CN111057539B - Single-chain collagen polypeptide probe induced by charge repulsion and preparation method thereof - Google Patents
Single-chain collagen polypeptide probe induced by charge repulsion and preparation method thereof Download PDFInfo
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- CN111057539B CN111057539B CN202010068364.9A CN202010068364A CN111057539B CN 111057539 B CN111057539 B CN 111057539B CN 202010068364 A CN202010068364 A CN 202010068364A CN 111057539 B CN111057539 B CN 111057539B
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Abstract
The invention belongs to the technical field of biological detection, and particularly relates to a single-chain collagen polypeptide probe induced by charge repulsion and a preparation method thereof. The single-chain collagen polypeptide probe provided by the invention contains a repeating sequence of (Gly-Pro-Hyp) n, (Gly-Pro-Pro) n or (Gly-Hyp-Hyp) n, a fluorescent signal molecule is modified on the sequence, and the C end of the sequence contains a plurality of amino acids with charges. The novel polypeptide probe provided by the invention does not contain unnatural amino acid and is simple to prepare. The novel polypeptide probe has pH sensitivity, forms a triple helix structure under an acidic condition, can keep a stable single-chain structure under a physiological pH condition, does not need any pretreatment, namely has the capability of specifically binding pathological collagen, and completely avoids the risk of damage to a tissue sample caused by pretreatment. The novel polypeptide probe provided by the invention can be used for detecting the content of pathological collagen in vitro, and has wide application prospect in early diagnosis and curative effect evaluation of collagen-related diseases such as tumors and the like.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a charge repulsion induced single-chain collagen polypeptide functional probe and a preparation method thereof.
Background
Collagen, the most abundant protein in the mammalian body, is a major constituent of the extracellular matrix and plays a key role in tissue formation and maintenance of homeostasis. In the normal development and tissue repair of a human body, a collagen remodeling process exists, namely, a triple-helical structure of natural collagen is deformed or degraded under the action of proteolytic enzyme or other external force, and if excessive collagen is generated in the collagen remodeling process, excessive collagen is accumulated in organs and is easy to generate tissue fibrosis, so that serious diseases such as tumors and the like are caused finally; if uncontrolled degradation of collagen occurs, degenerative diseases of collagen, such as arthritis, etc., may result. Therefore, how to quickly and accurately detect the content of the pathological change collagen in the body tissue has important significance for preventing and treating diseases related to collagen pathological changes.
The target recognition technology is a key technology for realizing early detection and accurate diagnosis of diseases such as tumors and the like, and is widely applied in the field of biomedicine. The collagen is simultaneously involved in basic life activities such as regulation and control of proliferation, differentiation and migration of cells, is closely related to major diseases such as osteogenesis imperfecta, arthritis and tumors, and can be used as a key biomarker for a plurality of diseases of a human body. Therefore, achieving targeted detection of collagen is critical for the early diagnosis and treatment of these diseases.
At present, the content of diseased collagen in body tissues is mainly detected by an antibody-antigen immunoassay or a kit with traditional dyes as cores, including an HE staining method, an V.G staining method, a Masson staining method and the like. These methods achieve staining of collagen fibers, however, they are generally complicated procedures, the staining process can cause damage to the tissue, and the stained tissue is not readily available for other staining. Moreover, these methods detect all collagens, including normal collagens, and do not specifically and specifically detect diseased collagens in tissues. With the continuous development of probe technology, it has been found that some polypeptide probes targeting collagen recognition, including natural amino acid polypeptides and non-natural amino acid polypeptides, can be used for in vitro collagen recognition and imaging. The synthesis cost of the non-natural amino acid polypeptide is high, and potential safety hazards exist when the non-natural amino acid polypeptide is applied to in vivo detection, so that a polypeptide probe completely consisting of natural amino acids becomes a research hotspot.
The research finds that the natural amino acid polypeptide consisting of GPO (Gly-Pro-Hyp) or GPP (Gly-Pro-Pro) repetitive sequence can be specifically combined with pathological collagen. Chinese patent CN107266562A discloses a polypeptide probe for identifying pathological collagen, wherein fluorescent substances are modified on a GPO or GPP repetitive sequence of the probe, and a triple helix structure is easily formed under the normal temperature condition; however, the triple helix structure may cause the polypeptide probe to lose the targeted binding ability of collagen. Therefore, a polypeptide probe having a triple helix structure must be pretreated with a high temperature or the like before use to maintain a single-stranded state, so that it can be bound to collagen in a targeted manner. However, the pretreatment process such as high temperature easily causes damage to the tissue to be detected, and the accuracy of the detection result is affected.
Aiming at the problems in the prior art, the inventor finds that a plurality of charged amino acids are introduced into a polypeptide containing a plurality of GPO repetitive sequences through a plurality of experimental researches, and the obtained novel polypeptide probe has obvious single-chain stability in a physiological pH environment and forms a triple helix structure under an acidic condition. After the polypeptide sequence modifies a signal molecule, the polypeptide sequence can be used as a polypeptide probe for targeted detection of pathological collagen. Compared with the existing collagen targeted polypeptide probe, the novel pH-sensitive polypeptide probe can keep a stable single-chain structure under physiological conditions, does not need any pretreatment, and has the capability of specifically binding pathological collagen. The novel polypeptide probe has no unnatural amino acid, is simple to prepare, and completely avoids the risk of damage to a tissue sample caused by pretreatment. The novel polypeptide probe can be used for detecting the content of pathological collagen in vitro, and has wide application prospect in early diagnosis and curative effect evaluation of collagen-related diseases such as tumors and the like.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a single-chain collagen polypeptide probe induced by charge repulsion, the collagen polypeptide can keep a stable single-chain structure in a physiological pH environment, and can be specifically combined with pathological collagen without pretreatment. The purpose of the invention is realized by the following technical scheme:
a single-chain collagen polypeptide probe induced by charge repulsion contains a plurality of polypeptide sequences of (Gly-Pro-Hyp), (Gly-Pro-Pro) or (Gly-Hyp-Hyp) in the middle, and functional substances are modified on the polypeptide sequences; the C-terminal of the polypeptide sequence is connected with a charged amino acid sequence Z to promote the polypeptide to keep a single-chain unfolded structure.
Preferably, the sequence of the collagen polypeptide probe is X- (Gly-Pro-Hyp) a-Z (a is an integer between 4 and 20), X- (Gly-Pro-Pro) a-Z (a is an integer between 4 and 20), X- (Gly-Hyp-Hyp) a-Z (a is an integer between 4 and 20), (Gly-Pro-Hyp) a-Gly-Pro-Amp (-X) - (Gly-Pro-Hyp) b-Z (a and b are integers between 1 and 20), (Gly-Pro-Pro) a-Gly-Pro-Amp (-X) - (Gly-Pro-Pro) b-Z (a and b are integers between 1 and 20) or (Gly-Hyp-Hyp) a-Gly-Pro-Amp (-X) - (Gly-Pro-Amp) -Hyp) b-Z (a and b are integers between 1 and 20); wherein X is a functional substance, Gly is glycine, Pro is proline, Hyp is hydroxyproline, Amp is 4-aminoproline, and Z is a sequence rich in charged amino acid.
Preferably, the functional substance X is one or more of fluorescein molecules, coumarin molecules, rhodamine molecules, cyanine dye molecules, BODIPY molecules, squaric acid molecules, phosphorescent molecules, semiconductor quantum dots, carbon quantum dots, silicon quantum dots, sulfur quantum dots, phosphorus quantum dots, perovskite quantum dots, noble metal clusters, up-conversion rare earth nanomaterials, long-afterglow nanomaterials, graphene oxide, molybdenum disulfide, tungsten disulfide, manganese dioxide, boron nitride, carbon nanotubes, ferroferric oxide nanoparticles, iron oxide nanoparticles, silicon nanoparticles, noble metal nanoparticles, MOF materials, silicon spheres, liposomes and magnetic beads; the functional substance is connected to the N end of the collagen polypeptide or is connected through a side chain of Amp (4-aminoproline) at a certain middle position of the collagen polypeptide.
Preferably, the functional substance X is carboxyfluorescein FAM or carboxy-Xrhodamine 6-ROX.
Preferably, the functional substance X is linked to the polypeptide sequence via a linker Ahx.
Preferably, the sequence Z comprises 1 to 15 identically or differently charged amino acids or charged derivatives of amino acids.
Preferably, the sequence Z comprises 1-6 charged amino acids.
Preferably, the charged amino acid is D.
Preferably, the single-chain collagen polypeptide probe contains 1-15 (Gly-Pro-Hyp), (Gly-Pro-Pro) or (Gly-Hyp-Hyp) repetitive sequences in the middle.
Preferably, the single-chain collagen polypeptide probe contains 7 (Gly-Pro-Hyp) in the middle.
Another object of the present invention is to provide a method for preparing a single-chain collagen polypeptide probe induced by charge repulsion, comprising the steps of:
a) adding 80-250mg of resin to a reactor with a sieve plate, swelling the resin with 2-8mL of dichloromethane;
b) removing the Fmoc protecting group at the N end from 15-25% piperidine/N, N-Dimethylformamide (DMF), and detecting the removal degree of the protecting group by color development reaction;
c) dissolving amino acid (4eq) with the N-terminal protected by Fmoc, HOBt (4eq) and HBTU (4eq) in DMF, activating at low temperature for 10-30min, dropwise adding DIEA (6eq) into the solution, mixing the solution uniformly, adding the solution into a reactor, and reacting for 1-6 hrs;
d) after the reaction is finished, extracting the reaction liquid from the reactor, washing the resin with 2-8mL of DMF and DCM for 2-4 times respectively, detecting complete condensation of amino acid through chromogenic reaction, treating the resin with 15-25% piperidine/DMF solution for 3 times, namely 5min, 5min and 15min respectively, washing the resin with 5mL of DMF and DCM for 3 times respectively, and detecting complete removal of a protecting group through chromogenic reaction;
e) thereafter repeating steps c) and d) until after synthesis of the collagen polypeptide of the target sequence;
f) weighing functional substances (4eq), HOBt (4eq) and HBTU (4eq), dissolving with DMF, activating at low temperature for 10-30min, adding DIEA (4-10eq) dropwise into the solution, adding the mixed solution into the polypeptide solution, and reacting for 12-48hrs in dark; or chelate substance (4eq), HOBt (4eq) and HBTU (4eq), dissolving with DMF, activating at low temperature for 10-30min, adding DIEA (4-10eq) dropwise into the solution, adding the mixture into the polypeptide solution, and reacting for 12-48hrs in dark;
g) washing the resin with DCM and methanol respectively for 2-5 times in turn, then draining the resin, adding cutting fluid, reacting for 1-6hrs, wherein the cutting fluid comprises TFA, TIS and water in a mass ratio of 95:2.5: 2.5;
h) adding the polypeptide into the reaction solution, precipitating the polypeptide, then collecting the precipitate by centrifugation, dissolving the precipitate with TFA, adding excessive ethyl acetate for secondary precipitation, collecting the precipitate by centrifugation, washing the precipitate with ethyl acetate for 2-4 times, and drying to obtain crude peptide, wherein the crude peptide is purified by reversed phase liquid chromatography to obtain pure peptide;
i) and (3) dissolving the chelate modified peptide into a buffer solution, adding the chelate modified peptide into the metal nano functional substance dissolved in the buffer solution, wherein the peptide: functional substance 1: 100, reacting for 2-12 hrs;
j) the reaction was dialyzed against the buffer for 24-48 hrs.
The invention has the beneficial effects that: the novel polypeptide probe provided by the invention has pH response characteristics, forms a triple helix structure under an acidic condition, maintains a stable single-chain structure under a physiological pH condition, and can be used for targeted detection of pathological collagen; the novel polypeptide probe provided by the invention can keep a stable single-chain state without pretreatment such as heating, and the risk of damage to a tissue sample caused by pretreatment is completely avoided; the novel polypeptide probe provided by the invention does not contain unnatural amino acid and is simple to prepare; the novel polypeptide probe provided by the invention can be used for detecting the content of pathological collagen in vitro, and has wide application prospect in early diagnosis and curative effect evaluation of collagen-related diseases such as tumors and the like.
Drawings
FIG. 1 is a graph of the thermal change of pH response of a polypeptide probe;
FIG. 2 is a colorimetric chart of a polypeptide probe solution;
FIG. 3 shows the binding selectivity of the polypeptide probe F-PCTP-D6 to diseased collagen;
FIG. 4 is a graph of staining of rat Achilles tendon and tail tissue with polypeptide probe F-PCTP-D6;
FIG. 5 is a graph showing the staining of rat skin tissue with the polypeptide probe R-PCTP-D6;
FIG. 6 is a graph showing the staining of tumor tissue by the polypeptide probe R-PCTP-D6;
FIG. 7 is a graph showing the staining of other tissues by the polypeptide probe R-PCTP-D6;
FIG. 8 shows a polypeptide probe F-PCTP- (GDD)3Tissue staining pattern of (a);
FIG. 9 shows a polypeptide probe F-PCTP- (GDD)2Tissue staining pattern of (a).
Detailed Description
In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the invention is further described with the specific embodiments. The scope of the invention is not limited to the examples described below.
GPO in any one of the following examples is Gly (glycine) -Pro (proline) -Hyp (hydroxyproline) and D is Asp (aspartic acid); the Ahx is aminoacetic acid, the FAM is carboxyfluorescein, and the 6-ROX is 6-carboxy-Xrhodamine.
Comparative example 1 collagen polypeptide probes F-PCTP, F-IPreparation of CTP
1. Design of collagen polypeptide probes
Polypeptide sequences of collagen polypeptides are respectively designed to be FAM-Ahx- (GPO)7-NH2、FAM-Ahx-LRELHLNNN-NH2。
2. Preparation of collagen polypeptide Probe
(1) 100mg of Rink ammonia resin was added to a reactor with sieve plate and the resin was swollen with 5mL of dichloromethane;
(2) removing the Fmoc protecting group at the N end by using 20% piperidine/N, N-Dimethylformamide (DMF) solution, and detecting complete removal of the protecting group by color reaction;
(3) dissolving amino acid (4eq) with the N-terminal protected by Fmoc, HOBt (4eq) and HBTU (4eq) in DMF, activating at low temperature for 20min, dropwise adding DIEA (6eq) into the solution, mixing the solution, adding the solution into a reactor, and reacting for 3 hrs;
(4) after the reaction is finished, the reaction solution is extracted from the reactor, the resin is washed by 5mL of DMF and DCM for 3 times respectively, the amino acid is completely condensed by color reaction detection, and the resin is treated by 20% piperidine/DMF solution for 3 times, namely 5min, 5min and 15min respectively; washing the resin with 5mL of DMF and DCM for 3 times respectively, and detecting complete removal of the protecting group through color reaction;
(5) repeat (R) toStep (3) and step (4) until the collagen polypeptide sequence Ahx- (GPO) is synthesized7And Ahx-LRELHLNNN;
(6) adding FAM (10eq) and HBTU (10eq) into a reactor, dropwise adding DIEA (16eq) into the solution, reacting for 20 hours at 37 ℃, detecting complete reaction by chromogenic reaction, and washing the resin by 5mL of DMF and DCM for 3 times respectively;
(7) the resin was washed 3 times with DCM and methanol in turn, drained and reacted for 3hrs with cleavage medium (TFA: TIS: water 95:2.5: 2.5);
(8) adding the reaction solution into glacial ethyl ether, precipitating the polypeptide, centrifuging, collecting the precipitate, dissolving the precipitate with a small amount of TFA, adding excessive glacial ethyl ether for re-precipitation, centrifuging, collecting the precipitate, washing the precipitate with glacial ethyl ether for 2 times, and air-drying to obtain crude peptide FAM-Ahx- (GPO)7-NH2And FAM-Ahx-LRELHLNNN-NH2(ii) a The crude peptide is purified by reverse phase liquid chromatography to obtain a polypeptide probe which is respectively named as F-PCTP and F-ICTP。
EXAMPLE 1 preparation of collagen polypeptide Probe F-PCTP-D1-6
1. Sequence design of collagen polypeptide probes
The polypeptide sequences of the collagen polypeptide probes are respectively designed as follows: FAM-Ahx- (GPO)7-(Asp)6-NH2、FAM-Ahx-(GPO)7-(Asp)5-NH2、FAM-Ahx-(GPO)7-(Asp)4-NH2、FAM-Ahx-(GPO)7-(Asp)3-NH2、FAM-Ahx-(GPO)7-(Asp)2-NH2、FAM-Ahx-(GPO)7-(Asp)1-NH2。
2. Preparation of collagen polypeptide Probe
(1) 100mg of Rink ammonia resin was added to a reactor with sieve plate, respectively, and 5mL of methylene chloride was used to swell the resin;
(2) removing the Fmoc protecting group at the N end by using 20% piperidine/N, N-Dimethylformamide (DMF) solution, and detecting complete removal of the protecting group by color reaction;
(3) dissolving amino acid (4eq) with the N-terminal protected by Fmoc, HOBt (4eq) and HBTU (4eq) in DMF, activating at low temperature for 20min, dropwise adding DIEA (6eq) into the solution, mixing the solution, adding the solution into a reactor, and reacting for 3 hrs;
(4) after the reaction is finished, extracting the reaction solution from the reactor, washing the resin with 5mL of DMF and DCM for 3 times respectively, detecting complete condensation of amino acid by color reaction, and treating the resin with 20% piperidine/DMF solution for 3 times, which are 5min, 5min and 15min respectively; washing the resin with 5mL of DMF and DCM for 3 times respectively, and detecting complete removal of the protecting group through color reaction;
(5) repeating the steps (3) and (4) until the collagen polypeptide Ahx- (GPO) is synthesized7-(Asp)6、Ahx-(GPO)7-(Asp)5、Ahx-(GPO)7-(Asp)4、Ahx-(GPO)7-(Asp)3、Ahx-(GPO)7-(Asp)2And Ahx- (GPO)7-(Asp)1；
(6) Respectively adding FAM (10eq) and HBTU (10eq) into a reactor, dropwise adding DIEA (16eq) into the solution, reacting for 20 hours at 37 ℃, detecting complete reaction by chromogenic reaction, and washing the resin for 3 times by 5mL of DMF and DCM respectively;
(7) the resin was washed 3 times with DCM and methanol in turn, drained and reacted for 3hrs with cleavage medium (TFA: TIS: water 95:2.5: 2.5);
(8) adding the reaction solution into glacial ethyl ether, precipitating the polypeptide, centrifuging, collecting the precipitate, dissolving the precipitate with a small amount of TFA, adding excessive glacial ethyl ether for re-precipitation, centrifuging, collecting the precipitate, washing the precipitate with glacial ethyl ether for 2 times, and air-drying to obtain crude peptide FAM-Ahx- (GPO)7-(Asp)6-NH2、FAM-Ahx-(GPO)7-(Asp)5-NH2、FAM-Ahx-(GPO)7-(Asp)4-NH2、FAM-Ahx-(GPO)7-(Asp)3-NH2、FAM-Ahx-(GPO)7-(Asp)2-NH2And FAM-Ahx- (GPO)7-(Asp)1-NH2. And purifying the crude peptide by reverse phase liquid chromatography to prepare polypeptide probes, wherein the names of the polypeptide probes are F-PCTP-D6, F-PCTP-D5, F-PCTP-D4, F-PCTP-D3, F-PCTP-D2 and F-PCTP-D1.
3. Thermal temperature profile determination of collagen polypeptide probes
Taking the polypeptide probe F-PCTP prepared in comparative example 1 and the polypeptide probes F-PCTP-D6, F-PCTP-D5, F-PCTP-D4, F-PCTP-D3, F-PCTP-D2 and F-PCTP-D1 prepared in example 1, respectively configuring into 300 mu M polypeptide probe solutions, using circular dichroism to characterize the strength of the polypeptide probe at 225nm, increasing the temperature of the polypeptide probe solution from 4 ℃ to 75 ℃, increasing the temperature rate to 1.0 ℃/min, balancing each temperature for 2min, determining the temperature change curves of the polypeptide probe under different pH conditions, and taking the first derivative of the curves on the temperature and normalizing the curves to obtain the normalized curves as shown in figure 1. At pH 7.4, the heat-altered temperature of the polypeptide probe gradually decreased as the amount of D in the polypeptide probe increased, indicating that the ability of the polypeptide probe to remain single-stranded was increased by the introduction of D under physiological conditions (shown in FIG. 1 a). When the pH is 2.7, the amount of D in the polypeptide probe has no influence on the heat-altered temperature of the polypeptide probe, which indicates that the introduction of D does not influence the heat-altered temperature of the polypeptide probe under acidic conditions, and the polypeptide probe still maintains the triple helix structure (shown in FIG. 1 b). The results show that the introduction of D enables the polypeptide probe to maintain a stable single-stranded structure under physiological conditions without pretreatment processes such as heating.
4. Colorimetric assay for collagen polypeptide probes
Taking polypeptide probes F-PCTP and F-PCTP-D6, and respectively preparing into 300 mu M polypeptide probe solution; and (3) respectively keeping the constant temperature of each polypeptide probe solution in a water bath at 25 ℃ for 20min and in an ice-water mixture at 0 ℃ for 24h, collecting picture data by a digital camera, and observing the color of the solution. As shown in FIG. 2, the polypeptide probe F-PCTP showed orange color after being treated at 0 deg.C (shown in FIG. 2 c) and 25 deg.C (shown in FIG. 2 d), i.e., the polypeptide probe F-PCTP maintained triple helix structure at room temperature and lost the targeted binding ability to collagen. The polypeptide probe F-PCTP-D6 prepared by the invention has orange color after being treated at 0 ℃ (figure 2a) and turns green at 25 ℃ (figure 2 b), i.e. the polypeptide probe F-PCTP-D6 can keep single-stranded structure at room temperature. The results show that the polypeptide probe provided by the invention can keep good single-chain stability at room temperature, and can be used for targeted detection of collagen.
5. Targeted binding capacity of collagen polypeptide probe to pathological collagen
96 micro-porous plates are respectively coated with different protein solutions (pathological collagen Dn-collagen), bovine serum albumin solution (BSA), human serum albumin solution (HSA) and Hemoglobin solution (Hemoglobin). And respectively adding 50 mu L of 15 mu M F-PCTP-D6(PBS solution) into the microporous plates coated with different proteins, reacting for 5h at room temperature, and washing for 5 times by PBST. Detecting by a microplate reader, and reading the fluorescence intensity of each microplate.
As shown in FIG. 3, the polypeptide probe F-PCTP-D6 has strong binding ability to pathological collagen (Dn-collagen), but has substantially no binding to Bovine Serum Albumin (BSA), Human Serum Albumin (HSA) and Hemoglobin (Hemoglobin). The result shows that the polypeptide probe F-PCTP-D6 provided by the invention can specifically recognize pathological collagen, and the introduction of charged amino acid does not influence the binding capacity of the polypeptide probe to the pathological collagen.
6. Rat Achilles tendon and tail injury tissue staining
Injured rat Achilles tendon tissue and tail tissue were prepared as frozen sections, sliced to a thickness of 4 μm, air-dried at room temperature, treated with 0.5mL of blocking solution (PBS containing 5% goat serum), and incubated at room temperature. Removing the blocking solution from the slide with absorbent paper; preparing polypeptide probes F-PCTP-D6 and F-PCTP solutions at a concentration of 15. mu.M in 10mM phosphate buffer (pH 7.4), respectively; mu.L of the polypeptide probe solution was applied to the tissue sections and incubated at 4 ℃ for 2 h. The solution on the slide was absorbed by absorbent paper. The slides were washed three times with 10mM phosphate buffer for 5 minutes. As shown in FIG. 4, the polypeptide probe F-PCTP-D6 directly stained the tissue without pretreatment (shown in FIGS. 4a and D); the polypeptide probe F-PCTP can stain tissues after being heated and pretreated (shown in figures 4b and e); whereas the polypeptide probe F-PCTP which had not been heat-treated failed to stain the tissue (FIGS. 4c and F). The result shows that the polypeptide probe F-PCTP-D6 provided by the invention can keep a stable single-chain structure without heating pretreatment, and can be used for specific detection of pathological collagen.
EXAMPLE 2 preparation of collagen polypeptide Probe R-PCTP-D6
1. Design of collagen polypeptide probes
The designed collagen polypeptide sequence is 6-ROX-Ahx- (GPO)7-(Asp)6-NH2Wherein 6-ROX is 6-carboxy-Xrhodamine; 2. preparation of collagen polypeptide Probe
(1) 100mg of Rink ammonia resin was added to a reactor with sieve plate and the resin was swollen with 5mL of dichloromethane;
(2) removing the Fmoc protecting group at the N end by using 20% piperidine/N, N-Dimethylformamide (DMF) solution, and detecting complete removal of the protecting group by color reaction;
(3) dissolving amino acid (4eq) with the N-terminal protected by Fmoc, HOBt (4eq) and HBTU (4eq) in DMF, activating at low temperature for 20min, dropwise adding DIEA (6eq) into the solution, mixing the solution, adding the solution into a reactor, and reacting for 3 hrs;
(4) after the reaction is finished, the reaction solution is extracted from the reactor, the resin is washed by 5mL of DMF and DCM for 3 times respectively, the amino acid is completely condensed by color reaction detection, and the resin is treated by 20% piperidine/DMF solution for 3 times, namely 5min, 5min and 15min respectively; washing the resin with 5mL of DMF and DCM for 3 times respectively, and detecting complete removal of the protecting group through color reaction;
(5) repeating the steps (3 and (4) until the collagen polypeptide Ahx- (GPO) is synthesized7-(Asp)6；
(6) Adding 6-ROX (10eq) and HBTU (10eq) into a reactor, dropwise adding DIEA (16eq) into the solution, reacting at room temperature for 24h, detecting complete reaction by chromogenic reaction, and washing the resin by 5mL of DMF and DCM for 3 times respectively;
(7) the resin was washed 3 times with DCM and methanol in turn, drained and reacted for 3hrs with cleavage medium (TFA: TIS: water 95:2.5: 2.5);
(8) adding the reaction solution into glacial ethyl ether, precipitating the polypeptide, centrifuging, collecting the precipitate, dissolving the precipitate with a small amount of TFA, adding excessive glacial ethyl ether, precipitating again, centrifuging, collecting the precipitate, washing the precipitate with glacial ethyl ether for 2 times, and air-drying to obtain crude peptide 6-Rox-Ahx- (GPO)7-(Asp)6-NH2(ii) a The crude peptide was purified by reverse phase liquid chromatography to obtain the polypeptide probe R-PCTP-D6.
3. Tissue-specific staining of injured skin of rat
Preparing normal skin tissue and scald model tissue of rat into frozen sections, slicing to 4 μm thickness, air drying at room temperature, and probing with polypeptide respectivelyNeedle R-PCTP-D6, polypeptide probe F-I CTP, DAPI solution, and mixed probe were stained at 4 ℃ for 4h, DAPI stained for 1min, washed three times with PBS, and unbound polypeptide probe and excess DAPI were removed. As shown in FIG. 5, the polypeptide probe R-PCTP-D6 only stained the collagen in the scald model tissue in orange color (shown in FIG. 5 e), but did not stain the collagen in the normal tissue (shown in FIG. 5 a); polypeptide probe F-ICTP stains type I collagen in normal tissues and scald tissues to green (shown in figures 5b and f); DAPI stained nuclei in both normal and scalded tissues as blue (shown in fig. 5c, g); the different polypeptide probe stained sites were superimposed and co-stained to show blue and green in normal and scalded tissues (fig. 5 d), and orange, blue and green in scalded model tissues (fig. 5 h). The above experimental results show that the polypeptide probe R-PCTP-D6 provided by the invention can only bind to collagen in the pathological tissue, but has no binding ability to collagen in the normal tissue.
4. Staining of tumor tissue
Lung, rectal, breast and cervical cancer tissues from patients at the first hospital of the university of langzhou were taken, fixed with 4% paraformaldehyde for 1h and embedded in paraffin. The tissue was cut to a thickness of 4 μm on a polylysine-treated slide. Each slide was dewaxed to water with xylene and graded ethanol. Each section was then treated with 0.5mL of blocking solution (PBS with 5% goat serum) and incubated at room temperature. The blocking solution was removed from the slide using absorbent paper. A solution of the polypeptide probe R-PCTP-D6 was prepared at a concentration of 15. mu.M in 10mM phosphate buffer (pH 7.4). Applying 100 mu L of polypeptide probe R-PCTP-D6 solution to the tissue section, covering with parafilm, and incubating at 4 ℃ for 2 h; 100 μ L of DAPI solution (10 μ g/mL) was applied to the tissue sections for 1 min; slides were washed three times with 10mM phosphate buffer for 5min, a drop of anti-quencher was added to the tissue sections, and sections were covered with coverslips. Stained tissue sections were imaged on a Zeiss Axio Z2 imager.
As shown in FIG. 6, R-PCTP-D6 was able to stain collagen in tumor tissue orange (shown in FIGS. 6 a-D); DAPI solution was able to stain nuclei in tumor tissue blue (shown in fig. 6 e-h); as a result of staining with the overlapping polypeptide probes R-PCTP-D6 and DAPI solution, collagen in tumor tissue was stained orange, and cell nuclei were stained blue (FIGS. 6 i-l). The polypeptide probe R-PCTP-D6 provided by the invention can specifically bind to pathological collagen in tumor tissues and does not influence the re-staining of other polypeptide probes or dyes.
5. Staining of other tissues
Intestinal tissue, tendon tissue, tail tissue, eye tissue, ear tissue and bladder tissue of 9-week-old rats were taken and decellularized with 1% SDS. The treated Tissue was fixed with 4% paraformaldehyde for 1 hour and then cryopreserved in Tissue-Tek o.c.t. The tissue was cut to a thickness of 4 μm on a glass slide. Tissue sections were infiltrated with cold methanol at-20 ℃ for 10min and incubated in 20mM PBS. Each section was then treated with 0.5mL of blocking solution (PBS containing 5% goat serum) and incubated at room temperature. The blocking solution was removed from the slide using absorbent paper. A solution of the polypeptide probe R-PCTP-D6 was prepared at a concentration of 15. mu.M in 10mM phosphate buffer (pH 7.4), 100. mu.L of the solution of the polypeptide probe R-PCTP-D6 was applied to the tissue sections, and incubated at 4 ℃ for 2 h. The solution on the slide was absorbed by absorbent paper. The slides were washed three times for 5min with 10mM phosphate buffer. A drop of anti-quencher was added to the tissue section and the section was covered with a coverslip. The photographs were observed using a fluorescence microscope.
As shown in FIG. 7, the polypeptide probe R-PCTP-D6 provided by the present invention was able to stain pathological collagen in intestinal tissue (shown in FIG. 7 a), tendon tissue (shown in FIG. 7 b), tail tissue (shown in FIG. 7 c), eye (shown in FIG. 7D), ear (shown in FIG. 7 e) and bladder tissue (shown in FIG. 7 f). The polypeptide probe R-PCTP-D6 provided by the invention can detect pathological collagen in various tissues and has broad spectrum.
EXAMPLE 3 collagen polypeptide Probe F-PCTP- (GDD)3Preparation of
1. Design of collagen polypeptide probes
Designed collagen polypeptide sequence is FAM-Ahx- (GPO)7-(Gly-Asp-Asp)3-NH2Wherein the FAM is carboxyfluorescein.
2. Preparation of collagen polypeptide Probe
(1) 100mg of Rink ammonia resin was added to a reactor with sieve plate and the resin was swollen with 5mL of dichloromethane;
(2) removing the Fmoc protecting group at the N end by using 20% piperidine/N, N-Dimethylformamide (DMF) solution, and detecting complete removal of the protecting group by color reaction;
(3) dissolving amino acid (4eq) with the N-terminal protected by Fmoc, HOBt (4eq) and HBTU (4eq) in DMF, activating at low temperature for 20min, dropwise adding DIEA (6eq) into the solution, mixing the solution, adding the solution into a reactor, and reacting for 3 hrs;
(4) after the reaction is finished, the reaction solution is extracted from the reactor, the resin is washed by 5mL of DMF and DCM for 3 times respectively, the amino acid is completely condensed by color reaction detection, and the resin is treated by 20% piperidine/DMF solution for 3 times, namely 5min, 5min and 15min respectively; washing the resin with 5mL of DMF and DCM for 3 times respectively, and detecting complete removal of the protecting group through color reaction;
(5) repeating the steps (3) and (4) until the collagen polypeptide sequence Ahx- (GPO) is synthesized7-(Gly-Asp-Asp)3；
(6) Adding FAM (10eq) and HBTU (10eq) into a reactor, dropwise adding DIEA (16eq) into the solution, reacting for 20 hours at 37 ℃, detecting complete reaction by chromogenic reaction, and washing the resin by 5mL of DMF and DCM for 3 times respectively;
(7) the resin was washed 3 times with DCM and methanol in turn, drained and reacted for 3hrs with cleavage medium (TFA: TIS: water 95:2.5: 2.5);
(8) adding the reaction solution into glacial ethyl ether, precipitating the polypeptide, centrifuging, collecting the precipitate, dissolving the precipitate with a small amount of TFA, adding excessive glacial ethyl ether for re-precipitation, centrifuging, collecting the precipitate, washing the precipitate with glacial ethyl ether for 2 times, and air-drying to obtain crude peptide FAM-Ahx- (GPO)7-(Gly-Asp-Asp)3-NH2(ii) a The crude peptide was purified by reverse phase liquid chromatography to obtain the polypeptide probe F-PCTP- (GDD) 3.
3. Tissue staining of injured rat tail
Rat lesion tail tissue was cut to a thickness of 4 μm on a glass slide. Frozen tissue sections were air dried at room temperature. The injured rat tail tissue was washed with PBS 2 times, and then blocked with 5% goat serum for 30min, and then stained with 15. mu.M of polypeptide probe F-PCTP- (GDD)3 at 4 ℃ for 2h, washed with PBS 3 times, and then the blocking solution was dropped, and the slide was covered and photographed by observing with a fluorescence microscope.
As shown in FIG. 8, the polypeptide probe F-PCTP- (GDD)3 provided by the present invention has a strong binding ability to the collagen in the damaged tail tissue of rats, and can stain the collagen in the damaged tail tissue of rats green. The polypeptide probe F-PCTP- (GDD)3 provided by the invention can be combined with pathological change collagen in a targeted manner, and can be used for specific detection of pathological change collagen in different types of tissues.
EXAMPLE 4 collagen polypeptide Probe F-PCTP- (GDD)2Preparation of
1. Design of collagen polypeptide probes
Designed collagen polypeptide sequence is FAM-Ahx- (GPO)7-(Gly-Asp-Asp)2-NH2Wherein FAM is carboxyfluorescein.
2. Preparation of collagen polypeptide Probe
(1) 100mg of Rink ammonia resin was added to a reactor with sieve plate and the resin was swollen with 5mL of dichloromethane;
(2) removing the Fmoc protecting group at the N end by using 20% piperidine/N, N-Dimethylformamide (DMF) solution, and detecting complete removal of the protecting group by color reaction;
(3) dissolving amino acid (4eq) with the N-terminal protected by Fmoc, HOBt (4eq) and HBTU (4eq) in DMF, activating at low temperature for 20min, dropwise adding DIEA (6eq) into the solution, mixing the solution, adding the solution into a reactor, and reacting for 3 hrs;
(4) after the reaction is finished, the reaction solution is extracted from the reactor, the resin is washed by 5mL of DMF and DCM for 3 times respectively, the amino acid is completely condensed by color reaction detection, and the resin is treated by 20% piperidine/DMF solution for 3 times, namely 5min, 5min and 15min respectively; washing the resin with 5mL of DMF and DCM for 3 times respectively, and detecting complete removal of the protecting group through color reaction;
(5) repeating the steps (3) and (4) until the collagen polypeptide sequence Ahx- (GPO) is synthesized7-(Gly-Asp-Asp)2；
(6) Adding FAM (10eq) and HBTU (10eq) into a reactor, dropwise adding DIEA (16eq) into the solution, reacting for 20 hours at 37 ℃, detecting complete reaction by chromogenic reaction, and washing the resin by 5mL of DMF and DCM for 3 times respectively;
(7) the resin was washed 3 times with DCM and methanol in turn, drained and reacted for 3hrs with cleavage medium (TFA: TIS: water 95:2.5: 2.5);
(8) adding the reaction solution into glacial ethyl ether, precipitating the polypeptide, centrifuging, collecting the precipitate, dissolving the precipitate with a small amount of TFA, adding excessive glacial ethyl ether for re-precipitation, centrifuging, collecting the precipitate, washing the precipitate with glacial ethyl ether for 2 times, and air-drying to obtain crude peptide FAM-Ahx- (GPO)7-(Gly-Asp-Asp)2-NH2(ii) a The crude peptide was purified by reverse phase liquid chromatography to obtain the polypeptide probe F-PCTP- (GDD) 2.
3. Tissue staining of injured rat tail
Rat lesion tail tissue was cut to a thickness of 4 μm on a glass slide. Frozen tissue sections were air dried at room temperature. The injured rat tail tissue was washed with PBS 2 times, and then blocked with 5% goat serum for 30min, and then stained with 15. mu.M of polypeptide probe F-PCTP- (GDD)2 at 4 ℃ for 2h, washed with PBS 3 times, and then the blocking solution was dropped, and the slide was covered and photographed by observing with a fluorescence microscope.
As shown in FIG. 9, the polypeptide probe F-PCTP- (GDD)2 provided by the present invention has a strong binding ability to the collagen in the damaged tail tissue of rats, and can stain the collagen in the damaged tail tissue of rats green. The polypeptide probe F-PCTP- (GDD)2 provided by the invention can be combined with pathological change collagen in a targeted manner, and can be used for specific detection of pathological change collagen in tissues.
The above description is only for details of a specific exemplary embodiment of the present invention, and it is obvious to those skilled in the art that various modifications and changes may be made in the present invention in the practical application process according to specific preparation conditions, and the present invention is not limited thereto. All that comes within the spirit and principle of the invention is to be understood as being within the scope of the invention.
Claims (2)
1. A charge repulsion induced single-chain collagen polypeptide functional probe, which is characterized in that: the collagen polypeptide probeThe sequence of the needles was: FAM-Ahx- (GPO)7-(Asp)6-NH2、6-ROX-Ahx-(GPO)7-(Asp)6-NH2、FAM-Ahx-(GPO)7-(Gly-Asp-Asp)3-NH2、FAM-Ahx-(GPO)7-(Gly-Asp-Asp)2-NH2。
2. The method for preparing a single-chain collagen polypeptide functional probe induced by charge repulsion according to claim 1, wherein: the method comprises the following steps:
a) adding 80-250mg of resin to a reactor with a sieve plate, swelling the resin with 2-8mL of dichloromethane;
b) removing the Fmoc protecting group at the N end from 15-25% piperidine/N, N-dimethylformamide DMF solution, and detecting the removal degree of the protecting group through color development reaction;
c) dissolving amino acid 4eq protected by Fmoc at the N end, HOBt 4eq and HBTU 4eq by DMF, activating at low temperature for 10-30min, dropwise adding DIEA 6eq into the solution, mixing the solution uniformly, adding the solution into a reactor, and reacting for 1-6 hrs;
d) after the reaction is finished, extracting the reaction liquid from the reactor, washing the resin with 2-8mL of DMF and DCM for 2-4 times respectively, detecting complete condensation of amino acid through chromogenic reaction, treating the resin with 15-25% piperidine/DMF solution for 3 times, namely 5min, 5min and 15min respectively, washing the resin with 5mL of DMF and DCM for 3 times respectively, and detecting complete removal of a protecting group through chromogenic reaction;
e) then repeating the steps c) and d) until collagen polypeptide of a target sequence is synthesized, then adding 20-30% acetic anhydride into the reactor, and after the color reaction detection reaction is completed, washing the resin for 2-4 times by 3-8mL of DMF and DCM respectively;
f) weighing functional substances 4eq, HOBt 4eq and HBTU 4eq, dissolving with DMF, activating at low temperature for 10-30min, adding DIEA4-10eq dropwise into the solution, adding the mixed solution into the polypeptide solution, and reacting for 12-48hrs in a dark place; or chelate substances 4eq, HOBt 4eq and HBTU 4eq are dissolved by DMF, after activation for 10-30min at low temperature, DIEA4-10eq is added into the solution, the mixed solution is added into the polypeptide solution, and the reaction is carried out for 12-48hrs in a dark place;
g) washing the resin with DCM and methanol respectively for 2-5 times in turn, then draining the resin, adding cutting fluid, reacting for 1-6hrs, wherein the cutting fluid comprises TFA, TIS and water in a mass ratio of 95:2.5: 2.5;
h) adding the polypeptide into the reaction solution, precipitating the polypeptide, then collecting the precipitate by centrifugation, dissolving the precipitate with TFA, adding excessive ethyl acetate for secondary precipitation, collecting the precipitate by centrifugation, washing the precipitate with ethyl acetate for 2-4 times, and drying to obtain crude peptide, wherein the crude peptide is purified by reversed phase liquid chromatography to obtain pure peptide;
i) and (3) dissolving the chelate modified peptide into a buffer solution, adding the chelate modified peptide into the metal nano functional substance dissolved in the buffer solution, wherein the peptide: functional substance 1: 100, reacting for 2-12 hrs;
j) the reaction was dialyzed against buffer for 24-48 hrs.
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