Infectious bursal disease virus VP3 phosphorylation-deficient virus, construction method, and application thereof

Abstract

This application relates to an infectious bursal disease virus (IBDV) VP3 phosphorylation-deficient virus, its construction method, and its applications. Specifically, this application provides an IBDV VP3 phosphorylation-deficient virus, including a mutant plasmid and a rescue vector carrying the mutant plasmid. The mutant plasmid has the following mutations: serine 130 to alanine and serine 163 to alanine in the VP3 protein. The IBDV VP3 phosphorylation-deficient virus provided in this application can serve as a powerful tool for studying the phosphorylation mechanism of IBDV VP3.

Description

Technical Field

[0001] The present application relates to the fields of molecular biology and biomedicine, and specifically to an infectious bursal disease virus VP3 phosphorylation-deficient virus, a construction method, and applications thereof. Background Art

[0002] Infectious bursal disease (IBD) is a highly acute, contagious avian disease caused by infection with the infectious bursal disease virus (IBDV). VP3 is the primary inner capsid protein of IBDV. Studies have shown that phosphorylation of VP3 plays a crucial regulatory role in its function. For example, studies have shown that IBDV infection induces upregulation of Cdc7 expression, which subsequently leads to phosphorylation of Serine 13 (S13) on the VP3 protein. This modification is crucial for efficient viral replication in host cells. Studying VP3 phosphorylation also provides potential antiviral targets, potentially inhibiting IBDV replication by interfering with the VP3 phosphorylation pathway. For example, developing inhibitors targeting specific VP3 phosphorylation sites could block viral replication and reduce pathogenicity, thereby providing new avenues for drug development against IBDV infection. Currently, research into the impact of VP3 phosphorylation on IBDV replication is hampered by a lack of clear VP3 phosphorylation sites and effective research tools. Summary of the Invention

[0003] The purpose of this application is to provide an infectious bursal disease virus VP3 phosphorylation-deficient virus, a construction method and its application, aiming to solve the problems of unclear VP3 phosphorylation sites and lack of effective research tools in the prior art.

[0004] To achieve the above application objectives, the technical solutions adopted in this application are as follows:

[0005] In the first aspect, the infectious bursal disease virus VP3 phosphorylation-deficient virus provided by the present application includes a mutant plasmid and a rescue vector carrying the mutant plasmid. The mutation points of the mutant plasmid are: the serine at position 130 of the VP3 protein mutates to alanine and / or the serine at position 163 mutates to alanine.

[0006] The infectious bursal disease virus VP3 phosphorylation-deficient virus provided in the first aspect of this application can be used as a powerful tool for studying the phosphorylation mechanism of infectious bursal disease virus VP3, because the rescue vector carries a mutant plasmid in which the serine at position 130 of the VP3 protein is mutated to alanine or / and the serine at position 163 is mutated to alanine, and provides a basis for studying infectious bursal disease virus vaccines.

[0007] In a second aspect, the present application provides an infectious bursal disease virus VP3 phosphorylation-deficient virus, in which the serine at position 130 of the VP3 protein is mutated to alanine and / or the serine at position 163 is mutated to alanine.

[0008] The second aspect of this application provides an infectious bursal disease virus VP3 phosphorylation-deficient virus. Due to the mutation of serine at position 130 of the VP3 protein to alanine, and the mutation of serine at position 163 of the VP3 protein to alanine, the VP3 protein phosphorylation site in the infectious bursal disease virus plasmid is deleted, which provides a strategy for subsequent studies on the effect of VP3 protein phosphorylation modification on the replication of infectious bursal disease virus.

[0009] In a third aspect, the present application provides a method for constructing an infectious bursal disease virus VP3 phosphorylation-deficient virus, comprising the following steps:

[0010] Step S20, mutating the serine at position 130 of the VP3 protein to alanine and / or mutating the serine at position 163 to alanine to obtain a mutant plasmid;

[0011] Step S30: transfect the mutant plasmid into the rescue vector using a first transfection reagent to obtain an infectious bursal disease virus VP3 phosphorylation-deficient virus.

[0012] The third aspect of the present application provides a method for constructing an infectious bursal disease virus VP3 phosphorylation-deficient virus, which mutates the serine at position 130 of the VP3 protein to alanine, and the serine at position 163 of the VP3 protein to alanine, thereby constructing an infectious bursal disease virus with a missing VP3 protein phosphorylation site.

[0013] In a fourth aspect, the present application provides an application of the infectious bursal disease virus VP3 phosphorylation-deficient virus or the infectious bursal disease virus VP3 phosphorylation-deficient virus prepared by the construction method described above in the preparation of vaccines, drugs and diagnostic reagents.

[0014] It is precisely because the infectious bursal disease virus VP3 phosphorylation-deficient virus in this application has undergone dual phosphorylation site mutations at S130 and S163, which leads to enhanced IBDV virus replication ability, enhanced dsRNA binding ability, and enhanced auxiliary polymerase activity. Therefore, it can be used as an IBDV candidate vaccine strain. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following briefly introduces the drawings required for use in the embodiments or descriptions of the prior art. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0016] Figure 1 The Phospho net3.1 online tool predicts the VP3 phosphorylation modification site map and the specific distribution map of high-scoring sites in VP3;

[0017] Figure 2A This is the immunoprecipitation staining diagram of VP3 phosphorylation modification sites;

[0018] Figure 2B This is the mass spectrometry prediction map of the VP3 phosphorylation modification site of S130A;

[0019] Figure 2C The mass spectrometry prediction map of the VP3 phosphorylation modification site of S163A;

[0020] Figure 3 This is a Western blot validation image of the predicted phosphorylation site mutation;

[0021] Figure 4 This is a Western blot verification diagram of VP3 phosphorylation modification sites;

[0022] Figure 5A This is a diagram showing the enhanced replication ability of viruses with mutations in the VP3 phosphorylation site;

[0023] Figure 5B This is a statistical graph of TCID50 determination results;

[0024] Figure 6 This is a diagram showing the enhanced interaction between VP3 phosphorylation site mutation and dsRNA;

[0025] Figure 7 A diagram showing that VP3 phosphorylation site mutations enhance VP1 polymerase activity. DETAILED DESCRIPTION

[0026] In order to make the technical problems, technical solutions and beneficial effects to be solved by this application more clearly understood, the present application is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain this application and are not intended to limit this application.

[0027] In a first aspect, an embodiment of the present application provides an infectious bursal disease virus VP3 phosphorylation-deficient virus, comprising a mutant plasmid, wherein the mutation points of the mutant plasmid are: serine at position 130 of the VP3 protein mutates to alanine and / or serine at position 163 mutates to alanine.

[0028] The infectious bursal disease virus VP3 phosphorylation-deficient virus provided in the embodiments of the present application can be used as a powerful tool for studying the phosphorylation mechanism of infectious bursal disease virus VP3, because the rescue vector carries a mutant plasmid in which the serine at position 130 of the VP3 protein is mutated to alanine or / and the serine at position 163 is mutated to alanine, and provides a basis for studying infectious bursal disease virus vaccines.

[0029] In some embodiments, the mutant plasmid includes a first mutant plasmid or a second mutant plasmid. Specifically, the first mutant plasmid includes Myc-VP3-S130A, Myc-VP3-S163A and Myc-VP3-SSAA; the sequence of Myc-VP3-S130A is shown in SEQ ID NO: 1, the sequence of Myc-VP3-S163A is shown in SEQ ID NO: 2, and the sequence of Myc-VP3-SSAA is shown in SEQ ID NO: 3. The second mutant plasmid includes T7-A VP3S130A 、T7-A VP3S163A and T7-A VP3SSAA ;T7-A VP3S130A The sequence of T7-A is shown in SEQ ID NO: 4. VP3S163A The sequence of T7-A is shown in SEQ ID NO: 5. VP3SSAA The sequence is shown in SEQ ID NO:6.

[0030] In a second aspect of an embodiment of the present application, an infectious bursal disease virus VP3 phosphorylation-deficient virus is provided, wherein the serine at position 130 of the VP3 protein is mutated to alanine and / or the serine at position 163 is mutated to alanine.

[0031] The infectious bursal disease virus VP3 phosphorylation-deficient virus provided in the examples of the present application has a deletion of the VP3 protein phosphorylation site in the infectious bursal disease virus plasmid due to the mutation of serine at position 130 of the VP3 protein to alanine and the mutation of serine at position 163 of the VP3 protein to alanine, thereby providing a strategy for subsequent studies on the effect of VP3 protein phosphorylation modification on the replication of infectious bursal disease virus.

[0032] A third aspect of the present invention provides a method for constructing an infectious bursal disease virus VP3 phosphorylation-deficient virus, comprising the following steps:

[0033] Step S20, mutating the serine at position 130 of the VP3 protein to alanine and / or mutating the serine at position 163 to alanine to obtain a mutant plasmid;

[0034] Step S30: transfect the mutant plasmid into the rescue vector using a first transfection reagent to obtain an infectious bursal disease virus VP3 phosphorylation-deficient virus.

[0035] In some embodiments, to determine the VP3 phosphorylation modification site, step S10 is further included before step S20:

[0036] Step S11, using the Netphospho3.1 online tool to predict the potential phosphorylation sites of the VP3 protein of the IBDV JX strain for the first time, and obtaining the first prediction result;

[0037] Step S12, constructing a recombinant plasmid using the VP3 gene sequence of the A segment of the IBDV JX strain as a template;

[0038] Step S13: Use immunoprecipitation and mass spectrometry methods to perform a second prediction of potential phosphorylation sites of the VP3 protein on the recombinant plasmid to obtain a second prediction result.

[0039] In the examples of this application, the results of identification using immunoprecipitation and mass spectrometry can more accurately and conveniently find the phosphorylation modification sites of the VP3 protein.

[0040] In some embodiments, in step S11, sites with a prediction score greater than 0.9 in the first prediction result are selected as potential phosphorylation sites of the first VP3 protein. In some specific embodiments, the amino acid sequence of VP3 is obtained based on the VP3 gene sequence of IBDV JX strain (GenBank: OR743230 and OR743231), and potential phosphorylation sites of the VP3 protein are predicted using the Netphospho3.1 online tool. Sites with prediction scores greater than 0.9 include: T92 (0.983), S105 (0.997), S130 (0.995), Y150 (0.973), S163 (0.992), and S253 (0.997).

[0041] In some embodiments, in step S12, the method for constructing a recombinant plasmid comprises the following steps:

[0042] Using the VP3 gene sequence of the A segment of IBDV JX strain as a template, PCR amplification was performed using the VP3-F and VP3-R primer pairs to obtain a B amplification product. The sequence of VP3-F is shown in SEQ ID NO:7, and the sequence of VP3-R is shown in SEQ ID NO:8. The gene sequences of the primer pairs are as follows:

[0043] VP3-F: 5'-ATGGCATCAGAGTTCAAAGAGACCC-3';

[0044] VP3-R: 5' -TTACTCAAGGTCCTCATCAGAGACG -3'.

[0045] Extract the DNA solution from the amplified product B, and then double-digest the DNA solution and the pCMV-Myc-N empty vector using an enzyme digestion system to obtain digested DNA fragments and digested vectors, respectively. Ligate the digested DNA fragments and digested vectors using T4 DNA ligase to obtain ligation products.

[0046] The ligation product was transformed into DH5α competent cells and cultured, and the plasmid was extracted to obtain the recombinant plasmid.

[0047] In some embodiments, in step S13, the method of performing co-immunoprecipitation and mass spectrometry comprises the following steps:

[0048] The recombinant plasmid and the Flag-MARK1 plasmid were co-transfected into 293T cells using the Express transfection reagent. The proteins were harvested and co-immunoprecipitated. The precipitated samples were subjected to SDS-PAGE electrophoresis and Coomassie Brilliant Blue staining. The VP3-specific band was excised and characterized by mass spectrometry. In some embodiments, in step S13, the VP3 phosphorylation modification sites can be substantially confirmed to be S130A and S163A.

[0049] In some embodiments, to further confirm the reliability of the VP3 phosphorylation modification sites S130A and S163A, step S10 further includes the step of verifying the second prediction result:

[0050] Step S14: designing third mutation primers based on the first prediction result and constructing a third mutation plasmid using the Myc-VP3 plasmid as a template, wherein the sequence of the Myc-VP3 plasmid is shown in SEQ ID NO: 33;

[0051] Step S15, transfecting the third mutant plasmid into HEK-293T cells using a second transfection agent;

[0052] Step S16: Based on the first prediction result, HEK-293T cells carrying the third mutant plasmid are subjected to Western blot verification to verify the second prediction result.

[0053] In the examples of this application, the phosphorylation modification sites were verified by gene site-directed mutagenesis and Western blot methods, which have the advantages of simple operation, low technical threshold and short cycle.

[0054] In some embodiments, in step S14, the third mutagenesis primer includes a third forward primer and a third reverse primer, the sequence of the third forward primer is at least one of SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, and SEQ ID NO: 31, and the sequence of the third reverse primer is at least one of SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, and SEQ ID NO: 32.

[0055] In some embodiments, in step S15, the third mutant plasmid is transfected into HEK-293T cells using a second transfection agent, and the second transfection agent is Exfect transfection reagent.

[0056] In some embodiments, step S16 , HEK-293T cells carrying the third mutant plasmid are subjected to Western blot verification based on the first prediction result to verify the second prediction result to verify whether the second prediction result is accurate.

[0057] The present invention provides a method for constructing a recombinant plasmid for infectious bursal disease virus. The method utilizes site-directed mutagenesis and Western blot analysis to verify phosphorylation sites, offering advantages such as ease of use, low technical barriers, and a short production cycle. Subsequently, the recombinant strain is depleted of VP3 phosphorylation by mutating the phosphorylation sites of the VP3 fragment on the rescue plasmid, demonstrating its ease of use. In a specific embodiment, step S16 further confirms that the VP3 phosphorylation sites are S130A and S163A.

[0058] In some embodiments, a first mutant plasmid is obtained based on the second prediction result. Specifically, the first mutant plasmid is directly screened from the third mutant plasmid based on the VP3 phosphorylation modification sites being S130A and S163A, and the first mutant plasmid includes Myc-VP3-S130A, Myc-VP3-S163A, and Myc-VP3-SSAA; the sequence of Myc-VP3-S130A is shown in SEQ ID NO: 1, the sequence of Myc-VP3-S163A is shown in SEQ ID NO: 2, and the sequence of Myc-VP3-SSAA is shown in SEQ ID NO: 3.

[0059] In another embodiment, based on the VP3 phosphorylation modification sites of S130A and S163A, the Myc-VP3 plasmid is used as a template, wherein the sequence of the Myc-VP3 plasmid is shown in SEQ ID NO: 33, and a first mutant primer is used for a first reverse amplification to obtain a first mutant plasmid, wherein the first mutant primer includes a first forward primer and a first reverse primer, the sequence of the first forward primer is at least one of SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, and SEQ ID NO: 15, and the sequence of the first reverse primer is at least one of SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 16.

[0060] Based on the above-mentioned VP3 phosphorylation modification sites being S130A and S163A, a second mutant plasmid was obtained, specifically comprising the following steps:

[0061] Step S40, mutating the serine at position 130 of the VP3 protein encoded in the T7-A plasmid to alanine and / or mutating the serine at position 163 to alanine to obtain a second mutant plasmid;

[0062] Step S50: transfect the second mutant plasmid and the T7-B plasmid into BSRT7 cells using a third transfection reagent to obtain an infectious bursal disease virus VP3 phosphorylation-deficient virus.

[0063] The IBDV rescue system provided in the embodiments of the present application is an IBDV rescue system based on the polymerase II type promoter - the T7 promoter, including two plasmids, T7-A and T7-B (T7-A is an expression plasmid in which IBDV Segment A is cloned downstream of the T7 promoter; T7-B is an expression plasmid in which IBDV Segment B is cloned downstream of the T7 promoter). In combination with BSRT7 cells (a BHK-21 stable cell line that can stably express T7 polymerase), infectious IBDV can be rescued, and an infectious bursal disease virus VP3 phosphorylation-deficient virus can be obtained. The IBDV two-plasmid rescue system based on the T7 promoter has the advantages of high rescue efficiency and high success rate.

[0064] In some embodiments, in step S40, a T7-A plasmid is used as a template, and the sequence of the T7-A plasmid is shown in SEQ ID NO:34. A second reverse amplification is performed using a second mutation primer to obtain a second mutant plasmid, wherein the second mutation primer includes a second forward primer and a second reverse primer, the sequence of the second forward primer is at least one of SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, and SEQ ID NO:15, and the sequence of the second reverse primer is at least one of SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, and SEQ ID NO:16;

[0065] In some embodiments, in step S50, the second mutant plasmid and the T7-B plasmid are transfected into BSRT7 cells using a third transfection reagent to obtain an infectious bursal disease virus VP3 phosphorylation-deficient virus.

[0066] The third aspect of the embodiments of the present application provides an infectious bursal disease virus VP3 phosphorylation-deficient virus or an infectious bursal disease virus VP3 phosphorylation-deficient virus prepared by the construction method, and its use in the preparation of vaccines, drugs and diagnostic reagents.

[0067] It is precisely because the infectious bursal disease virus VP3 phosphorylation-deficient virus in this application has undergone dual phosphorylation site mutations at S130 and S163, which leads to enhanced IBDV virus replication ability, enhanced dsRNA binding ability, and enhanced auxiliary polymerase activity. Therefore, it can be used as an IBDV candidate vaccine strain and can be used as a powerful research tool in the development of drugs and diagnostic reagents.

[0068] The following describes the details in conjunction with specific embodiments.

[0069] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples, unless otherwise specified, are conventional methods. The test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores. The quantitative tests in the following examples were all repeated three times, and the results were averaged.

[0070] <Cell Culture and Viruses>

[0071] HEK293T cells were purchased from CCTCC, DMEM medium used for culture was purchased from Gibco, and fetal bovine serum was purchased from ExCellBio; DF-1 cells and MEM medium and fetal bovine serum used for culture were purchased from Xiamen Yimo Biotechnology Co., Ltd.

[0072] All cell lines were cultured at 37°C in an atmosphere of 5% CO2. The IBDV JX strain used (GenBank: OR743230 and OR743231) was isolated and maintained in our laboratory. WT IBDV was a live virus rescued by transfecting T7-A and T7-B plasmids into BSRT7 cells. SSAA IBDV was a T7- AVP3SSAA Live virus rescued from transfection of the plasmid and T7-B plasmid into BSRT7 cells. WT-VP3 protein sample is the protein sample obtained from Myc-VP3. The tissue median infectious dose (TCID50) was measured in chicken embryo fibroblast (DF-1) cells.

[0073] <Antibodies>

[0074] Mouse monoclonal antibodies against Flag and Myc, as well as β-actin antibodies, were purchased from Proteintech. Anti-Flag agarose was purchased from Abmart. Polyclonal antibodies against VP1, VP2, and VP3 were produced in-house. Goat anti-rabbit secondary antibodies were purchased from KPL. NP-40 lysis buffer (containing 50 mM Tris [pH 7.4], 150 mM NaCl, and 1% NP-40) was purchased from Beyotime Biotechnology Co., Ltd. Protein loading buffer was purchased from Beijing Solebao Technology Co., Ltd.

[0075] <Plasmid construction>

[0076] The synthesis of primers (the first mutant primer, the second mutant primer, and the third mutant primer) was completed by Tsingke Biotechnology Co., Ltd. in Beijing. 2×Taq Master Mix, agarose for nucleic acids, nucleic acid maker, and DH5α competent cells were purchased from Tsingke Biotechnology Co., Ltd. in Beijing. The DNA gel extraction kit, PCR product purification kit, agarose, sodium chloride, yeast powder, and tryptone were purchased from Solarbio Science & Technology Co., Ltd. in Beijing. All restriction endonucleases (DpnⅠ enzyme, RNaseA) were purchased from Takara. T4 ligase was purchased from Beyotime Biotechnology Co., Ltd. The plasmid extraction kit was purchased from Tiangen. Exfect transfection reagent was purchased from Novoprotein. Protein pre-stained maker and PBS buffer were purchased from Sevier. The empty vector plasmids pCMV-Flag-N and pCMV-Myc-N were purchased from Wuhan Miaoling Biotechnology Co., Ltd.

[0077] <SDS-PAGE and Western blot>

[0078] Sodium dodecyl sulfate (SDS), electrophoresis buffer Tris 8.8, and electrophoresis buffer Tris 6.8 were purchased from Solarbio Science & Technology Co., Ltd. in Beijing. 40% acrylamide was purchased from Sangon Biotech (Shanghai) Co., Ltd. Coomassie brilliant blue staining solution, ammonium persulfate alternative (APS), and TEMED were purchased from Beyotime Biotechnology Co., Ltd. The nitrocellulose membrane (NC membrane) for immunoblotting (Western blot) was purchased from Merck; filter paper was purchased from Bio-Rad. PBST is a combination of phosphate buffer (PBS) and Tween-20.

[0079] In the results of immunoblotting (Western blot), for the light chain with a molecular weight below 25 kDa and the heavy chain with a molecular weight of 40 - 5525 kDa, p-VP3 indicates the phosphorylation of VP3. Flag-MARK1 or MARK1 is the gene / protein name.

[0080] <Other reagents>

[0081] Buffer P1 is a buffer solution, and its main components include 50 mM Tris-HCl (pH 8.0), 10 mM EDTA, and 100 μg / ml RNase A. Buffer P2 is a lysis buffer for purifying plasmid DNA. The main components are 0.2 mol / L NaOH and 1% SDS. Buffer P3 is a buffer solution. Wash buffer. Elution buffer (EB, Elution Buffer). Phanta Max Master Mix is a high-fidelity DNA polymerase purchased from Novoprotein Scientific Inc., Nanjing. Double-distilled water (ddH2O).

[0082] Example 1

[0083] Rescue of Recombinant Virus with Deletion of Phosphorylation Modification of IBDV VP3

[0084] <Prediction of Potential Phosphorylation Sites of VP3 Protein>

[0085] Based on the VP3 gene sequence of IBDV JX strain (GenBank: OR743230 and OR743231), the amino acid sequence of VP3 was obtained. The online tool Netphospho3.1 was used to predict the potential phosphorylation sites of VP3 protein. As Figure 1 shown, the sites with a prediction score greater than 0.9 are: T92(0.983), S105(0.997), S130(0.995), Y150(0.973), S163(0.992), S253(0.997).

[0086] <Construction of Recombinant Plasmid of VP3 Protein>

[0087] 1) Primer design: According to the VP3 gene sequence of the A segment of IBDV JX strain, primers for constructing eukaryotic expression vectors were designed. The primer sequences are as follows:

[0088] VP3-F: 5' -ATGGCATCAGAGTTCAAAGAGACCC -3' (nucleotide sequence shown in SEQ IDNO:7);

[0089] VP3-R: 5' -TTACTCAAGGTCCTCATCAGAGACG -3' (nucleotide sequence shown in SEQ IDNO:8).

[0090] 2) PCR Amplification: Using the VP3 gene sequence of the A segment of the IBDV JX strain as a template, PCR was performed using the VP3-F and VP3-R primer pairs to obtain the B amplification product (VP3 segment). The template DNA was selected from the VP3 gene sequence of the A segment of the IBDV JX strain and was stored in this laboratory. The PCR amplification reaction system is as follows:

[0091] 2×Taq Master Mix 25μL

[0092] Template DNA 1 μg

[0093] VP3-F 2μL

[0094] VP3-R 2μL

[0095] The system was balanced to 50 μL with ddH2O.

[0096] The reaction conditions were as follows: pre-denaturation at 95°C for 5 min, followed by 35 cycles of denaturation at 95°C for 30 s, annealing at 56°C for 30 s, and extension at 72°C for 1 min, with a total extension at 72°C for 10 min.

[0097] After the PCR amplification reaction is completed, gel electrophoresis is performed using 1% agarose gel. A VP3 band consistent with the size of the target fragment is observed. The gel is then cut and the gel is recovered using a Gel Extraction Kit. The specific steps are as follows:

[0098] (A) After electrophoresis on a 1% agarose gel, cut the gel under UV illumination and place the excised gel pieces in a 2.0 mL centrifuge tube. (B) Weigh the gel pieces and add 200 μL of Buffer A per 0.1 g of gel. Place the gel in a 55°C water bath for 20 min until the gel pieces are completely melted. (C) Add an equal volume of Buffer B to the melted gel solution and mix thoroughly. (D) Transfer the mixed solution from step (C) to a spin binding column equipped with a cannula. Centrifuge at 10,000 rpm for 30 s at room temperature. Discard the substrate waste and centrifuge 2-3 times until all the mixed solution is bound to the spin column. (E) Add 500 μL of Wash Buffer A to the centrifuge tube containing the binding column and centrifuge at 10,000 rpm for 30 seconds at room temperature. Discard the waste substrate solution. (F) Add 500 μL of Wash Buffer B to the centrifuge tube containing the binding column and centrifuge at 10,000 rpm for 30 seconds at room temperature. Discard the waste substrate solution. (G) Centrifuge the centrifuge column from step (F) at 10,000 rpm for 2 minutes and leave it uncovered for 2 minutes. (H) Place the centrifuge column in a sterile 1.5 mL centrifuge tube, add 50 μL of elution buffer, and centrifuge at 10,000 rpm for 1 minute at room temperature to obtain the purified DNA solution.

[0099] 3) Enzyme digestion of target fragment and vector: Use the enzyme digestion system to perform double enzyme digestion on the DNA solution and the pCMV-Flag-N and pCMV-Myc-N empty vector plasmids to obtain the digested DNA fragment and the digested vector, respectively.

[0100] The target fragment DNA solution and the pCMV-Flag / Myc-N empty vector solution were double-digested separately. The enzyme digestion system is as follows:

[0101] 10*Buffer 10 μL

[0102] EcoR 3 μL

[0103] Xho 3 μL

[0104] DNA / vector 5μg

[0105] ddH2O to 100 μL

[0106] Reaction conditions: 37°C water bath for 2 hours. Then clean and recover. The specific steps are as follows:

[0107] (A) After incubation, centrifuge the digested product briefly to remove any liquid from the tube wall. Add 400 μL of binding buffer and gently pipette to mix. (B) Transfer the mixed solution from the previous step to a spin column fitted with a cannula. Centrifuge at 12,000 rpm for 30 seconds at room temperature and discard the waste liquid. (C) Add 500 μL of Wash Buffer to the centrifuge tube containing the binding column. Centrifuge at 12,000 rpm for 30 seconds at room temperature and discard the waste liquid. (D) Repeat step C. (E) Centrifuge the tube at 12,000 rpm for 2 minutes and then leave it uncapped for 2 minutes. (F) Place the spin column in a sterile 1.5 mL centrifuge tube. Add 50 μL of heated elution buffer to the column. Let it sit for 2 minutes and then centrifuge at 12,000 rpm for 2 minutes to obtain the digested DNA fragments and vector.

[0108] 4) Ligation: Use T4 DNA ligase to ligate the digested DNA fragment and the digested vector to obtain the ligation product. The reaction system (10 μL) is as follows:

[0109] 10×T4 DNA ligase buffer 1 μL

[0110] T4 DNA ligase 0.5 μL

[0111] Recycled vector after enzyme digestion (50 ng / μL) 1 μL

[0112] Enzyme-digested target fragment (100 ng / μL) 2 μL

[0113] ddH2O 5.5 μL

[0114] Reaction conditions: 4°C overnight ligation;

[0115] 5) Transformation: Remove DH5α competent cells from a -80°C freezer and thaw on ice. Transform the ligation product into DH5α competent cells and mix thoroughly. Place the mixture on ice for 10 minutes. Heat shock the mixture in a 42°C water bath for 90 seconds. Remove the mixture and quickly place it on ice for 5 minutes. Add 1 mL of antibiotic-free LB medium to the mixture and incubate on a shaker at 37°C for 45 minutes. Centrifuge the mixture at low speed (3000 rpm) for 10 minutes to remove the competent cells from the tube. Discard the upper layer of LB medium, reserving approximately 100 μL. Resuspend the competent cells at the bottom of the tube with a pipette and add them to a pre-prepared plate of solid LB medium supplemented with ampicillin. Let it rest for 10 minutes. Invert the plate and incubate overnight at 37°C. Pick a single colony, expand it with shake culture, and send it to Universal Biotech for first-generation sequencing using a CMV-F primer in the forward direction.

[0116] 6) Plasmid extraction: Sequencing alignment is performed on the sequencing results returned by the sequencing company. The correct clones are those that match the VP3 template sequence. The bacterial solution of the correct clones is used for plasmid extraction to obtain the recombinant plasmid. The specific steps are as follows: (A) Pipette 100 μL of the original bacteria into a 100 mL sterilized conical flask, and add 20 mL of ampicillin-containing liquid LB medium to the conical flask for overnight culture. (B) Take 10 mL of the bacterial solution and transfer it to a 15 mL centrifuge tube. Centrifuge at 1000 r / min at room temperature for 5 min to centrifuge all the bacterial solution to the bottom of the tube, and discard the supernatant waste liquid. (C) Add 500 μL of Buffer P1 containing RNaseA to the centrifuge tube, resuspend the bacteria on both sides充分重悬左右菌体, and let it stand for 5 min;

[0117] (D) Add 500 μL of Buffer P2 to the centrifuge tube, gently invert the centrifuge tube 5 - 8 times until the liquid becomes clear and relatively transparent, and let it stand at room temperature for 5 min. (E) Add 700 μL of Buffer P3 to the centrifuge tube, gently invert the centrifuge tube 5 - 8 times until white flocculent substances appear, let it stand at room temperature for 5 min, and then centrifuge at 12000 r / min at high speed for 10 min at room temperature until all the flocculent substances precipitate to the bottom of the tube. (F) Take the supernatant in (E) and transfer it to the adsorption column installed with the bottom tube. Centrifuge at 12000 r / min at high speed for 30 s at room temperature, and repeat 2 - 3 times until all the supernatant passes through the adsorption column. (G) Add 500 μL of Wash buffer to the adsorption column, centrifuge at 12000 r / min at high speed for 30 s at room temperature, discard the waste liquid, and repeat the washing once. (H) Centrifuge the adsorption column at 12000 r / min at high speed for 1 min at room temperature, open the lid and let it stand at room temperature for 1 min. (I) Put the adsorption column into a new 1.5 mL sterile centrifuge tube, add 100 μL of EB (Elution Buffer), and let it stand at room temperature for 1 min. (J) Centrifuge the adsorption column at 12000 r / min at high speed for 1 min at room temperature to elute the recombinant plasmids (Flag-VP3 and Myc-VP3) on the adsorption column. After measuring the concentration, store it at -20°C for standby.

[0118] <Mass spectrometry identification of potential phosphorylation sites of VP3 protein>

[0119] It should be noted that there is an unclear expression "充分重悬左右菌体" in the original text, and it is translated as "重悬左右菌体" as accurately as possible. If there is a more accurate expression, it can be adjusted accordingly.1) Transfection: Use Exfect transfection reagent to transfect the recombinant plasmid into HEK-293T cells. Specific steps are as follows: (A) HEK-293T cells are cultured in a six-well plate and grown to a confluence of approximately 80% before transfection. (B) To set up experimental and control groups, prepare two sterile EP tubes and add 100 μL of serum-free DMEM medium to each. Add 10 μL of Exfect transfection reagent to each tube (A) and gently mix. Add 2 μg of Flag-VP3 plasmid to tube (B) of the control group and 2 μg each of Flag-VP3 plasmid and Flag-MARK1 plasmid to tube (B) of the experimental group, mix gently, and let stand for 5 minutes. (C) Add 10 μL of Exfect transfection reagent from one tube (A) to tube (B) of the experimental group, mix thoroughly, and add 10 μL of Exfect transfection reagent from the other tube (A) to tube (B) of the control group, mix thoroughly, and let stand for 15 minutes. (D) The mixed solution in tube B of the experimental group and the mixed solution in tube B of the control group were added dropwise into a 6-well plate and gently shaken to mix. After 6 hours, the culture medium was changed to DMEM containing 2% serum and cultured for another 48 hours to obtain transfected cells.

[0120] 2) Co-immunoprecipitation (Co-IP): Transfected cells and culture medium from the experimental group and cells and culture medium from the control group were collected into separate EP tubes and centrifuged at 3000 rpm for 5 min. The supernatant was discarded and 200 μL of NP-40 lysis buffer was added. Mix thoroughly by pipetting, place on ice for 10 min, and centrifuge at 12000 rpm for 10 min. The supernatant was removed and 5 μL of anti-Flag agarose was added. Mix gently and incubate on a shaker at 4°C for 3 h. Centrifuge at 1000 rpm for 3 min at 4°C. The supernatant was discarded and the agarose was washed with NP-40 lysis buffer. This wash was repeated three times. Appropriate amounts of NP-40 lysis buffer and protein loading buffer were added and the samples were boiled at 95°C for 10 min to obtain the target proteins for the experimental and control groups, respectively.

[0121] 3) SDS-PAGE and Coomassie Brilliant Blue Staining:

[0122] (A) Prepare a polyacrylamide gel containing 8% separating gel and 5% stacking gel. The formula is shown in Table 1 below:

[0123] Table 1

[0124] Name of each component 5% stacking gel 8% separation gel <![CDATA[H2O]]> 1.21 mL 2.6 mL 40% acrylamide 250 μL 1 mL Tris8.8 0 μL 1.25 Tris6.8 0.5 mL 0 μL 10% SDS 20 μL 50 μL 10%APS 20 μL 50 μL TEMED 2 μL 2 μL

[0125] Prepare the lower-layer gel according to the above formula. First, pour the lower-layer separating gel well, add isopropanol to the upper layer for wire pressing, and let it stand at room temperature. After the lower-layer gel solidifies, discard the upper-layer isopropanol and wash it three times with double-distilled water. Prepare the upper-layer stacking gel and pour it. Quickly insert the sample comb, and let it stand at room temperature for 1 h. After the upper-layer gel solidifies, gently take out the comb and perform the subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDA-PAGE) experiment; (B) Loading: Add 2 μL of protein prestained marker to the first lane; add the control group (Flag-VP3) sample to the second lane; add the experimental group (Flag-VP3+Flag-MARK1) sample to the third lane. (C) Electrophoresis: Use a Bio-Rad mini vertical electrophoresis system for the electrophoresis equipment. First, perform electrophoresis at a low voltage (80 V) for about 30 min. After the bromophenol blue enters the separating gel, increase the voltage to 120 V and continue electrophoresis until the bromophenol blue indicator reaches the bottom of the glass plate to stop electrophoresis. (D) Staining: After electrophoresis, disassemble the gel plate, cut off the excess part of the gel, and put the main part into the Coomassie Brilliant Blue staining solution and stain it on a shaker for 2 h. (E) Decolorization: Discard the staining solution, add an appropriate amount of protein decolorizing solution, decolorize it on a shaker, and change the decolorizing solution every 30 min until the blue color is completely removed. As Figure 2A shown, compared with the control group, the p-VP3 band (between 35 kDa and 40 kDa) in the experimental group is more obvious.

[0126] 4) Mass spectrometry identification: Cut the VP3 together with the p-VP3 (VP3 phosphorylation) band and send it to Nanjing Kangxuhe Biotechnology Co., Ltd. for mass spectrometry identification. The entire set of systems used for identification is the timsTOF Pro2 mass spectrometer (Bruker Daltonics) of the UltiMate 3000 system (Thermo Fisher Scientific, MA, USA) in tandem. The mass spectrometer operates in the DDAPASEF mode, and the mass spectrometry parameters are set as follows: Do 10 PASEF scans per cycle, with an accumulation time of 100 ms. The scanning range is from 100 to 1700 m / z, and the ion mobility (1 / K0) range is set to 0.6 - 1.6. The scanning charge number is set to 0 - 5, the target value is set to 10,000, and the dynamic exclusion time is 0.4 min. When m / z < 700, the isolation window width is set to 2 Th, and when m / z > 700, the isolation window width is set to 3 Th. The results Figure 2B and Figure 2C shown, the mass spectrometry results show that the S130A site and the S163A site have obvious mass spectrometry effects, and the information on the VP3 phosphorylation modification sites can be basically determined as the S130A site and the S163A site.

[0127] <Construction of VP3 Phosphorylation Site Mutant Plasmids>

[0128] 1) Primer design:

[0129] To further verify the accuracy of mass spectrometry for measuring VP3 phosphorylation modification sites, the Netphospho3.1 online tool was used to design primers for the third mutation site based on the first prediction results of T92 (0.983), S105 (0.997), S130 (0.995), Y150 (0.973), S163 (0.992), and S253 (0.997). The primer sequences are shown in Table 2 below:

[0130] Table 2

[0131] Serial number Primer name Primer sequence SEQ ID NO:9 S130A-F <![CDATA[GCACCGAGGGCCA GCC CCGGCCAGCTAAAG]]> SEQ ID NO: 10 S130A-R <![CDATA[CTTTAGCTGGCCGGG GGC TGGCCCTCGGTGC]]> SEQ ID NO:11 S130E-F <![CDATA[GCACCGAGGGCCA GAG CCGGCCAGCTAAAG]]> SEQ ID NO:12 S130E-R <![CDATA[CTTTAGCTGGCCGGG CTC TGGCCCTCGGTGC]]> SEQ ID NO:13 S163A-F <![CDATA[AGAAGAGCCGGTTGGCA GCA GAAGAACAAATC]]> SEQ ID NO:14 S163A-R <![CDATA[GATTTGTTCTT CTG CTGCCAACCGGCTCTTCT]]> SEQ ID NO:15 S163E-F <![CDATA[AGAAGAGCCGGTTGGCA GAG GAAGAACAAATC]]> SEQ ID NO:16 S163E-R <![CDATA[GATTTGTTCTT CCT CTGCCAACCGGCTCTTCT]]> SEQ ID NO: 17 T92A-F <![CDATA[GGAGGCTCGGGGCCCC GCA CCAGAGGAAGC]]> SEQ ID NO: 18 T92A-R <![CDATA[GCTTCCTCTGG TGC GGGGCCCCGAGCCTCC]]> SEQ ID NO: 19 T92E-F <![CDATA[GGAGGCTCGGGGCCCC GAA CCAGAGGAAGC]]> SEQ ID NO:20 T92E-R <![CDATA[GCTTCCTCTGG TTC GGGGCCCCGAGCCTCC]]> SEQ ID NO:21 S105A-F <![CDATA[GACACACGGATC GCA AAGAAGATGGAGACC]]> SEQ ID NO:22 S105A-R <![CDATA[GGTCTCCATCTTCTT TGC GATCCGTGTGTC]]> SEQ ID NO:23 S105E-F <![CDATA[GACACACGGATC GAA AAGAAGATGGAGACC]]> SEQ ID NO:24 S105E-R <![CDATA[GGTCTCCATCTTCTT TTC GATCCGTGTGTC]]> SEQ ID NO:25 Y150A-F <![CDATA[GACCCAAACGAGGAC GCT CTAGACTACGTG]]> SEQ ID NO:26 Y150A-R <![CDATA[CACGTAGTCTAG AGC GTCCTCGTTTGGGTC]]> SEQ ID NO:27 Y150E-F <![CDATA[GACCCAAACGAGGAC GAA CTAGACTACGTG]]> SEQ ID NO:28 Y150E-R <![CDATA[CACGTAGTCTAG TTC GTCCTCGTTTGGGTC]]> SEQ ID NO:29 S253A-F <![CDATA[GATCAGGACCGTC GCT GATGAGGACCTTGAG]]> SEQ ID NO:30 S253A-R <![CDATA[CTCAAGGTCCTCATC AGC GACGGTCCTGATC]]> SEQ ID NO:31 S253E-F <![CDATA[GATCAGGACCGTC GAA GATGAGGACCTTGAG]]> SEQ ID NO:32 S253E-R <![CDATA[CTCAAGGTCCTCATC TTC GACGGTCCTGATC]]>

[0132] 2) Reverse PCR: Using the Myc-VP3 plasmid as a template, the sequence of the Myc-VP3 plasmid is shown in SEQ ID NO:33: ATGGCATCAGAGTTCAAAGAGACCCCCGAACTCGAGAGTGCCGTCAGAGCAATGGAAGCAGCAGCCAACGTGGACCCACTATTCCAATCTGCACTCAGTGTGTTCATGTGGCTGGAAGAGAATGGGATTGTGACTGACATGGCCAACTTCGCACTCAGCGACCCGAACGCCCATCGGATGCGAAATTTTCTTGCAAACGCACCACAAGCAGGCAGCAAGTCGCAAAGGGCCAAGTACGGGACAGCAGGCTACGGAGTGGAGGCTCGGGGCCCCACACCAGAGGAAGCACAGAGGGAAAAAGACACACGGATCTCAAAGAAGATGGAGACCATGGGCATCTACTTTGCAACACCAGAATGGGTAGCACTCAATGGGCACCGAGGGCCAAGCCCCGGCCAGCTAAAGTACTGGCAGAACACACGAGAAATACCGGACCCAAACGAGGACTATCTAGACTACGTGCATGCAGAGAAGAGCCGGTTGGCATCAGAAGAACAAATCCTAAGGGCAGCTACGTCGATCTACGGGGCTCCAGGACAGGCAGAGCCACCCCAAGCTTTCATAGACGAAGTTGCCAAAGTCTATGAAATCAACCATGGACGTGGCCCAAACCAAGAACAGATGAAAGATCTGCTCTTGACTGCGATGGAGATGAAGCATCGCAATCCCAGGCGGGCTCTACCAAAGCCCAAGCCAAAACCCAATGCTCCAACACAGAGACCCCCTGGTCGGCTGGGCCGCTGGATCAGGACCGTCTCTGATGAGGACCTTGAGTAA。Reverse PCR is performed using a third mutation primer to obtain an A amplification product (mutant plasmid), wherein the third mutation primer includes a third forward primer and a third reverse primer, the sequence of the third forward primer is at least one of SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, and SEQ ID NO:31, and the sequence of the third reverse primer is at least one of SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, and SEQ ID NO:32.

[0133] The amplification system is: 2×Phanta Max Master Mix 10 μL,

[0134] Template DNA 100 ng

[0135] 1 μL each of forward and reverse primers,

[0136] The system was balanced to 20 μL with ddH2O.

[0137] The reaction conditions were as follows: pre-denaturation at 95°C for 5 min, followed by 20 cycles of denaturation at 95°C for 30 s, annealing at 56°C for 30 s, and extension at 72°C for 2 min, with a total extension at 72°C for 10 min. After the reaction, 1% agarose gel electrophoresis was used to verify successful amplification.

[0138] 3) Enzyme digestion: The PCR product obtained in the previous step is digested with Dpn I enzyme to remove the template plasmid and obtain the Dpn I enzyme digestion product. The reaction system is:

[0139] 10*Buffer 1 μL

[0140] DpnⅠ 0.5 μL

[0141] PCR product 8.5 μL

[0142] Reaction conditions: 37°C water bath for 1 h;

[0143] 4) Transformation: Remove DH5α competent cells from a -80°C freezer and thaw on ice. Transform the digestion product from the previous step into the DH5α competent cells and mix thoroughly. Place the mixture on ice for 10 minutes. Heat shock the mixture in a 42°C water bath for 90 seconds. Remove the mixture and quickly place it on ice for 5 minutes. Add 1 mL of antibiotic-free LB medium to the mixture and incubate on a shaker at 37°C for 45 minutes. Centrifuge the mixture at low speed (3000 rpm) for 10 minutes to remove all DH5α competent cells from the tube. Discard the upper layer of LB medium, reserving approximately 100 μL. Resuspend the DH5α competent cells at the bottom of the tube with a pipette and add them to a pre-prepared plate of solid LB medium supplemented with ampicillin. Let it rest for 10 minutes. Invert the plate and incubate overnight at 37°C. Pick a single colony, expand it with shake culture, and send it to Universal Biotech for first-generation sequencing using a CMV-F primer in the forward direction.

[0144] 5) Plasmid Extraction: Sequence alignment is performed on the sequencing results returned by the sequencing company. Single clones with successful mutations at the mutation site are considered correct. Select the correct clones for plasmid extraction. The specific steps are as follows: (A) Transfer 100 μL of the original bacteria to a 100 mL sterile Erlenmeyer flask. Add 20 mL of LB medium containing ampicillin and culture overnight. (B) Transfer 10 mL of the bacterial suspension to a 15 mL centrifuge tube and centrifuge at 1000 rpm for 5 minutes at room temperature. Pour the entire suspension to the bottom of the tube and discard the supernatant. (C) Add 500 μL of Buffer P1 containing RNase A to the tube to thoroughly resuspend the cells. Let stand for 5 minutes. (D) Add 500 μL of Buffer P2 to the tube and gently invert the tube 5-8 times until the liquid becomes clear and transparent. Let stand for 5 minutes at room temperature. (E) Add 700 μL of Buffer P3 to the centrifuge tube. Gently invert the tube 5-8 times until white flocs appear. Let stand at room temperature for 5 minutes. Centrifuge at 12,000 rpm for 10 minutes at room temperature until all the flocs have settled to the bottom of the tube. (F) Transfer the supernatant from (E) to an adsorption column with a bottom tube. Centrifuge at 12,000 rpm for 30 seconds at room temperature. Repeat 2-3 times until all the supernatant has passed through the column. (G) Add 500 μL of Wash Buffer to the adsorption column. Centrifuge at 12,000 rpm for 30 seconds at room temperature. Discard the waste solution and repeat the wash cycle once. (H) Centrifuge the adsorption column at 12,000 rpm for 1 minute at room temperature. Uncap and let stand at room temperature for 1 minute. (I) Place the adsorption column in a new 1.5 mL sterile centrifuge tube, add 100 μL of EB (Elution Buffer), and let stand at room temperature for 1 minute. (J) The adsorption column was centrifuged at 12,000 r / min for 1 min at room temperature to elute the plasmid on the adsorption column. After measuring the concentration, the plasmids were stored at -20°C for later use. The mutant plasmids Myc-VP3-T92A, Myc-VP3-T92E, Myc-VP3-S105A, Myc-VP3-S105E, Myc-VP3-T30A, Myc-VP3-130E, Myc-VP3-Y150A, Myc-VP3-Y150E, Myc-VP3-S253A, Myc-VP3-S253E, Myc-VP3-S163A, Myc-VP3-S163E and Myc-VP3-SSAA were obtained.

[0145] The sequence of Myc-VP3-S130A is shown in SEQ ID NO: 1:

[0146] ATGGCATCAGAGTTCAAAGAGACCCCCGAACTCGAGAGTGCCGTCAGAGCAATGGAAGCAGCAGCCAACGTGGACCCACTATTCCAATCTGCACTCAGTGTGTTCATGTGGCTGGAAGAGAATGGGATTGTGACTGACATGGCCAACTTCGCACTCAGCGACCCGAACGCCCATCGGATGCGAAATTTTCTTGCAAACGCACCACAAGCAGGCAGCAAGTCGCAAAGGGCCAAGTACGGGACAGCAGGCTACGGAGTGGAGGCTCGGGGCCCCACACCAGAGGAAGCACAGAGGGAAAAAGACACACGGATCTCAAAGAAGATGGAGACCATGGGCATCTACTTTGCAACACCAGAATGGGTAGCACTCAATGGGCACCGAGGGCCA GCC CCCGGCCAGCTAAAGTACTGGCAGAACACACGAGAAATACCGGACCCAAACGAGGACTATCTAGACTACGTGCATGCAGAGAAGAGCCGGTTGGCATCAGAAGAACAAATCCTAAGGGCAGCTACGTCGATCTACGGGGCTCCAGGACAGGCAGAGCCACCCCAAGCTTTCATAGACGAAGTTGCCAAAGTCTATGAAATCAACCATGGACGTGGCCCAAACCAAGAACAGATGAAAGATCTGCTCTTGACTGCGATGGAGATGAAGCATCGCAATCCCAGGCGGGCTCTACCAAAGCCCAAGCCAAAACCCAATGCTCCAACACAGAGACCCCCTGGTCGGCTGGGCCGCTGGATCAGGACCGTCTCTGATGAGGACCTTGAGTAA。

[0147] The sequence of Myc-VP3-S163A is shown in SEQ ID NO:2:

[0148] ATGGCATCAGAGTTCAAAGAGACCCCCGAACTCGAGAGTGCCGTCAGAGCAATGGAAGCAGCAGCCAACGTGGACCCACTATTCCAATCTGCACTCAGTGTGTTCATGTGGCTGGAAGAGAATGGGATTGTGACTGACATGGCCAACTTCGCACTCAGCGACCCGAACGCCCATCGGATGCGAAATTTTCTTGCAAACGCACCACAAGCAGGCAGCAAGTCGCAAAGGGCCAAGTACGGGACAGCAGGCTACGGAGTGGAGGCTCGGGGCCCCACACCAGAGGAAGCACAGAGGGAAAAAGACACACGGATCTCAAAGAAGATGGAGACCATGGGCATCTACTTTGCAACACCAGAATGGGTAGCACTCAATGGGCACCGAGGGCCAAGCCCCGGCCAGCTAAAGTACTGGCAGAACACACGAGAAATACCGGACCCAAACGAGGACTATCTAGACTACGTGCATGCAGAGAAGAGCCGGTTGGC AGC AGAAGAACAAATCCTAAGGGCAGCTACGTCGATCTACGGGGCTCCAGGACAGGCAGAGCCACCCCAAGCTTTCATAGACGAAGTTGCCAAAGTCTATGAAATCAACCATGGACGTGGCCCAAACCAAGAACAGATGAAAGATCTGCTCTTGACTGCGATGGAGATGAAGCATCGCAATCCCAGGCGGGCTCTACCAAAGCCCAAGCCAAAACCCAATGCTCCAACACAGAGACCCCCTGGTCGGCTGGGCCGCTGGATCAGGACCGTCTCTGATGAGGACCTTGAGTAA。

[0149] The sequence of Myc-VP3-SSAA is shown in SEQ ID NO:3;

[0150] ATGGCATCAGAGTTCAAAGAGACCCCCGAACTCGAGAGTGCCGTCAGAGCAATGGAAGCAGCAGCCAACGTGGACCCACTATTCCAATCTGCACTCAGTGTGTTCATGTGGCTGGAAGAGAATGGGATTGTGACTGACATGGCCAACTTCGCACTCAGCGACCCGAACGCCCATCGGATGCGAAATTTTCTTGCAAACGCACCACAAGCAGGCAGCAAGTCGCAAAGGGCCAAGTACGGGACAGCAGGCTACGGAGTGGAGGCTCGGGGCCCCACACCAGAGGAAGCACAGAGGGAAAAAGACACACGGATCTCAAAGAAGATGGAGACCATGGGCATCTACTTTGCAACACCAGAATGGGTAGCACTCAATGGGCACCGAGGGCCA GCC CCCGGCCAGCTAAAGTACTGGCAGAACACACGAGAAATACCGGACCCAAACGAGGACTATCTAGACTACGTGCATGCAGAGAAGAGCCGGTTGGC AGC AGAAGAACAAATCCTAAGGGCAGCTACGTCGATCTACGGGGCTCCAGGACAGGCAGAGCCACCCCAAGCTTTCATAGACGAAGTTGCCAAAGTCTATGAAATCAACCATGGACGTGGCCCAAACCAAGAACAGATGAAAGATCTGCTCTTGACTGCGATGGAGATGAAGCATCGCAATCCCAGGCGGGCTCTACCAAAGCCCAAGCCAAAACCCAATGCTCCAACACAGAGACCCCCTGGTCGGCTGGGCCGCTGGATCAGGACCGTCTCTGATGAGGACCTTGAGTAA。

[0151] <Verification of VP3 phosphorylation modification sites by immunoblotting (Western blot)>

[0152] 1) Transfection: Use Exfect transfection reagent to transfect the mutant plasmid (obtained in the plasmid extraction above) and the Myc-VP3 plasmid into HEK-293T cells. Specific steps are as follows: (A) HEK-293T cells are cultured in a 12-well plate and grown to a confluence of approximately 80% for transfection. (B) Prepare two sterile EP tubes for each group. Add 50 μL of serum-free DMEM medium to each tube. Add 4 μL of Exfect transfection reagent to tube A and gently mix. Add 2 μg of the corresponding mutant plasmid to tube B and gently mix. Incubate for 5 minutes. (C) Add the mixture from tube A to tube B and gently mix. Incubate for 15 minutes. (D) Add the mixed solution dropwise to each well of the 12-well plate and gently shake to mix. After 6 hours, switch to DMEM medium supplemented with 2% serum and continue incubation for 24 hours.

[0153] 2) Protein sample preparation: Collect the culture medium and cells into an EP tube and centrifuge at low speed to collect the cells. Discard the supernatant and add 100 μL of NP-40 lysis buffer. Lyse at 4°C for 20 min. Centrifuge at high speed for 10 min. Discard the precipitate and retain the supernatant. Add 25 μL of 5× protein loading buffer, mix thoroughly, and boil in a 100°C metal bath for 10 min. Store the prepared protein sample in a 4°C refrigerator for subsequent Western blot analysis to obtain the mutant VP3 protein. Similarly, prepare a control WT VP3 protein sample.

[0154] 3) Western blot detection:

[0155] (A) Sample loading: Add 2 μL of protein pre-stained Marker to the first well, add the WT-VP3 protein sample to the second well, and then sequentially add protein samples of different mutants (T92A, T92E, S105A, S105E, S130A, S130E, S163A, S163E, Y150A, Y150E, S253A, S253E) VP3. (B) Electrophoresis: The electrophoresis equipment uses a Bio-Rad mini vertical electrophoresis system. First, perform electrophoresis at a low voltage (80 V) for about 30 min. After the bromophenol blue enters the separating gel, increase the voltage to 120 V and continue electrophoresis until the bromophenol blue indicator reaches the bottom of the glass plate to stop electrophoresis. (C) Blotting: After electrophoresis, cut off the excess part of the gel. Soak the remaining gel in the transfer buffer. Prepare two thick transfer filter papers and a nitrocellulose membrane (NC membrane) that is relatively close to the size of the membrane. Soak the NC membrane and transfer filter papers in the transfer buffer for a few minutes to fully moisten. Stack the filter paper - NC membrane - gel - filter paper in sequence from bottom to top, place them neatly on the semi-dry blotting apparatus, gently press to remove the internal air, cover the lid of the blotting apparatus, limit the voltage to 25 V, keep the current constant at 0.2 mA, and transfer for 35 min. After the transfer is completed, take out the NC membrane. (D) Blocking: Soak the transferred NC membrane in PBS buffer containing 5% skim milk, let it stand at room temperature for 20 min, and then wash it 3 - 5 times with PBS buffer, 5 - 10 min each time. (E) Key step in primary antibody incubation: Detect the target protein by binding the mouse monoclonal antibody against Myc protein and β-actin antibody in the primary antibody reagent to the specific antigen: Dilute the mouse monoclonal antibody against Myc protein with the primary antibody diluent at a ratio of 1:3000 according to the instructions, and incubate the transferred NC membrane in it, shaking and incubating overnight at 4°C on a shaker. (F) Secondary antibody incubation: The next day, wash the NC membrane 3 - 5 times with PBST by shaking, 5 min each time. Dilute the goat anti-rabbit secondary antibody at a ratio of 1:10000, incubate it at room temperature by shaking for 1 h, and then wash it 3 - 5 times with PBST by shaking, 5 min each time. (G) Protein imaging and preservation: As Figure 3 shown, it can be seen that the phosphorylation levels of S130A and S163A are significantly decreased or absent compared with WT, while mutations at other sites have little effect, that is, other sites can be excluded and two sites, S130 and S163, are locked as the VP3 phosphorylation sites; as Figure 4 shown, it can be seen that the phosphorylation levels of S130A, S163A, and the SSAA double mutant samples are significantly decreased or absent compared with WT.

[0156] <Rescue and Identification of Recombinant Virus with VP3 Phosphorylation Deletion>

[0157] Construct a rescue plasmid of IBDV with phosphorylation site deletion:

[0158] 1) Construct T7-AVP3S130A and T7-A VP3S163A and T7-A VP3SSAA plasmids (i.e., corresponding mutations were made at the corresponding sites encoding the VP3 part in the T7-A plasmid), and the plasmids were extracted. Among them, the sequence of T7-A VP3S130A is shown in SEQ ID NO:4:

[0159] TGCATCAGAGTTCAAAGAGACCCCCGAACTCGAGAGTGCCGTCAGAGCAATGGAAGCAGCAGCCAACGTGGACCCACTATTCCAATCTGCACTCAGTGTGTTCATGTGGCTGGAAGAGAATGGGATTGTGACTGACATGGCCAACTTCGCACTCAGCGACCCGAACGCCCATCGGATGCGAAATTTTCTTGCAAACGCACCACAAGCAGGCAGCAAGTCGCAAAGGGCCAAGTACGGGACAGCAGGCTACGGAGTGGAGGCTCGGGGCCCCACACCAGAGGAAGCACAGAGGGAAAAAGACACACGGATCTCAAAGAAGATGGAGACCATGGGCATCTACTTTGCAACACCAGAATGGGTAGCACTCAATGGGCACCGAGGGCCA GCC CCCGGCCAGCTAAAGTACTGGCAGAACACACGAGAAATACCGGACCCAAACGAGGACTATCTAGACTACGTGCATGCAGAGAAGAGCCGGTTGGCA TCA GAAGAACAAATCCTAAGGGCAGCTACGTCGATCTACGGGGCTCCAGGACAGGCAGAGCCACCCCAAGCTTTCATAGACGAAGTTGCCAAAGTCTATGAAATCAACCATGGACGTGGCCCAAACCAAGAACAGATGAAAGATCTGCTCTTGACTGCGATGGAGATGAAGCATCGCAATCCCAGGCGGGCTCTACCAAAGCCCAAGCCAAAACCCAATGCTCCAACACAGAGACCCCCTGGTCGGCTGGGCCGCTGGATCAGGACCGTCTCTGATGAGGACCTTGAGTGA。

[0160] T7-A VP3S163AThe sequence is as shown in SEQ ID NO:5: TGCATCAGAGTTCAAAGAGACCCCCGAACTCGAGAGTGCCGTCAGAGCAATGGAAGCAGCAGCCAACGTGGACCCACTATTCCAATCTGCACTCAGTGTGTTCATGTGGCTGGAAGAGAATGGGATTGTGACTGACATGGCCAACTTCGCACTCAGCGACCCGAACGCCCATCGGATGCGAAATTTTCTTGCAAACGCACCACAAGCAGGCAGCAAGTCGCAAAGGGCCAAGTACGGGACAGCAGGCTACGGAGTGGAGGCTCGGGGCCCCACACCAGAGGAAGCACAGAGGGAAAAAGACACACGGATCTCAAAGAAGATGGAGACCATGGGCATCTACTTTGCAACACCAGAATGGGTAGCACTCAATGGGCACCGAGGGCCA AGC CCCGGCCAGCTAAAGTACTGGCAGAACACACGAGAAATACCGGACCCAAACGAGGACTATCTAGACTACGTGCATGCAGAGAAGAGCCGGTTGGCA GCA GAAGAACAAATCCTAAGGGCAGCTACGTCGATCTACGGGGCTCCAGGACAGGCAGAGCCACCCCAAGCTTTCATAGACGAAGTTGCCAAAGTCTATGAAATCAACCATGGACGTGGCCCAAACCAAGAACAGATGAAAGATCTGCTCTTGACTGCGATGGAGATGAAGCATCGCAATCCCAGGCGGGCTCTACCAAAGCCCAAGCCAAAACCCAATGCTCCAACACAGAGACCCCCTGGTCGGCTGGGCCGCTGGATCAGGACCGTCTCTGATGAGGACCTTGAGTGA。

[0161] T7-A VP3SSAAThe sequence is as shown in SEQ ID NO:6: TGCATCAGAGTTCAAAGAGACCCCCGAACTCGAGAGTGCCGTCAGAGCAATGGAAGCAGCAGCCAACGTGGACCCACTATTCCAATCTGCACTCAGTGTGTTCATGTGGCTGGAAGAGAATGGGATTGTGACTGACATGGCCAACTTCGCACTCAGCGACCCGAACGCCCATCGGATGCGAAATTTTCTTGCAAACGCACCACAAGCAGGCAGCAAGTCGCAAAGGGCCAAGTACGGGACAGCAGGCTACGGAGTGGAGGCTCGGGGCCCCACACCAGAGGAAGCACAGAGGGAAAAAGACACACGGATCTCAAAGAAGATGGAGACCATGGGCATCTACTTTGCAACACCAGAATGGGTAGCACTCAATGGGCACCGAGGGCCA GCC CCCGGCCAGCTAAAGTACTGGCAGAACACACGAGAAATACCGGACCCAAACGAGGACTATCTAGACTACGTGCATGCAGAGAAGAGCCGGTTGGCA GCA GAAGAACAAATCCTAAGGGCAGCTACGTCGATCTACGGGGCTCCAGGACAGGCAGAGCCACCCCAAGCTTTCATAGACGAAGTTGCCAAAGTCTATGAAATCAACCATGGACGTGGCCCAAACCAAGAACAGATGAAAGATCTGCTCTTGACTGCGATGGAGATGAAGCATCGCAATCCCAGGCGGGCTCTACCAAAGCCCAAGCCAAAACCCAATGCTCCAACACAGAGACCCCCTGGTCGGCTGGGCCGCTGGATCAGGACCGTCTCTGATGAGGACCTTGAGTGA。

[0162] 2) Inverse PCR: Using the T7-A plasmid as a template, the sequence of the T7-A plasmid is shown in SEQ ID NO:34, and a second reverse amplification is performed with mutant primers to obtain a second mutant plasmid, wherein the second mutant primers include a second forward primer and a second reverse primer, the sequence of the second forward primer is shown in at least one of SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, and SEQ ID NO:15, and the sequence of the second reverse primer is shown in at least one of SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, and SEQ ID NO:16.

[0163] The amplification system is: 2×Phanta Max Master Mix 10 μL,

[0164] Template DNA 100 ng

[0165] 1 μL each of forward and reverse primers

[0166] The system was balanced to 20 μL with ddH2O.

[0167] The reaction conditions were as follows: pre-denaturation at 95°C for 5 min, followed by 20 cycles of denaturation at 95°C for 30 s, annealing at 56°C for 30 s, and extension at 72°C for 2 min, with a total extension at 72°C for 10 min. After the reaction, 1% agarose gel electrophoresis was used to verify successful amplification.

[0168] 3) Enzyme digestion: The PCR product obtained in the previous step is digested with Dpn I enzyme to remove the template plasmid and obtain the Dpn I enzyme digestion product. The reaction system is:

[0169] 10*Buffer 1 μL

[0170] DpnⅠ 0.5 μL

[0171] PCR product 8.5 μL

[0172] Reaction conditions: 37°C water bath for 1 h;

[0173] 4) Transformation: Remove DH5α competent cells from a -80°C freezer and thaw on ice. Transform the Dpn I digested product from the previous step into the DH5α competent cells and mix thoroughly. Place the mixture on ice for 10 minutes. Heat shock the mixture in a 42°C water bath for 90 seconds. Remove the mixture and quickly place it on ice for 5 minutes. Add 1 mL of antibiotic-free LB medium to the mixture and incubate on a shaker at 37°C for 45 minutes. Centrifuge the mixture at low speed (3000 rpm) for 10 minutes to remove the competent cells from the tube. Discard the upper layer of LB medium, reserving approximately 100 μL. Resuspend the competent cells at the bottom of the tube with a pipette and add them to a pre-prepared plate of solid LB medium supplemented with ampicillin. Let it rest for 10 minutes. Invert the plate and incubate overnight at 37°C. Pick a single colony, expand it with shake culture, and send it to Universal Biotech for first-generation sequencing using a CMV-F primer in the forward direction.

[0174] 5) Plasmid Extraction: Sequence alignment is performed on the sequencing results returned by the sequencing company. Single clones with successful mutations at the mutation site are considered correct. Select the correct clones for plasmid extraction. The specific steps are as follows: (A) Transfer 100 μL of the original bacteria to a 100 mL sterile Erlenmeyer flask. Add 20 mL of LB medium containing ampicillin and culture overnight. (B) Transfer 10 mL of the bacterial suspension to a 15 mL centrifuge tube and centrifuge at 1000 rpm for 5 minutes at room temperature. Pour the entire suspension to the bottom of the tube and discard the supernatant. (C) Add 500 μL of Buffer P1 containing RNase A to the tube to thoroughly resuspend the cells. Let stand for 5 minutes. (D) Add 500 μL of Buffer P2 to the tube and gently invert the tube 5-8 times until the liquid becomes clear and transparent. Let stand for 5 minutes at room temperature. (E) Add 700 μL of Buffer P3 to the centrifuge tube. Gently invert the tube 5-8 times until white flocs appear. Let stand at room temperature for 5 minutes. Centrifuge at 12,000 rpm for 10 minutes at room temperature until all the flocs have settled to the bottom of the tube. (F) Transfer the supernatant from (E) to an adsorption column with a bottom tube. Centrifuge at 12,000 rpm for 30 seconds at room temperature. Repeat 2-3 times until all the supernatant has passed through the column. (G) Add 500 μL of Wash Buffer to the adsorption column. Centrifuge at 12,000 rpm for 30 seconds at room temperature. Discard the waste solution and repeat the wash cycle once. (H) Centrifuge the adsorption column at 12,000 rpm for 1 minute at room temperature. Uncap and let stand at room temperature for 1 minute. (I) Place the adsorption column in a new 1.5 mL sterile centrifuge tube, add 100 μL of EB (Elution Buffer), and let stand at room temperature for 1 minute. (J) Centrifuge the adsorption column at room temperature at 12000 r / min for 1 min to elute the plasmid on the adsorption column. After measuring the concentration, store it at -20°C for later use. VP3S130A 、T7-A VP3S163A and T7-A VP3SSAA Plasmid, T7-A VP3S130A The sequence of T7-A is shown in SEQ ID NO: 4. VP3S130A The sequence of T7-A is shown in SEQ ID NO: 5. VP3SSAA The sequence is shown in SEQ ID NO:6.

[0175] 6) Transfection (with IBDV VP3S130A For example): Use Exfect transfection reagent to transfect T7-A VP3S130AThe two plasmids, T7-A and T7-B, were transfected into BSRT7 cells. The specific operation steps were as follows: (A) BSRT7 cells were cultured in a 12-well cell culture plate. When the cell density reached about 80%, transfection could be carried out. (B) Prepare two sterile EP tubes, and add 50 μL of serum-free DMEM medium to each tube. Add 4 μL of Exfect transfection reagent to tube A and mix gently. Add 1 μg of each of the T7-A VP3S130A and T7-B plasmids to tube B and mix gently. Let it stand for 5 min. (C) Add the mixture in tube A to tube B and mix gently. Let it stand for 15 min. (D) Drop the mixed solution into the 12-well plate respectively, shake gently. After 6 h, change to DMEM medium containing 2% serum and continue culturing.

[0176] 7) Passage and blind passage: After 72 h, collect the medium and cells into an EP tube, freeze-thaw once and then centrifuge. Transfer the supernatant to DF-1 cells. After 2 h, discard the supernatant and change to MEM medium containing 2% serum. After 72 h, repeat the above steps and transfer to DF-1 cells again. When characteristic lesions are observed, the rescue is successful.

[0177] Example 2

[0178] Effect of VP3 phosphorylation-modification-deficient recombinant virus on IBDV replication

[0179] <Virus titer determination>

[0180] 1) Virus inoculation: DF-1 cells were cultured in a 96-well cell culture plate. After they grew to confluence, virus inoculation could be carried out. Dilute the virus solution in 2% MEM medium at ratios of 10-1, 10-2, 10-3, 10-4, 10-5, 10-6, 10-7, 10-8, 10-9, 10-10, 10-11, 10-12. The diluted virus-containing medium was used to culture DF-1 cells. Each group had 6 replicates, and 3 parallel experiments were conducted.

[0181] 2) Calculate the virus titer: After 72 h, observe the cytopathic effect, count the number of diseased wells, and calculate the TCID50.

[0182] <Western blot detection>

[0183] 1) Virus inoculation: Inoculate with a virus amount of 1 MOI. Determine the inoculation amount according to the TCID50 of different viruses. After the DF-1 cells in the 12-well plate grew to confluence, inoculate at different time points, and the inoculation times were 6 h, 12 h, and 18 h respectively.

[0184] 2) Sample collection: Collect the medium and cells into an EP tube, centrifuge at low speed to collect the cells, discard the supernatant, add 60 μL of NP-40 lysis buffer, lyse at 4°C for 20 min, then centrifuge at high speed for 10 min, discard the precipitate and retain the supernatant, add 15 μL of 5× protein loading buffer, mix well and boil in a metal bath at 100°C for 10 min. The prepared protein sample is temporarily stored in a 4°C refrigerator for subsequent WB detection;

[0185] 3) Western blot identification: As Figure 5A shown, perform Western blot detection according to the above method. The color development result shows that the viral protein bands of SSAA IBDV are thicker than those of WT IBDV at different virus inoculation times, indicating that the dephosphorylation modification of VP3 promotes the replication of IBDV.

[0186] <TCID50 Detection>

[0187] 1) Virus inoculation: Inoculate with a virus amount of 0.1 MOI, determine the inoculation amount according to the TCID50 of different viruses. After the DF-1 cells in the 12-well plate are confluent, inoculate at different time points, and the inoculation times are 12 h, 24 h, and 48 h respectively;

[0188] 2) Sample collection: Collect the medium and cells into an EP tube, freeze-thaw once, centrifuge at 12000 r / min for 10 min, and transfer the supernatant to a new EP tube;

[0189] 3) TCID50 determination: Culture DF-1 cells in a 96-well cell culture plate. After the cells are confluent, dilute the virus solution obtained in the previous step at ratios of 10-1, 10-2, 10-3, 10-4, 10-5, 10-6, 10-7, 10-8, 10-9, 10-10, 10-11, 10-12 in 2% MEM medium. The diluted virus-containing medium is used to culture DF-1 cells. Observe the cytopathic effect after 72 h, count the number of diseased wells, with 6 replicates in each group and 3 parallel experiments. Finally, summarize the data and calculate the TCID50. As Figure 5B shown, the TCID50 values of SSAA IBDV are higher than those of WT IBDV at different time points, indicating that the dephosphorylation modification of VP3 promotes the replication of IBDV.

[0190] Example 3

[0191] Effect of VP3 phosphorylation modification-deficient recombinant virus on its ability to bind dsRNA and assist polymerase activity

[0192] <The ability of VP3 phosphorylation modification-deficient recombinant virus to bind dsRNA is enhanced>

[0193] First, IBDV dsRNA was purified. Myc-VP3 and Myc-VP3SSAA plasmids were transfected into 293T cells. After 36 h, the dsRNA was co-incubated with NP-40 lysis buffer, and the interaction between VP3 and dsRNA was detected by IP using a dsRNA antibody. As Figure 6 shown, it can be seen that the amount of VP3SSAA binding to dsRNA increased significantly.

[0194] <Enhanced ability of the recombinant virus with VP3 phosphorylation modification deletion in the activity of the helper polymerase>

[0195] First, a plasmid for detecting the polymerase activity of VP3SSAA was constructed. The plasmid for detecting polymerase activity was co-transfected into 293T cells. After 72 h, protein samples were collected, and the expression of VP1 / VP2 / VP3 proteins was detected by Western blot. As Figure 7 shown, it can be seen that the expression level of viral proteins in the VP3SSAA group increased significantly.

[0196] In summary, the present invention discloses a method for constructing an infectious bursal disease virus VP3 protein phosphorylation site deletion virus. The IBDV VP3 protein was isolated and purified using immunoprecipitation technology, and the VP3 phosphorylation sites were identified as serine at position 130 and serine at position 163 by mass spectrometry. The codon at position 130 was mutated from AGC to GCC, corresponding to the mutation of serine to alanine. At the same time, the codon at position 163 was mutated from AGU to GCC, corresponding to the mutation of serine to alanine. A recombinant virus with double phosphorylation site mutations of the VP3 protein was rescued through a virus rescue system. The advantages of the present invention are as follows: Bioinformatics prediction combined with immunoprecipitation-mass spectrometry identification and experimental verification of the specific phosphorylation sites of IBDV VP3, and the double phosphorylation site mutations of VP3 lead to enhanced IBDV virus replication ability, enhanced dsRNA binding ability, and enhanced ability of the helper polymerase activity, which can be used as a candidate vaccine strain for IBDV.

[0197] The above are only the preferred embodiments of the present application and are not intended to limit the present application. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. An infectious bursal disease virus VP3 phosphorylation-deficient virus, characterized in that: The invention comprises a mutant plasmid and a rescue vector carrying the mutant plasmid, wherein the mutant plasmid comprises a mutant VP3 protein, wherein the mutation points of the mutant VP3 protein are: the serine at position 130 of the VP3 protein is mutated to alanine and the serine at position 163 is mutated to alanine; Wherein, the amino acid sequence of the VP3 protein is: MASEFKETPELESAVRAMEAAANVDPLFQSALSVFMWLEENGIVTDMANFALSDPNAHRMRNFLANAPQAGSKSQRAKYGTAGYGVEARGPTPEEAQREKDTRISKKMETMGIYFATPEWVALNGHRGP SPGQLKYWQNTREIPDPNEDYLDYVHAEKSRLASEEQILRAATSIYGAPGQAEPPQAFIDEVAKVYEINHGRGPNQEQMKDLLLTAMEMKHRNPRRALPKPKPKPNAPTQRPPGRLGRWIRTVSDEDLE.

2. The infectious bursal disease virus VP3 phosphorylation-deficient virus according to claim 1, characterized in that: The mutant plasmid includes a first mutant plasmid or a second mutant plasmid; The first mutant plasmid includes Myc-VP3-SSAA; the sequence of the Myc-VP3-SSAA is shown in SEQ ID NO: 3; The second mutant plasmid includes T7-A VP3SSAA ; the T7-A VP3SSAA The sequence is shown in SEQ ID NO:

6.

3. A method for constructing the infectious bursal disease virus VP3 phosphorylation-deficient virus according to claim 1 or 2, characterized in that: The steps include: Step S20, mutating the serine at position 130 of the VP3 protein to alanine and the serine at position 163 to alanine to obtain a mutant plasmid; Step S30, transfecting the mutant plasmid into a rescue vector using a first transfection reagent to obtain the infectious bursal disease virus VP3 phosphorylation-deficient virus; Wherein, the amino acid sequence of the VP3 protein is: MASEFKETPELESAVRAMEAAANVDPLFQSALSVFMWLEENGIVTDMANFALSDPNAHRMRNFLANAPQAGSKSQRAKYGTAGYGVEARGPTPEEAQREKDTRISKKMETMGIYFATPEWVALNGHRGP SPGQLKYWQNTREIPDPNEDYLDYVHAEKSRLASEEQILRAATSIYGAPGQAEPPQAFIDEVAKVYEINHGRGPNQEQMKDLLLTAMEMKHRNPRRALPKPKPKPNAPTQRPPGRLGRWIRTVSDEDLE.

4. The method for constructing the infectious bursal disease virus VP3 phosphorylation-deficient virus according to claim 3, characterized in that: The step S20 also includes the following steps of determining the VP3 phosphorylation modification site: The Netphospho3.1 online tool was used to predict the potential phosphorylation sites of the VP3 protein of the IBDV JX strain for the first time, and the first prediction results were obtained; The recombinant plasmid was constructed using the VP3 gene sequence of the A segment of IBDV JX strain as a template; The recombinant plasmid is subjected to a second prediction of potential phosphorylation sites of the VP3 protein using immunoprecipitation and mass spectrometry methods to obtain a second prediction result.

5. The method for constructing the infectious bursal disease virus VP3 phosphorylation-deficient virus according to claim 4, characterized in that: The method for constructing a recombinant plasmid comprises the following steps: Using the VP3 gene sequence of the A segment of the IBDV JX strain as a template, amplification was performed using the VP3-F and VP3-R primer pairs to obtain a B amplification product, wherein the VP3-F sequence includes the nucleotide sequence shown in SEQ ID NO: 7, and the VP3-R sequence includes the nucleotide sequence shown in SEQ ID NO: 8; Extracting a DNA solution from the amplified product B, and performing double enzyme digestion on the DNA solution and the empty vector using an enzyme digestion system to obtain enzyme-digested DNA fragments and enzyme-digested vectors, respectively; The digested DNA fragment and the digested vector are ligated using T4 DNA ligase to obtain a ligation product; The ligation product is transformed into DH5α competent cells, cultured, and the plasmid is extracted to obtain the recombinant plasmid.

6. The method for constructing the infectious bursal disease virus VP3 phosphorylation-deficient virus according to claim 4, characterized in that: The method further includes the step of verifying the second prediction result: Designing third mutation primers based on the first prediction result and constructing a third mutation plasmid using the Myc-VP3 plasmid as a template, wherein the sequence of the Myc-VP3 plasmid is shown in SEQ ID NO: 33; transfecting the third mutant plasmid into HEK-293T cells using a second transfection agent; Based on the first prediction result, HEK-293T cells carrying the third mutant plasmid were subjected to Western blot verification to verify the second prediction result.

7. The method for constructing the infectious bursal disease virus VP3 phosphorylation-deficient virus according to claim 6, characterized in that: The third mutation primer includes a third forward primer and a third reverse primer, The sequence of the third forward primer is shown in SEQ ID NO: 9, the sequence of the third reverse primer is shown in SEQ ID NO: 10, or The sequence of the third forward primer is shown in SEQ ID NO: 11, the sequence of the third reverse primer is shown in SEQ ID NO: 12, or The sequence of the third forward primer is shown in SEQ ID NO: 13, the sequence of the third reverse primer is shown in SEQ ID NO: 14, or The sequence of the third forward primer is shown in SEQ ID NO: 15, the sequence of the third reverse primer is shown in SEQ ID NO: 16, or The sequence of the third forward primer is shown in SEQ ID NO: 17, the sequence of the third reverse primer is shown in SEQ ID NO: 18, or The sequence of the third forward primer is shown in SEQ ID NO: 19, the sequence of the third reverse primer is shown in SEQ ID NO: 20, or The sequence of the third forward primer is shown in SEQ ID NO: 21, the sequence of the third reverse primer is shown in SEQ ID NO: 22, or The sequence of the third forward primer is shown in SEQ ID NO: 23, the sequence of the third reverse primer is shown in SEQ ID NO: 24, or The sequence of the third forward primer is shown in SEQ ID NO: 25, the sequence of the third reverse primer is shown in SEQ ID NO: 26, or The sequence of the third forward primer is shown in SEQ ID NO: 27, the sequence of the third reverse primer is shown in SEQ ID NO: 28, or The sequence of the third forward primer is shown in SEQ ID NO: 29, the sequence of the third reverse primer is shown in SEQ ID NO: 30, or The sequence of the third forward primer is shown in SEQ ID NO: 31, and the sequence of the third reverse primer is shown in SEQ ID NO:

32.

8. The method for constructing the infectious bursal disease virus VP3 phosphorylation-deficient virus according to claim 6 or 7, characterized in that: Step S20 further includes: Designing the mutant plasmid according to the second prediction result, wherein the mutant plasmid includes the first mutant plasmid or the second mutant plasmid; The step of designing the mutant plasmid according to the second prediction result includes: using the T7-A plasmid as a template and performing a second reverse amplification with a second mutant primer to obtain a second mutant plasmid; wherein the second mutant primer includes a second forward primer and a second reverse primer, the sequence of the second forward primer is shown in SEQ ID NO: 9 and the sequence of the second reverse primer is shown in SEQ ID NO: 10, or the sequence of the second forward primer is shown in SEQ ID NO: 11 and the sequence of the second reverse primer is shown in SEQ ID NO: 12, or the sequence of the second forward primer is shown in SEQ ID NO: 13 and the sequence of the second reverse primer is shown in SEQ ID NO: 14, or the sequence of the second forward primer is shown in SEQ ID NO: 15 and the sequence of the second reverse primer is shown in SEQ ID NO: 16; The second mutant plasmid and the T7-B plasmid are transfected into the rescue vector using a third transfection agent.

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