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Cell penetrating peptide hPP10 and use thereof

A technology of penetrating peptides and cells, which is applied in the field of biomedicine to achieve the effects of little possibility, few unsafe factors, and broad application prospects

Active Publication Date: 2012-12-19
深圳真实生物医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patented technology makes use of certain types of protein called hormone precursors (Hyp) which can be found naturally throughout our bodies or produced by genetic engineering techniques. These Hydroxyproline containing peptides have been shown effective at delivering therapeutic agents into cells without causing harmful side reactions like those caused by chemotherapy drugs used against cancer patients.

Problems solved by technology

Technologies aimed towards developing novel types of materials able to safely and efficiently introduce small substances like medicines inside living bodies without causing harmful health risks during use. Existing technologies involve encapsulating them within carriers but they lack specificity and potentially pose challenges when administering them outside the body. To address these technical requirements, there was developed a synthetic version of hollow particle carrying genetic material(NP)-related metabolites (CNMPs) containing negatively charged groups.

Method used

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  • Cell penetrating peptide hPP10 and use thereof
  • Cell penetrating peptide hPP10 and use thereof
  • Cell penetrating peptide hPP10 and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1, CPP primary structure, secondary structure analysis, prediction and identification of new human-derived CPP:

[0052] The secondary structure analysis uses the online analysis program of emboss (for the analysis program, please refer to the webpage: http: / / emboss.bioinformatics.nl / cgi-bin / emboss / pepwheel online analysis of the wheel structure of peptides; http: / / emboss. bioinformatics.nl / cgi-bin / emboss / online analysis of secondary structures (helices, folds, etc.). The schematic diagram of the wheel structure of hPP10, the schematic diagrams of the helical and folded structures are as follows figure 1 and figure 2 shown.

[0053] Chemical synthesis of green fluorescent labeled hPP10:

[0054] FITC-

[0055] Lys-Ile-Pro-Leu-Pro-Arg-Phe-Lys-Leu-Lys-Cys-Ile-Phe-Cys-Lys-Lys-Arg-Arg-Lys-Arg (hPP10-FITC)

[0056] and hPP10 without green fluorescent labeling:

[0057] Lys-Ile-Pro-Leu-Pro-Arg-Phe-Lys-Leu-Lys-Cys-Ile-Phe-Cys-Lys-Lys-Arg-Arg-Lys-Arg(hPP10)

...

Embodiment 2

[0121] Example 2, hPP10-mediated plasmid DNA transfection

[0122] The method of hPP10-mediated plasmid DNA transfection includes the following steps: (1) culture ECV-304 cells in the logarithmic growth phase, and use 1×10 5 The seeding density of cells / well was inoculated in a 12-well plate for routine culture, and corresponding positive and negative wells were set at the same time;

[0123] (2) Set at 37°C 5% CO 2 In the incubator, cultivate for 20~24h, and when the cell density reaches 70%-80%, replace the culture medium with serum-free RPMI-1640 culture medium, and continue to cultivate for 1 hour;

[0124] (3) Meanwhile prepare transfection samples:

[0125] a. Take a final concentration of 10 μM hPP10 and add it to a centrifuge tube containing 50 μl Opti-MEM, mix gently and incubate at room temperature for 5 minutes; b. Take 0.8 μg of the plasmid and add it to another centrifuge tube containing 50 μl Opti-MEM, Mix gently and incubate at room temperature for 5 min. Sl...

Embodiment 3

[0147] Example 3. Construction of pET15b-hPP10-GFP plasmid, expression and purification of fusion protein and research on its transmembrane efficacy

[0148] 3.1 Construction and identification of pET15b-hPP10-GFP recombinant plasmid

[0149] (1) Design two single-stranded cDNAs encoding hPP10, with NdeI and XhoI restriction sites on both sides, and submit them to Shanghai Shenggong Company to synthesize single-stranded oligonucleotide chains, and then mix the two single-stranded DNAs in equal amounts Add it into an aqueous solution, and cool it down to room temperature naturally for 5 minutes at 95°C to complete annealing to form a complementary double-stranded DNA fragment (hPP10); meanwhile, design a pair of primers and use pEGFP (purchased from Clontech) as a template to obtain GFP by PCR Protein gene fragment, with XhoI and BamHI restriction sites on both sides, purified PCR product for future use;

[0150] (2) Carry out double digestion with two restriction endonuclease...

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Abstract

The invention relates to the field of biomedicine, and in particular relates to novel human cell penetrating peptide hPP10 and a use of using the human cell penetrating peptide as an intracellular drug delivery carrier. The hPP10 deviates from a section of polypeptide sequence of lysine-specific demethylase 4A of human cell nuclear proteins, has a function of penetrating a cell membrane and can carry the proteins and nucleic acid (deoxyribonucleic acid DNA plasmid) to enter a plurality of cells in a transmembrane mode; and therefore, the cell penetrating peptide hPP10 is a transmembrane delivery carrier of bioactive molecules such as protein and nucleic acid which has good development prospect.

Description

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Claims

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Application Information

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Owner 深圳真实生物医药科技有限公司
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