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

[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...

[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...

[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...