CN1389269A - Compound of polypeptide-liposome and human vascular endothelial growth factor gene recombination plasmid and its use - Google Patents
Compound of polypeptide-liposome and human vascular endothelial growth factor gene recombination plasmid and its use Download PDFInfo
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Abstract
The present invention relates to a polypeptide-liposome and human vascular endothelial growth factor gene recombination plasmid compound and its use. Said compound is formed from human vascular endothelial growth factor gene recombination plasmid, polypeptide wrapping the exterior of said plasmid and liposome through compounding process, in which human vascular endothelial growth factor gene recombination plasmid is pVAX-VEGF 165, said polypeptide is a fatty substance which can wrap gene recombination plasmid or CNA, the polypeptide can be combined with human vascular endothelial growth factor gene recombination plasmid and liposome in non-covalent bond form. Said compound can be used for introducing human vascular endothelial growthf actor gene into starget cell to make blood vessel implement specific growth.
Description
Technical field
The present invention relates to biological technical field, relate to a kind of gene transfer system of non-virus carrier mediation specifically, more particularly relate to a kind of polypeptide-liposome and human vascular endothelial growth factor gene recombination plasmid complex and purposes.
Background technology
Cardiovascular disease is the first disease that threatens human health, and the number that cardiovascular and cerebrovascular disease is died from the whole world every year surpasses 1,500 ten thousand, accounts for 38% of total dead population.In China, the mortality rate of this disease also ranks first, and accounts for 34%, and morbidity has the trend of rejuvenation more.Wherein, the sickness rate of China CAD accounts for 0.649%, and the whole nation has more than 800 ten thousand CAD patients at least, and the trend that increases is year by year arranged.CAD has comprised the multiple heart disease that causes because of regional myocardial ischemia, and these cardiopathic causes have arteriosclerosis, blood to fasten and vasospasm.Present Therapeutic Method mainly comprises Drug therapy and operative treatment.The principle of Drug therapy is for reducing myocardium keto consumption and promoting local perfusion; Operative treatment mainly contains cardiovascular reshaping art (percutaneous transluminal coronary angioplasty), arteries and veins bypass aroused in interest (coronary artery bypass grafting) and myocardium laser reproduce blood vessel art (transmyocaradial laser revascularization), domestic commonly used have coronary bypass and scaffold tube technology.All these operations once can only solve the local stenosis at one to two place, and not only medical expense is very expensive, and the operation artery can occur narrow once more or stop up, and are serious postoperative problems.In addition, many patients are still arranged because a variety of causes and can not accept this invasive surgery treatment of having, and lose the chance of surviving.
Vascular endothelial cell growth factor (vascular endothelial growth factor, VEGF) be at present known to the clearest and the most definite main factor that can promote angiogenesis specifically.Its gene mapping is in No. 6 the short arm of a chromosome, and there are 6 members in this family, is respectively VEGF-A, B, C, D, E and placental growth factor.Wherein, because the different splicing forms of mRNA, VEGF-A can produce different VEGF hypotypes again, and main has: VEGF
165, VEGF
121, VEGF
145, VEGF
189, and VEGF
206In clinical trial, the angiogenesis function of VEGF is applied to treat the narrow ischemic disease that causes of peripheral arterial (Isner, et al.) at first.Early stage experimental result confirms that VEGF promoting aspect the angiogenesis positive effect is arranged, comprises that patient's pain relief, ischemic conditions alleviate, ulcer healing, and consistent patient's limbs are kept.Further investigation confirms that these clinical effects are that VEGF promotes the result that neovascularity generates.
Based on the result of this early stage clinical experiment, people have begun VEGF and have been used for the research (Losordo, et al.) that CAD treats.At present, the clinical gene therapy research of existing 46 FDA of the U.S. and the relevant promotion angiogenesis of RAC approval, research is used for the treatment of various forms of ischemic conditions, comprises narrow local disease that causes of peripheral arterial and CAD.The means that most clinical researches have adopted VEGF and FGF gene to import, wherein two clinical researches that import treatment CAD with the VEGF gene have proceeded to the clinical II phase and have studied (Berlex Lab and GenVec Inc.).Clinical research report has so far confirmed that all the VEGF gene imports the effectiveness of treatment, can obviously improve ischemic conditions.For example, (VEGF encodes with liposome DNA to 30 patients CAD
165) import and to carry out clinical treatment research, the result has 29 patients' angina pectoris to have significantly to alleviate; The treatment that 20 patients carried out six months is by a definite date observed, and the result has 12 patients' angina pectoris all to eliminate.10 patients observed the treatment phase at 12 months in addition, had 7 patients' angina pectoris all to eliminate (Symes, et al.).Other application VEGF
121Also reported similar clinical efficacy (Rosengart, et al., Grimes and Vale, et al.) with the research of FGF4 gene therapy CAD.In all treatment clinical courses, do not find serious adverse.Therefore it is safe and effective importing treatment CAD with the VEGF gene.Because these clinical effectivenesses show good prospect, have attracted more company and university to participate in further clinical research.
Different with the gene therapy of cancer, the gene therapy major part of short angiogenesis has adopted non-viral gene introduction method (39% adopts naked DNA, and 8% adopts liposome).One of its reason is that the expressed angiogenesis growth factor of gene (as VEGF and FGF gene) that is used for angiogenesis all is the protein of secreted, just be based on its secreted, the somatomedin of q.s can be expressed as long as can guarantee the gene that imports, under low relatively gene importing rate, also effective therapeutic outcome can be reached.The another one reason is the relative safety of non-viral gene introduction method, use the non-viral gene introduction method also to avoid owing to the existence or the generation of human body to antiviral antibody, and the gene that causes imports the unfavorable factor that efficient reduces, thereby can adopt multiple dosing to reach clinical therapeutic efficacy.
Number of research projects confirms in the past few years, compares with naked DNA, and the liposome gene introduction method can improve local loop because of importing rate (Lasic, et al.).Along with the exploitation of different liposome with to the formation Study on Mechanism of liposome DNA complex, its gene importing rate has obtained further raising (Templeton, et al.).This is comprising the liposome that is directed to specific cells being arranged, the exploitation of concealed liposome and polypeptide-liposome.However, still there is a big difference to shift this target apart from the differential high efficient mediated gene.
The restriction liposome is that gene imports the low and poor specificity of efficient in a main cause of field of gene broader applications at present.The exploitation of polypeptide utilizes the guiding function of polypeptide, then might increase substantially specificity and efficient that gene imports.
Summary of the invention
The present invention is intended to effectively improve specificity and the efficient that gene imports, and provide a kind of polypeptide-liposome and human vascular endothelial growth factor gene recombination plasmid complex, be used for the Mediated Human vascular endothelial growth factor gene and enter target cell, make the blood vessel-specific growth.The present invention also further provides the medical usage of this complex as gene therapy.
For achieving the above object, the invention provides a kind of polypeptide-liposome and human vascular endothelial growth factor gene recombination plasmid complex, this complex is by human vascular endothelial growth factor gene recombination plasmid and is wrapped in its outside polypeptide and liposome is composited.
Described human vascular endothelial growth factor gene recombination plasmid is pVAX-VEGF
165
Described polypeptide is the peptide section that is rich in hydrophobic amino acid of the synthetic of effective induced gene recombiant plasmid or DNA leap mammalian cell membrane and nuclear membrane structure, and its amino acid residue sequence and primary structure are: GNQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY
PKKKRKV. wherein, the GNQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY sequence is from heterogeneousnuclear ribonucleoprotein (hnRNP) A1 protein N LS sequence;
PKKKRKVSequence is from SV40 virus T antibody NLS sequence.
After being rich in the peptide sequence of hydrophobic amino acid, add other positively charged small peptide, can form complex polypeptide.
The character of polypeptide: not having cysteine in the aminoacid sequence, is the simple wire-form polypeptide.Contain the hydrophobic aminoacid of many neutrality (G, F, and Y) in the polypeptide.From SV40 virus T antibody NLS sequence, positively charged.
Described liposome is to wrap up gene recombination plasmid or DNA, and helps the fat material of the synthetic of recombiant plasmid or DNA leap mammalian cell membrane structure.
Polypeptide combines with human vascular endothelial growth factor gene recombination plasmid and liposome with the non-covalent bond form, and aminoacid of positively charged (K) and electronegative plasmid DNA interact in the sequence, thereby plasmid DNA is further condensed.This cohesion helps plasmid DNA and transmitted cell membrane, Cell sap and nuclear membrane.(nuclear pore complex, NPC) the importin beta protein on and polypeptide effect are transferred in the nucleus and then realize transcribing and translating of DNA in the plasmid DNA of following of the help of GTPase cohesion to be present in cell nucleopore complex.Hydrophobic amino acid in the polypeptide combines with fat on the liposome with hydrophobic bond.Like this, polypeptide plays simultaneously the function with plasmid DNA and liposome dual function.Form the very plasmid DNA of sealing therefrom, polypeptide and liposome complex, and then reach the highest gene conductivity.
The present invention also provides the medical usage of this complex, and this complex can be used for preparing the gene therapy medicament of IPVD and ischemic coronary heart disease.
Contribution of the present invention is, it efficiently solves, and gene importing efficient is hanged down and the problem of poor specificity, the present invention is by the polypeptide of some biogenetic derivation of screening, utilize the affinity and the nuclear localization signal effect of itself and people's certain detail after birth, mediated gene shifts, improve the specific while, improve transfection efficiency.These polypeptide combine with liposome and DNA, make polypeptide be presented on the liposome vesicle surface that is surrounded by target DNA of formation.By the polypeptide mediation, vesicle combines with purpose cell high-affinity, enter in the born of the same parents, and under the cooperation of polypeptide, DNA is released into endochylema, and under the position effect of appraising and deciding of polypeptide guided, DNA entered in the nuclear, reaches the purpose that improves gene transfer efficient subsequently.On this basis, the present invention is to the VEGF of polypeptide-liposome mediation
165The angiogenesis research that the gene inducing defecation by enema and suppository is used for ischemic tissue has also obtained positive effect.
Description of drawings
Fig. 1 is polypeptide-liposome of the present invention and human vascular endothelial growth factor gene recombination plasmid composite structure sketch map.
Fig. 2 is the fluorescence microscopy figure that simple lipid body mediation plasmid DNA pEGFP-N3 expresses at bovine aortic endothelial cells.
Fig. 3 is the fluorescence microscopy figure that polypeptide-liposome mediation plasmid DNA pEGFP-N3 expresses at bovine aortic endothelial cells.
Fig. 4 is an angiogenic growth test angiogram in the body of the present invention.
The specific embodiment
The following example is to further explanation of the present invention and explanation, and the present invention is not constituted any limitation.
Embodiment 1
The structure of this polypeptide-liposome and human vascular endothelial growth factor gene composite is seen Fig. 1, and 1 is human vascular endothelial growth factor gene recombination plasmid pVAX-VEGF among the figure
165, 2 is liposome, and 3 is polypeptide, and the average diameter of complex is 100nm.Complex with this structure is prepared by following technology path:
1, VEGF
165The acquisition of cDNA: the SMMC-7721 cell that the conventional method cracking is cultivated, extract total
RNA, method is extracted the test kit description with reference to RNA.With poly T as primer, will be wherein
The mRNA reverse transcription become cDNA.
2, EGF
165The acquisition of encoding gene: in VEGF
165The upstream and downstream of gene respectively designs a PCR primer, introduces restriction enzyme site (with reference to the multiple clone site of plasmid pVAX1).After the PCR reaction, target DNA is cloned among the specific sequencing vector pUC19, makees sequence analysis.Keep correct clone.
3, build VEGF
165Recombiant plasmid:, be cloned into (this plasmid will provide the poly A in T7 promoter and downstream) among the eukaryon expression plasmid pVAX1 with the correct sequence in the cloning vehicle.Positive colony is PCR and enzyme action is identified, obtains recombinant expression plasmid pVAX-VEGF
165.
4, VEGF
165Expression identify: with the pVAX-VEGF that builds
165The not VEGF expression of cultivating with the method transfection of liposome
165Cell line, screening after, detect VEGF with western-blot
165Expression.Keep pVAX-VEGF with high expressed ability
165Expression plasmid.
5, seed engineering bacterium and work engineering bacteria are set up: with recombiant plasmid pVAX-VEGF
165Transfection Escherichia coli DH5 α by fermentation, sets up engineering bacteria.Recombiant plasmid pVAX-VEGF is determined in test by going down to posterity
165Use plasmid content and determine the copy number of this plasmid.Plasmid purification adopts ultraviolet spectrophotometer to measure the purity of plasmid DNA, and the dna gel electrophoresis method is measured the ratio of super coiled DNA.
6, the high-affinity polypeptide obtains: the extracellular region of using the special memebrane protein of myocardial cell is as immobile phase, utilize phage surface to present the technology screening peptide library, after the enrichment of many wheels, obtain and myocardial cell high-affinity polypeptide, do gene sequencing and derivation amino acid sequence analysis.
7, yeast two-hybrid screening: the extracellular region of using the special memebrane protein of myocardial cell is done the yeast two-hybrid system screening as bait, and the first run is screened male sequence after verifying once more, analyzes its DNA sequence and amino acid sequence corresponding.
8, affinity is measured: the extracellular region of the special memebrane protein of vivoexpression myocardial cell, the polypeptide that the synthetic screening obtains carries out the two binding affinity with biosensor and measures, and selects the polypeptide of high-affinity to be used for the preparation of polypeptide-liposome complex.
9, the preparation of polypeptide-liposome complex: the aminoacid sequence according to screening obtains, add the nuclear localization signal sequence, adopt artificial synthesis to produce polypeptide, with plasmid pVAX-VEGF
165Mix,, form polypeptide-plasmid composite by the non-covalent bond combination.
10, VEGF
165The packing of gene preparation: the polypeptide-plasmid composite of the preparation of above-mentioned structure is mixed mixing naturally under the room temperature with liposome 2000.Make it to form this project product SBN-2 gene bridging plain (Gene Bypassin).See the finished product structural representation.
11, quality control: the finished product controlling index is mixed particle diameter and zeta potential, can use laser particle size instrument and zeta potential to measure respectively.
12, the transduction efficiency of gene transfer complex test: buy and contain reporter gene β-gal plasmid, with as the polypeptide-liposome complex of 9 preparations as the transduction medium, the transduction cultured vascular endothelial is done contrast with single with liposome, relatively transduction efficiency.
Embodiment 2
External transduction experiment of the present invention is as follows:
1, cell culture and transduction method: (WA) growth is paved with bovine aortic endothelial cells (BAEC), is passaged to 24 well culture plate continued growths 2 days for Cell SystemsCorporation, Kirkland, and reaching cell density is＞100,000/cm
2, then with the experiment of transduceing of liposome or polypeptide liposome.Cell growth medium is DMEM, contains 10% heat-inactivated hyclone.
2, transduction method: press liposome (lipofectamine-2000) and plasmid DNA pEGFP-N3 (Clontech Laboratories, Palo Alto, CA) weight ratio is mixing in 6: 1, promptly 3 μ l liposomees (2mg/ml) or 3 μ l liposome peptide complexes mix with 1 μ g plasmid DNA, add 96 μ l serum-free culture mediums (OPTIMEM Reduced Serum Medium, Gibco-BRL), 37 ℃ of incubation 45min.PEGFP-N3 contains CMV promoter, can express EGFP.Synthetic peptide (P9) 30 μ g are added in the 1 μ gpEGFP-N3 liposome mixture, 37 ℃ of incubation 15min.Then, add 150 μ l OPTIMEM serum-free culture mediums, soak cell (250 μ l per2cm fully
2Well), 5%CO
2, 37 ℃ of incubation 2hrs, add 10% serum, continued to hatch 2 days.At last, with the fluorescence microscope observed result of taking a picture.
3, result: simple lipid body (lipofectamine-2000) mediation plasmid DNA pEGFP-N3 enters BAEC, and the rare (see figure 2) of fluorescence speckle (expression of GFP) in the finding cell proves that transduction efficiency is relatively poor under the fluorescence microscope.Fig. 3 shows that the liposome-mediated plasmid DNA pEGFP-N3 of polypeptide enters BAEC, and the interior fluorescent spot point of finding cell obviously increases enhancing under the fluorescence microscope, and transduction efficiency improves about 10 times.
Embodiment 3
The angiogenic growth test is as follows in the body of the present invention:
By surgical operation, new zealand rabbit lower limb femoral artery is excised from the ilium outside to nest, cause severe lower extremity ischemia.Postoperative recovered animal 10 days, and collateral circulation is tentatively set up.After checking that the every physical signs of animal is normal, begin to do gene therapy.
Get polypeptide liposome-pVEG
165Composite article 500 μ g/2.5ml divide 5 somes injections, every 100 μ g/0.5ml to three muscular tissue two ends of ischemia.Note not injecting too deeply or injection being spilt.Before and after injection, with angiography with at microscopically, observe the blood vessel and the blood capillary regeneration situation of ishemic part, comprise the regenerated quantity of blood vessel and blood capillary, density, area, length and tissue blood perfusion state etc., to judge therapeutic effect.The result as shown in Figure 4, wherein Fig. 4 A for the contrast 1, postoperative first day; Fig. 4 B is contrast 2, and postoperative is after 30 days.As seen postoperative is after 30 days, and the blood vessel of ischemic tissue has generation reward property and increases slightly, but does not have obvious autonomy revascularization; Fig. 4 C demonstration pVEGF
165Inject cleft lip blood limbs after 30 days, the newborn situation of blood vessel.Blood vessel is obviously regenerated as seen from the figure.
Claims (7)
1, a kind of polypeptide-liposome and human vascular endothelial growth factor gene recombination plasmid complex is characterized in that, it is by human vascular endothelial growth factor gene recombination plasmid and is wrapped in its outside polypeptide and liposome is composited.
2, complex as claimed in claim 1 is characterized in that, described human vascular endothelial growth factor gene recombination plasmid is pVAX-VEGF
165
3, complex as claimed in claim 1; it is characterized in that; polypeptide wherein is the peptide section that is rich in hydrophobic amino acid of the synthetic of effective induced gene recombiant plasmid or DNA leap mammalian cell membrane and nuclear membrane structure, and its amino acid residue sequence and primary structure are: GNQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY
PKKKRKV. wherein, the GNQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY sequence is from heterogeneousnuclear ribonucleoprotein (hnRNP) A1 protein N LS sequence;
PKKKRKVSequence is from SV40 virus T antibody NLS sequence.
4, complex as claimed in claim 3 is characterized in that, adds other positively charged small peptide after being rich in the peptide sequence of hydrophobic amino acid, can form complex polypeptide.
5, complex as claimed in claim 1 is characterized in that, described liposome is to wrap up gene recombination plasmid or DNA, and helps the fat material of the synthetic of recombiant plasmid or DNA leap mammalian cell membrane structure.
6, complex as claimed in claim 1 is characterized in that, polypeptide combines with human vascular endothelial growth factor gene recombination plasmid and liposome with the non-covalent bond form.
As arbitrary described complex in the claim 1 to 6, it is characterized in that 7, this complex can be used for preparing the gene therapy medicament of IPVD and ischemic coronary heart disease.
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| CNB021343217A CN100431611C (en) | 2002-07-04 | 2002-07-04 | Compound of polypeptide-liposome and human vascular endothelial growth factor gene recombination plasmid and its use |
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| CNB021343217A CN100431611C (en) | 2002-07-04 | 2002-07-04 | Compound of polypeptide-liposome and human vascular endothelial growth factor gene recombination plasmid and its use |
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| CN100431611C CN100431611C (en) | 2008-11-12 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103393597A (en) * | 2013-07-02 | 2013-11-20 | 北京大学 | Hydrophobic peptide-modified long-circulation liposome drug delivery system for injection |
| US9616103B2 (en) | 2012-08-31 | 2017-04-11 | “Nextgen” Company Limited | Pharmaceutical composition for stimulation of angiogenesis |
| US9896493B2 (en) | 2015-05-26 | 2018-02-20 | “Nextgen” Company Limited | Nucleotide sequence and pharmaceutical composition based thereon with prolonged VEGF transgene expression |
-
2002
- 2002-07-04 CN CNB021343217A patent/CN100431611C/en not_active Expired - Lifetime
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9616103B2 (en) | 2012-08-31 | 2017-04-11 | “Nextgen” Company Limited | Pharmaceutical composition for stimulation of angiogenesis |
| CN103393597A (en) * | 2013-07-02 | 2013-11-20 | 北京大学 | Hydrophobic peptide-modified long-circulation liposome drug delivery system for injection |
| CN103393597B (en) * | 2013-07-02 | 2015-06-17 | 北京大学 | Hydrophobic peptide-modified long-circulation liposome drug delivery system for injection |
| US9896493B2 (en) | 2015-05-26 | 2018-02-20 | “Nextgen” Company Limited | Nucleotide sequence and pharmaceutical composition based thereon with prolonged VEGF transgene expression |
| US10040837B2 (en) | 2015-05-26 | 2018-08-07 | “Nextgen” Company Limited | Nucleotide sequence and pharmaceutical composition based thereon with prolonged VEGF transgene expression |
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| Publication number | Publication date |
|---|---|
| CN100431611C (en) | 2008-11-12 |
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