CN107043774B - Chimeric strong promoter and application thereof - Google Patents

Abstract

The invention belongs to the field of molecular biology, and relates to a chimeric strong promoter and application thereof. The promoter can efficiently express exogenous genes in a tumor cell in a broad spectrum manner, has higher expression activity in a series of tumor cells than the existing CAG promoter, and has stable sequence (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 in tumor cells in the process of tumor gene therapy.

Description

Chimeric strong promoter and application thereof

The invention relates to a divisional application of a parent case with the application number of 201410495099.7, the application date of the parent case is 9/25/2014, and the invention name is 'a tumor cell broad-spectrum high-activity promoter and application thereof'.

Technical Field

The invention belongs to the field of molecular biology, and relates to a tumor cell broad-spectrum high-activity promoter and application thereof. The promoter is an artificially synthesized chimeric promoter, and has broad-spectrum high activity in tumor cells. The invention also relates to a recombinant vector containing the promoter, a recombinant virus and application of the promoter in controlling therapeutic genes for gene therapy.

Background

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 are: 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 are: 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 are: 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.

In tumor gene therapy, it is very important to maintain the high-efficiency and stable expression of foreign genes. However, some constitutive promoters of viral origin, although highly active for transient expression (such as the CMV promoter), are susceptible to being turned off by epigenetic modifications; while some human-derived natural constitutive promoters or tumor-specific promoters have stable expression, the expression activity is relatively weak, and the gene therapy requirements are difficult to meet. Therefore, researchers have designed and constructed a series of artificial chimeric promoters, which contain some cis-regulatory elements, mainly including promoter core sequence capable of playing stable expression role, and upstream enhancer or downstream intron capable of enhancing expression efficiency, and the representative is chimeric promoter CAG (containing human CMV enhancer-chicken beta-actin promoter-rabbit beta-globin intron), which is widely applied to the expression of exogenous genes.

There is still a need to develop a promoter suitable for broad-spectrum high-efficiency expression of exogenous genes in tumor cells, which has higher expression activity in a series of tumor cells than CAG promoters, has stable sequence (no sequence loss in the process of transferring prokaryotic cells and eukaryotic cells), and can be suitable for driving the high-efficiency expression of exogenous genes in tumor cells.

Disclosure of Invention

The inventor designs and constructs a series of novel chimeric promoters through a large amount of tests and creative labor, and screens the promoters to obtain a promoter capable of efficiently expressing exogenous genes in tumor cells in a broad spectrum. The present inventors have surprisingly found that the promoter sequence is stable, does not undergo sequence loss following its transfer in prokaryotic or eukaryotic cells, and is capable of stably expressing a gene driving expression of a foreign gene. The following invention is thus provided:

one aspect of the present invention relates to an isolated polynucleotide comprising the following elements:

hCMV enhancer, beta-Actin promoter and mCMV enhancer;

in particular, it further comprises a Ubi enhancer; more specifically, the Ubi enhancer is located downstream of the 3 elements described above;

specifically, each of the 4 elements is independently 1, 2, 3, or 3 or more;

in particular, the polynucleotide has promoter function.

A polynucleotide according to any one of the present invention comprising, in order:

hCMV enhancer + beta-Actin promoter + mCMV enhancer, or

The mCMV enhancer + hCMV enhancer + β -Actin promoter;

preferably, it comprises in sequence:

hCMV enhancer + beta-Actin promoter + mCMV enhancer + Ubi enhancer, or

The mCMV enhancer + hCMV enhancer + β -Actin promoter + Ubi enhancer.

The polynucleotide according to any one of the present invention, wherein:

the nucleotide sequence of the hCMV enhancer is shown as SEQ ID NO: 17 is shown;

the nucleotide sequence of the beta-Actin promoter is shown as SEQ ID NO: 18 is shown in the figure;

the nucleotide sequence of the mCMV enhancer is shown as SEQ ID NO: 19 or SEQ ID NO: 20 is shown in the figure; preferably SEQ ID NO: 20;

the nucleotide sequence of the Ubi enhancer is shown as SEQ ID NO: shown at 21.

A polynucleotide according to any one of the present invention, with or without linker sequences or splice sites between the individual elements thereof; specifically, there are splice donors and splice acceptors upstream and downstream of the mCMV enhancer and/or hCMV enhancer and/or Ubi enhancer, respectively; more specifically, the nucleotide sequence of the splice donor is shown as SEQ ID NO: 22; the nucleotide sequence of the splicing acceptor is shown as SEQ ID NO: shown at 23.

Splice donor (splice donor, SD): AAAACAGGTAAGTCC (SEQ ID NO: 22)

Splice acceptor (splice acceptor, SA): GCCTCTACTAACCATGTTCATGTTTTCTTTTTTTTTCTACAGGTCCTGGGTGACGAACAG (SEQ ID NO: 23)

In one embodiment of the present invention, without being limited by theory, the two ends of the enhancer sequence behind the promoter contain a splicing site, which ensures that the enhancer sequence located at the downstream of the promoter can be accurately spliced after transcription without affecting the translation of the target protein; the remaining elements are directly connected.

Another aspect of the invention relates to an isolated polynucleotide comprising or being selected from the group consisting of a polynucleotide selected from any one of a-e:

seq ID NO: 1-SEQ ID NO: 4;

b. and SEQ ID NO: 1-SEQ ID NO: 4;

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.

The polynucleotide of the present invention is a promoter; specifically, it is a chimeric promoter.

SEQ ID NO: 1-SEQ ID NO: 4 the specific sequence is as follows:

CAC promoter (containing hCMV enhancer + β -Actin promoter + mCMV enhancer):

GGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGCGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTAAAACAGGTAAGTCCCTGAGTCATTAGGGACTTTCCAATGGGTTTTGCCCAGTACATAAGGTCAATAGGGGTGAATCAACAGGAAAGTCCCATTGGAGCCAAGTACACTGAGTCAATAGGGACTTTCCATTGGGTTTTGCCCAGTACAAAAGGTCAATAGGGGGTGAGTCAATGGGTTTTTCCCATTATTGGCACGTACATAAGGTCAATAGGGGTGAGTCATTGGGTTTTTCCAGCCAATTTAATTAAAACGCCATGTACTTTCCCACCATTGACGTCAATGGGCTATTGAAACTAATGCAACGTGACCTTTAAACGGTACTTTCCCATAGCTGATTAATGGGAAAGTACCGTTCGCCTCTACTAACCATGTTCATGTTTTCTTTTTTTTTCTACAGGTCCTGGGTGACGAACAG(SEQ ID NO：1)

CACU promoter (containing hCMV enhancer + β -Actin promoter + mCMV enhancer-Ubi enhancer):

GGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGCGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTAAAACAGGTAAGTCCCTGAGTCATTAGGGACTTTCCAATGGGTTTTGCCCAGTACATAAGGTCAATAGGGGTGAATCAACAGGAAAGTCCCATTGGAGCCAAGTACACTGAGTCAATAGGGACTTTCCATTGGGTTTTGCCCAGTACAAAAGGTCAATAGGGGGTGAGTCAATGGGTTTTTCCCATTATTGGCACGTACATAAGGTCAATAGGGGTGAGTCATTGGGTTTTTCCAGCCAATTTAATTAAAACGCCATGTACTTTCCCACCATTGACGTCAATGGGCTATTGAAACTAATGCAACGTGACCTTTAAACGGTACTTTCCCATAGCTGATTAATGGGAAAGTACCGTTCGGCCTCCGCGCCGGGTTTTGGCGCCTCCCGCGGGCGCCCCCCTCCTCACGGCGAGCGCTGCCACGTCAGACGAAGGGCGCAGCGAGCGTCCTGATCCTTCCGCCCGGACGCTCAGGACAGCGGCCCGCTGCTCATAAGACTCGGCCTTAGAACCCCAGTATCAGCAGAAGGACATTTTAGGACGGGACTTGGGTGACTCTAGGGCACTGGTTTTCTTTCCAGAGAGCGGAACAGGCGAGGAAAAGTAGTCCCTTCTCGGCGATTCTGCGGAGGGATCTCCGTGGGGCGGTGAACGCCGATGATGCCTCTACTAACCATGTTCATGTTTTCTTTTTTTTTCTACAGGTCCTGGGTGACGAACAG(SEQ ID NO：2)

CCAU promoter (containing mCMV enhancer + hCMV enhancer + β -Actin promoter + Ubi enhancer):

ACTGAGTCATTAGGGACTTTCCATTGGGTTTTGCCCAGTACAAAAGGTCAATAGGGGGTGAGTCAATGGGTTTTTCCCATTATTGGCACGTACATAAGGTCAATAGGGGTGAGTCATTGGGTTTTTCCAGCCAATTTAATTAAAACGCCATGTACTTTCCCACCATTGACGTCAATGGGCTATTGAAACTAATGCAACGTGACCTTTAAACGGTACTTTCCCATAGCTGATTAATGGGAAAGTACCGTTCGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGCGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTAAAACAGGTAAGTCCGGCCTCCGCGCCGGGTTTTGGCGCCTCCCGCGGGCGCCCCCCTCCTCACGGCGAGCGCTGCCACGTCAGACGAAGGGCGCAGCGAGCGTCCTGATCCTTCCGCCCGGACGCTCAGGACAGCGGCCCGCTGCTCATAAGACTCGGCCTTAGAACCCCAGTATCAGCAGAAGGACATTTTAGGACGGGACTTGGGTGACTCTAGGGCACTGGTTTTCTTTCCAGAGAGCGGAACAGGCGAGGAAAAGTAGTCCCTTCTCGGCGATTCTGCGGAGGGATCTCCGTGGGGCGGTGAACGCCGATGATGCCTCTACTAACCATGTTCATGTTTTCTTTTTTTTTCTACAGGTCCTGGGTGACGAACAG(SEQ ID NO：3)

4CAU promoter (containing mCMV enhancer + hCMV enhancer + β -Actin promoter + Ubi enhancer):

CTGAGTCATTAGGGACTTTCCAATGGGTTTTGCCCAGTACATAAGGTCAATAGGGGTGAATCAACAGGAAAGTCCCATTGGAGCCAAGTACACTGAGTCAATAGGGACTTTCCATTGGGTTTTGCCCAGTACAAAAGGTCAATAGGGGGTGAGTCAATGGGTTTTTCCCATTATTGGCACGTACATAAGGTCAATAGGGGTGAGTCATTGGGTTTTTCCAGCCAATTTAATTAAAACGCCATGTACTTTCCCACCATTGACGTCAATGGGCTATTGAAACTAATGCAACGTGACCTTTAAACGGTACTTTCCCATAGCTGATTAATGGGAAAGTACCGTTCGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCACTGAGTCATTAGGGACTTTCCAATGGGTTTTGCCCAGTACATAAGGTCAATAGGGGTGAATCAACAGGAAAGTCCCATTGGAGCCAAGTACACTGAGTCAATAGGGACTTTCCATTGGGTTTTGCCCAGTACAAAAGGTCAATAGGGGGTGAGTCAATGGGTTTTTCCCATTATTGGCACGTACATAAGGTCAATAGGGGTGAGTCATTGGGTTTTTCCAGCCAATTTAATTAAAACGCCATGTACTTTCCCACCATTGACGTCAATGGGCTATTGAAACTAATGCAACGTGACCTTTAAACGGTACTTTCCCATAGCTGATTAATGGGAAAGTACCGTTCGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGCGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTAAAACAGGTAAGTCCGGCCTCCGCGCCGGGTTTTGGCGCCTCCCGCGGGCGCCCCCCTCCTCACGGCGAGCGCTGCCACGTCAGACGAAGGGCGCAGCGAGCGTCCTGATCCTTCCGCCCGGACGCTCAGGACAGCGGCCCGCTGCTCATAAGACTCGGCCTTAGAACCCCAGTATCAGCAGAAGGACATTTTAGGACGGGACTTGGGTGACTCTAGGGCACTGGTTTTCTTTCCAGAGAGCGGAACAGGCGAGGAAAAGTAGTCCCTTCTCGGCGATTCTGCGGAGGGATCTCCGTGGGGCGGTGAACGCCGATGATGCCTCTACTAACCATGTTCATGTTTTCTTTTTTTTTCTACAGGTCCTGGGTGACGAACAG(SEQ ID NO：4)

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: 4, having a nucleotide sequence that hybridizes to the nucleotide sequence set forth in SEQ ID NO: 1-SEQ ID NO: 4, or similar promoter activity.

In the present invention, the pair of SEQ ID NOs: 1-SEQ ID NO: 4, means that substitution, deletion, addition modification of one or more bases is carried out on the 5 'end and/or the 3' end of the nucleotide sequence and/or inside the sequence, respectively or simultaneously, for example, not more than 2-45, or not more than 2-30, or not more than 3-20, or not more than 4-15, or not more than 5-10, or not more than 6-8 bases respectively expressed by one continuous integer are carried out.

In the present invention, the pair of SEQ ID NOs: 1-SEQ ID NO: 4 has a nucleotide sequence modified by the substitution, deletion and addition of one or more bases as shown in SEQ ID NO: 1-SEQ ID NO: 4, 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: 4 is at least 95% identical to the reference nucleotide sequence of seq id no: in SEQ ID NO: 1-SEQ ID NO: 4, 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: 4 comprises a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 1-SEQ ID NO: 4, e.g., 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: 4 has a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 1-SEQ ID NO: 4, or similar promoter activity.

Yet another aspect of the invention relates to a nucleic acid construct comprising a polynucleotide of any of the invention, and an operably linked foreign gene; specifically, the exogenous gene is selected from any one or more of p53, GM-CSF, IL-2, IL-12, IL-24, Trail, p16, TK, IFN gamma, TNF alpha, Rb, Sirp alpha and the like; specifically, the exogenous gene is SEQ ID NO: 12 and/or p53 shown in SEQ ID NO: 4 GM-CSF.

Yet another aspect of the invention relates to a recombinant vector comprising a polynucleotide according to any of the invention or a nucleic acid construct according to the invention; specifically, the recombinant vector is a recombinant viral vector; specifically, the recombinant vector is obtained by recombining the polynucleotide of any one of the present invention or the nucleic acid construct of the present invention with pDC315 plasmid or SG 655.

Cloning vectors suitable for constructing the recombinant vectors of the present invention include, but are not limited to, for example: pUC18, pUC19, pMD18-T, pMD19-T, pGM-T vector, pDC315 series vector and the like.

Suitable expression vectors for constructing the present invention include, but are not limited to, eukaryotic expression plasmids, recombinant viral vectors, and the like.

Such eukaryotic expression plasmids include, but are not limited to, for example: pCDNA3 series vectors, pCDNA4 series vectors, pCDNA5 series vectors, pCDNA6 series vectors, pRL series vectors, pDC315 series vectors, and the like. In one embodiment of the invention, the eukaryotic expression plasmid is a pDC315 vector.

Such recombinant viral vectors include, but are not limited to, for example: recombinant adenovirus vectors, recombinant adeno-associated virus vectors, recombinant retrovirus vectors, recombinant herpes simplex virus vectors, recombinant vaccinia virus vectors, and the like. 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 cell comprising a promoter of the invention or a nucleic acid construct of the invention or a vector of the invention. In particular, the cell is a mammalian cell. In one embodiment of the invention, the mammalian cell is a tumor cell. Such tumor cells include, but are not limited to, for example: primary liver cancer cells and liver cancer cell strains (Huh7, Hep3B, HepG2, PLC/PRF/5, SMMC-7721, BEL-7404, BEL7402, BEL-7405, L2.2.15, MHCC97-L, MHCC97-H, QGY-7701, HCCLM3 and SK-Hep-1); primary lung cancer cells and lung cancer cell strains (A549, NCI-H460, NCI-H446, NCI-H1299, NCI-H292 and NCI-H23); primary gastric cancer cells and gastric cancer cell strains (SGC-7901, BGC-823 and HGC-27); primary colon cancer cells, colon cancer cell lines (HT29, colo205, SW 620); primary breast cancer cells and breast cancer cell strains (MCF-7, MDA-MB-453, MDA-MB-468, HTB-122, HTB-133, SK-BR3 and T-47D); primary cervical cancer cells and cervical cancer cell strains (SK-OV3, HO-8910 and Hela); primary melanoma cells, melanoma cell lines (a375, a 431); primary glioma cells and glioma cell strains (A172 and U251); primary osteosarcoma cells and osteosarcoma cell strains (U2 OS); primary lymphoma cells and lymphoma cell strains (Raji, Jurkat, K562); primary pancreatic cancer cells and pancreatic cancer cell strains (PANC-1, Bxpc-3 and Su-86.86); primary prostate cancer cells, prostate cancer cell lines (PC-3, DU 145); primary bladder cancer cells, bladder cancer cell lines (T24); primary nasopharyngeal carcinoma cells, nasopharyngeal carcinoma cell strains (CNE-1) and the like.

Yet another aspect of the present invention relates to a method of introducing a polynucleotide (promoter) of the present invention or a nucleic acid construct of the present invention or a recombinant vector of the present invention into a mammalian cell; such methods include 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 of the present invention or a nucleic acid construct according to the present invention or a recombinant vector according to the present invention, and optionally a pharmaceutically acceptable excipient.

A further aspect of the present invention relates to the use of (1) or (2) selected from the group consisting of:

(1) the use of the polynucleotide of any one of the present invention or the nucleic acid construct of the present invention or the recombinant vector of the present invention for the preparation of a medicament for the treatment and/or prevention and/or adjuvant treatment of cancer or anti-tumor; 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 in the preparation of a medicament or 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; in a specific embodiment, the tumor cell is any one selected from the group consisting of Hep3B, Huh7, HepG2, PLC/PRF/5, BEL-7404, H460 and H1299 cells.

Yet another aspect of the present invention relates to a method of treatment and/or prevention and/or co-treatment of cancer or a tumor comprising the step of administering an effective amount of a polynucleotide of any of the present invention or a nucleic acid construct of the present invention or a recombinant vector of the present invention or a pharmaceutical composition 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.

Yet another aspect of the present invention relates to a method of inhibiting tumor cells in vivo or in vitro comprising the step of administering an effective amount 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 pharmaceutical composition of the present 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 a specific embodiment, the tumor cell is any one selected from the group consisting of Hep3B, Huh7, HepG2, PLC/PRF/5, BEL-7404, H460 and H1299 cells.

Yet another aspect of the invention relates to the use of a polynucleotide according to any one of the invention as a promoter.

Yet another aspect of the invention relates to an isolated polynucleotide comprising or being SEQ ID NO: 20 or a complement thereof. The invention also relates to the application of the polynucleotide in preparing a promoter; specifically, the promoter is a polynucleotide of any one of the preceding inventions having a 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.

ACTGAGTCATTAGGGACTTTCCATTGGGTTTTGCCCAGTACAAAAGGTCAATAGGGGGTGAGTCAATGGGTTTTTCCCATTATTGGCACGTACATAAGGTCAATAGGGGTGAGTCATTGGGTTTTTCCAGCCAATTTAATTAAAACGCCATGTACTTTCCCACCATTGACGTCAATGGGCTATTGAAACTAATGCAACGTGACCTTTAAACGGTACTTTCCCATAGCTGATTAATGGGAAAGTACCGTTC(SEQ ID NO：20)

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 invention, the gene is the human p53 gene and the GM-CSF gene the granulocyte-macrophage colony stimulating factor (GM-CSF) gene.

The promoters (polynucleotides) of the invention may be used in single and/or multiple copies, and may be used in combination with promoters known in the art.

Advantageous effects of the invention

The present invention provides a novel promoter. The promoter not only can efficiently express exogenous genes in a tumor cell in a broad spectrum manner, but also has higher expression activity in a series of tumor cells than CAG promoters, and has stable sequence (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 in tumor cells.

Drawings

FIG. 1: vector map of pDC 315-CCAU-RLuc.

FIG. 2: the activity of each promoter was determined using the adenovirus dual-luciferase reporter system.

FIG. 3: 11R-p53-2A-GM-CSF expression cassette.

FIG. 4: vector map of shuttle vector P74-Tp-GMP3 of virus SG655-GMP 3.

FIG. 5: western blotting assay of p53 expression following infection of 293 cells with the virus SG655-GMP 3. Blank, virus-uninfected 293 cells; 1, 2, 3, 4, 5 and 6 represent different virus clones respectively; ad-p53, infecting Ad-p53 virus.

FIG. 6: quantitative detection of hGM-CSF expression after infection of 293 cells by SG655-GMP3 of different clone (1# -6 #) virus.

FIG. 7: graph of killing activity of SG655-GMP against liver cancer cell Huh7 in vitro.

FIG. 8: graph of SG655-mGMP inhibition of growth in Huh7 transplants.

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 expression vectors carrying nucleotide sequences of respective promoters

1. 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 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 F1 and R1 are as follows:

f1: GCCgaattcGCCACCATGACTTCGAAAGT (SEQ ID NO: 5) in which the lower case letters represent the EcoRI cleavage site

R1: TGTgtcgacTTATTGTTCATTTTTGAGAACTCGCT (SEQ ID NO: 6), 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.

2. According to SEQ ID NO: 1-SEQ ID NO: 4, XbaI is introduced into the upstream of the vector, an enzyme cutting site EcoRI is introduced into the downstream of the vector, the vector is synthesized by Shanghai Jiehui biological company, and pDC315-CMV-Rluc vectors which are subjected to double enzyme cutting by XbaI and EcoRI (replaces CMV promoters in the original pDC315-CMV-Rluc vectors) are respectively named as pDC315-CAC-RLuc, pDC315-CACU-RLuc, pDC315-CCAU-RLuc (the specific vector map is shown in figure 1) and pDC315-4 CAU-RLuc.

3. Based on the sequence of the CAG promoter in the pCAGGS vector (purchased from Addgene plasmid library), a pair of PCR-specific amplification primers (an upstream primer F2, plus an NheI restriction site and a protective base; a downstream primer R2, plus an EcoRI restriction site and a protective base) were designed from the beginning to the end. The sequences of F2 and R2 are as follows:

f2: GCCgctagcTCTAGTTATTAATAGTAATCAATT (SEQ ID NO: 7) in which the lower case letters represent the NheI cleavage site

R2: TGTgaattcTTTGCCAAAATGATGAGACAGCAC (SEQ ID NO: 8), wherein the lower case letters represent the EcoRI cleavage site.

The nucleotide sequence of the CAG promoter is amplified by taking the pCAGGS plasmid as a template, and is loaded into a pDC315-Rluc vector (replacing a CMV promoter in the original pDC315-CMV-Rluc vector) which is subjected to double enzyme digestion by NheI + EcoRI, and the pDC315-Rluc vector is named as pDC315-CAG-RLuc respectively.

4. After transforming the above plasmids into DH5 α bacteria, shanghai was entrusted to determine the DNA sequences of the promoters, and as a result, it was found that there were some losses in the DNA sequences of the CAC, CACU, and 4CAU promoters (differences in bacterial clones), and the sequence loss occurred mainly in the mCMV enhancer segment; while the sequence of the CMV, CAG, CCAU promoters remained stable.

Example 2: construction of adenovirus dual-luciferase reporter system carrying nucleotide sequences of promoters

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 F3, an EcoRI restriction site and a protection base; a downstream primer R3, a SalI restriction site and a protection base) are designed at the beginning and the end. The sequences of F3 and R3 are as follows:

f3: GCCgaattcGCCACCATGGAAGACGCC (SEQ ID NO: 9) in which the lower case letters represent EcoRI cleavage sites;

r3: TGTgtcgacTTACACGGCGATCTTTCCGC (SEQ ID NO: 10), wherein the lower case letters represent SalI cleavage sites.

Using psi-CHECK2 plasmid as template to amplify FLuc gene coding sequence, EcoRI + SalI doubleDigested, filled with pENTR12 which was also digested doubly with EcoRI + SalI^TMVectors (purchased from Invitrogen, the expression of foreign genes driven by hCMV promoter, attL1 and attL2 sequences containing lambda phage site-specific recombination system) to obtain the pENTR12-Fluc vector. The BJ5183 bacteria was co-transformed with pENTR12-Fluc plasmid and pPE3 plasmid (purchased from Microbixbiosystems, Canada, containing attR1 and attR2 sequences of lambda phage site-specific recombination system, ccdB gene, and adenovirus type 5 right arm), and the backbone plasmid pPE3-Fluc carrying Fluc gene and adenovirus type 5 right arm was obtained by LR reaction recombination.

2. pDC315-CMV-Rluc, pDC315-CAG-Rluc, pDC315-CAC-RLuc, pDC315-CACU-RLuc, pDC315-CCAU-RLuc, pDC315-4CAU-RLuc and pPE3-Fluc were co-transfected into HEK293 cell strain (purchased from American Standard article Collection, ATCC) by Lipofectamine, respectively, in the specific procedures described in GIBCOBRL Co. And virus plaques appear 9-14 days after cotransfection, and the adenovirus dual-luciferase report system carrying nucleotide sequences of all promoters is obtained by purifying the virus plaques for three times and is named as Ad-CMV-2Luc, Ad-CAG-2Luc, Ad-CAC-2Luc, Ad-CACU-2Luc, Ad-CCAU-2Luc and Ad-4CAU-2Luc respectively.

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).

3. Extracting the genome of the adenovirus, and determining the DNA sequences of the promoters by entrusting Shanghai to the genome, wherein the DNA sequences of the promoters CAC, CACU and 4CAU are also lost to different degrees (different virus clones are different), and the sequence loss mainly occurs in the mCMV enhancer segment; while the sequence of the CMV, CAG, CCAU promoters remained stable.

Example 3: determination of the Activity of the promoters Using the Dual luciferase reporter System carrying adenovirus

Low generation HEK293, Hep3B, Huh7, HepG2, PLC/PRF/5, BEL-7404, H460 and H1299 with good growth stateThe cell lines (all purchased from ATCC) were arranged 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 Ad-CMV-2Luc, Ad-CAG-2Luc, Ad-CAC-2Luc, Ad-CACU-2Luc, Ad-CCAU-2Luc, Ad-4CAU-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 are shown in FIG. 2.

The results show that the activity of the CCAU promoter is most stable in a series of promoters, namely HEK293, Hep3B, Huh7, HepG2, PLC/PRF/5, BEL-7404, H460 and H1299 at the 2 nd, 1 st and 2 nd positions; the activity of the promoter is 2.8 times, 6.2 times, 2.0 times, 1.8 times, 2.5 times, 4.5 times, 3.5 times and 1.1 times of the activity of the CAG promoter in corresponding cells respectively (the specific results are shown in figure 2).

The result also shows that the promoter, particularly the CCAU promoter, can play the function of the promoter in various cells (tumor cells), has broad spectrum and is higher than the CAG promoter in the broad spectrum.

Example 4: construction of recombinant adenovirus SG655-GMP3

Reference is made to the chinese patent application publication No. CN103614416A (application No. 201310460980, published as 2014, 03/05). The method comprises the following specific steps:

1. an artificial first insulator (the specific sequence is shown in SEQ ID NO: 11) was synthesized, a multiple cloning site (BglII-XbaI-EcoRI-BamHI-SalI-NheI-HindIII-SpeI) was introduced upstream thereof, a cleavage site (EcoRV-BglII) was introduced downstream thereof, the synthesis was delegated to Shanghai Czeri Biotech, and the vector was inserted between the vector sites of the vector fragment pCA13 (BglII), and the vector thus constructed was named pCA 16. The pCA13 vector, purchased from Microbix biosystems Inc., Canada (Toronto), is an adenovirus shuttle vector containing the adenovirus type 5 left arm sequence (bp22-5790), with a deletion of the 342 to 3523bp fragment of E1 region, and can be used to clone foreign genes and packaged into an adenovirus vector with a vector containing the right arm of adenovirus.

2. The CAC promoter sequence, SEQ ID NO: the CCAU promoter sequences shown in 3 are respectively loaded into pCA16 vectors, and the successfully constructed vectors are respectively named pCA20 and pCA 21.

3. According to human 11R transmembrane peptide p53 gene (shown in SEQ ID NO: 12), human GM-CSF gene (abbreviated as hGM-CSF, SEQ ID NO: 13), mouse GM-CSF gene (abbreviated as mGM-CSF, SEQ ID NO: 14) and Furin-2A coding sequence (SEQ ID NO: 15), 11R-p53-F2A-hGM-CSF (abbreviated as hGMP, see FIG. 3) and 11R-p53-F2A-mGM-CSF are respectively synthesized, enzyme cutting sites (EcoRI + SalI) are introduced at two ends, the products are assigned to Shanghai Jirui biological company for whole gene synthesis and respectively filled into pCA20 and pCA21 vectors to respectively construct successful vectors which are named as pCA20-hGMP, pCA20-mGMP, pCA21-hGMP and pCA 21-mGMP.

4. According to SEQ ID NO: 16, synthesizing a DNA long sequence, wherein the sequence sequentially comprises a sequence in front of a adenovirus type 5 provirus E1A promoter, a second insulator, a human telomerase reverse transcriptase promoter, an E1A coding sequence and an E1A polyA tailing signal sequence, enzyme cutting sites EcoRI and BglII are arranged at two ends of the sequence, the fragment is connected to a pXC.1 vector (purchased from Microbix Biosystem Inc in Canada) subjected to double enzyme cutting by EcoRI and BglII after double enzyme cutting, and the constructed vector is named as p74-Tp, the human telomerase reverse transcriptase promoter is used for replacing the original E1A promoter of the adenovirus, and the sequences of coding regions of E1b 55KDa and 19KDa are deleted.

5. Plasmids pCA20-hGMP, pCA20-mGMP, pCA21-hGMP and pCA21-mGMP are respectively subjected to enzyme digestion (BglII), CAG-hGMP-SV40 PolyA-insulator, CAG-mGMP-SV40 PolyA-insulator, CCAU-hGMP-SV40 PolyA-insulator and CCAU-mGMP-SV40 PolyA-insulator expression frame fragments are recovered, the whole expression frame is put into a vector fragment p74-Tp (BglII) subjected to the same enzyme digestion, positive clones which are connected in a reverse direction are selected, and the successfully constructed vectors are respectively named as p74-Tp-GMP2, p74-Tp-mGMP2, p74-Tp-GMP3 (the vector map is shown in FIG. 4) and p74-Tp-mGMP 3.

6. The purified p74-Tp-GMP2, p74-Tp-mGMP2, p74-Tp-GMP3 and p74-Tp-mGMP3 were respectively co-transfected into HEK293 cells (purchased from ATCC) by Lipofectamine (purchased from Invitrogen) with a backbone plasmid pPE3 (purchased from Microbix Biosystem Inc, Canada) containing the right arm of the adenovirus genome to recombine, and recombinant oncolytic adenoviruses SG655-GMP2, SG655-mGMP2, SG655-GMP3 and SG655-mGMP3 were obtained.

First insulating subsequence:

AGAGAAATGTTCTGGCACCTGCACTTGCACTGGGGACAGCCTATTTTGCTAGTTTGTTTTGTTTCGTTTTGTTTTGATGGAGAGCGTATGTTAGTACTATCGATTCACACAAAAAACCAACACACAGATGTAATGAAAATAAAGATATTTTATT(SEQ ID NO：11)

human cell-penetrating peptide p53 gene:

ATGGGCCGCCGCAGGAGACGACGGCGACGGCGAAGAAGGGAGGAGCCGCAGTCAGATCCTAGCGTCGAGCCCCCTCTGAGTCAGGAAACATTTTCAGACCTATGGAAACTACTTCCTGAAAACAACGTTCTGTCCCCCTTGCCGTCCCAAGCAATGGATGATTTGATGCTGTCCCCGGACGATATTGAACAATGGTTCACTGAAGACCCAGGTCCAGATGAAGCTCCCAGAATGCCAGAGGCTGCTCCCCGCGTGGCCCCTGCACCAGCAGCTCCTACACCGGCGGCCCCTGCACCAGCCCCCTCCTGGCCCCTGTCATCTTCTGTCCCTTCCCAGAAAACCTACCAGGGCAGCTACGGTTTCCGTCTGGGCTTCTTGCATTCTGGGACAGCCAAGTCTGTGACTTGCACGTACTCCCCTGCCCTCAACAAGATGTTTTGCCAACTGGCCAAGACCTGCCCTGTGCAGCTGTGGGTTGATTCCACACCCCCGCCCGGCACCCGCGTCCGCGCCATGGCCATCTACAAGCAGTCACAGCACATGACGGAGGTTGTGAGGCGCTGCCCCCACCATGAGCGCTGCTCAGATAGCGATGGTCTGGCCCCTCCTCAGCATCTTATCCGAGTGGAAGGAAATTTGCGTGTGGAGTATTTGGATGACAGAAACACTTTTCGACATAGTGTGGTGGTGCCCTATGAGCCGCCTGAGGTTGGCTCTGACTGTACCACCATCCACTACAACTACATGTGTAACAGTTCCTGCATGGGCGGCATGAACCGGAGGCCCATCCTCACCATCATCACACTGGAAGACTCCAGTGGTAATCTACTGGGACGGAACAGCTTTGAGGTGCGTGTTTGTGCCTGTCCTGGGAGAGACCGGCGCACAGAGGAAGAGAATCTCCGCAAGAAAGGGGAGCCTCACCACGAGCTGCCCCCAGGGAGCACTAAGCGAGCACTGCCCAACAACACCAGCTCCTCTCCCCAGCCAAAGAAGAAACCACTGGATGGAGAATATTTCACCCTTCAGATCCGTGGGCGTGAGCGCTTCGAGATGTTCCGAGAGCTGAATGAGGCCTTGGAACTCAAGGATGCCCAGGCTGGGAAGGAGCCAGGGGGGAGCAGGGCTCACTCCAGCCACCTGAAGTCCAAAAAGGGTCAGTCTACCTCCCGCCATAAAAAACTCATGTTCAAGACAGAAGGGCCTGACTCAGAC(SEQ ID NO：12)

human GM-CSF gene:

ATGTGGCTGCAGAGCCTGCTGCTCTTGGGCACTGTGGCCTGCAGCATCTCTGCACCCGCCCGCTCGCCCAGCCCCAGCACGCAGCCCTGGGAGCATGTGAATGCCATCCAGGAGGCCCGGCGTCTCCTGAACCTGAGTAGAGACACTGCTGCTGAGATGAATGAAACAGTAGAAGTCATCTCAGAAATGTTTGACCTCCAGGAGCCGACCTGCCTACAGACCCGCCTGGAGCTGTACAAGCAGGGCCTGCGGGGCAGCCTCACCAAGCTCAAGGGCCCCTTGACCATGATGGCCAGCCACTACAAGCAGCACTGCCCTCCAACCCCGGAAACTTCCTGTGCAACCCAGATTATCACCTTTGAAAGTTTCAAAGAGAACCTGAAGGACTTTCTGCTTGTCATCCCCTTTGACTGCTGGGAGCCAGTCCAGGAGTGA(SEQ ID NO：13)

mouse GM-CSF gene:

ATGTGGCTGCAGAATTTACTTTTCCTGGGCATTGTGGTCTACAGCCTCTCAGCACCCACCCGCTCACCCATCACTGTCACCCGGCCTTGGAAGCATGTAGAGGCCATCAAAGAAGCCCTGAACCTCCTGGATGACATGCCTGTCACGTTGAATGAAGAGGTAGAAGTCGTCTCTAACGAGTTCTCCTTCAAGAAGCTAACATGTGTGCAGACCCGCCTGAAGATATTCGAGCAGGGTCTACGGGGCAATTTCACCAAACTCAAGGGCGCCTTGAACATGACAGCCAGCTACTACCAGACATACTGCCCCCCAACTCCGGAAACGGACTGTGAAACACAAGTTACCACCTATGCGGATTTCATAGACAGCCTTAAAACCTTTCTGACTGATATCCCCTTTGAATGCAAAAAACCAGGCCAAAAATGA(SEQ ID NO：14)

the coding sequence of Furin-2A:

CGTAAAAGGCGAGCTCCTGTTAAACAGACTTTGAATTTTGACCTTCTCAAGTTGGCGGGAGACGTCGAGTCCAACCCTGGGCCC(SEQ ID NO：15)

construction of the transition fragment sequence for the p74-TP vector:

GAATTCTCATGTTTGACAGCTTATCATCGATAAGCTTTAATGCGGTAGTTTATCACAGTTAAATTGCTAACGCAGTCAGGCACCGTGTATGAAATCTAACAATGCGCTCATCGTCATCCTCGGCACCGTCACCCTGGATGCTGTAGGCATAGGCTTGGTTATGCCGGTACTGCCGGGCCTCTTGCGGGATATCGTCCATTCCGACAGCATCGCCAGTCACTATGGCGTGCTGCTAGCGCTATATGCGTTGATGCAATTTCTATGCGCACCCGTTCTCGGAGCACTGTCCGACCGCTTTGGCCGCCGCCCAGTCCTGCTCGCTTCGCTACTTGGAGCCACTATCGACTACGCGATCATGGCGACCACACCCGTCCTGTGGATCCGGGCCCCCATTTCCCCTCCCTTCCAGCTCTCTGCCCCTTTTGGATTGAAGCCAATATGATAATGAGGGGGTGGAGTTTGTGACGTGGCGCGGGGCGTGGGAACGGGGCGGGTGACGTAGTAGTGTGGCGGAAGTGTGATGTTGCAAGTGTGGCGGAACACATGTAAGCGACGGATGTGGCAAAAGTGACGTTTTTGGTGTGCGCCGGTGTACACAGGAAGTGACAATTTTCGCGCGGTTTTAGGCGGATGTTGTAGTAAATTTGGGCGTAACCGAGTAAGATTTGGCCATTTTCGCGGGAAAACTGAATAAGAGGAAGTGAAATCTGAATAATTTTGTGTTACTCATAGCGCGTAATTTCTAGTAATAAAGGATCCTTTATTTTCATTGGATCCGTGTGTTGGTTTTTTGTGTGCGGCCGCGTACCGGTCACAGACGCCCAGGACCGCGCTTCCCACGTGGCGGAGGGACTGGGGACCCGGGCACCCGTCCTGCCCCTTCACCTTCCAGCTCCGCCTCCTCCGCGCGGACCCCGCCCCGTCCCGACCCCTCCCGGGTCCCCGGCCCAGCCCCCTCCGGGCCCTCCCAGCCCCTCCCCTTCCTTTCCGCGGCCCCGCCCTCTCCTCGCGGCGCGAGTTTCAGGCAGCGCTGCGTCCTGCTGCGCACGTGGGAAGCCCTGGCCCCGGCCACTCGAGGACGCACGTGGGTTCGAATGAAAATGAGACATATTATCTGCCACGGAGGTGTTATTACCGAAGAAATGGCCGCCAGTCTTTTGGACCAGCTGATCGAAGAGGTACTGGCTGATAATCTTCCACCTCCTAGCCATTTTGAACCACCTACCCTTCACGAACTGTATGATTTAGACGTGACGGCCCCCGAAGATCCCAACGAGGAGGCGGTTTCGCAGATTTTTCCCGACTCTGTAATGTTGGCGGTGCAGGAAGGGATTGACTTACTCACTTTTCCGCCGGCGCCCGGTTCTCCGGAGCCGCCTCACCTTTCCCGGCAGCCCGAGCAGCCGGAGCAGAGAGCCTTGGGTCCGGTTTCTATGCCAAACCTTGTACCGGAGGTGATCGATCTTACCTGCCACGAGGCTGGCTTTCCACCCAGTGACGACGAGGATGAAGAGGGTGAGGAGTTTGTGTTAGATTATGTGGAGCACCCCGGGCACGGTTGCAGGTCTTGTCATTATCACCGGAGGAATACGGGGGACCCAGATATTATGTGTTCGCTTTGCTATATGAGGACCTGTGGCATGTTTGTCTACAGTAAGTGAAAATTATGGGCAGTGGGTGATAGAGTGGTGGGTTTGGTGTGGTAATTTTTTTTTTAATTTTTACAGTTTTGTGGTTTAAAGAATTTTGTATTGTGATTTTTTTAAAAGGTCCTGTGTCTGAACCTGAGCCTGAGCCCGAGCCAGAACCGGAGCCTGCAAGACCTACCCGCCGTCCTAAAATGGCGCCTGCTATCCTGAGACGCCCGACATCACCTGTGTCTAGAGAATGCAATAGTAGTACGGATAGCTGTGACTCCGGTCCTTCTAACACACCTCCTGAGATACACCCGGTGGTCCCGCTGTGCCCCATTAAACCAGTTGCCGTGAGAGTTGGTGGGCGTCGCCAGGCTGTGGAATGTATCGAGGACTTGCTTAACGAGCCTGGGCAACCTTTGGACTTGAGCTGTAAACGCCCCAGGCCATAAGGTGTAAACCTGTGATTGCGTGTGTGGTTAACGCCTTTGTTTGCTGAATGAGTTGATGTAAGTTTAATAAAGGGTGAGATAATGTTTAACTTGCATGGCGTGTTAAATGGGGCGGGGCTTAAAGGGTATATAATGCGCCGTGGGCTAATCTTGGTTACATCTGACCTCATGGAGGCTTGGGAGTGTTAAAGATCT(SEQ ID NO：16)

example 5: identification of exogenous Gene expression Activity of recombinant adenovirus SG655-GMP3

HEK293 cells (from ATCC) at 5X 10⁵Cells/well were plated in 6-well plates and incubated at 37 ℃ with 5% CO₂Culturing, changing serum-free liquid 1ml on the next day, adding recombinant oncolytic adenovirus SG655-GMP3 according to MOI (molar equivalent of average molecular weight) 5, culturing for 90 min, washing twice with Phosphate Buffered Saline (PBS), washing virus, culturing with culture solution containing 5% fetal calf serum, culturing for 48 hr, lysing cells, collecting sample, detecting expression of p53 protein by Western Blotting experiment, and using housekeeping gene GAPDH as reference。

The results are shown in FIG. 5.

The results show that the recombinant oncolytic adenovirus SG655-GMP3 can express an 11R-P53 fusion protein with the size of about 55KDa in HEK293 cells, compared with a control virus (Ad-P53, purchased from Shenzhen Seeberuno Gene technology Limited, expressing a 53KDa product), as shown in FIG. 5.

The results also indicate that the recombinant oncolytic adenovirus SG655-GMP3 expresses 11R-P53 fusion protein in an amount higher than that of the P53 protein of the control virus.

2. Supernatants of each of recombinant oncolytic adenovirus SG655-GMP3 were diluted by a certain fold, and expression of hGM-CSF protein in HEK293 cells was detected using human GM-CSF ELISA MAX Deluxe detection kit (purchased from Biolegend). The results are shown in FIG. 6.

The results show that each clone of the recombinant oncolytic adenovirus SG655-GMP3 can express the hGM-CSF protein in HEK293 cells, the expression level is high, and the difference between the clones is not large.

Example 6: recombinant oncolytic adenoviruses SG655-GMP3 and SG655-GMP2 for cultured tumor cells in vitro Killing experiment

Hepatoma cell line Huh7 (purchased from Biochemical and cell research institute of Chinese academy of sciences) at a ratio of 1 × 10⁴Cells/well in 96-well plates, incubate 5% CO at 37 ℃₂Culturing, adding recombinant oncolytic adenovirus SG655-GMP2 and SG655-GMP3 (taking clone No. 5 as an example) according to different MOI value gradients on the next day, setting 3 times of each MOI value, and detecting a cell growth curve by using an MTT (3- (4, 5-dimethylthiazole-2) -2, 5-diphenyl tetrazole bromide salt purchased from SIGMA company) method after culturing for 7 days.

The results are shown in FIG. 7.

The result shows that SG655-GMP2 and SG655-GMP3 have good killing capacity on liver cancer cells, the killing effect of SG655-GMP3 is better than that of SG655-GMP2, and the in vitro effect of CCAU promoter for controlling therapeutic gene is better than that of CAG promoter.

Example 7: mobilization of recombinant oncolytic adenoviruses SG655-mGMP3 and SG655-mGMP2Physical experiment

Due to species differences, the human GM-CSF gene has no stimulatory activity on mouse DC cells and macrophages. In order to research the in vivo function of the virus, the invention simultaneously constructs experimental oncolytic virus SG655-mGMP carrying mouse GM-CSF gene. As known to those skilled in the art, the human GM-CSF gene has excellent stimulatory activity on human DC cells and macrophages. Therefore, for GM-CSF gene, the function of SG655-mGMP in mice can directly reflect the function of SG655-GMP in human body.

The first step is as follows: 50 BALB/C nude mice with the age of 4-6 weeks, the average weight of 22-27 g, are provided by a laboratory animal breeding center of Shanghai Zhongzhongji, and are raised in a clean-grade animal laboratory.

The second step is that: culturing human hepatocarcinoma cell Huh7 in vitro, collecting adherent growth cell in logarithmic growth phase, digesting with 0.25% pancreatin, centrifuging, collecting cell, resuspending with PBS solution, centrifuging at 3000g at room temperature for 2 min, discarding supernatant, resuspending with PBS solution, centrifuging, collecting cell, adjusting cell suspension concentration to 5 × 10⁷One per ml.

The third step: the dorsal part of the right rib of the nude mouse is inoculated with 0.1 ml/mouse of Huh7 cells subcutaneously. After inoculation for about three weeks, the inoculated part can grow hard grains under the skin, and the transplanted tumor model is established. The treatment is started when the tumor body grows to 7-9mm in diameter. 40 Huh7 transplants with subcutaneous tumors growing to 7-9mm were selected for treatment and nude mice were randomized into 4 groups. The administration route is direct intratumoral multipoint injection.

The fourth step: observing the living state of the mice every day, periodically measuring the tumor volume, periodically measuring the longest diameter (A) and the vertical diameter (B) of the tumor once, and obtaining the formula (A multiplied by B)²) And/2 calculating the volume of the tumor, and observing the curative effect of different dosage groups.

The results are shown in FIG. 8.

The results show that both genes, oncolytic adenovirus SG655-gmp, significantly inhibited the growth of transplantable tumors relative to placebo (PBS injection) (p < 0.05); the oncolytic adenovirus SG655-mGMP2 expressed by 11R-P53 and GM-CSF gene driven by CCAU promoter shows stronger tumor growth inhibition effect compared with the control oncolytic adenovirus SG655-mGMP2 driven by CAG promoter, the tumor volume is always obviously smaller than that of a control virus group, and the statistical difference (P <0.05) is significant, and is shown in figure 8. The anti-tumor effect of the gene-oncolytic adenovirus SG655-mGMP3 on a liver cancer animal model is better than that of SG655-mGMP2, and the in vivo effect of a CCAU promoter in tumor gene therapy is better than that of a CAG promoter.

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.

Sequence listing

<110> eastern hepatobiliary surgery Hospital of the second military medical university of the Chinese people liberation force

SHANGHAI ALLBRIGHT BIOTECH Co.,Ltd.

<120> a chimeric strong promoter and use thereof

<130> IDC170029

<160> 23

<170> PatentIn version 3.2

<210> 1

<211> 1093

<212> DNA

<213> Artificial sequence

<400> 1

ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc 60

ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca 120

ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta 180

tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta 240

tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat 300

cgctattacc atggtcgagg tgagccccac gttctgcttc actctcccca tctccccccc 360

ctccccaccc ccaattttgt atttatttat tttttaatta ttttgtgcag cgatgggggc 420

gggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg gggcggggcg 480

aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt tccttttatg 540

gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc gggagtcgct 600

gcgcgctgcc ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc gccccggctc 660

tgactgaccg cgttactaaa acaggtaagt ccctgagtca ttagggactt tccaatgggt 720

tttgcccagt acataaggtc aataggggtg aatcaacagg aaagtcccat tggagccaag 780

tacactgagt caatagggac tttccattgg gttttgccca gtacaaaagg tcaatagggg 840

gtgagtcaat gggtttttcc cattattggc acgtacataa ggtcaatagg ggtgagtcat 900

tgggtttttc cagccaattt aattaaaacg ccatgtactt tcccaccatt gacgtcaatg 960

ggctattgaa actaatgcaa cgtgaccttt aaacggtact ttcccatagc tgattaatgg 1020

gaaagtaccg ttcgcctcta ctaaccatgt tcatgttttc tttttttttc tacaggtcct 1080

gggtgacgaa cag 1093

<210> 2

<211> 1396

<212> DNA

<213> Artificial sequence

<400> 2

ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc 60

ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca 120

ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta 180

tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta 240

tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat 300

cgctattacc atggtcgagg tgagccccac gttctgcttc actctcccca tctccccccc 360

ctccccaccc ccaattttgt atttatttat tttttaatta ttttgtgcag cgatgggggc 420

gggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg gggcggggcg 480

aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt tccttttatg 540

gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc gggagtcgct 600

gcgcgctgcc ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc gccccggctc 660

tgactgaccg cgttactaaa acaggtaagt ccctgagtca ttagggactt tccaatgggt 720

tttgcccagt acataaggtc aataggggtg aatcaacagg aaagtcccat tggagccaag 780

tacactgagt caatagggac tttccattgg gttttgccca gtacaaaagg tcaatagggg 840

gtgagtcaat gggtttttcc cattattggc acgtacataa ggtcaatagg ggtgagtcat 900

tgggtttttc cagccaattt aattaaaacg ccatgtactt tcccaccatt gacgtcaatg 960

ggctattgaa actaatgcaa cgtgaccttt aaacggtact ttcccatagc tgattaatgg 1020

gaaagtaccg ttcggcctcc gcgccgggtt ttggcgcctc ccgcgggcgc ccccctcctc 1080

acggcgagcg ctgccacgtc agacgaaggg cgcagcgagc gtcctgatcc ttccgcccgg 1140

acgctcagga cagcggcccg ctgctcataa gactcggcct tagaacccca gtatcagcag 1200

aaggacattt taggacggga cttgggtgac tctagggcac tggttttctt tccagagagc 1260

ggaacaggcg aggaaaagta gtcccttctc ggcgattctg cggagggatc tccgtggggc 1320

ggtgaacgcc gatgatgcct ctactaacca tgttcatgtt ttcttttttt ttctacaggt 1380

cctgggtgac gaacag 1396

<210> 3

<211> 1305

<212> DNA

<213> Artificial sequence

<400> 3

actgagtcat tagggacttt ccattgggtt ttgcccagta caaaaggtca atagggggtg 60

agtcaatggg tttttcccat tattggcacg tacataaggt caataggggt gagtcattgg 120

gtttttccag ccaatttaat taaaacgcca tgtactttcc caccattgac gtcaatgggc 180

tattgaaact aatgcaacgt gacctttaaa cggtactttc ccatagctga ttaatgggaa 240

agtaccgttc ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 300

ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag 360

ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac 420

atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg 480

cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg 540

tattagtcat cgctattacc atggtcgagg tgagccccac gttctgcttc actctcccca 600

tctccccccc ctccccaccc ccaattttgt atttatttat tttttaatta ttttgtgcag 660

cgatgggggc gggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg 720

gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt 780

tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc 840

gggagtcgct gcgcgctgcc ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc 900

gccccggctc tgactgaccg cgttactaaa acaggtaagt ccggcctccg cgccgggttt 960

tggcgcctcc cgcgggcgcc cccctcctca cggcgagcgc tgccacgtca gacgaagggc 1020

gcagcgagcg tcctgatcct tccgcccgga cgctcaggac agcggcccgc tgctcataag 1080

actcggcctt agaaccccag tatcagcaga aggacatttt aggacgggac ttgggtgact 1140

ctagggcact ggttttcttt ccagagagcg gaacaggcga ggaaaagtag tcccttctcg 1200

gcgattctgc ggagggatct ccgtggggcg gtgaacgccg atgatgcctc tactaaccat 1260

gttcatgttt tctttttttt tctacaggtc ctgggtgacg aacag 1305

<210> 4

<211> 2048

<212> DNA

<213> Artificial sequence

<400> 4

ctgagtcatt agggactttc caatgggttt tgcccagtac ataaggtcaa taggggtgaa 60

tcaacaggaa agtcccattg gagccaagta cactgagtca atagggactt tccattgggt 120

tttgcccagt acaaaaggtc aatagggggt gagtcaatgg gtttttccca ttattggcac 180

gtacataagg tcaatagggg tgagtcattg ggtttttcca gccaatttaa ttaaaacgcc 240

atgtactttc ccaccattga cgtcaatggg ctattgaaac taatgcaacg tgacctttaa 300

acggtacttt cccatagctg attaatggga aagtaccgtt cggagttccg cgttacataa 360

cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata 420

atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag 480

tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc 540

cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta 600

tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac cactgagtca 660

ttagggactt tccaatgggt tttgcccagt acataaggtc aataggggtg aatcaacagg 720

aaagtcccat tggagccaag tacactgagt caatagggac tttccattgg gttttgccca 780

gtacaaaagg tcaatagggg gtgagtcaat gggtttttcc cattattggc acgtacataa 840

ggtcaatagg ggtgagtcat tgggtttttc cagccaattt aattaaaacg ccatgtactt 900

tcccaccatt gacgtcaatg ggctattgaa actaatgcaa cgtgaccttt aaacggtact 960

ttcccatagc tgattaatgg gaaagtaccg ttcggagttc cgcgttacat aacttacggt 1020

aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta 1080

tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg 1140

gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga 1200

cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt 1260

tcctacttgg cagtacatct acgtattagt catcgctatt accatggtcg aggtgagccc 1320

cacgttctgc ttcactctcc ccatctcccc cccctcccca cccccaattt tgtatttatt 1380

tattttttaa ttattttgtg cagcgatggg ggcggggggg ggggggggcg cgcgccaggc 1440

ggggcggggc ggggcgaggg gcggggcggg gcgaggcgga gaggtgcggc ggcagccaat 1500

cagagcggcg cgctccgaaa gtttcctttt atggcgaggc ggcggcggcg gcggccctat 1560

aaaaagcgaa gcgcgcggcg ggcgggagtc gctgcgcgct gccttcgccc cgtgccccgc 1620

tccgccgccg cctcgcgccg cccgccccgg ctctgactga ccgcgttact aaaacaggta 1680

agtccggcct ccgcgccggg ttttggcgcc tcccgcgggc gcccccctcc tcacggcgag 1740

cgctgccacg tcagacgaag ggcgcagcga gcgtcctgat ccttccgccc ggacgctcag 1800

gacagcggcc cgctgctcat aagactcggc cttagaaccc cagtatcagc agaaggacat 1860

tttaggacgg gacttgggtg actctagggc actggttttc tttccagaga gcggaacagg 1920

cgaggaaaag tagtcccttc tcggcgattc tgcggaggga tctccgtggg gcggtgaacg 1980

ccgatgatgc ctctactaac catgttcatg ttttcttttt ttttctacag gtcctgggtg 2040

acgaacag 2048

<210> 5

<211> 29

<212> DNA

<213> Artificial sequence

<400> 5

gccgaattcg ccaccatgac ttcgaaagt 29

<210> 6

<211> 35

<212> DNA

<213> Artificial sequence

<400> 6

tgtgtcgact tattgttcat ttttgagaac tcgct 35

<210> 7

<211> 33

<212> DNA

<213> Artificial sequence

<400> 7

gccgctagct ctagttatta atagtaatca att 33

<210> 8

<211> 33

<212> DNA

<213> Artificial sequence

<400> 8

tgtgaattct ttgccaaaat gatgagacag cac 33

<210> 9

<211> 27

<212> DNA

<213> Artificial sequence

<400> 9

gccgaattcg ccaccatgga agacgcc 27

<210> 10

<211> 29

<212> DNA

<213> Artificial sequence

<400> 10

tgtgtcgact tacacggcga tctttccgc 29

<210> 11

<211> 154

<212> DNA

<213> Artificial sequence

<400> 11

agagaaatgt tctggcacct gcacttgcac tggggacagc ctattttgct agtttgtttt 60

gtttcgtttt gttttgatgg agagcgtatg ttagtactat cgattcacac aaaaaaccaa 120

cacacagatg taatgaaaat aaagatattt tatt 154

<210> 12

<211> 1215

<212> DNA

<213> Artificial sequence

<400> 12

atgggccgcc gcaggagacg acggcgacgg cgaagaaggg aggagccgca gtcagatcct 60

agcgtcgagc cccctctgag tcaggaaaca ttttcagacc tatggaaact acttcctgaa 120

aacaacgttc tgtccccctt gccgtcccaa gcaatggatg atttgatgct gtccccggac 180

gatattgaac aatggttcac tgaagaccca ggtccagatg aagctcccag aatgccagag 240

gctgctcccc gcgtggcccc tgcaccagca gctcctacac cggcggcccc tgcaccagcc 300

ccctcctggc ccctgtcatc ttctgtccct tcccagaaaa cctaccaggg cagctacggt 360

ttccgtctgg gcttcttgca ttctgggaca gccaagtctg tgacttgcac gtactcccct 420

gccctcaaca agatgttttg ccaactggcc aagacctgcc ctgtgcagct gtgggttgat 480

tccacacccc cgcccggcac ccgcgtccgc gccatggcca tctacaagca gtcacagcac 540

atgacggagg ttgtgaggcg ctgcccccac catgagcgct gctcagatag cgatggtctg 600

gcccctcctc agcatcttat ccgagtggaa ggaaatttgc gtgtggagta tttggatgac 660

agaaacactt ttcgacatag tgtggtggtg ccctatgagc cgcctgaggt tggctctgac 720

tgtaccacca tccactacaa ctacatgtgt aacagttcct gcatgggcgg catgaaccgg 780

aggcccatcc tcaccatcat cacactggaa gactccagtg gtaatctact gggacggaac 840

agctttgagg tgcgtgtttg tgcctgtcct gggagagacc ggcgcacaga ggaagagaat 900

ctccgcaaga aaggggagcc tcaccacgag ctgcccccag ggagcactaa gcgagcactg 960

cccaacaaca ccagctcctc tccccagcca aagaagaaac cactggatgg agaatatttc 1020

acccttcaga tccgtgggcg tgagcgcttc gagatgttcc gagagctgaa tgaggccttg 1080

gaactcaagg atgcccaggc tgggaaggag ccagggggga gcagggctca ctccagccac 1140

ctgaagtcca aaaagggtca gtctacctcc cgccataaaa aactcatgtt caagacagaa 1200

gggcctgact cagac 1215

<210> 13

<211> 435

<212> DNA

<213> Artificial sequence

<400> 13

atgtggctgc agagcctgct gctcttgggc actgtggcct gcagcatctc tgcacccgcc 60

cgctcgccca gccccagcac gcagccctgg gagcatgtga atgccatcca ggaggcccgg 120

cgtctcctga acctgagtag agacactgct gctgagatga atgaaacagt agaagtcatc 180

tcagaaatgt ttgacctcca ggagccgacc tgcctacaga cccgcctgga gctgtacaag 240

cagggcctgc ggggcagcct caccaagctc aagggcccct tgaccatgat ggccagccac 300

tacaagcagc actgccctcc aaccccggaa acttcctgtg caacccagat tatcaccttt 360

gaaagtttca aagagaacct gaaggacttt ctgcttgtca tcccctttga ctgctgggag 420

ccagtccagg agtga 435

<210> 14

<211> 426

<212> DNA

<213> Artificial sequence

<400> 14

atgtggctgc agaatttact tttcctgggc attgtggtct acagcctctc agcacccacc 60

cgctcaccca tcactgtcac ccggccttgg aagcatgtag aggccatcaa agaagccctg 120

aacctcctgg atgacatgcc tgtcacgttg aatgaagagg tagaagtcgt ctctaacgag 180

ttctccttca agaagctaac atgtgtgcag acccgcctga agatattcga gcagggtcta 240

cggggcaatt tcaccaaact caagggcgcc ttgaacatga cagccagcta ctaccagaca 300

tactgccccc caactccgga aacggactgt gaaacacaag ttaccaccta tgcggatttc 360

atagacagcc ttaaaacctt tctgactgat atcccctttg aatgcaaaaa accaggccaa 420

aaatga 426

<210> 15

<211> 84

<212> DNA

<213> Artificial sequence

<400> 15

cgtaaaaggc gagctcctgt taaacagact ttgaattttg accttctcaa gttggcggga 60

gacgtcgagt ccaaccctgg gccc 84

<210> 16

<211> 2281

<212> DNA

<213> Artificial sequence

<400> 16

gaattctcat gtttgacagc ttatcatcga taagctttaa tgcggtagtt tatcacagtt 60

aaattgctaa cgcagtcagg caccgtgtat gaaatctaac aatgcgctca tcgtcatcct 120

cggcaccgtc accctggatg ctgtaggcat aggcttggtt atgccggtac tgccgggcct 180

cttgcgggat atcgtccatt ccgacagcat cgccagtcac tatggcgtgc tgctagcgct 240

atatgcgttg atgcaatttc tatgcgcacc cgttctcgga gcactgtccg accgctttgg 300

ccgccgccca gtcctgctcg cttcgctact tggagccact atcgactacg cgatcatggc 360

gaccacaccc gtcctgtgga tccgggcccc catttcccct cccttccagc tctctgcccc 420

ttttggattg aagccaatat gataatgagg gggtggagtt tgtgacgtgg cgcggggcgt 480

gggaacgggg cgggtgacgt agtagtgtgg cggaagtgtg atgttgcaag tgtggcggaa 540

cacatgtaag cgacggatgt ggcaaaagtg acgtttttgg tgtgcgccgg tgtacacagg 600

aagtgacaat tttcgcgcgg ttttaggcgg atgttgtagt aaatttgggc gtaaccgagt 660

aagatttggc cattttcgcg ggaaaactga ataagaggaa gtgaaatctg aataattttg 720

tgttactcat agcgcgtaat ttctagtaat aaaggatcct ttattttcat tggatccgtg 780

tgttggtttt ttgtgtgcgg ccgcgtaccg gtcacagacg cccaggaccg cgcttcccac 840

gtggcggagg gactggggac ccgggcaccc gtcctgcccc ttcaccttcc agctccgcct 900

cctccgcgcg gaccccgccc cgtcccgacc cctcccgggt ccccggccca gccccctccg 960

ggccctccca gcccctcccc ttcctttccg cggccccgcc ctctcctcgc ggcgcgagtt 1020

tcaggcagcg ctgcgtcctg ctgcgcacgt gggaagccct ggccccggcc actcgaggac 1080

gcacgtgggt tcgaatgaaa atgagacata ttatctgcca cggaggtgtt attaccgaag 1140

aaatggccgc cagtcttttg gaccagctga tcgaagaggt actggctgat aatcttccac 1200

ctcctagcca ttttgaacca cctacccttc acgaactgta tgatttagac gtgacggccc 1260

ccgaagatcc caacgaggag gcggtttcgc agatttttcc cgactctgta atgttggcgg 1320

tgcaggaagg gattgactta ctcacttttc cgccggcgcc cggttctccg gagccgcctc 1380

acctttcccg gcagcccgag cagccggagc agagagcctt gggtccggtt tctatgccaa 1440

accttgtacc ggaggtgatc gatcttacct gccacgaggc tggctttcca cccagtgacg 1500

acgaggatga agagggtgag gagtttgtgt tagattatgt ggagcacccc gggcacggtt 1560

gcaggtcttg tcattatcac cggaggaata cgggggaccc agatattatg tgttcgcttt 1620

gctatatgag gacctgtggc atgtttgtct acagtaagtg aaaattatgg gcagtgggtg 1680

atagagtggt gggtttggtg tggtaatttt ttttttaatt tttacagttt tgtggtttaa 1740

agaattttgt attgtgattt ttttaaaagg tcctgtgtct gaacctgagc ctgagcccga 1800

gccagaaccg gagcctgcaa gacctacccg ccgtcctaaa atggcgcctg ctatcctgag 1860

acgcccgaca tcacctgtgt ctagagaatg caatagtagt acggatagct gtgactccgg 1920

tccttctaac acacctcctg agatacaccc ggtggtcccg ctgtgcccca ttaaaccagt 1980

tgccgtgaga gttggtgggc gtcgccaggc tgtggaatgt atcgaggact tgcttaacga 2040

gcctgggcaa cctttggact tgagctgtaa acgccccagg ccataaggtg taaacctgtg 2100

attgcgtgtg tggttaacgc ctttgtttgc tgaatgagtt gatgtaagtt taataaaggg 2160

tgagataatg tttaacttgc atggcgtgtt aaatggggcg gggcttaaag ggtatataat 2220

gcgccgtggg ctaatcttgg ttacatctga cctcatggag gcttgggagt gttaaagatc 2280

t 2281

<210> 17

<211> 311

<212> DNA

<213> Artificial sequence

<400> 17

ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc 60

ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca 120

ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta 180

tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta 240

tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat 300

cgctattacc a 311

<210> 18

<211> 366

<212> DNA

<213> Artificial sequence

<400> 18

tggtcgaggt gagccccacg ttctgcttca ctctccccat ctcccccccc tccccacccc 60

caattttgta tttatttatt ttttaattat tttgtgcagc gatgggggcg gggggggggg 120

ggggcgcgcg ccaggcgggg cggggcgggg cgaggggcgg ggcggggcga ggcggagagg 180

tgcggcggca gccaatcaga gcggcgcgct ccgaaagttt ccttttatgg cgaggcggcg 240

gcggcggcgg ccctataaaa agcgaagcgc gcggcgggcg ggagtcgctg cgcgctgcct 300

tcgccccgtg ccccgctccg ccgccgcctc gcgccgcccg ccccggctct gactgaccgc 360

gttact 366

<210> 19

<211> 341

<212> DNA

<213> Artificial sequence

<400> 19

ctgagtcatt agggactttc caatgggttt tgcccagtac ataaggtcaa taggggtgaa 60

tcaacaggaa agtcccattg gagccaagta cactgagtca atagggactt tccattgggt 120

tttgcccagt acaaaaggtc aatagggggt gagtcaatgg gtttttccca ttattggcac 180

gtacataagg tcaatagggg tgagtcattg ggtttttcca gccaatttaa ttaaaacgcc 240

atgtactttc ccaccattga cgtcaatggg ctattgaaac taatgcaacg tgacctttaa 300

acggtacttt cccatagctg attaatggga aagtaccgtt c 341

<210> 20

<211> 250

<212> DNA

<213> Artificial sequence

<400> 20

actgagtcat tagggacttt ccattgggtt ttgcccagta caaaaggtca atagggggtg 60

agtcaatggg tttttcccat tattggcacg tacataaggt caataggggt gagtcattgg 120

gtttttccag ccaatttaat taaaacgcca tgtactttcc caccattgac gtcaatgggc 180

tattgaaact aatgcaacgt gacctttaaa cggtactttc ccatagctga ttaatgggaa 240

agtaccgttc 250

<210> 21

<211> 303

<212> DNA

<213> Artificial sequence

<400> 21

ggcctccgcg ccgggttttg gcgcctcccg cgggcgcccc cctcctcacg gcgagcgctg 60

ccacgtcaga cgaagggcgc agcgagcgtc ctgatccttc cgcccggacg ctcaggacag 120

cggcccgctg ctcataagac tcggccttag aaccccagta tcagcagaag gacattttag 180

gacgggactt gggtgactct agggcactgg ttttctttcc agagagcgga acaggcgagg 240

aaaagtagtc ccttctcggc gattctgcgg agggatctcc gtggggcggt gaacgccgat 300

gat 303

<210> 22

<211> 15

<212> DNA

<213> Artificial sequence

<400> 22

aaaacaggta agtcc 15

<210> 23

<211> 60

<212> DNA

<213> Artificial sequence

<400> 23

gcctctacta accatgttca tgttttcttt ttttttctac aggtcctggg tgacgaacag 60

Claims (22)

1. An isolated polynucleotide comprising the following elements:

hCMV enhancer, beta-Actin promoter and mCMV enhancer;

it further comprises a Ubi enhancer, which is located downstream of the 3 elements above;

and the polynucleotide comprises in order:

the mCMV enhancer + hCMV enhancer + β -Actin promoter + Ubi enhancer;

the polynucleotide has promoter function;

wherein,

the nucleotide sequence of the mCMV enhancer is shown as SEQ ID NO: 20 is shown in the figure;

each of the 4 elements is independently 1.

2. The polynucleotide of claim 1, wherein:

the nucleotide sequence of the hCMV enhancer is shown as SEQ ID NO: 17 is shown;

the nucleotide sequence of the beta-Actin promoter is shown as SEQ ID NO: 18 is shown in the figure;

the nucleotide sequence of the mCMV enhancer is shown as SEQ ID NO: 20 is shown in the figure;

the nucleotide sequence of the Ubi enhancer is shown as SEQ ID NO: shown at 21.

3. A polynucleotide according to any one of claims 1 to 2, with or without linker sequences or splice sites between the individual elements thereof.

4. The polynucleotide according to any one of claims 1 to 2, wherein there is a splice donor and a splice acceptor upstream and downstream of the mCMV enhancer and/or hCMV enhancer and/or Ubi enhancer, respectively.

5. The polynucleotide of claim 4, wherein the nucleotide sequence of the splice donor is as set forth in SEQ ID NO: 22; the nucleotide sequence of the splicing acceptor is shown as SEQ ID NO: shown at 23.

6. A nucleic acid construct comprising the polynucleotide of any one of claims 1 to 5, and an operably linked exogenous gene.

7. The nucleic acid construct according to claim 6, wherein the exogenous gene is selected from any one or more of p53, GM-CSF, IL-2, IL-12, IL-24, Trail, p16, TK, IFN γ, TNF α, Rb, Sirp α.

8. The nucleic acid construct of claim 6, wherein the exogenous gene is SEQ ID NO: 12 and/or p53 shown in SEQ ID NO: 13 or SEQ ID NO: GM-CSF as shown in FIG. 14.

9. 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 8.

10. The recombinant vector according to claim 9, wherein said recombinant vector is a recombinant viral vector.

11. The recombinant vector according to claim 9, wherein the recombinant vector is the polynucleotide of any one of claims 1 to 5 or the nucleic acid construct of any one of claims 6 to 8, recombined with pDC315 plasmid or SG 655.

12. A pharmaceutical composition comprising the polynucleotide of any one of claims 1 to 5 or the nucleic acid construct of any one of claims 6 to 8 or the recombinant vector of any one of claims 9 to 11, and optionally a pharmaceutically acceptable adjuvant.

13. Use of (1) or (2) selected from:

(1) use of the polynucleotide of any one of claims 1 to 5 or the nucleic acid construct of any one of claims 6 to 8 or the recombinant vector of any one of claims 9 to 11 for the preparation of a medicament for the treatment and/or prevention of cancer or an anti-tumor agent;

(2) use of the polynucleotide of any one of claims 1 to 5 or the nucleic acid construct of any one of claims 6 to 8 or the recombinant vector of any one of claims 9 to 11 in the manufacture of a medicament or agent for inhibiting tumor cells.

14. The use according to claim 13, wherein in item (1), 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, kidney cancer, glioma, melanoma, pancreatic cancer or prostate cancer.

15. The use according to claim 13, wherein, in item (2), the tumor cell is a cell of a tumor: 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.

16. The use according to claim 13, wherein in item (2), the tumor cell is any one selected from the group consisting of Hep3B, Huh7, HepG2, PLC/PRF/5, BEL-7404, H460 and H1299 cells.

17. A method of inhibiting tumor cells in vitro comprising the step of administering an effective amount of the polynucleotide of any one of claims 1 to 5 or the nucleic acid construct of any one of claims 6 to 8 or the recombinant vector of any one of claims 9 to 11.

18. The method of claim 17, wherein the tumor cell is a cell of a tumor: 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.

19. The method of claim 17, wherein the tumor cell is any one selected from the group consisting of Hep3B, Huh7, HepG2, PLC/PRF/5, BEL-7404, H460 and H1299 cells.

20. Use of the polynucleotide of any one of claims 1 to 5 as a promoter.

21. An isolated polynucleotide which is SEQ ID NO: 20 or a complement thereof.

22. Use of the polynucleotide of claim 21 in the preparation of a promoter; wherein the promoter is the polynucleotide of any one of claims 1 to 5 having a promoter function.

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