A v5 tag antibody and use thereof

Abstract

The present application relates to a kind of V5 label antibodies and its application, the antibody includes heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3, and light chain CDR1, light chain CDR2 and light chain CDR3;The amino acid sequence of the heavy chain CDR1 is as shown in SEQ ID NO:1;The amino acid sequence of heavy chain CDR2 is as shown in SEQ ID NO:2 sequence;The amino acid sequence of heavy chain CDR3 is as shown in SEQ ID NO:3 sequence;The amino acid sequence of light chain CDR1 is as shown in SEQ ID NO:4 sequence;The amino acid sequence of light chain CDR2 is as shown in SEQ ID NO:5;The amino acid sequence of light chain CDR3 is as shown in SEQ ID NO:6;Its ascites antibody titer can reach 2.07×10 6 , after purification antibody affinity reaches 1.425E-8, antibody activity is high, with great application potential.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more specifically to a V5 tag antibody and applications thereof. Background Art

[0002] Epitope tagging is a method of recombining proteins through genetic modification during the cloning process, allowing for detection and / or purification of expressed proteins. Epitope tags can localize gene products in a variety of cell types, investigate the topology of proteins and their complexes, and identify related proteins. They are particularly useful for characterizing newly identified, low-abundance, or poorly immunogenic proteins in the absence of protein-specific antibodies. Epitope tagging is typically performed by conjugating epitope tags to the C- or N-terminus of recombinant proteins. The V5 tag is derived from a small epitope (Pk) on the P and V proteins of the paramyxovirus simian virus 5 (SV5). The commonly used V5 tag contains all 14 amino acids (GKPIPNPLLGLDST), but a shorter 9-amino acid sequence (IPNPLLGLD) can also be used.

[0003] V5 tag antibodies are antibodies used to detect and purify V5-tagged proteins. They possess advantages such as high specificity, recognizing the V5 epitope at the C-terminus or N-terminus of recombinant proteins without being affected by the surrounding amino acid sequence. They are effective tools for detecting recombinant proteins containing the V5 epitope. They are commonly used to detect and purify target proteins carrying the V5 tag, especially when specific antibodies or other probes are unavailable. V5 tag antibodies are suitable for a variety of applications, such as enzyme-linked immunosorbent assays (ELISAs), western blotting (WBs), flow cytometry, immunoprecipitation (IP), and immunocytochemistry / immunofluorescence (ICC / IF).

[0004] While V5-tag antibodies offer advantages such as high specificity and versatility, providing researchers with effective tools for exploring protein function and interactions, the currently available V5-tag monoclonal antibodies are limited and expensive. Therefore, the development of novel V5-tag monoclonal antibodies has enormous potential for application, particularly in the immunoadsorption and purification of recombinant proteins, potentially expanding their broad application prospects. Summary of the Invention

[0005] In response to the above-mentioned defects or improvement needs of the prior art, the present invention provides a V5 tag antibody and its application, the purpose of which is to use a synthetic peptide of amino acid residues GKPIPNPLLGLDST (95-108) on the P and V proteins of simian virus 5 (SV5) paramyxovirus coupled with KLH as an antigen, immunostimulate mice to produce antibodies, obtain immune spleen cells from the mice and fuse them with SP2 / 0-Ag14 myeloma, and screen and obtain an antibody with the highest titer (1.96×10 4 ) hybridoma cell line, and use it to prepare V5-tagged monoclonal antibodies. By cloning and sequencing the variable region gene of the antibody, the amino acid sequences of the heavy and light chains of the antibody are obtained, and based on this, V5-tagged recombinant antibodies can be prepared, thereby solving the technical problems of the limited variety and high price of existing V5-tagged antibodies.

[0006] To achieve the above objectives, according to one aspect of the present invention, a V5-tagged antibody is provided, comprising three heavy chain complementarity determining regions: heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, and three light chain complementarity determining regions: light chain CDR1, light chain CDR2, and light chain CDR3;

[0007] The heavy chain CDR1 is the amino acid sequence SYSIH shown in SEQ ID NO: 1;

[0008] The heavy chain CDR2 is the amino acid sequence YINPASGYSAYNENFKB shown in SEQ ID NO: 2;

[0009] The heavy chain CDR3 is the amino acid sequence DKFYAYDY shown in SEQ ID NO: 3;

[0010] The light chain CDR1 is the amino acid sequence RASQSIVHKNGNTYLD shown in SEQ ID NO: 4;

[0011] The light chain CDR2 is the amino acid sequence RVAQKFS shown in SEQ ID NO: 5;

[0012] The light chain CDR3 is the sequence FNASHVPYT shown in SEQ ID NO:6.

[0013] Preferably, the V5-tagged antibody, wherein FR1-4 in the heavy chain variable region of the antibody is the following amino acid sequence:

[0014] comprising or consisting of the amino acid sequence shown in SEQ ID NO: 7, 8, 9, 10,

[0015] or a sequence having at least 80%, 85%, or 90% identity to the sequence shown in SEQ ID NO: 7, 8, 9, or 10,

[0016] Or a sequence having one or more amino acid mutations compared to the amino acid sequence shown in SEQ ID NO: 7, 8, 9, or 10.

[0017] Preferably, in the V5-tagged antibody, FR1-4 in the heavy chain variable region are the following amino acid sequences:

[0018] The amino acid sequences shown in SEQ ID NOs: 7, 8, 9, and 10,

[0019] 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,

[0020] Or an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid mutations compared to the amino acid sequence shown in SEQ ID NO: 7, 8, 9, 10; the amino acid mutation is a conservative mutation, including substitution, insertion or deletion.

[0021] Preferably, the V5-tagged antibody, wherein FR1-4 in the light chain variable region of the antibody is the following amino acid sequence:

[0022] comprising or consisting of the amino acid sequence shown in SEQ ID NO: 11, 12, 13, 14,

[0023] or a sequence having at least 80%, 85%, or 90% identity to the sequence shown in SEQ ID NO: 11, 12, 13, or 14,

[0024] Or a sequence having one or more amino acid mutations compared to the amino acid sequence shown in SEQ ID NO: 11, 12, 13, or 14.

[0025] Preferably, in the V5-tagged antibody, FR1-4 in the light chain variable region are the following amino acid sequences:

[0026] The amino acid sequences shown in SEQ ID NOs: 11, 12, 13, and 14,

[0027] 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,

[0028] Or an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid mutations compared to the amino acid sequence shown in SEQ ID NO: 11, 12, 13, 14; the amino acid mutation is a conservative mutation, including substitution, insertion or deletion.

[0029] Preferably, the V5 tag antibody, the antibody, its heavy chain amino acid sequence is shown as SEQ ID NO: 15, and the light chain amino acid sequence is shown as SEQ ID NO: 16.

[0030] According to another aspect of the present invention, a biological material containing a nucleic acid sequence encoding the V5 tag antibody of the present invention is also provided. The biological material includes an expression vector, an expression cassette, a host cell, an engineered bacterium or a hybridoma cell line.

[0031] According to another aspect of the present invention, there is also provided a use of the V5 tag antibody of the present invention in detecting, separating and purifying a V5-tagged target protein.

[0032] Preferably, the use is applied to enzyme-linked immunosorbent assay, immunoblotting, flow cytometry, immunoprecipitation, and / or immunocytochemistry / immunofluorescence.

[0033] According to another aspect of the present invention, a target protein detection kit is provided, which comprises the V5 tag antibody according to the present invention.

[0034] In general, the above technical solutions conceived by the present invention can achieve the following beneficial effects compared with the prior art:

[0035] The present invention uses a synthetic peptide of amino acid residues GKPIPNPLLGLDST (95-108) on the P and V proteins of simian virus 5 (SV5) paramyxovirus coupled with KLH as an antigen to stimulate mice to produce antibodies, obtain immune spleen cells, and prepare and screen high-titer V5 tag monoclonal antibodies using hybridoma technology. Experimental results show that the titer of the screened V5 tag monoclonal antibodies is as high as 2.07×10 6 (ascites antibody), affinity K D The activity is 1.425E-8 and is high, and can be used to detect, separate and purify target proteins labeled with V5 tags, especially for detecting target proteins without specific antibodies or other probes. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 are the heavy chain and light chain amino acid sequences of the V5 tag monoclonal antibody screened in Example 1;

[0037] Figure 2is the result of an activity assay of the V5 tag monoclonal antibody of Example 2. DETAILED DESCRIPTION

[0038] To further clarify the objects of the application, the technical solutions and effects adopted to achieve the objects of the present application are described below in detail with reference to the drawings and preferred embodiments.

[0039] The term "amino acid" refers to a naturally occurring or non-naturally occurring carboxyl alpha-amino acid. The term "amino acid" as used in the present application can 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 (Gin, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (lie, 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, homarginine, hydroxyproline, norleucine, pyridylalanine, sarcosine, and the like.

[0040] In the present application, peptides, polypeptides, and proteins are not strictly distinguished and in some instances can be used interchangeably, and generally refer to polymers of amino acids linked by peptide bonds, whether naturally produced or synthetic. Polypeptides can also contain non-amino acid components, such as carbohydrate groups, metal ions, or covalently attached carboxylate. Non-amino acid components can be added by the cell in which the polypeptide is expressed and can vary with the cell type. Polypeptides are defined in the present application with respect to their amino acid backbone structure or the nucleic acid encoding them. Addition of, for example, carbohydrate groups is typically not specified, but can be present. All polypeptide sequences are written in the commonly accepted convention with the alpha-N-terminal amino acid residue on the left and the alpha-C-terminal amino acid residue on the right. The term "N-terminal" when used in the present application refers to the free alpha-amino group of an amino acid in a polypeptide, and the term "C-terminal" refers to the free alpha-carboxylate end of an amino acid in a polypeptide. A polypeptide ending in a group at the N-terminus refers to a polypeptide carrying the group on the alpha-amino nitrogen of the N-terminal amino acid residue. An amino acid ending in a group at the N-terminus refers to an amino acid carrying the group on the alpha-amino nitrogen.

[0041] The present invention couples a synthetic peptide of amino acid residues GKPIPNPLLGLDST (95-108) from the P and V proteins of simian virus 5 (SV5) paramyxovirus with KLH as an antigen to immunostimulate mice to produce antibodies. Immune spleen cells from the mice are harvested and then fused with mouse myeloma cells SP2 / 0-Ag14 to obtain hybridoma cells. After cell culture and screening, multiple stable V5-tagged hybridoma cells are obtained. The selected hybridoma cell lines are injected into the peritoneal cavity of mice to screen for the ascites antibody (monoclonal antibody) with the highest titer. The antibody gene is detected, and its heavy chain amino acid sequence is shown in SEQ ID NO:15, and its light chain amino acid sequence is shown in SEQ ID NO:16. The complementarity determining regions of the heavy and light chains of the antibody are obtained, wherein the complementarity determining regions of the heavy chain of the antibody are as follows:

[0042] CDR1:SYSIH;

[0043] CDR2: YINPASGYSAYNENFKB;

[0044] CDR3: DKFYAYDY;

[0045] The complementarity determining regions of the light chain are as follows:

[0046] CDR1:RASQSIVHKNGNTYLD;

[0047] CDR2: RVAQKFS;

[0048] CDR3: FNASHVPYT;

[0049] V5-tagged recombinant antibodies were prepared according to the obtained antibody variable region gene sequence.

[0050] Based on this, the present invention provides a V5 tag antibody, which comprises VHCDR1, VHCDR2 and VHCDR3 with amino acid sequences as shown in SEQ ID NO:1-3, or consists of them; and comprises VLCDR1, VLCDR2 and VLCDR3 with amino acid sequences as shown in SEQ ID NO:4-6, or consists of them.

[0051] The amino acid sequence shown in SEQ ID NO: 1 is SYSIH, the amino acid sequence shown in SEQ ID NO: 2 is YINPASGYSAYNENFKB, and the amino acid sequence shown in SEQ ID NO: 3 is DKFYAYDY;

[0052] The amino acid sequence shown in SEQ ID NO:4 is RASQSIVHKNGNTYLD; the amino acid sequence shown in SEQ ID NO:5 is RVAQKFS; and the amino acid sequence shown in SEQ ID NO:6 is FNASHVPYT.

[0053] Preferably, in the antibody, FR1-4 in the heavy chain variable region are the following amino acid sequences:

[0054] comprising or consisting of the amino acid sequence shown in SEQ ID NO: 7, 8, 9, 10,

[0055] or a sequence having at least 80%, 85%, or 90% identity to the sequence shown in SEQ ID NO: 7, 8, 9, or 10;

[0056] or a sequence having one or more amino acid mutations compared to the amino acid sequence shown in SEQ ID NO: 7, 8, 9, or 10;

[0057] More preferably, in the antibody, FR1-4 in the heavy chain variable region are the following amino acid sequences:

[0058] The amino acid sequences shown in SEQ ID NOs: 7, 8, 9, and 10;

[0059] or a sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence shown in SEQ ID NO: 7, 8, 9, 10;

[0060] Or it has 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid mutations compared to the sequence shown in SEQ ID NO: 7, 8, 9, 10; the amino acid mutation is a conservative mutation, preferably a substitution, insertion or deletion.

[0061] Furthermore, in the antibody, FR1-4 in the light chain variable region are the following amino acid sequences:

[0062] comprising or consisting of the amino acid sequence shown in SEQ ID NO: 11, 12, 13, 14,

[0063] or a sequence having at least 80%, 85%, or 90% identity to the sequence shown in SEQ ID NO: 11, 12, 13, or 14; preferably a sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence shown in SEQ ID NO: 11, 12, 13, or 14;

[0064] Or a sequence having one or more amino acid mutations compared to the amino acid sequence shown in SEQ ID NO: 11, 12, 13, 14; preferably a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid mutations compared to the amino acid sequence shown in SEQ ID NO: 11, 12, 13, 14; the amino acid mutation is a conservative mutation, preferably a substitution, insertion or deletion.

[0065] In some embodiments, the V5 tag antibody has a heavy chain amino acid sequence as shown in SEQ ID NO: 15, and a light chain amino acid sequence as shown in SEQ ID NO: 16.

[0066] In some embodiments, the present invention also provides a nucleic acid sequence encoding a V5 tag antibody or antibody fragment thereof as described herein. In the present invention, the nucleic acid sequence includes conservatively substituted variants thereof (e.g., substitutions of degenerate codons) and complementary sequences. The terms "nucleic acid" and "polynucleotide" are synonymous and include genes, cDNA molecules, mRNA molecules, and fragments thereof, such as oligonucleotides.

[0067] In some embodiments, the present invention further provides a biological material containing a nucleic acid sequence encoding the V5 tag antibody of the present invention, wherein the biological material includes an expression vector, an expression cassette, a host cell, an engineered bacterium or a hybridoma cell line.

[0068] The nucleic acid sequence is operably linked to at least one regulatory sequence. "Operably linked" means that the coding sequence is linked to the regulatory sequence in a manner that allows expression of the coding sequence. Regulatory sequences are selected to direct expression of the desired protein in a suitable host cell and include promoters, enhancers, and other expression control elements.

[0069] In addition, the present invention also provides a use of the V5 tag antibody of the present invention in detecting, separating and purifying a target protein tagged with a V5 epitope.

[0070] The applications include enzyme linked immunosorbent assay (ELISA), Western blotting (WB), flow cytometry (Flow Cytometry), immunoprecipitation (IP) and immunocytochemistry / immunofluorescence (ICC / IF).

[0071] The present invention also provides a detection kit comprising the V5 tag antibody of the present invention.

[0072] The detection kit is used for detecting and / or purifying a target protein containing a V5 tag.

[0073] The following are examples

[0074] Example 1 Preparation and screening of V5-tagged monoclonal antibodies

[0075] (1) Antigen immunity

[0076] In this example, a synthetic peptide derived from amino acid residues GKPIPNPLLGLDST (95-108) from the P and V proteins of simian virus 5 (SV5) paramyxovirus was conjugated to KLH and injected subcutaneously in BALB / c mice at a dose of 0.15 mL. A booster dose was administered 14 days after the first immunization. After the fourth booster dose, tail blood was collected for titer determination, and the titer met the fusion requirement. Three days before fusion, another immunization was performed.

[0077] (2) Preparation of hybridoma cell lines

[0078] 1) Preparation of feeder cells

[0079] BALB / c mouse peritoneal macrophages were used as feeder cells. After killing the mouse, disinfecting the body surface, and fixing it, the abdomen was opened to expose the peritoneum. 5 mL of RPMI 1640 basal culture medium was injected. The abdomen was gently massaged for 1 minute, and the injected culture medium was then aspirated. The cells were centrifuged at 1000 rpm for 5 minutes, and the supernatant was removed. The cells were resuspended in RPMI 1640 culture medium containing HAT to adjust the concentration to 1×10 5 The cells were added into a 96-well cell culture plate at 150 μL / well and cultured overnight at 37°C in 5% CO2.

[0080] 2) Preparation of immune spleen cells

[0081] Three days after the last immunization, the spleen of the mouse was removed under sterile conditions, placed on a plate, washed with RPMI 1640 basal medium, and then ground and filtered through a nylon membrane to prepare a splenocyte suspension. After centrifugation, the supernatant was removed and the cells were resuspended in RPMI 1640 basal medium. This process was repeated three times to obtain immune splenocytes and count them.

[0082] 3) Preparation of myeloma cells

[0083] Mouse myeloma cells SP2 / 0-Ag14 were selected with 8-azaguanine and cultured to the logarithmic growth phase. Two large flasks were used to prepare a cell suspension. After centrifugation, the supernatant was removed and the cells were resuspended in RPMI 1640 basal medium. This was repeated three times to obtain myeloma cells, which were then counted.

[0084] 4) Cell fusion and HAT selection of hybridomas

[0085] Prepared myeloma cells were mixed with immune spleen cells at a ratio of 1:10, RPMI 1640 basal culture medium was added, centrifuged at 1200 rpm for 10 minutes, and the supernatant was removed. The cells were mixed well and fused by slowly adding 1 mL of 50% PEG 1500. After fusion for 1 minute, 15 mL of RPMI 1640 basal culture medium was added to terminate cell fusion. Centrifuged at 1000 rpm for 10 minutes and the supernatant was removed. Gently resuspended with 50 mL of RPMI 1640 culture medium, the cells were evenly plated in 10 96-well plates, 50 μL / well, and cultured at 37°C with 5% CO2. On the sixth day of culture, HAT culture medium (complete RPMI 1640 culture medium containing HAT) was replaced by aspirating 1 / 2 of the old culture medium in the culture well and replacing it with fresh medium twice.

[0086] 5) Hybridoma cell screening and cloning

[0087] V5 antigen was diluted to 1 μg / mL in 0.05M carbonate buffer (pH 9.6), added to a 96-well plate, and incubated at 37°C for 2 hours. Block the plate with 0.15 mL / well of 0.02M PBS (pH 7.2) containing 10% calf serum or 1% skim milk powder at 37°C for 2 hours before detection. Seven days after fusion, 0.1 mL of cell supernatant was transferred to a 96-well plate and incubated at 37°C for 30 minutes. After washing six times, secondary antibody was added and incubated at 37°C for 30 minutes. After washing, 100 μL of citric acid-phosphate buffer (pH 5.0) containing 0.1% (M / V) o-phenylenediamine and 0.1% (V / V) hydrogen peroxide was added to each well and incubated at 37°C for 15 minutes. Dilute sulfuric acid solution was then added (50 μL / well), and absorbance was measured at 450 nm.

[0088] In a 96-well culture plate, the antibody-secreting cells were cloned using the limiting dilution method at 1 cell / well, and the positive wells were screened and cloned three times in succession. After expansion, the cells were cryopreserved in a culture medium containing 10% DMSO at a cell density of 1×10 6 Five stable V5-tagged hybridoma cell lines were obtained and named A, B, C, D, and E.

[0089] (3) Preparation of V5 tag monoclonal antibody

[0090] Select healthy BALB / c mice of 6 to 8 weeks old and inject 0.5 mL of pristane into the peritoneal cavity of each mouse. Ten days later, inject 1×10 6Hybridoma cells. 7-10 days after inoculation, closely monitor the animal's health and signs of ascites. Once ascites have accumulated as much as possible and before the mouse is near death, sacrifice the mouse and aspirate the ascites into a test tube using a pipette. Typically, 5-10 mL of ascites fluid can be obtained from each mouse. Collect the ascites, centrifuge, and obtain the supernatant. Store in a refrigerator at -20°C. Dilute the supernatant with three volumes of PBS and filter through filter paper. Apply the resulting filtrate to a protein G affinity chromatography column equilibrated with PBS at a flow rate of 1 mL / min. Substances not adsorbed by protein G are then washed with PBS at a flow rate of 1 mL / min until the absorbance at 280 nm reaches baseline. Elute the antibody with 0.1 M glycine (pH 2.5) and recover the antibody. The recovered solution is neutralized with 0.1 M Tris (pH 8.8), adjusted to the appropriate antibody concentration by ultrafiltration, and stored frozen in aliquots at -20°C.

[0091] (4) Screening of V5 tag antibodies

[0092] The titers of the above five hybridoma cell lines and the secreted ascites antibodies were detected by indirect ELISA to screen the cell line with the highest titer. The specific experimental steps are as follows:

[0093] 1) Coating: Dilute V5 antigen to 1 μg / mL, add 100 μL / well to the ELISA plate, and incubate at 37°C for 2 hours or at 4°C overnight.

[0094] 2) Wash the plate five times using a plate washer, adding 350 μL of wash solution to each well each time, leaving the plate for 20 seconds; then pat dry.

[0095] 3) Wash away the coating solution with washing solution, block with blocking solution, 150 μL per well, and incubate at 37°C for 1.5-2 hours;

[0096] 4) Wash the plate 5 times using a plate washer, injecting 350 μL of wash solution into each well each time, leaving it for 20 seconds; then pat dry.

[0097] 5) Add samples: Add cell culture supernatant and ascites diluted to different gradients to the ELISA plate coated with V5 antigen, 100 μL / well, and react at 37°C for 1 hour (also prepare negative and positive control wells);

[0098] 6) Wash the plate 5 times using a plate washer, injecting 350 μL of wash solution into each well each time, leaving it for 20 seconds; then pat dry.

[0099] 7) Add horseradish peroxidase-labeled goat anti-mouse IgG enzyme-labeled secondary antibody (diluted 6000-fold with blocking buffer) at 100 μL / well and incubate at 37°C for 1 hour.

[0100] 8) Wash the plate 5 times using a plate washer, injecting 350 μL of wash solution into each well each time, leaving it for 20 seconds; then pat dry.

[0101] 9) Add TMB colorimetric solution: prepare immediately before use, 100 μL / well, incubate at 37°C in the dark for 30 minutes;

[0102] 10) Terminate the reaction: Add 2 M sulfuric acid to each reaction well, 50 μL / well;

[0103] 11) Microplate reader reading: 450nm, 630nm wavelength measurement.

[0104] The final measurement results are as follows:

[0105] Table 1 Comparison of titers of various cell lines

[0106] Cell lines Hybridoma cell culture supernatant titers Ascites antibody titers 4F6 <![CDATA[2.37×10 3 ]]> <![CDATA[2.83×10 5 ]]> 7C5 <![CDATA[2.71×10 3 ]]> <![CDATA[3.16×10 5 ]]> 5D8 <![CDATA[1.96×10 4 ]]> <![CDATA[2.07×10 6 ]]> 3E7 <![CDATA[3.34×10 3 ]]> <![CDATA[4.74×10 5 ]]> 6B10 1.82 x 10 3 ]]> <![CDATA[2.71×10 5 ]]>

[0107] From the results in Table 1, it can be seen that the V5 antibody secreted by cell line 5D8 has the highest titer, so the cell line 5D8 with the highest titer was screened out.

[0108] (5) Antibody variable region gene cloning and sequencing

[0109] Total RNA was extracted from the hybridoma cell line 5D8, which secretes the V5 monoclonal antibody, and first-strand cDNA was synthesized using the SMARTER™ RACE cDNA Amplification Kit and the included SMARTER II A Oligonucleotide and 5'-CDS primer. The resulting first-strand cDNA product was used as a template for PCR amplification. The light chain gene was amplified using Universal Primer A Mix (UPM), Nested Universal Primer A (NUP), and mIgG CKR primers, while the heavy chain gene was amplified using Universal Primer A Mix (UPM), Nested Universal Primer A (NUP), and mIgG CHR primers. The light chain primer pair amplified a target band of approximately 0.7 kb, while the heavy chain primer pair amplified a target band of approximately 1.5 kb. The product was purified and recovered by agarose gel electrophoresis, and the product was subjected to A addition reaction with rTaq DNA polymerase and inserted into the pMD-18T vector and transformed into DH5α competent cells. After the colonies grew, four clones of the Heavy Chain and Light Chain genes were taken and sent to Qingke Company for sequencing. The amino acid sequences of the heavy chain and light chain are shown in SEQ ID NO: 15 and SEQ ID NO: 16, respectively. The specific amino acid sequences are shown in Figure 1 As shown; after analysis, the complementarity determining region of the heavy chain:

[0110] CDR1:SYSIH;

[0111] CDR2: YINPASGYSAYNENFKB;

[0112] CDR3: DKFYAYDY;

[0113] Complementarity determining regions of light chains:

[0114] CDR1:RASQSIVHKNGNTYLD;

[0115] CDR2: RVAQKFS;

[0116] CDR3: FNASHVPYT.

[0117] Example 2 Monoclonal Antibody Affinity Analysis and Activity Determination

[0118] (1) Comparison of affinity of purified monoclonal antibodies with different V5 tags

[0119] V5-tag monoclonal antibodies were prepared and purified, and data were measured using an indirect enzyme immunoassay (ELISA) in the same manner as for activity assays. The coating concentrations included four gradients: 1 μg / mL, 0.5 μg / mL, 0.25 μg / mL, and 0.125 μg / mL. The antibody was diluted two-fold from 1000 ng / mL to 0.97656 ng / mL for loading. The corresponding OD values ​​for the different antibody concentrations at different coating concentrations were calculated. At the same coating concentration, plot the antibody concentration as the abscissa and the OD value as the ordinate on a logarithmic scale. Calculate the antibody concentration at 50% of the maximum OD value based on the fitting equation. Substitute the equation into the formula: K = (n-1) / (2*(n*Ab`-Ab)) to calculate the reciprocal of the affinity constant, where Ab and Ab` represent the antibody concentrations at 50% of the maximum OD value at the corresponding coating concentrations (Ag, Ag`), respectively, and n = Ag / Ag`. A K value can be calculated for every two coating concentrations, resulting in six K values. Take the average value, and then find the reciprocal to obtain the affinity constant, KD.

[0120] The affinity of the purified V5 monoclonal antibody was analyzed, and the results are shown in Table 2.

[0121] Table 2 Affinity determination results of purified V5-tagged monoclonal antibodies

[0122] Sample name <![CDATA[K D ]]> 4F6 6.446E-7 7C5 4.375E-6 5D8 1.425E-8 3E7 9.837E-7 6B10 7.106E-6

[0123] As shown in Table 2, the affinity constant KD of the antibody secreted by the 5D8 hybridoma cell line is 1.425E-8, which is significantly better than that of the antibodies secreted by the other four hybridoma cell lines.

[0124] (2) Activity identification

[0125] V5 antigen was diluted to 1 μg / mL with 50 mM carbonate buffer coating solution for microplate coating, 100 μL per well, incubated at 4°C overnight; the next day, the washing solution was washed twice with PBST and patted dry; blocking solution (20% BSA + 80% PBS) was added, 120 μL per well, incubated at 37°C for 1 hour, and patted dry; diluted V5 monoclonal antibody 5D8 was added, starting from 1000 ng / mL and diluted 5-fold, and the sample was loaded, 100 μL / well, incubated at 37°C for 3 hours. 0min (part of the supernatant 1h); wash 5 times with PBST washing solution, pat dry; add horseradish peroxidase-labeled goat anti-mouse IgG, 100μL per well, 37℃, 30min; wash 5 times with PBST washing solution, pat dry; add urea peroxide (50μL / well), add tetramethylbenzidine (50μL / well), 10min; add dilute hydrochloric acid to terminate the reaction, 50μL / well; read the OD value at 450nm (reference 620nm) on the microplate reader. The results are shown in Table 3 and Figure 2 shown.

[0126] Table 3 Activity identification of purified V5-tagged monoclonal antibodies

[0127] Sample concentration ng / mL 1000 200 40 8 1.6 0.32 0 V5 tag antibody 5D8 3.324 2.017 0.856 0.198 0.142 0.079 0.015

[0128] From Table 3 and Figure 2 It can be seen that the sample concentration can be detected as low as 0.32ng / ml, indicating its high activity.

[0129] Example 3 Use of V5 Tag Antibody

[0130] The V5 tag antibody of the present invention was compared with the V5 tag antibodies of other companies for anti-V5 monoclonal antibody affinity. The specific implementation method is as follows:

[0131] Activity was assessed using an indirect enzyme immunoassay (ELISA) assay. Different coating concentrations (1 μg / ml, 0.5 μg / ml, 0.25 μg / ml, and 0.125 μg / ml) of different V5 tag antibodies were used for each experiment. The antibody was then diluted 2-fold from an initial concentration of 1000 ng / ml to 0.97656 ng / ml for loading. The OD values ​​corresponding to each coating concentration were recorded.

[0132] Next, at the same coating concentration, a logarithmic plot is made with the antibody concentration as the horizontal axis and the OD value as the vertical axis. The antibody concentration at 50% of the maximum OD value is calculated using the fitting equation. Then, the reciprocal of the affinity constant is calculated according to the formula K = (n-1) / (2*(n*Ab`-Ab)), where Ab and Ab` represent the antibody concentrations at 50% of the maximum OD value at the corresponding coating concentration, and n is the coating concentration ratio (Ag / Ag`). Every two coating concentrations can be combined to calculate a K value, and finally six K values ​​are obtained. The average value is taken, and the reciprocal is the affinity constant KD.

[0133] Finally, the affinity analysis data of the purified anti-V5 monoclonal antibody are summarized in Table 4 below.

[0134] Table 4 Affinity determination results of anti-V5 monoclonal antibodies of different products

[0135]

[0136]

[0137] As shown in Table 4, the affinity of the anti-V5 monoclonal antibody of the present invention is significantly better than that of the other three V5 tag antibodies.

[0138] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Although the present invention is disclosed as above in terms of a preferred embodiment, it is not intended to limit the present invention. Any person skilled in the art can, without departing from the scope of the technical solution of the present invention, make some changes or modifications to equivalent embodiments using the technical contents disclosed above. However, any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A V5 tag antibody, characterized in that Includes three heavy chain complementarity determining regions: heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3, and three light chain complementarity determining regions: light chain CDR1, light chain CDR2 and light chain CDR3; The heavy chain CDR1 is the amino acid sequence SYSIH shown in SEQ ID NO: 1; The heavy chain CDR2 is the amino acid sequence YINPASGYSAYNENFKB shown in SEQ ID NO: 2; The heavy chain CDR3 is the amino acid sequence DKFYAYDY shown in SEQ ID NO: 3; The light chain CDR1 is the amino acid sequence RASQSIVHKNGNTYLD shown in SEQ ID NO: 4; The light chain CDR2 is the amino acid sequence RVAQKFS shown in SEQ ID NO: 5; The light chain CDR3 is the amino acid sequence FNASHVPYT shown in SEQ ID NO:

6.

2. The V5 tag antibody according to claim 1, wherein The antibody, wherein FR1-4 in the heavy chain variable region are the following amino acid sequences: comprising the amino acid sequence shown in SEQ ID NO: 7, 8, 9, 10 or the amino acid sequence shown in SEQ ID NO: 7, 8, 9, 10, or a sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 7, 8, 9, 10, Or a sequence having one or more amino acid mutations compared to the amino acid sequence shown in SEQ ID NO: 7, 8, 9, or 10.

3. The V5 tag antibody according to claim 2, wherein FR1-4 in the heavy chain variable region are the following amino acid sequences: The amino acid sequences shown in SEQ ID NOs: 7, 8, 9, and 10, or a sequence having at least 91% identity to the amino acid sequence shown in SEQ ID NO: 7, 8, 9, 10, Or an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid mutations compared to the amino acid sequence shown in SEQ ID NO: 7, 8, 9, 10; the amino acid mutation is a conservative mutation, including substitution, insertion or deletion.

4. The V5 tag antibody according to any one of claims 1 to 3, wherein The antibody, wherein FR1-4 in the light chain variable region are the following amino acid sequences: comprising the amino acid sequence shown in SEQ ID NO: 11, 12, 13, 14 or the amino acid sequence shown in SEQ ID NO: 11, 12, 13, 14, or a sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 11, 12, 13, 14, Or a sequence having one or more amino acid mutations compared to the amino acid sequence shown in SEQ ID NO: 11, 12, 13, or 14.

5. The V5 tag antibody according to claim 4, wherein FR1-4 in the light chain variable region are the following amino acid sequences: The amino acid sequences shown in SEQ ID NOs: 11, 12, 13, and 14, or a sequence having at least 91% identity to the amino acid sequence shown in SEQ ID NO: 11, 12, 13, 14, Or an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid mutations compared to the amino acid sequence shown in SEQ ID NO: 11, 12, 13, 14; the amino acid mutation is a conservative mutation, including substitution, insertion or deletion.

6. The V5 tag antibody 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 containing a nucleic acid sequence encoding the V5 tag antibody according to claim 6, characterized in that: The biological material includes an expression vector, an expression cassette, and a host cell.

8. Use of the V5 tag antibody according to claim 6 in preparing a reagent for detecting, separating and purifying a V5-tagged target protein.

9. The use according to claim 8, characterized in that For use in ELISA, immunoblotting, flow cytometry, immunoprecipitation, and / or immunocytochemistry / immunofluorescence.

10. A target protein detection kit, characterized in that: Comprising the V5 tag antibody as claimed in claim 6.

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