CN111303251B - A method for in vitro assembly of foot-and-mouth disease virus-like particles and its application - Google Patents
A method for in vitro assembly of foot-and-mouth disease virus-like particles and its application Download PDFInfo
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Abstract
本发明提供了一种口蹄疫病毒样颗粒体外组装的方法及其应用，涉及畜牧兽医技术领域。本发明利用口蹄疫病毒自身基因片段促进口蹄疫病毒样颗粒的体外组装，选择口蹄疫自身基因片段5′UTR和具有特殊二级结构的IRES作为组装病毒样颗粒的支架，模拟口蹄疫病毒的体内组装，以提高口蹄疫病毒样颗粒的体外组装效率。本发明所述5′UTR有利于病毒样颗粒(75S)的形成，而IRES更有利于五聚体(12S)的形成；病毒样颗粒对口蹄疫自身的基因片段具有保护作用；而且经组装后形成的组装产物与天然结构的病毒粒子结构类似，可用于制备口蹄疫相关疫苗。
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
Technical Field
The invention belongs to the technical field of animal husbandry and veterinary medicine, and particularly relates to a method for in-vitro assembly of foot-and-mouth disease virus-like particles and application thereof.
Background
Foot and Mouth Disease (FMD) is a highly contagious acute viral disease susceptible to artiodactyl animals. FMDV infection can cause acute or chronic asymptomatic, but persistent, signs of infection. The disease can limit the circulation and international trade of susceptible animals and products thereof, thereby causing huge economic loss to the breeding industry. At present, the inactivated vaccine is mainly used at home and abroad to carry out immune control on the foot-and-mouth disease, and has a better effect, but the vaccine may have incomplete inactivation in the production process, so that the immunized animal becomes a virus carrier and the biological safety problem of virus spreading exists. In addition, virus-like particles (VLPs) have great potential for use as a vaccine candidate in the form of a genetically engineered subunit that has recently emerged. The virus-like particles are hollow nanoparticles formed by self-assembly of structural proteins forming virus capsids, and have wide application prospects in the aspects of vaccine prevention and control, gene therapy, drug molecular vectors, material science and the like due to the characteristics of the virus-like particles.
The existing virus-like particles of foot-and-mouth disease virus produced by a prokaryotic expression system have low cost and simple and quick production process, but one of the challenges is that the assembly efficiency of the virus-like particles in an escherichia coli expression system is not very ideal. According to the research on icosahedral small RNA viruses, the assembly efficiency of virus-like particles of certain small RNA viruses in plant expression systems and eukaryotic expression systems is ideal, but the production cost is high.
Disclosure of Invention
In view of the above, the present invention aims to provide an in vitro assembly method of aftosa virus-like particles and an application thereof, wherein a foot-and-mouth disease self gene segment 5' UTR or IRES with a special secondary structure is used as a scaffold for assembling the virus-like particles to simulate the in vivo assembly of aftosa virus, so as to improve the in vitro assembly efficiency of the aftosa virus-like particles.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides an application of a foot-and-mouth disease virus self gene fragment in promoting foot-and-mouth disease virus-like particle in-vitro assembly, wherein the foot-and-mouth disease virus self gene fragment comprises 5'UTR or IRES, the nucleotide sequence of the 5' UTR is shown as SEQ ID NO.1, and the nucleotide sequence of the IRES is shown as SEQ ID NO. 2.
The invention provides a method for assembling foot-and-mouth disease virus-like particles in vitro, which comprises the following steps: mixing the foot-and-mouth disease virus-like particles with the gene fragment of the foot-and-mouth disease virus to form an assembly system, and then placing the assembly system in a dialysis bag of 10KDa for assembly; in the assembly system, the concentration ratio of the foot-and-mouth disease virus-like particles to the foot-and-mouth disease virus self gene fragments is 0.5 (3-20) multiplied by 10-5。
Preferably, the preparation method of the foot-and-mouth disease virus-like particles comprises the following steps: inducing recombinant escherichia coli containing the foot-and-mouth disease virus structural protein gene to express the foot-and-mouth disease virus structural protein, and carrying out ultrasonic crushing, extraction and purification, and then carrying out enzyme digestion assembly by utilizing SUMO to obtain the foot-and-mouth disease virus-like particles.
Preferably, the preparation method of the 5' UTR in the foot-and-mouth disease virus self gene fragment comprises the following steps: cloning the synthesized 5'UTR to pcDNA3.1+ vector, extracting plasmid of the cloned vector, and performing reverse transcription to obtain RNA fragment of the 5' UTR.
Preferably, the preparation method of the IRES in the foot-and-mouth disease virus self gene fragment comprises the following steps: and cloning the synthesized IRES to a pcDNA3.1+ vector, extracting a plasmid of the cloned vector, and performing reverse transcription to obtain the RNA fragment of the IRES.
Preferably, the synthesized gene is cloned between the NheI and BamHI cleavage sites of the pcDNA3.1+ vector.
The invention also provides the application of the assembly product obtained by the method in the preparation of foot-and-mouth disease vaccines.
The invention provides an application of a 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 5' UTR and IRES with a special secondary structure are selected as a bracket for assembling the virus-like particles, and the in-vivo assembly of the foot-and-mouth disease virus is simulated, so that the in-vitro assembly efficiency of the foot-and-mouth disease virus-like particles is improved. The embodiment proves that the foot-and-mouth disease self gene segment 5'UTR and IRES are used as a scaffold to induce the in vitro assembly of the foot-and-mouth disease virus-like particles, and the result shows that the 5' UTR is favorable for the formation of the virus-like particles (75S), and the IRES is more favorable for the formation of pentamer (12S); the virus-like particles have a protective effect on the gene fragment of the foot-and-mouth disease; and the assembled product formed after assembly is similar to the virus particle structure of the natural structure.
Drawings
FIG. 1 is the obtaining of foot and mouth disease virus-like particles, wherein M is 180KDa protein Marker, 1 is FMD structural protein (VP0-3) carrying His-Sumo tag, 2 is enzyme digestion assembly FMDV-VLP;
FIG. 2 is the acquisition of foot-and-mouth disease self gene segment 5'UTR, IRES, wherein M is 2K nucleic acid Marker, 1 is gene segment IRES, 2 is gene segment 5' UTR;
FIG. 3 is the verification of the co-assembly of the foot and mouth disease virus-like particle with the gene fragment 5'UTR, IRES of foot and mouth disease itself, wherein A is Zeta potential, 1 represents VLP, 2 represents VLP-5' UTR, 3 represents VLP-IRES; b is the difference of particle size; c is gel blocking and nuclease digestion profile, lane 1 shows 2K Marker, lane 2 shows VLP-5'UTR, lane 3 shows VLP-IRES, lane 4 shows 2K Marker, lane 5 shows VLP-5' UTR RnaseA digestion, lane 6 shows VLP-IRES RnaseA digestion;
FIG. 4 is a diagram showing the effect of size exclusion on the assembly of foot and mouth disease virus-like particles by 5'UTR and IRES, wherein A represents 146S purified by size exclusion, B represents VLP purified by size exclusion, C represents 5' UTR-VLP purified by size exclusion, D represents IRES-VLP purified by size exclusion, E represents peak area analysis of size exclusion, F represents 146S, 75S and 12S purified by size exclusion, G represents 146S observed by transmission electron microscope, H represents 75S observed by transmission electron microscope, and J represents 12S observed by transmission electron microscope.
Detailed Description
The invention provides an application of a foot-and-mouth disease virus self gene fragment in promoting foot-and-mouth disease virus-like particle in-vitro assembly, wherein the foot-and-mouth disease virus self gene fragment comprises 5'UTR or IRES, the nucleotide sequence of the 5' UTR is shown as SEQ ID NO.1, and the nucleotide sequence of the IRES is shown as SEQ ID NO. 2.
The foot-and-mouth disease self gene segment 5' UTR or the IRES with the special secondary structure can be used as a bracket 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, and after the assembly, the gene segment is used as the bracket to be wrapped inside the virus-like particles.
The invention provides a method for assembling foot-and-mouth disease virus-like particles in vitro, which comprises the following steps: after the foot-and-mouth disease virus-like particles and the foot-and-mouth disease virus self gene fragments are mixed into an assembly system, placing the assembly system in a dialysis bag of 10KDa for assembly; in the assembly system, the concentration ratio of the foot-and-mouth disease virus-like particles to the foot-and-mouth disease virus self gene fragments is 0.5 (3-20) multiplied by 10-5。
The preparation method of the foot-and-mouth disease virus-like particles preferably comprises the following steps: inducing recombinant escherichia coli containing the foot-and-mouth disease virus structural protein gene to express the foot-and-mouth disease virus structural protein, crushing, extracting and purifying, and performing enzyme digestion assembly by utilizing SUMO to obtain the foot-and-mouth disease virus-like particles. The method for 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 comprises the steps of inoculating a strain of the recombinant escherichia coli expressing the foot-and-mouth disease structural protein to an LB culture medium containing antibiotics, and culturing to OD600At 0.8, protein production was induced using IPTG. The volume ratio of the recombinant escherichia coli strain to the LB medium is preferably 1: 100. The LB culture medium comprises antibiotics, and the antibiotics comprise: 34mg/mL chloramphenicol, 50mg/mL ampicillin, and 10mg/mL kanamycin. The culture according to the present invention is preferably performed by shaking at 37 ℃ and 220rpmCulturing under shaking condition for 4 h. The final concentration of IPTG addition in the present invention is preferably 1 mmol/L. After the IPTG is added, the culture is preferably carried out for 16-18 h under the conditions of 16 ℃ and 200 rpm.
The crushing, extraction and purification of the present invention preferably comprises: the induced bacteria were collected by centrifugation at low temperature, resuspended in BufferA (300mmol/LNaCl, 40mmol/LTris-HCl, 5% glycerol, pH8.0) and disrupted by ultrasonication, then centrifuged at 11000rpm for 30min at 4 ℃ and the supernatant was transferred to Ni equilibrated with BufferA2+And (3) carrying out spin binding on the affinity chromatography resin in a chromatographic column at 4 ℃ for 1h, eluting the hybrid protein by using gradient Buffer C (5-30 mmol/L imidazole) for 10 column volumes respectively, and finally eluting the target protein by using Buffer B (500mmol/L imidazole, 300mmol/LNaCl, 40mmol/LTris-HCl, 5% glycerol, pH8.0) and collecting.
The obtained purified protein is assembled by utilizing SUMO enzyme digestion, preferably, the SUMO enzyme is added into the purified protein, the mixture is filled into a dialysis bag with the molecular weight cutoff of 10KD and is assembled by enzyme digestion overnight at 4 ℃ in an assembly Buffer D (300mmol/LNaCl, 20mmol/L Tris-HCl, pH 8.5) to obtain the foot-and-mouth disease virus-like particles. The invention preferably further comprises performing SDS-PAGE identification after said assembling.
The preparation method of the 5' UTR preferably comprises the following steps: and cloning the synthesized 5'UTR to a pcDNA3.1+ vector, extracting plasmids, and carrying out reverse transcription to obtain an RNA fragment of the 5' UTR. Preferably, the present invention entrusts Wuhan Kingkurui bioengineering Co., Ltd to prepare the 5' UTR. The present invention preferably clones the 5' UTR between NheI and BamHI cleavage sites on pcDNA3.1+ vector, then transforms it into a plate containing ampicillin to culture, and shakes it to extract plasmid for in vitro reverse transcription. In the in vitro reverse transcription of the invention, the plasmid obtained by the extraction is linearized by BamH1 and then gel is recovered as a template, preferably by using a T7 in vitro reverse transcription kit. The source of the kit for in vitro reverse transcription of T7 is not particularly limited in the present invention, and is preferably purchased from Promega corporation.
The preparation method of the IRES preferably comprises the following steps: the synthesized IRES is cloned to pcDNA3.1+ vector, plasmid is extracted and reverse transcription is carried out to obtain the fragment of the IRES. The preparation method of the IRES obtained by the present invention is completely the same as the preparation method of the 5' UTR (the synthesis of Wuhan Kingkunrei bioengineering Co., Ltd. is also entrusted), except that the cloning primers are different, and the details are not repeated herein.
The concentration ratio of the foot-and-mouth disease virus-like particles to the foot-and-mouth disease virus self gene fragments is preferably 0.5:3 × 10-5。
The invention also provides the application of the assembly product obtained by the method in the preparation of foot-and-mouth disease vaccines.
The following will explain the method for assembling foot-and-mouth disease virus-like particles in vitro and the application thereof provided by the present invention in detail with reference to the examples, but they should not be construed as limiting the scope of the present invention.
Example 1
1. Preparation of foot-and-mouth disease self-gene fragment and foot-and-mouth disease virus-like particle
Preparation of foot-and-mouth disease virus-like particles
The strain of Escherichia coli (BL21(DE3) pLysS recipient strain purchased from Solebao) expressing O type foot-and-mouth disease 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; and the nucleotide sequence of VP3 is shown in SEQ ID NO. 5) was inoculated into 1L autoclaved LB (10g sodium chloride, 10g tryptone, 5g yeast powder) medium containing 34mg/mL chloramphenicol, 50mg/mL ampicillin, 10mg/mL kanamycin at 37 ℃ and 220rpm, and cultured for about 4 hours at OD 100(μ L)600When the concentration reached about 0.8, an Inducer (IPTG) was added to the mixture at a final concentration of 1 mmol/L. Then culturing for 16-18 h at the temperature of 16 ℃ and at the rpm of 200. Centrifuging at low temperature, resuspending the strain in BufferA (300mmol/LNaCl, 40mmol/LTris-HCl, 5% glycerol, pH8.0) and disrupting the strain with a sonicator, then centrifuging at 11000rpm at 4 ℃ for 30min, transferring the supernatant to Ni equilibrated with BufferA2+Binding with affinity chromatography resin column at 4 deg.C for 1 hr, eluting with gradient Buffer C (5-30 mmol/L imidazole) for 10 column volumes each to elute heteroproteins, and eluting with Buffer B (500 mmol/imidazole, 300mmol/LNaCl, 40mmol/L Tris-HCl, 5% glycineOil, pH8.0) eluting the protein of interest and collecting. After the concentration is determined, glycerol is added for storage, and the solution is used for subsequent experiments. Adding the purified protein into SUMO enzyme, mixing, filling into a dialysis bag with cut-off molecular weight of 10KD, and performing enzyme digestion in assembly Buffer D (300mmol/L NaCl, 20mmol/L Tris-HCl, pH 8.5) at 4 ℃ overnight for assembly. After assembly, foot-and-mouth disease virus-like particles were obtained and characterized by SDS-PAGE (FIG. 1).
Secondly, 5' UTR and IRES genes are respectively synthesized and cloned to pcDNA3.1+ vector, and the cloning site is 5' NheI-3 ' BamHI.
The plasmids of gene fragment 5' UTR and IRES were transformed into a plate containing ampicillin and cultured, and plasmids were extracted by shake culture for in vitro reverse transcription. The 5' UTR and IRES plasmids were linearized with BamH1 and the gel was recovered as a template for in vitro reverse transcription, and the target RNA fragment was obtained by the procedure of T7 kit for in vitro reverse transcription (available from Promega corporation) and subjected to denaturing agarose gel electrophoresis to identify the target gene fragment (FIG. 2).
2. In-vitro co-assembly and identification of foot-and-mouth disease virus-like particles and foot-and-mouth disease self-gene fragments
The assembly of individual virus-like particles was used as a control, and the co-assembly of virus-like particles with the foot-and-mouth disease self gene fragment 5' UTR, IRES was used as a test group, and the amounts of virus-like particles in these three groups were identical except for the addition of the test group gene fragment. The concentration of virus-like particles is set to be 0.5mg/ml, and the concentration of nucleic acid after being evenly mixed (or vortexed) by blowing after adding 5' UTR and IRES of the foot-and-mouth disease self gene fragment is 30 ng/mu l. Then the mixed sample is put into a dialysis bag of 10KDa for dialysis assembly (4 ℃, the rotating speed of a magnetic stirrer is 350r/min), and the assembly is carried out for 28 hours. After the assembled product was collected and filtered using a 0.22 μm filter, the potentials and particle sizes of the three samples were measured using a Nano-particle sizer (Nano ZS).
Under the same assembly conditions, the Zeta potential measured after the assembly of the virus-like particles for 28h was-3.85 mV, the Zeta potential measured after the co-assembly of the virus-like particles with 5' UTR for 28h was-7.27 mV, the Zeta potential measured after the co-assembly of the virus-like particles with IRES for 28h was-9.135 mV (A in FIG. 3), and the potential of the co-assembly product of the virus-like particles and the gene segments was greater (absolute) than the potential of the virus-like particles, indicating the co-assembly of the virus-like particles and the nucleic acid segments.
Under the same assembly conditions, the Size measured after the assembly of the simplex virus-like particles for 28h was similar to the Size measured after the co-assembly of the virus-like particles with 5' UTR for 28h, the Size measured after the co-assembly of the virus-like particles with IRES for 28h was within the range of 20-35 nm (B in FIG. 3), indicating that the morphology and Size of the assembled product were similar.
Then, gel blocking experiments and nuclease digestion experiments are carried out for verification (C in FIG. 3), 20 μ l of each assembly product is mixed with 4 μ l of 6 × Loading and added into formaldehyde-denatured agarose gel, electrophoresis is carried out in 1 × MOPS buffer solution containing 3% formaldehyde (75V, 35min), and then observation is carried out on a gel ultraviolet imager, as the virus-like particles are assembled with the gene fragment of the foot-and-mouth disease, the gene fragment is wrapped inside the virus-like particles as a scaffold, and gel blocking phenomenon occurs in the agarose gel electrophoresis experiments, namely, the assembly product is retained in a sample adding hole ( lanes 2 and 5 of C in FIG. 3). At the same time, 30. mu.l of each assembly product is added with a proper amount of nuclease (RnaseA), incubated for 1 hour at 37 ℃, 20. mu.l of each undigested and digested assembly product is mixed with 4. mu.l of 6 Xloading and added into formaldehyde-denatured agarose gel to be electrophoresed in 1 XMOPS buffer solution containing 3% formaldehyde (75V, 35min), and then observed on a gel ultraviolet imager, because the gene fragment is wrapped inside the virus-like particle as a scaffold, nuclease digestion is not complete, further proving that the virus-like particle has protective effect on the gene fragment of the foot-and-mouth disease ( lanes 3 and 6 of C in FIG. 3).
3. Effect of foot-and-mouth disease self-gene fragments on the Assembly of Virus-like particles
Each assembly product was purified by Size Exclusion (SEC) separation (using Sephacryl high resolution gel column (GE Healthcare) on protein purifier aktap (GE Healthcare)), while inactivated FMDV (146S) was purified as a control (a in fig. 4) to determine the peak position of the virus-like particles (position marked with 146S in the figure).
OD in peak profile based on VLP-5' UTR, VLP-IRES and VLP260And OD280Difference (OD)260>OD280) It can be seen that the foot-and-mouth disease virus-like particles were co-assembled with the gene fragment of foot-and-mouth disease itself (B, C and D in FIG. 4).
Finally, the peak area analysis by peak spectrogram calculation of SEC compares the assembling effect of VLP-5' UTR, VLP-IRES and VLP assembling product, and finds that the co-assembly of virus-like particles and gene fragments is helpful for 75S and 12S assembly (E in figure 4).
Further, the different peak products separated and purified by size exclusion were subjected to circular dichroism spectroscopy, and the curves of the secondary structures of the separated virus-like particle (75S) and inactivated FMDV (146S) were found to be very close, demonstrating that the VLP-5' UTR, VLP-IRES assembly products are structurally similar to the naturally structured virions (F in FIG. 4).
The VLP-5' UTR, the assembled product of VLP-IRES and VLP were isolated and purified by SEC using transmission electron microscopy (G, H and J in FIG. 4), wherein 146S is a solid particle with a size of about 25nm, 75S is a hollow particle with a size similar to 146S, and 12S is a hollow particle with a size of about 15 nm.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> Lanzhou veterinary research institute of Chinese academy of agricultural sciences
<120> method for in vitro assembly of foot-and-mouth disease virus-like particles and application thereof
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Claims (4)
1. The method for assembling the foot-and-mouth disease virus-like particles in vitro is characterized by comprising the following steps: after the foot-and-mouth disease virus-like particles and the foot-and-mouth disease virus self gene fragments are mixed into an assembly system, placing the assembly system in a dialysis bag of 10KDa for assembly; in the assembly system, the concentration ratio of the foot-and-mouth disease virus-like particles to the foot-and-mouth disease virus self gene fragment is 0.5 (3-20) x 10-5；
The foot-and-mouth disease virus gene segment comprises 5'UTR or IRES, the nucleotide sequence of the 5' UTR is shown as SEQ ID NO.1, and the nucleotide sequence of the IRES is shown as SEQ ID NO. 2;
the preparation method of the 5' UTR comprises the following steps: cloning the synthesized 5'UTR to a pcDNA3.1+ vector, extracting plasmid of the cloned vector, and performing reverse transcription to obtain an RNA fragment of the 5' UTR;
a method of preparing the IRES, comprising: and cloning the synthesized IRES to a pcDNA3.1+ vector, extracting a plasmid of the cloned vector, and performing reverse transcription to obtain the RNA fragment of the IRES.
2. The method according to claim 1, wherein said foot-and-mouth disease virus-like particles are prepared by a method comprising: inducing recombinant escherichia coli containing the foot-and-mouth disease virus structural protein gene to express the foot-and-mouth disease virus structural protein, and carrying out ultrasonic crushing, extraction and purification, and then carrying out enzyme digestion assembly by utilizing SUMO to obtain the foot-and-mouth disease virus-like particles.
3. The method of claim 1, wherein the synthesized gene is cloned between NheI and BamHI cleavage sites of pcDNA3.1+ vector.
4. Use of the assembly product prepared by the method of any one of claims 1 to 3 in the preparation of a foot and mouth disease vaccine.
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| 口蹄疫病毒病毒样颗粒研究进展;张庆勋等;《中国畜牧兽医》;万方;20160506;第41卷(第11期);第258-262页 * |
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