CN106995487A - The polypeptides of CAV VP1 aa 23 43 are come from as the application of high efficiency cell cell-penetrating peptide - Google Patents
The polypeptides of CAV VP1 aa 23 43 are come from as the application of high efficiency cell cell-penetrating peptide Download PDFInfo
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- CN106995487A CN106995487A CN201710248386.1A CN201710248386A CN106995487A CN 106995487 A CN106995487 A CN 106995487A CN 201710248386 A CN201710248386 A CN 201710248386A CN 106995487 A CN106995487 A CN 106995487A
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Abstract
The polypeptides of CAV (Chicken anemia virus, CAV) VP1 aa 23 43 are come from as the application of high efficiency cell cell-penetrating peptide the present invention relates to one kind.The peptide sequences of VP1 aa 23 43 are as shown in SEQ ID NO.2.The polypeptide can carry FITC and enter 293T cells, the cells of HCT 116,3T3 cells, mdck cell, MSB1 cells in 30min, and wear membrane efficiency and being proportionate property of cell-penetrating peptide concentration, key features its can efficiently enter suspended culture cell.Shown with reference to laser confocal microscope and flow cytometry results, the polypeptides of CAV VP1 aa 23 43 at 10 μM with than TAT small peptide to wear membrane efficiency high more than 2 times.
Description
Technical field
CAV (Chicken anemia virus, CAV) VP1-aa is come from the present invention relates to one kind
The high efficiency cell cell-penetrating peptide of 23-43 polypeptides.The polypeptide can carry FITC, and that different cell is penetrated in 30min is (including adherent thin
Born of the same parents and suspension cell).With TAT's it was found that, the polypeptide is higher than TAT to FITC cell-penetrating function more than 2 times.Therefore,
A kind of high efficiency cell cell-penetrating peptide for coming from CAV VP1-aa 23-43 polypeptides that the present invention is obtained has certain
Application prospect and value.
Background technology
Cell-penetrating peptide (Cell penetrating peptides, CPPs) is that a class can carry large biological molecule entrance
The small peptide of cell, length is generally 5-30 and is rich in positively charged basic amino acid.It not only itself can efficiently be passed through
Cell membrane enters cell, can also by different types of exogenous molecules such as fluorescein, DNA, RNA, protein, quantum dot efficiently
It is carried along into the cell of different plant species.Because this new small peptide not only possesses, conduction efficiency is high, bio-toxicity is low, avoid exciting
Vivo immunization reaction and theca cell target is worn extensively and the characteristics of application is flexible, so being fast-developing one over nearly 20 years
Class novel exogenous genes or medicine conducting carrier.
According to the physicochemical property of structure of cell-penetrating peptide, 3 kinds of cationic, amphiphatic molecule type and hydrophobic type can be classified as.When
Preceding cationic CPPs representative TAT small peptides are to study one of most hot cell-penetrating peptide at present.But TAT small peptides include 2 Furin enzymes
Cleavage site (i.e. RKKR and RQRR) is recognized, therefore TAT can be hydrolyzed by Furin in membrane process is worn and be destroyed TAT structures, so that
Weaken it and wear film ability.A kind of new, safe and efficient cell-penetrating peptide needs to be mined out.
The content of the invention
It is an object of the invention to provide a kind of new safe and efficient cell-penetrating peptide.
The present invention carries out analysis rich in arginine sequence area (aa 1-60) to CAV VP1 albumen n ends and set
The polypeptide (aa 23-43) with potential cell-penetrating function is counted out, it is artificial synthesized after being marked through FITC.By the polypeptide of synthesis with
Different cells are incubated altogether, are identified using laser confocal microscope, flow cytometry and cell toxicity test and are verified that it wears film
Function and membrane property is worn, compare it and wear membrane efficiency, and evaluate its security.Data display, it can carry FITC and be worn in 30min
Enter different cells, including suspended culture cell.And found through comparative studies, the VP1 aa 23-43 under the conditions of same concentrations
The cell-penetrating efficiency of polypeptide is apparently higher than TAT.
The invention discloses come from CAV VP1-aa 23-43 polypeptides as high efficiency cell cell-penetrating peptide
Using the VP1-aa 23-43 peptide sequences are as shown in SEQ ID NO.2.
Application of the present invention, after specifically VP1-aa 23-43 polypeptides are marked through FITC, carries FITC in 30min
Penetrate different cells.The cell includes human colon cancer cell (HCT-116), MEC (3T3), people's kidney
Epithelial cell (293T), MDCK (MDCK), chicken Marek's disease lymphoma cell (MSB1).
(1) synthesis of CAV VP1-aa 23-43 polypeptides and the identification of transmembrane ability
Different Reference Strains CAV VP1N ends are rich in smart ammonia with reference to UniProt databases and line server MultiAlin
(aa 1-60) is compared and analyzed for acid region sequence area.According to the characteristic of cell-penetrating peptide (rich in 5-30 positive charge amino
Acid), it is designed and intends cell-penetrating peptide sequence (aa 23-43) with generation.It is artificial synthesized after being marked through FITC.Simultaneously with TAT
Cell-penetrating peptide is used as positive control.The different small peptides of synthesis are thin with 293T cells, HCT-116 cells, 3T3 cells, MDCK respectively
Born of the same parents, MSB1 cells are incubated altogether, and its transmembrane ability is identified using laser confocal microscope and fluorescence microscope.
(2) wearing for CAV VP1-aa 23-43 polypeptides and wears the comparison of membrane efficiency at membrane property
Respectively set CAV VP1aa 23-43 polypeptides under different concentration conditions with HCT116 cells
It is incubated, is imaged and Flow cytometry and the concentration dependent for evaluating polypeptide using laser confocal microscope altogether.Point
Not She Zhi VP1-aa 23-43 polypeptides be incubated altogether with HCT116 cells in different concentration, using TAT as control, using streaming
Cell art detection wears membrane efficiency from the different small peptides of evaluation.
(3) CAV VP1-aa 23-43 polypeptide cells safety evaluatios
CAV VP1-aa 23-43 polypeptides are set to be incubated altogether with HCT116 cells in different concentration
Educate, each concentration sets 3 multiple holes, co-culture after 12h, 24h, 48h, VP1 VP1-aa 23- are detected using cell toxicity test
Security of 43 polypeptides to cell.
One kind prepared by the present invention comes from CAV VP1-aa 23-43 polypeptide high efficiency cell cell-penetrating peptides can
Enter 293T cells, HCT-116 cells, 3T3 cells, mdck cell, MSB1 cells in 30min to carry FITC, and wear
Membrane efficiency and being proportionate property of cell-penetrating peptide concentration, key features its can efficiently enter suspended culture cell.With reference to laser co-focusing
Microscope and flow cytometry results show that CAV VP1-aa 23-43 polypeptides have than TAT at 10 μM
Small peptide to wear membrane efficiency high more than 2 times.
Brief description of the drawings
The comparison of Fig. 1 CAV VP1aa 1-60 multiple sequences
Grayish background alphabetic flag positive charge polar amino acid.
Fig. 2 laser confocal microscopes carry FITC transmembrane abilities to CAV VP1-aa 23-43 polypeptides
Detection.
Fig. 3 fluorescence microscopes carry FITC to CAV VP1-aa 23-43 polypeptides and enter MSB1 cell work(
The detection A of energy:VP1-aa 23-43 small peptides;B:TAT small peptides;C:MSB1 cells;D:VP1-M small peptides.
Fig. 4 laser confocal microscopes wear the detection of membrane property to CAV VP1-aa 23-43 polypeptides.
Fig. 5 flow cytometries wear the detection of membrane property to CAV VP1-aa 23-43 polypeptides
A:HCT116 cells;B:VP1-M(20μM);C:VP1-aa23-43(1μM);D:VP1-aa23-43(2μM);E:
VP1-aa23-43(5μM);F:VP1-aa23-43(10μM);G:VP1-aa23-43(20μM);X-axis:FITC fluorescence intensities;Y
Axle:Cell number.
Fig. 6 flow cytometries wear the comparison of membrane efficiency to CAV VP1-aa 23-43 polypeptides and tat peptide
X-axis:VP1-aa 23-43 and the TAT small peptides of various concentrations;Y-axis:Positive cell FITC fluorescence intensities.
Fig. 7 MTT experiments detect the cell security to CAV VP1-aa 23-43 polypeptides
X-axis:The VP1-aa 23-43 small peptides of various concentrations;Y-axis:Cell survival rate (%).
Embodiment
Content for a better understanding of the present invention, implementation below combination accompanying drawing explains one kind and comes from chicken infectious anemia
The preparation of the high efficiency cell cell-penetrating peptide of virus VP 1-aa 23-43 polypeptides and qualification process.
Implementation process
(1) preparation and synthesis of CAV VP1N ends small peptide (aa 23-43)
Use online database UniProt (http://www.uniprot.org/uniprot/), to from different CAV
The N-terminal (aa 1-60) of the VP1 sequences of Reference Strains carries out sequence alignment, analyzes and mark the positively charged included in sequence
The polar amino acid (such as Fig. 1) of lotus.According to the annotation information of Family&Domains modules, to the N-terminal (aa of CAV VP1 sequences
1-60) sequence carries out specificity analysis.As a result find, VP1N ends have well-conserved.Annotated according to UniProt database informations
Understand, VP1 N-terminal (aa 1-47) is one section and is rich in positively charged arginine region, and with being combined and appraised and decided with DNA
The characteristic of position.
CAV VP1N ends (aa 1-47) amino acid sequence is:
MARRARRPRGRFYAFRRGRWHHLKRLRRRYKFRHRRRQRYRRRAFRK(SEQ ID NO.1).We are according to arginic
Enrichment region (aa 23-43), is named as VP1-N2, is carried out after being modified through FITC artificial synthesized.In order to inquire into arginic deposit
In the influence to cell-penetrating peptide function, arginine contained therein is sported uncharged glycine (G) or non-by us
Polarity alanine (A) (the small peptide name VP1-M after mutation), in addition using TAT small peptides as positive control.Therefore, small peptide is synthesized
Sequence is respectively:VP1-aa 23-43:LKRLRRRYKFRHRRRQRYRRR(SEQ ID NO.2);TAT:YGRKKRRQRRR
(SEQ ID NO.3);VP1-M:LKRLGAGYKFAHGAGQGYGAG(SEQ ID NO.4);Small peptide is had by Jin Sirui biotechnologies
Limit company synthesizes.
(2) laser confocal microscope is to CAV VP1-aa 23-43 polypeptides transmembrane abilities and characteristic
Detection
1) cell climbing sheet after sterilizing is placed in 24 porocyte culture plates in advance, by 2 × 105293T cells, HCT-
116 cells, 3T3 cells and mdck cell are inoculated into 24 porocyte culture plates.
2) after cell inoculation 12h, by the different small peptides of synthesis be set to different concentration (5 μM, 10 μM, 20 μM, 40 μM)
It is mixed to join with 500 μ L Opti-MEM in cell, 37 DEG C, 5%CO2, under the conditions of, it is incubated altogether after 30min, with PBS 3
It is secondary, each 3min;Wash away the small peptide for not entering into cell.
3) Hoechst 33342 (10 μ g/mL) is dyed after 20min, is discarded after supernatant, PBS, adds 5 μ L 7-DDA
With 500 μ L distilled water, at room temperature lucifuge be incubated 15min.
4) glycerine mounting is finally used, transparent nail polish carries out solid piece, TCS SP8STED laser confocal scanning microscopes point
Not in different wavelength (561nm>488nm>405nm), it is scanned and takes pictures under the conditions of multichannel, as a result such as Fig. 2 and Fig. 4;
In cromogram, FITC is labeled as green;Hoechst 33342 is labeled as blueness.
(3) fluorescence microscope enters suspended culture cell function to CAV VP1-aa 23-43 polypeptides
Detection
1) by 5 × 105Individual MSB1 cells are inoculated into 12 orifice plates.
2) take 20 μM of small peptides to be mixed with 500 μ L Opti-MEM, be added in MSB1 cells.
3) 37 DEG C, 5%CO2, under the conditions of, it is incubated after 30min, is added to through cell mixture in 1.5mL centrifuge tubes, 1 altogether,
000 × g, centrifuges 3min, and cell is resuspended using 1mL PBS.PBS is cleaned 3 times altogether.
4) cell finally is resuspended with 500 μ L Opti-MEM, cell suspension is added in 12 orifice plates, under fluorescence microscope
Photographic analysis is carried out, as a result such as Fig. 3.
(4) flow cytometry membrane property and wears membrane efficiency to wearing for CAV VP1aa 23-43 polypeptides
Detection
1) by 5 × 105Individual HCT-116 cells are inoculated into 12 orifice plates, are treated that cell reaches 90% fusion after 12h, are used PBS
Cleaning 1 time.
2) small peptide of various concentrations and 500 μ L Opti-MEM are mixed to join in cell, to be not added with small peptide as blank
Control, 37 DEG C, 5%CO2, or under the conditions of 4 DEG C, 30min is incubated altogether.
3) PBS 3 times, each 3min.Add 200 μ L pancreatin and 100 μ L PBS are mixed and carried out digestion 2min.Add new
Fresh cell culture medium terminates digestion, and cell suspension is collected in 1.5mL centrifuge tubes, 800 × g centrifugations 2min.
4) supernatant is discarded.1mL PBS are added, cleaning is once.It is repeated once.PBSs of the 400 μ L containing 1%FBS is added to be resuspended carefully
Born of the same parents, fully mix, filter every solencyte respectively with cell sieve in streaming pipe, are carried out using CyAn ADP7 flow cytometers
Detection.Every group of sample is repeated parallel 3 times, and counts its positive percentage.Data are analyzed using FlowJo softwares, as a result such as
Fig. 5 and Fig. 6.
(5) MTT experiment
1) by 4 × 103Individual HCT116 cells are inoculated into 96 orifice plates, after cell attachment 12h, are separately added into various concentrations
Small peptide, each concentration sets 3 multiple holes.
2) 37 DEG C, 5%CO2, continue to cultivate after 12h, 24h, 48h, 20 μ L tetrazolium bromide solution (MTT, 5mg/ added to every hole
), mL and continue cultivate 3h.Then cell suspension is discarded.
3) dimethyl sulfoxide (DMSO) (DMSO, 100 μ L/ holes) is added.After room temperature 30min, with ELIASA at wavelength 490nm
Its light absorption value (OD490) is detected, not add the cell that small peptide is handled as negative control, and each experimental group cell survival rate is calculated
(Cell viability)。
4) cell survival rate (%)=experimental group cell absorbance/cellular control unit absorbance × 100%, as a result such as
Fig. 7.
SEQUENCE LISTING
<110>Yangzhou University
<120>CAV VP1-aa 23-43 polypeptides are come from as the application of high efficiency cell cell-penetrating peptide
<130>
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 47
<212> PRT
<213>CAV
<400> 1
Met Ala Arg Arg Ala Arg Arg Pro Arg Gly Arg Phe Tyr Ala Phe Arg
1 5 10 15
Arg Gly Arg Trp His His Leu Lys Arg Leu Arg Arg Arg Tyr Lys Phe
20 25 30
Arg His Arg Arg Arg Gln Arg Tyr Arg Arg Arg Ala Phe Arg Lys
35 40 45
<210> 2
<211> 21
<212> PRT
<213>Artificial sequence
<400> 2
Leu Lys Arg Leu Arg Arg Arg Tyr Lys Phe Arg His Arg Arg Arg Gln
1 5 10 15
Arg Tyr Arg Arg Arg
20
<210> 3
<211> 11
<212> PRT
<213>Artificial sequence
<400> 3
Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg
1 5 10
<210> 4
<211> 21
<212> PRT
<213>Artificial sequence
<400> 4
Leu Lys Arg Leu Gly Ala Gly Tyr Lys Phe Ala His Gly Ala Gly Gln
1 5 10 15
Gly Tyr Gly Ala Gly
20
Claims (3)
1. coming from CAV VP1-aa 23-43 polypeptides as the application of high efficiency cell cell-penetrating peptide, its feature exists
In the VP1-aa 23-43 peptide sequences are as shown in SEQ ID NO.2.
2. application according to claim 1, it is characterised in that after VP1-aa 23-43 polypeptides are marked through FITC, carries FITC
Different cells are penetrated in 30min.
3. application according to claim 2, it is characterised in that the cell is human colon cancer cell (HCT-116), mouse
Embryo fibroblast (3T3), people's renal epithelial cell (293T), MDCK (MDCK), chicken Marek's disease lymphoma cell
(MSB1)。
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Cited By (1)
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CN112457379A (en) * | 2020-11-23 | 2021-03-09 | 台州学院 | Cell-penetrating peptide derived from Cap protein of duck circovirus, and design method and application thereof |
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