CN107556383B - Preparation method of anti-human DLL4 monoclonal antibody 3F9 - Google Patents
Preparation method of anti-human DLL4 monoclonal antibody 3F9 Download PDFInfo
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- CN107556383B CN107556383B CN201710677649.0A CN201710677649A CN107556383B CN 107556383 B CN107556383 B CN 107556383B CN 201710677649 A CN201710677649 A CN 201710677649A CN 107556383 B CN107556383 B CN 107556383B
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Abstract
The invention discloses a preparation method of an anti-human DLL4 monoclonal antibody 3F9, which is secreted by a hybridoma cell strain, wherein the preservation information of the hybridoma cell strain is as follows: the preservation unit: china general microbiological culture Collection center (CGMCC), preservation address: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North; preservation time: 6 months and 7 days 2017; the preservation number is as follows: CGMCC No. 14283; and (3) classification and naming: and (3) a hybridoma cell strain 3F9 secreting an anti-human DLL4 molecular monoclonal antibody. The binding of monoclonal antibody 3F9 of the invention to DCs did not block the ability of DLL4+ DCs to induce meive T cells to differentiate in the Th1 direction compared to commercial clone MHD 4-46.
Description
Technical Field
The invention relates to a monoclonal antibody, in particular to a preparation method of an anti-human DLL4 monoclonal antibody 3F 9.
Background
The Notch signaling pathway is a highly evolutionarily conserved set of signaling systems that play important roles in cell proliferation, differentiation and apoptosis, as well as cell growth and various physiological functions. Notch signaling molecules are expressed in the vast majority of multicellular organisms. Mammals predominantly express four Notch receptors (Notch 1, 2, 3, 4, respectively) and five Notch ligands (DLL 1, DLL3, DLL4, Jagged1 and Jagged2, respectively).
Early studies showed that DLL4 is highly selectively expressed in vascular endothelial cells and is critical for regulating endothelial cell development. In 2004 Amsen and colleagues found that the stimulation of bone marrow cells containing antigen presenting cells by LPS could induce the expression of DLL 4. In 2007, Skokos and colleagues reported that CD8-DC can induce cells to differentiate toward Th1 by activating the Notch signaling pathway through DLL4 molecule, which is not IL-12 dependent. Subsequent studies have shown that DLL4 can modulate the pathogenesis of a number of diseases in experimental mouse models, such as inflammatory diseases, respiratory viral infections, experimental allergic colitis, experimental autoimmune encephalomyelitis, and mycobacterial granulomas in the lung. Studies by professor Zhang Yi have demonstrated that mouse Dendritic Cells (DCs) expressing DLL4 can enhance autoreactive T cell responses and mediate graft-versus-host responses. However, studies on human DLL4+ DCs have yet to be carried out.
Recently, studies by professor Zhang Yi et al reported a key role for human DLL4+ DCs in regulating T cell differentiation to Th1 and Th 17. CD1C + DCs and plasma cell-like DCs (pdcs) in peripheral blood from healthy humans do not express DLL4 molecules on the surface. In contrast, DLL4 mRNA levels expressed by DLL4+ CD1C + DCs in peripheral blood of patients undergoing allogeneic hematopoietic stem cell transplantation were 16-fold higher than those of healthy humans. The level of DLL4 expressed by CD1C + DCs from healthy humans was significantly upregulated after activation of TLR signaling. In contrast, pDCs were upregulated to a lesser extent. Activated DLL4+ DCs promoted Th1 and Th17 differentiation better than unstimulated DCs. Blocking Notch signaling using neutralizing antibodies to DLL4 during stimulation of activated T cells by DLL4+ DCs may reduce production of Th1 and Th17 cells. Thus, for human peripheral blood circulating DCs, DLL4 is an important functional molecule that induces T cell differentiation to Th1 and Th 17. These findings provide a possible approach for the treatment and intervention of inflammatory diseases in humans.
Since the discovery of the DLL4 molecule, it has been of interest to a large number of researchers, and many functional studies have been carried out on human DLL4 molecules. In many functional studies, DLL4+ cells of interest need to be sorted out by flow cytometry after antibody labeling. However, the commercial anti-human DLL4 monoclonal antibody used at present has certain limitations. The anti-human DLL4 fluorescent antibody currently used for flow detection by a plurality of known antibody companies has only one clone number (MHD 4-46), and the antibody of the clone number is clearly reported in the literature published by the professor Zhang Yi to have the function of blocking DLL4 signals. Currently, no commercial monoclonal antibody is available for labeling and flow cytometric sorting of DLL4+ DCs while retaining the signaling function of DLL 4. Therefore, the development of the anti-human DLL4+ functional monoclonal antibody is helpful for better researching the biological functions of the DLL4 molecule, and provides a new means for research.
Disclosure of Invention
The invention aims to provide a preparation method of an anti-human DLL4 monoclonal antibody 3F9, which is produced by a hybridoma cell strain of an anti-human DLL4 monoclonal antibody.
In order to achieve the purpose of the invention, the technical scheme adopted by the invention is as follows:
a preparation method of an anti-human DLL4 monoclonal antibody 3F9, wherein the anti-human DLL4 monoclonal antibody 3F9 is prepared from a hybridoma cell strain.
In the technical scheme, the hybridoma cell strain is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms, the preservation address is No. 3 of Xilu No.1 of Beijing Korean district, and the preservation number is CGMCC No. 14283.
In the technical scheme, hybridoma cell strains are inoculated in a hybridoma culture solution, and the culture solution is separated and purified to prepare the monoclonal antibody after culture.
In the technical scheme, hybridoma cell strains are inoculated in abdominal cavities of animals, and the ascites fluid of the animals is separated and purified to prepare the monoclonal antibody.
The invention also discloses a preparation method of the reagent for detecting the expression level of the DLL4 protein or the reagent for sorting the DLL4+ DC, hybridoma cell strains are inoculated in hybridoma culture solution, and the culture solution is separated and purified to prepare monoclonal antibodies after culture; and mixing the monoclonal antibody with a dispersion medium to prepare a reagent for detecting the expression level of the DLL4 protein or a reagent for sorting DLL4+ DC.
In the above technical solution, the dispersion medium includes a buffer solution.
In the technical scheme, the hybridoma cell strain is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms, the preservation address is No. 3 of Xilu No.1 of Beijing Korean district, and the preservation number is CGMCC No. 14283.
The invention also discloses a preparation method of the reagent for detecting the expression level of the DLL4 protein or the reagent for sorting the DLL4+ DC, which comprises the steps of inoculating a hybridoma cell strain in an animal abdominal cavity, and separating and purifying animal ascites fluid to prepare a monoclonal antibody; and mixing the monoclonal antibody with a dispersion medium to prepare a reagent for detecting the expression level of the DLL4 protein or a reagent for sorting DLL4+ DC.
In the above technical solution, the dispersion medium includes a buffer solution.
In the technical scheme, the hybridoma cell strain is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms, the preservation address is No. 3 of Xilu No.1 of Beijing Korean district, and the preservation number is CGMCC No. 14283.
The preparation method of the hybridoma cell strain disclosed by the invention comprises the following steps of:
(1) constructing transgenic cells highly expressing human DLL4 molecules: cloning the full-length sequence of CDS of human DLL4 into a eukaryotic expression vector; transfecting hamster ovary mother cell CHO cells, and screening by using a medicament and a flow cytometer to obtain a transgenic cell CHO/DLL4 highly expressing DLL4 molecules; immunizing BALB/C mice with transgenic cells CHO/DLL 4;
(2) obtaining a fused cell growth clone: taking spleen cells out of an immune qualified mouse aseptically as antigen-sensitized B cells, fusing the B cells with myeloma cell AG8 strain according to a conventional method, and then screening by using a conventional fusion cell HAT screening method to further obtain a fusion cell growth clone;
(3) after biochemical and immunological technologies such as Western Blot and flow cytometry are applied for screening and identification, hybridoma cell strains with high antibody secretion level are selected, and the hybridoma cell strains are preserved in China general microbiological culture collection center and classified and named as monoclonal antibody hybridoma cell strains 3F9 secreting anti-human DLL4 molecules.
The preservation information of the hybridoma cell strain is as follows: china general microbiological culture Collection center (CGMCC), preservation address: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North; preservation time: 6 months and 7 days 2017; the preservation number is as follows: CGMCC No. 14283; and (3) classification and naming: and (3) a mouse anti-human DLL4 molecule monoclonal antibody hybridoma cell strain 3F9 is secreted.
In the technical scheme, the transgenic cell CHO/DLL4 highly expressing the human DLL4 molecule in the step (1) has stronger immunogenicity, and the spatial configuration of the expressed antigen molecule can be exposed on the surface of a cell membrane in a natural state, so that the immune response of an organism can be more effectively stimulated.
In the above-described embodiment, in step (1), the method for preparing CHO/DLL4 cells can be performed by isolating genes, cleaving and ligating nucleotide fragments, constructing and amplifying Cloning and expression vectors, analyzing and identifying nucleotide sequences, transforming and culturing cells according to DNA manipulation techniques well known to those skilled in the art (see, for example, Sambrook et al, Molecular Cloning: A laboratory Manual, Cold Spring harbor, 1989).
The monoclonal antibody prepared from the hybridoma cell strain disclosed by the invention is an anti-human DLL4 monoclonal antibody and is named as monoclonal antibody 3F 9.
The invention also provides the heavy chain variable region amino acid sequence of the monoclonal antibody 3F 9: 1, SEQ ID no; light chain variable region amino acid sequence: SEQ ID No. 2.
SEQ.ID.NO:1：
PTTVPDEVVSIVLNISFNIQPENLERIKEEHRFSMAAENIVGDLLERSRGAGGGGTVYNSDFVSRVSISKDNSKSQVFLKMNSLQIDDTAIYYCVRDDDYDWFFDVWGAGTTVTVSS
Corresponding to:
Pro Thr Thr Val Pro Asp Glu Val Val Ser Ile Val Leu Asn Ile Ser PheAsn Ile Gln Pro Glu Asn Leu Glu Arg Ile Lys Glu Glu His Arg Phe Ser Met AlaAla Glu Asn Ile Val Gly Asp Leu Leu Glu Arg Ser Arg Gly Ala Gly Gly Gly GlyThr Val Tyr Asn Ser Asp Phe Val Ser Arg Val Ser Ile Ser Lys Asp Asn Ser LysSer Gln Val Phe Leu Lys Met Asn Ser Leu Gln Ile Asp Asp Thr Ala Ile Tyr TyrCys Val Arg Asp Asp Asp Tyr Asp Trp Phe Phe Asp Val Trp Gly Ala Gly Thr ThrVal Thr Val Ser Ser
SEQ.ID.NO:2：
DVLMTQTPLSLPVSLGDPASISCRSSQSLVHNNGNTYLHWYLQKPGQSPRLLIYKVFKRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPYTFGGGTKLEIK
corresponding to:
Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly AspPro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Asn Asn Gly Asn ThrTyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Arg Leu Leu Ile Tyr LysVal Phe Lys Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly ThrAsp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe CysSer Gln Ser Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
the reagent for detecting the expression level of the DLL4 protein or the reagent for sorting the DLL4+ DC is prepared by mixing the monoclonal antibody 3F9 and a dispersion medium; the dispersion medium includes a buffer, and can detect the expression level of DLL4 protein or sort DLL4+ DCs.
Compared with the prior art, the invention has the following advantages:
the anti-human DLL4 monoclonal antibody 3F9 prepared by the invention can identify different DLL4 molecular antigen binding sites; the kit has high titer, high specificity and high recognition capability on DLL4 protein on cells, and can be used for detecting the expression level of DLL4 protein by scientific research Western Blot. And in vitro experiments show that the binding of monoclonal antibody 3F9 to DC does not block the ability of DLL4+ DC to induce differentiation of Na-meive T cells to the Th1 direction compared to commercial clone MHD 4-46; can be used for sorting DLL4+ DC by flow cytometry, and does not influence the next functional experiment that DLL4+ DC induces differentiation of Na meive T cells to Th1 direction.
Drawings
FIG. 1 is a graph showing the results of flow cytometry analysis of the recognition of DLL4 molecules on transgenic cells CHO/DLL4 by a commercial anti-human DLL4 antibody in example one;
FIG. 2 is a nuclear type analysis diagram (1000-fold magnification) of chromosome of 3F9 hybridoma cell line in example I;
FIG. 3 is a graph showing the results of antigen recognition by Western Blot analysis of monoclonal antibody 3F9 in example I;
FIG. 4 is a graph showing the results of flow cytometry analysis of the recognition of DLL4 molecules on transgenic cells CHO/DLL4 by monoclonal antibody 3F9 in example one;
FIG. 5 is a graph showing the results of analyzing competitive inhibition of the antigenic site recognized by monoclonal antibody 3F9 by flow cytometry in the first example;
FIG. 6 is a flow cytometric analysis of the effect of anti-human DLL4 monoclonal antibody 3F9 on DLL4 positive mDC induced the process of differentiation of CD4+ Na meive T cell to the Th1 direction in example two.
Detailed Description
The invention will be further illustrated with reference to specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.
EXAMPLE preparation of anti-human DLL4 monoclonal antibody
1. Establishment of transgenic cell CHO/DLL4
(1) Cloning of the human DLL4 Gene
The plasmid containing the full length CDS fragment of human DLL4 was offered by korea home Huai laboratory, university of Xiamen. Carrying out PCR amplification by using a designed primer (table 1) with restriction enzyme sites under the reaction conditions of 94 ℃ denaturation for 60s, 55 ℃ annealing for 60s, 72 ℃ extension for 2 min, and after 35 cycles, carrying out extension at 72 ℃ for 5 min to obtain a full-length fragment; the PCR product was purified by recovery kit.
TABLE 1 amplification primer sequences
| Numbering | Nucleic acid sequences |
| Forward primer | 5’- CGCGGATCCATGGCGGCAGCGTCCC -3’ |
| Reverse primer | 5’- CCGGAATTCTTATACCTCCGTGGCAATGACAC -3’ |
(2) Construction of human DLL4 expression vector
Respectively cutting the recovered PCR product and the expression vector pcDNA3.1 by using restriction enzymes BamH I and EcoR I, separating the PCR product and the expression vector after reaction by agarose gel electrophoresis, cutting off the gel containing the target band, and recovering by using a recovery kit. And linking the PCR product with an expression vector under the action of T4 ligase, and transforming an allelopathic bacterium Top 10. Coating the transformed bacteria on a flat plate containing ampicillin, culturing overnight, selecting positive bacterial colonies, boiling the bacteria, carrying out PCR identification, keeping the seeds after eliminating false positive bacteria, and sequencing. And selecting clones with consistent sequences and without any mutation by Blast comparison on NCBI website. The constructed plasmid is extracted by using a plasmid extraction kit, and the expression vector is named pcDNA3.1/DLL 4.
(3) Construction of CHO transgenic cells stably expressing human DLL4
The expression vector pcDNA3.1/DLL4 was transfected into CHO cells pre-plated in 6-well plates by the liposome method, and the whole procedure was carried out according to the kit Lipofectamine (TM) 3000 operating manual. After the transfection overnight, the medium 1640 containing 10% FBS was replaced to 2 ml/well, and after further culture for 48 hours, a portion of the cells were taken to detect the GFP positive rate by flow cytometry. The efficiency of transfection of the expression vector was judged by the GFP positivity. Meanwhile, after part of cells are diluted according to a proper proportion, the cells are paved in a 6-well plate again, and selective culture medium containing 600mg/L G418 (the proper G418 concentration is determined through pre-screening) is used for screening culture; transgenic CHO cell populations highly expressing human DLL4 were sorted out by sorting flow cytometry using commercially available anti-human DLL4 antibody markers when transgenic cells to be resistant were grown to sufficient numbers. The cells obtained by sorting were subcloned, and a monoclonal cell line was selected. The expression level of human DLL4 was detected by flow cytometry on the selected monoclonal cell lines, and the clones with the highest positive rate and expression level were selected, see fig. 1.
2. Preparation of hybridoma cell strain secreting specific mouse anti-human DLL4 antibody
Balb/c mice (10) were immunized three times with the transgenic cells highly expressing human DLL4 molecule (CHO/DLL 4) obtained as above as immunogen 7500 ul/piece (3 weeks apart). On the fourth day after the last immunization, mouse spleen cells were taken and subjected to cell fusion with the P3X63Ag8 mouse myeloma cell line (10 total 96-well plates). CHO/DLL4 highly expressing human DLL4 molecule was used as a positive control and CHO/mock was used as a negative control, and the ratio of cells was 1:1, and performing primary screening on the hybridoma culture supernatant by using an indirect immunofluorescence method. Screening out the compound 1 of positive and negative proportion: 1 cloning of the characteristics. And after the positive clone is subjected to secondary screening and subcloning, a hybridoma cell strain 3F9 capable of stably secreting specific mouse anti-human DLL4 antibody is obtained.
The preservation information of the hybridoma cell strain is as follows: china general microbiological culture Collection center (CGMCC), preservation address: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North; preservation time: 6 months and 7 days 2017; the preservation number is as follows: CGMCC No. 14283; and (3) classification and naming: and (3) a mouse anti-human DLL4 molecule monoclonal antibody hybridoma cell strain 3F9 is secreted.
After continuous passage in vitro, the hybridoma cells can still stably secrete specific antibodies; chromosome analysis of hybridoma cell lines showed that the chromosome number of these two sets of hybridoma cells ranged from 80 to 110, see FIG. 2.
3. Production and characterization of anti-human DLL4 monoclonal antibody
(1) Production of monoclonal antibodies by ascites in vivo induction
Taking 6-8 week old female Balb/c mice, injecting Pristane (0.5 ml/mouse) into abdominal cavity, inoculating hybridoma cell (1 × 10) into abdominal cavity after one week6/one) while an equal volume of a mixture of Pristane and incomplete freund's adjuvant (0.2 ml/one) was again injected intraperitoneally. Ascites fluid was harvested after 5-10 days, and the supernatant was centrifuged and stored at-80 ℃.
The ascites fluid is purified by protein G affinity column chromatography after being processed by fibrin removal and salting out. Collecting the protein peak effluent, dialyzing against Phosphate Buffer Solution (PBS), and measuring the concentration of the antibody protein to be 0.8-1.8 mg/ml by using a 751 ultraviolet spectrophotometer. The SDS-PAGE results indicated that the murine anti-human DLL4 antibody secreted by 3F9 recognized the human DLL4 recombinant protein (see fig. 3). The result of the indirect immunofluorescence analysis shows that the titer of the purified monoclonal antibody is more than 1:10000 (see figure 4).
(2) Ig subclass identification
The Ig subclass was identified by rapid test using test paper (Argen corporation), which indicated that 3F9 is a mouse IgG2b antibody.
(3) Competitive inhibition assay for antibody recognition of antigenic sites
Human PBMC 24h after LPS and R848 stimulation activation (1X 10)6Tube) suspension monoclonal antibody 3F9 (1: 500, 2ug per tube), incubated at 4 ℃ for 30 minutes. After washing cells, Lin, HLA-DR, CD1C, CD123, and DLL4 fluorescent antibody were added in sequence and incubated at 4 ℃ for 30 minutes. After washing again, the cells were analyzed by flow cytometry, with positive and negative controls, and the results are shown in FIG. 5.
EXAMPLE in vitro biological Effect of Secondary mAb on DCs
This example describes the effect of anti-human DLL4 monoclonal antibodies of the invention on DLL4 positive mDC induced differentiation process of CD4+ Na meive Tcell to Th1 orientation
Human PBMCs were obtained from fresh human peripheral blood by Ficoll separation. Commercial sorting kit using MeitianNi (CD 1 c)+Dendritic Cell Isolation Kit), CD1c was sorted from PBMC according to the protocol provided by Meitianni+DC，The obtained cells are detected by a flow cytometer, and the purity of the obtained cells is over 90 percent. The culture was performed in RPMI-1640 medium containing 10% FBS, and the DCs were stimulated for 24h with the addition of R848 (final concentration of 1 ug/ml) and LPS (final concentration of 100 ng/ml).
Human PBMCs were obtained by Ficoll separation on day 2 using another fresh human peripheral blood. Commercial sorting kit using Stem Cell (EasySep Human CD 4)+T Cell Isolation Kit), sorting CD4 from PBMCs according to the protocol provided by Stem Cell+And (4) detecting the obtained cells by a flow cytometer, wherein the purity of the obtained cells is over 95 percent. The cells obtained from sorting were labeled using CFSE. After marking, according to T cell: DC is 10: 1, spreading on a U-shaped bottom 96 holes (CD 4)+T cell is 1x106/well) were mixed. Fluid replacement was performed on day 4 of culture. Three groups are set in the experiment, and are respectively: a control group; add 3F9 (2 ug/well); 6F12 (2 ug/well) was added.
After 7 days of culture, cells were harvested and stained for CD4 membrane prior to IFNg intracellular staining using commercial immobilisation and membrane disruption agents from eBioscience according to the protocol provided by eBioscience. Flow assay results showed no significant changes in the expression level and ratio of IFNg within cells of the CFSE Low population in T cells of the 3F9 antibody-affected group compared to the control group (see fig. 6), and independent replicates showed no statistical differences between groups. Indicating that monoclonal antibody 3F9 bound to DC and did not block DLL4+The ability of DCs to induce Na-meive T cells to differentiate towards the Th1 direction.
SEQUENCE LISTING
<110> Suzhou university subsidiary children hospital
<120> preparation method of anti-human DLL4 monoclonal antibody 3F9
<160>4
<170>PatentIn version 3.3
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<213> Artificial sequence
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Pro Thr Thr Val Pro Asp Glu Val Val Ser Ile Val Leu Asn Ile Ser Phe Asn Ile Gln
1 5 1015 20
Pro Glu Asn Leu Glu Arg Ile Lys Glu Glu His Arg Phe Ser Met Ala Ala Glu Asn Ile
25 30 35 40
Val Gly Asp Leu Leu Glu Arg Ser Arg Gly Ala Gly Gly Gly Gly Thr Val Tyr Asn Ser
45 50 55 60
Asp Phe Val Ser Arg Val Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys
65 70 75 80
Met Asn Ser Leu Gln Ile Asp Asp Thr Ala Ile Tyr Tyr Cys Val Arg Asp Asp Asp Tyr
85 90 95 100
Asp Trp Phe Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser
105 110 115
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<213> Artificial sequence
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Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly Asp Pro Ala Ser
1 5 10 15 20
Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Asn Asn Gly Asn Thr Tyr Leu His Trp
25 30 35 40
Tyr Leu Gln Lys Pro Gly Gln Ser Pro Arg Leu Leu Ile Tyr Lys Val Phe Lys Arg Phe
45 50 55 60
Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser Thr His Val Pro
85 90 95 100
Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
105 110
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CGCGGATCCATGGCGGCAGCGTCCC 25
<210>4
<211>32
<212>DNA
<213> Artificial sequence
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CCGGAATTCTTATACCTCCGTGGCAATGACAC 32
Claims (7)
1. The preparation method of the anti-human DLL4 monoclonal antibody 3F9 is characterized in that the anti-human DLL4 monoclonal antibody 3F9 is prepared from a hybridoma cell strain; the hybridoma cell strain is preserved in China general microbiological culture collection center with the preservation address of No. 3 of Xilu No.1 of Beijing Korean district, and the preservation number of CGMCC No. 14283; the heavy chain variable region amino acid sequence of the monoclonal antibody 3F9 is shown in a sequence table SEQ.ID.NO. 1, and the light chain variable region amino acid sequence is shown in a sequence table SEQ.ID.NO. 2.
2. The method for producing the anti-human DLL4 monoclonal antibody 3F9 of claim 1, wherein the monoclonal antibody is produced by inoculating a hybridoma cell line into a hybridoma culture medium, culturing, and then separating and purifying the culture medium.
3. The method for preparing the anti-human DLL4 monoclonal antibody 3F9 as claimed in claim 1, wherein the monoclonal antibody is prepared by inoculating a hybridoma cell line into the abdominal cavity of the animal, separating the ascites fluid of the animal, and purifying.
4. A preparation method of a reagent for detecting the expression level of DLL4 protein or a reagent for sorting DLL4+ DC is characterized in that hybridoma cell strains are inoculated in a hybridoma culture solution, and the culture solution is separated and purified to prepare monoclonal antibodies after culture; mixing the monoclonal antibody with a dispersion medium to prepare a reagent for detecting the expression level of DLL4 protein or a reagent for sorting DLL4+ DC; the hybridoma cell strain is preserved in China general microbiological culture collection center with the preservation address of No. 3 of Xilu No.1 of Beijing Korean district, and the preservation number of CGMCC No. 14283; the heavy chain variable region amino acid sequence of the monoclonal antibody 3F9 is shown in a sequence table SEQ.ID.NO. 1, and the light chain variable region amino acid sequence is shown in a sequence table SEQ.ID.NO. 2.
5. The method according to claim 4, wherein the dispersion medium comprises a buffer.
6. A preparation method of a reagent for detecting the expression level of DLL4 protein or a reagent for sorting DLL4+ DC is characterized in that hybridoma cell strains are inoculated in abdominal cavities of animals, and the ascites fluid of the animals is separated and purified to prepare monoclonal antibodies; mixing the monoclonal antibody with a dispersion medium to prepare a reagent for detecting the expression level of DLL4 protein or a reagent for sorting DLL4+ DC; the hybridoma cell strain is preserved in China general microbiological culture collection center with the preservation address of No. 3 of Xilu No.1 of Beijing Korean district, and the preservation number of CGMCC No. 14283; the heavy chain variable region amino acid sequence of the monoclonal antibody 3F9 is shown in a sequence table SEQ.ID.NO. 1, and the light chain variable region amino acid sequence is shown in a sequence table SEQ.ID.NO. 2.
7. The method according to claim 6, wherein the dispersion medium comprises a buffer.
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|---|---|---|---|---|
| EP2167542A1 (en) * | 2007-05-15 | 2010-03-31 | Smart Targeting Limited | Neutralizing monoclonal antibody against human dll4 |
| CN102264763A (en) * | 2008-09-19 | 2011-11-30 | 米迪缪尼有限公司 | Antibodies directed to dll4 and uses thereof |
| CN102741288A (en) * | 2009-08-29 | 2012-10-17 | 雅培制药有限公司 | Therapeutic dll4 binding proteins |
| CN105384818A (en) * | 2015-12-17 | 2016-03-09 | 中国药科大学 | Anti-human Delta like 4 monoclonal antibody and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2167542A1 (en) * | 2007-05-15 | 2010-03-31 | Smart Targeting Limited | Neutralizing monoclonal antibody against human dll4 |
| CN102264763A (en) * | 2008-09-19 | 2011-11-30 | 米迪缪尼有限公司 | Antibodies directed to dll4 and uses thereof |
| CN102741288A (en) * | 2009-08-29 | 2012-10-17 | 雅培制药有限公司 | Therapeutic dll4 binding proteins |
| CN105384818A (en) * | 2015-12-17 | 2016-03-09 | 中国药科大学 | Anti-human Delta like 4 monoclonal antibody and application thereof |
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