CN100352929C - Bitarget fibroblast growth factor acceptor and transgene carrier of integrated element - Google Patents
Bitarget fibroblast growth factor acceptor and transgene carrier of integrated element Download PDFInfo
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Abstract
The present invention provides a dual-target transgene carrier of a fibroblast growth factor receptor and integrin, which comprises a polypeptide CR16 specifically combined with a basic fibroblast growth factor receptor, and a gene transfer non-viral carrier composite system of polypeptide CP9 and CR16/CP9/cation polymer PEI/exogenous DNA specifically combined with the integrin. The carrier provided by the present invention can effectively lead the exogenous DNA in a tumor cell line and a tumor tissue with high expression of FGFRs to achieve the function of tumor growth inhibition. The carrier provided by the present invention have low transfection efficiency to tumor cells with no expression of FGFRs, so that the carrier has the tumor targeting performance. The transferable exogenous DNA range of the gene transfer non-viral carrier composite system provided by the present invention is from scores of bp to thousands of kb so as to overcome the size limit when the virus carriers are inserted in exogenous gene. The present invention can be used for preparing medicines for treating tumor by polygenic combined transfer and can also be used as a platform technology to prepare gene medicines for treating other diseases.
Description
Technical field
The invention belongs to biological technical field, made up a kind of with polymine (polyethylenimine, PEI) be basic framework, the associating target is in tumor cell surface fibroblast growth factor acceptor (fibroblast growth factor receptors, FGFRs) and integrate the non-viral transgene carrier of plain (integrin), be used for the expression plasmid of coding reporter gene and therapeutic gene is changed over to the tumour cell of high expression level FGFR and integrin receptor.The invention still further relates to the preparation method and the purposes of this non-viral transgene carrier.
Background technology
Gene therapy refers to such an extent that be meant and change foreign gene over to cell interior, by recovering or increasing genetic expression with the structure of correction people autogene or the entanglement on the function, the progress that stops pathology, kill the cell of pathology, or suppress duplicating of foreign pathogens genetic material, thereby reach the purpose of treatment disease.Gene therapy is the effective treatment measures of disease such as following treatment tumour.Gene therapy comprises three important steps, i.e. goal gene, transgene carrier and target cell.Wherein transgene carrier is that the core technology of gene therapy also is one of bottleneck of present gene therapy technology progress.Non-viral transgene carrier has safe for virus vector, immunogenicity is low, be easy to DNA is operated, preparation technology is simple relatively, advantages such as the easy control of influence factor, more and more paid attention to, and the shortcoming of non-virus carrier is that transfection efficiency is relatively low and lacks target, at present main policies is that to make up with the cell receptor be the non-viral transgene carrier system of target, utilize specific acceptor of cell surface expression or albumen, specific ligand molecular or fragment and carrier are connected to form the molecule couplet, make DNA can forward the cell of expressed receptor to target, pass through simultaneously to start receptor-mediated cell endocytic process, thereby reach the purpose that improves transfection efficiency.(polyethylenimine PEI) is the most frequently used cationic polymer non-viral gene rotaring carrier to polymine
[1,2], contain primary amine, secondary amine and tertiary amine ,-NH
2Has very strong DNA keying action; therefore PEI can form the mixture (complexes) of hundreds of nanometer sizes by coulombic interaction and electronegative dna molecular; enter cell by cell endocytic or phagolysis;-NH-,=very strong shock absorption is arranged after N-is protonated, can protect DNA in Cytolysosome not by nuclease degradation.The shock absorption of PEI has perviousness swelling effect, causes lysosome to break, and makes DNA enter endochylema, and promotes that DNA enters nucleus.
Fibroblast growth factor (fibroblast growth factors, FGFs) be the peptide family of a heparin-binding, at least comprised relevant polypeptide on 23 structures, bFGF (basic fibroblastgrowth factor wherein, bFGF or title FGF-2) closer with the tumour relation, bFGF can stimulate tumor growth, is strong angiogenic agent simultaneously, has vital role in the vascularization of tumour.Studies show that many cancerous cell line high expression level FGFR are as Chandler etc.
[3]Studied the expression of 9 dissimilar human tumor cell line FGFR of 60 strains, the result shows that 90% expresses more than one FGFR.In the nucleus of resting cell, almost detect less than bFGF and acceptor thereof.
In the virus vector transfectional cell, involve amboceptor mechanism.Need viral special acceptor on the one hand, need on the other hand in conjunction with integrating element, virus could enter cell effectively.Multiple virus is integrated plain the combination by the RGD primitive (RGD motif) of particle capsid protein with cell surface ground and is entered cell.Integrating element is cell surface adhesion molecule, participates in the adhesion process between cell-cell matrix; In tumor development, participate in adhesion, invasion and attack and the transfer of tumour, at endothelial cells in tumor neogenetic blood vessels surface high expression level.The amboceptor mediation mechanism that simulated virus carrier transfectional cell needs, comprise the native ligand that the RGD polypeptide of sequence can the simulation integration element, the transgene carrier of then uniting target FGFR has the associating targeting to genetic treatment of tumor, and the associating target can effectively improve target and transfection efficiency.
The part of dissimilar cell surface receptors for example carbohydrate residue, peptide, protein and antibody has been used for the target non-virus carrier, as Transferrins,iron complexes, seminose, epithelical cell growth factor, folic acid etc., the existing report of complete molecular targeted virus vector of bFGF and poly-lysine carrier
[4-10]At present aspect the structure of non-virus carrier, the application of polypeptide ligand because polypeptide synthetic easy, can reduce the space steric effect that complete molecule coupling brings and become one of research direction.Use the rgd peptide link coupled PEI of bFGF functional domain peptide molecule combined simulation integrin not appear in the newspapers as yet at present.
Summary of the invention
The invention provides the associating target in cell surface FGFR and the plain polymine transgene carrier system of integration, comprise and the design of the non-viral transgene carrier composite system of the design that contains rgd peptide CP9 of the design of the peptide C R16 of FGFR specific combination, the plain part of simulation integration, peptide C R16/ peptide C P9/PEI/ foreign DNA, the coupling rate and the synthesis technique of each assembly, and utilize this non-viral transgene carrier system mediate foreign gene to comprise application in the multiple malignant tumours such as liver cancer, prostate cancer, cervical cancer in treatment.
First part of the present invention provides and the peptide C R16 of cell surface basic fibroblast growth factor receptor specific combination and the peptide C P9 of analog cell surface integrin, we design and have synthesized bFGF ligand polypeptide CR16 sequence: NH2-Cys-Tyr-Arg-Ser-Arg-Lys-Tyr-Thr-Ser-Trp-Tyr-Val-Ala-Leu-Lys-Arg-OH, the plain part of synthetic simulation integration contain rgd peptide CP9 sequence: NH2-Cys-Tyr-Gly-Gly-Arg-Gly-Asp-Thr-Pro-OH.Polypeptide is synthetic by solid phase method, carries out on the Peptide synthesizer (431A) of American AB I company, adopts Fmoc (9-fluorenylmethyloxycarbonyl) scheme, and synthesis step carries out according to the polypeptide synthetic operation handbook of ABI company.Through high-efficient liquid phase chromatogram purification, purity＞95%, Sequence Identification is by mass spectroscopy.Polypeptide will be done crosslinkedly with the macromole of sulfhydrylation, and the halfcystine of synthetic back oligopeptides end keeps sulphydryl activity, avoids crosslinked between the polypeptide simultaneously, does not contain the sulfydryl reductive agent in the polypeptide, as DTT, 3-mercaptoethanol or dimercaptoethane etc.
Second section of the present invention provides the non-viral transgene carrier composite system of two polypeptide/PEI/ foreign DNA, wherein polypeptide is CR16 and CP9, cationic polymer is polymine PEI, and foreign DNA is that reporter gene is as the plasmid pEGFP of coding enhanced green fluorescence protein EGFP and the pCAG-Luc of coding Luci etc.The construction process of this non-virus carrier mixture: synthetic polypeptide end contains the halfcystine (Cysteine of a free sulfhydryl group, C), be used for forming disulfide linkage with isodigeranyl functional cross-link agent activatory PEI, guarantee that every polypeptide and PEI have only a crosslink sites, the coupled product of preparation polypeptide and PEI, by
1Physics and chemistry means such as H-NMR, infrared, ultraviolet are identified the successful coupling of polypeptide, mix forming the carrier complexes system again with electronegative foreign DNA, and the formation by confirmation composite system such as Electronic Speculum, gel retardation assasies.
To be the non-viral transgenic compound system that utilizes two polypeptide/PEI/ foreign DNA comprise application in the medicine of various malignant tumour tumours such as liver cancer, prostate cancer, cervical cancer in the preparation treatment to third part of the present invention, the coupling rate, N/P of determining transfection composite the best by experiment in cell in vitro transfection experiment and the body than and solvent.
Utilize above-mentioned carrier system transfection pEGFP plasmid, the pCAG-Luc plasmid is to monkey embryonic kidney cells COS-7 (the FGFR positive), human cervical carcinoma cell HeLa (the FGFR positive), Human Prostate Cancer Cells PC-3 (FGFR feminine gender), human liver cancer cell HepG2 (the FGFR positive) etc. also detect its transfection efficiency, experimental results show that CR16-PEI-CP9 (the coupling rate is mol ratio 0.25: 1: 0.25) can mediate pEGFP plasmid and pCAG-Luc plasmid FGFR male Hela effectively, COS-7, express in the cells such as HepG2, compare with free PEI and single polypeptide ligand link coupled PEI, transfection efficiency increases; PC-3 cell to the FGFR feminine gender then can not increase transfection efficiency.Set up the lotus knurl model of BALB/C nude mice, the carrier complexes system that foreign DNA is carried in injection in the knurl body, the expression of observation goal gene.Experiment showed, that CR16-PEI-CP9 transgenosis system can mediate the pCAG-Luc reporter gene and express effectively in the knurl body part of HepG2 liver cancer nude mice model.
The non-viral transgene carrier of the present invention system imports the tumour cell of expressing FGFRs with foreign DNA target ground in testing in vivo and in vitro, in the malignant tumour gene therapy, has application promise in clinical practice, for the application of clinical tumor genomic medicine provides the research basis.
Discharge medicine or transgenosis to tumour, FGFR is an attracting target site of potential, and most of cancer cells and tumor neogenetic blood vessels be high expression level FGFRs all.The polypeptide that contains RGD is second ligand function of simulated virus transfection effectively, obtains more fully proving on other carriers.The present invention utilizes the skeleton of PEI as non-viral transgene carrier system, utilize FGFR simultaneously and integrate plain characteristics at most of tumour cells and tumor neogenetic blood vessels high expression level, autonomous design can with the peptide C R16 and the CP9 of the corresponding receptors bind of tumor cell surface, utilize the chemically crosslinked technology with two polypeptide and PEI coupling, made up novel non-viral gene vector system, to increase its target, to aim to provide the research that is used for the preparation of malignant tumour gene therapy medicament to tumour cell and new vessel thereof.Utilize the acceptor of overexpression on the ligand polypeptide tumor cell film, start receptor-mediated cell endocytic approach, foreign DNA is optionally imported tumour cell, reach the purpose of treatment tumour by this approach.BFGF receptor binding domain polypeptide unite integrate plain combined function district polypeptide target to PEI do not see as yet and use and report.
Advantage of the present invention: the invention provides a kind of can with the synthetic peptide C R16 of tumor cell membrane surface fibroblast growth factor acceptor specific combination, the peptide C P9 of associating targeted integration element has made up CR16-CP9 peptide/cationic polymer PEI/ foreign DNA complex body non-viruse gene transferring vector; The non-viruse gene transferring vector of invention can import foreign DNA tumor cell line and the tumor tissues of high expression level FGFRs effectively, reach the effect that suppresses tumor growth, but the transfection efficiency to the tumour cell of not expressing FGFRs is very low, makes carrier system possess tumor-targeting; The transferable foreign DNA magnitude range of the non-viral transgene carrier system of the present invention can be from tens bp to several thousand kb, overcome the size restriction that virus vector inserts foreign gene, can be used for preparing polygenic medicine of uniting transfer, also can be used as platform technology simultaneously and be used to prepare the genomic medicine for the treatment of other diseases with the treatment tumour.
Figure of description
The 1H-NMR collection of illustrative plates of Fig. 1 CR16-PEI-CP9 (0.25: 1: 0.25);
The fourier transform infrared spectrometry collection of illustrative plates of Fig. 2 CR16-PEI-CP9 (0.25: 1: 0.25);
The thermogravimetric analysis collection of illustrative plates of Fig. 3 CR16-PEI-CP9 (0.25: 1: 0.25);
The uv-vis spectra of Fig. 4 CR16-PEI-CP9 (0.25: 1: 0.25);
Fig. 5 CR16-PEI-CP9/pEGFP dna gel electrophoresis result;
Fig. 6 CR16-PEI-CP9/pEGFP DNA mixture (N/P=10) Electronic Speculum result;
Fig. 7 CR16-PEI-CP9 (0.25: 1: 0.25)/DNA mixture particle size determination result;
Luciferase expression level behind Fig. 8 CR16-PEI-CP9 (0.25: 1: 0.25)/pCAG-Luc transfection COS-7, HepG2, the PC3 cell;
Fig. 9 CR16-PEI-CP9 (0.25: 1: 0.25) transfection pEGFP is to the fluorescence photo of COS-7 cell.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.
Embodiment one
Polypeptide and PEI are by isodigeranyl functional cross-link agent SPDP (Succinimidyl 3-(2-pyridyldithio) propionate) coupling.The amino effect of SPDP and PEI (pH 7～8) forms the PEI (PDP-PEI) that the pyridine dimercapto is modified; The latter can with the proteins react that contains sulfydryl.Polypeptide produces oligopeptides-PEI derivative that disulfide linkage connects by cysteine sulfydryl group and PDP-PEI reaction.Get CR16, CP9 peptide and PEI formation coupled product with the PEI different mol ratio, characterize mixture by various physics and chemistry means, compare with electronegative foreign DNA according to certain N/P again and be combined into CR16 peptide/CP9 peptide/PEI/ foreign DNA carrier complexes by electrostatic interaction, then this transfection composite is applied to external, the interior experiment of body, determines genetically modified effect and definite transgenosis condition.
1.CR16, the CP9 peptide synthetic:
Polypeptide is synthetic by solid phase method, through high-efficient liquid phase chromatogram purification, and purity＞95%, Sequence Identification is by mass spectroscopy.Polypeptide will be done crosslinkedly with the macromole of sulfhydrylation, and the halfcystine of synthetic back oligopeptides end keeps sulphydryl activity, avoids crosslinked between the polypeptide simultaneously, does not contain sulfydryl reductive agent (as DTT, 3-mercaptoethanol or dimercaptoethane etc.) in the polypeptide.The function of polypeptide is screened by polypeptide PEI conjugate/DNA mixture transfectional cell experiment and is verified.
The preparation of activation PEI and with the coupling of peptide:
Respectively add 2.78ml PEI (9mg/ml) solution in the 50ml round-bottomed flask, accurately taking by weighing 5.1mg SPDP is dissolved in the mixed solution of 1600 μ l physiological saline and 1600 μ lDMSO, be equally divided into four parts, every part 800 μ l, add respectively in the round-bottomed flask and mix, stir dark place reaction 3 hours under the room temperature with PEI solution.Stop the first step reaction, accurately taking by weighing the CR16 of corresponding different mol ratio and CP9 is dissolved in the 1500 μ l physiological saline (in the process of the present invention, the mol ratio of peptide and PEI was established 0.25: 1,0.5: 1,1: 1,2: 1 four ratios), be divided into four parts in proportion and add respectively in the above-mentioned flask, carry out the reaction of second step.Room temperature, nitrogen protection stir the dark place reaction down and spend the night.Stopped reaction places dialysis tubing (MWCO is 10000) with reaction product, flowing water dialysis 2 days, and frost drying 48hr obtains the white powder solid product then.
3. the evaluation of polypeptide-PEI conjugate
3.1
1H-NMR
Whether coupling is successful to identify polypeptide and PEI by NMR.Get part polypeptide-PEI solution to be determined and PEI solution for vacuum drying freely.Dried white powder is dissolved in water-d2 (deuterium oxide, D
2O), 0.22 μ m pin type miniature displacer filters, at Varian Inova 600 MHz spectrometers (Varian, Palo Alto, CA) the proton parametric measurement of last application standard
1The H-NMR wave spectrum, the chemical shift at peak is with reference to HDO (hydrogen deuderium oxide, resonance peak HDO) (4.8ppm).The result is referring to Fig. 1, and wherein scheming A is PEI's
1H-NMR collection of illustrative plates, figure B are CR16-PEI-CP9's (0.25: 1: 0.25)
1The H-NMR collection of illustrative plates.
3.2 fourier transform infrared spectrometry
The Potassium Bromide (KBr) of the solid sample of 1mg and 10～100 times grinds to form the fine powder mixture in agate mortar, get in right amount with the tabletting machine compressing tablet that reduces pressure, be pressed into diameter 10mm, thick 10mm thin slice, FT-IRBruber Vector 22 infrared spectrometers of packing into are measured.The PEI that takes a morsel is dissolved in and forms 1%～5% solution in the DMSO organic solvent.Drip a drop of liquid and be clipped in (sodium-chlor is transparent) between two sodium chloride salt sheets between 4000～625cm-1, FT-IR Bruber Vector 22 infrared spectrometers of packing into are measured, the result is referring to Fig. 2, and wherein reaching the standard grade is CR16-PEI-CP9 (0.25: 1: 0.25), and rolling off the production line is PEI
3.3 thermogravimetric analysis
Carry out thermogravimetric analysis with Universal V3.8B TA equipment.Sample with the speed of 10 ℃/min in nitrogen atmosphere (flow velocity=70ml/min) begin slowly to be warmed up to 600 ℃ from room temperature, the result is referring to Fig. 3, wherein scheming A is PEI, figure B is CR16-PEI-CP9 (0.25: 1: 0.25).
3.4 uv-vis spectra
Get wiring solution-forming in the pure water that a certain amount of sample is dissolved in about 5ml, detect with HITACHI U-3400 type ultraviolet spectrophotometer.The result is referring to Fig. 4, and wherein scheming A is PEI, and figure B is CR16-PEI-CP9 (0.25: 1: 0.25).
More than various physics and chemistry means show that all polypeptide successfully is coupled on the PEI.
4.CR16-CP9 the preparation of peptide/PEI/ foreign DNA complexes carrier, gel electrophoresis is identified, transmission electron microscope observing size and form, particle size determination.
4.1CR16-CP9 peptide/PEI/ foreign DNA complexes carrier gel electrophoresis is identified
Press PEI/DNAN/P=0,0.5,1.0,1.5,2.0,3.0,4.0,5.0, use 5%GS, prepare CR16-CP9 peptide/PEI/pEGFP DNA mixed solution respectively, get 10 μ l respectively and carry out agarose gel electrophoresis observation DNA retardance situation as solvent.Deposition condition: 1% agarose (containing 0.5 μ g/ml ethidium bromide), 1 * TAE damping fluid, voltage 5 V/cm, electrophoresis time 30min.The result shows N/P than plasmid DNA being arrested in the well fully greater than 3 o'clock.The results are shown in accompanying drawing 5.
4.2 CR16-CP9 peptide/PEI/ foreign DNA complexes carrier transmission electron microscope observing size and form
The size of CR16-CP9 peptide/PEI/ foreign DNA mixture and form detect by the negative staining transmission electron microscope.Prepare PEI/DNA mixture, CR16-CP9 peptide/PEI/pEGFP DNA mixture by N/P=10.Behind the incubation 30min, complex solution dropped in modestly to be covered with the contract copper/rhodium of methyl alcohol supporting film of polyvinyl alcohol online.Behind the 1min, copper/rhodium net is placed on negative staining 20s on the uranyl acetate solution.Excessive solution is carefully absorbed with thieving paper.Sample is observed under Philips TECNAI 10 transmission electron microscopes, voltage 80kV.The result shows that the mixture of formation is rounded, and size is about 200nm.The results are shown in accompanying drawing 6, and transmission electron microscope observing CR16-PEI-CP9 (0.25: 1: 0.25)/pEGFP plasmid composite physical form and size (N/P=10, scale=200nm).
4.3 the particle size determination of CR16-CP9 peptide/PEI/ foreign DNA complexes carrier
CR1 6-CP9 peptide/PEI and plasmid DNA solution are added in the eppendorf pipe of two 1.5ml than respectively by different N/P, with 0.9% physiological saline two solution all are diluted to final volume 250 μ l respectively.Dna solution is dropwise joined in vibration in CR16-CP9 peptide/PEI solution, place 30min behind the vibration 1min, measure the mixture particle diameter with 90Plus dynamic light scattering particle size analyzer then.0.9% physiological saline of adding about 2/3 adds mixture in the cuvette then in cuvette, places instrument to measure particle diameter.Measure 10 times, each 1min draws 10 data and gets its mean value.The result shows when carrier/DNA mixture is 10 at N/P, can form the particle of size about 200nm, and N/P continues to increase, and particle diameter further reduces, but the minimizing amplitude is not obvious.The results are shown in accompanying drawing 7.
5.CR16-CP9 peptide/PEI/ foreign DNA complexes carrier system is used for the gene therapy of the transfection experiment and the tumor bearing nude mice of COS-7 cell, tumour cell
5.1 the CR16-PEI-CP9 transfection pCAG-Luc of homopolypeptide coupling rate does not determine best coupling rate and transfection N/P ratio to COS-7, HepG2, PC3 cell.
COS-7, HepG2, PC3 cell are seeded in 48 orifice plates respectively, every hole 2.5 * 10
4Cell.Cultivate 24h, transfection when treating cell 60～80% fusions.Transfection 2 μ gpCAG-Luc plasmids in every hole are used PEI, CR16-PEI-CP9 (peptide and PEI mol ratio were respectively 0.25: 1,0.5: 1,1.0: 1,2.0: 1) preparation transfection composite respectively by PEI/DNAN/P=5, (7.5), 10,20,30.Cell culture fluid is removed in suction, and every hole adds 1ml serum-free DMEM, and transfection composite is added on the cell, and level is slowly shaken 30min, puts into cell culture incubator then and cultivates.After cultivating 6～8h, transfection liquid is removed in suction, after the DMEM that every hole adding 1ml contains 10% serum continues to cultivate 36h, outwell supernatant, add 200ulPBS,-80 ℃ of multigelations 3 times, obtained cell suspension is centrifugal, get supernatant 5ul, adopt the Luciferase detection kit of Promega company, according to test kit specification sheets, fluorescence intensity, the total protein concentration of quantitative respective volume supernatant, the luciferase expression amount of Units of Account albumen (mg), result show CR16-PEI-CP9 (0.25: 1: 0.25) in the time of N/P=10, and be the highest at the transfection efficiency of FGFRs male COS-7 and HepG2 cell, be higher than contrast PEI, otherwise in the PC3 of FGFRs feminine gender cell, transfection efficiency will be lower than PEI, the results are shown in accompanying drawing 8, wherein scheming A is the fluorescent value of CR16-PEI-CP9 transfection COS-7 cell, figure B is the fluorescent value of transfection HepG 2 cell, figure C is the fluorescent value of transfection PC3 cell.
5.2 CR16-PEI-CP9 (0.25: 1: 0.25) transfection pEGFP (N/P=10) detects to the fluorescin of COS-7 cell
Transfection process is with 5.1, and plasmid adopts pEGFP, and 36hr after the transfection adopts inverted fluorescence microscope to observe the green fluorescence that green fluorescent protein sends, and the result shows that pEGFP is transferred in the COS-7 cell, and expressing green fluorescent protein sends green fluorescence.The results are shown in accompanying drawing 9.
5.3 CR16-PEI-CP9 (0.25: 1: 0.25) is used for the interior transfection experiment of the body of tumor bearing nude mice
With the HepG2 cell strain, it is subcutaneous that the PC3 cell strain is inoculated in nude mice, set up tumor model, the knurl block length is during to the about 1.0cm of diameter, CR16-PEI-CP9 (0.25: 1: the 0.25) carrier complexes of pCAG-Luc plasmid is carried in injection in the knurl body, injection plasmid amount is 50ug, N/P=10 sacrifices nude mice behind the 36hr, take out tumour, make homogenate with 1mlPBS,-80 ℃ of multigelations 3 times, centrifugal, get the Luciferase detection kit that the 10ul supernatant adopts Promega company, according to the test kit specification sheets, fluorescence intensity, the total protein concentration of quantitative respective volume supernatant, the luciferase expression amount of Units of Account albumen (mg).Though the result shows intravital transfection efficiency and will be lower than experiment in vitro that CR16-PEI-CP9 (0.25: 1: 0.25) shows still that in vivo transfection efficiency is higher than the PEI contrast.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Claims (4)
1. two targets are in fibroblast growth factor acceptor and integrate plain polymine transgene carrier, form by polypeptide/poly ethyleneimine/exogenous plasmid dna, it is characterized in that: by the receptor binding domain peptide C R16 and the plain land peptide C P9 associating of the integration target of Prostatropin, with the poly ethyleneimine is the non-virus carrier composite system that skeleton and foreign DNA are formed, the aminoacid sequence that wherein said peptide C R16 has is NH2-Cys-Tyr-Arg-Ser-Arg-Lys-Tyr-Thr-Ser-Trp-Tyr-Val-Ala-Leu-Lys-Arg-OH, and the aminoacid sequence that described peptide C P9 has is NH2-Cys-Tyr-Gly-Gly-Arg-Gly-Asp-Thr-Pro-OH.
2. transgene carrier as claimed in claim 1 is characterized in that: described peptide C R16 is and cell surface fibroblast growth factor acceptor specific combination; Described peptide C P9 integrates plain specific combination with cell surface, and every polypeptide end contains a halfcystine, comprises sulfydryl freely, is used for and the coupling of linking agent activatory polymine.
3. transgene carrier as claimed in claim 1 or 2 is characterized in that: described foreign DNA is the eukaryotic expression recombination plasmid that contains reporter gene, cytokine gene, cancer suppressor gene.
4. as the application of the arbitrary described transgene carrier of claim 1-3 in the genomic medicine of preparation treatment tumour.
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| WO2004087931A1 (en) * | 2003-04-03 | 2004-10-14 | Korea Advanced Institute Of Science And Technology | Conjugate for gene transfer comprising oligonucleotide and hydrophilic polymer, polyelectrolyte complex micelles formed from the conjugate, and methods for preparation thereof |
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| WO2004087931A1 (en) * | 2003-04-03 | 2004-10-14 | Korea Advanced Institute Of Science And Technology | Conjugate for gene transfer comprising oligonucleotide and hydrophilic polymer, polyelectrolyte complex micelles formed from the conjugate, and methods for preparation thereof |
| CN1614027A (en) * | 2004-11-10 | 2005-05-11 | 浙江大学 | Polytheneimine transgened carrier of targeted fibroblast growth factor receptor |
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