CN115028731B

CN115028731B - anti-Flag-tag antibody and application thereof

Info

Publication number: CN115028731B
Application number: CN202210655919.9A
Authority: CN
Inventors: 吴川; 潘炜松; 陈刚; 徐超; 吕杨涛
Original assignee: Shenzhen Nuoda Biotechnology Co ltd
Current assignee: Shenzhen Nuoda Biotechnology Co ltd
Priority date: 2022-06-10
Filing date: 2022-06-10
Publication date: 2023-04-07
Anticipated expiration: 2042-06-10
Also published as: CN115028731A

Abstract

The invention discloses an anti-Flag-tag antibody, which comprises a heavy chain of a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3 or variants thereof, and a light chain of a light chain CDR1, a light chain CDR2, a light chain CDR3 or variants thereof; the heavy chain CDR1 comprises an amino acid sequence shown in SEQ ID NO. 1; the heavy chain CDR2 comprises an amino acid sequence shown as SEQ ID NO. 2; the heavy chain CDR3 comprises an amino acid sequence shown in SEQ ID NO. 3; the light chain CDR1 comprises an amino acid sequence shown in SEQ ID NO. 4; the light chain CDR2 comprises an amino acid sequence shown as SEQ ID NO. 5; and the light chain CDR3 comprises a sequence shown in SEQ ID NO. 6. The affinity of the recombinant monoclonal antibody tagged with Flag and the tagged protein reaches 5.219E-10, and the purified anti-Flag-tagged monoclonal antibody has high activity and great application potential.

Description

anti-Flag-tag antibody and application thereof

Technical Field

The invention belongs to the field of molecular immunology, and more particularly relates to an anti-Flag-tag antibody and application thereof.

Background

Flag is a short peptide consisting of 8 amino acids (Asp-Tyr-Lys-Asp-Lys) and is specially designed for immunoadsorption purification of fusion proteins. A high-efficiency detection and purification system based on the fusion polypeptide can be established by utilizing the Flag tag. The Flag tag has a short structure, and an enterokinase cutting site is arranged between the Flag tag and the fusion protein and can be removed by enterokinase cutting.

The Flag antigen tag technology has the advantages of simplicity and convenience. However, the types of the current commercial monoclonal antibodies for resisting the Flag tag protein are few, and most of the monoclonal antibodies need to be imported from foreign countries, so the price is very high. Therefore, the novel anti-Flag monoclonal antibody is developed, and has wide application prospect in immunoadsorption separation and purification of the fusion protein.

Disclosure of Invention

Aiming at the defects or the improvement requirements of the prior art, the invention provides an anti-Flag-tag antibody and application thereof, aiming at stimulating a mouse by a Flag antigen, finding a high-titer Flag antibody in the body of the mouse, obtaining the amino acid sequences of the heavy chain and the light chain of the antibody by cloning and sequencing the variable region gene of the antibody, and preparing a recombinant monoclonal Flag antibody on the basis of the amino acid sequences, thereby solving the technical problems of few types and high price of the existing Flag antibody.

To achieve the above object, according to one aspect of the present invention, there is provided an anti-Flag-tag antibody comprising a heavy chain of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3 or a variant thereof, and a light chain of light chain CDR1, light chain CDR2, light chain CDR3 or a variant thereof;

the heavy chain CDR1 comprises an amino acid sequence T-Y-T-I-H shown in SEQ ID NO. 1; the heavy chain CDR2 comprises an amino acid sequence Y-I-N-P-S-S-G-Y-A-Y-N-Q-N-F-K-D shown in SEQ ID NO. 2; the heavy chain CDR3 comprises an amino acid sequence E-K-F-Y-G-Y-D-Y shown in SEQ ID NO. 3;

the light chain CDR1 comprises an amino acid sequence R-S-S-Q-S-I-V-H-R-N-G-N-T-Y-L-E shown in SEQ ID NO. 4; the light chain CDR2 comprises an amino acid sequence K-V-S-N-R-F-S shown in SEQ ID NO. 5; the light chain CDR3 comprises a sequence F-Q-G-S-H-V-P-Y-T shown in SEQ ID NO. 6.

Preferably, the anti-Flag-tagged antibody, the variant heavy chain thereof, the heavy chain variable region thereof comprises any one of the amino acid sequences having more than 90% identity to the amino acid sequence set forth in SEQ ID NO. 7, or has one or more amino acid mutations compared to the amino acid sequence set forth in SEQ ID NO. 7; the light chain of the variant has a light chain variable region comprising any one of the amino acid sequences having 90% or more identity to the amino acid sequence shown in SEQ ID NO. 8, or having one or more amino acid mutations compared to the amino acid sequence shown in SEQ ID NO. 8.

Preferably, the anti-Flag-tagged antibody, the variant heavy chain thereof, the heavy chain variable region thereof comprises any one of the amino acid sequences having more than 90% identity with the amino acid sequence shown in SEQ ID NO. 7, or has less than 10 amino acid mutations compared with the amino acid sequence shown in SEQ ID NO. 7; the light chain variable region of the variant comprises any one amino acid sequence which has more than 90 percent of identity with the amino acid sequence shown in SEQ ID NO. 8, or has less than 10 amino acid mutations compared with the amino acid sequence shown in SEQ ID NO. 8.

Preferably, said amino acid mutation of said anti-Flag-tagged antibody is a conservative mutation, preferably a substitution, insertion or deletion of the amino acid sequence.

According to another aspect of the present invention, there is provided a nucleic acid sequence comprising a nucleic acid sequence encoding the anti-Flag tag antibody or fragment thereof of the present invention, the nucleic acid sequence of its heavy chain comprising SEQ ID NO 9, the nucleic acid sequence of its light chain comprising SEQ ID NO 10, or conservatively substituted variants and complements thereof.

Preferably, the nucleic acid sequence encoding the anti-Flag tag antibody or fragment thereof, the nucleic acid sequence thereof, includes genetic DNA molecules, cDNA molecules, mRNA molecules and their fragment oligonucleotides.

Preferably, the nucleic acid sequence encoding the anti-Flag tag antibody or fragment thereof, the nucleic acid sequence encoding the anti-Flag tag antibody, the nucleic acid sequence of the heavy chain thereof, including SEQ ID NO 9, and the nucleic acid sequence of the light chain, including SEQ ID NO 10.

According to another aspect of the invention, the biological material containing the nucleic acid sequence of the antibody or the fragment thereof encoding the anti-Flag tag of the invention comprises an expression vector, an expression cassette, a host cell, an engineering bacterium or a hybridoma cell strain.

According to another aspect of the invention, the invention also provides the application of the anti-Flag-tag antibody or the nucleic acid sequence or the biological material in the preparation of a Flag-tag fusion protein detection reagent.

According to another aspect of the present invention, there is also provided a detection kit comprising the anti-Flag-tag antibody according to the present invention.

In general, compared with the prior art, the above technical solution contemplated by the present invention can achieve the following beneficial effects:

the recombinant monoclonal antibody Flag-tagged antibody of the antibody is obtained by artificial modification on the basis of obtaining the amino acid sequence of the high-titer anti-Flag-tagged antibody in a mouse antigen immunity experiment, the affinity of the anti-Flag-tagged antibody provided by the invention and the tag protein reaches 5.219E-10, the purified anti-Flag-tagged monoclonal antibody has high activity, and the recombinant monoclonal antibody Flag-tagged antibody has wide application prospect in immunoadsorption separation and purification of fusion protein.

Drawings

FIG. 1 shows the heavy and light chain sequences of the antibody secreted by hybridoma cell line 6C10 in example 3.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. In addition, the technical features involved in the respective embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

Terms and definitions:

the following terms used in the present application have the following meanings, unless otherwise specified. A particular term should not be considered as ambiguous or unclear without special definition, but rather construed according to ordinary meaning in the art.

The term "amino acid" denotes a naturally occurring or non-naturally occurring carboxy alpha-amino acid. The term "amino acid" as used in this application may include both naturally occurring amino acids and non-naturally occurring amino acids. Naturally occurring amino acids include alanine (three letter code: ala, one 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, alpha-aminoadipic acid, aminobutyric acid, citrulline, homocitrulline, homoleucine, homoarginine, hydroxyproline, norleucine, pyridylalanine, sarcosine, and the like.

In the present invention, peptides, polypeptides, proteins are not strictly distinguished and may be used interchangeably in some cases, and generally refer to polymers of amino acids linked by peptide bonds, whether naturally occurring or synthetic. The polypeptide may also comprise non-amino acid components, such as carbohydrate groups, metal ions or carboxylic acid esters. The non-amino acid components may be added by the cell expressing the polypeptide and may vary with the cell type. The polypeptide is defined in the present invention with respect to its amino acid backbone structure or the nucleic acid encoding it. Such as the addition of carbohydrate groups, is not generally specified, however, may be present. All polypeptide sequences are written according to generally accepted practice with the alpha-N-terminal amino acid residue on the left and the alpha-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 terminus of an amino acid in a polypeptide. A polypeptide that ends with a group at the N-terminus refers to a polypeptide that carries a group on the alpha-amino nitrogen of the N-terminal amino acid residue. An amino acid ending with a group at the N-terminus is an amino acid bearing a group on the alpha-amino nitrogen.

The antibody variable region, i.e., the CDR region, is a region in which an antibody specifically recognizes an antigen, and generally has three segments; the tag antibody is an antibody capable of highly specifically recognizing a special epitope on the fusion protein constructed by the recombinant technology, wherein the anti-Flag-tag antibody (referred to as Flag antibody for short) is widely applied. The Flag antibody can be used for detecting the expression and intracellular localization of the Flag tag fusion expression protein, purifying, qualitatively or quantitatively detecting the Flag fusion expression protein and the like. When scientific researchers select the Flag antibody, the selection is mainly carried out according to the needs of the researchers, particularly the needs of experimental application, the titer of the Flag antibody is often an important reference factor and even a decisive factor, and the reason is mainly that the high titer Flag antibody not only influences the sensitivity of the experimental result, but also means higher cost performance, so that the finding of the high titer Flag antibody has great significance and good market prospect.

We stimulated mice with Flag antigen and found a high titer Flag antibody in their body. Specifically, a large number of hybridoma cell lines are prepared by immunizing a BALB/C mouse for multiple times by using Flag antigen, positive cells secreting antibodies are screened and cloned, five stable anti-Flag-labeled hybridoma cell lines are obtained, ascites is prepared and purified to obtain monoclonal antibodies, a hybridoma cell 6C10 with high titer of the secreted antibodies is screened out by measuring the titer of the antibodies, and the experiment result shows that the titer of ascites antibodies secreted by the hybridoma cell line 6C10 can reach 8.52 multiplied by 10 ⁶ Is obviously higher than other 4 hybridoma cell strains; the amino acid sequences of the heavy chain and the light chain of the antibody are obtained by cloning and sequencing the variable region gene of the antibody, and the complementarity determining regions of the heavy chain are analyzed as follows: CDR1: T-Y-T-I-H; CDR2: Y-I-N-P-S-S-G-Y-A-A-Y-N-Q-N-F-K-D; CDR3: E-K-F-Y-G-Y-D-Y; complementarity determining regions of its light chain: CDR1: R-S-S-Q-S-I-V-H-R-N-G-N-T-Y-L-E; CDR2: K-V-S-N-R-F-S; CDR3: F-Q-G-S-H-V-P-Y-T; and preparing a recombinant antibody according to the obtained amino acid sequences of the heavy chain and the light chain of the antibody, wherein the recombinant antibody is the antibody of the anti-Flag tag preferred by the invention.

The invention provides an anti-Flag-tagged antibody, which comprises a heavy chain of a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3 or a variant thereof, and a light chain of a light chain CDR1, a light chain CDR2, a light chain CDR3 or a variant thereof;

the heavy chain CDR1 comprises or consists of an amino acid sequence T-Y-T-I-H shown in SEQ ID NO. 1; the heavy chain CDR2 comprises or consists of an amino acid sequence Y-I-N-P-S-S-G-Y-A-Y-Q-N-F-K-D shown in SEQ ID NO. 2; the heavy chain CDR3 comprises or consists of an amino acid sequence E-K-F-Y-G-Y-D-Y shown in SEQ ID NO. 3;

the light chain CDR1 comprises or consists of an amino acid sequence R-S-S-Q-S-I-V-H-R-N-G-N-T-Y-L-E shown in SEQ ID NO. 4; the light chain CDR2 comprises or consists of an amino acid sequence K-V-S-N-R-F-S shown in SEQ ID NO. 5; the light chain CDR3 comprises or consists of a sequence F-Q-G-S-H-V-P-Y-T shown in SEQ ID NO. 6.

The heavy chain of said variant, whose heavy chain variable region comprises any one of the sequences having at least 90%, preferably at least 91%,92%,93%,94%,95%,96%, 97%,98%,99% or 100% sequence identity to the amino acid sequence depicted in SEQ ID NO. 7;

or having one or more amino acid mutations compared to the amino acid sequence shown in SEQ ID NO. 7; preferably with 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid mutations; the amino acid mutation, preferably conservative mutation, comprises a substituted, inserted or deleted amino acid sequence; more preferably the amino acid sequence shown as SEQ ID NO. 7.

The light chain of said variant having a light chain variable region comprising any one of a sequence having at least 90%, preferably 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. 8 or having one or more amino acid mutations compared to the amino acid sequence shown in SEQ ID No. 8; preferably having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid mutations, preferably conservative mutations, including amino acid sequences that are substitutions, insertions or deletions; more preferably, the amino acid sequence shown as SEQ ID NO. 8.

In some embodiments, the invention includes a nucleic acid sequence comprising an antibody or fragment thereof encoding an anti-Flag tag. For example, the nucleic acid sequence of its heavy chain, includes the nucleic acid sequence shown in SEQ ID NO. 9, and the nucleic acid sequence of its light chain, includes the nucleic acid sequence shown in SEQ ID NO. 10. In the present invention, the nucleic acid sequence comprises conservatively substituted variants thereof (e.g., substitution of degenerate codons) and complementary sequences. The terms "nucleic acid" and "polynucleotide" are synonymous and encompass genes, cDNA molecules, mRNA molecules, and fragments thereof such as oligonucleotides.

In some embodiments, the invention includes biomaterials comprising a nucleic acid sequence encoding an anti-Flag-tag antibody or fragment thereof, including expression vectors, expression cassettes, host cells, engineered bacteria, or hybridoma cell lines. Wherein 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 sequences in a manner that allows for expression of the coding sequence. Regulatory sequences are selected to direct the expression of the protein of interest in a suitable host cell and include promoters, enhancers and other expression control elements.

In addition, the invention also provides application of the anti-Flag-tag antibody or the nucleic acid sequence or the biological material in the preparation of a Flag-tag fusion protein detection reagent.

In another aspect, the present invention also provides a detection kit comprising the anti-Flag-tag antibody according to the present invention.

The following are examples:

example 1 amino acid sequence of anti-Flag tag antibody

The anti-Flag-tag antibody comprises a heavy chain CDR1, a heavy chain CDR2 and a heavy chain CDR3, wherein the heavy chain CDR1 comprises an amino acid sequence T-Y-T-I-H shown in SEQ ID NO. 1; the heavy chain CDR2 comprises an amino acid sequence Y-I-N-P-S-S-G-Y-A-Y-N-Q-N-F-K-D shown in SEQ ID NO. 2; the heavy chain CDR3 comprises an amino acid sequence E-K-F-Y-G-Y-D-Y shown in SEQ ID NO. 3;

and the antibody further comprises a light chain CDR1, a light chain CDR2, a light chain CDR3, the light chain CDR1 comprising the amino acid sequence R-S-S-Q-S-I-V-H-R-N-G-N-T-Y-L-E shown in SEQ ID NO. 4; the light chain CDR2 comprises an amino acid sequence K-V-S-N-R-F-S shown in SEQ ID NO. 5; the light chain CDR3 comprises a sequence F-Q-G-S-H-V-P-Y-T shown in SEQ ID NO. 6.

EXAMPLE 2 potency of monoclonal antibodies

1 antigen immunization

The Flag antigen (octaamino acid short peptide DYKDDDDK-coupled KLH) was mixed with Freund's complete adjuvant (appearance: amber cell suspension; component: paraffin Oil 85%, manide Monomate 15%, mycobacterium smegmatis 1 mg/mL) to give an oily emulsion. Injecting BALB/c mouse subcutaneously with 0.15mL dosage, performing intraperitoneal enhanced immunization (equal amount of antigen is mixed with Freund's incomplete adjuvant) 14 days after the first immunization, and collecting tail blood for titer detection after four needles, wherein the titer meets the fusion requirement. 3 days before fusion, the same amount of antigen is injected into abdominal cavity for additional immunization, and the immunization method is the same as above.

2 preparation of hybridoma cell line

(1) Preparation of feeder cells

BALB/c mouse peritoneal macrophages were used as feeder cells. 1 day before the fusion, BALB/c mice were killed by neck pulling, soaked in 75% alcohol, placed in an ultra clean bench, aseptically cut open the abdominal skin with scissors, the peritoneum was exposed, 5mL of RPMI1640 basic culture medium was injected into the abdominal cavity with a syringe, repeatedly washed, the washing solution was recovered, centrifuged at 1000rpm for 5 minutes, left to settle, the culture medium (in complete culture medium of RPMI1640 containing HAT) was resuspended in RPMI1640 screening medium, adjusted to a cell concentration of 1X 105 cells/mL, added to a 96-well plate, 150. Mu.L/well, incubated at 37 ℃ and 5% CO2 overnight.

(2) Preparation of immune spleen cells

Three days after the last immunization of the mice, the spleen is taken out under the aseptic condition, placed in a plate, washed once by RPMI1640 basic culture solution, placed on a nylon net of a small beaker, ground and filtered to prepare cell suspension. Centrifuging, discarding supernatant, resuspending RPMI1640 basic culture solution, repeating the steps for three times to obtain immune spleen cells, and counting.

(3) Preparation of myeloma cells

After 8-azaguanine screening, culturing mouse myeloma cells Sp2/0 to logarithmic growth phase, taking two bottles to prepare cell suspension, centrifuging, discarding supernatant, resuspending with RPMI1640 basic culture solution for three times if repeated to obtain myeloma cells, and counting.

(4) Cell fusion and HAT selection hybridomas

Myeloma cells and immune spleen cells were mixed at a ratio of 1. Discarding the supernatant, mixing the cells, slowly adding 1mL of 50% PEG 1500 for fusion, and adding 15mL of RPMI1640 basic culture solution to terminate the cell fusion after 1 minute of fusion. Centrifuge at 1000rpm for 5-10 minutes. Discard the supernatant, gently resuspend the selected culture medium in 50mL of RPMI1640, aliquot in 10 96-well plates at 50. Mu.L/well, 37 ℃ C., 5% CO2 culture. The culture was carried out until the sixth day, and HAT medium (complete HAT-containing RPMI1640 medium) was changed twice.

(5) Hybridoma cell selection

Flag antigen (DYKDDDDK coupled to BSA) was diluted with 0.05M pH 9.6 carbonate buffer to a final concentration of 1. Mu.g/mL. 0.1mL per well, 96 well polystyrene plates were added at 37 deg.C, 2 hours or 4 deg.C overnight. The next day, 0.02M pH 7.2 PBS containing 10% calf serum or 1% skim milk powder, 0.15mL/well, blocked at 37 ℃ for 2 hours, was used for detection. And on the seventh day after fusion, 0.1mL of cell supernatant is taken and put into the 96-well detection plate, the temperature is 37 ℃ for 30 minutes, after washing for six times, 2000-fold diluted goat anti-mouse IgG labeled by horseradish peroxidase is added, after washing for 30 minutes at 37 ℃,100 mu L of the goat anti-mouse IgG labeled by horseradish peroxidase is added into each well, 0.1% (M/V) o-phenylenediamine, 0.1% (V/V) hydrogen peroxide, pH 5.0 citric acid phosphate buffer solution is added, after 15 minutes at 37 ℃, diluted sulfuric acid solution is added, 50 mu L of each well, and the absorption value of 450nm is measured. The RPMI1640 complete culture solution is used as a negative control, and the ratio of the measured value to the control value is ≧ 2.0 as a positive cell well.

Antibody-secreting positive cell wells were cloned at 1 cell/well in 96-well plates by limiting dilution, positive wells were selected and cloned three times in succession according to the above method, and after extensive culture, they were cryopreserved using a culture medium containing 10% DMSO, at a cell density of 106 cells/mL. Five stable Flag-tag-resistant hybridoma cell strains are obtained and named as A, B, C, D and E respectively.

3 preparation of monoclonal antibodies

Selecting robust BALB/c mice with 6-8 weeks, and injecting 0.5mL of pristany into the abdominal cavity of each mouse; after 10 days, 1X 106 hybridoma cells were intraperitoneally injected. Ascites can be generated 7 to 10 days after the cells are inoculated, the health condition and the ascites symptoms of the animals are closely observed, the mice are sacrificed when the ascites is as much as possible and before the mice die frequently, the ascites is sucked into the test tube by a dropper, and generally, 5 to 10mL of ascites can be obtained by one mouse. Collecting ascites, centrifuging, collecting supernatant, and storing in refrigerator at-20 deg.C. The ascites supernatant was collected, diluted with 3 volumes of PBS and filtered through a filter paper. The resulting filtrate was applied to a protein G affinity column equilibrated with PBS at a flow rate of 1 mL/min. The material not adsorbed by protein G was then washed with PBS at a flow rate of 1mL/min until the absorbance at OD280 nm reached baseline. The antibody was then eluted with 0.1M glycine eluent (pH 2.5) and recovered. The recovered solution was neutralized with 0.1M Tris (pH 8.8), and the antibody concentration was adjusted to an appropriate concentration by ultrafiltration, and then frozen at-20 ℃.

4 measurement of antibody titer

The titers of the 5 hybridoma cells and the secreted ascites antibody were measured by an indirect ELISA method. The specific experimental steps are as follows:

(1) Coating: diluting Flag antigen (DYKDDDDK coupled BSA) to 1 μ g/mL, adding 100 μ L/well to the ELISA plate, and standing at 37 ℃ for 2 hours or 4 ℃ overnight;

(2) Washing the plate for 5 times by using a plate washing machine, injecting 350 mu L of washing liquid into each hole each time, and staying for 20 seconds; finally, patting to be dry;

(3) Washing with washing solution to remove coating solution, sealing with sealing solution at a temperature of 150 μ L per well, standing at 37 deg.C for 1.5-2 hr;

(4) Washing the plate for 5 times by the plate washing machine, injecting 350 mu L of washing liquid into each hole each time, and staying for 20 seconds; finally, patting to be dry;

(5) Adding a sample: respectively adding the diluted cell culture supernatant and the ascites with different gradients into an ELISA plate coated with Flag antigen, reacting at the temperature of 37 ℃ for 1 hour (simultaneously making a negative control hole and a positive control hole);

(6) Washing the plate for 5 times by using a plate washing machine, injecting 350 mu L of washing liquid into each hole every time, and staying for 20 seconds; finally, patting to be dry;

(7) Adding a goat anti-mouse IgG enzyme-labeled secondary antibody (6000 times diluted by a sealing solution) labeled by horseradish peroxidase, performing reaction at the temperature of 37 ℃ for 1 hour at 100 mu L/hole;

(8) Washing the plate for 5 times by the plate washing machine, injecting 350 mu L of washing liquid into each hole each time, and staying for 20 seconds; finally, patting to be dry;

(9) Adding a color development liquid TMB: the preparation is carried out immediately after use, 100 mu L/hole is carried out, and the reaction is carried out for 30 minutes at 37 ℃ in a dark place;

(10) And (3) terminating the reaction: 2M sulfuric acid was added to each reaction well at 50. Mu.L/well;

(11) Reading by a microplate reader: 450nm, 630nm. The antibody titer results are shown in table 1 below.

TABLE 1 results of different antibody titer determinations

Flag-tagged cell line	Hybridoma cell culture supernatant titer	Ascites antibody titer
			2E9	3.27×10 ³	4.57×10 ⁵
5B4	3.18×10 ³	6.24×10 ⁵
			6C10	6.29×10 ⁴	8.52×10 ⁶
2B11	4.24×10 ³	5.13×10 ⁵
			3G7	2.23×10 ³	4.19×10 ⁵

As can be seen from Table 1, the titer of the antibody secreted by hybridoma cell line 6C10 was 8.52X 10 ⁶ The antibody is obviously higher than antibodies secreted by other 4 hybridoma cell strains.

Example 3 preparation of recombinant antibody

(1) Antibody variable region gene cloning and sequencing

Total RNA was extracted from the hybridoma cell line 6C10 secreting Flag monoclonal antibody of example 2, and first strand cDNA synthesis was performed using SMARTERTM RACE cDNA Amplification Kit expression cassette and SMARTER IIA Oligonucleotide and 5' -CDS primer in the expression cassette, and the obtained first strand cDNA product was used as a PCR Amplification template. The light chain genes were amplified with Universal Primer A Mix (UPM), nested Universal Primer A (NUP), and mIgG CKR primers, and the heavy chain genes were amplified with Universal Primer A Mix (UPM), nested Universal Primer A (NUP), and mIgG CHR primers. Wherein the primer pair of the light chain can amplify a target band about 0.7KB, and the primer pair of the heavy chain can amplify a target band about 1.5 KB. Purifying and recovering by agarose gel electrophoresis, adding A into a pMD-18T vector after the product is subjected to an A adding reaction by rTaq DNA polymerase, transforming into DH5 alpha competent cells, taking 4 clones of heavy chain and light chain gene clones respectively after a colony grows out, and sending the 4 clones to a promoter company for sequencing.

The antibody obtained from hybridoma cell line 6C10 above was analyzed to have sequences such as the light and heavy chain sequences appended hereafter.

Upon analysis, the complementarity determining regions of the heavy chain:

CDR1：T-Y-T-I-H；

CDR2：Y-I-N-P-S-S-G-Y-A-A-Y-N-Q-N-F-K-D；

CDR3：E-K-F-Y-G-Y-D-Y。

complementarity determining regions of the light chain:

CDR1：R-S-S-Q-S-I-V-H-R-N-G-N-T-Y-L-E；

CDR2：K-V-S-N-R-F-S；

CDR3：F-Q-G-S-H-V-P-Y-T。

(2) Preparing recombinant antibody for affinity analysis and activity identification

(1) Affinity assay

Performing enzyme immunoassay indirect method data in the same way as activity identification, and coating to obtain four gradients of 1 μ g/ml, 0.5 μ g/ml, 0.25 μ g/ml and 0.125 μ g/ml; the antibody was diluted in a 2-fold gradient starting at 1000ng/ml and loaded at 0.97656 ng/ml. And obtaining the OD values corresponding to different antibody concentrations under the conditions of no coating concentration. Under the same coating concentration, the antibody concentration is used as an abscissa and the OD value is used as an ordinate, logarithmic mapping is carried out, and the antibody concentration at 50% of the maximum OD value is calculated according to a fitting equation; substitution into the formula: k = (n-1)/(2 × (n × Ab ') -Ab)), calculating the reciprocal of the affinity constant, where Ab and Ab' represent the antibody concentration at 50% of maximum OD value at the corresponding coating concentration (Ag, ag '), respectively, n = Ag/Ag'; every two coating concentrations can be combined to calculate a K value, and finally six K values can be obtained, the average value is taken, and the reciprocal value is obtained to be the affinity constant KD. The affinity analysis data of the purified anti-Flag mab are shown in table 2 below.

TABLE 2 affinity assay results of purified anti-Flag monoclonal antibody

Sample name	KD
		Flag-tagged antibody 6C10	5.219E-10
2E9	6.145E-9
		5B4	3.289E-8
2B11	9.118E-9
		3G7	7.834E-8

As can be seen from Table 2, the affinity of the purified anti-Flag monoclonal antibody C was significantly better than that of the antibody secreted from the other 4 hybridoma cells.

(2) Activity identification

Diluting Flag antigen (DYKDDDDK coupled BSA) to 1 μ g/mL by using 50mM carbonate buffer solution coating solution to carry out microplate coating, wherein each well is 100 μ L, and the temperature is kept overnight at 4 ℃; the next day, the wash solution was washed 2 times with PBST and patted dry; add blocking solution (20% BSA +80% PBS), 120 μ L per well, 37 deg.C, 1h, pat dry; adding the diluted anti-Flag monoclonal antibody 6C10, carrying out 5-fold dilution from 1000ng/ml, loading, 100 mu L/hole, 37 ℃ and 30min (partial supernatant is 1 h); washing with PBST cleaning solution for 5 times, and draining; adding horse radish peroxidase labeled goat anti-mouse IgG at 37 deg.C for 30min at a volume of 100 μ L per well; washing with PBST cleaning solution for 5 times, and draining; adding carbamide peroxide (50 μ L/well), adding tetramethyl benzidine (50 μ L/well), 10min; adding dilute hydrochloric acid to stop the reaction, wherein the concentration is 50 mu L/hole; OD readings were taken at 450nm (reference 630 nm) on the microplate reader. The activity of the purified anti-Flag tag monoclonal antibody is identified, and the result is shown in the following table 3.

TABLE 3 Activity identification of purified anti-Flag tag monoclonal antibody

Sample concentration ng/ml	1000	200	40	8	1.6	0.32	0
								Flag antibody 6C10	3.291	2.113	0.807	0.172	0.128	0.072	0.031

It will be understood by those skilled in the art that the foregoing is only a preferred embodiment of the present invention, and is not intended to limit the invention, and that any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.

SEQUENCE LISTING

<110> Changsha Nuo pine biotech partnership (limited partnership)

<120> anti-Flag-tag antibody and application thereof

<130> 2022-6-8

<160> 10

<170> PatentIn version 3.5

<210> 1

<211> 5

<212> PRT

<213> Artificial Synthesis

<400> 1

Thr Tyr Thr Ile His

1 5

<210> 2

<211> 17

<212> PRT

<213> Artificial Synthesis

<400> 2

Tyr Ile Asn Pro Ser Ser Gly Tyr Ala Ala Tyr Asn Gln Asn Phe Lys

1 5 10 15

Asp

<210> 3

<211> 8

<212> PRT

<213> Artificial Synthesis

<400> 3

Glu Lys Phe Tyr Gly Tyr Asp Tyr

1 5

<210> 4

<211> 16

<212> PRT

<213> Artificial Synthesis

<400> 4

Arg Ser Ser Gln Ser Ile Val His Arg Asn Gly Asn Thr Tyr Leu Glu

1 5 10 15

<210> 5

<211> 7

<212> PRT

<213> Artificial Synthesis

<400> 5

Lys Val Ser Asn Arg Phe Ser

1 5

<210> 6

<211> 9

<212> PRT

<213> Artificial Synthesis

<400> 6

Phe Gln Gly Ser His Val Pro Tyr Thr

1 5

<210> 7

<211> 117

<212> PRT

<213> Artificial Synthesis

<400> 7

Gln Val Gln Leu Gln Gln Ser Ala Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Thr Tyr

20 25 30

Thr Ile His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Ser Gly Tyr Ala Ala Tyr Asn Gln Asn Phe

50 55 60

Lys Asp Glu Thr Thr Leu Thr Ala Asp Pro Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Lys Phe Tyr Gly Tyr Asp Tyr Trp Gly Gln Gly Ala Thr

100 105 110

Leu Thr Val Ser Ser

115

<210> 8

<211> 115

<212> PRT

<213> Artificial Synthesis

<400> 8

Asp Val Leu Met Thr Gln Ile Pro Leu Ser Leu Pro Val Ser Leu Gly

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Arg

20 25 30

Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Leu Lys Pro Gly Gln Ser

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Arg

100 105 110

Arg Ala Asp

115

<210> 9

<211> 1383

<212> DNA

<213> Artificial Synthesis

<400> 9

atggaatggc ctttgatttt tttgtttttg ttgtctggaa ctgctggagt tcaatctcaa 60

gttcaattgc aacaatctgc tgctgaattg gctagacctg gagcttctgt taagatgtct 120

tgtaaggctt ctggatattc ttttactact tatactattc attgggttaa gcaaagacct 180

ggacaaggat tggaatggat tggatatatt aatccttctt ctggatatgc tgcttataat 240

caaaatttta aggatgaaac tactttgact gctgatcctt cttcttctac tgcttatatg 300

gaattgaatt ctttgacttc tgaagattct gctgtttatt attgtgctag agaaaagttt 360

tatggatatg attattgggg acaaggagct actttgactg tttcttctgc taagactact 420

cctccttctg tttatccttt ggctcctgga tctgctgctc aaactaattc tatggttact 480

ttgggatgtt tggttaaggg atattttcct gaacctgtta ctgttacttg gaattctgga 540

tctttgtctt ctggagttca tacttttcct gctgttttgc aatctgattt gtatactttg 600

tcttcttctg ttactgttcc ttcttctact tggccttctg aaactgttac ttgtaatgtt 660

gctcatcctg cttcttctac taaggttgat aagaagattg ttcctagaga ttgtggatgt 720

aagccttgta tttgtactgt tcctgaagtt tcttctgttt ttatttttcc tcctaagcct 780

aaggatgttt tgactattac tttgactcct aaggttactt gtgttgttgt tgatatttct 840

aaggatgatc ctgaagttca attttcttgg tttgttgatg atgttgaagt tcatactgct 900

caaactcaac ctagagaaga acaatttaat tctactttta gatctgtttc tgaattgcct 960

attatgcatc aagattggtt gaatggaaag gaatttaagt gtagagttaa ttctgctgct 1020

tttcctgctc ctattgaaaa gactatttct aagactaagg gaagacctaa ggctcctcaa 1080

gtttatacta ttcctcctcc taaggaacaa atggctaagg ataaggtttc tttgacttgt 1140

atgattactg atttttttcc tgaagatatt actgttgaat ggcaatggaa tggacaacct 1200

gctgaaaatt ataagaatac tcaacctatt atggatactg atggatctta ttttgtttat 1260

tctaagttga atgttcaaaa gtctaattgg gaagctggaa atacttttac ttgttctgtt 1320

ttgcatgaag gattgcataa tcatcatact gaaaagtctt tgtctcattc tcctggaaag 1380

tga 1383

<210> 10

<211> 720

<212> DNA

<213> Artificial Synthesis

<400> 10

atgagatgtt tggctgaatt tttgggattg ttggttttgt ggattcctgg agctattgga 60

gatgttttga tgactcaaat tcctttgtct ttgcctgttt ctttgggaga tcaagcttct 120

atttcttgta gatcttctca atctattgtt catagaaatg gaaatactta tttggaatgg 180

tatttgttga agcctggaca atctcctaag ttgttgattt ataaggtttc taatagattt 240

tctggagttc ctgatagatt ttctggatct ggatctggaa ctgattttac tttgaagatt 300

tctagagttg aagctgaaga tttgggagtt tattattgtt ttcaaggatc tcatgttcct 360

tatacttttg gaggaggaac taagttggaa attagaagag ctgatgctgc tcctactgtt 420

tctatttttc ctccttcttc tgaacaattg acttctggag gagcttctgt tgtttgtttt 480

ttgaataatt tttatcctaa ggatattaat gttaagtgga agattgatgg atctgaaaga 540

caaaatggag ttttgaattc ttggactgat caagattcta aggattctac ttattctatg 600

tcttctactt tgactttgac taaggatgaa tatgaaagac ataattctta tacttgtgaa 660

gctactcata agacttctac ttctcctatt gttaagtctt ttaatagaaa tgaatgttga 720

Claims

1. An anti-Flag-tagged antibody comprising a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, and a light chain CDR1, a light chain CDR2, a light chain CDR3;

the heavy chain CDR1 is an amino acid sequence T-Y-T-I-H shown in SEQ ID NO. 1; the heavy chain CDR2 has an amino acid sequence Y-I-N-P-S-S-G-Y-A-Y-Q-N-F-K-D shown in SEQ ID NO. 2; the heavy chain CDR3 has an amino acid sequence E-K-F-Y-G-Y-D-Y shown in SEQ ID NO. 3;

the light chain CDR1 has an amino acid sequence R-S-S-Q-S-I-V-H-R-N-G-N-T-Y-L-E shown in SEQ ID NO. 4; the light chain CDR2 is an amino acid sequence K-V-S-N-R-F-S shown in SEQ ID NO. 5; the light chain CDR3 is a sequence F-Q-G-S-H-V-P-Y-T shown in SEQ ID NO. 6.

2. The anti-Flag-tagged antibody according to claim 1, wherein the heavy chain, its heavy chain variable region comprises any one of the amino acid sequences having more than 90% identity to the amino acid sequence shown in SEQ ID No. 7, or has one or more amino acid mutations compared to the amino acid sequence shown in SEQ ID No. 7; the light chain, the light chain variable region of which comprises any one of the amino acid sequences having more than 90% identity with the amino acid sequence shown in SEQ ID NO. 8, or has one or more amino acid mutations compared with the amino acid sequence shown in SEQ ID NO. 8.

3. The anti-Flag-tagged antibody according to claim 1 or 2, wherein the heavy chain, its heavy chain variable region comprises any one of the amino acid sequences having 90% or more identity to the amino acid sequence shown in SEQ ID No. 7, or has 10 or less amino acid mutations compared to the amino acid sequence shown in SEQ ID No. 7; the light chain variable region of the light chain comprises any one amino acid sequence which has more than 90 percent of identity with the amino acid sequence shown in SEQ ID NO. 8, or has less than 10 amino acid mutations compared with the amino acid sequence shown in SEQ ID NO. 8.

4. The anti-Flag-tag antibody according to claim 2, wherein the amino acid mutation is a conservative mutation comprising a substitution, insertion or deletion of an amino acid sequence.

5. A nucleic acid sequence encoding the anti-Flag-tag antibody according to any one of claims 1 to 4, wherein the nucleic acid sequence, the nucleic acid sequence of the heavy chain thereof, comprises SEQ ID No. 9 or conservatively substituted variants or complements thereof; a nucleic acid sequence of a light chain comprising SEQ ID NO 10 or conservatively substituted variants or complements thereof.

6. The nucleic acid sequence encoding an anti-Flag-tag antibody of claim 5, wherein the nucleic acid sequence comprises a genomic DNA molecule, a cDNA molecule, an mRNA molecule, and fragments thereof.

7. The anti-Flag-tag antibody-encoding nucleic acid sequence of claim 6, wherein the anti-Flag-tag antibody-encoding nucleic acid sequence comprises the heavy chain nucleic acid sequence of SEQ ID NO. 9 and the light chain nucleic acid sequence comprises SEQ ID NO. 10.

8. A biomaterial containing a nucleic acid sequence encoding the anti-Flag-tag antibody or fragment thereof according to claim 1, wherein the biomaterial comprises an expression vector, an expression cassette, a host cell, an engineering bacterium or a hybridoma cell line.

9. Use of an anti-Flag-tag antibody according to any one of claims 1 to 4 or a nucleic acid sequence according to claim 5 or 6 or a biological material according to claim 8 for the preparation of a Flag-tag fusion protein detection reagent.

10. A detection kit comprising the anti-Flag-tagged antibody according to any one of claims 1 to 4.