Recombinant porcine interferon λ1 fusion protein, preparation method therefor and use thereof

Abstract

Provided are a fusion protein comprising recombinant porcine interferon λ1 and a use thereof, and a preparation method and use for the fusion protein. The fusion protein is formed by respectively linking porcine interferon λ1 to other fragments (such as porcine interferon γ) and / or porcine serum albumin (PSA) directly or indirectly by means of linker elements. The porcine interferon λ1 fusion protein can be prepared by using a mammalian cell expression system on the basis of genetic engineering technology. Compared with natural porcine interferon λ1, the provided recombinant porcine interferon λ1 fusion protein has a long half-life period and can be used for preparing drugs for preventing or treating porcine viral diseases.

Description

Recombinant porcine interferon lambda 1 fusion protein and preparation method and application thereof

[0001] Cross-reference to Related Applications

[0002] This application claims the benefit of Chinese Patent Application No. 202410591659.2, filed May 14, 2024, Chinese Patent Application No. 202410591755.7, filed May 14, 2024, and Chinese Patent Application No. 202410591789.6, filed May 14, 2024, the contents of which are incorporated herein by reference. TECHNICAL FIELD

[0003] The present application relates to the technical field of prevention and control of biological medicine and animal viral diseases, in particular to a recombinant porcine interferon lambda 1 fusion protein and a preparation method and application thereof. BACKGROUND

[0004] Porcine viral diseases present a variety of characteristics, and common ones include African swine fever, porcine epidemic diarrhea, porcine reproductive and respiratory syndrome, pseudorabies, and porcine circling, etc. At present, such diseases are mainly prevented by biological safety measures and vaccination. However, due to insufficient basic research, virus variation, and dependence on the state of the immune system, the vaccine effect of many porcine viral diseases is not ideal.

[0005] Porcine epidemic diarrhea virus (PEDV) vaccines face three difficulties. First, PEDV is a very fast mutating RNA coronavirus, especially the S protein (the main target of neutralizing antibodies), which mutates the most, so the vaccine is easily ineffective. Second, the death course of PEDV-infected piglets is very short, basically all die within 7 days of birth, while the vaccine usually starts to produce antibodies 10 days after injection, so there is no effect on piglets injected with PEDV vaccine. Third, PEDV vaccine injected into pregnant sows needs to be transmitted to piglets through the placenta and colostrum, but piglets obtain a limited amount of maternal antibodies, and there are large differences in the levels of maternal antibodies obtained by individuals, so the effect is very unstable.

[0006] Porcine reproductive and respiratory syndrome virus (PRRSV) also faces similar problems. PRRSV is a fast-mutating and frequently-recombining RNA virus. Inactivated vaccines have no effect on preventing PRRSV, and attenuated live vaccines often cause the emergence of anti-attenuated strains, leading to the transformation of originally negative pig farms into positive pig farms, causing the outbreak of PRRSV infectious diseases. In addition, many PRRSV virus lineages have been found, and multiple strains may exist in the same pig farm, making it difficult to choose an appropriate immunization scheme. There are many commercial PRRSV vaccines, but the cross-protection is limited, and some breeders lack professional knowledge, leading to the misuse and overuse of vaccines, and the long-term sub-health state of pig populations.

[0007] Interferons are an important class of cytokines in the cellular immune system, mainly including interferon a, interferon β, interferon γ and interferon λ. They can interfere with the replication and spread of viruses in the process of infecting host cells, thereby resisting viral infection. However, the half-life of natural interferon is short, and the plasma half-life is about 2-6 hours. For example, the half-life of porcine natural interferon cytokines in vivo is generally 2-4 hours, and the anti-viral prevention and treatment of pigs requires injection 1-2 times a day. In addition, the commercial pig IFN-α is produced by pig white blood cells induced by Newcastle disease virus (NDV), and the yield is insufficient and the quality is uneven. Interferon protein produced by E. coli system has been successful, but bacteria belong to prokaryotic system, which cannot correctly process and fold heterologous proteins, and often appears in the form of inclusion body, which increases the difficulty of operation.

[0008] Fusion protein technology can fuse functional protein molecules with biological activity with molecules such as Fc or albumin to produce new protein molecules. The half-life of the fused protein molecules is prolonged, which can reduce the frequency of administration, facilitate the use of operating personnel, reduce the stimulation to piglets, and improve the applicability of the product. SUMMARY

[0009] The purpose of the present application is to provide a new type of recombinant porcine interferon λ1 fusion protein and its preparation method and application.

[0010] The concept of the present application is as follows: using genetic engineering technology, porcine interferon λ1 is fused with other fragments, such as porcine interferon γ, porcine Fc, porcine Fc(KiH) and / or porcine serum albumin (PSA) to produce a new type of recombinant protein, so as to not only retain the high biological activity of porcine interferon λ1, but also obtain a longer half-life; at the same time, a high-purity, scalable recombinant protein is obtained. Using a mammalian expression system, especially Chinese hamster ovary cells (CHO) to express the recombinant protein can obtain a protein molecule closest to the natural protein molecule in terms of molecular structure, physicochemical properties and biological functions.

[0011] In a first aspect, the present application provides a fusion protein comprising recombinant porcine interferon λ1, wherein the porcine interferon λ1 is fused with porcine interferon γ, porcine Fc, porcine Fc(KiH) and / or porcine serum albumin (PSA).

[0012] Preferably, the porcine interferon λ1 is connected to each other through a linker or directly connected.

[0013] Further, the Linker is a flexible polypeptide consisting of 2-20 flexible amino acids selected from at least one of Gly, Ser, Ala, Thr; preferably, the Linker is (Gly-Gly-Gly-Gly-Ser)n, wherein n is an integer between 2-5, more preferably n is 3. The amino acid sequences of exemplary Linkers of the present application are shown in Linker 1 and Linker 2 of SEQ ID NO: 13 and 15, respectively, and the nucleotide sequences thereof are shown in SEQ ID NO: 14 and 16, respectively.

[0014] In some specific embodiments, the fusion protein comprises porcine interferon lambda 1 and PSA, which are connected to each other via a Linker or directly. For example, such a fusion protein comprises the porcine IFN-λ1 amino acid sequence shown in SEQ ID NO: 1 or a sequence with 90% or more homology thereto and having the same function; and the porcine PSA amino acid sequence shown in SEQ ID NO: 5 or a sequence with 90% or more homology thereto and having the same function. Preferably, such a fusion protein comprises the amino acid sequence shown in SEQ ID NO: 17; or a protein with 90% or more homology to the amino acid sequence shown in SEQ ID NO: 17 and having the same function.

[0015] In some specific embodiments, the fusion protein provided by the present application is a fusion protein of recombinant porcine interferon lambda 1, porcine interferon gamma and porcine Fc, which is connected by porcine interferon lambda 1, porcine interferon gamma and porcine Fc. For example, such a fusion protein comprises the porcine IFN-λ1 amino acid sequence shown in SEQ ID NO: 1 or a sequence with 90% or more homology thereto and having the same function; the porcine IFN-λ1 amino acid sequence shown in SEQ ID NO: 3 or a sequence with 90% or more homology thereto and having the same function; and the porcine Fc amino acid sequence shown in SEQ ID NO: 7 or a sequence with 90% or more homology thereto and having the same function.

[0016] Preferably, the porcine interferon lambda 1, the porcine interferon gamma and the porcine Fc comprise the following 3 ways:

[0017] ① The C-terminal of porcine interferon lambda 1, the N-terminal of porcine interferon gamma, the C-terminal of porcine interferon gamma, and the N-terminal of porcine Fc are sequentially connected.

[0018] ② The C-terminal of porcine interferon lambda 1, the N-terminal of porcine Fc, the C-terminal of porcine Fc, and the N-terminal of porcine interferon gamma are sequentially connected.

[0019] ③ The C-terminal of porcine interferon gamma, the N-terminal of porcine Fc, the C-terminal of porcine Fc, and the N-terminal of porcine interferon lambda 1 are sequentially connected.

[0020] Further preferably, the porcine interferon lambda 1, the porcine interferon gamma and the porcine Fc are connected with each other in Mode ②.

[0021] Preferably, the fusion protein is a protein comprising the amino acid sequence shown in SEQ ID NO: 19, SEQ ID NO: 21 or SEQ ID NO: 23, and further preferably a protein consisting of the amino acid sequence shown in SEQ ID NO: 21.

[0022] Preferably, the fusion protein is a protein having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 19, SEQ ID NO: 21 or SEQ ID NO: 23 and having the same function, and further preferably a protein having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 21 and having the same function.

[0023] In some specific embodiments, the fusion protein provided by the present application is a fusion protein comprising recombinant porcine interferon lambda 1, porcine interferon gamma and porcine PSA, which is connected by porcine interferon lambda 1, porcine interferon gamma and porcine serum albumin (PSA). For example, such a fusion protein comprises the porcine IFN-λ1 amino acid sequence shown in SEQ ID NO: 1 or a sequence having 90% or more homology therewith and having the same function; the porcine IFN-λ1 amino acid sequence shown in SEQ ID NO: 3 or a sequence having 90% or more homology therewith and having the same function; and the porcine PSA amino acid sequence shown in SEQ ID NO: 5 or a sequence having 90% or more homology therewith and having the same function.

[0024] Preferably, the porcine interferon lambda 1, the porcine interferon gamma and the porcine serum albumin (PSA) are connected by a Linker or directly connected.

[0025] Preferably, the porcine interferon lambda 1, the porcine interferon gamma and the porcine serum albumin (PSA) can include the following 2 modes:

[0026] ① The C-terminal of the porcine interferon lambda 1, the N-terminal of the porcine serum albumin (PSA), the C-terminal of the porcine serum albumin (PSA) and the N-terminal of the porcine interferon gamma are sequentially connected.

[0027] ② The C-terminal of the porcine interferon gamma, the N-terminal of the porcine serum albumin, the C-terminal of the porcine serum albumin and the N-terminal of the porcine interferon lambda 1 are sequentially connected.

[0028] Further preferably, the porcine interferon lambda 1, the porcine interferon gamma and the porcine serum albumin are connected with each other in Mode ①.

[0029] Preferably, the fusion protein is a protein consisting of the amino acid sequence shown in SEQ ID NO: 25 or SEQ ID NO: 27, and more preferably a protein consisting of the amino acid sequence shown in SEQ ID NO: 25.

[0030] Preferably, the fusion protein is a protein having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 25 or SEQ ID NO: 27 and having the same function, and more preferably a protein having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 25 and having the same function.

[0031] In some specific embodiments, the fusion protein provided by the present application is a fusion protein of recombinant porcine interferon lambda 1, porcine interferon gamma and porcine Fc(KiH), which is connected by porcine interferon lambda 1, porcine interferon gamma and porcine Fc(KiH). For example, such a fusion protein comprises the porcine IFN-λ1 amino acid sequence shown in SEQ ID NO: 1 or a sequence having 90% or more homology thereto and having the same function; the porcine IFN-λ1 amino acid sequence shown in SEQ ID NO: 3 or a sequence having 90% or more homology thereto and having the same function; the porcine Fc knob chain amino acid sequence shown in SEQ ID NO: 9 or a sequence having 90% or more homology thereto and having the same function; and the porcine Fc hole chain amino acid sequence shown in SEQ ID NO: 11 or a sequence having 90% or more homology thereto and having the same function.

[0032] Preferably, the porcine Fc is composed of a knob chain and a hole chain. Preferably, the porcine interferon lambda 1, the porcine interferon gamma are connected to the knob chain in the porcine Fc, the hole chain in the porcine Fc respectively by a linker or directly.

[0033] Preferably, the porcine interferon lambda 1, the porcine interferon gamma and the porcine Fc(KiH) are connected to each other in the following two ways:

[0034] ① The porcine interferon lambda 1 is connected to the knob chain in the porcine Fc, and the porcine interferon gamma is connected to the hole chain in the porcine Fc.

[0035] ② The porcine interferon gamma is connected to the knob chain in the porcine Fc, and the porcine interferon lambda 1 is connected to the hole chain in the porcine Fc.

[0036] More preferably, the porcine interferon lambda 1, the porcine interferon gamma and the porcine Fc(KiH) are connected to each other in the way ①.

[0037] Preferably, the fusion protein is a protein consisting of the amino acid sequence shown in SEQ ID NO: 29 and SEQ ID NO: 31, or a protein consisting of the amino acid sequence shown in SEQ ID NO: 33 and SEQ ID NO: 35.

[0038] Preferably, the fusion protein is a protein consisting of amino acids having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 29 and SEQ ID NO: 31 and having the same function, or a protein consisting of amino acids having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 33 and SEQ ID NO: 35 and having the same function.

[0039] The identity described above refers to amino acid sequence identity. The identity of the amino acid sequence can be determined using a homology search site on the Internet, such as the BLAST page of the NCBI homepage website.

[0040] Preferably, the 80% or more identity can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0041] In a second aspect, the present application provides a polynucleotide molecule encoding the fusion protein of the first aspect.

[0042] Preferably, the polynucleotide molecule comprises DNA and / or RNA, such as recombinant DNA, or mRNA.

[0043] Further preferably, the polynucleotide molecule comprises:

[0044] ① the nucleotide sequence shown in SEQ ID NO: 17, 19, 21, 23, 25, 27, 29, 31, 33, or 35.

[0045] ② the complementary, degenerate, or transcribed sequence of SEQ ID NO: 17, 19, 21, 23, 25, 27, 29, 31, 33, or 35;

[0046] ③ a DNA molecule or mRNA having 75% or more identity with the DNA molecule or mRNA defined in ① or ② and encoding the corresponding protein in the fusion protein.

[0047] The skilled person can easily mutate the nucleotide sequence encoding the fusion protein described above by using known methods, such as methods of directed evolution and point mutation. Those polynucleotides which have been artificially modified and have 75% or more identity with the nucleotide sequence encoding the fusion protein described above are derived from the nucleotide sequence of the present application and are equivalent to the sequence of the present application, as long as they encode the fusion protein described above and have the same function.

[0048] The identity refers to the sequence similarity with the compared nucleic acid sequence. The identity includes, for example, a nucleotide sequence having 75% or more, having 80% or more, or 85% or more, or 90% or more, or 95% or more identity with the nucleotide sequence shown in SEQ ID NO: 17, 19, 21, 23, 25, 27, 29, 31, 33 or 35 of the present application. The identity can be evaluated by naked eyes or computer software. Using computer software, the identity between two or more sequences can be expressed in percentage (%), which can be used to evaluate the identity between related sequences.

[0049] Preferably, the 75% or more identity can be 80%, 85%, 90% or 95% or more identity.

[0050] In a third aspect, the present application provides a vector comprising the polynucleotide molecule of the second aspect.

[0051] The vector refers to a vector capable of carrying foreign DNA, mRNA or a gene of interest into a host cell for amplification and expression. The vector can be a cloning vector or an expression vector, including but not limited to: plasmid, bacteriophage (such as lambda phage or M13 filamentous phage, etc.), cosmid (i.e. cos plasmid), Ti plasmid, viral vector (such as retrovirus (including lentivirus), adenovirus, adeno-associated virus, etc.). In a specific embodiment of the present application, the vector is plasmid pcDNA3.4.

[0052] In a fourth aspect, the present application provides a host cell comprising the polynucleotide molecule of the second aspect or the vector of the third aspect.

[0053] Preferably, the host cell (also referred to as recipient cell) is a eukaryotic cell. The host cell can be understood not only as a specific recipient cell, but also as the progeny of such a cell, which can not necessarily be identical to the original parent cell as a result of natural and accidental or deliberate mutations and / or changes, but still falls within the scope of the host cell.

[0054] Further preferably, the host cell can be a plant cell or an animal cell. Suitable host cells are known in the art. The plant cell can be a cell of Arabidopsis thaliana, Nicotiana tabacum, Zea mays, Oryza sativa, Triticum aestivum, etc., but is not limited thereto. The animal cell can be a mammalian cell, such as a Chinese hamster ovary cell (CHO cell), an African green monkey kidney cell (Vero cell), a baby hamster kidney cell (BHK cell), a mouse mammary carcinoma cell (C127 cell), a human embryonic kidney cell (HEK293 cell), a human HeLa cell, a fibroblast cell, a bone marrow cell line, a T cell, an NK cell, a pig kidney cell (PK15 cell), a pig lung alveolar macrophage cell (PAM cell), a pig small intestine epithelial cell (IPEC-1 cell), a pig testis cell (ST cell), an avian cell (e.g., a chicken or duck cell), an amphibian cell (e.g., a Xenopus laevis cell or an Andrias davidianus cell), a fish cell (e.g., a grass carp, a carp, a rainbow trout, or a catfish cell), an insect cell (e.g., an Sf21 cell or an Sf-9 cell), etc., but is not limited thereto. In one or more embodiments of the present application, the host cell is a CHO-K1 cell.

[0055] In a fifth aspect, the present application provides a method for preparing the fusion protein of the first aspect, the method comprising introducing the polynucleotide molecule of the second aspect or the vector of the third aspect into a host cell, and recovering the fusion protein of the first aspect from the cell or cell culture.

[0056] Preferably, the method comprises screening for a monoclonal cell strain with high and stable expression.

[0057] In a sixth aspect, the present application provides a pharmaceutical composition comprising the fusion protein of the first aspect, or the polynucleotide molecule of the second aspect, or the vector of the third aspect, or the host cell of the fourth aspect.

[0058] Optionally, the pharmaceutical composition further comprises pharmaceutically acceptable adjuvants and excipients.

[0059] In a seventh aspect, the present application provides use of the fusion protein, polynucleotide molecule, vector or host cell of any of the above aspects, the use comprising any of: (1) use in the manufacture of a product for preventing and / or treating a porcine viral disease; (2) use in a product for inducing an immune response to a porcine viral antigen; (3) use in preventing and / or treating a porcine viral disease; (4) use in inducing an immune response to a porcine viral antigen. Preferably, the porcine viral disease is PEDV or PRRSV infection.

[0060] In an eighth aspect, the present application provides a method of treating or preventing porcine epidemic diarrhea virus (PEDV) infection in a subject, the method comprising administering to the subject the fusion protein, polynucleotide molecule, vector or host cell of any of the above aspects or a medicament obtained or prepared therefrom. The subject is preferably a pig, more preferably a piglet, even more preferably a piglet not more than 2 months old. For example, the subject can be a piglet of about 1, 2, 3, 4, 5, 6, 7 days old, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks or 8 weeks old, and can also be in other age ranges. The administration comprises delivery to the subject by intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual or suppository administration, and the like standard administration techniques. Preferably, the delivered medicament can comprise 0.1 mg - 10 mg of the fusion protein of the present application per dose as an active ingredient, for example about 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg or any intermediate value as long as an effective response can be produced in the subject. Alternatively, the delivered medicament can comprise 100 μg / kg - 2000 μg / kg of the fusion protein of the present application per dose as an active ingredient based on the body weight of the subject, for example about 100 μg / kg, 200 μg / kg, 300 μg / kg, 400 μg / kg, 500 μg / kg, 600 μg / kg, 700 μg / kg, 800 μg / kg, 900 μg / kg, 1000 μg / kg, 1100 μg / kg, 1200 μg / kg, 1300 μg / kg, 1400 μg / kg, 1500 μg / kg, 1600 μg / kg, 1700 μg / kg, 1800 μg / kg, 1900 μg / kg, 2000 μg / kg or any intermediate value as long as an effective response can be produced in the subject.

[0061] The benefits of the present application include, but are not limited to:

[0062] The fusion protein of the present application can rapidly activate the immune system activity in pigs, activate the antiviral response, effectively resist the invasion of external viruses, and prevent the occurrence of pig viral diseases.

[0063] The fusion protein of the present application can inhibit the replication of viruses in pigs, can destroy the structure of viruses, and treat pig viral diseases.

[0064] Compared with the existing fusion protein, the fusion protein of the present application has low ED50 value and EC50 value, longer half-life, higher drug efficacy, and better safety, and the fusion protein of the present application has broad-spectrum antiviral function, can safely and efficiently prevent and control pig viral diseases. BRIEF DESCRIPTION OF DRAWINGS

[0065] Figure 1 shows the structure of various fusion proteins of pig IFN-λ1.

[0066] Figure 2 shows the SDS-PAGE electrophoretogram of the fusion protein of pig IFN-λ1. Figure A: lanes 1, 2 and 3 are non-reducing electrophoretic bands of rPoIFN-λ1 / IFN-γ / Fc, rPoIFN-λ1 / Fc / IFN-γ and rPoIFN-γ / Fc / IFN-λ1, respectively. Lane 4 is maker. Lanes 5, 6 and 7 are reducing electrophoretic bands of rPoIFN-λ1 / IFN-γ / Fc, rPoIFN-λ1 / Fc / IFN-γ and rPoIFN-γ / Fc / IFN-λ1, respectively. Figure B: lanes 1 and 2 are non-reducing electrophoretic bands of rPoIFN-λ1 / PSA / IFN-γ and rPoIFN-γ / PSA / IFN-λ1, respectively. Lane 3 is maker. Lanes 4 and 5 are reducing electrophoretic bands of rPoIFN-λ1 / PSA / IFN-γ and rPoIFN-γ / PSA / IFN-λ1, respectively. Figure C: lanes 1 and 2 are non-reducing electrophoretic bands of rPoIFN-λ1 / IFN-γ / Fc(KiH) and rPoIFN-γ / IFN-λ1 / Fc(KiH), respectively. Lane 3 is maker. Lanes 4 and 5 are reducing electrophoretic bands of rPoIFN-λ1 / IFN-γ / Fc(KiH) and rPoIFN-γ / IFN-λ1 / Fc(KiH), respectively.

[0067] Figure 3 shows the dose-effect curves of the fusion proteins of porcine IFN-λ1. Figures A-C show the dose-effect curves of rPoIFN-λ1 / IFN-γ / Fc, rPoIFN-λ1 / Fc / IFN-γ and rPoIFN-γ / Fc / IFN-λ1, respectively; rPoIFN-λ1 / PSA / IFN-γ and rPoIFN-γ / PSA / IFN-λ1; and rPoIFN-λ1 / IFN-γ / Fc(KiH) and rPoIFN-γ / IFN-λ1 / Fc(KiH).

[0068] Figure 4 shows the determination results of PRRSV viral titers in the treatment groups of PAM treated with different concentrations of fusion proteins of porcine IFN-λ1.

[0069] Figure 5 shows the determination results of PEDV viral titers in the treatment groups of PK1 treated with different concentrations of fusion proteins of porcine IFN-λ1.

[0070] Figure 6 shows the relative difference fold calculated according to the Cq values in the experiment of the regulation of the antiviral genes in the small intestines of piglets by the fusion proteins.

[0071] Figure 7 shows the results of the experiment of preventing epidemic diarrhea in piglets by the fusion protein rPoIFN-λ1 / PSA.

[0072] Figure 8 shows the diarrhea records of piglets in each group in the experiment of preventing epidemic diarrhea in piglets by the fusion protein rPoIFN-λ1 / PSA.

[0073] Figures 9A-9E show the results of the experiment of treating epidemic diarrhea in piglets by the fusion protein rPoIFN-λ1 / PSA. DETAILED DESCRIPTION

[0074] The technical solutions of the present application will be further described below in combination with the examples and the drawings, and the advantages and characteristics of the present application will be more apparent as the description proceeds. However, it should be understood that the examples are only exemplary and do not limit the scope of the present application.

[0075] The following examples are used to illustrate the present application but are not intended to limit the scope of the present application. If not specifically indicated, the examples are performed according to the conventional experimental conditions, such as Sambrook et al. Molecular Cloning: a Laboratory Manual (2001) or the conditions suggested by the manufacturer's instructions.

[0076] Example 1 Expression and purification of fusion proteins

[0077] The amino acid sequences of porcine IFN-λ1 (Uniprot ID: A0A287A6Q8), porcine IFN-γ (Uniprot ID: P17803), porcine serum albumin (Uniprot ID: P08835), and porcine IgG1 Fc (Gene ID: BAM66306.1) were retrieved, respectively. In some of the fusion proteins, the amino acid at position 147 of porcine IgG1 Fc was mutated from T to W to form an Fc knob, and the amino acids at positions 147, 149, and 190 of porcine IgG1 Fc were mutated from T, L, and Y to S, A, and V to form an Fc hole. Using a GS linker composed of glycine (G) and serine (S), the corresponding fusion protein molecules were designed, respectively. The structural schematic diagram of the fusion proteins is shown in FIG. 1. The specific amino acid and nucleotide sequences are shown in the sequence listing and the following table.

[0078] Table 1 Element / Construct Amino Acid Sequence

[0079] Table 2 Element / Construct Nucleotide Sequence

[0080] The corresponding nucleotide sequences of the fusion proteins were genetically synthesized, and XbaI and AgeI enzyme digestion sites were introduced at the 5' end and 3' end of the synthesized fragments, respectively. The synthesized fragments and the pcDNA3.4 vector were subjected to XbaI and AgeI double digestion, and after recovering the digestion products, the genetically synthesized fragments were inserted into the pcDNA3.4 vector using T4 ligase, respectively, to construct the recombinant expression plasmids of each recombinant protein based on pcDNA3.4.

[0081] The above recombinant expression plasmids were subjected to single digestion using PvuI enzyme, and linearized plasmids were obtained by recovering the digestion products. The linearized plasmids were transfected into CHO-K1 cells according to the Bio-Rad electroporator instruction manual. After 24 hours of transfection, 50 μM GS inhibitor L-amino sulfoxide methionine (MSX) was added to the culture medium. The cells were cultured under MSX selection pressure for 20 days, then the surviving cells were counted, and single clone cells were selected into 96-well plates. The cells were cultured for another 20 days, then the cell counting and single clone cell transfer to 96-well plates were repeated. After the above two rounds of single clone cell screening, it was basically ensured that the final cell strain was a single clone derived cell strain.

[0082] In the bioreactor, the cell culture expression liquid was obtained by fed-batch culture, and the cell culture supernatant was obtained by removing the cells and cell debris through deep filtration membrane filtration. The cell culture supernatant was purified by three steps of anion exchange chromatography, hydrophobic chromatography and complex mode anion exchange chromatography to obtain the target protein meeting the requirements. The purified pig IFN-λ1, pig IFN-γ and pig Fc fusion protein were subjected to SDS-PAGE protein electrophoresis to detect the electrophoretic purity. The results are shown in Figure 2.

[0083] As can be seen from Figure 2, the results show that the protein purity of rPoIFN-λ1 / Fc / IFN-γ is more than 95%, the protein purity of rPoIFN-λ1 / IFN-γ / Fc is 72%, and the protein purity of rPoIFN-γ / Fc / IFN-λ1 is 40%; the protein purity of rPoIFN-λ1 / PSA / IFN-γ is high, which is more than 90%, while rPoIFN-γ / PSA / IFN-λ1 has a hetero-band near the target band of the reduced electrophoresis, and the purity is about 70%; the protein purity of rPoIFN-λ1 / IFN-γ / Fc(KiH) and rPoIFN-γ / IFN-λ1 / Fc(KiH) is similar, both of which are about 70%.

[0084] Example 2 Biological activity detection of fusion protein (cytopathic effect inhibition method)

[0085] The anti-viral activity of each fusion protein was determined by cytopathic inhibition method. PK-15 cells were plated in 96-well cell culture plates at 10,000 cells per well, and then incubated at 37°C in a 5% CO2 cell incubator for 24 h. Then, 4-fold gradient dilutions of rPoIFN-λ1 / IFN-γ / Fc, rPoIFN-λ1 / Fc / IFN-γ, rPoIFN-γ / Fc / IFN-λ1 (100 ng / mL, 25 ng / mL, 6.25 ng / mL, 1.56 ng / mL, 0.39 ng / mL, 0.098 ng / mL, 0.024 ng / mL, 0.0061 ng / mL), rPoIFN-λ1 / PSA / IFN-γ and rPoIFN-γ / PSA / IFN-λ1 (100 ng / mL, 25 ng / mL, 6.25 ng / mL, 1.56 ng / mL, 0.39 ng / mL, 0.098 ng / mL, 0.024 ng / mL, 0.0061 ng / mL), rPoIFN-λ1 / IFN-γ / Fc(KiH) and rPoIFN-γ / IFN-λ1 / Fc(KiH) (100 ng / mL, 25 ng / mL, 6.25 ng / mL, 1.56 ng / mL, 0.39 ng / mL, 0.098 ng / mL, 0.024 ng / mL, 0.0061 ng / mL), and recombinant porcine interferon lambda 1 / pig serum albumin fusion protein (rPoIFN-λ1 / PSA; 1600 ng / mL, 400 ng / mL, 100 ng / mL, 25 ng / mL, 6.25 ng / mL, 1.56 ng / mL, 0.39 ng / mL, 0.098 ng / mL) were added to each well, with 8 replicates for each dilution. After 24 h of incubation at 37°C in a 5% CO2 cell incubator, the cell supernatant was discarded, and 100 TCID50 of VSV virus (vesicular stomatitis virus) was added to each well. After 24 h of incubation at 37°C in a 5% CO2 cell incubator, the cytopathic effect was observed, and the anti-viral activity (ED50) of each sample was calculated according to the Reed-Muench method. 50 VSV virus (vesicular stomatitis virus). A blank control group (without sample and virus) and a positive control (with only virus) were set up, with 8 replicates for each treatment. After 24 h of virus inoculation, the cytopathic effect was observed, and the anti-viral activity (ED 50 value) of each sample was calculated according to the Reed-Muench method.

[0086] The results showed that the ED 50 values (half maximal effective amount) of rPoIFN-λ1 / IFN-γ / Fc, rPoIFN-λ1 / Fc / IFN-γ, rPoIFN-γ / Fc / IFN-λ1, and rPoIFN-λ / PSA were 3.67 ng / mL, 0.89 ng / mL, 11.12 ng / mL, and 32 ng / mL, respectively; the ED 50The ED50 values of rPoIFN-λ1 / IFN-γ / Fc, rPoIFN-λ1 / Fc / IFN-γ, rPoIFN-γ / Fc / IFN-λ1, rPoIFN-λ1 / PSA / IFN-γ, rPoIFN-γ / PSA / IFN-λ1, rPoIFN-λ1 / IFN-γ / Fc(KiH), and rPoIFN-γ / IFN-λ1 / Fc(KiH) were 2.11 ng / mL, 4.01 ng / mL, 0.12 ng / mL, and 0.22 ng / mL, respectively. 50 The results showed that the fusion proteins had equivalent efficacy at a specific concentration and excellent antiviral activity. The half effective amount concentration of some fusion proteins was lower than that of other molecules, and the antiviral activity was better.

[0087] Example 3: Biological activity detection of fusion proteins (reporter gene method)

[0088] The biological activity of each fusion protein was detected by the reporter gene method. The plasmid containing the interferon-stimulated response element and the secreted alkaline phosphatase gene was transfected into HEK293 to construct a stable reporter cell line. HEK293 recombinant cells were inoculated into a 96-well cell culture plate at 50,000 cells per well. At the same time, 10-fold gradient dilutions of rPoIFN-λ1 / IFN-γ / Fc, rPoIFN-λ1 / Fc / IFN-γ, rPoIFN-γ / Fc / IFN-λ1, rPoIFN-λ1 / PSA / IFN-γ, rPoIFN-γ / PSA / IFN-λ1, rPoIFN-λ1 / IFN-γ / Fc(KiH), and rPoIFN-γ / IFN-λ1 / Fc(KiH) (2000 ng / mL, 200 ng / mL, 20 ng / mL, 2 ng / mL, 0.2 ng / mL, 0.02 ng / mL, 0.002 ng / mL, 0.0002 ng / mL, and 0.00002 ng / mL) and rPoIFN-λ1 / PSA (2000 ng / mL, 200 ng / mL, 20 ng / mL, 2 ng / mL, 0.2 ng / mL, and 0.02 ng / mL) were added, and the plate was incubated in a 37°C, 5% CO2 cell incubator for 24 h. Then, the cell supernatant was collected and alkaline phosphatase detection reagent (QUANTI-Blue TM , Invivogen, rep-qbs) was added. The absorbance value was read at 630 nm using a microplate reader, and the curve was fitted according to the four-parameter curve equation, and the EC 50 value was calculated. The results are shown in FIG. 3.

[0089] As shown in FIG. 3, the fusion proteins had biological activity, and the EC 50The values are: 1.743 ng / mL (rPoIFN-λ1 / IFN-γ / Fc), 0.1341 ng / mL (rPoIFN-λ1 / Fc / IFN-γ), 4.074 ng / mL (rPoIFN-γ / Fc / IFN-λ1), 0.328 ng / mL (rPoIFN-λ1 / PSA / IFN-γ), 0.801 ng / mL (rPoIFN-γ / PSA / IFN-λ1), 0.0230 ng / mL (rPoIFN-λ1 / IFN-γ / Fc(KiH)), 0.0343 ng / mL (rPoIFN-γ / IFN-λ1 / Fc(KiH)), and 15.88 ng / mL (rPoIFN-λ / PSA).

[0090] Example 4 Inhibition of PRRSV proliferation by fusion proteins in porcine primary alveolar macrophages

[0091] Fresh porcine lung was collected from a pig farm, and porcine primary alveolar macrophages (PAM) were collected. PAM cells were seeded in 96-well cell plates with RMPI1640 medium, and then the medium was replaced with RMPI1640 medium containing 1 μg / mL, 10 μg / mL or 100 μg / mL of rPoIFN-λ1 / Fc / IFN-γ, rPoIFN-λ1 / PSA / IFN-γ, rPoIFN-λ1 / IFN-γ / Fc(KiH), 100 μg / mL of rPoIFN-λ / PSA or negative control (PBS) containing 2% FBS, respectively, 3 replicates per group, and incubated in a 37°C, 5% CO2 cell incubator for 24 h. Then, PRRSV JXwn06 virus was added to each well (MOI = 0.01). After 24 hours of continuous incubation, the virus solution in the cell well was collected by repeated freezing and thawing 3 times (-80°C freezer freezing, slow thawing on ice), and the TCID50 of PRRSV was detected 50 .

[0092] The single-layer African green monkey embryo kidney cells (Marc-145 cells) were added with 100 μL of 10-fold diluted PRRSV virus liquid, 8 repeats for each dilution, cultured for 48 h, then pre-cooled anhydrous ethanol at -20 °C was added to fix the cells for 15 min; the anhydrous ethanol was discarded (Shanghai test, 10009218), the cells were washed with PBS solution for 3 times, then 2 mL of PRRSV N protein mouse monoclonal antibody (prepared by the Key Laboratory of Animal Epidemiology of the Ministry of Agriculture and Rural Affairs of China Agricultural University, diluted with PBS at 1:1000) was added to each well, and incubated in the incubator for 1 h; the N protein monoclonal antibody diluent was discarded, washed with PBS for 3 times, 2 mL of FITC-labeled goat anti-mouse IgG (Jackson ImmuResearch, #115-295-003, diluted with PBS at 1:200) was added to each well, and incubated in the 37 °C incubator for 1 h; the secondary antibody diluent was discarded, washed with PBS for 3 times, and observed under a fluorescence microscope with 488 nm excitation light, photographed and analyzed, and the TCID50 of the sample was calculated according to the Reed-Muench method. The results are shown in FIG. 4.

[0093] As can be seen from FIG. 4, the above-mentioned fusion protein can inhibit the proliferation of PRRSV virus, and the inhibitory effect increases with the increase of the dose.

[0094] Example 5 Inhibition of PEDV proliferation in porcine kidney cells by fusion protein

[0095] Porcine kidney cells (PK1 cells) were recovered and inoculated in a 6-well cell plate. After growing, the culture medium was replaced with DMEM medium (without FBS) containing 1 μg / mL, 10 μg / mL, 100 μg / mL of rPoIFN-λ1 / Fc / IFN-γ, rPoIFN-λ1 / PSA / IFN-γ, rPoIFN-λ1 / IFN-γ / Fc (KiH), 100 μg / mL, 100 μg / mL of rPoIFN-λ1 / PSA or negative control (PBS), 3 repeats for each group, and incubated in a 37 °C, 5% CO2 cell incubator for 36 h. Then, PEDV BJ2011C virus (MOI = 0.01) was added to each well. After continuing to incubate for 24 hours, the virus liquid in the 3 groups of cell wells was collected by repeated freezing and thawing 3 times (-80 °C refrigerator freezing, slow thawing on ice), and the TCID50 of PEDV in the virus liquid was detected 50 .

[0096] In single-layer African green monkey kidney cells (Vero cells), 100 μL of 10-fold diluted PEDV virus liquid was added, 8 replicates were made for each dilution, and cultured for 48 h. Then, -20°C pre-cooled anhydrous ethanol was added for 15 min for cell fixation; the anhydrous ethanol was discarded (Shanghai Chempur, 10009218), the cells were washed with PBS for 3 times, 2 mL of PEDV N protein mouse monoclonal antibody (prepared by the Key Laboratory of Animal Epidemiology of the Ministry of Agriculture and Rural Affairs of China Agricultural University, diluted with PBS at 1:1000) was added to each well, and incubated in a incubator for 1 h; the N protein monoclonal antibody diluent was discarded, washed with PBS for 3 times, 2 mL of FITC-labeled goat anti-mouse IgG (Beijing Zhongshanjinqiqiobitotechnology Co., Ltd., ZF-0312, diluted with PBS at 1:200) was added to each well, and incubated in a 37°C incubator for 1 h; the secondary antibody diluent was discarded, washed with PBS for 3 times, observed under a fluorescence microscope with 488 nm excitation light, photographed and analyzed, and the TCID of the sample was calculated according to the Reed-Muench method 50 The results are shown in Fig. 5.

[0097] As can be seen from Fig. 5, the above-mentioned fusion protein can inhibit the proliferation of PEDV virus, and the inhibition effect increases with the increase of the dosage. At the same time, the above-mentioned fusion protein can significantly inhibit the proliferation of PEDV in PK1, and when the concentration reaches 100 μg / mL, the PEDV virus is reduced by 1 titer. It shows that the fusion protein of the present application has significant anti-PEDV virus activity.

[0098] Example 6 Effect of fusion protein on antiviral gene regulation in small intestine of piglets

[0099] In order to explore whether the fusion protein of the present application can be delivered to the small intestinal epithelial cells of piglets to induce the expression of interferon effector genes, the following experiments were carried out.

[0100] Experimental steps:

[0101] Treatment

[0102] On Day 0 in the morning, 9 healthy piglets within 1 day of age were selected, regardless of gender, and randomly divided into 3 groups: a control group, a low-dose group and a high-dose group. Mark, weigh and record the body weight. After the birth of the 9 piglets, they were immediately injected with the drug at the same time by neck muscle injection. The low-dose group was injected with 1 mL of 0.1 mg of rPoIFN-λ1 / PSA per pig, the high-dose group was injected with 1 mL of 0.4 mg of rPoIFN-λ1 / PSA per pig, and the control group was injected with 1 mL of blank preparation per pig.

[0103] Feeding

[0104] Time: Day 0-Day 2.

[0105] The piglets were fed according to the daily feeding method.

[0106] Sampling

[0107] • Time: Day 2 morning.

[0108] • 48 hours after drug injection, the piglets were sacrificed. The jejunum of each pig was removed. The jejunum was placed in a 50 mL centrifuge tube containing cold PBS and placed on ice. The cells were isolated within 3 hours to extract RNA.

[0109] Detection

[0110] Intestinal epithelial cell isolation:

[0111] 1) Wash: After removing the mesentery, cut into 5-6 cm segments and rinse 3 times in a culture dish containing cold PBS to remove blood clots and dirt.

[0112] 2) Inversion: Insert the forceps into one end of the small intestine, and pierce and clamp the other end. Turn the small intestine upside down from the bottom of the forceps to expose the inside of the small intestine, and wash the contents of the small intestine in PBS.

[0113] 3) Scraping: Scrape the small intestine with a blade to obtain epithelial cells. Centrifuge in PBS at 2000 rpm for 10 minutes. Discard the supernatant and resuspend in 5 mL PBS.

[0114] RNA extraction

[0115] 1) (In a clean bench) extract RNA according to the instructions of the RNA extraction kit (Vazyme RC01), and finally elute with 50 μL water.

[0116] 2) Take 2.5 μL of each tube to detect the concentration. The remaining samples were stored at -80°C.

[0117] 3) Use a microspectrophotometer (Thermo Scientific NanoDrop) to detect the concentration, with a sample size of 2 μL.

[0118] Reverse transcription

[0119] 1) Take 2 ng of RNA and add RNase-Free water to 16 μL

[0120] 2) Add 4 μl of qPCR reverse transcription premix (Vazyme R323-01) and heat at 37 degrees for 15 minutes → 85 degrees for 5 seconds

[0121] qPCR

[0122] 1) Configure the reaction system according to the instructions of the probe quantitative PCR detection kit (Vazyme Q131-02)

[0123] 2) Quantification with a fluorescent quantitative PCR instrument (Bio-Rad CXF96) (dissociation curve uses machine default)

[0124] Experimental results

[0125] The relative difference fold calculated according to the Cq value is shown in Figure 6.

[0126] The results show that 48 hours after intraperitoneal injection of 0.1 mg or 0.4 mg rPoIFN-λ1 / PSA in 1-day-old piglets, the antiviral genes ISG56, OAS and MxA are significantly up-regulated, and the up-regulation fold of the high dose is higher. rPoIFN-λ1 / PSA can be delivered to the small intestinal epithelial cells of piglets to induce the expression of interferon effector genes.

[0127] Example 7 Effect of fusion protein on prevention of piglet epidemic diarrhea

[0128] In order to explore the preventive effect of the fusion protein of the present application on PEDV infection causing diarrhea in suckling piglets, the following experiments were carried out.

[0129] Test materials

[0130] Animals: 2-day-old newborn piglets, weighing about 1 kg

[0131] Strain: PEDV BJ2011C (Key Laboratory of Animal Epidemiology of the Ministry of Agriculture and Rural Affairs, China Agricultural University)

[0132] Drugs: rPoIFN-λ1 / PSA; porcine IFN-α (R&D systems 17105-1)

[0133] Test scheme

[0134] Preparation before challenge

[0135] Thirty healthy piglets with negative PEDV antigen and antibody detection were divided into six groups: control group, challenge group, IFN-α treatment group, low-dose rPoIFN-λ1 / PSA treatment group, medium-dose rPoIFN-λ1 / PSA treatment group and high-dose rPoIFN-λ1 / PSA treatment group. Label, weigh the body weight and record. Use milk powder to feed at 6:00, 12:00, 16:00, 20:00, 24:00 every day.

[0136] Drug treatment and challenge

[0137] The drug treatment and challenge are shown in Table 3.

[0138] Challenge method: oral. Slowly hit into the mouth by 10 mL syringe.

[0139] Table 3. Drug treatment and challenge schedule

[0140] Symptom observation

[0141] The diarrhea degree of piglets was scored and recorded during feeding, and the scoring criteria are shown in Table 4. The death of piglets was recorded every day.

[0142] Detection of toxin excretion

[0143] The anal swabs of piglets in each group were collected every day after challenge.

[0144] The swabs were placed in 1 mL PBS, vortexed, centrifuged at 10000 r / min for 5 min at 4°C, and the supernatant was taken to extract RNA. RT-PCR was performed to analyze the toxin excretion of piglets.

[0145] Table 4. Scoring criteria for diarrhea degree of piglets

[0146] Test results

[0147] Effect of drugs on diarrhea symptoms

[0148] The diarrhea of piglets in each test group was scored, and a diarrhea score graph was drawn (Figure 7A, where VJT-Low / Medium / High represents the low, medium, and high dose groups of rPoIFN-λ1 / PSA, respectively).

[0149] The results showed that there was no significant difference in the development and severity of diarrhea between the IFN-α treatment group and the challenge group; compared with the challenge group, the development and severity of diarrhea in piglets in the low-dose rPoIFN-λ1 / PSA treatment group were significantly reduced, and the degree of diarrhea in piglets in the high-dose treatment group was lighter than that in the challenge group within 5 days after challenge. The diarrhea record of piglets is shown in Figure 8.

[0150] Effect of drugs on survival of piglets

[0151] The survival of piglets in each experimental group is shown in Figure 7B.

[0152] The results showed that all piglets in the challenge group died 4-6 days after challenge (4 dpi-6 dpi), and the survival rate was 0% (0 / 5); piglets in the IFN-α treatment group started to die at 6 dpi, and the survival rate was 40% (2 / 5) at the end of the experiment (7 dpi); the survival rates of piglets in the low, medium, and high dose rPoIFN-λ1 / PSA treatment groups were 80% (4 / 5), 75% (3 / 4), and 80% (4 / 5), respectively. No piglets in the control group died by the end of the experiment.

[0153] Effect of drugs on toxin excretion of piglets

[0154] The anal swabs of the piglets in each test group were collected, and RT-PCR was used to detect the fecal shedding of the piglets, as shown in FIG. 3. The fecal shedding of the piglets in the challenge group was detected before death; the fecal shedding of the piglets in the low-dose rPoIFN-λ1 / PSA treatment group was not detected within 1 dpi, and the fecal shedding began to appear at 2 dpi, and only 2 piglets were detected to have the fecal shedding at 6 dpi-7 dpi; the fecal shedding of the piglets in the medium-dose rPoIFN-λ1 / PSA treatment group was detected in 2 piglets within 1 dpi, and the fecal shedding began to appear at 2 dpi, and the fecal shedding lasted to 6 dpi; the fecal shedding of the piglets in the high-dose rPoIFN-λ1 / PSA treatment group was not detected within 2 dpi and 5 dpi, and the fecal shedding of 1 piglet was detected at 3 dpi-4 dpi, and the fecal shedding of 1 piglet was still not detected at 6 dpi-7 dpi. The fecal shedding of the piglets in the IFN-α treatment group was detected within 2 dpi, and the fecal shedding disappeared at 3 dpi-4 dpi, and 1 piglet was still detected to have the fecal shedding among the 2 surviving piglets at the end of the experiment (7 dpi).

[0155] CONCLUSION

[0156] The treatment of rPoIFN-λ1 / PSA at different doses can delay the occurrence of diarrhea in piglets caused by PEDV infection to a certain extent, and significantly reduce the acute death of piglets caused by diarrhea. At the same time, the treatment of rPoIFN-λ1 / PSA can delay and reduce the fecal shedding of infected piglets through the intestinal tract.

[0157] Example 8 Effect of the fusion protein on the treatment of piglet epidemic diarrhea

[0158] The inventors studied the effect of the fusion protein of the present application on the treatment of piglet diarrhea caused by PEDV through a challenge model, and the following experiments were conducted.

[0159] Experimental animals

[0160] Animals: 7-day-old healthy piglets (PEDV, blue ear virus, African swine fever virus, transmissible gastroenteritis virus, and rotavirus negative).

[0161] PEDV strain

[0162] Strain: PEDV-JY.

[0163] Test drug

[0164] rPoIFN-λ1 / PSA, lyophilized powder injection, 4 mg / branch. Dissolved with sterile water for injection, and diluted with physiological saline.

[0165] Experimental scheme

[0166] Experimental grouping and piglet feeding

[0167] Thirty-two healthy piglets were grouped and numbered according to Table 5. The piglets were raised in a constant temperature isolator, and the temperature was set to 30°C. The piglets were fed with milk replacer at 6:00, 12:00, 16:00, 20:00 and 24:00 every day.

[0168] Challenge of piglets

[0169] The piglets were transported to the experimental site 4 days after birth and challenged at 7 days of age. The challenge was performed by oral administration of 5 mL of the virus diluted in culture medium (DMEM) at a dose of 1 x 10 3 TCID 50 The control piglets were orally administered with 5 mL of DMEM. The challenge time was recorded as Dpi 0.

[0170] Drug treatment

[0171] Immediately after the challenge of the challenge group, the piglets in the treatment group were intraperitoneally injected with 200 μg / kg (low dose group, Low), 1000 μg / kg (high dose group, High) of rPoIFN-λ1 / PSA or normal saline (challenge control group), and observed for 8 days after administration. The administration method was intraperitoneal injection. The administration dose was calculated according to the average body weight before administration.

[0172] Table 5. Grouping and treatment of test animals

[0173] Clinical observation

[0174] Survival rate

[0175] The survival of the piglets was observed and recorded at regular time intervals every day after the challenge until the end of the experiment (Dpi 8).

[0176] Autopsy

[0177] (1) On the second day after the challenge (Dpi 2), 2 piglets were selected from each group for autopsy, intestinal changes were observed, three sections of small intestine were taken, viral nucleic acid was detected, and tissue sections (HE and IHC) were prepared. Autopsy was performed on surviving piglets at the end of the experiment. Autopsy was not performed on piglets that died at other times.

[0178] (2) Spleen, liver, and intestine (three sections) were collected and frozen, interferon stimulating factor was detected by qPCR, and relative quantification method was used for quantification.

[0179] Diarrhea degree (diarrhea index)

[0180] The diarrhea of the piglets was observed and recorded at regular time intervals every day after the challenge, and was recorded as diarrhea or not until Dpi 8.

[0181] Detection of viral nucleic acid by anal swab

[0182] After challenge, anal swabs were collected from piglets at fixed times every day from Dpi 1 to 8 to detect PEDV virus.

[0183] Weighing

[0184] Weigh and record the weight of the piglets every two days.

[0185] Animal disposal

[0186] Animals that died during the experiment were disposed of in a harmless manner. The remaining animals were euthanized and disposed of in a harmless manner after the experiment.

[0187] Test results

[0188] Survival rate:

[0189] The results showed that 4 animals died in the challenge group, while 2 survived until the end of the experiment; 1 animal died in the low-dose group on the 7th day after challenge, while all animals in the high-dose group survived; therefore, it is suggested that the test product has a good therapeutic effect on PED (Figure 9A).

[0190] weight:

[0191] The average daily body weight of the control group increased steadily. The low-dose group showed a flat weight loss on day 6, while both the high-dose and challenge groups showed weight loss on day 4, but the challenge group experienced a greater weight loss. On day 6, the weight loss in the challenge group was less than that in the treatment group because individuals experiencing rapid weight loss died (Figure 9B).

[0192] Anal swab virus titer:

[0193] PEDV nucleic acid could be detected in the challenge group on day 1, and the Ct value decreased significantly after day 3; PEDV nucleic acid could not be detected in most of the treatment groups after day 3, and the Ct values ​​were all above 30 (Figure 9C).

[0194] Comparison of pathological changes:

[0195] Compared with the challenge group, the intestinal pathological changes of piglets examined on day 3 showed that the intestinal damage in the treatment group was significantly less (Figure 9D).

[0196] Relative quantification of JAK-STAT pathway-related genes (D3):

[0197] OALS mRNA was generally upregulated; the upregulation of this gene was significant in the duodenum; it was also upregulated in the jejunum and ileum, but mostly not significantly; in the liver and spleen, it was upregulated in the high-dose group. ISG15 mRNA expression was upregulated more significantly (Figure 9E).

[0198] in conclusion

[0199] Different doses of rPoIFN-λ1 / PSA treatment can delay the occurrence of diarrhea caused by PEDV infection in piglets to some extent, and significantly reduce the acute death of piglets due to diarrhea. rPoIFN-λ1 / PSA treatment can reduce the virus load of infected piglets through the intestinal tract, and the virus load under two doses is at a negative level. From the survival curve, administration (rPoIFN-λ1 / PSA) significantly reduces the mortality rate of PEDV. The weight loss of the administration group is delayed in time relative to the challenge control group. From the virus titer of anal swabs and intestinal lesions, administration can delay the infection rate of PEDV and reduce the degree of intestinal lesions. PEDV can inhibit the production of type I interferon in the host, and interferon-stimulated genes (ISGs) are the main effectors of interferon to play an antiviral role, and in this experiment, administration can significantly up-regulate the expression of ISG15 and OALS genes.

[0200] The preferred embodiments of the present application are described in detail above, but the present application is not limited to the specific details in the above-described embodiments, and various simple modifications can be made to the technical solutions of the present application within the technical concept of the present application, and these simple modifications all belong to the protection scope of the present application.

Claims

1. A fusion protein comprising recombinant porcine interferon lambda 1, characterized in that: The fusion protein further comprises porcine interferon gamma, porcine Fc, porcine Fc(KiH) and / or porcine serum albumin (PSA), wherein the porcine Fc(KiH) is composed of a knob chain and a hole chain.

2. The fusion protein of claim 1, wherein: The fusion protein is fused from recombinant porcine interferon lambda 1 and porcine serum albumin, fused from recombinant porcine interferon lambda 1, porcine interferon gamma and porcine Fc, fused from recombinant porcine interferon lambda 1, porcine interferon gamma and porcine serum albumin, or fused from recombinant porcine interferon lambda 1, porcine interferon gamma and porcine Fc(KiH).

3. The fusion protein of claim 1 or 2, wherein: The porcine interferon lambda 1 and the porcine serum albumin are sequentially connected from the N-terminus to the C-terminus of the fusion protein; or The porcine interferon lambda 1, the porcine interferon gamma and the porcine Fc are sequentially connected from the N-terminus to the C-terminus of the fusion protein, or the porcine interferon lambda 1, the porcine Fc and the porcine interferon gamma are sequentially connected from the N-terminus to the C-terminus of the fusion protein, or the porcine interferon gamma, the porcine Fc and the porcine interferon lambda 1 are sequentially connected from the N-terminus to the C-terminus of the fusion protein; or The porcine interferon lambda 1, the porcine serum albumin and the porcine interferon gamma are sequentially connected from the N-terminus to the C-terminus of the fusion protein, or the porcine interferon gamma, the porcine serum albumin and the porcine interferon lambda 1 are sequentially connected from the N-terminus to the C-terminus of the fusion protein; or The porcine interferon lambda 1 is connected to the knob chain of the porcine Fc(KiH), and the porcine interferon gamma is connected to the hole chain of the porcine Fc(KiH), or the porcine interferon gamma is connected to the knob chain of the porcine Fc(KiH), and the porcine interferon lambda 1 is connected to the hole chain of the porcine Fc(KiH), Wherein, each part in the fusion protein is connected to each other through a Linker or directly.

4. The fusion protein of any one of claims 1 to 3, wherein: The amino acid sequence of the porcine interferon lambda 1 is shown in SEQ ID NO: 1 or has at least 80% identity thereto; The amino acid sequence of the porcine interferon gamma is shown in SEQ ID NO: 3 or has at least 80% identity thereto; The amino acid sequence of the porcine serum albumin is shown in SEQ ID NO: 5 or has at least 80% identity thereto; The amino acid sequence of the porcine Fc is shown in SEQ ID NO: 7 or has at least 80% identity thereto; The amino acid sequence of the knob chain of the porcine Fc(KiH) is shown in SEQ ID NO: 9 or has at least 80% identity thereto; and / or The amino acid sequence of the hole chain of the porcine Fc(KiH) is shown in SEQ ID NO: 11 or has at least 80% identity thereto.

5. The fusion protein of any one of claims 1 to 4, wherein: The fusion protein has an amino acid sequence as set forth in SEQ ID NO: 17, 19, 21, 23, 25, 27, 29, 31, 33, or 35, or an amino acid sequence having at least 80% identity to the amino acid sequence as set forth in SEQ ID NO: 17, 19, 21, 23, 25, 27, 29, 31, 33, or 35.

6. A polynucleotide molecule, characterized by: The polynucleotide molecule encodes the fusion protein of any one of claims 1 to 5.

7. The polynucleotide molecule of claim 6, wherein: The polynucleotide molecule has a nucleotide sequence as set forth in SEQ ID NO: 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36; a complement, degenerate, or transcribed sequence of SEQ ID NO: 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36; or a nucleotide sequence having at least 75% identity to SEQ ID NO: 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36 or a complement, degenerate, or transcribed sequence thereof and encoding the corresponding protein in the fusion protein.

8. A vector, characterized by: The vector comprises the polynucleotide molecule of any one of claims 6 to 7.

9. A host cell, characterized in that: The host cell comprises the polynucleotide molecule of any one of claims 6 to 7 or the vector of claim 8.

10. A method of producing the fusion protein according to any one of claims 1 to 5, characterized by: The method of preparation comprises introducing the polynucleotide molecule of any one of claims 6 to 7 or the vector of claim 8 into a host cell, and recovering the fusion protein of any one of claims 1 to 5 from the cell or cell culture.

11. A pharmaceutical composition, characterized by: The pharmaceutical composition comprises the fusion protein of any one of claims 1 to 5, or the polynucleotide molecule of any one of claims 6 to 7, or the vector of claim 8.

12. Use of the fusion protein of any one of claims 1 to 5, the polynucleotide molecule of any one of claims 6 to 7, the vector of claim 8, or the host cell of claim 9, wherein the use comprises any one of: (1) use in preventing and / or treating a porcine viral disease; (2) use in inducing an immune response to a porcine viral antigen.

13. The use of claim 12, wherein the porcine viral disease comprises porcine epidemic diarrhea virus (PEDV) or porcine reproductive and respiratory syndrome virus (PRRSV) infection.

14. A method of treating or preventing porcine epidemic diarrhea virus (PEDV) infection in a subject, the method comprising administering to the subject the fusion protein of any one of claims 1 to 5.

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