A monoclonal antibody against c-Myc tag and its application

Abstract

The present invention discloses a monoclonal antibody against c-Myc tag and its application. The antibody comprises a heavy chain CDR1, a heavy chain CDR2 and a heavy chain CDR3, and a light chain CDR1, a light chain CDR2 and a light chain CDR3; the amino acid sequence of the heavy chain CDR1 is shown in SEQ ID NO: 1; the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID NO: 2; the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID NO: 3; the amino acid sequence of the light chain CDR1 is shown in SEQ ID NO: 4; the amino acid sequence of the light chain CDR2 is shown in SEQ ID NO: 5; and the amino acid sequence of the light chain CDR3 is shown in SEQ ID NO: 6; the ascites antibody titer thereof can reach 8.48×10 6 After purification, the antibody affinity reached 1.113E‑8, the antibody activity was high, and it has great application potential.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more particularly to an anti-c-Myc tag monoclonal antibody and applications thereof. Background Art

[0002] c-Myc protein is a transcription factor encoded by the c-Myc gene on human chromosome 8q24. c-Myc is commonly activated in various tumor cells and plays an important role in cell proliferation, differentiation, apoptosis, and cell cycle progression. Antibodies to the c-Myc tag interact with and specifically recognize the Myc-tagged target protein, enabling the successful detection of certain target proteins for which no specific antibodies are currently available. Existing c-Myc tag antibodies are limited in variety and expensive. Therefore, developing more monoclonal antibodies against the c-Myc tag will facilitate the detection of c-Myc fusion proteins or c-Myc expression levels in various immunoassays. These antibodies hold great promise for the detection, isolation, and purification of Myc-tagged target proteins. Summary of the Invention

[0003] In response to the above-mentioned deficiencies or improvement needs of the prior art, the present invention provides a monoclonal antibody against the c-Myc tag and its application. The purpose is to stimulate mice to produce antibodies by coupling c-Myc protein (a ten-amino acid short peptide EQKLISEEDL) with keyhole limpet hemocyanin (KLH) as antigens, thereby discovering a high-titer c-Myc tag antibody in vivo. The amino acid sequences of the heavy and light chain complementary determining regions of the antibody are obtained by cloning and sequencing the variable region genes of the antibody, and based on these sequences, a monoclonal antibody against the c-Myc tag is prepared, thereby solving the technical problem that existing c-Myc tag antibodies are relatively few in variety and expensive.

[0004] To achieve the above objectives, according to one aspect of the present invention, a monoclonal antibody against c-Myc tag is provided, comprising three heavy chain complementarity determining regions: heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, and three light chain complementarity determining regions: light chain CDR1, light chain CDR2, and light chain CDR3;

[0005] The heavy chain CDR1 is the amino acid sequence AYFVN shown in SEQ ID NO: 1;

[0006] The heavy chain CDR2 is the amino acid sequence KINPYNLYNFYNQKFKG shown in SEQ ID NO: 2;

[0007] The heavy chain CDR3 is the amino acid sequence EGEYSGTFPYGMDY shown in SEQ ID NO: 3;

[0008] The light chain CDR1 is the amino acid sequence KASQNVGNIIA shown in SEQ ID NO: 4;

[0009] The light chain CDR2 is the amino acid sequence LASYRYT shown in SEQ ID NO: 5;

[0010] The light chain CDR3 is the sequence NQYKNSPYT shown in SEQ ID NO:6.

[0011] Preferably, the monoclonal antibody against c-Myc tag, wherein FR1-4 in the heavy chain variable region of the antibody is the following amino acid sequence:

[0012] comprising or consisting of the amino acid sequence shown in SEQ ID NO: 7, 8, 9, 10,

[0013] or a sequence having at least 80%, 85%, or 90% identity to the sequence shown in SEQ ID NO: 7, 8, 9, or 10,

[0014] Or a sequence having one or more amino acid mutations compared to the amino acid sequence shown in SEQ ID NO: 7, 8, 9, or 10.

[0015] Preferably, in the monoclonal antibody against c-Myc tag, FR1-4 in the heavy chain variable region are the following amino acid sequences:

[0016] The amino acid sequences shown in SEQ ID NOs: 7, 8, 9, and 10,

[0017] or a sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 7, 8, 9, 10,

[0018] Or an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid mutations compared to the amino acid sequence shown in SEQ ID NO: 7, 8, 9, 10; the amino acid mutation is a conservative mutation, including substitution, insertion or deletion.

[0019] Preferably, the monoclonal antibody against c-Myc tag, wherein FR1-4 in the light chain variable region of the antibody is the following amino acid sequence:

[0020] comprising or consisting of the amino acid sequence shown in SEQ ID NO: 11, 12, 13, 14,

[0021] or a sequence having at least 80%, 85%, or 90% identity to the sequence shown in SEQ ID NO: 11, 12, 13, or 14,

[0022] Or a sequence having one or more amino acid mutations compared to the amino acid sequence shown in SEQ ID NO: 11, 12, 13, or 14.

[0023] Preferably, in the monoclonal antibody against c-Myc tag, FR1-4 in the light chain variable region are the following amino acid sequences:

[0024] The amino acid sequences shown in SEQ ID NOs: 11, 12, 13, and 14,

[0025] or a sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 11, 12, 13, 14,

[0026] Or an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid mutations compared to the amino acid sequence shown in SEQ ID NO: 11, 12, 13, 14; the amino acid mutation is a conservative mutation, including substitution, insertion or deletion.

[0027] Preferably, the monoclonal antibody against c-Myc tag has a heavy chain amino acid sequence as shown in SEQ ID NO: 15 and a light chain amino acid sequence as shown in SEQ ID NO: 16.

[0028] According to another aspect of the present invention, a biological material containing a nucleic acid sequence encoding the monoclonal antibody against c-Myc tag of the present invention is also provided. The biological material includes an expression vector, an expression cassette, a host cell, an engineered bacterium or a hybridoma cell line.

[0029] According to another aspect of the present invention, there is also provided a use of the anti-c-Myc tag monoclonal antibody of the present invention in detecting, separating and purifying Myc-tagged target proteins.

[0030] According to another aspect of the present invention, a detection kit is provided, which comprises the monoclonal antibody against c-Myc tag according to the present invention.

[0031] Preferably, the detection kit further comprises a c-Myc tag polypeptide.

[0032] In general, the above technical solutions conceived by the present invention, compared with the prior art, provide a monoclonal antibody against c-Myc tag, which can achieve the following beneficial effects:

[0033] The present invention stimulates mice by coupling c-Myc protein with KLH as an antigen, discovers a high-titer c-Myc tag antibody in the mice, clones and sequences the variable region genes of the antibody, obtains the amino acid sequences of the heavy chain and light chain complementary determining regions, and uses recombinant technology to prepare an anti-c-Myc tag monoclonal antibody. Measurements show that the anti-c-Myc tag monoclonal antibody provided by the present invention has an affinity KD of 1.113E-8 after purification, indicating that the purified anti-c-Myc tag monoclonal antibody has good affinity and high activity, and can be used to detect, separate and purify Myc-tagged target proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Schematic diagram of the amino acid sequences of the heavy and light chains of the monoclonal antibody against c-Myc tag in Example 4.

[0035] Figure 2 It is an assay for the activity of anti-c-Myc tag monoclonal antibodies. DETAILED DESCRIPTION

[0036] In order to further illustrate the technical means and effects adopted by the present invention to achieve the predetermined purpose of the invention, the specific implementation methods, structures, features and effects of the present invention are described in detail below in conjunction with the accompanying drawings and preferred embodiments.

[0037] The term "amino acid" refers to naturally occurring or non-naturally occurring carboxyl α-amino acids. The term "amino acid" as used in the present application may include naturally occurring amino acids and non-naturally occurring amino acids. Naturally occurring amino acids include alanine (three letter code: Ala, single letter code: A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine ​​(Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Val, V). Non-naturally occurring amino acids include, but are not limited to, α-aminoadipic acid, aminobutyric acid, citrulline, homocitrulline, homoleucine, homoarginine, hydroxyproline, norleucine, pyridylalanine, sarcosine, and the like.

[0038] In the present invention, the terms peptide, polypeptide, and protein are not strictly distinguished and are used interchangeably in some cases. They generally refer to polymers composed of amino acids linked by peptide bonds, whether naturally occurring or synthetic. Polypeptides may also contain non-amino acid components, such as carbohydrate groups, metal ions, or carboxylates. Non-amino acid components may be incorporated by the cell expressing the polypeptide and may vary with the cell type. Polypeptides are defined herein with respect to their amino acid backbone structure or the nucleic acid encoding them. For example, the addition of carbohydrate groups is generally not specified, but may be present. All polypeptide sequences are written according to generally accepted conventions, with the α-N-terminal amino acid residue on the left and the α-C-terminal amino acid residue on the right. As used herein, the term "N-terminus" refers to the free α-amino group of an amino acid in a polypeptide, and the term "C-terminus" refers to the free α-carboxylic acid end of an amino acid in a polypeptide. A polypeptide terminating in a group at the N-terminus refers to a polypeptide having a group on the α-amino nitrogen of the N-terminal amino acid residue. An amino acid terminating in a group at the N-terminus refers to an amino acid having a group on the α-amino nitrogen.

[0039] The entire antibody molecule can be divided into two parts: the constant region (C region) and the variable region (V region). The amino acid composition and arrangement sequence of the V region of different antibodies are different. Among them, the amino acid composition and arrangement sequence of three regions of VH and VL are particularly prone to change. These hypervariable regions in the variable regions of the heavy chain and light chain constitute the antigen (Ag) binding site of the antibody molecule. The hypervariable region is also called the complementarity-determining region (CDR) of the antibody molecule. That is, the amino acid sequences of the three CDR regions of the heavy chain and light chain determine the specificity of the antibody.

[0040] The present invention couples c-Myc protein (a ten-amino acid short peptide EQKLISEEDL) with KLH as an antigen and injects it into mice to stimulate antibody production in the body. As a result, a high-titer c-Myc tag antibody is discovered in the mice. The amino acid sequences of the heavy and light chains of the antibody are obtained by cloning and sequencing the variable region genes of the antibody. Analysis shows that the complementarity determining regions of the heavy chain: CDR1 comprises or consists of the amino acid sequence AYFVN shown in SEQ ID NO: 1; CDR2 comprises or consists of the amino acid sequence KINPYNLYNFYNQKFKG shown in SEQ ID NO: 2; and CDR3 comprises or consists of the amino acid sequence EGEYSGTFPYGMDY shown in SEQ ID NO: 3.

[0041] The complementarity determining regions of its light chain: CDR1 comprises or consists of the amino acid sequence KASQNVGNIIA shown in SEQ ID NO:4; CDR2 comprises or consists of the amino acid sequence LASYRYT shown in SEQ ID NO:5; CDR3 comprises or consists of the amino acid sequence NQYKNSPYT shown in SEQ ID NO:6.

[0042] The amino acid sequences of the heavy and light chains of the antibody were obtained by sequencing to prepare a monoclonal antibody, which is a preferred anti-c-Myc tag monoclonal antibody of the present invention. The heavy chain amino acid sequence is shown in SEQ ID NO: 15, and the light chain amino acid sequence is shown in SEQ ID NO: 16.

[0043] The present invention provides an anti-c-Myc tag monoclonal antibody, which comprises or consists of VHCDR1, VHCDR2 and VHCDR3 with amino acid sequences as shown in SEQ ID NOs: 1-3; and comprises or consists of VLCDR1, VLCDR2 and VLCDR3 with amino acid sequences as shown in SEQ ID NOs: 4-6.

[0044] The amino acid sequence shown in SEQ ID NO: 1 is AYFVN, the amino acid sequence shown in SEQ ID NO: 2 is KINPYNLYNFYNQKFKG, and the amino acid sequence shown in SEQ ID NO: 3 is EGEYSGTFPYGMDY;

[0045] The amino acid sequence shown in SEQ ID NO:4 is KASQNVGNIIA; the amino acid sequence shown in SEQ ID NO:5 is LASYRYT; and the amino acid sequence shown in SEQ ID NO:6 is NQYKNSPYT.

[0046] Preferably, in the antibody, FR1-4 in the heavy chain variable region are the following amino acid sequences:

[0047] comprising or consisting of the amino acid sequence shown in SEQ ID NO: 7, 8, 9, 10,

[0048] or a sequence having at least 80%, 85%, or 90% identity to the sequence shown in SEQ ID NO: 7, 8, 9, or 10;

[0049] or a sequence having one or more amino acid mutations compared to the amino acid sequence shown in SEQ ID NO: 7, 8, 9, or 10;

[0050] More preferably, in the antibody, FR1-4 in the heavy chain variable region are the following amino acid sequences:

[0051] The amino acid sequences shown in SEQ ID NOs: 7, 8, 9, and 10;

[0052] or a sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence shown in SEQ ID NO: 7, 8, 9, 10;

[0053] Or it has 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid mutations compared to the sequence shown in SEQ ID NO: 7, 8, 9, 10; the amino acid mutation is a conservative mutation, preferably a substitution, insertion or deletion.

[0054] Furthermore, in the antibody, FR1-4 in the light chain variable region are the following amino acid sequences:

[0055] comprising or consisting of the amino acid sequence shown in SEQ ID NO: 11, 12, 13, 14,

[0056] or a sequence having at least 80%, 85%, or 90% identity to the sequence shown in SEQ ID NO: 11, 12, 13, or 14; preferably a sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence shown in SEQ ID NO: 11, 12, 13, or 14;

[0057] Or a sequence having one or more amino acid mutations compared to the amino acid sequence shown in SEQ ID NO: 11, 12, 13, 14; preferably a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid mutations compared to the amino acid sequence shown in SEQ ID NO: 11, 12, 13, 14; the amino acid mutation is a conservative mutation, preferably a substitution, insertion or deletion.

[0058] In some embodiments, the anti-c-Myc tag monoclonal antibody has a heavy chain amino acid sequence as shown in SEQ ID NO: 15, and a light chain amino acid sequence as shown in SEQ ID NO: 16.

[0059] In some embodiments, the present invention also provides a nucleic acid sequence encoding a monoclonal antibody or antibody fragment thereof against c-Myc tag as described herein. In the present invention, the nucleic acid sequence includes conservatively substituted variants thereof (e.g., substitutions of degenerate codons) and complementary sequences. The terms "nucleic acid" and "polynucleotide" are synonymous and include genes, cDNA molecules, mRNA molecules, and fragments thereof, such as oligonucleotides.

[0060] In some embodiments, the present invention further provides a biological material containing a nucleic acid sequence encoding a monoclonal antibody against c-Myc tag as described in the present invention, wherein the biological material includes an expression vector, an expression cassette, a host cell, an engineered bacterium or a hybridoma cell line.

[0061] The nucleic acid sequence is operably linked to at least one regulatory sequence. "Operably linked" means that the coding sequence is linked to the regulatory sequence in a manner that allows expression of the coding sequence. Regulatory sequences are selected to direct expression of the desired protein in a suitable host cell and include promoters, enhancers, and other expression control elements.

[0062] In addition, the present invention also provides a use of the anti-c-Myc tag monoclonal antibody of the present invention in detecting, separating and purifying Myc-tagged target proteins.

[0063] The present invention also provides a detection kit comprising the monoclonal antibody against c-Myc tag according to the present invention.

[0064] The detection kit also includes a c-Myc tag polypeptide (also called a Myc tag polypeptide); the c-Myc tag is an epitope tag derived from the c-Myc gene, contains 10 amino acids, and its amino acid sequence is EQKLISEEDL.

[0065] c-Myc tag antibodies can interact with the Myc tag on the target protein and specifically recognize the Myc-tagged target protein, thereby successfully detecting certain target proteins for which there are currently no specific antibodies. They are suitable for detecting the expression level of c-Myc fusion proteins or c-Myc through various immunoassays.

[0066] The following are examples

[0067] Example 1 Preparation and screening of antibodies

[0068] (1) Antigen immunity

[0069] c-Myc protein (a ten-amino acid peptide, EQKLISEEDL, coupled to KLH) was mixed with Freund's complete adjuvant (appearance: an amber cell suspension; components: Paraffin Oil 85%, Mannide Monooleate 15%, Mycobacterium smegmatis 1 mg / mL) to produce an oily emulsion. BALB / c mice were subcutaneously injected with 0.15 mL of this emulsion 14 days after the first immunization. An intraperitoneal booster (equal amounts of antigen mixed with Freund's incomplete adjuvant) was administered. After four booster injections, tail blood was collected for titer testing to ensure that the titer met the fusion requirement.

[0070] Three days before fusion, booster immunization was performed by intraperitoneal injection of the same dose of antigen, using the same immunization method as above.

[0071] (2) Preparation of hybridoma cell lines

[0072] ① Preparation of feeder cells

[0073] BALB / c mouse peritoneal macrophages were used as feeder cells. The abdomen was cut open and 5 mL of RPMI 1640 basal culture medium was injected. The cells were rinsed repeatedly and collected. The cells were centrifuged at 1000 rpm for 5 minutes and the pellet was retained. The cells were resuspended and screened in RPMI 1640 culture medium containing HAT and the concentration was adjusted to 1×10 5 150 μL / well of 96-well plate was added and cultured at 37°C in 5% CO2 overnight.

[0074] ② Preparation of immune spleen cells

[0075] Three days after the last immunization, aseptically remove the mouse spleen and place it on a plate. Wash with RPMI 1640 basal medium, grind and filter on a nylon membrane to prepare a cell suspension. Centrifuge, remove the supernatant, and resuspend in RPMI 1640 basal medium. Repeat three times to obtain immune spleen cells and count them.

[0076] ③ Preparation of myeloma cells

[0077] Mouse myeloma Sp2 / 0 cells were selected with 8-azaguanine and cultured to the logarithmic growth phase. Cell suspensions were prepared from two large flasks and centrifuged. The supernatant was discarded and the cells were resuspended in RPMI 1640 basal medium. This process was repeated three times to obtain myeloma cells, which were then counted.

[0078] ④Cell fusion and HAT selection of hybridomas

[0079] Myeloma cells and immune spleen cells were mixed at a ratio of 1:10. Wash once with RPMI 1640 basal medium in a 50 mL plastic centrifuge tube and centrifuge at 1200 rpm for 10 minutes. Discard the supernatant, mix the cells thoroughly, and slowly add 1 mL of 50% PEG-1500 to allow fusion. After 1 minute of fusion, add 15 mL of RPMI 1640 basal medium to terminate cell fusion. Centrifuge at 1000 rpm for 5-10 minutes. Discard the supernatant, gently resuspend the cells in 50 mL of RPMI 1640 screening medium, and divide equally among ten 96-well plates, 50 μL / well, and incubate at 37°C, 5% CO2. On day six of culture, change the culture medium twice with HAT medium (RPMI 1640 complete medium containing HAT).

[0080] ⑤ Hybridoma cell screening

[0081] c-Myc protein was diluted to 1 μg / mL in 0.05M carbonate buffer (pH 9.6) and added to a 96-well plate for 2 hours at 37°C. Blocking was performed with 0.15 mL / well of 0.02M PBS (pH 7.2) containing 10% calf serum or 1% skim milk powder at 37°C for 2 hours for detection. Seven days after fusion, 0.1 mL of cell supernatant was transferred to a 96-well plate and incubated at 37°C for 30 minutes. After washing six times with water, the secondary antibody was added at 37°C for 30 minutes. After washing, 100 μL of citric acid-phosphate buffer (pH 5.0) containing 0.1% (M / V) o-phenylenediamine and 0.1% (V / V) hydrogen peroxide was added to each well for 15 minutes at 37°C. Then, 50 μL of dilute sulfuric acid solution was added to each well, and the absorbance was measured at 450 nm.

[0082] The antibody-secreting positive cells were cloned by limiting dilution method at 1 cell / well in 96-well culture plates. The positive wells were cloned three times continuously according to the above method. After expansion culture, they were frozen in culture medium containing 10% DMSO at a cell density of 10 6 A total of five hybridoma cell lines stably secreting anti-c-Myc tag monoclonal antibodies were obtained and named 2D9, 3B6, 4E7, 5A4, and 5C6.

[0083] (3) Antibody preparation

[0084] Select healthy BALB / c mice of 6 to 8 weeks old and inject 0.5 mL of pristane into each mouse intraperitoneally. Ten days later, inject 1×10 6Hybridoma cells (2D9, 3B6, 4E7, 5A4, 5C6) should develop ascites 7-10 days after inoculation. Closely monitor the animal's health and signs of ascites. Once ascites is as abundant as possible and before the mouse is on the verge of death, sacrifice the mouse and aspirate the ascites into a test tube using a pipette. Typically, 5-10 mL of ascites fluid will be obtained from each mouse. Collect the ascites, centrifuge, and remove the supernatant. Store in a -20°C refrigerator. Dilute the supernatant with three volumes of PBS and filter through filter paper. Apply the resulting filtrate to a protein G affinity chromatography column equilibrated with PBS at a flow rate of 1 mL / min. Substances not adsorbed by protein G are then washed with PBS at a flow rate of 1 mL / min until the absorbance at OD280 nm reaches baseline. Elute the antibody with 0.1 M glycine (pH 2.5) to recover the antibody. The recovered solution was neutralized with 0.1 M Tris (pH 8.8), the antibody concentration was adjusted to an appropriate concentration by ultrafiltration, and the solution was aliquoted and frozen at -20°C.

[0085] (4) Screening for high titer antibodies

[0086] The titers of the above five hybridoma cell lines and secreted ascites antibodies were detected by indirect ELISA to screen the cell lines and antibodies with the highest titers. The specific experimental steps are as follows:

[0087] ① Coating: Dilute c-Myc protein to 1 μg / mL, add 100 μL / well to the ELISA plate, and incubate at 37°C for 2 hours or at 4°C overnight;

[0088] ② Wash the plate 5 times with a plate washer, injecting 350 μL of wash solution into each well each time, leaving it for 20 seconds; finally, pat dry;

[0089] ③ Wash away the coating solution with washing solution, block with blocking solution, 150 μL per well, and place at 37°C for 1.5-2 hours;

[0090] ④ Wash the plate 5 times with a plate washer, injecting 350 μL of wash solution into each well each time, leaving it for 20 seconds; finally, pat dry;

[0091] ⑤ Add samples: Add cell culture supernatant and ascites diluted into different gradients to the ELISA plate coated with c-Myc protein, 100 μL / well, and react at 37°C for 1 hour (also make negative and positive control wells);

[0092] ⑥ Wash the plate 5 times with a plate washer, injecting 350 μL of wash solution into each well each time, leaving it for 20 seconds; finally, pat dry;

[0093] ⑦Add horseradish peroxidase-labeled goat anti-mouse IgG enzyme-labeled secondary antibody (diluted 6000 times with blocking solution), 100 μL / well, and react at 37°C for 1 hour;

[0094] ⑧Wash the plate 5 times with a plate washer, injecting 350 μL of wash solution into each well each time, leaving it for 20 seconds; finally, pat dry;

[0095] ⑨ Add TMB colorimetric solution: prepare immediately before use, 100 μL / well, incubate at 37°C in the dark for 30 minutes;

[0096] ⑩Terminate the reaction: Add 2M sulfuric acid to each reaction well, 50 μL / well;

[0097] Microplate reader reading: 450nm, 630nm wavelength measurement.

[0098] The results of the assays of five different hybridoma cells and antibody titers are shown in Table 1.

[0099] Table 1 Results of titer determination of different antibodies

[0100] cell lines Hybridoma cell culture supernatant titer Ascites antibody titer 2D9 <![CDATA[3.34×10 3 ]]> <![CDATA[4.78×10 5 ]]> 5A4 <![CDATA[8.73×10 3 ]]> <![CDATA[6.27×10 5 ]]> 5C6 <![CDATA[2.78×10 4 ]]> <![CDATA[8.48×10 6 ]]> 3B6 <![CDATA[8.89×10 3 ]]> <![CDATA[5.21×10 5 ]]> 4E7 <![CDATA[9.64×10 3 ]]> <![CDATA[6.62×10 5 ]]>

[0101] The data in Table 1 are the lowest concentrations of the antibody solution that can be detected by the microplate reader, i.e., the maximum dilution factor that can be used for the antibody solution. The results in Table 1 show that the antibody secreted by the 5C6 hybridoma cell line has the highest titer among the five antibodies obtained.

[0102] Example 2 Antibody variable region gene cloning and sequencing

[0103] (1) Antibody variable region gene cloning and sequencing

[0104] Total RNA was extracted from the hybridoma cell line 5C6, which secretes c-Myc monoclonal antibodies, and first-strand cDNA was synthesized using the SMARTER™ RACE cDNA Amplification Kit and the included SMARTER II A Oligonucleotide and 5'-CDS primers. The resulting first-strand cDNA product was used as a template for PCR amplification. The light chain gene was amplified using Universal Primer A Mix (UPM), Nested Universal Primer A (NUP), and mIgG CKR primers, while the heavy chain gene was amplified using Universal Primer A Mix (UPM), Nested Universal Primer A (NUP), and mIgG CHR primers. The light chain primer pair amplified a target band of approximately 0.7 kb, while the heavy chain primer pair amplified a target band of approximately 1.5 kb. The product was purified and recovered by agarose gel electrophoresis, and the product was inserted into the pMD-18T vector after A addition reaction with rTaq DNA polymerase. It was transformed into DH5α competent cells, and after colonies grew, 4 clones of the Heavy Chain and Light Chain genes were taken and sent to Qingke Company for sequencing.

[0105] After analysis, the complementarity determining region of its heavy chain:

[0106] CDR1:AYFVN;

[0107] CDR2:KINPYNLYNFYNQKFKG;

[0108] CDR3: EGEYSGTFPYGMDY;

[0109] Complementarity determining regions of light chains:

[0110] CDR1:KASQNVGNIIA;

[0111] CDR2: LASYRYT;

[0112] CDR3: NQYKNSPYT.

[0113] Its heavy chain amino acid sequence and light chain amino acid sequence are as follows Figure 1As shown, the anti-c-Myc tag monoclonal antibody provided by the present invention, its heavy chain CDR1 comprises the amino acid sequence AYFVN shown in SEQ ID NO: 1, or consists of it; the heavy chain CDR2 comprises the amino acid sequence KINPYNLYNFYNQKFKG shown in SEQ ID NO: 2, or consists of it; the heavy chain CDR3 comprises the amino acid sequence EGEYSGTFPYGMDY shown in SEQ ID NO: 3, or consists of it;

[0114] The light chain CDR1 comprises or consists of the amino acid sequence KASQNVGNIIA shown in SEQ ID NO:4; the light chain CDR2 comprises or consists of the amino acid sequence LASYRYT shown in SEQ ID NO:5; and the light chain CDR3 comprises or consists of the sequence NQYKNSPYT shown in SEQ ID NO:6.

[0115] Example 3 Monoclonal Antibody Affinity Analysis and Activity Identification

[0116] (1) Comparison of affinity of different purified anti-c-Myc tag monoclonal antibodies

[0117] Data were generated using the same enzyme immunoassay method as for activity assays. Four gradients of 1 μg / ml, 0.5 μg / ml, 0.25 μg / ml, and 0.125 μg / ml were coated. Antibody was diluted two-fold from 1000 ng / ml to 0.97656 ng / ml for loading. OD values ​​corresponding to different antibody concentrations at different coating concentrations were obtained.

[0118] At the same coating concentration, plot the antibody concentration as the horizontal axis and the OD value as the vertical axis on a logarithmic scale. Calculate the antibody concentration at 50% of the maximum OD value based on the fitting equation. Substitute this into the formula to calculate the reciprocal of the affinity constant. The specific formula is as follows:

[0119] K=(n-1) / (2*(n*Ab`-Ab))

[0120] Where Ab and Ab' represent the antibody concentrations at 50% of the maximum OD value under the corresponding coating concentrations (Ag and Ag'), n = Ag / Ag';

[0121] Each combination of two coating concentrations yields a K value, resulting in six K values. The average and reciprocal of these values ​​provide the affinity constant, KD. Affinity analysis data for the purified anti-c-Myc monoclonal antibody, along with affinity comparisons of antibodies secreted by 5C6 and four other hybridoma cell lines, are shown in Table 2.

[0122] Table 2 Affinity determination results of purified anti-c-Myc tag monoclonal antibodies

[0123] Sample name KD 5C6 1.113E-8 2D9 6.115E-7 3B6 4.118E-6 5A4 9.318E-7 4E7 7.834E-6

[0124] As shown in Table 2, the affinity constant KD of the antibody secreted by the 5C6 hybridoma cell line is 1.113E-8, ​​which is significantly better than that of the antibodies secreted by the other four hybridoma cell lines.

[0125] (2) Identification of monoclonal antibody activity

[0126] Dilute c-Myc protein to 1 μg / mL with 50 mM carbonate buffer coating solution for microplate coating, 100 μL per well, incubate at 4°C overnight; the next day, wash twice with PBST and pat dry; add blocking solution (20% BSA + 80% PBS) at 120 μL per well, incubate at 37°C for 1 hour, and pat dry; add diluted anti-c-Myc monoclonal antibody 5C6, starting from 1000 ng / mL and diluting 5-fold, load the sample, 100 μL / well, incubate at 37°C for 30 min (partial supernatant 1h); wash 5 times with PBST washing solution and pat dry; add horseradish peroxidase-labeled goat anti-mouse IgG, 100 μL per well, 37°C, 30 min; wash 5 times with PBST washing solution and pat dry; add urea peroxide (50 μL / well) and tetramethylbenzidine (50 μL / well) for 10 min; add dilute hydrochloric acid to terminate the reaction, 50 μL / well; read the OD value at 450 nm (reference 620 nm) on a microplate reader. The measurement results are shown in Table 3 and Figure 2 shown.

[0127] Table 3 Activity identification of purified anti-c-Myc tag monoclonal antibody 5C6

[0128]

[0129] From Table 3 and Figure 2 It can be seen that the sample concentration can be detected as low as 0.32ng / ml, indicating its high activity.

[0130] Example 4 Comparison of affinities of different anti-c-Myc tag monoclonal antibodies

[0131] The c-Myc tag monoclonal antibody of the present invention was compared with the c-Myc tag monoclonal antibodies of other companies to conduct anti-c-Myc monoclonal antibody affinity tests, as follows:

[0132] Activity was assessed using an indirect enzyme immunoassay (ELISA) assay using different c-Myc tag monoclonal antibodies at different coating concentrations (1 μg / ml, 0.5 μg / ml, 0.25 μg / ml, and 0.125 μg / ml). The antibody was then diluted 2-fold from a starting concentration of 1000 ng / ml to 0.97656 ng / ml for loading. The OD values ​​corresponding to each coating concentration were recorded.

[0133] Next, at the same coating concentration, plot the antibody concentration on the horizontal axis and the OD value on the vertical axis. Calculate the antibody concentration at 50% of the maximum OD value using the fitting equation. Then, calculate the inverse of the affinity constant using the formula:

[0134] K=(n-1) / (2*(n*Ab`-Ab)),

[0135] Wherein, Ab and Ab' represent the antibody concentrations at 50% of the maximum OD value under the corresponding coating concentration, and n is the coating concentration ratio (Ag / Ag').

[0136] Each combination of two coating concentrations yields a K value, resulting in six K values. The average value is then calculated, and its reciprocal is the affinity constant, KD. Finally, the affinity analysis data for the purified anti-c-Myc monoclonal antibody are summarized in Table 4.

[0137] Table 4 Affinity determination results of anti-c-Myc monoclonal antibodies of different products

[0138] Product Name KD Company A 8.219E-8 Company B 7.136E-8 Company C 9.238E-8 The present invention 6.248E-8

[0139] As shown in Table 4, the affinity of the anti-c-Myc monoclonal antibody of the present invention is significantly better than that of the c-Myc antibodies of the other three companies.

[0140] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Although the present invention is disclosed as above in terms of a preferred embodiment, it is not intended to limit the present invention. Any person skilled in the art can, without departing from the scope of the technical solution of the present invention, make some changes or modifications to equivalent embodiments using the technical contents disclosed above. However, any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A monoclonal antibody against c-Myc tag, characterized in that Includes three heavy chain complementarity determining regions: heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3, and three light chain complementarity determining regions: light chain CDR1, light chain CDR2 and light chain CDR3; The heavy chain CDR1 is the amino acid sequence AYFVN shown in SEQ ID NO: 1; The heavy chain CDR2 is the amino acid sequence KINPYNLYNFYNQKFKG shown in SEQ ID NO: 2; The heavy chain CDR3 is the amino acid sequence EGEYSGTFPYGMDY shown in SEQ ID NO: 3; The light chain CDR1 is the amino acid sequence KASQNVGNIIA shown in SEQ ID NO: 4; The light chain CDR2 is the amino acid sequence LASYRYT shown in SEQ ID NO: 5; The light chain CDR3 has the amino acid sequence NQYKNSPYT shown in SEQ ID NO:

6.

2. The anti-c-Myc tag monoclonal antibody according to claim 1, characterized in that The antibody, wherein FR1-4 in the heavy chain variable region are the following amino acid sequences: comprising the amino acid sequence shown in SEQ ID NO: 7, 8, 9, 10 or the amino acid sequence shown in SEQ ID NO: 7, 8, 9, 10, or a sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 7, 8, 9, 10, Or a sequence having one or more amino acid mutations compared to the amino acid sequence shown in SEQ ID NO: 7, 8, 9, or 10.

3. The anti-c-Myc tag monoclonal antibody according to claim 2, characterized in that FR1-4 in the heavy chain variable region are the following amino acid sequences: The amino acid sequences shown in SEQ ID NOs: 7, 8, 9, and 10, or a sequence having at least 91% identity to the amino acid sequence shown in SEQ ID NO: 7, 8, 9, 10, Or an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid mutations compared to the amino acid sequence shown in SEQ ID NO: 7, 8, 9, 10; the amino acid mutation is a conservative mutation, including substitution, insertion or deletion.

4. The anti-c-Myc tag monoclonal antibody according to any one of claims 1 to 3, characterized in that The antibody, wherein FR1-4 in the light chain variable region are the following amino acid sequences: comprising the amino acid sequence shown in SEQ ID NO: 11, 12, 13, 14 or the amino acid sequence shown in SEQ ID NO: 11, 12, 13, 14, or a sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 11, 12, 13, 14, Or a sequence having one or more amino acid mutations compared to the amino acid sequence shown in SEQ ID NO: 11, 12, 13, or 14.

5. The anti-c-Myc tag monoclonal antibody according to claim 4, characterized in that FR1-4 in the light chain variable region are the following amino acid sequences: The amino acid sequences shown in SEQ ID NOs: 11, 12, 13, and 14, or a sequence having at least 91% identity to the amino acid sequence shown in SEQ ID NO: 11, 12, 13, 14, Or an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid mutations compared to the amino acid sequence shown in SEQ ID NO: 11, 12, 13, 14; the amino acid mutation is a conservative mutation, including substitution, insertion or deletion.

6. The anti-c-Myc tag monoclonal antibody according to claim 5, characterized in that The heavy chain amino acid sequence of the antibody is shown in SEQ ID NO: 15, and the light chain amino acid sequence is shown in SEQ ID NO:

16.

7. A biological material containing a nucleic acid sequence encoding the monoclonal antibody against c-Myc tag according to claim 6, characterized in that: The biological material includes an expression vector, an expression cassette, and a host cell.

8. Use of the anti-c-Myc tag monoclonal antibody according to claim 6 in the preparation of reagents for detecting, separating and purifying Myc-tagged target proteins.

9. A detection kit, characterized in that The method comprises the anti-c-Myc tag monoclonal antibody according to claim 6.

10. The detection kit according to claim 9, wherein Also included are c-Myc tag polypeptides.

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