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Use of the cell-penetrating peptide lta2 from the lt subunit as an intracellular drug delivery vehicle

A drug and cell technology, applied in the direction of drug combination, medical preparations with non-active ingredients, medical preparations containing active ingredients, etc., to achieve the effect of high membrane penetration efficiency, less unsafe factors, and less quantity

Active Publication Date: 2018-02-06
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It can carry macromolecular substances with a maximum molecular weight of 120kDa and a diameter of 40mm, and there is currently no clear evidence that penetrating peptides have a loading limit

Method used

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  • Use of the cell-penetrating peptide lta2 from the lt subunit as an intracellular drug delivery vehicle
  • Use of the cell-penetrating peptide lta2 from the lt subunit as an intracellular drug delivery vehicle
  • Use of the cell-penetrating peptide lta2 from the lt subunit as an intracellular drug delivery vehicle

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1. pET24a-mCherry and pET24a-mCherry-linker-LTA 2 Construction of recombinant plasmids

[0030] Reagents and Kits: DNA Polymerase Pyrobest TM , restriction endonucleases (Nde I, XhoI, BamH I, Sal I, Not I), DNA ligation kit DNA Ligation Kit Ver.2.1, agarose gel extraction kit TaKaRaMiniBEST Agarose Gel DNA Extraction Kit Ver.4.0, plasmid The extraction kit TaKaRaMiniBEST Plasmid Purification Kit Ver.4.0 was purchased from Treasure Bioengineering Co., Ltd. (Dalian). Tryptone and yeast extract were purchased from Oxoid Company (UK). Kanamycin sulfate was purchased from Sigma-Aldrich (USA). Other biochemical reagents belong to domestic conventional analytical reagents.

[0031] Plasmids and strains: Plasmid pET-24a(+) (Novagen, USA), E.coliDH5α (Invirogen, USA) were used as plasmid amplification strains.

[0032] Reagent preparation:

[0033] LB liquid medium: Weigh 10 g of tryptone, 5 g of yeast extract, and 10 g of sodium chloride, dilute to 1 L with deioni...

Embodiment 2

[0090] Example 2. mCherry protein and mCherry-linker-LTA 2 Protein expression and purification

[0091] Reagents and kits: Isopropyl B-D-I-Thiogalactopyranoside (IPTG), kanamycin sulfate were purchased from Sigma-Aldrich (USA), Ni-NTA affinity column and filler Purchased from Biorad Company (USA), protein molecular weight standard product Premixed Protein Marker (Low), protein loading buffer 4×Protein SDS PAGE Loading Buffer, purchased from Bao Biological Engineering Co., Ltd. (Dalian). Bradford protein quantification kit was purchased from Tiangen Biochemical Technology Co., Ltd. (Beijing). Other biochemical reagents belong to domestic conventional analytical reagents.

[0092] Strains and plasmids: E. coli BL21(DE3) (Invirogen, USA) was used as a plasmid expression strain. Recombinant plasmid pET24a-mCherry, pET24a-mCherry-linker-LTA 2 Constructed for the above-mentioned stages of the present invention.

[0093] Reagent preparation:

[0094] 5× protein electrophoresis ...

Embodiment 3

[0109] Example 3.LTA 2 Transmembrane and translocation of proteins to cells

[0110] Reagents and cell lines: McCoy's 5A medium, RPMI-1640 medium, fetal bovine serum (FBS), trypsin were purchased from Gibco (USA), penicillin and streptomycin mixed solution was purchased from Suleibao (Beijing). Other biochemical reagents belong to domestic conventional analytical reagents. Hct-116 cell line, Bcap-37 cell line and A549 cell line were purchased from the Cell Bank of the Type Culture Collection Committee of the Chinese Academy of Sciences (Shanghai).

[0111] Reagent configuration:

[0112] Preparation of 10% fetal bovine serum medium: Measure 180ml of McCoy's 5A medium or 180ml of RPMI-1640 medium, add 20ml of fetal bovine serum, then add 2ml of 100× penicillin and streptomycin mixture, mix well and place at 4°C save.

[0113] Preparation of phosphate buffer: weigh 8NaCl, 1.42gNa 2 HPO 4 , 0.24g KHPO 4 , 0.2g KCl, after dissolving a small amount of ultrapure water, add ul...

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Abstract

The present invention discloses the application of the cell penetrating peptide LTA2 derived from the LT subunit as an intracellular drug delivery carrier, and the sequence of the LTA2 is shown in SEQ ID NO:1. And a method for preparing a fusion protein drug is provided, the fusion protein is obtained by linking the LTA2 peptide segment with a protein that can act as a drug through a connecting peptide Linker. The present invention found for the first time that the peptide segment LTA2 has the function of penetrating the cell membrane, and can carry macromolecules such as proteins into various cells in vitro through endocytosis. Transmembrane transport of molecules. LTA2 has high transmembrane efficiency, less cationic amino acids, is safe and non-cytotoxic, and has relatively few potential unsafe factors. The present invention is different from the previously discovered N-terminal membrane-penetrating peptides of recombinant proteins such as TAT ​​and MPG, and is a C-terminal membrane-penetrating peptide of a recombinant protein, adding a new option in the design of membrane-transmitting recombinant drug proteins.

Description

technical field [0001] The present invention relates to the field of biotechnology, more specifically, relates to a cell penetrating peptide LTA from LT subunit 2 Application as a transmembrane carrier. Background technique [0002] The cell membrane is an important channel for material exchange and information transmission between cells and the surrounding environment. It uses phospholipid bilayers as the basic skeleton, and various types of membrane proteins, sugars and lipids combined with membrane proteins are embedded in the skeleton. The special structure of the cell membrane makes it hydrophobic inside the membrane and hydrophilic on both sides of the membrane. In addition, the pores and transmembrane proteins with special functions in the membrane enable it to regulate the transport of substances inside and outside the cell. Due to its special selective permeability to substances, while protecting cells, it also blocks many potential biomacromolecular drugs out of c...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K47/42A61K47/64A61K38/00C07K14/245A61P35/00
Inventor 马兴元刘地郑文云张娜王天文王平
Owner EAST CHINA UNIV OF SCI & TECH
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