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Amphipathic polypeptide molecule capable of being used as gene vector

A gene carrier and amphipathic technology, applied in the field of genetic engineering, can solve the problems of non-degradability and strong cytotoxicity, and achieve the effects of efficient gene transfection, high transfection efficiency and high transfection efficiency.

Active Publication Date: 2017-07-04
CHINA UNIV OF PETROLEUM (EAST CHINA)
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are two outstanding problems in the use of cationic polymers as DNA aggregation reagents as gene carriers: first, cationic polymers are prone to dissociation from DNA under physiological conditions, while naked DNA molecules are easily degraded by nucleases, It is easy to be phagocytized and cleared by the reticuloendothelial system; secondly, although cationic polymers with high molecular weight and high concentration have ideal transfection efficiency, they have strong cell resistance because they are rich in positive charges and cannot be degraded in vivo toxicity

Method used

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  • Amphipathic polypeptide molecule capable of being used as gene vector
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  • Amphipathic polypeptide molecule capable of being used as gene vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Synthesis of amphiphilic polypeptide molecules that can be used as gene carriers (take the synthesis of 0.25mmol peptide molecules as an example)

[0031] 1. Materials

[0032] (1) Weigh 0.982g of MBHA resin with a loading capacity of 0.318mmol / g, and swell in DCM overnight;

[0033] (2) Prepare a DMF (dimethylformamide) solution of the following amino acids at a concentration of 0.2mol / L:

[0034] Fmoc-Ala-OH (N-fluorenylmethoxycarbonyl-alanine): volume 11mL, mass 0.82g;

[0035] Fmoc-Gly-OH (N-fluorenylmethoxycarbonyl-glycine): volume 56mL, mass 1.90g;

[0036] Fmoc-Pro-OH (N-fluorenylmethoxycarbonyl-proline): volume 11mL, mass 0.74g;

[0037] Fmoc-Ile-OH (N-fluorenylmethoxycarbonyl-isoleucine): volume 32mL, mass 2.26g;

[0038] Fmoc-Lys(Boc)-OH(N-fluorenylmethoxycarbonyl-N'-tert-butoxycarbonyl-lysine): volume 32mL, mass 3.00g;

[0039] Fmoc-Arg(Pbf)-OH(N-fluorenylmethoxycarbonyl-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl-arginine): volume 84mL, mass 10.9...

Embodiment 2

[0052] Morphology and secondary structure of amphiphilic polypeptide molecules that can be used as gene carriers

[0053] Prepare a series of Tris buffer (pH 7.0) for amphiphilic polypeptide molecules, the molecular sequence is as follows:

[0054] Ac-Arg-Gly-Asp-Gly-Pro-Leu-Gly-Leu-Ala-Gly-Ile-Ile-Ile-Gly-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-NH 2 , the concentration of which is 0.015mM, 0.040mM, 0.100mM, 0.250mM and 1.0mM, respectively, at room temperature for 3 days, observe the results of circular dichroism spectrum and atomic force microscope.

[0055] The results of the circular dichroism spectrum are as follows figure 1 As shown, it is found that the molecule is in the α-helical secondary structure within the concentration range of the prepared solution; the results of atomic force microscopy are as follows figure 2 As shown, it was found that many spherical aggregates existed in the solution below the concentration of 1.0mM, with a diameter of 5-100nm.

Embodiment 3

[0057] Morphology and structure exploration of amphiphilic polypeptide molecules that can be used as gene carriers after mixing with λ-DNA molecules

[0058] The amphiphilic polypeptide molecule, the molecular sequence is as follows:

[0059] Ac-Arg-Gly-Asp-Gly-Pro-Leu-Gly-Leu-Ala-Gly-Ile-Ile-Ile-Gly-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-NH 2 Mix with λ-DNA molecules, wherein the concentration of immobilized λ-DNA is 10 μg / mL, and the concentration of 1.54×10 3- mM ethidium bromide EtBr, control the concentration of amphiphilic polypeptide molecules, so that the ratio (+ / -) of the number of positive charges to the number of negative charges of λ-DNA molecules increases from small to large, and observe the mixed system at a wavelength of 600nm Fluorescence intensity changes (excitation wavelength: 520nm).

[0060] Fluorescence intensity changes as a result of image 3 As shown in a, the fluorescence intensity decreases with the increase of the ratio (+ / -), and reaches an equilibri...

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Abstract

The invention provides an amphipathic polypeptide molecule which can be used as a gene vector, and belongs to the field of gene engineering. The amphipathic polypeptide molecule can be combined with a gene molecule and introduces effective condensation thereof, and furthermore transfers the gene molecule into a cell so as to achieve high-effective gene transfection. A sequence of the amphipathic polypeptide molecule is represented as follows: Ac-Arg-Gly-Asp-Gly-Pro-Leu-Gly-Leu-Ala-Gly-Ile-Ile-Ile-Gly-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-NH2. The amphipathic polypeptide molecule can be combined with the gene molecule, so that the molecule has high cell combining capability and penetrability, and can be used as a gene vector and high-effectively applied to gene transfection.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to an amphiphilic polypeptide molecule which can be used as a gene carrier. Background technique [0002] Gene therapy is an advanced treatment method that has developed very rapidly in recent years. It is a biomedical technology that introduces therapeutic genes into human target cells through suitable vectors to treat diseases. Gene vectors include viral vectors and non-viral vectors. Viral vectors have great potential safety hazards, causing complications and even death of patients, while non-viral vectors have become a The most promising alternative to viral vectors. [0003] For a molecule to become a non-viral gene carrier, it not only needs to have efficient DNA / RNA condensation effect, but also needs to be able to effectively load DNA / RNA into the cell and release it successfully, so as to achieve effective transfection. Most of the currently studied DNA tran...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/00C12N15/87
CPCC12N15/87C07K14/00
Inventor 曹美文徐海赵文婧卢沙谢子龙
Owner CHINA UNIV OF PETROLEUM (EAST CHINA)
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