CN113308521A - Fluorescent PCR primer group for specifically recognizing TYGPVFMSL peptide fragment and application thereof - Google Patents

Abstract

The invention belongs to the technical field of genetic engineering, and particularly relates to a fluorescent PCR primer group for specifically recognizing TYGPVFMSL peptide fragments and application thereof. The primer group is designed according to HLAA24 typing and TCR of EBV virus antigenic peptide segment TYGPVFMSL, and comprises one or more of TCR primer pairs with sites of L24-3, L24-5 and L24-13. The primer sequence of the application is shown as SEQIDNO.1-6. The primer group can specifically recognize the TCR of the EBV virus antigen peptide segment TYGPVFMSL in the HLAA24 typing patient, and provides a basis for tracking and monitoring the TCR-T cell medicament in the patient.

Description

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

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a fluorescent PCR primer group for specifically recognizing TYGPVFMSL peptide fragments and application thereof.

Background

EBV is a member of the herpes virus family that can infect humans. EBV infection is associated with certain types of cancer. These cancer patients who are caused by EBV virus infection are expected to benefit from TCR-T cell therapy.

TCR-T cell therapy is a promising next generation immune cell therapy for the treatment of solid tumors. During TCR-T cell therapy, there is a need to constantly monitor the growth and persistence of the medicinal TCR-T cells infused into the patient. Among the technical means for achieving the purpose, the fluorescent quantitative PCR (qPCR) is a technical means with high detection sensitivity. The use of qPCR for the monitoring of TCR-T cells requires the design of qPCR primers for the specific TCR gene sequence of the TCR-T cells. However, the qPCR primer set designed aiming at the TCR gene sequence is not found in the prior art to be applied to detecting the growth and survival condition of the TCR-T cells in a patient body.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a primer group for specifically and sensitively tracking a TCR-T cell medicament, in particular to a fluorescent PCR primer group for specifically identifying TYGPVFMSL peptide fragments and application thereof. In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a primer group, which comprises one or more of TCR primer pairs with sites of L24-3, L24-5 and L24-13.

Preferably, the primer set is designed based on the TCR of HLAA24 typing, EBV virus antigenic peptide segment TYGPVFMSL.

Preferably, the sequence of the TCR primer pair with the L24-3 site is shown as SEQ ID NO. 1-2;

the sequence of the TCR primer pair with the locus L24-5 is shown as SEQ ID NO. 3-4;

the sequence of the TCR primer pair with the locus L24-13 is shown as SEQ ID NO. 5-6.

The invention also provides application of the primer group in preparing a TCR product for specifically detecting the EBV virus antigen peptide segment TYGPVFMSL in an HLAA24 typing patient.

Preferably, the product is a kit product.

Preferably, the kit is a fluorescent quantitative PCR kit.

The invention provides a fluorescent PCR primer group for specifically recognizing TYGPVFMSL peptide fragments and application thereof, wherein the primer group is obtained by typing according to HLAA2 and designing TCR of EBV virus antigen peptide fragments TYGPVFMSL. The primer group is prepared into a fluorescent quantitative PCR kit, and when the TCR-T cell medicament in a patient is tracked, the gene segment with the sensitivity of 10 copy numbers of the medicament can be specifically detected.

Drawings

FIG. 1 shows the amplification curves of plasmid DNA standards at L24-3 sites (10 dilutions of plasmid DNA standards from left to right, respectively)⁸Copy, 10⁷Copy, 10⁶Copy, 10⁵Copy, 10⁴Copy, 10³Copy, 10²Copy, 10¹A copied gene fragment).

FIG. 2 is an electrophoretogram of amplified products of plasmid DNA standards at site L24-3 (dilutions of plasmid DNA standards from left to right 10, respectively)⁸Copy, 10⁷Copy, 10⁶Copy, 10⁵Copy, 10⁴Copy, 10³Copy, 10²Copy, 10¹A copied gene fragment).

FIG. 3 is a standard curve diagram of plasmid DNA standard at L24-3 locus.

FIG. 4 is a graph showing the amplification curves of genomic DNA of the TCR-T cell drug at the L24-3 locus (from left to right, the dilutions of genomic DNA are stock solution, 10-fold dilution, 100-fold dilution, 1000-fold dilution, and 10000-fold dilution, respectively).

FIG. 5 is an electrophoresis diagram of the amplified product of the genomic DNA of the TCR-T cell drug at L24-3 locus (from left to right, the dilutions of genomic DNA are stock solution, 10-fold dilution, 100-fold dilution, 1000-fold dilution, and 10000-fold dilution, respectively).

Detailed Description

The construction method of the TCR plasmid standard substance in the embodiment of the invention comprises the following steps:

(1) cloning the TCR gene segment corresponding to each site into a plasmid vector shown by SEQ ID NO.7 to obtain a plasmid vector containing a specific TCR gene segment;

(2) amplifying the plasmid vector containing the specific TCR gene segment in escherichia coli, and purifying plasmid DNA to obtain a TCR plasmid standard product of a corresponding site.

In the examples of the present invention, the L24-3 site is taken as an example of the design scheme, and the detection methods of the other sites are the same.

The genomic DNA of the TCR-T cell drug described in the examples herein is genomic DNA extracted from the corresponding TCR-T drug cell.

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

Example 1 primer design

According to HLAA24 typing, a TCR primer pair is designed at the sites of L24-3, L24-5 and L24-13 of an EBV virus antigen peptide segment TYGPVFMSL, and the sequence of the obtained primer pair is shown as SEQ ID NO. 1-6.

Example 2 Standard Curve establishment

The L24-3 site of TCR is taken as an example for designing the scheme.

Taking 10 of plasmid DNA standard substance of L24-3 locus⁸Copy number dilution, 10⁷Copy number dilution, 10⁶Copy number dilution, 10⁵Copy number dilution, 10⁴Copy number dilution, 10³Copy number dilution, 10²Copy number dilution, 10¹The real-time fluorescent PCR was set up for each copy number dilution according to the reaction system and reaction program described below.

Reaction system: 10 mul of PCR premix; forward primer 0.4 μ l, primer concentration: 10 μm/. mu.l; reverse primer 0.4. mu.l, primer concentration: 10 μm/. mu.l; ROX Reference Dye (50X) 0.2. mu.l; 2 μ l of genomic DNA of TCR-T cell drug, 50ng/μ l of concentration, or 2 μ l of plasmid standard DNA of TCR; Nuclean-Free Water 7. mu.l; the total reaction volume was 20. mu.l.

PCR reaction procedure: at 95 ℃ for 30 s; (95 ℃, 5 s; 60 ℃, 20s)40 cycles. Setting reference fluorescence as ROX, selecting reaction type as SYBR Green Reagents, selecting fluorescence detection channel SYBR, and collecting fluorescence signals at 60 deg.C.

The amplification curve of the plasmid DNA standard at the L24-3 site amplified by real-time fluorescent PCR is shown in FIG. 1.

The amplification products of each dilution of the plasmid DNA standard were subjected to agarose gel electrophoresis, and the results are shown in FIG. 2.

And (3) establishing a standard curve by taking the Ct value of the amplification curve as the ordinate and the copy number of the gene fragment with the log of the plasmid DNA standard substance being the base 10 as the abscissa, wherein the result of the standard curve is shown in figure 3. The equation of the standard curve is generated by linear fitting of a computer, and R in the calculation formula²And the standard curve is more than or equal to 0.95, which proves that the standard curve of the embodiment is effective.

Example 3 sensitivity detection

The genomic DNA stock solution, 10-fold diluent, 100-fold diluent, 1000-fold diluent, 10000-fold diluent of the TCR-T cell drug for the L24-3 locus were amplified according to the reaction system and procedure of real-time fluorescent PCR of example 2. The amplification curve of the genomic DNA from the TCR-T cell drug against the L24-3 site is shown in FIG. 4. The amplification products were subjected to agarose gel electrophoresis, and the results are shown in FIG. 5.

The Ct value of the genomic DNA of the TCR-T cell drug obtained at each dilution was substituted into the standard curve of example 2, and the copy number of the genomic DNA of the TCR-T cell drug obtained at each dilution was calculated.

Through calculation, the copy number of a TCR-T cell drug genome DNA stock solution aiming at an L24-3 locus is 27423 copies, the copy number of a 10-time diluent is 3064 copies, the copy number of a 100-time diluent is 759 copies, the copy number of a 1000-time diluent is 70 copies, and the copy number of a 10000-time diluent is 6 copies. It can be concluded that the primer obtained in this example 1 can achieve the lowest detection limit of 6 copies for detecting the drug of TCR-T cells at the L24-3 site, but the sensitivity is set to 10 because the lowest value of the standard curve is 10. That is, in practical applications, the primers can detect 10 copies of the TCR gene fragment in the genomic DNA of the patient's peripheral blood sample.

The primers of the other sites are used for detecting the TCR-T cell drugs of the corresponding sites according to the detection method of the sensitivity of the embodiment 3, and the obtained sensitivity can reach 10 copies.

The above examples show that the invention provides a fluorescent PCR primer set for specifically recognizing TYGPVFMSL peptide fragment and application thereof, and the primer set is designed according to HLAA24 typing and TCR of EBV virus antigen peptide fragment TYGPVFMSL. The primer group is prepared into a fluorescent quantitative PCR kit, and the sensitivity of tracking the TCR-T cell medicament in a patient can reach 10 copies of the TCR-T cell medicament.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

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taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt 780

catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga 840

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agggttttcc cagtcacgac gttgtaaaac gacggccagt gaattagtac tctagcttaa 1740

gtaacgccat tttgcaaggc atggaaaata cataactgag aatagagaag ttcagatcaa 1800

ggttaggaac agagagacag cagaatatgg gccaaacagg atatctgtgg taagcagttc 1860

ctgccccggc tcagggccaa gaacagttgg aacagcagaa tatgggccaa acaggatatc 1920

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ccgccctcag cagtttctag agaaccatca gatgtttcca gggtgcccca aggacctgaa 2040

atgaccctgt gccttatttg aactaaccaa tcagttcgct tctcgcttct gttcgcgcgc 2100

ttctgctccc cgagctcaat aaaagagccc acaacccctc actcggcgcg ccagtcctcc 2160

gatagactgc gtcgcccggg tacccgtatt cccaataaag cctcttgctg tttgcatccg 2220

aatcgtggac tcgctgatcc ttgggagggt ctcctcagat tgattgactg cccacctcgg 2280

gggtctttca tttggaggtt ccaccgagat ttggagaccc ctgcccaggg accaccgacc 2340

cccccgccgg gaggtaagct ggccagcggt cgtttcgtgt ctgtctctgt ctttgtgcgt 2400

gtttgtgccg gcatctaatg tttgcgcctg cgtctgtact agttggctaa ctagatctgt 2460

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cgtcccagcg gcctcggggg cccgttttgt ggcccattct gtatcagtta acctacccga 2580

gtcggacttt ttggagctcc gccactgtcc gaggggtacg tggctttgtt gggggacgag 2640

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gctgacgcaa cccccactgg ctggggcatt gccaccacct gtcaactcct ttctgggact 5160

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tcctttccat ggctgctcgc ctgtgttgcc aactggatcc tgcgcgggac gtccttctgc 5340

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cggcctctcc cgcgtcttcg ctttcggcct ccgacgagtc ggatctccct ttgggccgcc 5460

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acctgaaatg accctgtgcc ttatttgaac taaccaatca gttcgcttct cgcttctgtt 6180

cgcgcgcttc tgctccccga gctcaataaa agagcccaca acccctcact cggcgcgcca 6240

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gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa 6480

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aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt 6660

cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga 6720

atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg 6780

taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa 6840

aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt 6900

tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct 6960

gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct 7020

cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc 7080

cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt 7140

atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc 7200

tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat 7260

ctgcgctctg ctgaagccag ttaccttcgg aaaaagagt 7299

Claims (6)

1. A TCR primer group is characterized by comprising one or more TCR primer pairs with sites of L24-3, L24-5 and L24-13.

2. The primer set of claim 1, wherein the primer set is designed based on the TCR of EBV virus antigenic peptide TYGPVFMSL typed according to HLAA 24.

3. The primer set of claim 1 or 2, wherein the sequence of the TCR primer pair with position L24-3 is represented by SEQ ID No. 1-2;

the sequence of the TCR primer pair with the locus L24-5 is shown as SEQ ID NO. 3-4;

the sequence of the TCR primer pair with the locus L24-13 is shown as SEQ ID NO. 5-6.

4. Use of the primer set of any one of claims 1 to 3 for preparing a TCR product for specifically detecting EBV virus antigenic peptide TYGPVFMSL in a patient with HLAA24 typing.

5. Use according to claim 4, wherein the product is a kit product.

6. The use according to claim 5, wherein the kit is a fluorescent quantitative PCR kit.

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