Peptide probe for identifying specific intestinal bacteria and preparation method thereof

Abstract

The invention belongs to the field of organic synthesis technology and biological fermentation, and particularly relates to a peptide probe for identifying special intestinal bacteria and a preparation method of the peptide probe. The FITC-AR-9 prepared by the invention can visualize the combination of the active peptide AR-9 and intestinal bacteria, not only can effectively improve the detection sensitivity and accuracy, but also can shorten the detection period and reduce the detection difficulty, and may become an important means for detecting whether the intestinal bacteria can utilize the active peptide or not in the future.

Description

Technical Field

[0001] The invention belongs to the field of organic synthesis, and in particular relates to a peptide probe for identifying specific intestinal bacteria and a preparation method thereof. Background Art

[0002] The intestinal flora is a huge ecosystem that contains more than 1,000 species of microorganisms and is called the "gut brain" of the human body. Under normal circumstances, the intestinal flora-host-environment maintains a state of balance. If this balance is broken, it will cause dysbiosis and promote the occurrence and development of multi-system diseases. Previous studies have confirmed that the AR-9 peptide isolated from the earthworm has a certain effect on the correction of intestinal flora, but it is not yet known which specific intestinal bacteria it acts on. Therefore, it is particularly important to explore and develop peptide probes that specifically bind to intestinal bacteria.

[0003] At present, there are many methods to determine whether intestinal bacteria can use active peptides to affect growth, such as 16s rRNA amplicon determination, metagenomic detection, strain sequencing, growth curve method, etc. These methods have their own advantages, but also have their own shortcomings. The 16s rRNA gene is the most useful and commonly used molecular clock in bacterial systematic classification research. Sequencing technology can be used to sequence one or several hypervariable regions (such as V1-V2, V3-V4, V4 or V5, etc.). The sequencing data can be compared with the 16S rRNA gene reference database (such as RDP, SILVA, GreenGene, etc.) to obtain sequence variation and relative abundance, and then obtain information such as sample species classification, species abundance, population structure, system evolution, community comparison, etc.; However, its resolution can only reach the relative abundance at the genus level, and different factors in the experimental process have a greater impact on the results. Metagenomic detection is a high-throughput sequencing of the total DNA of all microorganisms in environmental samples. Compared with 16s rRNA amplicon determination, it obtains more information and has a deeper identification depth. Strain sequencing can identify specific strain types and is one of the common methods for determining bacterial species; however, a large number of purification experiments are required in the early stages, which is time-consuming and labor-intensive. The growth curve method is to observe the regulation of intestinal bacteria by observing the logarithmic growth phase of the strain growth curve and the regulation of the influencing factors on its logarithmic growth phase; this method also requires a large number of parallel samples, time, and materials.

[0004] Therefore, how to provide a method that can accurately, efficiently and easily detect whether the bacterial flora can utilize AR-9 active peptides is a technical problem that needs to be solved urgently by those skilled in the art. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a peptide probe for identifying specific intestinal bacteria and a preparation method thereof.

[0006] It should be noted that compared with the current detection methods, the present invention designs a fluorescent probe. According to the special photophysical and photochemical properties of this probe, it can not only effectively improve the sensitivity and accuracy of detection, but also shorten the detection cycle and reduce the difficulty of detection. In the near future, it will become an important means to detect whether intestinal bacteria can utilize active peptides.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] The first technical purpose of the present invention is to provide a peptide probe for identifying specific intestinal bacteria, wherein the molecular formula of the peptide probe is C 113 H 123 N 15 O 32 , the structural formula is as follows:

[0009]

[0010] The second technical purpose of the present invention is to provide a method for preparing a peptide probe for identifying specific intestinal bacteria as described above, which is to link a fluorescent dye to a specific part of the AR-9 peptide so that the peptide carries fluorescence, and specifically comprises the following steps:

[0011] (1) A sufficient amount of Fmoc is dissolved in an alkaline solution with a pH of 7.2-9, and then 0.1-5 mol of various amino acids are added to synthesize Fmoc-amino acid-OH for later use;

[0012] (2) 2-Chlorotrityl Chloride Resin was eluted with DCM and then loaded into an elution column. 0.1-2 mol Fmoc-alanine (Ala)-OH was added to the resin at 25°C-50°C.

[0013] (3) adding sufficient amount of N,N-diisopropylethylamine (DIEA) and dimethylformamide (DMF) solution in sequence, shaking, blocking with methanol, and after 10-30 min, adding DMF solution containing 20%-30% piperidine (piperidine:DMF, v / v) for elution;

[0014] (4) adding 0.1-2 mol of Fmoc group-protected valine-OH and repeating step (3);

[0015] (5) Add 0.1-2 mol of Fmoc group-protected phenylalanine-OH and repeat step (3);

[0016] (6) Add 0.1-2 mol of Fmoc-protected proline-OH and repeat step (3);

[0017] (7) Add 0.1-2 mol of Fmoc group-protected lysine-OH and repeat step (3) to obtain a short peptide chain of AVFPK; the structural formula is as follows:

[0018]

[0019] (8) Add 0.1-4 mol FITC and repeat step (3) to form (Fmoc)OH-(FITC)-AVFRK-FITC with the short peptide chain, the structural formula of which is as follows:

[0020]

[0021] (9) Add 0.1-2 mol of Fmoc-protected serine-OH and repeat step (3);

[0022] (10) Add 0.1-2 mol of Fmoc-protected isoleucine-OH and repeat step (3);

[0023] (11) Add 0.1-2 mol of Fmoc-protected lysine-OH and repeat step (3);

[0024] (12) Add 0.1-4 mol FITC and repeat step (3) to generate (Fmoc)OH-(FITC)-AVFRK-(FITC)-SIK-(FITC); the structural formula is as follows:

[0025]

[0026] (13) Add 0.1-2 mol of Fmoc-protected valine-OH and repeat step (3);

[0027] (14) Add 0.1-2 mol of Fmoc-protected glycine -OH and repeat step (3);

[0028] (15) Add 0.1-2 mol of Fmoc-protected arginine-OH and repeat step (3) to synthesize (Fmoc)OH-(FITC)-AVFRK-(FITC)-SI-(FITC)-VGR, i.e. FITC-AR-9; the structural formula is as follows:

[0029]

[0030] The components on the resin were cut using an acidic cutting solution (pH 2.0-4.5), and the resulting FITC-AR-9 was systematically characterized using Fourier transform infrared spectrometer, circular dichroism spectrometer, and UHPLC-QExactive MS / MS.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] The FITC-AR-9 prepared by the present invention can visualize the combination of the active peptide AR-9 and intestinal bacteria, effectively improve the sensitivity and accuracy of detection, and can shorten the detection cycle and reduce the difficulty of detection. In the future, it may become an important means to detect whether intestinal bacteria can utilize active peptides. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0034] Figure 1 It is the HPLC spectrum of FITC-AR-9.

[0035] Figure 2 This is the mass spectrum of FITC-AR-9 under UHPLC-QExactive MS / MS.

[0036] Figure 3 This is a comparison of the labeling rate (A) and fluorescence intensity (B) of intestinal bacteria at different times.

[0037] Figure 4 It is the fluorescence image after 30 single colonies were incubated with fluorescent peptide probes. DETAILED DESCRIPTION

[0038] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0039] The word "embodiment" used here as an "exemplary" does not necessarily mean that any embodiment described is superior to or better than other embodiments. Unless otherwise specified, the performance index tests in the embodiments of this application are performed using conventional test methods in the art. It should be understood that the terms described in this application are only used to describe specific implementation methods and are not used to limit the content disclosed in this application.

[0040] Unless otherwise specified, the technical and scientific terms used in this document have the same meanings as commonly understood by ordinary technicians in the technical field to which this application belongs; other experimental methods and technical means not specifically specified in this application refer to experimental methods and technical means commonly used by ordinary technicians in this field.

[0041] In order to better illustrate the content of the present application, numerous specific details are provided in the specific examples below. It should be understood by those skilled in the art that the present application can also be implemented without certain specific details. In the embodiments, some methods, means, instruments, equipment, etc. well known to those skilled in the art are not described in detail in order to highlight the main purpose of the present application.

[0042] Under the premise of no conflict, the technical features disclosed in the embodiments of the present application can be combined arbitrarily, and the resulting technical solutions belong to the contents disclosed in the embodiments of the present application.

[0043] The invention discloses a peptide probe for identifying specific intestinal bacteria and a preparation method thereof.

[0044] In order to better understand the present invention, the present invention is further specifically described below through the following examples, but it should not be understood as a limitation of the present invention. Some non-essential improvements and adjustments made by technicians in this field based on the above invention content are also considered to fall within the protection scope of the present invention.

[0045] Example 1

[0046] (1) A sufficient amount of Fmoc is dissolved in an alkaline solution at a pH of 8.0, and then 0.2 mol of various amino acids are added to synthesize Fmoc-amino acid-OH, which is precipitated and set aside;

[0047] (2) 2-Chlorotrityl Chloride Resin was eluted with DCM and then loaded into an elution column. At 25°C, 0.1 mol (31.140 g) of Fmoc-alanine (Ala)-OH was added to the resin.

[0048] (3) Add sufficient amount of N,N-diisopropylethylamine (DIEA) and dimethylformamide (DMF) solution in sequence, shake, block with methanol, and after 15 min, add DMF solution containing 20% ​​piperidine (piperidine:DMF=2:8, v / v) for elution;

[0049] (4) Add 0.1 mol (33.943 g) of Fmoc-protected valine-OH and repeat step (3);

[0050] (5) Add 0.1 mol (38.743 g) of Fmoc-protected phenylalanine-OH and repeat step (3);

[0051] (6) Add 0.1 mol (33.741 g) of Fmoc-protected proline-OH and repeat step (3);

[0052] (7) Add 0.1 mol (36.846 g) of Fmoc-protected lysine-OH and repeat step (3) to obtain a short peptide chain of AVFPK;

[0053] (8) Add 0.2 mol (77.876 g) of FITC and repeat step (3) to form (Fmoc)OH-(FITC)-AVFRK-FITC with the short peptide chain;

[0054] (9) Add 0.1 mol (32.739 g) of Fmoc-protected serine-OH and repeat step (3);

[0055] (10) Add 0.1 mol (35.345 g) of Fmoc-protected isoleucine-OH and repeat step (3);

[0056] (11) Add 0.1 mol (36.846 g) of Fmoc-protected lysine-OH and repeat step (3);

[0057] (12) Add 0.2 mol (7.7876 g) of FITC and repeat step (3) to generate (Fmoc)OH-(FITC)-AVFRK-(FITC)-SIK-(FITC);

[0058] (13) Add 0.1 mol (33.943 g) of Fmoc-protected valine-OH and repeat step (3);

[0059] (14) Add 0.1 mol (29.738 g) of Fmoc-protected glycine -OH and repeat step (3);

[0060] (15) Add 0.1 mol (39.646 g) of Fmoc-protected arginine-OH and repeat step (3) to synthesize (Fmoc)OH-(FITC)-AVFRK-(FITC)-SI-(FITC)-VGR;

[0061] (16) The components on the resin were cut using an acidic cutting solution (TFA: 95%, deionized water: 1%, EDT: 2%, TIS: 2%).

[0062] Example 2

[0063] (1) A sufficient amount of Fmoc is dissolved in an alkaline solution at a pH of 8.5, and then 0.3 mol of various amino acids are added to synthesize Fmoc-amino acid-OH, which is precipitated and set aside;

[0064] (2) 2-Chlorotrityl Chloride Resin was eluted with DCM and then loaded into an elution column. At 37°C, 0.1 mol (31.140 g) of Fmoc-alanine (Ala)-OH was added to the resin.

[0065] (3) Add sufficient amount of N,N-diisopropylethylamine (DIEA) and dimethylformamide (DMF) solution in sequence, shake, block with methanol, and after 10 min, add DMF solution containing 25% piperidine (piperidine:DMF=1:3, v / v) for elution;

[0066] (4) Add 0.15 mol (50.915 g) of Fmoc-protected valine-OH and repeat step (3);

[0067] (5) Add 0.15 mol (58.115 g) of Fmoc-protected phenylalanine-OH and repeat step (3);

[0068] (6) Add 0.15 mol (50.612 g) of Fmoc-protected proline-OH and repeat step (3);

[0069] (7) Add 0.15 mol (55.269 g) of Fmoc-protected lysine-OH and repeat step (3) to obtain a short peptide chain of AVFPK;

[0070] (8) Add 0.03 mol (116.814 g) of FITC and repeat step (3) to form (Fmoc)OH-(FITC)-AVFRK-FITC with the short peptide chain;

[0071] (9) Add 0.15 mol (49.109 g) of Fmoc-protected serine-OH and repeat step (3);

[0072] (10) Add 0.15 mol (53.018 g) of Fmoc-protected isoleucine-OH and repeat step (3);

[0073] (11) Add 0.15 mol (55.269 g) of Fmoc-protected lysine-OH and repeat step (3);

[0074] (12) Add 0.03 mol (116.814 g) of FITC and repeat step (3) to generate (Fmoc)OH-(FITC)-AVFRK-(FITC)-SIK-(FITC);

[0075] (13) Add 0.15 mol (50.915 g) of Fmoc-protected valine-OH and repeat step (3);

[0076] (14) Add 0.15 mol (44.607 g) of Fmoc-protected glycine -OH and repeat step (3);

[0077] (15) Add 0.15 mol (59.469 g) of Fmoc-protected arginine-OH and repeat step (3) to synthesize (Fmoc)OH-(FITC)-AVFRK-(FITC)-SI-(FITC)-VGR;

[0078] (16) The components on the resin were cut using an acidic cutting solution (TFA: 98%, deionized water: 1%, EDT: 0.5%, TIS: 0.5%).

[0079] Example 3

[0080] (1) A sufficient amount of Fmoc is dissolved in an alkaline solution at a pH of 9.0, and then 0.4 mol of various amino acids are added to synthesize Fmoc-amino acid-OH, which is precipitated and set aside;

[0081] (2) 2-Chlorotrityl Chloride Resin was eluted with DCM and then loaded into an elution column. At 30°C, 0.1 mol (31.140 g) of Fmoc-alanine (Ala)-OH was added to the resin.

[0082] (3) Add sufficient amount of N,N-diisopropylethylamine (DIEA) and dimethylformamide (DMF) solution in sequence, shake, block with methanol, and after 30 minutes, add DMF solution containing 20% ​​piperidine (piperidine:DMF=2:8, v / v) for elution;

[0083] (4) Add 0.2 mol (67.886 g) of Fmoc-protected valine-OH and repeat step (3);

[0084] (5) Add 0.2 mol (77.487 g) of Fmoc-protected phenylalanine-OH and repeat step (3);

[0085] (6) Add 0.2 mol (67.483 g) of Fmoc-protected proline-OH and repeat step (3);

[0086] (7) Add 0.2 mol (73.692) of Fmoc-protected lysine-OH and repeat step (3) to obtain a short peptide chain of AVFPK;

[0087] (8) Add 0.4 mol (155.752 g) of FITC and repeat step (3) to form (Fmoc)OH-(FITC)-AVFRK-FITC with the short peptide chain;

[0088] (9) Add 0.2 mol (65.479 g) of Fmoc-protected serine-OH and repeat step (3);

[0089] (10) Add 0.2 mol (70.691 g) of Fmoc-protected isoleucine-OH and repeat step (3);

[0090] (11) Add 0.2 mol (73.692 g) of Fmoc-protected lysine-OH and repeat step (3);

[0091] (12) Add 0.4 mol (155.752 g) of FITC and repeat step (3) to generate (Fmoc)OH-(FITC)-AVFRK-(FITC)-SIK-(FITC);

[0092] (13) Add 0.2 mol (67.887 g) of Fmoc-protected valine-OH and repeat step (3);

[0093] (14) Add 0.2 mol (59.47 g) of Fmoc-protected glycine -OH and repeat step (3);

[0094] (15) Add 0.2 mol (79.292 g) of Fmoc-protected arginine-OH and repeat step (3) to synthesize (Fmoc)OH-(FITC)-AVFRK-(FITC)-SI-(FITC)-VGR;

[0095] (16) The components on the resin were cut using an acidic cutting solution (TFA: 90%, deionized water: 5%, EDT: 2.5%, TIS: 2.5%).

[0096] In order to further demonstrate the beneficial effects of the present invention and to better understand the present invention, the following characterization experiments and application tests are used to further illustrate the technical features disclosed in the present invention, but they should not be understood as limitations of the present invention. Other improvements made by those skilled in the art based on the above invention content without creative work are also considered to fall within the scope of protection of the present invention.

[0097] Characterization experiments:

[0098] The FITC-AR-9 obtained in Example 1 was detected by HPLC. The results are shown in the attached figure. Figure 1 shown.

[0099] A 4.6×250 mm, ChromCore 120C185u silica gel column was used, mobile phase A was 0.1% water, mobile phase B was 0.1% acetonitrile, and the mobile phase gradient was 0.1-25 min, 40%-65% B; 25-25.1 min, 65%-100% B; 25.1-30 min, 100% B. The flow rate was 1.0 ml / min, and the wavelength was 220 nm.

[0100] The FITC-AR-9 obtained in Example 1 was systematically characterized using UHPLC-QExactive MS / MS. The spectrum of the probe under UHPLC-QExactive MS / MS is shown in the attached figure. Figure 2 shown.

[0101] Use Waters ACQUITY UPLC BEH C18 column, ESI ion source, bias: +4.5kv, nebulizer gas oil: 1.5L / min, detector: 1.2kv, CDL: -20.0vT., CDL temperature of 250°C, mobile phase A is pure water, mobile phase B is acetonitrile, the mobile phase ratio is 40% B, and the flow rate is 0.2ml / min.

[0102] Application experiment:

[0103] The following experimental example further illustrates the application research uses of the peptide probe prepared by the present invention by observing the binding of the fluorescent probe to intestinal bacteria.

[0104] Experimental Example 1: Study on the binding of the fluorescent probe of the present invention (obtained from Example 1) to intestinal bacteria

[0105] 1. Test materials

[0106] FITC-AR-9 (hereinafter referred to as fluorescent probe), SD rat feces sample, PBS solution (1×), dimethyl sulfoxide (DMSO), GAM medium, agar, inoculation loop, flow cytometer, laser confocal microscope, anaerobic incubator, autoclave, etc.

[0107] 2. Drug preparation method: Before use, the above fluorescent probe was prepared into a uniform suspension with DMSO to a final concentration of 0.4 mM.

[0108] 3. Test methods

[0109] (1) Accurately weigh 1.0 g of rat feces and add it to 10.0 ml of 1× PBS solution. Stir with a sterilized glass rod to form a suspension, filter with gauze, and set aside the filtrate.

[0110] (2) The filtrate was centrifuged at 5000 r / min for 5 min, the supernatant was discarded, and the precipitate was resuspended in 10.0 ml of 1× PBS solution. This was repeated three times. Finally, the precipitate was resuspended in 2.5 ml of 1× PBS solution as the bacterial solution for later use.

[0111] (3) Take 1.0 ml of the bacterial solution and add it to 9.0 ml of sterilized GAM medium prepared in advance. Incubate in an anaerobic environment for 48 h. This is the first generation bacterial solution (F1).

[0112] (4) F1 was subcultured at a ratio of 1:9 and cultured under anaerobic conditions for 48 h to obtain F2.

[0113] 4. Determination of Fluorescence Labeling Rate and Fluorescence Intensity by Flow Cytometry

[0114] 1) F2 was centrifuged, the supernatant was discarded, and resuspended in 10.0 ml 1× PBS solution, and 100 μl of the fluorescent probe suspension was added thereto. The cells were then taken out after anaerobic culture for 0, 8, 16, 24, 32, 40, and 48 h.

[0115] 2) Centrifuge the bacterial solution at 5000r / min for 5min, discard the supernatant, and resuspend the precipitate in 2.0ml 1×PBS solution. Repeat 3 times. Finally, resuspend the precipitate in 2.0ml 1×PBS solution as a flow cytometer sample for testing. The results of fluorescence intensity and fluorescence labeling rate are shown in Figure 3 shown.

[0116] Experimental Example 2: Fluorescence analysis of a single intestinal bacteria

[0117] 1) Add 1.5% agar to the GAM medium during the preparation of the medium, pour it into a flat plate at above 50°C, cool it until solidified, turn it upside down to make a solid medium, and store it at 4°C.

[0118] 2) Place the solid culture medium in an anaerobic incubator to deoxygenate for 24 hours, return the temperature to 37°C, inoculate the F2 bacterial solution onto the solid culture medium using the zigzag streak method, and culture anaerobically for 48 hours.

[0119] 3) Pick out a single colony on the solid culture medium, transfer it to a liquid culture medium for culture, and obtain F1 after anaerobic culture for 48 hours.

[0120] 4) Repeat step 2) three times, pick a single colony, transfer it to liquid culture medium for culture, and obtain F1 after anaerobic culture for 48 hours, which is then subcultured to obtain F2.

[0121] 5) After F2 is cultured for a period of time, F2 is centrifuged, the supernatant is discarded, and the suspension is resuspended in 10.0 ml of 1×PBS solution, and 100 μl of the fluorescent probe suspension is added thereto. The suspension is then taken out after anaerobic culture for 48 hours.

[0122] 6) The bacterial solution was centrifuged at 5000 r / min for 5 min, the supernatant was discarded, the precipitate was fixed with 2.0 ml 70% ethanol at 4°C for 10 min, centrifuged at 5000 r / min for 1 min, and the supernatant was discarded.

[0123] 7) The precipitate was resuspended in 1×PBS solution, and the process was repeated 3 times. Finally, the precipitate was resuspended in 2.0 ml 1×PBS solution as a sample for laser confocal microscopy.

[0124] 8) A total of 30 single strains were co-incubated with the fluorescent peptide and numbered 1 to 30. Figure 4 shown.

[0125] In summary, FITC-AR-9 can visualize the combination of AR-9, an active peptide, and intestinal bacteria, which can not only effectively improve the sensitivity and accuracy of detection, but also shorten the detection cycle and reduce the difficulty of detection. In the future, it may become an important means to detect whether intestinal bacteria can utilize active peptides.

[0126] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A peptide probe for identifying specific intestinal bacteria, characterized in that: The peptide probe molecular formula is C 113 H 123 N 15 O 32 , the structural formula is as follows:

2. A method for preparing a peptide probe for identifying specific intestinal bacteria as claimed in claim 1, characterized in that: A fluorescent dye is linked to a specific part of the AR-9 peptide so that the peptide carries fluorescence; the method specifically comprises the following steps: (1) Alanine, valine, phenylalanine, proline and lysine are connected and eluted to form a short peptide chain of AVFPK, which is compound 1; the structural formula is as follows: (2) FITC reacts with the short peptide chain of AVFPK prepared in step (1) (compound 1) to form (Fmoc)OH-(FITC)-AVFRK-FITC, which is compound 2; the structural formula is as follows: (3) Serine, isoleucine and lysine react with the compound 2 prepared in step (2) to form (Fmoc)OH-(FITC)-AVFRK-(FITC)-SIK-(FITC), which is compound 3; the structural formula is as follows: (4) reacting valine, glycine, and arginine with the compound 3 prepared in step (3) to obtain (Fmoc)OH-(FITC)-AVFRK-(FITC)-SI-(FITC)-VGR is FITC-AR-9; the structural formula is as follows:

3. The method for preparing a peptide probe for identifying specific intestinal bacteria according to claim 2, characterized in that: The FITC dye is linked to the AR-9 peptide at a specific position using a lysine short chain as a medium, so that AR-9 carries the FITC group and exhibits green fluorescence under specific conditions (maximum emission wavelength 520-530nm).

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