T cell receptor capable of recognizing SAGE1 antigen, and nucleic acid encoding T cell receptor
A cell receptor and cell technology, applied in the field of TCR, can solve problems such as normal cell damage
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Embodiment 1
[0136] Example 1 Cloning of SAGE1-specific T cells
[0137] Peripheral blood lymphocytes (PBL) from healthy volunteers with genotype HLA-A2402 were stimulated with a synthetic short peptide (Beijing Saibaisheng Gene Technology Co., Ltd.) VFSTVPPAFI (SEQ ID NO.9, named PX149 in the present invention). The short peptide was annealed with biotin-labeled HLA-A*2402 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.
[0138] Due to the long time-consuming process of the entire experiment, many influencing factors, and the complexity of the experiment, the performance of the cells cannot be predicted at all. Even after layers of screening and strict testing, the success rate of obtaining the corresponding ...
Embodiment 2
[0141] Example 2 Construction of the TCR gene and vector for obtaining SAGE1-specific T cell clones
[0142] with Quick-RNA TM MiniPrep (ZYMO research) extracted the total RNA of the VFSTVPPAFI-specific and HLA-A2402-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 α-chain and β-chain of the TCR expressed by the double-positive clone are as follows: Figure 1a-Figure 1f with Figure 2a-Figure 2f as shown, Figure 1a , Figure 1b , Figure 1c with 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 w...
Embodiment 3
[0152] Example 3 Expression, refolding and purification of SAGE1 antigen short peptide-specific soluble TCR
[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 with Figure 4b As shown, the amino acid sequence and nucleotide sequence of its β chain are as follows Figure 5a with Figure 5b The introduced cysteine residues are shown in bold and underlined letters. By the standard method described in > (Molecular Cloning a Laboratory Manual) (third edition, Sambrook and Russell), the target gen...
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