CN106432421B - One can be with the polypeptide sequence and its application in conjunction with amylase - Google Patents
One can be with the polypeptide sequence and its application in conjunction with amylase Download PDFInfo
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- CN106432421B CN106432421B CN201611004671.0A CN201611004671A CN106432421B CN 106432421 B CN106432421 B CN 106432421B CN 201611004671 A CN201611004671 A CN 201611004671A CN 106432421 B CN106432421 B CN 106432421B
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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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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/924—Hydrolases (3) acting on glycosyl compounds (3.2)
- G01N2333/926—Hydrolases (3) acting on glycosyl compounds (3.2) acting on alpha -1, 4-glucosidic bonds, e.g. hyaluronidase, invertase, amylase
- G01N2333/928—Hydrolases (3) acting on glycosyl compounds (3.2) acting on alpha -1, 4-glucosidic bonds, e.g. hyaluronidase, invertase, amylase acting on alpha -1, 4-glucosidic bonds, e.g. hyaluronidase, invertase, amylase
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- Immunology (AREA)
- Urology & Nephrology (AREA)
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- General Physics & Mathematics (AREA)
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- Organic Chemistry (AREA)
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- Proteomics, Peptides & Aminoacids (AREA)
- Biotechnology (AREA)
- Food Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Genetics & Genomics (AREA)
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- Enzymes And Modification Thereof (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention relates to one to belong to polypeptide design and virus antigen detection field with the polypeptide sequence and its application in conjunction with amylase.The present invention is by means of molecular docking and virtual screening technology, on the basis of amylase crystals structure, pass through molecular docking technology, search in virtual peptide library with target proteins binding pattern and the optimal polypeptide ligand of affinity, finally obtaining can be with the polypeptide sequence in conjunction with amylase, its polypeptide sequence is HHWYIR, i.e. P615.Artificial synthesized P615, and affinity identification is carried out with amylase, the equilibrium dissociation constant KD to interact between P615 sequence and the amylase manually expressed is 4.94 × 10‑8M, i.e. 49.4nM illustrate that affinity is preferable.The P615 sequence that the present invention designs can be combined with artificial expression albumen, artificial purifying protein, except higher outer with amylase reactivity, intercrossing does not occur with other albumen and react, specific preferable.
Description
Technical field
The present invention relates to one to belong to polypeptide design and viral antigen with the polypeptide sequence and its application in conjunction with amylase
Detection field.
Background technique
Virtual screening technology based on molecular docking is interacted between one kind emerging research polypeptide and protein
Technological means.This technology mainly uses computer rapid computations to realize polypeptide and corresponding target proteins on space conformation
Docking, the molecule in virtual polypeptide database is docked with the given activity site of target proteins crystal structure one by one, is led to
Cross computer rapid computations and constantly position of the adjustment polypeptide in conjunction with target proteins, conformation, the rotatable key of intramolecule two
The amino acid residue side and skeleton of face angle and target proteins, find peptide molecule and target proteins are best on space structure
Conformation, and predict binding pattern between the two and affinity is picked out by score value close to native conformation and target egg
A kind of method of theoretical modeling intermolecular interaction of the white optimal polypeptide ligand of affinity.
Amylase, that is, serum amylase (AMS) in blood is the main parting of amylase in serum, belongs to glycosidic linkage water
Enzyme is solved, pancreas etc. is mainly derived from, in addition the organs such as proximal end duodenum, lung, uterus, mammary gland of lactation period also have a small amount of point
It secretes.Determination of amylase is mainly used for the diagnosis of acute pancreatitis in serum.Generally start to increase in 6-12h after the onset, 12-24h
Reach to peak value restores normal for 2-5 days, thus serum amyloid enzymatic activity significantly increases the early diagnosis to acute pancreatitis be worth compared with
Greatly.
Summary of the invention
The present invention passes through molecule on the basis of amylase crystals structure by means of molecular docking and virtual screening technology
Docking technique, search in virtual peptide library with target proteins binding pattern and the optimal polypeptide ligand of affinity, polypeptide sequence
For HHWYIR, i.e. P615.Artificial synthesized P615 carries out surface plasma body resonant vibration (Surface using the amylase of expression and purification
Plasmon Resonance, SPR) it is tested in conjunction with ELISA, the results showed that, artificial synthesized P615 has good with amylase
Thus binding ability proves, by the polypeptide that the present invention designs, can be used for amylase in serum and carry out qualitatively and quantitatively
Quickly detection.
To achieve the goals above, the technical scheme adopted by the invention is that:
One can be HHWYIR with the polypeptide sequence in conjunction with amylase, the polypeptide sequence.
It is described can with the polypeptide sequence in conjunction with amylase, it is any more to this including using the polypeptide sequence as core
The corresponding adjustment or modification that peptide sequence is carried out；Decorative material includes but is not limited in nano material, fluorescent material, enzyme, life
Object element and specific protein.
The polypeptide sequence is used for the detection of amylase in serum, including but be not limited to Enzyme-linked Immunosorbent Assay
Test (ELISA) detection.
Application of the polypeptide sequence in the quick detection of amylase described in a kind of, including but not limited to mammal
In serum, the amylase that artificial recombination expression or artificial means of purification obtain.
A kind of application of the polypeptide sequence in amylase quick detection qualitatively and quantitatively.
Beneficial effects of the present invention:
1, the present invention is by means of molecular docking and virtual screening technology, on the basis of amylase crystals structure, by dividing
Sub- docking technique is searched in virtual peptide library with target proteins binding pattern and the optimal polypeptide ligand of affinity, is finally obtained
It can be with the polypeptide sequence in conjunction with amylase, polypeptide sequence HHWYIR, i.e. P615.Artificial synthesized P615, and and amylase into
Row affinity identification, the equilibrium dissociation constant KD to interact between P615 sequence and the amylase manually expressed be 4.94 ×
10-8M, i.e. 49.4nM illustrate that affinity is preferable.
2, due to manually expressing the limitation of amylase, poor for the specificity of the antibody of amylase, the present invention is set
It counts obtained P615 sequence and avoids this problem well, realize rapid artificial synthesis and cheap testing cost.
3, the P615 sequence that designs of the present invention and artificial expression albumen, artificial purifying protein can combine, remove with
Amylase reactivity is higher outer, and intercrossing does not occur with other albumen and reacts, and specificity is preferably.
4, the present invention has simple, quick and at low cost feature compared with the screening of traditional phage polypeptide library；Pass through meter
Calculation machine auxiliary implements molecular docking, can provide preferable theoretical direction to realize that amylase structure function parses.
5, the present invention is immunized to have compared with obtaining for the process of protein antibodies with the albumen of artificial expression and purification
It is easy to operate, time saving and energy saving, the advantages such as expense is low；By being marked screening P615 sequence, it can be achieved that carrying out to amylase
Qualitative and quantitative quick detection.
Detailed description of the invention
Fig. 1 is that P615 sequence is shown with the result of docking of amylase.
Fig. 2 is the SPR affinity qualification result of P615 sequence and artificial expression amylase.Wherein, ordinate represents sensing
Signal value detected by device；The time that abscissa representative sample interacts in the sensor.In figure, each curve from top to bottom
Corresponding P615 solution concentration gradually decreases.
Fig. 3 is the ELISA qualification result of P615 sequence and artificial expression amylase.
Fig. 4 is the ELISA qualification result of P615 sequence and amylase positive serum.
Specific embodiment
Specific embodiments of the present invention will be described in further detail with reference to embodiments.
1 molecular docking of embodiment and the screening in virtual peptide library
1, the preparation of amylase
The crystal structure (4W93) that mammal amylase is searched from PDB database, by computer program to crystal
Structure is analyzed, and is selected the particular amino acid residue between its 50 to 300th amino acid residue and is used as docking setting regions, with
Carry out molecular docking.
2, the design of virtual peptide library
The space structure for establishing different aminoacids residue is realized and is criticized to input target polypeptides by computer program
Amount generates, to meet the automatic calling and processing of molecular docking computer program.Virtual peptide library is generated with linear form, no
Any side chain, head and the tail amino and carboxyl are modified.The generation of single virtual peptide library is to be no more than four amino acids residues
It is advisable.
3, judging for result is docked
Polypeptide and protein bound free energy, hydrogen chain are calculated separately, the mechanics parameters such as van der Waals carry out Comprehensive Assessment, with
This determines the selection result, and screens and obtain P615, and polypeptide sequence is that histidine-histidine-tryptophan-tyrosine-is different bright
Propylhomoserin-arginine (His-His-Trp-Tyr-Ile-Arg writes a Chinese character in simplified form HHWYIR) (SEQ ID NO.1), is docked with amylase
Interaction position result is shown in Fig. 1.
2 P615 sequence of embodiment and the affinity of artificial expression amylase are identified
1, the amylase of artificial expression and purification is diluted to 1 μ g/ml (albumen meter) using PBS buffer solution (pH 7.4),
Using active ester method, respectively by 1- (3- dimethylamino-propyl) -3- ethyl carbodiimide/n-hydroxysuccinimide (EDC/
NHS, 1:1), amylase injection be equipped in the SPR detector of amino chip, guarantee EDC/NHS and amylase respectively with amino
Chip interaction 5min, implements the coupling of amylase and amino chip.After the completion of coupling, which may be used for measuring
Interaction between amylase and P615 sequence.
2,250 μ l PBS buffer solution (pH 7.4) are injected, into sensor with maximum flow rate (150 μ l/min) runtime buffer
Liquid reaches signal base line, the flow velocity of buffer is down to 20 μ l/min, to obtain relatively stable baseline.
3, P615 dry powder that is artificial synthesized and modifying in aminoterminal biotinylation is used into PBS buffer solution (pH 7.4) respectively
Dilute at concentration is 343.39 μM, 171.97 μM, 42.29 μM, 85.98 μM, 21.51 μM, 10.74 μM, 5.38 μM of P615 solution,
Since the P615 solution for successively injecting 250 μ l low concentration into sensor, the stream that sample is all made of 20 μ l/min is injected every time
Speed and with sensor interact 5min, finally with PBS buffer solution (pH 7.4) rinse 5min.With obtained various concentration
The combination of P615 liquid and starch enzyme interacting and dissociation curve are foundation, and progress P615 sequence combines affine with amylase
Power analyzes (see Fig. 2).
The result shows that P615 sequence has in conjunction with preferable compatibility with the amylase manually expressed, between the two mutually
The equilibrium dissociation constant KD of effect is 4.94 × 10-8M, i.e. 49.4nM.
The ELISA of 3 P615 sequence of embodiment and artificial expression amylase is identified
1, the amylase of artificial expression and purification is subjected to ELISA Plate coating with 1 μ g/ml (albumen meter)；In the same way will
Different material, i.e. starch, glucan, lipase and maltose carry out ELISA Plate coating, as control.Wherein, envelope antigen is equal
It is diluted using carbonate (CBS) buffer, every 50 μ l of hole is added in 96 hole elisa Plates, under the conditions of being placed in 4 DEG C overnight, uses
PBST buffer is closed with the BSA liquid of mass fraction 2% again after washing 5 times.
2, PBS buffer solution (pH 7.4) is used to dilute in P615 dry powder that is artificial synthesized and modifying in aminoterminal biotinylation
At the concentration of 500ng/ml, it is added in above-mentioned ELISA Plate with the volume of every 50 μ l of hole, after mixing, is placed under the conditions of 37 DEG C, keeps away
Light incubates 30min.
3, it is washed 5 times with PBST buffer, and dries the liquid in ELISA Plate hole；It is peppery using the dilution coupling of PBST buffer
The Avidin (1:1000) of root peroxidase is added in the ELISA Plate of drying with the volume of every 50 μ l of hole, after mixing, is placed in 37
Under the conditions of DEG C, Incubation in dark 30min.
4, according to test aequum, TMB developing solution is added in above-mentioned ELISA Plate with the volume of every 100 μ l of hole, sufficiently
After mixing 30s, under room temperature, develop the color 10min.
5, the sulfuric acid terminate liquid of 2M is added in above-mentioned ELISA Plate with the volume of every 50 μ l of hole, after mixing well 30s,
On microplate reader device, light absorption value of each hole at 450nm is read, determines result.
The result shows that there is the amylase in P615 sequence and mammalian serum preferable compatibility and specificity to tie
It closes, does not react with other materials (see Fig. 3).
The ELISA of 4 P615 sequence of embodiment and serum identification
1, it takes mammalian (positive) to be centrifuged, removes haemocyte, serum is taken to carry out ELISA Plate coating；With same
Amylase standard items, negative serum are carried out ELISA Plate coating by mode, and with the PBS buffer solution of same volume simultaneously as control.
Wherein, envelope antigen is all made of carbonate (CBS) buffer and is diluted, and every 50 μ l of hole is added in 96 hole elisa Plates, is placed in 4 DEG C
Under the conditions of overnight, closed again with the BSA liquid of mass fraction 2% after washing 5 times with PBST buffer.
2, PBS buffer solution (pH 7.4) is used to dilute in P615 dry powder that is artificial synthesized and modifying in aminoterminal biotinylation
At the concentration of 500ng/ml, it is added in above-mentioned ELISA Plate with the volume of every 50 μ l of hole, after mixing, is placed under the conditions of 37 DEG C, keeps away
Light incubates 30min.
3, it is washed 5 times with PBST buffer, and dries the liquid in ELISA Plate hole；It is peppery using the dilution coupling of PBST buffer
The Streptavidin (1:1000) of root peroxidase is added in the ELISA Plate of drying with the volume of every 50 μ l of hole, after mixing, is set
Under the conditions of 37 DEG C, Incubation in dark 30min.
4, according to test aequum, TMB developing solution is added in above-mentioned ELISA Plate with the volume of every 100 μ l of hole, sufficiently
After mixing 30s, under room temperature, develop the color 10min.
5, the sulfuric acid terminate liquid of 2M is added in above-mentioned ELISA Plate with the volume of every 50 μ l of hole, after mixing well 30s,
On microplate reader device, light absorption value of each hole at 450nm is read, determines result.
The result shows that there is the amylase in P615 sequence and mammalian serum preferable compatibility and specificity to tie
It closes, does not react with other negative serums (see Fig. 4).
SEQUENCE LISTING
<110>the first affiliated hospital, Zhengzhou University
<120>one can be with the polypeptide sequence and its application in conjunction with amylase
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 6
<212> PRT
<213>artificial sequence
<400> 1
His His Trp Tyr Ile Arg
1 5
Claims (2)
1. one can be with the polypeptide sequence in conjunction with amylase, which is characterized in that the polypeptide sequence is HHWYIR.
2. a kind of polypeptide sequence as described in claim 1 answering in the quick detection reagent or kit for preparing amylase
With, including but not limited in mammalian serum, the amylase that artificial recombination expression or artificial means of purification obtain.
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| CN201611004671.0A CN106432421B (en) | 2016-11-15 | 2016-11-15 | One can be with the polypeptide sequence and its application in conjunction with amylase |
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| CN201611004671.0A CN106432421B (en) | 2016-11-15 | 2016-11-15 | One can be with the polypeptide sequence and its application in conjunction with amylase |
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| CN106432421B true CN106432421B (en) | 2019-03-15 |
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| CN113061160B (en) * | 2021-04-02 | 2023-06-02 | 河南农业大学 | Targeted Abeta inhibitory polypeptide and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0189485A1 (en) * | 1984-07-16 | 1986-08-06 | Vsesojuzny Kardiologichesky Nauchny Tsentr Akademii Meditsinskikh Nauk Sssr | Hexapeptide |
| CN102965359A (en) * | 2012-12-12 | 2013-03-13 | 江南大学 | Acid-resistant amylase mutant, and preparation method and application thereof |
| CN103232961A (en) * | 2013-04-21 | 2013-08-07 | 中国农业科学院植物保护研究所 | Uvioresistant bacillus thuringiensis strain and building method and application thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008021543A2 (en) * | 2006-08-17 | 2008-02-21 | Monsanto Technology, Llc | Transgenic plants with enhanced agronomic traits |
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2016
- 2016-11-15 CN CN201611004671.0A patent/CN106432421B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0189485A1 (en) * | 1984-07-16 | 1986-08-06 | Vsesojuzny Kardiologichesky Nauchny Tsentr Akademii Meditsinskikh Nauk Sssr | Hexapeptide |
| CN102965359A (en) * | 2012-12-12 | 2013-03-13 | 江南大学 | Acid-resistant amylase mutant, and preparation method and application thereof |
| CN103232961A (en) * | 2013-04-21 | 2013-08-07 | 中国农业科学院植物保护研究所 | Uvioresistant bacillus thuringiensis strain and building method and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| A synthetic peptide of the rab3a effector domain stimulates amylase release from permeabilized pancreatic acini;Padfield PJ 等;《Proc Natl Acad Sci U S A》;19920331;第89卷(第5期);第1656-1660页 * |
| 地衣芽孢杆菌α-淀粉酶信号肽的序列分析及其在大肠杆菌中的分泌特性;蔡恒 等;《华北农学报》;20080430;第23卷(第2期);第106-109页 * |
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