CN104288776B - A kind of self assembly polypeptide-apoptin gene composite nanometer particle and its preparation method and application - Google Patents
A kind of self assembly polypeptide-apoptin gene composite nanometer particle and its preparation method and application Download PDFInfo
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Abstract
The invention discloses a kind of self assembly polypeptide-apoptin gene composite nanometer particle and its preparation method and application.Self assembly polypeptide-apoptin gene composite nanometer particle of the present invention is as carrier by self assembly polypeptide, combined by forming ionic bond between the electric charge and the plasmid molecule for carrying apoptin gene of acidic amino acid and basic amino acid the institute band of itself, plasmid molecule secure adhesion forms composite nanometer particle in self assembly polypeptide surface.In threadiness, length is 100~200nm, a diameter of 10~20nm to the composite nanometer particle.The nano grain surface is rich in arginine, can be combined with surface of cell membrane arginine Receptor recognition and by cellular uptake, expresses the gene entrained by it in the cell, expressed apoptosis fibroin can specificity inducing apoptosis of tumour cell and to normal cytotoxic.Composite nanometer particle of the invention is a kind of new Antioncogene treatment biological agent, is had broad application prospects.
Description
Technical Field
The invention belongs to the technical field of gene therapy, and particularly relates to a self-assembled polypeptide-apoptin gene composite nanoparticle which can specifically induce tumor cell apoptosis and is nontoxic to normal cells, and a preparation method and application thereof.
Background
Apoptin (Apoptin), also known as VP3, is a small molecular protein derived from Chicken Anemia Virus (CAV), has a molecular weight of 13.6 kDa, consists of 121 amino acids, is rich in proline, basic amino acids, and a high proportion of silk/threonine residues. Apoptin has no sequence homology with any other animal or viral protein. The apoptin can specifically induce the apoptosis of tumor cells and cells with transformed phenotype without toxic effect on normal cells. So far, the existing research reports show that the apoptin can specifically induce over 70 tumor cell lines of human tumor cell types from more than 15 different tissues to die, and therefore, the apoptin has a broad-spectrum tumor cell apoptosis induction effect. Is an anti-tumor biological agent with a very promising application prospect.
In the past, the search for the application of apoptin includes plasmid delivery using adenovirus as a vector, targeted introduction of apoptin plasmid and sialoglycoprotein into liver tumor through polylysine linkage, inactivation of fowlpox virus as an apoptin plasmid vector, fusion of membrane-penetrating peptide TAT or PTD4 with apoptin protein, mutation of amino acid sequence of apoptin, development of application research of modification, and research using Phage nanobiotropic granule (Phage nanobioticle) as an apoptin delivery vector. These studies have led to a beneficial search for the clinical use of apoptin, but still face a series of challenges: firstly, the modified biological carrier is used for delivering apoptin plasmid or apoptin gene, the preparation process is complicated, the delivery efficiency is low, and the effect of inducing tumor cell apoptosis is not ideal; secondly, the safety of the modified virus serving as a gene vector needs to be further evaluated; the cell-penetrating peptide fusion apoptin protein is delivered, the preparation difficulty of the apoptin protein is high, the cost is high, a large amount of soluble protein is difficult to obtain, the apoptin protein is derived from virus and is easy to aggregate and precipitate in solution, and the apoptin protein can also cause a host to generate corresponding antibody when entering the body, thereby greatly reducing the effect of inducing the tumor cell to die.
At present, the development of nanotechnology provides a brand new technical platform for the biological treatment of tumors. Nanotechnology refers to the technology of studying and applying the structures and interactions of atoms and molecules within the scale range of 1-100 nm. In recent years, nano-drug delivery technology is increasingly applied to anti-tumor research. With the progress of research, it has been recognized that the application of nanotechnology can improve the physicochemical properties of the drug dosage form, and improve the targeting property of the drug, increase the blood concentration and sustained release property in solid tumors, and is one of the research hotspots in current tumor diagnosis and treatment, mainly focusing on the development of nanoparticle carriers carrying antitumor drugs or serving as tumor imaging agents.
With the rapid development of nano biotechnology, a novel non-viral vector system: the emergence of the nano gene vector system injects new vitality into the gene therapy of tumors. Compared with viral vectors, the method has the following advantages: low immunogenicity; high capacity performance; can protect the components inserted therein. Researchers have also conducted research using nanotechnology to deliver Small Interfering RNA (Si RNA) in vivo.
Recently, a novel in-situ hydrosol nano delivery material is developed, the system is based on self-assembly polypeptide (self-assembly peptide), the self-assembly peptide can be spontaneously assembled and crosslinked into nanofiber hydrogel with the diameter of 10-20 nm under physiological conditions, a stable beta-folded structure is formed in an aqueous solution containing ions, the immunogenicity is avoided, ultraviolet or chemical modification is not needed in the assembling and crosslinking process, the sequence is rich in arginine residues, the arginine residues are easy to combine with an arginine receptor on the surface of a cell membrane and transport to cells, and the self-assembly peptide can be used for packaging and delivering protein, micromolecular RNA and hydrophobic chemotherapeutic drug taxol. However, the packaging and related research of the recombinant plasmid DNA with large molecular weight and the self-assembled polypeptide has not been reported, and the conventional methods for delivering the recombinant plasmid DNA into cells include: liposome-mediated transfection, low temperature calcium phosphate transfection, DEAE dextran-mediated transfection, electroporation, microinjection, etc., each of which has its advantages and disadvantages. But all share a common property: has damage to cells.
Therefore, a self-assembly polypeptide-apoptin gene composite nano-particle, a preparation method and application thereof are researched and developed.
Disclosure of Invention
The first purpose of the invention is to provide a self-assembled polypeptide-apoptin gene composite nano-particle; the second aim is to provide a preparation method of the self-assembly polypeptide-apoptin gene composite nano-particle; the third purpose is to provide the application of the self-assembled polypeptide-apoptin gene composite nano-particles in anti-tumor treatment.
The first purpose of the invention is realized by that the self-assembly polypeptide-apoptin gene composite nano-particle takes the self-assembly polypeptide as a carrier, and ionic bond combination is formed between the charges carried by self acidic amino acid and basic amino acid and plasmid molecules carrying apoptin gene, and the plasmid molecules are firmly adhered to the surface of the self-assembly polypeptide to form the composite nano-particle.
The second object of the present invention is achieved by a method for preparing self-assembled polypeptide-apoptin gene composite nanoparticles, comprising the steps of:
(1) synthesizing a self-assembling polypeptide;
(2) constructing a plasmid containing an apoptin gene;
(3) preparing a self-assembly polypeptide solution;
(4) adding the plasmid solution constructed in the step (2) into the polypeptide solution, and fully acting to obtain a nano-particle suspension;
(5) and (4) detecting nanoparticles.
The third object of the invention is realized by the application of the self-assembly polypeptide-apoptin gene composite nano-particles in antitumor therapy.
Compared with the prior art, the invention has the beneficial effects that:
(1) the invention prepares the self-assembly polypeptide packaging apoptin coding recombinant plasmid (pcDNA 3-HA-apoptin) Forming a fibrous composite nano particle, wherein the chemical essence of the fibrous composite nano particle is a composite biological macromolecule formed by self-assembled polypeptide and plasmid DNA, the length is about 100-200 nm, and the diameter is about 10-20 nm;
(2) the composite nano-particles can be taken up by cells, do not damage the cells, can be taken up by the cells with high efficiency, the taking rate is more than 95 percent, the carried genes are expressed in the cells, and the expressed apoptin protein can specifically induce the apoptosis of tumor cells and has no toxicity to normal cells. Because the apoptin does not damage normal cells, the defect of insufficient targeting of therapeutic genes in the prior tumor gene therapy can be overcome, and the composite nano-particle is a novel biological preparation for anti-tumor gene therapy.
Drawings
FIG. 1 shows pcDNA3-HA-apoptinStructure diagram of plasmid molecule;
FIG. 2 shows a self-assembly polypeptide solution and a self-assembly polypeptide-pcDNA 3-HA-apoptinA composite nanoparticle suspension;
FIG. 3 shows the non-packaged pcDNA3-HA-apoptiWith the self-assembling polypeptide-pcDNA 3-HA-apoptinPerforming composite nanoparticle agarose gel electrophoresis;
in the figure, 1-DNA marker; 2-unpacked pcDNA3-HA-apoptin(ii) a 3-self-assembling polypeptide-pcDNA 3-HA-apoptinComposite nanoparticles;
FIG. 4 shows the self-assembly polypeptide-pcDNA 3-HA-apoptinComposite nanoparticle transmission electron microscopy pictures;
FIG. 5 shows that the immunofluorescence technique detects the expression of the apoptin gene in renal clear cell carcinoma 7860 (magnification: 10X 20);
in the figure, a-self-assembly polypeptide treatment, cells were free of apoptin protein expression; b-fluorescent dye DAPI shows cell nuclei; c-unpacked pcDNA3-HA-apoptinPlasmid treatment, cell without apoptin protein expression; d-fluorescent dye DAPI shows cell nucleus; e-self-assembling polypeptide-pcDNA 3-HA-apoptinComposite nanoparticle carried pcDNA3-HA-apoptinThe plasmid can be expressed in a kidney clear cell carcinoma 7860 cell; the F-fluorescent dye DAPI shows cell nucleus;
FIG. 6 shows that the immunofluorescence technique detects the expression localization (magnification: 10X 50) of the apoptin gene in the kidney clear cell carcinoma 7860 cell line and the renal tubular proximal epithelial cell HK2 cell line (normal cells);
in the figure, A-self-assembling polypeptide-pcDNA 3-HA-apoptinComposite nanoparticle carried pcDNA3-HA-apoptinThe plasmid is expressed in normal, and the apoptin protein is displayed by fluorescence; b-fluorescent dye DAPI shows cell nuclei; C-A, B overlap and amalgamate and show normal cell outline and cell nucleus, the apoptin protein expressed in normal cell is mainly distributed in cytoplasm; d-self-assembly polypeptide-pcDNA 3-HA-apoptinComposite nanoparticle carried pcDNA3-HA-apoptinThe plasmid is expressed in tumor cells, and the apoptin protein is displayed by fluorescence; e-fluorescent dye DAPI shows cell nuclei; F-D, E overlap and amalgamate and show normal cell outline and cell nucleus, the apoptin protein expressed in tumor cell is mainly distributed in the cell nucleus;
FIG. 7 shows the proliferation status of the renal clear cell carcinoma 7860 cell line and the renal tubular proximal epithelial cell HK2 cell line (normal cells) after nanoparticle treatment (magnification: 10X 20) by means of light microscopy;
FIG. 8 shows the optical microscope examination of 7860 renal clear cell carcinoma cells (magnification: 10X 40).
Detailed Description
The invention is further described with reference to the accompanying drawings, which are not intended to be limiting in any way, and any variations based on the teachings of the invention are intended to fall within the scope of the invention.
The self-assembly polypeptide-Apoptin (Apoptin) gene composite nanoparticle takes self-assembly polypeptide as a carrier, and ionic bond combination is formed between the charges carried by self acidic amino acid and basic amino acid and plasmid molecules carrying Apoptin gene, and the plasmid molecules are firmly adhered to the surface of the self-assembly polypeptide to form the composite nanoparticle. The composite nano-particles are fibrous, the length of the composite nano-particles is 100-200 nm, and the diameter of the composite nano-particles is 10-20 nm.
The sequence of the self-assembly polypeptide is shown as SEQ ID No. 1. The plasmid containing the apoptin gene is pcDNA3-HA-apoptinThe structure of the plasmid is shown in figure 1.
The preparation method of the self-assembly polypeptide-apoptin gene composite nano-particle comprises the following steps:
(1) synthesizing self-assembly polypeptide according to SEQ No. 1;
(2) construction of pcDNA3-HA-apoptinPlasmid: design, custom synthesis with pcDNA3 as carrierHA-apoptinPrimers, as pcDNA3-apoptinAs a template, amplificationHA-apoptinConstruction of the target Gene fragment pcDNA3-HA-apoptinA plasmid;HA-apoptinthe primer sequences are as follows:
HA-apoptinthe forward primer (underlined isBamHI cleavage site):
BamHI andXhoi double restriction enzyme plasmid vector pcDNA3 and target geneHA-apoptinIn the multiple cloning site of vector pcDNA3BamHI andXhoi accessHA-apoptinA gene fragment; t4 DNA ligase 16 ℃, 12h to connect the vector and the target gene fragment to obtain recombinant pcDNA3-HA-apoptinA eukaryotic expression plasmid;
(3) preparing a 5.26mg/mL self-assembly polypeptide solution, wherein the preparation process comprises the following steps: operating in an ultra-clean workbench, dissolving white powdery polypeptide in sterile PBS buffer solution, then placing in an ice-water bath, and using ultrasonic wave to assist dissolution, wherein the ultrasonic wave power is 4W/mL, the work time is 3s each time, the stop time is 5s, and the total duration time of the assist dissolution is 20 min;
(4) adding 2 μ g/μ L of pcDNA3-HA-apoptinPlasmid solution, the mass ratio of plasmid to polypeptide is 1: 45-1: 55. tightly covering the cover, reversing and uniformly mixing, sealing the opening and the cover with sealing glue after uniformly mixing, and placing in a shaking table at 150r/min for 12 h;
as a preferred mode of the technical scheme of the invention, the mass ratio of the plasmid to the polypeptide is 1: 50;
(5) the success of the composite nanoparticles was examined using 0.8% agarose gel electrophoresis and transmission electron microscopy.
The self-assembled polypeptide-apoptin gene composite nano-particle is applied to the anti-tumor treatment for preparing a gene medicament which can specifically induce tumor cells to die without damaging normal cells.
The self-assembly polypeptide-apoptin gene composite nano-particle is manufactured for the application in anti-tumor treatment, the surface of the nano-particle is rich in arginine, the nano-particle can be identified and combined with arginine receptor on the surface of cell membrane and taken up by cells, and the pcDNA 3-carried and packaged in the cellsHA-apoptinThe recombinant eukaryotic expression plasmid can smoothly express Apoptin (Apoptin) protein, and the expressed Apoptin can be specificThe composite nano-particle is a novel and specific anti-tumor gene medicine developed based on nano-carrier administration technology and tumor gene therapy.
Example 1: construction of recombinant pcDNA3-HA-apoptinEukaryotic expression plasmids
Step 1: PCR amplificationHA-apoptinGene
The reaction system is as follows:
| 10×Taq Buffer | 5 μL |
| 2 mM dNTP | 5 μL |
| 1 μ M upstream primer | 1 μL |
| 1 μ M downstream primer | 1 μL |
| Form panel | 2 μL |
| 25 mM Mg2+ | 2.5 μL |
| Taq DNA Polymerase | 1.3 μL |
| H2o | 32.2 μL |
| In total: | 50 μL |
wherein,HA-apoptinthe primer sequences are as follows:
HA-apoptinthe forward primer (underlined isBamHI cleavage site):
the PCR reaction program is: preheating at 95 ℃ for 300s, preheating at 95 ℃ for 30s → 55 ℃ for 30s → 72 ℃ for 40s, and circulating for 35 times, wherein the temperature is 72 ℃ for 300s, and the temperature is 4 ℃ for 1 h.
Step 2: recovery Using Omega Biotek kitHA-apoptinPCR products;
and step 3:HA-apoptingene fragment insertion into vector pcDNA3
BamHI andXhoi double-enzyme digestion vector pcDNA3 and target DNAHA-apoptinThe temperature was kept constant at 37 ℃ overnight. The cleavage system is shown in the following 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 d3H2O to 20 μ L of the total system | Supplement d3H2O to 20 μ L of the total system |
1% agarose gel electrophoresis quantitative cutting recovery of enzyme cutting pcDNA3 ring-opening plasmid; 2% agarose gel electrophoresis quantitative gel cutting recovery enzyme digestionHA-apoptinThe operation is the same as step 2.
Ligation of the double cleaved open circular plasmid pcDNA3 andHA-apoptinDNA fragment ligation reaction System 2. mu.L of 10 × Buffer, 0.5. mu.L of T4 Ligase, 100ng of vector plasmid, 80 ng of vector plasmidHA-apoptinDNA fragment of interest, supplement d3H2o to 20. mu.L total system. The vector and the target gene fragment are connected at 16 ℃ for 12 h.
And 4, step 4: screening for recombinant pcDNA3-HA-apoptinEukaryotic expression plasmids
Preparing escherichia coli competent cells by a low-temperature calcium chloride method, transforming the recombinant plasmid into escherichia coli, and specifically comprising the following steps: (1) adding 200 μ L of each competent bacteria, adding ligation product (using a volume of no more than 10 μ L, wherein the DNA content is less than 50 ng) to each tube, gently rotating to mix the contents, and standing on ice for 30 min; (2) placing the tube in a circulating water bath preheated to 42 ℃ and placing for 90s without shaking the tube; (3) taking out the tube, immediately putting the tube on ice, and cooling the bacteria for 1-2 min; (4) adding 800 μ LSOC culture medium (or LB) into each tube, heating to 37 deg.C in water bath incubator, and transferring the tubes to shaking table for incubation for more than 45 min; (5) the bacteria were sedimented by low speed centrifugation (about 500 g), retaining about 200. mu.L SOC (or LB), and after gentle mixing the bacteria were plated onto a 1.5% agarose plate medium containing ampicillin (1. mu.g/mL); (6) placing the plate in a super clean bench until the liquid is sucked dry (7), inverting the culture dish, culturing at 37 ℃, and growing a bacterial colony after 12-16 h; (8) after the colonies grow out, selecting a single colony to inoculate in a fresh culture medium containing antibiotics for amplification culture.
And 5: recombinant pcDNA3-HA-apoptinIdentification of
Plasmids were extracted using the Omega Biotek kit and identified by sequencing using the pcDNA3 sequencing universal primer. The sequencing result is consistent with the expected DNA sequence, and the target gene does not appearHA-apoptinPoint mutation, base insertion, and base deletion.
Finally, the obtained pcDNA3-HA-apoptinThe structure is shown in figure 1.
Example 2: preparation of self-assembled polypeptide-apoptin gene composite nano-particles
Synthesizing the self-assembly polypeptide according to the sequence SEQ No.1, wherein the synthesized self-assembly polypeptide is white powder and is prepared into a self-assembly polypeptide solution of 5.26 mg/mL. The preparation process comprises the steps of taking 0.95 mL of sterile PBS (pH7.4) buffer solution in a super clean bench, weighing 5 mg of self-assembly polypeptide powder, mixing and dissolving, placing in an ice-water bath, using ultrasonic wave to assist dissolution, wherein the ultrasonic wave power is 4W/mL, working for 3s each time, stopping for 5s, and the total duration of the assist dissolution is 20 min.
0.95 mL of 5.26mg/mL self-assembly polypeptide solution was added with 2. mu.g/. mu.L of pcDNA3-HA-apoptinThe plasmid solution is 50 mu L, the total volume of the mixed solution is 1 mL, the final mass concentration of the polypeptide is 5 mg/mL, the final mass concentration of the plasmid is 100 mu g/mL, and the mass ratio of the plasmid to the self-assembly polypeptide is 1: 50. Tightly covering the cover, reversing and uniformly mixing, sealing the opening and the cover with sealing glue after uniformly mixing, and placing in a shaking table at 150r/min for 12 h; the resulting nanoparticle solution is shown in fig. 2.
10 μ L of the nanoparticle solution was subjected to 0.8% agarose gel electrophoresis for detection with unpackaged pcDNA3-HA-apoptinPlasmid solution 10. mu.L was used as a control, and the results are shown in FIG. 3; the solution was examined by transmission electron microscopy, and the results are shown in FIG. 3. The electrophoresis results suggest that the plasmid has been packaged by the polypeptide, its molecular shape has been greatly changed compared to the unpacked plasmid, and its particle diameter is too large to enter the gel and remain at the origin of the loading well. The 3 bands appeared in the electrophoresis process of the unpacked plasmid are respectively in 3 structural forms of supercoiling, linearity and open loop appeared in the plasmid extraction process. FIGS. 3-4 show that the self-assembling polypeptide-pcDNA 3-HA-apoptinThe composite nano-particles are successfully prepared.
Example 3: detection of expression of apoptosis gene carried by nano particles in cells
Human renal clear cell carcinoma 7860 cells and renal tubular proximal epithelium HK2 cells (normal cells) were purchased from kunming animal institute, chinese academy of sciences). Both cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum (purchased from GIBCO), 100 IU/mL penicillin and 100. mu.g/mL streptomycin, 5% CO2And culturing at 37 ℃ in a conventional manner. When the kidney clear cell carcinoma 7860 cells and the renal tubular epithelial HK2 cells (normal cells) grow to 40 percent in culture, the self-assembly polypeptide and pcDNA3-HA-apoptinPlasmid and self-assembling polypeptide-pcDNA 3-HA-apoptinComposite nanoparticles. After 24h of culture, immunofluorescence staining is carried out to observe the expression of the apoptin gene and the location of the apoptin gene in cytoplasm and nucleus.
Immunofluorescence of cell slideThe operation process is as follows: self-assembled polypeptide, pcDNA3-HA-apoptinAnd self-assembling polypeptide-pcDNA 3-HA-apoptinThe kidney clear cell carcinoma 7860 cells and the renal tubular proximal epithelium HK2 cells (normal cells) treated by the composite nanoparticles respectively are precooled for 3 times by PBS (phosphate buffered saline) at 4 ℃; fixing with 4% paraformaldehyde at 4 deg.C for 20min, and washing with PBS for 3 times; breaking membrane with 0.5% Triton X-100 for 10min, washing with PBS 3 times; blocking with 5% BSA (dissolved in PBS) for 30min, discarding 5% BSA; diluting the mouse-derived monoclonal antibody Anti-HA-Tag in a 5% BSA solution at a dilution ratio of 1:500, and incubating overnight in a wet box at 4 ℃; taking out the slide the next day, and washing with PBS for 3 times; diluting a FITC-labeled secondary antibody (goat anti-mouse) in a 5% BSA solution at a dilution ratio of 1:1000, incubating for 1h at 37 ℃, keeping out of the sun, and selecting a place with darker light for subsequent operation after the secondary antibody is incubated; washing with PBS 3 times, staining with blue fluorescent dye DAPI, room temperature 15min, washing with PBS 3 times, and sealing with 50% glycerol. And detecting by a fluorescence microscope, and shooting to obtain an image.
As shown in FIGS. 5-6, the self-assembly polypeptide-pcDNA 3-HA-apoptinComposite nanoparticle carried pcDNA3-HA-apoptinThe plasmid can successfully express the apoptin protein in both normal cells and kidney clear cell carcinoma 7860 cells. But, in contrast, within tumor cells, apoptin proteins are predominantly distributed within the nucleus; within normal cells, apoptin proteins are mainly distributed in the cytoplasm (see fig. 6), and the nuclear localization of apoptin is a necessary condition for it to be modified by phosphorylation and to be able to induce apoptosis in tumor cells.
Example 4: specific expression regulation test of composite nano-particles in cells
Human renal clear cell carcinoma 7860 cells and renal tubular proximal epithelium HK2 cells (normal cells) were purchased from kunming animal institute, chinese academy of sciences). Both cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum (purchased from GIBCO), 100 IU/mL penicillin and 100. mu.g/mL streptomycin, 5% CO2And culturing at 37 ℃ in a conventional manner. 7860 cells of renal clear cell carcinoma and HK2 cells of renal tubular epithelium(Normal cells) when the cells were grown to about 30% in culture, the self-assembling polypeptide pcDNA3-HA-apoptinComposite nanoparticles. Treating cells, culturing for 24h, and inspecting cell proliferation and apoptosis with a common optical inverted microscope.
As shown in FIGS. 7-8, the self-assembly polypeptide-pcDNA 3-HA-apoptinThe composite nanoparticles have no inhibition effect on the growth of normal cells, and the renal tubular proximal epithelial cells HK2 can normally proliferate and divide; while the growth of renal clear cell carcinoma 7860 cells was significantly inhibited and exhibited morphological features of apoptosis (see fig. 8).
As can be seen from examples 1 to 4, the self-assembling polypeptide-pcDNA 3-HA-apoptinThe composite nano-particles can be taken up by cells, the carried genes are expressed in the cells, and the expressed apoptin protein can specifically induce tumor cells to die without toxicity to normal cells.
Sequence listing
<110> university of Kunming medical science
<120> self-assembled polypeptide-apoptin gene composite nanoparticle and preparation method and application thereof
<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 (4)
1. A self-assembly polypeptide-apoptin gene composite nano-particle is characterized in that self-assembly polypeptide is used as a carrier, the sequence of the self-assembly polypeptide is shown as SEQ ID No.1, and the self-assembly polypeptide is connected with pcDNA3-HA-apoptinThe plasmid molecules form ionic bond combination, and the plasmid molecules are firmly adhered to the surface of the self-assembly polypeptide to form composite nano-particles; the composite nano-particles are fibrous, the length of the composite nano-particles is 100-200 nm, and the diameter of the composite nano-particles is 10-20 nm.
2. A method for preparing the self-assembled polypeptide-apoptin gene composite nanoparticle of claim 1, which comprises the following steps:
(1) synthesizing a self-assembling polypeptide;
(2) constructing a plasmid containing an apoptin gene;
(3) preparing a self-assembly polypeptide solution with the mass concentration of 5.26 mg/mL: operating in an ultra-clean workbench, dissolving white powdery polypeptide in sterile PBS buffer solution, then placing in an ice-water bath, and using ultrasonic wave to assist dissolution, wherein the ultrasonic wave power is 4W/mL, the work time is 3s each time, the stop time is 5s, and the total duration time of the assist dissolution is 20 min;
(4) adding the plasmid solution constructed in the step (2) into the polypeptide solution, wherein the mass concentration of the plasmid solution is 2 mug/muL, and the mass ratio of the plasmid to the self-assembly polypeptide is 1: 45-1: 55; after the plasmids are added into the polypeptide solution, the cover is tightly covered, the mixture is inverted and uniformly mixed, the opening and the cover are sealed by using sealing glue after the mixture is uniformly mixed, and the mixture is placed in a shaking table for 150r/min for 12 hours to obtain a nano-particle suspension;
(5) and (4) detecting nanoparticles.
3. The method according to claim 2, wherein the nanoparticle in step (5) is detected by agarose gel electrophoresis and/or transmission electron microscopy.
4. An application of the self-assembled polypeptide-apoptin gene composite nano-particle of claim 1 in preparing anti-tumor drugs.
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