Anti-human DLL4 monoclonal antibody 3F9
A monoclonal antibody and hybridoma cell line technology, applied in the direction of anti-animal/human immunoglobulin, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, blood/immune system cells, etc., to achieve high The effect of specificity and high discrimination ability
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Embodiment 1
[0040] Example 1 Preparation of anti-human DLL4 monoclonal antibody
[0041] 1. Establishment of transgenic cells CHO / DLL4
[0042] (1) Cloning of human DLL4 gene
[0043] 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.
[0044] Numbering
nucleic acid sequence
5'-CGCGGATCCATGGCGGCAGCGTCCC-3'
5’-CCGGAATTCTTATACCTCCGTGGCAATGACAC-3’
[0045] (2) Construction of human DLL4 expression vector
[0046] The recovered PCR product and expression vector pcDNA3.1 were cut with restriction endonucleases BamH I and Eco...
Embodiment 2
[0061] Example 2 In vitro biological effect of monoclonal antibody on DC
[0062] 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
[0063] 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.
[0064] 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 Isolati...
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