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Fluorescent PCR primer group for specifically recognizing IYVLVMLVL peptide fragment and application of fluorescent PCR primer group
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A primer set and specific technology, applied in the field of genetic engineering, can solve problems such as undiscovered
Pending Publication Date: 2022-01-18
重庆天科雅生物科技有限公司
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Problems solved by technology
However, the prior art has not found that the qPCR primer set designed for the TCR gene sequence is used to detect the growth and survival of TCR-T cells in patients
Method used
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Embodiment 1
[0030] Example 1 Primer Design
[0031] According to the HLAA24 typing, the TCR primers were designed at the L24-2 and L24-49 sites of the EBV virus antigen peptide IYVLVMLVL. The sequences of the obtained primers are shown in SEQ ID NO.1-4 and SEQ ID NO.6-9.
Embodiment 2
[0032] Embodiment 2 standard curve is established
[0033] Take the L24-2 site of TCR as an example to design the scheme.
[0034] Take 10 of the plasmid DNA standard at the L24-2 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.
[0035] 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.
[0036] 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 SYB...
Embodiment 3
[0041] Embodiment 3 Sensitivity detection
[0042] 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 L24-2 site, according to the reaction system and program of real-time fluorescent PCR in Example 2 Amplify. Obtain the amplification curve of the genomic DNA of the TCR-T cell drug directed at the L24-2 site as Figure 4 shown. The amplified products were subjected to agarose gel electrophoresis, and the results were as follows: Figure 5 shown.
[0043] 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.
[0044]After calculation, the copy number of the TCR-T cell drug genomic DNA stock solution targeting the L24-2 site is 46766 copies, the copy number of the 10-fold dilution is 7998 copies, and the ...
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Abstract
The invention belongs to the technical field of genetic engineering and particularly relates to a fluorescent PCR primer group for specifically recognizing a IYVLVMLVL peptide fragment and application of the fluorescent PCR primer group. The primer group disclosed by the invention is designed according to an HLAA24 subtype and TCR of an EBV virus antigen peptide fragment IYVLVMLVL, and comprises a primer pair of which loci are L24-49 and / or L24-2. The primer group disclosed by the invention can be used for specifically recognizing the TCR of the EBV virus antigen peptide fragment IYVLVMLVL in a body of an HLAA24-subtype patient, and a basis is provided for tracking and monitoring TCR-T cell drugs in the body of the patient.
Description
[0001] The present invention requires the priority right of the Chinese patent with the application number 202110687266.8, the application date is June 21, 2021, and the title of the invention is "a fluorescent PCR primer set that specifically recognizes IYVLVMLVL peptide and its application". technical field [0002] The invention relates to the technical field of genetic engineering, in particular to a fluorescent PCR primer set for specifically recognizing IYVLVMLVL peptide and its application. Background technique [0003] EBV is a member of the herpesvirus family that can infect humans. EBV infection has been linked to certain types of cancer. These cancer patients caused by EBV virus infection are expected to benefit from TCR-T cell therapy. [0004] TCR-T cell therapy is a promising new generation of immune cell therapy for solid tumors. During TCR-T cell therapy, it is necessary to continuously monitor the growth and persistence of the medicinal TCR-T cells infused...
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