CN104262466B - Cell-penetrating peptides transfection reagent and its application - Google Patents
Cell-penetrating peptides transfection reagent and its application Download PDFInfo
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- CN104262466B CN104262466B CN201410275600.9A CN201410275600A CN104262466B CN 104262466 B CN104262466 B CN 104262466B CN 201410275600 A CN201410275600 A CN 201410275600A CN 104262466 B CN104262466 B CN 104262466B
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- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
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Abstract
The invention discloses a kind of cell-penetrating peptides transfection reagent and its application, peptide chain that described cell-penetrating peptides transfection reagent is made up of 23 amino acid, its amino acid sequence is SEQ ID NO.1.The cell-penetrating peptides transfection reagent of the present invention not only possesses the characteristics of simple in construction, high transfection efficiency and small cytotoxicity, and need not carry out culture medium replacement operation during transfection, is especially suitable for various high-flux cell screening experiments, convenient, quickly, efficiently.These features of the reagent creatively solve and efficiently convey the problem that various nucleic acid molecules arrive at.
Description
Technical field
The invention belongs to technical field of molecular biology, in particular to a kind of cell-penetrating peptides transfection reagent and its
Using.
Background technology
Cell membrane is the barrier for preventing ECM to be freely accessible to cell, and it ensure that the relatively steady of intracellular environment
It is fixed.Also exactly because the biological barrier effect of cell membrane prevents many large biological molecule materials to enter intracellular, so as to very big
Limit to degree application of these materials in therapy field.Therefore, how to guide these material penetration cell films to be one to compel
It is essential the problem of to be solved.
The method of mediation large biological molecule penetration cell film mainly includes cell-penetrating peptides (cell penetrating
Peptides, CPPs), liposome (liposome), adenovirus (adenovirus), nano particle (nanoparticle) etc..
Wherein, cell-penetrating peptides are a kind of with non-receptor-independent, polypeptide of the non-classical endocytosis mode directly through cell membrane into cell,
It is rich in basic amino acid, and amino acid sequence is generally positively charged.One important feature of cell-penetrating peptides be can carry it is more
The bioactive substance of kind of different size and property enters cell, including micromolecular compound, dyestuff, nucleic acid, DNA,
SiRNA, polypeptide, protein etc..Cell-penetrating peptides are the limitation of hypotoxicity and acellular type as the advantage of carrier, this
Property turns into the candidate vectors of targeted drug for it and provides possibility.Its practical application focuses mostly in various macromolecular substances, bag
Include the cell traffic of siRNA and DNA.
The cell-penetrating peptides being widely used at present include tat peptide (HIV-1 TAT protein functional areas), cell-penetrating peptide
(transportan), oligomerization arginine [poly (arginine) 9, R9], MPG (fusion HIV-1 gp41 protein function area and
The nuclear localization sequence of SV40T antigens) etc., although what cell-penetrating peptides in itself can be quickly and efficiently enters cell, its reality
Subject matter in be how optimization design cell-penetrating peptides, siRNA or DNA can be effectively combined, it is high
Effect enters cell, quickly breaks away from degraded caused by endosome or lysosome, siRNA or DNA is delivered to corresponding thin
The site of action of intracellular.However, existing cell-penetrating peptides transfection reagent transfection efficiency is not high, therefore how to obtain efficient thin
Born of the same parents' penetrating peptide transfection reagent is those skilled in the art's technical problem urgently to be resolved hurrily.
The content of the invention
In order to overcome prior art spy's defect, the present invention provides a kind of new cell-penetrating peptides transfection reagent and its should
With.In order to realize the purpose of the present invention, intend adopting the following technical scheme that：
The present invention relates to a kind of cell-penetrating peptides transfection reagent, it is characterised in that described cell-penetrating peptides transfection reagent is
The peptide chain being made up of 23 amino acid, its amino acid sequence are SEQ ID NO.1.
In a preferred embodiment of the present invention, described cell-penetrating peptides transfection reagent is to pass through Solid phase peptide synthesis
What method obtained.
Another aspect of the present invention further relates to the application in described cell-penetrating peptides transfection reagent nucleic acid molecules transfection.
In a preferred embodiment of the present invention, described transfection need not carry out culture medium in transfection and change behaviour
Make.
The cell-penetrating peptides transfection reagent of the present invention not only possesses simple in construction, high transfection efficiency and the small spy of cytotoxicity
Point, and culture medium replacement operation need not be carried out during transfection, it is especially suitable for various high-flux cell screening experiments, it is convenient, quickly,
Efficiently.These features of the reagent creatively solve and efficiently convey the problem that various nucleic acid molecules arrive at.
Brief description of the drawings
Fig. 1：It is in green fluorescence that the cell-penetrating peptides of FITC fluorescent labellings, which are distributed widely in cytoplasm,.(B, 20 μ gFAM-
The cell-penetrating peptides of mark are added in A549 cells after being mixed with 180 μ L cell culture mediums；A. it is same amount of as control
PBS solution is added to after being mixed with PBS in A549 cells, and blueness is presented after being dyed by DAPI in nucleus.)
Fig. 2：The siRNA of the peptide-mediated DY547 fluorescent labellings of cell-penetrating is distributed widely in cytoplasm the fluorescence that takes on a red color.
B is to be added to after cell-penetrating peptides mix with 180 μ L cell culture mediums in A549 cells.As control, same amount of PBS solution
It is added to after being mixed with PBS in A549 cells, redfree fluorescence shows in cytoplasm.Blueness is presented after being dyed by DAPI in nucleus
(A)。
Embodiment
The present invention is further described with reference to the accompanying drawings and examples.
Embodiment 1：
Peptide systhesis
After Peptide systhesis, efficient liquid phase is utilized in Jin Site Science and Technology Co., Ltd. of the U.S. (Piscataway, NJ)
Chromatography is purified (purity ＞ 95%).Prepare FAM (0.1mM), diisopropylethylamine (0.5mM) and dicarbamylamine
(DMF) mixed liquor, the polypeptide (0.1mM) that resin is fixed is incubated 12h in mixed liquor so that fluorescein group (FAM) passes through
Amion acetic acid marks the N-terminal in polypeptide.Mixtures of polypeptides is divided using reversed phase-high performance liquid chromatography in chromatographic column C18
Analysis is analyzed using electrospray ionization mass spectrometry (ESI-MS) after purification, by the polypeptide of purifying.
SiRNAs is synthesized
The siRNA of the DY547 marks of anti-PPIB albumen is synthesized by matching silent your science and technology of winged generation.The control siRNA of DY547 marks
Buy from the scientific and technological Dharmacon of the silent winged generation that of match.
Transhipment (Fig. 1) of the cell-penetrating peptides in A549 cells
The 20 μ l FAM cell-penetrating peptides marked are added in 180 μ l culture medium (DMEM), then added mixed liquor
Enter into A549 cells.As a control group, cell-penetrating peptides are replaced using the PBS of equivalent.At 37 DEG C, by A549 cell culture
24h, observed using fluorescence microscopy.
Transported in mouse NRK52 cells by the peptide-mediated siRNA of cell-penetrating
Under normal temperature, by the anti-PPIB of 100nM siRNA and control siRNA/ polypeptides respectively in mixed liquor [DMEM+2mM paddy ammonia
The nonessential amino acid of acid amides+1% (NEAA)+5-10% hyclones (FBS), Life Technologies] in be incubated jointly
15min.Meanwhile it will be grown on the coated octal chamber slides of collagen Ι (BD Biosciences, San Jose, CA)
The NRK52E cells for reaching~60% to Fusion Strain are cleaned 3 times with culture medium.Then, by cell and siRNA or polypeptide/
SiRNA mixed liquors are incubated 24h jointly, finally utilize fluorescence microscopy.
Fluorescence microscopy
At normal temperatures, the coated octal chamber slides of collagen Ι (BD Biosciences, San will be fixed on
Jose, CA) on cell utilize containing 3% paraformaldehyde PBS in clean 10min, drop is then had into anti-fluorescence decay
Agent and 4 ', 6-diamidino-2-phenylindole (DAPI, VECTOR Laboratories, Burlingame, CA) lid
Slide is covered on slide.Using Zeiss (Thornwood, NY) Axio Observer D1 microscopes carry out fluorescence signal and
IMAQ.The cell after fixation is taken pictures using inverted microscope LDA-Plan × 20/0.3Ph1 objective, transfection effect
Rate is more than 90%.
Described above is the preferred embodiments of the present invention, it is noted that is come for those skilled in the art
Say, on the premise of principle of the present invention is not departed from, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
Bibliography：
1.Vidal,P.,Chaloin,L.,Mry,J.,Lamb,N.,Lautredou,N.,Bennes,R.andHeitz,
F.(1996)J.Pep.Sci.,2,125–133.
2.Mery,J.,Granier,C.,Juin,M.and Brugidou,J.(1993)
.Int.J.PeptideProtein Res.,42,44–52.
3.Juliano R,Alam MR,Dixit V,KangH(2008)Mechanisms and strategies for
effectivedelivery of antisense and siRNA oligonucleotides.Nucleic Acids Res
36:4158–4171
4.Akhtar S,Benter IF(2007)Nonviral delivery of synthetic siRNAs in
vivo.J Clin Invest117:3623–3632
5.Lv H,Zhang S,Wang B,Cui S,Yan J(2006)Toxicity of cationic lipids
and cationic polymers in gene delivery.J Control Release 114:100–109
6.Meade BR,Dowdy SF(2008)Enhancing the cellular uptake of siRNA
duplexes following noncovalent packaging with protein transduction domain
peptides.Adv Drug Deliv Rev 60:530–536
7.Futaki S,Suzuki T,Ohashi W et al(2001)Arginine-rich peptides.An
abundant source of membrane-permeable peptides having potential as carriers
for intracellular protein delivery.J Biol Chem 276:5836–5840 。
Claims (4)
1. a kind of cell-penetrating peptides transfection reagent, it is characterised in that described cell-penetrating peptides transfection reagent is by 23 amino acid
The peptide chain of composition, its amino acid sequence are SEQ ID NO.1.
2. cell-penetrating peptides transfection reagent according to claim 1, described cell-penetrating peptides transfection reagent is by solid
What phase method of peptide synthesis obtained.
3. application of the cell-penetrating peptides transfection reagent in nucleic acid molecules transfection described in claim 1 or 2.
4. applying according to claim 3, described transfection need not carry out culture medium replacement operation in transfection.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2011157715A1 (en) * | 2010-06-14 | 2011-12-22 | F. Hoffmann-La Roche Ag | Cell-penetrating peptides and uses therof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011157715A1 (en) * | 2010-06-14 | 2011-12-22 | F. Hoffmann-La Roche Ag | Cell-penetrating peptides and uses therof |
| CN103096932A (en) * | 2010-06-14 | 2013-05-08 | 弗·哈夫曼-拉罗切有限公司 | Cell-penetrating peptides and uses therof |
Non-Patent Citations (1)
| Title |
|---|
| 细胞穿透肽的研究进展;李凤英，等;《中国新药杂志》;20131231;第22卷(第15期);第1761-1767页 * |
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