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.

CN106916209AActive Publication Date: 2017-07-04CHINA UNIV OF PETROLEUM (EAST CHINA)
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Patent Information

Authority / Receiving Office
CN · China
Patent Type
Applications(China)
Current Assignee / Owner
Publication Date
2017-07-04

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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.
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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...

Examples

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...