Fusion protein, preparation method therefor and use thereof

Abstract

Provided are a fusion protein, a preparation method therefor and the use thereof. The fusion protein contains a His tag, a SUMO tag, and natural canine interferon-α, wherein the His tag has an amino acid sequence as represented by SEQ ID NO: 11, the SUMO tag has an amino acid sequence as represented by SEQ ID NO: 12, and the natural canine interferon α has an amino acid sequence as represented by SEQ ID NO: 7. The fusion protein exhibits an extremely significant increase in expression level and yield, has preparation stability and biological activity significantly greater than those of a native canine interferon-α protein, and has good safety.

Description

A fusion protein, its preparation method and application

[0001] This application claims priority to Chinese patent application 2024115461555, filed on 2024 / 10 / 31. The entire contents of the aforementioned Chinese patent application are incorporated herein by reference. Technical Field

[0002] This invention belongs to the field of biomedicine, specifically relating to a fusion protein, its preparation method, and its application. Background Technology

[0003] Canine interferon-alpha (CaIFNα) is an endogenous protein with broad-spectrum antiviral, immunomodulatory, and antitumor activities, widely used in the treatment of canine viral diseases. However, the industrial production of natural canine interferon-alpha faces a series of limiting factors, severely restricting its large-scale application. First, low expression levels are a major problem; currently, the commonly used expression system for canine interferon-alpha is *E. coli*, and there is still significant room for improvement in expression levels. Second, canine interferon-alpha is expressed in inclusion bodies in the *E. coli* system, followed by denaturation and renaturation. Protein folding and stability issues are also limiting factors. The *E. coli* system cannot effectively form disulfide bonds, which are crucial for proper protein folding and stability. Proteins lacking disulfide bonds often cannot maintain their native conformation, thus affecting their biological activity and stability, ultimately leading to low activity recovery rates. This directly impacts its effectiveness in practical applications; low activity not only reduces therapeutic efficacy but also increases dosage and cost. Third, *E. coli* is the main source of endotoxins, making the purification process for removing endotoxins and pyrogens extremely complex and increasing production costs. To address these issues, designing and optimizing the molecular structure, developing efficient expression systems, and employing advanced preparation processes are crucial. Optimized molecular structures and efficient expression systems should ensure the activity of canine interferon-α while simultaneously improving product stability and expression levels. Efficient preparation processes should not only simplify production and reduce costs but also enhance product quality, activity, and recovery rates. Only by comprehensively addressing these challenges can we achieve cost reduction and efficiency improvement in canine interferon-α, enabling large-scale production and widespread application to meet the demands of the veterinary drug market.

[0004] As is well known, SUMO (small ubiquitin-like modifier) ​​is a small ubiquitin-like modified protein with wide applications in biology and bioengineering. SUMO is structurally similar to ubiquitin, but shares only about 20% amino acid sequence homology with ubiquitin. This tag protein can serve as a fusion tag and molecular chaperone for recombinant protein expression, increasing the expression level of fusion proteins. It also possesses anti-protease properties, promotes the correct folding of target proteins, and improves the solubility of recombinant proteins.

[0005] His tags are among the most popular tag proteins today. They can be inserted into the C-terminus or N-terminus of a target protein to facilitate its purification and identification.

[0006] To address the technical problems of low yield and activity recovery rate, and poor stability of natural canine interferon-α in existing technologies, this invention provides a fusion protein, its preparation method, and its applications. This invention fuses a His tag and a SUMO tag with natural canine interferon-α for expression, resulting in a significantly increased expression level and yield of the fusion protein. The formulation exhibits significantly better stability and biological activity than natural canine interferon-α, with good safety profiles. This lays a solid foundation for the commercial application of this molecule and enhances the market competitiveness of similar products. Summary of the Invention

[0007] To address the technical problems of low yield and activity recovery rate and poor stability of natural canine interferon α in existing technologies, this invention provides a fusion protein, its preparation method, and its application.

[0008] The first aspect of the present invention provides a fusion protein comprising: a His tag, a SUMO tag, and natural canine interferon α; the amino acid sequence of the His tag is shown in SEQ ID NO:11, the amino acid sequence of the SUMO tag is shown in SEQ ID NO:12, and the amino acid sequence of the natural canine interferon α is shown in SEQ ID NO:7.

[0009] In some embodiments, the fusion protein comprises, from the N-terminus to the C-terminus, a His tag, a SUMO tag, and natural canine interferon α.

[0010] A second aspect of the present invention provides an isolated nucleic acid that encodes a fusion protein as described in the first aspect of the present invention.

[0011] In some preferred embodiments, the nucleotide sequence is shown as SEQ ID NO:4 in the sequence listing.

[0012] A third aspect of the present invention provides a recombinant expression vector containing the isolated nucleic acid as described in the second aspect of the present invention.

[0013] In some preferred embodiments, the recombinant vector is a pSUMO vector.

[0014] A fourth aspect of the present invention provides a transformant comprising a recombinant expression vector as described in the third aspect of the present invention, wherein the transformant is a non-animal or non-plant variety.

[0015] In some preferred embodiments, the host cell of the transformant is Escherichia coli, such as Rosetta (DE3).

[0016] A fifth aspect of the present invention provides a pharmaceutical composition comprising a fusion protein as described in the first aspect of the present invention, and a pharmaceutically acceptable carrier.

[0017] A sixth aspect of the present invention provides a kit comprising a fusion protein as described in the first aspect of the present invention, an isolated nucleic acid as described in the second aspect of the present invention, a recombinant expression vector as described in the third aspect of the present invention, a transformant as described in the fourth aspect of the present invention, or a pharmaceutical composition as described in the fifth aspect of the present invention.

[0018] In some preferred embodiments, the kit further includes (i) a means of administering the fusion protein or pharmaceutical composition; and / or (ii) instructions for use.

[0019] The seventh aspect of the present invention provides a method for preparing a fusion protein, the method comprising the steps of: culturing a transformant as described in the fourth aspect of the present invention, and obtaining a fusion protein from the culture.

[0020] In some preferred embodiments, the culture is a cell growth culture and / or an induction phase culture; the cell growth culture is a non-induction culture, including a seed culture stage and a reactor culture stage, wherein the seed culture stage is preferably: the transformant is inoculated into a basal medium 2 containing antibiotics such as kanamycin and chloramphenicol at an inoculation ratio of 0.1% to 5%, for example 0.1% (v / v), the culture temperature is 37°C, the pH value is 7±0.2, and the culture is shaken until OD600 = 5.7-15.4 to obtain a seed solution; the reactor ... The seed culture is inoculated into the bioreactor, wherein the OD600 of the bacterial cells in the bioreactor after inoculation is 0.1, the culture temperature is 37℃, the pH is 7.00±0.20, and after the carbon source is depleted, C3 medium is added at a feeding rate of 60-90 g / kg / h, for example 60.00 g / kg / h. The preferred induction phase culture is as follows: the bacterial cells are cultured to OD600=150, the culture conditions of the transformant are adjusted to 25-35℃, for example 30℃, pH=7.00±0.2, and IPTG is added to induce protein expression for 11.0 h.

[0021] In some preferred embodiments, the basal culture medium 2 comprises 10.0 g / L glycerol, 8.0 g / L (NH4)2SO4, 1.7 g / L C6H8O7·H2O, 9.2 g / L KH2PO4, 0.11 g / L FeSO4·7H2O, 2.60 g / L NaOH, and 40.0 g / L yeast extract; the C3 culture medium comprises 270.0 g / L glycerol, 0.018 g / L FeSO4·7H2O, and 214.0 g / L yeast extract; and / or, the preparation method further includes a purification step; preferably, the purification process is as follows: lysis-inclusion body washing-denaturation-dilution-renaturation-deep filtration-concentration and solution replacement 1 (UF / DF-1)-affinity chromatography-anion exchange chromatography-endotoxin removal chromatography-concentration and solution replacement 2 (Final UF / DF). The eighth aspect of the present invention provides a fusion protein formulation comprising the fusion protein as described in the first aspect of the present invention, and preferably further comprising pharmaceutical excipients.

[0022] In some preferred embodiments, in the fusion protein formulation, the ratio of fusion protein to formulation excipients is 4:1, and the active unit of the fusion protein is 5 million IU.

[0023] In some embodiments, the excipient is 10% sucrose.

[0024] The ninth aspect of the present invention provides the use of a fusion protein as described in the first aspect of the present invention, an isolated nucleic acid as described in the second aspect of the present invention, a recombinant expression vector as described in the third aspect of the present invention, a transformant as described in the fourth aspect of the present invention, a pharmaceutical composition as described in the fifth aspect of the present invention, a kit as described in the sixth aspect of the present invention, or a fusion protein formulation as described in the eighth aspect of the present invention in the preparation of an antiviral drug for dogs.

[0025] In some preferred embodiments, the drug is a biological agent; the virus is canine parvovirus, canine distemper, and / or canine influenza.

[0026] The tenth aspect of the present invention provides a method for treating canine parvovirus, the method comprising administering to a dog infected with canine parvovirus an effective amount of a fusion protein as described in the first aspect of the present invention, an isolated nucleic acid as described in the second aspect of the present invention, a recombinant expression vector as described in the third aspect of the present invention, a transformant as described in the fourth aspect of the present invention, a pharmaceutical composition as described in the fifth aspect of the present invention, a kit as described in the sixth aspect of the present invention, or a fusion protein formulation as described in the seventh aspect of the present invention.

[0027] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0028] The reagents and raw materials used in this invention are all commercially available.

[0029] The positive and progressive effects of this invention are as follows:

[0030] 1. Improved formulation stability of the molecule: The initial design of this molecule was based on the His-SUMO dual tag, which can not only increase protein expression and achieve soluble expression, but also reduce purification difficulty. The final results showed that the expression level of the molecule was indeed significantly improved. Furthermore, it was unexpectedly found that when the SUMO tag was expressed and not excised, the stability of the molecule was significantly better than that of the natural canine interferon α (His-CaIFNα) without the SUMO tag, while maintaining the activity of the sample.

[0031] 2. Excellent molecular biological activity: Previous studies have shown that the fusion of other peptides or fusion proteins at the N-terminus or C-terminus of a protein can create steric hindrance to the binding of the target protein to the receptor, thereby reducing or even eliminating the activity of the target protein. However, the in vitro activity of the recombinant canine interferon α (His-SUMO-CaIFNα) prepared in this invention is superior to that of natural canine interferon α (His-CaIFNα). In vivo pharmacodynamic studies have shown that the molecule has an effective therapeutic effect on canine parvovirus disease. After administration once a day for 7 consecutive days, all experimental dogs in the control model group died, while the clinical manifestations of the test group returned to normal after the administration period, and no experimental dogs died.

[0032] 3. Significantly increased expression levels: Based on the molecular structure and the development of the fermentation process, the expression level is significantly increased. Specifically, using the same optimized process, the fermentation expression level of natural canine interferon α (His-CaIFNα) is 2.5 g / L; the expression level of the optimized recombinant canine interferon α molecule reaches 14.62 g / L, an increase of about 5 times; the quality of the samples prepared by the process meets the quality standards, and the endotoxin content is less than 10 EU / mg.

[0033] 4. Significantly increases the half-life of the molecule: According to PK research results, the half-life of natural canine interferon α (His-CaIFNα) is about 2.37h, while that of the modified molecule is about 6.15h, which is about 2.5 times longer. Attached Figure Description

[0034] Figure 1 shows the plasmid map of the recombinant canine interferon α expression vector pHis-SUMO-CaIFNα of the present invention.

[0035] Figure 2 shows the gene fragment identification during the construction of the recombinant canine interferon α plasmid of the present invention, where M: DNA Marker; 1: pSUMO-kana vector digested with enzymes; 2: PCR target fragment CaIFNα.

[0036] Figure 3 shows the PCR identification diagram of the recombinant canine interferon α colonies of the present invention, where M: DNA Marker; 1-5: 5 single colonies; NTC: negative control without template.

[0037] Figure 4 shows the SDS-PAGE diagram of the colony expression identification results of recombinant canine interferon α in this invention; where 1: expression of the correctly sequenced strain containing the recombinant pHis-SUMO-CaIFNα plasmid before IPTG induction, 2-9: expression of the correctly sequenced strain containing the pHis-SUMO-CaIFNα plasmid after single colony culture and IPTG induction, 10: expression of Rosetta(DE3) empty bacteria before IPTG induction, 11: expression of Rosetta(DE3) empty bacteria after IPTG induction, and M is the marker.

[0038] Figure 5 is an SDS-PAGE image of the colony expression identification results of recombinant canine interferon α in this invention; where M: Protein Marker; 1: whole bacteria after IPTG-induced bacterial disruption; 2: supernatant after IPTG-induced bacterial disruption; 3: precipitate after IPTG-induced bacterial disruption.

[0039] Figure 6 shows the SDS-PAGE expression levels of recombinant canine interferon α (His-SUMO-CaIFNα) and natural canine interferon α (His-CaIFNα) after fermentation; where M: Protein Marker; 1: whole cell culture of recombinant canine interferon α (His-SUMO-CaIFNα) fermentation broth; 2: whole cell culture of natural canine interferon α (His-CaIFNα) fermentation broth. The arrows indicate the target protein.

[0040] Figure 7 shows the downstream process flow diagram of recombinant canine interferon α.

[0041] Figure 8 shows the bacterial lysis diagram, where 1: Marker; 2: precipitate after 1 lysis; 3: supernatant after 1 lysis; 4: total sample after 1 lysis; 5: total sample after 2 lysis; 6: supernatant after 2 lysis; 7: precipitate after 2 lysis.

[0042] Figure 9 shows the non-reducibility diagram of inclusion body washing, where 1: Marker; 2: lysis-Total; 3: lysis supernatant; 4: lysis precipitate; 5: concentration reflux; 6: concentration permeate; 7: 1 DV of fluid exchange; 8: 2 DV of fluid exchange; 9: 3 DV of fluid exchange; 10: 4 DV of fluid exchange; 11: 5 DV of fluid exchange; 12: 6 DV of fluid exchange; 13: 7 DV of fluid exchange; 14: reflux of fluid exchange; 15: Marker.

[0043] Figure 10 shows the denaturation process over 1 hour, where: 1: Marker; 2: 4M-1h-10mM-Total; 3: 4M-1h-10mM-Supernatant; 4: 4M-1h-20mM-Total; 5: 4M-1h-20mM-Supernatant; 6: 4M-1h-30mM-Total; 7: 4M-1h-30mM-Supernatant; 8: 4M-1h-50mM-Total; 9: 4M-1h-50mM-Supernatant; 10: 6M-1h-10mM-Total; 11: 6M-1h-10mM-Supernatant.

[0044] Figure 11 shows the denaturation process over 2 hours, where: 1: Marker; 2: 4M-2h-10mM-Total; 3: 4M-2h-10mM-Supernatant; 4: 4M-2h-20mM-Total; 5: 4M-2h-20mM-Supernatant; 6: 4M-2h-30mM-Total; 7: 4M-2h-30mM-Supernatant; 8: 4M-2h-50mM-Total; 9: 4M-2h-50mM-Supernatant; 10: 6M-2h-10mM-Total; 11: 6M-2h-10mM-Supernatant.

[0045] Figure 12 shows the loading range for AC chromatography, where 1: Marker, 2: Load for affinity chromatography DBC experiment; 3-14 are the flow through FT1-12 for affinity chromatography DBC experiment, and 15: Standard STD.

[0046] Figure 13 shows the loading range for AC chromatography, where 1-13 are elutions for affinity chromatography DBC experiments, 13-1; 14 is the loading for affinity chromatography DBC experiments; and 15 is the standard STD.

[0047] Figure 14 shows the identification results of high-load elution in affinity chromatography, where 1-13 are Elution 13-1 of high-load elution in affinity chromatography; 14 is high-load loading in affinity chromatography; and 15 is the marker.

[0048] Figure 15 shows the identification results of low-load elution in affinity chromatography, where 1: Marker; 2: Low-load loading in affinity chromatography; 3-14 are elutions 1-12 in affinity chromatography, respectively.

[0049] Figure 16 shows the purification and identification results of anion exchange chromatography with high loading, where 1: loading in the anion exchange chromatography high loading experiment; 2: Marker; 3-12 are elutions 1-12 in the anion exchange chromatography high loading experiment, respectively.

[0050] Figure 17 shows the results of purification and identification by anion exchange chromatography underloading, where 1: Marker; 2: Loading in the anion exchange chromatography underloading experiment; 3-15 are the elutions in the anion exchange chromatography underloading experiment, respectively.

[0051] Figure 18 shows the SDS-PAGE images of the stability of recombinant canine interferon α protein molecules with different fusion tags according to the present invention. In the figure, M: Protein Marker, 1-3 are natural canine interferon α (His-CaIFNα) after 0 days, 4℃ for 14 days, and 40℃ for 14 days, respectively; 4-6 are recombinant canine interferon α (His-SUMO-CaIFNα) after 0 days, 4℃ for 14 days, and 40℃ for 14 days, respectively; 7-9 are recombinant canine interferon α (His-GST-CaIFNα) after 0 days, 4℃ for 14 days, and 40℃ for 14 days, respectively; 10-12 are recombinant canine interferon α (His-NusA-CaIFNα) after 0 days, 4℃ for 14 days, and 40℃ for 14 days, respectively.

[0052] Figure 19 shows the SDS-PAGE identification diagram of the stability study of the recombinant canine interferon α preparation of the present invention; where M: Protein Marker; 1: recombinant canine interferon α T0 (0 days); 2-5 are the concentrations and degradation status of recombinant canine interferon α T2W (14 days) in buffers a, b, c, and d, respectively; 6: natural canine interferon α T0 (0 days); 7-10 are the concentrations and degradation status of natural canine interferon α T2W (14 days) in buffers a, b, c, and d, respectively.

[0053] Figure 20 shows the pathological identification results of the in vivo anti-canine parvovirus activity study of the recombinant canine interferon α of the present invention.

[0054] Figure 21 is an SDS-PAGE diagram of the stability of the formulation of the present invention; where M: Protein Marker; the left figure shows the results after 0 days of incubation, with lanes 1-5 representing formulations 1-5 respectively; the right figure shows the results after 2 weeks (14 days) of incubation at 40°C, with lanes 1-5 representing formulations 1-5.

[0055] Figure 22 shows the SDS-PAGE plot of the stability of the formulation of the present invention; where M: Protein Marker, the left figure shows the results after 1 month (1 month) at 40℃, and lanes 1-5 are formulations 1-5 respectively; the right figure shows the results after 1 month at 4℃, and lanes 1-5 are formulations 1-5 respectively.

[0056] Figure 23 is a survival curve of dogs in an in vivo pharmacodynamic experiment of the recombinant canine interferon α (lyophilized type) of the present invention. Detailed Implementation

[0057] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0058] Example 1: Synthesis of Recombinant Canine Interferon α Gene, Construction and Identification of Expression Vector

[0059] 1. Synthesis and amplification of recombinant canine interferon α gene

[0060] The natural canine interferon α gene (nucleotide sequence: SEQ ID NO:1; amino acid sequence: SEQ ID NO:7) was synthesized by Shanghai Sangon Biotech Co., Ltd. after removing the signal peptide sequence according to GenBank sequence number XM_049116181.1 and then injected into the pUC57 vector to obtain pUC57-CaIFNα. Homologous recombination was used, and the empty pSUMO(kana+) vector was linearized using restriction endonucleases Bsa I and BamHI. Using pUC57-CaIFNα as a template, the target fragment of natural canine interferon α was amplified by PCR using the N-terminal primer CaIFNα-F (SEQ ID NO:5) and the C-terminal primer CaIFNα-R (SEQ ID NO:6). The underlined parts are homologous to the pSUMO-kana vector. The fragments were then separated by agarose gel electrophoresis, and the corresponding fragment products were recovered using a gel extraction kit (see Figure 2).

[0061] 2. Construction and validation of recombinant canine interferon-α expression vector

[0062] The target fragment was ligated into the pSUMO(kana+) vector (the amino acid sequence of the SUMO tag is SEQ ID NO:12) using the ClonExpress Ultra OneStep Cloning Kit. The ligation reaction was carried out in a 50°C circulating water bath for 5 min, and immediately placed on ice after the reaction (see Figure 1 for the map of the recombinant canine interferon α expression vector). 10 μl of the ligation product was added to 100 μl of DH 5α competent cells, incubated on ice for 30 min, heat-shocked in a 42°C water bath for 45 s, and then rapidly cooled on ice for 3–5 min. Then, 500 μl of antibiotic-free LB liquid medium was added and mixed well, and the mixture was incubated at 37°C with shaking for 45 min to recover. The recovered fragment was then spread onto LB solid medium plates containing a final concentration of 50 mg / L kanamycin and incubated overnight at 37°C. Then, single colonies were picked and cultured in LB liquid medium containing a final concentration of 50 mg / L kanamycin, and bacterial culture PCR was performed for identification (the identification results are shown in Figure 3). The bacterial cultures with positive results were sent to Shanghai Sangon Biotech for sequencing.

[0063] 3. Construction and identification of recombinant canine interferon α strain

[0064] The correctly sequenced recombinant pHis-SUMO-CaIFNα plasmid was transformed into competent cells of the expression strain Rosetta (DE3). Single colonies were picked and inoculated into 20 mL of LB broth containing both antibiotics (final concentrations of 50 mg / L kanamycin and 34 mg / L chloramphenicol resistance). The cells were activated at 37°C for 12 h. Then, at a 1% inoculation rate, the cells were transferred to another 20 mL of LB broth containing both antibiotics (final concentrations of 50 mg / L kanamycin and 34 mg / L chloramphenicol resistance). The cells were incubated at 37°C and 180 rpm for 2-3 h until the OD600 reached 0.4-0.6. Finally, a final concentration of [missing information - likely a specific concentration] was added. Induction was performed at 37°C for 4 hours with 0.25 mM IPTG, followed by sampling and identification. The positive clones with the highest expression levels were screened and inoculated into 20 mL of LB liquid medium containing double antibodies (final concentration of 50 mg / L kanamycin and final concentration of 34 mg / L chloramphenicol resistance). The medium was activated at 37°C for 12 hours, then transferred to 400 mL of medium at a 1% inoculation rate. The culture was carried out at 37°C and 180 rpm for 2-3 hours until the OD600 reached 0.4-0.6. IPTG was then added at a final concentration of 0.25 mM, and the culture was carried out at 25°C and 180 rpm for 24 hours. Sampling was performed, and the expression of the strains was identified by SDS-PAGE. The results are shown in Figures 4 and 5.

[0065] Natural CaIFNα gene nucleotide sequence (SEQ ID NO:1):

[0066] The amino acid sequence of the natural CaIFNα gene (SEQ ID NO:7):

[0067] CaIFNα-F (SEQ ID NO:5):

[0068] CaIFNα-R(SEQ ID NO:6):

[0069] The amino acid sequence of the His tag (SEQ ID NO:11):

[0070] His His His His His His His;

[0071] The amino acid sequence of the SUMO tag (SEQ ID NO:12):

[0072] Nucleotide sequence of His-SUMO fusion tag sequence (SEQ ID NO:3):

[0073] The amino acid sequence of the His-SUMO fusion tag (SEQ ID NO:9):

[0074] Example 2: Process Development and Sample Preparation of Recombinant Canine Interferon α

[0075] 1. Optimization and production of recombinant canine interferon α fermentation process

[0076] First, a platform fermentation process was adopted, in which 50 mg / L kanamycin and 34 mg / L chloramphenicol were added to the basal medium 1 (30 g / L glycerol, 6 g / L yeast extract, 4 g / L ammonium sulfate, 15 g / L KH2PO4, 1.5 g / L citric acid, 0.5 g / L NaCl, pH 7.0-7.2), and the culture was carried out at 37℃. 600 The induction temperature was lowered to 30.0℃, the inducer concentration (IPTG) was 0.25mM, the culture pH was 7.00±0.03, and the induction time was 6h. This study investigated the effects of recombinant canine interferon α (His-SUMO-CaIFNα, nucleotide sequence: SEQ ID NO:4; amino acid sequence: SEQ ID NO:10) and natural canine interferon α (His-CaIFNα, nucleotide sequence: SEQ ID NO:2; amino acid sequence: SEQ ID NO:10). NO:8) were fermented separately. The results showed that the recombinant canine interferon α (His-SUMO-CaIFNα) was expressed as inclusion bodies. Gray-scale analysis showed that it accounted for approximately 35% of the total host protein, with a target protein expression level of 2.8 g per liter of fermentation broth. Under the same fermentation conditions, the natural canine interferon α (His-CaIFNα) accounted for approximately 25% of the total host protein, with a target protein expression level of 0.6 g per liter of fermentation broth. The expression level of the fusion protein was approximately 5 times that of the natural canine interferon α (His-CaIFNα), indicating that the expression level of the modified fusion protein was significantly increased.

[0077] Nucleotide sequence of natural canine interferon α (His-CaIFNα) (SEQ ID NO:2):

[0078] The amino acid sequence of natural canine interferon α (His-CaIFNα) (SEQ ID NO:8):

[0079] Nucleotide sequence of recombinant canine interferon α (His-SUMO-CaIFNα) (SEQ ID NO:4):

[0080] The amino acid sequence of recombinant canine interferon α (His-SUMO-CaIFNα) (SEQ ID NO:10):

[0081] Next, the present invention systematically optimized the upstream fermentation process conditions, establishing a set of efficient and robust upstream fermentation production processes. Specific work is as follows: First, fed-batch culture medium screening, induction temperature, culture pH, inducer concentration optimization, and antibiotic-free fermentation evaluation were conducted. In the fed-batch culture medium optimization experiment, the effects of three different fed-batch media on the expression of recombinant canine interferon α (His-SUMO-CaIFNα) were investigated. The results (see Table 2 below) showed that when the fed-batch medium was C3 (270.0 g / L glycerol, 0.018 g / L FeSO4·7H2O, and 214.0 g / L yeast extract), the target protein expression level was the highest, reaching 13.41 g / L. In the induction temperature optimization experiment, the effects of four different induction temperatures (37.0℃, 34.0℃, 30.0℃, and 25.0℃) on cell growth and target protein expression were explored. The results (see Table 3 below) showed that the expression level and bacterial OD600 of recombinant canine interferon α (His-SUMO-CaIFNα) were lowest at an induction temperature of 25.0℃, at 6.12 g / L and 239.0, respectively. At induction temperatures of 37.0℃ and 34.0℃, the target protein expression levels were 10.27 g / L and 11 g / L, respectively, both higher than the control group (i.e., the level of recombinant interferon α protein at an induction temperature of 30℃ in Table 3). In the pH optimization experiment, the effects of two pH control ranges (6.80±0.20 and 7.00±0.20) on the expression of recombinant canine interferon α (His-SUMO-CaIFNα) were compared. It was found that under the two different pH control strategies, there was no significant difference in the expression level and bacterial biomass of recombinant canine interferon α (His-SUMO-CaIFNα), as detailed in Table 4 below. In the inducer concentration optimization experiment, the effects of three different IPTG concentrations (0.1 mM, 0.5 mM, and 1.0 mM) on bacterial growth and recombinant canine interferon α (His-SUMO-CaIFNα) expression were investigated. The results showed (see Table 5) that the IPTG concentrations of 0.1–1.0 mM had no significant effect on bacterial growth or the expression of the target protein. Finally, in the first round of optimization experiments, the effects of chloramphenicol-free and kanamycin-free fermentation on bacterial growth and recombinant canine interferon α (His-SUMO-CaIFNα) expression were also evaluated. It was found that antibiotic-free fermentation had no significant effect on bacterial growth or recombinant canine interferon α (His-SUMO-CaIFNα) expression.

[0082] Based on the results of the first round of process development, a second round of process development experiments was conducted in a 5L bioreactor, with a total of six 5L fermenters. The main focus was on investigating the effects of different feed rates, single-antibiotic fermentation (fermentation containing only kanamycin), and inducer concentration on cell growth and target protein production. In the feed rate optimization experiment, three different feed rates—60.00 g / kg / h, 77.93 g / kg / h, and 90.00 g / kg / h—were evaluated. The results (see Table 6) showed that high biomass could be obtained at all three feed rates. However, compared with the results of the first round of process optimization, the expression level of recombinant canine interferon α (His-SUMO-CaIFNα) was significantly reduced. Analysis indicated that there might be an interaction between the type of feed medium and the inducer concentration. Therefore, a supplementary experiment was conducted: the concentration of the inducer IPTG was adjusted to 0.5 mM, and the effects of two different post-induction feed rates (60.00 g / kg / h and 77.93 g / kg / h) on fermentation performance were investigated. The results (see Table 7) showed that increasing the inducer concentration significantly increased the expression level of recombinant canine interferon α (His-SUMO-CaIFNα), especially at a post-induction feeding rate of 77.93 g / kg / h, where the expression level of recombinant canine interferon α (His-SUMO-CaIFNα) reached its highest level of 18.12 g / L. However, under these conditions, fermentation foam was difficult to control in the later stages of induction. Considering the actual needs of process scale-up, it was finally decided to set the post-induction feeding rate to 60.00 g / kg / h, at which the expression level of recombinant canine interferon α (His-SUMO-CaIFNα) was 14.62 g / L.

[0083] After a second round of upstream fermentation process optimization, the final upstream process parameters were determined as follows:

[0084] Basic culture medium 2:

[0085] Add kanamycin and chloramphenicol to a final concentration of 50 mg / L in 10.0 g / L glycerol, 8.0 g / L (NH4)2SO4, 1.7 g / L C6H8O7·H2O, 9.2 g / L KH2PO4, 0.11 g / L FeSO4·7H2O, 2.60 g / L NaOH and 40.0 g / L yeast extract.

[0086] Feeding medium (C3):

[0087] 270.0 g / L glycerol, 0.018 g / L FeSO4·7H2O and 214.0 g / L yeast extract.

[0088] 1. Seed culture stage

[0089] The bacterial cells were inoculated into basal medium 2 containing kanamycin and chloramphenicol at an inoculation ratio of 0.1%, and cultured at 37°C and pH 7±0.2. After shaking culture until OD600=5.7-15.4, the seed culture was obtained.

[0090] 2. Reactor cultivation stage

[0091] The seed culture was inoculated into the bioreactor, wherein the OD600 of the bacteria in the bioreactor after inoculation was 0.1, the culture temperature was 37°C, the pH was 7.00±0.20, and after the carbon source was depleted, C3 medium was fed at a feeding rate of 60 g / kg / h.

[0092] 3. Induction phase culture

[0093] After 10.0–13.0 h of feeding, when the cell density reached the target OD 600 When OD600 = 150.0 ± 5.0, begin cooling. Set the program to reduce the temperature to 30.0 ± 1.0℃ in 1 hour. When OD600 = 175 ± 5, start inducing protein expression with 0.5 mM IPTG.

[0094] In summary, after two rounds of process development, the optimized process was finally locked in a 5L fermenter. This process showed good robustness and repeatability and will be used in subsequent GMP production.

[0095] Table 1 Comparison of the yields of recombinant canine interferon α and natural canine interferon α

[0096] Table 2. Effects of three fed culture media on bacterial growth and expression of recombinant canine interferon α (His-SUMO-CaIFNα).

[0097] Table 3. Effects of four different induction temperatures on bacterial growth and expression of recombinant canine interferon α (His-SUMO-CaIFNα).

[0098] Table 4. Effects of two different pH control strategies on bacterial growth and recombinant canine interferon α (His-SUMO-CaIFNα) expression.

[0099] Table 5. Effects of three different IPTG concentrations on bacterial growth and expression of recombinant canine interferon α (His-SUMO-CaIFNα).

[0100] Table 6. Effects of three different feeding rates on bacterial growth and expression of recombinant canine interferon α (His-SUMO-CaIFNα).

[0101] Table 7. Effects of two different feeding rates at 0.5 mM inducer concentration on bacterial growth and expression of recombinant canine interferon α (His-SUMO-CaIFNα).

[0102] Next, the optimized process was used to produce natural canine interferon α (His-CaIFNα). The fermentation production results showed that the recombinant canine interferon α was expressed as inclusion bodies, and grayscale analysis showed that it accounted for approximately 48% of the total host protein, with a target protein expression level of 14.62 g per liter of fermentation broth. Under the same fermentation conditions, natural canine interferon α (His-CaIFNα) was also expressed as inclusion bodies, with an expression level accounting for approximately 30% of the total host protein, and a target protein expression level of 2.5 g per liter of fermentation broth. Therefore, the expression level of the fusion protein was at least 5 times that of natural canine interferon α. ​​This indicates that the optimized fermentation process significantly improved the expression level of the target protein compared to the production of natural canine interferon α using the same optimized process before optimization, as shown in Figure 6 and Table 1.

[0103] 2. Establishment of downstream purification process and sample preparation for recombinant canine interferon α

[0104] To establish a robust and efficient complete production process, the downstream purification process for the recombinant canine interferon α (His-SUMO-CaIFNα) of this invention was systematically optimized, comprising 10 unit operations: lysis and lysis - inclusion body washing - denaturation - dilution and renaturation - deep filtration - concentration and buffer exchange 1 (UF / DF-1) - affinity chromatography - anion exchange chromatography - endotoxin removal chromatography - concentration and buffer exchange 2 (Final UF / DF). The downstream process flow chart is shown in Figure 7. The process confirmation batch yield of recombinant canine interferon α (His-SUMO-CaIFNα) was approximately 4.6 mg / g wet cells, and the drug substance (DS) met the quality standard release requirements after testing.

[0105] (1) Lysis. This invention optimized and confirmed parameters such as the number of lysis cycles in the homogenizer, the lysis pressure, and the ratio of cell dilution in the buffer solution. Lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 2 mM EDTA, pH 8.0) was added to the cells at a ratio of 1:6 (cells g: buffer g). The cells were then resuspended using a magnetic stirrer and subsequently homogenized at a pressure of 1000 bar. During lysis, the diluted bacterial solution was lysed once and twice, with the lysis temperature controlled at 4°C. The results are shown in Table 8 below. The SDS-PAGE results of the cell lysis are shown in Figure 8. The gray value of the band after lysis twice was 85% of that after lysis once, while the sample weight after lysis once was 87% of that after lysis twice, indicating that the protein yield from the two lysis cycles was roughly equivalent.

[0106] Table 8 Summary of Cell Lysis Development Parameters

[0107] However, lysing the sample twice is more conducive to subsequent microfiltration washing of inclusion bodies. Taking all factors into consideration, we chose to use a high-pressure homogenizer at 1000 bar, a 1:6 (bacterial weight: buffer weight) dilution ratio, and lysing twice.

[0108] (2) Inclusion body washing. The process parameters for inclusion body washing were optimized, including inlet shear force, permeate flux, concentration factor, replacement solution, and replacement factor. Experimental results showed that the critical flux was 15 LMH at 2000 and 3000 shear forces, and 20 LMH at 4000 shear force. To reduce the risk of membrane blockage, a safety factor of 70% was applied, ultimately determining the shear force range as 3000-4000 shear force and the permeate flux as 10 LMH. Considering both the sample solids content and system stability, a 2x concentration parameter was selected. The results showed that the process TMP was stable, and a 2x concentration was ultimately chosen. After determining the concentration factor, the replacement volume for inclusion body washing was determined. Permeate volumes of 1-7 DV were collected (Table 9). Non-reducing SDS-PAGE results (Figure 9) showed that impurities were largely removed after a replacement volume exceeding 5 DV.

[0109] Table 9 Summary of data on the development process of inclusion bodies

[0110] Based on the above results, the minimum volume of inclusion bodies in the hollow fiber membrane column washing was determined to be 5 DV, in order to better remove various impurities in the lysis solution.

[0111] (3) Denaturation. After confirming the inclusion body washing parameters, the final concentrations of guanidine hydrochloride and DTT in the denaturing solution were determined. The final concentrations of guanidine hydrochloride were controlled at 6M and 4M, and the final concentrations of DTT were 10mM, 20mM, 30mM, and 50mM. Denaturation was carried out for 1h and 2h respectively while maintaining uniform stirring with a magnetic stirrer. The results showed that in the 10mM DTT system, the total protein content and supernatant protein content of the 4M guanidine hydrochloride system were comparable. Considering the overall cost and scale-up, the final denaturation parameters were 4M guanidine hydrochloride, 10mM DTT, and denaturation for 1-2h (see Figures 10 and 11).

[0112] (4) Dilution and Refolding. The process development of this unit mainly consists of two parts: 1. Determining the denatured sample addition time and refolding time; 2. Determining the sample refolding dilution ratio. With a denatured sample: refolding buffer dilution ratio of 1:20 (v / v), different sample addition times and sample refolding times were set. The refolding effect of the target protein was evaluated by ELISA-Titer and activity detection. Then, the denatured sample dilution factor was further optimized. In summary, after optimization, the refolding unit operation has more comprehensive operating parameters for the denatured sample refolding system and stronger guidance for process scale-up. The dilution and refolding process is as follows: at 4℃, the denatured sample: refolding buffer dilution ratio (v / v) is 1:20, the denatured sample addition time is 60 min, and the refolding incubation time is 19 h.

[0113] (5) Depth filtration. Through optimization of the loading capacity and inlet flux of the depth filters (MX0HC054H1, MX0HC027H1, and MX0HC23CL3, Merck), the following parameters were confirmed: sample rewarming temperature 20-26℃, inlet flux ≤70 LMH, depth filter membrane area: 0.2 μm membrane area ratio ≤2, sample loading capacity 80-100 L / m³. 2 The pressure for deep and 0.2μm filtration is ≤14.5psi.

[0114] (6) Concentration and Liquid Replacement 1 (UF / DF-1). Based on the molecular characteristics of the sample, a 10kDa pore size, PES material, and Millipore membrane were used for concentration and liquid replacement. An inlet flux of 200-250 LMH was selected. The permeate flux of the sample under the initial state, the sample at a 2x concentration, and the sample at a 3DV liquid replacement was investigated as a function of TMP. Finally, the optimal inlet flux was selected at 200-250 LMH, with a 2x concentration and a 3DV liquid replacement. During the operation, the TMP was set at 10-15 psi, and the loading range was ≤164 L / m³. 2 .

[0115] (7) Affinity chromatography (IMAC bestarose FF, Bogleon). The process development in this unit mainly consists of two parts: 1. Determining the loading range; 2. Determining the elution method, washing method, and sample collection conditions. Dynamic binding capacity (DBC) experiments were used to develop the AC chromatography loading range. The results are shown in Figures 12 and 13. At 103.6 CV, no target protein flow was observed during sample loading in the gel image. Considering both loading time and loading capacity, 104 CV was chosen as the high loading capacity and 52 CV as the low loading capacity for further loading confirmation experiments. Based on the DBC experimental results, the washing conditions, one-step elution conditions, and elution and sample collection conditions were developed and confirmed after confirming the AC chromatography loading range. The final conditions were determined as follows: Loading capacity: target value 5 mg target protein / mL Resin, range 3.17-6.34 mg target protein / mL; elution buffer: 20 mM Tris-HCl, 0.1 M NaCl, 250 mM imidazole, 10% glycerol, pH 8.0; one-step elution with fixed elution buffer; peak collection range: 60 mAu / mm - peak - 60 mAu / mm; 3 CVs were collected. The identification results of high-loading affinity chromatography are shown in Figure 14, and the identification results of low-loading affinity chromatography are shown in Figure 15.

[0116] (8) Anion Exchange Chromatography (Diamond Q Mustang, Bogleon). The process development for this unit mainly consisted of two parts: 1. Determining the loading capacity; 2. Determining the elution method and sample collection range. The final AEX loading capacity was determined to be 30 mg / mL, within the range of 21-42 mg / mL. The elution buffer consisted of 20 mM Tris-HCl, 0.235 M NaCl, 10% glycerol, and pH 8.0. One-step elution with a fixed elution buffer was used, and the peak collection range was 100 mAu / mm - peak - 50 mAu / mm. The results of high-loading purification by anion exchange chromatography are shown in Figure 16, and the results of low-loading purification by anion exchange chromatography are shown in Figure 17.

[0117] (9) Endotoxicity removal chromatography (Etoxiclear, Astraea). The loading range for this process unit was determined based on key performance indicators (IPE) of endotoxicity removal, yield, and product quality. Etoxiclear packing material exhibits weak binding to the target protein; therefore, the high and low loading capacities were set at 100 mg target protein / mL Resin and 50 mg target protein / mL resin, respectively. Actual sample loading capacities for the high and low loading capacities were 98.1 mg / mL and 47.9 mg / mL, respectively. The collection UV was 25-25 mAu / mm. Sample loading, collection, and product quality are shown in Table 10 below.

[0118] Table 10 Information on EtoxiClear Chromatography Samples and Products

[0119] The endotoxin level of the sample after endotoxin removal chromatography was below 1 EU / mg, which meets the requirements. The yield of the target protein was greater than 80%, and the purity and activity of the product met the requirements. Therefore, this loading range can meet the process requirements.

[0120] (10) Concentration and buffer exchange 2 (UF / DF-1). Based on the parameters developed for the intermediate UF / DF sample, the operating parameters of the final UD / DF were further optimized. The Merck 10kDa PES membrane pack was still used in the experiment. The final inlet flow rate was selected as 200-250 LMH, TMP was controlled at 5-10 psi, the sample concentration was controlled in the range of 2.0-3.0 mg / mL, and the recommended buffer exchange was 7 DV (buffer buffer 20 mM Tris-HCl, pH 7.99, Cond.: 1.14 mS / cm).

[0121] Example 3: Study on the stability of recombinant canine interferon α protein with different fusion tags

[0122] The stability of recombinant canine interferon α protein with different fusion tags was studied using the following method.

[0123] A 20 mM Tris, pH 8.0 buffer solution was prepared. Recombinant canine interferon α molecules, including His-SUMO-CaIFNα, His-GST-CaIFNα, His-NusA-CaIFNα, and native canine interferon α (His-CaIFNα), were respectively added to the 20 mM Tris, pH 8.0 buffer solution and incubated at 4℃ and 40℃ for 2 weeks (14 days). The appearance, concentration, and degradation of the target proteins were observed. The results showed that at 4℃, compared to T0, the His-SUMO-CaIFNα molecule was the most stable after 14 days of incubation at 4℃, with no significant degradation. After 14 days at 4°C, His-GST-CaIFNα, His-NusA-CaIFNα, and natural canine interferon α (His-CaIFNα) molecules all showed slight precipitation. SDS-PAGE analysis of the supernatant after centrifugation revealed weakened target bands. Under the same buffer system, this indicates that the fused GST, NusA, and natural canine interferon molecules are unstable and exhibit some aggregation. At 40°C: compared to T0, the His-SUMO-CaIFNα molecule was the most stable after 2 weeks (14 days) at 40°C, showing no significant degradation. After being placed at 40°C for 14 days, the target bands of His-GST-CaIFNα, His-NusA-CaIFNα, and natural canine interferon α (His-CaIFNα) molecules were significantly weakened, indicating that the target protein was largely degraded. This suggests that recombinant expression using the SUMO fusion tag to fuse canine interferon α is more stable than that using the GST and NusA fusion tags, and is superior to that using natural canine interferon α (His-CaIFNα) without a fusion tag. The results are shown in Figure 18.

[0124] Example 4: Stability Study of Recombinant Canine Interferon α Molecules

[0125] The stability of recombinant canine interferon α was studied using the following method. The appropriate buffer solutions were prepared according to the composition of the buffer solutions in Table 11 below. Then, the recombinant canine interferon α protein (His-SUMO-CaIFNα) and natural canine interferon α (His-CaIFNα) were respectively replaced in different buffer solutions and placed at 4℃ and 40℃. The appearance, pH, concentration, and degradation of the target protein were observed at T0 (day 0) and T2W (day 14).

[0126] Table 11 List of different buffer systems

[0127] The results showed that, compared with T0, the recombinant canine interferon α molecule was the most stable in buffer d after being placed at 40℃ for 14 days without significant degradation. However, compared with T0, the band of natural canine interferon α was significantly weaker in all four buffers, and most of the target protein was degraded. This indicates that, in buffer d, the stability of recombinant canine interferon α after fusion expression modification was significantly improved, which was better than that of natural canine interferon α. ​​The results are shown in Figure 19.

[0128] Example 5: Determination of the half-life of recombinant canine interferon α in dogs.

[0129] To investigate the effect of the His-SUMO tag modification on the half-life of recombinant canine interferon α, natural canine interferon α (His-CaIFNα) was selected as the control group. Twelve healthy, susceptible beagle dogs aged 50–60 days were randomly divided into two groups of six each. Recombinant canine interferon α (His-SUMO-CaIFNα) and natural canine interferon α (His-CaIFNα) proteins were injected intramuscularly, respectively, at a single dose of 3 mg / kg. Serum samples were collected from each group at 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 18 h, and 24 h after administration. The concentration of the test sample in the serum was determined using the double-antibody sandwich ELISA method.

[0130] Table 12 Results of Interferon Half-Life Measurement

[0131] The experimental results show that the half-life of the recombinant canine interferon α (His-SUMO-CaIFNα) protein prepared in this invention is about 2.5 times that of the natural canine interferon α (His-CaIFNα). The results indicate that after fusing the His-SUMO tag, the half-life extension effect beyond the known function of the SUMO tag was obtained, as shown in Table 12.

[0132] Example 6: Determination of the biological activity of recombinant canine interferon α

[0133] 1. The biological activity of recombinant canine interferon α at the in vitro cellular level was detected using the A72 / VSV system.

[0134] Cell culture: A-72 cells were allowed to adhere and grow in the culture medium, and seeded into 96-well cell culture plates at 100 μl per well, and cultured at 37°C and 5% carbon dioxide.

[0135] Sample preparation: Dilute the test sample to 1 μg / mL with the assay medium as the stock solution. Then, perform a 4-fold serial dilution in a 96-well cell culture plate, with two wells for each dilution. Sample preparation must be performed under aseptic conditions.

[0136] Sample loading: Transfer the prepared test solution into a culture plate inoculated with A-72 cells, add 100 μl to each well, and incubate at 37°C and 5% carbon dioxide.

[0137] Challenge: Discard the supernatant in the cell culture plate, dilute the preserved vesicular stomatitis virus (VSV) with challenge culture medium (3% DMEM), 100 μl per well, and culture at 37°C and 5% carbon dioxide until more than 75% of the cells in the virus control well show lesions (with a few cells still surviving), and then determine the results.

[0138] Staining and Result Recording: Discard the supernatant in the cell culture plate, add 50 μl of 0.05% (w / v) crystal violet staining solution to each well, incubate at room temperature for 30 minutes, then carefully rinse off the staining solution with running water, shake off any remaining water, and visually inspect the plate. The degree of lesion is determined based on the integrity of the staining within the wells, and is represented by the following symbols:

[0139] — No cellular lesions;

[0140] + Less than 25% of cells are diseased;

[0141] ++ 25%–50% of the cells show lesions;

[0142] +++ 50%–75% of the cells show pathological changes;

[0143] ++++ More than 75% of the cells are diseased;

[0144] If the wells are completely and well-stained, showing a full purple coverage (like cell control wells), then it is judged as CPE (—). Interferon-protected wells should show a full purple coverage (—). 100% diseased cells are completely detached and therefore colorless. If both virus control wells and wells without interferon protection are colorless, then it is judged as CPE (++++).

[0145] The highest dilution (working unit) at which interferon can protect half (50%) of cells from viral damage was calculated using the Reed-Muench formula, as shown in Table 13 below. Then, in this study, recombinant human interferon α2b (427,000 IU / vial (2013) National Biosafety Standard No. 0027) was used as a standard to correct the levels of recombinant canine interferon α (His-SUMO-CaIFNα). The Reed-Muench formula is:

[0146] Table 13 Calculation of 50% Protected Cell Recombinant Canine Interference α (His-SUMO-CaIFNα) Dilution

[0147] Calculations show that the working unit for recombinant canine interferon α is 599.69 units / ml, and the working unit for the standard recombinant human interferon α2b is 73.52 units / ml. The revised working units are shown in Table 15. The corrected formula is as follows:

[0148] R1:R2 = X1:X2;

[0149] R1: Working unit of recombinant human interferon α2b standard;

[0150] R2: Working unit of recombinant human interferon α2b standard measured under the same conditions;

[0151] X2: Working unit for the determination of recombinant canine interferon α (His-SUMO-CaIFNα);

[0152] X1: Working unit for the determination of modified recombinant canine interferon α (His-SUMO-CaIFNα); see Tables 14 and 15.

[0153] Table 14 Interferon potency determination results (recorded with "+" and "-" signs) (procedure)

[0154] Table 15 Calculation of Interferon Potency in Working Units

[0155] Specific activity potency calculation: After correction with the recombinant human interferon α2b standard, the working unit of this 1 μg / ml recombinant canine interferon α (His-SUMO-CaIFNα) is 3.48E+06 IU / ml. The specific activity potency was calculated according to the specific activity potency formula. The final specific activities of recombinant canine interferon α (His-SUMO-CaIFNα) and natural canine interferon α (His-CaIFNα) are shown in Table 16. The specific activity potency calculation formula is:

[0156] Table 16 Results of in vitro activity assays of recombinant canine interferon α and natural canine interferon α

[0157] In summary, in vitro activity assays showed that the specific activity of recombinant canine interferon α (His-SUMO-CaINFα) was 3.48E+09 IU / mg, which is approximately 23.92 times that of natural canine interferon α (His-CaIFNα) (1.45E+08 IU / mg), indicating that the in vitro activity of the modified molecule is superior to that of natural canine interferon α.

[0158] 2. Study on the in vivo anti-canine parvovirus activity of recombinant canine interferon α protein.

[0159] To verify the efficacy of recombinant canine interferon-alpha against canine parvovirus, beagles were challenged with the virus via gavage. Six dogs were enrolled in each group, divided into an experimental group and a control group. The experimental group received recombinant canine interferon-alpha treatment 24 hours after challenge, with each dog receiving a subcutaneous injection of 400,000 IU / kg once daily for seven consecutive days. The control group received no treatment after challenge. Clinical symptoms were observed daily, and viral load was measured in anal swabs daily. All dogs were euthanized on the last day, and small intestine and mesenteric lymph nodes were collected for pathological examination.

[0160] The results (see Table 17) showed that all participants in the experimental group survived, while all participants in the control group died. The clinical symptoms in the experimental group were generally better during the experiment, while the control group developed abnormal symptoms such as lethargy, reduced appetite, porridge-like stools, and weight loss over time. Both the experimental and control groups showed tissue lesions in the small intestine and mesenteric lymph nodes, but the overall condition of the experimental group was significantly better than that of the control group (see Figure 20). The viral load in the anal swabs of both the experimental and control groups gradually increased after challenge. Based on the standard curve, the genomic copy concentration of CPV in each swab was calculated, showing that from the 4th day after challenge, the viral load in the experimental group was lower than that in the control group, and the copy concentration in the control group reached its peak. The copy concentration in the experimental group reached its peak on the 5th day after challenge (see Tables 18 and 19).

[0161] Table 17. Results of anal swab test strip testing

[0162] Table 18 Copy concentration in the test group

[0163] Table 19 Copy concentration in the control group

[0164] Therefore, this experiment demonstrates that recombinant canine interferon α can reduce and delay canine parvovirus infection, exhibiting good therapeutic effects.

[0165] Example 7: Screening of Recombinant Canine Interferon α Formulation

[0166] To test the stability of the recombinant canine interferon α (His-SUMO-CaIFNα) fusion protein formulation, different ratios of cryo-intervention mixtures (excipient mixtures without protein) were prepared, as shown in Table 20. The excipient ratios in Table 20 are based on the content per vial: weight (g) / volume (100mL). The mixture was sterilized by filtration through a 0.22μm filter membrane. 3ml packaging was used, with each vial containing 1ml. The volume ratio of the cryo-intervention mixture to the protein stock solution was 4:1, and the final protein concentration was 5 million IU / vial. The vials were filled and sealed with rubber stoppers, leaving a gap between the stopper and the vial. A placebo (control group) was prepared by adding only the cryo-intervention mixture and 20mM Tris at pH 8.0 and lyophilized. The lyophilized samples were examined for appearance, moisture content, HPLC purity, and in vitro activity, followed by high-temperature (40℃) and cold-stored (4℃) stability studies.

[0167] Table 20: Excipient Composition Table for Each Freeze-Dried Powder Formulation

[0168] Table 21 Stability Testing Items and Results of Freeze-Dried Powders with Different Excipient Components

[0169] The testing conditions, test items, and results are shown in Table 21. The results of Formula 2 after being placed at 40℃ for 2 weeks (14 days) and 1 month (1 month) were basically consistent with those after 0 days, indicating good stability. The SDS-PAGE results are shown in Figures 21 and 22.

[0170] Example 8: In vivo efficacy experiment of recombinant canine interferon α (lyophilized form)

[0171] To verify the efficacy of recombinant canine interferon alpha (lyophilized) against canine parvovirus enteritis, an in vivo efficacy study was designed in beagle dogs (2-3 months old, weighing 3.0-5.0 kg, with good mental and nutritional status; the experiment was commissioned to Changchun Xinuo Biotechnology Co., Ltd.). The CPV SD15 strain (source: Changchun Xinuo Biotechnology Co., Ltd., batch number: 20230309, original HA titer 1:2) was used. 9 After dilution, the HA titer is 1:2. 7The challenge method was gavage, 5 ml / dog / time, every 4 hours, for a total of 3 times per dog. Drug administration began 48 hours after the first challenge, once daily for 7 consecutive days. The experiment was divided into five groups, with 5 experimental dogs in each group (3 females and 2 males): model group, control veterinary drug (Zhongke Baike, 400,000 IU / kg body weight) + adjuvant drug group, 200,000 IU recombinant canine interferon α (lyophilized) / kg body weight + adjuvant drug group, 400,000 IU recombinant canine interferon α (lyophilized) / kg body weight, and 400,000 IU recombinant canine interferon α (lyophilized) / kg body weight + adjuvant drug group. The adjuvant medications included: ① Maropitan citrate injection (antiemetic): to be used upon the onset of vomiting; ② Enrofloxacin injection: to be used 48 hours after the first challenge; ③ Ethylphenidate phenolsulfonamide injection (hemostatic agent): to be used upon the onset of bloody stools; ④ 0.9% sodium chloride injection (fluid replacement): to be used upon the onset of loose stools; ⑤ Antipyretics: to be used upon the onset of fever. On the 10th day after challenge, the mortality rate was 80% in the model group; 80% in the control group (drug + adjuvant medication); 200,000 IU recombinant canine interferon α (lyophilized) / kg body weight + adjuvant medication group, with a mortality rate of 20%, and 2 animals near death; 60% in the group (400,000 IU recombinant canine interferon α (lyophilized) / kg body weight); and 20% in the group (400,000 IU recombinant canine interferon α (lyophilized) / kg body weight + adjuvant medication). The statistical results of the canine mortality rate are shown in Table 22.

[0172] Table 22. Mortality Statistics of Dogs in In Vivo Drug Efficacy Experiments

[0173] Experimental conclusions: The mortality rate in the model group was 80%, confirming the validity of the experiment; the group receiving 400,000 IU of recombinant canine interferon α (lyophilized) / kg body weight plus adjuvant drugs showed the best efficacy and the lowest mortality rate; the combined treatment of recombinant canine interferon α (lyophilized) and adjuvant drugs was significantly more effective than using recombinant canine interferon α (lyophilized) alone; the control veterinary drug showed no significant efficacy. The fusion protein described in this invention has a significant advantage in treating canine parvovirus compared to commercially available natural canine interferon α, indicating that this invention has achieved unexpected technical results. The canine survival curve is shown in Figure 23.

Claims

1. A fusion protein, characterized in that, The fusion protein comprises: a His tag, a SUMO tag, and natural canine interferon α; the amino acid sequence of the His tag is shown in SEQ ID NO:11, the amino acid sequence of the SUMO tag is shown in SEQ ID NO:12, and the amino acid sequence of the natural canine interferon α is shown in SEQ ID NO:

7.

2. The fusion protein as described in claim 1, characterized in that, The fusion protein contains a His tag, a SUMO tag, and natural canine interferon α sequentially from the N-terminus to the C-terminus.

3. The fusion protein as described in claim 1 or 2, characterized in that, The amino acid sequence of the fusion protein is shown in SEQ ID NO:

10.

4. An isolated nucleic acid, characterized in that, Its encoding is the fusion protein as described in any one of claims 1-3; Preferably, its nucleotide sequence is shown in SEQ ID NO:

4.

5. A recombinant expression vector, characterized in that, The recombinant expression vector contains the isolated nucleic acid as described in claim 4; Preferably, the recombinant expression vector is a pSUMO vector.

6. A transformant, characterized in that, The transformant comprises the recombinant expression vector as described in claim 5, wherein the transformant is a non-animal or plant variety; Preferably, the starting cell for the transformant is Escherichia coli, such as Rosetta (DE3).

7. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises a fusion protein as described in any one of claims 1-3, and a pharmaceutically acceptable carrier.

8. A reagent kit, characterized in that, It comprises the fusion protein as described in any one of claims 1-3, the isolated nucleic acid as described in claim 4, the recombinant expression vector as described in claim 5, the transformant as described in claim 6, or the pharmaceutical composition as described in claim 7; Preferably, the kit further includes (i) a means for administering the fusion protein or pharmaceutical composition; and / or (ii) instructions for use.

9. A method for preparing a fusion protein, characterized in that, The method includes the following steps: culturing the transformant as described in claim 6, and obtaining the fusion protein from the culture; Better place, The culture is a cell growth culture and / or an induction phase culture; the cell growth culture is a non-induction culture, including a seed culture stage and a reactor culture stage; the seed culture stage is preferably: the transformant is inoculated into a basal medium 2 containing antibiotics such as kanamycin and chloramphenicol at an inoculation ratio of 0.1% to 5%, for example 0.1% (v / v), the culture temperature is 37°C, the pH value is 7±0.2, and the culture is shaken until OD600 = 5.7-15.4 to obtain a seed solution; the reactor culture stage is preferably: the seed solution is inoculated into a... In the bioreactor, the inoculation is performed when the OD600 of the bacterial cells in the bioreactor is 0.1, the culture temperature is 37℃, the pH is 7.00±0.20, and after the carbon source is depleted, C3 medium is added at a feeding rate of 60-90 g / kg / h, for example, 60.00 g / kg / h. The preferred induction phase is as follows: the bacterial cells are cultured to OD600 = 150, the culture conditions of the transformant are adjusted to 25-35℃, for example, 30℃, pH = 7.00±0.2, and IPTG is added to induce protein expression for 11.0 h. More preferably, the preparation method further includes a purification step; the preferred purification process is as follows: lysis-inclusion body washing-denaturation-dilution-renaturation-deep filtration-concentration and solution replacement 1-affinity chromatography-anion exchange chromatography-endotoxin removal chromatography-concentration and solution replacement 2.

10. A fusion protein formulation, characterized in that, The fusion protein formulation comprises the fusion protein as described in any one of claims 1-3, and preferably further comprises formulation excipients; Preferably, in the fusion protein formulation, the ratio of fusion protein to formulation excipients is 4:1, and the active unit of the fusion protein is 5 million IU.

11. The fusion protein formulation of claim 10, characterized in that, The excipient is 10% sucrose.

12. The use of the fusion protein of any one of claims 1-3, the isolated nucleic acid of claim 4, the recombinant expression vector of claim 5, the transformant of claim 6, the pharmaceutical composition of claim 7, the kit of claim 8, or the fusion protein formulation of claim 10 in the preparation of an antiviral drug for dogs; Preferably, the drug is a biological agent; the virus is canine parvovirus, canine distemper, and / or canine influenza.

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