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Preparation method of anti-human DLL4 monoclonal antibody 6F12

A monoclonal antibody, hybridoma cell line technology, applied in biochemical equipment and methods, anti-animal/human immunoglobulin, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, etc. Specificity, high recognition ability, high titer effect

Active Publication Date: 2017-11-24
SOOCHOW UNIV AFFILIATED CHILDRENS HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no commercially available monoclonal antibody that can be used to label and sort DLL4+ DCs by flow cytometry while retaining the signaling function of DLL4

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Preparation of anti-human DLL4 monoclonal antibody

[0046] 1. Establishment of transgenic cells CHO / DLL4

[0047] (1) Cloning of human DLL4 gene

[0048] The plasmid containing the full-length CDS fragment of human DLL4 was donated by Han Jiahuai Laboratory of Xiamen University. PCR amplification was carried out with the designed primers with restriction enzyme sites (Table 1). The reaction conditions were denaturation at 94°C for 60s, annealing at 55°C for 60s, and extension at 72°C for 2min. The full-length fragment was obtained by extending for 5 min; the PCR product was purified by a recovery kit.

[0049] serial number

nucleic acid sequence

forward primer

5'-CGCGGATCCATGGCGGCAGCGTCCC-3'

reverse primer

5’-CCGGAATTCTTATACCTCCGTGGCAATGACAC-3’

[0050] (2) Construction of human DLL4 expression vector

[0051] The recovered PCR product and expression vector pcDNA3.1 were cut with restriction endonucleases BamH I and...

Embodiment 2

[0066] Example 2 In vitro biological effect of monoclonal antibody on DC

[0067] This example describes the effect of the anti-human DLL4 monoclonal antibody of the present invention on the differentiation process of DLL4-positive mDCs inducing CD4+ Naïve T cells to Th1

[0068]Human PBMCs were obtained from fresh human peripheral blood by Ficoll separation. A commercially available sorting kit (CD1c + Dendritic Cell Isolation Kit), CD1c was isolated from PBMC according to the experimental protocol provided by Meitini + DC, the obtained cells were detected by flow cytometry, and the purity was above 90%. Cultured in RPMI-1640 medium containing 10% FBS, DC was stimulated by adding R848 (final concentration 1ug / ml) and LPS (final concentration 100ng / ml) for 24h.

[0069] On the second day, another fresh human peripheral blood was used to obtain human PBMCs by Ficoll separation. Using a commercially available sorting kit from Stem Cell (EasySep Human CD4 + T cell Isolatio...

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Abstract

The invention discloses a preparation method of anti-human DLL4 monoclonal antibody 6F12. The anti-human DLL4 monoclonal antibody 6F12 is obtained by secreting a hybridoma cell strain. The preservation information of the hybridoma cell strain is that: preservation institute: China General Microbiological Culture Collection Center (CGMCC); preservation address: 3#, No. 1 courtyard, Beichen West Road, Chaoyang District, Beijing; preservation time: June, 7th, 2017; preservation number: CGMCC No. 14284; classification and naming: secreted anti-human DLL4 molecule monoclonal antibody hybridoma cell strain 6F12. Compared with commercial clonal MHD4-46, the combination of the monoclonal antibody 6F12 and DC cannot block the capability of inducing, by DLL4+DC, Naive T cells to be differentiated toward a Th1 direction.

Description

technical field [0001] The invention relates to a monoclonal antibody, in particular to a preparation method of the anti-human DLL4 monoclonal antibody 6F12. Background technique [0002] The Notch signaling pathway is a highly conserved signal system in evolution, which plays an important role in cell proliferation, differentiation and apoptosis, as well as cell growth and various physiological functions. Notch signaling molecules are expressed in most multicellular organisms. Mammals mainly express four Notch receptors (respectively: Notch1, 2, 3, 4) and five Notch ligands (respectively: DLL1, DLL3, DLL4, Jagged1 and Jagged2). [0003] Early studies have shown 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 stimulation of bone marrow cells containing antigen-presenting cells by LPS could induce the expression of DLL4. In 2007, Skokos and colleagu...

Claims

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Application Information

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IPC IPC(8): C07K16/28C12N5/20G01N33/68G01N33/577
CPCC07K16/28G01N33/577G01N33/6863G01N2333/705
Inventor 汪健李刚胡筱涵李毅平许云云赵赫周慧婷
Owner SOOCHOW UNIV AFFILIATED CHILDRENS HOSPITAL
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