Chimeric antigen receptor T cell capable of conducting allograft and preparation method
A chimeric antigen receptor and allogeneic transplantation technology, applied in the field of tumor treatment, can solve the problems of inability to effectively kill tumor cells, difficult separation of T cells, mixed tumor cells, etc., and achieve the effect of facilitating tumor treatment.
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Embodiment 1
[0046] Allograft TRAC-inactivated T cells combined with third-generation CAR to treat tumors
[0047] In this example, CRISPR / Cas9 technology was used to knock out the gene TRAC encoding the constant region of the alpha chain of TCR. For the exons of TRAC, including exon 1, exon 2, exon 3, and exon 4, exon 1 of TRAC was knocked out using CRISPR / Cas9 technology. According to the exon sequence of TRAC, especially the sequence of exon 1, the sequence designed to express sgRNA (singleguideRNA, sgRNA) includes: Seq-TRAC-EX1-1, Seq-TRAC-EX1-2, Seq-TRAC-EX1 -3, Seq-TRAC-EX1-4, Seq-TRAC-EX1-5.
[0048] Further, the above sequence, especially the sequence of Seq-TRAC-EX1-1 is reversely complementary to obtain the reverse complementary sequence of Seq-TRAC-EX1-1 (see sequence Seq-TRAC-EX1-1-R).
[0049] Furthermore, it is also necessary to add double restriction sites BamHI and EcoRI at both ends of Seq-TRAC-EX1-1 and Seq-TRAC-EX1-1-R to facilitate the construction of entry vectors af...
Embodiment 2
[0058] Allograft TCRBC1-inactivated T cells combined with third-generation CAR to treat tumors
[0059] In this example, the CRISPR / Cas9 technology was used to knock out the gene TCRBC1 encoding the constant region of the β chain of TCR. For exons of TCRBC1, including exon 1, exon 2, exon 3 and exon 4, exon 1 of TCRBC1 was knocked out by CRISPR / Cas9 technology. According to the exon sequence of TCRBC1, especially the sequence of exon 1, the sequences designed to express sgRNA (singleguideRNA, sgRNA) include: Seq-TCRBC1-EX1-1, Seq-TCRBC1-EX1-2, Seq-TCRBC1-EX1 -3, Seq-TCRBC1-EX1-4, Seq-TCRBC1-EX1-5.
[0060] Further, the above sequence, especially the sequence of Seq-TCRBC1-EX1-1 is reversely complementary to obtain the reverse complementary sequence of Seq-TCRBC1-EX1-1 (see sequence Seq-TCRBC1-EX1-1-R).
[0061] Further, it is also necessary to add double restriction sites BamHI and EcoRI at both ends of Seq-TCRBC1-EX1-1 and Seq-TCRBC1-EX1-1-R to facilitate the construction o...
Embodiment 3
[0070] Allograft TCRBC2-inactivated T cells combined with third-generation CAR to treat tumors
[0071] In this example, the CRISPR / Cas9 technology was used to knock out the gene TCRBC2 encoding the constant region of the β chain of TCR. For the exons of TCRBC2, including exon 1, exon 2, exon 3 and exon 4, exon 1 of TCRBC2 was knocked out by using CRISPR / Cas9 technology. According to the exon sequence of TCRBC2, especially the sequence of exon 1, the sequence designed to express sgRNA (singleguideRNA, sgRNA) includes: Seq-TCRBC2-EX1-1, Seq-TCRBC2-EX1-2, Seq-TCRBC2-EX1 -3, Seq-TCRBC2-EX1-4, Seq-TCRBC2-EX1-5.
[0072] Further, the above sequence, especially the sequence of Seq-TCRBC2-EX1-1 is reversely complementary to obtain the reverse complementary sequence of Seq-TCRBC2-EX1-1 (see sequence Seq-TCRBC2-EX1-1-R).
[0073] Further, it is also necessary to add double restriction sites BamHI and EcoRI at both ends of Seq-TCRBC2-EX1-1 and Seq-TCRBC2-EX1-1-R to facilitate the cons...
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