CN110511273A - A kind of preparation method and applications of high efficiency cell transmembrane polypeptide - Google Patents
A kind of preparation method and applications of high efficiency cell transmembrane polypeptide Download PDFInfo
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- CN110511273A CN110511273A CN201910627477.5A CN201910627477A CN110511273A CN 110511273 A CN110511273 A CN 110511273A CN 201910627477 A CN201910627477 A CN 201910627477A CN 110511273 A CN110511273 A CN 110511273A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
- C07K14/43577—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from flies
- C07K14/43581—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from flies from Drosophila
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C12N2800/00—Nucleic acids vectors
- C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
Abstract
The invention discloses a kind of preparation methods of high efficiency cell transmembrane polypeptide, comprising the following steps: changes angle homeosis domain (HDAntp) partial amino-acid residue in amino acid sequence, so that its total net positive charge and albumen quality ratio are higher than 1.6.The cell-penetrating polypeptide obtained using preparation method of the invention can efficiently wear the cytoplasm that film enters various mammalian cells.Preparation method of the invention can be on the basis of not influencing original albumen or polypeptide function, make polypeptide or albumen with film properties are worn by improving total net positive charge, this has the value and significance in great methodology to the transdermal delivery for the exogenous drugs albumen for needing to play specific function in the cell.
Description
Technical field
The present invention relates to bioengineering fields, and in particular, to a kind of high efficiency cell transmembrane polypeptide preparation method and
It is applied.
Background technique
Cell membrane is one of barrier of substance disengaging cell, is made of lipid bilayer, and many large biological molecules are such as more
Peptide, protein, DNA cannot freely cross over cell membrane and carry out substance and information transmitting.In recent years, some to be worn with cell membrane
The protein structure domain of saturating ability, such as human immunodeficiency virus's (Human immunodeficiency virus, HIV-1) are anti-
Formula activating transcription factor (Transactivating transcriptional activator, TAT), drosophila angle homeosis
Domain (Antennapedia homeodomain, HDAntp), herpes simplex virus I-type VP22 transcription factor not only have cell
Penetration capacity, and there are the macromoleculars such as mediation heterologous protein, oligonucleotide, liposome to be directed through cell membrane and enter cell
Function, referred to as protein transduction domain (protein transduction domain, PTD).Further study showed that mediating
Protein transduction domain transmembrane ability is the cell-penetrating for being located at the general amino acid residue by 20 or so shorter in PTD and forming
Polypeptide (cell-penetrating peptides, CPPs), such as 47~57 amino acids of TAT protein rise to film effect is worn
Key effect；The 43-58 residue segment of HDAntp is the main functional component for mediating HDAntp to wear film, is thus named as
Penetratin.Cell-penetrating peptide C PPs may pass through cell membrane and cell membrane is intact, be increasingly becoming research concern
Focus and the small peptide family constantly extended, including MPG, PEP-I, TAT, oligomerization arginine, oligomerization lysine etc..
A large number of studies show that the substance that CPPs is carried can be protein (green fluorescent protein, RNA enzyme, galactosidase
Deng), polypeptide (below 100 amino acid residues), DNA, chemical small molecule drug, oligonucleotides, antisense nucleic acid, peptide nucleic acid, receive
Rice grain, fluorescein, organic molecule, adenovirus vector, imaging substance, liposome and iron particle etc..CPPs can pass through chemistry
In conjunction with or the modes such as Gene Fusion carry various large biological molecules, the connection type of CPPs and drug includes non-covalent linking and altogether
Valence connection.
However, these transmembrane polypeptides are not high on wearing membrane efficiency.Large biological molecule is commonly currently imported into cell
Method has liposome mediated-method, viral vectors mediated method, electroporation and microinjection etc., but these methods have transhipment effect
The disadvantages of rate is low, cytotoxicity is big, safety is poor, targeting specific is poor, significantly limits its large-scale application.Therefore, it opens
The large biological molecule transmembrane transport technology of hair efficiently, safe becomes a significant challenge.
Summary of the invention
In order to solve the above-mentioned technical problem, inventor is largely explored, by increasing the band in HDAntp sequence just
The amino acid residue quantity of charge, so that total net positive charge and albumen quality ratio (Ratio of positive charges per
KDa) it is higher than 1.6, it was unexpectedly found that, it is dynamic into various lactations that improved HDAntp, i.e. HDSup+ can efficiently wear film
The cytoplasm of object cell, thereby completing the present invention.
The first aspect of the present invention provides a kind of preparation method of high efficiency cell transmembrane polypeptide, comprising the following steps:
Change the partial amino-acid residue in angle homeosis domain (HDAntp) amino acid sequence, so that its total net positive charge
It is higher than 1.6 with albumen quality ratio.
Further, the HDAntp has amino acid sequence shown in SEQ ID NO.1.
In some embodiments of the present invention, the partial amino-acid residue packet changed in HDAntp amino acid sequence
It includes and partial amino-acid residue is become into positively charged amino acid residue.Wherein, positively charged amino acid residue includes relying ammonia
Sour (K), arginine (R) and histidine (H), it is preferable that positively charged amino acid includes K and R.
In some specific embodiments of the invention, the partial amino-acid changed in HDAntp amino acid sequence is residual
Base refers to that the leucine (L) by the 14th in SEQ ID NO.1 changes into lysine (K), by the 35th and the 37th the third ammonia
Sour (A) changes into arginine (R), and the 39th cysteine (C) is changed into serine (S) to avoid cysteine residues
It forms cystine linkage and causes the generation of polypeptide dimer, to obtain amino acid sequence shown in SEQ ID NO.2.
The second aspect of the present invention provides a kind of high efficiency cell transmembrane polypeptide, is by changing HDAntp amino acid sequence
In partial amino-acid residue so that its total net positive charge and albumen quality are obtained than being higher than 1.6.
Further, the HDAntp has amino acid sequence shown in SEQ ID NO.1.
In some embodiments of the present invention, the partial amino-acid residue packet changed in HDAntp amino acid sequence
It includes and partial amino-acid residue is become into positively charged amino acid residue.Wherein, positively charged amino acid residue includes relying ammonia
Sour (K), arginine (R) and histidine (H), it is preferable that positively charged amino acid includes K and R.
In some specific embodiments of the invention, the cell-penetrating polypeptide has ammonia shown in SEQ ID NO.2
Base acid sequence is by the way that the 14th in SEQ ID NO.1 leucine (L) is changed into lysine (K), by the 35th and
37 alanine (A) change into arginine (R), and the 39th cysteine (C) changed into and is obtained after serine (S).
Third aspect present invention provides a kind of gene of cell-penetrating polypeptide described in second aspect of the present invention, the gene energy
Enough encode the cell-penetrating polypeptide.According to codon coding rule, the nucleotide sequence for encoding corresponding amino acid sequence be can be
It is multiple, but its function is consistent.
The fourth aspect of the present invention provides a kind of carrier comprising gene described in third aspect present invention.
In some embodiments of the present invention, the carrier is pET/RosettaTM2(DE3)pLysS。
It includes load described in gene described in third aspect present invention or this fourth aspect that the fifth aspect of the present invention, which provides a kind of,
The cell of body.
Further, the cell is prokaryotic cell, and further, the cell is Bacillus coli cells.
Sixth aspect present invention provide gene described in cell-penetrating polypeptide or the third aspect described in second aspect of the present invention or
The application of carrier described in fourth aspect or the 5th aspect cell in the drug that preparation needs to function in the cell.
In some embodiments of the present invention, the drug includes polypeptide.In certain preferred embodiments of the invention
In, the polypeptide is protein, and in some more preferreds of the invention, the polypeptide is antibody.
In some specific embodiments of the invention, the N-terminal or C-terminal and the cell of the amino acid sequence of the polypeptide
Transmembrane polypeptide fusion.
Beneficial effects of the present invention
Compared with the existing technology, the present invention has following effective effect:
Preparation method of the invention is by increasing the positively charged amino acid residue quantity in HDAntp sequence, so that always
Net positive charge and albumen quality ratio are higher than 1.6, to obtain the transformation version HDSup+ of HDAntp.Utilize preparation method of the invention
Efficiently worn the cytoplasm that film enters various mammalian cells.
Cell-penetrating polypeptide of the invention wears membrane efficiency higher than Penetratin, reaches as high as 5 times.
Preparation method of the invention can be total net by improving on the basis of not influencing original albumen or polypeptide function
Positive charge has polypeptide or albumen to wear film properties.This is to the exogenous drugs albumen for needing to play specific function in the cell
Transdermal delivery have the value and significance in great methodology.
Detailed description of the invention
Fig. 1 shows the Antennapdia homeodomain (HDAntp) and improved HDAntp that nature increases charge
(HDSup+).(A) amino acid sequence for being HDAntp and HDSup+.(B) HDAntp and HDSup+ specifically binds D4 enhancer
Probe；Wherein pmol represents the loading capacity of the compound of HDAntp or HDSup+ and D4 probe.
Fig. 2 shows HDSup+ compared with Penetratin is in Hela cell in terms of transfer efficiency.(A) Hela living
The confocal fluorescent microscopic of cell observes result.By cell in 2.5 μM of exoproteins, 37 DEG C are incubated for 1 hour, recombinate egg
White matter contains HDSup+, Penetratin and mCherry respectively.Fluorescence signal is shown that maximum is thrown by the flame color of ImageJ
Shadow z is superposed to 20 confocal slices, and scale bar represents 10 μm.(B) it is measured by fluorescence-activated cell sorting (FACS) various dense
Spend the quantity of lower cellular uptake.Data are the average value ± SD of three repeated experiments.
Fig. 3 shows the ratio of HDSup+ and Penetratin in SHSY5Y cell and NuMuMG cell in terms of transfer efficiency
Compared with.
Specific embodiment
In order to which the technical problems, technical solutions and beneficial effects solved by the present invention is more clearly understood, below in conjunction with
Embodiment, the present invention will be described in further detail.
Embodiment
Following example is used herein to demonstration the preferred embodiments of the invention.Those skilled in the art, it will be appreciated that under
State the technology disclosed in example represent inventor discovery can be used for implementing technology of the invention, therefore can be considered as implementation this
The preferred embodiment of invention.But those skilled in the art should be understood that specific reality disclosed herein according to this specification
Many modifications can be made by applying example, still can be obtained identical or similar as a result, rather than away from the spirit or scope of the present invention.
Unless otherwise defined, the term of all technologies as used herein and science, and the technology in fields of the present invention
Personnel institute is normally understood equivalent in meaning, and being disclosed reference and their materials of reference will all be incorporated.
Those skilled in the art will recognize or just will appreciate that by routine test many described here
Invention specific embodiment many equivalent technologies.These will equally be comprised in claims.
Institute is conventional method unless otherwise specified experimentally in following embodiments.It is as used in the following examples
Test material is unless otherwise specified to be commercially available from routine biochemistry reagent shop.
Mammal cell line Hela, SHSY5Y and NMuMG used containing 10% heat-inactivated fetal calf serum (FBS,
Invitrogen DMEM (the Dulbecco's Modified Eagle's with high W/Glutamax-I (Invitrogen))
Cultivated in Medium) (37 DEG C, 5%CO2)。
The preparation of embodiment 1HDSup+
As shown in Figure 1A, by increasing the positively charged total number of atnino acid in HDAntp sequence (SEQ ID NO.1)
Amount, changes into lysine (K) for the 14th in SEQ ID NO.1 leucine (L), by the 35th and the 37th alanine
(A) arginine (R) is changed into, the 39th cysteine (C) is changed into serine (S), to obtain with SEQ ID
The improved HDAntp of amino acid sequence shown in NO.2, i.e. HDSup+.The total net positive charge of HDSup+ and albumen quality ratio
(Ratio of positive charges per kDa) is higher than 1.6.
The preparation of 2 recombinant protein of embodiment
It is transferred to efficiency in order to compare HDSup+ and its transduction structural domain Penetratin, we are connect using identical
Head and direction merge them with mCherry fluorescin.Following (the SEQ ID of the amino acid sequence of mCherry fluorescin
NO.3):
MHHHHHHHHHHIEGRVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEI EGEGEGRPYEGTQTAKLKV
TKGGPLPFAWDILSPQFMYGSKAYVKHPADIPD YLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVK
LRGTNFPSD GPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTY KAKKPVQLPGAYNVNI
KLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKG
By Penetrain (DSLEFIASKLA, SEQ ID NO.4) and mCherry amalgamation and expression, recombinant expression egg is obtained
White mCherry-Penetratin, sequence are following (SEQ ID NO.5):
MHHHHHHHHHHIEGRVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEI EGEGEGRPYEGTQTAKLKV
TKGGPLPFAWDILSPQFMYGSKAYVKHPADIPD YLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVK
LRGTNFPSD GPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTY KAKKPVQLPGAYNVNI
KLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKG GDSLEFIASKLA
By HDAntp and mCherry amalgamation and expression, recombinant expression protein mCherry-HDAntp, the following (SEQ of sequence are obtained
ID NO.6):
MHHHHHHHHHHIEGRVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEI EGEGEGRPYEGTQTAKLKV
TKGGPLPFAWDILSPQFMYGSKAYVKHPADIPD YLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVK
LRGTNFPSD GPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTY KAKKPVQLPGAYNVNI
KLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKG GRKRGRQTYTRYQTLELEKEFHFNRYLTRRRRIEIAHALC
LTERQIKIWFQNR RMKWKKEN
By HDSup+ and mCherry amalgamation and expression, recombinant expression protein mCherry-HDSup+ is obtained, sequence recombinates as follows
Albumen mCherry-HDSup+ is expressed, sequence is following (SEQ ID NO.7):
MHHHHHHHHHHIEGRVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEI EGEGEGRPYEGTQTAKLKV
TKGGPLPFAWDILSPQFMYGSKAYVKHPADIPD YLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVK
LRGTNFPSD GPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTY KAKKPVQLPGAYNVNI
KLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKG GRKRGRQTYTRYQTKELEKEFHFNRYLTRRRRIEIRHRLS
LTERQIKIWFQNR RMKWKKER
Above-mentioned recombinant protein passes through escherichia expression system: pET/RosettaTM2 (DE3) pLysS carry out fusion table
It reaches.
2 electrophoretic mobility shift assay of embodiment
DNA ability is specifically bound in order to determine whether recombinant protein is able to maintain its, inventor is by [α -32P]
The D4 probe of dATP label is used for electrophoretic mobility shift assay (EMSA).D4 is made of 62 base pair dnas, corresponds to drosophila
Segment in middle no vertebra enhancer is combined during leg development by endogenous Antp and other total transcription factors.Specific behaviour
Make as follows:
According to the sequence for the D4 enhancer for being originated from drosophila, design probe is used for electrophoretic mobility shift assay as follows
(EMSA):
D4 forward direction probe sequence: (SEQ ID NO.8)
5’-AGTTTACCATTAAATTCCCATTTAGGCTGTCAATCATTTGCGCT-3’
The reversed probe sequence of D4: (SEQ ID NO.9)
5’-AAGCCGCCAAGAAAAATTAGCGCAAATGATTGACAGCCTAAATG GG-3’
Then use [α -32P] dATP that probe sequence is marked using the Klenow segment of DNA pol I.It is anti-every time
The oligonucleotide of 10ng annealing should be used.The label being not incorporated into is removed with illustra MicroSpin G-25 column (GE).
Poly (dI.dC) (Sigma) is for reducing non-specific binding.Pass through non denatured electrophoretic analysis using 6% acrylamide gel
Acquired solution, and developed with X-ray film.
As a result as shown in Figure 1B, recombination HDAntp and HDSup+ albumen can specifically bind 62bp D4 probe, HDSup+
The protein that albumen is formed with probe/DNA compound and the albumen quality of addition are positively correlated, show recombinate HDAntp and
HDSup+ can maintain the specific binding capacity with complementary dna sequence.
Embodiment 3HDSup+ and penetratin wear membrane efficiency and compare
1. living cells is imaged
Since the fixation of cell leads to the artificial redistribution of CPP, inventor is the monitoring cellular uptake weight in living cells
Histone.It seeds cells into containing (every hole 1 × 10 on 8 hole μ-Slide (ibidi) in 220 μ L culture mediums4It is a).22 is small
Shi Hou, washs that cell is primary with PBS, and is incubated for 30 minutes in the DMEM of serum-free, or with corresponding medical preconditioning, so
It is incubated for 60 minutes in each protein solution afterwards.After incubation, cell is washed three times to remove striping with the PBS of the heparin containing 20U/mL
Binding protein, and be imaged in the growth medium of preheating.Cell Leica SP5MP Laser Scanning Confocal Microscope using 60 × or
100 × camera lens is imaged on warm table, handles image using ImageJ software.
2. flow cytometry
It seeds cells on 24 orifice plates, every hole 6 × 104A cell.After 22 hours, it is imaged as described above for living cells
Then the same method incubated cell uses trypsin digestion, is resuspended in 200 μ l growth mediums and is placed on ice.In
(561nm) analyzes the mCherry internalization of cell on LSRII flow cytometer (BD Biosciences).Cell is gated
To obtain living cells, and analyze every kind of processing at least 10,000 living cells.With FlowJo software (Tree Star, Inc.) point
Analyse data.
As a result, it has been found that signal is more in the cell for signal ratio Penetratin fusion protein in the cell for HDSup+ fusion protein
(Fig. 2A) by force, and individually mCherry provides any detectable signal under the setting of identical microscope.
In order to further quantify the membrane efficiency of wearing of every kind of protein, inventor is before facs analysis, in identical processing
Under, fluorescence-assisted cell sorting (FACS) is applied to by Hela cell by other trypsin digestion step.Before FACS
The signal from dead cell is eliminated to the setting of lateral scattering door, these signals would generally generate strong error signal.In
Under 2.5 μM of extracellular protein concentration, HDSup+ can be to enter the efficiency that Hela cell is up to 5 times than Penetratin
It delivers mCherry (Fig. 2 B).And observe identical difference when down to 0.5 μM and up to 5 μM, although the latter HDSup+ and
Difference between Penetratin is contracted to 4 times (Fig. 2 B).In general, HDSup+ ratio Penetratin shows stronger wear
The ability of saturating Hela cell.
In order to exclude a possibility that difference is Hela cell-specific, inventor compares HDSup using other cell lines
+ membrane efficiency is worn between Penetratin.In SHSY5Y cell line and NuMuMG cell line, HDSup+ ratio is observed
Saturating element Penetratin, which is compared, wears membrane efficiency (Fig. 3) with higher.Show to wear membrane efficiency between HDSup+ and Penetratin
Difference do not influenced by cell type.
All references mentioned in the present invention is incorporated herein by reference, just as each document coverlet
It is solely incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art
Member can make various changes or modifications the present invention, and such equivalent forms equally fall within the application the appended claims and limited
Fixed range.
Sequence table
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Leu Lys Val Thr Lys Gly Gly Pro Leu Pro Phe Ala Trp Asp Ile Leu
65 70 75 80
Ser Pro Gln Phe Met Tyr Gly Ser Lys Ala Tyr Val Lys His Pro Ala
85 90 95
Asp Ile Pro Asp Tyr Leu Lys Leu Ser Phe Pro Glu Gly Phe Lys Trp
100 105 110
Glu Arg Val Met Asn Phe Glu Asp Gly Gly Val Val Thr Val Thr Gln
115 120 125
Asp Ser Ser Leu Gln Asp Gly Glu Phe Ile Tyr Lys Val Lys Leu Arg
130 135 140
Gly Thr Asn Phe Pro Ser Asp Gly Pro Val Met Gln Lys Lys Thr Met
145 150 155 160
Gly Trp Glu Ala Ser Ser Glu Arg Met Tyr Pro Glu Asp Gly Ala Leu
165 170 175
Lys Gly Glu Ile Lys Gln Arg Leu Lys Leu Lys Asp Gly Gly His Tyr
180 185 190
Asp Ala Glu Val Lys Thr Thr Tyr Lys Ala Lys Lys Pro Val Gln Leu
195 200 205
Pro Gly Ala Tyr Asn Val Asn Ile Lys Leu Asp Ile Thr Ser His Asn
210 215 220
Glu Asp Tyr Thr Ile Val Glu Gln Tyr Glu Arg Ala Glu Gly Arg His
225 230 235 240
Ser Thr Gly Gly Met Asp Glu Leu Tyr Lys Gly Gly Arg Lys Arg Gly
245 250 255
Arg Gln Thr Tyr Thr Arg Tyr Gln Thr Lys Glu Leu Glu Lys Glu Phe
260 265 270
His Phe Asn Arg Tyr Leu Thr Arg Arg Arg Arg Ile Glu Ile Arg His
275 280 285
Arg Leu Ser Leu Thr Glu Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg
290 295 300
Arg Met Lys Trp Lys Lys Glu Arg
305 310
<210> 8
<211> 44
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
agtttaccat taaattccca tttaggctgt caatcatttg cgct 44
<210> 9
<211> 46
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
aagccgccaa gaaaaattag cgcaaatgat tgacagccta aatggg 46
Claims (10)
1. a kind of preparation method of high efficiency cell transmembrane polypeptide, which comprises the following steps:
Change angle homeosis domain (HDAntp) partial amino-acid residue in amino acid sequence, so that its total net positive charge and egg
White mass ratio is higher than 1.6.
2. preparation method according to claim 1, which is characterized in that the HDAntpWith ammonia shown in SEQ ID NO. 1
Base acid sequence.
3. preparation method according to claim 1, which is characterized in that the change HDAntpPart in amino acid sequence
Amino acid residue includes that the amino acid residue of part aqueous is become positively charged amino acid residue.
4. preparation method according to claim 3, which is characterized in that the change HDAntpPart in amino acid sequence
Amino acid residue refers to that the leucine (L) by the 14th in SEQ ID NO. 1 changes into lysine (K), by the 35th and the 37th
The alanine (A) of position changes into arginine (R), the 39th cysteine (C) is changed into serine (S), to obtain SEQ
Amino acid sequence shown in ID NO. 2.
5. a kind of high efficiency cell transmembrane polypeptide, which is characterized in that it is by changing HDAntpPart ammonia in amino acid sequence
Base acid residue, so that its total net positive charge and albumen quality are obtained than being higher than 1.6.
6. cell-penetrating polypeptide according to claim 5, which is characterized in that the cell-penetrating polypeptide has SEQ ID
NO. amino acid sequence shown in 2.
7. a kind of gene for encoding the cell-penetrating polypeptide of claim 5 or 6, which is characterized in that the gene can encode
The cell-penetrating polypeptide.
8. application of the cell-penetrating polypeptide described in claim 5 in the drug that preparation needs to function in the cell.
9. application according to claim 8, which is characterized in that the drug includes polypeptide.
10. application according to claim 9, which is characterized in that the N-terminal or C-terminal of the amino acid sequence of the polypeptide and institute
State cell-penetrating peptide fusion.
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| CN111317826A (en) * | 2020-03-19 | 2020-06-23 | 上海交通大学 | Nucleic acid composite nano-drug constructed based on metal ion coordination self-assembly and preparation method and application thereof |
| CN114522246A (en) * | 2020-11-23 | 2022-05-24 | 上海洛启生物医药技术有限公司 | Pharmaceutical composition containing VEGF nano antibody and application thereof |
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| CN110511273B (en) | 2021-06-01 |
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