CN104804068A - Polypeptide, application thereof, and method for covalently connecting polypeptide to solid phase interface - Google Patents
Polypeptide, application thereof, and method for covalently connecting polypeptide to solid phase interface Download PDFInfo
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- CN104804068A CN104804068A CN201410038545.1A CN201410038545A CN104804068A CN 104804068 A CN104804068 A CN 104804068A CN 201410038545 A CN201410038545 A CN 201410038545A CN 104804068 A CN104804068 A CN 104804068A
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Abstract
The invention belongs to the field of biomolecule immobilization, and concretely relates to a polypeptide, an application thereof, and a method for covalently connecting the polypeptide to a solid phase interface. The amino acid sequence of the polypeptide is represented by SEQ ID No.1. The polypeptide can be used in qualitative/quantitative monitoring of cellulase endoglucanase I (EG I) as a target identification element. The polypeptide is immobilized through a symmetric vector to make the immobilized polypeptide have the characteristics of uniform orientation and high density.
Description
Technical field
The invention belongs to biomolecules fixed network, specifically a peptide species and application thereof and polypeptid covalence is connected to the method for solid phase interface.
Background technology
Along with completing of the Human Genome Project, protein will become the emphasis of life science research gradually.In view of protein and other biological molecule, as the interaction of albumen, sugar, fat etc., mostly all mediated by polypeptide.Therefore, polypeptide plays a part very important in these interact.In addition, the distinctive advantage of immobilization technology, makes polypeptide be fixed on the fields such as affinity purification, bio-sensing, biochip, novel material and has a wide range of applications.
Along with the development of technology, be connected to solid phase interface for polypeptid covalence, increasing method is developed.But these methods or need to modify, introduce the component of non-amino acid; Or be limited to the composition of peptide sequence, when especially containing Methionin and halfcystine in sequence.In addition, the low defect being also these methods and can not be ignored of the constant density of polypeptide.Finally, these methods are mostly time-consuming, effort, poor efficiency, greatly hinder the application of polypeptide in association area.
Summary of the invention
The object of the present invention is to provide a peptide species and application thereof and polypeptid covalence is connected to the method for solid phase interface.
For achieving the above object, the technical solution used in the present invention is:
One peptide species, peptide sequence is in SEQ ID No.1 shown in amino acid.
The application of one peptide species, described polypeptide is used for the monitoring cellulase endoglucanase i (EG I) of qualitative/quantitative as target recognition element.
Polypeptid covalence is connected to a method for solid phase interface, the polypeptide shown in SEQ ID No.1 is loaded into symmetrical carrier surface, then 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 the solid phase interface surface containing amino reaction active groups, after covalent reaction, realize the fixing of polypeptide.
Monitor cellulase endoglucanase i (EG I) with utilizing above-mentioned fixing polypeptide qualitative/quantitative.
Symmetrical carrier is organism or the synthetic materials with symmetrical structure.There is the organism of symmetrical structure as Ff phage, tobacco mosaic virus (TMV) etc.; There is the synthetic materials of symmetrical structure as Nano microsphere, shell, microballon, rod, line etc.
Polypeptide is loaded in the carrier of above-mentioned symmetry; The mode of loading by the genetic manipulation of standard, by the component in organism with symmetrical structure, can be integrated into organism surface, as the major capsid protein pVIII of Ff phage, realizes its high-density, symmetrical loading; The carrier surface that also directly can have a symmetrical structure at Nano microsphere, shell, microballon, rod, line etc. directly synthesizes.
Protein and other biological molecule, as the interaction of albumen, sugar, fat etc., mediated by peptide sequence usually.In view of peptide carrier itself has symmetric feature, peptide sequence can symmetrical on these carriers, distribute to high-density, and maintain amino group freely.Therefore, the solid phase interface using common amino reaction active groups (as epoxy group(ing), aldehyde radical, succinoamino) to activate carries out polypeptide when fixing, most polypeptide of carrier surface, due to away from these active groups, do not participate in reaction, thus farthest remain the function of polypeptide.
The advantage that the present invention has: the inventive method is easy, and the effective density of immobilized polypeptide is high.In addition, this fixing means also not subject polypeptide sequence composition impact, there is universality.Polypeptide probe is covalently attached to solid phase interface simultaneously, this is detection fibers element enzyme content, optimize the ratio of multienzyme component, develop efficient zymin etc. and lay the foundation.Finally, offering reference in the fields such as the functionalization of fixing, the material interface for other biological molecule, bio-sensing and biochip by this polypeptide fixed policy, brings new approaches.
Accompanying drawing explanation
Fig. 1 for the present invention be that polypeptide A EGSDPRMV high-density, the symmetry that example provides is showed in the organism surface figure with symmetrical structure.
Fig. 2 for the present invention be the schema that polypeptide A EGSDPRMV that example provides fixes by symmetrical carrier and applies
Fig. 3 is the contrast A of the present invention and traditional polypeptide fixed policy: chip point molding formula; B: chip scanning figure; The fluorescence signal intensity of two peptide species fixed policy in C: figure B.
Embodiment
For ease of implementing the present invention, object of the present invention, advantage will in conjunction with the embodiments, further illustrate with reference to accompanying drawing.
Embodiment 1:
Polypeptide is in SEQ ID No.1 shown in amino acid.
Sequence table SEQ ID No.1 is:
AEGSDPRMV
(a) sequence signature:
● length: 9
● type: aminoacid sequence
● chain: strand
● topological framework: linear
(b) molecule type: protein
C () is supposed: no
(d) antisense: no
E () is originated at first: engineer
Embodiment 2
Use Solid-phase synthesis peptides technology, at Wang resin (there is micro-sphere structure) surface synthesis SEQ IDNo.1, finally remove blocking group.But, maintain the covalently bound of polypeptide and interlaminar resin, namely obtain the resin of polypeptide SEQ ID No.1 symmetry loading.In view of density and the solid phase synthesis efficiency of polypeptide of Wang resin surface active group, peptide sequence SEQ ID No.1 can in Wang resin surface high-density, symmetrical covalently bound.
Embodiment 3
Peptide sequence SEQ ID No.1 is by the genetic recombination operation of prior art Plays, adopt Ff phage as axial symmetry carrier, by phage VIII type display system, by the aminoacid sequence in sequence table SEQ IDNo.1, be loaded into phage surface symmetrically, namely obtain the organism (as shown in Figure 1) that polypeptide SEQ ID No.1 symmetry is loaded.In view of the structure of Ff phage itself and the feature of VIII type display system, all by covalently bound a peptide sequence SEQ ID No.1 on every a major capsid protein pVIII, thus achieve the high-density of peptide sequence SEQ ID No.1, symmetrical loading.
Embodiment 4
By the organism (2.2 × 10 that the polypeptide SEQ ID No.1 symmetry obtained is loaded
12tU/mL) with the sampling liquid of same volume (containing the 300mM phosphate buffered saline buffer of 0.005% polysorbas20, pH8.5) after mixing, using chip point sample instrument (Chinese Bo Ao company) to select is formed on Slide H (German Schott company) substrate, then be transferred to 37 DEG C, react under the humidity condition of 75%, realize fixing (as the step in Fig. 2 is fixed) of polypeptide.
Embodiment 5
1. close.By in embodiment 4, the Slide H substrate of the functionalization finally obtained is placed in the borate buffer (50mM, pH9.2) containing 50mM thanomin.37 DEG C maintain 2 hours, close remaining amino reaction active groups in Slide H (as the step thanomin in Fig. 2 is closed).
2. hatch.After sealing treatment, at ambient temperature, PBST buffer solution for cleaning 3 times are used, each 5 minutes.Then deionized water is used to clean 5 minutes again.After centrifuge dripping, the EG I that fluorescence dye Cy3 marks is placed in solid phase surface, interacts with fixing polypeptide.37 DEG C maintain 2 hours (as the step in Fig. 2 is hatched).
3. signal interpretation.Under room temperature, use PBST buffer solution for cleaning 3 times, each 5 minutes.Then deionized water is used to clean 5 minutes again.After centrifuge dripping, Fluorescence Scanner is used to obtain fluorescence signal intensity (the step signal interpretation as in Fig. 2).Significantly can see that EG I and polypeptide SEQ ID No.1 symmetry load the combination of organism, it also can be found out from the right-hand part of Fig. 3 B.
Comparative example
Amino acid shown in SEQ ID No.1 in embodiment 1 is conventionally fixed.First, consider polypeptide SEQ ID No.1 fix after flexible variety and itself and EG I interacts time spatial orientation, GGGK sequence is loaded at the C-terminal of polypeptide SEQ ID No.1, namely the peptide sequence of last synthesis is AEGSDPRMVGGGK, wherein GGG provides the connexon that polypeptid covalence is fixed, and improves the degree of freedom of institute's immobilized polypeptide.K provides extra amino group, and polypeptide can be covalently attached on Slide H.
In contrast, by the organism in the polypeptide of above-mentioned synthesis and embodiment 3, be formed on same chip according to the flow points in embodiment 4, carry out the fixing of polypeptide.Subsequently, according to the step in embodiment 5, carry out signal interpretation.As shown in Figure 3, wherein Fig. 3 A is the some molding formula of chip to net result, and 1 is that 2 is the fixing polypeptide by gained organism in embodiment 3 based on the fixing polypeptide of traditional method; Fig. 3 B is last chip scans; Fig. 3 C is the quantized result of Fig. 3 B.Adopt based on the polypeptide chip prepared by the present invention, its fluorescence signal intensity is about 500 times of polypeptide chip prepared by traditional method, namely the present invention the polypeptide fixed, its efficiency is obviously better than traditional method.
Claims (5)
1. a peptide species, is characterized in that: peptide sequence is in SEQ ID No.1 shown in amino acid.
2. an application for polypeptide according to claim 1, is characterized in that: described polypeptide is used for the monitoring cellulase endoglucanase i (EG I) of qualitative/quantitative as target recognition element.
3. method polypeptid covalence being connected to solid phase interface according to claim 1, is characterized in that: the polypeptide shown in amino acid in SEQ ID No.1 is loaded into symmetrical carrier surface, is then covalently attached to solid phase interface again.
4. by method polypeptid covalence being connected to solid phase interface according to claim 3, it is characterized in that: the polypeptide shown in amino acid in SEQ ID No.1 is loaded into symmetrical carrier surface, then transfer them to the solid phase interface surface containing amino reaction active groups again, after covalent reaction, realize the fixing of polypeptide.
5. by method polypeptid covalence being connected to solid phase interface according to claim 4, it is characterized in that: monitor cellulase endoglucanase i (EG I) with utilizing above-mentioned fixing polypeptide qualitative/quantitative.
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|---|---|---|---|
| CN201410038545.1A CN104804068B (en) | 2014-01-26 | 2014-01-26 | A kind of polypeptide and its application and the method that polypeptid covalence is connected to solid phase interface |
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| CN201410038545.1A CN104804068B (en) | 2014-01-26 | 2014-01-26 | A kind of polypeptide and its application and the method that polypeptid covalence is connected to solid phase interface |
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| CN104804068A true CN104804068A (en) | 2015-07-29 |
| CN104804068B CN104804068B (en) | 2017-12-19 |
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Citations (3)
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| 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 |
| 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 |
| 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 |
-
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 |
Non-Patent Citations (4)
| Title |
|---|
| QI H等: "Phagemid vectors for phage display: properties, characteristics and construction.", 《JOURNAL OF MOLECULAR BIOLOGY 》 * |
| 梁波等: "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", 《中国化学会第28届学术年会第9分会场摘要集》 * |
| 殷龙等: "基于风景噬菌体文库的猪瘟病毒配体的高通量筛选", 《纪念中国微生物学会成立六十周年大会暨2012年中国微生物学会学术年会论文集》 * |
| 蒋超: "《STN检索报告》", 27 May 2017 * |
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