CN114891829B

CN114891829B - Liver-specific episomal expression vector, gene therapy vector and application thereof

Info

Publication number: CN114891829B
Application number: CN202210573172.2A
Authority: CN
Inventors: 张继红; 王天云; 张俊河; 王小引; 米春柳; 王琳; 徐婷婷; 赵凌冉; 董紫阳; 刘云霞; 钱昱如
Original assignee: Henan Punuoyi Biological Product Research Institute Co ltd; Xinxiang Medical University
Current assignee: Henan Punuoyi Biological Product Research Institute Co ltd; Xinxiang Medical University
Priority date: 2022-05-24
Filing date: 2022-05-24
Publication date: 2023-09-26
Anticipated expiration: 2042-05-24
Also published as: CN114891829A

Abstract

The invention belongs to the field of expression vectors, and particularly relates to a liver-specific episomal expression vector, a gene therapy vector and application thereof. The liver-specific episomal expression vector uses a liver-specific promoter to replace the EF-1 alpha promoter in the skeleton episomal expression vector pEMM, and the enhancer sequence is seamlessly cloned upstream of the liver-specific promoter sequence. The liver specificity free expression vector provided by the invention is constructed by taking pEMM as a basic vector, can obviously improve the expression level of a carried target gene, and under the same condition, the free expression vector of the liver specificity promoter modified by the enhancer can obviously improve the expression of the target gene compared with the vector without the enhancer.

Description

Liver-specific episomal expression vector, gene therapy vector and application thereof

Technical Field

The invention belongs to the field of expression vectors, and particularly relates to a liver-specific episomal expression vector, a gene therapy vector and application thereof.

Background

The liver is an important organ of the organism and plays the physiological roles of substance metabolism, amino acid utilization, bile acid synthesis, biosynthesis and transformation, oxidation protection, detoxification and the like. Many physical, chemical and biological factors can damage the liver, resulting in a series of liver diseases such as liver tumor, various hepatitis, fatty liver, cirrhosis, acute liver failure, etc. Along with the gradual maturation and perfection of DNA recombination technology, researchers aim at transgene therapy research so as to solve the problems of short protein half-life period, repeated injection of patients and the like in protein substitution therapy.

Gene expression vector systems play a key role in gene therapy. In recent years, researchers have developed a variety of different types of vector systems for therapeutic gene delivery, and viral vectors are widely used because of their higher transfection efficiency compared to plasmid vectors, but their transfection into host cells relies on interactions between viral vectors and host cell protein membranes, thus potentially causing carcinogenicity, autoimmunity, and the potential for altered cell pathology. In addition, the integrated vector system can enable the exogenous gene to be expressed in target cells with high efficiency and long term, and can easily cause insertion mutation and cytotoxicity due to random integration into host genome, and silencing phenomenon or transcriptional activation effect caused by the position effect of exogenous gene insertion. The episomal plasmid vector system is an independent extrachromosomal element in the host cell, and can effectively avoid adverse reactions caused by gene insertion into the host genome. Therefore, the development of a novel safe, efficient, sustained-expression, non-viral, non-integrated episomal plasmid vector is one of the problems that need to be addressed in the bottleneck and transgenic therapies in this research area.

Disclosure of Invention

The invention aims to provide a liver-specific episomal expression vector, which belongs to a novel non-viral liver-specific episomal gene therapy vector of human and mammal cells.

It is a second object of the present invention to provide a liver-specific episomal gene therapy vector.

A third object of the present invention is to provide the use of the above-mentioned liver-specific episomal expression vector and liver-specific episomal gene therapy vector.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:

a liver-specific episomal expression vector, wherein an enhancer sequence is seamlessly cloned upstream of a liver-specific promoter sequence using the liver-specific promoter in place of the EF-1 alpha promoter in the backbone episomal expression vector pEMM; the liver specific promoter is selected from one of ApoAI, AFP, AAT, ALB, and the nucleotide sequence of ApoAI, AFP, AAT, ALB is shown in SEQ ID NO: 1-4, wherein the enhancer sequence is selected from one of JSRV1, JSRV2, hCMV1, hCMV2, apoE1 and ApoE2, and the nucleotide sequence of the JSRV1, JSRV2, hCMV1, hCMV2, apoE1 and ApoE2 is shown as SEQ ID NO:5 to 10;

the backbone free expression vector pEMM is obtained by modifying a pEGFP-C1 plasmid vector, wherein the modification comprises inserting a synthesized MAR characteristic motif at the downstream of EGFP and the upstream of SV40 poly A, replacing a CMV promoter with an EF-1 alpha promoter, and inserting MAR1 at the downstream of SV40 poly A; the nucleotide sequence of the MAR characteristic motif is shown in SEQ ID NO:11, the nucleotide sequence of the EF-1 alpha promoter is shown as SEQ ID NO:12, the nucleotide sequence of MAR1 is shown in SEQ ID NO: shown at 13.

The liver specificity free expression vector provided by the invention is constructed by taking pEMM as a basic vector, can obviously improve the expression level of a carried target gene, and under the same condition, the free expression vector of the liver specificity promoter modified by the enhancer can obviously improve the expression of the target gene compared with the vector without the enhancer.

The expression vector can be constructed according to a conventional method in the field of genetic engineering.

From the standpoint of the percentage of positive cells and the expression level of the target protein, preferably, the liver-specific promoter is AAT, the nucleotide sequence of which is set forth in SEQ ID NO: 3. Preferably, the liver-specific promoter is ApoAI, the nucleotide sequence of which is shown in SEQ ID NO: 1.

To further optimise the expression level of the protein of interest, preferably the enhancer sequence is selected from hCMV1 or hCMV2, the nucleotide sequences of hCMV1, hCMV2 being as set out in SEQ ID NO:7 to 8.

A liver-specific episomal gene therapy vector is provided, wherein a functional gene is inserted into the MCS region of the liver-specific episomal expression vector.

A liver-specific episomal gene therapy vector is provided, wherein a functional gene is substituted for an EGFP reporter gene of the liver-specific episomal expression vector.

The liver-specific episomal gene therapy vector can provide a safe, efficient and stable gene transfer tool for the transgene therapy of clinical liver diseases and a favorable tool for the transgene therapy of liver related diseases.

The liver-specific episomal expression vector and the application of the liver-specific episomal gene therapy vector in preparing a reagent for efficiently expressing exogenous genes in liver cells.

The liver-specific episomal expression vector carrying the target gene is transformed into human liver cells, so that the high-efficiency expression of the target gene in host cells can be realized.

Drawings

FIG. 1 is a plasmid map of pEMM vector;

FIG. 2 is a Fluorescent In Situ Hybridization (FISH) analysis of a pEMM expression vector stably transfected with metaphase chromosomes of CHO cells in which the vector molecules (shown red) bind to metaphase chromosomes (shown blue) in low copy number free form (about 5 copies/cell);

FIG. 3 is a plasmid map of liver-targeting episomal expression vectors pApoAI-MM (a), pAFP-MM (b), pAAT-MM (c) and pALB-MM (d) in the present invention;

FIG. 4 is a plasmid map of enhancer modified liver targeting episomal expression vectors pJSRV1/AAT-MM (a), pJSRV2/AAT-MM (b), phCMV1/AAT-MM (c), phCMV2/AAT-MM (d), pApoE1/AAT-MM (e) and pApoE2/AAT-MM (f) of the present invention;

FIG. 5 shows the transient expression of EGFP 72h after transformation of human hepatocytes (HepG 2, HL-7702 and Huh-7) with the experimental group plasmids (free expression vectors pApoAI-MM, pAFP-MM, pAAT-MM and pALB-MM containing different liver-specific promoters) and the control group plasmid (pEMM) according to the invention, wherein: (a) Observing and photographing under an inverted fluorescence microscope to show the EGFP expression condition; (b) And (c) detecting MFI and positive cell ratio of EGFP for the flow cytometer, respectively;

FIG. 6 shows transient expression of EGFP 72h after transformation of human hepatoma cell lines HepG2 with experimental plasmids (liver-specific episomal expression vectors pJSRV1/AAT-MM, pJSRV2/AAT-MM, phCMV1/AAT-MM, phCMV2/AAT-MM, pApoE1/AAT-MM and pApoE2/AAT-MM containing different combinations of enhancers/AAT promoters) and control (pAAT-MM) according to the invention: (a) Observing and photographing under an inverted fluorescence microscope to show the EGFP expression condition; (b) Detecting EGFP expression and positive cell ratio by a flow cytometer;

FIG. 7 shows the Western blot analysis results of each of the experimental and control groups in the present invention, wherein: (a) EGFP protein expression levels of liver-specific free expression vectors containing different enhancer/AAT promoter combinations; (b) Image J software semi-quantitatively analyzed EGFP protein levels.

Detailed Description

The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Unless otherwise indicated, the examples are in accordance with conventional experimental conditions, such as the molecular cloning laboratory Manual of Sambrook et al (Sambrook J & Russell DW, molecular cloning: a laboratory manual, 2001), or in accordance with the manufacturer's instructions.

Coli DH 5. Alpha. Cells, plasmid vectors and reagents, and tool enzymes used in the examples below are commercially available.

Liver is an important target organ for gene therapy research by virtue of its huge protein synthesis capacity, and hydrodynamic transgenic technology (Hydrodynamics-based gene delivery, HGD) [ Kamimura K, kanefuji T, yokoo T, et al safety assessment of liver-targeted hydrodynamic gene delivery in digs. PLoS One,2014,9 (9): e107203.Nakamura S, maehara T, watanabe S, et al Liver lobe and strain difference in gene expression after Hydrodynamics-based gene delivery in mice. Anim Biotechnol,2015,26 (1): 51-57 ] can mediate efficient expression of exogenous genes in liver, becoming a favorable tool for transgene treatment of liver-related diseases. However, our earlier studies showed that this method mediated the expression of exogenous genes for a short period of time (6-24 h post injection), and repeated frequent injections tended to cause some damage to liver tissue. Therefore, the development of a novel safe, efficient and sustainable replication vector system is one of the scientific problems commonly focused by researchers in the clinical transgene treatment process.

The nuclear matrix binding region (matrix attachment region, MAR), which is a non-coding DNA sequence present in the chromosome of eukaryotic cells that specifically binds to the nuclear backbone (or nuclear matrix), is 300 to several thousand bp in length and often contains some characteristic motifs (sequence motifs), such as the yeast autonomous replication sequence ARS, drosophila topoisomerase II recognition site, and loose DNA that forms a protein recognition site, AT-rich regions, bent DNA, etc. [ Wang TY, yang R, qin C, et al enhanced expression of transgene in CHO cells using matrix attachment region.cell biolInt,2008,32 (10): 1279-1283 ].

We succeeded in obtaining the first generation episomal plasmid vector pCMV-M [ Lin Y, li ZX, wang TY et al MAR characteristic motifs mediate episomal vector in CHO cells.Gene,2015,559 (2): 137-143 ] of the present laboratory by inserting a DNA fragment (367 bp) containing a motif characteristic of MAR in the sequence of human interferon-beta MAR (GenBank M83137.1) (full length 2200 bp) downstream of the pEGFP-C1 vector reporter gene EGFP. On this basis, we optimized the regulatory element of the vector, the promoter, to obtain a second generation episomal plasmid vector pEF-1. Alpha. -M [ Xu ZJ, jia YL, wang M, et al effect of promoter, promoter mutation and enhancer on transgene expression mediated by episomal vectors in transfected HEK293, chang liver and primary cells.bioengineered,2019,10 (1): 548-560 ] driven by the EF-1. Alpha. Constitutive promoter for stably expressing exogenous genes. In addition, MAR elements mediate stable attachment of the vector to the nuclear matrix region, effectively inhibiting or attenuating transgene silencing by epigenetic effects and promoting mitotic stability, thereby increasing transgene expression levels. Based on this, we inserted a 791bp MAR1 sequence (Seq ID No. 13) downstream of EGFP expression cassette of the second generation vector, constituting the third generation episomal plasmid vector pEMM (FIG. 1), further enhancing the expression level of recombinant proteins. Therefore, the MAR modified episomal plasmid vector can effectively avoid the potential pathogenic hazard of the viral vector and the integrative vector in gene therapy, and is a safe and effective method for overcoming transgene silencing and improving transgene expression in clinical transgene therapy.

In addition, the liver-specific promoter has further accurate regulation and control on in vivo transgene treatment, and adverse effects caused by nonspecific transgenes are effectively avoided or minimized. Liver-specific promoters reported are Alpha Fetoprotein Promoter (AFP), alpha 1 antitrypsin promoter (hAAT), apolipoprotein promoter (ApoA 1), haptoglobin (HPGL), apolipoprotein enhancer (apoe) and the like [ references below:

Miao CH,Ye X,Thompson AR.High-level factor VIII gene expression in vivo achieved by nonviral liver-specific gene therapy vectors.Hum Gene Ther,2003,14(14):1297-1305.

Haase R,Magnusson T,Su B,et al.Generation of a tumor-and tissue-specific episomal non-viral vector system.BMC Biotechnol,2013,13:49.

Hu Y,Ren X,Wang H,et al.Liver-specific expression of an exogenous gene controlled by human apolipoprotein A-I promoter.Int J Pharm,2010,398(1-2):161-164.]。

the exploratory research of a liver-specific non-viral non-integrated episomal gene therapy plasmid vector provides a safe, efficient and stable gene transfer tool for the transgenic therapy of clinical liver diseases, and has certain theoretical and practical significance.

The pEMM episomal expression vector is a third generation episomal expression vector formed by inserting a DNA fragment (367 bp) of a synthetic MAR characteristic motif downstream of the EGFP of a pEGFP-C1 plasmid vector (purchased from Promega biosystems), replacing the CMV promoter with a highly efficient human EF-1 alpha promoter, and inserting MAR1 at the 3' -end of the EGFP expression cassette in the laboratory, and the vector map is shown in FIG. 1.

Among them, the Chinese patent application with publication number CN105802997A discloses constructing an attachment expression vector pEME containing a MAR characteristic motif with pEGFP-C1 as a starting vector and replacing CMV promoter with EF-1 alpha promoter. MAR1 was inserted into SV40 poly A by homologous recombination. MAR1 was inserted as a cis-regulatory element into the 3' end of EGFP expression cassette to further enhance the efficiency and stability of transgene expression.

As shown in FIG. 2, fluorescence In Situ Hybridization (FISH) analysis pEMM expression vectors stably transfected metaphase chromosomes in CHO cells, which exist in low copy number episomal form (about 5 copies/cell).

EXAMPLE 1 liver-specific episomal expression vectors

The liver-specific episomal expression vector of this example replaced the EF-1 a promoter in the backbone episomal expression vector pEMM with a liver-specific promoter, seamlessly cloning the enhancer sequence upstream of the liver-specific promoter sequence; the nucleotide sequence of the liver specificity promoter is selected from ApoAI, AFP, AAT, ALB, apoAI and AFP, AAT, ALB and is shown in SEQ ID NO: 1-4, wherein the nucleotide sequence of the enhancer sequence is selected from JSRV1, JSRV2, hCMV1, hCMV2, apoE1 and ApoE2, and the nucleotide sequence of the JSRV1, JSRV2, hCMV1, hCMV2, apoE1 and ApoE2 is shown as SEQ ID NO:5 to 10.

The specific construction process is as follows:

1. construction of pApoAI-MM, pAFP-MM, pAAT-MM and pALB-MM vectors

The DNA fragment ApoAI (GenBank: J04066.1), AFP (GenBank: NG_ 056260.1), AAT (GenBank: NT_ 187601.1) and ALB (GenBank: NC_ 000004.12) of the artificially synthesized liver-specific promoter were introduced at both ends with AgeI cleavage sites, and simultaneously the pEMM vector was digested singly with AgeI to replace the EF-1. Alpha. Promoter with the liver-specific promoter, and the gene fragment of the liver-specific promoter and pEMM after the cleavage was recovered in 1:5 (molar ratio) with T4 ligase, overnight at 16 ℃; then adding the connection product into E.coli DH5 alpha strain competent cell suspension, transforming, inoculating 200 mu l of bacterial liquid on LB plate containing 30 mu g/ml kanamycin, culturing overnight at 37 ℃, picking single colony for subculture, extracting recombinant plasmid, performing enzyme digestion verification, selecting plasmid with correct enzyme digestion verification, performing sequencing verification, and respectively naming the vectors with completely correct target gene sequences as pApoAI-MM, pAFP-MM, pAAT-MM and pALB-MM (plasmid map is shown in figure 3).

2. Enhancer/promoter combined liver targeting free vector construction

6 enhancer sequences were synthesized, JSRV1 (GenBank: AF 105220.1), JSRV2 (GenBank: AF 105220.1), hCMV1 (GenBank: K03104.1), hCMV2 (GenBank: K03104.1), apoE1 (GenBank: U32510.1) and ApoE2 (GenBank: U35114.1), seamlessly cloned upstream of the pAAT-MM vector AAT promoter, and enhancer/promoter combinations of liver-targeted episomes pJSRV1/AAT-MM, pJSRV2/AAT-MM, phCMV1/AAT-MM, phCMV2/AAT-MM, pApoE1/AAT-MM and pApoE2/AAT-MM were constructed (FIG. 4).

EXAMPLE 2 use of liver-specific episomal expression vectors

The application of the liver-specific episomal expression vector of this example is described below:

1. expression of pApoAI-MM, pAFP-MM, pAAT-MM and pALB-MM vectors in vitro cultured hepatocytes

1.1 transfection of hepatocytes with different liver-targeting episomal expression vectors

Human HepG2, HL-7702 and Huh-7 hepatocytes were incubated at 37℃with 5% CO ₂ Culturing in DMEM medium containing 10% foetal calf serum under conditions to about 1.25X10% ⁵ Cells were seeded in 24-well plates. Transfection was divided into 5 groups according to experimental design: control vector (pEMM) without liver specific promoter, episomal vectors pApoAI-MM, pAFP-MM, pAAT-MM and pALB-MM with liver specific promoter. Cell transfection was performed according to the instructions of the Biosharp company Lip2000 liposome transfection reagent. 72h after transfection, the cells were collected for detection by observation under an inverted fluorescence microscope and photographed, digested with 0.25% pancreatin.

1.2 detecting EGFP expression level by flow cytometry

Hepatocytes transfected with episomes of different liver-specific promoters were collected, 20000 cells were analyzed per sample, and flow cytometry detected EGFP mean fluorescence intensity (mean fluorescence intensity, MFI) and percent positive cells of the cells.

The results showed that the MFI of the transfected cells of AAT liver-specific promoter vector set reached the highest level in three human hepatocytes, hepG2, HL-7702 and Huh-7, and the percentage of EGFP positive cells was also at higher levels, 23%, 9% and 1.5%, respectively (fig. 5).

The liver-specific promoter can further accurately regulate and control in-vivo transgene treatment, and effectively avoid or minimize adverse effects caused by nonspecific transgene.

2. Expression of enhancer-modified pAAT-MM liver-targeting episomal expression vectors in vitro cultured hepatocytes

The methods for transfecting hepatocytes with the liver-targeting episomal expression vectors of different enhancer/promoter combinations are described in section 1.1.

2.1 detecting EGFP expression level by flow cytometry

The method of detecting EGFP expression levels by flow cytometry is the same as in section 1.2.

The results show that after the liver targeting episomal vectors of the different enhancer/promoter combinations are transfected into the human hepatoma cell HepG2, the MFI of the hCMV2/AAT enhancer/promoter combination reaches the highest value, which is about 1.32 times that of the control group without the enhancer. The second is the hCMV1/AAT vector group (1.21-fold). The EGFP-positive cell ratios of the two groups were 57.2% and 49.3%, respectively, and were also significantly higher than the control group (44.3%). However, the expression level of EGFP in HEpG2 human hepatoma cells was significantly lower 72h after each experimental group plasmid vector was transfected into HEK293E human embryonic kidney cells and HCT-116 human colon cancer cells (FIG. 6).

Meanwhile, the expression level of EGFP in human embryo kidney HEK293E cells and human colon cancer HCT-116 cells of AAT and enhancer modified AAT liver targeting free vector is obviously lower than that of HepG2 human liver cells (figure 6 a), and the expression of liver specificity is shown.

2.2, western blot method for analyzing EGFP protein expression quantity

The transfected cells of each group were fully lysed with cell lysate, centrifuged at 12 000r/min for 5min at 4℃and the supernatant was collected for quantification, 100. Mu.g of protein was loaded, electrophoresed on SDS-PAGE of 120g/L, after complete separation of the protein, transferred to PVDF membrane and in a blocking solution containing 50g/L skimmed milk powder overnight at 4 ℃. eGFP (BPI Co., ltd. AbM59003-6E 2-PU) and beta-actin (Boster, BM 0627) primary antibodies were diluted 1:3000 respectively, incubated at room temperature for 2h, goat anti-mouse secondary antibodies (Affinity Biosciences, S0002, cincinnati, OH, USA) were diluted 1:3000, incubated at room temperature for 1h, and the PVDF membrane was subjected to chromogenic reaction with ECL luminescent reagents, exposed to X-rays, and photographed. The expression level of EGFP protein was analyzed by Image J software, and the results showed that the EGFP protein level was highest in the cell transfected group of hCMV2/AAT enhancer/promoter (FIG. 7 a), about 5.1 times that of the control group (pEMM-AAT), followed by about 3.5 times that of the control group (FIG. 7 b), and the results were consistent with the observation by a fluorescence microscope and the results of flow assay.

EXAMPLE 3 liver-specific episomal Gene therapy vector and use

Based on the liver-specific episomal expression vector of example 1, a functional gene was inserted into the MCS region of the vector or an EGFP reporter gene was replaced, to obtain a liver-specific episomal gene therapy vector which is expected to exert the same effect of increasing the expression level of transgenes in the transgene therapy of the liver.

While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

<110> Henan Punuo Biopreparation Limited of New Country medical college

<120> a liver-specific episomal expression vector and gene therapy vector and use thereof

<160> 13

<170> PatentIn version 3.5

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<221> ApoAI promoter

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aggggaaggg gatgagtgca gggaaccccg accccacccg ggagacctgc aagcctgcag 60

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gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac 540

caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt 600

caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaa 654

<210> 9

<211> 774

<212> DNA

<221> APOE1 enhancer

<400> 9

ctgcaggctc agaggcacac aggagtttct gggctcaccc tgcccccttc caacccctca 60

gttcccatcc tccagcagct gtttgtgtgc tgcctctgaa gtccacactg aacaaacttc 120

agcctactca tgtccctaaa atgggcaaac attgcaagca gcaaacagca aacacacagc 180

cctccctgcc tgctgacctt ggagctgggg cagaggtcag agacctctct gggcccatgc 240

cacctccaac atccactcga ccccttggaa tttcggtgga gaggagcaga ggttgtcctg 300

gcgtggttta ggtagtgtga gagggtccgg gttcaaaacc acttgctggg tggggagtcg 360

tcagtaagtg gctatgcccc gaccccgaag cctgtttccc catctgtaca atggaaatga 420

taaagacgcc catctgatag ggtttttgtg gcaaataaac atttggtttt tttgttttgt 480

tttgttttgt tttttgagat ggaggtttgc tctgtcgccc aggctggagt gcagtgacac 540

aatctcatct caccacaacc ttcccctgcc tcagcctccc aagtagctgg gattacaagc 600

atgtgccacc acacctggct aattttctat ttttagtaga gacgggtttc tccatgttgg 660

tcagcctcag cctcccaagt aactgggatt acaggcctgt gccaccacac ccggctaatt 720

ttttctattt ttgacaggga cggggtttca ccatgttggt caggctggtc taga 774

<210> 10

<211> 535

<212> DNA

<221> APOE2 enhancer

<400> 10

ggatccaggg gtagagagaa agatttgaga gtggttttgg ggcttggtga cttagagaac 60

agagttgcag gctctgtttt tgggcccgcc ctgccccgtt ccgacctctt agttcctatc 120

ctccagcagc tgtttgtgtg ctgcctctga agtccaccct gaatgacctt cagcctgttc 180

ccgtccctga tatgggcaaa cattgcaagc agcaaacagc aaacacatag ccctccctgc 240

gtgctgacct tggagctgcg gcagaggtca gagacctctc agggcccata ccacttccaa 300

catccccttg atctcttgga ttttggtgga gaggggcaga ggttgtcctg gcctggttag 360

gtagtgtgag agggtcccgg ttcaaaacca ccacttgctg gttgaggagt cgtcagtaag 420

tggctgcgcc cccaccctga ggcttgtttc tccatctgta caatggaaat gatgaagatg 480

cccacctgat agggtttttg tggcaaataa gtaagtagtt tttttgtttt tcttt 535

<210> 11

<211> 367

<212> DNA

<221> characteristic motif of human interferon-beta MAR

<400> 11

atttagttta tatacatcta cagataaata catatcatat atttgaattc taatctccct 60

ctcaacccta cagtcaccca tttggtatat taaagatgtg ttgtctactg tctagtatcc 120

ctcaagcagt gtcaggaatt agtcatttaa atagtctgca agccaggagt ggtggctcat 180

gtctgtaatt ccagcacttg agaggtagaa gtgggaggac tgcttgagct caagagtttg 240

atattatcct ggacaacata gcaagacctc gtctctactt aaaaaaaaaa aattagccag 300

gcatgtgatg tacacctgta gtcccagcta ctcaggaggc cgaaatggga ggatcagatc 360

tggatcc 367

<210> 12

<211> 1335

<212> DNA

<221> EF-1 alpha promoter sequence

<400> 12

gagtaattca tacaaaagga ctcgcccctg ccttggggaa tcccagggac cgtcgttaaa 60

ctcccactaa cgtagaaccc agagatcgct gcgttcccgc cccctcaccc gcccgctctc 120

gtcatcactg aggtggagaa gagcatgcgt gaggctccgg tgcccgtcag tgggcagagc 180

gcacatcgcc cacagtcccc gagaagttgg ggggaggggt cggcaattga accggtgcct 240

agagaaggtg gcgcggggta aactgggaaa gtgatgtcgt gtactggctc cgcctttttc 300

ccgagggtgg gggagaaccg tatataagtg cagtagtcgc cgtgaacgtt ctttttcgca 360

acgggtttgc cgccagaaca caggtaagtg ccgtgtgtgg ttcccgcggg cctggcctct 420

ttacgggtta tggcccttgc gtgccttgaa ttacttccac gcccctggct gcagtacgtg 480

attcttgatc ccgagcttcg ggttggaagt gggtgggaga gttcgaggcc ttgcgcttaa 540

ggagcccctt cgcctcgtgc ttgagttgag gcctggcttg ggcgctgggg ccgccgcgtg 600

cgaatctggt ggcaccttcg cgcctgtctc gctgctttcg ataagtctct agccatttaa 660

aatttttgat gacctgctgc gacgcttttt ttctggcaag atagtcttgt aaatgcgggc 720

caagatctgc acactggtat ttcggttttt ggggccgcgg gcggcgacgg ggcccgtgcg 780

tcccagcgca catgttcggc gaggcggggc ctgcgagcgc ggccaccgag aatcggacgg 840

gggtagtctc aagctggccg gcctgctctg gtgcctggcc tcgcgccgcc gtgtatcgcc 900

ccgccctggg cggcaaggct ggcccggtcg gcaccagttg cgtgagcgga aagatggccg 960

cttcccggcc ctgctgcagg gagctcaaaa tggaggacgc ggcgctcggg agagcgggcg 1020

ggtgagtcac ccacacaaag gaaaagggcc tttccgtcct cagccgtcgc ttcatgtgac 1080

tccacggagt accgggcgcc gtccaggcac ctcgattagt tctcgagctt ttggagtacg 1140

tcgtctttag gttgggggga ggggttttat gcgatggagt ttccccacac tgagtgggtg 1200

gagactgaag ttaggccagc ttggcacttg atgtaattct ccttggaatt tgcccttttt 1260

gagtttggat cttggttcat tctcaagcct cagacagtgg ttcaaagttt ttttcttcca 1320

tttcaggtgt cgtga 1335

<210> 13

<211> 791

<212> DNA

<221> MAR1 sequence

<400> 13

tttaaaatca attgatcata aatgcaaaga ttatttctga actctcaatt tcatccttat 60

tccaacactg tcttgattac tgtacctttg tactaagttt tacaatgatg aagtgtgaat 120

acttcaactt tgttcttctt tttcaagatt gttttggata tcttaggttc ttgacatttc 180

catataaatt ttatttttat ttatttattt atttatttat ttatttattt attccatata 240

aattttagac taaggtgatc actttcaaca caaaagtaca ctggtatttt gattaggtat 300

gcactgaatc tttacatcaa tttagaaaaa tttgacataa taacaataat tgtgttatct 360

aatccatgaa catgttatat gtctctactg atttaggtct tatccagaat attcaggtag 420

gtgtagggtg gggggtgttg aggtttaagt aagcaaagta caagtacttt attagggtgg 480

caaaattatt ctgtatgata ttgcaatagc gaatacaaat tgtcaaaact catagaactt 540

tacaacacaa aacataaagg ttgtggacat attttgttac agttattttg cttttttggc 600

atggtattag caaaagaata gacccaagga tcagtcaaac agactacagg gcccagaaat 660

agacccacac aaatatagtc aactgatttt tgacaaagaa gcaaaggcaa tacaatggaa 720

aaagaatagt ctttccaaca aatggtgctg gaacaattga gtgtccatat gcaagcaagg 780

aactcaaaca c 791

Claims

1. A liver-specific episomal expression vector characterized in that an enhancer sequence is seamlessly cloned upstream of a liver-specific promoter sequence using the liver-specific promoter in place of the EF-1 alpha promoter in the backbone episomal expression vector pEMM; the liver specificity promoter is AAT, and the nucleotide sequence of the AAT is shown in SEQ ID NO:3, wherein the enhancer sequence is selected from hCMV1 or hCMV2, and the nucleotide sequences of hCMV1 and hCMV2 are shown in SEQ ID NO: 7-8;

the skeleton free expression vector pEMM is obtained by modifying a pEGFP-C1 plasmid vector, wherein the modification of the skeleton free expression vector pEMM comprises the steps of inserting a synthesized MAR characteristic motif at the downstream of EGFP and the upstream of SV40 poly A, replacing a CMV promoter by using an EF-1 alpha promoter, and inserting MAR1 at the downstream of SV40 poly A; the nucleotide sequence of the MAR characteristic motif is shown in SEQ ID NO:11, the nucleotide sequence of the EF-1 alpha promoter is shown as SEQ ID NO:12, the nucleotide sequence of MAR1 is shown in SEQ ID NO: shown at 13.

2. A liver-specific episomal gene therapy vector, characterized in that a functional gene is inserted into the MCS region of the liver-specific episomal expression vector of claim 1.

3. A liver-specific episomal gene therapy vector comprising a functional gene in place of the EGFP reporter gene of the liver-specific episomal expression vector of claim 1.

4. Use of the liver-specific episomal expression vector of claim 1, the liver-specific episomal gene therapy vector of claim 2 or 3 in the preparation of a reagent for expressing a foreign gene in hepatocytes.