CN101148681A

CN101148681A - Targeted coexpression p53 and Mda-7 recombination adenovirus

Info

Publication number: CN101148681A
Application number: CNA2006101230152A
Authority: CN
Inventors: 王尚武
Original assignee: Individual
Current assignee: Individual
Priority date: 2006-10-27
Filing date: 2006-10-27
Publication date: 2008-03-26

Abstract

The present invention is recombinant adenovirus for the expression box of targeting co-expressed tumor suppressor genes p53 and Mda-7 and its construction process, use and significance. The present invention features the mutant wild type tumor suppressor gene P53 with the No. 72 place amino acid changed from proline into arginine, the connection between the tumor suppressor gene P53 and the gene Mda-7 by means of the internal ribosome binding site, the tumor specific promoter PEG-3 to drive the expression box, and the recombinant serum A5 type adenovirus with copy defect. The new type of adenovirus has two kinds of targeting co-expressed anticancer genes, p53 and Mda-7, powerful biological function of apoptosis and no damage on health cell. The present invention is significant in the gene therapy of tumor, especially malignant tumor.

Description

The recombinant adenovirus of a kind of target coexpression p 53 and Mda-7

Technical field

The invention belongs to biological technical field.The present invention has narrated and a kind ofly has been structured in that specifically inside tumor cell duplicates and expresses the method for the novel recombinant adenovirus melanocyte differentiation associated gene, that can be applicable to therapy of tumor.

Background technology

Melanocyte differentiation associated gene (English name melanoma differentiation associated (mda) genes) claims interleukin II 4 (abbreviation IL-24, together following) again, belongs to a member of interleukin 10 family.The Mda-7/IL-24 gene is a gene conservative on the structure, a kind of glycoprotein that its expression product is made up of 206 amino acid.

Mda-7/IL-24 is a gene with specific tumor cell apoptosis of finding at application of difference hybrid method behind interferon-beta and mezerein (MEZ) processing melanocyte.It is main relevant with activating immune system that its antitumor mechanism is considered to.Its expression be limited to melanochrome wander cell with the relevant tissue of immune yarn system, as spleen, thymus gland and peripheral blood lymphocytes.Handle peripheral blood lymphocytes with greasiness sugar, can induce Mda-7/IL-24 genetic expression.On the other hand, reorganization Mda-7/IL-24 albumen with purifying is handled peripheral blood lymphocytes, can induce interleukin-6, interferon-, tumour necrosis factor-a, interleukin-1 ' beta ', interleukin 12 and granulocyte colony stimulating factor to express (Devanand Sarkar, et al:PNAS, 2005.105 (39): 14034-14039).The kinds of tumors animal model experiment studies have shown that this gene have the ability of distinguishing normal cell and cancer cells, can the inducing apoptosis of tumour cell function and suppress vascularization and the ability of tumor growth, adjusting organism immune response and make tumour cell to the more responsive and collaborative smelting treatment of chemical medicine, biotechnological formulation and radiation cure effect, and the more important thing is that normal cell is not had the overt toxicity effect.Satisfactory in the clinical verification curative effect at present.Gland cancer patient's clinical I phase and II phase show it is safe with well-tolerated in verifying late, and one time intratumor injection can cause most of apoptosis of tumor cells.Think that Mda-7 is a cytokine with dual function, its normal physiological function may be relevant with immune special aspects, its cross expression then cause the specific tumor cell apoptosis ( Fisher PB, et al: Cancer Biol Ther.2003Jul-Aug; 2 (4Suppl 1): S23-37; Fisher PBEt al: Curr Gene Ther.2006Feb; 6 (1): 73-91; Gupta P, et al: Pharmacol Ther.2006Sep; 111 (3): 596-628.Epub 2006Feb 7).Thereby Mda-7/IL-24 will be expected to become a kind of safe, effective, have the treatment tumour of great market prospect bio-pharmaceutical ( Fisher PB. Cancer Res.2005 Nov15; 65 (22): 10128-38; Chada S, et al: Cancer Gene Ther.2005Nov 11; ( Gupta P, et al: Pharmacol Ther.2006 Feb 3; Inoue S, et al: Curr Gene Ther.2006Feb; 6 (1): 73-91; : Miyahara R, et al: Cancer Gene Ther.2006 Mar 10).

The P53 gene is the main member in the tumor suppressor gene family, is the highest gene of dependency that discovery at present and human tumor take place.Studies have shown that there is common proline(Pro) (P72, down together)/arginine (R72, down together) polymorphism in wild-type P53 gene 72 sites, have very big meaning on the natural death of cerebral cells inducing function of this polymorphism for wild-type P53 gene.The P53 gene is the R72 type, and more than strong 2-15 times of its natural death of cerebral cells energy force rate P72 type (Murphy M, et al:2003,3 (3): 357-65, Nat.Genetics).China's wild-type P53 gene has been used for clinical gene therapy, obtains certain curative effect.But still the space that grows a lot.

Gene therapy is to use various carriers, especially virus vector, directly enters human body cell/tissue, and the delivery system of express therapeutic goal gene has great using value.In the treatment genetic treatment of tumor, be most widely used at present, have safety, effective, treat long-term characteristics with adenovirus.

Simultaneously because this virus vector self, as because of its cells infected must with acceptor coxsackie-adenovirus receptor on the cell (CAR, down with) in conjunction with just working, so its infect and cytolemma on acceptor quantity proportional.Therefore, for some histocyte, because acceptor quantity is few, especially the CAR acceptor of tumor cell surface is on the low side, and is limited to their infection, so this virus is difficult to infect and enter in the cell, effectively brings into play the therapeutic action that it carries gene.

Lack in order to overcome falling into of this carrier, in recent years the researchist is by the transgenation technology, with adenovirus carrier, several amino acid of the prominent knot of the fiber relevant with entering cell gene suddenly change, and skewer is gone into the amino acid polypeptide Arg-Gly-Asp (GRB) that combines with the integrin acceptor.Most of tumour cells that studies have shown that in the past are av-integrin positives, and the CAR of some terminal cancer expression is (Chattopadhyay N.etal:J.Exp.Ckin.Cancer Res, 20 (2001): 269-275 that descend; M.D.Sachs, et al:Urology 60 (2002) 531-536).Thereby, the adenovirus carrier of the prominent knot of fiber transgenation can improve the ability of cells infected greatly, and proving that using this adenovirus carrier reduces the above Yuka Okada of order of magnitude of adenovirus consumption at least, et al:BBA 16709 (2004): 172-180).Undoubtedly, targeting gene therapy be human in anticancer struggle dream not in the hope of a kind of sharp weapon.Targeting gene therapy is present and a very important developing direction of the research of cancer therapy in the future.

In recent years, a kind of tumor-specific promoters is found and clones.What is more important, this tumor-specific promoters does not have activity in normal cell.Recently, use this tumor-specific promoters in adenovirus carrier.Be built into a kind of adenovirus carrier that only in tumour cell, can carry out self-replication, form the adenovirus carrier (CRCA) of so-called conditionality replication.Therefore, be confirmed to be a kind of tumor-specific promoters (Haviv, Y.S.et al:Curr.Gene Ther.Adv, Drug.Delivery Rev.53,135-154; Haviv, Y.S.et al:Curr.Gene.Ther. (2003) 3,357-365; Su, ZZ.et al.PNAS (2005), 102 (4): 1059-1064; Devanand Sarkar, et al.NAS (2005), 102 (39): 14034-14039).

In sum, use the internal ribosome binding site and connect novel p53 and Mda-7 gene, structure drives above-mentioned dual expression of gene box with tumor-specific promoters, in conjunction with the prominent knot sudden change of adenovirus fiber, be built into a kind of weight that has stronger cell infection power, only in tumour cell, expresses again but the group adenovirus, for the clinical treatment malignant tumour provides a kind of adenovirus brand-new, the target therapy of tumor, the great market prospect be will have, immeasurable society, economic benefit produced.

Summary of the invention

The objective of the invention is to make up tumor-specific promoters and drive novel p53 of coexpression and Mda-7 expression of gene box, and, form the recombinant adenovirus of a kind of novel targeted property expression in conjunction with the prominent knot sudden change of adenovirus fiber.Make it become the adenovirus product of new generation for the treatment of malignant tumour clinically.Novel recombinant adenovirus structure of the present invention, its principal feature is:

1). the prominent knot of adenovirus fiber gene skewer is gone into amino acid peptide, and this amino acid peptide is made up of arginine, glycine and aspartoyl propylhomoserin, and other amino acid are formed and put in order constant;

2). the expression cassette that is driven expression male Mda-7 of novel p53 and poly VITAMIN B4 by tumor-specific promoters PEG-3 is contained in the A1E disappearance district of adenovirus carrier,

3). novel recombinant adenovirus has the tumour cell tropism that hits expresses novel p53 and Mda-7 function;

4). the sudden change of the prominent knot of adenovirus fiber gene makes its characteristics that tumorigenic infection power is stronger, the tumour cell pattern of infection is wider.

Description of drawings

The incision enzyme map of accompanying drawing 1 tumor-inhibiting factor p53 mutant R72: R72 mutant tumor-inhibiting factor p53 forms new Nucleotide restriction endonuclease smal1 site in the mutational site.After digesting R72 mutant and wild-type tumor-inhibiting factor p53 respectively with restriction endonuclease smal1, the R72 mutant produces two dna fragmentations; Wild-type only produces a dna fragmentation.Diagram electrophoresis first swimming lane is a landa/Hind111DNA molecular weight marker thing, and second swimming lane is an IkB dna molecular amount marker; The 3rd swimming lane is wild-type tumor-inhibiting factor P53; The 4th swimming lane R72 mutant tumor-inhibiting factor P53.

Accompanying drawing 2 wild-types and mutant p53 are to the influence of human cervical carcinoma Hela cell's growth.Wandered behind the cell 48 hours with equivalent wild-type and mutant P53 transfection, observe and the counting survival cells.This is the cell average counter result of three experiments, shows that people source cancer suppressor gene R72 type p53 has stronger apoptosis function than wild type p53.

The clone of accompanying drawing 3Mda-7 gene: through the RT-PCR reaction, clone p53AIP1: in the T-Easy carrier.First, second swimming lane is a dna molecular amount marker, and the 3rd swimming lane is that the EcoR1 enzyme of Mda-7 is cut the result, produces two fragments.The arrow indication is Mda-7: gene.

The clone of accompanying drawing 4 tumor-specific promoters: this result is the PCR reaction product behind the synthetic complementary DNA (cDNA).First swimming lane is that dna molecular standard, second, third and the 4th road are the PCR reaction product.Arrow is the PCR reaction product.

Accompanying drawing 5 constitutes collection of illustrative plates by the expression cassette that tumor-specific promoters drives: this expression cassette collection of illustrative plates shows by being positioned at tumor-specific promoters, novel p53, IRES, Mda-7 and the SV40 poly VITAMIN B4 that N-do not hold and forms, and is connected with different restriction enzyme sites.

Accompanying drawing 6 is expressed the recombinant adenovirus structure iron of the expression cassette that is driven by tumor-specific promoters: two auspicious left arm and the right arms that are respectively adenovirus in the structure iron, expression cassette is made up of tumor-specific promoters, R72 type tumor-inhibiting factor p53, internal ribosome binding site (IRES, down together), Mda-7 gene and SV40 polyadenylic acid.

Embodiment

Following embodiment is a content in order to demonstrate the invention, rather than limits the scope of the invention by any way.The used adenovirus carrier of the present invention is a Stratagene company product, comprises that adenovirus wears carrier PaDeasy-1.Shuttle plasmid pShuttle and pShuttle-IRES.

The formation of embodiment one, R72 type tumor-inhibiting factor p53: its method be with clone the human P 53 gene be template, adopt the transgenation technology, make the codon ccc (P72) of the 72nd proline(Pro) of wild-type be mutated into arginic codon cgg (R72), form new Nucleotide restriction endonuclease smal1 site at saltation zone.

1). people's wild-type tumor-inhibiting factor p53 gene 5 '-end Nco1 is to the mat woven of fine bamboo strips 72 bit codon fragment amplifications:

With N '-end-Nco1-saltation zone sheet in people's wild-type P72 tumor-inhibiting factor p53 gene cDNA

The Duan Zuowei template is carried out pcr amplification with artificial synthetic primer.

The PCR design of primers is as follows:

Primer 1:

N end: 5 '-gccttccgggtcactg

Aggagccg-3 '

30mer Nco1

Primer 2: ccg is the arginine codon

N end: 5 '-gaatgccagaggctgctccc

Gtggcccctgca-3 '

35mer

Pcr amplification product is connected to the T-easy carrier after tailing adds VITAMIN B4, behind the transform bacteria, amplification, extracting, purifying are determined through dna sequencing.

2). people's wild-type tumor-inhibiting factor p53 gene the 72nd password subarea is to C-end Nco1 fragment amplification:

With above-mentioned fragment district cDNA is template, carries out pcr amplification with the following primer of artificial synthetic.

The PCR design of primers is as follows:

Primer 3:ccg is the arginine codon

N holds 5-aggggccae

Gggagcagcctctggcattc-3

32mer

Primer 4:

N holds 5 '-gtagatgg

Cgcggacgcgggtg-3 '

28mer Nco1

With two products of pcr amplification by 1) method be attached to the T-easy carrier, correct through dna sequencing.

3). the splicing of above-mentioned two PCR fragment products forms Nco1～Nco1 fragment

With 1) and 2) in contain above-mentioned dna fragmentation the T-easy vector plasmid DNA use Nco1 and Smal1 complete degestion respectively, behind the purifying, be connected with the wild type p53 gene cDNA of cutting through the Nco1 enzyme again.Transform bacteria, plasmid extraction and through enzyme cut detect stitching direction correct after, be built into the wild type p53 gene expression plasmid pCMV-neo-p53 of R72 type.

Segmental synthetic of embodiment two, Mda-7DNA and clone thereof: root a tree name known sequence, the coding DNA fragment of technology synthetic Mda-7 routinely, and carry out pcr amplification with following primer.Primer 1 (43mer):

5’-agc

tatgaattttcaacagaggctgcaaagcctgtgg-3’

Smal1

Primer 2 (37mer):

5’-cgac

atcagagcttgtagaatttctgcatcc-3’

Xbal

The synthetic of embodiment three, tumor-specific promoters dna fragmentation and clone thereof: root a tree name known sequence, the dna fragmentation of technology synthetic tumor-specific promoters routinely, and carry out pcr amplification with following primer.

Primer 1:(is at terminal restriction enzyme site Nru1, Kpn1 and the Xba1 of introducing of N-)

5’-tgtcgcgaggtacctctagaatttcagtgttgttttcct-3’

Primer 2: (at terminal restriction enzyme site EcoR1 and the Stu1 of introducing of C-)

5′-gcgatatcaggcctgtccggttcggtttgccaaaagcg-3′

The structure of embodiment four, the expression cassette formed by tumor-specific promoters, novel p53, internal ribosome binding site (IRES) and Mda-7.The used Stratagene company product of present embodiment is pShuttle-IRES, is the framework construction expression cassette with this carrier.Transform and the order-checking confirmation by repeatedly different restriction endonuclease digestion, ligation, competence bacterium, finish this expression cassette and make up.Be that Sma1 restriction enzyme site and 3 '-end is that the Mda-7 gene fragment of Xba1 restriction enzyme site is spliced in the upstream of SV40 poly VITAMIN B4 promptly, and be positioned at the downstream of IRES with 5 '-end; With 5 '-end is that EcoR V and 3 '-end is the novel P53 fragment of Not1 restriction enzyme site, splices the upstream in IRES; With 5 '-end is that Nru1 and 3 '-end is the tumor-specific promoters PEG-3 fragment of EcoR1 restriction enzyme site, splices in the segmental upstream of novel P53, and so far, this expression cassette makes up just to accuse and finishes.

Embodiment five: adenovirus fiber gene H1 ring skewer is gone into the enforcement of arginine, glycine and aspartoyl propylhomoserin polypeptide, referring to " a kind of New-type adenovirus carrier that duplicates at specifically inside tumor cell " patent application.Application number is 200620063702.5.

Embodiment six, structure contain the adenovirus shuttle plasmid of this expression cassette

1). with restriction endonuclease Kpn1 and the above-mentioned pIRES plasmid DNA that contains expression cassette of Sal1 digestion, will produce two dna fragmentations of Kpn1-Kpn1 fragment and Kpn1-Sal1, thus, this expression cassette will comprise the poly adenine dna sequence of SV40.After the digestion, through 1.2% agarose electrophoresis separate, the required dna fragmentation of purifying is standby.

2). with restriction endonuclease Kpn1 and Sal1 digestion pShuttle shuttling expression plasmid vector DNA, through 1.2% agarose electrophoresis separate, the linearizing pShuttle carrier DNA of purifying is standby.

3). Kpn1-Kpn1 fragment and two dna fragmentations of Kpn1-Sal1 with above-mentioned linearizing pShuttle carrier DNA fragment and the generation of digestion expression cassette, under the ligase enzyme effect, carry out splicing reaction, and the transformed competence colibacillus bacterium, cultivate amplification.

4). screen the bacterium transformant, cultivate amplification, extract recombinant plasmid dna, cut digestion and dna sequencing confirmation through enzyme more at last.

The recombinant adenovirus of embodiment seven, the novel p53 of target coexpression and Mda-7 gene

1). the preparation linearizing contains the recombinant shuttle plasmid pShuttle-p53-Mda-7 of novel p53 and Mda-7 expression casette: get an amount of above-mentioned recombinant shuttle plasmid DNA, through Nucleotide restriction endonuclease Pme1 digestion, electrophoretic separation, purifying Pme1 linearization for enzyme restriction recombinant shuttle plasmid DNA, standby.

2). above-mentioned linearizing recombinant shuttle plasmid DNA electric shock is transformed the BJ5183-AD-1 bacterium of pre-inversion adenovirus carrier pAdEasy-1, carry out homologous recombination, and screen with the microbiotic kalamycin.The bacterium of gland-containing virus carrier pAdEasy-1 recombinant chou will be the thin strain of anti-kalamycin.

3). amplification gland-containing virus carrier pAdEasy-1 thin strain recombinant chou, anti-kalamycin, recombinant adenoviral vector pAdEasy-1DNA promptly increases.Extracting adenovirus carrier pAdEasy-1 recombinant DNA, with restriction endonuclease Pac1 digestion, linearizing adenovirus carrier pAdEasy-1 recombinant DNA, and separation and purification is standby.

4). press the method that Stratagene company is introduced about AdEasy XL Adenoviral Vector System instruction manual, the linearizing adenovirus carrier pAdEasy-1 recombinant DNA transfection AD-embryo kidney 293 cell that above-mentioned purifying is standby.

5). behind the transfection AD-embryo kidney 293 cell 7-10, prepare former generation recombinant adenovirus mother liquor, and optimize the condition of the infection AD-embryo kidney 293 cell of former generation recombinant adenovirus mother liquor, amplification recombinant adenovirus.

6). the high-titer recombinant adenovirus of amplification capacity is used for research and animal experiment.

Sequence table

Individual?Applicant

--------------------

Street: rooms 610, science town IBI A district

City: Guangzhou

State: Guangdong Province

Country: China

PostalCode：510633

PhoneNumber：020-32290208

FaxNumber：020-38491559

EmailAddress：shangwu_w@yahoo.com

＜110〉LastName: king

＜110〉FirstName: set great store by military affairs or martial arts

<110>MiddleInitial：

<110>Suffix：

Application?Project

--------------------

＜120〉Title: the recombinant adenovirus of novel p53 of target coexpression and Mda-7

<130>AppFi?leReference：

<140>CurrentAppNumber：1

<141>CurrentFilingDate：2006-10-26

Sequence

--------

<213>OrganismName：human

<400>PreSequenceString：

atcgatatcg?ccaccgtcca?gggagcaggt?agctgctggg?ctccggggac?actttgcgtt 60

cgggctggga?gcgtgctttc?cacgacggtg?acacgcttcc?ctggattggc?agccagactg 120

ccttccgggt?cactgccatg?gaggagccgc?agtcagatcc?tagcgtcgag?ccccctctga 180

gtcaggaaac?attttcagac?ctatggaaac?tacttcctga?aaacaacgtt?ctgtccccct 240

tgccgtccca?agcaatggat?gatttgatgc?tgtccccgga?cgatattgaa?caatggttca 300

ctgaagaccc?aggtccagat?gaagctccca?gaatgccaga?ggctgctccc?cgggtggccc 360

ctgcaccagc?agctcctaca?ccggcggccc?ctgcaccagc?cccctcctgg?cccctgtcat 420

cttctgtccc?ttcccagaaa?acctaccagg?gcagctacgg?tttccgtctg?ggcttcttgc 480

attctgggac?agccaagtct?gtgacttgca?cgtactcccc?tgccctcaac?aagatgtttt 540

gccaactggc?caagacctgc?cctgtgcagc?tgtgggttga?ttccacaccc?ccgcccggca 600

cccgcgtccg?cgccatggcc?atctacaagc?agtcacagca?catgacggag?gttgtgaggc 660

gctgccccca?ccatgagcgc?tgctcagata?gcgatggtct?ggcccctcct?cagcatctta 720

tccgagtgga?aggaaatttg?cgtgtggagt?atttggatga?cagaaacact?tttcgacata 780

gtgtggtggt?gccctatgag?ccgcctgagg?ttggctctga?ctgtaccacc?atccactaca 840

actacatgtg?taacagttcc?tgcatgggcg?gcatgaaccg?gaggcccatc?ctcaccatca 900

tcacactgga?agactccagt?ggtaatctac?tgggacggaa?cagctttgag?gtgcatgttt 960

gtgcctgtcc?tgggagagac?cggcgcacag?aggaagagaa?tctccgcaag?aaaggggagc 1020

ctcaccacga?gctgccccca?gggagcacta?agcgagcact?gcccaacaac?accagctcct 1080

ctccccagcc?aaagaagaaa?ccactggatg?gagaatattt?cacccttcag?atccgtgggc 1140

gtgagcgctt?cgagatgttc?cgagagctga?atgaggcctt?ggaactcaag?gatgcccagg 1200

ctgggaagga?gccagggggg?agcagggctc?actccagcca?cctgaagtcc?aaaaagggtc 1260

agtctacctc?ccgccataaa?aaactcatgt?tcaagacaga?agggcctgac?tcagactgac 1320

attctccact?tcttgttccc?cactgacagc?ctcccacccc?catctctccc?tcccctgcca 1380

ttttgggttt?tgggtctttg?aacccttgct?tgcaataggt?gtgcgtcaga?agcacccagg 1440

acttccattt?gctttgtccc?ggggctccac?tgaacaagtt?ggcctgcact?ggtgttttgt 1500

tgtggggagg?aggatgggga?gtaggacata?ccagcttaga?ttttaaggtt?tttactgtga 1560

gggatgtttg?ggagatgtaa?gaaatgttct?tgcagttaag?ggttagttta?caatcagcca 1620

cattctaggt?agggacccac?ttcaccgtac?taaccaggga?agctgtccct?cactgttgac 1680

tagtaattcg?cggccgcgtc?g 1701

<212>Type：DNA

<211>Length：1701

SequenceName：p53(72R)

SequenceDescription：

MetGluGluProGlnSerAspProSerValGluProProLeuSerGlnGluThrPheSer

1 5 10 15 20

AspLeuTrpLysLeuLeuProGluAsnAsnValLeuSerProLeuProSerGlnAlaMet

25 30 35 40

AspAspLeuMetLeuSerProAspAspIleGluGlnTrpPheThrGluAspProGlyPro

45 50 55 60

AspGluAlaProArgMetProGluAlaAlaProArgValAlaProAlaProAlaAlaPro

65 70 75 80

ThrProAlaAlaProAlaProAlaProSerTrpProLeuSerSerSerValProSerGln

85 90 95 100

LysThrTyrGlnGlySerTyrGlyPheArgLeuGlyPheLeuHisSerGlyThrAlaLys

105 110 115 120

SerValThrCysThrTyrSerProAlaLeuAsnLysMetPheCysGlnLeuAlaLysThr

125 130 135 140

CysProValGlnLeuTrpValAspSerThrProProProGlyThrArgValArgAlaMet

145 150 155 160

AlaIleTyrLysGlnSerGlnHisMetThrGluValValArgArgCysProHisHisGlu

165 170 175 180

ArgCysSerAspSerAspGlyLeuAlaProProGlnHisLeuIleArgValGluGlyAsn

185 190 195 200

LeuArgValGluTyrLeuAspAspArgAsnThrPheArgHisSerValValValProTyr

205 210 215 220

GluProProGluValGlySerAspCysThrThrIleHisTyrAsnTyrMetCysAsnSer

225 230 235 240

SerCysMetGlyGlyMetAsnArgArgProIleLeuThrIleIleThrLeuGluAspSer

245 250 255 260

SerGlyAsnLeuLeuGlyArgAsnSerPheGluValHisValCysAlaCysProGlyArg

265 270 275 280

AspArgArgThrGluGluGluAsnLeuArgLysLysGlyGluProHisHisGluLeuPro

285 290 295 300

ProGlySerThrLysArgAlaLeuProAsnAsnThrSerSerSerProGlnProLysLys

305 310 315 320

LysProLeuAspGlyGluTyrPheThrLeuGlnIleArgGlyArgGluArgPheGluMet

325 330 335 340

PheArgGluLeuAsnGluAlaLeuGluLeuLysAspAlaGlnAlaGlyLysGluProGly

345 350 355 360

GlySerArgAlaHisSerSerHisLeuLysSerLysLysGlyGlnSerThrSerArgHis

365 370 375 380

LysLysLeuMetPheLysThrGluGlyProAspSerAspTer

385 390

Sequence

<213>OrganismName：Rat

<400>PreSequenceString?：

tgaggctcgc?gaggtacctc?tagaatttca?gtgttgtttt?cctctctcca?cctttctcag 60

ggacttccga?aactccgcct?ctccggtgac?gtcagcatag?cgctgcgtca?gactataaac 120

tcccgggtga?tcgtgttggc?gcagattgac?tcagttcgca?gcttgtggaa?gattacatgc 180

gagaccccgc?gcgactccgc?atccctttgc?cgggacagcc?tttgcgacag?cccgtgagac 240

atcacgtccc?cgagccccac?gcctgagggc?gacatgaacg?cgctggcctt?gagagcaatc 300

cggacccacg?atcgcttttg?gcaaaccgaa?ccggacaggc?ctgctagccg?atatcgtgtt 360

ca 362

<212>Type：

<211>Length：362

SequenceName?：PEG-3Promotor

SequenceDescription：DNA

Sequence

<213>OrganismName：human

<400>PreSequenceString：

agcgggccca?tgaattttca?acagaggctg?caaagcctgt?ggactttagc?cagacccttc 60

tgccctcctt?tgctggcgac?agcctctcaa?atgcagatgg?ttgtgctccc?ttgcctgggt 120

tttaccctgc?ttctctggag?ccaggtatca?ggggcccagg?gccaagaatt?ccactttggg 180

ccctgccaag?tgaagggggt?tgttccccag?aaactgtggg?aagccttctg?ggctgtgaaa 240

gacactatgc?aagctcagga?taacatcacg?agtgcccggc?tgctgcagca?ggaggttctg 300

cagaacgtct?cggatgctga?gagctgttac?cttgtccaca?ccctgctgga?gttctacttg 360

aaaactgttt?tcaaaaacta?ccacaataga?acagttgaag?tcaggactct?gaagtcattc 420

tctactctgg?ccaacaactt?tgttctcatc?gtgtcacaac?tgcaacccag?tcaagaaaat 480

gagatgtttt?ccatcagaga?cagtgcacac?aggcggtttc?tgctattccg?gagagcattc 540

aaacagttgg?acgtagaagc?agctctgacc?aaagcccttg?gggaagtgga?cattcttctg 600

acctggatgc?agaaattcta?caagctctga?ttctagagtc?g 641

<212>Type：DNA

<211>Length：641

SequenceName：Mda-7

SequenceDescription：

MetAsnPheGlnGlnArgLeuGlnSerLeuTrpThrLeuAlaArgProPheCysProPro

5 10 15 20

LeuLeuAlaThrAlaSerGlnMetGlnMetValValLeuProCysLeuGlyPheThrLeu

25 30 35 40

LeuLeuTrpSerGlnValSerGlyAlaGlnGlyGlnGluPheHisPheGlyProCysGln

45 50 55 60

ValLysGlyValValProGlnLysLeuTrpGluAlaPheTrpAlaValLysAspThrMet

65 70 75 80

GlnAlaGlnAspAsnIleThrSerAlaArgLeuLeuGlnGlnGluValLeuGlnAsnVal

85 90 95 100

SerAspAlaGluSerCysTyrLeuValHisThrLeuLeuGluPheTyrLeuLysThrVal

105 110 115 120

PheLysAsnTyrHisAsnArgThrValGluValArgThrLeuLysSerPheSerThrLeu

125 130 135 140

AlaAsnAsnPheValLeuIleValSerGlnLeuGlnProSerGlnGluAsnGluMetPhe

145 150 155 160

SerIleArgAspSerAlaHisArgArgPheLeuLeuPheArgArgAlaPheLysGlnLeu

165 170 175 180

AspValGluAlaAlaLeuThrLysAlaLeuGlyGluValAspIleLeuLeuThrTrpMet

185 190 195 200

GlnLysPheTyrLysLeu

205

Claims

1. the recombinant adenovirus of novel p53 of target coexpression and Mda-7.The adenovirus carrier that the present invention uses is the product A dEasy-1 carrier of Stratagene company, is the adenovirus carrier of E1 and E3 district disappearance.The expression cassette that the present invention makes up is made up of tumour-specific mover, novel p53, IRES, Mda-7 and SV40 poly VITAMIN B4, and its principal character is:

A). the 72nd amino acids of people's wild type p53 cancer suppressor gene is by proline(Pro) sudden change becoming arginine, and other amino acid are formed and put in order constant;

B) the .P53 cancer suppressor gene at its N-end with restriction endonuclease EcoR V and C-end with restriction endonuclease Not1 fragment assembly in pIRES plasmid upstream;

C) the .Mda-7 gene at its N-end with restriction endonuclease Sma1 and C-end with restriction endonuclease Xba1 fragment assembly in pIRES plasmid downstream;

D). tumor-specific promoters at its N-end with restriction endonuclease Nru1 and C-end with restriction endonuclease EcoRV fragment assembly in the upstream of novel p53 gene;

After reorganization, this expression cassette is positioned at the E1 disappearance district of adenovirus carrier.This recombinant adenovirus has the effect of target coexpression p 53 and Mda-7, thereby has stronger apoptosis function, the wider biological function of anticancer spectrum.

2. root a tree name claim 1, a kind of tumor-specific promoters drives coexpression has Synergistic anti-cancer effect expression of gene box for two kinds, the deoxynucleoside acid sequence that it contains following amino acid sequences and has functional transcription:

A). the parent's amino acid sequence of polypeptide basically identical with human tumor suppressor gene p53, an amino acid whose replacement is only arranged, promptly the 72nd of this caused by tumor suppressor p 53 the different amino acid is replaced the proline(Pro) (P72) of wild-type P53 same position by arginine (R72).Described caused by tumor suppressor p 53 parental source is in the people;

B) a kind of parent comes from apoptosis-inducing albumen (p53AIP1) gene of people's p53 regulation and control;

C) tumor-specific promoters is the promotor of mouse source PEG-3 gene, and the zone that this promotor comprises is formed for-118 to+194 deoxynucleotides;

D) inner school ribosome bind site (IRES) comes from encephalomyocarditis virus;

3. root a tree name claim 1 and require 2 described recombinant adenovirus, it is characterized in that the promoter sequence that drives coexpression Novel Human caused by tumor suppressor p 53 and Mda-7 gene in the expression cassette is not limited only to mouse source tumour-specific PEG-3 gene promoter, also comprise other tumor-specific promoters, start and liver fetus first type glb promoter (AFP, tire first glb promoter) as human telomerase promotor, oestrogenic hormon and hypoxemia reaction promotor, the human prostate cancer specific factor;

4. root a tree name claim 1, the p53 gene of coexpression both can be positioned at the upstream of IRES in the expression cassette, also can be positioned at its downstream; Mda-7 is also like this.

5. root a tree name claim 1 and require 3 drives gene novel P53 both of the present invention coexpression, that be positioned at the IRES upstream (72R) by tumor-specific promoters, also can be p53 (46F); The gene that is positioned at the IRES downstream is Mda-7 gene both, also can be other short apoptosis genes, as Noxa, p53RFP and P27 (Kip1); Immune-regulating factor, as IL-2, IL-6, IFN-γ, granulocyte/macrophage colony stimulating factor (GMCSF) and TNF-a etc.;

6. root a tree name claim 1, the adenovirus carrier that the present invention uses is the product A dEasy-1 carrier of Stratagene company, is the replication-defective adenoviral vector of E1 and E3 district disappearance.But the adenovirus carrier that the present invention uses is not limited thereto replication defect type Ad5 type adenovirus, also comprise the adenovirus carrier that conditionality is duplicated, as the adenovirus carrier narrated of another patent " a kind of New-type adenovirus carrier that is used for the clinical tumor gene therapy " of my application.It is a kind ofly to drive adenoviral replication factor e1a gene by tumor-specific promoters and only express in tumour cell that the conditionality of this patents state is duplicated adenovirus carrier, thereby has the ability that only can duplicate in tumour cell.And its scleroproein AB ring, H1 ring and/or Shaft suddenly change, and have the advantages that infectivity is stronger, the cells infected spectrum is wider.This novel recombinant adenovirus also can only be expressed the Mda-7 gene.

7. root a tree name claim 1, the recombinant adenovirus of novel p53 of this target coexpression and Mda-7 is a kind of gene therapy medicament that is used for the treatment of multiple malignant cancer disease, and pharmaceutically acceptable carrier or vehicle.

8. gene therapy medicament as claimed in claim 7, it is characterized in that, the described recombinant adenovirus by tumor-specific promoters driving R72 type human P 53 gene and Mda-7 gene co-expressing of claim 1, described effective antitumor composition is apoptosis R72 type human P 53 gene with better function and Mda-7 expression of gene protein.

9. express medicinal composition as claim 1,5,6,7 and 8 gene therapy, its feature comprises that also tumor-specific promoters target coexpression thus contains the recombinant adenovirus of the expression cassette that other short apoptosis factor, cytokine and/or immune-regulating factors with anticancer therapy effect are connected with R72 type human P 53 gene or p53AIP1 gene by internal ribosome binding site (IRES) except that right requires the recombinant adenovirus of 1 described target coexpression R72 type human P 53 gene and Mda-7 gene.