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Preparation method of anti-human DLL4 monoclonal antibody 3F9

A monoclonal antibody and hybridoma cell line technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, anti-animal/human immunoglobulin, etc., to achieve high recognition ability, high specificity, and high titer effect

Active Publication Date: 2018-01-09
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 3F9
  • Preparation method of anti-human DLL4 monoclonal antibody 3F9
  • Preparation method of anti-human DLL4 monoclonal antibody 3F9

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Embodiment 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] Numbering

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 Isolation...

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Abstract

The invention discloses a preparation method of an anti-human DLL4 monoclonal antibody 3F9 which is obtained through secretion of a hybridoma cell strain preserved in CGMCC (China General Microbiological Culture Collection Center) at yard 3, No. 1 Beichen west road, Chaoyang District, Beijing City on June 7, 2017 with the preservation number of CGMCC No.14283. The anti-human DLL4 monoclonal antibody 3F9 is named as hybridoma cell strain 3F9 for secreting the anti-human DLL4 monoclonal antibody. The hybridoma cell strain has the classification and name of hybridoma cell strain 3F9 for secretingthe anti-human DLL4 monoclonal antibody. The capability of DLL4+DC for inducing Na-ve T cells to differentiate in the Th1 direction cannot be blocked through binding of the monoclonal antibody 3F9 and DC as compared with commercialized MHD4-46.

Description

technical field [0001] The invention relates to a monoclonal antibody, in particular to a preparation method of the anti-human DLL4 monoclonal antibody 3F9. 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 colleague...

Claims

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Application Information

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IPC IPC(8): C07K16/28C12N5/20C12N5/0784G01N33/68G01N33/577C12R1/91
Inventor 金美芳李刚周慧婷汪健宿广昊方芳谢翌
Owner SOOCHOW UNIV AFFILIATED CHILDRENS HOSPITAL
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