Recombinant protein combination of chicken infectious anemia virus vp1 and vp2 proteins and application in preparation of subunit vaccine

Abstract

The application belongs to the field of veterinary biotechnology and genetically engineered vaccine, and discloses a recombinant protein combination of chicken infectious anemia virus VP1 and VP2 proteins and application thereof in preparation of a subunit vaccine. An engineering cell strain capable of independently and efficiently secreting and expressing VP1 and VP2 proteins is screened, and the recombinant proteins in the supernatant of the suspension culture are purified and assembled into a subunit vaccine in vitro according to a specific ratio. The vaccine can induce chickens to produce high-titer neutralizing antibodies, and the protection rate against virus attack reaches 100%, which is significantly better than existing inactivated vaccines, and the vaccine is safe, has no risk of virus dissemination, and has a differential diagnosis DIVA function.

Description

Technical Field

[0001] This invention belongs to the field of veterinary biotechnology and genetic engineering vaccines, specifically relating to the recombinant protein combination of chicken infectious anemia virus VP1 and VP2 proteins and its application in the preparation of subunit vaccines. Background Technology

[0002] Chicken Infectious Anemia (CIA) is an acute, highly contagious disease in chicks caused by the chicken infectious anemia virus (CIAV). The disease is characterized by aplastic anemia, systemic lymphoid tissue atrophy (such as thymic atrophy and bone marrow jaundice), and severe immunosuppression. CIAV infection not only directly causes chick mortality but, more seriously, reduces the flock's immune response to vaccines against Newcastle disease, infectious bursal disease, and Marek's disease, making them highly susceptible to secondary bacterial (such as Escherichia coli and Staphylococcus aureus) and viral infections, resulting in enormous economic losses to the global poultry industry.

[0003] The CIAV genome primarily encodes three proteins: VP1 (capsid protein), VP2 (accessory protein), and VP3 (apoptin). VP1 (52 kDa) is the virus's only structural protein and its main protective antigen, containing abundant neutralizing epitopes. However, the native VP1 protein contains a nuclear localization signal (NLS), is primarily located in the cell nucleus in virus-infected cells, and exhibits strong hydrophobicity.

[0004] Currently, commercially available CIAV vaccines are primarily live attenuated vaccines. While live vaccines offer good protection, they also present significant safety risks:

[0005] 1) Risk of viral shedding and reversion: Attenuated vaccine strains can spread horizontally in chicken flocks and vertically to offspring, posing a risk of reversion to virulence or recombination with other strains.

[0006] 2) Immunosuppression: The live vaccine virus itself has a certain degree of pathogenicity, which may cause temporary immunosuppression after vaccination and interfere with the immune effect of other vaccines.

[0007] 3) Difficulty in differential diagnosis: Antibodies produced by live vaccine immunization are difficult to distinguish from antibodies produced by wild virus infection, which is not conducive to the disease eradication (DIVA) of breeder flocks.

[0008] Genetically engineered subunit vaccines have become a hot research topic due to their high safety profile and lack of risk of virus shedding. However, the development of existing CIAV subunit vaccines faces several bottlenecks:

[0009] 1) Defects in prokaryotic expression systems: VP1 expressed in *E. coli* is mostly in the form of inclusion bodies, which are difficult to renature and restore to their native conformation. It lacks post-translational modifications specific to eukaryotes (such as phosphorylation and glycosylation), resulting in a significant difference in antigenic conformation from natural viruses, and its immunogenicity is often inferior to that of live vaccines. VP2 (~24 kDa), as a scaffold protein, assists VP1 in correct folding during viral assembly. Studies have shown that the interaction between VP1 and VP2 is crucial for forming antigenic structures with high immunogenicity.

[0010] 2) Difficulty in controlling the co-expression ratio: Although some studies have attempted to co-express VP1 and VP2 in eukaryotic systems, it is difficult to precisely control the expression ratio of VP1 and VP2 in co-transfection or bicistronic vectors. Too low an expression of VP2 cannot effectively assist folding, while too high an expression may cause cytotoxicity or competition for cellular resources.

[0011] 3) Difficulty in purification: Natural VP1 and VP2 tend to remain in the cell or nucleus. If an intracellular expression method is used, the cells need to be broken for purification. This is not only cumbersome, but also easily introduces a large amount of host cell protein (HCP) and nucleic acid (HCD) contamination, which increases the cost and difficulty of downstream purification.

[0012] Therefore, developing a highly efficient CIAV subunit vaccine that utilizes a higher mammalian cell system, enables efficient secretory expression of VP1 and VP2, has controllable component ratios, and is simple to manufacture is an urgent need in the industry. Summary of the Invention

[0013] To address the problems of difficult secretion of VP1 protein, easy formation of inclusion bodies, and poor immunogenicity in existing CIAV subunit vaccines, the present invention aims to provide a recombinant protein combination of VP1 and VP2 proteins of chicken infectious anemia virus, wherein the VP1 protein is shown in SEQ ID NO.5 and the VP2 protein is shown in SEQ ID NO.3.

[0014] Another object of the present invention is to provide the application of the above-mentioned recombinant protein combination in the preparation of a chicken infectious anemia virus vaccine.

[0015] To achieve the above objectives, the present invention adopts the following technical solution:

[0016] To address the challenges of VP1 protein secretion difficulties, inclusion body formation, and poor immunogenicity in existing CIAV subunit vaccines, the applicant utilized Chinese hamster ovary (CHO) cells to efficiently secrete and express CIAV VP1 and VP2 proteins, respectively. Soluble secretory expression of VP2 protein was achieved by fusing a highly efficient eukaryotic secretion signal peptide to the N-terminus of the VP2 gene. Similarly, soluble and stable high expression of VP1 protein was achieved by fusing a highly efficient eukaryotic secretion signal peptide to the N-terminus of the VP1 gene and specifically mutating the nuclear localization signal (NLS) region at the N-terminus of the VP1 protein, eliminating its nuclear localization function while maintaining the stability of the N-terminal α-helix conformation. The VP1 protein is shown in SEQ ID NO. 5, and one of the genes encoding VP1 protein is shown in SEQ ID NO. 6; the amino acid sequence of the VP2 protein is shown in SEQ ID NO. 3, and one of the genes encoding VP2 protein is shown in SEQ ID NO. 4.

[0017] The scope of protection of this invention also includes:

[0018] The fusion protein obtained by fusing the VP1 protein shown in SEQ ID NO.5 with a protein purification tag.

[0019] The gene encoding the VP1 protein shown in SEQ ID NO.5 or the above-mentioned fusion protein.

[0020] The preferred encoding gene described above is the one shown in SEQ ID NO. 6.

[0021] Expression cassettes or recombinant vectors containing the aforementioned coding genes.

[0022] Recombinant microorganisms or isolated recombinant cells containing the above-mentioned coding genes.

[0023] The recombinant cells described above are preferably recombinant CHO cells.

[0024] The application of the gene encoding the VP1 protein or its fusion protein shown in SEQ ID NO.5, an expression cassette having the above-mentioned encoding gene, a recombinant vector, a recombinant microorganism or an ex vivo recombinant cell in the preparation of chicken infectious anemia virus VP1 protein.

[0025] The application of the VP1 protein shown in SEQ ID NO.5, the fusion protein, the gene encoding the VP1 protein shown in SEQ ID NO.5 or its fusion protein, expression cassettes having the above-mentioned encoding gene, recombinant vectors, recombinant microorganisms or ex vivo recombinant cells in the preparation of chicken infectious anemia subunit vaccines.

[0026] A recombinant protein combination comprising protein A and protein B, wherein protein A is one of the VP1 protein shown in SEQ ID NO.5 or a fusion protein thereof, and protein B is one of the VP2 protein shown in SEQ ID NO.3 or a fusion protein thereof.

[0027] In the recombinant protein combination described above, the mass ratio of protein A to protein B is 1:0.5 to 1:2.

[0028] The application of the above-mentioned recombinant protein combination in the preparation of chicken infectious anemia subunit vaccine.

[0029] The adjuvants used in the vaccines described above include oil adjuvants, such as ISA 201 and ISA 71, or aqueous adjuvants, such as aluminum hydroxide and carbomer.

[0030] Compared with the prior art, the present invention has the following advantages:

[0031] 1) In response to the problems of VP1 protein secretion difficulties, easy formation of inclusion bodies, and poor immunogenicity in existing CIAV subunit vaccines, the applicant eliminated the antagonistic effect between VP1 nuclear entry signal and secretion signal through NLS optimization strategy. By using the synergistic optimization of "charge elimination" and "structural stability", the secretion expression level of mutant VP1 was significantly higher than that of wild type by more than 3.5 times, which significantly reduced the production cost.

[0032] 2) The CHO system endows recombinant proteins with mammalian-characteristic glycosylation modifications and a proper disulfide bond formation environment. Correct expression of the VP2 and VP1 recombinant proteins allows VP2 to assist VP1 in correct folding and assembly during in vitro incubation, maintaining or restoring the stereoconformity of the natural viral capsid, thereby exposing more neutralizing epitopes. The antigen complex VP1-Ala / VP2 obtained after mixed incubation induced extremely high titers of neutralizing antibodies, with a GMT of 1:544, demonstrating not only the correctness of the assembly but also achieving a perfect translation from molecular design to clinical efficacy.

[0033] 3) The VP2 and VP1 recombinant proteins provided by this invention promote proper protein folding and secretion by lowering the temperature to 31-33℃ during the late logarithmic growth phase of the culture process, allowing for the harvesting of cell culture supernatant. The recombinant proteins directly enter the culture medium, avoiding the complex steps of cell disruption and inclusion body refolding required for intracellular expression, greatly reducing contamination from host proteins and nucleic acids, and simplifying downstream purification processes.

[0034] 4) The subunit vaccine prepared using the VP2 and VP1 recombinant protein composition of the present invention has a protection rate of 100% for chickens, which is much higher than that of the currently available CIAV inactivated vaccine.

[0035] 5) The composition of the present invention, as a subunit vaccine, completely eliminates the risk of virus shedding and reversion. Furthermore, it does not contain VP3 protein and has diagnostic differential diagnosis (DIVA) value. Attached Figure Description

[0036] Figure 1 The results of Western blotting and SDS-PAGE analysis of the supernatant of the recombinant cell line suspension culture in Example 2 of this invention are shown.

[0037] Wherein: lane M: Marker; 1: Gly control VP1; 2: mutant VP1; 3: wild-type VP1; 4: VP2.

[0038] Figure 2 The neutralizing antibody titers of each group of chickens after immunization in Example 3 of this invention are shown.

[0039] Figure 3 The hematocrit (HCT) values ​​of chickens in each group after challenge in Example 3 of this invention are given. Detailed Implementation

[0040] The following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0041] Example 1:

[0042] Screening and expression of recombinant proteins VP1 and VP2 from chicken infectious anemia virus:

[0043] Using CIAV HN1901 strain (GenBank OQ869186) as a template, the coding sequences of VP1 and VP2 proteins were optimized using Chinese hamster codons. The codon-optimized polynucleotide sequences were synthesized, and human IgGκ light chain signal peptides were uniformly fused to the 5' end of the above polynucleotides. The amino acid sequence of the VP1 protein fused with the signal peptide is shown in SEQ ID NO.1, and the polynucleotide encoding it is shown in SEQ ID NO.2; the amino acid sequence of the VP2 protein fused with the signal peptide is shown in SEQ ID NO.3, and the polynucleotide encoding it is shown in SEQ ID NO.4.

[0044] It was found that VP2 could be secreted into the supernatant of CHO cells and expressed after the addition of the signal peptide, while VP1 failed to be secreted and expressed. It is speculated that the nuclear localization signal peptide NLS affects the secretion and expression of VP1. Therefore, the applicant optimized and modified the VP1 sequence.

[0045] Ultimately, the applicant constructed a VP1 variant library containing different mutation combinations using site-directed saturation mutagenesis technology. Initial screening was performed using high-throughput transient transfection and supernatant ELISA detection, followed by secondary screening based on protein stability (stored at 4°C for 7 days) and conformational antibody binding ability. The protein VP1-Ala, as shown in SEQ ID NO. 5, was ultimately identified as having optimal secretory expression characteristics. The short alanine side chain and α-helix tendency can eliminate NLS positive charge interference while maintaining N-terminal secondary structure stability; glycine's excessive flexibility easily disrupts local conformation. Furthermore, experiments confirmed that replacing all identical sites with glycine (Gly) in the mutant (SEQ ID NO. 7) leads to protein misfolding and secretion failure. This indicates that achieving efficient secretion and correct folding requires more than simply eliminating the positive charge; a specific mutation strategy that simultaneously maintains N-terminal structural stability is needed. The 20-site alanine all-mutation scheme of this invention is the optimal solution, verified through the above screening, that simultaneously meets the above requirements.

[0046] 1) Construction of eukaryotic expression vectors

[0047] In this embodiment, the applicant presents some NLS protein mutants and controls obtained during the screening process to demonstrate the superiority of the recombinant VP1 protein obtained by the present invention:

[0048] (1) Using pCHO 1.0 (including GS screening system) as the backbone, the vector pCHO-spVP1-wt was obtained by double digestion with AvrII / BstZ17I and insertion of the sequence shown in SEQ ID NO.2.

[0049] (2) Using pCHO 1.0 (including GS screening system) as the backbone, the vector pCHO-spVP1-Ala was obtained by double digestion with AvrII / BstZ17I and insertion of the sequence shown in SEQ ID NO.6.

[0050] (3) Using pCHO 1.0 (including GS screening system) as the backbone, the vector pCHO-spVP1-Gly was obtained by double digestion with AvrII / BstZ17I and insertion of the sequence shown in SEQ ID NO.8.

[0051] (4) As a comparative study group, a VP1 truncated mutant eukaryotic expression vector with amino acids 1-40 removed from the N-terminus of the VP1 protein was constructed using pCHO 1.0 as the backbone and named pCHO-spVP1-ΔNLS.

[0052] (5) Using pCHO 1.0 (including GS screening system) as the backbone, the vector pCHO-spVP2 was obtained by double digestion with AvrII / BstZ17I and insertion of the sequence shown in SEQ ID NO.4.

[0053] All of the above-described insertion sequences have a 6×His (hexahistine tag) added before insertion.

[0054] All vectors were validated by Sanger sequencing.

[0055] 2) CHO eukaryotic expression

[0056] Suspended CHO-S cells were transfected with the above plasmids via electroporation. After 48 h, stable cell pools were established by selection under 25 μM MSX pressure. Single clones were selected using limiting dilution (≥200 wells per group) to obtain cell lines CHO-VP1-ΔNLS, CHO-VP2, CHO-VP1-wt, CHO-VP1-Gly, and CHO-VP1-Ala. Albumin content was detected by ELISA. Culture supernatant and cell lysate were collected for SDS-PAGE and Western Blot analysis. The results for CHO-VP2, CHO-VP1-wt, CHO-VP1-Gly, and CHO-VP1-Ala are shown in Table 1 and [Table data would be inserted here]. Figure 1 The VP1-ΔNLS truncated mutant expressed by CHO-VP1-ΔNLS lost the complete N-terminal conformational region, resulting in the protein's inability to fold normally and stably. Western blot analysis showed that it only had obvious low molecular weight degradation diffusion bands in cell lysates and supernatants, indicating that the direct truncation strategy failed.

[0057] Table 1. Expression of target proteins in different recombinant eukaryotic cell lines

[0058] .

[0059] The results of eukaryotic expression show that:

[0060] (1) The VP1-Gly mutant caused protein misfolding and intracellular aggregation due to the disruption of the N-terminal α-helix by glycine, which confirmed that the "basic to neutral" strategy needs to take into account structural stability and that only alanine substitution can achieve efficient secretion.

[0061] (2) Mass spectrometry confirmed that the supernatant of the VP1-Ala mutant contained the correct disulfide bond and glycosylation modification, indicating that it maintained the native conformation. Ten of the above CHO-VP1-Ala cells were used for expression, and the average expression level of VP1-Ala in a 5 L bioreactor was 280 mg / L. Ten of the above CHO-VP2 cells were used for expression, and the average expression level of VP2 in a 5 L bioreactor was 150 mg / L.

[0062] Example 2:

[0063] Large-scale preparation of VP1-Ala and VP2 proteins:

[0064] Fed-batch culture: CHO-VP1-Ala and CHO-VP2 cells prepared in Example 1 were seeded into a 5L bioreactor. Reactor parameters were set as follows: 37℃, rotation speed 80 rpm, dissolved oxygen 50%, pH 7.0. On the first day, the culture was carried out at a rate of 1.0 x 10⁻⁶ cells / year. 6 Inoculate at an initial density of cells / mL. Start feeding on the third day of culture, adding feed solution A at 2% of the culture volume and feed solution B at 0.2% of the culture volume. Start feeding on the fifth day of culture, cool down to 32℃, add feed solution A at 4% of the culture volume and feed solution B at 0.4% of the culture volume, and continue to culture for 10 days before harvesting and transferring to tank.

[0065] Basal culture medium: Eminence, catalog number: L10400.0100; Feed A solution: Eminence, catalog number: P11810.0050; Feed B solution: Eminence, catalog number: P11811.0005.

[0066] The supernatant was purified by Ni-NTA affinity chromatography, with VP1-Ala yield reaching 280 mg / L and VP2 yield reaching 150 mg / L.

[0067] Vaccine formulation and in vitro assembly: Purified VP1-Ala and VP2 proteins were adjusted to 0.4 mg / ml with PBS buffer (pH 7.4), mixed at a mass ratio of 1:1, and incubated at 4°C for 4 hours to allow VP2 to assist VP1 in forming the antigen complex VP1-Ala / VP2. Subsequently, it was mixed with ISA 201 VG adjuvant at a volume ratio of 1:1 and emulsified to prepare an oil-emulsion vaccine containing 30 μg of antigen per 0.3 ml dose, used in the following examples.

[0068] Example 3:

[0069] Application of recombinant protein combinations of VP1 and VP2 proteins from chicken infectious anemia virus in the preparation of subunit vaccines:

[0070] 1) Experimental Design

[0071] One-day-old SPF chickens were randomly divided into three groups of eight chickens each.

[0072] Feeding standards: Water first, then food; small amounts frequently; free access to food, always keep a small amount of fresh food in the trough, immunize at 7 days old.

[0073] Group A: VP1-Ala / VP2 vaccine prepared in Example 2 (0.3 mL / bird).

[0074] Group B: Commercially available inactivated vaccines.

[0075] Group C: PBS control, 0.3 mL / bird.

[0076] Detection Method (Neutralizing Antibody Assay): Serum was collected 21 days post-immunization (28 days of age). A micro-neutralization assay was performed on MDCC-MSB1 cells using the fixed virus-dilution serum method to determine the serum neutralizing titer. Results are as follows: Figure 2 As shown, the geometric mean titer (GMT) of neutralizing antibodies in chickens immunized with the vaccine of this invention (Group A) was as high as 1:544, significantly higher than that in the commercially available inactivated vaccine group (Group B, GMT=1:68). This ultra-high titer of neutralizing antibody response is direct functional evidence that the antigen complex successfully mimics the conformation of the natural virus and forms a highly immunogenic structure.

[0077] 2) Evaluation of Protective Efficacy Against Virus Challenge: Immediately after blood collection, all chickens were challenged with the self-isolated chicken infectious anemia virus, at a dose of 10... 4.8 TCID50 / bird, 0.2 mL of virus per chicken, and check the infection status of chickens on day 14 after challenge.

[0078] Results: Animals with neutralizing antibody levels below 1:64, hematocrit less than 0.4, and positive tissue qPCR were diagnosed with the disease. Based on these disease criteria, all 8 animals in Group A were protected (8 / 8, or 100%). In Group B, one animal developed the disease, while the remaining 7 were protected (7 / 8, or 87.5%). All 8 animals in Group C developed the disease.

[0079] The qPCR detection results are shown in Table 2, where the values ​​are the Ct values ​​for qPCR. The qPCR primer sequences are:

[0080] F:5'-GCA GGG GCA AGT AAT TTC AA-3', as shown in SEQ ID NO.9;

[0081] R:5'-GCC ACA CAG CGA TAG AGT GA-3', as shown in SEQ ID NO.10;

[0082] P:5':FAM-ACT GCA GAG AGA TCC GGA TTG GTA TCG-BHQ-3', as shown in SEQ ID NO.11.

[0083] On day 14 post-infection, the distribution of hematocrit (HCT) values ​​in each group of chickens is as follows: Figure 3 As shown, the average HCT in group A was 0.50, which was higher than the average HCT in group B (0.44). Decreased hematocrit is a characteristic clinical manifestation of infection with chicken infectious anemia virus; the lower the value, the more severe the anemia.

[0084] In summary, the subunit vaccine of the present invention can effectively protect chickens from the infection of chicken infectious anemia virus.

[0085] Table 2 qPCR detection data of tissues and organs of chickens in different groups

[0086] .

[0087] Note: - indicates no Ct value, Ct value < 30 indicates positive, 30 < Ct value < 35 indicates weak positive, and Ct value > 35 indicates negative.

[0088] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the present technical solution, and they should all be covered within the scope of the claims of the present invention.

Claims

1. A synthetically produced chicken infectious anemia virus VP1 protein, characterized in that, The VP1 protein is shown in SEQ ID NO.

5.

2. The fusion protein obtained by fusing the VP1 protein of claim 1 with a protein purification tag.

3. The gene encoding the VP1 protein of claim 1 or the fusion protein of claim 2.

4. An expression cassette or recombinant vector having the gene encoding as described in claim 3.

5. A recombinant microorganism or an isolated recombinant cell having the encoding gene of claim 3.

6. The use of the encoding gene of claim 3, the expression cassette or recombinant vector of claim 4, or the recombinant microorganism or ex vivo recombinant cell of claim 5 in the preparation of chicken infectious anemia virus VP1 protein.

7. A recombinant protein combination, characterized in that, It includes protein A and protein B, wherein protein A is one of the VP1 protein of claim 1 or the fusion protein of claim 2, and protein B is one of the protein shown in SEQ ID NO.3 or a fusion protein obtained by fusing with a protein purification tag, wherein the mass ratio of protein A to protein B is 1:0.5 to 1:

2.

8. The use of the recombinant protein combination of claim 7 in the preparation of a subunit vaccine for chicken infectious anemia.

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