CN108504688A - Regulate and control suicide gene steerable system specific expressed in liver tumor cells - Google Patents

Regulate and control suicide gene steerable system specific expressed in liver tumor cells Download PDF

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CN108504688A
CN108504688A CN201810186138.3A CN201810186138A CN108504688A CN 108504688 A CN108504688 A CN 108504688A CN 201810186138 A CN201810186138 A CN 201810186138A CN 108504688 A CN108504688 A CN 108504688A
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loxp
surv
stop
promoters
expression
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邹卫龙
童畅
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/45—Transferases (2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A61K48/0058—Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/01—Phosphotransferases with an alcohol group as acceptor (2.7.1)
    • C12Y207/01021—Thymidine kinase (2.7.1.21)
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    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/30—Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT

Abstract

The invention belongs to biotechnologys, specifically disclose a kind of steerable system that regulation and control suicide gene is specific expressed in liver tumor cells, including：(1) enzyme gene is recombinated by the Cre of AFP promoters driving expression；(2) by the suicide gene of SURV promoters driving expression；One section of LoxP Stop LoxP sequence is inserted between the SURV promoters and the suicide gene, Stop is transcription terminator, and LoxP is the unique identification sequence of Cre recombinases.Present invention utilizes AFP promoters to drive the expression of Cre recombinases in hepatoma Hep G 2 cells, Cre recombinases shear away the transcription terminator Stop between two LoxP, the SURV promoters of tumour-specific are allow to directly drive the expression of TK/GCV, cell of the specific induced liver apoptosis of tumor cells without damaging other any other types in vivo.

Description

Regulate and control suicide gene steerable system specific expressed in liver tumor cells
Technical field
The invention belongs to biotechnologys, specifically, it is efficient in liver tumor cells to be related to a kind of regulation and control suicide gene Specific expressed steerable system.
Background technology
The relevant death rate difference of incidence and disease of hepatocellular carcinoma (Hepatocellular carcinoma, HCC) The 6th and the 3rd of global cancer investigation is ranked, 55% is had more than in China, not in 70 Wan Xinfa liver cancer cases of the annual whole world The death in 3~6 months usually after clarifying a diagnosis through the patient for the treatment of.Hepatectomy and liver transfer operation are current most important HCC roots The property controlled treatment means, however, the hepatectomy patient of negative cutting edges still has 40% can be recurred in 1 year after surgery；Even if following most Stringent selection criteria (i.e. Milan standard：Single-shot diameter of tumor≤tumor number≤3 5cm or multiple and maximum gauge≤ 3cm, no big vessel invasion or extrahepatic metastases) it still has more than 10% patient and is recurred in 1 year after surgery, and then lead to almost institute There is the death of patients with recurrent.It is non-when effectively effecting a radical cure standard beyond surgery when gross tumor volume is larger, invades blood vessel or multiple transfer Operative treatment is also unsatisfactory；Though chemotherapy combine stereotactic radiotherapy in the past few years in effect, because cannot be smart Really control Radiotherapy dosimetry, increases liver and mucous membrane tissue injury, and remote difficulty reaches radical cure effect.
In the past 10 years, as molecular biology and technique for gene engineering are in progress, gene therapy is opened therapeutic field of tumor and is ground The new hot spot studied carefully, becomes the dominant direction of malignant tumour non-operative treatment.Wherein, it commits suiside by the thymidine kinase of carrier of adenovirus Genic system (Thymidine kinase/Ganciclovir, TK/GCV) research is carried out also the most extensive earliest, multinomial dynamic Object is tested and clinical research shows that the system is expected to effective selection as liver cancer treatment.But the self-replacation of adenovirus is special Property and the cytotropic uncontrollability of target, but limit effective application of the system in clinic.
Moreover, liver cancer suicide gene therapy majority is in animal and clinical experimental stage, restricts the main of its popularization Problem is safety and validity, including the selectivity of therapeutic gene expression is not high, not strong for the targeting of liver cancer cells, Suicide gene or carrier itself will produce toxic side effect etc. to normal liver cell.
Therefore, be badly in need of exploitation it is a kind of can selectively targeted liver cancer cells and can high efficiency regulatory expression of suicide gene manipulation system System.
Invention content
In order to solve the problems in the existing technology, the object of the present invention is to provide a kind of regulation and control suicide genes in liver Specific expressed steerable system in tumour cell.
In order to realize the object of the invention, technical scheme is as follows：
In a first aspect, the present invention provides a kind of manipulation system that regulation and control suicide gene is specific expressed in liver tumor cells System, the system comprises：
(1) enzyme gene is recombinated by the Cre of AFP (alpha-fetoprotein) promoter driving expression；
(2) by the suicide gene of SURV (survivin, Survivin) promoter driving expression；The SURV promoters and institute It states and is inserted into one section of LoxP-Stop-LoxP sequence between suicide gene, LoxP is the unique identification sequence of Cre recombinases, Stop It will be checked downstream for a kind of transcription terminator (alternatively referred to as stop code) when it is driven by SURV promoters expresses Expression of suicide gene.
Under recombinating enzyme effect in Cre, the end of the transcription between its specific recognition site is deleted by homologous recombination machinery Only sequence Stop, the transcription to release the driving of Stop sequence pair SURV promoters check effect, can make downstream Suicide gene is under the driving of SURV promoters, and specific efficient is expressed in liver tumor cells.
Preferably, the present invention uses TK genes as the suicide gene.
AFP promoters of the present invention, Cre recombinations enzyme gene, SURV promoters, LoxP, Stop, TK gene are this Genetic elements known to field.Present contribution to the art does not simultaneously lie in the sequence for changing these elements itself or property Matter, but a kind of new packaging strategy is proposed, a kind of high specific liver cancer targeting cell is obtained, and can realize control accurate Steerable system.
It should be noted that the system can embody in a variety of forms, for example, being embodied in the form of carrier combines.
Specifically, the system comprises two rAAV (recombinant adeno-associated virus) carriers：
First, containing AFP promoter elements specific expressed HCC, and the Cre of expression can be driven by AFP promoters Recombinate enzyme gene；It is named as rAAV-AFP-Cre；
Second, containing SURV promoter elements, and the LoxP-Stop-LoxP- of expression can be driven by SURV promoters TK/GCV expression cassettes, wherein LoxP-Stop-LoxP is located at the upstream of TK/GCV, is named as rAAV-SURV-LoxP-Stop- LoxP-TK。
The carrier, will using the conventional technical means of genetic engineering using AAV (adeno-associated virus) as the carrier that sets out Element/the expression cassette is inserted.
The present invention verifies the in vitro culture HepG2 cells that above two rAAV carriers are jointly processed by by Flow Cytometry Apoptosis rate and cytotoxicity, research find as rAAV-AFP-Cre and rAAV-SURV-LoxP-Stop-LoxP-TK while infecting When the HepG2 cells of GCV processing, since AFP promoters drive the expression of Cre recombinases in hepatoma Hep G 2 cells, Cre's The transcription terminator Stop between two LoxP is sheared away in expression so that the SURV promoters of tumour-specific can be straight The expression of driving TK/GCV is connect, specific induced liver apoptosis of tumor cells is without damaging the thin of other internal any other types Born of the same parents, tentative confirmation transfection efficiently, method it is stable, operable, repeatable.
Further, the present invention is by establishing people's HCC subcutaneous implantation tumor nude mice models, and by two kinds of rAVV of tail vein injection Carrier system further studies it and plants the apoptosis rate, tumour inhibiting rate and model survival state, Yi Jizheng of tumor to experimental model HCC kinds The extent of damage of normal cell and tissue, further verifies the stability, validity and safety of the system, to realize translational medicine And the accurate suicide gene therapies of HCC provide theoretical foundation and applied basic research.
Second aspect finds that the present invention provides aforementioned systems in terms of preparing tumor based on the studies above Application.
The tumour is preferably liver cancer；The drug specificity liver cancer targeting HepG2 cells, therefore it is directed to liver cancer HepG2 Cell, which has, especially significantly promotees apoptic effects.
The third aspect, the present invention provides the drug containing above system, reagent and kits.
The characteristics of high specific liver cancer targeting cell of above system is utilized, and the efficient of above system is utilized The characteristics of regulating and controlling the expression of TK genes, and the product produced in the form of drug, reagent or kit, belong to the present invention Protection domain.
The present invention relates to raw material or reagent be ordinary commercial products, the operation being related to is unless otherwise specified This field routine operation.
Beneficial effects of the present invention at least that：
The present invention is compared with previous hepatocellular carcinoma suicide gene therapy, and eye is in clinical liver cancer treatment is ineffective and late period liver Method of therapy for cancer is deficient, and viral vectors, gene knockout system, the specificity promoter that originality integrates existing maturation drive induction Hepatoma cell apoptosis and animal pattern anticarcinogenic effect.
The present invention lacks the characteristic of the of self-replication capacity and thermophilic liver cell using rAAV, and design carries suicide gene system It is extensive that rAAV carriers take precautions against suicide gene therapy；It is precisely knocked out using liver cancer-specific promoter driving Cre recombinases, montage The Stop sequences being inserted into LoxP sequences adjust downstream TK gene expressions, control the expression of its high selectivity.
Suicide gene is placed in containing by transcription terminator by the present invention in the past tumor suicide gene theoretical foundation LoxP read window downstream, rely on AFP driving the montage of Cre recombinases can transcriptional expression, play anticarcinogenic effect, be suicide base Because of innovation progress of the theory in terms of specificity, control.
The Technical comparings such as the present invention and existing therapy of tumor and cell therapy, utilize molecular biology, cell biological Learn and technique for gene engineering respectively drive Cre-LoxP inscribe enzyme systems using AFP, SURV double-promoter, high efficiency, specificity, Controllability, safety instruct HCC to treat, and are the cross discipline research of typical cell biology and genetic engineering, are conversions The early-stage study of medicine, also for hepatocellular carcinoma individuation, precisely medical treatment and clinical liver cancer treatment provide theoretical foundation.
Description of the drawings
Fig. 1 is the Vector map described in embodiment 1, wherein A rAAV-Survivin-LoxP-Stop-LoxP-TK Carrier；B is rAAV-AFP-Cre carriers.
Fig. 2 is the expression that rAAV-AFP-Cre and rAAV-SURV-LoxP-Stop-LoxP-TK steerable systems can drive TK.
Fig. 3 is the expression that rAAV-AFP-Cre and rAAV-SURV-LoxP-Stop-LoxP-TK steerable systems can drive TK.
Fig. 4 is rAAV-AFP-Cre/rAAV-SURV-LoxP-Stop-LoxP-TK and control AOV032 infects Huh7 respectively After cell, the killing situation of TK/GCV is detected.
Specific implementation mode
With reference to embodiment the present invention will be further explained explanation.It will be appreciated that following embodiment provides Merely to playing the purpose of explanation, it is not used to limit the scope of the present invention.Those skilled in the art is not In the case of spirit of the invention and spirit, the present invention can be carry out various modifications and be replaced.
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Embodiment 1
The present embodiment is used to illustrate the structure of system of the present invention.
1, the structure of rAAV-Survivin-LoxP-Stop-LoxP-TK carriers
According to information synthesis TK gene orders (Gene ID disclosed in NCBI：7083), and according to shown in SEQ ID NO.1 Sequent synthesis SURV promoters (Survivin).
It is utilized respectively restriction enzyme XbaI and KpnI, starts SURV with restriction enzyme BamHI and HindIII Son and TK it is gene constructed enter plasmid CN1005 (purchased from and first biotechnology (Shanghai) limited liability company) in, obtain rAAV- Survivin-LoxP-Stop-LoxP-TK carriers, collection of illustrative plates are as shown in Figure 1A.
The digestion and connection method use this field conventional means and condition.
2, the structure of rAAV-AFP-Cre carriers
According to information synthesis AFP promoters (Gene ID disclosed in NCBI：And Cre gene orders (Gene ID 174)： 2777477) restriction enzyme XbaI and BamHI, are utilized respectively, starts AFP with restriction enzyme BamH1 and Hind3 Son and Cre it is gene constructed enter plasmid AOV-021 (purchased from and first biotechnology (Shanghai) limited liability company) in, obtain rAAV- AFP-Cre carriers, collection of illustrative plates are as shown in Figure 1B.
Embodiment 2
The present embodiment is based on Cre-LoxP recombination system principles, the Cre weights of the AFP promoters driving of rAAV for illustrating Group enzyme, performance fixed point controllability knockout Survivin promoters driving, which carries, is inserted into LoxP open reading frame in TK suicide genes STOP intrones can drive the expression of downstream suicide gene TK.
1, by the rAAV-AFP-Cre of different MOI (also referred to as H5312) and rAAV-SURV-LoxP-Stop-LoxP-TK ( Claim H5315) co-infection HpG2 cells and Huh7 cells, detect the expression of TK genes.Viral dose is as follows：1#MOI 1* 105, i.e. H5312 and H5315 viruses respectively use 0.5*105；2#MOI 2*105, i.e. H5312 and H5315 viruses respectively use 1*105；3# MOI 1*106, i.e. H5312 and H5315 viruses respectively use 5*105.The results are shown in Figure 2, rAAV-AFP-Cre and rAAV-SURV- LoxP-Stop-LoxP-TK steerable systems can drive the expression of TK, and dependence is presented with viral dose in expression quantity.
2, rAAV-AFP-Cre (H5312), rAAV-SURV-LoxP-Stop-LoxP-TK (H5315) are infected respectively Huh7 cells.
First group：Independent virus infection rAAV-AFP-Cre (H5312), MOI 1*106；Second group：Independent virus infection RAAV-SURV-LoxP-Stop-LoxP-TK (H5315), MOI 1*106；Third group：Co-infection virus rAAV-AFP-Cre (H5312) and rAAV-SURV-LoxP-Stop-LoxP-TK (H5315), MOI 1*106That is rAAV-AFP-Cre (H5312) and RAAV-SURV-LoxP-Stop-LoxP-TK (H5315) viruses respectively use 5*105；Detect the expression of TK genes.As a result as schemed Shown in 3, rAAV-AFP-Cre and rAAV-SURV-LoxP-Stop-LoxP-TK steerable systems can drive the expression of TK.
Embodiment 3
RAAV-AFP-Cre/rAAV-SURV-LoxP-Stop-LoxP-TK and control AOV032 (are purchased from and the biological skill of member Art (Shanghai) limited liability company) Huh7 cells are infected respectively, the killing situation of TK/GCV is detected, viral dose is MOI 5* 106, rAAV-AFP-Cre and rAAV-SURV-LoxP-Stop-LoxP-TK viruses respectively use 2.5*106.The results are shown in Figure 4, In Huh7 cells, after virus infection, under the action of GCV, cell survival rate is continuously decreased with the increase of GCV drug concentrations, Docs-effect dependence is presented, in GCV2ug/ml and 10ug/ml drug concentration effect groups, p value is less than 0.05, has statistics Learn meaning.
Although above the present invention is described in detail with a general description of the specific embodiments, On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Sequence table
<110>Zou Weilong
<120>Regulate and control suicide gene steerable system specific expressed in liver tumor cells
<141> 2018-03-02
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
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<212> DNA
<213>Artificial sequence (Artificial Sequence)
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ccatattggc caggctggtc tcgaactcct gaccttgtga tctgcccacc tcagcctccc 60
aaagtcctgg gattacaggc gtgagccacc gtgcccagcc tgacccctct gccctttcaa 120
aaactatgtt cgttctctca cagccttctc ttgtcatatt aagtccacac cgcaggccta 180
atttgtccag tgaatgctat gcaaatattt catgcacctg ctgatcgcag gaatgatatg 240
tacttggtac gcactgatcg tacctcgggg tgggagaaga gagggcaagg aagcaaagaa 300
tagccccctc ctttcctggt gcaccttcag atgtgccgat ggggcccagg ctcgctgcag 360
atggccccct tcccagagac aggggaggat cctccaccca ctccccagcc tccaggacca 420
tcctgactcc tgccttcagg cactcaagtt atgcgtctag acatgcggat atattcaagc 480
tgggcacagc acagcagccc caccccaggc agcttgaaat cagagctggg gtccaaaggg 540
accacacccc gagggactgt gtgggggtcg gggcacacag gccactgctt ccccccgtct 600
ttctcagcca ttcctgaagt cagcctcact ctgcttctca gggatttcaa atgtgcagag 660
actctggcac ttttgtagaa gccccttctg gtcctaactt acacctggat gctgtggggc 720
tgcagctgct gctcgggctc gggaggatgc tgggggcccg gtgcccatga gcttttgaag 780
ctcctggaac tcggttttga gggtgttcag gtccaggtgg acacctgggc tgtccttgtc 840
catgcatttg atgacattgt gtgcagaagt gaaaaggagt taggccgggc atgctggctt 900
atgcctgtaa tcccagcact ttgggaggct gaggcgggtg gatcacgagg tcaggagttc 960
aataccagcc tggccaagat ggtgaaaccc cgtctctact aaaaatacaa aaaaattagc 1020
cgggcatggt ggcgggcgca tgtaatccca gctactgggg gggctgaggc agagaattgc 1080
tggaacccag gagatggagg ttgcagtgag ccaagattgt gccactgcac tgcactccag 1140
cctggcgaca gagcaagact ctgtctcaaa aaaaaaaaaa aaaagtgaaa aggagttgtt 1200
cctttcctcc ctcctgaggg caggcaactg ctgcggttgc cagtggaggt ggtgcgtcct 1260
tggtctgtgc ctgggggcca ccccagcaga ggccatggtg gtgccagggc ccggttagcg 1320
agccaatcag caggacccag gggcgacctg ccaaagtcaa ctggatttga taactgcagc 1380
gaagttaagt ttcctgattt tgatgattgt gttgtggttg tgtaagagaa tgaagtattt 1440
cggggtagta tggtaatgcc ttcaacttac aaacggttca ggtaaaccac ccatatacat 1500
acatatacat gcatgtgata tatacacata cagggatgtg tgtgtgttca catatatgag 1560
gggagagaga ctaggggaga gaaagtaggt tggggagagg gagagagaaa ggaaaacagg 1620
agacagagag agagcgggga gtagagagag ggaaggggta agagagggag aggaggagag 1680
aaagggagga agaagcagag agtgaatgtt aaaggaaaca ggcaaaacat aaacagaaaa 1740
tctgggtgaa gggtatatga gtattctttg tactattctt gcaattatct tttatttaaa 1800
ttgacatcgg gccgggcgca gtggctcaca tctgtaatcc cagcactttg ggaggccgag 1860
gcaggcagat cacttgaggt caggagtttg agaccagcct ggcaaacatg gtgaaacccc 1920
atctctacta aaaatacaaa aattagcctg gtgtggtggt gcatgccttt aatctcagct 1980
actcgggagg ctgaggcagg agaatcgctt gaacccgtgg cggggaggag gttgcagtga 2040
gctgagatca tgccactgca ctccagcctg ggcgatagag cgagactcag tttcaaataa 2100
ataaataaac atcaaaataa aaagttactg tattaaagaa tgggggcggg gtgggagggg 2160
tggggagagg ttgcaaaaat aaataaataa ataaataaac cccaaaatga aaaagacagt 2220
ggaggcacca ggcctgcgtg gggctggagg gctaataagg ccaggcctct tatctctggc 2280
catagaacca gagaagtgag tggatgtgat gcccagctcc agaagtgact ccagaacacc 2340
ctgttccaaa gcagaggaca cactgatttt ttttttaata ggctgcagga cttactgttg 2400
gtgggacgcc ctgctttgcg aagggaaagg aggagtttgc cctgagcaca ggcccccacc 2460
ctccactggg ctttccccag ctcccttgtc ttcttatcac ggtagtggcc cagtccctgg 2520
cccctgactc cagaaggtgg ccctcctgga aacccaggtc gtgcagtcaa cgatgtactc 2580
gccgggacag cgatgtctgc tgcactccat ccctcccctg ttcatttgtc cttcatgccc 2640
gtctggagta gatgcttttt gcagaggtgg caccctgtaa agctctcctg tctgactttt 2700
tttttttttt tagactgagt tttgctcttg ttgcctaggc tggagtgcaa tggcacaatc 2760
tcagctcact gcaccctctg cctcccgggt tcaagcgatt ctcctgcctc agcctcccga 2820
gtagttggga ttacaggcat gcaccaccac gcccagctaa tttttgtatt tttagtagag 2880
acaaggtttc accgtgatgg ccaggctggt cttgaactcc aggactcaag tgatgctcct 2940
gcctaggcct ctcaaagtgt tgggattaca ggcgtgagcc actgcacccg gcctgcacgc 3000
gttctttgaa agcagtcgag ggggcgctag gtgtgggcag ggacgagctg gcgcggcgtc 3060
gctgggtgca ccgcgaccac gggcagagcc acgcggcggg aggactacaa ctcccggcac 3120
accccgcgcc gccccgcctc tactcccaga aggccgcggg gggtggaccg cctaagaggg 3180
cgtgcgctcc cgacatgccc cgcggcgcgc cattaaccgc cagatttgaa tcgcgggacc 3240
cgttggcaga ggtggcggcg gcggcat 3267

Claims (9)

1. a kind of steerable system that regulation and control suicide gene is specific expressed in liver tumor cells, which is characterized in that the system System includes：
(1) enzyme gene is recombinated by the Cre of AFP promoters driving expression；
(2) by the suicide gene of SURV promoters driving expression；It is inserted into one between the SURV promoters and the suicide gene Section LoxP-Stop-LoxP sequences, Stop are transcription terminator, and LoxP is the unique identification sequence of Cre recombinases.
2. system according to claim 1, which is characterized in that the suicide gene is TK genes.
3. system according to claim 1 or 2, which is characterized in that the system comprises two rAAV carriers：
First, containing AFP promoter elements specific expressed HCC, and the Cre of expression can be driven to recombinate by AFP promoters Enzyme gene；
Second, containing SURV promoter elements, and the LoxP-Stop-LoxP-TK/GCV of expression can be driven by SURV promoters Expression cassette, wherein LoxP-Stop-LoxP is located at the upstream of TK/GCV.
4. application of claims 1 to 3 any one of them system in terms of preparing tumor.
5. application according to claim 4, which is characterized in that the tumour is liver cancer.
6. application according to claim 5, which is characterized in that the drug specificity liver cancer targeting HepG2 cells.
7. the drug containing any one of claims 1 to 3 system.
8. the reagent containing any one of claims 1 to 3 system.
9. the kit containing any one of claims 1 to 3 system.
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