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Production of cell-penetrating peptide hPP10 (human Pancreatic Polypeptide) and transfection method for mediated plasmid DNA (Deoxyribose Nucleic Acid) of hPP10

A technology of penetrating peptides and cells, applied in the field of biomedicine

Active Publication Date: 2014-07-30
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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  • Production of cell-penetrating peptide hPP10 (human Pancreatic Polypeptide) and transfection method for mediated plasmid DNA (Deoxyribose Nucleic Acid) of hPP10
  • Production of cell-penetrating peptide hPP10 (human Pancreatic Polypeptide) and transfection method for mediated plasmid DNA (Deoxyribose Nucleic Acid) of hPP10
  • Production of cell-penetrating peptide hPP10 (human Pancreatic Polypeptide) and transfection method for mediated plasmid DNA (Deoxyribose Nucleic Acid) of hPP10

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0094] Example 1, CPPs primary structure, secondary structure analysis, prediction and identification of new human-derived CPPs:

[0095] The secondary structure analysis adopts 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 peptides; http: / / emboss.bioinformatics.nl / cgi-bin / emboss / On-line analysis of secondary structure 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 with figure 2 shown.

[0096] First, artificially synthesize green fluorescently labeled hPP10:

[0097] FITC-

[0098] Lys-Ile-Pro-Leu-Pro-Arg-Phe-Lys-Leu-Lys-Cys-Ile-Phe-Cys-Lys-Lys-Arg-Arg-Lys-Arg (hPP10-FITC)

[0099] and hPP10 without green fluorescent labeling:

[0100] Lys-Ile-Pro-Leu-Pro-Arg-Phe-Lys-Leu-Lys-Cys-Ile-Phe-Cys-Lys-Lys-Arg-Arg-...

Embodiment 2

[0152] Example 2, hPP10-mediated plasmid DNA transfection

[0153] 1. The method for transfection of hPP10-mediated red luciferase expression plasmid pDsRed (purchased from Clontech), comprising the following steps:

[0154] (1) Culture cells ECV-304 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;

[0155] (2) Set at 37°C 5% CO 2 In the incubator, cultivate for 24 hours. 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;

[0156] (3) Meanwhile prepare transfection samples:

[0157] 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;

[0158] b. Take 0.8 μg of the plasmid and add it to another centrifuge tube con...

Embodiment 3

[0177] Example 3, Expression and purification of hPP10-GFP fusion protein and research on its transmembrane efficacy

[0178] The genetic engineering expression method adopts the prokaryotic expression method, comprising the following steps:

[0179] (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;

[0180] (2) Carry out double digestion with two restriction endo...

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Abstract

The invention relates to the field of biomedicine, in particular to production of a novel human cell-penetrating peptide hPP10 (human Pancreatic Polypeptide) and a transfection method for a mediated plasmid DNA (Deoxyribose Nucleic Acid) of the hPP10. The hPP10 is derived from human cell nucleoproteins; and one segment of polypeptide sequence of a lysine specific demethylated enzyme 4A has a cell penetrating function, can be used for carrying proteins, allows nucleic acids (DNA plasmids) to enter a plurality of types of cells in a transmembrane manner, and is a transmembrane transport carrier with an extremely good development prospect for bioactive molecules such as proteins, nucleic acids and the like.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to the production of a novel human-derived cell-penetrating peptide hPP10 and a method for mediating plasmid DNA transfection. 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 vivo, and molecules that need to function in cells are difficult 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....

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/08C07K1/06C07K1/04C12N15/12C12N15/70C12N15/63
CPCY02P20/55
Inventor 柳长柏马节兰蔡三金吴江锋张洁贺晓敏杨英桂
Owner 深圳真实生物医药科技有限公司
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