A nucleic acid and recombinant protein co-immune vaccine based on swine fever virus gene, preparation method and application

Abstract

The present invention discloses a nucleic acid and recombinant protein co-immune vaccine based on classical swine fever virus gene, a preparation method and an application thereof. The vaccine first obtains an optimized recombinant plasmid and a recombinant classical swine fever virus antigen protein by combining genetic engineering and cell engineering, and then compounds the nucleic acid sequence and the recombinant protein into different liquid phase layers of a biphasic adjuvant. The obtained co-immune vaccine has not only good immunogenicity but also a short immune blank period, and can quickly induce the body to produce high-level, high-affinity, and long-lasting antibody responses and immune memory, effectively protecting target animals from attacks by strong classical swine fever viruses, and can be used to prepare products for preventing / treating classical swine fever.

Description

Technical Field

[0001] The present invention relates to the technical field of vaccine preparation, and in particular to a nucleic acid and recombinant protein co-immune vaccine based on swine fever virus genes, a preparation method and an application thereof. Background Art

[0002] Classical swine fever (CSF) is caused by classical swine fever virus (CSFV), which mainly attacks domestic pigs and wild boars and can cause severe animal infectious diseases with high morbidity and mortality. The World Animal Health Code of the World Organization for Animal Health (OIE) lists it as a Class A epidemic disease, and the Implementation Rules of the Regulations on the Prevention and Control of Livestock and Poultry in my country stipulate that it is a Class I animal infectious disease. Due to its serious harm and wide distribution, it has become one of the hot spots in molecular virology and veterinary epidemic prevention research at home and abroad.

[0003] CSFV belongs to the Flaviviridae family and the Pestivirus genus. It is closely related to the bovine viral diarrhea virus (BVDV), sheep border disease virus (BDV), and giraffe plague virus of the same genus in terms of virus structure, antigenicity, and genetic characteristics. The genome of classical swine fever virus is a single-stranded positive-strand linear RNA of about 12.3 to 12.5 kb, consisting of an open reading frame and two short 5' and 3' non-coding regions on the flanks. Protective antibodies against E0 and E2 can be produced in infected animals, but the E0 protein-specific antibodies have a poor ability to neutralize classical swine fever virus. So far, antibodies against E1 glycoprotein have not been detected in naturally infected host animals, so E2 protein is the first choice for subunit vaccines.

[0004] CSFV envelope glycoprotein E2 (gpS5) is the main antigenic protein that induces the body to produce neutralizing antibodies and stimulate protective immune responses. Its interaction with cell surface receptors mediates the infection process of CSFV to cells.

[0005] Since 1950, my country has used live classical swine fever vaccine (rabbit-attenuated strain, abbreviated as HCLV strain or C strain) for large-scale immunization to control the large-scale epidemic of classical swine fever. However, the epidemic of the disease has also changed from the past characteristics of rapid onset, rapid spread, high morbidity and mortality to mild onset, atypical symptoms, low morbidity and mortality, and regional, sporadic and cyclical epidemics. In particular, chronic, atypical classical swine fever caused by latent infection and immune tolerance is an important form of clinical manifestation of classical swine fever in my country, posing a serious threat to the healthy development of the pig industry.

[0006] Studies have shown that under the high pressure of universal immunization with the C-strain live classical swine fever vaccine, the classical swine fever virus is undergoing escape mutations in the direction opposite to the protective effect of the vaccine, and the immune protection effect of the C-strain vaccine against the epidemic strains of the classical swine fever 2.1 subgroup is not ideal after vaccination.

[0007] In addition, commercially available live swine fever vaccines such as spleen lymph vaccine, bovine testicular cell vaccine, ST passage cell vaccine, etc. have the following disadvantages: (1) The production process is easily contaminated by exogenous viruses such as BVDV; (2) The virus content is titrated by using infected live rabbits to determine the rabbit infection dose. The test method is unscientific and is affected by the individual differences of the test rabbits, resulting in large batch differences in the vaccine and unstable quality; because it is a freeze-dried live vaccine, the temperature requirements during transportation, storage, and use are very strict, and the potency is easily reduced, affecting the immune effect; it is greatly interfered by maternal antibodies and has low protection for nursery pigs; it is easy to produce immune tolerance, affecting group immunity; it is impossible to perform differential diagnosis from wild virus-infected pigs, which increases the difficulty of purifying swine fever.

[0008] In view of this, the market is in urgent need of a new type of swine fever vaccine that has a broad protection spectrum, can solve immune tolerance problems, and is capable of differential diagnosis and elimination and purification.

[0009] In recent years, many domestic institutions have begun to develop recombinant subunit vaccines based on the E2 protein of classical swine fever virus. For example, the E2 subunit vaccine of Tiankang Biotechnology was successfully launched in 2017. Due to its high E2 protein content, the recombinant subunit vaccine can induce pigs to produce high levels of anti-CSFV neutralizing antibodies and can effectively protect against the prevalent strains of classical swine fever virus subgroups 1.1, 2.1, and 2.2. However, the vaccine cannot induce the body to produce good cellular immunity and has a long immune blank period, so it cannot be used for emergency immunization and treatment.

[0010] Nucleic acid vaccine, also known as gene vaccine, refers to a new type of vaccine that introduces a plasmid vector containing the gene sequence of the target protein into the host through intramuscular injection or microprojectile bombardment, and expresses the antigen protein through the host cells, inducing the host cells to produce an immune response to the antigen protein, so as to achieve the purpose of preventing and treating diseases. Studies have shown that nucleic acid vaccines not only have good immunogenicity and safety, but also because nucleic acids are easier to modify and transform, nucleic acid vaccines have greater flexibility than previous protein vaccines. In actual production, the preparation and purification process of nucleic acid vaccines is relatively simple, the production cycle is short, the production cost is low, and it is easy to make multivalent vaccines. Therefore, it is very likely to become the next new vaccine successfully applied to humans.

[0011] Nucleic acid vaccines mainly include DNA vaccines, viral vector vaccines and RNA vaccines, which are mainly composed of highly purified DNA or RNA fragments encoding specific antigenic proteins with appropriate buffers and adjuvants. The mechanism of action of nucleic acid vaccines is very different from that of traditional vaccines with antigenic proteins as active ingredients: after the body is vaccinated, the DNA or RNA fragments in the vaccine first enter the body's cells and use the host cell protein synthesis system to synthesize antigenic proteins. The antigenic proteins synthesized in situ are captured by immune cells after release, thereby stimulating an immune response.

[0012] Compared with traditional vaccines, nucleic acid vaccines have incomparable advantages. Nucleic acid vaccines can induce the body to produce both cellular immunity and humoral immunity, mainly through MHC class I and class II molecular pathways and B cell pathways to achieve immune protection. 8+ T cells, produce specific CTL effector-mediated cellular immunity; activate CD4+ T cells through MHC class II molecules 4+ T cells secrete cytokines such as IL-2, INF-γ, TNF-β, etc. to assist the body in producing specific humoral immunity; they stimulate and activate B cells through the B cell pathway, induce B cell proliferation and differentiation, produce a large number of specific antibodies, and induce humoral immune response.

[0013] Nucleic acid vaccines have the following advantages: (1) Good immune effect, inducing both cellular immunity and humoral immunity, and being more effective against chronic viral infections that rely on cellular immunity; (2) Good safety, low immune side effects, and containing only part of the pathogen genome sequence, without the risk of causing epidemics due to virulence reversion or residual virulent virus particles; (3) After vaccination, the protein antigens expressed by nucleic acid vaccines in host cells are similar to those in natural infection, so that the antigens are presented to the immune system in a natural conformation; (4) Easy to process, modify and transform, optimize the target gene in a timely manner according to the characteristics of the virus epidemic, select the dominant antigen epitope, and have a short construction cycle; (5) Simple production technology, low manufacturing cost, more economical than other vaccines; (6) Stable chemical properties, easy to store and transport, nucleic acid vaccines can be processed into dry small particles for easy storage and transportation, and dry DNA small particles are relatively stable at room temperature and do not require refrigeration equipment, so they are also more convenient for use in remote areas; (7) Convenient for combined immunization, that is, genes encoding different antigens can be constructed in the same plasmid or multiple plasmids of different antigen genes can be used in combination to prepare multivalent nucleic acid vaccines.

[0014] At present, nucleic acid vaccines have made great research progress in the prevention and treatment of various diseases in humans and animals. More than 10 nucleic acid vaccines such as hepatitis B vaccine have entered clinical trials (approved by the US FDA), and multiple viral nucleic acid vaccines (such as AIDS) have entered the preclinical stage. Multiple tumor nucleic acid vaccines (prostate cancer, lung cancer, breast cancer, etc.) are also in the research stage. However, the current immunity of nucleic acid vaccines is short-lived and the immune response is weak, which greatly limits the commercial application and promotion of this technology and needs to be further improved.

[0015] In view of this, the present invention is proposed. Summary of the invention

[0016] The purpose of the present invention is to provide a nucleic acid and recombinant protein co-immune vaccine based on classical swine fever virus genes. The vaccine is obtained by compounding nucleic acid and recombinant protein into different liquid phase layers of a biphasic adjuvant. The vaccine has a short immune blank period and can quickly induce the body to produce high-level, high-affinity, long-lasting antibody response and immune memory.

[0017] The present invention also provides a method for preparing a co-immune vaccine based on nucleic acid and recombinant protein of classical swine fever virus gene. The method successfully distributes the recombinant plasmid and recombinant classical swine fever virus antigen protein in different liquid phase layers by adopting a biphasic adjuvant, thereby realizing the control of the activation sequence of humoral immunity and cellular immunity in the body.

[0018] The present invention also provides the use of a nucleic acid and recombinant protein co-immune vaccine of a classical swine fever virus gene in the preparation of a product for preventing / treating classical swine fever. In the application, the nucleic acid and recombinant protein co-immune vaccine can cause the body to produce IFN-γ and / or IL-4 cytokines, and induce the body to secrete antibodies, and is particularly suitable for emergency immunization and prevention and control of classical swine fever.

[0019] The present invention is achieved in that:

[0020] The present invention provides a nucleic acid and recombinant protein co-immune vaccine based on the gene of classical swine fever virus, the co-immune vaccine comprises a biphasic adjuvant, and nucleic acid and recombinant protein distributed in different liquid phase layers of the biphasic adjuvant, the nucleic acid comprises a sequence encoding a binding site of classical swine fever virus antigen protein, and the recombinant protein comprises an amino acid sequence of the binding site of classical swine fever virus antigen protein. The binding site refers to an active site with immunogenicity in the classical swine fever virus antigen protein, which can induce an immune response in the body and endow the classical swine fever virus antigen protein with immunogenicity.

[0021] Classical swine fever virus antigen proteins mainly include E0, E1 and E2. Antibodies against E0 and E2 proteins have been detected in naturally infected host animals. Therefore, the above-mentioned classical swine fever virus antigen proteins include E2 and / or E0 proteins, among which E2 protein-specific antibodies have a stronger ability to neutralize classical swine fever virus.

[0022] The protein sequence encoded by the above nucleic acid sequence and the above recombinant protein correspond to the two antigen proteins E2 and E0 respectively. For example, the above nucleic acid sequence is derived from the E2 protein, and the above recombinant protein is derived from the E0 protein; or, the protein sequence encoded by the above nucleic acid sequence can correspond to the above recombinant protein. For example, the above nucleic acid sequence and the recombinant protein are both derived from the E2 protein. At this time, the antigens of the obtained co-immune vaccine that cause humoral immune response and cellular immune response are consistent, and the immune response effect obtained in the body is also more stable.

[0023] Preferably, the mass ratio of the nucleic acid sequence to the recombinant protein in the nucleic acid and recombinant protein co-immune vaccine is 1:(1.5-4.5).

[0024] Preferably, the mass ratio of the nucleic acid to the recombinant protein is 1:(2-4); more preferably, it is 1:2.5.

[0025] Preferably, the biphasic adjuvant comprises an aqueous phase layer, and the aqueous phase layer has at least two layers.

[0026] Preferably, the composition of the biphasic adjuvant includes any one of the following:

[0027] (a) at least one water-in-oil-in-water adjuvant;

[0028] (b) at least two water-in-oil adjuvants;

[0029] (c) at least one water-in-oil-in-water adjuvant and at least one water-in-oil adjuvant.

[0030] Preferably, the biphasic adjuvant comprises at least one of ISA 201VG, ISA 563VG, ISA 206VG or ISA 660VG, wherein the water-in-oil-in-water adjuvant ISA 201VG and the water-in-oil adjuvant ISA 563VG are used together to form a homogeneous and stable emulsion with good sustained release.

[0031] Preferably, the nucleic acid and the recombinant protein are distributed in different aqueous phase layers, respectively.

[0032] Since nucleic acid sequences and recombinant proteins have higher solubility in the aqueous phase, in the scheme that can achieve the distribution of nucleic acids and recombinant proteins in different liquid phase layers, the simplest liquid phase layer composition should be a three-layer structure of water-in-oil-in-water. Therefore, the above-mentioned biphasic adjuvant includes at least one of a water-in-oil-in-water adjuvant and an oil-in-water adjuvant, and includes at least two aqueous phase layers.

[0033] The present invention also provides a method for preparing a nucleic acid and recombinant protein co-immune vaccine based on classical swine fever virus genes, the method comprising respectively compounding the nucleic acid encoding the classical swine fever virus antigen protein binding site and the recombinant protein into different liquid phase layers of a biphasic adjuvant to obtain a nucleic acid and recombinant protein co-immune vaccine.

[0034] The above nucleic acid includes a recombinant plasmid constructed with a nucleic acid sequence encoding a classical swine fever virus antigen protein as the target gene.

[0035] Preferably, the recombinant plasmid includes a nucleic acid sequence encoding a binding site for the classical swine fever virus antigen protein E2 and / or E0 protein, and further, may include other gene sequences that enable the nucleic acid sequence encoding the active site for the classical swine fever virus antigen protein E2 and / or E0 protein to be smoothly expressed and translated; the above-mentioned recombinant protein includes an amino acid sequence for a binding site for the classical swine fever virus antigen protein E2 and / or E0 protein, and further, may include other auxiliary amino acid sequences, so that the classical swine fever virus antigen protein E2 and / or E0 protein has the properties of maintaining activity stability, being easy to separate and purify, or enhancing immunogenicity.

[0036] Preferably, a eukaryotic expression plasmid is selected for the construction of the recombinant plasmid. More preferably, plasmid pcDNA3.1 is selected for the construction of the recombinant plasmid. Plasmid pcDNA3.1 has the characteristics of high expression level and good compatibility, so that the recombinant E2 gene can be expressed efficiently.

[0037] Preferably, the above preparation method also includes, before constructing the recombinant plasmid, first performing codon modification on the nucleic acid sequence encoding the classical swine fever virus antigen protein to form a recombinant nucleic acid sequence.

[0038] The reason for the need to modify the codons is that there are new mutations in the circulating classical swine fever virus strains, and the recombinant plasmid constructed only based on the antigen protein gene sequence published on GenBank cannot guarantee that the body can produce an immune response against the circulating strains.

[0039] Therefore, it is necessary to use the published antigen protein gene sequences downloaded from GenBank and the antigen protein gene sequences of the epidemic strains as references to perform codon modification on the nucleic acid sequence encoding the classical swine fever virus antigen protein.

[0040] Preferably, the modification also includes inserting a signal peptide sequence at the 5' end of the nucleic acid sequence encoding the classical swine fever virus antigen protein. The signal peptide can guide the synthesized recombinant protein to be secreted extracellularly, which is beneficial to the separation and purification of the recombinant protein. At the same time, after mediating the transfer of ribosomes to the endoplasmic reticulum, the signal peptide can be automatically cut off from the recombinant protein and will not affect the activity of the recombinant protein.

[0041] And / or, the modification also includes removing the 3'-end transmembrane region sequence fragment of the nucleic acid sequence encoding the classical swine fever virus antigen protein. The transmembrane region of the protein is usually hydrophobic. After removing the 3'-end transmembrane region sequence fragment, the obtained recombinant protein does not contain a hydrophobic polypeptide segment, which is more conducive to the recombinant protein being compounded into the aqueous phase.

[0042] And / or, the modification also includes inserting a CpG sequence at the 3' end of the nucleic acid sequence encoding the classical swine fever virus antigen protein; preferably, the CpG sequence includes CpG19 and CpG2395 sequences, and the dimer structure of multiple glycosylation sites expressed by the CpG19 and CpG2395 sequences can enhance the immunogenicity of the classical swine fever virus antigen protein.

[0043] Preferably, the above modification also includes inserting a histidine tag at the 3' end of the nucleic acid sequence encoding the classical swine fever virus antigen protein. This histidine peptide segment can chelate with divalent metal ions (nickel, zinc, etc.), facilitating protein purification by metal chelate affinity chromatography.

[0044] Preferably, the modification further comprises inserting a Kozak sequence before the 5' end signal peptide sequence of the nucleic acid sequence encoding the classical swine fever virus antigen protein, wherein the Kozak sequence is located at the 5' end of the recombinant nucleic acid sequence and binds to the translation initiation factor to mediate the translation initiation of the mRNA containing the 5' cap structure.

[0045] Preferably, the Kozak sequence is GCCACC.

[0046] Preferably, the recombinant nucleic acid sequence is as shown in SEQ ID No. 1, wherein positions 1-6 are Kozak sequences, positions 7-72 ​​are signal peptide sequences, positions 73-1110 are codon-modified, truncated E2 genes, positions 1111-1278 are CpG sequences, and positions 1279-1296 are His tag sequences.

[0047] Preferably, restriction sites are inserted at both ends of the recombinant nucleic acid sequence to facilitate the construction of the recombinant plasmid.

[0048] Preferably, the method for preparing the nucleic acid and recombinant protein co-immune vaccine based on classical swine fever virus genes also includes transfecting the recombinant plasmid into a cell line to express an expression product including a recombinant protein and a recombinant plasmid, and then separating and purifying the recombinant protein and recombinant plasmid in the product respectively.

[0049] Preferably, the cell line used for transfection comprises CHO cells.

[0050] Preferably, the CHO cells include CHO-S or CHO-K1 cells.

[0051] Preferably, the transfection method comprises liposome transfection.

[0052] Preferably, chromatography is used to separate and purify the recombinant protein.

[0053] Preferably, the recombinant plasmid is separated and purified using a kit extraction method.

[0054] Preferably, the method of separately compounding the nucleic acid encoding the classical swine fever virus antigen protein binding site and the recombinant protein into different liquid phase layers of a biphasic adjuvant comprises selecting different biphasic adjuvants to construct at least two compounding systems, respectively compounding the obtained nucleic acid and recombinant protein into aqueous phase layers of biphasic adjuvants of different compounding systems, and then mixing the different compounding systems so that the nucleic acid and the recombinant protein are distributed in different liquid phase layers.

[0055] Preferably, after the different composite systems are mixed, a composite system comprising at least two aqueous phase layers is obtained.

[0056] Preferably, the nucleic acid and the recombinant protein are partitioned into different aqueous layers.

[0057] Preferably, the nucleic acid is located in the outer aqueous phase layer, and the recombinant protein is located in the inner aqueous phase layer.

[0058] Preferably, the nucleic acid comprises a recombinant plasmid constructed with a nucleic acid sequence encoding a classical swine fever virus antigen protein as the target gene.

[0059] Preferably, the recombinant protein is expressed by the recombinant plasmid.

[0060] Preferably, the two compound systems include a nucleic acid compound system and a recombinant protein compound, the mass content of nucleic acid in the nucleic acid compound system is 30-60 μg / g biphasic adjuvant, and the mass content of recombinant protein in the recombinant protein compound system is 80-140 μg / g biphasic adjuvant;

[0061] Preferably, the nucleic acid complex system and the recombinant protein complex are mixed in equal amounts.

[0062] The use of the above-mentioned co-immune vaccine or the co-immune vaccine prepared by the above-mentioned method in the preparation of products for preventing / treating classical swine fever.

[0063] Preferably, the application implements at least one of the functions AB:

[0064] A. Causes the body to produce IFN-γ.

[0065] B. Causes the body to produce IL-4 cytokine.

[0066] Preferably, the co-immune vaccine can also be used for emergency immunization and treatment when classical swine fever occurs in a pig farm.

[0067] The present invention has the following beneficial effects:

[0068] The nucleic acid and recombinant protein co-immune vaccine based on classical swine fever virus genes provided by the present invention is a co-immune vaccine obtained by compounding the nucleic acid sequence and the recombinant protein into different liquid phase layers of a biphasic adjuvant. The vaccine has not only good immunogenicity but also a short immune blank period. It can quickly induce the body to produce high-level, high-affinity, long-lasting antibody response and immune memory, and effectively protect target animals against attacks by strong classical swine fever viruses.

[0069] The present invention also provides a method for preparing a co-immune vaccine of nucleic acid and recombinant protein based on the classical swine fever virus gene. The method combines genetic engineering and cell engineering, and by adopting a biphasic adjuvant, successfully distributes the nucleic acid and recombinant protein in different liquid phase layers, thereby achieving control of the activation sequence of humoral immunity and cellular immunity in the body, so that the vaccine not only has an excellent preventive effect, but can also be used for emergency immunization and prevention and control of classical swine fever.

[0070] The present invention provides an application of a nucleic acid and recombinant protein co-immune vaccine of a classical swine fever virus gene in the preparation of a product for preventing / treating classical swine fever. In the application, the nucleic acid and recombinant protein co-immune vaccine can cause the body to produce IFN-γ and / or IL-4 cytokines and induce the body to secrete antibodies, and is particularly suitable for emergency immunization and prevention and control of classical swine fever.

[0071] The co-immune vaccine provided by the present invention integrates the advantages of nucleic acid vaccines and subunit vaccines, solves the problems existing in the clinical application of nucleic acid vaccines and subunit vaccines, and has the following characteristics: (1) when testing the quality of the vaccine, a nucleic acid quantifier and liquid chromatography technology can be used to quantify the effective ingredients in the vaccine, the quantitative method is scientific, the repeatability is good, and the difference between vaccine batches is small; (2) the nucleic acid and the recombinant protein have good stability, and can be stored at 2-8°C for 18 months after being emulsified with a biphasic adjuvant, and the vaccine has a stable potency and a long shelf life; (3) it is not interfered by maternal antibodies and has no immune tolerance problems; (4) it has the advantages of both nucleic acid vaccines and subunit vaccines, with a rapid immune response, a short immune blank period, and a long immune duration, and can be used as an emergency immune vaccine for disease prevention and control; (5) after immunizing the target animal, cellular immunity and humoral immunity can be generated simultaneously, and the protective efficacy is high; (6) it can be equipped with an antibody detection kit for swine fever virus antigen protein for differential diagnosis, and can distinguish wild virus-infected pigs from immune pigs, thereby eliminating wild virus-infected pigs and purifying swine fever in pig farms. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0073] Figure 1This is a flow chart of the method for preparing the co-immune vaccine provided in Example 4;

[0074] Figure 2 The homology analysis result of the optimized recombinant E2 gene sequence provided in Example 4;

[0075] Figure 3 The phylogenetic tree of the optimized recombinant E2 gene sequence and different subgroups of classical swine fever virus strains provided in Example 4;

[0076] Figure 4 The electrophoresis diagram of the products of three DNA sequence amplifications in the E2 gene optimization process in Example 4;

[0077] Figure 5 is the map of the pcDNA3.1-CSFV-E2 plasmid provided in Example 1;

[0078] Figure 6 This is the electrophoresis diagram after double restriction digestion of the pcDNA3.1-CSFV-E2 plasmid provided in Example 4, wherein 1 is the pcDNA3.1-CSFV-E2 plasmid, 2 is the pcDNA3.1 empty vector, and 3 is the modified E2 gene;

[0079] Figure 7 The results of SDS-PAGE and Western blot detection of the recombinant E2 protein provided in Example 4 after harvesting and purification denaturation, wherein lane 1 is before purification, lane 2 is the flow-through sample, and lane 3 is the purified sample;

[0080] Figure 8 The results of non-denaturing-PAGE and Western blot detection of the recombinant E2 protein purified in Example 4;

[0081] Fig. 9 The results of SDS-PAGE and Western blot detection of the recombinant E2 protein purified in Example 4 before and after deglycosylation, lane 1 is before deglycosylation, and lane 2 is after deglycosylation;

[0082] Fig.10 The electrophoresis detection results after the plasmid extract was amplified in Example 4, wherein lane 1 is the amplified product and lane 2 is the negative control;

[0083] Fig.11 The results of the determination of neutralizing antibodies against classical swine fever virus in mouse serum in Example 4;

[0084] Fig.12 The results of the IFN-γ assay in mouse serum in Example 4;

[0085] Fig.13The results of the IL-4 cytokine assay in mouse serum in Example 4;

[0086] Fig.14 The results of the determination of CD4+ and CD8+ lymphocyte subsets in the spleen of mice in Example 4;

[0087] Fig.15 The results of body temperature measurement of the target animals after the first immunization in Example 4;

[0088] Fig.16 The results of the determination of the neutralizing antibody titers in the serum of target animals in different immunization groups in Example 4;

[0089] Fig.17 The results of the determination of the neutralizing antibody titer in the serum of the target animals in the CCP vaccination group in Example 4;

[0090] Fig.18 The results of measuring the body temperature of target animals after the first immunization with different adjuvant co-immunization vaccines in Example 4;

[0091] Fig.19 These are the results of determining the neutralizing antibody titers in the serum of target animals after the first immunization with the co-immunization vaccines with different adjuvants in Example 4. DETAILED DESCRIPTION

[0092] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0093] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0094] Example 1

[0095] This embodiment provides a nucleic acid and recombinant protein co-immune vaccine based on classical swine fever virus gene, wherein the co-immune vaccine is a water-in-oil-in-water emulsion formed by compounding 1 g of biphasic adjuvants ISA 201VG and ISA 563VG, containing 20 μg of recombinant plasmid pcDNA3.1-CSFV-E2 and 50 μg of recombinant protein, wherein the recombinant protein is located in the inner aqueous phase, and its amino acid sequence is shown in SEQ ID No. 2, and the recombinant plasmid pcDNA3.1-CSFV-E2 is located in the outer aqueous phase, and the recombinant plasmid pcDNA3.1-CSFV-E2 map is shown in Figure 5As shown, a recombinant plasmid is formed by using plasmid pcDNA3.1 as a vector and integrating a gene sequence encoding a recombinant protein of an E2 binding site, wherein the gene sequence encoding a recombinant protein of an E2 binding site is shown in SEQ ID No.1.

[0096] Example 2

[0097] The difference between this example and Example 1 is only that the contents of the recombinant plasmid pcDNA3.1-CSFV-E2 and the recombinant protein are different. This example contains 15 μg of the recombinant plasmid pcDNA3.1-CSFV-E2 and 40 μg of the recombinant protein.

[0098] Example 3

[0099] The difference between this example and Example 1 is only that the contents of the recombinant plasmid pcDNA3.1-CSFV-E2 and the recombinant protein are different. This example contains 30 μg of the recombinant plasmid pcDNA3.1-CSFV-E2 and 70 μg of the recombinant protein.

[0100] The recombinant plasmids and recombinant proteins of the co-immune vaccine provided in the above three groups of embodiments are distributed in different aqueous phase layers inside and outside, which can control the activation sequence of humoral immunity and cellular immunity in the body, so that the vaccine not only has an excellent preventive effect, but can also be used for emergency immunization and prevention and control of swine fever.

[0101] Example 4

[0102] This example provides a method for preparing the nucleic acid and recombinant protein co-immune vaccine described in Example 1, and the flow chart is as follows: Figure 1 As shown, the specific steps include:

[0103] 1. Construction of recombinant plasmid

[0104] 1.1 Target gene optimization and synthesis

[0105] The published E2 gene sequences of classical swine fever virus and the E2 gene sequences of new strains prevalent in recent years were downloaded from GenBank for reference. Codon modification was performed on the basis of the E2 gene of strain C (the reference gene sequences include GenBank gene numbers: AY775178, AF333000, Z46258, AY805221, AF099102, and X87939), and a Kozak sequence (GCCACC) and a signal peptide sequence (SEQ ID No. 3) were added to the 5' end, part of the transmembrane region fragment was removed from the 3' end, and a His tag consisting of 6 histidines (SEQ ID No. 4) and CpG19 and CpG2395 sequences (SEQ ID No. 5) were connected to obtain SEQ The recombinant nucleic acid sequence shown in ID No. 1, positions 1-6 are Kozak sequences, positions 7-72 ​​are signal peptide sequences, positions 73-1110 are modified and truncated E2 genes (denoted as CSFV-HC), positions 1111-1278 are CpG sequences, positions 1279-1296 are His tag sequences, and finally restriction sites are added at both ends.

[0106] Protein structure prediction and analysis showed that the histidine and CpG sequences connected to the 3' end did not affect the synthesis and secretory expression of the E2 protein, and had little effect on the hydrophilicity, hydrophobicity and charge of the E2 protein.

[0107] The modified sequence was sent to Nanjing GenScript Company for synthetic gene sequence synthesis. After obtaining the synthetic gene, primers were designed to amplify the target gene, and sequencing verification was performed. The E2 gene sequence (CSFV-HC) in the measured target gene was compared with the E2 gene of the C strain and the reference strains of each subgroup. The results showed that the nucleotide homology of the E2 gene fragment finally obtained and the E2 gene of the C strain reached 94.64%. Figure 2 As shown in Figure 1, the amino acid sequence homology is 94.22%. Through cluster analysis, the phylogenetic tree of the final synthesized recombinant E2 protein gene (CSFV-HC) and different subgroups of swine fever virus strains was obtained, as shown in Figure 1. Figure 3 As shown in the figure, it can be seen that the synthesized recombinant E2 protein gene belongs to gene 1.1 subgroup.

[0108] PCR primers:

[0109] E2-F1:5'-CTGTCCTCTCAATACCATGGTGGC-3';

[0110] E2-R1:5'-CAGGGGCATCACCATCACCATCAC-3'.

[0111] The full length of the E2 gene sequence of strain C is 1119 bp, as shown in SEQ ID No.6.

[0112] The amino acid sequence encoded by the E2 gene of strain C includes 373AA, as shown in SEQ ID No.7.

[0113] The codon degeneracy optimized and truncated E2 gene sequence is 1038 bp in length, as shown in SEQ ID No.8.

[0114] The amino acid sequence encoded by the truncated E2 gene after codon degeneracy optimization includes 346AA, as shown in SEQ ID No.9,

[0115] After the synthesized SEQ ID No.6, SEQ ID No.1 and SEQ ID No.8 were diluted to 10 ppm / μl respectively, 2 μl was taken as template, and PCR reaction was carried out with corresponding primers and PCR reaction mixture. The amplification results were as follows Figure 4 As shown, lanes 1, 2 and 3 are electrophoresis bands of SEQ ID No.6, SEQ ID No.1 and SEQ ID No.8, respectively. It can be seen that the E2 gene sequence of strain C, the recombinant E2 gene sequence of this embodiment and the gene sequence of the modified E2 gene of strain C in this embodiment can be distinguished by electrophoresis, proving that the recombinant E2 gene sequence of this embodiment is successfully synthesized.

[0116] 1.2 Construction of recombinant plasmid pcDNA3.1-CSFV-E2

[0117] The recombinant E2 gene sequence synthesized in 1.1 was double-digested with HindⅢ and XhoⅠ, and then cloned and ligated into the pcDNA3.1(-) vector under the action of T4 ligase. The basic information of the vector is shown in Table 1. The recombinant plasmid pcDNA3.1-CSFV-E2 was constructed as follows: Figure 5 shown.

[0118] Table 1 Basic information of pcDNA3.1-CSFV-E2 vector

[0119]

[0120] 1.3 Recombinant plasmid transformation and identification

[0121] The pcDNA3.1-CSFV-E2 plasmid was transformed into DH5α competent cells by heat shock method, and evenly spread on LB agar plate containing ampicillin. After culturing at 37℃ for 12-16h, a single monoclonal colony was picked and inoculated into 5ml ampicillin-resistant LB liquid culture medium. The bacteria were cultured at 37℃ and 200rpm overnight, and the bacteria were collected by centrifugation and the recombinant plasmid was extracted for double enzyme digestion and electrophoresis verification. The results are as follows: Figure 6As shown, lane 1 is the recombinant plasmid after double enzyme digestion, lane 2 is the original plasmid pcDNA3.1, and lane 3 is the recombinant E2 gene sequence. It can be seen that the pcDNA3.1-CSFV-E2 plasmid was successfully obtained in this example. The plasmid with correct enzyme digestion was sent to Bao Biotechnology (Dalian) Co., Ltd. for sequencing verification, and the sequencing result was consistent with SEQ ID No.1.

[0122] 1.4 Recombinant plasmid transfection cells and drug pressure screening

[0123] The liposome transfection method was used to transfect the high-concentration, endotoxin-free eukaryotic expression recombinant plasmid into the host cell CHO-S cell line. The steps are as follows: (1) Take the positive clone verified by sequencing and inoculate it into a liquid culture medium containing ampicillin resistance, and shake the bacteria at 37°C overnight. Collect the bacteria by centrifugation, extract the plasmid with an endotoxin-free plasmid extraction kit, detect it by electrophoresis and determine the concentration, and then store it at -80°C for later use. (2) According to the instructions of Lipofectamine3000 of Thermofish Company, mix the recombinant plasmid pcDNA3.1-CSFV-E2 and the transfection reagent and incubate them; (3) Add the above mixture to 30ml CHO-S suspension cells (3×10 5 After 1-2 days of culture, transfection was performed, and the cell viability was about 95%. After 18-24 hours, the culture medium was replaced with 0.6 mg / ml geneticin (G418) for drug pressure screening. The cells were subcultured every 48-72 hours until the cell viability recovered to more than 85%.

[0124] 1.5 Screening of stable and high-yielding cell lines

[0125] When the suspension transfected cell pool is subcultured until the cell viability is greater than 85% and the expression level is stable, monoclonal cells are selected by limiting dilution or semi-solid culture medium combined with Clonepix. Monoclonal plated cells are placed in a 37°C, 5% CO2 incubator and cultured normally for about 15 days. When the cells are about 80% filled with 96 wells, 100μl of supernatant is taken and sent for ELISA; according to the ELISA test results, cells with positive OD values ​​and high expression levels are selected for expansion culture, and then Batch and Feed-Batch are used to test their protein production, and the cell line CHO-E2-5B11 with stable growth and good expression is selected, and a large number of frozen libraries are built, and small-scale process amplification is carried out.

[0126] 2. Preparation of nucleic acid-protein co-immune vaccine

[0127] 2.1 Feed batch of high-yield cell lines

[0128] After the high-yield cell line CHO-E2-5B11 was revived, it was subcultured for 3 to 5 generations in a medium containing G418. 6cells / mL were inoculated into an appropriate amount of culture medium without G418, and a feed batch was performed in a cell shake flask to express the recombinant E2 protein; the cell shake flask was placed in a constant temperature shaker at 37°C and 5% carbon dioxide at 100rpm, and samples were taken every 24 hours to determine the cell density and viability, and a blood glucose meter was used to determine the sugar concentration of the system. When the sugar was lower than 3g / L, glucose was added to 6g / L; on the 4th day of culture, the temperature was lowered from 37°C to 33°C, and Feed B was supplemented to 10% of the culture volume, and 2% CD Feed was added on the 4th, 6th, 8th, 10th, and 12th days of culture.

[0129] 2.2 Harvest and purification of recombinant E2 protein

[0130] On the 14th day of culture of the feed batch, the cell culture medium was collected aseptically, centrifuged at 8000g for 30 min, the supernatant was filtered through a 0.45μm filter, and protein was purified using a nickel column (the target gene has a His tag). The steps are as follows:

[0131] (1) Column equilibration: Take 4 mL of nickel affinity filler and equilibrate with Buffer A (20 mmol / L NaH2PO4, 500 mmol / L NaCl, 0.05% Tween 20, pH 7.4) for 2 to 3 column volumes at 5 ml / min;

[0132] (2) Loading: Load the cell supernatant onto the chromatography column at a rate of 1 ml / min and leave it on the column for 5 min;

[0133] (3) Washing: Wash the column with Buffer A containing 20 mmol / L imidazole at a flow rate of 2 ml / min to remove impurities until the OD280nm baseline is stable;

[0134] (4) Elution: Use Buffer A containing 250 mmol / L imidazole to elute the target protein at 2 ml / min until the baseline is reached, and collect the effluent; then use Buffer A containing 500 mmol / L imidazole for a second elution, and collect the effluent;

[0135] (5) Dialysis solution exchange: Pour the imidazole eluate containing the target protein into a dialysis bag and dialyze it 50 to 100 times with 1× PBS. Finally, take 100 μL, dilute it 500 to 1000 times, and then detect it using SDS-PAGE and Western blot.

[0136] Depend on Figure 7 It can be seen that the recombinant E2 protein expressed by the CHO-S system has a high content and few impurities. The purity after nickel affinity chromatography is not less than 95%, and the detected concentration is 0.8-1 mg / ml.

[0137] The purified recombinant E2 protein was subjected to non-denaturing PAGE and Western blot detection. The results of non-denaturing gel electrophoresis showed that the protein was a dimeric protein (about 95 kDa) in its natural state. For details, see Figure 8 The recombinant E2 protein was added with glycosidase and reacted at 37°C for 4 hours. The fragments on SDS-PAGE became smaller, proving that the recombinant protein had glycosylation sites and could be deglycosylated by glycosidase. Fig. 9 shown.

[0138] 2.3 Extraction of eukaryotic expression recombinant plasmid

[0139] The cell culture precipitate was resuspended in a solution containing 250 mM sucrose, 1 mM EDTA, and 10 mM Tris-HCl. The plasmid DNA was extracted using an endotoxin-removing plasmid extraction kit, and the nucleic acid concentration was quantified using an ultramicro spectrophotometer (NanoDrop2000, USA). The nucleic acid concentration was 1136 ± 233 ng / μl. The plasmid extract was amplified by PCR, and the obtained product was detected by electrophoresis. The results proved that the recombinant plasmid containing the recombinant E2 protein gene was successfully extracted, as shown in Figure 2 . Fig.10 shown.

[0140] 2.4 Preparation of co-immunization vaccines

[0141] Use nuclease-free water to dilute the recombinant plasmid into an aqueous phase with a content of 80 μg / ml, then measure 1 mL from it and mix it with 1 g ISA 201VG adjuvant for vaccination. Under the temperature-controlled condition of 30°C, stir at 150-200 r / min for 30-60 min until the antigen and adjuvant are fully mixed and emulsified into a two-phase water-in-oil-in-water emulsion with a recombinant plasmid concentration of 40 μg / g ISA 201VG.

[0142] The recombinant E2 protein was diluted with sterile PBS to an aqueous phase with a content of 200 μg / ml, and then 1 mL was measured and mixed with 1 g ISA 563VG adjuvant for vaccine preparation. Under the temperature-controlled condition of 25°C, a high-speed homogenizer was used for high-speed shearing at 12600 r / min for 5 to 8 minutes to emulsify and form an oil-in-water emulsion with uniformly distributed droplet particle size, thereby obtaining a two-phase oil-in-water emulsion containing 100 μg / g ISA 563VG.

[0143] Weigh 1g of each of the two emulsions, mix them evenly, add 0.05% of nonionic surfactant poloxamer (Pluronic F68) and 1% polyethylene glycol / ricinoleate (ELP), stir at 150-200r / min for 30-45min under 30°C temperature control conditions to fully mix the two dosage forms to obtain a swine fever virus E2 gene nucleic acid-protein co-immune vaccine. The vaccine is a biphasic water-in-oil-in-water emulsion in which the external aqueous phase is mainly a recombinant plasmid and the internal aqueous phase is mainly a recombinant E2 protein.

[0144] In this embodiment, a eukaryotic expression plasmid containing a classical swine fever virus gene and a porcine-preferred CpG sequence was constructed, and large-scale and efficient expression was performed using a CHO-S cell expression system to obtain a recombinant protein with a dimer structure having multiple glycosylation sites. The recombinant plasmid containing the recombinant nucleic acid was further induced, released and separated from the collected cell precipitate, and the recombinant plasmid was mixed with the purified recombinant protein in a mass ratio of 1:2.5, and then mixed with a biphasic adjuvant to prepare a classical swine fever co-immune vaccine for emergency immunization and prevention and control of classical swine fever, which is more efficient and safer.

[0145] 3. Evaluation of the immune effect of co-immunization vaccines in animal models

[0146] 3.1 Evaluation of immune effects in mouse models

[0147] Twenty BALB / C female mice were randomly divided into 4 groups, with 5 mice in each group. The grouping of experimental animals is shown in Table 2. The adjuvants used for the plasmid nucleic acid vaccine and the recombinant protein vaccine are both ISA 201VG adjuvant. The control group was prepared by emulsifying PBS with a pH of 7.2 to 7.4 and ISA 201VG adjuvant. The plasmid nucleic acid vaccine refers to a nucleic acid vaccine containing the above-mentioned recombinant plasmid.

[0148] Table 2 Grouping of experimental animals for vaccine safety and effectiveness

[0149]

[0150] The above four vaccines were used for subcutaneous immunization on the back, 100 μl / mouse, to evaluate the immune effect of the vaccine. Blood was collected from the submandibular vein of mice before the first vaccination and 2, 7, 14, 21, 28, and 35 days after the first vaccination, and the serum was separated aseptically. The blood samples taken before the first vaccination and 2 and 23 days after the vaccination were measured by commercial ELISA kits to measure the levels of IFN-γ and IL-4 cytokines in the serum, and the serum samples taken before the first vaccination and 7, 14, 21, 28, and 35 days after the vaccination were used to measure the neutralizing antibodies against classical swine fever virus. After blood was collected 2 weeks after the second vaccination, all mice were killed, and spleen lymphocytes were extracted according to the instructions of the kit. Take 10-20 μl for trypan blue staining and cell counting, and adjust the cell density to 10 6 / ml, take 500μl of cells and add 2μl of fluorescent labeled antibody in the dark, keep it away from light for 30min, and measure the results with flow cytometer.

[0151] 3.1.1 Results of neutralizing antibodies against classical swine fever virus in mouse serum

[0152] like Fig.11 As shown, the differences in antibody levels among the four test groups at different testing points were compared. Except for the control group, the antibodies in the other immunization groups increased after immunization; except before immunization, the neutralizing antibodies in the co-immunization vaccine group were significantly higher than those in the other three groups at the remaining monitoring points, followed by the recombinant protein vaccine group. The plasmid nucleic acid vaccine had the worst immunization effect, and the control group was always negative.

[0153] 3.1.2 Results of IFN-γ and IL-4 determination in mouse serum

[0154] Results of IFN-γ detection in mouse serum ( Fig.12 ) showed that IFN-γ was secreted in each immunization group after immunization. The IFN-γ level in the co-immunization vaccine group was the highest, which was significantly different from the other groups, followed by the plasmid nucleic acid vaccine, the recombinant protein vaccine was lower, and the control group was even lower.

[0155] Results of IL-4 cytokine assay in mouse serum ( Fig.13 ) showed that the IFN-γ level in the co-immune vaccine group was the highest, significantly higher than that in other groups, followed by the plasmid nucleic acid vaccine, the recombinant protein vaccine was lower, and the control group was the lowest.

[0156] 3.1.3 CD4 in spleen + and CD8 + Lymphocyte subsets assay results

[0157] CD4 in spleen + and CD8 + Lymphocyte subsets assay results ( Fig.14 ) showed that the CD4 + and CD8 + The number of lymphocytes was the largest, significantly higher than that of other groups, followed by plasmid nucleic acid vaccine, and the recombinant protein vaccine was lower, and the control group was the lowest.

[0158] CD4 + Th1 cells can secrete a variety of cytokines such as IL-1, IL-2, IFN-α and IFN-γ, and effectively promote the differentiation of Th0 to Th1 and inhibit the differentiation of Th2 cells. Among them, IFN-γ mainly mediates cellular immune response and produces specific cellular immunity. Th2 cells mainly secrete type II cytokines such as IL-4 and IL-5, which promote the differentiation of Th0 to Th2 and inhibit the proliferation of Th1 cells. IL-4 is the representative of them, which can induce IgE production and mediate humoral immunity.

[0159] Constant CD 4+ / CD 8+ The ratio maintains the balance of the body's cellular immune response. 4+ Lymphocytes play an important role in enhancing the body's humoral immunity. 8+ Lymphocytes will inhibit the immune response. Therefore, this study studied the serum neutralizing antibody levels, IFN-γ and IL-4 cytokine levels, and CD4+ T cells in different immunization groups. 4+ and CD 8+ The results showed that the serum neutralizing antibody, IFN-γ and IL-4 cytokine levels, CD 4+ and CD 8+ The levels were significantly higher than those in other experimental groups, indicating that the co-immunization vaccine can significantly improve the humoral immunity level of mice and induce the body to produce better cellular immune response to a certain extent.

[0160] 3.2 Evaluation of the immune effect of co-immunization vaccines on target animals

[0161] 3.2.1 Experimental animal grouping and immunization

[0162] Purified recombinant E2 protein and recombinant plasmid containing E2 gene were mixed with ISA201VG adjuvant and emulsified according to the preparation method in the table below to prepare a water-in-oil-in-water biphasic formulation of experimental vaccine for immunizing pigs to evaluate the safety and effectiveness of the vaccine. PBS with pH 7.2-7.4 was emulsified with ISA 201VG adjuvant as the control group. According to the "Standard for the Neutralization Test Method of Classical Swine Fever Virus" in the Notes of the Quality Standards for Classical Swine Fever Live Vaccine (Rabbit Source), 20 4-5 week old Classical Swine Fever antibody negative pigs were selected and randomly divided into 4 groups, 5 in each group. The experimental grouping and animal immunization scheme are shown in Table 3.

[0163] Table 3 Grouping of vaccine safety and efficacy tests in target animals

[0164]

[0165] 3.2.2 Security Check

[0166] After the first vaccination, the piglets were observed for 14 consecutive days and their temperatures were measured daily. All piglets ate normally and were in good spirits. No adverse reactions or deaths occurred, and their body temperatures did not exceed 40°C. Fig.15 shown.

[0167] 3.2.3 Determination of neutralizing antibodies

[0168] Blood was collected weekly before the first immunization until 5 weeks after the first immunization, and then every 5 weeks until 40 weeks. Serum was aseptically separated and used to measure neutralizing antibodies against classical swine fever virus.

[0169] According to the manufacturing and testing standards for the inactivated baculovirus vaccine of the E2 protein of classical swine fever virus, the neutralizing antibody titer of classical swine fever is not less than 1:32, which is considered as positive. Before immunization, the neutralizing antibody titer of pigs in each group was less than 1:4 (calculated as 1:4); one week after the first immunization, 3 / 5 of the neutralizing antibodies in the co-immunized vaccine group turned positive. Fig.17 As shown, 2 weeks after the first vaccination, all neutralizing antibodies turned positive and were not less than 1:64. 5-10 weeks after the first vaccination, the neutralizing antibody titer reached a peak value, which was not less than 1:2048. 40 weeks after the first vaccination, the neutralizing antibody was still not less than 1:32. That is, the co-immunization vaccine has a fast immune response, a short immune blank period, a long antibody peak period, and a long immune duration of not less than 40 weeks. Fig.16 As shown in the figure, the antibodies of the plasmid nucleic acid vaccine group all turned positive 4 weeks after the first vaccination (1 week after the second vaccination), and the antibodies reached the peak 5 weeks after the first vaccination, but the neutralizing antibodies were not higher than 1:128. By 10 weeks after the first vaccination, the antibodies had begun to turn negative, and the immune blank period was long and the immune duration was short. In the recombinant protein vaccine group, all neutralizing antibodies turned positive 3 weeks after the first vaccination, and the antibodies reached the peak 5 weeks after the first vaccination. By 40 weeks after the first vaccination, 3 / 5 antibodies were lower than 1:32, that is, the recombinant protein vaccine had a long immune blank period, and the immune duration could reach 40 weeks. The control group had negative antibodies for swine fever throughout. The above results show that the co-immunization vaccine group has the best immune effect, with a fast immune response and a long protection period.

[0170] The variance analysis method was used to compare the differences in antibody levels among the four test groups at different detection points. Except before immunization, the neutralizing antibodies in the co-immunization vaccine group were significantly higher than those in the other three groups at all other monitoring points; the antibodies in the recombinant protein vaccine group were significantly higher than those in the plasmid nucleic acid vaccine group and the control group, but lower than those in the co-immunization vaccine group; the antibodies in the plasmid nucleic acid vaccine group were significantly different from those in the control group only 7 to 105 days after immunization. The above results indicate that the immune effect of the co-immunization vaccine group is significantly higher than that of the recombinant protein vaccine group, followed by the recombinant protein vaccine group, and the plasmid nucleic acid vaccine has the worst immune effect, and the control group is always negative.

[0171] 3.3 Safety and efficacy tests of co-immune vaccines formulated with different types of adjuvants in target animals

[0172] 3.2.1 Experimental animal grouping and immunization

[0173] Purified recombinant E2 protein and recombinant plasmid containing E2 gene were mixed with biphasic adjuvant (same as Example 1), water adjuvant (Gel 01), and oil adjuvant (ISA 660VG) according to the preparation method in the table below, and emulsified according to the method provided by the adjuvant manufacturer to prepare three different dosage forms of vaccine for immunizing pigs, which were used to evaluate the safety and effectiveness of the three vaccines. PBS with pH 7.2-7.4 was emulsified with ISA 201VG adjuvant as a control group. According to the "Standard for Quality of Live Classical Swine Fever Vaccine (Rabbit Source)" in the notes, 20 4-5 week old Classical Swine Fever antibody negative pigs were selected and randomly divided into 4 groups, 5 in each group. The experimental grouping and animal immunization scheme are shown in Table 4.

[0174] Table 4 Grouping of safety and efficacy experiments of co-immunization vaccines with different adjuvants

[0175]

[0176] 3.2.2 Security Check

[0177] After the first vaccination, the piglets were observed for 14 days and their temperatures were measured daily. All piglets ate normally and were in good spirits. Except for the water adjuvant group, in which the body temperature of some pigs increased within 2 days after vaccination, no adverse reactions or deaths occurred in the other groups, and the body temperature did not exceed 40°C. Fig.18 shown.

[0178] 3.2.3 Determination of neutralizing antibodies

[0179] Blood was collected weekly before the first immunization until 5 weeks after the first immunization, and then every 5 weeks until 40 weeks. Serum was aseptically separated and used to measure neutralizing antibodies against classical swine fever virus.

[0180] According to the manufacturing and testing standards for the inactivated baculovirus vaccine of the E2 protein of classical swine fever virus, the neutralizing antibody titer of classical swine fever was not less than 1:32, which was considered as positive. Before immunization, the neutralizing antibody titer of pigs in each group was less than 1:4 (calculated as 1:4); one week after the first immunization of the biphasic adjuvant group, 4 / 5 of the neutralizing antibodies turned positive. Fig.19As shown, all turned positive 2 weeks after the first vaccination, the neutralizing antibody titer reached the peak 5-10 weeks after the first vaccination, and the neutralizing antibody titer was still not less than 1:32 40 weeks after the first vaccination, that is, the vaccine prepared with biphasic adjuvant had a fast immune response, a short immune blank period, a long antibody peak period, and a long immune duration of not less than 40 weeks; in the water adjuvant vaccine group, 3 / 5 antibodies turned positive 1 week after the first vaccination, and the antibody peaked 3-5 weeks after the first vaccination, but the neutralizing antibody titer was not higher than 1:724, and the antibody began to turn negative 20 weeks after the first vaccination, with a short immune blank period, but a short immune duration. In the recombinant protein vaccine group, all neutralizing antibodies turned positive 4 weeks after the first vaccination, reached the antibody peak 5-15 weeks after the first vaccination, and 2 / 5 antibodies were lower than 1:32 40 weeks after the first vaccination, that is, the recombinant protein vaccine had a long immune blank period, and the immune duration could reach 40 weeks. The control group had always been negative for swine fever antibodies. The above results show that the biphasic adjuvant group had the best immune effect, with a fast immune response and a long protection period.

[0181] The variance analysis method was used to compare the differences in antibody levels among the four test groups at different test points. Within 2 weeks after the first vaccination, the antibody difference between the biphasic adjuvant and water adjuvant vaccine groups was not significant, but the difference was significant with the other two groups; from 10 to 40 weeks after the first vaccination, the antibody difference between the biphasic adjuvant and oil adjuvant vaccine groups was not significant, but the difference was extremely significant with the other two groups. The above results show that the biphasic adjuvant group was superior to the other groups in the early, middle and late stages of immunization. The water adjuvant vaccine produced antibodies quickly in the early stage, but the duration was short; the oil adjuvant vaccine produced antibodies late, but the duration of immunity was long, and the control group was always negative.

[0182] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention. SEQUENCE LISTING <110> Tiankang Biotechnology Co., Ltd. <120> A nucleic acid and recombinant protein co-immune vaccine based on swine fever virus gene, preparation method and application <160> 9 <170> PatentIn version 3.5 <210> 1 <211> 1296 <212> DNA <213> Artificial sequence <400> 1 gccaccatgg tattgagagg acagatcgtg caaggtgtga tatggctgct attagtaact 60 ggggcgcaag gccggctagc ctgcaaggaa gactacaggt acgcaatatc gtcaaccgat 120 gagatagggc tacttggggc cggaggactc accaccacct ggaaagaata cacccacgat 180 ctgcagctga atgacgggac cgttaaggcc acttgcgtgg caggttcctt taaaatcaca 240 gcacttaatg cggtcagtag gaggtatttg gcatcactgc ataagaaggc tttacccact 300 tccgtgacat tcgaactcct gttcgacggg accaacccat caactgagga aatgggagat 360 gacttcgggt tcgggctgtg cccgtttgat acgagacccg ttgtcaaggg aaagtacaat 420 gcgaccttgg tgaacggtag tgctttctat cttgtctgcc caatagggtg gacgggtgtt 480 atagagtgca cagcagtgag cccaacaacc ctgagaacag aagtggtaaa gaccttcagg 540 agagacaagc cctttccgca cagaatgaat tgtgtgacca ccacagtgga aaatgaagac 600 ttattctact gtaagttggg gggcaactgg acatgtgtga aaggcgaacc agtggtctac 660 acaggggggt tagtgaaaca atgtagatgg tgtggcttcg acttcaacga gcctgatggg 720 cttccgcact accccatagg taagtgcatt ctggcaaacg agaccagtta cagagtagta 780 gattcaacgg actgtaacag agatggcgtt gtaatcagca cagaggggag tcatgagtgc 840 ttgattggta acacgactgt cagggtgcat gcatcagatg aaagattggg ccccatgcca 900 tgcagaccta aagagattgt ctctagtgca ggacctgcaa tgaaaacctc ctgtacattc 960 aattacgcaa aaactttgaa gaacaggtac tatgagccca gggacagcta cttccagcaa 1020 tacatgctta agggtgagta tcagtactgg tttgacctgg atgcgactga ccaccactca 1080 gattacttcg cagaatttgt tgtcttggtg tcgtcgtttt gtcgttttgt cgttggtgca 1140 tcgatgcagg ggtcgtcgtt ttgtcgtttt gtcgttggtg catcgatgca ggggtcgtcg 1200 ttttgtcgtt ttgtcgttgg tgcatcgatg caggggtcgt cgttttgtcg ttttgtcgtt 1260 ggtgcatcga tgcaggggca tcaccatcac catcac 1296 <210> 2 <211> 432 <212> PRT <213> Artificial Sequence <400> 2 Ala Thr Met Val Leu Arg Gly Gln Ile Val Gln Gly Val Ile Trp Leu 1 5 10 15 Leu Leu Val Thr Gly Ala Gln Gly Arg Leu Ala Cys Lys Glu Asp Tyr 20 25 30 Arg Tyr Ala Ile Ser Ser Thr Asp Glu Ile Gly Leu Leu Gly Ala Gly 35 40 45 Gly Leu Thr Thr Thr Trp Lys Glu Tyr Thr His Asp Leu Gln Leu Asn 50 55 60 Asp Gly Thr Val Lys Ala Thr Cys Val Ala Gly Ser Phe Lys Ile Thr 65 70 75 80 Ala Leu Asn Ala Val Ser Arg Arg Tyr Leu Ala Ser Leu His Lys Lys 85 90 95 Ala Leu Pro Thr Ser Val Thr Phe Glu Leu Leu Phe Asp Gly Thr Asn 100 105 110 Pro Ser Thr Glu Glu Met Gly Asp Asp Phe Gly Phe Gly Leu Cys Pro 115 120 125 Phe Asp Thr Arg Pro Val Val Lys Gly Lys Tyr Asn Ala Thr Leu Val 130 135 140 Asn Gly Ser Ala Phe Tyr Leu Val Cys Pro Ile Gly Trp Thr Gly Val 145 150 155 160 Ile Glu Cys Thr Ala Val Ser Pro Thr Thr Leu Arg Thr Glu Val Val 165 170 175 Lys Thr Phe Arg Arg Asp Lys Pro Phe Pro His Arg Met Asn Cys Val 180 185 190 Thr Thr Thr Val Glu Asn Glu Asp Leu Phe Tyr Cys Lys Leu Gly Gly 195 200 205 Asn Trp Thr Cys Val Lys Gly Glu Pro Val Val Tyr Thr Gly Gly Leu 210 215 220 Val Lys Gln Cys Arg Trp Cys Gly Phe Asp Phe Asn Glu Pro Asp Gly 225 230 235 240 Leu Pro His Tyr Pro Ile Gly Lys Cys Ile Leu Ala Asn Glu Thr Ser 245 250 255 Tyr Arg Val Val Asp Ser Thr Asp Cys Asn Arg Asp Gly Val Val Ile 260 265 270 Ser Thr Glu Gly Ser His Glu Cys Leu Ile Gly Asn Thr Thr Val Arg 275 280 285 Val His Ala Ser Asp Glu Arg Leu Gly Pro Met Pro Cys Arg Pro Lys 290 295 300 Glu Ile Val Ser Ser Ala Gly Pro Ala Met Lys Thr Ser Cys Thr Phe 305 310 315 320 Asn Tyr Ala Lys Thr Leu Lys Asn Arg Tyr Tyr Glu Pro Arg Asp Ser 325 330 335 Tyr Phe Gln Gln Tyr Met Leu Lys Gly Glu Tyr Gln Tyr Trp Phe Asp 340 345 350 Leu Asp Ala Thr Asp His His Ser Asp Tyr Phe Ala Glu Phe Val Val 355 360 365 Leu Val Ser Ser Phe Cys Arg Phe Val Val Gly Ala Ser Met Gln Gly 370 375 380 Ser Ser Phe Cys Arg Phe Val Val Gly Ala Ser Met Gln Gly Ser Ser 385 390 395 400 Phe Cys Arg Phe Val Val Gly Ala Ser Met Gln Gly Ser Ser Phe Cys 405 410 415 Arg Phe Val Val Gly Ala Ser Met Gln Gly His His His His His His 420 425 430 <210> 3 <211> 66 <212> DNA <213> Artificial sequence <400> 3 atggtattga gaggacagat cgtgcaaggt gtgatatggc tgctattagt aactggggcg 60 caaggc 66 <210> 4 <211> 18 <212> DNA <213> Artificial sequence <400> 4 catcaccatc accatcac 18 <210> 5 <211> 168 <212> DNA <213> Artificial sequence <400> 5 tcgtcgtttt gtcgttttgt cgtggtgca tcgatgcagg ggtcgtcgtt ttgtcgtttt 60 gtcgttggtg catcgatgca ggggtcgtcg ttttgtcgtt ttgtcgttgg tgcatcgatg 120 caggggtcgt cgtttgtcg ttttgtcgtt ggtgcatcga tgcagggg 168 <210> 6 <211> 1119 <212> DNA <213> classical swine fever virus <400> 6 cggctagcct gcaaggaaga ttacaggtac gcaatatcgt caaccgatga gatagggcta 60 cttggggccg gaggtctcac caccacctgg aaggaataca accacgattt gcaactgaat 120 gacgggaccg tcaaggccag ttgcgtggca ggttccttta aagtcacagc acttaatgtg 180 gtcagtagga ggtatttggc gtcattgcat aagaaggctt tacccacttc cgtgacattc 240 gagctcctgt tcgacgggac caacccatca actgaggaaa tgggagatga cttcaggtcc 300 gggctgtgcc cgtttgatac gagtcctgtt gttaagggaa agtacaatac gaccttgttg 360 aacggtagtg ctttctatct tgtctgccca atagggtgga cgggtgtcat agagtgcaca 420 gcagtgagcc caacaactct gaggacagaa gtggtaaaga ccttcaggag agacaagccc 480 tttccgcaca gaatggattg tgtgaccacc atagtggaaa atgaagattt attctattgt 540 aagttggggg gcaactggac atgtgtgaaa ggcgagccag tggtctacac agggggggta 600 gtaaaacaat gtagatggtg tggcttcgac ttcgatgggc ctgacggact cccgcattac 660 cccataggta agtgcatttt ggcaaatgag acaggttaca gaatagtaga ttcaacggac 720 tgtaacagag atggcgttgt aatcagcaca gaggggagtc atgagtgctt gatcggtaac 780 acgactgtca aggtgcatgc atcagatgaa agactgggcc ctatgccatg cagacctaaa 840 gagattgtct ctagtgctgg tcctgtaatg aaaacctcct gtacattcaa ctacacaaaa 900 actttgaaga acaggtacta tgagcccagg gacagctact tccagcaata tatgcttaag 960 ggtgagtatc agtactggtt tgacctggat gcgactgacc gccactcaga ttacttcgca 1020 gaatttgttg tcttggtggt ggtagcactg ttaggaggaa gatatgtcct gtggctgata 1080 gtgacctacg tagttctaac agaacaactc gccgctggt 1119 <210> 7 <211> 373 <212> PRT <213> classical swine fever virus <400> 7 Arg Leu Ala Cys Lys Glu Asp Tyr Arg Tyr Ala Ile Ser Ser Thr Asp 1 5 10 15 Glu Ile Gly Leu Leu Gly Ala Gly Gly Leu Thr Thr Thr Trp Lys Glu 20 25 30 Tyr Asn His Asp Leu Gln Leu Asn Asp Gly Thr Val Lys Ala Ser Cys 35 40 45 Val Ala Gly Ser Phe Lys Val Thr Ala Leu Asn Val Val Ser Arg Arg 50 55 60 Tyr Leu Ala Ser Leu His Lys Lys Ala Leu Pro Thr Ser Val Thr Phe 65 70 75 80 Glu Leu Leu Phe Asp Gly Thr Asn Pro Ser Thr Glu Glu Met Gly Asp 85 90 95 Asp Phe Arg Ser Gly Leu Cys Pro Phe Asp Thr Ser Pro Val Val Lys 100 105 110 Gly Lys Tyr Asn Thr Thr Leu Leu Asn Gly Ser Ala Phe Tyr Leu Val 115 120 125 Cys Pro Ile Gly Trp Thr Gly Val Ile Glu Cys Thr Ala Val Ser Pro 130 135 140 Thr Thr Leu Arg Thr Glu Val Val Lys Thr Phe Arg Arg Asp Lys Pro 145 150 155 160 Phe Pro His Arg Met Asp Cys Val Thr Thr Ile Val Glu Asn Glu Asp 165 170 175 Leu Phe Tyr Cys Lys Leu Gly Gly Asn Trp Thr Cys Val Lys Gly Glu 180 185 190 Pro Val Val Tyr Thr Gly Gly Val Val Lys Gln Cys Arg Trp Cys Gly 195 200 205 Phe Asp Phe Asp Gly Pro Asp Gly Leu Pro His Tyr Pro Ile Gly Lys 210 215 220 Cys Ile Leu Ala Asn Glu Thr Gly Tyr Arg Ile Val Asp Ser Thr Asp 225 230 235 240 Cys Asn Arg Asp Gly Val Val Ile Ser Thr Glu Gly Ser His Glu Cys 245 250 255 Leu Ile Gly Asn Thr Thr Val Lys Val His Ala Ser Asp Glu Arg Leu 260 265 270 Gly Pro Met Pro Cys Arg Pro Lys Glu Ile Val Ser Ser Ala Gly Pro 275 280 285 Val Met Lys Thr Ser Cys Thr Phe Asn Tyr Thr Lys Thr Leu Lys Asn 290 295 300 Arg Tyr Tyr Glu Pro Arg Asp Ser Tyr Phe Gln Gln Tyr Met Leu Lys 305 310 315 320 Gly Glu Tyr Gln Tyr Trp Phe Asp Leu Asp Ala Thr Asp Arg His Ser 325 330 335 Asp Tyr Phe Ala Glu Phe Val Val Leu Val Val Val Ala Leu Leu Gly 340 345 350 Gly Arg Tyr Val Leu Trp Leu Ile Val Thr Tyr Val Val Leu Thr Glu 355 360 365 Gln Leu Ala Ala Gly 370 <210> 8 <211> 1038 <212> DNA <213> Artificial Sequence <400> 8 cggctagcct gcaaggaaga ctacaggtac gcaatatcgt caaccgatga gatagggcta 60 cttggggccg gaggactcac caccacctgg aaagaataca cccacgatct gcagctgaat 120 gacgggaccg ttaaggccac ttgcgtggca ggttccttta aaatcacagc acttaatgcg 180 gtcagtagga ggtatttggc atcactgcat aagaaggctt tacccacttc cgtgacattc 240 gaactcctgt tcgacgggac caacccatca actgaggaaa tgggagatga cttcgggttc 300 gggctgtgcc cgtttgatac gagacccgtt gtcaagggaa agtacaatgc gaccttggtg 360 aacggtagtg ctttctatct tgtctgccca atagggtgga cgggtgttat agagtgcaca 420 gcagtgagcc caacaaccct gagaacagaa gtggtaaaga ccttcaggag agacaagccc 480 tttccgcaca gaatgaattg tgtgaccacc acagtggaaa atgaagactt attctactgt 540 aagttggggg gcaactggac atgtgtgaaa ggcgaaccag tggtctacac aggggggtta 600 gtgaaacaat gtagatggtg tggcttcgac ttcaacgagc ctgatgggct tccgcactac 660 cccataggta agtgcattct ggcaaacgag accagttaca gagtagtaga ttcaacggac 720 tgtaacagag atggcgttgt aatcagcaca gaggggagtc atgagtgctt gattggtaac 780 acgactgtca gggtgcatgc atcagatgaa agattgggcc ccatgccatg cagacctaaa 840 gagattgtct ctagtgcagg acctgcaatg aaaacctcct gtacattcaa ttacgcaaaa 900 actttgaaga acaggtacta tgagcccagg gacagctact tccagcaata catgcttaag 960 ggtgagtatc agtactggtt tgacctggat gcgactgacc accactcaga ttacttcgca 1020 gaatttgttg tcttggtg 1038 <210> 9 <211> 346 <212> PRT <213> classical swine fever virus <400> 9 Arg Leu Ala Cys Lys Glu Asp Tyr Arg Tyr Ala Ile Ser Ser Thr Asp 1 5 10 15 Glu Ile Gly Leu Leu Gly Ala Gly Gly Leu Thr Thr Thr Trp Lys Glu 20 25 30 Tyr Thr His Asp Leu Gln Leu Asn Asp Gly Thr Val Lys Ala Thr Cys 35 40 45 Val Ala Gly Ser Phe Lys Ile Thr Ala Leu Asn Ala Val Ser Arg Arg 50 55 60 Tyr Leu Ala Ser Leu His Lys Lys Ala Leu Pro Thr Ser Val Thr Phe 65 70 75 80 Glu Leu Leu Phe Asp Gly Thr Asn Pro Ser Thr Glu Glu Met Gly Asp 85 90 95 Asp Phe Gly Phe Gly Leu Cys Pro Phe Asp Thr Arg Pro Val Val Lys 100 105 110 Gly Lys Tyr Asn Ala Thr Leu Val Asn Gly Ser Ala Phe Tyr Leu Val 115 120 125 Cys Pro Ile Gly Trp Thr Gly Val Ile Glu Cys Thr Ala Val Ser Pro 130 135 140 Thr Thr Leu Arg Thr Glu Val Val Lys Thr Phe Arg Arg Asp Lys Pro 145 150 155 160 Phe Pro His Arg Met Asn Cys Val Thr Thr Thr Val Glu Asn Glu Asp 165 170 175 Leu Phe Tyr Cys Lys Leu Gly Gly Asn Trp Thr Cys Val Lys Gly Glu 180 185 190 Pro Val Val Tyr Thr Gly Gly Leu Val Lys Gln Cys Arg Trp Cys Gly 195 200 205 Phe Asp Phe Asn Glu Pro Asp Gly Leu Pro His Tyr Pro Ile Gly Lys 210 215 220 Cys Ile Leu Ala Asn Glu Thr Ser Tyr Arg Val Val Asp Ser Thr Asp 225 230 235 240 Cys Asn Arg Asp Gly Val Val Ile Ser Thr Glu Gly Ser His Glu Cys 245 250 255 Leu Ile Gly Asn Thr Thr Val Arg Val His Ala Ser Asp Glu Arg Leu 260 265 270 Gly Pro Met Pro Cys Arg Pro Lys Glu Ile Val Ser Ser Ala Gly Pro 275 280 285 Ala Met Lys Thr Ser Cys Thr Phe Asn Tyr Ala Lys Thr Leu Lys Asn 290,295,300 Arg Tyr Tyr Glu Pro Arg Asp Ser Tyr Phe Gln Gln Tyr Met Leu Lys 305 310 315 320 Gly Glu Tyr Gln Tyr Trp Phe Asp Leu Asp Ala Thr Asp His Ser 325 330 335 Asp Tyr Phe Ala Glu Phe Val Val Leu Val 340,345

Claims

1. A nucleic acid and recombinant protein co-immune vaccine based on swine fever virus gene, characterized in that: It comprises a biphasic adjuvant, and a recombinant eukaryotic expression plasmid and a recombinant protein distributed in different liquid phase layers of the biphasic adjuvant, wherein the recombinant eukaryotic expression plasmid comprises a gene sequence of a recombinant protein encoding an E2 binding site as shown in SEQ ID No. 1; and the amino acid sequence of the recombinant protein is shown in SEQ ID NO. 2; The co-immune vaccine is a water-in-oil-in-water emulsion formed by compounding a biphasic adjuvant, wherein the recombinant protein is located in the inner aqueous phase, and the recombinant eukaryotic expression plasmid is located in the outer aqueous phase.

2. The nucleic acid and recombinant protein co-immune vaccine based on classical swine fever virus gene according to claim 1, characterized in that: The mass ratio of the recombinant eukaryotic expression plasmid to the recombinant protein is 1:(1.5-4.5).

3. The nucleic acid and recombinant protein co-immune vaccine based on classical swine fever virus gene according to claim 2, characterized in that: The mass ratio of the recombinant eukaryotic expression plasmid to the recombinant protein is 1:(2-4).

4. The nucleic acid and recombinant protein co-immune vaccine based on classical swine fever virus gene according to claim 3, characterized in that: The mass ratio of the recombinant eukaryotic expression plasmid to the recombinant protein is 1:2.

5.

5. The nucleic acid and recombinant protein co-immune vaccine based on classical swine fever virus gene according to claim 1, characterized in that: The biphasic adjuvant includes at least one of ISA 201VG, ISA563VG, ISA206VG or ISA660VG.

6. The method for preparing a co-immune vaccine based on nucleic acid and recombinant protein of classical swine fever virus gene according to any one of claims 1 to 5, characterized in that: The eukaryotic expression plasmid and the recombinant protein are separately compounded into different liquid phase layers of the biphasic adjuvant to form a water-in-oil-in-water emulsion, in which the recombinant protein is located in the inner aqueous phase and the recombinant eukaryotic expression plasmid is located in the outer aqueous phase, thereby obtaining a nucleic acid and recombinant protein co-immune vaccine.

7. The preparation method according to claim 6, characterized in that: The recombinant eukaryotic expression plasmid includes plasmid pcDNA3.

1.

8. The preparation method according to claim 6, characterized in that: The preparation method also includes modifying the codons of the gene sequence encoding the recombinant protein of the E2 binding site before constructing the recombinant eukaryotic expression plasmid to form the gene sequence encoding the recombinant protein of the E2 binding site.

9. The preparation method according to claim 6, characterized in that: Restriction sites are inserted at both ends of the gene sequence encoding the recombinant protein of the E2 binding site.

10. The preparation method according to claim 6, characterized in that: The preparation method also includes transfecting the recombinant eukaryotic expression plasmid into a cell line to express an expression product including a recombinant protein and a recombinant eukaryotic expression plasmid, and then separating and purifying the recombinant protein and the recombinant eukaryotic expression plasmid in the product.

11. The preparation method according to claim 10, characterized in that: The cell lines include CHO cells.

12. The preparation method according to claim 11, characterized in that: The CHO cells include CHO-S or CHO-K1 cells.

13. The preparation method according to claim 10, characterized in that: The transfection method includes liposome transfection.

14. The preparation method according to claim 10, characterized in that: The recombinant protein was separated and purified by chromatography.

15. The preparation method according to claim 10, characterized in that: The recombinant eukaryotic expression plasmid was separated and purified using the kit extraction method.

16. The preparation method according to claim 6, characterized in that: The method of compounding the recombinant eukaryotic expression plasmid and the recombinant protein into different liquid phase layers of a biphasic adjuvant comprises selecting different biphasic adjuvants to construct two compounding systems, compounding the recombinant eukaryotic expression plasmid and the recombinant protein into the aqueous phase layers of the biphasic adjuvants of different compounding systems, and then mixing the different compounding systems so that the recombinant eukaryotic expression plasmid is located in the outer aqueous phase layer and the recombinant protein is located in the inner aqueous phase layer.

17. The preparation method according to claim 16, characterized in that: The two complex systems include a recombinant eukaryotic expression plasmid complex system and a recombinant protein complex. The mass content of nucleic acid in the recombinant eukaryotic expression plasmid complex system is 30-60 μg / g biphasic adjuvant, and the mass content of recombinant protein in the recombinant protein complex system is 80-140 μg / g biphasic adjuvant.

18. The preparation method according to claim 17, characterized in that: The complex system of the recombinant eukaryotic expression plasmid and the recombinant protein complex are mixed with equal mass.

19. Use of the co-immune vaccine described in any one of claims 1 to 5 or the co-immune vaccine prepared by the preparation method described in any one of claims 6 to 18 in the preparation of a vaccine for the prevention or treatment of classical swine fever.

20. The use according to claim 19, characterized in that The application implements at least one of the functions AB: A. Causes the body to produce IFN-γ; B. Causes the body to produce IL-4 cytokine.

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