Anti-cldn-5 antibody, and drug containing said antibody
An antibody, N-terminal technology, applied in the field of anti-CLDN-5 antibody and drugs containing the antibody, can solve the problems of low binding specificity, undisclosed, difficult development of blood-brain barrier regulation technology, etc.
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Embodiment 1
[0599] Example 1) Antibody production
Embodiment 1-1
[0600] Example 1-1) cell fusion
[0601] A DNA fragment comprising the coding sequence of wild-type human CLDN-5 protein (SEQ ID NO: 27) was inserted into a protein expression vector for mammalian cells, pcDNA3.1 (Thermo Fisher Scientific, V79020), to prepare a plasmid for immunization. Subcutaneous immunization was performed using the prepared plasmid for immunization. Through immunization, BXSB mice and Wistar rat individuals whose serum antibodies were elevated were finally immunized. After the final immunization, lymph node cells were collected from the animal according to a conventional method, and were subjected to cell fusion with mouse myeloma cells (P3U1). Seed the fused cells on ten 96-well plates in Medium 1* at 37°C, 10% CO 2 cultured for 10 days.
[0602] *Medium 1: Hybridoma SFM (Thermo Fisher Scientific, 12045084) + 1x BMcondimed H1 Hybridoma cloning supplement (Roche, 1088947), 1x HAT supplement (ThermoFisher Scientific, 21060017), 1x penicillin and strept...
Embodiment 1-2
[0603] Example 1-2) Preparation of Cells for Screening
[0604]A DNA fragment comprising the coding sequence of wild-type human CLDN-5 protein (SEQ ID NO: 27) was inserted into pCX4pur vector (manufactured by Osaka Bioscience Institute) to obtain a vector for retrovirus production. Inoculate 0.5×10 in each well of a 12-well plate 5 Packaging cells (Phoenix A cells) of 10 cells were cultured for 24 hours. Then, 0.5 μg of pCL ampho vector and 0.5 μg of retrovirus production vector were transfected into phoenix A cells using 3 μL of X-treme GENE HP DNA (Roche Diagnosis, 06366244001). The medium was changed after 24 hours and cultured for a further 24 hours. The culture supernatant containing the retrovirus was collected, filtered through a filter with a pore size of 0.45 μm to remove foreign substances, and polybrene (SigmaAldrich, H9268-5G) was added thereto to obtain 8 μg / mL. Using the obtained solution, human fibrosarcoma-derived cells (HT-1080 cells) were cultured for 24...
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