Duck interferon alpha and its mutant, preparation method and application

Abstract

The present invention discloses duck interferon alpha and its mutants, preparation methods and applications. The present invention optimizes duck interferon alpha and performs amino acid single-site mutation, double-site mutation and multi-site mutation on the basis thereof to obtain multiple duck interferon alpha mutants with improved antiviral activities. The present invention also relates to the application of the duck interferon alpha mutant in the preparation of drugs or reagents for preventing or treating duck viral diseases, belonging to the field of preparation and application of duck interferon alpha mutants. The present invention uses the silkworm baculovirus expression system to express the duck interferon alpha mutant in a silkworm bioreactor, and the antiviral activity is greatly improved, and the duck interferon alpha mutant can be used to prepare drugs or reagents for preventing or treating duck viral diseases, and has a defensive and inhibitory effect on viral infectious diseases that pose a major threat to the duck farming industry.

Description

[0001] This application is a divisional application of the following case. The original application filing date is June 13, 2019, the original application number is 201910510526.7, and the original application invention name is duck alpha interferon and its mutants, preparation methods and applications. Technical Field

[0002] The present invention relates to a duck interferon alpha mutant and also relates to a method for preparing the duck interferon alpha mutant by using a silkworm baculovirus expression system; the present invention also relates to the application of the duck interferon alpha mutant in the preparation of a medicine or reagent for preventing or treating duck viral diseases, belonging to the field of preparation and application of duck interferon alpha mutant. Background Art

[0003] Interferon is a cytokine with a wide range of biological activities, including broad-spectrum antiviral, anti-intracellular bacterial, anti-tumor, and immune regulation. The IFN protein family is divided into type I, type II, and type III interferons according to the sequence of its encoding gene, chromosomal location, and receptor specificity. Type I interferons include IFN-α, IFN-β, IFN-ω, IFN-δ, IFN-ε, IFN-ζ, IFN-τ, etc., and IFN-α and IFN-β are mainly found in mammals. Type I interferons have strong antiviral activity, mainly inhibiting the proliferation of viruses by interfering with their replication, and also have anti-tumor and immune regulation functions. Type II interferons have only one member, IFN-γ, also known as immune interferons, and their main function is to activate macrophages to kill microorganisms. Type III interferons are newly discovered cytokines, including λ1 (IL-29), λ2 (IL-28a), and λ3 (IL-28b). Type III interferon is closely related to type I interferon, but has special physiological functions, such as stimulating the activation and expression of major histocompatibility complex (MHC) molecules, regulating innate immunity and acquired immunity, etc. Since interferon has broad-spectrum antiviral and anti-tumor activities and strong immunomodulatory effects, it has become one of the research hotspots in related fields such as virology, cytology, molecular biology, clinical medicine, immunology, and oncology.

[0004] Interferon was first discovered in poultry, but compared with humans and other mammals, the research on duck interferon molecules started later, and compared with chickens, there are relatively few studies on duck interferon. In recent years, infectious diseases such as influenza, Newcastle disease, infectious bronchitis, infectious laryngotracheitis, infectious bursal disease, duck plague, duck hepatitis and Marek's disease caused by poultry virus infection have become more and more serious, causing huge economic losses to the poultry industry every year, seriously restricting the healthy development of the poultry industry. How to effectively prevent and treat viral infectious diseases in poultry has always been the focus of poultry disease prevention and treatment research.

[0005] The duck interferon-α gene was first obtained by German researchers Schultz et al. in 1995 by cloning and screening using the reported ChIFN-α gene as a probe. In 2000, domestic researcher Xia Chun reported the alpha interferon gene of Beijing ducks. Other researchers subsequently reported the alpha interferon genes of various duck breeds, including Muscovy ducks, Shaoxing ducks, and Mallards. In addition, researchers have successively reported the resistance of recombinant duck alpha interferon to various viruses. Schultz et al. confirmed that recombinant interferon has the effect of resisting vesicular stomatitis virus, influenza A virus, duck hepatitis virus and Newcastle disease virus; in 2007, Zhou Xue et al. injected Cherry Valley ducks with a plasmid carrying the duck alpha interferon gene and showed a certain ability to resist duck plague infection; in 2018, Pei Gao et al. used recombinant duck alpha interferon to treat 2-day-old ducks to study its resistance to the highly pathogenic avian influenza virus H5N1. The results showed that compared with the virus control group, the mortality rate of ducks in the experimental group dropped from 60% to 10%, indicating that recombinant duck alpha interferon has a resistance effect on 2-day-old ducks infected with avian influenza virus H5N1. Chen Bin cloned the IFN-α gene from Sichuan duck and detected its anti-VSV activity after prokaryotic expression, which was 150U / ml and the specific activity was 440U / mg. Gao Pei expressed rDuIFN-α, rDuIFN-γ and rDuIFNα-DuIFNγ in the prokaryotic expression system, and the anti-VSV activities were 2.1×10 5 U / mg, 3.6×10 4 U / mg, 1.6×10 7 U / mg. Since the prokaryotic expression system cannot perform complex post-translational processing and modification on the expressed recombinant protein, the titer of the expressed duck interferon-α is low. Therefore, this method uses the silkworm baculovirus expression system to express duck interferon-α in silkworm worms (pupae) to obtain a large amount of high-quality and low-cost recombinant duck interferon-α protein. Summary of the invention

[0006] The first technical problem to be solved by the present invention is to provide a duck interferon alpha and a duck interferon alpha mutant, wherein the duck interferon alpha mutant has high antiviral activity;

[0007] The second technical problem to be solved by the present invention is to provide a method for preparing the duck interferon alpha or duck interferon alpha mutant using the silkworm baculovirus expression system;

[0008] The third technical problem to be solved by the present invention is to provide the use of the duck interferon-alpha or duck interferon-alpha mutant in the preparation of drugs or reagents for preventing or treating duck viral diseases.

[0009] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0010] The present invention first discloses a duck alpha interferon, the amino acid sequence of which is shown in SEQ ID NO.1, and the polynucleotide sequence of its encoding gene is shown in (a) or (b) or (c):

[0011] (a) the polynucleotide sequence shown in SEQ ID No. 2; or

[0012] (b) a polynucleotide sequence that can hybridize with the complementary sequence of SEQ ID No. 2 under stringent hybridization conditions, and the protein encoded by the polynucleotide still has the function or activity of interferon; or

[0013] (c) a polynucleotide sequence having at least 80% homology with the polynucleotide sequence of SEQ ID No.2, and the protein encoded by the polynucleotide still has the function or activity of interferon; preferably, a polynucleotide sequence having at least 85% homology with the polynucleotide sequence of SEQ ID No.2, and the protein encoded by the polynucleotide still has the function or activity of interferon; more preferably, a polynucleotide sequence having at least 90% homology with the polynucleotide sequence of SEQ ID No.2, and the protein encoded by the polynucleotide still has the function or activity of interferon.

[0014] According to the codon preference of silkworm, the duck interferon α gene sequence was codon optimized, and the GC content, CpG dinucleotide content, codon preference, mRNA secondary structure, mRNA free energy stability, RNA instability motif, repeat sequence and other related parameters that affect gene transcription efficiency, translation efficiency and protein folding were optimized and designed, which is conducive to improving the transcription efficiency and translation efficiency of the optimized gene in silkworm, and keeping the final translated protein sequence unchanged; in order to improve the translation initiation efficiency in the silkworm baculovirus eukaryotic expression system, the Kozak sequence GCCAAC was added in front of the gene. In addition, the restriction enzyme sites such as BamHI, EcoRI, SmaI in the gene sequence were removed, BamHI was added upstream of the gene, and EcoRI restriction enzyme sites were added downstream of the gene; the new duck interferon α optimized sequence DuIFN-α-O was obtained, and its amino acid sequence is shown in SEQ ID NO.3, and the optimized gene nucleotide sequence is shown in SEQ ID NO.4.

[0015] The present invention further discloses a duck interferon alpha mutant, which is a mutant obtained by subjecting the duck interferon alpha mutant whose amino acid sequence is shown in SEQ ID NO.3 to any one of amino acid single point mutations of P2A, L19T, P25L, R35P, D38N, N51D, H64N, L73I, Q80R, D95K, H108D, Q117R, H123Y, R127Q, L136S, R142C, I148T, L159F, D170E or R182C; preferably, a mutant obtained by subjecting the duck interferon alpha mutant whose amino acid sequence is shown in SEQ ID NO.3 to any one of amino acid single point mutations of P25L, N51D, Q80R, H108D, L136S and D170E. The amino acid sequence of the mutant obtained by subjecting the duck interferon α mutant having the amino acid sequence shown in SEQ ID NO.3 to single-point mutation of amino acid N51D is shown in SEQ ID NO.5, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.6.

[0016] Among them, the amino acid single-point mutation P2A of the present invention means that the second amino acid of the duck α interferon mutant with the amino acid sequence shown in SEQ ID NO.3 is mutated from proline (P) to alanine (A); L19T means that the 19th amino acid is mutated from leucine (L) to threonine (T); and so on.

[0017] The present invention also discloses a duck interferon alpha mutant, which is a mutant obtained by subjecting the duck interferon alpha mutant having an amino acid sequence as shown in SEQ ID NO.3 to any one of P25L-N51D, P25L-Q80R, P25L-H108D, P25L-L136S, P25L-D170E, N51D-Q80R, N51D-H108D, N51D-L136S, N51D-D170E, Q80R-H108D, Q80R-L136S, Q80R-D170E, H108D-L136S, H108D-D170E or L136S-D170E double-site mutation; preferably, the duck interferon alpha mutant having an amino acid sequence as shown in SEQ ID NO.3 is subjected ... A mutant obtained by subjecting the duck interferon alpha mutant shown in NO.3 to any one of P25L-Q80R, N51D-H108D, and N51D-D170E amino acid double-site mutations. Among them, the amino acid sequence of the mutant obtained by subjecting the duck interferon alpha mutant shown in SEQ ID NO.3 to N51D-H108D amino acid double-site mutation is shown in SEQ ID NO.7, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.8.

[0018] Among them, the amino acid double-site mutation P25L-N51D of the present invention means that the 25th amino acid of the duck α interferon mutant with an amino acid sequence as shown in SEQ ID NO.3 is mutated from proline (P) to leucine (L), and the 51st amino acid is mutated from asparagine (N) to aspartic acid (D); the amino acid double-site mutation P25L-Q80R means that the 25th amino acid is mutated from proline (P) to leucine (L), and the 80th amino acid is mutated from glutamine (Q) to arginine (R); and so on.

[0019] The present invention also discloses a duck interferon alpha mutant, which is obtained by subjecting the duck interferon alpha mutant having the amino acid sequence shown in SEQ ID NO.3 to P25L-N51D-Q80R, P25L-Q80R-H108D, P25L-Q80R-D170E, P25L-N51D-H108D, N51D-Q80R-H108D, N51D-H108D-D170E, P25L-N51D-D170E, N51D- Q80R-D170E, P25L-N51D-Q80R-H108D, N51D-Q80R-H108D-D170E, P25L-N51D-H108D-D170E or P25L-N51D-Q80R-H108D-D170E any one amino acid multiple site mutation mutant; preferably, the duck interferon alpha mutant with the amino acid sequence shown in SEQ ID NO.3 is subjected to P25L-N51D-Q80R-H108D-D170E amino acid multiple site mutation mutant. Among them, the amino acid sequence of the mutant obtained by subjecting the duck interferon alpha mutant with the amino acid sequence shown in SEQ ID NO.3 to P25L-N51D-Q80R-H108D-D170E amino acid multiple site mutation is shown in SEQ ID NO.9, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.10.

[0020] Among them, the amino acid multi-site mutation P25L-N51D-Q80R of the present invention means that the 25th position of the duck α interferon mutant with the amino acid sequence shown in SEQ ID NO.3 is mutated from proline (P) to leucine (L), and the 51st amino acid is mutated from asparagine (N) to aspartic acid (D), and the 80th amino acid is mutated from glutamine (Q) to arginine (R); and so on.

[0021] The present invention analyzes all duck interferon-α amino acid sequences on NCBI, generates a consensus sequence, and uses the amino acid sequence shown in the consensus sequence as the original amino acid sequence of duck interferon-α (its amino acid sequence is shown in SEQ ID NO.1, and its nucleotide sequence of the encoding gene is shown in SEQ ID NO.2). The antiviral activity results show that the titer of DuIFN-α expressed in silkworm larvae is 8.70×10 5 U / mL.

[0022] Based on the original sequence of duck interferon alpha, the present invention optimizes its gene sequence according to the codon preference of silkworm, optimizes and designs various related parameters such as GC content, CpG dinucleotide content, codon preference, secondary structure of mRNA, mRNA free energy stability, RNA instability gene sequence, and repeat sequence that affect gene transcription efficiency, translation efficiency, and protein folding, and keeps the final translated protein sequence unchanged; in order to improve the translation initiation efficiency in the silkworm baculovirus eukaryotic expression system, the Kozak sequence GCCAAC is added in front of the gene ATG. In addition, the restriction enzyme sites such as BamHI, EcoRI, and SmaI in the gene sequence are removed, BamHI is added to the upstream of the gene, and EcoRI restriction enzyme sites are added to the downstream of the gene, so as to obtain a new duck interferon alpha mutant DuIFN-α-O mutant, whose amino acid sequence is shown in SEQ ID NO.3, and the optimized gene nucleotide sequence is shown in SEQ ID NO.4. The antiviral activity results show that the titer of DuIFN-α expressed in silkworm larvae is 1.14×10 6 U / mL.

[0023] The present invention uses the optimized gene sequence of DuIFN-α as a template to design multiple pairs of primers, and uses the fusion PCR method to perform amino acid single-site mutation, amino acid double-site mutation, and amino acid multi-site mutation to obtain multiple duck interferon α mutants. Among them, based on DuIFN-α-O, after performing single-site mutations at six sites, namely P25L, N51D, Q80R, H108D, L136S and D170E, the titer of the expressed duck interferon α is higher than the titer measured by the expression of DuIFN-α-O, and the antiviral titer reaches 7.80×10 4 U / mL~3.16×10 6 U / mL; while the titers of the other sites remained unchanged or even decreased after mutation, indicating that the mutations of these six sites are effective mutations and can achieve the purpose of improving antiviral activity. Among them, the DuIFN-α-O-N51D mutant obtained by N51D single-site mutation has the strongest antiviral effect.

[0024] The present invention further combines the single mutation sites P25L, N51D, Q80R, H108D, L136S and D170E with improved antiviral activity in pairs, and performs double-site mutation on DuIFN-α-O. The antiviral activity test results show that after the three groups of double mutations P25L-Q80R, N51D-H108D and N51D-D170E, the titer of duck α interferon expressed is higher than that measured by conservative sequence and single mutation sequence expression, and the antiviral titer reaches 8.01×10 4 U / mL~4.50×10 6 U / mL, while the titers of the other groups of sites remained unchanged or even decreased after mutation, indicating that the mutations of these three combined sites are effective mutations and can achieve the purpose of improving antiviral activity. Among them, the DuIFN-α-O-N51D-H108D mutant obtained by double-site mutation of N51D-H108D has the strongest antiviral effect.

[0025] The present invention further combines the obtained double mutation sites with high titer to perform amino acid multi-site mutation on the DuIFN-α-O mutant. The detection results of the mutant antiviral activity show that after the P25L-N51D-Q80R-H108D-D170E multi-site mutation, the titer of duck α interferon expressed is much higher than the titer measured by the conservative sequence, single mutation sequence, and double mutation sequence, which is 5.78×10 6 U / mL; while the titers of the other groups of sites remained unchanged or even decreased after mutation, indicating that the mutation of this combination of sites is an effective mutation, which can achieve the purpose of improving antiviral activity.

[0026] The present invention also discloses a recombinant vector or a recombinant host cell containing the coding gene of the duck interferon alpha or duck interferon alpha mutant, wherein the recombinant vector is a recombinant expression vector or a recombinant cloning vector.

[0027] The transfer vector constructed by the present invention comprises:

[0028] (1) a vector pVL-DuIFN-α-O-M1 containing the gene sequence of a mutant (DuIFN-α-O-M1 mutant) after amino acid single-site mutation of DuIFN-α-O;

[0029] (2) vector pVL-DuIFN-α-O-M1-M2 containing the gene sequence of the mutant (DuIFN-α-O-M1-M2 mutant) after double amino acid mutation of DuIFN-α-O;

[0030] (3) Vectors pVL-DuIFN-α-O-M1-M2-M3, pVL-DuIFN-α-O-M1-M2-M3-M4, and pVL-DuIFN-α-O-M1-M2-M3-M4-M5 containing the gene sequences of mutants of DuIFN-α-O after multiple amino acid mutations (DuIFN-α-O-M1-M2-M3, DuIFN-α-O-M1-M2-M3-M4, and DuIFN-α-O-M1-M2-M3-M4-M5 mutants).

[0031] The recombinant baculovirus obtained by the present invention includes: recombinant Bombyx mori nuclear polyhedrosis virus rBmBacmid (DuIFN-α), rBmBacmid (DuIFN-α-O, DuIFN-α-O-M1, DuIFN-α-O-M1-M2, DuIFN-α-O-M1-M2-M3, DuIFN-α-O-M1-M2-M3-M4, DuIFN-α-O-M1-M2-M3-M4-M5).

[0032] The invention also discloses the use of the duck alpha interferon or duck alpha interferon mutant in preparing medicines or reagents for preventing or treating duck viral diseases.

[0033] Among them, the duck viral diseases include: at least one of: duckling viral hepatitis, duck plague (duck viral enteritis), gosling plague, Muscovy duck parvovirus disease, Muscovy duck gosling plague, duck epidemic hemorrhagic disease (black feather disease), duck hepatitis, duck viral encephalitis, duck reovirus infection, duck adenovirus infection, duck infectious bursal disease, duck paramyxovirus disease (duck Newcastle disease), duck coronavirus infection, and duck influenza.

[0034] The present invention also discloses a method for preparing the duck interferon-alpha or duck interferon-alpha mutant, comprising the following steps: (1) cloning the coding gene of the duck interferon-alpha or the duck interferon-alpha mutant into a baculovirus transfer vector to construct a recombinant transfer vector; (2) co-transfecting the recombinant transfer vector and baculovirus DNA into insect cells to obtain a recombinant baculovirus; (3) infecting insect cells or insect hosts with the recombinant baculovirus, culturing the infected insect cells or insect hosts to express corresponding proteins, and purifying the obtained proteins.

[0035] Wherein, the baculovirus transfer vector is selected from AcRP23-lacZ, AcRP6-SC, AcUWl-lacZ, BacPAK6, Bac to Pac, Bacmid, BlucBacII (pETL), p2Bac, p2Blue, p89B310, pAc360, pAc373, pAcAB3, pAcAB 4, PAcAS3, pAcC129, pAcC4, DZI, pAcGP67, pAcIEl, pAcJPl, pAcMLF2, pAcMLF7, pAcMLF8, pAcMPl, pAcMP2, pAcRP23, pAcRP 25. pAcRW4, pAcsMAG, pAcUWl, pAcUW21, pAcUW2A, pAcUW2B, pAcUW3, pAcUW31, pAcUW41, pAcUW42, pAcUW43, pAcUW51, pAcVC2, pAcVC 3. pAcYMl, pAcJcC5, pBacl, pBac2, pBlueBacIII, pBlueBaSWis, pEV55, mXIV, pIEINeo, pJVETL, pJVNhel, pJVP10, pJVrsMAG, pMBac, pP10, pPAKl, pPBac, pSHONEX 1.1, pSYN XIV VI+, pSYNVI+wp, pSYNXIV VI-, pVL1391, pVL 1392, pVL 1393, pVL941, pVL 945, pVL 985, pVTBac, pBM030, or pUAC-5;

[0036] The baculovirus is selected from the Bombyx mori baculovirus parent strain BmBacmid, BmNPV, AcMNPV, ApNPV, HaNPV, HzNPV, LdMNPV, MbMNPV, OpMNPV, SlMNPV, SeMNPV or SpltNPV;

[0037] The insect host is selected from the group consisting of Bombyx mori, Bombyx mandarina, Philosamia cynthia ricim, Dictyoploca japanica, Philosamia cynthiapryeri, Antheraea pernyi, Antheraea yamamai, Antheraea polyphymus, Atographa califorica, Ectropis obliqua, Mamestra brassicae, Spodoptera littoralis, Spodoptera frugiperda, TrisWoplusia ni, Thaumetopoea wilkinsoni, Heliothis armigera, Heliothis zea, Heliothis budworm, and assulta), tobacco armyworm (Heliothis virescens), oriental armyworm (Pseudaletia separata), or gypsy moth (Lymantriadispar);

[0038] Preferably, the baculovirus transfer vector is pVL1393; the baculovirus is the Bombyx mori baculovirus parent strain BmBacmid; and the insect host is the silkworm (Bombyx mori).

[0039] The infection refers to the recombinant baculovirus infecting 1-5-year-old insect larvae or pupae by ingestion or penetration through the epidermis; preferably, the recombinant silkworm baculovirus infects silkworm cells or puncture-inoculates 1-5-year-old silkworm larvae or pupae, and collects body fluids or tissue homogenates of silkworm larvae or pupae containing various duck α interferon genes 3-6 days after infection; wherein the pupae are optimally early tender pupae of 1-2 days old.

[0040] Among them, SEQ ID NO.1: original amino acid sequence of duck interferon alpha;

[0041] MPGPSAPPPPAIYSALALLLLLTPPANAFSCSPLRLHDSAFAWDSLQLLRNMAPSPTQPCPQQHAPCSFPDTLLDTNDTQQAAHTALHLLQHLFDTLSSPSTPAHWLHTARHDLLNQLQHHIHHLERCFPADAARLHRRGPRNLHLSINKYFGCIQHFLQNHTYSPCAWDHVRLEAHACFQRIHRLTRTMR*

[0042] SEQ ID NO.2: Duck α-interferon original nucleotide sequence;

[0043] ATGCCTGGGCCATCAGCCCCACCACCACCAGCCATCTACAGCGCCCTGGCCCTCCTGCTCCTCCTCACGCCTCCCGCCAACGCCTTCTCCTGCAGCCCCCTGCGCCTCCACGACAGCGCCTTCGCCTGGGACAGCCTCCAGCTCCTCCGCAACATGGCTCCCAGCCCCACACAGCCCTGCCCGCAGCAACACGCGCCTTGCTCCTTCCCGGACACCCTCCTGGACACCAACGACACGCAGCAAGCCGCACACACCGCCCTCCACCTCCTCCAACACCTCTTCGACACCCTCAGCAGCCCCAGCACCCCCGCGCACTGGCTCCACACCGCACGCCACGACCTCCTCAACCAGCTTCAGCACCACATCCACCACCTCGAGCGCTGCTTCCCAGCCGACGCCGCGCGCCTCCACAGGCGAGGGCCCCGCAACCTTCACCTCAGCATCAACAAGTACTTCGGCTGCATCCAACACTTCCTCCAGAACCACACCTACAGCCCCTGCGCATGGGACCACGTCCGCCTCGAGGCTCACGCCTGCTTCCAGCGCATCCACCGCCTCACCCGCACCATGCGCTAA

[0044] SEQ ID NO.3: DuIFN-α-O amino acid sequence;

[0045] MPGPSAPPPPAIYSALALLLLLTPPANAFSCSPLRLHDSAFAWDSLQLLRNMAPSPTQPCPQQHAPCSFPDTLLDTNDTQQAAHTALHLLQHLFDTLSSPSTPAHWLHTARHDLLNQLQHHIHHLERCFPADAARLHRRGPRNLHLSINKYFGCIQHFLQNHTYSPCAWDHVRLEAHACFQRIHRLTRTMR*

[0046] SEQ ID NO.4: DuIFN-α-O nucleotide sequence;

[0047] ATGCCTGGACCATCAGCTCCTCCACCGCCCGCCATCTACTCTGCTTTGGCCCTGTTGCTCCTGTTGACCCCTCCAGCTAACGCCTTCAGCTGCTCCCCGTTGAGACTCCACGACTCGGCTTTCGCCTGGGATAGTCTGCAACTCCTGAGAAATATGGCTCCTAGCCCAACTCAACCTTGCCCACAACAGCATGCTCCGTGTTCCTTCCCCGACACATTGCTCGACACCAACGATACACAACAGGCTGCCCACACTGCTCTCCACCTGTTGCAACACCTCTTCGACACTCTGTCATCTCCTTCAACCCCAGCTCACTGGCTGCACACTGCCAGACACGATCTCCTGAATCAATTGCAGCACCACATACACCACCTCGAAAGATGCTTCCCTGCTGACGCTGCCAGATTGCACAGAAGAGGTCCAAGAAACCTGCACTTGTCAATCAACAAATACTTCGGATGTATTCAACACTTCCTCCAGAACCACACCTACTCTCCATGCGCCTGGGATCACGTGAGACTGGAAGCTCACGCCTGTTTCCAGAGAATCCACAGATTGACAAGAACTATGAGATAA

[0048] SEQ ID NO.5: DuIFN-α-O-N51D mutant amino acid sequence;

[0049] MPGPSAPPPPAIYSALALLLLLTPPANAFSCSPLRLHDSAFAWDSLQLLRDMAPSPTQPCPQQHAPCSFPDTLLDTNDTQQAAHTALHLLQHLFDTLSSPSTPAHWLHTARHDLLNQLQHHIHHLERCFPADAARLHRRGPRNLHLSINKYFGCIQHFLQNHTYSPCAWDHVRLEAHACFQRIHRLTRTMR*

[0050] SEQ ID NO.6: Nucleotide sequence of DuIFN-α-O-N51D mutant;

[0051] ATGCCTGGACCATCAGCTCCTCCACCGCCCGCCATCTACTCTGCTTTGGCCCTGTTGCTCCTGTTGACCCCTCCAGCTAACGCCTTCAGCTGCTCCCCGTTGAGACTCCACGACTCGGCTTTCGCCTGGGATAGTCTGCAACTCCTGAGAGATATGGCTCCTAGCCCAACTCAACCTTGCCCACAACAGCATGCTCCGTGTTCCTTCCCCGACACATTGCTCGACACCAACGATACACAACAGGCTGCCCACACTGCTCTCCACCTGTTGCAACACCTCTTCGACACTCTGTCATCTCCTTCAACCCCAGCTCACTGGCTGCACACTGCCAGACACGATCTCCTGAATCAATTGCAGCACCACATACACCACCTCGAAAGATGCTTCCCTGCTGACGCTGCCAGATTGCACAGAAGAGGTCCAAGAAACCTGCACTTGTCAATCAACAAATACTTCGGATGTATTCAACACTTCCTCCAGAACCACACCTACTCTCCATGCGCCTGGGATCACGTGAGACTGGAAGCTCACGCCTGTTTCCAGAGAATCCACAGATTGACAAGAACTATGAGATAA

[0052] SEQ ID NO.7: Amino acid sequence of DuIFN-α-O-N51D-H108D mutant;

[0053] MPGPSAPPPPAIYSALALLLLLTPPANAFSCSPLRLHDSAFAWDSLQLLRDMAPSPTQPCPQQHAPCSFPDTLLDTNDTQQAAHTALHLLQHLFDTLSSPSTPAHWLDTARHDLLNQLQHHIHHLERCFPADAARLHRRGPRNLHLSINKYFGCIQHFLQNHTYSPCAWDHVRLEAHACFQRIHRLTRTMR*

[0054] SEQ ID NO.8: Nucleotide sequence of DuIFN-α-O-N51D-H108D mutant;

[0055] ATGCCTGGACCATCAGCTCCTCCACCGCCCGCCATCTACTCTGCTTTGGCCCTGTTGCTCCTGTTGACCCCTCCAGCTAACGCCTTCAGCTGCTCCCCGTTGAGACTCCACGACTCGGCTTTCGCCTGGGATAGTCTGCAACTCCTGAGAGATATGGCTCCTAGCCCAACTCAACCTTGCCCACAACAGCATGCTCCGTGTTCCTTCCCCGACACATTGCTCGACACCAACGATACACAACAGGCTGCCCACACTGCTCTCCACCTGTTGCAACACCTCTTCGACACTCTGTCATCTCCTTCAACCCCAGCTCACTGGCTGGACACTGCCAGACACGATCTCCTGAATCAATTGCAGCACCACATACACCACCTCGAAAGATGCTTCCCTGCTGACGCTGCCAGATTGCACAGAAGAGGTCCAAGAAACCTGCACTTGTCAATCAACAAATACTTCGGATGTATTCAACACTTCCTCCAGAACCACACCTACTCTCCATGCGCCTGGGATCACGTGAGACTGGAAGCTCACGCCTGTTTCCAGAGAATCCACAGATTGACAAGAACTATGAGATAA

[0056] SEQ ID NO.9: Amino acid sequence of DuIFN-α-O-P25L-N51D-Q80R-H108D-D170E mutant;

[0057] MPGPSAPPPPAIYSALALLLLLTPLANAFSCSPLRLHDSAFAWDSLQLLRDMAPSPTQPCPQQHAPCSFPDTLLDTNDTRQAAHTALHLLQHLFDTLSSPSTPAHWLDTARHDLLNQLQHHIHHLERCFPADAARLHRRGPRNLHLSINKYFGCIQHFLQNHTYSPCAWEHVRLEAHACFQRIHRLTRTMR*

[0058] SEQ ID NO.10: Nucleotide sequence of DuIFN-α-O-P25L-N51D-Q80R-H108D-D170E mutant:

[0059] ATGCCTGGACCATCAGCTCCTCCACCGCCCGCCATCTACTCTGCTTTGGCCCTGTTGCTCCTGTTGACCCCTCTAGCTAACGCCTTCAGCTGCTCCCCGTTGAGACTCCACGACTCGGCTTTCGCCTGGGATAGTCTGCAACTCCTGAGAGATATGGCTCCTAGCCCAACTCAACCTTGCCCACAACAGCATGCTCCGTGTTCCTTCCCCGACACATTGCTCGACACCAACGATACACGACAGGCTGCCCACACTGCTCTCCACCTGTTGCAACACCTCTTCGACACTCTGTCATCTCCTTCAACCCCAGCTCACTGGCTGGACACTGCCAGACACGATCTCCTGAATCAATTGCAGCACCACATACACCACCTCGAAAGATGCTTCCCTGCTGACGCTGCCAGATTGCACAGAAGAGGTCCAAGAAACCTGCACTTGTCAATCAACAAATACTTCGGATGTATTCAACACTTCCTCCAGAACCACACCTACTCTCCATGCGCCTGGGAACACGTGAGACTGGAAGCTCACGCCTGTTTCCAGAGAATCCACAGATTGACAAGAACTATGAGATAA

[0060] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0061] The present invention analyzes all duck interferon alpha amino acid sequences on NCBI, performs sequence alignment and signal peptide analysis, generates a common sequence as the original sequence, and performs codon optimization on the original sequence. Multiple pairs of primers are designed using the codon-optimized sequence as a template to perform amino acid single-site mutation, amino acid double-site mutation, and amino acid multi-site mutation using a fusion PCR method, thereby obtaining multiple duck interferon alpha mutants. The present invention utilizes a silkworm baculovirus expression system to express the duck interferon alpha mutant in a silkworm bioreactor, and the antiviral activity of the expressed duck interferon alpha mutant is greatly improved, and has a relatively obvious antiviral activity. The method of the present invention is simple in process, and a large amount of safe and reliable duck interferon alpha can be quickly obtained. The duck interferon alpha mutant of the present invention can be used to prepare a drug or reagent for preventing or treating duck viral diseases, which is of great significance to the development of duck breeding industry.

[0062] Definitions of terms involved in this invention:

[0063] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0064] The term "polynucleotide" or "nucleotide" means deoxyribonucleotides, deoxyribonucleosides, ribonucleosides or ribonucleotides and polymers thereof in single-stranded or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides, which have binding properties similar to reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specifically limited, the term also means oligonucleotide analogs, which include PNA (peptide nucleic acid), DNA analogs used in antisense technology (phosphorothioates, phosphoramidates, etc.). Unless otherwise specified, a specific nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (including, but not limited to, degenerate codon substitutions) and complementary sequences as well as explicitly specified sequences. In particular, degenerate codon substitutions can be achieved by generating sequences in which position 3 of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Cassol et al., (1992); Rossolini et al., Mol Cell. Probes 8:91-98 (1994)).

[0065] The term "homology" refers to sequence similarity to a natural nucleic acid sequence. "Homology" includes nucleotide sequences that are preferably 85% or higher, more preferably 90% or higher, and most preferably 95% or higher identical to the nucleotide sequence of the regulatory fragment of the present invention. Homology can be evaluated by the naked eye or by computer software. Using computer software, the homology between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the homology between related sequences.

[0066] The term "complementary" herein refers to two nucleotide sequences including antiparallel nucleotide sequences that can pair with each other after forming hydrogen bonds between the complementary base residues of the antiparallel nucleotide sequences. It is known in the art that the nucleotide sequences of two complementary chains are reverse complementary to each other when both sequences are viewed from the 5' to 3' direction. It is also known in the art that two sequences that can hybridize to each other under a given set of conditions do not necessarily have to be 100% completely complementary.

[0067] The term "stringent hybridization conditions" means conditions of low ionic strength and high temperature as known in the art. Typically, under stringent conditions, the detectable degree of hybridization of a probe to its target sequence is higher than the detectable degree of hybridization to other sequences (e.g., at least 2 times greater than background). Stringent hybridization conditions are sequence-dependent and will be different under different environmental conditions, with longer sequences specifically hybridizing at higher temperatures. By controlling the stringency of hybridization or washing conditions, a target sequence that is 100% complementary to the probe can be identified. For detailed guidance on nucleic acid hybridization, reference may be made to the relevant literature (Tijssen, Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Probes," Overview of principles of hybridization and the strategy of nucleic acid assays. 1993). More specifically, the stringent conditions are usually selected to be about 5-10°C lower than the thermal melting point (Tm) of the specific sequence at a specified ionic strength and pH. Tm is the temperature (under specified ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence in a state of equilibrium (because the target sequence is present in excess, 50% of the probes are occupied in a state of equilibrium at Tm). The stringent conditions may be the following conditions: wherein at pH 7.0 to 8.3, the salt concentration is less than about 1.0 M sodium ion concentration, typically about 0.01 to 1.0 M sodium ion concentration (or other salts), and the temperature is at least about 30°C for short probes (including but not limited to 10 to 50 nucleotides) and at least about 60°C for long probes (including but not limited to greater than 50 nucleotides). Stringent conditions can also be achieved by adding destabilizing agents such as formamide. For selective or specific hybridization, a positive signal can be at least two times the background hybridization, optionally 10 times the background hybridization. Exemplary stringent hybridization conditions can be as follows: 50% formamide, 5×SSC and 1% SDS, incubated at 42°C; or 5×SSC, 1% SDS, incubated at 65°C, washed in 0.2×SSC and washed in 0.1% SDS at 65°C. The wash can be performed for 5, 15, 30, 60, 120 minutes or longer.

[0068] The terms "mutation" and "mutant" have their ordinary meanings herein and refer to genetic, naturally occurring or introduced changes in nucleic acid or polypeptide sequences, and their meanings are the same as those generally known to those skilled in the art.

[0069] The term "host cell" or "recombinant host cell" means a cell comprising a polynucleotide of the present invention, regardless of the method used for insertion to produce a recombinant host cell, such as direct uptake, transduction, f-mating, or other methods known in the art. The exogenous polynucleotide may be maintained as a non-integrating vector such as a plasmid or may be integrated into the host genome.

[0070] The term "transfection" refers to the process by which eukaryotic cells acquire new genetic markers as a result of the incorporation of foreign DNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] Figure 1 The fluorescence images corresponding to the cytopathic ratios are shown in Figure 1. A, “-”: no cytopathic ratio; B, “±”: a few cytopathic ratios; C, “+”: 20% to 30% cytopathic ratios; D, “++”: 50% to 60% cytopathic ratios;

[0072] Figure 2 It is the double enzyme digestion identification of the recombinant plasmid pVL-DuIFN-α; wherein, M: DNA molecular mass standard; 1: recombinant plasmid pVL-DuIFN-α double enzyme digestion product; 2: negative control;

[0073] Figure 3 The figures show various proportions of cells showing fluorescence; A: cells show no fluorescence after high-concentration interferon completely inhibits VSV virus; B: a large number of cells in the cell control group show fluorescence when infected with VSV virus; C: fluorescence shown by some cells infected with VSV virus when low-concentration interferon cannot completely inhibit VSV virus. DETAILED DESCRIPTION

[0074] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, it should be understood that the embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications or replacements all fall within the protection scope of the present invention.

[0075] 1. Test materials and reagents

[0076] Transfer vector pVL1393, E. coli strain TOP10, BmN cells, VSV-GFP virus, were all preserved and provided by the Institute of Biotechnology, Chinese Academy of Agricultural Sciences; the experimental silkworm varieties Qiufeng×Baiyu were provided by the Institute of Sericulture, Jiangsu University of Science and Technology, and the parent virus BmBacmid DNA was constructed according to the method disclosed in the literature (patent number: ZL 201110142492.4, authorization date: 2013.01.23). SPF duck embryos were purchased from Beijing Merial Viton Laboratory Animal Technology Co., Ltd., restriction endonucleases and T4 DNA ligase were purchased from Promega, LA Taq DNA polymerase and other related reagents used in PCR reactions were purchased from TakaRa, liposomes were purchased from Invitrogen, and DMEM cell culture medium and fetal bovine serum were products of GIBCO. For the preparation methods of solutions and culture media, please refer to relevant reference books (Joseph et al., Molecular Cloning Laboratory Guide, 3rd Edition, 2002; Osber et al., Compiled Molecular Biology Guide, 1998; David L. Spector, Cell Experiment Guide, 2001); unless otherwise stated, percentages and parts are calculated by weight.

[0077] 2. Experimental methods

[0078] The PHA induction of duck peripheral blood mononuclear cells in the experimental method was carried out according to the method described by Huang Ailong et al. (Analysis of duck α-interferon gene expression and diversity [J]. Chinese Journal of Immunology, 2000, 16(12): 644-647).

[0079] The fusion PCR method used for site-directed mutagenesis in the experimental method was carried out according to the method described by Kuang Feiting et al. (A new method for vector construction: recombinant fusion PCR, Genomics and Applied Biology, 2012, Vol. 31, No. 6, pp. 634-639).

[0080] The interferon titer was calculated using the DEF / VSV*GFP system and the Reed-Muench method. The specific operation was performed according to the method described by Liu Xingjian et al. (Expression and biological activity detection of cat ω-like interferon in silkworm, Progress in Biotechnology, 2015, 5(6): 441-445) and Summers MD et al. (A manual of methods for baculovirus vectors and insect cell culture procedures [R]. Texas Agricultural Experiment Station, 1987). The standard for judging cytopathic effect was based on Figure 1 .

[0081] The best improvement plan in each implementation case serves as the comparison standard for the improvement plan of the next implementation case.

[0082] Example 1 Expression and detection of the original sequence of DuIFN-α in a silkworm bioreactor

[0083] 1. Experimental methods

[0084] 1.1 Obtaining the original sequence of duck interferon-α

[0085] 1.1.1 In vitro stimulation of duck peripheral blood mononuclear cells (PBMC)

[0086] Duck peripheral anticoagulated blood was collected aseptically, and peripheral blood mononuclear cells and red blood cells were separated using lymphocyte separation fluid. Duck T cells were further separated using the specific adsorption of nylon wool. 1 mL of 8×10 6 mL -1 The above PBMCs were incubated at 37°C for 1-2 hours to allow antigen presenting cells (APCs) to adhere to the wall. Separated T lymphocytes (8×10 6 mL / well) and red blood cells (4×10 6 mL / well) and phytohemagglutinin (PHA) at a final concentration of 5 μg / mL, incubated at 37°C for 4 h, and the cells were collected.

[0087] 1.1.2 Extraction of total cell RNA

[0088] Take a certain amount of collected cells in 1mL Trizol reagent, blow and mix thoroughly, and let it stand at room temperature for 10min; add 200μL chloroform, vortex and shake for 15s, let it stand at room temperature for 5min; centrifuge at 4℃, 12000rpm for 15min; take the upper aqueous phase into a new EP tube, add 500μL isopropanol, and bathe on ice for 10min; centrifuge at 4℃, 12000rpm for 10min; wash the precipitate once with 70% ethanol, and centrifuge at 4℃, 12000rpm for 5min; dry the precipitate at room temperature, add an appropriate amount of DEPC-treated double distilled water or deionized formamide, and store at -20℃ for later use.

[0089] 1.1.3 RT-PCR reaction

[0090] Synthesis of the first chain of cDNA. Take 2μL RNA (≤1μg), add 2μL DuIFN-α-RT, add 13.75μL DEPC treated water, mix thoroughly and react at 70℃ for 5min, quickly put on ice for 5min; add 5μL 5×M-MLV buffer, 1.25μL 10mMdNTPs, 1μL M-MLV-RT (200U) to make the final volume 25μL, mix and place at room temperature for 10min; react at 42℃ for 1h; inactivate RTase at 70℃ for 2min, and store at -20℃ for use. The specific primers used for reverse transcription are as follows.

[0091] DuIFN-α-RT: 5'-TTATCTCATAGTTCTTGTCA-3' (SEQ ID NO.11)

[0092] PCR amplification of target fragment. Introduce BamHI and EcoRI recognition sites in the upstream and downstream primers respectively, and introduce the Kozak sequence GCCAAC before the start codon, and amplify the target fragment according to the conventional PCR method. The primers are as follows:

[0093] F:5'-CGGGATCC GCCAAC ATGCCTGGACCATCAGCTC-3'(SEQ ID NO.12)

[0094] R:5'-CGGAATTCTTATCTCATAGTTCTTGTCAATC-3'(SEQ ID NO.13)

[0095] 1.1.4 Purification and recovery of DNA fragments from glass milk

[0096] Prepare 1% (w / v) agarose gel and perform electrophoresis on the PCR amplified products; place the agarose gel under ultraviolet light, quickly cut out the gel containing a single target nucleic acid band, put it into a 1.5mL centrifuge tube, weigh it, add three times the volume of 6MNaI, and put it in a 37℃ constant temperature incubator to melt; add 8μL Glassmilk to the completely melted solution, mix well, ice bath for 5min, and shake twice in the middle; centrifuge at 8000rpm for 10s, and discard the supernatant; add 800μL New Wash to wash, gently flick and centrifuge, repeat 2 times; discard the supernatant, put the centrifuge tube in a 37℃ constant temperature incubator to dry for 2-3min; after drying, add 20μL 0.1×TE to dissolve, mix to fully dissolve the DNA, centrifuge at 12000rpm for 5min, take the supernatant and use it for connection immediately, and store the rest at -20℃.

[0097] 1.2 Construction of recombinant baculovirus transfer vector

[0098] The target fragment of DuIFN-α recovered by enzyme digestion was connected to the transfer vector pVL1393 treated with BamHI and EcoRI double enzyme digestion. Use T4 DNA ligase at 16°C for overnight connection. Transform the ligation product into E. coli competent cell TOP10, select the colony for culture, extract the plasmid, identify the positive clone with BamHI and EcoRI double enzyme digestion, and send the correctly identified recombinant plasmid to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The result is shown in SEQ ID NO.1. The obtained recombinant plasmid is named: pVL-DuIFN-α.

[0099] 1.3 Obtaining recombinant silkworm baculovirus

[0100] According to the methods reported in the literature, the recovery, passage and screening of recombinant viruses of BmN cells were carried out. When the BmN cells were cultured to a cell monolayer of about 80%, the old culture medium was discarded, and the cells were washed three times with serum-free TC-100 culture medium, and 1.5 mL of FBS-free culture medium was added. 1 μg of Bombyx mori baculovirus parent strain BmBacmid DNA, 2 μg of recombinant transfer plasmid pVL-DuIFN-α and 5 μL of liposomes were added to a sterilized tube in sequence, and the volume was made up to 60 μL with sterile double distilled water, and the mixture was gently mixed. After standing for 15 minutes, the mixture was added dropwise to the culture bottle for co-transfection. After culturing at 27°C for 4 hours, 1.5 mL of serum-free culture medium and 300 μL of FBS were added. The cells were cultured at a constant temperature of 27°C for 4 to 5 days until the cells fell off and floated, and the cell culture fluid was collected to obtain the recombinant virus rBmBacmid (DuIFN-α) containing the target gene.

[0101] The purification and amplification method of recombinant silkworm baculovirus is as follows: inoculate an appropriate amount of cells (about 70-80%) in a 35mm small plate. After the cells adhere to the wall, remove the culture medium, dilute the collected cell culture medium to different concentrations, take 1mL and add it to the adherent cells and distribute it evenly. After infection at 27℃ for 1h, remove the infection liquid, melt 2% low melting point agarose gel in a 60℃ water bath, cool to 40℃ and mix it evenly with 2×TC-100 culture medium (containing 20% ​​FBS) preheated at 40℃, add 4mL gel to each plate, seal it with Parafilm after solidification, invert and culture at 27℃ for 3-5d, and observe it under a microscope. Select the empty plaques without polyhedrons, repeat the above steps, and obtain pure recombinant silkworm baculovirus rBmBacmid (DuIFN-α) after 2-3 rounds of purification.

[0102] The recombinant silkworm baculovirus rBmBacmid (DuIFN-α) was used to infect the normally growing BmN cells, and the supernatant was collected after culturing for 3 days. The supernatant contained a large amount of recombinant virus rBmBacmid (DuIFN-α).

[0103] 1.4 Expression of duck interferon-α in silkworm

[0104] The recombinant virus culture medium was heated to 10 5 PFU / head was injected into silkworms from the fifth instar, and cultured at 27°C and 70% to 80% humidity. In the late growth stage of silkworm larvae, DuIFN-α was efficiently expressed under the action of the polyhedral gene promoter. About 3.5 to 4 days after inoculation and infection, symptoms such as swollen body segments, abnormal behavior, and decreased appetite were observed in silkworm larvae. When the larvae were observed to be significantly reduced in size and stopped eating, hemolymph was collected and stored at -20°C for later use.

[0105] 1.5 Preparation of duck embryo fibroblasts (DEF) and detection of antiviral activity of duck interferon-α

[0106] Take 10-12 day old duck embryos, sterilize the eggshells in a clean bench, break the eggshells from the air chamber, take out the embryos, cut off the head and limbs, rinse with HBSS three times, put the embryos into a sterile conical flask and cut into small pieces, rinse with HBSS three times, add trypsin to digest at 37°C for 40 min, discard the trypsin and rinse with HBSS three times to remove the residual trypsin, add 30 mL of complete culture medium, blow and shake the conical flask to disperse the cells, filter with 8 layers of sterile gauze to obtain a cell suspension, count the cells and dilute the cells to 10 6 Pieces / mL.

[0107] The antiviral activity of duck interferon-α expressed in silkworm hemolymph was detected by microcytopathic inhibition assay on DEF / VSV*GFP system. 5 The silkworm blood lymph that had been ultrasonically broken and filtered for sterilization was inoculated into solutions of different dilutions with M199 culture medium containing 50mL / L fetal bovine serum. The diluted samples were inoculated into culture wells that were already covered with DEF cells at 100μL / well. At least 12 replicate wells were set up for each dilution and control silkworm blood. At the same time, a cell control group without silkworm blood lymph and VSV*GFP and a virus control group with VSV*GFP were set up. The samples were cultured at 37°C and 5% CO2 for 18 to 24 hours. 50 VSV*GFP virus was added into the culture wells from which the supernatant had been aspirated at 100 μL / well and cultured at 37°C and 5% CO2. When observed under an inverted fluorescence microscope, when a large number of cells in each well of the virus control group showed fluorescence, while the cells in the cell control group still grew well without fluorescence, it indicated that the control system was completely qualified and a comprehensive observation could be made.

[0108] 2. Experimental results

[0109] 2.1 Identification of recombinant transfer vectors

[0110] The recombinant transfer vector pVL-DuIFN-α was double-digested with BamHI and EcoRI, and two fragments were separated by 1% agarose gel electrophoresis. The small fragment was located between 500-750 bp, which was consistent with the size of the target gene fragment of 576 bp, and the large fragment was located above 8000 bp, which was consistent with the size of the pVL1393 fragment of 9607 bp. The plasmid identified by enzyme digestion was sent to Beijing Qingke Xinye Biotechnology Co., Ltd. for nucleotide sequencing. The results of alignment with MegaAlign showed that the sequence was consistent with the originally designed sequence, indicating that the duck α interferon gene had been successfully inserted into the pVL1393 transfer vector between BamHI and EcoRI.

[0111] 2.2 Obtaining duck interferon-α recombinant virus and detecting the recombinant product

[0112] The antiviral activity of duck interferon-α expressed by silkworm larvae was detected by microcytopathic inhibition method on DEF / VSV*GFP system. Under an inverted fluorescence microscope, it was observed that the cells in the cell control group grew well and no fluorescence appeared; the cells in the virus-infected control group were lesioned, and most cells showed fluorescence. The cells added with recombinant duck interferon-α protein had the ability to resist viral infection. According to the protective effect of duck interferon-α on DEF cells, the degree of cell lesions was observed. When green fluorescent cells appeared, the cells in this well were recorded as "+", and the titer of interferon was calculated according to the Reed-Muench method. The test results showed that its titer reached 8.70×10 5 U / mL.

[0113] Example 2 Expression and detection of optimized sequence of DuIFN-α in silkworm bioreactor

[0114] 1.1 Construction of duck interferon-α codon-optimized mutant gene

[0115] By analyzing all duck interferon alpha amino acid sequences on NCBI, a consensus sequence was obtained. After comparison with the original amino acid sequence of duck interferon alpha cloned in Example 1, the consensus sequence amino acid sequence was consistent with the original amino acid sequence, and its amino acid sequence was shown in SEQ ID NO.1, and the nucleotide sequence of its encoding gene was shown in SEQ ID NO.2.

[0116] The present invention utilizes OptimumGene TMThe technology was used to optimize the above-mentioned duck α-interferon consensus sequence gene, and the gene sequence was modified according to the codon preference of the bioreactor silkworm. The GC content, CpG dinucleotide content, codon preference, mRNA secondary structure, mRNA free energy stability, RNA instability motif, repetitive sequence and other related parameters that affect gene transcription efficiency, translation efficiency and protein folding were optimized, which is beneficial to improve the transcription efficiency and translation efficiency of the optimized gene in silkworms and keep the final translated protein sequence unchanged.

[0117] In order to improve the efficiency of translation initiation in the Bombyx mori baculovirus eukaryotic expression system, the Kozak sequence GCCAAC was added in front of the gene ATG. In addition, the restriction enzyme sites such as BamHI, EcoRI, and SmaI within the gene sequence were removed, and BamHI and EcoRI restriction enzyme sites were added upstream and downstream of the gene, so that they could be subsequently cloned into the eukaryotic transfer vector pVL1393.

[0118] The optimized sequence of the designed duck α interferon gene was artificially synthesized by a biotechnology company and named DuIFN-α-O. Its nucleotide sequence is shown in SEQ ID NO.4. The synthesized gene fragment was inserted into the pUC57 vector to form the plasmid pUC57-DuIFN, named pUC57-DuIFN-α-O.

[0119] 1.2 Construction of recombinant baculovirus transfer vector

[0120] The specific operation was carried out with reference to Example 1, and the plasmid with correct sequencing was named pVL-DuIFN-α-O.

[0121] 1.3 Acquisition, purification and amplification of recombinant silkworm baculovirus

[0122] The recovery and passage of BmN cells were carried out as described in Example 1, and the recombinant virus rBmBacmid (DuIFN-α-O) containing the target gene was obtained by co-transfection.

[0123] The purification and amplification methods of the recombinant Bombyx mori baculovirus were similar to those in Example 1, and pure recombinant Bombyx mori baculovirus rBmBacmid (DuIFN-α-O) was obtained by purification.

[0124] The recombinant Bombyx mori baculovirus rBmBacmid (DuIFN-α-O) was used to infect the normally growing BmN cells, and the supernatant was collected after culturing for 3 days. The supernatant contained a large amount of recombinant virus rBmBacmid (DuIFN-α-O).

[0125] 1.4 Expression of duck interferon-α mutant in silkworm

[0126] Same as Example 1.

[0127] 1.5 Preparation of duck embryo fibroblasts (DEF) and detection of antiviral activity of duck interferon-α

[0128] Same as Example 1.

[0129] 2. Experimental results

[0130] 2.1 Identification of recombinant transfer vectors

[0131] The recombinant transfer vector pVL-DuIFN-α-O was double-digested with BamHI and EcoRI, and two fragments were separated by 1% agarose gel electrophoresis. The small fragment was located between 500-750 bp, which was consistent with the size of the target gene fragment of 576 bp, and the large fragment was located above 8000 bp, which was consistent with the size of the pVL1393 fragment of 9607 bp. The plasmid identified by enzyme digestion was sent to Beijing Qingke Xinye Biotechnology Co., Ltd. for nucleotide sequencing. The results of alignment with MegaAlign showed that the sequence was consistent with the originally designed sequence, indicating that the duck α interferon mutant gene had been successfully inserted into the pVL1393 transfer vector between BamHI and EcoRI.

[0132] 2.2 Obtaining duck interferon-α recombinant virus and detecting the recombinant product

[0133] The antiviral activity of duck interferon-α expressed in silkworm larvae was detected by the microcytopathic inhibition method on the DEF / VSV*GFP system. Under an inverted fluorescence microscope, it was observed that the cells in the cell control group grew well and no fluorescence appeared; the cells in the virus-infected control group became diseased, and most of the cells showed fluorescence. The cells added with recombinant duck interferon-α protein had the ability to resist viral infection. According to the protective effect of duck interferon-α on DEF cells, the degree of cell disease was observed, and when green fluorescent cells appeared, the cells in this well were recorded as "+", and the titer of interferon was calculated according to the Reed-Muench method. The test results are listed in Table 1. The results of the antiviral activity assay showed that DuIFN-α-O expressed in silkworm larvae had a more significant antiviral activity, with a titer of 1.14×10 6 U / mL, achieving the expected effect, indicating that the optimization method on duck α interferon gene to improve the antiviral activity of DuIFN-α is feasible and effective.

[0134] Table 1 Test results of antiviral activity of duck interferon α mutants after optimization

[0135]

[0136] Example 3 Expression and detection of DuIFN-α-O after amino acid single-site mutation in silkworm bioreactor

[0137] 1. Experimental methods

[0138] 1.1 Construction of duck interferon-α mutant gene

[0139] The present invention uses the gene sequence of DuIFN-α-O as a template and designs multiple pairs of primers to perform site-directed mutagenesis on the sequence. The site-directed mutagenesis is performed using a fusion PCR method. The fusion PCR method is described in the aforementioned "2. Experimental methods".

[0140] The mutation sites are P2A, L19T, P25L, R35P, D38N, N51D, H64N, L73I, Q80R, D95K, H108D, Q117R, H123Y, R127Q, L136S, R142C, I148T, L159F, D170E or R182C; the resulting duck interferon α mutant is named DuIFN-α-O-M1 (P2A, L19T, P25L, R35P, D38N, N51D, H64N, L73I, Q80R, D95K, H108D, Q117R, H123Y, R127Q, L136S, R142C, I148T, L159F, D170E or R182C) mutant.

[0141] Primers required for single-site, double-site and multi-site mutation of amino acid in IFN-α-O nucleotide sequence:

[0142] (1) Upstream and downstream primers on both sides:

[0143] F:TCATACCGTCCCACCATCGGGGCGCGGATCC GCCAAC ATGCCTGGACCATCAG(SEQ ID NO.14)

[0144] R:GATCTGCAGCGGCCGCTCCGGAATTCTTATCTCATAGTTCTTGTCAATC(SEQ ID NO.15)

[0145] (2) Intermediate upstream and downstream primers

[0146] 1.P2A primers are primers at both ends of the entire chain

[0147] F1: TCATACCGTCCCACCATCGGGGCGCGGATCC GCCAAC ATGGCTGGACCATCAG(SEQ ID NO.16)

[0148] R1:GATCTGCAGCGGCCGCTCCGGAATTCTTATCTCATAGTTCTTGTCAATC(SEQ ID NO.17)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 2.<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0150] <h2 style=";text-align:left;direction:ltr"> F2:GCTTTGGCCCTGACGCTCCTGTTGAC(SEQ ID NO.18)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0151] <h2 style=";text-align:left;direction:ltr"> R2:GTCAACAGGAGCGTCAGGCCAAAGC(SEQ ID NO.19)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 3.<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0153] <h2 style=";text-align:left;direction:ltr"> F3:GTTGACCCCTCTAGCTAACGCCTTCAGC(SEQ ID NO.20)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0154] <h2 style=";text-align:left;direction:ltr"> R3:GCTGAAGGCGTTAGCTAGAGGGGTCAAC(SEQ ID NO.21)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 4.<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0156] <h2 style=";text-align:left;direction:ltr"> F4:GCTGCTCCCCGTTGCCACTCCACGACTC(SEQ ID NO.22)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0157] <h2 style=";text-align:left;direction:ltr"> R4:GAGTCGTGGAGTGGCAACGGGGAGCAGC(SEQ ID NO.23)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 5.<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0159] <h2 style=";text-align:left;direction:ltr"> F5:GAGACTCCACAACTCGGCTTTCGCCTGG(SEQ ID NO.24)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0160] <h2 style=";text-align:left;direction:ltr"> R5:CAGGCGAAAGCCGAGTTGTGGAGTCTCAACG(SEQ ID NO.25)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 6.<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0162] <h2 style=";text-align:left;direction:ltr"> F6:GCAACTCCTGAGAGATATGGCTCCTAGC(SEQ ID NO.26)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0163] <h2 style=";text-align:left;direction:ltr"> R6:GCTAGGAGCCATATCTCTCAGGAGTTGC(SEQ ID NO.27)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 7.<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0165] <h2 style=";text-align:left;direction:ltr"> F7:CACAACAGAATGCTCCGTGTTCCTTCCCCGAC(SEQ ID NO.28)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0166] <h2 style=";text-align:left;direction:ltr"> R7:GAACACGGAGCATTCTGTTGTGGGCAAGGTTG(SEQ ID NO.29)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 8.<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0168] F8:CGACACAATACTCGACACCAACGATACACAAC(SEQ ID NO.30)

[0169] R8:CGTTGGTGTCGAGTATTGTGTCGGGGAAGGAAC(SEQ ID NO.31) 9.

[0171] F9:CAACGATACACGACAGGCTGCCCACACTGC(SEQ ID NO.32)

[0172] R9:GTGGGCAGCCTGTCGTGTATCGTTGGTGTCG(SEQ ID NO.33) 10.

[0174] F10:GCAACACCTCTTCAAGACTCTGTCATCTC(SEQ ID NO.34)

[0175] R10:GAGATGACAGAGTCTTGAAGAGGTGTTGC(SEQ ID NO.35) 11.

[0177] F11:GCTCACTGGCTGGACACTGCCAGACACG(SEQ ID NO.36)

[0178] R11:CGTGTCTGGCAGTGTCCAGCCAGTGAGC(SEQ ID NO.37) 12.

[0180] F12:CGATCTCCTGAATCGATTGCAGCACCACA(SEQ ID NO.38)

[0181] R12:GCTGCAATCGATTCAGGAGATCGTGTCTG(SEQ ID NO.39) 13.

[0183] F13:GCAGCACCACATATACCACCTCGAAAGATGC(SEQ ID NO.40)

[0184] R13:GCATCTTTCGAGGTGGTATATGTGGTGCTGC(SEQ ID NO.41) 14.

[0186] F14:CATACACCACCTCGAACAATGCTTCCCTGC(SEQ ID NO.42)

[0187] R14:GCGTCAGCAGGGAAGCATTGTTCGAGGTGG(SEQ ID NO.43) 15.

[0189] F15:CGCTGCCAGATCGCACAGAAGAGGTCCAAG(SEQ ID NO.44)

[0190] R15:CTCTTCTGTGCGATCTGGCAGCGTCAGCAG(SEQ ID NO.45) 16.

[0192] F16:GAGGTCCATGCAACCTGCACTTGTCAATC(SEQ ID NO.46)

[0193] R16:GTGCAGGTTGCATGGACCTCTTCTGTGC(SEQ ID NO.47) 17.

[0195] F17:GCACTTGTCAACCAACAAATACTTCGGATG(SEQ ID NO.48)

[0196] R17:CGAAGTATTTGTTGGTTGACAAGTGCAGG(SEQ ID NO.49) 18.

[0198] F18:GTATTCAACACTTCTTCCAGAACCACAC(SEQ ID NO.50)

[0199] R18:GTGGTTCTGGAAGAAGTGTTGAATACATC(SEQ ID NO.51) 19.

[0201] F19:CATGCGCCTGGGAACACGTGAGACTGGAAG(SEQ ID NO.52)

[0202] R19:CTCACGTGTTCCCAGGCGCATGGAGAGTAG(SEQ ID NO.53) 20.

[0204] F20:GCTCACGCCTGTTTCCAGTGCATCCACAG(SEQ ID NO.54)

[0205] R20: CTGTGGATGCACTGGAAACAGGCGTGAGC (SEQ ID NO.55)

[0206] 1.2 Construction of recombinant baculovirus transfer vector

[0207] The specific operation is the same as that in Example 1. The plasmid with correct sequencing was named pVL-DuIFN-α-O-M1 (P2A, L19T, P25L, R35P, D38N, N51D, H64N, L73I, Q80R, D95K, H108D, Q117R, H123Y, R127Q, L136S, R142C, I148T, L159F, D170E or R182C).

[0208] 1.3 Acquisition, purification and amplification of recombinant silkworm baculovirus

[0209] Same as Example 1.

[0210] The recombinant virus rBmBacmid (DuIFN-α-O-M1) containing the target gene was obtained by co-transfection.

[0211] Pure recombinant silkworm baculovirus rBmBacmid (DuIFN-α-O-M1) was obtained by purification.

[0212] The recombinant silkworm baculovirus rBmBacmid (DuIFN-α-O-M1) was used to infect the normally growing BmN cells, and the supernatant was collected after culturing for 3 days. The supernatant contained a large amount of recombinant virus rBmBacmid (DuIFN-α-O-M1).

[0213] 1.4 Expression of duck interferon-α mutant in silkworm

[0214] Same as Example 1.

[0215] 1.5 Preparation of duck embryo fibroblasts (DEF) and detection of antiviral activity of duck interferon-α

[0216] Same as Example 1.

[0217] 2. Experimental results

[0218] 2.1 Identification of recombinant transfer vectors

[0219] The recombinant transfer vector pVL-DuIFN-α-O-M1 was double-digested with BamHI and EcoRI, and two fragments were separated by 1% agarose gel electrophoresis. The small fragment was located between 500-750 bp, which was consistent with the size of the target gene fragment of 576 bp, and the large fragment was located above 8000 bp, which was consistent with the size of the pVL1393 fragment of 9607 bp. The plasmid identified by enzyme digestion was sent to Beijing Qingke Xinye Biotechnology Co., Ltd. for nucleotide sequencing. The results of alignment with MegaAlign showed that the sequence was consistent with the originally designed sequence, indicating that the duck α interferon mutant gene had been successfully inserted into the pVL1393 transfer vector between BamHI and EcoRI.

[0220] 2.2 Obtaining duck interferon-α recombinant virus and detecting the recombinant product

[0221] The antiviral activity of duck interferon-α expressed by silkworm larvae was detected by microcytopathic inhibition method on DEF / VSV*GFP system. Under an inverted fluorescence microscope, it was observed that the cells in the cell control group grew well and no fluorescence appeared; the cells in the virus-infected control group were lesioned, and most cells showed fluorescence. The cells added with recombinant duck interferon-α protein had the ability to resist viral infection. According to the protective effect of duck interferon-α on DEF cells, the degree of cell lesion was observed. When green fluorescent cells appeared, the cells in this well were recorded as "+". The interferon titer was calculated according to the Reed-Muench method. The test results are listed in Table 2. The titers of all duck interferon-α mutants were 7.80×10 4 U / mL~3.16×10 6 U / mL, among which P25L, N51D, Q80R, H108D, L136S and D170E mutations, the titer of duck interferon α expressed was slightly higher than the titer measured by conservative sequence expression, while the titer of the remaining sites remained unchanged or even decreased after mutation, indicating that the mutations of these 6 sites are effective mutations, which can achieve the purpose of improving the antiviral activity of DuIFN-α. Among them, the DuIFN-α-O-N51D mutant has the strongest antiviral effect, its amino acid sequence is shown in SEQ ID NO.5, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.6.

[0222] Table 2 Detection results of antiviral activity of recombinant duck interferon α single site mutations

[0223]

[0224]

[0225] Example 4 Expression and detection of the DuIFN-α-O-M1 mutant after double amino acid mutation in a silkworm bioreactor

[0226] 1. Experimental methods

[0227] 1.1 Construction of duck interferon-α mutant gene

[0228] In view of the results of Example 3, it is determined that the mutations at some sites are effective mutations, which can achieve the purpose of improving the antiviral activity of the DuIFN-α-O mutant. Considering that the order of amino acid arrangement is the primary structure of the protein and determines the higher-level structure of the protein, and the positions of some mutation sites of the amino acid single-site mutations performed in Example 3 may be interrelated, an attempt is made to perform amino acid double-site mutations. The present invention combines the single mutation sites P25L, N51D, Q80R, H108D, L136S and D170E with improved antiviral activity in pairs to perform double-site mutations. The double-site mutation is based on the single-site mutation sequence obtained in Example 3, using it (DuIFN-α-O-M1) as a template, using corresponding primers (see Example 3 for details), and performing a second site-directed mutation by a fusion PCR method, thereby obtaining a target fragment of a double-site mutation. The fusion PCR method is shown in the aforementioned "2. Experimental Methods".

[0229] The double mutation sites were P25L-N51D, P25L-Q80R, P25L-H108D, P25L-L136S, P25L-D170E, N51D-Q80R, N51D-H108D, N51D-L136S, N51D-D170E, Q80R-H108D, Q80R-L136S, Q80R-D170E, H108D-L136S, H108D-D170E or L136S-D170E, a total of 15 combinations, and the duck interferon α mutations obtained The mutants were designated as DuIFN-α-O-M1-M2 (P25L-N51D, P25L-Q80R, P25L-H108D, P25L-L136S, P25L-D170E, N51D-Q80R, N51D-H108D, N51D-L136S, N51D-D170E, Q80R-H108D, Q80R-L136S, Q80R-D170E, H108D-L136S, H108D-D170E or L136S-D170E) mutants.

[0230] 1.2 Construction of recombinant baculovirus transfer vector

[0231] The method is the same as that of Example 1. The correctly sequenced plasmid was named pVL-DuIFN-α-O-M1-M2 (P25L-N51D, P25L-Q80R, P25L-H108D, P25L-L136S, P25L-D170E, N51D-Q80R, N51D-H108D, N51D-L136S, N51D-D170E, Q80R-H108D, Q80R-L136S, Q80R-D170E, H108D-L136S, H108D-D170E or L136S-D170E).

[0232] 1.3 Acquisition, purification and amplification of recombinant silkworm baculovirus

[0233] Same as Example 1.

[0234] The recombinant virus rBmBacmid (DuIFN-α-O-M1-M2) containing the target gene was obtained by co-transfection.

[0235] Pure recombinant silkworm baculovirus rBmBacmid (DuIFN-α-O-M1-M2) was obtained by purification.

[0236] The recombinant silkworm baculovirus rBmBacmid (DuIFN-α-O-M1-M2) was used to infect the normally growing BmN cells. After culturing for 3 days, the supernatant was collected. The supernatant contained a large amount of recombinant virus rBmBacmid (DuIFN-α-O-M1-M2).

[0237] 1.4 Expression of duck interferon-α mutant in silkworm

[0238] Same as Example 1.

[0239] 1.5 Preparation of duck embryo fibroblasts (DEF) and detection of antiviral activity of duck interferon-α

[0240] Same as Example 1.

[0241] 2. Experimental results

[0242] 2.1 Identification of recombinant transfer vectors

[0243] The recombinant transfer vector pVL-DuIFN-α-O-M1-M2 was double-digested with BamHI and EcoRI, and two fragments were separated by 1% agarose gel electrophoresis. The small fragment was located between 500-750 bp, which was consistent with the size of the target gene fragment of 576 bp, and the large fragment was located above 8000 bp, which was consistent with the size of the pVL1393 fragment of 9607 bp. The plasmid identified by enzyme digestion was sent to Beijing Qingke Xinye Biotechnology Co., Ltd. for nucleotide sequencing. The results of alignment with MegaAlign showed that the sequence was consistent with the originally designed sequence, indicating that the duck α interferon mutant gene had been successfully inserted into the pVL1393 transfer vector between BamHI and EcoRI.

[0244] 2.2 Obtaining duck interferon-α recombinant virus and detecting the recombinant product

[0245] The antiviral activity of duck interferon-α expressed by silkworm larvae was detected by microcytopathic inhibition method on DEF / VSV*GFP system. Under an inverted fluorescence microscope, it was observed that the cells in the cell control group grew well and no fluorescence appeared; the cells in the virus-infected control group were lesioned, and most cells showed fluorescence. The cells added with recombinant duck interferon-α protein had the ability to resist viral infection. According to the protective effect of duck interferon-α on DEF cells, the degree of cell lesion was observed. When green fluorescent cells appeared, the cells in this well were recorded as "+". The interferon titer was calculated according to the Reed-Muench method. The test results are listed in Table 3. The titers of all duck interferon-α mutants were 8.01×10 4 U / mL~4.50×10 6 U / mL, among which P25L-Q80R, N51D-H108D, and N51D-D170E three groups of double mutations, the titer of duck interferon α expressed was slightly higher than the titer measured by conservative sequence and single mutation sequence expression, while the titer of the other groups of sites remained unchanged or even decreased after mutation, indicating that the mutations of these three combined sites are effective mutations, which can achieve the purpose of improving the antiviral activity of DuIFN-α-O-M1 mutant. Among them, the DuIFN-α-O-N51D-H108D mutant has the strongest antiviral effect, its amino acid sequence is shown in SEQ ID NO.7, and its encoding gene nucleotide sequence is shown in SEQ ID NO.8.

[0246] Table 3 Detection results of antiviral activity of recombinant duck interferon α double site mutation

[0247]

[0248] Example 5 Expression and detection of DuIFN-α-O-M1-M2 mutant after amino acid multi-site mutation in silkworm bioreactor

[0249] 1. Experimental methods

[0250] 1.1 Construction of duck interferon-α mutant gene

[0251] In view of the results of Example 4, considering that the arrangement order of amino acids is the primary structure of the protein and determines the higher-level structure of the protein, it is speculated that it may be because the positions of some mutation sites of the amino acid single-site mutations are close to each other and related to each other, so amino acid multi-site mutations are attempted. The present invention combines the obtained double mutation sites with high titers to determine the third mutation site. The multi-site mutation is based on the double-site mutation sequence obtained in Example 4, using it (DuIFN-α-O-M1-M2) as a template, using corresponding primers (see Example 3 for details), and performing site-directed mutation of the third position by fusion PCR, thereby obtaining the target fragment of multi-site mutation. The fusion PCR method is shown in the aforementioned "2. Experimental method".

[0252] The following combinations were obtained: P25L-N51D-Q80R, P25L-Q80R-H108D, P25L-Q80R-D170E, P25L-N51D-H108D, N51D-Q80R-H108D, N51D-H108D-D170E, P25L-N51D-D170E, N51D-Q80R-D170E, P25L-N51D-Q80R-H108D, N51D-Q80R-H108D-D170E, P25L-N51D-H108D-D170E, or P25L-N51D-Q80R-H108D-D170E. The obtained duck interferon-α mutants were named DuIFN-α-O-M1-M2-M3 (P25L-N51D-Q80R, P25L-Q80R-H108D, P25L-Q80R-D170E, P25L-N51D-H108D, N51D-Q80R-H108D, N51D-H108D-D170E, P25L-N51D-D170E, N51D-Q80R-H108D, OR-D170E) or DuIFN-α-O-M1-M2-M3-M4 (P25L-N51D-Q80R-H108D, N51D-Q80R-H108D-D170E, P25L-N51D-H108D-D170E) or DuIFN-α-O-M1-M2-M3-M4-M5 (P25L-N51D-Q80R-H108D-D170E) mutants.

[0253] 1.2 Construction of recombinant baculovirus transfer vector

[0254] Same as Example 1.

[0255] The correctly sequenced plasmids were named pVL-DuIFN-α-O-M1-M2-M3 (P25L-N51D-Q80R, P25L-Q80R-H108D, P25L-Q80R-D170E, P25L-N51D-H108D, N51D-Q80R-H108D, N51D-H108D-D170E, P25L-N51D-D170E, N51D-Q80R- D170E) or pVL-DuIFN-α-O-M1-M2-M3-M4 (P25L-N51D-Q80R-H108D, N51D-Q80R-H108D-D170E, P2 5L-N51D-H108D-D170E) or pVL-DuIFN-α-O-M1-M2-M3-M4-M5 (P25L-N51D-Q80R-H108D-D170E).

[0256] 1.3 Acquisition, purification and amplification of recombinant silkworm baculovirus

[0257] See Example 1 for specific operations.

[0258] Recombinant virus rBm-Bacmid (DuIFN-α-O-M1-M2-M3 or DuIFN-α-O-M1-M2-M3-M4 or DuIFN-α-O-M1-M2-M3-M4-M5) containing the target gene was obtained by co-transfection.

[0259] Pure recombinant silkworm baculovirus rBm-Bacmid (DuIFN-α-O-M1-M2-M3 or DuIFN-α-O-M1-M2-M3-M4 or DuIFN-α-O-M1-M2-M3-M4-M5) was obtained by purification.

[0260] Normally growing BmN cells were infected with recombinant silkworm baculovirus rBm-Bacmid (DuIFN-α-O-M1-M2-M3 or DuIFN-α-O-M1-M2-M3-M4 or DuIFN-α-O-M1-M2-M3-M4-M5). The supernatant was collected after 3 days of culture. The supernatant contained a large amount of recombinant virus rBm-Bacmid (DuIFN-α-O-M1-M2-M3 or DuIFN-α-O-M1-M2-M3-M4 or DuIFN-α-O-M1-M2-M3-M4-M5).

[0261] 1.4 Expression of duck interferon-α mutant in silkworm

[0262] Same as Example 1.

[0263] 1.5 Preparation of duck embryo fibroblasts (DEF) and detection of antiviral activity of duck interferon-α

[0264] Same as Example 1.

[0265] 2. Experimental results

[0266] 2.1 Identification of recombinant transfer vectors

[0267] The recombinant transfer vector pVL-DuIFN-α-O-M1-M2-M3 or pVL-DuIFN-α-O-M1-M2-M3-M4 or pVL-DuIFN-α-O-M1-M2-M3-M4-M5 was double-digested with BamHI and EcoRI, and two fragments were separated by 1% agarose gel electrophoresis. The small fragment was located between 500-750 bp, which was consistent with the size of the target gene fragment of 576 bp, and the large fragment was located above 8000 bp, which was consistent with the size of the pVL1393 fragment of 9607 bp. The plasmids identified by enzyme digestion were sent to Beijing Qingke Xinye Biotechnology Co., Ltd. for nucleotide sequencing. The results of alignment with MegaAlign showed that the sequence was consistent with the originally designed sequence, indicating that the duck α interferon mutant gene had been successfully inserted into the pVL1393 transfer vector between BamHI and EcoRI.

[0268] 2.2 Obtaining duck interferon-α recombinant virus and detecting the recombinant product

[0269] The antiviral activity of duck interferon-α expressed by silkworm larvae was detected by microcytopathic inhibition method on DEF / VSV*GFP system. Under an inverted fluorescence microscope, it was observed that the cells in the cell control group grew well and no fluorescence appeared; the cells in the virus-infected control group became lesions, and most cells showed fluorescence. The cells added with recombinant duck interferon-α protein had the ability to resist viral infection. According to the protective effect of duck interferon-α on DEF cells, the degree of cell lesions was observed. When green fluorescent cells appeared, the cells in this well were recorded as "+". The interferon titer was calculated according to the Reed-Muench method. The test results are listed in Table 4. The titers of all duck interferon-α mutants were 2.51×10 5 U / mL~5.78×10 6 U / mL, among which the titer of duck interferon-α expressed after the five-site P25L-N51D-Q80R-H108D-D170E mutation was much higher than that of the conservative sequence, single mutation sequence, and double mutation sequence, which was 5.78×10 6U / mL. The titers of the other groups of sites remained unchanged or even decreased after mutation, indicating that the mutation of this combination of sites is an effective mutation, which can achieve the purpose of improving the antiviral activity of the DuIFN-α-O-M1-M2 mutant. The amino acid sequence of the DuIFN-α-O-P25L-N51D-Q80R-H108D-D170E mutant is shown in SEQ ID NO.9, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.10.

[0270] Table 4 Detection results of antiviral activity of recombinant duck interferon α multi-site mutations

[0271]

[0272] Sequence Listing <110> Jiangsu University of Science and Technology <120> Duck alpha interferon and its mutant, preparation method and application <160> 55 <170> SIPOSequenceListing 1.0 <210> 1 <211> 191 <212> PRT <213> Duck <400> 1 Met Pro Gly Pro Ser Ala Pro Pro Pro Pro Ala Ile Tyr Ser Ala Leu 1 5 10 15 Ala Leu Leu Leu Leu Leu Thr Pro Pro Ala Asn Ala Phe Ser Cys Ser 20 25 30 Pro Leu Arg Leu His Asp Ser Ala Phe Ala Trp Asp Ser Leu Gln Leu 35 40 45 Leu Arg Asn Met Ala Pro Ser Pro Thr Gln Pro Cys Pro Gln Gln His 50 55 60 Ala Pro Cys Ser Phe Pro Asp Thr Leu Leu Asp Thr Asn Asp Thr Gln 65 70 75 80 Gln Ala Ala His Thr Ala Leu His Leu Leu Gln His Leu Phe Asp Thr 85 90 95 Leu Ser Ser Pro Ser Thr Pro Ala His Trp Leu His Thr Ala Arg His 100 105 110 Asp Leu Leu Asn Gln Leu Gln His His Ile His His Leu Glu Arg Cys 115 120 125 Phe Pro Ala Asp Ala Ala Arg Leu His Arg Arg Gly Pro Arg Asn Leu 130 135 140 His Leu Ser Ile Asn Lys Tyr Phe Gly Cys Ile Gln His Phe Leu Gln 145 150 155 160 Asn His Thr Tyr Ser Pro Cys Ala Trp Asp His Val Arg Leu Glu Ala 165 170 175 His Ala Cys Phe Gln Arg Ile His Arg Leu Thr Arg Thr Met Arg 180 185 190 <210> 2 <211> 576 <212> DNA <213> Duck <400> 2 atgcctgggc catcagcccc accaccacca gccatctaca gcgccctggc cctcctgctc 60 ctcctcacgc ctcccgccaa cgccttctcc tgcagccccc tgcgcctcca cgacagcgcc 120 ttcgcctggg acagcctcca gctcctccgc aacatggctc ccagccccac acagccctgc 180 ccgcagcaac acgcgccttg ctccttcccg gacaccctcc tggacaccaa cgacacgcag 240 caagccgcac acaccgccct ccacctcctc caacacctct tcgacaccct cagcagcccc 300 agcacccccg cgcactggct ccacaccgca cgccacgacc tcctcaacca gcttcagcac 360 cacatccacc acctcgagcg ctgcttccca gccgacgccg cgcgcctcca caggcgaggg 420 ccccgcaacc ttcacctcag catcaacaag tacttcggct gcatccaaca cttcctccag 480 aaccacacct acagcccctg cgcatgggac cacgtccgcc tcgaggctca cgcctgcttc 540 cagcgcatcc accgcctcac ccgcaccatg cgctaa 576 <210> 3 <211> 191 <212> PRT <213> Duck <400> 3 Met Pro Gly Pro Ser Ala Pro Pro Pro Pro Ala Ile Tyr Ser Ala Leu 1 5 10 15 Ala Leu Leu Leu Leu Leu Thr Pro Pro Ala Asn Ala Phe Ser Cys Ser 20 25 30 Pro Leu Arg Leu His Asp Ser Ala Phe Ala Trp Asp Ser Leu Gln Leu 35 40 45 Leu Arg Asn Met Ala Pro Ser Pro Thr Gln Pro Cys Pro Gln Gln His 50 55 60 Ala Pro Cys Ser Phe Pro Asp Thr Leu Leu Asp Thr Asn Asp Thr Gln 65 70 75 80 Gln Ala Ala His Thr Ala Leu His Leu Leu Gln His Leu Phe Asp Thr 85 90 95 Leu Ser Ser Pro Ser Thr Pro Ala His Trp Leu His Thr Ala Arg His 100 105 110 Asp Leu Leu Asn Gln Leu Gln His His Ile His His Leu Glu Arg Cys 115 120 125 Phe Pro Ala Asp Ala Ala Arg Leu His Arg Arg Gly Pro Arg Asn Leu 130 135 140 His Leu Ser Ile Asn Lys Tyr Phe Gly Cys Ile Gln His Phe Leu Gln 145 150 155 160 Asn His Thr Tyr Ser Pro Cys Ala Trp Asp His Val Arg Leu Glu Ala 165 170 175 His Ala Cys Phe Gln Arg Ile His Arg Leu Thr Arg Thr Met Arg 180 185 190 <210> 4 <211> 576 <212> DNA <213> Duck <400> 4 atgcctggac catcagctcc tccaccgccc gccatctact ctgctttggc cctgttgctc 60 ctgttgaccc ctccagctaa cgccttcagc tgctccccgt tgagactcca cgactcggct 120 ttcgcctggg atagtctgca actcctgaga aatatggctc ctagcccaac tcaaccttgc 180 ccacaacagc atgctccgtg ttccttcccc gacacattgc tcgacaccaa cgatacacaa 240 caggctgccc acactgctct ccacctgttg caacacctct tcgacactct gtcatctcct 300 tcaaccccag ctcactggct gcacactgcc agacacgatc tcctgaatca attgcagcac 360 cacatacacc acctcgaaag atgcttccct gctgacgctg ccagattgca cagaagaggt 420 ccaagaaacc tgcacttgtc aatcaacaaa tacttcggat gtattcaaca cttcctccag 480 aaccacacct actctccatg cgcctgggat cacgtgagac tggaagctca cgcctgtttc 540 cagagaatcc acagattgac aagaactatg agataa 576 <210> 5 <211> 191 <212> PRT <213> Duck <400> 5 Met Pro Gly Pro Ser Ala Pro Pro Pro Pro Ala Ile Tyr Ser Ala Leu 1 5 10 15 Ala Leu Leu Leu Leu Leu Thr Pro Pro Ala Asn Ala Phe Ser Cys Ser 20 25 30 Pro Leu Arg Leu His Asp Ser Ala Phe Ala Trp Asp Ser Leu Gln Leu 35 40 45 Leu Arg Asp Met Ala Pro Ser Pro Thr Gln Pro Cys Pro Gln Gln His 50 55 60 Ala Pro Cys Ser Phe Pro Asp Thr Leu Leu Asp Thr Asn Asp Thr Gln 65 70 75 80 Gln Ala Ala His Thr Ala Leu His Leu Leu Gln His Leu Phe Asp Thr 85 90 95 Leu Ser Ser Pro Ser Thr Pro Ala His Trp Leu His Thr Ala Arg His 100 105 110 Asp Leu Leu Asn Gln Leu Gln His His Ile His His Leu Glu Arg Cys 115 120 125 Phe Pro Ala Asp Ala Ala Arg Leu His Arg Arg Gly Pro Arg Asn Leu 130 135 140 His Leu Ser Ile Asn Lys Tyr Phe Gly Cys Ile Gln His Phe Leu Gln 145 150 155 160 Asn His Thr Tyr Ser Pro Cys Ala Trp Asp His Val Arg Leu Glu Ala 165 170 175 His Ala Cys Phe Gln Arg Ile His Arg Leu Thr Arg Thr Met Arg 180 185 190 <210> 6 <211> 576 <212> DNA <213> Duck <400> 6 atgcctggac catcagctcc tccaccgccc gccatctact ctgctttggc cctgttgctc 60 ctgttgaccc ctccagctaa cgccttcagc tgctccccgt tgagactcca cgactcggct 120 ttcgcctggg atagtctgca actcctgaga gatatggctc ctagcccaac tcaaccttgc 180 ccacaacagc atgctccgtg ttccttcccc gacacattgc tcgacaccaa cgatacacaa 240 caggctgccc acactgctct ccacctgttg caacacctct tcgacactct gtcatctcct 300 tcaaccccag ctcactggct gcacactgcc agacacgatc tcctgaatca attgcagcac 360 cacatacacc acctcgaaag atgcttccct gctgacgctg ccagattgca cagaagaggt 420 ccaagaaacc tgcacttgtc aatcaacaaa tacttcggat gtattcaaca cttcctccag 480 aaccacacct actctccatg cgcctgggat cacgtgagac tggaagctca cgcctgtttc 540 cagagaatcc acagattgac aagaactatg agataa 576 <210> 7 <211> 191 <212> PRT <213> Duck <400> 7 Met Pro Gly Pro Ser Ala Pro Pro Pro Pro Ala Ile Tyr Ser Ala Leu 1 5 10 15 Ala Leu Leu Leu Leu Leu Thr Pro Pro Ala Asn Ala Phe Ser Cys Ser 20 25 30 Pro Leu Arg Leu His Asp Ser Ala Phe Ala Trp Asp Ser Leu Gln Leu 35 40 45 Leu Arg Asp Met Ala Pro Ser Pro Thr Gln Pro Cys Pro Gln Gln His 50 55 60 Ala Pro Cys Ser Phe Pro Asp Thr Leu Leu Asp Thr Asn Asp Thr Gln 65 70 75 80 Gln Ala Ala His Thr Ala Leu His Leu Leu Gln His Leu Phe Asp Thr 85 90 95 Leu Ser Ser Pro Ser Thr Pro Ala His Trp Leu Asp Thr Ala Arg His 100 105 110 Asp Leu Leu Asn Gln Leu Gln His His Ile His His Leu Glu Arg Cys 115 120 125 Phe Pro Ala Asp Ala Ala Arg Leu His Arg Arg Gly Pro Arg Asn Leu 130 135 140 His Leu Ser Ile Asn Lys Tyr Phe Gly Cys Ile Gln His Phe Leu Gln 145 150 155 160 Asn His Thr Tyr Ser Pro Cys Ala Trp Asp His Val Arg Leu Glu Ala 165 170 175 His Ala Cys Phe Gln Arg Ile His Arg Leu Thr Arg Thr Met Arg 180 185 190 <210> 8 <211> 576 <212> DNA <213> Duck <400> 8 atgcctggac catcagctcc tccaccgccc gccatctact ctgctttggc cctgttgctc 60 ctgttgaccc ctccagctaa cgccttcagc tgctccccgt tgagactcca cgactcggct 120 ttcgcctggg atagtctgca actcctgaga gatatggctc ctagcccaac tcaaccttgc 180 ccacaacagc atgctccgtg ttccttcccc gacacattgc tcgacaccaa cgatacacaa 240 caggctgccc acactgctct ccacctgttg caacacctct tcgacactct gtcatctcct 300 tcaaccccag ctcactggct ggacactgcc agacacgatc tcctgaatca attgcagcac 360 cacatacacc acctcgaaag atgcttccct gctgacgctg ccagattgca cagaagaggt 420 ccaagaaacc tgcacttgtc aatcaacaaa tacttcggat gtattcaaca cttcctccag 480 aaccacacct actctccatg cgcctgggat cacgtgagac tggaagctca cgcctgtttc 540 cagagaatcc acagattgac aagaactatg agataa 576 <210> 9 <211> 191 <212> PRT <213> Duck <400> 9 Met Pro Gly Pro Ser Ala Pro Pro Pro Pro Ala Ile Tyr Ser Ala Leu 1 5 10 15 Ala Leu Leu Leu Leu Leu Thr Pro Leu Ala Asn Ala Phe Ser Cys Ser 20 25 30 Pro Leu Arg Leu His Asp Ser Ala Phe Ala Trp Asp Ser Leu Gln Leu 35 40 45 Leu Arg Asp Met Ala Pro Ser Pro Thr Gln Pro Cys Pro Gln Gln His 50 55 60 Ala Pro Cys Ser Phe Pro Asp Thr Leu Leu Asp Thr Asn Asp Thr Arg 65 70 75 80 Gln Ala Ala His Thr Ala Leu His Leu Leu Gln His Leu Phe Asp Thr 85 90 95 Leu Ser Ser Pro Ser Thr Pro Ala His Trp Leu Asp Thr Ala Arg His 100 105 110 Asp Leu Leu Asn Gln Leu Gln His His Ile His His Leu Glu Arg Cys 115 120 125 Phe Pro Ala Asp Ala Ala Arg Leu His Arg Arg Gly Pro Arg Asn Leu 130 135 140 His Leu Ser Ile Asn Lys Tyr Phe Gly Cys Ile Gln His Phe Leu Gln 145 150 155 160 Asn His Thr Tyr Ser Pro Cys Ala Trp Glu His Val Arg Leu Glu Ala 165 170 175 His Ala Cys Phe Gln Arg Ile His Arg Leu Thr Arg Thr Met Arg 180 185 190 <210> 10 <211> 576 <212> DNA <213> Duck <400> 10 atgcctggac catcagctcc tccaccgccc gccatctact ctgctttggc cctgttgctc 60 ctgttgaccc ctctagctaa cgccttcagc tgctccccgt tgagactcca cgactcggct 120 ttcgcctggg atagtctgca actcctgaga gatatggctc ctagcccaac tcaaccttgc 180 ccacaacagc atgctccgtg ttccttcccc gacacattgc tcgacaccaa cgatacacga 240 caggctgccc acactgctct ccacctgttg caacacctct tcgacactct gtcatctcct 300 tcaaccccag ctcactggct ggacactgcc agacacgatc tcctgaatca attgcagcac 360 cacatacacc acctcgaaag atgcttccct gctgacgctg ccagattgca cagaagaggt 420 ccaagaaacc tgcacttgtc aatcaacaaa tacttcggat gtattcaaca cttcctccag 480 aaccacacct actctccatg cgcctgggaa cacgtgagac tggaagctca cgcctgtttc 540 cagagaatcc acagattgac aagaactatg agataa 576 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <400> 11 ttatctcata gttcttgtca 20 <210> 12 <211> 33 <212> DNA <213> Artificial Sequence <400> 12 cgggatccgc caacatgcct ggaccatcag ctc 33 <210> 13 <211> 31 <212> DNA <213> Artificial Sequence <400> 13 cggaattctt atctcatagt tcttgtcaat c 31 <210> 14 <211> 52 <212> DNA <213> Artificial Sequence <400> 14 tcataccgtc ccaccatcgg gcgcggatcc gccaacatgc ctggaccatc ag 52 <210> 15 <211> 49 <212> DNA <213> Artificial Sequence <400> 15 gatctgcagc ggccgctccg gaattctttat ctcatagttc ttgtcaatc 49 <210> 16 <211> 52 <212> DNA <213> Artificial Sequence <400> 16 tcataccgtc ccaccatcgg gcgcggatcc gccaacatgg ctggaccatc ag 52 <210> 17 <211> 49 <212> DNA <213> Artificial Sequence <400> 17 gatctgcagc ggccgctccg gaattctttat ctcatagttc ttgtcaatc 49 <210> 18 <211> 26 <212> DNA <213> Artificial Sequence <400> 18 gctttggccc tgacgctcct gttgac 26 <210> 19 <211> 26 <212> DNA <213> Artificial Sequence <400> 19 gtcaacagga gcgtcagggc caaagc 26 <210> 20 <211> 28 <212> DNA <213> Artificial Sequence <400> 20 gttgacccct ctagctaacg ccttcagc 28 <210> twenty one <211> 28 <212> DNA <213> Artificial Sequence <400> twenty one gctgaaggcg ttagctagag gggtcaac 28 <210> twenty two <211> 28 <212> DNA <213> Artificial Sequence <400> twenty two gctgctcccc gttgccactc cacgactc 28 <210> twenty three <211> 28 <212> DNA <213> Artificial Sequence <400> twenty three gagtcgtgga gtggcaacgg ggagcagc 28 <210> twenty four <211> 28 <212> DNA <213> Artificial Sequence <400> twenty four gagactccac aactcggctttcgcctgg 28 <210> 25 <211> 31 <212> DNA <213> Artificial Sequence <400> 25 caggcgaaag ccgagttgtg gagtctcaac g 31 <210> 26 <211> 28 <212> DNA <213> Artificial Sequence <400> 26 gcaactcctg agagatatgg ctcctagc 28 <210> 27 <211> 28 <212> DNA <213> Artificial Sequence <400> 27 gctaggagcc atatctctca ggagttgc 28 <210> 28 <211> 32 <212> DNA <213> Artificial Sequence <400> 28 cacaacagaa tgctccgtgt tccttccccg ac 32 <210> 29 <211> 32 <212> DNA <213> Artificial Sequence <400> 29 gaacacggag cattctgttg tgggcaaggt tg 32 <210> 30 <211> 32 <212> DNA <213> Artificial Sequence <400> 30 cgacacaata ctcgacacca acgatacaca ac 32 <210> 31 <211> 33 <212> DNA <213> Artificial Sequence <400> 31 cgttggtgtc gagtattgtg tcggggaagg aac 33 <210> 32 <211> 30 <212> DNA <213> Artificial Sequence <400> 32 caacgataca cgacaggctg cccacactgc 30 <210> 33 <211> 31 <212> DNA <213> Artificial Sequence <400> 33 gtgggcagcc tgtcgtgtat cgttggtgtc g 31 <210> 34 <211> 29 <212> DNA <213> Artificial Sequence <400> 34 gcaacacctc ttcaagactc tgtcatctc 29 <210> 35 <211> 29 <212> DNA <213> Artificial Sequence <400> 35 gagatgacag agtcttgaag aggtgttgc 29 <210> 36 <211> 28 <212> DNA <213> Artificial Sequence <400> 36 gctcactggc tggacactgc cagacacg 28 <210> 37 <211> 28 <212> DNA <213> Artificial Sequence <400> 37 cgtgtctggc agtgtccagc cagtgagc 28 <210> 38 <211> 29 <212> DNA <213> Artificial Sequence <400> 38 cgatctcctg aatcgattgc agcaccaca 29 <210> 39 <211> 29 <212> DNA <213> Artificial Sequence <400> 39 gctgcaatcg attcaggaga tcgtgtctg 29 <210> 40 <211> 31 <212> DNA <213> Artificial Sequence <400> 40 gcagcaccac atataccacc tcgaaagatg c 31 <210> 41 <211> 31 <212> DNA <213> Artificial Sequence <400> 41 gcatctttcg aggtggtata tgtggtgctg c 31 <210> 42 <211> 30 <212> DNA <213> Artificial Sequence <400> 42 catacaccacctcgaacaatgcttccctgc 30 <210> 43 <211> 30 <212> DNA <213> Artificial Sequence <400> 43 gcgtcagcag ggaagcattg ttcgaggtgg 30 <210> 44 <211> 30 <212> DNA <213> Artificial Sequence <400> 44 cgctgccaga tcgcacagaa gaggtccaag 30 <210> 45 <211> 30 <212> DNA <213> Artificial Sequence <400> 45 ctcttctgtg cgatctggca gcgtcagcag 30 <210> 46 <211> 29 <212> DNA <213> Artificial Sequence <400> 46 gaggtccatg caacctgcac ttgtcaatc 29 <210> 47 <211> 28 <212> DNA <213> Artificial Sequence <400> 47 gtgcaggttg catggacctc ttctgtgc 28 <210> 48 <211> 30 <212> DNA <213> Artificial Sequence <400> 48 gcacttgtca accaacaaat acttcggatg 30 <210> 49 <211> 29 <212> DNA <213> Artificial Sequence <400> 49 cgaagtattt gttggttgac aagtgcagg 29 <210> 50 <211> 28 <212> DNA <213> Artificial Sequence <400> 50 gtattcaaca cttcttccag aaccacac 28 <210> 51 <211> 29 <212> DNA <213> Artificial Sequence <400> 51 gtggttctgg aagaagtgtt gaatacatc 29 <210> 52 <211> 30 <212> DNA <213> Artificial Sequence <400> 52 catgcgcctg ggaacacgtg agactggaag 30 <210> 53 <211> 30 <212> DNA <213> Artificial Sequence <400> 53 ctcacgtgtt cccaggcgca tggagagtag 30 <210> 54 <211> 29 <212> DNA <213> Artificial Sequence <400> 54 gctcacgcct gtttccagtg catccacag 29 <210> 55 <211> 29 <212> DNA <213> Artificial Sequence <400> 55 ctgtggatgc actggaaaca ggcgtgagc 29

Claims

1. A duck interferon-α mutant, characterized in that: The amino acid sequence of duck interferon is subjected to Q80R amino acid single point mutation to obtain the mutant; wherein the amino acid sequence of duck interferon is shown in SEQ ID NO.

1.

2. A duck interferon-α mutant, characterized in that: The amino acid sequence of duck interferon is subjected to Q80R-D170E amino acid double-site mutation to obtain the mutant; wherein the amino acid sequence of duck interferon is shown in SEQ ID NO.

1.

3. A recombinant vector containing the gene encoding the duck interferon-α mutant according to claim 1 or 2.

4. A recombinant host cell containing the gene encoding the duck interferon-α mutant according to claim 1 or 2.

5. Use of the gene encoding the duck interferon-α mutant according to claim 1 or 2 in the preparation of a drug or reagent for treating duck VSV virus disease.

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