CN101117635A - Fusion expression of PTD,HIF ODD and tumour inhibitory gene and uses thereof - Google Patents

Abstract

The present invention provides a fusion body of a protein transduction domain (PTD), an oxygen-dependent protein degradation domain (ODD) of a hypoxia-inducible factor (HIF) and a human tumor suppressor gene, the expression product of the present invention is the recombinant fusion protein, which can restrain the tumor cell growth, and can metabolize rapidly in the normal cells and slowly in the tumor cells, thus exerting the function of antitumor. The present invention also provides a manufacturing method of the recombinant fusion protein.

Description

The ODD of PTD, HIF and the amalgamation and expression of tumor suppressor gene and application thereof

Technical field

The present invention relates to genetically engineered and pharmaceutical field, be specifically related to utilize genetic engineering technique to express the fusion rotein of human caused by tumor suppressor p 53 and transduction peptide and oxygen dependence proteolytic degradation structural domain.

Background technology

As tumor suppressor gene, p53 participates in negative cell growth, the cell death inducing regulated.P53 is with tetrameric form apoptosis involvement, cell cycle inhibition, dna damage and processes such as reparation and aging.P53 albumen forms dead inducement signal mixture and activates caspases by inducing coding three transmembrane protein Fas, DR5 and PERP, causes apoptosis to take place, and forms outside apoptotic signal approach.Inner apoptotic signal approach is relevant with mitochondrial depolarization and cytochrome c release, is mainly finished by the Bc1-2 family protein.Bc1-2 family comprises Bc1-2, Bax, Noxa, PUMA, Bid etc., and it is controlling the release of mitochondrial cytochrome C.

Because the vital role of p53 in the growth of body, growth, differentiation, the p53 inactivation will have a strong impact on the stable of cellular genome, and transgenation is the most important mechanism of p53 inactivation, at least 50% the p53 gene alteration has taken place in the human malignancies, as: lung cancer, the esophageal carcinoma, cancer of the stomach, liver cancer, colorectal cancer, mammary cancer, ovarian cancer etc., about according to statistics 80% is missense mutation, 6% is nonsense mutation, 10% is disappearance and insertion, and cell can not synthesize correct p53 albumen, loses original biological function.Most p53 missense mutation are positioned at its DNA in conjunction with the territory, can cause p53 forfeiture and DNA bonded ability, and conventional treatment is produced resistant function.In addition, the p53 albumen of sudden change (mutant type p53, mt p53) also may obtain new function, if can combine with Daxx to suppress Fas inductive apoptosis pathway; With quinone oxidoreductase 1 mortise, thus the Degradation of the non-dependence of inhibition ubiquitinization.

The p53 Study on gene mutations provides the foundation for the p53 gene therapy, becomes for many years research focus based on the research of the tumour medicine of p53 function.For example: using adenoviral vectors carries out the p53 gene therapy, has all obtained the antitumor drug effect of certainty in experimentation on animals and clinical study.But be to use carrier security, gene expression dose regulation and control and problems such as patient's toxic action are also solved fully.Importing wt p53 albumen is the most direct means of p53 function of rebuilding in tumour cell.

P53 albumen all can carry out the prokaryotic expression preparation, and exogenous additional wild type p53 albumen or its active molecule of regulating can directly be brought into play their tumor inhibition effect.This method has been avoided the Vectors in Gene Therapy safety issue, and dosage is adjustable, can reach the antitumor of expression in vivo p53 gene too, and increase is put, the purpose of chemotherapy effect.Yet how the position that p53 plays a role enters tumour cell with the p53 transduction, and how to avoid it to Normocellular toxicity in cell, and prolonging their transformation period in tumour cell relatively becomes the key of its antitumor action of p53 target performance.

(protein transduction domin is that a kind of of discovered in recent years can efficiently pass biomembranous structural domain PTD) to protein transduction domain, is called the transduction peptide.Green in 1988 and Frankel reported first the trans-activator TAT of HIV-1 can stride film transfered cell inside, find that further its aa47-57 brings into play main effect as PTD in the protein transduction process.The PTD that has been found that is mainly derived from viral protein, as VP22 and TAT etc., is widely used in the transhipment of various biomolecules, as antigen peptide, and peptide nucleic acid(PNA), antisense oligonucleotide, full-length proteins, even nanoparticle and liposome.

It but is the beyond dispute fact that the transduction peptide can enter target cell with the various biomolecules transduction effectively.The protein of prokaryotic expression mostly is metaprotein, can prepare in a large number, be easy to purifying and storage, and sex change that protein molecular had was loose, the fixed space structure does not help the protein transduction peptide on the contrary its transduction is entered cell.Existing result shows that the efficient of transduction peptide TAT transduction metaprotein will be apparently higher than activated protein, and nonactive albumen enter after the cell can be by intracellular mechanism for correcting errors renaturation.

Utilize the spanning transduction membrane characteristics of PTD, the transduction of prokaryotic expression PTD-P53 fusion rotein is entered tumour cell, enhancing or the P53 function of rebuilding can be brought into play and regulate the cell cycle, the effect of inducing apoptosis of tumour cell.But we have also confirmed PTD and P53 fusion rotein induced tumor apoptosis in cell experiment.Yet, when the PTD translocator efficiently enters cell, lacking the selectivity that enters cell, P53 also may damage normal cell in killing tumor cell.

The important pathological characters that solid tumor cell malignant proliferation and blood supply are unbalance is an anoxic, existing clinically many evidences show, a series of adaptations take place in tumour cell under anaerobic environment, the indirect lethal effect of radioactive rays is weakened, make it radiotherapy, chemotherapy tolerance, and make tumour have more aggressive, distant metastasis takes place easily.(hypoxia inducible factor 1 HIF-1) is the important transcription factor of cell adapted anoxybiotic to oxygen deficient induction factor 1, the regulation and control of corresponding target genes when participating in anoxic.Can by the target gene of HIF-1 transcriptional activation all contain a hypoxemia response element (hypoxia reactive element, HRE) and the Cis effect regulate sequence, can find that in human tumor these gene products increase greatly.HIF-1 function therein can be divided into following several: 1. promote glycolysis-; 2. promote vasculogenesis; 3. relevant with tumor cell proliferation.

HIF-1 belongs to bHLH PAS albumen family, and by the assorted tetramer albumen that HIF-1 α and each two subunit of HIF-1 β form, two subunit molecular are respectively 120ku and 91-94ku.HIF-1 α is stronger to the dependency of oxygen, and when the oxygen concn of surrounding environment descended, the HIF-1 alpha expression increased.HIF-1 α albumen is not degraded easily during anoxic, and its transformation period was less than 5 minutes, and anoxic can increase the proteic stability of HIF-1 α, and the proteic degraded of HIF-1 α stops.The HIF-1 β of subunit to the dependency of oxygen a little less than, also essential in HIF-1, because only after two subunit's polymerizations and adaptations taking place, combine the competence exertion regulating effect with the HRE of its downstream factor that will regulate or enzyme (as VEGF, P53 and EPO).

When the degrading and regulating of research HIF-1, find, oxygen dependence proteolytic degradation structural domain (the oxygen-dependent degradation domain of HIF-1 α, ODD) be the stable critical elements of HIF-1 α, during non-anoxic condition, HIF-1 poly hydroxylase (HIF-1prolyhydroxylase, HIFPH) hydroxylation ODD core proline(Pro) (Pro 564), make pVHL (von Hippel-Lindau tumor suppressor gene product) combine with HIF-1 α subunit, and then poly ubiquitination HIF-1 α, finally make it by proteasome degradation, and under anoxia condition, make HIF PH inactivation owing to lack Sauerstoffatom (must from oxygen molecule), above reaction process is blocked.

The ODD of HIF-1 α contains 203 amino-acid residues, the aa548-603 of discovery ODD such as Harada can bring into play the degrading and regulating effect to its fusion rotein, wherein aa557-574 contains and regulates the fusion rotein necessary lease core composition of degrading, TAT-ODD-Gal is obviously increased in the intravital Gal activity of nude mice knurl of having transplanted people's pancreas cancer CF/PAC-1, and almost detect activity in the healthy tissues less than Gal, illustrate that ODD has brought into play the oxygen inducing action, quickened the degraded of foreign protein in healthy tissues, its fusion rotein can reduce the injury to healthy tissues to greatest extent in the performance antitumor action.

Neoplasm targeted therapy is the important content in the cancer therapy drug research, the present invention starts with from the characteristics of tumour cell, in TAT and p53 fusion rotein, introduce ODD, quicken the degraded of fusion rotein in normal cell, prolong its transformation period in tumour cell relatively and reach the antineoplastic purpose of P53 targeting proteins.

Summary of the invention

The gene that the present invention is intended to have a potential therapeutic action with the protein transduction domain that can transducer enters cell, quicken fusion rotein metabolic oxygen dependence proteolytic degradation structural domain in healthy tissues and organically merge, by the prokaryotic expression carrier expressed fusion protein, through proteic purifying and renaturation, provide a kind of and can enter tumour cell, tachymetabolism in healthy tissues, and in anoxybiotic solid tumor tissue the fusion rotein of cell death inducing, reach the prevention or/and the treatment tumour purpose.

For achieving the above object, the invention provides the syzygy of a kind of protein transduction domain, oxygen dependence proteolytic degradation structural domain and human tumor suppressor gene.Described human tumor suppressor gene can be to have in the gene of tumor inhibition effect any, its most preferably gene be the p53 gene.In preferred embodiments, this syzygy is defined as PTD-ODD-p53, and its sequence is:

ATGTACGGCCGTAAAAAGAGACGCCAACGTAGACGTATGAACCCATTTTCTACTCAGGACACAGATTTAGACTTGGAGATGTTAGCTCCCTATATCCCAATGGATGATGACTTCCAGTTACGTTCCTTCGATCAGTTGTCACCATTAGAAAGCAGTTCCGCAAGCCCTGAAAGCGCAAGTCCTCAAAGCACAGTTACAGTATTCCAGATGGAGGAGCCGCAGTCAGATCCTAGCGTCGAGCCCCCTCTGAGTCAGGAAACATTTTCAGACCTATGGAAACTACTTCCTGAAAACAACGTTCTGTCCCCCTTGCCGTCCCAAGCAATGGATGATTTGATGCTGTCCCCGGACGATATTGAACAATGGTTCACTGAAGACCCAGGTCCAGATGAAGCTCCCAGAATGCCAGAGGCTGCTCCCCGCGTGGCCCCTGCACCAGCAGCTCCTACACCGGCGGCCCCTGCACCAGCCCCCTCCTGGCCCCTGTCATCTTCTGTCCCTTCCCAGAAAACCTACCAGGGCAGCTACGGTTTCCGTCTGGGCTTCTTGCATTCTGGGACAGCCAAGTCTGTGACTTGCACGTACTCCCCTGCCCTCAACAAGATGTTTTGCCAACTGGCCAAGACCTGCCCTGTGCAGCTGTGGGTTGATTCCACACCCCCGCCCGGCACCCGCGTCCGCGCCATGGCCATCTACAAGCAGTCACAGCACATGACGGAGGTTGTGAGGCGCTGCCCCCACCATGAGCGCTGCTCAGATAGCGATGGTCTGGCCCCTCCTCAGCATCTTATCCGAGTGGAAGGAAATTTGCGTGTGGAGTATTTGGATGACAGAAACACTTTTCGACATAGTGTGGTGGTGCCCTATGAGCCGCCTGAGGTTGGCTCTGACTGTACCACCATCCACTACAACTACATGTGTAACAGTTCCTGCATGGGCGGCATGAACCGGAGGCCCATCCTCACCATCATCACACTGGAAGACTCCAGTGGTAATCTACTGGGACGGAACAGCTTTGAGGTGCGTGTTTGTGCCTGTCCTGGGAGAGACCGGCGCACAGAGGAAGAGAATCTCCGCAAGAAAGGGGAGCCTCACCACGAGCTGCCCCCAGGGAGCACTAAGCGAGCACTGCCCAACAACACCAGCTCCTCTCCCCAGCCAAAGAAGAAACCACTGGATGGAGAATATTTCACCCTTCAGATCCGTGGGCGTGAGCGCTTCGAGATGTTCCGAGAGCTGAATGAGGCCTTGGAACTCAAGGATGCCCAGGCTGGGAAGGAGCCAGGGGGGAGCAGGGCTCACTCCAGCCACCTGAAGTCCAAAAAGGGTCAGTCTACCTCCCGCCATAAAAAACTCATGTTCAAGACAGAAGGGCCTGACTCAGACTAA ( SEQ ID NO：1 ) ,：1-36HIV-147-57 ( TAT ) ； Residue 37-207 is the hypoxia inducible factor sequence; Residue 208-1386 is the p53 gene order; Residue 1387-1389 is the termination codon subsequence.

The fusion rotein of above-mentioned syzygy coding is:

MYGRKKRRQRRRMNPFSTQDTDLDLEMLAPYIPMDDDFQLRSFDQLSPLESSSASP ESASPQSTVTVFQMEEPQSDPSVEPPLSQETFSDLWKLLPENNVLSPLPSQAMDDL MLSPDDIEQWFTEDPGPDEAPRMPEAAPRVAPAPAAPTPAAPAPAPSWPLSSSVPS QKTYQGSYGFRLGFLHSGTAKSVTCTYSPALNKMFCQLAKTCPVQLWVDSTPPPGT RVRAMAIYKQSQHMTEVVRRCPHHERCSDSDGLAPPQHLIRVEGNLRVEYLDDRNT FRHSVVVPYEPPEVGSDCTTIHYNYMCNSSCMGGMNRRPILTIITLEDSSGNLLGR NSFEVRVCACPGRDRRTEEENLRKKGEPHHELPPGSTKRALPNNTSSSPQPKKKPL DGEYFTLQIRGRERFEMFRELNEALELKDAQAGKEPGGSRAHSSHLKSKKGQSTSR HKKLMFKTEGPDSD (SEQIDNO:2), wherein: residue 1-12 is the 47-57 position peptide (TAT) of the trans-activator of HIV-1; Residue 13-69 is a hypoxia inducible factor dependence protein degrading texture territory sequence; Residue 70-462 is the p53 protein sequence.

The present invention also inserts described syzygy in the expression vector.This expression vector is that the prokaryotic expression carrier by the dna clone technique construction is combined with above-mentioned antigen-4 fusion protein gene expression cassette, is built into an energy and increases in prokaryotic cell prokaryocyte, breeds, the fusion sequence of the tumor suppressor protein of expression.

This expression vector can be any of prokaryotic expression carrier, and its preferred vector is pET28a.In a preferred embodiment, syzygy of the present invention obtains pET-PTD-ODD-P53 after inserting carrier.

The invention still further relates to described Expression of Fusion Protein, purifying and refolding method, comprise: with the plasmid of encoding said fusion protein and His label, as the pET plasmid, transform host bacterium BL21 (DE3), cultivation is at 25 ℃～42 ℃, take out activation bacterium liquid, ratio with 5%～20% is cultured to the OD600 value in fresh culture be 0.5～2.0, and adding concentration is the inductor I PTG of 0.4～1mM, and induction time is 3～24h, collect the thalline of abduction delivering, PBS cleans, with the resuspended thalline of 10mmol/L Tris damping fluid of pH8.5～11.5, and the vibration mixing, put the broken bacterium of ultrasonic 10～15min on ice, reclaim inclusion body then.With the dissolving of 6-10M urea, with nickel post or chromatography column purifying inclusion body.Adopt continuous gradient method renaturation target protein.

Also on the other hand, the invention provides the purposes that described fusion rotein is used for preparing the medicine for the treatment of solid tumor.The present invention be more particularly directed to contain the pharmaceutical composition of described fusion rotein, for being selected from the formulation that is suitable for intravenous injection, intra-arterial injection, intratumor injection, intramuscular injection, subcutaneous injection, organ injection and thoracic cavity, intraperitoneal injection.

Description of drawings

Fig. 1: pET-PTD-ODD-P53 makes up synoptic diagram;

Fig. 2: pET-PTD-ODD-P53 is through EcoR I and the evaluation of Sal I double digestion, wherein

Label 1, pET-P53; 2, pET-PTD-P53; 3, pET-PTD-ODD-P53

M, DNA Marker is respectively 500,1000,2000,3500,5500,7000bp;

Fig. 3: the pET-PTD-ODD-P53 expression of results, wherein label 1, contains the pET28a bacterium; 2,, the standard protein molecular weight;

Fig. 4: PTD-ODD-P53 Western result, wherein TOP is PTD-ODD-P53, TP is PTD-P53.

Embodiment

The present invention will be more clearly described by the following example.These embodiment are illustrative, not should be understood to limit the scope of the invention.

Embodiment 1 preparation p53 gene

Get the total RNA 1 μ g of people's tire, add reacted constituent successively by cDNA synthesis system operational guidance.Reaction cumulative volume 20 μ l, behind 42 ℃ of reaction 15min, 95 ℃ of deactivation 5min are as pcr template.With P7:GAGAATTCATGGAGGAGCCGCAGTC (SEQ ID NO:3); P4:CCGTCGACTTAGTCTGAGTCAGGC (SEQ ID NO:4) is a primer, PCR people's total length p53 gene.

Get carrier pET28a and p53PCR fragment respectively, carry out double digestion with EcoR I and SalI, 37 ℃ of 3h reclaim test kit with gel again and reclaim.PET carrier segments and p53 fragment add the T4 ligase enzyme of 3U with 1: 3 mixed, and 16 ℃ of reaction 16h transform reaction product in the competence escherichia coli DH5a, and picking transforms successful bacterium colony, upgrading grain PCR evaluation.Positive colony called after pET-p53.The pET-p53 plasmid is transformed in the competence e. coli bl21.

Embodiment 2 preparation PTD-ODD-P53 genes

With p53PCR fragment among the embodiment 1 is template, with P8:GAGAATTCATGTACGGCCGTAAAAAGAGACGCCAACGTAGACGTGCTATGGAG GAGCC (SEQ ID NO:5); P4 is that primer carries out PCR, and gel reclaims fragment (PTD-P53) behind 1% electrophoresis.

With EcoR I and Sal I double digestion fragment PTD-P53, reclaim test kit with gel behind 37 ℃ of 3h and reclaim.The pET carrier segments of EcoR I and Sal I double digestion and PTD-P53 fragment add the T4 ligase enzyme with 1: 3 mixed, and 16 ℃ of reaction 16h transform reaction product in the competence escherichia coli DH5a, and picking transforms successful bacterium colony, upgrading grain PCR evaluation.Positive colony called after pET-PTD-P53.The pET-PTD-P53 plasmid is transformed in the competence e. coli bl21.

Adopt overlapping extension to make up the PTD-ODD-P53 gene

With P1:GAGAATTCATGTACGGCCGTAAAAAGAGACGCCAACGTAGACG (SEQ IDNO:6); P2:

TCATCATCCATTGGGATATAGGGAGCTAACATCTCCAAGTCTAAAGCACGTCTACG TTGGCG (SEQ ID NO:7) balanced mix adds the PCR reaction system, reacts 30 times, and gel reclaims fragment (P1+P2) behind 2% electrophoresis.

With p53PCR fragment among the embodiment 1 is template, with P3:

TCCCAATGGATGATGACTTCCAGTTAGCTATGGAGGAGCCGCAGTC(SEQ?ID?NO：8)；

P4 is that primer carries out PCR, and gel reclaims fragment (P3+p53) behind 1% electrophoresis.

With fragment (P1+P2) and fragment (P3+p53) balanced mix, be that primer carries out PCR with P1, P4, gel reclaims fragment (PTD-ODD-P53) behind 1% electrophoresis.

With EcoR I and Sal I double digestion fragment PTD-ODD-P53, reclaim test kit with gel behind 37 ℃ of 3h and reclaim.The pET carrier segments of EcoR I and Sal I double digestion and PTD-ODD-P53 fragment were with 1: 3 mixed, add the T4 ligase enzyme, 16 ℃ of reaction 16h transform reaction product in the competence escherichia coli DH5a, picking transforms successful bacterium colony, and upgrading grain PCR identifies (Fig. 1, Fig. 2).Positive colony called after pET-PTD-ODD-P53, wherein the sequence of PTD-ODD-P53 is shown in SEQ ID NO:1.The pET-PTD-ODD-P53 plasmid is transformed in the competence e. coli bl21.

Embodiment 3. preparation PTD-ODD-P53 fusion roteins

1.PTD-ODD-P53 expression

The picking positive bacterium colony that transforms pET-PTD-ODD-P53 is put the activation that (contains kalamycin 60g/ml) in the LB nutrient solution respectively, is inoculated in the 2YT nutrient solution by 5-20% again, and being cultured to OD600 is 0.5～2.0 o'clock, adds 0.4～1mM IPTG abduction delivering (Fig. 3).Collect the thalline of abduction delivering 3～24h, PBS cleans.

2.PTD-ODD-P53 Western identify

The fusion rotein that obtains through abduction delivering is through polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed: target protein is arranged in the precipitation, and its molecular weight conforms to theoretical value.Through the Western trace, be PTD-ODD-P53 (Fig. 4) with P53 monoclonal antibody identification and analysis.Order-checking shows that it has the aminoacid sequence of SEQID NO:2.

3. inclusion body washing

Resuspended with phosphoric acid buffer (pH 7.6), carrying out ultrasonic bacteria breaking, 4 ℃ of 10000rpm are centrifugal, and the precipitation part is used 0.5-4M urea, 100mmol/L NaCl, 20mmol/L Tris-HCl (pH8.0) washing is spent the night for 4 ℃.

4. solubilization of inclusion bodies and renaturation

Centrifugal 15 minutes with 10000 rev/mins, collecting precipitation with 6-10mol/L urea (being dissolved in 20mmol/L tri methylol amino methane pH of buffer 8.0-12.0) dissolving, slowly adds the equal-volume renaturation solution and (contains 20mmol/L Tris, 1mmol/LEDTA, 0.1mmol/LGSH; 0.01mol/L GSSG), 12000 rev/mins centrifugal 15 minutes, get the supernatant 8000-12000 dialysis tubing of packing into, put and adopt the continuous gradient dialysis method to dialyse in 4-12 ℃, with normal saline dialysis 3 times to renaturation solution.

Embodiment 4.PTD-ODD-P53 determination of activity

1) PTD-ODD-P53 crosses the cytolemma test

The PTD-ODD-P53 fusion rotein that embodiment 3 is obtained adds among the SW480, be that an anti-immunohistochemistry of doing detects with anti-P53 monoclonal antibody behind the 2h, control group adds among the SW480 with PTD-P53, P53 respectively, the result shows, PTD-ODD-P53, PTD-P53 can enter in the cell, and to be distributed as the master in the nucleus.

Also studied the influence of PTD-ODD-P53 pair cell under the normal oxygen condition, the influence of PTD-ODD-P53 pair cell under the weary oxygen condition, and PTD-ODD-P53 is to the influence of tumor animal.

The invention effect

Utilize the means such as genetic engineering, protein purification, prepare and have the film activity of wearing, lacking Stable and the active multiple active peptide of other biological and guides to prepare to have medicine in the oxygen environment With the bio-carrier medicine that is worth, it is thin that PTD-ODD-P53 can suppress tumour at the hypoxia in vitro environment Intracellular growth can suppress the growth of transplantable tumor in Mice Body in vivo, prolongs the survival of mouse Time. Be used for the treatment of human multiple solid tumor, have important medical research and clinical use Be worth.

Sequence table

＜110〉Inst of Toxic Medicinal Materials, P.L.A. Academy of Military Medical Sciences

＜120〉amalgamation and expression of the ODD of PTD, HIF and tumor suppressor gene and application thereof

<160>8

<210>1

<211>1389

<212>DNA

＜213〉sequence of genetic fusant PTD-ODD-p53

<400>1

ATGTACGGCC?GTAAAAAGAG?ACGCCAACGT?AGACGTATGA?ACCCATTTTC?TACTCAGGAC

ACAGATTTAG?ACTTGGAGAT?GTTAGCTCCC?TATATCCCAA?TGGATGATGA?CTTCCAGTTA

CGTTCCTTCG?ATCAGTTGTC?ACCATTAGAA?AGCAGTTCCG?CAAGCCCTGA?AAGCGCAAGT

CCTCAAAGCA?CAGTTACAGT?ATTCCAGATG?GAGGAGCCGC?AGTCAGATCC?TAGCGTCGAG

CCCCCTCTGA?GTCAGGAAAC?ATTTTCAGAC?CTATGGAAAC?TACTTCCTGA?AAACAACGTT

CTGTCCCCCT?TGCCGTCCCA?AGCAATGGAT?GATTTGATGC?TGTCCCCGGA?CGATATTGAA

CAATGGTTCA?CTGAAGACCC?AGGTCCAGAT?GAAGCTCCCA?GAATGCCAGA?GGCTGCTCCC

CGCGTGGCCC?CTGCACCAGC?AGCTCCTACA?CCGGCGGCCC?CTGCACCAGC?CCCCTCCTGG

CCCCTGTCAT?CTTCTGTCCC?TTCCCAGAAA?ACCTACCAGG?GCAGCTACGG?TTTCCGTCTG

GGCTTCTTGC?ATTCTGGGAC?AGCCAAGTCT?GTGACTTGCA?CGTACTCCCC?TGCCCTCAAC

AAGATGTTTT?GCCAACTGGC?CAAGACCTGC?CCTGTGCAGC?TGTGGGTTGA?TTCCACACCC

CCGCCCGGCA?CCCGCGTCCG?CGCCATGGCC?ATCTACAAGC?AGTCACAGCA?CATGACGGAG

GTTGTGAGGC?GCTGCCCCCA?CCATGAGCGC?TGCTCAGATA?GCGATGGTCT?GGCCCCTCCT

CAGCATCTTA?TCCGAGTGGA?AGGAAATTTG?CGTGTGGAGT?ATTTGGATGA?CAGAAACACT

TTTCGACATA?GTGTGGTGGT?GCCCTATGAG?CCGCCTGAGG?TTGGCTCTGA?CTGTACCACC

ATCCACTACA?ACTACATGTG?TAACAGTTCC?TGCATGGGCG?GCATGAACCG?GAGGCCCATC

CTCACCATCA?TCACACTGGA?AGACTCCAGT?GGTAATCTAC?TGGGACGGAA?CAGCTTTGAG

GTGCGTGTTT?GTGCCTGTCC?TGGGAGAGAC?CGGCGCACAG?AGGAAGAGAA?TCTCCGCAAG

AAAGGGGAGC?CTCACCACGA?GCTGCCCCCA?GGGAGCACTA?AGCGAGCACT?GCCCAACAAC

ACCAGCTCCT?CTCCCCAGCC?AAAGAAGAAA?CCACTGGATG?GAGAATATTT?CACCCTTCAG

ATCCGTGGGC?GTGAGCGCTT?CGAGATGTTC?CGAGAGCTGA?ATGAGGCCTT?GGAACTCAAG

GATGCCCAGG?CTGGGAAGGA?GCCAGGGGGG?AGCAGGGCTC?ACTCCAGCCA?CCTGAAGTCC

AAAAAGGGTC?AGTCTACCTC?CCGCCATAAA?AAACTCATGT?TCAAGACAGA?AGGGCCTGAC

TCAGACTAA

1389

<210>2

<211>462

＜212〉aminoacid sequence

＜213〉fusion rotein of PTD-ODD-p53 coding

<400>2

MYGRKKRRQR?RRMNPFSTQD?TDLDLEMLAP?YIPMDDDFQL?RSFDQLSPLE?SSSASPESAS

PQSTVTVFQM?EEPQSDPSVE?PPLSQETFSD?LWKLLPENNV?LSPLPSQAMD?DLMLSPDDIE

QWFTEDPGPD?EAPRMPEAAP?RVAPAPAAPT?PAAPAPAPSW?PLSSSVPSQK?TYQGSYGFRL

GFLHSGTAKS?VTCTYSPALN?KMFCQLAKTC?PVQLWVDSTP?PPGTRVRAMA?IYKQSQHMTE

VVRRCPHHER?CSDSDGLAPP?QHLIRVEGNL?RVEYLDDRNT?FRHSVVVPYE?PPEVGSDCTT

IHYNYMCNSS?CMGGMNRRPI?LTIITLEDSS?GNLLGRNSFE?VRVCACPGRD?RRTEEENLRK

KGEPHHELPP?GSTKRALPNN?TSSSPQPKKK?PLDGEYFTLQ?IRGRERFEMF?RELNEALELK

DAQAGKEPGG?SRAHSSHLKS?KKGQSTSRHK?KLMFKTEGPD?SD 462

<210>3

<211>25

<212>DNA

＜213〉primer P7

<400>3

GAGAATTCAT?GGAGGAGCCG?CAGTC 25

<210>4

<211>24

<212>DNA

＜213〉primer P4

<400>4

CCGTCGACTT?AGTCTGAGTC?AGGC 24

<210>5

<211>58

<212>DNA

＜213〉primer P8

<400>5

GAGAATTCAT?GTACGGCCGT?AAAAAGAGAC?GCCAACGTAG?ACGTGCTATG?GAGGAGCC 58

<210>6

<211>43

<212>DNA

＜213〉primer P1

<400>6

GAGAATTCAT?GTACGGCCGT?AAAAAGAGAC?GCCAACGTAG?ACG 43

188

<210>7

<211>62

<212>DNA

＜213〉primer P2

<400>7

TCATCATCCA?TTGGGATATA?GGGAGCTAAC?ATCTCCAAGT?CTAAAGCACG?TCTACGTTGG

CG 62

<210>8

<211>46

<212>DNA

＜213〉primer P3

<400>8

TCCCAATGGA?TGATGACTTC?CAGTTAGCTA?TGGAGGAGCC?GCAGTC 46

Claims (13)

1. genetic fusant, it is made up of protein transduction domain (PTD), the oxygen dependence proteolytic degradation structural domain (ODD) of hypoxia inducible factor (HIF-1), human tumor suppressor gene.

2. genetic fusant as claimed in claim 1, wherein PTD is selected from protein transduction district (TAT), fruit bat feeler albumen homology structural domain and the proteic protein transduction sequence of hsv VP22 in the HIV-1 trans-activator.

3. genetic fusant as claimed in claim 1, wherein ODD can be the full length sequence of oxygen dependence proteolytic degradation structural domain or any a part of sequence fragment wherein.

4. genetic fusant as claimed in claim 1, wherein human tumor suppressor gene is p53.

5. genetic fusant as claimed in claim 1, it has the sequence of SEQ ID NO:1.

6. by the fusion rotein of each genetic fusant coding among the claim 1-5.

7. the fusion rotein of claim 6, it has the sequence of SEQ ID NO:2.

8. gene construct, it contains among the claim 1-5 each genetic fusant, and expression vector.

9. the gene construct of claim 8, wherein said expression vector is a prokaryotic expression carrier, preferred pET28a.

10. claim 8 or 9 gene construct, wherein the upstream of genetic fusant is eukaryotic cell promotor, prokaryotic cell prokaryocyte promotor or viral promotors.

11. the purposes of the fusion rotein of claim 6 or 7 in the medicine of the various malignant entity tumors of preparation treatment.

12. the purposes of the fusion rotein of claim 6 or 7 in the medicine of transfer for preparing prophylaxis of tumours and the tumor regrowth behind the excision.

13. a pharmaceutical composition contains the fusion rotein of claim 6 or 7, is preferably the formulation that is suitable for carrying out intravenous injection, intra-arterial injection, intratumor injection, intramuscular injection, subcutaneous injection, organ injection and thoracic cavity, intraperitoneal injection.

Images