CN101126100A - Tumour bi-target adenovirus AdCN103 and its construction method and application - Google Patents
Tumour bi-target adenovirus AdCN103 and its construction method and application Download PDFInfo
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Abstract
The invention pertains to biological technique and gene treatment field, and discloses a dual tumor targeting replicative oncolytic adenouiruses, which is dually targeting, and controls the expression of virus early protein EIA and replication of virus by the synergism of specific tumor promoter and a CR2 region of a target deleted Rb protein. The invention also discloses a novel a specific tumor promoter hTERT-4DEB and a construction method thereof, the promoter adds three E-boxes behind an hTERT promoter -255-+40nt nuclear region, thus specifically enhancing the tumor target of the promoter. The invention takes AdCN103 as an example, and the oncolytic adenouiruses controls the expression of the CR2 region deleted E1A protein by modified hTERT-4DEB promoter, so the replicative oncolytic adenouiruses has dual targets. The invention also discloses a construction method of the use in preparing medicines for tumor treatment of the dual tumor targeting replicative oncolytic adenouiruses AdCN103; the dual tumor targeting replicative oncolytic adenouiruses AdCN103 can be used for the treatment of a plurality of tumors, which can be a new effective virus-gene anti-cancer medicine for the treatment of tumor.
Description
Technical field
The invention belongs to biotechnology and field of gene, specifically, is tumor double-target virus vector AdCN103 and construction process and the application about a kind of proliferated specifically inside tumor cell.
Background technology
Cancer for most of kinds, what still adopt at present is traditional remedies such as surgical operation, radiotherapy, chemotherapy etc., and in the face of a lot of advanced malignant tumors, it is at a loss what to do that traditional remedies seems, show that mainly curative ratio is low, recurrence rate and mortality ratio height, prognosis are relatively poor.Gene therapy is a kind of biological high-technology scheme for the treatment of disease by modifying gene, and people lodge at high enthusiasm to genetic treatment of tumor, and 66% gene therapy clinical trial all is used for oncotherapy.Up to the present, genetic treatment of tumor research has obtained impressive progress, for malignant tumour, gene therapy can utilize the difference of molecular level between tumour cell and normal cell, significantly widen " the treatment window " of tumour, efficiently, optionally killing tumor cell or the transfer that stops growth of tumour cell and prevent tumour.
Yet through more than ten years more than 600 clinical trial protocol find that although gene therapy is very safe, its anti-tumour effect is also very low, have in addition do not have any therapeutic action, this mainly is the problem of present stage gene therapy vector.Present carrier system can not all tumour cells of specificity transfection in human body, therapeutic gene is efficiently expressed in tumour, and tumour is to comprise the rapid process of multistep that several genes changes, and the modification by term single gene is difficult to utterly destroy tumour.These a series of problems seriously hamper the clinical efficacy of therapy of tumor.Therefore development carrier system efficient, target transfection tumor cell is most important to present genetic treatment of tumor.
Become the focus of scientific research with the viral therapy malignant tumour, and obtained new breakthrough.Several viruses that clinical experiment is recently used, as adenovirus ONYX-015, CV706, hsv G207 and G1716, result of treatment is encouraging.These viruses can optionally be duplicated in tumour cell, be bred, the kill tumor cell, the progeny virus that discharges is diffused into the tumour cell that closes on, and causes a kind of chain reaction in tumor tissues, the whole tumours of final elimination, and in normal cell, do not duplicate.This virus is called " tumour-specific virus of proliferation ", is called " melting tumor virus " again.
Implement the gene viruses treatment of tumour, at first will make up and can specificly in tumour cell, breed, and to the harmless virus of normal cell.Virogene is suddenlyd change, make up tomour specific proliferous type virus and mainly contain two kinds of methods, the one, utilize the necessary gene of tumor-specific promoters control virus replication, thus viral the duplicating of control.The 2nd, institute in normal cell is essential with virus replication, and unwanted gene lacks in tumour cell, and virus can only be duplicated in tumour cell.This project is intended to make up a kind of targeting gonad virus of two regulation and control, and these two kinds of methods are applied in the same adenovirus, improves the target of adenovirus, make adenovirus carrier more effective and safe be applied to clinical.
Utilizing the necessary gene of tumor-specific promoters control virus replication is one of method that makes up the tumour-specific virus of proliferation, these genes can only be expressed in tumour cell, and in normal cell, do not express, thereby make virus can only in tumour cell, duplicate, breed.The adenovirus e1a gene is the gene of expressing the earliest behind the adenovirus infection cell.The proteic function of E1A is very complicated, and it can promote cell to enter the S phase, i.e. the synthesis phase of cell cycle, help duplicating of virus; Simultaneously, E1A albumen can be used as trans-acting factor, activates the gene of virus, and other gene transcription in the cell, promotes duplicating of virus.In a word, e1a gene is that virus replication is necessary.Therefore use the expression of tumor-specific promoters control e1a gene, just can reach the purpose of control virus replication.In recent years studies show that Telomerase may be find so far one the new mark of tumour the most widely, plays an important role in cell immortalityization and tumor development process.Under physiological condition, normal cell does not mostly have telomerase activation; And the tumour cell Telomerase Activity height about 90%.And its active height is relevant with the grade malignancy of tumour, and the prompting Telomerase can be used as the good target spot of oncotherapy.Reverse transcriptase of telomere, promptly hTERT (human telomerase reverse transcriptase) is the nucleus of Telomerase mixture, its expression status and telomerase activation significant correlation are the key links of cell regulate and control telomerase activation.The hTERT expression of gene mainly is from the transcriptional level adjusted, transcribes rise in the tumour cell about 90%, and does not transcribe in the quiescent stage cell.Therefore, the hTERT promotor is used to control expression of exogenous gene, improves the target of therapy of tumor.
The hTERT promotor is a tumor-specific promoters widely, and expression of exogenous gene is limited in the tumour cell, so we think that the hTERT promotor is the outstanding candidate's promotor that makes up the tumour-specific virus of proliferation.This project is transformed wild-type adenovirus with the method for gene clone, the promotor that replaces e1a gene self with the hTERT promotor, made up a new tumour-specific replicative adenovirus, can make e1a gene specific expressed in tumour cell, and in normal cell, do not express, thereby in tumour cell, duplicate, breed, thus the kill tumor cell, and in normal cell, do not duplicate.
On this basis, we make e1a gene CR2 district disappearance 24bp with gene clone method simultaneously, E1A CR2 target Rb albumen, its effect is behind the adenovirus infection cell, with the Rb protein binding, discharge E2F albumen, promote that cell enters the S phase, for virus replication provides various associated protein and control environment.Rb protein mutation in 80% tumour cell, E1ACR2 albumen need not with it in conjunction with just duplicating, and in normal cell if lack this sequence, virus can't be duplicated.These two kinds of targets have broad spectrum and selectivity concurrently, studies have shown that cervical cancer, and lung cancer, liver cancer, colorectal carcinoma etc. all have certain curative effect.But also there is the high expression level phenomenon in these two kinds of methods also each defectiveness: hTERT in stem cell; Some virus infection can cause normal cell Rb approach sudden change (can cause the sudden change of normal liver cell Rb approach as the hepatitis B viruses (HBV) infection).Therefore, there is certain hidden danger in the security of the oncolytic adenovirus of single target, on this research basis, can use safely clinically in order to ensure the oncolytic adenovirus carrier, and we have made up the oncolytic adenovirus carrier of two targets of a new generation.The E1A albumen that contains genome sequence 922-947bp disappearance with the hTERT promoter regulation after improving, control duplicating of virus simultaneously at gene transcription level and protein translation level, confirm after deliberation, this method can largely improve the selectivity of adenovirus, thereby improves the security of adenovirus carrier.
Summary of the invention
The objective of the invention is to two kinds of cancer target advantages of tumor-specific promoters and deleted adenovirus E1ACR2 zone are combined in, the common proteic expression of regulating and controlling adenovirus E1A, thus a kind of safer tumour bi-target adenovirus carrier is provided.
Another object of the present invention is to make up a kind of novel hTERT promotor hTE1, and the tumor-targeting specificity of this promotor improves.
Another object of the present invention is to provide the construction process of a kind of novel hTERT promotor hTE1.
Another object of the present invention is to provide the construction process of a kind of tumour bi-target adenovirus carrier A dCN103.
Another object of the present invention is to provide a kind of tumour bi-target adenovirus carrier A dCN103 to be used for the treatment of the purposes of tumour.
For achieving the above object, the invention provides a kind of tumour bi-target adenovirus carrier A dCN103, this carrier has dual tumor-targeting, can be as a kind of better gene-virus treatment carrier platform.
The invention provides the construction process of a kind of hTERT promotor hTE1, its step comprises:
The A PCR method is a template with the DNA of HEKC HEK293, uses the hTERT Auele Specific Primer, obtain the PCR product and be hTERT promotor core-255～+ the 40nt sequence
B designs primer, introduces three E-box sequences in downstream primer
The present invention also provides the construction process of anticancer tumor double-target virus vector AdCN103, and its step comprises:
A, use PCR method, behind nucleus-255-+40nt of hTERT, insert three E-BOX and polyA sequence, be built into a kind of hTE1 of tumor-specific promoters efficiently;
B, use PCR method, obtain containing the plasmid vector of viral 947-1354bp, by EcoRV/Xba I double digestion, obtain an end for flat terminal, one section is sticking terminal fragment, uses Cla I (Klnow) and Xba I enzyme to cut the plasmid vector that contains the E1A complete sequence equally, obtain an end for flat terminal, one section big fragment for sticking end connects both with ligase enzyme, constituted the 24bp disappearance in E1A district.
C, the plasmid pCN103 that obtains and the plasmid pTG3602 that contains complete sequence adenovirus skeleton DNA are recombinated in intestinal bacteria BJ5183, produce E1A district by the plasmid vector control of hTERT promotor and that lack the complete adenovirus skeleton DNA of 24bp.
The method of structure bi-target adenovirus of the present invention can be used to develop the PTS of effective treatment tumour.
The present invention has following beneficial effect:
1, the present invention organically combines the method for two kinds of target tumors, has further improved the target and the security of adenovirus carrier, can be used for the treatment of kinds of tumors;
2, the invention provides the construction process of a kind of novel tumor specificity promoter hTE1, the tumour-specific of this promotor significantly improves.
3, the invention provides two target AdCN103 adenovirus construction methods, this method is easy to grasp;
4, the bi-target adenovirus AdCN 103 energy that makes up of the present invention and other non-replicatings adenovirus carrier synergy that has gene is for gene-virus combination therapy is from now on laid a solid foundation.
5, the bi-target adenovirus AdCN 103 of the present invention's structure proves optionally kill tumor cell through animal experiment, and does not influence normal cell; This novel bi-target adenovirus treatment can suppress tumor growth effectively, for the treatment cancer provides a good new technique platform.
Description of drawings
The structure sketch of four kinds of different hTERT promotors of Fig. 1
Fig. 2 is that hTE1 promotor and the transcriptional activity of other three kinds of hTERT promotors in tumour cell and normal cell compare
Fig. 3 shows that the hTE1 promotor does not have transcriptional activity in the normal mouse liver
Fig. 4 A is the structure synoptic diagram of bi-target adenovirus AdCN 103 of the present invention.
Fig. 4 B is the evaluation synoptic diagram of bi-target adenovirus AdCN 103 of the present invention.
Fig. 5 is the replication synoptic diagram of bi-target adenovirus AdCN 103 in normal cell or tumour cell.
Fig. 6 is mtt assay detect tumour cell and normal cell after the different virus of 10 MOI (multiplicity of infection) is handled 4 days survival rate.
Fig. 7 helps the specificity increment in tumour cell of non-replicability adenovirus for bi-target adenovirus AdCN 103 and the non-replicability adenovirus synergy of carrying green fluorescent protein.
Fig. 8 is the result schematic diagram of bi-target adenovirus AdCN 103 in nude mouse internal therapy tumor cell transplantation knurl.
Embodiment
The invention will be further described below in conjunction with specific embodiment, should be understood that following examples only are used to the present invention is described and are not used in the scope of the present invention that limits.
The A plasmid construction of being correlated with
Design following primer earlier:
255-P1：5’-CTGCGCTGTCGGGGCCAGGC-3’
255-P2：5’-ATCGGCCAGGGCTTCCCACG-3’
255-4DEB-P2：5’-CCCACGTGCGCCCACGTGCGCCCACGTGCGATCGGCCAGGGCTTCCCACG-3’
408-P1：5’-CCACATCATGGCCCCTCCCT-3’
408-P2：5’-GCTGCCTGAAACTCGCGCCG-3’
1125-P1：5’-CTGTCCTGCGGTTGTGCCGG-3’
Using PCR method, is template with the DNA of HEKC HEK293, primer 2 55-P1,255-P2, obtain PCR product hTERT1, for hTERT promotor core-255～+ the 40nt sequence, primer 408-P1,408-P2 obtains PCR product hTERT3, for hTERT promotor core-408～+ the 40nt sequence, primer 1125-P1,408-P2, obtain PCR product hTERT3, for hTERT promotor core-1125～+ the 40nt sequence
Use PCR method, primer 2 55-P1,255-4DEB-P2 is a template with hTERT1, core-255～+ add three E-box sequences after the 40nt sequence, obtain PCR product hTERT2
With hTERT1, hTERT2, hTERT3, hTERT4 are cloned on the T carrier respectively, difference called after pBT-255, pBT-2554DEB, pBT-418, pBT-SE
The following two groups of primers of B design
Ad195’-GAGTGCCAGCAGTAGAGTT-3’
Ad205’-GAAAATCCAGCAGGTACCCC-3’
Ad215’-GGGGTACCTGCTGGATTTTC-3’
Ad225’-GAATTCTCAATCTGTATCTTCATC-3’
With two step PCR method, with pXC1 (Microbix, Toronto, Canada) plasmid is a template, at first respectively with Ad19/Ad20, two groups of primers of Ad21/Ad22, obtain two groups of PCR products, then two groups of PCR products are mixed into template, with Ad19/Ad22 is primer, obtain adenoviral gene group 504-3510bp segment, correspond to adenovirus E 1 zone complete sequence. with the PCR product cloning in the pGEM-T-EASY plasmid, called after pGEM-T-E1, order-checking confirms that sequence is correct, the success of structure plasmid. subsequently, cut from the pGEM-T-E1 enzyme and to obtain E1 district segment, be cloned into the Not I site of pcDNA3, be configured to pCMV-E1A, further E1 district segment is cloned into the EcoRV site that the pBluescript KS plasmid of two BGH polyA sequences is arranged in BamH I site then, wherein BGH polyA sequence can intercept the effect of residual E1A promotor. and four EcoRV sites that hTERT promotor segment is inserted this plasmid respectively that will before make up are configured to pBT-255-E1A, pBT-2554DEB-E1A, pBT-418-E1A, pBT-SE-E1A. plasmid pBT-2554DEB-E1A is pBT-hTE1-E1A.
B virus recombination to construct
With Sal I and Spe I respectively from pBT-255-E1A, pBT-2554DEB-E1A, pBT-418-E1A, pBT-SE-E1A. downcut the hTERT-E1A segment, be cloned into the plasmid pTGC1960 (Microbix of gland-containing virus homology arm, Ontario, Canada) in, with these shuttle plasmids respectively with adenovirus skeleton packaging plasmid pBGH10 (Microbix, Ontario, Canada) cotransfection N52.E6 cell, behind the plaque purifying, obtain virus of A dBT-255-E1A, AdBT-2554DEB-E1A, AdBT-418-E1A, AdBT-SE-E1A.
Reporter gene plasmid pCMV β-ga1 is available from Clontech company, and beta-galactosidase enzymes is expressed in the control of CMV promotor down; The analysis of Photinus pyralis LUC reporter gene with plasmid pGL3-Basic (not containing promotor and enhanser), pGL3-Control (containing SV40 promotor/enhanser) available from Promega company.
Four kinds of hTERT promotors are cut with the KpnI+BglII enzyme, be cloned into the pGL3-Basic plasmid, called after pGL3-BT-255, pGL3-BT-2554DEB, pGL3-BT-418, pGL3-SE.In this plasmid, the hTERT promotor starts Photinus pyralis LUC (Luciferase) expression of gene.Cell is by 1.0 * 10
5Be inoculated in 6 well culture plates, behind the 24h, will contain Photinus pyralis LUC reporter gene plasmid 1 μ g, interior mark beta-galactosidase enzymes reporter gene pCMV-β Gal 0.1 μ g transfectional cell with LipofectAMINE (GIBCO BRL).Behind the 48h, collecting cell, PBS washes twice, and cracking is in Reporter Lysis Buffer (Promega company).A part and Photinus pyralis LUC substrate (LuciferaseAssay test kit, Promega company) reaction (10sec), another part and beta-galactosidase enzymes chemical luminous substrate (Clontech company) reaction (37 ℃ of 30min) is gone up at photofluorometer (BG-P company) respectively and is measured the luminous photon counting of 10sec (RLU).Calculate luciferase RLU/ tilactase RLU and promptly get the plain enzymic activity of relative fluorescence.
Shown in figure two A, B, in tumour cell, though the activity of pGL3-BT-255 is the highest, pGL3-BT-2554DEB's is active lower slightly, and in various normal cells, the activity of pGL3-BT-2554DEB is starkly lower than the activity of other three kinds of promotors.The data sheet oolemma has the hTE1 promotor of three extra E-box sequences to have good tumor cell specific.
Per four to six Balb/c nude mices are one group, totally six groups.The 20ug plasmid DNA is dissolved in the 1.6ml physiological saline intravenous injection nude mice respectively.Put to death nude mice after 24 hours, after liver was collected, PLB (Promega) solution that contains Ultra-TurraxT25 with 1.5ml was made homogenate with liver.Collection is through 14, and the supernatant solution after 4 ℃ of centrifugal 10 minutes centrifugal backs of 000rpm or the filtration is measured the fluorescence activity of respectively organizing sample.
Shown in figure three, have only pGL3-BT-2554DEB in the nude mice liver, can't detect luciferase expression fully, show that its hTE1 promotor tumor-targeting is very remarkable, normal liver tissue is had no side effect substantially.
A, at E1A district disappearance CR224bp
Design following primer earlier:
Ad49：5’-CACCCAGTGACGACGAGGATGAA-3’
Ad50：5’-TATTGCATTCTCTAGACACAGGTG-3
PTG3602 is the plasmid vector that contains 5 type adenovirus complete sequences.At first use Bgl II digested plasmid pTG3602, obtain one section fragment about the 6k that contains the E1A district after enzyme is cut, connect transformed into escherichia coli Gm2163 certainly, called after pCN001.With pTG3602 for touching plate, obtain adenoviral gene group fragment 947-1354bp with primer Ad49/Ad50, after being cloned into into the T carrier, with plasmid called after pTE/Ad947-1354, behind EcoRV+Xba I double digestion, wherein the 400bp endonuclease bamhi be connected the plasmid pCN101 that obtains in E1A district 922-947bp disappearance through the 6kb fragment that Cla I (Klenow)+Xba I double digestion pCN001 obtains.
The plasmid pCN101 that obtains is 922-947bp disappearance wherein after measured, and the result is consistent with gross data, shows that plasmid construction successfully.
The structure of B, hTE1 promotor:
1 disappearance wild-type E1A district promotor
Ad55：5’-GGATCCGAATTCTTAATTAA-3’
Ad56：5’-GCGGCCGCAAATATTACGCGCTATGAGT-3’
Ad52：5’-GCGGCCGCCTCGAGGATATCGAATTCGTCGAC?GCCGCTCCGACACCGGGACT-3’
Ad51：5’-ATCGATCACCTCCGGTACAA-3’
With the pTG3602 plasmid is the template of PCR reaction, and primer Ad55, Ad56 and Ad52, Ad51 carry out the PCR reaction respectively and (see Molecular Cloning:A Laboratory Manual for details, 3
RdEd., Joseph Sambrook andDavid W.Russell), fragment is reclaimed in the leakage of electricity swimming, obtains the PCR product.Two kinds of PCR products are cut with Not I enzyme respectively, connect into E1A1-922 (Δ) fragment of disappearance E1A promotor, this fragment is cloned into arbitrarily on the T carrier, cut with BamH I/Cla I enzyme, the 779bp fragment links to each other with the big fragment that plasmid pCN101 that identical enzyme is cut obtains, be built into novel plasmid pCN002.
PBlueScript KS plasmid (available from Stratagene) contains 2 BGH poly A tailing signals in BamH I site, downcut the poly A of 115bp with Not I/XhoI, insert the disconnected place of pCN002 wild-type E1A promoter deletion, further block the effect of wild-type E1A promotor as insulator, be configured to pCN003.
2 insert the hTE1 promotor
With plasmid pBT-2554DEB is template, primer: upstream 5 '-GTGCAGGTGCCAGAACATTTCT-3 ', downstream 5 '-CAGTACCGGATATCCAAGCTCCCA-3 ' (comprising the EcoRV restriction enzyme site), the PCR product inserts the multiple clone site before the pBlueScript polyA after the HindIII/EcoRV enzyme is cut, be built into pBlueScript-TE1, after further cutting with the XhoI/.EcoRV enzyme, cutting product with the enzyme of pCN003 is connected, it is consistent with expected results through the order-checking evaluation to be built into complete two targeting vector phTE1-delat24, shows the vector construction success.
The structure of the structure of C, two target virus of A dCN103
PhTE1-delat24 and pTG3602 after the reorganization, obtain complete bi-target adenovirus carrier pCN103 in intestinal bacteria BJ5183 after ClaI enzyme tangent typeization, transform HEKC HEK293, packaging virus.(consult He, T.C., Zhou, S., da Costa, L.T., Yu, J., Kinzler, K.W., and Vogelstein, B.A simplifiedsystem for generating recombinant adenoviruses.Proc.Natl.Acad.Sci.USA, 95:2509-2514,1998) viral amplification, evaluation: 293 cells are laid on a small amount of amplicon virus in 24 orifice plates.Viral DNA obtains with Qiagen Blood Kit, utilizes round pcr, identified gene virus hTERT promotor and 24bp disappearance.It is synthetic to identify that the primer is given birth to the worker by Shanghai.
HTERT upstream: 5 '-AGTAGTAGTGTGGCGGAAGT-3 '
HTERT downstream: 5 '-ACGAATTCGATATCCAAGCTCCCA-3 '
24bp lacks upstream: 5 '-TTACCTGCCACGAGGCTGGCTTTC-3 '
24bp lacks downstream 5 '-T CTGCTGTGCGTAACTTG-3 '
24bp positive control upstream 5 '-CCGTCTTTTGGACCAGC-3 '
24bp positive control downstream 5 '-TCTGCTGTGCGTAACTTG-3 '
As template, simultaneously in contrast, carry out the PCR reaction with the viral DNA of Qiagen Blood Kit extracting gained with above-mentioned three groups of primers with wild-type virus DNA.The PCR condition: 94 ℃ * 1min, 55 ℃ * 1min, 72 ℃ * 2min15s.The PCR product is detected, contain hTE1 as the PCR product, do not contain 24bp wild-type adenovirus DNA, virus packets is dressed up merit.Repeat this process once, obtain the correct adenovirus of recombinating.
Bi-target adenovirus AdCN 103 is breeding in a large number in 293 cells, uses the caesium chloride density gradient centrifugation purified virus.Concrete operation method is seen the operation instructions of Microbix Biosystem Inc..
With 3 * 10
5Normal cell or tumour cell be laid on 6 orifice plates, behind the 24h, the AdCN101 (single target virus) that adds 5MOI, AdCN102v (single target virus), AdCN103 (two target virus), WtAd5 infects hepatoma cell strain BEL7404 respectively, SMMC7721, Hep3B cell (available from the Shanghai Inst. of Life Science, CAS cell bank), MHCC-97 (available from last marine mountain hospital cell bank) and normal human embryonic lung cell's strain NHLF, WI-38 (available from ATCC) normal people lung MRC-5 cell strain (available from the Shanghai Inst. of Life Science, CAS cell bank), normal liver cell's strain L02 (available from the Shanghai Inst. of Life Science, CAS cell bank).Behind the 48h, collecting cell supernatant and cell ,-20 ℃ with 37 ℃ of multigelations 3 times with releasing virus.With viral dilution, detect virus titer.293 cells are laid on 60mmdish, and cell adds and contains different extent of dilution (10 near covering with behind the 24h
-1-10
-8) virus, 37 ℃ were infected after 2 hours, shop 8ml low melting point glue (5%FBS, 1.25%Agarose).Numeration about 9 days.Calculate the virus numbers that every PFU virus produces.The result is shown in figure five.
By the result of figure five as seen, in tumour cell, AdCN101 (single target virus), AdCN102 (single target virus), AdCN103 (two target virus), the replication of WtAd5 is roughly the same.And in normal cell, the replication of AdCN103 (two target virus) significantly reduces, and WtAd5 is not obvious at tumour cell and Normocellular replication difference, does not have selectivity; And AdCN101 (single target virus), AdCN102 (single target virus), AdCN103 (two target virus) can optionally duplicate in tumour cell, and the selectivity of AdCN103 (two target virus) is best.
The survival rate of cell after virus treated detects (Cancer Research, 1989,49 (17): 4785-90) by mtt assay.Step is as follows: with liver cancer cell strain BEL7404, SMMCC7721 and normal human embryonic lung cell NHLF and normal people's pneumonocyte MRC-5 spread into 96 orifice plates with the amount in 5000 every holes, cultivate the virus that adds 10MOI after 24 hours respectively, act on 1 day, then once a day, the nutrient solution that will contain virus in continuous four days is removed, change the normal nutrient solution that contains 5mg/ml MTT into, cultivating the nutrient solution that will contain MTT after 4 hours removes, with the DMSO cracking, shaking up, is that reference is measured 595nm place absorbancy with 655nm place absorbancy then.
Cell survival rate (%)=A595 (sample)/A595 (contrast) * 100%.
The result is shown in figure six, and two target virus of A dCN103 have certain lethal effect to tumour cell, and very little to normal cytotoxicity, have tumor-selective.
Embodiment 7, two target replication competent adenovirus virus of A dCN103 and the synergy of non-replicating virus of A d-EGFP in tumour cell
Prepare two and contain 1 * 10
6The Tissue Culture Dish of the 10cm of BEL7404 is used non-replication competent adenovirus Ad-EGFP and two target replication competent adenovirus virus of A dCN103 co-infected tumour cells respectively, and virus absorption was changed clean DMEM nutrient solution after two hours, hatched three days.Cell is collected two kinds of cell conditioned medium liquid respectively through three 37 ℃/-20 ℃ multigelation releasing virus then, infects off-the-shelf two once more respectively and contains 1 * 10
6The Tissue Culture Dish of the 10cm of BEL7404 is used fluorescence microscope virus replication situation two days later.Experimental result shows that under two target replication competent adenovirus virus of A dCN103 synergies, non-replicating virus of A d-EGFP can duplicate propagation in tumour cell.
With the 4-5 nude mice subcutaneous vaccination hepatoma cell strain BEL7404 in age in week, laggard action thing grouping in 12 days.The treatment group gives 1 * 10
9The AdCN103 of pfu (two target virus) treats, five groups of contrast components, the 1st group is phosphoric acid buffer (PBS) treatment group, the 2nd, 3, use AdCN101 (single target virus), AdCN102 (single target virus), the WtAd5 virus of same dose to treat respectively for 4 groups, test-results is shown in figure eight.
By the result of figure eight as seen, the AdCN103 curative effect approaches WtAd5 and two kinds of single target viruses, can suppress growth of tumor, prolongs the tumor bearing nude mice life cycle.
Claims (4)
1. tumor double-target rf oncolytic adenovirus, it is characterized in that, with the proteic expression of E1A in any one tumor-specific promoters (as the hTERT promotor, AFP promotor etc.) control disappearance CR2 zone, this rf oncolytic adenovirus has dual-target.
2. a novel modified tumor-specific promoters hTE1 is characterized in that: add three E-box in hTERT promotor-255-+40 core sequence downstream, improve the selectivity of hTERT promotor to tumour cell
3. tumor double-target rf oncolytic adenovirus AdCN103 as claimed in claim 1, it is characterized in that, with the proteic expression of E1A that will try to achieve tumor-specific promoters hTE1 control disappearance CR2 zone as right 2, this rf oncolytic adenovirus has dual-target.
4. the construction process of a tumor double-target rf oncolytic adenovirus AdCN103 as claimed in claim 1, it is characterized in that: described method comprises the following steps: successively
A, use PCR method, behind nucleus-255-+40nt of hTERT, insert three E-BOX and polyA sequence, be built into a kind of hTE1 of tumor-specific promoters efficiently;
B, use PCR method, obtain containing the plasmid vector of viral 947-1354bp, by EcoRV/Xba I double digestion, obtain an end for flat terminal, one section is sticking terminal fragment, uses Cla I (Klnow) and Xba I enzyme to cut the plasmid vector that contains the E1A complete sequence equally, obtain an end for flat terminal, one section big fragment for sticking end connects both with ligase enzyme, constituted the 24bp disappearance in E1A district.
C, the E1A district that uses the hTE1 promotor to replace wild-type adenovirus E1A district promotor control 24bp to lack, obtain two target plasmid pCN103 and in intestinal bacteria BJ5183, recombinate, produce the plasmid vector of E1A district by the complete adenovirus skeleton DNA control of hTERT promotor and disappearance 24bp with the plasmid pTG3602 that contains complete sequence adenovirus skeleton DNA.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105002145A (en) * | 2015-07-01 | 2015-10-28 | 天津农学院 | Method for constructing recombinant adenovirus by using mTERT and mTyr double promoters to jointly regulate and control HN gene, recombinant adenovirus and application |
| CN114703186A (en) * | 2022-03-31 | 2022-07-05 | 四川大学 | Tumor specific promoter and application thereof |
| CN116036313A (en) * | 2023-02-02 | 2023-05-02 | 中国医学科学院血液病医院(中国医学科学院血液学研究所) | A composition for treating liver cancer based on mesenchymal stem cells |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105002145A (en) * | 2015-07-01 | 2015-10-28 | 天津农学院 | Method for constructing recombinant adenovirus by using mTERT and mTyr double promoters to jointly regulate and control HN gene, recombinant adenovirus and application |
| CN105002145B (en) * | 2015-07-01 | 2017-12-15 | 天津农学院 | Method and recombined adhenovirus and application using the gene constructed recombined adhenovirus of mTERT and mTyr double-promoter combined regulating HN |
| CN114703186A (en) * | 2022-03-31 | 2022-07-05 | 四川大学 | Tumor specific promoter and application thereof |
| CN114703186B (en) * | 2022-03-31 | 2023-08-29 | 四川大学 | Tumor-specific promoters and their applications |
| CN116036313A (en) * | 2023-02-02 | 2023-05-02 | 中国医学科学院血液病医院(中国医学科学院血液学研究所) | A composition for treating liver cancer based on mesenchymal stem cells |
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