Cat interferon omega-RTB fusion protein

Abstract

The invention provides a cat interferon omega-RTB recombinant fusion protein. The cat interferon omega-RTB recombinant fusion protein comprises cat interferon omega protein and ricin B chain protein, the amino acid sequence of the recombinant fusion protein is SEQ ID NO: 5, and the nucleotide sequence of the coding gene corresponding to the recombinant fusion protein is SEQ ID NO: 6. The fusion protein provided by the invention can be used for preparing antiviral products. According to the invention, the gene of the cat interferon omega is connected with the RTB sequence, so that the obtained fusion protein has longer half-life period and high antiviral activity.

Description

Technical Field

[0001] The invention belongs to the technical field of cytokine recombinant fusion protein expression, and particularly relates to a feline interferon ω-RTB fusion protein. Background Art

[0002] With the development of the pet industry, cats are becoming increasingly popular as companion animals. However, feline viral diseases, such as feline panleukopenia caused by feline parvovirus, feline stomatitis caused by feline calicivirus, and feline rhinotracheitis caused by feline herpesvirus, pose a significant threat to the pet industry. Currently, there are no effective vaccines for cat diseases, making research on feline pharmaceuticals highly valuable. Interferon ω is induced by viral infection and exhibits strong antiviral activity against various RNA viruses. Feline interferon ω (FeIFN-ω) is primarily produced by white blood cells. Feline interferon ω (FeIFN-ω) is the most potent type I interferon with antiviral activity in cats and is widely found in mammals, but IFN-ω has not been found in dogs or mice. Due to its excellent antiviral efficacy, FeIFN-ω has been widely used in cytokine-based vaccine formulations. Current research has shown that feline interferon ω primarily inhibits canine parvovirus, feline HIV, and feline leukemia virus, and can also be used as an adjunctive therapy for calicivirus, herpesvirus, coronavirus, and rotavirus. Studies have shown that feline interferon-ω is effective in treating canine dermatitis and canine parvovirus enteritis. It can also be added to eye drops or oral sprays for the treatment of gingivitis and keratitis. Natural interferons are often glycosylated, but FeIFN-ω is not, allowing it to be produced using a prokaryotic expression system. However, interferon's pharmacokinetics and short half-life make it less effective in practical applications. Summary of the Invention

[0003] The purpose of the present invention is to provide a feline interferon ω-RTB fusion protein, thereby making up for the deficiencies of the prior art.

[0004] The present invention first provides a fusion protein, which comprises feline interferon ω protein and ricin B chain protein;

[0005] Furthermore, the amino acid sequence of the ω protein is as follows:

[0006] MCALPGSHAQVSRDNLVLLGQMRRLSPFLCLRARKDFRFPREMLEGGQLREAQAAAAVLRELLQQ TFNLLHTERSSAAWSPAPLHGLRSGLHRQLEALDACLLQATGEGERAPGMHGPALAIKRYFQDIRVYLED EGYSDCAWEIVRLEIMRALSSSATLQDSLAIKDGDLASS(SEQ ID NO:1);

[0007] The nucleotide sequence of one of its encoding genes is as follows:

[0008] ATGTGTGCACTGCCGGGCAGCCATGCACAGGTTAGTCGTGATAATCTGGTTCTGCTGGGCCAGAT

[0009] GCGCCGCCTGAGCCCTTTTCTGTGTCTGCGCGCCCGCAAAGATTTTCGCTTTCCGCGCGAAATGCTGG

[0010] AAGGTGGTCAGCTGCGCGAAGCACAGGCAGCAGCAGCCGTGCTGCGCGAACTGCTGCAGCAGACCT

[0011] TTAATCTGCTGCATACCGAACGTAGTAGTGCCGCATGGAGCCCGGCCCCGCTGCATGGTCTGCGTAGC

[0012] GGTCTGCATCGTCAGCTGGAAGCACTGGATGCATGTCTGCTGCAGGCAACCGGTGAAGGTGAACGCG

[0013] CACCGGGCATGCATGGTCCGGCACTGGCAATTAAGCGCTATTTTCAGGATATTCGTGTTTATCTGGAAG

[0014] ATGAAGGTTATAGTGATTGCGCATGGGAAATTGTGCGTCTGGAAATTATGCGTGCCCTGAGCAGTAGC

[0015] GCCACCCTGCAGGATAGTCTGGCCATTAAGGATGGTGACCTGGCCAGCAGT(SEQ ID NO:2);

[0016] The amino acid sequence of the ricin B chain protein is as follows:

[0017] ADVCMDPEPIVRIVGRNGLCVDVRDGRFHNGNAIQLWPCKSNTDANQLWTLKRDNTIRSNGKCLTT

[0018] YGYSPGVYVMIYDCNTAATDATRWQIWDNGTIINPRSSLVLAATSGNSGTTLTVQTNIYAVSQGWLPTNN

[0019] TQPFVTTIVGLYGLCLQANSGQVWIEDCSSEKAEQQWALYADGSIRPQQNRDNCLTSDSNIRETVVKILSC

[0020] GPASSGQRWMFKNDGTILNLYSGLVLDVRASDPSLKQIILYPLHGDPNQIWLPLF(SEQ ID NO:3);

[0021] The nucleotide sequence of one of its coding genes is as follows:

[0022] GCTGATGTGTGTATGGACCCTGAACCGATTGTGCGTATTGTTGGTCGCAATGGCCTGTGTGTGGAT

[0023] GTTCGTGATGCCGTTTTCATAATGGCAATGCCATTCAGCTGTGGCCGTGCAAAAGTAATACCGATGCA

[0024] AATCAGCTGTGGACCCTGAAACGTGATAATACATTCGTAGTAATGGTAAATGTCTGACCACCTATGGC

[0025] TATAGTCCCGGGTGTTTATGTGATGATCTATGATTGTAATACCGCAGCAACCGATGCCACCCGTTGGCAG

[0026] ATTTGGGATAATGGCACCATTATTAATCCGCGTAGTAGTCTGGTTCTGGCAGCCACCAGTGGTAATAGT

[0027] GGCACCACCCTGACCGTTCAGACCAATATCTATGCAGTTAGCCAGGGTTGGCTGCCGACCAATAATAC

[0028] CCAGCCGTTTGTTACCACCATTGTTGGCCTGTATGGCCTGTGCCTGCAGGCCAATAGTGGCCAGGTTT

[0029] GGATTGAAGATTGCAGTAGCGAAAAAGCCGAACAGCAGTGGGCCCTGTATGCCGATGGTAGTATTCG

[0030] TCCGCAGCAGAATCGCGATAATTGTCTGACCAGTGATAGTAATATTCGCGAAACCGTTGTGAAAATTCT

[0031] GAGTTGTGGCCCGGCAAGCAGTGGTCAGCGCTGGATGTTTAAAAATGATGGCACCATTCTGAATCTGT

[0032] ATAGTGGCCTGGTGCTGGATGTTCGTGCCAGTGATCCGAGCCTGAAACAGATTATTCTGTATCCGCTGC

[0033] ATGGCGATCCGAATCAGATTTGGCTGCCGCTGTTT (SEQ ID NO: 4).

[0034] As a specific record of the embodiment, the fusion protein has a specific amino acid sequence as follows:

[0035] MCALPGSHAQVSRDNLVLLGQMRRLSPFLCLRARKDFRFPREMLEGGQLREAQAAAAVLRELLQQTFNLLHTERSSAAWSPAPLHGLRSGLHRQLEALDACLLQATGEGERAPGMHGPALAIKRYFQDIRVYLEDEGYSDCAWEIVRLEIMRALSSSATLQDSLAIKDGDLASSGGGGSGGGGSGGGGSADVCMDPEPIVRIVGRNGLCVDVRDGRFHNGNAIQLWPCKSNTDANQLWTLKRDNTIRSNGKCLTTYGYSPGVYVMIYDCNTAATDATRWQIWDNGTIINPRSSLVLAATSGNSGTTLTVQTNIYAVSQGWLPTNNTQPFVTTIVGLYGLCLQANSGQVWIEDCSSEKAEQQWALYADGSIRPQQNRDNCLTSDSNIRETVVKILSCGPASSGQRWMFKNDGTILNLYSGLVLDVRASDPSLKQIILYPLHGDPNQIWLPLF(SEQ ID NO:5).

[0036] The nucleotide sequence of its corresponding coding gene is as follows:

[0037] ATGTGTGCACTGCCGGGTAGTCATGCCCAGGTGAGTCGTGATAATCTGGTGCTGCTGGGTCAGAT

[0038] GCGCCGTCTGAGCCCGTTTCTGTGTCTGCGTGCCCGTAAAGATTTTCGTTTTCCGCGTGAAATGCTGG

[0039] AAGGCGGTCAGCTGCGTGAAGCCCAGGCCGCAGCAGCAGTGCTGCGTGAACTGCTGCAGCAGACCT

[0040] TTAATCTGCTGCATACCGAACGTAGTAGTGCAGCATGGAGCCCGGCACCGCTGCATGGCCTGCGTAGT

[0041] GGTCTGCATCGTCAGCTGGAAGCCCTGGATGCCTGTCTGCTGCAGGCAACCGGCGAAGGTGAACGCG

[0042] CACCGGGTATGCATGGTCCGGCCCTGGCAATTAAGCGTTATTTTCAGGATATTCGTGTTTATCTGGAAG

[0043] ATGAAGGTTATAGTGATTGCGCCTGGGAAATTGTTCGTCTGGAAATTATGCGCGCCCTGAGCAGCAGT

[0044] GCCACCCTGCAGGATAGTCTGGCCATTAAGGATGGTGACCTGGCCAGCAGCGGTGGTGGTGGCAGCG

[0045] GTGGTGGCGGTAGCGGTGGTGGTGGTAGTGCTGATGTGTGTATGGACCCTGAACCGATTGTGCGTATT

[0046] GTTGGTCGCAATGGCCTGTGTGTGGATGTTCGTGATGGCCGTTTTCATAATGGCAATGCCATTCAGCTG

[0047] TGGCCGTGCAAAAGTAATACCGATGCAAATCAGCTGTGGACCCTGAAACGTGATAATACCATTCGTAG

[0048] TAATGGTAAATGTCTGACCACCTATGGCTATAGTCCGGGTGTTTATGTGATGATCTATGATTGTAATACC

[0049] GCAGCAACCGATGCCACCCGTTGGCAGATTTGGGATAATGGCACCATTATTAATCCGCGTAGTAGTCTG

[0050] GTTCTGGCAGCCACCAGTGGTAATAGTGGCACCACCCTGACCGTTCAGACCAATATCTATGCAGTTAG

[0051] CCAGGGTTGGCTGCCGACCAATAATACCCAGCCGTTTGTTACCACCATTGTTGGCCTGTATGGCCTGTG

[0052] CCTGCAGGCCAATAGTGGCCAGGTTTGGATTGAAGATTGCAGTAGCGAAAAAGCCGAACAGCAGTGG

[0053] GCCCTGTATGCCGATGGTAGTATTCGTCCGCAGCAGAATCGCGATAATTGTCTGACCAGTGATAGTAAT

[0054] ATTCGCGAAACCGTTGTGAAAATTCTGAGTTGTGGCCCGGCAAGCAGTGGTCAGCGCTGGATGTTTA

[0055] AAAATGATGGCACCATTCTGAATCTGTATAGTGGCCTGGTGCTGGATGTTCGTGCCAGTGATCCGAGC

[0056] CTGAAACAGATTATTCTGTATCCGCTGCATGGCGATCCGAATCAGATTTGGCTGCCGCTGTTT (SEQ ID NO: 6).

[0057] The present invention also provides a recombinant engineering strain for recombinantly expressing the above fusion protein.

[0058] The present invention also provides a use of the fusion protein, which is use in preparing antiviral products.

[0059] In another aspect, the present invention provides an antiviral product, wherein the active component comprises the above-mentioned fusion protein.

[0060] The invention connects the gene of feline interferon omega with the RTB sequence, and the obtained fusion protein has a better half-life and high antiviral activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 : PCR amplification diagram of target gene FeIFNω-RTB; M in the figure: DL 2000 DNA Marker; 1-2: target fragment FeIFNω-RTB

[0062] Figure 2 : PCR identification results of PET28a-FeIFNω-RTB bacterial solution; M: DL 2000 DNA Marker; 1-5: PET28a-FeIFNω-RTB; 6: PET28a (+) (empty control)

[0063] Figure 3: PET28a-FeIFNω-RTB plasmid double enzyme digestion results; M in the figure: DL 10000 DNA Marker; 1-3: PET28a-FeIFNω digestion products

[0064] Figure 4 : SDS-PAGE identification of the recombinant fusion protein PET28a-FeIFNω-RTB; M in the figure: Protein Marker; 1: PET28a(-) 2: PET28a(+) 3: Uninduced bacterial solution 4: Induced whole bacterial solution 5: Bacterial solution supernatant 6: Bacterial solution precipitate

[0065] Figure 5 :Western Blot detection of recombinant fusion protein PET28a-FeIFNω-RTB; M: Protein Marker; 1: bacterial precipitate; 2: PET28a empty vector

[0066] Figure 6 :SDS-PAGE identification of recombinant fusion protein PET28a-FeIFNω-RTB; M: Protein Marker; 1: PET28a(-) 2: PET28a(+) 3: Uninduced bacterial solution 4: 0.2mM 5: 0.4mM 6: 0.6mM 7: 0.8mM

[0067] Figure 7 : SDS-PAGE identification of recombinant fusion protein PET28a-FeIFNω-RTB; M in the figure: Protein Marker; 1: PET28a(-) 2: PET28a(+) 3: uninduced bacterial solution 4: 15℃ 5: 26℃ 6: 37℃

[0068] Figure 8 : Purification results of recombinant fusion protein PET28a-FeIFNω-RTB; M: Protein Marker; 1-8: Purified target protein PET28a-FeIFNω-RTB

[0069] Figure 9 :BCA standard curve

[0070] Figure 10 :Toxic effect of FeIFNω-RTB on F81 cells DETAILED DESCRIPTION

[0071] The coding genes of the optimized feline interferon ω and RTB sequences of the present invention are connected in series through the linker (GGGS) 3 to obtain a fusion protein. The toxicity of the recombinant fusion protein to feline kidney cells (F81 cells) was detected by the CCK8 method, and the antiviral activity titer of the two recombinant feline interferon ω expressed was determined by the microcytopathic effect inhibition method. The test results showed that FeIFNω-RTB had no toxicity to F81 cells at 100μg / ml-1pg / ml, and the antiviral titer was 10 5.75 IU / ml. To determine the half-life of feline interferon ω, the fusion product of feline interferon ω and RTB, the expressed FeIFNω and FeIFNω-RTB were subcutaneously injected into rats. Plasma drug concentrations were measured by ELISA, and pharmacokinetic parameters were calculated using Phoenix WinNonlin 8.1. The results showed that the half-lives of FeIFNω and FeIFNω-RTB were 14.248518 hours and 21.48186 hours, respectively. FeIFNω-RTB has a half-life 1.5 times that of FeIFNω.

[0072] The present invention is described in detail below with reference to the embodiments and accompanying drawings.

[0073] Example 1: Construction and identification of recombinant plasmid PET28a-FeIFNω-RTB

[0074] 1.1 Design and synthesis of FeIFNω and FeIFNω-RTB target genes

[0075] Based on the feline interferon ω and RTB genes, the two genes were concatenated by introducing a flexible linker (GGGGS) 3, removing the original signal peptide sequence, and optimizing the sequence based on the codon preference of E. coli. The amino acid sequence of the determined fusion protein is SEQ ID NO: 5, and the nucleotide sequence of the gene encoding it is SEQ ID NO: 6.

[0076] 1.2 Primer design and synthesis

[0077] Based on the PET-28a(+) vector and the synthetic FeIFNω-RTB plasmid, primers for homologous recombination were designed using SnapGene software. The relevant primer sequences are detailed in Table 1.

[0078] Table 1: Primer sequences

[0079]

[0080] 1.3 Amplification of target genes

[0081] Using the synthetic plasmid FeIFNω-RTB as a DNA template, polymerase chain reaction (PCR) amplification was performed using the primers listed in Table 2-1. The amplification process followed the following protocol: 95°C for 3 minutes, 95°C for 15 seconds, 55°C for 15 seconds, 72°C for 15 seconds, and 72°C for 5 minutes. The specific amplification reaction system for 35 cycles is shown in Table 2.

[0082] Table 2: PCR amplification reaction system

[0083]

[0084] The amplified product was detected by agarose gel electrophoresis, and the expected band of FeIFNω-RTB was observed at 1356 bp. Figure 1 shown.

[0085] 1.4 Vector linearization

[0086] The pET-28a(+) vector was double-digested with BamHI and XhoI restriction enzymes to obtain a linearized vector. The specific enzyme digestion reaction system is shown in Table 3.

[0087] Table 3: Double enzyme digestion system

[0088]

[0089] 1.5 Ligation of the target gene FeIFNω-RTB and the PET-28a vector

[0090] The target gene FeIFNω-RTB amplified in 1.3 and the linearized vector pET-28a(+) obtained by double enzyme digestion as described in Section 1.4 were analyzed by agarose gel electrophoresis. Subsequently, the target gene fragment and linearized vector were purified and recovered using a gel recovery kit. The purified target gene and linearized vector were ligated using the ligation system and reaction conditions listed in Table 4.

[0091] Table 4: Connection system table

[0092]

[0093] 1.6 Transformation of ligation products

[0094] (1) Remove competent DH5α cells stored at -80°C and thaw on ice for 5 minutes. After the cells are thawed, add 1 μL of DNA ligation product to the cells and gently stir the bottom of the EP tube to mix. Then, let it stand on ice for 25 minutes.

[0095] (2) Preheat the water bath to 42°C. Heat shock the sample in the water bath for 45 seconds, then immediately place on ice and let it stand for 2 minutes. Minimize vibration during this process, as vibration may reduce transformation efficiency.

[0096] (3) Add 700 μL of LB liquid medium without antibiotics to the centrifuge tube, mix thoroughly, and place on a shaker at 37°C and 200 rpm / min to recover for 60 min.

[0097] (4) After centrifugation at 5000 rpm / min for one minute, the cells were collected. Then, approximately 100 μl of the supernatant was carefully removed and gently pipetted to resuspend the cells. The cells were then evenly spread on a plate containing Kan. + on LB solid medium plates.

[0098] (5) Place the plate upside down in a 37°C incubator for overnight culture.

[0099] 1.7 Identification of the recombinant plasmid PET28a-FeIFNω-RTB

[0100] Select a single clone from section 2.1.6 and inoculate it with a sterile white pipette tip into a tube containing Kan + The bacteria were then shaken in a 20 ml LB liquid culture medium containing 50 μg / mL of LB. The container was then placed in a shaker at 37°C and 220 rpm / min for 12 hours. Subsequently, PCR identification was performed using the bacterial solution. The specific reaction system is shown in Table 5. Agarose gel electrophoresis analysis revealed a specific band at 1356 bp, consistent with the expected size. Figure 2 .

[0101] Next, the plasmid was extracted using a plasmid extraction kit, and the recombinant plasmid was double-digested with restriction endonucleases BamHI and XhoI. The enzyme digestion reaction system is detailed in Table 6. The enzyme digestion products were identified by agarose gel electrophoresis. Figure 3 As shown in Figure 2 , the PET28a-FeIFNω-RTB plasmid showed the expected target bands at 1369 and 5329 bp. The plasmid was then sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing. After sequence alignment analysis, the plasmid, which was confirmed to be correct, was named PET28a-FeIFNω-RTB.

[0102] Table 5: Bacterial liquid PCR amplification system

[0103]

[0104] Table 6: Double enzyme digestion system

[0105]

[0106]

[0107] Example 2: Prokaryotic expression of the recombinant fusion protein PET28a-FeIFNω-RTB

[0108] 2.1 Transformation of the recombinant fusion protein PET28a-FeIFNω-RTB

[0109] The recombinant plasmid with correct sequencing was transformed into BL21 competent cells, and the transformation method was the same as 1.6. Subsequently, a single colony was picked using a sterile white pipette tip and inoculated into 4 mL LB liquid culture medium containing Kan+ (concentration of 50 μg / mL) at a ratio of 1:100. The culture was placed in a constant temperature shaker at 37°C and 200 rpm for shaking culture for about 12 hours, until the OD 600 value of the bacterial solution was between 0.6 and 0.8. At this time, IPTG at a concentration of 0.5 mM was added to the bacterial solution and cultured at 37°C for 12 hours. After the culture was completed, the cells were separated by centrifugation after 10 minutes of crushing at 40% power, the supernatant after crushing was taken for sample preparation, and the precipitated cells were resuspended in PBS buffer. Finally, the samples were collected for SDS-PAGE gel electrophoresis to identify whether the recombinant fusion protein was correctly expressed.

[0110] 2.2SDS-PAGE gel electrophoresis

[0111] (1) Assemble the glass panels. To ensure the sealing of the assembled glass panels, inject distilled water into them for leakage detection. If leakage is found, reassemble and repeat the detection until there is no leakage.

[0112] (2) Prepare a polyacrylamide gel electrophoresis system containing 12.5% ​​protein: First, take 2.7 ml of the bottom gel solution and 2.7 ml of the bottom gel buffer, place them in a beaker, add 60 μl of the catalyst, and shake gently to mix. Next, take 0.75 ml of the top gel solution and 0.7 ml of the top gel buffer, place them in another beaker, add 15 μl of the catalyst, and shake gently to mix.

[0113] (3) Pour the solution prepared in step 2 into the glass plate used for glue preparation, first injecting the bottom layer of glue and then covering it with the top layer of glue. Insert a comb vertically above the liquid surface, taking care to prevent the formation of bubbles. Then, place the glass plate containing the glue at room temperature for 40 minutes before use.

[0114] (4) Install the cured rubber sheet into the electrophoresis tank and secure it. Then, inject electrophoresis fluid into the inner tank to check for leakage. If there is no leakage, add electrophoresis fluid to the outer tank.

[0115] (5) Slowly remove the comb, add the sample and marker into the well, and perform electrophoresis at a constant voltage of 80V until the upper colloid separates. Then increase the voltage to 120V and continue electrophoresis until the marker is completely separated. At this point, stop the electrophoresis process.

[0116] (6) Remove the glass plate, take the protein gel and place it in a dish containing Coomassie Brilliant Blue stain. Then, place the dish on a shaker and shake it slowly for 5 minutes. After staining is completed, pour out the staining solution and rinse the surface stain with clean water to prepare for observation of the results in the nucleic acid electrophoresis gel imaging system. By comparing the width of the target bands of different samples, the optimal conditions for protein expression can be determined.

[0117] The expression was induced by IPTG, and the bacterial precipitate and lysis supernatant were analyzed by SDS-PAGE gel electrophoresis. Figure 4 As shown, the target protein PET28a-FeIFNω-RTB was correctly expressed mainly in the form of inclusion bodies.

[0118] 2.3 Western Blot Identification

[0119] (1) Cut out the portion of the strip to be transferred and soak it in transfer buffer.

[0120] (2) Cut a 0.22 μm PVDF membrane of appropriate size, then activate it by immersing it in methanol for 10 seconds, and then transfer it to the transfer buffer to ensure that the membrane does not dry out.

[0121] (3) Following the basic principle that substances migrate from the negative electrode to the positive electrode, assemble the transfer plate in the prescribed order, namely the black plate, sponge pad, filter paper, protein gel, PVDF membrane, filter paper, sponge pad, and red plate, and place it in the transfer tank. Subsequently, a constant current of 200mA was used for transfer, and the duration was 40min. After the transfer was completed, the PVDF membrane was removed and placed in a 5% skim milk powder solution and blocked overnight at 37°C.

[0122] (4) Wash the PVDF membrane in 1×TBST and shake it on a shaker at 92 rpm / min for 15 min, for a total of three washes.

[0123] (5) Dilute the primary antibody (Anti-His Tag) at a ratio of 1:5000 with TBST and incubate the PVDF membrane in the primary antibody at room temperature for 4 h.

[0124] (6) Repeat step 5.

[0125] (7) Dilute the secondary antibody (HRP-conjugated His-tag) in 1×TBST at a ratio of 1:3000, then incubate the PVDF membrane in the secondary antibody at room temperature for 1 h, gently shaking it at 45 rpm / min during incubation.

[0126] (8) Repeat step 5.

[0127] (9) Place the PVDF membrane on a protein imaging instrument for analysis, and evenly drip chemiluminescence on top of the membrane.

[0128] After the bacterial solution was cultured at 37°C and 200 rpm, Western Blot analysis was performed using the bacterial pellet and lysis supernatant. Figure 5 As shown, the target protein PET28a-FeIFNω-RTB was correctly expressed mainly in the form of inclusion bodies.

[0129] Example 3 Optimization of IPTG expression conditions for the recombinant fusion protein PET28a-FeIFNω-RTB

[0130] 3.1 Optimization of IPTG induction concentration for recombinant fusion protein PET28a-FeIFNω-RTB

[0131] In order to determine the optimal induction concentration of the recombinant fusion protein, the bacterial solution was added to 20 mL of LB liquid medium at a ratio of 1:100 and cultured overnight at 37°C and 220 rpm in a shaker. The induction concentration of IPTG inducer was set to 0.2 mM, 0.4 mM, 0.6 mM, and 0.8 mM respectively for induction expression. The optimal induction concentration was detected by SDS-PAGE gel electrophoresis. The results showed that ( Figure 6 ), PET28a-FeIFNω-RTB had the best induction effect at 0.6 mM.

[0132] 3.2 Optimization of IPTG induction temperature for recombinant fusion protein PET28a-FeIFNω-RTB

[0133] In order to determine the optimal induction concentration of the recombinant fusion protein, the bacterial solution was added to 20 mL of LB liquid medium at a ratio of 1:100 and cultured overnight at 37°C and 220 rpm in a shaker. The induction temperature was set to 15°C, 26°C, and 37°C for expression, and the optimal expression temperature was detected by SDS-PAGE gel electrophoresis. Figure 7 As shown, the induction effect of PET28a-FeIFNω-RTB was best at 37°C.

[0134] 3.3 Optimization of induction time of recombinant fusion protein PET28a-FeIFNω-RTBIPTG

[0135] In order to determine the effect of induction time on protein expression, the bacterial liquid was added to 20 mL LB liquid culture medium at a ratio of 1:100 and cultured on a shaker at 37°C and 220 rpm. Samples were taken at 3 h, 6 h, 9 h, 12 h, and 24 h, and the optimal induction time was detected by SDS-PAGE gel electrophoresis. The results showed that the expression effect was best at 9 h.

[0136] 3.4 Purification and refolding of recombinant fusion proteins

[0137] The recombinant proteins PET28a-FeIFNω and PET28a-FeIFNω-RTB were expressed in large quantities using the optimal expression conditions identified above, and purified using a His-tagged affinity chromatography nickel column protein purification kit. After purification, they were identified by SDS-PAGE gel electrophoresis.

[0138] 3.4.1 Purification of recombinant protein

[0139] (1) Place the overnight induced culture in a centrifuge at 10,000 rpm for 15 minutes at 4°C. After centrifugation, discard the supernatant and resuspend the pellet in 1 mL of 1× PBS solution, then adjust the volume to 20 mL. Subsequently, disrupt the culture using an ultrasonic cell disruptor for 10 minutes and centrifuge again at 10,000 rpm for 15 minutes at 4°C.

[0140] (2) After centrifugation, discard the supernatant and resuspend the pellet in denaturing lysis buffer and dilute to 16 ml. Centrifuge at 10,000 rpm / min for 30 min at 4°C. Keep the supernatant in an ice bath for use in subsequent experimental steps.

[0141] (3) Take 2.5 mL of denaturation resistance agent, centrifuge at 10,000 rpm at 4°C for 10 s, discard the storage solution, add 2 mL of deformation lysis solution, centrifuge at 10,000 rpm at 4°C for 10 s, discard the waste solution, repeat the centrifugation 1-2 times, and discard the waste solution.

[0142] (4) Mix the gel and supernatant and place in a shaker at 4°C and 60 rpm to mix thoroughly so that the protein can fully bind to the column. Remove the mixture after 1 hour.

[0143] (5) Place the mixture of lysate and denaturation-resistant formulation into an appropriate empty column tube, open the bottom plug and lid, collect the flow-through, and repeat the column loading 5 times to fully bind the target protein.

[0144] (6) Open the column lid and allow the liquid in the column to slowly flow out under the action of gravity. Collect the flowing liquid for subsequent analysis.

[0145] (7) Wash the column five times with lysis buffer, adding 1 mL each time, and collect the lysis buffer for subsequent use (optional).

[0146] (8) Wash the column five times with denaturing washing solution to remove impurities, adding 1 mL each time and collecting the washing solution twice.

[0147] (9) Elute the target protein 10 times with 1 mL of eluent each time, and collect the eluent in a new centrifuge tube. The collected eluate is the target protein.

[0148] like Figure 8 As shown, after purification, the target band appeared at around 50 kd.

[0149] 3.4.2 Renaturation of recombinant protein

[0150] (1) Gently remove the dialysis bag soaked in PBS and rinse it in a container filled with deionized water.

[0151] (2) Ensure that one end of the dialysis bag is clamped and then tested for leaks. Once there is no leakage, pipette the target protein into the pre-prepared dialysis bag and securely seal the other end. Then, gently flip the dialysis bag upside down to observe its tightness and confirm that there is no leakage.

[0152] (3) Place the dialysis bag containing the target protein into a beaker containing the refolding solution (see the refolding solution formula in the table). Place the beaker on a magnetic stirrer. The magnetic stirrer should not rotate too fast. Place a rotor in the beaker. Change the solution once in the morning and evening (every 12 hours). The urea concentrations of the refolding solution are: 6M, 4M, 2M, and 1M. (See Table 7)

[0153] Table 7: Dialysis fluid formula

[0154]

[0155] The purified target protein was placed in a dialysis bag with a molecular weight cutoff of 10KDa for dialysis. After dialysis, the concentration of the renatured protein solution was determined using a BCA protein quantification kit to establish a standard curve. The results are as follows: Figure 9 , the concentration of the recombinant fusion protein FeIFNω-RTB was determined to be 0.4 mg / ml.

[0156] Example 4: Determination of the half-life of the recombinant fusion protein FeIFNω-RTB

[0157] Nine female Sprague-Dawley rats weighing approximately 350 g were fasted for 24 hours with free access to water before drug administration. They were then randomly divided into three groups of three: the FeIFNω group, the FeIFNω-RTB group, and a saline control group. First, 100 μL of blood was drawn from the rat's tail vein, and then 1 ml of a 100 μg / kg drug dose was injected subcutaneously in the neck. At different time points (1, 3, 5, 7, 9, 12, 24, 48, 72, 96, 120, 144, and 168 hours), 100 μL of blood was drawn from the rat's tail vein for testing. All blood samples were refrigerated at 4°C for 1 hour, then centrifuged at 3000 rpm / min for 10 minutes. Serum was then collected for FeIFNω and FeIFNω-RTB concentration determination.

[0158] The toxicity of the recombinant fusion protein to feline kidney cells (F81 cells) was detected by CCK8 method, and the antiviral activity of the two recombinant feline interferon ω was determined by microcytopathic inhibition assay. The test results showed that FeIFNω-RTB had no toxicity to F81 cells at 100μg / ml-1pg / ml. Figure 10 ), the antiviral titer is 10 5.75 IU / ml (Table 8).

[0159] To determine the half-life of feline interferon ω after fusion with RTB, the expressed FeIFNω and FeIFNω-RTB were subcutaneously injected into rats. Plasma drug concentrations were measured by ELISA, and pharmacokinetic parameters were calculated using Phoenix WinNonlin 8.1. The results showed that the half-lives of FeIFNω and FeIFNω-RTB were 14.248518 h and 21.48186 h, respectively. The half-life of FeIFNω-RTB was 1.5 times that of FeIFNω (Table 9).

[0160] Table 8: Antiviral potency table

[0161]

[0162] Table 9: Half-life results of FeIFNω and FeIFNω-RTB

[0163]

[0164] In summary, the present invention genetically fuses feline interferon omega with ricin B chain (RTB), uses feline interferon omega as a control, and expresses both in an E. coli expression system. It was determined that the E. coli expression system expresses a recombinant feline interferon omega fusion protein with high antiviral activity and a long half-life.

Claims

1. A fusion protein, characterized in that The fusion protein contains cat interferon ω protein and ricin B chain protein.

2. The fusion protein according to claim 1, wherein The amino acid sequence of the ω protein is SEQ ID NO:

1.

3. The fusion protein according to claim 2, wherein The nucleotide sequence of the gene encoding the ω protein is SEQ ID NO:

2.

4. The fusion protein according to claim 1, wherein The amino acid sequence of the ricin B chain protein is SEQ ID NO:

3.

5. The fusion protein according to claim 4, wherein The nucleotide sequence of the gene encoding the ricin B chain protein is SEQ ID NO:

4.

6. The fusion protein according to claim 1, wherein The amino acid sequence of the fusion protein is SEQ ID NO:

5.

7. The fusion protein according to claim 6, wherein The nucleotide sequence of the gene encoding the fusion protein is SEQ ID NO:

6.

8. A recombinant engineered strain, characterized in that: The recombinant engineering strain is used for recombinantly expressing the fusion protein according to claim 1.

9. Use of the fusion protein according to claim 1 in the preparation of antiviral products.

10. An antiviral product, characterized in that The active component in the antiviral product comprises the fusion protein according to claim 1.

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