CN111233984B

CN111233984B - O-type foot-and-mouth disease virus-like particle antigen, vaccine composition thereof, preparation method and application

Info

Publication number: CN111233984B
Application number: CN201811446135.5A
Authority: CN
Inventors: 田克恭; 张素玲; 孙进忠; 张许科
Original assignee: Pulaike Biological Engineering Co Ltd
Current assignee: Pulaike Biological Engineering Co Ltd
Priority date: 2018-11-29
Filing date: 2018-11-29
Publication date: 2022-08-12
Anticipated expiration: 2038-11-29
Also published as: CN111233984A

Abstract

The invention provides a foot-and-mouth disease virus-like particle antigen which is assembled by VP0, VP1 and VP3 antigen proteins, wherein the VP0 antigen protein is encoded by Seq ID No.1 or a degenerate sequence thereof, the VP1 antigen protein is encoded by Seq ID No.3 or a degenerate sequence thereof, and the VP3 antigen protein is encoded by Seq ID No.2 or a degenerate sequence thereof. The foot-and-mouth disease virus-like particle antigen has good immunogenicity, the vaccine composition prepared by the foot-and-mouth disease virus-like particle antigen can protect the currently epidemic O-type Ind strain through one-time immunization, and the immune protection period is long. The invention also provides a vaccine composition prepared by using the foot-and-mouth disease virus-like particle antigen, a preparation method and application of the vaccine.

Description

O-type foot-and-mouth disease virus-like particle antigen, vaccine composition thereof, preparation method and application

Technical Field

The present invention belongs to a viral antigen and a medical preparation of the antigen. Specifically, the invention relates to an O-type foot-and-mouth disease virus-like particle antigen, a vaccine composition prepared from the same, a preparation method and an application.

Background

Foot-and-mouth disease (FMD), an acute, highly contagious and rapidly remotely transmissible disease of animals, is the most contagious disease in mammals, with cloven-hoof infections causing significant economic losses worldwide. Animals suffering from foot and mouth disease include cattle, sheep and pigs. The pathogenic factor is foot-and-mouth disease virus (FMDV), which is a aphtha virus in the picornavirus family. The virus is divided into 7 serotypes (A, O, C, Asial, SAT1, SAT2 and SAT 3), wherein the foot and mouth disease virus type O is most prevalent. Vaccine immunization is an effective measure for controlling the disease and protecting livestock from harm. However, no vaccine against the new pandemic strain of the Ind strain of type O is currently commercially available for type O vaccines.

However, O type Ind vaccine strain aiming at the current epidemic strain has weak autoimmunity, and the prepared inactivated vaccine can not induce the animal body to generate enough immunity. Therefore, how to improve the immunogenicity of the antigen to prevent the current epidemic strains is urgent, and the antigen also meets the requirements of effective prevention and control of major animal epidemic diseases and guarantee the healthy and sustainable development of animal husbandry proposed by the nation.

Virus-like particles (VLPs) are viroid particles that are capable of self-packaging into a viral coat structure upon expression in vitro and/or in vivo, and are pseudoviruses that have a similar coat structure to a virus but do not have the ability to replicate a virus. The VLPs vaccine can effectively stimulate the organism to generate anti-infection and anti-tumor immunity, but VLPs with stable structures are not formed, namely the immunogenicity of the VLPs vaccine can not be effectively exerted, so that the vaccine has poor immune effect. No example of the success of the type O foot-and-mouth disease Ind strain VLPs vaccine exists at present.

Disclosure of Invention

In order to solve the problem that no vaccine is available for O-type Ind strain epidemic strain foot-and-mouth disease virus infection in the prior art, the invention provides a foot-and-mouth disease virus-like particle antigen, wherein the foot-and-mouth disease virus-like particle antigen is assembled by VP0, VP1 and VP3 antigen proteins, wherein the VP0 antigen protein is encoded by a nucleotide sequence shown as Seq ID No.1 or a degenerate sequence thereof, the VP1 antigen protein is encoded by a nucleotide sequence shown as Seq ID No.3 or a degenerate sequence thereof, and the VP3 antigen protein is encoded by a nucleotide sequence shown as Seq ID No.2 or a degenerate sequence thereof.

The foot-and-mouth disease virus-like particle antigen has good immunogenicity, and the vaccine composition prepared by the antigen can provide complete protection for the current Ind strain epidemic foot-and-mouth disease virus only through one-time immunization. Meanwhile, the foot-and-mouth disease virus-like particle antigen has good stability, is placed at 4 ℃ for 3 months, and is still full and free of aggregation through phosphotungstic acid negative staining and electron microscope observation.

The O type Ind strain foot-and-mouth disease virus-like particle antigen is derived from the current epidemic strain, can generate complete protection aiming at the current epidemic wild strain, and makes up the defect that the inactivated vaccine in the prior art can not carry out immune protection.

The invention also provides a vaccine composition, wherein the vaccine composition comprises an immunizing amount of the foot-and-mouth disease virus-like particle antigen and a pharmaceutically acceptable carrier.

The O type Ind strain foot-and-mouth disease virus-like particle vaccine can reach an antibody titer of more than 1:128 on 14 th day after immunization, can maintain a high-titer antibody titer for a long period, and can protect against the whole fattening period.

As one embodiment of the invention, in the vaccine composition of the invention, the content of the foot-and-mouth disease virus-like particle antigen is 160-240 μ g/ml.

The O type Ind strain foot-and-mouth disease virus-like particle antigen content can be selected from 160 mu g/ml, 170 mu g/ml, 180 mu g/ml, 190 mu g/ml, 200 mu g/ml, 210 mu g/ml, 220 mu g/ml, 230 mu g/ml and 240 mu g/ml.

Even when the content of the O type Ind strain foot-and-mouth disease virus-like particle antigen is only 160 mu g/ml, the 14 th day after immunization can achieve the antibody titer of more than 1:128, namely, the immunoprotection can be generated, and the antibody titer with high titer can be maintained for a long period of time.

In a preferred embodiment of the present invention, the vaccine composition of the present invention comprises 200 μ g/ml of the foot-and-mouth disease virus-like particle antigen.

As an embodiment of the present invention, in the vaccine composition of the present invention, the pharmaceutically acceptable carrier includes an adjuvant selected from the group consisting of: (1) white oil, alumina gel adjuvant, saponin, alfvudine, DDA; (2) water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion; or (3) a copolymer of a polymer of acrylic acid or methacrylic acid, maleic anhydride and an alkenyl derivative; and one or more of RIBI adjuvant system, Block co-polymer, SAF-M, monophosphoryl lipid A, Avridine lipid-amine adjuvant, Escherichia coli heat-labile enterotoxin, cholera toxin, IMS 1314, muramyl dipeptide, Montanide ISA 206, and Gel adjuvant; preferably, the saponin is Quil A, QS-21, GPI-0100; preferably, the adjuvant is ISA 206 adjuvant.

The adjuvant content is 5% -60% V/V, preferably from 30% -60% V/V, more preferably 50% V/V.

As one embodiment of the present invention, the pharmaceutically acceptable carrier includes drugs, immunostimulants, antioxidants, surfactants, colorants, volatile oils, buffers, dispersants, propellants, and preservatives; the immunostimulant includes alpha-interferon, beta-interferon, gamma-interferon, granulocyte macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), and interleukin 2(IL 2).

To prepare such compositions, methods well known in the art may be used.

The invention also relates to a method for preparing the vaccine composition, wherein the method comprises the following steps: respectively cloning and recombining the gene of the VP0 antigen protein, the gene of the VP1 antigen protein and the gene of the VP3 antigen protein to the same tandem expression vector; transforming the recombinant expression vector obtained in the step (1) to a host, and performing soluble expression on VP0 antigen protein, VP1 antigen protein and VP3 antigen protein, wherein the expressed VP0 antigen protein, VP1 antigen protein and VP3 antigen protein are self-assembled into virus-like particle antigen; and (3) separating and purifying the virus-like particle antigen in the step (2), and adding an adjuvant to obtain the vaccine composition.

According to the invention, by expressing the O-type Ind strain foot-and-mouth disease virus VP0, VP3 and VP1 antigen proteins, stable self-assembled virus-like particles can be obtained, and the immune effect of the particles can realize complete protection of pigs. According to the invention, the O type Ind strain foot-and-mouth disease virus VP0, VP3 and VP1 antigen proteins are expressed in series, and the expressed active proteins are self-assembled into the O type Ind strain foot-and-mouth disease virus-like particle antigen, so that subsequent antigen purification and separation are facilitated.

As an embodiment of the invention, in the method of the invention, the tandem expression vector of the step (1) is pET28 a; the host of the step (2) is E.coli BL21(DE 3).

According to the invention, tandem expression is carried out on VP0, VP3 and VP1 antigen proteins by selecting a tandem expression vector and host bacteria, and the expressed soluble protein has biological activity and is self-assembled into a virus-like particle antigen, so that subsequent antigen separation and purification are facilitated, and the procedure is simplified.

As a preferred embodiment of the present invention, in the method of the present invention, IPTG is added after the host cells are amplified in the step (2), and the protein expression is induced.

In a preferred embodiment of the present invention, in the separation and purification step of step (3), the virus-like particle antigen is purified by ammonium sulfate fractionation and chromatography by taking the supernatant after disruption of the bacterial cells.

The invention utilizes an escherichia coli expression system to produce the foot-and-mouth disease virus-like particles, and has the advantages of high yield, low production cost, good immunogenicity, no biological safety risk and the like. The virus-like particle vaccine composition prepared by the invention can provide protective activity for O type Ind strain foot-and-mouth disease, and has the advantages of fast antibody generation, high antibody generation level, long immune duration and capability of maintaining long-time immune protection.

The invention also relates to application of the vaccine composition in preparing a medicament for preventing the O-type foot-and-mouth disease.

The subject of the invention for preparing the medicament for preventing and/or treating foot-and-mouth disease virus infection comprises pigs.

The vaccine composition maintains the antibody titer of more than 1:128 for 133 days, and completely protects the pig body, and the time period can cover the whole fattening period.

Detailed Description

Hereinafter, embodiments of the present invention will be described.

Definition of

The foot-and-mouth disease virus belongs to the family of picornaviridae, the genus of foot-and-mouth disease virus, which has O, A, C, SAT1, SAT2, SAT3 (namely south Africa foot-and-mouth disease virus types 1, 2 and 3) and Asia1 (Asia type 1) 7 serotypes, no cross protection reaction exists among the types, and each type has a plurality of subtypes. A positive-stranded RNA, which is single-stranded in the center of the virus, consists of about 8000 bases and is the basis for infection and inheritance; the surrounding protein determines the antigenicity, immunity and serological reaction capability of the virus; the viral coat is a symmetrical 20-sided body. Foot-and-mouth disease virus is a highly infectious disease of even-hoof animals, namely, the pathogeny of foot-and-mouth disease, the foot-and-mouth disease is listed as the first infectious disease list of A animals by the International animal epidemic department, the foot-and-mouth disease is listed as the infectious disease of quarantine of entry animals by China, the foot-and-mouth disease is prevented and treated in China, the prevention is mainly inoculated through vaccine injection, and the foot-and-mouth disease is killed when the foot-and-mouth disease occurs.

"Antigen" refers to a substance that induces an immune response in the body, i.e., a substance that is specifically recognized and bound by an Antigen receptor (TCR/BCR) on the surface of T/B lymphocytes, activates T/B cells, proliferates and differentiates the T/B cells, produces an immune response product (sensitized lymphocytes or antibodies), and specifically binds to the corresponding product in vitro or in vivo.

"Virus-like particles (VLPs)" are particles assembled from one or more viral structural proteins and have similar external structure and antigenicity to viral particles, but do not contain viral genes.

"foot-and-mouth disease virus VP0, VP3, VP1 antigen protein": FMDV structural protein precursor protein P1 is catalytically processed by protease 3C into VP0, VP1 and VP3, and this 3 proteins self-assemble into an icosahedral viral capsid. The VP0 protein is an intermediate after cleavage of P1 by protease 3C, and VP0 is cleaved mature to VP2 and VP4 in the final stage of virion formation.

The terms "vaccine", "vaccine composition" as used herein refer to a pharmaceutical composition comprising a foot and mouth disease virus-like particle antigen which induces, stimulates or enhances an immune response in pigs against foot and mouth disease only.

The term "immunizing amount" shall be understood as an "immunologically effective amount," also referred to as an immunoprotective amount or an amount effective to produce an immune response, of antigen effective to induce an immune response in a recipient, sufficient to prevent or ameliorate the signs or symptoms of disease, including adverse health effects or complications thereof. The immune response may be sufficient for diagnostic purposes or other testing, or may be suitable for use in preventing signs or symptoms of disease, including adverse health consequences or complications thereof caused by infection by a pathogen. Humoral immunity or cell-mediated immunity or both can be induced. The immune response of an animal to an immunogenic composition can be assessed indirectly, for example, by measuring antibody titers, lymphocyte proliferation assays, or directly by monitoring signs or symptoms after challenge with a wild-type strain, while the protective immunity provided by the vaccine can be assessed by measuring, for example, clinical signs such as mortality, reduction in morbidity, temperature values, overall physiological condition of the subject, and overall health and performance. The immune response may include, but is not limited to, induction of cellular and/or humoral immunity.

The term "pharmaceutically acceptable carrier" refers to all other ingredients in the vaccine composition of the present invention, except for the foot-and-mouth disease virus antigen, which do not stimulate the body and do not hinder the biological activity and properties of the compound used, or a diluent, preferably an adjuvant. The term "adjuvant" may include an alumina gel adjuvant; saponins (saponin), such as Quil A, QS-21(Cambridge Biotech Incorporation, Cambridge MA), GPI-0100(Galenica Pharmaceuticals Incorporation, Birmingham AL); a water-in-oil emulsion; an oil-in-water emulsion; a water-in-oil-in-water emulsion; polymers of acrylic acid or methacrylic acid; maleic anhydride and alkenyl (alkenyl) derivatives. The term "emulsion" may be based in particular on light liquid paraffin oil (European Pharmacopea type); isoprenoid oils (isoprenoid oils) resulting from the oligomerization of olefins, such as squalane (squalane) or squalene oil (squalene oil), in particular isobutene or decene; linear alkyl-containing esters of acids or alcohols, more particularly vegetable oils, ethyl oleate, propylene glycol di- (caprylate/caprate), glycerol tri- (caprylate/caprate) or propylene glycol dioleate; esters of branched fatty acids or alcohols, especially isostearic acid esters. The oil is used in combination with an emulsifier to form an emulsion. The emulsifiers are preferably nonionic surfactants, in particular esters of sorbitan, of mannide (such as, for example, anhydrous mannitol oleate), of aliphatic diols (glycols), of polyglycerols, of propylene glycol and of oleic acid, of isostearic acid, of ricinoleic acid or of hydroxystearic acid, which are optionally ethoxylated, and also polyoxypropylene-polyoxyethylene block copolymers, in particular the Pluronic products, in particular L121. See The description of The same and The reactive application of adjuvants by Hunter et al (Ed. by DES Stewart-Tull, John Wiley and Sons, New York,1995:51-94) and The description of Vaccine by Todd et al (1997,15: 564-570). For example, the SPT emulsion described on page 147 and the MF59 emulsion described on page 183 of Vaccine design, the Subunit and adivant propaach (Plenum Press,1995) written by Powell M and Newman M can be used. The term "polymer of acrylic or methacrylic acid" is preferably a crosslinked polymer of acrylic or methacrylic acid, in particular a polyalkenyl ether or polyalcohol crosslinked with a sugar (sugar), these compounds being known under the name Carbomer (Carbopol, trade name Carbopol) (Phameuropa,1996,8 (2)). Those skilled in the art can also see US2909462, which describes such acrylic polymers crosslinked with polyhydroxylated compounds having at least 3 hydroxyl groups, preferably not more than 8, wherein the hydrogen atoms of at least 3 hydroxyl groups are substituted by unsaturated aliphatic hydrocarbon groups (aliphatic radial) having at least 2 carbon atoms. Preferred groups are those containing 2 to 4 carbon atoms, such as vinyl, allyl and other ethylenically unsaturated groups (ethylenically unsaturated groups). The unsaturated groups may themselves contain other substituents, such as methyl. These products are sold under the name carbopol, (BF Goodrich, Ohio, USA) are particularly suitable. They are crosslinked with allyl sucrose or with allyl pentaerythritol. Among these, mention may be made of carbopols 974P, 934P and 971P, the most preferred being the use of carbopol 971P. The term "copolymers of maleic anhydride and alkenyl derivative" also contemplates the maleic anhydride and ethylene copolymers ema (monsanto), which are dissolved in water to give an acidic solution, neutralized, preferably to physiological pH, in order to give an adjuvant solution into which the immunogenic, immunogenic or vaccinal composition itself can be incorporated. The term "adjuvant" also includes, but is not limited to, the RIBI adjuvant system (Ribi Incorporation), Block co-polymer (CytRx, Atlanta GA), SAF-M (Chiron, Emeryville CA), monophosphoryl lipid A (monophosphoryl lipid A), Avridine lipoamine adjuvant, E.coli heat labile enterotoxin (recombinant or otherwise), cholera toxin, IMS 1314, muramyl dipeptide, Gel adjuvant, and the like. Preferably, the adjuvant comprises one or more of white oil, an alumina Gel adjuvant, a saponin, a water-in-oil emulsion, an oil-in-water emulsion, a water-in-oil-in-water emulsion, a polymer of acrylic acid or methacrylic acid, a copolymer of maleic anhydride and an alkenyl (alkenyl) derivative, a RIBI adjuvant system, a Block co-polymer, SAF-M, a monophosphoryl lipid A, Avridine lipid-amine adjuvant, escherichia coli heat labile enterotoxin, cholera toxin, IMS 1314, muramyl dipeptide, Montanide ISA 206 or Gel adjuvant.

"degenerate sequence": in molecular biology, the phenomenon that the same amino acid has two or more codons is called degeneracy of the codon (degeneracy), and such a sequence is called degenerate sequence.

"Gene recombination": refers to the recombination of genes that control different traits. Modern genetic engineering techniques carry out genetic recombination, also called recombinant DNA, in vitro by artificial design, with the aim of transferring a genetic gene in one individual cell to another individual cell DNA molecule of a different character, causing genetic variation. After the target gene from the donor is transferred into the recipient bacterium, the expression of the gene product can be carried out, thereby obtaining a product which is difficult to obtain by a common method.

"transformation" refers to the acquisition of a new genetic phenotype in a cell or a cultured recipient cell by the automated acquisition or artificial supply of exogenous DNA.

"transduction" means that when the virus is released from an infected (donor) cell and re-infects another (recipient) cell, the DNA transfer and gene recombination that occurs between the donor and recipient cells is transduction.

The term "preventing and/or treating" when referring to foot and mouth disease virus infection means inhibiting replication of foot and mouth disease virus, inhibiting spread of foot and mouth disease virus or preventing colonization of foot and mouth disease virus in its host, and alleviating symptoms of foot and mouth disease virus infected diseases or conditions. Treatment is considered to be therapeutically effective if the viral load is reduced, the condition is reduced and/or the food intake and/or growth is increased.

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.

The chemical reagents used in the examples of the present invention are all analytical reagents and purchased from the national pharmaceutical group.

In order that the invention may be more readily understood, reference will now be made to the following examples. The experimental methods are conventional methods unless specified otherwise; the biomaterial is commercially available unless otherwise specified.

Example 1 expression of type O Ind Strain foot-and-mouth disease Virus-like particles

1. Construction of expression vectors

VP0 gene fragment shown in sequence table SEQ ID NO.1, VP3 gene fragment shown in sequence table SEQ ID NO.2 and VP1 gene fragment shown in sequence table SEQ ID NO.3 are synthesized by Jinwei corporation, and are respectively connected with pBLUE-T Vector, the successfully connected recombinant clones are respectively digested by BamH I/EcoR I, Sac I/Sal I and Hind III/Xho I to obtain fragments, and the fragments are connected with pET28a Vector which is digested by the same enzyme, so as to obtain positive clone pET28a-VP0-VP3-VP1 inserted with O type Ind strain foot and mouth disease virus VP0, VP3 and VP1 genes. Transformation of the ligated plasmid into CaCl ₂ The prepared DH5 alpha competent cells were plated on a kanamycin-resistant solid LB medium, and when colonies of the monoclonal were clearly visible, the monoclonal was picked up into a kanamycin-containing LB liquid medium, cultured at 37 ℃ at 230 rpm for 12 hours overnight, and the recombinant plasmid was extracted.

2. Construction of expression Strain

Mu.l of the positive clone pET28a-VP0-VP3-VP 140. mu.l of the gene inserted into the O-type Ind strain foot-and-mouth disease virus VP0, VP3 and VP1 was transformed into competent Escherichia coli BL21(DE3) prepared by calcium chloride method, applied to kanamycin-resistant solid LB medium, and left to stand at 37 ℃ for 10 to 12 hours until the single colony was clearly visible, the single clone was picked up into a test tube containing 4ml of kanamycin-resistant liquid LB medium, subjected to shaking culture at 37 ℃ at 230 rpm for 12 hours, and 1ml of the culture solution was freeze-dried at-80 ℃ for storage.

3. Expression and characterization of proteins

Taking out Escherichia coli strain with recombinant plasmid pET28a-VP0-VP3-VP1 from-80 deg.C, inoculating 50ml LB liquid culture medium with kanamycin resistance, shaking culturing at 37 deg.C and 230 rpm for 12 hr, transferring into 1L LB liquid culture medium, performing large scale amplification at 37 deg.C, and waiting for OD ₆₀₀ After reaching the value of 0.6, 0.1mM IPTG was added to induce protein expression overnight at 20 ℃.

The fermentation tank was a 50L fermentation tank of Shanghai Baoxing Bio Inc., 30L of the medium was prepared and charged into the fermentation tank, and sterilized at 121 ℃ for 30 minutes. The next day, 5L of seed liquid is inoculated into a fermentation tank, and the concentration of the culture liquid reaches OD ₆₀₀ About 10 hours later, the culture temperature was lowered to 25 ℃ and 4g of IPTG was added to induce culture for 12 hours. OD ₆₀₀ About 40 cells were collected by centrifugation.

Resuspend the cells and crush the cells 4 times with a homogenizer at 800bar pressure. 13500rpm, centrifuging for 40min, collecting the supernatant, and detecting by 15% SDS-PAGE electrophoresis, wherein the expression level of each of the three proteins expressed in series in the supernatant is about 20%. The protein is roughly purified by adopting an ammonium sulfate fractional precipitation method, then chromatographic purification is carried out, and the purified protein is subjected to SDS-PAGE electrophoresis, so that the target protein is purified and enriched.

Foot-and-mouth disease protein is formed into virus-like particles through phosphotungstic acid negative staining and electron microscope observation, and the formed virus-like particles are full, high in assembly efficiency and free of aggregation. The foot-and-mouth disease virus-like particles are placed at 4 ℃ for 3 months, and then phosphotungstic acid negative staining and electron microscope observation show that the virus-like particles are still full and have no aggregation phenomenon. It is demonstrated that the foot-and-mouth disease protein prepared by the sequence screened by the invention forms stable virus-like particles.

EXAMPLE 2 preparation of O type Ind Strain foot-and-mouth disease Virus-like particle vaccine composition

Slowly adding the virus-like particles prepared in the example 1 into an adjuvant, continuously stirring for 12min by using an emulsifying machine with the rotating speed of 800rpm in the adding process, uniformly mixing, and storing at 4 ℃ to obtain the foot-and-mouth disease virus-like particle vaccine composition. The specific ratio is shown in table 1. Adjuvants suitable for use in the present invention may be those known to those skilled in the art. In this example, the adjuvant ISA 206 (france seebeck) was used.

TABLE 1 component ratio of O type Ind strain foot-and-mouth disease virus-like particle vaccine composition

Components	Vaccine 1	Vaccine 2	Vaccine 3
				Foot-and-mouth disease antigen (mu g/ml)	160	200	240
ISA 206 adjuvant (V/V%)	50％	50％	50％

EXAMPLE 3 immunogenicity testing of Ind O-type strains foot-and-mouth disease Virus-like particle vaccine compositions

Selecting 20 healthy susceptible piglets which are negative to O-type foot-and-mouth disease virus antigens and antibodies and have the weight of about 40kg, and randomly dividing the piglets into 4 groups, wherein each group has 5 piglets. Groups 1-3 are vaccine 1, vaccine 2, and vaccine 3 immunization groups prepared in example 2 of the present invention, respectively, and group 4 is a control group. The immunization group was administered by intramuscular injection of 2ml into the neck, and the control group was immunized with an equal amount of PBS. Blood is collected from each pig before immunization of the vaccine, and blood is collected on 7 th day, 14 th day, 21 th day and 28 th day after immunization.

And (3) detecting related antibodies of the collected serum by using an O-type Ind strain foot-and-mouth disease antibody ELISA detection kit. The result shows that all the antibodies of the pigs before the vaccine immunization are negative, and the 14 th day after 1 immunization can reach more than 1: 128. Control group pigs were negative for antibody and did not change. The specific results are shown in Table 2.

TABLE 2 ELISA antibody levels following immunization of O-type Ind strain foot-and-mouth disease Virus-like particle vaccine compositions of the present invention

The virus-like particle antigen prepared by the invention can quickly form high-level specific antibody, and even if the antigen content is only 160 mu g/ml, the virus-like particle antigen can play a good immune protection role on O type Ind strain foot-and-mouth disease on 14 days after one-time immunization.

Example 4 duration of immunization test of O type Ind Strain foot-and-mouth disease Virus-like particle vaccine compositions

Selecting 10 healthy susceptible piggy-stocks with negative O-type foot-and-mouth disease virus antigen and antibody and weight of about 40kg, and randomly dividing the healthy susceptible piggy-stocks into 2 groups with 5 pigs in each group. The 5 th group was the vaccine 2 immunization group prepared in example 2 of the present invention, and the 6 th group was the control group. The immunization approach of the group 5 immunization group is to inject 2ml of PBS into neck muscle, the group 6 control group is immunized with PBS with the same amount, the immunization is performed uniformly, each pig is subjected to blood collection before vaccine immunization, and the blood collection is performed 21 days, 28 days, 35 days, 77 days, 105 days and 133 days after immunization.

The results show that all the antibodies of the pigs before the vaccine immunization are negative, the vaccine 2 immunization group on the 21 st day after 1 immunization can reach more than 1:128, and the high antibody level is still maintained after 1 immunization for 133 days. Control group pigs were negative for antibody and did not change. The specific results are shown in Table 3.

TABLE 3 ELISA antibody levels for the duration of immunization of O-type Ind strain foot-and-mouth disease virus-like particle vaccine compositions of the present invention

The experimental data show that the virus-like particle vaccine composition prepared by the invention has the advantages of fast antibody generation, high antibody level, good immune protection effect only through one-time immunization, long immune duration, capability of maintaining long-time immune protection and capability of covering the whole fattening period.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.

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<213> foot-and-mouth disease Virus Ind Strain (foot-and-mouth disease Virus, strain Ind)

<400> 3

accacctcta ccggtgaatc tgctgacccg gttaccacca ccgttgaaaa ctacggtggt 60

gaaacccagg ttcagcgtcg tcagcacacc gacgtttctt tcatcctgga ccgtttcgtt 120

aaagttaccc cgaaagacca gatcaacgtt ctggacctga tgcagacccc ggctcacacc 180

ctggttggtg ctctgctgcg taccgctacc tactacttcg ctgacctgga agttgctgtt 240

aaacacaaag gtaacctgac ctgggttccg aacggtgctc cggaagctgc tctggacaac 300

accaccaacc cgaccgctta ccacaaagct ccgctgaccc gtctggctct gccgtacacc 360

gctccgcacc gtgttctggc taccgtttac aacggtaact gcaaatacgg tgaatctaac 420

gttccgaacg ttcgtggtga cctgcaggtt ctggctcaga aagctgctcg tccgctgccg 480

acctctttca actacggtgc tatcaaagct acccgtgtta ccgaactgct gtaccgtatg 540

aaacgtgctg aaacctactg cccgcgtccg ctgctggcta tccacccgtc tgaagctcgt 600

cacaaacaga aaatcgttgc tccggttaaa cag 633

Claims

1. The foot-and-mouth disease virus-like particle antigen is assembled by VP0, VP1 and VP3 antigen proteins, wherein the VP0 antigen protein is encoded by a nucleotide sequence shown in Seq ID No.1, the VP1 antigen protein is encoded by a nucleotide sequence shown in Seq ID No.3, and the VP3 antigen protein is encoded by a nucleotide sequence shown in Seq ID No. 2.

2. A vaccine composition comprising an immunizing amount of the foot-and-mouth disease virus-like particle antigen of claim 1 and a pharmaceutically acceptable carrier.

3. The vaccine composition according to claim 2, wherein the foot-and-mouth disease virus-like particle antigen content is 160-240 μ g/ml.

4. The vaccine composition according to claim 3, wherein the foot-and-mouth disease virus-like particle antigen content is 200 μ g/ml.

5. The vaccine composition of claim 2, wherein the pharmaceutically acceptable carrier comprises an adjuvant selected from the group consisting of: (1) alumino-gel adjuvant, saponin, avridine, DDA; (2) water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion; or (3) a copolymer of a polymer of acrylic acid or methacrylic acid, maleic anhydride and an alkenyl derivative; and one or more of RIBI adjuvant system, Block co-polymer, SAF-M, monophosphoryl lipid A, Avridine lipid-amine adjuvant, Escherichia coli heat-labile enterotoxin, cholera toxin, IMS 1314, muramyl dipeptide and Gel adjuvant.

6. The vaccine composition of claim 5, wherein the saponin is Quil a, QS-21, or GPI-0100.

7. The vaccine composition of claim 5, wherein the adjuvant is ISA 206 adjuvant.

8. The vaccine composition according to claim 5, wherein the concentration of the adjuvant ranges from 5% to 60% V/V.

9. The vaccine composition according to claim 8, wherein the concentration of the adjuvant ranges from 30% to 60% V/V.

10. The vaccine composition of claim 9, wherein the adjuvant is at a concentration of 50% V/V.

11. A method of preparing the vaccine composition of claim 2, wherein the method comprises:

respectively cloning and recombining the gene of the VP0 antigen protein, the gene of the VP1 antigen protein and the gene of the VP3 antigen protein to the same tandem expression vector;

transforming the recombinant expression vector obtained in the step (1) to a host, and performing soluble expression on VP0 antigen protein, VP1 antigen protein and VP3 antigen protein, wherein the expressed VP0 antigen protein, VP1 antigen protein and VP3 antigen protein are self-assembled into virus-like particle antigen;

and (3) separating and purifying the virus-like particle antigen obtained in the step (2), and adding an adjuvant to obtain the vaccine composition.

12. The process of claim 11, wherein the tandem expression vector of step (1) is pET28 a; the host of the step (2) is E.coli BL21(DE 3).

13. The method according to claim 12, wherein the protein expression is induced in step (2) by adding IPTG after the host cell is expanded.

14. The method according to claim 11, wherein the separation and purification step (3) comprises disrupting the bacterial cells, collecting the supernatant, and purifying the virus-like particle antigen by ammonium sulfate fractionation and chromatography.

15. Use of a vaccine composition according to any one of claims 2 to 10 in the manufacture of a medicament for the prevention of foot and mouth disease type O.