CN106749519A - Peptide aglucon sequences Design and application that CSFVE2 targeting proteins based on computer simulation are combined - Google Patents
Peptide aglucon sequences Design and application that CSFVE2 targeting proteins based on computer simulation are combined Download PDFInfo
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- CN106749519A CN106749519A CN201611004956.4A CN201611004956A CN106749519A CN 106749519 A CN106749519 A CN 106749519A CN 201611004956 A CN201611004956 A CN 201611004956A CN 106749519 A CN106749519 A CN 106749519A
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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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- 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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Abstract
The present invention relates to be based on peptide aglucon sequences Design and the application that the CSFV E2 targeting proteins of computer simulation are combined, belong to polypeptide design and virus antigen detection field.Wherein, described peptide is FYWRWMMK with basic sequence.The present invention is by means of molecular docking and virtual screening technology, on the basis of flaviviridae E2 albumin crystal structures, by molecular docking technology, search in virtual peptide library with target proteinses binding pattern and the optimal peptide aglucon of affinity, the peptide for finally giving specific binding CSFV E 2 protein matches somebody with somebody basic sequence, its peptide is FYWRWMMK, i.e. Pep3 with basic sequence.The equilibrium dissociation constant KD of the interphase interaction of the CSFVE2 albumen that Pep3 sequences reach with labor statement is 7.54 × 10‑6M, i.e., 7.54 μM, illustrate that affinity is preferable.The Pep3 sequences that the present invention is designed can be combined with artificial infection CSFV, labor statement up to CSFV E2 albumen, except with BVDVE2 albumino reactions it is higher in addition to, do not occur with other virus proteins intercrossing reaction, specificity preferably.
Description
Technical field
The present invention relates to be based on peptide aglucon sequences Design and the application that the CSFV E2 targeting proteins of computer simulation are combined,
Belong to polypeptide design and virus antigen detection field.
Background technology
Virtual screening technology based on molecular docking is the interphase interaction of a kind of emerging research polypeptide and protein
Technological means.This technology is 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 position, conformation, the two of the rotatable key of intramolecule that polypeptide is combined with target proteinses
The amino acid residue side and skeleton of face angle and target proteinses, find peptide molecule optimal on space structure with target proteinses
Conformation, and binding pattern between the two and affinity are predicted, picked out close to native conformation and target egg by score value
A kind of method of the theoretical modeling intermolecular interaction of the optimal polypeptide ligand of white affinity.
CSFV (Classical Swine Fever Virus, CSFV) is to cause swinery to break out hog cholera, China
The referred to as main pathogen of rinderpest.It is that hyperpyrexia, hemorrhagic focus and leukopenia occur with the swinery that falls ill be principal character
A kind of high incidence and the death rate deadly infectious disease.The virus is widely distributed in worldwide, to the cultivation in the whole world
Industry brings strong influence with loss.CSFV is flaviviridae, one of member of pestivirus, with bovine viral diarrhea virus
(Bovine Viral Diarrhea Virus, BVDV) has similitude very high in structure.CSFV belongs to the list for having cyst membrane
Stock positive chain RNA virus, its genome is about 12.3kb, formed a big ORFs (open reading frame,
ORF), a polyprotein being made up of 3989 amino acid is encoded.E2 albumen is a kind of cyst membrane for being typically located at transmembrane region
Glycoprotein, vital effect is played in viruses adsorption and during invading host cell.Meanwhile, it exempts from as CSFV's
Epidemic disease immunodominant proteins, can induce body to produce the neutralizing antibody level of high titre, so that body possesses can resist velogen strain sense
The ability of dye, is to develop the first-selected target protein for CSFV new generation vaccines, therefore E2 albumen also turns into many researchers
Main study subject.
The content of the invention
The present invention by means of molecular docking and virtual screening technology, on the basis of flaviviridae E2 albumin crystal structures
On, by molecular docking technology, with target proteinses binding pattern and the optimal peptide aglucon of affinity in the virtual peptide library of search, its
Peptide is FYWRWMMK, i.e. Pep3 with basic sequence.Artificial synthesized Pep3, surface etc. is carried out using the CSFV E2 albumen of expression and purification
Ion resonance body (Surface Plasmon Resonance, SPR) and ELISA binding tests, as a result show, artificial synthesized
Pep3 has good binding ability with CSFV E2 albumen, thus proves that the peptide aglucon designed by the present invention can be used for
Quick detection qualitatively and quantitatively is carried out to CSFV antigen.
To achieve these goals, the technical solution adopted in the present invention is：
The peptide that CSFV E2 targeting proteins based on computer simulation are combined matches somebody with somebody basic sequence, and described peptide is with basic sequence
FYWRWMMK。
The peptide that the described CSFV E2 targeting proteins based on computer simulation are combined matches somebody with somebody basic sequence, including with described peptide
It is core with basic sequence, any corresponding adjustment or modification carried out with basic sequence to the peptide；Decorative material includes but does not limit
In nano material, fluorescent material, enzyme, biotin and specific protein.
Described peptide is used for the identification of the E2 albumen of CSFV or bovine viral diarrhea virus with basic sequence, wherein
Including but not limited to EUSA (ELISA) detection.
A kind of described peptide is with basic sequence in the quick detection of CSFV or bovine viral diarrhea virus E2 albumen
Application.
A kind of described peptide matches somebody with somebody application of the basic sequence in qualitatively and quantitatively being detected to CSFV antigen.
Beneficial effects of the present invention：
1st, it is of the invention by means of molecular docking and virtual screening technology, in the base of flaviviridae E2 albumin crystal structures
On plinth, by molecular docking technology, with target proteinses binding pattern and the optimal peptide aglucon of affinity in the virtual peptide library of search,
The peptide of specific binding CSFV E 2 protein is finally given with basic sequence, its peptide is FYWRWMMK, i.e. Pep3 with basic sequence.People
Work synthesizes Pep3, and carries out affinity identification with CSFV E 2 protein, the CSFV E2 albumen that Pep3 sequences reach with labor statement it
The equilibrium dissociation constant KD of interphase interaction is 7.54 × 10-6M, i.e., 7.54 μM, illustrate that affinity is preferable.
2nd, due to the limitation of CSFV purification technique, the antibody for CSFV is difficult, present invention design
The Pep3 sequences for obtaining avoid this problem well, realize rapid artificial synthesis and cheap testing cost.
3rd, the Pep3 sequences and artificial infection CSFV, labor statement that the present invention is designed can be tied up to CSFV E2 albumen
Close, except higher outer with BVDV E2 albumino reactions, intercrossing reaction does not occur with other virus proteins, specificity is preferable.
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 CSFV E 2 protein structure function is parsed.
5th, compared with the albumen with artificial expression and purification of the invention is immunized to obtain the process for protein antibodies, have
It is simple to operate, time saving and energy saving, the low advantage of expense；It is marked by screening Pep3 sequences, is capable of achieving to CSFV E2
Albumen carries out qualitative and quantitative quick detection.
Brief description of the drawings
Fig. 1 is that Pep3 sequences show with the result of docking of E2 albumen.
Fig. 2 is the SPR affinity qualification results that Pep3 sequences reach CSFV E2 albumen with labor statement.Wherein, ordinate is represented
Signal value detected by sensor；The time that abscissa representative sample interacts in the sensor.In figure, from top to bottom respectively
The corresponding Pep3 solution concentrations of curve are gradually reduced.
Fig. 3 is the ELISA qualification results that Pep3 sequences reach CSFV E2 albumen with labor statement.
Fig. 4 is the ELISA qualification results of Pep3 sequences and artificial infection CSFV.
Specific embodiment
Specific embodiment of the invention is described in further detail with reference to embodiments.
The molecular docking of embodiment 1 and the screening in virtual peptide storehouse
1st, the preparation of E2 albumen
The crystal structure (4JNT) of flaviviridae E2 albumen is searched from PDB databases, by computer program pair
Crystal structure is analyzed, and its 800 to 900th amino acid residue is selected as docking setting regions, to 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 criticizing input target polypeptides
Amount generation, to meet calling and process automatically for molecular docking computer program.Virtual peptide library is generated with linear form, no
Any side chain and head and the tail amino and carboxyl are modified.The generation of single virtual peptide library is with no more than four amino acids residues
It is advisable.
3rd, judging for result is docked
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 judges the selection result, and screening obtains Pep3, and its polypeptide sequence is phenylalanine-tyrosine-tryptophan-arginine-color
Propylhomoserin-methionine-methionine-lysine (Phe-Tyr-Trp-Arg-Trp-Met-Met-Lys writes a Chinese character in simplified form FYWRWMMK) (SEQ
ID NO.1), the interaction position result that it is docked with E2 is shown in Fig. 1.
The Pep3 sequences of embodiment 2 are identified with labor statement up to the affinity of E2 albumen
1st, the CSFV E2 albumen of artificial expression and purification is diluted to 1 μ g/ml (protein contents using PBS (pH 7.4)
Meter), using active ester method, respectively by 1- (3- dimethylamino-propyls) -3- ethyl carbodiimides/N-hydroxy-succinamide
(EDC/NHS, 1:1), CSFV E2 albumen injection is provided with the SPR detectors of amino chip, it is ensured that EDC/NHS and CSFV E2
Albumen respectively with amino chip interaction 5min, implement the coupling of CSFV E2 albumen and amino chip.After the completion of coupling, should
Sensor may be used for measuring the interaction between CSFV E2 albumen and Pep3 sequences.
2nd, to 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 into 20 μ l/min, to obtain relatively stable baseline.
3rd, artificial synthesized and in the modification of aminoterminal biotinylation Pep3 dry powder is used into PBS (pH 7.4) difference
It is dilute into concentration be 269.04 μM, 134.52 μM, 67.26 μM, 33.63 μM, 8.41 μM, 4.23 μM, 2.11 μM, 1.05 μM of Pep3
Solution, since the Pep3 solution for injecting 250 μ l low concentration into sensor successively, injection sample uses 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 Pep3 solution and CSFV E2 protein-interactings and dissociation curve are foundation, carry out Pep3 sequences and CSFV E2 eggs
The white affinity analysis being combined (see Fig. 2).
Result shows that there is the CSFV E2 albumen that Pep3 sequences reach with labor statement preferable compatibility to be combined, between the two
The equilibrium dissociation constant KD of interaction is 7.54 × 10-6M, i.e., 7.54 μM.
The Pep3 sequences of embodiment 3 are identified with labor statement up to the ELISA of E2 albumen
1st, the CSFV E2 albumen of artificial expression and purification is carried out into ELISA Plate coating with 1 μ g/ml (albumen gauge)；With same
Mode is by the albumen of different virus expression and purification, i.e. bovine viral diarrhea virus E2 albumen (BVDV-E2), encephalitis B virus E2 albumen
(JEV-E2), PCV-II Cap protein (PCV-Cap), Pseudorabies virus gE albumen (PRV-gE), and the ox blood of mass fraction 2%
Pure albumen (BSA), PBS carry out ELISA Plate coating, used as control.Wherein, envelope antigen uses carbonate (CBS)
Buffer solution is diluted, and is added per the μ l of hole 50 in 96 hole elisa Plates, under the conditions of being placed in 4 DEG C overnight, is washed with PBST buffer solutions 5 times
Closed with the BSA liquid of mass fraction 2% again afterwards.
2nd, artificial synthesized and in the modification of aminoterminal biotinylation Pep3 dry powder is diluted using PBS (pH 7.4)
Into the concentration of 500ng/ml, it is added in above-mentioned ELISA Plate with the volume of the μ l of every hole 50, after mixing, is placed under the conditions of 37 DEG C, is kept away
Light incubates 30min.
3rd, washed 5 times with PBST buffer solutions, and dry the liquid in ELISA Plate hole；It is peppery coupling to be diluted using PBST buffer solutions
The Streptavidin (1 of root peroxidase:1000), added with the volume of the μ l of every hole 50 in the ELISA Plate of drying, after mixing, put
Under the conditions of 37 DEG C, Incubation in dark 30min.
4th, according to experiment aequum, TMB nitrite ions are added in above-mentioned ELISA Plate with the volume of the μ l of every hole 100, fully
Mix after 30s, under room temperature condition, develop the color 10min.
5th, in the sulfuric acid terminate liquid of 2M being added into above-mentioned ELISA Plate with the volume of the μ l of every hole 50, after fully mixing 30s,
On ELIASA device, light absorption value of each hole at 450nm, result of determination are read.
Result shows that Pep3 sequences have preferable compatibility and specific binding with labor statement up to CSFV E2 albumen, remove
It is higher with the cross reaction of bovine viral diarrhea virus E2 albumen outer, (see Fig. 3) is not reacted with other virus proteins.
The Pep3 sequences of embodiment 4 are identified with the ELISA of artificial infection CSFV
(1) the PK-15 cell culture fluids for being inoculated with CSFV are carried out into ultrasonication, then with 200TCID50(viral level meter)
Carry out ELISA Plate coating；In the same fashion by different virus nutrient solution, i.e. bovine viral diarrhea virus (BVDV), encephalitis B virus
(JEV), PCV-II (PCV), the nutrient solution of Pseudorabies virus (PRV), and the non-virus inoculation of equal volume PK-15 cells
Nutrient solution carries out ELISA Plate coating, used as control.Wherein, envelope antigen is diluted using CBS buffer solutions, with the μ l of every hole 50
Volume add 96 hole elisa Plates, under the conditions of being placed in 4 DEG C overnight, again with mass fraction 2% after wash 5 times with PBST buffer solutions
BSA fluid-tights close.
(2) will be artificial synthesized and dilute using PBS (pH 7.4) in the Pep3 dry powder of aminoterminal biotinylation modification
The concentration of 500ng/ml is interpreted into, is added in above-mentioned ELISA Plate with the volume of the μ l of every hole 50, after mixing, be placed under the conditions of 37 DEG C,
Incubation in dark 30min.
(3) washed 5 times with PBST buffer solutions, and dry the liquid in ELISA Plate hole；Diluted using PBST buffer solutions and be coupled
The Streptavidin (1 of horseradish peroxidase:1000), added per the μ l of hole 50 in the ELISA Plate for drying, after mixing, be placed in 37 DEG C
Under the conditions of, Incubation in dark 30min.
(4) according to experiment aequum, TMB nitrite ions are added in above-mentioned ELISA Plate with the volume of the μ l of every hole 100, fully
Mix after 30s, under room temperature condition, develop the color 10min.
(5) in the sulfuric acid terminate liquid of 2M being added into above-mentioned ELISA Plate with the volume of the μ l of every hole 50, after fully mixing 30s,
On ELIASA device, light absorption value of each hole at 450nm, result of determination are read.
Result shows that Pep3 sequences with the CSFV cell culture fluids of artificial infection there is preferable compatibility to be tied with specificity
Close, in addition to having cross reaction higher with bovine viral diarrhea virus cell culture fluid, do not occur with other virus-culturing fluids anti-
Should be (see Fig. 4).
SEQUENCE LISTING
<110>Henan Academy of Agricultural Sciences
<120>Peptide aglucon sequences Design and application that CSFV E2 targeting proteins based on computer simulation are combined
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 8
<212> PRT
<213>Artificial sequence
<400> 1
Phe Tyr Trp Arg Trp Met Met Lys
1 5
Claims (5)
1. the peptide that the CSFV E2 targeting proteins based on computer simulation are combined matches somebody with somebody basic sequence, it is characterised in that described peptide aglucon
Sequence is FYWRWMMK.
2. the peptide that the CSFV E2 targeting proteins based on computer simulation according to claim 1 are combined matches somebody with somebody basic sequence, and it is special
Levy and be, including basic sequence is matched somebody with somebody as core with described peptide, any corresponding adjustment or modification carried out with basic sequence to the peptide；
Decorative material includes but is not limited in nano material, fluorescent material, enzyme, biotin and specific protein.
3. the peptide that the CSFV E2 targeting proteins based on computer simulation according to claim 1 are combined matches somebody with somebody basic sequence, and it is special
Levy and be, described peptide is used for the identification of the E2 albumen of CSFV or bovine viral diarrhea virus with basic sequence, wherein
Including but not limited to EUSA (ELISA) detection.
4. a kind of peptide as described in claim any one of 1-3 matches somebody with somebody basic sequence in CSFV or bovine viral diarrhea virus E2
Application in the quick detection of albumen.
5. a kind of peptide as described in claim any one of 1-3 is qualitatively and quantitatively examined with basic sequence to CSFV antigen
Application in survey.
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Citations (5)
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| US5965134A (en) * | 1994-12-20 | 1999-10-12 | Akzo Nobel, N.V. | Immunogenic composition against Classical Swine Fever Virus (CSFV) |
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| CN102532281A (en) * | 2012-01-17 | 2012-07-04 | 江苏省农业科学院 | Classical swine fever virus recombinant E2 protein and IgM (immune globulin M) antibody ELISA (enzyme-linked immunosorbent assay) test kit thereof |
| CN104597256A (en) * | 2015-02-14 | 2015-05-06 | 河南省农业科学院 | Polypeptide sequence combined with bovine viral diarrhea E2 protein and application of polypeptide sequence |
| CN104650185A (en) * | 2015-02-14 | 2015-05-27 | 河南省农业科学院 | Polypeptide sequence bound with swine fever E0 protein and application thereof |
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2016
- 2016-11-15 CN CN201611004956.4A patent/CN106749519B/en active Active
Patent Citations (5)
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|---|---|---|---|---|
| US5965134A (en) * | 1994-12-20 | 1999-10-12 | Akzo Nobel, N.V. | Immunogenic composition against Classical Swine Fever Virus (CSFV) |
| UA98620C2 (en) * | 2006-05-30 | 2012-06-11 | Зе Юнайтед Стейтс Ов Амеріка, Ез Репресентед Бай Зе Секретарі Ов Егрікалчер | Attenuated classic swine fever virus containing modified glycoprotein e2 |
| CN102532281A (en) * | 2012-01-17 | 2012-07-04 | 江苏省农业科学院 | Classical swine fever virus recombinant E2 protein and IgM (immune globulin M) antibody ELISA (enzyme-linked immunosorbent assay) test kit thereof |
| CN104597256A (en) * | 2015-02-14 | 2015-05-06 | 河南省农业科学院 | Polypeptide sequence combined with bovine viral diarrhea E2 protein and application of polypeptide sequence |
| CN104650185A (en) * | 2015-02-14 | 2015-05-27 | 河南省农业科学院 | Polypeptide sequence bound with swine fever E0 protein and application thereof |
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| Title |
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| QIUYING YU等: "Comparison of two docking methods for peptide-protein interactions", 《J SCI FOOD AGRIC》 * |
| 杨苏珍等: "猪O型FMDV主要抗原表位多肽的合成与鉴定", 《西北农业学报》 * |
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