A fluorescent protein probe for non-invasive detection of cardiomyocytes and application thereof

Abstract

The application discloses a fluorescent protein probe for noninvasive detection of myocardial cells and application thereof, and belongs to the field of protein and gene engineering. The application obtains an affinity peptide for myocardial cells by using a phage display dodecapeptide library, combines the affinity peptide with EGFP, and then is transferred into an expression vector, so that the expression is realized by using an E. coli expression system to obtain the fluorescent protein probe. The fluorescent protein probe can be used for detecting myocardial cells, real-time myocardial cell level detection is realized, and the defects and barriers existing in traditional detection methods are broken.

Description

Technical Field

[0001] This invention belongs to the field of protein and genetic engineering technology, specifically relating to a non-invasive fluorescent protein probe for detecting cardiomyocytes and its application. Background Technology

[0002] Cardiac cardiomyocytes are key cells in cell therapy for heart failure and are widely used in fields such as tissue engineering and regenerative medicine. Although significant progress has been made in basic research and clinical translation of cardiomyocyte detection technology, there are still many technical defects that cannot be ignored, which restrict the reliability and widespread application of its results.

[0003] Because in vitro models generally lack key components such as the coronary artery system, neural regulation, and immune cells, the simulated cardiac microenvironment is highly simplified, often resulting in discrepancies between preclinical data and clinical trial results. More critically, there is currently no globally unified standard for evaluating cardiomyocyte differentiation maturity and functional testing specifications. Different research teams use vastly different evaluation index systems, making it difficult to effectively compare and integrate experimental data across platforms and laboratories.

[0004] Secondly, current detection methods are all traditional, such as immunological detection using antigens and antibodies. Current methods for detecting cardiomyocytes suffer from poor specificity, remain at the level of dead cells, and cannot achieve non-invasive detection.

[0005] Therefore, developing three-dimensional models that more closely resemble physiological states, higher-throughput and non-destructive precision detection technologies, and establishing unified industry standards to comprehensively enhance the clinical application value of myocardial cell detection technology have become important issues that urgently need to be addressed. Summary of the Invention

[0006] Therefore, the purpose of this invention is to provide a non-invasive fluorescent protein probe for detecting cardiomyocytes and its application, in order to solve the problems existing in the prior art. This fluorescent protein probe can achieve non-invasive detection of cardiomyocytes at the cellular level.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] In a first aspect, the present invention provides a non-invasive fluorescent protein probe for detecting cardiomyocytes, the amino acid sequence of which is shown in SEQ ID NO.2.

[0009] Based on the above technical solution, the fluorescent protein probe further contains an affinity peptide.

[0010] Based on the above technical solution, the amino acid sequence of the affinity peptide is further shown in SEQ ID NO.1.

[0011] Based on the above technical solution, the affinity peptide can specifically recognize and bind to cardiomyocytes.

[0012] Secondly, the present invention provides a gene encoding the fluorescent protein probe.

[0013] Thirdly, the present invention provides a recombinant expression vector into which the gene is inserted.

[0014] Fourthly, the present invention provides a recombinant engineered bacterium carrying the recombinant expression vector.

[0015] Fifthly, the present invention provides the application of the fluorescent protein probe, the gene, the recombinant expression vector, or the recombinant engineered bacteria in the preparation of non-invasive detection products for cardiomyocytes.

[0016] Sixthly, the present invention provides a non-invasive method for detecting cardiomyocytes, wherein the fluorescent protein probe is co-incubated with the cardiomyocytes to be tested, the fluorescence signal of the cells is detected, and the presence or number of cardiomyocytes is determined.

[0017] Based on the above technical solution, the co-incubation time is further 0.5~2h.

[0018] Compared with the prior art, the present invention has the following beneficial effects: This invention uses cardiomyocytes as targets for affinity peptide ligand screening to obtain highly specific binding affinity peptides. By combining these affinity peptides with on-feed fluorescent protein technology, a target fluorescent protein probe is designed and constructed. This probe enables simple detection by switching fluorescence on when cardiomyocytes are present and off when they are absent. This achieves real-time, non-invasive detection at the cardiomyocyte level, breaking through the technical barriers of traditional detection methods. Attached Figure Description

[0019] To more clearly illustrate the embodiments of the present invention, the accompanying drawings involved in the embodiments will be briefly described below.

[0020] Figure 1 This is a flowchart of the whole-cell subtraction method for screening myocardial cell affinity peptides using bacteriophages in Example 1 of the present invention.

[0021] Figure 2 This is a graph showing the results of ELISA detection of the binding affinity of the phage corresponding to the affinity peptide to target cells and negative cells in Example 1 of the present invention.

[0022] Figure 3 This is a graph showing the results of further detection of the real-time binding strength between the affinity peptide and the target cell using QCM-D in Example 1 of the present invention.

[0023] Figure 4This is a schematic diagram of the probe designed to bind the affinity peptide to the fluorescent protein in Example 2 of the present invention.

[0024] Figure 5 The image shows the fluorescence spectrum detection results of the fluorescent protein probe in Example 3 of this invention after incubation with AC16, MSCs and fibroblasts, respectively.

[0025] Figure 6 This is a diagram showing the fluorescence labeling results detected by fluorescence microscopy after incubation of the fluorescent protein probe with cardiomyocytes in Example 4 of the present invention.

[0026] Figure 7 This is a flow cytometry result of the fluorescent labeling of cardiomyocytes after incubation with the fluorescent protein probe in Example 4 of the present invention. Detailed Implementation

[0027] The present invention will be described in detail below with reference to the embodiments. However, the implementation of the present invention is not limited thereto. Obviously, the embodiments described below are only some embodiments of the present invention. For those skilled in the art, other similar embodiments can be obtained without creative effort and all fall within the protection scope of the present invention.

[0028] Example 1 Screening of affinity peptides Cardiomyocytes (AC16), mesenchymal stem cells (MSCs), and fibroblasts purchased from ATCC were seeded into three 60mm × 15mm culture dishes and cultured in a 37℃ incubator. When the cell density reached 80%, screening was performed. The screening procedure is as follows: Figure 1 As shown.

[0029] First, take an AC16 dish, add 1 mL of 5% BSA-DMEM / F12 blocking buffer, block for 1 h, discard the buffer, and wash the cells three times with PBS; then add 1 mL of a phage peptide library (titer approximately 1.0 × 10⁻⁶). 11 Incubate cells in DMEM / F12 medium (PFU) for 1 h. Discard the solution and wash cells 6 times with TBST (0.1%). Add 1 mL of glycine elution buffer (pH 2.2), shake on a shaker for 40 min, and aspirate the eluent into a 1.5 mL centrifuge tube. Add 150 μl of Tris-1 to the 1.5 mL centrifuge tube. Neutralize and preserve with HCl (pH 9.1); transfer the neutralized preservation eluent to MSCs blocked with 5% BSA-DMEM for 1 hour, incubate for 1 hour, then aspirate the supernatant and transfer it again to fibroblasts blocked with 5% BSA-α-MEM for 1 hour, incubate for 1 hour. The supernatant at this point is the phage library obtained in the first round of screening. Infect host bacteria. E. coliER2738 was used to determine the screening titer and amplify the phage library for the next round of screening. After three rounds of screening, the phage titer was determined, and 25 phage clones were randomly selected from plates with fewer than 100 phage clone blue spots and sent for sequencing.

[0030] ELISA was used to detect the affinity of the sequencing-derived affinity peptides for AC16, MSCs, and fibroblasts. When the three cell types reached 80% confluence in 96-well plates, they were fixed with 4% paraformaldehyde for 15 min, washed three times with 0.05% PBST, and then fixed with 0.1% Triton X. Treatment with 100 mg / L solution for 10 min, followed by washing three times with 0.05% PBST; blocking with 2% BSA for 1 h, then adding approximately 1.5 × 10⁻⁶ drops. 11 Phages were incubated at 37°C for 2 hours, washed three times with 0.05% PBST; M13 antibody labeled with shangen peroxidase (1:5000 dilution) was added, incubated for 1 hour, washed three times with 0.05% PBST; ABTS colorimetric reagent was added, and readings were taken at 410 nm using a multi-functional microplate reader. Unbound phages from the first round were used as controls. Results are as follows. Figure 2 As shown.

[0031] QCM-D further detected the real-time binding strength of the two preferred affinity peptides to the target cells. PBS was introduced into each of the two detection channels. After the PBS remained stable at the gold tablet for 5 minutes, AC16 cardiomyocytes were introduced into each channel. After the AC16 remained stable at the gold tablet for a period of time, the preferred affinity peptides P1 and P3 were introduced into each channel. The results are as follows. Figure 3 As shown, the affinity peptide P1 has a stronger binding force than P3, so P1 was selected as the AC16 affinity peptide.

[0032] The amino acid sequence of the affinity peptide is shown below (SEQ ID NO.1): TIPNLTRVSNIV Example 2: Construction, Expression, and Purification of Fluorescent Protein Probe Plasmids Using open-ended fluorescent protein technology, a fluorescent protein probe was designed and synthesized by binding the affinity peptide obtained in Example 1 to EGFP with a flexible linker L1 and a rigid linker L2. The probe structure is shown in the schematic diagram below. Figure 4 As shown in the image. The gene encoding the fluorescent protein probe was then transferred into the pRset-B expression vector to construct a recombinant plasmid. The successfully constructed recombinant plasmid was then transferred into *E. coli* BL21 competent cells, and the target fluorescent protein probe was obtained through routine extraction and purification experiments.

[0033] The amino acid sequence of the fluorescent protein probe is shown below (SEQ ID NO.2): MRGSHHHHHHHHGMMVSKGEELFTGVVPILVELDGDVNGHKFSVYGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAAMPEGYTQERTIFFEDDGNYKTRAEVKFEGDT LVNRIELKGIDFKEDGNILGHKLEYNLPDGTIPNLTRVSNIVAANNVYIMADKEKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGPVLLPDNHYLSVQSRLSKDPNEKRDHMVLLEFVTAAGITLGMDELYM Example 3: Detection of the effectiveness of fluorescent protein probes The fluorescent protein probes prepared in Example 2 were directly loaded into AC16, MSCs and fibroblasts at a concentration of 10 ng / mL, respectively. Using cell-free DMEM / F12 medium as a control, the cells were incubated at 37°C for 1 hour, and the fluorescence intensity emitted at a wavelength of 515 nm was detected by a multi-functional microplate reader.

[0034] like Figure 5 As shown, the fluorescence intensity of the fluorescent protein probe increased 4.5-fold after the addition of AC16, while the fluorescence intensity of MSCs and fibroblasts was almost identical to that of the control group without cells. This demonstrates that the fluorescent protein probe prepared in this invention can be used to detect AC16.

[0035] Example 4: Detection of fluorescent protein probe fluorescent labeling The probe protein prepared in Example 2 was added directly to AC16 at 10 ng / mL and incubated at 37°C for 1 h. Fluorescence was excited at 488 nm, and the fluorescence labeling efficiency was detected by fluorescence confocal microscopy and flow cytometry.

[0036] Test results as follows Figure 6 and Figure 7 As shown, the fluorescent labeling rate of the fluorescent protein probe can reach over 98%.

[0037] Therefore, in subsequent tissue engineering, the fluorescent protein probes prepared in this invention can be used for real-time non-invasive detection and purification of differentiated cardiomyocytes using equipment such as fluorescence microscopes, flow cytometry, and fluorescence spectrometers for detection or sorting.

[0038] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A non-invasive fluorescent protein probe for detecting cardiomyocytes, characterized in that, The amino acid sequence of the fluorescent protein probe is shown in SEQ ID NO.

2.

2. The fluorescent protein probe according to claim 1, characterized in that, The fluorescent protein probe contains an affinity peptide; the amino acid sequence of the affinity peptide is shown in SEQ ID NO.

1.

3. The fluorescent protein probe according to claim 2, characterized in that, The affinity peptide can specifically recognize and bind to cardiomyocytes.

4. A gene encoding the fluorescent protein probe of claim 1.

5. A recombinant expression vector for inserting the gene of claim 4.

6. A recombinant engineered bacterium carrying the recombinant expression vector of claim 5.

7. The use of the fluorescent protein probe of claim 1, the gene of claim 4, the recombinant expression vector of claim 5, or the recombinant engineered bacteria of claim 6 in the preparation of a non-invasive detection product for cardiomyocytes.

8. A non-invasive method for detecting cardiomyocytes, characterized in that, The fluorescent protein probe of claim 1 is co-incubated with the cardiomyocytes to be tested, and the fluorescence signal of the cells is detected to determine the presence or number of cardiomyocytes.

9. The method according to claim 8, characterized in that, The co-incubation time is 0.5~2 hours.

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