Monoclonal antibody recognizing human transcription factor Snail and its preparation and application

Abstract

The present application provides a monoclonal antibody that recognizes human transcription factor Snail, whose heavy chain CDR1 amino acid sequence is SEQ ID NO.1; heavy chain CDR2 amino acid sequence is SEQ ID NO.2; heavy chain CDR3 amino acid sequence is SEQ ID NO.3; light chain CDR1 amino acid sequence is SEQ ID NO.6; light chain CDR2 amino acid sequence is SEQ ID NO.7; light chain CDR3 amino acid sequence is SEQ ID NO.8. The present application also provides the detection application and preparation method of the above monoclonal antibody. The monoclonal antibody of the present application has good specificity and high sensitivity, has good application value in actual production, and provides material support for the diagnosis, monitoring and early warning of diseases such as cancer.

Description

Technical Field

[0001] The present application belongs to the field of immunity and biological detection. Specifically, the present application provides a monoclonal antibody that recognizes human transcription factor Snail and its preparation and application. Background Art

[0002] Snail is a zinc finger transcription factor that plays a negative regulatory role. Its sequence and function are very conserved in different species of animals. Snail superfamily members play an important role in physiological or pathological processes such as embryo implantation, embryogenesis, tumorigenesis, cell fate determination, cell cycle regulation, left-right asymmetric development and wound healing. Further research on Snail can not only clarify the mechanism of action of the Snail superfamily, but also provide an important theoretical basis for exploring Snail-related tumor treatment strategies. This is of great significance for revealing the cellular and molecular biological mechanisms of tumor occurrence and development, and it also opens up ideas and provides a strong basis for new methods and approaches for tumor treatment.

[0003] At present, there are few reports on Snail antibodies, especially monoclonal antibodies, and they are often insufficient in sensitivity and specificity. There is a demand in the field for research on new Snail monoclonal antibodies. Summary of the invention

[0004] In response to the above situation, on the one hand, the present application provides a monoclonal antibody that recognizes human transcription factor Snail, the heavy chain CDR1 amino acid sequence of the monoclonal antibody is SEQ ID NO.1; the heavy chain CDR2 amino acid sequence is SEQ ID NO.2; the heavy chain CDR3 amino acid sequence is SEQ ID NO.3; the light chain CDR1 amino acid sequence is SEQ ID NO.6; the light chain CDR2 amino acid sequence is SEQ ID NO.7; and the light chain CDR3 amino acid sequence is SEQ ID NO.8.

[0005] Furthermore, the heavy chain variable region sequence of the monoclonal antibody is SEQ ID NO.4, and / or the light chain variable region sequence of the monoclonal antibody is SEQ ID NO.9.

[0006] On the other hand, the present application provides the use of the above monoclonal antibody in the preparation of a reagent for detecting human transcription factor Snail.

[0007] The reagent can be used for diagnosing tumors and the like.

[0008] Furthermore, the reagent is a western bolt detection reagent, an immunofluorescence detection reagent, an immunoprecipitation detection reagent, an ELISA detection reagent, a colloidal gold detection reagent or a chemiluminescence immunoassay reagent.

[0009] In another aspect, the present application provides a non-diagnostic method for detecting human transcription factor Snail, wherein the method uses the above monoclonal antibody.

[0010] On the other hand, the present application provides a nucleic acid encoding the above monoclonal antibody, whose nucleotide sequence comprises SEQ ID NO.5 and SEQ ID NO.10.

[0011] In another aspect, the present application provides a vector comprising the above nucleic acid.

[0012] In another aspect, the present application provides a host cell comprising the above-mentioned vector.

[0013] In another aspect, the present application provides a method for producing the above monoclonal antibody, the method comprising the step of culturing the above host cell.

[0014] The host cell can be selected from various yeasts, Escherichia coli, Bacillus subtilis and cell-free expression systems, etc. Those skilled in the art can also select a suitable commercial vector according to the host and perform insertion and other operations according to the operating instructions.

[0015] When the antibody CDR region or variable region is known, those skilled in the art can routinely design the framework region and other steps, and design the corresponding gene sequence according to the host preference.

[0016] Beneficial effects of the present invention:

[0017] (1) The present invention prepares a monoclonal antibody that can specifically recognize the human transcription factor Snail.

[0018] (2) The Snail monoclonal antibody and preparation method thereof provided by the present invention have good specificity, high efficiency, accuracy, high sensitivity and low cost, and have great application value in actual production, providing material support for the diagnosis, monitoring and early warning of diseases such as cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the result of Western Blotting detection of immunoprecipitated Snail protein.

[0020] Figure 2 This is the detection result of endogenous Snail protein.

[0021] Figure 3 The results of immunofluorescence detection. DETAILED DESCRIPTION

[0022] The test materials used in the examples of the present invention are all conventional test materials in the art and can be purchased through commercial channels.

[0023] Example 1: Preparation of Snail monoclonal antibody

[0024] (1) Mix 100 ng of plasmid and 20 ul of competent cells, place on ice for 30 min; heat shock for 45 s, place on ice for 2 min, culture at 37°C, 200 rpm for 1 h, centrifuge at 2000 rpm, and keep a small amount of supernatant to coat Kana resistance plates. Culture overnight at 37°C incubator; the next day, expand the culture, induce with 1 mM IPTG, and 37°C for 4 h; after successful expression, increase the volume of the culture, perform protein purification, and detect the protein expression and purification effect by SDS-PAGE. The results are as follows Figure 1 shown.

[0025] (2) Rapidly immunize mice with recombinant human Snail protein to obtain immune mice, use lymphocytes from the immune mice to fuse with myeloma cells, and then screen positive cells and perform subcloning.

[0026] The mouse immunization process is as follows:

[0027] First immunization: 100 μg recombinant human Snail protein and complete Freund's adjuvant were completely mixed at a volume ratio of 1:1 and injected subcutaneously into the hind foot pad of mice;

[0028] Second immunization: The second immunization was performed 2 weeks after the first immunization. 100 μg of recombinant human Snail protein and incomplete Freund's adjuvant were completely mixed at a volume ratio of 1:1 and injected subcutaneously into the hind foot pad of mice.

[0029] The third immunization: 2 weeks after the second immunization, 100 μg recombinant human Snail protein and PBS buffer were mixed at a volume ratio of 1:1 and injected intraperitoneally;

[0030] Ten days after the third immunization, periocular blood of mice was collected for titer testing;

[0031] Booster immunization: Three days before fusion, 100 μg of recombinant human Snail protein was mixed with PBS buffer at a volume ratio of 1:1 and then injected into the tail vein of mice.

[0032] The specific operation of the fusion experiment of lymphocytes and myeloma cells is as follows:

[0033] Myeloma cells and lymphocytes were mixed at a ratio of 1:5 to obtain a cell mixture; the cell mixture was placed in a 50 ml centrifuge tube, diluted with RPMI-1640 basal medium, and then centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, the centrifuge tube was shaken to make the cells uniform, 1 ml of 50% PEG was slowly added, reacted for 90 seconds, and then 10 ml of RPMI-1640 medium was added to terminate PEG, the fused cells were placed in a 37° C. water bath to react for 10 minutes, centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and the RPMI-1640 complete medium was added to resuspend the cells;

[0034] The fused cells were added to a 96-well plate, 100 μl per well; the cell culture plate was then placed in a C02 incubator for culture, and after 10 days of fusion, the cells in the positive wells were screened for fusion.

[0035] The specific operation of the subcloning is as follows:

[0036] Blow the cells in the positive wells, take 10μl and count as N, add PBS buffer to the centrifuge tube according to the limiting dilution method, take 100μl of cell suspension into the centrifuge tube, blow it evenly, take 30μl, add 500μl RPMI-1640 complete medium, blow it evenly and inoculate it on the semi-solid medium, select the monoclonal cell strain visible to the naked eye and inoculate it on the 96-well plate after 6-8 days, and then screen the positive monoclonal cell strain. Prepare ascites.

[0037] (3) Large-scale preparation and purification of monoclonal antibodies:

[0038] Preparation of mouse ascites: 6-week-old healthy BALB / c female mice were sensitized by intraperitoneal injection of 0.5 mL of liquid paraffin. One to two weeks later, the mice were intraperitoneally injected with 10 6 After 7-10 days, the abdominal cavity of the mice was obviously swollen and the ascites was collected. The ascites was centrifuged at 4000 rpm for 15 minutes at room temperature, and the supernatant was the monoclonal antibody ascites.

[0039] (4) Screening:

[0040] The strongly positive cell line obtained after subcloning in step (3) is subjected to ascites preparation and purification. The specific operation of the ascites purification is as follows:

[0041] The protein A agarose gel medium was loaded into a nickel ion affinity chromatography column, and the ascites and PBS were mixed in equal amounts at a volume ratio of 1:1 and then slowly loaded. After the antibody was bound, it was eluted with glycine elution buffer, and finally 10 monoclonal antibodies were purified.

[0042] (5) Saturated ammonium sulfate was slowly added dropwise to half saturation under stirring at 4°C, and stirring was continued for 30 min. Centrifuged at 13000 rpm for 30 min at 4°C, and the supernatant was discarded. The precipitate was dissolved in an appropriate amount of PBS (0.01M, pH7.4); saturated ammonium sulfate was slowly added dropwise to 33% under stirring at 4°C, and stirring was continued for 30 min. Centrifuged at 13000 rpm for 30 min at 4°C, and the supernatant was discarded; the precipitate was dissolved in an appropriate amount of PBS (0.01M, pH 7.4), and dialyzed at 4°C overnight. After ammonium sulfate precipitation, the protein G column was used for purification, and the concentration of the purified antibody was measured by the BCA method, and the monoclonal antibody was finally purified.

[0043] (6) Screening of specific recognition monoclonal antibodies.

[0044] (6.1) Immunoprecipitation experiment of the antibody

[0045] Sample collection

[0046] 293T cells were cultured in a 10 cm culture dish. When the cell density was about 50%, PEI reagent was added to transfect 10 ug plasmid to express snail protein, which was fused with Flag tag. Two days after transfection, the cells were collected and lysed with np40 lysis buffer to set up three experimental groups, with 200 ug of cell lysate taken for each group.

[0047] Immunoprecipitation

[0048] The first group was added with normal mouse IgG and Protein A / G resin; the second group was added with Snail antibody (purchased from Cell Signaling, catalog number: 3879S) and Protein A / G resin; the third group was added with antibody 6-G12-1 described in this patent and Protein A / G resin. After incubation, the resin was collected by centrifugation and the protein in the precipitation reaction was collected by boiling.

[0049] Sample processing

[0050] Add an appropriate amount of concentrated SDS-PAGE protein loading buffer to the collected protein sample, such as 2X or 5X SDS-PAGE protein loading buffer. Heat at 100°C or in a boiling water bath for 3 to 5 minutes to fully denature the protein.

[0051] Sample loading and electrophoresis

[0052] After cooling to room temperature, the protein sample can be directly loaded into the purchased SDS-PAGE gel loading hole. Bio-Rad's standard electrophoresis device usually sets the voltage to 100V and sets the timer to 90-120 minutes. Usually, the electrophoresis can be stopped when the bromophenol blue reaches the bottom of the gel, or it can be stopped when the target protein has been properly separated according to the electrophoresis of the pre-stained protein molecular weight standard.

[0053] Transfer

[0054] Choose PVDF membrane or nitrocellulose membrane (NC membrane), use Bio-Rad's standard wet transfer device, set the transfer current to 300-400mA, and the transfer time to 30-60 minutes. During the transfer process, there is usually very serious heating, so place the transfer tank in an ice bath for transfer.

[0055] Blocking

[0056] After the transfer is completed, immediately place the protein membrane in the pre-prepared Western washing solution and rinse for 1 to 2 minutes to wash off the transfer solution on the membrane. Add Western blocking solution, shake slowly on a shaker, and block at room temperature for 60 minutes.

[0057] Primary antibody incubation

[0058] Dilute the Flag antibody with Western primary antibody diluent at a ratio of 1:1000-10000. Incubate for one hour on a shaker at room temperature. Recover the primary antibody. Add Western wash solution and wash for 5 to 10 minutes on a shaker. After the wash solution is completely absorbed, add wash solution again and wash for 5 to 10 minutes. Wash 3 times in total.

[0059] Secondary antibody incubation

[0060] Purchased horseradish peroxidase-labeled goat anti-mouse IgG (H+L) (A0216), dilute with Western secondary antibody diluent in appropriate proportions, and incubate slowly on a shaker at room temperature or 4°C for one hour. Recover the secondary antibody. Add Western washing solution, and wash slowly on a shaker for 5 to 10 minutes. After the washing solution is completely absorbed, add washing solution again and wash for 5 to 10 minutes. Wash 3 times in total.

[0061] The results are as follows Figure 1 As shown, the co-precipitation effect of the antibody of the present invention is significantly better than that of the control antibody.

[0062] (6.2) Endogenous detection experiment of the antibody

[0063] Sample collection

[0064] 293T cells and SUM159 cells were inoculated in different wells of a 6-well plate. Two days later, the cells were harvested, lysed with np40 lysis buffer, and the lysate was collected for subsequent detection.

[0065] Sample processing

[0066] Add an appropriate amount of concentrated SDS-PAGE protein loading buffer to the collected protein sample, such as 2X or 5X SDS-PAGE protein loading buffer. Heat at 100°C or in a boiling water bath for 3 to 5 minutes to fully denature the protein.

[0067] Sample loading and electrophoresis

[0068] After cooling to room temperature, the protein sample can be directly loaded into the purchased SDS-PAGE gel loading hole. Bio-Rad's standard electrophoresis device usually sets the voltage to 100V and sets the timer to 90-120 minutes. Usually, the electrophoresis can be stopped when the bromophenol blue reaches the bottom of the gel, or it can be stopped when the target protein has been properly separated according to the electrophoresis of the pre-stained protein molecular weight standard.

[0069] Transfer

[0070] Choose PVDF membrane or nitrocellulose membrane (NC membrane), use Bio-Rad's standard wet transfer device, set the transfer current to 300-400mA, and the transfer time to 30-60 minutes. During the transfer process, there is usually very serious heating, so place the transfer tank in an ice bath for transfer.

[0071] Blocking

[0072] After the transfer is completed, immediately place the protein membrane in the pre-prepared Western washing solution and rinse for 1 to 2 minutes to wash off the transfer solution on the membrane. Add Western blocking solution, shake slowly on a shaker, and block at room temperature for 60 minutes.

[0073] Primary antibody incubation

[0074] Dilute the antibody 6-G12-1 of the present invention with Western primary antibody diluent at a ratio of 1:1000-10000, and incubate for one hour on a shaker at room temperature. Recover the primary antibody. Add Western washing solution and wash for 5 to 10 minutes on a shaker. After the washing solution is completely absorbed, add washing solution again and wash for 5 to 10 minutes. Wash 3 times in total.

[0075] Secondary antibody incubation

[0076] Purchased horseradish peroxidase-labeled goat anti-mouse IgG (H+L) (A0216), dilute with Western secondary antibody diluent in appropriate proportions, and incubate slowly on a shaker at room temperature or 4°C for one hour. Recover the secondary antibody. Add Western washing solution, and wash slowly on a shaker for 5 to 10 minutes. After the washing solution is completely absorbed, add washing solution again and wash for 5 to 10 minutes. Wash 3 times in total.

[0077] The results are as follows Figure 2 As shown: the endogenous Snail protein level in 293T cells is very low, while the Snail protein level in Suml59 cells is high.

[0078] (6.3) Immunofluorescence detection experiment of the antibody

[0079] Prepare cell samples. Use conventional cell culture techniques to plant cells on glass-bottomed dishes and ensure that the cells grow adherently. Incubate cells in the logarithmic growth phase with the optimal working concentration of ECL antibody and control antibody for 24 hours.

[0080] Cell fixation: Fix the cells with polymethanol at room temperature for 20 minutes to stabilize intracellular proteins. Wash the cells after fixation to remove excess fixative. Wash with PBST three times, 5 minutes each time.

[0081] Cell permeabilization: Use 0.5% Triton X-100 to permeabilize the cells for 10 minutes at room temperature so that the antibodies can enter the cells and bind to the target protein. After permeabilization, wash the cells to remove excess chemical reagents. Wash 3 times with PBST, 5 minutes each time.

[0082] Blocking: Block the cells with BSA for 60 min at room temperature. Wash the cells to remove excess blocking solution.

[0083] Antibody incubation. Incubate the cells with the antibody against the target protein (6-G12-1) at 4°C overnight to allow the antibody to bind to the target protein. Ensure antibody specificity and low background. After incubation, wash the cells to remove excess antibody. Wash 3 times with PBST, 5 minutes each time.

[0084] Fluorescence labeling. Incubate the cells with green fluorescent labeled mouse secondary antibody in a humidified box at room temperature in the dark for 1 hour to allow the fluorescent labeled secondary antibody to bind to the bound antibody. Wash the cells to remove excess fluorescent labeled secondary antibody. Wash 3 times with PBST, 5 minutes each time.

[0085] Counterstain the nucleus. Add DAPI and incubate in the dark for 5 min. Wash 4 times with PBST, 5 min each time. Add anti-fluorescence quenching stain to seal the slide.

[0086] Observation and analysis. Use a fluorescence microscope to observe the experimental results, and use image analysis software for quantitative and qualitative analysis as needed. Figure 3 shown.

[0087] Example 2: Sequence and structural analysis of monoclonal antibodies 6-G12-1 and

[0088] Monoclonal Antibody 6-G12-1:

[0089] The heavy chain variable region of monoclonal antibody 6-G12-1 contains three CDR regions, as follows:

[0090] VHCDR1:GHTMN (SEQ ID NO: 1);

[0091] VHCDR2: LIDPNNGGTGYNQRFRD (SEQ ID NO: 2);

[0092] VHCDR3: WGYYRFDGGGLDY (SEQ ID NO: 3);

[0093] The amino acid sequence of the heavy chain variable region of monoclonal antibody 6-G12-1 is:

[0094]

[0095] Nucleotide sequence:

[0096]

[0097] The light chain variable region of monoclonal antibody 6-G12-1 contains three CDR regions, as follows:

[0098] VLCDR1: RSSQSIVHSNGNTYLE (SEQ ID NO: 6);

[0099] VLCDR2: KVSTRFS (SEQ ID NO: 7);

[0100] VLCDR3: FQGSHVPPT (SEQ ID NO: 8);

[0101] The amino acid sequence of the light chain variable region of monoclonal antibody 6-G12-1 is:

[0102]

[0103] Nucleotide sequence:

[0104]

[0105] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A monoclonal antibody that recognizes human transcription factor Snail, characterized in that: The heavy chain CDR1 amino acid sequence of the monoclonal antibody is SEQ ID NO.1; the heavy chain CDR2 amino acid sequence is SEQ ID NO.2; the heavy chain CDR3 amino acid sequence is SEQ ID NO.3; the light chain CDR1 amino acid sequence is SEQ ID NO.6; the light chain CDR2 amino acid sequence is SEQ ID NO.7; and the light chain CDR3 amino acid sequence is SEQ ID NO.

8. 2 . The monoclonal antibody according to claim 1 , wherein the heavy chain variable region sequence of the monoclonal antibody is SEQ ID NO.

4.

3. The monoclonal antibody according to claim 1 or 2, wherein the light chain variable region sequence of the monoclonal antibody is SEQ ID NO.

9.

4. Use of the monoclonal antibody according to any one of claims 1 to 3 in the preparation of a reagent for detecting human transcription factor Snail.

5. The use according to claim 4, wherein the reagent is a western bolt detection reagent, an immunofluorescence detection reagent, an immunoprecipitation detection reagent, an ELISA detection reagent, a colloidal gold detection reagent or a chemiluminescence immunoassay reagent.

6. A non-diagnostic method for detecting human transcription factor Snail, characterized in that The method uses the monoclonal antibody according to any one of claims 1 to 3.

7. A nucleic acid encoding a monoclonal antibody according to any one of claims 1 to 3, characterized in that: The nucleotide sequence of the nucleic acid comprises SEQ ID NO.5 and SEQ ID NO.

10.

8. A carrier, characterized in that The vector comprises the nucleic acid according to claim 7.

9. A host cell, characterized in that The host cell comprises the vector according to claim 8.

10. A method for producing a monoclonal antibody according to any one of claims 1 to 3, characterized in that: The method comprises the step of culturing the host cell according to claim 9.

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