Anti-CD22 antibody and application thereof
A technology of antibody and chimeric antigen receptor, which is applied in the field of biomedicine to achieve efficient targeting, easy preparation, and simple structure
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Embodiment 1
[0104] In this example, a phage nanobody library was constructed and panning and ELISA preliminary screening were performed.
[0105] 1. Construction of phage nanobody library
[0106] (1) Bactrian camels were immunized with CD22-Fc expressing the extracellular region, and after the titer was verified by ELISA, 200 mL of peripheral blood was drawn;
[0107] (2) Sorting lymphocytes, obtaining peripheral blood mononuclear lymphocyte precipitates, and extracting RNA;
[0108] (3) use III reverse transcriptase uses RNA as a template to synthesize the first-strand cDNA, and then uses nested PCR to amplify the VHH gene;
[0109] (4) Insert the VHH gene into the pMECS phage display vector, and after electrotransformation of TG1 competent cells, take the bacterial liquid for library identification, spread all the remaining cultures evenly on the LB / AMPGLU plate, collect the bacterial lawn after the bacterial growth, and add 1 / 3 of the volume of 50% glycerin, mixed well, stored at ...
Embodiment 2
[0118] In this example, fluorescence activated cell sorting (FACS) candidate clones were performed.
[0119] Nalm6(CD22 + ), Raji (CD22 + ), K562 (CD22 - ) (both purchased from ATCC) and SK-hep-1 (CD22 - , purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences), the cells were digested with trypsin to prepare CD22-positive and negative cell suspensions, centrifuged (300×g, 5min) to remove the culture medium, and then resuspended the cells to 2×10 6 cell / mL, add 2×10 5 For the cell suspension of 1 cell, remove the supernatant after centrifugation at 300×g for 5 minutes, add the crude extract of VHH antibody to resuspend the cells, and incubate at 4°C for 1 hour, remove the supernatant after centrifugation at 300×g for 5 minutes, resuspend the cells in Flow Buffer, Dilute the APC anti-his antibody to 2 μg / mL with Flow Buffer, resuspend the cells in 100 μL per well, incubate at 4 °C for 1 h, wash the cells with Flow Buffer three times, resuspend the cells wi...
Embodiment 3
[0121] In this example, VHH-mIgG2a Fc nanobody expression, purification and antibody affinity determination were carried out.
[0122] In order to further identify the antibodies screened in Example 2, the antibodies need to be expressed in mammalian cells. Therefore, a plasmid vector C-4pCP.Stuffer-mCg2a-FC with a mouse Fc tag expressing VHH was first constructed, The build method includes the following steps:
[0123] (1) PCR amplification of CD22 VHH: CD22-28 and CD22-29, the reagents used are shown in Table 1, the primers are shown in Table 2, and the system and PCR reaction conditions are shown in Table 3;
[0124] Table 1
[0125]
[0126] Table 2
[0127]
[0128] table 3
[0129]
[0130] (2) The enzyme digestion system and reaction conditions are shown in Table 4 respectively. The PCR purification kit was used for purification, and the air-dried DNA was dissolved in 20 μL of water to detect the concentration of DNA;
[0131] Table 4
[0132]
[0133...
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