A mutant of porcine alpha interferon and its preparation method and application

Abstract

The present application relates to a kind of pig alpha interferon mutant and its preparation method and application, the mutant amino acid sequence is as shown in SEQ ID NO.2.Preparation, with original PIFN-alpha gene as template, according to CHO-K1 cell codon bias optimization gene sequence, again with murine IGk signal peptide replaces traditional CHO signal peptide, while the glutamine of N-terminal 155th is replaced by the alanine with anti-degradation characteristics, by introducing N-terminal regular anti-degradation site to extend protein half-life, finally, the nucleotide sequence encoding the mutant is obtained by directional optimization.It is second, the present application is by constructing the recombinant expression vector pCHO1.0-PIFN-Bip containing double promoter synergistic expression system, and it is transferred into CHO cell and is expressed, successfully realize the efficient expression of pig alpha interferon in CHO cell, and it also has significant effect to be applied to the treatment of pig viral infectious disease.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a porcine α-interferon mutant, its preparation method, and its application. Background Technology

[0002] Porcine interferon-alpha (PIFN-α), a naturally occurring type I cytokine in pigs, is primarily regulated by viral infection or immune stimulation and can be secreted by various immune cells, including macrophages and T lymphocytes. This factor possesses two core functions: first, broad-spectrum antiviral activity, which, by binding to the IFNAR receptor, activates the JAK-STAT signaling pathway, thereby inducing the expression of hundreds of interferon-stimulating genes (ISGs) and exerting a comprehensive inhibitory effect at all stages of the viral life cycle; second, immunomodulatory function, enhancing the activity of natural killer (NK) cells, promoting dendritic cell maturation, precisely regulating the balance between Th1 and Th2 helper T cells, and effectively inhibiting the excessive release of inflammatory factors. Currently, porcine interferon-alpha has been widely used in the prevention and control of major viral diseases such as porcine reproductive and respiratory syndrome (PRRS), porcine epidemic diarrhea (PED), and porcine circovirus disease (PCV2). Especially when used in combination with vaccines, it can significantly enhance the immune protection effect, becoming an indispensable core biological agent for ensuring the health of pigs.

[0003] Porcine reproductive and respiratory syndrome (PRRS) is an acute infectious disease caused by PRRSV virus, characterized by reproductive disorders in sows (abortion, stillbirth, etc.) and severe respiratory symptoms in piglets (dyspnea, high mortality). It is mainly transmitted through contact, air, and vertical transmission, and has a wide susceptible population. Current treatments for PRRS primarily rely on antibiotics (such as tilmicosin and tylosin) to control secondary infections, supplemented by antiviral traditional Chinese medicine, interferon, and immune enhancers to improve resistance. Antipyretics and analgesics are also used to alleviate symptoms. However, these treatments have drawbacks, including antibiotic overuse leading to drug resistance, immunosuppressants (such as dexamethasone) exacerbating the disease, slow onset of action of traditional Chinese medicine, and false advertising in the market.

[0004] Porcine alpha interferon can directly inhibit the replication of porcine reproductive and respiratory syndrome virus (PRRSV) by activating interferon receptors to synthesize antiviral proteins, while enhancing the activity of natural killer cells and regulating the immune response. Clinical application can significantly improve the treatment efficacy and reduce the mortality rate. It also has a broad-spectrum antiviral effect against a variety of variant strains and has the advantages of low drug residue and wide range of applications. It is an important biological agent for the prevention and control of PRRS.

[0005] Natural PIFN-α in pigs is present in low concentrations and exhibits unstable activity, making it difficult to meet clinical needs. Genetic engineering techniques have been used to produce recombinant PIFN-α through heterologous expression systems (such as E. coli, yeast, and CHO cells), significantly improving yield and activity. Among these, Chinese hamster ovary cells (CHO) have become the mainstream production system for recombinant porcine alpha interferon (PIFN-α) due to their eukaryotic expression characteristics; however, current technologies still face the following challenges: (1) Low expression efficiency: In the traditional CHO system, exogenous genes are randomly integrated, resulting in low expression levels (<2g / L), requiring long-term screening of high-yield clones; (2) Severe protein inactivation: lack of eukaryotic glycosylation modification, and low proportion of active protein due to protease degradation in CHO cells; (3) High cost: The culture medium contains high-titer serum (such as fetal bovine serum), and purification requires 3 to 4 steps of chromatography; (4) Application limitations: Existing products are limited to injection formulations, which cannot meet the convenient drug administration needs of large-scale farming.

[0006] Therefore, it is of great significance to research and develop a recombinant PIFN-α CHO cell expression system with high expression efficiency, high activity stability, low cost, and scalable production, as well as a convenient drug delivery formulation. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention aims to provide a porcine α-interferon mutant, its preparation method, and its applications. This invention optimizes the PIFN-α gene sequence based on the codon preference of CHO-K1 cells. Secondly, it optimizes the signal peptide by replacing the traditional CHO signal peptide with a mouse-derived IGk signal peptide. Simultaneously, it replaces glutamine at position 155 with alanine, which has anti-degradation properties, and extends the protein's half-life by introducing a regular anti-degradation site at the N-terminus. Ultimately, this achieves targeted optimization of the PIFN-α gene sequence, significantly improving its expression efficiency and stability in CHO cells.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: The first objective of this invention is to provide a porcine α-interferon mutant, the amino acid sequence of which is shown in SEQ ID NO.2.

[0009] Preferably, the nucleotide sequence encoding the porcine α-interferon mutant is shown in SEQ ID NO.1.

[0010] A second objective of this invention is to provide a recombinant expression vector pCHO1.0-PIFN-Bip, with a vector backbone of pCHO1.0, comprising the following elements: (1) The encoding gene PIFN-α of the porcine α-interferon mutant mentioned above; (2) The CMV promoter, located at the 5' end of the pig α-interferon mutant encoding gene PIFN-α, drives its expression; (3) The molecular chaperone Bip gene, whose nucleotide sequence is shown in SEQ ID NO.3; (4) The EF1α promoter, located at the 5' end of the Bip gene, drives its expression; The CMV promoter and the EF1α promoter form a dual promoter system, which achieves the co-expression of the coding gene PIFN-α and the molecular chaperone Bip gene through promoter strength matching; the strength ratio of CMV:EF1α is set to 2.5~3.5:1.

[0011] A third objective of this invention is to provide a method for producing porcine α-interferon mutants in a CHO cell expression system, comprising the following steps: S1. Genes that synthesize porcine α-interferon mutants as described above and the molecular chaperone Bip gene; S2. The gene of the porcine α-interferon mutant described in step S1 is ligated into the pCHO1.0 vector that has been cut by a single enzyme using homologous recombination to construct the recombinant expression vector pCHO1.0-PIFN-α. S3. The molecular chaperone Bip gene described in step S1 is ligated into the recombinant expression vector pCHO1.0-PIFN-α, which has been cut by a single enzyme, by homologous recombination to construct the recombinant expression vector pCHO1.0-PIFN-Bip; S4. Transform the recombinant expression vector pCHO1.0-PIFN-Bip into CHO cells to construct a CHO cell expression system containing the porcine α-interferon mutant gene; S5. Express the porcine α-interferon mutant in the CHO cell expression system and obtain the culture supernatant. S6. The culture supernatant was separated and purified using a low-temperature purification process to obtain the porcine α-interferon mutant.

[0012] Preferably, in step S4, the recombinant expression vector pCHO1.0-PIFN-Bip is transfected into CHO cells using electroporation, liposome transfection, or polyethyleneimine transfection.

[0013] Preferably, in step S4, the culture conditions of the CHO cell expression system are: temperature 36.5~37.5℃, pH 7.2~7.4, and dissolved oxygen 30~50%.

[0014] Preferably, the low-temperature purification process in step S6 includes: anion exchange chromatography using Q Sepharose FF resin at 2-8°C; after equilibration with a buffer, gradient elution with an elution buffer containing 0-30% B solution; collecting the elution peak containing the porcine α-interferon mutant; subsequently, the collected target component is replaced with buffer and concentrated using a 10kD tangential flow ultrafiltration system; finally, it is further processed using a 100kD ultrafiltration system, controlling the transmembrane pressure to <20psi, to complete the purification of the porcine α-interferon mutant in the culture supernatant; The equilibration buffer solution was 20 mM Tris-HCl, pH 7.5; The eluent A for the gradient elution is an equilibrium buffer, and the eluent B is 20 mM Tris-HCl + 1 M NaCl, pH 7.5. The replacement buffer is 20 mM sodium phosphate, pH 6.0.

[0015] Another object of the present invention is to provide a CHO cell comprising the above-described recombinant expression vector pCHO1.0-PIFN-Bip and expressing a porcine α-interferon mutant with the amino acid sequence shown in SEQ ID NO.2.

[0016] Another object of the present invention is to provide a porcine α-interferon mutant as described above; the recombinant expression vector pCHO1.0-PIFN-Bip as described above; the method described above; or the use of the CHO cells described above in the preparation of drugs for treating porcine viral infectious diseases.

[0017] Preferably, the porcine viral infectious disease includes porcine reproductive and respiratory syndrome (PRRS).

[0018] Compared with the prior art, the present invention has the following beneficial effects: This invention obtains a porcine α-interferon mutant through gene sequence optimization (codon preference optimization, signal peptide substitution, and introduction of anti-degradation sites) and its synergistic expression using a CMV / EF1α dual promoter system. Under shake-flask culture conditions, the expression level of the porcine α-interferon mutant reaches 5.1 mg / mL (i.e., 5.1 g / L, detected by BCA method), which is more than 2.5 times higher than that of the conventional CHO expression system (<2 g / L). Under large-scale culture conditions in a CHO cell bioreactor, the viable cell density reaches a maximum of 15 × 10⁶ cells / mL. 6 The cell / mL ratio achieved high-efficiency expression in CHO cells.

[0019] Secondly, the porcine α-interferon mutant prepared in this invention also has significant effects in treating porcine viral infectious diseases. Attached Figure Description

[0020] Figure 1Gel electrophoresis image of the target gene amplification; Figure 2 This is a gel electrophoresis image of homologous recombination. Figure 3 Gel electrophoresis image for SDS-PAGE identification. Detailed Implementation

[0021] Unless otherwise specified, the experimental methods described in the following embodiments of the present invention are generally performed under conventional conditions or as recommended by the manufacturer. All commonly used chemical reagents used in the embodiments are commercially available products.

[0022] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention.

[0023] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that these descriptions are merely exemplary and not intended to limit the scope of the invention. Furthermore, descriptions of well-known structures and technologies are omitted in the following description to avoid unnecessarily obscuring the concepts of the invention.

[0024] The following embodiments further describe the present invention, but these embodiments are not intended to limit the scope of protection of the present invention.

[0025] 1. Reagents and raw materials EcoRV, AvrII restriction endonuclease, High-Fidelity DNA Polymerase (catalog number 10140ES60), and Hieff Clone® Plus One Step Cloning Kit were all purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.; EmCD104 basal medium, EmCD CHO® supplement A, and EmCD CHO® supplement B were purchased from Emines (Suzhou) Biotechnology Co., Ltd.; CD CHO medium was purchased from Thermo Fisher Scientific; the blood / cell / tissue genomic DNA extraction kit and gel extraction kit were purchased from Tiangen Biotech (Beijing) Co., Ltd.; the DL10000 and DL5000 Markers were purchased from TAKARA; the 2×Taq Master Mix, PageRuler™ pre-stained protein marker, and homologous recombination kit were purchased from Nanjing Novizan Biotechnology Co., Ltd.; and the BCA protein detection kit was purchased from Beyotime.

[0026] 2. Instruments and Equipment The clean bench was purchased from Shanghai Zuoyan Instrument Technology Co., Ltd., model XBQ-2H; the carbon dioxide incubator was purchased from Shenzhen Ruiwode Life Technology Co., Ltd., model D180-P; the nuclear transfection instrument was purchased from Lonza Group, model 4D-Nucleofector; the cell counter was purchased from Suzhou Quiketai Biotechnology Co., Ltd., model ADAM™ MC2; and the inverted microscope was purchased from Olympus (China) Co., Ltd., model IX73.

[0027] Example 1: PIFN-α gene optimization and vector construction 1. Targeted optimization of gene sequences Gene synthesis process: The original PIFN-α gene sequence (GenBank: NM_214393.1) was obtained from the GenBank database. The complete sequence is 570 bp in length, and the corresponding PIFN-α gene consists of 189 amino acids. To improve the expression efficiency and activity stability of the PIFN-α gene in CHO cells, this invention selected CHO-K1 cells as the recombinant expression host, and performed codon optimization and sequence modification on the PIFN-α gene sequence based on the codon preference of CHO-K1 cells. (1) Codon preference optimization: Remove rare codons that are used less frequently in CHO-K1 cells and replace them with codons that are used more frequently in the host cell, thereby improving the translation efficiency of the target gene in CHO cells and increasing the yield of the target protein.

[0028] (2) Signal peptide replacement optimization: The recombinant expression signal peptide was replaced with a mouse IGk signal peptide. Because the signal peptide commonly used in traditional CHO expression has different structural and functional characteristics from the mouse IGk signal peptide, the original CHO signal peptide coding sequence was removed and replaced with a mouse IGk signal peptide coding sequence during gene optimization, so as to better promote the secretion of the target protein into the extracellular space and improve the secretion efficiency of the protein.

[0029] (3) Stability Enhancement and Optimization: The stability and half-life of a protein in the cell directly affect its biological function and expression yield. In order to improve the stability of PIFN-α protein in the cell and prolong its half-life, this invention introduces an anti-degradation site that conforms to the N-terminal rule during the gene optimization stage. The glutamine at position 155 of the protein is replaced with alanine, which has anti-degradation properties. This can effectively enhance the stability of the C-terminus of the protein, reduce its degradation in the cell, and thus prolong the protein half-life and increase the effective content of the protein.

[0030] In summary, this invention has achieved targeted optimization of the PIFN-α gene sequence through three targeted modifications: codon preference optimization in CHO-K1 cells, replacement of the traditional CHO signal peptide with a mouse-derived IGk signal peptide, and introduction of a regular anti-degradation site at the N-terminus (replacing glutamine at position 155 with alanine). This significantly improves the expression efficiency of the gene and the stability of the expression product in CHO cells.

[0031] The optimized PIFN-α gene sequence is approximately 564 bp in length, containing a 63 bp signal peptide coding region, a 495 bp mature region, a 3 bp mutation site coding region, and a 3 bp terminator. Its nucleotide sequence is shown in SEQ ID NO.1, where a single wavy underline represents the signal peptide coding region, a single straight underline represents the mature region, double straight underlines represent the mutation site coding region, and double wavy underlines represent the terminator. The corresponding amino acid sequences are shown in SEQ ID NO.2. This invention commissioned a biotechnology company to synthesize the optimized sequence and insert it into the pCHO1.0 vector.

[0032]

[0033] MAPTSAFLTALVLLSCNAICSLGCDLPQTHSLAHTRALRLLAQMRRISPFSCLDHRDFGSPHEAFGGNQVQKAQAMALVHEMLQQTFQLFSTEGSAAAWNESLLHQFCTGLDQQLRDLEACVMQEAGLEGTPLLEEDSILAVRKYFHRLTLYLQEKSYSPCAWEIVRAEVMRSFSSSRNLQDRLRKKE (SEQ ID NO. 2).

[0034] 2. Carrier Construction Using pCHO1.0 as the vector backbone, the optimized porcine α-interferon mutant encoding gene PIFN-α was cloned into the vector via homologous recombination to construct the pCHO1.0-PIFN-Bip recombinant plasmid. This recombinant plasmid contains a dual promoter system, the specific components of which are as follows: (1) Target gene: PIFN-α, the encoding gene of porcine α-interferon mutant; (2) CMV promoter: located at the 5' end of the PIFN-α gene, responsible for driving the expression of the target gene; (3) Molecular chaperone Bip gene: nucleotide sequence as shown in SEQ ID NO.3; (4) EF1α promoter: located at the 5' end of the Bip gene, responsible for driving the expression of the molecular chaperone Bip gene; The CMV promoter and the EF1α promoter form a dual promoter system, which achieves the co-expression of the coding gene PIFN-α and the molecular chaperone Bip gene through promoter strength matching. The strength ratio of CMV to EF1α is set to 3:1.

[0035]

[0036] 2.1 Construction of the recombinant expression vector pCHO1.0-PIFN-α The specific construction process of the recombinant expression vector pCHO1.0-PIFN-α is as follows: (1) Vector single enzyme digestion and recovery The pCHO1.0 vector was digested with EcoRV restriction endonuclease at 37℃. The reaction system is shown in Table 1 below.

[0037] Table 1 Restriction endonuclease reaction system

[0038] After the enzyme digestion reaction was completed, nucleic acid gel electrophoresis was performed and the pCHO1.0 single enzyme digestion product was recovered using a gel recovery kit (Tiangen Biotech, catalog number DP204) for later use.

[0039] (2) PCR amplification and verification of PIFN gene Based on the optimized PIFN-α gene sequence, specific primers were designed, and PCR amplification was performed using the synthesized target gene as a template. The primer design information is shown in SEQ ID NO.4-5 below, and the reaction system is shown in Table 2 below.

[0040] Forward primer (F): 5'-CTCAAGCTTGATATCATGGAGACCGACACTCTC-3' (SEQ ID NO.4); Reverse primer (R): 5'-ATTAACGCCGATATCTCATTCCTTCTTCCGAAGA-3' (SEQ ID NO.5).

[0041] Table 2 PCR reaction system (50 μL)

[0042] The PCR reaction program was set as follows: 98℃ pre-denaturation for 1 min; 98℃ denaturation for 10 s, 60℃ annealing for 30 s, 72℃ extension for 30 s, repeated 30 cycles; 72℃ extension for 3 min. After amplification, the PCR products were analyzed by electrophoresis on a 1% agarose gel, and the expected fragment length was approximately 594 bp. The electrophoresis results are as follows. Figure 1 As shown, the size of the electrophoretic bands is consistent with the expected size, indicating that the synthesized target gene is correct.

[0043] (3) Homologous recombination ligation and verification of recombinant plasmids Homologous recombination ligation was performed using the Hieff Clone® Plus One Step Cloning Kit (catalog number: 10911ES20). The pCHO1.0 vector fragment recovered in step (1) was ligated with the target gene fragment recovered from the gel in step (2) through homologous recombination. The recombination reaction conditions were incubation at 50℃ for 5 min, followed by storage at 4℃ after the reaction. The specific reaction system is shown in Table 3 below.

[0044] Table 3. Homologous recombination system (total volume 10µL)

[0045] After the recombination reaction was completed, the recombinant plasmid pCHO1.0-PIFN-α was transferred into competent intestinal cells. The recombinant plasmid was identified by plating, picking single clones, and sequencing. The universal primers are as follows: Universal primer (F): 5'-GGTGTCGTGAGGAATTTCAG-3' (SEQ ID NO.6); Universal primer (R): 5'-GAGGCAGCCGGATCATAATC-3' (SEQ ID NO.7).

[0046] 2.2 Construction of the recombinant expression vector pCHO1.0-PIFN-Bip The specific construction process of the recombinant expression vector pCHO1.0-PIFN-Bip is as follows: (1) Single enzyme digestion of recombinant plasmid pCHO1.0-PIFN-α The above positive clones were selected and propagated using LB (kanamycin resistance). Then, the recombinant plasmid pCHO1.0-PIFN-α was extracted using a plasmid extraction kit (Tiangen Biotech, catalog number DP103-02), and digested with AvrII single enzyme (Yisheng Biotech, 15002ES). The enzyme digestion system is shown in Table 4 below. The digestion was carried out at 37℃ for 15 min. The digested products were directly subjected to electrophoresis, and the single enzyme digestion products were recovered using a gel recovery kit for later use.

[0047] Table 4. Recombinant plasmid pCHO1.0-PIFN-α digestion system

[0048] (2) PCR amplification and verification of the Bip gene Specific primers were designed based on the Bip gene sequence, and PCR amplification was performed using the synthesized target gene as a template. The primer design information is shown in SEQ ID NO.8-9 below, and the reaction system is shown in Table 5 below.

[0049] The PCR reaction program was as follows: 98℃ pre-denaturation for 1 min; 98℃ denaturation for 10 s, 60℃ annealing for 30 s, 72℃ extension for 1 min, repeated for 35 cycles; 72℃ extension for 5 min. After amplification, the PCR products were analyzed by electrophoresis on a 1% agarose gel, with the expected fragment length being approximately 2 kb. The electrophoresis results are shown below. Figure 2 As shown, the size of the electrophoretic bands is consistent with expectations, indicating that the synthesized target gene is correct. The target fragment was then recovered using a gel extraction kit for later use.

[0050] Forward primer (F): 5'-TCCGGGCCGCCTAGGATGAAGCTGTCCCTGGT-3' (SEQ ID NO.8); Reverse primer (R): 5'-GAGTATACAGTCCTAGGCTACAACTCATCTTTGTC-3' (SEQ ID NO.9).

[0051] Table 5 PCR reaction system (50 μL)

[0052] (3) Homologous recombination ligation and verification with recombination plasmid pCHO1.0-PIFN-Bip Homologous recombination ligation was performed using the Hieff Clone® Plus One Step Cloning Kit (catalog number: 10911ES20). The pCHO1.0-PIFN-α vector fragment recovered in step (1) was ligated with the Bip gene fragment recovered from gel cloning in step (2). The recombination reaction was carried out at 50°C for 5 min, and the mixture was stored at 4°C after the reaction. The specific reaction system is shown in Table 6 below.

[0053] Table 6. Homologous recombination system (total volume 10µL)

[0054] After the recombination reaction was completed, the recombinant plasmid pCHO1.0-PIFN-Bip was transferred into competent E. coli cells. Positive clones were selected by plating, picking single clones, PCR, or sequencing (using universal primers for the vector) and amplified in 200 ml LB medium. Then, the plasmid was extracted using the endotoxin-free plasmid extraction kit (Tiangen, catalog number DP117) for subsequent cell transfection.

[0055] Example 2: Screening and Validation of CHO Cell Lines The transfection and screening process is as follows: (1) Cell resuscitation and transfection: CHO-K1 host cells were resuscitated and the recombinant plasmid pCHO1.0-PIFN-Bip constructed above was introduced into the host cells by electrotransfection.

[0056] (2) Mini Pool screening: Transfected cells were seeded into 96-well plates at a density of 4000 cells / well and cultured in CD CHO Medium containing 25 μM MTX for 21 days. Mini pools with good growth were selected and gradually expanded to 24-well plates for 3-5 days. Mini pools with good growth were screened again and transferred to 6-well plates for 3-5 days. Finally, all qualified Mini Pools were expanded into 125 mL shake flasks and cultured at 37℃, 5% CO2, and 120 r / min on a shaker. At the same time, cryopreservation and expression evaluation were performed.

[0057] (3) Expression assessment of Mini Pool cells: Mini Pool cells were expressed at a concentration of 0.5 × 10⁻⁶. 6 Cells were seeded at a density of 3.0 × 10⁶ cells / mL in EmCD104 basal medium and cultured in fed-batch mode at 37°C, 5% CO₂, and a shaker speed of 120 rpm. When the cell density reached 3.0 × 10⁶ cells / mL... 6 ~5.0×10 6 When the protein concentration reached 1000 mg / mL, fed-batch culture was performed: daily additions of 2.5% EmCD CHO® Feed A and 0.25% EmCD CHO® Feed B were added to the culture medium; simultaneously, the glucose concentration in the culture medium was measured daily, and glucose was added as needed to maintain a concentration of 2.0–6.0 g / L, for a total of 14 days. After culture, the supernatant was collected by centrifugation, and the total protein content was determined using the BCA method. Protein identification was performed using SDS-PAGE electrophoresis, and the results were recorded.

[0058] Experimental results are as follows Figure 3 As shown in the band of sample a, the total protein content was 5.1 mg / mL as determined by the BCA method.

[0059] Example 3: Large-scale culture and purification 1. Large-scale culture in a bioreactor (50L system) A serum-free culture system was used, with EmCD104 containing 20 mmol MTX as the basal medium, supplemented by EmCD CHO® feed A and B media using a combined feeding strategy. Large-scale culture was completed in a 50L bioreactor for 14 days, achieving a final viable cell density of 15 × 10⁶ cells / day. 6 cells / mL. The specific procedure is as follows: (1) Preparation of culture medium and seed cells Culture medium: The basal medium is Em104 medium containing 20 mmol MTX, and the feed is EmCD CHO® Feed A medium and EmCD CHO® Feed B medium.

[0060] Seed cell expansion: Resuscitate frozen working cell bank cells, rapidly thaw them in a 37°C water bath, and then seed them into 125mL shake flasks. Incubate at 37°C with 5% CO2. Continue incubation until the cell density reaches 2.0 × 10⁻⁶ cells / mL. 6 When cells / mL and viability >95%, administer at 0.5 × 10⁻⁶. 6 The cells / mL inoculation density was used for subculturing, with the volume gradually increased with each subculturing until the seed quantity met the inoculation requirements of a 50L reactor (based on a 20L inoculation volume, 0.5×10⁻⁶ cells / mL). 6 (cells / mL density inoculation).

[0061] (2) Bioreactor inoculation and parameter setting Aseptic inoculation: Transfer the seed suspension to a 50L glass / disposable reactor, with the final inoculation volume controlled at 20-30L, to achieve an initial cell density of 0.5 × 10⁻⁶ cells / year. 6 cells / mL.

[0062] Parameter control: The culture temperature was maintained at 36.5~37.5℃, and the pH value was stabilized at 7.2~7.4 using a CO2 and sodium bicarbonate buffer system; the dissolved oxygen (DO) was controlled at 30~50%, regulated by air and O2 ventilation, and a stirring speed of 80~120 rpm was used to ensure uniform dissolved oxygen.

[0063] (3) Culture cycle and feeding strategy (total cycle 14 days) A time-segmented, gradient feeding strategy is adopted, with the feeding time and dosage as follows: EmCD CHO® Feed A Medium: On days 3, 5, 7, 9, 11, and 13 of culture, add 2% (v / v), 3% (v / v), 3% (v / v), 3% (v / v), 3% (v / v), and 2% (v / v) of the initial culture volume, respectively. EmCD CHO® Feed B Medium: On days 3, 5, 7, 9, 11, and 13 of culture, add 0.2% (v / v), 0.3% (v / v), 0.3% (v / v), 0.3% (v / v), 0.3% (v / v), and 0.2% (v / v) of the initial culture volume, respectively.

[0064] Sugar supplementation strategy: Starting from the third day, control the sugar concentration to no less than 4.0 g / L. If the sugar concentration is lower than 4.0 g / L after supplementation, add 40% glucose to bring it up to 6.0 g / L. The culture was maintained at 37°C until day 5. From day 6, the temperature was adjusted to 33°C, and the culture was completed on day 14. The final viable cell density reached 15 × 10⁶ cells / day. 6 cells / mL.

[0065] (4) Harvest cell culture supernatant After the culture is completed, the supernatant is collected by aseptic centrifugation (5000×g, 15min) or deep filtration (0.45μm filter cartridge) and temporarily stored at 4℃ for purification.

[0066] 2. Purification process (anion exchange chromatography + virus inactivation) (1) Anion exchange chromatography (Q Sepharose FF resin) Column preparation: Take an appropriate amount of Q Sepharose FF resin (wet volume), rinse with purified water and pack into the column. Equilibrate the column with equilibration buffer (20mM Tris-HCl, pH 7.5) until the conductivity and pH of the eluent are consistent with the buffer and the baseline remains stable.

[0067] Sample loading: The protein concentration of the harvested supernatant was determined using the BCA method, ensuring a loading volume ≤ 60 mg protein / mL resin. The pH of the supernatant was adjusted to 7.0–7.5, and the sample was loaded at a flow rate of 1–2 mL / min, while simultaneously collecting unbound components.

[0068] Washing and Elution: 1) Washing: Rinse the column with equilibration buffer for 3-5 column volumes (CV) to remove unbound contaminating proteins until UV detection (UV280) values ​​return to baseline. 2) Elution: Use a gradient elution method. Solution A is equilibration buffer, and solution B is 20mM Tris-HCl + 1M NaCl (pH 7.5). Elute 30 column volumes with 0-30% solution B, collect the elution peaks, and combine the target protein fractions.

[0069] Buffer replacement: Using a tangential flow ultrafiltration system (molecular weight cutoff 10kD), the buffer solution of the target component was replaced with 20mM sodium phosphate (pH 6.0) and concentrated to 10~20mL, and stored at 4℃.

[0070] (2) Virus inactivation (ultrafiltration + endotoxin control) Ultrafiltration concentration and endotoxin removal: Buffer replacement is performed using an ultrafiltration system (molecular weight cutoff 100kD) to control the transmembrane pressure to <20psi, and the mixture is concentrated to 1 / 5 to 1 / 10 of its original volume.

[0071] Endotoxin activity assay: Endotoxin was detected using the Limulus Amebocyte Lysate (LAL) gel electrophoresis. After mixing the filtrate with LAL, incubation at 37°C for 60 min showed no gel formation, indicating endotoxin levels <0.05 EU / mL. Protein activity was detected using the cytopathic effect inhibition assay, with a result of 2.33 × 10⁻⁶. 8 IU / mL.

[0072] The purified product was then verified by gel electrophoresis, and the results are as follows: Figure 3 As shown in the sample b band, there are no obvious contaminating protein bands, confirming that the purification process is efficient and the purity of the target protein meets the standards.

[0073] Example 4: Blue Ear Disease Control Trial Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious disease caused by porcine reproductive and respiratory syndrome virus (PRRSV). This study aims to evaluate the efficacy of recombinant porcine alpha interferon (PIFN-α) administered by injection in the prevention and control of highly pathogenic PRRSV-infected piglets, and to compare it with a positive control, providing a scientific basis for PRRS control.

[0074] 1. Experimental Design Experimental animals: Twenty healthy 30-day-old piglets (PRRSV antigen / antibody double negative, African swine fever negative, purchased from Hangzhou Daguanshan Breeding Pig Co., Ltd.) were randomly divided into two groups of 10 piglets each. The specific grouping and treatment are as follows: ①Experimental group: basal diet + recombinant PIFN-α (1×10) 4 IU / kg body weight, administered intramuscularly twice daily for 5 consecutive days. Administration began 10 days after administration, followed by challenge treatment after administration, and continued observation for 28 days.

[0075] ② Positive control group: basal diet; after 10 days of feeding, the same dose of physiological saline was injected intramuscularly, and after the end of the period, the group was challenged with the virus and observed for 28 days.

[0076] 2. Infectious Disease Treatment Virus strain: PRRSV WH-1 strain was used, with a challenge dose of 10. 3 TCID 50 / head; Route of infection: Intramuscular injection (5 mL / head).

[0077] 3. Detection indicators Viral load: Blood samples were collected from piglets weekly, and the CT value of PRRSV RNA was detected using RT-qPCR technology to assess viral replication in vivo.

[0078] Body temperature and clinical symptoms: The body temperature of piglets was measured at regular intervals every day, the respiratory rate was recorded, and the incidence of diarrhea and cough was statistically analyzed to assess the impact of the disease on the physiological state of piglets.

[0079] Immune markers: The levels of IL-6 (Cusabio, CSB-E06700p), TNF-α (Cloud-Clone Corp, SEA133Po), and IFN-γ (Cusabio, CSB-E06876p) in serum were detected by ELISA to assess the body's immune response status.

[0080] 4. Test Results (1) Viral load control Experimental group: On day 14 after challenge, the viral load in 80% of piglets dropped below the detection limit (CT≥40), and on day 21, the viral load in all piglets turned negative. Positive control group: The virus continued to replicate actively in the piglets (CT≈28-30), and no individuals turned negative. The difference between the two groups was extremely significant (p<0.01).

[0081] (2) Relief of clinical symptoms Thermoregulation: The peak body temperature of piglets in the experimental group was 41.2℃, which was significantly lower than that of the positive control group (42.8℃), and the duration of fever was shortened by 60% compared with the control group.

[0082] Respiratory symptoms: The total incidence of cough and diarrhea in the experimental group piglets was 13.3%, which was only 20% of that in the positive control group (66.7%).

[0083] (3) Immunomodulatory effects Inhibition of pro-inflammatory factors: Compared with the positive control group, the serum levels of IL-6 and TNF-α in the experimental group decreased by 58% and 63%, respectively; the IFN-γ level increased by 2.1 times, which can effectively activate the mucosal immune response.

[0084] Differences in antibody response: IgA antibody levels (OD) in the experimental group 630 =1.2) was significantly higher than that of the positive control group (OD). 630 =0.8), indicating that recombinant PIFN-α can clear the virus through a non-antibody-dependent pathway.

[0085] (4) Recovery of production performance Daily weight gain: The daily weight gain of piglets in the experimental group was 0.28 kg / day, which was 87% higher than that of the positive control group (0.15 kg / day); Material weight ratio: The material weight ratio of the experimental group was 1.52, which was 34% better than that of the positive control group (2.31); Mortality rate: The mortality rate of piglets in the experimental group was 0%, while the mortality rate in the positive control group was 33.3%.

[0086] 5. Analysis of Experimental Results The above experimental results show that the recombinant PIFN-α intramuscular injection provided by the present invention has a significant preventive and therapeutic effect on highly pathogenic PRRSV-infected piglets, effectively reducing viral load, alleviating clinical symptoms, regulating immune response, and promoting recovery of production performance, and its effect is better than conventional antibiotic treatment.

[0087] In summary, this invention significantly improves the efficiency, product stability, and process reproducibility of porcine alpha interferon expression in CHO cells through gene sequence optimization, construction of a CMV and EF1α dual-promoter co-expression system, application of a low-cost serum-free culture system, and optimization of low-temperature targeted purification processes. The porcine alpha interferon prepared by this invention exhibits significant preventive and therapeutic effects against highly pathogenic PRRSV-infected piglets, providing a new and effective strategy for the control of porcine reproductive and respiratory syndrome (PRRS).

[0088] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A mutant of porcine alpha interferon, characterized in that, The amino acid sequence of the porcine α-interferon mutant is shown in SEQ ID NO.

2.

2. The porcine alpha interferon mutant according to claim 1, characterized in that, The nucleotide sequence encoding the porcine α-interferon mutant is shown in SEQ ID NO.

1.

3. A recombinant expression vector pCHO1.0-PIFN-Bip, characterized in that, The carrier skeleton is pCHO1.0 and contains the following components: (1) The encoding gene PIFN-α of the porcine α-interferon mutant as described in claim 1 or 2; (2) The CMV promoter, located at the 5' end of the pig α-interferon mutant encoding gene PIFN-α, drives its expression; (3) The molecular chaperone Bip gene, the nucleotide sequence of which is shown in SEQ ID NO.3; (4) The EF1α promoter, located at the 5' end of the Bip gene, drives its expression; The CMV promoter and the EF1α promoter form a dual promoter system, which achieves the co-expression of the coding gene PIFN-α and the molecular chaperone Bip gene through promoter strength matching; the strength ratio of CMV:EF1α is set to 2.5~3.5:

1.

4. A method for producing porcine α-interferon mutants in a CHO cell expression system, characterized in that, Includes the following steps: S1. Synthesize the gene and molecular chaperone Bip gene of the porcine α-interferon mutant as described in claim 1 or 2; S2. The gene of the porcine α-interferon mutant described in step S1 is ligated into the pCHO1.0 vector that has been cut by a single enzyme using homologous recombination to construct the recombinant expression vector pCHO1.0-PIFN-α. S3. The molecular chaperone Bip gene described in step S1 is ligated into the recombinant expression vector pCHO1.0-PIFN-α, which has been cut by a single enzyme, by homologous recombination to construct the recombinant expression vector pCHO1.0-PIFN-Bip; S4. Transform the recombinant expression vector pCHO1.0-PIFN-Bip into CHO cells to construct a CHO cell expression system containing the porcine α-interferon mutant gene; S5. Express the porcine α-interferon mutant in the CHO cell expression system and obtain the culture supernatant. S6. The culture supernatant was separated and purified using a low-temperature purification process to obtain the porcine α-interferon mutant.

5. The method according to claim 4, characterized in that, In step S4, the recombinant expression vector pCHO1.0-PIFN-Bip is transfected into CHO cells using electroporation, liposome transfection, or polyethyleneimine transfection.

6. The method according to claim 4, characterized in that, In step S4, the culture conditions for the CHO cell expression system are: temperature 36.5~37.5℃, pH 7.2~7.4, and dissolved oxygen 30~50%.

7. The method according to claim 4, characterized in that, The low-temperature purification process in step S6 includes: anion exchange chromatography using Q Sepharose FF resin at 2-8°C; after equilibration with a buffer, gradient elution with an elution buffer containing 0-30% B solution; collecting the elution peak containing the porcine α-interferon mutant; subsequently, the collected target component is replaced with buffer and concentrated using a 10kD tangential flow ultrafiltration system; finally, it is further processed using a 100kD ultrafiltration system, controlling the transmembrane pressure to <20psi, to complete the purification of the porcine α-interferon mutant in the culture supernatant. The equilibration buffer solution was 20 mM Tris-HCl, pH 7.5; The eluent A for the gradient elution is an equilibrium buffer, and the eluent B is 20 mM Tris-HCl + 1 M NaCl, pH 7.

5. The replacement buffer is 20 mM sodium phosphate, pH 6.

0.

8. A CHO cell, characterized in that, The CHO cells comprise the recombinant expression vector pCHO1.0-PIFN-Bip as described in claim 3, and express a porcine α-interferon mutant with the amino acid sequence shown in SEQ ID NO.

2.

9. A porcine α-interferon mutant as described in claim 1 or 2; the recombinant expression vector pCHO1.0-PIFN-Bip as described in claim 3; the method described in any one of claims 4 to 7; or the use of CHO cells as described in claim 8 in the preparation of drugs for treating porcine viral infectious diseases.

10. The application according to claim 9, characterized in that, The viral infectious diseases of pigs mentioned include porcine reproductive and respiratory syndrome (PRRS).

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