CN101671670B - Hepatocellular carcinoma targeting gene expression element AP and applications thereof - Google Patents
Hepatocellular carcinoma targeting gene expression element AP and applications thereof Download PDFInfo
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Abstract
The invention provides a hepatocellular carcinoma targeting gene expression element AP and applications thereof, and the gene expression element AP has a nucleotide sequence indicated in SEQ ID No.1. After the expression element AP is guided into Alpha-fetoprotein positive tumor cells, the artificial microRNA aiming at DNA polymerase can be expressed in the cell and the expression of the DNA polymerase in the cell can be effectively inhibited, the copy of the DNA in the cell is further affected; the cell cycle of is blocked finally, the multiplication of the AFP positive hepatoma cell is specially inhibited and the expression element AP can be applied to the preparation of the targeting gene curing medicine of AFP (alpha fetal protein) positive liver cancer.
Description
Technical field
The invention belongs to biotechnology; Relate to gene expression regulation; Specifically; Relate to a specific specificity can suppress archaeal dna polymerase in the liver cancer cell effectively to design, the preparation of the targeted gene expression element AP of alph-fetoprotein positive liver cancer and after specifically expressing, its transcription product processs in the liver cancer cell of alph-fetoprotein positive in cell expression, make that dna replication dna can't carry out because of archaeal dna polymerase lacks in the cell, thereby reach the effect of inhibition hepatoma cell proliferation.The present invention can be used for preparing target property therapy of tumor medicine.
Background technology
Malignant tumour is human at present mainly one of " killer ", the change of Along with people's mode of life and environment in recent years, and its sickness rate is in rising trend, has become first cause of death in many cities, is at the second place in the rural area.Liver cancer is one of modal very harmful malignant tumour in China, compares with other common cancers such as lung cancer, gastroenteric tumor, breast cancers, and the curative effect and the prognosis of usual manners such as present operation and chemotherapy are all undesirable.
The biotherapy that is called as the oncotherapy new model is just demonstrating more and more good prospects for application at present.In various tumor biotherapy means; Gene therapy is a main direction all the time, China development first formally gets into the genomic medicine of clinical use, promptly is its representative to the adenovirus injection liquid " Gendicine " cancer suppressor gene p53, that can express wild type p53 of undergoing mutation in many tumour cells in the world.Along with the develop rapidly of Medical Molecular Biology technology and knowledge, various advanced persons' molecular biology method all applies in the genetic treatment of tumor research.But because the incidence and development of tumour relates to numerous genes; And present research is mostly only to indivedual target genes; Therefore, press down the knurl effect though these researchs all demonstrate in the body of external tumor killing effect even tumor animal model of cell levels preferably, actual effect is unsatisfactory.Therefore, explore, attempt new therapy of tumor means and have crucial meaning.
Normal cell changes under the effect of various carcinogenic factors until last generation canceration gradually, has considerable gene to show the phenomenon of over-expresses, and especially some promote the overexpression of malignant phenotypes' such as cell growth, invasion and attack gene.To the gene of unconventionality expression in the tumour, people have designed the method for many targeting gene therapies, and the research that wherein promotes Expression of Related Genes such as tumor growth, invasion and attack with sealing is a quite popular field.In a large amount of in the past research; People utilize sense-rna (antisense RNA) that the tumour cell cance high-expression gene is sealed; But because the restriction that sense-rna itself exists; Though this type research has obtained bigger achievement at aspects such as suppressing growth of tumour cell, the migration of control cell invasion, the effect of its sealing genetic expression is still undesirable.
SiRNA (small interfering RNA, siRNA) be found to be that expression of gene provides a strong instrument in the human intervention cell.Utilize the siRNA of the double chain form of chemosynthesis; Or drive little hair fastener appearance RNA (the small hairpin RNA of the similar transcribe by III type promotor; ShRNA); Can pass through sequence-specific matching principle degraded target gene, thereby target gene expression is effectively suppressed, therefore also attempted being applied to gene therapy.But because the siRNA cost of chemosynthesis is too high, and the expression of shRNA is unfavorable for regulation and control, and these factors have limited this The Application of Technology widely.
Microrna (microRNA) is naturally occurring a kind of microRNA in the cell; It is a kind of important way that level is carried out expression regulation after genetic transcription; Target gene mRNA can degrade under the situation that sequence is mated fully; Also can under the situation of the non-coupling fully of sequence, suppress the translation of target gene; Be a research focus in the present life science, aspect the relevant microRNA of tumour big quantity research arranged also, and mostly concentrating on discovery and the Function Identification of expressing discrepant various natural microRNA between normal cell and the tumour cell.
Because natural microRNA is driven by II type promotor to transcribe; If so microRNA molecule like the artificial design class; Also should transcribe by the guiding of II type promotor; Can regulate and control and the characteristics of II type promotor are its transcripting starting functions, so the expression of the artificial mi RNA in its downstream should receive artificial control, and some nearest bibliographical informations of the mode of action of microRNA have confirmed this imagination.
Our this important step of dna replication dna of selecting in the cell cycle is a target spot in the present invention, designs the sequence of artificial microRNA, suppresses the expression of the key enzyme in the dna replication dna, duplicates through blocking dna the cell cycle is stopped.Because duplicating of DNA is the process of a complicacy and accurate regulation and control; Mainly by archaeal dna polymerase and etc. enzyme catalysis, be the semiconservative replication that template is carried out with original DNA chain; Therefore will effectively block the dna replication dna of cell to the inhibition of archaeal dna polymerase; Thereby the cell cycle is stopped because of DNA can't duplicate, and then suppress the propagation of cell.
In order to reach target property effect to tumour cell; To China occurred frequently, high, the curative effect of grade malignancy and all relatively poor this knurl kind of liver cancer of prognosis; Be utilized in do not express in the normal cell but in liver cancer cell the liver cancer cell specificity ALPHA-FP (alpha-fetoprotein of high expression level; AFP) characteristics, the expression regulation sequence of the AFP upstream region of gene of having recombinated is used for the expression of microRNA of artificial design.This gene expression element does not have any expression after importing normal cell; And after importing AFP male liver cancer cell; Can give expression to corresponding microRNA and block the dna replication dna in the liver cancer cell effectively, thereby reach the growth of target property inhibition liver cancer cell.
Based on thinking of the present invention; Can also carry out the design of artificial microRNA with other site of dna polymerase gene as target sequence; Or other important gene is carried out the blocking-up that cell DNA duplicates as target gene in duplicating with cell DNA, and then cell growth inhibiting and propagation.
Summary of the invention
The purpose of this invention is to provide a kind of hepatocellular carcinoma targeting gene expression element AP, be a kind of dna molecular, have the nucleotide sequence shown in the SEQ ID No:1.Wherein the 7th to the 827th is the-4113 to the-3292 at human a-fetoprotein (AFP) the genetic transcription starting point upper reaches, and a plurality of enhancement factor and HNF binding sites of transcribing are contained in this zone; The 831st to the 1012nd is the-182 to the-1 at human a-fetoprotein (AFP) the genetic transcription starting point upper reaches, and the basic promoter function of AFP is contained in this zone; The AFP positive cell expression regulation sequence of forming reorganization from the 1st to the 1012nd 1012bp fragment; The 1019th to the 1828th microRNA that the manual work to the human DNA polymerase gene that is 6 series connection connect designs; The 1829th to the 2177th is bovine growth hormone gene polyadenylic acid signaling zone, for genetic expression provides transcription termination signal.
Another object of the present invention provides the application of this Expression element AP in the hepatoma-targeting gene therapy medicament of preparation alph-fetoprotein positive.Described Expression element AP is after importing AFP male tumour cell; Can in cell, express the artificial microRNA to archaeal dna polymerase, and effectively suppress the expression of archaeal dna polymerase in the cell, and then have influence on duplicating of DNA in the cell; The final blocking-up cell cycle, suppress cell proliferation.
Described Expression element AP can make up the carrier of targeting gene therapy medicine, is used for the target gene therapy of AFP male liver cancer.Recombinant AFP expression regulation element among this Expression element AP can be used for regulating and control other expression of gene, can be used for other expression vector to the artificial microRNA sequence of human DNA polymerase reaches the expression purpose that inhibitory phase is answered the cell archaeal dna polymerase.
Utilize Expression element AP provided by the invention can prepare gene therapy medicament; Its characteristics be with the embryonal antigen AFP that liver cancer cell specificity is expressed be tumor cell specific expression regulation means, with tumour cell fast the required key protein archaeal dna polymerase of cell cycle dna replication dna in the stage of growth be that action target spot carries out expression inhibiting; Block dna replication dna in the cell effectively, suppress the propagation of AFP male liver cancer cell specifically.
Description of drawings
Fig. 1 is that recombinant AFP positive cell expression regulation sequence makes up PCR product electrophorogram.
Fig. 2 makes up PCR product electrophorogram for the artificial microRNA to archaeal dna polymerase.
Fig. 3 cuts the evaluation electrophorogram for the pDC312-AP enzyme.
Fig. 4 is that Western blot method detects the inhibition that AP expresses archaeal dna polymerase.
Fig. 5 is the mtt assay detected result.
Embodiment
The present invention combines accompanying drawing and specific embodiment to be described further.These embodiment only are used for explanation, but do not limit the present invention.
Embodiment 1:
The clone of recombinant AFP positive cell expression regulation sequence
At first obtain mRNA sequence (the GenBank accession number: NM_001134) of people AFP gene from the GenBank retrieval; And then the online genome compare of analysis method of warp obtains No. 4 chromosomal genome sequence (the GenBank accession number: NT_006216.14) that the people comprises the AFP gene from GenBank; And be benchmark with AFP gene mRNA sequence; With its 5 ' end as AFP genetic transcription starting point; Genome sequence design synthetic primer 1:5 '-AGA TCT CAG ATT GAA TTA TTTGCC TGT CA-3 ', primer 2 according to its upper reaches: 5 '-GGA TCC TAG GAA GTT TTC GCA ATA ATAC-3 ', primer 3:5 '-AGATCT GCC CCA AAG AGC TCT GTG T-3 ' and primer 4:5 '-GGATCC AAA TCA TGC TGA AAT TCT TTT ATA CTC-3 '; Utilize polymerase chain reaction technique (Polymerase Chain Reaction, PCR) genomic dna with human liver cancer cell SMMC7721 is a template, carries out nucleic acid amplification (primer 1 and primer 2, primer 3 and primer 4) with above-mentioned primer; Obtain amplified production A (821bp) and B (180bp); Referring to Fig. 1, wherein swimming lane 1,4 is 1kb DNA Ladder; Swimming lane 2 is amplified production A (821bp), and swimming lane 3 is amplified production B (180bp).
Amplified production A, B are cloned into pGEM T-easy carrier (Promega company, the U.S.) respectively with T-A clone's mode, through determined dna sequence and with its exactness of genome sequence (NT_006216.14) check verify.Then A is connected to the Bgl II site of the 5 ' end of B behind restriction enzyme BamH I and BglII double digestion, becomes the AFP positive cell expression regulation sequence of reorganization.
Embodiment 2:
Clone to the artificial microRNA of human DNA polymerase
Obtain mRNA sequence (the GenBank accession number: NM_016937) of human DNA polymerase gene from the GenBank retrieval; Utilize suitable site on this mRNA sequence of online software analysis; Confirm the action target spot of artificial microRNA, design synthetic primer 1:5 '-TGC TGT TGA CAG TGA GCGACC AAT TTA GAG TTC ATC ATT ATA GTG AAG CCA CAG ATG TA-3 ', primer 2: 5 '-TCC GAG GCA GTA GGC ACC CAA TTT AGA GTT CAT CAT TAT ACATCT GTG GCT TCA CTA TA-3 ', primer 3:5 '-AGA TCT GAT CCAAGAAGG TATATT GCT GTT GAC AGT GAG CG-3 ' and primer 4:5 '-GGA TCC ATC GTA GCCCTT GAA GTC CGA GGC AGT AGG CA-3 '.Earlier primer 1 and primer 2 are carried out overlapping extension PCR; Obtaining the amplified production C of 97bp, is template with this amplified production C again, carries out pcr amplification with primer 3 and primer 4; Obtain the amplified production D of 142bp; Referring to Fig. 2, wherein swimming lane 1 is DL2000DNALadder, and swimming lane 2 is amplified production D (142bp).
Mode with the T-A clone is cloned into pGEM T-easy carrier, through its exactness of dna sequencing checking.This promptly is directed against the artificial microRNA sequence of human DNA polymerase.In order to strengthen its action effect, the BamH I site with reclaiming this manual work microRNA sequence and insert identical carrier again behind restriction enzyme BamH I and the Bgl II double digestion obtains 6 artificial microRNA sequences that are connected in series.
Embodiment 3:
The structure of hepatocellular carcinoma targeting gene expression element AP and the preparation of adenovirus carrier
At first make up the pDC312-BGHpA carrier: with adenovirus shuttle plasmid pDC312 carrier (Microbix company; The U.S.) open with restriction enzyme Xba I single endonuclease digestion; Mend flatly with the Klenow enzyme, use restriction enzyme HindIII single endonuclease digestion again, remove Xba I to the part between the HindIII (from HindIII, Sac I, Ecl136II, Acc I, Sal I to Xba I); Recovery part one end is a flush end, and the other end is the HindIII sticky end.PcDNA3.1 (+) carrier (Invitrogen company; The U.S.) with restriction enzyme Pvu II and HindIII double digestion; Recovery comprises the fragment of HindIII, Asp718, Kpn I, BamH I, BstXI, EcoR I, EcoRV, BstXI, Not I, Xho I, Xba I, Dra II, Apa I and Pme I MCS and ox growth factor gene polyadenylic acid signal (BGHpA); One end is the HindIII sticky end, and the other end is the flush end of PvuII.Above-mentioned two fragments are coupled together, the pDC312-BGHpA carrier, can on MCS, insert promotor and goal gene coding region.
Again the recombinant AFP positive cell expression regulation sequence among the embodiment 1 is reclaimed after with restriction enzyme BamH I and Bgl II double digestion from carrier; And the BamH I site of inserting pDC312-BGHpA, obtain having the adenovirus shuttle plasmid of recombinant AFP positive cell expression regulation sequence and BGHpA.At last with 6 reclaiming after from carrier of being connected in series with restriction enzyme BamH I and Bgl II double digestion to the artificial microRNA sequences of human DNA polymerase; Insert the BamH I site of this shuttle plasmid; Obtain having the adenovirus shuttle plasmid of complete hepatoma targeting character Expression element AP; Enzyme is cut qualification result and is conformed to fully with expection, and referring to Fig. 3, wherein swimming lane 1 is 1Kb Plus DNA Marker; Swimming lane 2 is cut through BamH I, Bgl I, EcoR I enzyme for pDC312-AP.
With above-mentioned adenovirus shuttle plasmid and adenovirus skeleton plasmid pBHGlox (delta) E1 that has complete hepatoma targeting character Expression element AP; 3cre (Microbix company; The U.S.) cotransfection adenovirus packaging cell 293 cells obtain having the adenovirus carrier of Expression element AP.
Embodiment 4:
Gene expression element AP is to the target property inhibition of AFP masculine liver cancer cell
A, Western blot method detect the inhibition that AP expresses archaeal dna polymerase
The AFP male liver cancer cell Hep3B cell of taking the logarithm vegetative period is by 3 * 10
5The density of cells/well is inoculated in 6 orifice plates, cultivates to make it adherent and reach 80% degree of converging in 24 hours.With the above-mentioned adenovirus that has Expression element AP of substratum dilution, (multiplicity of infection is 100pfu/cell and 10pfu/cell cells infected MOI), termination effect behind the 48hr with infection multiplicity; Extract total protein of cell, carry out SDS-polyacrylamide gel electrophoresis (10% separation gel, 5% concentrates glue); Then albumen is gone to pvdf membrane, sealing back and 1: the 1000 anti-archaeal dna polymerase antibody of dilution (Santa Cruz Biotechnology, Inc.; The U.S., DNApol (G-16): sc-5921) combine, behind the room temperature 2hr; Wash film 3 times with TBST, combine the anti-sheep IgG of HRP mark antibody (Bioisystech Co., Ltd of China fir Golden Bridge in Beijing) again, wash film once more; Use chemical luminescence reagent kit (Roche company, the U.S.) to develop X-ray sheet exposure then.With GAPDH is internal reference, with the same combination of GAPDH antibody (go up the Haikang and become Bioisystech Co., Ltd), development and the exposure of HRP mark.The band green fluorescent protein is established in experiment, and (green fluorescent protein, adenovirus infection GFP) compares with the cell of no adenovirus infection.The result shows that referring to Fig. 4 the archaeal dna polymerase expression among the AFP masculine liver cancer cell Hep3B that infects the adenovirus that has Expression element AP obviously is suppressed.G among the figure: archaeal dna polymerase was expressed after band AP adenovirus MOI was respectively 100 and 10 infection Hep3B liver cancer cells; GFP: archaeal dna polymerase was expressed after band GFP adenovirus MOI was respectively 100 and 10 infection Hep3B liver cancer cells; N: do not infect in the Hep3B liver cancer cell of adenovirus archaeal dna polymerase and express.
B, in-vitro cell growth inhibition test (mtt assay)
The liver cancer cell Hep3B that takes the logarithm vegetative period, HepG2, SMMC7721 and breast cancer cell Bcap37 (non-liver cancer cell) are by 2 * 10
3The density of cells/well is inoculated in 96 orifice plates, cultivates to make it adherent in 24 hours.With the adenovirus of band AP is 200pfu/cell, 100pfu/cell, 50pfu/cell, 10pfu/cell, 1pfu/cell and 0pfu/cell cells infected with MOI.After 4 days, every hole adds MTT solution (5mg/ml) 20ul at virus infection, and 37 ℃ are continued to cultivate 4hr, end to cultivate.Discard culture supernatant liquid, every hole adds DMSO 200ul, and vibration 10min fully dissolves crystallisate, on enzyme-linked immunosorbent assay instrument, measures each hole 570nm wavelength OD value.Cell survival rate=(not effect group of virus function group OD value/virus OD value) * 100%.Set up 5 multiple holes, repeat 3 times for every group.The result is referring to Fig. 5, shows that the adenovirus of the band AP of different MOI all has inhibition in various degree to the growth of different liver cancer cell Hep3B, HepG2 and SMMC7721, and Bcap37 does not then have obvious inhibition to breast cancer cell (non-liver cancer cell).
The sequence that the present invention relates to
< 120>a kind of hepatocellular carcinoma targeting gene expression element AP and application thereof
<160>9
<210>1
<211>2177
<212>DNA
< 213>artificial sequence
<220>
< 223>alph-fetoprotein positive liver cancer cell specificity Expression element AP sequence
<400>1
DNASIS
SEQ
agatctcaga?ttgaattatt?tgcctgtcat?acagctaata?attgaccata?agacaattag?60
atttaaatta?gttttgaatc?tttctaatac?caaagttcag?tttactgttc?catgttgctt?120
ctgagtggct?tcacagactt?atgaaaaagt?aaacggaatc?agaattacat?caatgcaaaa?180
gcattgctgt?gaactctgta?cttaggacta?aactttgagc?aataacacat?atagattgag?240
gattgtttgc?tgttagtata?caaactctgg?ttcaaagctc?ctctttattg?cttgtcttgg?300
aaaatttgct?gttcttcatg?gtttctcttt?tcactgctat?ctatttttct?caaccactca?360
catggctaca?ataactgtct?gcaagcttat?gattcccaaa?tatctatctc?tagcctcaat?420
cttgttccag?aagataaaaa?gtagtattca?aatgcacatc?aacgtctcca?cttggagggc?480
ttaaagacgt?ttcaacatac?aaaccgggga?gttttgcctg?gaatgtttcc?taaaatgtgt?540
cctgtagcac?atagggtcct?cttgttcctt?aaaatctaat?tacttttagc?ccagtgctca?600
tcccacctat?ggggagatga?gagtgaaaag?ggagcctgat?taataattac?actaagtcaa?660
taggcataga?gccaggactg?tttgggtaaa?ctggtcactt?tatcttaaac?taaatatatc?720
caaaactgaa?catgtactta?gttactaagt?ctttgacttt?atctcattca?taccactcag?780
ctttatccag?gccacttatt?tgacagtatt?attgcgaaaa?cttcctagga?tctgccccaa?840
agagctctgt?gtccttgaac?ataaaataca?aataaccgct?atgctgttaa?ttattggcaa?900
atgtcccatt?ttcaacctaa?ggaaatacca?taaagtaaca?gatataccaa?caaaaggtta?960
ctagttaaca?ggcattgcct?gaaaagagta?taaaagaatt?tcagcatgat?ttggatctga?1020
tccaagaagg?tatattgctg?ttgacagtga?gcgaccaatt?tagagttcat?cattatagtg?1080
aagccacaga?tgtataatga?tgaactctaa?attgggtgcc?tactgcctcg?gacttcaagg?1140
gctacgatgg?atctgatcca?agaaggtata?ttgctgttga?cagtgagcga?ccaatttaga?1200
gttcatcatt?atagtgaagc?cacagatgta?taatgatgaa?ctctaaattg?ggtgcctact?1260
gcctcggact?tcaagggcta?cgatggatct?gatccaagaa?ggtatattgc?tgttgacagt?1320
gagcgaccaa?tttagagttc?atcattatag?tgaagccaca?gatgtataat?gatgaactct?1380
aaattgggtg?cctactgcct?cggacttcaa?gggctacgat?ggatctgatc?caagaaggta?1440
tattgctgtt?gacagtgagc?gaccaattta?gagttcatca?ttatagtgaa?gccacagatg?1500
tataatgatg?aactctaaat?tgggtgccta?ctgcctcgga?cttcaagggc?tacgatggat?1560
ctgatccaag?aaggtatatt?gctgttgaca?gtgagcgacc?aatttagagt?tcatcattat?1620
agtgaagcca?cagatgtata?atgatgaact?ctaaattggg?tgcctactgc?ctcggacttc?1680
aagggctacg?atggatctga?tccaagaagg?tatattgctg?ttgacagtga?gcgaccaatt?1740
tagagttcat?cattatagtg?aagccacaga?tgtataatga?tgaactctaa?attgggtgcc?1800
tactgcctcg?gacttcaagg?gctacgatgg?atccactagt?ccagtgtggt?ggaattctgc?1860
agatatccag?cacagtggcg?gccgctcgag?tctagagggc?ccgtttaaac?ccgctgatca?1920
gcctcgactg?tgccttctag?ttgccagcca?tctgttgttt?gcccctcccc?cgtgccttcc?1980
ttgaccctgg?aaggtgccac?tcccactgtc?ctttcctaat?aaaatgagga?aattgcatcg?2040
cattgtctga?gtaggtgtca?ttctattctg?gggggtgggg?tggggcagga?cagcaagggg?2100
gaggattggg?aagacaatag?caggcatgct?ggggatgcgg?tgggctctat?ggcttctgag?2160
gcggaaagaa?ccagctg?2177
<210>2
<211>29
<212>DNA
< 213>artificial sequence
<220>
< 223>the AFP upper reaches expression regulation sequence amplification primers 1 that designs according to the AFP genome sequence
<400>2
agatctcaga?ttgaattatt?tgcctgtca?29
<210>3
<211>28
<212>DNA
< 213>artificial sequence
<220>
< 223>the AFP upper reaches expression regulation sequence amplification primers 2 that designs according to the AFP genome sequence
<400>3
ggatcctagg?aagttttcgc?aataatac?28
<210>4
<211>25
<212>DNA
< 213>artificial sequence
<220>
< 223>the AFP upper reaches expression regulation sequence amplification primers 3 that designs according to the AFP genome sequence
<400>4
agatctgccc?caaagagctc?tgtgt?25
<210>5
<211>33
<212>DNA
< 213>artificial sequence
<220>
< 223>the AFP upper reaches expression regulation sequence amplification primers 4 that designs according to the AFP genome sequence
<400>5
ggatccaaat?catgctgaaa?ttcttttata?ctc?33
<210>6
<211>59
<212>DNA
< 213>artificial sequence
<220>
< 223>to the artificial microRNA amplification primers 1 of archaeal dna polymerase a gene design
<400>6
tgctgttgac?agtgagcgac?caatttagag?ttcatcatta?tagtgaagcc?acagatgta?59
<210>7
<211>59
<212>DNA
< 213>artificial sequence
<220>
< 223>to the artificial microRNA amplification primers 2 of archaeal dna polymerase a gene design
<400>7
tccgaggcag?taggcaccca?atttagagtt?catcattata?catctgtggc?ttcactata?59
<210>8
<211>41
<212>DNA
< 213>artificial sequence
<220>
< 223>to the artificial microRNA amplification primers 3 of archaeal dna polymerase a gene design
<400>8
agatctgatc?caagaaggta?tattgctgtt?gacagtgagc?g?41
<210>9
<211>38
<212>DNA
< 213>artificial sequence
<220>
< 223>to the artificial microRNA amplification primers 4 of archaeal dna polymerase a gene design
<400>9
ggatccatcg?tagcccttga?agtccgaggc?agtaggca?38
Claims (2)
1. hepatocellular carcinoma targeting gene expression element AP; Its nucleotide sequence is shown in SEQ ID No:1; Wherein the 7th to the 827th is the-4113 to the-3292 at the human a-fetoprotein gene transcripting start point upper reaches, and a plurality of enhancement factor and HNF binding sites of transcribing are contained in this zone; The 831st to the 1012nd is the-182 to the-1 at the human a-fetoprotein gene transcripting start point upper reaches, and the basic promoter function of ALPHA-FP is contained in this zone; The alph-fetoprotein positive cell expressing regulating and controlling sequence of forming reorganization from the 1st to the 1012nd 1012bp fragment; The 1019th to the 1828th is 6 microRNA that the manual work to the human DNA polymerase gene that is connected in series designs; The 1829th to the 2177th is bovine growth hormone gene polyadenylic acid signaling zone, for genetic expression provides transcription termination signal.
2. the application of a kind of hepatocellular carcinoma targeting gene expression element AP according to claim 1 in the target gene therapy medicine of preparation alph-fetoprotein positive liver cancer.
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| CN1410546A (en) * | 2002-11-12 | 2003-04-16 | 浙江大学医学院附属第二医院 | Real time fluorescence quantitative RT-PCR detection human alpha-fetoprotein reagent box |
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| CN1410546A (en) * | 2002-11-12 | 2003-04-16 | 浙江大学医学院附属第二医院 | Real time fluorescence quantitative RT-PCR detection human alpha-fetoprotein reagent box |
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