CN116589572B - Monoclonal antibody resisting HA tag and application thereof - Google Patents
Monoclonal antibody resisting HA tag and application thereof Download PDFInfo
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- CN116589572B CN116589572B CN202310771383.1A CN202310771383A CN116589572B CN 116589572 B CN116589572 B CN 116589572B CN 202310771383 A CN202310771383 A CN 202310771383A CN 116589572 B CN116589572 B CN 116589572B
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1018—Orthomyxoviridae, e.g. influenza virus
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/51—Complete heavy chain or Fd fragment, i.e. VH + CH1
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/515—Complete light chain, i.e. VL + CL
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses an anti-HA tag monoclonal antibody and application thereof, comprising a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2 and a light chain CDR3; the heavy chain CDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO. 1; the heavy chain CDR2 comprises or consists of the sequence shown in SEQ ID NO. 2; the heavy chain CDR3 comprises or consists of the sequence shown in SEQ ID NO. 3; light chain CDR1 comprises or consists of the sequence shown in SEQ ID NO. 4; light chain CDR2 comprises or consists of the sequence shown in SEQ ID NO. 5; the light chain CDR3 comprises or consists of the sequence shown in SEQ ID NO. 6; its potency can be up to 1.48×10 6 The affinity reaches 1.333E-8, the antibody activity is high, and the application potential is great.
Description
Technical Field
The invention belongs to the field of molecular immunology, and in particular relates to an anti-HA tag monoclonal antibody and application thereof.
Background
The HA tag (HA-tag) is a protein tag based on human influenza virus Hemagglutinin (HA) antigen, the chemical nature of the HA tag is a short sequence (amino acid sequence: YPYDVPDYA) from 98 th to 106 th amino acids of influenza virus Hemagglutinin (HA) protein, and the molecular weight of the HA tag is 1.1kDa, and the HA tag is one of the epitope tags widely used at present. The HA tag is fused to the C end or N end of the target protein by a molecular biological means, and then the fusion protein with the HA tag at the C end or N end is specifically identified by the HA tag antibody, so that the HA tag antibody can be generally used for detecting and expressing the HA tag fusion expression protein, positioning in cells, purifying, qualitatively or quantitatively detecting the HAtag fusion expression protein and the like, namely the HA tag antibody can be used for detecting, separating and purifying the HA-tagged target protein without protein specific antibodies or probes. Currently, HA-tagged antibodies are one of the most commonly used antibodies in protein expression and cell biology research. However, currently there are few monoclonal antibodies to HA tag proteins commercially available and they are very expensive. Therefore, more monoclonal antibodies resisting the HA tag are researched, and the method HAs wide application prospects in expression detection, positioning, immunoadsorption, separation and purification of fusion proteins.
Disclosure of Invention
Aiming at the defects or improvement demands of the prior art, the invention provides an anti-HA-tag monoclonal antibody and application thereof, and aims to prepare the anti-HA-tag monoclonal antibody based on the amino acid sequences of heavy chain and light chain complementarity determining regions of the antibody by coupling synthetic peptide of amino acid residues YPYDVPDYA (98-106) of influenza virus Hemagglutinin (HA) with keyhole limpet hemocyanin (Keyhole Limpet Hemocyanin, KLH) as antigen to stimulate mice to generate the antibody, and discovering the high-titer HA antibody in the body of the mouse, and cloning and sequencing the variable region genes of the antibody to obtain the anti-HA-tag monoclonal antibody based on the amino acid sequences of the heavy chain and the light chain complementarity determining regions of the antibody, thereby solving the technical problems of less types and high price of the existing anti-HA-tag monoclonal antibody.
To achieve the above object, according to one aspect of the present invention, there is provided an anti-HA-tagged monoclonal antibody comprising 3 complementarity determining regions of heavy chains: heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, and complementarity determining regions of 3 light chains: light chain CDR1, light chain CDR2, and light chain CDR3;
the heavy chain CDR1 comprises or consists of an amino acid sequence S-Y-G-M-S shown in SEQ ID NO. 1;
the heavy chain CDR2 comprises or consists of an amino acid sequence Y-I-S-D-G-G-V-R-T-Y-Y-P-D-T-I-K-G shown in SEQ ID NO. 2;
the heavy chain CDR3 comprises or consists of an amino acid sequence H-R-G-Y-D-G-M-D-Y shown in SEQ ID NO. 3;
the light chain CDR1 comprises or consists of an amino acid sequence R-A-S-A-S-V-I-A-M-H shown in SEQ ID NO. 4;
the light chain CDR2 comprises or consists of an amino acid sequence A-T-S-S-L-A-S shown in SEQ ID NO. 5
The light chain CDR3 comprises or consists of the amino acid sequence Q-H-W-S-R-N-P-L-T shown in SEQ ID NO. 6.
Preferably, the anti-HA-tagged monoclonal antibody, the antibody thereof, the heavy chain variable region thereof comprises the amino acid sequence:
comprising or consisting of the amino acid sequence shown in SEQ ID NO. 7,8,9, 10, a sequence having at least 80%,85%,90% identity to the 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, 10.
Preferably, the anti-HA-tagged monoclonal antibody, the heavy chain variable region of which comprises the amino acid sequence:
comprises an amino acid sequence as shown in SEQ ID NO. 7,8,9, 10, a sequence having at least 91%,92%,93%,94%,95%,96%,97%,98%,99% or 100% sequence identity to the amino acid sequence as 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 to the amino acid sequence as shown in SEQ ID NO. 7,8,9, 10; the amino acid mutation is a conservative mutation, including substitution, insertion or deletion.
Preferably, the monoclonal antibody against the HA tag, the antibody thereof, the light chain variable region thereof comprises the amino acid sequence:
comprising or consisting of the amino acid sequence shown in SEQ ID NO. 11, 12, 13, 14, a sequence having at least 80%,85%,90% identity to the 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, 14.
Preferably, the anti-HA-tagged monoclonal antibody, the light chain variable region of which comprises the amino acid sequence:
comprises an amino acid sequence as shown in SEQ ID NO. 11, 12, 13, 14, a sequence having at least 91%,92%,93%,94%,95%,96%,97%,98%,99% or 100% sequence identity to the amino acid sequence as 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 to the amino acid sequence as shown in SEQ ID NO. 11, 12, 13, 14; the amino acid mutation is a conservative mutation, including substitution, insertion or deletion.
Preferably, the heavy chain amino acid sequence of the monoclonal antibody against the HA tag is shown as SEQ ID NO. 15, and the light chain amino acid sequence of the monoclonal antibody is shown as SEQ ID NO. 16.
According to another aspect of the present invention there is also provided a nucleic acid sequence encoding an anti-HA-tagged monoclonal antibody or antibody fragment thereof according to the present invention, wherein the nucleic acid sequence comprises oligonucleotides of genes, cDNA molecules, mRNA molecules and fragments thereof.
According to another aspect of the present invention there is also provided a biological material comprising a nucleic acid sequence encoding a monoclonal antibody or antibody fragment thereof against an HA tag according to the present invention, the biological material comprising an expression vector, an expression cassette, a host cell, an engineered bacterium or a hybridoma cell line.
According to another aspect of the invention there is also provided the use of an anti-HA-tagged monoclonal antibody according to the invention for the detection, isolation and purification of HA-tagged target proteins.
According to another aspect of the present invention there is also provided a detection kit comprising a monoclonal antibody against an HA tag according to the present invention.
In general, compared with the prior art, the above technical solution contemplated by the present invention HAs the following beneficial effects, since the present invention stimulates mice by combining HA and KLH as antigens, and finds an anti-HA antibody with high potency in the body:
the invention stimulates mice through HA antigen, and finds an ascites antibody with high titer in the mice, and the titer can reach 1.48 multiplied by 10 6 The amino acid sequences of the heavy chain and light chain complementarity determining regions are obtained through gene cloning and sequencing of the antibody variable region, the anti-HA-tag monoclonal antibody is prepared by adopting a recombination technology, and the affinity KD of the anti-HA-tag monoclonal antibody provided by the invention is 1.333E-8 through determination, and the purified anti-HA-tag monoclonal antibody HAs good affinity, high activity and wide market prospect application.
Drawings
FIG. 1 is the amino acid sequences of the heavy and light chains of an anti-HA-tagged monoclonal antibody.
Detailed Description
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention. In addition, the technical features of the embodiments of the present invention described below may be combined with each other as long as they do not collide with each other.
The whole antibody molecule can be divided into a constant region (C region) and a variable region (V region), and the amino acid composition and arrangement sequence of the different antibody V regions are different, wherein the amino acid composition and arrangement sequence of each of VH and VL has 3 regions, which are called HVR, are particularly easy to change. The three HVRs of VH and VL together constitute the antigen binding site of Ig, i.e., the hypervariable regions within the heavy and light chain variable regions constitute the antigen (Ag) binding site of the antibody molecule. Thus, the hypervariable region is also known as the complementarity-determining region (CDR) region of an antibody molecule, the amino acid sequence of which determines the specificity of an antibody.
The invention uses synthetic peptide of amino acid residue YPYYDVPDYA (98-106) of human influenza virus Hemagglutinin (HA) to couple with KLH as antigen, and injects mice to stimulate the antibody to generate in vivo, as a result, a high-titer HA antibody is found in the mice, the amino acid sequences of the heavy chain and the light chain of the antibody are obtained through the gene cloning and sequencing of the variable region of the antibody, and the complementarity determining region of the heavy chain is analyzed: CDR1: S-Y-G-M-S; CDR2: Y-I-S-D-G-G-V-R-T-Y-Y-P-D-T-I-K-G; CDR3: H-R-G-Y-D-G-G-M-D-Y; complementarity determining regions of the light chain: CDR1: R-A-S-A-S-V-I-A-M-H; CDR2: A-T-S-S-L-A-S CDR3: Q-H-W-S-R-N-P-L-T; and preparing a recombinant antibody according to the amino acid sequences of the heavy chain and the light chain of the antibody, namely the preferred anti-HA monoclonal antibody.
The invention provides a monoclonal antibody resisting an HA tag, which comprises 3 complementarity determining regions of a heavy chain and 3 complementarity determining regions of a light chain, wherein the complementarity determining regions of the heavy chain are respectively a heavy chain CDR1, a heavy chain CDR2 and a heavy chain CDR3, and the complementarity determining regions of the light chain are respectively a light chain CDR1, a light chain CDR2 and a light chain CDR3;
the heavy chain CDR1 comprises or consists of an amino acid sequence S-Y-G-M-S shown in SEQ ID NO. 1;
the heavy chain CDR2 comprises or consists of an amino acid sequence Y-I-S-D-G-G-V-R-T-Y-Y-P-D-T-I-K-G shown in SEQ ID NO. 2;
the heavy chain CDR3 comprises or consists of an amino acid sequence H-R-G-Y-D-G-M-D-Y shown in SEQ ID NO. 3;
the light chain CDR1 comprises or consists of an amino acid sequence R-A-S-A-S-V-I-A-M-H shown in SEQ ID NO. 4;
the light chain CDR2 comprises or consists of an amino acid sequence A-T-S-S-L-A-S shown in SEQ ID NO. 5
The light chain CDR3 comprises or consists of the amino acid sequence Q-H-W-S-R-N-P-L-T shown in SEQ ID NO. 6.
Preferably, the anti-HA-tagged monoclonal antibody, the heavy chain variable region thereof, further comprises or consists of the amino acid sequence:
comprises amino acid sequences shown as SEQ ID NO. 7,8,9 and 10;
or a sequence having at least 80%,85%,90%, preferably 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;
or a sequence having one or more amino acid mutations compared to the amino acid sequences shown in SEQ ID NO. 7,8,9, 10; preferably 1, 2, 3, 4, 5, 6, 7,8,9 or 10 amino acids; the amino acid mutation is preferably a conservative mutation, more preferably a substitution, insertion or deletion.
The light chain variable region thereof further comprises or consists of the amino acid sequence:
comprises amino acid sequences shown as SEQ ID NO. 11, 12, 13 and 14;
or a sequence having at least 80%,85%,90%, preferably 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, 14;
or a sequence having one or more amino acid mutations compared to the amino acid sequences shown in SEQ ID NOs 11, 12, 13, 14; preferably 1, 2, 3, 4, 5, 6, 7,8,9 or 10 amino acids; the amino acid mutation is preferably a conservative mutation, more preferably a substitution, insertion or deletion.
In some embodiments, the anti-HA tag monoclonal antibody HAs a heavy chain amino acid sequence shown in SEQ ID NO. 15 and a light chain amino acid sequence shown in SEQ ID NO. 16.
In some embodiments, the invention also provides a nucleic acid sequence encoding an anti-HA-tagged monoclonal antibody or antibody fragment thereof according to the invention. In the present invention, a nucleic acid sequence comprises a variant (e.g., substitution of degenerate codons) of its conservative substitution and a complementary sequence. The terms "nucleic acid" and "polynucleotide" are synonymous and include genes, cDNA molecules, mRNA molecules and fragments thereof, e.g., oligonucleotides.
In some embodiments, the invention also provides a biological material comprising a nucleic acid sequence encoding a monoclonal antibody or antibody fragment thereof against an HA tag of the invention, including an expression vector, an expression cassette, a host cell, an engineered bacterium, or a hybridoma cell line.
Wherein the nucleic acid sequence is operably linked to at least one regulatory sequence. "operably linked" refers to a coding sequence being linked to regulatory sequences in a manner that allows for the expression of the coding sequence. Regulatory sequences are selected to direct 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 monoclonal antibody of the anti-HA tag in detection, separation and purification of the HA-tagged target protein.
The invention also provides a detection kit comprising the monoclonal antibody against the HA tag.
The following are examples:
EXAMPLE 1 monoclonal antibodies against the HA tag
The heavy and light chain amino acid sequences of the anti-HA-tagged monoclonal antibodies of this example are shown in fig. 1, the heavy chain comprising heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3, and the light chain comprising light chain CDR1, light chain CDR2 and light chain CDR3;
wherein the heavy chain CDR1 consists of an amino acid sequence S-Y-G-M-S shown in SEQ ID NO. 1; heavy chain CDR2, the amino acid sequence shown by SEQ ID NO. 2
Y-I-S-D-G-G-V-R-T-Y-Y-P-D-T-I-K-G; heavy chain CDR3, by SEQ ID NO 3 shows the amino acid sequence H-R-G-Y-D-G-G-M-D-Y composition;
the light chain CDR1 is composed of an amino acid sequence R-A-S-A-S-V-I-A-M-H shown in SEQ ID NO. 4; the light chain CDR2 consists of an amino acid sequence A-T-S-S-L-A-S shown in SEQ ID NO. 5 The light chain CDR3 consists of the amino acid sequence Q-H-W-S-R-N-P-L-T shown in SEQ ID NO. 6.
Example 2
1. Antigen immunization
HA is a hapten, and needs to be coupled with keyhole limpet hemocyanin (Keyhole Limpet Hemocyanin, KLH) to form an antigen, and BALB/c mice are subcutaneously injected with a synthetic peptide derived from amino acid residues YPYDVPDYA (98-106) of human influenza virus Hemagglutinin (HA) combined with KLH in the dosage of 0.15 mL. After 14 days of the first immunization, the immunization is enhanced, and after the fourth immunization, tail blood is collected for detection, and the titer meets the fusion requirement. Immunization was continued three days prior to fusion.
2. Preparation of hybridoma cell lines
(1) Preparation of feeder cells
BALB/c murine peritoneal macrophages were used as feeder cells. The abdomen was cut, 5mL of RPMI 1640 basal medium was injected, repeatedly rinsed, and collected. 1000rpm,5 minutes, a precipitate was left. Resuspension screening with RPMI 1640 culture solution containing HAT to adjust concentration to 1×10 5 mu.L/well of 96-well plate, 150. Mu.L/well, 37℃and 5% CO were added per mL 2 Culturing overnight.
(2) Preparation of immune spleen cells
3 days after the last immunization, spleens of mice were placed in a plate under aseptic conditions, and were washed with RPMI 1640 basal medium, ground on nylon membrane, and filtered to prepare a cell suspension. Centrifuging, removing supernatant, re-suspending the RPMI 1640 basic culture solution, repeating for three times to obtain immune spleen cells, and counting.
(3) Preparation of myeloma cells
After 8-azaguanine screening, the mouse myeloma cells Sp2/0 are cultured to the logarithmic phase, two large bottles are taken to prepare cell suspension, the cell suspension is centrifuged, the supernatant is discarded, the cell suspension is resuspended by using RPMI 1640 basic culture solution, and myeloma cells are obtained by repeating the steps for three times if needed, and the number of the myeloma cells is counted.
(4) Cell fusion and HAT selection hybridomas
Myeloma cells and immune splenocytes were mixed at a ratio of 1:10, washed 1 time with RPMI 1640 basal medium in a 50mL plastic centrifuge tube, and centrifuged at 1200rpm for 10 minutes. The supernatant was discarded, the cells were mixed well, 1mL of 50% PEG 1500 was slowly added for fusion, and after 1 minute of fusion, 15mL of RPMI 1640 basal medium was added to terminate the cell fusion. 1000rpm, and centrifuging for 5-10 minutes. The supernatant was discarded, and the culture broth was gently resuspended in 50mL of RPMI 1640, and plated in 10 96-well plates at 50. Mu.L/well at 37℃with 5% CO 2. Culturing was carried out until the sixth day, and the HAT medium (the complete medium of RPMI 1640 containing HAT) was changed twice.
(5) Hybridoma cell screening
HA antigen was diluted to 1. Mu.g/mL with 0.05M pH 9.6 carbonate buffer, added to 96-well plates at 37℃for 2 hours. 0.02M pH 7.2PBS,0.15mL/well containing 10% calf serum or 1% skimmed milk powder was blocked for 2 hours at 37℃for detection. On day 7 after fusion, 0.1mL of cell supernatant was taken into 96-well plates, 37℃for 30min, washed 6 times with water, and then secondary antibody was added for 30min at 37 ℃. After washing, 100. Mu.L of a phosphate buffer containing 0.1% (M/V) o-phenylenediamine, 0.1% (V/V) hydrogen peroxide and pH 5.0 was added to each well, a diluted sulfuric acid solution was added at 37℃for 15 minutes, and an absorption value of 450nm was measured.
The positive cell holes of the secreted antibody are cloned by a limiting dilution method on a 96-well culture plate with 1 cell/well, the positive holes are screened and cloned three times continuously according to the upper method, after the expansion culture, the positive cells are frozen in a culture solution containing 10% DMSO, and the cell density is 10 6 And each mL. Five stable HA-tag-resistant hybridoma cell lines, designated A, B, C, D, E, were obtained in total.
3. Preparation of monoclonal antibodies
Selecting BALB/c mice with 6-8 weeks of robustness, and injecting 0.5mL of pristane into the abdominal cavity of each mouse; intraperitoneal injection 1X 10 after 10 days 6 And a hybridoma cell. Ascites can be generated 7-10 days after inoculating cells, the health condition and the symptoms of the ascites of animals are closely observed, the mice are killed before death as much as possible, the ascites is sucked into a test tube by a dropper, and generally 5-10 mL of ascites can be obtained by one mouse. Collecting ascites, centrifuging to obtain supernatant, and storing in a refrigerator at-20deg.C. The ascites supernatant was diluted with 3 volumes of PBS and filtered through filter paper. The resulting filtrate was applied to a protein G affinity chromatography column equilibrated with PBS at a flow rate of 1 mL/min. The non-protein G adsorbed material was then washed with PBS at a flow rate of 1mL/min until the absorbance at OD280 nm reached baseline. The antibody was eluted with a 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 sub-packaged and frozen at-20 ℃.
4. Potency determination
The titers of the 5 hybridoma cells and the secreted ascites antibodies are detected by an indirect ELISA method, and the specific experimental steps are as follows:
(1) Coating: HA antigen was diluted to 1. Mu.g/mL, 100. Mu.L/Kong Jiazhi ELISA plate, 37℃for 2 hours or 4℃overnight;
(2) Washing the plate with a plate washing machine for 5 times, injecting 350 mu L of washing liquid into each hole, and staying for 20 seconds; finally, beating to dryness;
(3) Washing off the coating liquid with washing liquid, sealing with sealing liquid, placing 150 μl of each hole at 37deg.C for 1.5-2 hr;
(4) The plate washer washes the plate 5 times, and each time, each hole is filled with 350 mu L of washing liquid, and the plate stays for 20 seconds; finally, beating to dryness;
(5) Adding a sample: respectively adding cell culture supernatant and ascites diluted into different gradients into an ELISA plate coated with HA antigen, reacting at 37 ℃ for 1 hour (simultaneously making a negative control hole and a positive control hole) at 100 mu L/hole;
(6) The plate washing machine washes the plate 5 times, injects 350 mu L of washing liquid into each hole, stays for 20 seconds, and finally shoots;
(7) Adding horseradish peroxidase-labeled goat anti-mouse IgG enzyme-labeled secondary antibody (diluted 6000 times with blocking solution), and reacting at 37 ℃ for 1 hour at 100 mu L/hole;
(8) The plate washing machine washes the plate 5 times, injects 350 mu L of washing liquid into each hole, stays for 20 seconds, and finally shoots;
(9) Adding a color development liquid TMB: the preparation is ready to use, 100 mu L/hole and is reacted for 30 minutes at 37 ℃ in a dark place;
(10) Terminating the reaction: 2M sulfuric acid, 50. Mu.L/well, was added to each reaction well;
(11) Microplate reader reading: 450nm,630nm wavelength.
The results are shown in Table 1, and cell lines with higher titers were selected.
Table 15 hybridoma cells secrete ascites antibodies at a lower cost
The data in table 1 are the lowest concentration of the antibody solution that can be detected by the microplate reader, i.e., the maximum dilution factor available for the antibody solution. As is clear from Table 1, among the above 5 hybridoma cells, the ascites antibody secreted by hybridoma cell 6C6 was the highest, and its titer was 1.48×10 6 。
5. Antibody variable region gene cloning and sequencing
Total RNA is extracted from the hybridoma cell strain 6C6 secreting the HA monoclonal antibody, SMARTER II A Oligonucleotide and 5' -CDS primer in a SMARTERTM RACE cDNA Amplification Kit kit are used for first-strand cDNA synthesis, and the obtained first-strand cDNA product is used as a PCR amplification template. The light chain genes were amplified with Universal Primer AMix (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 amplifies about 0.7KB of the target band, and the primer pair of the heavy chain amplifies about 1.5KB of the target band. Purifying and recovering by agarose gel electrophoresis, adding A reaction of the product by rTaq DNA polymerase, inserting into pMD-18T vector, transforming into DH5 alpha competent cells, taking 4 clones of heavy chain and light chain gene clone respectively after bacterial colony growth, and sequencing by the company of the family Praeparatae.
The complementarity determining regions of the heavy chain were analyzed:
CDR1：S-Y-G-M-S
CDR2：Y-I-S-D-G-G-V-R-T-Y-Y-P-D-T-I-K-G
CDR3：H-R-G-Y-D-G-G-M-D-Y
complementarity determining regions of the light chain:
CDR1：R-A-S-A-S-V-I-A-M-H
CDR2：A-T-S-S-L-A-S
CDR3：Q-H-W-S-R-N-P-L-T
6. preparation of recombinant antibodies for affinity analysis and Activity characterization
The enzyme indirect method is used as data, and the coating is used as four gradients of 1 mug/ml, 0.5 mug/ml, 0.25 mug/ml and 0.125 mug/ml; antibodies were loaded from a 2-fold gradient dilution of 1000ng/ml to 0.97656 ng/ml. The OD values corresponding to the different antibody concentrations at the coating-free concentration were obtained. Under the same coating concentration, the antibody concentration is plotted with the abscissa, the OD value is plotted with the ordinate, the antibody concentration at 50% of the maximum OD value is calculated according to a fitting equation, and the reciprocal of the affinity constant is calculated by taking into the formula, wherein the specific formula is as follows:
K＝(n-1)/(2*(n*Ab`-Ab))；
wherein Ab and Ab ' respectively represent the antibody concentration at 50% of the maximum OD value at the corresponding coating concentration (Ag, ag '), 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 obtained, and the reciprocal is obtained to be the affinity constant KD, and the result is shown in the following table 2.
Table 25 affinity comparison of antibodies secreted by hybridoma cells
| Sample name | KD |
| 6C6 | 1.333E-8 |
| 2D9 | 6.245E-7 |
| 5A4 | 4.298E-6 |
| 3B6 | 9.328E-7 |
| 4E7 | 7.645E-6 |
As can be seen from Table 2, the affinity analysis data of the purified anti-HA monoclonal antibody shows significantly better affinity with the antibodies secreted by the other 4 hybridoma cells (the antibodies secreted by the cloned hybridoma cells 6C 6).
Activity assay
The HA antigen was diluted to 1. Mu.g/mL with 50mM carbonate buffer coating solution for microwell plate coating, 100. Mu.L per well, overnight at 4 ℃; the next day, the washing liquid is washed for 2 times by PBST, and is patted dry; blocking solution (20% BSA+80% PBS) was added, 120. Mu.L per well, 37℃for 1h, and the mixture was dried by shaking; adding diluted anti-HA monoclonal antibody 6C6, performing 5-fold specific dilution from 1000ng/ml, loading, 100 mu L/hole, and performing 30min (part of supernatant 1 h) at 37 ℃; washing with PBST washing solution for 5 times, and drying; adding horseradish peroxidase-labeled goat anti-mouse IgG (immunoglobulin G) into each hole at 100 mu L and 37 ℃ for 30min; washing with PBST washing solution for 5 times, and drying; urea peroxide (50 μl/well) was added, and tetramethylbenzidine (50 μl/well) was added for 10min; adding dilute hydrochloric acid to terminate the reaction, wherein the concentration is 50 mu L/hole; OD values were read on the microplate reader at 450nm (reference 620 nm) and the results are shown in the following table:
| sample concentration ng/ml | 1000 | 200 | 40 | 8 | 1.6 | 0.32 | 0 |
| OD value | 3.262 | 2.003 | 0.812 | 0.166 | 0.128 | 0.062 | 0.01 |
As is clear from the above table, the sample concentration was still detectable at low concentrations of 0.32ng/ml, indicating high activity.
It will be readily appreciated by those skilled in the art that the foregoing description is merely a preferred embodiment of the invention and is not intended to limit the invention, but any modifications, equivalents, improvements or alternatives falling within the spirit and principles of the invention are intended to be included within the scope of the invention.
Claims (9)
1. An anti-HA-tagged monoclonal antibody comprising 3 heavy chain complementarity determining regions: heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, and complementarity determining regions of 3 light chains: light chain CDR1, light chain CDR2, and light chain CDR3;
the heavy chain CDR1 is an amino acid sequence S-Y-G-M-S shown in SEQ ID NO. 1;
the heavy chain CDR2 is an amino acid sequence Y-I-S-D-G-G-V-R-T-Y-Y-P-D-T-I-K-G shown in SEQ ID NO. 2;
the heavy chain CDR3 is an amino acid sequence H-R-G-Y-D-G-M-D-Y shown in SEQ ID NO 3;
the light chain CDR1 is an amino acid sequence R-A-S-A-S-V-I-A-M-H shown in SEQ ID NO. 4;
the light chain CDR2 is an amino acid sequence A-T-S-S-L-A-S shown in SEQ ID NO. 5
The light chain CDR3 is an amino acid sequence Q-H-W-S-R-N-P-L-T shown in SEQ ID NO. 6.
2. The monoclonal antibody against the HA tag of claim 1, wherein the antibody HAs the following amino acid sequence from FR1 to 4 in the heavy chain variable region:
comprises or consists of the amino acid sequences shown as SEQ ID NO. 7,8,9 and 10,
or a sequence having at least 80%,85%,90% identity to the sequences shown in SEQ ID NO. 7,8,9, 10,
or a sequence having one or more amino acid mutations compared with the amino acid sequences shown in SEQ ID NO. 7,8,9, 10.
3. The monoclonal antibody against the HA tag of claim 2, wherein FR1-4 in the heavy chain variable region HAs the following amino acid sequence in sequence:
amino acid sequences shown in SEQ ID NO. 7,8,9 and 10,
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,
or an amino acid sequence having 1, 2, 3, 4, 5, 6, 7,8,9 or 10 amino acid mutations with 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. A monoclonal antibody against an HA tag according to any one of claims 1 to 3, wherein the antibody HAs the following amino acid sequence in the light chain variable region FR 1-4:
comprises or consists of the amino acid sequences shown as SEQ ID NO. 11, 12, 13, 14,
or a sequence having at least 80%,85%,90% identity to the 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 sequences shown in SEQ ID NOS.11, 12, 13, 14.
5. The monoclonal antibody against the HA tag of claim 4, wherein FR1-4 in the light chain variable region HAs the following amino acid sequence in sequence:
amino acid sequences shown in SEQ ID NO. 11, 12, 13 and 14,
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,
or an amino acid sequence having 1, 2, 3, 4, 5, 6, 7,8,9 or 10 amino acid mutations with the amino acid sequence shown in SEQ ID NOs 11, 12, 13, 14; the amino acid mutation is a conservative mutation, including substitution, insertion or deletion.
6. The monoclonal antibody against the HA tag according to claim 5, wherein 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 comprising a nucleic acid sequence encoding the anti-HA-tagged monoclonal antibody of claim 6, wherein the biological material comprises an expression vector, an expression cassette, a host cell, an engineered bacterium, or a hybridoma cell line.
8. Use of the anti-HA-tagged monoclonal antibody of claim 6 for detecting, isolating and purifying HA-tagged target proteins.
9. A test kit comprising the monoclonal antibody of claim 6 directed against an HA tag.
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