Method for preparing and use of vaccine diluent

Abstract

The present application relates to the field of immunology, in particular to a vaccine diluent preparation method and application. The present application provides two fusion proteins, and the composition of the combination of the two fusion proteins can have the effect of improving the immune effect of the vaccine, effectively prolonging the immune duration of the vaccine, providing more efficient immune protection for the animal body, and the vaccine diluent production process is simple and low in cost.

Description

Technical Field

[0001] The present invention relates to the field of immunological technology, in particular to a preparation method and application of a vaccine diluent. Background Art

[0002] Commonly used vaccines are categorized as freeze-dried, dry powder, and liquid vaccines. Vaccine diluents refer to the solvents or reagents used to dilute vaccines. Depending on the vaccine type, such as freeze-dried, dry powder, or liquid, the required diluents vary. These diluents not only help the vaccine disperse better throughout the body but also provide certain protective and adjuvant benefits. Each vaccine manufacturer uses its own dedicated diluents to ensure the vaccine's immune effectiveness.

[0003] At present, the research and development of vaccine diluents is constantly developing with the advancement of vaccine technology. Currently, the research and development of vaccine diluents mainly focuses on the following aspects:

[0004] Optimize the composition of the diluent: According to the characteristics of different vaccines, researchers optimize the composition of the diluent, such as adding immune enhancers, adjuvants, protective agents, etc., to improve the immune effect and stability of the vaccine.

[0005] Improving diluent manufacturing processes: To improve the quality and production efficiency of diluents, many vaccine manufacturers are committed to improving the manufacturing processes of diluents, including increasing the degree of automation and refinement of production.

[0006] Expanding the application areas of vaccine diluents: With the continuous emergence of new vaccines, the application areas of vaccine diluents are also expanding. In addition to the traditional animal vaccine field, some vaccine diluents have also begun to be used in the field of human vaccines.

[0007] Research and development of new diluents: To meet the specific needs of different vaccines, researchers are constantly developing new diluents that meet higher standards in terms of composition, stability, and safety.

[0008] Although significant progress has been made in the research of vaccine diluents, current diluents still have some shortcomings, such as a short half-life or weak immune support. Furthermore, different types of vaccines often require different types of diluents, which brings inconvenience to clinical use. Summary of the Invention

[0009] In view of this, the technical problem to be solved by the present invention is to provide a method for preparing a vaccine diluent and its application.

[0010] The present invention provides two fusion proteins, which are respectively referred to as fusion protein A and fusion protein B.

[0011] The fusion protein A provided by the present invention is composed of at least two of the IL-2 protein, the NPS protein and the WT1 protein.

[0012] For example, the fusion protein A in the present invention is composed of IL-2 protein and NPS protein, or IL-2 protein and WT1 protein, or NPS protein and WT1 protein, or IL-2 protein, NPS protein and WT1 protein.

[0013] The present invention does not limit the sources of IL-2 protein, NPS protein and WT1 protein. For example, in the embodiments of the present invention, they are derived from chicken.

[0014] In a specific embodiment, the amino acid sequence of the IL-2 protein is as shown in SEQ ID NO. 1; or a sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in SEQ ID NO. 1; or a sequence having an identity of more than 80% with the amino acid sequence shown in any of the preceding items.

[0015] In a specific embodiment, the amino acid sequence of the NPS protein is as shown in SEQ ID NO.2; or a sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in SEQ ID NO.2; or a sequence having an identity of more than 80% with the amino acid sequence shown in any of the preceding items.

[0016] In a specific embodiment, the amino acid sequence of the WT1 protein is as shown in SEQ ID NO. 3; or a sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in SEQ ID NO. 3; or a sequence having an identity of more than 80% with the amino acid sequence shown in any of the preceding items.

[0017] In the fusion protein A provided by the present invention, the connection order of IL-2 protein, NPS protein and WT1 protein is: rIL-2-NPS-WT1, rIL-2-WT1-NPS, rNPS-IL-2-WT1, rNPS-WT1-IL-2, rWT1-IL-2-NPS or rWT1-NPS-IL-2. In addition, the fusion protein A provided by the present invention can also be rIL-2-NPS, rNPS-IL-2, rIL-2-WT1 or rWT1-IL-2.

[0018] The present invention connects IL-2 with the NPS gene and the WT1 gene in series, and the obtained fusion protein can help the body to more effectively eliminate pathogens and / or tumor cells and improve immune defense capabilities. The fusion protein A in the present invention takes the protein from chicken as an example. After being injected into the chicken body, it can stimulate the high level of expression of IL-2R receptors, promote the binding of IL-2 to IL-2R, and promote the proliferation and differentiation of T cells by activating multiple signaling pathways, thereby enhancing the immune response. Secondly, the increase in the expression level of IL-2R receptors is also crucial for the activation and function of NK cells. IL-2 activates NK cells by binding to IL-2R on NK cells, promotes their secretion of cytokines such as gamma interferon, thereby enhancing the immune response. At the same time, the modified interleukin-2 protein has higher biological activity and stability and lower side effects, and its half-life is significantly extended.

[0019] Experiments have shown that combining IL-2, NPS, and WT1 proteins in series is more effective than combining IL-2 with NPS or IL-2 with WT1. The order in which the proteins are combined also has a significant impact on the immune response, with experiments demonstrating that IL-2-NPS-WT1 is more effective than other combinations.

[0020] In fusion protein A, two different proteins are connected by a linker. The present invention has tested various linkers, such as (G4S)n and (AG)n. The results show that the linker affects the physiological activity and in vivo half-life of fusion protein A. Among them, (G4S)n as a linker is more effective in prolonging the duration of vaccine immunity. Preferably, the IL-2 protein and NPS protein are linked by GGGGSGGGGS, and the NPS protein and WT1 protein are linked by GGGGSGGGGS.

[0021] The present invention also provides a small peptide with an amino acid sequence of RRRYY (SEQ ID NO. 13). Experiments have shown that inserting this small peptide into a protein, particularly at the N-terminus, can further enhance the biological activity of the protein, particularly by extending its half-life.

[0022] The present invention provides the use of a small peptide with an amino acid sequence of RRRYY in improving the physiological activity of a protein containing an IL-2 fragment.

[0023] The fusion protein A provided by the present invention further comprises a small peptide having an amino acid sequence of RRRYY. Preferably, the N-terminus of the fusion protein A comprises a small peptide having an amino acid sequence of RRRYY. More preferably, the N-terminus of the IL-2 protein further comprises an RRRYY fragment.

[0024] In some specific embodiments, the amino acid sequence of the fusion protein A is as shown in SEQ ID NO. 4; or a sequence comprising one or more amino acids substituted, deleted, added, and / or replaced based on the amino acid sequence of SEQ ID NO. 4; or a sequence having at least 80% identity to the amino acid sequence of any of the preceding items (denoted as IL-2*-NPS-WT1).

[0025] Furthermore, the present invention also provides a fusion protein B, which is composed of CaIFN-γ protein and GATA3 protein.

[0026] The present invention does not limit the sources of the CaIFN-γ protein and the GATA3 protein. For example, in the embodiments of the present invention, they are derived from chicken.

[0027] In a specific embodiment, the amino acid sequence of the Calfn-γ protein is as shown in SEQ ID NO.5; or a sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in SEQ ID NO.5; or a sequence having more than 80% identity with the amino acid sequence shown in any of the preceding items.

[0028] The amino acid sequence of the GATA3 protein is as shown in SEQ ID NO.6; or a sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in SEQ ID NO.6; or a sequence having an identity of more than 80% with the amino acid sequence shown in any of the preceding items.

[0029] In the fusion protein B provided by the present invention, the connection sequence of the CaIFN-γ protein and the GATA3 protein is: rCaIFN-γ-GATA3 or rGATA3-CaIFN-γ.

[0030] The present invention connects CaIFN-γ protein and GATA3 protein in series, and the biological activity of the obtained fusion protein is significantly improved compared with ChIFN-γ, and the half-life is also significantly prolonged, indicating that the fusion protein can help the body to more effectively eliminate pathogens and / or tumor cells and improve immune defense capabilities.

[0031] Experiments have shown that the tandem sequence of CaIFN-γ protein and GATA3 protein has an important influence on the improvement of immune effect, and the effect of rCaIFN-γ-GATA3 is better.

[0032] In fusion protein B, the CaIFN-γ protein and the GATA3 protein are connected by a linker. The present invention has tested various linkers, such as (G4S)n and (AG)n. The results show that the linker affects the physiological activity and in vivo half-life of fusion protein B. Among them, (G4S)n as a linker is more effective in prolonging the duration of vaccine immunity. Preferably, the CaIFN-γ protein and the GATA3 protein are connected by GGGGSGGGGS.

[0033] In some specific embodiments, the amino acid sequence of the fusion protein B is as shown in SEQ ID NO. 7; or a sequence comprising one or more amino acids substituted, deleted, added, and / or replaced based on the amino acid sequence of SEQ ID NO. 7; or a sequence having at least 80% identity to the amino acid sequence of any of the preceding items (referred to as rCaIFN-γ-GATA3).

[0034] Furthermore, the present invention also provides a composition comprising the fusion protein A as described above and the fusion protein B as described above.

[0035] The present invention combines modified IL-2-NPS-WT1 with CaIFN-γ-GATA3, creating a synergistic effect between IL-2 and IFN-γ. The recombinant IL-2 and IFN-γ are continuously released, and IL-2 promotes T cell proliferation and differentiation, providing more effector cells for IFN-γ production. The vaccine diluent of the present invention can significantly enhance the immune efficacy of the vaccine, effectively prolong the duration of the vaccine's immunity, and provide more efficient immune protection to animals. The vaccine diluent also features a simple production process and low cost.

[0036] In the present invention, the mass ratio of the fusion protein A to the fusion protein B is 1:(0.01-100,000). For example, the mass ratio of the fusion protein A to the fusion protein B is 1:0.01, or 1:0.1, or 1:1, or 1:10, or 1:100, or 1:1000, or 1:10,000, or 1:20,000, or 1:30,000, or 1:40,000, or 1:50,000, or 1:60,000, or 1:70,000, or 1:80,000, or 1:90,000, or 1:100,000.

[0037] In the composition of the present invention, fusion protein A and fusion protein B may be mixed or independently present, and the present invention does not limit this. In the composition, fusion protein A and fusion protein B may be in the form of powder or solution, and the present invention does not limit this.

[0038] Furthermore, the present invention also provides a biomaterial comprising at least one of the following I) to IV):

[0039] 1), a nucleic acid encoding the aforementioned fusion protein A and / or a nucleic acid encoding the aforementioned fusion protein B;

[0040] II), a plasmid vector containing the nucleic acid described in I);

[0041] III) a host cell having the nucleic acid molecule described in I) integrated into its genome;

[0042] IV), transforming or transfecting the host cell with the plasmid vector described in II).

[0043] Among the nucleic acids described herein, any nucleic acid encoding the aforementioned fusion protein A or B is within the scope of protection of the present invention. These nucleic acids may be wild-type sequences or codon-optimized sequences, and the present invention is not limited thereto. The nucleic acids described herein may comprise only the reading frame of the aforementioned fusion protein or may comprise nucleic acid sequences for restriction sites and / or selection markers, and the present invention is not limited thereto either.

[0044] In some embodiments, the nucleic acid sequence encoding the IL-2 protein is as shown in SEQ ID NO.8; or a sequence in which one or more nucleic acids are substituted, deleted, added and / or replaced based on the nucleic acid sequence shown in SEQ ID NO.8; or a sequence having more than 80% identity with the nucleic acid sequence shown in any of the preceding items.

[0045] In some embodiments, the nucleic acid sequence encoding the NPS protein is as shown in SEQ ID NO.9; or a sequence in which one or more nucleic acids are substituted, deleted, added and / or replaced based on the nucleic acid sequence shown in SEQ ID NO.9; or a sequence having an identity of more than 80% with the nucleic acid sequence shown in any of the preceding items.

[0046] In some embodiments, the nucleic acid sequence encoding the WT1 protein is as shown in SEQ ID NO.10; or a sequence in which one or more nucleic acids are substituted, deleted, added and / or replaced based on the nucleic acid sequence shown in SEQ ID NO.10; or a sequence having an identity of more than 80% with the nucleic acid sequence shown in any of the preceding items.

[0047] In some embodiments, the nucleic acid sequence encoding the Calfn-γ protein is as shown in SEQ ID NO. 11; or a sequence in which one or more nucleic acids are substituted, deleted, added and / or replaced based on the nucleic acid sequence shown in SEQ ID NO. 11; or a sequence having more than 80% identity with the nucleic acid sequence shown in any of the preceding items.

[0048] In some embodiments, the nucleic acid sequence encoding the GATA3 protein is as shown in SEQ ID NO.12; or a sequence in which one or more nucleic acids are substituted, deleted, added and / or replaced based on the nucleic acid sequence shown in SEQ ID NO.12; or a sequence having more than 80% identity with the nucleic acid sequence shown in any of the preceding items.

[0049] In the present invention, the plasmid vector is used for the storage and amplification of the nucleic acid or for the expression of the fusion protein A or B, which is not limited in the present invention. In some embodiments, the vector is a plasmid vector, which includes a pUC series plasmid vector, a pBR322 plasmid vector, a pGEM series plasmid vector, a pET series plasmid vector, a Yeast series plasmid vector or a Gateway plasmid vector.In some embodiments, the fusion protein is expressed using a pET series plasmid vector. As a feasible example, the pET series expression vector is selected from the group consisting of: pET-23c(+), pET-23(+), pET-12b(+), pET-12c(+), pET-12a(+), pET-11b(+), pET-11a(+), pET-11c(+), pET-50b(+), pET-49b(+), pET-48b(+), pET-47b(+), pET-26b(+), pET-32a(+), pET-21b(+), pET-22b(+), pET-14b, pET-16b, pET-15b, pET-19b, pET-20b(+), pET-21d(+), pET-21c(+), pET-21b(+), pET-21a(+), pET-24a(+), pET-24d(+), pET-25b(+), pET-27b(+), pET-28a(+), pET-30a(+), pET-42a(+), pET-43.1c(+), pET-43.1b(+), pET-43.1a(+), pET-44a(+), pET-44c(+), pET-46pET-37b(+), pET303 / CT-His, pET302 / NT-His, pET300 / NT-DEST, pET301 / CT-DEST, pET-5b(+), pET-17b, pET102 / D-TOPO, pET-5a(+), pET-31b(+), pET-3b(+), pET-43.1, pET-41, pET-41a(+), pET-28b(+), pET-42b(+), pET-3a(+), pET-23d(+), pET-41b(+), pET-44b(+), pET-42c(+), pET-41c(+), pET-45b(+), pET-33b(+), pET-39b(+), pET-32, pET-40b(+), pET-32c(+), pET-32b(+), pET-30, pET-32, pET-30c(+), pET-29c(+), pET-29b(+), pET-30, pET-24c(+), pET-24b(+), pET-24(+), pET-29a(+) or pET-11d(+).

[0050] In the present invention, the host is used for the storage and amplification of the plasmid vector, or for the expression of the fusion protein, as described above. In the present invention, the host is a eukaryotic host or a prokaryotic host. Eukaryotic hosts include, but are not limited to, yeast, insect cells, and renal epithelial cells, while prokaryotic hosts include, but are not limited to, Escherichia coli. In the embodiments of the present invention, the host is E. coli BL21 (DE3), BL21 (DE3) pLysS, DH5α, JM109, JM110, TOP10, HB101, or Xl1-Blue.

[0051] Furthermore, the present invention also provides a method for preparing the fusion protein as described above, which comprises culturing the host as described above to obtain a product containing the fusion protein. Preferably, the preparation method further comprises the steps of enrichment and purification.

[0052] Furthermore, the present invention also provides the use of the fusion protein A, the fusion protein B, the composition and / or the biological material as described in any of the preceding items in preparing a vaccine diluent.

[0053] Furthermore, the vaccine diluent provided by the present invention comprises: the fusion protein A as described above, the fusion protein B as described above and / or the combination as described above.

[0054] The vaccine diluent of the present invention further comprises a buffer, an aqueous phase and an oil phase.

[0055] In the present invention, the buffer is phosphate buffered saline (PBS), acetate buffer (HAc-NaAc), citrate buffer, borate buffer, barbital buffer, Tris buffer, carbonate buffer, or HEPES buffer. In some embodiments, the buffer is phosphate buffer. In a specific embodiment, the phosphate buffer is composed of water and the following components at the following concentrations: 8.0 g / L NaCl, 0.2 g / L KCl, 1.44 g / L Na2HPO4, and 0.24 g / L KH2PO4;

[0056] In the present invention, the aqueous phase includes water and may also include other excipients. In some embodiments, the aqueous phase includes water and glutamine, bezoar, glucose oxidase, vitamin C, and Tween; wherein the Tween is Tween 20, Tween 40, Tween 60, or Tween 80. In a specific embodiment, the aqueous phase is composed of water and the following components at the following concentrations: 10 g / L glutamine, 0.2 g / L bezoar, 1 g / L glucose oxidase, 5 mg / L vitamin C, and 4 wt% Tween.

[0057] In the present invention, the oil phase includes mineral oil and may further include lauric acid monoglyceride and vitamin E. In a specific embodiment, in the oil phase, the concentration of lauric acid monoglyceride is 0.5 mg / L, and the concentration of vitamin E is 0.5 mg / L.

[0058] In the present invention, the mass ratio of the buffer, aqueous phase and oil phase is (1-10):(1-10):(1-10). For example, the mass ratio of the buffer, aqueous phase and oil phase is (1-10):(1-10):1, or (1-10):(1-10):2, or (1-10):(1-10):3, or (1-10):(1-10):5, or (1-10):(1-10):6, or (1-10):(1-10):7, or (1-10):(1-10): 10):8, or (1~10): (1~10):9, or (1~10): (1~10):10, or (1~10):1: (1~10), or (1~10):2: (1~10), or (1~10):3: (1~10), or (1~10):4: (1~10), or (1~10):5: (1~10), or (1~10):6:(1~10), or (1~10):7:(1~10), or (1~10):8:(1~10), or (1~10):9:(1~10), or (1~10):10:(1~10), or 1:(1~10):(1~10), or 2:(1~10):(1~10), or 3:(1~10) :(1-10), or 4:(1-10):(1-10), or 5:(1-10):(1-10), or 6:(1-10):(1-10), or 7:(1-10):(1-10), or 8:(1-10):(1-10), or 9:(1-10):(1-10), or 10:(1-10):(1-10). In some embodiments, the mass ratio of the buffer, aqueous phase, and oil phase is 3:2:1.

[0059] In the present invention, the concentration of fusion protein A in the diluent is 1×10 -4 mg / L~2×10 -4 mg / L, the concentration of fusion protein B is 1-5 mg / L. In some embodiments, the concentration of fusion protein A in the dilution is 1.34×10 -4 mg / L, and the concentration of fusion protein B was 2.67 mg / L.

[0060] Furthermore, the present invention also provides a method for preparing the vaccine diluent as described above, comprising:

[0061] dissolving the aforementioned composition in a buffer solution to obtain a protein solution;

[0062] After mixing the oil phase with the water phase, emulsification is performed to obtain an emulsion;

[0063] The emulsion is mixed with the protein solution to prepare the vaccine diluent.

[0064] In the present invention, the vaccine diluent comprises three components: a recombinant protein composition, a buffer, and an emulsion. Once these three components are prepared, the vaccine diluent can be immediately prepared, without limitation in the present invention. In this case, the vaccine diluent has a shelf life of 12 months. Preferably, the three components of the vaccine diluent can be stored independently and then prepared immediately upon mixing with the vaccine. In this case, the shelf life of each component, and therefore the vaccine diluent, is 24 months.

[0065] In the present invention, the emulsification can be performed using a common emulsification method in the art. In an embodiment, the emulsification is performed using a high shear emulsifier. Preferably, the emulsification conditions include: high shear emulsification at 4000 rpm for 30 minutes.

[0066] Furthermore, the present invention provides the use of the vaccine diluent or the vaccine diluent prepared by the preparation method in diluting vaccines.

[0067] In the present invention, the vaccine is a live vaccine, an inactivated vaccine, a recombinant vaccine, a subunit vaccine or a nucleic acid vaccine.

[0068] In the present invention, the vaccine is a vaccine for humans, mammals, or poultry. For example, in the present invention, the vaccine is administered to mammals, including bovines, equines, ovines, porcines, canines, felines, rodents, and primates. Alternatively, the vaccine is administered to poultry, including chickens, ducks, geese, turkeys, pigeons, quails, guinea fowl, cormorants, peacocks, swans, ostriches, parrots, partridges, pelicans, cranes, grouse, mallards, pheasants, doves, and / or emus.

[0069] In the present invention, the vaccine is a vaccine against infectious diseases and / or a vaccine against tumors.

[0070] The vaccines for infectious diseases include but are not limited to avian influenza vaccine, Newcastle disease vaccine, fowl pox vaccine, infectious bursal disease vaccine, avian cholera vaccine, Marek's disease vaccine, goose parvovirus vaccine, Newcastle disease vaccine, tuberculosis vaccine, duck viral hepatitis vaccine, avian encephalomyelitis vaccine, duck plague vaccine, gosling plague vaccine, Muscovy duck parvovirus vaccine, avian mycoplasmosis vaccine, avian aspergillosis vaccine, chicken coccidiosis vaccine, turkey rhinotracheitis vaccine, infectious laryngotracheitis vaccine or avian salmonellosis vaccine.

[0071] The tumor-targeting vaccines include, but are not limited to, cervical cancer vaccines, prostate cancer vaccines, or lung cancer vaccines.

[0072] As a feasibility example, the live vaccine is a live vaccine of infectious bursal disease of chickens, a live vaccine of infectious bronchitis of chickens and / or a live vaccine of Newcastle disease of chickens;

[0073] As a feasibility case, the inactivated vaccine is an inactivated avian influenza virus vaccine and / or an inactivated Newcastle disease vaccine;

[0074] As a feasibility case, the subunit vaccine is a genetically engineered subunit vaccine of avian influenza virus and / or a genetically recombinant subunit vaccine of Newcastle disease virus.

[0075] The present invention also provides a vaccine, which includes the vaccine diluent as described above or the vaccine diluent prepared by the preparation method as described above.

[0076] In the vaccine described in the present invention, the diluent is as described above, and the immunogen is selected from any type of immunogen in the art, such as live vaccines, inactivated vaccines, recombinant proteins and / or nucleic acids, which are limited by the present invention.

[0077] The method for preparing the vaccine of the present invention comprises diluting the vaccine with the vaccine diluent as described above or the vaccine diluent prepared by the preparation method as described above.

[0078] The present invention also provides a method for preventing and treating a disease, which comprises administering the vaccine as described above.

[0079] The control of the present invention includes prevention and / or treatment. The administration method includes injection and / or oral administration.

[0080] The present invention provides two fusion proteins, the combined composition of which can effectively improve the immune effect of the vaccine, effectively prolong the duration of vaccine immunity, and provide more efficient immune protection to the animal body. At the same time, the production process of the vaccine diluent is simple and low-cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0081] Figure 1 shows the electrophoresis diagram of the pET-IL-2-NPS-WT1 plasmid, where M: DL5000 marker; 1: enzyme-digested fragment; 2: water control;

[0082] Figure 2 The WB results of protein expression of IL-2-NPS-WT1 are shown, where M: protein marker; 1: recombinant bacteria; 2: uninduced expression bacteria;

[0083] Figure 3 The electrophoresis diagram of pET-CaIFN-γ-GATA3 plasmid is shown, where M: DL5000 marker; 1: enzyme-digested fragment; 2: water control;

[0084] Figure 4 This is the WB result of protein expression of CaIFN-γ-GATA3, where M is protein marker; 1 is recombinant bacteria; 2 is uninduced expression bacteria;

[0085] Figure 5 Shows the technical roadmap of the present invention. DETAILED DESCRIPTION

[0086] The present invention provides methods and applications for preparing vaccine diluents. Those skilled in the art can refer to the contents herein and appropriately modify the process parameters to achieve the desired results. It should be noted that all similar substitutions and modifications will be apparent to those skilled in the art and are considered to be encompassed by the present invention. The methods and applications of the present invention have been described through preferred embodiments, and it is apparent that those skilled in the art can modify or appropriately alter and combine the methods and applications herein to implement and apply the technology of the present invention without departing from the content, spirit, and scope of the present invention.

[0087] Unless otherwise defined herein, scientific and technical terms related to the present invention shall have the meanings that are understood by those of ordinary skill in the art.

[0088] In this application, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single or plural items.

[0089] The terms "include," "comprising," and "having" are used interchangeably herein and are intended to indicate the inclusiveness of a solution, meaning that the solution may contain other elements in addition to the listed elements. It should also be understood that the use of "include," "comprising," and "having" in this document also provides a "consisting of" solution.

[0090] The term "and / or" as used herein includes the meanings of "and," "or," and "all or any other combination of elements linked by the associated term."

[0091] Interleukin-2 (IL-2) is a very important immunomodulatory factor with a wide range of biological activities. It is a Th1 lymphocyte factor secreted by T lymphocytes or T lymphocyte lines activated by specific antigens or mitogens. It can activate T cells and promote B cell differentiation and antibody secretion. It also induces T cells and NK cells to secrete interferon-gamma, enhances the killing activity of monocytes and natural killer (NK) cells, and improves the cellular and humoral immunity of animals. It plays a key regulatory role in the body's immune response.

[0092] The neuropeptide S (NPS) gene, a neuropeptide composed of a specific amino acid sequence, plays an important role in genetics and biological research, including its involvement in immune regulation. This study found that NPS can promote or inhibit the secretion of certain cytokines by regulating the activity or functional state of immune cells.

[0093] WT1 (Wilms' tumor gene 1) is a multifunctional gene expressed in various tissues and organs. It interacts with other genes and signaling pathways to regulate key processes such as cell proliferation, differentiation, and apoptosis. WT1 may regulate immune responses by regulating T cell differentiation and activation, affecting the expression of cytokines such as interferon (IFN) and interleukin (IL).

[0094] Interferon-gamma (Chicken interferon-gamma, ChIFN-γ) is a type II interferon. It is primarily produced by lymphocyte T cells, with a smaller portion produced by NK cells. IFN-γ itself cannot directly kill viruses. Instead, it acts on target cells, activating genes within them to synthesize proteins with antiviral properties, thus exerting its broad-spectrum antiviral activity. It also has immunomodulatory effects, primarily through promoting the interaction between antigen-presenting cells and lymphocyte T cells via major histocompatibility complex II (MHC II) molecules. It can inhibit the proliferation of tumor viruses, promote tumor cell apoptosis, and activate innate and specific immune responses, thereby exerting its anti-tumor effects.

[0095] GATA3 (GATA-binding protein 3) is an important transcription factor. It plays a key role in the development and differentiation of T lymphocytes in chickens. It promotes the differentiation of T cells into Th2 cells (helper T type 2 cells), regulates the expression of various immune-related cytokines, and indirectly modulates the expression and function of IFN-γ.

[0096] The term "identity" herein can be calculated in the following manner: to determine the "identity" percentage of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., spaces can be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal comparison, or non-homologous sequences can be discarded for comparison purposes). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, the molecules are identical at that position. The percentage of identity between the two sequences varies as the number of identical positions shared by the sequences changes, taking into account the number of spaces that need to be introduced and the length of each space for optimal comparison of the two sequences.

[0097] Mathematical algorithms can be used to compare sequences and calculate percent identity between two sequences. For example, the Needlema and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm, which has been integrated into the GAP program in the GCG software package (available at www.gcg.com), is used with a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6 to determine the percent identity between two amino acid sequences. For another example, the GAP program in the GCG software package (available at www.gcg.com) is used with a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6 to determine the percent identity between two nucleotide sequences. A particularly preferred parameter set (and the one that should be used unless otherwise stated) is the Blossum62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0098] The percent identity between two amino acid or nucleotide sequences can also be determined using the algorithm of E. Meyers and W. Miller, (1989) CABIOS, 4: 11-17), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weighted remainder table, a gap length penalty of 12, and a gap penalty of 4.

[0099] In the present invention, the identity of 80% or more means an identity greater than 85%, or greater than 90%, or greater than 95%, or greater than 96%, or greater than 97%, or greater than 98%, or greater than 98%, or greater than 99%, or greater than 99.5%, or greater than 99.8%, or greater than 99.9%.

[0100] The numerical ranges and parameters involved in this disclosure are presented as precisely as possible in the specific examples. However, any numerical value inherently and inevitably contains standard deviations resulting from individual testing methods. Therefore, unless otherwise expressly stated, it should be understood that all numerical ranges or specific data used in this disclosure are subject to reasonable deviation within a certain range, for example, within ±10%, ±5%, ±1%, or ±0.5%.

[0101] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution. Some or all of the steps can be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0102] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0103] The present invention will be further described below in conjunction with the embodiments:

[0104] Example

[0105] 1 Design and synthesis of IL-2-NPS-WT1 gene

[0106] Refer to the three gene sequences published in GenBank: chicken IL-2 gene (Genbank: AM231331.1), chicken NPS gene sequence (Genbank: XM_046920688.1) and WT1 gene sequence (Genbank: NM_205216.2):

[0107] The amino acid sequence of IL-2 is:

[0108] MCKVLIFGCISVAMLMTTAYGASLSSAKRKPLQTLIKDLEILENIKNKIHLELYTPTETQECTQQTLQCYLGEVVTLKKETEDDTEIKEEFVTAIQNIEKNLKSLTGLNHTGSECKICGANNKKKFPDFLHELTNFVRYLQK (SEQ ID NO.1)

[0109] The codon-optimized nucleic acid sequence encoding IL-2 is:

[0110] ATGTGCAAAGTTCTGATCTTCGGCTGTATTTCTGTAGCAATGCTGATGACTACCGCTTATGGTGCATCTCTGTCTTCCGCAAAAAGGAAACCGCTGCAGACCTTAATCAAGGATTTAGAAATCTTGGAAAATATCAAGAACAAGATTCATCTGGAGCTGTACACCCCAACTGAGACCCAGGAGTGCACCCAGCAAACTCTGCAGTGTTACCTGGG TGAAGTGGTTACTCTGAAGAAAGAAACTGAAGATGACACTGAAATTAAAGAAGAATTCGTAACTGCTATTCAGAACATCGAAAAGAACCTGAAGAGCCTGACGGGTCTGAACCACACCGGTAGCGAATGCAAGATCTGTGGTGCTAACAACAAGAAAAAAATTTCCGGATTTTCTGCATGAACTGACCAACTTTGTGAGATATCTGCAGAAA (SEQ ID NO.8)

[0111] The amino acid sequence of chicken NPS is:

[0112] MISLCRLNLLFILWMSAMFVCSGYPVGPSMSSNPFYLNCQLYGKSDYCLVLLNSCLAKVGRSEEVALLEPHLEMPFNKRSFRNGVGSGIKKTSFRRAKS (SEQ ID NO.2)

[0113] The codon-optimized chicken NPS nucleic acid sequence is:

[0114] ATGATCAGCCTGTGCAGGCTGAACCTGCTGTTCATCCTGTGGATGTCTGCTATGTTTGTGTGCTCTGGTTACCCAGTTGGCCCATCCATGAGCAGCAACCCGTTCTATTTGAACTGCCAGCTGTACGGTAAATCTGATTACTGCCTGGTGCTGCTGAACAGCTGCTTAGCCAAGGTGGGCAGGAGCGAAGAGGTGGCTCTGCTGGAGCCGCACCTGGAGATGCCGTTCAACAAACGTTCCTTTCGCAACGGTGTGGGATCGGGTATTAAAAAAACTTCCTTTCGTAGAGCAAAGTCG (SEQ ID NO.9)

[0115] The amino acid sequence of WT1 is:

[0116] MGSDVRDLNALLPSVPSLPGNSNCAMPVSSAAQWAPVLDFPPGASYGSLGPHSFIKQEPSWNGSDPHEEQYLSAFTVHFSGQFTGTAGACRYGPFGAPPPSQPPSGQARMFPNGPYLPNCLESQQAIRNQGYGTVAFDGTPSYGHTPSHHAAQFTNHSFKHEDPMSQQPSLGDQQYSVPPPVYGCHTPTDSCTGSQALLLRTPYNSDNLYQMTSQLECMTWNQMNLGSTLKGHTTGYENENHSAPMLYSCGAQYRIHTHGVFRGIQDVRRVPGVAPTIVRSASETNEKRPFMCAYPGCNKRYFKLSHLQMHSRKHTGEKPYQCDFKDCERRFSRSDQLKRHQRRHTGVKPFQCKTCQRKFSRSDHLKTHTRTHTGKTSEKPFSCRWPSCQKKFARSDELVRHHNMHQRNMTKLQLAL (SEQ ID NO.3)

[0117] The nucleic acid sequence encoding WT after codon optimization is

[0118]

[0119] The chicken IL-2, NPS and WT1 gene sequences were concatenated in different orders, with the linker GGGGSGGGGS (GGCGGCGGCGGCAGCGGCGGCGGCG CAGC), and the resulting fragments were:

[0120] 1) rIL-2-NPS-WT1

[0121] 2) rIL-2-WT1-NPS

[0122] 3) rNPS-IL-2-WT1

[0123] 4) rNPS-WT1-IL-2

[0124] 5) rWT1-IL-2-NPS

[0125] 6) rWT1-NPS-IL-2

[0126] also,

[0127] The chicken IL-2 gene sequence was modified by inserting CGTCGTCGTTATTAT after the start codon. The modified chicken IL-2, NPS and WT1 gene sequences were concatenated using the linker GGGGSGGGGS (GGCGGCGGCGGCAGCGGCGGCGGCAGC) to obtain the fragment rIL-2*-NPS-WT1.

[0128] Taking rIL-2*-NPS-WT1 as an example, its nucleic acid sequence is ( The underlined sequence indicates the insertion sequence. mark is a linker, with a total length of 2055 bp)

[0129] ATG cgtcgtcgttattatATGTGCAAAGTTCTGATCTTCGGCTGTATTTCTGTAGCAATGCTGATGACTACCGCTTATGGTGCATCTCTGTCTTCCGCAAAAAGGAAACCGCTGCAGACCTTAATCAAGGATTTAGAAATCTTGGAAAATATCAAGAACAAGATTCATCTGGAGCTGTACACCCCAACTGAGACCCAGGAGTGCACCCAGCAAACTCTGCAGTGTTACCTGGGTGAAGTG4GTTACTCTGAAGAAAGAAACTGAAGATGACACTGAAATTAAAGAAGAATTCGTAACTGCTATTCAGAACATCGAAAAGAACCTGAAGAGCCTGACGGGTCTGAACCACACCGGTAGCGAATGCAAGATCTGTGGTGCTAACAACAAGAAAAAATTTCCGGATTTTCTGCATGAACTGACC4AACTTTGTGAGATATCTGCAGAAA gg cggcggcggcagcggcggcggcggcagc ATGATC4AGCCTGTGCAGGCTGAACCTGCTGTTCATCCTGTGGATGTCTGCTATGTTTGTGTGCTCT4GGTTACCCAGTTGGCCCATCCATGAGCAGCAACCCGTTCTATTTGAACTGCCAGCTGTACGGTAAATCTGATTACTGCCTGGTGCTGCTGAACAGCTGCTTAGCCAAGGTGGGCAGGAGCGAAGAGGTGGCTCTGCTGGAGCCGCACCTGGAGATGCCGTTCAACAAACGTTCCTTTCGCAACGGTGTGGGATCGGGTATTAAAAAAACTTCCTTTCGTAGAGCAAAGTCG ggcggcggcggcagcggcggcggcggcagc

[0130] rIL-2*-NPS-WT1 amino acid sequence

[0131] M rrryy MCKVLIFGCISVAMLMTTAYGASLSSAKRKPLQTLIKDLEILENIKNKIHLELYTPTETQECTQQTLQCYLGEVVTLKKETEDDTEIKEEFVTAIQNIEKNLKSLTGLNHTGSECKICGANNKKKFPDFLHELTNFVRYLQK ggggsggggs MISLCRLNLLFILWMSAMFVCSGYPVGPSMSSNPFYLNCQLYGKSDYCLVLLNSCLAKVGRSEEVALLEPHLEMPFNKRSFRNGVGSGIKKTSFRRAKS ggggsggggs MGSDVRDLNALLPSVPSLPGNSNCAMPVSSAAQWAPVLDFPPGASYGSLGPHSFIKQEPSWNGSDPHEEQYLSAFTVHFSGQFTGTAGACRYGPFGAPPPSQPPSGQARMFPNGPYLPNCLESQQAIRNQGYGTVAFDGTPSYGHTPSHHAAQFTNHSFKHEDPMSQQPSLGDQQYSVPPPVYGCHTPTDSCTGSQALLLRTPYNSDNLY QMTSQLECMTWNQMNLGSTLKGHTTGYENENHSAPMLYSCGAQYRIHTHGVFRGIQDVRRVPGVAPTIVRSASETNEKRPFMCAYPGCNKRYFKLSHLQMHSRKH TGEKPYQCDFKDCERRFSRSDQLKRHQRRHTGVKPFQCKTCQRKFSRSDHLKTHTRTHTGKTSEKPFSCRWPSCQKKFARSDELVRHHNMHQRNMTKLQLAL (SEQ ID NO.4)

[0132] The NdeI restriction enzyme site CATATG and the XhoI restriction enzyme site CTCGAG were added to the 5' and 3' ends of the fragment obtained by the above ligation, respectively.

[0133] 2 Design and synthesis of CaIFN-γ-GATA3 protein gene

[0134] Reference is made to the chicken interferon gamma (CaIFN-γ) gene sequence (Genbank: NM_205149.2) and GATA3 gene sequence (Genbank: NM_001397223.1):

[0135] The amino acid sequence of chicken interferon gamma (CaIFN-γ) is:

[0136] MTCQTYNLFVLSVIMIYYGHTASSLNLVQLQDDIDKLKADFNSSHSDVADGGPIIVEKLKNWTERNEKRIILSQVSMYLEMLENTDKSKPHIKHISEELYTLKNNLPDGVKKVKDIMDLAKLPMNDLRIQRKAANELFSILQKLVDPPSFKRKRSQSQRRCNC (SEQ ID NO.5)

[0137] After codon optimization, the nucleic acid sequence encoding chicken gamma interferon Caifn-γ

[0138] ATGACTTGCCAGACTTACAACTTGTTTGTTCTGTCTGTCATCATGATTTATTATGGACATACTGCATCTTCTCTGAACCTGGTTCAGCTGCAGGATGATATCGACAAACTGAAAGCTGACTTTAACTCTAGTCATTCTGATGTAGCTGACGGTGGTCCGATTATTGTAGAGAAACTGAAAAACTGGACCGAGAGAAACGAAAAGGATCATCCTGAGCCAGATTGTTTCGATGTACCTGGAAATGCT GGAAAACACTGACAAGTCTAAGCCGCACATCAAACACATCTCTGAGGAGCTGTATACTCTGAAAAACAACCTGCCGGATGGCGTGAAGAAGGTGAAAGATATCATGGACCTGGCCAAGCTCCCG ATGAACGACCTGCGTATCCAGCGCAAAGCCGCGAACGAACTCTTCAGCATCCTGCAGAAGCTGGTGGATCCGCCGAGCTTCAAACGTAAACGTAGCCAGTCTCAGCGTCGTTGCAACTGC (SEQ ID NO.11)

[0139] The amino acid sequence of GATA3 is

[0140] MEVSTDQPRWVSHHHPAVLNGQHPDSHHPTLGHTYMDPTQYPLAEEVDVLFNIDGQGNPVPPYYGNSVRATVQRYPTAHHGSQVCRPPLLHGSLPWLDGSKALSSHHSASPWNLSPFSKTSIHHSSPGPLSVYPPASSSTLSAGHSSPHLFTFPPTPPKDVSPDPSISTPGSTGSTRQDEKECIKYQVSLADTMKLESSHSRSSMASLGGATSSAHHPITTYPPYVPEYSSGLFPPSSLLGGSPTGFGCKSRPKARSSTGRECVNCGATSTPLWRRDGTGHYLCNACGLYHKMNGQNRPLIKPKRRLSAARRAGTSCANCQTTTTTLWRRNANGDPVCNACGLYYKLHNINRPLTMKKEGIQTRNRKMSSKSKKCKKVHDNLEDFPKSSSFNPAALSRHMSSISHISPFSHSSHMLTTPTPMHPPSSLSFGPHHPSSMVTAMG (SEQ ID NO.6)

[0141] The nucleic acid sequence encoding GATA3 is

[0142]

[0143] The Califn-γ and Gata3 gene sequences were concatenated in different orders using the linker GGGGSGGGGS (GG CGGCGGCGGCAGCGGCGGCGGCG CAGC). Codon optimization was performed based on the preference of Escherichia coli. The NdeI restriction site CATATG was added to the 5' end, and the XhoI restriction site CTCGAG was added to the 3' end. The resulting fragments were recorded as:

[0144] 1) rCaIFN-γ-GATA3

[0145] 2) rGATA3-CaIFN-γ

[0146] Taking CaIFN-γ-GATA3 as an example, its gene synthesis sequence is as follows: ( Marked as linker, 1854 bp)

[0147] ATGACTTGCCAGACTTACAACTTGTTTGTTCTGTCTGTCATCATGATTTATTATGGACATACTGCATCTTCTCTGAACCTGGTTCAGCTGCAGGATGATATCGACAAACTGAAAGCTGACTTTAACTCTAGTCATTCTGATGTAGCTGACGGTGGTCCGATTATTGTAGAGAAACTGAAAAACTGGACCGAGAGAAACGAGAAAAGGATCATCCTGAGCCAGATTGTTTCGATGTACCTGGAAATG CTGGAAAACACTGACAAGTCTAAGCCGCACATCAAACACATCTCTGAGGAGCTGTATACTCTGAAAAACAACCTGCCGGATGGCGTGAAGAAGGTGAAAGATATCATGGACCTGGCCAAGCTCCCGATGAACGACCTGCGTATCCAGCGCAAAGCCGCGAACGAACTCTTCAGCATCCTGCAGAAGCTGGTGGATCCGCCGAGCTTCAAACGTAAACGTAGCCAGTCTCAGCGTCGTTGCAACTGC ggcggcggcggcagcggcggcggcggcagc

[0148] Amino acid sequence

[0149] MTCQTYNLFVLSVIMIYYGHTASSLNLVQLQDDIDKLKADFNSSHSDVADGGPIIVEKLKNWTERNEKRIILSQIVSMYLEMLENTDKSKPHIKHISEELYTLKNNLPDGVKKVKDIMDLAKLPMNDLRIQRKAANELFSILQKLVDPPSFKRKRSQSQRRCNC ggggsggggs MEVSTDQPRWVSHHHPAVLNGQHPDSHHPTLGHTYMDPTQYPLAEEVDVLFNIDGQGNPVPPYYGNSVRATVQRYPTAHGSQVCRPPLLHGSLPWLDGSKALSSHHSASP WNLSPFSKTSIHHSSPGPLSVYPPASSSTLSAGHSSPHLFTFPPTPPKDVSPDPSISTPGSTGSTRQDEKECIKYQVSLADTMKLESSHSRSSMASLGGATSSAHHPITTYP PYVPEYSSGLFPPSSLLGGSPTGFGCKSRPKARSSTGRECVNCGATSTPLWRRDGTGHYLCNACGLYHKMNGQNRPLIKPKRRLSAARRAGTSCANCQTTTTTLWRRNANGD PVCNACGLYYKLHNINRPLTMKKEGIQTRNRKMSSKSKKCKKVHDNLEDFPKSSSFNPAALSRHMSSISHISPFSHSSHMLTTPTPMHPPSSLSFGPHHPSSMVTAMG (SEQ ID NO.7)

[0150] 3 Prokaryotic expression and purification of IL-2-NPS-WT1 protein

[0151] 3.1 Construction of IL-2-NPS-WT1 fusion expression vector

[0152] (1) Enzyme digestion of target gene: The synthetic IL-2-NPS-WT1 gene was double-digested with restriction endonucleases Nde I and Xho I. The digestion system (20µl) was as follows: 1µl IL-2-NPS-WT1 gene, 1µl Nde I, 1µl Xho I, 2µl 10× Buffer, 15µl ddH2O. The reaction was performed on ice. After mixing, the digestion was incubated at 37°C for 2h. The digested fragments were separated by agarose gel electrophoresis and recovered and purified using a gel recovery kit.

[0153] (2) Enzyme digestion of vector: Select vector pET30a(+) and double-enzyme digest it with restriction endonucleases Nde I and Xho I. The enzyme digestion system (20µl): pET30a(+) 1µl, Nde I 1µl, Xho I 1µl, 10× Buffer 2µl, ddH2O 15µl. Operate on ice, mix and digest at 37℃ for 2h.

[0154] (3) Ligation of target gene and vector: The IL-2-NPS-WT1 gene digestion product and the vector pET30a(+) digestion product were ligated with T4 ligase. The ligation system (10µl) was as follows: 10× Buffer 1µl, pET30a(+) digestion product 2µl, interferon gene digestion product 2µl, T4 DNA ligase 1µl, ddH2O 4µl. The operation was performed on ice. After mixing, the ligation was carried out at 16°C overnight.

[0155] (4) Transformation: Take 10µl of the ligation product and add it to a centrifuge tube containing 100µl of Escherichia coli DH5α competent cells. Mix well and place in an ice bath for 30 minutes. Transfer to a 42℃ constant temperature water bath for heat shock for 90 seconds. Take it out and immediately place in an ice bath for 2 minutes. Add 500µl of LB liquid culture medium to each centrifuge tube and culture at 37℃, 200 r / min for 1 hour. Take 100µl and spread it on a plate containing kanamycin. Incubate it in an inverted manner at 37℃ overnight for 14 hours.

[0156] (5) Plasmid extraction: Pick a single colony from the plate and inoculate it into LB liquid medium containing kanamycin. Incubate at 37°C, 200 rpm for 12 h. Extract the plasmid according to the instructions of the plasmid extraction kit. Double-digest the recombinant plasmid with Nde I and Xho I. Agarose electrophoresis shows a pET30a(+) vector band of about 5 kb and an exogenous fragment of 2055 bp. The size is completely consistent with the expectation. The electrophoresis results are shown in Figure 1 The recombinant plasmid was named pET-IL-2-NPS-WT1.

[0157] 3.2 Fermentation Culture The pET-IL-2-NPS-WT1 plasmid was transformed into BL21 (DE3), and a single colony was picked and inoculated into an appropriate amount of LB medium containing 50 μg / ml kanamycin. The culture was shaken at 200 r / min at 37°C overnight. The bacterial liquid was inoculated into a sterile fermenter at 2% of the culture volume, and the culture was aerated at 37°C. The fermenter was controlled with a stirring speed of 500-700 r / min, dissolved oxygen of 60%-90%, and pH 7.0. After the bacteria grew to the mid-logarithmic growth period, IPTG was added to a final concentration of 1 mmol / L, and the cells were induced at 37°C for 5 h.

[0158] 3.3 After the bacterial cell culture is completed, the bacterial pellet is collected by centrifugation, washed twice with PBS, and prepared into a 10% PBS suspension. The bacteria are broken by a high-pressure homogenizer at 2-8°C. The broken bacterial suspension is centrifuged at 8000 rpm for 15 minutes to collect the inclusion bodies.

[0159] 3.4 Purification of recombinant protein The inclusion bodies were washed with a buffer containing 2M urea, washed with a magnetic stirrer for 30 minutes, centrifuged at 8000 rpm for 10 minutes at 4°C, and the supernatant was removed and washed again. 8 mol / L urea was added to dissolve and wash the inclusion bodies, ultrasonically cleaned in an ice bath for 30 minutes, and centrifuged at 8000 rpm for 20 minutes to remove the precipitate. The supernatant was the inclusion body solution. The solution was stored at 4°C overnight and renatured on an ultrafiltration refolding device. The solution was exchanged at 5-10 volumes. After renaturation, the solution was centrifuged at 8000 rpm to remove the precipitate. The supernatant was the desired protein solution. The protein solution was added to the chromatography column at a rate of 0.3 ml / min and linearly eluted with pH 7.0 phosphate buffer at a rate of 1 ml / min. The target elution peak was detected by UV detector at a wavelength of 280 nm. The target elution peak was collected and sterilized by filtration through a 0.22 μm filter. The protein content was determined to be ≥3.8 mg / ml by UV spectrophotometry.

[0160] 3.5 Western-blot Identification of Recombinant Proteins After SDS-PAGE electrophoresis, the recombinant proteins were transferred to PVDF membranes at 100V for 70min, washed twice with TBST, blocked with 1% BSA blocking solution at 37°C for 1.5h, and then washed three times with TBST. Rabbit IL-2 monoclonal antibody was used as the primary antibody, diluted 1:1000, and incubated at 4°C overnight. The membranes were washed three times with TBST, and then a 1:4000 dilution of goat anti-rabbit IgG (HRP) secondary antibody was added. The membranes were washed three times with TBS and detected with a DAB colorimetric kit. The size of the recombinant protein in the recombinant bacteria after induction was 75.3kDa, which was consistent with the size of the IL-2-NPS-WT1 fusion protein ( Figure 2 ).

[0161] 4. Prokaryotic expression and purification of CaIFN-γ-GATA3 protein

[0162] 4.1 Construction of CaIFN-γ-GATA3 vector

[0163] (1) Enzyme digestion of target gene: The synthetic CaIFN-γ-GATA3 gene was double-digested with restriction endonucleases Nde I and Xho I. The digestion system (20µl) was as follows: CaIFN-γ-GATA3 gene 1µl, Nde I 1µl, Xho I 1µl, 10× Buffer 2µl, ddH2O 15µl. The reaction was performed on ice. After mixing, the digestion was incubated at 37°C for 2h. The digested fragments were separated by agarose gel electrophoresis and recovered and purified using a gel recovery kit.

[0164] (2) Enzyme digestion of vector: Select vector pET30a(+) and double-enzyme digest it with restriction endonucleases Nde I and Xho I. The enzyme digestion system (20µl): pET30a(+) 1µl, Nde I 1µl, Xho I 1µl, 10× Buffer 2µl, ddH2O 15µl. Operate on ice, mix and digest at 37℃ for 2h.

[0165] (3) Ligation of target gene and vector: The digested product of CaIFN-γ-GATA3 gene and the digested product of vector pET30a(+) were ligated with T4 ligase. The ligation system (10µl) was as follows: 10× Buffer 1µl, pET30a(+) digested product 2µl, interferon gene digested product 2µl, T4 DNA ligase 1µl, ddH2O 4µl. The operation was performed on ice. After mixing, the ligation was carried out at 16°C overnight.

[0166] (4) Transformation: Take 10µl of the ligation product and add it to a centrifuge tube containing 100µl of Escherichia coli DH5α competent cells. Mix well and place in an ice bath for 30 minutes. Transfer to a 42℃ constant temperature water bath for heat shock for 90 seconds. Take it out and immediately place in an ice bath for 2 minutes. Add 500µl of LB liquid culture medium to each centrifuge tube and culture at 37℃, 200 r / min for 1 hour. Take 100µl and spread it on a plate containing kanamycin. Incubate it in an inverted manner at 37℃ overnight for 14 hours.

[0167] (5) Plasmid extraction: Pick a single colony from the plate and inoculate it into LB liquid medium containing kanamycin. Incubate at 37°C, 200 rpm for 12 h. Extract the plasmid according to the instructions of the plasmid extraction kit. Double-digest the recombinant plasmid with Nde I and Xho I. Agarose electrophoresis shows a pET30a(+) vector band of about 5 kb and an exogenous fragment of 1854 bp. The size is completely consistent with the expectation. The electrophoresis results are shown in Figure 3 The recombinant plasmid was named pET-CaIFN-γ-GATA3.

[0168] 4.2 Fermentation Culture BL21 (DE3) cells were transformed with the pET-CaIFN-γ-GATA3 plasmid, and a single colony was picked and inoculated into an appropriate amount of LB medium containing 50 µg / ml kanamycin. The culture was shaken at 200 rpm at 37°C overnight. The bacterial suspension was inoculated into a sterile fermenter at 2% of the culture volume and cultured at 37°C with aeration. The fermenter was controlled with an agitation speed of 500-700 rpm, a dissolved oxygen level of 60%-90%, and a pH of 7.0. After the cells reached the mid-logarithmic growth phase, IPTG was added to a final concentration of 1 mmol / L and induced at 37°C for 5 h.

[0169] 4.3 After the bacterial cell culture is completed, the bacterial pellet is collected by centrifugation, washed twice with PBS, and prepared into a 10% PBS suspension. The bacteria are then broken using a high-pressure homogenizer at 2-8°C. The broken bacterial suspension is centrifuged at 8000 rpm for 15 min to collect the inclusion bodies.

[0170] 4.4 Purification of recombinant protein: Inclusion bodies were washed with a buffer containing 2M urea, washed with a magnetic stirrer for 30 min, centrifuged at 8000 rpm for 10 min at 4°C, and the supernatant was removed and washed again. 8 mol / L urea was added to dissolve and wash the inclusion bodies, ultrasonically cleaned in an ice bath for 30 min, and centrifuged at 8000 rpm for 20 min to remove the precipitate. The supernatant was the inclusion body solution. The solution was stored at 4°C overnight and renatured on an ultrafiltration refolding device. The solution was exchanged at 5-10 volumes. After renaturation, the solution was centrifuged at 8000 rpm to remove the precipitate. The supernatant was the desired protein solution. The protein solution was added to the chromatography column at a rate of 0.3 ml / min and linearly eluted with pH 7.0 phosphate buffer at a rate of 1 ml / min. The target elution peak was detected by UV detection at a wavelength of 280 nm. The target elution peak was collected and sterilized by filtration through a 0.22 μm filter. The protein content was determined to be ≥3.8 mg / ml by UV spectrophotometry.

[0171] 4.5 Western-blot Identification of Recombinant Proteins After SDS-PAGE electrophoresis, the recombinant proteins were transferred to PVDF membranes at 100V for 70min, washed twice with TBST, blocked with 1% BSA blocking solution at 37°C for 1.5h, and then washed three times with TBST. Rabbit anti-CaIFN-γ was used as the primary antibody, diluted 1:1000, and incubated at 4°C overnight. The membranes were washed three times with TBST, and then goat anti-rabbit IgG-HRP secondary antibody diluted 1:4000 was added. The membranes were washed three times with TBS and detected with a DAB colorimetric kit. The size of the recombinant protein in the recombinant bacteria after induction was 67.8kd, which was consistent with the expected protein size ( Figure 4 ).

[0172] 5. Protein biological activity assay

[0173] 5.1 rIL-2-NPS-WT1 protein bioactivity assay

[0174] The bioactivity of the recombinant proteins obtained by combining IL-2 with NPS and WT1 in different combinations and concatenations was assayed. The effects of linker 1 (GGGGSGGGGS (GG CGGCGGCGGCAGCGGCGGCGGCAGC)) and linker 2 (AGAGAG (GCCGGTGCCGGTGCCGGT)) on the biological activity of the fusion proteins were also compared.

[0175] The national standard for the biological activity assay of recombinant human interleukin-2 was used as the reference substance. Under sterile conditions, the reference substance, rIL-2-NPS-WT1, rIL-2*-NPS-WT1, rIL-2-WT1-NPS, rNPS-IL-2-WT1, rNPS-WT1-IL-2, rWT1-IL-2-NPS, rWT1-NPS-IL-2, rIL-2-NPS, rNPS-IL-2, rIL-2-WT1, rWT1-IL-2, and rIL-2 protein prepared using linker1 and rIL-2-NPS-WT1, rIL-2*-NPS-WT1, rIL-2-WT1-NPS, rNPS-IL-2-WT1, rNPS-WT1-IL-2, rWT1-IL-2-NPS , rWT1-NPS-IL-2, rIL-2-NPS, rNPS-IL-2, rIL-2-WT1, rWT1-IL-2, and rIL-2 proteins were diluted with basal culture medium to contain approximately 200 IU per ml. Two-fold serial dilutions were made in a 96-well cell culture plate, for a total of 10 dilutions, with 2 wells for each dilution.

[0176] CTLL-2 cells were cultured in complete culture medium at 37°C and 5% CO2 until sufficient cells were obtained. CTLL-2 cells were collected by centrifugation, washed three times with RPMI 1640 culture medium, and then resuspended in basal culture medium to prepare a solution containing 6.0×10 5A cell suspension of 100 cells was prepared and stored at 37°C, 5% CO2. 50 µl of the cell suspension was added to each well of a 96-well cell culture plate containing the standard solution and the test solution. The plates were incubated at 37°C, 5% CO2 for 18-24 hours. Subsequently, 20 µl of MTT solution was added to each well. The plates were incubated at 37°C, 5% CO2 for 4-6 hours. After that, 150 µl of lysis buffer was added to each well. The plates were incubated at 37°C, 5% CO2 for 18-24 hours. All procedures were performed under sterile conditions. The plate was mixed thoroughly and the absorbance was measured at 570 nm using a reference wavelength of 630 nm in a microplate reader. The results were recorded. The biological activity assay results are shown in Tables 1 and 2.

[0177] Table 1 Biological activity assay results (link 1)

[0178]

[0179] Note: * indicates that this IL-2 protein has not been modified and does not contain the CGTCGTCGTTATTAT insertion sequence.

[0180] Table 2 Biological activity assay results (link 2)

[0181]

[0182] Note: * indicates that this IL-2 protein has not been modified and does not contain the CGTCGTCGTTATTAT insertion sequence.

[0183] The biological activity of the rIL-2-NPS-WT1 protein of the present invention is significantly higher than that of rIL-2*-NPS-WT1, rIL-2-WT1-NPS, rNPS-IL-2-WT1, rNPS-WT1-IL-2, rWT1-IL-2-NPS, rWT1-NPS-IL-2, rIL-2-NPS, rNPS-IL-2, rIL-2-WT1, rWT1-IL-2, and rIL-2 proteins. In the study, by comparing linker 1 and linker 2, it was found that the biological activity of the fusion protein was better when linker 1 was used, and linker 1 was selected as the linker of the present invention.

[0184] The experiment determined that the optimal combination was to use linker1 (GGGGSGGGGS (GGCGGCGGCGGCAGCGGCGG CGGCGGCAGC)), the tandem mode was rIL-2-NPS-WT1, and the combination effect was best when CGTCGTCGTTATTAT was inserted after the start codon of the chicken IL-2 gene sequence.

[0185] 5.2 Determination of biological activity of rCaIFN-γ-GATA3 protein

[0186] The biological activity of recombinant proteins obtained by various tandem conjugation strategies of CaIFN-γ and GATA3 was assayed. The effects of linker 1 (GGGGSGGGGS (GG CGGCGGCGGCAGCGGCGGCGGCG CAGC)) and linker 2 (AGAGAG (GCCGGTGCCGGTGCCGGT)) on the biological activity of the fusion proteins were also compared. Under sterile conditions, a control interferon standard, rCaIFN-γ-GATA3, rGATA3-CaIFN-γ, and rCaIFN-γ prepared using linker 1, and rCaIFN-γ-GATA3, rGATA3-CaIFN-γ, and rCaIFN-γ prepared using linker 2 were diluted in assay medium to 1000 units per ml and serially diluted four-fold in 96-well cell culture plates for a total of 10 dilutions, with two wells plated for each dilution. CEF cells were grown adherently in culture medium, passaged at a ratio of 1:2 to 1:4, and maintained in complete medium. Take the cultured cells and discard the culture medium. Wash them twice with PBS, digest and collect the cells, and prepare a complete culture medium containing 2.5×10 5 ~3.5×10 5 The cell suspension of 100 cells was inoculated into a 96-well cell culture plate, 100 μl per well, and incubated at 37°C, 5% CO2 for 4-6 hours. The diluted interferon product to be tested and the reference interferon product solution were transferred to the culture plate inoculated with CEF cells, 100 μl per well, and incubated at 37°C, 5% CO2 for 24 hours. The supernatant in the cell culture plate was discarded, and the stored vesicular stomatitis virus (VSV, stored at -70°C) was diluted to 1000 TCID with the challenge culture medium. 50 / ml, 100µl per well, incubate at 37℃, 5% CO2 for 24h (50% of the lesion points of the test interferon product solution are in IU / ml under microscopic examination). When CPE in the interferon-protected wells no longer progresses, the results can be observed.

[0187] The cell plate cover was opened, the liquid in each well was discarded and placed in disinfectant, 1 to 2 drops of crystal violet dye were added to each well, and after standing at room temperature for 30 minutes, the residual dye in the well was rinsed with a thin stream of water, and the residual water was aspirated to record the results. The results of the biological activity assay are shown in Tables 3 and 4.

[0188] Table 3 Biological activity assay results (linker1)

[0189]

[0190] Table 4 Biological activity assay results (linker2)

[0191]

[0192] The biological activity of the rCaIFN-γ-GATA3 of the present invention is significantly higher than that of rCaIFN-γ. In the study, by comparing linker 1 and linker 2, it was found that the biological activity of the fusion protein was better when linker 1 was used, and linker 1 was selected as the linker of the present invention.

[0193] The experiment determined that the optimal combination was to use linker1 (GGGGSGGGGS (GGCGGCGGCGGCAGCGGCGG CGGCGGCAGC)), and the combination effect was best when the series connection was rCaIFN-γ-GATA3.

[0194] 6 Determination of half-life of recombinant protein in chickens

[0195] 6.1 Determination of the half-life of rIL-2-NPS-WT1 protein in chickens

[0196] The in vivo half-life of recombinant proteins obtained by combining IL-2 with NPS and WT1 in different combinations and concatenations was measured in chickens. The effects of linker 1 (GGGGSGGGGS (GGCGGCGGCGGCAGCGGCGGCGGCAGC)) and linker 2 (AGAGAG (GCCGGTGCCGGTGCCGGT)) on the in vivo half-life of the fusion protein were also compared. rIL-2-NPS-WT1, rIL-2*-NPS-WT1, rIL-2-WT1-NPS, rNPS-IL-2-WT1, rNPS-WT1-IL-2, rWT1-IL-2-NPS, rWT1-NPS-IL-2, rIL-2-NPS, rNPS-IL-2, rIL-2-WT1, rWT1-IL-2, and rIL-2 proteins prepared using linker1 and rIL-2-NPS-WT1, rIL-2*-NPS-WT1, rIL-2-WT1-NPS, rNPS-IL-2-WT1, rNPS-WT1-IL-2, rWT1-IL-2-NPS prepared using linker2 , rWT1-NPS-IL-2, rIL-2-NPS, rNPS-IL-2, rIL-2-WT1, rWT1-IL-2, and rIL-2 proteins were tested simultaneously. Three- to four-week-old SPF chickens were divided into 24 groups of 10 chickens each. Each group received an intramuscular injection of 24 proteins at a dose of 100,000 units per kg body weight. Blood samples were collected before injection and at 1, 2, 4, 6, 8, 12, 48, 72, 96, 120, 144, 168, and 192 hours after injection. The IL-2 concentration in the blood was determined by HPLC. The results are shown in Tables 5 and 6.

[0197] Table 5 Results of serum IL-2 concentration determination (ng / ml) (linker 1)

[0198]

[0199] Note: (1) * indicates that this IL-2 protein has not been modified and does not contain the CGTCGTCGTTATTAT insertion sequence.

[0200] The IL-2 concentrations shown in the table are the arithmetic mean values ​​of 10 chickens.

[0201] Table 6 Results of serum IL-2 concentration determination (ng / ml) (linker 2)

[0202]

[0203] Note: (1) * indicates that this IL-2 protein has not been modified and does not contain the CGTCGTCGTTATTAT insertion sequence.

[0204] The IL-2 concentrations shown in the table are the arithmetic mean values ​​of 10 chickens.

[0205] Results: The half-life of the rIL-2-NPS-WT1 protein of the present invention was significantly higher than that of rIL-2*-NPS-WT1, rIL-2-WT1-NPS, rNPS-IL-2-WT1, rNPS-WT1-IL-2, rWT1-IL-2-NPS, rWT1-NPS-IL-2, rIL-2-NPS, rNPS-IL-2, rIL-2-WT1, rWT1-IL-2, and rIL-2. By comparing linker1 and linker2, the study found that the two linkers had no significant effect on the half-life of the fusion protein in chickens. By 192 hours after injection, the concentration of IL-2 in the serum was 39 and 32 ng / ml, while the concentration of IL-2 in the serum of the rIL-2 without any modification had dropped to 2 and 3 ng / ml 12 hours after injection, and was completely undetectable by 48 hours.

[0206] Comprehensive studies comparing linker 1 and linker 2 on the biological activity of the fusion protein and experimental half-life in chickens revealed that linker 1 was selected as the linker of the present invention. The experiments determined that the optimal combination was linker 1 (GGGGSGGGGS (GGCGGCGGCGGCAGCGGCGG CGGCGG CAGC)) in a tandem configuration of rIL-2-NPS-WT1, with CGTCGTCGTTATTAT inserted after the start codon of the chicken IL-2 gene sequence.

[0207] 6.2 Determination of the half-life of rCaIFN-γ-GATA3 protein in chickens

[0208] The in vivo half-life of recombinant proteins obtained by tandemly linking Califn-γ and GATA3 in different ways was determined in chickens. The effects of linker 1 (GGGGSGGGGS (GG CGGCGGCGGCAGCGGCGGCGGCG CAGC)) and linker 2 (AGAGAG (GCCGGTGCCGGTGCCGGT)) on the in vivo half-life of the fusion protein were also compared. The rCaIFN-γ-GATA3, rGATA3-CaIFN-γ, and rCaIFN-γ proteins prepared using linker 1 of the present invention and rCaIFN-γ-GATA3, rGATA3-CaIFN-γ, and rCaIFN-γ proteins prepared using linker 2 were simultaneously tested for half-life. 3-4 week old SPF chickens were divided into 6 groups of 10 chickens each, and the 6 groups of proteins were injected intramuscularly at a dose of 100,000 units per kg body weight. Blood was collected before and 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 18 hours, 24 hours, 30 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, and 96 hours after injection, and the concentration of recombinant interferon in the blood was determined by HPLC. The results are shown in Tables 7 and 8.

[0209] Table 7 Results of interferon concentration determination in serum (ng / ml) (linker 1)

[0210]

[0211] Note: The interferon concentrations shown in the table are the arithmetic mean of 10 chickens.

[0212] Table 8 Results of interferon concentration determination in serum (ng / ml) (linker 2)

[0213]

[0214] Note: The interferon concentrations shown in the table are the arithmetic mean of 10 chickens.

[0215] Results: By comparing linker1 and linker2, the study found that the two linkers had no significant effect on the half-life of the fusion protein in chickens. The serum interferon concentrations of rCaIFN-γ-GATA3 protein, rGATA3-CaIFN-γ and rCaIFN-γ protein were basically the same at 1 hour and 2 hours after injection. However, with the extension of the detection time after injection, the interferon concentration in the serum of rCaIFN-γ-GATA3 gradually increased, reaching 102 and 100 ng / ml in the serum 12 hours after injection. During the same period, the interferon concentration in the serum of the rGATA3-CaIFN-γ injection group had dropped to 41 and 37 ng / ml, and the interferon concentration in the serum of the rCaIFN-γ injection group had dropped to 5 and 3 ng / ml. At 96 hours after injection, interferon was still detectable in the serum of the rCaIFN-γ-GATA3 injection group, indicating a significant extension of the half-life of rCaIFN-γ-GATA3 protein.

[0216] Comprehensive studies comparing linker 1 and linker 2 on the biological activity of the fusion protein and the in vivo half-life in chickens revealed that linker 1 was selected as the linker of the present invention. The experiments determined that the optimal combination was linker 1 (GGGGSGGGGS (GGCGGCGGCGGCAGCGGCGG CGGCGG CAGC)), with the rCaIFN-γ-GATA3 tandem structure providing the best results.

[0217] 7 Preparation of vaccine diluent ( Figure 5 )

[0218] X powder: rIL-2*-NPS-WT1 and rCaIFN-γ-GATA3 protein powder were mixed at a mass ratio of 1:20000.

[0219] Solution Y is phosphate buffer: 8.0 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4, 0.24 g KH2PO4, dissolved in 800 mL distilled water, adjusted to 7.4 with HCl, and finally added distilled water to 1 L.

[0220] Z Liquid

[0221] Aqueous phase: Add 10g of glutamine, 0.2g of artificial bezoar, 1g of glucose oxidase, and 5mg of vitamin C per liter of water. After the above substances are completely dissolved, take 94 parts and add 4 parts of Tween-80. Filter and sterilize and set aside.

[0222] Oil phase: Sterilize the mineral oil at 121°C and 100 kPa, then add 0.5 mg of lauric acid monoglyceride and 0.5 mg of vitamin E per liter for later use.

[0223] Emulsification: Add water phase to the emulsion tank, slowly add oil phase into the water phase and stir, the ratio of oil phase to water phase is 1:2, after the oil phase is added, use high shear emulsifier 4000r / min emulsification for 30min to obtain Z liquid.

[0224] When using the diluent, dissolve powder X in liquid Y (add 5.35 mg of powder X per liter of liquid Y). After dissolution, mix thoroughly with liquid Z in a 1:1 volume ratio.

[0225] 8 Determination of half-life in vaccine dilution fluid

[0226] When using the diluent, dissolve powder X in solution Y. After dissolution, mix thoroughly with solution Z in a 1:1 volume ratio. Ten 3- to 4-week-old SPF chickens were injected intramuscularly with 0.3 ml of the vaccine diluent per chicken. Blood was collected before injection and at 1, 2, 4, 6, 8, 12, 48, 72, 96, 120, 144, 168, 192, and 216 hours after injection. IL-2 and CaIFN-γ concentrations in the blood were determined by HPLC. The results are shown in Table 9.

[0227] Table 9 Results of serum interleukin and interferon concentration determination (ng / ml)

[0228]

[0229] 9 Application of vaccine diluent

[0230] The diluent of the present invention can be used as a diluent for live vaccines, inactivated vaccines, and genetically engineered vaccines such as subunit vaccines and nucleic acid vaccines. It can effectively enhance the immune function of animals and effectively improve the immune efficacy of conventional vaccines and prolong the duration of immunity.

[0231] 9.1 Application of dilutions of live seedlings

[0232] 9.1.1 Application of Vaccine Diluent for Live Infectious Bursal Disease Vaccine

[0233] 9.1.1.1 Application method

[0234] According to the instructions for the live Infectious Bursal Disease (freeze-dried) vaccine, the live Infectious Bursal Disease (IBD) vaccine of the present invention was diluted with a vaccine diluent and sterile saline solution, respectively. Sixty 10-day-old SPF chickens were selected: a first group of 20 chickens received the vaccine diluent, and a second group of 20 chickens received the saline solution dilution. Each chicken received 0.3 ml of the vaccine by eyedrop or oral administration. A blank control group of 20 chickens was selected and kept in isolation. Blood was collected before immunization and at 7, 14, and 21 days, as well as at 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, and 7 months after immunization. Serum neutralizing antibody titers were determined, and antibody changes were statistically analyzed. At 6 and 7 months, 10 chickens were challenged with an IBD virus strain, each receiving at least 10 MID of virulent virus by eyedrop. All chickens were culled and dissected 72 hours later to analyze and analyze changes in the IBD virus.

[0235] 9.1.1.2 Results

[0236] Antibody levels in the immunized chickens gradually increased after immunization. Antibody production in the vaccine diluent group was earlier than in the saline group. Antibody levels in the vaccine diluent group were higher than those in the saline group over the same period. Antibody test results are shown in Table 10.

[0237] Table 10 Neutralizing antibody test results for each chick test group

[0238]

[0239] Note: The data in the table are the arithmetic mean of 20 chickens.

[0240] When challenged with the virus 6 months after immunization, the chickens in the control group showed mental lethargy, ruffled feathers, slow movements, and white loose stools, and 12 / 20 died; no clinical symptoms were shown in the chickens in the vaccine diluent immunization group; two chickens in the saline dilution immunization group showed mental lethargy and white loose stools; the chickens in each group were autopsied to observe the bursal lesions. The results showed that the bursal protection rate of the chickens in the vaccine diluent immunization group was 20 / 20; the bursal protection rate of the chickens in the saline dilution immunization group was 18 / 20; the bursal lesions of the chickens in the control group were all positive. Seven months after immunization, when challenged with the virus, the control group exhibited lethargy, ruffled feathers, sluggish movements, and loose white stools, with 20 / 20 chickens dying. Two chickens in the vaccine diluent-immunized group exhibited lethargy and loose white stools, while 12 chickens in the saline-immunized group exhibited lethargy, ruffled feathers, sluggish movements, and loose white stools, with 8 dying. Necropsy of the chickens in each group revealed bursal lesions. The results showed that 18 / 20 chickens in the vaccine diluent-immunized group and 8 / 20 chickens in the saline-immunized group had bursal protection. All chickens in the control group showed positive bursal lesions. The challenge results showed that the peak antibody period in the vaccine diluent-immunized group lasted longer than in the saline-immunized group, and the duration of immunity was also longer. The challenge statistical results are shown in Table 11.

[0241] Table 11 Statistical results of immune protection in each experimental group

[0242]

[0243] 9.1.2 Application of vaccine diluent for live infectious bronchitis vaccine

[0244] 9.1.2.1 Application method

[0245] According to the instructions for the avian infectious bronchitis live vaccine (freeze-dried), the vaccine was diluted with the vaccine diluent of the present invention and sterile saline, respectively. Sixty 21-day-old SPF chickens were selected: 20 chickens in the vaccine diluent group (Group 1) and 20 chickens in the saline dilution group (Group 2). Each chicken received 0.3 ml of the vaccine intratracheally. A separate control group of 20 chickens remained unvaccinated and were kept in isolation. Blood was collected before immunization and at 7, 14, 21 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, and 7 months after immunization. Serum HI antibody titers were measured, and antibody changes were statistically analyzed. At 6 and 7 months, 10 chickens were challenged with the infectious bronchitis virus strain, with 1 to 2 drops administered intranasally. The chickens were observed for 10 consecutive days, and mortality in each experimental group was statistically analyzed.

[0246] 9.1.2.2 Results

[0247] Neutralizing antibodies gradually increased after immunization, with those in the vaccine diluent group consistently higher than those in the saline diluent group over the same period, and the peak duration of the antibody response was prolonged. Antibody titers reached as high as 1:31.56 in the vaccine diluent group. Antibody test results for each group are shown in Table 12.

[0248] Table 12 HI antibody test results of each chick test group

[0249]

[0250] Note: The data in the table are the arithmetic mean of 20 chickens.

[0251] Six months after immunization, when challenged with the virus, 20 / 20 chickens in the control group died; 20 / 20 chickens in the vaccine diluent-immunized group were protected against infectious bronchitis; and 16 / 20 chickens in the saline-immunized group were protected against bursal disease. Seven months after immunization, when challenged with the virus, 20 / 20 chickens in the control group died; 16 / 20 chickens in the vaccine diluent-immunized group were protected against infectious bronchitis; and 4 / 20 chickens in the saline-immunized group were protected against bursal disease. The challenge results showed that the peak antibody period and duration of immunity were longer in the vaccine diluent-immunized group than in the saline-immunized group. The challenge statistical results are shown in Table 13.

[0252] Table 13 Statistical results of immune protection in each experimental group

[0253]

[0254] 9.1.3 Application of vaccine diluent for Newcastle disease live vaccine

[0255] 9.1.3.1 Application method

[0256] According to the instructions of the live Newcastle disease vaccine (freeze-dried type), the vaccine was diluted with the vaccine diluent of the present invention and sterile saline respectively; 60 SPF chickens aged 30 to 60 days were selected, 20 chickens were in the first vaccine diluent group; 20 chickens were in the second saline dilution group; each chicken was inoculated with 0.3 ml of vaccine by nasal drops, and another 20 chickens were not inoculated as blank control groups, and they were isolated and raised respectively. Blood was collected before immunization and 7 days, 14 days, 21 days, 1 month, 2 months, 3 months, 4 months, and 5 months after immunization to determine the serum HI antibody titer, and the antibody change pattern was counted and analyzed. At 4 months and 5 months, 10 chickens were challenged with the virulent Beijing strain of Newcastle disease virus (CVCC AV1611) and each chicken was injected intramuscularly with 0.5 ml (10 4.0 ELD 50) , observed for 14 days, and the mortality of chickens in each experimental group was counted.

[0257] 9.1.3.2 Results

[0258] After immunization, the HI antibody levels in the chickens gradually increased. The antibody levels in the vaccine diluent group were higher than those in the saline diluent group over the same period, and the peak antibody levels lasted longer. The antibody test results for each group are shown in Table 14.

[0259] Table 14 HI antibody test results of each chick test group

[0260]

[0261] Note: The data in the table are the arithmetic mean of 20 chickens.

[0262] Four months after immunization, during the 14-day observation period, 20 / 20 of the control group chickens died after developing symptoms such as depression, loss of appetite, purple combs, nodding like rice pecking, motor incoordination, and loose yellow-green and yellow-white feces. All chickens in the vaccine diluent-immunized group survived, with a protection rate of 20 / 20. Four chickens in the saline diluent-immunized group died, while the remaining chickens survived, with a protection rate of 16 / 20. Five months after immunization, during the 14-day observation period, 20 / 20 of the control group chickens died after developing symptoms such as depression, loss of appetite, purple combs, nodding like rice pecking, motor incoordination, and loose yellow-green and yellow-white feces. Two chickens in the vaccine diluent-immunized group died, with a protection rate of 18 / 20. Twelve chickens in the saline diluent-immunized group died, with a protection rate of 8 / 20. The challenge results showed that the peak antibody period and the duration of immunity were longer in the vaccine diluent-immunized group than in the saline-immunized group. The challenge statistical results are shown in Table 15.

[0263] Table 15 Statistical results of immune protection in each experimental group

[0264]

[0265] 9.2 Application of inactivated vaccine dilutions

[0266] 9.2.1 Application of vaccine diluents in inactivated avian influenza virus vaccines

[0267] 9.2.1.1 Application method

[0268] The inactivated avian influenza virus vaccine was diluted 1:1 using the vaccine diluent of the present invention and sterile physiological saline respectively; 60 SPF chicks aged 3 to 4 weeks were selected, 20 chicks were in the first vaccine diluent group; 20 chicks were in the second physiological saline dilution group; the diluted vaccine was inoculated twice according to the vaccine instructions, 0.6 ml per chick. Another 20 chicks were selected as blank control groups and kept in isolation. 14 days after the first immunization, a booster immunization was performed once using the same method, and blood was collected before the second immunization, 7 days, 14 days, 21 days, 1 month, 2 months, 3 months, 4 months, 5 months, and 6 months after the second immunization to determine the serum neutralizing antibody titer, and the antibody change pattern was counted and analyzed. Ten chicks were attacked with the avian influenza virus strain at 5 months and 6 months respectively, and each chick was intravenously injected with 0.1 ml (containing 10 8.0 ELID 50 ), 5 days after infection, cloacal swabs were collected from each chicken and inoculated into 9- to 11-day-old SPF chicken embryos for virus isolation, and the positive rate of virus isolation was calculated.

[0269] 9.2.1.2 Results

[0270] Antibody levels in the immunized chickens gradually increased after immunization. Antibody production in the vaccine diluent group was earlier than in the saline group. Antibody levels in the diluent groups were higher than those in the saline group over the same period. Antibody test results are shown in Table 16.

[0271] Table 16 HI antibody test results of each chick test group

[0272]

[0273] Note: The data in the table are the arithmetic mean of 20 chickens.

[0274] Five months after immunization, when challenged with the virus, 20 / 20 isolates of the blank control group were positive for avian influenza virus, 0 / 20 isolates of the vaccine diluent group were positive, and 2 / 20 isolates of the saline dilution group were positive. Six months after immunization, when challenged with the virus, 20 / 20 isolates of the blank control group were positive for avian influenza virus, 4 / 20 isolates of the vaccine diluent group were positive, and 14 / 20 isolates of the saline dilution group were positive. Specific antibody test results are shown in Table 17.

[0275] Table 17 Statistical results of immune protection in each experimental group

[0276]

[0277] 9.2.2 Application of vaccine diluent for inactivated Newcastle disease virus vaccine

[0278] 9.2.2.1 Application method

[0279] The inactivated Newcastle disease vaccine was diluted 1:1 using the vaccine diluent of the present invention and sterile physiological saline respectively; 60 SPF chicks aged 3 to 4 weeks were selected, 20 chicks were in the first vaccine diluent group, and 20 chicks were in the second physiological saline dilution group; the diluted vaccine was inoculated twice according to the vaccine instructions. Each chick was injected intramuscularly with 2 / 10 dose, and another 20 chicks were not vaccinated as blank control groups, and they were isolated and raised separately. 14 days after the first vaccination, a booster immunization was performed once using the same method, and blood was collected before the second immunization, 7 days, 14 days, 21 days, 1 month, 2 months, 3 months, 4 months, 5 months, and 6 months after the second immunization to determine the serum HI antibody titer, and the antibody change pattern was counted and analyzed. At 4 months and 5 months, the Newcastle disease virus strain was used to attack the virus, and 0.5 ml (10 4.0 ELD 50 ), and observe for 14 days, and count the deaths of chickens in each experimental group.

[0280] 9.2.2.2 Results

[0281] After immunization, HI antibody levels in the chickens gradually increased. The antibody levels in the vaccine diluent group were higher than those in the saline dilution group over the same period, and the peak duration of the antibody level was longer. The antibody test results for each group of chickens are shown in Table 18.

[0282] Table 18 HI antibody test results of each chick test group

[0283]

[0284] Note: The data in the table are the arithmetic mean of 20 chickens.

[0285] During the challenge four months after immunization, 20 / 20 chickens in the control group died after 14 days of observation. All chickens in the vaccine diluent-immunized group survived, with a protection rate of 20 / 20. Two chickens in the saline diluent-immunized group died, with a protection rate of 18 / 20. During the challenge five months after immunization, 20 / 20 chickens in the control group died after 14 days of observation. Two chickens in the vaccine diluent-immunized group died, with a protection rate of 18 / 20. Sixteen chickens in the saline diluent-immunized group died, while the remaining chickens survived, with a protection rate of 4 / 20. The challenge results showed that the peak antibody period in the vaccine diluent-immunized group lasted longer than that in the saline-immunized group, and the duration of immunity was also longer. The challenge statistical results are shown in Table 19.

[0286] Table 19 Statistical results of immune protection in each experimental group

[0287]

[0288] 9.3 Use of Subunit Vaccine Dilutions

[0289] 9.3.1 Application of Vaccine Diluent in Genetically Engineered Avian Influenza Virus Subunit Vaccines

[0290] 9.3.1.1 Application method

[0291] The genetically engineered subunit vaccine of avian influenza virus was diluted 1:1 using the vaccine diluent of the present invention and sterilized physiological saline respectively; 60 SPF chicks aged 21 to 35 days were selected, 20 chicks were in the first vaccine diluent group; 20 chicks were in the second physiological saline dilution group; the diluted vaccine was inoculated twice according to the vaccine instructions, and 0.6 ml was injected intramuscularly for each chick. Another 20 chicks were selected as blank control groups and isolated and raised separately. 14 days after the first vaccination, a booster vaccination was performed using the same method and dosage. Blood was collected before the second vaccination, 7 days, 14 days, 21 days, 1 month, 2 months, 3 months, 4 months, and 5 months after the second vaccination to determine the serum HI antibody titer, and the antibody change pattern was counted and analyzed. At 4 and 5 months, the avian influenza virus strain was used to attack the virus, and each chick was intravenously injected with 0.5 ml (containing 10 5.0 ELID 50), 5 days after infection, cloacal swabs were collected from each chicken and inoculated into 9- to 11-day-old SPF chicken embryos for virus isolation, and the positive rate of virus isolation was calculated.

[0292] 9.3.1.2 Results

[0293] Antibody levels in the immunized chickens gradually increased after immunization. Antibody production in the vaccine diluent group began earlier than in the saline group. Antibody levels in both diluent groups were higher than those in the saline group over the same period. Antibody test results are shown in Table 20.

[0294] Table 20 HI antibody test results of each chick test group

[0295]

[0296] Note: The data in the table are the arithmetic mean of 20 chickens.

[0297] When challenged 4 months after immunization, 20 / 20 isolates of avian influenza virus were positive in the blank control group, 0 / 20 in the vaccine diluent group, and 4 / 20 in the saline dilution group. When challenged 5 months after immunization, 20 / 20 isolates of avian influenza virus were positive in the blank control group, 2 / 20 in the vaccine diluent group, and 18 / 20 in the saline dilution group. Antibody test results are shown in Table 21.

[0298] Table 21 Statistical results of immune protection in each experimental group

[0299]

[0300] 9.3.2 Application of vaccine diluents in Newcastle disease virus recombinant subunit vaccines

[0301] 9.3.2.1 Application method

[0302] The vaccine diluent of the present invention and sterilized physiological saline were used to dilute the Newcastle disease virus gene recombinant subunit vaccine in a 1:1 ratio; 60 SPF chickens aged 4 to 5 weeks were selected, 20 chickens were in the first vaccine diluent group, and 20 chickens were in the second physiological saline dilution group; the diluted vaccine was inoculated twice according to the vaccine instructions. Each chicken was injected intramuscularly with 0.6 ml, and another 20 chickens were selected as blank control groups without vaccination, and they were isolated and raised separately. 14 days after the first vaccination, a booster immunization was performed once with the same method and dosage, and blood was collected before the second immunization, 7 days, 14 days, 21 days, 1 month, 2 months, 3 months, 4 months, and 5 months after the second immunization to determine the serum HI antibody titer, and the antibody change pattern was counted and analyzed. At 4 months and 5 months, 10 chickens were attacked with the Newcastle disease virus strain, and each chicken was injected intramuscularly with 0.5 ml (10 5.0 ELD 50), 5 days after the infection, collect cloacal swabs from each chicken and inoculate them into 9-11 day old SPF chicken embryos for virus isolation, and calculate the virus isolation positive rate. For samples with negative virus isolation, the samples should be blindly propagated for one generation and then judged again.

[0303] 9.3.2.2 Results

[0304] After immunization, HI antibody levels in the chickens gradually increased. The antibody levels in the vaccine diluent group were higher than those in the saline diluent group over the same period, and the peak duration of the antibody level was longer. The antibody test results for each group of chickens are shown in Table 22.

[0305] Table 22 HI antibody test results of each chick test group

[0306]

[0307] Note: The data in the table are the arithmetic mean of 20 chickens.

[0308] When challenged 4 months after immunization, 20 / 20 isolates of the blank control group were positive for avian influenza virus, 0 / 20 were positive for avian influenza virus in the vaccine diluent group, and 6 / 20 were positive for avian influenza virus in the saline dilution group. When challenged 5 months after immunization, 20 / 20 isolates of the blank control group were positive for avian influenza virus, 2 / 20 were positive for avian influenza virus in the vaccine diluent group, and 16 / 20 were positive for avian influenza virus in the saline dilution group. The challenge results showed that the peak antibody period in the vaccine diluent group lasted longer than in the saline-immunized group, and the duration of immunity was also longer. The challenge statistical results are shown in Table 23.

[0309] Table 23 Statistical results of immune protection in each experimental group

[0310]

[0311] The above are only preferred embodiments of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Fusion protein A, composed of sequentially linked IL-2 protein, NPS protein, and WT1 protein; The amino acid sequence of the IL-2 protein is shown in SEQ ID NO.1; The amino acid sequence of the NPS protein is shown in SEQ ID NO.2; The amino acid sequence of the WT1 protein is shown in SEQ ID NO.

3.

2. The fusion protein A according to claim 1, characterized in that The IL-2 protein is linked to the NPS protein via GGGGSGGGGS, and the NPS protein is linked to the WT1 protein via GGGGSGGGGS.

3. The fusion protein A according to claim 1, characterized in that The N-terminus of the IL-2 protein also includes a RRRYY fragment.

4. The fusion protein A according to any one of claims 1 to 3, characterized in that Its amino acid sequence is shown in SEQ ID NO.

4.

5. A composition comprising the fusion protein A and the fusion protein B according to any one of claims 1 to 4; The fusion protein B is composed of sequentially connected CaIFN-γ protein and GATA3 protein; The amino acid sequence of the CaIFN-γ protein is shown in SEQ ID NO.5; The amino acid sequence of the GATA3 protein is shown in SEQ ID NO.

6.

6. The composition according to claim 5, characterized in that The Calfn-γ protein is linked to the GATA3 protein via GGGGSGGGGS.

7. The composition according to claim 5, characterized in that The amino acid sequence of the fusion protein B is shown in SEQ ID NO.

7.

8. The composition according to any one of claims 5 to 7, characterized in that The mass ratio of the fusion protein A to the fusion protein B is 1:20000.

9. A biological material comprising at least one of the following I) to IV): 1), a nucleic acid encoding the composition according to any one of claims 5 to 8; II), a plasmid vector containing the nucleic acid described in I); III) a host cell having the nucleic acid molecule described in I) integrated into its genome; IV), transforming or transfecting the host cell with the plasmid vector described in II).

10. The biomaterial according to claim 9, characterized in that In the nucleic acid: The nucleic acid sequence encoding the IL-2 protein is shown in SEQ ID NO.8; The nucleic acid sequence encoding the NPS protein is shown in SEQ ID NO.9; The nucleic acid sequence encoding the WT1 protein is shown in SEQ ID NO.10; The nucleic acid sequence encoding the CaIFN-γ protein is shown in SEQ ID NO.11; The nucleic acid sequence encoding the GATA3 protein is shown in SEQ ID NO.

12.

11. Use of the composition according to any one of claims 5 to 8 and / or the biological material according to claim 9 or 10 in preparing a poultry vaccine diluent.

12. A poultry vaccine diluent, characterized in that include: The composition according to any one of claims 5 to 8.

13. The poultry vaccine diluent according to claim 12, characterized in that The concentration of fusion protein A was 1.34×10 -4 mg / L, and the concentration of fusion protein B was 2.67 mg / L.

14. A method for preparing the poultry vaccine diluent according to claim 12 or 13, comprising: dissolving the composition according to any one of claims 5 to 8 in a buffer solution to obtain a protein solution; The oil phase is mixed with the water phase, and then emulsified and mixed with the protein solution to prepare the vaccine diluent.

15. The preparation method according to claim 14, characterized in that The emulsification conditions include: high shear emulsification treatment at 4000 r / min for 30 minutes.

16. Use of the poultry vaccine diluent according to claim 12 or 13 in the preparation of diluted poultry vaccines.

17. The use according to claim 16, characterized in that The poultry vaccine is a live vaccine, an inactivated vaccine, a recombinant vaccine, a subunit vaccine or a nucleic acid vaccine.

18. The use according to claim 17, characterized in that The live vaccine is a live vaccine of infectious bursal disease of chickens, a live vaccine of infectious bronchitis of chickens and / or a live vaccine of Newcastle disease of chickens; The inactivated vaccine is an inactivated avian influenza virus vaccine and / or an inactivated Newcastle disease vaccine; The subunit vaccine is a genetically engineered subunit vaccine of avian influenza virus and / or a genetically recombinant subunit vaccine of Newcastle disease virus.

19. A poultry vaccine comprising the vaccine diluent according to claim 12 or 13 or the vaccine diluent prepared by the preparation method according to claim 14 or 15.

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