Bidirectional activated costimulatory molecule acceptor and use thereof
A costimulatory signal, molecular technology, applied in the field of cell biology and immunology, which can solve the problems of lack of bystander function and inability to activate surrounding T cells
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Embodiment 1
[0336] Example 1-(1): 5 recombinant plasmids, pNB328-meso CAR G1, pNB328-meso G2 CAR, PS328b Construction of 137DCR1, PS328b 137DCR2 and PS328b 137DCR3
[0337] 1. Artificially synthesized 137DCR1 gene (SEQ ID NO:15), 137DCR2 gene (SEQ ID NO:16), 137DCR3 gene (SEQ ID NO:17), meso G1 CAR gene (SEQ ID NO:24) and meso G2 CAR gene (SEQ ID NO: 26), the schematic diagram of its structure is as follows: Figure 1A-1 , Figure 1B-1 , Figure 1 C-1, Figure 1E-1 and Figure 1F-1 shown. The 5 synthesized genes were respectively loaded into the PNB328 vector and the PS328b vector, between the EcoRI and SalI restriction sites,
[0338] The pNB328 vector contains elements such as the EF1α promoter and the PB transposon. For the construction of the pNB328 vector, please refer to Example 2 of WO2017054647A1. PS328b is an artificially synthesized sequence, synthesized by Shanghai Jierui Bioengineering Co., Ltd., and the sequence is shown in SEQ ID NO:27.
[0339] The constructed reco...
Embodiment 1-
[0344] Example 1-(3): 5 kinds of recombinant plasmids, namely pNB328-EGFR G1 CAR, pNB328-EGFR G2 CAR, PS328b Construction of 40DCR1, PS328b 40DCR2 and PS328b 40DCR3
[0345] 1. Artificially synthesized 40DCR1 gene (SEQ ID NO:65), 40DCR2 gene (SEQ ID NO:66), 40DCR3 gene (SEQ ID NO:67), EGFR G1CAR gene (SEQ ID NO:74) and EGFR G2 CAR gene (SEQ ID NO: 75), its structural schematic diagram is respectively as follows Figure 1A-3 , Figure 1B-3 , Figure 1C-3 , Figure 1F-3 and Figure 1D-3 shown. The 5 synthetic genes were respectively loaded into the pNB328 vector and the PS328b vector, between the EcoRI and SalI restriction sites,
[0346] The pNB328 vector contains elements such as the EF1α promoter and the PB transposon. For the construction of the pNB328 vector, please refer to Example 2 of WO2017054647A1. PS328b is an artificially synthesized sequence, synthesized by Shanghai Jierui Bioengineering Co., Ltd., and the sequence is shown in SEQ ID NO:27.
[0347] The c...
Embodiment 2-
[0348] Example 2-(1): Nine kinds of chimeric antigen receptor modified T cells, namely recombinant cells meso G1CAR, meso G2 CAR, meso G1 CAR-137DCR1, meso G1 CAR-137DCR2, meso G1 CAR-137DCR3, 137DCR1, Construction and Identification of 137DCR2, 137DCR3 and Mock T
[0349] (1) Construction of 9 kinds of recombinant cells
[0350] Peripheral blood mononuclear cells (PBMCs) were adhered and cultured for 2-4 hours, and the unattached suspension cells were naive T cells. Collect the suspended cells into a 15ml centrifuge tube, centrifuge at 1200rmp for 3min, discard the supernatant; add normal saline, centrifuge at 1200rmp for 3min, discard the normal saline, and repeat the steps of "adding normal saline, centrifuging at 1200rmp for 3min, discarding the normal saline" 3 times.
[0351] Take 8 1.5ml centrifuge tubes and add 5×10 6 The above-mentioned cells, numbered a, b, c, d, e, f, g, h, were centrifuged at 1200rmp for 3min, the supernatant was discarded, and the electrotr...
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