CN106084003A - Polypeptide monomer molecule based on desmoenzyme catalytic polymerization and the application in building nano material thereof - Google Patents
Polypeptide monomer molecule based on desmoenzyme catalytic polymerization and the application in building nano material thereof Download PDFInfo
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- CN106084003A CN106084003A CN201610408076.7A CN201610408076A CN106084003A CN 106084003 A CN106084003 A CN 106084003A CN 201610408076 A CN201610408076 A CN 201610408076A CN 106084003 A CN106084003 A CN 106084003A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
Abstract
The present invention relates to a kind of polypeptide monomer molecule based on desmoenzyme catalytic polymerization and the application in building nano material especially class elastin laminin nano material thereof, described polypeptide monomer molecule is repeated polypeptide by functional molecular, polymerization activity site and class elastin laminin and constitutes, described polypeptide monomer molecule can be polymerized by transglutaminase-catalyzed efficiently, the polymer formed has temperature-sensing property, the parameters such as the three dimensional structure of polymer, conversion temperature, pore size can be regulated by changing the peptide sequence of monomer molecule；This polymer can in situ controlledly synthesis in living cells, and form nanostructured；The preparation method of this nano material can realize real-time in-situ imaging or height in living cells and put and stay the medicine of rate to deliver, and has a good application prospect.
Description
Technical field
The present invention relates to nano material manufacture technology field, be specifically related to a kind of polypeptide monomer molecule and building nanometer
Application in material, particularly relates to a kind of polypeptide monomer molecule based on desmoenzyme catalytic polymerization and is building class elasticity egg
Application in white nano material.
Background technology
Controlled nano material preparation is the pursuit that Material Field is consistent, this controlled is not only embodied in the equal of nanoscale
Evenness, the uniformity of surface chemical property, be also embodied in the controllability of preparation method.In recent years, for the application of biological field,
Nano material preparation in vitro also has biological function and is widely developed, but biological safety therein, material
Prominent the constraining the most to a certain extent of the problems such as stability under complex physiologic environment and the metabolism of nano material is received
The development of rice material.But, the assemble in situ nano material in live body or living cells proposed for the problems referred to above, and have
The strategy of biological function becomes the effective means solving above-mentioned bottleneck problem.This strategy not only embodies little molecule in vivo
The advantage of high efficiency infiltration during distribution, also embodies nano material in lesions position imaging or the advantage for the treatment of, becomes
The research direction of tool potentiality.
In this system, the assembly system based on polypeptide is due to features such as its good biocompatibility, synthetic method maturations
Become material system first.In the Research foundation of early stage, the self assembly chief motivation of polypeptide is weak active force, example
As: hydrogen bond, Van der Waals force, electrostatic interaction, π-π interaction etc..Polypeptide nano material the most in vitro has been prepared
Quite ripe, portion of material has been used to clinical research.But in vivo polypeptide nano material in situ assembles is nearly 5 years in
Just occur and develop.Typically case such as Xu seminar utilizes intracellular phosphatase to make the polypeptide dephosphorylation into born of the same parents,
The hydrophobic interaction strengthened, causes in the intracellular assembling that there occurs gelation, it is achieved that the in situ imaging of desmoenzyme or swollen
The apoptosis-promoting effect of oncocyte.Rao seminar utilizes a kind of bio-orthogonal reaction, is successfully realized peptide molecule intracellular
Supramolecular Assembling, can be used to monitor the activity of caspase-3 enzyme.Also have to study and devise, in early stage, the polypeptide that chlorophyll is modified
Material, is achieved the Supramolecular Assembling of nanofibrous structures in vivo, is used successfully to work by the molecule shearing of gelatinase
Body imaging.
Although existing above-mentioned achievement case, but the Supramolecular Assembling that polypeptide is in vivo still suffers from controllability
The problems such as packaging efficiency is the highest under difference, complex environment.Therefore, development covalency assembly system will effectively solve above-mentioned faced being stranded
Difficult.In conjunction with the endogenous catalyst-enzyme in organism, set up covalency enzyme-catalyzed polymerization system, will effectively realize in complex physiologic
Controllable polymerization and molecule under environment assemble, and provide new approaches and new method, for biological field by the preparation for nano material
Application provides new technique.
Summary of the invention
For solving the deficiencies in the prior art, the invention provides a kind of polypeptide monomer molecule and building nanometer
Application in material, in particular, provides a kind of polypeptide monomer molecule based on desmoenzyme catalytic polymerization and is building class bullet
Application in property protein nano material, present invention also offers described nano material at intracellular transglutaminase expression
Application in the delivery of imaging or polypeptide slow releasing pharmaceutical.
The polypeptide monomer molecule that the present invention provides can be polymerized under intracellular transglutaminase-catalyzed, shape
The poly-peptide become has class elastin laminin character, and distortion and the winding of polymer chain can occur in the environment changing temperature, real
Now nano-supermolecule in situ assembles and controllably in intracellular preparation class elastin laminin nano material, and be implemented as or
The function of disease treatment.
For reaching this purpose, the present invention by the following technical solutions:
First aspect, the invention provides a kind of polypeptide monomer molecule based on desmoenzyme catalytic polymerization, described polypeptide
Monomer molecule is repeated polypeptide by functional molecular, polymerization activity site and class elastin laminin and constitutes.
The present invention is by functional molecular, polymerization activity site and class elastin laminin by using polypeptide monomer MOLECULE DESIGN
Repeating the structure that polypeptide is constituted, the poly-peptide after this MOLECULE DESIGN mode not only ensure that polymerization has the character of class elastin laminin together
Time also ensure that the modification density of entrained functional molecular.
Heretofore described polypeptide monomer molecule, its polymerization activity site is the spy of catalyzing enzyme transglutaminase
Opposite sex catalytic site, holds including glutamine (Q) end and lysine (K), can be realized by the catalytic coupling of two active ends
Enzyme-catalyzed polymerization.
According to the present invention, the concrete structure of described polypeptide monomer molecule is: functional molecular, the glutamy in polymerization activity site
Amine end, class elastin laminin repeat polypeptide and the lysine end in polymerization activity site, and it is sequentially connected with composition.By polymerization activity site
Designing and just can realize enzyme-catalyzed polymerization at the two ends of class elastin laminin repetition polypeptide, the poly-peptide after simultaneously keeping polymerization has class
The character of elastin laminin；The polymerization activity site design of two ends cannot change.
According to the present invention, described glutamine terminal sequence is expressed as XQY, and its expression has 3 amino acid whose polypeptide；Its
In, X represents the aminoacid in addition to proline, preferably hydrophobic amino acid；Q represents glutamine；Y represents except electronegative
Aminoacid beyond aminoacid, preferably hydrophobic amino acid.
X and Y in glutamine terminal sequence of the present invention can use hydrophobic amino acid simultaneously, and such selection can
To be greatly increased enzymatic selectivity and enzyme catalysis efficiency, the molecular weight of the polymer of synthesis is had an impact.
According to the present invention, described glutamine terminal sequence specifically can use the sequence as shown in one of SEQ ID NO.1-2
Row, it is possible to use each sequence particularized below: YQR, YQW, YQY, RQW, RQY etc., are not specially limited at this, it is emphasized that
It is that the glutamine terminal sequence of the present invention preferably employs the sequence as shown in one of SEQ ID NO.1-2, and it is in the sequence of XQY
Middle Enzyme-catalyzed Selective is the highest, and the polymer molecular weight that catalysis obtains is maximum.
SEQ ID NO.1:LQR
SEQ ID NO.2:RQL
The present invention has the above-mentioned consequent glutamine sequences having shown in general formula X QY by employing, i.e. uses glutamine
It is designed to centre position Q, Q+1 position is designed as X aminoacid, Q-1 position is designed as Y aminoacid, it is advantageous that
Keep while Enzyme-catalyzed Selective, reduce the shadow of temperature sensitive response to the poly-peptide of class elastin laminin after polymerization as far as possible
Ring.Therefore the design of three aminoacid XQY can realize above-mentioned functions simultaneously.
According to the present invention, described class elastin laminin repeats can contain in the sequence of polypeptide 5-8 aminoacid, but the most only
It is limited to this；Described class elastin laminin repeats shown in one of sequence such as SEQ ID NO.3-30 of polypeptide, preferably SEQ ID
Sequence shown in NO.24-30, the class elastin laminin character of the poly-peptide that described sequence is formed preferably (sensitive temperature-responsive,
Transition temperature can cell cultivate 30-37 DEG C between and repeat number just can well more than 20 repetitives
Show the character of class elastin laminin).
SEQ ID NO.3:GVGVP
SEQ ID NO.4:GVGHP
SEQ ID NO.5:GVGAP
SEQ ID NO.6:AVPGVG
SEQ ID NO.7:TVPGVG
SEQ ID NO.8:VAPGVG
SEQ ID NO.9:GVPGVG
SEQ ID NO.10:VHPGVG
SEQ ID NO.11:VPVGVG
SEQ ID NO.12:APVGVG
SEQ ID NO.13:VPAVG
SEQ ID NO.14:GPAVG
SEQ ID NO.15:TPAVG
SEQ ID NO.16:VPHVG
SEQ ID NO.17:GPHVG
SEQ ID NO.18:TPHVG
SEQ ID NO.19:RGDSPYQG
SEQ ID NO.20:RGDAPYQG
SEQ ID NO.21:RGDSPYG
SEQ ID NO.22:RGDSPFG
SEQ ID NO.23:RGDSPHG
SEQ ID NO.24:VPHVG
SEQ ID NO.25:GVGFP
SEQ ID NO.26:VHPGVG
SEQ ID NO.27:GRGDSPFG
SEQ ID NO.28:GRDGSPYG
SEQ ID NO.29:GRGESPYG
SEQ ID NO.30:GRGDSPYG
According to the present invention, described functional molecular is imaging molecule and/or drug molecule.
Preferably, any during described imaging molecule is fluorescence imaging molecule, photoacoustic imaging molecule or polar sensitive molecule
Any one in a kind of or combination of at least two, preferably C.I. Natural Red 8 18, burnt pheophytin, Cy5-Cy7 or DBD molecule
Or the combination of at least two.
Preferably, described drug molecule is chemotherapy of tumors molecule and/or antibiotic molecule, preferably amycin and/or ten thousand
Ancient mycin.
Second aspect, present invention also offers the polymerization of polypeptide monomer molecule as described in relation to the first aspect, described many
The polymerization of peptide monomer molecule is to be polymerized by enzymic catalytic reaction in living cells.
The polypeptide monomer molecule that the present invention provides can be polymerized by enzymic catalytic reaction in living cells, by changing
The temperature of polyreaction so that polymer chain is distorted or is wound around, thus realize nano-supermolecule assemble in situ and can
Control ground is in intracellular preparation class elastin laminin nano material.
According to the present invention, the temperature of described catalytic reaction is 37-40 DEG C, can be such as 37 DEG C, 38 DEG C, 38.5 DEG C, 39
DEG C or 40 DEG C, preferably 37 DEG C.
According to the present invention, the time of described catalytic reaction is 4-12h, can be such as 4h, 6h, 7h, 8h, 10h, 11h or
12h, preferably 4h.The catalytic reaction time of the present invention is preferably 4h, and it can ensure that the poly-peptide molecular weight after enzyme-catalyzed polymerization
While, reduce the response time as far as possible, say, that during catalysis 4h, enzyme-catalyzed polymerization speed reaches plateau.
According to the present invention, the concentration of described polypeptide monomer molecule is 10-50 μM, can be such as 10 μMs, 20 μMs, 25 μMs,
30 μMs, 35 μMs, 40 μMs or 50 μMs, preferably 50 μMs.
According to the present invention, the concentration of described enzyme is 1-3U, can be such as 1U, 1.2U, 1.5U, 1.8U, 2U, 2.5U or
3U。
According to the present invention, described enzyme is 1:5-1:10 with the reaction mol ratio of polypeptide monomer molecule, enzyme and the polypeptide of catalysis
The molecular size range of the poly-peptide after catalysis, than difference, is had an impact by substrate.
According to the present invention, described enzyme is transglutaminase.
According to the present invention, described transglutaminase includes carrying in recombiant protein that antibacterial extracts, human or animal tissues
The TG2 taken.
The third aspect, present invention also offers a kind of desmoenzyme catalytic polymerization build nano material method, including with
Lower step:
(1) polypeptide monomer molecule as described in relation to the first aspect is configured to solution；
(2) the polypeptide monomer molecular solution that step (1) obtains is joined in enzymatic solution, under the conditions of temperature is 4-40 DEG C
Reaction 4-12h, obtains nano material.
The nano material obtained in the present invention is class elastin laminin nano material.
According to the present invention, step (1) described polypeptide monomer molecular solution uses HEPES buffer solution to prepare；Wherein,
In described polypeptide monomer molecular solution, the concentration of polypeptide monomer molecule is 100-1000 μM, can be such as 100 μMs, 300 μMs,
500 μMs, 600 μMs, 700 μMs, 800 μMs or 1000 μMs, preferably 600 μMs.
According to the present invention, the temperature of step (2) described reaction is 37-40 DEG C, can be such as 37 DEG C, 38 DEG C, 38.5 DEG C,
39 DEG C or 40 DEG C, preferably 37 DEG C.
According to the present invention, the time of step (2) described reaction is 4-12h, such as, can be 4h, 6h, 7h, 8h, 10h, 11h
Or 12h, preferably 4h.
Fourth aspect, present invention also offers the nano material that a kind of method as described in the third aspect prepares, institute
Stating nano material is one-dimensional and/or 3-D nano, structure.
According to the present invention, the polymerization monomer two ends of described one-dimentional structure are held by glutamine (Q) end and lysine (K) respectively
Composition；The polymerization monomer two ends of described three dimensional structure are respectively by glutamine-lysine (QK) end and lysine-glutamine
(KQ) end composition.
According to the present invention, shown in one of described glutamine terminal sequence such as SEQ ID NO.1-2；Described glutamine-rely
Propylhomoserin terminal sequence is as shown in SEQ ID NO.31；Described lysine-glutamine terminal sequence is as shown in SEQ ID NO.32.
SEQ ID NO.31:RQLK
SEQ ID NO.32:KRQL
5th aspect, present invention also offers the purposes of nano material as described in fourth aspect, and described nano material can
For intracellular transglutaminase expression imaging or the delivery of polypeptide slow releasing pharmaceutical, wherein said medicine can be used for resisting
Tumor or infection.
Compared with prior art, the present invention at least has the advantages that
The polypeptide monomer molecule that the present invention provides can be polymerized under intracellular transglutaminase-catalyzed, shape
The poly-peptide become has class elastin laminin character, and distortion and the winding of polymer chain can occur in the environment changing temperature, real
Now nano-supermolecule in situ assembles and controllably in intracellular preparation class elastin laminin nano material, and be implemented as or
The function of disease treatment.
Accompanying drawing explanation
Fig. 1 is the structural representation of polypeptide monomer molecule of the present invention；
Fig. 2 is the polypeptide monomer molecule of the present invention schematic diagram in intracellular generation polyreaction；
Fig. 3 is the MALDI-TOF figure being polymerized monomer molecule described in embodiment 1
Fig. 4 is the nanostructured scanning electron microscope that described in embodiment 1, poly-polypeptide assembles in solution；
Fig. 5 is that Micelle-like Nano-structure of Two described in embodiment 1 is in the application of cell imaging；
Fig. 6 is the scanning electron microscope of nanogel described in embodiment 2；
Fig. 7 be nanogel described in embodiment 2 for the destruction of cytoskeleton, wherein Fig. 7 (a) represents that TG2 is positive thin
Born of the same parents；Fig. 7 (b) represents TG2 negative cells.
Detailed description of the invention
The polypeptide monomer molecule based on desmoenzyme catalytic polymerization provided in the present invention, it is by functional molecular, polymerization
Avtive spot and class elastin laminin repeat polypeptide and constitute, and concrete structure is: functional molecular, the glutamine in polymerization activity site
End, class elastin laminin repeat the lysine end in polypeptide and polymerization activity site and are sequentially connected with, and its concrete structure is as shown in Figure 1.
The polypeptide monomer molecule based on desmoenzyme catalytic polymerization provided in the present invention, its in living cells by enzyme,
Preferably be polymerized by glutamine enzymic catalytic reaction, by change polyreaction temperature so that polymer chain be distorted or
It is wound around, thus realizes the assemble in situ of nano-supermolecule and controllably in intracellular preparation class elastin laminin nano material, its
Concrete polymerization process is as shown in Figure 2.
Below in conjunction with embodiment, the present invention will be further described, and theme protection domain involved in the present invention the most only limits
In these embodiments.
Embodiment 1
Polypeptide monomer molecule based on desmoenzyme catalytic polymerization, its structure is designed as: functional molecular, polymerization activity site
Glutamine end, class elastin laminin repeats the lysine end in polypeptide and polymerization activity site and is sequentially connected with.
Wherein, functional molecular is DBD fluorescence signal molecule；The glutamine terminal sequence in polymerization activity site is SEQ ID
Sequence RQL shown in NO.2；It is the sequence shown in SEQ ID NO.25: GVGFP that class elastin laminin repeats peptide sequence；Polymerization is lived
The lysine end in property site is K (Lys), and the structure of this polypeptide monomer molecule is as follows.
Embodiment 2
Polypeptide monomer molecule based on desmoenzyme catalytic polymerization, its structure is designed as: functional molecular, polymerization activity site
Glutamine end, class elastin laminin repeats the lysine end in polypeptide and polymerization activity site and is sequentially connected with.
Wherein, functional molecular is DBD fluorescence signal molecule；The glutamine terminal sequence in polymerization activity site is SEQ ID
Sequence LQR shown in NO.1；It is the sequence shown in SEQ ID NO.24: VPHVG that class elastin laminin repeats peptide sequence；Polymerization is lived
The lysine end in property site is K (Lys).
Embodiment 3
Polypeptide monomer molecule based on desmoenzyme catalytic polymerization, its structure is designed as: functional molecular, polymerization activity site
Glutamine end, class elastin laminin repeats the lysine end in polypeptide and polymerization activity site and is sequentially connected with.
Wherein, functional molecular is DBD fluorescence signal molecule；The glutamine terminal sequence in polymerization activity site is SEQ ID
Sequence RQL shown in NO.2；It is the sequence shown in SEQ ID NO.3: GVGVP that class elastin laminin repeats peptide sequence；Polymerization is lived
The lysine end in property site is K (Lys).
Embodiment 4
Polypeptide monomer molecule based on desmoenzyme catalytic polymerization, its structure is designed as: functional molecular, polymerization activity site
Glutamine end, class elastin laminin repeats the lysine end in polypeptide and polymerization activity site and is sequentially connected with.
Wherein, functional molecular is amycin；The glutamine terminal sequence in polymerization activity site is shown in SEQ ID NO.1
Sequence LQR；It is the sequence shown in SEQ ID NO.27: GRGDSPFG that class elastin laminin repeats peptide sequence；Polymerization activity site
Lysine end be K (Lys).
Embodiment 5
Polypeptide monomer molecule based on desmoenzyme catalytic polymerization, its structure is designed as: functional molecular, polymerization activity site
Glutamine end, class elastin laminin repeats the lysine end in polypeptide and polymerization activity site and is sequentially connected with.
Wherein, functional molecular is vancomycin；The glutamine terminal sequence in polymerization activity site is shown in SEQ ID NO.2
Sequence RQL；It is the sequence shown in SEQ ID NO.29: GRGESPYG that class elastin laminin repeats peptide sequence；Polymerization activity position
The lysine end of point is K (Lys).
Embodiment 6
Polypeptide monomer molecule based on desmoenzyme catalytic polymerization, its structure is designed as: functional molecular, polymerization activity site
Glutamine end, class elastin laminin repeats the lysine end in polypeptide and polymerization activity site and is sequentially connected with.
Wherein, functional molecular is Cy5-Cy7；The glutamine terminal sequence in polymerization activity site is shown in SEQ ID NO.1
Sequence LQR；It is the sequence shown in SEQ ID NO.29: GRGESPYG that class elastin laminin repeats peptide sequence；Polymerization activity position
The lysine end of point is K (Lys).
Embodiment 7
Polypeptide monomer molecule based on desmoenzyme catalytic polymerization, its structure is designed as: functional molecular, polymerization activity site
Glutamine end, class elastin laminin repeats the lysine end in polypeptide and polymerization activity site and is sequentially connected with.
Wherein, functional molecular is Cy5-Cy7；The glutamine terminal sequence in polymerization activity site is shown in SEQ ID NO.2
Sequence RQL；It is the sequence shown in SEQ ID NO.30: GRGDSPYG that class elastin laminin repeats peptide sequence；Polymerization activity position
The lysine end of point is K (Lys).
Embodiment 8
The polymerization of the polypeptide monomer molecule based on desmoenzyme catalytic polymerization in embodiment 1, including: at living cells
The interior transglutaminase that passes through is at 37 DEG C, and catalytic reaction 4h is polymerized.
Embodiment 9
The polymerization of the polypeptide monomer molecule based on desmoenzyme catalytic polymerization in embodiment 2, including: at living cells
The interior transglutaminase that passes through is at 37 DEG C, and catalytic reaction 6h is polymerized.
Embodiment 10
The polymerization of the polypeptide monomer molecule based on desmoenzyme catalytic polymerization in embodiment 4, including: at living cells
The interior transglutaminase that passes through is at 39 DEG C, and catalytic reaction 12h is polymerized.
Embodiment 11
The polymerization of the polypeptide monomer molecule based on desmoenzyme catalytic polymerization in embodiment 6, including: at living cells
The interior transglutaminase that passes through is at 40 DEG C, and catalytic reaction 8h is polymerized.
Embodiment 12
Utilize the method that the polypeptide monomer molecule based on desmoenzyme catalytic polymerization of embodiment 1 prepares nano material, bag
Include following steps:
(1) synthesis of connecting peptides
Employing Fmoc solid-phase synthesis synthesis connecting peptides:
Synthesis selects 0.35mM to modify the Wang resin of density, and wherein the N end of first aminoacid (lysine) is by Fmoc
Protection, C end is fixed on resin.Slough the Fmoc protection of N end with the DMF solution of the hexahydropyridine of 20% (v/v), then use indenes
Triketone method of testing test deprotection result.Then by the 4-methyl morpholine (NMM) and 10 of next amino acid whose carboxyl 0.4M
Times amino acid whose BTA-N, the DMF solution activation of N, N', N'-tetramethylurea hexafluorophosphoric acid ester (HBTU), and add
2h is reacted in de-de-protected resin.According to said method, remaining all aminoacid are all passed through condensation reaction and connects up, shape
Become to be fixed on the connecting peptides of resin.
(2) synthesis of fluorescence signal molecule DBD connecting peptides
After step (1) completes all amino acid whose synthesis, using the DBD fluorescence signal molecule that activated as last
Aminoacid repeat the above steps is coupled at the N end of connecting peptides；Then with containing 2.5% water and 2.5% tri isopropyl silane
Synthetic polypeptide is removed from resin by trifluoroacetic acid solution, removes amino acid whose side chain protected simultaneously；Trifluoroacetic acid is used
Rotary evaporation is removed, and then the crude product of polypeptide absolute ether precipitates, and washs and is dried；Finally select anti-phase preparation liquid phase
Chromatograph, by peptide purification.
The condition of purge process is: flowing is the acetonitrile containing 0.1% trifluoroacetic acid mutually and contains 0.1% trifluoroacetic acid
Distilled water；Parameter be gradient elution from 5% acetonitrile/95% water to 60% acetonitrile/40% water, flow velocity is 10ml/min, during process
Between be 30min.The MALDI-TOF collection of illustrative plates of the polymerization monomer molecule obtained by said method is as shown in Figure 3.
(3) polypeptide polymerization enzymatically.
Synthetic polymerization monomer molecule HEPES buffer solution is dissolved, joins the glutamine transfer prepared
In enzymatic solution, 37 DEG C, shaking table about 4h under 150rmp, transglutaminase can be with amino and paddy on catalytic polypeptide chain lysine
There is covalent cross-linking in glutamine between hydroxyl amide groups, thus formed the heat sensitive poly-peptide of class elastin laminin, the polymerization of formation
Thing molecular weight is about 30000, and the degree of polymerization is 27-28.This polymer nanometer in vitro assembles pattern scanning electron microscope and characterizes, as
Shown in Fig. 4.
(4) assemble method of this in-situ nano material is for monitoring the activity of transglutaminase.
Polymerization monomer molecule is joined in cell culture medium with the ultimate density that concentration is 600 μMs, quiescent culture 12h
After, with PBS, cell is cleaned three times repeatedly, then with Hochest (blue) and Dil (red) respectively to nucleus and cell membrane
Dyeing, and use confocal microscope imaging, imaging temperature is 4 DEG C (as shown in Figure 5).
Embodiment 13
Utilize the method that polypeptide monomer molecule based on desmoenzyme catalytic polymerization prepares nano material, including following step
Rapid:
With sequence VPHVG shown in SEQ ID NO.24 for elastin polypeptide fragment, Gln-Lys and Lys-Gln is respectively
For Q end and K end, molecular sequences is QKVPHVGQK, carries out the synthesis of class elastin laminin nanogel of the present invention as follows:
(1) synthesis of connecting peptides
Employing Fmoc solid-phase synthesis synthesis connecting peptides:
Synthesis selects 0.35mM to modify the Wang resin of density, and wherein the N end of first aminoacid (lysine) is by Fmoc
Protection, C end is fixed on resin.Slough the Fmoc protection of N end with the DMF solution of the hexahydropyridine of 20% (v/v), then use indenes
Triketone method of testing test deprotection result.Then by the 4-methyl morpholine (NMM) and 10 of next amino acid whose carboxyl 0.4M
Times amino acid whose BTA-N, the DMF solution activation of N, N', N'-tetramethylurea hexafluorophosphoric acid ester (HBTU), and add
2h is reacted in de-de-protected resin.According to said method, remaining all aminoacid are all passed through condensation reaction and connects up, shape
Become to be fixed on the connecting peptides of resin.
(2) polypeptide polymerization enzymatically.
Synthetic polymerization monomer molecule HEPES buffer solution is dissolved, joins the glutamine transfer prepared
In enzymatic solution, 37 DEG C, shaking table about 4h under 150rmp, transglutaminase can be with amino and paddy on catalytic polypeptide chain lysine
There is covalent cross-linking in glutamine between hydroxyl amide groups, thus formed heat sensitive class elastin laminin nanogel, gelation
Matter is as shown in Figure 6.
(3) assemble method of this in-situ nano material is used for promoting apoptosis.
Polymerization monomer molecule is joined in cell culture medium with the ultimate density that concentration is 600 μMs, quiescent culture 12h
After, with PBS, cell is cleaned three times repeatedly, then with the actin of the phalloidin dye cell of Cy5 labelling, and with fluorescence altogether
Focusing microscope imaging, imaging temperature is 37 DEG C, it can be seen that contrast does not has the cell that transglutaminase is expressed, and intracellular is coagulated
The formation of glue and collapsing so that actin is by considerable damage, promotes apoptosis (as shown in Fig. 7 (a)-(b)).
By above-described embodiment it can be seen that the polypeptide monomer molecule that the present invention provides can be at intracellular glutamine
Being polymerized under transferring enzyme catalysis, the poly-peptide of formation has class elastin laminin character, can occur in the environment changing temperature
The distortion of polymer chain and winding, it is achieved nano-supermolecule in situ assembles and controllably at intracellular preparation class elastin laminin
Nano material, and it is implemented as picture or the function of disease treatment.
Applicant states, the present invention illustrates the present invention by above-mentioned case study on implementation, but the invention is not limited in above-mentioned,
I.e. do not mean that the present invention has to rely on above-mentioned could implement.Person of ordinary skill in the field is it will be clearly understood that to the present invention
Any improvement, to raw material selected by the present invention equivalence replace and the interpolation of auxiliary element, concrete way choice etc., all fall
Within the scope of protection scope of the present invention and disclosure.
Claims (10)
1. a polypeptide monomer molecule based on desmoenzyme catalytic polymerization, it is characterised in that described polypeptide monomer molecule is by merit
Energy molecule, polymerization activity site and class elastin laminin repeat polypeptide and constitute.
2. polypeptide monomer molecule as claimed in claim 1, it is characterised in that described polymerization activity site includes glutamine end
With lysine end；
Preferably, described polypeptide monomer molecule is repeated by functional molecular, the glutamine end in polymerization activity site, class elastin laminin
The lysine end in polypeptide and polymerization activity site is sequentially connected with composition.
3. polypeptide monomer molecule as claimed in claim 2, it is characterised in that described glutamine terminal sequence is expressed as XQY；Its
In, X represents the aminoacid in addition to proline, preferably hydrophobic amino acid；Q represents glutamine；Y represents except electronegative
Aminoacid beyond aminoacid, preferably hydrophobic amino acid；
Preferably, shown in one of described glutamine terminal sequence such as SEQ ID NO.1-2；
Preferably, described class elastin laminin repeats shown in one of sequence such as SEQ ID NO.3-30 of polypeptide, preferably SEQ ID
Sequence shown in NO.24-30.
4. the polypeptide monomer molecule as described in one of claim 1-3, it is characterised in that described functional molecular is imaging molecule
And/or drug molecule；
Preferably, any one during described imaging molecule is fluorescence imaging molecule, photoacoustic imaging molecule or polar sensitive molecule
In the combination of at least two, preferably C.I. Natural Red 8 18, burnt pheophytin, Cy5-Cy7 or DBD molecule any one or extremely
The combination of few two kinds；
Preferably, described drug molecule is chemotherapy of tumors molecule and/or antibiotic molecule, preferably amycin and/or the most mould
Element.
5. the polymerization of the polypeptide monomer molecule as described in one of claim 1-4, it is characterised in that described polypeptide monomer divides
The polymerization of son is to be polymerized by enzymic catalytic reaction in living cells.
6. method as claimed in claim 5, it is characterised in that the temperature of described catalytic reaction is 37-40 DEG C, preferably 37
℃；
Preferably, the time of described catalytic reaction is 4-12h, preferably 4h；
Preferably, the concentration of described polypeptide monomer molecule is 10-50 μM, preferably 50 μMs；
Preferably, the concentration of described enzyme is 1-3U；
Preferably, described enzyme is 1:5-1:10 with the reaction mol ratio of polypeptide monomer molecule；
Preferably, described enzyme is transglutaminase；
Preferably, the TG2 that described transglutaminase extracts in including recombiant protein that antibacterial extracts, human or animal tissues.
7. the method that a desmoenzyme catalytic polymerization builds nano material, it is characterised in that comprise the following steps:
(1) the polypeptide monomer molecule as described in one of claim 1-4 is configured to solution；
(2) the polypeptide monomer molecular solution that step (1) obtains is joined in enzymatic solution, react under the conditions of temperature is 4-40 DEG C
4-12h, obtains nano material.
8. method as claimed in claim 7, it is characterised in that step (1) described polypeptide monomer molecular solution uses HEPES to delay
Dissolved liquid is prepared；
Preferably, in step (1) described polypeptide monomer molecular solution, the concentration of polypeptide monomer molecule is 100-1000 μM, is preferably
600μM；
Preferably, the temperature of step (2) described reaction is 37-40 DEG C, preferably 37 DEG C；
Preferably, the time of step (2) described reaction is 4-8h, preferably 4h.
9. the nano material that method prepares as claimed in claim 7 or 8, it is characterised in that described nano material is
Dimension and/or 3-D nano, structure；
Preferably, the polymerization monomer two ends of described one-dimentional structure are made up of glutamine end and lysine end respectively；Described three-dimensional
The polymerization monomer two ends of structure are made up of glutamine-lysine end and lysine-glutamine end respectively；
Preferably, shown in one of described glutamine terminal sequence such as SEQ ID NO.1-2；Described glutamine-lysine end sequence
Row are as shown in SEQ ID NO.31；Described lysine-glutamine terminal sequence is as shown in SEQ IDNO.32.
10. the purposes of nano material as claimed in claim 9, it is characterised in that described nano material is used for intracellular paddy ammonia
Amide transferase expression imaging or the delivery of polypeptide slow releasing pharmaceutical.
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| CN107022048A (en) * | 2017-05-22 | 2017-08-08 | 国家纳米科学中心 | A kind of temperature sensitive driver of macromolecular and its preparation method and application |
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| CN107648618A (en) * | 2017-09-27 | 2018-02-02 | 国家纳米科学中心 | A kind of drug delivery system and preparation method and application |
| CN110343146A (en) * | 2019-07-12 | 2019-10-18 | 上海应用技术大学 | A kind of crab flavour mushroom flavor peptide and its preparation method and application |
| CN110343146B (en) * | 2019-07-12 | 2022-09-30 | 上海应用技术大学 | Hypsizygus marmoreus flavor-developing peptide and preparation method and application thereof |
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