CN112358547A - Anti-human CXCL-2 monoclonal antibody 3-D3 and coding gene and application thereof - Google Patents
Anti-human CXCL-2 monoclonal antibody 3-D3 and coding gene and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of bioengineering, and relates to an anti-human CXCL-2 monoclonal antibody 3-D3, and a coding gene and application thereof. The monoclonal antibody or the fragment thereof is combined with an extracellular region containing a chemotactic factor CXCL-2 and has the function of blocking the CXCL-2, and the invention relates to an antihuman CXCL-2 monoclonal antibody and preparation thereof, a variable region amino acid sequence and a gene coding sequence and application thereof. The invention newly finds a monoclonal antibody capable of blocking the function of human chemotactic factor CXCL-2 and a coding gene thereof, the antibody can be combined with the extracellular region of human CXCL-2, can specifically block the combination of CXCL-2 and a ligand thereof, and inhibits signal transduction, so that the monoclonal antibody can be used as a blocking agent of a CXCL-2 signal channel, thereby becoming a novel antibody medicine for treating tumor immunotherapy, acute and chronic infectious diseases and autoimmune diseases.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and relates to an anti-human CXCL-2 monoclonal antibody, a preparation method thereof, a variable region amino acid sequence, a gene coding sequence and application thereof, wherein the monoclonal antibody or a fragment thereof is combined with an extracellular region containing a chemotactic factor CXCL-2 and has the function of blocking the CXCL-2 function.
Background
Chemokines are a class of proteins that regulate leukocyte migration and play an important role in inflammatory responses, and in 1988, Cerami et al performed purification analysis of two heparin-binding proteins released from macrophages stimulated by LPS and found MIP-1[ Wolpe SD, Davatelis G, Sherry B, Beutler B, Hesse DG, Nguyen HT, et al. macrophages secrete a novel heparin-binding protein with affinity and neutrophilic chemical properties. the Journal of experimental media.1988; 167:570-81 ], the next year, CXCL-2 was isolated and its molecular structure and biological activity were tested [ Wolpe SD, Sherry B, Juers D, Davatelis G, Yurt RW, Cerami A. identification and characterization of macromolecular in fluorescence protein 2.Proceedings of the National Academy of science of the United States of America.1989; 86:612-6.]. Unlike MIP-1, CXCL-2 is a basic heparin binding protein with a molecular weight of about 6000 Kd. The murine CXCL-2 molecule has 4 exons and three introns, a structure typical of the platelet factor 4 (PF 4) subfamily, but partial N-terminal amino acid sequence analysis of the CXCL-2 molecule reveals that the mouse CXCL-2 sequence has 62.5% and 68.7% homology with human and hamster GROs, respectively [8 ]. Murine CXCL-2 is a neutrophil chemokine that belongs to the IL-8 family, and in 1993, Watanabe K et al found that the neutrophil chemotactic cytokine CXCL-2 in LPS-induced inflammatory rat inflammatory secretions has a higher similarity to human gro/MGSA than to IL-8[9 ].
CXCL-2 is a main chemotactic factor and an activating factor of neutrophilic granulocyte, and researches show that the expression of CXCL-2mRNA is remarkably increased in a rat inflammation model and is positively correlated with the chemotactic infiltration quantity of the neutrophilic granulocyte in a rat body, and the secretion of TNF-alpha in the abdominal cavity of the rat can be remarkably increased by injecting recombinant CXCL-2 protein into the abdominal cavity of the rat [36 ]. Blockade of CXCL-2 in vivo can lead to a decrease in the number of neutrophil infiltrates and a reduction in the inflammatory response of the body [ 37 ]. In acute pneumonia rat model CXCL-2 can chemotaxis neutrophils to lung inflammation position and activated, and participates in lung tissue inflammation [ 39 ]. In a mouse model of pseudomonas aeruginosa infection, CXCL-2 secreted by inflammatory tissues can lead to long-term recruitment and chemotaxis of polymorphonuclear neutrophils, constituting irreversible destruction of tissue [ 40 ]. The chemokine CXCL-2 can participate in the migration of neutrophils to the liver of a mouse through a P2Y2 receptor, and the CXCL-2 antibody can inhibit the chemotaxis of the neutrophils in the peritoneal exudate of a peritonitis mouse [ 34, 41 ]. Taken together, CXCL-2 plays an important role in the recruitment of neutrophils to the chemotactic process of the liver and other organs in inflammatory states.
The existing CXCL-2 antibodies are low in affinity, and a blocking CXCL-2 antibody with high affinity is still absent.
Disclosure of Invention
In order to solve the problems, the invention discloses a monoclonal antibody or a fragment thereof for resisting human CXCL-2, wherein the monoclonal antibody or the fragment thereof can be specifically combined with human CXCL-2 protein and has a functional effect of blocking a CXCL-2 protein signal channel.
The monoclonal antibody comprises a light chain and a heavy chain, the amino acid variable region sequences of the monoclonal antibody are respectively shown as SEQ ID NO 7 and SEQ ID NO 8 in a sequence table, and the coding gene sequences of the monoclonal antibody are respectively shown as SEQ ID NO 9 and SEQ ID NO 10 in the sequence table.
Amino acid sequence of light chain variable region of NO 7
1
AspIleGlnLeuThrGlnSerProAlaSerLeuAlaValSerLeuGlyGlnArgAlaThr
21
IleSerTyrArgAlaSerLysSerValSerThrSerGlyTyrSerTyrMETHisTrpAsn
41
GlnGlnLysProGlyGlnProProArgLeuLeuIleTyrLeuValSerAsnLeuGluSer
61
GlyValProAlaArgPheSerGlySerGlySerGlyThrAspPheThrLeuAsnIleHis
81
ProValGluGluGluAspAlaAlaThrTyrTyrCysGlnHisIleArgGluLeuThrArg
101 SerGluGlyGlyProSerTrpArgSerAsn
Amino acid sequence of heavy chain variable region of NO 8
1
ValLysLeuGlnGlnSerGlyProGluLeuLysLysProGlyGluThrValLysIleSer
21
CysLysAlaSerGlyTyrThrPheThrHisTyrGlyIleAsnTrpValThrGlnAlaPro
41
GlyLysGlyLeuLysTrpMETGlyTrpIleAsnThrAsnThrGlyGluProThrTyrAla
61
GluGluPheLysGlyArgPheAlaPheSerLeuGluMETSerAlaSerThrAlaTyrLeu
81
GlnIleAsnAsnLeuLysAsnGluAspThrAlaThrTyrPheCysAlaArgLeuGluVal
101 ArgGlyTyrAlaMETAspTyrTrpGlyGlnGlyThrThrValThrValSerSer
NO 9 light chain variable region DNA sequence
10 heavy chain variable region DNA sequence
The amino acid sequences of the complementarity determining region CDR1-3 of the monoclonal antibody light chain variable region are respectively shown in sequence tables SEQ ID NO:1 and SEQ ID NO: 2. SEQ ID NO. 3. The amino acid sequences of the complementarity determining region CDR1-3 of the heavy chain variable region of the monoclonal antibody are respectively shown in the sequence tables SEQ ID NO:4 and SEQ ID NO: 5. SEQ ID NO. 6.
LysSerValSerThrSerGlyTyrSerTyr
Amino acid sequence of complementarity determining region 2(CDR2) in light chain variable region of NO 2
LeuValSerAsn
Amino acid sequence of complementarity determining region 3(CDR3) in light chain variable region of NO 3
GlnHisIleArgGluLeuThrArg
Amino acid sequence of complementarity determining region 1(CDR1) of heavy chain variable region of NO 4
GlyTyrThrPheThrHisTyrGly
Amino acid sequence of complementarity determining region 2(CDR2) of heavy chain variable region of NO 5
TrpIleAsnThrAsnThrGly
Amino acid sequence of complementarity determining region 3(CDR3) of heavy chain variable region of NO 6
CysAlaArgLeuGluValArgGlyTyrAlaMETAsp
The monoclonal antibody or an antibody fragment thereof of the present invention may be any of a single-chain antibody, a diabody, a chimeric antibody, a humanized antibody, and a derivative, a functional equivalent or a homologue of the above antibody, as long as it can bind to a human CXCL-2 protein and has a functional effect capable of blocking the CXCL-2 pathway.
The invention discloses a monoclonal antibody for blocking the function of human chemotactic factor CXCL-2, and a coding gene and application thereof. It comprises a light chain and a heavy chain, wherein the amino acid sequence of the light chain is shown as (NO:7), and the amino acid sequence of the heavy chain is shown as (NO: 8). The invention newly finds a monoclonal antibody capable of blocking the function of human chemotactic factor CXCL-2 and a coding gene thereof, the antibody can be combined with the extracellular region of human CXCL-2, can specifically block the combination of CXCL-2 and a ligand thereof, and inhibits signal transduction, so that the monoclonal antibody can be used as a blocking agent of a CXCL-2 signal channel, thereby becoming a novel antibody medicine for treating tumor immunotherapy, acute and chronic infectious diseases and autoimmune diseases.
Drawings
FIG. 1 is an agarose gel electrophoresis of the light and heavy chain variable region gene of anti-human CXCL-2 monoclonal antibody 3-D3 amplified by RT-PCR, wherein M is DM1000 DNA marker, L represents the light chain variable region gene, and H represents the heavy chain variable region gene.
FIG. 2 shows an animal experiment of a mouse with hepatitis of 3-D3 monoclonal antibody against human CXCL-2, in which a galactosamine-induced acute hepatitis mouse model treated with 3-D3 antibody has reduced ALT and AST contents in peripheral blood of the mouse and significantly reduced inflammation of the mouse.
Detailed Description
Example 1: construction of hybridoma cell lines
1. Material
DNA immunoadjuvants were purchased from Sigma; fetal bovine serum and PRMI1640 medium were purchased from Gibco; SP2/0 cells were purchased from ATCC; BALB/c mice were provided by the Experimental animals center of university of Zhejiang.
2. The method comprises the following steps:
(1) plasmid construction
The primers are designed according to the CXCL-2 gene sequence searched at NCBI, the DNA sequence of the CXCL-2 gene is amplified by taking human cDNA as a template, and the DNA sequence is constructed on pcDNA3.1 plasmid by utilizing a molecular biology method.
(2) Immunization of mice
BALB/c mice of 4-6 weeks old are selected, the constructed CXCL-2 gene recombinant plasmid is dissolved in PBS and mixed with adjuvant in equal volume for emulsification, and subcutaneous and abdominal multipoint immunization is carried out. Every 2 weeks after the first immunization, the plasmid and the adjuvant with the same dose are mixed and emulsified respectively, and then injected for boosting immunization. After 7 days of 3 rd immunization, tail vein blood was collected and subjected to gradient dilution by ELISA method to detect the antibody production. 3 days before fusion, the same dose of plasmid was mixed with adjuvant and boosted 1 time.
(3) Cell fusion
After the immunized mouse is killed, the spleen of the mouse is picked up aseptically, a single cell suspension is prepared by grinding, and the number of cells is counted after the single cell suspension is washed twice by RPMI1640 culture solution; mixing mouse spleen lymphocyte and myeloma cell SP2/0 at a ratio of 1:5-1:10, slowly adding 1ml of 50% PEG solution dropwise in a short time, placing in 37 deg.C water bath for 1min, adding RPMI1640 culture solution, centrifuging at 800r/min for 5min, discarding supernatant, adding 1640 culture medium containing 20% FBS to suspend cells, mixing the cell suspension with equal volume of feedMixing cultured cells (mouse abdominal cavity macrophages), subpackaging into 96-well cell culture plates, standing at 37 deg.C with 5% CO2Culturing in an incubator. After 3d of culture, HAT 1640 containing 20% FBS was added for selection medium culture.
(4) Screening of hybridoma cells
When the cell colony of the 96-well plate grows to a proper size, the cell culture supernatant is extracted and detected by an indirect ELISA method, and positive hybridoma cells are screened.
(5) Hybridoma cell cloning
When the hybridoma cells identified as positive were cultured to good condition, the cells were diluted and plated into 96-well plates by limiting dilution so that only one cell was present in each well, and the cells were incubated at 37 ℃ in 5% CO2Culturing in an incubator. Observing the cell growth condition in 5d-10d, taking the supernatant in the hole forming single cell clone, detecting by using an indirect ELISA method, screening and identifying positive clone; and repeating the subcloning for 3-5 times until the positive porosity of the hybridoma cells reaches 100%, and determining the stability of the cells.
Example 2: antibody subclass identification and stability test
The subclass of the antibody is identified by referring to the specification of the antibody subclass identification kit, and as a result, the heavy chain class is IgG1 type, and the light chain is Kappa type.
Continuously subculturing the hybridoma cell strain in vitro for 3 months, and measuring the titer of the antibody in the supernatant; the cryopreserved hybridoma cell strain is recovered after 4 months, and the antibody titer in the supernatant is detected without obvious change, which shows that the obtained hybridoma cell strain generating the antibody has stable performance.
Example 3: experiment for inhibiting migration of neutrophils by anti-CXCL-2 monoclonal antibody
1. Material
2. Method and results
(1) Matrix glue common plate: the upper surface of the bottom membrane of the Transwell cell was coated by diluting with Matrigel 1:8 from BD, and the Matrigel was polymerized into a gel at 37 ℃ for 30min before use to hydrate the substrate membrane.
(2) Preparation of cell suspensions
(3) Seeding cells
100ul of the decellularized suspension was placed in a Transwell chamber, and 600ul of 20% FBS-containing medium was typically placed in the lower chamber of a 24-well plate, and cultured for 24 hours
(4) And (5) counting the results. The Transwell chamber was removed, the medium from the wells was discarded, washed 2 times with calcium-free PBS, fixed with formaldehyde for 30 minutes, and the chamber was appropriately air-dried. 0.1% crystal violet staining for 20min, gently rubbing off the upper layer of non-migrated cells with a cotton swab, washing 3 times with PBS, observing the cells with 5 visual fields under 400-fold microscope, and counting.
Example 4: cloning of heavy and light chains of the CXCL-2 monoclonal antibody
1. Material
Designing amplification primers of heavy chain and light chain according to the gene sequence of the vector in Hangzhou Ongke Biotechnology GmbH; the DNA fragment purification kit and the plasmid extraction kit are purchased from QIAGEN company; pMD-18T kit, reverse transcriptase and Fidelity PCR enzymes were purchased from TAKARA; competent cells were purchased from promega; trizol reagent was purchased from Invitrogen corporation.
2. Method and results
(1) Take 5X 106-107And centrifuging the monoclonal antibody hybridoma cells to remove supernatant, adding 1ml of Trizol reagent, and repeatedly blowing and beating until the cells are fully lysed. Standing at room temperature for 5min, adding 0.2ml chloroform, mixing by turning upside down, and standing at room temperature for 2-3 min. Centrifuge at 12000 × g for 15min at 4 ℃. Carefully suck the supernatant and mix with another centrifuge tube, add 500. mu.l isopropanol, mix well by inversion, then stand at room temperature for 10min, centrifuge at 12000 × g at 4 ℃ for 10 min. Adding 1ml of precooled 75% ethanol to wash the precipitate, centrifuging 7500 x g for 5min, then discarding the supernatant, drying at room temperature for 5min, and adding RNase-free deionized water to dissolve the precipitate.
(2) Adding 1 mu L of random primer into 1 mu g of RNA, supplementing the random primer to 6 mu L with RNase-free water, quickly placing on ice for 2min after incubating for 10min at 70 ℃, adding 5M-MLV Buffer 2 mu L, dNTP mix 0.5 mu L, RNase inhibitor 0.25 mu L, M-MLV enzyme 1 mu L and RNase-free water to 10 mu L, incubating for 1h at 42 ℃, incubating for 15min at 70 ℃, and cooling on ice to obtain a product which is a first strand cDNA.
(3) In an amplification system of 25. mu.L, 0.5. mu.L of each of two pairs of heavy chain and light chain PCR amplification primers, 2. mu.L of a reverse transcription product, 0.5. mu.L of a high fidelity PCR amplification enzyme, 2. mu.L of dNTP and 5. mu.L of Buffer are respectively added, and water is used for complementing the mixture to 25. mu.L. The PCR amplification parameters were: pre-denaturation at 94 ℃ for 2min, cyclic procedure: 35 cycles of 98 ℃ 10s 55 ℃ 30s 68 ℃ 30 s.
(4) PCR amplification products run agarose gel, the desired target products are reclaimed by tapping, after being reclaimed by a DNA fragment reclaiming kit, the DNA fragments and the carrier are mixed according to the molar ratio of 10:1 according to the specification of a pMD-18T kit, and then ligase and connecting Buffer are added, the total volume is 10 mu L, and the overnight connection is carried out at 16 ℃.
(5) Adding 10 μ L of the connecting liquid into 200 μ L of TOP 10F' competent bacteria, ice-cooling for 30min, heat-shocking at 42 ℃ for 90s, rapidly placing on ice for 2min, adding 800 μ L of LB culture liquid, shaking and culturing at 37 ℃ and 180rpm/min for 45min, centrifuging at 5000rpm for 2min, discarding about 800 μ L of supernatant, blowing the precipitate uniformly with the rest liquid, spreading on LB solid culture medium plate containing ampicillin, and inversely culturing the plate in a 37 ℃ culture box for 12-16 h. Selecting a single bacterial clone from a plate, inoculating the bacterial clone into an LB liquid culture medium containing ampicillin, carrying out shaking culture at 37 ℃ and 220rpm/min for overnight, removing part of bacterial liquid, cracking, carrying out PCR amplification verification by using light chain and heavy chain primers, and amplifying bacterial liquid with fragments with required sizes to carry out sequencing. After the bacterial liquid with correct sequencing is transferred, a DNA plasmid extraction kit is used for extracting plasmids which carry genes of heavy chains and light chains of variable regions of antibodies.
Example 5: hepatitis mouse animal experiment of anti-human CXCL-2 monoclonal antibody 3-D3
Male C57BL/6 mice, 7 weeks old and 18-20g in weight, were prepared and fasted for 12h before the start of the experiment without water deprivation. The mice were weighed according to the labeled numbers to record the body weights, and were divided into 4 groups, namely a blank control group, an acute hepatitis mouse model group, a high concentration antibody group (100 ug/mouse), and a low concentration antibody group (25 ug/mouse). Anti-human CXCL-2 monoclonal antibodies were prepared using physiological saline, and the antibodies were injected into the mice of the high concentration antibody group (100 ug/mouse) and the low concentration antibody group (25 ug/mouse) by intraperitoneal injection of the mice. After 1 hour, LPS/D-GaIN mixed solutions with final concentrations of 1ug/ml and 100mg/kg were prepared, and the LPS/D-GaIN mixed solutions were injected into mice of the acute hepatitis mouse model group, the high concentration antibody group (100 ug/mouse), and the low concentration antibody group (25 ug/mouse) by intraperitoneal injection. After the model building is finished according to the time needed by the model building, 5 percent sodium pentobarbital is injected into the abdominal cavity of the anesthetized mouse in sequence, the eyeball takes blood, the supernatant is obtained by centrifugation, and the concentration of alanine Aminotransferase (ALT) and aspartate Aminotransferase (AST) in the serum is detected.
Sequence listing
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<120> antihuman CXCL-2 monoclonal antibody 3-D3, and coding gene and application thereof
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gggctggata aacaccaaca ctggagagcc aacatatgct gaagagttca agggacgatt 180
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Claims (4)
1. An antihuman CXCL-2 monoclonal antibody or an antibody fragment thereof has amino acid variable region sequences shown as SEQ ID NO 7 and SEQ ID NO 8 in a sequence table respectively, and encoding gene sequences shown as SEQ ID NO 9 and SEQ ID NO 10 in the sequence table respectively.
2. An anti-human CXCL-2 monoclonal antibody or an antibody fragment thereof, characterized in that: the monoclonal antibody or antibody fragment comprises an antibody light chain comprising the complementarity determining region CDR1-3 and an antibody heavy chain comprising CDR 1-3:
light chain CDR1 has the amino acid sequence shown in SEQ ID NO. 1;
light chain CDR2 has the amino acid sequence shown in SEQ ID NO. 2;
light chain CDR3 has the amino acid sequence shown in SEQ ID NO. 3;
heavy chain CDR1 has the amino acid sequence shown in SEQ ID NO. 4;
heavy chain CDR2 has the amino acid sequence shown in SEQ ID NO. 5;
heavy chain CDR3 has the amino acid sequence shown in SEQ ID NO. 6.
3. The monoclonal antibody or antibody fragment thereof of claim 1 or 2, characterized in that: the monoclonal antibody or antibody fragment thereof includes single chain antibodies, diabodies, chimeric antibodies, humanized antibodies, and derivatives, functional equivalents or homologs of the foregoing.
4. Use of the monoclonal antibody or antibody fragment thereof according to claim 1 or 2 for the preparation of a medicament for the treatment of an immunological or infectious disease.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011066422.0A CN112358547A (en) | 2020-09-30 | 2020-09-30 | Anti-human CXCL-2 monoclonal antibody 3-D3 and coding gene and application thereof |
| PCT/CN2020/136276 WO2022068078A1 (en) | 2020-09-30 | 2020-12-14 | Anti-human cxcl-2 monoclonal antibody 3-d3, and coding gene and use thereof |
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| CN202011066422.0A CN112358547A (en) | 2020-09-30 | 2020-09-30 | Anti-human CXCL-2 monoclonal antibody 3-D3 and coding gene and application thereof |
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| WO (1) | WO2022068078A1 (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20070160574A1 (en) * | 2000-04-12 | 2007-07-12 | Ahmed Merzouk | Design of CXC chemokine analogs for the treatment of human diseases |
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| WO2009006359A2 (en) * | 2007-06-29 | 2009-01-08 | Centocor, Inc. | Anti- mcp-1 antibodies, compositions, methods and uses |
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2020
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