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

Inactive Publication Date: 2021-08-06
GUANGDONG TCRCURE BIOPHARMA TECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are no qPCR primers designed for these 15 TCR gene sequences in the prior art, which can monitor the growth and survival of TCR-T cells in patients

Method used

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  • TCR primer group for specifically recognizing EBV peptide fragment with immune subtype of HLAA02 and application of TCR primer group
  • TCR primer group for specifically recognizing EBV peptide fragment with immune subtype of HLAA02 and application of TCR primer group
  • TCR primer group for specifically recognizing EBV peptide fragment with immune subtype of HLAA02 and application of TCR primer group

Examples

Experimental program
Comparison scheme
Effect test

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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PUM

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Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to a TCR primer group for specifically recognizing an EBV peptide fragment with immune typing of HLAA02 and application of the TCR primer group. The primer group provided by the invention is designed according to HLAA02 typing and TCR of an EBV virus antigen peptide fragment FLYALALLL, and comprises one or more of TCR primer pairs with the loci of L2-1, L2-2, L2-3, L2-5, L2-6, L2-9, L2-10, L2-11, L2-12, L2-13, L2-16, L2-19, L2-23, L2-24 and L2-25. The sequences of the primers disclosed by the invention are as shown in SEQ ID NO. 1-30. The primer group disclosed by the invention can be used for specifically recognizing the TCR of the EBV antigen peptide fragment FLYALALL in the body of an HLAA02 typing patient.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a TCR primer set for specifically recognizing EBV virus peptides immunotyped as HLAA02 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....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6881C12Q1/6851C12N15/11
CPCC12Q1/6881C12Q1/6851C12Q2531/113C12Q2563/107C12Q2545/114
Inventor 张立峰窦浪贾杨彦圣
Owner GUANGDONG TCRCURE BIOPHARMA TECHNOLOGY CO LTD
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