Recombinant vector comprising gene encoding feline interferon lambda-1 protein, and method for preparing same

Abstract

A recombinant vector comprising a gene encoding a recombinant feline interferon lambda-1 (rFeIFN-λ1) protein, of the present invention, has excellent expression efficiency and the effect of inducing strong antiviral activity without being cytotoxic, and thus can be used as a safe and effective immunomodulator in the field of feline calicivirus infection treatment and prevention.

Description

Recombinant vector containing a gene encoding feline interferon lambda-1 protein and method for producing the same

[0001] The present invention relates to a recombinant vector comprising a gene encoding feline interferon lambda-1 (rFeIFN-λ1) protein and a method for producing the same.

[0002] Feline Calicivirus (FCV) is a non-enveloped RNA virus belonging to the Caliciviridae family and is one of the major pathogens that causes upper respiratory disease in cats. FCV is transmitted through cat secretions such as saliva, nasal discharge, and tears, and infected cats can shed the virus even without showing any symptoms, making it highly contagious. After infection, the virus can be shed for several weeks or months, and some cats remain as lifelong carriers and can infect other cats. FCV is known to have strong survival in the outdoor environment, making it difficult to disinfect, and therefore, the risk of infection spreading in group housing environments is known to be high.

[0003] There are various strains of FCV, which can lead to different symptoms and severity of infection. Typical FCV infections present with symptoms such as nasal discharge, sneezing, and oral ulcers, and some cats may develop arthritis. In particular, the highly pathogenic strain, Virulent Systemic FCV (VS-FCV), causes systemic symptoms such as fever, facial and limb swelling, skin ulcers, and jaundice, with a reported mortality rate of over 60%.

[0004] The main symptoms of FCV infection are upper respiratory symptoms and oral lesions. Early symptoms include sneezing, runny nose, and conjunctivitis, followed by oral ulcers, gingivitis, drooling, and loss of appetite. Some cats may experience arthritis, causing lameness or pain, but these symptoms are usually temporary. Infection with highly pathogenic variants can result in rapid systemic symptoms and rapid death. Furthermore, infected cats can continue to shed the virus even after recovery, potentially spreading the infection to other cats.

[0005] Treatment of feline calicivirus infection primarily focuses on alleviating symptoms and preventing secondary bacterial infections. Currently, no specific treatment has been developed that can directly eliminate the virus itself, and conservative treatment, including antibiotics, immunomodulators, and fluid therapy, is primarily administered. Some studies have shown that drugs such as nitazoxanide and mizoribine can inhibit FCV replication, but their clinical application remains limited. Furthermore, immunomodulators such as recombinant feline interferon omega (RFeIFN-ω) are being used experimentally, but their efficacy is limited and their cost and administration complexity prevent widespread use. These limitations make complete eradication of FCV infection difficult, and some cats remain carriers who shed the virus even after recovery, perpetuating the risk of further spread of infection. Therefore, the development of effective treatments for FCV is urgently needed.

[0006] Interferons are cytokines induced by various physiological stimuli, including viral infections, tumors, and immune responses. They are broadly classified into type 1, type 2, and type 3 interferons. Among these, interferon lambda (IFN-λ), a type 3 interferon, acts primarily through a specific receptor complex (IFNLR1 / IL10R2) expressed on epithelial cells. Due to the restricted distribution of these receptors, interferon lambda exhibits local antiviral effects and has minimal systemic side effects. In particular, it is known to induce an effective immune response against viral infections in respiratory and digestive epithelial cells.

[0007] Against this backdrop, the inventors of the present invention completed the present invention by producing feline interferon lambda-1 protein in E. coli using genetic recombination technology and confirming that rFeIFN-λ1 (feline interferon lambda 1) induces antiviral gene expression in FCV-infected cells and significantly inhibits viral replication without cytotoxicity.

[0008] [Prior Art Literature]

[0009] [Patent Document]

[0010] Republic of Korea Patent Publication No. 10-2022-0169275 (December 27, 2022)

[0011] Republic of Korea Patent Publication No. 10-2024-0015036 (February 2, 2024)

[0012] An object of the present invention is to provide a recombinant vector comprising a gene encoding feline interferon lambda-1 protein.

[0013] Another object of the present invention is to provide a cell transformed with a recombinant vector.

[0014] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating feline calicivirus infection comprising the recombinant vector.

[0015] Another object of the present invention is to provide a method for producing a recombinant vector.

[0016] Another object of the present invention is to provide a feline interferon lambda-1 protein expressed from the recombinant vector.

[0017] Another object of the present invention is to provide a vaccine composition comprising, as an active ingredient, the recombinant vector, the feline interferon lambda-1 protein expressed from the recombinant vector, a cell into which the recombinant vector has been introduced, or a recombinant virus produced from the cell.

[0018] Another object of the present invention is to provide a method for preventing or treating a calicivirus infection disease, comprising administering the vaccine composition to a subject in need thereof.

[0019] To achieve the above purpose, the present invention provides a recombinant vector comprising a gene encoding feline interferon lambda-1 protein.

[0020] The present invention also provides a cell transformed with the recombinant vector.

[0021] The present invention also provides a pharmaceutical composition for preventing or treating feline calicivirus infection comprising the recombinant vector.

[0022] The present invention also comprises the steps of (a) preparing a recombinant vector by cloning a gene encoding feline interferon lambda-1 protein into a vector;

[0023] (b) a step of transforming a cell with the recombinant vector;

[0024] (c) a step of culturing the cells to express the recombinant vector; and

[0025] (d) a step of purifying the expressed vector;

[0026] A method for producing a recombinant vector comprising:

[0027] The present invention also provides a feline interferon lambda-1 protein expressed from a recombinant vector.

[0028] The present invention also provides a vaccine composition comprising, as an active ingredient, a recombinant vector, a feline interferon lambda-1 protein expressed from the recombinant vector, a cell into which the recombinant vector has been introduced, or a recombinant virus produced from the cell.

[0029] The present invention also provides a method for preventing or treating a calicivirus infection disease, comprising administering a vaccine composition to a subject in need thereof.

[0030] The recombinant vector comprising a gene encoding the recombinant feline interferon lambda-1 (rFeIFN-λ1) protein of the present invention can express the feline interferon lambda-1 protein with high efficiency, is non-cytotoxic, and exhibits excellent antiviral activity against feline calicivirus. In particular, the effect is maximized when treated before virus infection, and can contribute to infection suppression by increasing the expression of interferon-induced genes to induce an innate immune response.

[0031] Figure 1 shows a schematic diagram of the production of recombinant feline IFN-λ1 (rFeIFN-λ1).

[0032] Figure 2a shows the results of Western blot analysis using a 6x His tag antibody to identify recombinant feline IFN-λ1 (rFeIFN-λ1) protein (M: protein molecular weight standard marker (7-240 kDa). Protein induction time: 0, 2.5, 5 hours. W1, W2, W3: protein fractions after the first, second, and third washes, respectively. E1, E2, E3, E4: first, second, third, and fourth eluates, respectively).

[0033] Figure 2b shows the signal peptide prediction results of the rFeIFN-λ1 protein.

[0034] Figure 3(a) shows the optimal codon frequency of rFeIFN-λ1, and Figure 3(b) shows the results of comparing the GC values ​​of feline interferon lambda.

[0035] Figure 4 shows the original base sequence and optimized base sequence alignment of rFeIFN-λ1.

[0036] Figure 5 shows the translated amino acids of rFeIFN-λ1.

[0037] Figure 6 shows the protein structure prediction results of the original and optimized base sequences of rFeIFN-λ1.

[0038] Figure 7 shows the results of cytotoxicity analysis after rFeIFN-λ1 treatment.

[0039] Figures 8(A) and 8(B) show the results of plaque assay performed to confirm the antiviral activity of rFeIFN-λ1. * P<0.05; ** P<0.01; *** P<0.001).

[0040] Figure 9(A) shows the results of detecting viral antigens by immunofluorescence staining (IFA) after treating CRFK cells with rFeIFN-λ1 at concentrations of 10 or 100 ng / mL at -1, 0, and +1 dpi, and Figure 9(B) shows the virus reduction rate as a bar graph.* P<0.05; ** P<0.01; *** P<0.001).

[0041] Figure 10(A) shows the results of RT-PCR analysis of the antiviral activity of rFeIFN-λ1 and rFeIFN-ω, and Figure 10(B) shows the results of Western blot analysis. * P<0.05; ** P<0.01; *** P<0.001).

[0042] Figure 11(A) shows the results of comparison of the expression of interferon-induced gene (ISG) mRNA after rFeIFN-λ1 or rFeIFN-ω treatment. * P<0.05; ** P<0.01; *** P<0.001).

[0043] Hereinafter, the present invention will be described in detail.

[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Generally, the nomenclature used herein is well known and commonly used in the art.

[0045] In the present invention, when it is said that a component or a step “includes”, this does not mean that other components or other steps are excluded, but rather that other components or other steps may be further included, unless specifically stated otherwise.

[0046] In the present invention, “prevention” refers to any act of suppressing or delaying the onset of a viral disease by administering a composition according to the present invention, and “treatment” refers to any act of improving or beneficially changing the symptoms of a viral disease by administering a composition according to the present invention.

[0047] The term "vector" as used herein refers to a DNA construct containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing the DNA in a suitable host. The vector may be a plasmid, a phage particle, or simply a potential genomic insert. Once transformed into a suitable host, the vector can replicate and function independently of the host genome, or in some cases, integrate into the genome itself. Since plasmids are currently the most commonly used form of vector, the terms "plasmid" and "vector" are sometimes used interchangeably herein.

[0048] In the present invention, the term "recombinant vector" means a bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus, or other vector.

[0049] In the present invention, the term "transformation" means any act of inducing a genetically stable inheritance that allows the genetic construct or recombinant expression vector according to the present invention to move into the genome of a host cell and express a desired protein. Any transformation method may be used as the transformation method, and can be easily performed according to a method commonly used in the art. Common transformation methods include transformation using CRISPR / Cas9, CaCl2 precipitation, electroporation, calcium phosphate precipitation, protoplast fusion, stirring using silicon carbide fiber, Agrobacterium-mediated transformation, transformation using PEG, dextran sulfate, lipofectamine, and desiccation / inhibition-mediated transformation methods.

[0050] The amino acid sequence and base sequence described in the present invention can be interpreted to be extended to a sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homology with the provided sequence.

[0051]

[0052] The present invention provides a recombinant vector comprising a gene encoding feline interferon lambda-1 protein.

[0053] Feline interferon lambda is a type of cytokine that plays a crucial role in the cat's immune system, particularly as a key element of the innate immune response to viral infections. Interferons are broadly classified into three types (types 1, 2, and 3), and interferon lambda belongs to type 3 interferon. Unlike the existing type 1 (alpha, beta) interferons, type 3 lambda acts primarily on epithelial cells, resulting in fewer side effects and the advantage of selectively acting on target tissues. Currently, omega (ω) interferon is mainly used for viral diseases among animal interferon preparations both domestically and internationally, and clinical application of lambda-series interferons in cats is still limited.

[0054] The above feline interferon lambda-1 protein may comprise the amino acid sequence of sequence number 1.

[0055] The gene encoding the above feline interferon lambda-1 protein may include the base sequence of SEQ ID NO: 2.

[0056] The above vector may be, but is not limited to, pET-21a(+).

[0057] The above pET-21a(+) vector is a bacterial expression vector designed for high-efficiency protein expression in E. coli, and has elements optimized for experiments, such as a strong T7 promoter, various cloning sites, antibiotic resistance, and a purification tag. It is capable of high-level target protein expression under IPTG induction, and contains a sequence that can attach a 6xHis tag to the C-terminus, making it easy to purify and detect the expressed protein.

[0058] The above vector may include, but is not limited to, a T7 promoter.

[0059] The T7 promoter, derived from bacteriophage T7, is a powerful promoter widely used for highly efficient expression of recombinant proteins in Escherichia coli. This promoter is recognized only by T7 RNA polymerase, enabling robust gene transcription. Furthermore, the timing of expression can be precisely controlled using inducers such as IPTG, making it useful for mass-producing desired proteins.

[0060] The above recombinant vector can be constructed using a prokaryotic cell or a eukaryotic cell as a host. For example, when the vector used is an expression vector and a prokaryotic cell is used as a host, it generally includes a strong promoter capable of initiating transcription (e.g., pLλ promoter, CMV promoter, trp promoter, lac promoter, tac promoter, T7 promoter, etc.), a ribosome binding site for initiating translation, and a transcription / translation termination sequence. When a eukaryotic cell is used as a host, the replication origin that operates in a eukaryotic cell included in the vector includes, but is not limited to, the f1 replication origin, the SV40 replication origin, the pMB1 replication origin, the adeno replication origin, the AAV replication origin, and the BBV replication origin. Additionally, promoters derived from the genome of mammalian cells (e.g., metallothionine promoter) or promoters derived from mammalian viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter, tk promoter of HSV, etc.) can be used, and generally have a polyadenylation sequence as a transcription termination sequence.

[0061] The present invention also provides a cell transformed with the recombinant vector.

[0062] The above cell may be, but is not limited to, E. coli.

[0063] During the process of producing recombinant proteins, various unexpected isoforms or variants can be generated due to the host cell's translation and protein folding mechanisms, as well as mRNA processing. In particular, when expressing genes in a heterologous host, there is a possibility that proteins with structural and functional differences from the originally intended protein may be produced due to differences in the host's unique post-translational modifications or processing mechanisms. The present invention has overcome the aforementioned limitations and confirmed that the rFeIFN-λ1 protein can be successfully expressed.

[0064]

[0065] The present invention also provides a pharmaceutical composition for preventing or treating feline calicivirus infection comprising the recombinant vector.

[0066] The composition may be administered intravenously, intramuscularly, intraperitoneally, orally, or subcutaneously, but is not limited thereto.

[0067] The composition can increase the expression of any one or more interferon-induced genes selected from the group consisting of ISG15, OAS1, PKR and Mx1.

[0068] The expression of the above interferon-stimulated genes (ISGs) is complexly regulated by various factors, including cell type, the type and status of viral infection, the subtype and concentration of interferon, and the intrinsic characteristics of the host cell. In particular, heterologous proteins may exhibit reduced binding affinity and signaling efficiency with host cell receptors, resulting in minimal or no induction of ISG expression.

[0069] The above-mentioned ISG15 (Interferon-stimulated gene 15) is a ubiquitin-like protein that regulates the function of other intracellular proteins by conjugating them (ISGylation). ISG15 has antiviral activity and is known to be involved in various cellular processes, such as intracellular signaling, protein stability, and immune response regulation.

[0070] The above OAS1 (2'-5'-oligoadenylate synthetase 1) is an enzyme that is activated during viral infection and synthesizes an oligonucleotide called 2'-5'-oligoadenylate (2-5A). 2-5A is known to inhibit viral replication by activating an endoribonuclease called RNase L, which degrades viral RNA and cellular RNA.

[0071] The above-mentioned PKR (Protein kinase R) is a protein kinase activated by intracellular double-stranded RNA (dsRNA). dsRNA generated during viral infection activates PKR, which phosphorylates the translation initiation factor eIF2α, thereby inhibiting protein synthesis and blocking viral replication. It is also known to be involved in activating cell death and immune signaling pathways.

[0072] The above Mx1 (Myxovirus resistance protein 1) is a guanylate-binding protein (GTPase) that functions to inhibit the early stages of viral replication. Mx1 is known to exhibit antiviral activity by interfering with nucleocapsid transport of certain viruses or inhibiting viral RNA synthesis.

[0073] The pharmaceutical composition of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

[0074] The pharmaceutical dosage form of the composition according to the present invention can be used in the form of their pharmaceutically acceptable salts, and can also be used alone or in combination with other pharmaceutically active compounds as well as in an appropriate combination.

[0075] The pharmaceutical composition according to the present invention can be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injection solutions, respectively, according to conventional methods. Carriers, excipients, and diluents that can be included in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate mixtures, etc. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations can be manufactured by mixing at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc., with the pharmaceutical composition. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, syrups, etc. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, suspending agents, emulsions, lyophilized preparations, and preservatives can be included. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspending agents can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases that can be used include Wetepsol, Macrogol, Tween 61, cocoa butter, laurin butter, and glycerogelatin.

[0076] The preferred dosage of the composition of the present invention varies depending on the patient's condition and weight, the degree of disease, the drug form, the route of administration, and the period of administration, but can be appropriately selected by a person skilled in the art.

[0077]

[0078] The present invention also comprises the steps of (a) preparing a recombinant vector by cloning a gene encoding feline interferon lambda-1 protein into a vector;

[0079] (b) a step of transforming a cell with the recombinant vector;

[0080] (c) a step of culturing the cells to express the recombinant vector; and

[0081] (d) a step of purifying the expressed vector;

[0082] A method for producing a recombinant vector comprising:

[0083]

[0084] The present invention also provides a feline interferon lambda-1 protein expressed from the recombinant vector.

[0085] The present invention also provides a vaccine composition comprising, as an active ingredient, the recombinant vector, the feline interferon lambda-1 protein expressed from the recombinant vector, a cell into which the recombinant vector has been introduced, or a recombinant virus produced from the cell.

[0086] The present invention also provides a method for preventing or treating a calicivirus infection disease, comprising administering the vaccine composition to a subject in need thereof.

[0087]

[0088] The description of the above manufacturing method, interferon lambda-1 protein, vaccine composition, and method for preventing or treating calicivirus infection, its effects, and all related descriptions are the same as described above, and therefore, the description thereof is omitted to avoid excessive complexity of this specification due to duplicate description.

[0089]

[0090] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0091]

[0092] Example 1. Experimental materials and methods

[0093]

[0094] 1-1. Cell culture and virus propagation

[0095]

[0096] Crandell-Rees Feline Kidney (CRFK) cells (American Type Cell Collection) were used to evaluate the antiviral activity of feline interferon lambda-1 (IFN-λ1; Feline IFN-λ1). Cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing 8% heat-inactivated fetal bovine serum (FBS) and antibiotic-antimycotic solution (penicillin and streptomycin). Feline calicivirus (FCV17D03-3P) was obtained from the Animal and Plant Quarantine Agency of the Republic of Korea and cultured by inoculating CRFK cells at a multiplicity of infection (MOI) of 0.01.

[0097]

[0098] 1-2. Cloning and expression of recombinant feline IFN-λ1

[0099]

[0100] The mRNA sequence of feline IFN-λ1 (GenBank Accession No.: XM_006941288) was cloned into the pET-21a(+) vector with a 6x His tag, using primers of the sequences 5'-GTT GGA TGA ACT ACC CGC CAA TC-3' (SEQ ID NO: 3) and 5'-CAT ATG CGG CTC TGA TGG CTT GAA ACT G-3' (SEQ ID NO: 4).

[0101]

[0102] Escherichia coli was cultured in Luria-Bertani medium at 37°C, and cells were harvested when the optical density at 600 nm (OD600) reached 0.6–0.7. The cells were then resuspended in xTractor Buffer (Takara, Japan; 20 mL per 1 g of cells) and sonicated. The resulting lysate was centrifuged, and the supernatant was used for protein purification.

[0103]

[0104] 1-3. rFeIFN-λ1 purification

[0105]

[0106] Recombinant IFN-λ1 was purified by gravity-flow using nickel nitrilotriacetic acid (Ni-NTA) agarose. The solution was mixed with Ni-NTA slurry, reacted, and loaded onto a column. The bound protein was washed twice with washing buffer (Takara, Japan), and the 6xHis-tagged rFeIFN-λ1 protein was eluted from the column using elution buffer (Takara, Japan). After purification, the protein was dialyzed overnight at 4°C against 1 L of PBS using cellulose membrane tubing with a molecular weight of 10,000. The protein concentration was measured using a BCA protein quantitation kit (Thermo Scientific, USA).

[0107]

[0108] 1-4. Recombinant feline interferon omega

[0109]

[0110] The antiviral activity of commercially available feline interferon omega (Virbagen Omega, France) was evaluated compared with rFeIFN-λ1.

[0111]

[0112] 1-5. Cell Viability and Plaque Assay

[0113]

[0114] Cell viability was evaluated using the Cell Counting Kit-8 (CCK-8) assay (Sigma-Aldrich, USA), and experiments were conducted by adding rFeIFN-λ1 at concentrations of 1, 10, 100, 1000, and 10000 ng / mL. To evaluate antiviral activity, cells were treated with rFeIFN-λ1 at concentrations of 10 ng / mL and 100 ng / mL. Treated cells were fixed with formaldehyde and stained with crystal violet, and the virus reduction rate according to interferon treatment was confirmed by comparison with the positive control.

[0115]

[0116] 1-6. RT-QPCR

[0117]

[0118] Cellular mRNA was extracted from FCV-infected CRFK cells treated with or without rFeIFN-λ1 or rFeIFN-ω at concentrations of 1, 10, and 100 ng / mL, respectively. mRNA extraction was performed using the Xenopure PF-Total RNA Purification Kit (Xenohelix, Korea). Quantitative RT-qPCR analysis was performed using the One Step TB Green PrimeScript RT-PCR Kit (Takara, Japan), using ISG and GAPDH primers (Table 1). Data analysis was performed using the delta-delta Ct method.

[0119]

[0120] Target gene Primer sequence (5' to 3') Size (bp) Reference ISG15 Forward TCC TGG TGA GGA ACC ACA AGG G125 X. Zhang et al. (2016) Reverse TTC AGC CAG AAC AGG TCG GCOAS1 Forward AAC GTT TGC AGT GCA GTT TG166 designed in this study Reverse TCT GGG GTC AGG TCT GTA GGPKR Forward GGA AGG CAG GTG AAG TAA66 X. Zhang et al. (2017) Reverse TTGT AGT AGT GAA CGA TAT TTG GGT GATMx1 Forward TTC GGA GGT GGA GGC AAT C134 Y. Wang et al. (2022) Reverse CAG GGA GGT CTA TCA GGG TCA GAT CGAPDH Forward GTC CCC GAG ACA CGA TGG T60 Safi et al. (2017) Reverse CCA TAA CCC GCG GAC CFCV Forward GTT GGA TGA ACT ACC CGC CAA TC122 Kummrow et al. (2005) Reverse CAT ATG CGG CTC TGA TGG CTT GAA ACT G

[0121] 1-7. Western blot analysis

[0122]

[0123] Cell lysates were prepared by centrifugation, mixed with Laemmli buffer (Sigma-Aldrich, USA), and subjected to SDS-PAGE. Proteins were transferred to membranes, reacted with FCV-1 (ab33990, Abcam, USA) and GAPDH (ab8245, Abcam, USA) antibodies, and detected using a chemiluminescent substrate (Thermo Scientific, USA).

[0124]

[0125] 1-8. Immunofluorescence analysis

[0126]

[0127] CRFK cells were fixed with 4% paraformaldehyde for 10 min and divided into three groups according to the timing of interferon treatment: (i) 24 h before infection, (ii) simultaneously with infection, and (iii) 24 h after infection. Counterstaining with 4′6′-diamidino-2-phenylindole (DAPI) was performed to visualize cell nuclei.

[0128]

[0129] 1-9. Statistical Analysis

[0130]

[0131] Data were analyzed using GraphPad Prism (version 8.0.2; GraphPad Software, USA). Differences were analyzed using paired t-tests and one-way ANOVA, and a p-value <0.05 was considered statistically significant. qPCR data were analyzed using the delta-delta Ct method, with GAPDH used as the normalization standard. All in vitro experiments were performed at least three times.

[0132]

[0133] Example 2. Purification and codon optimization of rFeIFN-λ1

[0134]

[0135] The mRNA sequence of feline interferon lambda-1 (Felis catus interferon lambda-1, LOC101091490) (NCBI Reference Sequence: XM_006941288) was used to design PCR primers, and then mRNA was extracted from the CRFK cell line, RT-PCR was performed, and the interferon lambda-1 gene was amplified. Of the total 570 bp of the amplified gene, 510 bp, excluding the signal peptide (20 amino acids) coding region containing the transmembrane domain, was selected and cloned into the expression vector pET21a+. The plasmid containing the gene was then transformed into E. coli BL21 (DE3), and the selected transformant was used for expression analysis of the rFeIFN-λ1-His6 protein. Cloning was confirmed by PCR, and the size and homogeneity of the expressed recombinant protein were confirmed through SDS-PAGE and Western blot analysis. As a result, the molecular weight of the purified rFeIFN-λ1 protein was found to be 17.8 kDa as expected (Fig. 2).

[0136]

[0137] To maximize the expression efficiency of the recombinant protein, codon optimization was performed on the Feline interferon lambda-1 gene. Codon usage considering expression efficiency in E. coli was applied to the entire 570-bp gene sequence, resulting in an increase in the Codon Adaptation Index (CAI) from 0.68 to 0.97 (Fig. 3(a)). Generally, a CAI value of 0.8 or higher is known to indicate high expression efficiency. Furthermore, the GC content was reduced from 64.4% to 50.0%, ensuring favorable conditions for stable expression (Fig. 3(b)). Furthermore, while repetitive base sequences can form stem-loop structures within mRNA, inhibiting expression, no repetitive regions were identified in this gene (Fig. 4). Predictive analysis was also used to compare the protein structures before and after codon optimization, revealing no structural changes (Figs. 5 and 6).

[0138] This optimization technique improves protein expression efficiency by selecting codons preferred by the host cell among multiple codons corresponding to the same amino acid. It is widely used in nucleic acid therapeutics and antibody production. Because codon usage varies across species, optimization has been reported to improve recombinant protein production efficiency by up to several thousand times (Gustafsson, Claes, et al., "Engineering genes for predictable protein expression." Protein expression and purification 83.1 (2012): 37-46).

[0139]

[0140] Example 3. Evaluation of cytotoxicity of recombinant proteins in CRFK cells

[0141]

[0142] The cytotoxicity of rFeIFN-λ1 was assessed prior to infection of CRFK cells with feline calicivirus. Cells were treated with a 10-fold dilution of the recombinant protein for 3 days, and cytotoxicity levels were measured. As a result, rFeIFN-λ1 was confirmed to not induce cytotoxicity up to a concentration of 1,000 ng / mL (Fig. 7).

[0143]

[0144] Example 4. Evaluation of Reduction in Viral Replication Using Plaque Assay

[0145]

[0146] Plaque assays were performed to investigate the antiviral activity of rFeIFN-λ1.

[0147] Treatment of calicivirus-infected CRFK cells with rFeIFN-λ1 at concentrations of 10 ng / mL or 100 ng / mL at -1, 0, and +1 dpi, respectively, resulted in a significant decrease in viral load (P<0.01, Fig. 8(A) and Fig. 8(B)). In particular, in cells treated with rFeIFN-λ1 at -1 dpi, the number of plaques was reduced by approximately 60% compared to the positive control group, and similar reduction rates were observed at both concentrations.

[0148]

[0149] Example 5. Evaluation of reduced viral replication using immunofluorescence analysis.

[0150]

[0151] The reduction in viral antigen levels in cells treated with rFeIFN-λ1 was confirmed by immunofluorescence analysis (IFA) 36 hours after virus infection. The most pronounced inhibitory effect was observed at -1 dpi. As expected, no fluorescent signal was observed in the negative control group, whereas a green fluorescent signal was observed in the positive control group (Fig. 9). A concentration-dependent decrease in fluorescent signal was observed in virus-infected cells treated with rFeIFN-λ1. These results suggest that rFeIFN-λ1 plays an important role in suppressing FCV protein expression.

[0152]

[0153] Example 6. Comparison of antiviral activities of rFeIFN-λ1 and rFeIFN-ω

[0154]

[0155] Since rFeIFN-ω is the only feline interferon currently commercially available, we compared its antiviral activity with that of rFeIFN-λ1. When calicivirus-infected cells were treated with 100 ng / mL rFeIFN-λ1 and rFeIFN-ω at -1, 0, and +1 dpi, respectively, a significant decrease in viral replication was observed in the -1 dpi treatment group as determined by RT-PCR (P<0.001, Fig. 10(A)). Furthermore, Western blot analysis of cells treated with 100 ng / mL rFeIFN-λ1 and rFeIFN-ω at -1 dpi revealed a similar virus-reducing effect as observed by RT-qPCR (Fig. 10(B)).

[0156]

[0157] Example 7. Confirmation of ISG mRNA expression levels

[0158]

[0159] The mRNA expression levels of ISG15, OAS1, PKR, and Mx1 in cells treated with rFeIFN-λ1 and rFeIFN-ω were analyzed using RT-qPCR. Cells were treated with 1, 10, and 100 ng / mL of rFeIFN-λ1 or rFeIFN-ω, and cell lysates were collected daily for 3 days and analyzed. As a result, in cells treated with rFeIFN-λ1, the mRNA expression of ISG15, OAS1, PKR, and Mx1 tended to increase in a time- and concentration-dependent manner (Fig. 11(A)). In contrast, in cells treated with rFeIFN-ω, the mRNA expression of ISG genes tended to increase in a concentration-dependent manner, but the pattern of gradual increase in expression over time was not clear (Fig. 11(B)).

[0160]

[0161] Feline_IFNL1_amino acid sequence (SEQ ID NO: 1):

[0162] MAAAWVLVLVAAGLSLASAGPVPTSKPTTAWTDCDFGRFKSLSPRELEAFKEARDALENSLKSWSCTTRPFPRNRDLRQLQVWERPVALEAELALTLKVLGAMADASQGDILDQPLHTLRHMHSELQACVSAQPTAGPQPQGRLHHWLHRLQEASRKESQGCLEASVLFNLFRLLKKDLECVASGDLCV

[0163]

[0164] Feline_IFNL1_DNA base sequence (SEQ ID NO: 2):

[0165] ATGGCAGCAGCATGGGTTCTGGTTCTGGTTGCAGCAGGTCTGAGTCTGGCAAGTGCAGGTCCGGTTCCGACCTCTAAACCGACCACCGCATGGACCGATTGTGATTTTGGTCGTTTTAAAAGTCTGAGCCCTCGTGAACTGGAAGCCTTTAAAGAAGCACGTGATGCATTAGAAAATAGTCTGAAATCATGGAGTTGTACCACACGTCCGTTTCCGCGTAATCGTGATCTGCGTCAGCTGCAGGTTTGGGAACGTCCAGTTGCACTGGAAGCAGAATTAGCACTGACCCTGAAAGTTCTGGGTGCGATGGCTGATGCATCACAGGGTGATATTCTGGATCAGCCGCTGCATACCCTGCGCCATATGCATTCTGAACTGCAAGCATGTGTTAGTGCACAGCCGACAGCAGGTCCGCAGCCGCAGGGTCGTCTGCATCATTGGCTGCATCGTCTGCAGGAAGCAAGTCGTAAAGAAAGTCAGGGTTGTCTGGAAGCAAGCGTTCTGTTTAATTTATTTCGTCTGCTGAAAAAAGATCTGGAATGTGTTGCAAGTGGTGATCTGTGTGTTTAA

Claims

1. A recombinant vector containing a gene encoding feline interferon lambda-1 protein.

2. In paragraph 1, A vector characterized in that the above feline interferon lambda-1 protein comprises an amino acid sequence of sequence number 1.

3. In paragraph 1, A vector characterized in that the gene encoding the above feline interferon lambda-1 protein comprises the base sequence of sequence number 2.

4. A vector according to claim 1, characterized in that the vector is pET-21a(+).

5. A vector according to claim 1, characterized in that the vector comprises a T7 promoter.

6. Cells transformed with the recombinant vector of paragraph 1.

7. A cell according to claim 6, characterized in that the cell is E. coli.

8. A pharmaceutical composition for preventing or treating feline calicivirus infection comprising the recombinant vector of paragraph 1.

9. A composition according to claim 8, characterized in that the composition is administered intravenously, intramuscularly, intraperitoneally, orally, or subcutaneously.

10. A composition according to claim 8, characterized in that the composition increases the expression of at least one interferon-induced gene selected from the group consisting of ISG15, OAS1, PKR, and Mx1. 11.(a) A step of producing a recombinant vector by cloning a gene encoding feline interferon lambda-1 protein into a vector; (b) a step of transforming a cell with the recombinant vector; (c) a step of culturing the cells to express the recombinant vector; and (d) a step of purifying the expressed vector; A method for producing a recombinant vector, comprising:

12. Feline interferon lambda-1 protein expressed from a recombinant vector according to paragraph 1.

13. A vaccine composition comprising, as an active ingredient, a recombinant vector according to claim 1, a feline interferon lambda-1 protein expressed from the recombinant vector, a cell into which the recombinant vector has been introduced, or a recombinant virus produced from the cell.

14. A method for preventing or treating a calicivirus infection disease, comprising administering a vaccine composition according to Article 13 to an individual in need thereof.

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