A fluorescent protein probe for non-invasive detection of mesenchymal stem cells and its application

Abstract

The present invention discloses a fluorescent protein probe for non-invasive detection of mesenchymal stem cells and its application, belonging to the field of protein and genetic engineering. The affinity peptide M12 of mesenchymal stem cells is obtained by using a phage display peptide library (12 peptides), which is combined with cyclized mCherry and transferred into an expression vector, and then expressed by an Escherichia coli expression system to obtain the target fluorescent protein probe. Through experimental detection, the fluorescent protein probe can be used to detect mesenchymal stem cells, realizing the detection of mesenchymal stem cell levels in real time, breaking the defects and barriers of traditional detection methods.

Description

Technical Field

[0001] The invention relates to the fields of protein and gene engineering, and in particular to a fluorescent protein probe for non-invasively detecting mesenchymal stem cells and an application thereof. Background Art

[0002] Mesenchymal stem cells are currently the hottest stem cells and are widely used in fields such as tissue engineering and regenerative medicine. However, there have always been many problems with their identification. First, it has multiple markers, not a single marker. Second, the detection methods are all traditional methods, such as immunological detection with the help of antigens and antibodies. The current methods for detecting mesenchymal stem cells have poor specificity, the detection methods remain at the level of dead cells, and non-destructive detection cannot be achieved. Therefore, a new method for detecting mesenchymal stem cells is urgently needed, which is of great significance to solving the problems existing in the existing technology. Summary of the invention

[0003] The purpose of the present invention is to provide a fluorescent protein probe for non-invasive detection of mesenchymal stem cells and its application to solve the problems existing in the above-mentioned prior art. The fluorescent protein probe can realize non-invasive and non-destructive detection of mesenchymal stem cells in real time.

[0004] To achieve the above object, the present invention provides the following solutions:

[0005] The present invention provides a fluorescent protein probe for non-invasively detecting mesenchymal stem cells. The amino acid sequence of the fluorescent protein probe is shown in SEQ ID NO:2.

[0006] The present invention also provides a nucleic acid molecule, which is a nucleic acid encoding the fluorescent protein probe.

[0007] The invention also provides a recombinant plasmid comprising the nucleic acid molecule.

[0008] The present invention also provides a recombinant bacterium comprising the recombinant plasmid.

[0009] The present invention also provides the use of the fluorescent protein probe, the nucleic acid molecule, the recombinant plasmid or the recombinant bacteria in preparing a product for non-invasive detection of mesenchymal stem cells.

[0010] The present invention also provides a method for non-invasively detecting mesenchymal stem cells, comprising the following steps: incubating the fluorescent protein probe with the mesenchymal stem cells to be detected, and then detecting the fluorescence intensity of the cells to determine the presence and / or quantity of the mesenchymal stem cells.

[0011] Preferably, the co-incubation conditions are: incubation at 37°C for 0.5-1h

[0012] The present invention discloses the following technical effects:

[0013] The present invention uses mesenchymal stem cells as a whole as a target to screen affinity peptide ligands and obtain unique and specifically labeled affinity peptides. The affinity peptide is combined with cyclized fluorescent protein technology, combined with cyclized mCherry and transferred into an expression vector, and then expressed using an Escherichia coli expression system to obtain a target fluorescent protein probe. The fluorescent protein probe can be used to detect stem cells, realizing a simple detection in which the fluorescence is turned on when the stem cells are present and the fluorescence is turned off when they are not present. The present invention realizes real-time mesenchymal stem cell level detection, breaking the technical barriers of traditional detection methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0015] Figure 1 The results of ELISA assay; the horizontal axis 1-4 represents the numbers of different groups, negative represents the negative control; the vertical axis is the normalized OD value of the assay;

[0016] Figure 2 Schematic diagram of the binding of affinity peptide M12 to cyclized Mcherry;

[0017] Figure 3 The fluorescence spectrum detection results of the fluorescent protein probe in the presence or absence of MSCs;

[0018] Figure 4 The fluorescence spectrum detection results of adding fluorescent protein probes to different cells;

[0019] Figure 5 This is the result of fluorescent labeling of MSCs with fluorescent protein probe. DETAILED DESCRIPTION

[0020] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0021] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0022] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0023] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0024] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0025] Example 1

[0026] 1. Acquisition of M12 gene

[0027] 1.1 Experimental Materials

[0028] NEB phage display kit, P5 umbilical cord mesenchymal stem cells (MSCs), chondrocytes, and endothelial cells (ECs) were purchased from the American Cell Culture Collection (ATCC).

[0029] 1.2 Experimental methods

[0030] When MSCs, ECs and chondrocytes grew to 90%, they were digested with 2.5% trypsin and the cell density was adjusted at 10 5 cells / dish were inoculated in a 60 mm × 15 mm culture dish and cultured in a 37°C, 5% CO2 incubator. The 2nd day was used for screening. MSCs were first cultured in serum-free α-MEM for 2 h, then blocked with 5% BSA for 1 h, and the phage peptide library (titer of about 1.5 × 10 11pfu), incubated at 37℃ for 1h. Remove the supernatant, rinse 6 times with TBST (0.1%), add 1mL of glycine elution buffer (pH 2.2), and after 10min, aspirate the eluate and add 150ul of Tris-HCl (pH 9.1) to neutralize; transfer the eluate to ECs cells that have been cultured with serum-free DMEM for 2h and blocked with 5% BSA at 37℃ for 1h, incubate at 37℃ for 1h, then aspirate the supernatant and transfer it to chondrocytes that have been cultured with serum-free DMEM for 2h and blocked with 5% BSA at 37℃ for 1h, incubate at 37℃ for 1h, and the supernatant is the phage library obtained in the first round of screening. Infect the host bacteria E.coli ER2738, determine the titer, amplify, and use it for the next round of screening. After 3 rounds of screening, determine the phage titer, randomly pick a batch of phage monoclones, and send them for sequencing.

[0031] Phage peptide ELISA assay MSCs and chondrocytes were divided into 1×10 4 The cells were inoculated in a 96-well plate at a density of 10 cells / well and used on the second day. The cells were treated with serum-free water for 1 hour, fixed with 4% paraformaldehyde for 20 minutes, washed with 0.05% PBST for 3 times, 1 minute / time, treated with 0.1% Triton X-100 for 10 minutes, washed with 0.05% PBST for 3 times, 1 minute / time; blocked with 2% BSA for 1 hour, and added with a titer of about 10 12 Phages were incubated at 37℃ for 2h, washed 3 times with 0.05% PBST, 1min / time; HRP-anti M13 antibody (1:5000 dilution) was added, incubated at 37℃ for 1h, washed 3 times with 0.05% PBST, 1min / time; TMB was used for color development, HCl was used to terminate the reaction, and the microplate reader was used to read at 450nm. The blue spots of the unbound phages in the first round were randomly selected as controls, and P / N>2 was positive.

[0032] ELISA test results Figure 1 As shown, the peptide corresponding to the phage with the highest OD value was selected as the MSCs affinity peptide (SEQ ID NO: 1): SPDDKWFFESPM, named: M12.

[0033] 2. LV-McpM plasmid construction

[0034] By connecting chains L1 and L2, M12 is bound to the circularized mCherry (see Figure 2, inserted between the 150th and 151st amino acids of mCherry) to synthesize the probe DNA sequence, and then transferred into the PRsetB expression vector to construct a recombinant plasmid (named LV-McpM plasmid, this part of the work was completed by Sangon Biotech (Shanghai) Co., Ltd.). After the sequencing was correct, the successfully constructed recombinant vector was transferred into Escherichia coli DH5α and subjected to conventional screening, purification and other experiments to obtain the target fluorescent protein probe. The amino acid sequence of the fluorescent protein probe is shown in SEQ ID NO: 2:

[0035] LVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEAFQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYIKHPADIPDYFKLSFPEGFRWERVMNFEDGGIIHVNQDSSLQDGVFIYKV KLRGTNFPPPDGPVMQKKTMGWEQWRDGSPDDKWFFESPMETVSERMYPEDGALKSEIKKGLRLKDGGHYAAEVKTTYKAKKPVQLPGAYIVDIKLDIVSHNEDYTIVEQCERAEGRHTGGMDELYK.

[0036] 3. Detection of the effectiveness of fluorescent protein probes

[0037] The fluorescent protein probe prepared above was directly loaded into P5 cultured MSCs and control group cells ECs and soft cells (cell confluence was 80%) at 10 ng / mL, incubated at 37°C for 1 h, and excited at 580 nm using an ELISA reader to detect the fluorescence intensity emitted at a wavelength of 600 nm.

[0038] like Figure 3 and Figure 4 As shown, the fluorescence intensity of the fluorescent protein probe increased by 6.5 times after adding MSCs, while the fluorescence intensity of ECs and soft cells was consistent with that of the control group without cells, indicating that the fluorescent protein probe prepared by the present invention can be used to detect MSCs.

[0039] 4. Fluorescent protein probe cell fluorescence detection

[0040] The probe protein was directly loaded into MSCs cells cultured at 80% density at P5, incubated at 37°C for 1 h, and detected using a laser confocal fluorescence microscope with an excitation wavelength of 580 nm and a receiving wavelength of 600 nm.

[0041] Test results such as Figure 5 As shown, the fluorescence labeling rate of the fluorescent protein probe can reach 99%.

[0042] Therefore, in the subsequent study of stem cells MSCs in tissue engineering, the fluorescent protein probe prepared by the present invention can be used for detection using equipment such as a fluorescence microscope, flow cytometer, and fluorescence spectrometer.

[0043] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A fluorescent protein probe for non-invasive detection of mesenchymal stem cells, characterized in that: The amino acid sequence of the fluorescent protein probe is shown in SEQ ID NO:

2.

2. A nucleic acid molecule, characterized in that It is a nucleic acid encoding the fluorescent protein probe according to claim 1.

3. A recombinant plasmid, characterized in that: Comprising the nucleic acid molecule of claim 2.

4. A recombinant bacterium, characterized in that: Comprising the recombinant plasmid according to claim 3.

5. Use of the fluorescent protein probe according to claim 1, the nucleic acid molecule according to claim 2, the recombinant plasmid according to claim 3, or the recombinant bacteria according to claim 4 in the preparation of a product for non-invasive detection of mesenchymal stem cells.

6. A method for non-invasively detecting mesenchymal stem cells, characterized in that: The method comprises the following steps: incubating the fluorescent protein probe described in claim 1 with the mesenchymal stem cells to be tested, and then detecting the fluorescence intensity of the cells to determine the existence and / or quantity of the mesenchymal stem cells.

7. The method according to claim 6, characterized in that The co-incubation conditions are: incubation at 37° C. for 0.5-1 h.

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