CN101381742A - Oncolytic adenovirus pCN305 vector targeting late promoter regulation and its construction method and application - Google Patents
Oncolytic adenovirus pCN305 vector targeting late promoter regulation and its construction method and application Download PDFInfo
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Abstract
本发明公开了一种晚期启动子靶向性调控溶瘤腺病毒pCN305载体及其构建方法与应用；该载体是在具有靶向性的pCN103载体的基础上加以构建，通过引入内部核糖体进入位点IRES和多克隆位点，利用病毒自身的晚期启动子来调控表达所有具有杀伤和抑制各类癌症的外源基因如IL－24、TRAIL、SOCS3、cpp－SOCS3、SOCS1、cpp－SOCS1，且通过在体内外实验均显现出很好的抗癌效果。应用本发明构建的所有双靶向复制型溶瘤腺病毒如AdCN305－IL－24、AdCN305－TRAIL、AdCN305－SOCS3、AdCN305－cpp－SOCS3、AdCN305－SOCS1和AdCN305－cpp－SOCS1都可用于治疗各类肿瘤，并可以开发出有效的治疗肿瘤的病毒-基因抗癌新药。The present invention discloses an oncolytic adenovirus pCN305 carrier with late promoter targeting regulation and its construction method and application; Point IRES and multiple cloning sites, use the late promoter of the virus itself to regulate and express all exogenous genes that can kill and inhibit various cancers, such as IL-24, TRAIL, SOCS3, cpp-SOCS3, SOCS1, cpp-SOCS1, and Both in vivo and in vitro experiments showed good anticancer effects. All dual-targeting replication-type oncolytic adenoviruses constructed by the present invention, such as AdCN305-IL-24, AdCN305-TRAIL, AdCN305-SOCS3, AdCN305-cpp-SOCS3, AdCN305-SOCS1 and AdCN305-cpp-SOCS1, can be used to treat various Tumor-like tumors, and effective virus-gene anti-cancer new drugs for tumor treatment can be developed.
Description
Technical field
The invention belongs to field of gene, particularly a kind ofly utilize two targets (cancer target) oncolytic adenovirus construction of carrier of self late promoter efficiently expressing exogenous gene and carry the application that all have killing and wounding, suppress foreign gene such as IL-24, TRAIL, SOCS3, cpp-SOCS3, SOCS1 and the cpp-SOCS1 of cancer.
Background technology
Malignant tumour serious harm human health, clinically most late tumor patients are still lacked effective treatment means at present, and conventional treatment is except having certain toxic side effect and resistance, also have in killing tumor cell, the difficult problem that healthy tissues and cell are suffered damage is especially particularly serious to the infringement of hemopoietic tissue and cell.Therefore, medical work personnel and patient expect to have a kind of new effective methods of treatment for the treatment of tumour to occur.
Obtain develop rapidly when Celluar and Molecular Biology is theoretical with technology, gene therapy tumour strategy arises at the historic moment.And along with virusology and oncology fast development, people attempt improved virus is used for the research work of oncotherapy again.
The target gene of cancer-viral therapy strategy is to utilize oncolytic virus to improve strong lethal effect or the restraining effect of antioncogene to cancer cells in specifically inside tumor cell cracking, propagation.This virus vector with the traditional tumour gene therapy has in essence different, and the latter is a kind of non-replicability virus, as just launch vehicle antioncogene is transported in tissue or the cell, and no target of carrier itself and replication does not have therapeutic action yet.Target gene-viral therapy makes full use of that viral volume is little, reproducible and the strong advantage of diffusibility, can at the tumour primary lesion and metastasis is local forms high concentration virus, and excites immunity system to reply to strengthen knurl effect extremely; Antioncogene target ground can also be transported in the tumour cell simultaneously, antioncogene can increase copy number along with duplicating of virus, improves the expression amount of antioncogene, thereby both act synergistically and further improve antineoplastic treatment
Be divided into virus type and non-virus type two big classes as Vectors in Gene Therapy at present.Virus vector comprises: adenovirus, adeno-associated virus, retrovirus, slow virus and simplexvirus etc.Virus vector transfection efficiency height, expression time is long, but immunogenicity is strong, and certain risk is arranged.Non-virus carrier comprises: the DNA of naked DNA, liposome and other material parcel.Non-virus carrier then immunogenicity is little and security good, but transfection efficiency is low, and gene stability is poor, and expression time is short.Use at most still virus vector at present.And adenovirus is wide with its host range in numerous virus vector, and pathogenic low, bale capacity is big, and producing the titre advantages of higher becomes most widely used gene therapy vector.According to the recent statistics of http://www.wiley.co.uk/genmed, the whole world has 1145 gene therapy schemes and enters clinically, and wherein 67% is used for treatment for cancer, and is that the research of carrier is the most extensively and up to 25% with the adenovirus.
But most of carrier systems still exist therapeutic gene is efficiently expressed specifically in tumour cell, thereby can not thoroughly kill tumour cell and seriously hamper the clinical efficacy of therapy of tumor.In the tumour patient body, if can only kill the part tumour cell, residual small part cell causes tumor recurrence with very fast growth.Therefore development carrier system efficient, target transfection tumor cell is most important to present genetic treatment of tumor.
Summary of the invention
The objective of the invention is at the deficiencies in the prior art, a kind of structure and application thereof of late promoter targeting regulation oncolytic adenovirus pCN 305 carrier are provided.
The objective of the invention is to be achieved through the following technical solutions:
A kind of late promoter targeting regulation oncolytic adenovirus pCN 305 carrier, it has the gene order of SEQ ID No.1.
A kind of above-mentioned late promoter targeting regulation oncolytic adenovirus pCN 305 construction of carrier may further comprise the steps:
(1) design following primer:
Ad66:5’CCAGAATATTGGAACTTTAG?3’
BstXI
Ad67:5’GGATCCATCGAGGATCCTCATTCTTGGGCAATGTATGAA?3’
BamHI
Ad68:5’GGATCCGTCGACTCGCGAGAATCGTTTGTGTTATGTT?3’
BamHI
Ad69:5’CTTAAGTGAGCTGCCCGGGGA?3’
AflII；
(2) make up transfer vector pCZ301:
With HindIII digested plasmid pTG1801, enzyme cuts the fragment that the 2937bp size is reclaimed in the leakage of electricity swimming of full back, with this fragment with same with the HindIII enzyme cut digestion also the carrier pGEM-11Zf (+) of dephosphorization be connected, transform, get correct positive colony, called after pCZ301;
(3) make up transfer vector pCZ302:
With plasmid pTG3602 is template, carries out PCR with primer Ad66, Ad67 and Ad68, Ad69 respectively, and electrophoresis reclaims product and difference called after A8 and A9, and A8 is 341bp, and A9 is 339bp; To be connected, transform with A8 and the carrier pGEM-Teasy that the BamHI enzyme is cut digestion with BstXI, get correct clone and called after pGEM-T/A8.Similarly, will be connected, transform with A9 and the carrier pGEM-Teasy that the AflII enzyme is cut digestion, get correct clone and called after pGEM-T/A9 with BamHI.Cut digested plasmid pGEM-T/A8 with BstXI and BamHI enzyme then, Segment A 8 is reclaimed in leakage of electricity swimming, and it is cut the plasmid pGEM-T/A9 that digested with BstXI with the BamHI enzyme and be connected, transform with same, gets correct positive colony called after pGEM/A8-A9.Cut the plasmid pGEM/A8-A9 that obtains with BstXI and AflII enzyme, electrophoresis reclaims the Segment A 8-A9 of 650bp, and it is cut the plasmid pCZ301 that digested with BstXI with the AflII enzyme and be connected, transform with same, gets correct positive colony called after pCZ302;
(4) make up transfer vector pCZ303:
With SphI digested plasmid pTG1801, enzyme cuts the leakage of electricity swimming of full back and reclaims the fragment of 10248bp, and makes it from connecting, transforming, and gets correct positive colony called after pCZ303.
(5) make up transfer vector pCZ304:
The HindIII enzyme is cut pCZ302, and electrophoresis reclaims the fragment of 2937bp size.It is connected, transforms with the same plasmid pCZ303 that cuts also dephosphorization with the HindIII enzyme, get correct positive colony called after pCZ304.
(6) make up transfer vector pCZ305:
SphI single endonuclease digestion pTG3602, electrophoresis reclaims the fragment of 6126bp.It is connected, transforms with the same carrier pCZ304 that cuts also dephosphorization with the SphI enzyme, get correct positive colony called after pCZ305, pCZ305 has the gene order of SEQ ID No.1.
A kind of method of using the above-mentioned two target rf oncolytic adenovirus of late promoter targeting regulation oncolytic adenovirus pCN 305 carrier acquisition is characterized in that, may further comprise the steps:
(1) internal ribosome entry site IRES and termination signal polyA are cloned on the carrier pBluescript-sk, form the transfer vector pBC/polyA that contains IRES, MCS and polyA expression cassette.
(2) carry out homologous recombination by the electricity method of changeing the adenovirus skeleton plasmid pCN103 that in bacterium, makes two targets and recombinant plasmid pCZ305-IL-24, the pCZ305-TRAIL, pCZ305-SOCS3, pCZ305-cpp-SOCS3, pCZ305-SOCS1, the pCZ305-cpp-SOCS1 that contain cancer suppressor gene IL-24, TRAIL, SOCS3, cpp-SOCS3, SOCS1, cpp-SOCS1, obtain recombinant plasmid pCN305-IL-24, pCN305-TRAIL, pCN305-SOCS3, pCN305-cpp-SOCS3, pCN305-SOCS1, pCN305-cpp-SOCS1.
(3) enzyme is cut the recombinant adenovirus plasmid pCN305-IL-24, the pCN305-TRAIL that expose packaging signal after the digestion, pCN305-SOCS3, pCN305-cpp-SOCS3pCN305-SOCS1, pCN305-cpp-SOCS1 respectively transfection in 293 cells, make it be packaged into two target rf oncolytic adenovirus AdCN305-IL-24, AdCN305-TRAIL, AdCN305-SOCS3, AdCN305-cpp-SOCS3, AdCN305-SOCS1 and AdCN305-cpp-SOCS1; Make it efficiently express IL-24, TRAIL, SOCS3, cpp-SOCS3, SOCS1 and cpp-SOCS1 protein molecular.
A kind ofly use above-mentioned method and obtain the purposes that two target rf oncolytic adenovirus are used to prepare the medicine for the treatment of tumour.
The invention has the beneficial effects as follows:
1. the present invention combines the virus therapy and the gene therapy of tumour effectively, but efficiently expressing exogenous gene has strengthened kill capability to tumour with respect to simple virus therapy.
2. the present invention has adopted a kind of strategy of dual-target tumour, has strengthened the security of adenoviral gene treatment carrier greatly.
3. the present invention utilizes viral late body promotor to come the regulating and expressing foreign gene, has guaranteed only just expression under the situation of virus replication of foreign gene, with respect to exogenous promotor, has increased the specificity of genetic expression.
4. the present invention makes all kill and wound the gene such as IL-24, TRAIL, SOCS3, cpp-SOCS3, SOCS1 and the cpp-SOCS1 that suppress cancer by the New-type adenovirus gene therapy vector that makes up and has brought into play tumor-killing effect preferably.
Description of drawings
Fig. 1 makes up schema for shuttle plasmid pCZ305;
Fig. 2 is the structure schema of shuttle plasmid pBC/polyA;
Fig. 3 carries the structure schema of cancer suppressor gene IL-24 for two target rf oncolytic adenovirus PCN305 carriers;
Fig. 4 is for adopting SOCS3 behind the resulting virus infection of Western-blot method and the ability to express figure in the cpp-SOCS3 cell;
Fig. 5 is anticancer factor S OCS3 and cpp-SOCS3 inducing apoptosis of tumour cell synoptic diagram;
Fig. 6 is that virus of A dCN305-SOCS3 and cpp-SOCS3 are at normal cell and the replication synoptic diagram in tumour cell;
(a)～(e) of Fig. 7 is respectively mtt assay and detects virus of A dCN305-SOCS3 and AdCN305-cpp-SOCS3 at normal cell and tumour cell 5 days survival rate synoptic diagram after the different virus of 10MOI (multiplicity of infection) is handled;
(a) of Fig. 8 and (b) be respectively AdCN305-SOCS3 and AdCN305-cpp-SOCS3 result schematic diagram in nude mouse internal therapy tumor cell transplantation knurl.
Embodiment
The invention provides and a kind ofly utilize self late promoter regulating and expressing all kill and wound construction process and the application of two target rf oncolytic adenovirus gene therapy vector PCN305 of the foreign gene that suppresses cancer such as SOCS1, cpp-SOCS1, SOCS3, cpp-SOCS3, TRAIL and IL-24.
This invention provides for example construction process of pCN305-IL-24, pCN305-TRAIL, pCN305-SOCS3, pCN305-cpp-SOCS3, pCN305-SOCS1 and pCN305-cpp-SOCS1 of carrier. and its step comprises:
One, by the termination signal in deleted adenovirus Fiber gene downstream, is built into the metastasis transplanting physique grain pCZ305 that contains homology arm with pCN103.
The method that makes up metastasis transplanting physique grain pCZ305 is specific as follows:
1. at first design following primer:
Ad66:5’CCAGAATATTGGAACTTTAG?3’
BstXI
Ad67:5’GGATCCATCGAGGATCCTCATTCTTGGGCAATGTATGAA?3’
BamHI
Ad68:5’GGATCCGTCGACTCGCGAGAATCGTTTGTGTTATGTT?3’
BamHI
Ad69:5’CTTAAGTGAGCTGCCCGGGGA?3’
AflII
2. make up transfer vector pCZ301:
With HindIII digested plasmid pTG1801, enzyme cuts the fragment that the 2937bp size is reclaimed in the leakage of electricity swimming of full back.With this fragment with same with the HindIII enzyme cut digestion also the carrier pGEM-11Zf (+) of dephosphorization be connected, transform, get correct positive colony, called after pCZ301.
3. make up transfer vector pCZ302:
With plasmid pTG3602 is template, carries out PCR with primer Ad66, Ad67 and Ad68, Ad69 respectively and (sees Molecular Cloning:A Laboratory Manual for details, 3
RdEd., Joseph Sambrookand David W.Russell), electrophoresis reclaims product and difference called after A8 (341bp) and A9 (339bp).To be connected, transform with A8 and the carrier pGEM-Teasy that the BamHI enzyme is cut digestion with BstXI, get correct clone and called after pGEM-T/A8.Similarly, will be connected, transform with A9 and the carrier pGEM-Teasy that the AflII enzyme is cut digestion, get correct clone and called after pGEM-T/A9 with BamHI.Cut digested plasmid pGEM-T/A8 with BstXI and BamHI enzyme then, Segment A 8 is reclaimed in leakage of electricity swimming, and it is cut the plasmid pGEM-T/A9 that digested with BstXI with the BamHI enzyme and be connected, transform with same, gets correct positive colony called after pGEM/A8-A9.Cut the plasmid pGEM/A8-A9 that obtains with BstXI and AflII enzyme, electrophoresis reclaims the Segment A 8-A9 of 650bp, and it is cut the plasmid pCZ301 that digested with BstXI with the AflII enzyme and be connected, transform with same, gets correct positive colony called after pCZ302.
4. make up transfer vector pCZ303:
With SphI digested plasmid pTG1801, enzyme cuts the leakage of electricity swimming of full back and reclaims the fragment of 10248bp, and makes it from connecting, transforming, and gets correct positive colony called after pCZ303.
5. make up transfer vector pCZ304:
The HindIII enzyme is cut pCZ302, and electrophoresis reclaims the fragment of 2937bp size.It is connected, transforms with the same plasmid pCZ303 that cuts also dephosphorization with the HindIII enzyme, get correct positive colony called after pCZ304.
6. make up transfer vector pCZ305:
SphI single endonuclease digestion pTG3602, electrophoresis reclaims the fragment of 6126bp.It is connected, transforms with the same carrier pCZ304 that cuts also dephosphorization with the SphI enzyme, get correct positive colony called after pCZ305, pCZ305 has the gene order of SEQ ID No.1.
Two, internal ribosome entry site (IRES) and termination signal polyA are cloned on the carrier pBluescript-sk, form the transfer vector pBC/polyA that contains IRES, MCS and polyA expression cassette.
Three, in bacterium, make the adenovirus skeleton plasmid pCN103 of two targets and contain cancer suppressor gene such as IL-24 by the electricity method of changeing, TRAIL, SOCS3, cpp-SOCS3, SOCS1 and cpp-SOCS1 (above-mentioned range gene all can be found in gene pool) recombinant plasmid pCZ305-IL-24, pCZ305-TRAIL, pCZ305-SOCS3, pCZ305-cpp-SOCS3, pCZ305-SOCS1 and pCZ305-cpp-SOCS1 carry out homologous recombination, obtain recombinant plasmid pCN305-IL-24, pCN305-TRAIL, pCN305-SOCS3, pCN305-cpp-SOCS3, pCN305-SOCS1 and pCN305-cpp-SOCS1.
Four, enzyme is cut the recombinant adenovirus plasmid that exposes packaging signal after the digestion such as pCN305-IL-24, pCN305-TRAIL, pCN305-SOCS3, pCN305-cpp-SOCS3pCN305-SOCS1 and pCN305-cpp-SOCS1 respectively transfection in 293 cells, make it be packaged into virus and it efficiently expressed as IL-24, TRAIL, SOCS3, cpp-SOCS3, SOCS1 and cpp-SOCS1 protein molecular as AdCN305-IL-24, AdCN305-TRAIL, AdCN305-SOCS3, AdCN305-cpp-SOCS3, AdCN305-SOCS1 and AdCN305-cpp-SOCS1.
The invention will be further described below in conjunction with specific embodiment, should understand following examples and only be used to the present invention is described and be not used in the scope of the present invention that limits.
Describe the present invention in detail with specific embodiment with reference to the accompanying drawings below, it is more obvious that purpose of the present invention and effect will become.
Be example with cancer suppressor gene IL-24 and SOCS3 below, two target rf oncolytic adenovirus PCN305 construction of carrier be described and be used to prepare the structure flow process and the result of treatment of the medicine for the treatment of tumour.
Embodiment 1:
The structure of the adenovirus carrier carrier PCN305 of the cancer target of portability foreign gene and virus are as acquisitions such as AdCN305-IL-24, AdCN305-SOCS3 and AdCN305-cpp-SOCS3.The structure of A, transferring plasmid pCZ305:
At first design following primer:
Ad66:5’CCAGAATATTGGAACTTTAG?3’
BstXI
Ad67:5’GGATCCATCGAGGATCCTCATTCTTGGGCAATGTATGAA?3’
BamHI
Ad68:5’GGATCCGTCGACTCGCGAGAATCGTTTGTGTTATGTT?3’
BamHI
Ad69:5’CTTAAGTGAGCTGCCCGGGGA?3’
AflII
1. make up transfer vector pCZ301:
With HindIII digested plasmid pTG1801, enzyme cuts the fragment that the 2937bp size is reclaimed in the leakage of electricity swimming of full back.With this fragment with same with HindI II enzyme cut digestion also the carrier pGEM-11Zf (+) of dephosphorization be connected, transform, get correct positive colony, called after pCZ301.
2. make up transfer vector pCZ302:
With plasmid pTG3602 is template, carries out PCR with primer Ad66, Ad67 and Ad68, Ad69 respectively and (sees Molecular Cloning:A Laboratory Manual for details, 3
RdEd., Joseph Sambrookand David W.Russell), electrophoresis reclaims product and difference called after A8 (341bp) and A9 (339bp).To be connected, transform with A8 and the carrier pGEM-Teasy that the BamHI enzyme is cut digestion with BstXI, get correct clone and called after pGEM-T/A8.Similarly, will be connected, transform with A9 and the carrier pGEM-Teasy that the AflII enzyme is cut digestion, get correct clone and called after pGEM-T/A9 with BamHI.Cut digested plasmid pGEM-T/A8 with BstXI and BamHI enzyme then, Segment A 8 is reclaimed in leakage of electricity swimming, and it is cut the plasmid pGEM-T/A9 that digested with BstXI with the BamHI enzyme and be connected, transform with same, gets correct positive colony called after pGEM/A8-A9.Cut the plasmid pGEM/A8-A9 that obtains with BstXI and AflII enzyme, electrophoresis reclaims the Segment A 8-A9 of 650bp, and it is cut the plasmid pCZ301 that digested with BstXI with the AflII enzyme and be connected, transform with same, gets correct positive colony called after pCZ302.
3. make up transfer vector pCZ303:
With SphI digested plasmid pTG1801, enzyme cuts the leakage of electricity swimming of full back and reclaims the fragment of 10248bp, and makes it from connecting, transforming, and gets correct positive colony called after pCZ303.
4. make up transfer vector pCZ304:
The HindIII enzyme is cut pCZ302, and electrophoresis reclaims the fragment of 2937bp size.It is connected, transforms with the same plasmid pCZ303 that cuts also dephosphorization with the HindIII enzyme, get correct positive colony called after pCZ304.5. make up transfer vector pCZ305:
SphI single endonuclease digestion pTG3602, electrophoresis reclaims the fragment of 6126bp.It is connected, transforms with the same carrier pCZ304 that cuts also dephosphorization with the SphI enzyme, get correct positive colony called after pCZ305.
The structure of B, transferring plasmid pBC/polyA:
Design one section and contain restriction enzyme site NcoI, the Linker of the polyA of SacI:
5’-CATGGTTGCGGCCGCGGATCCTCTAGAAATAAAAGATCTTAAGTTTCATTAGATCTGTGTGTTGGTTTTTTGTGTGGTCGACGAGCT-3’
With EcoRI and XbaI enzyme cutting carrier pCITE-1 (containing the IRES sequence), enzyme cuts full rear electrophoresis and reclaims the 1752bp fragment.With this fragment be connected, transform with the carrier pBluescript-Sk that XbaI enzyme cutting digested with EcoRI, get correct positive colony, called after pBC.With NcoI and SacI digested plasmid pBC, electrophoresis reclaims the fragment of 3495bp, and it is connected conversion with Linker, gets correct positive colony and is transferring plasmid pBC/polyA.
C, pCN305-IL-24, pCN305-SOCS3, pCN305-cpp-SOCS3 make up:
1. transfer vector pCZ305-IL-24, pCZ305-SOCS3 and pCZ305-cpp-SOCS3 make up:
Use NcoI and NotI digested plasmid pMD/IL-24, pMD/SOCS3, pMD/-cpp-SOCS3, pMD/SOCS1 and pMD/-cpp-SOCS1 respectively, enzyme cuts full rear electrophoresis and reclaims 691bp, 680bp and 720bp band respectively.With its with equally cut the plasmid pBC/polyA that digested with the NotI enzyme and be connected, transform with NcoI, get correct positive colony and be named as pBC/polyA-IL-24pBC/polyA-SOCS3 and pBC/polyA-cpp-SOCS3 respectively.Cut the plasmid pBC/polyA-IL-24 that this obtains with the SalI enzyme, enzyme cuts the fragment that full rear electrophoresis reclaims.With itself and PCZ305 connection, the conversion of cutting also dephosphorization equally with the SalI enzyme, get correct positive colony and be recombinant plasmid pCZ305-IL-24, pCZ305-SOCS3 and pCN305-cpp-SOCS3.
2. homologous recombination in the bacterium:
Use the SnaBI enzyme to cut digested plasmid pCZ305-IL-24, pCZ305-SOCS3 and pCZ305-cpp-SOCS3 respectively, enzyme cuts full back and reclaims big fragment with dehydrated alcohol sodium-acetate precipitation.Plasmid pCN103 (is made up by this laboratory, with hTERT replaced the promotor of E1A and lack on the E1A can with the interactional CR2 of Rb district) with reclaim the fragment that obtains and in competent cell BJ5183, carry out homologous recombination by the method that electricity changes, get correct positive colony and be recombinant plasmid pCN305-IL-24, pCN305-SOCS3 and pCN305-cpp-SOCS3.
D, virus of A dCN305-IL-24, AdCN305-SOCS3 and AdCN305-cpp-SOCS3 packing:
Use pacI digested plasmid pJN103/IL-24, pJN103/SOCS3 and pJN103/cpp-SOCS3 (exposing the packaging signal of adenovirus) respectively, enzyme cuts full back and reclaims big fragment with dehydrated alcohol sodium-acetate precipitation, by transfection it can be packed in 293 cells (containing the E1A district) and forms virus.This kind method packaging virus can not produce the pollution of wild-type virus.Virus is carried out a large amount of breedings at 293 cells, use the caesium chloride density gradient centrifugation purified virus.
Embodiment 2: the ability to express behind the virus infection in the SOCS3 cell.
Virus of A dCN305-SOCS3 is pressed 5MOI respectively infects MRC5, BEL7404, Hep3B, H460 and five kinds of cells of Bcap37, behind the 48hr, extract the total protein of cell, by Western-blot to SOCS3 proteic expression analyze.As shown in Figure 4, represent respectively that from left to right AdCN305-SOCS3 expresses expression SOCS3 protein level among SOCS3, the human breast cancer cell strain Bcap37 in human embryonic lung cell MRC5, human hepatoma cell strain BEL7404, human hepatoma cell strain Hep3B, human lung carcinoma cell line H460.In normal cell MRC5, the SOCS3 protein expression level is lower, illustrates that the virus replication rate is low.And in four kinds of tumour cells, the SOCS3 protein expression level is higher, illustrates that the virus replication rate is higher.
Embodiment 3:SOCS3 inducing apoptosis of tumour cell.
Virus of A d-wt, AdCN305-SOCS3 and AdCN305-cpp-SOCS3 are pressed 5MOI infected person lung carcinoma cell H460, behind the effect 48hr, detect the ability of SOCS3 inducing apoptosis of tumour cell by the Hochest staining.As shown in Figure 5, wherein, a represents PBS blank group Hochest dyeing, b represents that Ad-wt acts on Hochest dyeing behind the H460 cell, c represents that AdCN305-SOCS3 acts on Hochest dyeing behind the H460 cell, and d represents that AdCN305-cpp-SOCS3 acts on Hochest dyeing behind the H460 cell.As seen, virus of A dCN305-SOCS3 and AdCN305-cpp-SOCS3 can produce apoptosis by inducing tumor cell, and PBS and Ad-wt do not have inducing apoptosis of tumour cell.(the arrow indication is an apoptotic cell)
The replication analysis in normal cell and tumour cell of embodiment 4:AdCN305-SOCS3 and AdCN305-cpp-SOCS3 recombinant adenovirus.
With 3 * 10
5Normal cell or tumour cell be laid on 6 orifice plates, behind the 24h, the virus that adds 5MOI is pressed 5MOI infection MRC5, BEL7404, Hep3B, H460 and five kinds of cells of Bcap37 with Ad-wt, AdCN305-HcRed, AdCN305-SOCS3 and four kinds of viruses of AdCN305-cpp-SOCS3.After 48 hours, 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 60mm dish, and cell adds and contains different extent of dilution (10 near covering with after 24 hours
-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.
By Fig. 6 result as seen, in normal cell MRC5, wild-type virus Ad-wt has higher replication, and 7 our improved targeting type viruses present very low levels of replication.In four kinds of tumour cells, our recombinant virus and wild-type virus all have higher replication, and be very nearly the same.These have illustrated the security of recombinant virus and the ability that specificity is duplicated in tumour cell.And the influence that the insertion of foreign gene does not also cause from the replication in tumour cell virus.Also can obtain identical experimental result with above-mentioned experimental technique AdCN305-TRAIL with AdCN305-IL-24.
Embodiment 5: virus of A dCN305-SOCS3 and AdCN305-cpp-SOCS3 detect in external kill capability to normal cell and tumour cell.
The survival rate of cell after virus treated detected by mtt assay.Step is as follows: liver cancer cell strain BEL7404, SMMCC7721 and normal human embryonic lung cell NHLF and normal people's pneumonocyte MRC-5 are 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/mlMTT 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 as shown in Figure 7, AdCN305-SOCS3 and AdCN305-cpp-SOCS3 have very strong lethal effect to tumour cell, and very little to normal cytotoxicity, have tumor-selective.Use the same method and also can produce identical result of treatment with AdCN305-TRAIL virus of A dCN305-IL-24.
Embodiment 6: virus of A dCN305-SOCS3 and AdCN305-cpp-SOCS3 are in nude mouse internal therapy tumor cell transplantation knurl.
With the nude mice subcutaneous vaccination hepatoma cell strain BEL7404 in 4～5 ages in week, laggard action thing grouping in 12 days.The treatment group gives 1 * 10
9The AdCN305-SOCS3 of pfu treats, test-results is shown in Fig. 8-1, simple wild-type virus Ad-wt or empty carrier virus of A dCN305-HcRed can show certain oncolytic effect, but the AdCN305-SOCS3 and the AdCN305-cpp-SOCS3 that carry therapeutic gene can suppress growth of tumor significantly, and have merged the virus inhibition better effects if of the SOCS3 that wears the film peptide.It is identical with AdCN305-TRAIL nude mouse internal therapy tumor cell transplantation knurl curative effect to AdCN305-IL-24 to use the same method.
Embodiment 7: the pathology and the immunohistochemical analysis of virus of A dCN305-SOCS3 and AdCN305-cpp-SOCS3 transplanted tumor and liver
Get the nude mice tumor tissues of injecting virus after 7 days, routinely H﹠amp; The E dyeing process carries out, dewaters respectively and paraffin embedding, and the thick 4um of paraffin section, after conventional dewaxing, the gradient alcohol aquation, bush uniformly dyeing 10min, the hydrochloride alcohol differentiation several seconds, washing, 3min is dyed in Yihong, washing, dehydration, transparent, the neutral gum sealing.Experimental result is shown in Fig. 8-2: simple wild-type virus Ad-wt or empty carrier virus of A dCN305-HcRed can show certain oncolytic effect, but the AdCN305-SOCS3 and the AdCN305-cpp-SOCS3 that carry therapeutic gene can suppress growth of tumor significantly, and have merged the virus inhibition better effects if of the SOCS3 that wears the film peptide.By pathology and immunohistochemistry the liver of injection tumour and mouse is analyzed, AdCN305-SOCS3 and AdCN305-cpp-SOCS3 have suppressed the expression level of phospho-Stat3 in the tumour and have made tumour a large amount of necrosis occur.The virion of wild-type virus Ad-wt can be detected in liver, and AdCN305-SOCS3 and AdCN305-cpp-SOCS3 do not show the toxicity to liver, further understands the security of recombinant virus.
Sequence table
＜110〉Institutes Of Technology Of Zhejiang
＜120〉late promoter targeting regulation oncolytic adenovirus pCN 305 carrier and construction process thereof and application
<160>1
<170>PatentIn?version?3.1
<210>1
<211>1357
<212>DNA
<400>1
Claims (4)
1. a late promoter targeting regulation oncolytic adenovirus pCN 305 carrier is characterized in that, it has the gene order of SEQ ID No.1.
2. the described late promoter targeting regulation oncolytic adenovirus pCN 305 construction of carrier of claim 1 is characterized in that, may further comprise the steps:
(1) design following primer:
Ad66:5’CCAGAATATTGGAACTTTAG?3’
BstXI
Ad67:5’GGATCCATCGAGGATCCTCATTCTTGGGCAATGTATGAA?3’
BamHI
Ad68:5’GGATCCGTCGACTCGCGAGAATCGTTTGTGTTATGTT?3’
BamHI
Ad69:5’CTTAAGTGAGCTGCCCGGGGA?3’
AflII。
(2) make up transfer vector pCZ301:
With HindIII digested plasmid pTG1801, enzyme cuts the fragment that the 2937bp size is reclaimed in the leakage of electricity swimming of full back, with this fragment with same with the HindIII enzyme cut digestion also the carrier pGEM-11Zf (+) of dephosphorization be connected, transform, get correct positive colony, called after pCZ301.
(3) make up transfer vector pCZ302:
With plasmid pTG3602 is template, carries out PCR with primer Ad66, Ad67 and Ad68, Ad69 respectively, and electrophoresis reclaims product and difference called after A8 and A9, and A8 is 341bp, and A9 is 339bp; To be connected, transform with A8 and the carrier pGEM-Teasy that the BamHI enzyme is cut digestion with BstXI, get correct clone and called after pGEM-T/A8.Similarly, will be connected, transform with A9 and the carrier pGEM-Teasy that the AflII enzyme is cut digestion, get correct clone and called after pGEM-T/A9 with BamHI.Cut digested plasmid pGEM-T/A8 with BstXI and BamHI enzyme then, Segment A 8 is reclaimed in leakage of electricity swimming, and it is cut the plasmid pGEM-T/A9 that digested with BstXI with the BamHI enzyme and be connected, transform with same, gets correct positive colony called after pGEM/A8-A9.Cut the plasmid pGEM/A8-A9 that obtains with BstXI and AflII enzyme, electrophoresis reclaims the Segment A 8-A9 of 650bp, and it is cut the plasmid pCZ301 that digested with BstXI with the AflII enzyme and be connected, transform with same, gets correct positive colony called after pCZ302.
(4) make up transfer vector pCZ303:
With SphI digested plasmid pTG1801, enzyme cuts the leakage of electricity swimming of full back and reclaims the fragment of 10248bp, and makes it from connecting, transforming, and gets correct positive colony called after pCZ303.
(5) make up transfer vector pCZ304:
The HindIII enzyme is cut pCZ302, and electrophoresis reclaims the fragment of 2937bp size.It is connected, transforms with the same plasmid pCZ303 that cuts also dephosphorization with the HindIII enzyme, get correct positive colony called after pCZ304.(6) make up transfer vector pCZ305:
SphI single endonuclease digestion pTG3602, electrophoresis reclaims the fragment of 6126bp.It is connected, transforms with the same carrier pCZ304 that cuts also dephosphorization with the SphI enzyme, get correct positive colony called after pCZ305, pCZ305 has the gene order of SEQ ID No.1.
3. an application rights requires 1 described late promoter targeting regulation oncolytic adenovirus pCN 305 carrier to obtain the method for two target rf oncolytic adenovirus, it is characterized in that, may further comprise the steps:
(1) internal ribosome entry site IRES and termination signal polyA are cloned on the carrier pBluescript-sk, form the transfer vector pBC/polyA that contains IRES, MCS and polyA expression cassette.
(2) carry out homologous recombination by the electricity method of changeing the adenovirus skeleton plasmid pCN103 that in bacterium, makes two targets and recombinant plasmid pCZ305-IL-24, the pCZ305-TRAIL, pCZ305-SOCS3, pCZ305-cpp-SOCS3, pCZ305-SOCS1, the pCZ305-cpp-SOCS1 that contain cancer suppressor gene IL-24, TRAIL, SOCS3, cpp-SOCS3, SOCS1, cpp-SOCS1, obtain recombinant plasmid pCN305-IL-24, pCN305-TRAIL, pCN305-SOCS3, pCN305-cpp-SOCS3, pCN305-SOCS1, pCN305-cpp-SOCS1.
(3) enzyme is cut the recombinant adenovirus plasmid pCN305-IL-24, the pCN305-TRAIL that expose packaging signal after the digestion, pCN305-SOCS3, pCN305-cpp-SOCS3 pCN305-SOCS1, pCN305-cpp-SOCS1 respectively transfection in 293 cells, make it be packaged into virus of A dCN305-IL-24, AdCN305-TRAIL, AdCN305-SOCS3, AdCN305-cpp-SOCS3, AdCN305-SOCS1 and AdCN305-cpp-SOCS1; Make it efficiently express IL-24, TRAIL, SOCS3, cpp-SOCS3, SOCS1 and cpp-SOCS1 protein molecular.
4. an application rights requires two target rf oncolytic adenovirus of 3 described methods acquisitions to be used to prepare the purposes of the medicine for the treatment of tumour.
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