CN117903241A - Polypeptide probe for detecting tumor lesion collagen and preparation method and application thereof - Google Patents
Polypeptide probe for detecting tumor lesion collagen and preparation method and application thereof Download PDFInfo
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- 108010017349 glycyl-prolyl-hydroxyproline Proteins 0.000 claims abstract description 165
- HVIBGVJOBJJPFB-OFQRNFBNSA-N Gly-Pro-Hyp Chemical compound NCC(=O)N1CCC[C@H]1C(=O)N1[C@H](C(O)=O)C(O)CC1 HVIBGVJOBJJPFB-OFQRNFBNSA-N 0.000 claims abstract description 78
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
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- Peptides Or Proteins (AREA)
Abstract
The invention belongs to the technical field of biological detection, and in particular relates to a polypeptide probe for detecting tumor lesion collagen, a preparation method and application thereof, wherein the sequence of the polypeptide is as follows: (Gly-Pro-Hyp) a-(Gly-Amp-Pro)b-(Gly-Pro-Hyp)c, wherein a is an integer between 3 and 10, b is an integer between 1 and a, c is an integer between a-1 and a+1; the polypeptide probe can keep a stable triple helix structure in physiological pH, and becomes a single-chain structure in weak acidic pH of tumor, thereby realizing specific recognition of tumor lesion collagen. The polypeptide probe has the advantages of simple preparation method, convenient detection, good specificity and strong binding capacity. Has wide application prospect in the fields of early diagnosis, curative effect evaluation, treatment and the like of tumors.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a polypeptide probe for detecting tumor lesion collagen, and a preparation method and application thereof.
Background
Collagen is an important biomarker in the development process of tumors, and is involved in regulating proliferation, differentiation, invasion and metastasis of tumor cells. The overexpression and crosslinking of fibroblast collagen results in hardening of the tissue while promoting proliferation, invasion and diffusion of surrounding tumor cells. Tumor cells secrete large amounts of matrix metalloproteinases during tumor invasion, leading to pathological remodeling of the tumor extracellular matrix to form tumor lesion collagen. The production of large amounts of tumor lesion collagen in the tumor microenvironment is a hallmark event of tumor metastasis and invasion. Therefore, the specific detection of the collagen of the tumor lesions has important significance for accurate diagnosis, pathological analysis, targeted treatment and prognosis evaluation of tumors.
At present, several short peptides and antibodies have been developed for collagen recognition and imaging. Collagen targeting peptides such as TKKTLRT, LRELHLNNN have been identified using domains of other proteins that bind collagen. Polypeptides KLWVLPK that specifically bind type IV collagen have been successfully screened using phage display technology. The above studies have focused mainly on the targeted recognition of natural collagen, and the above polypeptides often suffer from the disadvantages of low affinity and non-specific binding due to the specific structure of the three peptide chains of natural collagen closely packed.
The triple helix structure of the pathological collagen can be partially de-linked, and the exposed unfolded part provides a target point for identifying the pathological collagen. The polypeptides of (Gly-Pro-Hyp) n and (Gly-Hyp-Hyp) n sequences can be specifically combined with the unfolded collagen chain, and a new direction is pointed out for the design of a lesion collagen targeting probe. Single-chain (Gly-fPro-Hyp) n derivative probes containing unnatural amino acids facilitate in vivo imaging studies of diseased collagen. Meanwhile, a complete single-chain pathological change collagen targeting polypeptide probe (Gly-Hyp-Pro) n constructed based on a sequence reconstruction strategy realizes in-vitro imaging of tumor tissues. However, the single-stranded probe can be non-selectively combined with pathological collagen in any disease, lacks specificity on pathological collagen of tumor, and cannot meet the requirements of high specificity and accuracy of tumor diagnosis.
In view of the problems of the prior art, the inventors have found through experiments that a polypeptide containing a (Gly-Pro-Hyp) a-(Gly-Amp-Pro)b-(Gly-Pro-Hyp)c sequence can maintain a stable triple helix structure in physiological pH, but becomes a single chain structure in weak acidic pH of tumors. By utilizing the characteristic, the specific recognition of the tumor lesion collagen is realized, and the lesion collagen in other environments can not be combined. The probe can be effectively used for specific imaging of tumor lesion collagen in vivo and in vitro. Has wide application prospect in the fields of early diagnosis, curative effect evaluation, treatment and the like of tumors.
Disclosure of Invention
The primary aim of the invention is to provide a polypeptide for detecting tumor lesion collagen, wherein the sequence of the polypeptide is as follows: (Gly-Pro-Hyp) a-(Gly-Amp-Pro)b-(Gly-Pro-Hyp)c, wherein a is an integer of 3 to 10, b is an integer of 1 to a, and c is an integer of a-1 to a+1.
The second object of the present invention is to provide a polypeptide probe for detecting collagen of tumor lesions, wherein the polypeptide probe comprises the polypeptide and a signal molecule F, and the signal molecule F is connected with the N end or a side chain of a polypeptide sequence.
Preferably, the signal molecule F is linked to the polypeptide sequence N via the linker amino acid Ahx.
Preferably, the signal molecule F is one or more of fluorescein dye, coumarin dye, rhodamine dye, cyanine dye, BODIPY dye, tetraphenyl ethylene dye (TPE), hexaphenyl silyl dye (HPS), stilbene anthracene Dye (DSA) and rare earth ion complex.
Preferably, the signal molecule F is carboxyfluorescein.
The third object of the present invention is to provide a method for preparing the polypeptide probe, which comprises the following steps:
(1) 80-120mg of resin was charged to a reactor with a sieve plate, and 2-8mL of dichloromethane was used to swell the resin; removing the N-end Fmoc protecting group from the piperidine/N, N-dimethylformamide solution;
(2) Dissolving amino acid with the N end protected by Fmoc, HOBt and HBTU by DMF, activating at low temperature for 10-30min, dripping DIEA into the solution, uniformly mixing the solution, adding the solution into a reactor, and reacting for 1-6 hours;
(3) After the reaction is finished, the reaction solution is pumped out of the reactor, the resin is washed 2 to 4 times by DMF and DCM respectively, the resin is treated 3 times by 15 to 25 percent piperidine/DMF solution, and the resin is washed 3 times by DMF and DCM respectively after that until the protecting group is completely removed;
(4) Repeating steps (2) and (3) until a polypeptide of the target sequence is synthesized;
(5) Washing resin with DCM and methanol for 2-4 times, pumping resin, adding cutting fluid, and reacting for 1-6 hours;
(6) Precipitating the reaction solution by using glacial ethyl ether, centrifugally collecting the precipitate, washing the precipitate with the glacial ethyl ether for 3 times, and drying to obtain a crude polypeptide product;
(7) Purifying the crude polypeptide product by a C18 chromatographic column and a reversed-phase high performance liquid chromatography method, freeze-drying and preserving at-20 ℃;
(8) In the preparation of the functionalized polypeptide, after the step (4), the resin is dispersed in DMF, the signal molecules, HOBt and HBTU are weighed, dissolved by DMF, activated for 10-30min at low temperature, DIEA is added into the solution and fully mixed, and the mixed solution is added into the resin dispersion for reaction for 12-48 hours in a dark place. The polypeptides were purified in steps (6) and (7) as in step (5) for the resin.
Preferably, the cutting fluid in the step (5) is TFA: TIS: water=95:2.5:2.5.
The fourth object of the invention is to provide the application of the polypeptide probe in preparing a detection reagent for detecting tumor, and/or a kit and/or an imaging reagent.
A fifth object of the present invention is to provide a detection reagent comprising the polypeptide probe.
The sixth object of the present invention is to provide a detection kit containing the polypeptide probe.
It is a seventh object of the present invention to provide an in vitro imaging agent comprising said polypeptide probe.
An eighth object of the present invention is to provide a living body imaging agent containing the polypeptide probe.
The beneficial effects of the invention are as follows: the invention provides a polypeptide probe for detecting tumor lesion collagen, which can keep a stable triple helix structure in physiological pH, and change into a single-chain structure in weak acidic pH of tumor, thereby realizing specific recognition of the tumor lesion collagen. The polypeptide probe has the advantages of simple preparation method, convenient detection, good specificity and strong binding capacity, and has wide application prospect in the fields of early diagnosis, curative effect evaluation, treatment and the like of tumors.
Drawings
The invention is further described below with reference to the drawings and examples.
FIG. 1 is a representation of the thermal stability of a probe according to the present invention: a: the probe was heat-set at pH 7.4 (blue) and pH 6.5 (red), respectively; b: the probe was subjected to a thermal temperature difference between pH 7.4 and pH 6.5.
FIG. 2 shows the unfolding ratio of the probe according to the present invention
FIG. 3 is a representation of probe responsiveness of the present invention
FIG. 4 is a specific cell imaging injection of a probe of the invention: p3-8 Co-imaging of FITC-labeled type I collagen-cultured 4T1-3D cells
FIG. 5 is a 3D tumor model imaging of the probe of the present invention: probes FAM-P3-8 imaged HeLa (a-c), huH7 (D-f), AGS (g-i) 3D cells.
FIG. 6 is an in vivo image of a probe of the present invention
Detailed Description
The following embodiments are further described in detail to illustrate the present invention, but the scope of the present invention is not limited to the following embodiments.
The polypeptide probes P1-6, P1-7, P1-8, P2-7, P2-8, P2-9, P3-7, P3-8 and P3-9 prepared in the following examples can specifically bind to tumor lesion collagen, and imaging studies are specifically carried out by taking P2-8 and P3-8 as examples, but the polypeptide probes of the invention are not limited to P2-8 and P3-8, and other probes also have good staining ability and can specifically bind to tumor lesion collagen.
Example 1 preparation of polypeptide probes P1-6
1. Design of polypeptide probes
The polypeptide probe sequence designed at this time is as follows: FAM-Ahx- (Gly-Pro-Hyp) 3-(Gly-Amp-Pro)1-(Gly-Pro-Hyp)3, wherein FAM is carboxyfluorescein.
2. Solid phase Synthesis of polypeptide sequences (Gly-Pro-Hyp) 3-(Gly-Amp-Pro)1-(Gly-Pro-Hyp)3 and Ahx- (Gly-Pro-Hyp) 3-(Gly-Amp-Pro)1-(Gly-Pro-Hyp)3
(1) 100Mg Rink ammonia resin was charged to a reactor with a sieve plate, and 5mL of dichloromethane was used to swell the resin;
(2) Removing N-end Fmoc protecting groups by 20% piperidine/N, N-Dimethylformamide (DMF) solution, and detecting that the protecting groups are completely removed by a color reaction;
(3) The amino acid (4 eq) with the N-terminal protected by Fmoc, HOBt (4 eq) and HBTU (4 eq) are dissolved by DMF, activated for 20min at low temperature, DIEA (6 eq) is added dropwise to the solution, and the solution is mixed and then added into a reactor for reaction for 3h.
(4) After the reaction was completed, the reaction mixture was withdrawn from the reactor, and the resin was washed 3 times with 10mL of DMF and 2 times with 10mL of DCM, respectively. The reaction was followed by 3 treatments of the resin with 20% piperidine/DMF solution for 5min, 5min and 15min, respectively. The resin is washed 3 times by 10mL of DMF and 2 times by 10mL of DCM respectively, and the removal of the protecting group is detected to be complete by the color reaction;
(5) Steps (3) and (4) were repeated until polypeptides (Gly-Pro-Hyp) 3-(Gly-Amp-Pro)1-(Gly-Pro-Hyp)3 and Ahx-(Gly-Pro-Hyp)3-(Gly-Amp-Pro)1-(Gly-Pro-Hyp)3.(Gly-Pro-Hyp)3-(Gly-Amp-Pro)1-(Gly-Pro-Hyp)3 of the synthetic target sequence were capped by treatment with 25% acetic anhydride for 30 minutes for the preparation of unmodified P1-6.
3. Signal molecule modified polypeptide sequence
(1) Weighing carboxyl fluorescein (10 eq), HOBt (10 eq) and HBTU (10 eq), dissolving by DMF, activating at low temperature for 20min, dripping DIEA (16 eq) into the solution, adding the mixed solution into synthetic polypeptide Ahx- (Gly-Pro-Hyp) 3-(Gly-Amp-Pro)1-(Gly-Pro-Hyp)3 resin, and reacting for 24h in a dark place;
(2) The resin was washed 3 times with DCM and methanol, respectively, the resin was drained and cleavage liquid (TFA: TIS: water=95:2.5:2.5) was added and reacted for 2.5h;
(3) The reaction solution was added to glacial ethyl ether, and the polypeptide was precipitated. And centrifuging to collect precipitate, dissolving the precipitate with a small amount of TFA, adding excessive glacial ethyl ether for reprecipitation, centrifuging to collect precipitate, washing the precipitate with the glacial ethyl ether for 2 times, and then air-drying to obtain a crude polypeptide product FAM-Ahx- (Gly-Pro-Hyp) 3-(Gly-Amp-Pro)1-(Gly-Pro-Hyp)3 -NH2. Purifying the crude polypeptide product by a C18 chromatographic column and a reversed-phase high performance liquid chromatography method to obtain the polypeptide probes P1-6.
Example 2 preparation of polypeptide probes P1-7
1. Design of polypeptide probes
The polypeptide probe sequence designed at this time is as follows: FAM-Ahx- (Gly-Pro-Hyp) 4-(Gly-Amp-Pro)1-(Gly-Pro-Hyp)3, wherein FAM is carboxyfluorescein.
2. Solid phase Synthesis of polypeptide sequences (Gly-Pro-Hyp) 4-(Gly-Amp-Pro)1-(Gly-Pro-Hyp)3 and Ahx- (Gly-Pro-Hyp) 4-(Gly-Amp-Pro)1-(Gly-Pro-Hyp)3
(1) 100Mg Rink ammonia resin was charged to a reactor with a sieve plate, and 5mL of dichloromethane was used to swell the resin;
(2) Removing N-end Fmoc protecting groups by 20% piperidine/N, N-Dimethylformamide (DMF) solution, and detecting that the protecting groups are completely removed by a color reaction;
(3) The amino acid (4 eq) with the N-terminal protected by Fmoc, HOBt (4 eq) and HBTU (4 eq) are dissolved by DMF, activated for 20min at low temperature, DIEA (6 eq) is added dropwise to the solution, and the solution is mixed and then added into a reactor for reaction for 3h.
(4) After the reaction was completed, the reaction mixture was withdrawn from the reactor, and the resin was washed 3 times with 10mL of DMF and 2 times with 10mL of DCM, respectively. The reaction was followed by 3 treatments of the resin with 20% piperidine/DMF solution for 5min, 5min and 15min, respectively. The resin is washed 3 times by 10mL of DMF and 2 times by 10mL of DCM respectively, and the removal of the protecting group is detected to be complete by the color reaction;
(5) Steps (3) and (4) were repeated until polypeptides (Gly-Pro-Hyp) 4-(Gly-Amp-Pro)1-(Gly-Pro-Hyp)3 and Ahx-(Gly-Pro-Hyp)4-(Gly-Amp-Pro)1-(Gly-Pro-Hyp)3.(Gly-Pro-Hyp)4-(Gly-Amp-Pro)1-(Gly-Pro-Hyp)3 of the synthetic target sequence were capped by treatment with 25% acetic anhydride for 30 minutes for the preparation of unmodified P1-7.
3. Signal molecule modified polypeptide sequence
(1) Weighing carboxyl fluorescein (10 eq), HOBt (10 eq) and HBTU (10 eq), dissolving by DMF, activating at low temperature for 20min, dripping DIEA (16 eq) into the solution, adding the mixed solution into synthetic polypeptide Ahx- (Gly-Pro-Hyp) 4-(Gly-Amp-Pro)1-(Gly-Pro-Hyp)3 resin, and reacting for 24h in a dark place;
(2) The resin was washed 3 times with DCM and methanol, respectively, the resin was drained and cleavage liquid (TFA: TIS: water=95:2.5:2.5) was added and reacted for 2.5h;
(3) The reaction solution was added to glacial ethyl ether, and the polypeptide was precipitated. And centrifuging to collect precipitate, dissolving the precipitate with a small amount of TFA, adding excessive glacial diethyl ether for reprecipitation, centrifuging to collect precipitate, washing the precipitate with the glacial diethyl ether for 2 times, and then air-drying to obtain a crude polypeptide product FAM-Ahx- (Gly-Pro-Hyp) 4-(Gly-Amp-Pro)1-(Gly-Pro-Hyp)3-NH2. Purifying the crude polypeptide product by a C18 chromatographic column and a reversed-phase high performance liquid chromatography method to obtain the polypeptide probe P1-7.
Example 3 preparation of polypeptide probes P1-8
1. Design of polypeptide probes
The polypeptide probe sequence designed at this time is as follows: FAM-Ahx- (Gly-Pro-Hyp) 4-(Gly-Amp-Pro)1-(Gly-Pro-Hyp)4, wherein FAM is carboxyfluorescein.
2. Solid phase Synthesis of polypeptide sequences (Gly-Pro-Hyp) 4-(Gly-Amp-Pro)1-(Gly-Pro-Hyp)4 and Ahx- (Gly-Pro-Hyp) 4-(Gly-Amp-Pro)1-(Gly-Pro-Hyp)4
(1) 100Mg Rink ammonia resin was charged to a reactor with a sieve plate, and 5mL of dichloromethane was used to swell the resin;
(2) Removing N-end Fmoc protecting groups by 20% piperidine/N, N-Dimethylformamide (DMF) solution, and detecting that the protecting groups are completely removed by a color reaction;
(3) The amino acid (4 eq) with the N-terminal protected by Fmoc, HOBt (4 eq) and HBTU (4 eq) are dissolved by DMF, activated for 20min at low temperature, DIEA (6 eq) is added dropwise to the solution, and the solution is mixed and then added into a reactor for reaction for 3h.
(4) After the reaction was completed, the reaction mixture was withdrawn from the reactor, and the resin was washed 3 times with 10mL of DMF and 2 times with 10mL of DCM, respectively. The reaction was followed by 3 treatments of the resin with 20% piperidine/DMF solution for 5min, 5min and 15min, respectively. The resin is washed 3 times by 10mL of DMF and 2 times by 10mL of DCM respectively, and the removal of the protecting group is detected to be complete by the color reaction;
(5) Steps (3) and (4) were repeated until polypeptides (Gly-Pro-Hyp) 4-(Gly-Amp-Pro)1-(Gly-Pro-Hyp)4 and Ahx-(Gly-Pro-Hyp)4-(Gly-Amp-Pro)1-(Gly-Pro-Hyp)4.(Gly-Pro-Hyp)4-(Gly-Amp-Pro)1-(Gly-Pro-Hyp)4 of the synthetic target sequence were capped by treatment with 25% acetic anhydride for 30 minutes for the preparation of unmodified P1-8.
3. Signal molecule modified polypeptide sequence
(1) Weighing carboxyl fluorescein (10 eq), HOBt (10 eq) and HBTU (10 eq), dissolving by DMF, activating at low temperature for 20min, dripping DIEA (16 eq) into the solution, adding the mixed solution into synthetic polypeptide Ahx- (Gly-Pro-Hyp) 4-(Gly-Amp-Pro)1-(Gly-Pro-Hyp)4 resin, and reacting for 24h in a dark place;
(2) The resin was washed 3 times with DCM and methanol, respectively, the resin was drained and cleavage liquid (TFA: TIS: water=95:2.5:2.5) was added and reacted for 2.5h;
(3) The reaction solution was added to glacial ethyl ether, and the polypeptide was precipitated. And centrifuging to collect precipitate, dissolving the precipitate with a small amount of TFA, adding excessive glacial diethyl ether for reprecipitation, centrifuging to collect precipitate, washing the precipitate with the glacial diethyl ether for 2 times, and then air-drying to obtain a crude polypeptide product FAM-Ahx- (Gly-Pro-Hyp) 4-(Gly-Amp-Pro)1-(Gly-Pro-Hyp)4-NH2. Purifying the crude product of the polypeptide by a C18 chromatographic column and a reversed-phase high performance liquid chromatography method to obtain the polypeptide probe P1-8.
Example 4 preparation of polypeptide Probe P2-7
1. Design of polypeptide probes
The polypeptide probe sequence designed at this time is as follows: FAM-Ahx- (Gly-Pro-Hyp) 4-(Gly-Amp-Pro)2-(Gly-Pro-Hyp)3, wherein FAM is carboxyfluorescein.
2. Solid phase Synthesis of polypeptide sequences (Gly-Pro-Hyp) 4-(Gly-Amp-Pro)2-(Gly-Pro-Hyp)3 and Ahx- (Gly-Pro-Hyp) 4-(Gly-Amp-Pro)2-(Gly-Pro-Hyp)3
(1) 100Mg Rink ammonia resin was charged to a reactor with a sieve plate, and 5mL of dichloromethane was used to swell the resin;
(2) Removing N-end Fmoc protecting groups by 20% piperidine/N, N-Dimethylformamide (DMF) solution, and detecting that the protecting groups are completely removed by a color reaction;
(3) The amino acid (4 eq) with the N-terminal protected by Fmoc, HOBt (4 eq) and HBTU (4 eq) are dissolved by DMF, activated for 20min at low temperature, DIEA (6 eq) is added dropwise to the solution, and the solution is mixed and then added into a reactor for reaction for 3h.
(4) After the reaction was completed, the reaction mixture was withdrawn from the reactor, and the resin was washed 3 times with 10mL of DMF and 2 times with 10mL of DCM, respectively. The reaction was followed by 3 treatments of the resin with 20% piperidine/DMF solution for 5min, 5min and 15min, respectively. The resin is washed 3 times by 10mL of DMF and 2 times by 10mL of DCM respectively, and the removal of the protecting group is detected to be complete by the color reaction;
(5) Steps (3) and (4) were repeated until polypeptides (Gly-Pro-Hyp) 4-(Gly-Amp-Pro)2-(Gly-Pro-Hyp)3 and Ahx-(Gly-Pro-Hyp)4-(Gly-Amp-Pro)2-(Gly-Pro-Hyp)3.(Gly-Pro-Hyp)4-(Gly-Amp-Pro)2-(Gly-Pro-Hyp)3 of the synthetic target sequence were capped by treatment with 25% acetic anhydride for 30 minutes for the preparation of unmodified P2-7.
3. Signal molecule modified polypeptide sequence
(1) Weighing carboxyl fluorescein (10 eq), HOBt (10 eq) and HBTU (10 eq), dissolving by DMF, activating at low temperature for 20min, dripping DIEA (16 eq) into the solution, adding the mixed solution into synthetic polypeptide Ahx- (Gly-Pro-Hyp) 4-(Gly-Amp-Pro)2-(Gly-Pro-Hyp)3 resin, and reacting for 24h in a dark place;
(2) The resin was washed 3 times with DCM and methanol, respectively, the resin was drained and cleavage liquid (TFA: TIS: water=95:2.5:2.5) was added and reacted for 2.5h;
(3) The reaction solution was added to glacial ethyl ether, and the polypeptide was precipitated. And centrifuging to collect precipitate, dissolving the precipitate with a small amount of TFA, adding excessive glacial diethyl ether for reprecipitation, centrifuging to collect precipitate, washing the precipitate with the glacial diethyl ether for 2 times, and then air-drying to obtain a crude polypeptide product FAM-Ahx- (Gly-Pro-Hyp) 4-(Gly-Amp-Pro)2-(Gly-Pro-Hyp)3-NH2. Purifying the crude polypeptide product by a C18 chromatographic column and a reversed-phase high performance liquid chromatography method to obtain the polypeptide probe P2-7.
Example 5 preparation of polypeptide Probe P2-8
1. Design of polypeptide probes
The polypeptide probe sequence designed at this time is as follows: FAM-Ahx- (Gly-Pro-Hyp) 4-(Gly-Amp-Pro)2-(Gly-Pro-Hyp)4, wherein FAM is carboxyfluorescein.
2. Solid phase Synthesis of polypeptide sequences (Gly-Pro-Hyp) 4-(Gly-Amp-Pro)2-(Gly-Pro-Hyp)4 and Ahx- (Gly-Pro-Hyp) 4-(Gly-Amp-Pro)2-(Gly-Pro-Hyp)4
(1) 100Mg Rink ammonia resin was charged to a reactor with a sieve plate, and 5mL of dichloromethane was used to swell the resin;
(2) Removing N-end Fmoc protecting groups by 20% piperidine/N, N-Dimethylformamide (DMF) solution, and detecting that the protecting groups are completely removed by a color reaction;
(3) The amino acid (4 eq) with the N-terminal protected by Fmoc, HOBt (4 eq) and HBTU (4 eq) are dissolved by DMF, activated for 20min at low temperature, DIEA (6 eq) is added dropwise to the solution, and the solution is mixed and then added into a reactor for reaction for 3h.
(4) After the reaction was completed, the reaction mixture was withdrawn from the reactor, and the resin was washed 3 times with 10mL of DMF and 2 times with 10mL of DCM, respectively. The reaction was followed by 3 treatments of the resin with 20% piperidine/DMF solution for 5min, 5min and 15min, respectively. The resin is washed 3 times by 10mL of DMF and 2 times by 10mL of DCM respectively, and the removal of the protecting group is detected to be complete by the color reaction;
(5) Steps (3) and (4) were repeated until polypeptides (Gly-Pro-Hyp) 4-(Gly-Amp-Pro)2-(Gly-Pro-Hyp)4 and Ahx-(Gly-Pro-Hyp)4-(Gly-Amp-Pro)2-(Gly-Pro-Hyp)4.(Gly-Pro-Hyp)4-(Gly-Amp-Pro)2-(Gly-Pro-Hyp)4 of the synthetic target sequence were capped by treatment with 25% acetic anhydride for 30 minutes for the preparation of unmodified P2-8.
3. Signal molecule modified polypeptide sequence
(1) Weighing carboxyl fluorescein (10 eq), HOBt (10 eq) and HBTU (10 eq), dissolving by DMF, activating at low temperature for 20min, dripping DIEA (16 eq) into the solution, adding the mixed solution into synthetic polypeptide Ahx- (Gly-Pro-Hyp) 4-(Gly-Amp-Pro)2-(Gly-Pro-Hyp)4 resin, and reacting for 24h in a dark place;
(2) The resin was washed 3 times with DCM and methanol, respectively, the resin was drained and cleavage liquid (TFA: TIS: water=95:2.5:2.5) was added and reacted for 2.5h;
(3) The reaction solution was added to glacial ethyl ether, and the polypeptide was precipitated. And centrifuging to collect precipitate, dissolving the precipitate with a small amount of TFA, adding excessive glacial diethyl ether for reprecipitation, centrifuging to collect precipitate, washing the precipitate with the glacial diethyl ether for 2 times, and then air-drying to obtain a crude polypeptide product FAM-Ahx- (Gly-Pro-Hyp) 4-(Gly-Amp-Pro)2-(Gly-Pro-Hyp)4-NH2. Purifying the crude product of the polypeptide by a C18 chromatographic column and a reversed-phase high performance liquid chromatography method to obtain the polypeptide probe P2-8.
Example 6 preparation of polypeptide Probe P2-9
1. Design of polypeptide probes
The polypeptide probe sequence designed at this time is as follows: FAM-Ahx- (Gly-Pro-Hyp) 5-(Gly-Amp-Pro)2-(Gly-Pro-Hyp)4, wherein FAM is carboxyfluorescein.
2. Solid phase Synthesis of polypeptide sequences (Gly-Pro-Hyp) 5-(Gly-Amp-Pro)2-(Gly-Pro-Hyp)4 and Ahx- (Gly-Pro-Hyp) 5-(Gly-Amp-Pro)2-(Gly-Pro-Hyp)4
(1) 100Mg Rink ammonia resin was charged to a reactor with a sieve plate, and 5mL of dichloromethane was used to swell the resin;
(2) Removing N-end Fmoc protecting groups by 20% piperidine/N, N-Dimethylformamide (DMF) solution, and detecting that the protecting groups are completely removed by a color reaction;
(3) The amino acid (4 eq) with the N-terminal protected by Fmoc, HOBt (4 eq) and HBTU (4 eq) are dissolved by DMF, activated for 20min at low temperature, DIEA (6 eq) is added dropwise to the solution, and the solution is mixed and then added into a reactor for reaction for 3h.
(4) After the reaction was completed, the reaction mixture was withdrawn from the reactor, and the resin was washed 3 times with 10mL of DMF and 2 times with 10mL of DCM, respectively. The reaction was followed by 3 treatments of the resin with 20% piperidine/DMF solution for 5min, 5min and 15min, respectively. The resin is washed 3 times by 10mL of DMF and 2 times by 10mL of DCM respectively, and the removal of the protecting group is detected to be complete by the color reaction;
(5) Steps (3) and (4) were repeated until polypeptides (Gly-Pro-Hyp) 5-(Gly-Amp-Pro)2-(Gly-Pro-Hyp)4 and Ahx-(Gly-Pro-Hyp)5-(Gly-Amp-Pro)2-(Gly-Pro-Hyp)4.(Gly-Pro-Hyp)5-(Gly-Amp-Pro)2-(Gly-Pro-Hyp)4 of the synthetic target sequence were capped by treatment with 25% acetic anhydride for 30 minutes for the preparation of unmodified P2-9.
3. Signal molecule modified polypeptide sequence
(1) Weighing carboxyl fluorescein (10 eq), HOBt (10 eq) and HBTU (10 eq), dissolving by DMF, activating at low temperature for 20min, dripping DIEA (16 eq) into the solution, adding the mixed solution into synthetic polypeptide Ahx- (Gly-Pro-Hyp) 5-(Gly-Amp-Pro)2-(Gly-Pro-Hyp)4 resin, and reacting for 24h in a dark place;
(2) The resin was washed 3 times with DCM and methanol, respectively, the resin was drained and cleavage liquid (TFA: TIS: water=95:2.5:2.5) was added and reacted for 2.5h;
(3) The reaction solution was added to glacial ethyl ether, and the polypeptide was precipitated. And centrifuging to collect precipitate, dissolving the precipitate with a small amount of TFA, adding excessive glacial diethyl ether for reprecipitation, centrifuging to collect precipitate, washing the precipitate with the glacial diethyl ether for 2 times, and then air-drying to obtain a crude polypeptide product FAM-Ahx- (Gly-Pro-Hyp) 5-(Gly-Amp-Pro)2-(Gly-Pro-Hyp)4-NH2. Purifying the crude polypeptide product by a C18 chromatographic column and a reversed-phase high performance liquid chromatography method to obtain the polypeptide probe P2-9.
Example 7 preparation of polypeptide Probe P3-7
1. Design of polypeptide probes
The polypeptide probe sequence designed at this time is as follows: FAM-Ahx- (Gly-Pro-Hyp) 4-(Gly-Amp-Pro)3-(Gly-Pro-Hyp)3, wherein FAM is carboxyfluorescein.
2. Solid phase Synthesis of polypeptide sequences (Gly-Pro-Hyp) 4-(Gly-Amp-Pro)3-(Gly-Pro-Hyp)3 and Ahx- (Gly-Pro-Hyp) 4-(Gly-Amp-Pro)3-(Gly-Pro-Hyp)3
(1) 100Mg Rink ammonia resin was charged to a reactor with a sieve plate, and 5mL of dichloromethane was used to swell the resin;
(2) Removing N-end Fmoc protecting groups by 20% piperidine/N, N-Dimethylformamide (DMF) solution, and detecting that the protecting groups are completely removed by a color reaction;
(3) The amino acid (4 eq) with the N-terminal protected by Fmoc, HOBt (4 eq) and HBTU (4 eq) are dissolved by DMF, activated for 20min at low temperature, DIEA (6 eq) is added dropwise to the solution, and the solution is mixed and then added into a reactor for reaction for 3h.
(4) After the reaction was completed, the reaction mixture was withdrawn from the reactor, and the resin was washed 3 times with 10mL of DMF and 2 times with 10mL of DCM, respectively. The reaction was followed by 3 treatments of the resin with 20% piperidine/DMF solution for 5min, 5min and 15min, respectively. The resin is washed 3 times by 10mL of DMF and 2 times by 10mL of DCM respectively, and the removal of the protecting group is detected to be complete by the color reaction;
(5) Steps (3) and (4) were repeated until polypeptides (Gly-Pro-Hyp) 4-(Gly-Amp-Pro)3-(Gly-Pro-Hyp)3 and Ahx-(Gly-Pro-Hyp)4-(Gly-Amp-Pro)3-(Gly-Pro-Hyp)3.(Gly-Pro-Hyp)4-(Gly-Amp-Pro)3-(Gly-Pro-Hyp)3 of the synthetic target sequence were capped by treatment with 25% acetic anhydride for 30 minutes for the preparation of unmodified P3-7.
3. Signal molecule modified polypeptide sequence
(1) Weighing carboxyl fluorescein (10 eq), HOBt (10 eq) and HBTU (10 eq), dissolving by DMF, activating at low temperature for 20min, dripping DIEA (16 eq) into the solution, adding the mixed solution into synthetic polypeptide Ahx- (Gly-Pro-Hyp) 4-(Gly-Amp-Pro)3-(Gly-Pro-Hyp)3 resin, and reacting for 24h in a dark place;
(2) The resin was washed 3 times with DCM and methanol, respectively, the resin was drained and cleavage liquid (TFA: TIS: water=95:2.5:2.5) was added and reacted for 2.5h;
(3) The reaction solution was added to glacial ethyl ether, and the polypeptide was precipitated. And centrifuging to collect precipitate, dissolving the precipitate with a small amount of TFA, adding excessive glacial diethyl ether for reprecipitation, centrifuging to collect precipitate, washing the precipitate with the glacial diethyl ether for 2 times, and then air-drying to obtain a crude polypeptide product FAM-Ahx- (Gly-Pro-Hyp) 4-(Gly-Amp-Pro)3-(Gly-Pro-Hyp)3-NH2. Purifying the crude product of the polypeptide by a C18 chromatographic column and a reversed-phase high performance liquid chromatography method to obtain the polypeptide probe P3-7.
Example 8 preparation of polypeptide Probe P3-8
1. Design of polypeptide probes
The polypeptide probe sequence designed at this time is as follows: X-Ahx- (Gly-Pro-Hyp) 4-(Gly-Amp-Pro)3-(Gly-Pro-Hyp)4, wherein X is carboxyfluorescein or ROX or Cy7.5 or dansyl chloride (Dy).
2. Solid phase Synthesis of polypeptide sequences (Gly-Pro-Hyp) 4-(Gly-Amp-Pro)3-(Gly-Pro-Hyp)4 and Ahx- (Gly-Pro-Hyp) 4-(Gly-Amp-Pro)3-(Gly-Pro-Hyp)4
(1) 100Mg Rink ammonia resin was charged to a reactor with a sieve plate, and 5mL of dichloromethane was used to swell the resin;
(2) Removing N-end Fmoc protecting groups by 20% piperidine/N, N-Dimethylformamide (DMF) solution, and detecting that the protecting groups are completely removed by a color reaction;
(3) The amino acid (4 eq) with the N-terminal protected by Fmoc, HOBt (4 eq) and HBTU (4 eq) are dissolved by DMF, activated for 20min at low temperature, DIEA (6 eq) is added dropwise to the solution, and the solution is mixed and then added into a reactor for reaction for 3h.
(4) After the reaction was completed, the reaction mixture was withdrawn from the reactor, and the resin was washed 3 times with 10mL of DMF and 2 times with 10mL of DCM, respectively. The reaction was followed by 3 treatments of the resin with 20% piperidine/DMF solution for 5min, 5min and 15min, respectively. The resin is washed 3 times by 10mL of DMF and 2 times by 10mL of DCM respectively, and the removal of the protecting group is detected to be complete by the color reaction;
(5) Steps (3) and (4) were repeated until polypeptides (Gly-Pro-Hyp) 4-(Gly-Amp-Pro)3-(Gly-Pro-Hyp)4 and Ahx-(Gly-Pro-Hyp)4-(Gly-Amp-Pro)3-(Gly-Pro-Hyp)4.(Gly-Pro-Hyp)4-(Gly-Amp-Pro)3-(Gly-Pro-Hyp)4 of the synthetic target sequence were capped by treatment with 25% acetic anhydride for 30 minutes for the preparation of unmodified P3-8.
3. Signal molecule modified polypeptide sequence
(1) Weighing carboxyl fluorescein (10 eq) or ROX (10 eq) or Cy7.5 (10 eq), HOBt (10 eq) and HBTU (10 eq), dissolving by DMF, activating at low temperature for 20min, or weighing Dy (10 eq) dissolving by DMF, dripping DIEA (16 eq) into the solution, adding the mixture into synthetic polypeptide Ahx- (Gly-Pro-Hyp) 4-(Gly-Amp-Pro)3-(Gly-Pro-Hyp)4 resin, and reacting for 24h in the dark;
(2) The resin was washed 3 times with DCM and methanol, respectively, the resin was drained and cleavage liquid (TFA: TIS: water=95:2.5:2.5) was added and reacted for 2.5h;
(3) The reaction solution was added to glacial ethyl ether, and the polypeptide was precipitated. The precipitate was collected by centrifugation, dissolved with a small amount of TFA, reprecipitated with an excess of glacial ethyl ether and collected by centrifugation, washed with glacial ethyl ether for 2 times and air-dried to obtain crude polypeptide products FAM-Ahx- (Gly-Pro-Hyp) 4-(Gly-Amp-Pro)3-(Gly-Pro-Hyp)4-NH2 or ROX-Ahx- (Gly-Pro-Hyp) 4-(Gly-Amp-Pro)3-(Gly-Pro-Hyp)4-NH2 or Cy7.5-Ahx- (Gly-Pro-Hyp) 4-(Gly-Amp-Pro)3-(Gly-Pro-Hyp)4-NH2 or Dy-Ahx- (Gly-Pro-Hyp) 4-(Gly-Amp-Pro)3-(Gly-Pro-Hyp)4-NH2. Purifying the crude product of the polypeptide by a C18 chromatographic column and a reversed-phase high performance liquid chromatography method to obtain the polypeptide probe P3-8.
Example 9 preparation of polypeptide Probe P3-9
1. Design of polypeptide probes
The polypeptide probe sequence designed at this time is as follows: FAM-Ahx- (Gly-Pro-Hyp) 5-(Gly-Amp-Pro)3-(Gly-Pro-Hyp)4, wherein FAM is carboxyfluorescein.
2. Solid phase Synthesis of polypeptide sequences (Gly-Pro-Hyp) 5-(Gly-Amp-Pro)3-(Gly-Pro-Hyp)4 and Ahx- (Gly-Pro-Hyp) 5-(Gly-Amp-Pro)3-(Gly-Pro-Hyp)4
(1) 100Mg Rink ammonia resin was charged to a reactor with a sieve plate, and 5mL of dichloromethane was used to swell the resin;
(2) Removing N-end Fmoc protecting groups by 20% piperidine/N, N-Dimethylformamide (DMF) solution, and detecting that the protecting groups are completely removed by a color reaction;
(3) The amino acid (4 eq) with the N-terminal protected by Fmoc, HOBt (4 eq) and HBTU (4 eq) are dissolved by DMF, activated for 20min at low temperature, DIEA (6 eq) is added dropwise to the solution, and the solution is mixed and then added into a reactor for reaction for 3h.
(4) After the reaction was completed, the reaction mixture was withdrawn from the reactor, and the resin was washed 3 times with 10mL of DMF and 2 times with 10mL of DCM, respectively. The reaction was followed by 3 treatments of the resin with 20% piperidine/DMF solution for 5min, 5min and 15min, respectively. The resin is washed 3 times by 10mL of DMF and 2 times by 10mL of DCM respectively, and the removal of the protecting group is detected to be complete by the color reaction;
(5) Steps (3) and (4) were repeated until polypeptides (Gly-Pro-Hyp) 5-(Gly-Amp-Pro)3-(Gly-Pro-Hyp)4 and Ahx-(Gly-Pro-Hyp)5-(Gly-Amp-Pro)3-(Gly-Pro-Hyp)4.(Gly-Pro-Hyp)5-(Gly-Amp-Pro)3-(Gly-Pro-Hyp)4 of the synthetic target sequence were capped by treatment with 25% acetic anhydride for 30 minutes for the preparation of unmodified P3-9.
3. Signal molecule modified polypeptide sequence
(1) Weighing carboxyl fluorescein (10 eq), HOBt (10 eq) and HBTU (10 eq), dissolving by DMF, activating at low temperature for 20min, dripping DIEA (16 eq) into the solution, adding the mixed solution into synthetic polypeptide Ahx- (Gly-Pro-Hyp) 5-(Gly-Amp-Pro)3-(Gly-Pro-Hyp)4 resin, and reacting for 24h in a dark place;
(2) The resin was washed 3 times with DCM and methanol, respectively, the resin was drained and cleavage liquid (TFA: TIS: water=95:2.5:2.5) was added and reacted for 2.5h;
(3) The reaction solution was added to glacial ethyl ether, and the polypeptide was precipitated. And centrifuging to collect precipitate, dissolving the precipitate with a small amount of TFA, adding excessive glacial diethyl ether for reprecipitation, centrifuging to collect precipitate, washing the precipitate with the glacial diethyl ether for 2 times, and then air-drying to obtain a crude polypeptide product FAM-Ahx- (Gly-Pro-Hyp) 5-(Gly-Amp-Pro)3-(Gly-Pro-Hyp)4-NH2. Purifying the crude product of the polypeptide by a C18 chromatographic column and a reversed-phase high performance liquid chromatography method to obtain the polypeptide probe P3-9.
Comparative example 1 preparation of polypeptide probes P0-8
1. Design of polypeptide probes
The polypeptide probe sequence designed at this time is as follows: X-Ahx- (Gly-Pro-Hyp) 8, wherein X is carboxyfluorescein or dansyl chloride.
2. Solid phase synthesis of polypeptide sequence Ahx- (Gly-Pro-Hyp) 8
(1) 100Mg Rink ammonia resin was charged to a reactor with a sieve plate, and 5mL of dichloromethane was used to swell the resin;
(2) Removing N-end Fmoc protecting groups by 20% piperidine/N, N-Dimethylformamide (DMF) solution, and detecting that the protecting groups are completely removed by a color reaction;
(3) The amino acid (4 eq) with the N-terminal protected by Fmoc, HOBt (4 eq) and HBTU (4 eq) are dissolved by DMF, activated for 20min at low temperature, DIEA (6 eq) is added dropwise to the solution, and the solution is mixed and then added into a reactor for reaction for 3h.
(4) After the reaction was completed, the reaction mixture was withdrawn from the reactor, and the resin was washed 3 times with 10mL of DMF and 2 times with 10mL of DCM, respectively. The reaction was followed by 3 treatments of the resin with 20% piperidine/DMF solution for 5min, 5min and 15min, respectively. The resin is washed 3 times by 10mL of DMF and 2 times by 10mL of DCM respectively, and the removal of the protecting group is detected to be complete by the color reaction;
(5) Repeating the steps (3) and (4) until the polypeptide Ahx- (Gly-Pro-Hyp) 8 of the target sequence is synthesized.
3. Signal molecule modified polypeptide sequence
(1) Weighing carboxyl fluorescein (10 eq), HOBt (10 eq) and HBTU (10 eq), dissolving by DMF, activating at low temperature for 20min, or weighing Dy (10 eq) dissolving by DMF, dripping DIEA (16 eq) into the solution, adding the mixed solution into synthetic polypeptide Ahx- (Gly-Pro-Hyp) 8 resin, and reacting for 24h in the dark;
(2) The resin was washed 3 times with DCM and methanol, respectively, the resin was drained and cleavage liquid (TFA: TIS: water=95:2.5:2.5) was added and reacted for 2.5h;
(3) The reaction solution was added to glacial ethyl ether, and the polypeptide was precipitated. And centrifuging to collect precipitate, dissolving the precipitate with a small amount of TFA, adding excessive glacial diethyl ether for reprecipitation, centrifuging to collect precipitate, washing the precipitate with the glacial diethyl ether for 2 times, and air-drying to obtain a polypeptide crude product FAM-Ahx- (Gly-Pro-Hyp) 8-NH2 or Dy-Ahx- (Gly-Pro-Hyp) 8-NH2. Purifying the crude product of the polypeptide by a C18 chromatographic column and a reversed-phase high performance liquid chromatography method to obtain the polypeptide probe P0-8.
Example 10 Performance measurement of polypeptide Probe
1. Stability detection
(1) Round two chromatography: the unmodified polypeptide was prepared as a 1mg/mL solution in 50mM phosphate buffer, pH6.5 and pH 7.4, respectively, and equilibrated at 4℃for more than 24 hours after heating at 85℃for 30 minutes. Circular dichroism was characterized by an Aviv 400 spectrophotometer (Applied Photophysics Ltd, england), cuvette slit width 10mm, wavelength scan range from 190 to 280nm, increment of 0.5nm per step, and measurement repeated three times each. The thermal profile of the polypeptide was obtained by monitoring the signal at 225nm as a function of temperature. The temperature was adjusted using a Peltier temperature controller, the temperature range was set to 4℃to 85℃and the average heating rate was 1.0℃per minute, with an equilibration time of 2 minutes. Calculating and smoothing the first derivative of the thermal change curve to obtain a thermal change temperature, wherein the thermal change temperature (Tm) is an extremum of the first derivative. The Tm of P1-6, P1-7, P1-8, P2-7, P2-8, P2-9, P3-7, P3-8 and P3-9 at pH6.5 and 7.4, respectively, was obtained, and the difference in Tm (ΔTm) was obtained in both environments.
As shown in FIG. 1, the heat altered temperatures of the polypeptides P1-6, P1-7, P1-8, P2-7, P2-8, P2-9, P3-7, P3-8 and P3-9 at pH 6.5 were 17, 31, 49, 19, 31, 42, 17, 27 and 36℃respectively, while the heat altered temperatures of the polypeptides P1-6, P1-7, P1-8, P2-7, P2-8, P2-9, P3-7, P3-8 and P3-9 at pH 7.4 were 22, 36, 54, 30, 41, 52, 33, 43 and 51℃respectively (FIG. 1 a). By comparing the heat altered temperatures of the above polypeptide sequences at two pH values and their differences (FIG. 1 b), it was demonstrated that the heat altered temperature of the polypeptide increased with increasing number of repeated sequences (Gly-Pro-Hyp); while the more (Gly-Amp-Pro) sequences, the more the difference in heat-altered temperature between pH 6.5 and pH 7.4 increases. Wherein, the heat changing temperatures of P2-8 and P3-8 are respectively 31 and 27 ℃ at pH 6.5, and are both less than 37 ℃; the heat-altered temperatures at pH 7.4 were 41 and 43℃respectively, both greater than 37 ℃.
(2) Fluorescence spectrum: taking FAM modified polypeptide probes P0-8, P2-8 and P3-8, and respectively preparing 300 mu M probe solutions in buffer solutions with pH value of 6.5 and pH value of 7.4; the probe solutions are kept at the constant temperature of 85 ℃ in a water bath for 20min, the heated probe solutions are placed in an ice-water mixture at the temperature of 0 ℃ for 12h, and a fluorescence spectrum diagram of the probe solutions at the temperature of 37 ℃ is measured by a fluorescence spectrophotometer; wherein the excitation wavelength is 497nm, and the scanning range of the emission spectrum is 500-700nm.
The experimental results are shown in FIG. 2, and the unfolding ability of the probe was further studied by monitoring the fluorescence intensities of probes of pH 6.5 and pH7.4 at 37℃at an emission wavelength of 525nm by fluorescence spectrum and calculating the folding rate of the probe (FIG. 2). The proportion of P3-8 triple helix unfolding at pH 6.5 is much greater than that of P3-8 at pH7.4 and greater than that of P2-8 at pH 6.5, as compared to P2-8. The results show that the thermal stability of P2-8 and P3-8 are regulated by pH, and the length of the sequence influences the degree of influence of pH on the thermal stability.
PH response Properties
The fluorescence spectrum of the Dy modified polypeptide probe was recorded on an RF-6000 fluorescence spectrometer (ShimadzuCorporation, kyoto, japan), the excitation wavelength was 330nm, the emission spectrum was scanned over a range of 450 to 650nm, and the scanning interval was 1nm. The polypeptide probes which need to be subjected to pH-fluorescence titration are respectively prepared into solutions with the same concentration at pH 7.8、pH 7.6、pH 7.4、pH 7.25、pH 7.15、pH 7.05、pH 6.95、pH 6.85、pH 6.72、pH 6.63、pH 6.55、pH 6.41、pH 6.24 and pH 6.01, incubated at 37 ℃ before each measurement, balanced for 5min, and the titration curve of the fluorescence intensity of the probes along with the change of pH is completed through the measurement of fluorescence spectrum. Each set of experiments was repeated three times.
As shown in FIG. 3, the pH response of the probe P3-8 rapidly occurred near pH 6.8, and almost all of the probe was converted from the triple helix structure to the single strand structure at pH 6.5. While the fluorescence intensity of the probe P0-8 in the pH range of 6 to 8 is hardly changed. The titration of pH-fluorescence intensity demonstrated that probes P3-8 responded sensitively and rapidly to pH in the range from neutral to weakly acidic, whereas negative control probes P0-8 did not.
Example 11 imaging experiment
1. Staining of 3D tumor models with polypeptide probes
Type I collagen dissolved by acetic acid was thoroughly mixed with Fluorescein Isothiocyanate (FITC) solution at a molar ratio of 18:1 and reacted at 4℃for 24 hours in the absence of light. The fully reacted FITC-type I collagen solution was dialyzed through a dialysis bag of 1000 cutoff in 20mM PBS solution at pH 7.4 until fluorescence was undetectable in the dialysate, indicating complete removal of unreacted FITC small molecules. FITC-labeled type I collagen was used for 3D cell culture of tumor cells, and ROX-modified P3-8 probes were used to stain 3D tumor cells. 3D cells were imaged by a laser confocal microscope (inverted) (Olympus FV3000, japan) using 405nm wavelength to excite blue fluorescence to observe nuclei, 488nm wavelength to excite green fluorescence to observe FITC-labeled type I collagen, 561nm wavelength to excite red fluorescence to observe tumor lesion collagen.
The experimental results are shown in FIG. 4, in which FITC-I collagen shows strong green fluorescence, while the extracellular matrix of tumor shows clear red fluorescence, and the green fluorescence partially overlaps with that of FITC-I collagen. Staining of Hoechst 33342 showed a distribution of nuclei. Imaging results prove that the probe P3-8 specifically recognizes pathological collagen in tumors.
Meanwhile, probe FAM-P3-8 was further used for 3D tumor model imaging of HeLa, huH7 and AGS cells and staining of nuclei using Hoechst 33342. The results are shown in fig. 5, where the outer matrix in each of the three models showed strong green fluorescence (fig. 5a, d and g). Blue fluorescence clearly localizes the cell distribution (FIGS. 5b, e and h), and green fluorescence is distributed around the blue fluorescence (FIGS. 5c, f and i). The results show that the probe P3-8 has targeting capability on tumor lesion collagen generated by different tumor types, and the universality of the P3-8 polypeptide probe is further proved.
2. Living imaging of tumor model mice
Prior to performing live imaging of mice, the mice were anesthetized with pentobarbital (10% w/v) by intraperitoneal injection, and then near infrared imaging photographs of the mice at this time were taken as pre-imaging images. A40 nmol solution of the polypeptide probe P3-8 modified by Cy7.5 in PBS was injected subcutaneously in situ in 4T1 tumor-bearing mice, and imaging images at different time points were acquired with a VISQUE compact in vivo real-time small animal imaging system. Each mouse took the same posture and instrument scan parameters.
The experimental results are shown in FIG. 6, and a distinct fluorescent signal was observed 20min after probe injection. Over time, the fluorescence signal at the tumor site gradually increases. Until a strong fluorescence signal is observed at the tumor part 30h after injection, the position of the fluorescence signal is consistent with the actual position of the tumor, and no fluorescence signal is generated at other parts; whereas normal mice have no obvious fluorescent signal. The in-vivo imaging result of the mouse shows that the probe P3-8 can specifically target the tumor part of the tumor mouse, thereby realizing the specific imaging of the tumor.
In summary, the invention provides a polypeptide probe for detecting tumor lesion collagen, which can keep a stable triple helix structure in physiological pH, and becomes a single-chain structure in weak acidic pH of tumor, thereby realizing specific recognition of tumor lesion collagen. Experimental results show that the polypeptide probe can specifically identify pathological change collagen in tumors, has targeting capability on the pathological change collagen of the tumors generated by different tumor types, and the mouse living body imaging results show that the polypeptide probe can specifically target tumor parts of tumor mice so as to realize specific imaging of the tumors. The polypeptide probe has the advantages of simple preparation method, convenient detection, good specificity and strong binding capacity, and has wide application prospect in the fields of early diagnosis, curative effect evaluation, treatment and the like of tumors.
The above description of the embodiments is only for the understanding of the method of the present invention and its core ideas. It should be noted that other embodiments based on the inventive concept will also fall within the scope of the claims of the present invention for a person skilled in the art without departing from the principles of the present invention.
Claims (10)
1. A polypeptide for detecting tumor lesion collagen, wherein the polypeptide has the sequence: (Gly-Pro-Hyp) a-(Gly-Amp-Pro)b-(Gly-Pro-Hyp)c, wherein a is an integer of 3 to 10, b is an integer of 1 to a, and c is an integer of a-1 to a+1.
2. A polypeptide probe for detecting tumor lesion collagen, which is characterized by comprising the polypeptide of claim 1 and a signal molecule F, wherein the signal molecule F is connected with the N end or a side chain of a polypeptide sequence.
3. The polypeptide probe of claim 2, wherein the signal molecule F is linked to the N-terminus of the polypeptide sequence via a linker amino acid Ahx.
4. The polypeptide probe as set forth in claim 2, wherein the signal molecule F is one or more of fluorescein dye, coumarin dye, rhodamine dye, cyanine dye, BODIPY dye, tetraphenyl ethylene dye (TPE), hexaphenyl silyl dye (HPS), stilbene anthracene Dye (DSA), and rare earth ion complex.
5. The method of preparing a polypeptide probe as claimed in any one of claims 2 to 4, wherein the method comprises the steps of:
(1) 80-120mg of resin was charged to a reactor with a sieve plate, and 2-8mL of dichloromethane was used to swell the resin; removing the N-end Fmoc protecting group from the piperidine/N, N-dimethylformamide solution;
(2) Dissolving amino acid with the N end protected by Fmoc, HOBt and HBTU by DMF, activating at low temperature for 10-30min, dripping DIEA into the solution, uniformly mixing the solution, adding the solution into a reactor, and reacting for 1-6 hours;
(3) After the reaction is finished, the reaction solution is pumped out of the reactor, the resin is washed 2 to 4 times by DMF and DCM respectively, the resin is treated 3 times by 15 to 25 percent piperidine/DMF solution, and the resin is washed 3 times by DMF and DCM respectively after that until the protecting group is completely removed;
(4) Repeating steps (2) and (3) until a polypeptide of the target sequence is synthesized;
(5) Washing resin with DCM and methanol for 2-4 times, pumping resin, adding cutting fluid, and reacting for 1-6 hours;
(6) Precipitating the reaction solution by using glacial ethyl ether, centrifugally collecting the precipitate, washing the precipitate with the glacial ethyl ether for 3 times, and drying to obtain a crude polypeptide product;
(7) Purifying the crude polypeptide product by a C18 chromatographic column and a reversed-phase high performance liquid chromatography method, freeze-drying and preserving at-20 ℃;
(8) In the preparation of the functionalized polypeptide, after the step (4), dispersing the resin in DMF, weighing signal molecules, HOBt and HBTU, dissolving the signal molecules, the HOBt and the HBTU in DMF, activating the solution at a low temperature for 10-30min, adding DIEA into the solution, fully mixing, adding the mixed solution into the resin dispersion, and carrying out light-shielding reaction for 12-48 hours; the polypeptides were purified in steps (6) and (7) as in step (9) for the resin.
6. The method of claim 5, wherein the cutting fluid in step (5) is TFA: TIS: water=95:2.5:2.5.
7. Use of a polypeptide probe according to any one of claims 2-4 for the preparation of a detection reagent for detecting a tumor, and/or a kit, and/or an imaging reagent.
8. A detection reagent comprising the polypeptide probe of any one of claims 2-4.
9. A test kit comprising the polypeptide probe of any one of claims 2-4.
10. An in vitro/in vivo imaging agent comprising the polypeptide probe of any one of claims 2-4.
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