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Tumor cell targeting penetrating peptide

A technology of tumor cells and membrane-penetrating peptides, which is applied in the direction of anti-tumor drugs, peptides, and decapeptides, and can solve problems such as affecting biological functions, loss, and reduced binding activity

Inactive Publication Date: 2014-11-12
ZHEJIANG REACHALL PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Any deletion or large deletion mutation of these conservative amino acids will lead to the reduction or complete loss of the binding activity of these growth factors to their receptors, thereby affecting their biological functions

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0100] Example 1: Expression plasmid construction

[0101] 1. Construction of EH (recombinant protein with green fluorescent protein-HBD)

[0102] Synthesize primers with Nde I and BamH I restriction sites, carry out PCR amplification of the EGFP gene in the existing EGFP-pMD18T plasmid (Xiao Wei et al., Food and Drugs, 2010, 12(5):158), add Insert restriction sites Nde I and BamH I, insert the amplified fragment into HBD-pET28a, construct EH-pET28a plasmid, referred to as EH, and the expression product is: EGFP-HBD (see image 3 )

[0103] (1) Using EGFP-pMD18T as a template, according to the EGFP gene sequence, design the following primers for PCR amplification. The forward and reverse primers respectively introduce Nde I and BamH I restriction sites:

[0104] Primer 1: 5'-AAA CATATG GTGAGCAAGGGCGAGGAGCTG-3'

[0105] Primer 2: 5'-AAA GGATCC GAGTCCGGACTTGTACAGCTCGTCCATG-3'

[0106] (2) PCR amplification reaction conditions: The reaction conditions of PCR amplification...

Embodiment 2

[0130] Embodiment 2: Recombinant protein expression

[0131] 1. Expression and purification of EH recombinant protein

[0132] (1) Pick a single colony transformed with the EGFP-HBD-pET28a plasmid from the preserved solid LB medium plate and culture it in 3ml LB liquid medium containing kanamycin at 37°C until OD 600 About 0.5.

[0133] (2) Take the bacterial culture solution, insert 1% of the inoculum into a certain volume of medium containing kanamycin and continue to expand the culture until the OD 600 About 0.5-1.0.

[0134] (3) Adjust the culture temperature to 37°C, add an appropriate amount of IPTG to induce the target protein, continue to culture overnight, and collect the bacteria by centrifugation.

[0135] (4) The collected cells were resuspended with 20mM Tris-HCl buffer (pH8.5) and ultrasonically disrupted. Centrifuge to collect the supernatant containing the target protein.

[0136] (5) The collected supernatant was separated by affinity chromatography on a ...

Embodiment 3

[0154] Example 3: Research on the Effect of Penetrating the Membrane

[0155] Incubate ESH recombinant protein in various cells (including tumor cells and normal cells), conduct fluorescence microscope and laser confocal microscope observation and flow cytometry analysis, and use EH recombinant protein and ES recombinant protein as controls to conduct transfusion Membrane activity comparison

[0156] A. Fluorescence microscopy study comparison of transmembrane activity

[0157] (1) HeLa tumor cell line was used to investigate the incubation concentration and incubation time of ESH recombinant protein, and the final concentration of ESH recombinant protein was 40 μg / ml. The experimental results show that the transmembrane efficiency of the ESH recombinant protein is time-dependent. The experimental results are as follows: Figure 15 As shown, the intracellular fluorescence intensity increases as the incubation time prolongs, and the fluorescence in HeLa cells can be clearly o...

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PUM

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Abstract

The invention relates to the biotechnical field, and concretely relates to a tumor cell targeting penetrating peptide, a polynucleotide molecule and a use thereof, and a targeting drug delivery system containing the tumor cell targeting penetrating peptide. The tumor cell targeting penetrating peptide is characterized in that the tumor cell targeting penetrating peptide contains a CPPs structure domain having cell penetrating peptide activity and an EFG third ring sample mimic peptide structure domain with specific targeting affinity to tumor cells. The tumor cell targeting penetrating peptide has a substantial cell penetrating effect, has the novel characteristics of cell specificity and species specificity, and can be used to diagnose tumor diseases and develop medicines required in the treatment process.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a tumor cell-targeted penetrating peptide, a polynucleotide molecule, its use and a targeted drug delivery system containing it. 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-transcription activator (TAT), fruit Antler angle homeomorphic domain (AN...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/00A61K47/42A61K45/00A61K49/00A61P35/00
CPCC07K14/00A61K47/62C07K2319/00
Inventor 赵健王富军傅云龙张涛铸单含文任伟扬谢先文
Owner ZHEJIANG REACHALL PHARMA
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