Polypeptide nucleic acid vector, preparation method and uses thereof

A nucleic acid carrier and nucleic acid technology, applied in the field of polypeptide nucleic acid carriers

Active Publication Date: 2016-04-06
INST OF PHARMACOLOGY & TOXICOLOGY ACAD OF MILITARY MEDICAL SCI P L A
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since TAT and Rn lack the ability to escape endosomes, it is difficult to achieve ideal transfection efficiency as a vector for delivering DNA alone

Method used

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  • Polypeptide nucleic acid vector, preparation method and uses thereof
  • Polypeptide nucleic acid vector, preparation method and uses thereof
  • Polypeptide nucleic acid vector, preparation method and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] 1. G(LLKK) 3 Synthesis of G-TAT

[0084] According to G(LLKK) 3 Amino acid sequence of G-TAT, Ac-Gly-Leu-Leu-Lys-Lys-Leu-Leu-Lys-Lys-Leu-Leu-Lys-Lys-Gly-Gly-Arg-Lys-Lys-Arg-Arg-Gln -Arg-Arg-Arg-NH 2 , with Rink-amide resin as the solid phase carrier and HBTU-HOBt as the condensing agent, the target peptide sequence was synthesized using a microwave peptide synthesizer (CEM, USA) using the standard Fmoc strategy. Use 20ml of trifluoroacetic acid: thioanisole: m-cresol: ethanedithiol: water (8.25:0.5:0.5:0.25:0.5, volume ratio) as the lysate, react at 0°C for 30 minutes, and at room temperature for 120 minutes, The peptide is deprotected and cleaved from the resin. The solution was purified by RP-HPLC, RP-HPLC conditions, phase A: 0.05% TFA / water; phase B: 0.05% TFA / 70% ACN / water; column: C8; MALDI-Tof-MS: 2999.84.

[0085] 2. G(LLKK) 3 Circular Dichroism Characterization of G-TAT

[0086] In order to verify whether the polypeptide can form α-helix, the circular di...

Embodiment 2

[0101] 1. C (LLKK) 3 Synthesis of C-TAT

[0102]According to C (LLKK) 3 Amino acid sequence of C-TAT, Ac-Cys-Leu-Leu-Lys-Lys-Leu-Leu-Lys-Lys-Leu-Leu-Lys-Lys-Cys-Gly-Arg-Lys-Lys-Arg-Arg-Gln -Arg-Arg-Arg-NH 2 , with Rink-amide resin as the solid phase carrier and HBTU-HOBt as the condensing agent, the target peptide sequence was synthesized using a microwave peptide synthesizer (CEM, USA) using the standard Fmoc strategy. Use 20ml of trifluoroacetic acid: thioanisole: m-cresol: ethanedithiol: water (8.25:0.5:0.5:0.25:0.5, volume ratio) as the lysate, react at 0°C for 30 minutes, and at room temperature for 120 minutes, The peptide is deprotected and cleaved from the resin. The solution was purified by RP-HPLC, RP-HPLC conditions, phase A: 0.05% TFA / water; phase B: 0.05% TFA / 70% ACN / water; column: C8; MALDI-Tof-MS: 3093.26.

[0103] 2. C (LLKK) 3 Circular Dichroism Characterization of C-TAT

[0104] In order to verify whether the polypeptide can form α-helix, the circular ...

Embodiment 3

[0119] 1. C18-G(LLKK) 3 Synthesis of G-TAT

[0120] According to C18-G (LLKK) 3 Amino acid sequence of G-TAT, C18-Gly-Leu-Leu-Lys-Lys-Leu-Leu-Lys-Lys-Leu-Leu-Lys-Lys-Gly-Gly-Arg-Lys-Lys-Arg-Arg-Gln -Arg-Arg-Arg-NH 2 (where C18 is stearic acid), using Rink-amide resin as a solid phase carrier, HBTU-HOBt as a condensing agent, and using a standard Fmoc strategy, the target peptide sequence was synthesized using a microwave peptide synthesizer (CEM, USA). Use 20ml of trifluoroacetic acid: thioanisole: m-cresol: ethanedithiol: water (8.25:0.5:0.5:0.25:0.5, volume ratio) as the lysate, react at 0°C for 30 minutes, and at room temperature for 120 minutes, The peptide is deprotected and cleaved from the resin. The solution was purified by RP-HPLC, RP-HPLC conditions, phase A: 0.05% TFA / water; phase B: 0.05% TFA / 70% ACN / water; column: C8; MALDI-Tof-MS: 3225.84.

[0121] 2. C 18 -G(LLKK) 3 Circular Dichroism Characterization of G-TAT

[0122]In order to verify whether the polyp...

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Abstract

The present invention relates to a nucleic acid vector, particularly to a peptide nucleic acid vector containing a cell-penetrating peptide and an antibacterial peptide having an alpha-helix amphiphilic structure. The present invention further relates to a complex containing the nucleic acid vector and nucleic acid molecules. The present invention further relates to preparation methods and uses of the nucleic acid vector and the nucleic acid vector / nucleic acid molecule complex. According to the present invention, the nucleic acid vector has characteristics of high transfection efficiency, simple preparation method, low cytotoxicity and the like, and provides effective treatment way for gene therapy.

Description

technical field [0001] The present invention relates to a nucleic acid carrier, in particular to a polypeptide nucleic acid carrier. The present invention also relates to the complex containing the nucleic acid carrier and the nucleic acid molecule, as well as the preparation method and application of the nucleic acid carrier and the nucleic acid carrier / nucleic acid molecule complex. Background technique [0002] The US Food and Drug Administration (FDA) defines gene therapy as a method of treating diseases by transcribing or translating, transporting and / or integrating foreign genes into the host genome. Gene therapy is potentially the most effective treatment for cancer, monogenic diseases, cardiovascular and neurological diseases, etc. Since 1990, when gene therapy entered clinical trials for the first time, more than 1,800 gene therapy clinical trials have been carried out around the world. In 2004, my country became the first country to introduce gene medicine (Gendi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C07K19/00C12N5/10A61K48/00A61K47/48A61P35/00
CPCA61K47/50A61K48/00C07K19/00C12N5/10C12N15/63
Inventor 刘克良栾亮徐亮孟庆斌许笑宇贾启燕
Owner INST OF PHARMACOLOGY & TOXICOLOGY ACAD OF MILITARY MEDICAL SCI P L A
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