CN112683867B - Method for detecting mesothelin D on living cells in real time and application thereof - Google Patents
Method for detecting mesothelin D on living cells in real time and application thereof Download PDFInfo
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Abstract
The invention relates to a method for detecting the mesothelin D on living cells in real time and application thereof, belonging to the technical field of medical detection. According to the invention, through constructing the GSDMD over-expression plasmid containing double fluorescent label marks, two fluorescent protein sequences with different colors are respectively arranged outside the N end sequence and the C end sequence of the GSDMD, and then the over-expression plasmid is directly transfected to target cells through transfection; dynamically observing fluorescence change in real time through a fluorescence microscope or a laser confocal microscope to reflect activation of GSDMD and time-space positioning change; therefore, the method can detect the expression, the cutting, the activation and the positioning of the GSDMD on living cells in real time, and simultaneously has the advantages of simple and high efficiency of the detection process and relatively low detection cost, and has important significance for the related research of immune inflammation.
Description
Technical Field
The invention relates to a method for detecting the mesothelin D on living cells in real time and application thereof, belonging to the technical field of medical detection.
Background
Deregulation of inflammatory cell death is a key factor in many inflammatory diseases. Pyrodeath is a highly inflammatory form of cell death that uses pores created within cells to disrupt electrolyte homeostasis and perform cell death. Apoptosis is a manifestation of many diseases that worsen to some extent. Deuterin D (GSDMD) is a pore-forming effector protein of pyrosis that coordinates membrane cleavage and release of high inflammatory molecules, such as interleukin-1 (IL-1), enhancing the overactivation of innate immune responses. However, to date, there is no pharmacological mechanism that disrupts pyrodeath.
Studies show that after GSDMD is cut, an amino terminal (N-terminal) fragment of the GSDMD can be positioned on the surface of a cell membrane to form a pore canal, and the permeability of the cell membrane is changed, so that substances inside and outside the cell can freely pass through, at the moment, extracellular water can freely enter the cell, so that the cell expands and breaks, and finally, cell death is caused, and the phenomenon is called cell coke death. GSDMD is therefore the final effector of cell apoptosis. In addition, studies have shown that the N-terminal fragment of GSDMD (GSDMD-N) can localize to the mitochondrial membrane and form a pore channel, altering the permeability of the mitochondrial membrane, thereby allowing the mitochondria to communicate with the internal environment of the cytoplasm, from which harmful substances such as cytochrome C, mitochondrial DNA, reactive oxygen species, etc. can be released, causing cell damage. The evidence above suggests that GSDMD expression, cleavage, activation, localization have important effects on cell function and cell fate. However, the current means for observing and detecting GSDMD is limited, the expression of GSDMD in tissues or cells mainly depends on Western blotting (Western blot), and if further detection of GSDMD and its cleavage fragments in mitochondria or cytoplasm is desired, it is necessary to separate the mitochondria from cytoplasmic components and then detect them separately. In addition, the distribution and positioning of GSDMD and its cleaved fragments in cells and subcellular structures (organelles) of tissues or cells can also be observed using immunoelectron microscopy techniques. In addition, expression and localization of GSDMD and its cleaved fragments within tissues or cells after fixation with formaldehyde or paraformaldehyde can also be observed by immunochemical or immunofluorescent staining. However, none of the above methods can observe and detect GSDMD expression, cleavage, activation, localization in real time on living or living cells; moreover, all the above methods need to rely on antigen-antibody reactions, and the sensitivity and specificity of the methods have certain problems; in addition, the detection method has more investment in time or cost, such as more steps of Western blot, immunochemistry/fluorescent staining and mitochondrial protein extraction, more complicated process and more expensive acquisition of related antibodies; the preparation process of the sample preparation and detection of the electron microscope is longer, and the detection cost is higher. Therefore, developing a method which can detect the expression, cleavage, activation and localization of GSDMD on living cells in real time and simultaneously has the advantages of simple and high efficiency of the detection process and relatively low detection cost is of great significance for the research on immune inflammation.
Disclosure of Invention
The invention aims to solve the technical problems of how to detect the expression, cutting, activating and positioning of GSDMD on living cells in real time, and simultaneously, the detection process is simple and efficient and the detection cost is relatively low.
In order to solve the above-mentioned problem, the technical scheme adopted by the invention is to provide a method for detecting the mesothelin D on living cells in real time; the method comprises the following steps:
step 1: constructing GSDMD overexpression plasmid containing double fluorescent label marks;
step 2: cell transfection with the plasmid obtained in step 1 above; performing drug intervention on the transfected cells to induce activation of inflammatory bodies and downstream GSDMD cleavage;
step 3: observing the fluorescence intensity and the color change in the cells by a fluorescence microscope or a laser confocal microscope;
step 4: mitochondria are marked by mitochondrial fluorescent staining, and the localization of GSDMD active fragments on mitochondria is observed by fluorescence co-localization.
Preferably, in the step 1, the dual fluorescent label marks GSDMD are provided that two fluorescent protein sequences with different colors are arranged outside the N-terminal and C-terminal sequences of the GSDMD.
Preferably, a blue fluorescent protein BFP (Blue Fluorescence Protein) sequence is arranged outside the N-terminal sequence of the GSDMD.
Preferably, a yellow fluorescent protein EYFP (enhanced yellow fluorescent protein) sequence is arranged outside the C-terminal sequence of the GSDMD.
Preferably, the mitochondrial fluorescence staining label in step 4 above uses a mitochondria mitochondrial green fluorescent probe.
The invention provides an application of a method for detecting the mesothelin D on living cells in real time in a cell inflammatory response induction model.
The invention provides an application of a method for detecting the mesothelin D on living cells in real time in detection of inflammatory reaction of medicine intervention cells.
The invention provides an application of a method for detecting the mesothelin D on living cells in real time in a cell inflammatory reaction detection kit.
Compared with the prior art, the invention has the following beneficial effects:
a. the expression, cutting, activating and positioning of GSDMD can be detected on living cells in real time.
b. The method can be used for realizing the simplicity, high efficiency and relatively low detection cost of the detection process.
c. Through gene overexpression, GSDMD molecules are directly connected with the bicolor fluorescent protein, and complicated experimental techniques such as additional fixed cells, immunofluorescence staining and the like are not needed.
d. Provides a good visual research tool for researching the functionality of GSDMD in cells.
Drawings
FIG. 1 shows fluorescence change of a GSDMD overexpressing plasmid of the invention after HEK-293T cell transfection without inducing GSDMD activation state;
wherein panel a is the non-induced GSDMD activated state following HEK-293T cell transfection of GSDMD overexpressing plasmids in the present invention. Control cells in the figure: blue is GSDMD-N color development, yellow is GSDMD-C color development, and red is mitochondrial Mitothecker color development. The blue and yellow distribution is consistent, indicating that GSDMD-N and GSDMD-C fragments are not cleaved.
Panel B shows the fluorescence change of GSDMD after transfection of HEK-293T cells with the GSDMD overexpression plasmid of the invention; blue in the figure is GSDMD-N color development, yellow is GSDMD-C color development, and red is mitochondrial Mitothecker color development. The blue and yellow distributions are not consistent, indicating that GSDMD-N and GSDMD-C fragments are cleaved and separated, and GSDMD-N (blue) and Mitotracker (red) are fluorescent fused (purple), indicating co-localization.
FIG. 2 is a schematic block diagram of a method process provided by the present invention;
Detailed Description
In order to make the invention more comprehensible, preferred embodiments accompanied with the accompanying drawings are described in detail as follows:
as shown in fig. 1 and 2, the present invention provides a method for detecting the mesothelin D on living cells in real time; the method comprises the following steps:
step 1: constructing GSDMD overexpression plasmid containing double fluorescent label marks;
step 2: cell transfection with the plasmid obtained in step 1 above; performing drug intervention on the transfected cells to induce activation of inflammatory bodies and downstream GSDMD cleavage;
step 3: observing the fluorescence intensity and the color change in the cells by a fluorescence microscope or a laser confocal microscope;
step 4: mitochondria are marked by mitochondrial fluorescent staining, and the localization of GSDMD active fragments on mitochondria is observed by fluorescence co-localization.
In the step 1, the dual fluorescent label marks GSDMD are arranged outside the N-end and C-end sequences of the GSDMD in such a way that two fluorescent protein sequences with different colors are arranged outside the N-end and C-end sequences of the GSDMD. And a blue fluorescent protein BFP sequence is arranged outside the N-end sequence of the GSDMD. The outside of the C-terminal sequence of GSDMD is provided with a yellow fluorescent protein EYFP sequence.
The mitochondrial fluorescence staining label in the step 4 adopts a mitochondria mitochondrial green fluorescent probe.
The invention provides an application of a method for detecting the mesothelin D on living cells in real time in a cell inflammatory response induction model.
The invention provides an application of a method for detecting the mesothelin D on living cells in real time in detection of inflammatory reaction of medicine intervention cells.
The invention provides an application of a method for detecting the mesothelin D on living cells in real time in a cell inflammatory reaction detection kit.
GSDMD has a molecular weight of 60kD, and can be sheared into two fragments by Caspase-1 or Caspase-4, namely GSDMD-N with a molecular weight of 31kD at the N end and GSDMD-C with a molecular weight of 29kD at the C end. The current view suggests that GSDMD-N plays a major role in the activation-mediated cell scorch process of inflammatory bodies, whereas GSDMD-C has no detailed or thought to play no role. The invention utilizes two fluorescent proteins with different colors, such as Blue Fluorescent Protein (BFP) and yellow fluorescent protein (EYFP), to respectively mark the N end and the C end of GSDMD to form BFP-GSDMD-EYFP fusion protein, living cell cells express the fusion protein, and GSDMD protein is not cut in a baseline state (without inducing activation of inflammatory bodies), so that the BFP and EYFP have consistent fluorescence distribution, and blue fluorescence and yellow fluorescence are co-localized under a fluorescence microscope to generate fusion fluorescence. After intervening cells induce activation of inflammatory bodies, GSDMD is cut into two fragments, namely BFP-GSDMD-N fragment and EYFP-GSDMD-C fragment, and the N-terminal fragment can be positioned on cell membranes or tectorial membrane organelles so as to be separated from the C-terminal fragment, at the moment, fluorescence co-positioning phenomenon does not exist any more, and yellow-blue fusion fluorescence is weakened or vanished. Therefore, the two ends of the GSDMD molecule are marked by bicolor fluorescence, and the fluorescence change is dynamically observed in real time through a fluorescence microscope or a laser confocal microscope, so that the activation of the GSDMD and the time-space positioning change are reflected.
The design scheme of the invention is as follows:
a. taking the yellow Lan Yingguang combination as an example, a vector plasmid containing the BFP-M_GSDMD-EYFP over-expression sequence was first constructed: firstly, designing a primer to amplify a target gene fragment from an original plasmid, and recombining the target gene fragment onto an enzyme-cut overexpression vector through seamless cloning recognition sites at two ends of the primer; transferring the connection product into the prepared bacterial competent cells, carrying out sequencing identification on the monoclonal colonies, and comparing the correct clones to obtain the successfully constructed overexpression plasmid.
b. The scheme comprises the following steps: designing and synthesizing a primer; double enzyme cutting of the carrier; amplifying the target fragment; ligation of the overexpression vector to the fragment of interest (seamless cloning); cell transformation; and (5) sequencing and identifying.
GSDMD acts as an effector molecule downstream of the inflammatory body, and its functional activation can directly mediate cell apoptosis, which is an important cell death mode and becomes a hotspot in the research field of inflammation immunity. The cleavage state of GSDMD and the distribution and localization of its activated fragments determine the realization of its function, but there is currently no visual research tool concerning the activation process of GSDMD in cells and its localization in subcellular structures and cell membranes. Based on the method, the expression, the cutting, the activation and the positioning of the GSDMD can be detected on living cells in real time, and meanwhile, the method which has the advantages of simple and high efficiency of the detection process and relatively low detection cost is of great significance to the research on the related immune inflammation.
Examples
1. Reagents used in the experiments:
2. instrument for experiment
| Voltage stabilizing electrophoresis apparatus | Bio-Rad |
| Gel imaging instrument | Haimen Kylin-Bell Lab Instruments Co.,Ltd. |
| Bacteria cradle | SHANGHAI YIHENG INSTR Co.,Ltd. |
| Bacteria incubator | SHANGHAI YIHENG INSTR Co.,Ltd. |
| PCR instrument | Eppendorf |
| High-speed centrifuge | Thermo |
| Disposable plate | Shanghai Pre-dust BioCo Ltd |
Taking the yellow-blue fluorescent combined double-labeled fluorescent protein as an example:
a. creating a vector of the over-expressed sequence: firstly, selecting a proper plasmid as a vector; the bacterial liquid containing BFP-EYFP carrier plasmid is cultured overnight, and 3-5ml of fresh bacterial liquid is taken to extract plasmid. Specific methods refer to QIAGEN plasmid extraction instructions. 1. Mu.g of fresh plasmid was taken and digested with the corresponding restriction enzymes at 37℃for about 3 hours. And (3) carrying out agarose gel electrophoresis on the enzyme digestion product, and after the electrophoresis is finished, carrying out gel recovery, wherein the steps are as follows: the strip containing the fragment of interest was cut under UV light. The total weight was weighed with a balance and the weight of the empty tube was subtracted to calculate the weight of the gel, the volume of the gel was calculated as 100mg = 100 μl, and a binding Solution of 1 gel volume was added and placed in a 65 ℃ water bath to thoroughly melt the gel. During which the EP tube is properly shaken to accelerate the dissolution of the gel. All the above liquid was transferred to a filter column and centrifuged at 13000 rpm for 30S (which can be repeated once). The liquid in the tube WAs then discarded, 500. Mu.L of WA Solution WAs added to the column, and centrifuged at 13000 rpm for 30S. The liquid in the tube was discarded, and 500. Mu.L of Wash Solution was added to the column and centrifuged at 13000 rpm for 30S (which can be repeated once). Then the mixture was left free for 3min. The filter column was placed in a new 1.5mL EP tube and dried at room temperature. Finally, 35. Mu.L of ddH2O was added to the column, and the mixture was left for 5min and centrifuged at 13000 rpm for 1.5min. To increase recovery, the dissolved DNA may be added again to the column and centrifuged for one minute. The column was discarded, i.e., the recovered carrier fragment, and the concentration was determined.
Amplification of gsdmd fragment of interest: designing and synthesizing primers, namely PCR amplified fragment primers, and introducing homologous sequences at the tail ends of the linearization cloning vectors at the 5' ends of the primers, so that the 5' and 3' end-most sequences of amplified products are completely consistent with the two end sequences of the linearization cloning vectors respectively. And (3) artificially synthesizing the designed primer sequence. The synthesized primer was diluted into a stock solution having a final concentration of 10. Mu. Mol/L. PCR amplification was performed using the diluted primers and templates. After the PCR, agarose gel electrophoresis was performed, and the GSDMD gene of interest was recovered.
c. Ligation of the overexpression vector to the fragment of interest (seamless cloning): the concentration of the recovered vector and the fragment of interest was determined. The optimal cloning vector of the Hieff CloneTM recombination reaction system is 0.03pmol; the molar ratio of the optimum cloning vector to the insert was 1:2, i.e., the optimum insert was used in an amount of 0.06pmol. The DNA mass corresponding to these moles can be calculated from the following formula: optimal cloning vector usage= [0.02×cloning vector base pair number ] ng (0.03 pmol); optimal insert usage = [0.04 x insert base pair number ] ng (0.06 pmol).
d. Cell transformation and antibiotic selection: after the competent cells were naturally thawed on ice (4 ℃), 10. Mu.l of the ligation product of the vector and the target gene fragment was added to the competent cells and left on ice (4 ℃) for 30min. Then heat-shocked in a water bath at 42 ℃ for 90S. Then rapidly placing on ice (4 ℃) for 2-3min. Add 500. Mu.L of antibiotic-free SOC culture based on shaking culture at 37℃and 225rpm for 45min. Centrifuging at 3000 rpm for 2min, removing 900 μl supernatant, blowing off the bacterial solution at the bottom of the tube, adding into culture plate containing corresponding resistance (ampicillin or Canada etc.) on the carrier, homogenizing with sterilized coater (the temperature of the coater cannot be too high to avoid scalding dead bacterial cells), and culturing in a constant temperature incubator at 37deg.C overnight.
e. Sequencing and identification: two of each clone were selected for sequencing identification.
f. By comparison, the sequence of the inserted fragment in the recombinant clone is completely consistent with the sequence of the target fragment, so that the plasmid construction is successful.
g. Plasmid transfection: the cells required for research are selected, in the scheme, HEK-293T tool cells are taken as an example, and plasmids are transferred into the cells according to the operation instructions through a plasmid transfection commercial kit (the specific operation method is shown in a kit instruction).
h. Drug intervention 24-36 hours after plasmid transfection induced inflammatory body activation and downstream GSDMD cleavage.
i. The fluorescence intensity and the color change are observed by a fluorescence microscope or a laser confocal microscope.
j. Furthermore, after labeling mitochondria by fluorescence staining of mitochondria (e.g., mitotracker), localization of GSDMD active fragments on mitochondria can be observed by fluorescence co-localization. Similarly, co-localization with GSDMD-N can also be observed after labeling with specific fluorochromes of other organelles.
(9) The invention is used as follows:
the invention establishes GSDMD over-expression plasmid containing double fluorescent label marks, and fluorescent protein sequences with two different colors are respectively positioned at the N end and the C end of GSDMD. The target cells are directly transfected by using the transfection reagent, after successful transfection, a cell intervention model required by a researcher is established, and fluorescence changes of BFP fluorescent protein and EYFP fluorescent protein are detected by a fluorescence microscope or a laser confocal microscope, so that the aims of observing the expression, cutting, activating and positioning of GSDMD on living cells in real time are fulfilled. FIG. 1 shows fluorescence change of non-induced GSDMD activation state and induced GSDMD activation state after HEK-293T cells are transfected with the GSDMD overexpression plasmids of the invention.
While the invention has been described with respect to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Equivalent embodiments of the present invention will be apparent to those skilled in the art having the benefit of the teachings disclosed herein, when considered in the light of the foregoing disclosure, and without departing from the spirit and scope of the invention; meanwhile, any equivalent changes, modifications and evolution of the above embodiments according to the essential technology of the present invention still fall within the scope of the technical solution of the present invention.
Claims (8)
1. A method for detecting the mesothelin D on living cells in real time; the method is characterized by comprising the following steps of:
step 1: constructing GSDMD overexpression plasmid containing double fluorescent label marks;
step 2: cell transfection with the plasmid obtained in step 1 above; performing drug intervention on the transfected cells to induce activation of inflammatory bodies and downstream GSDMD cleavage;
step 3: observing the fluorescence intensity and the color change in the cells by a fluorescence microscope or a laser confocal microscope;
step 4: mitochondria are marked by mitochondrial fluorescent staining, and the localization of GSDMD active fragments on mitochondria is observed by fluorescence co-localization.
2. A method for real-time detection of desetin D on living cells according to claim 1, characterized in that: in the step 1, the dual fluorescent label marks GSDMD are arranged in such a way that two fluorescent protein sequences with different colors are arranged outside the N-end and C-end sequences of the GSDMD.
3. A method for real-time detection of desetin D on living cells according to claim 2, characterized in that: and a blue fluorescent protein BFP sequence is arranged outside the N-end sequence of the GSDMD.
4. A method for real-time detection of desetin D on living cells according to claim 3, characterized in that: and a yellow fluorescent protein EYFP sequence is arranged outside the C-terminal sequence of the GSDMD.
5. A method for real-time detection of desetin D on living cells according to claim 4, wherein: and in the step 4, a mitochondria mitochondrial green fluorescent probe is adopted for the mitochondrial fluorescent staining label.
6. Use of a method for real-time detection of desetin D on living cells according to any of the claims 1 to 5 for inducing a model of cellular inflammatory response.
7. Use of a method for detecting in real time the presence of desetin D on living cells according to any of the claims 1 to 5 for the detection of inflammatory reactions in cells mediated by drugs.
8. Use of a method for detecting in real time the presence of desetin D on living cells according to any of the claims 1 to 5 in a kit for detecting cellular inflammatory reactions.
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