CN112439057B

CN112439057B - Self-assembly ferritin nano-antigen particle, swine fever vaccine prepared from same and application of swine fever vaccine

Info

Publication number: CN112439057B
Application number: CN201910803838.7A
Authority: CN
Inventors: 张志芳; 李轶女; 易咏竹; 刘兴健; 胡小元; 魏珍珍; 宋浩志
Original assignee: Biotechnology Research Institute of CAAS
Current assignee: Biotechnology Research Institute of CAAS
Priority date: 2019-08-28
Filing date: 2019-08-28
Publication date: 2022-06-03
Anticipated expiration: 2039-08-28
Also published as: CN112439057A

Abstract

The invention discloses a self-assembly ferritin-based nano antigen particle, a swine fever vaccine prepared from the self-assembly ferritin-based nano antigen particle and application of the swine fever vaccine. The invention fuses the CSFV envelope E2 protein and the self-assembly ferritin nanoparticle subunit to obtain the fusion protein. The invention carries out single-site, double-site and multi-site mutation on the CSFV envelope E2 protein, and the soluble expression quantity and the expression efficiency of the obtained mutant are obviously improved. The invention utilizes a prokaryotic expression system, a silkworm and AcMNPV-insect cell eukaryotic expression system to express recombinant protein or carries out gene presentation in a vertebrate body through recombinant baculovirus to generate antigen-induced antibody. The vaccine provided by the invention displays the CSFV envelope E2 protein on the surface of the helicobacter pylori ferritin cage structure, so as to cause a widely neutralizing anti-CSFV antibody, improve the immune efficacy, expand the immune range and have the potential of becoming a general vaccine with cross immune efficacy.

Description

Self-assembly ferritin-based nano antigen particle, swine fever vaccine prepared from self-assembly ferritin-based nano antigen particle and application of swine fever vaccine

Technical Field

The invention relates to a self-assembly ferritin-based nano antigen particle, in particular to a nano antigen particle formed by fusing swine fever virus envelope E2 protein and a monomeric ferritin subunit and a swine fever vaccine prepared from the nano antigen particle, belonging to the field of preparation and application of swine fever vaccines.

Background

Classical Swine Fever (CSF) is called European swine fever (European swine fever), Hog cholera (Hog cholera) and rotten bowel disease (CSFV) in China, is a viral infectious disease of pigs caused by CSFV, and can be infected and developed by domestic pigs and wild pigs in all ages. Swine fever may first occur in the mid-west united states in the thirties of the nineteenth century, and was not revealed until the early twentieth century by analysis of the etiology of humoral swine fever infecting swine. Hog cholera is a systemic disease accompanied by bleeding injury, and clinical manifestations can be classified into acute, subacute, chronic and delayed types, depending on the virulence of the hog cholera virus strain, the dose of viral infection and the host conditions. Acute infection is the most serious and high in fatality rate, is mostly seen in young livestock, has main symptoms of fever, inappetence, conjunctivitis, constipation accompanied by diarrhea, nervous symptoms, skin and other organ bleeding and the like, and secondary infection can also cause damage to the intestines and the respiratory system. The research shows that the swine fever is also accompanied with cytopenia, especially leukopenia. The delayed swine fever is the congenital infected swine fever virus, and the infected piglet does not produce specific antibody but carries the virus for the lifetime, thus being the natural infection source.

Classical Swine Fever Virus (CSFV) together with Bovine Viral Diarrhea Virus (BVDV) and ovine Border Disease Virus (BDV) constitute the genus Pestivirus (Pestivirus) of the family Flaviviridae. The classical swine fever virus particles are of a 40-60 nm spherical structure, and contain single-stranded positive-strand polar RNA, the peripheries of the virus particles are wrapped with lipoprotein envelope, and 6-8 nm spica-like protrusions are formed on the surfaces of the virus particles. Hog cholera virus infectious c DNA has been isolated in the laboratory at a genome size of about 12.3 kb. Viral RNA encodes 4 structural genes and 8 non-structural genes. The classical swine fever virus is extremely stable in a humid environment and fresh meat products, but the classical swine fever virus is heat-resistant, can completely inactivate the virus at 56 ℃ for 60min and 60 ℃ for 10min, is stable at a pH value of 5-10, but has reduced virus toxicity in an environment with a p H value lower than 3, and can inactivate the virus by treatment with detergents, fat solvents, peptidases and conventional disinfectants. China is a big pig-raising country in the world, and swine fever is a disease which is relatively easy to infect pigs of all ages, so that the swine fever becomes one of important factors for restricting the development of the pig-raising industry in China. The best treatment for the disease is no longer preventive, but vaccines for various diseases are formally preventive. Most of the traditional vaccines are attenuated live vaccines, including natural attenuated strains, gene recombinant attenuated strains and the like. The biggest drawback of using attenuated strain vaccines is the risk of the attenuated strain recovering the virulence effects in the susceptible population. Therefore, the production of safe, efficient and inexpensive genetically engineered vaccines is a new need.

In recent years, Ferritin (Ferritin) has been used as a nanomaterial in a variety of fields. Ferritin is a cage-like protein widely found in organisms, the protein shell of which is self-assembled from 24 ferritin subunits, wherein every third subunit constitutes a trimeric subunit. The N-terminal of ferritin extends to the outer surface, and is easy to carry out gene modification and fuse protein polypeptide. Highly ordered repetitive antigens tend to induce strong T cell-dependent antibody responses when distributed at 5-10nm intervals on the surface of microorganisms. Ferritin is very stable and can tolerate high temperatures (70-80 ℃) and a variety of denaturants without affecting the native protein structure. Ferritin is pH sensitive, the protein shell disintegrates under acidic conditions at pH2.0, and when the pH returns to physiological conditions (pH 7.0), the disintegrated protein subunits can again reassemble into intact ferritin. The research on ferritin in recent years has mainly focused on: (1) coating a specific medicine in the ferritin shell or promoting the synthesis of a nano material by modifying the inner surface of the ferritin; (2) the outer surface of the ferritin is modified and connected with PEG or antibody to expand new functions; (3) the self-assembly of ferritin is controlled by the modification of the ferritin outer surface or the interface between subunits. The ferritin nanoparticles display the antigen, can remarkably enhance the immunogenicity of the antigen, and cause stronger humoral and cellular immune reactions, so ferritin is an ideal nano vaccine platform.

The ferritin nano-particle formed by self-assembly of 24 subunits is used as an antigen presentation and vaccine development platform to develop a swine fever vaccine, and has important application value for prevention and treatment of swine fever.

Disclosure of Invention

One of the objects of the present invention is to provide a self-assembled ferritin nano-antigen particle comprising a fusion protein of CSFV envelope E2 protein;

the other purpose of the invention is to carry out mutation of site of the fusion protein so as to improve the expression quantity or the expression efficiency of the fusion protein;

the invention also aims to provide a swine fever vaccine obtained based on self-assembled ferritin nano antigen particles;

the fourth object of the present invention provides a method for efficiently expressing the fusion protein;

the fifth purpose of the invention is to provide a method for presenting a fusion gene constructed by self-assembled ferritin nanoparticles and CSFV envelope E2 protein to an animal body and presenting antigen in the animal body to induce and generate anti-CSFV antibody.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

the invention firstly provides a nano antigen particle of fusion protein containing swine fever virus envelope E2 protein, wherein the fusion protein is obtained by connecting the C end of swine fever virus envelope E2 with the N end of a monomer ferritin subunit; preferably, the swine fever virus envelope E2 protein and the N-terminal of the monomeric ferritin subunit are connected through a connecting peptide SGG to obtain the fusion protein.

The monomeric ferritin subunit includes, but is not limited to, any of bacterial ferritin, plant ferritin, algal ferritin, insect ferritin, fungal ferritin, or mammalian ferritin; preferably, the monomeric ferritin subunit is a helicobacter pylori ferritin monomer, and the amino acid sequence of the monomeric ferritin subunit is a sequence shown as a GenBank sequence number WP _000949190 on NCBI.

The selected region of the CSFV envelope E2 protein comprises a region selected from the group consisting of: a signal peptide at the N terminal, a transmembrane region at the C terminal and a middle amino acid region; preferably, the hog cholera virus envelope E2 protein is selected from a complete envelope E2 protein; most preferably, the amino acid sequence of the CSFV envelope E2 protein is the sequence shown in the GenBank sequence number ART84247.1 on NCBI.

In order to improve the expression quantity of the fusion protein obtained by connecting the CSFV envelope E2 protein and the Ferritin monomer, the invention further carries out mutation optimization on the homologous sequences of the two original fusion proteins (CSFV E2-Ferritin), carries out glycosylation site analysis after the sequence optimization to eliminate glycosylation sites to increase soluble expression, and further carries out amino acid single-site mutation, double-site mutation and multi-site mutation after the homologous sequence optimization to improve the soluble expression quantity and the expression efficiency:

specifically, the inventor finds out a most universal homologous sequence as an antigen gene of a corresponding strain by analyzing 20 swine fever virus envelope E2 protein amino acid sequences which are popular in different regions in the latest year and performing comparison analysis, so as to obtain the optimal protection effect; on the basis, the invention further utilizes OptimumGene^TMThe technology optimizes the amino acid sequence of the hog cholera virus envelope E2 protein, modifies the amino acid sequence of the E2 protein and the amino acid sequence of the ferritin monomer subunit according to the codon preference of escherichia coli, optimizes and designs various related parameters which influence the transcription efficiency and the translation efficiency of the gene, the GC content of protein folding, the CpG dinucleotide content, the codon preference, the secondary structure of mRNA, the free energy stability of mRNA, the RNA unstable gene sequence, the repetitive sequence and the like, and keeps the protein sequence translated finally unchanged. In addition, in order to increase the amount of ferritin expressed and increase soluble expressionPoint mutation N19Q was performed on the ferritin monomer subunit. Finally, the nucleotide obtained by the nucleotide sequence (SEQ ID NO.2) of the homologous sequence (SEQ ID NO.1) of the original fusion protein according to the optimization mode is the optimized gene sequence shown in SEQ ID NO. 3.

According to the invention, the optimized homologous sequence is expressed in a silkworm expression system, and the expression quantity of the homologous sequence after codon optimization is obviously improved compared with that before optimization according to the ELISA titer result of a gene expression product.

The invention obtains the CSFV E2-Ferritin-C mutant, takes the gene sequence of the CSFV E2-Ferritin-C mutant codon optimized as a template, designs a plurality of pairs of primers to carry out site-directed mutagenesis on the conserved sequence:

multiple single-site mutants are obtained by carrying out amino acid single-site mutation on an amino acid sequence shown in SEQ ID NO.1 according to the modes of I13H, G23M, N35A, S48K, L59N, Y66C, S78T, D86R, D97N, S109V, Y116W, A125V, E139F, V152H, D167G, N175P, V202I, P215Q, I227D or D238E;

the amino acid single-site mutation 'I13H' of the invention refers to the mutation of the 13 th amino acid of the amino acid sequence shown in SEQ ID NO.1 from I to H; the expression of the remaining single-site mutations is analogized.

The invention expresses the mutated single-site mutants in a silkworm expression system, and according to the expression result, the expression method comprises the following steps: after the transmembrane region of the amino acid sequence shown in SEQ ID NO.1 is removed, the titer of the expression products of the 6 mutants obtained by the amino acid single-site mutation mode of N35A, D86R, Y116W, E139F, D167G and I227D is remarkably improved; wherein, the titer of the mutant obtained by mutating the amino acid sequence shown in SEQ ID NO.1 according to D86R amino acid single site is improved most obviously.

Based on that the determined mutation of part of the single sites is effective mutation, the aim of improving the expression quantity of the CSFV E2-Ferritin-C-O-M mutant can be achieved; the present invention further performs amino acid double site mutation considering that the sequence of amino acids is the primary structure of the protein and determines the higher order structure of the protein, and the positions of the partial mutation sites of the above-described amino acid single site mutation may be correlated with each other. The invention combines single mutation sites which can improve the expression quantity in pairs to carry out double-site mutation, and the method comprises the following specific steps:

on the basis of single-site mutation, 15 double-site mutations are obtained by amino acid double-site mutation modes of an amino acid sequence shown in SEQ ID NO.1 according to N35A-D86R, N35A-Y116W, N35A-E139F, N35A-D167G, N35A-I227D, D86R-Y116W, D86R-E139F, D86R-D167G, D86R-I227D, Y116W-E139F, Y116W-D167G, Y116W-I227D, E139F-D167G, E139F-I227D or D167G-I227D.

The amino acid double-site mutation 'N35A-D86R' of the invention refers to the mutation of the amino acid at the 35 th position of the amino acid sequence shown in SEQ ID NO.1 from N to A and the mutation of the amino acid at the 86 th position from D to R; the remainder of the two-site mutations are described in analogy.

The 15 double-site mutants obtained are respectively expressed in a silkworm expression system, and according to the expression result, the expression results are as follows: the titer of the expression product of the 3-site mutant obtained by the amino acid sequence shown in SEQ ID NO.1 according to the amino acid double-site mutation mode of D86R-Y116W, E139F-D167G and E139F-I227D is remarkably improved, wherein the titer of the mutant obtained by the amino acid shown in SEQ ID NO.1 according to the amino acid double-site mutation mode of D86R-Y113W is remarkably improved.

Considering that the partial double-site mutation can effectively improve the titer after the expression amount, and considering that the arrangement sequence of amino acids is the primary structure of the protein and determines the high-level structure of the protein, the presumption is probably that the positions of partial mutation points of the amino acid single-site mutation are close to each other and are related to each other, the invention further tries to carry out the amino acid multi-site mutation. The invention obtains 6 single mutation sites by analyzing glycosylation sites to effectively improve the expression quantity of target genes, therefore, the multi-site mutation is based on the effective double-site mutation sequence obtained above, the site-directed mutation of the multi-mutation sites is carried out by a fusion PCR method, and the specific mutation mode is as follows: the multi-site mutant is obtained by carrying out multi-site mutation on the amino acid sequence shown in SEQ ID NO.1 according to the amino acid multi-site mutation mode of N35A-D86R-Y116W-E139F-D167G-I227D.

The amino acid multi-site mutation 'N35A-D86R-Y116W-E139F-D167G-I227D' of the invention indicates that the amino acid sequence shown in SEQ ID NO.1 is subjected to the following mutations simultaneously: the amino acid at the 35 th position is mutated from N to A, the amino acid at the 86 th position is mutated from D to R, the amino acid at the 116 th position is mutated from Y to W, the amino acid at the 139 th position is mutated from E to F, the amino acid at the 167 th position is mutated from D to G, and the amino acid at the 227 th position is mutated from I to D.

The obtained multi-site mutant is expressed in a silkworm expression system, and according to an expression result, the expression method comprises the following steps: compared with the expression levels of the single mutant and the double mutants, the expression levels of the two multi-site mutants are obviously improved. The expression products of the two multi-site mutants in the silkworm expression system are further preliminarily purified and then observed by adopting an electron microscope, the observation result shows that the size of the product is consistent with the expected nano particles, the diameter of the cage body is about 12 nanometers, and the antenna-shaped protrusion is observed by careful observation.

The obtained multi-site mutant coding gene is cloned into an expression vector of baculovirus mammals to construct recombinant baculovirus presenting genes; the recombinant baculovirus is presented to mice, and the result shows that the titer of the antibody generated by the mice is obviously higher than that of a healthy silkworm pupa control and a traditional vaccine.

Therefore, the self-assembled ferritin nano antigen particle containing the fusion protein provided by the invention can be applied to preparation of swine fever vaccines, and the application method comprises the following steps:

expressing the fusion protein coding gene in prokaryotic cells by adopting a prokaryotic expression system to obtain nano antigen particles, purifying the expressed nano antigen particle product, and splicing the purified nano antigen particle product with a medically acceptable carrier to obtain the swine fever vaccine;

for reference, the step of expressing the nano-antigen particles in prokaryotic cells using a prokaryotic expression system comprises:

(1) cloning the original sequence of the fusion protein or the sequence of the fusion protein after mutation optimization to an expression vector pET28a to obtain a recombinant plasmid pET28a-CSFV E2-Ferritin;

(2) the recombinant plasmid pET28a-CSFV E2-Ferritin is transformed into BL21(DE3) competent cells for expression, and then purified by a nickel column to obtain the recombinant plasmid.

(II) expressing the fusion protein coding gene in eukaryotic cells by adopting a eukaryotic expression system, purifying the expressed antigen product, and splicing the purified antigen product with a medically acceptable carrier to obtain the swine fever vaccine.

For reference, the method for expressing the fusion protein encoding gene in eukaryotic cells by using a eukaryotic expression system comprises the following steps:

expressing the fusion protein coding gene in a silkworm expression system, and collecting and purifying the expressed antigen; preferably, the fusion protein coding gene is constructed into a silkworm baculovirus expression vector to prepare a recombinant silkworm baculovirus; amplifying the recombinant silkworm baculovirus in silkworm cells and expressing the amplified recombinant silkworm baculovirus in silkworms or silkworm pupas;

or the fusion protein coding gene is expressed in an AcMNPV-insect cell eukaryotic expression system, and the expressed antigen is collected and purified; preferably, the fusion protein coding gene is cloned into a baculovirus transfer vector to construct a recombinant baculovirus transfer vector; co-transfecting the recombinant baculovirus transfer vector and baculovirus DNA into an insect cell to obtain a recombinant baculovirus; infecting the recombinant baculovirus into an insect host or an insect cell, culturing the infected insect cell or the insect host to express a corresponding antigen, and purifying to obtain the recombinant baculovirus;

(III) the fusion protein coding gene can be cloned to a gene presenting vector to construct a recombinant baculovirus transfer vector presenting exogenous genes to vertebrate cells or individuals, and the recombinant baculovirus transfer vector is transfected to silkworm cells to obtain recombinant viruses; the resulting recombinant virus presents antigen in animals and induces antibodies in animals by injection or orally.

The invention further provides a vaccine for preventing and treating swine fever, which comprises the following components: a prophylactically or therapeutically effective amount of a self-assembled ferritin nano-antigen particle comprising a fusion protein and a pharmaceutically acceptable carrier.

The vaccines of the present invention may be formulated in a variety of different pharmaceutically acceptable carriers. They may include salts and buffers to provide physiological ionic strength and pH, surfactants such as polysorbates 20 and 80 to prevent antigen aggregation, stabilizers for antigen stabilization such as PEG, trehalose and gelatin and polymers for sustained release such as CMC, HEC and dextran. Vaccines can also be formulated with controlled release or enhanced display systems such as hydrogels, virosomes, nanoparticles, and emulsions. The vaccine may also be formulated with adjuvants to further increase the cross-reactive immune response and cross-protection, suitable adjuvants may be selected from polysaccharides such AS lipopolysaccharides and saponins, nucleic acids such AS CpG and poly I: C, lipids such AS MPL (monophosphoryl lipid a), proteins such AS bacterial flagellin, inorganic salts such AS aluminium salts and calcium phosphate, emulsions such AS freund's incomplete adjuvant, MF59 and AS03 and various Toll-like receptor ligands. Different adjuvants can be tested with the treated antigen to identify suitable adjuvants that produce higher levels of cross-reactive immune response and cross-protection, including complete or 100% protection, at appropriate adjuvant doses.

The swine fever vaccine of the present invention may be administered by various routes, such as intramuscular, subcutaneous, intranasal, topical, sublingual, or oral administration.

The vaccine provided by the invention can display the hog cholera virus envelope E2 protein tripolymer structure on the surface of the helicobacter pylori ferritin cage structure, thereby being capable of causing the widely neutralizing hog cholera antibody. The vaccine induces a neutralizing antibody generated by an individual to increase the immune efficacy, increase the immune range and immunize homotypic and heterotypic classical swine fever viruses in different years.

Compared with the prior art, the invention has the following advantages and effects:

1. according to the invention, the prokaryotic expression system escherichia coli, silkworm baculovirus and AcMNPV-insect cell are used for expressing the recombinant protein vaccine, live harmful viruses are not involved in the vaccine preparation process, and compared with the traditional method for preparing the swine fever vaccine, the method is safer and simpler to operate and is suitable for rapid large-scale production;

2. the nano swine fever vaccine provided by the invention can induce a swine fever antibody with broad spectrum property, and lays a foundation for preparing a general swine fever vaccine.

3. The nano swine fever vaccine provided by the invention has the advantage that the level of anti-swine fever antibodies induced by immunizing animals with the nano swine fever vaccine is obviously higher than that of the traditional vaccine.

Definitions of terms to which the invention relates

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.

The words "antigen" and "immunogen" are used interchangeably and refer to a molecule, substance, protein, glycoprotein, or live virus capable of inducing specific humoral (antibody) and cellular immune responses.

The term "antigenicity" refers to the ability of an antibody to react or bind to a specific antigen; the term "immunogenicity" refers to the ability of an antigen or vaccine to induce a specific immune response; the term "immune response" refers to both humoral or antibody-mediated and cell-mediated immune responses against antigens, vaccines or infectious agents; the term "vaccine" refers to a composition comprising an antigen for the therapeutic treatment or prophylactic immunization against an infectious or non-infectious disease; the term "immunization" refers to an immune response generated by vaccination or infection that provides protection against infectious or foreign agents; the term "recombinant protein or antigen" refers to a protein or antigen produced by recombinant DNA techniques that can be used to clone and express genes to produce proteins in a variety of hosts including bacteria, mammalian cells, insect cells, and plants. The term "potency" refers to the amount of antigen in an antigen preparation or vaccine as measured by a specified potency assay.

The terms "mutation" and "mutant" have their usual meanings herein, and refer to a genetic, naturally occurring or introduced change in a nucleic acid or polypeptide sequence, which has the same meaning as is commonly known to those of skill in the art.

The term "host cell" or "recombinant host cell" means a cell comprising a polynucleotide of the invention, regardless of the method used for insertion to produce the recombinant host cell, e.g., direct uptake, transduction, f-pairing or other methods known in the art. The exogenous polynucleotide may remain as a non-integrating vector, such as a plasmid, or may integrate into the host genome.

The term "transfection" refers to the process by which eukaryotic cells acquire a new genetic marker due to the incorporation of foreign DNA.

Drawings

FIG. 1 is a polyacrylamide gel electrophoresis diagram of an expression product of CSFV E2-Ferritin in a prokaryotic expression system; m is Marker; 1-6 is a CSFV E2-Ferritin prokaryotic expression sample; a prokaryotic expression sample with no load as a pET-28a carrier; the samples were not induced to be uninduced prokaryotic expression samples.

FIG. 2 CSFV E2-Ferritin-C-O-M₆Western blotting detection image of expression product in silkworm expression system; a is CSFV E2-Ferritin-C-O-M₆Silkworm expression products; b is a negative control.

FIG. 3 CSFV E2-Ferritin-C-O-M₆Electron micrograph of the expression product in the silkworm expression system.

Detailed Description

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

1. Test materials and reagents

(1) Strains, strains and vectors: prokaryotic expression vector pET-28a (+), escherichia coli TOP10 strain, transfer vector pVL1393, prokaryotic expression strain BL21(DE3), silkworm cell BmN, silkworm nuclear polyhedrosis virus parent strain BmBacmid and silkworm variety JY1 are all stored in molecular microorganism laboratories of the institute of biotechnology of the Chinese academy of agricultural sciences;

(2) ferritin sequence and hog cholera virus envelope E2 protein gene sequence: the consensus sequence obtained by analysis was sent to the Kisry company for synthesis and cloned into the prokaryotic expression vector pUC 57.

(3) Enzymes and reagents: restriction enzymes, T4 DNA ligase and corresponding buffers were purchased from Promega; LA Taq polymerase and buffer were purchased from TaKaRa; DNA and protein molecular weight standards of various specifications are products of TranGen Biotech company; 2K Plus II DNA Marker was purchased from Beijing Quanjin Biotechnology Ltd; goat anti-rabbit IgG secondary antibody labeled by horseradish peroxidase was purchased from MBL company; DEPC, M-MLV-Rtase (reverse transcriptase) from Promega; ferritin primary antibody was provided to the laboratory;

(4) biochemical reagents: tris, Ampicillin, Kanamycin, IPTG, SDS, urea, imidazole, TritonX-100, TEMED (N, N, N ', N' -tetramethylenethylene diamine), Ammonium Persulfate (Ammonium Persulfate), Kanamycin (Kanamycin) were purchased from Sigma; bisacrylamide, acrylamide, IPTG, X-Gal were purchased from Promega; agarose is a product of Sunbiotech company; yeast Extract (Yeast Extract), tryptone were purchased from OXOID, UK; 0.2um, 0.45um filters were purchased from Gelman Sciences; ethidium Bromide (EB), Coomassie Brilliant blue R-250 from Fluka; Ni-NTA Agarose, Proteinase K, fetal bovine serum were purchased from Invitrogen; bovine serum albumin was purchased from roche; the others are all domestic or imported analytical pure reagents. The primer synthesis and gene sequencing are completed by the biotechnology limited of Beijing Optimalaceae New industry.

(5) Culture medium: the Escherichia coli culture medium is LB culture medium; the silkworm insect cell culture medium is TC-100 purchased from Applichem company;

(6) animal experiments of the nano vaccine constructed by fusing the hog cholera virus envelope E2 protein and ferritin are carried out in an isolation laboratory.

2. Fusion PCR method for site-directed mutagenesis in experimental methods

Refer to Kuang Jandine et al (a new method for vector construction: recombinant fusion PCR method, genomics and applied biology, 2012, 31, 6, 634-639).

Example 1 preparation and potency assay of CSFV E2-Ferritin original sequence nanoparticle vaccine

1 arrangement of solutions and culture media

Reference is made to the relevant tool book for the preparation of solutions and media (Joseph et al, third edition of the molecular cloning guidelines, 2002; Oseber, et al, eds. molecular biology guidelines, 1998).

2, synthesizing the hog cholera virus envelope E2 protein gene sequence and the ferritin gene sequence.

In order to realize better fusion expression of the CSFV envelope E2 protein and ferritin, the amino acid sequence of CSFV envelope E2 protein was analyzed by signal peptide analysis software (SignalP) and transmembrane domain analysis software (TMHMM), and we used a consensus sequence without signal peptide region, in which the extracellular domain is the first 246 amino acids.

In order to promote the expression efficiency of the fusion nano-particles of the classical swine fever virus and the ferritin and improve the soluble expression, the 19 th asparagine (N) in the amino acid sequence of the helicobacter pylori ferritin is mutated into glutamine (Q) so as to eliminate the glycosylation site. Wherein the CSFV envelope E2 protein sequence is connected with the ferritin sequence by a connecting peptide (SGG), the first 4 amino acids of the ferritin amino acid sequence are removed, and then the connecting peptide is connected with the 5 th amino acid at the N end of the ferritin.

In order to improve the translation initiation efficiency of a target gene in a silkworm baculovirus eukaryotic expression system, a Kozak sequence AAC is added in front of the gene, and in order to improve the translation termination efficiency, a termination codon is changed into TAA. In addition, restriction sites for BamHI, EcoRI and the like within the gene sequence were removed, BamHI was added upstream of the gene, and EcoRI restriction sites were added downstream of the gene, for subsequent cloning into the eukaryotic transfer vector pVL 1393.

The signal peptide was removed from the target gene sequence, and ATG on pET-28a (+) vector was used to initiate translation. In addition, restriction sites for BamHI, EcoRI and the like within the gene sequence were removed, BamHI was added upstream of the gene, and EcoRI restriction sites were added downstream of the gene, for subsequent cloning into the prokaryotic vector pET-28a (+).

The designed CSFV envelope E2 protein gene sequence and ferritin sequence are artificially synthesized.

Plasmid construction of 3 hog cholera virus and ferritin fusion protein

3.1 PCR amplification of the fusion protein of classical swine fever Virus and ferritin

The classical swine fever virus and the ferritin are fused together by a fusion PCR technology. The specific experimental method is shown in the experimental method 2. 3.1.1 PCR amplification of E.coli expression plasmids

PCR amplification of CSFV E2 ectodomain sequence: plasmid pUC57-CSFV E2 is used as a template

F1	5’-CGGGATCCATGCGGCTAGCCTGTAAGGA-3’
		R1	5’-GCCACCGGAGCCATCTCTGTTACAGTCCG-3

PCR amplification of CSFV E2 ectodomain sequence: plasmid pUC57-CSFV E2-Ferritin is used as a template'

PCR amplification of Ferritin sequence: using pUC57-Ferritin as a template,

F2	5’-TTCAGCAGCTTGATGATGTCGCCACCGGA-3’
		R2	5’-CGGAATTCTTAGCTCTTGCGGGACTTGG-3’

taking PCR products CSFV E2 and Ferritin as templates, and performing Overlap-PCR amplification to obtain CSFV E2-Ferritin

F1	5’-CGGGATCCATGCGGCTAGCCTGTAAGGA-3’
		R2	5’-CGGAATTCTTAGCTCTTGCGGGACTTGG-3’

3.1.2 PCR amplification of expression plasmids in silkworm expression systems

F3	5’-CGGGATCCAATATGCGGCTAGCCTGTAAGGA-3’
		R3	5’-GCCACCGGAGCCATCTCTGTTACAGTCCG-3

PCR amplification of Ferritin sequence: using pUC57-Ferritin as a template,

F4	5’-TTCAGCAGCTTGATGATGTCGCCACCGGA-3’
		R4	5’-CGGAATTCTTAGCTCTTGCGGGACTTGG-3’

F3	5’-CGGGATCCAATATGCGGCTAGCCTGTAAGGA-3’
		R4	5’-CGGAATTCTTAGCTCTTGCGGGACTTGG-3’

The PCR reaction system is shown in table 1:

TABLE 1 PCR reaction System

Setting PCR parameters:

3.2 purification and recovery of DNA fragments from glass milk

Preparing 1% (w/v) agarose gel, and carrying out electrophoresis on the PCR amplification product; placing the agarose gel under an ultraviolet lamp, quickly cutting the gel containing a single target nucleic acid strip, placing the gel into a centrifugal tube of 1.5mL, weighing, adding 6M NaI with three times of volume, and placing the gel in a constant-temperature incubator at 37 ℃ for melting; adding 8 μ L of Glassmik into the completely melted solution, mixing, ice-cooling for 5min, and shaking twice; centrifuging at 8000rpm for 10s, and discarding the supernatant; adding 800 mu L of New Wash to Wash, slightly bouncing, centrifuging, and repeating for 2 times; removing the supernatant, and drying the centrifuge tube in a 37 ℃ constant-temperature incubator for 2-3 min; after drying, 20. mu.L of 0.1 XTE was added to dissolve, the DNA was mixed and dissolved thoroughly, centrifuged at 12000rpm for 5min, the supernatant was immediately used for ligation, and the rest was stored at-20 ℃.

3.3 cleavage of the target Gene PCR product

And (3) running glue on the PCR product, recovering a correct product from the glue, and performing double enzyme digestion reaction on the product by using restriction enzymes BamH I and EcoR I to obtain a target fragment CSFV E2-Ferritin. The enzyme system is shown in the following table 2:

TABLE 2 enzyme digestion System

3.4 Mini-Production of competent cells

Coli Top10 competent cells were prepared and stored at-80 ℃.

3.5 ligation and transformation of the Gene of interest to pET-28a (+) vector and pVL1393 vector

3.5.1 enzymatic digestion of pET-28a (+) and pVL1393 vectors

The transferred transformants pVL1393 and pET-28a (+) were digested simultaneously with restriction enzymes BamH I and EcoRI, inactivated at 65 ℃ for 20min and stored at-20 ℃ for further use.

3.5.2 joining

The target fragment recovered by enzyme digestion is connected with the transfer vector pVL1393 and pET-28a (+) after double enzyme digestion treatment by BamHI/EcoRI. By T₄DNA ligase, 16 ℃ and ligation overnight. Is connected withThe linker system is shown in table 3 below:

TABLE 3 connection System

3.5.3 transformation

Taking competent cells stored at-80 ℃, rapidly melting half, adding 3 mu L of the ligation product, and standing on ice for half an hour; placing the mixture in a constant-temperature water bath kettle at 42 ℃ for 90s, and quickly placing the mixture on ice for 3-5 min; adding a proper amount of 1mL LB culture medium into the tube, and standing and culturing for 60min in a constant temperature incubator at 37 ℃; after centrifugation, most of the supernatant was discarded, and 200. mu.L of the supernatant was applied to LB plates (100. mu.g/mL Amp), and cultured in a 37 ℃ incubator for 30min in the upright position and then in the inverted position overnight.

3.6 Rapid extraction of nucleic acids Positive clones

Picking a single colony on an LB plate, inoculating the single colony in an LB liquid culture medium (100 mu g/mL Amp), placing the single colony in a constant-temperature shaking incubator at 37 ℃, setting the rotating speed to be 220rpm, and culturing overnight; taking 500 mu L of bacterial liquid in a centrifugal tube, and collecting thalli; adding 30 mu L of Loading Buffer and 20 mu L of phenol/chloroform (1:1), and fully mixing by using a vortex shaker to resuspend the thalli; centrifugation was carried out at 12000rpm for 3min, and 8. mu.L of the supernatant was subjected to agarose gel electrophoresis, while an empty vector treated in the same manner was used as a control. Observing the band under an ultraviolet lamp of the gel imaging system, and selecting bacterial liquid with the plasmid band obviously retreated to extract the plasmid.

3.7 SDS alkaline lysis method for extracting plasmid DNA

3mL of bacterial liquid is collected in a centrifuge tube, plasmid DNA is extracted by an SDS alkaline lysis method, and the plasmid DNA is stored at the temperature of minus 20 ℃ for standby.

3.8 restriction enzyme digestion and sequencing identification of Positive clones

The cleavage system is shown in Table 4:

TABLE 4 enzyme digestion System

After reaction at 37 ℃ for 2 hours, 7. mu.L of the reaction mixture was subjected to electrophoresis using 1% agarose. And (3) carrying out DNA sequencing on the plasmid with correct enzyme digestion detection, wherein the result is consistent with the target gene, and the obtained recombinant plasmid is named as pET28a-CSFV E2-Ferritin and pVL1393-CSFV E2-Ferritin.

4 expression and purification of recombinant plasmids

4.1 inducible expression of recombinant plasmids in E.coli

The correctly identified recombinant expression plasmid pET28a-CSFV E2-Ferritin is transformed into BL21 competent cells, under the conditions of 37 ℃ and IPTG final concentration of 0.5mM, 1h, 2h, 3h, 4h and 5h are respectively induced, bacteria liquid is collected, SDS-PAGE electrophoresis is used for analyzing expression conditions, a specific band appears at the position of about 52kDa of pET28a-CSFV E2-Ferritin, the size of the specific band is consistent with that of the expected recombinant protein with His, the specific band is not generated by the non-induced recombinant expression vector, the fusion protein is successfully expressed in escherichia coli, the expression quantity is gradually increased 1-4 h after IPTG is added, and the amount of the induced 6h and the induced 8h of multi-accumulated recombinant protein is almost the same. The bacterial cells are crushed by ultrasonic waves, the supernatant is found to have a small amount of target protein, and the precipitate has obvious target bands, which indicates that the recombinant protein His-CSFV E2-Ferritin mainly exists in the form of insoluble inclusion bodies. The electrophoresis pattern of the polypropylene gel is shown in FIG. 1.

4.2 Mass expression of recombinant proteins and treatment of Inclusion body protein samples

Streaking the strain with high expression quantity stored at-80 ℃, culturing overnight at 37 ℃, selecting a single colony, inoculating the single colony in 4mL LB liquid medium (50 mu g/mL Kan), and culturing overnight at 37 ℃; transferring 1% of the bacterial solution into 200mL LB liquid medium (50. mu.g/mL Kan), shaking and culturing at 37 ℃ until OD value reaches about 0.6, adding IPTG (final concentration of 0.5mM), and continuously culturing at 37 ℃ for 4 h; centrifuging at 4 deg.C and 5000rpm for 10min to collect thallus, and sterilizing with sterile ddH₂O washing for 2 times, and centrifuging to collect thalli. Resuspending the thallus with lysis buffer solution with dosage of 100 μ L lysate/mL bacterial solution, ice-bathing for 30min, and breaking the thallus with ultrasonic wave on ice; centrifuging at 12000rpm for 10min at 4 deg.C, discardingClearing and precipitating to obtain a recombinant protein inclusion body; resuspending and washing the precipitate with a proper amount of inclusion body washing solution I and an appropriate amount of inclusion body washing solution II, and discarding the supernatant; the pellet was resuspended in the appropriate amount of urea NTA-0Buffer and dissolved overnight at 4 ℃.

4.3 Nickel column affinity chromatography purification of recombinant proteins

Centrifuging the overnight dissolved inclusion body solution at 4 ℃ and 12000rpm for 15min, taking the supernatant, and filtering with a 0.45 mu m membrane; purifying the expressed protein by using a Ni-NTA resin chromatographic column, collecting eluent in 5 gradients of urea NTA-25, urea NTA-50, urea NTA-100, urea NTA-250 and urea NTA-500, collecting penetration liquid and eluent, collecting an NTA volume in each tube, and determining the binding condition of the protein and the distribution condition of the target protein in the eluent by SDS-PAGE analysis. Protein electrophoresis showed the most protein eluted at 25mM imidazole concentration. After SDS-PAGE electrophoresis, the purified recombinant protein is observed to have correct size and single protein band, and the content of the purified protein is 6 mg/mL.

4.4 preparation of polyclonal antibodies

Quantifying the purified His-Ferritin protein, collecting 1.5mg protein, cutting off gel containing target protein after SDS-PAGE electrophoresis, cutting up the gel as much as possible, drying at 37 ℃, grinding into powder, diluting the antigen protein to 2 times of final concentration by using normal saline, fully mixing the adjuvant, taking out the required dosage under aseptic condition, and mixing the required dosage with the antigen protein according to the volume ratio of 1:1, mixing the mixture quickly, injecting the mixture into an immune mouse through hind leg and calf muscles, collecting all serum after two immunizations, and measuring the antibody titer of the serum.

5 recombinant plasmid is expressed and purified in a silkworm eukaryotic expression system

5.1 reproduction of parent strain BmBacmid of Bombyx mori nuclear polyhedrosis virus and preparation of virus DNA

Preparing a1 XTC-100 culture medium according to the product specification of Applichem company, adjusting the pH to 6.22 by using 2M NaOH, supplementing 10 percent fetal bovine serum to the culture medium after filtration sterilization, and culturing the bombyx mori cell BmN at 27 ℃. Infecting about 50mL of cells in logarithmic growth phase with parent strain of bombyx mori nuclear polyhedrosis virus, collecting virus infection liquid after 3-4 d, centrifuging at 10000rpm for 10min, removing precipitate, centrifuging the supernatant at 25000rpm for 1h, removing the supernatant, suspending virus particles with 1mL of virus DNA extract (1L containing 12.1g of Tris, 33.6g of EDTA, 14.1g of KCl and pH 7.5), transferring to a 1.5mL centrifuge tube, adding proteinase K to a final concentration of 50 μ g/mL, keeping the temperature at 50 ℃ for 2h, adding 35% of Sarkorsel to a final concentration of 1%, keeping the temperature at 50 ℃ for 2h, extracting with equal volumes of saturated phenol, chloroform (1:1) and chloroform sequentially, transferring the upper aqueous phase to a new tube, adding 1/10 volume of 3M NaCl, adding 2 times of absolute ethanol, standing at-20 ℃ for more than 2h to precipitate virus DNA, centrifuging at 5000rpm for 10min, washing the precipitate with 75% ethanol, and freeze drying. Dissolved in 100. mu.l of TE Buffer and stored at 4 ℃ until use.

5.2 recombinant Bombyx mori baculovirus rBmBacmid (P)_PHConstruction and obtaining of-CSFV E2-Ferritin)

Inoculation of about 1X 10⁶Cells at 15cm²After the cells were attached to the wall in the flask, the medium containing Fetal Bovine Serum (FBS) was removed, washed three times with FBS-free medium, and 1.5ml FBS-free medium was added. Mu.g of bombyx mori baculovirus parent strain BmBcBmid DNA (patent number: ZL201110142492.4), 2 mu.g of recombinant transfer plasmid pVL1393-CSFV E2-Ferritin and 5 mu.l of liposome are sequentially added into a sterilizing tube, the volume is complemented to 60 mu.l by sterile double distilled water, the mixture is gently mixed, the mixture is kept stand for 15min and then the mixture is dropwise added into a culture bottle for cotransfection. After 4h incubation at 27 ℃ 1.5ml serum free medium and 300. mu.l FBS were supplemented. Culturing at 27 ℃ for 4-5 days at constant temperature, collecting supernatant for recombinant virus rBmBacmid (P)_PH-CSFVE 2-Ferritin). Inoculating a proper amount of cells (about 70-80%) in a small 35mm dish, sucking out the culture medium after the cells adhere to the wall, diluting the co-transfection supernatant at different concentrations, and adding 1ml of co-transfection solution into the adherent cells for uniform distribution. After infection for 1h at 27 ℃, absorbing infection liquid, melting 2% low melting point agarose gel in water bath at 60 ℃, cooling to 40 ℃, uniformly mixing with 2 XTC-100 culture medium (containing 20% FBS) preheated at 40 ℃, adding 4ml of the gel into each dish, sealing with Parafilm after solidification, carrying out inverted culture at 27 ℃ for 3-5 d, and observing by using a microscope. Selecting out the plaques without polyhedra, repeating the steps, and obtaining the pure recombinant silkworm baculovirus rBmBacmid (P) through 2-3 rounds of purification_PH-CSFV E2-Ferritin)。

5.3 recombinant Virus rBmBacmid (P)_PH-CSFV E2-Ferritin) amplification in Bombyx mori cells

Recombinant bombyx mori baculovirus rBmBacmid (P)_PHCSFV E2-Ferritin) to infect the normal growth BmN cells, after 3 days of culture, the supernatant containing a large amount of recombinant virus rBmBacmid (P)_PH-CSFV E2-Ferritin)。

5.4 identification of recombinant viruses

Exogenous gene integration was analyzed by PCR. The extraction method of free virus genome DNA is as follows: 150 μ L of virus supernatant was added with 150 μ L (0.5mol/L) of NaOH and mixed well, 20 μ L (8mol/L) of ammonium acetate was added, mixed well and extracted once with equal volume of phenol and chloroform, and DNA was dissolved with 20 μ L of TE after alcohol precipitation.

Taking 1 mul of the virus genome DNA for PCR amplification, wherein the reaction conditions are as follows: denaturation at 94 deg.C for 5min, denaturation at 94 deg.C for 1min, denaturation at 58 deg.C for 1min, and denaturation at 72 deg.C for 3min for 30 cycles, and final extension at 72 deg.C for 10 min. Electrophoresis analysis was performed on 15. mu.l of the reaction product, and the result confirmed that the recombinant virus was obtained.

5.5 expression of CSFV E2-Ferritin in Bombycis and pupa Bombycis

The silkworm pupae used are high-expression variety JY1 (stored in the laboratory). The breeding of the JY1 variety silkworm is carried out according to the conventional method of China sericulture (Shanghai science and technology publishing Co., 1991) compiled by Luhong Yin. Selecting silkworm with the same average weight 48h after the food in the area and selecting 15 silkworm pupas with the same average weight seven days after cocooning, wherein each silkworm pupa and silkworm are inoculated with about 1.0 multiplied by 10⁵pfu rBmBacmid(P_PH-CSFV E2-Ferritin), collecting the silkworm pupae with disease and silkworm blood after 4-5 days, and freezing and storing at-20 ℃ for ELISA detection.

5.6 Collection and purification of CSFV E2-Ferritin viroid

Silkworm pupae containing the gene of interest were ground with precooled PBS (1: 9 ratio) in a homogenizer and then filtered through a 0.45um filter. In 30% sucrose solution, 1.5X 10⁵g ultra-high speed centrifugation for 2 h. The pellet was reconstituted to volume with Tris-HCl (pH 7.0) containing 0.1M NaCl and passed through cation exchange chromatography packing SP (GE Corp.)) 0.5M NaCl in Tris-HCl (pH 7.0). Then, the mixture was subjected to molecular sieve chromatography S200 (GE). The purity can reach 95%, and the yield can reach more than 40%.

6 Western blotting detection

Diluting 10 times of ultrasonic waves by PBS (pH 7.4) to break silkworm hemolymph infected by recombinant virus, carrying out SDS-PAGE gel electrophoresis, carrying out gel concentration of 5% and separation gel concentration of 15%, transferring protein to a polyvinylidene fluoride (PVDF) membrane by a semi-dry transfer method, preparing 3% BSA (bovine serum albumin) by PBST for blocking, preparing a mouse-derived H protein monoclonal antibody as a primary antibody (1:1000 for dilution, self-made in a laboratory), using goat anti-mouse IgG marked by HRP as a secondary antibody (1:5000 for dilution), finally carrying out color development by DAB (diaminobenzidine), terminating by deionized water, and detecting a result. Western blotting results showed that a specific band of 48kDa (CSFV E2-Ferritin) size was detectable in the supernatant of a silkworm hemolymph sample after infection with the recombinant virus.

7 ELISA assay

Diluting the silkworm hemolymph sample to be detected by using a coating solution in a proper multiple proportion, taking a silkworm hemolymph sample infected by a parent virus as a negative control, only adding the coating solution as a blank control, adding 100 mu L of the coating solution into each hole of an enzyme label plate, and standing overnight at 4 ℃. The well was quickly drained and washed 3 times with PBST. mu.L of 3% BSA blocking solution was added to each well, acted on at 37 ℃ for 3h, and washed 3 times with PBST. Diluting a His-Ferritin polyclonal antibody prepared in a laboratory by 1:1000, 100. mu.L per well, 1.5h at 37 ℃ and 4 washes with PBST. 100 μ LHRP-labeled goat anti-mouse (1: 5000) was added to each well, incubated at 37 ℃ for 45-60 min, and washed 4 times with PBST. Then adding 100 mu L of freshly prepared OPD (o-phenylenediamine) color developing solution, and developing for 10-30 min at room temperature in a dark place. The reaction was terminated by adding 50. mu.L of 2M sulfuric acid to each reaction well. The OD value is measured by the wavelength at 492nm on a microplate reader, the OD value of each well is measured after the blank control well is zeroed, and the positive is determined by the P/N value (the OD value of the positive well minus the OD value of the blank control well/the OD value of the negative well) being more than or equal to 2.1.

And (3) judging an ELISA result: the positive result is that the P/N value (the OD value of the positive hole minus the OD value of the blank control hole/the OD value of the negative hole) is more than or equal to 2.1, and the result shows that the ELISA titer of the CSFV E2-Ferritin gene expression product can reach 1: 32.

table 5 shows the ELISA titer experimental data of the CSFV E2-Ferritin original gene sequence expression product, and the results show that the ELISA titer of the CSFV E2-Ferritin gene expression product can reach 1: 32.

TABLE 5 ELISA titers of expression products of CSFV E2-Ferritin original sequence

Group of	Potency of the drug
		CSFV E2-Ferritin	1：32
Silkworm blood sample infected with parental virus (negative control)	1：4

Example 2 preparation and potency assay of nanoparticle vaccine optimized by homology sequence design of CSFV E2-Ferritin original sequence

1 arrangement of solutions and culture media

The specific solution and culture medium preparation method is shown in example 1.

2 acquisition of Gene of conserved sequence of envelope E2 protein of classical swine fever virus

The original amino acid sequence of the CSFV envelope E2 protein in example 1 is compared with other 20 amino acid sequences obtained from NCBI to obtain a homologous sequence. The isosensory sequence is optimized, and further utilizes OptimumGene^TMThe technology optimizes the swine fever virus envelope E2 protein amino acid sequence, modifies the optimized swine fever virus envelope E2 protein amino acid sequence and ferritin monomer subunit amino acid sequence according to silkworm codon preference, and influences gene transcription efficiency, translation efficiency andthe GC content of protein folding, the CpG dinucleotide content, the codon preference, the secondary structure of mRNA, the stability of mRNA free energy, RNA instability gene sequences, repetitive sequences and other related parameters are optimized and designed, and the finally translated protein sequence is kept unchanged. The specific optimization procedure for the nomenclature CSFVE2-Ferritin-C-O is shown in example 1.

3 plasmid construction of fusion proteins

See example 1 for a specific experimental procedure.

CSFV E2-Ferritin-C-O fusion PCR primer:

hog cholera virus envelope E2 protein ectodomain PCR primers:

F5:5’-CGGGATCCAATATGCGGCTAGCCTGTAAGGA-3’

R5:5’-GCCATCTCTGTTACAGTCCG-3’

ferritin PCR primers:

F6：5’-GACATCATCAAGCTGCTGAA-3’

R6：5’-TTCAGCAGCTTGATGATGTC-3’

Over-lapPCR primers:

F5:5’-CGGGATCCAATATGCGGCTAGCCTGTAAGGA-3’

R5:5’-CGGAATTCTTAGCTCTTGCGGGACTTGG-3’

3.1 ligation and transformation of the target Gene with the pVL1393 vector

3.2 Rapid extraction of nucleic acids Positive clones

See example 1 for a specific experimental procedure.

3.3 SDS alkaline lysis method for extracting plasmid DNA

See example 1 for a specific experimental procedure.

3.4 enzyme digestion and sequencing identification of Positive clones

See example 1 for a specific experimental procedure.

4 the recombinant plasmid pVL1393-CSFV E2-Ferritin-C-O is expressed and purified in a silkworm expression system

4.1 reproduction of parent strain BmBacmid of Bombyx mori nuclear polyhedrosis virus and preparation of virus DNA

See example 1 for a specific experimental procedure.

4.2 construction and acquisition of recombinant Bombyx mori baculovirus rBmBacmid

See example 1 for a specific experimental procedure.

4.3 amplification of recombinant Virus rBmBacmid in silkworm cells

See example 1 for a specific experimental procedure.

4.4 identification of recombinant viruses

See example 1 for a specific experimental procedure.

4.5 expression of CSFV E2-Ferritin-C-O in Bombycis and pupa

See example 1 for a specific experimental procedure.

4.6 Collection and purification of CSFV E2-Ferritin-C-O viroid

See example 1 for a specific experimental procedure.

5 Western blotting and ELISA detection

See example 1 for a specific experimental procedure.

6 results identification

And (3) judging an ELISA result: the positive result is that the P/N value (the OD value of the positive hole minus the OD value of the blank control hole/the OD value of the negative hole) is more than or equal to 2.1, and the result shows that the ELISA titer of the CSFV E2-Ferritin-C-O gene expression product can reach 1: 128.

as can be seen from the titer detection results in Table 6, the ELISA titer of the CSFV E2-Ferritin-C gene expression product can reach 1: 64, the ELISA titer of the CSFV E2-Ferritin-C-O gene expression product can reach 1: 128. therefore, the expression level of the consensus sequence after codon optimization is greatly improved, which indicates that the modification and optimization work of the embodiment is successful.

TABLE 6 ELISA Titers of CSFV E2-Ferritin-C-O Gene expression products

Group of	Potency of the drug
		CSFV E2-Ferritin	1：32
CSFV E2-Ferritin-C	1：64
		CSFV E2-Ferritin-C-O	1：128
Parent Virus infected silkworm blood sample (negative control)	1：4

Example 3 preparation and potency assay of nanoparticle vaccine after single-site mutation of amino acid in CSFV E2-Ferritin-C-O mutant

1 method of experiment

1.1 construction of single-site mutant gene of CSFV E2-Ferritin-C-O amino acid sequence

Based on the result of example 2, the invention obtains CSFV E2-Ferritin-C mutant, takes the gene sequence of CSFV E2-Ferritin-C mutant codon optimized as a template, designs a plurality of pairs of primers to perform site-specific mutagenesis on the conserved sequence, the site-specific mutagenesis is performed by using a fusion PCR method, and the fusion PCR method is shown in example 1.

The mutation sites are respectively CSFV E2: I13H, G23M, N35A, S48K, L59N, Y66C, S78T, D86R, D97N, S109V, Y116W, a125V, E139F, V152H, D167G, N175P, V202I, P215Q, I227D, D238E. The obtained mutant is named as CSFV E2-Ferritin-C-O-M (I13H, G23M, N35A, S48K, L59N, Y66C, S78T, D86R, D97N, S109V, Y116W, A125V, E139F, V152H, D167G, N175P, V202I, P215Q, I D27 2, D238E).

On the basis, the invention provides a single mutation site CSFV E2 which can improve the expression quantity: combining N35A, D86R, Y116W, E139F, D167G or I227D in pairs to carry out double-site mutation; the double site mutation is based on the single site mutation sequence, and the target fragment of the double site mutation is obtained by performing the second site-directed mutation by using a fusion PCR method by using a corresponding primer and taking the single site mutation sequence (CSFV E2-Ferritin-C-O-M) as a template, wherein the fusion PCR method is shown in example 1.

The double mutation site is CSFV E2: the mutants obtained by 15 combinations of N35-D86, N35-Y116, N35-E139, N35-D167, N35-I227, D86-Y116, D86-E139, D86-D167, D86-I227, Y116-E139, Y116-D167, Y116-I227, E139-D167, E139-I227 or D167-I227 are named as CSFV E-Ferritin-C-O-D (N35-D86, N35-Y116, N35-E139, N35-D167, N35-I227, D86-Y116, D86-E139, D86-D167, D86-I227, Y116-E139, Y116-D167, Y116-I167, E139-D227, E139-I227 or D167-I227).

The invention obtains 6 single mutation sites by analyzing glycosylation sites, which can effectively improve the expression quantity of target genes, so that the multi-site mutation is based on a double-site mutation sequence, and the multi-site mutation is carried out on site-specific mutation of the multi-site mutation sites by taking the multi-site mutation sites (CSFV E2-Ferritin-C-O-D) as a template and utilizing corresponding primers through a fusion PCR method, thereby obtaining a multi-site mutated target fragment, and the fusion PCR method is shown in example 1.

The multi-site mutant is obtained by carrying out multi-site mutation on the amino acid sequence shown in SEQ ID NO.1 according to the amino acid multi-site mutation mode of N35A-D86R-Y116W-E139F-D167G-I227D.

The following combinations were obtained: CSFV E2 (N35A-D86R-Y116W-E139F-D167G-I227D); named as CSFVE2-Ferritin-C-O-M₆(N35A-D86R-Y116W-E139F-D167G-I227D)。

Mixing CSFV E2-Ferritin-C-O-M₆Expressing in silkworm eukaryotic expression system and AcMNPV-insect cell expression system separately.

Primers required for amino acid single-site mutation of CSFV E2-Ferritin-C-O:

CSFV E2-Ferritin-C-O:

(1) primers for upstream and downstream on both sides:

F：AATATGCGGCTAGCCTGTAAGGA

R：TTAGCTCTTGCGGGACTTGG

(2) middle upstream and downstream primers:

1.

F：TTGGGTAACGCCAGGCACCCAGTCACGACG

R：CGTCGTGACTGGGAAGTGGGCGTTACCCAA

2.

F：ACGTTGTAAAACGACATGTGCCAAGCTTGC

R：GCAAGCTTGGCACTGCATGTTTTACAACGT

3.

F：CCTGCAGGTCGACGAAACGACTTAGCCATC

R：GATGGCTAAGTCGACGTTTCGACCTGCAGG

4.

F：ACAGTCCGTTGAATCAAGTCTGTAACCTGT

R：ACAGGTTACAGAATACTTTCAACGGACTGT

5.

F：TTTAAAGTCACAGCACTTAATGTGGTCAGTAGG

R：CCTACTGACCACATTAAGTGCTGTGACTTTAAA

6.

F：GTGGTCAGTAGGAGGGCGGCGTCACTGCAT

R：ATGCAGTGACGCCAACGCCCTCTGACCAC

7.

F：AAGGCTTTACCCACTACCGTGACATTCGAGCTC

R：GAGCTCGAATGTCACGGTAGTGGGTAAAGCCTT

8.

F：TTCGAGCTCCTGTTCTGCGGGACCAACCCATCA

R：TGATGGGTTGGTCCCGCAGAACAGGAGCTCGAA

9.

F：ACTGAGGAAATGGGACTTGACTTCAGGTCCGGG

R：CCCGGACCTGAAGTCAAGTCCCATTTCCTCAGT

10.

F：TGCCCGTTTGATACGGTGCCTGTTGTTAAGGGA

R：TCCCTTAACAACAGGCACCGTATCAAACGGGCA

11.

F：GTTGTTAAGGGAAAGGCAAATACGACCTTGTTG

R：CAACAAGGTCGTATTTGCCTTTCCCTTAACAAC

12.

F：TTGTTGAACGGTAGTGTGTTCTATCTTGTCTGC

R：GCAGACAAGATAGAACACACTACCGTTCAACAA

13.

F：TGGACGGGTGTCATATTCTGCACAGCAGTGAGC

R：GCTCACTGCTGTGCAGAATATGACACCCGTCCA

14.

F：ACTCTGAGGACAGAACACGTAAAGACCTTCAGG

R：CCTGAAGGTCTTTACGTGTTCTGTCCTCAGAGT

15.

F：TTTCCGCACAGAATGGGCTGTGTGACCACCACA

R：TGTGGTGGTCACACAGCCCATTCTGTGCGGAAA

16.

F：ACCACCACAGTGGAAGCCGAAGATTTATTCTAT

R：ATAGAATAAATCTTCGGCTTCCACTGTGGTGGT

17.

F：TACACAAGGGGGGTAAGGGAACAACGTAGATGG

R：CCATCTACGTTGTTCCCTTACCCCCCTTGTGTA

18.

F：TTCGACTTCGATGGGCAGGACGGACTCCCGCAT

R：ATGCGGGAGTCCGTCCTGCCCATCGAAGTCGAA

19.

F：CCCATAGGTAAGTGCGACTTGGCAAATGAGACA

R：TGTCTCATTTGCCAAGTCGCACTTACCTATGGG

20.

F：GGTTACAGAATAGTA TCAACGGACTGTAAC

R：GTTACAGTCCGTTGACTCTACTATTCTGTAACC

plasmid construction of 2 CSFV E2-Ferritin-C-O-M mutant

See example 1 for a specific experimental procedure.

3 transformation and identification of recombinant plasmid

See example 1 for a specific experimental procedure.

4 the recombinant plasmid is expressed and purified in a silkworm expression system and an AcMNPV-insect cell expression system

The specific procedure is the same as in example 1.

In addition, construction and preparation of AcBacmid DNA: the insect bioreactor expressing multiple exogenous genes and a construction method and application thereof (Zhangyifang, Lianecdou, Yi Yong bamboo, and the like) are prepared according to the following method (P)

Identification and expression of recombinant virus rAcBacmid in insect cells: exogenous gene integration was analyzed by PCR. Extracting virus genome DNA. Taking 1 mu L of the virus genome DNA for PCR amplification, taking 15 mu L of reaction product for electrophoretic analysis, and the result proves that the recombinant virus rAcBacmid-CSFV E2-Ferritin-C-O-M is obtained₆. The recombinant virus rAcBacmid-CSFV E2-C-O-M₆Culture solution according to 10^6-7pfu was infected with 100mL of insect cells, and after 96 hours the infected cells were harvested and frozen at-20 ℃ for ELISA detection.

5 identification of results

Determination standard of ELISA results: positive results were obtained with a P/N value (OD of positive wells minus OD of blank wells/OD of negative wells) of 2.1 or higher, although the highest ELISA value was around 512, since the assay was performed using the threshold and dilution factor as quantitative indicators, and the amount of the sample was different depending on the magnitude of the P/N value.

As can be seen from the titer detection data in Table 7, the amino acid single-site mutation was performed on the basis of the consensus sequence, and the expression level of the expression products of the obtained mutants (N35A, D86R, Y116W, E139F, D167G and I227D) was significantly improved compared with that of the consensus sequence.

TABLE 7 ELISA Titers of expression products of the CSFV E2-Ferritin-C-O-M mutant

Group of	Potency of the drug
		CSFV E2-Ferritin	1：32
CSFV E2-Ferritin-C-O	1：128
		CSFV E2-Ferritin-C-O-I13H	1：64
CSFV E2-Ferritin-C-O-G23M	1：32
		CSFV E2-Ferritin-C-O-N35A	1:256
CSFV E2-Ferritin-C-O-S48K	1：64
		CSFV E2-Ferritin-C-O-L59N	1：32
CSFV E2-Ferritin-C-O-Y66C	1：64
		CSFV E2-Ferritin-C-O-S78T	1：64
CSFV E2-Ferritin-C-O-D86R	1：512
		CSFV E2-Ferritin-C-O-D97N	1：32
CSFV E2-Ferritin-C-O-S109V	1：64
		CSFV E2-Ferritin-C-O-Y116W	1：256
CSFV E2-Ferritin-C-O-A125V	1：64
		CSFV E2-Ferritin-C-O-E139F	1：256
CSFV E2-Ferritin-C-O-V152H	1：32
		CSFV E2-Ferritin-C-O-D167G	1：256
CSFV E2-Ferritin-C-O-N175P	1：64
		CSFV E2-Ferritin-C-O-V202I	1：32
CSFV E2-Ferritin-C-O-P215Q	1：64
		CSFV E2-Ferritin-C-O-I227D	1：256
CSFV E2-Ferritin-C-O-D238E	1：64
		Silkworm blood sample infected with parental virus (negative control)	1：4

According to the results, the single mutation site is effective, so that 6 single mutation sites with obviously improved potency are further selected to be combined pairwise for double site mutation; the ELISA titer test results are shown in Table 8.

Determination standard of ELISA results: positive was obtained by setting the P/N value (OD value of positive well minus OD value of blank well/OD value of negative well) to 2.1 or more.

As shown in the titer detection data in Table 8, in the amino acid double-site mutation of the homologous sequence, the expression level of three double mutants (D86R-Y116W, E139F-D167G and E139F-I227D) obtained by carrying out amino acid double-site mutation on the basis of the homologous sequence (SEQ ID NO.1) is obviously improved compared with that of the homologous sequence.

TABLE 8 ELISA Titers of expression products of the CSFV E2-Ferritin-C-O-D mutant

Group of	Potency of the drug
		CSFV E2-Ferritin	1：32
CSFV E2-Ferritin-C-O-M	1：512
		CSFV E2-Ferritin-C-O-N35A-D86R	1：128
CSFV E2-Ferritin-C-O-N35A-Y116W	1：64
		CSFV E2-Ferritin-C-O-N35A-E139F	1：128
CSFV E2-Ferritin-C-O-N35A-D167G	1：64
		CSFV E2-Ferritin-C-O-N35A-I227D	1：64
CSFV E2-Ferritin-C-O-D86R-Y116W	1：2048
		CSFV E2-Ferritin-C-O-D86R-E139F	1：128
CSFV E2-Ferritin-C-O-D86R-D167G	1：64
		CSFV E2-Ferritin-C-O-D86R-I227D	1：64
CSFV E2-Ferritin-C-O-Y116W-E139F	1：128
		CSFV E2-Ferritin-C-O-Y116W-D167G	1：32
CSFV E2-Ferritin-C-O-Y116W-I227D	1：64
		CSFV E2-Ferritin-C-O-E139F-D167G	1：1024
CSFV E2-Ferritin-C-O-E139F-I227D	1：1024
		CSFV E2-Ferritin-C-O-D167G-I227D	1：128
Silkworm blood sample infected with parental virus (negative control)	1：4

The above results obtained three double mutants (D86R-Y116W, E139F-D167G and E139F-I227D) with significantly improved expression levels, and considering that the sequence of amino acids is the primary structure of the protein and determines the higher structure of the protein, it is presumed that the positions of the partial mutation points of the single-site mutation of amino acids are close to each other, and therefore, the above 6 mutation sites were tried to be mutated at the same time to obtain a multi-site mutant; the results of ELISA titer detection of the multi-site mutations are shown in Table 8.

According to the potency test results in Table 9, CSFV E2-Ferritin-C-O-M₆The ELISA titer of the gene expression product can reach 1: 4096.

TABLE 9 CSFV E2-Ferritin-C-O-M₆ELISA detection of expression product of mutant in silkworm and AcMNPV-insect cell expression system

Group of	Potency of the drug
		CSFV E2-Ferritin-C-O-D	1：2048
CSFV E2-Ferritin-C-O-M₆(silkworm)	1：4096
		AcCSFV E2-Ferritin-C-O-M₆(AcMNPV-insect cell)	1：1024
Silkworm blood sample infected with parental virus (negative control)	1：4

6 Western blotting detection

See example 1 for a specific experimental procedure. Western blotting results showed that a specific band of 48kDa (CSFV E2-Ferritin-C-O-M6) size was detectable in the supernatant of silkworm hemolymph samples after recombinant virus infection (see FIG. 1).

7 Electron microscopy

A1 ml syringe was used to aspirate a certain amount of 1% uranium acetate for future use, and another syringe was used to aspirate a certain amount of distilled water. Respectively preliminarily purifying CSFV E2-Ferritin-C-O-M6 nanoparticle silkworm hemolymph, diluting with suspension, dripping the suspended sample on a sealing film to form a small liquid bead, clamping a carrier net with the tip of a tweezers, allowing the side with the film to face downwards, dipping the sample, then blotting the sample with filter paper, washing off redundant suspended substances, and washing for 5 times. After the drying, the carrying net is placed on the liquid drop of the 1% uranium acetate dye liquor, dyeing is carried out for 3 minutes, the filter paper is used for sucking the redundant dye liquor from the edge of the copper net, the process is repeated for 2-3 times, and microscopic examination is carried out after the drying. As a result, as shown in FIG. 3, nanoparticles having a size corresponding to the expected size were observed, the diameter of the cage was about 12 nm, and antenna-like protrusions were observed carefully.

Example 4 pVLCAG-CSFV E2-Ferritin-C-O-M₆Construction of recombinant virus for baculovirus mammalian expression and animal experiment

1. Construction of pVLCAG vector

Specific experimental methods reference is made to the method of expressing a foreign gene in animal cells or animal tissues [ P ]. china: ZL 201210408558.4 ], zhang (zhanqiang, yao, anecdotal et al) to construct recombinant baculovirus transfer vectors that present a foreign gene in vertebrate cells or individuals.

2. Construction of recombinant viruses presenting reporter genes

2.1 preparation of CSFV E2-Ferritin-C-O-M₆Cloning of genes into Gene presenting transfer vectors

The CSFV E2-Ferritin-C-O-M with the restriction enzyme cutting site in example 3₆The gene fragment is cut by enzyme, recovered and connected to pVLCAG vector treated by the same enzyme, and pVLCAG-CSFV E2-Ferritin-C-O-M is obtained after correct identification₆。

2.2 construction of recombinant viruses for Gene presentation and preparation thereof in Large quantities

Respectively using pVLCAG-CSFV E2-Ferritin-C-O-M₆The transfer vector uses rebmBac to co-transfect BmN cells to obtain recombinant virus Bm-CAG CSFV E2-Ferritin-C-O-M₆The pVL1393-Luc was still required as a control during the co-transfection procedure to determine the success of the co-transfection and the virus purification procedure was as above.

Infecting larva of a 5-year-old silkworm with the recombinant virus, and harvesting silkworm hemolymph after 4-5 days, wherein the silkworm hemolymph contains a large amount of amplified recombinant virus.

Silkworm hemolymph was diluted with PBS and sonicated (10 s.times.10 times), and then centrifuged at 12000rpm for 10 minutes to remove cell debris, followed by 15X 10⁴g centrifuging for 3h, removing supernatant, and resuspending the precipitate with appropriate amount of PBS to obtain virus particles of primarily purified recombinant baculovirus, wherein the recombinant virus of 10mL silkworm blood is resuspended with 2mL LPBS after centrifugation, and the amount of the recombinant virus after resuspension is about 2.5 × 10¹²PFU/mL (about 5X 10)¹²viral genes (vg)/mL, viral copy number was calculated by fluorescent quantitative PCR using BmNPV viral DNA backbone sequence primers, GJ-1F (CGAACGGAGACGATGGATGGATGGGATC) and GJ-1R (GTGCCGAGCGATTGTAAGGGATC).

3 expression of recombinant viruses in mammalian cells recombinant viruses Bm-CAG PCV-II CAP-Ferritin-C-O-M were targeted for gene presentation using VERO cells₆And Bm-CAG PCV-III CAP-Ferritin-C-O-M₆100MOI of each virus was taken for study. The method comprises the following steps:

1) six well plates were seeded with VERO cells (1X 10)⁶cell/well), adherent culture at 37 ℃ for 8-12h

2) Take 1X 10⁸PFU purified recombinant virus Bm-CAG PCV-II CAP-Ferritin-C-O-M₆And Bm-CAG PCV-III CAP-Ferritin-C-O-M₆Adding into six-well plate cells, and incubating at 37 deg.C for 1h

3) After incubation, removing a culture medium containing viruses, replacing a normal DMEM serum-containing culture medium, treating cells for about 42 hours, collecting an expression product, and performing ELISA detection on titer of 1: 1024.

4 animal test

4.1 preparation of CSFV E2-Ferritin-C-O-M₆Expression product immunization of animals

The optimal sequence CSFV E2-Ferritin-C-O-M obtained by analysis₆The silkworm pupae obtained by expressing in a silkworm eukaryotic expression system are injected into animals according to the amount of 25 mu g/animal, and 30 parts/g silkworm pupae vaccine is prepared.

The preparation method comprises the following steps: respectively weighing 10g of expression CSFV E2-Ferritin-C-O-M₆Adding 90mL PBS buffer solution into silkworm pupas with nano-particle antigens, stirring for 5-10 min by a stirrer to fully mix the solution uniformly, preparing a mother solution, and putting the mother solution into a sterilization bottle. The 206 adjuvant is sterilized in advance and then placed in an incubator at 30 ℃ for heat preservation. An appropriate amount of the mother liquor is put on ice and adjusted, when the mother liquor is mixed with the adjuvant, 3mL of the adjuvant is added into a 15mL centrifuge tube, 3mL of the mother liquor is slowly dropped, and the homogenate is carried out for 3min by a homogenizer. Ciprofloxacin hydrochloride was added. The vaccine is milk white, a small amount of the vaccine can be taken out when the quality of the vaccine is detected, the vaccine is centrifuged at 3000rpm for 15min, and the qualified vaccine is obtained when the vaccine is not layered. Treating healthy pupa Bombycis by the same method to obtainThe vaccine served as a control.

The optimal sequence CSFV E2-Ferritin-C-O-M obtained by analysis₆The resulting cell pellet was expressed in an AcBacmid-insect cell eukaryotic expression system and injected into animals at 25. mu.g/cell.

The preparation method comprises the following steps: the antigen expressed by insect cells is prepared by mixing corresponding adjuvant after the cell precipitation amount of a unit required for preparing the vaccine is determined and is subjected to ultrasonic disruption.

After 50 SPF mice are taken and adaptively raised for one week, the SPF mice are randomly divided into 5 groups of 10 mice, and the two groups of mice are respectively injected with CSFV E2-Ferritin-C-O-M intraperitoneally or intramuscularly₆1 part (0.2mL) of vaccine prepared by expressing the product in a silkworm eukaryotic expression system and 1 part (0.2mL) of vaccine prepared by expressing the product in an AcMNPV-insect cell expression system. The vaccine prepared by inoculating 10 healthy silkworm pupas is used as a negative silkworm pupa immune group, 10 silkworm pupas are used as a normal control group without immune treatment, and 10 silkworm pupas are inoculated with a traditional vaccine strain and used as a negative control. After 15 days of inoculation, blood is collected from the orbit, about 1mL of blood is collected, the blood is placed in a test tube in an inclined mode, the test tube is placed at 37 ℃ for 2 hours, and then the test tube is turned to the room temperature to be overnight. Transferring the serum into a centrifuge tube for 2000rpmin and 10min, collecting the serum, and detecting the antibody titer in the serum by respectively using the protein expressed by pET-28a-CSFV E2-Ferritin pronucleus as target protein. The antibody titer of the negative silkworm pupa immune group should be not higher than 1: 4, the antibody titer of the traditional vaccine strains is 1: 64-128 and CSFV E2-Ferritin-C-O-M₆The antibody titer of the expression sample group in the silkworm eukaryotic expression system was 1: more than 512, CSFV E2-Ferritin-C-O-M₆The antibody titers of the expression sample sets in the AcMNPV-insect cell expression system were 1: above 128.

4.2 presentation of foreign genes into mice Using recombinant viruses

4.2.1 presents the form of CSFV E2-Ferritin-C-O-M in mice₆Gene

Purified recombinant virus Bm-CAG CSFV E2-Ferritin-C-O-M₆By tail vein injection (1X 10)¹²vg/mouse) and perfusion (1X 10)¹³vg/mouse) was administered to mice weighing about 25 g. Mouse sera were collected at 5d, 11d, 17d, 21d, respectively, and used with the prokaryotic protein pET-28a-CAP-Ferritin and PCV-III CAP-Ferritin are used as detection target proteins for detecting the titer of the antibody.

5 antibody titer

The neutralizing antibody titer was highest at day 21, see above for the specific experimental procedures, and the specific results are shown in table 10. As can be seen from the data in Table 10, the mutants after the multi-site mutation of the amino acids in the fusion protein presented to mice with better antibody potency than the healthy silkworm pupa control and the conventional vaccine.

TABLE 10 CSFV E2-Ferritin-C-O-M₆Mouse serum antibody titer (21 days)

Composition of	Potency of the drug
		Healthy silkworm pupa control (mouse)	1：4
Traditional vaccine (mouse)	1：256
		CSFV E2-Ferritin-C-O-M₆Mouse serum (injection)	1：512
CSFV E2-Ferritin-C-O-M₆Mouse serum (perfusion)	1：1024

Sequence listing

<110> institute of biotechnology of Chinese academy of agricultural sciences

<120> self-assembly ferritin-based nano antigen particle, swine fever vaccine prepared from same and application

<130> BJ-2002-190804A

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 412

<212> PRT

<213> Artifical sequence

<400> 1

Met Arg Leu Ala Cys Lys Glu Asp Tyr Arg Tyr Ala Ile Ser Ser Thr

1 5 10 15

Asp Glu Ile Gly Leu Leu Gly Ala Gly Gly Leu Thr Thr Thr Trp Lys

20 25 30

Glu Tyr Asn His Asp Leu Gln Leu Asn Gly Gly Thr Val Lys Ala Ser

35 40 45

Cys Val Ala Gly Ser Phe Lys Val Thr Ala Leu Asn Val Val Ser Arg

50 55 60

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

65 70 75 80

Phe Glu Leu Leu Phe Asp Gly Thr Asn Pro Ser Thr Glu Glu Met Gly

85 90 95

Asp Asp Phe Arg Ser Gly Leu Cys Pro Phe Asp Thr Ser Pro Val Val

100 105 110

Lys Gly Lys Tyr Asn Thr Thr Leu Leu Asn Gly Ser Ala Phe Tyr Leu

115 120 125

Val Cys Pro Ile Gly Trp Thr Gly Val Ile Glu Cys Thr Ala Val Ser

130 135 140

Pro Thr Thr Leu Arg Thr Glu Val Val Lys Thr Phe Arg Arg Asp Lys

145 150 155 160

Pro Phe Pro His Arg Met Asp Cys Val Thr Thr Thr Val Glu Asn Glu

165 170 175

Asp Leu Phe Tyr Cys Lys Leu Gly Gly Asn Trp Ala Cys Val Lys Gly

180 185 190

Glu Pro Val Val Tyr Thr Arg Gly Val Val Glu Gln Arg Arg Trp Cys

195 200 205

Gly Phe Asp Phe Asp Gly Pro Asp Gly Leu Pro His Tyr Pro Ile Gly

210 215 220

Lys Cys Ile Leu Ala Asn Glu Thr Gly Tyr Arg Ile Val Asp Ser Thr

225 230 235 240

Asp Cys Asn Arg Asp Gly Ser Gly Gly Asp Ile Ile Lys Leu Leu Asn

245 250 255

Glu Gln Val Asn Lys Glu Met Gln Ser Ser Asn Leu Tyr Met Ser Met

260 265 270

Ser Ser Trp Cys Tyr Thr His Ser Leu Asp Gly Ala Gly Leu Phe Leu

275 280 285

Phe Asp His Ala Ala Glu Glu Tyr Glu His Ala Lys Lys Leu Ile Ile

290 295 300

Phe Leu Asn Glu Asn Asn Val Pro Val Gln Leu Thr Ser Ile Ser Ala

305 310 315 320

Pro Glu His Lys Phe Glu Gly Leu Thr Gln Ile Phe Gln Lys Ala Tyr

325 330 335

Glu His Glu Gln His Ile Ser Glu Ser Ile Asn Asn Ile Val Asp His

340 345 350

Ala Ile Lys Ser Lys Asp His Ala Thr Phe Asn Phe Leu Gln Trp Tyr

355 360 365

Val Ala Glu Gln His Glu Glu Glu Val Leu Phe Lys Asp Ile Leu Asp

370 375 380

Lys Ile Glu Leu Ile Gly Asn Glu Asn His Gly Leu Tyr Leu Ala Asp

385 390 395 400

Gln Tyr Val Lys Gly Ile Ala Lys Ser Arg Lys Ser

405 410

<210> 2

<211> 1239

<212> DNA

<213> Artifical sequence

<400> 2

atgcggctag cctgtaagga agattacagg tacgcaatat cgtcaaccga tgagataggg 60

ctacttgggg ccggaggtct caccaccacc tggaaggaat acaaccacga tttgcaactg 120

aatggcggga ccgtcaaggc cagttgcgtg gcaggttcct ttaaagtcac agcacttaat 180

gtggtcagta ggaggtattt ggcgtcactg cataagaagg ctttacccac ttccgtgaca 240

ttcgagctcc tgttcgacgg gaccaaccca tcaactgagg aaatgggaga tgacttcagg 300

tccgggctgt gcccgtttga tacgagtcct gttgttaagg gaaagtacaa tacgaccttg 360

ttgaacggta gtgccttcta tcttgtctgc ccaatagggt ggacgggtgt catagagtgc 420

acagcagtga gcccaacaac tctgaggaca gaagtggtaa agaccttcag gagagacaag 480

ccctttccgc acagaatgga ttgtgtgacc accacagtgg aaaatgaaga tttattctat 540

tgtaagttgg ggggcaactg ggcatgtgtg aaaggcgagc cagtggtcta cacaaggggg 600

gtagtagaac aacgtagatg gtgtggcttc gacttcgatg ggcctgacgg actcccgcat 660

taccccatag gtaagtgcat tttggcaaat gagacaggtt acagaatagt agattcaacg 720

gactgtaaca gagatggctc cggtggcgac atcatcaagc tgctgaacga acaggtgaac 780

aaggagatgc agtccagcaa cctgtacatg tctatgtctt catggtgcta cacccactca 840

ctggacggag ctggtctgtt cctgttcgac cacgctgccg aggaatacga acacgccaag 900

aagctgatca tcttcctgaa cgagaacaac gtgcctgtcc agctgacctc catcagcgct 960

cccgaacaca agttcgaggg tctgactcaa atcttccaga aggcctacga acacgagcag 1020

cacatctctg aatcaatcaa caacatcgtg gaccacgcta tcaagagcaa ggaccacgcc 1080

actttcaact tcctgcaatg gtacgtggct gagcagcacg aggaagaggt cctgttcaag 1140

gacatcctgg acaagatcga actgatcggc aacgagaacc acggactgta cctggctgac 1200

cagtacgtca agggcatcgc caagtcccgc aagagctaa 1239

<210> 3

<211> 738

<212> DNA

<213> Artifical sequence

<400> 3

atgtgctgca aggcgattaa gttgggtaac gccagggttt tcccagtcac gacgttgtaa 60

aacgacggcc agtgccaagc ttgcatgcct gcaggtcgac gattcgtcga cttagccatc 120

tctgttacag tccgttgaat ctactattct gtaacctgtc tcatttgcca aaatgcactt 180

acctatgggg taatgcggga gtccgtcagg cccatcgaag tcgaagccac accatctacg 240

ttgttctact accccccttg tgtagaccac tggctcgcct ttcacacatg cccagttgcc 300

ccccaactta caatagaata aatcttcatt ttccactgtg gtggtcacac aatccattct 360

gtgcggaaag ggcttgtctc tcctgaaggt ctttaccact tctgtcctca gagttgttgg 420

gctcactgct gtgcactcta tgacacccgt ccaccctatt gggcagacaa gatagaaggc 480

actaccgttc aacaaggtcg tattgtactt tcccttaaca acaggactcg tatcaaacgg 540

gcacagcccg gacctgaagt catctcccat ttcctcagtt gatgggttgg tcccgtcgaa 600

caggagctcg aatgtcacgg aagtgggtaa agccttctta tgcagtgacg ccaaatacct 660

cctactgacc acattaagtg ctgtgacttt aaaggaacct gccacgcaac tggccttgac 720

ggtcccgcca ttcagttg 738

Claims

1. The optimized gene of the homologous sequence of the fusion protein is obtained by connecting swine fever virus envelope E2 protein and a monomeric ferritin subunit, the amino acid sequence of the homologous sequence of the fusion protein is shown in SEQ ID No.1, and the optimized gene is characterized in that the nucleotide sequence of the optimized gene is shown in SEQ ID No. 3.

2. A mutant of a consensus sequence of a fusion protein, which is a single-site mutant obtained by mutating an amino acid sequence represented by SEQ ID NO.1 in any one of N35A, D86R, Y116W, E139F, D167G, and I227D.

3. Mutant variants of the consensus sequence of the fusion protein, characterized by a two-site mutant obtained by two-site mutation of the amino acid sequence shown in SEQ ID No.1 according to any of the positions D86R-Y116W, E139F-D167G or E139F-I227D.

4. The mutant of the homologous sequence of the fusion protein is characterized in that the multi-site mutant is obtained by carrying out multi-site mutation on the amino acid sequence shown in SEQ ID NO.1 according to the N35A-D86R-Y116W-E139F-D167G-I227D.

5. Use of the optimized gene of claim 1 in the preparation of a swine fever vaccine.

6. Use of the mutant of any one of claims 2-4 in the preparation of a vaccine for swine fever.

7. Use according to claim 5, comprising: expressing the optimized gene of claim 1 in a prokaryotic expression system of escherichia coli, collecting and purifying the expressed antigen;

or, expressing the optimized gene of claim 1 in a silkworm expression system or an AcMNPV-insect cell eukaryotic expression system, collecting and purifying the expressed antigen;

or cloning the optimized gene of claim 1 into an expression vector of baculovirus mammal to obtain recombinant baculovirus; recombinant baculoviruses are genetically presented to produce antigens in tissues of vertebrate animals.

8. Use according to claim 7, wherein the optimized gene is cloned into a baculovirus transfer vector to construct a recombinant transfer vector; co-transfecting the recombinant transfer vector and baculovirus DNA into an insect cell to obtain recombinant baculovirus; infecting the recombinant baculovirus into insect host or cell, culturing the infected insect cell or insect host to express corresponding antigen, and purifying to obtain the recombinant baculovirus.

9. Use according to claim 6, comprising: expressing the gene encoding the mutant of any one of claims 2 to 4 in a prokaryotic expression system of E.coli, collecting and purifying the expressed antigen;

or, expressing the gene encoding the mutant of any one of claims 2 to 4 in a silkworm expression system or an AcMNPV-insect cell eukaryotic expression system, collecting and purifying the expressed antigen;

or, cloning the gene encoding the mutant of any one of claims 2 to 4 into an expression vector of baculovirus mammal to obtain recombinant baculovirus; the recombinant baculovirus is gene-presented in tissues in vertebrates to produce antigen.

10. Use according to claim 9, comprising: cloning the coding gene of the mutant of any one of claims 2-4 into a baculovirus transfer vector to construct a recombinant transfer vector; co-transfecting the recombinant transfer vector and baculovirus DNA into an insect cell to obtain recombinant baculovirus; infecting the recombinant baculovirus into insect host or cell, culturing the infected insect cell or insect host to express corresponding antigen, and purifying to obtain the recombinant baculovirus.

11. A hog cholera vaccine comprising an effective amount of the mutant of any one of claims 2-4 and a pharmaceutically acceptable adjuvant or carrier.