TCR primer group for specifically recognizing EBV peptide fragment with immune subtype of HLAA02 and application of TCR primer group
A primer set, specific technology, applied in the field of genetic engineering
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Embodiment 1
[0039] Example 1 Primer Design
[0040] According to HLAA02 typing, L2-1, L2-2, L2-3, L2-5, L2-6, L2-9, L2-10, L2-11, L2-12, Primer pairs were designed for L2-13, L2-16, L2-19, L2-23, L2-24 and L2-25 sites. The obtained primer sequences are shown in SEQ ID NO.1-30.
Embodiment 2
[0041] Embodiment 2 standard curve is established
[0042] Take the L2-1 site of TCR as an example to design the scheme.
[0043] Take 10 of the plasmid DNA standard at the L2-1 site 8 copy number dilution, 10 7 copy number dilution, 10 6 copy number dilution, 10 5 copy number dilution, 10 4 copy number dilution, 10 3 copy number dilution, 10 2 copy number dilution, 10 1 copy number dilutions, and set up real-time fluorescent PCR according to the following reaction system and reaction procedures.
[0044] Reaction system: PCR master mix 10 μl; forward primer 0.4 μl, primer concentration: 10 μm / μl; reverse primer 0.4 μl, primer concentration: 10 μm / μl; ROX Reference Dye (50×) 0.2 μl; TCR-T cell drug Genomic DNA 2μl, concentration 50ng / μl, or TCR plasmid standard DNA 2μl; Nuclease-Free Water 7μl; total reaction system 20μl.
[0045] PCR reaction program: 95°C, 30s; (95°C, 5s; 60°C, 20s) 40 cycles. Set the reference fluorescence to ROX, select the reaction type as SYBR ...
Embodiment 3
[0050] Embodiment 3 Sensitivity detection
[0051] The genomic DNA stock solution, 10-fold dilution, 100-fold dilution, 1000-fold dilution, and 10000-fold dilution of the TCR-T cell drug directed at the L2-1 site, according to the reaction system and program of real-time fluorescent PCR in Example 2 Amplify. The amplification curve of the genomic DNA of the obtained TCR-T cell drug against the L2-1 site is as follows Figure 4 shown. The amplified products were subjected to agarose gel electrophoresis, and the results were as follows: Figure 5 shown.
[0052] Substitute the obtained Ct value of the genomic DNA of each dilution of the TCR-T cell drug into the standard curve in Example 2, and the copy number of the genomic DNA of each dilution of the TCR-T cell drug can be calculated.
[0053] After calculation, the copy number of the TCR-T cell drug genomic DNA stock solution targeting the L2-1 site is 19079 copies, the copy number of the 10-fold dilution is 1721 copies, a...
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