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Cell penetrating peptide and method for delivering biologically active substance using same

一种生物活性物质、穿透肽的技术，应用在生物活性物质输送到细胞内的系统领域，能够解决副作用、连接效率低、不需要免疫应答等问题

Active Publication Date: 2016-12-21
IUCF HYU (IND UNIV COOP FOUND HANYANG UNIV)
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Since these existing cell-penetrating peptides are derived from proteins of viruses such as HIV-1, derived from proteins expressed by other species of Drosophila, or artificially synthesized proteins based on amino acid sequence analysis of previously known cell-penetrating peptides, When used in humans, it can cause side effects such as an immune response
[0008] Also, since it is composed of relatively long chains of amino acids, it is more likely to elicit an unwanted immune response
Also, since it can affect the structure and function of the protein to be delivered, the efficiency of attachment to biologically active substances to be delivered into cells is usually low

Method used

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  • Cell penetrating peptide and method for delivering biologically active substance using same
  • Cell penetrating peptide and method for delivering biologically active substance using same
  • Cell penetrating peptide and method for delivering biologically active substance using same

Examples

Experimental program
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Effect test

preparation Embodiment 1

[0065] Preparation Example 1: Synthesis and Purification of Peptides

[0066] Peptides having the amino acid sequences of SEQ ID NO 1 to SEQ ID NO 12 were synthesized.

[0067] After synthesizing sense oligodeoxynucleotides and antisense oligodeoxynucleotides corresponding to the amino acid sequences, followed by removal of secondary or tertiary structures (denaturation) at 95°C for 3 minutes, by changing the temperature to 50°C and then Double-stranded DNA was prepared for 72°C. For insertion into the pRSET-b vector, restriction enzyme specific sequences were inserted into the 5' site and the 3' site except for the sense oligodeoxynucleotide and the antisense oligodeoxynucleotide. The sequence was then amplified in large quantities by transformation into E. coli. After confirming the integrity of the sequence, expression was induced in E. coli.

[0068] In order to fuse a peptide having the amino acid sequence of SEQ ID NO 1 (hereinafter also referred to as "AP") and enhan...

preparation Embodiment 2

[0069] Preparation Example 2: Preparation of double-stranded DNA encoding AP having EGFP linked at N-terminus

[0070] A forward primer was constructed by adding a DNA base sequence encoding a peptide having an amino acid sequence of SEQ ID NO 1 to a DNA base sequence encoding an N-terminal portion of enhanced green fluorescent protein (hereinafter also referred to as "EGFP").

[0071] The forward primer of SEQ ID NO 13 contains an NheI restriction enzyme recognition site for DNA cloning at the 5' end and a BamHI restriction enzyme recognition site between the base sequences of AP and EGFP. Meanwhile, a reverse primer of SEQ ID NO 14 was constructed for amplifying AP-EGFP by PCR. The reverse primer contains the DNA base sequence encoding the terminal portion of EGFPC. For DNA cloning, a HindIII restriction enzyme recognition site was inserted into the 5' end of the primer.

[0072] PCR was performed using the pRSETb vector containing the EGFP gene as a template and using the...

preparation Embodiment 3

[0074] Preparation Example 3: Preparation of pRSETb vector inserted with AP-EGFP

[0075] In order to express the AP-EGFP protein, the 789-bp DNA fragment prepared in Preparation Example 2 was inserted into the protein expression vector pRSETb using restriction enzymes and ligases.

[0076] The DNA fragment amplified in Preparation Example 2 was treated with NheI and HindIII (NEB) enzymes to make the 5' / 3' ends of the DNA sticky. At the same time, pRSETb was treated with the same restriction enzymes to prepare a linear pRSETb vector having NheI and HindIII insertion sites. After each enzymatic reaction, products were isolated using a PCR purification kit (Cosmo Genetech).

[0077] The isolated AP-EGFP double-stranded DNA fragment and pRSET-b vector were treated with T4 ligase (NEB) for 2 hours at 25°C. The concentration of AP-EGFP double-stranded DNA fragment and pRSET-b vector was analyzed by 1% agarose gel electrophoresis ( figure 2 ).

[0078] The obtained pRSETb vecto...

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Abstract

The purpose of the present invention is to provide: a new cell penetrating peptide; and a composition for delivery of a biologically active substance, a composition for gene therapy, a method for delivery of the biologically active substance, and a method for the gene therapy using the same. The cell penetrating peptide of the present invention can effectively deliver a protein into human cell lines and tissues, can deliver a protein with higher efficiency in comparison with a TAT peptide that is commercially used as a cell penetrating peptide, and can also be usefully used in the delivery of biologically active substances such as proteins, genetic materials, chemical compounds etc. which may be used for therapeutic purposes in a cell.

Description

technical field [0001] The present disclosure relates to systems for delivering biologically active substances that function in cells into cells. Background technique [0002] In general, bioactive polymeric substances such as proteins and DNA cannot enter cells through cell membranes because they cannot pass through phospholipid bilayers. However, cell penetrating peptides are known to be able to cross cell membranes without the aid of receptors or other molecules. [0003] Cell penetrating peptides are also known as PTD (protein transduction domain) or MTS (membrane translocation sequence), which can associate or mix with cargo such as protein, DNA, RNA, etc., and transport the cargo through the cell membrane into the cell and cytoplasm , organelles, and within the nucleus (Endoh and Ohtsuki, 2010; Joliot and Prochiantz, 2004; Mogi and Kondo, 2010). [0004] During HIV-1 (Human Immunodeficiency Virus-1) infection, tat was the first protein found to penetrate cell membran...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/06C07K7/08C07K19/00A61K48/00
CPCC07K2319/10C07K2319/60C07K14/43595A61K38/00A61K48/0075C07K7/06
Inventor 崔济民赵贤贞金沅柱金度炫具滋贤李贞雅李哄均林相昊
Owner IUCF HYU (IND UNIV COOP FOUND HANYANG UNIV)
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