A genetically engineered subunit vaccine of getah virus and its preparation method and application

Abstract

The application discloses a Gitta virus genetically engineered subunit vaccine and a preparation method and application thereof, and belongs to the technical field of vaccine preparation. The Gitta virus P6E recombinant protein is obtained by using a eukaryotic expression system, and is further loaded on the surface of a nanoparticle skeleton to form a recombinant nanoparticle. The obtained recombinant protein and the recombinant nanoparticle are respectively compounded with an adjuvant to prepare the genetically engineered subunit vaccine. After twice immunization, the vaccines of the two components can significantly induce the host to produce strong humoral immune and cellular immune responses, thereby providing effective protection against GETV infection. The technical scheme of the application provides an important technical basis and application reference for research and optimization of the Gitta virus vaccine.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a Getta virus genetically engineered subunit vaccine and its preparation method and application. Background Technology

[0002] Getah virus (GETV), belonging to the alphavirus genus, is an RNA virus primarily transmitted by mosquitoes. In recent years, its prevalence in pig farms in my country has been on the rise, causing symptoms such as fever and diarrhea in piglets and abortion in sows, resulting in significant losses to pig herd health and the livestock industry. Due to the expanding host range of GETV, controlling its spread has become a crucial need for my country's livestock industry. However, current control measures for Getah virus still mainly rely on biosecurity management, and no commercially available Getah virus vaccine is yet on the market, severely limiting the active immunization capabilities of pig herds.

[0003] In recent years, genetically engineered subunit vaccines have attracted considerable attention due to their well-defined components, high safety profile, near-absence of host proteins, avoidance of potential biosafety risks associated with inactivation or attenuation processes, and ability to precisely present protective antigens through molecular design. However, traditional subunit vaccines often exhibit weak immunogenicity, necessitating structural optimization or the application of novel delivery vectors to enhance their immunogenicity. Protein nanoparticle-based subunit vaccines demonstrate significant advantages in this regard: protein nanoparticles possess highly regular and stable three-dimensional structures, capable of self-assembling into uniform particles, and presenting multiple copies of antigenic epitopes on their surface in a highly repetitive and high-density manner. This not only significantly enhances the cross-linking effect of B cell receptors and promotes a robust humoral immune response but also effectively activates dendritic cells and enhances cellular immune responses, thereby comprehensively improving the immunogenicity of subunit vaccines. Therefore, the design of optimized subunit antigens combined with protein nanoparticle presentation strategies has become an important trend in current vaccine development. However, there are very few publicly reported studies on the application of nanoparticle technology to Getta virus subunit vaccines, especially recombinant nanoparticle vaccines based on precise antigen design and controllable assembly strategies. The only report is in authorization number CN117964722B, which describes the fusion expression of GETV E2 protein and ferritin in a baculovirus system to form self-assembled nanoparticles.

[0004] Therefore, based on the advantages of the aforementioned subunit vaccines and protein nanoparticle carriers, constructing a GETV genetic engineering subunit platform based on structural optimization, and synergistically integrating the characteristics of both, can not only further enhance the immunogenicity and protective effect of GETV antigens, but also provide a safer and more efficient new vaccine technology route for the prevention and control of porcine Gettavirus, which has important scientific significance and application value. Summary of the Invention

[0005] To overcome the shortcomings of existing technologies in Geta virus (GETV) prevention and control, this invention aims to provide a Geta virus genetically engineered subunit vaccine, its preparation method, and its application. This vaccine uses a Geta virus P6E recombinant protein prepared via a mammalian cell expression system as an antigen. This antigen is covalently coupled to the surface of mi3 nanoparticles, forming recombinant nanoparticles capable of displaying multiple copies of the P6E antigen. The obtained Geta virus P6E recombinant protein and P6E nanoparticles are then combined with adjuvants to prepare Geta virus subunit vaccines and Geta virus nanoparticle vaccines, respectively. Both vaccines can effectively induce humoral and cellular immune responses against Geta virus, thereby achieving effective prevention of Geta virus infection.

[0006] The above-mentioned objectives of the present invention are achieved through the following technical solutions:

[0007] In a first aspect, the present invention claims protection for a Getavirus P6E recombinant protein, which is formed by linking Getavirus p62 protein and E1 protein (after removing the transmembrane domain) via a linker sequence; said P6E recombinant protein is at least one protein selected from (a1)-(a2) below:

[0008] (a1) A protein with the amino acid sequence shown in SEQ ID NO:2;

[0009] (a2) A fusion protein with the same function obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein described in (a1).

[0010] Secondly, the present invention claims protection for a recombinant nanoparticle loaded with the Geta virus P6E recombinant protein as described above, wherein the recombinant nanoparticle is obtained by displaying the P6E recombinant protein on the surface of the nanoparticle via a SpyTag003 / SpyCatcher003 covalent linking system.

[0011] Furthermore, the nanoparticles are SpyCatcher003-mi3 nanoparticles or SpyCatcher003-ferritin nanoparticles; the amino acid sequence of the SpyCatcher003-mi3 nanoparticles is shown in SEQ ID NO: 4.

[0012] Thirdly, the present invention claims protection for a getta virus genetically engineered vaccine comprising a pharmaceutically acceptable adjuvant and an antigenic component selected from the following (b1), (b2), or (b3):

[0013] (b1) The Gettervirus P6E recombinant protein as described above;

[0014] (b2) Recombinant nanoparticles loaded with Geyta virus P6E recombinant protein as described above;

[0015] (b3) A composition comprising the recombinant Getavirus P6E protein of (b1) and the recombinant nanoparticles loaded with the Getavirus P6E protein of (b2).

[0016] Going further,

[0017] When the antigen component is combination (b1), the content of the Geta virus P6E recombinant protein is 50-200 μg / mL;

[0018] When the antigen component is combination (b2), the content of the recombinant nanoparticles loaded with Geyta virus P6E recombinant protein is 100-200 μg / mL;

[0019] When the antigen component is combination (b3), the content of the Geyta virus P6E recombinant protein is 50-200 μg / mL, and the content of the recombinant nanoparticles loaded with Geyta virus P6E recombinant protein is 100-200 μg / mL.

[0020] Furthermore, the adjuvant is an oil-in-water adjuvant (e.g., SMMMIT). TM (Adjuvants, ISA adjuvants, etc.), wherein the volume ratio of the adjuvant to the antigen component is 1:4.

[0021] Fourthly, the present invention claims protection for a nucleic acid molecule encoding the above-mentioned Getavirus P6E recombinant protein, the nucleotide sequence of which is shown in SEQ ID NO:1.

[0022] Fifthly, the present invention claims protection for an expression cassette, recombinant vector, or host cell containing the aforementioned nucleic acid molecules.

[0023] Sixthly, the present invention claims a method for preparing recombinant nanoparticles as described above, comprising the following steps: under in vitro conditions, covalently coupling purified Geta virus P6E recombinant protein with purified nanoparticles via a SpyTag003 / SpyCatcher003 system to obtain recombinant nanoparticles loaded with Geta virus P6E recombinant protein.

[0024] Furthermore, the nanoparticles in the above method are SpyCatcher003-mi3 nanoparticles or SpyCatcher003-ferritin nanoparticles; the amino acid sequence of the SpyCatcher003-mi3 nanoparticles is shown in SEQ ID NO: 4.

[0025] In a seventh aspect, the present invention seeks protection for the use of the above-described Getavirus P6E recombinant protein or the above-described recombinant nanoparticles in the preparation of a medicament for preventing Getavirus infection.

[0026] Seventhly, the present invention seeks protection for the use of the above-described Gettavirus genetically engineered vaccine in the preparation of a medicament for the prevention of Gettavirus infection.

[0027] Eighthly, the present invention claims protection for the use of the above-described nucleic acid molecules in any of the following:

[0028] (1) Preparation of Gettavirus genetically engineered vaccine;

[0029] (2) Prepare drugs to prevent Getta virus infection.

[0030] Ninthly, the present invention claims protection for the use of the above-described expression cassette, recombinant vector, or host cell in any of the following:

[0031] (1) Preparation of Gettavirus genetically engineered vaccine;

[0032] (2) Prepare drugs to prevent Getta virus infection.

[0033] In a specific embodiment of the present invention, the Linker sequence is preferably a 4×GGGGS Linker sequence.

[0034] The protein tag mentioned is a Strep II tag, specifically, a Strep II tag is introduced at the C-terminus of the E1 protein to prepare a fusion protein.

[0035] The nucleotide sequence encoding the Getta virus P6E recombinant protein is shown in SEQ ID NO:1; the nucleotide sequence encoding the SpyCatcher003-mi3 nanoparticles is shown in SEQ ID NO:3.

[0036] In a specific embodiment of the present invention, the method for preparing the above-mentioned Getavirus P6E recombinant protein includes the following steps:

[0037] Step 1: Take the gene fragment with the nucleotide sequence shown in SEQ ID NO:1, ligate it with the expression vector, transform it into competent cells, extract the recombinant plasmid, transfect suspension cells, culture at 37℃ for 3-5 days, and collect the supernatant.

[0038] Step 2: Bind the collected supernatant to strep agarose beads at 4°C for 4-6 hours. Collect the agarose beads by passing the supernatant through a column, remove impurities with Buffer W, and elute the target protein with 1x BXT. Concentrate the eluted target protein through a Millipore ultrafiltration tube, replace it with binding buffer (50 mM Tris-HCl, 150 mM NaCl, pH 8.0), and store at -80°C for later use.

[0039] Preferably, the expression vector in step 1 is pcDNA3.4(+) vector, the competent cells are DH5α competent cells, and the suspension cells are CHO cells.

[0040] In a specific embodiment of the present invention, the preparation method of the SpyCatcher003-mi3 nanoparticles includes the following steps:

[0041] Step 1: Take the gene fragment with the nucleotide sequence shown in SEQ ID NO:3, ligate it with the expression vector, transform it into DH5α competent cells, extract the plasmid and then transform it into competent cells of the expression strain.

[0042] Step 2: Select single colonies for induced expression. After expression, collect the bacterial cells, sonicate to disrupt, centrifuge to remove the precipitate, and purify the supernatant by attaching it to a nickel column. Collect the purified protein after washing and elution. The eluted target protein is then replaced with binding buffer (50 mM Tris-HCl, 150 mM NaCl, pH 8.0) through a Millipore ultrafiltration tube and stored at 4°C.

[0043] Preferably, the expression vector in step 1 is the pCold II vector, and the expression strain used is BL21(DE3) with the addition of the TF16 molecular chaperone.

[0044] Compared with the prior art, the present invention has the following beneficial effects:

[0045] (1) The genetically engineered subunit vaccine prepared in this invention includes GETV-P6E recombinant protein and its derived nanoparticles. The antigen portion expresses Getavirus GETV-P6E recombinant protein using a mammalian cell expression system. This expression system has advantages such as high expression level, simple purification process, high protein purity, suitability for large-scale production, and easy control of product quality. Experimental results show that the P6E protein obtained by this system has good immunogenicity and safety. The nanoparticle backbone portion is expressed solublely using a prokaryotic expression system, resulting in high purity and large yield of the backbone protein, while maintaining the integrity and self-assembly characteristics of the nanoparticle structure. The antigen portion and the nanoparticle backbone are efficiently covalently coupled using the SpyTag003 / SpyCatcher003 system. This system has advantages such as high connection efficiency, uniform antigen display, mild reaction conditions, and stable structure of the resulting complex, which can significantly improve the display effect of the antigen on the surface of the nanoparticles.

[0046] (2) The antigenic portion of this genetically engineered subunit vaccine is a recombinant Getta virus P6E protein expressed in eukaryotic cells. This protein has a structure similar to the membrane protein on the surface of the virus, and contains immunogenic p62 and E1 proteins. It has high affinity for antibodies, ensuring the natural spatial structure and modification. The eukaryotically expressed protein has glycosylation modifications similar to viral proteins, further enhancing the antigenicity of the fusion protein. The separate expression of the antigenic portion and the nanoparticle backbone, coupled with the covalent coupling strategy of the SpyTag003 / SpyCatcher003 system, not only allows for the selection of the most suitable expression system to ensure the correct folding of the antigenic protein and the efficient and soluble expression of the nanoparticle backbone, but also avoids the problems of misfolding and hindered particle assembly caused by fusion expression. By preparing them separately and performing precise covalent coupling on the surface of the nanoparticles, the antigen can be displayed uniformly and at a high density in a near-native conformation, ensuring good immunogenicity.

[0047] (3) Currently, there are no commercially available GETV vaccines. This invention utilizes a mammalian cell expression system to prepare GETV-P6E recombinant protein and efficiently loads it onto the surface of a nanoparticle backbone to construct a recombinant nanoparticle structure that presents multiple copies of the antigen. The resulting recombinant protein and recombinant nanoparticles, respectively, are combined with adjuvants to form two genetically engineered subunit vaccines, which can significantly enhance the immunogenicity of the antigen and induce a strong and sustained humoral and cellular immune response. Compared with traditional inactivated vaccines, the two vaccines of this invention have significant advantages in antigen display density, immune response strength, and protective effect, effectively reducing the risk of GETV infection in pig herds, and have significant promotional value and industrial application prospects. Attached Figure Description

[0048] Figure 1 SDS-PAGE analysis of Gettavirus P6E recombinant protein and its P6E nanoparticles.

[0049] Figure 2 This study evaluates the immunogenicity of the GETV genetically engineered subunit vaccine in mice. Specifically, a) is the assay for IgG antibodies after vaccine immunization; b) is the assay for neutralizing antibodies after vaccine immunization; and c) is the assay for spleen lymphocyte proliferation after vaccine immunization.

[0050] Figure 3 This study describes antibody detection in pigs immunized with the GETV genetically engineered subunit vaccine. Specifically, a) represents the measurement of IgG antibodies after vaccine immunization; and b) represents the measurement of neutralizing antibodies after vaccine immunization. Detailed Implementation

[0051] The following examples further illustrate the content of the present invention, but should not be construed as limiting the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from its spirit and essence are within the scope of the invention. Reagents or instruments used without specifying a manufacturer are considered to be conventional products that can be purchased on the market.

[0052] Example 1: Preparation of GETV P6E recombinant protein

[0053] The gene fragment with the nucleotide sequence shown in SEQ ID NO:1 was synthesized into the pcDNA3.4(+) vector (between the XbaI and EcoRV restriction enzyme sites), transformed into DH5α competent cells, and positive bacteria that were correctly identified by bacterial PCR and sequencing were selected to extract the plasmid, and the recombinant plasmid pcDNA3.4-GETV-P6E was obtained.

[0054] CHO suspension cells were cultured in culture flasks at 37°C, 8% CO2, and a shaker speed of 130 rpm / min. The target cell count for the cultured CHO suspension cells was 2.5 × 10⁻⁶. 6 At a cell / mL concentration, the constructed positive recombinant plasmid was transfected into CHO cells. The transfection system consisted of: 100 μg plasmid, 1 mL Opti-MEM, and 300 μL PEI. After mixing the plasmid and transfection reagent, the mixture was incubated at room temperature for 15 min, then slowly added dropwise to the cell culture flask, shaken well, and placed in a 37°C CO2 incubator for further culture. 24 h after transfection, 3.5 mL / 0.1 L of CHO cell culture enhancement medium was added, and the cells were cultured for another 3 to 4 days. After culture, the cells and culture medium were collected, centrifuged at 6000 rpm / min for 10 min at 4°C, and the cell culture supernatant was collected. The recombinant protein was purified using IBA strep II affinity chromatography.

[0055] The specific steps for purifying the recombinant P6E protein using the IBA strep II tag affinity chromatography method (IBA Strep-Tactin®XT 4flow®) described above are as follows:

[0056] ① Add 5 volumes of PBS solution (10 mM, pH=7.4) to the affinity chromatography column to replace its storage buffer.

[0057] ② After centrifuging the collected cell culture supernatant at 4°C and 8000 rpm for 20 min, add the supernatant to the affinity chromatography column and incubate overnight on a rotating shaker.

[0058] ③ Release the supernatant from the culture medium after binding and wash the column with 10 times the volume of washing buffer. The washing buffer formula is: 100mM Tris, 150mM NaCl, 1mM EDTA.

[0059] ④ Add 2-4 mL of biotin-containing eluent for elution. The eluent formulation is: 100 mM Tris, 150 mM NaCl, 1 mM EDTA, 50 mM biotin. Collect the eluent and concentrate it to replace the buffer.

[0060] ⑤ After concentrating the harvested purified protein sample using a Millipore ultrafiltration tube, 15 mL of binding buffer (50 mM Tris-HCl, 150 mM NaCl, pH 8.0) was added to replace the storage solution. This process was repeated twice, and the final concentration was brought to 200-300 μL. The purified protein concentration was determined using the Bradford method, and then aliquoted and stored at -80°C for later use.

[0061] After the above expression and purification steps, purified recombinant GETV P6E protein was obtained. SDS-PAGE analysis showed a purity of over 90%. The SDS-PAGE molecular weight (e.g., [details omitted]) was [details omitted] Figure 1 (abbreviated as P6E) is approximately 100 kDa, which is in line with the expected size.

[0062] Example 2: Preparation of SpyCatcher003-mi3 nanoparticles

[0063] (1) The gene fragment with the nucleotide sequence shown in SEQ ID NO:3 was directly synthesized between the NdeI and XbaI restriction endonuclease sites of the pCold II vector and then transformed into DH5α competent cells. A single positive colony was picked and the plasmid was extracted. 0.5 μg of the plasmid was transformed into BL21(DE3) competent cells with TF16 molecular chaperone. Single colonies were picked and inoculated into LB liquid medium containing ampicillin and chloramphenicol antibiotics and activated overnight. The next day, the cells were inoculated into LB liquid medium containing ampicillin and chloramphenicol resistance at a volume ratio of 1:100, and arabinose was added to a final concentration of 2 g / L. The cells were cultured at 37°C and 200 rpm with shaking until OD. 600 The concentration was increased to 0.6, and then the culture system was cooled to 16°C. IPTG was added to a final concentration of 0.1 mM to induce expression, and the expression was continued at 16°C for 12–16 h to ensure that the target protein was fully soluble and expressed.

[0064] (2) After induction, the bacterial cells were collected by centrifugation at 4°C and 8000 rpm for 30 min, and resuspended in pre-cooled lysis buffer (20 mM Tris-HCl, 300 mM NaCl, 20 mM imidazole, pH 8.0), with an appropriate amount of protease inhibitor added. The bacterial cells were lysed by sonication, and the lysate was centrifuged at 4°C and 10000 rpm for 30 min. The supernatant was collected as the crude soluble protein extract.

[0065] The supernatant was filtered through a 0.45 μm filter and then loaded into pre-equilibrated Ni. 2+ -NTA affinity chromatography column, using His tag for affinity purification. After removing non-specifically bound proteins with wash buffer containing 80 mM imidazole, the target protein is obtained by stepwise elution with elution buffer containing 250–300 mM imidazole. The eluted product is further filtered through Millipore ultrafiltration tubes to change the buffer to binding buffer (50 mM Tris-HCl, 150 mM NaCl, pH 8.0). The concentrated protein is stored at 4°C for later use.

[0066] After the above expression and purification steps, purified SpyCatcher003-mi3 nanoparticles were obtained. SDS-PAGE analysis showed a purity of over 90%. The SDS-PAGE molecular weight (e.g., [missing information]) was [missing information]). Figure 1 (abbreviated as mi3) is approximately 45kDa, which is in line with the expected size.

[0067] Example 3: In vitro coupling and assembly of nanoparticles

[0068] The GETV-P6E recombinant protein obtained in Example 1 and the SpyCatcher003-mi3 protein obtained in Example 2 were respectively replaced in the same reaction buffer system (50 mM Tris-HCl, 150 mM NaCl, pH 8.0). The antigen protein was added to the mi3 nanoparticles at a 1:1 molar ratio of mi3 nanoparticles to antigen protein for in vitro coupling reaction. The mixture was incubated at 4°C for 6–8 h to allow the antigen to form stable covalent isopeptide bonds with the mi3 surface via the SpyTag003 / SpyCatcher003 system. After the reaction, the mixture was centrifuged at 8000 rpm for 30 min to remove a small amount of insoluble matter. The resulting supernatant was loaded onto a pre-equilibrated gel filtration column (Superose 6 Increase) for separation and purification. High molecular weight peaks were collected and combined to obtain successfully loaded GETV-P6E-mi3 nanoparticles. The obtained nanoparticles were further subjected to ultrafiltration to a buffer solution of 50 mM Tris-HCl, 150 mM NaCl, and pH 8.0. The integrity of the particle structure and the antigen loading were verified by SDS-PAGE and transmission electron microscopy, thus obtaining recombinant nanoparticles with uniform structure and stable antigen presentation.

[0069] The GETV-P6E-mi3 recombinant nanoparticles, purified by in vitro coupling and molecular sieve gel filtration, had a molecular weight of approximately 145 kDa according to SDS-PAGE analysis, and the protein size was as expected (e.g., Figure 1 The GETV-P6E-mi3 nanoparticles (abbreviated as P6E-mi3) have a purity greater than 90%. Transmission electron microscopy analysis shows that the GETV-P6E-mi3 nanoparticles have obvious protrusions on their surface, uniform particle size, and a diameter of about 35 nm.

[0070] Example 4: Preparation of GETV genetically engineered subunit vaccine

[0071] The GETV P6E recombinant protein prepared in Example 1 and the GETV-P6E-mi3 recombinant nanoparticles prepared in Example 3 were diluted to 125 μg / mL and 181.5 μg / mL, respectively. 40 mL of the diluted recombinant nanoparticles were then added to 10 mL of SMMIT. TM The adjuvant, after thorough mixing, yields the GETV nanoparticle vaccine, containing GETV P6E recombinant protein at concentrations of 100 μg / mL and GETV-P6E-mi3 recombinant nanoparticles at concentrations of 145 μg / mL. Sterility testing is performed according to the current Chinese Veterinary Pharmacopoeia, and the qualified vaccine is stored at 2–8°C for later use.

[0072] Example 5: Immunogenicity assessment of GETV genetically engineered subunit vaccine in mice

[0073] Three-week-old female ICR mice were randomly divided into 4 groups of 15 mice each. The mice were immunized by intramuscular injection (GETV genetically engineered subunit vaccine prepared in Example 4). A booster immunization was performed on day 14 after the first immunization. The specific immunization grouping is shown in Table 1 below.

[0074] Table 1. Mouse Immunization Groups

[0075]

[0076] (1) Blood samples were collected on days 7, 14, 21, 28 and 35 after immunization to collect serum for antibody titer testing. The GETV antibody level was detected using a self-developed GETV antibody detection indirect ELISA kit, and the GETV neutralizing antibody titer was determined by neutralization assay.

[0077] (2) On the 14th day after the second immunization, the spleens of 3 mice from each group were isolated for spleen cell proliferation experiments. The specific steps are as follows:

[0078] Spleen cells were aseptically isolated using a 70-mesh cell sieve, treated twice with erythrocyte lysis buffer, and then diluted to 2.5 × 10⁶ cells in RPMI 1640 medium. 6 Cells were seeded at a density of 100 μL / mL in 96-well plates. 100 μL of RPMI 1640 medium was added to each well as a blank control. Each sample consisted of three replicates. Subsequently, 10 μL of GETV-P6E recombinant protein (10 μg / mL) or RPMI 1640 medium (control) was added to each well. Cells were incubated at 37 °C for 36 h. Then, 10 μL of CCK8 reagent was added to each well, and the cells were incubated at 37 °C for 4 h. The OD450 values ​​of different wells were measured using a multi-mode microplate reader. The spleen cell stimulation index (SI) was calculated as follows: SI index = (OD value of recombinant protein-stimulated wells - OD value of blank control wells) / (OD value of RPMI 1640 medium-stimulated wells - OD value of blank control wells).

[0079] (3) On day 14 after the second immunization, 5 mice from each group were randomly selected for GETV challenge. A total of 20 mice in the challenge group were subcutaneously inoculated with 100 μL of 10 6 TCID 50 The GETV-HN strain of virus was used. Blood was collected daily for 3 days after challenge to monitor viremia, and mice were necropsy on day 3 after challenge to detect viral load in the spleen.

[0080] The results showed that the levels of specific IgG antibodies against GETV from both vaccines increased rapidly after the second immunization, reaching their highest level 14 days after the second immunization, and then the antibody levels remained at a high level. Figure 2 In the group of nanoparticles, the neutralizing antibody titer reached its highest level 14 days after the second immunization, with an average neutralizing titer of 1:560, which was significantly higher than that of the control group. Figure 2 In (b) of the study, the antibody levels produced by the recombinant protein group were slightly lower than those in the nanoparticle group, and the early antibody production levels were significantly lower than those in the nanoparticle vaccine group. These results indicate that mice immunized with the GETV genetically engineered subunit vaccine exhibited a significant humoral immune response and high antibody titers.

[0081] The results of the lymphocyte proliferation experiment are shown below. Figure 2 In the c-cell group, stimulation with the GETV recombinant protein resulted in significant spleen cell proliferation in both the recombinant protein and nanoparticle vaccine groups, with stimulation indices greater than 1.5, showing a significant difference compared to the control group. These results indicate that immunization of mice with the GETV genetically engineered subunit vaccine can trigger a favorable cellular immune response.

[0082] GETV challenge results showed that no GETV viremia was detected in the GETV engineered subunit vaccine group within 1-3 days post-challenge, while significant viremia was detected in the control group. Furthermore, no infectious GETV was detected in the spleen of the GETV engineered subunit vaccine group on the third day post-challenge, while the control group showed a higher viral load in the spleen. These results indicate that the GETV engineered subunit vaccine effectively protects mice from GETV challenge.

[0083] Example 6: Safety Trial of GETV Genetically Engineered Subunit Vaccine

[0084] Ten GETV nanoparticle-vaccinated 3-week-old antibody-negative piglets were divided into two groups. Each piglet received a 1 mL injection of the vaccine via intramuscular injection in the neck. Five piglets received the recombinant protein vaccine and five received the nanoparticle vaccine. A control group of five piglets received PBS solution (10 mM, pH=7.4). The piglets' body temperature and weight were monitored daily after injection. The piglets were also observed for adverse reactions such as swelling and nodules at the injection site. The health status of the piglets was monitored daily for 28 consecutive days.

[0085] The results showed that the genetically engineered subunit vaccine group was safe and well-absorbed at the injection site, with no adverse reactions such as swelling or nodules. The pigs' body temperature remained between 38.5℃ and 39.6℃, and there were no abnormal symptoms such as fever. During the 28-day observation period, all pigs had normal body temperature and no high fever; their mental state was good, their appetite was normal, and no abnormal symptoms were observed.

[0086] Example 7: Evaluation of the protective efficacy of GETV nanoparticle vaccine in pigs

[0087] Serum samples were collected from each group of immunized pigs in Example 6 at 7, 14, 21, 28, and 35 days post-immunization, and antibody levels were detected using a self-developed GETV antibody detection ELISA kit. Piglets immunized in Example 6 were used for a GETV challenge experiment. Each piglet in the recombinant protein vaccine group, nanoparticle vaccine group, and control group was injected with one dose (each dose containing 5 x 10^6 viruses). 7 The P1 generation of GETV-HN strain (TCID50) virus was used to observe clinical symptoms and changes in body temperature daily for 14 days after challenge, and serum viremia was monitored from day 1 to day 7 after challenge (nucleic acid positive or negative was identified by PCR).

[0088] Post-immunization antibody test results Figure 3 The specific IgG antibody against GETV in the genetically engineered vaccine group increased rapidly after immunization, stabilized 14 days after the second immunization, and remained at a high level thereafter. The average neutralizing antibody in the nanoparticle group reached 1:480, which was slightly higher than that in the recombinant protein group, while no GETV antibody was detected in the control group and the neutralizing titer was less than 1:10.

[0089] After GETV challenge, none of the pigs in the genetically engineered vaccine group showed obvious clinical symptoms, and their body temperature and mental state were normal, with no detectable viremia (Table 2). Significant viremia was detected in the serum of the control group pigs. These results indicate that the GETV nanoparticle vaccine can effectively resist GETV challenge in pigs and has a good immunoprotective effect.

[0090] Table 2. Results of viremia detection after challenge.

[0091]

[0092] Note: "+" represents positive, and "-" represents negative.

[0093] Comparative Example 1

[0094] The patent application CN117964722B employs a strategy of fusing GETV E2 protein with ferritin for expression. The method involves cloning the fusion gene into the baculovirus expression vector pFastBac I, transposing it via DH10Bac to obtain a recombinant baculovirus plasmid, which is then transfected into Sf9 cells, and the recombinant baculovirus solution is harvested for protein expression. After infecting Sf9 cells with this baculovirus system, the recombinant protein is obtained, and the cell culture supernatant is inactivated and directly mixed with adjuvant to prepare a vaccine. However, the post-translational modifications of this type of baculovirus / insect cell expression system differ significantly from those in mammalian cells, particularly in glycosylation type and glycan structure, which are not entirely consistent with the natural viral antigen. A single E2 protein is insufficient to reflect the true viral particle structure, potentially affecting the native conformation, stability, and immunogenicity of the recombinant protein. Furthermore, directly inactivating unpurified culture supernatant before use may also disrupt the antigenic conformation, thereby affecting the vaccine's immunization efficacy.

Claims

1. A recombinant P6E protein of Getta virus, characterized in that, The recombinant P6E protein of this geta virus is at least one of the following proteins (a1)-(a2): (a1) A protein with the amino acid sequence shown in SEQ ID NO:2; (a2) A fusion protein with the same function obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein described in (a1).

2. A recombinant nanoparticle loaded with the Getavirus P6E recombinant protein as described in claim 1, characterized in that, The recombinant nanoparticles were obtained by displaying the P6E recombinant protein on the surface of the nanoparticles via a SpyTag003 / SpyCatcher003 covalent linking system.

3. The recombinant nanoparticles according to claim 2, characterized in that, The nanoparticles are SpyCatcher003-mi3 nanoparticles or SpyCatcher003-ferritin nanoparticles; the amino acid sequence of the SpyCatcher003-mi3 nanoparticles is shown in SEQ ID NO:

4.

4. A Gettavirus genetically engineered vaccine, characterized in that, The vaccine contains a pharmaceutically acceptable adjuvant and an antigenic component selected from the following (b1), (b2), or (b3): (b1) The Getavirus P6E recombinant protein as described in claim 1; (b2) The recombinant nanoparticles loaded with Geyta virus P6E recombinant protein as described in claim 2; (b3) A composition comprising the recombinant Getavirus P6E protein of (b1) and the recombinant nanoparticles loaded with the Getavirus P6E protein of (b2).

5. The genetically engineered vaccine according to claim 4, characterized in that: When the antigen component is combination (b1), the content of the Geta virus P6E recombinant protein is 50-200 μg / mL; When the antigen component is combination (b2), the content of the recombinant nanoparticles loaded with Geyta virus P6E recombinant protein is 100-200 μg / mL; When the antigen component is combination (b3), the content of the Geyta virus P6E recombinant protein is 50-200 μg / mL, and the content of the recombinant nanoparticles loaded with Geyta virus P6E recombinant protein is 100-200 μg / mL.

6. The genetically engineered vaccine according to claim 4, characterized in that, The adjuvant is an oil-in-water adjuvant, and the volume ratio of the adjuvant to the antigen component is 1:

4.

7. A nucleic acid molecule encoding the Getavirus P6E recombinant protein of claim 1, characterized in that, The nucleotide sequence of this nucleic acid molecule is shown in SEQ ID NO:

1.

8. An expression cassette, recombinant vector, or host cell containing the nucleic acid molecule of claim 7.

9. A method for preparing the recombinant nanoparticles of claim 2, characterized in that, Includes the following steps: Under in vitro conditions, purified Getavirus P6E recombinant protein and purified nanoparticles were covalently coupled using the SpyTag003 / SpyCatcher003 system to obtain recombinant nanoparticles loaded with Getavirus P6E recombinant protein.

10. The use of the Getavirus P6E recombinant protein of claim 1 or the recombinant nanoparticles of claim 2 in the preparation of a drug for preventing Getavirus infection.

11. The use of the Gettavirus genetically engineered vaccine according to claim 4 in the preparation of a drug for preventing Gettavirus infection.

12. The use of the nucleic acid molecule according to claim 7 in the preparation of a getta virus genetically engineered vaccine.

13. The use of the expression cassette, recombinant vector, or host cell as described in claim 8 in the preparation of a gettavirus genetically engineered vaccine.

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