Method for purifying foot-and-mouth disease virus-like particles expressed by pichia pastoris by adopting affinity chromatography mode

Abstract

The invention discloses a method for purifying foot-and-mouth disease virus-like particles expressed by pichia pastoris by adopting an affinity chromatography mode. The method comprises the following steps: expressing plasmids cloned with foot-and-mouth disease virus structural protein VP0, VP3 and VP1-GHH genes, plasmids cloned with foot-and-mouth disease virus structural protein VP0, VP3-CH and VP1 genes or plasmids cloned with foot-and-mouth disease virus structural protein VP0, VP3 and VP1-CH genes through pichia pastoris to obtain foot-and-mouth disease virus-like particles, and then purifying by adopting affinity chromatography. The inventor finds a place where a His6 affinity label can be inserted by exploring the structure of the foot-and-mouth disease virus-like particle, so that the aim of purifying the virus-like particle by utilizing an affinity chromatography method is fulfilled. The establishment of the method provides a new technical means for research on preparation of foot-and-mouth disease virus-like particles by a pichia pastoris expression system and purification and industrial production of the virus-like particles.

Description

Technical Field

[0001] The present invention relates to a method for preparing and purifying foot-and-mouth disease virus-like particles, and particularly to a method for purifying foot-and-mouth disease virus-like particles expressed by Pichia pastoris by means of affinity chromatography. The present invention belongs to the technical field of biology and medicine. Background Art

[0002] Foot-and-mouth disease is an acute and highly contagious disease that mainly infects cloven-hoofed animals such as pigs, cattle, and sheep. After animals are infected with foot-and-mouth disease, the production capacity of the animals is severely reduced, which has an adverse impact on the local animal husbandry and economic development. At present, except for some developed countries that have achieved the eradication of foot-and-mouth disease, foot-and-mouth disease in Asia, Europe, Africa, and South America has not stopped. Foot-and-mouth disease virus (FMDV) belongs to the Picornaviridae family, the Foot-and-mouth disease virus genus, and is a single-stranded positive-strand RNA virus without an envelope. The virus particle has a diameter of about 30 nm, and the genome size is 8500 nts, mainly encoding a structural protein precursor P1 and non-structural proteins P2 and P3. The precursor protein P1 is cleaved by 3C protease to generate VP0, VP3, and VP1 proteins. VP0 is cleaved into VP2 and VP4 during the assembly of virus particles. 60 copies of VP1-VP4 assemble into VLPs, and VP4 is located inside the VLPs.

[0003] Virus-Like Particles (VLPs for short) can mimic the morphological structure of virus particles but lack nucleic acids, and are a very safe and ideal vaccine type. At present, there are reports of successful preparation of foot-and-mouth disease virus-like particles in mammalian cells, insect cells, and plant cells. Previously, when preparing VLPs using mammalian cells or insect cells, the method of expressing P1-2A-3C was usually adopted. However, due to the non-specific cleavage ability of 3C, it can not only cleave P1 during the expression process, but also cleave host proteins, resulting in transcriptional termination or even death of host cells. As the eukaryotic expression system with the lowest cost, Pichia pastoris has a very high cost performance and is widely used in subunit vaccines of various viruses, especially virus-like particle vaccines, such as hepatitis B vaccine and human papillomavirus vaccine. Although there have been multiple studies attempting to obtain VLPs of picornaviruses using Pichia pastoris, such as EV71, CA16, PV, etc. However, there are few studies on preparing foot-and-mouth disease virus-like particles using Pichia pastoris at present.

[0004] During the assembly of FMDV VLPs, the N-termini of VP0, VP3, and VP1 proteins are inside the VLPs, the C-terminus of VP0 is at the secondary axis interface, and the protonation of a large number of histidines in this region under acidic conditions is the main inducement affecting the instability of VLPs. The C-termini of VP3 and VP1 are on the surface of the VLP. Inserting the GH Loop of VP1 into the flag tag does not affect the assembly of VLPs and can display the flag on the surface of VLPs. Currently, chemical precipitation and sucrose density gradient purification methods are generally used to purify foot-and-mouth disease virus-like particles. This method generally requires a centrifuge with a high specification and the amount of purification each time is very small. Therefore, it is very necessary to find a suitable purification method according to the properties of foot-and-mouth disease virus-like particles. Summary of the Invention

[0005] The object of the present invention is to provide a method for purifying foot-and-mouth disease virus-like particles expressed by Pichia pastoris by affinity chromatography.

[0006] To achieve the above object, the present invention adopts the following technical means:

[0007] A method for purifying foot-and-mouth disease virus-like particles expressed by Pichia pastoris by affinity chromatography according to the present invention includes the steps of expressing plasmids cloned with foot-and-mouth disease virus structural protein VP0, VP3, and VP1-GHH genes, plasmids cloned with foot-and-mouth disease virus structural protein VP0, VP3-CH, and VP1 genes, or plasmids cloned with foot-and-mouth disease virus structural protein VP0, VP3, and VP1-CH genes by Pichia pastoris to obtain foot-and-mouth disease virus-like particles, and then purifying them by affinity chromatography; wherein the protein encoded by the VP1-GHH gene inserts a His-tag polypeptide (His6) at the 136th amino acid of the foot-and-mouth disease virus structural protein VP1, the protein encoded by the VP1-GHH gene inserts a His-tag polypeptide (His6) at the C-terminus of the foot-and-mouth disease virus structural protein VP1, and the protein encoded by the VP3-CH gene inserts a His-tag polypeptide (His6) at the C-terminus of the foot-and-mouth disease virus structural protein VP3.

[0008] Preferably, the nucleotide sequences of the VP0, VP3, VP1, VP1-GHH, VP3-CH, and VP1-CH genes are respectively as shown in SEQ ID NO.1-6.

[0009] Among them, preferably, the plasmids cloned with the foot-and-mouth disease virus structural protein VP0, VP3, VP1-GHH genes, the plasmids cloned with the foot-and-mouth disease virus structural protein VP0, VP3-CH, VP1 genes, or the plasmids cloned with the foot-and-mouth disease virus structural protein VP0, VP3, VP1-CH genes are obtained by inserting their respective genes into the pPink-HC vector, and are named pPink-VP1-GHH / VP3 / VP0, pPink-VP1 / VP3-CH / VP0 plasmid pPink-VP1-CH / VP3 / VP0 respectively.

[0010] Among them, preferably, the nucleotide sequence of the pPink-VP1-GHH / VP3 / VP0 plasmid is as shown in SEQ ID NO.7, the nucleotide sequence of the pPink-VP1-CH / VP3 / VP0 plasmid is as shown in SEQ ID NO.8, and the nucleotide sequence of the pPink-VP1 / VP3-CH / VP0 plasmid is as shown in SEQ ID NO.9.

[0011] Among them, preferably, the method includes the following steps:

[0012] (1) Express foot-and-mouth disease virus-like particles through Pichia pastoris

[0013] 1) Plasmid construction

[0014] Synthesize the VP0, VP3, and VP1 genes encoding the foot-and-mouth disease virus structural protein, and insert a His-tag tag polypeptide (His6) at the 136th amino acid of the VP1 gene-encoded protein, denoted as VP1-GHH, insert a His-tag tag polypeptide (His6) at the C-terminus of the VP1 gene-encoded protein, denoted as VP1-CH, and insert a His-tag tag polypeptide (His6) at the C-terminus of the VP 3 gene-encoded protein, denoted as VP3-CH; among them, the nucleotide sequences of the VP0, VP3, VP1, VP1-GHH, VP3-CH, and VP1-CH genes are as shown in SEQ ID NO.1-6 respectively;

[0015] 2) Transformation

[0016] Insert VP0, VP3, VP1, VP1-GHH, VP3-CH, and VP1-CH into the pPink-HC vector respectively to construct plasmids pPink-VP0, pPink-VP3, pPink-VP1, pPink-VP1-GHH, pPink-VP3-CH, and pPink-VP1-CH. Then, insert the expression cassette of VP3 into the pPink-VP0 vector through Bgl II / BamH I to obtain the pPink-VP3 / VP0 plasmid. Next, insert the expression cassette of VP1-GHH into this plasmid to finally obtain pPink-VP1-GHH / VP3 / VP0 (GHH). Obtain the pPink-VP1 / VP3-CH / VP0 (3CH) plasmid and the pPink-VP1-CH / VP3 / VP0 (1CH) plasmid in the same way. After linearizing the successfully constructed plasmids with Spe I, electrotransform them into the Pichia pastoris strain 1;

[0017] 3) Induced expression

[0018] Inoculate the strains containing the plasmids pPink-VP1-GHH / VP3 / VP0 (GHH), pPink-VP1 / VP3-CH / VP0 (3CH), and pPink-VP1-CH / VP3 / VP0 (1CH) obtained in step 2) into the BMGY medium and culture them overnight until the OD600 reaches between 2 and 6. Then, centrifuge to collect the cell pellet and transfer it to the BMMY medium with a pH of 7 respectively to make the initial OD600 reach 2. Subsequently, add 0.5% v / v methanol for induction for 72 hours;

[0019] (2) Purification of VLPs

[0020] After induction, ultrasonically disrupt the strains, centrifuge at 12000 rpm for 30 min to collect the supernatant. Then, dialyze the supernatant in the assembly buffer overnight. Place the dialyzed sample in a centrifuge at 4°C and centrifuge at 12000 rpm for 30 min. After centrifugation, perform affinity chromatography on the supernatant, wash the miscellaneous proteins with a 10 mM imidazole solution, collect the target protein with a 300 mM imidazole solution, ultrafilter and concentrate it with PBS, and then detect the VLP content in the sample.

[0021] Preferably, the assembly buffer contains 300 mM NaCl, 10 mM tris, 50 mM KCl, 2 mM MgCl, 1% v / v Triton X-100, 0.1 mM PMSF, and pH 8.0.

[0022] Among them, preferably, the nucleotide sequence of the pPink-VP1-GHH / VP3 / VP0 plasmid is shown in SEQ ID NO.7, the nucleotide sequence of the pPink-VP1-CH / VP3 / VP0 plasmid is shown in SEQ ID NO.8, and the nucleotide sequence of the pPink-VP1 / VP3-CH / VP0 plasmid is shown in SEQ ID NO.9.

[0023] The foot-and-mouth disease virus-like particles prepared by the method described in any one of the above are also within the protection scope of the present invention.

[0024] Furthermore, the present invention also provides the use of the foot-and-mouth disease virus-like particles in the preparation of reagents for detecting foot-and-mouth disease virus.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0026] The present invention provides a method for purifying foot-and-mouth disease virus-like particles expressed by Pichia pastoris by affinity chromatography. The inventors of the present invention explored the structure of the foot-and-mouth disease virus-like particles to find a place where the His6 affinity tag can be inserted, so as to achieve the purpose of purifying the virus-like particles by affinity chromatography. The establishment of this method provides a new technical means for the research on the preparation of foot-and-mouth disease virus-like particles by the Pichia pastoris expression system, as well as the purification and industrial production of virus-like particles. Description of the Drawings

[0027] Figure 1 For the purification and electron microscopy observation of pPink-130 VLPs;

[0028] Among them, a WB detection of the sample after sucrose gradient centrifugation; b electron microscopy observation of the purified pPink-130 sample; the yeast after induced expression was ultrasonically broken, and after the broken solution was dialyzed, sucrose density gradient purification was carried out.

[0029] Figure 2 For the optimization of the expression conditions of pPink-130;

[0030] Among them, a WB detection of pPink-130 at different time points and different pH values; b OD values of the pPink-130 strain under different conditions; the expression levels of VP0 (c), VP3 (d), and VP1 (e) in the WB detection results were quantified by Image J under different conditions;

[0031] Figure 3 For the optimization of the VLP purification method;

[0032] Among them, the tertiary structures of the protomers, pentamers and secondary axis interfaces of a FMDV VLPs are shown; VP0 is magenta, VP3 is cyan, VP1 is green, the C-terminus of VP1 is yellow, the C-terminus of VP3 is red, and 136-137 of VP1 is blue; WB detection of the samples after affinity chromatography of pPink-GHH(b), pPink-3CH(c) and pPink-1CH(d);

[0033] Figure 4 Assembly detection of VLPs produced by pPink-GHH, pPink-3CH and pPink-1CH;

[0034] Among them, a electron microscopy observation of VLPs of pPink-GHH, pPink-3CH and pPink-1CH after affinity chromatography; b DLS detection; c determination of the average particle size and d antigen content per unit volume. Specific embodiments

[0035] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer with the description. However, it should be understood that the described embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that the details and forms of the technical solutions of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but such modifications or replacements all fall within the protection scope of the present invention.

[0036] Example 1 Preparation and purification of foot-and-mouth disease virus-like particles expressed by Pichia pastoris

[0037] 1. Materials and methods

[0038] 1.1 Plasmids and strains

[0039] The VP0 gene (H2145Y), VP3 gene, VP1 gene (N17D), VP1-GHH gene, VP1-1CH gene and VP3-3CH gene were synthesized by GenScript and then inserted into the pPink-HC plasmid respectively to construct the following vectors: pPink-VP0, pPink-VP3, pPink-VP1, pPink-VP1-GHH, pPink-VP1-1CH, pPink-VP3-3CH; the nucleotide sequences of the VP0 gene (H2145Y), VP3 gene, VP1 gene (N17D), VP1-GHH gene, VP1-1CH gene and VP3-3CH gene are shown in SEQ ID NO.1-6 respectively. The pPink-HC plasmid and the O-type FMDV polyclonal serum are stored in the Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences. PichiaPink TMStrain1 was purchased from Invitrogen. Anti-His6 monoclonal antibody was purchased from Solarbio.

[0040] 1.2 Plasmid construction and transformation

[0041] First, the expression cassette of VP3 was cut from the vector pPink-VP3 by Bgl II / BamH I and then inserted into the pPink-VP0 vector to obtain the pPink-VP3 / VP0 plasmid. The gene expression cassette of VP1 was obtained by double digestion of the pPink-VP1 plasmid with Bgl II / BamH I. Subsequently, pPink-VP3 / VP0 was linearized with Bgl II, and the gene expression cassette of VP1 was inserted into the vector through this site to obtain pPink-VP1 / VP3 / VP0 (pPink-130). The same operation steps were used to obtain the pPink-VP1-GHH / VP3 / VP0 (pPink-GHH) plasmid (the plasmid sequence is shown in SEQ ID NO.7) and the pPink-VP1-CH / VP3 / VP0 (pPink-1CH) plasmid (the plasmid sequence is shown in SEQ ID NO.8). If the expression cassette VP3-CH was inserted into the pPink-VP0 vector to obtain pPink-VP3-CH / VP0, and then the expression cassette of VP1 was inserted into this vector, the pPink-VP1 / VP3-CH / VP0 (pPink-3CH) plasmid (the plasmid sequence is shown in SEQ ID NO.9) could be obtained. After the successfully constructed plasmids were linearized with Spe I, they were electrotransformed into the Pichia pastoris strain 1. The electrotransformation steps and strain screening refer to the operation manual, and the obtained strains were named pPink-130, pPink-GHH, pPink-1CH, and pPink-3CH respectively.

[0042] 1.3 Optimization of the expression conditions of pPink-130

[0043] The expression conditions were optimized using pPink-130 as a representative. First, the strain containing pPink-130 was inoculated into the BMGY medium and cultured overnight until its OD 600 reached between 2 and 6. Then, the cell pellet was collected by centrifugation and transferred to the BMMY medium with a pH of 5 - 8 so that its initial OD 600 reached 2. Subsequently, 0.5% methanol was added for induction. 200 uL of the bacterial solution was taken every 12 hours to measure the OD 600 . At the same time, 100 uL of the bacterial solution was taken each time, the cell pellet was collected by centrifugation, and an equal volume of acid-washed glass beads was added and vortexed for disruption (vortex for 30 seconds, ice bath for 30 seconds, repeat 8 times). After that, the supernatant was collected by centrifugation at 12000 rpm for 10 minutes for WB detection.

[0044] 1.4 Purification of VLPs

[0045] After the induction, the strains pPink-130, pPink-GHH, pPink-1CH, and pPink-3CH were ultrasonically disrupted (350 w, working for 3 s, intermittent for 3 s, disrupted for 20 min), and the supernatant was collected by centrifugation at 12,000 rpm for 30 min. Then, the supernatant was dialyzed overnight in the assembly buffer (300 mM NaCl, 10 mM tris, 50 mM KCl, 2 mM MgCl, 1% Triton X-100, 0.1 mM PMSF, pH 8.0). The dialyzed sample was placed in a centrifuge at 4 °C and centrifuged at 12,000 rpm for 30 min. After centrifugation, the supernatant was subjected to affinity chromatography, and the impurity proteins were washed with a 10 mM imidazole solution, and the target protein was collected with a 300 mM imidazole solution. After ultrafiltration and concentration with PBS, the VLP content in the sample was detected.

[0046] 1.5 ELISA Antigen Quantification

[0047] For the quantification of FMDV VLPs, the previous description of the laboratory can be referred to (CN117825701A). Briefly, 100 μL / well (0.5 μg) of the M170 antibody diluted with carbonate (pH 9.6) was added to the 96-well plate and coated overnight at 4 °C. The next day, a 1% BSA solution was added and blocked at 37 °C for 1 hour. Subsequently, the gradient-diluted VLP antigen sample was added to the 96-well plate and incubated at 37 °C for 1 hour. Subsequently, a polyclonal antibody against FMDV from guinea pigs and an HRP-conjugated rabbit anti-guinea pig antibody were added successively and incubated at 37 °C for 30 minutes. Finally, TMB was added for staining for 15 minutes, and the absorbance at 450 nm was measured. According to the standard curve generated from the known concentration of FMDV virus particles, the relative antigen content in the sample was determined, and then the VLP content in the sample to be tested was calculated.

[0048] 2. Results

[0049] 2.1 Expression Verification of Foot-and-Mouth Disease Virus Structural Proteins in Pichia pastoris

[0050] After inducing expression, the yeast cells were disrupted by sonication. After dialysis of the disrupted solution, sucrose density gradient purification was carried out. 1 mL of the supernatant was added to a sucrose gradient solution of 15 - 50% (w / v), and centrifuged at 35000 rpm for 3 hours. The centrifuged solution was separated from top to bottom in volumes of 500 μL / tube, and the purified VLPs were detected by double antibody sandwich ELISA. The results showed that the absorbance value of component 9 of pPink-130 was the highest, indicating the presence of correctly assembled VLPs. Samples of components 5 - 12 with detected absorbance values were subjected to WB detection, and it was found that the structural proteins VP0, VP3, and VP1 proteins were co-precipitated at the same concentration, indicating that the precipitated VLPs were assembled from these three subunits( Figure 1 a). Subsequently, the purified sample of pPink-130 was observed by electron microscopy, and uniform particles with a diameter of about 30 nm could be seen( Figure 1 b).

[0051] 2.2 Optimization of pPink-130 expression conditions

[0052] By detecting the OD 600 value and protein expression at different time points, it was found that with the increase of time, the growth of the strain and the protein expression showed a positive correlation. After more than 72 hours, the OD of the strain no longer increased. The density of yeast cells was not much different at pH 5 and 6, and when pH was 7, its OD value decreased by about 2. When pH was 8, the growth of the strain was severely affected and death occurred( Figure 2 a and 2b). The WB detection results showed that within a certain time, the protein expression increased with the increase of time. After inducing for 72 hours, the protein expression reached the peak. When pH was 8, the target protein was hardly detected( Figure 2 c - e). Through comprehensive comparison, when pH ≤ 7 and induced for 72 hours, the pPink-130 strain reached the best expression level. Strains pPink-GHH, pPink-3CH, and pPink-1CH containing plasmids pPink-VP1-GHH / VP3 / VP0 (GHH), pPink-VP1 / VP3-CH / VP0 (3CH), and pPink-VP1-CH / VP3 / VP0 (1CH) were also induced for expression under the same conditions.

[0053] 2.3 Optimization of FMDV VLPs purification method

[0054] Previously, for the purification of VLPs, the sucrose density gradient method was generally used, and the operation steps were cumbersome. Through the structural analysis of VP0, VP3, and VP1, it was found that positions 136 and the C-terminus of VP1 and the C-terminus of VP3 were all on the surface of VLPs, while the C-terminus of VP0 was at the secondary axis interface, and this region contained a large number of histidines that affected the stability of VLPsFigure 3 a). After the pPink-GHH, pPink-3CH, and pPink-1CH strains were induced for expression, they were sonicated, centrifuged at 12,000 rpm for 30 minutes, the supernatant was collected, and dialyzed overnight. After affinity chromatography, the eluate was collected and detected by WB. The results showed that when detected with His6 antibody, pPink-GHH( Figure 3 b), pPink-3CH( Figure 3 c), and pPink-1CH( Figure 3 d) could all detect a single target band at the corresponding position. When detected with anti-FMDV polyclonal antibody, 3 target bands could be detected at 25 - 35 kDa. These results indicate that inserting His6 at the appropriate position can achieve the purpose of purifying 3 target proteins simultaneously using one His6 tag.

[0055] 2.4 Inserting His6 tags at the C-terminus of VP1 and VP3 and the GH loop region of VP1 does not affect assembly

[0056] The results showed that spherical particles with a diameter of approximately 30 nm could be seen in all samples purified by affinity chromatography( Figure 4 a). DLS analysis showed that pPink-GHH, pPink-3CH, and pPink-1CH all had particles with uniform particle sizes, with diameters of 31.24 nm, 30.67 nm, and 30.29 nm respectively( Figure 4 b and 4c). The VLPs in all samples were detected by the ELISA quantification method established in this laboratory. Compared with pPink-130, after affinity chromatography, the antigen content per unit volume of pPink-GHH and pPink-3CH did not decrease, but was slightly higher; however, the antigen content of pPink-1CH decreased by 50% after affinity purification( Figure 4 d). These results indicate that by inserting His6 tags on the surface of VLPs and utilizing the self-assembly ability between VP0, VP3, and VP1 of FMDV to assemble them into VLPs, it is feasible to achieve the purpose of purifying VLPs, and the effects of pPink-GHH and pPink-3CH are better than that of pPink-1CH.

Claims

1. A method for purifying foot-and-mouth disease virus-like particles (VLPs) expressed by Pichia pastoris by means of affinity chromatography, characterized in that, The method described includes expressing plasmids cloned with the foot-and-mouth disease virus structural protein VP0, VP3, and VP1-GHH genes, plasmids cloned with the foot-and-mouth disease virus structural protein VP0, VP3-CH, and VP1 genes, or plasmids cloned with the foot-and-mouth disease virus structural protein VP0, VP3, and VP1-CH genes through Pichia pastoris to obtain foot-and-mouth disease virus-like particles, and then purifying them by affinity chromatography; wherein the protein encoded by the VP1-GHH gene inserts a His-tag polypeptide (His6) at the 136th amino acid of the foot-and-mouth disease virus structural protein VP1, the protein encoded by the VP1-GHH gene inserts a His-tag polypeptide (His6) at the C-terminus of the foot-and-mouth disease virus structural protein VP1, and the protein encoded by the VP3-CH gene inserts a His-tag polypeptide (His6) at the C-terminus of the foot-and-mouth disease virus structural protein VP3.

2. The method according to claim 1, characterized in that, The nucleotide sequences of the VP0, VP3, VP1, VP1-GHH, VP3-CH, and VP1-CH genes are shown in SEQ ID NO.1-6 respectively.

3. The method according to claim 1, wherein The plasmids cloned with the foot-and-mouth disease virus structural protein VP0, VP3, and VP1-GHH genes, the plasmids cloned with the foot-and-mouth disease virus structural protein VP0, VP3-CH, and VP1 genes, or the plasmids cloned with the foot-and-mouth disease virus structural protein VP0, VP3, and VP1-CH genes are obtained by inserting their respective foot-and-mouth disease virus structural protein genes into the pPink-HC vector, and are named pPink-VP1-GHH / VP3 / VP0, pPink-VP1 / VP3-CH / VP0, and pPink-VP1-CH / VP3 / VP0 respectively.

4. The method according to claim 3, wherein The nucleotide sequence of the pPink-VP1-GHH / VP3 / VP0 plasmid is shown in SEQ ID NO.7, the nucleotide sequence of the pPink-VP1-CH / VP3 / VP0 plasmid is shown in SEQ ID NO.8, and the nucleotide sequence of the pPink-VP1 / VP3-CH / VP0 plasmid is shown in SEQ ID NO.

9.

5. The method according to any one of claims 1-3, characterized in that, It includes the following steps: (1) Express foot-and-mouth disease virus-like particles through Pichia pastoris 1) Plasmid construction Synthesize the VP0, VP3, and VP1 genes encoding the foot-and-mouth disease virus structural protein. At the same time, insert a His-tag polypeptide (His6) at the 136th amino acid of the protein encoded by the VP1 gene, denoted as VP1-GHH, insert a His-tag polypeptide (His6) at the C-terminus of the protein encoded by the VP1 gene, denoted as VP1-CH, and insert a His-tag polypeptide (His6) at the C-terminus of the protein encoded by the VP3 gene, denoted as VP3-CH; wherein the nucleotide sequences of the VP0, VP3, VP1, VP1-GHH, VP3-CH, and VP1-CH genes are shown in SEQ ID NO.1-6 respectively; 2) Transformation Insert VP0, VP3, VP1, VP1-GHH, VP3-CH, and VP1-CH into the pPink-HC vector respectively to construct plasmids pPink-VP0, pPink-VP3, pPink-VP1, pPink-VP1-GHH, pPink-VP3-CH, and pPink-VP1-CH. Then, insert the expression cassette of VP3 into the pPink-VP0 vector through Bgl II / BamH I to obtain the pPink-VP3 / VP0 plasmid. Next, insert the expression cassette of VP1-GHH into pPink-VP3 / VP0 to finally obtain pPink-VP1-GHH / VP3 / VP0 (GHH). Obtain the pPink-VP1 / VP3-CH / VP0 (3CH) plasmid and pPink-VP1-CH / VP3 / VP0 (1CH) plasmid in the same way. After linearizing the successfully constructed plasmids with Spe I, electrotransform them into Pichia pastoris strain 1; 3) Induced expression Inoculate the strains containing the plasmids pPink-VP1-GHH / VP3 / VP0 (GHH), pPink-VP1 / VP3-CH / VP0 (3CH), and pPink-VP1-CH / VP3 / VP0 (1CH) obtained in step 2) into BMGY medium and culture overnight until the OD 600 reaches between 2 and 6. Then, centrifuge to collect the cell pellet and transfer it respectively to BMMY medium with a pH of 7 to make the initial OD 600 reach 2. Subsequently, add 0.5% v / v methanol for induction and induce for 72 hours; (2) Purification of VLPs After induction, the strain is ultrasonically disrupted, and the supernatant is collected by centrifugation at 12,000 rpm for 30 min. Then, the supernatant is dialyzed overnight in the assembly buffer. The dialyzed sample is placed in a centrifuge at 4°C and centrifuged at 12,000 rpm for 30 min. After centrifugation, the supernatant is subjected to affinity chromatography. The impurity proteins are washed with a 10 mM imidazole solution, and the target protein is collected with a 300 mM imidazole solution. After ultrafiltration and concentration with PBS, the VLP content in the sample is detected.

6. The method according to claim 5, wherein The nucleotide sequence of the pPink-VP1-GHH / VP3 / VP0 plasmid is shown in SEQ ID NO.7, the nucleotide sequence of the pPink-VP1-CH / VP3 / VP0 plasmid is shown in SEQ ID NO.8, and the nucleotide sequence of the pPink-VP1 / VP3-CH / VP0 plasmid is shown in SEQ ID NO.

9.

7. The method according to claim 5, wherein The assembly buffer contains 300 mM NaCl, 10 mM tris, 50 mM KCl, 2 mM MgCl2, 1% v / v Triton X-100, 0.1 mM PMSF, pH 8.

0.

8. Foot-and-mouth disease virus-like particles prepared by the method according to any one of claims 1-7.

9. Use of the foot-and-mouth disease virus-like particles according to claim 8 in the preparation of reagents for detecting foot-and-mouth disease virus.

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