Kit for cell apoptosis detection and product thereof

Abstract

The invention relates to the technical field of cell apoptosis detection, in particular to a kit for cell apoptosis detection and a product thereof. The Annexin V-FITC provided by the invention can well realize the detection of apoptosis, has obvious linear correlation with the apoptosis rate, and can realize the high-sensitivity detection of the occurrence of apoptosis. The method and the kit for detecting cell apoptosis can effectively identify phosphatidylserine exposed on the outer surface of apoptotic cells, and have more detection advantages compared with Annexin V full length. The sensitivity and the cell apoptosis rate of the detection method have a good linear relationship, and the detection method has a relatively great application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of cell apoptosis detection, and in particular to a kit for cell apoptosis detection and a product thereof. Background Art

[0002] Apoptosis, or programmed cell death, is one of the built-in defense mechanisms of cells and plays an important role in the normal development of organisms and disease resistance. Abnormalities in the apoptosis process and pathways can lead to a variety of diseases, including tumors. As an important part of the cell life cycle, the process of apoptosis is accompanied by a series of changes in biomolecules and cell morphology.

[0003] Phosphatidylserine (PS) in the lipid bilayer of the cell membrane flips from the inner layer of the cell membrane to the outer layer and is then exposed to the cell surface. This is an early event in apoptosis, occurs before the morphological changes of apoptotic cells, and is also the initial event of the apoptotic cascade. As a sign of apoptotic cells being recognized by phagocytes, the eversion of PS will further cause phenomena such as cytoplasmic shrinkage, chromatin condensation, and nuclear DNA degradation. Therefore, the everted PS is a target for apoptosis detection with high timeliness and can detect apoptosis at an early stage. Annexin V is a member of the intracellular protein annexin family and binds to phosphatidylserine (PS) in a calcium-dependent manner. In the early stages of apoptosis, membrane asymmetry is lost and PS translocates to the cell surface. Fluorescently labeled Annexin V can specifically bind to it, indicating that the cell is an apoptotic cell.

[0004] However, although apoptosis detection methods based on fluorescently labeled Annexin V have been reported, there are major problems with their detection sensitivity and detection line. Most detection results are average and cannot accurately determine whether cells are apoptotic. Summary of the invention

[0005] In view of the technical problems existing in the prior art, the present invention provides a kit for detecting cell apoptosis and its products. The present invention is based on the active binding domain of Annexin V, involves fluorescently labeled Annexin V, and can well detect cell apoptosis, has an obvious linear correlation with the cell apoptosis rate, can realize highly sensitive detection of cell apoptosis, and has great application prospects.

[0006] Specifically, the present invention first provides a kit for detecting cell apoptosis, wherein the kit comprises a fluorescently labeled Annexin V active binding domain.

[0007] Preferably, the Annexin V active binding domain contains the amino acid sequence shown in SEQ ID NO.1; Preferably, the Annexin V active binding domain has an amino acid sequence as shown in SEQ ID NO.1; Furthermore, the present invention also provides a fluorescently labeled Annexin V active binding domain, wherein the Annexin V active binding domain is labeled with a fluorescent group.

[0008] Preferably, the fluorescent label provided by the present invention is a fluorescent group label such as FITC, FAM, Rhodamine B, TAMRA, Cy3, Cy5, etc.

[0009] Preferably, the fluorescent label provided by the present invention is FITC.

[0010] Furthermore, the present invention also provides a product for detecting cell apoptosis, wherein the product is the fluorescently labeled Annexin V active binding domain.

[0011] Furthermore, the present invention also provides a method for fluorescent detection of cell apoptosis for non-diagnostic purposes, which comprises contacting the test kit or the fluorescently labeled Annexin V active binding domain or the product with the cell lysate of the sample to be tested, and after the incubation, observing the results under a fluorescence microscope or reading the fluorescence value with an enzyme-labeled instrument.

[0012] Furthermore, the present invention also provides the use of the fluorescently labeled Annexin V active binding domain in the preparation of a kit for detecting cell apoptosis.

[0013] The advantages of the present invention are as follows: the Annexin V-FITC provided by the present invention can well realize the detection of cell apoptosis, has a significant linear correlation with the cell apoptosis rate, and can realize highly sensitive detection of cell apoptosis. The method and kit for detecting cell apoptosis constructed by the present invention can effectively identify phosphatidylserine exposed on the outer surface of apoptotic cells, and has a greater detection advantage than the full length of Annexin V. The sensitivity of the detection method of the present invention has a good linear relationship with the cell apoptosis rate, and has a great application prospect. DETAILED DESCRIPTION

[0014] The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand the present invention more clearly.

[0015] The following embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Based on the specific embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without creative work belong to the protection scope of the present invention.

[0016] In the examples of the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art; in the examples of the present invention, unless otherwise specified, the technical means used are conventional means well known to those skilled in the art.

[0017] Example 1 Preparation of Annexin V-FITC Annexin V active binding site screening: Annexin V (GenPept: AAB60648.1) included in NCBI was used as the target, and its active binding domain binding to phosphatidylserine (PS) was analyzed. The hydrophilicity, charge, polarity and length of the active binding domain were comprehensively considered, and the active binding domain sequence was finally selected as vvgdtsgyyq rmlvvllqan (SEQ ID NO.1).

[0018] The coding sequence of SEQ ID NO.1 was artificially synthesized and used to construct the prokaryotic expression plasmid pET-N-GST-PreScission (Beijing Biolab Technology Co., Ltd., product number YT971), and the Annexin V recombinant protein containing a GST tag at the N-terminus was expressed in Escherichia coli DH5α. The recombinant protein was purified by glutathione agarose gel affinity chromatography, and then the GST tag was enzymatically removed by PreScission, and the Annexin V short peptide was collected. SDS-PAGE electrophoresis detection and Western blot identification confirmed that the target protein Annexin V short peptide was correctly expressed, and the protein concentration was adjusted to 15 mg / mL with PBS solution for standby use.

[0019] Take 100μg / mL Annexin V solution, dialyze overnight in 0.5mol / L carbonate buffer with pH 9.5, add dimethyl sulfoxide (DMSO) containing FITC at a mass ratio of 10:1 between FITC and antibody; place the antibody solution on a magnetic stirrer and stir gently until no foaming occurs. Add FITC-DMSO solution dropwise to the monoclonal antibody solution according to the above ratio, stir for 12 hours at 4℃ in the dark with a magnetic stirrer; after addition, measure the pH value of the stirring solution with a test paper at any time. If it is lower than 9.0, adjust it with sodium carbonate solution. Place at 4℃ and stir for 12 hours. Filter Annexin V-FITC with a Sephadex G25 column to remove free fluorescein.

[0020] The OD values ​​of Annexin V-FITC at 280nm and 495nm were measured by UV spectrophotometer. According to the formula F / P = (2.87 × OD 495 ) / (OD 280 -0.35×OD 495) Calculate the binding rate of fluorescent pigment to Annexin V and evaluate the labeling effect of Annexin V-FITC.

[0021] The results showed that the optical density of Annexin V-FITC at 280nm was 0.856 and the optical density at 495nm was 0.469. The F / P value of the fluorescent antibody was calculated to be 1.946 according to the formula, indicating that there was no over-labeling of Annexin V-FITC in this experiment.

[0022] Example 2 Cell apoptosis detection based on Annexin V-FITC In vitro apoptosis model construction: Taking L-02 cells as an example, 1×10 5 The cells were inoculated in RPMI-1640 medium containing 10% FBS. After 24 hours of overnight culture, the supernatant was discarded, the culture medium was replaced and 0.6mM H2O2 was added. After 1 hour of action, the culture supernatant in the 24-well plate was collected to detect AST activity and MDA content, and the cell apoptosis in the culture plate was observed under a microscope. The results showed that the AST activity and MDA content detected in the culture supernatant in the model group were significantly increased, and the cell apoptosis under the microscope reached 85.2%, confirming that the in vitro cell apoptosis model of the present invention was successfully constructed.

[0023] Cell apoptosis detection method based on Annexin V-FITC: L-02 cell lysates treated with different concentrations of H2O2 (6mM, 0.6mM, 0.06mM, 6μM, 0.6μM, 0.06μM and 0μM) were collected and cultured respectively, and the Annexin V-FITC at a concentration of 50μg / mL was added, incubated at 37°C for 60min, washed 3 times with PBS, and recorded on a microplate reader with an excitation wavelength of 460nm and an emission wavelength of 520nm.

[0024] The control group was based on the full-length Annexin V, and FITC-labeled Annexin V was prepared using the method described in Example 1 of the present invention.

[0025] The results are shown in Table 1 below.

[0026] Table 1: Test results

[0027] According to the analysis of fluorescence detection data, the Annexin V-FITC provided by the present invention can well detect apoptosis, and has a significant linear correlation with the apoptosis rate, and can detect apoptosis with high sensitivity. In the control group, the apoptosis detection rate was low, and its fluorescence labeling effect was not as good as that of the experimental group. It is further proved that the Annexin V active binding fragment provided by the present invention is more advantageous than the full length in detecting apoptosis.

[0028] It is necessary to point out that the above embodiments are limited to further elaboration and explanation of the technical solution of the present invention, and are not further limitations of the technical solution of the present invention. The method of the present invention is only a preferred implementation scheme, and is not used to limit the protection scope of the present invention. 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 kit for detecting cell apoptosis, characterized in that: The kit contains a fluorescently labeled Annexin V active binding domain.

2. The kit according to claim 1, characterized in that The Annexin V active binding domain contains the amino acid sequence shown in SEQ ID NO.

1.

3. The kit according to claim 1, characterized in that The Annexin V active binding domain has an amino acid sequence as shown in SEQ ID NO.

1.

4. The kit according to any one of claims 1 to 3, characterized in that The fluorescent label includes but is not limited to one or more of FITC, FAM, Rhodamine B, TAMRA, Cy3, and Cy5.

5. The kit according to claim 4, characterized in that The fluorescent marker is FITC.

6. A fluorescently labeled Annexin V active binding domain, characterized in that: The Annexin V active binding domain is labeled with a fluorescent group, wherein the Annexin V active binding domain contains the amino acid sequence shown in SEQ ID NO.

1.

7. The fluorescently labeled Annexin V active binding domain according to claim 6, characterized in that The fluorescent label includes but is not limited to one or more of FITC, FAM, Rhodamine B, TAMRA, Cy3, and Cy5.

8. The fluorescently labeled Annexin V active binding domain according to claim 7, characterized in that: The fluorescent marker is FITC.

9. A product for detecting cell apoptosis, characterized in that: The product comprises the fluorescently labeled Annexin V active binding domain according to any one of claims 6 to 8.

10. A method for detecting cell apoptosis by fluorescence for non-diagnostic purposes, characterized in that: The detection method comprises contacting the test kit according to any one of claims 1 to 5, the fluorescently labeled Annexin V active binding domain according to any one of claims 6 to 8, or the product according to claim 9 with the cell lysate of the sample to be tested, and after the incubation, observing the result under a fluorescence microscope or reading the fluorescence value with an enzyme-labeled instrument.

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