Biological probes for detecting Pin1 isomeric activity and their applications

Abstract

The present invention belongs to the field of biotechnology, and specifically relates to a biological probe for detecting Pin1 isomerase activity and its application. The biological probe consists of a donor-acceptor pair, a linker, and a Pin1 recognition domain; wherein the donor is a green fluorescent protein called mClover3, and the acceptor is a red fluorescent protein called mRuby3; the recognition domain is a peptide segment containing the potential recognition site Ser / Thr-Pro of Pin1; and the linker is a short peptide rich in hydrophobic amino acid residues. Based on the principle of fluorescence resonance energy transfer, this patent proposes for the first time to use the Ser / Thr-Pro motif as a recognition domain to construct a FRET biological probe that can detect Pin1 isomerase activity in different living cells. This patent not only provides a new method for studying the pathogenesis and early diagnosis of cancer and AD, but can also evolve into a high-throughput screening device for anti-cancer drugs and anti-AD drugs.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a biological probe for detecting Pin1 isomerization activity and an application thereof. Background Art

[0002] Peptidyl-prolyl cis-trans isomerase 1 (Pin1) is a peptide prolyl cis-trans isomerase that interacts with the protein NIMA and is the only known enzyme catalyzing cis-trans isomerization. Pin1 consists of an N-terminal WW domain, a central flexible linker, and a C-terminal PPIase domain. Each domain has distinct ligand-binding sites, but only the PPIase domain catalyzes substrate isomerization. The occurrence and prognosis of human metabolic diseases, cardiovascular diseases, neurodegenerative diseases, and cancer are associated with abnormal intracellular peptidyl-prolyl cis-trans isomerase 1 activity. In particular, Pin1 can promote cancer development, proliferation, invasion, and metastasis, making it an important target for cancer therapy. Pin1-targeted drug screening, as well as the diagnosis, prognosis, and prognosis of cancer and Alzheimer's disease (AD), all rely on the detection of intracellular Pin1 activity.

[0003] With the exception of a few reports, current methods for detecting Pin1 in cells mostly use PCR, quantitative PCR, and various immunological experimental methods to identify the expression and activity of Pin1 in cells at the transcriptional and translational levels, such as immunohistochemistry, immunoblotting, and immunofluorescence. However, these methods are not only cumbersome and require a lot of manpower and material resources, but also require cell disruption for detection. In particular, the results obtained by these traditional detection methods can only indicate the abundance of Pin1 protein or mRNA, or the interaction between Pin1 and its substrate, but cannot explain the changes in Pin1 isomeric activity. Previous methods for detecting Pin1 catalytic substrate isomers have only been found in Tau (pThr231-Pro232) in neurons, and methods for detecting Pin1 isomeric activity in other cells have rarely been reported.

[0004] Fluorescence resonance energy transfer (FRET) technology has a unique advantage in detecting changes in tiny spatial distances between molecules. In previous studies, we found that the distance between the two ends of the Pin1 target site (pSer / Thr-Pro) changes with the conformational changes of pSer / Thr-Pro. Based on the principle of fluorescence resonance energy transfer (FRET), this patent proposes for the first time to use the Ser / Thr-Pro motif as a recognition domain to construct a FRET bioprobe that can detect Pin1 isomeric activity and the direction of conformational changes in different living cells. This probe is used to explore the mechanism of Pin1 isomeric activity and conformational change response in cells with different Pin1 expression levels. This patent lays the foundation for subsequent research on the mechanisms of cancer and AD pathogenesis, early diagnosis, and high-throughput screening equipment for anticancer drugs. Summary of the Invention

[0005] One of the purposes of the present invention is to provide a biological probe based on the principle of fluorescence resonance energy transfer, which can be transferred into cells through transgenic technology and convert the heterogeneous activity of Pin1 in cells into fluorescent signals of different wavelengths, thereby realizing non-destructive detection of heterogeneous activity of Pin1 in cells.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] A biological probe based on the principle of fluorescence resonance energy transfer, the biological probe consists of a donor-acceptor pair, a linker and a Pin1 recognition domain; the donor-acceptor pair is two fluorescent proteins of different colors; the Pin1 recognition domain is a peptide segment containing the potential recognition site Ser / Thr-Pro of Pin1; the linker is a short peptide rich in hydrophobic amino acid residues.

[0008] Preliminary studies of this patent found that Pin1 can specifically recognize and cause conformational changes in exogenous probes; at the same time, studies have shown that cancer factors can specifically recognize and cause conformational changes in exogenous probes.

[0009] Furthermore, the donor is a green fluorescent protein called mClover3, and the acceptor is a red fluorescent protein called mRuby3.

[0010] Furthermore, the sequence of Pin1 is shown as SEQ ID No.1.

[0011] Furthermore, the connector includes connector 1 and connector 2; the connector 1 is connected to the donor and the Pin1 recognition domain; the connector 2 is connected to the receptor and the Pin1 recognition domain; the amino acid residue sequences of the connector 1 and the connector 2 are the same, as shown in SEQ ID No. 2.

[0012] Furthermore, the spatial distance between the donor and the acceptor is 1 to 10.08 nm.

[0013] Furthermore, different Pin1 recognition domains were designed based on the two different conformations of Pin1 produced after Pin1 binds to the substrate molecule, namely trans and cis. The amino acid sequence of the trans-recognition domain is shown in SEQ ID No. 3; the amino acid sequence of the cis-recognition domain is shown in SEQ ID No. 4.

[0014] The recognition domain is a crucial core component of the probe. The success of its design directly impacts the ability of FRET bioprobes to be recognized by intracellular biomolecules and to convert intracellular biological information into fluorescent signals that can be received and analyzed by extracellular instruments such as confocal fluorescence microscopy. This patent study found that the optimal FRET effect is achieved when the spatial distance between the donor and acceptor fluorescent groups is between 1 and 10.08 nm. As the only enzyme discovered in mammalian cells that catalyzes protein cis-trans isomerization, Pin1 requires that its substrate protein recognition sites, Ser-Pro or Thr-Pro, be recognized and phosphorylated by intracellular kinases in order to exert its isomerization effect. However, for transgenic exogenous probe proteins, although the Ser-Pro or Thr-Pro residues in the probe recognition domain are also potential binding and isomerization sites for Pin1, not all probe recognition domains are recognized and phosphorylated by intracellular kinases due to the influence of the FRET donor-acceptor fluorescent pair, thereby enabling Pin1 to recognize and bind, and undergo isomerization at the binding site.

[0015] Our previous studies have found that after the probe of the invention is introduced into cells with high levels of Pin1 expression, the Ser / Thr-Pro isomerization of the probe recognition domain (SEQ ID No. 3 and SEQ ID No. 4) will cause the distance between the central carbon atoms at both ends to change by about 0.1 nm, and the peptide bond to twist by 180°. The "linker 1-recognition domain-linker 2" component used to construct the FRET biological probe is composed of about 30 amino acid residues, and the maximum linear space distance between the donor and acceptor fluorescent groups at both ends (calculated based on the average distance between amino acid residues of 0.36 nm) is 10.08 nm. Therefore, the range of variation in the distance between the donor-acceptor fluorescent pair of the probe of the present invention is basically consistent with the effective distance range of the FRET effect (1-10 nm). In addition, the previous studies of the present invention also found that when the isomerization activity of Pin1 in the cell changes, the FRET value between the donor and acceptor will also change accordingly. The difference is that the FRET value of the SEQ ID No. 3 recognition domain decreases in cells with high Pin1 expression, indicating an increase in trans conformation molecules; while the SEQ ID No. 4 recognition domain exhibits the opposite behavior, increasing the FRET value in cells with high Pin1 expression, indicating an increase in cis conformation molecules. The changes in FRET values caused by both modifications can be observed using instruments such as confocal fluorescence microscopy, so probes containing both recognition domains can be used to detect intracellular Pin1 isoform activity.

[0016] Furthermore, the method for using the biological probe to detect Pin1 isomerase activity in vitro cells is as follows: synthesizing the encoding gene DNA fragment of the biological probe fusion protein by chemical synthesis; then using DNA recombination technology to insert the DNA fragment into the multiple cloning site of the eukaryotic gene expression plasmid, and after the recombinant plasmid is confirmed to be correct by sequencing alignment and open reading frame (ORF) analysis, the recombinant plasmid is introduced into the cancer cell line; 48 hours after the transgenic, the 488nm and 543nm wavelength excitation light images of the cancer cell line are observed by laser confocal microscopy, and FRET analysis is performed based on the fluorescence intensity values at different wavelengths.

[0017] Furthermore, the amino acid sequences of the coding gene DNA fragments are shown in SEQ ID No. 5 and SEQ ID No. 6; SEQ ID No. 5 contains a DNA fragment of a trans-recognition domain, and SEQ ID No. 6 contains a DNA fragment of a cis-recognition domain.

[0018] Furthermore, the transgenic agent includes liposomes and the like.

[0019] Furthermore, the cancer cell lines include but are not limited to SGC-7901, MCS, MKN-45 and MGC-803 gastric cancer cell lines; in previous studies, the present invention found that SGC-7901 and MCS cell lines are gastric cancer cells with relatively low Pin1 expression levels, and MGC-803 and MKN-45 cell lines are gastric cancer cells with relatively high Pin1 expression levels.

[0020] Furthermore, the method specifically comprises the following steps: first, chemically synthesizing DNA fragments encoding the probe fusion protein, the sequences of which are set forth in SEQ ID No. 5 and SEQ ID No. 6. Sequence No. 5 contains the trans-recognition domain, and SEQ ID No. 6 contains the cis-recognition domain. The synthesized DNA fragments are then inserted into the multiple cloning site of a eukaryotic gene expression plasmid using DNA recombination technology. After the recombinant plasmids are confirmed to be correct through sequencing alignment and open reading frame (ORF) analysis, the recombinant plasmids are then introduced into SGC-7901 and MGC-803 gastric cancer cell lines using transgenic agents such as liposomes (preliminary studies of this patent have shown that the SGC-7901 cell line is a gastric cancer cell line with relatively low Pin1 expression levels, while the MGC-803 cell line is a gastric cancer cell line with relatively high Pin1 expression levels). Forty-eight hours after transfection, images of the two cell lines were observed using a laser confocal microscope under 488 nm and 543 nm excitation wavelengths, and FRET analysis was performed based on the fluorescence intensity values at different wavelengths.

[0021] A second object of the present invention is to provide a method for preparing model cells for high-throughput screening of potential anticancer drugs.

[0022] High-level expression of Pin1 in cells will activate the expression of more than 50 oncogenes while inhibiting the expression of more than 20 tumor suppressor genes. Therefore, Pin1 has become an effective anti-cancer target recognized by the academic community. Previous studies of the present invention have found that high-level expression of Pin1 in cells can significantly promote the proliferation, invasion and metastasis of gastric cancer, while RNAi and Pin1 inhibitors can inhibit Pin1 activity and thus inhibit the proliferation, invasion and metastasis of gastric cancer cells. Therefore, Pin1 inhibitors are a class of potential anti-cancer drugs with great application prospects. Screening of Pin1 inhibitors is inseparable from the detection of Pin1 activity in cells. The biological probe of the present invention can realize non-destructive detection of intracellular Pin1 isomeric activity. The probe of the invention can not only detect the extent of the drug's effect on Pin1 isomeric activity, but also simplify the complex Pin1 expression level detection process to the observation of optical signals, which is conducive to the high-throughput screening of targeted Pin1 inhibitors. Since the chemical nature of the probe is a fusion protein, the coding gene of the fusion protein can be introduced into any eukaryotic cell (including various cancer cells) for biosynthesis. If the introduced coding gene fragment can be integrated into the genomic DNA of cancer cells and passed to daughter cells as the cancer cells divide, then various cancer cells containing the probes described in the invention can be mass-produced using in vitro cell culture methods. After further combining these different types of cancer cells with cell chip preparation technology to make cell arrays, high-throughput screening of anti-cancer drugs targeting Pin1 inhibitors can be carried out according to the technical scheme described in one of the objectives of the present invention.

[0023] To achieve the above object, the present invention adopts the following technical solutions:

[0024] A method for preparing model cells for high-throughput screening of potential anticancer drugs comprises: chemically synthesizing a DNA fragment as shown in SEQ ID No. 5 and / or SEQ ID No. 6, inserting the fragment into the multiple cloning site of a lentiviral vector plasmid, packaging the recombinant lentiviral vector plasmid into replication-defective lentiviral particles, and then transfecting various cancer cells; after the lentiviral particles are transfected, screening and purifying cell lines stably expressing the biological probe integrin using a screening method corresponding to a screening marker gene to obtain model cells for high-throughput screening of potential anticancer drugs.

[0025] Furthermore, the lentiviral vector plasmid includes but is not limited to PLVX and / or PCDH, etc.

[0026] Furthermore, the cancer cells include but are not limited to gastric cancer cells, lung cancer cells, liver cancer cells, intestinal cancer cells and / or pancreatic cancer cells.

[0027] Furthermore, the method is specifically as follows: after chemically synthesizing the DNA fragment of SEQ ID No. 5 or SEQ ID No. 6, it is inserted into the multiple cloning site of the lentiviral vector plasmid, and the recombinant lentiviral recombinant vector plasmid is packaged into replication-defective lentiviral particles and then transfected into various cancer cells. Since the lentiviral-mediated transgenic operation will integrate the DNA fragment of SEQ ID No. 5 or SEQ ID No. 6 and the eukaryotic selection marker gene into the genomic DNA of the cancer cell, and pass it to the daughter cells as the cancer cell divides, the daughter cells will express the selection marker gene at the same time when expressing the probe gene. Therefore, after the lentiviral particles are transfected, the cell line stably expressing the probe integration protein can be screened and purified by the screening method corresponding to the selection marker gene, and then the high-throughput screening of anticancer drugs targeting Pin1 can be carried out according to the technical solution described in one of the objectives of the present invention.

[0028] A third object of the present invention is to provide a method for preparing model cells for high-throughput screening of potential AD therapeutic drugs.

[0029] In contrast to cancer cells, Pin1 activity in hippocampal neurons of AD patients is low. There are a large number of pathogenic cis-Tau (Thr231-Pro) proteins in the hippocampal neurons of AD patients' brains. Pin1 can accelerate the conversion of cis-Tau (Thr231-Pro) to trans-Tau (Thr231-Pro), thereby preventing the accumulation of pathogenic cis-Tau (Thr231-Pro) in AD, reducing NFTs and preventing the occurrence of neurodegenerative diseases such as AD. Therefore, Pin1 activators are a class of potential AD therapeutic drugs with great application prospects. Since the recognition domain (SEQ ID No. 3) of the probe of the present invention can be recognized by Pin1 and related biological molecules in AD pathological cells and model cells, and can produce and simulate the conformational change process of cis-Tau (Thr231-Pro) to trans-Tau (Thr231-Pro), the Pin1 activity detection technology of the present invention can also achieve high-throughput screening of potential AD therapeutic drugs.

[0030] To achieve the above object, the present invention adopts the following technical solutions:

[0031] A method for preparing model cells for high-throughput screening of potential AD therapeutic drugs comprises: chemically synthesizing a DNA fragment with an amino acid sequence as shown in SEQ ID No. 5, inserting the fragment into the multiple cloning site of a lentiviral vector plasmid, packaging the recombinant lentiviral vector plasmid into replication-defective lentiviral particles, and then transfecting various AD lesion model cells; after the lentiviral particles are transfected, cell lines stably expressing the probe integrin are screened and purified using a screening method corresponding to a screening marker gene, thereby obtaining model cells for high-throughput screening of potential AD therapeutic drugs.

[0032] Furthermore, the lentiviral vector plasmid includes PLVX and / or PCDH.

[0033] Furthermore, the AD lesion model cells include but are not limited to HPPNCs cells, SK-N-SH cells, SH-SY5Y cells, NG108-15 cells and / or HEK293 cells.

[0034] Furthermore, the method is specifically as follows: after synthesizing the DNA fragment described in SEQ ID No. 5 by a chemical method, inserting it into the multiple cloning site of the lentiviral vector plasmid, and packaging the recombinant lentiviral recombinant vector plasmid into replication-defective lentiviral particles and then transfecting various AD pathology model cells. Since the lentiviral-mediated transgenic operation will integrate the DNA fragment described in SEQ ID No. 5 and the eukaryotic screening marker gene into the genomic DNA of the cancer cells, and pass it to the daughter cells as the cancer cells divide, the daughter cells will express the screening marker gene at the same time when expressing the probe gene. Therefore, after the lentiviral particles are transfected, the cell line stably expressing the probe integration protein can be screened and purified by the screening method corresponding to the screening marker gene, and then the high-throughput screening of anti-AD drugs can be carried out according to the technical solution described in one of the purposes of the present invention.

[0035] The beneficial effects of the present invention are:

[0036] 1. This patent, for the first time, utilizes the principle of spatial distance changes caused by peptide bond isomerization at the Pin1 substrate protein binding site. It innovatively proposes using Pin1's potential recognition and binding site, Ser / Thr-Pro, to construct a FRET bioprobe. Transgenic technology is then used to deliver the FRET bioprobe into cells. The probe interacts with Pin1 through its recognition domain and converts Pin1's isomerized activity signal into a fluorescent signal, thereby enabling real-time dynamic observation of changes in Pin1 isomerized activity without damaging the cells. This patent lays the foundation for research on cell proliferation, differentiation, apoptosis, and high-throughput screening of anti-tumor drugs.

[0037] 2. This patent utilizes the "pSer / Thr-Pro" site of the FRET biosensor junction domain stably expressed within cells to sense changes in Pin1 activity caused by cell mutations. Through the isomerization reaction occurring at the pSer / Thr-Pro site, changes in Pin1's catalytic activity are converted into fluorescent signals of varying wavelengths and intensities. This solves the challenge of timely and dynamic observation of changes in Pin1 activity and cis-isomer levels outside cells. This patent provides a new method for studying protein conformation and function in the biological field.

[0038] 3. This patent not only provides a practical method for detecting Pin1 isomeric activity and the direction of substrate conformational changes in living cells, but also provides a FRET biological probe that is stably expressed in cells. This biological probe can be mass-produced through in vitro culture, simplifying the probe production process and saving time and costs, thereby laying the foundation for subsequent research and industrial promotion and application of the mechanism of Pin1 regulating cell life activities, early diagnosis of cancer and AD, and high-throughput screening of anti-cancer drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 Schematic diagram of the structure of the FRET bioprobe used for Pin1 substrate conformation detection;

[0040] Figure 2 The graph shows the expression detection results of Pin1 in gastric cancer cell lines and gastric cancer specimens of patients. Figure 2 A is the relative expression level of Pin1 mRNA in gastric cancer cell lines detected by qPCR; Figure 2 B is the Western blot detection result of Pin1 protein expression level in different gastric cancer cell lines; Figure 2 C is the qPCR detection results of the relative expression levels of Pin1 mRNA in gastric cancer tissues and adjacent tissues of different patients; Figure 2 D is the statistical result of the relative expression level of Pin1 mRNA in gastric cancer tissues and adjacent tissues of different patients; Figure 2 E is the Western blot detection result of Pin1 protein expression level in gastric cancer tissues and adjacent tissues of different patients;

[0041] Figure 3 The figure shows the results of the responsiveness observation of the biological probe in the MGC-803 gastric cancer cell group; Figure 3 A is the fluorescence microscopic image of each group of cells under 488nm and 543nm wavelength excitation light; Figure 3 B is the variance analysis results of the FRET values of each group of cells under 488nm and 543nm wavelength excitation light;

[0042] Figure 4Observation of the responsiveness of the biological probe in the MKN-45 gastric cancer cell group; Figure 4 A is the fluorescence microscopic image of each group of cells under 488nm and 543nm wavelength excitation light; Figure 4 B is the variance analysis results of the FRET values of each group of cells under 488nm and 543nm wavelength excitation light;

[0043] Figure 5 Observation of the responsiveness of the bioprobe in SGC-7901 gastric cancer cells; Figure 5 A is the fluorescence microscopic image of each group of cells under 488nm and 543nm wavelength excitation light; Figure 5 B is the variance analysis results of the FRET values of each group of cells under 488nm and 543nm wavelength excitation light;

[0044] Figure 6 To observe the responsiveness of the biological probe in AGS gastric cancer cells; Figure 6 A is the fluorescence microscopic image of each group of cells under 488nm and 543nm wavelength excitation light; Figure 6 B is the variance analysis results of the FRET values of each group of cells under 488nm and 543nm wavelength excitation light. DETAILED DESCRIPTION

[0045] The technical solutions of the present invention will be further described in detail below with reference to specific embodiments. Obviously, the embodiments described are only a portion of the embodiments of the present invention, not all of them. Therefore, all other embodiments derived by those skilled in the art based on the embodiments of the present invention without inventive effort are intended to fall within the scope of protection of the present invention.

[0046] Example

[0047] (1) Design of FRET bioprobes

[0048] The FRET bioprobe constructed in this example consists of three parts: a donor-acceptor fluorescent group pair (DA pair), a linker, and a Pin1 recognition domain. The structural diagram of the FRET bioprobe is shown in FIG. Figure 1 As shown in the figure, the probe of the present invention is composed of a donor fluorescent protein mClover3, a linker 1, a Pin1 recognition domain, a connection system and an acceptor fluorescent protein mRuby3 from left to right.

[0049] 1) Donor-acceptor fluorescent group pair: mutant green fluorescent protein (mClover3) as the donor and mutant red fluorescent protein (mRuby3) as the acceptor;

[0050] 2) Linker: Includes Linker 1 and Linker 2. Linker 1 connects the donor and Pin1 recognition domains, and its amino acid residue composition is: "-GGSGGGGSGG-." Linker 2 connects the receptor and Pin1 recognition domains, and its amino acid residue composition is the same as Linker 1.

[0051] 3) Recognition domain: A peptide consisting of approximately 10 amino acid residues containing the potential Pin1 recognition site, Ser-Pro or Thr-Pro, in the center. Different Pin1 recognition domains were designed based on the "trans" and "cis" conformations produced by Pin1 upon probe binding. The amino acid sequence of the trans-recognition domain is shown in SEQ ID No. 3, and the amino acid sequence of the cis-recognition domain is shown in SEQ ID No. 4.

[0052] (2) Design of FRET bioprobe fusion gene and construction of expression vector

[0053] The genes encoding the three different recognition domain FRET fusion proteins were codon-optimized to avoid hairpin structures that affect fusion gene expression. Whole-gene chemical synthesis was then used to construct a eukaryotic gene expression vector plasmid expressing the FRET fusion protein gene.

[0054] (3) Pin1 expression detection results in gastric cancer cell lines and clinical gastric cancer tissue specimens

[0055] To detect the expression level of PIN1 in gastric cancer cells and clinical specimens, we detected the mRNA and protein expression levels of PIN1 in gastric cancer cell lines AGS, HGC-27, MKN-28, MKN-45, MKN-74, SGC-7901, MGC803, BGC-823 and immortalized normal gastric epithelial cell line GES. Figure 2 A is the fluorescence microscopic image of each group of cells under 488nm and 543nm wavelength excitation light. Figure 2 A shows that in the cell group transferred with cis-probe, T / A mutation probe cell group, P / A mutation probe cell group and trans-probe cell group, due to the occurrence of fluorescence energy resonance, the cells exhibited yellow fluorescence (composed of the superposition of green fluorescence and red fluorescence) under 488nm excitation light. Among them, the yellow fluorescence of the cis-probe cell group was stronger than that of the T / A mutation probe cell group and the P / A mutation probe cell group, while that of the trans-probe cell group was weaker than that of the T / A mutation probe cell group and the P / A mutation probe cell group; no yellow fluorescence was observed in the mClover3 and mRuby3 control group cells. Figure 2 B is the variance analysis result of the FRET values of each group of cells under 488nm and 543nm wavelength excitation light. Figure 2B shows that the cis-probe in the cell underwent an isomerization reaction in response to the change in Pin1 activity, making its FRET value significantly higher than that of the T / A mutation probe and P / A mutation probe cell groups; on the contrary, the FRET value of the trans-probe in cells with high Pin1 expression activity was significantly lower than that of the T / A mutation probe and P / A mutation probe cell groups. The above results show that the mRNA and protein expression levels of PIN1 are higher in most gastric cancer cell lines than in GES cells. In addition, we collected cancer and adjacent tissues from 24 clinical patients and detected the Pin1 expression level by qPCR. We found that Pin1 was also highly expressed in the cancer tissues of most patients. We further verified the qPCR results by WB experiments. Figure 2 C~ Figure 2 As shown in E, the results are consistent with expectations.

[0056] (4) Observation of the responsiveness of the bioprobe in MGC-803 gastric cancer cell lines

[0057] To observe the responsiveness of trans-probes and cis-probes in the MGC-803 gastric cancer cell group with a high level of Pin1 expression, we introduced plasmid vectors expressing donor fluorescent protein (mClover3) (negative control group), cis-probe (experimental group 1), recognition domain Pin1 binding site T / A mutation probe (Pin1 negative control group 1), recognition domain Pin1 binding site G / A mutation probe (Pin1 negative control group 2), trans-probe (experimental group 2) and receptor fluorescent protein (positive control) into MGC-803 gastric cancer cell lines. The fluorescence intensity of these cells under irradiation with 488nm and 543nm wavelength laser was observed using a laser confocal microscope, and the Fret value of each cell group was calculated and analyzed. The experimental results are shown in Figure 2. Figure 3 A and Figure 3 As shown in B. Figure 3 A is a fluorescence microscopic image of each cell group under 488nm and 543nm excitation light. As can be seen in the figure, in the cis-probe cell group, the T / A mutation probe cell group, the P / A mutation probe cell group, and the trans-probe cell group, fluorescence energy resonance occurs, resulting in yellow fluorescence (a superposition of green and red fluorescence) under 488nm excitation light. The yellow fluorescence of the cis-probe cell group is stronger than that of the T / A mutation probe cell group and the P / A mutation probe cell group, while that of the trans-probe cell group is weaker than that of the T / A mutation probe cell group and the P / A mutation probe cell group. No yellow fluorescence was observed in the mClover3 and mRuby3 control cells. Figure 3B shows the results of the analysis of variance analysis of the FRET values for each cell group under 488nm and 543nm excitation light. The figure shows that the cis-probe undergoes an isomerization reaction in response to changes in Pin1 activity, resulting in significantly higher FRET values than those in the T / A and P / A mutant probe groups. Conversely, in cells expressing high levels of Pin1, the FRET values of the trans-probe group are significantly lower than those in the T / A and P / A mutant probe groups. This suggests that, under Pin1 catalysis, the cis-probe undergoes a molecular conformational transition from trans to cis, while the reverse occurs for the trans-probe.

[0058] (5) Observation of the responsiveness of the bioprobe in MKN-45 gastric cancer cell lines

[0059] To further observe the responsiveness of trans-probes and cis-probes in the MKN-45 gastric cancer cell line with a high level of Pin1 expression, we introduced plasmid vectors expressing donor fluorescent protein (mClover3) (negative control group), cis-probe (experimental group 1), recognition domain Pin1 binding site T / A mutation probe (Pin1 negative control group 1), recognition domain Pin1 binding site G / A mutation probe (Pin1 negative control group 2), trans-probe (experimental group 2) and acceptor fluorescent protein (positive control) into MKN-45 gastric cancer cell lines and observed their fluorescence at 488 nm and 543 nm using laser confocal microscopy.

[0060] The fluorescence intensity under the irradiation of wavelength laser was calculated and analyzed, and the FRET value of each cell group was obtained. Figure 4 A and Figure 4 As shown in B. Figure 4 A is a fluorescence microscopic image of each group of cells under 488nm and 543nm wavelength excitation light. As can be seen in the figure, in the cis-probe cell group, T / A mutation probe cell group, P / A mutation probe cell group and trans-probe cell group, due to fluorescence energy resonance, the cells exhibit yellow fluorescence (composed of green fluorescence and red fluorescence superposition) under 488nm excitation light. Among them, the yellow fluorescence of the cis-probe cell group is stronger than that of the T / A mutation probe cell group and the P / A mutation probe cell group, and the yellow fluorescence of the trans-probe cell group is weaker than that of the T / A mutation probe cell group and the P / A mutation probe cell group; no yellow fluorescence was observed in the mClover3 and mRuby3 control group cells. Figure 4B is the variance analysis result of the FRET values of each group of cells under 488nm and 543nm wavelength excitation light. As can be seen in the figure, the cis-probe in the cells underwent an isomerization reaction in response to the change in Pin1 activity, making its FRET value significantly higher than that of the T / A mutant probe cell group and the P / A mutant probe cell group; on the contrary, in cells with high-level Pin1 expression activity, the FRET value of the trans-probe was significantly lower than that of the T / A mutant probe cell group and the P / A mutant probe cell group.

[0061] The results of (4) and (5) show that under the catalysis of Pin1, the molecular conformation of the cis-probe will switch from trans to cis, while the opposite is true for the trans-probe.

[0062] (6) Observation of the responsiveness of the bioprobe in SGC-7901 gastric cancer cells

[0063] To observe whether trans-probes and cis-probes are responsive in the SGC-7901 gastric cancer cell group with low Pin1 expression levels, we introduced plasmid vectors expressing donor fluorescent protein (mClover3) (negative control group), cis-probe (experimental group 1), recognition domain Pin1 binding site T / A mutation probe (Pin1 negative control group 1), recognition domain Pin1 binding site G / A mutation probe (Pin1 negative control group 2), trans-probe (experimental group 2) and receptor fluorescent protein (positive control) into MKN-45 gastric cancer cell lines. The fluorescence intensity of these probes under 488nm and 543nm wavelength laser irradiation was observed using a laser confocal microscope, and the Fret value of each cell group was calculated and analyzed. The experimental results are shown in Figure 2. Figure 5 A and Figure 5 As shown in B. Figure 5 A is a fluorescence microscopic image of each group of cells under 488nm and 543nm wavelength excitation light. As can be seen in the figure, in the cis-probe cell group, T / A mutation probe cell group, P / A mutation probe cell group and trans-probe cell group, due to fluorescence energy resonance, the cells exhibited yellow fluorescence (composed of green fluorescence and red fluorescence superposition) under 488nm excitation light. No yellow fluorescence was observed in the mClover3 and mRuby3 control group cells. Figure 5B shows the results of analysis of variance for the FRET values of each cell group under 488 nm and 543 nm excitation. As shown in the figure, the yellow fluorescence intensity of the cis-probe and trans-probe cell groups was not significantly different from that of the T / A mutant probe and P / A mutant probe cell groups. These results indicate that low intracellular Pin1 activity results in no significant difference in the FRET values of the cis-probe and trans-probe cell groups compared with the T / A mutant probe and P / A mutant probe cell groups. This suggests that under conditions of low Pin1 activity, the conformational changes of the cis-probe and trans-probe probe molecules are not significant.

[0064] (7) Observation of the responsiveness of biological probes in AGS gastric cancer cells

[0065] To further investigate the responsiveness of trans-probes and cis-probes in AGS gastric cancer cells with low Pin1 expression levels, we introduced plasmid vectors expressing donor fluorescent protein (mClover3) (negative control group), cis-probe (experimental group 1), recognition domain Pin1 binding site T / A mutation probe (Pin1 negative control group 1), recognition domain Pin1 binding site G / A mutation probe (Pin1 negative control group 2), trans-probe (experimental group 2), and receptor fluorescent protein (positive control) into MKN-45 gastric cancer cell lines. We then observed their fluorescence intensities under 488 nm and 543 nm laser irradiation using a laser confocal microscope, and calculated and analyzed the Fret values of each cell group. The experimental results are shown in Figure 2. Figure 6 A and Figure 6 As shown in B. Figure 6 A is a fluorescence microscopic image of each group of cells under 488nm and 543nm wavelength excitation light. As can be seen in the figure, in the cis-probe cell group, T / A mutation probe cell group, P / A mutation probe cell group and trans-probe cell group, due to fluorescence energy resonance, the cells exhibited yellow fluorescence (composed of green fluorescence and red fluorescence superposition) under 488nm excitation light. No yellow fluorescence was observed in the mClover3 and mRuby3 control group cells. Figure 6 B shows the results of the variance analysis of the FRET values of each cell group under 488nm and 543nm excitation light. As can be seen in the figure, the yellow fluorescence intensity of the cis-probe cell group and the trans-probe cell group was not significantly different from that of the T / A mutation probe cell group and the P / A mutation probe cell group. These results further indicate that low intracellular Pin1 activity results in no significant difference in the FRET values of the cis-probe cell group and the trans-probe cell group compared with the T / A mutation probe cell group and the P / A mutation probe cell group.

[0066] The results of (6) and (7) show that under the condition of low Pin1 activity, the conformational changes of cis-probe and trans-probe molecules are not obvious.

Claims

1. A biological probe based on the principle of fluorescence resonance energy transfer, characterized in that: The biological probe consists of a donor-acceptor pair, a connector and a Pin1 recognition domain; the donor-acceptor pair is two fluorescent proteins of different colors; the Pin1 recognition domain is a peptide segment containing the potential recognition site Ser / Thr-Pro of Pin1; the connector is a short peptide rich in hydrophobic amino acid residues; the nucleotide sequence of the gene DNA fragment encoding the biological probe is shown in SEQ ID No. 5 and SEQ ID No. 6.

Images