CN107383195B

CN107383195B - Preparation method of anti-human DLL4 monoclonal antibody 6F12

Info

Publication number: CN107383195B
Application number: CN201710677650.3A
Authority: CN
Inventors: 汪健; 李刚; 胡筱涵; 李毅平; 许云云; 赵赫; 周慧婷
Original assignee: Affiliated Childrens Hospital of Soochow University
Current assignee: Affiliated Childrens Hospital of Soochow University
Priority date: 2017-08-09
Filing date: 2017-08-09
Publication date: 2020-04-21
Anticipated expiration: 2037-08-09
Also published as: CN107383195A

Abstract

The invention discloses a preparation method of an anti-human DLL4 monoclonal antibody 6F12, 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. 14284; and (3) classification and naming: and (3) a hybridoma cell strain 6F12 secreting an anti-human DLL4 molecular monoclonal antibody. Binding of monoclonal antibody 6F12 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

Preparation method of anti-human DLL4 monoclonal antibody 6F12

Technical Field

The invention relates to a monoclonal antibody, in particular to a preparation method of an anti-human DLL4 monoclonal antibody 6F 12.

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 6F12, 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 6F12, wherein the anti-human DLL4 monoclonal antibody 6F12 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. 14284.

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.

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.

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 techniques 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 the China general microbiological culture Collection center and are classified and named as monoclonal antibody hybridoma cell strains 6F12 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. 14284; and (3) classification and naming: and (3) a mouse anti-human DLL4 molecule monoclonal antibody hybridoma cell strain 6F12 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 6F 12.

The invention also provides the heavy chain variable region amino acid sequence of the monoclonal antibody 6F 12: 1, SEQ ID no; light chain variable region amino acid sequence: SEQ ID No. 2.

SEQ.ID.NO:1：

EVHVKQSGPELVKPGASVKMSCKASGYTFTSYLLHWVKQKPGQGLEWIGYIIPYNDGTRYNEKFKGKATLTSDKSSNTAYMELSSLTSEDSAVYYCAREGTGTGAFDYWGQGTSLTVSS

Corresponding to:

Glu Val His Val Lys Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala SerVal Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Leu Leu His TrpVal Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile Gly Tyr Ile Ile Pro Tyr AsnAsp Gly Thr Arg Tyr Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ser Asp LysSer Ser Asn Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala ValTyr Tyr Cys Ala Arg Glu Gly Thr Gly Thr Gly Ala Phe Asp Tyr Trp Gly Gln GlyThr Ser Leu Thr Val Ser Ser

SEQ.ID.NO:2：

DIVLTQSPAIMSASPGEKVTMTCRASSSVNYMYWYQQKPGSSPRLLIYDTSNLASGVPVRFSGSGSGTSYSLTISRMEAEDAATYYCQQWSSYPYTFGGGTKLEIK

corresponding to:

Asp Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly GluLys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Asn Tyr Met Tyr Trp Tyr GlnGln Lys Pro Gly Ser Ser Pro Arg Leu Leu Ile Tyr Asp Thr Ser Asn Leu Ala SerGly Val Pro Val Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr IleSer Arg Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser TyrPro 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 6F12 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 6F12 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 6F12 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 6F12 hybridoma cell line in example I;

FIG. 3 is a graph showing the results of antigen recognition by Western Blot analysis of monoclonal antibody 6F12 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 6F12 in example one;

FIG. 5 is a graph showing the results of analyzing competitive inhibition of the antigenic site recognized by monoclonal antibody 6F12 by flow cytometry in the first example;

FIG. 6 is a flow cytometric analysis of the effect of anti-human DLL4 monoclonal antibody 6F12 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 6F12 capable of stably secreting specific mouse anti-human DLL4 antibody is obtained.

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

Female Balb/c mice, 6-8 weeks old, were injected intraperitoneally with Pristane (0.5 ml/mouse). One week later, the abdominal cavity was inoculated with hybridoma cells (1X 10)⁶/one) while an equal volume of a mixture of Pristane and incomplete freund's adjuvant (0.2 ml/one) was again injected intraperitoneally. Harvesting ascites after 5-10 days, centrifuging, collecting supernatant, and storing at-80 deg.C。

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 6F12 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 a test paper (Argen corporation), which indicated that 6F12 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 6F12 (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 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），CD4 was sorted from PBMC 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 6F12 (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 6F12 antibody-affected group compared to the control group (see fig. 6), and independent replicates showed no statistical differences between groups. Indicating that monoclonal antibody 6F12 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 6F12

<160>4

<170>PatentIn version 3.3

<210>1

<211>119

<212>PRT

<213> Artificial sequence

<400>1

Glu Val His Val Lys Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser Val Lys Met

1 5 10 15 20

Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Leu Leu His Trp Val Lys Gln Lys

25 30 35 40

Pro Gly Gln Gly Leu Glu Trp Ile Gly Tyr Ile Ile Pro Tyr Asn Asp Gly Thr Arg Tyr

45 5055 60

Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Asn Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Glu Gly

85 90 95 100

Thr Gly Thr Gly Ala Phe Asp Tyr Trp Gly Gln Gly Thr Ser Leu Thr Val Ser Ser

105 110 115

<210>2

<211>106

<212>PRT

<213> Artificial sequence

<400>2

Asp Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr

1 5 10 15 20

Met Thr Cys Arg Ala Ser Ser Ser Val Asn Tyr Met Tyr Trp Tyr Gln Gln Lys Pro Gly

25 30 35 40

Ser Ser Pro Arg Leu Leu Ile Tyr Asp Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg

45 50 55 60

Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Met Glu Ala Glu

65 70 75 80

AspAla Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Tyr Pro Tyr Thr Phe Gly Gly Gly

85 90 95 100

Thr Lys Leu Glu Ile Lys

105

<210>3

<211>25

<212>DNA

<213> Artificial sequence

<400>3

CGCGGATCCATGGCGGCAGCGTCCC 25

<210>4

<211>32

<212>DNA

<213> Artificial sequence

<400>4

CCGGAATTCTTATACCTCCGTGGCAATGACAC 32

Claims

1. The preparation method of the anti-human DLL4 monoclonal antibody 6F12 is characterized in that the anti-human DLL4 monoclonal antibody 6F12 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. 14284.

2. The method for producing the anti-human DLL4 monoclonal antibody 6F12 of claim 1, wherein a hybridoma cell line is inoculated into a hybridoma culture medium, and the culture medium is separated and purified after culture to produce the monoclonal antibody; the heavy chain variable region amino acid sequence of monoclonal antibody 6F 12: 1, SEQ ID no; light chain variable region amino acid sequence: SEQ ID No. 2.

3. The method for preparing the anti-human DLL4 monoclonal antibody 6F12 as claimed in claim 1, wherein a hybridoma cell line is inoculated into the abdominal cavity of the animal, and the ascites fluid of the animal is separated and purified to prepare the monoclonal antibody; the heavy chain variable region amino acid sequence of monoclonal antibody 6F 12: 1, SEQ ID no; light chain variable region amino acid sequence: SEQ ID No. 2.

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. 14284.

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. 14284.

7. The method according to claim 6, wherein the dispersion medium comprises a buffer.