A method for detecting the activity of a leptin-related protein or analog thereof

Abstract

The application discloses a method for detecting the activity of leptin related protein or its analog. The application utilizes the stable cell strain BAF3-EF1a-LepR with high expression of leptin antibody to detect samples with the activity of leptin protein such as leptin related protein and leptin protein analog. The activity of the target protein is determined through the cell proliferation promoting effect of leptin.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for detecting the activity of leptin-related protein or its analog, belonging to the technical field of biological detection. BACKGROUND

[0002] Leptin is an important protein hormone in vivo, which acts on multiple target organs and has a wide range of functions. Its main physiological functions include: ① inhibiting food intake and increasing energy consumption; ② regulating growth and development; ③ regulating inflammatory response and immune function; ④ promoting epithelial cell and blood vessel growth; ⑤ regulating neuroendocrine; ⑥ protecting digestive system function; ⑦ maintaining normal lipid metabolism, etc. It can also participate in functions such as reproduction, blood pressure, hematopoiesis, fetal growth and development, and endocrine regulation. Its main function is to regulate energy balance and is a basic hormone for maintaining normal body weight. Since the discovery of the leptin gene, people have conducted a large number of studies on the biological effects of leptin. In recent years, the role of leptin and its receptor in the occurrence and development of tumors has also been gradually studied. In particular, leptin can stimulate the proliferation of various tumor cells, and its mechanism may be related to increasing the invasiveness of tumor cells to the basement membrane and promoting the formation of new blood vessels in tumors, so leptin is considered a new tumor growth factor. Experiments have shown that leptin and leptin receptor are expressed in breast cancer, lung adenocarcinoma, and digestive tract tumor tissues and tumor cell lines. In recent years, it has been found that Leptin, as a growth factor, can promote the proliferation of various cells. Based on the above, the research on leptin-related proteins and their analogs is currently very popular. Therefore, the activity detection of leptin protein analogs is particularly important.

[0003] Most of the existing technologies currently use double antibody sandwich enzyme-linked immunosorbent assay detection technology (ELISA) to detect leptin protein. That is, specific anti-human leptin antibodies are pre-coated on high-affinity enzyme-labeled plates. Standard, sample to be tested and biotinylated detection antibody are added to the enzyme-labeled plate wells, incubated, and the leptin present in the sample is combined with the solid-phase antibody and the detection antibody. After washing to remove unbound substances, streptavidin-HRP labeled with horseradish peroxidase is added. After washing, color developing substrate TMB is added, and color development is avoided. The depth of the color reaction is proportional to the concentration of leptin in the sample. The stop solution is added to stop the reaction, and the absorbance value is measured at 450 nm wavelength (reference wavelength 570-630 nm).

[0004] The existing technology is aimed at qualitatively and quantitatively detecting the binding activity of leptin antibodies. Enzyme-linked immunosorbent assay is usually used for detection, which can only qualitatively and quantitatively confirm the binding of protein and antibody, and cannot simulate the binding state of protein in a complex cell environment. SUMMARY

[0005] To solve the above technical problems, the present application uses a stable cell strain BAF3-EF1a-LepR with high expression of leptin antibody (LepR) to detect leptin-related protein, leptin protein analogs and other samples with leptin protein activity. The activity of the target protein is determined by the cell proliferation effect of leptin.

[0006] The first object of the present application is to provide a method for detecting the activity of leptin-related protein or its analogs, which uses different concentrations of leptin-related protein or its analogs to co-culture with cell strain BAF3-EF1a-LepR, and detects the activity of leptin-related protein or its analogs according to the proliferation of cell strain BAF3-EF1a-LepR.

[0007] Further, the leptin-related protein or its analogs are leptin protein, leptin protein analogs or leptin fusion protein.

[0008] Further, the different concentrations of leptin-related protein or its analogs are gradiently set at several different concentrations of leptin-related protein or its analogs solution in the concentration range of 0.01 ng / ml to 333 ng / ml.

[0009] Further, the cell strain BAF3-EF1a-LepR is a LepRb gene regulated by pSMPUW as a carrier, EF-1a promoter, IL-3 signal peptide and post-transcriptional regulatory element WPRE in mouse BAF3 cells.

[0010] Further, the co-culture time is 48 to 96 hours.

[0011] Further, the co-culture temperature is 35 to 38℃.

[0012] Further, during co-culture, the cell strain BAF3-EF1a-LepR is a cell strain cultured to the logarithmic growth phase with cell viability greater than 95%.

[0013] Further, during co-culture, the cell strain is cultured in a detection medium, and the detection medium is obtained by adding 2% FBS and 1% PS to culture medium RPMI 1640.

[0014] Further, the proliferation of cell strain BAF3-EF1a-LepR is determined by adding indicator Alamar-blue for incubation and color development, and then detecting the fluorescence value to determine the proliferation of cell strain BAF3-EF1a-LepR.

[0015] Further, the method specifically comprises the following steps:

[0016] S1, recover cell strain BAF3-EF1α-LepR, and stably culture to logarithmic growth phase to obtain cell strain for activity detection;

[0017] S2, prepare solutions of different concentrations of the leptin-related protein or analogs thereof to be detected, and respectively co-culture with the cell strain of S1 step for 48-96 hours;

[0018] S3, add indicator Alamar-blue to the cell culture solution after co-culture, and after 2-4 hours of incubation, measure the fluorescence value;

[0019] S4, use the logarithmic value of the concentration of the leptin-related protein or analogs thereof to be detected as the abscissa, and the fluorescence value as the ordinate, fit the curve, and judge the activity of the leptin-related protein or analogs thereof.

[0020] The beneficial effects of the present application are:

[0021] The present application uses the stable cell strain BAF3-EF1α-LepR with high expression of leptin antibody (LepR) to detect samples with leptin protein activity of leptin-related proteins, leptin protein analogs, etc. The activity of the target protein is determined through the cell proliferation promoting effect of leptin, and the detection sensitivity reaches 0.061 ng / ml-14.77 ng / ml. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 It is a sample distribution diagram for a 96-well plate.

[0023] Figure 2 It is the proliferation of cells detected by fitting an S-type curve with the logarithmic value of the concentration of the leptin protein as the abscissa and the fluorescence value as the ordinate after stimulating BAF3-EF1α-LepR cells with different concentrations of leptin protein for 72 hours.

[0024] Figure 3 It is the proliferation of cells detected by fitting an S-type curve with the logarithmic value of the concentration of the leptin protein as the abscissa and the fluorescence value as the ordinate after stimulating BAF3-EF1α-LepR cells with different concentrations of leptin protein and leptin fusion protein for 72 hours. Among them, "leptin" is a positive control protein, and "sample 1" and "sample 2" are leptin fusion protein detection samples. DETAILED DESCRIPTION

[0025] The present application will be further described below in conjunction with specific embodiments, so that those skilled in the art can better understand the present application and implement it, but the embodiments are not limiting to the present application.

[0026] The BAF3-EF1a-LepR cell strain used in the present application is obtained by expressing LepRb gene in mouse BAF3 cells using pSMPUW as a carrier, and cloning EF-1a promoter, IL-3 signal peptide and post-transcriptional regulatory element WPRE on the expression vector to regulate the expression of LepRb gene (for specific construction method, refer to patent CN 110157734A). The cell strain has high expression level of leptin antibody (LepR) on the cell surface, and the expression is stable, which is a better tool cell for detection.

[0027] Example 1:

[0028] 1. BAF3-EF1a-LepR cell recovery:

[0029] 1) Prepare complete culture medium RPMI 1640 + 10% fetal bovine serum (FBS) + 1% penicillin / streptomycin (PS) and preheat to 37°C for standby;

[0030] 2) Take the cells from the liquid nitrogen container and quickly melt at 37°C.

[0031] 3) Add the cells to a centrifuge tube containing 10 ml of complete culture medium, mix well, and centrifuge at 800 rpm for 5 min at room temperature;

[0032] 5) Discard the supernatant, resuspend the cells with complete culture medium, and add leptin to the culture medium to a final concentration of 0.4 μg / ml. Place the cells in a 37°C, 5% CO2 incubator and incubate;

[0033] 6) The next day, replace the culture medium and add leptin to a final concentration of 25 nM (0.4 μg / ml). The cell culture density is maintained at about 5×10 5 -2×10 6 / ml, and the medium is replaced according to the cell state and density during the period.

[0034] 2. Cell plating:

[0035] 1) The activity detection experiment can be carried out after the cells are recovered and stabilized for one week (the cells enter the logarithmic growth phase). Ensure that the cell viability is greater than 95% before the experiment;

[0036] 2) Prepare the detection culture medium (RPMI 1640 + 2% FBS + 1% PS) before the experiment, and preheat the detection culture medium and DPBS to room temperature for standby;

[0037] 3) Take out the cells in logarithmic phase from the incubator, measure the cell density and cell viability (more than 95% is preferred), take the required number of cells in the centrifuge tube, centrifuge at 800 rpm for 5 min, discard the supernatant; add 5 ml of phosphate buffer (DPBS) to resuspend the cells, centrifuge at 800 rpm for 5 min, discard the supernatant, repeat the above steps 3 times. Finally, resuspend the cells with the detection medium, and the cell density is 1 x 10 5 / ml;

[0038] 4) Prepare a black-walled transparent-bottom 96-well plate, add 60 μl of cell suspension (6 x 10 3 / well) in step 3) to each well, and spread the cells in the 96-well plate according to the following table, "1" is the different concentration experimental well, 10 concentration gradients are set for each sample, and 3 replicate wells are set for each concentration. "Medium" is the well containing only the detection medium, and "0" is the well with a sample concentration of 0. Add 100 μl of DPBS to the cells in rows A, E and the connected honeycombs to ensure the uniformity of the liquid and cell state in each well. Figure 1

[0039] 5) Place the 96-well plate with cells in the incubator.

[0040] 6) Use the detection medium to prepare different concentrations of leptin protein solution (Leptin): 333, 111, 37, 12.3, 4.1, 1.4, 0.45, 0.15, 0.05, 0.01 ng / ml. Take 40 μl of each concentration sample and add it to the 96-well plate to obtain experimental wells with a sample concentration starting from 133 ng / ml, three-fold dilution, and a final volume of 100 μl per well, numbered from high to low concentration as 1-1#-1-10#;

[0041] 7) After adding the sample, place the 96-well plate in the incubator (37°C, 5% CO2) for incubation and culture for 72 h, and observe the cell state every day during the culture period.

[0042] 3. Fluorescence detection

[0043] 1) Prepare the detection medium with the appropriate concentration of Alamar-blue color reagent, and the working concentration of Alamar-blue is 5%. Filter Alamar-blue 0.22 μm membrane in advance to remove the precipitate in the reagent. According to the concentration ratio, accurately add 10 μL of Alamar-blue to each well (make sure that the gun head is below the liquid surface when adding the sample). Gently shake the cell plate in front, back, left and right to mix the color reagent in the culture medium.

[0044] Place the 96-well plate in the incubator for constant temperature incubation for 3 h.

[0045] ​2) Using a microplate reader, the fluorescence value of each well was measured at Excitation / Emission = 540 nm / 590 nm.

[0046] 4. Experimental results and calculations

[0047] Using the logarithmic value of the concentration of the test substance as the abscissa and the fluorescence value as the ordinate, an S-shaped curve was fitted, and the half-effective concentration of the test substance was calculated as Figure 2 (EC50), by Figure 2 It can be concluded that the EC50 of leptin in this example is = 0.64 ng / ml, and the detection sensitivity is: 0.061 ng / ml-14.77 ng / ml.

[0048] Example 2:

[0049] The activities of protein sample 1 (Sample 1) and sample 2 (Sample 2) were detected according to the experimental method in Example 1 as shown in Figure 3 .

[0050] Among them, Sample 1 and Sample 2 are leptin fusion protein samples constructed in the laboratory, Leptin is a positive control in the experiment, and a group of negative control samples are additionally added. Using the logarithmic value of the concentration of the test substance as the abscissa and the fluorescence value as the ordinate, an S-shaped curve was fitted, and the half-effective concentration of the test substance was calculated as follows (EC50). The EC50 of Sample 1, Sample 2 and Leptin positive control is 0.73 ng / ml, 0.60 ng / ml and 0.64 ng / ml, respectively. It can be seen that Sample 1 and Sample 2 can bind to leptin antibody and promote the proliferation of BAF3-EF1α-LepR cells. Through the experiment, it can be reflected that Sample 1 and Sample 2 are active protein samples.

[0051] The above-described examples are only preferred examples for fully illustrating the present application, and the protection scope of the present application is not limited thereto. The equivalent substitutions or transformations made by the person skilled in the art on the basis of the present application are within the protection scope of the present application. The protection scope of the present application is subject to the claims.

Claims

1. A method for detecting the activity of a leptin-related protein or an analog thereof, characterized by, The method is to co-culture the leptin-related protein or its analogues with different concentrations with the cell strain BAF3-EF1a-LepR, and then detect the activity of the leptin-related protein or its analogues according to the proliferation of the cell strain BAF3-EF1a-LepR; The cell strain BAF3-EF1a-LepR is to express the LepRb gene in the mouse BAF3 cells by taking pSMPUW as a carrier, and taking the EF-1a promoter, IL-3 signal peptide and the post-transcriptional regulatory element WPRE for regulation; The co-culture time is 48-96 hours; The co-culture temperature is 35-38 DEG C; During the co-culture, the cell strain BAF3-EF1a-LepR is a cell strain cultured to the logarithmic growth phase and having a cell viability of greater than 95%; During the co-culture, the cell strain is cultured in a detection culture medium, and the detection culture medium is obtained by adding 1-3% FBS and 0.5-1.5% PS to the culture medium RPMI 1640; The proliferation of the cell strain BAF3-EF1a-LepR is determined by adding the indicator Alamar-blue for incubation and color development, and then by detecting the fluorescence value to determine the proliferation of the cell strain BAF3-EF1a-LepR.

2. The method of claim 1, wherein, The leptin-related protein or its analogues are leptin protein, leptin protein analogues or leptin fusion protein.

3. The method of claim 1, wherein, The leptin-related protein or its analogues with different concentrations are a plurality of leptin-related protein or its analogues solution with different concentrations set in a gradient in the concentration range of 0.01 ng / ml-333 ng / ml.

4. The method of claim 1, wherein, The method specifically comprises the following steps: S1, resuscitate the cell strain BAF3-EF1a-LepR, and stably culture to the logarithmic growth phase to obtain the cell strain for activity detection; S2, prepare the leptin-related protein or its analogues solution with different concentrations, and co-culture with the cell strain of S1 for 48-96 hours; S3, add the indicator Alamar-blue to the cell culture solution after the co-culture, incubate for 2-4 hours, and then detect the fluorescence value; S4, take the logarithmic value of the concentration of the leptin-related protein or its analogues as the abscissa, and take the fluorescence value as the ordinate, fit the curve, and determine the activity of the leptin-related protein or its analogues.

Images