Chicken alpha interferon polypeptide, coding gene thereof, recombinant bacterium thereof and application of chicken alpha interferon polypeptide

Abstract

The invention belongs to the technical field of microbial genetic engineering, and discloses a chicken interferon-alpha polypeptide, a coding gene thereof, a recombinant bacterium thereof and application thereof, the chicken interferon-alpha polypeptide is any one of the following: 1) the amino acid sequence of the chicken interferon-alpha polypeptide is shown as SEQ ID NO.5, and leucine at the 35th site is mutated into threonine; 2) the amino acid sequence of the gene is shown as SEQ ID NO.7, leucine at the 35th site is mutated into threonine, and tyrosine at the 122nd site is mutated into glycine; 3) the amino acid sequence of the chicken interferon-alpha polypeptide is shown as SEQ ID NO.9, leucine at the 35th site is mutated into threonine, glutamine at the 82nd site is mutated into arginine, and tyrosine at the 122nd site is mutated into glycine. The coding gene of the chicken interferon-alpha polypeptide is any one of the following genes: 1) the nucleotide sequence of the chicken interferon-alpha polypeptide is shown as SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.18, SEQ ID NO.20 or SEQ ID NO.22;

Description

Technical Field

[0001] This invention relates to the field of microbial genetic engineering technology, and in particular to a chicken α-interferon polypeptide, its encoding gene, its recombinant strain, and its applications. Background Technology

[0002] my country is the world's largest poultry farmer, with an annual output of over 12 billion broilers, accounting for more than 35% of the global total. However, viral diseases such as avian influenza and Newcastle disease cause direct economic losses exceeding 10 billion yuan annually. Chicken interferon is currently widely used for the prevention and treatment of viral diseases in poultry. Since the complete ban on growth-promoting antibiotics in 2020, interferon has been included as an alternative in the "Action Plan for Reducing the Use of Veterinary Antibiotics." Therefore, research on chicken interferon is not only a strategic need for disease control but also a core lever for industrial upgrading and international competitiveness.

[0003] Chicken interferon-alpha (ChIFNα) is an important antiviral cytokine in the avian immune system. It activates the JAK-STAT signaling pathway by binding to receptors on the cell membrane (a heterodimer formed by IFNAR1 and IFNAR2), inducing the expression of a series of antiviral proteins (such as 2',5'-oligoadenylate synthase), thereby inhibiting the proliferation of viruses such as avian influenza, Newcastle disease, and infectious bursal disease, thus exerting its antiviral effect. Studies have shown that chicken interferon-alpha can reduce antibiotic use by more than 50% while improving poultry farming efficiency. Henan Province, Shandong Province, and other regions have listed chicken interferon-alpha as a key green additive for promotion, enjoying research and development subsidies and tax incentives.

[0004] However, wild-type chicken alpha interferon has limited affinity for its receptor, making it difficult to achieve ideal antiviral efficiency. Furthermore, it is easily degraded by proteases in poultry, limiting its application in aquaculture.

[0005] In existing technologies, there are many attempts to modify ChIFNα through random mutation or truncation, but these methods have problems such as high randomness in mutation sites and insignificant improvement in activity.

[0006] Therefore, the existing chicken alpha interferon needs further improvement. Summary of the Invention

[0007] To address the aforementioned problems, this invention provides a modified chicken alpha interferon polypeptide, its encoding gene, its recombinant strain, and its applications. The chicken alpha interferon polypeptide is a wild-type mutant with high specific binding affinity to IFNAR2 and significantly enhanced antiviral activity.

[0008] To address the above problems, this application provides the following technical solution: In a first aspect, this application provides a chicken alpha interferon polypeptide, wherein the polypeptide is any one of the following: 1) Its amino acid sequence is shown in SEQ ID NO.5, with leucine (L) at position 35 mutated to threonine (T); 2) Its amino acid sequence is shown in SEQ ID NO.7, where leucine (L) at position 35 is mutated to threonine (T) and tyrosine (Y) at position 122 is mutated to glycine (G). 3) Its amino acid sequence is shown in SEQ ID NO.9, where leucine (L) at position 35 is mutated to threonine (T), glutamine (Q) at position 82 is mutated to arginine (R), and tyrosine (Y) at position 122 is mutated to glycine (G).

[0009] Secondly, this application provides the encoding gene for the aforementioned chicken α-interferon polypeptide, which is any one of the following: 1) Its nucleotide sequence is shown in SEQ ID NO.6, encoding a chicken α-interferon polypeptide with an amino acid sequence shown in SEQ ID NO.5; 2) Its nucleotide sequence is shown in SEQ ID NO.8, encoding a chicken α-interferon polypeptide with an amino acid sequence shown in SEQ ID NO.7; 3) Its nucleotide sequence is shown in SEQ ID NO.10, which encodes a chicken α-interferon polypeptide with an amino acid sequence shown in SEQ ID NO.9.

[0010] The above sequence was obtained by codon optimization based on the codon preference of tobacco, then replacing the self-signal peptide with the tobacco secretion signal peptide (PR1a) at the N-terminus, and removing the terminator at the end of the sequence.

[0011] The IFNAR2 extracellular domain gene fragment (amino acids 30-243) was codon-optimized according to the codon preference of tobacco, and then the tobacco secretion signal peptide (PR1a) was added to the N-terminus, and the terminator was removed from the end of the sequence.

[0012] Thirdly, this application also provides another encoding gene for chicken α-interferon polypeptide, which is obtained by removing the signal peptide from the aforementioned encoding gene and optimizing it according to the codon preference of Pichia pastoris. Its nucleotide sequence is shown in any one of SEQ ID NO.18, SEQ ID NO.20 or SEQ ID NO.22.

[0013] The wild-type and ChIFNα-I, ChIFNα-II, and ChIFNα-III mutant sequences were obtained by removing the signal peptide and the terminator from the end of the sequence, and then optimizing according to the codon preference of Pichia pastoris.

[0014] The polypeptides encoding the genes described above are the polypeptide sequences obtained by removing the signal peptide from the protein sequences of claim 1. The tobacco secretion signal peptide (PR1a) is MGFVLFSQLPSFLLVSTLLLFLVISHSCRA.

[0015] Fourthly, this application also provides a recombinant vector, which is an expression vector containing the encoding gene of the aforementioned chicken α-interferon polypeptide.

[0016] Preferably, the expression vector is the pPIC9K vector.

[0017] Fifthly, this application also provides a recombinant bacterium, which is obtained by transforming a host bacterium with the aforementioned recombinant vector.

[0018] Preferably, the host bacterium is Pichia pastoris. The recombinant bacterium is a recombinant expression strain of Pichia pastoris.

[0019] Sixthly, this application also provides the use of the above-mentioned chicken α-interferon polypeptide, the encoding gene of the above-mentioned chicken α-interferon polypeptide, the above-mentioned recombinant vector or the above-mentioned recombinant bacteria in the preparation of chicken α-interferon polypeptide.

[0020] Seventhly, this application also provides a method for preparing the above-mentioned chicken α-interferon polypeptide, the method comprising the following steps: fermenting the aforementioned recombinant bacteria, inducing expression, and then separating and purifying to obtain the chicken α-interferon polypeptide.

[0021] Preferably, the recombinant strain is Pichia pastoris.

[0022] Eighthly, this application also provides the use of the above-mentioned chicken α-interferon polypeptide, the encoding gene of the above-mentioned chicken α-interferon polypeptide, the above-mentioned recombinant vector or the above-mentioned recombinant bacteria in the preparation of drugs for the prevention and treatment of avian viral diseases.

[0023] Chicken alpha interferon, as a cytokine with antiviral activity, is of great value in the prevention and treatment of avian viral diseases, especially in the poultry industry. Therefore, the encoding gene of the aforementioned chicken alpha interferon peptide, the aforementioned recombinant vector, or the aforementioned recombinant bacteria can be used in the preparation of chicken alpha interferon peptides and in the preparation of avian viral disease drugs with chicken alpha interferon peptides as the active ingredient.

[0024] The present invention has the following beneficial effects: 1. This invention provides a chicken alpha interferon mutant. Compared with the wild type, the chicken alpha interferon mutant can enhance the binding ability to the chicken alpha interferon receptor IFNAR2, and after expression in Pichia pastoris, the antiviral (VSV) activity of the chicken alpha interferon mutant is increased by 10 to 100 times compared with the wild type. Specifically, the activity relative to the wild type is 3.2 × 10⁻⁶. 5IU / ml, ChIFNα-I mutant titer at 1.8×10 5 IU / mL; ChIFNα-II mutant titer at 3.8 × 10⁻⁶ 7 IU / mL; ChIFNα-III mutant titer at 2.1 × 10⁻⁶ 6 IU / mL. This indicates that the ChIFNα-II mutant exhibits the best antiviral activity.

[0025] 2. This application also provides a method for producing chicken α-interferon mutants in Pichia pastoris. The method is simple and easy to operate, and can be used for the industrial production of chicken α-interferon mutants. Attached Figure Description

[0026] Figure 1 The results of the interaction between wild-type chicken alpha interferon and its receptor IFNAR2 in a two-molecule luciferase assay are shown; A shows the fluorescence images of the positive and negative controls and the interaction between wild-type chicken alpha interferon and its receptor IFNAR2; the top left image shows ASK1-LUC. N LUC C -Coil; LUC is in the upper right corner. N LUC C -ChIFNα; LUC is located in the lower left corner. C LUC N -IFNAR2; IFNAR2-LUC is located in the lower right corner. N LUC C -ChIFNα; B represents the relative luciferase activity value; Figure 2 The results of the interaction between wild-type chicken α-interferon, ChIFNα-I, ChIFNα-II, and ChIFNα-III mutant proteins and the receptor IFNAR2 in a two-molecule luciferase assay are shown; A shows fluorescence images of the interaction between the four chicken α-interferon and their receptors, with the IFNAR2-LUC protein in the upper left corner. N LUC C -ChIFNα-I; top right is IFNAR2-LUC N LUC C -ChIFNα-II; IFNAR2-LUC (bottom left) N LUC C -ChIFNα; IFNAR2-LUC is located at the bottom right. N LUC C -ChIFNα-III; B represents the luciferase activity value; Figure 3The results of the interaction between wild-type chicken α-interferon, ChIFNα-III, ChIFNα-IV, and ChIFNα-V mutant proteins and the receptor IFNAR2 in a two-molecule luciferase assay are shown; A shows fluorescence images of the interaction between the four chicken α-interferon and their receptors, with the top left showing IFNAR2-LUC. N LUC C -ChIFNα; Bottom left: IFNAR2-LUC N LUC C -ChIFNα-V; Top right: IFNAR2-LUC N LUC C -ChIFNα-IV; Bottom right: IFNAR2-LUC N LUC C -ChIFNα-III; B represents the luciferase activity value; Figure 4 The SDS-PAGE results are for purified wild-type chicken α-interferon and three mutant chicken α-interferon proteins; where 1 is ChIFNα protein; 2 is ChIFNα-I mutant protein; 3 is ChIFNα-II mutant protein; and 4 is ChIFNα-III mutant protein. Detailed Implementation

[0027] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. In the present invention, unless otherwise specified, the equipment and raw materials used can be purchased from the market or are commonly used in the art. Unless otherwise specified, the methods in the following embodiments are conventional methods in the art.

[0028] Example 1: Mutation site analysis and mutant screening of chicken alpha interferon 1.1 Bioinformatics analysis of potential mutation sites in wild-type ChIFNα Using publicly available online databases and related software, the crystal structures of chicken alpha interferon and its receptor IFNAR2 were simulated and homology modeled. A molecular docking structure model was obtained using Z-DOCK. Alanine-scanning mutations were performed on the full-length sequence of chicken alpha interferon, and changes in the binding affinity between interferon and receptor before and after mutation were analyzed. Key amino acid residues at the receptor-binding interface of chicken alpha interferon were identified, and potential mutation sites were screened. Analysis revealed that the key amino acid residues of chicken alpha interferon are located at the N-terminus (positions 29-35), the middle (positions 80-100), and the C-terminus (positions 120-150), respectively.

[0029] Three mutation sites were selected for the amino acid residues of chicken alpha interferon: L→T at position 35 (to increase hydrophilic interaction), Q→R at position 82 (to increase positive charge and enhance electrostatic interaction with the negatively charged region of IFNAR2), and Y→G at position 122 (to mutate into a small-volume residue and reduce steric hindrance).

[0030] Based on the above three mutation sites, seven candidate mutation schemes for chicken alpha interferon were designed, namely: single mutation at position 35, single mutation at position 82, single mutation at position 122, double mutation at positions 35 and 82 / 35 and 122 / 82 and 122, and triple mutation at positions 35, 82 and 122.

[0031] PolyPhen-2 software was used to predict the functional effects of mutations. The prediction results were categorized as benign, potentially harmful, and highly likely harmful. Among them, the single mutation at position 82 and the double mutation at positions 35 and 82 were deemed potentially harmful and were therefore eliminated. Finally, five candidate mutation combinations were identified for the next step of designing the target gene for chicken alpha interferon.

[0032] 1.2 Design and synthesis of the target gene Based on the wild-type ChIFNα sequence, whose accession number in GenBank is DQ226093.1, the aforementioned five mutation designs were performed: a single point mutation at L35T; a double-site mutation at L35T and Y122G; a single-site mutation at Y122G; a double-site mutation at Q82R and Y122G; and a multi-site mutation at L35T, Q82R, and Y122G. These chicken α-interferon mutants were named ChIFNα-I mutant, ChIFNα-II mutant, ChIFNα-III mutant, ChIFNα-IV mutant, and ChIFNα-V mutant, respectively.

[0033] Example 2: Construction of tobacco recombinant expression vector and recombinant bacteria for chicken α-interferon 2.1 Synthesis of Chicken α-interferon sequences adapted to tobacco expression The wild-type and these five mutant ChIFNα sequences were subjected to the following operations: codon optimization was performed based on tobacco codon preference, then the signal peptide was replaced with the tobacco signal peptide (PR1a), all end-terminal terminators were removed, and the final designed sequences were sent to BGI Genomics Co., Ltd. for gene synthesis. The optimized sequences of wild-type and ChIFNα-I~ChIFNα-V are shown in the sequence listing as SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12 and SEQ ID NO.14, respectively.

[0034] The IFNAR2 extracellular domain gene fragment, amino acids 30-243 (GenBank accession number EU477528.1), was codon optimized according to the codon preference of tobacco. Then, the tobacco secretion signal peptide (PR1a) was added to the N-terminus, and the terminator was removed from the end of the sequence. The fragment was sent to BGI Genomics Co., Ltd. for gene synthesis. Its nucleotide sequence is shown in SEQ ID NO.2 of the sequence listing.

[0035] 2.2 Construction of expression vector and recombinant bacteria The pCambia1300-CLuc vector was double-digested with EcoR1 / Kpn1, recovered by gel, and ligated with the wild-type ChIFNα and the five ChIFNα mutant genes mentioned above. The ligation was then performed on DH5α competent E. coli cells, plated on LB agar plates containing 100 μg / mL kanamycin, and cultured overnight. Single colonies grown on LB plates were shaken, and plasmids were extracted for double-enzyme digestion and identification. After confirmation of correct sequencing, positive strains were preserved.

[0036] Similarly, the pCambia1300-NLuc vector was digested with EcoR1 / Kpn1 double enzymes, ligated with the IFNAR2 (chicken alpha interferon receptor) target gene, transformed into E. coli, and the recombinant plasmid was sent for sequencing to obtain positive strains for preservation.

[0037] The positive control group used Coil-pCambia1300-CLuc and Ask1-pCambia1300-NLuc provided by our laboratory, while the negative control group used empty vectors pCambia1300-CLuc and pCambia1300-NLuc.

[0038] Example 3: Further screening of chicken α-interferon mutants In this embodiment, the interaction strength between five chicken alpha interferon mutants and IFNAR2 (chicken alpha interferon receptor) was verified on a constructed bimolecular luciferase tobacco transient conversion system, thereby enabling further screening and functional verification of the mutants.

[0039] 3.1 Experimental Methods (1) Preparation of Agrobacterium competent cells Agrobacterium strain (GV3101) was picked and inoculated into antibiotic-free YEB liquid medium (containing 50 μg / mL rifampicin) and cultured at 28°C with shaking until OD600 = 0.5-0.6. The bacterial culture was then collected in 50 mL centrifuge tubes and incubated on ice for 30 min, followed by centrifugation at 4°C and 5000 rpm for 5 min. The supernatant was discarded, and the precipitate was resuspended in pre-chilled 0.02 M CaCl2. The resuspending was repeated twice, and finally, the precipitate was resuspended in 1 mL of pre-chilled 0.02 M CaCl2 solution and aliquoted into 1.5 mL sterile centrifuge tubes. The tubes were then flash-frozen in liquid nitrogen and stored at -80°C.

[0040] (2) Freeze-thaw method to transform competent states Thaw one competent cell on ice, add 1 μg of recombinant plasmid DNA, gently tap to mix, incubate on ice for 5 min, flash freeze in liquid nitrogen for 5 min, heat shock at 37°C for 5 min, then place on ice for 5 min, and finally add YEB liquid medium and incubate at 30°C for 2-3 h. Spread the bacterial culture on YEB plates containing rifampicin (Rif+) and kanamycin (Kan+) resistance; incubate at 30°C until single colonies grow, pick a single colony, and verify the successful introduction of the recombinant plasmid into Agrobacterium using colony PCR. After successful identification, store the bacteria at -80°C.

[0041] (3) Agrobacterium-mediated transient transformation expression in tobacco Agrobacterium containing the recombinant plasmid was streaked onto a YEB plate containing Kan+ and Rif for activation. Single colonies were picked and cultured in 5 ml of YEB (containing Kan+ and Rif) liquid medium (28℃ / 220 rpm). The culture was then diluted 1:20 with 20 ml of IM induction medium and cultured for approximately 8 hours (28℃ / 220 rpm). The culture was collected in 50 ml centrifuge tubes, centrifuged, and the cells were resuspended twice with 10 mM MS-MES (pH 5.6) solution. Finally, the cells were treated with MS-10 mM MES (pH 5.6)-150 μM acetylsylgenin (AS). The bacterial cells were resuspended in the solution and the OD value was adjusted to 0.5. The Agrobacterium tumefaciens bacterial solution prepared in the previous step was mixed in a 1:1 ratio according to the N-terminus and C-terminus binding principle. The solution was placed at room temperature for about 1 hour to allow the bacterial solution to mix thoroughly. Then, on the surface of a healthy and flat tobacco leaf, a 1 ml syringe was used to make a hole, and an appropriate amount of the mixed bacterial solution was injected into the leaf surface until a circle was formed. The tobacco leaves injected with the bacterial solution were placed in the dark and cultured at normal temperature for 48 hours, and then exposed to light for 16 hours.

[0042] (4) CCD imaging and LUC activity detection Spray 1 mM luciferase onto the surface of tobacco leaves, gently tap to ensure even coating, and treat in the dark for 5 minutes. Use a CCD imaging device pre-cooled to -120℃ to capture LUC images. The exposure time for each leaf LUC image is 7 minutes. The conditions for capturing autofluorescence imaging of leaf chloroplasts are 1 minute of light exposure and 2 minutes of exposure. Finally, the relative luciferase activity value is calculated.

[0043] 3.2 Experimental Results and Analysis like Figure 1 As shown in Figure A, with positive and negative controls as references, wild-type ChIFNα protein can emit a fluorescent signal when it binds to IFNAR2 receptor protein, thus confirming that protein interaction has occurred.

[0044] like Figure 2 As shown in Figure A, with wild-type ChIFNα protein as a positive control, the ChIFNα-I, ChIFNα-II, and ChIFNα-III mutant proteins can interact with the IFNAR2 receptor protein to varying degrees.

[0045] like Figure 3 As shown in A, wild-type ChIFNα and mutant ChIFNα-III were used as positive controls, while ChIFNα-IV and ChIFNα-V could not interact with the IFNAR2 receptor protein.

[0046] Figure 1 B Figure 2 B Figure 3 B shows the relative luciferase activity values ​​measured using imageJ software. The results show that, in the verification of the interaction strength between the five ChIFNα mutants and the IFNAR2 protein, the mutant ChIFNα-II has the strongest interaction with the IFNAR2 receptor protein and the strongest fluorescence signal, which is significantly higher than that of wild-type ChIFNα.

[0047] Therefore, based on the results of this embodiment, the ChIFNα-IV and ChIFNα-V mutant proteins failed to interact with the IFNAR2 receptor protein, while the ChIFNα-I, ChIFNα-II, and ChIFNα-III mutants interacted with the IFNAR2 receptor protein to varying degrees. Therefore, these three mutants (ChIFNα-I, ChIFNα-II, and ChIFNα-III) were used to construct a recombinant yeast expression strain, prepare chicken α-interferon mutant protein, and determine its biological activity.

[0048] Example 4: Pichia pastoris recombinant expression vector and recombinant strain of chicken α-interferon 4.1 Design and Synthesis of Chicken α-Interferon Sequence The signal peptide was removed from the sequences of wild-type (amino acid sequence as shown in SEQ ID NO. 3 in the sequence listing) and ChIFNα-I mutant, ChIFNα-II mutant, and ChIFNα-III mutant (amino acid sequences as shown in SEQ ID NO. 5, 7, and 9 in the sequence listing), and the terminator was removed from the end of the sequence. The sequences were then optimized according to the codon preference of Pichia pastoris to obtain the nucleotide sequences shown in SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, and SEQ ID NO. 22 in the sequence listing, respectively. These sequences were then sent to BGI Genomics Co., Ltd. for gene synthesis.

[0049] 4.2 Construction of Pichia pastoris recombinant expression vector and recombinant strain The pPIC9K vector was double-digested with EcoR1 / Not1 enzymes. After gel recovery, the large fragments of the vector were digested and ligated to the wild-type, ChIFNα-I, ChIFNα-II, or ChIFNα-III mutant genes with the signal peptide removed in the previous step. The ligation products were transformed into DH5α competent E. coli cells, plated on LB agar plates containing kanamycin, and cultured overnight. Single colonies grown on the plates were shaken, and plasmids were extracted for double digestion and identification. After correct sequencing, positive strains were preserved.

[0050] Example 5: Secretory expression and biological activity detection of wild-type chicken α-interferon and its mutants in Pichia pastoris 5.1 Experimental methods (1) Electroconversion of recombinant plasmids and resistance screening The pPIC9K vector recombinant plasmid containing ChIFNα, ChIFNα-I, ChIFNα-II and ChIFNα-III mutants prepared in Example 4 was linearized with SalI and then transformed into Pichia pastoris host strain GS115.

[0051] Take 5 μg of linearized plasmid vector, add it to 80 μL of GS115 competent cells, transfer it to a pre-cooled 0.2 cm electroporation cuvette, mix well, and incubate on ice for 5 min. Place the cuvette on an electroporator for electroporation conversion. Electroporation conditions: voltage 1.5 kV, capacitance 25 μF, resistance 200 Ω.

[0052] Immediately after electroporation, add 1 ml of pre-cooled 1M sorbitol and transfer to a 1.5 ml EP tube. Incubate at 30°C for 1 h. Take 200 μl and spread it onto YPD plates containing 0.5 mg / ml, 0.75 mg / ml, 1 mg / ml, and 2 mg / ml genimycin, respectively. Incubate at 30°C for 3-5 days. High-copy strains are screened by different concentrations of resistance.

[0053] (2) PCR identification of positive transformants Pick a single colony growing on a YPD plate, resuspend it in 10 μL of sterile water, add 1 U of lysozyme litycase, incubate at 30°C for 10 min, freeze at -80°C for 10 min, and then separate the supernatant.

[0054] Using the supernatant obtained above as a DNA template, PCR was performed. The following 20ul PCR reaction system was set up: 10ul of 2×Hieff PCR Master Mix, 1ul of 5'AOX1 primer, 1ul of 3'AOX1 primer, 2ul of DNA template, and 6ul of sterile water.

[0055] The PCR procedure is as follows: 95℃ for 5 minutes 94℃ for 1 minute 54℃ for 1 minute 72℃ for 1 minute 72℃ for 7 minutes, 30 cycles; After amplification, 10 μL was taken for agarose gel electrophoresis, which yielded a 2.2 kb band and a 1032 bp band, confirming that high-copy positive transformants with the Mut+ phenotype were screened.

[0056] (3) Induction of expression in high-copy positive strains High-copy-positive single colonies of ChIFNα wild-type, ChIFNα-I, ChIFNα-II, and ChIFNα-III mutants were picked from YPD plates and inoculated into BMGY medium, incubated at 30°C and 220 rpm until OD500. 600 Collect bacterial cells by centrifugation at 4000 rpm for 5 min for 2-6 minutes, then transfer them to BMMY medium and adjust the OD value. 600 Induction was initiated at 1, 28°C, and 220 rpm. Induction lasted three days, with 1% (volume percentage) methanol added every 24 hours. The fermentation supernatant was collected after 72 hours.

[0057] (4) Purification of the product The fermentation supernatant was purified by nickel column chromatography.

[0058] 1) Take 1 ml of well-mixed 50% His-tag, centrifuge at 4℃, 1000g, for 30s and discard the stock solution. Add 0.5 ml of non-denaturing lysis buffer to the gel and mix well to equilibrate the gel. Centrifuge at 4℃, 1000g, for 30s and discard the liquid. Repeat the equilibration process twice.

[0059] 2) Bind 5 ml of secretory protein supernatant to a 50% His-tag gel, transfer it to a 10 ml centrifuge tube, and incubate at 4°C for 1 h.

[0060] 3) Load the protein supernatant and 50% His-tag gel mixture into an empty affinity chromatography column tube. Open the bottom cap of the purification column and allow the liquid inside the column to flow out under gravity. Collect about 200 μL of the eluent for subsequent analysis (the eluent can be collected and loaded onto the column multiple times).

[0061] 4) Discard the eluent, add 0.5-1 ml of non-denaturing washing buffer to wash the column 5 times, and collect the washing buffer (W1-W5) after each column penetration for subsequent detection.

[0062] 5) Elute the target protein 6-10 times, using 0.5 ml of non-denaturing elution buffer each time. Collect each elution buffer into centrifuge tubes (E1-E10), which are the purified protein samples.

[0063] (5) Protein verification The purified protein was validated by SDS-PAGE.

[0064] 5.2 Experimental Results and Analysis SDS-PAGE electrophoresis results are as follows: Figure 2 As shown, the wild-type ChIFNα, ChIFNα-I, ChIFNα-II, and ChIFNα-III mutant strains all expressed the target band (chicken α-interferon) normally. However, due to partial degradation of the target protein, two bands were expressed, ranging in size from 26KD to 33KD, significantly larger than the theoretical value (20KD). This suggests that the expressed target protein (chicken α-interferon) underwent glycosylation. Further experiments were conducted to detect the activity of the target protein.

[0065] Example 6: Detection of the biological activity of chicken α-interferon protein expressed in Pichia pastoris This experiment detected and compared the biological activities of wild-type chicken alpha interferon protein with those expressed by three mutant chicken alpha interferon, thereby evaluating the activity of the mutant chicken alpha interferon expressed in this application.

[0066] 6.1 Experimental Methods This experiment was set up with four groups, using wild-type chicken α-interferon protein prepared in Example 5 and chicken α-interferon expressed by three mutants as samples, and the following experiments were conducted using the cytopathic effect inhibition method: Take 9-11 day old SPF chicken embryos, remove the head, limbs and internal organs, wash 3 times with PBS, cut into small pieces, digest with trypsin, then stop digestion with DMEM medium, sieve and count, then plate in 96-well plates and culture at 37°C for about 12 hours until a monolayer of cells is formed.

[0067] Remove the culture medium, add 10-fold serially diluted chicken interferon protein stock solution to each well, incubate for 24 hours, and then use 100 TCID50 cells / well. 50The cells were inoculated with a dose of vesicular stomatitis virus (VSV), and a healthy cell control group (interferon only, no virus) and a virus control group (no interferon, only virus) were set up. After 24 hours of culture, the results were determined, and the activity titer was calculated according to the Reed-Muench method.

[0068] 6.2 Experimental Results and Analysis The activity results are shown in Table 1 below. From the results in Table 1, it can be seen that the wild-type chicken α-interferon (ChIFNα) has an antiviral activity of 3.2 × 10⁻⁶. 5 The antiviral activity of the ChIFNα-I mutant was 1.8 × 10 IU / ml. 5 IU / ml, comparable to wild-type activity; the ChIFNα-III mutant showed antiviral activity of 2.1 × 10⁻⁶. 6 The antiviral activity of the ChIFNα-II mutant was 10 times higher than that of wild-type chicken α-interferon at IU / ml; while the antiviral activity of the ChIFNα-II mutant was 3.8 × 10⁻⁶. 7 IU / ml, with 100 times higher antiviral activity than wild-type chicken alpha interferon.

[0069] Therefore, the site-directed mutation of chicken alpha interferon in this application significantly increases its ability to bind to the receptor IFNAR2, thereby enhancing its antiviral activity and demonstrating promising application prospects. However, the results of the aforementioned mutant ChIFNα-I indicate that not every predicted mutant can enhance the antiviral activity of chicken alpha interferon, and that a higher number of mutation sites does not necessarily lead to a greater increase in antiviral activity.

[0070] Table 1. Results of activity assay of chicken α-interferon protein in each group

[0071] It is understood that those skilled in the art can make equivalent substitutions or modifications to the technical solutions and concepts of this invention, and all such substitutions or modifications should fall within the protection scope of the appended claims.

Claims

1. A chicken alpha interferon polypeptide, characterized in that, It can be any of the following: 1) Its amino acid sequence is shown in SEQ ID NO.5, with leucine at position 35 mutated to threonine; 2) Its amino acid sequence is shown in SEQ ID NO.7, with leucine at position 35 mutated to threonine and tyrosine at position 122 mutated to glycine; 3) Its amino acid sequence is shown in SEQ ID NO.9, with leucine at position 35 mutated to threonine, glutamine at position 82 mutated to arginine, and tyrosine at position 122 mutated to glycine.

2. A gene encoding the chicken α-interferon polypeptide as described in claim 1, characterized in that, It can be any of the following: 1) Its nucleotide sequence is shown in SEQ ID NO.6, encoding a chicken α-interferon polypeptide with an amino acid sequence shown in SEQ ID NO.5; 2) Its nucleotide sequence is shown in SEQ ID NO.8, encoding a chicken α-interferon polypeptide with an amino acid sequence shown in SEQ ID NO.7; 3) Its nucleotide sequence is shown in SEQ ID NO.10, which encodes a chicken α-interferon polypeptide with an amino acid sequence shown in SEQ ID NO.

9.

3. A gene encoding the chicken α-interferon polypeptide as described in claim 1, characterized in that, The nucleotide sequence obtained by removing the signal peptide from the encoding gene of claim 2 and optimizing it according to the codon preference of Pichia pastoris is shown in any one of SEQ ID NO.18, SEQ ID NO.20 or SEQ ID NO.

22.

4. A recombinant vector, characterized in that, It carries the encoding gene for the chicken α-interferon polypeptide as described in claim 2.

5. A recombinant bacterium, characterized in that, The recombinant vector as described in claim 2 or 3 is used to transform the host bacterial strain.

6. The recombinant bacteria according to claim 5, characterized in that, The host bacteria are Pichia pastoris or Agrobacterium.

7. The use of the chicken α-interferon polypeptide as described in claim 1, the encoding gene of the chicken α-interferon polypeptide as described in claim 2 or 3, the recombinant vector as described in claim 4, or the recombinant bacteria as described in claim 5 in the preparation of the chicken α-interferon polypeptide.

8. A method for preparing the chicken α-interferon polypeptide as described in claim 1, characterized in that, Includes the following steps: The recombinant bacteria as described in claim 5 are fermented and induced to express the protein, and then chicken α-interferon polypeptide is isolated and purified; preferably, the recombinant bacteria is Pichia pastoris.

9. The use of the chicken α-interferon polypeptide as described in claim 1, the encoding gene of the chicken α-interferon polypeptide as described in claim 2 or 3, the recombinant vector as described in claim 4, or the recombinant bacteria as described in claim 5 in the preparation of drugs for the prevention and treatment of avian viral diseases.

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