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Fluorescent PCR primer group for specifically recognizing TYGPVFMSL peptide fragment and application thereof

A primer set and specific technology, applied in the field of genetic engineering, can solve problems such as undiscovered

Inactive Publication Date: 2021-08-27
重庆天科雅生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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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  • Fluorescent PCR primer group for specifically recognizing TYGPVFMSL peptide fragment and application thereof
  • Fluorescent PCR primer group for specifically recognizing TYGPVFMSL peptide fragment and application thereof
  • Fluorescent PCR primer group for specifically recognizing TYGPVFMSL peptide fragment and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Primer Design

[0026] According to HLAA24 typing, TCR primer pairs were designed at the L24-3, L24-5 and L24-13 sites of the EBV virus antigen peptide TYGPVFMSL, and the sequences of the obtained primer pairs are shown in SEQ ID NO.1-6.

Embodiment 2

[0027] Embodiment 2 standard curve is established

[0028] Take the L24-3 site of TCR as an example to design the scheme.

[0029] Take 10 of the plasmid DNA standard at the L24-3 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.

[0030] 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.

[0031] 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

[0035] Embodiment 3 Sensitivity detection

[0036] 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 L24-3 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 L24-3 site as Figure 4 shown. The amplified products were subjected to agarose gel electrophoresis, and the results were as follows: Figure 5 shown.

[0037] 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.

[0038] After calculation, the copy number of the TCR-T cell drug genomic DNA stock solution targeting the L24-3 site is 27423 copies, the copy number of the 10-fold dilution is 3064 copies, and the...

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Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to a fluorescent PCR primer group for specifically recognizing a TYGPVFMSL peptide fragment and application thereof. The primer group disclosed by the invention is designed according to HLAA24 typing and TCR of an EBV virus antigen peptide fragment TYGPVFMSL, and comprises one or more of TCR primer pairs of which the loci are L24-3, L24-5 and L24-13. The primer sequences provided by the invention are as shown in SEQ ID NO.1-6. The primer group provided by the invention can specifically recognize the TCR of the EBV virus antigen peptide fragment TYGPVFMSL in the body of an HLAA24 typing patient, and provides a basis for tracking and monitoring TCR-T cell drugs in the body of the patient.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a fluorescent PCR primer set for specifically recognizing TYGPVFMSL peptide and its application. Background technique [0002] 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. [0003] 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 into the patient. Among the technical means that can achieve this goal, fluorescent quantitative PCR (qPCR) is a technical means with high detection sensitivity. To monitor TCR-T cells by qPCR, it is necessary to design qPCR primers for the specific TCR gene sequence of TCR-T cells. However, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6851C12Q1/6881C12N15/11C12R1/93
CPCC12Q1/6851C12Q1/6881C12Q2531/113C12Q2563/107C12Q2545/114
Inventor 邓大川周杰吴海阳
Owner 重庆天科雅生物科技有限公司
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