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Self-assembled polypeptide-apoptin gene composite nanoparticle, and preparation method and application thereof

A composite nanoparticle and nanoparticle technology are applied in the field of self-assembled polypeptide-apoptin gene composite nanoparticles and their preparation to achieve the effect of overcoming the lack of targeting of therapeutic genes

Active Publication Date: 2015-01-21
KUNMING MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there is no report on the packaging and related research of large molecular weight recombinant plasmid DNA and self-assembled polypeptides. In the past, the commonly used methods for delivering recombinant plasmid DNA into cells include: liposome-mediated transfection, low-temperature calcium phosphate transfection, DEAE glucose Glycan-mediated transfection, electroporation, microinjection, etc. Each of the above methods has its own advantages and disadvantages

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  • Self-assembled polypeptide-apoptin gene composite nanoparticle, and preparation method and application thereof
  • Self-assembled polypeptide-apoptin gene composite nanoparticle, and preparation method and application thereof
  • Self-assembled polypeptide-apoptin gene composite nanoparticle, and preparation method and application thereof

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preparation example Construction

[0034] The preparation method of the self-assembling polypeptide-apoptin gene composite nanoparticle of the present invention comprises the following steps:

[0035] (1) According to the sequence of SEQ No.1, synthesize the self-assembling polypeptide;

[0036] (2) Construction of pcDNA3- HA - apoptin Plasmid: Design and custom synthesis with pcDNA3 as vector HA - apoptin Primers to pcDNA3- apoptin as a template, amplified HA - apoptin Target gene fragment, construct pcDNA3- HA - apoptin plasmid; HA - apoptin The primer sequences are as follows:

[0037] HA - apoptin Upstream primers (underlined as Bam HI restriction site):

[0038]

[0039] Bam HI and xho I Double digestion plasmid vector pcDNA3 and target gene HA - apoptin , in the vector pcDNA3 multiple cloning site Bam HI and xho Access at I HA - apoptin Gene fragment; T4 DNA ligase at 16°C, 12h to connect the vector and the target gene fragment to obtain recombinant pcDNA3- HA - ...

Embodiment 1

[0046] Example 1: Construction of recombinant pcDNA3- HA - apoptin eukaryotic expression plasmid

[0047] Step 1: PCR Amplification HA - apoptin Gene

[0048] 10×Taq Buffer 5 μL 2 mM dNTPs 5 μL 1 μM upstream primer 1 μL 1 μM downstream primer 1 μL template 2 μL 25 mM Mg 2+ 2.5 μL Taq DNA Polymerase 1.3 μL h 2 o 32.2 μL total: 50 μL

[0049] in, HA - apoptin The primer sequences are as follows:

[0050] HA - apoptin Upstream primers (underlined as Bam HI restriction site):

[0051]

[0052] The PCR reaction program was: 95°C preheating for 300s, 95°C for 30s→55°C for 30s→72°C for 40s, 35 cycles, 72°C for 300s, 4°C for 1h.

[0053] Step 2: Recovery using Omega Biotek Kit HA - apoptin PCR product;

[0054] Step 3: HA-apoptin The gene fragment was inserted into the vector pcDNA3

[0055] pcDNA3 HA-apoptin xho I 1 μL xho I 1 μL Bam HI 1μL Bam HI 1...

Embodiment 2

[0063] Example 2: Preparation of self-assembling polypeptide-apoptin gene composite nanoparticles

[0064] The self-assembling polypeptide was synthesized according to the sequence SEQ No.1, and the synthesized self-assembling polypeptide was in the form of white powder, which was prepared into a 5.26 mg / mL self-assembling polypeptide solution. The preparation process is to take 0.95 mL sterile PBS (pH7.4) buffer solution in an ultra-clean bench, weigh 5 mg of self-assembled peptide powder, mix and dissolve it, place it in an ice-water bath, and use ultrasonic waves to aid in dissolution, with an ultrasonic power of 4 W / mL, work for 3 s each time, stop for 5 s, and the total duration of aiding solution is 20 min.

[0065] Take 0.95 mL of 5.26 mg / mL self-assembling peptide solution, add 2 μg / μL pcDNA3- HA - apoptin The plasmid solution is 50 μL, the total volume of the mixed solution is 1 mL, the final mass concentration of the polypeptide is 5 mg / mL, the final mass concentr...

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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 [0001] The invention belongs to the technical field of gene therapy, and specifically relates to a self-assembled polypeptide-apoptin gene composite nanoparticle capable of specifically inducing apoptosis of tumor cells and nontoxic to normal cells, as well as its preparation method and application. Background technique [0002] Apoptin, also known as VP3, is a small molecule protein derived from chicken anemia virus (CAV), with a molecular weight of 13.6 kDa, composed of 121 amino acids, rich in proline, basic amino acids and High ratio of ser / threonine residues. Apoptin has no sequence homology to any other animal or viral protein. Apoptin can specifically induce apoptosis of tumor cells and cells with transformed phenotypes, but has no toxic effect on normal cells. So far, the existing research reports have shown that apoptin can specifically induce the apoptosis of more than 70 tumor cell lines from more than 15 different tissue-derived human tumor cell...

Claims

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Application Information

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IPC IPC(8): A61K47/48A61K48/00A61P35/00
Inventor 狄勇彭传梅
Owner KUNMING MEDICAL UNIVERSITY
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