Vaccine for preventing and treating swine Japanese encephalitis virus and application

Abstract

The invention discloses a vaccine for preventing and treating swine Japanese encephalitis virus and application, and belongs to the field of biological pharmacy for livestock. The CHO stable expression cell line expressing the E protein Domain III is constructed, transcription and protein expression levels are remarkably improved, the cell line grows under the glutamine-free condition, accumulation of metabolites such as ammonia is reduced, and cell activity and long-term culture stability are improved. The FC fusion protein is added after the ED3 gene sequence through the flexible Linker, so that the affinity chromatography purification process is simplified, the recovery rate and the purity are improved, and the production cost is reduced; meanwhile, the high-efficiency expression of CHO cells is combined, so that the purified protein yield is higher than 2g / L, which is far better than that of a traditional method, and the large-scale requirement of veterinary vaccines is met. The vaccine provided by the invention has no pathogen residual risk, the safety is higher than that of the traditional inactivated or attenuated live vaccine, and the vaccine which is safe, reliable and good in immunogenicity is provided for preventing and treating infection of the swine Japanese encephalitis virus.

Description

Technical Field

[0001] This invention relates to the field of veterinary biopharmaceuticals, specifically to a vaccine for the prevention and treatment of swine encephalitis virus infection and its application. Background Technology

[0002] Japanese encephalitis virus (JEV) is a mosquito-borne zoonotic virus that causes Japanese encephalitis and poses a threat to pigs, horses, birds, and humans. In pigs, the disease typically manifests as reproductive failure, such as abortion, stillbirth, and mummified fetuses, while in humans it can cause severe encephalitis. Japanese encephalitis is classified as a Class II animal disease in my country.

[0003] The JEV genome is a single-molecule linear positive-sense RNA, approximately 11 kb in length, starting at position 96 (5′) of the ATG and ending at position 10393 (3′), forming an ORF of approximately 10.3 kb encoding a polyprotein precursor of 3432 amino acids. This polyprotein precursor cleaves within the host cell, forming three structural proteins (C, PrM, and E proteins) and seven non-structural proteins, including NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5. In the Flaviviridae genus, the E protein is the major structural protein, closely related to viral particle adsorption, penetration, pathogenicity, induction of host immune responses, and the production of neutralizing antibodies. Over 90% of the antibodies induced by the E protein are neutralizing antibodies that specifically bind to functional sites of the E protein (such as the receptor-binding region of Domain III), blocking viral binding to host cells or inhibiting membrane fusion, thereby neutralizing the virus. The E protein can be structurally divided into three independent β-barrel domains: Domain I: the central domain, which is involved in the assembly and conformational changes of the E protein dimer; Domain II: the extended domain, which contains a highly conserved fusion peptide at the end and is responsible for membrane fusion; and Domain III: the immunoglobulin-like domain, which is considered to be the main region for receptor binding and is also the main target of neutralizing antibodies.

[0004] CHO expression systems are currently the most important mammalian cell expression platforms in the biopharmaceutical field, producing the vast majority of therapeutic proteins (such as monoclonal antibodies and recombinant proteins). CHO cells can perform complex glycosylation of proteins, which is crucial for protein stability, half-life, potency, and immunogenicity. Through various screening and amplification systems, engineered cell lines capable of stably and at high levels expressing target proteins can be obtained, and these cells can be cultured in suspension in serum-free media with well-defined chemical compositions. This is essential for controlled and scalable production in large-scale bioreactors. Furthermore, CHO cells themselves are not infected by most human pathogenic viruses, significantly reducing the risk of viral contamination. In CHO expression systems, adding an Fc fusion tag to the target protein is a common and highly efficient strategy that can significantly increase protein expression levels, enhance protein stability and solubility, prolong serum half-life, and simplify downstream purification processes.

[0005] This study selected the JEV E protein Domain III, the main target of neutralizing antibodies, and linked an Fc fusion tag via a flexible linker. The target antigen was secreted and expressed using CHO suspension cells, and purified by affinity chromatography. This provides a new paradigm for the application of CHO cell expression systems in the field of veterinary biological products. Summary of the Invention

[0006] To achieve the above-mentioned technical objectives, the first objective of this invention is to provide an antigenic protein for preventing and treating porcine Japanese encephalitis virus, which is a recombinant ED3 protein; the amino acid sequence of the recombinant ED3 protein is SEQ ID NO.1. Furthermore, the gene encoding the recombinant protein comprises a nucleotide molecule as shown in SEQ ID NO.2.

[0007] The present invention also provides a recombinant vector containing the coding gene, wherein the recombinant vector is a CHO eukaryotic cell expression vector.

[0008] Furthermore, the recombinant vector includes one of the pEE series, pcDNA series, and pCHO series vectors.

[0009] Furthermore, the recombinant vector includes one of the pEE series vectors.

[0010] A second objective of this invention is to provide a vaccine for the prevention and treatment of swine encephalitis virus, the vaccine comprising the recombinant protein described above, as well as pharmaceutically acceptable adjuvants, stabilizers, and buffers.

[0011] Furthermore, the adjuvant is ISA201 VG.

[0012] The third objective of this invention is to provide a method for preparing a vaccine against swine encephalitis virus, comprising the following steps: (1) The coding gene is cloned into a eukaryotic expression vector to obtain a recombinant vector; (2) Transfect host cells with the recombinant vector and screen host cells that express the recombinant protein stably and efficiently in suspension, then culture them at a lower temperature, and then separate and purify the recombinant protein from the cell supernatant; (3) Dilute the purified protein with physiological saline to the concentration for vaccine preparation, mix evenly, preheat, add adjuvant for emulsification, dispense quantitatively, seal, and store at 2~8℃ to obtain the vaccine.

[0013] Furthermore, the eukaryotic expression vector includes one of the pEE series vectors, preferably the pEE12.4 vector.

[0014] Furthermore, the host cell is a CHO eukaryotic cell.

[0015] Furthermore, the concentration of the recombinant protein obtained after isolation and purification is ≥2 g / L.

[0016] The recombinant protein provided by this invention can be used in the preparation of genetically engineered subunit vaccines for the prevention and treatment of swine encephalitis virus.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) This invention solves the problem of low JEV antigen expression in the prior art. After the ED3 gene sequence, FC fusion protein is added through a flexible linker, which simplifies the affinity chromatography purification process, improves the recovery rate and purity, and reduces the production cost. At the same time, combined with the high-efficiency expression of CHO cells, the purified protein yield is higher than 2g / L, which is much higher than conventional methods and meets the needs of large-scale production.

[0018] (2) This invention utilizes the efficient protein synthesis capabilities of CHO eukaryotic cells to ensure correct protein folding and post-translational modification, thereby improving vaccine immunogenicity. CHO cells have a mature industrial application foundation, and the glycosylation modification of the expression products is close to that of natural proteins, making them suitable for vaccine production. The obtained stable cell lines expressing ED3 protein can grow under glutamine-free conditions, reducing the accumulation of metabolites such as ammonia, improving cell activity and long-term culture stability, reducing culture medium costs, and reducing metabolic stress during the culture process, making them more suitable for industrial production.

[0019] (3) Through glutamine-free culture and purification optimization of CHO cell lines, a high-efficiency, stable and low-cost vaccine production system was formed, providing a safe, reliable and immunogenic vaccine for the prevention and control of swine encephalitis virus infection. Attached Figure Description

[0020] Figure 1 This is the result of enzyme digestion identification.

[0021] Figure 2 SDS-PAGE was used to express ED3 protein under cooling conditions.

[0022] Figure 3 SDS-PAGE of purified ED3 protein.

[0023] Figure 4 To detect ELISA antibody levels in the serum of immunized mice.

[0024] Figure 5 Survival rate of mice after challenge with the virus. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0026] Example 1: Construction of the recombinant eukaryotic expression vector pEE12.4-ED3-FC The codons of JEV E protein Domain III (GenBank: GQ495005.1) were optimized using bioinformatics software. An Fc fusion tag was then linked using a flexible linker. Finally, Hind III and EcoRI restriction sites were added to form a nucleotide molecule as shown in SEQ ID NO.2, which was then ligated into the pUC57 vector to obtain the pUC57-ED3-FC plasmid vector.

[0027] (1) The vector pEE12.4 and plasmid pUC57-ED3-FC were digested with Hind III and EcoRI at 37℃ for 3 hours. The digestion reaction system was as follows: 5 μg vector / plasmid; 5 μl Hind III and 5 μl EcoRI each; 4 μl 10×Buffer; and H2O was added to 40 μl.

[0028] (2) The above enzyme digestion products were recovered by agarose gel extraction, and the target fragment was then cut under ultraviolet light for gel extraction.

[0029] (3) The purified product was ligated at 22°C for 4 hours, and the total system was 20 μl, including 13 μl of ED3-FC fragment, 4 μl of purified pEE12.4, 1 μl of T4 ligase, and 2 μl of 10×Buffer.

[0030] (4) Add the ligation product to DH5α competent cells and mix well. Incubate on ice for 30 minutes, then at 42°C for 90 seconds. Then quickly transfer to an ice bath and incubate on ice for 3 minutes. Add 300 μl of LB medium and incubate on a shaker at 37°C for 1 hour. Spread the mixture onto a plate containing ampicillin antibiotic resistance and invert it to incubate overnight at 37°C.

[0031] (5) Extraction and identification: Select a single colony for amplification, and then extract according to the instructions of the plasmid extraction kit.

[0032] The enzyme digestion and identification reaction system for the recombinant plasmid was as follows: pEE12.4-ED3-FC 2 μl; Hind III and EcoRI 1 μl each; 10× Buffer 1 μl; H2O 5 μl; digestion at 37℃ for 1 hour. Subsequently, 5 μl of the digestion product was subjected to agarose gel electrophoresis for detection (see...). Figure 1 The positive plasmids were sent to Kexin Technology for sequencing. The plasmids with correct sequencing results were amplified to obtain the eukaryotic expression plasmid pEE12.4-ED3-FC.

[0033] Example 2 Construction and screening of recombinant CHO cells (1) Plasmid linearization and recovery: The plasmid pEE12.4-ED3-FC was linearized using Pvu I enzyme and incubated at 37°C for 3 hours. The enzyme digestion system contained: 100 ng plasmid; 10 μl Pvu I; 20 μl 10× Buffer; and H2O to 200 μl. The digested product was purified and recovered using a gel extraction kit. Finally, sterile water was added to dissolve the DNA, and the product was stored at -20°C for later use.

[0034] (2) Cell transfection and cell line selection: CHO cells were centrifuged at 1000 rpm for 5 minutes to collect the pellet. The cells were gently resuspended in glutamine-free CD-CHO-AGT medium, and the cells were washed to remove residual glutamine. The cells were centrifuged at 1000 rpm for 5 minutes to remove the medium. The cells were gently resuspended in electroporation buffer and the density was adjusted to 3 × 10⁻⁶ cells / mL. 7 cells / mL. Take two sterile 1.5mL EP tubes, add 0.5mL of cell suspension resuspended in electroporation buffer to each, then add 5μg of plasmid to each, mix gently, and incubate at room temperature for 15 minutes, gently tapping to mix every 5 minutes. After incubation, incubate on ice for 5 minutes. Add 0.5mL of the cell and plasmid mixture to each transfer cuvette, place in the electroporator, and start the electroporation program. After electroporation, transfer the cells to a 20mL Erlenmeyer flask containing CD-AGT medium, and then incubate at 37°C on a shaker.

[0035] After 48 hours of culture, the cell culture medium was replaced with CD-CHO-AGT medium containing 50 μM MSX, and the cells were continued to be cultured in a shaker at 37°C. Cell density and viability were observed starting from day 7, with cell density increasing from 1.2 × 10⁻⁶ cells / day. 6 The cell count / mL initially decreased, and around day 10-15, the cell density began to increase. When the cell density reached 1.0 × 10⁻⁶, the cell density returned to normal. 6 After reaching a cell / mL density, the cells were passaged to a density of approximately 1.0 × 10⁶ cells / mL using CD-CHO-AGT medium containing 50 μM MSX. 5 Cells / mL were cultured and transferred to CHO-2 suspension culture for scale-up production. After acclimatization, the cells were cryopreserved to ultimately obtain a cell line that highly expresses ED3 protein.

[0036] (3) PAGE identification of ED3 protein: The supernatant of the protein expressed by the cell line was collected by centrifugation and subjected to SDS-PAGE detection on the supernatant and precipitate of the cooled expression (see Figure 2 ).

[0037] Example 3: Purification of Recombinant Protein Proteins were purified using affinity chromatography. Binding / washing buffer, elution buffer, and neutralization buffer were prepared in advance. 100 μl of neutralization buffer was added to each target protein collection tube, with 1 mL of sample to be collected per tube. The sample was diluted with an equal volume of binding buffer and filtered through a 0.22 μm filter. rProtein G Beads 4FF were packed into a suitable chromatography column, and the column was equilibrated with 5 column volumes of binding buffer. The sample was added to the equilibrated rProtein G Beads 4FF, and washed with 10-15 column volumes of washing buffer to remove non-specifically adsorbed proteins; the wash buffer was collected. Elution was performed with 5-10 column volumes of elution buffer, and the eluent, representing the target protein fraction, was collected in a collection tube. Its amino acid sequence is shown in SEQ ID NO. 1. The purified protein was analyzed by SDS-PAGE (see [link to SDS-PAGE analysis]). Figure 3 The protein was purified, and the total protein content was determined using a BCA protein concentration assay kit. The concentration of ED3-FC recombinant protein was 2.12 mg / mL.

[0038] Example 4 Vaccine Preparation (1) Aqueous phase preparation: Dilute the purified protein with physiological saline to the concentration for seed preparation, prepare two groups, one with a protein antigen content of 160 μg / mL and the other with a protein antigen content of 80 μg / mL, mix evenly and preheat to 32℃.

[0039] (2) Emulsification: Preheat ISA201 VG adjuvant to 32°C, and slowly add the aqueous phase to ISA201 VG adjuvant at a ratio of 1:1 (mass ratio) of aqueous phase to oil phase, and emulsify at 100~120r / min for 60 minutes.

[0040] (3) Dispensing: Dispense in quantitative amounts, 1 mL per head, seal with stopper, affix label, and store at 2~8℃.

[0041] They received 80ug / dose and 40ug / dose of vaccine, respectively.

[0042] Example 5: Safety Trial of JEV Genetically Engineered Subunit Vaccine Thirty Balb / c mice aged 6-8 weeks were randomly divided into three groups. Group 1 was immunized with JEV subunit vaccine (40 μg / dose), Group 2 with JEV subunit vaccine (80 μg / dose), and Group 3 with PBS. Each mouse received an intramuscular injection of 0.2 mL of vaccine in its hind leg. The mice were observed for 14 consecutive days, and their mental state, respiration, behavior, and food intake were recorded. The injection site was also observed for redness, swelling, induration, and ulceration. The results are shown in Table 1.

[0043] Table 1. Safety test results of JEV subunit vaccine in mice.

[0044] Example 6: Efficacy Trial of JEV Genetically Engineered Subunit Vaccine (1) Vaccine immunization Forty Balb / c mice aged 6-8 weeks were randomly divided into four groups. The first group was immunized with a commercially available live porcine Japanese encephalitis vaccine (Keyijing, Wuhan Keqian Biotechnology). While this live vaccine provides strong immunoprotection, its safety profile is relatively low. The second group was immunized with JEV subunit vaccine (40 μg / dose), the third group with JEV subunit vaccine (80 μg / dose), and the fourth group with PBS. Each mouse received an intramuscular injection of 0.1 mL of vaccine in its hind leg. A booster immunization was administered 14 days after the initial immunization.

[0045] (2) Antibody detection and challenge test Fourteen days after the second immunization, tail vein blood was collected for ELISA antibody testing. The results showed that the ELISA antibody level in the immunized vaccine group was higher than that in the immunized PBS group, while the ELISA antibody level in the commercially available vaccine group was lower than that in the subunit vaccine group (see...). Figure 4 Simultaneously, using porcine Japanese encephalitis virus strain WH-1 (100LD) 50Mice were injected intraperitoneally with the PBS vaccine. After infection, their survival and clinical symptoms were observed daily for 14 days. Results showed that all mice in the PBS-immunized group died on day 4. After immunization with both the subunit vaccine and the commercially available vaccine, the survival rate of mice was higher than 80% (see [link to relevant documentation]). Figure 5 This demonstrates that the subunit vaccine has a good protective effect.

[0046] Although the above embodiments have described the present invention and its implementation in detail, it should be noted that for those skilled in the art, any changes, modifications, substitutions, combinations, simplifications, etc., made to the corresponding conditions without departing from the technical principles of the present invention should be considered as equivalent substitutions, and these improvements should also be considered within the scope of protection of the present invention.

Claims

1. An antigen protein for preventing and treating swine encephalitis B virus, characterized in that, It is a recombinant ED3 protein; The amino acid sequence of the ED3 recombinant protein is SEQ ID NO.

1.

2. The gene encoding the recombinant protein as described in claim 1, characterized in that, The encoding gene includes nucleotide molecules as shown in SEQ ID NO.

2.

3. A recombinant vector, characterized in that, It contains the encoding gene as described in claim 2, and the recombinant vector is a CHO eukaryotic cell expression vector.

4. The recombinant vector according to claim 3, characterized in that, This includes one of the pEE series, pcDNA series, and pCHO series vectors.

5. The recombinant vector according to claim 3, characterized in that, This includes one of the pEE series carriers.

6. A vaccine for preventing and treating porcine Japanese encephalitis virus, characterized in that, The vaccine comprises the recombinant protein of claim 1, as well as pharmaceutically acceptable adjuvants, stabilizers, and buffers.

7. A method for preparing a vaccine against swine encephalitis virus, characterized in that, Includes the following steps: (1) The coding gene described in claim 2 is cloned into a eukaryotic expression vector to obtain a recombinant vector; (2) Transfect host cells with the recombinant vector and screen host cells that express the recombinant protein stably and efficiently in suspension, then culture them at a lower temperature, and then separate and purify the recombinant protein from the cell supernatant; (3) Dilute the purified protein with physiological saline to the concentration for vaccine preparation, mix evenly, preheat, add adjuvant for emulsification, dispense quantitatively, seal, and store at 2~8℃ to obtain the vaccine.

8. The method for preparing a vaccine according to claim 7, characterized in that, The eukaryotic expression vector includes one of the pEE series vectors, preferably the pEE12.4 vector.

9. The recombinant protein according to claim 1, characterized in that, Application in the preparation of genetically engineered subunit vaccines for the prevention and treatment of swine encephalitis virus.

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