CN106749519B - Peptide ligand sequence design and application based on computer simulation and used for CSFV E2 protein targeted combination - Google Patents
Peptide ligand sequence design and application based on computer simulation and used for CSFV E2 protein targeted combination Download PDFInfo
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Abstract
The invention relates to computer simulation-based peptide ligand sequence design and application of CSFV E2 protein targeted combination, belonging to the field of polypeptide design and virus antigen detection. Wherein, the peptide ligand sequence is FYWRWMMK. The invention searches a peptide ligand with the best combination mode and affinity with a target protein in a virtual polypeptide library by a molecular docking and virtual screening technology on the basis of a crystal structure of a flavivirus E2 protein through the molecular docking technology, and finally obtains a peptide ligand sequence specifically combined with classical swine fever virus E2 protein, wherein the peptide ligand sequence is FYWRWMMK, namely Pep 3. The equilibrium dissociation constant KD of the interaction between the Pep3 sequence and the artificially expressed CSFVE2 protein is 7.54 multiplied by 10‑6M, i.e., 7.54. mu.M, indicates better affinity. The Pep3 sequence designed by the invention can be combined with artificially inoculated CSFV and artificially expressed CSFV E2 protein, has higher reactivity with BVDVE2 protein, does not have cross reaction with other virus proteins, and has better specificity.
Description
Technical Field
The invention relates to computer simulation-based peptide ligand sequence design and application of CSFV E2 protein targeted combination, belonging to the field of polypeptide design and virus antigen detection.
Background
A virtual screening technology based on molecular docking is an emerging technical means for researching the interaction between polypeptide and protein. The technology mainly uses computer fast operation to realize the butt joint of polypeptide and corresponding target protein on spatial conformation, butt joints the molecules in a virtual polypeptide database with specific active sites of a target protein crystal structure one by one, searches the optimal conformation of the polypeptide molecules and the target protein on the spatial structure through computer fast operation and continuously adjusts the position and conformation of the combination of the polypeptide and the target protein, the dihedral angle of rotatable bonds in the molecules and the amino acid residue side chain and the skeleton of the target protein, predicts the combination mode and the affinity between the two, and selects a polypeptide ligand which is close to the natural conformation and has the optimal affinity with the target protein through score evaluation.
Classical Swine Fever Virus (CSFV) is a main pathogen causing outbreak of hog cholera in swinery, also called as gastrointestinal plague in China. It is a virulent infectious disease with high morbidity and mortality, which is mainly characterized by hyperpyrexia, hemorrhagic focus and leukopenia of the sick pig herds. The virus is widely distributed all over the world, and brings great influence and loss to the breeding industry all over the world. CSFV is a member of the Flaviviridae family, pestivirus genus, and has a high structural similarity to Bovine Viral Diarrhea Virus (BVDV). CSFV belongs to enveloped single-stranded positive-strand RNA virus, the genome of which is about 12.3kb in length and forms a large Open Reading Frame (ORF) encoding a polyprotein of 3989 amino acids. The E2 protein is a typical envelope glycoprotein located in the transmembrane region and plays a crucial role in viral attachment and invasion into host cells. Meanwhile, the E2 protein is used as an immunodominant protein of CSFV, can induce an organism to generate a high-titer neutralizing antibody level, so that the organism has the capability of resisting the infection of a virulent strain, and is a preferred target protein for developing a novel vaccine against CSFV, so the E2 protein also becomes a main research object of many researchers.
Disclosure of Invention
The invention searches a peptide ligand with the best combination mode and affinity with a target protein in a virtual polypeptide library by means of molecular docking and virtual screening technologies on the basis of a crystal structure of a flavivirus E2 protein through the molecular docking technology, wherein the peptide ligand sequence is FYWRWMMK, namely Pep 3. The Pep3 is artificially synthesized, and the binding test of Surface Plasmon Resonance (SPR) and ELISA is carried out by using the expressed and purified CSFV E2 protein, and the result shows that the artificially synthesized Pep3 and the CSFV E2 protein have good binding capacity, so that the peptide ligand designed by the invention can be used for carrying out quantitative and qualitative rapid detection on the hog cholera virus antigen.
In order to achieve the purpose, the invention adopts the technical scheme that:
based on a peptide ligand sequence targeted and combined by the CSFV E2 protein in computer simulation, the peptide ligand sequence is FYWRWMMK.
The peptide ligand sequence for the CSFV E2 protein targeted combination based on the computer simulation comprises any corresponding adjustment or modification to the peptide ligand sequence by taking the peptide ligand sequence as a core; modifying materials include, but are not limited to, nanomaterials, fluorescent materials, enzymes, biotin, and specific proteins.
The peptide ligand sequence is used for identifying the E2 protein of the hog cholera virus or the bovine viral diarrhea virus, and the detection comprises but is not limited to enzyme-linked immunosorbent assay (ELISA).
The application of the peptide ligand sequence in the rapid detection of classical swine fever virus or bovine viral diarrhea virus E2 protein.
The application of the peptide ligand sequence in quantitative and qualitative detection of the hog cholera virus antigen is provided.
The invention has the beneficial effects that:
1. the invention relates to a method for detecting the flavivirus E2 in the Flaviviridae family by means of molecular docking and virtual screening technologyOn the basis of the protein crystal structure, a peptide ligand with the best binding mode and affinity with a target protein in a virtual polypeptide library is searched through a molecular docking technology, and a peptide ligand sequence specifically binding to classical swine fever virus E2 protein is finally obtained, wherein the peptide ligand sequence is FYWRWMMK, namely Pep 3. Pep3 is artificially synthesized, and the affinity identification is carried out on the Pep3 and the CSFV E2 protein, the equilibrium dissociation constant KD of the interaction between the Pep3 sequence and the CSFV E2 protein which is artificially expressed is 7.54 multiplied by 10-6M, i.e., 7.54. mu.M, indicates better affinity.
2. Due to the limitation of the classical swine fever virus purification technology, the antibody against the classical swine fever virus is difficult to obtain, and the Pep3 sequence designed by the invention well avoids the problem and realizes rapid artificial synthesis and low detection cost.
3. The Pep3 sequence designed by the invention can be combined with artificially inoculated CSFV and artificially expressed CSFV E2 protein, has higher reactivity with BVDV E2 protein, does not have cross reaction with other virus proteins, and has better specificity.
4. Compared with the traditional phage polypeptide library screening, the method has the characteristics of simplicity, rapidness and low cost; by means of computer-aided molecular docking, better theoretical guidance can be provided for the structural function analysis of the classical swine fever virus E2 protein.
5. Compared with the process of obtaining the protein-specific antibody by immunizing the artificially expressed and purified protein, the method has the advantages of simple operation, time and labor saving, low cost and the like; by marking the screened Pep3 sequence, the qualitative and quantitative rapid detection of the classical swine fever virus E2 protein can be realized.
Drawings
FIG. 1 shows the docking results of the sequence of Pep3 and the E2 protein.
FIG. 2 shows the SPR affinity identification result of Pep3 sequence and artificially expressed CSFV E2 protein. Wherein the ordinate represents the signal value detected by the sensor; the abscissa represents the time of interaction of the sample in the sensor. In the figure, the concentration of the solution of Pep3 gradually decreases from the top to the bottom of each curve.
FIG. 3 shows the result of the Pep3 sequence and ELISA identification of the artificially expressed CSFV E2 protein.
FIG. 4 shows the result of ELISA identification of Pep3 sequence and artificially inoculated CSFV.
Detailed Description
The following examples further illustrate the embodiments of the present invention in detail.
Example 1 molecular docking and screening of virtual peptide libraries
1. Preparation of E2 protein
The crystal structure (4JNT) of the E2 protein of the Flaviviridae virus was searched from the PDB database, and the crystal structure was analyzed by a computer program to select the 800 th to 900 th amino acid residues as docking setting regions for molecular docking.
2. Design of virtual polypeptide libraries
The spatial structure of different amino acid residues is established, and the input target polypeptide is generated in batch by means of a computer program so as to meet the requirement of automatic calling and processing of a molecular docking computer program. The virtual polypeptide library is generated in a straight chain form, and any side chain, head-tail amino group and carboxyl group are not modified. Preferably, a single library of virtual polypeptides is generated with no more than four amino acid residues.
3. Assessment of docking results
Respectively calculating the binding free energy of the polypeptide and the protein, hydrogen chain, Van der Waals force and other mechanical parameters to carry out comprehensive evaluation, thereby judging the screening result and screening to obtain Pep3, wherein the polypeptide sequence is phenylalanine-tyrosine-tryptophan-arginine-tryptophan-methionine-lysine (Phe-Tyr-Trp-Arg-Trp-Met-Met-Lys, abbreviated as FYWRWMMK) (SEQ ID NO.1), and the result of the interaction position of the polypeptide and the E2 in butt joint is shown in figure 1.
Example 2 identification of the affinity of Pep3 sequence to the artificially expressed E2 protein
1. Diluting the artificially expressed and purified CSFV E2 protein to 1 mu g/ml (protein amount) by PBS buffer solution (pH 7.4), respectively injecting 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide/N-hydroxysuccinimide (EDC/NHS, 1:1) and CSFV E2 protein into an SPR detector provided with an amino chip by an active ester method, ensuring that the EDC/NHS and CSFV E2 proteins respectively interact with the amino chip for 5min, and coupling the CSFV E2 protein with the amino chip. After the coupling is completed, the sensor can be used for measuring the interaction between the CSFV E2 protein and the Pep3 sequence.
2. The sensor was filled with 250 μ l PBS buffer (pH 7.4), the buffer was run at maximum flow rate (150 μ l/min) to reach the signal baseline, and the flow rate of the buffer was reduced to 20 μ l/min to obtain a more stable baseline.
3. Artificially synthesized and biotinylated modified Pep3 dry powder at the amino terminal was diluted to Pep3 solutions with concentrations of 269.04. mu.M, 134.52. mu.M, 67.26. mu.M, 33.63. mu.M, 8.41. mu.M, 4.23. mu.M, 2.11. mu.M and 1.05. mu.M using PBS buffer (pH 7.4), 250. mu.l of Pep3 solution was injected into the sensor in sequence from low concentration, the flow rate of 20. mu.l/min was used for each injection and the interaction with the sensor was carried out for 5min, and finally the sensor was washed with PBS buffer (pH 7.4) for 5 min. And (3) carrying out affinity analysis on the combination of the Pep3 sequence and the CSFV E2 protein based on the obtained combination and dissociation curve of the interaction between the solutions with different concentrations of Pep3 and the CSFV E2 protein (see figure 2).
The result shows that the Pep3 sequence has better affinity combination with the artificially expressed CSFV E2 protein, and the equilibrium dissociation constant KD of the interaction between the two is 7.54 multiplied by 10-6M, i.e., 7.54. mu.M.
Example 3 sequence of Pep3 and ELISA identification of artificially expressed E2 protein
1. Coating the artificially expressed and purified CSFV E2 protein by an ELISA plate at 1 mu g/ml (protein amount); different viruses were expressed and purified in the same manner as the control, i.e., bovine viral diarrhea virus E2 protein (BVDV-E2), Japanese encephalitis virus E2 protein (JEV-E2), circovirus Cap protein (PCV-Cap), pseudorabies virus gE protein (PRV-gE), and Bovine Serum Albumin (BSA), PBS buffer, with a mass fraction of 2%, were coated with an ELISA plate. Wherein, the coating antigens are diluted by Carbonate (CBS) buffer solution, 50 mul of each hole is added into a 96-hole enzyme label plate, the 96-hole enzyme label plate is placed at 4 ℃ for overnight, and the 96-hole enzyme label plate is washed by PBST buffer solution for 5 times and then is blocked by BSA solution with the mass fraction of 2 percent.
2. Artificially synthesized and amino-terminal biotinylated modified Pep3 dry powder was diluted to a concentration of 500ng/ml using PBS buffer (pH 7.4), added to the above enzyme plate in a volume of 50 μ l per well, mixed well, placed at 37 ℃, and incubated for 30min in the absence of light.
3. Washing with PBST buffer solution for 5 times, and spin-drying the liquid in the holes of the enzyme-labeled plate; streptavidin (1:1000) coupled with horseradish peroxidase was diluted with PBST buffer, added to the spin-dried microplate in a volume of 50. mu.l per well, mixed well, and incubated at 37 ℃ for 30min in the absence of light.
4. According to the required amount of the test, TMB developing solution is added into the enzyme label plate in a volume of 100 mul per hole, and after fully mixing for 30s, the color development is carried out for 10min at room temperature.
5. Adding 2M sulfuric acid stop solution into the enzyme label plate in a volume of 50 mu l per hole, fully and uniformly mixing for 30s, reading the light absorption value of each hole at 450nm on an enzyme label instrument, and judging the result.
The result shows that the Pep3 sequence has better affinity and specificity combination with the artificially expressed CSFV E2 protein, and does not react with other viral proteins except the cross reaction with the bovine viral diarrhea virus E2 protein is higher (see figure 3).
Example 4 ELISA identification of Pep3 sequence and artificially inoculated CSFV
(1) CSFV inoculated PK-15 cell culture fluid is subjected to ultrasonication and then 200TCID50Coating an enzyme label plate (virus content); in the same manner, different virus culture solutions, i.e., culture solutions of Bovine Viral Diarrhea Virus (BVDV), Japanese Encephalitis Virus (JEV), circovirus (PCV), pseudorabies virus (PRV), and the same volume of non-inoculated virus PK-15 cell culture solution were subjected to microplate coating as a control. Wherein, the coating antigens are diluted by CBS buffer solution, added into a 96-hole enzyme label plate with the volume of 50 mul per hole, placed at 4 ℃ for overnight, washed for 5 times by PBST buffer solution and then blocked by BSA solution with the mass fraction of 2%.
(2) Artificially synthesized and amino-terminal biotinylated modified Pep3 dry powder was diluted to a concentration of 500ng/ml using PBS buffer (pH 7.4), added to the above enzyme plate in a volume of 50 μ l per well, mixed well, placed at 37 ℃, and incubated for 30min in the absence of light.
(3) Washing with PBST buffer solution for 5 times, and spin-drying the liquid in the holes of the enzyme-labeled plate; streptavidin (1:1000) coupled with horseradish peroxidase was diluted with PBST buffer, 50. mu.l of each well was added to a spin-dried microplate, mixed well, placed at 37 ℃ and incubated for 30min in the dark.
(4) According to the required amount of the test, TMB developing solution is added into the enzyme label plate in a volume of 100 mul per hole, and after fully mixing for 30s, the color development is carried out for 10min at room temperature.
(5) Adding 2M sulfuric acid stop solution into the enzyme label plate in a volume of 50 mu l per hole, fully and uniformly mixing for 30s, reading the light absorption value of each hole at 450nm on an enzyme label instrument, and judging the result.
The result shows that the sequence of Pep3 has better affinity and specificity combination with the artificially inoculated CSFV cell culture solution, and does not react with other virus culture solutions except that the cross reaction with the bovine viral diarrhea virus cell culture solution is higher (see figure 4).
SEQUENCE LISTING
<110> agricultural science institute of Henan province
<120> peptide ligand sequence design and application based on computer simulation and in CSFV E2 protein targeted combination
<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 (2)
1. A peptide ligand sequence for the targeted binding of CSFV E2 protein based on computer simulation, which is characterized in that the peptide ligand sequence is FYWRWMMK.
2. Use of a peptide ligand sequence as defined in claim 1 in the preparation of a reagent or kit for rapid detection of classical swine fever virus E2 protein or bovine viral diarrhea virus E2 protein.
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- 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 |
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