Antigen fusion protein and application thereof in preparation of live vaccine for preventing infectious bursal disease

Abstract

The invention discloses an antigen fusion protein and application thereof in preparation of a live vaccine for preventing infectious bursal disease. Specifically disclosed are antigen fusion proteins comprising a VP2 protein, a trimer tag, and a C3d protein. The invention also discloses recombinant Eimeria heap constructed by using the antigen fusion protein and application of the recombinant Eimeria heap in preparation of IBDV live vaccines. The oocyst of the recombinant Eimeria heap can effectively induce humoral immune response aiming at IBDV as a vaccine active ingredient, the level of an induced antibody is obviously higher than that of an insect strain expressed by a single VP2 antigen, viruses can be more effectively neutralized, the immune protection efficacy is improved, the safety is high, and the oocyst has the potential of serving as a live vector genetic engineering vaccine. The IBDV live vaccine can be orally taken through drinking water or feed, vaccination is simple, large-scale immunization of chicken flocks can be achieved, and the IBDV live vaccine is economical and efficient and has a wide prospect of being applied to prevention and control of the IBDV of the chicken flocks.

Description

Technical Field

[0001] The present invention belongs to the field of veterinary biological products and relates to the technical field of veterinary vaccines, and particularly to an antigen fusion protein and its application in preparing a live vaccine for preventing infectious bursal disease. Background Art

[0002] Infectious bursal disease (IBD) is a highly contagious disease of young chickens caused by the infectious bursal disease virus (IBDV). Its clinical symptoms are characterized by edema, hemorrhage, necrosis, and atrophy of the bursa of Fabricius. With a high incidence rate, IBD is currently one of the most important diseases in the poultry industry. IBDV primarily attacks the bursa of Fabricius, causing apoptosis of immature B lymphocytes, which in turn leads to immunosuppression, increased susceptibility to other pathogens, and even mortality.

[0003] Vaccination is one of the main measures for preventing and controlling infectious bursal disease. The research and development of new IBDV vaccines mainly focuses on the VP2 protein, which contains antigenic determinants and can induce the production of neutralizing antibodies, but is conformation-dependent. Eimeria is a unicellular eukaryotic organism that can maintain the natural structure of exogenous antigens through post-translational modification, which has obvious advantages for vaccine development. Kelleher, Tomley and others first achieved transient transfection of exogenous genes in Eimeria tenella. Liu et al. then used restriction enzyme-mediated integration (REMI) technology to transfect exogenous genes into coccidian sporozoites, increasing the transfection efficiency of Eimeria by at least 200 times. At present, the M2e protein of avian influenza virus and the CjaA protein of Campylobacter jejuni have been successfully expressed in Eimeria tenella. Eimeria acervulina, a highly prolific coccidia pathogen, is a key component of live oocyst vaccines for chicken coccidia. Expressing a single copy of IBDV-VP2, it can stimulate a specific humoral immune response in the host, but antibody levels are low. Therefore, developing a live oocyst-vectored vaccine with high safety and efficacy is of great significance. Summary of the Invention

[0004] The technical problem addressed by the present invention is how to enhance the humoral immune response against IBDV and more safely and effectively prevent IBDV infection. The technical problem to be solved is not limited to the technical subject matter described herein; those skilled in the art will readily understand other technical subjects not described herein through the following description.

[0005] To solve the above technical problems, the present invention first provides an antigen fusion protein, which can be named VP2-Trimer-tag-C3d. The antigen fusion protein comprises VP2 protein, a trimer tag and a C3d protein. The amino acid sequence of the VP2 protein can be shown as positions 1-453 of SEQ ID NO:1, and the amino acid sequence of the C3d protein can be shown as positions 484-813 of SEQ ID NO:1.

[0006] Furthermore, the antigen fusion protein comprises VP2 protein, trimer tag and C3d protein in sequence from N-terminus to C-terminus.

[0007] The trimer tag may be non-limiting, and its purpose is to make the VP2 protein fused with the trimer tag form a trimer. The trimer tag may be any polypeptide or protein that can make the VP2 protein form a trimer.

[0008] Furthermore, the trimer tag includes but is not limited to the T4 phage fibrous protein (foldon) folding trimer domain, the isoleucine zipper and coiled coil trimer domain derived from the yeast transcription activator GCN4, the procollagen C-propeptide domain (Trimer-Tag), the catalytic subunit of Escherichia coli aspartate transcarbamylase (ATCase), the trimer domain of collagen XV, the trimer domain of collagen XVIII, the coiled coil trimer domain of eukaryotic heat shock transcription factor, etc.

[0009] Furthermore, the trimer tag may be a T4 phage fiber protein fold trimer domain.

[0010] The T4 phage fiber protein fold trimer domain (abbreviated as T4 foldon) is well known to those skilled in the art. Furthermore, the amino acid sequence of the T4 foldon may be positions 454-479 of SEQ ID NO: 1.

[0011] Furthermore, the antigen fusion protein further comprises a linker for connecting the trimer tag described herein and the C3d protein.

[0012] The linker may be non-restrictive, and its purpose is to prevent steric hindrance. Those skilled in the art can select a conventional linker, as long as the linker has a certain flexibility to allow the polypeptides or proteins on both sides to perform their respective independent functions.

[0013] Furthermore, the linker may be a flexible peptide linker, for example a peptide linker comprising glycine, serine, proline and / or lysine residues. The peptide linker may be composed of 1-40 amino acids.

[0014] Furthermore, the linker includes, but is not limited to, MHGS, SAIG, (G)n, (S)n, (GxS)n, (SxG)n, (GSSGG)n, (GGSGG)n, (GSGGSG)n, (GSGSGS)n, (GGQGG)n, and (EAAAK)n, and various combinations thereof, wherein n can be any integer between 1 and 10, and x can be any integer between 1 and 6.

[0015] Furthermore, the linker may be a peptide linker MHGS or GGGGS.

[0016] The antigen fusion protein described herein may comprise, from N-terminus to C-terminus, the following: the VP2 protein described herein, the trimer tag described herein, the linker described herein, and the C3d protein described herein.

[0017] Furthermore, the antigen fusion protein may be any of the following:

[0018] A1) a protein whose amino acid sequence is positions 1-813 of SEQ ID NO: 1;

[0019] A2) a protein having at least 80% identity with the protein of A1) and having the same function as the protein of A1) obtained by substitution, deletion, and / or addition of amino acid residues of the amino acid sequence of SEQ ID NO: 1;

[0020] A3) A fusion protein having the same function obtained by connecting a tag to the N-terminus and / or C-terminus of A1) or A2);

[0021] A4) A fusion protein obtained by linking a signal peptide to the N-terminus of A1), A2) or A3).

[0022] To facilitate the separation, purification, detection, and / or localization of the protein described in A1) or A2), a tag may be attached to the amino or carboxyl terminus of the protein described in A1) or A2). The tag includes, but is not limited to, a GST (glutathione sulfhydryltransferase) tag, a Trx (thioredoxin) tag, a nitrogen utilization substrate A (NusA) tag, a His-tag, a Strep tag, an MBP (maltose binding protein) tag, a Flag tag, a SUMO (small ubiquitin-like modifier) ​​tag, an HA (influenza hemagglutinin) tag, a Myc tag, a LacZ tag, a CBD (cellulose binding domain) tag, a bacteriophage T7 protein kinase (T7PK) tag, a GFP (green fluorescent protein) tag, a CFP (cyan fluorescent protein) tag, a YFP (yellow fluorescent protein) tag, an mCherry (monomeric red fluorescent protein) tag, an AviTag tag, or a HiBiT tag (VSGWRLFKKIS), or a combination of the above tags. Those skilled in the art will know how to select an appropriate tagged protein based on the desired purpose. The use of a tag does not alter the function of the target protein; its purpose is to separate, purify, detect, or trace. Therefore, the tagged proteins suitable for this application are not limited to a specific type. The tag can be separated from the target protein by chemical cleavage methods known in the art or enzymatic methods (e.g., introducing a protease cleavage site to remove the tag using TEV protease).

[0023] Furthermore, the tag may be a 3FLAG tag.

[0024] Furthermore, the amino acid sequence of the 3FLAG tag may be positions 816-837 of SEQ ID NO: 1.

[0025] The signal peptide described in A4) is non-limiting and can be any signal peptide that effectively secretes the antigen fusion protein of the present invention. Those skilled in the art are aware that signal peptides function to direct newly synthesized proteins into the secretory pathway and are often cleaved during this process. By introducing a signal peptide, the antigen fusion protein of the present invention can be directed to cross the cell membrane for secretory expression without affecting the immunogenicity of the antigen fusion protein. Signal peptides are known to those skilled in the art, and those skilled in the art can select an appropriate signal peptide as needed. Suitable signal peptides can be found at http: / / www.signalpeptide.de / .

[0026] Furthermore, the signal peptide may be the Toxoplasma gondii dense granule protein GRA8 signal peptide.

[0027] Furthermore, the amino acid sequence of the GRA8 signal peptide may be SEQ ID NO: 3.

[0028] Furthermore, the amino acid sequence of the fusion protein described in A3) may be as shown in SEQ ID NO: 1, and / or the amino acid sequence of the signal peptide described in A4) may be as shown in SEQ ID NO: 3.

[0029] Furthermore, the amino acid sequence of the fusion protein described in A4) may be any of the following:

[0030] (1) an amino acid sequence obtained by directly linking the amino acid sequence set forth in SEQ ID NO: 3 with the amino acid sequence set forth in positions 1 to 813 of SEQ ID NO: 1;

[0031] (2) The amino acid sequence obtained by directly linking the amino acid sequence shown in SEQ ID NO: 3 and the amino acid sequence shown in SEQ ID NO: 1.

[0032] The present invention also provides a biomaterial, which may be any of the following:

[0033] B1) a nucleic acid molecule encoding any one of the antigen fusion proteins described herein;

[0034] B2) an expression cassette containing the nucleic acid molecule described in B1);

[0035] B3) a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0036] B4) a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3);

[0037] B5) A recombinant host cell containing the nucleic acid molecule of B1), or a recombinant host cell containing the expression cassette of B2), or a recombinant host cell containing the recombinant vector of B3).

[0038] In the above biological materials, the recombinant vector can be a cloning vector or an expression vector.

[0039] The recombinant vector can be constructed using a cloning vector. Constructing a cloning vector is generally for the purpose of amplifying the target gene in large quantities, restriction enzyme digestion, sequencing, facilitating long-term storage of the target gene, and ready use. Common cloning vectors include pUC series vectors, pGEM series vectors, pMD18-T, pBluescript, and pBR322. Those skilled in the art can select a suitable cloning vector as needed.

[0040] The recombinant vector can be constructed using an expression vector. The structure of the expression vector is well known to those skilled in the art, and the expression vector generally contains elements required for the expression of the target gene such as promoter, multiple cloning site, terminator, ribosome binding site, etc., and can also contain screening marker genes (such as kanamycin resistance gene kanr, neomycin resistance gene neo, hygromycin resistance gene hyg, chloramphenicol resistance gene cat, streptomycin resistance gene str, bleomycin resistance gene ble, etc.). The expression vector can be constructed using any method known in the art (such as recombinant technology, synthetic technology, etc.), or can be purchased commercially, and those skilled in the art can select suitable expression vectors as needed.

[0041] Furthermore, the recombinant vector can be a recombinant expression vector obtained by cloning the coding gene of the antigen fusion protein of the present invention into an expression vector (including a prokaryotic expression vector and a eukaryotic expression vector). The expression vector is not limited to a specific vector, and those skilled in the art can use a suitable expression vector as long as the expression vector can express the antigen fusion protein.

[0042] The gene encoding the antigen fusion protein described herein can be any gene capable of encoding the antigen fusion protein. Taking into account the degeneracy of codons and the codon preferences of different species, those skilled in the art can use codons suitable for expression in a specific species as needed. For example, the nucleotide sequence of the gene encoding the antigen fusion protein can be SEQ ID NO: 2, positions 1-2439 or SEQ ID NO: 2.

[0043] Furthermore, the recombinant vector may contain the following elements:

[0044] (1) Eimeria tenella EtMic2 promoter (SEQ ID NO. 5), used to drive the expression of the gene encoding the antigen fusion protein of the present invention;

[0045] (2) GRA8 signal peptide (amino acid sequence is SEQ ID NO: 3), used to promote the stable expression of the antigen fusion protein of the present invention in the host dense granules;

[0046] (3) Proline tRNA synthetase (PRS), as a drug selection marker, whose expression is regulated by a coccidian promoter (such as the EtMic2 promoter);

[0047] (4) Enhanced yellow fluorescent protein (EYFP), as a fluorescent screening marker, whose expression is regulated by a coccidian promoter (such as the EtMic2 promoter).

[0048] In one or more embodiments of the present invention, the recombinant vector is pMic2GRA8VP2TrilinkerC3dPEA. The recombinant vector pMic2GRA8VP2TrilinkerC3dPEA comprises, operably linked from the N-terminus to the C-terminus, the EtMic2 promoter (SEQ ID NO: 5), the gene encoding the GRA8 signal peptide (SEQ ID NO: 4), the gene encoding the antigen fusion protein VP2-Trimer-tag-C3d-Flag (SEQ ID NO: 2), the gene encoding the self-cleavage peptide P2A (SEQ ID NO: 7), the PRS gene (SEQ ID NO: 6), the EYFP gene, and the Actin gene.

[0049] Furthermore, the nucleotide sequence of the recombinant vector pMic2GRA8VP2TrilinkerC3dPEA can be Figure 10 The nucleotide sequence shown and Figure 11 The nucleotide sequence shown was directly linked to the resulting sequence (consisting of 11,726 nucleotides).

[0050] Methods for introducing a recombinant vector carrying a target gene into a host (such as a coccidia) so that the protein encoded by the target gene can be expressed in the host are well known to those skilled in the art. As long as the target gene can be expressed in the host, it will suffice.

[0051] Furthermore, a recombinant vector carrying the antigen fusion protein encoding gene of the present invention can be integrated into the genome of Eimeria acervulina. For example, a recombinant vector carrying the antigen fusion protein encoding gene of the present invention can be integrated into a conserved region of the Eimeria acervulina genome (such as the non-coding region of the EtMic2 gene) by homologous recombination. Southern blot can be used to verify single-site integration, and the number of integrated copies can be 1-3 copies.

[0052] Furthermore, the recombinant microorganism in B4) includes recombinant coccidia. Furthermore, the recombinant microorganism in B4) includes recombinant Eimeria acervulina.

[0053] In the above biological material, the nucleotide sequence of the nucleic acid molecule in B1) may be as shown in positions 1 to 2439 of SEQ ID NO: 2 or SEQ ID NO: 2.

[0054] Those skilled in the art can easily mutate the nucleotide sequence encoding the antigen fusion protein of the present invention using known methods, such as site-directed mutagenesis (including oligonucleotide primer-mediated site-directed mutagenesis, PCR-mediated site-directed mutagenesis, and cassette mutagenesis) or directed evolution (including error-prone PCR, DNA shuffling, and in vitro random priming recombination). Artificially modified nucleotide sequences that are 75% or more identical to the nucleotide sequence encoding the antigen fusion protein of the present invention (such as positions 1-2439 of SEQ ID NO: 2 or SEQ ID NO: 2) are all derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention, as long as they encode the antigen fusion protein and have the same function as the antigen fusion protein.

[0055] The present invention also provides a recombinant Eimeria acervulina, wherein the recombinant Eimeria acervulina stably expresses any one of the antigen fusion proteins or nucleic acid molecules described herein.

[0056] Furthermore, the recombinant Eimeria acervulina can be obtained by integrating the gene encoding any one of the antigen fusion proteins described herein into the following site of the Eimeria acervulina genome: positions 24035-31025 of GenBank Accession No. HG671014.

[0057] The present invention also provides a method for constructing a recombinant Eimeria acervulina, which comprises integrating a gene encoding any one of the antigen fusion proteins described herein into the genome of Eimeria acervulina to obtain a recombinant Eimeria acervulina; or integrating a gene encoding any one of the antigen fusion proteins described herein into the following site of the Eimeria acervulina genome to obtain a recombinant Eimeria acervulina: positions 24035-31025 of GenBank Accession No. HG671014.

[0058] Furthermore, the recombinant Eimeria acervulina can stably express the antigen fusion protein.

[0059] Furthermore, the method may include the following steps:

[0060] (1) cloning the gene encoding the antigen fusion protein of the present invention into an expression vector to obtain a recombinant expression vector;

[0061] (2) The recombinant expression vector is introduced into the genome of wild-type Eimeria acervulina, and recombinant Eimeria acervulina stably expressing the antigen fusion protein is obtained through screening and identification.

[0062] The step (2) can further include introducing the recombinant expression vector into the wild-type Eimeria acervulina genome at the following site: GenBank Accession No. HG671014, positions 24035-31025, and obtaining recombinant Eimeria acervulina stably expressing the antigen fusion protein through screening and identification.

[0063] The screening and identification methods are well known to those skilled in the art. For example, transgenic strains can be verified by fluorescence screening, PCR, and Western blot, and monoclonal strains (such as Ea-VTC) that stably express the antigen fusion protein of the present invention can be screened.

[0064] Furthermore, the screening step may include the following steps:

[0065] (1) Sorting antigen-expressing coccidian gametocytes by flow cytometry (FACS);

[0066] (2) After continuous passage for more than 5 generations, the antigen expression stability was tested by Western blot to ensure that the expression of the antigen fusion protein in each generation did not fluctuate by more than ±15%.

[0067] Furthermore, the introduction method can be based on homologous recombination or gene editing. Those skilled in the art can use well-known homologous recombination, gene editing and other methods to site-specifically integrate the gene encoding the antigen fusion protein of the present invention into the genome of Eimeria acervulina for stable expression.

[0068] Furthermore, the method may include the following steps:

[0069] (1) constructing a recombinant expression vector containing the gene encoding the antigen fusion protein of the present invention;

[0070] (2) introducing the recombinant expression vector into wild-type Eimeria acervulina sporozoites by homologous recombination or gene editing;

[0071] (3) Select stably integrated recombinant Eimeria acervulina at the sporulation stage.

[0072] The recombinant Eimeria acervulina described herein may have at least one of the following characteristics:

[0073] (1) capable of stably expressing the antigen fusion protein of the present invention;

[0074] (2) The pattern of oocyst excretion was similar to that of wild-type Eimeria acervulina (Ea-WT), but the fecundity was significantly reduced;

[0075] (3) Compared with wild-type Eimeria acervulina (Ea-WT), it can enhance the humoral immune response against infectious bursal disease virus.

[0076] The present invention also provides the use of any one of the antigen fusion proteins, the biological material, the recombinant Eimeria acervulina, or the recombinant Eimeria acervulina obtained by the method for constructing recombinant Eimeria acervulina in any of the following:

[0077] C1) Use in the preparation of a product for enhancing the humoral immune response against infectious bursal disease virus;

[0078] C2) Use in the preparation of a product for preventing and / or treating infectious bursal disease virus infection or infectious bursal disease.

[0079] The product described herein can be a vaccine, a reagent, a kit, a formulation, a product, a drug or a pharmaceutical composition.

[0080] The improvement of humoral immune response against infectious bursal disease virus can be reflected in the improvement of VP2 protein antibody level in the host after immunization, thereby significantly improving the immune protection efficacy against IBDV.

[0081] The present invention also provides a vaccine composition, characterized in that the vaccine composition comprises any one of the following:

[0082] D1) the recombinant Eimeria acervulina described herein;

[0083] D2) recombinant Eimeria acervulina obtained by the method for constructing recombinant Eimeria acervulina described herein;

[0084] D3) live oocysts of the recombinant Eimeria acervulina described in D1) or D2);

[0085] D4) sporulated oocysts of the recombinant Eimeria acervulina described in D1) or D2).

[0086] The vaccine composition can be used to prevent infectious bursal disease virus infection or infectious bursal disease in avian animals (such as chickens).

[0087] Furthermore, the vaccine composition may further comprise one or more pharmaceutically or veterinarily acceptable carriers.

[0088] The pharmaceutically or veterinarily acceptable carrier includes, but is not limited to, a wetting agent, a dispersing agent, an emulsifier, a buffer, a stabilizer, a diluent, an isotonic agent and / or a preservative (such as thimerosal).

[0089] Pharmaceutically or veterinarily acceptable carriers are well known to those skilled in the art and may include components such as water, saline, buffered saline such as phosphate-buffered saline, or any other physiologically acceptable medium.

[0090] The vaccine composition of the present invention can also be used in combination with an inactivated infectious bronchitis virus (IBV) vaccine to enhance the immune protection efficacy against IBDV and IBV.

[0091] The recombinant Eimeria acervulina oocysts of the present invention can be stored for 1-2 years in an environment of 2-8° C. and remain active.

[0092] The survival time of the recombinant Eimeria acervulina oocysts in the chicken intestine does not exceed 7 days, and the oocysts do not have the ability to spread over a long period of time in the natural environment.

[0093] In vaccine development, the antigenic structure of the trimer is closer to the natural virus particle (such as the trimerization of HIV gp140), which can better simulate the natural form of the virus, thereby inducing a stronger immune response. The trimer form can provide more binding sites or expand the surface area to enhance activity. The C-terminal domain (foldon) of T4 fiber protein can be used as an artificial trimerization domain. Its natural structure consists of a trimeric β-hairpin propeller. The VP2 protein of IBDV is the main structural protein of its capsid and is naturally present in the viral capsid trimer. The VP2 trimer plays a key role in the assembly of virus particles and the conformational integrity of the antigenic determinant. The present invention promotes VP2 to exist in the correct trimeric form through a trimer tag (Trimer-tag), structurally better mimicking the natural structure of the virus and thereby improving immunogenicity.

[0094] C3d is a cleavage product of complement component 3 and plays a key role in foreign antigen recognition. It links innate immunity with the adaptive immune response by binding to the complement receptor (CD21, formerly known as CR2). After the complement cascade is activated by foreign proteins, C3d covalently binds to the activating antigen. In turn, C3d can bind to CR2 present on B cells and follicular dendritic cells (FDC), leading to B cell activation and the initiation of the adaptive immune response. The present invention fuses C3d with trimerized VP2 to further enhance the immunogenicity of VP2, particularly during recognition by B cells and FDCs (follicular dendritic cells). By binding to the CR2 receptor, C3d promotes the specific recognition and presentation of the trimeric VP2 antigen, helping to initiate a stronger immune response. This fusion not only improves the stability and folding efficiency of the antigen but also promotes a rapid immune response through complement receptors.

[0095] The present invention first designs an antigen fusion protein VP2-Trimer-tag-C3d (positions 1-813 of SEQ ID NO: 1), and then introduces the coding gene of the antigen fusion protein into the genome of wild-type Eimeria acervulina to obtain recombinant Eimeria acervulina (e.g., Ea-VTC) that can stably express the antigen fusion protein VP2-Trimer-tag-C3d. Live oocysts (sporulated oocysts) of the recombinant Eimeria acervulina Ea-VTC are used as a live vaccine to orally immunize SPF chickens. The results show that the number of oocysts excreted by chickens infected with the recombinant Eimeria acervulina Ea-VTC of the present invention is significantly reduced. Compared with wild-type Eimeria acervulina (Ea-WT) or a strain expressing the VP2 antigen alone (Ea-VP2), the recombinant Eimeria acervulina Ea-VTC of the present invention has significantly reduced fecundity and is safer. In the immune challenge experiment, ELISA was used to monitor the specific humoral immune response against IBDV-VP2. The results showed that after immunizing chicks with the recombinant Eimeria acervulina Ea-VTC of the present invention, the level of VP2 protein antibodies in the serum was significantly higher than that of chicks immunized with wild-type Eimeria acervulina (Ea-WT) and the insect strain Ea-VP2 expressing the VP2 antigen alone, thereby significantly improving the immune protection efficacy against IBDV.

[0096] In summary, the present invention provides a novel antigen fusion protein VP2-Trimer-tag-C3d (positions 1-813 of SEQ ID NO: 1), and the coding gene of the antigen fusion protein is introduced into the genome of wild-type Eimeria acervulina to obtain recombinant Eimeria acervulina, and the antigen fusion protein is continuously expressed during the endogenous developmental stage of the recombinant Eimeria acervulina, thereby fully stimulating the host's immune system and inducing the host to produce a humoral immune response against IBDV VP2. The live oocysts of the recombinant Eimeria acervulina can be prepared into a live IBDV vaccine, which can significantly improve the humoral immune response after immunizing chickens and significantly improve the level of VP2 protein antibodies produced in the host chickens. Compared with wild-type Eimeria acervulina or a strain expressing the VP2 antigen alone, the recombinant Eimeria acervulina of the present invention has a significantly reduced reproductive capacity while maintaining the regularity of oocyst excretion, thereby improving the safety of the vaccine and reducing the potential harm to the host caused by excessive reproduction of coccidia. Furthermore, the recombinant Eimeria acervulina (e.g., Ea-VTC) of the present invention can effectively induce a humoral immune response against IBDV, with the induced antibody levels significantly higher than strains expressing only the VP2 antigen. This more effectively neutralizes the virus and enhances immune protection, demonstrating its potential as a live vector genetically engineered vaccine. The live IBDV vaccine of the present invention can be administered orally through drinking water or feed, is simple to administer, and can be used to vaccinate large flocks of chickens. It is economical and highly effective, and holds broad promise for its application in the prevention and control of IBDV in chickens.

[0097] Definition of terms

[0098] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. At the same time, in order to better understand the present invention, the definitions and explanations of relevant terms are provided below.

[0099] The term "expression cassette" generally refers to a nucleic acid construct comprising nucleic acid elements sufficient to express a gene of interest. A typical expression cassette comprises a promoter, an MCS (multiple cloning site) and / or a terminator. An expression cassette may also include a gene of interest, a marker gene (such as a TK gene, a DHFR gene, a CAT gene, and a NEO gene), a ribosome recognition and binding site (SD), a transcription factor binding site (TFBS), an enhancer, a silencer, a repressor, an intron, a poly (A) signal sequence, and / or an mRNA splicing signal sequence. The elements in the expression cassette may be directly connected or indirectly connected via a linker.

[0100] The term "vector" generally refers to a vehicle capable of transporting exogenous DNA or a gene of interest into host cells for amplification and / or expression. Such a vector can be a cloning vector or an expression vector. A vector can be introduced into a host cell via transformation, transduction, or transfection, allowing the genetic material it carries to be amplified and / or expressed in the host cell. Those skilled in the art can select an appropriate vector based on the purpose of the genetic engineering project and the properties of the recipient cell. The vector includes, but is not limited to, a plasmid, a phage (e.g., lambda phage or M13 phage), a cosmid (i.e., cosmid), a phagemid, a shuttle vector (e.g., a yeast expression vector), a Ti plasmid, an artificial chromosome (e.g., a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC), a P1 artificial chromosome (PAC), or a Ti plasmid artificial chromosome (TAC)), a viral vector (e.g., a baculovirus vector, a retrovirus (including a lentivirus), adenovirus, adeno-associated virus, poxvirus, papillomavirus, papillomavirus (e.g., SV40), or a herpes virus (e.g., herpes simplex virus)). A vector may contain a variety of elements for controlling expression, including, but not limited to, a promoter sequence, a transcription initiation sequence, an enhancer sequence, a selection element, and a reporter gene. In addition, the vector may also contain a replication initiation site.

[0101] The term "microorganism" generally includes bacteria, viruses, fungi, actinomycetes, rickettsia, mycoplasma, chlamydia, spirochetes, algae, coccidia, etc. For example, the bacteria can be from the genus Corynebacterium sp. (such as Corynebacterium glutamicum, Corynebacterium pekinensis, Corynebacterium crenulate, etc.), Brevibacterium sp. (such as Brevibacterium lactofermentum, Brevibacterium flavum, Brevibacterium ammoniaphagoides, etc.), Escherichia sp. (such as Escherichia coli), Erwinia sp., Agrobacterium sp. (such as Agrobacterium tumefaciens), Flavobacterium sp., Alcaligenes sp., Pseudomonas sp. and Bacillus sp. (such as Bacillus), etc. The virus may include rotavirus, baculovirus, retrovirus (such as lentivirus), adenovirus, adeno-associated virus, poxvirus, papillomavirus, influenza virus, papillomavirus (such as SV40) and herpes virus (such as herpes simplex virus), etc. The fungus may be from the genus Saccharomyces (such as Saccharomyces cerevisiae, Candida, Methanol yeast, Pichia pastoris), Fusarium (Fusarium sp.), Rhizoctonia (Rhizoctonia sp.), Verticillium (Verticillium sp.), Penicillium (Penicillium sp.), Aspergillus (Aspergillus sp.) and Cephalosporium (Cephalosporium sp.), etc. The actinomycete may be from the genus Streptomyces (Streptomyces sp.) (such as Streptomyces). The algae may be from Cyanophyta (such as Cyanobacteria), Fucus sp., Achnanthes sp., Amphiprora sp., Amphora sp., Ankistrodesmus sp., Asteromonas sp., and Boekelovia sp., etc. The coccidia may be Eimeria acervulina.

[0102] The term "host cell," also referred to as a recipient cell, generally refers to any type of cell into which a vector can be introduced, such as a plant cell and / or an animal cell. The host cell is understood to refer not only to a specific recipient cell but also to the progeny of such a cell. Due to natural, accidental, or intentional mutations and / or changes, such progeny may not necessarily be completely identical to the original parent cell, but are still included within the scope of host cells. Suitable host cells are known in the art, wherein: the plant cell can be, but is not limited to, plant cells such as Arabidopsis thaliana, tobacco (Nicotiana tabacum), corn (Zea mays), rice (Oryza sativa), and wheat (Triticum aestivum); the animal cell can be a mammalian cell (e.g., Chinese hamster ovary cells (CHO cells), Chinese hamster ovary cell substrain (CHO-K1 cells), African green monkey kidney cells (Vero cells), SV40-transformed African green monkey kidney cells (COS cells), baby hamster kidney cells (BHK cells), mouse breast cancer cells (C127 cells), human embryonic kidney cells (HEK293 cells), human HeLa cells, fibroblasts, bone marrow cell lines, T cells, or NK cells), avian cells (e.g., chicken or duck cells), amphibian cells (e.g., African clawed frog (Xenopus laevis) cells, or giant salamander (Andrias davidianus) cells). davidianus) cells), fish cells (such as grass carp, carp, rainbow trout or catfish cells), insect cells (such as Sf21 cells, Sf-9 cells or Hi-5 cells), etc., but are not limited thereto.

[0103] The term "recombinant vector" generally refers to a recombinant DNA molecule constructed by connecting an exogenous target gene to a vector in vitro. It can be constructed in any suitable manner, as long as the constructed recombinant vector can carry the exogenous target gene into the recipient cell and provide the exogenous target gene with the ability to replicate, integrate, amplify and / or express in the recipient cell.

[0104] The term "recombinant microorganism" generally refers to a microorganism whose genes have been manipulated and modified to produce a functionally altered recombinant microorganism. This can be achieved by introducing an exogenous gene of interest or a recombinant vector into the microorganism, or by directly editing the endogenous genes of the microorganism.

[0105] The term "recombinant host cell" generally refers to a recombinant host cell whose genes have been manipulated and modified to produce functionally altered recombinant host cells. This can include introducing an exogenous gene of interest or a recombinant vector into a host cell, or directly editing the endogenous genes of the host cell.

[0106] The term "identity" generally refers to the degree to which two (nucleotide or amino acid) sequences have the same residue at the same position in an alignment, and is generally expressed as a percentage. Identity as described herein may refer to the identity of an amino acid sequence or a nucleotide sequence. Two copies having exactly the same sequence have 100% identity. Those skilled in the art will appreciate that the identity of an amino acid sequence or a nucleotide sequence can be determined using an identity search site on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, by using blastp as a program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as a Matrix, setting the Gap existence cost, Perresidue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values), respectively, and performing a search to calculate the identity of the amino acid sequence, the value (%) of identity can then be obtained. In addition, sequence analysis software (such as CLC Main Workbench and MegAlign TM ), for example, using the computer program BLAST, in particular BLASTP or TBLASTN, with default parameters. As used herein, greater than 75% identity may be at least 75%, 80%, 85%, 90%, or greater than 95% identity. As used herein, greater than 80% identity may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or greater than 99% identity.

[0107] The term "link" generally refers to the association of two or more molecules. The link can be covalent or non-covalent. The link described herein can be directly connected by a peptide bond or connected by a linker (joint).

[0108] The term "linker" may also be referred to as a connecting peptide, peptide linker or connector, and is used to fuse, couple, link or join two proteins or polypeptides to prevent steric hindrance. A linker is an amino acid chain that acts as a connector between two fusion proteins and has a certain flexibility to allow the proteins on both sides to perform their respective independent functions. It should be understood that the presence of a linker is optional and that the length of the flexible linker can be adjusted to allow the fusion protein to fold correctly or to achieve optimal biological activity. The characteristics of the linker and its suitability for a specific purpose are known in the art, and those skilled in the art can independently select and / or optimize each peptide linker.

[0109] The term "trimer tag" generally refers to a tag polypeptide that can trimerize any target protein. The trimer formed by fusion of the target protein with the trimer tag can mimic the structure of the target protein in vivo. The trimer tag can form a trimer upon fusion of any target protein with the trimer tag, i.e., form a biomolecular complex in which three identical molecules aggregate into a single trimer.

[0110] The term "import" generally refers to transferring an exogenous gene into a host. The method of importing is not particularly limited, and any known transformation method is sufficient to transfer the target gene (e.g., the coding gene of the antigen fusion protein of the present invention) into a host (e.g., coccidia). The DNA molecule imported can be a single copy or multiple copies. The method of importing can include transferring the target gene or a recombinant vector containing the target gene into a host by chemical transformation (e.g., Ca ion-induced transformation, polyethylene glycol-mediated transformation, or metal cation-mediated transformation, etc.) or physical transformation (e.g., electroporation transformation).

[0111] The term "stable expression" generally refers to the transfer of exogenous genes into the host genome for expression. During stable expression, the exogenous gene is stably inserted into the host genome (e.g., chromosome), allowing the host to stably express the exogenous protein over a long period of time.

[0112] The term "oocysts" or "live oocysts" generally refers to live coccidian oocysts, including sporulated oocysts. Live oocysts can be prepared by methods known to those skilled in the art, such as saturated salt water flotation, sodium hypochlorite solution treatment, and the like.

[0113] The term "sporulated oocysts" may refer to oocysts that have matured naturally or been artificially manipulated to be capable of infecting a susceptible host. Sporulated oocysts can be obtained by dissolving the oocysts in a potassium dichromate solution and then sporulating them. For example, oocysts can be suspended in a 2%-3% potassium dichromate solution and incubated at 25-30°C with aeration for 48 hours to obtain sporulated oocysts.

[0114] The term "humoral immune response" generally refers to an immune response mediated by B cells and encompasses immune responses mediated by antibody molecules.

[0115] The term "comprising" is not intended to be limiting, but rather inclusive and means that there may be additional elements other than the listed elements and can be interpreted as "including but not limited to". The term "comprising" also encompasses the terms "consisting of" and "consisting essentially of". The terms "comprising" and "including" are used interchangeably herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0116] Figure 1 Schematic diagram of the recombinant plasmid for fusion expression of trimerized VP2 antigen in Example 2 of the present invention.

[0117] Figure 2 Schematic diagram of the recombinant plasmid for fusion expression of trimerized VP2 antigen and C3d in Example 2 of the present invention.

[0118] Figure 3 This figure shows the PCR identification results of the Ea-VTC strain in Example 4 of the present invention, where Ea-WT represents the wild-type Eimeria acervulina strain, and Ea-VP2 represents the transgenic Eimeria acervulina strain expressing only VP2. Ea-VTC is the transgenic Eimeria acervulina strain in Example 4.

[0119] Figure 4 This is a Western blot image of the Ea-VTC strain described in Example 4. Western blot analysis of the VP2-trimer-tag-C3d fusion protein in Ea-VTC using an anti-Flag tag mouse monoclonal antibody as the primary antibody revealed a protein band of approximately 204 kDa in Ea-VTC.

[0120] Figure 5 This is an IFA identification image of the Ea-VTC strain in Example 4 of the present invention. The expression location of the VP2-trimer-tag-C3d fusion protein in Ea-VTC was identified by indirect immunofluorescence using an anti-Flag tag mouse monoclonal antibody as the primary antibody and a Cy3-labeled goat anti-mouse IgG antibody as the secondary antibody. The results were compared with Ea-WT. The results showed that the fusion protein was expressed in the sporozoite sporozoite sporozoite, mainly located at the apex.

[0121] Figure 6 This is a graph showing the oocyst excretion curve of the Ea-VTC strain in Example 5 of the present invention.

[0122] Figure 7 This is a graph showing the changes in the amount of oocysts produced during the initial and secondary infection with the Ea-VTC strain in Example 5 of the present invention.

[0123] Figure 8 Schematic diagram of the experimental procedure for the immune protection experiment of 7-day-old SPF chickens in Example 6 of the present invention.

[0124] Figure 9 These are the results of IBDV-VP2-specific antibody detection in the sera of 7-day-old SPF chickens immunized at different times in Example 6 of the present invention.

[0125] Figure 10 It is the 1st to 5912th nucleotides of plasmid pMic2GRA8VP2TrilinkerC3dpeA.

[0126] Figure 11It is the 5913-11726 nucleotides of plasmid pMic2GRA8VP2TrilinkerC3dpeA.

[0127] Figure 12 It is the 1st to 4382nd nucleotides of plasmid pMic2GRA8VP2TrilinkerC3dpeA.

[0128] Figure 13 It is the 4383-8477th nucleotides of plasmid pMic2GRA8VP2TrilinkerC3dpeA. DETAILED DESCRIPTION

[0129] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0130] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0131] The methods for extracting sporozoites of wild-type Eimeria acervulina (Ea-WT) (Ea-WT sporozoites) in Example 2 and sporozoites of recombinant Eimeria acervulina (Ea-VTC) in Example 4 are as follows:

[0132] According to experimental requirements, a certain number of sporulated oocysts were collected from wild-type Eimeria acervulina (Ea-WT) and recombinant Eimeria acervulina (Ea-VTC), and washed three times with PBS (3000 rpm, 3 min); 1-2 volumes of PBS and an appropriate amount of cellophane were added to the oocyst precipitate in a 15 ml tube, mixed and then vortexed to break the oocyst wall for 30 s. Microscopic examination showed that about 90% of the oocysts were sporangia and oocyst walls; the sporangium suspension (containing a small amount of oocysts and oocyst walls) was transferred to a 1.5 ml centrifuge tube and washed twice with PBS (3000 rpm, 3 min); 1 ml of 50% Percoll solution was added to the precipitate, the resuspended precipitate was centrifuged (10000 g, 1 min), the supernatant was discarded, and the precipitate was retained. Add 1 ml of PBS; the slide should now be free of oocyst walls. Wash twice with PBS (3000 rpm, 3 min). Resuspend the sporangium pellet in excystment buffer (generally, 1-1.5 ml is sufficient for 10 million oocysts) and incubate in a 42°C water bath for approximately 5 minutes, inverting and mixing every minute. Excystment of sporozoites is microscopically examined after 5 minutes. Incubation is stopped when few sporangia are visible. Centrifuge (3000 rpm, 3 min) to remove the excystment buffer. Add 1 ml of 55% Percoll solution to the pellet, mix thoroughly, and then centrifuge (10,000 g, 1 min). Discard the supernatant and wash twice with PBS (3000 rpm, 3 min). Pure sporozoites can be obtained.

[0133] Among them, the wild-type Eimeria acervulina is the Beijing strain (BJ), which is available to the public from the National Protozoa Laboratory.

[0134] IBDV B87 used in the following examples was purchased from Yibang Biological Company (veterinary drug registration number 150132026).

[0135] Example 1. Design and sequence of antigen fusion protein

[0136] After extensive and in-depth research, the inventors of this application have designed and developed an antigen fusion protein. When the gene encoding this antigen fusion protein is introduced into wild-type Eimeria acervulina, the resulting live oocysts of the transgenic coccidia can significantly enhance humoral immune responses. This can be used to prepare a live vaccine for the prevention and / or treatment of infectious bursal disease virus (IBDV) infection or infectious bursal disease. The design and sequence of the antigen fusion protein are as follows:

[0137] Infectious bursal disease virus (IBDV) VP2 protein (SEQ ID NO: 1, position 1-453) and red junglefowl (Gallus gallus) were selected.

[0138] C3d (SEQ ID NO: 1, position 484-813) was used to construct the antigen fusion protein. VP2 protein was fused to C3d via a trimer tag and a linker.

[0139] The resulting antigen fusion protein is sequentially composed of VP2-trimer tag-linker-C3d from N-terminus to C-terminus. This antigen fusion protein is named VP2-Trimer-tag-C3d.

[0140] A trimer tag is a protein tag that can trimerize any target protein. By introducing a trimer tag, VP2 is promoted to exist in the correct trimer form, structurally mimicking the native structure of the virus and thereby improving immunogenicity. In this embodiment, the exemplary trimer tag is the T4 bacteriophage fiber protein (foldon) fold trimer domain (YIPEAPRDGQAYVRKDGEWVLLSTFL, SEQ ID NO: 1, positions 454-479, referred to as T4 foldon).

[0141] Specifically, the amino acid sequence of the antigen fusion protein VP2-Trimer-tag-C3d is positions 1-813 of SEQ ID NO: 1, wherein: positions 1-453 of SEQ ID NO: 1 are the infectious bursal disease virus (IBDV) VP2 protein; positions 454-479 of SEQ ID NO: 1 are the trimer tag (T4 foldon); positions 480-483 of SEQ ID NO: 1 are the linker; and positions 484-813 of SEQ ID NO: 1 are C3d.

[0142] The gene encoding the antigen fusion protein VP2-Trimer-tag-C3d is named VP2-Trimer-tag-C3d gene, and the nucleotide sequence of the VP2-Trimer-tag-C3d gene is SEQ ID NO: 2, positions 1-2439. Among them, positions 1-1359 of SEQ ID NO: 2 are the gene encoding the VP2 protein; positions 1360-1437 of SEQ ID NO: 2 are the gene encoding the trimer tag (T4foldon); positions 1438-1449 of SEQ ID NO: 2 are the gene encoding the linker; and positions 1450-2439 of SEQ ID NO: 2 are the gene encoding C3d.

[0143] To facilitate the purification and detection of the antigen fusion protein, a 3FLAG tag (DYKDHDGDYKDHDIDYKDDDDK, positions 816-837 of SEQ ID NO: 1) was further fused to its C-terminus. To facilitate the separation of the 3FLAG tag, a restriction enzyme cleavage site (KpnI) was designed between the antigen fusion protein and the 3FLAG tag.

[0144] The antigen fusion protein fused with the 3FLAG tag was named VP2-Trimer-tag-C3d-Flag, and its amino acid sequence was SEQ ID NO: 1.

[0145] The gene encoding the antigen fusion protein fused with the 3FLAG tag is named VP2-Trimer-tag-C3d-Flag gene, and its nucleotide sequence is SEQ ID NO: 2.

[0146] Furthermore, in order to promote the stable expression of the antigen fusion protein in the host dense granules, the GRA8 signal peptide (MALPLRVSATVFVVFAVFGVARAMNGPL, SEQ ID NO: 3) was fused to its N-terminus. The nucleotide sequence of the gene encoding the GRA8 signal peptide is SEQ ID NO: 4.

[0147] This example also designed a control protein without C3d fusion, named VP2-Trimer-tag-Flag. The amino acid sequence of the control protein, compared to SEQ ID NO: 1, only lacks the C3d sequence. Specifically, the control protein's amino acid sequence is the amino acid sequence obtained by directly concatenating positions 1-483 of SEQ ID NO: 1 with positions 814-837 of SEQ ID NO: 1.

[0148] Example 2: Construction of recombinant Eimeria acervulina stably expressing VP2-Trimer-tag-C3d

[0149] In this example, wild-type Eimeria acervulina (Ea-WT) was used as the base strain to construct a recombinant Eimeria acervulina stably expressing VP2-Trimer-tag-C3d. The specific steps are as follows:

[0150] 1. Construction of recombinant plasmid pMic2GRA8VP2TrilinkerC3dpeA

[0151] (1) Recombinant plasmid design: Based on the plasmid pMic2GRA8VP2TriEA (containing enhanced yellow fluorescent protein EYFP to express trimerized VP2 (VP2-Trimer-tag-Flag) transgenic Eimeria acervulina Figure 1), designed a plasmid expressing trimerized VP2 fused to C3d (VP2-Trimer-tag-C3d-Flag) - pMic2GRA8VP2TrilinkerC3dPEA ( Figure 2 ). Among them, EtMic2 at the 5' end serves as a promoter, GRA8ss serves as a signal localization sequence, the VP2 fusion trimer-tag tag is connected to C3d through a soft peptide linker, Flag serves as a tag, P2A is a short peptide with self-cleavage function from porcine teschovirus, PRS is a halofuginone drug screening gene, Actin at the 3' end serves as a terminator, and SnaBI (5'-TACGTA-3') restriction sites are added to both ends. The nucleotide sequence of the EtMic2 promoter is SEQ ID NO: 5. The nucleotide sequence of the PRS gene is SEQ ID NO: 6. The nucleotide sequence of the self-cleavage peptide P2A (GSGATNFSLLKQAGDVEENPGPTS, SEQ ID NO: 7) is SEQ ID NO: 8.

[0152] (2) Fragment amplification

[0153] pMic2GRA8VP2TrilinkerC3dPEA was amplified in four fragments, and the primer sequences were:

[0154] Clip 1:

[0155] Upstream primer: linkerC3d-1Fw: 5′-CTTTCTACCTTTTTAATGCATGGCTCAACAAAGGTTTCTA-3′;

[0156] Downstream primer: C3d3fp-1Rv: 5′-GCCCTTGCTCACCATGTAGCTCCGGCCAAACAAACACCAA-3′;

[0157] Clip 2:

[0158] Upstream primer: peA-2Fw: 5′-GGCCGGAGCTACATGGTGAGCAAGGGCGAGGAGCTGTTCA-3′;

[0159] Downstream primer: A-2Rv: 5′-GAAGCTGCCCTTTACGTATCTAGAAACCTACAATTACCTG-3′;

[0160] Clip 3:

[0161] Upstream primer: Backbone-3Fw: 5′-TCTAGATACGTAAAGGGCAGCTTCAATTCGCCCTATAGTG-3′;

[0162] Downstream primer: Backbone-3Rv: 5′-ACTGCGATCTTACGTAAAGGGCAGCTTGGCGTAATCATGG-3′;

[0163] Clip 4:

[0164] Upstream primer: pEtmicGraVP2-4Fw: 5′-GCCAAGCTGCCCTTTACGTAAGATCGCAGTGTGTCTGGAA-3′;

[0165] Downstream primer: TrilinkerC3d-4Rv: 5′-ACCTTTGTTGAGCCATGCATTAAAAAGGTAGAAAGCAATACC-3′;

[0166] All the above DNA fragments were synthesized using NEB's Q5 high-fidelity DNA polymerase ( High-Fidelity DNA Polymerase) was used for amplification, and the PCR reaction system was shown in Table 1.

[0167] Table 1. PCR reaction system

[0168]

[0169] The PCR reaction conditions are shown in Table 2.

[0170] Table 2. PCR reaction conditions

[0171]

[0172] Note: Q5 High-Fidelity DNA Polymerase extends at least 2 kb per minute, so the extension time is determined by the specific length of the amplified fragment.

[0173] After the PCR reaction, the target fragment was recovered by running the gel and purified using the gel recovery kit from TransGen Biotech ( Quick Gel Extraction Kit) to purify and recover each DNA fragment.

[0174] (3) Fragment connection

[0175] The purified DNA products were ligated using the TransGen Biotech Multi-Fragment Seamless Cloning Kit ( Seamless Cloning and Assembly Kit). The reaction system is shown in Table 3.

[0176] Table 3. Multi-fragment ligation reaction conditions

[0177]

[0178] The reaction system was gently mixed and allowed to react at 50°C for 15 minutes. After completion, the reaction was cooled on ice. The product was transferred to 50 μL of Trans1-T1 competent cells (TransGen Biotech, Trans1-T1 Phage Resistant Chemically Competent Cell), gently mixed, and placed on ice for 30 minutes. Heat shock was performed in a 42°C metal bath for 1 minute, and then immediately transferred to ice for 2 minutes. 500 μL of LB medium was added and the cells were incubated on a shaker at 37°C at 250 rpm for 1 hour. 100 μL of the solution was evenly spread on an ampicillin-resistant plate. After 24 hours, several single clones were selected for sequencing. The sequencing primers used were universal primers M13F and M13R, and one reaction each. The sequencing results showed that the sequence was correct and there were no errors at the plasmid junction. The plasmid pMic2GRA8VP2TrilinkerC3dpeA was successfully constructed.

[0179] The nucleotide sequence of plasmid pMic2GRA8VP2TrilinkerC3dpeA is Figure 10 The nucleotide sequence shown and Figure 11 The nucleotide sequences shown are directly linked to the resulting sequence. Plasmid pMic2GRA8VP2TrilinkerC3dpeA consists of 11726 nucleotides. Figure 10 The nucleotide sequence shown is nucleotides 1-5912 of plasmid pMic2GRA8VP2TrilinkerC3dpeA; Figure 11 The nucleotide sequence shown is nucleotides 5913-11726 of plasmid pMic2GRA8VP2TrilinkerC3dpeA.

[0180] The plasmid pMic2GRA8VP2TrilinkerC3dpeA contains two SnaBIs and is transfected into the genome of wild-type Eimeria acervulina based on restriction endonuclease-mediated integration technology (REMI).

[0181] The plasmid pMic2GRA8VP2TrilinkerC3dpeA was extracted using a PL14-large plasmid extraction kit (Aidlab biotechnologies CO. Ltd) for later use.

[0182] Plasmid linearization by enzyme digestion: Prepare 500 μL of a plasmid digestion system, including 100 μL of the pre-assembled plasmid (generally at a concentration of 2000–4000 ng / μL), 5 μL of SnaBI restriction enzyme, 50 μL of 10× rCutsmart, and 345 μL of ddH2O. Mix thoroughly by pipetting and incubate in a 37°C enzyme digestion apparatus or a thermostatic metal bath for 3 hours to obtain the linearized plasmid.

[0183] (4) Plasmid purification: Add 500 μL of DNA extract (i.e., the linearized plasmid in step (3)) to a 1.5 mL centrifuge tube, invert to mix, and let stand at room temperature for 5 min; centrifuge at 4°C and 12,000 r / min for 15 min until a white film appears in the middle, and aspirate the upper liquid into a new 1.5 mL centrifuge tube; add 500 μL of isopropanol solution, invert to mix, and let stand at -20°C for 1 h; centrifuge at 4°C and 12,000 r / min for 10 to 15 min, and slowly discard the supernatant; add 1 mL of 75% ethanol to each tube, centrifuge at 4°C and 12,000 r / min for 3 min, remove the supernatant along the side wall with a pipette, and place in a 50°C metal bath to evaporate the ethanol; add 20 to 50 μL of ddH2O to dissolve the precipitate, and store at -20°C.

[0184] 2. Transfection of transgenic Eimeria acervulina strains

[0185] (1) The 7 The PBS suspension of freshly obtained Ea-WT sporozoites was added to a 1.5 mL centrifuge tube, centrifuged at 2500 rpm for 5 min, and the supernatant was discarded.

[0186] (2) Mix Buffer I and Buffer II at a ratio of 1:50 by pipetting, prepare and use immediately, and aspirate 80 μL of the resuspended sporozoite pellet.

[0187] (3) Add 15 μL of linearized plasmid and 5 μL of SnaBI restriction enzyme, pipette to mix well, and transfer to the nuclear transfer cup.

[0188] (4) Set the nuclear transfer instrument program to U-033, place the nuclear transfer cup and confirm the start. When "OK" appears on the screen, it means the nuclear transfer program is completed.

[0189] (5) Add 1 mL of DMEM medium twice along the side wall of the nuclear transfer cup, gently pipette to mix, and transfer to a 1.5 mL centrifuge tube.

[0190] 3. Passaging and screening of transgenic Eimeria acervulina strains

[0191] (1) Using a 1 mL syringe, the sporozoite suspension transfected in step 2 was inoculated into coccidia-free chickens through the sub-wing vein, and then the Eimeria acervulina oocysts were expanded and purified.

[0192] (2) Observe the first generation of sporulated oocysts under an inverted fluorescence microscope. If they are green, it means that the plasmid has been successfully transfected into Ea-WT sporozoites. Otherwise, nuclear transfection needs to be repeated.

[0193] (3) The purified oocysts or their sporangia are extracted and screened using fluorescence activated cell sorting (FACS).

[0194] (4) The selected oocysts or sporocysts were orally inoculated into coccidia-free chickens. The Eimeria acervulina oocysts were then propagated and purified, and the in vivo passage and screening process was repeated in chicks to increase the proportion of transgenic coccidia expressing EYFP. After six generations of flow cytometric screening, a recombinant Eimeria acervulina stably expressing VP2-Trimer-tag-C3d was obtained, designated Ea-VTC, with a luminescence efficiency of >90%.

[0195] The recombinant Eimeria acervulina stably expressing the control protein obtained by transfecting the control plasmid pMic2GRA8VP2TriEA into the wild type Eimeria acervulina (Ea-WT) was named Ea-VP2. Figure 12 The nucleotide sequence shown and Figure 13 The nucleotide sequence shown is directly connected to the resulting sequence. The control plasmid pMic2GRA8VP2TriEA consists of 8477 nucleotides. Figure 12 The nucleotide sequence shown is nucleotides 1-4382 of plasmid pMic2GRA8VP2TrilinkerC3dpeA; Figure 13 The nucleotide sequence shown is nucleotides 4383-8477 of plasmid pMic2GRA8VP2TrilinkerC3dpeA.

[0196] Example 3: Determination of the integration site of the Ea-VTC strain

[0197] 1. Obtaining Ea-VTC and Extracting Genomic DNA

[0198] Take 4×10 5 14-day-old AA coccidia-free broiler chickens were orally inoculated with sporulated oocysts of Ea-VTC. Cecum was dissected 120 hours after inoculation, and the cecal mucosa scrapings were digested with 0.5% taurodeoxycholate solution and 0.25% pancreatic enzyme, and merozoites were collected by centrifugation.

[0199] The genomic DNA of merozoites was extracted using the CTAB method. Resuspend the merozoites in 65°C preheated CTAB extract and proteinase K, mix by inversion, and incubate at 60°C for 2 h; add RNase at 37°C and incubate for 30 min; add an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1), mix gently, and centrifuge at 12,000 rpm at room temperature for 10 min. After transferring the supernatant, add an equal volume of chloroform:isoamyl alcohol (24:1), mix by inversion, and centrifuge at 12,000 rpm at room temperature for 5 min; remove the supernatant, add 1-2 volumes of -20°C precooled isopropanol, mix gently, let stand at -20°C for 30 min, and centrifuge at 12,000 rpm for 15 min; wash the precipitate with 75% ethanol and 0.2 mol / L sodium acetate, and centrifuge at 12,000 rpm at room temperature for 10 min, repeat once; add an appropriate amount of water to dissolve the precipitate, determine the concentration, and store at -20°C until use.

[0200] The purity and integrity of the DNA were checked by 0.8% agarose gel electrophoresis, and the DNA concentration was determined using Qubit 4.0.

[0201] 2. Ea-VTC genome resequencing

[0202] After the sample's genomic DNA passed the test, it was mechanically sheared (ultrasound-assisted) to fragment the DNA. The fragments were then purified, end-repaired, triple-end-addition DNA was added, and sequencing adapters were ligated. Fragments were then size-selected by agarose gel electrophoresis and amplified by PCR to create a sequencing library. The constructed library underwent quality control. Those that passed the control were sequenced using the Illumina NovaSeq platform with a paired-end read length of 150 bp. To ensure the accuracy of subsequent analysis, a sequencing depth of 100× was used to generate at least 6 GB of data, with a data quality standard of Q20 >90%. Whole-genome resequencing was commissioned by Shanghai Paisonno Biotechnology Co., Ltd.

[0203] 3. Data Analysis

[0204] Raw data were filtered using Fastp. Filtering criteria included: 1) removing adapter sequences, 2) setting a low quality value of 20 (Q20) to filter out reads with a low-quality base content greater than 30%, and 3) removing reads with a length less than 50.

[0205] The quality-controlled sequencing data were aligned to the Eimeria reference genome (https: / / www.ncbi.nlm.nih.gov / assembly / GCA_905310635.1) using Bowtie2 software. The randomness of the next-generation sequencing was assessed based on the depth and coverage of the sequencing data. The sequencing data were then aligned with the transgenic vector sequence, removing reads that did not map to the T-DNA and generating chimeric reads (reads with one end mapping to the T-DNA and the other to the Tender reference genome). Sequences of these chimeric reads were then aligned to ToxoDB to determine the specific location of the T-DNA on the genome, indicating the possible insertion site. The results indicated that the T-DNA insertion site was located at HG671014:24035-31025.

[0206] Sequencing identification showed that the coding gene of the fusion protein VP2-Trimer-tag-C3d of the present invention was integrated into the 24035-31025 positions of GenBank Accession No. HG671014 in the genome of wild-type Eimeria acervulina.

[0207] Example 4: Identification of recombinant Eimeria acervulina expressing VP2-Trimer-tag-C3d

[0208] 1. PCR identification of transgenic coccidia Ea-VTC genome

[0209] Containing 1×10 7 DNA was extracted from sporulated oocysts of recombinant-positive Eimeria acervulina (Tiangen Biochemical Technology (Beijing) Co., Ltd., Blood / Cell / Tissue Genomic DNA Extraction Kit) for identification. The DNA extraction method followed the cell DNA extraction method of the kit. The identification primers are as follows:

[0210] VP2-1Fw: 5'-ATGACAAACCTGCAGGATCA-3';

[0211] VP2-1Rv: 5'-TCTTCTAATAGCTCTAATAA-3';

[0212] C3d-2Fw: 5'-GCCAAGAGATGGGCAAGCTTACG-3';

[0213] C3d-2Rv: 5'-CTTTGTAATCAATATCATGATCCT-3';

[0214] EYFP-3Fw: 5'-GGTGAGCAAGGGCGAGGAGCT-3';

[0215] EYFP-3Rv: 5'-GTACAGCTCGTCCATGCCGAGA-3'.

[0216] The identification results of recombinant transgenic Eimeria acervulina expressing fusion of trimerized VP2 and C3d are as follows Figure 3 As shown, there are no target bands in PCR1, PCR2 and PCR3 of Ea-WT strain, target bands in PCR1 of Ea-VP2 strain, and target bands in PCR1, PCR2 and PCR3 of Ea-VTC strain, indicating that the recombinant plasmid has been successfully transferred into the recombinant strain.

[0217] 2. Western blot identification of Ea-VTC transgenic strains

[0218] Western blot was used to identify the fusion protein VP2-Trimer-tag-C3d in Ea-VTC. The specific implementation method is as follows:

[0219] ①Absorb at least 5×10 6 A PBS suspension of recombinant Eimeria acuminata (Ea-VTC) sporozoites was placed in a 1.5 mL centrifuge tube and centrifuged at 2500 rpm for 5 min. The supernatant was discarded and the sporozoite pellet was resuspended in 100 μL RIPA lysis buffer and placed on ice for 1 h. 20 μL 6× Protein loading buffer was added and the tube was heated in a 100°C water bath for 10 min.

[0220] ② Take a 1.5mm gel plate and select a gel of appropriate concentration according to the size of the target protein for SDS-PAGE. First prepare the lower layer of separation gel and seal it with distilled water, then prepare the upper layer of concentration gel.

[0221] ③ Centrifuge the protein sample at 10,000 rpm for 5 min, add 40 μL of supernatant and 10 μL of protein marker to the lane, and add 40 μL of 1× protein loading buffer to each of the remaining lanes.

[0222] ④ Add 1× Running buffer to the electrophoresis tank, adjust the voltage to 70V constant voltage electrophoresis for 20 minutes, and then adjust the voltage to 120V constant voltage electrophoresis for 60-90 minutes.

[0223] ⑤ Cut the gel according to the size of the target protein and cut out the PVDF membrane of the corresponding size. Soak it in methanol for 1 minute. Place the sponge and filter paper on the two side clamps in turn. Place the gel block on the filter paper of the negative electrode and the PVDF membrane on the gel block. Scrape it gently to avoid bubbles between the gel block and the membrane. Close the clamps and insert it into the transfer tank. Add transfer solution and transfer the membrane at a constant voltage of 70V for 3 hours.

[0224] ⑥ Soak the PVDF membrane in PBST solution and wash it on a shaker three times, 5 minutes each time. Pour away the PBST solution, add 5% skim milk, block it on a shaker at room temperature for 1 hour or at 4°C overnight, and wash it twice with PBST solution, 5 minutes each time.

[0225] ⑦ Use primary antibody diluent to dilute the anti-Flag tag mouse monoclonal antibody (purchased from Sigma, product number F1804) 2000 times and the anti-GAPDH mouse monoclonal antibody (purchased from Zhongke Maichen, product number A01622-40) 2000 times, incubate on a shaker at room temperature for 1 hour or at 4°C overnight, recover the antibody, and wash with PBST solution 5 times, 5 minutes each time.

[0226] ⑧ Dilute HRP-goat anti-mouse secondary antibody (purchased from Zhongke Maichen Company, product number IS001) 2000 times with primary antibody diluent, incubate at room temperature on a shaker for 1 hour, recover the antibody, and wash with PBST solution 5 times, 5 minutes each time.

[0227] ⑨ Referring to the ECL chemiluminescence kit, take equal volumes of solution A and solution B, mix them by pipetting, evenly drop them on the PVDF membrane, and place it in a Tanon 5200 imager for development.

[0228] The results showed that a protein band of approximately 204 kDa appeared in Ea-VTC ( Figure 4 ), which is consistent with the expected protein band size.

[0229] 3. Identification of the expression location of the fusion protein VP2-Trimer-tag-C3d in Ea-VTC transgenic coccidia

[0230] Take at least 1×10 6 Sporozoites of recombinant Eimeria acuminata (Ea-VTC) were inoculated into 12-well plates filled with HFF cells (human foreskin fibroblasts, purchased from ATCC) (a sterile cell slide was placed before the cells were inoculated). Four hours after inoculation, uninvaded sporozoites were washed away and the cells were cultured in an incubator. After 12 hours of culture, an IFA test was performed as follows:

[0231] ① Connect the chromatography column to a constant flow pump, adjust the cross flow rate to 50-60 Hz, first fill the chromatography column with distilled water to rinse, then fill the chromatography column with glycine solution to rinse; add DE-52 cellulose solution, and continue to add glycine solution. When the cellulose precipitation reaches 1 cm in the chromatography column, add the extracted sporozoites, and continue to add glycine solution. Adjust the cross flow rate to 30-40 Hz, and collect the sporozoite solution in a 50 mL centrifuge tube. During this period, drop the solution on a slide for microscopic examination. When no sporozoites are observed in the solution, turn off the constant flow pump, stop the column, centrifuge at 2500 r / min for 5 minutes, discard the supernatant, and resuspend the precipitate with PBS.

[0232] ②1×10 6 The sporozoites were inoculated into 24-well plates cultured with HFF cells, 100× double antibody was added, and the cells were gently pipetted to mix. The cells were placed in a cell culture incubator for 4-6 hours of invasion, the culture medium was discarded, 1 mL of PBS was added to wash once, and the supernatant was discarded.

[0233] ③ Use tweezers to pick up the glass slide inoculated with sporozoites from the 24-well plate, place it in a new 24-well plate, add 1 mL of 4% paraformaldehyde solution, let it stand for 30 minutes, add 1 mL of PBS to wash once, and discard the supernatant.

[0234] ④ Add 1 mL of 0.25% Triton X-100, let it stand for 30 min, add 1 mL of PBS to wash once, and discard the supernatant.

[0235] ⑤ Add 1 mL of 3% BSA, let it stand for 30 min, add 1 mL of PBS to wash once, and discard the supernatant.

[0236] ⑥ Dilute the anti-Flag tag mouse monoclonal antibody 200 times with 3% BSA and drop it on a sealing film of appropriate size. Place the 24-well plate with the sporozoite slide inoculated face down in a humidified chamber and incubate at 37°C for 1 hour. Add 1 mL of PBS to wash and repeat 3 times.

[0237] ⑦ Dilute Cy3-labeled goat anti-mouse IgG antibody 200 times and Hoechst 33258 100 times with 3% BSA, drop them on a sealing film of appropriate size, place the 24-well plate with the sporozoite slide inoculated face down in a humidified chamber, incubate at 37°C for 1 hour, add 1 mL PBS to wash, and repeat 5 times.

[0238] ⑧ Add 5 μL of anti-fluorescence quencher on the slide, place the slide inoculated with sporozoites in the 24-well plate with the front side facing down to avoid bubbles, seal the slide with mounting medium, place in a humidified box, and store at 4°C.

[0239] The results are as follows Figure 5As shown, after sporozoites were grown in HFF cells for 12 h, the fusion protein was expressed in the sporoplasm of the sporozoites and was mainly located at the apex of the sporozoites.

[0240] Example 5: Detection of Ovulation Cyst Patterns in Recombinant Eimeria acervulina Expressing VP2-Trimer-tag-C3d

[0241] Sporulated oocysts were obtained from wild-type Eimeria acervulina Ea-WT, recombinant Eimeria acervulina Ea-VP2, and recombinant Eimeria acervulina Ea-VTC. Oocysts are excreted in chicken feces and are considered unsporulated and incapable of infecting chickens. Under appropriate conditions of temperature, humidity, and oxygen, unsporulated oocysts undergo sporulation, forming sporulated oocysts. Each oocyst contains four sporocysts, each of which contains two sporozoites. These oocysts then become capable of infecting chickens. The collected oocysts were connected to an aerator and placed in a 28°C incubator or a homemade incubator for aerated sporulation for 48 hours (note that water should be added after 24 hours to compensate for evaporation during aeration). After sporulation, the oocysts were labeled and stored at 4°C.

[0242] (1) Twelve one-week-old SPF chickens were randomly divided into three groups, with 4 chickens in each group. Each chicken was orally inoculated with 5×10 2 The chickens in the Ea-WT, Ea-VP2 and Ea-VTC groups were kept in an environment free of coccidia contamination and given sufficient feed and drinking water.

[0243] (2) Remove the feces from the first 3 days after inoculation, and collect the feces from 4 to 14 days after inoculation in a ziplock bag every 24 hours, add appropriate amount of clean water and stir evenly, and count the number of ovulation sacs every day using a McMaster counting plate.

[0244] (3) 14 days after primary infection (PI), each chicken was orally inoculated with 5×10 3 The sporulated oocysts of Ea-WT were collected and feces were collected 5 to 7 days after secondary infection (SI) using a ziplock bag.

[0245] (4) Add appropriate amount of clean water and stir evenly, and use a McMaster counting plate to count the number of ovulation sacs at the peak period.

[0246] (5) Take 2 g of feces and add it to a 100 mL beaker. Add 60 mL of saturated saline and mix well with a rubber-tipped pipette. Let "n1" and "n2" represent the total number of oocysts in the two counting chambers of the McMaster counting plate, respectively. The number of oocysts per gram of feces (OPG) = [(n1 + n2) / (2 × 0.15)] × 60 / 2.

[0247] Ea-VTC starts to release ovulation sacs in chicks from 4 dpi, with the peak at 6 dpi, and then the amount of ovulation sacs gradually decreases ( Figure 6 ); The amount of oocysts released by chicks in the Ea-VTC group was significantly lower than that in the Ea-WT group 5-7 days after the initial infection (P < 0.0001). Only a small amount of oocysts was released 5-7 days after the secondary infection ( Figure 7 ). It can be seen that compared with the wild-type Eimeria acervulina (Ea-WT) or the strain expressing VP2 antigen alone (Ea-VP2), the recombinant Eimeria acervulina (Ea-VTC) of the present invention has significantly reduced fecundity and is safer.

[0248] Example 6: Detection of the immune efficacy of recombinant Eimeria acervulina Ea-VTC in chickens

[0249] The Ea-VTC strain was used to immunize 7-day-old SPF chickens. The experimental procedures of immunization, challenge and detection are shown in the figure below. Figure 8 The specific method is as follows:

[0250] (1) Grouping: Forty SPF chickens were randomly divided into four groups: the IBDV-only infected group, also known as the unimmunized and challenged control (UCC) group, the Ea-WT group, the Ea-VP2 group, and the Ea-VTC group.

[0251] (2) Immunization: The first immunization was performed on 7-day-old chickens. Each chicken in the UCC group was orally inoculated with 200 μL PBS solution, and each chicken in the other three groups was orally inoculated with 2×10 3 The chickens were immunized again at 21 days of age. Each chicken in the UCC group was orally inoculated with 200 μL of PBS solution, and each chicken in the other three groups was orally inoculated with 1×10 5 (200 μL) corresponding to fresh sporulated oocysts.

[0252] (3) Virus challenge: The chickens were challenged at 35 days of age. Each chicken was orally infected with 1×10 5 EID 50 The mice were challenged with IBDV B87 strain at a dose of 1 mg / kg.

[0253] (4) Sampling: Blood was collected from chickens at 7, 21, 35, and 42 days of age.

[0254] (5)ELISA

[0255] 1) Place the collected blood in a 37°C incubator for 1 hour, then in a 4°C refrigerator for 2 hours. Centrifuge at 5000 rpm for 5 minutes. Aliquot 30 μL into each PCR tube, label it, and store at -80°C until use.

[0256] 2) Dilute the serum 1:50 with sample diluent. At the same time, dilute the 100× Normal Control solution and 100× Positive Control solution in the kit to 1× for use. Prepare them immediately before use.

[0257] 3) Add 50 μL of sample diluent to a 96-well plate coated with IBD antigen. Add 50 μL of 1× Normal Control to wells A2, H10, and H12; add 50 μL of 1× Positive Control to wells A1, A3, and H11; add diluted serum samples to the remaining wells and let stand at room temperature for 30 minutes.

[0258] 4) Discard the liquid in the wells and add 300 μL of 1× Wash Solution to each well. Let it stand for 3 minutes and then discard the liquid in the wells. Repeat three times. Finally, turn the well upside down on absorbent paper and gently tap off any residual liquid.

[0259] 5) Add 100 μL of 1× HRP-Conjugate Solution to each well and let it stand at room temperature for 30 minutes.

[0260] 6) Same as step 4).

[0261] 7) Add 100 μL of substrate solution to each well and let it stand at room temperature for 20 minutes.

[0262] 8) Add 100 μL of 1× Stop Solution to each well and let it stand at room temperature for 10 minutes.

[0263] 9) Measure the OD value at a wavelength of 405-410 nm using a microplate reader. Corrected positive value = average OD value of the positive control - average OD value of the negative control; S / P = (OD value of a single sample - average OD value of the negative control) / corrected positive value; Log10 titer = (1.172 × Log10 S / P) + 3.614; refer to the kit instructions; when the antibody titer is >999, it is considered a positive serum sample.

[0264] (6) Statistical analysis of data

[0265] GraphPad Prism 8.3.0 software was used for statistical plotting, and one-way ANOVA was used to analyze the significance of antibody titers at different time points. * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, **** indicates P < 0.0001, and ns indicates no significant difference.

[0266] The antibody levels of IBDV VP2 protein in the serum of chicks were detected by ELISA. It was found that 14 days after the first vaccination, the VP2 protein antibody levels in the serum of chicks in the Ea-VTC group and the Ea-VP2 group were significantly higher than those in the Ea-WT group (P < 0.0001), and the antibody level in the Ea-VTC group was significantly higher than that in the Ea-VP2 group (P < 0.001). 28 days after the first vaccination, the VP2 protein antibody levels in the serum of chicks in the Ea-VTC group and the Ea-VP2 group showed an extremely significant difference (P < 0.0001). 35 days after the first vaccination (7 days after the challenge), the antibody titers in the serum of the Ea-VTC group and the Ea-VP2 group were still significantly higher than those in the Ea-WT group (P < 0.0001). Figure 9 ).

[0267] In summary, the present invention provides a recombinant transgenic Eimeria acervulina live oocyst vector vaccine expressing trimerized VP2 fused to C3d. The trimerized VP2 fused to C3d antigen (i.e., the antigen fusion protein VP2-Trimer-tag-C3d) expressed by this coccidia maintains regular oocyst excretion, but although fertility is reduced, it can effectively enhance the humoral immune response against IBDV-VP2 after oral immunization. The antibody levels induced by this vaccine are significantly higher than those of strains expressing a single VP2 antigen, further validating the potential application of Ea-VTC live oocysts as a live IBDV vaccine.

[0268] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using conventional techniques known in the art.

Claims

1. An antigen fusion protein, characterized in that: The antigen fusion protein comprises VP2 protein, a trimer tag and C3d protein, the amino acid sequence of the VP2 protein is shown as positions 1-453 of SEQ ID NO: 1, and the amino acid sequence of the C3d protein is shown as positions 484-813 of SEQ ID NO:

1.

2. The antigen fusion protein according to claim 1, characterized in that The trimeric tag is a T4 phage fiber protein fold trimeric domain.

3. The antigen fusion protein according to claim 1 or 2, characterized in that The antigen fusion protein is any one of the following: A1) a protein whose amino acid sequence is positions 1-813 of SEQ ID NO: 1; A2) a protein having at least 80% identity with the protein of A1) and having the same function as the protein of A1) obtained by substitution, deletion, and / or addition of amino acid residues of the amino acid sequence of SEQ ID NO: 1; A3) A fusion protein having the same function obtained by connecting a tag to the N-terminus and / or C-terminus of A1) or A2); A4) A fusion protein obtained by linking a signal peptide to the N-terminus of A1), A2) or A3).

4. The antigen fusion protein according to claim 3, characterized in that The amino acid sequence of the fusion protein in A3) is shown as SEQ ID NO: 1, and / or the amino acid sequence of the signal peptide in A4) is shown as SEQ ID NO:

3.

5. Biomaterial, characterized in that The biological material is any one of the following: B1) a nucleic acid molecule encoding the antigen fusion protein according to any one of claims 1 to 4; B2) an expression cassette containing the nucleic acid molecule described in B1); B3) a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3); B5) A recombinant host cell containing the nucleic acid molecule of B1), or a recombinant host cell containing the expression cassette of B2), or a recombinant host cell containing the recombinant vector of B3).

6. Recombinant Eimeria acervulina, characterized in that The recombinant Eimeria acervulina stably expresses the antigen fusion protein according to any one of claims 1 to 4 or the nucleic acid molecule according to claim 5.

7. The recombinant Eimeria acervulina according to claim 6, characterized in that The recombinant Eimeria acervulina is obtained by integrating the gene encoding the antigen fusion protein according to any one of claims 1 to 4 into the following site of the Eimeria acervulina genome: positions 24035 to 31025 of GenBank Accession No. HG671014.

8. A method for constructing a recombinant Eimeria acervulina, characterized in that: The method comprises integrating the gene encoding the antigen fusion protein of any one of claims 1 to 4 into the genome of Eimeria acervulina to obtain recombinant Eimeria acervulina; or integrating the gene encoding the antigen fusion protein of any one of claims 1 to 4 into the following site of the genome of Eimeria acervulina to obtain recombinant Eimeria acervulina: positions 24035-31025 of GenBank Accession No. HG671014.

9. Use of the antigen fusion protein according to any one of claims 1 to 4, the biological material according to claim 5, the recombinant Eimeria acervulina according to claim 6 or 7, or the recombinant Eimeria acervulina obtained by the method according to claim 8 in any of the following: C1) Use in the preparation of a product for enhancing the humoral immune response against infectious bursal disease virus; C2) Use in the preparation of a product for preventing and / or treating infectious bursal disease virus infection or infectious bursal disease.

10. A vaccine composition, characterized in that The vaccine composition comprises any one of the following: D1) the recombinant Eimeria acervulina according to claim 6 or 7; D2) recombinant Eimeria acervulina obtained by the method according to claim 8; D3) live oocysts of the recombinant Eimeria acervulina described in D1) or D2); D4) sporulated oocysts of the recombinant Eimeria acervulina described in D1) or D2).

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