CN107245099A - Dendroid Humanized cell cell-penetrating peptide hPP7K, production and its mediation plasmid DNA transfection method - Google Patents
Dendroid Humanized cell cell-penetrating peptide hPP7K, production and its mediation plasmid DNA transfection method Download PDFInfo
- Publication number
- CN107245099A CN107245099A CN201710046566.1A CN201710046566A CN107245099A CN 107245099 A CN107245099 A CN 107245099A CN 201710046566 A CN201710046566 A CN 201710046566A CN 107245099 A CN107245099 A CN 107245099A
- Authority
- CN
- China
- Prior art keywords
- cell
- penetrating peptide
- hpp7k
- lys
- drained
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- 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
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
Abstract
The present invention relates to biomedical sector, a kind of new dendroid Humanized cell cell-penetrating peptide hPP7K, production and its mediation plasmid DNA transfection method are disclosed.HPP7K is new dendroid Humanized cell cell-penetrating peptide, in C-terminal or N-terminal covalently or non-covalently linker or loading molecule, and carries label or cargo molecule enters cell through cell membrane.The invention has the advantages that：Cell-penetrating peptide hPP7K belongs to the improvement and modification of Humanized cell cell-penetrating peptide, and immunogenicity is low, safety and low toxicity；Wear film effect significantly, efficiency is higher than hPP10；Cell-penetrating peptide hPP7K is obtained by synthesis in solid state, and cost is relatively low, is easy to quality management and control.Label can be carried or cargo molecule cross-film enters various kinds of cell, be a kind of transdermal delivery carrier of great DEVELOPMENT PROSPECT.
Description
Technical field
The present invention relates to biomedical sector, it is related to a kind of Humanized cell cell-penetrating peptide, production and its mediation DNA and turns
The method of dye.
Background technology
At this stage, most of diseases are required for carrying out diagnoses and treatment in molecular level, and in face of cell membrane, many has body
Outer bioactive macromolecule medicine cannot be introduced into it is intracellular, so that its due effect can not be played.To overcome macromolecular in body
Interior transit barrier, people develop various technologies.Use viral vector, although transport efficacy is high, but its potential toxicity is not
It can ignore.Non-viral method, as the methods such as electroporation, microinjection have necessarily traumatic to cell, may be damaged very
To destruction cell membrane.And the method for Non-Invasive such as liposome, it is desirable to make film loss of stability under low acid environment to discharge
Medicine enters endochylema, has certain limitation to pharmaceutically-active research in human physiological environment.
In recent years, increasingly paid attention to as the dendritic macromole of new drug carrier by scientist.Dendroid is big
Molecule has retention time of the extension effective treatment concentration of medicine in blood circulation as carrier, protects a drug from biocycle
The advantages of border is destroyed, toxic side effect is small and easy to use, so as to significantly improve the utilization rate of medicine.In the early 1990s,
The research such as Haensler shows that dendritic can carry out efficient gene transfer.The surface of dendritic macromole is highdensity
Amino can be combined and spatially high compression with the large biological molecule such as DNA, as non-viral type carrier by DNA and oligonucleotides
Efficiently it is transported in cell Deng inhereditary material, so that the function with regulation Antisense Suppression.
In in the past few decades, it has been found that some peptides and protein energy penetration cell film enter intracellular, and
A variety of transport molecules can also be connected with these peptides and protein and transposition enters intracellular.These peptides and protein carrier are constituted
A kind of new very promising medicament transport carrier, i.e. cell-penetrating peptide (cell-penetrating peptides, CPPs),
It is the small peptide that a major class is made up of 10-30 amino acid, also referred to as protein transduction domain (protein translocation
domain,PTD).These peptide molecules will not produce the permanent damage of cell membrane, and toxicity is low.Particularly humanized wears film
Peptide (hCPP), it is compared with the CPPs of other biological source place, and hCPP causes the possibility of the immune response of human body smaller, potential
Relatively fewer (such as hPP10 publication numbers of unsafe factor：102863516A), but for hPP10, it mediates DNA
What is transfected is less efficient, influences it as the application prospect of medicine intracellular delivery vehicle.It is high that the present invention combines dendritic macromole
Effect transfection and the advantage of hPP10 low toxicities, a kind of synthetically produced new dendroid Humanized cell cell-penetrating peptide.
The content of the invention
The technical problems to be solved by the invention are to provide a kind of dendroid Humanized cell cell-penetrating peptide, production and its mediated
Plasmid DNA transfection method, to overcome the shortcomings of that the transfection efficiency that existing cell-penetrating peptide is present is low, potential danger factor.
In order to solve the above technical problems, the present invention is carried except a kind of new dendroid Humanized cell cell-penetrating peptide first
HPP7K, its sequence of its amino acid is expressed as follows：
The dendroid Humanized cell cell-penetrating peptide hPP7K of the present invention, can covalently or non-covalently be acted on biological micromolecule
It is combined with each other, and carries biological micromolecule and enters cell through cell membrane.
The dendroid Humanized cell cell-penetrating peptide hPP7K is in C-terminal or N-terminal covalently or non-covalently linker or loading
Molecule, and carry label or cargo molecule enters cell through cell membrane.
It is preferred that, the label is selected from the group being made up of fluorescein, biotin, affinity groups；
It is preferred that, the cargo molecule is selected from micro- by carbohydrate, polypeptide, albumen, drug molecule precursor, nano-particle, nanometer
The group that ball is constituted.
The dendroid Humanized cell cell-penetrating peptide hPP7K production methods of the present invention are as follows：
(1) a certain amount of Fmoc-Arg (PBF)-Wang resins or H-Arg (PBF) -2cl resins are weighed, glass reaction is poured into
In device, proper amount of dichloromethane (DCM) immersion is added until resin swelling, is drained；
(2) appropriate deprotection solution is added into reactor, logical nitrogen is agitated 0.5-2 hours, drained, adds into reactor
Enter appropriate dimethylformamide (DMF), nitrogen is agitated, and is drained, and is repeated 1-10 times；
(3) weigh and often walk the amino acid that feeds intake, the carbon two of 1~10 times of mole equivalent to 1~10 times of mole of resin
Hypotype condensing agent or BTA salt form condensing agent, which are dissolved in dimethylformamide (DMF) or DCM or tetrahydrofuran (THF), to be added
Enter reactor, react 1-4h, ninhydrin method detection is until stop reaction when not developing the color；
(4) solution in reactor is drained, plus appropriate DMF is washed, nitrogen is agitated, drained, repeated washing 1-5 times；
(5) appropriate liquid of raising one's hat is added into reactor, nitrogen is agitated 1-3h, drained, ninhydrin method tests positive；
(6) solution in reactor is drained, plus appropriate DMF is washed, nitrogen is agitated, drained, repeated washing 1-10 times；
(7) aforesaid operations are completed up to polypeptide chain is synthesized repeatedly, obtain Lys2-Lys) 2-Lys-Lys-Ile-Pro-Leu-
Pro-Arg-Phe-Lys-Leu-Lys-Cys-Ile-Phe-Cys-Lys-Lys-Arg-Arg-Lys-Arg-OH；
(8) dried peptide-resin is fitted into suitable centrifuge tube, adds the cutting liquid 4-30 degree constant temperature prepared in right amount
Mechanical agitation 1-4h, is washed after filtering with TFA, filtering, and filtrate is all collected into flask, is directly added into 5-60 times of volume
Ice absolute ether, places polypeptide crude product needed for being obtained after 1-4h, high speed centrifugation；
(9) identification and purifying of polypeptide are carried out with HPLC/MS or ultrafiltration or high performance capillary electrophoresis, is finally given
Required polypeptide and the polypeptide chain hPP7K for adding green fluorescent label.
The dendroid Humanized cell cell-penetrating peptide hPP7K of present invention mediation plasmid DNA transfection method is as follows：
(i) cell-penetrating peptide hPP7K is obtained according to above-mentioned chemical synthesis process, and plasmid pEGFP or PdsRED is worn with above-mentioned
Film peptide is mixed in phosphate buffer (PBS) respectively；
(ii) the N/P ratios (NH+3/PO-4) between plasmid and cell-penetrating peptide content are calculated, N/P ratios between the two are adjusted；
(ii) cell overnight is supported, is washed with the culture medium without serum twice, DNA is added and cell-penetrating peptide compound adds
Enter into 500ul culture mediums；
(iv) 6 as a child removed culture medium and add the fresh culture containing 10%FCS；
(v) 1-4 hours are stood.
The present invention mainly has advantages below and beneficial effect：
1) the cell-penetrating peptide hPP7K belongs to the improvement and modification of Humanized cell cell-penetrating peptide, and immunogenicity is low, safety
Low toxicity；
2) cell-penetrating peptide hPP7K wear film effect significantly, efficiency be higher than hPP10；
3) the cell-penetrating peptide hPP7K is obtained by synthesis in solid state, and cost is relatively low, is easy to quality management and control.
It can be widely used for producing the practical application of medicine, health products, beauty or skin care item, transfection reagent or diagnostic reagent.
Brief description of the drawings
Technical scheme is further described in detail with reference to the accompanying drawings and detailed description.
Fig. 1 is cell-penetrating peptide hPP7K of the present invention wheel-like structure schematic diagram；
Fig. 2 is cell-penetrating peptide hPP7K of the present invention spiral, folded structural representation；
Fig. 3 is that cell-penetrating peptide hPP7K of the present invention is tested from DNA with combining rear agar electrophoresis under conditions of different N/P ratios
Figure；
After Fig. 4 is combined from DNA under conditions of different N/P ratios respectively for cell-penetrating peptide hPP7K and hPP10 of the present invention,
Green fluorescence design sketch after being incubated 4 hours in BHK21 cell lines；
After Fig. 5 is combined from DNA under conditions of different N/P ratios respectively for cell-penetrating peptide hPP7K and hPP10 of the present invention,
Green fluorescence design sketch after being incubated 4 hours in B16 cell lines；
Fig. 6 be cell-penetrating peptide hPP7K and hPP10 of the present invention in the range of various concentrations, incubation is 4 small in BHK21 cell lines
When after green fluorescence intensity figure；
Fig. 7 be cell-penetrating peptide hPP7K and hPP10 of the present invention in the range of various concentrations, in B16 cell lines incubation 4 hours
Green fluorescence intensity figure afterwards；
Fig. 8 be cell-penetrating peptide hPP7K and hPP10 of the present invention in the range of various concentrations, incubation is 4 small in BHK21 cell lines
Shi Hou, the cell survival rate result figure detected by MTT；
Fig. 9 be cell-penetrating peptide hPP7K and hPP10 of the present invention in the range of various concentrations, in B16 cell lines incubation 4 hours
Afterwards, the cell survival rate result figure detected by MTT；
Embodiment
Dendroid Humanized cell cell-penetrating peptide hPP7K amino acid its sequence of the present invention is：
Dendroid Humanized cell cell-penetrating peptide hPP7K can covalently or non-covalently be acted on biological micromolecule to be combined with each other,
And carry biological micromolecule and enter cell through cell membrane.
Dendroid Humanized cell cell-penetrating peptide hPP7K is in C-terminal or N-terminal covalently or non-covalently linker or loading point
Son, and carry label or cargo molecule enters cell through cell membrane.Label is selected from by fluorescein, biotin, affine base
The constituted group of group；Cargo molecule is selected from by carbohydrate, polypeptide, albumen, drug molecule precursor, nano-particle, nanoparticle institute group
Into group.
Embodiment 1：
(1) a certain amount of Fmoc-Arg (PBF)-Wang resins or H-Arg (PBF) -2cl resins are weighed, glass reaction is poured into
In device, proper amount of dichloromethane (DCM) immersion is added until resin swelling, is drained；
(2) appropriate deprotection solution is added into reactor, logical nitrogen is agitated 0.5-2 hours, drained, adds into reactor
Enter appropriate dimethylformamide (DMF), nitrogen is agitated, and is drained, and is repeated 1-10 times；
(3) weigh and often walk the amino acid that feeds intake, the hypotype of carbon two condensation of 1 times of mole equivalent to 1 times of mole of resin
Agent (such as dicyclohexylcarbodiimide (DCC)) or BTA salt form condensing agent (such as O- BTAs-tetramethylurea hexafluoro
Phosphate (HBTU) or O- BTAs-N, N, N', N'- tetramethylurea tetrafluoroborate (TBTU)) it is dissolved in dimethyl formyl
Reactor is added in amine (DMF) or DCM or tetrahydrofuran (THF), 1-4h is reacted, ninhydrin method detection when not developing the color until stop
Reaction；
(4) solution in reactor is drained, plus appropriate DMF is washed, nitrogen is agitated, drained, repeated washing 1-5 times；
(5) appropriate liquid of raising one's hat is added into reactor, nitrogen is agitated 1-3h, drained, ninhydrin method tests positive；
(6) solution in reactor is drained, plus appropriate DMF is washed, nitrogen is agitated, drained, repeated washing 1-10 times；
(7) aforesaid operations are completed up to polypeptide chain is synthesized repeatedly, obtain Lys2-Lys) 2-Lys-Lys-Ile-Pro-Leu-
Pro-Arg-Phe-Lys-Leu-Lys-Cys-Ile-Phe-Cys-Lys-Lys-Arg-Arg-Lys-Arg-OH；
(8) dried resin is fitted into suitable centrifuge tube, adds the cutting liquid 4-30 degree constant temperature machines prepared in right amount
Tool stirs 1-4h, is washed after filtering with TFA, filters, filtrate is all collected into flask, the ice of 5-60 times of volume is directly added into
Absolute ether, places polypeptide crude product needed for being obtained after 1-4h, high speed centrifugation；
(9) identification and purifying of polypeptide are carried out with HPLC/MS or ultrafiltration or high performance capillary electrophoresis, is finally given
Required polypeptide and the polypeptide chain for adding green fluorescent label；
Embodiment 2：
(1) a certain amount of Fmoc-Arg (PBF)-Wang resins or H-Arg (PBF) -2cl resins are weighed, glass reaction is poured into
In device, proper amount of dichloromethane (DCM) immersion is added until resin swelling, is drained；
(2) appropriate deprotection solution is added into reactor, logical nitrogen is agitated 0.5-2 hours, drained, adds into reactor
Enter appropriate DMF, nitrogen is agitated, and is drained, and is repeated 1-10 times；
(3) weigh and often walk the amino acid that feeds intake, the hypotype of carbon two contracting of 10 times of moles equivalent to 10 times of moles of resin
Mixture (such as dicyclohexylcarbodiimide (DCC)) or BTA salt form condensing agent (such as O- BTAs-tetramethylurea six
Fluorophosphate (HBTU) or O- BTAs-N, N, N', N'- tetramethylurea tetrafluoroborate (TBTU)) it is dissolved in dimethyl methyl
Reactor is added in acid amides (DMF) or DCM or tetrahydrofuran (THF), 1-4h is reacted, ninhydrin method detection when not developing the color until stop
Only react；
(4) solution in reactor is drained, plus appropriate DMF is washed, nitrogen is agitated, drained, repeated washing 1-5 times；
(5) appropriate liquid of raising one's hat is added into reactor, nitrogen is agitated 1-3h, drained, ninhydrin method tests positive；
(6) solution in reactor is drained, plus appropriate DMF is washed, nitrogen is agitated, drained, repeated washing 1-10 times；
(7) aforesaid operations are completed up to polypeptide chain is synthesized repeatedly, obtain Lys2-Lys) 2-Lys-Lys-Ile-Pro-Leu-
Pro-Arg-Phe-Lys-Leu-Lys-Cys-Ile-Phe-Cys-Lys-Lys-Arg-Arg-Lys-Arg-OH；
(8) dried resin is fitted into suitable centrifuge tube, adds the cutting liquid 4-30 degree constant temperature machines prepared in right amount
Tool stirs 1-4h, is washed after filtering with TFA, filters, filtrate is all collected into flask, the ice of 5-60 times of volume is directly added into
Absolute ether, places polypeptide crude product needed for being obtained after 1-4h, high speed centrifugation；
(9) identification and purifying of polypeptide are carried out with HPLC/MS or ultrafiltration or high performance capillary electrophoresis, is finally given
Required polypeptide and the polypeptide chain for adding green fluorescent label.
Embodiment 3：
(1) a certain amount of Fmoc-Arg (PBF)-Wang resins or H-Arg (PBF) -2cl resins are weighed, glass reaction is poured into
In device, proper amount of dichloromethane (DCM) immersion is added until resin swelling, is drained；
(2) appropriate deprotection solution is added into reactor, logical nitrogen is agitated 0.5-2 hours, drained, adds into reactor
Enter appropriate DMF, nitrogen is agitated, and is drained, and is repeated 1-10 times；
(3) weigh and often walk the amino acid that feeds intake, the hypotype of carbon two condensation of 5 times of moles equivalent to 5 times of moles of resin
Agent (such as dicyclohexylcarbodiimide (DCC)) or BTA salt form condensing agent (such as O- BTAs-tetramethylurea hexafluoro
Phosphate (HBTU) or O- BTAs-N, N, N', N'- tetramethylurea tetrafluoroborate (TBTU)) it is dissolved in dimethyl formyl
Reactor is added in amine (DMF) or DCM or tetrahydrofuran (THF), 1-4h is reacted, ninhydrin method detection when not developing the color until stop
Reaction；
(4) solution in reactor is drained, plus appropriate DMF is washed, nitrogen is agitated, drained, repeated washing 1-5 times；
(5) appropriate liquid of raising one's hat is added into reactor, nitrogen is agitated 1-3h, drained, ninhydrin method tests positive；
(6) solution in reactor is drained, plus appropriate DMF is washed, nitrogen is agitated, drained, repeated washing 1-10 times；
(7) aforesaid operations are completed up to polypeptide chain is synthesized repeatedly, obtain Lys2-Lys) 2-Lys-Lys-Ile-Pro-Leu-
Pro-Arg-Phe-Lys-Leu-Lys-Cys-Ile-Phe-Cys-Lys-Lys-Arg-Arg-Lys-Arg-OH；
(8) dried resin is fitted into suitable centrifuge tube, adds the cutting liquid 4-30 degree constant temperature machines prepared in right amount
Tool stirs 1-4h, is washed after filtering with TFA, filters, filtrate is all collected into flask, the ice of 5-60 times of volume is directly added into
Absolute ether, places polypeptide crude product needed for being obtained after 1-4h, high speed centrifugation；
(9) identification and purifying of polypeptide are carried out with HPLC/MS or ultrafiltration or high performance capillary electrophoresis, is finally given
Required polypeptide and the polypeptide chain for adding green fluorescent label.
Embodiment 4：
(1) a certain amount of Fmoc-Arg (PBF)-Wang resins or H-Arg (PBF) -2cl resins are weighed, glass reaction is poured into
In device, proper amount of dichloromethane (DCM) immersion is added until resin swelling, is drained；
(2) appropriate deprotection solution is added into reactor, logical nitrogen is agitated 0.5-2 hours, drained, adds into reactor
Enter appropriate DMF, nitrogen is agitated, and is drained, and is repeated 1-10 times；
(3) weigh and often walk the amino acid that feeds intake, the hypotype of carbon two condensation of 7 times of moles equivalent to 3 times of moles of resin
Agent (such as dicyclohexylcarbodiimide (DCC)) or BTA salt form condensing agent (such as O- BTAs-tetramethylurea hexafluoro
Phosphate (HBTU) or O- BTAs-N, N, N', N'- tetramethylurea tetrafluoroborate (TBTU)) it is dissolved in dimethyl formyl
Reactor is added in amine (DMF) or DCM or tetrahydrofuran (THF), 1-4h is reacted, ninhydrin method detection when not developing the color until stop
Reaction；
(4) solution in reactor is drained, plus appropriate DMF is washed, nitrogen is agitated, drained, repeated washing 1-5 times；
(5) appropriate liquid of raising one's hat is added into reactor, nitrogen is agitated 1-3h, drained, ninhydrin method tests positive；
(6) solution in reactor is drained, plus appropriate DMF is washed, nitrogen is agitated, drained, repeated washing 1-10 times；
(7) aforesaid operations are completed up to polypeptide chain is synthesized repeatedly, obtain Lys2-Lys) 2-Lys-Lys-Ile-Pro-Leu-
Pro-Arg-Phe-Lys-Leu-Lys-Cys-Ile-Phe-Cys-Lys-Lys-Arg-Arg-Lys-Arg-OH；
(8) dried resin is fitted into suitable centrifuge tube, adds the cutting liquid 4-30 degree constant temperature machines prepared in right amount
Tool stirs 1-4h, is washed after filtering with TFA, filters, filtrate is all collected into flask, the ice of 5-60 times of volume is directly added into
Absolute ether, places polypeptide crude product needed for being obtained after 1-4h, high speed centrifugation；
(9) identification and purifying of polypeptide are carried out with HPLC/MS or ultrafiltration or high performance capillary electrophoresis, is finally given
Required polypeptide and the polypeptide chain for adding green fluorescent label.
Embodiment 5：
(1) a certain amount of Fmoc-Arg (PBF)-Wang resins or H-Arg (PBF) -2cl resins are weighed, glass reaction is poured into
In device, proper amount of dichloromethane (DCM) immersion is added until resin swelling, is drained；
(2) appropriate deprotection solution is added into reactor, logical nitrogen is agitated 0.5-2 hours, drained, adds into reactor
Enter appropriate DMF, nitrogen is agitated, and is drained, and is repeated 1-10 times；
(3) weigh and often walk the amino acid that feeds intake, the hypotype of carbon two condensation of 3 times of moles equivalent to 7 times of moles of resin
Agent (such as dicyclohexylcarbodiimide (DCC)) or BTA salt form condensing agent (such as O- BTAs-tetramethylurea hexafluoro
Phosphate (HBTU) or O- BTAs-N, N, N', N'- tetramethylurea tetrafluoroborate (TBTU)) it is dissolved in dimethyl formyl
Reactor is added in amine (DMF) or DCM or tetrahydrofuran (THF), 1-4h is reacted, ninhydrin method detection when not developing the color until stop
Reaction；
(4) solution in reactor is drained, plus appropriate DMF is washed, nitrogen is agitated, drained, repeated washing 1-5 times；
(5) appropriate liquid of raising one's hat is added into reactor, nitrogen is agitated 1-3h, drained, ninhydrin method tests positive；
(6) solution in reactor is drained, plus appropriate DMF is washed, nitrogen is agitated, drained, repeated washing 1-10 times；
(7) aforesaid operations are completed up to polypeptide chain is synthesized repeatedly, obtain Lys2-Lys) 2-Lys-Lys-Ile-Pro-Leu-
Pro-Arg-Phe-Lys-Leu-Lys-Cys-Ile-Phe-Cys-Lys-Lys-Arg-Arg-Lys-Arg-OH；
(8) dried resin is fitted into suitable centrifuge tube, adds the cutting liquid 4-30 degree constant temperature machines prepared in right amount
Tool stirs 1-4h, is washed after filtering with TFA, filters, filtrate is all collected into flask, the ice of 5-60 times of volume is directly added into
Absolute ether, places polypeptide crude product needed for being obtained after 1-4h, high speed centrifugation；
(9) identification and purifying of polypeptide are carried out with HPLC/MS or ultrafiltration or high performance capillary electrophoresis, is finally given
Required polypeptide and the polypeptide chain for adding green fluorescent label.
(10) cell-penetrating peptide hPP7K is obtained according to above-mentioned chemical synthesis process, and plasmid pEGFP or PdsRED is worn with above-mentioned
Film peptide is mixed in phosphate buffer (PBS) respectively；
(11) the N/P ratios (NH between plasmid and cell-penetrating peptide content is calculated+ 3/PO- 4), adjustment between the two N/P ratios respectively 0,1,
2、3、5、10、20、40、80；
(12) by it is above-mentioned adjust N/P than mixed liquor carry out agarose electrophoresis experiment, determine optimal N/P ratios, as a result such as
Shown in Fig. 3, N/P is at 5, and plasmid is bound preferably with cell-penetrating peptide.
CPPs primary structures, secondary structure analysis, predict, identify novel human-derived property CPPs：
The cell-penetrating peptide hPP7K obtained to the present invention secondary structure is analyzed, and employs online point of emboss
(its analysis program refers to webpage to analysis program：http://emboss.bioinformatics.nl/cgi-bin/emboss/
The wheel-like structure of pepwheel on-line analysis polypeptides；http://emboss.bioinformatics.nl/cgi-bin/
The spiral of emboss/ on-line analysis secondary structures, folded etc.).HPP7K wheel-like structure schematic diagram, spiral, folded structural representation
Figure difference is as depicted in figs. 1 and 2.
Cell-penetrating peptide hPP7K combined with DNA after wear film effect experiment：
(1) the synthesized cell-penetrating peptide hPP7K and pEGFP or PdsRED of the present invention is mixed by 0,5,10,20,40,80 N/P ratios
Close, room temperature or 37 DEG C of incubation half an hours or a hour；Cell-penetrating peptide hPP10 and pEGFP or PdsRED are by 0,5,10,20,
40th, 80 N/P is than mixing, room temperature or 37 DEG C of incubation half an hours or a hour.Positive controls are respectively obtained to use2000, experimental method is shown to specifications；
(2) experimental cell is respectively BHK21 and B16 cell lines.Cell pellet overnight is cultivated, two are washed with the culture medium without serum
It is secondary, add DNA and cell-penetrating peptide compound is added in 500ul culture mediums；
(3) 6, which as a child removed culture medium, adds the fresh culture containing 10% hyclone；
Micro- sem observation after (4) 4 hours, in BHK21 cell lines, as a result as shown in figure 4, hPP7K and DNA are in N/
P ratios are that it can bring DNA into intracellular, and when N/P ratios are 10, it wears membrane efficiency highest when combining under conditions of 5,
But it is still below2000.And hPP10 N/P ratios be 40 when, it could bring DNA into intracellular, and wear
Membrane efficiency is far below hPP7K.In B16 cell lines, as a result as shown in figure 5, hPP7K and matter similar with BHK21 cell line situations
Grain DNA is when N/P ratios are to combine under conditions of 10, and its transfection efficiency is higher, but wears membrane efficiency and be less than
2000.And hPP10 is when N/P ratios are 40, it could bring DNA into intracellular, and wear membrane efficiency far below hPP7K.
(5) BHK21 or B16 cell line cells obtain fluorescent quantitation knot through flow cytomery after above-mentioned processing 4h
Really, in BHK21 cell lines, as shown in fig. 6, hPP7K is in 8uM, its fluorescence intensity is most strong, and in 0-16uM concentration range
Interior, its fluorescence intensity is far longer than hPP10 fluorescence intensity, but is respectively less than2000；In B16 cells
In system, as shown in fig. 7, hPP7K is in 8uM, its fluorescence intensity is most strong, and in 0-16uM concentration range, its fluorescence intensity
It is far longer than hPP10 fluorescence intensity, but is respectively less than2000。
(6) in summary, in CHR1-6 serial cell-penetrating peptide, CHR5 cell-penetrating efficiency optimization.
Cytotoxicity experiment：
(1) take the logarithm growth period culture cell BHK21 and B16, with every hole 1 × 104Individual cell is inoculated in 96 orifice plates, 37
DEG C, 5% CO2gas incubator culture 24 hours makes cell attachment；
(2) to exponential phase, change the nutrient solution of serum-free into, continue to cultivate 1 hour；
(3) the cell-penetrating peptide hPP7K and hPP10 of various concentrations are configured, while setting three negative control holes and positive control
Hole (2000), Positive control wells (2000) operation is carried out in fact to specifications
Test.37 DEG C, 5% CO2gas incubator culture 1-24h；
(4) after incubation time terminates, PBS washings are added per hole；
(5) attached cell adds 20ul MTT (0.5%) per hole, continues to discard nutrient solution after incubation 4-6h, adds per hole
Enter 150ul DMSO (dimethyl sulfoxide (DMSO)), shake 10min；
(6) colorimetric：490nm or 570nm wavelength is selected, absorbance value is determined in ELIASA immune detector, number is handled
According to cell survival rate is obtained, for BHK21 cell lines, as a result as shown in figure 8, cell-penetrating peptide hPP7K of the present invention is in 1-
In 16umol/L concentration range, cell survival rate is above positive controls and hPP10 treatment groups；For B16 cell lines, knot
Fruit is as shown in figure 9, cell-penetrating peptide hPP7K of the present invention is in 1-16umol/L concentration range, and cell survival rate is above sun
Property control group, and it is little with hPP10 treatment group difference.Above-mentioned the results show cell-penetrating peptide of the present invention is low safely
Poison, its toxicity is below2000, and the toxicity change of each concentration is little, no concentration dependent.
It should be noted last that, above embodiment is merely illustrative of the technical solution of the present invention and unrestricted,
Although the present invention is described in detail with reference to preferred embodiment, it will be understood by those within the art that, can be right
Technical scheme is modified or equivalent substitution, and without departing from the spirit and scope of technical solution of the present invention, its is equal
It should cover among scope of the presently claimed invention.
Claims (7)
1. a kind of dendroid Humanized cell cell-penetrating peptide hPP7K, it is characterised in that the amino acid sequence of the cell-penetrating peptide
For：
2. dendroid Humanized cell cell-penetrating peptide hPP7K according to claim 1, it is characterised in that can be small with biology
Molecule covalent or noncovalent interaction be combined with each other, and carry biological micromolecule and enter cell through cell membrane.
3. the dendroid Humanized cell cell-penetrating peptide hPP7K according to claim 1, it is characterised in that the cell-penetrating peptide is in C
End or N-terminal covalently or non-covalently linker or loading molecule, and carry label or cargo molecule through cell membrane entrance
Cell.
4. dendroid Humanized cell cell-penetrating peptide hPP7K according to claim 3, it is characterised in that the label is selected from
The group being made up of fluorescein, biotin, affinity groups.
5. cell-penetrating peptide hPP7K according to claim 3, it is characterised in that the cargo molecule is selected from by carbohydrate, many
The group that peptide, albumen, drug molecule precursor, nano-particle or nanoparticle are constituted.
6. dendroid Humanized cell cell-penetrating peptide hPP7K production method described in a kind of claim 1, it is characterised in that including
Following steps：
(1) a certain amount of Fmoc-Arg (PBF)-Wang resins or H-Arg (PBF) -2cl resins are weighed, is poured into glass reactor,
Proper amount of dichloromethane (DCM) immersion is added until resin swelling, is drained；
(2) appropriate deprotection solution is added into reactor, logical nitrogen is agitated 0.5-2 hours, drained, and is added into reactor suitable
Dimethylformamide (DMF) is measured, nitrogen is agitated, and is drained, and is repeated 1-10 times；
(3) weigh and often walk the amino acid that feeds intake, the hypotype of carbon two of 1~10 times of mole equivalent to 1~10 times of mole of resin
Condensing agent or BTA salt form condensing agent, which are dissolved in dimethylformamide (DMF) or DCM or tetrahydrofuran (THF), to be added instead
Device is answered, 1-4h is reacted, ninhydrin method detection is until stop reaction when not developing the color；
(4) solution in reactor is drained, plus appropriate DMF is washed, nitrogen is agitated, drained, repeated washing 1-5 times；
(5) appropriate liquid of raising one's hat is added into reactor, nitrogen is agitated 1-3h, drained, ninhydrin method tests positive；
(6) solution in reactor is drained, plus appropriate DMF is washed, nitrogen is agitated, drained, repeated washing 1-10 times；
(7) aforesaid operations are completed up to polypeptide chain is synthesized repeatedly, obtain Lys2-Lys) 2-Lys-Lys-Ile-Pro-Leu-Pro-
Arg-Phe-Lys-Leu-Lys-Cys-Ile-Phe-Cys-Lys-Lys-Arg-Arg-Lys-Arg-OH；
(8) dried peptide-resin is fitted into suitable centrifuge tube, adds the cutting liquid 4-30 degree constant temperature machinery prepared in right amount
Stir 1-4h, washed after filtering with TFA, filter, filtrate is all collected into flask, be directly added into the ice of 5-60 times of volume without
Water ether, places polypeptide crude product needed for being obtained after 1-4h, high speed centrifugation；
(9) identification and after purification of polypeptide is carried out with HPLC/MS or ultrafiltration or high performance capillary electrophoresis, obtains required many
Peptide chain hPP7K.
7. dendroid Humanized cell cell-penetrating peptide hPP7K mediates plasmid DNA transfection method, its feature according to claim 1
It is, comprises the following steps：
(i) cell-penetrating peptide hPP7K is obtained according to above-mentioned chemical synthesis process, and by plasmid pEGFP or PdsRED and above-mentioned cell-penetrating peptide
It is mixed in respectively in phosphate buffer (PBS)；
(ii) the N/P ratios (NH+3/PO-4) between plasmid and cell-penetrating peptide content are calculated, N/P ratios between the two are adjusted；
(ii) cell overnight is supported, is washed with the culture medium without serum twice, DNA is added and cell-penetrating peptide compound is added to
In 500ul culture mediums；
(iv) 6 as a child removed culture medium and add the fresh culture containing 10%FCS；
(v) 1-4 hours are stood.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710046566.1A CN107245099B (en) | 2017-01-20 | 2017-01-20 | Dendritic human cell penetrating peptide hPP7K, production and plasmid DNA transfection mediated method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710046566.1A CN107245099B (en) | 2017-01-20 | 2017-01-20 | Dendritic human cell penetrating peptide hPP7K, production and plasmid DNA transfection mediated method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107245099A true CN107245099A (en) | 2017-10-13 |
| CN107245099B CN107245099B (en) | 2020-12-29 |
Family
ID=60016823
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710046566.1A Active CN107245099B (en) | 2017-01-20 | 2017-01-20 | Dendritic human cell penetrating peptide hPP7K, production and plasmid DNA transfection mediated method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107245099B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107261112A (en) * | 2017-07-25 | 2017-10-20 | 四川大学 | A kind of chiral tree-shaped peptides macromolecular induces the application of peptide medicament as autophagy |
| 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 |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102827254A (en) * | 2012-07-30 | 2012-12-19 | 三峡大学 | Cell penetrating peptide hPP10 and use thereof |
| CN102863516A (en) * | 2012-07-30 | 2013-01-09 | 三峡大学 | Production of cell-penetrating peptide hPP10 (human Pancreatic Polypeptide) and transfection method for mediated plasmid DNA (Deoxyribose Nucleic Acid) of hPP10 |
| CN104073516A (en) * | 2014-05-27 | 2014-10-01 | 浙江大学 | Gene vector as well as preparation method and application thereof |
| WO2014197835A3 (en) * | 2013-06-06 | 2015-02-26 | The General Hospital Corporation | Methods and compositions for the treatment of cancer |
| US20160361402A1 (en) * | 2010-11-18 | 2016-12-15 | Mayo Foundation For Medical Education And Research | Enhancing t cell activation using altered mhc-peptide ligands |
-
2017
- 2017-01-20 CN CN201710046566.1A patent/CN107245099B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20160361402A1 (en) * | 2010-11-18 | 2016-12-15 | Mayo Foundation For Medical Education And Research | Enhancing t cell activation using altered mhc-peptide ligands |
| CN102827254A (en) * | 2012-07-30 | 2012-12-19 | 三峡大学 | Cell penetrating peptide hPP10 and use thereof |
| CN102863516A (en) * | 2012-07-30 | 2013-01-09 | 三峡大学 | Production of cell-penetrating peptide hPP10 (human Pancreatic Polypeptide) and transfection method for mediated plasmid DNA (Deoxyribose Nucleic Acid) of hPP10 |
| WO2014197835A3 (en) * | 2013-06-06 | 2015-02-26 | The General Hospital Corporation | Methods and compositions for the treatment of cancer |
| CN104073516A (en) * | 2014-05-27 | 2014-10-01 | 浙江大学 | Gene vector as well as preparation method and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| KRISTEN SADLER: "Peptide dendrimers: applications and synthesis", 《REIEWS IN MOLECULAR BIOTECHNOLOGY》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107261112A (en) * | 2017-07-25 | 2017-10-20 | 四川大学 | A kind of chiral tree-shaped peptides macromolecular induces the application of peptide medicament as autophagy |
| CN107261112B (en) * | 2017-07-25 | 2020-04-21 | 四川大学 | Application of chiral dendritic peptide macromolecules as autophagy-inducing peptide drugs |
| 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 |
| CN108707187B (en) * | 2018-06-12 | 2021-01-29 | 肽泽（武汉）生物科技有限公司 | Cell-penetrating peptide and preparation method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107245099B (en) | 2020-12-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103550781B (en) | Dendrimer self assembly pharmaceutical carrier and its preparation method and application | |
| CN106800592A (en) | A kind of cell-penetrating peptide and preparation method thereof, application | |
| CN109355310B (en) | ROS (reactive oxygen species) -responsive gene delivery vector as well as preparation method and application thereof | |
| CN108728496B (en) | Polycation gene vector, preparation method and application thereof | |
| CN106916209B (en) | Amphiphilic polypeptide molecule capable of being used as gene vector | |
| CN103172701B (en) | A kind of novel cell-penetrating peptide and application thereof | |
| CN106916207A (en) | The method of the plasmid DNA transfection of cell-penetrating peptide hPP chol, production and its mediation | |
| CN101402965A (en) | Nano-golden particle-containing non-virogene carrier, production method and uses thereof | |
| CN111116904A (en) | Phenylboronic acid modified fluorine-containing high polymer material and application thereof in intracellular delivery of protein | |
| CN107629118A (en) | Targeting cell-penetrating peptide carrier and purposes based on histidine | |
| CN101085356A (en) | Non-virogene transfection carrier, complex particles of the same and plasmid DNA, preparing method and using method | |
| CN103990143B (en) | Multi-walled carbon nano-tubes medicine carrying composite of a kind of hepatoma-targeting and preparation method thereof | |
| CN106890343A (en) | A kind of targeting type polypeptide nano genophore compound | |
| CN105859990B (en) | The polymer of side chain sulfur-bearing caprylyl, its preparation method and polymer vesicle prepared therefrom and its application | |
| CN111544598B (en) | Ferritin nanoparticle loaded with Survivin double inhibitors as well as preparation method and application thereof | |
| CN107245099A (en) | Dendroid Humanized cell cell-penetrating peptide hPP7K, production and its mediation plasmid DNA transfection method | |
| CN101781373B (en) | Salbutamol modified guanidinated chitosan and preparation method and application thereof | |
| CN102140171A (en) | Glutathione-modified chitosan copolymer serving as non-viral gene carrier material and preparation and application thereof | |
| CN107296962A (en) | Chemical drug/gene cotransports the preparation method and application of functionalized carbon nano-tube | |
| CN102266566B (en) | Magnetic compound, and preparation method and purpose thereof | |
| CN104650192B (en) | One class can be used to repair self-assembled short peptide and its application of uterus and protection cardiac muscle | |
| CN103990138A (en) | Layer-by-layer assembled nanogold composite drug delivery carrier system, preparation method and application thereof | |
| CN106086079A (en) | The load gene composite of multiple targeting modification and preparation method and application | |
| CN102010508B (en) | Cationic polymer and preparation method and application thereof | |
| CN108707187A (en) | A kind of cell-penetrating peptide and preparation method thereof, application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: Dendritic human cell membrane penetrating peptide hpp7k, its production and method for mediating plasmid DNA transfection Effective date of registration: 20211119 Granted publication date: 20201229 Pledgee: Bank of Hankou Limited by Share Ltd. Sales Department Pledgor: TAIZE (WUHAN) BIOLOGICAL TECHNOLOGY CO.,LTD. Registration number: Y2021420000127 |