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Genetically recombinant efficient cell penetrating peptide RTP as well as preparation method and application thereof

A gene recombination and membrane-penetrating peptide technology, applied in the field of genetic engineering, can solve the problems of few hydrophobic cell membrane-penetrating peptides, unclear specific mechanisms, and controversial mechanism hypotheses, and achieve good biological safety, good industrial application value and Prospect of industrialization, wide range of application effects

Active Publication Date: 2021-03-09
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Hydrophobic cell-penetrating peptides mainly rely on their hydrophobic groups to exert their membrane-penetrating effect, but there are relatively few reports on hydrophobic cell-penetrating peptides, and the specific mechanism is still unclear
There have been relatively many research advances on the mechanism of cell-penetrating peptides, but some mechanism hypotheses are still controversial

Method used

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  • Genetically recombinant efficient cell penetrating peptide RTP as well as preparation method and application thereof
  • Genetically recombinant efficient cell penetrating peptide RTP as well as preparation method and application thereof
  • Genetically recombinant efficient cell penetrating peptide RTP as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Construction and Expression of Expression Engineering Bacteria pET32a-RTP / BL21(DE3)

[0059] Specific steps are as follows:

[0060] (1) Utilize PCR technology to synthesize RTP gene:

[0061] The RTP gene was synthesized by PCR reaction with a pair of complementary primers:

[0062] Primer design:

[0063] Primer 1:

[0064]

[0065] Primer 2:

[0066]

[0067] GGTACC is the KpnI restriction site, CTCGAG It is the XhoI restriction site.

[0068] The amount of each component of annealing: (primer concentration is 1OD dissolved in 400μL ddH 2 O, reaction system: 20μL)

[0069] Primer 1 2μL, Primer 2 2μL, T4 PNK 1μL (10U), ATP 20mM, ddH 2 O replenish water to 20 μL;

[0070] PCR reaction program: 95°C, 5min; 95°C, 1min; 56°C, 1min; 72°C, 1min; 30 cycles; 72°C, 10min;

[0071] Naturally cooled to room temperature, the PCR product was set aside.

[0072] (2) Construction of recombinant vector pET32a-RTP:

[0073] Use KpnI enzyme and XhoI enzyme to carry...

Embodiment 2

[0079] Purification, Preparation and Identification of Recombinant Human Cell Penetrating Peptide RTP

[0080] Pipette and mix 2mL of medium (Ni-NTA column, GenScript Biotechnology Co., Ltd.) into the column (10mL column volume), add 4 times the column volume of equilibration buffer to balance the chromatography medium; The supernatant was loaded onto the column at a flow rate of 1.0mL / min; with 8 times the column volume of washing buffer (50mM Na 2 HPO 4 , 0.3M NaCl, 10~50mM imidazole, pH=8.0) to pass through the column at 1.0mL / min to wash away impurity proteins or unaffinity-bound fusion proteins, and use 10 times the column volume of elution buffer to Min passes through the column, collects the eluate, takes the sample effluent, washes the effluent, and elutes the effluent and carries out SDS-PAGE electrophoresis to detect the purification effect of the fusion protein Trx-6His-RTP (the results are as follows: figure 2 Shown), the experimental results show that in the 25...

Embodiment 3

[0089] Fluorescent Intensity Detection of Cell Penetrating Activity of Recombinant Human Cell Penetrating Peptide RTP

[0090] Culture HEK-293A cells (purchased from Kunming Cell Bank, Chinese Academy of Sciences), adjusted to 5×10 4 / mL, seeded in 12-well plate, 1 mL per well, cultured in the cell incubator until the cells grew to 80%-90% confluence, discarded the old medium, added serum-free cell culture medium, and added to the 12-well plate respectively Gene recombined human cell penetrating peptide RTP and the culture medium in which the plasmid pEGFP complex was added at an N / P ratio of 40, the concentration of RTP was 25, 50, 75, 100 μM, and the penetrating peptide TAT 47-57 (its amino acid sequence: YGRKKRRQRRR ) as a control group were treated under the same conditions as above. After incubation for 4 hours, the fluorescence quantitative results of each group were obtained by microplate reader detection, see Figure 4 . The experimental results show that the fluores...

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Abstract

The invention discloses a genetically recombinant efficient cell penetrating peptide RTP as well as a preparation method and application thereof, and belongs to the technical field of genetic engineering. The cell penetrating peptide RTP is genetically recombined, and is small in molecular weight, easy to prepare, higher in cell penetration efficiency, lower in cytotoxicity, high in biological activity and easier to penetrate through skin epithelial tissues compared with other skin penetration enhancers, thereby making it easier for carried drugs and the like to be absorbed and utilized in a transdermal manner; and moreover, the cell penetrating peptide RTP is an efficient penetration enhancer in the aspect of transdermal transport of biomacromolecules and hydrophilic drugs, and has bettersafety without obvious toxic or side effects, so that the cell penetrating peptide RTP can be used in the fields of cosmetology, cosmetics and the like. Thus, the cell penetrating peptide RTP has a wide application range, as well as good industrial application values and industrial prospects. The preparation method of the cell penetrating peptide RTP is safe, simple, efficient, high in yield andlow in production cost; and the produced small peptide is high in stability and wide in application prospects.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, in particular to a gene recombined high-efficiency cell-penetrating peptide RTP and its preparation method and application. Background technique [0002] Cell-penetrating peptides, as the name implies, are a class of polypeptides that can penetrate cell membranes or tissue barriers. The first reported cell-penetrating peptide was the human immunodeficiency virus (HIV) trans-transcription activator (TAT) discovered in 1988, which can penetrate the cell membrane and enter the cell. In the following decades, various research teams discovered that many different types of polypeptides have the ability to cross cell membranes, and such polypeptides are named cell-penetrating peptides. A cell-penetrating peptide is defined as a polypeptide molecule composed of several to forty amino acids, which has the ability to independently pass through the cell membrane without relying on cell membrane...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/06C12N15/11C12N15/62C12N15/70C12N1/21C07K1/22C07K1/16C07K1/14A61K8/64A61Q19/00C12R1/19
CPCC07K7/06C12N15/70A61K8/64A61Q19/00C07K2319/10
Inventor 马义
Owner JINAN UNIVERSITY
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