Application of swine antiviral protein as immunopotentiator in hog cholera virus inactivated vaccine

Abstract

According to the invention, swine antiviral proteins IFITM1, IFITM3 and Viperin are used as immunopotentiators and are applied to the swine fever virus E2 protein antiviral baculovirus inactivated vaccine. When swine antiviral proteins IFITM1, IFITM3 and Viperin serve as immunopotentiators and are used for immunization together with the swine fever virus E2 protein antiviral baculovirus inactivated vaccine, immunized mice can be promoted to generate higher-content swine fever virus serum IgG antibodies. At present, the inactivated vaccine of the antiviral protein immunopotentiator for the pigs is not put into use in the domestic market. However, the swine immunopotentiator adopted in the scheme is matched with the swine fever virus E2 protein antiviral baculovirus inactivated vaccine, so that the swine fever can be more effectively prevented and controlled, and the blank in the aspect of immunopotentiators in the current pig raising market is filled.

Description

Technical Field

[0001] The present invention relates to the technical field of veterinary biological products, and in particular to the application of a porcine antiviral protein as an immunopotentiator in an inactivated swine fever virus vaccine. Background Art

[0002] Classical swine fever is a highly acute, febrile, contagious disease caused by the classical swine fever virus. The disease is classified into the most acute, acute, subacute, chronic, and mild forms. Its clinical characteristics are rapid onset, high fever, degeneration of small blood vessel walls, generalized petechial hemorrhages, and splenic infarction. The classical swine fever virus can be transmitted through various routes, including the digestive and respiratory tracts. Infected sows can also infect their fetuses vertically through the placenta. It can occur year-round, with high morbidity and mortality rates. It can be devastating to the pig industry, making it one of the most serious infectious diseases affecting the sector. Therefore, strengthening the prevention and control of this disease is of vital importance to the livestock industry.

[0003] Currently, in response to the prevalence and harm of swine fever, various countries have adopted various methods and measures to control the spread of the disease. Traditional inactivated vaccines still dominate the market for immunization against the disease. However, commercial vaccines offer limited protection against emerging strains and variants. Furthermore, new influenza vaccines targeting the swine fever virus, such as subunit and antiviral vaccines, have also failed to gain market acceptance due to various issues, including poor immune efficacy. Therefore, to address the shortcomings of current inactivated swine fever vaccines and newer vaccines, such as poor immunogenicity, weak immune responses, and short immune memory, the research on safe and effective immune enhancers is particularly urgent.

[0004] Vaccine adjuvants, as auxiliary substances, can improve vaccine efficiency by combining with specific antigens to enhance antigen delivery, reduce antigen dosage requirements, and enhance vaccine-induced host immune responses. Vaccine adjuvants play a vital role in improving vaccine efficiency, especially in the preparation of vaccines with weak immunogenicity, such as inactivated vaccines, synthetic peptide vaccines, subunit vaccines, and DNA vaccines. These vaccines often have difficulty stimulating a sufficient immune response after vaccination due to insufficient immunogenicity of the antigens, so adjuvants are needed to enhance their effectiveness. Currently, adjuvants have been widely used in the preparation of vaccines, especially inactivated vaccines or new genetically engineered vaccines, which require the cooperation of adjuvants.

[0005] Numerous recent studies have revealed the presence of numerous ISGs (internal growth regulators) within host cells with unique antiviral functions. These ISGs imbue cells with potent antiviral capabilities, effectively defending against foreign viral invasion. For example, when foreign microorganisms such as viruses and bacteria invade, interferon genes are activated, inducing the expression of IFITMs. IFITMs can inhibit the membrane antiviral response between viruses and target cells by altering the lipid order within the cell, thereby modifying membrane rigidity and fluidity. IFITMs can inhibit the entry of many viruses, thereby blocking the viral infection process; viperin has been shown to inhibit viral release and the replication of multiple viruses. More importantly, viperin exhibits antiviral activity against a wide range of viruses, mediating immune-related signaling pathways, T cell development, and regulating cellular metabolism, thereby comprehensively enhancing the body's antiviral capacity. These properties hold great promise for the application of IFITMs and viperin as vaccine adjuvants.

[0006] Currently, there are no porcine antiviral protein-based immunopotentiators on the market in my country. Therefore, combining host proteins with broad-spectrum antiviral activity, such as Viperin, with an inactivated baculovirus vaccine targeting the Classical Swine Fever virus E2 protein not only addresses the immunodeficiency of existing vaccines but also meets the modern livestock industry's demand for highly effective prevention and control technologies. This has important practical implications for advancing Classical Swine Fever prevention and control technologies and the development of related products. Summary of the Invention

[0007] Based on the above, the purpose of the present invention is to propose a veterinary immunopotentiator that is reliable and effective to implement, easy to prepare, and can be used to enhance the immune efficacy of the inactivated baculovirus vaccine against the E2 protein of the classical swine fever virus.

[0008] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0009] In a first aspect, the present invention proposes the use of antiviral proteins IFITM1, IFITM3 or Viperin as immune enhancers in the preparation of inactivated baculovirus vaccines against classical swine fever virus E2 protein.

[0010] In some embodiments, the method for preparing an inactivated baculovirus vaccine comprising the antiviral protein IFITM1, IFITM3 or Viperin as an immunopotentiator comprising the steps of:

[0011] In a sterile container, the inactivated baculovirus vaccine containing the E2 protein of classical swine fever virus and a vaccine adjuvant are added at a ratio of 1:2.81, and the antiviral proteins IFITM1, IFITM3 or Viperin are added to a concentration of 167 μg / mL in the vaccine;

[0012] Use HR-500 dispersing emulsifier to gradually emulsify from gear A to gear E for 30 minutes.

[0013] In some embodiments, the antiviral proteins IFITM1, IFITM3, and Viperin are obtained by prokaryotic expression in Escherichia coli.

[0014] In some embodiments, the method for obtaining the antiviral proteins IFITM1, IFITM3, and Viperin comprises the following steps:

[0015] 1) The antiviral plasmids pCold-TF-IFITM1, pCold-TF-IFITM3, and pCold-TF-Viperin were transformed into competent cells respectively. The transformed cells were spread on a solid LB culture dish containing ampicillin resistance and cultured at 37°C for 14 hours.

[0016] 2) Select a single positive colony from the above culture dish and inoculate it into liquid culture medium containing antibiotics. Cultivate the culture at 37°C and 200 rpm with shaking until the OD600 reaches 0.8-1.2. Then, add IPTG to the culture solution to a final IPTG concentration of 0.1-0.5 mmol / L.

[0017] 3) The bacterial solution containing IPTG was then induced and cultured in a constant temperature shaker at 16°C and 200 rpm for 16 h;

[0018] 4) Wet the precipitated cells with 5 mL of 1× PBS buffer, mix thoroughly by pipetting, and then place in an ice bath for ultrasonic disruption;

[0019] 5) The bacterial suspension obtained after ultrasonic disruption was centrifuged at 8000 rpm at 4°C for 15 min, and the supernatant was retained;

[0020] 6) Purify the antiviral proteins IFITM1, IFITM3, and Viperin using Ni-NTA affinity chromatography;

[0021] Alternatively, the antiviral proteins IFITM1, IFITM3, and Viperin can be purified using ammonium sulfate precipitation.

[0022] In some embodiments, the ultrasonic fragmentation conditions in step 4) are set as follows: 20% power, 5s working time, 5s interval, cumulative working time of 20min, and a total of 40min.

[0023] In some embodiments, the antiviral proteins IFITM1, IFITM3, and Viperin are purified using a Ni-NTA affinity chromatography purification method, specifically comprising the following steps:

[0024] The antiviral proteins IFITM1, IFITM3, and Viperin were purified by Ni-NTA affinity chromatography, and 500 mmol / L imidazole was used to elute the antiviral proteins IFITM1, IFITM3, and Viperin to obtain the purified antiviral proteins IFITM1, IFITM3, and Viperin.

[0025] In some embodiments, the antiviral proteins IFITM1, IFITM3, and Viperin are purified using ammonium sulfate precipitation, specifically comprising the following steps:

[0026] Ammonium sulfate was added to the supernatant, the mixture was slowly shaken at 10°C for 2 h, centrifuged at 8500 rpm for 15 min, the supernatant was discarded, 15 mL of 1× PBS buffer was added to resuspend the precipitate, and centrifuged again at 8500 rpm for 15 min. The supernatant was taken for ultrafiltration to obtain the purified antiviral proteins IFITM1, IFITM3, and Viperin.

[0027] In some embodiments, in step 5), the bacterial pellet is simultaneously resuspended with an equal volume of 1×PBS buffer and thoroughly mixed to prepare a protein sample, which is then sampled for SDS-PAGE electrophoresis to analyze the solubility of the antiviral protein in the bacteria.

[0028] In a second aspect, the present invention also proposes the use of antiviral proteins IFITM1, IFITM3 or Viperin as veterinary immunopotentiators in the preparation of veterinary vaccines.

[0029] The beneficial effects of the invention are:

[0030] 1. The immunopotentiator provided by the present invention can enhance the effect of inactivated swine fever vaccine on the production of higher levels of serum antibodies in pigs, and can effectively enhance the immune effect of the vaccine when combined with the inactivated antiviral vaccine.

[0031] 2. Currently, there are no porcine antiviral protein immunopotentiators available in China. Therefore, developing highly effective immunopotentiators for use with inactivated vaccines meets the current market demands of the livestock industry and is of great significance for both vaccine development and the prevention and control of porcine-derived diseases in my country. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0033] Figure 1 Double enzyme digestion identification and product electrophoresis of porcine antiviral prokaryotic expression vectors pCold-TF-IFITM1, pCold-TF-IFITM3, and pCold-TF-Viperin;

[0034] Figure 2 Purification of the antiviral porcine antiviral proteins IFITM1, IFITM3, and Viperin was performed by SDS-PAGE assay;

[0035] Figure 3 The effects of antiviral proteins IFITM1, IFITM3, and Viperin on the expression level of PRV gE protein in PK-15 cells;

[0036] Figure 4 This is a comparison chart of the mouse and porcine homology of IFITM1 sequences;

[0037] Figure 5 This is a comparison chart of the mouse and porcine homology of IFITM3 sequences;

[0038] Figure 6 This is a comparison chart of the mouse and porcine homology of Viperin sequences;

[0039] Figure 7 is the expression level of NP protein of H3N2 at the mRNA level in the antiviral protein IFITM1, IFITM3 and Viperin treatment groups;

[0040] Figure 8 The effects of vaccine group, IFITM1+ vaccine group, IFITM3+ vaccine group and Viperin+ vaccine group on specific IgG antibodies in the serum of immunized mice;

[0041] Figure 9 The effects of vaccine group, IFITM1+ vaccine group, IFITM3+ vaccine group and Viperin+ vaccine group on the proliferation rate of spleen T lymphocytes in immunized mice;

[0042] Figure 10 The induced expression of antiviral proteins IFITM1, IFITM3 and Viperin. DETAILED DESCRIPTION

[0043] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0044] Example 1. Screening and preparation of antiviral protein Viperin as an immunopotentiator

[0045] 1. Construction and identification of prokaryotic expression vectors for the antiviral proteins IFITM1, IFITM3, and Viperin

[0046] The porcine IFITM1, IFITM3 and Viperin gene fragments and the prokaryotic expression vector pCold-TF were double-digested using restriction endonucleases XhoI and XbaI. The gene fragments and vectors recovered after double digestion and purification were ligated with T4 DNA ligase, transformed and the antiviral plasmids were extracted. Three antiviral plasmids, pCold-TF-IFITM1, pCold-TF-IFITM3 and pCold-TF-Viperin, were successfully constructed. Figure 1 As shown, the antiviral plasmids were identified by double enzyme digestion, and the target gene fragments with the expected size were obtained. The sequencing of pCold-TF-IFITM1, pCold-TF-IFITM3 and pCold-TF-Viperin was confirmed to be correct. Figure 1 , indicating that three antiviral prokaryotic expression vectors pCold-TF-IFITM1, pCold-TF-IFITM3 and pCold-TF-Viperin were successfully constructed.

[0047] 2. Inducible Expression and Solubility Analysis of Antiviral Proteins IFITM1, IFITM3, and Viperin

[0048] The prokaryotic expression vectors pCold-TF-IFITM1, pCold-TF-IFITM3 and pCold-TF-Viperin were transformed into competent E. coli. The positive clone strains were selected and inoculated into LB liquid culture medium for culture. First, pre-culture was carried out in a 37°C constant temperature shaker environment until the OD600 value reached 0.8-1.2; IPTG was added to make the final concentration of IPTG in the bacterial solution 0.1mmol / L and the shaking conditions were adjusted to 16°C and 200rpm to induce expression for 16 hours. The uninduced bacterial solution and the induced bacterial solution were analyzed by SDS-PAGE to detect the expression status of the antiviral proteins IFITM1, IFITM3 and Viperin. The antiviral proteins IFITM1, IFITM3 and Viperin can be successfully expressed and specific bands are expressed near 66kDa, 69kDa and 93kDa, respectively, which are consistent with the expected antiviral protein size, indicating that the induction expression of the antiviral proteins IFITM1, IFITM3 and Viperin is successful (such as Figure 10 shown).

[0049] 3. Purification of antiviral proteins IFITM1, IFITM3, and Viperin

[0050] The above experiments have verified that the antiviral proteins IFITM1, IFITM3, and Viperin can be expressed as soluble proteins. Next, the antiviral proteins IFITM1, IFITM3, and Viperin were purified by Ni-NTA affinity chromatography and eluted with 500 mmol / L imidazole. The results are shown in Figure 2. Figure 2 As shown, relatively pure antiviral proteins IFITM1, IFITM3 and Viperin were obtained after Ni-NTA purification.

[0051] In addition to the Ni-NTA affinity chromatography purification method, a more efficient and convenient ammonium sulfate precipitation method was also used. The specific steps are as follows: After IPTG-induced bacterial culture, ultrasonically disrupt it, take the supernatant, add a certain amount of ammonium sulfate, slowly shake it at low temperature (10°C) for 2 hours, centrifuge it at 8500 rpm for 15 minutes, discard the supernatant, add 15 mL of 1× PBS buffer to resuspend the precipitate, centrifuge it again at 8500 rpm for 15 minutes, and take the supernatant for ultrafiltration to obtain the antiviral proteins IFITM1, IFITM3, and Viperin with high purity.

[0052] Example 2: Detection of the Effects of Antiviral Proteins IFITM1, IFITM3, and Viperin on the Replication Capacity of Various Viruses

[0053] 1. Antiviral proteins IFITM1, IFITM3, and Viperin inhibit PRV replication in PK-15 cells

[0054] Pseudorabies virus (PRV) Min-A strain (MOI = 1) was adsorbed onto porcine kidney PK-15 cells for 2 hours. Antiviral proteins at concentrations of 1 ng / mL, 10 ng / mL, and 100 ng / mL were then added to the PK-15 cells for 24 hours. The results showed that the expression of PRV gE protein mRNA decreased in an antiviral protein concentration-dependent manner in the IFITM1, IFITM3, and Viperin treatment groups. At an antiviral protein concentration of 100 ng / mL, IFITM1 and IFITM3 significantly inhibited PRV gE protein expression (p < 0.05), and Viperin highly significantly inhibited PRV gE protein expression (p < 0.01). These results indicate that IFITM1, IFITM3, and Viperin all inhibit PRV replication in PK-15 cells, with Viperin showing the strongest inhibitory effect.

[0055] 2. Effects of antiviral proteins IFITM1, IFITM3, and Viperin on PRV replication in other cells

[0056] The mouse and pig homology of IFITM1, IFITM3 and Viperin sequences were compared through NCBI. Primers were designed with reference to the CDS region nucleotide sequences of porcine (Sus scrofa) IFITM1, IFITM3 and Viperin in Genbank (NCBI numbers are: 100127358, 100518544, 396752 respectively). Using PK-15 cells 24 hours after PRV infection as templates, the porcine IFITM1, IFITM3 and Viperin genes were cloned, respectively, hereinafter abbreviated as IFITM1, IFITM3 and Viperin. The mouse and pig homology of IFITM1, IFITM3 and Viperin sequences were compared through NCBI. The results showed that the mouse and pig homology of IFITM1 gene was 75%, the homology of IFITM3 was 84%, and the homology of Viperin was 85%, as shown in Figure 2. Figure 4-6 The present invention relates to three antiviral proteins, all of which have high homology.

[0057] The three antiviral proteins involved in the present invention have high homology. Therefore, PRV was used to infect porcine cells 3D4 / 21 and mouse cells 3T3 in addition to PK-15 cells. The results are as follows: Figure 3It was shown that the antiviral Viperin could significantly inhibit the expression level of PRV gE protein in porcine and mouse cells (p<0.01).

[0058] Effects of antiviral proteins IFITM1, IFITM3, and Viperin on H3N2 replication in PK-15 cells

[0059] At the same time, the present invention also used other susceptible pig viruses to infect PK-15 cells to detect the antiviral function of antiviral IFITM1, IFITM3 and Viperin. First, PK-15 cells were infected with swine influenza H3N2 for 2 hours, and then incubated with IFITM1, IFITM3 and Viperin antiviral proteins at a concentration of 100 ng / mL for 48 hours to detect the replication of H3N2 in PK-15 cells. The results are as follows Figure 7 As shown, the expression levels of H3N2 NP protein at the mRNA level were significantly inhibited in the antiviral protein IFITM3 and Viperin treatment groups.

[0060] Example 3: Detection of the immune efficacy of the antiviral proteins IFITM1, IFITM3, and Viperin as immune enhancers in combination with the inactivated baculovirus vaccine containing the swine fever virus E2 protein.

[0061] In a sterile container, the Classical Swine Fever Virus E2 protein antiviral baculovirus inactivated vaccine and vaccine adjuvant were added at a ratio of 1:2.81, and the antiviral protein Viperin was added to a concentration of 167 μg / mL in the vaccine. The HR-500 dispersing emulsifier was used to gradually emulsify from gear A to gear E for 30 minutes to complete the preparation of the Viperin+ vaccine group. The same method was used to prepare the IFITM1+ vaccine group and the IFITM3+ vaccine group containing the antiviral proteins IFITM1 and IFITM3 as immune enhancers. The same method was used to prepare the vaccine group without adding antigen (sterile PBS replaced the antigen). The above-mentioned porcine antiviral protein was used as an immune enhancer and was co-prepared with the Classical Swine Fever Virus E2 protein antiviral baculovirus inactivated vaccine to form an inactivated vaccine, which was then tested for the inactivated vaccine:

[0062] 1. Appearance: Place 5 mL of the vaccine into a clean glass tube and observe whether the vaccine has any color change or is free of impurities.

[0063] 2. Dosage form: Use a clean pipette to draw up a small amount of vaccine and drop it onto the surface of cold water. Observe whether the vaccine spreads.

[0064] 3. Stability: Pipette 10 mL of each batch of vaccine into a centrifuge tube and centrifuge at 3000 rpm for 15 minutes to observe whether the vaccine has stratification.

[0065] 4. Sterility test: According to the appendix of the current Chinese Veterinary Pharmacopoeia, no bacteria grew.

[0066] 5. Pure inspection: Inspection is carried out according to the appendix of the current "Chinese Veterinary Pharmacopoeia", and all regulations are met.

[0067] 6. Determination of formaldehyde residue: The test was carried out according to the appendix of the current "Chinese Veterinary Pharmacopoeia". The results showed that it complies with the provisions of the General Rules for Veterinary Biological Products.

[0068] 7. Safety Assessment: Ten 5-week-old BALB / c mice were subcutaneously injected with 0.5 mL of the inactivated vaccine containing the immunopotentiator described herein. The mice were observed for 28 days, with weight changes measured every 7 days. All mice showed no significant changes in mental state, food intake, or drinking habits. The vaccine did not induce any local or systemic adverse reactions.

[0069] The results of the effects of antiviral IFITM1, IFITM3 and Viperin antiviral proteins on the body weight of mice are shown in Table 1. The body weight of each group increased after immunization. Compared with the blank control group, there was no significant difference in the other groups (p>0.05); compared with the vaccine group, there was no significant difference in the other groups (p>0.05).

[0070] Table 1 Effects of antiviral proteins IFITM1, IFITM3 and Viperin on the body weight of immunized mice

[0071]

[0072] At the same time, the mice were dissected on the 7th day (i.e., the 28th day) after the second immunization, and the spleen coefficient and thymus coefficient of the mice were detected. The effects of antiviral IFITM1, IFITM3 and Viperin antiviral proteins on the organ coefficients of mice are shown in Table 2. Compared with the blank control group, the spleen coefficients of each group showed an increasing trend after immunization, and the difference in the vaccine group was not significant (p>0.05). The IFITM1, IFITM3 and Viperin protein groups increased extremely significantly (p<0.01). The thymus coefficients of each group showed an increasing trend, but the differences were not significant (p>0.05).

[0073] Compared with the vaccine group, the spleen coefficients of the IFITM1, IFITM3 and Viperin protein groups were significantly increased (p<0.01), while the thymus coefficients were not significantly different (p>0.05).

[0074] This indicates that antiviral IFITM1, IFITM3 and Viperin antiviral proteins affect the spleen coefficient of immunized mice, but have no significant effect on the thymus coefficient of immunized mice.

[0075] Table 2 Effects of antiviral proteins IFITM1, IFITM3 and Viperin on organ coefficients of immunized mice

[0076]

[0077] Compared with the blank control group, ** indicates p < 0.01, *** indicates p < 0.001; compared with the vaccine group, ## indicates p < 0.01, ### indicates p < 0.001

[0078] 8. Effectiveness test:

[0079] ①Effect on specific IgG antibodies in the serum of immunized mice

[0080] On the 7th day after the second immunization, blood was collected from each group of mice by eye sampling, serum was separated, and the IgG expression level in the serum of each group of mice was detected using a mouse serum IgG detection kit. Figure 8 As shown in the data, compared with the blank group, the expression level of IgG in the vaccine group, IFITM1+ vaccine group, IFITM3+ vaccine group and Viperin+ vaccine group was extremely significantly increased (p<0.01); compared with the vaccine group, the expression level of IgG in the IFITM1+ vaccine group was significantly increased (p<0.05), and there was an increasing trend in the IFITM3+ vaccine group and Viperin+ vaccine group.

[0081] ②Effect on the proliferation rate of spleen T lymphocytes in immunized mice

[0082] To further test the proliferation rate of spleen lymphocytes, during the immunization period, 3 mice were randomly killed in each group every 7 days, spleen lymphocytes were isolated, and spleen T lymphocytes of the immunized mice were stimulated with Con A for 24 hours. The proliferation rate of spleen T lymphocytes of mice was tested. During the immunization period, the proliferation rate of spleen T lymphocytes of mice in the group with added antiviral protein was higher than that of the vaccine group and the control group. Figure 9 As shown, on day 14 after immunization, the proliferation rate of spleen T lymphocytes in the IFITM1+ vaccine group was significantly increased compared with the blank control group (p<0.01), and all other groups showed significant increases (p<0.05). Compared with the vaccine group, the proliferation rate of spleen T lymphocytes in the IFITM1+ vaccine group was significantly increased (p<0.05), and all other groups showed an increasing trend. On day 28 after immunization, i.e., day 7 after the second immunization, the proliferation rate of spleen T lymphocytes in all groups was significantly increased compared with the blank control group or the vaccine group (p<0.01), with the Viperin+ vaccine group showing the greatest increase. This indicates that the antiviral IFITM1, IFITM3, and Viperin can all promote the proliferation of spleen T lymphocytes in immunized mice, with Viperin having the best effect.

[0083] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. Application of antiviral proteins IFITM1, IFITM3 or Viperin as immune enhancers in the preparation of inactivated baculovirus vaccines against classical swine fever virus E2 protein.

2. The use according to claim 1, characterized in that The method for preparing an inactivated baculovirus vaccine against swine fever virus E2 protein using the antiviral protein IFITM1, IFITM3 or Viperin as an immunopotentiator comprises the following steps: In a sterile container, the inactivated baculovirus vaccine containing the E2 protein of classical swine fever virus and a vaccine adjuvant are added at a ratio of 1:2.81, and the antiviral proteins IFITM1, IFITM3 or Viperin are added to a concentration of 167 μg / mL in the vaccine; Use HR-500 dispersing emulsifier to gradually emulsify from gear A to gear E for 30 minutes.

3. The use according to claim 1, characterized in that The antiviral proteins IFITM1, IFITM3 and Viperin are obtained through prokaryotic expression in Escherichia coli.

4. The use according to claim 3, characterized in that The method for obtaining the antiviral proteins IFITM1, IFITM3, and Viperin comprises the following steps: 1) The antiviral plasmids pCold-TF-IFITM1, pCold-TF-IFITM3, and pCold-TF-Viperin were transformed into competent cells respectively. The transformed cells were spread on a solid LB culture dish containing ampicillin resistance and cultured at 37°C for 14 hours. 2) Select a single positive colony from the above culture dish and inoculate it into liquid culture medium containing antibiotics. Cultivate the culture at 37°C and 200 rpm with shaking until the OD600 reaches 0.8-1.

2. Then, add IPTG to the culture solution to a final IPTG concentration of 0.1-0.5 mmol / L. 3) The bacterial solution containing IPTG was then induced and cultured in a constant temperature shaker at 16°C and 200 rpm for 16 h; 4) Wet the precipitated cells with 5 mL of 1× PBS buffer, mix thoroughly by pipetting, and then place in an ice bath for ultrasonic disruption; 5) The bacterial suspension obtained after ultrasonic disruption was centrifuged at 8000 rpm at 4°C for 15 min, and the supernatant was retained; 6) Purify the antiviral proteins IFITM1, IFITM3, and Viperin using Ni-NTA affinity chromatography; Alternatively, the antiviral proteins IFITM1, IFITM3, and Viperin can be purified using ammonium sulfate precipitation.

5. The use according to claim 4, characterized in that In step 4), the ultrasonic disruption conditions were set as follows: 20% power, 5 s operation, 5 s interval, cumulative working time of 20 min, and a total of 40 min.

6. The use according to claim 4, characterized in that The antiviral proteins IFITM1, IFITM3, and Viperin were purified using Ni-NTA affinity chromatography, which specifically includes the following steps: The antiviral proteins IFITM1, IFITM3, and Viperin were purified by Ni-NTA affinity chromatography, and 500 mmol / L imidazole was used to elute the antiviral proteins IFITM1, IFITM3, and Viperin to obtain the purified antiviral proteins IFITM1, IFITM3, and Viperin.

7. The use according to claim 4, characterized in that The antiviral proteins IFITM1, IFITM3, and Viperin were purified using ammonium sulfate precipitation, which specifically includes the following steps: Ammonium sulfate was added to the supernatant, the mixture was slowly shaken at 10°C for 2 h, centrifuged at 8500 rpm for 15 min, the supernatant was discarded, 15 mL of 1× PBS buffer was added to resuspend the precipitate, and centrifuged again at 8500 rpm for 15 min. The supernatant was taken for ultrafiltration to obtain the purified antiviral proteins IFITM1, IFITM3, and Viperin.

8. The use according to claim 4, characterized in that In step 5), the bacterial pellet was simultaneously resuspended with an equal volume of 1×PBS buffer and thoroughly mixed to prepare a protein sample, which was then sampled for SDS-PAGE electrophoresis to analyze the solubility of the antiviral protein in the bacteria.

9. Application of antiviral proteins IFITM1, IFITM3 or Viperin as veterinary immunopotentiators in the preparation of veterinary vaccines.

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