Targeted VEGFR-2 and/or VEGFR-3 chimeric antigen receptor and application thereof
A VEGFR-2, VEGFR-3 technology, applied in the field of biomedicine, can solve the problems of recurrence, metastasis, drug resistance, etc.
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Embodiment 1
[0080] Example 1: Cloning of Antigen Recognition Region VEGF-Cf in Chimeric Antigen Receptor
[0081] 1. Collect the MSCs of the cultured AML patients and extract the total RNA of the cells: at 5×10 6 Add 1 ml of RNA isoPlus (Takara) to the cells, and mix well by pipetting. Add 200 μl of chloroform, invert up and down, and vortex to mix. Centrifuge at 12000 rpm for 5 minutes at 4°C. Pipette the supernatant into a 1.5ml EP tube, add the same volume of isopropanol, and mix by gently inverting up and down. Centrifuge at 12000 rpm for 15 minutes at 4°C. Pre-cool 75% ethanol to precipitate RNA at 4°C, and dissolve total RNA in 50 μl DEPC water.
[0082] 2. Synthesize the first strand of cDNA by reverse transcription: Prepare the PCR reaction system (20 μl) as follows: Oligo d(T)15Primers: 2 μl; M-MLV (200u / μl): 1 μl; dNTP (each 2.5mM): 1 μl; DTT ( 0.1M): 2μl; First strand buffer (5×): 4μl; MSC-RNA: 2μg; DEPC water: make up to 20μl. Reaction conditions: 37°C, 60 minutes, 70°C,...
Embodiment 2
[0087] Example 2: Construction of Chimeric Antigen Receptor Vector
[0088] 1. Use BamH I and EcoR I endonucleases to digest the previously constructed plasmid containing the CD8α-CD28-CD3ζ fragment (such as "Construction of a new anti-CD19 chimeric antigen receptor and the anti-leukemia function study of the transduced T cells ") to obtain the CD8α-CD28-CD3ζ fragment, the amino acid sequence of which is shown in SEQ ID NO.5.
[0089] 2. The VEGF-Cf fragment obtained in Example 1 is connected to the destination vector to construct the pCDH-VEGF-Cf-CD8α-CD28-CD3ζCAR destination vector. BamHI and EcoRI were used for enzyme digestion identification. The result is as figure 2 As shown, the enzyme digestion results showed that the positive clone contained the target band and was correctly identified by sequencing. The schematic diagram of the carrier is as image 3 shown.
Embodiment 3
[0090] Example 3: Preparation of Chimeric Antigen Receptor VEGF-Cf-CD8α-CD28-CD3ζ Lentiviral Modified T Cells
[0091] 1. The pCDH-VEGF-Cf-CD8α-CD28-CD3ζ expression plasmid and packaging plasmids psPAX2 and pMD2.G were extracted using EndoFree Plasmid Maxi Plasmid Extraction Kit (QIAGEN Company). The three plasmids were transfected with Turbofect Transfection Reagent (Thermo Company) at a ratio of 4:3:1 (for specific methods, see Turbofect Transfection Reagent Manual). The virus supernatant was collected 48 hours and 72 hours after transfection, centrifuged at 4°C, 3000rpm for 10 minutes, filtered through a 0.45μm filter, concentrated 10 times by ultracentrifugation at 50000g, 4°C for 2 hours, and then transferred to - Store at 80°C.
[0092] 2. Preparation of T cells: Take 10 ml of fresh healthy human peripheral blood, and use RosetteSep T cell enrichment Cocktail (Stemcell) and Ficoll-Paque PLUS (GE Healthcare) to extract T cells (specific steps follow RosetteSep T cell enr...
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