CN103088029B - Double-enhancer alpha fetoprotein (AFP) recombination promoter and application thereof - Google Patents

Abstract

The invention provides a double-enhancer alpha fetoprotein (AFP) recombination promoter. The double-enhancer alpha fetoprotein recombination promoter is a gene expression regulating element and is a DNA (deoxyribonucleic acid) molecule, and a nucleotide sequence of the double-enhancer alpha fetoprotein recombination promoter is shown as SEQIDNo:1. The gene expression regulating element is a double-enhancer recombinant human fetoprotein promoter provided with a CMV (cytomegalovirus) early transcription enhancer and a fetoprotein enhancer and can specifically efficiently promote expression of a target gene in a hepatoma carcinoma cell. Promotion efficiency of a fetoprotein gene promoter driven by two enhancers is greatly improved compared with the effect of a single enhancer, and a foreign gene can be expressed in quantity in the hepatoma carcinoma cell after a foreign gene expression carrier is constructed and introduced into the hepatoma carcinoma cell. The double-enhancer alpha fetoprotein recombination promoter provided by the invention can be applied to construction of the foreign gene expression carrier and can also be applied to preparation of a targeted gene therapeutic drug used for treating live cancer with positive AFP.

Description

A kind of two enhanser alpha-fetoprotein recombinant promoter and application thereof

Technical field

The invention belongs to biotechnology, relate to gene expression regulation, specifically, relate to the structure of a kind of pair of enhanser alpha-fetoprotein recombinant promoter, this recombinant promoter can in the liver cancer cell of alph-fetoprotein positive high expression goal gene.The present invention is used in expression alien gene and preparation targeting gene therapy of liver cancer medicine in liver cancer cell.

Background technology

In recent years along with the change of people life style and environment, the sickness rate of Several Kinds of Malignancy is all in rising trend, and mortality ratio also remains high, and malignant tumour has become first cause of death in many cities, is at the second place in rural area.Outside the means such as operation, chemicotherapy of routine, need badly and will develop new treatment means to improve result for the treatment of, reduce mortality ratio.

Tumor biotherapy is a kind of new tumor treatment model, wherein gene therapy is a kind of mode of carrying out comparatively early research, China have approved first Adenovirus " Gendicine " formally entering the genomic medicine expression wild type p53 of Clinical practice and oncolytic virus medicine restructuring human adenovirus type 5 (H101) injection liquid " An Kerui " got the Green Light subsequently in the world, external also listing has this medicine of retroviral gene therapy medicine Rexin G(carrying Cyclin G target tumor and metastasis for the treatment of solid tumors of Chemoresistance to be treatment osteosarcoma in the U.S. at present, " orphan's medicine (the orphan drug) " of soft tissue sarcoma and carcinoma of the pancreas.

Target gene therapy is the most important aspect of current therapy of tumor research.Target gene therapy relates to multiple link, enter tumour cell, foreign gene targeting earth's surface in tumour cell with comprising carrier targeting reach, and the foreign gene targeting ground of expressing suppresses, killing tumor cell, wherein foreign gene targeting earth's surface in tumour cell reaches is a study hotspot.

Normal cell changes until finally there is canceration gradually under the effect of various carcinogenic factor, has and much not to express or the gene of low expression level all shows the phenomenon of overexpression in healthy tissues.Why these genes can have abnormal high expression in tumour cell, are because the promotor of this gene is activated and has abundant transcription factor, the activating transcription factor relevant to this promotor in cell in tumour cell.Therefore the promotor of this genoid usually by as tumor-specific promoters (tumor-specific promoter) for regulating and controlling the targeted expression of foreign gene in tumour cell in gene therapy.

Liver cancer is one of modal very harmful malignant tumour in China, and M & M all correspondingly accounts for global half.Compared with other common cancer, hepatocarcinoma early diagnosis is more difficult, and grade malignancy is high, and curative effect and the prognosis of the usual manners such as current operation and interventional therapy are all undesirable, liver transplantation expense not only high and but also still can recur, therefore gene therapy of liver cancer research has very important significance.

Alpha-fetoprotein (alpha fetoprotein, AFP) be the tumor markers of liver cancer, unconventionality expression in liver cancer cell and not expressing in normal liver cell, therefore the startup of a-fetoprotein gene is a desirable liver cancer-specific promotor, is all used as the expression regulation element of exogenous therapeutic gene in a lot of hepatoma-targeting gene therapy.Such as drive specific expressed [7] of suicide gene in liver cancer cell and design liver cancer-specific oncolytic virus [8] etc.The gene promoters such as hTERT, Survivin of the wide spectrum conventional with some other experimental tumor gene therapy research are compared, expression due to alpha-fetoprotein is different from the genes such as hTERT, Survivin also positive expression in tumour cell and many normal tissue stem cell, alpha-fetoprotein is only limitted to embryonic tissue or adult liver cancer (comprising a small amount of cancer of the stomach), therefore the specificity based on the gene therapy of afp promoter is better, less to the side effect of other healthy tissues.

But afp promoter also exists and comprises carcinomebryonic antigen (carcinoembryonic antigen, CEA) in the problem that interior some other tissue-specific promoter is the same, namely expression efficiency is lower comparatively speaking, and to have height to have low due to the expression level of alpha-fetoprotein in different liver cancer cells, the transcripting starting efficiency of afp promoter in the liver cancer cell that these are different is differed, thus the expression efficiency affecting the goal gene of its regulation and control and the effect played, the effect of the targeting gene therapy of liver cancer based on afp promoter is finally caused to differ, especially poor to the gene therapy effect of the lower liver cancer cell of some alpha-fetoprotein expression levels.Therefore, from the present Research of current targeting gene therapy of liver cancer, the effect of targeting gene therapy of liver cancer be improved, the transcripting starting efficiency of specificity promoter alpha-fetoprotein must be improved.

Promotor is the basic controlling element of genetic expression, and enhanser (enhancer) then can significantly improve the expression regulation element of the starting efficiency of promotor.5 ' flank of the a-fetoprotein gene promotor of people has an enhanser region, has multiple liver cell idiosyncratic transcription factor binding site, can improve the efficiency of afp promoter greatly specifically.

A nearest functional study based on the early transcription enhanser of cytomegalovirus (cytomegalovirus, CMV) shows, this enhanser greatly can improve the transcripting starting efficiency of a lot of gene promoter.

In order to improve the transcripting starting efficiency of a-fetoprotein gene promotor, at present in the research work utilizing a-fetoprotein gene promotor to carry out, have and adopt some enhansers to improve the report of its starting efficiency, but employing being all single enhanser.

Summary of the invention

The object of this invention is to provide a kind of gene expression regulation element, i.e. a kind of two enhanser alpha-fetoprotein recombinant promoter, for a kind of DNA molecular, its nucleotide sequence is as shown in SEQ ID No:1, wherein the 7th to the 537th is CMV early transcription enhancer sequence, 544th is human a-fetoprotein gene enhancer sequence to the 1364th, and the 1370th is human a-fetoprotein gene promoter sequence to the 1549th.

Another object of the present invention is to provide a kind of two enhanser alpha-fetoprotein recombinant promoter (gene expression regulation element) and is building the application in exogenous gene expression carrier.This gene expression regulation element for build exogenous gene expression carrier and after importing liver cancer cell can in liver cancer cell huge amount ground expression alien gene.

Another object of the present invention is to provide a kind of two enhanser alpha-fetoprotein recombinant promoter (gene expression regulation element) application in the target gene therapy medicine of the liver cancer for the preparation of the AFP positive.Build when gene expression regulation element provided by the invention is used for gene therapy vector and then when preparing gene therapy medicament, can be used for the target gene therapy of the liver cancer of the AFP positive.

We are according to the character feature of different enhanser and principle of work, devise the thinking improving a-fetoprotein gene promotor with two kinds of enhansers, one is the enhanser adopting a-fetoprotein gene itself, this enhanser is positioned at a-fetoprotein gene upstream, containing multiple hepatocyte neclear factor binding site, there is liver cell specificity; Another adopts CMV early transcription enhanser because CMV promoter is the promotor that in current mammalian cell, starting efficiency is the highest, its enhancers upstream to transcribe enhancement very strong.

Therefore, we construct a kind of afp promoter of the artificial recombination with CMV early transcription enhanser and alpha-fetoprotein autospecific enhanser in the present invention, specificity can start the expression of goal gene expeditiously in liver cancer cell.The starting efficiency of the a-fetoprotein gene promotor that two enhanser drives can be greatly improved than the effect of single enhanser.

The present invention compared with the existing technology has the following advantages and effect:

Utilize gene expression element provided by the invention can prepare the gene therapy medicament of the liver cancer of the AFP positive, its feature is that the embryonal antigen alpha-fetoprotein expressed with liver cancer cell specificity is tumor cell specific expression regulation means, improves the efficiency of afp promoter with CMV early transcription enhanser and a-fetoprotein gene self enhanser two enhansers simultaneously.The present invention may be used for the research and development of hepatocarcinoma gene targeting gene therapy medicine, also may be used for the foreign recombinant proteins that some needs of great expression synthesize in liver cancer cell in liver cancer cell.

Accompanying drawing explanation

Fig. 1 is that two enhanser alpha-fetoprotein recombinant promoter builds schematic diagram.

The clone of Fig. 2 CMV early transcription enhanser.

The clone of Fig. 3 alpha-fetoprotein enhanser.

The clone of Fig. 4 afp promoter.

The clone of Fig. 5 alpha-fetoprotein enhanser-promotor.

The qualification of the Luciferase Expression Vectors plasmid that Fig. 6 alpha-fetoprotein recombinant promoter drives.

In Fig. 7 liver cancer cell, two enhanser afp promoter is to the raising effect of luciferase gene expression.

Embodiment

The present invention is described further with specific embodiment by reference to the accompanying drawings.These embodiments only for illustration of, but do not limit the present invention.

Embodiment 1: the structure of two enhanser alpha-fetoprotein recombinant promoter

We have cloned CMV early transcription enhanser, AFP enhanser and AFP promotor respectively according to the schematic diagram shown in Fig. 1, then construct two enhanser alpha-fetoprotein recombinant promoter.

1, the clone of CMV early transcription enhanser

With mammalian expression vector pcDNA3.1 (+) plasmid of Invitrogen company for template, adopt polymerase chain reaction (polymerase chain reaction, PCR) the CMV early transcription enhanser of sequence as shown in SEQ No:2 in region, 241-771 position on method amplification plasmid, 5 ' primer is: 5 '- aGA TCT(SEQ No:3, italic is introduce for cloning further to GAC ATT GAT TAT TGA CTA GT-3 ' bgliI site), 3 ' primer is: 5 '- gGA TCC(SEQ No:4, italic is introduce for cloning further to ATG GGG CGG AGT TGT TAC GA-3 ' bamHi site), PCR condition is: 95 DEG C, 5 min; 95 DEG C, 30 s, 45 DEG C, 45 s, 72 DEG C, 45 s, totally 30 circulations; Last continues reaction 10 min in 72 DEG C after having circulated.1.2% agarose gel electrophoresis is separated PCR primer, the band of 543 bp is cut under ultraviolet lamp, reclaim test kit with the Qiaquick gel of Qiagen company and operate this PCR primer of recovery to specifications, and to specifications recovery product is connected with the pMD19-T carrier of Takara company, spend the night in 16 DEG C of connections, transform competent E. coli DH5 , coat the LB agar plate containing penbritin, picking list bacterium colony shaking culture in LB liquid medium is spent the night, and collects thalline, extracts plasmid, bgliI and bamh I double digestion is identified, has plasmid (Fig. 2: wherein M:1kb plus DNA molecular weight marker, 1:CMV early transcription enhanser amplified production, the 2:pMD19-T/CMVenhancer plasmid use of positive Insert Fragment bgliI and bamhI double digestion) carry out DNA sequencing, the correct person of sequence is the plasmid pMD19-T/CMVenhancer of CMV early transcription enhanser.

2, the clone of people AFP genetic enhancer-promotor

With reference to related documents, the genome sequence (GenBank accession number: NT_006216.14) that people comprises the rice chromosome of AFP gene is obtained from GenBank, this sequence is determined the AFP enhanser (SEQ No:5) of 821bp and AFP promotor (SEQ No:6) region of 180bp, design primer respectively, AFP enhanser upstream primer is: 5'- aGA TCTcAG ATT GAA TTA TTT GCC TGT CA-3'(SEQ No:7, italic is introduce for cloning further bgliI site), downstream primer is: 5'- gGA TCCtAG GAA GTT TTC GCA ATA ATA C-3'(SEQ No:8, italic is introduce for cloning further bamh I site), amplified production overall length should be 833 bp; AFP promotor upstream primer is: 5'- aGA TCTgCC CCA AAG AGC TCT GTG T-3'(SEQ No:9, italic is introduce for cloning further bgliI site), downstream primer is: 5'- gGA TCCaAA TCA TGC TGA AAT TCT TTT ATA CTC-3'(SEQ No:10, italic is introduce for cloning further bamh I site), amplified production overall length should be 191 bp.Human liver cancer cell HepG2 is incubated in the RPMI1640 nutrient solution containing 10% foetal calf serum, 5% CO ₂, 37 DEG C.Collect the vigorous HepG2 cell of growth, extract genomic dna also quantitatively by reference.With this genomic dna for template, adopt PCR method to increase respectively the enhanser of people AFP gene and promotor, PCR condition is: 95 DEG C of sex change 5 min, then carries out the amplifications of 30 circulations: 95 DEG C, 30 s, 58 DEG C, 30 s, 72 DEG C of 30 s.Last continues reaction 10 min in 72 DEG C after having circulated.Obtain AFP enhanser fragment (833 bp) and AFP promoter fragment (191 bp), see M:DL2000 DNA molecular weight marker in Fig. 3,4(Fig. 3,1:AFP enhanser amplified production, 2:pGEM-T Easy/AFPenhancer plasmid is used bgliI and bamhI double digestion.M:DL2000 DNA molecular weight marker in Fig. 4, the sub-amplified production of 1:AFP promotor, 2:pGEM-T Easy/AFPpromoter plasmid is used bgliI and bamhI double digestion).Sepharose with 1.5% carries out electrophoretic separation PCR primer, the same gel reclaims trial-production box and operates the object PCR primer reclaiming expection size to specifications, and connect system to clone respective segments with pGEM-T Easy carrier according to operation instruction, spend the night in 16 DEG C of connections, transform competent E. coli DH5 , coat the LB agar plate containing penbritin, extract transformant plasmid DNA, use bgliI He bamh I double digestion detects the Insert Fragment with or without corresponding size, delivers to the order-checking of Invitrogen company, sequencing result and its exactness of genome sequence (NT_006216.14) check verify by there being the plasmid DNA of expection size Insert Fragment.The correct person of sequence respectively called after pGEM-T Easy/AFPenhancer(containing AFP enhanser) and pGEM-T Easy/AFPpromoter(contain AFP promotor).With bgliI He bamh I double digestion pGEM-T Easy/AFPenhancer, the same electrophoretic separation also reclaims AFP enhanser fragment, inserts before the AFP promotor of pGEM-T Easy/AFPpromoter plasmid bgliI site, cuts qualification through enzyme and obtains plasmid pGEM T-Easy/AFPenhancer-promoter, its bgliI He bamthe size being acquisition between H I is the recombinant AFP genetic enhancer-promotor (AFPenhancer-promoter) of 1018bp, see Fig. 5, (M:DL2000 DNA molecular weight marker in figure, 1:pGEM-T Easy/AFPenhancer-promoter plasmid is used bgliI and bamhI double digestion).

3, the structure of two enhanser AFP recombinant promoter

With bgliI He bamh I double digestion has the plasmid pMD19-T/CMVenhancer of CMV early transcription enhanser, the same electrophoretic separation also reclaims the CMV early transcription enhanser of 543bp, inserts pGEM T-Easy/AFPenhancer-promoter plasmid obtained above bgliI site, cuts qualification through enzyme, obtains plasmid pGEM-T Easy/CMVenhancer-AFPenhancer-promoter, its bgliI He bamtwo enhanser AFP recombinant promoters (SEQ No:1, CMVenhancer-AFPenhancer-promoter) of the 1555bp that between H I, namely sequence obtains.

Embodiment 2: the structure of the Luciferase Expression Vectors that alpha-fetoprotein recombinant promoter drives

First use bgliI He bamh I is double digestion plasmid pGEM-T Easy/AFPenhancer-promoter and pGEM-T Easy/CMVenhancer-AFPenhancer-promoter respectively, electrophoretic separation also reclaims the CMVenhancer-AFPenhancer-promoter fragment of AFPenhancer-promoter and 1567bp of 1018bp respectively, inserts pGL4.10's separately bgliI site, uses bgliI He hind III double digestion qualification direction of insertion, 4223 bp should be there are in the plasmid of correct insertion AFPenhancer-promoter, 653 bp and 384 bp tri-bands, 4223 bp should be there are in the plasmid of correct insertion CMVenhancer-AFPenhancer-promoter, 927 bp and 653 bp tri-bands, result (Fig. 6, wherein M:1kb plus DNA molecular weight marker, use by 1:pGL4.10/AFP plasmid bgliI and hindIII double digestion, 2:pGL4.10/CMVenhancer-AFP plasmid is used bgliI and hindIII double digestion) show, two carriers all correctly build, and therefore obtain the luciferase reporter plasmid pGL4.10/AFP with AFPenhancer-promoter and the luciferase reporter plasmid pGL4.10/CMVenahncer-AFP with CMVenhancer-AFPenhancer-promoter.

Embodiment 3: two enhanser AFP recombinant promoter is to the raising of luciferase expression

Human liver cancer cell Hep3B, HepG2 and SMMC7721 are incubated in the RPMI RPMI-1640 containing 10% foetal calf serum, 5% CO ₂, 37 DEG C.Get eugonic above-mentioned cell and be adjusted to 4 × 10 ⁵cell/mL, is inoculated in 24 orifice plates, 2 × 10 ⁵μ L/ hole, cell/500, overnight incubation.Extraction, purifying two kinds of luciferase reporter plasmid pGL4.10/AFP and pGL4.10/CMVenahncer-AFP, be dissolved in sterilized water, ultraviolet spectrophotometer measure 260 nm place absorbance values and carries out quantitatively.Respectively get the luciferase reporter vector pGL4.10/AFP of the band a-fetoprotein gene enhanser-promotor of the above-mentioned purifying of 1 μ g respectively, the pGL4.10 plasmid of pGL4.10/CMVenahncer-AFP and not tape starting, mix with the internal reference plasmid pGL4.74 of 10 ng respectively, 50 μ L are adjusted to serum-free medium, respectively get 2 μ L transfection reagent Lipofectamine 2000 again and be diluted to 50 μ L with serum-free medium, the DNA of dilution and transfection reagent are mixed, room temperature is placed after 20 min form transfection composite and is dropwise added in the cell of 24 orifice plates, mix in rearmounted incubator and cultivate.Three wells is established in often kind of transfection.

According to Dual-Luciferase activity detection kit illustrate carry out cotransfection reporter plasmid and internal reference plasmid after the Activity determination of Photinus pyralis LUC (Firefly luciferase) and renilla luciferase (Renilla luciferase) in cell: the PBS of the cell precooling after transfection 24 h washes twice, then uses lysing cell 15 min under 100 μ L Passive Lysis Buffer ice baths; Move to Eppendorf tube and fully vibration, centrifugal 15 s of 13000 r/min at 4 DEG C; Get 20 μ L supernatant to 100 μ L LAR II mixings, in chemiluminescence detector GloMax 20/20(Promega company) upper detection 10 s, record Photinus pyralis LUC is active; Add 100 μ L Stop & Glo reagent, detect 10 s again after mixing, record renilla luciferase is active.Using firefly luciferin activity/sea pansy luciferase activities as relative luciferase activity unit (Relative Luciferase Unit, RLU), with the relative luciferase activity of the not pGL4.10 transfectional cell of tape starting for background, calculate the relative luciferase activity used respectively in the various cells of pGL4.10/AFP and pGL4.10/CMVenhancer-AFP two kinds of reporter gene transfections, analyze CMV early transcription enhanser to a-fetoprotein gene enhanser-promoter transcription efficiency and specific impact.As can be seen from Figure 7, the activity that two enhanser a-fetoprotein gene recombinant promoter can improve luciferase widely in human liver cancer cell Hep3B, HepG2 with SMMC7721 (is compared with the effect of single a-fetoprotein gene enhanser, improve 33.07 times, 134.22 times and 465.18 times respectively), in these cells, namely substantially increase the expression of the luciferase gene that it regulates and controls.

<110> Zhejiang University

The two enhanser alpha-fetoprotein recombinant promoter of <120> mono-kind and application thereof

<160> 10

 

<210> 1

<211> 1555

<212> DNA

<213> artificial sequence

<220>

The two enhanser alpha-fetoprotein recombinant promoter of <223>

<400> 1

AGATCTGACA TTGATTATTG ACTAGTTATT AATAGTAATC AATTACGGGG TCATTAGTTC 60

ATAGCCCATA TATGGAGTTC CGCGTTACAT AACTTACGGT AAATGGCCCG CCTGGCTGAC 120

CGCCCAACGA CCCCCGCCCA TTGACGTCAA TAATGACGTA TGTTCCCATA GTAACGCCAA 180

TAGGGACTTT CCATTGACGT CAATGGGTGG AGTATTTACG GTAAACTGCC CACTTGGCAG 240

TACATCAAGT GTATCATATG CCAAGTACGC CCCCTATTGA CGTCAATGAC GGTAAATGGC 300

CCGCCTGGCA TTATGCCCAG TACATGACCT TATGGGACTT TCCTACTTGG CAGTACATCT 360

ACGTATTAGT CATCGCTATT ACCATGGTGA TGCGGTTTTG GCAGTACATC AATGGGCGTG 420

GATAGCGGTT TGACTCACGG GGATTTCCAA GTCTCCACCC CATTGACGTC AATGGGAGTT 480

TGTTTTGGCA CCAAAATCAA CGGGACTTTC CAAAATGTCG TAACAACTCC GCCCCATGGA 540

TCTCAGATTG AATTATTTGC CTGTCATACA GCTAATAATT GACCATAAGA CAATTAGATT 600

TAAATTAGTT TTGAATCTTT CTAATACCAA AGTTCAGTTT ACTGTTCCAT GTTGCTTCTG 660

AGTGGCTTCA CAGACTTATG AAAAAGTAAA CGGAATCAGA ATTACATCAA TGCAAAAGCA 720

TTGCTGTGAA CTCTGTACTT AGGACTAAAC TTTGAGCAAT AACACATATA GATTGAGGAT 780

TGTTTGCTGT TAGTATACAA ACTCTGGTTC AAAGCTCCTC TTTATTGCTT GTCTTGGAAA 840

ATTTGCTGTT CTTCATGGTT TCTCTTTTCA CTGCTATCTA TTTTTCTCAA CCACTCACAT 900

GGCTACAATA ACTGTCTGCA AGCTTATGAT TCCCAAATAT CTATCTCTAG CCTCAATCTT 960

GTTCCAGAAG ATAAAAAGTA GTATTCAAAT GCACATCAAC GTCTCCACTT GGAGGGCTTA 1020

AAGACGTTTC AACATACAAA CCGGGGAGTT TTGCCTGGAA TGTTTCCTAA AATGTGTCCT 1080

GTAGCACATA GGGTCCTCTT GTTCCTTAAA ATCTAATTAC TTTTAGCCCA GTGCTCATCC 1140

CACCTATGGG GAGATGAGAG TGAAAAGGGA GCCTGATTAA TAATTACACT AAGTCAATAG 1200

GCATAGAGCC AGGACTGTTT GGGTAAACTG GTCACTTTAT CTTAAACTAA ATATATCCAA 1260

AACTGAACAT GTACTTAGTT ACTAAGTCTT TGACTTTATC TCATTCATAC CACTCAGCTT 1320

TATCCAGGCC ACTTATTTGA CAGTATTATT GCGAAAACTT CCTAGGATCT GCCCCAAAGA 1380

GCTCTGTGTC CTTGAACATA AAATACAAAT AACCGCTATG CTGTTAATTA TTGGCAAATG 1440

TCCCATTTTC AACCTAAGGA AATACCATAA AGTAACAGAT ATACCAACAA AAGGTTACTA 1500

GTTAACAGGC ATTGCCTGAA AAGAGTATAA AAGAATTTCA GCATGATTTG GATCC 1555

 

 

<210> 2

<211> 531

<212> DNA

<213> artificial sequence

<220>

<223>CMV early transcription enhanser

<400> 2

GACATTGATT ATTGACTAGT TATTAATAGT AATCAATTAC GGGGTCATTA GTTCATAGCC 60

CATATATGGA GTTCCGCGTT ACATAACTTA CGGTAAATGG CCCGCCTGGC TGACCGCCCA 120

ACGACCCCCG CCCATTGACG TCAATAATGA CGTATGTTCC CATAGTAACG CCAATAGGGA 180

CTTTCCATTG ACGTCAATGG GTGGAGTATT TACGGTAAAC TGCCCACTTG GCAGTACATC 240

AAGTGTATCA TATGCCAAGT ACGCCCCCTA TTGACGTCAA TGACGGTAAA TGGCCCGCCT 300

GGCATTATGC CCAGTACATG ACCTTATGGG ACTTTCCTAC TTGGCAGTAC ATCTACGTAT 360

TAGTCATCGC TATTACCATG GTGATGCGGT TTTGGCAGTA CATCAATGGG CGTGGATAGC 420

GGTTTGACTC ACGGGGATTT CCAAGTCTCC ACCCCATTGA CGTCAATGGG AGTTTGTTTT 480

GGCACCAAAA TCAACGGGAC TTTCCAAAAT GTCGTAACAA CTCCGCCCCA T 531

 

<210> 3

<211> 26

<212> DNA

<213> artificial sequence

<220>

<223>CMV early transcription enhanser amplification upstream primer

<400> 3

AGATCTGACA TTGATTATTG ACTAGT 26

 

<210> 4

<211> 26

<212> DNA

<213> artificial sequence

<220>

<223>CMV early transcription enhanser amplification downstream primer

<400> 4

GGATCCATGG GGCGGAGTTG TTACGA 26

 

<210> 5

<211> 821

<212> DNA

<213>Homo sapien

<220>

<223> human a-fetoprotein gene enhanser

<400> 5

CAGATTGAAT TATTTGCCTG TCATACAGCT AATAATTGAC CATAAGACAA TTAGATTTAA 60

ATTAGTTTTG AATCTTTCTA ATACCAAAGT TCAGTTTACT GTTCCATGTT GCTTCTGAGT 120

GGCTTCACAG ACTTATGAAA AAGTAAACGG AATCAGAATT ACATCAATGC AAAAGCATTG 180

CTGTGAACTC TGTACTTAGG ACTAAACTTT GAGCAATAAC ACATATAGAT TGAGGATTGT 240

TTGCTGTTAG TATACAAACT CTGGTTCAAA GCTCCTCTTT ATTGCTTGTC TTGGAAAATT 300

TGCTGTTCTT CATGGTTTCT CTTTTCACTG CTATCTATTT TTCTCAACCA CTCACATGGC 360

TACAATAACT GTCTGCAAGC TTATGATTCC CAAATATCTA TCTCTAGCCT CAATCTTGTT 420

CCAGAAGATA AAAAGTAGTA TTCAAATGCA CATCAACGTC TCCACTTGGA GGGCTTAAAG 480

ACGTTTCAAC ATACAAACCG GGGAGTTTTG CCTGGAATGT TTCCTAAAAT GTGTCCTGTA 540

GCACATAGGG TCCTCTTGTT CCTTAAAATC TAATTACTTT TAGCCCAGTG CTCATCCCAC 600

CTATGGGGAG ATGAGAGTGA AAAGGGAGCC TGATTAATAA TTACACTAAG TCAATAGGCA 660

TAGAGCCAGG ACTGTTTGGG TAAACTGGTC ACTTTATCTT AAACTAAATA TATCCAAAAC 720

TGAACATGTA CTTAGTTACT AAGTCTTTGA CTTTATCTCA TTCATACCAC TCAGCTTTAT 780

CCAGGCCACT TATTTGACAG TATTATTGCG AAAACTTCCT A 821

 

<210> 6

<211> 180

<212> DNA

<213>Homo sapien

<220>

<223> human a-fetoprotein gene promotor

<400> 6

TGCCCCAAAG AGCTCTGTGT CCTTGAACAT AAAATACAAA TAACCGCTAT GCTGTTAATT 60

ATTGGCAAAT GTCCCATTTT CAACCTAAGG AAATACCATA AAGTAACAGA TATACCAACA 120

AAAGGTTACT AGTTAACAGG CATTGCCTGA AAAGAGTATA AAAGAATTTC AGCATGATTT 180

 

<210> 7

<211> 29

<212> DNA

<213> artificial sequence

<220>

<223> human a-fetoprotein gene enhanser amplification upstream primer

<400> 7

AGATCTCAGA TTGAATTATT TGCCTGTCA 29

 

<210> 8

<211> 28

<212> DNA

<213> artificial sequence

<220>

<223> human a-fetoprotein gene enhanser amplification downstream primer

<400> 8

GGATCCTAGG AAGTTTTCGC AATAATAC 28

 

 

<210> 9

<211> 25

<212> DNA

<213> artificial sequence

<220>

<223> human a-fetoprotein gene promotor amplification upstream primer

<400> 9

AGATCTGCCC CAAAGAGCTC TGTGT 25

 

<210> 10

<211> 33

<212> DNA

<213> artificial sequence

<220>

<223> human a-fetoprotein gene promotor amplification downstream primer

<400> 10

GGATCCAAAT CATGCTGAAA TTCTTTTATA CTC 33

Claims (2)

1. a two enhanser alpha-fetoprotein recombinant promoter is building the application in exogenous gene expression carrier, it is characterized in that, described pair of enhanser alpha-fetoprotein recombinant promoter, for a kind of DNA molecular, its nucleotide sequence is as shown in SEQ ID No:1, wherein the 7th to the 537th is CMV early transcription enhancer sequence, and the 544th is human a-fetoprotein gene enhancer sequence to the 1364th, and the 1370th is human a-fetoprotein gene promoter sequence to the 1549th.

2. the two application of enhanser alpha-fetoprotein recombinant promoter in the target gene therapy medicine of the liver cancer for the preparation of the AFP positive, it is characterized in that, described pair of enhanser alpha-fetoprotein recombinant promoter, for a kind of DNA molecular, its nucleotide sequence is as shown in SEQ ID No:1, wherein the 7th to the 537th is CMV early transcription enhancer sequence, 544th is human a-fetoprotein gene enhancer sequence to the 1364th, and the 1370th is human a-fetoprotein gene promoter sequence to the 1549th.