CN111303251A - A method for in vitro assembly of foot-and-mouth disease virus-like particles and its application - Google Patents

Abstract

The invention provides a method for assembling foot-and-mouth disease virus-like particles in vitro and application thereof, and relates to the technical field of animal husbandry and veterinary medicine. In the present invention, the self-gene fragment of foot-and-mouth disease virus is used to promote the in vitro assembly of the foot-and-mouth disease virus-like particle, and the 5'UTR of the foot-and-mouth disease self-gene fragment and the IRES with special secondary structure are selected as the scaffold for assembling the virus-like particle, and the in vivo assembly of the foot-and-mouth disease virus is simulated to improve the In vitro assembly efficiency of foot-and-mouth disease virus-like particles. The 5'UTR of the present invention is conducive to the formation of virus-like particles (75S), while IRES is more conducive to the formation of pentamers (12S); the virus-like particles have a protective effect on the gene fragments of foot-and-mouth disease itself; and formed after assembly The assembled product is similar to the virion structure of the natural structure, and can be used to prepare a vaccine related to foot-and-mouth disease.

Description

A method for in vitro assembly of foot-and-mouth disease virus-like particles and its application

technical field

The invention belongs to the technical field of animal husbandry and veterinary medicine, and particularly relates to a method for assembling foot-and-mouth disease virus-like particles in vitro and its application.

Background technique

Foot-and-mouth disease (FMD) is a highly contagious acute viral disease of susceptible cloven-hoofed animals. FMDV infection can cause acute or long-term asymptomatic but persistent infection. Because the disease can restrict the circulation and international trade of susceptible animals and their products, it has caused huge economic losses to the breeding industry. At present, inactivated vaccines are mainly used at home and abroad to prevent and control foot-and-mouth disease, which has good results. However, such vaccines may have incomplete inactivation during the production process, resulting in the immunized animals becoming virus carriers and the biological safety of spreading the virus. question. In addition, virus-like particles (VLPs) have great potential as a new candidate vaccine in the form of genetically engineered subunits in recent years. Virus-like particles are hollow nanoparticles formed by self-assembly of structural proteins that make up the viral capsid. Due to their own characteristics, virus-like particles have broad application prospects in vaccine prevention and control, gene therapy, drug molecular carriers, and materials science.

At present, the virus-like particles of foot-and-mouth disease virus produced by prokaryotic expression system have low cost and simple and rapid production process, but one challenge is that the assembly efficiency of virus-like particles in E. coli expression system is not very ideal. According to the research on icosahedral picornaviruses, the assembly efficiency of virus-like particles of some picornaviruses in plant expression systems and eukaryotic expression systems is ideal, but the production cost is high.

SUMMARY OF THE INVENTION

In view of this, the object of the present invention is to provide a method for assembling FMD virus-like particles in vitro and its application, using the FMD self gene fragment 5'UTR or an IRES with a special secondary structure as a scaffold for assembling virus-like particles, Mimic the in vivo assembly of foot-and-mouth disease virus to improve the in vitro assembly efficiency of foot-and-mouth disease virus-like particles.

In order to achieve the above-mentioned purpose of the invention, the present invention provides the following technical solutions:

The present invention provides the application of foot-and-mouth disease virus self-gene fragment in promoting the in vitro assembly of foot-and-mouth disease virus-like particles, wherein the foot-and-mouth disease virus self-gene fragment comprises 5'UTR or IRES, and the nucleotide sequence of the 5'UTR is as shown in SEQ ID NO.1 As shown, the nucleotide sequence of the IRES is shown in SEQ ID NO.2.

The invention provides a method for in vitro assembly of foot-and-mouth disease virus-like particles, comprising the following steps: after mixing the foot-and-mouth disease virus-like particles with the gene fragments of the foot-and-mouth disease virus itself to form an assembly system, placing them in a 10KDa dialysis bag for assembly; In the assembly system, the concentration ratio of the foot-and-mouth disease virus-like particles and the gene fragments of the foot-and-mouth disease virus itself is 0.5:(3-20)×10 ^-5 .

Preferably, the preparation method of the FMD virus-like particles comprises: inducing the recombinant Escherichia coli containing the FMD virus structural protein gene to express the FMD virus structural protein, and after ultrasonication, extraction and purification, SUMO digestion and assembly are used to obtain the obtained result. Foot-and-mouth disease virus-like particles.

Preferably, the preparation method of the 5'UTR in the gene fragment of the foot-and-mouth disease virus itself comprises: cloning the synthesized 5'UTR into a pcDNA3.1+ vector, extracting the plasmid of the cloned vector and performing reverse transcription, Obtain the RNA fragment of the 5'UTR.

Preferably, the preparation method of the IRES in the gene fragment of the foot-and-mouth disease virus itself comprises: cloning the synthesized IRES into a pcDNA3.1+ vector, extracting the plasmid of the cloned vector and performing reverse transcription to obtain the IRES RNA fragments.

Preferably, the synthesized gene is cloned between the NheI and BamHI restriction sites of the pcDNA3.1+ vector.

The invention also provides the application of the assembled product obtained by the method in the preparation of a foot-and-mouth disease vaccine.

The invention provides the application of foot-and-mouth disease virus self-gene fragment in promoting the in vitro assembly of foot-and-mouth disease virus-like particles. The 5'UTR of the foot-and-mouth disease self-gene fragment and IRES with special secondary structure are selected as scaffolds for assembling virus-like particles, and the in vivo simulation of the foot-and-mouth disease virus is provided. assembly to improve the in vitro assembly efficiency of foot-and-mouth disease virus-like particles. It is verified by the examples that the 5'UTR and IRES of the foot-and-mouth disease self-gene fragment are used as scaffolds to induce the in vitro assembly of the FMD virus-like particles, and it is found that the 5'UTR is conducive to the formation of virus-like particles (75S), while the IRES is more conducive to the pentamer. (12S) formation; virus-like particles have a protective effect on the gene fragments of foot-and-mouth disease; and the assembled product formed after assembly is similar to the natural structure of the virus particle structure.

Description of drawings

Figure 1 shows the acquisition of foot-and-mouth disease virus-like particles, wherein M is a 180KDa protein Marker, 1 is an FMD structural protein (VP0-3) carrying a His-Sumo tag, and 2 is an enzyme digestion to assemble FMDV-VLP;

Fig. 2 is the acquisition of foot-and-mouth disease self-gene fragment 5'UTR, IRES, wherein M is 2K nucleic acid Marker, 1 is gene fragment IRES, and 2 is gene fragment 5'UTR;

Fig. 3 is the verification after the FMD virus-like particle co-assembles with the gene fragment 5'UTR and IRES of FMD itself, where A is Zeta potential, 1 is VLP, 2 is VLP-5'UTR, 3 is VLP-IRES; B is Particle size difference; C is the graph of gel retardation and nuclease digestion, lane 1 represents 2K Marker, lane 2 represents VLP-5'UTR, lane 3 represents VLP-IRES, lane 4 represents 2K Marker, and lane 5 represents VLP-5 'UTR RnaseA digestion, lane 6 represents VLP-IRES RnaseA digestion;

Figure 4 shows the effects of 5'UTR and IRES on the assembly of FMD virus-like particles by using size exclusion method to separate and purify the assembled product, wherein A represents 146S separated and purified by size exclusion, and B represents 146S separated and purified by size exclusion. VLP, C represents 5'UTR-VLP purified by size exclusion separation, D represents IRES-VLP purified by size exclusion separation, E represents peak area analysis of size exclusion separation and purification, F represents circular dichroism spectrum analysis Size exclusion separation and purification of 146S, 75S and 12S, G represents 146S observed by TEM, H represents 75S observed by TEM, J represents 12S observed by TEM.

Detailed ways

The present invention provides the application of foot-and-mouth disease virus self-gene fragment in promoting the in vitro assembly of foot-and-mouth disease virus-like particles, wherein the foot-and-mouth disease virus self-gene fragment comprises 5'UTR or IRES, and the nucleotide sequence of the 5'UTR is as shown in SEQ ID NO.1 As shown, the nucleotide sequence of the IRES is shown in SEQ ID NO.2.

The 5'UTR of the foot-and-mouth disease self-gene fragment or the IRES with special secondary structure of the present invention can be used as a scaffold for assembling virus-like particles to simulate the in vivo assembly of the foot-and-mouth disease virus, so as to improve the in vitro assembly efficiency of the foot-and-mouth disease virus-like particles. After assembly, the gene fragments are encapsulated inside virus-like particles as scaffolds.

The invention provides a method for in vitro assembly of foot-and-mouth disease virus-like particles, comprising the following steps: after mixing the foot-and-mouth disease virus-like particles with the gene fragments of the foot-and-mouth disease virus itself to form an assembly system, put them in a 10KDa dialysis bag for assembly; In the assembly system, the concentration ratio of the foot-and-mouth disease virus-like particles and the gene fragments of the foot-and-mouth disease virus itself is 0.5:(3-20)×10 ^-5 .

The preparation method of the foot-and-mouth disease virus-like particle of the present invention preferably includes: inducing a recombinant Escherichia coli containing the foot-and-mouth disease virus structural protein gene to express the foot-and-mouth disease virus structural protein, and after crushing, extracting and purifying, using SUMO to cut and assemble to obtain the Foot-and-mouth disease virus-like particles. According to the present invention, inducing the recombinant Escherichia coli containing the foot-and-mouth disease virus structural protein gene to express the foot-and-mouth disease virus structural protein preferably includes connecting the strain of the recombinant Escherichia coli expressing the foot-and-mouth disease structural protein to the LB medium containing antibiotics, and culturing to OD ₆₀₀ At 0.8, IPTG was used to induce protein production. The volume ratio of the recombinant Escherichia coli strain of the present invention to the LB medium is preferably 1:100. The LB medium of the present invention contains antibiotics, and the antibiotics include: 34 mg/mL chloramphenicol, 50 mg/mL ampicillin and 10 mg/mL kanamycin. The culture in the present invention is preferably cultured at 37°C and under shaking conditions of 220 rpm for 4 hours. The added final concentration of IPTG according to the present invention is preferably 1 mmol/L. In the present invention, after adding the IPTG, it is preferable to culture at 16° C. and 200 rpm for 16-18 hours.

Preferably, the crushing, extraction and purification of the present invention include: collecting the induced bacteria by centrifugation under low temperature conditions, resuspending the bacteria with Buffer A (300mmol/LNaCl, 40mmol/L Tris-HCl, 5% glycerol, pH8.0) and using The cells were broken by ultrasonication, and then centrifuged at 11,000 rpm for 30 min at 4 °C. The supernatant was transferred to a column of Ni ²⁺ affinity chromatography resin equilibrated with Buffer A, and then rotated at 4 °C for 1 h, using gradient Buffer C. (5～30mmol/L imidazole) wash 10 column volumes each to elute the impurity protein, and finally use Buffer B (500mmol/L imidazole, 300mmol/LNaCl, 40mmol/LTris-HCl, 5% glycerol, pH8.0) to elute the target protein and collect.

In the present invention, the obtained purified protein is digested and assembled by SUMO, preferably including adding SUMO enzyme to the purified protein, mixing and then loading it into a dialysis bag with a molecular weight cut-off of 10KD, and assembling Buffer D (300mmol/LNaCl, 20mmol/L) Tris-HCl, pH 8.5) was digested overnight at 4°C and assembled to obtain the FMD virus-like particles. The present invention preferably further includes performing SDS-PAGE identification after the assembly.

The method for preparing the 5'UTR of the present invention preferably includes: cloning the synthesized 5'UTR into a pcDNA3.1+ vector, extracting the plasmid and performing reverse transcription to obtain the RNA fragment of the 5'UTR. The present invention preferably entrusts Wuhan Jinkarui Bioengineering Co., Ltd. to complete the preparation of the 5'UTR. In the present invention, the 5'UTR is preferably cloned between the NheI and BamHI restriction sites on the pcDNA3.1+ vector, and then transformed into a plate containing ampicillin for culture, and the plasmid is extracted by shaking bacteria for in vitro reverse transcription. When performing the in vitro reverse transcription in the present invention, the plasmid obtained by the above extraction is linearized with BamH1 and recovered as a template, preferably using a T7 in vitro reverse transcription kit. The source of the T7 in vitro reverse transcription kit is not particularly limited in the present invention, and it is preferably purchased from Promega.

The preparation method of the IRES of the present invention preferably includes: cloning the synthesized IRES into a pcDNA3.1+ vector, extracting the plasmid and performing reverse transcription to obtain the fragment of the IRES. Except for the different cloning primers, the preparation method of the present invention to obtain the IRES is exactly the same as the preparation method of the above-mentioned 5'UTR (also entrusted to Wuhan Jinkarui Bioengineering Co., Ltd. to synthesize), which will not be repeated here.

The concentration ratio of the foot-and-mouth disease virus-like particles of the present invention and the gene fragments of the foot-and-mouth disease virus itself is preferably 0.5:3×10 ⁻⁵ .

The invention also provides the application of the assembled product obtained by the method in the preparation of a foot-and-mouth disease vaccine.

The method for in vitro assembly of the FMD virus-like particles provided by the present invention and its application are described in detail below with reference to the examples, but they should not be construed as limiting the protection scope of the present invention.

Example 1

1. Preparation of FMD gene fragments and FMD virus-like particles

① Preparation of foot-and-mouth disease virus-like particles

According to the ratio of 1:100 (μl), the O-type FMD structural protein (wherein the nucleotide sequence of VP0 is shown in SEQ ID NO.3; the nucleotide sequence of VP1 is shown in SEQ ID NO.4; the nucleotide sequence of VP3 is shown in SEQ ID NO. The bacterial strain of Escherichia coli (BL21(DE3)pLysS acceptor strain was purchased from Solebao) whose nucleotide sequence is shown in SEQ ID NO. /mL kanamycin in 1L autoclaved LB (10g sodium chloride, 10g tryptone, 5g yeast powder) medium at 37°C, 220rpm for about 4h, when the OD ₆₀₀ reaches about 0.8, add the final Inducer (IPTG) at a concentration of 1 mmol/L. After that, the cells were cultured at 200 rpm for 16-18 h at 16°C. The bacteria were harvested by low-temperature centrifugation, resuspended in Buffer A (300mmol/LNaCl, 40mmol/L Tris-HCl, 5% glycerol, pH 8.0) and disrupted by a sonicator, then centrifuged at 4°C and 11000rpm for 30min, and the supernatant was transferred to In the chromatography column of Ni ²⁺ affinity chromatography resin equilibrated with Buffer A, rotate and bind at 4°C for 1 h, wash with gradient Buffer C (5-30 mmol/L imidazole) for 10 column volumes each to elute the impurity protein, and finally use Buffer B (500 mmol/imidazole, 300 mmol/L NaCl, 40 mmol/L Tris-HCl, 5% glycerol, pH 8.0) eluted the target protein and collected it. After the concentration is determined, add glycerol and store it for subsequent experiments. The above-mentioned purified protein was added to SUMO enzyme, mixed and put into a dialysis bag with a molecular weight cut-off of 10KD, and then assembled overnight at 4° C. in assembly Buffer D (300 mmol/L NaCl, 20 mmol/L Tris-HCl, pH 8.5). After assembly, FMD virus-like particles were obtained and identified by SDS-PAGE (Fig. 1).

②The 5'UTR and IRES genes were synthesized and cloned into pcDNA3.1+ vector, the cloning site was 5'NheI-3'BamHI.

The plasmids of gene fragments 5'UTR and IRES were transformed into plates containing ampicillin, and the plasmids were extracted by shaking bacteria for in vitro reverse transcription. The 5'UTR and IRES plasmids were linearized with BamH1 and recovered as templates for in vitro reverse transcription. The target RNA fragments were obtained according to the instructions of the T7 in vitro reverse transcription kit (purchased from Promega), and were subjected to denaturing agarose gelation. The target gene fragment was identified by gel electrophoresis (Figure 2).

2. In vitro co-assembly and identification of FMD virus-like particles and FMD gene fragments

The assembly of the virus-like particles alone was used as a control, and the co-assembly of the virus-like particles with the 5′UTR and IRES gene fragments of foot-and-mouth disease itself was used as the test group. The amount of virus-like particles in these three groups was the same, except for the addition of gene fragments in the test group. . The concentration of virus-like particles was set as 0.5mg/ml, and the nucleic acid concentration after adding FMD self gene fragment 5'UTR and IRES after pipetting and mixing (or vortexing) were all 30ng/μl. Then, the mixed samples were put into a 10KDa dialysis bag for dialysis assembly (4° C., magnetic stirrer rotation speed 350 r/min), and assembly was performed for 28 h. After the assembled products were collected and filtered through a 0.22 μm filter, the potential and particle size of the three samples were determined using a Nano ZS.

Under the same assembly conditions, the zeta potential measured after 28h of VLP assembly was -3.85mV, the zeta potential measured after 28h co-assembly of VLP and 5'UTR was -7.27mV, and the VLP and IRES co-assembled The Zeta potential measured after 28h was -9.135mV (A in Figure 3), the potential of the co-assembly product of virus-like particles and gene fragments was larger than that of pure virus-like particles (absolute value), indicating that virus-like particles and nucleic acid fragments co-assembly.

Under the same assembly conditions, the size determined after 28h of assembly of pure virus-like particles was similar to the size determined after 28h of co-assembly of virus-like particles and 5′UTR, and the size determined after 28h of co-assembly of virus-like particles and IRES. Within the range of 20-35 nm (B in Figure 3), it shows that the morphology and size of the assembled products are all similar.

Afterwards, gel retardation experiments and nuclease digestion experiments were performed to verify (C in Figure 3), and 20 μl of each assembled product was mixed with 4 μl of 6×Loading and added to a formaldehyde-denatured agarose gel in 1×MOPS buffer containing 3% formaldehyde. Electrophoresis (75V, 35min) was carried out in the liquid, and then observed on a gel UV imager. Since the virus-like particles co-assembled with the gene fragments of FMD itself, the gene fragments were encapsulated inside the virus-like particles as scaffolds, and were displayed on agarose gel. Gel retardation occurs in electrophoresis experiments, that is, the assembled product remains in the wells (lanes 2 and 5 of C in Figure 3). At the same time, take 30 μl of each assembly product, add an appropriate amount of nuclease (RnaseA), incubate at 37°C for 1 hour, take 20 μl of each undigested and digested assembly product and mix 4 μl of 6×Loading and add it to a formaldehyde-denatured agarose gel containing Electrophoresis was performed in 1×MOPS buffer of 3% formaldehyde (75V, 35min), and then observed on a gel UV imager, because the gene fragment was encapsulated inside the virus-like particle as a scaffold, and the nuclease digestion was incomplete, which further proved that the virus The like particles were protective against the gene fragment of FMD itself (lanes 3 and 6 of C in Figure 3).

3. The effect of FMD's own gene fragments on the assembly of virus-like particles

The assembled products were separated and purified by size exclusion method (SEC) (separated and purified on a protein purifier AKTApure (GE Healthcare) using a Sephacryl high-resolution gel column (GE Healthcare)), and at the same time, the inactivated FMDV (146S) was separated and purified. As a control (A in Figure 4), the peak position of virus-like particles (the position marked 146S in the figure) was determined.

According to the difference of OD ₂₆₀ and OD ₂₈₀ in the peak spectra of VLP-5′UTR, VLP-IRES and VLP (OD ₂₆₀ >OD ₂₈₀ ), it can be seen that the FMD virus-like particle co-assembles with the gene fragments of FMD itself (Fig. 4). B, C and D).

Finally, the SEC peak spectrum was used to calculate the peak area analysis to compare the assembly effect of VLP-5′UTR, VLP-IRES and VLP assembly products. middle E).

In addition, the different peak products purified by size exclusion were subjected to circular dichroism spectroscopy analysis, and it was found that the curve of the secondary structure of the isolated virus-like particles (75S) was very close to that of the inactivated FMDV (146S), which proved that VLP-5′ The assembled product of UTR and VLP-IRES is similar to the virion structure of the natural structure (F in Figure 4).

The assembly products of VLP-5'UTR, VLP-IRES and VLP were separated and purified by SEC (G, H and J in Figure 4), and 146S was a solid particle with a size of about 25 nm, and 75S was similar in size to 146S. 12S is a hollow particle with a size of about 15nm.

The above are only the preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, without departing from the principles of the present invention, several improvements and modifications can be made. It should be regarded as the protection scope of the present invention.

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cagtaa 666

Claims (7)

1. the application of the gene fragment of foot-and-mouth disease virus itself in promoting the in vitro assembly of foot-and-mouth disease virus-like particles, the gene fragment of the foot-and-mouth disease virus itself comprises 5 ' UTR or IRES, and the nucleotide sequence of the 5 ' UTR is such as SEQ ID NO. 1, the nucleotide sequence of the IRES is shown in SEQ ID NO.2. 2. a method for assembling foot-and-mouth disease virus-like particles in vitro, it is characterized in that comprising the following steps: after foot-and-mouth disease virus-like particles and the described foot-and-mouth disease virus self-gene fragment of claim 1 are mixed into an assembly system, place in the dialysis bag of 10KDa and assemble ; In the assembly system, the concentration ratio of the foot-and-mouth disease virus-like particles to the gene fragments of the foot-and-mouth disease virus itself is 0.5:(3-20)×10 ^-5 . 3. method according to claim 2, is characterized in that, the preparation method of described foot-and-mouth disease virus-like particle comprises: inducing, the recombinant Escherichia coli that comprises the foot-and-mouth disease virus structural protein gene expresses the foot-and-mouth disease virus structural protein, through ultrasonic fragmentation, extraction After purification, the FMD virus-like particles are obtained by digesting and assembling with SUMO enzyme. 4. method according to claim 2 is characterized in that, the preparation method of the 5 ' UTR in described foot-and-mouth disease virus self-gene fragment, comprises: the described 5 ' UTR of synthesis is cloned on the pcDNA3.1+ carrier, extracts After cloning, the plasmid of the vector is reverse transcribed to obtain the RNA fragment of the 5'UTR. 5. method according to claim 2, is characterized in that, the preparation method of the IRES in described foot-and-mouth disease virus self gene fragment, comprises: the described IRES of synthesis is cloned on the pcDNA3.1+ carrier, extracting the back of the cloned carrier After the plasmid is reverse transcribed, the RNA fragment of the IRES is obtained. 6. The method according to claim 4 or 5, wherein the synthesized gene is cloned between the NheI and BamHI restriction sites of pcDNA3.1+ vector. 7. The application of the assembled product prepared by the method according to any one of claims 2 to 6 in the preparation of a foot-and-mouth disease vaccine.

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