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Fluorescent PCR primer group for specifically recognizing IYVLVMLVL 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: 2021-07-30
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 IYVLVMLVL peptide fragment and application thereof
  • Fluorescent PCR primer group for specifically recognizing IYVLVMLVL peptide fragment and application thereof
  • Fluorescent PCR primer group for specifically recognizing IYVLVMLVL peptide fragment and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 primer design

[0026] The TCR primers were designed in accordance with the HLAA24 classification, EBV viral antigen peptide IYVLVMLVL L24-2 and L24-49 sites. The sequences of the obtained primers are shown in SEQ ID NO.1 to 4.

Embodiment 2

[0027] Example 2 Standard Curve Establishment

[0028] Taking the L24-2 site of TCR as an example design.

[0029] Take 1 L24-2 site of plasmid DNA standard 10 8 Copy number diluent, 10 7 Copy number diluent, 10 6 Copy number diluent, 10 5 Copy number diluent, 10 4 Copy number diluent, 10 3 Copy number diluent, 10 2 Copy number diluent, 10 1 A copy number diluent is provided in real time fluorescent PCR according to the following reaction system and reaction procedure.

[0030] Reaction system: PCR premix 10 μL; positive primer 0.4 μL, primer concentration: 10 μm / μl; reverse primer: 10 μm / μl; ROX Reference Dye (50 ×) 0.2 μL; TCR-T cell medicine Genomic DNA2 μL, concentration 50 ng / μL, or TCR plasmid standard DNA2 μL; Nuclease-Free Water 7 μL; total reaction system is 20 μL.

[0031] PCR react procedures: 95 ° C, 30S; (95 ° C, 5S; 60 ° C, 20S) 40 cycles. Set the reference fluorescence to ROX, select the type of reaction to Sybr Green Reagents, select the fluorescence detectio...

Embodiment 3

[0036] Example 3 Sensitivity detection

[0037] The genomic DNA stock, 10-fold dilution, 100-fold dilution, 1000-fold dilution, 10000-fold dilution, 1000-fold dilution, 10000 times dilution, 1000-fold dilution, 10000 times dilution, 1000-fold dilution, 10000 times dilution, reaction system and procedure of real-time fluorescent PCR according to Example 2 Making amplification. Amplification curve of genomic DNA for TCR-T cell medicines for L24-2 sites Figure 4 Indicated. The amplified product is subjected to agarose gel electrophoresis. Figure 5 Indicated.

[0038] The CT value of the genomic DNA of each dilution of the TCR-T cell medicine was produced into the standard curve of Embodiment 2, and the copy number of the genomic DNA of the TCR-T cell medicine of each dilution can be calculated.

[0039] After calculation, the copy number of the TCR-T cell medicine genome DNA stock solution for the L24-2 site is 46,766 copies, and the number of copies of the 10-fold dilution is 7998 c...

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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 fluorescent PCR primer group for specifically recognizing an IYVLVMLVL peptide fragment and application thereof. The primer group provided by the invention is a primer pair which is designed according to HLAA24 typing and TCR of an EBV virus antigen peptide fragment IYVLVMLVL and comprises a site of L24-49 and / or L24-2. The sequence of the primer group is as shown in SEQ ID NO.1-4. The primer group can specifically recognize the TCR of the EBV virus antigen peptide IYVLVMLVL 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 present invention relates to the field of genetic engineering techniques, and more particularly to a fluorescent PCR primer group specific to identify the IYVLVMLVL peptide. Background technique [0002] EBV is a member of the herpes virus family that can infect humans. EBV infection is related to certain types of cancers. These cancer patients caused by EBV virus infections are expected to benefit in TCR-T cell therapy. [0003] TCR-T cell therapy is a new generation of immunocyte therapy expected to treat solid tumors. In the process of TCR-T cell therapy, it is necessary to continuously monitor the growth and renewability of medicinal TCR-T cells in the patient's body. In this paper, the fluorescent quantitative PCR (QPCR) is a technique for detecting high sensitivity. TCR-T cells were monitored by QPCR, requiring QPCR primers for specific TCR gene sequences for TCR-T cells. However, prior art did not find that the QPCR primer group designed for TCR ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6851C12N15/11
CPCC12Q1/6851C12Q2531/113C12Q2563/107C12Q2537/16
Inventor 银霜周杰吴海阳
Owner 重庆天科雅生物科技有限公司