CN110256539B

CN110256539B - Novel genetic engineering subunit vaccine of O-type foot-and-mouth disease virus

Info

Publication number: CN110256539B
Application number: CN201910620895.1A
Authority: CN
Inventors: 曹文龙; 孔迪; 滕小锘; 易小萍; 张大鹤
Original assignee: Suzhou Shinuo Biotechnology Co ltd
Current assignee: Suzhou Womei Biology Co ltd
Priority date: 2019-07-10
Filing date: 2019-07-10
Publication date: 2020-04-10
Anticipated expiration: 2039-07-10
Also published as: CN110256539A

Abstract

The invention discloses an immune composition, which comprises: structural proteins VP3 and VP1 of type O foot-and-mouth disease virus, and structural proteins VP2 and/or VP4 of type O foot-and-mouth disease virus. Further, the immune composition can also comprise a structural protein VP0 of the foot-and-mouth disease virus type O. The immune composition can be used for preparing novel genetic engineering subunit vaccines of the O-type foot-and-mouth disease virus, the antigenicity, the immunogenicity and the functions of the vaccines are similar to those of natural proteins, the expression level is higher, the immunogenicity is strong, and the vaccines have no pathogenicity to animals.

Description

Novel genetic engineering subunit vaccine of O-type foot-and-mouth disease virus

Technical Field

The invention relates to the technical field of animal immunity drugs, in particular to a novel genetic engineering subunit vaccine of O-type foot-and-mouth disease virus.

Background

Foot-and-mouth disease (FMD), a disease caused by the infection of animals of the cloven-footed type (such as pigs, cows, sheep, deer, etc.) by foot-and-mouth disease virus (FMDV), is characterized in that blisters appear on the skin of the mouth, feet, etc. of the infected cloven-footed type, thereby causing the death of some animals.

FMDV belongs to the family Picornaviridae (Picornaviridae) aphtha virus (Aphthovius) and is a pathogen of highly infectious diseases (foot-and-mouth disease) of artiodactyls. 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. FMDV immunization is a T-cell dependent B-cell response, and vaccination mainly induces the production of neutralizing antibodies. FMDV comprises A, O, C, Asia1 and 7 serotypes including SAT1, SAT2, SAT3 and the like without cross immune protection, more than 80 subtypes are formed in long-term mutual transmission infection, and a serious challenge is brought to prevention and control of foot-and-mouth disease.

Animals with foot and mouth disease experience symptoms such as fever, lameness, and bubbly rash on the skin and skin mucosa. For domestic animals, the symptoms of foot-and-mouth disease are mainly high fever that declines rapidly after two to three days; blisters in the mouth cause excessive secretion of viscous or foamy saliva and flow out of the mouth; the blisters on the foot can break and cause disability. Adult animals may experience unrecoverable weight loss for months after infection and swollen testicles in adult males, with a significant reduction in milk production for cows. Although most animals recover themselves after disease, the disease can also lead to myocarditis (heart muscle infection) or death when severe, particularly in neonatal animals. Some infected animals may be asymptomatic, they do not develop fever or any symptoms of illness; however, these animals are also foot and mouth disease vectors, which also transmit the disease.

Foot and mouth disease infections are typically regional, with the virus being transmitted to susceptible animals by direct contact with the infected animals, or by contaminated barns or trucks transporting the animals. Sick and virulent animals are the main sources of infection, and they can be transmitted to susceptible animals by both direct and indirect contact infections (e.g., secretions, excretions, animal products, contaminated air, feed, etc.). The outer coat or skin of an animal management person (e.g., farm worker), water to which the animal has come into contact, and uncooked food scraps and feed additives containing infected animal products can all be sources of viral load.

In the past, foot and mouth disease has occurred in many regions of the world (including europe, africa, asia, and south america), and the widespread and rapid spread of this disease has raised widespread international society attention. The world animal health organization lists the animal infectious diseases in the A-class animal disease list, and the government of China also ranks the animal infectious diseases in the first class. As the first pork producing and selling country in China, the pork safety problem is closely related to the benign sustainable development of the animal husbandry in China. With the continuous development and progress of molecular biology and immunology, novel vaccines with high cost performance and good safety are imperative.

At present, some researches are carried out by respectively expressing three proteins of VP1, VP3 and VP0 by using escherichia coli and then assembling in vitro, the method has high difficulty in purifying the proteins and very low efficiency in assembling the proteins in vitro; there are other studies in which VP1, VP0 and VP3 proteins are expressed separately using a plurality of baculoviruses and then Sf9 cells are co-infected with a plurality of viruses to co-express the three proteins, which requires a large number of viruses to be infected, and 2 or more viruses to be simultaneously infected into the same cell, which is problematic in that the efficiency is very low.

Disclosure of Invention

The present invention is directed to an immunological composition that solves the problems of the prior art.

In order to achieve the above object, according to one aspect of the present invention, there is provided an immunological composition comprising:

the structural protein VP3 protein of the O-type foot-and-mouth disease virus encoded by the nucleic acid molecule with the sequence shown as SEQ ID NO.1 or the nucleic acid molecule which is 95% identical to the nucleotide sequence of SEQ ID NO. 1;

the structural protein VP1 protein of O-type foot-and-mouth disease virus encoded by nucleic acid molecule with the sequence as shown in SEQ ID NO. 12 or nucleic acid molecule with 95% of the same nucleotide sequence as SEQ ID NO. 12;

and one or two of the structural protein VP2 protein of the foot-and-mouth disease virus type O or the structural protein VP4 protein of the foot-and-mouth disease virus type O can be selected in any combination.

The further technical scheme of the invention is as follows: the structural protein VP3 protein of the type O foot-and-mouth disease virus comprises an amino acid sequence of SEQ ID NO. 19 or an amino acid sequence which is more than 95% identical with the full-length amino acid sequence of SEQ ID NO. 19;

the structural protein VP1 protein of the O type foot-and-mouth disease virus comprises an amino acid sequence of SEQ ID NO. 22 or an amino acid sequence which is more than 95% identical with the full-length amino acid sequence of SEQ ID NO. 22.

The further technical scheme of the invention is as follows: the immune composition further comprises one or any combination of more than two of structural proteins VP2 and VP4 of the foot-and-mouth disease virus type O; wherein the content of the first and second substances,

the structural protein VP2 protein of the O type foot-and-mouth disease virus comprises an amino acid sequence of SEQ ID NO. 21 or an amino acid sequence which is more than 95% identical with the full-length amino acid sequence of SEQ ID NO. 21;

the structural protein VP4 protein of the O type foot-and-mouth disease virus comprises the amino acid sequence of SEQ ID NO. 23 or the amino acid sequence which is more than 95% identical with the full-length amino acid sequence of SEQ ID NO. 23.

The further technical scheme of the invention is as follows: wherein the content of the first and second substances,

the O type foot-and-mouth disease virus structural protein VP2 protein is obtained by encoding a nucleic acid molecule with a sequence shown as SEQ ID NO. 8 or a nucleic acid molecule with the same nucleotide sequence of more than 95% of SEQ ID NO. 8;

the structural protein VP4 protein of the O-type foot-and-mouth disease virus is obtained by encoding a nucleic acid molecule with the sequence shown as SEQ ID NO. 16 or a nucleic acid molecule which is 95% identical to the nucleotide sequence of the SEQ ID NO. 16.

The further technical scheme of the invention is as follows: the immune composition further comprises an O-type foot-and-mouth disease virus structural protein VP0, wherein the O-type foot-and-mouth disease virus structural protein VP0 is obtained by encoding a nucleic acid molecule with a sequence shown as SEQ ID NO. 4 or a nucleic acid molecule with 95% of nucleotide sequence identity with the SEQ ID NO. 4.

The further technical scheme of the invention is as follows: the structural protein VP0 protein of the type O foot-and-mouth disease virus comprises an amino acid sequence of SEQ ID NO. 20 or an amino acid sequence which is more than 95% identical with the full-length amino acid sequence of SEQ ID NO. 20.

The further technical scheme of the invention is as follows: the immune composition is a composition containing the structural proteins VP3, VP1 and VP2 of the type O foot-and-mouth disease virus. The further technical scheme of the invention is as follows: the immune composition is a composition containing the structural proteins VP3, VP1 and VP4 of the type O foot-and-mouth disease virus. The further technical scheme of the invention is as follows: the immune composition is a composition comprising the structural proteins VP3, VP1, VP2 and VP0 of the type O foot-and-mouth disease virus. The further technical scheme of the invention is as follows: the immune composition is a composition containing the structural proteins VP3, VP1, VP2 and VP4 of the O-type foot-and-mouth disease virus.

The further technical scheme of the invention is as follows: the immune composition is a composition comprising the structural proteins VP3, VP1, VP4 and VP0 of the type O foot-and-mouth disease virus.

The further technical scheme of the invention is as follows: the immune composition is a composition containing the O type foot-and-mouth disease virus structural proteins VP3, VP1, VP2, VP4 and VP0 proteins.

Another object of the present invention is to provide a method for preparing the immunological composition, the method comprising the steps of:

s1, cloning the gene of the O-type foot-and-mouth disease virus structural protein to a corresponding shuttle vector to obtain a recombinant shuttle vector;

s2, transforming the recombinant shuttle vector into DH10Bac bacteria containing a baculovirus genome, and directionally inserting a target gene expression frame in the recombinant shuttle vector into the baculovirus genome plasmid to obtain a recombinant baculovirus genome plasmid containing the target gene expression frame;

s3, transfecting the recombinant baculovirus genome plasmid into an insect cell to obtain a recombinant baculovirus;

s4, inoculating the obtained recombinant baculovirus into insect cells, and producing O-type foot-and-mouth disease virus structural protein in a reactor in a large scale;

s5, adding the O type foot-and-mouth disease virus structural protein obtained in the step S4 into an adjuvant to obtain the immune composition.

The further technical solution of the present invention is that step S1 includes: cloning coding genes of structural proteins VP3 and VP1 of the O-type foot-and-mouth disease virus and any one, two or three expression frames selected from VP2, VP4 and VP0 to the same shuttle vector to obtain the recombinant shuttle vector.

Preferably, step S1 includes: and cloning coding genes of structural proteins VP3 and VP1 of the O-type foot-and-mouth disease virus and expression frames selected from any one or two of structural proteins VP2 and VP4 of the O-type foot-and-mouth disease virus to the same shuttle vector to obtain the recombinant shuttle vector.

More preferably, step S1 includes: cloning the coding genes of the structural proteins VP3 and VP1 of the O-type foot-and-mouth disease virus, the expression frame of any one or two of the structural proteins VP2 and VP4 of the O-type foot-and-mouth disease virus and the expression frame of the structural protein VP0 of the O-type foot-and-mouth disease virus to the same shuttle vector to obtain the recombinant shuttle vector.

Particularly preferably, the step S1 includes: genes of VP1 protein, VP3 protein, VP0 protein, VP2 protein and VP4 protein are all cloned to the same shuttle vector to obtain the recombinant shuttle vector.

The further technical scheme of the invention is as follows: the shuttle vector is pFastBac Dual.

The further technical scheme of the invention is as follows: the insect cell is selected from any one of Sf9, High Five, S2 or Sf21 cell, but is not limited to the above.

In the foregoing embodiment of the present invention, one virus is used to co-express VP1 and VP3 proteins and any one, two or three proteins selected from VP2, VP4 and VP0 simultaneously. More preferably, five proteins of VP1, VP3, VP2, VP4 and VP0 are co-expressed at the same time by using one virus. Wherein VP2 and VP4 can be cleaved from VP0 protein. The VP1, VP3 and VP0 proteins can be automatically assembled into VLPs, and VP1, VP3, VP2 and VP4 proteins can also be automatically assembled into VLPs, so that a plurality of proteins are co-expressed using one baculovirus, so that the assembly efficiency of VLPs is greatly improved.

It is another object of the present invention to provide a use of said immunological composition for the manufacture of a medicament for inducing an immune response against a foot and mouth disease virus type O antigen in a subject animal.

It is another object of the present invention to provide a use of said immunological composition for the manufacture of a medicament for preventing infection of animals with foot and mouth disease virus type O.

It is still another object of the present invention to provide a nucleic acid molecule composition comprising: the structural protein VP3 protein for encoding type O foot-and-mouth disease virus comprises a sequential nucleotide sequence of SEQ ID NO.1 or a sequential nucleotide sequence which is 95% identical to the nucleotide sequence of SEQ ID NO. 1;

the structural protein VP1 protein for encoding type O foot-and-mouth disease virus comprises the sequential nucleotide sequence of SEQ ID NO. 12 or the sequential nucleotide sequence which is 95% identical to the nucleotide sequence of SEQ ID NO. 12.

Still another object of the present invention is to provide a use of said nucleic acid molecule composition for the manufacture of a medicament for inducing an immune response against a foot and mouth disease virus type O antigen in a subject animal.

The invention also aims to provide application of the nucleic acid molecule composition in producing a medicament for preventing animals from being infected by the type-O foot-and-mouth disease virus. For example, the medicament can be a novel genetic engineering subunit vaccine of the O-type foot-and-mouth disease virus.

It is still another object of the present invention to provide a protein composition comprising:

19 and the full-length amino acid sequence of 19 SEQ ID NO:19 are more than 95 percent of the same protein; 22 and the full-length amino acid sequence of the SEQ ID NO. 22 are more than 95 percent of the same protein.

In a preferred embodiment, in step S3, the medium used in the fermentation culture is a subculture medium.

The invention discloses a preparation method and application of an O-type foot-and-mouth disease virus recombinant subunit vaccine expressed by Sf9 cells, and proves that the vaccine can generate stronger humoral immunity in pigs and cattle, and immunized mammals such as pigs and cattle can resist the infection of the O-type foot-and-mouth disease virus, belonging to the technical field of animal vaccines and biological products for livestock and aiming at providing a preparation method of the O-type foot-and-mouth disease virus recombinant subunit vaccine capable of being industrially produced in a large scale.

After adopting the scheme, compared with the prior art, the invention has the following outstanding advantages and effects:

the novel genetic engineering subunit vaccine of the O-type foot-and-mouth disease virus has antigenicity, immunogenicity and functions similar to those of natural protein, high expression level, strong immunogenicity and no pathogenicity to animals, and can be prepared through large-scale serum-free suspension culture in a bioreactor, thereby greatly reducing the production cost of the vaccine.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention. In the drawings:

FIG. 1 shows a gel electrophoresis result of a PCR product obtained by PCR amplification of the VP3 protein gene, in which a band of about 0.7kbp is observed; wherein 1 is FMDV-VP3 gene, 2 is negative control, and M is molecular weight marker;

FIG. 2 shows the results of gel electrophoresis of PCR products obtained after PCR amplification of a plurality of colony samples transformed with the VP3 protein gene, showing that a positive sample was present in the vicinity of a 0.7kbp band. Wherein 1-5 are products obtained after PCR amplification of colony samples transformed by VP3 protein genes, 6 is a non-positive sample, and M is a molecular weight marker;

FIG. 3 shows a gel electrophoresis result of a PCR product obtained by PCR amplification of the VP0 protein gene, in which a band of about 0.9kbp is observed; wherein 1 is FMDV-VP0 gene, 2 is negative control, and M is molecular weight marker;

FIG. 4 shows the results of gel electrophoresis of PCR products obtained after PCR amplification of a plurality of colony samples transformed with the VP0 protein gene, showing that a positive sample was present in the vicinity of the 0.9kbp band. Wherein 1-5 are products obtained after PCR amplification of colony samples transformed by VP0 protein genes, 6 is a non-positive sample, and M is a molecular weight marker;

FIG. 5 shows a gel electrophoresis result of a PCR product obtained by PCR amplification of the VP2 protein expression cassette, in which a band of about 1.4kbp was observed; wherein 1 is FMDV-VP2 protein expression frame gene, 2 is negative control, and M is molecular weight marker;

FIG. 6 shows the results of gel electrophoresis of PCR products obtained after PCR amplification of a colony sample transformed with a plurality of VP2 protein expression cassettes, showing a positive sample in the vicinity of a 1.4kbp band. 1-5 are products obtained after PCR amplification of colony samples transformed by VP2 protein expression frames, 6 is a non-positive sample, and M is a molecular weight marker;

FIG. 7 shows a gel electrophoresis result of a PCR product obtained by PCR amplification of the VP1 protein expression cassette, in which a band of about 1.3kbp was observed; wherein 1 is FMDV-VP1 protein expression frame gene, 2 is negative control, and M is molecular weight marker;

FIG. 8 shows the results of gel electrophoresis of PCR products obtained after PCR amplification of a colony sample transformed with a plurality of VP1 protein expression cassettes, showing a positive sample in the vicinity of the 1.3kbp band. 1-5 are products obtained after PCR amplification of colony samples transformed by VP1 protein expression frames, 6 is a non-positive sample, and M is a molecular weight marker;

FIG. 9 shows a gel electrophoresis result of a PCR product obtained by PCR amplification of the VP4 protein expression cassette, in which a band of about 1.0kbp was observed; wherein 1 is FMDV-VP4 protein expression frame gene, 2 is negative control, and M is molecular weight marker;

FIG. 10 shows the results of gel electrophoresis of PCR products obtained after PCR amplification of a colony sample transformed with a plurality of VP4 protein expression cassettes, showing a positive sample in the vicinity of a 1.0kbp band. 1-5 are products obtained after PCR amplification of colony samples transformed by VP4 protein expression frames, 6 is a non-positive sample, and M is a molecular weight marker;

FIG. 11 is a diagram of constructed transfer vector Dual-VP3-VP0-VP2-VP1-VP4 containing a target gene;

FIG. 12 shows the SDS-PAGE gel electrophoresis of the cell culture supernatant harvested in example 3, wherein the cell culture of the VP3-VP0-VP2-VP1-VP4 recombinant protein showed the desired bands around 21kDa, 33kDa, 26kDa, 24kDa and 9kDa, respectively; wherein 1 is a negative control, 2 is the cell culture harvested in example 3, and M is a molecular weight marker;

FIG. 13 shows the results of WesternBlot detection of the product after SDS-PAGE in example 4; wherein 1 is a recombinant baculovirus expression sample, 2 is a negative control, and M is a molecular weight marker;

FIG. 14 shows the results of immunofluorescence assays;

FIG. 15 shows the results of electron microscope observation;

FIG. 16 shows the results after protein purification.

Detailed Description

It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, 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.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

The present invention provides an immunological composition comprising:

the structural protein VP3 protein of O-type foot-and-mouth disease virus encoded by the nucleic acid molecule of SEQ ID NO.1 or the nucleic acid molecule which is 95% identical to the nucleotide sequence of SEQ ID NO. 1;

the structural protein VP1 protein of O-type foot-and-mouth disease virus encoded by the nucleic acid molecule of SEQ ID NO. 12 or the nucleic acid molecule which is 95% identical to the nucleotide sequence of SEQ ID NO. 12;

The invention also relates to a method of inducing an immune response against a foot and mouth disease virus type O antigen, said method comprising administering to a subject animal a vaccine of the invention.

The present invention also relates to a method for protecting a test animal against infection by foot and mouth disease virus type O, said method comprising administering to said test animal a vaccine of the present invention.

The vaccine of the present invention may be a plasmid comprising the above-described nucleic acid molecule, the nucleic acid molecule may be incorporated into a virus particle, the vaccine may further comprise an adjuvant molecule, the adjuvant may be IL-12, IL-15, IL-28, CTACK, TECK, platelet-derived growth factor (PDGF), TNF α, TNF β, GM-CSF, Epidermal Growth Factor (EGF), IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-18, IL-21, IL-31, IL-33, or a combination thereof, and in some embodiments may be IL-12, IL-15, IL-28, or TES.

The vaccines of the present invention comprise a protein molecule composition. Provided herein are proteins selected from the group consisting of: a protein comprising SEQ ID NO 19 or 22 or 21 or 23 or 20; a protein that is 95% identical over the entire length of the amino acid sequence of SEQ ID NO 19 or 22 or 21 or 23 or 20; fragments of SEQ ID NO 19 or 22 or 21 or 23 or 20; a protein that is 95% identical to a fragment of SEQ ID NO 19 or 22 or 21 or 23 or 20.

Also provided herein is a protein composition selected from the group consisting of: (a) 19 or 22 or 21 or 23 or 20; (b) a protein that is 95% identical over the entire amino acid sequence length of the full-length sequence as set forth in SEQ ID NO 19 or 22 or 21 or 23 or 20; (c) an immunogenic fragment of SEQ ID NO 19 or 22 or 21 or 23 or 20 comprising 20 or more amino acids of SEQ ID NO 19 or 22 or 21 or 23 or 20; and (d) an immunogenic fragment comprising 20 or more amino acids of a protein that is 95% identical over the entire length of the amino acid sequence of SEQ ID NO 19 or 22 or 21 or 23 or 20. Of course, also provided herein is a protein composition further comprising a protein molecule or a combination of protein molecules selected from the group consisting of: (a) 20 or 21 or 23; (b) a protein that is 95% identical over the entire amino acid sequence length of the full-length sequence as set forth in SEQ ID NO 20 or 21 or 23; (c) an immunogenic fragment of SEQ ID NO 20 or 21 or 23 comprising 20 or more amino acids of SEQ ID NO 20 or 21 or 23; and (d) an immunogenic fragment comprising 20 or more amino acids of a protein that is 95% identical over the entire length of the amino acid sequence of SEQ ID NO:20 or 21 or 23.

The present invention provides nucleic acid molecule compositions comprising sequences encoding one or more of the protein molecules described above. In some embodiments, the nucleic acid molecule comprises a sequence selected from the group consisting of seq id no:1 or 12 or 8 or 16 or 4; a nucleic acid sequence that is 95% identical over the entire length of the nucleotide sequence of

SEQ ID NO

1 or 12 or 8 or 16 or 4; 1 or 12 or 8 or 16 or 4; a nucleotide sequence which is 95% identical to a fragment of

SEQ ID NO

1 or 12 or 8 or 16 or 4. The nucleic acid molecule composition further comprising one or a combination of nucleic acid molecules selected from the group consisting of: 4 or 8 or 16; a nucleic acid sequence that is 95% identical over the entire length of the nucleotide sequence of SEQ ID NO 4 or 8 or 16; a fragment of SEQ ID NO 4 or 8 or 16; a nucleotide sequence 95% identical to a fragment of SEQ ID NO 4 or 8 or 16.

Some aspects of the invention provide methods of inducing an immune response against foot and mouth disease virus type O, comprising the steps of: administering to the individual a foot and mouth disease virus type O antigen and/or a composition thereof.

Further aspects of the invention provide methods of protecting an individual from infection by foot-and-mouth disease virus type O. The method comprises the following steps: administering to the individual a prophylactically effective amount of a nucleic acid molecule or composition comprising such a nucleic acid sequence; wherein the nucleic acid sequence is expressed in cells of the individual and induces a protective immune response against a protein encoded by the nucleic acid sequence.

Some aspects of the invention provide a method of inducing an immune response against a foot and mouth disease virus type O antigen, the method comprising administering to a subject animal a nucleic acid molecule of the invention.

Some aspects of the invention provide a method of protecting a subject animal from infection by foot and mouth disease virus type O, comprising administering to said subject animal a nucleic acid molecule of the invention.

Some aspects of the invention provide a vaccine suitable for use in generating an immune response against foot and mouth disease virus type O in a subject animal, the vaccine comprising a nucleic acid molecule of the invention and an adjuvant molecule, the adjuvant may be IL-12, IL-15, IL-28, CTACK, TECK, Platelet Derived Growth Factor (PDGF), TNF α, TNF β, GM-CSF, Epidermal Growth Factor (EGF), IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-18, IL-21, IL-31, IL-33, or a combination thereof, and in some embodiments, may be IL-12, IL-15, IL-28, or RANTES.

The vaccine of the invention also comprises one or more nucleic acid molecules as described above and one or more proteins encoded by said nucleic acid molecules.

1. And (4) defining.

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used in the specification and in the claims, the singular form of "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

To the extent that numerical ranges are recited herein, each intervening number between equal degrees of precision is explicitly recited. For example, for the range of 6-9, the numbers 7 and 8 are encompassed in addition to 6 and 9, and for the range of 6.0-7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are expressly encompassed.

An "adjuvant" as used herein means any molecule added to the vaccines described herein to enhance the immunogenicity of the antigen encoded by the encoding nucleic acid sequence described below.

"antibody" as used herein means an antibody of the type IgG, IgM, IgA, IgD or IgE, or a fragment, fragment or derivative thereof, including Fab, F (ab')2, Fd, as well as single chain antibodies, diabodies, bispecific antibodies, bifunctional antibodies and derivatives thereof. The antibody may be an antibody isolated from a serum sample of an animal, a polyclonal antibody, an affinity purified antibody, or a mixture thereof that exhibits sufficient binding specificity for the desired epitope or a sequence derived therefrom.

"coding sequence" or "coding nucleic acid" as used herein means a nucleic acid (RNA or DNA molecule) comprising a nucleotide sequence encoding a protein. The coding sequence may further include initiation and termination signals operably linked to regulatory elements including a promoter and polyadenylation signals capable of directing expression in the cells of the subject or animal to which the nucleic acid is administered.

"complement" or "complementary" as used herein means that a nucleic acid can refer to Watson-Crick (e.g., A-T/U and C-G) or Hoogsteen base pairing between nucleotides or nucleotide analogs of the nucleic acid molecule.

As used herein, "consensus" or "consensus sequence" means a polypeptide sequence of multiple subtypes based on analysis of a cohort of specific type O foot-and-mouth disease virus antigens. Nucleic acid sequences encoding the consensus polypeptide sequence may be prepared. Vaccines comprising proteins comprising consensus sequences and/or nucleic acid molecules encoding these proteins may be used to induce broad immunity against multiple subtypes or serotypes of a particular type O foot-and-mouth disease virus antigen.

"electroporation," "electro-permeabilization," or "electrokinetic enhancement" ("EP") as used interchangeably herein means the use of transmembrane electric field pulses to induce microscopic pathways (pores) in a biological membrane; their presence allows biomolecules such as plasmids, oligonucleotides, siRNA, drugs, ions and water to flow from one side of the cell membrane to the other.

"fragment" as used herein with respect to a nucleic acid sequence means a nucleic acid sequence or a portion thereof encoding a polypeptide capable of eliciting an immune response in an animal that is cross-reactive with the full-length wild-type strain, type O foot-and-mouth disease virus antigen. The fragment may be a DNA fragment selected from at least one of various nucleotide sequences encoding protein fragments described below.

By "fragment" or "immunogenic fragment" with respect to polypeptide sequences is meant a polypeptide capable of eliciting an immune response in an animal that is cross-reactive with the full-length wild-type strain type O foot-and-mouth disease virus antigen. A fragment of a protein may comprise at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the protein. In some embodiments, a fragment of a protein may comprise at least 20 amino acids or more, at least 30 amino acids or more, at least 40 amino acids or more, at least 50 amino acids or more, at least 60 amino acids or more, at least 70 amino acids or more, at least 80 amino acids or more, at least 90 amino acids or more, at least 100 amino acids or more, at least 110 amino acids or more, at least 120 amino acids or more of the protein, at least 130 amino acids or more, at least 140 amino acids or more, at least 150 amino acids or more, at least 160 amino acids or more, at least 170 amino acids or more, at least 180 amino acids or more, at least 190 amino acids or more, at least 200 amino acids or more, at least 210 amino acids or more, at least 220 amino acids or more, at least 230 amino acids or more, or at least 240 amino acids or more.

The term "genetic construct" as used herein refers to a DNA or RNA molecule comprising a nucleotide sequence encoding a protein. The coding sequence comprises an initiation signal and a termination signal operably linked to regulatory elements including a promoter and polyadenylation signal capable of directing expression in the cells of the individual to whom the nucleic acid molecule is administered. The term "expression form" as used herein refers to a genetic construct containing the necessary regulatory elements operably linked to a coding sequence encoding a protein such that the coding sequence will be expressed when present in the cells of the individual.

The term "homology" as used herein refers to the degree of complementarity. There may be partial homology or complete homology (i.e., identity). Partial complementary sequences that at least partially inhibit hybridization of a fully complementary sequence to a target nucleic acid are referred to using the functional term "substantially homologous". The term "substantially homologous" as used herein when used with respect to a double-stranded nucleic acid sequence, such as a cDNA or genomic clone, means that the probe can hybridize to a strand of the double-stranded nucleic acid sequence under conditions of low stringency. The term "substantially homologous" as used herein when used with respect to a single-stranded nucleic acid sequence means that the probe can hybridize to the single-stranded nucleic acid template sequence (i.e., is the complement of the single-stranded nucleic acid template sequence) under low stringency conditions.

In the case of two or more nucleic acid or polypeptide sequences, "identical" or "identity" as used herein means that the sequences have a specified percentage of identical residues in a specified region. The percentage may be calculated by: optimally aligning the two sequences, comparing the two sequences over a specified region, determining the number of positions of the identical residue in the two sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions within the specified region, and multiplying the result by 100 to yield the percentage of sequence identity. Where two sequences are of different lengths or the alignment produces one or more staggered ends and the specified regions of comparison include only a single sequence, the residues of the single sequence are included in the denominator of the calculation rather than in the numerator. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. Identity can be performed manually or by using a computer sequence algorithm such as BLAST or BLAST 2.0.

As used herein, "immune response" means the activation of the immune system of a host (e.g., the immune system of an animal) in response to the introduction of an antigen, such as a foot and mouth disease virus type O consensus antigen. The immune response may be in the form of a cellular response or a humoral response or both.

As used herein, a "nucleic acid" or "oligonucleotide" or "polynucleotide" means at least two nucleotides covalently linked together. The description of single strands also defines the sequence of the complementary strand. Thus, nucleic acids also encompass the complementary strand of the single strand described. Many variants of a nucleic acid can be used for the same purpose as a given nucleic acid. Thus, nucleic acids also encompass substantially the same nucleic acids and their complements. Single strands provide probes that can hybridize to a target sequence under stringent hybridization conditions. Thus, nucleic acids also encompass probes that hybridize under stringent hybridization conditions.

The nucleic acid may be single-stranded or double-stranded or may contain portions of both double-stranded or single-stranded sequences. The nucleic acid can be DNA, both genomic and cDNA, RNA, or a hybrid, wherein the nucleic acid can contain a combination of deoxyribonucleotides and ribonucleotides, as well as a combination of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine, and isoguanine. The nucleic acid may be obtained by chemical synthesis methods or by recombinant methods.

The expression of the gene is carried out under the control of a promoter which is spatially linked thereto. Under its control, the promoter may be positioned 5 '(upstream) or 3' (downstream) of the gene. The distance between the promoter and the gene may be about the same as the distance between the promoter and the gene it controls in the gene from which the promoter is derived. As is known in the art, this change in distance can be adjusted without loss of promoter function. By "promoter" is meant a molecule of synthetic or natural origin that is capable of conferring, activating or enhancing expression of a nucleic acid in a cell. The promoter may comprise one or more specific transcriptional regulatory sequences to further enhance expression and/or alter spatial and/or temporal expression thereof. A promoter may also contain distal enhancer or repressor elements, which can be located as much as several thousand pairs of base pairs from the start of transcription. Promoters may be obtained from sources including viruses, bacteria, fungi, plants, insects, and animals. A promoter may regulate expression of a gene component either substantially or differentially with respect to the cell, tissue or organ in which expression occurs or with respect to the developmental stage at which expression occurs or in response to an external stimulus such as a physiological stress, pathogen, metal ion or inducer. Representative examples of promoters include a bacteriophage T7 promoter, a bacteriophage T3 promoter, an SP6 promoter, a lactose operon-promoter, a tac promoter, an SV40 late promoter, an SV40 early promoter, an RSV-LTR promoter, a CMVIE promoter, an SV40 early promoter or an SV40 late promoter, and a CMVIE promoter.

"Signal peptide" and "leader sequence" refer to amino acid sequences that may be attached to the amino terminus of the foot and mouth disease virus type O protein described herein. The signal peptide/leader sequence is generally indicative of the location of the protein. The signal peptide/leader sequence used herein preferably facilitates secretion of the protein from the cell in which it is produced. The signal peptide/leader sequence is often cleaved from the remainder of the protein, which is often referred to as the mature protein after secretion from the cell. The signal peptide/leader sequence is linked to the N-terminus of the protein.

By "stringent hybridization conditions" is meant conditions under which a first nucleic acid sequence (e.g., a probe) will hybridize to a second nucleic acid sequence (e.g., a target), such as in a complex mixture of nucleic acids. Stringent conditions are sequence dependent and will be different in different circumstances. Stringent conditions may be selected to be about 5-10 ℃ lower than the thermodynamic melting point (Tm) of the particular sequence at a defined ionic strength pH. The Tm can be the temperature (under defined ionic strength, pH, and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (at Tm, 50% of the probes are occupied at equilibrium because the target sequence is present in excess). Stringent conditions may be those in which the salt concentration is less than about 1.0M sodium ion, such as about 0.01-1.0M sodium ion concentration (or other salts) at pH7.0 to 8.3, and the temperature is at least about 30 ℃ for short probes (e.g., about 10-50 nucleotides) and at least about 60 ℃ for long probes (e.g., greater than about 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For a selected or specific hybridization, the positive signal can be at least 2 to 10 times the background hybridization. Exemplary stringent hybridization conditions include the following: 50% formamide, 5XSSC and 1% SDS, incubated at 42 ℃ or 5XSSC, 1% SDS, incubated at 65 ℃ washed with 0.2XSSC and 0.1% SDS at 65 ℃.

"substantially complementary" as used herein means that a first sequence is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the complement of a second sequence over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 180, 270, 360, 450, 540 or more nucleotides or amino acids, or that two sequences hybridize under stringent hybridization conditions.

"substantially identical" as used herein means that the first and second sequences are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100, 180, 270, 360, 450, 540 or more nucleotides or amino acid regions at least 60%, 65%, 70%, 95%, 97%, 98% or 99% identical, or, in the case of nucleic acids, if the first and second sequences are substantially complementary, so are the first and second sequences, within 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 85, 90, 95, 80, 85, 540 or more nucleotides or amino acid regions.

"subtype" or "serotype": as used interchangeably herein and with respect to type O foot and mouth disease virus, means a genetic variant of type O foot and mouth disease virus such that one subtype is recognized by the immune system and separated from different subtypes.

"variant" as used herein with respect to a nucleic acid means (i) a portion or fragment of a reference nucleotide sequence; (ii) a complement of a reference nucleotide sequence or a portion thereof; (iii) a nucleic acid that is substantially identical to a reference nucleic acid or a complement thereof; or (iv) a nucleic acid that hybridizes under stringent conditions to a reference nucleic acid, its complement, or a sequence substantially identical thereto.

"variants" in the case of peptides or polypeptides differ in amino acid sequence by insertion, deletion or conservative substitution of amino acids, but retain at least one biological activity. A variant also means a protein having substantially the same amino acid sequence as a reference protein having an amino acid sequence that retains at least one biological activity. Conservative substitutions of amino acids, i.e., the replacement of an amino acid with a different amino acid of similar characteristics (e.g., hydrophilicity, extent and distribution of charged regions) are believed in the art to typically involve minor changes. As understood in the art, these minor changes may be identified in part by considering the hydropathic index of amino acids. Kate (Kyte), et al, J.Mol.biol., 157:105-132 (1982). The hydropathic index of the amino acid is based on considerations of its hydrophobicity and charge. It is known in the art that amino acids of similar hydropathic indices can be substituted and still retain protein function. In one aspect, amino acids with a hydropathic index of ± 2 are substituted. The hydrophilicity of amino acids can also be used to reveal substitutions that will result in proteins that retain biological function. Considering the hydrophilicity of amino acids in the case of peptides allows the calculation of the maximum local average hydrophilicity of the peptide, which is a useful measure that has been reported to correlate well with antigenicity and immunogenicity. As is understood in the art, substitution of amino acids with similar hydrophilicity values can result in peptides that retain biological activity (e.g., immunogenicity). Substitutions may be made with amino acids having hydrophilicity values within ± 2 of each other. Both the hydropathic index and the hydropathic value of an amino acid are affected by the specific side chain of the amino acid. Consistent with the observations, amino acid substitutions that are compatible with biological function are understood to depend on the relative similarity of these amino acids, and in particular the side chains of those amino acids, as revealed by hydrophobicity, hydrophilicity, charge, size, and other properties.

"vector" as used herein means a nucleic acid sequence containing an origin of replication. The vector may be a viral vector, a bacteriophage, a bacterial artificial chromosome, or a yeast artificial chromosome. The vector may be a DNA or RNA vector. The vector may be a self-replicating extrachromosomal vector, and is preferably a DNA vector.

2. Vaccine

The vaccines of the present invention can be designed to control the extent or intensity of an immune response in a subject animal against one or more serotypes of type O foot-and-mouth disease virus. The vaccine may comprise elements or agents that inhibit its integration into the chromosome. The vaccine may be RNA encoding the structural protein of type O foot-and-mouth disease virus. An RNA vaccine can be introduced into the cells. The vaccine of the present invention may comprise a foot and mouth disease virus type O structural protein. The foot and mouth disease virus type O structural proteins are targets for immune-mediated viral clearance by inducing 1) a Cytotoxic T Lymphocyte (CTL) response, 2) a T helper cell response and/or 3) a B cell response, or preferably all of the above mentioned responses, to achieve cross presentation.

The antigens may comprise protein epitopes that make them particularly effective as immunogens against which an immune response against foot and mouth disease virus type O can be induced. The type O foot and mouth disease virus antigen may include full length translation products, variants thereof, fragments thereof, or combinations thereof.

Some embodiments relate to nucleic acid molecules encoding immunogenic proteins that are 95% homologous to the nucleic acid coding sequences herein. Some embodiments relate to nucleic acid molecules encoding immunogenic proteins having 96% homology to the nucleic acid coding sequences herein. Some embodiments relate to nucleic acid molecules encoding immunogenic proteins that have 97% homology to the nucleic acid coding sequences herein. Some embodiments relate to nucleic acid molecules encoding immunogenic proteins that have 98% homology to the nucleic acid coding sequences herein. Some embodiments relate to nucleic acid molecules encoding immunogenic proteins having 99% homology to the nucleic acid coding sequences herein. In some embodiments, a nucleic acid molecule having a coding sequence disclosed herein that is homologous to a coding sequence of a protein disclosed herein comprises a sequence encoding an IgE leader sequence linked to the 5' end of the coding sequence encoding the homologous protein sequence disclosed herein.

In some embodiments, the nucleic acid sequence does not contain a coding sequence that encodes a leader sequence. In some embodiments, the nucleic acid sequence does not contain a coding sequence that encodes an IgE leader.

Some embodiments relate to fragments of

SEQ ID NO

1 or 12 or 8 or 16 or 4. A fragment may be at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, or at least 55% at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of SEQ ID No.1 or 12 or 8 or 16 or 4. Fragments may be at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to fragments of SEQ ID NO.1 or 12 or 8 or 16 or 4. The fragment may be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a fragment of SEQ ID No.1 or 12 or 8 or 16 or 4. In some embodiments, a fragment comprises a sequence encoding a leader sequence, e.g., an immunoglobulin leader, such as an IgE leader. In some embodiments, a fragment does not contain a coding sequence that encodes a leader sequence. In some embodiments, the fragment does not contain a coding sequence that encodes a leader sequence, such as, for example, an IgE leader.

Some embodiments relate to proteins homologous to SEQ ID NO 19 or 22 or 21 or 23 or 20. Some embodiments relate to immunogenic proteins having 95% homology to the protein sequence as set forth in SEQ ID NO 19 or 22 or 21 or 23 or 20. Some embodiments relate to immunogenic proteins having 96% homology to the protein sequence as set forth in SEQ ID NO 19 or 22 or 21 or 23 or 20. Some embodiments relate to immunogenic proteins having 97% homology to the protein sequence as set forth in SEQ ID NO 19 or 22 or 21 or 23 or 20. Some embodiments relate to immunogenic proteins having 98% homology to the protein sequence as set forth in SEQ ID NO 19 or 22 or 21 or 23 or 20. Some embodiments relate to immunogenic proteins having 99% homology to the protein sequence as set forth in seq id NO 19 or 22 or 21 or 23 or 20.

Some embodiments relate to the same protein as SEQ ID NO 19 or 22 or 21 or 23 or 20. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 80% identical over the entire amino acid sequence length of the full length consensus amino acid sequence as set forth in SEQ ID NOs 19 or 22 or 21 or 23 or 20. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 85% identical over the entire amino acid sequence length of the full length consensus amino acid sequence as set forth in SEQ id nos 19 or 22 or 21 or 23 or 20. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 90% identical over the entire amino acid sequence length of the full length consensus amino acid sequence as set forth in SEQ ID NOs 19 or 22 or 21 or 23 or 20. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 91% identical over the entire amino acid sequence length of the full length consensus amino acid sequence as set forth in SEQ ID NOs 19 or 22 or 21 or 23 or 20. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 92% identical over the entire amino acid sequence length of the full length consensus amino acid sequence as set forth in SEQ ID NOs 19 or 22 or 21 or 23 or 20. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 93% identical over the entire amino acid sequence length of the full-length consensus amino acid sequence as set forth in seq id NOs 19 or 22 or 21 or 23 or 20. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 94% identical over the entire amino acid sequence length of the full length consensus amino acid sequence as set forth in SEQ ID NOs 19 or 22 or 21 or 23 or 20. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 95% identical over the entire amino acid sequence length of the full length consensus amino acid sequence as set forth in SEQ ID NOs 19 or 22 or 21 or 23 or 20. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 96% identical over the entire amino acid sequence length of the full length consensus amino acid sequence as set forth in SEQ ID NOs 19 or 22 or 21 or 23 or 20. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 97% identical over the entire amino acid sequence length of the full length consensus amino acid sequence as set forth in SEQ ID NOs 19 or 22 or 21 or 23 or 20. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 98% identical over the entire amino acid sequence length of the full length consensus amino acid sequence as set forth in SEQ ID NOs 19 or 22 or 21 or 23 or 20. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 99% identical over the entire amino acid sequence length of the full length consensus amino acid sequence as set forth in SEQ ID NOs 19 or 22 or 21 or 23 or 20.

In some embodiments, the protein does not contain a leader sequence. In some embodiments, the protein does not contain an IgE leader. A fragment of a protein may comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, or at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the protein. Immunogenic fragments of SEQ ID NO 19 or 22 or 21 or 23 or 20 may be provided. An immunogenic fragment may comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% or at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of SEQ ID NO 19 or 22 or 21 or 23 or 20. In some embodiments, the fragment includes a leader sequence, such as, for example, an immunoglobulin leader, such as an IgE leader. In some embodiments, the fragment does not contain a leader sequence. In some embodiments, the fragment does not contain a leader sequence, such as, for example, an IgE leader.

Immunogenic fragments of proteins having amino acid sequences homologous to the immunogenic fragments of SEQ ID NO 19 or 22 or 21 or 23 or 20 may be provided. The immunogenic fragment may comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% or at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the protein homologous to SEQ ID NO 19 or 22 or 21 or 23 or 2095%. Some embodiments relate to immunogenic fragments that are 96% homologous to the immunogenic fragments of protein sequences herein. Some embodiments relate to immunogenic fragments that are 97% homologous to the immunogenic fragments of protein sequences herein. Some embodiments relate to immunogenic fragments that are 98% homologous to the immunogenic fragments of protein sequences herein. Some embodiments relate to immunogenic fragments that are 99% homologous to the immunogenic fragments of protein sequences herein. In some embodiments, the fragment comprises a leader sequence, such as, for example, an immunoglobulin leader sequence, such as an IgE leader. In some embodiments, the fragment does not contain a leader sequence. In some embodiments, the fragment does not contain a leader sequence, such as, for example, an IgE leader.

Immunogenic fragments of proteins having the same amino acid sequence as the immunogenic fragment of SEQ ID NO 19 or 22 or 21 or 23 or 20 may be provided. The immunogenic fragment may comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% or at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the proteins that are 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical over the entire length of the amino acid sequence set forth in SEQ ID No. 19 or 22 or 21 or 23 or 20. In some embodiments, the fragment includes a leader sequence, such as, for example, an immunoglobulin leader, such as an IgE leader. In some embodiments, the fragment does not contain a leader sequence. In some embodiments, the fragment does not contain a leader sequence, such as, for example, an IgE leader.

3. Vaccine constructs and plasmids

Vaccines may include nucleic acid constructs or plasmids encoding the structural proteins of type O foot and mouth disease virus, the antigens of type O foot and mouth disease virus, and combinations of structural proteins/antigens of type O foot and mouth disease virus. Provided herein are genetic constructs that may comprise a nucleic acid sequence encoding a foot and mouth disease virus type O antigen disclosed herein, including a protein sequence, a sequence homologous to a protein sequence, a fragment of a protein sequence, and a sequence homologous to a fragment of a protein sequence. Additionally, provided herein are genetic constructs that can comprise a nucleic acid sequence encoding a foot and mouth disease virus type O surface antigen disclosed herein (including protein sequences, sequences homologous to protein sequences, fragments of protein sequences, and sequences homologous to fragments of protein sequences). The genetic construct may be present as a functional extrachromosomal molecule. The genetic construct may be a linear minichromosome comprising a centromere, telomere or plasmid or cosmid.

The genetic construct may also be part of the genome of a recombinant viral vector, including recombinant adenovirus, recombinant adeno-associated virus, and recombinant vaccinia. The genetic construct may be part of the genetic material in a recombinant microbial vector in a live attenuated microorganism or in a cell.

The genetic construct may comprise regulatory elements for gene expression of the coding sequence of the nucleic acid. The regulatory element may be a promoter, enhancer, start codon, stop codon or polyadenylation signal.

The nucleic acid sequence may constitute a genetic construct which may be a vector. The vector is capable of expressing an antigen in cells of an animal in an amount effective to elicit an immune response in the animal. The vector may be recombinant. The vector may comprise a heterologous nucleic acid encoding an antigen. The vector may be a plasmid. The vector may be suitable for transfecting cells with nucleic acid encoding an antigen, the transformed host cells being cultured and maintained under conditions in which expression of the antigen occurs.

The coding sequence can be optimized for stability and high levels of expression. In some cases, the codons are selected to reduce the formation of RNA secondary structures, such as those due to intramolecular bonds.

The vector may comprise a heterologous nucleic acid encoding an antigen, and may further comprise a start codon that may be upstream of the antigen encoding sequence and a stop codon that may be downstream of the antigen encoding sequence. The initiation codon and the stop codon can be in frame with the antigen coding sequence. The vector further comprises a promoter operably linked to the antigen coding sequence. The promoter operably linked to the antigen-encoding sequence may be a promoter from simian virus 40(SV40), mouse mammary virus (MMTV) promoter, Human Immunodeficiency Virus (HIV) promoter such as the Bovine Immunodeficiency Virus (BIV) Long Terminal Repeat (LTR) promoter, Moloney (Moloney) virus promoter, Avian Leukemia Virus (ALV) promoter, Cytomegalovirus (CMV) promoter such as the CMV immediate early promoter, Epstein Barr Virus (EBV) promoter, or Rous Sarcoma Virus (RSV) promoter. The promoter may also be a promoter from a human gene such as human actin, human myosin, human heme, human muscle creatine or human metallothionein. The promoter may also be a tissue-specific promoter, such as a natural or synthetic muscle or skin-specific promoter.

The vector may further comprise a polyadenylation signal, which may be downstream of the foot and mouth disease virus type O core protein coding sequence, which may be the SV40 polyadenylation signal, the LTR polyadenylation signal, the bovine growth hormone (bGH) polyadenylation signal, the human growth hormone (hGH) polyadenylation signal, or the human β -globin polyadenylation signal the SV40 polyadenylation signal may be the polyadenylation signal from the pCEP4 vector (Invitrogen, San Diego, CA).

The vector may also comprise an enhancer upstream of the consensus type O foot-and-mouth disease virus core protein coding sequence or the consensus type O foot-and-mouth disease virus surface antigen protein coding sequence. The enhancer is necessary for DNA expression. The enhancer may be human actin, human myosin, human hemoglobin, human muscle creatine or a viral enhancer such as one from CMV, HA, RSV or EBV.

The vector may also comprise an animal origin of replication, in order to maintain the vector extrachromosomally and produce multiple copies of the vector in the cell. The vector may be pVAX1, pCEP4 or pREP4 from Invitrogen (San Diego, CA), which may contain the replication origin of epstein-barr virus and the nuclear antigen EBNA-1 coding region, which may produce high copy episomal replication without integration. The vector may be a pVAX1 or a pVAX1 variant, such as a variant plasmid described herein, with a variation. The variant pVax1 plasmid is a 2998 base pair variant of the backbone vector plasmid pVax1(Invitrogen, Carlsbad CA). The CMV promoter is located at base 137-724. The T7 promoter/initiation site was located at base 664-683. The multiple cloning site is located at bases 696-811. The bovine GH polyadenylation signal is at base 829-1053. The Kanamycin (Kanamycin) resistance gene is at base 1226-containing 2020. The pUC origin is at base 2320-2993.

The vector may be pSE420(Invitrogen, San Diego, Calif), which can be used to produce proteins in e. The vector may be pYES2(Invitrogen, San Diego, Calif.) which can be used to produce proteins in a Saccharomyces cerevisiae strain of yeast (Saccharomyces cerevisiae strain). The vector may also have a MAXBACTM complete baculovirus expression system (Invitrogen, San Diego, Calif.), which can be used to produce proteins in insect cells. The vector may also be pcDNAI or pcDNA3(Invitrogen, san Diego, Calif.), which may be used to produce proteins in animal cells such as the Sf9 cell line. The vector may be an expression vector or system for producing a protein by conventional techniques and readily available starting materials, including Sambrook et al, Molecular Cloning and Laboratory Manual, 2 nd edition, Cold spring Harbor (1989).

The promoter for the baculovirus expression system transfer vector in the recombinant baculovirus vector is any of the pFastBacDual.five proteins, such as the P10 promoter, the PH promoter, the shrimp β -actin gene promoter, the OpIE promoter, but not limited to these four promoters, and possibly other baculovirus promoters, the transcription termination signals for the Five proteins are any of the SV40polyA transfection termination signal, the HSV tk polyA transfection termination signal, or the OpIE A termination signal, and should not be limited to the use of these three transcription termination signals, but may also be other transcription termination signals, the insect cell line may be Sf9, gh Five, S2, or Sf21 cells, preferably Sf 9. the invention is preferably used for animals, particularly sheep, cattle, farm animals, cattle.

The principle of the present invention lies in that a recombinant Baculovirus shuttle plasmid is constructed, which contains expression genes expressing any three or four or five of the five proteins VP, VP and VP (wherein VP and VP proteins are essential), VP and VP proteins and VP, VP and VP three protein expression cassettes can be expressed using any one of a P promoter, a PH promoter, a shrimp-actin gene promoter, an opine promoter (which should not be limited to using these four promoters, and may also be other promoters), and the transcription termination signals of VP and VP proteins and VP, VP and VP three protein expression cassettes can be any one of SV polyA transfection termination signals, HSV tk polyA transfection termination signals, opine polyA termination signals (which should not be limited to using these three transcription termination signals, and may also be other transcription termination signals), so that these five proteins or three or four thereof are simultaneously expressed on one vector, but VP and Baculovirus proteins are necessary, and efficiency is greatly improved, and recombinant Baculovirus transformation bacdh 10 is constructed to obtain recombinant virus midid (VP, VP and VP, VP and VP are automatically transfected into recombinant Baculovirus cells, and VLP proteins, and VP are automatically expressed into recombinant Baculovirus.

Specifically, optimized and synthesized VP and VP protein encoding genes of FMDV are firstly cloned to the PH promoter and the P promoter of a pFastBacDual vector respectively, a Dual-VP-VP vector is constructed, then a VP protein expression cassette is inserted at the SnaB I cleavage site of the Dual-VP vector, the expression cassette comprises a prawn-actin gene promoter, an optimized VP gene and SV40polyA transcription termination signals, so that a Dual-VP-VP-VP vector is constructed, then a VP protein expression cassette is inserted at the Avr II cleavage site of the Dual-VP-VP vector, the expression cassette comprises an OpIE promoter, which is a second early regulator promoter in Orgyia pseudofollowid multicapus nuclear virus (OpMNPV) virus, the optimized VP gene and the OpIE polyA transcription termination signals, so that a Dual-VP-VP-VP gene and VP protein expression cassette are constructed, and a recombinant VP protein expression cassette is constructed after the optimized VP-VP protein expression cassette is transfected by the recombinant vector, and the recombinant VP-VP-VP-VP-VP protein expression cassette is constructed, and recombinant vector is constructed, so that the gene expression cassette is obtained after the strain is transfected by the strain.

Thus, five proteins, namely optimized protein sequences of VP1, VP0, VP3, VP2 and VP4, are simultaneously expressed by using one baculovirus and Sf9 cell, the five expressed proteins can be spontaneously assembled into VLP, and the VLP can be better assembled with VP1 and VP3 due to the fact that the interior of the VLP contains VP0, VP2 and VP4, and the assembly efficiency is high. The invention simultaneously expresses five proteins of VP1, VP3, VP2, VP4 and VP0, VP1, VP3 and VP0 can be assembled into VLP, VP1, VP3, VP2 and VP4 can also be assembled into VLP, and the five proteins are co-expressed, so that the assembly efficiency of VLP is greatly improved. The antigenicity, immunogenicity and function of the formed FMDV VLP are similar to those of natural protein, the expression level is high, the immunogenicity is strong, and the VLP has no pathogenicity to pigs.

EXAMPLE 1 construction and characterization of the transfer vector Dual-VP3-VP0-VP2-VP1-VP4

1. Construction and identification of transfer vector Dual-VP3

1.1VP3 Gene amplification and purification A codon-optimized VP3 gene (SEQ ID NO:1) was synthesized by Nanjing Kinshire and cloned into a pUC17 vector to obtain a pUC-VP3 plasmid vector. PCR amplification was carried out using pUC-VP3 plasmid as template and VP3-F, VP3-R as upstream and downstream primers (gene sequences of VP3-F, VP3-R are shown in SEQ ID NO.2 and 3), and the amplification system is shown in Table 1.

TABLE 1VP3 Gene amplification System

The reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 45 seconds, annealing at 54 ℃ for 45 seconds, extension at 72 ℃ for 1 minute, 35 cycles; extension at 72 ℃ for 10 min.

The size of the target gene was verified by subjecting the PCR product to gel electrophoresis, and as shown in FIG. 1, a band of interest appeared at a position of about 0.7kbp, and the target gene was successfully amplified, and then recovered and purified using a gel recovery and purification kit.

1.2 digestion and purification the PCR amplification products of pFastBacDual plasmid and VP3 gene were digested simultaneously for 3 hours at 37 ℃ with BamHI and Hind III, and the specific digestion reaction systems are shown in tables 2 and 3.

And (3) performing gel electrophoresis on the enzyme digestion product, and purifying the enzyme digestion pFastBacDual plasmid and the VP3 gene fragment by using a gel recovery and purification kit respectively.

TABLE 2 enzyme digestion reaction system of VP3 gene

TABLE 3pFastbacDual plasmid digestion reaction System

1.3 ligation the double-digested pFastBac Dual plasmid and the product of the VP3 gene digestion were ligated using T4DNA ligase in a water bath at 16 ℃ overnight. The specific ligation reaction system is shown in Table 4.

TABLE 4 VP3 Gene and pFastBac Dual plasmid ligation System

1.4 transformation 10. mu.l of the ligation product was added to 100. mu.l of DH5 α competent cells and mixed well, ice-cooled for 30 minutes, heat shocked in a water bath at 42 ℃ for 90 seconds, ice-cooled for 2 minutes again, 900. mu.l of LB liquid medium without Amp was added, and cultured at 37 ℃ for 1 hour, 1.0ml of the bacterial solution was concentrated to 100. mu.l and spread on LB solid medium with Amp, and cultured at 37 ℃ for 16 hours.

1.5 colony PCR and sequencing identification Single colonies on the picked plates were inoculated respectively to LB liquid medium, cultured at 37 ℃ for 2 hours, colony PCR identification was performed with the bacterial solution as template and VP3-F and VP3-R as primers, the PCR product was subjected to gel electrophoresis to verify the size of the target gene, as shown in FIG. 2, the sample with 0.7kbp band appeared was a positive sample. And (4) sending the bacteria liquid with positive identification to a sequencing company for sequencing, and selecting the bacteria liquid with correct sequencing for storage.

2. Construction and identification of transfer vector Dual-VP3-VP0

2.1VP0 Gene amplification and purification A codon-optimized VP0 gene (SEQ ID NO:4) was synthesized by Nanjing Kinshire and cloned into a pUC17 vector to obtain a pUC-VP0 plasmid vector. PCR amplification was carried out using pUC-VP0 plasmid as template and VP0-F, VP0-R as upstream and downstream primers (gene sequences of VP0-F, VP0-R are shown in SEQ ID NO.5 and 6), and the amplification system is shown in Table 5.

TABLE 5 VP0 Gene amplification System

The size of the target gene was verified by subjecting the PCR product to gel electrophoresis, and as shown in FIG. 3, a band of interest appeared at a position of approximately 0.9kbp, and the target gene was successfully amplified, and then recovered and purified using a gel recovery and purification kit.

2.2 digestion and purification the PCR amplification product of Dual-VP3 plasmid and VP0 gene was digested simultaneously with Xho I and Kpn I at 37 ℃ for 3 hours, and the specific digestion reaction systems are shown in tables 6 and 7.

And (3) performing gel electrophoresis on the enzyme digestion product, and purifying the enzyme digestion pFastBacDual plasmid and the VP0 gene fragment by using a gel recovery and purification kit respectively.

TABLE 6 enzyme digestion reaction system of VP0 gene

TABLE 7 Dual-VP3 plasmid digestion reaction System

2.3 ligation the double digested Dual-VP3 plasmid and the VP0 gene digest were ligated using T4DNA ligase in a water bath at 16 ℃ overnight. The specific ligation reaction system is shown in Table 8.

TABLE 8 connection System of VP0 Gene and Dual-VP3 plasmid

2.4 transformation 10. mu.l of the ligation product was added to 100. mu.l of DH5 α competent cells and mixed well, ice-cooled for 30 minutes, heat shocked in a water bath at 42 ℃ for 90 seconds, ice-cooled for 2 minutes again, 900. mu.l of LB liquid medium without Amp was added, and cultured at 37 ℃ for 1 hour, 1.0ml of the bacterial solution was concentrated to 100. mu.l and spread on LB solid medium with Amp, and cultured at 37 ℃ for 16 hours.

2.5 colony PCR and sequencing identification Single colonies on the picked plates were inoculated into LB liquid medium, cultured at 37 ℃ for 2 hours, colony PCR identification was performed using the bacterial solution as template, VP0-F and VP0-R as primers, the PCR product was subjected to gel electrophoresis to verify the size of the target gene, as shown in FIG. 4, and the sample with 0.9kbp band appeared was a positive sample. And (4) sending the bacteria liquid with positive identification to a sequencing company for sequencing, and selecting the bacteria liquid with correct sequencing for storage.

3. Construction and identification of transfer vector Dual-VP3-VP0-VP2

3.1 amplification and purification of VP2 Gene expression cassette the VP2 gene expression cassette (SEQ ID NO:7) was synthesized by Nanjing Kinshire and cloned into pUC17 vector to obtain pUC-VP2 plasmid vector, which included the promoter of prawn β -actin gene, codon optimized FMDV VP2 gene and SV40poly A stop signal sequence, PCR amplification was performed using pUC-VP2 plasmid as template and VP2-F, VP2-R as upstream and downstream primers (the gene sequences of VP2-F, VP2-R are shown in SEQ ID NO.9 and 10), and the amplification system is shown in Table 9.

TABLE 9 VP2 Gene amplification System

The size of the target gene was verified by subjecting the PCR product to gel electrophoresis, and as shown in FIG. 5, a band of interest appeared at a position of about 1.4kbp, and the target gene was successfully amplified, and then recovered and purified using a gel recovery and purification kit.

3.2 digestion and purification the PCR amplification products of the Dual-VP3-VP0 plasmid and the VP2 gene expression cassette were digested with SnaBI at 37 ℃ for 3 hours, and the specific digestion reaction systems are shown in tables 10 and 11.

And (3) performing gel electrophoresis on the enzyme digestion product, and purifying the PCR amplification product of the enzyme digestion Dual-VP3-VP0 plasmid and VP2 gene expression cassette by using a gel recovery purification kit respectively.

TABLE 10 enzyme digestion reaction system of VP2 gene

TABLE 11 Dual-VP3-VP0 plasmid digestion reaction System

3.3 ligation the double digested Dual-VP3-VP0 plasmid and the VP2 gene expression cassette cleavage product were ligated using T4DNA ligase in a water bath at 16 ℃ overnight. The specific ligation reaction system is shown in Table 12.

TABLE 12 PCR product of gene expression cassette for VP2 and Dual-VP3-VP0 plasmid ligation System

3.4 transformation 10. mu.l of the ligation product was added to 100. mu.l of DH5 α competent cells and mixed well, ice-cooled for 30 minutes, heat shocked in a water bath at 42 ℃ for 90 seconds, ice-cooled for 2 minutes again, 900. mu.l of LB liquid medium without Amp was added, and cultured at 37 ℃ for 1 hour, 1.0ml of the bacterial solution was concentrated to 100. mu.l and spread on LB solid medium with Amp, and cultured at 37 ℃ for 16 hours.

3.5 colony PCR and sequencing identification Single colonies on the picked plates were inoculated respectively to LB liquid medium, cultured at 37 ℃ for 2 hours, colony PCR identification was performed using the bacterial solution as template, VP2-F and VP2-R as primers, the PCR product was subjected to gel electrophoresis to verify the size of the target gene, as shown in FIG. 6, the sample with a 1.4kbp band appeared was a positive sample. And (4) sending the bacteria liquid with positive identification to a sequencing company for sequencing, and selecting the bacteria liquid with correct sequencing for storage.

4. Construction and identification of transfer vector Dual-VP3-VP0-VP2-VP1

4.1 amplification and purification of VP1 Gene expression cassette the VP1 gene expression cassette (SEQ ID NO:11) was synthesized by Nanjing Kinshire and cloned into pUC17 vector to obtain pUC-VP1 plasmid vector, the VP1 gene expression cassette comprising OpIE promoter, codon optimized FMDVVP1 gene and OpIEpolyA transcription termination signal sequence. PCR amplification was carried out using pUC-VP1 plasmid as template and VP1-F, VP1-R as upstream and downstream primers (gene sequences of VP1-F, VP1-R are shown in SEQ ID NO.13 and 14), and the amplification system is shown in Table 13.

TABLE 13 VP1 Gene expression cassette amplification System

The size of the target gene was verified by subjecting the PCR product to gel electrophoresis, and as shown in FIG. 7, a band of interest appeared at a position of about 1.3kbp, and the target gene was successfully amplified, and then recovered and purified using a gel recovery and purification kit.

4.2 digestion and purification the PCR amplification products of the Dual-VP3-VP0-VP2 plasmid and the VP1 gene expression cassette were digested simultaneously for 3 hours at 37 ℃ with AvrII endonuclease, and the specific digestion reaction systems are shown in tables 14 and 15.

And (3) performing gel electrophoresis on the enzyme digestion product, and purifying the enzyme digestion Dual-VP3-VP0-VP2 plasmid and VP1 gene expression cassette PCR amplification product by using a gel recovery purification kit respectively.

TABLE 14 enzyme digestion reaction system for PCR product of gene expression cassette VP1

TABLE 15 Dual-VP3-VP0-VP2 plasmid digestion reaction System

4.3 ligation the double digested Dual-VP3-VP0-VP2 plasmid and the VP1 gene expression cassette digest were ligated using T4DNA ligase in a water bath at 16 ℃ overnight. The specific ligation reaction system is shown in Table 16.

TABLE 16 VP1 Gene expression cassette and Dual-VP3-VP0-VP2 plasmid ligation System

4.4 transformation 10. mu.l of the ligation product was added to 100. mu.l of DH5 α competent cells and mixed well, ice-cooled for 30 minutes, heat shocked in a water bath at 42 ℃ for 90 seconds, ice-cooled for 2 minutes again, 900. mu.l of LB liquid medium without Amp was added, and cultured at 37 ℃ for 1 hour, 1.0ml of the bacterial solution was concentrated to 100. mu.l and spread on LB solid medium with Amp, and cultured at 37 ℃ for 16 hours.

4.5 colony PCR and sequencing identification Single colonies on the picked plates were inoculated into LB liquid medium, cultured at 37 ℃ for 2 hours, colony PCR identification was performed using the bacterial solution as template, VP1-F and VP1-R as primers, the PCR product was subjected to gel electrophoresis to verify the size of the target gene, as shown in FIG. 8, and the sample with a 1.3kbp band was found to be a positive sample. And (4) sending the bacteria liquid with positive identification to a sequencing company for sequencing, and selecting the bacteria liquid with correct sequencing for storage.

Construction of Dual-VP3-VP0-VP2-VP1-VP4 vector

5.1 amplification and purification of VP4 Gene expression cassette VP4 Gene expression cassette (SEQ ID NO:15) was synthesized by Nanjing Kinshire and cloned into pUC17 vector to obtain pUC-VP4 plasmid vector, where the VP4 gene expression cassette includes prawn β -actin, codon-optimized FMDV VP4 gene and SV40poly A transcription termination signal sequence, PCR amplification was performed using pUC-VP4 plasmid as template and VP4-F, VP4-R as upstream and downstream primers (the gene sequences of VP4-F, VP4-R are shown in SEQ ID NO.17, 18), and the amplification system is shown in Table 17.

TABLE 17 VP4 Gene expression cassette amplification System

The size of the target gene was verified by subjecting the PCR product to gel electrophoresis, and as shown in FIG. 9, a band of interest appeared at a position of about 1.0kbp, and the target gene was successfully amplified and recovered and purified using a gel recovery and purification kit.

5.2 digestion and purification the Dual-VP3-VP0-VP2-VP1 plasmid and the VP4 gene expression cassette PCR amplification product were digested simultaneously with BsrGI endonuclease at 37 ℃ for 3 hours, and the specific digestion reaction systems are shown in tables 18 and 19.

And (3) performing gel electrophoresis on the enzyme digestion product, and purifying the enzyme digestion product of the Dual-VP3-VP0-VP2-VP1 plasmid and the VP4 gene expression frame PCR amplification product by using a gel recovery and purification kit respectively.

TABLE 18 enzyme digestion reaction system for PCR product of gene expression cassette of VP4

TABLE 19 Dual-VP3-VP0-VP2-VP1 plasmid digestion reaction System

5.3 ligation the double digested Dual-VP3-VP0-VP2-VP1 plasmid and the VP4 gene expression cassette cleavage product were ligated using T4DNA ligase in a water bath at 16 ℃ overnight. The specific ligation reaction system is shown in Table 20.

TABLE 20 plasmid ligation System of the expression cassette of the VP4 Gene with Dual-VP3-VP0-VP2-VP1

5.4 transformation 10. mu.l of the ligation product was added to 100. mu.l of DH5 α competent cells and mixed well, ice-cooled for 30 minutes, heat shocked in a water bath at 42 ℃ for 90 seconds, ice-cooled for 2 minutes again, 900. mu.l of LB liquid medium without Amp was added, and cultured at 37 ℃ for 1 hour, 1.0ml of the bacterial solution was concentrated to 100. mu.l and spread on LB solid medium with Amp, and cultured at 37 ℃ for 16 hours.

5.5 colony PCR and sequencing identification Single colonies on the picked plates were inoculated respectively to LB liquid medium, cultured at 37 ℃ for 2 hours, colony PCR identification was performed using the bacterial solution as template, VP4-F and VP4-R as primers, the PCR product was subjected to gel electrophoresis to verify the size of the target gene, as shown in FIG. 10, the sample with a 1.0kbp band was found to be a positive sample. And (4) sending the bacteria liquid with positive identification to a sequencing company for sequencing, and selecting the bacteria liquid with correct sequencing for storage. The constructed transfer vector containing the target gene is Dual-VP3-VP0-VP2-VP1-VP4, and a schematic diagram thereof is shown in FIG. 11.

Example 2 construction of recombinant baculovirus genome Bac-VP3-VP0-VP2-VP1-VP4

DH10Bac strain transformation mu.l of Dual-VP3-VP0-VP2-VP1-VP4 plasmid from example 1 was added to 100. mu.l of DH10Bac competent cells and mixed well, ice-cooled for 30 minutes, heat-shocked in 42 ℃ water bath for 90 seconds, ice-cooled for 2 minutes, added 900. mu.l each of LB liquid medium without Amp, and cultured at 37 ℃ for 5 hours. After 100. mu.l of the diluted bacterial solution was diluted 81 times, 100. mu.l of the diluted bacterial solution was applied to LB solid medium containing gentamicin, kanamycin, tetracycline, X-gal and IPTG, and cultured at 37 ℃ for 48 hours.

2. Selecting single colony, streaking on LB solid culture medium containing gentamicin, kanamycin, tetracycline, X-gal and IPTG, culturing at 37 deg.c for 48 hr, selecting single colony, inoculating LB liquid culture medium containing gentamicin, kanamycin and tetracycline, culturing, preserving strain and extracting plasmid. Obtaining the recombinant plasmid Bacmid-VP3-VP0-VP2-VP1-VP 4.

Example 3 recombinant baculovirus transfection

Six well plates were seeded 0.8X 10 per well⁶The confluency of Sf9 cells is 50-70%. The following complexes were prepared for each well: diluting 4. mu.l of Cellffectin transfection reagent with 100. mu.l of transfection medium T1, and shaking briefly with vortex; mu.g of the recombinant Bacmid-VP3-VP0-VP2-VP1-VP4 plasmid from example 2 was diluted with 100. mu.l of transfection culture T1 medium, and the diluted transfection reagent and plasmid were mixed and gently blown down to prepare a transfection mixture. And adding the transfection compound after the cells adhere to the wall, incubating for 5 hours at 27 ℃, removing the supernatant, adding 2ml of SF-SFM fresh culture medium, and culturing for 4-5 days at 27 ℃ to obtain the supernatant. Obtaining recombinant baculovirus rBac-VP3-VP0-VP2-VP1-VP4, detecting virus titer of the harvested P1 generation recombinant baculovirus by using an MTT relative efficacy method, wherein the titer of the rBac-VP3-VP0-VP2-VP1-VP4P1 virus is 5.4 multiplied by 10⁷pfu/mL. The amplified recombinant baculovirus rBac-VP3-VP0-VP2-VP1-VP4 is used as seed virus for standby.

Example 4 SDS-PAGE detection

The cell cultures harvested in example 3 were subjected to SDS-PAGE detection while using Sf9 cells infected with empty baculovirus as negative controls, respectively. The specific operation is as follows: mu.l of the harvested cell culture was taken, 10. mu.l of 5 × loading buffer was added, the mixture was centrifuged in a boiling water bath for 5 minutes at 12000r/min for 1 minute, the supernatant was subjected to SDS-PAGE gel (12% strength gel) electrophoresis, and the gel was stained and decolored after electrophoresis to observe the band. As shown in FIG. 12, bands appeared around molecular weights of about 21kDa, 33kDa, 26kDa, 24kDa and 9kDa, and the negative control had no band at the corresponding position.

Example 5 Western Blot identification

The product after SDS-PAGE electrophoresis in example 4 was transferred to an NC (nitrocellulose) membrane, blocked with 5% skim milk for 2 hours, incubated with swine anti-FMDV positive serum for 2 hours, rinsed, incubated with secondary goat anti-swine polyclonal antibody labeled with HRP for 2 hours, rinsed, added dropwise with an enhanced chemiluminescent fluorogenic substrate, and photographed using a chemiluminescent imager. As shown in FIG. 13, the recombinant baculovirus expression sample had a band of interest, and the negative control had no band of interest, indicating that the antigen protein of interest was correctly expressed in Sf9 cells.

Example 6 Indirect immunofluorescence assay

Separately adding transfected rBac-V into 96-well cell culture plateSf9 cell suspension of P3-VP0-VP2-VP1-VP4, 100. mu.l/well (cell concentration 2.5X 10)⁵～4.0×10⁵One/ml), 4 wells were inoculated, left at 27 ℃ for 15 minutes, Sf9 cells were attached to the bottom wall of the plate, and 10. mu.l of a 10-fold diluted seed was added to each well. Meanwhile, a blank cell control is set. After inoculation, the cells are placed in a constant temperature incubator at 27 ℃ for culture for 72-96 hours, the culture solution is discarded, and cold methanol/acetone (1:1) is used for fixation. Firstly reacting with swine anti-FMDV multi-antiserum, then reacting with FITC labeled goat anti-porcine IgG, and observing the result by an inverted fluorescence microscope. As shown in FIG. 14, no fluorescence could be observed by the inoculated empty baculovirus Sf9 cells, while fluorescence could be observed by the inoculated recombinant baculovirus Sf9 cells, indicating that the target antigen protein was correctly expressed in Sf9 cells and the recombinant baculovirus was correctly constructed.

Example 7 Electron microscopy

Carrying out ultrasonic disruption on the recombinant baculovirus cell culture, centrifuging for 30 minutes at 12000r/min, taking the supernatant, filtering by using a 0.22-micron filter membrane, removing impurities, and concentrating by 10 times by using an ultrafiltration tube with the molecular weight cutoff of 3 kDa. 10ml of a 40% sucrose solution was added to each centrifuge tube, then 2.0ml of the ultrafiltration concentrated sample was added, ultracentrifugation was performed at 29000r/min for 2 hours, the supernatant was discarded, and the pellet was resuspended in 2.0ml PBS. Then the suspension is centrifuged by sucrose with gradient concentration of 50 percent, 60 percent, 70 percent and 80 percent respectively, ultracentrifuged at 29000r/min for 2 hours, and then strips positioned at the junction of 60 percent to 70 percent concentration are collected. The collected product is observed by phosphotungstic acid negative staining, virus-like particles with the same size and similar morphology with the O-type FMDV virus particles are observed under an electron microscope, and the result of the electron microscope is shown in figure 15.

EXAMPLE 8 bioreactor serum-free suspension culture of insect cells and quantification of VP3-VP0-VP2-VP1-VP4 expression

Aseptically culturing Sf9 insect cells in 1000ml shake flask for 3-4 days until the concentration reaches 3-5X 10⁶cell/mL, when the activity is more than 95%, inoculating the cells into a 5L bioreactor, wherein the inoculation concentration is 3-8 × 10⁵cell/mL. When the cell concentration reaches 3-55X 10⁶At cell/mL, cells were seeded into a 50L bioreactor until the cells grew to a concentration of 3-55X 10⁶cell/mL, inoculated into 500L organismsIn the reactor, the cell concentration is 2-85X 10⁶When the cell is in the volume of one mL, the recombinant baculovirus rBac-VP3-VP0-VP2-VP1-VP4 is inoculated, and the culture conditions of the reactor are that the pH value is 6.0-6.5, the temperature is 25-27 ℃, the dissolved oxygen is 30-80%, and the stirring speed is 100-. In view of the optimum conditions for cell culture, it is preferable to set pH6.2, the temperature at the stage of cell culture at 27 ℃, the dissolved oxygen at 50%, and the stirring speed at 100-180 rpm. Culturing for 5-9 days after infection, adding one-thousandth final concentration BEI, acting at 37 deg.C for 48 hr, adding two-thousandth final concentration Na₂S₂O₃The inactivation is terminated. Cell culture supernatant is obtained by centrifugation or hollow fiber filtration, and the vaccine stock solution is stored at 2-8 ℃.

Example 9 protein purification

Purifying the harvested stock solution by cation exchange chromatography

The particle exchange chromatography was performed using strong cation particle chromatography packing POROS50HS, which was sterilized with 0.5M NaOH before use. The vaccine stock was then equilibrated with microfiltration buffer at room temperature, and then loaded onto the column at a rate of 125mL/min, followed by elution with rinse bufferA (0.05M MOPS (sodium salt), pH7.0, 0.5M NaCl) for 8 column volumes. Elution was then performed with a linear gradient from 0% buffer a to 100% buffer B (0.05M MOPS (sodium salt), pH7.0, 1.5M NaCl), where a total of 10 column volumes were eluted with linear elution, and then the 10 column volumes were harvested on average. After linear elution was complete, 2 column volumes were eluted with bufferB and collected separately. The collected sample was placed in a 2L sterile plastic bottle at 4 ℃. The fractions collected under the last elution peak (A280) were then stored sterile filtered at 4 ℃.

2.3 hydroxyapatite hydrophobic chromatography

Using a pre-packed hydroxyapatite Column (CHT)^TMCeramic hydroxide Type II Media) was first equilibrated with 50mM MOPS (sodium salt), pH7.0, 1.25M NaCl, and then the above preliminary purified sample was loaded at 90cm/hour and eluted with 8 volumes of the equilibration solution until the UV value was zero. Then gradient elution is carried out using eluent (0.2M phosphate, pH7.0, 1.25M nacl), the concentration of eluent is from 0% to 100%,the speed was still 90cm/h and the elution volume was 4 column volumes. Purifying to obtain the target protein.

The purified single-sided target protein is quantified by using BCA total protein, and then the purity of the target protein is determined by combining gray scanning, and the purified protein is shown in figure 16, and the concentration of the target protein is 312ug/mL, and the purity is 92%.

EXAMPLE 10 preparation of vaccine

Adding appropriate amount of purified O type foot-and-mouth disease virus VP3-VP0-VP2-VP1-VP4 protein into MONTANIDE ISA206 VG adjuvant (volume ratio is 1:1) to make protein final concentration be 100ug/mL, emulsifying, placing at 4 deg.C after quality inspection is qualified.

Example 11 immunization experiment

Pigs of about 1 month old (antibody titer <1:16), 21 pigs, randomly divided into 3 groups of 7 pigs each, and group A injected with the genetic engineering vaccine prepared in example 10; group B normal saline injection; group C was injected with a brand of commercially available O-type FMDV whole virus inactivated vaccine. Injecting 2ml vaccine into neck muscle, enhancing immunity 28 days after first immunization, collecting blood 1 time at2 weeks interval, separating serum, and detecting antibody titer. Antibody detection was performed using a liquid phase blocking ELISA detection kit for foot-and-mouth disease type O antibodies purchased from the lanzhou veterinary institute. The experimental results are as follows

1. And (4) judging a result: the titer of the antibody is more than or equal to 1:128, and the foot-and-mouth disease O-type antibody is judged to be positive; when the ratio is 1: 64-1: 128, the result is judged to be suspicious; <1:64, determined to be negative. The suspicious serum sample can be retested, and the retested antibody titer is more than or equal to 1:128 and is judged to be positive, and the retested antibody titer is less than 1:128 and is judged to be negative.

As can be seen from the results on the graph, the antibody was negative throughout the experiment in the negative control group. The immune recombinant genetic engineering vaccine is used for the swine to turn positive at 21 days after immunization, the antibody level is improved very high seven days after the second immunization, and the immune recombinant genetic engineering vaccine is continued until 56 days after the first immunization. The antibody excitation level and the antibody duration are superior to those of a certain brand of complete virus inactivated vaccine sold in the market.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

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<213> Artificial sequence (Artificial sequence)

<400>11

cctaggtcat gatgataaac aatgtatggt gctaatgttg cttcaacaac aattctgttg 60

aactgtgttt tcatgtttgc caacaagcac ctttatactc ggtggcctcc ccaccaccaa 120

cttttttgca ctgcaaaaaa acacgctttt gcacgcgggc ccatacatag tacaaactct 180

acgtttcgta gactatttta cataaatagt ctacaccgtt gtatacgctc caaatacact 240

accacacatt gaaccttttt gcagtgcaaa aaagtacgtg tcggcagtca cgtaggccgg 300

ccttatcggg tcgcgtcctg tcacgtacga atcacattat cggaccggac gagtgttgtc 360

ttatcgtgac aggacgccag cttcctgtgt tgctaaccgc agccggacgc aactccttat 420

cggaacagga cgcgcctcca tatcagccgc gcgttatctc atgcgcgtga ccggacacga 480

ggcgcccgtc ccgcttatcg cgcctataaa tacagcccgc aacgatctgg taaacacagt 540

tgaacagcat ctgttatgac aacaagtacc ggagagagcg ccgatccagt gacagccaca 600

gtggagaact acggaggaga aacacaggtg cagcgtcgcc accataccga cgtgtccttc 660

atcctggacc gcttcgtgaa agtgaccccc aaggacagca tcaacgtgct ggacctgatg 720

cagaccccca gtcataccct ggtgggagct ctgctgcgta ccgccaccta ctacttcgcc 780

gatctggagg tcgccgtgaa gcacgaggga gatctgactt gggtgcccaa cggagctcca 840

gaagccgctc tggataacac caccaacccc accgcctacc ataaggctcc actgacacgc 900

ctggctctgc catataccgc tccacatcgc gtgctggcta ccgtgtacaa cggcaattgc 960

aagtacgccg gaggaagcct gccaaacgtg cgaggagatc tgcaagtgct ggcccaaaaa 1020

gccgctcgtc cactgccaac cagcttcaac tacggagcca tcaaggccac acgcgtgaca 1080

gagctgctgt accgcatgaa gcgcgccgag acctattgcc cacgtccatt gttggccgtg 1140

catccaagcg ccgcccgtca taagcagaag atcgtggctc ccgtgaagca gtaaatctta 1200

gtttgtattg tcatgtttta atacaatatg ttatgtttaa atatgttttt aataaatttt 1260

ataaaataat ttcaactttt attgtaacaa cattgtccat ttacacactc ctttcaagcg 1320

cgtgcctagg 1330

<210>12

<211>639

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400>12

atgacaacaa gtaccggaga gagcgccgat ccagtgacag ccacagtgga gaactacgga 60

ggagaaacac aggtgcagcg tcgccaccat accgacgtgt ccttcatcct ggaccgcttc 120

gtgaaagtga cccccaagga cagcatcaac gtgctggacc tgatgcagac ccccagtcat 180

accctggtgg gagctctgct gcgtaccgcc acctactact tcgccgatct ggaggtcgcc 240

gtgaagcacg agggagatct gacttgggtg cccaacggag ctccagaagc cgctctggat 300

aacaccacca accccaccgc ctaccataag gctccactga cacgcctggc tctgccatat 360

accgctccac atcgcgtgct ggctaccgtg tacaacggca attgcaagta cgccggagga 420

agcctgccaa acgtgcgagg agatctgcaa gtgctggccc aaaaagccgc tcgtccactg 480

ccaaccagct tcaactacgg agccatcaag gccacacgcg tgacagagct gctgtaccgc 540

atgaagcgcg ccgagaccta ttgcccacgt ccattgttgg ccgtgcatcc aagcgccgcc 600

cgtcataagc agaagatcgt ggctcccgtg aagcagtaa 639

<210>13

<211>34

<212>DNA

<213> Artificial primer (Artificial sequence)

<400>13

atacctaggt catgatgata aacaatgtat ggtg 34

<210>14

<211>33

<212>DNA

<213> Artificial primer (Artificial sequence)

<400>14

atacctaggc acgcgcttga aaggagtgtg taa 33

<210>15

<211>987

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400>15

tgtacaaaaa tgaggcggcg gcaatgattt acgggcatat attcggtcga ggaggacgaa 60

atattctgaa atgggacgaa aggggatgac gcggcgcggc tctcgtcttc ccgcctcgca 120

ttcaacgctc ggctcgacca atcagcggcc gagttttgcg ctatgaccat ataaggcgat 180

acgtttgtcc gggtggggtg ggacgagcca ttgcggctta tcgcgcgggg gagtaccctc 240

tcaaaatgca ctatgcactg ccgtaacact ctttcggaaa gaatataata catcagtaga 300

tacctcttga aaattaggat ccgatgcata ccataaatcc ccaaattaga gagaataaaa 360

ggggttaatt cgatcgagag taatgacact tggaacgacc tcccctctgg agaaagtcga 420

cgatccgaga ggtggagtaa gcgccctact cactctctca tgggagctgg acaaagtagc 480

ccagctaccg gaagccagaa tcagagcgga aacaccggca gcatcatcaa caactactac 540

atgcagcagt accagaacag catggacacc cagctgggcg ataatgccat cagcggagga 600

agcaacgagg gcagcacaga taccaccagc acccacacca ccaacaccca gaacaacgat 660

tggttcagca agctggctag cagcgccttt agcggactgt tcggagctct gctggcctaa 720

tgagtcgaga agtactagag gatcataatc agccatacca catttgtaga ggttttactt 780

gctttaaaaa acctcccaca cctccccctg aacctgaaac ataaaatgaa tgcaattgtt 840

gttgttaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat 900

ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat 960

gtatcttatc atgtctggat ctgtaca 987

<210>16

<211>264

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400>16

atgggagctg gacaaagtag cccagctacc ggaagccaga atcagagcgg aaacaccggc 60

agcatcatca acaactacta catgcagcag taccagaaca gcatggacac ccagctgggc 120

gataatgcca tcagcggagg aagcaacgag ggcagcacag ataccaccag cacccacacc 180

accaacaccc agaacaacga ttggttcagc aagctggcta gcagcgcctt tagcggactg 240

ttcggagctc tgctggccta atga 264

<210>17

<211>36

<212>DNA

<213> Artificial primer (Artificial sequence)

<400>17

atatgtacaa aaatgaggcg gcggcaatga tttacg 36

<210>18

<211>39

<212>DNA

<213> Artificial primer (Artificial sequence)

<400>18

atatgtacag atccagacat gataagatac attgatgag 39

<210>19

<211>221

<212>PRT

<213> Artificial sequence (Artificial sequence)

<400>19

Met Gly Ile Phe Pro Val Ala Cys Ser Asp Gly Tyr Gly Gly Leu Val

1 5 10 15

Thr Thr Asp Pro Lys Thr Ala Asp Pro Val Tyr Gly Lys Val Phe Asn

20 25 30

Pro Pro Arg Asn Met Leu Pro Gly Arg Phe Thr Asn Leu Leu Asp Val

35 40 45

Ala Glu Ala Cys Pro Thr Phe Leu His Phe Asp Gly Asp Val Pro Tyr

50 55 60

Val Thr Thr Lys Thr Asp Ser Asp Arg Val Leu Ala Gln Phe Asp Leu

65 70 75 80

Ser Leu Ala Ala Lys His Met Ser Asn Thr Phe Leu Ala Gly Leu Ala

85 90 95

Gln Tyr Tyr Thr Gln Tyr Ser Gly Thr Val Asn Leu His Phe Met Phe

100 105 110

Thr Gly Pro Thr Asp Ala Lys Ala Arg Tyr Met Ile Ala Tyr Ala Pro

115 120 125

Pro Gly Met Glu Pro Pro Lys Thr Pro Glu Ala Ala Ala His Cys Ile

130 135 140

His Ala Glu Trp Asp Thr Gly Leu Asn Ser Lys Phe Thr Phe Ser Ile

145 150 155 160

Pro Tyr Leu Ser Ala Ala Asp Tyr Ala Tyr Thr Ala Ser Asp Ala Ala

165 170 175

Glu Thr Thr Asn Val Gln Gly Trp Val Cys Leu Phe Gln Ile Thr His

180 185 190

Gly Lys Ala Glu Gly Asp Ala Leu Val Val Leu Ala Ser Ala Gly Lys

195 200 205

Asp Phe Glu Leu Arg Leu Pro Val Asp Ala Arg Gln Gln

210 215 220

<210>20

<211>304

<212>PRT

<213> Artificial sequence (Artificial sequence)

<400>20

Met Gly Ala Gly Gln Ser Ser Pro Ala Thr Gly Ser Gln Asn Gln Ser

1 5 10 15

Gly Asn Thr Gly Ser Ile Ile Asn Asn Tyr Tyr Met Gln Gln Tyr Gln

20 25 30

Asn Ser Met Asp Thr Gln Leu Gly Asp Asn Ala Ile Ser Gly Gly Ser

35 40 45

Asn Glu Gly Ser Thr Asp Thr Thr Ser Thr His Thr Thr Asn Thr Gln

50 55 60

Asn Asn Asp Trp Phe Ser Lys Leu Ala Ser Ser Ala Phe Ser Gly Leu

65 70 75 80

Phe Gly Ala Leu Leu Ala Asp Lys Lys Thr Glu Glu Thr Thr Leu Leu

85 90 95

Glu Asp Arg Ile Leu Thr Thr Arg Asn Gly His Thr Thr Ser Thr Thr

100 105 110

Gln Ser Ser Val Gly Ile Thr His Gly Tyr Ala Thr Ala Glu Asp Phe

115 120 125

Val Asn Gly Pro Asn Thr Ser Gly Leu Glu Thr Arg Val Val Gln Ala

130 135 140

Glu Arg Phe Phe Lys Thr His Leu Phe Asp Trp Val Thr Ser Asp Pro

145 150 155 160

Phe Gly Arg Cys Tyr Leu Leu Glu Leu Pro Thr Asp His Lys Gly Val

165 170 175

Tyr Gly Ser Leu Thr Asp Ser Tyr Ala Tyr Met Arg Asn Gly Trp Asp

180 185 190

Val Glu Val Thr Ala Val Gly Asn Gln Phe Asn Gly Gly Cys Leu Leu

195 200 205

Val Ala Met Val Pro Glu Leu Cys Ser Ile GluArg Arg Glu Leu Phe

210 215 220

Gln Leu Thr Leu Phe Pro His Gln Phe Ile Asn Pro Arg Thr Asn Met

225 230 235 240

Thr Ala His Ile Lys Val Pro Phe Val Gly Val Asn Arg Tyr Asp Gln

245 250 255

Tyr Lys Val His Lys Pro Trp Thr Leu Val Val Met Val Val Ala Pro

260 265 270

Leu Thr Val Asn Thr Glu Gly Ala Pro Gln Ile Lys Val Tyr Ala Asn

275 280 285

Ile Ala Pro Thr Asn Val His Val Ala Gly Glu Phe Pro Ser Lys Glu

290 295 300

<210>21

<211>219

<212>PRT

<213> Artificial sequence (Artificial sequence)

<400>21

Met Asp Lys Lys Thr Glu Glu Thr Thr Leu Leu Glu Asp Arg Ile Leu

1 5 10 15

Thr Thr Arg Asn Gly His Thr Thr Ser Thr Thr Gln Ser Ser Val Gly

20 25 30

Ile Thr His Gly Tyr Ala Thr Ala Glu Asp Phe Val Asn Gly Pro Asn

35 40 45

Thr Ser Gly Leu Glu Thr Arg Val Val Gln Ala Glu Arg Phe Phe Lys

50 55 60

Thr His Leu Phe Asp Trp Val Thr Ser Asp Pro Phe Gly Arg Cys Tyr

65 70 75 80

Leu Leu Glu Leu Pro Thr Asp His Lys Gly Val Tyr Gly Ser Leu Thr

85 90 95

Asp Ser Tyr Ala Tyr Met Arg Asn Gly Trp Asp Val Glu Val Thr Ala

100 105 110

Val Gly Asn Gln Phe Asn Gly Gly Cys Leu Leu Val Ala Met Val Pro

115 120 125

Glu Leu Cys Ser Ile Glu Arg Arg Glu Leu Phe Gln Leu Thr Leu Phe

130 135 140

Pro His Gln Phe Ile Asn Pro Arg Thr Asn Met Thr Ala His Ile Lys

145 150 155 160

Val Pro Phe Val Gly Val Asn Arg Tyr Asp Gln Tyr Lys Val His Lys

165 170 175

Pro Trp Thr Leu Val Val Met Val Val Ala Pro Leu Thr Val Asn Thr

180 185 190

Glu Gly Ala Pro Gln Ile Lys Val Tyr Ala Asn Ile Ala Pro Thr Asn

195 200 205

Val His Val Ala Gly Glu Phe Pro Ser Lys Glu

210 215

<210>22

<211>212

<212>PRT

<213> Artificial sequence (Artificial sequence)

<400>22

Met Thr Thr Ser Thr Gly Glu Ser Ala Asp Pro Val Thr Ala Thr Val

1 5 10 15

Glu Asn Tyr Gly Gly Glu Thr Gln Val Gln Arg Arg His His Thr Asp

20 25 30

Val Ser Phe Ile Leu Asp Arg Phe Val Lys Val Thr Pro Lys Asp Ser

35 40 45

Ile Asn Val Leu Asp Leu Met Gln Thr Pro Ser His Thr Leu Val Gly

50 55 60

Ala Leu Leu Arg Thr Ala Thr Tyr Tyr Phe Ala Asp Leu Glu Val Ala

65 70 75 80

Val Lys His Glu Gly Asp Leu Thr Trp Val Pro Asn Gly Ala Pro Glu

85 90 95

Ala Ala Leu Asp Asn Thr Thr Asn Pro Thr Ala Tyr His Lys Ala Pro

100 105 110

Leu Thr Arg Leu Ala Leu Pro Tyr Thr Ala Pro His Arg Val Leu Ala

115120 125

Thr Val Tyr Asn Gly Asn Cys Lys Tyr Ala Gly Gly Ser Leu Pro Asn

130 135 140

Val Arg Gly Asp Leu Gln Val Leu Ala Gln Lys Ala Ala Arg Pro Leu

145 150 155 160

Pro Thr Ser Phe Asn Tyr Gly Ala Ile Lys Ala Thr Arg Val Thr Glu

165 170 175

Leu Leu Tyr Arg Met Lys Arg Ala Glu Thr Tyr Cys Pro Arg Pro Leu

180 185 190

Leu Ala Val His Pro Ser Ala Ala Arg His Lys Gln Lys Ile Val Ala

195 200 205

Pro Val Lys Gln

210

<210>23

<211>86

<212>PRT

<213> Artificial sequence (Artificial sequence)

<400>23

Met Gly Ala Gly Gln Ser Ser Pro Ala Thr Gly Ser Gln Asn Gln Ser

1 5 10 15

Gly Asn Thr Gly Ser Ile Ile Asn Asn Tyr Tyr Met Gln Gln Tyr Gln

20 25 30

Asn Ser Met Asp Thr Gln Leu Gly Asp Asn Ala Ile Ser Gly Gly Ser

3540 45

Asn Glu Gly Ser Thr Asp Thr Thr Ser Thr His Thr Thr Asn Thr Gln

50 55 60

Asn Asn Asp Trp Phe Ser Lys Leu Ala Ser Ser Ala Phe Ser Gly Leu

65 70 75 80

Phe Gly Ala Leu Leu Ala

85

Claims

1. A method of preparing an immunogenic composition, comprising:

s1, sequentially cloning the coding genes of the structural proteins VP3 and VP0 of the O-type foot-and-mouth disease virus after codon optimization and the gene expression frames of the structural proteins VP2, VP1 and VP4 of the O-type foot-and-mouth disease virus to the same pFastBac Dual shuttle vector to obtain a recombinant shuttle vector;

s2, transforming the recombinant shuttle vector into DH10Bac bacteria containing the baculovirus genome plasmid, and directionally inserting a target gene expression frame in the recombinant shuttle vector into the baculovirus genome plasmid to obtain the recombinant baculovirus genome plasmid containing the target gene expression frame;

s4, inoculating the obtained recombinant baculovirus into insect cells, and producing recombinant O-type foot-and-mouth disease virus-like particles in a reactor in a large scale;

s5, adding the recombinant O-type foot-and-mouth disease virus-like particles obtained in the step S4 into an adjuvant to obtain an immune composition;

the gene expression cassettes of the VP3 and VP0 proteins are respectively shown as SEQ ID NO.1 and SEQ ID NO. 4, the gene expression cassette of the VP1 protein is shown as SEQ ID NO. 11, the gene expression cassette of the VP1 protein comprises an OpIE promoter, a codon-optimized coding gene shown as SEQ ID NO. 12 and an OpIE polyA transcription termination signal sequence, the gene expression cassette of the VP2 protein is shown as SEQ ID NO. 7, the gene expression cassette of the VP2 protein comprises a prawn β -actin gene promoter, a codon-optimized coding gene shown as SEQ ID NO. 8 and an SV40polyA termination signal sequence, the gene expression cassette of the VP4 protein is shown as SEQ ID NO. 15, and the gene expression cassette of the VP4 protein comprises a prawn β -actin gene promoter, a codon-optimized coding gene shown as SEQ ID NO. 16 and an SV40polyA termination signal sequence.

2. The preparation method according to claim 1, wherein the amino acid sequence of the VP3 protein is shown as SEQ ID NO. 19, the amino acid sequence of the VP1 protein is shown as SEQ ID NO. 22, the amino acid sequence of the VP2 protein is shown as SEQ ID NO. 21, the amino acid sequence of the VP4 protein is shown as SEQ ID NO. 23, and the amino acid sequence of the VP0 protein is shown as SEQ ID NO. 20.

3. Use of the immunological composition prepared by the method of claim 1 or 2 for the manufacture of a medicament for inducing an immune response against a foot and mouth disease virus type O antigen in a test animal.

4. Use of the immunological composition prepared by the method of claim 1 or 2 for the manufacture of a medicament for preventing infection of animals with foot and mouth disease virus type O.