A kind of isolated protein binding antigen psma and its use
A technology for binding proteins and chimeric antigen receptors, applied in the field of biomedicine, can solve the problems of unknown epitope information, lack of cross-germline cross-reactivity, etc., and achieve the effect of relieving and/or treating tumors
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Embodiment 1
[0181] Example 1. Generation and screening of monoclonal hybridoma cells
[0182] 1.1 Preparation of CHO-K1 / cyno PSMA cell stable transfection line
[0183] Lentiviral particles (Jikai Gene, cat#LVCON335) packaged with the nucleic acid sequence encoding cynomolgus monkey PSMA (the Genbank accession number of the corresponding cynomolgus PSMA amino acid sequence is XP_ 005579379) were packed with M.O.I.=100 ((M.O.I. = (slow Virus particle titer * volume) / number of infected cells) to infect CHO-K1 cells (ATCC, cat# CCL-61). Cells after 48 hours of infection were subcultured at a ratio of 1:10 with 8 µg / ml Screening resistance and F2K medium with 10% (w / v) fetal bovine serum for about 1 week until CHO-K1 in the uninfected control group was screened for resistance and killed, while CHO-K1 cells in the infected group still survived. CHO-K1 cells in the lentivirus infection group were digested and limitedly diluted to 0.5 cells per well in a 96-well plate, and then cultured with ...
Embodiment 2
[0190] Example 2. Sequencing, Expression and Purification of Monoclonal PSMA Antibody
[0191] The monoclonal PSMA antibody screened in Example 1 was sequenced, and the amino acid sequence is shown in Table 1.
[0192] Table 1. Sequencing results of monoclonal PSMA antibodies
[0193]
[0194] The heavy chain variable region sequence of the above monoclonal PSMA antibody was subcloned into pTT5 expression vector containing signal peptide and human heavy chain IgG1 constant region (SEQ ID NO: 19). The light chain variable region sequence of the monoclonal PSMA antibody was subcloned into an expression vector containing the signal peptide and the human antibody light chain kappa constant region (SEQ ID NO: 20). After the recombinant plasmid was confirmed by sequencing, the plasmid was extracted with a large extraction kit (Macherey-Nagel, NucleoBond® Xtra Midi) to improve the purity and quality of the recombinant plasmid, and the plasmid was filtered through a 0.22 µm filter...
Embodiment 3
[0200] Example 3. Binding Ability of Monoclonal PSMA Antibody to Cell Surface PSMA
[0201] HEK293T cells overexpressing human PSMA (HEK293T / human PSMA, Kangyuan Bochuang, cat#KC-1005) or CHO-K1 cells overexpressing cynomolgus PSMA (CHO-K1 / cyno PSMA) or overexpressing human PSMA Tumor cells (LNCAP) were cultured and expanded in T-75 culture flasks. After reaching 90% confluency, the medium was aspirated and the cells were washed twice with PBS. Cells were treated with trypsin (Invitrogen, cat#15050065) for about 1 minute, and the trypsin was neutralized with culture medium. Then the cells were washed twice with PBS, the cell count was determined, and the cells were resuspended in PBS to 2×10 6 cells / ml. Add 100 µl of cell suspension to each well of a 96-well V-plate. Incubate with different concentrations of purified PSMA antibody or isotype control antibody for 1 hour on ice. Cells were washed twice with PBS and incubated with goat anti-human (H+L)-Alexa Fluor 647 (Life t...
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