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Gene transfer vector and preparation method as well as application thereof

A gene transfection carrier and gene technology, applied in the field of gene transfection carrier and its preparation, can solve the problems of unstable transfection effect, no improvement, complex synthesis steps, etc., achieve stable transfection effect and stable material composition The effect of simple control and synthesis process

Active Publication Date: 2013-09-11
EAST CHINA NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, gene transfection vectors based on amino-containing polymers generally face high material costs, complicated synthesis steps, unstable chemical composition of polymers, unstable transfection effects, and the need for multiple modifications to improve gene transfection. A series of problems such as high transfection efficiency and high cytotoxicity accompanied by high gene transfection efficiency have not been improved or resolved.

Method used

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  • Gene transfer vector and preparation method as well as application thereof
  • Gene transfer vector and preparation method as well as application thereof
  • Gene transfer vector and preparation method as well as application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] Example 1: Preparation and Characterization of Gene Transfection Material G5-DAT66 Based on Dendrimer and Diaminotriazine Functional Group

[0085] Take the gene transfection material G5-DAT66 based on dendrimer and diaminotriazine functional groups as an example, such as figure 1 As shown, G5 is the fifth generation polyamide-amine dendrimer, the central core is ethylenediamine, the terminal group is a primary amino group, n is 6, m is 2; DAT is 2-chloro-4, 6-diamino-1,3,5-triazine; 66 is 2-chloro-4,6-diamino-1,3,5-triazine covalently attached to the 5th generation polyamidoamine dendrimer The number of surfaces, whose structural formula is:

[0086] Formula (4);

[0087] In formula (4), R is the 5th generation polyamide-amine dendrimer G5; its structure is as shown in formula (5):

[0088] Formula (5);

[0089] In formula (5), M is the central core of the fifth generation polyamide-amine dendrimer G5: ethylenediamine.

[0090] In each specific embodim...

Embodiment 2

[0093] Example 2: Combination and characterization of gene transfection material G5-DAT66 and DNA.

[0094] (1) Using agarose gel electrophoresis to detect the combination of gene transfection material G5-DAT66 and DNA to form a complex

[0095] The gene transfection reagent G5-DAT66 prepared in Example 1 and the green fluorescent protein particle DNA (pEGFP-1) form a complex at room temperature, and the complex DNA of the gene transfection material G5-DAT66 is detected by agarose gel electrophoresis. ability.

[0096] The specific method is as follows: the gene transfection material G5-DAT66 and the green fluorescent protein particle DNA (pEGFP-1) were mixed according to different nitrogen and phosphorus ratios (0:1, 0.5:1, 1:1, 2:1, 4:1, respectively). 1, 8:1) mixed at room temperature, incubated for 30 minutes, then diluted with DNA loading buffer, and finally the sample was subjected to agarose gel electrophoresis at 90 volts for 50 minutes, and the agarose gel was dissol...

Embodiment 3

[0101] Embodiment 3: Gene transfection experiment of gene transfection material G5-DAT66 on 293T cells

[0102] The gene transfection material G5-DAT66 prepared in Example 1 and the green fluorescent protein plasmid or luciferase plasmid form a complex at room temperature, and then transfect on 293T cells, by detecting the green fluorescent protein or luciferase The gene transfection efficiency of the material was evaluated by the expression level. The specific method is: culture 293T cells in a 24-well plate and incubate for 24 hours, mix 1.6 micrograms of green fluorescent protein or luciferase plasmid with G5-DAT66 at different nitrogen-to-phosphorus ratios, and then add them to the medium and mix well; together with the cells After 6 hours of incubation, 500 microliters of medium containing 10% serum was added, and the cells were treated 42 hours later. The expression of green fluorescent protein was quantitatively analyzed by flow cytometry to analyze the efficiency of t...

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Abstract

The invention provides a gene transfer vector shown in a formula (1) in the specification. The vector comprises a dendrimer skeleton and a diamino triazine functional group, wherein the diamino triazine group is covalently connected on the surface of the dendrimer skeleton; a dendrimer comprises a polyamidoamine dendrimer and a poly(propyleneimine) dendrimer; and a primary amine group is arranged on the surface of the dendrimer. The invention also provides a preparation method of the gene transfer vector and application of the gene transfer vector as a nucleic acid molecule conveying vector in vitro or in vivo. The invention also provides a compound comprising the gene transfer vector. The gene transfer vector is low in synthetic cost, has low cytotoxicity, can effectively and safely convey gene molecules to cells and has the advantages of efficiency, low toxicity, low cost and the like.

Description

technical field [0001] The invention relates to the technical fields of polymer chemistry, organic synthesis and biological materials, and in particular to a novel gene transfection carrier and its preparation method and application. Background technique [0002] Gene transfection refers to the process of introducing foreign genes into cells to obtain new genetic traits. Gene transfection vector is the core of gene transfection technology. An ideal gene transfection vector should have the following characteristics: high transfection efficiency, low cytotoxicity, good biological safety, and low price. Currently used gene transfection vectors mainly include viral vectors and non-viral vectors. The main application research still uses viral vectors with high transfection efficiency, but viral vectors have problems such as limited ability to carry genes and potential safety hazards. [0003] Polymers containing amino groups have been widely used in gene transport. The amino gr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C08G83/00C08G73/04C12N15/87
Inventor 程义云邵乃敏
Owner EAST CHINA NORMAL UNIV
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