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Cell-penetrating peptide hPP8 and application thereof

A cell membrane, penetrating peptide technology, applied in the field of biomedicine, to achieve the effects of no toxic side effects, broad application prospects, and few unsafe factors

Active Publication Date: 2015-12-23
深圳真实生物医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

It has also been reported that application of TAT drug-carrying upper airway nebulizers triggered severe lung pathology

Method used

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  • Cell-penetrating peptide hPP8 and application thereof
  • Cell-penetrating peptide hPP8 and application thereof
  • Cell-penetrating peptide hPP8 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1, CPP primary structure, secondary structure analysis, prediction and identification of new human-derived CPP:

[0037] 1. The secondary structure of the cell membrane-penetrating peptide hPP8 obtained in the present invention was analyzed by using the online analysis program of emboss (for the analysis program, please refer to the web page: http: / / emboss.bioinformatics.nl / cgi-bin / emboss / pepwheel Online analysis of the round structure of polypeptides; http: / / emboss.bioinformatics.nl / cgi-bin / emboss / help / garnier online analysis of secondary structure helices, folds, etc.). The schematic diagram of the wheel structure of hPP8, the schematic diagrams of the helical and folded structures are as follows image 3 and Figure 4 shown.

[0038] In order to facilitate the study of the cell-penetrating function of hPP8:

[0039] Chemically synthesized green fluorescently labeled hPP8:

[0040] Arg-Arg-Arg-Arg-Lys-Arg-Ser-Leu-Val-Met-His-Lys-Arg-Arg-Arg-Arg-FITC (hPP8-...

Embodiment 2

[0096] Example 2, hPP8 has little effect on cell viability

[0097] (1) Culture cells ECV-304 and HepG2 in the logarithmic growth phase, with 1×10 4 The seeding density of each cell / well was inoculated in a 96-well plate for routine culture, 100 μl per well, and 3 replicate wells were set up for each cell, and cultured at 37°C;

[0098] (2) In the logarithmic growth phase, the culture medium was replaced with serum-free RPMI-1640 culture medium, and the cultivation was continued for 1 h;

[0099] (3) Replace with serum-free RPMI-1640 medium with hPP8 concentration gradients of 10 μM, 20 μM, 30 μM, 40 μM and 50 μM, respectively, and continue culturing for 24 hours;

[0100] (4) After the incubation time is over, add 100 μl of PBS to each well to wash, 2 min×3 times;

[0101] (5) Add 80 μl of serum-containing normal culture solution and 20 μl of MTT (mother solution concentration 5 mg / ml, ie 0.5% MTT) solution to each well, continue culturing at 37° C. for 4 h, and aspirate th...

Embodiment 3

[0105] Example 3. Construction of pET15b-hPP8-GFP plasmid, expression and purification of fusion protein and research on its transmembrane efficacy

[0106] 3.1 Construction and identification of pET15b-hPP8-GFP recombinant plasmid

[0107] (1) Design two single-stranded cDNAs encoding hPP8, with XhoI and NdeI restriction sites on both sides, and hand them over 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, cool to room temperature naturally at 95°C for 5 minutes, and anneal to complete to form a complementary double-stranded DNA fragment (hPP8); at the same time, design a pair of primers and use pEGFP (purchased from Clontech) as a template to obtain GFP protein by PCR The gene fragment has XhoI and BamHI restriction sites on both sides, and the purified PCR product is ready for use;

[0108] (2) Carry out double digestion with two restriction endo...

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Abstract

The invention belongs to the field if biological medical science, and relates to a cell-penetrating peptide hPP8 and application thereof. The provided cell-penetrating peptide hPP8 possesses function of penetrating cell membrane, and is capable of entering into multiple cells in a transmembrane manner while carrying protein and other macro-molecule. Because the peptide comes from human protein, the possibility that the peptide causes human immunity response is small, potential unsafe factors are relatively less, and thus hPP8 is a transmembrane transportation carrier which possesses exploitation prospect and is applicable to protein, polypeptide and other biological active molecules, and is applicable as an intracellular medicine transportation carrier.

Description

technical field [0001] The present invention relates to the field of biomedicine, in particular to a cell membrane penetrating peptide and its application. Background technique [0002] With the completion of the Human Genome Project and the rise of proteomics, more and more biomolecules such as proteins, polypeptides, and nucleic acids are found to be likely to become therapeutic drugs. However, unlike traditional drugs, these therapeutic molecules have low stability in the body, and it is difficult for molecules that need to function in cells to enter cells, which limits their promotion and application as drugs. Therefore, it is an urgent problem to develop an economical and safe carrier system that can effectively carry these therapeutic biomacromolecules into target cells. [0003] In recent years, non-viral drug delivery vehicles have been placed with high hopes due to their safety, low toxicity, and low immune response. So far, the most commonly used methods for intr...

Claims

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Application Information

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IPC IPC(8): C07K14/47C07K19/00C12N15/12C12N15/62A61K47/48A61K38/00A61K8/64A61K49/00A61P35/00C12N15/87
CPCA61K8/64A61K47/642A61K49/0056C07K14/4702C07K2319/10C12N15/87
Inventor 柳长柏王琥邹黎黎马节兰杨英桂
Owner 深圳真实生物医药科技有限公司
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