Protease detection method

Abstract

The invention discloses a protease detection method, which comprises the following specific implementation processes: constructing reporter gene plasmids and dEcCas6-barnase variant plasmids, transforming and extracting plasmids, culturing and transfecting cells, carrying out fluorescence imaging and flow analysis, and detecting the protease. Specific and high-sensitivity protease detection is achieved through the specific cleavage activity of protease and the mRNA recognition and degradation function of the dEcCas6-barnase variant, the method is low in cost and easy to operate, intracellular detection of protease can be achieved, and the method can also be applied to detection of tumor cells.

Description

Technical Field

[0001] The invention belongs to the technical field of protease detection, and particularly relates to a protease detection method. Background Art

[0002] Proteases can catalyze the hydrolysis of specific peptide bonds in target proteins and are involved in regulating important physiological and pathological processes. The dysregulation of proteases is closely related to a variety of physiological activities. Therefore, the development of protease detection methods is of great significance. Cathepsin B (CaB) is a cysteine ​​cathepsin belonging to the cathepsin family. It has been confirmed to be highly expressed in a variety of tumor tissues such as lung cancer, liver cancer, breast cancer and cervical cancer cells, and is a valuable tumor marker.

[0003] Traditional detection methods for proteases include enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance based on antigen-antibody interaction, and gel electrophoresis. These methods have limitations in quantitative measurements due to the involvement of expensive antibody proteins. A series of methods based on active protease cleavage of substrate peptides have been developed, including mass spectrometry, magnetic resonance imaging, colorimetric analysis, fluorescence resonance energy transfer, etc. Although these methods show good selectivity due to the high specificity of protease cleavage sequences, they have complex steps and can only be applied to in vitro detection, not intracellular detection. Summary of the invention

[0004] In order to solve the deficiencies in the research of the above-mentioned field, the purpose of the present invention is to provide a method for detecting proteases, which has low cost and simple operation, and can not only realize the intracellular detection of proteases, but also be applied to the detection of tumor cells.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] A method for detecting protease, characterized in that it comprises the following steps:

[0007] 1) constructing a reporter gene plasmid and a dEcCas6-barnase variant plasmid, wherein the reporter gene plasmid is a green fluorescent protein plasmid, and its 5' end is connected to a dEcCas6 protein binding sequence (CBS); the dEcCas6-barnase variant plasmid comprises a dEcCas6 amino acid sequence as shown in SEQ ID NO.1 and a barnase variant amino acid sequence as shown in SEQ ID NO.2, which are connected by a substrate sequence of the protease to be tested; when the protease to be tested is tobacco etch virus protease TEVp, the amino acid sequence of the dEcCas6-barnase variant plasmid used is shown in SEQ ID NO.3; when the protease to be tested is recombinant human rhinovirus 3C protease HRVp, the amino acid sequence of the dEcCas6-barnase variant plasmid used is shown in SEQ ID NO.4; when the protease to be tested is cathepsin B, the amino acid sequence of the dEcCas6-barnase variant plasmid used is shown in SEQ ID NO.5.

[0008] 2) Transformation and extraction of plasmid: The recombinant plasmid was transformed into E.coli DH5α competent cells, then smeared on ampicillin plates, and placed in a 37°C incubator for overnight culture. Single colonies were picked for colony PCR verification and sequencing, and the correct bacterial solution was transferred to 4mL LB liquid culture medium, and 4μL ampicillin was added, and it was placed in a shaker at 37°C and 220rpm for 12-14h. 1mL of the overnight cultured bacterial solution was placed in a 2mL centrifuge tube, centrifuged at 12000rpm for 1min, the supernatant was discarded, and then 1mL of 20% glycerol was added and mixed by pipetting, and stored at -80°C for use. The remaining bacterial solution was extracted with an endotoxin-free plasmid extraction kit.

[0009] 3) Cell culture and transfection: The cells to be tested were inoculated in a cell culture plate, 500 μL of DMEM medium was added to each well, and the cells were cultured in a cell culture incubator until the cell confluence rate reached 60-70%; 50 μL of Opti-MEM was added to a centrifuge tube, and the reporter gene plasmid and dEcCas6-barnase variant plasmid were added to the tube in sequence, mixed, and Lipofectamine 8000 transfection reagent was added, pipetted and mixed, and then directly added to the above cells;

[0010] 4) Fluorescence imaging: After the transfected cells were incubated in a cell culture incubator for 48 h, the fluorescence in the cells was observed and photographed under an inverted fluorescence microscope;

[0011] 5) Flow cytometry analysis: aspirate the culture medium in the cells, add 500 μL PBS buffer to each well, resuspend the cells in a 1.5 mL centrifuge tube by pipetting, centrifuge at 5000 g in a 4°C refrigerated centrifuge for 5 min, aspirate the supernatant, add 500 μL PBS buffer to each tube again to resuspend the cells, perform flow cytometric analysis on the cell resuspension, and select the fluorescence channel to detect the fluorescence intensity of the cells.

[0012] The beneficial effects of the present invention are:

[0013] 1) The present invention has low cost, simple and convenient plasmid construction, and does not require complicated processing, expensive modification and synthesis;

[0014] 2) The present invention has strong specificity and universality. The present invention utilizes the specific recognition between proteases and substrates, and can detect multiple proteases by changing the substrate sequence of the protease between the dEcCas6-barnase variant plasmids;

[0015] 3) The present invention has simple steps and can directly detect proteases in cells without the need to separate proteases from cells;

[0016] 4) The present invention can realize the detection of tumor cells. Cathepsin B (CaB) is a cysteine ​​cathepsin belonging to the cathepsin family. It has been confirmed to be highly expressed in various tumor tissues such as lung cancer, liver cancer, breast cancer and cervical cancer cells. It is a valuable tumor marker. Therefore, the present invention can realize the detection of tumor cells by detecting cathepsin B in tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a schematic diagram of the detection method of the protease in Example 1 of the present invention;

[0018] Figure 2 This is a schematic diagram of the tumor cell detection method in Example 2 of the present invention;

[0019] Figure 3 The protease detection results of TEVp, HRVp and CaB in Example 1 of the present invention;

[0020] Figure 4 This is the detection result of tumor cells in Example 2 of the present invention. DETAILED DESCRIPTION

[0021] It should be noted that the following detailed descriptions are all illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0022] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0023] As introduced in the background technology, in order to solve the defects of high detection cost and duplication of steps in the prior art protease detection method, the present invention provides a protease detection method, which has low cost and simple operation, and can not only realize the intracellular detection of protease, but also can be applied to the detection of tumor cells.

[0024] In the reporter gene plasmid written in the embodiment of the present invention, the reporter gene is displayed with green fluorescent protein (GFP), and its 5' end is connected to a dEcCas6 protein binding sequence (CBS). The dEcCas6-barnase variant plasmid includes a dEcCas6 amino acid sequence as shown in SEQ ID NO.1 and a barnase variant amino acid sequence as shown in SEQ ID NO.2, which is connected by a substrate sequence of a protease to be tested, and the dEcCas6-barnase variant plasmid can be translated into a dEcCas6-barnase variant protein in a cell, and the dEcCas6 protein can be combined with a dEcCas6 protein binding sequence (CBS), and the barnase variant protein has an mRNA degradation function, and can degrade the mRNA of green fluorescent protein.

[0025] The reagents and consumables required for the test include LB culture medium, ampicillin, transfection reagent Lipofectamine8000 transfection reagent, Opti-MEM, DMEM culture medium, PBS buffer, centrifuge tubes, cell culture dishes, and well plates.

[0026] The instruments required for detection include cell culture incubator, biological safety cabinet, 4°C refrigerated centrifuge, inverted fluorescence microscope and flow cytometer.

[0027] Example 1

[0028] This embodiment provides a method for detecting TEVp, HRVp, and CaB in 293T cells. Figure 1As shown, the detection principle is to use the specific cleavage activity of the protease and the mRNA recognition and degradation function of the dEcCas6-barnase variant to regulate the expression of green fluorescent protein. A protease cleavage sequence is added between the dEcCas6 sequence and the barnase variant sequence. When there is no protease in the cell, the dEcCas6-barnase variant binds to the dEcCas6 protein binding sequence (CBS) to exert the mRNA degradation function, degrade the mRNA of the green fluorescent protein, and the green fluorescent protein is not expressed; when there is a protease in the cell, the substrate sequence will be cut to separate dEcCas6 and barnase, hindering its mRNA degradation function, and the green fluorescent protein is expressed normally. The detection method comprises the following steps:

[0029] (1) Preparation of green fluorescent protein plasmid and dEcCas6-barnase variant plasmid

[0030] First, the gene sequence fragments of green fluorescent protein and dEcCas6-barnase variant were obtained by PCR reaction, and then they were constructed into eukaryotic vectors (including CMV promoter, polyA tail, and ampicillin resistance gene) by homologous recombination reaction. The dEcCas6-barnase variant sequence of TEVp is as follows: dEcCas6-GGS-ENLYFQS-GGS-barnase variant (TEVcs), as shown in SEQ ID NO.3; the dEcCas6-barnase variant sequence of HRVp is as follows: dEcCas6-GGS-LEVLFQGP-GGS-barnase variant (HRVcs), as shown in SEQ ID NO.4; the dEcCas6-barnase variant sequence of CaB is as follows: dEcCas6-GGS-KGFLGK-GGS-barnase variant (CaBcs), as shown in SEQ ID NO.5.

[0031] (2) Plasmid transformation and extraction

[0032] The recombinant plasmid was transformed into E.coli DH5α competent cells, then smeared on an ampicillin plate and placed in a 37°C incubator for overnight culture. A single colony was picked for colony PCR verification and sequencing. The bacterial solution with correct sequencing was transferred to 4mL LB liquid culture medium, and 4μL ampicillin was added. It was placed in a 37°C, 220rpm shaker for 12h. 1mL of the overnight cultured bacterial solution was placed in a 2mL centrifuge tube, centrifuged at 12000rpm for 1min, the supernatant was discarded, and then 1mL of 20% glycerol was added and mixed by pipetting, and stored at -80°C for use. The remaining bacterial solution was extracted with an endotoxin-free plasmid extraction kit (ABclonaAFTSpinEndoFreePlasmid Mini Kit).

[0033] (3) Cell culture and transfection

[0034] The protease-infected 293T cells were inoculated in a 24-well cell culture plate as an experimental group, and the protease-uninfected 293T cells were inoculated in a 24-well cell culture plate as a control group. 500 μL of DMEM medium was added to each well, and the cells were cultured in a cell culture incubator until the cell confluence rate reached 60-70%. 50 μL of Opti-MEM, green fluorescent protein plasmid and dEcCas6-barnase variant plasmid were added to a centrifuge tube, mixed, and then Lipofectamine 8000 transfection reagent was added. After pipetting and mixing, the cells were directly added dropwise to the above two groups of cells.

[0035] (4) Fluorescence imaging

[0036] After culturing the cells in the 24-well plate for 48 hours, take photos and analyze them with a fluorescent inverted microscope. Green fluorescent protein is excited by blue light. According to the order of instrument use, make adjustments in sequence. First, turn on the mercury lamp, select the corresponding excitation light, place the cell 24-well plate on the stage, select the 10× objective lens for observation, then open the computer software, use the wells without protease as the control for adjustment, determine the gain and exposure time, and start taking photos of the wells with protease.

[0037] (5) Flow cytometry

[0038] The culture medium of the cells in the 24-well plate was aspirated, 500 μL PBS buffer was added to each well, and the cells were resuspended in a 1.5 mL centrifuge tube by pipetting. The cells were centrifuged at 5000 g in a 4°C refrigerated centrifuge for 5 min, the supernatant was aspirated, and 500 μL PBS buffer was added to each tube to resuspend the cells again. The cell resuspension was analyzed by flow cytometer, and the green fluorescence channel was selected to detect the green fluorescence intensity of the cells.

[0039] like Figure 3As shown, obvious green fluorescence signals appeared in 293T cells containing TEVp, HRVp, and CaB. Flow cytometry analysis showed that the green fluorescence intensities were 25.8, 42.6, and 12.6 times those without TEVp, HRVp, and CaB, respectively.

[0040] Example 2

[0041] This embodiment provides a method for detecting HepG2, Hela, MCF-7 and A549 tumor cells. Figure 2 As shown, the detection principle is to use the specific cleavage activity of protease and the mRNA recognition and degradation function of dEcCas6-barnase variants to regulate the expression of green fluorescent protein. A cathepsin B cleavage sequence (CaBcs) and a cathepsin B incleavage sequence (CaBncs) are added between the dEcCas6 sequence and the barnase variant sequence. In HepG2, Hela, MCF-7 and A549 tumor cells, cathepsin B is overexpressed and can cleave CaBcs, separating dEcCas6 and barnase, hindering their mRNA degradation function, and normal expression of green fluorescent protein. However, cathepsin B cannot cleave CaBncs, and the dEcCas6-barnase variant binds to the dEcCas6 protein binding sequence (CBS), exerts mRNA degradation function, degrades the mRNA of green fluorescent protein, and green fluorescent protein is not expressed.

[0042] The specific steps are as follows:

[0043] (1) Preparation of green fluorescent protein plasmid and dEcCas6-barnase variant plasmid

[0044] First, green fluorescent protein and dEcCas6-barnase variant gene sequence fragments were obtained by PCR reaction, and then they were respectively constructed into eukaryotic vectors (including CMV promoter, polyA tail, and ampicillin resistance gene) by homologous recombination reaction. The dEcCas6-barnase variant sequence of CaB is as follows: dEcCas6-GGS-KGFLGK-GGS-barnase variant (CaBcs), and the dEcCas6-barnase variant sequence that cannot be cut by CaB is as follows: dEcCas6-GGS-HGGGGH-GGS-barnase variant (CaBncs), as shown in SEQ ID NO.6.

[0045] (2) Plasmid transformation and extraction

[0046] The recombinant plasmid was transformed into E.coli DH5α competent cells, then smeared on an ampicillin plate and placed in a 37°C incubator for overnight culture. A single colony was picked for colony PCR verification and sequencing. The bacterial solution with correct sequencing was transferred to 4mL LB liquid culture medium, and 4μL ampicillin was added. It was placed in a 37°C, 220rpm shaker for 12h. 1mL of the overnight cultured bacterial solution was placed in a 2mL centrifuge tube, centrifuged at 12000rpm for 1min, the supernatant was discarded, and then 1mL of 20% glycerol was added and mixed by pipetting, and stored at -80°C for use. The remaining bacterial solution was extracted with an endotoxin-free plasmid extraction kit (ABclonaAFTSpinEndoFreePlasmid Mini Kit).

[0047] (3) Cell culture and transfection

[0048] HepG2, Hela, MCF-7 and A549 tumor cells were inoculated in a 24-well cell culture plate, 500 μL DMEM medium was added to each well, and the cells were cultured in a cell culture incubator until the cell confluence rate reached 60-70%; 50 μL Opti-MEM was added to the centrifuge tube, the green fluorescent protein plasmid and dEcCas6-barnase variant plasmid (CaBncs) were added to the tube of the control group, and the green fluorescent protein plasmid and dEcCas6-barnase variant plasmid (CaBcs) were added to the centrifuge tube of the experimental group in sequence, and after mixing, Lipofectamine 8000 transfection reagent was added, and after pipetting and mixing, it was directly added dropwise to the above two groups of cells.

[0049] (4) Fluorescence imaging

[0050] After culturing the cells in the 24-well plate for 48 hours, take photos and analyze them with a fluorescent inverted microscope. Green fluorescent protein is excited by blue light. According to the order of instrument use, make adjustments in sequence. First, turn on the mercury lamp, select the corresponding excitation light, place the cell 24-well plate on the stage, select the 10× objective lens for observation, then open the computer software, use the wells without protease as the control for adjustment, determine the gain and exposure time, and start taking photos of the wells with protease.

[0051] (5) Flow cytometry

[0052] The culture medium of the cells in the 24-well plate was aspirated, 500 μL PBS buffer was added to each well, and the cells were resuspended in a 1.5 mL centrifuge tube by pipetting. The cells were centrifuged at 5000 g in a 4°C refrigerated centrifuge for 5 min, the supernatant was aspirated, and 500 μL PBS buffer was added to each tube to resuspend the cells again. The cell resuspension was analyzed by flow cytometer, and the green fluorescence channel was selected to detect the green fluorescence intensity of the cells.

[0053] like Figure 4 As shown, the results showed that obvious green fluorescence signals appeared in HepG2, Hela, MCF-7 and A549 tumor cells containing the CaB-cleavable dEcCas6-barnase variant (CaBcs). Flow cytometry analysis showed that the green fluorescence intensity was 9.4, 7.9, 7.6 and 6.5 times that of the CaB-incapable dEcCas6-barnase (CaBncs), respectively.

[0054] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for detecting protease, characterized in that: The steps include: 1) Construction of reporter gene plasmid and dEcCas6-barnase variant plasmid; 2) Transformation and extraction of plasmids; 3) Cell culture and transfection: The cells to be tested were inoculated in a cell culture plate, 500 μL of DMEM medium was added to each well, and the cells were cultured in a cell culture incubator until the cell confluence rate reached 60-70%; 50 μL of Opti-MEM was added to a centrifuge tube, and the reporter gene plasmid and dEcCas6-barnase variant plasmid were added to the tube in sequence, mixed, and Lipofectamine 8000 transfection reagent was added, pipetted and mixed, and then directly added to the above cells; 4) Fluorescence imaging: After the transfected cells were incubated in a cell culture incubator for 48 h, the fluorescence in the cells was observed and photographed under an inverted fluorescence microscope; 5) Flow cytometry analysis: aspirate the culture medium in the cells, add 500 μL PBS buffer to each well, resuspend the cells in a 1.5 mL centrifuge tube by pipetting, centrifuge at 5000 g in a 4°C refrigerated centrifuge for 5 min, aspirate the supernatant, add 500 μL PBS buffer to each tube again to resuspend the cells, perform flow cytometric analysis on the cell resuspension, and select the fluorescence channel to detect the fluorescence intensity of the cells.

2. A method for detecting a protease according to claim 1, characterized in that: The reporter gene plasmid is a green fluorescent protein plasmid, and the 5' end of the plasmid is connected to the dEcCas6 protein binding sequence (CBS).

3. The method for detecting a protease according to claim 1, characterized in that: The dEcCas6-barnase variant plasmid includes the dEcCas6 amino acid sequence shown in SEQ ID NO.1 and the barnase variant amino acid sequence shown in SEQ ID NO.2, which are connected by the substrate sequence of the protease to be tested.

4. A method for detecting a protease according to claim 3, characterized in that: When the protease to be tested is tobacco etch virus protease TEVp, the amino acid sequence of the dEcCas6-barnase variant plasmid used is shown in SEQ ID NO.3; when the protease to be tested is recombinant human rhinovirus 3C protease HRVp, the amino acid sequence of the dEcCas6-barnase variant plasmid used is shown in SEQ ID NO.4; when the protease to be tested is cathepsin B, the amino acid sequence of the dEcCas6-barnase variant plasmid used is shown in SEQ ID NO.

5.

5. A method for detecting a protease according to claim 4, characterized in that: The specific operation process of step 2) is as follows: transform the recombinant plasmid into E.coli DH5α competent cells, then apply it to the ampicillin plate, place it in a 37°C incubator for overnight culture, pick a single colony for colony PCR verification and sequencing, transfer the correctly sequenced bacterial solution to 4mL LB liquid culture medium, add 4μL ampicillin, and place it at 37°C, 220rpm shaker for 12-14h. Take 1mL of the overnight cultured bacterial solution and place it in a 2mL centrifuge tube, centrifuge it at 12000rpm for 1min, discard the supernatant, then add 1mL 20% glycerol to mix it by blowing, store it at -80°C for use, and extract the plasmid with the remaining bacterial solution using an endotoxin-free plasmid extraction kit.

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