Preparation method and application of vaccine diluent

Abstract

The invention relates to the technical field of immunity, in particular to a preparation method and application of a vaccine diluent. The invention provides two fusion proteins, and a composition formed by combining the two fusion proteins has a good effect of improving the immune effect of the vaccine, effectively prolongs the immune duration of the vaccine and provides more efficient immune protection for an animal body, and meanwhile, a vaccine diluent is simple in production process and low in cost.

Description

Technical Field

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

[0002] Commonly used vaccines are divided into three types: "freeze-dried vaccines", "dry powder vaccines" and "liquid vaccines". Vaccine diluent refers to the solvent or reagent used to dilute the vaccine. Depending on the type of vaccine, such as freeze-dried vaccines, dry powder vaccines and liquid vaccines, the required diluents are also different. These diluents can not only help the vaccine to disperse better in the body, but also provide certain protection and auxiliary effects for the vaccine. When producing vaccines, each vaccine manufacturer has its own dedicated diluent to ensure the immune effect of the vaccine.

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

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

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

[0006] Expanding the application areas of 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 human vaccine field.

[0007] Research and development of new diluents: In order to meet the special needs of different vaccines, researchers are constantly developing new diluents. These new diluents have higher standards in terms of composition, stability, safety, etc.

[0008] Although significant progress has been made in the research of vaccine diluents, current diluents still have some disadvantages, such as short half-life or weak auxiliary effect on immune effect, etc. Moreover, different types of diluents are still required for different types of vaccines, 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 an application thereof.

[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 is composed of IL-2 protein and WT1 protein, or is composed of NPS protein and WT1 protein, or is composed of 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 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 as shown in SEQ ID NO.1; or a sequence having an identity of more than 80% with the amino acid sequence as 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 NPS gene and 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 ability. The fusion protein A in the present invention takes the protein from chicken as an example. After being injected into the chicken, it can stimulate the high-level expression of IL-2R receptor, 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 receptor 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 the effect of combining IL-2 protein, NPS protein and WT1 protein in series is better than that of combining IL-2 and NPS in series, or combining IL-2 and WT1 in series. The order of protein series also has an important influence on the improvement of immune effect. Experiments have shown that compared with other series sequences, IL-2-NPS-WT1 has a better effect.

[0020] In fusion protein A, two different proteins are connected by a linker. The present invention has tried a variety of linkers, such as (G4S)n or (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 can more effectively prolong the duration of vaccine immunity. Preferably, the IL-2 protein is connected to the NPS protein by GGGGSGGGGS, and the NPS protein is connected to the WT1 protein by GGGGSGGGGS.

[0021] The present invention also provides a small peptide, whose amino acid sequence is RRRYY (SEQ ID NO. 13). Experiments show that the insertion of the small peptide into a protein, especially the N-terminus of the protein, is conducive to further improving the biological activity of the protein, especially to further extending the half-life.

[0022] The present invention provides application 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 with an amino acid sequence of RRRYY. Preferably, the N segment of the fusion protein A comprises a small peptide with an amino acid sequence of RRRYY. More preferably, the N-terminus of the IL-2 protein further comprises a 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 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.4; or a sequence having an identity of more than 80% with the amino acid sequence shown in 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 from chicken.

[0027] In a specific embodiment, the amino acid sequence of the CaIFN-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 as shown in SEQ ID NO.5; or a sequence having an identity of more than 80% with the amino acid sequence as 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 as shown in SEQ ID NO.6; or a sequence having an identity of more than 80% with the amino acid sequence as 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 tried a variety of linkers, such as (G4S)n or (AG)n. The results show that the linker affects the physiological activity and in vivo half-life of the fusion protein B. Among them, (G4S)n as a linker can more effectively prolong 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 in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence as shown in SEQ ID NO.7; or a sequence having an identity of more than 80% with the amino acid sequence as shown in any of the preceding items. (denoted as rCaIFN-γ-GATA3).

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

[0035] The present invention uses the modified IL-2-NPS-WT1 and CaIFN-γ-GATA3 in combination, IL-2 and IFN-γ act synergistically with each other, recombinant IL-2 and IFN-γ are continuously released, and IL-2 provides more effector cells for the production of IFN-γ by promoting the proliferation and differentiation of T cells. The vaccine diluent of the present invention can significantly improve the vaccine immune effect, effectively prolong the duration of vaccine immunity, and provide more efficient immune protection to the animal body. At the same time, the vaccine diluent production process is simple and low in cost.

[0036] In the present invention, the mass ratio of the fusion protein A to the fusion protein B is 1:(0.01-100000). 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 1:10000, or 1:20000, or 1:30000, or 1:40000, or 1:50000, or 1:60000, or 1:70000, or 1:80000, or 1:90000, or 1:100000.

[0037] In the composition of the present invention, fusion protein A and fusion protein B can be mixed or exist independently of each other, which is not limited by the present invention. In the composition, fusion protein A and fusion protein B can be in the form of powder or solution independently, which is not limited by the present invention.

[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 fusion protein A as described above and / or a nucleic acid encoding the fusion protein B as described above;

[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 a host cell with the plasmid vector described in II).

[0043] Among the nucleic acids described in the present invention, any nucleic acid that can encode the fusion protein A or B as described above is within the scope of protection of the present invention. It can be a wild-type sequence or a codon-optimized sequence, and the present invention does not limit this. Among the nucleic acids described in the present invention, it can only include the reading frame of the fusion protein as described above, or it can include the nucleic acid sequence of the restriction site and / or the screening marker, and the present invention does not limit this 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 as shown in SEQ ID NO.8; or a sequence having more than 80% identity with the nucleic acid sequence as 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 more than 80% identity 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 as shown in SEQ ID NO.10; or a sequence having an identity of more than 80% with the nucleic acid sequence as shown in any of the preceding items.

[0047] In some embodiments, the nucleic acid sequence encoding the CaIFN-γ protein is as shown in SEQ ID NO.11; or a sequence in which one or more nucleic acids are replaced, deleted, added and / or substituted based on the nucleic acid sequence as shown in SEQ ID NO.11; or a sequence having more than 80% identity with the nucleic acid sequence as 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 replaced, deleted, added and / or substituted based on the nucleic acid sequence as 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, and the present invention does not limit this. 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, etc.In some embodiments, the fusion protein is expressed using a pET series plasmid vector. As a feasible case, the pET series expression vector is selected from: 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 preservation, amplification or expression of the fusion protein of the plasmid vector as described above. In the present invention, the host is a eukaryotic host or a prokaryotic host, the eukaryotic host includes but is not limited to yeast, insect cells, kidney epithelial cells, and the prokaryotic host includes but is not limited to Escherichia coli. In the embodiment of the present invention, the host is BL21 (DE3), BL21 (DE3) pLysS, DH5α, JM109, JM110, TOP10, HB101, Xl1-Blue of Escherichia coli.

[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 the preparation of 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 buffer (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: 8.0 g / L NaCl, 0.2 g / L KCl, 1.44 g / L Na2HPO4, 0.24 g / LKH2PO4;

[0056] In the present invention, the aqueous phase includes water and may also include other auxiliary materials. In some embodiments, the aqueous phase includes water and glutamine, bezoar, glucose oxidase, vitamin C and Tween; wherein 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: glutamine 10g / L, bezoar 0.2g / L, glucose oxidase 1g / L, vitamin C 5mg / L and 4wt% 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, the aqueous phase and the oil phase is (1-10): (1-10): (1-10). For example, the mass ratio of the buffer, the aqueous phase and the 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): 8. 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, the aqueous phase, and the 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, and the concentration of fusion protein B is 1-5 mg / L. In some embodiments, the concentration of fusion protein A in the diluent 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 above-mentioned composition in a buffer 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 has three components: a recombinant protein composition, a buffer, and an emulsion. After the three components are prepared, the vaccine diluent can be immediately prepared, and the present invention does not limit this. At this time, the shelf life of the vaccine diluent is 12 months. Preferably, the three components of the vaccine diluent can be stored independently and then prepared when mixed with the vaccine, that is, prepared immediately before use. At this time, the shelf life of each component is 24 months, that is, the shelf life of the vaccine diluent is 24 months.

[0065] In the present invention, the emulsification may be carried out by a common emulsification method in the art. In the embodiment, the emulsification is carried out by a high shear emulsifier. Preferably, the emulsification conditions include: 4000r / min high shear emulsification treatment for 30min.

[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 subject is poultry, including chickens, ducks, geese, turkeys, pigeons, quails, guinea fowls, cormorants, peacocks, swans, ostriches, parrots, partridges, pelicans, cranes, grouse, wild ducks, 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 mycoplasma 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, lung cancer vaccines, and the like.

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

[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 for animals. At the same time, the production process of the vaccine diluent is simple and the cost is low. BRIEF DESCRIPTION OF THE DRAWINGS

[0081] Figure 1 shows the electrophoresis diagram of 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: protein marker; 1: recombinant bacteria; 2: uninduced expression bacteria;

[0085] Figure 5 The technical roadmap of the present invention is shown. DETAILED DESCRIPTION

[0086] The present invention provides a method for preparing a vaccine diluent and an application. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

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

[0088] In this application, "at least one" means one or more, "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 the solution, meaning that the solution may have other elements in addition to the listed elements. At the same time, it should be understood that the use of "include", "comprising" and "having" descriptions herein 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 corresponding term".

[0091] The interleukin-2 (IL-2) is one of the most important immunomodulatory factors with a wide range of biological activities. It is a Th1 lymphocyte factor secreted by T-lymphocytes or T lymphocytes activated by specific antigens or mitogens. It can activate T cells and promote B cell differentiation and antibody secretion, and induce T cells and NK cells to secrete interferon-γ, enhance the killing activity of monocytes and natural killer cells (NK), and improve the cellular immunity and humoral immunity of animals. It plays an important regulatory role in the body's immune response.

[0092] Neuropeptide S (NPS) gene is a neuropeptide composed of a specific amino acid sequence. It plays an important role in genetics and biological research, including participating 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 that is expressed in multiple tissues and organs, interacts with other genes and signaling pathways, and participates in the regulation of key processes such as cell proliferation, differentiation, and apoptosis. WT1 gene may participate in the regulation of immune response by regulating the differentiation and activation of T cells and affecting the expression levels of cytokines such as interferon (IFN) and interleukin (IL).

[0094] Interferon-γ (Chicken interferon-gamma, ChIFN-γ) belongs to type II interferon. It is mainly produced by lymphocyte T cells, and a small part is produced by NK cells. IFN-γ itself cannot directly kill viruses, but acts on target cells, activates genes in target cells, and then synthesizes proteins with antiviral effects to exert its activity, so it has broad-spectrum antiviral activity. It also has an immunomodulatory effect, mainly through major histocompatibility complex II (MHC II) class molecules to promote the interaction between antigen presenting cells and lymphocyte T cells to exert immunomodulatory effects. It can inhibit the proliferation of tumor viruses, promote tumor cell apoptosis, and activate innate immunity and specific immune responses, thereby exerting anti-tumor effects.

[0095] GATA3 (GATA-binding protein 3, GATA3) is an important transcription factor. It has many important functions in the growth and development of chickens. GATA3 plays a key regulatory role in the development and differentiation of T lymphocytes. It can promote the differentiation of T cells into Th2 cells (helper T cells type 2), regulate the expression of various immune-related cytokines, and indirectly regulate the expression or 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 this position. The percent identity between the two sequences varies with the number of identical positions shared by the sequences, taking into account the number of spaces that need to be introduced for optimal comparison of the two sequences and the length of each space.

[0097] Mathematical algorithms can be used to compare sequences between two sequences and calculate percent identity. For example, the Needlema and Wunsch ((1970) J. Mol. Biol. 48: 444-453) algorithm (available at www.gcg.com) that has been integrated into the GAP program of the GCG software package is used, using 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, using the 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 that the identity is 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 the present invention have been presented in the specific embodiments as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual testing methods. Therefore, unless otherwise explicitly stated, it should be understood that all numerical ranges or specific data used in this disclosure may have certain reasonable deviations 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, and 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 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 nucleic acid sequence encoding IL-2 after codon optimization 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 three gene sequences of chicken IL-2, NPS and WT1 were concatenated in different orders, and the linker was GGGGSGGGGS (GGCGGCGGCGGCAGCGGCGGCGGCGG CAGC). The obtained 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 connected in series using the linker GGGGSGGGGS (GGCGGCGGCGGCAGCGGCGGCGGCGGCAGC) 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 site CATATG and the XhoI restriction site CTCGAG were added to the 5' end and 3' end of the fragment obtained by the above ligation, respectively.

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

[0134] Refer to the chicken interferon-γ 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] ATGACTTGCCAGACTTACAACTTGTTTGTTCTGTCTGTCATCATGATTTATTATGGACATACTGCATCTTCTCTGAACCTGGTTCAGCTGCAGGATGATATCGACAAACTGAAAGCTGACTTTAACTCTAGTCATTCTGATGTAGCTGACGGTGGTCCGATTATTGTAGAGAAACTGAAAAACTGGACCGAGAGAAACGAGAAAAGGATCATCCTGAGCCAGATTGTTTCGATGTACCTGGAAATGCT 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 CaIFN-γ and GATA3 gene sequences were concatenated in different orders with linker GGGGSGGGGS (GG CGGCGGCGGCAGCGGCGGCGGCGG CAGC), and codon optimization was performed according to the preference of E. coli. The NdeI restriction site CATATG was added at the 5' end, and the XhoI restriction site CTCGAG was added at the 3' end. The obtained fragment was 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) Target gene digestion: The synthetic IL-2-NPS-WT1 gene was double digested with restriction endonucleases Nde I and Xho I. The digestion system (20 µl) was as follows: 1 µl of IL-2-NPS-WT1 gene, 1 µl of Nde I, 1 µl of Xho I, 2 µl of 10× Buffer, and 15 µl of ddH2O. The digestion was performed on ice and mixed and digested at 37°C for 2 h. The digested fragments were separated by agarose gel electrophoresis and recovered and purified using a gel recovery kit.

[0153] (2) Vector digestion: Select the vector pET30a(+) and double digest it with restriction endonucleases Nde I and Xho I. The digestion system (20µl): pET30a(+) 1µl, Nde I 1µl, Xho I 1µl, 10× Buffer 2µl, ddH2O 15µl. Perform the digestion on ice and mix well. 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 connected using 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 10 µl of the ligation product and add it to a centrifuge tube containing 100 µl of E. coli DH5α competent cells. Mix well and place in an ice bath for 30 min. Transfer to a 42°C constant temperature water bath for heat shock for 90 s. Take out and immediately place in an ice bath for 2 min. Add 500 µl of LB liquid culture medium to each centrifuge tube. Incubate at 37°C, 200 rpm for 1 h. Take 100 µl and spread on a plate containing kanamycin. Incubate inverted at 37°C overnight for 14 h.

[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 r / min 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. After agarose electrophoresis, a pET30a(+) vector band of about 5 kb and an exogenous fragment of 2055 bp appeared. The size was 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 and 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 37°C and 200 r / min overnight. The bacterial liquid was inoculated into a sterile fermenter at 2% of the volume of the culture medium, and aeration was cultured at 37°C. The fermenter was stirred at 500-700 r / min, the dissolved oxygen was 60%-90%, and the pH was 7.0. When the bacteria grew to the middle logarithmic growth period, IPTG was added at a final concentration of 1 mmol / L, and induced at 37°C for 5 h.

[0158] 3.3 After the bacterial cell disruption culture is completed, the bacterial precipitate is collected by centrifugation, washed twice with PBS, and made into a 10% PBS suspension. The bacteria are disrupted by a high-pressure homogenizer at 2-8°C. The disrupted bacterial solution is centrifuged at 8000r / min for 15min 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 30min, centrifuged at 8000r / min for 10min at 4℃, and washed again after removing the supernatant. 8mol / L urea was added to dissolve and wash the inclusion bodies, ultrasonically cleaned in an ice bath for 30min, and centrifuged at 8000r / min for 20min to remove the precipitate. The supernatant was taken as the inclusion body solution. The solution was kept at 4℃ overnight, and the solution was renatured on an ultrafiltration refolding device. The solution was replaced by 5-10 volumes. After the refolding was completed, the solution was centrifuged at 8000r / min to remove the precipitate. The supernatant was collected as the desired protein solution. The protein solution was added to the chromatography column at a speed of 0.3ml / min, and linear gradient elution was performed at a speed of 1ml / min with pH 7.0 phosphate buffer. The target elution peak was collected and sterilized by 0.22μm filter membrane filtration. The protein content was determined by UV spectrophotometry to be ≥3.8 mg / ml.

[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℃ for 1.5h, and washed three times with TBST. Rabbit IL-2 monoclonal antibody was used as the primary antibody, and a 1:1000 dilution was added. The membranes were incubated at 4℃ overnight, washed three times with TBST, and 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 induction recombinant bacteria 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) Target gene digestion: The synthetic CaIFN-γ-GATA3 gene was double digested with restriction endonucleases Nde I and Xho I. The digestion system (20 µl) was as follows: CaIFN-γ-GATA3 gene 1 µl, Nde I 1 µl, Xho I 1 µl, 10× Buffer 2 µl, ddH2O 15 µl. The digestion was performed on ice and mixed and digested at 37°C for 2 h. The digested fragments were separated by agarose gel electrophoresis and recovered and purified using a gel recovery kit.

[0164] (2) Vector digestion: Select the vector pET30a(+) and double digest it with restriction endonucleases Nde I and Xho I. The digestion system (20µl): pET30a(+) 1µl, Nde I 1µl, Xho I 1µl, 10× Buffer 2µl, ddH2O 15µl. Perform the digestion on ice and mix well. 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 connected with T4 ligase. The ligation system (10µl) is as follows: 1µl 10×Buffer, 2µl pET30a(+) digested product, 2µl interferon gene digested product, 1µl T4 DNA ligase, 4µl ddH2O. The operation was performed on ice. After mixing, the ligation was carried out at 16°C overnight.

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

[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 r / min 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 and culture The pET-CaIFN-γ-GATA3 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 37°C and 200 r / min overnight. The bacterial liquid was inoculated into a sterile fermenter at 2% of the volume of the culture medium, and aeration was cultured at 37°C. The fermenter was stirred at 500-700 r / min, the dissolved oxygen was 60%-90%, and the pH was 7.0. When the bacteria grew to the middle logarithmic growth period, IPTG was added at a final concentration of 1 mmol / L, and induced at 37°C for 5 h.

[0169] 4.3 After the bacterial cell disruption, the bacterial precipitate was collected by centrifugation, washed twice with PBS, and made into a 10% PBS suspension. The bacteria were disrupted by a high-pressure homogenizer at 2-8°C. The disrupted bacterial solution was centrifuged at 8000r / min for 15min to collect the inclusion bodies.

[0170] 4.4 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 8000r / min for 10 minutes at 4°C, and washed again after removing the supernatant. 8mol / L urea was added to dissolve and wash the inclusion bodies, ultrasonically cleaned in an ice bath for 30 minutes, and centrifuged at 8000r / min for 20 minutes to remove the precipitate. The supernatant was taken as the inclusion body solution. The solution was kept at 4°C overnight, and the solution was renatured on an ultrafiltration refolding device. The solution was replaced by 5 to 10 volumes. After the refolding was completed, the solution was centrifuged at 8000r / min to remove the precipitate. The supernatant was collected as the desired protein solution. The protein solution was added to the chromatography column at a speed of 0.3ml / min, and linear gradient elution was performed at a speed of 1ml / min with pH 7.0 phosphate buffer. The target elution peak was collected and sterilized by 0.22μm filter membrane filtration. The protein content was determined by UV spectrophotometry to be ≥3.8 mg / ml.

[0171] 4.5 Western-blot identification of recombinant proteins After SDS-PAGE electrophoresis, the above recombinant proteins were transferred to PVDF membranes at 100V for 70min, washed twice with TBST, blocked with 1% BSA blocking solution at 37℃ for 1.5h, and washed three times with TBST. Rabbit anti-CaIFN-γ was used as the primary antibody, and a 1:1000 dilution was added. The cells were incubated at 4℃ overnight, washed three times with TBST, and then a 1:4000 dilution of goat anti-rabbit IgG-HRP secondary antibody was added. The membrane was washed three times with TBS and detected with a DAB colorimetric kit. The size of the recombinant protein in the inducible recombinant bacteria 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 biological activity assay

[0174] The biological activity of the recombinant proteins obtained by different combinations and concatenations of IL-2, NPS and WT1 during the experiment was determined. The effects of linker1 (GGGGSGGGGS (GG CGGCGGCGGCAGCGGCGGCGGCGGCAGC)) and linker2 (AGAGAG (GCCGGTGCCGGTGCCGGT)) on the biological activity of the fusion protein 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, rIL-2 proteins prepared using linker2 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 all diluted with basic culture medium to contain approximately 200 IU in 1 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 volume was reached. 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 5The cell suspension of each cell was stored at 37°C and 5% CO2 for later use. In a 96-well cell culture plate with standard solution and test solution, 50 µl of cell suspension was added to each well and cultured at 37°C and 5% CO2 for 18 to 24 hours. Then, 20 µl of MTT solution was added to each well and cultured at 37°C and 5% CO2 for 4 to 6 hours. Then, 150 µl of lysis solution was added to each well and kept at 37°C and 5% CO2 for 18 to 24 hours. All the above operations were performed under sterile conditions. The liquid in the cell plate was mixed and placed in an ELISA reader. The absorbance was measured at a wavelength of 570 nm with 630 nm as the reference wavelength, and the measurement results were recorded. The specific biological activity measurement 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 linker1 and linker2, it was found that the biological activity of the fusion protein was better when linker1 was used, and linker1 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 the recombinant proteins obtained by different tandem methods of CaIFN-γ and GATA3 during the experiment was determined. The effects of linker1 (GGGGSGGGGS (GG CGGCGGCGGCAGCGGCGGCGGCGG CAGC)) and linker2 (AGAGAG (GCCGGTGCCGGTGCCGGT)) on the biological activity of the fusion protein were also compared. Under sterile conditions, the control interferon standard, rCaIFN-γ-GATA3, rGATA3-CaIFN-γ, rCaIFN-γ prepared using linker1, and rCaIFN-γ-GATA3, rGATA3-CaIFN-γ, and rCaIFN-γ prepared using linker2 were diluted to 1000U per 1ml in the assay culture medium, and a 4-fold serial dilution was made in a 96-well cell culture plate, with a total of 10 dilutions, and 2 wells were made for each dilution. CEF cells grew adherently in the culture medium, were passaged at a ratio of 1:2 to 1:4, and grown in complete culture 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 10 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 into 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 under microscopic examination are at IU / ml), and observe the results when CPE in the interferon-protected wells no longer progresses.

[0187] Open the cover of the cell plate, discard the liquid in each well and put it in disinfectant, add 1-2 drops of crystal violet dye to each well, and place it at room temperature for 30 minutes. Then, rinse the residual dye in the well with a thin stream of water, dry the residual water and record the results. The results of biological activity determination 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 linker1 and linker2, it was found that the biological activity of the fusion protein was better when linker1 was used, and linker1 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 chicken

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

[0196] The half-life of the recombinant proteins obtained by different combinations and tandems of IL-2, NPS and WT1 in chickens was measured. The effects of linker1 (GGGGSGGGGS (GGCGGCGGCGGCAGCGGCGGCGGCAGC)) and linker2 (AGAGAG (GCCGGTGCCGGTGCCGGT)) on the half-life of the fusion protein in chickens were also compared. The 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 the 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, rIL-2 proteins, and half-life detection were performed at the same time. 3-4 week old SPF chickens were selected and divided into 24 groups, 10 in each group, and 24 groups of proteins were injected intramuscularly. The injection dose was 100,000 units per kg body weight. Blood was collected before and after injection at 1h, 2h, 4h, 6h, 8h, 12h, 48h, 72h, 96h, 120h, 144h, 168h, and 192h, and the concentration of IL-2 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) (linker2)

[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, it was found that the two linkers had no significant effect on the half-life of the fusion protein in chickens. At 192 hours after injection, the concentration of IL-2 in serum was 39 and 32 ng / ml, while the concentration of IL-2 in serum of rIL-2 without any modification had dropped to 2 and 3 ng / ml at 12 hours after injection, and was completely undetectable at 48 hours.

[0206] In a comprehensive study, linker1 and linker2 were compared for the biological activity of the fusion protein and the experimental data of the half-life in chickens, and linker1 was selected as the linker of the present invention. The experiment determined that the optimal combination was to use linker1 (GGGGSGGGGS (GGCGGCGGCGGCAGCGGCGG CGGCGG CAGC)), the tandem mode was rIL-2-NPS-WT1, and CGTCGTCGTTATTAT was 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 half-life of recombinant proteins obtained by different tandem methods of CaIFN-γ and GATA3 during the experiment was determined in chickens. At the same time, the effects of linker1 (GGGGSGGGGS (GG CGGCGGCGGCAGCGGCGGCGGCGG CAGC)) and linker2 (AGAGAG (GCCGGTGCCGGTGCCGGT)) on the half-life of fusion proteins in chickens were also compared. The rCaIFN-γ-GATA3, rGATA3-CaIFN-γ, rCaIFN-γ proteins prepared by linker1 of the present invention and rCaIFN-γ-GATA3, rGATA3-CaIFN-γ, rCaIFN-γ proteins prepared by linker2 were simultaneously tested for half-life, 3-4 week old SPF chickens were selected and divided into 6 groups, 10 in each group, and 6 groups of proteins were injected intramuscularly, and the injection dose was 100,000 units per kg body weight, and blood was collected before and after injection at 1h, 2h, 4h, 6h, 8h, 12h, 18h, 24h, 30h, 36h, 48h, 60h, 72h, and 96h, 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) (linker1)

[0210]

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

[0212] Table 8 Results of interferon concentration determination in serum (ng / ml) (linker2)

[0213]

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

[0215] Results By comparing linker1 and linker2, it was found that the two linkers had no significant effect on the half-life of the fusion protein in chickens. The interferon concentrations in the serum of rCaIFN-γ-GATA3 protein, rGATA3-CaIFN-γ and rCaIFN-γ protein were basically the same at 1h and 2h after injection. However, with the extension of the detection time after injection, the interferon concentration in the serum of rCaIFN-γ-GATA3 gradually increased, resulting in serum interferon concentrations of 102 and 100 ng / ml at 12h after injection, while the interferon concentrations in the serum of the rGATA3-CaIFN-γ injection group had dropped to 41 and 37 ng / ml, and the interferon concentrations in the serum of the rCaIFN-γ injection group had dropped to 5 and 3 ng / ml. At 96h after injection, interferon could still be detected in the serum of the rCaIFN-γ-GATA3 injection group, and the half-life of rCaIFN-γ-GATA3 protein was significantly prolonged.

[0216] In a comprehensive study, linker1 and linker2 were compared for the biological activity of the fusion protein and the experimental data of the half-life in chicken, and linker1 was selected as the linker of the present invention. The experiment determined that the optimal combination was to use linker1 (GGGGSGGGGS (GGCGGCGGCGGCAGCGGCGG CGGCGG CAGC)), and the combination effect was best when the series connection mode was rCaIFN-γ-GATA3.

[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: 8.0g NaCl, 0.2g KCl, 1.44g Na2HPO4, 0.24g KH2PO4, dissolved in 800mL distilled water, adjusted the solution to 7.4 with HCl, and finally added distilled water to 1L.

[0220] Z Liquid

[0221] Aqueous phase: Add 10g glutamine, 0.2g artificial bezoar, 1g glucose oxidase, and 5mg 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 for later use.

[0222] Oil phase: After sterilizing the mineral oil at 121°C and 100 kPa pressure, add 0.5 mg of monolaurin and 0.5 mg of vitamin E per liter for later use.

[0223] Emulsification: Add water phase into 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 at 4000r / min for 30min to obtain Z liquid.

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

[0225] 8 Determination of half-life of vaccine in diluent

[0226] When using the diluent, use Y liquid to dissolve X powder, and then mix it with Z liquid at a volume ratio of 1:1. 10 SPF chickens aged 3 to 4 weeks were selected, and 0.3 ml of vaccine diluent was injected intramuscularly. Blood was collected before injection and 1h, 2h, 4h, 6h, 8h, 12h, 48h, 72h, 96h, 120h, 144h, 168h, 192h, and 216h after injection. HPLC was used to determine the concentration of IL-2 and CaIFN-γ in the blood. 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, subunit vaccines, nucleic acid vaccines and other genetic engineering 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 of the chicken infectious bursal live vaccine (freeze-dried type), the chicken infectious bursal live vaccine was diluted with the vaccine diluent of the present invention and sterilized physiological saline respectively; 60 SPF chicks aged 10 days were selected, 20 chicks were in the first vaccine diluent group; 20 chicks were in the second physiological saline dilution group; 0.3 ml of vaccine was inoculated into each chick’s eyes or orally, and 20 chicks were selected as blank control groups without inoculation, and they were isolated and raised respectively. Blood was collected before immunization, 7 days, 14 days, 21 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, and 7 months after immunization, and the titer of serum neutralizing antibodies was measured, and the antibody variation rule was counted and analyzed. 10 chicks were attacked with chicken infectious bursal virus strains at 6 months and 7 months, and each chick was attacked with at least 10 MID of strong virus by eye drop, and all chickens were killed and dissected at 72 hours to detect and count the changes in Fabricius.

[0235] 9.1.1.2 Results

[0236] The antibody level of the chickens in the immunized group gradually increased after immunization. The vaccine diluent group produced antibodies earlier than the saline immunized group, and the antibody level of the vaccine diluent group was higher than that of the saline dilution group in the same period. The antibody test results are shown in Table 10.

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

[0238]

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

[0240] When challenged with virus 6 months after immunization, the chickens in the control group showed 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; 2 chickens in the saline dilution immunization group showed lethargy and white loose stools; the chickens in each group were autopsied to observe the bursal lesions, and 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. When challenged 7 months after immunization, the control group chickens showed mental depression, ruffled feathers, slow movements, and white loose stools, and 20 / 20 died; 2 chickens in the vaccine diluent immunization group showed mental depression and white loose stools; 12 chickens in the saline dilution immunization group showed mental depression, ruffled feathers, slow movements, and white loose stools, and 8 died; the lesions of the bursal lesions of the test chickens in each group were observed by autopsy; the results showed that the bursal protection rate of the test chickens in the vaccine diluent immunization group was 18 / 20; the bursal protection rate of the test chickens in the saline dilution immunization group was 8 / 20; the bursal lesions of the test chickens in the control group were all positive. The challenge results showed that the peak period of antibodies in the vaccine diluent group lasted longer than that in the saline immunization 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 of the live vaccine of infectious bronchitis in chickens (freeze-dried type), the live vaccine of infectious bronchitis in chickens was diluted with the vaccine diluent of the present invention and sterilized physiological saline respectively; 60 SPF chickens aged 21 days were selected, 20 in the first vaccine diluent group; 20 in the second physiological saline dilution group; 0.3 ml of vaccine was inoculated into each trachea, and 20 were selected as blank control groups without inoculation, and they were isolated and raised respectively. Blood was collected before immunization, 7 days, 14 days, 21 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, and 7 months after immunization, and the titer of serum HI antibody was determined, and the antibody change rule was counted and analyzed. At 6 months and 7 months, 10 chickens were attacked with the infectious bronchitis virus strain of chickens, and 1 to 2 drops were dripped into the nose of each chicken, and the chickens were observed continuously for 10 days, and the death of chickens in each test group was counted.

[0246] 9.1.2.2 Results

[0247] After immunization, the neutralizing antibodies of the test chickens gradually increased, and the antibodies in the vaccine diluent group were higher than those in the saline dilution group during the same period, and the peak duration of the antibodies was longer. The antibody titer of the vaccine diluent group after immunization can be as high as 1:31.56. The antibody test results of each group of test chickens 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] When challenged with poison 6 months after immunization, 20 / 20 chickens in the control group died; the protection rate of chickens in the vaccine diluent immunization group against infectious bronchitis was 20 / 20; the protection rate of chickens in the saline dilution immunization group against bursal disease was 16 / 20; when challenged with poison 7 months after immunization, 20 / 20 chickens in the control group died; the protection rate of chickens in the vaccine diluent immunization group against infectious bronchitis was 16 / 20; the protection rate of chickens in the saline dilution immunization group against bursal disease was 4 / 20; the results of the challenge showed that the peak duration of antibodies in the vaccine diluent group was longer than that in the saline immunization group, and the duration of immunity was also longer. The statistical results of the challenge 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 in the first vaccine diluent group; 20 in the second saline dilution group; each chicken was inoculated with 0.3 ml of the vaccine by nasal drops, and another 20 chickens were selected as blank control groups without inoculation, and they were isolated and raised separately. 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 with 0.5 ml (10 4.0 ELD 50) , observed for 14 days, and counted the deaths of chickens in each experimental group.

[0257] 9.1.3.2 Results

[0258] After immunization, the HI antibody of the test chickens gradually increased, and the antibody of the vaccine diluent group was higher than that of the physiological saline dilution group during the same period, and the peak period of the antibody lasted longer. The antibody test results of each group of test chickens 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] When the virus was challenged 4 months after immunization, the control group chickens showed symptoms such as depression, loss of appetite, purple comb, nodding like pecking rice, incoordination, and yellow-green and yellow-white loose feces after the virus was challenged, and 20 / 20 died; the vaccine diluent immunization group chickens were all alive and well, with a protection rate of 20 / 20, and 4 chickens died in the saline dilution group, and the rest of the chickens were alive and well, with a protection rate of 16 / 20; when the virus was challenged 5 months after immunization, the observation was 14 days, and the results showed that the control group chickens showed symptoms such as depression, loss of appetite, purple comb, nodding like pecking rice, incoordination, and yellow-green and yellow-white loose feces after the virus was challenged, and 20 / 20 died; the vaccine diluent immunization group chickens died, with a protection rate of 18 / 20, and the saline dilution group chickens died, with a protection rate of 8 / 20; the virus challenge results showed that the vaccine diluent group had a longer antibody peak period than the saline immunization group, and the immune duration was also longer. The virus 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 diluent in inactivated avian influenza virus vaccine

[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 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 saline dilution group; the diluted vaccine was inoculated twice according to the vaccine instructions, 0.6 ml / chicken. 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 with 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 challenged 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 virus isolation positive rate was calculated.

[0269] 9.2.1.2 Results

[0270] The antibody level of the chickens in the immunized group gradually increased after immunization. The vaccine diluent group produced antibodies earlier than the saline immunized group, and the antibodies in the diluent group were higher than those in the saline dilution group during the same period. The 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] When challenged 5 months after immunization, 20 / 20 of the avian influenza virus separations in the blank control group were positive, the positive rate of avian influenza virus separation in the vaccine diluent group was 0 / 20, and the positive rate of avian influenza virus separation in the saline dilution group was 2 / 20. When challenged 6 months after immunization, 20 / 20 of the avian influenza virus separations in the blank control group were positive, the positive rate of avian influenza virus separation in the vaccine diluent group was 4 / 20, and the positive rate of avian influenza virus separation in the saline dilution group was 14 / 20. The 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 Newcastle disease virus inactivated 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 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 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 selected as blank control groups without vaccination, and they were raised in isolation. 14 days after the first vaccination, a booster immunization was performed once with 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, and the serum HI antibody titer was determined, 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 each chick was injected intramuscularly with 0.5 ml (10 4.0 ELD 50 ), observed for 14 days, and counted the mortality of chickens in each test group.

[0280] 9.2.2.2 Results

[0281] After immunization, the HI antibody of the test chickens gradually increased, and the antibody of the vaccine diluent group was higher than that of the saline dilution group during the same period, and the peak period of the antibody lasted longer. The antibody test results of each group of test 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] When the virus was challenged 4 months after immunization, 20 / 20 chickens in the control group died after 14 days of observation; all the chickens in the vaccine diluent immunization group were alive and well, with a protection rate of 20 / 20, and 2 chickens died in the saline dilution group, with a protection rate of 18 / 20; when the virus was challenged 5 months after immunization, 20 / 20 chickens in the control group died after 14 days of observation; 2 chickens in the vaccine diluent immunization group died, with a protection rate of 18 / 20, and 16 chickens died in the saline dilution group, and the rest of the chickens were alive and well, with a protection rate of 4 / 20; the results of the virus challenge showed that the vaccine diluent group had a longer antibody peak period than the saline immunization group, and the duration of immunity was also longer. The statistical results of the virus challenge 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 diluents

[0289] 9.3.1 Application of vaccine diluent in avian influenza virus genetically engineered subunit vaccines

[0290] 9.3.1.1 Application method

[0291] The vaccine diluent of the present invention and sterile saline were used to dilute the genetically engineered subunit vaccine of avian influenza virus in a 1:1 ratio; 60 SPF chicks aged 21 to 35 days were selected, 20 chicks were in the first vaccine diluent group, and 20 chicks were in the second saline dilution group; the diluted vaccine was inoculated twice according to the vaccine instructions, and 0.6 ml was injected intramuscularly to each chick. Another 20 chicks were selected as blank control groups and kept in isolation. 14 days after the first vaccination, a booster vaccination was performed once with 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, and the serum HI antibody titer was measured. 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 virus isolation positive rate was calculated.

[0292] 9.3.1.2 Results

[0293] The antibody level of the chickens in the immunized group gradually increased after immunization. The vaccine diluent group produced antibodies earlier than the saline immunized group, and the antibodies in the diluent group were higher than those in the saline dilution group during the same period. The 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 of the avian influenza virus in the blank control group were positive, the positive rate of avian influenza virus in the vaccine diluent group was 0 / 20, and the positive rate of avian influenza virus in the saline dilution group was 4 / 20. When challenged 5 months after immunization, 20 / 20 of the avian influenza virus in the blank control group were positive, the positive rate of avian influenza virus in the vaccine diluent group was 2 / 20, and the positive rate of avian influenza virus in the saline dilution group was 18 / 20. The 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 diluent in Newcastle disease virus gene recombinant subunit vaccine

[0301] 9.3.2.1 Application method

[0302] The Newcastle disease virus gene recombinant subunit vaccine was diluted 1:1 using the vaccine diluent of the present invention and sterile saline respectively; 60 SPF chicks aged 4 to 5 weeks were selected, 20 in the first vaccine dilution group; 20 in the second saline dilution group; the diluted vaccine was inoculated twice according to the vaccine instructions. Each chick was injected intramuscularly with 0.6 ml, and another 20 chicks were selected as blank control groups without vaccination, and they were raised in isolation. 14 days after the first vaccination, a booster immunization was performed once with 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, 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 chicks were attacked with the Newcastle disease virus strain respectively, and each chick was injected intramuscularly with 0.5 ml (10 5.0 ELD 50), 5 days after the virus attack, 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, the HI antibody of the test chickens gradually increased, and the antibody of the vaccine diluent group was higher than that of the physiological saline dilution group during the same period, and the peak period of the antibody lasted longer. The specific results of the antibody test results of each group of test 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 the virus was challenged 4 months after immunization, 20 / 20 of the avian influenza virus isolations in the blank control group were positive, the positive rate of avian influenza virus isolation in the vaccine diluent group was 0 / 20, and the positive rate of avian influenza virus isolation in the saline dilution group was 6 / 20. When the virus was challenged 5 months after immunization, 20 / 20 of the avian influenza virus isolations in the blank control group were positive, the positive rate of avian influenza virus isolation in the vaccine diluent group was 2 / 20, and the positive rate of avian influenza virus isolation in the saline dilution group was 16 / 20. The results of the virus challenge showed that the peak duration of the antibody in the vaccine diluent group was longer than that in the saline immunization group, and the duration of immunity was also longer. The statistical results of the virus challenge 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, It is characterized in that The IL-2 protein is connected to the NPS protein via GGGGSGGGGS, and the NPS protein is connected to the WT1 protein via GGGGSGGGGS.

3. The fusion protein A according to claim 1, It is 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, It is 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 a sequentially connected CaIFN-γ protein and a 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, It is characterized in that The CaIFN-γ protein is connected to the GATA3 protein via GGGGSGGGGS.

7. The composition according to claim 5, It is 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, It is 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 fusion protein A according to any one of claims 1 to 4 and / or a nucleic acid encoding the fusion protein B according to any one of claims 5 to 7; 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 a host cell with the plasmid vector described in II).

10. The biomaterial according to claim 9, It is 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 fusion protein A according to any one of claims 1 to 4, the composition according to any one of claims 5 to 7 and / or the biological material according to claim 9 or 10 in preparing a vaccine diluent.

12. Vaccine diluent, It is characterized in that include: The fusion protein A according to any one of claims 1 to 4 and / or the composition according to any one of claims 5 to 7.

13. The vaccine diluent according to claim 12, It is characterized in that Also includes: buffer, aqueous phase and oil phase, wherein: The buffer is a phosphate buffer; The aqueous phase comprises water, glutamine, bezoar, glucose oxidase, vitamin C and Tween; The oil phase includes mineral oil, monolaurin and vitamin E.

14. The vaccine diluent according to claim 13, It is characterized in that The phosphate buffer solution is composed of water and the following components: 8.0 g / L NaCl, 0.2 g / L KCl, 1.44 g / L Na 2 HPO 4 , 0.24g / L KH 2 PO 4 ; The aqueous phase is composed of water and the following components in concentration: 10 g / L glutamine, 0.2 g / L bezoar, 1 g / L glucose oxidase, 5 mg / L vitamin C and 4 wt% Tween; 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.

15. The vaccine diluent according to claim 13, It is characterized in that in, The mass ratio of buffer, aqueous phase and oil phase was 3:2:

1.

16. The vaccine diluent according to any one of claims 12 to 15, It is 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.

17. A method for preparing a vaccine diluent according to any one of claims 12 to 16, wherein include: Dissolving the composition according to any one of claims 5 to 7 in a buffer to obtain a protein solution; The oil phase is mixed with the water phase and then mixed with the protein solution through emulsification to prepare the vaccine diluent.

18. The preparation method according to claim 17, It is characterized in that The emulsification conditions include: high shear emulsification treatment at 4000 r / min for 30 min.

19. Use of the vaccine diluent according to any one of claims 12 to 16 or the vaccine diluent prepared by the preparation method according to claim 17 or 18 in diluting a vaccine.

20. The use according to claim 19, It is characterized in that The vaccine is a live vaccine, an inactivated vaccine, a recombinant vaccine, a subunit vaccine or a nucleic acid vaccine.

21. The use according to claim 20, It is characterized in that The live vaccine is a live vaccine of infectious bursal disease of chicken, a live vaccine of infectious bronchitis of chicken and / or a live vaccine of Newcastle disease of chicken; 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.

22. A vaccine comprising the vaccine diluent according to any one of claims 12 to 16 or the vaccine diluent prepared by the preparation method according to claim 17 or 18.

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