E2 fusion protein of hog cholera virus and preparation method and application of vaccine

Abstract

The invention provides a hog cholera virus E2 fusion protein and a preparation method and application of a vaccine. Compared with the prior art, the E2 fusion protein provided by the invention has the beneficial effects that the C terminal of the E2 protein is replaced by the fusion protein of a porcine epidemic diarrhea virus transmembrane region, the blocking rate of the fusion protein to the hog cholera virus is relatively high, and polynucleotide synthesized by depending on the fusion protein can efficiently express the fusion protein; the mRNA vaccine prepared on the basis of the polynucleotide has a good immune protection effect on pigs, is not influenced by the existence of maternal antibodies, is high in safety and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the field of veterinary biomedicine, and in particular to a preparation method and application of an E2 fusion protein and vaccine of a swine fever virus. Background Art

[0002] Classical swine fever is a multi-system hemorrhagic infectious disease caused by classical swine fever virus (CSFV). Domestic pigs and wild boars are susceptible animals. It is highly contagious and lethal. The virus is an enveloped RNA virus. The virus particles are approximately spherical with a size of 34-50nm. It is composed of core protein C and envelope proteins E0, E1 and E2. The core wraps a single-stranded positive-strand RNA of about 12.3kb in size. The two sides are untranslated regions. The middle coding region encodes a precursor protein of 3898 amino acids. After the precursor protein is processed by cellular proteases and viral proteases, 12 proteins can be obtained, including the above-mentioned four structural proteins and eight non-structural proteins. The genetic diversity of classical swine fever virus does not produce new serotypes, so it will not affect the efficacy of the vaccine. At present, classical swine fever vaccines are mainly divided into attenuated live vaccines and recombinant protein vaccines, both of which have good safety and effectiveness. my country first developed a rabbit-based attenuated swine fever vaccine in 1956. It has the advantages of high safety, high effectiveness and low cost, and has played a very important role in the prevention and control of swine fever in my country. However, attenuated vaccines have safety risks, and it is difficult to distinguish between vaccine immunity and natural infection during swine fever purification.

[0003] The E2 protein is the main envelope protein of CSFV. It can form a homodimer with a molecular weight of 100kD by itself, or a heterodimer with E1 with a molecular weight of 75kD. It is present on the surface of viral particles and infected cells. The E2 protein plays a variety of important functions in the viral life cycle. First, E2 is involved in the entry and attachment of the virus to host cells. It binds to specific receptors (such as CD163) on the surface of susceptible cells, thereby promoting viral internalization. Secondly, E2 is the main target of the host immune response. It contains multiple antigenic sites that can be recognized by neutralizing antibodies, which is important for preventing infection. Third, E2 is also involved in the assembly and maturation of the virus. It interacts with other viral proteins to form the viral envelope and promote the release of the virus from infected cells. Overall, the E2 protein is essential for the replication and pathogenesis of CSFV, making it a target for the development of vaccines and antiviral therapies. The conserved regions contained in E2 are relatively stable, making it effective against different CSFV strains. E2 does not cause any obvious side effects or adverse reactions to vaccinated animals and is considered a safe antigen for vaccines.

[0004] Due to the immunogenicity, protective efficacy, conserved regions and safety of E2, it is currently proven to be the antigen with the best immune protection effect, and E2 recombinant protein vaccine is also widely used. However, recombinant protein vaccines have defects such as high production costs and inability to induce effective cellular immunity in the body. In recent years, the development of mRNA technology has made it achieve good results in the field of infectious disease vaccines. By delivering mRNA encoding viral proteins into the body, the viral proteins are expressed by the body's own cells, which can effectively activate humoral immunity and cellular immunity. The advantages of mRNA vaccines are: 1) mRNA vaccines use cells in the body to express, do not need to purify proteins, and do not need to consider protein degradation and conformational changes. The development speed is fast. If the mRNA vaccine can achieve high expression and small dosage, the cost can be reduced. 2) The cellular immune response activated by mRNA is strong, which can provide relatively long-term protection for the body, and stand out among various types of new crown vaccines. 3) It is easy to iterate, and the combination with other vaccines in the future can reduce costs, including injection costs. 4) It is not integrated into the genome and has good safety. In view of the above situation, an E2 fusion protein with good immunogenicity and high expression level, E2 encoding gene, etc. are needed to improve the immune protection effect of pigs and better develop mRNA vaccine products. Summary of the invention

[0005] 1. Technical issues to be resolved

[0006] The present invention provides a method for preparing and applying an E2 fusion protein and vaccine of classical swine fever virus which can be applied on a large scale and has good immune protection effect against classical swine fever virus and has high safety, broad application prospects and low cost.

[0007] (II) Technical solution

[0008] In the first aspect, the present invention provides an E2 fusion protein of classical swine fever virus, wherein the E2 fusion protein comprises an E2 protein fragment of classical swine fever virus lacking a C-terminal domain, obtaining the amino acid sequence of the E2 protein fragment as shown in SEQ ID NO: 2, and replacing the protein fragment of the C-terminal domain of the E2 protein with a porcine epidemic diarrhea virus transmembrane region to obtain the E2 fusion protein.

[0009] Furthermore, the E2 fusion protein satisfies at least one of the following conditions:

[0010] (1) The porcine epidemic diarrhea virus transmembrane region has an amino acid sequence as shown in SEQ ID NO: 7;

[0011] (2) The C-terminus of the E2 fusion protein further comprises a polypeptide tag; preferably, the polypeptide tag is connected to the C-terminus of the E2 fusion protein via a linker;

[0012] (3) the E2 fusion protein has the amino acid sequence shown in SEQ ID NO: 6;

[0013] (4) The N-terminus of the E2 fusion protein includes a signal peptide.

[0014] In a second aspect, the present invention provides a method for preparing an E2 fusion protein, which comprises expressing a polynucleotide encoding the E2 fusion protein as claimed in claim 1 or 2 in an organism or a biological cell to obtain the E2 fusion protein.

[0015] In a third aspect, the present invention provides a biomaterial, wherein the biomaterial is at least one of the following (1) to (3):

[0016] (1) A polynucleotide encoding the E2 fusion protein according to claim 1 or 2;

[0017] (2) a recombinant expression vector containing the polynucleotide described in (1);

[0018] (3) A transformant containing the polynucleotide described in (1) or the recombinant expression vector described in (2).

[0019] Furthermore, the polynucleotide is mRNA; preferably, the mRNA further comprises a 5' cap structure, a 5' non-coding region, a 3' non-coding region and a polyA tail, and the mRNA has a sequence as shown in SEQ ID NO:5.

[0020] Furthermore, the mRNA contains modified nucleotides; preferably, the modification is a pseudouridine modification; more preferably, the uracil nucleotide of the mRNA is a pseudouridine.

[0021] In a fourth aspect, the present invention provides a vaccine comprising E2 fusion protein or biological material.

[0022] Furthermore, the vaccine is an mRNA vaccine, which includes a polynucleotide encoding the E2 fusion protein as described in claim 1 or 2 and a delivery vector; preferably, the delivery vector is a liposome.

[0023] In a fifth aspect, the present invention provides the use of E2 fusion protein, or biological material, or vaccine in the preparation of drugs for preventing and treating classical swine fever virus infection.

[0024] In a fifth aspect, the present invention also proposes a method for preventing and treating classical swine fever virus infection, the method comprising administering an effective amount of E2 fusion protein or the biological material or vaccine to a subject; the subject is a pig.

[0025] (III) Beneficial effects

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows: the present invention provides an E2 fusion protein, in which the C-terminus of the E2 protein is replaced with a fusion protein of the porcine epidemic diarrhea virus transmembrane region, the fusion protein has a high blocking rate for classical swine fever virus, the polynucleotide synthesized depending on the fusion protein can efficiently express the fusion protein, and the mRNA vaccine prepared based on the polynucleotide can have a good immune protection effect on pigs, is not affected by the presence of maternal antibodies, has high safety, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Schematic diagram of the process for preparing mRNA for in vitro transcription.

[0028] Figure 2 The results of immunoblotting to detect the expression of three mRNAs, E2_EX, E2-TMPEDV and E2-TMHA, in 293T cells transfected with lip2000. The detection antibody is anti-E2 protein antibody. Each lane is from the same PVDF membrane, and irrelevant lanes are removed by cutting; NC: negative control without transfection.

[0029] Figure 3 Schematic diagram of the encapsulation process of nanolipid particles.

[0030] Figure 4 The results are shown for the expression of three mRNAs, E2_EX, E2-TMPEDV and E2-TMHA, in 293T cells encapsulated with lipid nanoparticles. The detection antibody is anti-E2 antibody.

[0031] Figure 5 Comparison of E2-specific antibody levels at different time points in pigs immunized with three mRNA vaccines, E2_EX, E2-TMPEDV and E2-TMHA.

[0032] Figure 6 The results of the duration of immunity were observed in pigs immunized with E2-TMPEDV mRNA vaccine and subunit vaccine.

[0033] Figure 7 Results of an IFN-γ elispot experiment in pigs immunized with E2-TMPEDV mRNA vaccine and subunit vaccine.

[0034] Figure 8 The results of the second IFN-γ elispot experiment in pigs immunized with E2-TMPEDV mRNA vaccine and subunit vaccine.

[0035] Fig. 9Comparison of E2-specific antibody levels at different time points in pigs immunized with E2-TMPEDV mRNA vaccine and subunit vaccine in the presence of maternal antibodies.

[0036] Fig.10 Experimental flow chart A for immunizing pigs with E2-TMPEDV mRNA vaccine.

[0037] Fig.11 Figure B is the survival curve of pigs immunized with E2-TMPEDV mRNA vaccine;

[0038] Fig.12 Figure C shows the temperature detection data of pigs immunized with E2-TMPEDV mRNA vaccine;

[0039] Fig.13 Figure D shows the clinical symptom scores of pigs immunized with E2-TMPEDV mRNA vaccine;

[0040] Fig.14 This is the viral load monitoring chart of pigs immunized with E2-TMPEDV mRNA vaccine; among them, E, F, G, and H are the monitoring of viral loads of blood, oral swabs, nasal swabs, and anal swabs on the 2nd, 4th, 6th, 8th, 10th, 12th, 14th, and 16th days after infection, respectively.

[0041] Fig.15 This is the viral load detection chart of the heart, liver, spleen, lung and kidney of pigs immunized with E2-TMPEDV mRNA vaccine on the 16th day after infection. DETAILED DESCRIPTION

[0042] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The test methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0043] The antigen design of this vaccine is mainly centered around the envelope glycoprotein E2 of classical swine fever. The source of the E2 sequence is the CSFV C strain (GENBANK accession number: AY805221.1). The sequences constructed in the embodiment include the secretory E2_EX with a truncation of the C-terminal of the E2 protein, and two replacement designs of the C-terminal transmembrane region, E2-TMHA and E2-TMPEDV (Table 1). In order to achieve high expression in pig cells, the untranslated region (UTR) and the coding region are codon-optimized for pigs. Furthermore, the N-terminus of the E2 fusion protein constructed in the above three schemes includes a signal peptide, which can promote the secretory expression of the E2 fusion protein in the host. The signal peptide is tissue plasminogen activator (t-PA). The C-terminus of the E2 fusion protein also includes a polypeptide tag to facilitate the separation and purification of the E2 fusion protein. The polypeptide tags include but are not limited to 6xHIS, Flag, GST, MBP, Strep, etc., and the polypeptide tag is preferably a 6*His tag (amino acid sequence: HHHHHH); preferably, the polypeptide tag is connected to the C-terminus of the E2 fusion protein through a linker, and the linker is a GS linker (such as the amino acid sequence: GGGGS or GSSGS).

[0044] Table 1 Design of Classical Swine Fever E2 Antigen

[0045]

[0046] Example 1: In vitro synthesis of classical swine fever E2 sequence mRNA

[0047] like Figure 1 As shown, three E2 sequences (E2_EX, E2-TMHA and E2-TMPEDV) were optimized for porcine codons. The optimized sequences were used as templates for mRNA synthesis, and UTRs were added to their 3' and 5' ends, respectively. They were cloned into the downstream of the T7 promoter on the pcDNA3.1 vector, and used as templates for PCR amplification to add Poly (A) tails. After obtaining the DNA template, in vitro transcription was performed, and the cap analog EZ-Cap (Cat. No.: B8176, purchased from APExBIO) was used to cap the RNA at the 5' end. In order to eliminate the interference of the DNA template, the transcription product was digested with DNase, and purified and recovered to obtain the synthesized mRNA.

[0048] In order to reduce the immunogenicity of mRNA, nucleotides carrying pseudouridine (Ψ) were incorporated into the transcription to obtain pseudouridine-modified mRNA, and the introduction and replacement of pseudouridine-modified nucleotides during synthesis were 100%. The pseudouridine (Ψ) modified group was compared with the non-modified (UTP) group.

[0049] After the concentration of purified mRNA was measured, Lipofectamine TM The mRNA was packaged and transfected into HEK293T cells using Lip2000 transfection reagent (Cat. No. 11668019, purchased from Thermo Fisher Scientific). After culturing in a 37°C, 5% CO2 incubator for 24 hours, cell samples were collected using lysis buffer and Western blotted with anti-E2 antibody (source: China Veterinary Drug Administration) to verify the expression of the encoded protein. The results are shown in the figure. Figure 2 As shown: the expression levels of the three sequences are all very high, and the size is about 60kD, which is consistent with expectations; from the expression level, E2-TMPEDV>E2_EX>E2-TMHA; E2-TMPEDV and E2-TMHA were both detected in the Ψ group and the UTP group, and from the expression level, the UTP group>Ψ group. This shows that this example successfully obtained three mRNA sequences that can efficiently express CSFV E2.

[0050] Example 2: Using lipids to encapsulate mRNA to obtain nanolipid nanoparticles (mRNA-LNP)

[0051] In order to achieve effective presentation of mRNA in vivo, it is selected to be encapsulated with lipids to obtain LNP. The lipid molecules used here include four types: cationic lipid RL151 (article number: W211-YB211202, purchased from: Zhejiang Shenzhou Pharmaceutical), cholesterol, distearoyl phosphatidylcholine (DSPC) and polyethylene glycol-modified lipid molecules (PEG2000-DMG). The four lipids of RL151: cholesterol: DSPC: PEG2000-DMG are dissolved in ethanol at a molar ratio of 50:38.5:10:1.5, and the ethanol solution of the mixed lipids and the mRNA obtained in Example 1 are controlled to have a nitrogen-phosphorus ratio of ionizable lipids to mRNA of (3-15):1 to prepare lipid nanoparticles. The lipids dissolved in ethanol and the mRNA obtained in Example 1 are quickly mixed and encapsulated by a microfluidic device, and then allowed to stand, dialyzed, filtered, and then self-packaged to obtain lipid nanoparticles LNP ( Figure 3 ), and the particle size, PDI and encapsulation efficiency of LNP were determined (Table 2).

[0052] Table 2. Characterization of E2 nanolipid particles

[0053]

[0054]

[0055] Subsequently, the expression of lipid nanoparticles described in Table 2 in porcine cells was evaluated in HEK293T cells. After transfecting HEK293T cells with the three E2 sequences encapsulated by LNP, they were placed in a 37°C, 5% CO2 incubator for 24 hours, and cell samples were collected with lysis buffer and the expression of the encoded proteins was verified by Western blot. The results are shown in Figure 4 As shown: consistent with the mRNA expression results, all three sequences encapsulated by LNP were expressed. In terms of expression level, the E2-TMPEDV group had the highest expression level, and for the three sequences, the UTP group was > the Ψ group.

[0056] Example 3: Verification of the immunogenicity and protective effect of swine fever mRNA vaccine in animal models

[0057] 42-day-old piglets (CSFV, PCV, PRRSV, PEDV, FMDV antigen and antibody negative) were selected for animal testing. They were raised in the test site for 7 days and their body temperature was measured every day. The results showed that the body temperature of the experimental animals was between 38.0℃ and 40.0℃, which is within the normal body temperature range of pigs and meets the test requirements. During the feeding period, the test animals were in good spirits, had a strong appetite, and had no abnormal clinical symptoms, which met the test requirements.

[0058] Animal study 1: Comparison of the immunogenicity of three mRNA vaccines, E2_EX, E2-TMPEDV and E2-TMHA, in pigs ( Figure 5 ). 12 pigs were randomly divided into 3 groups (n=4), and immunized twice on day 0 and day 14, with 60μg vaccine injected respectively. Serum was collected on days 7, 14, 21, 28, and 35, and the antibody blocking rate of the E2 protein of the classical swine fever virus was detected using the "Horse Fever Virus Antibody Diagnostic ELISA Kit" (Cat. No.: 99-43220) produced by IDEXX. The blocking rate ≥40% was positive for antibodies, the blocking rate ≤30% was negative, and the blocking rate between the two was suspicious. The experiment found that on days 21, 28, and 35 (i.e., days 7, 14, and 21 after the second immunization), the antibody blocking rate of the E2 protein in the E2-TMPEDV group (E2tm group in the figure) could reach about 90%, and the individual differences were very small. The average antibody blocking rate of the E2 protein in the E2_EX group and the E2-TMHA group was between 55% and 75%, which was significantly different from the E2-TMPEDV group (p<0.0001). Therefore, the immunogenicity of E2-TMPEDV vaccine is superior to that of E2_EX vaccine and E2-TMHA vaccine.

[0059] In subsequent experiments, the focus was on studying the immunogenicity of the E2-TMPEDV vaccine and its protective effect on infected pigs.

[0060] Animal study 2: Comparison of antibody levels and duration between E2-TMPEDV vaccine and subunit vaccine.

[0061] Preparation of Classical Swine Fever E2 Subunit Vaccine: The CHO-K1 cell line (stored by Jinyu Baoling Biological Pharmaceutical Co., Ltd.) that efficiently expresses the Classical Swine Fever virus E2 protein was taken out from the liquid nitrogen tank and quickly thawed in a 37°C water bath. Resuspend the cells in 302 serum-free medium (purchased from Sigma), inoculate the cells in a cell shaker of appropriate volume, and culture them in a shaker at 37°C and 5CO2 for 48 to 72 hours. After expanding the culture, harvest the cell supernatant, filter and concentrate the E2 protein using a hollow fiber ultrafiltration concentration system. After inactivation with β-propiolactone, add oil adjuvant for emulsification to prepare the E2 subunit vaccine.

[0062] The test was conducted on 6 heads in each group. The test set up several groups including E2-TMPEDV (Ψ) 60μg, E2-TMPEDV (UTP) 60μg, E2-TMPEDV (Ψ) 150μg, E2-TMPEDV (UTP) 150μg, subunit vaccine, and blank control. Similarly, two immunizations were conducted on day 0 and day 14, with 1ml of classical swine fever E2-mRNA vaccine each time. Classical swine fever E2 subunit vaccine immunization 2ml / head. Sera were collected every 7 days until day 56. The antibody blocking rate of classical swine fever virus E2 protein was detected using the "Classic swine fever virus antibody diagnostic ELISA kit" (catalog number: 99-43220) produced by IDEXX. A blocking rate ≥40% was considered antibody positive, a blocking rate ≤30% was considered negative, and anything in between was considered suspicious. Figure 6 The statistical results show that the serum antibodies of all pigs immunized with mRNA vaccines were all positive 7 days after the secondary immunization (i.e., day 21). Comparison of the mRNA vaccine group with the subunit vaccine group and the blank control group showed that the mRNA vaccine can stimulate the body to produce specific E2 antibodies, achieving the same effect as the subunit vaccine, and the produced E2 antibodies are stable and persistent in the pigs. Judging from the antibody blocking rate, the effect of the 150μg dose is more stable than that of the 60μg dose, and the effects of the Ψ-modified and UTP-modified types at the same dose are similar.

[0063] Animal experiment 3: Detection of cellular immune response produced by E2-TMPDEV mRNA vaccine ( Figure 7 , Figure 8). Anticoagulated blood was collected from the immune pigs 14 days after the first immunization and 21 days after the second immunization. The anticoagulated blood was diluted with 2% FBS + 1% double antibody + PBS (the double antibody was Gibco culture medium, item number 15140-122). The cells were separated using the Dayu porcine lymphocyte separation kit, and the PBMC cells of the immune pigs were stimulated with CSFV E2 protein. The experimental results showed that the number of IFN-γ spots did not increase when PBS was added to the negative control group; a large number of IFN-γ spots were stimulated when ConA was added to the negative control group, indicating that the experiment was established. Each vaccine group was stimulated with E2 protein, and the IFN-γ level produced 14 days after the first immunization was very low (the average number of spots was <10 / 5e 5 Cells), and the average number of IFN-γ spots in each vaccine group increased compared with the blank control group 21 days after the second vaccination, and the level of increase in the E2-TMPEDV (Ψ) 150μg group was the highest, higher than the subunit vaccine group. Therefore, the E2-TMPEDV mRNA vaccine can produce cellular immunity, and the E2-TMPEDV (Ψ) 150μg group is the best.

[0064] Animal study 4: Comparison of antibody levels between E2-TMPEDV vaccine and subunit vaccine in the presence of maternal antibodies ( Figure 8 ). Maternal antibodies come from sows and can be transferred to embryos through the placenta, colostrum, etc., so that newborn piglets are protected. However, the presence of maternal antibodies will interfere with and reduce the immune response of newborn piglets to the primary immunization vaccine, thereby weakening the effect of the primary immunization. This example uses 12 7-day-old piglets, which are randomly divided into 3 groups (n=4). The piglets are immunized with 60μg E2-TMPEDV vaccine, 60μg of a certain E2 subunit vaccine, and PBS, respectively. The sera of the immunized pigs were collected on days 0, 7, 14 and 21 after immunization, and the antibody blocking rate of the E2 protein was detected. The results showed that the antibody blocking rate of all groups on day 0 was about 50%, and the PBS group gradually decreased to negative levels on days 7 and 14. The antibody blocking rate of the E2-TMPEDV vaccine group increased significantly on the 7th day after immunization, reaching about 90%, and remained at a very high level on days 14 and 21, indicating that the immunogenicity of this group of vaccines was not interfered with by maternal antibodies. The antibody blocking rate of the E2 subunit vaccine injection group decreased on the 7th day, and then gradually increased to about 70%. These results show that the E2-TMPEDV vaccine can induce faster and stronger E2 antibodies without being affected by maternal antibodies.

[0065] Animal experiment 5: Detection of the challenge response of the E2-TMPDEV mRNA vaccine group ( Fig. 9 On the 42nd day of immunization, 5 pigs in each mRNA vaccine group were injected subcutaneously in the neck with 1 mL (10 6infection dose), and the blank control group (PBS group) was injected subcutaneously in the neck with 1 mL of Shimen blood poisoning (10 6 Infectious dose) was used as the control, and the negative control group was not challenged. The body temperature of all pigs was recorded and clinical symptoms were monitored daily. Clinical samples of serum and oral swabs, nasal swabs and anal swabs were collected on days 0, 2, 4, 6, 8, 10, 12, 14, and 16 after the challenge, and all surviving pigs were euthanized and autopsied on day 58. The number of viral RNA copies in swabs, blood and tissue samples was determined by fluorescent quantitative qRT-PCR. The primers and probes for real-time fluorescent RT-PCR of classical swine fever virus in the method of "Real-time fluorescent RT-PCR detection method for classical swine fever virus" (GB / T 27540-2011) were used, and the upstream primer F: 5'-TACAGGACAGTCGTCAGTAGTTCGA-3'; downstream primer R: 5'-CCGCTAGGGTTAAGGTGTGTCT-3'; probe P: 5'-FAM-CCCACCTCGAGATGCTATGTGGACGA-TAMRA-3', all of which were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0066] The body temperature of the pigs in the control group began to rise 3 days after the virus challenge, and all died on the 9th day. However, the body temperature of the pigs immunized with the E2-TMPEDV mRNA vaccine was normal, and all survived during the 16-day observation period, indicating that these vaccines can provide 100% protection.

[0067] Combined with Figures 10 to 15 In order to explore the detoxification of immunized pigs, oral swabs, nasal swabs, anal swabs and blood were collected for fluorescence quantitative detection 2, 4, 6, 8 and 10 days after the virus challenge. The number of virus copies in the blood, oral, nasal and anal swabs of the control group after the virus challenge reached 10 6 ~10 7 / mL. The highest number of virus copies in pigs immunized with E2-TMPEDV mRNA vaccine was only 10 3 / mL, and over time, the number of viral copies decreased to 10 2 / mL until it disappeared. On the 16th day after the challenge, the pigs in each mRNA vaccine group were killed and the viral loads in the heart, liver, spleen, lung, and kidney of the organs were analyzed and compared with those of the control group that had died previously. Viruses were detected in all organs of the control group. 4 -10 7 / mg. The viral load of each organ in the E2-TMPEDV(Ψ)60μg group was 10 1 -10 2The viral load of each organ in the E2-TMPEDV (UTP) 60μg group was about 10 copies / mg, the viral load of each organ in the E2-TMPEDV (Ψ) 150μg group was <10 copies / mg, and no virus was detected in each organ in the E2-TMPEDV (UTP) 150μg group. The above results show that E2-TMPEDV mRNA vaccine, whether modified or unmodified, and whether the dose is 60μg or 150μg, can effectively inhibit the detoxification of infected pigs, and 150μg is more effective.

[0068] In general, the embodiment designs three mRNA vaccines E2_EX, E2-TMHA and E2-TMPEDV for E2 protein, among which the E2-TMPEDV vaccine has a high expression level in cultured cells, and the level of in vivo specific antibodies produced after immunization of pigs is the highest. The specific antibody levels of immunized pigs were continuously monitored, and it was found that at different time points, whether E2-TMPEDV was a modified (Ψ) vaccine or a non-modified (UTP) vaccine, the effect of a 150 μg dose was comparable to that of a subunit vaccine. From the perspective of cellular immunity, the effect of a 150 μg dose of the E2-TMPEDV modified (Ψ) vaccine is even higher than that of a subunit vaccine. In the presence of maternal antibodies, the E2-TMPEDV vaccine can be unaffected by maternal antibodies, and can induce faster and stronger E2 antibodies than subunit vaccines. The challenge experiment found that E2-TMPEDV mRNA vaccine, whether it is a modified (Ψ) vaccine or a non-modified (UTP) vaccine, whether it is a 60μg or 150μg dose, can produce 100% protection against challenged pigs, and effectively inhibit the excretion of toxins in infected pigs, with 150μg having a better effect.

[0069] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. 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.

Claims

1. The E2 fusion protein of classical swine fever virus, characterized in that The E2 fusion protein comprises an E2 protein fragment of classical swine fever virus lacking a C-terminal domain, obtaining the amino acid sequence of the E2 protein fragment as shown in SEQ ID NO: 2, and replacing the protein fragment of the C-terminal domain of the E2 protein with the porcine epidemic diarrhea virus transmembrane region to obtain the E2 fusion protein.

2. The E2 fusion protein according to claim 1, characterized in that The E2 fusion protein satisfies at least one of the following conditions: (1) The porcine epidemic diarrhea virus transmembrane region has an amino acid sequence as shown in SEQ ID NO: 7; (2) The C-terminus of the E2 fusion protein further comprises a polypeptide tag; preferably, the polypeptide tag is connected to the C-terminus of the E2 fusion protein via a linker; (3) the E2 fusion protein has the amino acid sequence shown in SEQ ID NO: 6; (4) The N-terminus of the E2 fusion protein includes a signal peptide.

3. A method for preparing the E2 fusion protein according to claim 1 or 2, characterized in that: The polynucleotide encoding the E2 fusion protein as claimed in claim 1 or 2 is expressed in an organism or biological cell to obtain the E2 fusion protein.

4. A biomaterial, characterized in that: The biological material is at least one of the following (1) to (3): (1) A polynucleotide encoding the E2 fusion protein according to claim 1 or 2; (2) a recombinant expression vector containing the polynucleotide described in (1); (3) A transformant containing the polynucleotide described in (1) or the recombinant expression vector described in (2).

5. The biomaterial according to claim 4, characterized in that The polynucleotide is mRNA; preferably, the mRNA further comprises a 5' cap structure, a 5' non-coding region, a 3' non-coding region and a polyA tail, and the mRNA has a sequence as shown in SEQ ID NO:

5.

6. The biomaterial according to claim 5, characterized in that The mRNA comprises modified nucleotides; preferably, the modification is a pseudouridine modification; more preferably, the uracil nucleotide of the mRNA is a pseudouridine.

7. A vaccine comprising the E2 fusion protein according to claim 1 or 2 or the biological material according to any one of claims 4 to 6.

8. The vaccine according to claim 7, characterized in that The vaccine is an mRNA vaccine, which includes a polynucleotide encoding the E2 fusion protein as described in claim 1 or 2 and a delivery vector; preferably, the delivery vector is a liposome.

9. Use of the E2 fusion protein according to claim 1 or 2, or the biological material according to any one of claims 4 to 6, or the vaccine according to claim 7 or 8 in the preparation of a medicament for preventing and treating classical swine fever virus infection.

10. A method for preventing and treating classical swine fever virus infection, characterized in that: The method comprises administering an effective amount of the E2 fusion protein of claim 1 or 2, or the biological material of any one of claims 4 to 6, or the vaccine of claim 7 or 8 to a subject; the subject is a pig.

Images