CN106518969A - Peptide ligand sequence target-binding to lipase, and application thereof - Google Patents
Peptide ligand sequence target-binding to lipase, and application thereof Download PDFInfo
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- CN106518969A CN106518969A CN201611004667.4A CN201611004667A CN106518969A CN 106518969 A CN106518969 A CN 106518969A CN 201611004667 A CN201611004667 A CN 201611004667A CN 106518969 A CN106518969 A CN 106518969A
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- lipase
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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
-
- 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)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
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- Hematology (AREA)
- Urology & Nephrology (AREA)
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- Analytical Chemistry (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- General Physics & Mathematics (AREA)
- 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 invention relates to a peptide ligand sequence target-binding to lipase, and an application thereof, and belongs to the field of polypeptide design and virus antigen detection. A peptide ligand binding to the lipase is finally obtained by searching optimum peptide ligands binding to a target protein and having optimum affinity in a virtual polypeptide database through a molecular docking technology on the basis of the crystal structure of the lipase by means of molecular docking and virtual screening technologies, and the sequence of the peptide ligand is RRRDCW, that is P19. The P19 is artificially synthesized, the P19 and the lipase undergo affinity identification, and the interaction balance dissociation constant KD between the sequence of the P19 and the artificially expressed lipase is 3.653 * 10<-8> M, that is 36.53 nM, so the affinity is good. The synthesized P19 sequence can bind to artificially expressed proteins and artificially purified proteins, has high response to the lipase, has no cross response to other proteins, and has good specificity.
Description
Technical field
The present invention relates to the peptide for combining is targetted with lipase matches somebody with somebody basic sequence and its application, belong to polypeptide design and viral antigen
Detection field.
Background technology
It is the interphase interaction of a kind of emerging research polypeptide and protein based on the virtual screening technology of molecular docking
Technological means.This technology mainly realizes polypeptide with corresponding target proteinses on space conformation with computer rapid computations
Docking, the molecule in virtual many peptide databases is docked with the given activity site of target proteinses crystal structure one by one, is led to
Cross computer rapid computations and constantly adjust polypeptide combined with target proteinses position, conformation, the two of the rotatable key of intramolecule
The amino acid residue side and skeleton of face angle and target proteinses, finds peptide molecule optimal on space structure with target proteinses
Conformation, and predict binding pattern between the two and affinity, picked out by score value be close to native conformation with target egg
A kind of method of the theoretical modeling intermolecular interaction of the optimal polypeptide ligand of white 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 are easily removed by kidney.But, when the acute pancreas of generation
During adenositis, after morbidity, in 4-8h, in serum, lipase active is raised, 24h peakings, general to continue 8-14 days.Lipase can rise to
Normal reference value upper limit 2-50 times.Fatty enzymic change is generally parallel with amylase, but raises earlier than amylase, and decline is more late,
And elevation amplitude is big, therefore the diagnosis than amylase to acute pancreatitis is more sensitive, more has diagnostic significance.
The content of the invention
The present invention by means of molecular docking and virtual screening technology, on the basis of lipase crystal structure, by molecule
Docking technique, matches somebody with somebody motif with target proteinses binding pattern and the optimal polypeptide ligand of affinity, its peptide in searching virtual peptide library
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) and ELISA binding tests, as a result showing, artificial synthesized P19 has good with lipase
Binding ability, thus proves, by the peptide aglucon that the present invention is designed, in can be used for serum, lipase is carried out qualitatively and quantitatively
Quick detection.
To achieve these goals, the technical solution adopted in the present invention is：
The peptide for combining is targetted with lipase and matches somebody with somebody basic sequence, described peptide is RRRDCW with basic sequence.
The described peptide for combining that targets with lipase matches somebody with somebody basic sequence, matches somebody with somebody basic sequence as core including with described peptide, any
The corresponding adjustment carried out with basic sequence by the peptide or modification；Decorative material include but be not limited in nano material, fluorescent material,
Enzyme, biotin and specific protein.
By described peptide with basic sequence be used for serum in lipase detection, including but be not limited to enzyme linked immunological suction
Adhesion test (ELISA) is detected.
A kind of described peptide matches somebody with somebody application of the basic sequence in the quick detection of lipase, dynamic including but not limited to lactation
In thing serum, the lipase that obtains of artificial recombination expression or artificial means of purification.
A kind of application of described peptide with basic sequence in lipase quick detection qualitatively and quantitatively.
Beneficial effects of the present invention：
1st, it is of the invention by means of molecular docking and virtual screening technology, on the basis of lipase crystal structure, by dividing
Sub- docking technique, with target proteinses binding pattern and the optimal peptide aglucon of affinity in the virtual peptide library of search, finally giving can
The peptide aglucon combined with lipase, its peptide are RRRDCW, i.e. P19 with basic sequence.Artificial synthesized P19, and parent is carried out with lipase
Identify with power, the equilibrium dissociation constant KD of the interphase interaction of the lipase that P19 sequences are reached with labor statement is 3.653 × 10-8M,
That is 36.53nM, illustrates that affinity is preferable.
2nd, due to labor statement up to lipase limitation, for lipase antibody it is specific poor, the present invention sets
The P19 sequences that meter is obtained avoid this difficult problem well, realize rapid artificial synthesis and cheap testing cost.
3rd, the P19 sequences that design of the present invention can be combined with artificial expressing protein, artificial purifying protein, except with fat
Fat enzyme reaction is higher outer, and intercrossing reaction does not occur with other albumen, and specificity is preferably.
4th, it is of the invention compared with traditional phage polypeptide storehouse is screened, with simple, the characteristics of quick and low cost；By meter
Calculation machine auxiliary implements molecular docking, can provide preferable theoretical direction to realize that lipase structures function is parsed.
5th, the albumen with artificial expression and purification of the invention carries out immunity, compared with obtaining the process for protein antibodies, to have
It is simple to operate, time saving and energy saving, the low advantage of expense；By being marked to screening P19 sequences, it is capable of achieving lipase is carried out determining
Property and quantitative quick detection.
Description of the drawings
Fig. 1 is that P19 sequences are shown with the result of docking of lipase.
Fig. 2 is the SPR affinity qualification results that P19 sequences and labor statement reach lipase.Wherein, ordinate representative sensor
Detected signal value；The time that abscissa representative sample is interacted in the sensor.In figure, each curve pair from top to bottom
The P19 concentration answered is gradually lowered.
Fig. 3 is the ELISA qualification results of P19 sequences and different samples.
Specific embodiment
With reference to embodiments the specific embodiment of the present invention is described in further detail.
1 molecular docking of embodiment and the screening in virtual peptide storehouse
1st, the preparation of lipase
The crystal structure (5DMK) of mammal fat enzyme is searched from PDB databases, by computer program to crystal
Structure is analyzed, select its 70 to 240th amino acid residue between particular amino acid residue as docking setting regions, with
Carry out molecular docking.
2nd, the design of virtual peptide library
The space structure of different aminoacids residue is set up, by computer program, realizes carrying out criticizing to being input into target polypeptides
Amount is generated, to meet calling automatically and processing for 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 with less than four amino acids residues
It is advisable.
3rd, dock judging for result
Calculating the mechanics parameters such as polypeptide and protein bound free energy, hydrogen chain, Van der Waals power respectively carries out Comprehensive Assessment, with
This is judging the selection result, and screening obtains P19, and its peptide 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), which is docked with lipase
Interaction position result is shown in Fig. 1.
Embodiment 2P19 sequence is identified up to the affinity of lipase with labor statement
1st, the lipase of artificial expression and purification is diluted to into 1 μ g/ml (albumen gauge) using PBS (pH 7.4),
Using active ester method, respectively by 1- (3- dimethylamino-propyls) -3- ethyl carbodiimides/N-hydroxy-succinamide (EDC/
NHS, 1:1), lipase injection be provided with the SPR detectors of amino chip, it is ensured that EDC/NHS and lipase respectively with amino
Chip interaction 5min, implements the coupling of lipase and amino chip.After the completion of coupling, the sensor may be used for measurement
Interaction between lipase and P19 sequences.
2nd, 250 μ l PBSs (pH 7.4) are injected in sensor, with Peak Flow Rate (150 μ l/min) runtime buffer
Liquid, reaches signal base line, and the flow velocity of buffer solution is down to 20 μ l/min, to obtain relatively stable baseline.
3rd, artificial synthesized and in the modification of aminoterminal biotinylation P19 dry powder is distinguished using PBS (pH 7.4)
It is dilute into concentration be 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, starts to inject the P19 solution of 250 μ l successively in sensor from low concentration, and injection sample adopts 20 μ l/min every time
Flow velocity and with sensor interaction 5min, finally with PBS (pH 7.4) rinse 5min.It is different dense with what is obtained
The combination of degree P19 solution and fatty enzyme interacting and dissociation curve are foundation, carry out the parent that P19 sequences are combined with lipase
Analyze (see Fig. 2) with power.
As a result show, P19 sequences there is preferable compatibility to be combined with the lipase that labor statement reaches, between the two phase interaction
Equilibrium dissociation constant KD is 3.653 × 10-8M, i.e. 36.53nM.
The ELISA identifications of the lipase of embodiment 3P19 sequence and serum
1st, take mammalian (positive) to be centrifuged, remove haemocyte, taking serum carries 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, used as control.Wherein, envelope antigen is carried out dilute using carbonate (CBS) buffer solution
Release, in adding 96 hole elisa Plates per 50 μ l of hole, under the conditions of being placed in 4 DEG C overnight, with after PBST buffer solutions 5 times again with quality point
The BSA liquid of number 2% is closed.
2nd, artificial synthesized and in the modification of aminoterminal biotinylation P19 dry powder is diluted using PBS (pH 7.4)
Into the concentration of 500ng/ml, it is added in above-mentioned ELISA Plate, after mixing with the volume of 50 μ l of every hole, is placed under the conditions of 37 DEG C, keeps away
Light incubates 30min.
3rd, with PBST buffer solutions 5 times, and dry the liquid in ELISA Plate hole；It is coupled using the dilution of PBST buffer solutions peppery
The Streptavidin (1 of root peroxidase:1000), added in the ELISA Plate for drying with the volume of 50 μ l of every hole, after mixing, put
Under the conditions of 37 DEG C, Incubation in dark 30min.
4th, according to test aequum, TMB nitrite ions are added in above-mentioned ELISA Plate, fully with the volume of 100 μ l of every hole
After mixing 30s, under room temperature condition, develop the color 10min.
5th, the sulfuric acid terminate liquid of 2M is added in above-mentioned ELISA Plate with the volume of 50 μ l of every hole, after fully mixing 30s,
On ELIASA device, light absorption value of each hole at 450nm, result of determination are read.
As a result show, P19 sequences have preferable compatibility and specific binding with the lipase in mammalian blood serum,
(see Fig. 3) is not reacted with other negative serums.
SEQUENCE LISTING
<110>The first affiliated hospital of Zhengzhou University
<120>The peptide for combining is targetted with lipase and matches somebody with somebody 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 (5)
1. the peptide for combining is targetted with lipase and matches somebody with somebody basic sequence, it is characterised in that described peptide is RRRDCW with basic sequence.
2. the peptide for combining that targets with lipase according to claim 1 matches somebody with somebody basic sequence, it is characterised in that include with described
Peptide is core with basic sequence, any to match somebody with somebody corresponding adjustment or the modification carried out by basic sequence to the peptide；Decorative material includes but does not limit
System is in nano material, fluorescent material, enzyme, biotin and specific protein.
3. the peptide for combining that targets with lipase according to claim 1 matches somebody with somebody basic sequence, it is characterised in that match somebody with somebody described peptide
Basic sequence be used for serum in lipase detection, including but be not limited to EUSA (ELISA) detection.
4. a kind of peptide as described in any one of claim 1-3 matches somebody with somebody application of the basic sequence in the quick detection of lipase, wherein
Including but not limited in mammalian blood serum, the lipase that obtains of artificial recombination expression or artificial means of purification.
5. a kind of peptide as described in any one of claim 1-3 with basic sequence in lipase quick detection qualitatively and quantitatively
Using.
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| Application Number | Priority Date | Filing Date | Title |
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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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| 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 true CN106518969A (en) | 2017-03-22 |
| CN106518969B CN106518969B (en) | 2019-03-15 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108690838A (en) * | 2018-05-30 | 2018-10-23 | 华南理工大学 | A kind of marine source monoglyceride lipase and its crystal structure and preparation method |
Citations (4)
| 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 |
| US20140296161A1 (en) * | 2011-09-21 | 2014-10-02 | King Abdullah University Of Science And Technology | Didemnin biosynthetic gene cluster in tistrella mobilis |
-
2016
- 2016-11-15 CN CN201611004667.4A patent/CN106518969B/en active Active
Patent Citations (4)
| 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 |
| US20140296161A1 (en) * | 2011-09-21 | 2014-10-02 | King Abdullah University Of Science And Technology | Didemnin biosynthetic gene cluster in tistrella mobilis |
| 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 |
|---|
| PASMANTIER R 等: "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", 《ENDOCRINOLOGY》 * |
| 宁江海 等: "纤维连接蛋白模拟肽P19的实验研究", 《解放军医学杂志》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108690838A (en) * | 2018-05-30 | 2018-10-23 | 华南理工大学 | A kind of marine source monoglyceride lipase and its crystal structure and preparation method |
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