CN102051358B - Mosaic tumor specific gene expression regulation kit and application thereof - Google Patents
Mosaic tumor specific gene expression regulation kit and application thereof Download PDFInfo
- Publication number
- CN102051358B CN102051358B CN2009101985758A CN200910198575A CN102051358B CN 102051358 B CN102051358 B CN 102051358B CN 2009101985758 A CN2009101985758 A CN 2009101985758A CN 200910198575 A CN200910198575 A CN 200910198575A CN 102051358 B CN102051358 B CN 102051358B
- Authority
- CN
- China
- Prior art keywords
- gene
- tumour
- specific
- gene expression
- end non
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Images
Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention belongs to the technical field of biomedicines, and in particular provides a mosaic tumor specific gene expression regulation kit, which comprises a tumor specific gene promoter, a 5' untranslated region of a tumor specific translational control component, a polyclonal site, a gene expression enhancer and a polyadenylic acid signal region, wherein the tumor specific gene promoter, the 5' untranslated region of the tumor specific translational control component, the gene expression enhancer and the polyadenylic acid signal region are connected in turn; and the polyclonal site is positioned between the 5' untranslated region of the tumor specific translational control component and the polyadenylic acid signal region and is independent of the gene expression enhancer. The mosaic tumor specific gene expression regulation kit effectively regulates the expression of tumor therapy genes to limit the genes in tumor cells, and obviously improves the specificity of the expression of the therapy genes so as to effectively improve the safety of tumor gene therapy and reduce the harm of the tumor gene therapy to normal tissues and cells.
Description
Technical field
The invention belongs to the medical biotechnology field; Be specifically related to a kind of novel tumor gene therapy targeted expression technology; Promptly through making up a kind of chimeric tumour-specific gene expression regulation box; Use the expression of this regulation and control box modulate tumor therapeutic gene, and therefore construct the gene therapy system of tumour-specific.
Background technology
Along with the development of modern genetics and genomics, gene therapy has become possibility as a kind of new disease treatment mode.Gene therapy is generation, development and the process of directly intervening disease through the operation and the intervention of gene level.Compare with strategy with existing other treatment method; The advantage of gene therapy is directly to utilize people in the new technology of molecular level to the cause of disease of disease, pathogenetic newly discovered, new knowledge and biology field, repairs even replace Disease-causing gene pointedly or correct gene expression regulation unusual.Gene therapy will be the revolution of medical science and pharmaceutical field, be one of most important milestone of current biomedical development, also will become important medicinal industry of 21 century simultaneously.The development of gene therapy is extremely rapid; According to statistics; Begin from first gene therapy clinical trial of nineteen ninety U.S. FDA official approval; By in March, 2009, existing 1537 each phase gene therapy clinical study schemes (http://www.wiley.co.uk/genmed/clinical/) have contained various fields such as inherited disease, metabolic disease, tumour, transmissible disease in the world wide; Wherein III phase clinical study has 52, and Chinese in addition SFDA has also ratified 2 gene therapy new drug listings in the world first.But; Generally speaking, gene therapy technology and product also are in the initial stage of its development, are faced with problems and challenge; It is wherein main that to show as gene import system efficient lower; Lack target property, and the therapeutic gene that imports lacked effectively regulation and control etc., these problems also be to restrict the major cause that develops effective and safe gene therapy.Matter of utmost importance in the gene therapy gordian technique is selectivity therapeutic gene to be transported to specified target tissue (or target organ) (or relative specificity), makes it to get into target cell, in target cell, realizes the selective expression simultaneously.Target gene therapy is the most important research direction of current gene therapy.
Therapy of tumor is most important field in the gene therapy research, and its clinical study scheme has reached 993 at present, accounts for 64.6% of all gene therapy clinical protocol.The same with the governing principle of other drug research and technological assessment, gene therapy medicament also requires to accomplish safe, effective, quality controllable.At present, the key areas of therapy of tumor research also concentrates on the target gene therapy mostly.In general, target gene therapy mainly comprises two aspects: the one, and the target property of gene therapy vector transduction even gene therapy vector imports the tumour target cell specifically, and does not get into normal cell; The one, the target property expression of therapeutic gene is even the goal gene of treatment is realized specific expressed in the tumour target cell and in normal cell, do not expressed.Wherein, the target property expression of therapeutic gene is basic.
Usually, expression of gene receives the dual regulation and control of transcribing with translation skill.For the oncotherapy gene, its target property expression is also nothing more than these two aspects.At present, the target property expression of oncotherapy gene mainly concentrates on and utilizes tumour-specific and/or tissue-specific gene promoter on transcriptional level, to regulate and control, comprise ALPHA-FP AFP (alpha-fetoprotein) gene promoter in liver cancer; CEACAMS CEA (carcinoembryonic antigen) gene promoter in colorectal carcinoma; Estrogen response element (Estrogen-response element), anti-epithelium Saliva Orthana MUC1 gene promoters such as (Mucin 1) in mammary cancer; Probasin, PSA PSA gene promoters such as (prostate-specific antigen) in prostate cancer; SLPI SLP I (secretoryleukoprotease inhibitor) gene promoter in lung cancer; Tyrosylprotein kinase Tyr (Tyrosinase) gene promoter in melanoma; Have a liking for plain CgA (Chromogranin A) gene promoter of chromium in carcinoma of the pancreas; Uroplakin II gene promoter in bladder cancer; Musashi-1, nidogen gene promoters such as (Nestin) in the cancer of the brain; Plain (Mesothelin) gene promoter of mesothelium in mesothelioma; Cyclooxygenase 2 (Cyclooxygenase-2), hypoxia inducible factor HIF (Hypoxia-inducible factor), Oct-3/4 response element (Oct-3/4 response elements), E2F response element (E2F-responsiveelement), progress improve gene PEG-3 (Progression elevated gene-3), midkine (Midkine), Survivn Survivin, reverse transcriptase of telomere TERT (human telomerasereverse transcriptase), early growth response factor 1 (early growth response 1, Egr-1) wait gene promoter in kinds of tumors etc.
Usually; The complete mRNA molecule of a coded protein comprises three parts in the mammalian cell: 5 ' UTR, coding region and 3 ' UTR; Wherein 5 ' of highly structural UTR and 3 ' UTR participate in the regulation and control of genetic expression on translation skill; Control the translation speed of mRNA, and safeguard the stability of mRNA.In recent years, to utilize 5 ' end non-translational region (5 ' Untranslated Region, 5 ' UTR) of highly structural to regulate and control the translation skill of therapeutic gene specific expressed in tumour cell to realize it for existing report.This is by the structures shape of 5 ' UTR; It has comprised the element of some regulation and control mRNA translation, and comprising: (1) m7GpppG cap is the decisive factor of translation efficiency; (eukaryotic initiation factor 4E eIF-4E) discerns for cap combines complex body; (2) secondary structure (claiming the stem ring again) is come the reverse translation of regulating through combination or the migration that hinders rrna 40S subunit; (3) the special element that acts on mutually with 5 ' UTR modulin (5 '-UTR-interacting protein) suppresses translation through the mode that is similar to secondary structure; (4) upper reaches ORF (Upstream ORFs, uORF) with upper reaches AUG (upstream AUGs, uAUG), usually so that the translation that mode is reduced main ORF of alternate initiation site to be provided; (5) (Internalribosome entry sites IRES), promotes the translation initiation that non-cap relies on to the ribosome internal entry site.Generally, 5 ' UTR can stop the translation of mRNA.But, in tumour cell, 5 ' UTR can be elF-4E to the restraining effect of translating and reverses.ElF-4E be positioned on human No. four karyomit(e), the coding molecule amount is the oncogene of 25KD polypeptide; It can combine with terminal cap-like structure (m7GpppN) specificity of mRNA 5 ', makes the secondary structure of mRNA 5 ' terminal non-coding region (5 ' UTR) become flexible, untwist and forms the mRNA of one section strand.The mRNA of single-stranded structure makes initiator codon be able to scanning, starts mRNA and combines with rrna, promotes proteinic synthetic.Therefore, the eIF-4E the most effectively speed limit regulatory factor that is considered to translate.Simultaneously, the transhipment of eIF-4E and eukaryotic cell mRNA and degrade in close relations.EIF-4E has high expression level in multiple malignant tumor tissue, the aspects such as ability that the vicious transformation of pair cell, division and acquisition are outwards corroded, shifted are important influence all.Therefore, the mRNA that 5 ' UTR can effectively limit the oncotherapy gene only translates in tumour cell, and in normal cell, does not translate.
Likewise, 3 ' UTR has also comprised some translational control elements: (1) modulin recognition site element.The regulation and control of common 3 ' UTR need modulin complex body rather than single albumen; (2) antisense microRNA suppresses interpretative function through the complementary sequence in the target 3 ' UTR; (3) (Cytoplasmicpolyadenylation elements CPE) with six polynucleotide AAUAAA, activates the prolongation of mRNA polyA tail to CPE, and CPE suppresses also to have effect to translation simultaneously; (4) PolyA tail, but outer polyA conjugated protein (poly (A)-binding protein, PABP) molecule, the promotion translation of the length supplementary quota of increase polyA tail.3 ' UTR has multiple actions such as regulation and control mRNA translation, the stability of keeping mRNA and Subcellular Localization.
In eukaryotic cells, the regulation and control of multiple levels such as expression of gene is transcribed, translation.Yet, in the targeted expression technology of present oncotherapy gene, be to concentrate on the targeted expression level of transcribing more, the mRNA stability under its regulation and control is still waiting to improve with expression efficiency.Therefore, have only the expression and regulation mechanism of complete simulates real nuclear gene,, just might in the tumour target cell, realize the highest specifically expressing from transcribing and translate common regulation and control therapeutic gene on the dual level.
Summary of the invention
Main purpose of the present invention is design and makes up a kind of chimeric tumour-specific gene expression regulation box, uses this regulation and control box modulate tumor therapeutic gene and makes it in the tumour target cell, to realize specifically expressing, and therefore construct the gene therapy system of tumour-specific.
The present invention is directed to the targeted expression technology of oncotherapy gene; Design also provides a kind of chimeric tumour-specific gene expression regulation box; Transcribe and translate regulation and control oncotherapy expression of gene on the dual level to be implemented in; Make it to express being confined in the tumour target cell, and in other normal cells, do not express.
The invention provides a kind of chimeric tumour-specific gene expression regulation box, it comprises tumour-specific gene promoter, tumour-specific translational control element 5 ' end non-translational region, MCS, gene expression enhancer and polyadenylic acid signaling zone; Tumour-specific gene promoter, tumour-specific translational control element 5 ' end non-translational region, gene expression enhancer are connected with the polyadenylic acid signaling zone successively; MCS is positioned between tumour-specific translational control element 5 ' end non-translational region and the polyadenylic acid signaling zone, and is separate with gene expression enhancer.
In the above-mentioned tumour-specific gene expression regulation box, described tumour-specific gene promoter is that telomerase reverse transcriptase gene promotor, Survivn gene promoter, cyclooxygenase 2 gene promoters, hypoxia inducible factor gene promoter, Oct-3/4 response element, E2F response element, progress improve gene promoter, early growth response factor 1 gene promoter, midkine gene promoter, a-fetoprotein gene promotor, CEACAMS CEA gene promoter, estrogen response element, anti-epithelium mucin gene promotor, Probasin gene promoter, PSA gene promoter, SLPI gene promoter, tyrosine kinase gene promotor, have a liking in chromium plain gene promotor, Uroplakin II gene promoter, Musashi-1 gene promoter, entactin gene promoter or the mesothelium plain gene promotor any one.
In the above-mentioned tumour-specific gene expression regulation box, one of 5 ' the end non-translational region that described tumour-specific translational control element 5 ' end non-translational region can be a Prostatropin 2,5 ' the end non-translational region of people's IAP hBcl-XL, 5 ' the end non-translational region of people's hypoxia inducible factor hHIF-1 or 5 ' end non-translational region of VEGF-2.For example, described tumour-specific translational control element 5 ' end non-translational region is that 5 ' of Prostatropin 2 is held non-translational region.
In the above-mentioned tumour-specific gene expression regulation box, described gene expression enhancer is any one perhaps several kinds in hepatitis B virus enhanser, SV40 enhanser, cytomegalovirus enhanser, ALPHA-FP enhanser or the CEACAMS enhanser.
In the above-mentioned tumour-specific gene expression regulation box, also has tumour-specific translational control element 3 ' end non-translational region before the polyadenylic acid signaling zone of this tumour-specific gene expression regulation box.
In the above-mentioned tumour-specific gene expression regulation box, 3 ' the end non-translational region that described tumour-specific translational control element 3 ' end non-translational region can be an EGF-R ELISA, 3 ' the end non-translational region of cancer suppressor gene p53 or 3 ' the end non-translational region of people VEGF.For example, described tumour-specific translational control element 3 ' end non-translational region is that 3 ' of EGF-R ELISA is held non-translational region.
The present invention also provides the preparation method of above-mentioned tumour-specific gene expression regulation box, promptly prepares each integral part of tumour-specific gene expression regulation box respectively, then it is connected successively.
The concrete steps that make up chimeric tumour-specific gene expression regulation box are following:
Utilize conventional molecular biology and molecule clone technology; Set up chimeric tumour-specific gene expression regulation box, this regulation and control box is made up of tumour-specific gene promoter, tumour-specific translational control element 5 ' end non-translational region, enhanser (enhancer) and/or 3 ' end non-translational region and polyadenylic acid signaling zone etc.
Above-mentioned tumour-specific gene promoter includes but not limited to: and reverse transcriptase of telomere TERT (humantelomerase reverse transcriptase, hTERT) gene promoter, Survivn (Survivin) gene promoter, cyclooxygenase 2 (Cyclooxygenase-2) gene promoter, hypoxia inducible factor HIF (Hypoxia-inducible factor) gene promoter, early growth response factor Egr-1 (Earlygrowth response 1) gene promoter, Oct-3/4 response element (Oct-3/4 responseelements), E2F response element (E2F-responsive element), progress improve gene PEG-3 (Progression elevated gene-3) gene promoter, midkine (Midkine) gene promoter, ALPHA-FP AFP (alpha-fetoprotein) gene promoter, CEACAMS CEA (carcinoembryonic antigen) gene promoter, estrogen response element (Estrogen-responseelement), anti-epithelium Saliva Orthana MUC1 (Mucin 1) gene promoter, Probasin gene promoter, PSA PSA (prostate-specific antigen) gene promoter, SLPI SLPI (secretory leukoprotease inhibitor) gene promoter, Tyrosylprotein kinase Tyr (Tyrosinase) gene promoter, have a liking for plain CgA (Chromogranin A) gene promoter of chromium, Uroplakin II gene promoter, Musashi-1 gene promoter, nidogen (Nestin) gene promoter, plain (Mesothel in) gene promoter of mesothelium etc.;
Above-mentioned 5 ' end non-translational region includes but not limited to: Prostatropin 2 (basicfibroblast growth factor 2; BFGF-2) 5 ' the end non-translational region of 5 ' end non-translational region, 5 ' the end non-translational region of people's IAP hBcl-XL, people's hypoxia inducible factor hHIF-1, VEGF-2 (Vascular endothelial growth factor, 5 ' end non-translational region VEGF) etc.;
Above-mentioned enhanser includes but not limited to: hepatitis B virus enhanser WRE (Woodchuck hepatitisvirus post-transcriptional regulatory element or Woodchuck ResponseElement; WRE), SV40 (simian virus 40) enhanser, cytomegalovirus (cytomegalovirus, CMV) enhanser, ALPHA-FP enhanser, CEACAMS enhanser etc.;
Above-mentioned 3 ' end non-translational region includes but not limited to: EGF-R ELISA (Epidermal growthfactor receptor, 3 ' the end non-translational region of 3 ' end non-translational region EGFR), 3 ' the end non-translational region of cancer suppressor gene p53, people VEGF etc.;
Above-mentioned polyadenylic acid signaling zone includes but not limited to: and Polisac polyadenylic acid signaling zone (bovinegrowth hormone polyadenylation signal, BGHpolyA), SV40 ployA etc.
The major function of MCS is to insert exogenous dna fragment, is made up of a series of excision enzymes usually, can connected and got by the excision enzyme of some routines, SalI for example, HindIII, EcoRI; SacI, KpnI, EcoRV, NotI, SphI, NcoI; XhoI, SmaI, PacI, NheI, BamHI, or the like.
Utilize chimeric tumour-specific gene expression regulation box of the present invention can make up the genetic expression reporting system under its regulation and control.
Utilize conventional molecular biology and molecule clone technology; Make up the genetic expression reporting system that above-mentioned chimeric tumour-specific gene expression regulation box is regulated and control, include but not limited to: strengthen green fluorescence protein gene reporting system EGFP (Enhance Green Fluorescence Protein) and quantitative luciferase gene reporting system (Luciferase reporter) qualitatively.
Thus, can further make up the oncotherapy gene and the gene therapy system thereof of chimeric tumour-specific gene expression regulation box regulation and control.
Utilize conventional molecular biology and molecule clone technology, make up oncotherapy gene and gene therapy system thereof that above-mentioned chimeric tumour-specific gene expression regulation box is regulated and control.
Tumour-specific gene expression regulation box of the present invention can be applied to genetic treatment of tumor, is about to the MCS that the oncotherapy gene inserts tumour-specific gene expression regulation box, is used to prepare the Antioncogene healing potion.
Described oncotherapy gene is any one or a few in bacteriotoxin gene, suicide gene, apoptosis-inducing gene, anti-new vessel formation gene, radioactive rays enhanced sensitivity gene, tumor suppressor gene, the immune-regulating factor gene.
The oncotherapy gene that above-mentioned chimeric tumour-specific gene expression regulation box is regulated and control includes but not limited to:
(1) bacteriotoxin gene; Include but not limited to: diphtheria toxin (diphtheria toxin, DT), the Pseudomonas aeruginosa extracellular toxin (pseudomonas exotoxin, PE), Toxins,exo-, cholera (choleratoxin; CT), shiga toxin (shiga toxin; ST), Toxins, pertussis (pertussis toxin, PT), (staphylococcal enterotoxin B SEB) waits gene to Staphylococcal enterotoxin B gene;
(2) kill gene; Include but not limited to: herpes simplex virus thymidine kinase (Herpes SimplexVirus Thymidine Kinase; HSV-TK), varicella Acyclovir virus thymidine kinase (Varicella-Zoster Virus Thymidine Kinase; VZV-TK), coli cytosine deaminase (E.coli Cytosine Deaminase; CD), intestinal bacteria deoxycytidine kinase (E.coliDeoxycytidine Kinase; DCK), the intestinal bacteria xanthine-guanine phosphoribosyl transferase (E.colihypoxanthine-guanine phosphoribosyl transferase, HGPRT) and cytopigment p450 (Cytochromo P450 CYP) waits gene;
(3) apoptosis-inducing gene; Include but not limited to: B cell lymphoma/white blood disease-2 (B-celllymphoma/leukemia-2; Bcl-2) gene family, Fatty acid synthetase (Fatty acid synthase; Fas) and part (Fas-ligand, Fas-L), gene such as bax, caspase;
(4) anti-new vessel forms gene; Include but not limited to: Endostatin (Endostatin), angiostatin (angiostatin), VEGF (vascular endothelial growthfactor; VEGF), Prostatropin (basic fibroblast growth factor, bFGF; Angiogenin (angiopoietin-2, Ang-2), matrix metalloprotease-7 (MatrixMetalloproteinases 7, MMP-7), HIF-1 (hypoxia induced factor-1, HIF-1), Survivn genes such as (Survivin);
(5) radiation sensitization gene; Include but not limited to: dna dependent protein kinase (DNA dependentprotein kinase; DNA-PK), (ataxia-telangiectasia mutant ATM) waits gene for Ku70, Ku80, ataxia telangiectasia mutator gene;
(6) tumor suppressor gene; Include but not limited to: cancer suppressor gene p53, Rb, PTEN (phosphataseand tensin homolog; PTEN), APC (adenomatous polyposis coli), BRCA1 and 2 genes such as (breast cancer 1 ,-2, early onset);
(7) immune-regulating factor includes but not limited to: cell interleukin-2 ,-12 and-24 (Interleukin2 ,-12 ,-24; IL-2, IL-12, IL-24), GM-CSF (Granulocyte-Macrophage Colony-Stimulating Factor, GM-CSF), interferon-' alpha ';-β ,-γ (Interferon alpha ,-beta;-gama, IFN-α ,-β;-γ), tumour necrosis factor (tumornecrosis factor, TNF) and the relevant apoptosis induction ligand of tumour necrosis factor (TNF-relatedapoptosis inducing ligand TRAIL) waits gene;
(8) fusion gene of said gene;
(9) the crucial Yin Jiyin of virus replication includes but not limited to: genes such as adenovirus early gene E1A, hsv utmost point early gene ICP4 and ICP27.
Tumour-specific gene expression regulation box of the present invention can be used to prepare pUC pUC, recombinant virus system or the oncolytic virus system of Antioncogene treatment.
The therapy of tumor system that above-mentioned chimeric tumour-specific gene expression regulation box is regulated and control includes but not limited to:
(1) pUC pUC; Include but not limited to: the unloaded plasmid of eukaryotic gene expression, reporter gene plasmid and the therapeutic gene plasmid that comprise above-mentioned chimeric tumour-specific gene expression regulation box; Above-mentioned plasmid and/or with liposome, polyethyene diamine (Polyethylenimine; PEI), POLYACTIC ACID (poly-lactic acid, PLA), POLYACTIC ACID-glycolic acid (poly (lactic-co-glycolic acid), PLGA), chitosan nanoparticles such as (Chitosan) forms the therapy of tumor system;
(2) recombinant virus system includes but not limited to: comprise above-mentioned chimeric tumour-specific gene expression regulation box and regulate and control the recombinant adenovirus of the various replication defect types of reporter gene, oncotherapy gene and zero load thereof, recombinant herpes simplex virus, recombinant poxvirus etc.;
(3) oncolytic virus system includes but not limited to: comprise above-mentioned chimeric tumour-specific gene expression regulation box and regulate and control the oncolytic adenovirus of the various selection rfs of reporter gene, oncotherapy gene and zero load thereof, oncolytic hsv, oncolytic poxvirus etc.
The invention belongs to the biological medicine technology field, a kind of chimeric tumour-specific gene expression regulation box specifically is provided.Use the expression of this regulation and control box modulate tumor therapeutic gene, and therefore construct the gene therapy system of tumour-specific.The present invention makes it to be confined in the tumour cell through the expression of effective modulate tumor therapeutic gene, has significantly improved the specificity that therapeutic gene is expressed, and then the security that effectively improves therapy of tumor, reduces its infringement to healthy tissues and cell.With therapeutic gene sHSV1-tk is example, and what chimeric tumour-specific gene expression regulation box was regulated and control improves 50 to 800 times to tumour cell target property kill capability than normal cell.The recombinant adenovirus system that utilizes this chimeric tumour-specific gene expression regulation box to be built into will improve 300 to 1800 times to tumour cell target property kill capability.Can effectively improve the tumor-bearing mice survival time 2-5 month, and the tumor-bearing mice curative ratio can reach 50-80%.
Description of drawings
The tumour-specific gene expression regulation box that Fig. 1 is chimeric and the reporter gene of regulation and control thereof, therapeutic gene synoptic diagram.Be respectively: skeleton plasmid pBA-MCS; Unloaded expression plasmid phTUW-MCS and phTUWR-MCS; Reporter gene plasmid phTUW-EGFP and phTUWR-EGFP; Therapy of tumor plasmid phTUW-sHSV1-tk and phTUWR-sHSV1-tk.
Embodiment
Embodiment 1
Separate and clone's tumour-specific gene expression regulation box controlling element
The A.hTERT gene promoter
Telomerase is the DNA polymerase that a kind of RNA relies on, and the activation of Telomerase is the important mechanisms of being immortalized of cell and malignant change.Research shows that Telomerase presents positive expression in the tumour cell more than 85%, and in nearly all one-tenth human body cell, presents feminine gender.The telomerase catalytic subunit of hTRT's gene hTERT coding is the deciding factor of telomerase activation.The activity of hTERT self is mainly regulated at transcriptional level, and the transcriptional activity of hTERT gene promoter is played an important role in this course.Because the hTERT promotor only has transcriptional activity in tumour cell, so hTERT realizes that therapeutic gene is at the tumour cell desirable transcriptional regulatory element that the tropism expresses that hits.The embodiment of the invention is used conventional round pcr, is template with human cell DNA, and amplification hTRT's hTERT gene promoter fragment (SEQ No.1) is inserted among the TA cloning vector pCR2.1 (Invitrogen) then, through this sequence of order-checking conclusive evidence.
B.bFGF-25’UTR
In recent years, utilize translation skill that 5 ' UTR of highly structural regulates and control therapeutic gene to realize its specific expressed reports that have in tumour cell more.Under normal conditions, 5 ' UTR can stop the translation of mRNA.But in tumour cell, can reverse the restraining effect of 5 ' UTR to translation because of high expression level eIF-4E.The embodiment of the invention adopts conventional RT-PCR technology, is template with the total RNA of rat cell, and the 5 ' UTR (SEQ No.2) of amplification rat bFGF-2 is inserted among the TA cloning vector pCR2.1 (Invitrogen) then, through this sequence of order-checking conclusive evidence.
C.WRE
Usually its transcriptional activity of gene promoter of tumour-specific relatively a little less than, need come further to improve its transcriptional capability by enhanser.The embodiment of the invention is used conventional round pcr, is template with hepatitis B virogene group DNA, and amplification hepatitis B virus enhanser WRE (SEQ No.3) is inserted among the TA cloning vector pCR2.1 (Invitrogen) then, through this sequence of order-checking conclusive evidence.
D.EGFR?3’UTR
In eukaryotic cells, 3 ' UTR has multiple actions such as regulation and control mRNA translation, the stability of keeping mRNA and Subcellular Localization.The embodiment of the invention is used conventional RT-PCR technology, is template with the total RNA of human cell, and the 3 ' UTR (SEQ No.4) of amplification Human epidermal growth factor receptor EGFR is inserted among the TA cloning vector pCR2.1 (Invitrogen) then, through this sequence of order-checking conclusive evidence.
E.BGHpolyA
In eukaryotic cells, the Transcription Termination of its mRNA need be by the polyadenylic acid signal.The embodiment of the invention is used conventional round pcr; With plasmid pCDNA3.1 (+) is template (Invitrogen); The polyadenylic acid signaling zone BGHpolyA (SEQ No.5) of amplification Polisac is inserted among the TA cloning vector pCR2.1 (Invitrogen) then, through this sequence of order-checking conclusive evidence.
Make up chimeric tumour-specific gene expression regulation box
Above-mentioned separation and clone's various tumour-specific gene expression regulation box controlling elements need follow a definite sequence and put combination, are built into chimeric tumour-specific gene expression regulation box.Embodiment of the invention applied molecular clone technology ultimate principle; Design and chemosynthesis MCS MCS (Multiple Cloning Sites; MCS) (SEQ No.6); Behind PacI and XhoI double digestion, be inserted into formation skeleton plasmid pBA-MCS (Figure 1A) among the pORF-MCS (Invivogen) through same double digestion.Among the embodiment 1 through the hTERT gene promoter sequence of order-checking conclusive evidence with EcoRI and HindIII double digestion, bFGF-25 ' UTR with HindIII and BamHI double digestion, WRE with HindIII and SalI double digestion also benefit put down end, EGFR 3 ' UTR with the BglII enzyme cut, BGHpolyA is with BglII and XhoI double digestion; Be inserted into above-mentioned skeleton plasmid pBA-MCS successively, be built into the expression plasmid phTUW-MCS and the phTUWR-MCS (Figure 1B) that comprise different chimeric tumour-specific gene expression regulation boxes respectively.
Embodiment 3
Set up the reporter gene of chimeric tumour-specific gene expression regulation box regulation and control
Can the chimeric tumour-specific gene expression regulation box that comprise among the expression plasmid phTUW-MCS of above-mentioned structure and the phTUWR-MCS play a role, and the further application report gene of need is analyzed in clone and verified.The embodiment of the invention uses NcoI and the XbaI double digestion cuts out enhancing green fluorescence protein gene EGFP from plasmid pFUGW (Addgene); Be inserted into respectively in expression plasmid phTUW-MCS and phTUWR-MCS that same enzyme is cut, be built into qualitative gene reporter plasmid phTUW-EGFP and phTUWR-EGFP (Fig. 1 C).Use the various tumor cell line of the further transfection of above-mentioned reporter gene system, prove conclusively the dual regulate gene expression effect of its tumour-specific.(like normal liver cell L-02) compares with normal cell, and the specificity that the reporter gene EGFP of these chimeric tumour-specific gene expression regulation box regulation and control expresses in tumour cell (like hepatocellular carcinoma cells QGY-7703 and SMMC-7721 and liver cell metastasis cancer cell MHCC97-H etc.) will improve 100 to 2000 times.
Embodiment 4
Set up the oncotherapy gene plasmid system of chimeric tumour-specific gene expression regulation box regulation and control
The chimeric tumour-specific gene expression regulation box that comprises among the expression plasmid phTUW-MCS of above-mentioned structure and the phTUWR-MCS is modulate tumor therapeutic gene specific expressed effectively, can further analyze with the cell in vitro killing experiments and verify.Embodiment of the invention application NcoI and NheI double digestion cut out the sHSV1-tk gene of solubility from plasmid pMOD-HSV1tk (Invivogen); Be inserted into respectively in the expression plasmid phTUW-MCS and phTUWR-MCS of NcoI and XbaI double digestion, be built into therapy of tumor gymnoplasm grain phTUW-sHSV1-tk and phTUWR-sHSV1-tk (Fig. 1 D).Use the various tumor cell line of the further transfection of above-mentioned therapeutic gene gymnoplasm grain system, with conventional its specific expressed and target property fragmentation effect in tumour cell of mtt assay conclusive evidence.(like normal liver cell L-02) compares with normal cell, and the therapeutic gene sHSV1-tk of these chimeric tumour-specific gene expression regulation box regulation and control will improve 50 to 800 times to tumour cell (like hepatocellular carcinoma cells QGY-7703 and SMMC-7721 and liver cell metastasis cancer cell MHCC97-H etc.) target property kill capability.
Embodiment 5
Set up the oncotherapy gene recombinant adenovirus system of chimeric tumour-specific gene expression regulation box regulation and control
The neoplasm targeted therapy effect of the therapy of tumor gymnoplasm grain that above-mentioned chimeric tumour-specific gene expression regulation box is regulated and control can be further enhanced in recombinant virus system and oncolytic virus system and improve.The embodiment of the invention is used AdEasy adenovirus system (AdEasy Adenoviral Vector System; Stratagene) make up the recombinant adenovirus system; At first Using P acI and XhoI double digestion cut out the therapeutic gene expression cassette of complete dual-target regulation and control from therapy of tumor gymnoplasm grain phTUW-sHSV1-tk and phTUWR-sHSV1-tk; Be inserted into then among the shuttle vectors pShuttle; In E.coli BJ5183, carry out homologous recombination with adenoviral gene group skeleton plasmid pAdEasy-1 again; Obtain recombinant plasmid pAd-hTUW/sHSV1-tk and pAd-hTUWR/sHSV1-tk, this recombinant plasmid obtains recombinant adenovirus Ad-hTUW/sHSV1-tk and Ad-hTUWR/sHSV1-tk through further rotaring redyeing 293 cell.Use the various tumor cell line of the further Infection in Vitro of above-mentioned recombinant adenovirus system, with conventional its specific expressed and target property fragmentation effect in tumour cell of mtt assay conclusive evidence.(like normal liver cell L-02) compares with normal cell, and the therapeutic gene sHSV1-tk of these chimeric tumour-specific gene expression regulation box regulation and control will improve 300 to 1800 times to tumour cell (like hepatocellular carcinoma cells QGY-7703 and SMMC-7721 and liver cell metastasis cancer cell MHCC97-H etc.) target property kill capability.Above-mentioned recombinant adenovirus is treatment various bearing mouse model (like above-mentioned hepatocellular carcinoma transplanted tumor model) further, can effectively improve the tumor-bearing mice survival time 2-5 month, and the tumor-bearing mice curative ratio can reach 50-80%.
Sequence table
<210>1
<211>1087
<212>DNA
<213>Artificial
<400>1
cagaattcct?gggaagtcct?cagctgtcct?gcggttgtgc?cggggcccca?ggtctggagg 60
ggaccagtgg?ccgtgtggct?tctactgctg?ggctggaagt?cgggcctcct?agctctgcag 120
tccgaggctt?ggagccaggt?gcctggaccc?cgaggttgcc?ctccaccctg?tgcgggcggg 180
atgtgaccag?atgttggcct?catctgccag?acagagtgcc?ggggcccagg?gtcaaggccg 240
ttgtggctgg?tgtgaggcgc?ccggtgcgcg?gccagcagga?gcgcctggct?ccatttccca 300
ccctttctcg?acgggaccgc?cccggtgggt?gattaacaga?tttggggtgg?tttgctcatg 360
gtggggaccc?ctcgccgcct?gagaacctgc?aaagagaaat?gacgggcctg?tgtcaaggag 420
cccaagtcgc?ggggaagtgt?tgcagggagg?cactccggga?ggtcccgcgt?gcccgtccag 480
ggagcaatgc?gtcctcgggt?tcgtccccag?ccgcgtctac?gcgcctccgt?cctccccttc 540
acgtccggca?ttcgtggtgc?ccggagcccg?acgccccgcg?tccggacctg?gaggcagccc 600
tgggtctccg?gatcaggcca?gcggccaaag?ggtcgccgca?cgcacctgtt?cccagggcct 660
ccacatcatg?gcccctccct?cgggttaccc?cacagcctag?gccgattcga?cctctctccg 720
ctggggccct?cgctggcgtc?cctgcaccct?gggagcgcga?gcggcgcgcg?ggcggggaag 780
cgcggcccag?acccccgggt?ccgcccggag?cagctgcgct?gtcggggcca?ggccgggctc 840
ccagtggatt?cgcgggcaca?gacgcccagg?accgcgcttc?ccacgtggcg?gagggactgg 900
ggacccgggc?acccgtcctg?ccccttcacc?ttccagctcc?gcctcctccg?cgcggacccc 960
gccccgtccc?gacccctccc?gggtccccgg?cccagccccc?tccgggccct?cccagcccct 1020
ccccttcctt?tccgcggccc?cgccctctcc?tcgcggcgcg?agtttcaggc?agcgctgcaa 1080
gcttatc 1087
<210>2
<211>575
<212>DNA
<213>Artificial
<400>2
gataagcttg?caccccattc?ctggcctctg?tctcccgcac?cctatccctt?cacagcctgt 60
gctctagggg?actggagatt?tccaaaacct?gacccgatcc?ctccccagtt?cagttccttc 120
tactgctttg?ggtggaaggc?tggtcgttgt?gttaaaaggc?aggaagggag?aaagttgcat 180
ttaaacttta?ggagctgcgt?cacggc8gtc?tcctggagaa?agctccgccg?aacgggacag 240
attctttttg?caacttggag?gcgccgggcg?tggggaggag?gcggcgcgcg?gggcgggggc 300
gcgcggggcc?ggggtgcagg?cggggacgcg?gggtgacgcg?ggcccgggcc?gctgtagcac 360
acaggggctc?ggtctctcgg?cttcaggcgg?agtccggctg?cactaggctg?ggagcgcggc 420
gggacgcgaa?ccgggaggct?ggcagcccgc?gggcgagccg?cgctgggggg?ccgaggccgg 480
ggtcggggcc?ggggagcccc?gagagctgcc?gcagcggggt?cccggggccg?cggaggggcc 540
atggctgccg?gcagcatcac?ttcgcttgga?tcctt 575
<210>3
<211>621
<212>DNA
<213>Artificial
<400>3
caaagcttat?cgataatcaa?cctctggatt?acaaaatttg?tgaaagattg?actggtattc 60
ttaactatgt?tgctcctttt?acgctatgtg?gatacgctgc?tttaatgcct?ttgtatcatg 120
ctattgcttc?ccgtatggct?ttcattttct?cctccttgta?taaatcctgg?ttgctgtctc 180
tttatgagga?gttgtggccc?gttgtcaggc?aacgtggcgt?ggtgtgcact?gtgtttgctg 240
acgcaacccc?cactggttgg?ggcattgcca?ccacctgtca?gctcctttcc?gggactttcg 300
ctttccccct?ccctattgcc?acggcggaac?tcatcgccgc?ctgccttgcc?cgctgctgga 360
caggggctcg?gctgttgggc?actgacaatt?ccgtggtgtt?gtcggggaaa?tcatcgtcct 420
ttccttggct?gctcgcctgt?gttgccacct?ggattctgcg?cgggacgtcc?ttctgctacg 480
tcccttcggc?cctcaatcca?gcggaccttc?cttcccgcgg?cctgctgccg?gctctgcggc 540
ctcttccgcg?tcttcgcctt?cgccctcaga?cgagtcggat?ctccctttgg?gccgcctccc 600
cgcatcgata?ccgtcgacct?c 621
<210>4
<211>1677
<212>DNA
<213>Artificial
<400>4
gaagatctag?tatgagccct?aaaaatccag?actctttcga?tacccaggac?caagccacag 60
caggtcctcc?atcccaacag?ccatgcccgc?attagctctt?agacccacag?actggttttg 120
caacgtttac?accgactagc?caggaagtac?ttccacctcg?ggcacatttt?gggaagttgc 180
attcctttgt?cttcaaactg?tgaagcattt?acagaaacgc?atccagcaag?aatattgtcc 240
ctttgagcag?aaatttatct?ttcaaagagg?tatatttgaa?aaaaaaaaaa?agtatatgtg 300
aggattttta?ttgattgggg?atcttggagt?ttttcattgt?cgctattgat?ttttacttca 360
atgggctctt?ccaacaagga?agaagcttgc?tggtagcact?tgctaccctg?agttcatcca 420
ggcccaactg?tgagcaagga?gcacaagcca?caagtcttcc?agaggatgct?tgattccagt 480
ggttctgctt?caaggcttcc?actgcaaaac?actaaagatc?caagaaggcc?ttcatggccc 540
cagcaggccg?gatcggtact?gtatcaagtc?atggcaggta?cagtaggata?agccactctg 600
tcccttcctg?ggcaaagaag?aaacggaggg?gatggaattc?ttccttagac?ttacttttgt 660
aaaaatgtcc?ccacggtact?tactccccac?tgatggacca?gtggtttcca?gtcatgagcg 720
ttagactgac?ttgtttgtct?tccattccat?tgttttgaaa?ctcagtatgc?tgcccctgtc 780
ttgctgtcat?gaaatcagca?agagaggatg?acacatcaaa?taataactcg?gattccagcc 840
cacattggat?tcatcagcat?ttggaccaat?agcccacagc?tgagaatgtg?gaatacctaa 900
ggatagcacc?gcttttgttc?tcgcaaaaac?gtatctccta?atttgaggct?cagatgaaat 960
gcatcaggtc?ctttggggca?tagatcagaa?gactacaaaa?atgaagctgc?tctgaaatct 1020
cctttagcca?tcaccccaac?cccccaaaat?tagtttgtgt?tacttatgga?agatagtttt 1080
ctccttttac?ttcacttcaa?aagcttttta?ctcaaagagt?atatgttccc?tccaggtcag 1140
ctgcccccaa?accccctcct?tacgctttgt?cacacaaaaa?gtgtctctgc?cttgagtcat 1200
ctattcaagc?acttacagct?ctggccacaa?cagggcattt?tacaggtgcg?aatgacagta 1260
gcattatgag?tagtgtggaa?ttcaggtagt?aaatatgaaa?ctagggtttg?aaattgataa 1320
tgctttcaca?acatttgcag?atgttttaga?aggaaaaaag?ttccttccta?aaataatttc 1380
tctacaattg?gaagattgga?agattcagct?agttaggagc?ccaccttttt?tcctaatctg 1440
tgtgtgccct?gtaacctgac?tggttaacag?cagtcctttg?taaacagtgt?tttaaactct 1500
cctagtcaat?atccacccca?tccaatttat?caaggaagaa?atggttcaga?aaatattttc 1560
agcctacagt?tatgttcagt?cacacacaca?tacaaaatgt?tccttttgct?tttaaagtaa 1620
tttttgactc?ccagatcagt?cagagcccct?acagcattgt?taagaaagta?gatctac 1677
<210>5
<211>254
<212>DNA
<213>Artificial
<400>5
gatagatctt?agttgccagc?catctgttgt?ttgcccctcc?cccgtgcctt?ccttgaccct 60
ggaaggtgcc?actcccactg?tcctttccta?ataaaatgag?gaaattgcat?cgcattgtct 120
gagtaggtgt?cattctattc?tggggggtgg?ggtggggcag?gacagcaagg?gggaggattg 180
ggaagacaat?agcaggcatg?ctggggatgc?ggtgggctct?atggcttctg?aggcggaaag 240
aaccagctcg?agat 254
<210>6
<211>78
<212>DNA
<213>Artificial
<400>6
ccttaattaa?gaattcgata?tcaagcttga?cggatcccca?tgggtcgact?ctagaattta 60
aatagatctc?tcgagcag 78
Claims (5)
1. a chimeric tumour-specific gene expression regulation box is characterized in that, it comprises tumour-specific gene promoter, tumour-specific translational control element 5 ' end non-translational region, MCS, gene expression enhancer and polyadenylic acid signaling zone; Tumour-specific gene promoter, tumour-specific translational control element 5 ' end non-translational region, gene expression enhancer are connected with the polyadenylic acid signaling zone successively; MCS is positioned between tumour-specific translational control element 5 ' end non-translational region and the polyadenylic acid signaling zone, and is separate with gene expression enhancer; Also has tumour-specific translational control element 3 ' end non-translational region before the polyadenylic acid signaling zone; .
The sequence of described tumour-specific gene promoter is shown in SEQ ID NO 1; The sequence of tumour-specific translational control element 5 ' end non-translational region shown in SEQ ID NO 2, the sequence of MCS shown in SEQ ID NO 6, the sequence of gene expression enhancer shown in SEQ ID NO 3, the sequence of polyadenylic acid signaling zone is shown in SEQ ID NO 5;
3 ' the end non-translational region that described tumour-specific translational control element 3 ' end non-translational region is an EGF-R ELISA, 3 ' the end non-translational region of cancer suppressor gene p53 or 3 ' the end non-translational region of people VEGF.
2. the preparation method of the tumour-specific gene expression regulation box of claim 1 is characterized in that, prepares each integral part of tumour-specific gene expression regulation box respectively, then it is connected successively.
3. the application of the tumour-specific gene expression regulation box of claim 1 in the preparation anti-tumor agents is about to the MCS that the oncotherapy gene inserts tumour-specific gene expression regulation box.
4. application according to claim 3; It is characterized in that described oncotherapy gene is any one or a few in bacteriotoxin gene, suicide gene, apoptosis-inducing gene, anti-new vessel formation gene, radioactive rays enhanced sensitivity gene, tumor suppressor gene, the immune-regulating factor gene.
5. application according to claim 3 is characterized in that, described application is pUC pUC, recombinant virus system or the oncolytic virus system that described tumour-specific gene expression regulation box is used to prepare the Antioncogene treatment.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009101985758A CN102051358B (en) | 2009-11-10 | 2009-11-10 | Mosaic tumor specific gene expression regulation kit and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009101985758A CN102051358B (en) | 2009-11-10 | 2009-11-10 | Mosaic tumor specific gene expression regulation kit and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102051358A CN102051358A (en) | 2011-05-11 |
| CN102051358B true CN102051358B (en) | 2012-08-22 |
Family
ID=43956157
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2009101985758A Expired - Fee Related CN102051358B (en) | 2009-11-10 | 2009-11-10 | Mosaic tumor specific gene expression regulation kit and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102051358B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110669740B (en) * | 2019-07-16 | 2021-10-08 | 伍泽堂 | Oncolytic virus, application thereof and medicine for treating cancer |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1258742A (en) * | 1998-12-25 | 2000-07-05 | 华西医科大学 | Recombined toxin adenovirus mutant for treating tumor caused by tumor suppressor p53 defect |
| CN101157934A (en) * | 2007-09-20 | 2008-04-09 | 复旦大学 | Gene clone plasmid based on Phi BT1 integrase and Phi C31 integrase |
-
2009
- 2009-11-10 CN CN2009101985758A patent/CN102051358B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1258742A (en) * | 1998-12-25 | 2000-07-05 | 华西医科大学 | Recombined toxin adenovirus mutant for treating tumor caused by tumor suppressor p53 defect |
| CN101157934A (en) * | 2007-09-20 | 2008-04-09 | 复旦大学 | Gene clone plasmid based on Phi BT1 integrase and Phi C31 integrase |
Non-Patent Citations (2)
| Title |
|---|
| 方煜翔,薛京伦,田 聆.翻译起始的调控与肿瘤靶向基因治疗.《中国肿瘤生物治疗杂志》.2008,第15卷(第4期),396-400. * |
| 韦炜, 薛京伦, 田聆.载体重靶向与肿瘤基因治疗.《癌症》.2009,第28卷(第1期),107-112. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102051358A (en) | 2011-05-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| RU2361611C2 (en) | Designing of carcinolytic adenovirus recombinant, specifically expressing immunomodulatory factor gm-csf in tumoral cells and its application | |
| US10280420B2 (en) | Cancer specific-splicing ribozyme and use thereof | |
| CN104212802B (en) | A kind of tumour cell wide spectrum high activity promoter and application thereof | |
| US20240148807A1 (en) | Oncolytic virus (oncolytic immunotherapy) capable of effectively treating even metastatic cancer while ensuring safety, with expression control system providing optimal expression level of mounted immunogenic gene | |
| Palmer et al. | Cancer gene-therapy: clinical trials | |
| EP3512533B1 (en) | Oncolytic viral vectors for use in the treatment of retinoblastoma | |
| HRP20110059T1 (en) | Adenovirus/alphavirus hybrid vector for the effective administration and expression of therapeutic genes in tumour cells | |
| JP7387675B2 (en) | Gene expression cassette and expression vector containing the same | |
| CN101120093B (en) | Vectors comprising novel regulatory elements | |
| CN104178460B (en) | It is a kind of transcribed and transcribed after double regulation and control oncolytic adenovirus and its construction method | |
| US20040038403A1 (en) | Use of spliceosome mediated RNA trans-splicing to confer cell selective replication to adenoviruses | |
| CN108342366B (en) | Recombinant oncolytic gene-adenovirus of targeted cancer and construction method and application thereof | |
| JP2020146033A (en) | Cancer specific trans-splicing ribozymes and uses thereof | |
| CN102051358B (en) | Mosaic tumor specific gene expression regulation kit and application thereof | |
| CN105755043A (en) | Double-copy human p53 gene recombinant adenovirus and preparation method thereof | |
| CN100500222C (en) | Cancer targeted double gene-virus, its structure method and application thereof | |
| EP2062971A1 (en) | Chimeric adenovirus, method for producing the same and pharmaceutical using the same | |
| US20020165173A1 (en) | TCF responsive element | |
| Annan et al. | Gene therapy in the treatment of human cancer | |
| CN110684743A (en) | Virus for specifically killing tumor cells and tumor therapeutic drug | |
| EP1662004B1 (en) | Method of preparing a proliferation-regulated recombinant adenoviral vector, kit and vector | |
| WO1999044423A1 (en) | Amplification of gene transfer and gene therapy by controlled replication | |
| Akbulut et al. | Cancer gene therapy | |
| CN101880688B (en) | Method for selectively replicating replication-defective adenovirus and application | |
| Abaan et al. | Gene therapy in human breast cancer |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20120822 Termination date: 20151110 |
|
| EXPY | Termination of patent right or utility model |