A fusion protein comprising a classical swine fever virus E2 protein and a porcine circovirus type 2 Cap protein and a bivalent subunit vaccine

Abstract

This invention provides a fusion protein comprising classical swine fever virus (CSFV) E2 protein and porcine circovirus type 2 (PCV2) Cap protein, and a bivalent subunit vaccine, relating to the field of veterinary biological products technology. This invention fuses and expresses the E2, Fc, and Cap proteins, and incorporates a TEV protease digestion motif into the fusion protein. This invention also provides a recombinant vector containing the fusion protein and a recombinant cell line expressing the fusion protein, enabling the simultaneous production of subunit vaccines for both viruses using a single expression process. The purification process is simple, utilizing commercially available Protein A packing material for protein purification, eliminating the need for complex processes such as protein refolding and ion exchange chromatography. In the bivalent subunit vaccine formed using the fusion protein of this invention, CSFV E2 exists in a dimer form, and PCV2 Cap protein exists in the form of VLPs (virtual protein chains).

Description

Technical Field

[0001] This invention belongs to the field of veterinary biological products technology, specifically relating to a fusion protein and bivalent subunit vaccine containing classical swine fever virus E2 protein and porcine circovirus type 2 Cap protein. Background Technology

[0002] Swine fever virus (CSFV) is an enveloped virus, 40–60 nm in size, with single-stranded positive-sense RNA. The CSFV genome is approximately 123 kb long and contains only one large open reading frame (ORF). This ORF is translated into a polymeric protein containing 3898 amino acid residues with a molecular weight of approximately 438 kDa, and further processed into a mature protein by viral and host cell proteases. Among the structural proteins, E0 and E2 are the most valuable for immunoprophylactic research, especially E2, which is currently the preferred protein for subunit vaccines.

[0003] The E2 protein is an envelope glycoprotein and a major antigenic protein of the virus. It is also the least conserved and most easily mutated of the three viral glycoproteins. The E2 protein typically exists as a 100 kDa homodimer on the surface of viral particles and cells infected with classical swine fever virus. The E2 protein can induce the production of antiviral neutralizing antibodies. Hulst immunized pigs with an E2 biphasic oil-in-water emulsion, which resisted challenge with the virulent 100 LD50 CSVBrescia strain. The E2 protein backbone consists of 370 amino acids (690–1060 amino acid residues encoded by the ORF), and is anchored to the membrane by its C-terminal 40 hydrophobic amino acids.

[0004] Porcine circovirus (PCV) belongs to the genus Circovirus in the family Circoviridae. It is a non-enveloped, single-stranded, negative-stranded circular DNA virus and one of the smallest animal viruses. Traditionally, it was believed that there were two genotypes of porcine circovirus: PCV1 and PCV2, with PCV2 being pathogenic. The N-terminus of the Cap protein in PCV2 contains an arginine-rich nuclear localization sequence responsible for mediating the interaction between the Rep complex and nucleic acid, thereby participating in viral DNA replication. Studies have shown that the nuclear localization sequence has a significant impact on the immunogenicity of the Cap protein; its deletion significantly reduces its ability to induce neutralizing antibodies. Although both full-length and truncated N-terminal 32-amino acid Cap proteins have been expressed in baculoviruses and can form virus-like particles (VLPs), the expression of the complete Cap protein affects its expression level in the expression system. However, the deletion of the entire segment may affect the surface characteristics and stability of the virus-like particles, thereby affecting their immunogenicity.

[0005] While existing technologies contain bivalent vaccines for classical swine fever and porcine circovirus type 2, they all employ a scheme of expressing each virus separately and then mixing them, resulting in cumbersome purification steps. Therefore, there is an urgent need for a scheme that can simultaneously produce subunit vaccines of both viruses through a single expression process and solve the problem of limited expression levels. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide a fusion protein and a bivalent subunit vaccine comprising classical swine fever virus E2 protein and porcine circovirus type 2 Cap protein, which can simultaneously produce subunit vaccines of two viruses in a single expression process, and the expression level is not affected by the nuclear localization signal region rich in basic amino acids at the N-terminus of the Cap protein. The protein purification is simple and does not require complex processes such as protein refolding and ion exchange chromatography.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] This invention provides a fusion protein comprising classical swine fever virus E2 protein and porcine circovirus type 2 Cap protein. The structure of the fusion protein from the N-terminus to the C-terminus includes: a signal peptide, classical swine fever virus E2 protein with the C-terminal intracellular region removed, Fc, a TEV restriction motif, and porcine circovirus Cap protein.

[0009] Preferably, the source of the signal peptide includes mouse IgG kappa chain signal peptide and / or silkworm immunoglobulin signal peptide;

[0010] The source of the Fc includes porcine IgG Fc.

[0011] This invention provides a fusion protein expressed in CHO cells, wherein the structure of the fusion protein from the N-terminus to the C-terminus includes: mouse IgG kappa chain signal peptide, classical swine fever virus E2 protein with the C-terminal intracellular region removed, porcine IgG Fc, TEV restriction enzyme motif, and porcine circovirus type 2 Cap protein.

[0012] The nucleotide sequence of the mouse IgG kappa chain signal peptide is shown in SEQ ID NO.1, the nucleotide sequence of the classical swine fever virus E2 protein with the C-terminal intracellular region removed is shown in SEQ ID NO.2, the nucleotide sequence of the porcine IgG Fc is shown in SEQ ID NO.3, the nucleotide sequence of the TEV restriction motif is shown in SEQ ID NO.4, and the nucleotide sequence of the porcine circovirus type 2 Cap protein is shown in SEQ ID NO.5.

[0013] Preferably, the amino acid sequence of the fusion protein is shown in SEQ ID NO.7.

[0014] The present invention also provides the above-mentioned fusion protein expressed by insect cells, wherein the structure of the fusion protein from the N-terminus to the C-terminus includes: silkworm immunoglobulin signal peptide, classical swine fever virus E2 protein with the C-terminal intracellular region removed, porcine IgG Fc, TEV restriction enzyme motif and porcine circovirus type 2 Cap protein;

[0015] The nucleotide sequence of the silkworm immunoglobulin signal peptide is shown in SEQ ID NO.8, the nucleotide sequence of the classical swine fever virus E2 protein with its C-terminal intracellular region removed is shown in SEQ ID NO.9, the nucleotide sequence of the porcine IgG Fc is shown in SEQ ID NO.10, the nucleotide sequence of the TEV restriction enzyme motif is shown in SEQ ID NO.11, and the nucleotide sequence of the porcine circovirus type 2 Cap protein is shown in SEQ ID NO.12.

[0016] Preferably, the amino acid sequence of the fusion protein is shown in SEQ ID NO.14.

[0017] Preferably, the fusion protein is secreted into the culture supernatant after the signal peptide is removed, and the amino acid sequence after the signal peptide is removed is shown in SEQ ID NO.15.

[0018] The present invention also provides a recombinant vector comprising the above-described fusion protein.

[0019] The present invention also provides a recombinant cell line expressing the above-mentioned fusion protein.

[0020] The present invention also provides the application of the above-mentioned fusion protein, the above-mentioned recombinant vector, or the above-mentioned recombinant cell line in the preparation of a bivalent subunit vaccine against classical swine fever and porcine circovirus type 2.

[0021] The present invention also provides a bivalent subunit vaccine against classical swine fever and porcine circovirus type 2, using the fusion protein expressed by the above-mentioned recombinant cell line as the antigen.

[0022] Beneficial Effects: This invention provides a fusion protein comprising classical swine fever virus (CSFV) E2 protein and porcine circovirus type 2 (PCV2) Cap protein. The addition of an FC tag to the fusion protein induces dimerization of the CSFV E2 protein, prolonging its half-life and promoting antigen presentation. This invention fuses the E2, Fc, and Cap proteins, placing the nuclear localization signal in the middle of the fusion protein, eliminating the adverse effects of its location at the N-terminus. This ensures that the expression level is unaffected by the basic amino acid-rich nuclear localization signal region at the N-terminus of the Cap protein, thus eliminating the need for specific sequence deletion. Furthermore, this invention incorporates a TEV protease digestion motif into the fusion protein. The PCV2 Cap protein produced by TEV digestion is most similar to the natural virus.

[0023] This invention also provides a recombinant vector containing the fusion protein and a recombinant cell line expressing the fusion protein. This allows for the simultaneous production of subunit vaccines for two viruses using a single expression process. The purification process is simple, utilizing commercially available Protein A packing material for protein purification, eliminating the need for complex processes such as protein refolding and ion exchange chromatography. In the bivalent subunit vaccine formed using the fusion protein of this invention, classical swine fever E2 exists in a dimer form, while porcine circovirus type 2 Cap protein exists in the form of VLPs. Attached Figure Description

[0024] Figure 1 Results of ELISA antibody detection for classical swine fever virus; Detailed Implementation

[0025] This invention provides a fusion protein comprising classical swine fever virus E2 protein and porcine circovirus type 2 Cap protein. The structure of the fusion protein from the N-terminus to the C-terminus includes: a signal peptide, classical swine fever virus E2 protein with the C-terminal intracellular region removed, Fc, a TEV restriction motif, and porcine circovirus type 2 Cap protein.

[0026] The signal peptides described in this invention are preferably derived from mouse IgG kappa chain signal peptides and / or silkworm immunoglobulin signal peptides; the Fc is preferably derived from porcine IgG Fc.

[0027] When the fusion protein described in this invention is expressed using different cell lines, codon optimization is required based on the codon preferences of the expressing cells. Therefore, although the structure of the fusion protein is the same, its nucleotide sequence is slightly different based on different cell expression systems.

[0028] This invention fuses Cap protein with classical swine fever E2 and IgG FC tags, placing the nuclear localization signal in the middle of the fusion protein and eliminating the adverse effects of its location at the N-terminus. Using the fusion protein described in this invention as an antigen, it can effectively activate antigen-presenting cells (APCs). Since APCs express FcR on their surface, the antigen-Fc fusion protein can serve as an antigen delivery vehicle, targeting and binding to APCs via the Fc fragment, shortening the antigen's free time in plasma, reducing protease degradation, and increasing the antigen's half-life, thereby enhancing antigen presentation.

[0029] When the fusion protein described in this invention is expressed using different cell lines, codon optimization is required based on the codon preferences of the expressing cells. Therefore, although the structure of the fusion protein is the same, its nucleotide sequence is slightly different based on different cell expression systems.

[0030] This invention provides a fusion protein expressed in CHO cells, referred to as CHO-E2-FC-Cap, whose structure from the N-terminus to the C-terminus includes: mouse IgG kappa chain signal peptide, classical swine fever virus E2 protein with the C-terminal intracellular region removed, porcine IgG Fc, TEV restriction motif, and porcine circovirus type 2 Cap protein.

[0031] The nucleotide sequence of the mouse IgG kappa chain signal peptide is shown in SEQ ID NO.1, the nucleotide sequence of the classical swine fever virus E2 protein with the C-terminal intracellular region removed is shown in SEQ ID NO.2, the nucleotide sequence of the porcine IgG Fc is shown in SEQ ID NO.3, the nucleotide sequence of the TEV restriction motif is shown in SEQ ID NO.4, and the nucleotide sequence of the porcine circovirus type 2 Cap protein is shown in SEQ ID NO.5.

[0032] In this invention, the sequences shown in SEQ ID NO.1 to SEQ ID NO.5 are all codon-optimized sequences. The mouse IgG kappa chain signal peptide sequence is 63 bases in length, and its nucleotide sequence is shown in SEQ ID NO.1; the classical swine fever virus (CSF) Shimen strain E2 protein sequence with the C-terminal intracellular region removed is 999 bases in length, and its nucleotide sequence is shown in SEQ ID NO.2; the porcine IgG FC sequence is 681 nucleotides in length, and its nucleotide sequence is shown in SEQ ID NO.3; the TEV restriction enzyme motif is 21 bases in length, and its nucleotide sequence is shown in SEQ ID NO.4; and the porcine circovirus type 2 Cap protein sequence is 696 bases in length, and its nucleotide sequence is shown in SEQ ID NO.5. Preferably, after synthesizing the CHO-E2-FC-Cap sequence, this invention further includes designing HindIII and EcoRI restriction enzyme sites at both ends of the sequence, with a total length of 2478 bases, and its nucleotide sequence is shown in SEQ ID NO.6, encoding the amino acid sequence shown in SEQ ID NO.7.

[0033] The present invention also provides the above-mentioned fusion protein expressed by insect cells, wherein the structure of the fusion protein from the N-terminus to the C-terminus includes: silkworm immunoglobulin signal peptide, classical swine fever virus E2 protein with the C-terminal intracellular region removed, porcine IgG Fc, TEV restriction enzyme motif and porcine circovirus type 2 Cap protein;

[0034] The nucleotide sequence of the silkworm immunoglobulin signal peptide is shown in SEQ ID NO.8, the nucleotide sequence of the classical swine fever virus E2 protein with its C-terminal intracellular region removed is shown in SEQ ID NO.9, the nucleotide sequence of the porcine IgG Fc is shown in SEQ ID NO.10, the nucleotide sequence of the TEV restriction enzyme motif is shown in SEQ ID NO.11, and the nucleotide sequence of the porcine circovirus type 2 Cap protein is shown in SEQ ID NO.12.

[0035] The present invention does not specifically limit the type of insect cell. The example of insect cell SF9 is used in the embodiments for illustration, but it should not be considered as the entire scope of protection of the present invention. In this invention, the fusion protein expressed based on insect cells is abbreviated as SF9-E2-FC-Cap, and its sequence is as follows: a silkworm immunoglobulin signal peptide sequence, 60 bases in size, whose nucleotide sequence is shown in SEQ ID NO.8; a classical swine fever strain E2 protein sequence with the C-terminal intracellular region removed, 999 bases in size, whose nucleotide sequence is shown in SEQ ID NO.9; a porcine IgG FC sequence, 681 nucleotides in size, whose nucleotide sequence is shown in SEQ ID NO.10; a TEV restriction enzyme sequence, 21 bases in size, whose nucleotide sequence is shown in SEQ ID NO.11; a porcine circovirus type 2 Cap protein sequence, 696 bases in size, whose nucleotide sequence is shown in SEQ ID NO.12; and the synthesized SF9-E2-FC-Cap sequence, which contains BamHI and HindIII restriction enzyme sites at both ends, with a full length of 2469 bases, as shown in SEQ ID NO.13, and the encoded amino acid sequence is shown in SEQ ID NO.14.

[0036] The CHO-E2-FC-Cap or SF9-E2-FC-Cap of this invention is secreted into the culture supernatant after the signal peptide is cleaved. The amino acid sequence after cleavage of the signal peptide is shown in SEQ ID NO.15:

[0037] RLACKEDYRYAISSTNEIGLAGGLTTTWKEYSHDLQLNDGTVKAICVAGSFKVTALNVVSRRYLASLHKGALLTSVTFELLFDGTNPSTEEMGDDFGFGLCPFDTSPVVKGKYNTTLLNGSAFYLVCPIGWTGVIECTAVSPTTLRTEVVKTFRREKPFPHRMDCVTTTVENEDLFYCKLGGNWTCVKGEPVVYTGG QVKQCKWCGFDFNEPDGLPHYPIGKCILANETGYRIVDSTDCNRDGVVISAEGSHECLVGNTTVKVHASDERLGPMPCRPKEIVSSAGPVRKTSCTFNYA KTLKNKYYEPRDSYFQQYMLKGEYQYWFDLDVTDVGRPCPICPACEGPGPSAFIFPPKPKDTLMISRTPQVTCVVVDVSQENPEVQFSWYVDGVEVHTAQ TRPKEAQFNSTYRVVSVLPIQHEDWLKGKEFECKVNNKDLPAPITRIISKAKGPSREPQVYTLSPSAEELSRSKVSITCLVTGFYPPDIDVEWKSNGQP EPEGNYRTTPPQQDVDGTYFLYSKLAVDKASWQRGDPFQCAVMHEALHNHYTQKSVSKTQGKENLYFQGYPRRRYRRRRHRPRSHLGQILRRRPWLLHPR HRYRWRRKNGIFNTRLSRTFGYTIKRTTVKTPSWAVDMMRFNINDFLPPGGGSNPRSVPFEYYRIRKVKVEFWPCSPITQGDRGVGSSAVILDDNFVTKA TALTYDPYVNYSSRHTITQPFSYHSRYFTPKPVLDSTIDYFQPNNKRNQLWLRLQTAGNVDHVGLGTAFENSIYDQEYNIRVTMYVQFREFNLKDPPLNP

[0038] The present invention also provides a recombinant vector comprising the above-described fusion protein.

[0039] In this invention, different recombinant vectors are designed for different sequences. For example, the CHO-E2-FC-Cap sequence and pEE12.4 are ligated and transformed and cloned. After correct sequencing, the CHO-E2-FC-Cap-pEE12.4 recombinant plasmid is constructed.

[0040] When the fusion protein is SF9-E2-FC-Cap, the present invention preferably uses BamHI and HindIII to digest the SF9-E2-FC-Cap sequence and the pFastBac1 vector sequence, recovers the digested SF9-E2-FC-Cap and pFastBac1 sequence fragments, performs ligation transformation and cloning, and after sequencing verification, constructs the SF9-E2-FC-Cap-pFastBac1 transfer vector.

[0041] The present invention also provides a recombinant cell line expressing the above-mentioned fusion protein.

[0042] This invention does not specifically limit the construction method of the recombinant cell line; conventional construction methods in the art can be used. For example, after constructing the CHO-E2-FC-Cap-pEE12.4 recombinant plasmid, CHO-K1 cell transfection and high-expression cell pool screening are used to screen for stable cell lines (high-expression cell lines), and the high-expression cell lines are subjected to serum-free suspension acclimation to obtain the recombinant cell line. Preferably, this invention involves shaking-flask fermentation of the recombinant cell line. After fermentation, the cell supernatant is harvested, and the protein is purified using commercially available Protein A packing material. After obtaining the purified protein, this invention preferably further includes enzymatic digestion with 1U TEV protease added at a ratio of 8μg protein to 8μg protein. The digested protein is then placed in a dialysis bag for VLP self-assembly. The liquid in the dialysis bag is removed, and the sample is loaded onto a nickel column to remove the His-tagged TEV enzyme. The flow-through is the antigen solution containing classical swine fever virus E2 protein dimer and porcine circovirus type 2 VLPs, which can be used to prepare a bivalent subunit vaccine for classical swine fever and porcine circovirus type 2.

[0043] After obtaining the SF9-E2-FC-Cap-pFastBac1 transfer vector, this invention preferably further includes transforming it into competent DH10Bac cells, culturing at 37°C, screening using a blue-white screening method, and identifying by PCR to obtain the SF9-E2-FC-Cap-Bacmid recombinant baculovirus. The SF9-E2-FC-Cap-Bacmid recombinant baculovirus is then transfected into SF9 cells in logarithmic growth phase. After culturing, the P1 generation of recombinant baculovirus SF9-E2-FC-Cap-rBV is harvested and continuously passaged to the P3 generation. The P3 generation virus is centrifuged, and the supernatant is the viral fluid. High Five cells are infected with the P3 generation virus, and after culturing, the supernatant is collected. The supernatant contains the target protein. The purification, enzyme digestion, and antigen preparation methods for the target protein are all the same in this invention and will not be described in detail here.

[0044] The present invention also provides the application of the above-mentioned fusion protein, the above-mentioned recombinant vector, or the above-mentioned recombinant cell line in the preparation of a bivalent subunit vaccine against classical swine fever and porcine circovirus type 2.

[0045] The applications described in this invention are preferably the same as those described above, and will not be repeated here.

[0046] The present invention also provides a bivalent subunit vaccine against classical swine fever and porcine circovirus type 2, using the fusion protein expressed by the above-mentioned recombinant cell line as the antigen.

[0047] After obtaining the above-mentioned antigen solution, the present invention preferably further includes diluting the antigen solution with PBS buffer to a final concentration of 60 μg / mL, and emulsifying it with ISA201 VG adjuvant at a volume ratio of 46:54, which is a bivalent subunit vaccine against classical swine fever and porcine circovirus type 2.

[0048] The following detailed description, in conjunction with embodiments, of a fusion protein and bivalent subunit vaccine comprising classical swine fever virus E2 protein and porcine circovirus type 2 Cap protein provided by the present invention, should not be construed as limiting the scope of protection of the present invention.

[0049] Example 1

[0050] CHO cell expression sequence synthesis

[0051] Based on the gene sequences of the E2 protein of the classical swine fever strain from Shimen and the Cap protein of the domestically prevalent porcine circovirus type 2 strain, the codons were optimized according to the codon preferences of CHO cells to synthesize the CHO-E2-FC-Cap sequence. The sequence contains HindIII and EcoRI restriction sites at both ends, and is 2478 bases in length, as shown in SEQ ID NO. 6. The amino acid sequence encoded by the CHO-E2-FC-Cap sequence is shown in SEQ ID NO. 7.

[0052] Example 2

[0053] Constructing the CHO-E2-FC-Cap-pEE12.4 recombinant plasmid

[0054] The CHO-E2-FC-Cap sequence and pEE12.4 vector synthesized in Example 1 were digested with HindIII and EcoRI. The digested CHO-E2-FC-Cap and pEE12.4 sequence fragments were recovered, ligated, transformed, and cloned. After correct sequencing, the recombinant plasmid CHO-E2-FC-Cap-pEE12.4 was constructed.

[0055] Example 3

[0056] CHO-K1 cell transfection and screening of high-expressing cell lines

[0057] 1. Prepare cells:

[0058] Take one 10cm cell culture dish of CHO-K1 cells that have been passaged for 24 hours and have a confluence of 80% to 90%. Discard the culture medium, wash the cells once with 10mL of PBS, discard the PBS, add 15mL of serum-free and antibiotic-free DMEM / F12 medium, and incubate in a 37℃ 5% CO2 cell culture incubator.

[0059] 2. Transfection plasmid

[0060] according to According to the 2000 instructions, 24 μg of the E2-FC-Cap-pEE12.4 recombinant plasmid and 60 μL of serum-free and antibiotic-free DMEM / F12 medium were diluted separately. 2000, mix well and let stand at room temperature for 5 minutes. Mix the two tubes of liquid and let stand at room temperature for 20 minutes. Take the prepared CHO-K1 cells, add the above-mentioned mixed reagent dropwise, and then place them in a 37℃ 5% CO2 cell culture incubator. After culturing for 4-6 hours, discard the culture medium, add 10 mL of DMEM / F12 medium (10% serum, 1% penicillin and streptomycin, the same below, glutamine-free), and place in a 37℃ 5% CO2 cell culture incubator.

[0061] 3. Pressure screening

[0062] 24 hours after transfection, take one dish each of transfected CHO-K1 cells and untransfected CHO-K1 cells (negative control), discard the supernatant medium, add 10 mL of DMEM / F12 medium (10% serum + 25 μM MSX, 1% penicillin antibody, glutamine-free), and select under pressure for 7 days. Observe the cells during this period. If there are many dead cells, change the medium to the same medium.

[0063] 4. Screening of stable cell lines

[0064] After approximately 7 days of pressure selection using 25 μM MSX until the negative control cells are almost completely dead, cell line selection begins. Transfected CHO-K1 cells are collected, the culture medium is discarded, and the cells are washed once with PBS. 500 μL of 0.25% trypsin is added, and the cells are digested at room temperature for 2–5 minutes until single cells are formed. The digestion reaction is stopped by adding 10 mL of DMEM / F12 medium (10% serum + 25 μM MSX, 1% penicillin-dextrin, glutamine-free). Cells are dispersed using a pipette and counted. The cells are then diluted to 10⁻¹⁰ with DMEM / F12 medium (10% serum + 25 μM MSX, 1% penicillin-dextrin, glutamine-free). 4 Cells / mL were transferred to 96-well plates, 200 μL per well, and incubated in a 37°C, 5% CO2 cell culture incubator. Once the 96-well plates were confluent, the supernatant was collected and analyzed by ELISA. Highly expressed positive cell lines were further cultured and cryopreserved.

[0065] Example 4

[0066] Serum-free suspension acclimatization of high-expression cell lines

[0067] Resuscitate the high-expressing cell line in DMEM / F12 medium (10% serum, 1% penicillin-dextrose antibody, glutamine-free) and continue passage in this medium 2 to 3 times until cell growth is stable, and the cell density reaches 2 × 10⁶ cells / year. 6 After reaching a cell density of 0.3-0.5 × 10⁶ cells / mL, the cells were passaged, with the passage density controlled at 0.3-0.5 × 10⁶ cells / mL. 5 cells / mL, culture medium was DMEM / F12 medium with 10% serum and The basal culture medium (purchased from Jianshun Biotechnology) was mixed at a ratio of 75:25, and the cells were cultured in a constant temperature shaker at 37℃, 5% CO2, and 110 rpm. When the cell density reached 2 × 10⁶ cells / day after 3–4 days... 6 Cells / mL and cell viability >90%, passaged in DMEM / F12 medium with 10% serum and The basal culture medium was mixed at a 50:50 ratio, and the cell density was controlled at 0.3–0.5 × 10⁻⁶ cells / year. 6 cells / mL. If cell growth is slow, centrifuge the supernatant, retain 20% of the original culture medium, and add 80% DMEM / F12 medium containing 10% serum. Continue culturing with a 75:25 basal medium mixture; repeat the aforementioned steps, gradually increasing the amount of medium. The proportion of basal culture medium is up to 100%. Basic culture medium.

[0068] Example 5

[0069] Cell shake-flask fermentation

[0070] Remove the cells from the shake flask from the constant temperature shaker and dilute the cells to 0.3–0.5 × 10⁻⁶. 5 30 mL cells / mL were inoculated. The basal culture medium was transferred to a 125 mL shake flask and incubated in a constant temperature shaker at 37°C, 5% CO2, and 110 rpm. Glucose concentration was monitored daily; when the glucose concentration was less than 4 g / L, glucose solution was added to the culture medium to a glucose concentration of 4 g / L. 3% glucose solution was added on days 3, 5, 7, and 9. Feed culture medium. On day 12, harvest the cell culture supernatant.

[0071] Example 6

[0072] Insect cell expression sequence synthesis

[0073] Referring to the E2 protein gene sequence of the classical swine fever strain Shimen strain and the Cap protein gene sequence of the domestically prevalent porcine circovirus type 2 strain, the codons were optimized according to the codon preference of insect cells, and the nucleotide sequence of the synthesized SF9-E2-FC-Cap sequence is shown in SEQ ID NO. 13, and the encoded amino acid sequence is shown in SEQ ID NO. 14.

[0074] Example 7

[0075] Construction of baculovirus transfer vector

[0076] The SF9-E2-FC-Cap sequence and pFastBac1 vector sequence synthesized in Example 6 were digested with BamHI and HindIII. The digested SF9-E2-FC-Cap and pFastBac1 sequence fragments were recovered, ligated, transformed, and cloned. After correct sequencing, the SF9-E2-FC-Cap-pFastBac1 transfer vector was constructed.

[0077] Example 8

[0078] Rod pellet preparation

[0079] The SF9-E2-FC-Cap-pFastBac1 transfer vector prepared in Example 7 was transformed into competent DH10Bac cells. After culturing at 37°C, the cells were screened using the blue-white screening method and identified by PCR to obtain recombinant SF9-E2-FC-Cap-Bacmids. The transformation and screening methods were performed according to the Invitrogen Bac-to-Bac™ Baculovirus Expression System User Guide.

[0080] Example 9

[0081] Recombinant baculovirus harvest

[0082] The SF9-E2-FC-Cap-Bacmid recombinant rod-like particles obtained in Example 8 were transfected using a transfection reagent. SF9 cells in logarithmic growth phase were transfected, and after 72 hours of culture, P1 generation recombinant baculovirus SF9-E2-FC-Cap-rBV was harvested. The harvested P1 generation recombinant baculovirus was passaged in SF9 cells to the P3 generation. The P3 generation virus was centrifuged, and the supernatant was used as the viral fluid. The viral titer of the P3 generation was determined by plaque assay. High Five cells were infected with the P3 generation virus at an inoculation density of 1 MOI. After 96 hours of culture, the cell culture supernatant contained the target protein.

[0083] Example 10

[0084] Protein purification

[0085] Collect the cell culture supernatant from Example 5 or 9, centrifuge at 8000g for 30 min at 4°C, collect the supernatant, and filter through a 0.8 μm filter membrane. Equilibrate the Protein A column with 5–10 column volumes of PBS. Repeat the column loading with the treated cell supernatant 3 times. Wash the column with 10 times PBST, wash the column with 2 times PBS, and elute the protein with 2 column volumes of 0.1M glycine (pH 3.0). Collect the eluent and neutralize to pH 7.5 with 1M Tris (pH 9.0). This is the purified protein.

[0086] Example 11

[0087] Enzymatic protein digestion and VLP self-assembly

[0088] Following the instructions of the BCA protein quantification kit (purchased from Shanghai Sangon Biotech), the concentration of purified protein was determined, and the total protein mass was calculated. 1 U of TEV protease (His-tag, purchased from Beyotime Biotechnology) was added per 8 μg of protein, along with 10× restriction enzyme buffer (500 mM NaH₂PO₄, 150 mM NaCl, 10 mM EDTA, 10 mM DTT, 1% Tween-20, pH 8.0). Digestion was performed at 4°C for 12–16 h. After digestion, the digested protein was placed in a dialysis bag (3500D) with a dialysis buffer of 50 mM NaH₂PO₄, 500 mM NaCl, pH 8.0, and dialyzed for 12–16 h, changing the buffer 2–3 times. After the first dialysis, the mixture was incubated at 4°C for 8–12 h to allow VLPs to self-assemble. The dialysis buffer was then changed to PBS buffer, and dialyzed for 12–16 h, changing the buffer 2–3 times. After the third dialysis, the liquid in the dialysis bag was removed, and the sample was loaded onto a nickel column to remove the His-tagged TEV enzyme. The flow-through solution contained the classical swine fever virus E2 protein dimer and porcine circovirus type 2 VLPs. The nickel column was eluted with PBS buffer containing 200 mM imidazole, and the eluent was collected as the recovered TEV protease. After enzyme activity assay, it can be directly used in the next production enzymatic digestion process.

[0089] Example 12

[0090] vaccine preparation

[0091] The antigen solution from Example 11 was diluted with PBS buffer to a final concentration of 60 μg / mL, and then emulsified with ISA201VG adjuvant at a volume ratio of 46:54 to obtain a bivalent subunit vaccine for classical swine fever and porcine circovirus disease.

[0092] Example 13

[0093] Swine fever virus immune challenge experiment

[0094] 1. Immunization program

[0095] Ten healthy, susceptible piglets aged 21–28 days were selected (blocking ELISA for classical swine fever antibodies, with a blocking rate of less than 30%) and randomly divided into two groups: a blank control group (5 piglets) and a vaccine-immunized group (5 piglets). The vaccine-immunized group was immunized with a bivalent subunit vaccine against classical swine fever and porcine circovirus disease, while the blank control group was immunized with PBS. Each immunization was administered via intramuscular injection 1 mL behind the ear, with a booster immunization 21 days after the first immunization. Serum samples were collected before and 21 days after the second immunization to measure classical swine fever neutralizing antibodies (see Example 14) and ELISA antibodies (IDEXX classical swine fever virus antibody detection kit).

[0096] 2. Criteria for Infection and Onset of Disease

[0097] 21 days after the first vaccination, piglets were injected intramuscularly with 1 ml (not less than 10 ml) of Shimen lineage blood toxin. 5 Minimum lethal dose (purchased from the China Institute of Veterinary Drug Control), observed for 14 consecutive days.

[0098] Criteria for determining disease onset after challenge with the Shimen strain of classical swine fever hemorrhagic virus:

[0099] ① Death.

[0100] ② Elevated body temperature (≥41℃) for at least 3 days.

[0101] ③ Clinical symptoms include lethargy and loss of appetite.

[0102] If symptoms meet ① or both ② and ③, the disease can be diagnosed.

[0103] 3. Experimental Results

[0104] Results of the ELISA antibody blocking rate test for classical swine fever virus are as follows: Figure 1 As shown, the pre-immunization blocking rate was less than 30%, all of which were negative for classical swine fever virus antibodies. The blocking rate in the vaccine-immunized group 21 days after the second immunization was no less than 60%, with a maximum of 93.31%, indicating that the bivalent subunit vaccine against classical swine fever and porcine circovirus disease has good immunogenicity. Table 1 shows the results of the classical swine fever virus neutralizing antibody and challenge experiment 21 days after the second immunization. The results indicate that the neutralizing antibody titer against classical swine fever virus in the vaccine-immunized group was no less than 1:64, and the bivalent subunit vaccine against classical swine fever and porcine circovirus disease can provide 100% protection against classical swine fever virus for piglets.

[0105] Table 1. Results of neutralizing antibodies against classical swine fever virus and challenge experiments.

[0106]

[0107]

[0108] Example 14

[0109] Experiment on neutralizing antibodies against classical swine fever virus

[0110] Classical swine fever virus strain CVCCAV65 (purchased from the China Institute of Veterinary Drug Control) and PK15 cells were used as the experimental virus and cells, respectively. FITC-labeled WH303 monoclonal antibody was used as the immunofluorescence staining antibody. The day before the experiment, PK15 cells were digested with trypsin, resuspended in MEM containing 10% FBS, and seeded into 96-well cell culture plates at a density of 2 × 10⁶ cells / well. 5 Cells / mL, seeded at 0.1 mL per well, and cultured at 37°C and 5% CO2 until a cell monolayer with a confluence of 70%–80% is formed (1–2 days). The serum sample to be tested is inactivated at 56°C for 30 minutes and diluted with serum-free MEM cell culture medium to a dilution of 21 days after the second immunization. 1 ,2 2 ,2 3 ,2 4 ,2 5 ,2 6 ,2 7 ,2 8 Mix thoroughly. Dilute the classical swine fever virus solution to 200 TCID values ​​using MEM cell culture medium containing 2% FBS, according to the determined viral titer. 50 / 0.1ml. Take 100μL of serum diluent and mix it with an equal volume of classical swine fever virus (200 TCID50). 50 Mix thoroughly, the final toxic dose is 100 TCID. 50 0.1 mL of serum-virus mixture was added and incubated at 37°C in a 5% CO2 incubator for 1–2 hours. The cell monolayer was washed with serum-free MEM cell culture medium, and then 0.1 mL of serum-virus mixture was added to each well, with two replicates for each dilution. Normal negative control cells without neutralizing virus were also included. The cells were incubated at 37°C in a 5% CO2 incubator for 1 hour. The reaction mixture was aspirated, and MEM maintenance medium (containing 2% fetal bovine serum (without BVDV antibody, inactivated at 56°C for 30 minutes, and 1% penicillin antibody) was added. The cells were incubated at 37°C for 72 hours. Indirect immunofluorescence was used for staining and result determination. The 96-well plate was removed, the liquid in the wells was discarded, and the cells were washed three times with PBS buffer, patted dry on absorbent paper, and dried in a fume hood for 5 minutes. 100 μL of pre-chilled fixative (methanol and acetone 1:1 mixture) was added to each well, and the plate was incubated at -20°C for at least 30 minutes. The fixative was discarded, and the cells were washed three times with PBS buffer. Dilute the FITC-labeled WH303 monoclonal antibody to the working concentration, add 50 μL to each well, and incubate at 37°C in the dark for 1 hour. Discard the liquid in the wells, wash three times with PBS buffer, and observe the results under a fluorescence inverted microscope in a 96-well plate. A bright green fluorescence in the cell cytoplasm indicates a positive staining result; no staining in the cytoplasm indicates a negative staining result. The highest dilution in which both replicates of serum show positive staining is the neutralizing antibody titer of the serum sample.

[0111] Example 15

[0112] Porcine circovirus type 2 immune challenge experiment

[0113] 1. Immunization program

[0114] Fifteen healthy, susceptible piglets aged 14–21 days, with negative PRRSV ELISA antibodies (detected using the IDEXX ELISA kit), negative PCV2 ELISA antibodies (see Example 16), and negative results from a porcine circovirus type 2 fluorescent quantitative PCR detection kit (purchased from Hunan Guanmu Biotechnology Co., Ltd.), were randomly divided into three groups of five piglets each. Group 1 received a 1 mL intramuscular injection of a bivalent subunit vaccine against classical swine fever and porcine circovirus disease in each piglet via the neck. A booster immunization was administered 21 days after the first immunization, following the same route and dosage. Group 2 served as a non-immunized challenge control, receiving a 1 mL intramuscular injection of PBS in each piglet via the neck. Group 3 served as a non-immunized, non-challenge blank control. Blood samples were collected before immunization and 21 days after the second immunization to measure ELISA antibody levels (see Example 16).

[0115] 2. Criteria for Infection and Onset of Disease

[0116] Twenty-one days after the second immunization, all piglets were weighed. Piglets in groups 1 and 2 were each injected with 2.5 mL of porcine circovirus type 2 ZJ / C strain (virus content 1.0 × 10⁻⁶) via nasal drops and intramuscular injection. 7 TCID 50 / mL (provided by Hangzhou Youben Biological Vaccine Co., Ltd.). On days 4 and 7 post-challenge, all pigs were inoculated with keyhole hemocyanin (KLH / ICFA, 0.5 mg / mL) emulsified with Freund's incomplete adjuvant at four sites: both armpits and both buttocks. 1 mL was inoculated at each site, and 10 mL / pig was simultaneously injected intraperitoneally with thioglycolic acid medium. On days 11 and 19 post-challenge, 10 mL / pig was injected intraperitoneally again with thioglycolic acid medium. Pigs were observed continuously for 28 days post-challenge. The outcome was determined based on the relative growth rate and viral antigen detection results. At least four pigs in the immunized challenge group were protected, and at least four pigs in the non-immunized challenge control group developed the disease.

[0117] Criteria for determining disease onset after challenge with porcine circovirus type 2:

[0118] ① Relative growth rate

[0119] If the relative growth rate of piglets in the challenge test groups (groups A and B) is ≥5.0% compared with that in the blank control group (group C), the piglets are considered to have poor growth and development and clinical symptoms caused by porcine circovirus type 2.

[0120] Relative growth rate (%) = (a - b) / c × 100%

[0121] a. Average daily weight gain of the blank control group = (Sum of weights of piglets in the blank control group 28 days after challenge - Sum of weights of piglets in the blank control group on the day of challenge) / (28 days × 5 piglets)

[0122] Average daily weight gain of piglets in the challenge test group b = (sum of weights of piglets in the challenge test group 28 days after challenge - sum of weights of piglets in the challenge test group on the day of challenge) / (28 days × 5 piglets)

[0123] c. Average daily weight gain of piglets in the challenge experiment = (piglet's weight 28 days after challenge - piglet's weight on the day of challenge) / 28 days

[0124] ② Viral antigen detection

[0125] Twenty-eight days after challenge, blood was collected from the anterior vena cava to separate serum, and the Ct value was detected by porcine circovirus type 2 fluorescent quantitative PCR with a value ≤35.

[0126] If either ① or ② is met, the disease can be diagnosed.

[0127] 3. Experimental Results

[0128] Table 2 shows the results of ELISA antibody detection and challenge for porcine circovirus type 2 (PCV2). Before immunization, the S / P value was less than 0.2, indicating negative PCV2 ELISA antibodies in all cases. Twenty-one days after the second immunization, the S / P values ​​in the vaccine-immunized group ranged from a minimum of 0.791 to a maximum of 1.343, indicating positive antibodies in all cases. Twenty-eight days after challenge, all piglets in the vaccine-immunized PCV2 challenge group were negative for the virus antigen (Ct value > 35), with a relative weight gain rate of less than 5%, showing 5 / 5 protection. In the PBS-immunized PCV2 challenge group, 5 / 5 developed the disease. This indicates that the bivalent subunit vaccine against classical swine fever and porcine circovirus disease has excellent immunogenicity, providing 100% protection against PCV2 in piglets.

[0129] Table 2. Results of ELISA antibody detection and challenge for porcine circovirus type 2.

[0130]

[0131]

[0132] Example 16

[0133] ELISA antibody detection of porcine circovirus type 2

[0134] The Protein A column was equilibrated with PBS buffer. The antigen solution containing classical swine fever virus E2 protein dimer and porcine circovirus type 2 (VLPs) described in Example 11 was loaded three times repeatedly. The flow-through buffer was the antigen solution containing only porcine circovirus type 2 (VLPs). The concentration was quantified using the BCA method, and the solution was diluted with PBS buffer to 0.2 μg / mL to coat the ELISA plate at 100 μL / well, incubating at 4°C for 16 h. After coating, the liquid in the wells was discarded, and 300 μL of PBST was added to each well for rinsing once. 200 μL of freshly prepared blocking buffer (5% skim milk, PBS) was added to each well, and the plate was blocked at 37°C for 2 h. After blocking, the liquid in the wells was discarded, and 300 μL of PBST was added to each well for rinsing once. The plate was then patted dry on absorbent filter paper. The serum to be tested was diluted 100-fold with PBS buffer, added to the antigen-coated plate, and incubated at 37°C for 1 h. Discard the liquid in each well, add 300 μL of washing buffer to each well, and rinse 3 times. Dilute the rabbit anti-bovine HRP secondary antibody 10000-fold with PBS containing 5% skim milk, add 100 μL to each well, and incubate at 37°C for 1 h. Discard the liquid in each well, add 300 μL of PBST washing buffer to each well, and rinse 3 times. Add 100 μL of TMB chromogenic solution to each well, and incubate at room temperature in the dark for 10 min. Add 50 μL of stop solution to each well, and read the absorbance at 450 nm using a microplate reader. The criteria for judging antibody positivity are: S / P ≥ 0.4, P > 1.0.

[0135] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A fusion protein expressed by CHO cells, characterized in that, The structure of the fusion protein from N-terminus to C-terminus is: mouse IgG kappa chain signal peptide, classical swine fever virus E2 protein with C-terminal intracellular region removed, porcine IgG Fc, TEV restriction motif and porcine circovirus type 2 Cap protein. The nucleotide sequence of the mouse IgG kappa chain signal peptide is shown in SEQ ID NO.1, the nucleotide sequence of the classical swine fever virus E2 protein with the C-terminal intracellular region removed is shown in SEQ ID NO.2, the nucleotide sequence of the porcine IgG Fc is shown in SEQ ID NO.3, the nucleotide sequence of the TEV restriction motif is shown in SEQ ID NO.4, and the nucleotide sequence of the porcine circovirus type 2 Cap protein is shown in SEQ ID NO.

5.

2. A fusion protein expressed using insect cells, characterized in that, The structure of the fusion protein from N-terminus to C-terminus is: silkworm immunoglobulin signal peptide, classical swine fever virus E2 protein with C-terminal intracellular region removed, porcine IgG Fc, TEV restriction motif and porcine circovirus type 2 Cap protein. The nucleotide sequence of the silkworm immunoglobulin signal peptide is shown in SEQ ID NO.8, the nucleotide sequence of the classical swine fever virus E2 protein with its C-terminal intracellular region removed is shown in SEQ ID NO.9, the nucleotide sequence of the porcine IgG Fc is shown in SEQ ID NO.10, the nucleotide sequence of the TEV restriction enzyme motif is shown in SEQ ID NO.11, and the nucleotide sequence of the porcine circovirus type 2 Cap protein is shown in SEQ ID NO.

12.

3. A fusion protein, characterized in that, The fusion protein of claim 1 or 2 is secreted into the culture supernatant after the signal peptide is removed, and the amino acid sequence after the signal peptide is removed is shown in SEQ ID NO.

15.

4. A recombinant vector comprising the encoding gene of the fusion protein according to any one of claims 1 to 3.

5. A recombinant cell line expressing the fusion protein of any one of claims 1 to 3.

6. The use of the fusion protein according to any one of claims 1 to 3, the recombinant vector according to claim 4, or the recombinant cell line according to claim 5 in the preparation of a bivalent subunit vaccine against classical swine fever and porcine circovirus type 2, characterized in that, The active ingredients of the bivalent subunit vaccine are classical swine fever virus E2 protein dimer and porcine circovirus type 2 VLPs, which are generated by enzymatic digestion of the fusion protein shown in SEQ ID No. 15 with TEV protease.

7. A bivalent subunit vaccine against classical swine fever and porcine circovirus type 2, characterized in that, The active ingredients of the bivalent subunit vaccine are classical swine fever virus E2 protein dimer and porcine circovirus type 2 VLPs, which are generated by enzymatic digestion of the fusion protein shown in SEQ ID No. 15 with TEV protease.

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