Human IL1RAP (IL-1 receptor accessory protein) specific CAR (chimeric antigen receptor) and application thereof
A specific, co-stimulatory molecule technology, applied in the biological field, can solve the problems of reducing anti-tumor ability and low T cell activation rate, and achieve the effect of increasing T cell activation rate and enhancing killing ability
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[0032] Example 1 V H and V L Gene cloning and identification
[0033] V H Gene refers to the variable region gene of the antibody heavy chain, V L Gene refers to the variable region gene of the antibody light chain, V H Gene and V L After the genes are connected by Linker, scFv is formed. V H and V L Genetically Encoded Antibody V H and V L The fragment is the key site for specific recognition and binding to the antigen, so the antibody V that specifically recognizes human IL1RAP protein was cloned and identified H and V L Genes are the first step.
[0034] Adopt Trizol (purchased from Invitrogen) reagent method to extract 10D8A7 hybridoma cell total RNA, carry out agarose gel electrophoresis detection to the total RNA of extraction, the result is as follows figure 1 Shown in B.
[0035] The first strand of cDNA was synthesized by reverse transcription with reverse transcriptase SuperScriptTMⅢ (purchased from Invitrogen), and stored at -20°C for future use.
[00...
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[0039] Example 2 Construction of anti-human IL1RAP scFv
[0040] pMD18-10D8A7V H and pMD18-10D8A7V L The sequence is used as a template to amplify V H and V L Gene, amplified by overlap extension PCR and ligated as anti-human IL1RAP scFv, named 10D8A7scFv. The primer sequence is SEQ ID NO: 6-9.
[0041] Reaction conditions: Denaturation at 94°C for 1 rnin, annealing at 58°C for 1 rnin, extension at 72°C for 1.5 min, a total of 30 cycles, and finally 72°C for 10 min.
[0042] The total RNA of anti-human IL1RAP hybridoma cells was extracted and identified by agarose gel electrophoresis. The upper two bands are bright, clear and sharp, and the brightness of the first band (28S) is about twice the brightness of the second band (18S), and the lowermost band (5S) is the lightest, in line with Characterization of total RNA. The VH and VL gene fragments of two anti-human IL1RAP antibodies were amplified from total RNA of hybridoma cell lines. The results of agarose gel electro...
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[0043] Example 3 Construction and Identification of Anti-human IL1RAP CAR Stable Expression Plasmid
[0044] Refer to the research of Uherek C et al. to design the gene sequence of CAR and synthesize ICD, including CD8α transmembrane segment-CD137 intracellular segment-TCRζ chain, such as figure 2 As shown in middle A, CAR is composed of anti-human IL1RAP antibody scFv, CD8α transmembrane segment, CD137 intracellular segment and TCRζ chain, and BamHI and SalI restriction enzyme sites are respectively introduced at both ends of the sequence, and BamHI site Then introduce hCD8αSignal peptide, and then introduce Stop codon before the SalI site.
[0045] Ligate the CAR into the LV-Lac vector, and the ligation product pattern is as follows figure 2 Shown in B. Transform the ligation product into competent cells DH5α, spread LB homogeneous medium (containing 150 μg / mL ampicillin) on a plate and culture at 37°C for 16 hours, pick a single clone colony in LB medium (containing 150...
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