CN106518969B - The peptide combined with lipase targeting is with basic sequence and its application - Google Patents
The peptide combined with lipase targeting is with basic sequence and its application Download PDFInfo
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- CN106518969B CN106518969B CN201611004667.4A CN201611004667A CN106518969B CN 106518969 B CN106518969 B CN 106518969B CN 201611004667 A CN201611004667 A CN 201611004667A CN 106518969 B CN106518969 B CN 106518969B
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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)
- G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
- G01N2333/918—Carboxylic ester hydrolases (3.1.1)
- G01N2333/92—Triglyceride splitting, e.g. by means of lipase
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- Life Sciences & Earth Sciences (AREA)
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- Urology & Nephrology (AREA)
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- Microbiology (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The present invention relates to the peptides combined with lipase targeting with basic sequence and its application, belongs to polypeptide design and virus antigen detection field.The present invention is by means of molecular docking and virtual screening technology, on the basis of lipase crystal structure, pass through molecular docking technology, search in virtual peptide library with target proteins binding pattern and the optimal peptide aglucon of affinity, finally obtaining can be with the peptide aglucon in conjunction with lipase, its peptide is RRRDCW, i.e. P19 with basic sequence.Artificial synthesized P19, and affinity identification is carried out with lipase, the equilibrium dissociation constant KD to interact between P19 sequence and the lipase manually expressed is 3.653 × 10‑8M, i.e. 36.53nM illustrate that affinity is preferable.The P19 sequence that the present invention designs can be combined with artificial expression albumen, artificial purifying protein, except higher outer with lipase reactivity, intercrossing does not occur with other albumen and react, specific preferable.
Description
Technical field
The present invention relates to the peptides combined with lipase targeting with basic sequence and its application, belongs to polypeptide design and viral antigen
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.
Lipase is a kind of enzyme of hydrolysis of long chain fatty acid glyceride, is mainly derived from pancreas, is the digestion of pancreatic secretion
One of enzyme.In normal blood, only a small amount of lipase, the lipase in blood is easily removed by kidney.But when the acute pancreas of generation
When adenositis, lipase active is increased in serum in 4-8h after the onset, for 24 hours reach to peak value, general to continue 8-14 days.Lipase can rise to
2-50 times of the normal reference value upper limit.Fatty enzymic change is usually parallel with amylase, but increases earlier than amylase, declines later,
And elevation amplitude is big, therefore more more sensitive than diagnosis of the amylase to acute pancreatitis, more there is diagnostic significance.
Summary of the invention
The present invention passes through molecule on the basis of lipase crystal structure by means of molecular docking and virtual screening technology
Docking technique is searched with target proteins binding pattern and the optimal polypeptide ligand of affinity in virtual peptide library, and peptide is with motif
It is classified as RRRDCW, i.e. P19.Artificial synthesized P19 carries out surface plasma body resonant vibration (Surface using the lipase of expression and purification
Plasmon Resonance, SPR) it is tested in conjunction with ELISA, the results showed that, artificial synthesized P19 has good with lipase
Thus binding ability proves, by the peptide aglucon that the present invention designs, can be used for lipase in serum and carry out qualitatively and quantitatively
Quick detection.
To achieve the goals above, the technical scheme adopted by the invention is that:
The peptide combined with lipase targeting is RRRDCW with basic sequence with basic sequence, the peptide.
The described peptide combined with lipase targeting is any including matching basic sequence as core using the peptide with basic sequence
The corresponding adjustment or modification that the peptide is carried out with basic sequence；Decorative material include but be not limited in nano material, fluorescent material,
Enzyme, biotin and specific protein.
The peptide is used for the detection of lipase in serum with basic sequence, including but be not limited to enzyme linked immunological and inhale
Adhesion test (ELISA) detection.
Application of the peptide with basic sequence in the quick detection of lipase a kind of described in, it is dynamic including but not limited to lactation
In object serum, the lipase that artificial recombination expression or artificial means of purification obtain.
A kind of application of the peptide with basic sequence in lipase 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 lipase crystal structure, by dividing
Sub- docking technique is searched with target proteins binding pattern and the optimal peptide aglucon of affinity in virtual peptide library, and finally obtaining can
Peptide aglucon in conjunction with lipase, peptide are RRRDCW, i.e. P19 with basic sequence.Artificial synthesized P19, and parent is carried out with lipase
It is identified with power, the equilibrium dissociation constant KD to interact between P19 sequence and the lipase manually expressed is 3.653 × 10-8M,
That is 36.53nM illustrates that affinity is preferable.
2, due to manually expressing the limitation of lipase, poor for the specificity of the antibody of lipase, the present invention is set
It counts obtained P19 sequence and avoids this problem well, realize rapid artificial synthesis and cheap testing cost.
3, the P19 sequence that designs of the present invention and manually express albumen, artificial purifying protein can combine, remove and rouge
Fat enzyme reaction 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 lipase structures 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 P19 sequence, it can be achieved that determining lipase
Property and quantitative quick detection.
Detailed description of the invention
Fig. 1 is that P19 sequence is shown with the result of docking of lipase.
Fig. 2 is the SPR affinity qualification result of P19 sequence and artificial expression lipase.Wherein, ordinate representative sensor
Detected signal value；The time that abscissa representative sample interacts in the sensor.In figure, each curve pair from top to bottom
The P19 concentration answered gradually decreases.
Fig. 3 is the ELISA qualification result of P19 sequence and different samples.
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 lipase
The crystal structure (5DMK) that mammal fat enzyme is searched from PDB database, by computer program to crystal
Structure is analyzed, and is selected the particular amino acid residue between its 70 to 240th 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 P19, and polypeptide sequence is-half Guang of Arg-Arg-argin-ine-aspartic acid
Propylhomoserin-tryptophan (Arg-Arg-Arg-Asp-Cys-Trp writes a Chinese character in simplified form RRRDCW) (SEQ ID NO.1), is docked with lipase
Interaction position result is shown in Fig. 1.
Embodiment 2P19 sequence and the affinity of artificial expression lipase are identified
1, the lipase 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), lipase injection be equipped in the SPR detector of amino chip, guarantee EDC/NHS and lipase respectively with amino
Chip interaction 5min, implements the coupling of lipase and amino chip.After the completion of coupling, which may be used for measuring
Interaction between lipase and P19 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, P19 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 180.01 μM, 90.07 μM, 45.03 μM, 22.51 μM, 11.25 μM, 5.63 μM, 2.81 μM, 1.41 μM of P19
Solution injects sample every time and is all made of 20 μ l/min since the P19 solution for successively injecting 250 μ l low concentration into sensor
Flow velocity and with sensor interact 5min, finally with PBS buffer solution (pH 7.4) rinse 5min.It is dense with obtained difference
The combination and dissociation curve for spending P19 solution and fatty enzyme interacting are foundation, carry out the parent that P19 sequence is combined with lipase
(see Fig. 2) is analyzed with power.
The result shows that in conjunction with P19 sequence with the lipase manually expressed has preferable compatibility, phase interaction between the two
Equilibrium dissociation constant KD is 3.653 × 10-8M, i.e. 36.53nM.
The ELISA of the lipase of embodiment 3P19 sequence and serum is identified
1, it takes mammalian (positive) to be centrifuged, removes haemocyte, serum is taken to carry out ELISA Plate coating；With same
Mode is by different samples, i.e. lipase standard items, negative serum, heat shock protein (HSP), bovine serum albumin(BSA) (BSA) and ovum
Albumin (OVA) carries out ELISA Plate coating, as control.Wherein, it is dilute to be all made of the progress of carbonate (CBS) buffer for envelope antigen
It releases, every 50 μ l of hole is added in 96 hole elisa Plates, under the conditions of being placed in 4 DEG C overnight, again with quality point after washing 5 times with PBST buffer
The BSA liquid of number 2% is closed.
2, PBS buffer solution (pH 7.4) is used to dilute in P19 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 the lipase in P19 sequence and mammalian serum has preferable compatibility and specific binding,
It does not react with other negative serums (see Fig. 3).
SEQUENCE LISTING
<110>the first affiliated hospital, Zhengzhou University
<120>peptide combined with lipase targeting is with basic sequence and its application
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 6
<212> PRT
<213>artificial sequence
<400> 1
Arg Arg Arg Asp Cys Trp
1 5
Claims (2)
1. the peptide combined with lipase targeting is with basic sequence, which is characterized in that the peptide is RRRDCW with basic sequence.
2. a kind of peptide as described in claim 1 matches basic sequence answering in the quick detection reagent or kit for preparing lipase
With, including but not limited in mammalian serum, the lipase that artificial recombination expression or artificial means of purification obtain.
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| CN201611004667.4A CN106518969B (en) | 2016-11-15 | 2016-11-15 | The peptide combined with lipase targeting is with basic sequence and its application |
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| CN106518969A CN106518969A (en) | 2017-03-22 |
| CN106518969B true CN106518969B (en) | 2019-03-15 |
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| CN108690838B (en) * | 2018-05-30 | 2021-09-21 | 华南理工大学 | Marine-source monoglyceride lipase, crystal structure and preparation method thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0407033A2 (en) * | 1989-06-05 | 1991-01-09 | Rhone-Poulenc Inc. | Lipase and isozymes, notably of Candida rugosa, and their use |
| CN1345963A (en) * | 2000-09-29 | 2002-04-24 | 上海博德基因开发有限公司 | Novel polypeptide-UDP glycosyltransferase (UGT) and cobalamin conjugated protein 9.57 and polynucleotide for encoding said polypeptide |
| CN103305489A (en) * | 2012-03-08 | 2013-09-18 | 中国农业科学院饲料研究所 | Lipase, coding gene and application thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9644005B2 (en) * | 2011-09-21 | 2017-05-09 | King Abdullah University Of Science And Technology | Didemnin biosynthetic gene cluster in Tistrella mobilis |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0407033A2 (en) * | 1989-06-05 | 1991-01-09 | Rhone-Poulenc Inc. | Lipase and isozymes, notably of Candida rugosa, and their use |
| CN1345963A (en) * | 2000-09-29 | 2002-04-24 | 上海博德基因开发有限公司 | Novel polypeptide-UDP glycosyltransferase (UGT) and cobalamin conjugated protein 9.57 and polynucleotide for encoding said polypeptide |
| CN103305489A (en) * | 2012-03-08 | 2013-09-18 | 中国农业科学院饲料研究所 | Lipase, coding gene and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| P19, a hormonally regulated phosphoprotein of peptide hormone-producing cells: secretagogue-induced phosphorylation in AtT-20 mouse pituitary tumor cells and in rat and hamster insulinoma cells;Pasmantier R 等;《Endocrinology》;19860930;第119卷(第3期);第1229-1238页 * |
| 纤维连接蛋白模拟肽P19的实验研究;宁江海 等;《解放军医学杂志》;20050630;第30卷(第6期);第475-478页 * |
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