A foot-and-mouth disease virus capsid fusion protein solublely expressed in Escherichia coli and its preparation method

Abstract

This invention provides a foot-and-mouth disease virus capsid fusion protein that can be solublely expressed in *Escherichia coli* and its preparation method. The fusion protein contains foot-and-mouth disease virus capsid protein P1-2A, a chaperone protein, and n arginine residues; wherein the chaperone protein is selected from one of TF protein, ProS2 protein, GB1 protein, MBP protein, GST protein, and SUMO protein, and n = 5-10. This invention enables the direct expression of soluble P1-2A in *E. coli*, which ensures that the elements (VP0, VP1, VP3) in the virus-like particles are expressed in approximately equimolar proportions.

Description

Technical Field

[0001] This invention relates to a soluble and soluble foot-and-mouth disease virus capsid fusion protein expressed in Escherichia coli, its preparation method and application, belonging to the field of veterinary biological products technology. Background Technology

[0002] Foot-and-mouth disease (FMD) is an acute, febrile, and highly contagious animal disease caused by the foot-and-mouth disease virus. It is an acute and deadly infectious disease of cloven-hoofed animals that can spread rapidly over long distances. Pigs, cattle, sheep, and other cloven-hoofed animals are susceptible. The World Organisation for Animal Health (OIE) lists FMD as the top legally notifiable animal infectious disease, and my country also ranks it first among Class I animal infectious diseases.

[0003] Foot-and-mouth disease (FMD) is caused by the foot-and-mouth disease virus, which is a single-stranded RNA virus. Currently, based on serotypes, it is classified into seven types: A, O, C, Asia1, SAT1, SAT2, and SAT3. Although the symptoms caused by each type of virus are the same, there is no cross-immunity between different serotypes. Animals that have recovered from FMD or have been immunized can still be infected with other serotypes of the virus and develop the disease.

[0004] FMDV has an icosahedral symmetry structure, a diameter of 25 nm, and contains a positively polar single-stranded RNA molecule composed of approximately 8500 nucleotides. The protein encoded by this RNA molecule is as follows: Figure 1 As shown, during maturation, protein P1 is cleaved by protease 3C with the assistance of protein 2A into three proteins called VP0, VP1, and VP3. Single copies of VP0, VP1, and VP3 form the 5S primordium. Five copies of the 5S primordium subsequently form 12S pentamers, and twelve 12S pentamers assemble into an icosahedral 75S empty capsid. After the formation of the 5S primordium, during RNA encapsulation, VP0 cleaves into VP4 and VP2. However, a viral capsid can also form when the RNA molecule is not present inside the viral capsid; this empty viral capsid can also be called a foot-and-mouth disease virus-like particle (VLP).

[0005] Currently, traditional vaccines are all inactivated virus vaccines, but these vaccines have many shortcomings in the prevention and control of foot-and-mouth disease, mainly in the following aspects: it is difficult to differentiate between infected animals and immunized animals; during the production process of vaccines, a large number of natural viruses are proliferated, requiring strict facilities to prevent the spread of the virus, but there is still a possibility of virus escape and incomplete inactivation leading to the shedding of the virus.

[0006] Foot-and-mouth disease virus-like particles (VLPs) possess the same immunological characteristics as intact viruses but do not pose a risk of viral shedding. Therefore, the production of empty capsids for FMDV using various expression systems has been a research hotspot both domestically and internationally. Currently, extensive research has been conducted on two main expression systems: insect baculovirus expression systems and Escherichia coli expression systems. However, the insect baculovirus expression system suffers from relatively low yields, high cell culture costs, and the risk of baculovirus contamination (requiring inactivation, but incomplete inactivation also carries the risk). Therefore, significant challenges remain for its industrial application. The E. coli expression system, on the other hand, offers relatively high yields and has very low culture costs, making it a more promising candidate for industrial application.

[0007] Currently, eukaryotic expression systems generally express P1-2A directly, followed by enzymatic digestion with PP3C enzyme and subsequent self-assembly into VLPs. However, due to the toxic side effects of PP3C enzyme, the expression level is low. There are no reports of direct soluble expression of P1-2A in *E. coli*; direct expression of P1-2A in *E. coli* results in inclusion bodies with folding errors. Current *E. coli* expression systems primarily involve constructing three separate expression vectors (VP0, VP3, and VP2) and then merging them into a single expression vector via tandem. These vectors are then transformed into *E. coli* for co-expression and co-purification. During vector construction, a SUMO tag protein and a suitable SUMO restriction site are inserted at the 5' end of the target nucleotide to improve the soluble expression of the target protein (see CN201410609214.9 for details). This preparation method has the following problems: the expression level and molar ratio between proteins cannot be accurately controlled during expression (although the separately constructed vectors are tandemly incorporated into one expression vector, each target protein has a separate ribosome binding site during expression, which will prevent the subsequent target proteins from being expressed in a 1:1:1 molar ratio), which is not conducive to subsequent VLP assembly and quality control; the enzyme suitable for cleaving the SUMO restriction site (ULP1 (Ubl-specific protease 1)) is very expensive, resulting in relatively high costs in industrial production; in addition, it cannot solve the problem of complete solubility, and the presence of inclusion bodies affects the protein yield.

[0008] No effective solution has yet been found to address the problem of inclusion bodies. Summary of the Invention

[0009] Based on existing technologies, in order to overcome many defects in existing technologies (such as high preparation cost of insect baculovirus, inability to directly solublely express P1-2A in Escherichia coli, inability to control the equimolar ratio expression of the target protein, and relatively high cost), this invention provides a method for directly solublely preparing foot-and-mouth disease virus capsid fusion protein in Escherichia coli and the corresponding preparation method.

[0010] According to one aspect of the present invention, the present invention provides a foot-and-mouth disease virus capsid fusion protein that is solublely expressed in Escherichia coli, the foot-and-mouth disease virus capsid fusion protein containing foot-and-mouth disease virus capsid proteins P1-2A, chaperone proteins and n arginine residues; wherein: the chaperone protein is selected from one of TF protein, ProS2 protein, GB1 protein, MBP protein, GST protein and SUMO protein, and n = 5-10.

[0011] In a preferred embodiment of the present invention, preferably, the chaperone protein in the fusion protein is located at the N-terminus of the foot-and-mouth disease virus capsid protein P1-2A, and also contains an enzyme cleavage site protein that removes the chaperone protein.

[0012] In a preferred embodiment of the present invention, preferably, n arginine residues in the fusion protein are located at the C-terminus of the foot-and-mouth disease virus capsid protein P1-2A, where n = 6.

[0013] In a preferred embodiment of the present invention, the fusion protein is preferably a chaperone protein-enzyme cleavage site-P1-2A-6Arg.

[0014] In a preferred embodiment of the present invention, the C-terminus of the fusion protein preferably contains 6 histidine residues.

[0015] In a preferred embodiment of the present invention, preferably, P1-2A in the fusion protein is a derivative protein that has one or more amino acids replaced, deleted, or added based on its amino acid sequence and has the same function.

[0016] In a preferred embodiment of the present invention, preferably, the enzyme cleavage site protein in the fusion protein is selected from one of the following: TEV protease cleavage site, Factor Xa protease cleavage site, HRV 3C protease cleavage site, and Enterokinase cleavage site.

[0017] In a preferred embodiment of the present invention, preferably, the enzyme cleavage site protein in the fusion protein is a TEV protease cleavage site, and the amino acid sequence of the TEV protease cleavage site is shown in SEQ ID NO9.

[0018] In a preferred embodiment of the present invention, the chaperone protein is preferably a TF protein, the amino acid sequence of which is shown in SEQ ID NO.8.

[0019] According to a second aspect of the present invention, the present invention also provides a method for preparing the foot-and-mouth disease virus capsid fusion protein, the method comprising the following steps:

[0020] 1) Insert the gene coding sequence of a chaperone protein and the gene coding sequence of an enzyme cleavage site into the 5' end of the nucleotide sequence of the foot-and-mouth disease virus capsid protein P1-2A after codon optimization, to obtain the nucleotide sequence opti-chaperone protein-cleavage site-P1-2A-nArg.

[0021] 2) Insert a gene coding sequence of 6 histidines into the 3' end of the nucleotide sequence of opti-chaperone protein-restriction site-P1-2A-nArg described in step 1) to obtain the nucleotide sequence of opti-chaperone protein-restriction site-P1-2A-nArg-6His.

[0022] 3) Insert the nucleotide sequence of opti-chaperone protein-restriction site-P1-2A-nArg-6His from step 2) into a prokaryotic expression plasmid, wherein the prokaryotic expression plasmid is a pCold plasmid or a pET plasmid, to obtain a recombinant vector containing the nucleotide sequence of opti-chaperone protein-restriction site-P1-2A-nArg-6His;

[0023] 4) Transform the recombinant vector described in step 3) into Escherichia coli to obtain an E. coli strain containing the nucleotide sequence of the recombinant vector containing the opti-chaperone protein-cleavage site-P1-2A-nArg-6His;

[0024] 5) The *E. coli* strain from step 4) was fermented and cultured. After IPTG induction, the soluble chaperone protein-restriction site-P1-2A-nArg-6His fusion protein was expressed; and

[0025] 6) The supernatant after the E. coli cells were recovered from fermentation and lysed. The chaperone protein-enzyme site-P1-2A-nArg-6His fusion protein was obtained by nickel column affinity chromatography.

[0026] In a preferred embodiment of the present invention, preferably, the nArg in the fusion protein is n = 5-10, and more preferably 6.

[0027] Compared with existing technologies, the expression sequence, expression vector, and corresponding preparation method disclosed in this invention overcome the defects of existing technologies, such as the high cost of preparing insect baculoviruses (we use Escherichia coli to prepare virus-like particles, firstly because the expression yield is high, reaching 400 mg / L, and secondly because the fermentation culture cycle of Escherichia coli is short and the cost is much lower than that of cell culture), the inability to directly express P1-2A in soluble Escherichia coli, the inability to control the equimolar ratio of the target protein, and the relatively high cost. However, this invention can directly express P1-2A in soluble Escherichia coli, which can ensure that the elements (VP0, VP1, VP3) in the virus-like particles are expressed in a basically equimolar ratio. Attached Figure Description

[0028] Figure 1 Indicates the molecular structure of FMDV;

[0029] Figure 2 pCold-opti-TF- TEV -P1-2A-6Arg-6His double enzyme digestion identification results;

[0030] Figure 3 This indicates that SDS-PAGE detects TF- TEV Results of induction of expression of the P1-2A-6Arg-6His fusion protein;

[0031] Figure 4 This indicates that SDS-APGE detects TF- TEV The result of purification of the P1-2A-6Arg-6His fusion protein;

[0032] Figure 5 This indicates that SDS-PAGE detects TF- TEV Results of induction of expression of the P1-2A-6His fusion protein;

[0033] Figure 6 This indicates the results of SDS-PAGE detection of GST-2TEV-P1-2A-6Arg-6His fusion protein induced expression;

[0034] Figure 7 This indicates the results of SDS-PAGE detection of SUMO-TEV-P1-2A-6Arg-10His fusion protein induced expression.

[0035] Figure 8 This indicates the results of SDS-PAGE detection of TF-TEV-P1-2A-6Arg-6His fusion protease digestion;

[0036] Figure 9 This indicates that SDS-PAGE detects TF- TEVThe result after purification by digestion of the P1-2A-6Arg-6His fusion protease. Detailed Implementation

[0037] The present invention will be further described below with reference to the accompanying drawings and embodiments. The embodiments of the present invention are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention.

[0038] All reagents used were domestically produced and commercially available products.

[0039] Example 1 TF- TEV Preparation of -P1-2A-6Arg-6His

[0040] 1.1 Selection of foot-and-mouth disease P1-2A protein

[0041] The foot-and-mouth disease virus (FMDV) structural protein precursor P1-2A, after cleavage with PP3C, forms capsid proteins VP0, VP3, and VP1, which then self-assemble into VLPs. Co-expression of P1-2A and its viral gene-encoded 3C protease (PP3C) in eukaryotic systems (such as insect baculoviruses) is currently the main technique for preparing FMDV-like particles. Similarly, in prokaryotic systems, FMDV VLPs can also be self-assembled intracellularly by co-expressing P1-2A and PP3C; or P1-2A and PP3C can be expressed separately, cleaved, and then FMDV VLPs can be self-assembled in vitro. However, there are currently no reports of soluble expression of P1-2A directly in prokaryotic expression systems as in eukaryotic systems, which is an important technical problem that this invention aims to solve.

[0042] 1.2 Codon Optimization of Foot-and-Mouth Disease P1-2A Protein

[0043] Using the O / GSLX / 2010 sequence (GenBank: JQ900581.1), a prevalent foot-and-mouth disease (FMD) strain in China in recent years, as a template, our laboratory performed codon optimization on the nucleotide sequence of FMD P1-2A, obtaining the opti-P1-2A sequence, as shown in SEQ ID NO.1. This work was commissioned to Nanjing GenScript Biotech Co., Ltd. 21.6% of the nucleotide sequence differed before and after optimization. The nucleotide sequence of the P1-2A protein before codon optimization is shown in SEQ ID NO.2.

[0044] 1.3 Construction and Validation of Expression Vectors

[0045] 1.3.1 PCR amplification of the target fragment

[0046] 1.3.1.1 PCR reaction

[0047] (1) Primer design and synthesis

[0048] Upstream primer: 5'- accctcgagGAGAACCTGTACTTCCAGGGCGCGGGTCAAAGC-3'

[0049] Downstream primer: 5'-CCCAAGCTTTCAGTGATGATGATGATGATGTCGCCTTCGACGCCTGCGGCCCGGGTTGCTCTCAACATC-3'

[0050] (2) The sample loading system is 50 μL, as shown in the table below:

[0051]

[0052] 1.3.1.2 PCR amplification program:

[0053]

[0054] 1.3.1.3 Gel recovery of PCR products: Follow the instructions in the kit (kit purchased from Tiangen Biotech Co., Ltd., catalog number DP214-02).

[0055] 1.3.2 Double enzyme digestion reaction of PCR products and vector

[0056] (1) Label the 200μL PCR tubes to be used, and add samples and mix them according to the table below: 50μL reaction system

[0057]

[0058] (2) Place the PCR tube from step (1) in a constant temperature water bath at the optimal temperature of the corresponding enzyme and incubate for 1-2 hours.

[0059] Double enzyme digestion product gel recovery: Take out the above double enzyme digestion system and perform agarose gel electrophoresis to recover the DNA fragments therein, using the same method as the PCR product gel recovery in 1.3.1.3.

[0060] 1.3.3 Connection Reaction

[0061] (1) Prepare several clean 200μL PCR tubes, label them, and place them on the EP tube rack for later use.

[0062] (2) Add the sample to a 200 μL PCR tube and mix according to the table below.

[0063]

[0064] (3) After completing the sample addition according to the table in step (2), place each 10 μl reaction system in a PCR instrument at 16℃ for 2 h;

[0065] (4) Remove the EP tube from step (3) and store it at 4°C.

[0066] 1.3.4 Transformation Reaction

[0067] Add 10 μL of ligation reaction solution rapidly to 100 μL of competent cells, mix well by pipetting, and incubate on ice for 30 min. Remove the sample tube, place it in a 42℃ water bath for 100 s, and then immediately incubate on ice for 2 min. Remove the sample tube, add 600 μL of liquid LB medium to the sample tube in a clean bench, and then place the sample tube in a 37℃ constant temperature shaker at 220 rpm / min for 1 h. Plate the transformation: Remove the sample tube from the previous step, centrifuge at room temperature at 8,000 rpm / min for 2 min, remove 600 μL of supernatant, resuspend the bacterial cells at the bottom of the tube in the remaining supernatant, place the resuspended bacterial solution in the center of the corresponding transformation plate, and spread the bacterial solution evenly in the center of the transformation plate using a spreading stick. Place the plate from the previous step upright in a biochemical constant temperature incubator and incubate at 37℃ for 1 h, then invert the transformation plate and incubate for 15 h. Observe the transformation results.

[0068] 1.3.5 Plasmid extraction and double enzyme digestion identification

[0069] 1.3.5.1 Plasmid extraction: Perform the procedure according to the instructions of the kit (the kit was purchased from Sangon Biotech (Shanghai) Co., Ltd., catalog number SK8192).

[0070] 1.3.5.2 Double enzyme digestion identification

[0071] (1) Label the 200μL PCR tubes to be used and add samples according to the table below: 10μL reaction system

[0072]

[0073] (2) Place the 200 μL PCR tube and 10 μL reaction system from step (1) in a 37°C constant temperature water bath and incubate for 1 h.

[0074] (3) Perform agarose gel electrophoresis on the double enzyme digestion system samples from step (2) to check if the inserted fragment size is correct; the experimental results are shown in […]. Figure 2 As shown, 1, 2, 3, and 4 are pCold-opti-TF- TEV The plasmid -P1-2A-6Arg-6His was double-digested with HindIII / Xho I, and the band sizes were approximately 2298bp and 5733bp, indicating that the plasmid digestion was correct.

[0075] (4) Select a clone with the correct insertion fragment and send it to a sequencing company for sequencing.

[0076] 1.4 TF- TEV-P1-2A-6Arg-6His fusion protein expression

[0077] 1.4.1 Transformation of Escherichia coli BL21(DE3)

[0078] Add 1 μl of plasmid to 100 μl of BL21(DE3) competent cells and incubate on ice for 30 min; heat shock at 42℃ for 90 s; incubate on ice for 2 min; add 500 μl of antibiotic-free LB medium in a clean bench; shake at 37℃ and 220 rpm for 1 h; plate 100 μl of bacterial culture on kanamycin-resistant LB agar plates and incubate overnight at 37℃; pick single clones and add glycerol to store at -80℃ for later use.

[0079] 1.4.2 Small-scale induction of expression

[0080] Activation of strains from glycerol tube preservation tubes: Take E. coli BL21 pCold-opti-TF- TEV Thaw the glycerol culture tube of strain P1-2A-6Arg-6His and streak the bacterial suspension from the glycerol tube onto an ampicillin-resistant plate (100 μg / mL) using an inoculation loop. Incubate overnight at 37°C. Activation: Pick a single colony from the cultured plate and transfer it to 3 mL of ampicillin-resistant LB medium. Incubate at 37°C and 220 rpm for 5–6 h with a shaker until the OD600 reaches 0.5–0.8. Fermentation inoculation: Inoculate 150 μL of the activated bacterial suspension into 15 mL of ampicillin-resistant LB medium and incubate at 37°C and 220 rpm. Cooling induction: When the OD600 reaches 0.8… 600 When the bacterial culture reaches 0.6-0.8, take 5 mL of the culture, centrifuge at 12000 rpm for 5 min, and store the cells at -20℃; this is the "pre-induction" stage. Place the remaining culture in an ice-water bath for 10 min, then add 2 μL of 1M IPTG to a final concentration of 0.2 mM IPTG. Lower the shaker temperature to 20℃ and induce fermentation for 9 h. Cell collection: After fermentation, measure the OD. 600 Collect an equal amount of bacterial cells as before induction, centrifuge at 12000 r / min for 5 min, and store the collected bacterial cells at -20℃; this is the "post-induction" state.

[0081] The results of SDS-PAGE detection of induced expression are as follows: Figure 3 As shown, 1 is the marker, 2 is the supernatant before induction, 3 is the precipitate before induction, 4 is the supernatant after induction, and 5 is the precipitate after induction. The arrows point to TF- TEV -P1-2A-6Arg-6His fusion protein. As can be seen from the figure, our prepared TF- TEV -P1-2A-6Arg-6His fusion protein is expressed in a soluble form.

[0082] Additionally, following the steps in 1.3, we also constructed pCold-opti-TF- TEV The plasmid P1-2A-6His was used, and after transformation and low-level induction expression, TF-1 was found to be generated using this plasmid. TEV The P1-2A-6His fusion protein is expressed in soluble form (approximately 70%), but 30% is expressed as inclusion bodies. Therefore, in subsequent experiments and production, we selected pCold-opti-TF- TEV -P1-2A-6Arg-6His positive strain.

[0083] 1.4.3 Extensive Induction of Expression

[0084] Activation of strains from glycerol tube preservation tubes: Take pCold-opti-TF- TEV Thaw the glycerol preservation tubes of strain P1-2A-6Arg-6His. Use an inoculation loop to pick up the bacterial suspension from the glycerol tubes and streak it onto an ampicillin-resistant plate. Incubate overnight at 37°C. Activation: Pick single colonies from the cultured plate and transfer them to 3 mL of ampicillin-resistant LB medium. Incubate at 37°C and 220 rpm for 5–6 h until the OD600 reaches 0.5–0.8. Seed culture: Inoculate 150 μL of the activated bacterial suspension into 150 mL of ampicillin-resistant LB medium. Incubate at 37°C and 220 rpm for 9–10 h. Fermentation medium preparation: Prepare fermentation medium component 1 according to the fermentation medium formula in a 5 L fermenter. Install the combined fermenter. Prepare fermentation medium component 2 and feed medium in blue-mouth bottles and autoclave at 121°C for 20 min. Fermentation parameters: Agit 400r / min; Temperature 37℃; pH 7.00; DO 40; Air 100%; Gasflow 2.0. Fermentation inoculation: Add 450mL of fermentation medium component 2, 1mL of fermentation medium component 3, 200μL of antifoaming agent, and 3mL of ampicillin antibiotic (100mg / mL) to the fermenter through the inoculation port; inoculate 150mL of the prepared seed culture into 3L of fermentation medium for scale-up culture in the fermenter, and culture for 5-6 hours until OD reaches zero. 600 Values ​​were adjusted to 12-14. Cooling induction: Temperature parameters were set, and the fermenter temperature was lowered to 20℃. A sample was taken, and 0.9 mL of IPTG (1M) was added until the final IPTG concentration was 0.3 mmol / L. Induction culture was carried out at 20℃ for 8 hours. Fed culture: When the OD600 reached 17-19, fed culture medium was continuously added at a rate of 5% (components 1 and 2 of the fed culture medium were mixed first). Cell collection: After fermentation, the fermentation broth was collected, centrifuged at 8000 r / min for 10 min, and the collected cells were stored at -20℃.

[0085] The culture media used in the above process are as follows:

[0086] Fermentation medium component 1: yeast extract 10 g / L, tryptone 20 g / L, KH2PO4 1.14 g / L, K2HPO4 0.9 g / L, (NH4)2SO4 3.0 g / L, MgSO4·7H2O 0.3 g / L, NaCl 5 g / L, pH 7.0; Fermentation medium component 2: glycerol 30 g / L; Fermentation medium component 3: VB1 2 mg / L; Fed medium component 1: yeast extract 16.67 g / L; tryptone 33.33 g / L; Fed medium component 2: glycerol 100 g / L.

[0087] 1.5 TF- TEV Purification of P1-2A-6Arg-6His fusion protein

[0088] Cell disruption: Add lysis buffer (10 ml / g wet weight) to the cells and mix thoroughly. Pour the cell sample into the sample chamber of a cell homogenizer, and prepare a beaker for sample collection at the outlet. One cycle is defined as 90% of the sample flowing out of the outlet. After one cycle, return the sample collected in the beaker to the sample chamber. Perform a total of 4 cycles. Centrifugation: Aliquot the completely disrupted sample from the previous step into 250 mL Beckman centrifuge tubes and centrifuge at 12,000 rpm, 4°C for 30 min. Use the supernatant as the loading sample. Nickel column equilibration: Equilibrate with 2–3 column volumes (CV) of ultrapure water, removing 20% ​​of the ethanol preservation solution; then equilibrate with lysis buffer for 2–3 column volumes (CV). Loading: Take the supernatant from the disrupted cells and mix it with the nickel column packing material. Mix thoroughly on a roller bottle for 1 h, then perform flow-through and collect the flow-through liquid. Endotoxin removal: Wash the column with 20 column volumes (CV) of washing buffer component 1 to remove endotoxins. Removal of Triton X-114: Wash the column with 10 column volumes (CV) of elution buffer component 1 (Triton X-114-free) to reduce Triton X-114 residue. Elution: Wash with 50 mM imidazole elution buffer until no blue light is detected by Coomassie Brilliant Blue G250; elute the target protein with 500 mM imidazole elution buffer until no blue light is detected by Coomassie Brilliant Blue G250, and collect the target protein.

[0089] Results: The purification results are as follows. Figure 4 As shown, 1 is the marker, and 2 is the purified TF- TEV -P1-2A-6Arg-6His fusion protein. As can be seen from the figure, the purified TF- TEVThe SDS-PAGE purity of the P1-2A-6Arg-6His fusion protein can reach over 85%; calculations show that the expression level can reach over 400 mg / L without optimized fermentation conditions.

[0090] The solutions required for protein purification are as follows:

[0091] Bacterial lysis buffer: Composition: 50 mM NaH₂PO₄, 500 mM NaCl, 1 mM β-ME, 0.05% Tween-20, 0.2% Triton X-114, adjusted to pH 8.0. Wash buffer 1: 50 mM NaH₂PO₄, 1 M NaCl, 0.05% Tween-20, 1 mM β-ME, 0.2% Triton X-114, adjusted to pH 7.0. Elution buffer 1: 50 mM NaH₂PO₄, 500 mM NaCl, adjusted to pH 8.0. Elution buffer 2: 50 mM NaH₂PO₄, 500 mM NaCl, 500 mM imidazole, adjusted to pH 8.0.

[0092] Example 2 TF- TEV Preparation of -P1-2A-6His

[0093] Following the steps in Example 1, we also constructed pET-opti-TF- TEV -P1-2A-6His plasmid and pCold-opti-TF- TEV The plasmid P1-2A-6His was used, and the plasmid was transformed and induced to express at a low level. It was found that TF-... prepared using this plasmid... TEV The -P1-2A-6His fusion protein is expressed in soluble form (approximately 70%), but 30% is expressed as inclusion bodies. Figure 5 As shown.

[0094] Example 3: Preparation of GST-2TEV-P1-2A-6Arg-6His

[0095] Following the steps in Example 1, we also constructed the pGEX-4T-2TEV-P1-2A-6Arg-6His plasmid, and transformed and induced small-scale expression of this plasmid. We found that nearly 25% of the GST-2TEV-P1-2A-6Arg-6His fusion protein prepared using this plasmid was soluble in the supernatant, but 75% was expressed as inclusion bodies. Figure 6 As shown.

[0096] Example 4: Preparation of SUMO-TEV-P1-2A-6Arg-10His

[0097] Following the steps in Example 1, we also constructed pCold-SUMO- TEV The plasmid P1-2A-6Arg-10His was transformed and induced to express at low levels. It was found that nearly 50% of the SUMO-TEV-P1-2A-6Arg-10His fusion protein prepared using this plasmid was soluble in the supernatant, but 50% was expressed as inclusion bodies. Figure 7 As shown.

[0098] Example 5 TF- TEV Enzyme digestion and purification of -P1-2A-6Arg-6His

[0099] The TF-TEV-P1-2A-6Arg-6His fusion protein obtained in Example 1 was mixed with 5% PP3C enzyme (w / w) and 5% TEV enzyme (w / w) according to the mass of the fusion protein. The mixture was then digested at 30±1℃ for 16±2 hours. The digested product was then purified by molecular sieve chromatography after dialysis (50mM phosphate buffer, pH 8.0, 0.5M NaCl).

[0100] Take 20 μL of the sample from this enzyme digestion method, add 5 μL of 5x loading buffer, mix well, and incubate at 80°C for 10 min. Then, take 3 μL, 5 μL, and 10 μL of the sample and electrophoresis them on a 12% SDS-polyacrylamide gel at 120 V for 90 min. Subsequently, stain with Coomassie Brilliant Blue to visualize the electrophoretic bands. The electrophoresis results are shown below. Figure 8 As shown in Figure 9, where M is the marker, 1 is the PP3C enzyme control, 2 is the TEV enzyme control, 3 is the sample before enzyme digestion, and 4 is the sample after enzyme digestion. From Figure 8 As can be seen, the precursor protein P1-2A is completely cleaved, and the foot-and-mouth disease virus capsid proteins (VP0, VP1, VP3) are stable after cleavage. Figure 9 After purification to remove impurities such as TF, the ratio of VP0:VP1:VP3 was approximately 1:1:1, indicating an equimolar expression ratio.

[0101] The present invention has been illustrated by the above embodiments; however, it should be understood that the present invention is not limited to the specific examples and embodiments described herein. The purpose of including these specific examples and embodiments is to assist those skilled in the art in practicing the present invention. Any person skilled in the art can readily make further improvements and modifications without departing from the spirit and scope of the present invention; therefore, the present invention is limited only by the content and scope of the claims, and is intended to cover all alternatives and equivalents included within the spirit and scope of the present invention as defined by the appended claims. sequence list <110> Zhejiang Hailong Biotechnology Co., Ltd. <120> A foot-and-mouth disease virus capsid fusion protein solublely expressed in Escherichia coli and its preparation method <160> 9 <170> SIPOSequenceListing 1.0 <210> 4 <211> 2256 <212> DNA <213> The codon-optimized nucleotide sequence (DNA) of the P1-2A protein. <400> 4 ggcgcgggtc aaagcagccc ggcgaccggt agccagaacc aaagcggtaa caccggcagc 60 atcatcaaca actactacat gcagcaatac cagaacagca tggacaccca actgggtgat 120 aacgcgatca gcggtggcag caacgaaggc agcaccgata ccaccagcac ccacaccacc 180 aacacccaga acaacgactg gttcagcaag ctggcgagca gcgcgttcag cggtctgttt 240 ggcgcgctgc tggcggataa gaaaaccgag gaaaccaccc tgctggagga ccgtatcctg 300 accacccgta acggtcacac caccagcacc acccagca gcgtgggtat tacccacggc 360 tacgcgaccg cggaagattt tgtgagcggt ccgaacacca gcggtctgga gacccgtgtt 420 atccaagcgg aacgtttctt taagacccac ctgttcgact gggtgaccag cgatccgttt 480 ggccgttgct acctgctgga gctgccgacc gaccacaaag gtgtttatgg cagcctgacc 540 gatagctacg cgtatatgcg taacggttgg gacgttgagg tgaccgcggt tggcaaccag 600 ttcaacggtg gctgcctgct ggttgcgatg gttccggaac tgtgcagcat tgagcgtcgt 660 gaactgtttc agctgaccct gttcccgcac caatttatca acccgcgtac caacatgacc 720 gcgcacatta aagtgccgtt cgtgggtgtt aaccgttacg accagtataa ggttcacaaa 780 ccgtggaccc tggtggttat ggtggttgcg ccgctgaccg tgaacaccga aggtgcgccg 840 caaatcaagg tttatgcgaa cattgcgccg accaacgtgc acgttgcggg cgagtttccg 900 agcaaagaag gcatctttcc ggtggcgtgc agcgacggtt atggcagcct ggttaccacc 960 gacccgaaga ccgcggatcc ggtgtacggc aaagttttca acccgccgcg taacatgctg 1020 ccgggtcgtt ttaccaacct gctggacgtg gcggaagcgt gcccgacctt cctgcacttt 1080 gacggcgatg tgccgtatgt taccaccaag accgacagcg atcgtgttct ggcgcaattc 1140 gatctgagcc tggcggcgaa acacatgagc aacacctttc tggcgggtct ggcgcagtac 1200 tataccaat acagcggcac catcaacctg cacttcatgt ttaccggtcc gaccgacgcg 1260 aaggcgcgtt acatgattgc gtatgctccg ccgggtatgg agccgccgaa aaccccggaa 1320 gcggcggcgc actgcatcca cgcggagtgg gataccggtc tgaacagcaa gttcaccttt 1380 agcattccgt atctgagcgc ggcggactac gcgtataccg cgagcgatgc ggcggaaacc 1440 accaacgtgc agggctgggt ttgcctgttc caaattaccc acggcaaggc ggagggtgat 1500 gcgctggttg tgctggcgag cgcgggtaaa gactttgaac tgcgtctgcc ggtggatgcg 1560 cgtcagcaaa ccaccagcac cggtgaaagc gcggacccgg tgaccgcgac cgttgagaac 1620 tatggtggcg aaacccaggt gcaacgtcgt caccacaccg acgttagctt catcctggat 1680 cgttttgtga aggttacccc gaaagacagc attaacgtgc tggatctgat gcaaaccccg 1740 ccgcacaccc tggttggtgc gctgctgcgt accgcgacct actattttgc ggacctggaa 1800 gtggcggtta agcacgaagg tgatctgacc tgggtgccga acggtgcgcc ggaggcggcg 1860 ctggataaca ccaccaaccc gaccgcgtac cacaaagcgc cgctgacccg tctggcgctg 1920 ccgtataccg cgccgcaccg tgtgctggcg accgtttaca acggtgactg caagtatgcg 1980 ggtggcagcc tgaccaacgt gcgtggtgat ctgcaggttc tggcgcaaaa agcggcgcgt 2040 ccgctgccga ccagcttcaa ctacggcgcg atcaaggcga cccgtgtgac cgagctgctg 2100 taccgtatga aacgtgcgga aacctattgc ccgcgtccgc tgctggcggt tcacccgagc 2160 gcggcgcgtc acaagcagaa aattgtggcg ccggttaagc aaagcctgaa ctttgacctg 2220 ctgaaactgg cgggtgatgt tgagagcaac ccgggc 2256 <210> 4 <211> 2256 <212> DNA <213> Nucleotide sequence of P1-2A protein (DNA) before codon optimization <400> 4 ggagccggac aatccagtcc ggctactggg tcacagaacc aatcaggcaa caccgggagt 60 atcatcaaca actactacat gcagcagtac cagaactcca tggacaccca acttggtgac 120 aatgctatca gcggaggctc caacgaggga tccacagaca caacttccac ccacacaacc 180 aacactcaga acaatgactg gttttcaaag ttggccagct ctgccttcag cggtcttttc 240 ggcgccctcc tcgccgataa gaaaaccgag gagaccactc ttctcgagga ccgcatcctc 300 accacccgaa acggacacac cacctcgaca acccagtcga gtgttggcat aacgcacggg 360 tacgcaacag ctgaggattt tgtgagcggg ccaaacacct ctggtcttga gaccagagtt 420 atccaggcgg aacggttctt taaaacccac ctgttcgact gggtcaccag tgatccgttc 480 ggacggtgct acttgttgga gctcccgact gaccacaaag gtgtctacgg cagcctgacc 540 gactcgtacg cctacatgag aaacggttgg gacgttgaag tcaccgctgt ggggaatcag 600 ttcaacggag gctgcctact ggtggccatg gtgcctgaac tttgttccat cgagcggaga 660 gagctgttcc agcttacgct cttcccccac cagttcatca acccccggac gaacatgaca 720 gcccacatca aggtgccctt tgttggcgtc aaccgttacg atcagtacaa ggtacacaag 780 ccgtggaccc ttgtggttat ggtcgtagcc ccactgactg tcaacaccga aggcgctccg 840 cagatcaagg tgtatgccaa catcgcaccc accaacgtgc acgtcgcggg tgagttccct 900 tccaaagagg ggattttccc tgtggcctgt agcgacggtt atggcagttt ggtgacaact 960 gacccaaaga cggctgaccc cgtttacggc aaagtgttca accccccccg caacatgttg 1020 ccgggacggt tcaccaacct cctggacgtg gctgaggctt gccccacgtt tctgcacttc 1080 gatggcgacg taccgtatgt gaccactaag acggattcgg acagggtgct cgcacaattt 1140 gacttgtctt tggcagcaaa acacatgtca aacaccttcc ttgcaggtct tgcccaatac 1200 tacacgcagt acagcggcac catcaacctg cacttcatgt tcacaggtcc cactgacgcg 1260 aaagcgcgtt acatgattgc gtatgcccct ccgggcatgg agccgcccaa aacacctgag 1320 gctgctgctc actgcattca cgcagagtgg gacacgggtc tgaactcaaa gtttaccttt 1380 tccatcccct acctctcggc ggctgattac gcgtacaccg cgtctgacgc tgctgaaacc 1440 acaaatgttc agggatgggt ctgcttattt caaataacac acgggaaagc tgagggtgac 1500 gctcttgtcg tgctggccag tgctggcaaa gactttgagc tgcgcctgcc tgtggacgct 1560 cggcaacaga ccacttcgac aggcgagtcg gctgaccccg tgactgccac cgttgagaat 1620 tacggcggcg agacacaggt ccagaggcgc caccacacag acgtctcatt catattggac 1680 agatttgtga aagtcacacc aaaagactca ataaatgtat tggacctgat gcagaccccc 1740 ccccacaccc tagtaggggc gctcctccgc actgccactt actatttcgc tgatctagag 1800 gtggcagtga aacacgaggg ggaccttacc tgggtgccaa atggagcacc tgaagcagcc 1860 ttggacaaca ccaccaaccc aacggcgtac cataaggcgc cgcttacccg gcttgcattg 1920 ccctacacgg caccacaccg tgttttggcc accgtttaca acggggattg caaatacgcc 1980 gggggttcac tgaccaacgt gagaggcgat ctccaagtgc tggctcagaa ggcggcgagg 2040 ccgctgccta cttctttcaa ctacggtgcc atcaaggcca ctcgggtgac agaactgctg 2100 taccgcatga agagggccga gacgtactgt cctcggcccc tcttggctgt tcacccgagt 2160 gcggctagac acaaacagaa aatagtggca cctgtaaagc agtccttgaa ctttgatctg 2220 ctcaagttgg caggggacgt ggagtccaac cctggg 2256 <210> 4 <211> 1413 <212> DNA <213> TF protein gene coding sequence (TF protein nucleotide sequence DNA) <4百> 4 atgaatcaca aagtgatgca agtttcagtt gaaaccactc aaggccttgg ccgccgtgta 60 acgattacta tcgctgctga cagcatcgag accgctgtta aaagcgagct ggtcaacgtt 120 gcgaaaaaag tacgtattga cggcttccgc aagggcaaag tgccaatgaa tatcgttgct 180 It should be noted that the translation of "4百" as "<4百>" is a bit unclear in the context. It might be better to clarify or correct this part if possible. cagcgttatg gcgcgtctgt acgccaggac gttctgggtg acctgatgag ccgtaacttc 240 attgacgcca tcattaaaga aaaaatcaat ccggctggcg caccgactta tgttccgggc 300 gaatacaagc tgggtgaaga cttcacttac tctgtagagt ttgaagttta tccggaagtt 360 gaactgcaag gtctggaagc gatcgaagtt gaaaaaccga tcgttgaagt gaccgacgct 420 gacgttgacg gcatgctgga tactctgcgt aaacagcagg cgacctggaa agaaaaagac 480 ggcgctgttg aagcagaaga ccgcgtgacc atcgacttca ccggttctgt agacggcgaa 540 gagttcgaag gcggtaaagc gtctgatttc gtactggcga tgggccaggg tcgtatgatc 600 ccgggctttg aagacggtat caaaggccac aaagctggcg aagagttcac catcgacgtg 660 accttcccgg aagaatacca cgcagaaaac ctgaaaggta aagcagcgaa attcgctatc 720 aacctgaaga aagttgaaga gcgtgaactg ccggaactga ccgcagagtt catcaaacgt 780 ttcggcgttg aagatggttc cgtagaaggt ctgcgcgctg aagtgcgtaa aaacatggag 840 cgcgagctga agagcgccat ccgtaaccgc gttaagtctc aggcgatcga aggtctggta 900 aaagctaacg acatcgacgt accggctgcg ctgatcgaca gcgaaatcga cgttctgcgt 960 cgccaggctg cacagcgttt cggtggcaac gaaaaacaag ctctggaact gccgcgcgaa 1020 ctgttcgaag aacaggctaa acgccgcgta gttgttggcc tgctgctggg cgaagttatc 1080 cgcaccaacg agctgaaagc tgacgaagag cgcgtgaaag gcctgatcga agagatggct 1140 tctgcgtacg aagatccgaa agaagttatc gagttctaca gcaaaaacaa agaactgatg 1200 gacaacatgc gcaatgttgc tctggaagaa caggctgttg aagctgtact ggcgaaagcg 1260 aaagtgactg aaaaagaaac cactttcaac gagctgatga accagcaggc gtccgcgggt 1320 ctggaagttc tgttccaggg gccctccgcg ggtctggtgc cacgcggtag tggtggtatc 1380 gaaggtaggc atatggagct cggtaccctc gag 1413 <210> 4 <211> twenty one <212> DNA <213> The gene coding sequence of the TEV enzyme cleavage site (DNA nucleotide sequence of the TEV enzyme cleavage site) <400> 4 gagaacctgt actttcaggg c 21 <210> 4 <211> 18 <212> DNA <213> 6Arg gene coding sequence (6Arg nucleotide sequence DNA) <400> 4 cgcaggcgtc gaaggcga 18 <210> 4 <211> 18 <212> DNA <213> 6His gene coding sequence (6His nucleotide sequence DNA) <400> 4 catcatcatc atcatcac 18 <210> 4 <211> 752 <212> PRT <213> The amino acid sequence (PRT) of the P1-2A protein. <400> 4 Gly Ala Gly Gln Ser Ser Pro Ala Thr Gly Ser Gln Asn Gln Ser Gly 1 5 10 15 Asn Thr Gly Ser Ile Ile Asn Asn Tyr Tyr Met Gln Gln Tyr Gln Asn 20 25 30 Ser Met Asp Thr Gln Leu Gly Asp Asn Ala Ile Ser Gly Gly Ser Asn 35 40 45 Glu Gly Ser Thr Asp Thr Thr Ser Thr His Thr Thr Asn Thr Gln Asn 50 55 60 Asn Asp Trp Phe Ser Lys Leu Ala Ser Ser Ala Phe Ser Gly Leu Phe 65 70 75 80 Gly Ala Leu Leu Ala Asp Lys Lys Thr Glu Glu Thr Thr Leu Leu Glu 85 90 95 Asp Arg Ile Leu Thr Thr Arg Asn Gly His Thr Thr Ser Thr Thr Gln 100 105 110 Ser Ser Val Gly Ile Thr His Gly Tyr Ala Thr Ala Glu Asp Phe Val 115 120 125 Ser Gly Pro Asn Thr Ser Gly Leu Glu Thr Arg Val Ile Gln Ala Glu 130 135 140 Arg Phe Phe Lys Thr His Leu Phe Asp Trp Val Thr Ser Asp Pro Phe 145 150 155 160 Gly Arg Cys Tyr Leu Leu Glu Leu Pro Thr Asp His Lys Gly Val Tyr 165 170 175 Gly Ser Leu Thr Asp Ser Tyr Ala Tyr Met Arg Asn Gly Trp Asp Val 180 185 190 Glu Val Thr Ala Val Gly Asn Gln Phe Asn Gly Gly Cys Leu Leu Val 195 200 205 Ala Met Val Pro Glu Leu Cys Ser Ile Glu Arg Arg Glu Leu Phe Gln 210 215 220 Leu Thr Leu Phe Pro His Gln Phe Ile Asn Pro Arg Thr Asn Met Thr 225 230 235 240 Ala His Ile Lys Val Pro Phe Val Gly Val Asn Arg Tyr Asp Gln Tyr 245 250 255 Lys Val His Lys Pro Trp Thr Leu Val Val Met Val Val Ala Pro Leu 260 265 270 Thr Val Asn Thr Glu Gly Ala Pro Gln Ile Lys Val Tyr Ala Asn Ile 275 280 285 Ala Pro Thr Asn Val His Val Ala Gly Glu Phe Pro Ser Lys Glu Gly 290 295 300 Ile Phe Pro Val Ala Cys Ser Asp Gly Tyr Gly Ser Leu Val Thr Thr 305 310 315 320 Asp Pro Lys Thr Ala Asp Pro Val Tyr Gly Lys Val Phe Asn Pro Pro 325 330 335 Arg Asn Met Leu Pro Gly Arg Phe Thr Asn Leu Leu Asp Val Ala Glu 340 345 350 Ala Cys Pro Thr Phe Leu His Phe Asp Gly Asp Val Pro Tyr Val Thr 355 360 365 Thr Lys Thr Asp Ser Asp Arg Val Leu Ala Gln Phe Asp Leu Ser Leu 370 375 380 Ala Ala Lys His Met Ser Asn Thr Phe Leu Ala Gly Leu Ala Gln Tyr 385 390 395 400 Tyr Thr Gln Tyr Ser Gly Thr Ile Asn Leu His Phe Met Phe Thr Gly 405 410 415 Pro Thr Asp Ala Lys Ala Arg Tyr Met Ile Ala Tyr Ala Pro Pro Gly 420 425 430 Met Glu Pro Pro Lys Thr Pro Glu Ala Ala Ala His Cys Ile His Ala 435 440 445 Glu Trp Asp Thr Gly Leu Asn Ser Lys Phe Thr Phe Ser Ile Pro Tyr 450 455 460 Leu Ser Ala Ala Asp Tyr Ala Tyr Thr Ala Ser Asp Ala Ala Glu Thr 465 470 475 480 Thr Asn Val Gln Gly Trp Val Cys Leu Phe Gln Ile Thr His Gly Lys 485 490 495 Ala Glu Gly Asp Ala Leu Val Val Leu Ala Ser Ala Gly Lys Asp Phe 500 505 510 Glu Leu Arg Leu Pro Val Asp Ala Arg Gln Gln Thr Thr Ser Thr Gly 515 520 525 Glu Ser Ala Asp Pro Val Thr Ala Thr Val Glu Asn Tyr Gly Gly Glu 530 535 540 Thr Gln Val Gln Arg Arg His His Thr Asp Val Ser Phe Ile Leu Asp 545 550 555 560 Arg Phe Val Lys Val Thr Pro Lys Asp Ser Ile Asn Val Leu Asp Leu 565 570 575 Met Gln Thr Pro Pro His Thr Leu Val Gly Ala Leu Leu Arg Thr Ala 580 585 590 Thr Tyr Tyr Phe Ala Asp Leu Glu Val Ala Val Lys His Glu Gly Asp 595 600 605 Leu Thr Trp Val Pro Asn Gly Ala Pro Glu Ala Ala Leu Asp Asn Thr 610 615 620 Thr Asn Pro Thr Ala Tyr His Lys Ala Pro Leu Thr Arg Leu Ala Leu 625 630 635 640 Pro Tyr Thr Ala Pro His Arg Val Leu Ala Thr Val Tyr Asn Gly Asp 645 650 655 Cys Lys Tyr Ala Gly Gly Ser Leu Thr Asn Val Arg Gly Asp Leu Gln 660 665 670 Val Leu Ala Gln Lys Ala Ala Arg Pro Leu Pro Thr Ser Phe Asn Tyr 675 680 685 Gly Ala Ile Lys Ala Thr Arg Val Thr Glu Leu Leu Tyr Arg Met Lys 690 695 700 Arg Ala Glu Thr Tyr Cys Pro Arg Pro Leu Leu Ala Val His Pro Ser 705 710 715 720 Ala Ala Arg His Lys Gln Lys Ile Val Ala Pro Val Lys Gln Ser Leu 725 730 735 Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn Pro Gly 740 745 750 <210> 4 <211> 471 <212> PRT <213> TF protein amino acid sequence (PRT) <400> 4 Met Asn His Lys Val Met Gln Val Ser Val Glu Thr Thr Gln Gly Leu 1 5 10 15 Gly Arg Arg Val Thr Ile Thr Ile Ala Ala Asp Ser Ile Glu Thr Ala 20 25 ३० Val Lys Ser Glu Leu Val Asn Val Ala Lys Lys Val Arg Ile Asp Gly 35 40 45 Phe Arg Lys Gly Lys Val Pro Met Asn Ile Val Ala Gln Arg Tyr Gly 50 55 60 Ala Ser Val Arg Gln Asp Val Leu Gly Asp Leu Met Ser Arg Asn Phe 65 70 75 80 Ile Asp Ala Ile Ile Lys Glu Lys Ile Asn Pro Ala Gly Ala Pro Thr 85 90 95 Tyr Val Pro Gly Glu Tyr Lys Leu Gly Glu Asp Phe Thr Tyr Ser Val 100 105 110 Glu Phe Glu Val Tyr Pro Glu Val Glu Leu Gln Gly Leu Glu Ala Ile It should be noted that there is a Chinese character "३०" in the original text which is likely a mistake. It is translated as "30" in the English translation above.115 120 125 Glu Val Glu Lys Pro Ile Val Glu Val Thr Asp Ala Asp Val Asp Gly 130 135 140 Met Leu Asp Thr Leu Arg Lys Gln Gln Ala Thr Trp Lys Glu Lys Asp 145 150 155 160 Gly Ala Val Glu Ala Glu Asp Arg Val Thr Ile Asp Phe Thr Gly Ser 165 170 175 Val Asp Gly Glu Glu Phe Glu Gly Gly Lys Ala Ser Asp Phe Val Leu 180 185 190 Ala Met Gly Gln Gly Arg Met Ile Pro Gly Phe Glu Asp Gly Ile Lys 195 200 205 Gly His Lys Ala Gly Glu Glu Phe Thr Ile Asp Val Thr Phe Pro Glu 210 215 220 Glu Tyr His Ala Glu Asn Leu Lys Gly Lys Ala Ala Lys Phe Ala Ile 225 230 235 240 Asn Leu Lys Lys Val Glu Glu Arg Glu Leu Pro Glu Leu Thr Ala Glu 245 250 255 Phe Ile Lys Arg Phe Gly Val Glu Asp Gly Ser Val Glu Gly Leu Arg 260 265 270 Ala Glu Val Arg Lys Asn Met Glu Arg Glu Leu Lys Ser Ala Ile Arg 275 280 285 Asn Arg Val Lys Ser Gln Ala Ile Glu Gly Leu Val Lys Ala Asn Asp 290 295 300 Ile Asp Val Pro Ala Ala Leu Ile Asp Ser Glu Ile Asp Val Leu Arg 305 310 315 320 Arg Gln Ala Ala Gln Arg Phe Gly Gly Asn Glu Lys Gln Ala Leu Glu 325 330 335 Leu Pro Arg Glu Leu Phe Glu Glu Gln Ala Lys Arg Arg Val Val Val 340 345 350 Gly Leu Leu Leu Gly Glu Val Ile Arg Thr Asn Glu Leu Lys Ala Asp 355 360 365 Glu Glu Arg Val Lys Gly Leu Ile Glu Glu Met Ala Ser Ala Tyr Glu 370 375 380 Asp Pro Lys Glu Val Ile Glu Phe Tyr Ser Lys Asn Lys Glu Leu Met 385 390 395 400 Asp Asn Met Arg Asn Val Ala Leu Glu Glu Gln Ala Val Glu Ala Val 405 410 415 Leu Ala Lys Ala Lys Val Thr Glu Lys Glu Thr Thr Phe Asn Glu Leu 420 425 430 Met Asn Gln Gln Ala Ser Ala Gly Leu Glu Val Leu Phe Gln Gly Pro 435 440 445 Ser Ala Gly Leu Val Pro Arg Gly Ser Gly Gly Ile Glu Gly Arg His 450 455 460 Met Glu Leu Gly Thr Leu Glu 465 470 <210> 4 <211> 7 <212> PRT <213> The amino acid sequence of the TEV enzyme cleavage site (PRT) <400> 4 Glu Asn Leu Tyr Phe Gln Gly 1 5

Claims

1. A foot-and-mouth disease virus capsid fusion protein solublely expressed in Escherichia coli, characterized in that, The foot-and-mouth disease virus capsid fusion protein is a TF-TEV-P1-2A-6Arg-6His fusion protein, wherein TF is a chaperone protein with the amino acid sequence SEQ ID NO: 8; TEV is an enzyme cleavage site protein with the amino acid sequence SEQ ID NO: 9; P1-2A is the foot-and-mouth disease virus capsid protein with the optimized nucleotide sequence SEQ ID NO: 1; Arg is arginine, and His is histidine.

2. A method for preparing the foot-and-mouth disease virus capsid fusion protein according to claim 1, characterized in that, The method includes the following steps: 1) In the nucleotide sequence of the foot-and-mouth disease virus capsid protein P1-2A after codon optimization ， End Insertion The gene coding sequence of a chaperone protein and the gene coding sequence of the restriction site of a restriction enzyme protein were used to obtain the nucleotide sequence opti-chaperone protein-restriction site protein-P1-2A-6Arg; the chaperone protein is TF, and its amino acid sequence is SEQ ID NO: 8; the restriction enzyme site protein is TEV, and its amino acid sequence is SEQ ID NO: 9; the P1-2A is the foot-and-mouth disease virus capsid protein, and its optimized nucleotide sequence is SEQ ID NO: 1; 2) The nucleotide sequence of the opti-chaperone protein-cleavage site protein-P1-2A-6Arg described in step 1) is 3 ， The gene coding sequence with 6 histidine residues was inserted at the end to obtain the nucleotide sequence of opti-chaperone protein-cleavage site protein-P1-2A-6Arg-6His; 3) Insert the nucleotide sequence of the opti-chaperone protein-restriction site protein-P1-2A-6Arg-6His from step 2) into a prokaryotic expression plasmid, wherein the prokaryotic expression plasmid is a pCold plasmid or a pET plasmid, to obtain a recombinant vector containing the nucleotide sequence of the opti-chaperone protein-restriction site protein-P1-2A-6Arg-6His; 4) Transform the recombinant vector described in step 3) into Escherichia coli to obtain an E. coli strain containing the nucleotide sequence of the recombinant vector of opti-chaperone protein-restriction site protein-P1-2A-6Arg-6His; 5) The *E. coli* strain from step 4) was fermented and cultured. After IPTG induction, the soluble chaperone protein-restriction site protein-P1-2A-6Arg-6His fusion protein was expressed; and 6) The supernatant after the fermentation of E. coli cells was recovered and purified by nickel column affinity chromatography to obtain the chaperone protein-enzyme site protein-P1-2A-6Arg-6His fusion protein.

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