CN116675778A - Recombinant protein, foot-and-mouth disease virus-like particle vaccine, and preparation method and application thereof - Google Patents
Recombinant protein, foot-and-mouth disease virus-like particle vaccine, and preparation method and application thereof Download PDFInfo
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- CN116675778A CN116675778A CN202310363668.1A CN202310363668A CN116675778A CN 116675778 A CN116675778 A CN 116675778A CN 202310363668 A CN202310363668 A CN 202310363668A CN 116675778 A CN116675778 A CN 116675778A
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Abstract
The invention discloses a recombinant protein, foot-and-mouth disease virus-like particle vaccine and a preparation method and application thereof, and belongs to the technical field of biological medicines. The invention successfully constructs pET-11a-Dps-VP1 recombinant plasmid, and the recombinant protein Dps-VP1 can be stably expressed by the induction expression of escherichia coli, and can be self-assembled into virus-like particles (about 9 nm) and can be stored for at least 6 months at room temperature; the recombinant protein Dps-VP1 is emulsified to prepare vaccine, and the vaccine can be used for immunizing a balb/c mouse (50 mug/mouse) and effectively inducing the mouse to generate FMDV specific antibodies.
Description
Technical Field
The invention relates to the technical field of biological medicine, in particular to a recombinant protein, foot-and-mouth disease virus-like particle vaccine, and a preparation method and application thereof.
Background
Foot-and-mouth disease (FMD) is an acute, hot, highly contagious and rapidly spread animal epidemic disease in a long distance caused by Foot-and-mouth disease virus (Foot-and Mouth Disease Virus, FMDV), and the symptoms are blisters at the parts of animal mouth, nose, hooves, female animal nipple and the like, or ulcers or scabs formed after the blisters are broken, so that the animal mouth, nose, hooves, female animal nipple and the like can infect various main domestic animals such as pigs, cattle, sheep and the like, cause huge economic loss, seriously threaten the development of the global animal husbandry, and the world animal health Organization (OIE) also lists the animal epidemic disease as the first part of a class A animal epidemic disease list.
FMDV belongs to the genus foot-and-mouth disease virus of the family picornaviridae, and includes A, O, C, asia 1 and 7 serotypes of SAT1, SAT2, SAT3, etc. that are not cross-immunoprotected, there is no cross-protection between the serotypes, and genetic variation between different strains within a type can lead to antigenic differences, with type O, type a and type Asia 1 being the most prevalent serotypes. FMDV virions are about 20-30 nm in diameter and are icosahedral, and mainly comprise RNA and capsids, wherein the capsids mainly comprise VP1, VP2, VP3 and VP4 structural proteins, 1A, 1B, 1C and 1D 4 structural proteins are respectively encoded, and the 4 structural polypeptides participate in immunogenicity. Among them, the structural proteins VP1, VP2, VP3 are the main proteins constituting the viral capsid, and are also the most main antigen proteins, and most of the antigen epitopes (antigenic epitope) are located on FMDV capsid proteins, determining the antigenicity and immunogenicity of FMDV to a host. Structural protein VP1 is related to neutralizing antibody and virus infection, and is a main component for inducing neutralizing antibody, and is also key to antigenic site and antigenic variation.
The prevention and treatment of foot-and-mouth disease in China adopts the conventional inactivated vaccine for immunoprophylaxis, but adverse reactions such as anorexia, vomiting, body temperature rise and the like often occur after the immunization of the inactivated vaccine, and some of the adverse reactions even cause death and have the problem of biological potential safety hazard. Therefore, the development of a safer, more effective and more practical novel vaccine is a main hot spot for the research of the foot-and-mouth disease at present.
Disclosure of Invention
The invention aims to provide a recombinant protein, a foot-and-mouth disease virus-like particle vaccine, and a preparation method and application thereof, so as to solve the problems in the prior art.
In order to achieve the above object, the present invention provides the following solutions:
the invention provides a recombinant protein, and the amino acid sequence of the recombinant protein is shown as SEQ ID NO. 1.
Nucleotide sequence of Dps-VP1 gene of the coding recombinant protein.
Preferably, the nucleotide sequence is shown as SEQ ID NO. 2.
The invention also provides a recombinant plasmid, which is pET-11a-Dps-VP1 recombinant plasmid.
The invention also provides a foot-and-mouth disease virus-like particle vaccine comprising the recombinant protein of claim 1 and other pharmaceutically acceptable immunological adjuvants.
Preferably, the immunoadjuvant is an ISA201 adjuvant.
The invention also provides a preparation method of the foot-and-mouth disease virus-like particle vaccine, which comprises the following steps:
(1) Constructing pET-11a-Dps-VP1 recombinant plasmid;
(2) Introducing the recombinant plasmid into competent cells, and performing colony PCR identification;
(3) Amplifying and culturing the colony with positive identification result, and inducing the expression of the recombinant plasmid;
(4) Crushing the thalli and purifying to obtain the recombinant protein as defined in claim 1;
(5) Slowly adding the recombinant protein with the same volume into the adjuvant, swirling for 4-8min, standing at 18-22 ℃ for 40-80min, and obtaining the foot-and-mouth disease virus-like particle vaccine.
Preferably, the competent cells are competent BL21 cells.
Preferably, the primers identified by colony PCR comprise an upstream primer shown as SEQ ID NO.3 and a downstream primer shown as SEQ ID NO. 4.
Preferably, the culture medium for the expansion culture is LB liquid culture medium containing Amp.
Preferably, the induction of expression of the recombinant plasmid is specifically comprised in the culture medium OD 600 When reaching 0.5-0.7, IPTG with a final concentration of 1mM was added to induce expression at 37℃for 6h.
The invention also provides an application of the recombinant protein or the foot-and-mouth disease virus-like particle vaccine in preparing medicines for treating and/or preventing foot-and-mouth disease.
The invention discloses the following technical effects:
the DNA-binding protein from starved cells (Dps) is a nanoparticle protein present only in bacteria and archaea, consisting of The sequence of 2:3, wherein the twelve-polymer nano particles composed of 12 identical subunits are piled up, the diameter is about 9nm, the N end and the C end of the twelve-polymer nano particles can be combined with DNA at the same time, the function of combining DNA is lost at 65 ℃, but the twelve-polymer nano particles can not be dissociated with DNA even at 100 ℃ under the state of combining the twelve-polymer nano particles with DNA, and the DNA combining activity is maintained. Traditional antigens, especially multi-epitope combined antigens, have small molecular weight and poor immunogenicity, and are unfavorable for recognition and presentation of antigen molecules. Therefore, the invention finds the region which has the least influence on the structure and the function and is positioned on the outer surface in the dodecamer structure by analyzing the gene sequence of the Dps protein, inserts the exogenous antigen gene sequence, expresses and prepares the epitope fusion antigen molecule, displays the antigen molecule on the surface of the dodecamer by self-assembly to form the granular antigen with extremely strong immunogenicity, not only increases the contact area of the antigen molecule and the antibody, but also increases the molecular weight of the antigen, thereby improving the immunogenicity of the antigen and being beneficial to exciting the immune response of an organism.
The invention successfully constructs pET-11a-Dps-VP1 recombinant plasmid, and the recombinant protein Dps-VP1 can be stably expressed by the induction expression of escherichia coli, and can be self-assembled into virus-like particles (about 9 nm) and can be stored for at least 6 months at room temperature; the recombinant protein Dps-VP1 is emulsified to prepare vaccine, and the vaccine can be used for immunizing a balb/c mouse (50 mug/mouse) and effectively inducing the mouse to generate FMDV specific antibodies.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the restriction enzyme digestion of recombinant plasmids pET-11a-Dps-VP1, pET-11a-Dps; wherein 1 represents a DNA Marker; 2. 4 is double enzyme cutting of recombinant plasmids pET-11a-Dps-VP1 and pET-11a-Dps; 3.5 is recombinant plasmids pET-11a-Dps-VP1 and pET-11a-Dps;
FIG. 2 is a PCR identification of colonies pET-11a-Dps-VP1, pET-11a-Dps; wherein 1 represents a DNA Marker;2 represents ddH 2 O;3 represents BL21 (DE 3); 4 represents pET-11a;5-8 represent Dps-VP1, lane 9-12 and Dps;
FIG. 3 is SDS-PAGE identification of recombinant protein Dps-VP1, dps supernatant; wherein 1 represents a protein Marker;2 represents Dps-VP1 is not induced; 3 represents Dps-VP1 induction; 4 represents Dps is not induced; 5 represents Dps induction;
FIG. 4 shows Western blot identification of recombinant proteins Dps-VP1 and Dps; wherein 1 represents a protein Marker;2-3 represents recombinant proteins Dps-VP1 and Dps;
FIG. 5 shows the purification results of recombinant proteins Dps-VP1 and Dps by Ni-NTA; wherein a is the SDS-PAGE result of the flowing liquid after the combination of the recombinant protein Dps-VP1 and the Ni-NTA, b is the SDS-PAGE result of the collected liquid after the elution of the recombinant protein Dps-VP1 by 500mM imidazole, c is the SDS-PAGE result of the flowing liquid after the combination of the recombinant protein Dps and the Ni-NTA, and d is the SDS-PAGE result of the collected liquid after the elution of the recombinant protein Dps by 400mM imidazole;
FIG. 6 shows transmission electron microscope identification of recombinant proteins Dps-VP1, dps; wherein a is recombinant protein Dps-VP1; b is recombinant protein Dps;
FIG. 7 shows HE staining results of different organ sections of mice;
FIG. 8 is a graph showing the results of body weight monitoring after immunization of mice;
FIG. 9 is the detection levels of mouse specific antibodies;
FIG. 10 shows stability assays for recombinant proteins Dps-VP1 and Dps.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, with reference to the examples using conventional methods, unless otherwise indicated, and with reference to reagents, either conventional commercial reagents or reagents configured using conventional methods. The detailed description is not to be taken as limiting, but is to be understood as a more detailed description of certain aspects, features, and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
Example 1
1.1 Gene Synthesis and construction of recombinant plasmid pET-11a-Dps-VP1/pET-11a-Dps
According to 129 th-169 th amino acid and E.coli Dps gene for binding protein (167 aa) of the swine O-type foot-and-mouth disease VP1 protein recorded by NCBI, the Dps amino acid sequence and the VP1 amino acid sequence are connected in series to obtain the recombinant protein shown as SEQ ID NO. 1.
SEQ ID NO.1：MSTAKLVKSKATNLLYTRNDVSDSEKKATVELLNRQVIQFIDLS LITKQAHWNMRGANFIAVHEMLDGFRTALIDHLDTMAERAVQLGGVALGTTQVINS KTPLKSYPLDIHNVQDHLKELADRYAIVANDVRKAIGEAKDDDTADILTAASRDLDK FLWFIESNIEVYNGNCKYGESPVTNVRGDLQVLAQKAARTLPTSFNYGAIK。
The codons were optimized according to the preference of E.coli, 6 XHis tags were added to the N-terminal, bamH I and Nde I cleavage sites were added to the N, C terminal, respectively, and the gene sequences were synthesized and sequenced. The detection result of the gene sequence is shown as SEQ ID NO. 2.
SEQ ID NO.2：CATATGCACCACCACCACCACCACTCAACAGCTAAACTAGTAAAGAGT AAGGCGACCAACCTGCTGTACACCCGTAACGATGTTAGCGACAGCGAGAAGAAAGCGACCGTGGAACTGCTGAACCGTCAGGTTATCCAATTCATTGATCTGAGCCTGATCACCAAACAGGCGCACTGGAACATGCGTGGTGCGAACTTCATTGCGGTGCACGAGATGCTGGACGGCTTTCGTACCGCGCTGATCGATCACCTGGACACCATGGCGGAACGTGCGGTGCAGCTGGGTGGCGTTGCGCTGGGTACCACCCAAGTGATCAACAGCAAGACCCCGCTGAAAAGCTACCCGCTGGATATTCACAACGTTCAAGACCACCTGAAGGAGCTGGCGGATCGTTATGCGATCGTGGCGAACGACGTTCGTAAGGCGATTGGCGAAGCGAAAGACGATGACACCGCGGATATTCTGACCGCGGCGAGCCGTGATCTGGACAAGTTCCTGTGGTTTATCGAGAGCAACATTGAAGTGTACAACGGTAACTGCAAATATGGCGAGAGCCCGGTGACCAACGTTCGTGGTGACCTGCAGGTTCTGGCGCAAAAGGCGGCGCGTACCCTGCCGACCAGCTTTAACTATGGCGCGATCAAATAATGAGGATCC。
1.2 extraction and restriction enzyme identification of recombinant plasmids
The TOP10 clone strains containing recombinant plasmids pET-11a-Dps-VP1 and pET-11a-Dps synthesized by the company are streaked on an LB solid culture plate (containing Amp), and placed in a constant temperature incubator at 37 ℃ for 12 hours; picking single colony and inoculating into 5mL LB liquid medium containing AmpCulturing at 37 ℃ at 220rpm for 12 hours; recombinant plasmids were extracted according to the Plasmid Mini Kit protocol. Double cleavage of the recombinant plasmid with BamHI and NdeI endonucleases, H 2 O and pET-11a (+) plasmids were used as controls, and the reaction system is shown in Table:
the reaction is carried out for 2 hours at 37 ℃ in a PCR instrument, 1% agarose gel electrophoresis is carried out on the product after enzyme digestion, the voltage is 120V for 20 minutes, and a gel imager is used for scanning gel.
1.3 transformation of recombinant plasmids
Adding 5 mu L of recombinant plasmids pET-11a-Dps-VP1 and pET-11a-Dps into competent cells BL21 (DE 3), respectively, gently mixing with a pipette, and ice-bathing for 30min; heat-shocking at 42 deg.C for 90s, immediately ice-bathing for 2min, slowly adding 1mL LB liquid culture medium along the tube wall, culturing at 37 deg.C and 200rpm for 1h; 150. Mu.L of the bacterial liquid was added dropwise to an LB solid culture plate containing Amp resistance, the mixture was gently spread with a sterile glass rod, and after standing at 37℃for 1 hour, the plate was cultured for 12 hours in an inverted state.
1.4 PCR identification of recombinant bacteria
The primer adopts the universal primer of pET series carrier,
SEQ ID NO.3：T7：5’-TAATACGACTCACTATAGGG-3’；
SEQ ID NO.4：T7t：5’-GCTAGTTATTGCTCAGCGG-3’。
6 single colonies were randomly selected from each of the four plates, and each single colony was picked up with a sterile gun head and dissolved in 20. Mu.LddH 2 In O, 1. Mu.L of the template was extracted and H was used 2 O and pET-11a were used as controls, and the upstream and downstream primers were each 0.8. Mu.L, 2 XTaq PCR Mix 10. Mu.L, 8.4. Mu.L ddH 2 O, the reaction procedure is: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, annealing at 56℃for 30s,35 cycles; extending at 72 ℃ for 40s; renaturation at 72℃for 7min.
The PCR products were subjected to 1% agarose gel electrophoresis at 120V for 20min and the gel was scanned with a gel imager.
1.5 prokaryotic expression and identification of recombinant protein Dps-VP1, dps
Selecting PCR to identify correct single colonies, respectively inoculating the single colonies into 5mL LB liquid medium containing Amp, culturing at 37 ℃ and 200rpm overnight, and taking the single colonies as seed liquid; the seed liquid is prepared according to the following steps of 1:100 is added into 300mL LB liquid medium containing Amp at 37 ℃ and 200rpm until OD 600 Reaching about 0.6, adding IPTG with the final concentration of 1mM, and inducing expression at 37 ℃ for 6h; centrifuging at 4000rpm at 4deg.C for 30min, discarding supernatant, re-suspending bacterial pellet with sterile PBS solution, centrifuging at 4000rpm at 4deg.C for 10min, repeating for three times, and re-suspending bacterial cells with 30mL PBS; crushing thallus with ultrasonic crusher at 400W and 5s intermittently until thallus is clear, centrifuging at 12000rpm for 20min at 4deg.C, collecting supernatant, suspending precipitate with equal volume PBS, adding 4×loading Buffer, denaturing at 95deg.C in constant temperature metal bath for 10min, and performing SDS-PAGE detection; western blot identification was performed by incubation with Anti-His tag murine monoclonal antibody and the coat Anti-mouse HRP IgG secondary antibody.
1.6 purification of recombinant proteins Dps-VP1, dps
(1) NaCl, tris-HCl and imidazole are added into the supernatant collected after crushing, and the final concentrations are respectively as follows: 0.5mol/L, 0.02mol/L and 0.005mol/L, and filtering with a 0.45 μm filter to remove impurities;
(2) Connecting the XK16/20 chromatographic column with a constant flow pump, adding distilled water to check tightness, and then adding 10mL ProteinPure Ni-NTA resin into the chromatographic column to naturally settle; adding 10 times of balancing solution to balance the column;
(3) Flowing the supernatant of (1) through a chromatographic column at a flow rate of 6mL/min, and collecting the flow-through; balancing the column with 8 times of volume of balancing solution again, and collecting eluent;
(4) Eluting the combined proteins by using 8 times of imidazole solutions with different concentrations, collecting 8mL multiplied by 8 tubes, adding a proper amount of 20% ethanol to collect Ni-NTA resin, preserving at 4 ℃, adding the collected liquid into 4×loading buffer, denaturing at 95 ℃, and performing SDS-PAGE gel electrophoresis;
(5) Mixing eluates containing target proteins only, adding into a Millipore ultrafiltration centrifuge tube, centrifuging at 4000rpm for 30min at room temperature, concentrating, placing into a 3.5kDa dialysis bag, dialyzing with PBS dialysate containing 1M NaCl and pH 8.0 at room temperature for 6 hr, and changing dialysate every 2 hr;
(6) Collecting dialyzed protein, sterilizing with 0.22 μm filter, measuring target protein concentration with BCA protein concentration measuring kit, packaging into sterile EP tube, sealing with sealing film, and storing at room temperature.
1.7 identification of recombinant protein Dps-VP1 nanoparticles by Transmission Electron microscopy
And (3) absorbing 10 mu L of purified proteins Dps-VP1 and Dps, dripping the obtained product on a copper mesh to precipitate for 1min, absorbing floating liquid by using filter paper, absorbing 10 mu L of uranyl acetate, dripping the obtained product on the copper mesh to precipitate for 1min, absorbing the floating liquid by using filter paper, drying the obtained product at normal temperature for 5-10min, and performing electron microscopy detection imaging at 80-120kv to obtain a transmission electron microscopy imaging result.
1.8 preparation of vaccine
Heating MONTANIDETM ISA201 adjuvant, PBS and recombinant proteins Dps-VP1 and Dps to 31 ℃ in a water bath, slowly adding equal volumes of PBS or recombinant proteins Dps-VP1 and Dps into the ISA201 adjuvant, swirling for 5min on a vortex oscillator, and standing in the water bath at 20 ℃ for 1h to form the water-in-oil-in-water two-phase oil emulsion vaccine.
1.9 immunization
The 6-8 week old balb/c mice were randomly divided into 4 groups, 6 groups (3 female and male) respectively, PBS group, 10 μg Dps-VP1 group, 50 μg Dps-VP1 group, 100 μl intramuscular injection of the inner side of the rear leg of the mice, two immunization intervals of two weeks, the mental state and death of the mice were observed, and the mice were sacrificed 14d after the second immunization, and heart, liver, spleen, lung and kidney were dissected, and pathological changes of the viscera were observed by HE staining.
The 6-8 week old balb/c females were randomly divided into 6 groups, 6/group, PBS group, 50 μg Dps-VP1 group (Dps-VP 1H), 10 μg, 30 μg, 50 μg Dps-VP1+ISA201 group (Dps-VP 1H+ISA201, dps-VP1M+ISA201, dps-VP1 L+ISA201), 50 μg Dps+ISA201 group, and a bivalent vaccine group of foot-and-mouth disease, which was injected into 100 μl of inner muscle of hind leg of mice, and the bivalent vaccine of foot-and-mouth disease was injected 15 μl according to the inter-species dose conversion, and serum was collected from the orbit vein of the mice at two-week intervals, and the specific antibodies in mice were detected by indirect ELISA.
1.10 recombinant protein stability experiments
And (3) placing the purified recombinant proteins Dps-VP1 and Dps at room temperature for preservation, measuring the concentration of the recombinant proteins every month, and detecting the stability of the recombinant proteins under different room temperature environments.
Test results:
the result of the digestion is shown in figure 1, and after the recombinant plasmids pET-11a-Dps-VP1 and pET-11a-Dps are digested with double enzymes, the target bands which are consistent with the expectations are respectively 657bp and 534bp, which indicates that the recombinant expression plasmid inserted with the target gene is successfully constructed. The recombinant plasmids pET-11a-Dps-VP1 and pET-11a-Dps are introduced into competent cells BL21 (DE 3) and colony PCR is carried out, and the result is shown in figure 2, the target fragments with specific length are respectively 850bp and 727bp except for single colony No.2 of the recombinant colony pET-11a-Dps-VP1, and the target fragments with specific length are respectively identified as positive clones, so that the induction expression of the recombinant protein can be carried out.
The PCR positive single bacteria are respectively taken to be placed at 37 ℃ and 200rpm, the amp concentration is 100 mug/mL, the induction expression is carried out for 6 hours in LB culture medium with the IPTG final concentration of 1mM, after the culture medium is crushed by an ultrasonic crusher, the supernatant is taken to carry out SDS-PAGE detection, the result is shown in figure 3, when the recombinant proteins Dps-VP1 and Dps are not induced by the IPTG, the expression of the target protein is not seen, the target band appears at 23.9kDa after the recombinant protein Dps-VP1 is induced, the dimer appears at 47.8kDa, the target band appears at 19.6kDa by the Dps, the target band appears at the same position by the binding of his label antibody as shown in figure 4, and the stable expression of the recombinant proteins Dps-VP1 and Dps in an escherichia coli expression system can be confirmed.
The supernatant after collecting the induced expression is purified by Ni-NTA, and recombinant proteins Dps-VP1 and Dps with higher purity are obtained in 500mM and 400mM imidazole eluates respectively, which reach more than 95% (shown in figure 5).
After protein was dialyzed against PBS (pH 8.0) containing 1M NaCl, negative staining transmission electron microscopy was performed. As can be seen from FIG. 6, the recombinant proteins Dps-VP1 and Dps each form round particles with a size of about 9nm and have a smooth surface as the natural foot-and-mouth disease virus.
After the mice were first immunized, the growth rate and mental state of the group with different concentration of Dps-VP1 were normal compared with the PBS group, and no death of the mice occurred until 14d after the second immunization. The HE staining results of heart, liver, spleen, lung, kidney showed (as shown in FIG. 7), that no obvious histopathological changes such as neutrophil infiltration and tissue structure loss were observed in both 50 μg and 100 μg Dps-VP1 groups compared with PBS inoculated group, demonstrating that Dps-VP1 has no toxic effect on mice, and that 100 μg Dps-VP1 is within a safe concentration range.
After immunization, there was no significant difference in the change in body weight of each group of mice (as shown in fig. 8).
As shown in FIG. 9, VP 1-specific antibodies were produced in mice of both bivalent vaccine group and DPS-VP 1-different immunized group with increasing immunization times, and the level of VP 1-specific IgG induced by DPS-VP 1-different immunized group and bivalent vaccine group was significantly increased compared to the blank control group, PBS group and DPS+ISA201 group at 42dpi, and the level of specific IgG produced by DPS-VP1H+ISA201 group mice was significantly higher than that produced by DPS-VP1H group mice, and there was no significant difference between DPS-VP1H+ISA201 group and bivalent vaccine group.
As shown in FIG. 10, the recombinant proteins Dps-VP1 and Dps were stored at room temperature for six months without significant changes in protein concentration.
In conclusion, the invention successfully constructs pET-11a-Dps-VP1/pET-11a-Dps recombinant plasmid, and the recombinant proteins Dps-VP1 and Dps can be stably expressed through the induction expression of escherichia coli and can be self-assembled into virus-like particles (about 9 nm), and can be stored for at least 6 months at room temperature; the recombinant protein Dps-VP1 is emulsified to prepare vaccine, and the vaccine can be used for immunizing a balb/c mouse (50 mug/mouse) and effectively inducing the mouse to generate FMDV specific antibodies.
The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.
Claims (10)
1. The recombinant protein is characterized in that the amino acid sequence of the recombinant protein is shown as SEQ ID NO. 1.
2. Nucleotide sequence of Dps-VP1 gene of the coding recombinant protein.
3. The nucleotide sequence according to claim 2, wherein the nucleotide sequence is shown as SEQ ID No. 2.
4. The recombinant plasmid is characterized by being pET-11a-Dps-VP1 recombinant plasmid.
5. A foot-and-mouth disease virus-like particle vaccine comprising the recombinant protein of claim 1 and other pharmaceutically acceptable immunoadjuvants.
6. The foot and mouth disease virus like particle vaccine of claim 5, wherein the immunoadjuvant is ISA201 adjuvant.
7. A method of preparing a foot and mouth disease virus-like particle vaccine according to any one of claims 5 to 6, comprising the steps of:
(1) Constructing pET-11a-Dps-VP1 recombinant plasmid;
(2) Introducing the recombinant plasmid into competent cells, and performing colony PCR identification;
(3) Amplifying and culturing the colony with positive identification result, and inducing the expression of the recombinant plasmid;
(4) Crushing the thalli and purifying to obtain the recombinant protein as defined in claim 1;
(5) Slowly adding the recombinant protein with the same volume into the adjuvant, swirling for 4-8min, standing at 18-22 ℃ for 40-80min, and obtaining the foot-and-mouth disease virus-like particle vaccine.
8. The method of claim 5, wherein the competent cells are competent BL21 cells; the primers identified by colony PCR comprise an upstream primer shown as SEQ ID NO.3 and a downstream primer shown as SEQ ID NO. 4.
9. The method of claim 5, wherein inducing expression of the recombinant plasmid comprises OD in a medium 600 When reaching 0.5-0.7, IPTG with a final concentration of 1mM was added to induce expression at 37℃for 6h.
10. Use of the recombinant protein of claim 1 or the foot-and-mouth disease virus-like particle vaccine of any one of claims 5-6 in the manufacture of a medicament for the treatment and/or prevention of foot-and-mouth disease.
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