TW201410707A - Canine fusion interferon - Google Patents

Abstract

This invention relates to a canine fusion interferon. The canine fusion interferon comprises a canine interferon and the Fc region of canine immunoglobulin G (IgG). The canine interferon and the Fc region of canine immunoglobulin G can be further fused with a linker. This invention also relates to a polynucleotide that encodes the canine fusion interferon and the use of the canine fusion interferon.

Description

Canine fusion interferon

The present invention relates to the field of animal health, and in particular to a canine fusion interferon having antiviral activity.

Interferon was first discovered in 1957 by British scholars Alick Isaacs and Jean Lindenmann in the influenza virus test. When a cell is infected with a virus, it immediately creates a cytokine that induces adjacent cells to produce antiviral proteins that interfere with viral replication. . This cytokine was subsequently named Interferon (IFN).

Interferon is a glycoprotein, mainly composed of interferon α, β, γ, which can be induced by different pathogens, such as viruses, bacteria, rickettsia, protozoa and double-stranded nucleotides; The interferon does not act directly on the pathogen, but instead produces antiviral proteins by inducing antiviral proteins, including dsRNA-dependent protein kinase (PKR), Mx protein. (Mx protein) and 2', 5'-oligoadenylate synthetase (RNase L, OAS). PKR is a protein kinase that phosphorylates eIF2, allowing host cells to stop translating viral proteins. Mx protein is a guanylate triphosphate hydrolase (GTPase) that binds to viral proteins and interferes with viral replication and protein. Transport; OAS can hydrolyze the double-stranded RNA of the virus. on the other hand, Interferon also induces the production of antiviral proteins in neighboring cells, which prevents the virus from infecting other cells, and allows the cells to establish an antiviral state and achieve the effect of interfering with viral infections. In addition to its antiviral effect, interferon also has anti-tumor, cell differentiation and immune regulation.

Currently, interferon preparations on the market are mostly designed for humans, for example, for treating viral diseases such as human type B and hepatitis C, and Kaposi's sarcoma (KS) and melanoma (malignant melanoma). ) Interferon on tumor diseases.

In recent years, affected by the aging and declining social trends, the number of pets has increased, which has led to the development of many pet-related industries. The most common pet-dog has gradually played an important role in human work. According to the United States Pet Products Manufacturers Association (APPMA) survey, the value of the US pet-related industry has reached 52 billion US dollars in 2009, of which pet medical care accounts for about 40% (about 20.8 billion US dollars), pet medicine accounts for about 24% (about 124.8) In the pet medicine, antibiotic preparations and skin care products account for about 70%, and the remaining 30% are vaccines and therapeutic drugs. Among them, there are few types of therapeutic drugs but high prices, which is a market with great development potential. . The present invention is directed to the development of a specific interferon for dogs as a therapeutic drug.

The present invention provides a canine fusion interferon in the first part. Since the interferon belongs to a small molecule protein and has a short half-life (about 2 to 8 hours) in vivo and is unstable, the canine fusion interferon provided by the present invention is a canine interferon protein and a canine immunoglobulin IgG Fc having a long half-life. The fragments are fused to form a more stable canine fusion interferon. In a preferred embodiment, the canine interferon protein and the canine immunoglobulin IgG Fc fragment are linked by a linker consisting of glycine (Glycine, G) and serine (Srine). Connected together. In one embodiment, the canine interferon protein is canine interferon alpha 2, having the amino acid sequence set forth in SEQ ID No: 2; the canine immunoglobulin The white IgG Fc fragment has the amino acid sequence set forth in SEQ ID No: 4; and the linker has the amino acid sequence set forth in SEQ ID No: 6; the canine fusion interferon has SEQ ID No: 8 The amino acid sequence shown.

The present invention provides a polynucleotide encoding the above canine fusion interferon in the second part. The canine fusion interferon provided by the present invention is obtained by gene transfer technology. First, a DNA sequence encoding a canine interferon protein, and a DNA sequence encoding a canine immunoglobulin IgG Fc fragment are selected into a expression vector system to form a plastid containing a DNA sequence encoding a canine fusion interferon, and the plastid is then ligated. Transgenic into the expression system, can be obtained by inducing protein expression to obtain canine fusion interferon.

In a preferred embodiment, the DNA sequence encoding the canine interferon protein and the DNA sequence encoding the canine immunoglobulin IgG Fc fragment are selected into a performance vector system and the coding consists of glycine and serine. The DNA sequence of the linker is cloned into the expression vector system to ligate the DNA sequence encoding the canine interferon protein and the DNA sequence encoding the canine immunoglobulin IgG Fc fragment. In one embodiment, the DNA sequence encoding the canine interferon protein has the sequence set forth in SEQ ID No: 1, and the DNA sequence encoding the canine immunoglobulin IgG Fc fragment has the sequence set forth in SEQ ID No: 3. And the DNA sequence encoding the linker consisting of glycine and serine has a sequence as shown in SEQ ID No: 5; the DNA sequence encoding the canine fusion interferon has SEQ ID No: 7 The sequence shown.

The expression vector can be a prokaryotic expression vector or a eukaryotic expression vector. The prokaryotic expression vector includes, but is not limited to, a pET series expression vector and a pGEX series expression vector. The eukaryotic expression vector includes, but is not limited to, a pcDNA series expression vector.

The expression system can be a prokaryotic expression system (eg, a bacterium) or a eukaryotic expression system (eg, yeast, insect cells, plant cells, and mammalian cells, etc.). In one embodiment, the expression system is Escherichia coli . In another embodiment, the expression system is a mammalian cell. Mammalian cells useful for the expression of canine fusion interferon of the present invention include, but are not limited to, 3T3 cells, Chinese hamster ovary cells (CHO cells), baby hamster kidney cells (BHK cells), humans. Cervical cancer cells (HeLa cells), and human hepatoma cells (HepG2 cells).

The invention provides in a third part the use of a canine fusion interferon for the preparation of a canine antiviral drug. It has been proved by experiments that the canine fusion interferon provided by the invention has the function of inducing cells to produce antiviral protein to achieve antiviral effect. In one embodiment, the canine fusion interferon (CIFNα2-IgG Fc) of the present invention induces the production of antiviral proteins in dog kidney cells (MDCK) by Western blotting and real-time RT-PCR, respectively. Analysis of the amount of antiviral protein and its gene expression, the results show that the canine fusion interferon (CIFNα2-IgG Fc) of the present invention can induce MDCK cells to produce antiviral protein Mx protein, and induce MDCK cells to increase antiviral protein Mx protein, Gene expression of PKR protein and OAS protein. Therefore, the canine fusion interferon provided by the present invention can induce the production of antiviral proteins and is used to prepare canine antiviral drugs.

The term "preventing, protecting, combating" means that an animal or cell sample of the canine fusion interferon of the present invention has a higher survival rate than an animal or cell sample not using the canine fusion interferon of the present invention, and The number of viruses in the sample is low.

The term "treating" means preventing or partially preventing a disease, a condition, a condition, and/or partially or completely curing or ameliorating a disease, a symptom, a condition, or an adverse effect caused by the disease.

The term "inhibiting" means a decrease in quality or quantity compared to the baseline. For example, in the context of the present invention, inhibition of viral replication refers to a reduction in viral replication compared to baseline. less. Similarly, inhibition of viral infection refers to a reduction in viral infection compared to baseline.

All of the technical and scientific terms described in this specification, unless otherwise defined, are intended to be common to those of ordinary skill in the art.

The present invention is exemplified by the following examples, but the present invention is not limited by the following examples.

Figure 1 shows the canine fusion interferon (CIFNα2-IgG Fc) expressed by E. coli harboring pET24a-CIFNα2-IgG Fc plastid by western blot method; M: Protein Marker; Protein 1; Analysis by mouse anti 6x His antibody; lane 2: analysis with rabbit anti canine IFN antibody; lane 3: analysis with rabbit anti canine IgG antibody; lane 4: analysis with mouse anti canine IFN antibody.

Figure 2A shows the results of IFA analysis after transfection of the pcDNA3 vector into CHO cells with Zeocin antibiotics; Figure 2B shows the transfection of CHO cells with plastids encoding the canine fusion interferon gene (CIFNα2-IgG Fc) After the screening with Zeocin antibiotics, the results were analyzed by IFA.

Figure 3 shows canine fusion interferon (CIFNα2-IgG Fc) expressed by CHO cells with pcDNA3-CIFNα2-IgG Fc plastid by Western blot method; M: Protein Marker; Protein 1; Analysis by mouse anti canine IFN antibody; lane 2: analysis with rabbit anti canine IFN antibody; lane 3: analysis with rabbit anti canine IgG antibody; lane 4: analysis with mouse anti 6x His antibody.

Figure 4 shows the antiviral protein induced by canine fusion interferon (CIFNα2-IgG Fc) by western blot method; A is the antiviral protein Mx egg detected by rabbit anti Mx protein antibody White; B is α-tubulin (intra-control group); lane 1: untreated MDCK cells (negative control group); lane 2: MDCK cells treated with interferon inducer polyI:C Lane 3: MDCK cells treated with canine fusion interferon (CIFNα2-IgG Fc).

Figure 5 is a graph showing the gene expression of anti-viral protein Mx protein in MDCK cells induced by canine fusion interferon (CIFNα2-IgG Fc) by real-time PCR.

Figure 6 is a graph showing the gene expression of the anti-viral protein PKR protein in MDCK cells induced by canine fusion interferon (CIFNα2-IgG Fc) by real-time PCR.

Figure 7 shows the gene expression of the anti-viral protein OAS protein in MDCK cells induced by canine fusion interferon (CIFNα2-IgG Fc) by real-time PCR.

Example 1 Selection of Canine Interferon α2 Gene (CIFNα2) and Construction of Its Expression Vector

4x10 ⁵ dog kidney cells (Madin-Darby Canine Kidney, MDCK) were inoculated into 6-well cell culture plates and transfected with interferon inducer-polyinosinic: polycytidylic acid for 24 hours. , polyI: C) 2 μg / well, then collect the cells once every 24 hours, and extract total RNA (total RNA) by guanidine thiocyanate (GTC) method. The extracted total RNA is then subjected to reverse polymerase chain reaction (RT-PCR); 20 μl of the extracted total RNA is applied at 70 ° C for 3 minutes, and then 15 μl of total RNA and 1 μl of the positive phase are taken. Primer, 1 μl reverse primer and 3 μl distilled water and AMV reverse transcription reagent (AMV RT Kit), the total volume is 20 μl, mixed uniformly and placed in a PCR reactor (Applied Biosystems GeneAmp PCR system 2400), the reaction conditions are First, cDNA synthesis was carried out at 42 ° C for 30 seconds, followed by PCR reaction to proliferate canine interferon α2 gene fragment, and 35 cycles were performed at 93 ° C for 30 seconds, 53 ° C for 30 seconds, and 72 ° C for 1 minute, and finally PCR was completed at 72 ° C for 5 minutes. reaction. Among them, the specific primer sequence of the canine interferon α2 gene (CIFNα2) is as follows:

Forward primer (CIFNα2/F1): (SEQ ID NO: 9)

Reverse primer (CIFNα2/R1): (SEQ ID NO: 10).

The PCR reaction product was analyzed by agarose electrophoresis to confirm the size of the product fragment, followed by purification of the PCR product using a nucleic acid extraction kit (Gel/PCR DNA Fragments Extraction kit, Geneaid, Taiwan). Next, the DNA fragment of the RT-PCR product was cloned with a vector kit (yT&A Cloning Vector Kit, Yeastern Biotech, Taiwan), and 8 μl of PCR product, 3 μl of distilled water, 2 μl of yT&A cloning vector, and 1 μl of T4 ligase buffer were taken. 1 μl of T4 ligase, mixed uniformly, placed in a 14 ° C water bath for ligation for 12 hours, and then transformed into the host E. coli , after overnight culture, selected with CIFNα2 / The PCR product sequence of yT&A plastid Escherichia coli and sequence-confirmed proliferation is indeed the canine interferon alpha 2 gene (CIFNα2), the canine interferon alpha 2 gene sequence is shown in SEQ ID No: 1, and the amino acid sequence is SEQ. ID No: 2 is shown.

Next, the CIFNα2/yT&A plastid was extracted and purified from the above-mentioned Escherichia coli carrying the CIFNα2/yT&A plastid using a Rapid Plasmid Extraction Kit (High-Speed Plasmid Mini Kit, Geneaid, Taiwan). The CIFNα2/yT&A plastid, the expression vector pET24a and the expression vector pcDNA3 were digested with restriction enzymes Eco RI and Xho I, respectively, and purified by a nucleic acid extraction kit (Geneaid, Taiwan). Gene (CIFNα2) and expression vector, followed by ligation, the canine interferon gene (CIFNα2) was separately selected into pET24a vector and pcDNA3 vector to form pET24a-CIFNα2 plastid and pcDNA3-CIFNα2 plastid, and pET24a-CIFNα2 The plastids of pcDNA3-CIFNα2 were transformed into E. coli and transfection into Chinese hamster ovary cell lines (CHO cells), and Escherichia coli with pET24a-CIFNα2 plastid was selected. Chinese hamster ovary cell line (CHO cells) with pcDNA3-CIFNα2 plastid.

Example 2 Canine immunoglobulin IgG Fc fragment gene selection

Fresh dog spleens were taken and total RNA was extracted by GTC method. The extracted total RNA is then subjected to reverse transcription polymerase chain reaction (RT-PCR); 20 μl of the extracted total RNA is applied at 70 ° C for 3 minutes, then 15 μl of total RNA, 1 μl of forward primer, and 1 μl of anti- To the primer and 3 μl of distilled water and AMV reverse transcription reagent (AMV RT Kit), the total volume was 20 μl, and the mixture was uniformly mixed and placed in a PCR reactor (Applied Biosystems GeneAmp PCR system 2400) under the reaction conditions of 42 ° C. cDNA synthesis was performed in seconds, followed by PCR reaction to proliferate the canine immunoglobulin IgG Fc fragment gene, and 35 cycles were performed at 93 ° C for 30 seconds, 53 ° C for 30 seconds, and 72 ° C for 1 minute, and finally the PCR reaction was completed at 72 ° C for 5 minutes. Among them, the specific primer sequence of the canine immunoglobulin IgG Fc fragment gene (Canine IgG Fc) is as follows:

Forward primer (Canine IgG Fc/F1): (SEQ ID NO: 11)

Reverse primer (Canine IgG Fc/R1): (SEQ ID NO: 12).

The PCR reaction product was analyzed by gel electrophoresis to confirm the size of the product fragment, followed by purification of the PCR product using a nucleic acid extraction kit (Geneaid, Taiwan). Next, the DNA fragment of the RT-PCR product was cloned with yT&A Cloning Vector Kit (Yeastern Biotech, Taiwan), and 8 μl of PCR product, 3 μl of distilled water, 2 μl of yT&A cloning vector, 1 μl of T4 ligase buffer, and 1 μl of T4 ligase were taken. After mixing, it was placed in a 14 ° C water bath for bonding for 12 hours, and then transformed into host E. coli . After overnight culture, Escherichia coli with Canine IgG Fc/yT&A plastid was selected and subjected to The sequence of the PCR product confirmed by sequencing is indeed the canine immunoglobulin IgG Fc fragment gene (Canine IgG Fc), the canine immunoglobulin IgG Fc fragment gene sequence is shown in SEQ ID No: 3, and the amino acid sequence is SEQ ID No: 4 is shown.

Example 3 Construction of canine fusion interferon (CIFNα2-IgG Fc) expression vector

In the present example, the canine interferon alpha 2 gene (CIFNα2) (SEQ ID No: 1) obtained in Example 1 and the canine immunoglobulin IgG Fc fragment gene (Canine IgG-Fc) obtained in Example 2 (SEQ ID) No: 3) The DNA sequence (SEQ ID No: 5) of a linker consisting of glycine (Glycine, G) and serine (Serine, S) was ligated to construct a canine fusion interferon (CIFNα- DNA sequence of IgG Fc) (SEQ ID No: 7).

First, the canine interferon alpha 2 gene (CIFNα2) (SEQ ID No: 1) and the canine immunoglobulin IgG Fc fragment gene (Canine IgG-Fc) (SEQ ID No: 3) were amplified by PCR, and PCR primers were used for design. The DNA sequence (SEQ ID No: 5) of the linker consisting of glycine and serine was segmented together with the canine interferon alpha 2 gene (CIFNα2) and the canine immunoglobulin IgG Fc fragment gene (Canine IgG-Fc). The PCR reaction increased. Among them, canine interferon alpha 2 gene (CIFNα2) The specific primer sequence is as follows:

Forward introduction (CIFNα2/F2): (SEQ ID NO: 13)

Reverse primer (CIFNα2-linker/R): (SEQ ID NO: 14);

The specific primer sequence of the canine immunoglobulin IgG Fc fragment gene is as follows: forward primer (Linker-IgG/F): AGAGAAAATGGA-3' (SEQ ID NO: 15)

Reverse primer (IgG-Fc/R2): (SEQ ID NO: 16).

Add 0.3 ng of plastid DNA (CIFNα2/yT&A plastid or Canine IgG Fc/yT&A plastid) to the PCR reaction tube, 5 μl of 10x PCR reaction solution, 8 μl of 1.25 mM dNTP, 1 μl of 50 μM 5'-end forward primer 1 μl of 50 μM 3' reverse primer, 33 μl of sterilized water, 1 μl of Taq polymerase, mixed well and placed in a PCR reactor (Applied Biosystems GeneAmp PCR system 2400) under the conditions of 94 ° C for 5 minutes. The DNA was denatured, followed by 30 cycles of 94 ° C for 30 seconds, 57 ° C for 30 seconds, and 72 ° C for 30 seconds, and finally the PCR reaction was completed at 72 ° C for 5 minutes.

The PCR reaction product was analyzed by gel electrophoresis to confirm the size of the product fragment, followed by purification of the PCR product using a nucleic acid extraction kit (Geneaid, Taiwan). The purified PCR product canine interferon α2 gene (CIFNα2) was digested with restriction enzymes Kpn I and Bam HI, and the purified PCR product canine immunoglobulin IgG Fc fragment gene (Canine IgG-Fc) was restricted. The enzymes Bam HI and Xho I were digested, and the expression vector pcDNA3 was digested with restriction enzymes Kpn I and Xho I, and then the PCR product was purified by nucleic acid extraction kit (Geneaid, Taiwan). Expression vector, followed by ligation reaction, the PCR product was cloned into pcDNA3 vector to form pcDNA3-CIFNα2-IgG Fc plastid, and pcDNA3-CIFNα2-IgG Fc plastid was transfected into Chinese hamster ovary cell line (CHO cells). The Chinese hamster ovary cell line (CHO cells) carrying the pcDNA3-CIFNα2-IgG Fc plastid was selected, and the PCR product sequence which confirmed the proliferation was confirmed to be the DNA of the canine fusion interferon (CIFNα2-IgG Fc) of the present example. The sequence (SEQ ID No: 7), and the amino acid sequence of the canine fusion interferon (CIFNα2-IgG Fc) of the present example is shown in SEQ ID No: 8.

In addition, the pcDNA3-CIFNα2-IgG Fc plastid was used as a template for the PCR reaction, and the canine fusion interferon (CIFNα2-IgG Fc) DNA sequence of the present example was propagated with the following primers:

Forward primer (CIFNα2/F1): (SEQ ID NO: 9)

Reverse primer (CIFNα2-IgG/R): (SEQ ID NO: 17).

The PCR reaction conditions were first denatured at 94 ° C for 5 minutes, followed by 94 ° C for 30 seconds, 55 ° C 30 The cycle was carried out for 30 cycles at 72 ° C for 30 seconds, and finally the PCR reaction was completed at 72 ° C for 5 minutes.

Next, the PCR reaction product was analyzed by gel electrophoresis to confirm the size of the product fragment, followed by purification of the PCR product using a nucleic acid extraction kit (Geneaid, Taiwan). The purified PCR product canine fusion interferon (CIFNα2-IgG Fc) was digested with the expression vector pET24a with restriction enzymes Eco RI and Xho I, and then purified by nucleic acid extraction kit (Geneaid, Taiwan). PCR product and expression vector, followed by ligation reaction, the PCR product was cloned into pET24a vector to form pET24a-CIFNα2-IgG Fc plastid, and pET24a-CIFNα2-IgG Fc plastid was transformed into the expression host E. coli BL21 The PCR product sequence in which E. coli BL21 carrying the pET24a-CIFNα2-IgG Fc plastid was selected and sequenced to confirm proliferation was indeed the DNA sequence of the canine fusion interferon (CIFNα2-IgG Fc) of the present example (SEQ ID No: 7).

Example 4 Performance of canine fusion interferon (CIFNα-IgG Fc) in prokaryotes

The pET24a-CIFNα-IgG Fc plastid obtained in Example 3 was transformed into the expression host E. coli BL21. First, take 200 μl of competent cells and add 5 μl of the pET24a-CIFNα-IgG Fc plastid obtained in Example 3. Place on ice for 30 minutes, then immediately put it into a 42 °C water bath for 1 minute and 30 seconds for heat shock. Then, it was placed in ice water, and after standing for 5 minutes, 800 μl of LB medium was further added, and the mixture was incubated at 37 ° C for 1 hour in a water bath. Centrifuge at room temperature 3000xg for 5 minutes, remove 600 μl of the supernatant, and disperse the remaining LB and bacteria in the lower layer. Add 200 μl of the bacterial solution to 30 μg/ml kanamycin or 50 μg. /ml ampicillin (ampicillin) was applied to LB solid medium, and cultured overnight at 37 ° C, a single colony was selected for culture and sequencing confirmed that the transformation was correct.

The expression host E. coli BL21 carrying the pET24a-CIFNα-IgG Fc plastid obtained in Example 3 was inoculated to an LB medium containing 30 μg/ml kanamycin for 12 hours, and then 1% by volume was taken. The inoculum was inoculated to 250 ml of LB medium containing 30 μg/ml kanamycin, and cultured at 37 ° C until the concentration of the solution reached OD _600nm of 0.6-0.8, and 1 mM isopropyl-β- was added. Induced by Isopropyl-beta-D-thiogalactopyranoside (IPTG), the bacterial samples were collected at 4, 6, and 8 hours after induction, and after 8 hours, centrifuged at 13,000 x g, 4 ° C for 10 minutes to remove The supernatant was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) by 12.5% or 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the dog was detected by Western blotting method. Fusion of interferon (CIFNα2-IgG Fc) protein expression.

The protein on the colloid analyzed by the above SDS-PAGE was transferred onto a PVDF membrane, and the transferred PVDF membrane was placed in a blocking buffer (5% Skim milk dissolved in TBST) at room temperature. The effect was 1 hour to remove the non-specific reaction, and then washed three times with TBST (10 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.3% Tween 20) for 5 minutes each, followed by the addition of mouse anti-6xHis (mouse anti 6xHis) Monoclonal antibody (Invitrogen, USA), rabbit anti-canine IFN antibody, rabbit anti-canine IgG antibody, and mouse anti-canine interferon (mouse anti) -canine IFN) multiple antibodies were used as primary antibodies to detect canine fusion interferon; after addition of antibody, the cells were washed at room temperature for 1 hour, washed twice with TBST for 5 minutes, and then labeled with alkaline phosphatase (AP). Goat anti-mouse antibody (Sigma, USA) or goat anti-rabbit antibody (Sigma, USA) labeled with alkaline phosphatase (AP) as a secondary antibody, The antibody was diluted 2000 times with TBST containing 0.5% skin milk and gently shaken at room temperature. After 1 hour, the cells were washed 6 times with TBST for 5 minutes, and then added with AP-treated NBT/BCIP (Bio-Rad) for about 1 to 2 minutes. Then, the developer was poured off and rinsed with water to terminate the color reaction.

Western blot analysis results are shown in Figure 1. The E. coli bacterium with canine fusion interferon gene can be murine anti-6xHis (mouse anti 6xHis) monoclonal antibody, rabbit anti-canine IFN (rabbit anti-canine IFN) Multiple antibody, rabbit anti-canine IgG antibody and mouse anti-canine IFN antibody were identified, indicating that the Escherichia coli contains canine fusion interferon, and this embodiment The molecular weight of the dog fusion interferon is about 51 KDa, which is consistent with the Western blot analysis.

Example 5 Performance of canine fusion interferon (CIFNα2-IgG Fc) in eukaryotes

The pcDNA3-CIFNα2-IgG Fc plastid obtained in Example 3 was transfected into Chinese hamster ovary cell line (CHO cells). 3 μg of pcDNA3-CIFNα2-IgG Fc plastid DNA was added to antibiotic-free and serum-free VP medium (Invitrogen) for 15 seconds (mixture A); 6 μg of Lipofectamine reagent (Invitrogen) was added to antibiotic-free and serum-free In VP medium (mixture B), it was allowed to act at room temperature for 5 minutes; then, the mixture A was added to the mixture B, shaken for 15 seconds, and then allowed to act at room temperature for 30 minutes. The above mixture (A+B) was uniformly added to the overnight cultured CHO cells, and the cells were placed in a 37 ° C, 5% CO ₂ incubator for 6 hours, and then the mixture was removed and added with 10% fetal bovine serum. The F12 medium of (FBS) was cultured in a 37 ° C, 5% CO ₂ incubator for 48 hours.

Next, CHO cells harboring the canine fusion interferon gene were screened with Zeocin. The transfected CHO cell line was subcultured in a 24-well cell culture dish containing 10% FBS, 100 Units/ml Penicillin, 100 Units/ml Streptomycin, and 700 μg. /ml Bocomycin (Zeocin) was cultured in F12 medium for screening. Then, the cells were washed twice with phosphate buffered saline (PBS) and then added with 0.125% trypsin (Trypsin) for digestion. After the cells were rounded, the cells were shaken to shake off the cells, and the cells were dispersed and suspended in a medium, and the cells were cultured. In a 37 ° C, 5% CO ₂ incubator, after subculture twice, when the cells to be alive remained about one to two percent, the cell culture medium was replaced with 50 μg/ml of Zeocin and 10% FBS. F12 medium, after the cells were restored to the original growth rate, the cells were confirmed by the Indirect Immunofluorescence Assay (IFA) method and the western blot method to confirm whether the cells contained the canine fusion interferon (CIFN-IgG Fc) gene and expressed the recombination. protein.

1. Detection of porcine fusion recombinant interferon by indirect immunofluorescence staining (IFA)

The transfected CHO cells were seeded in a 24-well cell culture dish (2×10 ⁵ cells/well) in F12 medium containing 10% FBS for 2 days, washed three times with PBS and then added with 80% acetone (-20 ° C). And fixed at 4 ° C for 30 minutes for cell fixation, and then washed three times with PBS, then added 5,000 times diluted rabbit anti canine IgG antibody (300 μl / well), placed in a 37 ° C incubator in the dark After washing for 30 minutes in PBS, 300 μl of goat anti rabbit-FITC antibody diluted 2000 times with PBS was added to each well, and placed in a 37 ° C incubator for 30 minutes in the dark, and then observed under a fluorescent microscope.

The results of the fluorescence microscope observation are shown in Figure 2. The CHO cells surviving by Zeocin have a canine fusion interferon gene, and the fluorescent signal can be observed by indirect immunofluorescence staining (IFA) analysis (as shown in Figure 2B); CHO cells infected with the pcDNA3-CIFNα2 vector did not have a fluorescent signal (as shown in Figure 2A).

2. Detection of porcine fusion recombinant interferon by Western blotting

The experimental procedure of the Western blotting method was as described in Example 4, using mouse anti-6xHis (mouse anti 6xHis) monoclonal antibody (Invitrogen, USA), rabbit anti-canine interferon (rabbit). Anti-canine IFN) polyclonal antibody, rabbit anti-canine IgG antibody and mouse anti-canine IFN antibody were used as primary antibodies to detect transfection of CHO cells. Whether the animal contains canine fusion interferon (CIFNα2-IgG Fc); and goat anti-mouse antibody (Sigma, USA) or labeled alkaline phosphatase (labeled alkaline phosphatase (AP)) AP) goat anti-rabbit antibody (Sigma, USA) as a secondary antibody; results shown in Figure 3. CHO cell secretion with canine fusion interferon gene can be inhibited by mouse anti-6xHis (mouse anti 6xHis) monoclonal antibody, rabbit anti-canine IFN antibody, rabbit anti-canine IgG antibody and mouse anti-canine IFN) multiple antibody recognition, showing that the CHO cells contain canine fusion interferon, and the protein molecular weight of the canine fusion interferon of this example is about 51 KDa, which is consistent with the Western blot analysis.

Example 6 Activity analysis of canine fusion interferon (CIFNα2-IgG Fc) induced by cells to produce antiviral protein

1. Inducing antiviral protein in dog kidney cells (MDCK) by canine interferon (CIFNα2), canine fusion interferon (CIFNα2-IgG Fc) and interferon inducer (polyI:C)

First, the dog kidney cells (MDCK) were seeded in a 6-well cell culture tray (4× ^{10 5} cells/well), cultured at 37° C. under 5% CO ₂ for 24 hours, and the supernatant culture solution was removed and washed three times with PBS. Add 100 μg of canine interferon alpha 2 (Canine IFNα2), 100 μg of canine fusion interferon (CIFNα2-IgG Fc) and premixed 2.5 μg of polyI:C and lipofectamine 2000 mixture at 37 ° C, 5 Incubate for 1 hour under % CO ₂ condition, supplement 0.5% DMEM medium to 3 ml, then incubate in a 37 ° C, 5% CO ₂ incubator, and collect MDCK cell total protein and total cellular RNA for analysis after 24 hours. (3 repetitions per process).

2. Extraction of total protein from dog kidney cells (MDCK)

Each of the above groups of MDCK cells was washed three times with PBS, and cell lysis buffer (100 μl/well) was added, and the cell culture plate was placed at room temperature, and shaken uniformly with a shaker at 120 rpm. When shaking, every 3 to 5 minutes, gently pat the cell culture plate to detach the cells, collect the cell mass and solution, centrifuge at 14000 xg for 20 minutes at 4 ° C, and centrifuge the supernatant (cytoplasmic protein) and the lower cells. The pieces were placed at -20 ° C for use. The total protein in the cytoplasm was quantified using the Protein Assay kit (Bio-Rad).

3. Extraction of total RNA from dog kidney cells (MDCK)

The total RNA of MDCK cells was extracted with a nucleic acid extraction kit (Nucleic Acid Extraction Kit I, FAVORGEN). First add 570 μl VNE buffer to disperse the cell pellet and let it sit at room temperature for 10 minutes, then add 570 μl of 99.9% ethanol, mix it evenly with a vortex, and then add the liquid at room temperature to the VNE column. Centrifuge at 8000xg for 1 minute, remove the liquid in the cannula, add 400μl W1 buffer, centrifuge at 8000xg for 1 minute, remove the cannula fluid, add 600μl Wash buffer, centrifuge at 8000xg for 1 minute, remove the cannula fluid, and add 600μl Wash buffer again. Centrifuge at 8000xg for 1 minute, remove the cannula fluid, centrifuge at 13000xg for 3 minutes, remove the cannula fluid, add 50μl of RNase free water, and centrifuge at 13000xg for 3 minutes, then place the RNA at -20 °C. .

4. Analysis of the performance of antiviral proteins by Western blotting

The Western blot method was tested as described in Example 4; rabbit anti-Mx1 antibody (GeneTex, USA) diluted 500-fold with 0.05% TBST as primary antibody, and labeled with horseradish Oxidase (HRP) goat anti-rabbit (HRP) antibody (configured in 0.05% TBST) (KPL, USA) as secondary antibody, respectively It was detected whether the anti-viral protein Mx protein was contained in MDCK cells treated with canine fusion interferon (CIFNα2-IgG Fc) and interferon inducer (polyI:C) expressed in CHO cells.

As a result, as shown in Fig. 4, MK protein was contained in canine fusion interferon (CIFNα2-IgG Fc) expressed by CHO cells and MDCK cells treated with interferon inducer (polyI: C), and MDCK of untreated group was obtained. There is no production of Mx protein in the cells, indicating that MDCK cells can induce the production of antiviral protein-Mx protein after treatment with the canine fusion interferon of the present invention (as shown in Figure 4A).

5. Analysis of gene expression of antiviral proteins by real-time RT-PCR

The total RNA extracted (20 ng) was subjected to real-time RT-PCR using a QuantiFast SYBR Green RT-PCR Handbook kit (QIAGEN, The Netherlands) to analyze antiviral proteins (Mx protein, PKR protein, OAS protein). Gene expression. The above extracted total RNA was diluted to 5 ng/μl, followed by 12.5 μl of 2x QuantiFast SYBR Green RT-PCR Master Mix, 1 μl forward primer, 1 μl reverse primer, QuantiFast RT Mix, template RNA and RNase. -free water for real-time RT-PCR reactions; the specific primer sequences for each antiviral protein are as follows:

Mx protein forward primer (Mx real-time/F): 5'-ATGAGCCATGACGAGGTTTC-3' (SEQ ID NO: 18)

Mx protein reverse primer (Mx real-time/R): 5'-TTCAGGAGCCAGCTGTAGGT-3' (SEQ ID NO: 19)

PKR protein forward primer (PKR real-time/F): 5'-TGAGCAATGCCAGATACAGTG-3' (SEQ ID NO: 20)

PKR protein reverse primer (PKR real-time/R): 5'-CCATATCCACCTGAGCCAAT-3' (SEQ ID NO: 21)

OAS protein forward primer (OAS real-time/F): 5'-AGCTCGAGAAACGAGGACAG-3' (SEQ ID NO: 22)

OAS protein reverse primer (OAS real-time/R): 5'-ACTTCAGGGTTGGGTCTGTG-3' (SEQ ID NO: 23)

In addition, the expression amount of actin of the housekeeping gene was used as a control group: Actin real-time/F: 5'-GCGCAAGTACTCTGTGTGGAT-3' (SEQ ID NO: 24)

Actin protein reverse primer (Actin real-time/R): 5'-GTCGTACTCCTGCTTGCTGAT-3' (SEQ ID NO: 25)

Real-time RT-PCR reaction conditions were: Reverse transcription 50 ° C for 10 minutes, followed by PCR initiation step 95 ° C for 5 minutes, Denaturation (95 ° C for 10 seconds, bonding / extension (annealing / extension) The reaction was completed after a total of 35 cycles at 60 ° C for 30 seconds. The results of the experiment were calculated by the following formula and analyzed by Sigmastat software. The formula is as follows: 2 ^-△Ct =2 ^{-{(CT gene of interest-CT internal control)-(CT gene of interest-CT intemal control)}} The real-time RT-PCR results of each antiviral protein are shown in Figure 5 to Figure 7 shows.

As shown in Figure 5, purified canine interferon alpha 2 (CIFNα2), canine fusion interferon (CIFNα2-IgG Fc) and CHO cells were purified by canine fusion interferon (CIFNα2-IgG Fc). After 24 hours of MDCK cells, the Mx protein gene induced by CHO cell-purified canine fusion interferon (CIFNα2-IgG Fc) treatment group was the highest, which was treated with canine interferon α2 (CIFNα2) purified by E. coli. The Mx protein gene expression of the group was 7.9 times, and was about 41 times that of the Mx protein gene in the untreated group (negative control group) ( p < 0.05).

As shown in Fig. 6, purified canine interferon α2 (CIFNα2), canine fusion interferon (CIFNα2-IgG Fc) and CHO cells were purified by canine fusion interferon (CIFNα2-IgG Fc). After 24 hours of MDCK cells, the PKR protein gene was induced to the highest in the canine interferon alpha 2 (CIFNα2)-treated group, while the PKR protein gene expression was higher in the untreated group (negative control group). Purified PKR protein was also expressed in E. coli-expressing canine fusion interferon (CIFNα2-IgG Fc), CHO cells expressing purified canine fusion interferon (CIFNα2-IgG Fc), and interferon inducer polyI:C-treated MDCK cells. Gene performance ( p <0.05).

As shown in Figure 7, purified canine interferon alpha 2 (CIFNα2), canine fusion interferon (CIFNα2-IgG Fc) and CHO cells were purified by canine fusion interferon (CIFNα2-IgG Fc). After 24 hours of MDCK cells, the purified canine fusion interferon (CIFNα2-IgG Fc)-treated group showed the highest OAS protein gene expression; whereas the OAS protein gene was compared to the untreated group (negative control group). The amount of expression, E. coli expression purified canine interferon alpha 2 (CIFNα2), canine fusion interferon (CIFNα2-IgG Fc) and interferon inducer polyI:C-treated MDCK cells still have gene expression of induced OAS protein ( p <0.05).

It can be seen from the above examples that the canine fusion interferon provided by the invention can induce the expression of antiviral proteins in canine cells, thereby achieving the antiviral effect.

The detailed description of the preferred embodiments of the present invention is intended to be limited to the scope of the invention, and is not intended to limit the scope of the invention. The patent scope of this case.

In summary, the technical features disclosed in this case have fully complied with the statutory invention patent requirements of novelty and progressiveness. If you apply in accordance with the law, you are requested to approve the application for this invention patent to encourage invention.

<110> Fuda Biotech Co., Ltd.

<120> Canine fusion interferon

<130> P13-0200

<150> CN201210319688.0

<151> 2012-09-03

<160> 25

<170> PatentIn version 3.5

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Claims (9)

A canine fusion interferon comprising a canine interferon and a canine immunoglobulin Fc fragment. The canine fusion interferon of claim 1, wherein the canine interferon and the canine immunoglobulin Fc fragment are linked by a linker. The canine fusion interferon of claim 1 or 2, wherein the canine interferon has the sequence set forth in SEQ ID No: 2. The canine fusion interferon of claim 1 or 2, wherein the canine immunoglobulin Fc fragment has the sequence set forth in SEQ ID No: 4. The canine fusion interferon of claim 2, wherein the linker has the sequence set forth in SEQ ID No: 6. The canine fusion interferon of claim 2, wherein the canine fusion interferon has the sequence set forth in SEQ ID No: 8. A polynucleotide encoding a canine fusion interferon as described in claim 1 of the patent application. The polynucleotide of claim 7, wherein the polynucleotide has the sequence set forth in SEQ ID No: 7. A use of canine fusion interferon as described in claim 1 of the patent application for the preparation of a canine antiviral drug.