus.

Claims (6)

1. an antibacterial agent FAM-AMP containing a gelatinase cleavage sequence is characterized in that, the antibacterial agent is the antibacterial peptide containing a gelatinase cleavage site marked by a fluorescent agent. 2 . The antibacterial agent according to claim 1 , wherein the fluorescent agent is 5-FAM with a maximum absorption of 492 nm and a fluorescence emission of 518 nm. 3 . 3. The antibacterial agent according to claim 1, wherein the amino acid sequence of the antimicrobial peptide AMP containing the gelatinase cleavage site is GKRWWKWWRRPLGVRGC, wherein, GKRWWKWWRR is the antimicrobial peptide, and PLGVRG is the gelatinase cleavage sequence. 4. antibacterial agent according to claim 1, is characterized in that, described AMP adopts conventional Fmoc solid-phase method to synthesize, promptly exposes amino group after the monomer amino acid deprotection by Fmoc protection on solid-phase resin, by condensation reaction and with. The carboxyl groups of the amino acids in solution form peptide bonds, thereby linking the amino acids to the resin, extending the peptide chain from the C-terminus to the N-terminus until the desired peptide chain is synthesized. 5. antibacterial agent according to claim 1, is characterized in that, the method that fluorescent agent 5-FAM marks AMP is: take by weighing 5-FAM, HOBT, EDC equivalent to the resin with polypeptide 3 times molar amount and dissolve in 5-FAM, HOBT, EDC. In DMF, DIEA was added to react overnight in the dark, and finally the labeled polypeptide was cleaved from the resin with a cleavage solution, purified by HPLC, and analyzed by mass spectrometry. 6 . The application of an antibacterial agent according to claim 1 , wherein the antibacterial agent FAM-AMP is used as an antibacterial agent, and is used to detect the bacterial concentration of S. aureus by fluorescence capillary electrophoresis. 7 .

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