CN107629118A - Targeting cell-penetrating peptide carrier and purposes based on histidine - Google Patents
Targeting cell-penetrating peptide carrier and purposes based on histidine Download PDFInfo
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- CN107629118A CN107629118A CN201711047238.XA CN201711047238A CN107629118A CN 107629118 A CN107629118 A CN 107629118A CN 201711047238 A CN201711047238 A CN 201711047238A CN 107629118 A CN107629118 A CN 107629118A
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- cell
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Abstract
The invention discloses a kind of targeting cell-penetrating peptide carrier and purposes based on histidine, the amino acid sequence of the targeting cell-penetrating peptide carrier based on histidine is REDV YGRKKRRQRRR PKKKRKV Hm, m=4 12.The targeting cell-penetrating peptide carrier based on histidine of the present invention, has more preferable biocompatibility.Possess transmembrane ability and appraise and decide bit function, Human Umbilical Vein Endothelial Cells target function and possess certain pH buffering effects, can be easier to escape from endosome, so as to improve gene delivery effect, realize the purpose for promoting cell migration propagation and promoting vascularization.To solve current non-virus carrier problems faced.
Description
Technical field
The invention belongs to gene vector material technical field, and in particular to be carried to the targeting cell-penetrating peptide based on histidine
Body.
Background technology
It is exactly to be delivered target gene by gene delivery vector that gene therapy, which promotes the quick endothelialization in vascular graftses surface,
To endothelial cell, promote the division growth of endothelial cell by effective expression of the therapeutic gene in endothelial cell, from
And quickly form the process of endodermis.In in the past few decades, with cationic polymer, liposome and dendritic
Certain development has been obtained for the non-viral gene vector of representative.People have prepared a variety of with compared with high gene transfer performance
Carrier, but due to this kind of carrier generally existing biocompatibility is not high, lack bioactivity the deficiencies of, also to a certain extent
Limit its application and development in terms of gene therapy in vivo.
At present, polypeptide genophore increasingly causes the concern of people, particularly rich in lysine, histidine and smart ammonia
The cationic polypeptide of acid, on the basis of using peptide carrier good biological degradability and biocompatibility, have biology living
Property polypeptide material more turn into nowadays genophore research important directions.
Quick endothelialization is realized on artificial blood vessel surface, the long-term patency rates of artificial blood vessel, anti-tampon shape can be improved
Into.Many researchs are directed to developing different methods to realize the quick endothelialization of artificial blood vessel at present.Research shows, Arg-
Glu-Asp-Val (REDV) peptide sequence can the specific integrin receptors of 4 β of α 1 identification by endothelial cell surface.Therefore,
REDV polypeptides are frequently used for modifying artificial blood vessel surface, realize specific recognition and the adhesion of Human Umbilical Vein Endothelial Cells.At present, also lack
A kind of high efficiency gene carrier for vascular endothelial cell.
As typical cell-penetrating peptide, the 47-57 sites (Tyr-Gly-Arg-Lys-Lys-Arg- of TAT polypeptides
Arg-Gln-Arg-Arg-Arg), arginine content is higher.Contained 2 lysines and 6 arginine are in TAT polypeptides
Basic amino acid, effectively it can be combined with DNA.
At present, genophore is modified using the cell-penetrating characteristic of TAT polypeptides and has obtained certain result.But
Its endosome escape capability is poor, and then have impact on the effect of gene delivery.
Have a many genophores of document report, but these carriers have for tumour cell and smooth muscle cell it is ideal
Transfection, and there is no efficient gene carrier also for vascular endothelial cell, need badly researched and developed for vascular endothelial cell it is a kind of
High efficiency gene carrier is, it is necessary to research and develop the method for promoting vascularization.
At present, there has been no with hypotoxicity, targeting, nuclear location and endosome escape function loaded gene based on
The report of the targeting cell-penetrating peptide carrier of histidine.
The content of the invention
The purpose of the present invention is overcome the deficiencies in the prior art, there is provided the targeting cell-penetrating peptide carrier based on histidine.
Second object of the present invention is to provide the purposes of the targeting cell-penetrating peptide carrier based on histidine.
Technical scheme is summarized as follows：
Targeting cell-penetrating peptide carrier based on histidine, the amino acid sequence of the carrier are
REDV-YGRKKRRQRRR-PKKKRKV-Hm, m=4-12.
The purposes of the above-mentioned carrier loaded gene of targeting cell-penetrating peptide based on histidine.
It is an advantage of the invention that：
(1) the targeting cell-penetrating peptide carrier based on histidine of the invention, has more preferable biocompatibility.
(2) the targeting cell-penetrating peptide carrier based on histidine of the invention, possess transmembrane ability and appraise and decide bit function, internally
The target function of chrotoplast simultaneously possesses certain pH buffering effects, can be easier to escape from endosome, so as to improve base
Because delivering effect, the purpose for promoting cell migration propagation and promoting vascularization is realized.Faced with solving current non-virus carrier
Problem.
Brief description of the drawings
Fig. 1 is agarose gel electrophoresis retardance figure.
Fig. 2 is the versus cell vigor figure of Human umbilical vein endothelial cells.
Fig. 3 is Transwell cell migration figures.
Fig. 4 is that extracorporeal blood vessel forms figure.
Embodiment
ZNF580 gene sources：ZNF580 be by People's Armed Police's logistics institute's physiology and Pathology Lab Zhang Wencheng seminars first
Clone and in the C2H2 type transcription factor new genes of Genbank registrations, number of registration AF184939.The nucleotide sequence of the gene
Shown in SEQ ID NO.5.
PEGFP is commercially available.
The present invention is expanded on further with reference to specific embodiment.It should be understood that the embodiment be merely to illustrate the present invention and
It is not used in limitation the scope of the present invention.In addition, after the content that the present invention lectures is read, those skilled in the art can be to this
Invention, which is made, to be changed or changes, and the equivalent form of value equally falls within claims hereof limited range.
Embodiment 1
Targeting cell-penetrating peptide carrier based on histidine, the amino acid sequence of the carrier are
REDV-YGRKKRRQRRR-PKKKRKV-HHHH, shown in SEQ ID NO.1.
Embodiment 2
Targeting cell-penetrating peptide carrier based on histidine, the amino acid sequence of the carrier are
REDV-YGRKKRRQRRR-PKKKRKV-HHHHHHHH, shown in SEQ ID NO.2.
Embodiment 3
Targeting cell-penetrating peptide carrier based on histidine, the amino acid sequence of the carrier are
REDV-YGRKKRRQRRR-PKKKRKV-HHHHHHHHHHHH, shown in SEQ ID NO.3.
Reference examples
REDV-YGRKKRRQRRR-PKKKRKV, shown in SEQ ID NO.4.
Embodiment 4
Targeting cell-penetrating peptide carrier (abbreviation genophore) adsorption compression pEGFP-ZNF580 based on histidine (is abbreviated as
PZNF580 ability and purposes)
Genophore (embodiment 1-3) is compound with pZNF580, prepare different w/w ratios (genophore and pZNF580's
Weight ratio) gene composite：
Step is：Under agitation, 15 microlitres of the pZNF580 aqueous solution that concentration is 200 mcg/mls is taken, by w/w ratios
For 0.5,1,2,3,4,5 is added drop-wise in the genophore aqueous solution, stirs 1 hour, obtains gene composite.
Reference examples, sequence shown in SEQ ID NO.4 is compound with pZNF580, prepare different w/w than crt gene it is compound
Thing.
Step is：Under agitation, 15 microlitres of the pZNF580 aqueous solution that concentration is 200 mcg/mls is taken, by w/w ratios
For 0.5,1,2,3,4,5 is added drop-wise in crt gene carrier aqueous solution, stirs 1 hour, obtains crt gene compound.
The ability of agarose gel electrophoresis experiment exam genophore and the carrier loaded gene of crt gene.
Step is as follows：Prepare different w/w than gene composite, with 6 × loadingbuffer mix after, sample is 1
× TAE cushioning liquid, 0.8% Ago-Gel, electrophoresis is carried out under conditions of 100 volts of voltage after 30 minutes, in ultra violet lamp
Lower observation plasmid pZNF580 distributing positions in gel electrophoresis simultaneously photograph to record.
Analysis result：Fig. 1 is agarose gel electrophoresis figure.Wherein,
A is the gene load diagram of crt gene carrier；
B is the gene load diagram of the genophore of embodiment 1；
C is the gene load diagram of the genophore of embodiment 2；
D is the gene load diagram of the genophore of embodiment 3.
Genophore has the ability of loaded gene.Crt gene carrier, embodiment 1, genophore prepared by embodiment 2
W/w=2 be can complete loaded gene, embodiment 3 prepare genophore being capable of complete loaded gene in w/w=3.
Embodiment 5
Targeting cell-penetrating peptide carrier (abbreviation genophore) based on histidine prepares w/w=5 base with pZNF580 genes
Because of compound：
Step is：Under agitation, 15 microlitres of the pZNF580 aqueous solution that concentration is 200 mcg/mls is taken, by w/w=
5 are added drop-wise in the genophore aqueous solution, stir 1 hour, obtain gene composite.
Crt gene carrier prepares w/w than the gene composite for 5 with pZNF580 genes.
Step is：Under agitation, 15 microlitres of the pZNF580 aqueous solution that concentration is 200 mcg/mls is taken, by w/w=
5 are added drop-wise in crt gene carrier aqueous solution, stir 1 hour, obtain crt gene compound.
Genophore, crt gene carrier, gene composite and crt gene compound cytotoxicity by MTT (3- (4,
5- dimethylthiazoles -2) -2,5- diphenyltetrazolium bromide bromides) colorimetric method evaluated.
Step is as follows：Human umbilical vein endothelial cells are inoculated into 96 orifice plates (1 × 104Cells/well) in, cell growth to 90%
Afterwards, complete medium (DMEM containing 10%FBS) is changed to plasma-free DMEM medium, carries out Nature enemy 12 hours.Afterwards
Re-replace as plasma-free DMEM medium, the genophore of various concentrations and the gene composite aqueous solution are added to culture medium
In, it is well mixed, discards culture medium after 4 hours, be replaced by complete medium, continues culture 24 hours.Then into each hole
20 microlitres of the MTT solution of 5 mg/mls is added, continues culture 4 hours, makes first a ceremonial jade-ladle, used in libation sufficient crystallising.Careful discard in hole is cultivated
Base, and 150 microlitres of DMSO are added in backward hole, low-speed oscillation 10 minutes on shaking table are put, crystal is fully dissolved.Exempted from enzyme-linked
Epidemic disease detector measures the OD value (OD) in each hole in 490 nanometer wave strong points.Versus cell activity is calculated using equation below
(%)：
Wherein, OD490 '：Experimental group subtracts the absorbance of zeroing group, avg (OD490C ')：Control group after correction is inhaled
Luminosity average value.
Analysis result：Fig. 2 is cytoactive, determine respectively crt gene carrier, crt gene compound, embodiment 1,
Genophore and its gene composite prepared by embodiment 2 and embodiment 3.
The toxicity of targeting cell-penetrating peptide carrier based on histidine is very low, and cytoactive is very high, and in higher concentrations still
With very high cytoactive.For genophore, after gene composite processing cell, cell has higher work
Property, this is due to that pZNF580 can promote endothelial cell proliferation and migration.
Embodiment 6
Turned using Cell migration assay to evaluate Human umbilical vein endothelial cells through gene composite and crt gene compound
Transfer ability after dye.
Step is as follows：Different genes compound and crt gene compound (w/w=5) transfected with human navel are utilized in 24 orifice plates
After venous endothelial cell 24 hours, it is hungry 12 hours to be replaced by plasma-free DMEM medium.Cell dissociation is got off and washed with PBS
It is dispersed in again in serum free medium after 3 times, then takes 100 microlitres of cell suspension inoculations in interior (1.2 on transwell
×105Cells/well), the complete medium of indoor 500 microlitres of addition under transwell.It is put into incubator that to continue culture 6 small
When.Take out transwell upper chambers and wash 3 times nonadherent cells of removing with PBS, then fixed with 4% paraformaldehyde.
The upper indoor cells of transwell are dabbed off with aseptic cotton carrier, and the adhesion for migrating upper chamber bottom filter membrane arrival lower surface is thin
Born of the same parents were with eosin stains 8 minutes.After washing away loose colour with PBS, just putting micro- Microscopic observation and photographing to record.Parallel test takes three times
6 visuals field, the migration of Human umbilical vein endothelial cells after being transfected by Image-ProPlus6.0 softwares to different genes compound
Ability carries out statistical analysis.
Analysis result：Fig. 3 moves for the cell migration figure (1) after 6 hours and by the Image-ProPlus6.0 cells calculated
Move quantity (2).Wherein,
A is the migration quantity of crt gene compound transfectional cell；
B is the migration quantity of the gene composite transfectional cell of genophore prepared by embodiment 1；
C is the migration quantity of the gene composite transfectional cell of genophore prepared by embodiment 2；
D is the migration quantity of the gene composite transfectional cell of genophore prepared by embodiment 3.
Relative to crt gene compound, after the targeting cell-penetrating peptide gene composite transfectional cell based on histidine, carefully
The transfer ability enhancing of born of the same parents.Relative to embodiment 1 and the gene composite of the genophore of embodiment 2, the genophore of embodiment 3
Gene composite transfectional cell after, the migration quantity of cell is most.This is due to that histidine contains imidazole radicals, passes through proton sea
Continuous effect so that the targeting cell-penetrating peptide gene composite containing more histidines realizes more preferable endosome escape, improves
PZNF580 efficiency gene transfections, and then promote endothelial cell migration propagation.
Embodiment 7
The vascularization ability of Human umbilical vein endothelial cells after experimental evaluation transfection is formed by extracorporeal blood vessel.
Step is as follows：Experimental implementation is carried out according to CorningMatrigelMatrix specifications.First by Matrigel glue
Fully melt overnight at 4 DEG C.Matrigel (ultimate density is 10 mg/mls) after 50 microlitres of dilutions is slowly dropped to
In 96- orifice bores, pay attention to avoiding the generation of bubble.Then 96- orifice plates are put into incubator and be incubated 1 hour, make Matrigel
Glue fully gelation.It is again that the Human umbilical vein endothelial cells after different genes compound and the transfection of crt gene compound are uniform
Ground is seeded in Matrigel glue surface (4 × 104Cells/well), cultivate cell under 37 DEG C, 5% carbon dioxide conditions.6 hours
Observe vascularization under an optical microscope afterwards and photograph to record.Each sample carries out 3 parallel laboratory tests, and passes through Image-
ProPlus6.0 softwares count to vascular circle number.
Analysis result：Fig. 4 is that extracorporeal blood vessel forms figure (1) and the blood vessel number (2) calculated by Image-ProPlus6.0.
Wherein,
A is the vascular circle quantity of crt gene compound transfectional cell；
B is the vascular circle quantity of the gene composite transfectional cell of genophore prepared by embodiment 1；
C is the vascular circle quantity of the gene composite transfectional cell of genophore prepared by embodiment 2；
D is the vascular circle quantity of the gene composite transfectional cell of genophore prepared by embodiment 3.
Relative to crt gene compound, the gene composite transfectional cell of the targeting cell-penetrating peptide carrier based on histidine
Afterwards, cell forms the ability enhancing of blood vessel.The gene composite of the genophore prepared relative to embodiment 1 and embodiment 2, it is real
After the gene composite transfectional cell for applying the genophore of the preparation of example 3, plastidogenetic vascular circle quantity is most.REDV polypeptides have
There is the ability of targeting endothelial cell, simultaneously as the endosome escape capability of histidine so that gene prepared by embodiment 3 carries
The gene composite of body improves cellular uptake and pZNF580 efficiency gene transfections, and the overexpression of pZNF580 genes promotes people's navel quiet
The migration propagation of arteries and veins endothelial cell and the ability of vascularization.
ZNF580 gene orders, it is artificial synthesized
Sequence table
<110>University Of Tianjin
<120>Targeting cell-penetrating peptide carrier and purposes based on histidine
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 26
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 1
Arg Glu Asp Val Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro
1 5 10 15
Lys Lys Lys Arg Lys Val His His His His
20 25
<210> 2
<211> 30
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 2
Arg Glu Asp Val Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro
1 5 10 15
Lys Lys Lys Arg Lys Val His His His His His His His His
20 25 30
<210> 3
<211> 34
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 3
Arg Glu Asp Val Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro
1 5 10 15
Lys Lys Lys Arg Lys Val His His His His His His His His His His
20 25 30
His His
<210> 4
<211> 22
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 4
Arg Glu Asp Val Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro
1 5 10 15
Lys Lys Lys Arg Lys Val
20
<210> 5
<211> 519
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 5
atgctgctgc tgcctccgcg cccaccgcat ccgcgttctt cttctccaga agcaatggac 60
ccgccgcctc cgaaagcccc accgttcccg aaagctgaag gcccgtcctc tactccgtct 120
agcgccgctg gcccgcgtcc gccacgcctg ggtcgtcacc tgctgatcga tgccaacggt 180
gtaccgtaca cctacactgt tcagctggaa gaggaaccac gtggcccgcc gcaacgtgaa 240
gcacctccgg gtgaaccggg ccctcgtaaa ggttattcct gcccggaatg tgcacgtgtg 300
ttcgcatctc cgctgcgtct gcagagccac cgcgttagcc actccgacct gaagccgttc 360
acctgcggcg cgtgcggtaa agctttcaaa cgtagctccc acctgtctcg tcaccgtgcg 420
acccaccgcg ctcgtgcggg tccgccgcat acgtgcccgc tgtgtccacg tcgctttcag 480
gatgctgcgg agctggcgca gcacgtccgc ctgcattaa 519
Claims (2)
1. the targeting cell-penetrating peptide carrier based on histidine, it is characterized in that the amino acid sequence of the carrier is REDV-
YGRKKRRQRRR-PKKKRKV-Hm, m=4-12.
2. the purposes of the carrier loaded gene of targeting cell-penetrating peptide based on histidine of claim 1.
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| CN108379595A (en) * | 2018-02-14 | 2018-08-10 | 天津大学 | Multifunctional targeted property genophore and Preparation method and use |
| CN108404136A (en) * | 2018-01-30 | 2018-08-17 | 天津大学 | A kind of ternary genes delivery system and its application based on cell-penetrating peptide |
| CN108864311A (en) * | 2018-08-03 | 2018-11-23 | 中国人民解放军第四军医大学 | A kind of inhibition MD2 and the protein bound small peptide of CIRP and its application |
| CN112773904A (en) * | 2019-11-04 | 2021-05-11 | 天津大学 | Nanoscale double-gene delivery system with synergistic expression function and preparation method and application thereof |
| CN112972701A (en) * | 2019-12-12 | 2021-06-18 | 天津大学 | Polypeptide/gene co-delivery material, preparation method and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108404136A (en) * | 2018-01-30 | 2018-08-17 | 天津大学 | A kind of ternary genes delivery system and its application based on cell-penetrating peptide |
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| CN112773904A (en) * | 2019-11-04 | 2021-05-11 | 天津大学 | Nanoscale double-gene delivery system with synergistic expression function and preparation method and application thereof |
| CN112773904B (en) * | 2019-11-04 | 2022-04-26 | 天津大学 | Nanoscale double-gene delivery system with synergistic expression function and preparation method and application thereof |
| CN112972701A (en) * | 2019-12-12 | 2021-06-18 | 天津大学 | Polypeptide/gene co-delivery material, preparation method and application |
| CN112972701B (en) * | 2019-12-12 | 2022-09-23 | 天津大学 | Polypeptide/gene co-delivery material, preparation method and application |
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Application publication date: 20180126 |