Recombinant porcine interferon fusion protein as well as preparation method and application thereof

Abstract

The invention discloses a recombinant porcine interferon fusion protein as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The recombinant porcine interferon fusion protein is obtained by operably connecting porcine interferon lambda and a protein C activating factor through a linker; wherein the amino acid sequence of the protein C activating factor is as shown in SEQ ID NO: 5, and the amino acid sequence of the linker is as shown in SEQ ID NO: 7. After the porcine interferon lambda and the protein C activating factor are operably connected, the enrichment capacity of the obtained fusion protein in pichia pastoris is remarkably improved; meanwhile, after multi-copy expression is carried out on a target gene, enrichment of the fusion protein in pichia pastoris can be further improved, and in addition, the fusion protein is long in half-decay length and good in stability, so that the fusion protein has a good application prospect in prevention and treatment of porcine viral epidemic diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to a recombinant porcine interferon fusion protein and a preparation method and application thereof. Background Art

[0002] Interferon (IFN) is a multifunctional secretory protein produced by animals, involved in antiviral, cell growth regulation, and immune activation. Interferon has multiple biological activities, including enhancing the activity of natural killer cells (NK cells), macrophages, and T lymphocytes, as well as antiviral, anti-tumor, and immunomodulatory functions. It is widely used as a broad-spectrum antiviral agent. Interferon does not directly kill or inhibit viruses. Instead, it inhibits viral replication by interacting with cell surface receptors, inducing the production of antiviral proteins.

[0003] Interferons are primarily classified into types I, II, and III. Type III interferons primarily include IFN-λ1 (IL-29), IFNλ-2 (IL-28α), and IFN-λ3 (IL-28β). Their functional receptor complex is a heterodimer composed of IL-28Rα and IL-10Rβ (also known as CRF2-4). A fourth type III interferon, IFN-λ4, was later discovered, located upstream of IFN-λ3. Porcine type III interferon (PoIFN-λ) is primarily composed of two subtypes: IFN-λ1 and IFN-λ3.

[0004] The swine industry requires extensive use of exogenous PoIFN-λ to enhance pigs' antiviral resistance. To date, a variety of exogenous protein expression systems have been used to enhance the production and activity of PoIFN-λ, including Escherichia coli, Lactobacillus casei, Pichia pastoris, and mammalian cells. Pichia pastoris is a promising host cell due to its eukaryotic nature, efficient secretion capacity, and ability to cultivate high cell densities. Furthermore, under the regulated AOX1 pathway, methanol as the sole carbon and energy source can further reduce industrial production costs. Furthermore, the simplified protein purification process further reduces production costs.

[0005] Currently, Pichia pastoris has been used to express PoIFN-λ, but there are problems such as the interferon PoIFN-λ cannot be enriched in Pichia pastoris. Summary of the Invention

[0006] The purpose of the present invention is to provide a recombinant porcine interferon fusion protein and its preparation method and application, so as to solve the problem that when PoIFN-λ is expressed in Pichia pastoris, the interferon PoIFN-λ cannot be enriched in Pichia pastoris.

[0007] In a first aspect, the present invention provides a recombinant porcine interferon fusion protein, which is obtained by operably linking porcine interferon λ and protein C activator through a linker; wherein the amino acid sequence of the protein C activator is shown in SEQ ID NO: 5, and the amino acid sequence of the linker is shown in SEQ ID NO: 7.

[0008] In the present invention, the inventors have found that after operably linking porcine interferon λ and protein C activator, the ability of the resulting fusion protein to be enriched in Pichia pastoris is significantly improved.

[0009] In some embodiments, the C-terminus of protein C activator is operably linked to the N-terminus of porcine interferon λ via a linker.

[0010] In some embodiments, the fusion protein is selected from any of the following: A1) having an amino acid sequence as shown in SEQ ID NO: 1; A2) a protein having the same biological activity obtained by changing the amino acid sequence as shown in SEQ ID NO: 1 by one or more amino acid residues, wherein the change includes at least one of the following: B1) substitution; B2) deletion; B3) addition.

[0011] The above-mentioned fusion protein provided by the present invention can be a natural, recombinant or synthetic active polypeptide, and the active polypeptide can be a naturally purified product, a chemically synthesized product, or a product produced using recombinant technology from a prokaryotic host (such as Escherichia coli) or a eukaryotic host (such as yeast, higher plants).

[0012] In a second aspect, the present invention provides a nucleic acid molecule encoding any one of the above-mentioned recombinant porcine interferon fusion proteins.

[0013] In some embodiments, the nucleic acid molecule is any of the following: 1) a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO: 2; 2) a nucleic acid molecule that hybridizes with the nucleic acid molecule defined in 1) under stringent conditions and encodes any of the above-mentioned fusion proteins; 3) a nucleic acid molecule that has more than 90% sequence identity with the nucleic acid molecule defined in 1) or 2) and encodes any of the above-mentioned fusion proteins.

[0014] The nucleic acid molecules provided by the present invention can usually be obtained by PCR amplification or artificial synthesis.

[0015] As used herein, the term "hybridize under stringent conditions" refers to the hybridization of two nucleic acid molecule fragments under standard hybridization conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (1989) (Cold Spring Lane Laboratories, New York, USA), in the section "Expression of Cloned Genes in Escherichia coli." Such conditions include, for example, hybridization in 6.0×SSC at 45°C, followed by a wash step at 2×SSC at 50°C. To select the stringency, the salt concentration in the wash step can be selected, for example, between 2.0×SSC at 50°C for low stringency and 2.0×SSC at 50°C for high stringency. In addition, the temperature in the wash step can be varied between room temperature of approximately 22°C for low stringency and 65°C for high stringency.

[0016] As used herein, the term "sequence identity" can be assessed visually or using computer software (e.g., the software program described in Ausubel et al., eds. (2007), in Current Protocols in Molecular Biology). When a position in the compared sequences is occupied by the same base or amino acid, the molecules are identical at that position. The identity between two or more sequences can be expressed as a percentage (%), which can be used to assess the identity between related sequences. A polynucleotide sequence or amino acid sequence that has a certain percentage (e.g., 90%, 95%, 98%, or 99%) of "sequence identity" with another sequence means that, when the sequences are aligned, that percentage of bases or amino acids are the same in the two sequences being compared.

[0017] In a third aspect, the present invention provides a recombinant vector comprising the above nucleic acid molecule.

[0018] The recombinant vectors in the present invention include cloning vectors and expression vectors. The cloning vectors are used to replicate related sequences, and the expression vectors are used to express related genes. The vector used in constructing the expression vector can be the PoHBM905BDM vector.

[0019] In some embodiments, the gene of interest in the PoHBM905BDM vector may contain one or more copies.

[0020] In some preferred embodiments, the gene of interest in the PoHBM905BDM vector comprises four copies.

[0021] In the present invention, the inventors further discovered that the enrichment of the fusion protein in Pichia pastoris can be further improved after expressing multiple copies of the target gene.

[0022] In a fourth aspect, the present invention provides a recombinant cell comprising the aforementioned nucleic acid molecule or the aforementioned recombinant vector.

[0023] In some embodiments, the method for preparing a recombinant cell comprises the step of transforming the above-mentioned recombinant vector into an expression host cell.

[0024] In a fifth aspect, the present invention provides a method for preparing a recombinant porcine interferon fusion protein, comprising the following steps: culturing the above-mentioned recombinant cells, inducing expression to obtain a culture; and isolating any one of the above-mentioned fusion proteins from the culture.

[0025] In the present invention, there are no special requirements for the culture method and culture conditions, as long as the recombinant cells can grow normally. In addition, the methods for isolating the above-mentioned fusion protein from the culture are all conventional methods in the art.

[0026] In some embodiments, the culture medium used in the method for preparing the fusion protein is a culture medium that can express proteins in the art, preferably BMMY medium.

[0027] In a sixth aspect, the present invention provides a pharmaceutical composition comprising any one of the above-mentioned fusion proteins, any one of the above-mentioned nucleotide molecules, the above-mentioned recombinant vector, the above-mentioned recombinant cell, or the fusion protein prepared by the above-mentioned preparation method.

[0028] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0029] As used herein, the term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material. Exemplarily, pharmaceutically acceptable carriers include buffers, sterile water for injection, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are compatible with drug administration. Each carrier must be "acceptable," meaning compatible with the other ingredients of the composition and not harmful to the patient.

[0030] In the seventh aspect, the present invention provides the use of any of the above-mentioned fusion proteins, any of the above-mentioned nucleotide molecules, the above-mentioned recombinant vectors, the above-mentioned recombinant cells or the fusion proteins prepared by the above-mentioned preparation methods in the preparation of products for preventing and / or treating porcine viral diseases.

[0031] In some embodiments, the product comprises a pharmaceutical.

[0032] The beneficial effects of the present invention are as follows: different from the prior art, after the porcine interferon λ and protein C activator are operably linked in the present invention, the ability of the obtained fusion protein to be enriched in Pichia pastoris is significantly improved; at the same time, by expressing multiple copies of the target gene, the enrichment of the fusion protein in Pichia pastoris can be further improved. In addition, the half-life of the fusion protein is long and it has good stability. Therefore, it has good application prospects in the prevention and treatment of porcine viral diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is an SDS-PAGE image of the target protein after transformation and induction expression of the recombinant plasmids containing different copy numbers prepared in Example 1 of the present invention, wherein lane M: Marker, lane 1: deglycosylation enzyme, lane 2: target protein in the supernatant of single-copy expression, lane 3: target protein in the supernatant of single-copy expression treated with deglycosylation enzyme, lane 4: target protein in the supernatant of 2-copy expression, lane 5: target protein in the supernatant of 2-copy expression treated with deglycosylation enzyme, lane 6: target protein in the supernatant of 3-copy expression, lane 7: target protein in the supernatant of 3-copy expression treated with deglycosylation enzyme, lane 8: target protein in the supernatant of 4-copy expression, lane 9: target protein in the supernatant of 4-copy expression treated with deglycosylation enzyme, lane 10: target protein in the supernatant of 5-copy expression, and lane 11: target protein in the supernatant of 5-copy expression treated with deglycosylation enzyme; Figure 2A This is an SDS-PAGE image of the antibiotic (PoIFN-λ) in Example 2 of the present invention, wherein lane M: Marker, lane 1: target protein in the supernatant after 1 day of induction, lane 2: target protein in the supernatant after 2 days of induction, lane 3: target protein in the supernatant after 3 days of induction, lane 4: target protein in the supernatant after 4 days of induction, lane 5: target protein in the supernatant after 5 days of induction, and lane 6: target protein in the supernatant after 6 days of induction; Figure 2B This is an SDS-PAGE image of the fusion protein (PoIFN-p-λ) in Example 2 of the present invention, wherein lane M: Marker, lane 1: target protein in the supernatant after 1 day of induction, lane 2: target protein in the supernatant after 2 days of induction, lane 3: target protein in the supernatant after 3 days of induction, lane 4: target protein in the supernatant after 4 days of induction, lane 5: target protein in the supernatant after 5 days of induction, and lane 6: target protein in the supernatant after 6 days of induction; Figure 3 This is a graph showing the antiviral (VSV) activity test results of the fusion protein (PoIFN-p-λ) in Example 3 of the present invention; Figure 4This is a graph showing the antiviral (VSV) activity results of different fusion protein (PoIFN-p-λ) concentrations in MDBK cells in Example 4 of the present invention; Figure 5A This is a graph showing the protein concentration of the fusion protein (PoIFN-p-λ) in Example 5 of the present invention after being stored in preservation solution A for different periods of time; Figure 5B This is a graph showing the protein concentration of the fusion protein (PoIFN-p-λ) in Example 5 of the present invention after being stored in preservation solution B for different periods of time. DETAILED DESCRIPTION

[0034] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0035] Experimental procedures in the examples, where specific conditions are not specified, were generally performed in accordance with conventional methods in molecular biology, including but not limited to those described in M.R. Green's Molecular Cloning: A Laboratory Manual and Robert F. Weaver's Molecular Biology, or according to the recommendations of kit and instrument manufacturers. Unless otherwise specified, reagents and biological materials used in the examples were commercially available.

[0036] Example 1 Expression and purification of recombinant porcine interferon fusion protein Porcine interferon λ (amino acid sequence shown in SEQ ID NO: 3) and protein C activator (amino acid sequence shown in SEQ ID NO: 5) were operably linked using a linker (amino acid sequence shown in SEQ ID NO: 7) to produce a recombinant porcine interferon fusion protein. The amino acid sequence of the recombinant porcine interferon fusion protein is shown below: Amino acid sequence of recombinant porcine interferon fusion protein (SEQ ID NO: 1) MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKREAEAEFTTIDEWLLKEAKEKAIEELKKAGITSDYYFDLINKAKTVEGVNALKDEILKAGGGSGGGSVPVPEALRAL PGARGCHLAQFKSLSPQALQAFKRAKDAFEESLLEDWNCSSRIFPRSRDLKQLQVWERPVALEAEVALTLSVLGSLANSSLHSSLDQPLHTLRHIHAQLQACVPAQPMAGPRPRGRLHHWLHRLQEAQKKEPQSCLEASVMFNLFRLLLTRDLKCVASGDLCV Based on the preference of Pichia pastoris, the nucleotide sequence of the recombinant porcine interferon fusion protein was obtained, as shown below: Nucleotide sequence of recombinant porcine interferon fusion protein (SEQ ID NO: 2) ATGAGATTCCCATCTATTTTCACTGCTGTTTTGTTTGCTGCTTCTTCTGCTTTGGCTGCTCCTGTTAACACTACTACTGAAGATGAAACTGCTCAAATTCCAGCTGAAGCTGTTATTGGTTATTCTGATTTGGAAGGTGATTTCGATGTTGCTGTTTTGCCATTTTCTAATTCTACTAATAACGGTTTGCTGTTCATTAACACTACTATTGCTTCTATTGCTGCTAAAGAAGAAGGTGTTTCTTTGGAAAAGAGAGAAGCTGAAGCTGAATTTACTACTATTGATGAATGGTTGTTGAAGGAAGCTAAGGAAAAGGCTATTGAAGAATTGAAAAAGGCTGGTATTACTTCTGATTACTACTTCGATTTGATCAATAAGGCTAAGACTGTTGAAGGTGTTAATGCTTTGAAAGATGAAATTTTGAAGGCTGGCGGCGGCAGTGGCGGCGGCAGTGTTCCAGTTCCAGAAGCTTTGAGAGCTTTGC CAGGTGCTAGAGGTTGTCATTTGGCTCAATTTAAATCTTTGTCTCCACAAGCTTTGCAAGCTTTCAAAAGAGCTAAAGATGCTTTCGAAGAATCTTTGTTGGAAGATTGGAATTGTTCTTCTCGTATTTTTCCAAGATCTCGTGATTTGAAACAATTGCAAGTTTGGGAAAGACCAGTTGCTTTGGAAGCTGAAGTTGCTTTGACTTTGTCTGTTTTGGGTTCTTTGGCTAACTCTTCTTTGCATTCTTCTTTGGATCAACCATTGCATACTTTGAGACATATTCATGCTCAATTGCAAGCTTGTGTTCCTGCTCAACCAATGGCTGGTCCAAGACCTAGAGGTAGATTGCATCATTGGTTGCATAGATTGCAAGAAGCTCAAAAGAAGGAACCACAATCTTGTTTGGAAGCTTCTGTTATGTTCAACTTGTTTAGATTGTTGACTAGAGATTTGAAGTGTGTTGCTTCTGGTGATTTGTGTGTT The amino acid sequences and nucleotide sequences of porcine interferon λ, linker, and protein C activator are shown below: Amino acid sequence of porcine interferon λ (SEQ ID NO: 3) VPVPEALRALPGARGCHLAQFKSLSPQALQAFKRAKDAFEESLLEDWNCSSSRIFPRSRDLKQLQVWERPVALEAEVALTLSVLGSLANSSLHSSLDQPLHTLRHIHAQLQACVPAQPMAGPRGRLHHWLHRLQEAQKKEPQSCLEASVMFNLFRLLLTRDLKCVASGDLCV Nucleotide sequence of porcine interferon λ (SEQ ID NO: 4) GTTCCAGTTCCAGAAGCTTTGAGAGCTTTGCCAGGTGCTAGAGGTTGTCATTTGGCTCAATTTAAATCTTTGTCTCCACAAGCTTTGCAAGCTTTCAAAAGAGCTAAAGATGCTTTCGAAGAATCTTTG TTGGAAGATTGGAATTGTTCTTCTCGTATTTTTCCAAGATCTCGTGATTTGAAACAATTGCAAGTTTGGGAAAGACCAGTTGCTTTGGAAGCTGAAGTTGCTTTGACTTTGTCTGTTTTGGGTTCTTTG GCTAACTCTTCTTTGCATTCTTCTTTGGATCAACCATTGCATACTTTGAGACATATTCATGTCAATTGCAAGCTTGTGTTCCTGCTCAACCAATGGCTGGTCCAAGACCTAGAGGTAGATTGCATCAT TGGTTGCATAGATTGCAAGAAGCTCAAAAGAAGGAACCACAATCTTGTTTGGAAGCTTCTGTTATGTTCAACTTGTTTAGATTGTTGACTAGAGATTTGAAGTGTGTTGCTTCTGGTGATTTGTGTGTT Amino acid sequence of protein C activator (SEQ ID NO: 5) MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKREAEAEFTTIDEWLLKEAKEKAIEELKKAGITSDYYFDLINKAKTVEGVNALKDEILKA Nucleotide sequence of protein C activator (SEQ ID NO: 6) ATGAGATTCCCATCTATTTTCACTGCTGTTTTGTTTGCTGCTTCTTCTGCTTTGGCTGCTCCTGTTAACACTACTGAAGATGAAACTGCTCAAATTCCAGCTGAAGCTGTTATTGGTTATTCTGATTTGGAAGGTGATTTCGATGTTGCTGTTTTGCCATTTTCTAATTCTACTAATAACGGTTTGCTGTTCATTAACACTACTATTGCTT CTATTGCTGCTAAAGAAGAAGGTGTTTCTTTGGAAAAGAGAGAAGCTGAAGCTGAATTTACTACTATTGATGAATGGTTGTTGAAGGAAGCTAAGGAAAAGGCTATTGAAGAATTGAAAAAGGCTGGTATTACTTCTGATTACTACTTCGATTTGATCAATAAGGCTAAGACTGTTGAAGGTGTTAATGCTTTGAAAGATGAAATTTTGAAGGCT Linker amino acid sequence (SEQ ID NO: 7) GGGSGGGS Linker nucleotide sequence (SEQ ID NO: 8) GGCGGCGGCAGTGGCGGCGGCAGT The nucleotide sequence of the above-mentioned recombinant porcine interferon fusion protein was fully synthesized, and the synthesized gene fragment and the pHBM905BDM vector were digested and ligated by enzymes to obtain the pHBM905BDM-PoIFN-p-λ recombinant plasmid. After verification by colony PCR, it was sent for sequencing to obtain the pHBM905BDM-PoIFN-p-λ recombinant plasmid with the correct sequence.

[0037] Furthermore, the same tail enzyme method was used to construct a recombinant plasmid containing multiple copies.

[0038] Specifically, using restriction endonucleases Spe Ⅰ. Bam HI, Xba Ⅰ Three enzymes are used for digestion, according to Spe Ⅰ and Xba Ⅰ The principle of complementary pairing of sticky ends, respectively through Xba Ⅰ. Bam The PoIFN-p-λ expression cassette fragment was obtained by HⅠ digestion and Spe Ⅰ. Bam The PoHBM905BDM vector fragment containing the PoIFN-p-λ expression cassette was obtained by HⅠ enzyme digestion. The above fragment was placed in a 37°C incubator for enzyme digestion reaction. After the reaction, it was detected by 0.8% agarose gel. After correct enzyme digestion, 1% agarose gel was used for gel recovery to obtain the PoIFN-p-λ expression cassette fragment and the pHBM905BDM vector fragment containing the PoIFN-p-λ expression cassette.

[0039] Mix 1 μL of PoIFN-p-λ expression cassette fragment and 3 μL of pHBM905BDM vector fragment containing the PoIFN-p-λ expression cassette, and perform a ligation reaction under the action of T4 DNA Ligase. Then add Gold competent cells, ice bath for 30 minutes, heat shock for 45 seconds at 42°C, ice bath for 2-3 minutes, add NZY medium, shake at 37°C for 50 minutes, and coat the plate.

[0040] Pick a single colony for culture, then harvest the bacteria to extract the plasmid and use Xba Ⅰ. Bam The result was verified by HⅠ double enzyme digestion and two copies of pHBM905BDM-PoIFN-p-λ-2 recombinant plasmid were obtained.

[0041] Similarly, the above method was continued to obtain 3 copies of pHBM905BDM-PoIFN-p-λ-3 recombinant plasmid, 4 copies of pHBM905BDM-PoIFN-p-λ-4 recombinant plasmid and 5 copies of pHBM905BDM-PoIFN-p-λ-5 recombinant plasmid respectively.

[0042] The recombinant plasmids obtained above (pHBM905BDM-PoIFN-p-λ recombinant plasmid, pHBM905BDM-PoIFN-p-λ-2 recombinant plasmid, pHBM905BDM-PoIFN-p-λ-3 recombinant plasmid, pHBM905BDM-PoIFN-p-λ-4 recombinant plasmid and pHBM905BDM-PoIFN-p-λ-5 recombinant plasmid) were respectively used SalI enzyme digestion was performed to obtain linearized recombinant plasmids, which were then added to Pichia pastoris competent cells (GS115) and electroporated at 1.5Kv. 200μL of sorbitol and YPD medium were added, respectively. The cells were shaken at 28°C for 2h, and MD plates were spread. Single colonies were picked and added to a 24-well plate containing BMGY medium. The plates were shaken at 28°C for 2d, centrifuged, the supernatant discarded, and the cells were resuspended in BMMY medium. 1% methanol was added for induction every 24h. After 5d of induction, the supernatant was dialyzed through an ultrafiltration membrane and then subjected to SDS-PAGE detection. The results are shown in the figure. Figure 1 shown.

[0043] from Figure 1 As can be seen in lanes 2, 4, 6, 8, and 10, a diffuse band is present at the 25-60 kDa position. This is because proteins secreted by the Pichia pastoris expression system typically undergo glycosylation in the endoplasmic reticulum and Golgi apparatus. Depending on the retention time, recombinant proteins are prone to heterogeneous glycosylation. Therefore, the diffuse banding observed on SDS-PAGE is a typical characteristic. After deglycosylation treatment with glycosylase, the target proteins in these lanes all yielded single bands with molecular weights consistent with the expected molecular weight of the fusion protein (PoIFN-p-λ). These results demonstrate that the fusion protein (PoIFN-p-λ) can be secreted and expressed in Pichia pastoris. Furthermore, the strain containing four copies of the pHBM905BDM-PoIFN-p-λ-4 recombinant plasmid expressed the highest level of the fusion protein (PoIFN-p-λ) extracellularly. These results indicate that increasing the copy number of the target gene can increase the expression of the target protein.

[0044] Example 2 Enrichment of recombinant porcine interferon fusion protein in Pichia pastoris The nucleotide sequence of porcine interferon λ was fully synthesized, and the synthesized gene fragment and pHBM905BDM vector were digested and ligated by enzymes to obtain the pHBM905BDM-PoIFN-λ recombinant plasmid. After colony PCR verification, it was sent for sequencing to obtain the pHBM905BDM-PoIFN-λ recombinant plasmid with the correct sequence.

[0045] The pHBM905BDM-PoIFN-p-λ-4 recombinant plasmid and pHBM905BDM-PoIFN-λ recombinant plasmid prepared in Example 1 were transformed into Pichia pastoris competent cells by the method in Example 1. 1% methanol was added every 24 h for induction for different time periods. The supernatants were collected on days 1, 2, 3, 4, 5, and 6, and purified by ultrafiltration dialysis and liquid exchange. SDS-PAGE analysis was performed, and the results were as follows: Figure 2A and 2B shown.

[0046] from Figure 2AAs can be seen from the figure, after the first day of induction, a diffuse band of the antibiotic (PoIFN-λ) was present at the position of 15-45 kDa. From the third day onwards, no target protein band appeared. The results showed that the antibiotic (PoIFN-λ) could not be enriched when expressed in the Pichia pastoris system. Figure 2B It can be seen that after the first day of induction, a diffuse band of the fusion protein (PoIFN-p-λ) exists at the position of 25-60 kDa, and as the induction time increases, the expression level of the fusion protein (PoIFN-p-λ) of the present invention increases. The results show that the fusion protein of the present invention can be enriched in Pichia pastoris.

[0047] Example 3 Detection of antiviral (VSV) activity of fusion protein (PoIFN-p-λ) 1) The well-grown MDBK cells were digested with trypsin, centrifuged, and resuspended to prepare a cell suspension. After counting with a counting plate, the cells were counted at a concentration of 1×10 5 Cells were seeded with 100 μL of culture medium per well on a 16-well plate of an RTCA S16 real-time label-free cell function analyzer. The cells were incubated at 37°C with 5% CO2 to allow them to adhere to the plate as a monolayer. 2) Add 100 μL / well of complete culture medium containing 0.2 ng, 0.4 ng, 0.8 ng, 1.6 ng, and 3.2 ng of the fusion protein (PoIFN-p-λ) to the cells in a 16-well plate, with two wells for each concentration. Incubate in a 37°C, 5% CO2 incubator for 20-24 h. 3) Add 100 μL of TCID50 to each well. -9.67 -10 -4.66 / 0.1mL VSV virus solution was placed in an incubator at 37℃ and 5% CO2. At the same time, 3 wells were set for virus control and cell control. When the virus blank control cells basically adhered to the wall and approached 0, data statistics were performed. The results are as follows Figure 3 shown.

[0048] from Figure 3 As can be seen, VSV exhibited significant differences in cell viability between MDBK cells treated with different concentrations of PoIFN-p-λ and the control group. Data analysis showed that after infection, cell viability in groups treated with different concentrations of PoIFN-p-λ was significantly different from that in the control group, indicating that increasing the concentration of the fusion protein (PoIFN-p-λ) exhibited greater inhibitory activity against VSV.

[0049] Example 4 Antiviral (VSV) Activity of Different Fusion Protein (PoIFN-p-λ) Concentrations in MDBK Cells 1) The well-grown MDBK cells were digested with trypsin, centrifuged, and resuspended to prepare a cell suspension. After counting with a counting plate, the cells were counted at a concentration of 1×10 5 100 cells / mL were inoculated into 24-well plates and cultured in a 37°C, 5% CO2 incubator until the cells adhered to the wall. 2) Add 1 mL / well of complete culture medium containing 0.2 ng, 0.4 ng, 0.8 ng, 1.6 ng, 3.2 ng, and 6.4 ng of the fusion protein (PoIFN-p-λ) to the cells in a 24-well plate, with 3 wells for each concentration. Incubate in a 37°C, 5% CO2 incubator for 20-24 h, then discard the culture medium. 3) Wash once with serum-free culture medium, add 1 mL of VSV virus solution to each well, and culture in a 37°C, 5% CO2 incubator. Set up 3 wells for virus control and 3 wells for cell control. When the virus blank control cells are basically attached to the wall and close to 0, remove the 24-well plate, discard the supernatant, stain with crystal violet for 30 minutes, decolorize at room temperature for 3 minutes, and then use a microplate reader to measure the A of each well. 570nm The cell survival rate was calculated and the results were as follows: Figure 4 shown.

[0050] from Figure 4 It can be seen that as the concentration of the fusion protein (PoIFN-p-λ) in MDBK cells increases, the cell survival rate increases. The results show that increasing the concentration of the fusion protein (PoIFN-p-λ) has better inhibitory activity against VSV virus.

[0051] Example 5 The fusion protein (PoIFN-p-λ) prepared above was placed in preservation solution A (3wt% mannitol, 4wt% histidine, and the rest was water) and preservation solution B (3wt% sucrose, 4wt% glycerol, and the rest was water), respectively. The samples in preservation solution A were then stored at -20°C and 4°C, respectively, while the samples in preservation solution B were stored at 25°C and 37°C, respectively. At certain time intervals, samples were taken to detect the concentration and activity of the protein to evaluate the stability of the protein samples and the effects of storage conditions on protein activity. The results are shown below. Figure 5A and 5B shown.

[0052] from Figure 5A It can be seen that the protein concentration hardly decreases when stored at -20℃ and 4℃ for 3 months; Figure 5BAs can be seen, the protein concentration barely decreased after one month of storage at both 25°C and 37°C. After two months of storage, the protein concentration decreased somewhat, but remained relatively high. These results demonstrate that the fusion protein (PoIFN-p-λ) of the present invention exhibits excellent stability and a long half-life.

[0053] In summary, after the porcine interferon λ and protein C activator are operably linked in the present invention, the ability of the obtained fusion protein to be enriched in Pichia pastoris is significantly improved. In addition, the half-life of the fusion protein is long and the stability is good.

[0054] It should be noted that the above embodiments all belong to the same inventive concept, and the description of each embodiment has its own focus. For any details not described in individual embodiments, reference may be made to the description in other embodiments.

[0055] The above-described embodiments merely illustrate the implementation methods of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make various modifications and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A recombinant porcine interferon fusion protein, characterized in that: The fusion protein is obtained by operably connecting porcine interferon λ and protein C activating factor via a linker; Among them, the amino acid sequence of the protein C activator is shown in SEQ ID NO: 5, and the amino acid sequence of the linker is shown in SEQ ID NO:

7.

2. The fusion protein according to claim 1, characterized in that The C-terminus of the protein C activator is operably connected to the N-terminus of porcine interferon λ via a linker.

3. The fusion protein according to claim 1, characterized in that The fusion protein is selected from any one of the following: A1) has the amino acid sequence shown in SEQ ID NO: 1; A2) A protein having the same biological activity obtained by changing one or more amino acid residues in the amino acid sequence as shown in SEQ ID NO: 1, wherein the change includes at least one of the following: B1) Replacement; B2) missing; B3) Add.

4. A nucleic acid molecule encoding the recombinant porcine interferon fusion protein according to any one of claims 1 to 3.

5. The nucleic acid molecule according to claim 4, characterized in that The nucleic acid molecule is any of the following: 1) A nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO: 2; 2) a nucleic acid molecule that hybridizes with the nucleic acid molecule defined in 1) and encodes the fusion protein according to any one of claims 1 to 3; 3) A nucleic acid molecule having a sequence identity of 90% or more with the nucleic acid molecule defined in 1) or 2) and encoding the fusion protein according to any one of claims 1 to 3.

6. A recombinant vector, characterized in that: Comprising the nucleic acid molecule according to claim 4 or 5.

7. A recombinant cell, characterized in that Comprising the nucleic acid molecule according to claim 4 or 5 or the recombinant vector according to claim 6.

8. A method for preparing a recombinant porcine interferon fusion protein, characterized in that: The steps include: Cultivating the recombinant cell according to claim 7, inducing expression, and obtaining a culture; The fusion protein of any one of claims 1 to 3 is isolated from the culture.

9. A pharmaceutical composition, characterized in that The invention comprises the fusion protein according to any one of claims 1 to 3, the nucleotide molecule according to any one of claims 4 to 5, the recombinant vector according to claim 6, the recombinant cell according to claim 7 or the fusion protein prepared by the preparation method according to claim 8.

10. Use of the fusion protein according to any one of claims 1 to 3, the nucleotide molecule according to any one of claims 4 to 5, the recombinant vector according to claim 6, the recombinant cell according to claim 7 or the fusion protein prepared by the preparation method according to claim 8 in the preparation of products for preventing and / or treating porcine viral diseases.

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