CN104288776A - Self-assembled polypeptide-apoptin gene composite nanoparticle, and preparation method and application thereof - Google Patents
Self-assembled polypeptide-apoptin gene composite nanoparticle, and preparation method and application thereof Download PDFInfo
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- CN104288776A CN104288776A CN201410303298.3A CN201410303298A CN104288776A CN 104288776 A CN104288776 A CN 104288776A CN 201410303298 A CN201410303298 A CN 201410303298A CN 104288776 A CN104288776 A CN 104288776A
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Abstract
The invention discloses a self-assembled peptide-apoptin gene composite nanoparticle, and a preparation method and an application thereof. A self-assembled polypeptide is used as a carrier, charges carried by acidic amino acids and alkaline amino acids of the carrier and plasmid molecules carrying the apoptin gene form ionic bonds, and the plasmid molecules are firmly adhered to the surface of the self-assembled polypeptide to form the self-assembled polypeptide-apoptin gene composite nanoparticle. The composite nanoparticle is fibrous, the length is 100-200nm, and the diameter is 10-20nm. The surface of the nanoparticle is rich in arginine, so the nanoparticle can be identified by and combined with an arginine receptor on the surface of a cell membrane, and can be taken by the cell, the gene carried by the nanoparticle can be expressed in the cell, and an expressed apoptosis protein can specifically induce tumor cell apoptosis and is nontoxic to normal cells. The composite nanoparticle is a novel antitumor gene treatment bio-preparation and has a broad application prospect.
Description
Technical field
The invention belongs to gene therapy technology field, be specifically related to a kind of can specificity inducing apoptosis of tumour cell and to self assembly polypeptide-apoptin gene composite nanometer particle of not normal cells and its preparation method and application.
Background technology
Apoptosis element (Apoptin) is also known as VP3, a kind of small molecular protein being derived from chicken anaemia virus (chicken anemia virus, CAV), molecular weight 13.6 kDa, be made up of 121 aminoacid, proline rich, basic amino acid and a high proportion of serine/threonine residue.Apoptosis element does not have sequence homology with other any animals or virus protein.Apoptosis element can specificity inducing tumor cell and have transformation phenotype apoptosis and to not normal cells effect.So far, existing research report shows, apoptosis element the human tumor cells type of specific induction more than 15 kinds different tissue sources more than totally 70 can plant tumor cell line apoptosis, and apoptosis element has the tumor cell induction apoptosis effect of wide spectrum as can be seen here.It is a kind of antineoplastic biologic preparation having very much application prospect.
Had with adenovirus in the exploration of apoptosis element application is that the plasmid of carrier is delivered, apoptosis quality grain and sialoglycoprotein links targeting by poly-D-lysine imported liver neoplasm, take deactivation fowlpox virus as apoptosis element plasmid vector, merges with permeable membrane peptide TAT or PTD4 and apoptosis fibroin in the past, or the research suddenling change to the aminoacid sequence of apoptosis element on this basis, transform expansion applied research, also have the plain delivery carrier using phage nano biological granule (Phage Nanobioparticle) as apoptosis.These researchs are that useful exploration has been made in the clinical practice of apoptosis element, but face an a series of difficult problem namely: 1. deliver apoptosis quality grain or apoptin gene with the bio-carrier of transformation, it is low that preparation process is loaded down with trivial details, it delivers efficiency, and inducing apoptosis of tumour cell effect is undesirable; 2. using improved virus as genophore, its safety needs to be assessed further; 3. merge apoptosis cellulose protein with permeable membrane peptide to deliver, apoptosis cellulose protein is prepared that difficulty is large, cost is high, is difficult to obtain great amount of soluble protein, because apoptosis fibroin is derived from its easy aggregate and precipitate in the solution of virus, and apoptosis cellulose protein enters in body and host also can be made to produce corresponding antibodies, greatly reduces the effect of its inducing apoptosis of tumour cell.
At present, the Biotherapeutics developing into tumor of nanotechnology provides brand-new technology platform.Nanotechnology refers to studies atom, the structure of molecule and interaction thereof and the technology be applied in 1 ~ 100 nm range scale.In recent years, nanometer medicine-feeding technology has more and more been applied in antitumor research.Along with going deep into of research, now have recognized that, applying nano technology can improve the physicochemical property of pharmaceutical dosage form, and improve the targeting of medicine, increase the inner advantage such as blood drug level, slow-releasing of solid tumor, be one of study hotspot of current cancer Diagnosis and Treat, mainly concentrate on development and carry antitumor drug or the nanoparticle vector as tumor imaging reagent.
Along with the develop rapidly of nanometer biotechnology, a kind of novel non-viral carrier systems: the gene therapy appearing as tumor of nano-gene carrier system is filled with new vitality.It has following advantage compared with viral vector: reduced immunogenicity; High power capacity; Good protective effect can be had to the material composition inserted wherein.Also scholar is had to utilize nanotechnology to deliver Small Interfering RNA(Si RNA in vivo) explore.
Recently, a kind of novel original position hydrosol nanometer is delivered material and is developed, this system is based on self assembly polypeptide (self-assembling peptide), it spontaneous assembling can be cross-linked into diameter 10 ~ 20 nm nanofiber hydrogels in physiological conditions, stable beta sheet structure is formed in containing the aqueous solution of ion, non-immunogenicity, without the need to through ultraviolet or chemical modification in assembling cross-linking process, arginine residues is rich in its sequence, be easy to be transported in cell with surface of cell membrane arginine receptors bind, can be used for protein, microRNA, the packaging of hydrophobicity chemotherapeutic drug Paclitaxel is delivered.But packaging and the correlational study of the recombinant plasmid dna of macromolecule and self assembly polypeptide there is no report, the method in the past delivering recombinant plasmid dna conventional in cell has: liposome mediated transfection, low temperature calcium phosphate transfection, deae dextran mediated transfection, electroporation, microinjection etc., said method respectively has its own pluses and minuses.But all deposit a general character: have damage to cell.
For this reason, a kind of self assembly polypeptide-apoptin gene composite nanometer particle and its preparation method and application is developed.
Summary of the invention
The present invention first object is to provide a kind of self assembly polypeptide-apoptin gene composite nanometer particle; Second object is the preparation method providing a kind of self assembly polypeptide-apoptin gene composite nanometer particle; 3rd object is to provide a kind of self assembly polypeptide-application of apoptin gene composite nanometer particle in antineoplaston.
The present invention first object realizes like this, described self assembly polypeptide-apoptin gene composite nanometer particle, using self assembly polypeptide as carrier, by self acidic amino acid and basic amino acid with electric charge with form ionic bond between the plasmid molecule carrying apoptin gene and be combined, plasmid molecule secure adhesion, in self assembly polypeptide surface, forms composite nanometer particle.
The present invention second object realizes like this, and the preparation method of described self assembly polypeptide-apoptin gene composite nanometer particle, comprises the steps:
(1) self assembly polypeptide is synthesized;
(2) plasmid containing apoptin gene is built;
(3) self assembly polypeptide solution is prepared;
(4) in polypeptide solution, add the plasmid solution that step (2) builds, fully obtain nano-particle suspension after effect;
(5) nano-particle detects.
The present invention the 3rd object realizes like this, the described self assembly polypeptide-application of apoptin gene composite nanometer particle in antineoplaston.
Compared with prior art, beneficial effect of the present invention is:
(1) the present invention is first with the amount matter ratio between suitable self assembly polypeptide and macromole recombiant plasmid DAN, preparation self assembly polypeptide packaging apoptosis element coding recombiant plasmid (pcDNA3-
hA-
apoptin), form a kind of fibrous composite nanometer particle, its chemical nature is the compound bio macromole that self assembly polypeptide and plasmid DNA are formed, length degree about 100 ~ 200nm, and diameter about 10 ~ 20nm is not etc.;
(2) composite nanometer particle of the present invention can by cellular uptake, not damaging cells, can be absorbed by cell high-efficient rate, uptake ratio is greater than 95%, at cell inner expression gene entrained by it, expressed apoptosis fibroin energy specificity inducing apoptosis of tumour cell and to not normal cells.Due to apoptosis element not injuring normal cell, can overcome the shortcoming of therapeutic gene targeting deficiency in therapy of tumor in the past, this composite nanometer particle is a kind of novel Antioncogene treatment biological preparation.
Accompanying drawing explanation
Fig. 1 is pcDNA3-
hA-
apoptinplasmid molecule structure chart;
Fig. 2 is self assembly polypeptide solution and self assembly polypeptide-pcDNA3-
hA-
apoptincomposite nanometer particle suspension;
Fig. 3 is unpackaged pcDNA3-
hA-
apoptiwith self assembly polypeptide-pcDNA3-
hA-
apoptincomposite nanometer particle agarose gel electrophoresis;
In figure, 1-DNA marker; The unpackaged pcDNA3-of 2-
hA-
apoptin; 3-self assembly polypeptide-pcDNA3-
hA-
apoptincomposite nanometer particle;
Fig. 4 is self assembly polypeptide-pcDNA3-
hA-
apoptincomposite nanometer particle transmission electron microscope picture;
Fig. 5 is that immunofluorescence technique detection apoptin gene expresses (amplification: 10 × 20) in kidney clear cell carcinoma 7860;
In figure, the process of A-self assembly polypeptide, cell is expressed without apoptosis fibroin; B-fluorescent dye DAPI showed cell core; The unpacked pcDNA3-of C-
hA-
apoptinplasmid process, cell is expressed without apoptosis fibroin; D-fluorescent dye DAPI showed cell core; E-self assembly polypeptide-pcDNA3-
hA-
apoptinthe pcDNA3-that composite nanometer particle carries
hA-
apoptinplasmid can at kidney clear cell carcinoma 7860 cell inner expression; F-fluorescent dye DAPI showed cell core;
Fig. 6 is that immunofluorescence technique detects apoptin gene expression and localization (amplification: 10 × 50) in kidney clear cell carcinoma 7860 cell strain and renal tubules near-end epithelial cell HK2 cell strain (normal cell);
In figure, A-self assembly polypeptide-pcDNA3-
hA-
apoptinthe pcDNA3-that composite nanometer particle carries
hA-
apoptinplasmid is expressed in normal, fluorescence display apoptosis fibroin; B-fluorescent dye DAPI showed cell core; The overlapping split display normal cell profile of C-A, B and nucleus, the apoptosis fibroin given expression in normal cell is mainly distributed in Cytoplasm; D-self assembly polypeptide-pcDNA3-
hA-
apoptinthe pcDNA3-that composite nanometer particle carries
hA-
apoptinplasmid at expression in tumor cells, fluorescence display apoptosis fibroin; E-fluorescent dye DAPI showed cell core; The overlapping split display normal cell profile of F-D, E and nucleus, the apoptosis fibroin gone out at expression in tumor cells is mainly distributed in nucleus;
Fig. 7 is that light microscopic detects kidney clear cell carcinoma 7860 cell strain and renal tubules near-end epithelial cell HK2 cell strain (normal cell) proliferative conditions (amplification: 10 × 20) after nano-particle process;
Fig. 8 is that light microscopic detects kidney clear cell carcinoma 7860 cell (amplification: 10 × 40).
Detailed description of the invention
Below in conjunction with accompanying drawing, the invention will be further described, but limited the present invention never in any form, based on any conversion that training centre of the present invention is done, all falls into scope.
Self assembly polypeptide of the present invention-apoptosis element (Apoptin) gene composite nanometer particle is using self assembly polypeptide as carrier, by self acidic amino acid and basic amino acid with electric charge with form ionic bond between the plasmid molecule carrying apoptin gene and be combined, plasmid molecule secure adhesion, in self assembly polypeptide surface, forms composite nanometer particle.This composite nanometer particle is in threadiness, and length is 100 ~ 200nm, and diameter is 10 ~ 20nm.
The sequence of self assembly polypeptide of the present invention is as shown in SEQ ID No.1.The described plasmid containing apoptin gene is pcDNA3-
hA-
apoptinplasmid, its structure as shown in Figure 1.
The preparation method of self assembly polypeptide-apoptin gene composite nanometer particle of the present invention, comprises the steps:
(1) according to sequence SEQ No.1, synthesis self assembly polypeptide;
(2) pcDNA3-is built
hA-
apoptinplasmid: take pcDNA3 as carrier, design, customization synthesis
hA-
apoptinprimer, with pcDNA3-
apoptinfor template, amplification
hA-
apoptingenes of interest fragment, builds pcDNA3-
hA-
apoptinplasmid;
hA-
apoptinprimer sequence is as follows:
hA-
apoptin(underscore is forward primer
bamhI restriction enzyme site):
bamhI and
xhoi double digestion plasmid vector pcDNA3 and genes of interest
hA-
apoptin, in carrier pcDNA3 multiple clone site
bamhI and
xhoi place accesses
hA-
apoptingenetic fragment; T4 DNA ligase 16 DEG C, 12h connection carrier and genes of interest fragment, obtain restructuring pcDNA3-
hA-
apoptineukaryon expression plasmid;
(3) the self assembly polypeptide solution of 5.26 mg/mL is prepared, the detailed process of preparation is: operation in superclean bench, white powder polypeptide is dissolved in aseptic PBS buffer solution, then ice-water bath is placed in, and using ultrasound wave hydrotropy, ultrasonic power is 4 W/mL, every task 3 s, stop 5 s, hydrotropy total duration is 20 min;
(4) in self assembly polypeptide solution, add the pcDNA3-of 2 μ g/ μ L
hA-apoptinplasmid solution, the mass ratio of plasmid and polypeptide is 1:45 ~ 1:55.Cover tightly lid, put upside down mixing, mixing bonnet mouth and lid with sealing compound sealing, put shaking table 150 r/min, 12 h again;
One as technical solution of the present invention is preferred, and the mass ratio of plasmid and polypeptide is 1:50;
(5) use 0.8% agarose gel electrophoresis and transmission electron microscope to detect composite nanometer particle whether to be successfully prepared.
Self assembly polypeptide-apoptin gene composite nanometer particle of the present invention being applied as in antineoplaston manufactures can the genomic medicine of specificity inducing apoptosis of tumour cell and not injuring normal cell.
Self assembly polypeptide-apoptin gene composite nanometer particle of the present invention, manufacture for the application in antineoplaston, this nano grain surface is rich in arginine, can be combined and by cellular uptake with surface of cell membrane arginine Receptor recognition, in cell packaged by it, the pcDNA3-that carries
hA-apoptineukaryotic expression recombinant plasmid can give expression to apoptosis element (Apoptin) albumen smoothly, expressed apoptosis element can specificity inducing apoptosis of tumour cell and not injuring normal cell, and this composite nanometer particle is a kind of novel, specific Antioncogene medicine researched and developed based on nano-carrier medicine-feeding technology and therapy of tumor.
Embodiment 1: build restructuring pcDNA3-
hA-
apoptineukaryon expression plasmid
Step 1:PCR increases
hA-
apoptingene
Reaction system is:
| 10×Taq Buffer | 5 μL |
| 2 mM dNTP | 5 μL |
| 1 μM of forward primer | 1 μL |
| 1 μM of downstream primer | 1 μL |
| Template | 2 μL |
| 25 mM Mg 2+ | 2.5 μL |
| Taq DNA Polymerase | 1.3 μL |
| H 2o | 32.2 μL |
| Amount to: | 50 μL |
Wherein,
hA-
apoptinprimer sequence is as follows:
hA-
apoptin(underscore is forward primer
bamhI restriction enzyme site):
PCR response procedures is: 95 DEG C of preheating 300s, 95 DEG C of 30s → 55 DEG C 30s → 72 DEG C 40s, 35 circulations, 72 DEG C of 300s, 4 DEG C of 1h.
Step 2: utilize Omega Biotek test kit to reclaim
hA-
apoptinpCR primer;
Step 3:
hA-apoptingenetic fragment insertion vector pcDNA3
bamhI and
xhoi double digestion carrier pcDNA3 and target DNA
hA-apoptin, 37 DEG C of constant temperature spend the night.Enzyme action system is as shown in the table:
| pcDNA3 | HA-apoptin |
| Xho I 1μL | Xho I 1μL |
| Bam HI 1μL | Bam HI 1μL |
| 10×Buffer 2μL | 10×Buffer 2μL |
| pcDNA3 2μg | HA-apoptin 2μg |
| Supplement d 3H 2The total system of O to 20 μ L | Supplement d 3H 2The total system of O to 20 μ L |
1% agarose gel electrophoresis quantitatively cuts the enzyme action pcDNA3 open circular plasmid that glue reclaims; The enzyme action that glue reclaims quantitatively is cut in 2% agarose gel swimming
hA-apoptin, operate same step 2.
Connect pcDNA3 open circular plasmid after double digestion and
hA-apoptindNA fragmentation coupled reaction system: the 10 × Buffer of 2 μ L, the T4 Ligase of 0.5 μ L, 100ng vector plasmid; 80 ng
hA-apoptintarget DNA fragment, supplements d
3h
2the total system of o to 20 μ L.Put 16 DEG C, 12h connection carrier and genes of interest fragment.
Step 4: screening restructuring pcDNA3-
hA-
apoptineukaryon expression plasmid
Low temperature chlorination calcium legal system is for competent escherichia coli cell, recombinant plasmid transformed is entered escherichia coli, concrete steps are: (1) every part of competence bacteria 200 μ L, often pipe adds and connects product (with the volume being no more than 10 μ L, wherein DNA content is less than 50ng), rotate gently to mix content, place 30min on ice; (2) pipe is put into the circulator bath being preheated to 42 DEG C, accurately place 90s, do not shake pipe; (3) by pipe take out put immediately make on ice antibacterial cool 1 ~ 2min; (4) often pipe adds 800 μ L SOC culture medium (or LB), heats to 37 DEG C, then pipe is transferred to more than incubation 45min on shaking table in water bath incubator; (5) low-speed centrifugal (about 500g) sedimentation antibacterial, retains about 200 μ L SOC(or LB), after soft mixing, antibacterial bed board is extremely contained in 1.5% agarose plate culture medium of ampicillin (1 μ g/mL); (6) put by flat board until liquid is blotted (7) be inverted culture dish in super-clean bench, 37 DEG C of cultivations, can grow bacterium colony after 12 ~ 16h; (8) after bacterium colony grows, the single colony inoculation of picking is in containing amplification culture in antibiotic fresh culture.
Step 5: restructuring pcDNA3-
hA-apoptinqualification
Omega Biotek test kit is utilized to extract plasmid, recycling pcDNA3 order-checking universal primer order-checking qualification.Sequencing result conforms to expection DNA sequence, does not occur genes of interest
hA-apoptinpoint mutation, base are inserted, base deletion phenomenon.
Finally, the pcDNA3-obtained
hA-
apoptinstructure as shown in Figure 1.
Embodiment 2: preparation self assembly polypeptide-apoptin gene composite nanometer particle
Synthesize self assembly polypeptide according to sequence SEQ No.1, the self assembly polypeptide of synthesis is white powder, is mixed with the self assembly polypeptide solution of 5.26 mg/mL.Process for preparation for getting the aseptic PBS(pH7.4 of 0.95 mL in super-clean bench) buffer, take 5 mg self assembly polypeptide powder, mixed dissolution is placed in ice-water bath, and using ultrasound wave hydrotropy, ultrasonic power is 4 W/mL, every task 3 s, stop 5 s, hydrotropy total duration is 20 min.
Get self assembly polypeptide solution 0.95 mL of 5.26 mg/mL, add the pcDNA3-of 2 μ g/ μ L
hA-
apoptinplasmid solution 50 μ L, mixed solution cumulative volume 1 mL, the whole mass concentration of polypeptide is the whole mass concentration of 5 mg/mL plasmids is 100 μ g/mL, and the mass ratio of plasmid and self assembly polypeptide is 1:50.Cover tightly lid, put upside down mixing, mixing bonnet mouth and lid with sealing compound sealing, put shaking table 150 r/min, 12 h again; The nanoparticles solution obtained as shown in Figure 2.
The nanoparticles solution of getting 10 μ L carries out 0.8% agarose gel electrophoresis detection, with the pcDNA3-of unpackaged process
hA-
apoptinin contrast, result as shown in Figure 3 for plasmid solution 10 μ L; Carry out transmission electron microscope detection to solution again, result as shown in Figure 3.This electrophoresis result prompting plasmid is packed by polypeptide, and its molecular shape there occurs larger change compared with unpackaged plasmid, and its particle diameter is excessive causes it to fail enter gel and rest on loading hole initial point.3 bands occurred in unpackaged plasmid electrophoresis process are respectively the superhelix occurred in plasmid extraction process, linear and open loop 3 kinds of versions.Fig. 3 ~ 4 show, self assembly polypeptide-pcDNA3-
hA-
apoptincomposite nanometer particle is successfully prepared.
Embodiment 3: the apoptin gene that nano-particle carries detects at cell inner expression
People's kidney clear cell carcinoma 7860 cell and renal tubules near-end epithelium HK2 cell (normal cell) are purchased from Kunming Institute of Zoology, Chinese Academy of Sciences).Two kinds of cells are containing 10% hyclone (purchased from GIBCO), in the DMEM high glucose medium of 100 IU/mL penicillins and 100 μ g/mL streptomycins, and 5%CO
2, 37 DEG C of cellar cultures.When kidney clear cell carcinoma 7860 cell and renal tubules near-end epithelium HK2 cell (normal cell) incubation growth to 40%, in cultured cell, add self assembly polypeptide, pcDNA3-respectively
hA-
apoptinplasmid and self assembly polypeptide-pcDNA3-
hA-
apoptincomposite nanometer particle.Cultivate after 24h, carry out immunofluorescence dyeing observing apoptosis plain gene and to express and at Cytoplasm, apoptotic nueleolus.
The operating process of cell climbing sheet immunofluorescence is as follows: through self assembly polypeptide, pcDNA3-
hA-
apoptinwith self assembly polypeptide-pcDNA3-
hA-
apoptinkidney clear cell carcinoma 7860 cell that composite nanometer particle processes respectively and renal tubules near-end epithelium HK2 cell (normal cell), pre-cooling 4 DEG C of PBS wash cell 3 times; 4 DEG C, 4% paraformaldehyde fixes 20 min, and PBS washes 3 times; 0.5 %Triton X-100 rupture of membranes 10min, PBS washes 3 times; 5% BSA(is dissolved in PBS) close 30 min, abandon 5%BSA; Mouse monoclonal antibody Anti-HA-Tag is diluted in 5%BSA solution, dilution ratio 1:500, overnight incubation in 4 DEG C of wet boxes; Next day takes out creep plate, and PBS washes 3 times; FITC labelling two anti-(sheep anti-Mouse) is diluted in 5%BSA solution, and dilution ratio 1:1000, hatches 1h, lucifuge for 37 DEG C, two anti-hatch after select the place of dark to carry out follow-up operation; PBS washes 3 times, and blue fluorescent dyes DAPI contaminates core, room temperature 15min; PBS develops a film 3 times, 50% glycerol mounting.Detect through fluorescence microscope, take acquisition image.
As shown in Fig. 5 ~ 6, self assembly polypeptide-pcDNA3-
hA-
apoptinthe pcDNA3-that composite nanometer particle carries
hA-
apoptinplasmid all successful expression can go out apoptosis fibroin in normal cell and kidney clear cell carcinoma 7860 cell.But unlike, in tumor cell, apoptosis fibroin is mainly distributed in nucleus; In normal cell, apoptosis fibroin is mainly distributed in (see figure 6) in Cytoplasm, and the apoptotic nueleolus of apoptosis element to be it be phosphorylated modifies and can the essential condition of inducing apoptosis of tumour cell.
Embodiment 4: composite nanometer particle is tested at cell internal specific expression regulation
People's kidney clear cell carcinoma 7860 cell and renal tubules near-end epithelium HK2 cell (normal cell) are purchased from Kunming Institute of Zoology, Chinese Academy of Sciences).Two kinds of cells are containing 10% hyclone (purchased from GIBCO), in the DMEM high glucose medium of 100 IU/mL penicillins and 100 μ g/mL streptomycins, and 5%CO
2, 37 DEG C of cellar cultures.Until kidney clear cell carcinoma 7860 cell and renal tubules near-end epithelium HK2 cell (normal cell) incubation growth to about 30% time, in two kinds of cultured cells, add self assembly polypeptide-pcDNA3-
hA-
apoptincomposite nanometer particle.Process cell, after cultivating 24h, ordinary optical inverted microscope checks cell proliferation, apoptosis situation.
As shown in Fig. 7 ~ 8, self assembly polypeptide-pcDNA3-
hA-
apoptincomposite nanometer particle to Normocellular growth unrestraint effect, renal tubules near-end epithelial cell HK2 can normal proliferative, division; And the growth of kidney clear cell carcinoma 7860 cell is significantly inhibited, and show apoptotic morphological characteristic (see figure 8).
From embodiment 1 ~ 4, self assembly polypeptide-pcDNA3-
hA-
apoptincomposite nanometer particle can by cellular uptake, at cell inner expression gene entrained by it, and expressed apoptosis fibroin energy specificity inducing apoptosis of tumour cell and to not normal cells.
Sequence table
<110> Kunming Medical University
<120> self assembly polypeptide-apoptin gene composite nanometer particle and its preparation method and application
<130> 2014
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 16
<212> PRT
<213> artificial sequence
<400> 1
Arg Ala Asp Ala Arg Ala Asp Ala Arg Ala Asp Ala Arg Ala Asp Ala
1 5 10 15
Claims (10)
1. self assembly polypeptide-apoptin gene composite nanometer particle, it is characterized in that using described self assembly polypeptide as carrier, by self acidic amino acid and basic amino acid with electric charge with form ionic bond between the plasmid molecule carrying apoptin gene and be combined, plasmid molecule secure adhesion, in self assembly polypeptide surface, forms composite nanometer particle.
2. self assembly polypeptide-apoptin gene composite nanometer particle according to claim 1, it is characterized in that described composite nanometer particle is in threadiness, length is 100 ~ 200nm, and diameter is 10 ~ 20nm.
3. self assembly polypeptide-apoptin gene composite nanometer particle according to claim 1, is characterized in that the sequence of described self assembly polypeptide is as shown in SEQ ID No.1.
4. self assembly polypeptide-apoptin gene composite nanometer particle according to claim 1, is characterized in that the described plasmid carrying apoptin gene is pcDNA3-
hA-
apoptinplasmid.
5. a preparation method for the arbitrary described self assembly polypeptide-apoptin gene composite nanometer particle of Claims 1 to 4, is characterized in that comprising the steps:
(1) self assembly polypeptide is synthesized;
(2) plasmid containing apoptin gene is built;
(3) self assembly polypeptide solution is prepared;
(4) in polypeptide solution, add the plasmid solution that step (2) builds, fully obtain nano-particle suspension after effect;
(5) nano-particle detects.
6. the preparation method of self assembly polypeptide-apoptin gene composite nanometer particle according to claim 5, it is characterized in that the mass concentration of the self assembly polypeptide solution described in step (3) is 5.26 mg/mL, the detailed process of preparation is: operation in superclean bench, white powder polypeptide is dissolved in aseptic PBS buffer solution, is then placed in ice-water bath, and use ultrasound wave hydrotropy, ultrasonic power is 4 W/mL, every task 3 s, stops 5 s, and hydrotropy total duration is 20 min.
7. the preparation method of self assembly polypeptide-apoptin gene composite nanometer particle according to claim 5, it is characterized in that the mass concentration of the plasmid solution described in step (4) is 2 μ g/ μ L, the mass ratio of plasmid and self assembly polypeptide is 1:45 ~ 1:55.
8. the preparation method of self assembly polypeptide-apoptin gene composite nanometer particle according to claim 5, it is characterized in that fully acting as after add plasmid in polypeptide solution described in step (4), cover tightly lid, put upside down mixing, mixing bonnet mouth and lid seal with sealing compound again, put shaking table 150 r/min, 12h.
9. the preparation method of self assembly polypeptide-apoptin gene composite nanometer particle according to claim 5, is characterized in that the method that the nano-particle described in step (5) detects is that agarose gel electrophoresis detects and/or transmission electron microscope detects.
10. the arbitrary described self assembly polypeptide-application of apoptin gene composite nanometer particle in antineoplaston of Claims 1 to 4.
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| CN104906045A (en) * | 2015-06-25 | 2015-09-16 | 中国科学院过程工程研究所 | Low-molecular-weight heparin slow-release preparation as well as preparation method and application thereof |
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