pSFV-p54 replicon particles and preparation method and application thereof

Abstract

The present invention belongs to the field of genetic engineering technology, and discloses a pSFV-p54 replicon particle and a preparation method and application thereof. The preparation method of the pSFV-p54 replicon particle comprises the following steps: (1) using pSFV-sp6-EGFP plasmid and flag-p54 gene as materials, constructing a pSFV-sp6-p54 plasmid; the flag-p54 gene is as shown in SEQ ID NO: 1; (2) linearizing the SFV-helper auxiliary plasmid and the pSFV-sp6-p54 plasmid respectively and performing in vitro transcription, and co-electrotransferring the obtained mRNA into BHK cells or Vero cells to obtain pSFV-p54 replicon particles. The present invention uses Semliki Forest virus as a vector, inserts the African swine fever p54 gene, and the prepared pSFV-p54 replicon particles can efficiently express the African swine fever virus p54 protein, and have high biosafety, and can be used to develop African swine fever related vaccines.

Description

Technical Field

[0001] The invention belongs to the technical field of gene engineering, and in particular relates to a pSFV-p54 replicon particle and a preparation method and application thereof. Background Art

[0002] African swine fever virus (ASFV) belongs to the order DNA virus (dsDNA), family Asfivirus, genus Asfivirus in virus classification. It is the only member of the family Asfivirus. Domestic pigs and various wild boars infected with ASFV can cause an acute, hemorrhagic, and highly contagious disease - African swine fever. The clinical symptoms of African swine fever are mainly characterized by high fever, loss of appetite, bleeding in the skin and internal organs, and high mortality. There is currently no effective vaccine for prevention. Once discovered, it can only be culled on a large scale, causing huge losses to the pig farming industry. The virus particles of African swine fever virus are icosahedral symmetric, with an average diameter of about 200nm. The structure of the virus particle from inside to outside is the nucleoplasm, nucleocapsid, and envelope (divided into outer and inner layers): the nucleoplasm is mainly composed of the viral genome, DNA binding proteins (p10, p14, p37, p34, etc.) and enzymes required for early gene transcription; the nucleocapsid mainly contains structural proteins p72 and p17, and is composed of about 2000 capsomers; the inner envelope comes from the host's endoplasmic reticulum, combined with p12, p22, p54, p54 and CD2v proteins, and the outer envelope is a byproduct of viral budding, loosely surrounding it. The ASFV genome is a linear double-stranded DNA molecule, and the full length of different strains varies, ranging from 170-193kb, encoding 150-175 proteins. The genome consists of a central conserved region (about 125kb), variable regions on both sides (38-48kb and 13-22kb, respectively), and an inverted repeat sequence at the end of the genome (about 2.1-2.5kb). Among them, p54 is a protein encoded by the ASFV open reading frame E183L gene. It is a type I transmembrane protein that spans the inner envelope of the virus particle and forms homodimers through the cysteine ​​in the cavity. The p54 protein helps the virus to quickly bind to susceptible cells, stimulates the body to produce specific antibodies, and has certain antigenicity. In ASFV-infected cells, the p54 protein binds to the endoplasmic reticulum and promotes the transformation of the endoplasmic reticulum membrane into the precursor of the virus particle envelope. The p54 protein can undergo a special cross-reaction with the cytoplasmic motor protein DLC8 to mediate the intracellular transport of the virus. It plays an important role in the process of cellular internalization of the virus and the processing of the virus in the cell. In addition, the p54 protein replaces Bim (bcl-2interacting mediator of cell death) from the microtubules, relocates it to the mitochondria, activates the intracellular apoptotic pathway, and thus causes apoptosis.

[0003] In the past 20 years, ASFV subunit vaccines and recombinant vaccines have been widely studied, and various viral proteins involved in ASFV infection, such as p72, p54, and p54, have become target proteins for research. However, the subunit vaccines and recombinant vaccines currently developed still cannot provide sufficient protection to the body, and can only reduce the level of viremia to a certain extent and delay the onset of clinical symptoms. Therefore, the present invention hopes to propose a method that can efficiently express viral proteins and has high safety, in order to obtain vaccine products with better performance. Summary of the invention

[0004] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention proposes a pSFV-p54 replicon particle and a preparation method and application thereof. By using Semliki forest virus (SFV) as a vector and inserting the African swine fever p54 gene, the prepared pSFV-p54 replicon particle can efficiently express the African swine fever virus p54 protein and has high biosafety, and can be used to develop African swine fever related vaccines.

[0005] The present invention provides a method for preparing pSFV-p54 replicon particles, comprising the following steps:

[0006] (1) Using pSFV-sp6-EGFP plasmid and flag-p54 gene as materials, constructing pSFV-sp6-p54 plasmid; the flag-p54 gene is shown in SEQ ID NO: 1;

[0007] (2) The SFV-helper plasmid and the pSFV-sp6-p54 plasmid are linearized and transcribed in vitro, and the obtained mRNA is co-electroporated into BHK cells or Vero cells to obtain pSFV-p54 replicon particles.

[0008] The present invention uses Semliki forest virus (SFV, a kind of alphavirus) as a replicon vector to construct a packaging system. The packaging system includes two independent RNA molecules, one RNA molecule is an RNA replicon vector (transcribed from pSFV-sp6-p54 plasmid) containing a flag-tag and a p54 fusion protein gene inserted therein; the other RNA molecule is an auxiliary RNA (transcribed from SFV-helper auxiliary plasmid), which includes a 5'-3' replication signal and a subgenomic promoter and a gene encoding a structural protein. The two mRNA molecules are co-transfected into cells to be packaged into replicon particles, which can replicate autonomously and efficiently express African swine fever virus p54 protein (ASFV p54), and will not integrate with the genome, so it has good biosafety.

[0009] Preferably, the step (1) comprises the following steps:

[0010] The pSFV-sp6-EGFP plasmid is double-digested with BamHI / SmaI endonucleases, and the digested product is homologously recombined with the flag-p54 gene to obtain a recombinant product; the recombinant product is introduced into competent cells, identified, and extracted to obtain the pSFV-sp6-p54 plasmid.

[0011] More preferably, the recombination product is identified using primer tongyong-F and primer p54-R as shown in SEQ ID NO: 2-3.

[0012] More preferably, the competent cells are DH5α cells or Trans1-T1 Phage Resistant chemically competent cells.

[0013] Preferably, step (1) also includes enzyme digestion identification and sequencing of the pSFV-sp6-p54 plasmid.

[0014] Preferably, in step (2), the pSFV-sp6-p54 plasmid is linearized by digestion with SpaI, and the SFV-helper plasmid is linearized by digestion with SpeI.

[0015] The present invention also provides a pSFV-p54 replicon particle prepared by the above preparation method.

[0016] The present invention also provides the use of the pSFV-p54 replicon particles in expressing African swine fever virus p54 protein.

[0017] The present invention also provides the use of the pSFV-p54 replicon particles in the preparation of RNA vaccines.

[0018] The present invention also provides an African swine fever vaccine, comprising the pSFV-p54 replicon particles.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] The present invention uses SFV replicon as an expression vector, inserts the African swine fever p54 gene, and the obtained pSFV-p54 replicon particles have the advantages of wide host selection range, high expression efficiency, etc., can replicate autonomously and efficiently express African swine fever virus p54 protein (ASFV p54), and will not integrate with the genome, so it has good biosafety. The pSFV-p54 replicon particle vaccine can be used to prepare African swine fever vaccine, which can effectively induce the body's immune system to produce an immune protection response. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is the plasmid map of pSFV-SP6-p54;

[0022] Figure 2 The results of double restriction digestion of pSFV-sp6-EGFP;

[0023] Figure 3 This is the identification result of the recombinant plasmid bacterial solution;

[0024] Figure 4 The results of enzyme digestion identification of the recombinant plasmid;

[0025] Figure 5 The results of in vitro transcription of linearized pSFV-sp6-p54 and pSFV-helper;

[0026] Figure 6 Results of indirect immunofluorescence assay to detect ASFV p54 protein expression

[0027] Figure 7 Western blot results of ASFV p54;

[0028] Figure 8 The results of SDS-PAGE experiments of ASFV p54;

[0029] Fig. 9 This is the result of measuring the concentration of purified ASFV p54 protein using the BCA method. DETAILED DESCRIPTION

[0030] In order to make those skilled in the art more clearly understand the technical solution of the present invention, the following examples are listed for illustration. It should be pointed out that the following examples are only preferred embodiments of the present invention and do not constitute a limitation on the protection scope of the present invention. Any modification, substitution, and combination made without violating the spirit and principle of the present invention are included in the protection scope of the present invention.

[0031] Unless otherwise specified, the raw materials, reagents or devices used in the following examples can be obtained from conventional commercial sources or by existing known methods.

[0032] Experimental reagents

[0033] Taq DNA polymerase, 10×PCR Buffer, MgCl2 (25 mM), dNTP (1 mM), Marker, 6×DNA loading buffer, 10×TAE, one-step rapid competent cell preparation kit, SanPrep column plasmid DNA small amount extraction kit, agarose, DNA column gel recovery kit were all purchased from Sangon Biotechnology Co., Ltd.; 4S Red Plus nucleic acid stain was purchased from BBI; fetal bovine serum (Fetal Bovine Serum), pancreatin (0.25% Trypsin-EDTA), DMEM medium (DMEM / High glucose), 0.25% Trypsin-EDTA were all purchased from Gibco; tissue genomic DNA extraction kit was purchased from Tiangen; 2×qPCR Mix was purchased from Novazon; Vector, SYBR Premix ExTaqTM (Tli RNAseH plus) (RR420A), PrimescriptTM RT reagent kit with gDNA Eraser (Perfect Real Time) (RR047A), Gel Extraction Kit, Stbl3 competent cells, BamHI / AscI restriction endonucleases were all purchased from Bao Biotechnology (Dalian) Co., Ltd.; RNAprep pure Tissue Kit (DP431) was purchased from TIANGEN; Plasmid Mini Kit was purchased from OMEGA; transfection reagent Lipofectamine 3000Regeant was purchased from Invitrogen; pSFVCs-lacz (#92076) was purchased from addgene; EGFP-C1 vector plasmid (obtained from Laboratory 114, Building 9, South China Agricultural University), Anti-Flag Affinity Gel (Biyuntian); pSFV-sp6-EGFP plasmid (prepared according to the invention patent with application number 2021108223525, and its sequence is shown in SEQID NO: 4); SFV-helper (prepared according to the invention patent with application number 2021108224072, and its sequence is shown in SEQID NO: 5); BCA protein concentration determination kit (enhanced) (Biyuntian); FITC-labeled goat anti-swine IgG; African swine fever positive serum (Zhaoqing Dahuanong Biopharmaceutical Co., Ltd.). PageRuler Prestained Protein Ladder (26619, Thermo); BeyoBlue TMCoomassie Brilliant Blue Ultrafast Staining Solution (Beyotime); SDS-PAGE Gel Preparation Kit (Taraka).

[0034] Experimental Instruments

[0035] Biosafety cabinet 1300SERIES A2 (Thermo); constant temperature water bath DK-8D (Yiheng / Shanghai); CO2 incubator Forma 371 (Thermo); biochemical incubator DHP-9162 (Yiheng / Shanghai); high-performance high-speed desktop refrigerated centrifuge 5804R (Eppendorf / Germany); electric constant temperature incubator DHP-9162 (Yiheng / Shanghai); vortex oscillator Vortex (Thermo); pipette Research plus (Eppendorf / Germany); inverted microscope DMI1 (Leica, Germany); countstar cell counter IC-1000 (Shanghai Ruiyu Biotechnology Co., Ltd.); MicroAmp Optical 96-Well Reaction Plate N8010560 (ABI); MicroAmpTM Optical Adhesive Film4311971 (ABI); clean bench SW-CJ-2FD (Sujing Antai); fluorescence microscope (Leica, Germany); CO2 incubator HF240 (Likang); centrifuge NeoFuge 1600R (Likang); high-speed centrifuge centrifuge 5840R (Eppendorf, Germany); DYY-6C steady-current and steady-voltage electrophoresis instrument (Beijing Liuyi); H6-1 micro electrophoresis tank (Shanghai Jingyi Organic Glass Products Instrument Factory); FR980 gel imaging system (Shanghai Furi Technology Co., Ltd.); SMA4000 micro-spectrophotometer (merinton); PCR reaction amplifier (BIO).

[0036] Example 1: Construction of pSFV-SP6-p54 plasmid

[0037] This embodiment provides a pSFV-SP6-p54 plasmid, and its map is as follows Figure 1 The construction method of the above-mentioned pSFV-SP6-p54 plasmid comprises the following steps:

[0038] (1) Gene synthesis and primer design

[0039] The flag-p54 fragment (its sequence is shown in SEQ ID NO: 1) was synthesized according to the gene sequence of ASFV p54 logged in GenBank, and the sequence was synthesized by Jinweizhi Biotechnology Co., Ltd.; the primers tongyong-F and p54-R were designed using homologous recombination technology for the identification of the recombinant product, and its nucleotide sequence is shown in Table 1.

[0040] Table 1 Primers for identification of recombinant products

[0041] Primer name Primer sequence (5'-3') Serial number Tongyong-F ggtttaatgatcctcgaagatc SEQ ID NO: 2 p54-R ttcaattaattacccgggttacaattctgcttttg SEQ ID NO: 3

[0042] Primers tongyong-F and p54-R were synthesized by Genewise Technology Co., Ltd. (Suzhou).

[0043] (2) BamHI / SmaI digested plasmid pSFV-sp6-EGFP

[0044] The pSFV-sp6-EGFP plasmid (whose sequence is shown in SEQ ID NO: 4) was double-digested with BamHI / SmaI endonucleases. The enzyme digestion system was 300 μL in total and divided into 6 tubes, each with 50 μL. The system is shown in Table 2.

[0045] Table 2 Enzyme digestion system

[0046]

[0047]

[0048] After the enzyme digestion, 1% nucleic acid gel electrophoresis was performed to identify the results. Figure 2 As shown in the figure (M: the band sizes from top to bottom are 15000, 10000, 7500, 5000, 3000, 1500, 1000, 500), after double enzyme digestion, obvious target bands appear around 726bp and 11045bp. Cut the gel of the band around 11045bp under ultraviolet light, and try to make the cut gel size just contain all the target bands, not too large, cut the gel quickly, and turn off the ultraviolet light immediately after cutting to prevent the target fragment from being degraded by excessive ultraviolet radiation. Transfer the cut gel to a 2mL EP tube, recover the gel, and measure the DNA concentration.

[0049] (3) Homologous recombination

[0050] The flag-p54 fragment synthesized by the above gene was homologously recombined with the large fragment of the digested vector obtained by double digestion, and the following reaction system was prepared on ice, as shown in Table 3:

[0051] Table 3 Homologous recombination reaction system

[0052] Element Volume (μL) Flag-p54 fragment 1 Enzyme-cut vector large fragment 2 5×CE 4 En 2 <![CDATA[ddH20]]> 11

[0053] After gently shaking the above system, incubate it in a 37°C water bath for 30 min, and then cool it at 4°C or on ice to obtain the recombinant product;

[0054] (4) Transformation of recombinant products

[0055] Thaw the chemically competent DH5α cells for cloning on ice, take 10 μL of the recombinant product and add it to 100 μL of competent cells, flick the tube wall to mix (do not oscillate to mix), and let it stand on ice for 30 minutes.

[0056] Heat shock in a 42℃ water bath for 45s, then immediately cool on ice for 2-3min. Add 900μL SOC and shake at 37℃ for 1h (speed 200-250rpm). Preheat the corresponding LB plate solid culture medium containing chloramphenicol resistance in a 37℃ incubator. Centrifuge at 5000rpm for 5min, and discard 900μL supernatant. Resuspend the bacteria with the remaining culture medium, spread it on the plate containing chloramphenicol resistance with a sterile coating stick, and culture it upside down in a 37℃ incubator for 12-16h.

[0057] (5) Identification of recombinant products

[0058] After overnight culture, check the colonies on the plate and pick 10 colonies for bacterial liquid identification. Add 2 mL EP tubes with LP culture medium containing chloramphenicol resistance and culture in a shaker at 37°C for about 5 hours. Then, identify the bacterial liquid. The identification fragment is 1310 bp. The identification PCR system is shown in Table 4:

[0059] Table 4 Bacterial liquid identification PCR system

[0060] Element Volume (μL) Bacterial liquid 1 Tongyong-F 1 p54-R 1 Ex Taq 10 <![CDATA[ddH20]]> 7

[0061] Take 100 μL of the positive bacterial solution and put it into a 50 mL centrifuge tube with LP medium containing chloramphenicol resistance and shake it overnight. Figure 3 As shown (M: the band sizes from top to bottom are 2000, 1000, 750, 500, 250, 100), wherein the sizes of lanes 1, 2, 4, 5, 6, 7, 9 and 10 are all about 1310 bp, which is consistent with the theoretical value.

[0062] (6) Extraction of plasmid

[0063] 1. Column equilibration step: add 500 μL of equilibration solution BL to the adsorption column CP3 (the adsorption column is placed in the collection tube), centrifuge at 12000 rpm for 1 min, pour out the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

[0064] 2. Take 15 mL of overnight cultured bacterial solution and add it to a centrifuge tube. Use a regular desktop centrifuge and centrifuge at 12,000 rpm for 1 min. Remove the supernatant as much as possible.

[0065] 3. Add 500 μL of solution P1 to the centrifuge tube containing the bacterial pellet and use a pipette or vortex oscillator to thoroughly suspend the bacterial pellet.

[0066] 4. Add 500μL of solution P2 to the centrifuge tube and gently flip it upside down 6-8 times to fully lyse the bacteria. Note: Mix gently and do not shake violently to avoid interrupting the genomic DNA and causing the extracted plasmid to be mixed with genomic DNA fragments. At this time, the bacterial solution should become clear and viscous. The time used should not exceed 5 minutes to avoid damage to the plasmid. If it does not become clear, it may be due to excessive bacteria and incomplete lysis. The amount of bacteria should be reduced.

[0067] 5. Add 700μL of solution P3 to the centrifuge tube, and immediately and gently turn it upside down 6-8 times to mix thoroughly. A white flocculent precipitate will appear. Centrifuge at 12000rpm for 10min. Note: Mix immediately after adding P3 to avoid local precipitation. If there is still a tiny white precipitate in the supernatant, centrifuge again and take the supernatant.

[0068] 6. Transfer the supernatant collected in the previous step to the adsorption column CP3 with a pipette, centrifuge at 12000rpm for 30-60s, pour out the waste liquid in the collection tube, and place the adsorption column CP3 in the collection tube.

[0069] 7. Add 600 μL of rinsing solution PW to the adsorption column CP3, centrifuge at 12000 rpm for 30-60 seconds, pour out the waste liquid in the collection tube, and place the adsorption column CP3 in the collection tube; repeat this step once.

[0070] 8. Place the adsorption column CP3 in the collection tube and centrifuge at 12000rpm for 2min to remove the residual rinse solution in the adsorption column. Open the adsorption column CP3 and place it at room temperature for several minutes to completely dry the residual rinse solution in the adsorption material.

[0071] 9. Place the adsorption column CP3 in a clean centrifuge tube, add 50-100 μL of elution buffer EB to the middle part of the adsorption membrane, place at room temperature for 2 minutes, centrifuge at 12000 rpm for 2 minutes, and collect the solution containing the recombinant plasmid (pSFV-sp6-p54 plasmid) into the centrifuge tube.

[0072] (7) Enzyme digestion identification and sequencing:

[0073] The obtained solution containing the recombinant plasmid (pSFV-sp6-p54 plasmid) was double-digested using SmaI and BamHI endonucleases. The enzyme digestion system was 20 μL in total. The system is shown in Table 5:

[0074] Table 5 Enzyme digestion system

[0075] Element Volume (μL) SmaI 0.25 B H 0.25 Recombinant plasmid 1.5 Quicker buffer 2 <![CDATA[ddH2O]]> 20

[0076] After the enzyme digestion, 1% nucleic acid gel electrophoresis was performed to identify the results. Figure 4 As shown (M: the band sizes from top to bottom are 8000, 5000, 3000, 1500, 1000, and 500), the obtained fragment sizes were 563 bp and 11557 bp, respectively, which are consistent with the theoretical values; they were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing, and the sequencing results were exactly the same.

[0077] Example 2: In vitro transcription of the replicon plasmid pSFV-sp6-p54 and the helper plasmid SFV-heleper (whose sequence is shown in SEQ ID NO: 5)

[0078] (1) The pSFV-sp6-p54 plasmid was linearized by digestion with SpaI, and the SFV-helper plasmid was linearized by digestion with SpeI. The reaction system is shown in Table 6-7:

[0079] Table 6 SpaI digestion of pSFV-sp6-p54 plasmid

[0080] Element Volume (μL) SpeI 8 pSFV-sp6-p54 plasmid 49.1 Quicker buffer 30 <![CDATA[ddH20]]> 212.9

[0081] Table 7 SpeI digestion of SFV-helper plasmid

[0082] Element Volume (μL) SpeI 9 SFV-helper plasmid 53 Quicker buffer 30 <![CDATA[ddH20]]> 208

[0083] (2) The linearized pSFV-sp6-p54 and SFV-heleper were transcribed in vitro to obtain the corresponding mRNA; in vitro transcription was performed using the mMESSAGE mMACHINE SP6 transcription kit (AM1340, purchased from Thermo Fisher Scientific), and the reaction system was shown in Table 8-9 below:

[0084] Table 8 pSFV-sp6-p54 in vitro transcription system

[0085] Element Volume (μL) Nuclease-free Water 3 2×NTP / CAP 10 10×Reaction Buffer 2 Linearization of pSFV-sp6-p54 2 Enzyme Mix 2

[0086] Table 9 SFV-helper in vitro transcription system

[0087] Element Volume (μL) Nuclease-free Water 4 2×NTP / CAP 10 10×Reaction Buffer 2 Linearization SFV-helper 1 Enzyme Mix 2

[0088] After the above system is prepared, transcribe at 37℃ for 40min, then add 1μL GTP and continue transcription at 37℃ for 2h; then add 1μL TURBO DNase and react at 37℃ for 15min. After the transcription is completed, take 1μL of the transcription product and add 4μL Nuclease-free Water and 6× loading buffer for dilution. Perform sterile operation to prevent RNA degradation, and then perform electrophoresis experiment in 1% SDS-page to verify whether the band is correct.

[0089] The test results are as follows Figure 5 As shown (M: 8000, 5000, 3000, 1500, 1000, 500), lane 3 is the in vitro transcription result of pSFV-sp6-p54 replicon plasmid, and lanes 4 and 5 are the in vitro transcription results of pSFV-helper auxiliary plasmid. The above results show that the transcription results are consistent with the theoretical values.

[0090] (3) Electroporate pSFV-SP6-p54 and SFV-heleper in vitro transcribed mRNA into BHK cells

[0091] 1. Transfer BHK cells to T75 flasks in advance, and conduct experiments when the cell confluence reaches 80%;

[0092] 2. Pre-cool 1.5 mL EP tubes and PBS on ice, and pre-cool 4 mm cuvettes (electrode cups) in a -20°C refrigerator;

[0093] 3. Preheat 6 ml of culture medium (DMEM containing 1% FBS) at 37°C;

[0094] 4. Trypsinize the cells in the T25 flask and resuspend them in 5 ml of complete medium;

[0095] 5. Centrifuge at 500g, 4°C for 5 minutes;

[0096] 6. Discard the supernatant and wash the cell pellet with 10 mL of ice-cold PBS;

[0097] Centrifuge at 7.500 g and 4°C for 5 min, and repeat the PBS wash again;

[0098] 8. Discard the supernatant and completely remove the residual PBS, and resuspend the cells with 260 μL pre-cooled PBS. After mixing, pipette 260 μL of PBS cell resuspension into pre-cooled 1.5 mL EP tubes;

[0099] 9. Add 10 μL of pSFV-sp6-p54 and SFV-heleper mRNA transcribed in vitro to the PBS cell resuspension and place on ice;

[0100] 10. Transfer the above system to a pre-cooled electrode cup (4 mm), set the electroporator conditions to 100 V, 20 ms, shock once, and place on ice for 1 min after shock;

[0101] 11. Add about 400 μL of DMEM containing 1% FBS to each electrode cup and mix well by pipetting. Then transfer to a T25 square bottle and add 6 mL of DMEM containing 1% FBS. Place in a 37°C, 5% CO2 incubator for culture overnight.

[0102] 12. Observe the cell growth after electroporation. Collect the supernatant after 36 hours and store it at -80℃. The obtained cells can produce pSFV-p54 replicon particles.

[0103] Example 3 Indirect immunofluorescence experiment

[0104] 1. Collect the cells into which mRNA was co-electrotransfected in Example 2 and add them to a culture plate, fix them with methanol precooled to 4°C overnight, and wash them three times with PBST; normal BHK cells were used as blank controls;

[0105] 2. Continue to add 1 mL of 5% BSA, incubate at 37°C for 1 hour, and then wash three times with PBST;

[0106] 3. Add 1500-fold diluted African swine fever serum to each well, incubate at 37°C for 1 hour, and then wash three times with PBST;

[0107] 4. Add 200-fold diluted FITC-labeled goat anti-pig IgG to each well, incubate at 37°C for 1 hour, and wash three times with PBST, keeping away from light;

[0108] 5. The culture plate was observed under a fluorescence microscope (×200) and compared with normal cells.

[0109] The test results are as follows Figure 6 As shown (A is the experimental group, B is the negative control group), the results showed that the experimental group had strong fluorescence expression, while the negative control group had no fluorescence expression. The above results indicate that ASFV p54 protein is expressed.

[0110] Example 4 Determination of TCID50 (tissue culture infectious dose) of pSFV-p54 replicon particles

[0111] The pSFV-p54 replicon particles were diluted 10-fold in series using DMEM medium. -1 , 10 -2 , …, 10 -8100 μL of each dilution was added to a 96-well cell culture plate, followed by 100 μL of BHK-21 cells digested with 0.25% trypsin (cell content was 10 5 / mL is appropriate), and 8 replicates are made for each dilution, and a blank cell culture control is set. Place in a 37°C, 5% CO2 incubator. Observe the cytopathic effect daily and record the number of cytopathic wells until the control cells age and fall off. Calculate the TCID50 of the virus according to the Reed-Muench method, and the titer is 10 -3.5 TCID 50 / 0.1mL , the titer is sufficient to meet the experimental requirements.

[0112] Example 5 ASFV p54 protein expression

[0113] The pSFV-p54 replicon particles were taken out from the -80°C refrigerator and placed at room temperature until completely thawed. 10 μL of pSFV-p54 replicon particles were inoculated into a 10 cm cell culture dish and cultured in advance until 80% of the BHK cells were grown. After 36 hours, the cell supernatant was removed, and the cells were washed with PBS three times. 600 μL of IP cell lysis buffer (Biyuntian, containing PMSF) was added, and the cells were gently pipetted several times to allow the lysis buffer and cells to fully contact. The cells were scraped and collected on ice with a pipette tip, and 50 μL was taken for total protein Western blot experiments (the primary antibody was African swine fever serum, the dilution multiple was 1:1000, and the secondary antibody was FITC-labeled goat anti-swine IgG, the dilution multiple was 1:600); the remaining 550 μL was used for subsequent experiments such as purification of Flag fusion proteins, and all were stored in a -80°C refrigerator for use.

[0114] Western blot test results are as follows Figure 7 As shown, lane 1 is the target protein band, about 30KDa, which is consistent with the theoretical value.

[0115] Example 6 ASFV p54 protein purification

[0116] 1. Fully resuspend Anti-Flag Affinity Gel (Biyuntian) to form a uniform gel suspension.

[0117] 2. Take the remaining 550 μL of the sample stored in the -80°C refrigerator in Example 5, and add 120 μL of the well-mixed gel suspension.

[0118] 3. Incubate on ice with shaking at 4°C overnight.

[0119] 4. Centrifuge at 6000g for 30 seconds at 4°C and carefully remove the supernatant.

[0120] 5. Wash three times by adding 0.5 mL TBS and placing on a shaker for 5 min each time, then centrifuge at 6000 g for 30 s and carefully remove the supernatant.

[0121] 7. Add 100 μL of 3× Flag peptide elution buffer (150 μg / mL) to the obtained sample, incubate with shaking on ice for 2 hours, centrifuge at 4°C, 6000g for 30 seconds, and carefully transfer the supernatant to a new tube. The supernatant is the eluted Flag fusion protein.

[0122] 8. Store the eluted Flag fusion protein at -80℃ for long-term storage for subsequent SDS-PAGE and Western blot experiments. Take 15μL of the purified protein stored at -80℃, mix it with an equal amount of Loading buffer (2×), boil it in 100℃ boiling water for 10min, then centrifuge it at 12000rpm and 4℃ for 5min, take 20μL of the supernatant and load it for SDS-PAGE experiment. The results of SDS-PAGE experiment are shown in the figure. Figure 8 As shown: Lane 1 is the target protein band, the band size is around 30KDa, which is consistent with the theoretical size range, the band is relatively bright and single without any other bands.

[0123] Example 7 BCA method to determine the concentration of purified ASFV p54 protein

[0124] The concentration of purified ASFV p54 protein was determined using the Bio-Tech BCA Protein Concentration Assay Kit (Enhanced), including the following steps:

[0125] (1) Add 0, 1, 2, 4, 8, 12, 16, and 20 μL of the standard into the standard wells of a 96-well plate, and add standard diluent to make up to 20 μL. That is, the concentrations of the standard are 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / mL, respectively.

[0126] (2) Add an appropriate volume of the ASFV p54 protein sample to be tested to the sample wells of the 96-well plate. If the sample volume is less than 20 μL, add the standard diluent to make it up to 20 μL and record the specific amount of sample volume added.

[0127] (3) Add 200 μL of BCA working solution to each well and place at 37°C for 20-30 minutes. Alternatively, place at room temperature for 2 hours or at 60°C for 30 minutes. When the BCA method is used to determine protein concentration, the color will deepen over time, and the color development reaction will accelerate as the temperature rises. If the concentration is low, it is appropriate to incubate at a higher temperature or extend the incubation time appropriately.

[0128] (4) Measure the absorbance of A562 using an ELISA reader.

[0129] (5) Calculate the protein concentration of the ASFV p54 protein sample to be tested based on the standard curve and the sample volume used.

[0130] The protein concentration was tested by BCA protein concentration assay kit. The test results were as follows: Fig. 9 The measured standard curve is y = 0.8935x-0.1153 (R 2 =0.9973), thus knowing that the concentration of ASFV p54 protein after purification is 300 μg / mL. It can be used for subsequent coating of ELISA plates to detect serum sample IgG, and as a stimulus for ELISPOT experiments to detect cytokines.

[0131] The embodiments of the present application are described in detail above in conjunction with the accompanying drawings, but the present application is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present application. In addition, the embodiments of the present application and the features in the embodiments can be combined with each other without conflict. SEQUENCE LISTING <110> Huanong (Zhaoqing) Bio-Industry Technology Research Institute Co., Ltd. <120> pSFV-p54 replicon particles and preparation method and application thereof <130> 1 <160> 5 <170> PatentIn version 3.5 <210> 1 <211> 588 <212> DNA <213> Artificial sequence <400> 1 gactacaagg acgacgatga taagtccgga tccatggatt ctgaattttt tcaaccggtt 60 tatccgcggc attatggtga gtgtttgtca ccagtcacta caccaagctt cttctccaca 120 catatgtata ctattctcat tgctatcgtg gtcttagtca tcattatcat cgttctaatc 180 tatctattct cttcaagaaa gaaaaaagct gctgctattg aggaggaaga tatacagttt 240 ataaatcctt atcaagatca gcagtgggta gaagtcactc cacaaccagg tacctctaaa 300 ccagctggag cgactacagc aagtgtaggc aagccagtca cgggcagacc ggcaacaaac 360 agaccagcaa caaacaaacc agttacggac aacccagtta cggacagact agtcatggca 420 actggcgggc cggcggccgc acctgcggcc gcgagtgctc ctgctcatcc ggctgagcct 480 tacacgacag tcactactca gaacactgct tcacaaacaa tgtcggctat tgaaaattta 540 cgacaaagaa acacctatac gcataaagac ctagaaaact ccttgtaa 588 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <400> 2 ggtttaatga tcctcgaaga tc 22 <210> 3 <211> 35 <212> DNA <213> Artificial Sequence <400> 3 ttcaattaat tacccgggtt acaattctgc ttttg 35 <210> 4 <211> 11771 <212> DNA <213> Artificial Sequence <400> 4 atggccgcca aagtgcatgt tgatattgag gctgacagcc cattcatcaa gtctttgcag aaggcatttc cgtcgttcga ggtggagtca ttgcaggtca caccaaatga ccatgcaaat gccagagcat tttcgcacct ggctaccaaa ttgatcgagc aggagactga caaagacaca ctcatcttgg atatcggcag tgcgccttcc aggagaatga tgtctacgca caaataccac 300. tgcgtatgcc ctatgcgcag cgcagaagac cccgaaaggc tcgtatgcta cgcaaagaaa ctggcagcgg cctccggga ggtgctggat agagagatcg caggaaaaat caccgacctg cagaccgtca tggctacgcc agacgctga tctcctacct tttgcctgca cagacgtc acgtgtcgta cggcagccga agtggccgta taccaggacg tgtatgctgt acatgcacca 480 acatcgctgt accatcaggc gatgaaaggt gtcagaacgg cgtattggat tgggtttgac 540 accaccccgt ttatgtttga cgcgctagca ggcgcgtatc caacctacgc cacaaactgg 600 gccgacgagc aggtgttaca ggccaggac ataggactgt gtgcagcatc cttgactgag 660 720. ggaagactcg gcaaactgtc cattctccgc aagaagcaat tgaaaccttg cgacacagtc atgttctcgg taggatctac attgtacact gagagcagaa agctactgag gagctggcac 780 ttaccctccg tattccacct gaaaggtaaa caatccttta cctgtaggtg cgataccatc 840 gtatcatgtg aagggtacgt agttaagaaa atcactatgt gccccggcct gtacggtaaa 900 acggtagggt acgccgtgac gtatcacgcg gagggattcc tagtgtgcaa gaccacagac 960 actgtcaaag gagaaagagt ctcattccct gtatgcacct acgtcccctc aaccatctgt 1020 gatcaaatga ctggcatact agcgaccgac gtcacaccgg aggacgcaca gaagttgtta 1080 gtgggattga atcagaggat agttgtgaac ggaagaacac agcgaaacac taacacgatg 1140 aagaactatc tgcttccgat tgtggccgtc gcatttagca agtgggcgag ggaatacaag 1200 gcagaccttg atgatgaaaa acctctgggt gtccgagaga ggtcacttac ttgctgctgc 1260 ttgtgggcat ttaaaacgag gaagatgcac accatgtaca agaaaccaga cacccagaca 1320 atagtgaagg tgccttcaga gtttaactcg ttcgtcatcc cgagcctatg gtctacaggc 1380 ctcgcaatcc cagtcagatc acgcattaag atgcttttgg ccaagaagac caagcgagag 1440 ttaatacctg ttctcgacgc gtcgtcagcc agggatgctg aacaagagga gaaggagagg 1500 ttggaggccg agctgactag agaagcctta ccacccctcg tccccatcgc gccggcggag 1560 acgggagtcg tcgacgtcga cgttgaagaa ctagagtatc acgcaggtgc aggggtcgtg 1620 gaaacacctc gcagcgcgtt gaaagtcacc gcacagccga acgacgtact actaggaaat 1680 tacgtagttc tgtccccgca gaccgtgctc aagagctcca agttggcccc cgtgcaccct 1740 ctagcagagc aggtgaaaat aataacacat aacgggaggg ccggccgtta ccaggtcgac 1800 ggatatgacg gcagggtcct actaccatgt ggatcggcca ttccggtccc tgagtttcaa 1860 gctttgagcg agagcgccac tatggtgtac aacgaaaggg agttcgtcaa caggaaacta 1920 taccatattg ccgttcacgg accgtcgctg aacaccgacg aggagaacta cgagaaagtc 1980 agagctgaaa gaactgacgc cgagtacgtg ttcgacgtag ataaaaaatg ctgcgtcaag 2040 agagaggaag cgtcgggttt ggtgttggtg ggagagctaa ccaacccccc gttccatgaa 2100 ttcgcctacg aagggctgaa gatcaggccg tcggcaccat ataagactac agtagtagga 2160 gtctttgggg ttccgggatc aggcaagtct gctattatta agagcctcgt gaccaaacac 2220 gatctggtca ccagcggcaa gaaggagaac tgccaggaaa tagtcaacga cgtgaagaag 2280 caccgcggac tggacatcca ggcaaaaaca gtggactcca tcctgctaaa cgggtgtcgt 2340 cgtgccgtgg acatcctata tgtggacgag gctttcgctt gccattccgg tactctgcta 2400 gccctaattg ctcttgttaa acctcggagc aaagtggtgt tatgcggaga ccccaagcaa 2460 tgcggattct tcaatatgat gcagcttaag gtgaacttca accacaacat ctgcactgaa 2520 gtatgtcata aaagtatatc cagacgttgc acgcgtccag tcacggccat cgtgtctacg 2580 ttgcactacg gaggcaagat gcgcacgacc aacccgtgca acaaacccat aatcatagac 2640 accacaggac agaccaagcc caagccagga gacatcgtgt taacatgctt ccgaggctgg 2700 gtaaagcagc tgcagttgga ctaccgtgga cacgaagtca tgacagcagc agcatctcag 2760 ggcctcaccc gcaaaggggt atacgccgta aggcagaagg tgaatgaaaa tcccttgtat 2820 gcccctgcgt cggagcacgt gaatgtactg ctgacgcgca ctgaggatag gctggtgtgg 2880 aaaacgctgg ccggcgatcc ctggattag gtcctatca acatccaca gggtacttt 2940 acggccacat tggagaatg gcagagaaa cacgacaaaa tatgaggt tgaagg 3000 ccggctgcgc ctgtggacgc gttccagaac aaagcgaacg tgtgttgggc gaaagcctg 3060 gtgcctgtcc tggacactgc cggaatcaga ctcactgcag agagtggag cacgattatt 3120 acagcattta aggaggacag agcttactct ccagtggtgg ccttgaatga aatttgcacc 3180 aagtactatg gagttgacct ggacagtgc ctgttttctg ccccgaaggt gtccctgtat 3240 tacgagaaca accactggga taacagacct ggtggaagga tgtaggatt caatgccgca 3300 acagctgcca ggctggaagc tagacatacc ttcctgaagg ggcagtggca tacggcaag 3360 caggcagtta tcgcagaaag aaaaatccaa ccgctttctg tgctggacaa tgtattcct 3420 atcaccgca ggctgccgca cgccctgtg gctgagtaca agacggttaa aggcagtagg 3480 gttgagtggc tggtcaataa agtaagaggg taccacgtcc tgctggtgg tgagtacaac 3540 ctggctttgc ctcgacgcag gtcacttgg tgtcaccgc tgaatgtcac aggcccgat 3600 aggtgctacg acctaagttt aggactgccg gctgacgccg gcaggttcga cttggtcttt 3660 gtgaacattc acacggaatt cagaatccac cactaccagc agtgtgtcga ccacgccatg 3720 aagctgcaga tgcttggggg agatgcgcta cgactgctaa aacccggcgg cagcctcttg 3780 atgagagctt acggatacgc cgataaaatc agcgaagccg ttgtttcctc cttaagcaga 3840 aagttctcgt ctgcaagagt gttgcgcccg gattgtgtca ccagcaatac agaagtgttc 3900 ttgctgttct ccaacttcga taacggaaag aggccctcaa ccttacacca gatgaatacc 3960 aagctgagtg ccgtgtatgc cggagaagcc atgcacacgg ccgggtgtgc accatcctac 4020 agagttaaga gagcagacat agccacgtgc acagaagcgg ctgtggttaa cgcagctaac 4080 gcccgtggaa ctgtagggga tggcgtatgc agggccgtgg cgaagaaatg gccgtcagcc 4140 tttaagggag aagcaacacc agtgggcaca attaaaacag tcatgtgcgg ctcgtacccc 4200 gtcatccacg ctgtagcgcc taatttctct gccacgactg aagcggaagg ggaccgcgaa 4260 ttggccgctg tctaccgggc agtggccgcc gaagtaaaca gactgtcact gagcagcgta 4320 gccatcccgc tgctgtccac aggagtgttc agcggcggaa gagataggct gcagcaatcc 4380 ctcaaccatc tattcacagc aatggacgcc acggacgctg acgtgaccat ctactgcaga 4440 gacaaaagtt gggagaagaa aatccaggaa gccatagaca tgaggacggc tgtggagttg 4500 ctcaatgatg acgtggagct gaccacagac ttggtgagag tgcacccgga cagcagcctg 4560 gtgggtcgta agggctacag taccactgac gggtcgctgt actcgtactt tgaaggtacg 4620 aaattcaacc aggctgctat tgatatggca gagatactga cgttgtggcc cagactgcaa 4680 gaggcaaacg aacagatatg cctatacgcg ctgggcgaaa caatggacaa catcagatcc 4740 aaatgtccgg tgaacgattc cgattcatca acacctccca ggacagtgcc ctgcctgtgc 4800 cgctacgcaa tgacagcaga acggatcgcc cgccttaggt cacaccaagt taaaagcatg 4860 gtggtttgct catcttttcc cctcccgaaa taccatgtag atggggtgca gaaggtaaag 4920 tgcgagaagg ttctcctgtt cgacccgacg gtaccttcag tggttagtcc gcggaagtat 4980 gccgcatcta cgacggacca ctcagatcgg tcgttacgag ggtttgactt ggactggacc 5040 accgactcgt cttccactgc cagcgatacc atgtcgctac ccagtttgca gtcgtgtgac 5100 atcgactcga tctacgagcc aatggctccc atagtagtga cggctgacgt acaccctgaa 5160 cccgcaggca tcgcggacct ggcggcagat gtgcatcctg aacccgcaga ccatgtggac 5220 ctcgagaacc cgattcctcc accgcgcccg aagagagctg cataccttgc ctcccgcgcg 5280 gcggagcgac cggtgccggc gccgagaaag ccgacgcctg ccccaaggac tgcgtttagg 5340 aacaagctgc ctttgacgtt cggcgacttt gacgagcacg aggtcgatgc gttggcctcc 5400 gggattactt tcggagactt cgacgacgtc ctgcgactag gccgcgcggg tgcatatatt 5460 ttctcctcgg acactggcag cggacattta caacaaaaat ccgttaggca gcacaatctc 5520 cagtgcgcac aactggatgc ggtcgaggag gagaaatgt acccgccaaa attggatact 5580 gagagggaga agctgttgct gctgaaaatg cagatgcacc catcggaggc taataagagt 5640 cgataccagt ctcgcaaagt ggagaacatg aaagccacgg tggtggacag gctcacatcg 5700 ggggccagat tgtacacggg agcggacgta ggccgcatac caacatacgc ggttcggtac 5760 ccccgccccg tgtactcccc taccgtgatc gaaagattct caagccccga tgtagcaatc 5820 gcagcgtgca acgaatacct atccagaaat tacccaacag tggcgtcgta ccagataaca 5880 gatgaatacg acgcatactt ggacatggtt gacgggtcgg atagttgctt ggacagagcg 5940 acattctgcc cggcgaagct ccggtgctac ccgaaacatc atgcgtacca ccagccgact 6000 gtacgcagtg ccgtcccgtc accctttcag aacacactac agaacgtgct agcggccgcc 6060 accaagagaa actgcaacgt cacgcaaatg cgagaactac ccaccatgga ctcggcagtg 6120 ttcaacgtgg agtgcttcaa gcgctatgcc tgctccggag aatattggga agaatatgct 6180 aaacaaccta tccggataac cactgagaac atcactacct atgtgaccaa attgaaaggc 6240 ccgaaagctg ctgccttgtt cgctaagacc cacaacttgg ttccgctgca ggaggttccc 6300 atggacagat tcacggtcga catgaaacga gatgtcaaag tcactccagg gacgaaacac 6360 acagaggaaa gacccaaagt ccaggtaatt caagcagcgg agccattggc gaccgcttac 6420 ctgtgcggca tccacaggga attagtaagg agactaaatg ctgtgttacg ccctaacgtg 6480 cacacattgt ttgatatgtc ggccgaagac tttgacgcga tcatcgcctc tcacttccac 6540 ccaggagacc cggttctaga gacggacatt gcatcattcg acaaaagcca ggacgactcc 6600 ttggctctta caggtttaat gatcctcgaa gatctagggg tggatcagta cctgctggac 6660 ttgatcgagg cagcctttgg ggaaatatcc agctgtcacc taccaactgg cacgcgcttc 6720 aagttcggag ctatgatgaa atcgggcatg tttctgactt tgtttattaa cactgttttg 6780 aacatcacca tagcaagcag ggtactggag cagagactca ctgactccgc ctgtgcggcc 6840 ttcatcggcg acgacaacat cgttcacgga gtgatctccg acaagctgat ggcggagagg 6900 tgcgcgtcgt gggtcaacat ggaggtgaag atcattgacg ctgtcatggg cgaaaaaccc 6960 ccatattttt gtgggggatt catagttttt gacagcgtca cacagaccgc ctgccgtgtt 7020 tcagacccac ttaagcgcct gttcaagttg ggtaagccgc taacagctga agacaagcag 7080 gacgaagaca ggcgacgagc actgagtgac gaggttagca agtggttccg gacaggcttg 7140 ggggccgaac tggaggtggc actaacatct aggtatgagg tagagggctg caaaagtatc 7200 ctcatagcca tggccacctt ggcgagggac attaaggcgt ttaagaaatt gagaggacct 7260 gttatacacc tctacggcgg tcctagattg gtgcgttaat acacagaatt ctgattatag 7320 cgcactatta tagcaccgga tcccatggtg agcaagggcg aggagctgtt caccggggtg 7380 gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc 7440 gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc 7500 aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc 7560 agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc 7620 tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag 7680 gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag 7740 gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat 7800 atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc 7860 gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc 7920 cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc 7980 aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc 8040 ggcatggacg agctgtacaa gcccgggtaa ttaattgaat tacatcccta cgcaaacgtt 8100 ttacggccgc cggtggcgcc cgcgcccggc ggcccgtccc tggccgttgc aggccactcc 8160 ggtggctccc gtcgtccccg acttccaggc ccagcagatg cagcaactca tcagcgccgt 8220 aaatgcgctg acaatgagac agaacgcaat tgctcctgct aggcctccca aaccaaagaa 8280 gaagaagaca accaaaccaa agccgaaaac gcagcccaag aagatcaacg gaaaaacgca 8340 gcaagcaaaag aagaaagaca agcaagccga caagaagaag aagaaacccg gaaaaagaga 8400 aagaatgtgc atgaagattg aaaatgactg tatctatgcg gctagccaca gtaacgtagt 8460 gtttccagac atgtcgggca ccgcactatc atgggtgcag aaaatctcgg gtggtctggg 8520 ggccttcgca atcggcgcta tcctggtgct ggttgtggtc acttgcattg ggctccgcag 8580 ataagttagg gtaggcaatg gcattgatat agcaagaaaa ttgaaaacag aaaaagttag 8640 ggtaagcaat ggcatataac cataactgta taacttgtaa caaagcgcaa caagacctgc 8700 gcaattggcc ccgtggtccg cctcacggaa actcggggca actcatattg acacattaat 8760 tggcaataat tggaagctta cataagctta attcgacgaa taattggatt tttattttat 8820 tttgcaattg gtttttaata tttccaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 8880 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaactagt ctgcattaat gaatcggcca 8940 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc 9000 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg 9060 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa 9120 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga 9180 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag 9240 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct 9300 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg 9360 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc 9420 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt 9480 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta 9540 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac 9600 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc 9660 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat 9720 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc 9780 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt 9840 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta 9900 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct 9960 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg 10020 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga 10080 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt 10140 atccgcctcc atccagtcta ttaattgttg ccgggaagct agatagat gttcgccagt 10200 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt 10260 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat 10320 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc 10380 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc 10440 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat 10500 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag 10560 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaaactct caaggatctt 10620 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc 10680 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa 10740 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg 10800 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa 10860 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac 10920 cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtctcgc 10980 gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc 11040 ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg 11100 cgggtgtcgg ggctggctta actatgcggc atcagagcag attgtactga gagtgcacca 11160 ttcgacgctc tcccttatgc gactcctgca ttaggaagca gcccagtagt aggttgaggc 11220 cgttgagcac cgccgccgca aggaatggtg catgcaagga gatggcgccc aacagtcccc 11280 cggccacggg gcctgccacc atacccacgc cgaaacaagc gctcatgagc ccgaagtggc 11340 gagcccgatc ttccccatcg gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg 11400 cgccggtgat gccggccacg atgcgtccgg cgtagaggat ctggctagcg atgaccctgc 11460 tgattggttc gctgaccatt tccgggtgcg ggacggcgtt accagaaact cagaaggttc 11520 gtccaaccaa accgactctg acggcagttt acgagagaga tgatagggtc tgcttcagta 11580 agccagatgc tacacaatta ggcttgtaca tattgtcgtt agaacgcggc tacaattaat 11640 acataacctt atgtatcata cacatacgat ttaggtgaca ctatagatgg cggatgtgtg 11700 acatacacga cgccaaaaga ttttgttcca gctcctgcca cctccgctac gcgagagatt 11760 aaccacccac g 11771 <210> 5 <211> 8192 <212> DNA <213> Artificial sequence <400> 5 atttaggtga cactatagat ggcggatgtg tgacatacac gacgccaaaa gattttgttc 60 cagctcctgc cacctccgct acgcgagaga ttaaccaccc acgatggccg ccaaagtgca 120 tgttgatatt gaggctgaca gcccattcat caagtctttg cagaaggcat ttccgtcgtt 180 cgaggtggag tcattgcagg tcacaccaaa tgaccatgca aatgccagag cattttcgca 240 cctggctacc aaattgatcg agcaggagac tgacaaagac acactcatct tggatatcgg 300 cagtgcgcct tccaggagaa tgatgtcgac atgaaacgag atgtcaaagt cactccaggg 360 acgaaacaca cagaggaaag acccaaagtc caggtaattc aagcagcgga gccattggcg 420 accgcttacc tgtgcggcat ccacagggaa ttagtaagga gactaaatgc tgtgttacgc 480 cctaacgtgc acacattgtt tgatatgtcg gccgaagact ttgacgcgat catcgcctct 540 cacttccacc caggagaccc ggttctagag acggacattg catcattcga caaaagccag 600 gacgactcct tggctcttac aggtttaatg atcctcgaag atctaggggt ggatcagtac 660 ctgctggact tgatcgaggc agcctttggg gaaatatcca gctgtcacct accaactggc 720 acgcgcttca agttcggagc tatgatgaaa tcgggcatgt ttctgacttt gtttattaac 780 actgttttga acatcaccat agcaagcagg gtactggagc agagactcac tgactccgcc 840 tgtgcggcct tcatcggcga cgacaacatc gttcacggag tgatctccga caagctgatg 900 gcggagaggt gcgcgtcgtg ggtcaacatg gaggtgaaga tcattgacgc tgtcatgggc 960 gaaaaacccc catatttttg tgggggattc atagtttttg acagcgtcac acagaccgcc 1020 tgccgtgttt cagacccact taagcgcctg ttcaagttgg gtaagccgct aacagctgaa 1080 gacaagcagg acgaagacag gcgacgagca ctgagtgacg aggttagcaa gtggttccgg 1140 acaggcttgg gggccgaact ggaggtggca ctaacatcta ggtatgaggt agagggctgc 1200 aaaagtatcc tcatagccat ggccaccttg gcgagggaca ttaaggcgtt taagaaattg 1260 agaggacctg ttacacct ctacggcggt cctagattgg tgcgttaata cacagaattc 1320 tgattatagc gcactattat agcaccatga attacatccc tacgcaaacg ttttacggcc 1380 gccggtggcg cccgcgccg gcggcccgtc cctggccgtt gcaggccact cggtggctc 1440 ccgtcgtccc cgacttccag gcccagcaga tgcagcaact catcagcgcc gtaaatgcgc 1500 tgacaatgag acagaacgca attgctcctg ctaggcctcc caaaccaaag aagaagaaga 1560 caaccaaacc aaagccgaaa acgcagccca agaagatcaa cggaaaaacg cagcagcaaa 1620 agaagaaaga caagcaagcc gacaagaaga agaagaaacc cggaaaaaga gaaagaatgt 1680 gcatgaagat tgaaaatgac tgtatcttcg aagtcaaaca cgaaggaaag gtcactgggt 1740 acgcctgcct ggtgggcgac aaagtcatga aacctgccca cgtgaaagga gtcatcgaca 1800 acgcggacct ggcaaagcta gctttcaaga aatcgagcaa gtatgacctt gagtgtgccc 1860 agataccagt tcacatgagg tcggatgcct caaagtacac gcatgagaag cccgagggac 1920 actataactg gcaccacggg gctgttcagt acagcggagg taggttcact ataccgacag 1980 gagcgggcaa accgggagac agtggccggc ccatctttga caacaagggt agggtagtcg 2040 ctatcgtcct gggcggggcc aacgagggct cacgcacagc actgtcggtg gtcacctgga 2100 acaaagatat ggtgactaga gtgaccccg aggggtccga agagtggtcc gccccgctga 2160 ttactgccat gtgtgtcctt gccaatgcta ccttcccgtg cttccagccc ccgtgtgtac 2220 cttgctgcta tgaaaacaac gcagaggcca cactacggat gctcgaggat aacgtggata 2280 ggccagggta ctacgacctc cttcaggcag ccttgacgtg ccgaaacgga acaagacacc 2340 ggcgcagcgt gtcgcaacac ttcaacgtgt ataaggctac acgcccttac atcgcgtact 2400 gcgccgactg cggagcaggg cactcgtgtc atagccccgt agcaattgaa gcggtcaggt 2460 ccgaagctac cgacgggatg ctgaagattc agttctcggc acaaattggc atagataaga 2520 gtgacaatca tgactacacg aagataaggt acgcagacgg gcacgccatt gagaatgccg 2580 tccggtcatc tttgaaggta gccacctccg gagactgttt cgtccatggc acaatgggac 2640 atttcatact ggcaaagtgc ccaccgggtg aattcctgca ggtctcgatc caggacacca 2700 gaaacgcggt ccgtgcctgc agaatacaat atcatcatga ccctcaaccg gtgggtagag 2760 aaaaatttac aattagacca cactatggaa aagagatccc ttgcaccact tatcaacaga 2820 ccacagcgaa gaccgtggag gaaatcgaca tgcatatgcc gccagatacg ccggacagga 2880 cgttgctatc acagcaatct ggcaatgtaa agatcacagt cggaggaaag aaggtgaaat 2940 acaactgcac ctgtggaacc ggaaacgttg gcactactaa ttcggacatg acgatcaaca 3000 cgtgtctaat agagcagtgc cacgtctcag tgacggacca taagaaatgg cagttcaact 3060 caccttcgt cccgagagcc gacgaaccgg ctagaaaagg caaagtccat atcccattcc 3120 cgttggacaa catcacatgc agagttccaa tggcgcgcga accaaccgtc atccacggca 3180 aaaagaagt gacactgcac cttcacccag atcatcccac gctcttttcc taccgcacac 3240 3300 ccgtaccagt ggacgggatg gagtaccact ggggaaaaa cgacccagtg aggctttggt 3360 ctcaactcac cactgaaggg aaaccgcacg gctggccgca tcagatcgta footctact 3420 atgggcttta cccggccgct acagtatccg cggtcgtcgg gatgagctta ctggcgttga 3480 tatcgatctt cgcgtcgtgc tacatgctgg ttgcggcccg cattaagtgc ttgacccctt 3540 atgctttaac accaggagct gcagttccgt ggacgctggg gatactctgc tgcgccccgc 3600 gggcgcacgc agctagtgtg gcagagacta tggcctactt gtgggaccaa aaccaagcgt 3660 tgttctggtt ggagtttgcg gcccctgttg cctgcatcct catcatcacg tattgcctca 3720 gaaacgtgct gtgttgctgt aagagccttt cttttttagt gctactgagc ctcggggcaa 3780 ccgccagagc ttacgaacat tcgacagtaa tgccgaacgt ggtggggttc ccgtataagg 3840 ctcacattga aaggccagga tatagcccc tcactttgca gatgcaggtt gttgaaacca 3900 gcctcgaacc aacccttaat ttggaataca taacctgtga gtacaagacg gtcgtcccgt 3960 cgccgtacgt gaagtgctgc ggcgcctcag agtgctccac taaagagaag cctgactacc 4020 aatgcaaggt ttacacaggc gtgtacccgt tcatgtgggg aggggcatat tgcttctgcg 4080 actcagaaaa cacgcaactc agcgaggcgt acgtcgatcg atcggacgta tgcaggcatg 4140 atcacgcatc tgcttacaaa gcccatacag catcgctgaa ggccaaagtg agggttatgt 4200 acggcaacgt aaaccagact gtggatgttt acgtgaacgg agaccatgcc gtcacgatag 4260 ggggtactca gttcatattc gggccgctgt catcggcctg gaccccgttc gacaacaaga 4320 tagtcgtgta caaagacgaa gtgttcaatc aggacttccc gccgtacgga tctgggcaac 4380 cagggcgctt cggcgacatc caaagcagaa cagtggagag taacgacctg tacgcgaaca 4440 cggcactgaa gctggcacgc ccttcacccg gcatggtcca tgtaccgtac acacagacac 4500 cttcagggtt caaatattgg ctaaaggaaa aagggacagc cctaaatacg aaggctcctt 4560 ttggctgcca aatcaaaacg aaccctgtca gggccatgaa ctgcgccgtg ggaaacatcc 4620 ctgtctccat gaatttgcct gacagcgcct ttacccgcat tgtcgaggcg ccgaccatca 4680 ttgacctgac ttgcacagtg gctacctgta cgcactcctc ggatttcggc ggcgtcttga 4740 cactgacgta caagaccgac aagaacgggg actgctctgt acactcgcac tctaacgtag 4800 ctactctaca ggaggccaca gcaaaagtga agacagcagg taaggtgacc ttacacttct 4860 ccacggcaag cgcatcacct tcttttgtgg tgtcgctatg cagtgctagg gccacctgtt 4920 cagcgtcgtg tgagcccccg aaagaccaca tagtcccata tgcggctagc cacagtaacg 4980 tagtgtttcc agacatgtcg ggcaccgcac tatcatgggt gcagaaaatc tcgggtggtc 5040 tgggggcctt cgcaatcggc gctatcctgg tgctggttgt ggtcacttgc attgggctcc 5100 gcagataagt tagggtaggc aatggcattg atatagcaag aaaattgaaa acagaaaaag 5160 ttagggtaag caatggcata taaccataac tgtataactt gtaacaaagc gcaacaagac 5220 ctgcgcaatt ggccccgtgg tccgcctcac ggaaactcgg ggcaactcat attgacacat 5280 taattggcaa taattggaag cttacataag cttaattcga cgaataattg gatttttatt 5340 ttattttgca attggttttt aatatttcca aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 5400 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaac tagtctgcat taatgaatcg 5460 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg 5520 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa 5580 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc 5640 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc 5700 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat 5760 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc 5820 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct 5880 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg 5940 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc 6000 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga 6060 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactaga gacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa aggttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc 6240 attacgcg cagaaaaaaa ggatctcaag aagatccttt gatctttct acggggtctg 6300 acgctcagtg gaacgaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg 6540 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc 6600 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc 6720. cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc 6840 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt 6900 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc 6960 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt 7020 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata 7080 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga 7140 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag 7200 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa 7260 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt 7320 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga 7380 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag 7440 aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc 7500 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 7620 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 7680 accattcgac gctctccctt atgcgactcc tgcattagga agcagcccag tagtagttg 7740 aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagattggc gcccaacagt cccccggcca cggggcctgc cccccc acgccgaaac aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc aaccgcacct 7920 gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatctggct agcgatgacc 7980 ctgctgattg gttcgctgac catttccggg tgcgggacgg cgttaccaga aactcagaag gttcgtccaa ccaaaccgac tctgacggca gttcgaga gagatgatag ggtctgcttc attack atgctacaca attaggcttg cgttagacg cggctacaat father ccttatgtat catacacata cg

Claims

1. A method for preparing pSFV-p54 replicon particles, characterized in that: The following steps are involved: (1) Using pSFV-sp6-EGFP plasmid and flag-p54 gene as materials, constructing pSFV-sp6-p54 plasmid; the flag-p54 gene is shown in SEQ ID NO: 1, and the pSFV-sp6-EGFP plasmid sequence is shown in SEQ ID NO: 4; (2) linearizing the SFV-helper plasmid and the pSFV-sp6-p54 plasmid respectively and performing in vitro transcription to obtain corresponding mRNA, and co-electrotransfecting the obtained mRNA into BHK cells or Vero cells to obtain pSFV-p54 replicon particles; the sequence of the SFV-helper plasmid is shown in SEQ ID NO: 5; The step (1) comprises the following steps: using BamHI / SmaI endonucleases to double-digest the pSFV-sp6-EGFP plasmid, and homologously recombining the digestion product with the flag-p54 gene to obtain a recombinant product; introducing the recombinant product into competent cells, identifying, and extracting to obtain the pSFV-sp6-p54 plasmid; Using primers as shown in SEQ ID NO: 2-3 to identify the recombinant product; The competent cells are DH5α cells or Trans1-T1 Phage Resistant chemical competent cells; Step (1) also includes the steps of performing restriction enzyme digestion, identification and sequencing on the pSFV-sp6-p54 plasmid; In step (2), the pSFV-sp6-p54 plasmid is linearized by digestion with SpaI, and the SFV-helper plasmid is linearized by digestion with SpeI.

2. A pSFV-p54 replicon particle, characterized in that Prepared by the preparation method described in claim 1.

3. Use of the pSFV-p54 replicon particles according to claim 2 in the preparation of a product for expressing African swine fever virus p54 protein.

4. Use of the pSFV-p54 replicon particles according to claim 2 in the preparation of African swine fever virus RNA vaccine.

5. An African swine fever vaccine, characterized in that: Comprising the pSFV-p54 replicon particles according to claim 2.

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