CN107312068B

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Info

Publication number: CN107312068B
Application number: CN201710432805.7A
Authority: CN
Inventors: 李新; 蒋权; 卢应梅; 邓亚萍; 蒋国军; 韩峰; 胡永洲
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2017-06-09
Filing date: 2017-06-09
Publication date: 2021-02-09
Anticipated expiration: 2037-06-09
Also published as: CN107312068A

Abstract

Translated fromChinese

本发明提供一种检测脯氨酸异构酶活性的荧光探针。该探针在生理环境中荧光较弱，可以特异性地在脯氨酸异构酶的催化作用下发生构型翻转，生成荧光信号增强的产物，因此该探针可用于脯氨酸异构酶的活性评价。可在检测细胞或组织蛋白质提取液中脯氨酸异构酶活性中的应用；以及在检测活细胞中脯氨酸异构酶活性中的应用。本发明提供的荧光探针及检测方法具有以下有益效果：(1)探针稳定性好，能够长期保存使用；(2)检测方法简便，检测体系中加入探针孵育10分钟测荧光强度，即可给出结果，无需其它试剂辅助；(3)能实现活细胞中脯氨酸异构酶活性的在体检测，而其它报导方法仅能用于细胞匀浆液。所述探针的具结构：

The present invention provides a fluorescent probe for detecting proline isomerase activity. The probe has weak fluorescence in the physiological environment, and can specifically reverse its configuration under the catalysis of proline isomerase to generate a product with enhanced fluorescence signal, so the probe can be used for proline isomerase activity evaluation. It can be used in detecting proline isomerase activity in cell or tissue protein extract; and in detecting proline isomerase activity in living cells. The fluorescent probe and detection method provided by the present invention have the following beneficial effects: (1) the probe has good stability and can be stored and used for a long time; (2) the detection method is simple and convenient, and the probe is added to the detection system and incubated for 10 minutes to measure the fluorescence intensity, namely The results can be given without the aid of other reagents; (3) in vivo detection of proline isomerase activity in living cells can be achieved, while other reporting methods can only be used in cell homogenates. The structure of the probe:

Description

Fluorescent probe for detecting activity of proline isomerase, preparation and application thereof

Technical Field

The invention belongs to the field of biological detection, and relates to a fluorescent probe for detecting the activity of proline isomerase, a preparation method thereof and application thereof in detecting the activity of proline isomerase in a biological sample.

Background

Proline is a special amino acid, and the amino group in the structure of proline is disubstituted secondary amine. In the protein secondary structure, the normal alpha helical backbone is disrupted by the presence of proline, which forms the beta turn. The proline isomerase is a highly conserved immunophilin family, which is composed of three subfamilies, namely an FK506 binding Family (FKBPs), a cyclophilins Family (FKBPs) and a parvulins family, and is expressed in various microorganisms, such as prokaryotes and eukaryotes, and animals and plants. In many tissue sites of the human body, expression is also abundant and can regulate various functions of cells, such as calcium-related signal regulation, protein folding and gene expression. The catalytic principle of the proline isomerase is mainly that the proline of a substrate protein is isomerized by acting on the proline of the substrate protein through the active structural domain of the proline isomerase, so that the protein conformation is changed, and the function of the protein is changed.

The storage and release of intracellular calcium signals can lead to the activation of a variety of signaling pathways, involving a variety of response pathways for autophagy, apoptosis and necrosis of cells. While the endoplasmic reticulum serves as a reservoir of intracellular calcium ions, which are regulated by the ryanodine (ryanodine) receptor and inositol triphosphate (IP 3). Meanwhile, related research reports indicate that FKBPs can bind to related receptors on endoplasmic reticulum membrane, regulate calcium release, and thus cause activation of cell signals.

There is increasing evidence that FKBPs are involved in numerous cellular processes, such as the cell cycle, cell survival and apoptotic pathways, particularly in cancer. Because in cancer tissues, the expression of FKBPs is found to be abnormal. Thus, proline isomerase plays an important role in tumor neogenesis, radiotherapy and chemotherapy reactions, and it can inhibit oncogenes or tumors in various tissues. Numerous clinical data also indicate that the proline isomerase FKBPs can be used as biomarkers for cancer diagnosis, treatment and prognosis.

At present, no detection reagent for activity and content of proline isomerase exists in the market and clinically, but the detection for activity of proline isomerase recombinant protein generally adopts a double-enzyme-linked enzyme digestion method for detection, the method mainly combines chymotrypsin digestion with activity of recombinant protein proline isomerase, and meanwhile, the method is only suitable for in vitro detection and cannot be used for detection of living cells and in vivo, so that the application range of the method is limited.

Disclosure of Invention

The invention aims to provide a fluorescent probe for detecting the activity of proline isomerase, which can quickly detect the activity of proline isomerase in vitro and in living cells. Has a structure shown in formula I:

wherein R is₁Is hydrogen or a benzene ring;

R₂is a fluorophore such as naphthoic acid, fluorescein, rhodamine, BODIPY, etc.

Still another object of the present invention is to provide a method for synthesizing a compound represented by formula I, which comprises the following steps: (1) preparing N-Fmoc-phenylalanyl-p-nitroaniline by taking phenylalanine and p-nitroaniline protected by amino Fmoc as raw materials; (2) preparation of N-terminal R by solid phase synthesis₂A modified "alanine-glycine (or phenylalanine) -proline" tripeptide chain; (3) removing Fmoc protecting group from the N-Fmoc-phenylalanyl-p-nitroaniline prepared in the step and then reacting the Fmoc protecting group with the R₂And condensing the marked tripeptide chain in a dried dichloromethane solution to obtain the fluorescent probe shown in the formula I.

Still another object of the present invention is to provide the use of the compound represented by formula I for detecting proline isomerase activity in a cell or tissue protein extract, which is achieved by the following steps:

1. tissue sample extraction: 1) collecting a tissue sample of a specific part, and storing the tissue sample at-80 ℃; 2) preparing a lysate (10mM Tris,150mM NaCl,1mM EDTA, pH7.5, adding 0.1mg/ml PMSF, 10 mu g/ml Leuteptin and 20 mu g/ml Aprotitin before use); 3) adding a certain amount of lysate according to the sample mass of 10mg/50 mu l, and grinding and cracking by using a grinding rod; 4) the cells were lysed on ice for 30min, centrifuged at 12000g for 10min, the supernatant was pipetted into a new EP tube, the protein extract was quantitatively analyzed using a kit (Bio-Rad, cat. 5000111), and finally the lysate was supplemented to a final concentration of 1. mu.g/. mu.l.

2. Cell sample extraction: 1) when the cells had grown to 90% density, the supernatant was discarded and 3ml of PBS (8.00g NaCl,0.20g KCl,3.63g Na) was added₂HPO₄·12H₂O,0.24g KH₂PO₄Adding water to a constant volume of 1000ml), cleaning for 3 times, adding 1.5ml of PBS solution, collecting cells by using a clean cell spatula, sucking the solution into a centrifuge tube, centrifuging for 4min at 1200rpm, discarding all supernatant, keeping bottom cells, and freezing at-80 ℃ for subsequent use; 2) lysates (10mM Tris,150mM NaCl,1mM EDTA, pH7.5) were prepared. Adding PMSF 0.1mg/ml, Leuteptin 10 mug/ml and Aprotitin 20 mug/ml before use); 3) adding a certain amount of lysate according to the number of cells and the ratio of 6 pore plates to 20 mul; 4) the cells were lysed on ice for 30min, centrifuged at 12000g for 10min, the supernatant was pipetted into a new EP tube, the protein extract was quantitatively analyzed using a kit (Bio-Rad, cat. 5000111), and finally the lysate was supplemented to a final concentration of 1. mu.g/. mu.l.

3. Proline isomerase activity assay in tissue/cell samples: 1) preparing a 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES) solution with the concentration of 100mM and the pH value of 8.0; 2) preparing probe I with the concentration of 5mM, and dissolving the probe I in 70% DMSO; 3) in a 96-well all-black plate, the set is HEPES + ddH₂O + Probe I, HEPES + ddH₂Adding HEPES and ddH into the O + probe I + protein extract in sequence₂O, 10 mu g of protein extracting solution and 100 mu L of probe I in total; 4) the fluorescence value was measured every 20s for 10min using a M5 microplate reader (SpectraMax) with shaking 5s before measurement, EX 360 and EM 460. And after the detection is finished, data are exported, and the fluorescence value change trend result is counted.

The fourth purpose of the invention is to provide the application of the compound shown in the formula I in detecting the activity of proline isomerase in living cells. The method is realized by the following steps: the compound of formula I was added to a DMEM high-glucose medium (brand: Gibco, cat # 12800017) to a final concentration of 5. mu.M, incubated at 37 ℃ for 30 minutes, and the fluorescence intensity of the cells was observed and recorded. The fluorescent probe provided by the invention is characterized in that the fluorescent probe only has weak fluorescence in a physiological environment, but can change the configuration under the catalytic action of proline isomerase to generate a strong-fluorescence product, and the fluorescence intensity is positively correlated with the activity of the proline isomerase, so that the specificity detection and quantitative analysis of the proline isomerase are realized.

The fluorescent probe and the detection method provided by the invention have the following beneficial effects: (1) the probe has good stability and can be stored and used for a long time; (2) the detection method is simple and convenient, the detection system is added with the probe to be incubated for 10 minutes to measure the fluorescence intensity, and the result can be given without the assistance of other reagents; (3) in vivo detection of proline isomerase activity in living cells could be achieved, whereas other reported methods could only be used for cell homogenates.

Drawings

FIG. 1 shows that the fluorescence of the HBVP cell protein extract gradually increases after the probe represented by formula Ia is added.

FIG. 2 shows that the fluorescence of the HBVP cell protein extract gradually increases when a probe represented by formula Ib is added.

FIG. 3 shows that intracellular fluorescence gradually increases after the probe represented by formula Ia is added to EA.Hy926 cell culture medium.

Detailed Description

The following examples are further illustrative of the present invention and are not intended to limit the scope of the present invention.

Example 1: synthesis of Probe molecule Ia

Removing Fmoc protecting group from N-Fmoc-phenylalanyl-p-nitroaniline, dissolving the Fmoc protecting group in a dried dichloromethane methane solution, adding equivalent alanine-phenylalanine-proline tripeptide chain marked by N, N-dimethylnaphthoic acid and equivalent EDC, condensing HOBt, and separating a prepared liquid phase to obtain the compound shown in the formula Ia. MS: 798.3550.

example 2: synthesis of Probe molecule Ib

The N-Fmoc-phenylalanyl-p-nitroaniline removes Fmoc protecting groups, is dissolved in a dried dichloromethane methane solution, and is added with an equivalent of an alanine-glycine-proline tripeptide chain marked by N, N-dimethylnaphthoic acid and an equivalent of EDC and HOBt for condensation, and the product is separated by a preparation liquid phase to obtain the compound shown in the formula Ia. MS: 722.3333.

example 3: probe molecule Ia for detecting proline isomerase activity in HBVP cell protein extracting solution

Preparing a cell protein extracting solution: preparing cell lysate (10mM Tris,150mM NaCl,1mM EDTA, pH7.5), adding 100. mu.l lysate to each bottle of cells, adding corresponding protease inhibitor Cocktail, performing lysis on ice for 30min, centrifuging at 12000g for 10min, sucking supernatant into a new EP tube, performing quantitative analysis on protein extract by using a kit (Bio-Rad company, Cat. 5000111), and finally supplementing the lysate to the final concentration of the protein extract of 1. mu.g/. mu.l.

And adding Ia into phosphate buffer salt solution to enable the final concentration to be 10 micromoles, adding 20 microliters of protein extracting solution, and placing a fluorescence spectrophotometer to record the change rule of the fluorescence emission intensity at 460nm along with time under the 363nm excitation condition. The experimental results show that: the fluorescence value of the proline isomerase fluorescent probe can be improved by adding the protein extract, so that the fact that the protein extract contains proline isomerase is proved, and the proline isomerase fluorescent probe can change the fluorescence value to respond to the proline isomerase fluorescent probe is proved. See fig. 1.

Example 4: probe molecule Ib for detecting activity of proline isomerase in HBVP cell protein extracting solution

Ib is added into phosphate buffer salt solution to enable the final concentration to be 10 micromole, 20 microliter of protein extracting solution is added, and a fluorescence spectrophotometer is arranged to record the change rule of the fluorescence emission intensity at 460nm along with time under the 363nm excitation condition. The experimental results show that: the fluorescence value of the proline isomerase fluorescent probe can be improved by adding the protein extract, so that the fact that the protein extract contains proline isomerase is proved, and the proline isomerase fluorescent probe can change the fluorescence value to respond to the proline isomerase fluorescent probe is proved. See fig. 2.

Example 5: probe molecule Ia detection of proline isomerase in EA.Hy926 live cell

When the cells grew to 90% density, they were passaged to a glass petri dish at a ratio of 1: 5. After 48 hours, adding the compound shown in the formula I into the cell culture medium to ensure that the final concentration is 5 mu M, and uniformly mixing; and (3) using a fluorescence microscope, starting a 405nm laser, collecting a fluorescence signal of the compound shown in the formula I, and recording the fluorescence of the cells in real time. The results are shown in FIG. 3.

Claims

Translated fromChinese

1.一种检测脯氨酸异构酶活性的荧光探针，其特征在于，具有式 I 所示的结构：1. A fluorescent probe for detecting proline isomerase activity, characterized in that it has the structure shown in formula I:

其中，R₁为氢或者苯环；Wherein, R₁ is hydrogen or a benzene ring;

R₂为荧光团，选自萘甲酸；所述荧光探针通过以下步骤实现：R₂ is a fluorophore, selected from naphthoic acid; the fluorescent probe is achieved through the following steps:

（1）以氨基 Fmoc 保护的苯丙氨酸及对硝基苯胺为原料制备N-Fmoc-苯丙氨酰对硝基苯胺；（2）固相合成法制备N端R₂修饰的“丙氨酸-甘氨酸或苯丙氨酸-脯氨酸”三肽链；(1) N-Fmoc-phenylalanyl-p-nitroaniline was prepared from amino-Fmoc-protected phenylalanine and p-nitroaniline; (2) N-terminal R₂ modified "alanine" was prepared by solid-phase synthesis Acid-glycine or phenylalanine-proline" tripeptide chain;

（3）将上述步骤制备的“N-Fmoc-苯丙氨酰对硝基苯胺”脱除 Fmoc保护基后与“R₂标记的三肽链”在干燥的二氯甲烷溶液中缩合，得到式I所示荧光探针。(3) The "N-Fmoc-phenylalanyl-p-nitroaniline" prepared in the above steps is decondensed with the "R₂ -labeled tripeptide chain" after removing the Fmoc protective group in a dry dichloromethane solution to obtain the formula Fluorescent probe shown in I.

2.根据权利要求 1 所述的式I化合物在制备检测组织蛋白质提取液中脯氨酸异构酶活性的试剂中的应用；其特征在于，通过以下步骤实现：2. The application of the compound of formula I according to claim 1 in the preparation of a reagent for detecting proline isomerase activity in tissue protein extracts; it is characterized in that, it is achieved by the following steps:

（1）组织样本提取：a）收集特定部位组织样本，储存于-80℃；b）配制裂解液；c）根据样本质量按10mg/50μl加入裂解液，使用研磨棒进行研磨裂解；d)冰上裂解 30min，12000g 离心10min，吸取上清至新的EP管中，使用Bio-Rad公司的试剂盒对蛋白质提取液进行定量分析，最后补充裂解液至蛋白质提取液终浓度为1μg/μl；所述裂解液为10 mM Tris， 150mMNaCl， 1mM EDTA， pH7.5，临用前加入PMSF 0.1mg/ml，Leupeptin 10μg/ml，Aprotinin 20μg/ml；(1) Tissue sample extraction: a) Collect tissue samples from specific parts and store at -80°C; b) Prepare lysis solution; c) Add 10 mg/50 μl of lysis solution according to the sample quality, and use a grinding rod to grind and lyse; d) Ice Lysis was performed for 30 min, centrifuged at 12000g for 10 min, the supernatant was transferred to a new EP tube, and the protein extract was quantitatively analyzed using a kit from Bio-Rad, and finally the lysate was supplemented to a final concentration of 1 μg/μl of the protein extract; The lysate was 10 mM Tris, 150 mM NaCl, 1 mM EDTA, pH 7.5, PMSF 0.1 mg/ml, Leupeptin 10 μg/ml, Aprotinin 20 μg/ml were added before use;

（2）细胞样本提取：a）待细胞长至90%密度时，弃上清，加入3ml的PBS溶液清洗3次，最后加入1.5 ml PBS溶液，使用干净的细胞刮铲将细胞收集，吸取溶液至离心管中，1200 rpm离心4 min，弃掉所有上清，保留底部细胞，冻存-80℃便于后续使用；b)配制裂解液；c）根据细胞数量按6孔板/20μl加入裂解液；d)冰上裂解30 min，12000 g离心10 min，吸取上清至新的EP管中，使用Bio-Rad公司的试剂盒对蛋白质提取液进行定量分析，最后补充裂解液至蛋白质提取液终浓度为1μg/μl；所述PBS溶液为：8.00 g NaCl， 0.20 g KCl，3.63gNa₂HPO₄•12H₂O，0.24g KH₂PO₄加水定容至1000ml；配制的裂解液为10mM Tris，150 mM NaCl，1 mM EDTA， pH7.5，临用前加入PMSF 0.1mg/ml，Leupeptin 10μg/ml，Aprotinin 20μg/ml；(2) Extraction of cell samples: a) When the cells grow to 90% density, discard the supernatant, add 3 ml of PBS solution to wash 3 times, and finally add 1.5 ml of PBS solution, use a clean cell scraper to collect the cells and aspirate the solution Transfer to a centrifuge tube, centrifuge at 1200 rpm for 4 min, discard all supernatant, keep the bottom cells, and freeze at -80°C for subsequent use; b) Prepare lysate; c) Add lysate to 6-well plate/20μl according to the number of cells ;d) Lyse on ice for 30 min, centrifuge at 12,000 g for 10 min, pipette the supernatant into a new EP tube, use Bio-Rad's kit for quantitative analysis of the protein extract, and finally supplement the lysate to the end of the protein extract. The concentration is 1 μg/μl; the PBS solution is: 8.00 g NaCl, 0.20 g KCl, 3.63 g Na₂ HPO₄ 12H₂ O, 0.24 g KH₂ PO₄ and water to make up to 1000 ml; the prepared lysis solution is 10 mM Tris, 150 mM NaCl, 1 mM EDTA, pH 7.5, PMSF 0.1 mg/ml, Leupeptin 10 μg/ml, Aprotinin 20 μg/ml were added immediately before use;

（3）组织/细胞样品中脯氨酸异构酶活性测试：a）配制浓度为100 mM，pH=8.0的4-羟乙基哌嗪乙磺酸溶液；b）配制浓度为5 mM 的探针I，溶解于70% DMSO中；c）在96孔全黑色板中，设置组别为4-羟乙基哌嗪乙磺酸+ddH₂O+探针I，4-羟乙基哌嗪乙磺酸+ddH₂O+探针I+蛋白质提取液，依次加入4-羟乙基哌嗪乙磺酸溶液，ddH₂O，蛋白质提取液10μg，探针I，总体系为100μL；d）使用M5多功能酶标仪，设置检测前晃动5s，EX=360，EM=460，每20s检测一次荧光值，共检测10 min，检测完成后，导出数据，统计荧光值变化趋势结果。(3) Test of proline isomerase activity in tissue/cell samples: a) 4-hydroxyethylpiperazine ethanesulfonic acid solution with a concentration of 100 mM and pH=8.0; b) a probe with a concentration of 5 mM. Needle I, dissolved in 70% DMSO; c) In a 96-well full black plate, set the group to 4-hydroxyethylpiperazineethanesulfonic acid + ddH₂ O + probe I, 4-hydroxyethylpiperazineethane Sulfonic acid + ddH₂ O + probe I + protein extract solution, followed by adding 4-hydroxyethylpiperazine ethanesulfonic acid solution, ddH₂ O, 10 μg of protein extract solution, and probe I, the total system is 100 μL; d) Use M5 polyamide The functional microplate reader is set to shake for 5s before detection, EX=360, EM=460, and the fluorescence value is detected every 20s for a total of 10 minutes.

3.根据权利要求 1 所述的式I化合物在制备检测活细胞中脯氨酸异构酶活性的试剂中的应用。3. The use of the compound of formula I according to claim 1 in the preparation of a reagent for detecting proline isomerase activity in living cells.

4.根据权利要求3所述的应用，其特征在于，通过以下步骤实现：在DMEM高糖培养基中加入式I化合物，使其终浓度为5μM，37℃下孵育30分钟，观察记录细胞荧光强度；式I化合物的荧光强度与脯氨酸异构酶的活性之间存在正向相关，从而实现对脯氨酸异构酶的特异性检测和定量分析。4. The application according to claim 3, characterized in that, it is realized by the following steps: adding the compound of formula I in DMEM high-glucose medium, making its final concentration 5 μM, incubating at 37°C for 30 minutes, observing and recording cell fluorescence Intensity; there is a positive correlation between the fluorescence intensity of the compound of formula I and the activity of proline isomerase, thereby realizing the specific detection and quantitative analysis of proline isomerase.