CN111220810B - Polypeptide probe for targeted recognition of denatured collagen and detection method thereof - Google Patents
Polypeptide probe for targeted recognition of denatured collagen and detection method thereof Download PDFInfo
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Abstract
The invention discloses a polypeptide probe for targeted recognition of denatured collagen and a detection method thereof. The polypeptide probe comprises a targeting polypeptide sequence and a luminescent substance X for modifying the N end of the targeting polypeptide, wherein the targeting polypeptide sequence comprises (Hyp-Hyp-Gly) N. The novel polypeptide probe designed by the invention has good binding capacity to denatured collagen, good biocompatibility, strong specificity, simple synthesis process and convenient operation flow. The probe provides a new potential detection method for the molecular level diagnosis of important diseases such as cancer, cardiovascular diseases, organ fibrosis and the like.
Description
Technical Field
The invention relates to a polypeptide probe for targeted recognition of denatured collagen and a detection method thereof, belonging to the technical field of biological detection.
Background
Collagen is the highest protein content in the human body, is the main component of extracellular matrix, and forms a molecular scaffold to provide structural integrity and mechanical strength for connective tissues such as skin, bones, tendons and the like. Collagen has a very specific amino acid sequence, which requires that every third amino acid must have a glycine, forming the characteristic repeat sequence of Gly-X-Y. The collagen of normal connective tissue presents a unique triple helix structure, and three peptide chains are precisely packed together due to the interaction of inter-chain hydrogen bonds. Under a plurality of pathological conditions such as tumor, arthritis and the like, the triple helix structure of collagen molecules can be damaged by protease, and denatured collagen with a partial unfolded structure is generated, so that the normal physiological function of the collagen is influenced. Therefore, the denatured collagen is a key biomarker of a plurality of important diseases, and the establishment of a high-efficiency detection method of the denatured collagen is very important for the analysis and accurate diagnosis of the pathological mechanism of the diseases.
At present, the collagen detection method mainly comprises a tissue staining method, including an HE staining method, an V.G staining method, a Masson staining method and the like. Other methods include antibodies to collagen, peptides derived from collagen binding proteins, and peptides selected from phage expression. These methods have the disadvantages of complicated operation, weak probe binding ability, poor specificity, etc., and they are generally used for simultaneous detection of all collagens, including native collagens and denatured collagens having triple helical structures. Only denatured collagen is a key factor causing collagen-related diseases, and therefore, it is urgently required to establish a detection method with high specificity for denatured collagen. A novel polypeptide probe with specific binding capacity to denatured collagen is designed and synthesized, and the method has important significance for detection and curative effect evaluation of a plurality of diseases such as cancer, cardiovascular diseases, organ fibrosis and the like.
Wherein, the natural amino acid polypeptide consisting of the GPO (Gly-Pro-Hyp) repetitive sequence can be specifically combined with pathological collagen. For example, chinese patent CN107266562A discloses a polypeptide probe for recognizing pathological collagen, which modifies fluorescent substance on the GPO repeat sequence to easily form triple helix structure at room temperature. However, there are currently a few types of polypeptide probes that specifically bind to diseased collagen. Therefore, research and preparation of a novel polypeptide probe for recognizing pathological collagen can provide more choices for specific detection of collagen.
Hydroxyproline (Hyp) was found to be important for the formation of the robust collagen signature Gly-X-Y, where proline hydroxylation leads to 4R-hydroxy-L-proline (Hyp)R) Selectively occurs at the Y position of the collagen characteristic sequence Gly-X-Y, and is used as post-translational modification to endow the collagen characteristic sequence Gly-X-Y with additional stability; but also independently favours the formation of hydrogen bonds mediated by water, and hence favours folding into a triple helix structure, of hydroxyproline in the 4R configuration.
However, in the collagen characteristic sequence Gly-X-Y, the arrangement of Hyp generally has the following problems: firstly, since Hyp has a hydroxyl group in addition to the pyrrole ring having Pro, it is not easily synthesized due to the existence of steric hindrance in the synthesis; ② in the X position with CγPro residue of-endo generally stabilizes the triple helix, with CγPro residues of exo fold destabilize triple helix, while Hyp favors CγExo ring folds, thus excessively increasing the torsion angle of the X position of the collagen triple helix, which is detrimental to the formation of the triple helix structure; ③ the GOO (Gly-Hyp-Hyp) sequence is almost absent in the type I collagen chain and does not belong to the natural collagen composition sequence, the technicians in the field can generally select the natural amino acid sequence as the probe, but can not select the non-natural sequence to design the probe.
The invention adopts the non-natural amino acid sequence GOO and synthesizes the collagen protein mimic peptide with the GOO repetitive sequence by a solid phase synthesis method. The obtained GOO repetitive sequence not only has good capability of being folded into a triple helix, but also can be hybridized with a pathological collagen melting point, and can be used as a novel collagen hybrid peptide to perform targeted imaging and detection on pathological collagen.
Disclosure of Invention
In view of the above, the present invention provides a polypeptide probe for targeted recognition of denatured collagen and a detection method thereof. The method utilizes the polypeptide rich in (Hyp-Hyp-Gly) n repeated amino acid sequence to have good binding capacity to denatured collagen, and the designed novel polypeptide probe has good biocompatibility, strong specificity, simple synthesis process and convenient detection. The probe provides a new potential detection method for the molecular level diagnosis of important diseases such as cancer, cardiovascular diseases, organ fibrosis and the like. The purpose of the invention is realized by the following technical scheme:
a polypeptide probe for targeted recognition of denatured collagen comprises a targeting polypeptide sequence and a luminescent substance X for modifying the N end of the targeting polypeptide, wherein the targeting polypeptide sequence comprises (Hyp-Hyp-Gly) N.
Preferably, the 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, up-conversion rare earth nano materials and long-afterglow nano materials.
Preferably, X is carboxyfluorescein FAM.
Preferably, the sequence of the polypeptide probe is X- (Gly) m- (Hyp-Hyp-Gly) n, m is an integer between 0 and 8, and n is an integer between 4 and 20.
Preferably, m is 1, and n is an integer between 8 and 10.
Preferably, n is 8.
Preferably, n is 10.
A preparation method of a polypeptide probe for targeted recognition of denatured collagen comprises the following steps: synthesizing a target polypeptide sequence by solid phase synthesis, and modifying the luminescent material to the N end of the target polypeptide sequence.
Preferably, the method for solid phase synthesis of the target polypeptide sequence is:
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 through color 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% of 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 a polypeptide of the target sequence is synthesized. 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 luminescent 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 a dark place;
g) washing the resin with DCM and methanol for 2-5 times in turn, draining the resin, adding cutting fluid with TFA to water volume ratio of 95:5, and reacting for 1-6 hrs;
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.
An application of a polypeptide probe for targeted recognition of denatured collagen in preparation of an imaging reagent.
Preferably, the imaging agent comprises an in vitro tissue imaging agent and an in vivo imaging agent.
An application of a polypeptide probe for identifying denatured collagen in a targeted manner in detecting the content of diseased collagen in vitro.
Preferably, the detection method comprises: adding 0.2-0.8mL of blocking solution to the tissue section, standing for 10-50min, sucking off the liquid, staining with 80-120 μ L of probe solution, incubating at 2-6 deg.C for 1-3hrs, sucking off the staining solution, staining with 80-120 μ L of DAPI diluent for 0.5-1.5min, washing away unbound probe and excess DAPI diluent with 1 XPBS, dripping the blocking solution, covering with a glass slide, and taking a picture by observing with a fluorescence microscope.
Preferably, the tissue section is a frozen section or a paraffin section.
Preferably, the tissue section includes a model animal tissue section and a pathological tissue section.
Preferably, the pathological tissue section comprises a benign tumor tissue section, a malignant tumor tissue section, an orthopedic tissue disease section, a fibrosis tissue section, an inflammation tissue section, a hyperplasia tissue section, an atherosclerosis tissue section, a liver cirrhosis tissue section, a tissue section after eye laser correction surgery, a skin photodamage tissue section, a skin thermal damage tissue section, a skin acid-base corrosion tissue section and a radiation damage tissue section.
An application of a polypeptide probe for identifying denatured collagen in a targeted manner in detecting the content of diseased collagen in vivo.
Preferably, the detection method is as follows: injecting the collagen polypeptide probe into a living body for experiment, then placing the probe into an imaging device for imaging experiment after anesthesia.
Preferably, the polypeptide probes are also dissolved in sterilized cysteine-containing 1X PBS prior to injection.
The invention has the beneficial effects that: the invention provides a novel polypeptide probe rich in (Hyp-Hyp-Gly) n sequence, and provides a novel method for detecting and diagnosing denatured collagen; the polypeptide probe provided by the invention has good biocompatibility, strong specificity and good targeting capability on denatured collagen; the polypeptide probe provided by the invention has simple synthesis process and convenient detection; the probe provides a new potential detection method for the molecular level diagnosis of important diseases such as cancer, cardiovascular diseases, organ fibrosis and the like.
Drawings
FIG. 1 polypeptide sequence G (OOG) according to the invention8Thermal change curves and first derivative plots of;
FIG. 2 shows FAM-G (OOG) as a probe according to the present invention8A binding profile selective for diseased collagen;
FIG. 3 shows FAM-G (OOG) as a probe according to the present invention8A microscopic image of normal rat tendon tissue and decellularized rat tendon tissue by fluorescent staining;
FIG. 4 shows FAM-G (OOG) as a probe according to the present invention8Fluorescence micrographs of injured tissues from different parts of rats;
FIG. 5 shows FAM-G (OOG) as a probe according to the present invention8Fluorescence microscopy of pathological sections of leiomyoma, liver fibrosis, stomach cancer and rectal cancer tissues;
FIG. 6 shows FAM-G (OOG) probes according to the present invention8Fluorescence microscopic picture of pathological section of esophageal cancer, breast cancer, liver cancer and lung cancer tissues;
FIG. 7 shows FAM-G (OOG) probes according to the present invention10Fluorescence microscopy of pathological sections of mouse intestinal tissue
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.
Example 1 polypeptide Probe FAM-G (OOG)8Preparation of
The designed polypeptide sequence is FAM-Gly- (Hyp-Hyp-Gly)8Wherein FAM is carboxyfluorescein.
The preparation steps of the probe are as follows:
(1) 100mg 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 N-terminal protected by Fmoc, HOBt (4eq) and HBTU (4eq) in DMF, activating at low temperature for 20min, adding DIEA (6eq) dropwise into the solution, mixing the solution, adding into a reactor, and reacting for 3 hrs.
(4) After the reaction, the reaction solution was taken out of the reactor, and the resin was washed 3 times with 5mL of DMF and DCM, respectively. The amino acid condensation was complete as detected by color reaction, and the resin was treated with 20% piperidine/DMF solution 3 times for 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 polypeptide Gly- (Hyp-Hyp-Gly) of the target sequence is synthesized8. FAM (4eq), HOBt (4eq) and HBTU (4eq) were added to the reactor, DIEA (6eq) was added dropwise to the solution, the reaction was carried out at 30 ℃ for 20 hours, the reaction was detected by color reaction and the resin was washed 3 times with 5mL of DMF and DCM, respectively.
(6) The resin was washed 3 times with DCM and MeOH, respectively, in turn. The resin was drained and cleavage medium (TFA: water: 95:5) was added and reacted for 3 hrs.
7(7) adding the reaction solution to glacial ethyl ether to precipitate the polypeptide. And (3) centrifuging to collect precipitate, dissolving the precipitate with a small amount of TFA, adding excessive ethyl glacial ether for re-precipitation, centrifuging to collect precipitate, washing the precipitate with ethyl glacial ether for 3 times, and air-drying to obtain the crude peptide. The crude peptide was purified by reverse phase liquid chromatography to obtain polypeptide probe FAM-G- (OOG)8。
Example 2 polypeptide Probe FAM-G (OOG)10Preparation of
The designed polypeptide sequence is FAM-Gly- (Hyp-Hyp-Gly)10Wherein FAM is carboxyfluorescein.
The preparation steps of the probe are as follows:
(1) 100mg 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 N-terminal protected by Fmoc, HOBt (4eq) and HBTU (4eq) in DMF, activating at low temperature for 20min, adding DIEA (6eq) dropwise into the solution, mixing the solution, adding into a reactor, and reacting for 3 hrs.
(4) After the reaction, the reaction solution was taken out of the reactor, and the resin was washed 3 times with 5mL of DMF and DCM, respectively. The amino acid condensation was complete as detected by color reaction, and the resin was treated with 20% piperidine/DMF solution 3 times for 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 polypeptide Gly- (Hyp-Hyp-Gly) of the target sequence is synthesized10. FAM (4eq), HOBt (4eq) and HBTU (4eq) were added to the reactor, DIEA (6eq) was added dropwise to the solution, the reaction was carried out at 30 ℃ for 20 hours, the reaction was detected by color reaction and the resin was washed 3 times with 5mL of DMF and DCM, respectively.
(6) The resin was washed 3 times with DCM and MeOH, respectively, in turn. The resin was drained and cleavage medium (TFA: water: 95:5) was added and reacted for 3 hrs.
(7) The reaction solution was added to ethyl acetate to precipitate the polypeptide. And (3) centrifuging to collect precipitate, dissolving the precipitate with a small amount of TFA, adding excessive ethyl glacial ether for re-precipitation, centrifuging to collect precipitate, washing the precipitate with ethyl glacial ether for 3 times, and air-drying to obtain the crude peptide. The crude peptide was purified by reverse phase liquid chromatography to obtain the polypeptide probe FAM-G (OOG)10。
Comparative example 1 polypeptide Probe FAM-G (POG)8Preparation of
The designed polypeptide sequence is FAM-Gly- (Pro-Hyp-Gly)8Wherein FAM is carboxyfluorescein.
The preparation steps of the probe are as follows:
(1) 100mg 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 N-terminal protected by Fmoc, HOBt (4eq) and HBTU (4eq) in DMF, activating at low temperature for 20min, adding DIEA (6eq) dropwise into the solution, mixing the solution, adding into a reactor, and reacting for 3 hrs.
(4) After the reaction, the reaction solution was taken out of the reactor, and the resin was washed 3 times with 5mL of DMF and DCM, respectively. The amino acid condensation was complete as detected by color reaction, and the resin was treated with 20% piperidine/DMF solution 3 times for 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 polypeptide Gly- (Pro-Hyp-Gly) of the target sequence is synthesized8. FAM (4eq), HOBt (4eq) and HBTU (4eq) were added to the reactor, DIEA (6eq) was added dropwise to the solution, the reaction was carried out at 30 ℃ for 20 hours, the reaction was detected by color reaction and the resin was washed 3 times with 5mL of DMF and DCM, respectively.
(6) The resin was washed 3 times with DCM and MeOH, respectively, in turn. The resin was drained and cleavage medium (TFA: water: 95:5) was added and reacted for 3 hrs.
(7) The reaction solution was added to ethyl acetate to precipitate the polypeptide. And (3) centrifuging to collect precipitate, dissolving the precipitate with a small amount of TFA, adding excessive ethyl glacial ether for re-precipitation, centrifuging to collect precipitate, washing the precipitate with ethyl glacial ether for 3 times, and air-drying to obtain the crude peptide. The crude peptide was purified by reverse phase liquid chromatography to obtain the polypeptide probe FAM-G (POG)8。
Comparative example 2 preparation of polypeptide Probe FAM-sGPO
The designed random polypeptide sequence is FAM-Pro-Pro-Pro-Gly-Gly-Gly-Hyp-Hyp-Pro-Gly-Gly-Gly-Hyp-Hyp-Pro-Pro-Gly, wherein FAM is carboxyl fluorescein.
The preparation steps of the probe are as follows:
(1) 100mg 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 N-terminal protected by Fmoc, HOBt (4eq) and HBTU (4eq) in DMF, activating at low temperature for 20min, adding DIEA (6eq) dropwise into the solution, mixing the solution, adding into a reactor, and reacting for 3 hrs.
(4) After the reaction, the reaction solution was taken out of the reactor, and the resin was washed 3 times with 5mL of DMF and DCM, respectively. The amino acid condensation was complete as detected by color reaction, and the resin was treated with 20% piperidine/DMF solution 3 times for 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) and (4) repeating the steps (3) and (4) until the polypeptide Pro-Pro-Pro-Gly-Gly-Hyp-Hyp-Pro-Gly-Gly-Hyp-Hyp-Pro-Pro-Gly of the target sequence is synthesized. FAM (4eq), HOBt (4eq) and HBTU (4eq) were added to the reactor, DIEA (6eq) was added dropwise to the solution, the reaction was carried out at 30 ℃ for 20 hours, the reaction was detected by color reaction and the resin was washed 3 times with 5mL of DMF and DCM, respectively.
(6) The resin was washed 3 times with DCM and MeOH, respectively, in turn. The resin was drained and cleavage medium (TFA: water: 95:5) was added and reacted for 3 hrs.
(7) The reaction solution was added to ethyl acetate to precipitate the polypeptide. And (3) centrifuging to collect precipitate, dissolving the precipitate with a small amount of TFA, adding excessive ethyl glacial ether for re-precipitation, centrifuging to collect precipitate, washing the precipitate with ethyl glacial ether for 3 times, and air-drying to obtain the crude peptide. The crude peptide was purified by reverse phase liquid chromatography to obtain the polypeptide probe FAM-sGPO.
Comparative example 3 polypeptide Probe FAM-G (POG)10Preparation of
The designed polypeptide sequence is FAM-Gly- (Pro-Hyp-Gly)10Wherein FAM is carboxyfluorescein.
The preparation steps of the probe are as follows:
(1) 100mg 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 N-terminal protected by Fmoc, HOBt (4eq) and HBTU (4eq) in DMF, activating at low temperature for 20min, adding DIEA (6eq) dropwise into the solution, mixing the solution, adding into a reactor, and reacting for 3 hrs.
(4) After the reaction, the reaction solution was taken out of the reactor, and the resin was washed 3 times with 5mL of DMF and DCM, respectively. The amino acid condensation was complete as detected by color reaction, and the resin was treated with 20% piperidine/DMF solution 3 times for 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 polypeptide G of the target sequence is synthesized(POG)10. FAM (4eq), HOBt (4eq) and HBTU (4eq) were added to the reactor, DIEA (6eq) was added dropwise to the solution, the reaction was carried out at 30 ℃ for 20 hours, the reaction was detected by color reaction and the resin was washed 3 times with 5mL of DMF and DCM, respectively.
(6) The resin was washed 3 times with DCM and MeOH, respectively, in turn. The resin was drained and cleavage medium (TFA: water: 95:5) was added and reacted for 3 hrs.
(7) The reaction solution was added to ethyl acetate to precipitate the polypeptide. And (3) centrifuging to collect precipitate, dissolving the precipitate with a small amount of TFA, adding excessive ethyl glacial ether for re-precipitation, centrifuging to collect precipitate, washing the precipitate with ethyl glacial ether for 3 times, and air-drying to obtain the crude peptide. The crude peptide was purified by reverse phase liquid chromatography to obtain the polypeptide probe FAM-G (POG)10。
Example 3 polypeptide Probe FAM-G (OOG)8Determination of temperature profile of thermal change
The polypeptide probe FAM-G (OOG) prepared in example 1 was sampled8And configuring a 300 mu M polypeptide probe solution, using circular dichroism chromatography to characterize the intensity of the polypeptide probe at 225nm, increasing the temperature of the polypeptide probe solution from 4 ℃ to 75 ℃, increasing the temperature at a rate of 1.0 ℃/min, balancing each temperature for 2min, determining the thermal change temperature curve (shown as a in figure 1) of the polypeptide probe under different pH conditions, and performing first derivative on the temperature according to the curve and normalizing to obtain a normalized curve (shown as b in figure 1).
Example 4 polypeptide Probe FAM-G (OOG)8Specifically binds to the diseased collagen
6uL of gelatin dissolved in 70 ℃, 10mM PBS (pH7.4) was added to 96-well plates and incubated for 20 minutes at 4 ℃ to ensure complete gelation. MES buffer solution (pH 4.7) containing 2mM NHS and 10mM EDC was added to the gelatin film to crosslink it. After shaking overnight at room temperature, it was washed 3 times with 10mM PBS (pH7.4), 10 minutes each. Carboxyfluorescein-labeled peptide solution (20. mu.M, 50. mu.L) prepared in 10mM PBS (pH7.4) was added to the wells with gelatin film and bound at 4 ℃ for 5 hours. In the "heating +" group, the classical polypeptide probe FAM-G (POG) prepared in comparative example 1 was taken8Random sequence polypeptide Probe FAM-sGPO prepared in comparative example 2 and polypeptide Probe FAM-G (OOG) prepared in example 18The solution is at 80 deg.CAfter heating for 15 minutes, the mixture was quenched in ice water for 30 seconds, and then diluted for use. In the "heat-" group, pre-equilibrated FAM-G (POG) at 4 ℃ can be diluted directly without any pretreatment8FAM-sGPO and FAM-G (OOG)8And (3) solution. After binding was complete, the plates were washed 3 times with 10mM PBS (pH7.4), 10 minutes each. Fluorescence (ex: 493nm, em: 533nm) was measured using Infinite M200(TECAN Corporation, Switzerland). Each experiment was repeated three times.
The collagen solution (type I) dissolved in 0.5M acetic acid was denatured by heating at 70 ℃ for 15 minutes, and then added to a 96-well plate. After the well plate was air-dried, the denatured collagen membrane was neutralized by washing with 10mM PBS (pH 7.4). HSA (human serum albumin), BSA (bovine serum albumin), hemoglobin, pepsin and trypsin dissolved in 10mM PBS (pH7.4) were added to the well plates and dried. FAM-GOO (FAM-G (OOG)) by preheating and quenching8) With FAM-GPO (FAM-G (POG))8) After 5 hours of staining with the polypeptide probe, the ELISA plate was washed 3 times with PBS buffer. Fluorescence intensity (ex: 493nm, em: 533nm) was measured using Infinite M200(TECAN Corporation, Switzerland). Each experiment was repeated three times.
The experimental results are shown in FIG. 2, wherein a is FAM-G (OOG)8With classical polypeptide probes FAM-G (POG)8And random sequence control peptide FAM-sGPO fluorescence levels of gelatin films treated with the heat-quench step (+) or direct staining at 4 ℃ (-). b is FAM-G (POG) subjected to preheating treatment8Fluorescence levels binding to denatured collagen HSA, BSA, hemoglobin, trypsin and pepsin; c is FAM-G (OOG)8Fluorescence levels binding denatured collagen, HSA, BSA, hemoglobin, trypsin and pepsin. The results show that the polypeptide probe FAM-G- (OOG) provided by the invention8Has strong binding capacity to pathological collagen (Dn-collagen), basically has no binding to Bovine Serum Albumin (BSA), Human Serum Albumin (HSA) and Hemoglobin (Hemoglobin), and is combined with a classical polypeptide probe FAM-G (POG)8Has equivalent binding capacity to pathological collagen.
Example 5 polypeptide Probe FAM-G (OOG)8Tissue imaging of
0.5mL of confining liquid was added to the frozen sections of normal rat tendon tissue and decellularized rat tendon tissue, respectively, and the sections were left for 30min and then aspirated. FAM-G (OOG) using 100. mu.L of 15. mu.M probe8Staining the solution and incubating at 4 ℃ for 2 hrs. After blotting, the staining solution was washed 3 times with 1 × PBS to wash away unbound probes. Dropping the mounting solution, covering the glass slide, and taking a photograph by observing with a fluorescence microscope, as shown in FIG. 3, the polypeptide probe FAM-G (OOG)8After the preheating treatment, only the pathological rat tendon tissue (decellularized rat tendon tissue) can be specifically stained, and the normal tissue is not stained.
Adding 0.5mL of confining liquid on rat ocular corneal tissue, bladder tissue, stomach tissue, intestinal tissue, ear tissue and kidney tissue frozen section, standing for 30min, and sucking off liquid. Staining was performed using 100. mu.L of 15. mu.M probe solution and incubation was performed at 4 ℃ for 2 hrs. After blotting, the staining solution was washed 3 times with 1 × PBS to wash away unbound probes. Dropping the mounting solution, covering the glass slide, and taking a photograph by observing with a fluorescence microscope, as shown in FIG. 4, the polypeptide probe FAM-G (OOG)8Has binding ability to pathological collagen in various tissues, can be used for staining various tissues, and has broad spectrum.
Leiomyoma (Leiomyoma), Liver Fibrosis (Liver Fibrosis), gastric cancer (Stomach cancer), Rectal cancer (renal cancer), Esophageal cancer (Esophageal cancer), Breast cancer (Breast cancer), Liver cancer (Liver cancer) and Lung cancer (Lung cancer) tissues from patients at the first Hospital of Lanzhou university were 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. Preparation of polypeptide Probe FAM-G (OOG) at a concentration of 15. mu.M in 10mM phosphate buffer (pH7.4)8And (3) solution. Mixing 100 μ L of polypeptide probe FAM-G (OOG)8Applying the solution to tissue slices, covering with parafilm, and incubating at 4 deg.C for 2 h; 100 μ L of DAPI solution (10 μ g/mL) was applied to the tissue sections for 1 min; slides were buffered with 10mM phosphateLiquid wash 5min, three times, add a drop of anti-quencher to the tissue section, and cover the section with coverslip. Stained tissue sections were imaged on a Zeiss Axio Z2 imager.
As a result, as shown in FIGS. 5 and 6, FAM-G (OOG)8The ability to stain collagen in various tumor tissues green (shown in FIGS. 5a-d and 6 a-d); DAPI solution was able to stain nuclei in various tumor tissues blue (shown in fig. 5e-h and fig. 6 e-h); stacked polypeptide probes FAM-G (OOG)8And DAPI solution staining, the diseased collagen in each tumor tissue was stained green and the nuclei were stained blue (FIGS. 5i-l and 6 i-l). Description of the polypeptide Probe of the present invention FAM-G (OOG)8Can specifically bind to pathological collagen in tumor tissue, and does not affect the re-staining of cell nucleus by other polypeptide probes or dyes.
Example 6 polypeptide Probe FAM-G (OOG)10Tissue imaging
0.5mL of confining liquid is added on the frozen section of the intestinal tissue of the mouse respectively, and the liquid is sucked away after the section is placed for 30 min. Using 100. mu.L, 15. mu.M, respectively, of the polypeptide probe FAM-G (OOG) prepared in example 210Solution and polypeptide Probe FAM-G (OOG) prepared in comparative example 310Staining and incubation at 4 ℃ for 2 hrs. After blotting, the staining solution was washed 3 times with 1 × PBS to wash away unbound probes. Dropping the mounting solution, covering the glass slide, and taking pictures by observing with a fluorescence microscope, as shown in FIG. 7, wherein a and b are polypeptide probes FAM-G (OOG)10C and d are polypeptide probes FAM-G (POG)10The results of the experiments showed that the polypeptide probe FAM-G (OOG)10After the preheating treatment, the intestinal tissue of the lesion mouse can be specifically stained, and the staining result is compared with a polypeptide probe FAM-G (POG)10The dyeing results were comparable.
Finally, the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting, although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all of them should be covered in the claims of the present invention.
Claims (7)
1. The polypeptide probe for targeted recognition of denatured collagen is characterized by comprising a targeted polypeptide sequence Gly- (Hyp-Hyp-Gly) N and a luminescent substance X for modifying the N end of the targeted polypeptide, wherein the sequence of the polypeptide probe is X-Gly- (Hyp-Hyp-Gly) N, and N is an integer between 8 and 10.
2. The polypeptide probe for targeted recognition of denatured collagen according to claim 1, wherein 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, up-conversion rare earth nanomaterials and long-afterglow nanomaterials.
3. The polypeptide probe for targeted recognition of denatured collagen according to claim 2, wherein X is carboxyfluorescein FAM.
4. The method for preparing a polypeptide probe for targeted recognition of denatured collagen according to any one of claims 1 to 3, comprising the steps of: synthesizing a target polypeptide sequence by solid phase synthesis, and modifying the luminescent material to the N end of the target polypeptide sequence.
5. The method of claim 4, wherein the solid phase synthesis of the targeting polypeptide sequence comprises:
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 through color 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% of 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 the polypeptide of the target sequence is synthesized, and washing the resin for 2-4 times by 3-8mL of DMF and DCM respectively after the chromogenic reaction detection reaction is completed;
f) weighing luminescent materials 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 a dark place;
g) washing the resin with DCM and methanol for 2-5 times in turn, draining the resin, adding cutting fluid with TFA to water volume ratio of 95:5, and reacting for 1-6 hrs;
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.
6. Use of a polypeptide probe targeted to recognize denatured collagen according to any one of claims 1 to 3 in the preparation of an imaging agent.
7. The use of claim 6, wherein the imaging agent comprises an in vitro tissue imaging agent and an in vivo imaging agent.
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| CN113151604A (en) * | 2021-04-29 | 2021-07-23 | 兰州大学 | PNA probe structure for detecting novel coronavirus and synthetic method thereof |
| CN114958338B (en) * | 2022-05-11 | 2023-07-14 | 北京兴德通医药科技股份有限公司 | Method for detecting protein kinase A by adopting carbon oxide nanohorn quenching fluorescent probe |
| CN115925988B (en) * | 2022-09-14 | 2023-08-22 | 中山大学附属第五医院 | Denatured collagen targeted antibacterial peptide and preparation method and application thereof |
| CN115931517A (en) * | 2022-12-05 | 2023-04-07 | 中山大学附属第五医院 | Dyeing method of unwinding collagen in bone and cartilage |
| CN116298316A (en) * | 2023-01-18 | 2023-06-23 | 中山大学附属第五医院 | Method for inducing collagen hybridization peptide to maintain single-chain collagen hybridization capability and application |
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