CN113388612A - Primer designed for TCR with epitope point of IYVLVMLVL and application thereof - Google Patents

Abstract

The invention relates to the technical field of plasmid cloning. The invention provides a primer designed for TCR with an epitope point of IYVLVMLVL and application thereof, wherein the primer pair comprises one or two of a primer pair of a L24-49 site and a primer pair of a L24-2 site. The primer pair is designed aiming at the TCR of the EBV virus antigen peptide segment in the patient with HLA A24 and IYVLVMLVL epitope, and can realize the cloning of specific plasmids.

Description

Primer designed for TCR with epitope point of IYVLVMLVL and application thereof

Technical Field

The invention relates to the technical field of plasmid cloning, in particular to a primer designed for TCR with an epitope point of IYVLVMLVL and application thereof.

Background

A vector is a vehicle (vehicle) by which a polynucleotide sequence (e.g., a foreign gene) can be introduced into a host cell in order to transform the host and facilitate expression (e.g., transcription and translation) of the introduced sequence. Vectors include plasmids, phages, viruses, etc. The most common type of vector is a plasmid, which refers to a closed circular double stranded DNA loop into which additional DNA segments containing the gene of interest can be ligated. Another type of vector is a viral vector, in which the nucleic acid construct to be transported is ligated into the viral genome. Viral vectors may replicate autonomously in the host cell into which they are introduced, or may integrate themselves into the genome of the host cell, and thereby replicate together with the host genome. In addition, certain vectors can direct the expression of genes to which they are operably linked.

To clone a target gene fragment into a plasmid vector, primers must be present that match the sequence of the target gene fragment. I cloned 2 TCRs against IYVLVMLVL epitope of EBV viral protein in a patient immunophenotyped with HLA A24 and applied for patent protection for the gene sequences of these 2 TCRs. However, no primers have been available in the prior art for cloning the gene sequences of these 2 TCRs into a plasmid vector.

Disclosure of Invention

The invention aims to provide a primer pair for cloning plasmids.

In order to achieve the above object, the present invention provides the following technical solutions.

The invention provides a primer pair, which comprises one or two of a primer pair at a position L24-49 and a primer pair at a position L24-2.

Preferably, the primer pair is designed for the TCR of the EBV virus antigenic peptide fragment in a patient classified as HLA a24, epitope IYVLVMLVL.

Preferably, the sequences of the primer pair at the L24-49 sites are shown as SEQ ID NO.1 and SEQ ID NO. 2.

Preferably, the sequences of the primer pair at the L24-2 site are shown as SEQ ID NO.3 and SEQ ID NO. 4.

The invention also provides application of the primer pair in plasmid cloning.

The invention provides a primer designed for TCR with an epitope point of IYVLVMLVL and application thereof, wherein the primer pair comprises one or two of a primer pair of a L24-49 site and a primer pair of a L24-2 site. The primer pair is designed aiming at the TCR of the EBV virus antigen peptide segment in the patient with HLA A24 and IYVLVMLVL epitope, and can realize the cloning of specific plasmids.

Drawings

FIG. 1 shows the gel recovery electrophoresis results after TCRPCR amplification;

FIG. 2 is a backbone vector plasmid map;

FIG. 3 shows the result of electrophoresis of the TCR and the recombinant plasmid;

FIG. 4 is a graph of sequencing peaks of TCR and vector recombinant plasmid;

FIG. 5 shows the transfection efficiency of TCR transfected Jurkat cells by flow assay;

FIG. 6 shows the transfection efficiency of TCR transfected Jurkat cells by flow assay;

FIG. 7 is a functional validation flow assay for IFN- γ following TCR transfection of Jurkat cells;

FIG. 8 is a functional validation flow assay for IFN- γ following TCR transfection with Jurkat cells.

Detailed Description

The invention provides a primer pair, which comprises one or two of a primer pair at a position L24-49 and a primer pair at a position L24-2.

In the present invention, the primer pair is designed for the TCR of the EBV virus antigenic peptide fragment in a patient classified as HLA a24, epitope IYVLVMLVL.

In the invention, the sequences of the primer pair at the L24-49 sites are shown as SEQ ID NO.1 and SEQ ID NO. 2.

In the invention, the sequences of the primer pair at the L24-2 site are shown as SEQ ID NO.3 and SEQ ID NO. 4.

The invention also provides application of the primer pair in plasmid cloning.

In the present invention, the plasmid is preferably MP 71.

In the present invention, the plasmid backbone is public available information.

In the invention, the sequence of the MP71 is shown as SEQ ID NO. 5.

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 pair design examples: take SEQ ID NO.1 and SEQ ID NO.2 as an example.

Forwardprimer(SEQ ID NO.1):GCCGCGCCACCATGGCCACA；

Reverse primer(SEQ ID NO.2):GTAGGTCCTCCAGAACCAGCAG。

Example 2 vector construction

The method for constructing the vector comprises the following steps:

1. PCR amplification is carried out after the TCR gene is synthesized, the synthesized TCR gene is taken as a template, and the PCR amplification is carried out by using SEQ ID NO.1 and SEQ ID NO. 2; the PCR reaction system is as follows: PCR premix 25. mu.l, forward primer 5. mu.l, primer concentration: 10 μm/. mu.l, reverse primer 5 μ l, primer concentration: 10 μm/. mu.l, synthetic TCR gene sequence 1 μ l, concentration: 10 ng/ul, 14 ul of nucleic-Free Water, and 50 ul of total reaction system; PCR reaction procedure: at 98 ℃ for 2 min; (98 ℃, 15 s; 64 ℃, 15 s; 72 ℃, 20s)30 cycles; 72 ℃ for 2 min.

2. PCR product gel recovery

Performing Agarose Gel electrophoresis on the PCR product in the step 1, then recovering Gel, preparing Agarose Gel according to the instruction of a Kit TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0, and performing electrophoresis on the recovered product fragment, wherein the result is shown in figure 1.

3. Homologous recombination of linearized vector and recovered gel fragment

The molar ratio of the gel recovery PCR product fragment in the step 2 to the linearized vector recombination is 1:1, then 10 μ l of 2X Seamless Cloning Mix is added and supplemented to 20 μ l with water, the reaction is carried out for 30min at 50 ℃, the nucleic acid sequence of the vector skeleton is shown as SEQ ID NO.7, and the map information of the recombinant plasmid is shown as FIG. 2.

4. Recombinant plasmid vector transformation of DH5 alpha competent bacteria

Adding 5 μ l of the reaction sample of step 4 into 50 μ l of competent cells such as DH5 α, mixing, and placing the mixture on ice for 30 min; carrying out heat shock in a water bath at 42 ℃ for 90 seconds, then quickly putting back to an ice bath, and standing for 3-5 min; adding 300 mul of LB culture solution without antibiotics, mixing evenly, and shake culturing for 1h at 37 ℃; then, the whole was spread evenly on LB plates containing the appropriate antibiotics and cultured overnight in an incubator at 37 ℃.

5. Plasmid extraction

Picking the monoclonal colony in the step 4, and shaking the colony overnight; taking 5ml of overnight cultured bacterial liquid, adding into a centrifuge tube, centrifuging at 12,000rpm for 1min, and removing supernatant by suction; adding 250 mul of solution P1 into the centrifuge tube with the thallus precipitate, and blowing off the precipitate; adding 250 mul of solution P2 into a centrifuge tube, and gently turning the centrifuge tube up and down for 6-8 times to fully crack the thalli; adding 350 μ l of solution P3 into the centrifuge tube, immediately turning gently up and down for 6-8 times, mixing well, centrifuging at 12,000rpm for 10min to obtain white flocculent precipitate; transferring the supernatant collected in the last step into an adsorption column by a liquid transfer device, centrifuging at 12,000rpm for 30-60s, pouring off waste liquid in the collection tube, and putting the adsorption column into the collection tube; adding 600 μ l of rinsing liquid PW into the adsorption column, centrifuging at 12,000rpm for 30-60s, pouring off waste liquid in the collection tube, and introducing the adsorption column into the collection tube; repeating the previous step; placing the adsorption column C in a collection tube, and centrifuging at 12,000rpm for 2 min; the adsorption column was placed in a clean centrifuge tube, 50. mu.l of elution buffer was added dropwise to the middle of the adsorption membrane, the membrane was left at room temperature for 2min, and the plasmid solution was collected by centrifugation at 12,000rpm for 2 min.

6. Identification of recombinant plasmids

And (3) carrying out double enzyme digestion and sequencing identification on the plasmid extracted in the step (5), wherein the enzyme digestion electrophoresis result and a sequencing peak map are shown in a figure 3 and a figure 4.

EXAMPLE 3 use of the vector

The process comprises preparation of virus solution and transfection of cells, and the detailed process is as follows:

1. preparation of virus liquid

Lipo3000 transfected 293FT cells, the day before transfection, at appropriate cell density into 6-well plates, Lipo3000 reagent transfection protocol details see reagent Invitrogen^TMLipofectamine^TM3000Transfection Reagent；37℃、5％CO₂Culturing for 48 hours, and sucking out the culture medium supernatant for transfection;

2. virus transfection of Jurkat cells

The day after 293T cell transfection, 24-well culture plates were coated with Retronectin and kept overnight at 4 ℃ for future use; adding virus liquid into a 24-hole culture plate coated by Retronectin, centrifuging for 2h at 32 ℃ at 2000g, and removing supernatant; adding a proper amount of transfected Jurkat cells into a 24-hole culture plate, centrifuging at 30 ℃ for 30min and 600g to ensure that the cells are attached to the bottom wall; after centrifugation, the plates were placed at 37 ℃ in 5% CO₂Culturing in an incubator. After 48h, taking a proper amount of cells, centrifuging for 5min at 500g, and removing the supernatant of the culture medium; resuspending in PBS, 1ul each of hCD3(PE)/mTCRbeta (PB) flow dyes, staining for 15min in the dark at room temperature, adding 1ml PBS, centrifuging for 5min at 500g, and removing the supernatant; after resuspension with appropriate amount of PBS or FASC Buffer, flow cytometry was performed, and the flow charts are shown in FIG. 5 and FIG. 6.

Example 4: result verification

The detailed procedure for result verification is as follows:

collecting K562 cells, resuspending and counting, performing stimulation co-culture in a 24-well plate, adding 4e 5K 562 cells per well, and adding corresponding antigen peptide to a final concentration of 10 ug/ml; incubating for 1.5h in an incubator, and centrifuging a 24-pore plate to remove a culture medium supernatant; fresh supplement medium and 2e per well⁵Transfected Jurkat cells, incubated overnight; hCD3(PE), mtcrbeta (pb), hCD69(APC), staining for 15min at room temperature in the dark; after washing with PBS, flow cytometry was performed, and the flow charts of the results are shown in FIGS. 7 and 8.

As can be seen from the above examples, the present invention provides a primer designed for TCR with epitope point IYVLVMLVL and its application, wherein the primer pair includes one or both of the primer pair at position L24-49 and the primer pair at position L24-2. The primer pair is designed aiming at the TCR of the EBV virus antigen peptide segment in the patient with HLA A24 and IYVLVMLVL epitope, and can realize the cloning of specific plasmids.

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

<110> Chongqing Tianke Biotechnology Ltd

<120> primer designed for TCR with epitope point IYVLVMLVL and application thereof

<160> 5

<170> SIPOSequenceListing 1.0

<210> 1

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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gccgcgccac catggccaca 20

<210> 2

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

gtaggtcctc cagaaccagc ag 22

<210> 3

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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gccgcgccac catggccaca 20

<210> 4

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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gtaggtcctc cagaactgtc agtctgg 27

<210> 5

<211> 5979

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

gaggacctac gtaacgtgac ccctcccaag gtgtccctgt tcgagcctag caaggccgag 60

atcgccaaca agcagaaggc cacactcgtc tgcctggcta gaggcttctt cccagaccac 120

gtggagctgt cttggtgggt gaacggcaag gaggtgcact caggagtgtc taccgaccct 180

caggcctaca aggagagcaa ctacagctac tgcctgtcct ccagactcag ggtctgcgcc 240

accttttggc acaaccctcg gaaccacttc cgctgtcagg tccagttcca cggcctgtcc 300

gaggaagaca agtggccaga gggctctcct aagccagtga cacagaacat cagcgccgag 360

gcttggggaa gagccgattg cggaatcacc agcgcctctt accagcaggg agtgctgtca 420

gctaccatcc tgtacgagat cctgctgggc aaggccacac tgtacgcagt gctggtgtcc 480

actctggtcg tgatggctat ggtgaagcgg aagaacagct aggaattcga gcatcttacc 540

gccatttatt cccatatttg ttctgttttt cttgatttgg gtatacattt aaatgttaat 600

aaaacaaaat ggtggggcaa tcatttacat tttatgggat atgtaattac tagttcaggt 660

gtattgccac aagacaaaca tgttaagaaa ctttcccgtt atttacgctc tgttcctgtt 720

aatcaacctc tggattacaa aatttgtgaa agattgactg atattcttaa ctatgttgct 780

ccttttacgc tgtgtggata tgctgcttta atgcctctgt atcatgctat tgcttcccgt 840

acggctttcg ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg 900

tggcccgttg tccgtcaacg tggcgtggtg tgctctgtgt ttgctgacgc aacccccact 960

ggctggggca ttgccaccac ctgtcaactc ctttctggga ctttcgcttt ccccctcccg 1020

atcgccacgg cagaactcat cgccgcctgc cttgcccgct gctggacagg ggctaggttg 1080

ctgggcactg ataattccgt ggtgttgtcg gggaagctga cgtcctttcc atggctgctc 1140

gcctgtgttg ccaactggat cctgcgcggg acgtccttct gctacgtccc ttcggctctc 1200

aatccagcgg acctcccttc ccgaggcctt ctgccggttc tgcggcctct cccgcgtctt 1260

cgctttcggc ctccgacgag tcggatctcc ctttgggccg cctccccgcc tgtttcgcct 1320

cggcgtccgg tccgtgttgc ttggtcgtca cctgtgcaga attgcgaacc atggattcca 1380

ccgtgaactt tgtctcctgg catgcaaatc gtcaacttgg catgccaaga ataattcgga 1440

tccaagctta ggcctgctcg ctttcttgct gtcccatttc tattaaaggt tcctttgttc 1500

cctaagtcca actactaaac tgggggatat tatgaagggc cttgagcatc tggattctgc 1560

ctagcgctaa gcttaacacg agccatagat agaataaaag attttattta gtctccagaa 1620

aaagggggga atgaaagacc ccacctgtag gtttggcaag ctagcttaag taacgccatt 1680

ttgcaaggca tggaaaatac ataactgaga atagagaagt tcagatcaag gttaggaaca 1740

gagagacagc agaatatggg ccaaacagga tatctgtggt aagcagttcc tgccccggct 1800

cagggccaag aacagttgga acagcagaat atgggccaaa caggatatct gtggtaagca 1860

gttcctgccc cggctcaggg ccaagaacag atggtcccca gatgcggtcc cgccctcagc 1920

agtttctaga gaaccatcag atgtttccag ggtgccccaa ggacctgaaa tgaccctgtg 1980

ccttatttga actaaccaat cagttcgctt ctcgcttctg ttcgcgcgct tctgctcccc 2040

gagctcaata aaagagccca caacccctca ctcggcgcgc cagtcctccg atagactgcg 2100

tcgcccgggt acccgtgttc tcaataaacc ctcttgcagt tgcatccgac tcgtggtctc 2160

gctgttcctt gggagggtct cctctgagtg attgactgcc cacctcgggg gtctttcatt 2220

ctcgagcagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct 2280

cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg 2340

agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct 2400

gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg 2460

gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc 2520

ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg 2580

aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct 2640

ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca 2700

gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct 2760

cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc 2820

gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt 2880

tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc 2940

cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc 3000

cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg 3060

gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc 3120

agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag 3180

cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga 3240

tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat 3300

tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag 3360

ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat 3420

cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc 3480

cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat 3540

accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag 3600

ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg 3660

ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc 3720

tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca 3780

acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg 3840

tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc 3900

actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta 3960

ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc 4020

aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg 4080

ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc 4140

cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc 4200

aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat 4260

actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag 4320

cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc 4380

ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa 4440

taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg 4500

acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca 4560

agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta actatgcggc 4620

atcagagcag attgtactga gagtgcacca tatgcggtgt gaaataccgc acagatgcgt 4680

aaggagaaaa taccgcatca ggcgccattc gccattcagg ctgcgcaact gttgggaagg 4740

gcgatcggtg cgggcctctt cgctattacg ccagctggcg aaagggggat gtgctgcaag 4800

gcgattaagt tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa cgacggccag 4860

tgaattagta ctctagctta agtaacgcca ttttgcaagg catggaaaat acataactga 4920

gaatagagaa gttcagatca aggttaggaa cagagagaca gcagaatatg ggccaaacag 4980

gatatctgtg gtaagcagtt cctgccccgg ctcagggcca agaacagttg gaacagcaga 5040

atatgggcca aacaggatat ctgtggtaag cagttcctgc cccggctcag ggccaagaac 5100

agatggtccc cagatgcggt cccgccctca gcagtttcta gagaaccatc agatgtttcc 5160

agggtgcccc aaggacctga aatgaccctg tgccttattt gaactaacca atcagttcgc 5220

ttctcgcttc tgttcgcgcg cttctgctcc ccgagctcaa taaaagagcc cacaacccct 5280

cactcggcgc gccagtcctc cgatagactg cgtcgcccgg gtacccgtat tcccaataaa 5340

gcctcttgct gtttgcatcc gaatcgtgga ctcgctgatc cttgggaggg tctcctcaga 5400

ttgattgact gcccacctcg ggggtctttc atttggaggt tccaccgaga tttggagacc 5460

cctgcccagg gaccaccgac ccccccgccg ggaggtaagc tggccagcgg tcgtttcgtg 5520

tctgtctctg tctttgtgcg tgtttgtgcc ggcatctaat gtttgcgcct gcgtctgtac 5580

tagttggcta actagatctg tatctggcgg tcccgcggaa gaactgacga gttcgtattc 5640

ccggccgcag cccctgggag acgtcccagc ggcctcgggg gcccgttttg tggcccattc 5700

tgtatcagtt aacctacccg agtcggactt tttggagctc cgccactgtc cgaggggtac 5760

gtggctttgt tgggggacga gagacagaga cacttcccgc ccccgtctga atttttgctt 5820

tcggttttac gccgaaaccg cgccgcgcgt cttgtctgct gcagcatcgt tctgtgttgt 5880

ctctgtctga ctgtgtttct gtatttgtct gaaaattagc tcgacaaagt taagtaatag 5940

tccctctctc caagctcact tacaggcggc cgcgccacc 5979

Claims (5)

1. A primer pair, comprising one or both of a primer pair at position L24-49 and a primer pair at position L24-2.

2. The primer pair of claim 1, wherein the primer pair is designed for TCR of EBV virus antigenic peptide fragment in a patient classified as HLA A24 and having epitope IYVLVMLVL.

3. The primer pair of claim 2, wherein the sequences of the primer pair at positions L24-49 are shown as SEQ ID No.1 and SEQ ID No. 2.

4. The primer pair of any one of claims 1 to 3, wherein the sequence of the primer pair at the L24-2 site is shown as SEQ ID NO.3 and SEQ ID NO. 4.

5. Use of the primer pair of any one of claims 1 to 4 in plasmid cloning.

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