Polypeptide sequence combined with classical swine fever virus E0 protein and application thereof
阅读说明：本技术 一条结合猪瘟病毒e0蛋白的多肽序列及应用 (Polypeptide sequence combined with classical swine fever virus E0 protein and application thereof ) 是由 张改平 王爱萍 王方雨 刘东民 赵建国 陈玉梅 刘红亮 丁培杨 冯景 于 2020-12-25 设计创作，主要内容包括：本发明主要涉及猪瘟病毒E0蛋白亲和多肽及应用,该多肽利用计算机辅助由虚拟分子对接技术设计而成,序列为WRHDFQ(P-3)。本发明以牛病毒性腹泻病毒E0蛋白同源建模获得的猪瘟病毒E0蛋白晶体结构为模板,利用分子对接技术获得多肽序列P-3；合成多肽后,采用等离子共振试验和酶联免疫吸附测定试验,鉴定该序列与猪瘟E0蛋白相互作用的亲和力和特异性,结果表明P-3序列与猪瘟E0蛋白具有较高的亲和力和特异性；本发明可通过对多肽进行标记,从而对猪瘟抗原进行快速定性和定量检测,并可通过多肽对蛋白的结合能力,利用多肽对猪瘟E0蛋白进行富集,为蛋白纯化提供新思路。(The invention mainly relates to a classical swine fever virus E0 protein affinity polypeptide and application thereof, wherein the polypeptide is designed by a virtual molecule docking technology with the assistance of a computer, and the sequence is WRHDFQ (P) 3 ). The invention uses the classical swine fever virus E0 protein crystal structure obtained by bovine viral diarrhea virus E0 protein homologous modeling as a template, and utilizes a molecular docking technology to obtain a polypeptide sequence P 3 (ii) a After the polypeptide is synthesized, the affinity and the specificity of the interaction of the sequence and the hog cholera E0 protein are identified by adopting a plasma resonance test and an enzyme-linked immunosorbent assay test, and the result shows that P 3 The sequence has higher affinity and specificity with the hog cholera E0 protein; the invention can carry out rapid qualitative and quantitative detection on the swine fever antigen by marking the polypeptide, and can enrich the swine fever E0 protein by utilizing the polypeptide through the binding capacity of the polypeptide to the protein, thereby providing a new thought for protein purification.)
1. A polypeptide sequence capable of being combined with swine fever E0 protein, wherein the polypeptide sequence is a linear polypeptide and has the sequence P3：WRHDFQ。
2. The polypeptide sequence of claim 1, comprising the amino acid sequence P3Sequence as core, any pair P3The sequence is adjusted or modified accordingly, and the modified material includes but is not limited to biotin, avidin, magnetic beads, nanomaterials, fluorescent materials, enzymes, and specific proteins.
3. The polypeptide sequence of claim 1, wherein P is3The sequence is used for rapid qualitative or quantitative detection and identification related to any classical swine fever virus, and the sequence comprises but is not limited to enzyme-linked immunosorbent assay detection, plasma resonance assay detection and polyacrylamide gel electrophoresis.
4. Use of a polypeptide sequence according to claim 1 in the preparation of a reagent or kit for rapid detection of classical swine fever virus.
Technical Field
The invention mainly relates to design of classical swine fever virus E0 protein affinity polypeptide and detection of E0 binding capacity of the polypeptide, and belongs to classical swine fever virus detection application.
Background
Classical Swine Fever (CSF) is an acute, febrile and highly contagious disease caused by Classical Swine Fever Virus (CSFV). The classical swine fever virus E0 protein is also called Erns protein and is the first envelope glycoprotein of CSFV, 9 potential glycosylation sites exist in the protein amino acid sequence, the molecular weight of the protein varies with the glycosylation in the protein translation process, and the E0 protein is the only protein which can be secreted into cell supernatant in all structural proteins of classical swine fever virus, and can stimulate the organism to generate neutralizing protective antibody for resisting CSFV infection. The E0 protein has important influence on the proliferation and infection of CSFV and plays an important role in mediating the interaction between virus and host cell. In addition, the E0 protein also has RNase activity and plays an important role in neurotoxicity, tumor resistance and immune regulation.
The unprecedented development of bioinformatics and computer molecular simulation technologies in the 90 s of the 20 th century provided a broader platform for the analysis of protein functions and higher-level structures, enabling more and more researchers to further understand the interaction pattern between polypeptides and proteins from the molecular level. Compared with the traditional polypeptide screening method, the computer virtual screening technology has the advantages of saving production cost, saving time, not needing physical synthesis of the polypeptide and the like, and is widely applied at present. Molecular docking is one of the commonly used computer simulation techniques.
Disclosure of Invention
On the basis of the homologous modeled CSFV E0 protein structure, the invention combines the simulated polypeptide and the target CSFV E0 protein on a computer by a virtual molecule docking technology simulated by the computer to obtain a polypeptide with the best affinity, the sequence of the polypeptide is WRHDFQ and is named as P3. Synthesis of P3And then, identifying the affinity and specificity between the polypeptide and the target protein through a Local Surface Plasmon Resonance (LSPR) test and an enzyme-linked immunosorbent assay (ELISA) and detection. The results show that P3Has stronger affinity and specificity with the classical swine fever virus E0 protein. Therefore, the polypeptide sequence designed by the invention can be used for related researches such as virus detection, protein purification and the like based on the combination with the hog cholera E0 protein.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
one polypeptide sequence capable of being combined with swine fever E0 protein is a linear polypeptide, and the sequence is P3：WRHDFQ。
The polypeptide sequence comprises P3Sequence as core, any pair P3The sequence is adjusted or modified accordingly, and the modified material includes but is not limited to biotin, avidin, magnetic beads, nanomaterials, fluorescent materials, enzymes, and specific proteins.
The P is added3The sequence is used for rapid qualitative or quantitative detection and identification related to any classical swine fever virus, and the sequence comprises but is not limited to enzyme-linked immunosorbent assay detection, plasma resonance assay detection and polyacrylamide gel electrophoresis.
The polypeptide sequence is applied to the preparation of a reagent or a kit for rapidly detecting the hog cholera virus.
The invention has the following positive beneficial effects:
(1) the invention obtains a polypeptide sequence P capable of being combined with classical swine fever virus E0 protein by using a virtual molecule docking technology on the basis of the classical swine fever virus E0 protein (CSFV-E0) which is homologously modeled by bovine viral diarrhea virus E0 protein (BVDV-E0)3The affinity of the polypeptide and classical swine fever virus E0 protein is higher, and the equilibrium dissociation constant K of the interaction between the polypeptide and the classical swine fever virus E0 proteinDIs 2.86X 10-7M, 286 nM.
(2) P of the invention3The polypeptide has the advantages of stable property, simple structure, no immunogenicity, easy synthesis and modification, easy water solubility and the like.
(3) P of the invention3The sequence can be combined with the artificially expressed CSFV E0 protein and artificially inoculated CSFV.
(4) P of the invention3The sequence has good specificity, and has low cross reaction with other proteins except high cross reaction with bovine viral diarrhea virus E0 protein.
(5) Compared with the traditional phage polypeptide screening, the method has the advantages of simple operation, low screening strength, short research and development period, low production cost and the like, can realize virtual molecular docking through computer simulation, realize the targeted combination of specific sites of the swine fever E0 protein, provide better theoretical support for realizing the structural function analysis of the swine fever E0 protein, and provide better theoretical support for P3The sequence is marked, so that the qualitative and quantitative rapid detection of the classical swine fever virus E0 protein can be realized.
Drawings
FIG. 1 is a diagram showing the structure of the homologous modeling activity pocket of CSFV E0 protein.
FIG. 2 is P3The results of the sequence alignment with classical swine fever virus E0 protein are shown.
FIG. 3 is P3And identifying the SPR affinity of the sequence and the artificially expressed and purified classical swine fever virus E0 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, P corresponds to each curve from top to bottom3The concentration of the solution is gradually reduced; the corresponding concentrations of each curve are: 2.5. mu.M, 1.25. mu.M, 625nM, 312.5nM, 156.25nM, 78 nM.
FIG. 4 is P3And (3) an ELISA identification result of the sequence combined with the artificially expressed CSFV E0 protein and other virus proteins.
FIG. 5 is P3And (3) an ELISA identification result of the combination of the sequence and artificially inoculated classical swine fever virus and virus.
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 E0 protein
Homology modeling was performed based on the crystal structure of bovine viral diarrhea virus E0 protein (PDB ID: 4DWC) in the PDB database, and the crystal structure was analyzed by a computer program to select a docking specific region (see FIG. 1) for molecular docking.
2. Design of virtual polypeptide libraries
The optimal amino acid residue for docking is selected firstly by means of computer aided technology and the number of amino acids is increased successively with the amino acid residue as core until the optimal docking result is simulated. The virtual polypeptide is generated in a straight chain form, the side chain and two ends of a polypeptide sequence are not modified, and the polypeptide generated by the virtual polypeptide library preferably has 2-9 amino acid residues.
3. Assessment of docking results
Respectively calculating the van der Waals force, hydrogen chain, hydrophobic acting force, electrostatic acting force and other mechanical parameters of the combination of the polypeptide and the protein by utilizing an MOLCAD module in the molecular docking software to judge a screening result, and screening to obtain P3The polypeptide sequence is WRHDFQ (SEQ ID NO.1), P3The interaction site results of the docking with hog cholera E0 protein are shown in FIG. 2.
Example 2P3Sequence and artificial expression E0 protein affinity identification
1. Using EDC/NHS activated ester method, 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) were dissolved in activation buffer and dispensed into 100. mu.L/tube. Mixing EDC and NHS (EDC/NHS, volume ratio 1:1) in one tube, injecting into SPR detector equipped with carboxyl chip, interacting for 5min, displaying carboxyl on the chip on the surface, and activating carboxyl group on the chip.
2. Diluting the artificially expressed hog cholera E0 protein to 1m by PBSg/mL (protein amount), diluting the protein to 150 μ g/mL by PBS in a 200 μ L system, adding 50 μ L chip activation buffer, injecting the swine fever E0 protein into an SPR detector to ensure the interaction between the protein and a carboxyl chip for 5min, coupling the swine fever E0 protein to the carboxyl chip, and injecting 200 μ L chip matched sealing solution for sealing. After the coupling is finished, the sensing chip is used for measuring the hog cholera E0 protein and P3The interaction between sequences.
3. The buffer was run at a flow rate of 150 μ L/min and 200 μ L of PBS solution (pH 7.4) was injected into the sensor, and after the signal baseline was reached, the running flow rate of the buffer was reduced to 20 μ L/min to obtain a more stable baseline.
4. To be synthesized P3The powder was diluted with ultrapure water to 10mg/mL, and then sequentially diluted with PBS buffer to 2.5. mu.M, 1.25. mu.M, 625nM, 312.5nM, 156.25nM, 78nM P3The solution is prepared by mixing a solvent and a solvent,
injecting 200 mul of polypeptide solution into the sensor from low concentration to high concentration in sequence, wherein the operation flow rate of the injected sample is 20 mul/min, the injected sample interacts with the sensor for 5min, and then washing the chip with buffer solution for 5min to dissociate the polypeptide. After the end of the injection cycle of each concentration of the sample, the remaining polypeptide P bound to the protein was completely dissociated with 250. mu.L of 0.25% SDS solution3. Finally, the P is measured according to the obtained combination and dissociation curves of the polypeptide and the protein with different concentrations3Sequence and E0 protein were subjected to affinity analysis.
The results show that P3The sequence has better affinity combination with the artificially expressed hog cholera E0 protein, and the equilibrium dissociation constant K of the interaction between the sequence and the proteinDThe value was 2.86X 10-7M is 286nM (see FIG. 3).
Example 3P3Sequence and ELISA identification of artificially expressed E0 protein
1. Diluting the artificially expressed hog cholera E0 protein with a coating solution at a concentration of 10 mug/mL (protein amount), adding 50 muL of the protein into each hole for enzyme label plate coating, incubating for 2h at 37 ℃, after washing the plate for four times by PBST, sealing for 2h by a 5% skimmed milk PBST solution, and washing the plate for 4 times by PBST for later use; the purified proteins expressed by different viruses were coated in the same manner, i.e., bovine viral diarrhea virus E0 protein (BVDV-E0), pseudorabies virus gD protein (PRV-gD), porcine reproductive and respiratory syndrome virus M protein (PRRSV-M), circovirus Cap protein (PCV-Cap), and Japanese encephalitis virus E protein (JEV-E), with 2% Bovine Serum Albumin (BSA) as a negative control and PBS as a blank control.
2. Artificially synthesized and biotinylated modified P at amino terminal3Diluting the dry powder to a concentration of 10mg/mL by using ultrapure water, then diluting the polypeptide solution to 500 mu g/mL by using PBS (pH 7.4), adding the diluted polypeptide solution into the coated enzyme label plate in a volume of 50 mu L/hole, uniformly mixing, incubating for 1h at 37 ℃, washing the plate for 4 times by using PBST, and drying the liquid in the hole of the enzyme label plate.
3. Adding a horseradish peroxidase-avidin secondary antibody diluted by 1000 times by PBST buffer solution, adding the secondary antibody into an ELISA plate according to the volume of 50 mu L/hole, uniformly mixing, incubating for 30min at the temperature of 37 ℃, and washing the plate for 4 times by PBST.
4. Adding TMB color development solution into the above enzyme labeling plate at a volume of 100 μ L/hole, mixing well for 30s at room temperature, and developing in dark for 10 min.
5. Adding 2M sulfuric acid solution stop solution into the ELISA plate at the volume of 50 mu L/hole, fully and uniformly mixing for 30s, placing the ELISA plate on an ELISA reader, reading the light absorption value of each hole at 450nm, and analyzing the result.
The results show that P3The sequence has better affinity and specificity with the artificially expressed and purified classical swine fever E0 protein, and has no reaction with other viral proteins except high cross reaction with bovine viral diarrhea virus E0 protein (see figure 4).
Example 4P3Sequence and ELISA identification of artificially inoculated CSFV
1. Using coating buffer solution as antigen coating solution, carrying out ultrasonication on PK-15 cell culture solution infected with CSFV, and then using 200TCID50Coating enzyme label plate (virus content); different virus cultures, porcine circovirus type 2 (PCV2), Porcine Epidemic Diarrhea Virus (PEDV) and uninfected PK-15 cell cultures were coated with microtiter plates in the same manner as controls. The same is added into an enzyme label plate with the volume of 50 mu L/hole and placedIncubate at 37 ℃ for 2h and wash the plate 4 times with PBST.
2. 5% skimmed milk PBST solution was blocked, incubated at 37 ℃ for 2h, and the plates were washed 4 times with PBST.
3. Artificially synthesized and biotinylated modified P at amino terminal3The dry powder is diluted to the concentration of 10mg/mL by ultrapure water, then the polypeptide solution is diluted to 500 mu g/mL by PBS buffer solution (pH 7.4), the polypeptide solution is added into the coated enzyme label plate in the volume of 50 mu L/hole, the mixture is evenly mixed and incubated for 1h at the temperature of 37 ℃, and the plate is washed by PBST for 4 times.
4. Adding a secondary horseradish peroxidase-avidin antibody diluted by 1000 times by PBST, adding the secondary horseradish peroxidase-avidin antibody into an ELISA plate with the volume of 50 mu L/hole, incubating for 30min at the temperature of 37 ℃, and washing the plate for 4 times by PBST.
5. Adding TMB color development liquid into the ELISA plate at a volume of 100 μ L/hole, mixing uniformly for 30s, and developing in dark for 10min at room temperature.
6. Adding 2M sulfuric acid solution stop solution into an ELISA plate at the volume of 50 mu L/hole, fully and uniformly mixing for 30s, placing the ELISA plate on an ELISA reader to read the light absorption value of each hole at 450nm, and analyzing the result.
The results show that P3The sequence has better affinity and specificity with artificially inoculated CSFV cell culture solution, and does not react with other virus culture solution except the BVDV cell culture solution with higher homology (see figure 5).
Sequence listing
<110> Henan Zhongze bioengineering, Inc
<120> one swine fever virus E0 protein-binding polypeptide sequence and application
<130> virtual molecule docking technique
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<170> SIPOSequenceListing 1.0
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<211> 6
<212> PRT
<213> Artificial Synthesis ()
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Trp Arg His Asp Phe Gln
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