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Novel cell-penetrating peptide

A membrane-penetrating peptide and cell technology, which is applied to peptides, bacteria, cells modified by introducing foreign genetic material, etc., can solve the problem of low membrane penetration efficiency

Inactive Publication Date: 2014-07-02
ZHEJIANG REACHALL PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This domain can bind to heparin-like polysaccharides on the cell surface [Karlsson K, Marklund SL. Lab Invest. 1989, 60:659-666], and can achieve transmembrane transduction by endocytosis, and localize it to the nucleus, but its transmembrane efficiency is not high

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Embodiment 1: HBD mutant expression plasmid construction

[0069] We constructed expression plasmids fused with EGFP and various mutated HBD sequences: EGFP-CS1-pET28a, EGFP-CS2-pET28a, EGFP-CS3-pET28a, EGFP-CS4-pET28a, EGFP-CS5-pET28a .

[0070] 1. Construction of EGFP-CS1 recombinant protein expression plasmid

[0071] Using the EGFP-pET28a plasmid as a template, primers were designed, and the sequence of CS1 was amplified by PCR to obtain the target band of EGFP with CS1, which was cloned into the pMD18T vector for amplification. The amplified plasmid DNA was extracted, and the target fragment was recovered by double digestion with NdeI and BamHI, and ligated with pET28a recovered by double digestion with the same two enzymes to construct a plasmid into an EGFP-CS1-pET28a expression vector ( figure 2 ).

[0072] PCR amplification primers for CS1

[0073] Forward: 5'-AAA CATATG GTGAGCAAGGGCGAGGAGC-3'

[0074] Reverse: 5'-AAA GGATCC TTAGCGCCGCCGCTTCTTGCGCTCTG...

Embodiment 2

[0108] Embodiment 2: Expression and purification of EGFP-HBD recombinant protein

[0109] Expression and purification of EGFP-CS1 (amino acid sequence described in SEQ ID NO.9), EGFP-CS2 (amino acid sequence described in SEQ ID NO.10), EGFP-CS3 (amino acid sequence described in SEQ ID NO.11) , EGFP-CS4 (amino acid sequence described in SEQ ID NO.8), EGFP-CS5 (amino acid sequence described in SEQ ID NO.12), EGFP-CS recombinant protein (amino acid sequence described in SEQ ID NO.13) , EGFP-TAT recombinant protein (amino acid sequence as described in SEQ ID NO.14).

[0110] Taking EGFP-CS4 (amino acid sequence as described in SEQ ID NO.8) as an example, transfer the plasmid EGFP-CS4-pET28a into BL21 (DE3), and culture it in 200ml medium at 37°C until OD 600 0.4-0.6, add IPTG (final concentration: 0.5mM), induce expression at 15°C for 12h, and ultrasonically break the cells. The ultrasound conditions are: 40w ultrasound for 4s, rest for 5s, ultrasound 70 times. The supernatant ...

Embodiment 3

[0113] Example 3: Research on the Effect of Penetrating the Membrane

[0114] 3.1 Membrane penetration of EGFP-HBD recombinant protein

[0115] A549 lung cancer cells or HeLa cells in good growth state were treated with 1×10 5 / ml inoculated into the cell culture plate (put a sterile cover glass in each well), 2ml per well. Then place the cell plate at 37°C, 5% CO 2 Culture in an incubator with saturated humidity to make the cells adhere to the wall well.

[0116] When the cell coverage rate reaches about 50%, discard the medium in the culture plate, rinse the adherent cells with PBS three times, then add 2ml of new medium, and add the corresponding EGFP-CS mutant fusion protein to each well to The same amount of EGFP recombinant protein was added as a negative control, and the sample well without drug was used as a blank control, and placed in an incubator at 37°C and 5% CO2 saturated humidity for 13 hours. The medium was then aspirated, rinsed three times with PBS, and t...

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Abstract

The invention relates to the technical field of biology and specifically relates to a novel cell-penetrating peptide, a gene, a recombinant vector, a transformant, an application thereof and a cell-penetrating drug delivery system. The invention discloses a novel cell-penetrating peptide which is characterized in that the novel cell-penetrating peptide is a polypeptide with the amino acid sequence as shown in SEQ ID NO. 1, or a polypeptide which contains the amino acid residue as shown in SEQ ID NO. 1 and has the cell-penetrating activity in addition to an EC-SOD carboxyl terminal protein transduction domain as shown in SEQ ID NO. 2. The invention firstly discloses the novel cell-penetrating peptide which can reduce the number of fused AA, reduce the molecular weight, avoid the defect that wild type HBD is easy to form a polymer and improve the cell-penetrating efficiency of the cell-penetrating peptide, wherein the cell-penetrating efficiency is 2 times higher than the wild type HBD. A new idea is provided for developing cell-penetrating drugs.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a novel cell-penetrating peptide, gene, recombinant vector, transformant and use thereof and a membrane-penetrating drug delivery system. Background technique [0002] Cell membranes consist of lipid bilayers with an interior of hydrophobic, nonpolar molecules. The cell membrane is a barrier for substances to enter and exit the cell. Many biological macromolecules such as polypeptides, proteins, and DNA cannot freely cross the cell membrane for material and information transfer. Although this barrier has a protective effect on the body, it also makes many promising drugs be abandoned. [0003] In recent years, foreign scholars have successively discovered a class of protein domains in the study of some virus transfection characteristics, such as: human immunodeficiency virus (human immunodeficiency virus, HIV-1) trans-transcriptional activator (Tram activating transcriptional activa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/06C07K7/08C07K19/00A61K47/48A61K45/00C12N15/11C12N15/62C12N15/63C12N1/21C12N1/19C12N5/10C12R1/19
Inventor 赵健汪雅雯王富军傅龙云张涛铸吕稼锋单含文
Owner ZHEJIANG REACHALL PHARMA
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