CN106995487B - Application derived from Chicken Infectious Anemia Virus VP1-aa 23-43 polypeptide as high efficiency cell cell-penetrating peptide - Google Patents
Application derived from Chicken Infectious Anemia Virus VP1-aa 23-43 polypeptide as high efficiency cell cell-penetrating peptide Download PDFInfo
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Abstract
The present invention relates to one kind to be derived from Chicken Infectious Anemia Virus (Chicken anemia virus, the CAV) application of VP1-aa 23-43 polypeptide as high efficiency cell cell-penetrating peptide.The VP1-aa 23-43 polypeptide sequence is as shown in SEQ ID NO.2.The polypeptide can carry FITC and enter 293T cell, HCT-116 cell, 3T3 cell, mdck cell, MSB1 cell in 30min, and wear membrane efficiency and being positively correlated property of cell-penetrating peptide concentration, key features its can efficiently enter suspended culture cell.Shown in conjunction with laser confocal microscope and flow cytometry results, Chicken Infectious Anemia Virus VP1-aa 23-43 polypeptide at 10 μM with than TAT small peptide to wear membrane efficiency high more than 2 times.
Description
Technical field
The present invention relates to one kind to be derived from Chicken Infectious Anemia Virus (Chicken anemia virus, CAV) VP1-aa
The high efficiency cell cell-penetrating peptide of 23-43 polypeptide.The polypeptide can carry FITC, and that different cells is penetrated in 30min is (including adherent thin
Born of the same parents and suspension cell).It was found that, the polypeptide is to the cell-penetrating function ratio TAT high of FITC more than 2 times with TAT's.Therefore,
A kind of high efficiency cell cell-penetrating peptide derived from Chicken Infectious Anemia Virus VP1-aa 23-43 polypeptide that the present invention obtains has certain
Application prospect and value.
Background technique
Cell-penetrating peptide (Cell penetrating peptides, CPPs) is that one kind can carry large biological molecule entrance
The small peptide of cell, length are generally 5-30 rich in positively charged basic amino acid.Not only itself can efficiently be passed through for it
Cell membrane enters cell, can also be efficient by different types of exogenous molecules such as fluorescein, DNA, RNA, protein, quantum dot etc.
It is carried along into the cell of different plant species.Because this novel small peptide not only has, conduction efficiency is high, bio-toxicity is low, avoids exciting
Vivo immunization reaction and the extensive and flexible in application feature of theca cell target is worn, so being fast-developing in the past 20 years one
Class novel exogenous genes or drug conducting carrier.
According to the physicochemical property of structure of cell-penetrating peptide, cationic, amphiphatic molecule type and 3 kinds of hydrophobic type can be classified as.When
The representative TAT small peptide of preceding cationic CPPs is most hot one of the cell-penetrating peptide of current research.But TAT small peptide includes 2 Furin enzymes
It identifies cleavage site (i.e. RKKR and RQRR), therefore TAT can be hydrolyzed by Furin in wearing membrane process and be destroyed TAT structure, thus
Weaken it and wears film ability.Novel, the safe and efficient cell-penetrating peptide of one kind needs to be mined out.
Summary of the invention
The object of the present invention is to provide a kind of new safe and efficient cell-penetrating peptides.
The present invention carries out analysis rich in arginine sequence area (aa 1-60) to Chicken Infectious Anemia Virus VP1 albumen n end and sets
Meter provides the polypeptide (aa 23-43) of potential cell-penetrating function, artificial synthesized after FITC is marked.By the polypeptide of synthesis with
Different cells are incubated for altogether, are identified using laser confocal microscope, flow cytometry and cell toxicity test and are verified it and wear film
Function and membrane property is worn, compares it and wear membrane efficiency, and evaluate its safety.Data are shown, can be carried FITC and be worn in 30min
Enter different cells, including suspended culture cell.And it is found through comparative studies, VP1 aa 23-43 under the conditions of same concentrations
The cell-penetrating efficiency of polypeptide is apparently higher than TAT.
The invention discloses be derived from Chicken Infectious Anemia Virus VP1-aa 23-43 polypeptide as high efficiency cell cell-penetrating peptide
Using the VP1-aa 23-43 polypeptide sequence is as shown in SEQ ID NO.2.
Application of the present invention, specifically VP1-aa 23-43 polypeptide carry FITC in 30min after FITC is marked
Penetrate different cells.The cell includes human colon cancer cell (HCT-116), mouse embryonic fibroblasts (3T3), people's kidney
Epithelial cell (293T), canine kidney cells (MDCK), chicken Marek's disease lymphoma cell (MSB1).
(1) identification of the synthesis of Chicken Infectious Anemia Virus VP1-aa 23-43 polypeptide and transmembrane ability
Smart ammonia is rich in the different ends Reference Strains CAV VP1N in conjunction with UniProt database and line server MultiAlin
(aa 1-60) is compared and analyzes for acid region sequence area.(5-30 positive charge amino is rich according to the characteristic of cell-penetrating peptide
Acid), it is designed and generates quasi- cell-penetrating peptide sequence (aa 23-43).It is artificial synthesized after FITC is marked.Simultaneously with TAT
Cell-penetrating peptide is as positive control.The different small peptides of synthesis are thin with 293T cell, HCT-116 cell, 3T3 cell, MDCK respectively
Born of the same parents, MSB1 cell carry out total incubation, identify its transmembrane ability using laser confocal microscope and fluorescence microscope.
(2) Chicken Infectious Anemia Virus VP1-aa 23-43 polypeptide wears membrane property compared with wearing membrane efficiency
Be respectively set Chicken Infectious Anemia Virus VP1aa 23-43 polypeptide under different concentration conditions with HCT116 cell
Total incubation is carried out, using the concentration dependent of laser confocal microscope imaging and Flow cytometry and evaluation polypeptide.Point
Not She Zhi VP1-aa 23-43 polypeptide in different concentration and HCT116 cell carry out total incubation, be control with TAT, using streaming
Cell art detection small peptides different from evaluation wear membrane efficiency.
(3) Chicken Infectious Anemia Virus VP1-aa 23-43 polypeptide cells safety evaluatio
Chicken Infectious Anemia Virus VP1-aa 23-43 polypeptide is arranged to be incubated altogether in different concentration and HCT116 cell
It educates, 3 multiple holes are arranged in each concentration, co-culture 12h, for 24 hours, after 48h, detect VP1 VP1-aa 23- using cell toxicity test
Safety of 43 polypeptides to cell.
One kind prepared by the present invention can derived from Chicken Infectious Anemia Virus VP1-aa 23-43 polypeptide high efficiency cell cell-penetrating peptide
Enter 293T cell, HCT-116 cell, 3T3 cell, mdck cell, MSB1 cell in 30min to carry FITC, and wears
Membrane efficiency and being positively correlated property of cell-penetrating peptide concentration, key features its can efficiently enter suspended culture cell.In conjunction with laser co-focusing
Microscope and flow cytometry results show that Chicken Infectious Anemia Virus VP1-aa 23-43 polypeptide has at 10 μM than TAT
Small peptide to wear membrane efficiency high more than 2 times.
Detailed description of the invention
The comparison of Fig. 1 Chicken Infectious Anemia Virus VP1aa 1-60 multiple sequence
Grayish background alphabetic flag positive charge polar amino acid.
Fig. 2 laser confocal microscope carries FITC transmembrane ability to Chicken Infectious Anemia Virus VP1-aa 23-43 polypeptide
Detection.
Fig. 3 fluorescence microscope carries FITC to Chicken Infectious Anemia Virus VP1-aa 23-43 polypeptide and enters MSB1 cell function
The detection A:VP1-aa 23-43 small peptide of energy；B:TAT small peptide；C:MSB1 cell；D:VP1-M small peptide.
Fig. 4 laser confocal microscope wears the detection of membrane property to Chicken Infectious Anemia Virus VP1-aa 23-43 polypeptide.
Fig. 5 flow cytometry wears the detection of membrane property to Chicken Infectious Anemia Virus VP1-aa 23-43 polypeptide
A:HCT116 cell；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 (20 μM)；X-axis: FITC fluorescence intensity；Y
Axis: cell number.
Fig. 6 flow cytometry wears the comparison of membrane efficiency to Chicken Infectious Anemia Virus VP1-aa 23-43 polypeptide and tat peptide
X-axis: VP1-aa 23-43 and the TAT small peptide of various concentration；Y-axis: positive cell FITC fluorescence intensity.
Fig. 7 MTT experiment detects the cell safety to Chicken Infectious Anemia Virus VP1-aa 23-43 polypeptide
X-axis: the VP1-aa 23-43 small peptide of various concentration；Y-axis: cell survival rate (%).
Specific embodiment
Content for a better understanding of the present invention, following implementation combination attached drawing are illustrated a kind of derived from chicken infectious anemia
The preparation of the high efficiency cell cell-penetrating peptide of virus VP 1-aa 23-43 polypeptide and qualification process.
Implementation process
(1) preparation and synthesis of the end Chicken Infectious Anemia Virus VP1N small peptide (aa 23-43)
Using online database UniProt (http://www.uniprot.org/uniprot/), to from different CAV
The N-terminal (aa 1-60) of the VP1 sequence of Reference Strains carries out sequence alignment, analyzes and marks and is positively charged included in sequence
The polar amino acid (such as Fig. 1) of lotus.According to the annotation information of Family&Domains module, to the N-terminal (aa of CAV VP1 sequence
1-60) sequence carries out specificity analysis.As a result, it has been found that the end VP1N is with well-conserved.It is annotated according to UniProt database information
It is found that the N-terminal (aa 1-47) of VP1 is one section rich in positively charged arginine region, and has in conjunction with DNA and appraise and decide
The characteristic of position.
The end CAV VP1N (aa 1-47) amino acid sequence are as follows: MARRARRPRGRFYAFRRGRWHHLKRLRRRYKFRHRR
RQRYRRRAFRK(SEQ ID NO.1).We are named as VP1-N2 according to arginic enrichment region (aa 23-43), warp
It is carried out after FITC modification artificial synthesized.In order to inquire into the arginic influence existed to cell-penetrating peptide function, we will wherein
Contained arginine sports uncharged glycine (G) or non-polar-alanine (A) (the small peptide name VP1- after mutation
M), in addition using TAT small peptide as positive control.Therefore, the sequence for synthesizing small peptide is respectively as follows: 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 synthesized by Jin Sirui Biotechnology Co., Ltd.
(2) laser confocal microscope is to Chicken Infectious Anemia Virus VP1-aa 23-43 polypeptide transmembrane ability and characteristic
Detection
1) cell climbing sheet after sterilizing is placed in 24 porocyte culture plates in advance, by 2 × 105293T cell, HCT-
116 cells, 3T3 cell and mdck cell are inoculated into 24 porocyte culture plates.
2) after cell inoculation 12h, by the different small peptides of synthesis be set as different concentration (5 μM, 10 μM, 20 μM, 40 μM)
It is mixed to join in cell with 500 μ L Opti-MEM, 37 DEG C, 5%CO2, under the conditions of, after being incubated for 30min altogether, 3 are cleaned with PBS
It is secondary, each 3min；Wash away the small peptide for not entering into cell.
3) it after Hoechst 33342 (10 μ g/mL) dyes 20min, discards supernatant and 5 μ L 7-DDA is added after PBS cleaning
With 500 μ L distilled water, it is protected from light is incubated for 15min at room temperature.
4) glycerol mounting is finally used, transparent nail polish carries out solid piece, TCS SP8STED laser confocal scanning microscope point
It is not scanned and takes pictures under the conditions of different wavelength (561nm > 488nm > 405nm), multichannel, as a result such as Fig. 2 and Fig. 4；In
In cromogram, FITC is labeled as green；Hoechst 33342 is labeled as blue.
(3) fluorescence microscope enters suspended culture cell function to Chicken Infectious Anemia Virus VP1-aa 23-43 polypeptide
Detection
1) by 5 × 105A MSB1 cell inoculation is into 12 orifice plates.
2) it takes 20 μM of small peptides to be mixed with 500 μ L Opti-MEM, is added in MSB1 cell.
3) 37 DEG C, 5%CO2, under the conditions of, after being incubated for 30min altogether, it is added in 1.5mL centrifuge tube through cell mixture, 1,
000 × g is centrifuged 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 Chicken Infectious Anemia Virus VP1aa 23-43 polypeptide
Detection
1) by 5 × 105A HCT-116 cell inoculation reaches into 12 orifice plates, after 12h to cell 90% fusion, uses PBS
Cleaning 1 time.
2) small peptide of various concentration and 500 μ L Opti-MEM are mixed to join in cell, small peptide is not added as blank
Control, 37 DEG C, 5%CO2Or under the conditions of 4 DEG C, it is incubated for 30min altogether.
3) PBS is cleaned 3 times, each 3min.200 μ L pancreatin are added and 100 μ L PBS mixing carries out digestion 2min.It is added new
Fresh cell culture medium terminates digestion, and cell suspension is collected in 1.5mL centrifuge tube, and 800 × g is centrifuged 2min.
4) supernatant is discarded.1mL PBS is added, cleaning is primary.It is repeated once.PBS of the 400 μ L containing 1%FBS is added to be resuspended carefully
Born of the same parents mix well, and filter every solencyte respectively in streaming pipe with cell sieve, are carried out using CyAn ADP7 flow cytometer
Detection.Every group of sample repeats parallel 3 times, and counts its positive percentage.Data are analyzed using FlowJo software, as a result such as
Fig. 5 and Fig. 6.
(5) MTT experiment
1) by 4 × 103A HCT116 cell inoculation after the adherent 12h of cell, is separately added into various concentration into 96 orifice plates
Small peptide, each concentration set 3 multiple holes.
2) 37 DEG C, 5%CO2, continue cultivate 12h, for 24 hours, after 48h, to every hole be added 20 μ L thiazolyl blue solution (MTT, 5mg/
), mL and continue to cultivate 3h.Then cell suspension is discarded.
3) dimethyl sulfoxide (DMSO, 100 holes μ L/) are added.After room temperature 30min, with microplate reader at wavelength 490nm
Its light absorption value (OD490) is detected, not add the cell of small peptide processing as negative control, and calculates each experimental group cell survival rate
(Cell viability)。
4) cell survival rate (%)=experimental group cell absorbance value/cellular control unit absorbance value × 100%, as a result such as
Fig. 7.
SEQUENCE LISTING
<110>Yangzhou University
<120>it is derived from application of the Chicken Infectious Anemia Virus VP1-aa 23-43 polypeptide as high efficiency cell cell-penetrating peptide
<130>
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 47
<212> PRT
<213>Chicken Infectious Anemia Virus
<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 (2)
1. existing derived from Chicken Infectious Anemia Virus VP1-aa 23-43 polypeptide preparing the application in cell-penetrating peptide, feature
In the VP1-aa 23-43 polypeptide sequence is as shown in SEQ ID NO.2；The cell is human colon cancer cell, mice embryonic
Fibroblast, people's renal epithelial cell, canine kidney cells, chicken Marek's disease lymphoma cell.
2. application according to claim 1, it is characterised in that VP1-aa 23-43 polypeptide carries FITC after FITC is marked
Different cells is penetrated in 30min.
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