CN106084003B - Polypeptide monomer molecule based on desmoenzyme catalytic polymerization and its application in building nano material - Google Patents
Polypeptide monomer molecule based on desmoenzyme catalytic polymerization and its application in building nano material Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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- 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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- 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
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Abstract
The present invention relates to a kind of polypeptide monomer molecule based on desmoenzyme catalytic polymerization and its applications in building nano material especially class elastin laminin nano material, the polypeptide monomer molecule repeats polypeptide by functional molecular, polymerization activity site and class elastin laminin and constitutes, the polypeptide monomer molecule can efficiently be polymerize by transglutaminase-catalyzed, the polymer of formation has temperature-sensing property, the parameters such as the three-dimensional structure, conversion temperature, pore size of polymer can be adjusted by the polypeptide sequence of change monomer molecule；The polymer can the controlledly synthesis in situ in living cells, and form nanostructure；The preparation method of the nano material may be implemented real-time in-situ imaging or height in living cells and set the drug delivery for staying rate, have a good application prospect.
Description
Technical field
The present invention relates to nano material manufacture technology fields, and in particular to a kind of polypeptide monomer molecule and its in building nanometer
Application in material more particularly to a kind of polypeptide monomer molecule based on desmoenzyme catalytic polymerization and its in building class elasticity egg
Application in white nano material.
Background technique
Controllable nano material preparation is the consistent pursuit of Material Field, this to be controllably not only embodied in the equal of nanoscale
Evenness, the uniformity of surface chemical property, are also embodied in the controllability of preparation method.In recent years, for the application of biological field,
Nano material preparation in vitro simultaneously there is biological function to be widely developed, but biological safety therein, material
The protrusion of the problems such as metabolism of stability and nano material under complex physiologic environment also constrains to a certain extent to be received
The development of rice material.However, the assemble in situ nano material in living body or living cells proposed regarding to the issue above, and have
The strategy of biological function becomes the effective means for solving above-mentioned bottleneck problem.The strategy not only embodies small molecule in vivo
The advantage of high efficiency infiltration when distribution also embodies the advantage that nano material is imaged or treats in lesions position, becomes at present most
Has the research direction of potentiality.
In this system, assembly system based on polypeptide due to its good biocompatibility, synthetic method is mature the features such as
As material system first.In the Research foundation of early period, the self assembly chief motivation of polypeptide is weak active force, example
Such as: hydrogen bond, Van der Waals force, electrostatic interaction, π-π interaction.Wherein polypeptide nano material in vitro is prepared
Quite mature, some materials have been used to clinical research.But in vivo the polypeptide nano material assembling of original position is in nearly 5 years
Just occurs and develop.Typical case such as Xu seminar makes the polypeptide dephosphorylation into born of the same parents using intracellular phosphatase,
The hydrophobic effect of enhancing leads to the assembling that gelation has occurred in the cell, realizes the in situ imaging or swollen of desmoenzyme
The apoptosis-promoting effect of oncocyte.Rao seminar utilizes a kind of bio-orthogonal reaction, is successfully realized peptide molecule in the cell
Supramolecular Assembling can be used to monitor the activity of caspase-3 enzyme.Also research devises the polypeptide of chlorophyll modification in early period
Material realizes the Supramolecular Assembling of nanofibrous structures in vivo by the molecule shearing of gelatinase, is used successfully to live
Body imaging.
Although having above-mentioned achievement case, the Supramolecular Assembling of polypeptide in vivo still suffers from controllability
The problems such as inefficient is assembled under difference, complex environment.Therefore, above-mentioned faced be stranded will effectively be solved by developing covalent assembly system
It is difficult.In conjunction with the intracorporal endogenous catalyst-enzyme of biology, covalent enzyme-catalyzed polymerization system is established, will effectively be realized in complex physiologic
Controllable polymerization and molecule assembling under environment, will provide new approaches and new method for the preparation of nano material, are biological field
Using offer new technology.
Summary of the invention
To solve the deficiencies in the prior art, the present invention provides a kind of polypeptide monomer molecule and its in building nanometer
Application in material in particular, provides a kind of polypeptide monomer molecule based on desmoenzyme catalytic polymerization and its in building class bullet
Application in property protein nano material, the present invention also provides nano material transglutaminase expression quantity in the cell
Application in imaging or the delivering of polypeptide slow releasing pharmaceutical.
Polypeptide monomer molecule provided by the invention can polymerize under transglutaminase-catalyzed in the cell, shape
At poly- peptide there is class elastin laminin property, the distortion and winding of polymer chain can occur in the environment for changing temperature, it is real
Nano-supermolecule assembling now in situ and controllably prepare class elastin laminin nano material in the cell, and realize imaging or
The function of disease treatment.
To achieve this purpose, the present invention adopts the following technical scheme:
In a first aspect, the present invention provides a kind of polypeptide monomer molecule based on desmoenzyme catalytic polymerization, the polypeptide
Monomer molecule repeats 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 by polypeptide monomer MOLECULE DESIGN
The structure that polypeptide is constituted is repeated, which not only ensure that the poly- peptide after polymerization has the property of class elastin laminin same
When also ensure the modification density of entrained functional molecular.
Heretofore described polypeptide monomer molecule, polymerization activity site are catalyzing enzyme-transglutaminase spies
Anisotropic catalytic site, including glutamine (Q) end and the end lysine (K), can be realized by the catalytic coupling at two active ends
Enzyme-catalyzed polymerization.
According to the present invention, the specific structure of the polypeptide monomer molecule are as follows: functional molecular, polymerization activity site glutamy
Amine end, class elastin laminin repeat the lysine end of polypeptide and polymerization activity site, are sequentially connected composition.By polymerization activity site
It designs and enzyme-catalyzed polymerization just may be implemented at the both ends that class elastin laminin repeats polypeptide, while keeping the poly- peptide after polymerization that there is class
The property of elastin laminin；The polymerization activity site design of two ends cannot change.
According to the present invention, the glutamine terminal sequence is expressed as XQY, indicates the polypeptide with 3 amino acid；Its
In, X indicates the amino acid in addition to proline, preferably hydrophobic amino acid；Q indicates glutamine；Y is indicated except electronegative
Amino acid other than amino acid, 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 greatly increase the selectivity and enzymatic efficiency of enzymatic, have an impact to the molecular weight of the polymer of synthesis.
According to the present invention, the glutamine terminal sequence can specifically use the sequence as shown in one of SEQ ID NO.1-2
Column, each sequence particularized below can also be used: YQR, YQW, YQY, RQW, RQY etc. are not specially limited herein, it is emphasized that
It is that glutamine terminal sequence of the invention preferably uses the sequence as shown in one of SEQ ID NO.1-2, in the sequence of XQY
Middle Enzyme-catalyzed Selective highest, the polymer molecular weight being catalyzed are maximum.
SEQ ID NO.1:LQR
SEQ ID NO.2:RQL
The present invention has consequent glutamine sequences shown in general formula X QY by using with above-mentioned, that is, uses glutamine
It is designed to middle position Q, X amino acid is designed as on the position Q+1, Y amino acid is designed as on the position Q-1, it is advantageous that
While keeping Enzyme-catalyzed Selective, the shadow to the temperature sensitive responsiveness of the poly- peptide of class elastin laminin after polymerization is reduced as far as possible
It rings.Therefore the design of three amino acid XQY can be realized simultaneously above-mentioned function.
According to the present invention, the class elastin laminin repeats that 5-8 amino acid can be contained in the sequence of polypeptide, but not only
It is limited to this；The class elastin laminin repeats the sequence of polypeptide as shown in one of SEQ ID NO.3-30, preferably SEQ ID
Sequence shown in NO.24-30, the sequence be formed by poly- peptide class elastin laminin property preferably (sensitive temperature-responsive,
Transition temperature can be between 30-37 DEG C of cell culture and repetition number can be good more than 20 repetitive units
Show the property 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, the functional molecular is imaging molecule and/or drug molecule.
Preferably, the imaging molecule is any in fluorescence imaging molecule, photoacoustic imaging molecule or polar sensitive molecule
It is a kind of or at least two combination, preferably purpurine 18, burnt pheophytin, any one in Cy5-Cy7 or DBD molecule
Or at least two combination.
Preferably, the drug molecule is chemotherapy of tumors molecule and/or antibiotic molecule, preferably adriamycin and/or ten thousand
Ancient mycin.
Second aspect is described more the present invention also provides the polymerization of polypeptide monomer molecule as described in relation to the first aspect
The polymerization of peptide monomer molecule is to be polymerized in living cells by enzymic catalytic reaction.
Polypeptide monomer molecule provided by the invention can be polymerized in living cells by enzymic catalytic reaction, pass through change
The temperature of polymerization reaction so that polymer chain is distorted or winds, thus realize nano-supermolecule assemble in situ and can
Control ground prepares class elastin laminin nano material in the cell.
According to the present invention, the temperature of the catalysis reaction is 37-40 DEG C, such as can be 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, it is described catalysis reaction time be 4-12h, such as can be 4h, 6h, 7h, 8h, 10h, 11h or
12h, preferably 4h.The catalysis reaction time of the invention is preferably 4h, the poly- peptide molecular weight after can guaranteeing enzyme-catalyzed polymerization
While, reduce the reaction time as far as possible, that is to say, that enzyme-catalyzed polymerization rate reaches plateau when catalysis 4h.
According to the present invention, the concentration of the polypeptide monomer molecule be 10-50 μM, such as can be 10 μM, 20 μM, 25 μM,
30 μM, 35 μM, 40 μM or 50 μM, preferably 50 μM.
According to the present invention, the concentration of the enzyme be 1-3U, such as can be 1U, 1.2U, 1.5U, 1.8U, 2U, 2.5U or
3U。
According to the present invention, the enzyme is 1:5-1:10, the polypeptide of enzyme and catalysis with the molar ratio of reacting of polypeptide monomer molecule
Substrate has an impact to the molecular size range of the poly- peptide after catalysis than difference.
According to the present invention, the enzyme is transglutaminase.
According to the present invention, the transglutaminase include bacterium extract recombinant protein, mention in human or animal tissues
The TG2 taken.
The third aspect, the present invention also provides a kind of methods of desmoenzyme catalytic polymerization building nano material, 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 added in enzyme solutions, under the conditions of temperature is 4-40 DEG C
4-12h is reacted, nano material is obtained.
Nano material obtained in the present invention is class elastin laminin nano material.
According to the present invention, step (1) the polypeptide monomer molecular solution is prepared using HEPES buffer solution；Wherein,
In the polypeptide monomer molecular solution concentration of polypeptide monomer molecule be 100-1000 μM, such as can be 100 μM, 300 μM,
500 μM, 600 μM, 700 μM, 800 μM or 1000 μM, preferably 600 μM.
According to the present invention, the temperature of step (2) described reaction be 37-40 DEG C, such as can be 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, the nano material that the method as described in the third aspect that the present invention also provides a kind of is prepared, institute
Stating nano material is one-dimensional and/or 3-D nano, structure.
According to the present invention, the polymerized monomer both ends of the one-dimentional structure are held by the end glutamine (Q) and lysine (K) respectively
Composition；The polymerized monomer both ends of the three-dimensional structure are respectively by the end glutamine-lysine (QK) and lysine-glutamine
(KQ) end composition.
According to the present invention, the glutamine terminal sequence is as shown in one of SEQ ID NO.1-2；The glutamine-relies
Propylhomoserin terminal sequence is as shown in SEQ ID NO.31；The lysine-glutamine terminal sequence is as shown in SEQ ID NO.32.
SEQ ID NO.31:RQLK
SEQ ID NO.32:KRQL
5th aspect, the present invention also provides the purposes of the nano material as described in fourth aspect, the nano material can
With for intracellular transglutaminase expression quantity imaging or polypeptide slow releasing pharmaceutical delivering, wherein the drug can be used for resisting
Tumour is anti-infective.
Compared with prior art, the present invention at least has the advantages that
Polypeptide monomer molecule provided by the invention can polymerize under transglutaminase-catalyzed in the cell, shape
At poly- peptide there is class elastin laminin property, the distortion and winding of polymer chain can occur in the environment for changing temperature, it is real
Nano-supermolecule assembling now in situ and controllably prepare class elastin laminin nano material in the cell, and realize imaging or
The function of disease treatment.
Detailed description of the invention
Fig. 1 is the structural schematic diagram of polypeptide monomer molecule of the present invention；
Fig. 2 is the schematic diagram that polymerization reaction occurs in the cell for polypeptide monomer molecule of the present invention；
Fig. 3 is the MALDI-TOF figure of polymerized monomer molecule described in embodiment 1
Fig. 4 is the nanostructure scanning electron microscope that poly- polypeptide described in embodiment 1 assembles in solution；
Fig. 5 is application of the Micelle-like Nano-structure of Two described in embodiment 1 in cell imaging；
Fig. 6 is the scanning electron microscope of nanogel described in embodiment 2；
Fig. 7 is destruction of the nanogel described in embodiment 2 for cytoskeleton, and wherein Fig. 7 (a) indicates that TG2 is positive thin
Born of the same parents；Fig. 7 (b) indicates TG2 negative cells.
Specific embodiment
Polypeptide monomer molecule provided in the present invention based on desmoenzyme catalytic polymerization is by functional molecular, polymerization
Active site and class elastin laminin repeat polypeptide and constitute, specific structure are as follows: functional molecular, polymerization activity site glutamine
End, class elastin laminin repeat polypeptide and the lysine end in polymerization activity site is sequentially connected, and specific structure is as shown in Figure 1.
Polypeptide monomer molecule provided in the present invention based on desmoenzyme catalytic polymerization passes through enzyme in living cells,
It is preferred that by glutamine enzymatic reactive polymeric, by changing the temperature of polymerization reaction so that polymer chain be distorted or
Winding, thus realize the assemble in situ of nano-supermolecule and controllably prepare class elastin laminin nano material in the cell,
Specific polymerization process is as shown in Figure 2.
Below with reference to embodiment, the present invention will be further described, and theme protection scope according to the present invention not only limits
In these embodiments.
Embodiment 1
Polypeptide monomer molecule based on desmoenzyme catalytic polymerization, structure design are as follows: functional molecular, polymerization activity site
Glutamine end, class elastin laminin repeats the lysine end in polypeptide and polymerization activity site and is sequentially connected.
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 sequence shown in SEQ ID NO.25: GVGFP that class elastin laminin, which repeats polypeptide sequence,；Polymerization is lived
Property site lysine end be K (Lys), the structure of the polypeptide monomer molecule is as follows.
Embodiment 2
Polypeptide monomer molecule based on desmoenzyme catalytic polymerization, structure design are as follows: functional molecular, polymerization activity site
Glutamine end, class elastin laminin repeats the lysine end in polypeptide and polymerization activity site and is sequentially connected.
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 sequence shown in SEQ ID NO.24: VPHVG that class elastin laminin, which repeats polypeptide sequence,；Polymerization is lived
Property site lysine end be K (Lys).
Embodiment 3
Polypeptide monomer molecule based on desmoenzyme catalytic polymerization, structure design are as follows: functional molecular, polymerization activity site
Glutamine end, class elastin laminin repeats the lysine end in polypeptide and polymerization activity site and is sequentially connected.
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 sequence shown in SEQ ID NO.3: GVGVP that class elastin laminin, which repeats polypeptide sequence,；Polymerization is lived
Property site lysine end be K (Lys).
Embodiment 4
Polypeptide monomer molecule based on desmoenzyme catalytic polymerization, structure design are as follows: functional molecular, polymerization activity site
Glutamine end, class elastin laminin repeats the lysine end in polypeptide and polymerization activity site and is sequentially connected.
Wherein, functional molecular is adriamycin；The glutamine terminal sequence in polymerization activity site is shown in SEQ ID NO.1
Sequence LQR；It is sequence shown in SEQ ID NO.27: GRGDSPFG that class elastin laminin, which repeats polypeptide sequence,；Polymerization activity site
Lysine end be K (Lys).
Embodiment 5
Polypeptide monomer molecule based on desmoenzyme catalytic polymerization, structure design are as follows: functional molecular, polymerization activity site
Glutamine end, class elastin laminin repeats the lysine end in polypeptide and polymerization activity site and is sequentially connected.
Wherein, functional molecular is vancomycin；The glutamine terminal sequence in polymerization activity site is shown in SEQ ID NO.2
Sequence RQL；It is sequence shown in SEQ ID NO.29: GRGESPYG that class elastin laminin, which repeats polypeptide sequence,；Polymerization activity position
The lysine end of point is K (Lys).
Embodiment 6
Polypeptide monomer molecule based on desmoenzyme catalytic polymerization, structure design are as follows: functional molecular, polymerization activity site
Glutamine end, class elastin laminin repeats the lysine end in polypeptide and polymerization activity site and is sequentially connected.
Wherein, functional molecular Cy5-Cy7；The glutamine terminal sequence in polymerization activity site is shown in SEQ ID NO.1
Sequence LQR；It is sequence shown in SEQ ID NO.29: GRGESPYG that class elastin laminin, which repeats polypeptide sequence,；Polymerization activity position
The lysine end of point is K (Lys).
Embodiment 7
Polypeptide monomer molecule based on desmoenzyme catalytic polymerization, structure design are as follows: functional molecular, polymerization activity site
Glutamine end, class elastin laminin repeats the lysine end in polypeptide and polymerization activity site and is sequentially connected.
Wherein, functional molecular Cy5-Cy7；The glutamine terminal sequence in polymerization activity site is shown in SEQ ID NO.2
Sequence RQL；It is sequence shown in SEQ ID NO.30: GRGDSPYG that class elastin laminin, which repeats polypeptide 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, comprising: in living cells
It is interior by glutaminase at 37 DEG C, catalysis reaction 4h be polymerized.
Embodiment 9
The polymerization of the polypeptide monomer molecule based on desmoenzyme catalytic polymerization in embodiment 2, comprising: in living cells
It is interior by glutaminase at 37 DEG C, catalysis reaction 6h be polymerized.
Embodiment 10
The polymerization of the polypeptide monomer molecule based on desmoenzyme catalytic polymerization in embodiment 4, comprising: in living cells
It is interior by glutaminase at 39 DEG C, catalysis reaction 12h be polymerized.
Embodiment 11
The polymerization of the polypeptide monomer molecule based on desmoenzyme catalytic polymerization in embodiment 6, comprising: in living cells
It is interior by glutaminase at 40 DEG C, catalysis reaction 8h be polymerized.
Embodiment 12
Utilize the method for the polypeptide monomer molecule preparation nano material based on desmoenzyme catalytic polymerization of embodiment 1, packet
Include following steps:
(1) synthesis of connecting peptides
Connecting peptides are synthesized using Fmoc solid-phase synthesis:
The Wang resin of 0.35mM modification density is selected in synthesis, wherein the N-terminal of first amino acid (lysine) is by Fmoc
Protection, C-terminal are fixed on resin.The Fmoc protection that N-terminal is sloughed with the DMF solution of the hexahydropyridine of 20% (v/v), then uses indenes
Triketone method of testing test deprotection result.Then by the carboxyl of next amino acid the 4- methyl morpholine (NMM) of 0.4M and 10
The DMF solution activation of benzotriazole-N, N, N', N'- the tetramethylurea hexafluorophosphoric acid ester (HBTU) of times amino acid, and be added
2h is reacted to taking off in de-protected resin.According to said method, remaining all amino acid are all got on by condensation reaction connection, shape
At the connecting peptides for being fixed on resin.
(2) synthesis of fluorescence signal molecule DBD connecting peptides
After the synthesis that step (1) completes all amino acid, using activated DBD fluorescence signal molecule as the last one
Amino acid, which repeats the above steps, is coupled at the N-terminal of connecting peptides；Then with containing 2.5% water and 2.5% tri isopropyl silane
Trifluoroacetic acid solution removes synthetic polypeptide from resin, while removing the side chain protection of amino acid；Trifluoroacetic acid is used
Rotary evaporation removal, then the crude product of polypeptide is precipitated with anhydrous ether, is washed and is dried；Finally reverse phase is selected to prepare liquid phase
Chromatography, by peptide purification.
The condition of purification process are as follows: mobile phase is the acetonitrile containing 0.1% trifluoroacetic acid and containing 0.1% trifluoroacetic acid
Distilled water；Parameter be gradient elution from 5% acetonitrile/95% water to 60% acetonitrile/40% water, flow velocity 10ml/min, when processing
Between be 30min.The MALDI-TOF map of the obtained polymerized monomer molecule of the above method is as shown in Figure 3.
(3) polymerization of polypeptide enzymatically.
Synthetic polymerized monomer molecule HEPES buffer solution is dissolved, prepared glutamine transfer is added to
In enzyme solutions, 37 DEG C, shaking table 4h or so under 150rmp, transglutaminase can be with amino and paddy on catalytic polypeptide chain lysine
Covalent cross-linking occurs between hydroxyl amide groups in glutamine, to be formed to the thermo-responsive poly- peptide of class elastin laminin, the polymerization of formation
Object molecular weight is about 30000, degree of polymerization 27-28.The nanometer assembling pattern of the polymer in vitro is characterized with scanning electron microscope, such as
Shown in Fig. 4.
(4) assemble method of the in-situ nano material is used to monitor the activity of transglutaminase.
Polymerized monomer molecule is added in cell culture medium with the ultimate density that concentration is 600 μM, stationary culture 12h
Afterwards, cell is cleaned three times repeatedly with PBS, then with Hochest (blue) and Dil (red) respectively to nucleus and cell membrane
It is dyed, and is imaged with confocal microscope, imaging temperature is 4 DEG C (as shown in Figure 5).
Embodiment 13
Utilize the method for the polypeptide monomer molecule preparation nano material based on desmoenzyme catalytic polymerization, including following step
It is rapid:
Using sequence VPHVG shown in SEQ ID NO.24 as elastin polypeptide segment, Gln-Lys and Lys-Gln difference
For the end Q and the end K, molecular sequences QKVPHVGQK carries out the synthesis of class elastin laminin nanogel of the present invention as follows:
(1) synthesis of connecting peptides
Connecting peptides are synthesized using Fmoc solid-phase synthesis:
The Wang resin of 0.35mM modification density is selected in synthesis, wherein the N-terminal of first amino acid (lysine) is by Fmoc
Protection, C-terminal are fixed on resin.The Fmoc protection that N-terminal is sloughed with the DMF solution of the hexahydropyridine of 20% (v/v), then uses indenes
Triketone method of testing test deprotection result.Then by the carboxyl of next amino acid the 4- methyl morpholine (NMM) of 0.4M and 10
The DMF solution activation of benzotriazole-N, N, N', N'- the tetramethylurea hexafluorophosphoric acid ester (HBTU) of times amino acid, and be added
2h is reacted to taking off in de-protected resin.According to said method, remaining all amino acid are all got on by condensation reaction connection, shape
At the connecting peptides for being fixed on resin.
(2) polymerization of polypeptide enzymatically.
Synthetic polymerized monomer molecule HEPES buffer solution is dissolved, prepared glutamine transfer is added to
In enzyme solutions, 37 DEG C, shaking table 4h or so under 150rmp, transglutaminase can be with amino and paddy on catalytic polypeptide chain lysine
Covalent cross-linking occurs between hydroxyl amide groups in glutamine, to be formed to thermo-responsive class elastin laminin nanogel, gelation
Matter is as shown in Figure 6.
(3) assemble method of the in-situ nano material is for promoting Apoptosis.
Polymerized monomer molecule is added in cell culture medium with the ultimate density that concentration is 600 μM, stationary culture 12h
Afterwards, cell is cleaned three times with PBS repeatedly, the actin of cell is then contaminated with the phalloidine of Cy5 label, and total with fluorescence
Focusing microscope imaging, imaging temperature are 37 DEG C, it can be seen that the cell of no transglutaminase expression is compared, it is intracellular solidifying
It the formation of glue and collapses so that actin is by considerable damage, promotes Apoptosis (shown in such as Fig. 7 (a)-(b)).
It can be seen from above-described embodiment that polypeptide monomer molecule provided by the invention can glutamine in the cell
Transfer enzymatic issues raw polymerization, and the poly- peptide of formation has class elastin laminin property, can occur in the environment for changing temperature
The distortion and winding of polymer chain realize nano-supermolecule assembling in situ and controllably prepare class elastin laminin in the cell
Nano material, and realize the function of imaging or disease treatment.
The Applicant declares that the present invention illustrates the present invention by above-mentioned case study on implementation, but the invention is not limited to above-mentioned,
Not meaning that the present invention must rely on above-mentioned could implement.It should be clear to those skilled in the art, to the present invention
Any improvement, addition, selection of concrete mode of equivalence replacement and auxiliary element to raw material selected by the present invention etc. are fallen
Within the scope of protection and disclosure of the present invention.
Claims (21)
1. a kind of polypeptide monomer molecule based on desmoenzyme catalytic polymerization, which is characterized in that the polypeptide monomer molecule is by function
Can molecule, the glutamine end in polymerization activity site, class elastin laminin repeat the lysine end in polypeptide and polymerization activity site according to
It is secondary to connect and compose；
The glutamine terminal sequence is as shown in one of SEQ ID NO.1-2；The sequence that the class elastin laminin repeats polypeptide is
Sequence shown in SEQ ID NO.24-30 is any；The lysine end is lysine residue；
The functional molecular is imaging molecule and/or drug molecule；The imaging molecule is fluorescence imaging molecule, photoacoustic imaging point
Son or polar sensitive molecule in any one or at least two combination；The drug molecule be chemotherapy of tumors molecule and/or
Antibiotic molecule.
2. polypeptide monomer molecule as described in claim 1, which is characterized in that the fluorescence imaging molecule is purpurine 18, coke
In pheophytin, Cy5-Cy7 or DBD molecule any one or at least two combination.
3. polypeptide monomer molecule as described in claim 1, which is characterized in that the chemotherapy of tumors molecule is adriamycin.
4. polypeptide monomer molecule as described in claim 1, which is characterized in that the antibiotic molecule is vancomycin.
5. the polymerization of the polypeptide monomer molecule as described in one of claim 1-4, which is characterized in that the polypeptide monomer point
The polymerization of son is to be polymerized in vitro living cells by enzymic catalytic reaction.
6. method as claimed in claim 5, which is characterized in that the temperature of the catalysis reaction is 37-40 DEG C.
7. method as claimed in claim 6, which is characterized in that the temperature of the catalysis reaction is 37 DEG C.
8. method as claimed in claim 5, which is characterized in that the time of the catalysis reaction is 4-12h.
9. method according to claim 8, which is characterized in that the time of the catalysis reaction is 4h.
10. a kind of method for constructing nano material, which comprises 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 added in transglutaminase solution, is 4-40 in temperature
4-12h is reacted under the conditions of DEG C, obtains nano material.
11. method as claimed in claim 10, which is characterized in that step (1) the polypeptide monomer molecular solution uses HEPES
Buffer solution is prepared.
12. method as claimed in claim 10, which is characterized in that the concentration of step (1) the polypeptide monomer molecule is 10-50
μM。
13. method as claimed in claim 12, which is characterized in that the concentration of step (1) the polypeptide monomer molecule is 50 μM.
14. method as claimed in claim 10, which is characterized in that step (2) the enzyme solutions glutamine transferase
Concentration is 1-3U.
15. method as claimed in claim 10, which is characterized in that transglutaminase described in step (2) and polypeptide list
The reaction molar ratio of body molecule is 1:5-1:10.
16. method as claimed in claim 10, which is characterized in that the transglutaminase includes the recombination that bacterium is extracted
The TG2 extracted in albumen, human or animal tissues.
17. method as claimed in claim 10, which is characterized in that the temperature of step (2) described reaction is 37-40 DEG C.
18. method as claimed in claim 17, which is characterized in that the temperature of step (2) described reaction is 37 DEG C.
19. method as claimed in claim 10, which is characterized in that the time of step (2) described reaction is 4-8h.
20. method as claimed in claim 19, which is characterized in that the time of step (2) described reaction is 4h.
21. the nano material that method as claimed in claim 10 is prepared, which is characterized in that the nano material is one-dimensional
And/or 3-D nano, structure；
The polymerized monomer both ends of the one-dimentional structure are made of glutamine end and lysine end respectively；The three-dimensional structure is gathered
Monomer both ends are closed to be made of glutamine-lysine end and lysine-glutamine end respectively；
The glutamine terminal sequence is as shown in one of SEQ ID NO.1-2；The glutamine-lysine terminal sequence such as SEQ
Shown in ID NO.31；The lysine-glutamine terminal sequence is as shown in SEQ ID NO.32.
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