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A kind of cell-penetrating peptide derived from cap protein of duck circovirus and its design method and application

A membrane-penetrating peptide and cell technology, applied in the field of cell-penetrating peptides, can solve the problems of weakening immune stimulation effect and prolonging residence time

Active Publication Date: 2022-05-17
TAIZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since the two carry the same charge, Poly(I:C) and CpG ODN cannot actively and efficiently enter the target cells, and can only rely on endocytosis to stimulate the generation of cascade reactions. This process is due to Poly(I:C) C) and CpG ODN have prolonged extracellular residence time and are easily decomposed by nucleases, which will inevitably weaken their immunostimulatory effect

Method used

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  • A kind of cell-penetrating peptide derived from cap protein of duck circovirus and its design method and application
  • A kind of cell-penetrating peptide derived from cap protein of duck circovirus and its design method and application
  • A kind of cell-penetrating peptide derived from cap protein of duck circovirus and its design method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Bioinformatics analysis and synthesis of candidate cell-penetrating peptides of duck circovirus Capsid aa 18-37

[0038] Use the online server Multalin (http: / / www.sacs.ucsf.edu / cgi-bin / multalin.py) to compare and analyze the Capsid of different duck circovirus reference strains in GenBank, such as figure 1 Its N-terminal sequence (aa18-37) is shown to be highly conserved and rich in positively charged arginine (R), marked with a black border.

[0039] Use the online server PEP-FOLD 3 ( https: / / bioserv.rpbs.univ -paris-diderot.

[0040] fr / services / PEP-FOLD3 / ) to simulate the three-dimensional structure of its N-terminal sequence (aa 18-37), such as figure 1 It was found that it has a similar α-helical structure characteristic to the commonly used cell-penetrating peptide TAT.

[0041] specific, figure 1 Middle: A: N-terminal sequence comparison of Capsid of different duck circovirus reference strains; Consensus: Consensus of different sequence alignments...

Embodiment 2

[0045] Example 2 Laser Confocal Microscopy Detection of Du-Cap aa18-37's Cell Transmembrane Function and Characteristics

[0046] (1) Place sterile cell slides in a 12-well cell culture plate, and place 5×10 5 Hela cells, HD11 cells and 3D4 / 21 cells were seeded into cell culture plates.

[0047] (2) After overnight culture, take the synthetic short peptide (Du-Cap aa 18-37) and dilute it to 5 μM, mix it with 500 μL serum-free medium and add it to the cell wells. Use TAT as a positive control and FITC dye as a negative control . Place the cell mixture at 37°C, 5% CO 2 Incubate in an incubator for 20 min. After incubation, wash with 1mL of PBS each time, a total of 3 times, to remove short peptides that did not enter the cells.

[0048] (3) Stain with Hoechst 33342, a nuclear marker dye, for 5 minutes, and wash with PBS.

[0049] (4) The cell slides were taken out, placed upside down on a glass slide, sealed with transparent nail polish, and scanned and photographed under d...

Embodiment 3

[0051] Example 3 Flow cytometry detection of different concentrations of Du-Cap aa18-37 and TAT transmembrane efficiency comparison

[0052] (1) Divide 5×10 5 Hela cells were seeded into 12-well plates and washed once with PBS after overnight culture.

[0053] (2) Short peptides of different concentrations (0.1 μM / L, 1 μM / L and 10 μM / L) and 500 μL Opti-MEM medium were added to the cells, and no short peptide was used as a negative control, 37 ° C, 5% CO 2 , under the condition of incubation for 20min.

[0054] (3) Wash with PBS 3 times, 1 min each time. Add 200 μL trypsin and 100 μL PBS and mix well to digest the cells for 3 minutes. Add fresh cell culture medium containing serum to terminate the digestion, collect the cell suspension in a 1.5mL centrifuge tube, and centrifuge at 400×g for 2min.

[0055] (4) Discard the supernatant. Add 1mL PBS, wash once; repeat wash once; add 400μL PBS to resuspend the cells, mix thoroughly, filter each tube of cells into the flow tube...

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Abstract

The present application discloses a cell-penetrating peptide derived from Cap protein of duck circovirus and its design method and application. The amino acid sequence of the cell-penetrating peptide is: LRRRRRRRRLRIARPRRRF; the cell-penetrating peptide of this application is derived from the Capsid structural protein of duck circovirus, which can significantly improve the transmembrane efficiency of transport targets such as nucleic acid immunopotentiators. It mediates Poly(I:C) and CpG ODN into macrophages to induce interferon IFN-β transcription levels to increase nearly 4 times that of TAT, and 8 times that of immunostimulators alone. The cell-penetrating peptide Du-Cap aa18-37 of the present application is safe and efficient, can significantly improve the transmembrane efficiency, further enrich the types of cell-penetrating peptides, and can be developed into a safe and efficient gene drug and nucleic acid immunity Ideal delivery vehicle for stimulants.

Description

technical field [0001] The invention relates to a cell-penetrating peptide, in particular to a cell-penetrating peptide derived from duck circovirus Cap protein and its application. Background technique [0002] Immunopotentiators (immune adjuvants) are a class of immunoregulatory factors, compounds or macromolecular complexes that can improve the immunogenicity of antigens. Immune adjuvants assist the antigen response by stimulating the body's innate immune system. The innate immune system recognizes pathogen-associated molecular patterns (PAMPs) expressed by pathogenic microorganisms through pattern recognition receptors (PRR) in the body. Toll-like receptor (TLR) is an important receptor in pattern recognition receptors. There are currently 11 types discovered, such as TLR3 that mainly recognizes Poly(I:C), TLR9 that recognizes CpG ODN, and bacterial LPS TLR4. Poly(I:C) is a double-stranded RNA analogue of artificially synthesized polyinosine nucleotides and polycytosi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/01A61K39/39A61K48/00A61K47/42A61P31/04A61P31/12
CPCC07K14/005A61K39/39A61K48/0025A61K47/42A61P31/12A61P31/04C12N2750/10022A61K2039/55561Y02A50/30
Inventor 呼高伟付永前马哲潘苑霞
Owner TAIZHOU UNIV
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