TCR capable of recognizing short peptide of PASD1 antigen
A variable, β-chain technology, applied in the field of TCR, can solve problems such as normal cell damage
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Embodiment 1
[0137] Example 1 Clone-specific T cells
[0138] Peripheral blood lymphocytes (PBL) from healthy volunteers with genotype HLA-A02 were stimulated with the synthetic short peptide QLEERTWLL (Nanjing Jinsikang Biotechnology Co., Ltd.). The QLEERTWLL short peptide was refolded with biotin-labeled HLA-A*0201 to prepare pHLA monomers. 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
[0139] Example 2 Obtaining the construction of the TCR gene and vector of PASD1-specific T cell clones
[0140] with Quick-RNA TM MiniPrep (ZYMO research) extracted the total RNA of the QLEERTWLL-specific, HLA-A02-restricted T cell clones 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 , Figure 2b , Figure 2c and Figure 2d They are the amino aci...
Embodiment 3
[0150] Example 3 Expression, refolding and purification of PASD1 antigen short peptide-specific soluble TCR
[0151] 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 vector pET28a+ (Novagene ...
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