A TCR that recognizes short peptides of Prame antigen
A variable, amino acid technology, applied in the field of TCR, can solve problems such as normal cell damage
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Embodiment 1
[0146]Example 1 Cloning PRAME antigen shorthaft specific T cell
[0147]The peripheral blood lymphocytes (PBL) from health volunteers from genotypes are HLA-A0201 were stimulated using synthetic short peptide AVLDGLDVLL (SEQ ID NO.9; Beijing Sai Sheng Gene Technology Co., Ltd.). The AVLDGLDVLL short peptide was represented by HLA-A0201 with biotin-labeled biotoxia, and prepared a PHLA haplot. These haploids were combined with PE-labeled streptavidin (BD) into PE-labeled tetramers, sorting the tetrametics and anti-CD8-APC dicycle. The sorted cells were amplified, and the secondary sorting was carried out in the above method, followed by monoclonal using a limited dilution method. Monoclonal cells were stained with tetramers, screening double-positive clones, screening double positive clones,Figure 15 Indicated.
[0148]The function and specificity of the Sclerotic T cell clones were further detected by ELISPOT experiments. Those skilled in the art are well known to detect the method of det...
Embodiment 2
[0152]Example 2 Acquisition of TCR gene and carrier of PRAME antigen shorthaft specific T cell clones
[0153]Quick-RNATM Miniprep (Zymo Research) extracted the total RNA of antigen short peptide AVLDGLDVLL-screened in Example 1, HLA-A0201 restricted T cell cloned total RNA. The synthesis of cDNA adopts Clontech's Smart Race cDNA amplification kit, which is the primer designed in the C-terminal conserved area of the human TCR gene. Sequence clones were sequenced to T-carriers (TAKARA). It should be noted that the sequence is a complementary sequence, and does not contain introns. Determination, the α chain and β chain sequence structure of the TCR expressed in the double positive cloning are shown in Figures 1 and 2, respectively.Figure 1A ,Figure 1B ,Figure 1C ,Figure 1D ,Figure 1e withFigure 1f The TCRα chain variable domain amino acid sequence, TCRα chain can be variable domain nucleotide sequence, TCRα chain amino acid sequence, TCRα chain nucleotide sequence, TCRα chain amino ac...
Embodiment 3
[0163]Example 3 Expression, rejection and purification of PRAME antigen short peptide-specific soluble TCR
[0164]In order to obtain a soluble TCR molecule, the α and β chains of the TCR molecules of the present invention may contain only their variable domains and partial constant domains, and a cysteine residue is introduced in the constant domain of the α and β chains. To form an artificial chain disulfide bond, the position of the introduction of the cysteine residue is THR48 and TRBC2 * 01 of the TRAC * 01 exon 1 of the SER57 of the SER57 of the Exisseon 1; the amino acid sequence of the α chain and nucleotide Sequence is asFigure 3A withFigure 3B As shown, the amino acid sequence of the beta chain and the nucleotide sequence is respectivelyFigure 4A withFigure 4b As shown, the introduced cysteine residue is expressed in a bold and a crossed letter. The target gene sequence of the above TCRα and the beta chain is inserted into the expression vector PET28A + after the synthe...
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