Method for producing chimeric antigen receptor modified gamma delta T cell
A chimeric antigen receptor and cell technology, applied in the biological field, can solve the problems of low cell content, low purity of the final product, low transfection rate, etc.
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Embodiment 1
[0070] Example 1. A method for expanding γδT cells
[0071] 1. Construction of K562-shFPPS tumor cell line with reduced expression of FPPS
[0072] 1. Construction of lentiviral recombinant plasmid
[0073] (1) Preparation of recombinant plasmid
[0074] The U6-based shRNA construction system of the Vectorbuilder system of Saiye Biotechnology Co., Ltd. was used to construct the lentiviral recombinant plasmid. Specific steps are as follows:
[0075] Design and synthesize a specific shRNA coding sequence (shFPPS) targeting FPPS as the experimental group: (Sequence 1); wherein, the bold 6bp sequence in the middle is the stem-loop sequence, its left 21bp sequence is the sense sequence, and the right 21bp sequence is the antisense sequence; at the same time, the following Scramble-shRNA coding sequence (shSRB) was synthesized as a negative control Group: Among them, the bold 6bp sequence in the middle is the stem-loop sequence, the left 21bp sequence is the sense sequence, a...
Embodiment 2
[0115] Example 2. Production method of CAR-γδT cells
[0116] 1. Preparation of CAR22 lentiviral particles
[0117] In the present invention, the CAR structure is used to genetically modify γδT cells. The CAR structure from the amino terminal to the carboxyl terminal is: ScFv(CD22)-Hinge(CD8)-TM(CD8)-CD137-CD3ζ, that is, from the amino terminal to the carboxyl terminal in sequence It is: single-chain variable region, CD8a hinge region and transmembrane region, CD137 signal domain and CD3ζ chain intracellular region derived from CD22 monoclonal antibody (clone number: M971). Its amino acid sequence is shown in sequence 3 of the sequence listing, and its nucleotide sequence is shown in sequence 4 of the sequence listing. Insert the DNA molecule shown in Sequence 4 into the Senl_pLenti-EF1 vector (the Senl_pLenti-EF1 vector is a vector obtained by adding restriction sites PacI and SpeI on both sides of the original plasmid cloning site, and the original plasmid name is LV-pRRLEF...
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