CN104804068B - A kind of polypeptide and its application and the method that polypeptid covalence is connected to solid phase interface - Google Patents
A kind of polypeptide and its application and the method that polypeptid covalence is connected to solid phase interface Download PDFInfo
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- CN104804068B CN104804068B CN201410038545.1A CN201410038545A CN104804068B CN 104804068 B CN104804068 B CN 104804068B CN 201410038545 A CN201410038545 A CN 201410038545A CN 104804068 B CN104804068 B CN 104804068B
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Abstract
The invention belongs to biomolecule fixation field, specifically a kind of polypeptide and its method applied and polypeptid covalence is connected to solid phase interface.Peptide sequence is shown in amino acid in SEQ ID No.1.The polypeptide is used for the monitoring cellulase endoglucanase i of qualitative/quantitative as target recognition element（EG I）.The present invention realizes the fixation of polypeptide by symmetrical carrier so that fixed polypeptide has the characteristics that orientation is homogeneous, density is high.
Description
Technical field
The invention belongs to biomolecule fixation field, specifically a kind of polypeptide and its application connect with by polypeptid covalence
To the method for solid phase interface.
Background technology
With the completion of the Human Genome Project, protein will be increasingly becoming the emphasis of life science research.In view of
The interaction of protein and other biological molecule, such as albumen, sugar, fat, is all by how peptide-mediated mostly.Therefore, it is more
Peptide plays very important effect in these interactions.In addition, the distinctive advantage of immobilization technology so that polypeptide is fixed on
The fields such as affinity purification, bio-sensing, biochip, new material have a wide range of applications.
With the development of technology, solid phase interface is connected to for polypeptid covalence, increasing method is developed.But
It is these methods or needs to modify, introduces the component of non-amino acid；Or it is limited to the composition of peptide sequence, especially sequence
In containing in the case of lysine and cysteine.In addition, the constant density of polypeptide is low and what these methods can not be ignored lacks
Fall into.Finally, these methods are mostly time-consuming, laborious, poorly efficient, greatly hinder application of the polypeptide in association area.
The content of the invention
The method of solid phase interface is connected to it is an object of the invention to provide a kind of polypeptide and its application and by polypeptid covalence.
To achieve the above object, the technical solution adopted by the present invention is：
A kind of polypeptide, peptide sequence is shown in amino acid in SEQ ID No.1.
A kind of application of polypeptide, the polypeptide are used for as target recognition element in the monitoring cellulase of qualitative/quantitative
Cut dextranase I（EG I）.
A kind of method that polypeptid covalence is connected to solid phase interface, the polypeptide shown in SEQ ID No.1 is loaded into pair
The carrier surface of title, then it is covalently attached to solid phase interface.
Specifically, the polypeptide shown in SEQ ID No.1 is loaded into symmetrical carrier surface, then transfers them to and contain
There is the solid phase interface surface of amino reaction active groups, after covalent reaction, realize the fixation of polypeptide.
Using above-mentioned fixation polypeptide qualitative/quantitative monitor cellulase endoglucanase i（EG I）.
Symmetrical carrier is the organism with symmetrical structure or synthetic material.Organism with symmetrical structure such as Ff bites
Thalline, tobacco mosaic virus (TMV) etc.；Synthetic material with symmetrical structure such as nanoparticle, shell, microballon, rod, line etc..
Polypeptide is loaded in above-mentioned symmetrical carrier；The mode of loading can be by the genetic manipulation of standard, by life
There is the component of symmetrical structure in object, be integrated into organism surface, such as the major capsid protein pVIII of Ff bacteriophages,
Realize its high density, symmetrical loading；Also directly there can be the carrier table of symmetrical structure in nanoparticle, shell, microballon, rod, line etc.
Face directly synthesizes.
The interaction of protein and other biological molecule, such as albumen, sugar, fat, is generally mediated by peptide sequence.In view of
Peptide carrier has the characteristics of symmetry in itself, peptide sequence can it is symmetrical on these carriers, be distributed to high-density, and maintain
Free amino group.Therefore, using common amino reaction active groups（Such as epoxy radicals, aldehyde radical, succinoamino）Activation
Solid phase interface when carrying out polypeptide fixation, most polypeptides of carrier surface are due to away from these active groups, being not involved in anti-
Should, so as to farthest remain the function of polypeptide.
Advantage for present invention：The inventive method is easy, and the effective density of immobilized polypeptide is high.In addition, this is solid
Determine the method also not influence of subject polypeptide sequence composition, there is universality.Polypeptide probe is covalently attached to solid phase interface simultaneously, this
Laid the foundation to detect cellulase content, the ratio for optimizing multienzyme component, the efficient enzyme preparation of exploitation etc..Finally, the polypeptide is consolidated
Fixed strategy will provide for fields such as the fixation of other biological molecule, the functionalization of material interface, bio-sensing and biochips to be borrowed
Mirror, brings new approaches.
Brief description of the drawings
Fig. 1 is that the present invention is the polypeptide A EGSDPRMV high density of example offer, is symmetrically showed in the life with symmetrical structure
Body surface figure.
Fig. 2 is that the present invention is the flow chart that the polypeptide A EGSDPRMV that example provides is fixed and applied by symmetrical carrier
Fig. 3 is the of the invention and contrast A of traditional polypeptide fixed policy：Chip point molding formula；B：Chip scanning figure；C：Scheme B
In two kinds of polypeptide fixed policies fluorescence signal intensity.
Embodiment
For ease of implementing the present invention, the purpose of the present invention, advantage will further illustrate referring to the drawings in conjunction with the embodiments.
Embodiment 1：
Polypeptide is shown in amino acid in SEQ ID No.1.
Sequence table SEQ ID No.1 are：
AEGSDPRMV
（a）Sequence signature：
● length：9
● type：Amino acid sequence
● chain：It is single-stranded
● topological structure：Linearly
（b）Molecule type：Protein
（c）Assuming that：It is no
（d）Antisense：It is no
（e）Initial source：Engineer
Embodiment 2
Using Solid-phase synthesis peptides technology, SEQ ID No.1 are synthesized on Wang resins (there is micro-sphere structure) surface, finally
Remove blocking group.But maintain covalent attachment of the polypeptide with interlaminar resin, that is, obtain polypeptide SEQ ID No.1 symmetry dress
The resin of load.In view of the density and solid phase synthesis efficiency of polypeptide of Wang resin surface active groups, peptide sequence SEQ ID
No.1 can be in Wang resin surfaces high density, symmetrical covalent attachment.
Embodiment 3
Peptide sequence SEQ ID No.1 are by the genetic recombination operations of prior art Plays, using Ff bacteriophage conducts
Axial symmetry carrier, by bacteriophage VIII type display systems, by the amino acid sequence in sequence table SEQ ID No.1, symmetrically
Ground is loaded into phage surface, that is, obtains the organism of polypeptide SEQ ID No.1 symmetry loading（As shown in Figure 1）.In view of Ff
The characteristics of structure of bacteriophage in itself is with VIII type display systems, often it will be all covalently attached on a major capsid protein pVIII
A peptide sequence SEQ ID No.1, it is achieved thereby that peptide sequence SEQ ID No.1 high density, symmetrical loading.
Embodiment 4
The organism that the polypeptide SEQ ID No.1 symmetry of acquisition is loaded（2.2×1012TU/mL）With same volume
Sampling liquid（300mM phosphate buffers containing 0.005% polysorbas20, pH8.5）After mixing, chip point sample instrument is used（Rich Austria of China
Company）Select and be formed on Slide H (German Schott companies) substrate, be then transferred to 37 DEG C, carry out under 75% damp condition it is anti-
Should, realize the fixation of polypeptide（As the step in Fig. 2 is fixed）.
Embodiment 5
1. closing.The Slide H substrates of the functionalization in embodiment 4, finally given are placed in containing 50mM monoethanolamines
Borate buffer（50mM,pH9.2）In.37 DEG C maintain 2 hours, remaining amino reaction active groups in closing Slide H（Such as
Step monoethanolamine closing in Fig. 2）.
2. it is incubated.After Seal treatment, at ambient temperature, PBST buffer solution for cleaning 3 times, every time 5 minutes is used.Then make
Cleaned again with deionized water 5 minutes.After centrifuge dripping, the EG I that fluorescent dye Cy3 is marked are placed in solid phase surface, it is and fixed
More peptide interactions.37 DEG C maintain 2 hours（As the step in Fig. 2 is incubated）.
3. signal interpretation.At room temperature, PBST buffer solution for cleaning 3 times, every time 5 minutes is used.Then using deionized water again
Cleaning 5 minutes.After centrifuge dripping, fluorescence signal intensity is obtained using Fluorescence Scanner（Such as the step signal interpretation in Fig. 2）.Energy
Enough significantly to see that EG I and polypeptide SEQ ID No.1 symmetry load the combination of organism, it also can be from Fig. 3 B right side
Find out.
Comparative example
Amino acid in embodiment 1 shown in SEQ ID No.1 is conventionally fixed.First, it is contemplated that more
Peptide SEQ ID No.1 fix after flexible variety and its spatial orientation when being interacted with EG I, in polypeptide SEQ ID
No.1 C-terminal loading GGGK sequences, that is, the peptide sequence being finally synthesizing is AEGSDPRMVGGGK, and wherein GGG provides polypeptide and is total to
The connexon that valency is fixed, improve the free degree of institute's immobilized polypeptide.K provides extra amino group so that polypeptide can covalently connect
It is connected on Slide H.
As control, by the organism in the polypeptide of above-mentioned synthesis and embodiment 3, according to the flow points system in embodiment 4
In on same chip, the fixation of polypeptide is carried out.Then, according to the step in embodiment 5, signal interpretation is carried out.Final result is such as
Shown in Fig. 3, wherein Fig. 3 A are the point molding formula of chip, and 1 is the polypeptide fixed based on conventional method, and 2 is by institutes in embodiment 3
Obtain the polypeptide of organism and fixation；Fig. 3 B are last chip scans；Fig. 3 C are Fig. 3 B quantized result.Using based on this
The prepared polypeptide chip of invention, its fluorescence signal intensity is approximately 500 times of polypeptide chip prepared by conventional method, i.e., of the invention
The polypeptide fixed, its efficiency are substantially better than conventional method.
Claims (5)
- A kind of 1. polypeptide, it is characterised in that：Peptide sequence is shown in SEQ ID No.1.
- A kind of 2. application of the polypeptide described in claim 1, it is characterised in that：It is fixed that immobilized polypeptide is used for as target recognition element The monitoring cellulase endoglucanase i of property/quantitative（EG I）.
- A kind of 3. method that polypeptid covalence is connected to solid phase interface, it is characterised in that：Polypeptide shown in SEQ ID No.1 is filled Symmetrical carrier surface is loaded in, is then covalently attached to solid phase interface again.
- 4. the method that polypeptid covalence is connected to solid phase interface as described in claim 3, it is characterised in that：By SEQ ID Polypeptide shown in No.1 is loaded into symmetrical carrier surface, then transfers them to the solid phase containing amino reaction active groups again Interface surface, after covalent reaction, realize the fixation of polypeptide.
- 5. the method that polypeptid covalence is connected to solid phase interface as described in claim 4, it is characterised in that：Utilize above-mentioned fixation Polypeptide qualitative/quantitative monitor cellulase endoglucanase i（EG I）.
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| CN103348244A (en) * | 2011-01-31 | 2013-10-09 | 株式会社日立制作所 | Oligopeptide sequence specifically bonding to phenylboronic acid group |
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2014
- 2014-01-26 CN CN201410038545.1A patent/CN104804068B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101023187A (en) * | 2004-03-05 | 2007-08-22 | 伯乐实验室有限公司 | KSV-2 type specific immunoassays using glycoprotein G2 peptides |
| CN103348244A (en) * | 2011-01-31 | 2013-10-09 | 株式会社日立制作所 | Oligopeptide sequence specifically bonding to phenylboronic acid group |
| CN103087969A (en) * | 2011-10-28 | 2013-05-08 | 中国科学院青岛生物能源与过程研究所 | Bacterial surface demonstrating system for xylose dehydrogenase based on ice nucleating protein and application of system |
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| Phagemid vectors for phage display: properties, characteristics and construction.;Qi H等;《Journal of Molecular Biology 》;ELSEVIER;20120330;第417卷(第3期);第129-143页 * |
| 基于冰核蛋白的木糖脱氢酶细菌展示系统的构建及其在D-木糖检测中的应用;梁波等;《中国化学会第28届学术年会第9分会场摘要集》;CNKI;20120413;第1页 * |
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