TCR (T-cell receptor) capable of recognizing short peptide of SAGE1 (sarcoma antigen 1)
A VFSTVPPAFI-HLAA2402, variable technology, applied in the field of TCR, can solve problems such as normal cell damage
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Embodiment 1
[0139] Example 1 Cloning of Antigen Short Peptide-Specific T Cells
[0140] Peripheral blood lymphocytes (PBL) from healthy volunteers with genotype HLA-A2402 were stimulated with a synthetic short peptide SAGE1PX149 597-606VFSTVPPAFI (Beijing Saibaisheng Gene Technology Co., Ltd.). SAGE1PX149597-606VFSTVPPAFI short peptide was refolded with biotin-labeled HLA-A*2402 to prepare pHLA monomer. These monomers were combined with PE-labeled streptavidin (BD Company) to form PE-labeled tetramers, and the tetramers and anti-CD8-APC double-positive cells were sorted. Sorted cells were expanded and subjected to secondary sorting as described above, followed by monoclonal culture by limiting dilution. Monoclonal cells were stained with tetramers, and the double-positive clones screened were as follows: image 3 shown.
Embodiment 2
[0141] Example 2 Obtaining the construction of the TCR gene and carrier of the antigen short peptide-specific T cell clone of the present invention
[0142] with Quick-RNA TM MiniPrep (ZYMO research) extracted the total RNA of the SAGE1PX149597-606VFSTVPPAFI-specific, HLA-A2402-restricted T cell clone screened in Example 1. The cDNA was synthesized using clontech's SMART RACE cDNA amplification kit, and the primers used were designed at the C-terminal conserved region of the human TCR gene. The sequence was cloned into T vector (TAKARA) for sequencing. After sequencing, the sequence structures of the TCR α chain and β chain expressed by the double-positive clone are shown in Figure 1 and Figure 2, respectively. Figure 1a , Figure 1b , Figure 1c and Figure 1d They are the amino acid sequence of TCRα chain variable domain, the nucleotide sequence of TCRα chain variable domain, the amino acid sequence of TCRα chain and the nucleotide sequence of TCRα chain; Figure 2a ,...
Embodiment 3
[0152] Example 3 Expression, refolding and purification of antigenic short peptide-specific soluble TCR of the present invention
[0153] In order to obtain a soluble TCR molecule, the α and β chains of the TCR molecule of the present invention may only include their variable domains and part of the constant domains respectively, and a cysteine residue is introduced into the constant domains of the α and β chains respectively To form an artificial interchain disulfide bond, the positions of the introduced cysteine residues are Thr48 of TRAC*01 exon 1 and Ser57 of TRBC2*01 exon 1; the amino acid sequence and nucleotides of the α chain sequence as Figure 4a and Figure 4b As shown, the amino acid sequence and nucleotide sequence of its β chain are as follows Figure 5a and Figure 5b The introduced cysteine residues are shown in bold and underlined letters. The target gene sequences of the above TCRα and β chains were synthesized and inserted into the expression vecto...
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