Novel antibody specific for CLEC14A and uses thereof
一种抗体、特异性的技术，应用在抗体、抗体模拟物/支架、非有效成分的医用配制品等方向，能够解决分子机制未被清楚地鉴定等问题
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Embodiment 1
[0171] Example 1: Cell culture and transfection
[0172] Human umbilical vein endothelial cells (HUVEC, Lonza, Baltimore, MD, USA) were maintained in Endothelial Growth Medium-2 (EGM-2). Mouse aortic endothelial cells (MAEC) and COS-7 cells were cultured in Dulbecco's modified Eagle's medium containing 10% (v / v) fetal bovine serum and 1% (v / v) penicillin / streptomycin. Maintain cells at 37 °C and 5% CO 2 Humid CO 2 - In a controlled incubator (Sanyo, Panasonic Healthcare Company, Secaucus, NJ, USA). at 37°C and 8% CO 2 , HEK293F cells were maintained in Freestyle with 1% (v / v) penicillin / streptomycin in a humidified shaking incubator shaker (Infors HT, Bottmingen, Switzerland). TM 293 expression medium (Invitrogen). HUVEC and COS-7 cells were transfected with vectors encoding GFP, clec14a-GFP (wild-type clec14a) or clec14aΔCTLD-GFP (clec14a CTLD deletion mutant) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions .
Embodiment 2
[0173] Embodiment 2: Construct and prepare the CTLD Fc fusion protein of human and mouse
[0174] DNA encoding amino acid residues 31-172 of hCTLD was amplified using primers 5'-TCGCGCGGCCGCTGCTCGGCCTCGGGGGCCTGC-3' (SEQ ID NO: 1) and 5'-TCGCCTCGAGCTTGCACAGGTAGCCGTTGG-3' (SEQ ID NO: 2). DNA encoding the same residues of mCTLD was amplified using primers 5'-TCGCGGCCCAGGCGGCCTGTTCGGCCTCGGGGGCTTG-3' (SEQ ID NO: 3) and 5'-TCGCGGCCGGCCTGGCCCTTGCATAGGTAGCCATCGG-3' (SEQ ID NO: 4). The PCR fragment (NEB, Ipswich, MA, USA) was digested with Sfi I (NEB, Ipswich, MA, USA) and cloned into the modified mammalian expression vector pCEP4 (Invitrogen), wherein pCEP4 was located at 3 of the cloning site. ' region encoding the hinge and CH2-CH3 domains of human IgG1 (a gift from Dr. Chung, Seoul National University, Seoul, South Korea). The ligation product was transformed into competent Escherichia coli DH5α cells, and plasmid DNA was prepared. 0.75 mg of various DNAs were transfected into HE...
Embodiment 3
[0175] Example 3: Pre-cleaning of synthetic human scFv libraries
[0176] The synthetic human scFv library was reamplified (Barbas CF. Phage display: a laboratory manual. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press; 2001). For pre-washing of Fc binders, human immunoglobulin-G (Green Cross Pharma Derivatives Corp, Yong-in, Korea) was immobilized on protein A Sepharose, and antibody-protein A complexes were incubated with the library at 37°C for 2 hours . After brief centrifugation, collect the supernatant and discard the pellet. Repeat this process twice. The final supernatant was analyzed by phage ELISA to assess the extent of washing.
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