Amplification method for T cell receptor
A technology of cell receptors and nuclear cells, applied in the field of biomedicine, can solve the problems of low amplification efficiency, achieve the effect of improving the amplification efficiency and purifying T cell receptors
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[0059] Preparation of lentivirus containing TCR chain
[0060] After the above-mentioned TCR gene that was initially verified by IMGT was synthesized into a gene fragment by gene synthesis, it was assembled into a complete TCR chain by the method of overlapPCR, in which the constant region of TCR was replaced by the constant region of mouse origin to avoid the imported TCR Mismatches with endogenous TCR genes and facilitate subsequent testing of TCR gene expression. The PCR product was cut with NotI and NheI and then subcloned into the pHAGE vector (pHAGE vector is a plasmid, the TCR gene is placed on this vector to be packaged into a lentivirus together with psPAX2 and pMD2.G 3 plasmids, so that the TCR gene into lymphocytes). After the correct sequence was verified by sequencing, 293T cells were co-transfected with psPAX2 and pMD2.G 3 plasmid packaging system. 24 hours after transfection, collect the first batch of lentiviral supernatant; 48 hours later, collect the second...
Embodiment 1
[0065] Example 1 Expansion of T cell receptors
[0066] Fresh peripheral blood was drawn from healthy donors. After spraying with alcohol, transfer to a sterile 50ml centrifuge tube. Dilute it with DPBS (full name: Dulbecco’s Phosphate-buffered saline, Chinese name: phosphate-buffered saline) (purchased from Life Technologies) at a ratio of 1:1 before use. Take 2 sterile 50ml centrifuge tubes, and add 15ml of lymphocyte separation medium (purchased from GE) to each tube. Add 25ml of 1:1 diluted peripheral blood to each tube of lymphocyte separation medium, and centrifuge at 20°C for 30min with a relative centrifugal force of 400g, where g is the acceleration of gravity. After centrifugation, the liquid in the centrifuge tube is divided into 4 layers, from top to bottom: yellow plasma layer, buffy coat layer, colorless and transparent lymphocyte separation liquid layer, and red and black mixed cell layer. Carefully draw the buffy coat into a new 50ml centrifuge tube, add DPB...
Embodiment 2
[0072] Example 2 Preparation of lentivirus containing TCR chain
[0073] After the above-mentioned TCR gene that was initially verified by IMGT was synthesized into a gene fragment by gene synthesis, it was assembled into a complete TCR chain by the method of overlapPCR, in which the constant region of TCR was replaced by the constant region of mouse origin to avoid the imported TCR Mismatches with endogenous TCR genes and facilitate subsequent testing of TCR gene expression. The PCR product was cut with NotI and NheI and then subcloned into the pHAGE vector. After the correct sequence was verified by sequencing, 293T cells were co-transfected with psPAX2 and pMD2.G 3 plasmid packaging system. 24 hours after transfection, collect the first batch of lentiviral supernatant; 48 hours later, collect the second batch of lentiviral supernatant.
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