Anti-PD-1 antibody and application thereof
A PD-1, PD-L1 technology, applied in the medical field, can solve problems such as the inability to effectively stimulate T cell immunity, and achieve the effect of improving the level and inhibiting tumor growth.
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Embodiment 16
[0043] The preparation of embodiment 1.6# antibody
[0044] 1. Construction of PD-1 recombinant expression plasmid
[0045] Using Origene's PD-1 cDNA cloning plasmid SC117011 as a template, design two primers and introduce restriction sites SgfI and MluI respectively, and clone them into the expression vector pCMV6-Entry to establish a recombinant eukaryotic expression plasmid for PD-1 full-length protein .
[0046] The cloned primer sequences are as follows:
[0047] Upstream amplification primer sequence: CAC GCGATCGC ATGCAGATCCCACAGGCGC
[0048] Downstream amplification primer sequence: ACCG ACGCGT GAGGGGCCAAGAGCAGT
[0049] 2. Expression and purification of PD-1 recombinant protein
[0050] 1) Transfection of HEK293T cells: HEK293T cells were transferred to culture dishes at a ratio of 1:3 to continue culture; take 7.5mL DMEM (without serum and antibiotics) into a 50mL tube, add 300μL polyetherimide (PEI) MegaTran 1.0 and mix well ;Add 75μg PD-1 recombinant plasmi...
Embodiment 2
[0081] Example 2. PD-1 blocking antibody screening and affinity analysis
[0082] Mice were immunized with the PD-1 protein expressed in vitro by 293T cells, and the obtained monoclonal antibodies were screened for antibodies that could specifically block the interaction between PD-1 and PD-L1 by blocking experiments on PD-1 and PD-L1.
[0083] 1. Preparation of 293T cells expressing full-length PD-L1
[0084] In this example, by transfecting 293T cells (ATCC) with the full length of PD-L1 (pEGFP-N1 carrier-GFP tag plasmid, PD-L1-GFP-p (Clontech Company), the PD-L1 expressing the full length 293T cells. One day before transfection, according to 0.5~2×10 5 The cells were inoculated into each well of a 24-well culture plate, and 500 μl of complete DMEM medium (GIBCO Company) without antibiotics was added to ensure that the cells reached 70-80% confluence during transfection. 1 μg of PD-L1-GFP-p plasmid was diluted in 50 μl of medium without serum and antibiotics, and mixed gen...
Embodiment 3
[0087] Example 3. The ability of PD-1 blocking antibody to activate influenza-specific T cells
[0088] Blocking the combination of PD-1 and PD-L1 can release T cell activity and increase the proliferation and cytokine secretion of specific T cells. Therefore, in this example, the peripheral blood of healthy individuals was collected, and influenza-specific T cells were expanded and cultured in vitro, and then the influenza-specific T cells were detected by enzyme-linked immunospot test (ELISPOT) after adding PD-1 blocking antibodies. The effect on the level of responsive T cells and nonspecific T cell responses was evaluated.
[0089] 1. Isolation of peripheral blood lymphocytes (PBMCs) from healthy volunteers:
[0090] The lymphocytes used in the present invention come from the venous peripheral blood of healthy individuals. After the selected individuals pass the physical examination of the clinician, the experimenter will inform the specific project process and the amoun...
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