CN114672515A - Transduction plasmid, lentiviral vector system containing same and application thereof - Google Patents

Abstract

The invention discloses a transduction plasmid, a lentiviral vector containing the same and application thereof, wherein the transduction plasmid comprises the following functional region elements from 5 'end to 3' end: a PGK promoter, TCR β, paired alkaline protease cleavage site, amino acid spacer, P2A, and TCR α; wherein the TCR alpha and the TCR beta are human and mouse chimeric TCR, the variable region of the TCR alpha and the TCR beta is a humanized sequence, and the constant region is a mouse sequence. The constructed transduction plasmid has high transduction efficiency, strong continuous expression capability, safety and stability, and is suitable for clinical application.

Description

Transduction plasmid, lentiviral vector system containing same and application thereof

Technical Field

The invention relates to the technical field of biomedicine, in particular to a transduction plasmid, a lentivirus vector system containing the transduction plasmid and application of the transduction plasmid.

Background

Adoptive Cell Immunotherapy (ACT) belongs to the Immunotherapy of tumors, and in adoptive T Cell Immunotherapy, Chimeric Antigen Receptor T-Cell Immunotherapy (Chimeric Antigen Receptor T-Cell Immunotherapy) and T Cell Receptor genetically engineered T-Cell Immunotherapy (T Cell Receptor-gene engineered T-Cell Immunotherapy) are classified according to the type of T cells that are returned. Adoptive T cell therapy is a treatment mode of infusing specific T cells with anti-tumor activity to a tumor patient, killing tumor cells by directly killing the tumor or exciting the immune reaction of an organism, and finally achieving the purpose of removing the tumor. The back-transfused T lymphocytes include tumor-infiltrating lymphocytes (TILs), T cell receptor genetically engineered T cells (TCR-T), and Chimeric antigen receptor T cells (CAR-T).

Most TCR gene transduction uses gamma-retroviral-based vectors (γ -retroviruses-based vectors) that require full activation and proliferation of T cells during transduction for stable insertional integration into the host cell genome and affect the expression of homing-related molecules (CD62L) or co-activating molecules (CD 28).

Another commonly used means of gene transduction in adoptive immunotherapy is an adenovirus-based vector system, but it is not commonly used in the construction of TCR-T because it cannot continuously express the transduced gene in host cells, the length of the gene that can be transduced is short, and its packaging cells are already lysed prior to virus collection, plus the cumbersome manipulation of packaging collection.

Lentiviruses are single-stranded RNA viruses. Lentivirus (Lentivirus) vectors are gene therapy vectors developed based on HIV-1 (human immunodeficiency virus type I). Studies have shown that lentiviral vectors have higher transduction efficiency than retroviral vectors, can insert longer genes, and can infect slower growing cells such as Tumor Infiltrating Lymphocytes (TILs). Later, studies have demonstrated that lentiviral vectors can efficiently transduce both Cytotoxic T Lymphocytes (CTLs) and minimally activated Peripheral Blood Lymphocytes (PBLs) with anti-tumor activity.

Lentiviral vectors are also used in a wide variety of clinical applications. In 2003, VIR SYS began the first clinical trial using lentiviral vectors, and CD4+ T cells obtained from HIV-1 patients were transduced into a lentiviral vector comprising an antisense sequence against the HIV-1 envelope. In 2013, the viral load of 64 patients was reduced after treatment, and various adverse reactions were not caused. Other clinical trials of diseases have also achieved good results, including sickle cell anemia and beta-thalassemia.

The second generation lentiviral vector systems contained three plasmids, a packaging plasmid (enveloping plasmid) and a transduction plasmid (transfer plasmid). The packaging plasmid carries the mutated HIV-1 provirus, but because of some missing proteins, cannot be packaged into a virus. The envelope plasmid contains a viral envelope to determine tropism, i.e., which cell the virus can infect, and since the viral envelope from HIV-1 can only infect CD4+ T cells, it is replaced with the viral envelope from VSV-G, making it more infectious. The transduction plasmid encodes the target gene to be transduced by inserting the Long Terminal Repeats (LTRs) of HIV-1.

The effectiveness of adoptive cellular immunotherapy depends largely on the efficiency of gene transduction by lentiviruses and the ability of TCR persistence of TCR expression by TCR-T cells after gene transduction.

Factors influencing the continuous expression of the specific TCR sequence of the TCR-T cell constructed by the lentivirus comprise the selection of a promoter with high-efficiency continuous expression, the selection of a high-efficiency 2A peptide segment and the mismatching degree of internal and external TCR sequences.

In comparison of the various lentiviral promoters MSCV (the U3 promoter from tissue stem virus) promoter, PGK (phosphoglycerate kinase) promoter, CMV (cytomegavirus) promoter with EF-1 α (electroluminescence factor-1 α) promoter, CAG (a composite promoter sequence complex of the CMV promoter and ports of the chip b-active promoter and the ribbon b-ligand gene) promoter, SV40/CD43(a composite cellular promoter complex of the SV40 enhancer, the human CD43 promoter, and the MSUS 150 untranslated region UTR promoter), the promoters have very good expression efficiency and enable the gene to be expressed more efficiently in cells than is possible in other host cells under certain conditions. Since the α and β chains of the TCR need to be expressed separately in the cell, folded, and then assembled for transport to the cell surface, the genes encoding the TCR α and β chains can be driven by two different promoters, or an internal ribosome entry site can be introduced between the two. There are many ways to solve this problem, including increasing internal ribosome entry sites or dual promoters. A novel bicistronic lentiviral vector is researched and developed, the MSCV U3 promoter is combined with a furin cleavage site and an amino acid spacer after 2A self-cleavage of polypeptide, and experiments prove that the T2A sequence with the amino acid spacer can effectively construct a modified PBL with stronger anti-melanoma activity.

The internal self-cleavage mechanism mediated by the 2A sequence is that the ribosome skips the structure of a glycyl-prolyl peptide bond at the C-terminal of the 2A sequence, and the C-terminal of different 2A sequences a conserved sequence GDVEXNPGP, which is necessary for the formation of the steric hindrance phenomenon and the ribosome skipping.

The 2A sequence is found in a variety of viruses, including F2A (foot-and-mouth disease virus), E2A (organ rhinitis A virus), P2A (pore teschovirus-12A), and T2A (thrombus asigna virus 2A). Evaluation of the cleavage efficiency of different 2A in the dicistronic environment and the resulting mixed polypeptide results, some studies showed the highest efficiency of T2A, and others demonstrated the highest efficiency of P2A.

The exogenous TCR is transferred into the T cell and has the problem of mismatch with the endogenous TCR, the expression of the exogenous TCR on the T cell is influenced, and even the functional damage, off-target reaction and autoimmune disease of the T cell can be caused. There are several approaches to promote proper TCR sequence pairing, one of which is to engineer the exogenous TCR sequence to replace the human TCR constant region with murine (mouse Cb1 or mouse Cb2), and others which involve the use of leucine zipper structure fusion proteins to optimize the exogenous TCR sequence, the use of single chain TCR sequences linking a variable α domain to a variable β domain, and the introduction of an additional disulfide bond in the TCR sequence. Through comparison, research reports that the variable region transferred into the TCR is connected with the constant region of a mouse, so that the expression efficiency of the exogenous TCR can be effectively improved, and mismatching is reduced.

The above prior art has the disadvantages that the transduction efficiency of the optimized lentiviral vector system can be improved due to the fact that the transduction efficiency is optimized only for one element.

In addition, the current adoptive T cell immunotherapy has a drawback in practical clinical application that the reinfusion of TCR-T also recognizes normal tissues containing the same antigen as tumor cells, thereby causing serious side effects, and therefore, in practical clinical application, the expression intensity of TCR is required to be not so high as not to damage normal tissues while ensuring its effectiveness.

Therefore, it is urgently needed to construct a novel lentiviral vector which has high transduction efficiency, proper TCR expression strength and continuous expression, so as to improve the application value of the lentiviral vector in the immunotherapy.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a transduction plasmid, a lentiviral vector system containing the transduction plasmid and application of the transduction plasmid, in order to overcome the defects of low lentivirus transduction efficiency and short TCR continuous expression time in the prior art.

Since in practical clinical applications of adoptive T cell immunotherapy, too high TCR expression intensity can cause the reinfused TCR-T cells to recognize normal tissues containing the same antigen as the tumor target cells, causing serious side effects. Therefore, in the process of TCR transduction, the transduction efficiency is high, and the expression intensity is not too high to avoid side reaction.

In order to solve the above technical problems, one of the technical solutions of the present invention is: a transduction plasmid comprising the following functional region elements, in order from the 5 'end to the 3' end: a PGK promoter, TCR β, paired alkaline protease cleavage site, amino acid spacer, P2A, and TCR α; the TCR alpha and the TCR beta are human and mouse chimeric TCR, the variable region of the TCR alpha and the TCR beta is a humanized sequence, and the constant region of the TCR alpha and the TCR beta is a mouse sequence.

Preferably, the nucleotide sequence of the PGK promoter is shown as SEQ ID No. 1;

the nucleotide sequence of the paired alkaline protease cleavage sites is shown as SEQ ID No. 5;

the nucleotide sequence of the amino acid spacer is shown as SEQ ID No. 6;

the nucleotide sequence of the P2A is shown as SEQ ID No. 4;

the nucleotide sequence of the TCR alpha constant region is shown as SEQ ID No. 7; the nucleotide sequence of the TCR beta constant region is shown as SEQ ID No. 8.

Preferably, the variable regions of TCR α and TCR β are human melanoma associated antigen-specific TCRs; the human melanoma-associated antigen is preferably MART-1.

Preferably, the nucleotide sequence of the TCR alpha variable region is shown as SEQ ID No. 9; the nucleotide sequence of the TCR beta variable region is shown as SEQ ID No. 10.

Preferably, the nucleotide sequence of the vector of the transduction plasmid is shown as SEQ ID NO. 11.

The second technical scheme of the invention is as follows: a transformant comprising the transduction plasmid according to one of the above technical means.

The third technical scheme of the invention is as follows: a lentiviral vector system comprising the transduction plasmid of any of the above claims, preferably further comprising PsPAX2 and the pmd2.g plasmid.

The fourth technical scheme of the invention is as follows: the use of a transduction plasmid according to one of the claims for the preparation of a reagent for TCR infection; the agent is preferably CD8 infection⁺A T cell agent or an agent that infects Jurkat cells.

On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.

The reagents and starting materials used in the present invention are commercially available.

The positive progress effects of the invention are as follows: the transduction plasmid constructed by the invention has high transduction efficiency, and the exogenous TCR is transferred into the T cell, so that the assembly and expression efficiency of the exogenous TCR can be effectively improved, and the mismatching of the internal and exogenous TCR can be reduced; the constructed transduction plasmid TCR expression strength is not too high, and the TCR continuous expression capability is strong, safe and stable, thus being extremely suitable for clinical application.

Drawings

FIG. 1 is a map of TCR lentiviral shuttle plasmid vector construction.

FIG. 2 shows the TCR expression efficiency 4 days after lentiviral infection of Jurkat cells with three promoters.

FIG. 3 is a graph showing the TCR expression efficiency 10 days after infection of Jurkat cells with lentiviruses from three promoters by flow assay.

FIG. 4 shows a comparison of lentivirus infection expression efficiency and persistence of the three promoter vectors in a 0.1. mu.L lentivirus infection group.

FIG. 5 is a comparison of TCR expression efficiency 5 days after infection of CD8+ T cells with lentiviruses from three promoters detected by flow.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.

Example 1 construction of TCR Lentiviral transduction plasmid vectors for different promoters

The TCR sequence used in the invention is a MART-1 specific TCR that specifically recognizes the melanoma tumor associated antigen MART-1. The MART-1 specific TCR alpha variable region (SEQ ID No:9) and TCR beta variable region (SEQ ID No:10) sequences are respectively fused with a mouse TCR alpha constant region (SEQ ID No:7) and a TCR beta 2 constant region (SEQ ID No:8) to obtain human and mouse chimeric TCR alpha and TCR beta chains, the human and mouse chimeric TCR alpha and TCR beta chains are connected by a 2A sequence (P2A, Porcine teschovir-12A, the nucleotide sequence is shown as SEQ ID No: 4) of an internal self-splitting Porcine teschovirus (Porcine teschovirus), and a pairing basic amino acid protease cleavage site (furin cleavage site, SEQ ID No:5) and an amino acid spacer region (SEQ ID No:6) are introduced in front of the 2A sequence, as shown in FIG. 1. The invention places TCR beta before 2A sequence, because the length of TCR beta is larger than TCR alpha, the TCR beta gene can be favorable to TCR expression and assembly before 2A sequence based on literature report. The invention clones exogenous TCR gene between BamHI and EcoRI restriction enzyme sites of pRRLSIN. cPPT. PGK-GFP. WPRE lentivirus transduction plasmid vector (purchased from Addgene, the nucleotide sequence is shown as SEQ ID No:11) through gene synthesis and BamHI and EcoRI restriction enzyme digestion, and replaces EGFP gene in the vector with TCR gene. This lentiviral transduction plasmid vector was designated 5T-4 PGK.

The nucleotide sequence of P2A is shown in SEQ ID No. 4:

GCAACCAATTTCAGCCTGCTGAAGCAGGCAGGCGATGTGGAGGAGAACCCTGGACCA。

the nucleotide sequence of the paired basic amino acid protease cleavage sites is shown in SEQ ID No. 5: AGGGCAAAGCGG are provided.

The nucleotide sequence of the amino acid spacer is shown as SEQ ID No. 6: AGCGGCTCTGGA is added.

The nucleotide sequence of the mouse TCR alpha constant region is shown as SEQ ID No. 7:

GACATCCAGAACCCCGAGCCTGCCGTGTACCAGCTGAAGGACCCAAGATCCCAGGATAGCACCCTGTGCCTGTTCACCGACTTTGATTCTCAGATCAATGTGCCCAAGACCATGGAGAGCGGCACCTTTATCACAGACAAGACCGTGCTGGATATGAAGGCCATGGACAGCAAGTCCAACGGCGCCATCGCCTGGAGCAATCAGACATCCTTCACCTGCCAGGATATCTTTAAGGAGACAAACGCCACCTACCCTTCTAGCGACGTGCCATGTGATGCCACCCTGACAGAGAAGTCCTTCGAGACAGACATGAACCTGAATTTTCAGAACCTGTCTGTGATGGGCCTGAGAATCCTGCTGCTGAAGGTGGCCGGCTTCAATCTGCTGATGACCCTGAGACTGTGGTCCTCT。

the nucleotide sequence of the mouse TCR beta constant region is shown as SEQ ID No. 8:

GAGGATCTGAGAAACGTGACCCCCCCTAAGGTGTCTCTGTTTGAGCCTAGCAAGGCCGAGATCGCCAATAAGCAGAAGGCCACCCTGGTGTGCCTGGCAAGGGGCTTCTTTCCAGATCACGTGGAGCTGAGCTGGTGGGTGAACGGCAAGGAGGTGCACTCCGGCGTGTCTACAGACCCCCAGGCCTATAAGGAGAGCAATTACTCCTATTGCCTGTCTAGCCGGCTGAGAGTGTCCGCCACCTTCTGGCACAACCCCCGGAATCACTTCAGATGTCAGGTGCAGTTTCACGGCCTGTCCGAGGAGGATAAGTGGCCTGAGGGCTCTCCAAAGCCCGTGACACAGAACATCAGCGCCGAGGCATGGGGAAGAGCAGACTGTGGCATCACCTCTGCCAGCTACCACCAGGGCGTGCTGAGCGCCACAATCCTGTATGAGATCCTGCTGGGCAAGGCCACCCTGTACGCCGTGCTGGTGTCCGGACTGGTGCTGATGGCCATGGTGAAGAAGAAGAACTCT。

the nucleotide sequence of the human TCR alpha variable region is shown as SEQ ID No. 9:

ATGATGAAATCCTTGAGAGTTTTACTAGTGATCCTGTGGCTTCAGTTGAGCTGGGTTTGGAGCCAACAGAAGGAGGTGGAGCAGAATTCTGGACCCCTCAGTGTTCCAGAGGGAGCCATTGCCTCTCTCAACTGCACTTACAGTGACCGAGGTTCCCAGTCCTTCTTCTGGTACAGACAATATTCTGGGAAAAGCCCTGAGTTGATAATGTTCATATACTCCAATGGTGACAAAGAAGATGGAAGGTTTACAGCACAGCTCAATAAAGCCAGCCAGTATGTTTCTCTGCTCATCAGAGACTCCCAGCCCAGTGATTCAGCCACCTACCTCTGTGCCGTGAACTTCGGAGGAGGAAAGCTTATCTTCGGACAGGGAACGGAGTTATCTGTGAAACCCAAT。

the nucleotide sequence of the human TCR beta variable region is shown as SEQ ID No. 10:

ATGAGAATCAGGCTCCTGTGCTGTGTGGCCTTTTCTCTCCTGTGGGCAGGTCCAGTGATTGCTGGGATCACCCAGGCACCAACATCTCAGATCCTGGCAGCAGGACGGCGCATGACACTGAGATGTACCCAGGATATGAGACATAATGCCATGTACTGGTATAGACAAGATCTAGGACTGGGGCTAAGGCTCATCCATTATTCAAATACTGCAGGTACCACTGGCAAAGGAGAAGTCCCTGATGGTTATAGTGTCTCCAGAGCAAACACAGATGATTTCCCCCTCACGTTGGCGTCTGCTGTACCCTCTCAGACATCTGTGTACTTCTGTGCCAGCAGCCTAAGTTTCGGCACTGAAGCTTTCTTTGGACAAGGCACCAGACTCACAGTTGTA。

MSCV (shown in sequence SEQ ID No: 2) and EF 1-alpha/HTLV (shown in sequence SEQ ID No: 3) promoter nucleic acid fragments are obtained through gene synthesis. Compared with a wild type EF-1a promoter, the EF1-a/HTLV hybrid promoter is more beneficial to the stability of RNA after transcription and improves the translation level. Designing primers with two overlapped ends, respectively carrying out PCR amplification on EF1-a/HTLV and MSCV promoter fragments, carrying out linear amplification on a 5T-4PGK lentiviral transduction plasmid by using a PCR method, and respectively cloning EF1-a/HTLV and MSCV promoter fragments onto lentiviral vectors by using One-step cloning kit seamless cloning. And obtaining the plasmid vector with successfully replaced promoter through monoclonal gene sequencing identification. Named 5T-4MSCV and 5T-4EF1-a respectively.

The nucleotide sequence of the PGK promoter contained in the vector is shown as SEQ ID No. 1:

GGGGTTGGGGTTGCGCCTTTTCCAAGGCAGCCCTGGGTTTGCGCAGGGACGCGGCTGCTCTGGGCGTGGTTCCGGGAAACGCAGCGGCGCCGACCCTGGGTCTCGCACATTCTTCACGTCCGTTCGCAGCGTCACCCGGATCTTCGCCGCTACCCTTGTGGGCCCCCCGGCGACGCTTCCTGCTCCGCCCCTAAGTCGGGAAGGTTCCTTGCGGTTCGCGGCGTGCCGGACGTGACAAACGGAAGCCGCACGTCTCACTAGTACCCTCGCAGACGGACAGCGCCAGGGAGCAATGGCAGCGCGCCGACCGCGATGGGCTGTGGCCAATAGCGGCTGCTCAGCAGGGCGCGCCGAGAGCAGCGGCCGGGAAGGGGCGGTGCGGGAGGCGGGGTGTGGGGCGGTAGTGTGGGCCCTGTTCCTGCCCGCGCGGTGTTCCGCATTCTGCAAGCCTCCGGAGCGCACGTCGGCAGTCGGCTCCCTCGTTGACCGAATCACCGACCTCTCTCCCCAG。

the nucleotide sequence of the MSCV promoter is shown as SEQ ID No. 2:

TGAAAGACCCCACCTGTAGGTTTGGCAAGTTAGCTTAAGTAACGCCATTTTGCAAGGCATGGAAAATACATAACTGAGAATAGAGAAGTTCAGATCAAGGTTAGGAACAGAGAGACAGCAGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGATGGTCCCCAGATGCGGTCCCGCCCTCAGCAGTTTCTAGCGAACCATCAGATGTTTCCAGGGTGCCCCAAGGACCTGAAATGACCCTGTGCCTTATTTGAACTAACCAATCAGTTTGCTTCTTGCTTCTGTTTGTGTGCTTCTGCTCCCTGAGCTCAATAAAAGAGCCCACAACCCCTCACTTGGTGGGCCAGTCCTCTGATAGACTGTGTCCCCTGGATACCCGTAT。

the nucleotide sequence of the EF1-a/HTLV promoter is shown as SEQ ID No. 3:

GCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGCTGAAGCTTCGAGGGGCTCGCATCTCTCCTTCACGCGCCCGCCGCCCTACCTGAGGCCGCCATCCACGCCGGTTGAGTCGCGTTCTGCCGCCTCCCGCCTGTGGTGCCTCCTGAACTGCGTCCGCCGTCTAGGTAAGTTTAAAGCTCAGGTCGAGACCGGGCCTTTGTCCGGCGCTCCCTTGGAGCCTACCTAGACTCAGCCGGCTCTCCACGCTTTGCCTGACCCTGCTTGCTCAACTCTACGTCTTTGTTTCGTTTTCTGTTCTGCGCCGTTACAGATCCAAGCTGTGACCGGCGCCTAC。

pRRLSIN. cPPT. PGK-GFP. WPRE lentivirus transduction plasmid vector sequence (SEQ ID NO: 11): AGCTTAATGTAGTCTTATGCAATACTCTTGTAGTCTTGCAACATGGTAACGATGAGTTAGCAACATGCCTTACAAGGAGAGAAAAAGCACCGTGCATGCCGATTGGTGGAAGTAAGGTGGTACGATCGTGCCTTATTAGGAAGGCAACAGACGGGTCTGACATGGATTGGACGAACCACTGAATTGCCGCATTGCAGAGATATTGTATTTAAGTGCCTAGCTCGATACATAAACGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTGGCGCCCGAACAGGGACTTGAAAGCGAAAGGGAAACCAGAGGAGCTCTCTCGACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGCGATGGGAAAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAAACATATAGTATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAGGCTGTAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAAGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAGATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGACCACCGCACAGCAAGCGGCCGCTGATCTTCAGACCTGGAGGAGGAGATATGAGGGACAATTGGAGAAGTGAATTATATAAATATAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAGAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTGACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGATTTGGAATCACACGACCTGGATGGAGTGGGACAGAGAAATTAACAATTACACAAGCTTAATACACTCCTTAATTGAAGAATCGCAAAACCAGCAAGAAAAGAATGAACAAGAATTATTGGAATTAGATAAATGGGCAAGTTTGTGGAATTGGTTTAACATAACAAATTGGCTGTGGTATATAAAATTATTCATAATGATAGTAGGAGGCTTGGTAGGTTTAAGAATAGTTTTTGCTGTACTTTCTATAGTGAATAGAGTTAGGCAGGGATATTCACCATTATCGTTTCAGACCCACCTCCCAACCCCGAGGGGACCCGACAGGCCCGAAGGAATAGAAGAAGAAGGTGGAGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGGATCTCGACGGTATCGGTTAACTTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAATTACAAAAATTCAAAATTTTATCGATCACGAGACTAGCCTCGAGAAGCTTGATATCGAATTCCACGGGGTTGGGGTTGCGCCTTTTCCAAGGCAGCCCTGGGTTTGCGCAGGGACGCGGCTGCTCTGGGCGTGGTTCCGGGAAACGCAGCGGCGCCGACCCTGGGTCTCGCACATTCTTCACGTCCGTTCGCAGCGTCACCCGGATCTTCGCCGCTACCCTTGTGGGCCCCCCGGCGACGCTTCCTGCTCCGCCCCTAAGTCGGGAAGGTTCCTTGCGGTTCGCGGCGTGCCGGACGTGACAAACGGAAGCCGCACGTCTCACTAGTACCCTCGCAGACGGACAGCGCCAGGGAGCAATGGCAGCGCGCCGACCGCGATGGGCTGTGGCCAATAGCGGCTGCTCAGCAGGGCGCGCCGAGAGCAGCGGCCGGGAAGGGGCGGTGCGGGAGGCGGGGTGTGGGGCGGTAGTGTGGGCCCTGTTCCTGCCCGCGCGGTGTTCCGCATTCTGCAAGCCTCCGGAGCGCACGTCGGCAGTCGGCTCCCTCGTTGACCGAATCACCGACCTCTCTCCCCAGGGGGATCCACCGGTCGCCACCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAAGCGGCCGCGTCGACAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAGCTGACGTCCTTTCCATGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCCTGGAATTCGAGCTCGGTACCTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCCACTTTTTAAAAGAAAAGGGGGGACTGGAAGGGCTAATTCACTCCCAACGAAGACAAGATCTGCTTTTTGCTTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTAGTAGTTCATGTCATCTTATTATTCAGTATTTATAACTTGCAAAGAAATGAATATCAGAGAGTGAGAGGAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGCTCTAGCTATCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGGACGTACCCAATTCGCCCTATAGTGAGTCGTATTACGCGCGCTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTTAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCGCGCAATTAACCCTCACTAAAGGGAACAAAAGCTGGAGCTGCA are provided.

Example 2 packaging and Collection of lentiviruses

The lentiviruses were packaged separately with the three lentivirus transduction plasmids constructed in 1, using 293t cell line (purchased from ATCC) and were passaged one day before transfection and the next day when the confluency reached about 70%. Firstly, carrying out liquid changing treatment, and then carrying out liquid changing treatment according to the following steps of 4: 3: 2 to 500. mu.L of Opti-MEM solution were added three plasmids: 5T-4PGK transduction plasmids, PsPAX2, and pMD2.G (purchased from Addgene). Polyethyleneimine (PEI) with the mass 3 times that of the plasmid was added to 500. mu.L of LOpti-MEM solution, and the two solutions were mixed well and incubated at room temperature for 20 min. Gently adding into the culture medium, and shaking.

Collecting the first batch of virus at 48 hr, centrifuging culture medium supernatant at 2000RPM for 10min, passing through 0.45 μ M filter membrane, treating the virus solution collected at 64 hr, centrifuging the filtered virus supernatant with Beckman ultracentrifuge, Type 45Ti rotor at 35000 RPM, 4 deg.C, and 90 min.

After centrifugation, the supernatant was removed, 200. mu.L of serum-free T009 medium was resuspended, mixed and aliquoted, and stored at-80 ℃.

Example 3 detection of TCR Positive Rate of Jurkat cell line infected by different promoter lentiviral vectors

To compare the expression efficiency and persistence of the TCRs of the three lentiviruses infected cells prepared in example 2, the virus was infected in a gradient diluted form into Jurkat cell line (purchased from ATCC).

The specific experimental process is as follows:

prior to infection, cell concentration was adjusted to 1X 10 counts⁶One per ml. The cells were plated in 96-well plates at 100. mu.L/well and the cell resuspension medium was supplemented with polybrene as a pro-infection reagent at a final concentration of 6. mu.g/ml. Setting three gradients of lentivirus addition, namely three gradients of 0.1, 1 and 10 mu L, adding lentivirus, uniformly mixing with a liquid transferring device, centrifuging at 800g for 30min to make the virus and cells fully contact, uniformly mixing with a liquid transferring device, placing at 37 ℃ and 5% CO₂Culturing in an incubator. Since this experiment examined the persistent expression function of the lentiviral vector promoter, TCR positive rate detection was performed on days 4 and 10, respectively.

Tetramer synthesis was performed by diluting the Mart-1 polypeptide stock solution to 400. mu.M, 20. mu.L of diluted polypeptide and 20. mu.M monomer (Flex-TTM monomer, BioLegend, 200. mu.g/ml) per reaction, adding both to a 96-well U-plate, and mixing. Incubating the plate, centrifuging at 3300g for 2min at 4 deg.C, removing the seal, and irradiating with ultraviolet (365nm) for 30min on ice with the ultraviolet lamp tube at a distance of 2-5cm from the sample. The plates were sealed again and incubated in the dark at 37 ℃ for 30 min. The reacted replacement peptide monomers were removed to a new 1.5ml centrifuge tube, 1.1. mu.L of LAPC/PE fluorescence-coupled conjugated streptavidin (fluorescence-coupled streptavidin (BioLegend) was added to 10. mu.L of other brand fluorescence-coupled streptavidin at a molar ratio of 6:1), the tube was mixed by inverting up and down or blowing with a pipette gun, incubated in the dark on ice for 30min, stop buffer (1.6. mu.L of 50mM D-Biotin and 6. mu.L of 10% (w/v) NaN3 to 192.4. mu.L PBS) was added to 10. mu.L, and the reaction was stopped by inverting up and down or blowing with a pipette gun. Sealing the plate, and carrying out dark incubation on ice for 30min to obtain the Mart-1tetramer antibody.

The basic steps of the flow dyeing are as followsCells were harvested 4 and 10 days after lentivirus infection and counted, and the number of cells removed for flow sorting was 5X 10⁵200 μ L/tube, prepared MART-1tetramer (0.5 μ L/5X 10) was added in groups to a flow tube⁵Cells) and anti-murine TCR β constant region antibody (3 μ/5X 10)⁵Cells), mixed evenly and reacted for 30min at 4 ℃; after the reaction time was complete, 3ml of PBSA (0.5% FBS in PBS) was added for resuspension, 400g at 4 ℃ for 5min, the supernatant carefully discarded, 200. mu.L of PBSA was added for resuspension, and the mixture was placed on ice for flow cytometry.

The flow detection and analysis results are shown in FIGS. 2-4, wherein 5T-4PGK,5T-4MSCV and 5T-4EF-1 respectively refer to the groups of Jurkat cells infected by the three lentiviral vectors prepared in 2; detection was performed on day 4 and day 10 post infection, respectively; 10. mu.L, 1. mu.L, 0.1. mu.L were applied to the groups infected with the corresponding volume of virus, respectively. As can be seen from the test results, 10 μ L of virus infection is basically saturated, the positive rate of TCR expression of each group is more than 98%, and the positive rate of TCR expression of 1 μ L and 0.1 μ L of virus infection of each group is higher than that of the other two groups when the positive rate of PGK group is compared with that of TCR expression of the other two groups on day 4 or day 10. More specifically, a comparison analysis of the TCR expression rate and strength of 0.1 μ L virus infection revealed that the TCR expression rate of the lentiviral vector infected with the three promoters was increased from day 4 to day 10 after infection, and the expression strength was decreased, as shown in FIG. 4. The PGK promoter was more efficiently infected with lentivirus than the other groups, and the expression intensity decreased the least from day 4 to day 10. In practical clinical applications, the treatment period of adoptive T cell immunotherapy is usually as long as several months, so the sustained expression capability of TCR is crucial, as shown in fig. 4, the expression intensity of PGK promoter decreases the least from day 4 to day 10, indicating that the PGK promoter lentiviral vector constructed by us is most beneficial to the sustained expression of TCR in several promoter comparisons.

Example 4 detection of TCR Positive Rate of Primary T cells infected with lentiviral vectors with different promoters

To examine the TCR expression efficiency of primary T cells infected with several promoter lentiviral vectors, CD8 was infected with MOI 3 of each of the three lentiviruses prepared in 2⁺T cells, exogenous TCR stained with tetramer, positive rate was detected by flow.

The specific experimental process is as follows:

10ml of HLA-A0201 typed healthy human peripheral blood was collected and subjected to Ficoll density gradient centrifugation to obtain Peripheral Blood Mononuclear Cells (PBMC). From the isolated PBMCs, CD8 was sorted using anti-CD 8 antibody coated magnetic beads (Miltenyi)⁺T cells. Sorting the obtained CD8⁺T cells were activated with anti-CD 3 and anti-CD 28 coated magnetic beads (Miltenyi), 48 hours later CD8 was infected with MOI 3 using three lentiviruses prepared in 2, respectively⁺T cells. The MOI (Multiplicity of infection) represents the number of virus particles corresponding to one cell. After 24 hours of infection, the transduced cells were expanded by culturing in a medium containing IL-2, IL-7, IL-15 cytokines. TCR expression positive rate was detected 5 days post infection with tetramer staining. The procedure for infection and flow detection was the same as for Jurkat cells in 3.

As shown in FIG. 5, three promoter lentiviral vectors infected CD8⁺The TCR expression rate and PGK promoter lentivirus infection efficiency of the T cells are higher than those of other groups, which shows that the PGK promoter lentivirus vector constructed by the inventor is more favorable for expressing TCR in primary T cells among several promoters.

Sequence listing

<110> Shenzhen Huada clinical verification center

<120> transduction plasmid, lentiviral vector system containing same and application thereof

<130> P20016141C

<160> 11

<170> PatentIn version 3.5

<210> 1

<211> 511

<212> DNA

<213> Artificial Sequence

<220>

<223> PGK promoter

<400> 1

ggggttgggg ttgcgccttt tccaaggcag ccctgggttt gcgcagggac gcggctgctc 60

tgggcgtggt tccgggaaac gcagcggcgc cgaccctggg tctcgcacat tcttcacgtc 120

cgttcgcagc gtcacccgga tcttcgccgc tacccttgtg ggccccccgg cgacgcttcc 180

tgctccgccc ctaagtcggg aaggttcctt gcggttcgcg gcgtgccgga cgtgacaaac 240

ggaagccgca cgtctcacta gtaccctcgc agacggacag cgccagggag caatggcagc 300

gcgccgaccg cgatgggctg tggccaatag cggctgctca gcagggcgcg ccgagagcag 360

cggccgggaa ggggcggtgc gggaggcggg gtgtggggcg gtagtgtggg ccctgttcct 420

gcccgcgcgg tgttccgcat tctgcaagcc tccggagcgc acgtcggcag tcggctccct 480

cgttgaccga atcaccgacc tctctcccca g 511

<210> 2

<211> 412

<212> DNA

<213> Artificial Sequence

<220>

<223> MSCV promoter

<400> 2

tgaaagaccc cacctgtagg tttggcaagt tagcttaagt aacgccattt tgcaaggcat 60

ggaaaataca taactgagaa tagagaagtt cagatcaagg ttaggaacag agagacagca 120

gaatatgggc caaacaggat atctgtggta agcagttcct gccccggctc agggccaaga 180

acagatggtc cccagatgcg gtcccgccct cagcagtttc tagcgaacca tcagatgttt 240

ccagggtgcc ccaaggacct gaaatgaccc tgtgccttat ttgaactaac caatcagttt 300

gcttcttgct tctgtttgtg tgcttctgct ccctgagctc aataaaagag cccacaaccc 360

ctcacttggt gggccagtcc tctgatagac tgtgtcccct ggatacccgt at 412

<210> 3

<211> 532

<212> DNA

<213> Artificial Sequence

<220>

<223> EF1-a/HTLV promoter

<400> 3

gctccggtgc ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg 60

gaggggtcgg caattgaacc ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg 120

atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag 180

tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag ctgaagcttc 240

gaggggctcg catctctcct tcacgcgccc gccgccctac ctgaggccgc catccacgcc 300

ggttgagtcg cgttctgccg cctcccgcct gtggtgcctc ctgaactgcg tccgccgtct 360

aggtaagttt aaagctcagg tcgagaccgg gcctttgtcc ggcgctccct tggagcctac 420

ctagactcag ccggctctcc acgctttgcc tgaccctgct tgctcaactc tacgtctttg 480

tttcgttttc tgttctgcgc cgttacagat ccaagctgtg accggcgcct ac 532

<210> 4

<211> 57

<212> DNA

<213> Artificial Sequence

<220>

<223> P2A

<400> 4

gcaaccaatt tcagcctgct gaagcaggca ggcgatgtgg aggagaaccc tggacca 57

<210> 5

<211> 12

<212> DNA

<213> Artificial Sequence

<220>

<223> paired alkaline amino acid protease cleavage site

<400> 5

agggcaaagc gg 12

<210> 6

<211> 12

<212> DNA

<213> Artificial Sequence

<220>

<223> amino acid spacer

<400> 6

agcggctctg ga 12

<210> 7

<211> 411

<212> DNA

<213> Artificial Sequence

<220>

<223> TCR alpha constant region

<400> 7

gacatccaga accccgagcc tgccgtgtac cagctgaagg acccaagatc ccaggatagc 60

accctgtgcc tgttcaccga ctttgattct cagatcaatg tgcccaagac catggagagc 120

ggcaccttta tcacagacaa gaccgtgctg gatatgaagg ccatggacag caagtccaac 180

ggcgccatcg cctggagcaa tcagacatcc ttcacctgcc aggatatctt taaggagaca 240

aacgccacct acccttctag cgacgtgcca tgtgatgcca ccctgacaga gaagtccttc 300

gagacagaca tgaacctgaa ttttcagaac ctgtctgtga tgggcctgag aatcctgctg 360

ctgaaggtgg ccggcttcaa tctgctgatg accctgagac tgtggtcctc t 411

<210> 8

<211> 519

<212> DNA

<213> Artificial Sequence

<220>

<223> TCR beta constant region

<400> 8

gaggatctga gaaacgtgac cccccctaag gtgtctctgt ttgagcctag caaggccgag 60

atcgccaata agcagaaggc caccctggtg tgcctggcaa ggggcttctt tccagatcac 120

gtggagctga gctggtgggt gaacggcaag gaggtgcact ccggcgtgtc tacagacccc 180

caggcctata aggagagcaa ttactcctat tgcctgtcta gccggctgag agtgtccgcc 240

accttctggc acaacccccg gaatcacttc agatgtcagg tgcagtttca cggcctgtcc 300

gaggaggata agtggcctga gggctctcca aagcccgtga cacagaacat cagcgccgag 360

gcatggggaa gagcagactg tggcatcacc tctgccagct accaccaggg cgtgctgagc 420

gccacaatcc tgtatgagat cctgctgggc aaggccaccc tgtacgccgt gctggtgtcc 480

ggactggtgc tgatggccat ggtgaagaag aagaactct 519

<210> 9

<211> 399

<212> DNA

<213> Artificial Sequence

<220>

<223> TCR alpha variable region

<400> 9

atgatgaaat ccttgagagt tttactagtg atcctgtggc ttcagttgag ctgggtttgg 60

agccaacaga aggaggtgga gcagaattct ggacccctca gtgttccaga gggagccatt 120

gcctctctca actgcactta cagtgaccga ggttcccagt ccttcttctg gtacagacaa 180

tattctggga aaagccctga gttgataatg ttcatatact ccaatggtga caaagaagat 240

ggaaggttta cagcacagct caataaagcc agccagtatg tttctctgct catcagagac 300

tcccagccca gtgattcagc cacctacctc tgtgccgtga acttcggagg aggaaagctt 360

atcttcggac agggaacgga gttatctgtg aaacccaat 399

<210> 10

<211> 393

<212> DNA

<213> Artificial Sequence

<220>

<223> TCR beta variable region

<400> 10

atgagaatca ggctcctgtg ctgtgtggcc ttttctctcc tgtgggcagg tccagtgatt 60

gctgggatca cccaggcacc aacatctcag atcctggcag caggacggcg catgacactg 120

agatgtaccc aggatatgag acataatgcc atgtactggt atagacaaga tctaggactg 180

gggctaaggc tcatccatta ttcaaatact gcaggtacca ctggcaaagg agaagtccct 240

gatggttata gtgtctccag agcaaacaca gatgatttcc ccctcacgtt ggcgtctgct 300

gtaccctctc agacatctgt gtacttctgt gccagcagcc taagtttcgg cactgaagct 360

ttctttggac aaggcaccag actcacagtt gta 393

<210> 11

<211> 7388

<212> DNA

<213> Artificial Sequence

<220>

<223> pRRLSIN. cPPT. PGK-GFP. WPRE lentivirus transduction plasmid vector

<400> 11

agcttaatgt agtcttatgc aatactcttg tagtcttgca acatggtaac gatgagttag 60

caacatgcct tacaaggaga gaaaaagcac cgtgcatgcc gattggtgga agtaaggtgg 120

tacgatcgtg ccttattagg aaggcaacag acgggtctga catggattgg acgaaccact 180

gaattgccgc attgcagaga tattgtattt aagtgcctag ctcgatacat aaacgggtct 240

ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt 300

aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac 360

tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc 420

gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc 480

ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa 540

ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg 600

ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata 660

aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc 720

tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga 780

caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc 840

aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca 900

aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg 960

agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga accattagga 1020

gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata 1080

ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg 1140

acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg 1200

ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag 1260

ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt 1320

tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt 1380

aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt 1440

aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag 1500

aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata 1560

acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta 1620

agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta 1680

tcgtttcaga cccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa 1740

gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc tcgacggtat 1800

cggttaactt ttaaaagaaa aggggggatt ggggggtaca gtgcagggga aagaatagta 1860

gacataatag caacagacat acaaactaaa gaattacaaa aacaaattac aaaaattcaa 1920

aattttatcg atcacgagac tagcctcgag aagcttgata tcgaattcca cggggttggg 1980

gttgcgcctt ttccaaggca gccctgggtt tgcgcaggga cgcggctgct ctgggcgtgg 2040

ttccgggaaa cgcagcggcg ccgaccctgg gtctcgcaca ttcttcacgt ccgttcgcag 2100

cgtcacccgg atcttcgccg ctacccttgt gggccccccg gcgacgcttc ctgctccgcc 2160

cctaagtcgg gaaggttcct tgcggttcgc ggcgtgccgg acgtgacaaa cggaagccgc 2220

acgtctcact agtaccctcg cagacggaca gcgccaggga gcaatggcag cgcgccgacc 2280

gcgatgggct gtggccaata gcggctgctc agcagggcgc gccgagagca gcggccggga 2340

aggggcggtg cgggaggcgg ggtgtggggc ggtagtgtgg gccctgttcc tgcccgcgcg 2400

gtgttccgca ttctgcaagc ctccggagcg cacgtcggca gtcggctccc tcgttgaccg 2460

aatcaccgac ctctctcccc agggggatcc accggtcgcc accatggtga gcaagggcga 2520

ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca 2580

caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa 2640

gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac 2700

ctacggcgtg cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa 2760

gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa 2820

ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct 2880

gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta 2940

caacagccac aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt 3000

caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa 3060

cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc 3120

cgccctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac 3180

cgccgccggg atcactctcg gcatggacga gctgtacaag taaagcggcc gcgtcgacaa 3240

tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc 3300

ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat 3360

ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg 3420

gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg 3480

ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat 3540

tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt 3600

gggcactgac aattccgtgg tgttgtcggg gaagctgacg tcctttccat ggctgctcgc 3660

ctgtgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa 3720

tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg 3780

ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcctg gaattcgagc 3840

tcggtacctt taagaccaat gacttacaag gcagctgtag atcttagcca ctttttaaaa 3900

gaaaaggggg gactggaagg gctaattcac tcccaacgaa gacaagatct gctttttgct 3960

tgtactgggt ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg 4020

aacccactgc ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt 4080

ctgttgtgtg actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc 4140

tctagcagta gtagttcatg tcatcttatt attcagtatt tataacttgc aaagaaatga 4200

atatcagaga gtgagaggaa cttgtttatt gcagcttata atggttacaa ataaagcaat 4260

agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc 4320

aaactcatca atgtatctta tcatgtctgg ctctagctat cccgccccta actccgccca 4380

tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt 4440

ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag 4500

gcttttttgg aggcctaggg acgtacccaa ttcgccctat agtgagtcgt attacgcgcg 4560

ctcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa 4620

tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga 4680

tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc 4740

attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct 4800

agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg 4860

tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga 4920

ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt 4980

ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg 5040

aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc 5100

ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat 5160

attaacgctt acaatttagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt 5220

ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg 5280

cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt 5340

cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta 5400

aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc 5460

ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa 5520

gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc 5580

cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt 5640

acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact 5700

gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac 5760

aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata 5820

ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta 5880

ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg 5940

gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat 6000

aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt 6060

aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga 6120

aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa 6180

gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag 6240

gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac 6300

tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc 6360

gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat 6420

caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat 6480

actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct 6540

acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt 6600

cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg 6660

gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta 6720

cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg 6780

gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg 6840

tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc 6900

tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg 6960

gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat 7020

aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc 7080

agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg 7140

cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt 7200

gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc tttacacttt 7260

atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac 7320

agctatgacc atgattacgc caagcgcgca attaaccctc actaaaggga acaaaagctg 7380

gagctgca 7388

Claims (10)

1. A transduction plasmid, characterized in that it comprises the following functional region elements, in order from the 5 'end to the 3' end: a PGK promoter, TCR β, paired alkaline protease cleavage site, amino acid spacer, P2A, and TCR α;

wherein the TCR alpha and the TCR beta are human and mouse chimeric TCR, the variable region of the TCR alpha and the TCR beta is a humanized sequence, and the constant region is a mouse sequence.

2. The transduction plasmid of claim 1, wherein the nucleotide sequence of the PGK promoter is set forth in SEQ ID No: 1;

the nucleotide sequence of the paired alkaline protease cleavage sites is shown as SEQ ID No. 5;

the nucleotide sequence of the amino acid spacer is shown as SEQ ID No. 6;

the nucleotide sequence of the P2A is shown as SEQ ID No. 4;

the nucleotide sequence of the constant region of the TCR alpha is shown as SEQ ID No. 7; the nucleotide sequence of the constant region of the TCR beta is shown as SEQ ID No. 8.

3. The transduction plasmid of claim 1, wherein the variable regions of TCR α and TCR β are human melanoma-associated antigen-specific TCRs; preferably, the human melanoma associated antigen is MART-1.

4. The transduction plasmid of claim 1, wherein the variable region nucleotide sequence of TCR α is set forth in SEQ ID No. 9; the nucleotide sequence of the variable region of the TCR beta is shown as SEQ ID No. 10.

5. The transduction plasmid of any one of claims 1 to 4, wherein the vector has the nucleotide sequence shown in SEQ ID NO. 11.

6. A transformant comprising the transduction plasmid according to any one of claims 1 to 5.

7. A lentiviral vector system comprising a transduction plasmid according to any one of claims 1 to 5.

8. The lentiviral vector system of claim 7, further comprising PsPAX2 and the pMD2.G plasmid.

9. Use of a transduction plasmid according to any one of claims 1 to 5 in the preparation of a reagent for TCR infection.

10. The use of claim 9, wherein the agent is infected with CD8⁺A T cell agent or an agent that infects Jurkat cells.

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