Coding gene of anti-B7-H3 chimeric antigen receptor, preparation method, plasmid with gene, immune cell and application thereof
A chimeric antigen receptor, B7-H3 technology, applied in the field of genes, can solve the problems of lack of safe and effective specific tumor antigen targets, obstacles, and poor results
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Embodiment 1
[0055] The anti-B7-H3 chimeric antigen receptor coding gene at least contains artificial nucleotide sequences expressing IL15 and CCL21. It also includes antigen binding region, transmembrane structure region, co-stimulatory signal transduction region and T cell signal transduction region functional domain.
[0056] In this embodiment, the anti-B7-H3 chimeric antigen receptor includes sequentially linked
[0057] (1) artificial sequence of leader nucleic acid (SEQ ID NO.2)
[0058] (2) Anti-B7-H3 antigen antibody single chain Fv antibody (scFv) nucleic acid artificial sequence (SEQ ID NO.3)
[0059] (3) CD8 Hinge region nucleic acid artificial sequence (SEQ ID NO.4)
[0060] (4) CD8 transmembrane region nucleic acid artificial sequence (SEQ ID NO.5)
[0061] (5) CD226 intracellular nucleic acid artificial sequence (SEQ ID NO.6)
[0062] (6) CD3ζ intracellular region nucleic acid artificial sequence (SEQ ID NO.7)
[0063] (7) T2A-IL15-CCL21 nucleic acid artificial sequence...
Embodiment 2
[0066] Example of preparation of anti-B7-H3 chimeric antigen receptor coding gene.
[0067] The preparation method of the anti-B7-H3 chimeric antigen receptor coding gene of this embodiment comprises the following steps:
[0068] (1) According to the artificial nucleic acid sequence of the fusion gene fragment guider Leader, the artificial nucleic acid sequence of Anti-B7-H3, the artificial nucleic acid sequence of CD8, the artificial nucleic acid sequence of linker, the artificial nucleic acid sequence of CD8TM, and the artificial nucleic acid sequence of CD226 , CD3ζ nucleic acid artificial sequence, and T2A-IL15-CCL21 nucleic acid artificial sequence entrust Sangon Bioengineering (Shanghai) Co., Ltd. to synthesize the entire expression cassette, insert it into pLent-EF1α-FH-CMV-GFP-P2A-Puro, and obtain pLent-EF1α-CAR(B7-H3)-IL15-CCL21;
[0069] (2) Carry out double enzyme digestion of pLent-EF1α-CAR(B7-H3)-IL15-CCL21, use agar electrophoresis to cut out the agar part of th...
Embodiment 3
[0079] Example of a plasmid with an anti-B7-H3 chimeric antigen receptor encoding gene.
[0080] A plasmid with an anti-B7-H3 chimeric antigen receptor coding gene is prepared by a method comprising the following steps: respectively according to the fusion gene fragment Leader-scFv(B7-H3)-CD8-CD226-4-1BB-CD3ζ-T2A- The sequence of Leader-scFv(CTLA4)-CD8-CD3ζ was inserted into the NotI-AsiSI site of pLent-EF1α-FH-CMV-GFP-P2A-Puro vector (Invitrogen), transformed into E.coli(DH5α), and identified by bacterial liquid PCR Finally, use the plasmid extraction kit of Qiagen company to extract the plasmid, after the plasmid is sequenced correctly, the recombinant plasmid with the correct sequencing result is named pLent-EF1α-CAR(B7-H3)-IL15-CCL21, as figure 1 shown.
[0081] In this embodiment, the more detailed steps are:
[0082] Insert the pLent-EF1α-FH-CMV-GFP-P2A-Puro vector (Invitrogen) NotI-AsiSI in the order of the fusion gene fragment Leader-scFv(Anti-B7-H3)-CD8-CD226-CD3ζ-T...
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