CN106046115B - A kind of novel polypeptide ligand for modifying quantum dot - Google Patents
A kind of novel polypeptide ligand for modifying quantum dot Download PDFInfo
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- CN106046115B CN106046115B CN201610375715.4A CN201610375715A CN106046115B CN 106046115 B CN106046115 B CN 106046115B CN 201610375715 A CN201610375715 A CN 201610375715A CN 106046115 B CN106046115 B CN 106046115B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/08—Tripeptides
- C07K5/0802—Tripeptides with the first amino acid being neutral
- C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
- C07K5/0806—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/08—Tripeptides
- C07K5/0802—Tripeptides with the first amino acid being neutral
- C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
- C07K5/081—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing O or S as heteroatoms, e.g. Cys, Ser
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Abstract
The invention discloses a kind of novel polypeptide ligands (ATTO-NH) for modifying quantum dot, belong to field of nano biotechnology.Novel polypeptide ligand in the present invention includes being combined for the dyestuff ATTO590 (1) of FRET to occur with quantum dot, improves polypeptide water solubility and negatively charged sequence (2), the asparagine for incorporating quantum point and histidine intervening sequence (3) between each sequence with peptide bond.The ligand synthetic method is simple, and C-terminal in conjunction with quantum dot, is substantially increased the association rate and stability of ligand and quantum dot, can be used as namo fluorescence probe and be widely used in biomarker by 6 groups of asparagines and histidine intervening sequence.
Description
Technical field
The present invention relates to field of nano biotechnology, and in particular to a kind of novel polypeptide ligand for modifying quantum dot.
Background technique
Metal cation invents a kind of detection metal of novel sensitivity because its toxicity causes seriously to pollute to environment
The method of ion is necessary.Molecule sensor based on special metal ion has become the hot spot of Recent study, and is based on
The development of the sensor of quantum dot (QDs) is highly dependent on the self assembly of Novel Ligands and its.The quantum dot of affine metal driving
Because its high controllability effectively applies in functional quantum point.Research shows that histidine number (N≤6) is to polypeptide ligand
Have an impact with quantum dot self assembly, it is found that long histidine sequences can reinforce binding affinity.However, due to steric hindrance, more than 6
The polypeptide sequence of a histidine residues is difficult to improve binding force (Wang, J.H., Li, J.Y., Chen, Y., et
al.Electrophoresis 2015,36,2419–2424.).Therefore, the quantity and structure of histidine play polypeptide ligand
Vital effect is of great significance for designing efficient polypeptide ligand and quantum dot self assembly.In addition, although histidine
Label is one of most popular fusion tag, but it is possible to influence the activity of target protein and their tertiary structure.
Summary of the invention
The technical problem to be solved by the present invention is in order to overcome, existing polypeptide ligand speed in conjunction with QDs is slow, stability is poor
The problems such as, its universal use in biological field is limited, it is more more than 6 histidine residues to solve due to steric hindrance
Peptide sequence be difficult improve binding force, although and histidine tag is one of most popular fusion tag, it is possible to influence
The technical issues of activity of target protein and their tertiary structure, it is fast that the present invention provides one kind speed in conjunction with QDs, Er Qiewen
Qualitative good novel polypeptide ligand.
The technical solution adopted by the present invention to solve the technical problems is: a kind of novel polypeptide ligand for modifying quantum dot,
It is characterized by comprising dyestuff ATTO 590 (1), the raising polypeptide for FRET to occur with quantum dot are water-soluble and negatively charged
Sequence (2), the asparagine for incorporating quantum point and histidine intervening sequence (3) are combined with peptide bond between each sequence.
The dyestuff ATTO 590 (1) for FRET to occur with quantum dot can receive the excitation of quantum dot as receptor
Light issues the transmitting light of new wavelength again；The described raising polypeptide is water-soluble and negatively charged sequence (2) is 2 glutamic acid and one
A glycine；The histidine sequences (3) are the polypeptide sequence of 6 groups of asparagines and histidine interval.
Quantum dot of the present invention is the quantum dot containing Zn, such as CdSe/ZnS, CdTe/ZnS, CdSe/ZnSe or
CdTe/ZnSe。
After above-mentioned technical solution, the beneficial effect that the present invention obtains is: the present invention is devised containing asparagine
(N) and novel polypeptide ligand (the ATTO 590-E of histidine (H)2G(NH)6, ATTO-NH).Pass through asparagine separation group ammonia
Acid, this novel structure will make all histidines and be incorporated in Zn2+On the surface of quantum dot, steric hindrance can be greatly reduced,
Thus it is shown that better binding affinity.In addition, the novel polypeptide ligand of modification quantum dot provided by the invention, synthetic method
Simply, repeatability is high, and speed is fast in conjunction with quantum dot, and stability is high, solves existing quantum dot polypeptide ligand stability not
Foot and lead to its in vivo unstable, further expanded quantum dot --- polypeptide complex is as namo fluorescence probe
Application in biology.
Detailed description of the invention
Fig. 1 is the schematic diagram that FRET occurs for quantum dot and novel polypeptide ligand (ATTO-NH), in figure:
1 for the dyestuff ATTO 590 of FRET to occur with quantum dot；
2 improve polypeptide water solubility and negatively charged sequence；
3 are used for the asparagine and histidine intervening sequence of incorporating quantum point.
Fig. 2 is the electrophoretogram with fluorescent capillary electrophoresis tube detection ATTO-NH in conjunction with QDs, in which:
A, QDs；B, ATTO-NH:QD=8:1；C, ATTO-NH:QD=16:1；D, ATTO-NH:QD=32:1；e,ATTO-
NH:QD=64:1.
Fig. 3 is that stability curve of the ATTO-NH in conjunction with QDs is detected in capillary with various concentration imidazoles.
Fig. 4 is that stability curve of the ATTO-NH in conjunction with QDs is detected in capillary with various concentration histidine.
Fig. 5 is with various concentration polypeptide containing histidine tag H6G6It is steady in conjunction with QDs that ATTO-NH is detected in capillary
Qualitative curve.
Specific embodiment
The present invention will be described further with regard to following embodiment, however, it should be noted that these embodiments are only to illustrate
It is used, and is not necessarily to be construed as the limitation implemented to the present invention.
Embodiment 1
Method of the present invention is using conventional solid phase Fmoc method, i.e., by fmoc-protected monomer amino on solid-phase resin
Expose amino after acid deprotection, peptide bond is formed by the carboxyl of amino acid in condensation reaction and solution, so that amino acid be connected
Onto resin, extend peptide chain from C-terminal to N-terminal.
1, basic material:
(1) resin and connection molecule: the resin that solid phase Fmoc method selects is RinkTree
Rouge.This resin has extraordinary swellability, can make preferably to carry out condensation reaction between peptide chain, and have enough networks
Space meets ever-increasing peptide chain.Using HBTU and HOBt as connection molecule, peptide molecule is fixed on resin.
(2) monomer: synthesizing monomer used is the a-amino acid through chemical modification.
2, reaction step:
First amino acid is covalently attached on resin by the first step
Condensing agent appropriate such as HBTU and HOBt is added, makes to form total rouge by protected amino acid c-terminus and resin to complete
The fixation of amino acid；
Second step, deprotection
Using the Fmoc on 20% piperidines of basic solvent removal amino, amino is exposed.
Third step, activation and crosslinking
Carboxyl on next amino is activated using activator HBTU and HOBt, is crosslinked with the amino on resin, peptide is formed
Key.
4th step repeats second step and third step, and iterative cycles add single amino acid, according to E2G(NH)6
The sequence of sequence synthesizes from right to left, until synthesis is completed.
3, it synthesis post-processing: (1) elutes and is deprotected: being washed peptide chain from resin with deprotection agent trifluoroacetic acid (TFA)
It takes off, and deprotection base.
(2) HPLC analysis purifying, freeze-drying save.
Pass through the above method, synthetic peptide sequence ATTO 590-E2G(NH)6(ATTO-NH)
4, QDs and ATTO-NH Effect study in capillary:
In capillary, first sample introduction QDs 10s is spaced 10s, then sample introduction ATTO-NH 10s.The experimental results showed that ATTO-
The association reaction of NH and QDs reaches balance (such as Fig. 2, curve d) in 32:1.
In order to detect stability of the novel polypeptide ligand in conjunction with QDs, after QDs and ATTO-NH sample introduction, then sample introduction replaces
Molecule Im, His, H6G6.The experimental results showed that after a large amount of substitution molecules are added, under part ATTO-NH can be substituted from the surface QDs
Come (Fig. 3,4,5).
Test method:
With the combination of fluorescent capillary electrophoresis tube detection ATTO-NH and QDs, deposition condition: 25mM borate buffer (pH
9.3), voltage 18kV.[QD]=2.5 μM.Detection wavelength, solid line are that 565nm detects QDs, and dotted line is that 625nm detects ATTO
590。
Stability of the ATTO-NH in conjunction with QDs is detected in capillary with various concentration imidazoles.Deposition condition: 25mM boron
Acid buffer (pH 9.3), voltage 18kV.ATTO-NH:QD=32:1, [QD]=2.5 μM.
Stability of the ATTO-NH in conjunction with QDs is detected in capillary with various concentration histidine.Deposition condition: 25mM
Borate buffer (pH 9.3), voltage 18kV.ATTO-NH:QD=32:1, [QD]=2.5 μM.
With various concentration polypeptide containing histidine tag H6G6Stability of the ATTO-NH in conjunction with QDs is detected in capillary.
Deposition condition: 25mM borate buffer (pH 9.3), voltage 18kV.ATTO-NH:QD=32:1, [QD]=2.5 μM.
Embodiment 2
Peptide systhesis step is referring to embodiment 1.According to DDG (NH)6The sequence of sequence synthesizes from right to left, and synthesizing new is more
Peptide ligand sequence is as follows:
ATTO-DDG(NH)6
Embodiment 3
Peptide systhesis step is referring to embodiment 1.According to EES (NH)6The sequence of sequence synthesizes from right to left, and synthesizing new is more
Peptide ligand sequence is as follows:
ATTO-EES(NH)6
The polypeptide sequence that embodiment 2~3 is obtained according to the method for embodiment 1 respectively with QDs capillary electrophoresis analysis,
Testing conditions are same as Example 1, and testing result finds that they can be with QDs stable bond.
It can be seen from above-described embodiment that quantum dot and polypeptide ligand may be implemented in novel polypeptide ligand of the invention
It quickly combines, and stability is high, it is easy to operate.
Taking the above-mentioned ideal embodiment according to the present invention as inspiration, through the above description, relevant staff is complete
Various changes and amendments can be carried out without departing from the scope of the technological thought of the present invention' entirely.The technology of this invention
Property range is not limited to the contents of the specification, it is necessary to which the technical scope thereof is determined according to the scope of the claim.
Claims (5)
1. a kind of novel polypeptide ligand for modifying quantum dot, it is characterised in that: for the dyestuff ATTO of FRET to occur with quantum dot
Polypeptide water solubility and negatively charged sequence (2), the asparagine for incorporating quantum point and histidine interval sequence are improved in 590 (1)
It arranges (3) and forms polypeptide ligand, combined between each sequence with peptide bond.
2. a kind of novel polypeptide ligand for modifying quantum dot according to claim 1, it is characterised in that: described to be used for and measure
The exciting light that the dyestuff ATTO 590 (1) that FRET occurs for son point can receive quantum dot as receptor issues the transmitting of new wavelength again
Light.
3. a kind of novel polypeptide ligand for modifying quantum dot according to claim 1, it is characterised in that: the raising is more
Peptide water solubility and negatively charged sequence (2) are 2 glutamic acid or aspartic acid and a glycine or serine any combination sequence
Column.
4. a kind of novel polypeptide ligand for modifying quantum dot according to claim 1, it is characterised in that: the histidine
Sequence (3) is the polypeptide sequence of 6 groups of asparagines and histidine interval.
5. a kind of novel polypeptide ligand for modifying quantum dot according to claim 1, it is characterised in that: the quantum dot
For the quantum dot containing Zn, such as CdSe/ZnS, CdTe/ZnS, CdSe/ZnSe or CdTe/ZnSe.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102961761A (en) * | 2012-11-02 | 2013-03-13 | 常州大学 | Quantum dot targeting probe for colorectal cancer tumor tissue identification and preparation method thereof |
| CN103497231A (en) * | 2013-09-18 | 2014-01-08 | 常州大学 | Method for marking protein polymer by using quantum dots |
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- 2016-05-31 CN CN201610375715.4A patent/CN106046115B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102961761A (en) * | 2012-11-02 | 2013-03-13 | 常州大学 | Quantum dot targeting probe for colorectal cancer tumor tissue identification and preparation method thereof |
| CN103497231A (en) * | 2013-09-18 | 2014-01-08 | 常州大学 | Method for marking protein polymer by using quantum dots |
Non-Patent Citations (3)
| Title |
|---|
| A capillary electrophoresis method to explore the self-assembly of a novel polypeptide ligand with quantum dots;Wang Jianhao等;《Electrophoresis》;20160623(第37期);第7962-7966页 |
| Capillary electrophoretic studies on quantum dots and histidine appended peptides self-assembly;Wang Jianhao等;《Electrophoresis》;20150818(第36期);第2419-2424页 |
| Fast Self-Assembly Kinetics of Quantum Dots and a Dendrimeric Peptide Ligand;Wang Jianhao等;《Langmuir》;20120514(第28期);第2156-2162页 |
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