A method for purifying a foot-and-mouth disease virus capsid protein virus-like particle

Abstract

The application provides a purification method of foot-and-mouth disease virus capsid protein virus-like particles, and the purification method comprises the following steps: a fusion protein containing P1-2A is subjected to enzymolysis by 3C and TEV proteases to obtain an enzymolysis liquid; the enzymolysis liquid is subjected to nickel column purification by a nickel column to obtain foot-and-mouth disease virus capsid protein virus-like particle 5S protomers; and then foot-and-mouth disease virus capsid protein virus-like particles VLPs can be obtained by self-assembly. The purity of the foot-and-mouth disease virus capsid protein virus-like particles obtained by the purification method of the application is more than 90%, and the yield is more than 95%.

Description

Technical Field

[0001] This invention relates to a method for purifying foot-and-mouth disease virus capsid protein virus-like particles, 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 an RNA single-stranded 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 the disease 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) have the same immunological characteristics as the intact virus, but do not pose a risk of spreading the virus. Therefore, the production of empty capsids of FMDV using various expression systems has always been a research hotspot both domestically and internationally.

[0007] CN109601007B discloses a method of first concentrating foot-and-mouth disease VLPs using ultrafiltration with a hollow fiber column, and then purifying them by ultracentrifugation with a gradient sucrose solution. Alternatively, the ultrafiltration concentrate can be purified by two column chromatography processes: the first using a Sepharose 6 FF column and the second using a CaptoQ ImpRes ion exchange chromatography column. However, the gradient sucrose solution ultracentrifugation purification method is not conducive to large-scale production and is cumbersome. In addition, the two-stage chromatography purification method is also cumbersome and the protein yield is not high.

[0008] CN110777160A discloses a method for the preliminary purification of proteins from cell lysate containing P1-2A using ammonium sulfate fractionation precipitation, followed by affinity chromatography to obtain P1-2A. This P1-2A is then digested with 3C protease, and the resulting digest is dialyzed in 20mM phosphate buffer (pH 8.0) and 150mM NaCl to assemble into foot-and-mouth disease virus-like particles. These particles are then purified by molecular sieve chromatography. However, molecular sieve purification results in a small sample loading capacity, which is not conducive to large-scale production.

[0009] CN104404074B discloses a method of obtaining SUMO-tagged foot-and-mouth disease virus capsid protein (VP3, VP1, VP0) samples, which are then subjected to enzymatic digestion at 4°C for 12 hours before further purification by molecular sieve chromatography. However, molecular sieve purification involves a small sample loading capacity, which is not conducive to large-scale production.

[0010] CN110981946A discloses a method for expressing three proteins (smtVP0, smtVP3, and smtVP1) with the sumo tag in *E. coli*, followed by nickel column purification. These proteins were then digested with a small ubiquitin-like modified protease, and finally assembled into virus-like particles via cyclic dialysis using an 8–20 kJ membrane. However, this method, which uses membrane dialysis, cannot effectively remove the sumo tag and small ubiquitin-like modified protease after digestion, potentially affecting product quality.

[0011] CN101914501B discloses the expression of three proteins with a sumo tag in E. coli. After purification by nickel column, the proteins are digested by a small ubiquitin-like modified protease, and then the small ubiquitin-like modified protease is removed by nickel column. The assembly of virus-like particles is carried out by dialysis. However, the dialysis method is not suitable for large-scale production.

[0012] In summary, the purification of foot-and-mouth disease virus capsid proteins (VP0, VP1, VP3) using existing technologies is complex, unfavorable for large-scale production, and results in low purity and yield. Summary of the Invention

[0013] Based on the existing technology, in order to overcome the many defects existing in the existing technology, the present invention provides a purification method for foot-and-mouth disease virus capsid protein virus-like particles. The purification method includes the following steps: 1) digesting the fusion protein containing P1-2A with 3C and TEV proteases to obtain an enzyme digestion solution; 2) purifying the enzyme digestion solution obtained in step 1) by passing it through a nickel column to obtain the original foot-and-mouth disease virus capsid protein virus-like particle 5S; 3) removing imidazole from the original foot-and-mouth disease virus capsid protein virus-like particle 5S obtained in step 2) and then self-assembling it into foot-and-mouth disease virus capsid protein virus-like particles (VLPs) by desalting the original foot-and-mouth disease virus capsid protein virus-like particle 5S.

[0014] In the purification method of the present invention, preferably, in step 1), the mass of the 3C protease added is 5-10% of the total mass of the fusion protein containing P1-2A, preferably 7%; the amount of TEV protease added is 4-6% of the total mass of the fusion protein containing P1-2A, preferably 5%.

[0015] In the purification method of the present invention, preferably, in step 1), the fusion protein containing P1-2A is TF- TEV -P1-2A-6Arg-6His fusion protein or TF- TEV -P1-2A-6Arg fusion protein.

[0016] In the purification method of the present invention, preferably, in step 2), the nickel column packing is GE FF NiSepharose 6 Fast Flow, Cytiva FF Ni Sepharose 6 Fast Flow, or Ni-TED purose 6 Fast Flow.

[0017] In the purification method of the present invention, preferably, in step 2), after the enzyme digestion solution passes through a nickel column GEFF Ni Sepharose 6 Fast Flow or Cytiva FF Ni Sepharose 6 Fast Flow, the nickel column is further eluted with a low concentration of imidazole elution buffer to obtain an elution buffer containing the foot-and-mouth disease virus capsid protein virus-like particle 5S; wherein, the low concentration of imidazole elution buffer is a 50mM phosphate buffer containing 10mM-50mM imidazole.

[0018] In the purification method of the present invention, preferably, the phosphate buffer is NaH2PO4, pH=7.4, 0.5M NaCl.

[0019] In the purification method of the present invention, preferably, the concentration of imidazole in the low-concentration imidazole elution buffer is 20-30 mM.

[0020] In the purification method of the present invention, preferably, the eluent containing the foot-and-mouth disease virus capsid protein virus-like particle 5S original is further dialyzed and concentrated to remove imidazole, and the foot-and-mouth disease virus capsid protein virus-like particle 5S original is obtained; the 5S original can self-assemble to obtain foot-and-mouth disease virus capsid protein virus-like particles (VLPs) after removing imidazole by passing through a desalting column.

[0021] In the purification method of the present invention, preferably, in step 2), after the enzyme digestion solution passes through a nickel column Ni-TED purose 6 Fast Flow, the flow-through solution containing the foot-and-mouth disease virus capsid protein virus-like particles 5S is collected.

[0022] In the purification method of the present invention, preferably, the flow-through solution can self-assemble to obtain foot-and-mouth disease virus capsid protein virus-like particles (VLPs).

[0023] Based on existing technologies, in order to overcome the many defects in existing technologies, this invention unexpectedly discovered that foot-and-mouth disease virus capsid proteins (VP0, VP1, VP3) can bind to specific nickel columns with affinity and can be eluted with low concentrations of imidazole. After purification, the purity can reach over 90%, and the yield is over 95%. It can also remove endotoxins. This solves the problems of existing technologies, such as complex operation which is not conducive to large-scale production, and low purity and yield. Attached Figure Description

[0024] Figure 1 This represents the molecular structure of FMDV.

[0025] Figure 2 This indicates that SDS-PAGE detects TF- TEV The result after digestion by the P1-2A-6Arg-6His fusion protease.

[0026] Figure 3 This indicates that SDS-PAGE detects TF- TEV The result after purification by digestion of the P1-2A-6Arg-6His fusion protease.

[0027] Figure 4 This indicates the distribution of the target protein at each stage after using GE FF packing material.

[0028] Figure 5 The results of molecular sieve analysis of the 20mM imidazole eluent (before dialysis) show a peak at 17.5, which is the 5S virus-like particle of the foot-and-mouth disease virus capsid protein.

[0029] Figure 6The results of molecular sieve analysis of the 20mM imidazole eluent (after dialysis) show a peak at 17.5, which is the 5S virus-like particle of the foot-and-mouth disease virus capsid protein.

[0030] Figure 7 This indicates the analysis results of molecular sieve samples taken after assembly.

[0031] Figure 8 This indicates the distribution of the target protein at each stage after using TED packing material.

[0032] Figure 9 This indicates the detection results of purified foot-and-mouth disease virus-like particles detected by electron microscopy.

[0033] Figure 10 This indicates the distribution of the target protein at various stages after using GE FF filler, which contains SUMO lysolytic protein.

[0034] Figure 11 This indicates the presence of SUMO lysin, and the results of molecular sieve analysis after purification, assembly, and sampling. Detailed Implementation

[0035] 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.

[0036] All reagents used were domestically produced and commercially available products.

[0037] TF- prepared according to patent application CN2021111316714.X TEV -P1-2A-6Arg-6His or TF- TEV -P1-2A-6Arg fusion protein.

[0038] TEV enzyme preparation: For specific preparation methods, please refer to the literature (Tropea JE1, Cherry S, Waugh DS, Expression and Purification of Soluble His6-Tagged TEV Protease. Methods MolBiol. 2009; 498:297-307).

[0039] Preparation of 3C protease: For specific preparation methods, please refer to the literature (James R.Birtley, Stephen R.Knox, et al., Crystal Structure of Foot-and-Mouth Disease Virus 3C Protease. THE JOURNALOF BIOLOGICAL CHEMISTRY Vol.280, No.12.11520–11527, 2005).

[0040] Example 1 TF- TEV -P1-2A-6Arg-6His fusion protein digestion

[0041] The TF-TEV-P1-2A-6Arg-6His fusion protein prepared according to the patent application CN2021111316714.X was collected. Based on the total mass of the fusion protein, 7% (w / w) of 3C protease and 5% (w / w) of TEV enzyme were added. After mixing, the mixture was digested at 30±1℃ for 16±2 hours to obtain the digestion solution, which was then purified by nickel column chromatography.

[0042] Enzyme digestion identification: Take 20 μl each of 3C protease, TEV protease, pre-digestion sample, and post-digestion sample, add 5 μl of 5× loading buffer to each, mix well, boil for 10 min, then take 5 μl of each and spot onto a 12% SDS-polyacrylamide gel. Run the stacking gel at 80V and the separating gel at 120V. Then, stain with Coomassie Brilliant Blue to visualize the electrophoretic bands. The electrophoresis results are shown in the figure. Figure 2 As shown, M is the marker, 1 is the 3C protease control, 2 is the TEV protease control, 3 is the sample before digestion, and 4 is the sample after digestion. From Figure 2 As can be seen, the precursor protein P1-2A is completely digested by enzymes, and the foot-and-mouth disease virus capsid proteins (VP0, VP1, VP3) are stable after enzyme digestion.

[0043] Example 2: Purification of enzyme digestion buffer using nickel column (GE FF Ni Sepharose 6 Fast Flow, Cytiva FF Ni Sepharose 6 Fast Flow)

[0044] Through extensive experimental exploration, we discovered that foot-and-mouth disease capsid proteins (VP0, VP1, VP3) without the 6His tag after enzymatic digestion can bind to a nickel column (GE FF Ni Sepharose 6 Fast Flow or Cytiva FF Ni Sepharose 6 Fast Flow, manufactured by Jiangsu Qianchun Biotechnology Co., Ltd.), and can be eluted with a low concentration of imidazole (generally 10mM-50mM, 20mM-30mM used in this preferred embodiment). The low-concentration imidazole eluent is a 50mM phosphate buffer containing 10mM-50mM imidazole. The phosphate buffer is NaH2PO4, pH=7.4, 0.5M NaCl. Furthermore, the purity after purification can reach over 90%, the yield is over 95%, and endotoxins can be removed, such as... Figure 3 As shown. The specific steps for purifying the sample after enzyme digestion are as follows: After equilibration on a nickel column, the enzyme-digested sample is loaded onto the column, then washed with 50mM phosphate buffer for equilibration, and then the target protein is eluted with 20mM-30mM imidazole elution buffer (50mM phosphate buffer, 20mM-30mM imidazole) to obtain the 5S original virus-like particles of foot-and-mouth disease virus capsid protein.

[0045] Results identification: 20 μl of protein purified by nickel column was added to 5 μl of 5× loading buffer, mixed well, and boiled for 10 min. Then, 5 μl of the mixture was spotted onto a 12% SDS-polyacrylamide gel, and the gel was run at a constant voltage of 80 V for stacking and 120 V for separating. Coomassie brilliant blue staining was then used to visualize the electrophoretic bands. The electrophoresis results are shown below. Figure 3 , where 1 is the marker and 2 is the protein purified by nickel column.

[0046] Depend on Figure 3 Electrophoresis results showed that the enzyme digestion products could be completely separated from the TF chaperone protein by nickel column purification. The purity of the foot-and-mouth disease virus capsid protein (VP0, VP1, VP3) after enzyme digestion was over 90%. Calculations showed that the yield after enzyme digestion and purification was over 95%, with almost no loss (by comparing the molar ratio of the target protein before and after purification), and the endotoxin content was qualified.

[0047] from Figure 4 It can be seen that the foot-and-mouth disease virus capsid proteins (VP0, VP1, VP3) after enzyme digestion were mainly obtained by elution with low concentration of imidazole elution buffer.

[0048] from Figure 5 and Figure 6 It can be seen that the target protein after enzyme digestion automatically assembles into the 5S protozoan.

[0049] Assembly: After removing imidazole from the eluted sample by passing it through a desalting column, VLPs can be assembled automatically to obtain foot-and-mouth disease virus capsid protein virus-like particles (VLPs). Molecular sieve analysis chromatograms are shown below. Figure 6 According to the spectrum, the peak position of VLPs is around 8.3, and the content of VLPs is 63.4%. Moreover, the obtained VLPs are stable and can still maintain their activity at 37.5℃ without depolymerization.

[0050] Example 3: TF- TEV -P1-2A-6Arg fusion protein digestion

[0051] The conditions for TF- in Example 1 are as follows. TEV The P1-2A-6Arg fusion protein was digested with enzymes to obtain a digestion solution containing foot-and-mouth disease virus capsid proteins (VP0, VP1, VP3).

[0052] Example 4: Purification of enzyme digestion buffer using a nickel column (Ni-TED purose 6 Fast Flow)

[0053] After enzymatic digestion, the foot-and-mouth disease capsid proteins (VP0, VP1, VP3) were passed through a nickel column (Ni-TED purose 6 Fast Flow, manufactured by Jiangsu Qianchun Biotechnology Co., Ltd.) and remained in the flow-through. However, TF chaperone proteins, TEV protease, 3C protease, and other miscellaneous proteins could bind to the nickel column (Ni-TED purose 6 Fast Flow). Therefore, the purity of the target protein in the flow-through could reach over 90%, and the yield was also over 90%. Figure 7 As shown. The specific steps for sample purification after enzyme digestion are as follows: After equilibration of the nickel column, the enzyme-digested sample is loaded and the flow-through is collected.

[0054] Results identification: 20 μL of the flow-through protein purified from the nickel column was added to 5 μL of 5× loading buffer, mixed well, and boiled for 10 min. Then, 5 μL of the mixture was spotted onto a 12% SDS-polyacrylamide gel, and the gel was run at a constant voltage of 80 V for the stacking gel and 120 V for the separating gel. The electrophoretic bands were then visualized by Coomassie Brilliant Blue staining. The electrophoresis results are shown below. Figure 8 .

[0055] Depend on Figure 8 Electrophoresis results showed that the TF chaperone proteins could be completely separated after nickel column purification of the enzyme digestion products. The purity of the foot-and-mouth disease virus capsid proteins (VP0, VP1, VP3) after enzyme digestion was over 90%, and the yield after enzyme digestion and purification was over 90%. A small amount of the product remained on the column and could be eluted with a low concentration of imidazole elution buffer, but the endotoxin content was found to be excessive.

[0056] Assembly: The flow-through solution can spontaneously assemble VLPs to obtain foot-and-mouth disease virus capsid protein virus-like particles (VLPs); and the obtained VLPs are stable and can still maintain their activity at 37.5℃ without depolymerization.

[0057] Example 5: Electron Microscopy Detection of Purified Foot-and-Mouth Disease Virus-like Particles

[0058] 10 μL of the assembled foot-and-mouth disease virus capsid proteins (VP0, VP1, VP3) were added to a 200-mesh copper grid and allowed to adsorb for 1 min. Excess sample was then blotted away with filter paper. The sample was washed twice with ultrapure water, once with 1% uranium acetate, and negatively stained for 1 min. Observation was performed using a transmission electron microscope. Figure 9 The results show that over 90% of the prepared samples formed VLPs with a diameter of approximately 30 nm. The number of individual particles was relatively small, and most virus-like particles existed in the form of dimers, trimers, and polymers.

[0059] Example 6: Subunit Vaccine Preparation and Immunization Experiment

[0060] 6.1 ISA 201VG adjuvant (purchased from SEPPIC, France) vaccine preparation

[0061] Aqueous phase preparation: Based on the target protein content in the vaccine, dilute the target protein to an appropriate concentration using PBS (or physiological saline) to obtain the aqueous phase. Oil phase preparation: Based on the total amount of vaccine prepared, measure an appropriate amount of ISA 201VG adjuvant according to an antigen-to-adjuvant weight ratio of 1:1 and a volume ratio of 46:54. Emulsification: Preheat both the aqueous and oil phases to 33°C. Slowly add the aqueous phase to the oil phase, stir at 200–500 rpm for 20–30 minutes, let stand at 20°C for 1 hour, and then incubate overnight at 4°C. Dispensing and storage: Dispense as needed, and store at 4°C for later use after passing inspection.

[0062] 6.2 Immunoassay

[0063] See 6.1 for the preparation of three concentrations of vaccine: vaccine 1 (30 μg / dose), vaccine 2 (60 μg / dose), and vaccine 3 (100 μg / dose).

[0064] The above three vaccines were tested according to the safety and efficacy testing methods on page 53 of Part III of the 2015 edition of the Veterinary Pharmacopoeia of the People's Republic of China. Since foot-and-mouth disease virus is a nationally controlled virus, no challenge experiment was conducted for efficacy testing; only neutralizing antibodies were tested 28 days after the first immunization.

[0065] Safety test results: All guinea pigs and pigs that were immunized were normal, with no deaths or obvious local or systemic adverse reactions caused by the vaccine; therefore, all three batches of vaccines are safe.

[0066] Efficacy test results: The neutralizing antibody test results are shown in the table below. Using a neutralizing antibody ratio ≥1:32 as the criterion for protection, the PD50 after vaccine immunization was calculated. Vaccine 1 had a PD50 of 6.4, while vaccines 2 and 3 both had PD50s of 7.6, meeting the current national standard for foot-and-mouth disease vaccine efficacy (PD50 ≥ 6). Therefore, all three batches of vaccines passed the efficacy test. Furthermore, vaccines 2 and 3 showed consistent protection rates, both better than vaccine 1. In terms of potency, while the potency increased with increasing immunization dose, the increase was not significant, especially for vaccines 2 and 3. Therefore, considering both cost and effectiveness, vaccine 2 was selected as the ideal vaccine for further research and industrial production.

[0067]

[0068]

[0069] Comparative example: SUMO- TEV -VP0 / SUMO- TEV -VP1 / SUMO- TEV -VP3 purification

[0070] SUMO- was prepared according to CN101914501B. TEV -VP0 / SUMO- TEV -VP1 / SUMO- TEV The fusion protein of -VP3 was digested with enzymes under the conditions of Example 1 to obtain an enzyme digest containing foot-and-mouth disease virus capsid proteins (VP0, VP1, VP3).

[0071] The enzyme-digested sample was purified using a nickel column under the conditions described in Example 2. The purity of the foot-and-mouth disease virus capsid proteins (VP0, VP1, VP3) obtained was above 90%. Figure 10 .

[0072] Assembly: The eluted sample was passed through a desalting column to remove imidazole for VLP assembly, yielding foot-and-mouth disease virus capsid protein virus-like particles (VLPs). Molecular sieve analysis chromatograms are shown below. Figure 11 According to the spectrum, the peak position of VLPs is around 8.6, the content of VLPs is 15.7%, and a large number of 5S precursors failed to assemble into VLPs. The obtained VLPs depolymerized at 37.5℃ and could not maintain the VLPs morphology.

[0073] 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.

Claims

1. A method for purifying foot-and-mouth disease virus capsid protein virus-like particles, characterized in that, The purification method includes the following steps: 1) The fusion protein containing P1-2A is digested with 3C protease and TEV protease to obtain a digestion solution; wherein, the mass of the 3C protease added is 5-10% of the total mass of the fusion protein containing P1-2A, and the amount of TEV protease added is 4-6% of the total mass of the fusion protein containing P1-2A; 2) The enzyme digestion solution obtained in step 1) is then purified by passing it through a nickel column to obtain the 5S primary form of foot-and-mouth disease virus capsid protein virus-like particles. The nickel column packing material is GE FF Ni Sepharose 6 Fast Flow, Cytiva FF Ni Sepharose 6 Fast Flow, or Ni-TED purose 6 Fast Flow. After the enzyme digestion solution passes through the GE FF Ni Sepharose 6 Fast Flow or Cytiva FF Ni Sepharose 6 Fast Flow nickel column, the column is further eluted with a low-concentration imidazole elution buffer to obtain an eluent containing the 5S primary form of foot-and-mouth disease virus capsid protein virus-like particles. The low-concentration imidazole elution buffer is a 50mM phosphate buffer containing 10mM~50mM imidazole. After the enzyme digestion solution passes through the Ni-TED purose 6 Fast Flow nickel column, the flow-through containing the 5S primary form of foot-and-mouth disease virus capsid protein virus-like particles is collected. 3) The foot-and-mouth disease virus capsid protein virus-like particle 5S primordium described in step 2) self-assembles to obtain foot-and-mouth disease virus capsid protein virus-like particles (VLPs).

2. The purification method according to claim 1, characterized in that, In step 1), the mass of the 3C protease added is 7% of the total mass of the fusion protein containing P1-2A, and the amount of TEV protease added is 5% of the total mass of the fusion protein containing P1-2A.

3. The purification method according to claim 1, characterized in that, In step 1), the fusion protein containing P1-2A is TF- TEV -P1-2A-6Arg-6His fusion protein or TF- TEV -P1-2A-6Arg fusion protein.

4. The purification method according to claim 1, characterized in that, The phosphate buffer solution is NaH2PO4, pH=7.4, 0.5M NaCl.

5. The purification method according to claim 1, characterized in that, The concentration of imidazole in the low-concentration imidazole elution buffer is 20-30 mM.

6. The purification method according to claim 1, characterized in that, The eluent was further dialyzed to remove imidazole, and the foot-and-mouth disease virus capsid protein virus-like particles 5S were obtained. Then, the particles were self-assembled to obtain foot-and-mouth disease virus capsid protein virus-like particles (VLPs).

7. The purification method according to claim 1, characterized in that, The flow-through fluid can self-assemble into foot-and-mouth disease virus capsid protein virus-like particles (VLPs).

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