CN104745581B - A kind of promoter and application thereof of T cell high activity - Google Patents
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Abstract
The invention belongs to molecular biology fields, are related to a kind of promoter and application thereof of T cell high activity.Specifically, the promoter of the T cell high activity includes element 1 and element 2, wherein the element 1 is EF1 α promoter and/or the EF1 α promoter containing introne；The element 2 be in mCMV enhancer, hCMV enhancer and CD3e enhancer any one, two or three.Promoter of the invention can in T cell efficiently expressing exogenous gene, and sequence stablize, will not occur in prokaryotic cell and eukaryocyte transfer process sequence lose.The promoter is applicable to during adoptive cellular immunotherapy, and driving includes foreign gene especially high efficient expression of the full length antibody gene in T cell.
Description
Technical Field
The invention belongs to the field of molecular biology, relates to a promoter, and particularly relates to an artificially synthesized chimeric promoter, wherein the promoter has high activity in T cells. The invention also relates to a recombinant vector containing the promoter, a recombinant virus and application of the promoter in controlling exogenous genes in transgenic T cell treatment.
Background
Immunotherapy for malignant tumors has been rapidly developed in recent years, and has a remarkable clinical efficacy. In 2011, the same review article entitled "the coming of tumor immunotherapy" was published in Nature and JCO, the top journal of clinical tumor (Nature.2011; 480(7378): 480; J Clin Oncol.2011; 29(36):4828), and it is considered that tumor immune cell therapy is about to come up with a new round of research climax and probably occupies a significant position in tumor therapy in the future. In the industrialization field, the us prosperity report of 8 months in 2013, eight major field innovations that may change the world, cancer immunotherapy rank two, which is expected to start only in the market of $ 100-. This therapy is still currently used for the treatment of patients with advanced tumors, but it may become the first line of cancer treatment in the future, like chemotherapy. Recently, douglas bank analysts believe that 60% of advanced cancer patients will be treated with immunotherapy, costing $ 350 billion in the united states, in the next 10 years.
Currently, two major hot spots of immunotherapy are: 1. immune checkpoint monoclonal antibody therapy, including CTLA-4 monoclonal antibody and PD-1 monoclonal antibody; 2. transgenic CAR-T cell therapy (T cells modified with a chimeric antigen receptor gene) or transgenic TCR-T cell therapy (T cells modified with a T cell receptor gene). Commercial mergers and investment cases around these two hotspots are frequently pursued. Therefore, if a monoclonal antibody (e.g., an immune checkpoint monoclonal antibody) with functional activity is expressed by using T cells through a transgenic modification means, the dual effects of anti-tumor cellular immunity (mainly mediated by killer T cells) and humoral immunity (mainly mediated by antibodies) can be synergistically exerted, and a better anti-tumor effect can be exerted. To achieve this goal, it is critical to construct a suitable full-length antibody expression system.
A promoter is a component of a gene, usually located upstream of the 5' end of a structural gene, and is a DNA sequence that is recognized, bound, and transcribed by RNA polymerase. The promoter can guide holoenzyme (holoenzyme) to be correctly combined with a template, activate RNA polymerase and start gene transcription, thereby controlling the starting time and the expression degree of gene expression (transcription). The promoter is one of important factors influencing the expression efficiency of the transgene, and the selection of the high-efficiency promoter is the key for expressing the exogenous gene with high efficiency.
Promoters can be classified into 3 types according to their transcription patterns: constitutive promoters, tissue or organ specific promoters and inducible promoters. The constitutive promoter refers to a constitutive promoter, which is called because the gene expression of different tissues, organs and developmental stages is not obviously different under the control of the constitutive promoter.
Commonly used constitutive promoters in mammals include those of viral origin: murine or human Cytomegalovirus (CMV) promoters (mCMV and hCMV for short, respectively), simian vacuolating virus SV40 promoter; the human genome is naturally derived: EF1 alpha promoter, Ubiquitin promoter (Ubi), beta-actin promoter, PGK-1 promoter, Rosa26 promoter, HSP70 promoter, GAPDH promoter, eIF4A1 promoter, Egr1 promoter, FerH promoter, SM22 alpha promoter, Endothelin-1 promoter, etc.
Commonly used tissue-specific promoters for mammals include: the B29 promoter (B cell specific), CD14 promoter (monocyte specific), CD43 promoter (lymphocyte and platelet specific), CD45 and INF-beta promoter (hematopoietic cell specific), CD68 promoter (macrophage specific), Desmin and Myoglobin promoter (muscle cell specific), CD105 and CD102 promoter (endothelial cell specific), GFAP promoter (astrocyte specific), GPIIb promoter (megakaryocyte specific), Surfactant Protein B promoter (lung specific), NSE promoter (mature neuronal cell specific), Alb promoter (liver specific), and the like.
Commonly used tumor-specific promoters include: AFP promoter (liver cancer specificity), CCKAR promoter (pancreatic cancer specificity), PSA promoter (prostate cancer specificity), Tyr promoter (melanoma specificity), hTERT promoter/CEA promoter/Survivin promoter/CXCR 4 promoter/COX-2/L-plastin promoter/epididymis protein 4 promoter/E2F 1 promoter (tumor broad spectrum specificity) and the like.
Commonly used inducible promoters include: inducible promoters based On the tetracycline (Tet) system (including Tet-On or Tet-off), inducible promoters of the ecdysone-type inducible system, inducible promoters of the FK506 regulatory system, inducible promoters of the rapamycin inducible system, inducible promoters of the RU486 inducible system, and the like.
Although in previous studies we have established an adenovirus-mediated full-length antibody gene expression system and preliminarily achieved expression of functionally active full-length antibodies in T cells, antibody expression levels were difficult to achieve therapeutic concentrations due to the lack of a suitable promoter. Therefore, in order to realize high-efficiency expression of foreign genes, particularly full-length antibody genes, in T cells, it is urgently needed to design and construct a novel high-activity promoter in the T cells.
Disclosure of Invention
The inventor designs and constructs a series of novel chimeric promoters through a great deal of tests and creative labor, and screens the promoters to obtain a group of promoters capable of being efficiently expressed in T cells. The present inventors have surprisingly found that, unlike other chimeric promoters constructed in parallel, the promoter of the present invention is stable and does not lose sequence following its transfer in prokaryotic or eukaryotic cells. The promoter is particularly suitable for driving the high-efficiency expression of exogenous genes (such as full-length antibody genes) in T cells. The following invention is thus provided:
one aspect of the present invention relates to an isolated polynucleotide comprising element 1 and element 2,
wherein,
the element 1 is an EF1 alpha promoter and/or an EF1 alpha promoter containing introns;
the element 2 is any one, two or three selected from the group consisting of the mCMV enhancer, the hCMV enhancer and the CD3e enhancer.
In particular, the polynucleotide is a promoter; more specifically, chimeric promoters.
In one embodiment of the invention, said polynucleotide consists of said element 1 and said element 2.
In one embodiment of the invention, the element 1 is in single or multiple copies, and/or the element 2 is in single or multiple copies.
In one embodiment of the invention, the mCMV enhancer, hCMV enhancer or CD3e enhancer selected in element 2 is independently single copy or multiple copy.
Without being bound by theory, the mCMV enhancer and hCMV enhancer are derived from the same type of viral genome of different species (murine cytomegalovirus, and human cytomegalovirus, respectively), and have similar functions and higher homology with the corresponding DNA sequences. Therefore, even if it is considered that these elements are used in combination, the sequences of both elements are unstable and easily lost because of their high degree of sequence similarity, as a result of previous studies, they are generally used alone as cis-acting elements for replacing functions. An important reason is that whether prokaryotic or eukaryotic expression vectors are involved in the replication of DNA polymerases in the host, it is possible that some of the repeat units may be missed during replication of the repeat sequences due to the complexity of the template, resulting in loss of the sequence. This is a consensus in the field of gene manipulation. Corresponding references refer to the mismatch phenomenon of DNA polymerase during PCR process when amplifying repeated sequence templates. Bias and artifacts in multitemplatepolylase chain reactions (PCR). J Biosci Bioeng.2003; 96(4):317-23.
The polynucleotide of any one of the above wherein element 2 is located upstream (5' of) element 1.
A polynucleotide according to any one of the above which comprises, in order:
(1) the mCMV enhancer + hCMV enhancer + EF1 alpha promoter,
(2) the CD3e enhancer + EF1 alpha promoter,
(3) the CD3e enhancer + mCMV enhancer + hCMV enhancer + EF1 alpha promoter,
(4) the mCMV enhancer + hCMV enhancer + intron-containing EF1 a promoter,
(5) the CD3e enhancer + the EF1 alpha promoter containing introns, or
(6) CD3e enhancer + mCMV enhancer + hCMV enhancer + intron-containing EF1 α promoter.
The polynucleotide according to any one of the above, wherein,
the nucleotide sequence of the mCMV enhancer is shown as SEQ ID NO: 17 is shown;
the nucleotide sequence of the hCMV enhancer is shown as SEQ ID NO: 18 is shown in the figure;
the nucleotide sequence of the CD3e enhancer is shown as SEQ ID NO: 19 is shown in the figure;
the nucleotide sequence of the EF1 alpha promoter is shown as SEQ ID NO: 12 is shown in the specification; and/or
The nucleotide sequence of the EF1 alpha promoter containing the intron is shown as SEQ ID NO: shown at 13.
mCMV enhancer (stable sequence):
ACTGAGTCATTAGGGACTTTCCATTGGGTTTTGCCCAGTACAAAAGGTCAATAGGGGGTGAGTCAATGGGTTTTTCCAGCCAATTTAATTAAAACGCCATGTACTTTCCCACCATTGACGTCAATGGGCTATTGAAACTAATGCAACGTGACCTTTAAACGGTACTTTCCCATAGCTGATTAATGGGAAAGTACCGTTC(SEQ ID NO：17)
in a preferred embodiment of the invention, the above-described mCMV enhancer sequence (SEQ ID NO: 17) is used differently from the following wild-type mCMV enhancer sequence (SEQ ID NO: 20). The wild-type mCMV enhancer sequence is selected in previous researches, but after the wild-type mCMV enhancer sequence is fused with the EF1 alpha promoter, the sequence loss phenomenon occurs in the process of intracellular recombination transformation. Applicants also found in multiple experiments that partial sequence loss of wild-type mCMV is often random, often resulting in reduced or even lost enhancer function. However, NO partial sequence loss occurs during recombinant transformation in cells using the preferred mCMV enhancer of the present invention (SEQ ID NO: 17).
Wild-type mCMV enhancer:
CTGAGTCATTAGGGACTTTCCAATGGGTTTTGCCCAGTACATAAGGTCAATAGGGG ACTGAGTCAATAGGGACTTTCCATTGGGTTTTGCCCAGTACAAAAGGTCAATAGGGGGTG TGAGTCATTGGGTTTTTCCAGCCAATTTAATTAAAACGCCATGTACTTTCCCACCATTGACGTCAATGGGCTATTGAAACTAATGCAACGTGACCTTTAAACGGTACTTTCCCATAGCTGATTAATGGGAAAGTACCGTTC (SEQ ID NO: 20, where the boxes indicate one of the major parts of the sequence that is missing)
hCMV enhancer:
GGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCA(SEQ ID NO：18)
the CD3e enhancer:
AAGTTTCCATGACATCATGAAAGTTTCCATGACATCATGAAAGTTTCCATGACATCATGAAAGTTTCCATGACATCATGAAAGTTTCCATGACATCATGACAAATTAATTAAAAATAAAGAACATGATGCAAATTAATTAAAAATAAAGAACATGATGCAAATTAATTAAAAATAAAGAACATGATG(SEQ ID NO：19)
the nucleotide sequence of the EF1 a promoter and the nucleotide sequence of the EF1 a promoter with introns are shown in example 1 in SEQ ID NO: 12 and SEQ ID NO: 13.
the invention also relates to an isolated polynucleotide comprising or being selected from any one of the following a-e:
seq ID NO: 1-SEQ ID NO: 6;
b. and SEQ ID NO: 1-SEQ ID NO: 6;
c. a polynucleotide which is capable of hybridizing with the polynucleotide a or b under high stringency conditions and has a promoter function;
d. a polynucleotide having a promoter function, which is obtained by modifying the polynucleotide represented by a or b by substitution, deletion or addition of one or more bases; and
e. a polynucleotide having at least 90% identity to the polynucleotide of a or b above and having promoter function;
specifically, the polynucleotide in a or b has a promoter function.
CCEF promoter (containing mCMV enhancer + hCMV enhancer + EF1 α promoter):
ACTGAGTCATTAGGGACTTTCCATTGGGTTTTGCCCAGTACAAAAGGTCAATAGGGGGTGAGTCAATGGGTTTTTCCAGCCAATTTAATTAAAACGCCATGTACTTTCCCACCATTGACGTCAATGGGCTATTGAAACTAATGCAACGTGACCTTTAAACGGTACTTTCCCATAGCTGATTAATGGGAAAGTACCGTTCGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCAAGGATCTGCGATCGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACGGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGCTGAAGCTTCGAGGGGCTCGCATCTCTCCTTCACGCGCCCGCCGCCCTACCTGAGGCCGCCATCCACGCCGGTTGAGTCGCGTTCTGCCGCCTCCCGCCTGTGGTGCCTCCTGAACTGCGTCCGCCGTCTAGGTAAGTTTAAAGCTCAGGTCGAGACCGGGCCTTTGTCCGGCGCTCCCTTGGAGCCTACCTAGACTCAGCCGGCTCTCCACGCTTTGCCTGACCCTGCTTGCTCAACTCTACGTCTTTGTTTCGTTTTCTGTTCTGCGCCGTTACAGATCCAAGCTGTGACCGGCGCCTAC(SEQ ID NO：1)
TEF promoter (containing CD3e enhancer + EF1 α promoter):
AAGTTTCCATGACATCATGAAAGTTTCCATGACATCATGAAAGTTTCCATGACATCATGAAAGTTTCCATGACATCATGAAAGTTTCCATGACATCATGACAAATTAATTAAAAATAAAGAACATGATGCAAATTAATTAAAAATAAAGAACATGATGCAAATTAATTAAAAATAAAGAACATGATGAGGATCTGCGATCGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACGGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGCTGAAGCTTCGAGGGGCTCGCATCTCTCCTTCACGCGCCCGCCGCCCTACCTGAGGCCGCCATCCACGCCGGTTGAGTCGCGTTCTGCCGCCTCCCGCCTGTGGTGCCTCCTGAACTGCGTCCGCCGTCTAGGTAAGTTTAAAGCTCAGGTCGAGACCGGGCCTTTGTCCGGCGCTCCCTTGGAGCCTACCTAGACTCAGCCGGCTCTCCACGCTTTGCCTGACCCTGCTTGCTCAACTCTACGTCTTTGTTTCGTTTTCTGTTCTGCGCCGTTACAGATCCAAGCTGTGACCGGCGCCTAC(SEQ ID NO：2)
TCEF promoter (containing CD3e enhancer + mCMV enhancer + hCMV enhancer + EF1 α promoter):
AAGTTTCCATGACATCATGAAAGTTTCCATGACATCATGAAAGTTTCCATGACATCATGAAAGTTTCCATGACATCATGAAAGTTTCCATGACATCATGACAAATTAATTAAAAATAAAGAACATGATGCAAATTAATTAAAAATAAAGAACATGATGCAAATTAATTAAAAATAAAGAACATGATGACTGAACTGAGTCATTAGGGACTTTCCATTGGGTTTTGCCCAGTACAAAAGGTCAATAGGGGGTGAGTCAATGGGTTTTTCCAGCCAATTTAATTAAAACGCCATGTACTTTCCCACCATTGACGTCAATGGGCTATTGAAACTAATGCAACGTGACCTTTAAACGGTACTTTCCCATAGCTGATTAATGGGAAAGTACCGTTCGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCAGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGAGGAATTAGC(SEQID NO：3)
CCEFI promoter (EF 1 a promoter containing mCMV enhancer + hCMV enhancer + intron):
ACTGAGTCATTAGGGACTTTCCATTGGGTTTTGCCCAGTACAAAAGGTCAATAGGGGGTGAGTCAATGGGTTTTTCCAGCCAATTTAATTAAAACGCCATGTACTTTCCCACCATTGACGTCAATGGGCTATTGAAACTAATGCAACGTGACCTTTAAACGGTACTTTCCCATAGCTGATTAATGGGAAAGTACCGTTCGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCAGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGAGGAATTAGC(SEQ ID NO：4)
TEFI promoter (CD 3e enhancer + intron-containing EF1 α promoter):
AAGTTTCCATGACATCATGAAAGTTTCCATGACATCATGAAAGTTTCCATGACATCATGAAAGTTTCCATGACATCATGAAAGTTTCCATGACATCATGACAAATTAATTAAAAATAAAGAACATGATGCAAATTAATTAAAAATAAAGAACATGATGCAAATTAATTAAAAATAAAGAACATGATGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGAGGAATTAGC(SEQ ID NO：5)
TCEFI promoter (EF 1 a promoter with CD3e enhancer + mCMV enhancer + hCMV enhancer + intron):
AAGTTTCCATGACATCATGAAAGTTTCCATGACATCATGAAAGTTTCCATGACATCATGAAAGTTTCCATGACATCATGAAAGTTTCCATGACATCATGACAAATTAATTAAAAATAAAGAACATGATGCAAATTAATTAAAAATAAAGAACATGATGCAAATTAATTAAAAATAAAGAACATGATGACTGAGTCATTAGGGACTTTCCATTGGGTTTTGCCCAGTACAAAAGGTCAATAGGGGGTGAGTCAATGGGTTTTTCCAGCCAATTTAATTAAAACGCCATGTACTTTCCCACCATTGACGTCAATGGGCTATTGAAACTAATGCAACGTGACCTTTAAACGGTACTTTCCCATAGCTGATTAATGGGAAAGTACCGTTCGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCAGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGAGGAATTAGC(SEQ IDNO：6)
typically, "hybridization conditions" are classified according to the degree of "stringency" of the conditions used to measure hybridization. The degree of stringency can be based, for example, on the melting temperature (Tm) of the nucleic acid binding complex or probe. For example, "maximum stringency" typically occurs at about Tm-5 ℃ (5 ℃ below the Tm of the probe); "higher stringency" occurs at about 5-10 ℃ below Tm; "moderate stringency" occurs about 10-20 ℃ below the Tm of the probe; "Low stringency" occurs at about 20-25 ℃ below the Tm. Alternatively, or further, hybridization conditions may be based on hybridization salt or ionic strength conditions and/or one or more stringency washes. For example, 6 × SSC is extremely low stringency; 3 × SSC — low to medium stringency; 1 × SSC to medium stringency; high stringency with 0.5 × SSC. Functionally, conditions of maximum stringency can be used to determine nucleic acid sequences that are strictly identical or nearly strictly identical to the hybridization probes; and nucleic acid sequences having about 80% or more sequence identity to the probe are determined using conditions of high stringency.
For applications requiring high selectivity, it is typically desirable to employ relatively stringent conditions to form the hybrid, e.g., relatively low salt and/or high temperature conditions are selected. Hybridization conditions including medium and high stringency are provided by Sambrook et al (Sambrook, J. et al (1989) molecular cloning, A laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y.).
For ease of illustration, suitable moderately stringent conditions for detecting hybridization of a polynucleotide of the invention to another polynucleotide include: prewashing with 5 XSSC, 0.5% SDS, 1.0mM EDTA (pH8.0) solution; hybridization in 5 XSSC at 50-65 ℃ overnight; followed by two washes with 2X, 0.5X and 0.2 XSSC containing 0.1% SDS at 65 ℃ for 20 minutes each. One skilled in the art will appreciate that hybridization stringency can be readily manipulated, such as by varying the salt content of the hybridization solution and/or the hybridization temperature. For example, in another embodiment, suitable high stringency hybridization conditions include those described above, except that the hybridization temperature is increased, for example, to 60-65 ℃ or 65-70 ℃.
In the present invention, the nucleotide sequence that hybridizes with SEQ ID NO: 1-SEQ ID NO: 6, having a nucleotide sequence that hybridizes to the nucleotide sequence set forth in SEQ ID NO: 1-SEQ ID NO: 6, or similar promoter activity.
In the present invention, the pair of SEQ ID NOs: 1-SEQ ID NO: 6 refers to a nucleotide sequence which is subjected to substitution, deletion and addition modification of one or more bases respectively or simultaneously at the 5 'end and/or the 3' end of the nucleotide sequence and/or inside the sequence, such as substitution, deletion and addition modification of not more than 2-45 bases, or not more than 2-30 bases, or not more than 3-20 bases, or not more than 4-15 bases, or not more than 5-10 bases, or not more than 6-8 bases respectively expressed by one continuous integer.
In the present invention, the pair of SEQ ID NOs: 1-SEQ ID NO: 6 has a nucleotide sequence which is modified by the substitution, deletion and addition of one or more bases as shown in SEQ ID NO: 1-SEQ ID NO: 6, or similar promoter activity.
By a polynucleotide having a nucleotide sequence that is substantially identical to, for example, the nucleotide sequence set forth in SEQ ID NO: 1-SEQ ID NO: 6 is at least 95% identical to the reference nucleotide sequence in that: in SEQ ID NO: 1-SEQ ID NO: 6, the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the nucleotide sequence of the polynucleotide differs by up to 5 nucleotides. In other words, up to 5% of the nucleotides in a reference sequence may be deleted or replaced by another nucleotide in order to obtain a polynucleotide whose nucleotide sequence is at least 95% identical to the reference nucleotide sequence; or some nucleotides may be inserted into the reference sequence, wherein the inserted nucleotides may be up to 5% of the total nucleotides of the reference sequence; or in some nucleotides there is a combination of deletions, insertions and substitutions, wherein the nucleotides are up to 5% of the total nucleotides of the reference sequence. These mutations of the reference sequence may occur at the 5 'or 3' terminal positions of the reference nucleotide sequence, or anywhere between these terminal positions, either interspersed individually within the nucleotides of the reference sequence, or in one or more contiguous groups within the reference sequence.
In the present invention, algorithms for determining sequence identity and percent sequence similarity are, for example, the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al (1977) Nucl.acid.Res.25: 3389 3402 and Altschul et al (1990) J.mol.biol.215: 403-410. BLAST and BLAST 2.0 can be used to determine percent nucleotide sequence identity according to the invention using, for example, the parameters described in the literature or default parameters. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (NCBI).
In the present invention, the nucleotide sequence shown in SEQ ID NO: 1-SEQ ID NO: 6 comprises a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 1-SEQ ID NO: 6, such as those sequences that contain at least 90% sequence identity, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity compared to a polynucleotide sequence of the invention when using the methods described herein (e.g., BLAST analysis using standard parameters).
In the present invention, the nucleotide sequence shown in SEQ ID NO: 1-SEQ ID NO: 6 has at least 90% sequence identity with the nucleotide sequence set forth in SEQ ID NO: 1-SEQ ID NO: 6, or similar promoter activity.
Yet another aspect of the invention relates to a nucleic acid construct comprising the polynucleotide of any of the above, and operably linked one or more of the same or different exogenous genes; specifically, the foreign gene encodes a monoclonal antibody having an antitumor effect, particularly a full length of a monoclonal antibody having an antitumor effect; specifically, the monoclonal antibody having an anti-tumor effect can be expressed in T cells (for example, CTLA-4 monoclonal antibody or PD-1 monoclonal antibody); specifically, the exogenous gene comprises or is SEQ ID NO: 16.
In the present invention, the term "operably linked" refers to a functional spatial arrangement of two or more nucleotide regions or nucleic acid sequences. In the nucleic acid construct of the present invention, for example, a promoter is placed at a specific position of the nucleic acid sequence of the gene, for example, a promoter is placed at a position upstream of the nucleic acid sequence of the gene, so that transcription of the nucleic acid sequence is guided by the promoter region, whereby the promoter region is "operably linked" to the nucleic acid sequence of the gene. The gene is generally any nucleic acid sequence for which increased transcription is desired, or the promoter and gene of the invention may be designed so as to down-regulate a particular nucleic acid sequence. I.e.by linking the promoter to the gene in the antisense orientation.
Said "operably linked" may be achieved by means of genetic recombination, in particular, the nucleic acid construct is a recombinant nucleic acid construct. In a specific embodiment of the present invention, the gene is a luciferase (luciferase) gene; in another specific embodiment of the present invention, the gene is a full length antibody gene against human PD1 protein.
Yet another aspect of the invention relates to a recombinant vector comprising a polynucleotide according to any one of the invention or a nucleic acid construct according to the invention;
specifically, the recombinant vector is a recombinant cloning vector, a recombinant eukaryotic expression plasmid or a recombinant virus vector;
in particular, the amount of the solvent to be used,
the recombinant cloning vector is a recombinant vector obtained by recombining the polynucleotide of any one of the invention or the nucleic acid construct of the invention with pUC18, pUC19, pMD18-T, pMD19-T, pGM-T vector or pDC315 series vector;
the recombinant expression vector is a recombinant vector obtained by recombining the polynucleotide of any one of the invention or the nucleic acid construct of the invention with a pCDNA3 series vector, a pCDNA4 series vector, a pCDNA5 series vector, a pCDNA6 series vector, a pRL series vector or a pDC315 series vector;
the recombinant virus vector is a recombinant adenovirus vector, a recombinant adeno-associated virus vector, a recombinant retrovirus vector, a recombinant herpes simplex virus vector or a recombinant vaccinia virus vector.
In one embodiment of the invention, the recombinant viral vector is a recombinant adenoviral vector.
Yet another aspect of the invention relates to a recombinant host cell, wherein said cell comprises a polynucleotide according to any one of the invention or a nucleic acid construct of the invention or a recombinant vector of the invention; in particular, the cell is a recombinant mammalian cell; in particular, the cells are recombinant T cells; specifically, the recombinant T cell is a recombinant Jurkat cell, a K562 cell, or a primary culture T cell.
Yet another aspect of the invention relates to methods of introducing a promoter of the invention or a nucleic acid construct of the invention or a vector of the invention into a mammalian cell, including virus-mediated transformation, microinjection, particle bombardment, biolistic transformation, electroporation, and the like. In one embodiment of the invention, the method is virus-mediated transformation, more specifically adenovirus-mediated transformation.
Yet another aspect of the invention relates to a pharmaceutical composition comprising a polynucleotide according to any one of the invention or a nucleic acid construct of the invention or a recombinant vector of the invention or a recombinant host cell of the invention, and optionally a pharmaceutically acceptable adjuvant.
A further aspect of the present invention relates to the use of (1) or (2) selected from the group consisting of:
(1) use of a polynucleotide of any one of the invention or a nucleic acid construct of the invention or a recombinant vector of the invention or a recombinant host cell of the invention in the manufacture of a medicament for the treatment and/or prevention and/or co-treatment of cancer or an anti-tumour; specifically, the cancer or tumor is lung cancer, hepatocellular carcinoma, lymphoma, colon cancer, colorectal cancer, breast cancer, ovarian cancer, cervical cancer, gastric cancer, bile duct cancer, gallbladder cancer, esophageal cancer, renal cancer, glioma, melanoma, pancreatic cancer or prostate cancer;
(2) use of a polynucleotide of any one of the present invention or a nucleic acid construct of the present invention or a recombinant vector of the present invention or a recombinant host cell of the present invention in the preparation of a medicament or an agent for inhibiting a tumor cell; specifically, the tumor cell is a cell of the following tumors: lung cancer, hepatocellular carcinoma, lymphoma, colon cancer, colorectal cancer, breast cancer, ovarian cancer, cervical cancer, gastric cancer, cholangiocarcinoma, gallbladder cancer, esophageal cancer, renal cancer, glioma, melanoma, pancreatic cancer, or prostate cancer.
Yet another aspect of the invention relates to a method of inhibiting a tumor cell in vivo or in vitro comprising the step of administering an effective amount of a polynucleotide according to any one of the invention or a nucleic acid construct of the invention or a recombinant vector of the invention or a recombinant host cell of the invention; specifically, the tumor cell is a cell of the following tumors: lung cancer, hepatocellular carcinoma, lymphoma, colon cancer, colorectal cancer, breast cancer, ovarian cancer, cervical cancer, gastric cancer, cholangiocarcinoma, gallbladder cancer, esophageal cancer, renal cancer, glioma, melanoma, pancreatic cancer, or prostate cancer.
In one embodiment of the invention, the method of inhibiting tumor cells in vivo or in vitro is of non-therapeutic interest.
Yet another aspect of the present invention relates to a method for the treatment and/or prevention and/or co-treatment of cancer or anti-tumor comprising the step of administering an effective amount of a composition comprising an effective amount of a polynucleotide according to any of the present invention or a nucleic acid construct of the present invention or a recombinant vector of the present invention or a recombinant host cell of the present invention; specifically, the cancer or tumor is lung cancer, hepatocellular carcinoma, lymphoma, colon cancer, colorectal cancer, breast cancer, ovarian cancer, cervical cancer, gastric cancer, bile duct cancer, gallbladder cancer, esophageal cancer, renal cancer, glioma, melanoma, pancreatic cancer or prostate cancer. In one embodiment of the invention, the method is adoptive cellular immunotherapy.
A further aspect of the invention relates to the use of a polynucleotide according to any one of the invention as a promoter.
To achieve the above object, the promoter of the present invention can be used in single copy and/or multiple copies, and can be used in combination with promoters known in the art.
Yet another aspect of the invention relates to an isolated polynucleotide comprising or being SEQ ID NO: 17 or the complement thereof. In particular, it is a stable mCMV enhancer.
Yet another aspect of the invention relates to SEQ ID NO: 17 in the preparation of a promoter; in particular, the promoter is a polynucleotide of any of the preceding invention having promoter function.
The present inventors found that the promoter sequence prepared from the polynucleotide is stable, does not undergo sequence loss accompanying its transfer in prokaryotic or eukaryotic cells, and can stably express and drive the expression of foreign genes.
Advantageous effects of the invention
The present invention provides a novel promoter. The promoter can not only efficiently express exogenous genes in T cells, but also has stable sequence (for example, the sequence can not be lost in the transfer process of prokaryotic cells and eukaryotic cells). The promoter can be suitable for driving the high-efficiency expression of exogenous genes, particularly full-length antibody genes in T cells.
Drawings
FIG. 1: pattern of recombinant adenovirus carrying dual luciferase expression cassettes.
FIG. 2: the activity of a strong promoter in a T cell line is often tested. 2A, and detecting the activity of dual luciferase in K562 cells. 2B, results of dual luciferase activity assay in Jurkat cells.
FIG. 3: pattern diagram of a newly constructed strong promoter for T cells.
FIG. 4: pDC315-CCEF-anti-PD1 vector map.
FIG. 5: and (3) detecting the activity of the newly constructed strong T cell promoter in the T cell strain. 5A, and detecting the activity of the dual luciferase in K562 cells. 5B, results of dual luciferase Activity assay in Jurkat cells.
FIG. 6: expression of Ad35-anti-PD1 in Jurkat-T cell line was examined. And 6A, detecting by Western blotting. 6B, ELISA assay standard curve. And 6C, detecting the expression level of the anti-PD1 antibody in K562 cells. 6D, detection result of anti-PD1 antibody expression in Jurkat cells.
Detailed Description
Embodiments of the present invention will be described in detail with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples do not show the specific techniques or conditions, and the techniques or conditions are described in the literature in the art (for example, refer to molecular cloning, a laboratory Manual, third edition, scientific Press, written by J. SammBruker et al, Huang Petang et al) or according to the product instructions. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Example 1: construction of dual-luciferase detection system with commonly-used strong promoter
1. According to the coding sequence of Firefly luciferase (Fluc) gene in psi-CHECK2 plasmid (purchased from Promega), a pair of PCR specific amplification primers (an upstream primer F1, an EcoRI restriction site and a protection base; a downstream primer R1, a SalI restriction site and a protection base) are designed at the beginning and the end. The sequences of F1 and R1 are as follows:
f1: GCCgaattcGCCACCATGGAAGACGCC (SEQ ID NO: 7), wherein lower case letters represent EcoRI cleavage sites;
r1: TGTgtcgacTTACACGGCGATCTTTCCGC (SEQ ID NO: 8), wherein the lower case letters represent SalI cleavage sites.
Using psi-CHECK2 plasmid as template, amplifying FLuc gene coding sequence, cutting by EcoRI + SalI double enzyme, loading into pENTR cut by EcoRI + SalI double enzymeTMVector (available from Invitrogen corporation, pENTR)TMattL1 and attL2 sequences containing a lambda phage site-specific recombination system) to obtain pENTR-Fluc vectorAnd (3) a body. pENTR-Fluc plasmid and pPE35 plasmid (constructed on the basis of pPE3 from Microbixbiosystems, Canada, which contains attR1 and attR2 sequences of lambda phage site-specific recombination system, ccdB gene and 5/35 chimeric adenovirus right arm; the specific construction method is disclosed in Chinese patent application with the granted patent number ZL 201010149683.9.) are co-transformed into BJ5183 bacteria (from Invitrogen), and the backbone plasmid pPE35-Fluc carrying the Fluc gene and 5/35 chimeric adenovirus right arm is obtained through LR reaction recombination.
2. A pair of PCR specific amplification primers (an upstream primer F2, an EcoRI restriction site and a protection base; a downstream primer R2, a SalI restriction site and a protection base) are designed from the beginning to the end according to the coding sequence of the Renilla luciferase (RLuc) gene in a psi-CHECK2 plasmid (purchased from Promega). The sequences of F2 and R2 are as follows:
f2: GCCgaattcGCCACCATGACTTCGAAAGT (SEQ ID NO: 9) with lower case letters representing EcoRI cleavage sites.
R2: TGTgtcgacTTATTGTTCATTTTTGAGAACTCGCT (SEQ ID NO: 10), wherein the lower case letters represent SalI cleavage sites.
The RLuc gene coding sequence is amplified by taking psi-CHECK2 plasmid as a template, and is subjected to EcoRI + SalI double enzyme digestion, and then is loaded into an adenovirus shuttle vector pDC315 (purchased from Beijing Benyuan Zhengyang company and containing an adenovirus left arm, an adenovirus packaging signal and a long terminal repetitive sequence) which is also subjected to EcoRI + SalI double enzyme digestion, so that a pDC315-CMV-Rluc vector (the pDC315 contains a CMV promoter) is obtained.
3. According to the following SEQ ID NO: 11-SEQ ID NO: 15 (a) nucleotide sequences of a CMVi promoter (a CMV promoter containing a CMV intron sequence), an EF1 a promoter, an EF1 a i promoter (an EF1 a promoter containing an EF1 a intron sequence), a CAG promoter and a CCAU promoter (patent 201410495099.7 of the invention) were introduced upstream thereof, an XbaI site was introduced downstream thereof, and the synthesis was performed by Shanghai Jiri Bio-Co Ltd, and pDC315-CMV-Rluc vectors (replacing the CMV promoter in the original pDC315-CMV-Rluc vector) which had been subjected to double digestion with XbaI + EcoRI were loaded and named pDC315-CMVi-RLuc, pDC315-EF1 a-RLuc, pDC 315-1 a i-RLuc, pDC315-CAG-RLuc and pDC315-CCAU-RLuc, respectively.
CMVi promoter:
GAGTCATTAGGGACTTTCCAATGGGTTTTGCCCAGTACATAAGGTCAATAGGGGTGAATCAACAGGAAAGTCCCATTGGAGCCAAGTACACTGAGTCAATAGGGACTTTCCATTGGGTTTTGCCCAGTACAAAAGGTCAATAGGGGGTGAGTCAATGGGTTTTTCCCATTATTGGCACGTACATAAGGTCAATAGGGGTGAGTCATTGGGTTTTTCCAGCCATTTAATTAAAACGCCATGTACTTTCCCACCATTGACGTCAATGGGCTATTGAAACTAATGCAACGTGACCTTTAAACGGTACTTTCCCATAGCTGATTAATGGGAAAGTACCGTTCTCGAGCCAATACACGTCAATGGGAAGTGAAAGGGCAGCCAAAACGTAACACCGCCCCGGTTTTCCCCTGGAAATTCCATATTGGCACTCATTCTATTGGCTGAGCTGCGTTCTACGTGGGTATAAGAGGCGCGACCAGCGTCGGTACCGTCGCAGTCTTCGGTCTGACCACCGTAGAACGCAGATCGAATTGGGCGTCTAACCAGTCACAGTCGCAAGGTAGGCTGAGCACCGTGGCGGGCGGCAGCGGGTGGCGGTCGGGGTTGTTTCTGGCGGAGGTGCTGCTGATGATGTAATTAAAGTAGGCGGTCTTGAGACGGCGGATGGTCGAGGTGAGGTGTGGCAGGCTTGAGATCGATCTGGCCATACACTTGAGTGACAATGACATCCACTTTGCCTTTCTCTCCACAGGTGTCCACTCCC(SEQ ID NO：11)
EF1 α promoter:
AGGATCTGCGATCGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACGGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGCTGAAGCTTCGAGGGGCTCGCATCTCTCCTTCACGCGCCCGCCGCCCTACCTGAGGCCGCCATCCACGCCGGTTGAGTCGCGTTCTGCCGCCTCCCGCCTGTGGTGCCTCCTGAACTGCGTCCGCCGTCTAGGTAAGTTTAAAGCTCAGGTCGAGACCGGGCCTTTGTCCGGCGCTCCCTTGGAGCCTACCTAGACTCAGCCGGCTCTCCACGCTTTGCCTGACCCTGCTTGCTCAACTCTACGTCTTTGTTTCGTTTTCTGTTCTGCGCCGTTACAGATCCAAGCTGTGACCGGCGCCTAC(SEQ ID NO：12)
EF1 α i promoter:
GCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGAGGAATTAGC(SEQ ID NO：13)
the CAG promoter:
TATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGACTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGTTGCCTTCGCCCCGTGCCCCGCTCCGCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTTAATGACGGCTCGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTAAAGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGGAGCGGCTCGGGGGGTGCGTGCGTGTGTGTGTGCGTGGGGAGCGCCGCGTGCGGCCCGCGCTGCCCGGCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGCTTTGTGCGCTCCGCGTGTGCGCGAGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCGGGGGGGCTGCGAGGGGAACAAAGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGGGGTGTGGGCGCGGCGGTCGGGCTGTAACCCCCCCCTGCACCCCCCTCCCCGAGTTGCTGAGCACGGCCCGGCTTCGGGTGCGGGGCTCCGTGCGGGGCGTGGCGCGGGGCTCGCCGTGCCGGGCGGGGGGTGGCGGCAGGTGGGGGTGCCGGGCGGGGCGGGGCCGCCTCGGGCCGGGGAGGGCTCGGGGGAGGGGCGCGGCGGCCCCGGAGCGCCGGCGGCTGTCGAGGCGCGGCGAGCCGCAGCCATTGCCTTTTATGGTAATCGTGCGAGAGGGCGCAGGGACTTCCTTTGTCCCAAATCTGGCGGAGCCGAAATCTGGGAGGCGCCGCCGCACCCCCTCTAGCGGGCGCGGGCGAAGCGGTGCGGCGCCGGCAGGAAGGAAATGGGCGGGGAGGGCCTTCGTGCGTCGCCGCGCCGCCGTCCCCTTCTCCATCTCCAGCCTCGGGGCTGCCGCAGGGGGACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACAGCTCCTGGGCAACGTGCTGGTTGTTGTGCTGTCTCATCATTTTGGCAAAGAATTC(SEQ ID NO：14)
CCAU promoter (containing mCMV enhancer + hCMV enhancer + β -Actin promoter + Ubi enhancer):
ACTGAGTCATTAGGGACTTTCCATTGGGTTTTGCCCAGTACAAAAGGTCAATAGGGGGTGAGTCAATGGGTTTTTCCCATTATTGGCACGTACATAAGGTCAATAGGGGTGAGTCATTGGGTTTTTCCAGCCAATTTAATTAAAACGCCATGTACTTTCCCACCATTGACGTCAATGGGCTATTGAAACTAATGCAACGTGACCTTTAAACGGTACTTTCCCATAGCTGATTAATGGGAAAGTACCGTTCGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGCGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTAAAACAGGTAAGTCCGGCCTCCGCGCCGGGTTTTGGCGCCTCCCGCGGGCGCCCCCCTCCTCACGGCGAGCGCTGCCACGTCAGACGAAGGGCGCAGCGAGCGTCCTGATCCTTCCGCCCGGACGCTCAGGACAGCGGCCCGCTGCTCATAAGACTCGGCCTTAGAACCCCAGTATCAGCAGAAGGACATTTTAGGACGGGACTTGGGTGACTCTAGGGCACTGGTTTTCTTTCCAGAGAGCGGAACAGGCGAGGAAAAGTAGTCCCTTCTCGGCGATTCTGCGGAGGGATCTCCGTGGGGCGGTGAACGCCGATGATGCCTCTACTAACCATGTTCATGTTTTCTTTTTTTTTCTACAGGTCCTGGGTGACGAACAG(SEQ ID NO：15)
pDC315-CMV-Rluc, pDC315-CMVi-RLuc, pDC315-EF1 alpha-RLuc, pDC315-EF1 alpha i-RLuc, pDC315-CAG-RLuc, pDC315-CCAU-RLuc and pPE35-Fluc were co-transfected into HEK293 cell strain (purchased from American Standard article Collection, ATCC) by Lipofectamine, respectively, the specific methods of which are described in the instructions of GIBCOBRL company. After cotransfection, virus plaques appear 9-14 days later, and the adenovirus dual-luciferase report system carrying nucleotide sequences of all promoters is obtained after three times of virus plaque purification, and is named as Ad35-CMV-2Luc, Ad35-CMVi-2Luc, Ad35-EF1 alpha-2 Luc, Ad35-EF1 alpha i-2Luc, Ad35-CAG-2Luc and Ad35-CCAU-2Luc (the specific pattern diagram is shown in figure 1).
The recombinant adenovirus is propagated in HEK293 cells in large quantities, the adenovirus is purified in large quantities by cesium chloride gradient centrifugation, and then the virus titer is determined by 50% tissue culture infectious dose (TCID50) (see AdEasy (TM) operating manual of Qbiogene, USA).
Example 2: activity detection of commonly used strong promoters in T cell lines
The low generation Jurkat T and K562 cell lines (both purchased from ATCC) with good growth status were expressed at 1X 104cells/well were plated on 96-well plates at 37 ℃ with 5% CO2Incubating for 24 hours; respectively infecting 6 recombinant viruses such as Ad35-CMV-2Luc, Ad35-CMVi-2Luc, Ad35-EF1 alpha-2 Luc, Ad35-EF1 alpha i-2Luc, Ad35-CAG-2Luc, Ad35-CCAU-2Luc and the like according to the virus infection complex MOI which is 5, and setting 4 multiple holes in each group; then placing at 37 ℃ and 5% CO2Culturing an incubator; after 24 hours, the cells were lysed, and the enzyme activity of RLuc was measured in a microplate reader using a dual-luciferase assay kit (purchased from Promega) with the enzyme activity of FLuc as an internal reference, to obtain the relative ratio of RLuc/FLuc. The specific operation steps are completed according to the kit instruction.
The results showed (see FIGS. 2A-2B) that, among the various strong promoters, the strong promoter of the EF1 alpha line (including EF1 alpha and EF1 alpha i) had stronger activity in T cell lines than the strong promoter of the CMV line (including CMV and CMVi) and the artificially constructed chimeric promoter (including CAG and CCAU).
Example 3: construction of T cell strong promoter based on EF1 alpha promoter
According to SEQ ID NO: 1-SEQ ID NO: the nucleotide sequences of CCEF promoter, TEF promoter, TCEF promoter, CCEFi promoter, TEFi promoter and TCEFi promoter shown in 6 (a promoter pattern is shown in figure 3), XbaI is introduced into the upstream of the nucleotide sequences, EcoRI is introduced into the downstream of the nucleotide sequences, the DNA sequences are synthesized by Shanghai Jiyu biology company, and pDC315-CMV-Rluc vectors (replacing CMV promoters in original pDC315-CMV-Rluc vectors) which are subjected to double digestion by XbaI + EcoRI are loaded into the DNA sequences and named pDC315-CCEF-RLuc, pDC315-TEF-RLuc, pDC315-TCEF-RLuc, pDC315-CCEFi-RLuc, pDC315-TEFi-Rluc and pDC315-TCEFi-Rluc respectively (a vector pattern is shown in figure 4). The DNA sequences of the promoters were determined by Shanghai's work after transformation of DH 5. alpha. bacteria with the plasmids described above, and it was found that the sequences of the promoters were stable.
pDC315-CCEF-RLuc, pDC315-TEF-RLuc, pDC315-TCEF-RLuc, pDC315-CCEFi-RLuc, pDC315-TEFi-Rluc, pDC315-TCEFi-Rluc and pPE35-Fluc were co-transfected into HEK293 cell strain (purchased from American Standard collections of articles, ATCC) by Lipofectamine, respectively, in the specific methods described in the instructions of GIBCOBRL company. And (3) generating virus plaques 9-14 days after cotransfection, and purifying the virus plaques for three times to obtain the adenovirus dual-luciferase report system carrying nucleotide sequences of each promoter, which is named as Ad35-CCEF-2Luc, Ad35-TEF-2Luc, Ad35-TCEF-2Luc, Ad35-CCEFi-2Luc, Ad35-TEFi-2Luc and Ad35-TCEFi-2 Luc. The genome of the adenovirus was extracted, and the DNA sequence of each promoter was determined by Shanghai's work, and it was found that the sequence of each promoter was stable.
Example 4: activity detection of newly constructed promoters in T cell lines
The low passage Jurkat and K562 cell lines (both purchased from ATCC) with good growth status were expressed at 1X 104cells/well were plated on 96-well plates at 37 ℃ with 5% CO2Incubating for 24 hours; respectively infecting 8 recombinant viruses such as Ad35-EF1 alpha-2 Luc, Ad35-CCEF-2Luc, Ad35-TEF-2Luc, Ad35-TCEF-2Luc, Ad35-EF1 alpha i-2Luc, Ad35-CCEFi-2Luc, Ad35-TEFi-2Luc, Ad35-TCEFi-2Luc and the like according to the virus infection complex MOI which is 5, and setting 4 complex holes in each group; then placing at 37 ℃ and 5% CO2Culturing an incubator; after 24 hours, the cells were lysed and lysedA dual-luciferase assay kit (purchased from Promega) is placed in an enzyme-labeling instrument to measure the enzyme activity of RLuc, and the relative ratio of RLuc to FLuc is obtained by taking the enzyme activity of FLuc as an internal reference. The specific operation steps are completed according to the kit instruction.
The results show (see FIGS. 5A-5B) that CCEF, TEF, TCEF are more active in T cell lines than the EF1 alpha promoter; the activities of CCEFi, TEFi, TCEFi were higher in T cell lines relative to the EF1 α i promoter. The expression activity of the promoter sequence of the EF1 alpha line in T cells can be obviously enhanced after the CMV enhancer and/or the CD3e enhancer sequence is added.
Example 5: construction of recombinant adenovirus Ad35-anti-PD1
According to the following SEQ ID NO: 16 (containing heavy chain, F2A and light chain genes of the anti-PD1 full-length antibody gene) and introducing EcoRI at the upstream and introducing SalI at the downstream, entrusting the synthesis of the anti-PD1 full-length antibody gene by Shanghai Jiyu biological company, respectively loading pDC315-EF1 alpha-RLuc, pDC315-CCEF-RLuc, pDC315-TEF-RLuc, pDC315-TCEF-RLuc, pDC315-EF1 alpha i-RLuc, pDC315-CCEFi-RLuc, pDC315-TEFi-Rluc and pDC315-TCEFi-Rluc vectors (replacing the original Rluc coding gene sequence), and constructing pDC315-EF1 alpha-anti-PD 3535 1, pDC 315-CCEF-anti-PD-1, pDC 315-TEF-anti-1, pDC 315-TCEFi-Rluc-1, antEF-1-31 alpha-RCE, pDC 315-TCEF-35 alpha-RLuc, pDC315-CCEFi-anti-PD1, pDC315-TEFi-anti-PD1, pDC315-TCEFi-anti-PD 1.
gaattcGCCACCATGGAAGCCCCAGCTCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCACCGGACAGGTGTACTTGGTAGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTAACTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCACTTATATGGTATGATGGAAGTAATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGACCAGTCTGAGAGTCGAGGACACGGCTGTGTATTATTGTGCGAGCAACGTTGACCATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTTCCACCAAGGGCCCATCCGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCACCATGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAACGTAAAAGGCGAGCTCCTGTTAAACAGACTTTGAATTTTGACCT TCTCAAGTTGGCGGGAGACGTCGAGTCCAACCCTGGGCCCATGGAAGCCCCAGCTCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCACCGGAGAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGTAGTTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGAGTAGCAACTGGCCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAAgtcgac (SEQ ID NO: 16), in which the lower case letters represent the cleavage site and the F2A coding sequence is underlined.
pDC315-EF1 alpha-anti-PD 1, pDC315-CCEF-anti-PD1, pDC315-TEF-anti-PD1, pDC315-TCEF-anti-PD1, pDC315-EF1 alpha i-anti-PD1, pDC315-CCEFi-anti-PD1, pDC315-TEFi-anti-PD1, pDC315-TCEFi-anti-PD1 and pPE35 were co-transfected into HEK293 cell line (purchased from American Standard article Collection, ATCC) by Lipofectamine, which was specifically described in the instructions of GIBCOBRL company. After cotransfection, virus plaques appear in 9-14 days, and the recombinant adenovirus carrying the PD1 full-length antibody gene expression frame is obtained after three virus plaque purifications and is named as Ad35-EF1 alpha-anti-PD 1, Ad35-CCEF-anti-PD1, Ad35-TEF-anti-PD1, Ad35-TCEF-anti-PD1, Ad35-EF1 alpha i-anti-PD1, Ad35-CCEFi-anti-PD1, Ad35-TEFi-anti-PD1 and Ad35-TCEFi-anti-PD 1.
Example 6: detection of recombinant adenovirus Ad35-anti-PD1 antibody expression in T cell strain
Human T cell lines K562 and Jurkat were 5X 10, respectively5cells/well 6-well plate, put at 37 deg.C and 5% CO224H, infecting recombinant adenovirus Ad35-EF1 alpha-anti-PD 1, Ad35-CCEF-anti-PD1, Ad35-TEF-anti-PD1, Ad35-TCEF-anti-PD1, Ad35-EF1 alpha i-anti-PD1, Ad35-CCEFi-anti-PD1, Ad35-TEFi-anti-PD1, Ad35-TCEFi-anti-PD1 respectively according to MOI 20, collecting cell supernatant after 48 hours, and quantitatively detecting the expression of anti-PD-1 protein by Western blotting (using murine anti-human IgG4(H + L) as a primary antibody) and a double sandwich ELISA method (using goat anti-human polyclonal antibody, murine anti-human IgG for detection, using a cetuximab as a standard substance and measuring after the sample is diluted by 100 times).
Western Blotting experiments show that after recombinant adenovirus Ad35-EF1 alpha-anti-PD 1, Ad35-CCEF-anti-PD1, Ad35-TEF-anti-PD1, Ad35-TCEF-anti-PD1, Ad35-EF1 alpha i-anti-PD1, Ad35-CCEFi-anti-PD1, Ad35-TEFi-anti-PD1 and Ad35-TCEFi-anti-PD1 infect human Jurkat and K562 cell strains, PD1 full-length antibody genes can be normally expressed in cells, and the light-heavy chain gene sizes of the antibodies are consistent with the expectation (FIG. 6A, a sample infected with Ad35-CCEF-anti-PD1 is taken as a representative).
ELISA results show that after infection with Ad35-CCEF-anti-PD1, Ad35-TEF-anti-PD1, Ad35-TCEF-anti-PD1, Ad35-CCEFi-anti-PD1, Ad35-TEFi-anti-PD1 and Ad35-TCEFi-anti-PD1, PD1 antibodies can be expressed in K562 and Jurkat cell strains, and the expression amounts are all obviously higher than those of control viruses Ad35-EF1 alpha-anti-PD 1 and Ad35-EF1 alpha i-anti-PD1 (FIGS. 6B, 6C and 6D).
Although specific embodiments of the invention have been described in detail, those skilled in the art will appreciate. Various modifications and substitutions of those details may be made in light of the overall teachings of the disclosure, and such changes are intended to be within the scope of the present invention. The full scope of the invention is given by the appended claims and any equivalents thereof.
Claims (29)
1. An isolated polynucleotide comprising element 1 and element 2,
wherein,
the element 1 is an EF1 alpha promoter and/or an EF1 alpha promoter containing introns;
the element 2 comprises the mCMV enhancer and the hCMV enhancer;
the polynucleotide is a promoter;
wherein said element 2 further comprises the CD3e enhancer;
and, the isolated polynucleotide, in order, comprising:
(1) the CD3e enhancer + mCMV enhancer + hCMV enhancer + EF1 alpha promoter, or
(2) CD3e enhancer + mCMV enhancer + hCMV enhancer + intron-containing EF1 α promoter;
wherein,
the nucleotide sequence of the mCMV enhancer is shown as SEQ ID NO: 17 is shown;
the nucleotide sequence of the hCMV enhancer is shown as SEQ ID NO: 18 is shown in the figure;
the nucleotide sequence of the CD3e enhancer is shown as SEQ ID NO: 19 is shown in the figure;
the nucleotide sequence of the EF1 alpha promoter is shown as SEQ ID NO: 12 is shown in the specification;
the nucleotide sequence of the EF1 alpha promoter containing the intron is shown as SEQ ID NO: shown at 13.
2. The isolated polynucleotide of claim 1, which is a chimeric promoter.
3. The isolated polynucleotide of claim 1, wherein the polynucleotide consists of said element 1 and said element 2.
4. The polynucleotide of any one of claims 1 to 3, wherein element 2 is upstream of element 1.
5. An isolated polynucleotide selected from any one of a-b:
seq ID NO: 1. SEQ ID NO: 3. SEQ ID NO: 4 and SEQ ID NO: 6;
b. a polynucleotide complementary to any one of the sequences in a;
wherein the polynucleotide in a or b has a promoter function.
6. A nucleic acid construct comprising the polynucleotide of any one of claims 1 to 5, and operably linked one or more identical or different exogenous genes.
7. The nucleic acid construct according to claim 6, wherein the foreign gene encodes a monoclonal antibody having an antitumor effect.
8. The nucleic acid construct according to claim 6, wherein the foreign gene encodes the full length of a monoclonal antibody having an antitumor effect.
9. The nucleic acid construct according to claim 7 or 8, wherein the monoclonal antibody having an anti-tumor effect is capable of being expressed in T cells.
10. The nucleic acid construct according to claim 9, wherein the monoclonal antibody having an anti-tumor effect is CTLA-4 mab or PD-1 mab.
11. The nucleic acid construct of claim 6, wherein the exogenous gene is SEQ ID NO: 16.
12. A recombinant vector comprising the polynucleotide of any one of claims 1 to 5 or the nucleic acid construct of any one of claims 6 to 11.
13. The recombinant vector according to claim 12, wherein said recombinant vector is a recombinant cloning vector, a recombinant eukaryotic expression plasmid or a recombinant viral vector.
14. The recombinant vector according to claim 13, wherein the recombinant cloning vector is a polynucleotide according to any one of claims 1 to 5 or a nucleic acid construct according to any one of claims 6 to 11, which is recombined with pUC18, pUC19, pMD18-T, pMD19-T, pGM-T vector or pDC315 series vector.
15. The recombinant vector according to claim 13, wherein the recombinant eukaryotic expression plasmid is the polynucleotide of any one of claims 1 to 5 or the nucleic acid construct of any one of claims 6 to 11, which is obtained by recombining the polynucleotide with a pCDNA3 series vector, a pCDNA4 series vector, a pCDNA5 series vector, a pCDNA6 series vector, a pRL series vector or a pDC315 series vector.
16. The recombinant vector according to claim 13, wherein the recombinant viral vector is a recombinant adenoviral vector, a recombinant adeno-associated viral vector, a recombinant retroviral vector, a recombinant herpes simplex viral vector or a recombinant vaccinia viral vector.
17. A recombinant host cell comprising the polynucleotide of any one of claims 1 to 5 or the nucleic acid construct of any one of claims 6 to 11 or the recombinant vector of any one of claims 12 to 16.
18. The recombinant host cell of claim 17, which is a recombinant mammalian cell.
19. The recombinant host cell of claim 17, which is a recombinant T cell.
20. The recombinant host cell according to claim 19, wherein said recombinant T cell is a recombinant Jurkat cell or a primary culture T cell.
21. The recombinant host cell of claim 17, which is a recombinant K562 cell.
22. A pharmaceutical composition comprising the nucleic acid construct of any one of claims 6 to 11 or the recombinant vector of any one of claims 12 to 16 or the recombinant host cell of any one of claims 17 to 21 and a pharmaceutically acceptable excipient.
23. Use of the polynucleotide of any one of claims 1 to 5 or the nucleic acid construct of any one of claims 6 to 11 or the recombinant vector of any one of claims 12 to 16 or the recombinant host cell of any one of claims 17 to 21 in the manufacture of a medicament for the treatment and/or prevention of cancer.
24. The use according to claim 23, wherein the cancer or tumour is lung cancer, hepatocellular cancer, lymphoma, colorectal cancer, breast cancer, ovarian cancer, cervical cancer, gastric cancer, cholangiocarcinoma, gallbladder cancer, oesophageal cancer, renal cancer, glioma, melanoma, pancreatic cancer or prostate cancer.
25. The use according to claim 24, wherein the colon cancer.
26. Use of the polynucleotide of any one of claims 1 to 5 or the nucleic acid construct of any one of claims 6 to 11 or the recombinant vector of any one of claims 12 to 16 or the recombinant host cell of any one of claims 17 to 21 in the manufacture of a medicament for inhibiting a tumor cell.
27. The use of claim 26, wherein the tumor cell is a cell of a tumor: lung cancer, hepatocellular carcinoma, lymphoma, colorectal cancer, breast cancer, ovarian cancer, cervical cancer, gastric cancer, cholangiocarcinoma, gallbladder cancer, esophageal cancer, renal cancer, glioma, melanoma, pancreatic cancer, or prostate cancer.
28. The use according to claim 27, wherein the colon cancer.
29. Use of the polynucleotide of any one of claims 1 to 5 as a promoter.
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| CN105796597A (en) * | 2016-03-11 | 2016-07-27 | 江苏三特生物科技有限公司 | Application of CAR-T cell carrying PD-L1 and CTLA-4 antibody genes in tumor immunology |
| CN107523548A (en) * | 2016-06-20 | 2017-12-29 | 上海细胞治疗研究院 | A kind of T cell of high efficiency stable expression antibody and application thereof |
| CN107523545A (en) | 2016-06-20 | 2017-12-29 | 上海细胞治疗研究院 | A kind of lethal cell of high efficiency stable expression antibody and application thereof |
| CN109988759A (en) * | 2017-12-29 | 2019-07-09 | 上海细胞治疗研究院 | A kind of chimeric promoters in T cell with high transcriptional activity |
| CN109486815B (en) * | 2018-11-02 | 2021-12-31 | 成都大学 | Artificial chimeric promoter and construction method thereof |
| CN112143700B (en) * | 2019-06-26 | 2024-07-19 | 上海细胞治疗集团股份有限公司 | Method for preparing immune effector cell over-expressing exogenous gene |
| CN110592014A (en) * | 2019-08-14 | 2019-12-20 | 广东美赛尔细胞生物科技有限公司 | Method for continuously removing feeder cells in vitro and in vivo without irradiation in NK cell therapy |
| CN112813099A (en) * | 2019-11-15 | 2021-05-18 | 上海细胞治疗集团有限公司 | A promoter with high activity in activated T cells |
| CN115044585B (en) * | 2022-06-22 | 2023-01-24 | 浙江欧赛思生物科技有限公司 | Eukaryotic cell promoter CF1 and application thereof in cell gene expression |
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