CN108794579A - A kind of cell-penetrating peptide and preparation method thereof, application - Google Patents
A kind of cell-penetrating peptide and preparation method thereof, application Download PDFInfo
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- CN108794579A CN108794579A CN201810604034.XA CN201810604034A CN108794579A CN 108794579 A CN108794579 A CN 108794579A CN 201810604034 A CN201810604034 A CN 201810604034A CN 108794579 A CN108794579 A CN 108794579A
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Abstract
The invention discloses a kind of cell-penetrating peptide and preparation method thereof, applications, and the cell-penetrating peptide includes following sequence：RRRRRKQARRPRRRRAR.And the cell-penetrating peptide safety, wear film significant effect.
Description
Technical field
The present invention relates to biomedical sector more particularly to a kind of Humanized cell cell-penetrating peptide and preparation method thereof, applications.
Background technology
Body cell envelope barrier only allows non-fat-soluble molecule of the molecular weight less than 600Da to enter living cells, though to machine
Body has important protective effect, but many is also made to have the drug of application prospect to be abandoned.Cell-penetrating peptide (cell-
Penetrating peptides, CPPs), it is the small peptide that some are less than 30 amino acid, it is bigger than itself molecular weight can be carried
The molecule of manyfold enters cell.There are many mode classifications for cell-penetrating peptide, can be divided into cation, parents according to physicochemical property
Property and hydrophobicity cell-penetrating peptide.
In vivo, the sequence of protein belt positive charge can enhance protein and the combination of RNA, to play to RNA
The function of regulation and control or alternative splicing.Therefore, film can be worn to screen cation by albumen of the search with such function
Peptide.But cationic cell-penetrating peptide with the above function is not yet developed at present.
Invention content
The defects of for the above-mentioned prior art, the present invention provides a kind of Humanized cell cell-penetrating peptide and its preparation sides
Method, application, and cell-penetrating peptide safety, wear film significant effect.
The technical solution that the present invention is provided to solve above-mentioned technical problem is as follows：
On the one hand, a kind of cell-penetrating peptide is provided comprising following sequence：RRRRRKQARRPRRRRAR.
On the other hand, a kind of preparation method of above-mentioned cell-penetrating peptide is additionally provided comprising following steps：
S1, resin is provided, and the resin is dipped to the resin swelling in the first solvent；
S2, amino acid, condensing agent are dissolved in the second solvent, form mixture；
S3, reaction system, reaction time 1-4h are formed in the resin that swelling is added in the mixture；
Solution after S4, removal reaction in the reaction system, washs residue 1-5 times, gas is agitated, and is drained；
S5, liquid of raising one's hat is added in the residue, gas is agitated, drained；
S6, removal are washed 1-10 times, gas is agitated, and is drained, and is thus obtained via the solution in the reaction system after step S5
The polypeptide chain that must be synthesized；
S7, cutting liquid is added in the polypeptide chain, stirs 1-4h at 4-30 DEG C, filters to get filtrate, and described in washing
Filtrate；
S8, the filtrate of washed mistake is extracted, centrifugation obtains cell-penetrating peptide crude product；
And S9, the cell-penetrating peptide crude product is identified and purified.
Preferably, the resin includes Fmoc-Wang resins.
Preferably, in step s 2, the condensing agent includes benzotriazole salt form condensing agent；In step sl, described
First solvent includes：One or more of dichloromethane, dimethylformamide, dichloromethane and tetrahydrofuran；In step S5
In, the liquid of raising one's hat includes：Dimethyl formamide solution, and the piperazine of 6%W/V that is dissolved in dimethyl formamide solution
With 0.1M HOBT；In the step s 7, the cutting liquid includes：By weight percentage, 95% TFA, 2% TIS and
3% H2O。
Preferably, the benzotriazole salt form condensing agent includes O- benzotriazole-tetramethylurea hexafluorophosphate, O-
One or more of benzotriazole-N, N, N', N'- tetramethylurea tetrafluoroborate.
Preferably, the mole ratio that feeds intake of each amino acid and the resin is (1:1)-(10:1)；The condensing agent
The mole ratio that feeds intake with the resin is (1:1)-(10:1).
Preferably, in the step S8, the extraction includes：Anhydrous ether is added in the filtrate of washed mistake, places
1-4h；And the volume ratio of the anhydrous ether and filtrate is (5:1)-(60:1).
On the other hand, a kind of application process of above-mentioned cell-penetrating peptide is also provided, the cell-penetrating peptide be used in C-terminal or
N-terminal covalently or non-covalently linker or loading molecule, and carry the marker or loading molecules across cellular membranes enter
Cell.
Preferably, it carries the marker or loading molecules across cellular membranes enters the process of cell and include：The cell
Cell-penetrating peptide is combined to mediate Plasmid DNA with Plasmid DNA.
Preferably, it includes following step that the cell-penetrating peptide, which is combined with Plasmid DNA for mediating the process of Plasmid DNA,
Suddenly：
S1, Plasmid DNA and the cell-penetrating peptide are respectively mixed in phosphate buffer；
S2, the adjustment Plasmid DNA and the N/P ratios of the cell-penetrating peptide between the two are 0-80；
The cell of S3, overnight incubation are washed with the culture medium of serum-free, will contain the Plasmid DNA and cell-penetrating peptide
Phosphate buffer be added in culture medium；
And remove culture medium after S4,4-6h, the culture medium containing 10-15%FCS is added, 1-4h is stood.
Effect caused by technical solution of the present invention：
The Humanized cell cell-penetrating peptide of the present invention wears film significant effect, wears membrane efficiency height；And immunogenicity is low, safety is low
Poison；Preparation method is easy to operate and is convenient for quality management and control.
Description of the drawings
Fig. 1 be the present invention cell-penetrating peptide combined with Plasmid DNA rear optimal N/P than experimental result picture.
Fig. 2 is that the cell-penetrating peptide of the present invention is used for BHK21 cell transfecting result schematic diagrams.
Fig. 3 is that the cell-penetrating peptide of the present invention is used for B16 cell transfecting result schematic diagrams.
Fig. 4 is that the cell-penetrating peptide of the present invention is used for the fluorescent value that BHK21, B16 are transfected.
Fig. 5 a are that the cell-penetrating peptide of the present invention is used for BHK21MTT result schematic diagrams.
Fig. 5 b are that TAT is used for BHK21MTT result schematic diagrams.
Fig. 6 a are that the cell-penetrating peptide of the present invention is used for B16MTT result schematic diagrams.
Fig. 6 b are that TAT is used for B16MTT result schematic diagrams
Specific implementation mode
In order to make the purpose , technical scheme and advantage of the present invention be clearer, with reference to the accompanying drawings and embodiments, right
The present invention is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, and
It is not used in the restriction present invention.
Embodiment one：
For inventor by carrying out retrieval analysis to human protein database, hair finds m6A reading codes in the nucleus of the mankind
The small peptide (RRRRRKQARRPRRRRAR) of one section long 17 amino acid of device YTHDC1 albumen is rich in arginine, has very strong
Positive charge, thus it is speculated that this section of small peptide may be a novel amphiphilic cell-penetrating peptide of humanized with autonomous transmembrane ability, thus
Provide the Humanized cell cell-penetrating peptide (abbreviation YTHDC1) in the present embodiment comprising following sequence：
RRRRRKQARRPRRRRAR, amino acid sequence is as shown in sequence table；Wherein, the P is selected from proline residue, and the R is selected from
Arginine residues, the A are selected from alanine residue, and the Q is selected from glutamine residue, and the K is selected from lysine residue.
Further, the preparation method of above-mentioned cell-penetrating peptide includes the following steps：
S1, resin (such as Fmoc-Wang resins) is weighed, poured into reactor, the first solvent of addition (including dichloromethane
(DCM), one or more of dimethylformamide (DMF) and tetrahydrofuran (THF)) it impregnates, until the resin is first
It is dipped to the resin swelling in solvent, then appropriate deprotection solution is added into reactor, logical nitrogen is agitated 0.5-2 hours
(preferably 1.5h), is drained, then appropriate DMF is added into reactor, logical nitrogen is agitated, and is drained；Repeatedly above-mentioned plus deprotection liquid,
Operation 1-10 times (preferably 5 times) that logical nitrogen is agitated, drains and added appropriate DMF, logical nitrogen to agitate, drains；
S2, amino acid and condensing agent are weighed, and each amino acid (including arginine, alanine, glutamine, dried meat ammonia
Acid and lysine) mole be 1 times of the resin mole, the mole of the condensing agent is the resin mole
3 times；Preferably, the condensing agent includes benzotriazole salt form condensing agent, and the benzotriazole salt form condensing agent packet
Include O- benzotriazole-tetramethylurea hexafluorophosphate (HBTU), O- benzotriazole-N, N, N', N'- tetramethylurea tetrafluoro boron
One or more of hydrochlorate (TBTU)；Amino acid, condensing agent are dissolved in again be contained in reactor the second solvent (including
One or more of DMF, DCM, THF) in, form mixture；
S3, reaction system being formed in the resin that swelling is added in the mixture, the reaction time is 1-4h (preferably 3h),
Ninhydrin method detection is until stop reaction when not developing the color；
S4, the solution in the reaction system after removal is reacted is drained, appropriate DMF is added to wash 1-5 times (preferably to residue
It is 3 times), logical nitrogen is agitated, and is drained；
S5, liquid of raising one's hat is added in the residue, logical nitrogen is agitated 1-3h (preferably 2h), drained, until ninhydrin
Method tests positive；Preferably, the liquid of raising one's hat includes：DFM solution, and to be dissolved in the dimethylformamide (DFM) molten
The piperazine and 0.1M HOBT of 6%W/V in liquid；
S6, removal is drained via the solution in the reaction system after step S5, add appropriate DMF to wash 1-10 times, lead to nitrogen
It agitates, drains, thus to obtain containing Arg-Arg-Arg-Arg-Arg-Lys-Gln-Ala-Arg-Arg-Pro-Arg-Arg-Arg-
The polypeptide chain of Arg-Ala-Arg；
S7, the resin after drying is fitted into suitable centrifuge tube, cutting liquid is added in the polypeptide chain, at 4-30 DEG C
Constant temperature mechanical agitation 1-4h (preferably 3h), filters to get filtrate, and wash the filtrate with TFA under (preferably 18 DEG C)；It is described
Cutting liquid includes：By weight percentage, the H of 95% TFA, 2% TIS and 3%2O；
S8, the filtering filtrate, filtrate is all collected into flask；The filtrate of washed mistake is extracted, high speed
Centrifugation obtains cell-penetrating peptide crude product；The extraction includes：Anhydrous ether is added in the filtrate of washed mistake, places 1-4h
(preferably 3h)；And the volume ratio of the anhydrous ether and filtrate is (5:1)-(60:1) (preferably 30:1)；
And S9, the identification and purifying that carry out with HPLC/MS or ultrafiltration or high performance capillary electrophoresis polypeptide, finally
Obtain required polypeptide.
In addition, the present embodiment also provides a kind of application process of above-mentioned cell-penetrating peptide, specifically, the cell-penetrating peptide
For in C-terminal or N-terminal covalently or non-covalently linker or loading molecule, and carry the marker or loading molecule passes through
Cell membrane enters cell.In the present embodiment, the one kind or several of the marker in fluorescein, biotin and affinity groups
Kind；The one kind or several of the loading molecule in carbohydrate, polypeptide, albumen, drug molecule precursor, nano-particle and nanoparticle
Kind.
Wherein, it carries the marker or loading molecules across cellular membranes enters the process of cell and include：The cell is worn
Film peptide is combined to mediate Plasmid DNA with Plasmid DNA；In the present embodiment, the plasmid is selected from pEGFP or PdsRED.
Specifically, it includes following step that the cell-penetrating peptide, which is combined with Plasmid DNA for mediating the process of Plasmid DNA,
Suddenly：
S1, Plasmid DNA and the cell-penetrating peptide are respectively mixed in phosphate buffer；
S2, the adjustment Plasmid DNA and N/P (the i.e. NH of the cell-penetrating peptide between the two+3/PO-4) than being 0-80；It is excellent
Choosing, N/P ratios are respectively 0,1,3,5,10,20,40,80 between the two for adjustment；By it is above-mentioned adjust N/P than Plasmid DNA and institute
It states cell-penetrating peptide mixed liquor and carries out agarose electrophoresis experiment, determine optimal N/P ratios, the results are shown in Figure 1, and N/P is 5 or so
When, Plasmid DNA is combined preferably with cell-penetrating peptide, and therefore, the above-mentioned ratios of N/P between the two can be more preferably 5；
The cell of S3, overnight incubation are washed with the culture medium of serum-free, will contain the Plasmid DNA and cell-penetrating peptide
Phosphate buffer be added in culture medium；
And remove culture medium, training of the addition containing 10-15%FCS (i.e. fetal calf serum) after S4,4-6h (preferably 5h)
Base is supported, 1-4h (preferably 3h) is stood.
Embodiment two：
The present embodiment the difference is that only with embodiment one, in the step S2 of the preparation method of cell-penetrating peptide, often
The mole of kind amino acid (including arginine, alanine, glutamine, proline and lysine) is the resin mole
5 times, the mole of the condensing agent is 10 times of the resin mole.
Embodiment three：
The present embodiment the difference is that only with embodiment one, in the step S2 of the preparation method of cell-penetrating peptide, often
The mole of kind amino acid (including arginine, alanine, glutamine, proline and lysine) is the resin mole
1 times, the mole of the condensing agent is 10 times of the resin mole.
Example IV：
The present embodiment the difference is that only with embodiment one, in the step S2 of the preparation method of cell-penetrating peptide, often
The mole of kind amino acid (including arginine, alanine, glutamine, proline and lysine) is the resin mole
5 times, the mole of the condensing agent is 1 times of the resin mole.
Embodiment five：
The present embodiment the difference is that only with embodiment one, in the step S2 of the preparation method of cell-penetrating peptide, often
The mole of kind amino acid (including arginine, alanine, glutamine, proline and lysine) is the resin mole
10 times, the mole of the condensing agent is 10 times of the resin mole.
Embodiment six：
The present embodiment the difference is that only with embodiment one, in the step S2 of the preparation method of cell-penetrating peptide, kind
The mole of amino acid (including arginine, alanine, glutamine, proline and lysine) is the resin mole
7 times, the mole of the condensing agent is 5 times of the resin mole.
It shows as follows and wears film effect experiment process after the cell-penetrating peptide YTHDC1 of the present invention is combined with Plasmid DNA,
It specifically comprises the following steps：
(1) experimental group be the present invention cell-penetrating peptide, by the present invention cell-penetrating peptide YTHDC1 and pEGFP (or
PdsRED 0,5,10,20,40,80 N/P ratios mixing) is pressed, room temperature or 37 DEG C are incubated half an hours or a hour；Control group
For cell penetrating peptide Tat, equally mixed respectively by 0,5,10,20,40,80 N/P ratios with pEGFP (or PdsRED), incubation time with
And temperature condition is identical as experimental group；
(2) it cultivates cell (being respectively BHK21 and B16 cell lines) overnight, is washed twice with the culture medium of not serum；Experiment
In group, Plasmid DNA and cell-penetrating peptide YTHDC1 are added in cell, forms experimental group mixture, then takes the experimental group mixed
500 μ l of object are closed to be added in culture medium；
Remove former culture medium after (3) 6 hours, adds the fresh culture containing 10% fetal calf serum；
(4) micro- sem observation after 1-4 hours；As shown in Fig. 2, in BHK21 cell lines, cell-penetrating peptide of the invention
When YTHDC1 is combined with Plasmid DNA under conditions of N/P ratios are 10, Plasmid DNA can be brought into intracellular, be 20 in N/P ratios
When, the membrane efficiency of wearing that membrane efficiency can be more than Tat is worn, and when N/P ratios are 80, wear membrane efficiency highest, far above Tat's
Wear membrane efficiency.Similar, as shown in figure 3, in B16 cell lines, cell-penetrating peptide YTHDC1 of the invention is with Plasmid DNA in N/
When P ratios combine under conditions of being 5, Plasmid DNA can be brought into intracellular, when N/P ratios are 40, wear membrane efficiency highest, and
Membrane efficiency is worn far above Tat.
(5) BHK21 or B16 cell line cells detect after above-mentioned processing 4h through microplate reader, obtain fluorescent quantitation as a result, such as
Shown in Fig. 4, in BHK21 and B16 cell lines, for cell-penetrating peptide YTHDC1 of the invention on each concentration value, fluorescence is strong
Degree is better than the fluorescence intensity of Tat, and at 20 μM, fluorescence intensity is most strong.
The following cell-penetrating peptide YTHDC1 cytotoxicity experiment processes for showing the present invention, specifically include following step
Suddenly：
(1) logarithmic growth phase culture cell BHK21 and B16, with every hole 1 × 104A cell inoculation is in 96 orifice plates, 37 DEG C
Under cultivated 24 hours in 5% carbon dioxide incubator, keep cell adherent；
(2) culture solution for changing serum-free when reaching exponential phase into continues culture 1 hour；
(3) the cell-penetrating peptide YTHDC1 of various concentration is configured, while three negative control holes and Positive control wells are set
(Tat), Positive control wells (Tat) operate tested to specifications；In 5% carbon dioxide incubator culture 1- at 37 DEG C
24h；
(4) after incubation time, PBS washings are added per hole；
(5) 20 μ l MTT (0.5%) are added per hole for attached cell, continue to discard culture solution after incubation 4-6h, add per hole
Enter 150 μ l DMSO (dimethyl sulfoxide (DMSO)), shakes 10min；
(6) colorimetric：490nm or 570nm wavelength is selected, absorbance value is measured in microplate reader immune detector, handles number
According to obtaining cell survival rate；As a result as shown in Fig. 5 a-5b, for BHK21 cell lines, cell-penetrating peptide YTHDC1 of the present invention is in 0-
In the concentration range of 16 μm of ol/L, cell survival rate is with positive controls without significant difference；It is thin for B16 as shown in Fig. 6 a-6b
Born of the same parents are, cell-penetrating peptide YTHDC1 of the present invention in the concentration range of 0-16 μm of ol/L, cell survival rate and positive controls without
Significant difference；Above-mentioned the results show cell-penetrating peptide safety and low toxicity of the present invention, toxicity and Tat indifferences, and
The toxicity variation of each concentration is little, no concentration dependent.
In conclusion the cell-penetrating peptide YTHDC1 of the present invention has the following advantages that：Molecular weight is small, amino acid residue number
It is few, film significant effect is worn, efficiency is apparently higher than cell penetrating peptide Tat；Immunogenicity is low, safety and low toxicity, toxicity and Tat indifferences；It can
It is obtained by solid phase synthesis process, the synthetic method can both be carried out in laboratory level or in industrial level, easy to operate
And it is convenient for quality management and control.
The foregoing is merely presently preferred embodiments of the present invention, is not intended to limit the invention, it is all the present invention spirit and
Within principle, any modification, equivalent replacement, improvement and so on should all be included in the protection scope of the present invention.
Claims (10)
1. a kind of cell-penetrating peptide, it is characterised in that：The cell-penetrating peptide includes following sequence：
RRRRRKQARRPRRRRAR。
2. a kind of preparation method of cell-penetrating peptide as described in claim 1, which is characterized in that include the following steps：
S1, resin is provided, and the resin is dipped to the resin swelling in the first solvent；
S2, amino acid, condensing agent are dissolved in the second solvent, form mixture；
S3, the resin of swelling is added in the mixture to form reaction system, reaction time 1-4h；
Solution after S4, removal reaction in the reaction system, washs residue 1-5 times, gas is agitated, and is drained；
S5, liquid of raising one's hat is added in the residue, gas is agitated, drained；
S6, removal are washed 1-10 times, gas is agitated, and is drained, thus to obtain conjunction via the solution in the reaction system after step S5
At polypeptide chain；
S7, cutting liquid is added in the polypeptide chain, stirs 1-4h at 4-30 DEG C, filters to get filtrate, and wash the filtrate；
S8, the filtrate of washed mistake is extracted, centrifugation obtains cell-penetrating peptide crude product；
And S9, the cell-penetrating peptide crude product is identified and purified.
3. preparation method as claimed in claim 2, which is characterized in that the resin includes Fmoc-Wang resins.
4. preparation method as claimed in claim 2, which is characterized in that in step s 2, the condensing agent includes benzotriazole
Salt form condensing agent；In step sl, first solvent includes：Dichloromethane, dimethylformamide, dichloromethane and tetrahydrochysene
One or more of furans；In step s 5, the liquid of raising one's hat includes：Dimethyl formamide solution, and it is dissolved in diformazan
The piperazine and 0.1M HOBT of 6%W/V in base formamide solution；In the step s 7, the cutting liquid includes：With weight percent
Number meter, 95% TFA, 2% TIS and 3% H2O。
5. preparation method as claimed in claim 4, which is characterized in that the benzotriazole salt form condensing agent includes O- benzos
Triazole-tetramethylurea hexafluorophosphate, one kind in O- benzotriazole-N, N, N', N'- tetramethylurea tetrafluoroborate or
It is several.
6. preparation method as claimed in claim 2, which is characterized in that the mole ratio that feeds intake of each amino acid and the resin
Example is (1:1)-(10:1)；The mole ratio that feeds intake of the condensing agent and the resin is (1:1)-(10:1).
7. preparation method as claimed in claim 2, which is characterized in that in the step S8, the extraction includes：Washed
Anhydrous ether is added in the filtrate crossed, places 1-4h；And the volume ratio of the anhydrous ether and filtrate is (5:1)-(60:1).
8. a kind of application process of cell-penetrating peptide as described in claim 1, which is characterized in that the cell-penetrating peptide is used for
In C-terminal or N-terminal covalently or non-covalently linker or loading molecule, and carry the marker or loading molecule passes through cell
Film enters cell.
9. application process as claimed in claim 8, which is characterized in that carry the marker or loading molecules across cellular membranes
Process into cell includes：The cell-penetrating peptide is combined to mediate Plasmid DNA with Plasmid DNA.
10. such as claim 9 any one of them application process, which is characterized in that the cell-penetrating peptide is combined with Plasmid DNA
For mediating the process of Plasmid DNA to include the following steps：
S1, Plasmid DNA and the cell-penetrating peptide are respectively mixed in phosphate buffer；
S2, the adjustment Plasmid DNA and the N/P ratios of the cell-penetrating peptide between the two are 0-80；
The cell of S3, overnight incubation are washed with the culture medium of serum-free, by the phosphorus containing the Plasmid DNA and cell-penetrating peptide
Phthalate buffer is added in culture medium；
And remove culture medium after S4,4-6h, the culture medium containing 10-15%FCS is added, 1-4h is stood.
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| CN108059655A (en) * | 2017-12-25 | 2018-05-22 | 肽泽（武汉）生物科技有限公司 | A kind of cell-penetrating peptide and preparation method thereof, application |
| CN108707187A (en) * | 2018-06-12 | 2018-10-26 | 肽泽（武汉）生物科技有限公司 | A kind of cell-penetrating peptide and preparation method thereof, application |
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| CN107987129A (en) * | 2017-12-25 | 2018-05-04 | 肽泽（武汉）生物科技有限公司 | A kind of cell-penetrating peptide and preparation method thereof, application |
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Application publication date: 20181113 |