Hybridoma cell line 105d11, antibody and application thereof
A hybridoma cell line and monoclonal antibody technology, which is applied in the fields of application, antibody, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, etc., can solve problems such as listing and unavailable products, and promote phagocytosis Effect, high affinity, good specificity
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Embodiment 1
[0028]Example 1: Construction of CHO-K1 stable cell lines overexpressing human CD47 and cynomolgus monkey CD47
[0029] 1) Shuttle carrier construction
[0030] Human CD47 protein sequence
[0031] NP_001768leukocyte surface antigen CD47 isoform 1precursor[Homosapiens]
[0032] MWPLVAALLLGSACCGSAQLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSWFSPNENILIVIFPIFAILLFWGQFGIKTLKYRSGGMDEKTIALLVAGLVITVIVIVGAILFVPGEYSLKNATGLGLIVTSTGILILLHYYVFSTAIGLTSFVIAILVIQVIAYILAVVGLSLCIAACIPMHGPLLISGLSILALAQLLGLVYMKFVASNQKTIQPPRKAVEEPLNAFKESKGMMNDE
[0033] 35,214Da, 323aa, 1-18 are signal peptides, 19-141,198-207,257-268 are extracellular domains, 142-162,177-197,208-228,236-256,269-289 are transmembrane helices, 163-176,229-235,290-323 are intracellular domains .
[0034] Cynomolgus monkey CD47 protein sequence
[0035] XP_005548289PREDICTED:leukocyte surface antigen CD47 isoform X1[Macaca fascicularis]
[0036] MWPLVAALLL...
Embodiment 2
[0059] Example 2: Animal immunity and antiserum ELISA titer and flow cytometry assay
[0060] Five 6- to 8-week-old Balb / c mice (Beijing Speifu) were selected and housed in the same cage, numbered #1, #2, #3, #4, and #5 respectively. Four days before the first immunization, about 0.05ml of blood was collected from each mouse through the tail vein, placed at 4°C for half an hour, then centrifuged at 10,000rpm at 4°C for 10min, and the serum was separated as negative control serum for subsequent experiments. For the first immunization, the immunogen of recombinant human CD47-mFc fusion protein (Acro Biosystems CD7-H52A5) was fully mixed with an equal volume of Freund's complete adjuvant (SIGMA), and the phacoemulsification was complete, and two subcutaneous and two intraperitoneal injections were taken. For 50 μg of immunogen per mouse, the injection volume was 0.2 ml. The second and third immunizations were carried out at intervals of 14 days and 35 days respectively. The diff...
Embodiment 3
[0064] The preparation of embodiment 3 hybridoma monoclonal cell lines
[0065] 1. Cell Fusion
[0066] Cell fusion was performed using the polyethylene glycol method. The specific operation is as follows:
[0067]1) One week before fusion, revive SP2 / 0-Ag14 myeloma cells (Beijing Beina). Two days before cell fusion, expand the culture of SP2 / 0-Ag14 so that it is in logarithmic growth phase on the day of fusion.
[0068] 2) Half an hour before cell fusion, pre-treat SP2 / 0-Ag14 cells, resuspend SP2 / 0-Ag14 cells and count, take 2-3×10 7 The cells were placed in a 37°C water bath for later use.
[0069] 3) Collect blood from the heart of the mouse to be fused, collect serum in the same manner as above, and store it at -20°C, which can be used as a positive control for screening after fusion. The mice were killed by neck dislocation, soaked in 75% alcohol, and transferred to the cell room. The spleen was taken for grinding, and filtered through a 70 μm sieve to make a single...
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