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CN116554338A - His tag-resistant monoclonal antibody and application thereof - Google Patents

His tag-resistant monoclonal antibody and application thereof Download PDF

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CN116554338A
CN116554338A CN202310182881.2A CN202310182881A CN116554338A CN 116554338 A CN116554338 A CN 116554338A CN 202310182881 A CN202310182881 A CN 202310182881A CN 116554338 A CN116554338 A CN 116554338A
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heavy chain
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acid sequence
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CN116554338B (en
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徐超
熊潇然
潘炜松
吴川
陈刚
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Hunan Nuohe New Biotechnology Co ltd
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    • 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

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Abstract

The invention discloses an anti-His tag monoclonal antibody and application thereof, wherein the antibody comprises a heavy chain of a heavy chain CDR1 or a variant thereof, a heavy chain of a heavy chain CDR2 or a variant thereof, and a heavy chain of a heavy chain CDR3 or a variant thereof; and a light chain of light chain CDR1 or variant thereof, a light chain of light chain CDR2 or variant thereof, and a light chain of light chain CDR3 or variant thereof; the heavy chain CDR1 comprises or consists of an amino acid sequence shown in SEQ ID NO. 1; the heavy chain CDR2 comprises or consists of an amino acid sequence shown in SEQ ID NO. 2; the heavy chain CDR3 comprises or consists of an amino acid sequence shown in SEQ ID NO. 3; the light chain CDR1 comprises or consists of an amino acid sequence shown in SEQ ID NO. 4; the light chain CDR2 comprises or consists of an amino acid sequence shown in SEQ ID NO. 5; the light chain CDR3 comprises or consists of a sequence shown in SEQ ID NO. 6. Its antibody titer can be up to 7.59X10 6 Affinity with His tag protein and high activity, and in fusion proteinHas wide application prospect in immunoadsorption separation and purification of the cytoplasm.

Description

His tag-resistant monoclonal antibody and application thereof
Technical Field
The invention belongs to the field of molecular immunology, and in particular relates to an anti-His tag monoclonal antibody and application thereof.
Background
His Tag is a Tag frequently used in protein recombination technology and consists of 6-10 consecutive histidine residues. The most commonly used His tag is a short peptide consisting of 6 histidines (His-His-His-His-His or HHHHHH), which is characterized by a small molecular weight of 0.8kDa, and does not substantially alter the biological structure of the protein after fusion to the recombinant protein, nor alter the solubility of the protein. Since His tag can be bound to various metal ions (such as Ca 2+ 、Mg 2+ 、Ni 2+ 、Cu 2+ ，Fe 2+ Etc.), the characteristic of the protein surface can be utilized to make the protein adsorbed on a gel column, thereby achieving the purpose of separating the protein, while the His-tagged monoclonal antibody can detect or purify His-tagged target protein, and the protein-specific antibody or probe is not required to be utilized, and is commonly used for detecting and purifying recombinant protein.
However, monoclonal antibodies against His tag proteins are currently commercially available in a small number of classes and are very expensive. Therefore, a novel anti-His tag monoclonal antibody is developed, and has wide application prospect in immunoadsorption, separation and purification of fusion protein.
Disclosure of Invention
In order to overcome the above defects or improvements in the prior art, the present invention provides an anti-His tag monoclonal antibody and its application, and aims to find an anti-His tag monoclonal antibody with higher titer, wherein the antibody titer can reach 7.59X10 6 The anti-His tag monoclonal antibody is prepared by purification and recombination, so that the technical problems of few varieties and high price of the existing anti-His tag monoclonal antibody are solved.
To achieve the above object, according to one aspect of the present invention, there is provided an anti-His tag monoclonal antibody comprising a heavy chain of a heavy chain CDR1 or variant thereof, a heavy chain of a heavy chain CDR2 or variant thereof, and a heavy chain of a heavy chain CDR3 or variant thereof;
and a light chain of light chain CDR1 or variant thereof, a light chain of light chain CDR2 or variant thereof, and a light chain of light chain CDR3 or variant thereof;
the heavy chain CDR1 comprises or consists of an amino acid sequence G-Y-K-F-T-D-Y-W-I shown in SEQ ID NO. 1; the heavy chain CDR2 comprises or consists of an amino acid sequence I-L-C-G-Y-E-T-T shown in SEQ ID NO. 2; the heavy chain CDR3 comprises or consists of an amino acid sequence A-R-D-G-K-H-A-M-D-Y shown in SEQ ID NO 3;
the light chain CDR1 comprises or consists of an amino acid sequence Q-D-V-L-N-S-G-H-Q-K-N-Y shown in SEQ ID NO. 4; the light chain CDR2 comprises or consists of an amino acid sequence W-A shown in SEQ ID NO. 5; the light chain CDR3 comprises or consists of a sequence Q-N-D-H-R-Y-P-L-T shown in SEQ ID NO. 6.
Preferably, the anti-His tag monoclonal antibody, the heavy chain of the variant thereof, the heavy chain variable region thereof comprises the amino acid sequence shown in SEQ ID NOs 7,8,9, 10, or a sequence having at least 80%,85%,90% identity to the sequence shown in SEQ ID NOs 7,8,9, 10, or a sequence having one or more amino acid mutations compared to the sequence shown in SEQ ID NOs 7,8,9, 10.
Preferably, the anti-His tag monoclonal antibody, the light chain of the variant thereof, the light chain variable region thereof comprises the amino acid sequence shown in SEQ ID nos. 11, 12, 13, 14, or a sequence having at least 80%,85%,90% identity to the sequence shown in SEQ ID nos. 11, 12, 13, 14, or a sequence having one or more amino acid mutations compared to the amino acid sequence shown in SEQ ID nos. 11, 12, 13, 14.
Preferably, the anti-His tag monoclonal antibody, the heavy chain of the variant thereof, the heavy chain variable region thereof comprises the amino acid sequence shown in SEQ ID No. 7,8,9, 10, or a sequence having at least 91%,92%,93%,94%,95%,96%,97%,98%,99% or 100% identity to the sequence shown in SEQ ID No. 7,8,9, 10, or a sequence having 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 or 10, said amino acid mutations being conservative mutations, including substitutions, insertions or deletions of amino acids.
Preferably, the anti-His tag monoclonal antibody, the light chain of the variant thereof, the light chain variable region thereof comprises the amino acid sequence shown in SEQ ID No. 11, 12, 13, 14, or a sequence having at least 91%,92%,93%,94%,95%,96%,97%,98%,99% or 100% identity to the sequence shown in SEQ ID No. 11, 12, 13, 14, or 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, said amino acid mutations being conservative mutations, including substitutions, insertions or deletions of amino acids.
Preferably, the heavy chain amino acid sequence of the anti-His tag monoclonal antibody is shown as SEQ ID NO. 15, and the light chain amino acid sequence of the 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-His tag monoclonal antibody or a fragment thereof according to the present invention, including genes, cDNA molecules, mRNA molecules and fragments thereof.
According to another aspect of the present invention, there is also provided an expression vector of an anti-His tag monoclonal antibody, which expresses a nucleic acid sequence encoding the anti-His tag monoclonal antibody or an antibody fragment thereof according to the present invention, comprising an expression cassette, a host cell, an engineering bacterium or a hybridoma cell line.
According to another aspect of the invention, there is also provided the use of an anti-His tag monoclonal antibody according to the invention for the preparation of a reagent for detecting, purifying or enriching His-tagged proteins.
According to another aspect of the invention, there is also provided a detection kit comprising an anti-His tag monoclonal antibody according to the invention.
In general, compared with the prior art, the above technical solution contemplated by the present invention can achieve the following beneficial effects due to providing an anti-His tag monoclonal antibody:
the anti-His tag monoclonal antibody with higher titer is obtained through a mouse antigen immunization experiment, and is prepared through gene detection and cloning, the KD value of the anti-His tag monoclonal antibody is 5.73E-7, and the OD value can reach 2.94 when the sample concentration is 1000ng/ml, so that the anti-His tag monoclonal antibody has high affinity and activity with His tag protein, and has wide application prospect in immunoadsorption separation purification of fusion protein.
Drawings
FIG. 1 is a schematic representation of the heavy chain amino acid sequence of an anti-His tag monoclonal antibody;
FIG. 2 is a schematic representation of the amino acid sequence of the light chain of an anti-His tag 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 term "amino acid" refers to a naturally occurring or non-naturally occurring carboxy alpha-amino acid. The term "amino acid" as used herein may include 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, homocysteine, homoleucine, homoarginine, hydroxyproline, norleucine, pyridylalanine, sarcosine, and the like.
In the present invention, peptides, polypeptides, proteins are not strictly distinguished and may in some cases be used interchangeably, generally referring to polymers composed 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 component may be added by the cell expressing the polypeptide and may vary with cell type. A 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, but may be present. All polypeptide sequences are written according to commonly accepted practices, with the α -N-terminal amino acid residue on the left and the α -C-terminal amino acid residue on the right. The term "N-terminus" as used herein refers to the free alpha-amino group of an amino acid in a polypeptide, and the term "C-terminus" refers to the free alpha-carboxylic acid end of an amino acid in a polypeptide. A polypeptide ending 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 refers to an amino acid bearing a group on the alpha-amino nitrogen.
The terms "nucleic acid" and "polynucleotide" are synonymous and include genes, cDNA molecules, mRNA molecules and fragments thereof, e.g., oligonucleotides. 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 invention provides an anti-His tag monoclonal antibody, which comprises a heavy chain of a heavy chain CDR1 or a variant thereof, a heavy chain of a heavy chain CDR2 or a variant thereof, and a heavy chain of a heavy chain CDR3 or a variant thereof;
and a light chain of light chain CDR1 or variant thereof, a light chain of light chain CDR2 or variant thereof, and a light chain of light chain CDR3 or variant thereof;
the heavy chain CDR1 comprises or consists of an amino acid sequence G-Y-K-F-T-D-Y-W-I shown in SEQ ID NO. 1; the heavy chain CDR2 comprises or consists of an amino acid sequence I-L-C-G-Y-E-T-T shown in SEQ ID NO. 2; the heavy chain CDR3 comprises or consists of an amino acid sequence A-R-D-G-K-H-A-M-D-Y shown in SEQ ID NO 3;
the light chain CDR1 comprises or consists of an amino acid sequence Q-D-V-L-N-S-G-H-Q-K-N-Y shown in SEQ ID NO. 4; the light chain CDR2 comprises or consists of an amino acid sequence W-A shown in SEQ ID NO. 5; the light chain CDR3 comprises or consists of the sequence Q-N-D-H-R-Y-P-L-T shown in SEQ ID NO. 6.
The heavy chain, heavy chain variable region of the variant comprises the amino acid sequence shown in SEQ ID No. 7,8,9, 10 or a sequence having at least 80%,85%,90% identity, 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 an amino acid sequence having one or more amino acid mutations, preferably conservative mutations, preferably substitutions, insertions or deletions, compared to the amino acid sequence shown in SEQ ID NO. 7,8,9, 10; more preferably 1, 2, 3, 4, 5, 6, 7,8,9 or 10 amino acid mutations.
The light chain, light chain variable region of the variant comprises the amino acid sequence shown in SEQ ID nos. 11, 12, 13, 14 or a sequence having at least 80%,85%,90% identity, preferably at least 91%,92%,93%,94%,95%,96%,97%,98%,99% or 100% sequence identity to the sequence shown in SEQ ID nos. 11, 12, 13, 14; or an amino acid sequence having one or more amino acid mutations, preferably conservative mutations, preferably substitutions, insertions or deletions, compared to the amino acid sequence shown in SEQ ID NO. 11, 12, 13, 14; more preferably 1, 2, 3, 4, 5, 6, 7,8,9 or 10 amino acid mutations.
More preferably, the heavy chain amino acid sequence of the anti-His tag monoclonal antibody is shown as SEQ ID NO. 15, and the light chain amino acid sequence is shown as SEQ ID NO. 16.
In some embodiments, the invention also includes nucleic acid sequences comprising antibodies or antibody fragments thereof encoding anti-6 XHis tags as described herein. In this context, a nucleic acid sequence comprises variants of its conservative substitutions (e.g., substitutions of degenerate codons) and the complement. 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 includes expression vectors comprising expression cassettes, host cells, engineered bacteria or hybridoma cell lines that express nucleic acid sequences encoding antibodies or antibody fragments thereof against the 6XHis tag of the invention.
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 anti-His tag monoclonal antibody in preparation of a reagent for detecting, purifying or enriching His-tagged proteins.
The invention also provides a detection kit comprising the anti-His tag monoclonal antibody.
The following are examples:
EXAMPLE 1 screening of anti-His tag monoclonal antibodies
1-1 antigen immunization
The 6XHIS antigen (hexaamino acid short peptide HHHHH-coupled OVA) was mixed with Freund's complete adjuvant (appearance: amber cell suspension; composition: 85% paraffin oil, 15% mannitol monooleate, 1mg/mL Mycobacterium tuberculosis) to give an oily emulsion. The emulsion is subcutaneously injected into a BALB/c mouse at a dosage of 0.15mL, the immunity is enhanced in the abdominal cavity (equal amount of antigen is mixed with Freund's incomplete adjuvant) after 14 days of the first immunization, and after the immunity is enhanced to four needles, tail blood is collected for potency detection, and the potency meets the fusion requirement.
3 days before fusion, the same dose of antigen is used for intraperitoneal injection to boost immunity, and the immunization method is the same.
Preparation of 1-2 hybridoma cell lines
(1) Preparation of feeder cells
BALB/c murine peritoneal macrophages were used as feeder cells. 1 day before fusion, BALB/c mice were sacrificed by pulling the neck, immersed in 75% alcohol whole body, and in an ultra clean bench withoutCutting abdominal skin with scissors under fungus operation, exposing peritoneum, injecting 5mL of RPMI1640 basic culture solution into abdominal cavity with syringe, repeatedly washing, recovering washing solution, centrifuging at 1000rpm for 5 min, collecting precipitate, re-suspending with RPMI1640 screening culture solution (containing HAT in RPMI1640 complete culture solution), and adjusting cell 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
Three days after the last immunization of the mice, the spleens were taken out under aseptic conditions, placed in a plate, rinsed once with RPMI1640 basal culture solution, placed on a nylon mesh of a small beaker, and ground and filtered to prepare a cell suspension. Centrifuging, discarding supernatant, re-suspending RPMI1640 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 RPMI1640 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 RPMI1640 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 RPMI1640 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 RPMI1640, halved in 10 96-well plates, 50. Mu.L/well, 37℃and 5% CO 2 Culturing. Culturing was carried out until the sixth day, and the HAT medium (the complete medium of RPMI1640 containing HAT) was changed twice.
(5) Screening of anti-6 XHIS tag hybridoma cells
The 6XHIS antigen (HHHHH coupled BSA) was diluted with 0.05M pH 9.6 carbonate buffer solution to a final concentration of 1. Mu.g/mL. 0.1mL per well, 96 well polystyrene plates were added at 37℃for 2 hours or at 4℃overnight. The following day, 0.02M pH 7.2PBS,0.15mL/well containing 10% calf serum or 1% skim milk powder was blocked at 37 ℃ for 2 hours for detection. On the seventh day after fusion, 0.1mL of cell supernatant was taken out in the 96-well assay plate, washed with water for 30 minutes at 37℃for six times, then 2000-fold diluted horseradish peroxidase-labeled goat anti-mouse IgG was added, and after 30 minutes at 37℃and the same wash, 100. Mu.L of a phosphate buffer containing 0.1% (M/V) o-phenylenediamine, 0.1% (V/V) hydrogen peroxide, pH 5.0 citric acid phosphate buffer solution, 15 minutes at 37℃was added, 50. Mu.L of a diluted sulfuric acid solution was added per well, and the absorption value at 450nm was measured. RPMI1640 complete culture solution is used as a negative control, and the ratio of the measured value to the control value is more than or equal to 2.0, which is used as a positive cell hole.
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 6 XHis-resistant tag hybridoma cell lines were obtained in total, and were designated as A, B, C, D, E.
1-3 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 ℃.
EXAMPLE 2 evaluation of antibody titers and cloning of recombinant antibodies
(1) Antibody titer determination
The titers of the 5 hybridoma cells obtained in example 1 and the secreted ascites antibodies were measured by an indirect ELISA method, and the specific experimental procedures were as follows:
(1) Coating: 6XHIS antigen (HHHHH coupled BSA) 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 6XHIS 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 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;
(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 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;
(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 measurement results are shown in table 1 below.
Table 1 antibody titer determination
6XHIS tag cell strain Hybridoma cell culture supernatant titers Ascites antibody titre
4F9 2.38×10 3 3.75×10 5
6B2 2.66×10 3 4.28×10 5
3G7 4.88×10 4 7.59×10 6
3E10 3.26×10 3 4.01×10 5
2D6 1.79×10 3 2.66×10 5
As can be seen from Table 1, the anti-His tag monoclonal antibody produced by cell line 3G7 was selected to have the highest titer.
(2) Cloning
Cloning and sequencing of the variable region gene of the anti-His tag monoclonal antibody generated by the cell strain 3G7, specifically comprises the following steps:
total RNA was extracted from hybridoma cell line 3G7 secreting 6XHIS monoclonal antibody using SMART TM RACE cDNAAmplification Kit kitFirst-strand cDNA synthesis is carried out by SMARTER II AOligonucleoteide and 5' -CDS primer in the kit, and the obtained first-strand cDNA product is used as a PCR amplification template. The Light Chain gene was amplified with Universal Primer AMix (UPM), nested Universal Primer A (NUP) and mIgG CKR primers and the Heavy Chain gene was amplified with Universal Primer AMix (UPM), nested Universal Primer A (NUP) and mIgG CHR primers. Wherein the primer pair of Light Chain amplifies about 0.7KB of the target band, and the primer pair of 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, and respectively taking the Heavy Chain and Light Chain gene clone of 4 clones of the Heavy Chain and Light Chain gene clone for sequencing by the engine company after colony growth.
The antibody obtained by the hybridoma cell strain 3G7 has the sequences such as light chain and heavy chain sequences attached later.
The complementarity determining regions of the heavy chain were analyzed:
CDR1：G-Y-K-F-T-D-Y-W-I；
CDR2：I-L-C-G-Y-E-T-T；
CDR3：A-R-D-G-K-H-A-M-D-Y。
complementarity determining regions of the light chain:
CDR1：Q-D-V-L-N-S-G-H-Q-K-N-Y；
CDR2：W-A；
CDR3：Q-N-D-H-R-Y-P-L-T。
(3) Preparation of recombinant antibodies and Activity characterization
Enzyme-free indirect method was used as data in the same manner as for activity identification, and the coating was used as four gradients of 1. Mu.g/ml, 0.5. Mu.g/ml, 0.25. Mu.g/ml, 0.125. Mu.g/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 antibody concentration as an abscissa and the OD value as an ordinate, and the antibody concentration at the maximum OD value of 50% is calculated according to a fitting equation; the formula is introduced: k= (n-1)/(2 x (n x Ab '-Ab)), where Ab and Ab' represent the antibody concentration at 50% of the 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, finally six K values can be obtained, the average value is obtained, and the reciprocal is obtained as an affinity constant KD, and the result is shown in Table 2.
TABLE 2 affinity constant KD determination results
As can be seen from table 2, the affinity analysis data of the purified anti-6 XHis monoclonal antibody showed significantly better affinity than the affinity control of the antibodies secreted by the other 4 hybridoma cells.
Activity identification:
microwell plate coating was performed by diluting 6XHis antigen (hhhhhhh conjugated BSA) to 1 μg/mL with 50mM carbonate buffer coating solution, 100 μl 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 the diluted anti-6 XHIS monoclonal antibody 3G7, performing 5-fold specific dilution from 1000ng/ml, loading the sample, 100 mu L/well, and performing at 37 ℃ for 30min (part of supernatant for 1 h); 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 at 450nm (reference 620 nm) on a microplate reader and the results are shown in Table 3.
TABLE 3 identification of 3G7 Activity of purified anti-6 XHIS Label mAb
Sample concentration ng/ml 1000 200 40 8 1.6 0.32 0
OD value 2.941 2.003 0.926 0.162 0.107 0.061 0.021
Example 2 amino acid sequence of anti-His tag monoclonal antibody
The amino acid sequence of the anti-His tag monoclonal antibody is shown in the figure 1 (SEQ ID NO: 15), and the amino acid sequence of the heavy chain is shown in the figure 2 (SEQ ID NO: 16);
wherein the heavy chain CDR1 is composed of an amino acid sequence G-Y-K-F-T-D-Y-W-I shown in SEQ ID NO. 1; heavy chain CDR2, by SEQ ID NO 2 shows the amino acid sequence I-L-C-G-Y-E-T-T composition; heavy chain CDR3, by SEQ ID NO 3 shows the amino acid sequence A-R-D-G-K-H-A-M-D-Y composition;
light chain CDR1, is by SEQ ID NO 4 shown in the amino acid sequence Q-D-V-L-N-S-G-H-Q-K-N-Y composition; light chain CDR2, consisting of the amino acid sequence W-A shown in SEQ ID NO. 5; light chain CDR3, by SEQ ID NO. 6 shows the amino acid sequence Q-N-D-H-R-Y-P-L-T composition.
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 (10)

1. An anti-His tag monoclonal antibody comprising a heavy chain of a heavy chain CDR1 or variant thereof, a heavy chain of a heavy chain CDR2 or variant thereof, and a heavy chain of a heavy chain CDR3 or variant thereof;
and a light chain of light chain CDR1 or variant thereof, a light chain of light chain CDR2 or variant thereof, and a light chain of light chain CDR3 or variant thereof;
the heavy chain CDR1 comprises or consists of an amino acid sequence G-Y-K-F-T-D-Y-W-I shown in SEQ ID NO. 1; the heavy chain CDR2 comprises or consists of an amino acid sequence I-L-C-G-Y-E-T-T shown in SEQ ID NO. 2; the heavy chain CDR3 comprises or consists of an amino acid sequence A-R-D-G-K-H-A-M-D-Y shown in SEQ ID NO 3;
the light chain CDR1 comprises or consists of an amino acid sequence Q-D-V-L-N-S-G-H-Q-K-N-Y shown in SEQ ID NO. 4; the light chain CDR2 comprises or consists of an amino acid sequence W-A shown in SEQ ID NO. 5; the light chain CDR3 comprises or consists of a sequence Q-N-D-H-R-Y-P-L-T shown in SEQ ID NO. 6.
2. The anti-His tag monoclonal antibody of claim 1, wherein the heavy chain of the variant comprises the amino acid sequence set forth in SEQ ID NOs 7,8,9, 10, or a sequence having at least 80%,85%,90% identity to the sequence set forth in SEQ ID NOs 7,8,9, 10, or a sequence having one or more amino acid mutations compared to the sequence set forth in SEQ ID NOs 7,8,9, 10.
3. The anti-His tag monoclonal antibody of claim 1 or 2, wherein the variant light chain comprises the amino acid sequence set forth in SEQ ID NOs 11, 12, 13, 14, or a sequence having at least 80%,85%,90% identity to the sequence set forth in SEQ ID NOs 11, 12, 13, 14, or a sequence having one or more amino acid mutations compared to the amino acid sequence set forth in SEQ ID NOs 11, 12, 13, 14.
4. The anti-His tag monoclonal antibody of claim 2, wherein the heavy chain variable region of the variant comprises the amino acid sequence set forth in SEQ ID No. 7,8,9, 10, or a sequence having at least 91%,92%,93%,94%,95%,96%,97%,98%,99% or 100% identity to the sequence set forth in SEQ ID No. 7,8,9, 10, or a sequence having 1, 2, 3, 4, 5, 6, 7,8,9 or 10 amino acid mutations, including substitutions, insertions or deletions, as compared to the sequence set forth in SEQ ID No. 7,8,9 or 10.
5. The anti-His tag monoclonal antibody of claim 3, wherein the variant has a light chain variable region comprising the amino acid sequence set forth in SEQ ID No. 11, 12, 13, 14, or a sequence having at least 91%,92%,93%,94%,95%,96%,97%,98%,99% or 100% identity to the sequence set forth in SEQ ID No. 11, 92%, 94%,95%, 97%,98%,99% or 100% identity to the sequence set forth in SEQ ID No. 11, 12, 13, 14, or a sequence having a mutation of 1, 2, 3, 4, 5, 6, 7,8,9 or 10 amino acids, said mutation being a conservative mutation, including a substitution, insertion or deletion.
6. The anti-His tag monoclonal antibody of any one of claims 1-5, 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 nucleic acid encoding an anti-His tag monoclonal antibody or antibody fragment thereof according to any one of claims 1 to 6, comprising a gene, a cDNA molecule, an mRNA molecule, and fragments thereof.
8. An expression vector of an anti-His tag monoclonal antibody, wherein a nucleic acid sequence encoding the anti-His tag monoclonal antibody or antibody fragment thereof of any one of claims 1 to 6 is expressed, comprising an expression cassette, a host cell, an engineered bacterium, or a hybridoma cell line.
9. Use of an anti-His-tag monoclonal antibody of any one of claims 1-6 for the preparation of a reagent for detecting, purifying or enriching a His-tagged protein.
10. A test kit comprising an anti-His tag monoclonal antibody according to any one of claims 1 to 6.
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