CN114853875B - Duck alpha interferon, mutant thereof, preparation method and application - Google Patents

Duck alpha interferon, mutant thereof, preparation method and application Download PDF

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CN114853875B
CN114853875B CN202210479170.7A CN202210479170A CN114853875B CN 114853875 B CN114853875 B CN 114853875B CN 202210479170 A CN202210479170 A CN 202210479170A CN 114853875 B CN114853875 B CN 114853875B
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易咏竹
王朋
沈兴家
唐顺明
胡小元
魏珍珍
曾振
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Jiangsu University of Science and Technology
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Abstract

The invention discloses duck alpha interferon, a mutant thereof, a preparation method and application thereof. The invention optimizes the duck alpha interferon and carries out amino acid single-site mutation, double-site mutation and multi-site mutation on the basis of the duck alpha interferon to obtain a plurality of duck alpha interferon mutants with improved antiviral activity. The invention also relates to application of the duck alpha interferon mutant in preparing a medicament or a reagent for preventing or treating duck viral diseases, and belongs to the field of preparation and application of the duck alpha interferon mutant. The invention utilizes the silkworm baculovirus expression system to greatly improve the antiviral activity of the duck alpha interferon mutant expressed in the silkworm bioreactor, can be used for preparing medicines or reagents for preventing or treating duck viral diseases, and has the effects of defending and inhibiting viral infectious diseases with great threat in the duck breeding industry.

Description

Duck alpha interferon, mutant thereof, preparation method and application
The application is a divisional application of the following cases, and the application date of the original application is: 2019, 6 months and 13 days, the original application number: 201910510526.7, name of the original application: duck alpha interferon and mutant, preparation method and application thereof.
Technical Field
The invention relates to a duck alpha interferon mutant and a method for preparing the duck alpha interferon mutant by using a silkworm baculovirus expression system; the invention also relates to application of the duck alpha interferon mutant in preparing a medicament or a reagent for preventing or treating duck viral diseases, and belongs to the field of preparation and application of the duck alpha interferon mutant.
Background
The interferon is a cytokine with broad-spectrum antiviral, anti-parasitic bacteria in cells, anti-tumor, immunity regulating and other wide biological activities. The IFN family of proteins is classified into type i, type ii and type iii interferons according to the sequence, chromosomal location and receptor specificity of the genes encoding them. Type I interferons include IFN- α, IFN- β, IFN- ω, IFN- δ, IFN- ε, IFN- ζ, IFN- τ, and the like, predominantly IFN- α and IFN- β in mammals. The I-type interferon has strong antiviral activity, inhibits proliferation of viruses mainly by interfering with replication of the viruses, and also has the functions of resisting tumor and regulating immunity. Type II interferon has only one member of IFN-gamma, also called immunity interferon, and has the main function of activating macrophage to kill microbe. Type III interferons are newly discovered cytokines, including λ1 (IL-29), λ2 (IL-28 a), and λ3 (IL-28 b). Type III interferons are closely related to type I interferons, but have specific physiological functions, such as stimulating activation and expression of major histocompatibility complex (major histocompatibility complex, MHC) molecules, modulating innate and acquired immunity, and the like. Because of the broad-spectrum antiviral and antitumor activities and the powerful immunoregulatory effects, the interferon has become one of research hotspots in related fields such as virology, cytology, molecular biology, clinical medicine, immunology, oncology and the like.
Interferon was first found in birds, but duck interferon molecular levels were studied later than in humans and other mammals, and relatively less was studied in terms of duck interferon than in chickens. In recent years, infectious diseases such as influenza, newcastle disease, infectious bronchitis, infectious laryngotracheitis, infectious bursal disease, duck plague, duck hepatitis, marek and the like caused by poultry virus infection are increasingly severe, huge economic losses are brought to the poultry industry each year, the healthy development of the poultry industry is severely restricted, and how to effectively prevent and treat the viral infectious diseases of poultry is always an important point in the prevention and treatment research of the poultry diseases.
The duck interferon-alpha gene was obtained by cloning and screening by German researchers Schultz equal to the reported CHIFN-alpha gene as a probe in 1995 at the earliest. The domestic researchers report the alpha 0 interferon genes of Beijing ducks in summer and spring 2000, other researchers report the alpha interferon genes of various ducks such as Muscovy ducks, shaoxing ducks, sheldrake and the like subsequently, in addition, the researchers report the resistance of the recombinant duck alpha interferon to various viruses successively, and Schultz and the like prove the effect of the recombinant interferon on resisting vesicular stomatitis virus, influenza A virus, duck hepatitis virus and newcastle disease virus; zhou Xue and the like show a certain capability of resisting duck plague virulent infection after the cherry valley ducks are injected by adopting plasmids with duck alpha interferon genes in 2007; the recombinant duck alpha interferon is used for treating 2-day-old ducks in 2018 of Pei Gao et al to study the resistance to the highly pathogenic avian influenza virus H5N1, and the result shows that the death rate of the ducks in the test group is reduced from 60% to 10% compared with the virus control group, which indicates that the recombinant duck alpha interferon has the resistance to the avian influenza virus H5N1 infected by the 2-day-old ducks. Chen cloning IFN-alpha gene from Sichuan sheldrake, detecting its anti-VSV activity of 150U/ml after prokaryotic expression, specific activity of 440U/mg, gao Pei expressing rDuIFN-alpha, rDuIFN-gamma and rDuIFN alpha-DuIFN gamma on prokaryotic expression system, and anti-VSV activity of 2.1α110 5 U/mg、3.6×10 4 U/mg、1.6×10 7 U/mg. Because the prokaryotic expression system can not carry out complex post-translational processing modification on the expressed recombinant protein, the titer of the expressed duck alpha interferon is low, the method adopts the silkworm baculovirus expression system to express the duck alpha interferon in the body of silkworm (pupa) so as to obtain a large amount of high-quality and low-cost recombinant duck alpha interferon proteins.
Disclosure of Invention
The first technical problem to be solved by the invention is to provide the duck alpha interferon and the duck alpha interferon mutant, wherein the duck alpha interferon mutant has high antiviral activity;
the second technical problem to be solved by the invention is to provide a method for preparing the duck alpha interferon or the duck alpha interferon mutant by using a silkworm baculovirus expression system;
the third technical problem to be solved by the invention is to provide the application of the duck alpha interferon or the duck alpha interferon mutant in preparing a medicament or a reagent for preventing or treating duck viral diseases.
In order to solve the technical problems, the invention adopts the following technical scheme:
the invention firstly discloses a duck alpha interferon, the amino acid sequence of which is shown as SEQ ID NO.1, and the polynucleotide sequence of the encoding gene of which is shown as (a) or (b) or (c):
(a) A polynucleotide sequence shown in SEQ ID No. 2; or (b)
(b) A polynucleotide sequence capable of hybridizing with the complement of SEQ ID No.2 under stringent hybridization conditions, the protein encoded by the polynucleotide still having interferon function or activity; or (b)
(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, the polynucleotide sequence has at least more than 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, the polynucleotide sequence has at least more than 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.
Carrying out codon optimization on duck alpha interferon gene sequence according to the codon preference of silkworm, carrying out optimization design on various related parameters which influence the transcription efficiency, translation efficiency and GC content of protein folding, cpG dinucleotide content, codon preference, secondary structure of mRNA, free energy stability of mRNA, RNA instability motif, repeated sequence and the like, thereby being beneficial to improving the transcription efficiency and translation efficiency of the optimized gene in the silkworm and keeping the protein sequence finally translated unchanged; in order to improve the translation initiation efficiency in a eukaryotic expression system of the silkworm baculovirus, a Kozak sequence GCCAAC is added in front of the gene. In addition, the restriction enzyme sites such as BamHI, ecoRI, smaI in the gene sequence are removed, bamHI is added at the upstream of the gene, and EcoRI restriction enzyme sites are added at the downstream of the gene; the novel duck alpha interferon optimized sequence DuIFN-alpha-O is obtained, the amino acid sequence of the DuIFN-alpha-O is shown as SEQ ID NO.3, and the optimized gene nucleotide sequence is shown as SEQ ID NO. 4.
The invention further discloses a duck alpha interferon mutant, which is obtained by carrying out P2A, L19T, P25L, R35P, D38N, N51D, H64N, L I, Q80R, D95K, H108D, Q117R, H123Y, R127Q, L136S, R142C, I148T, L159F, D E or any one of R182C amino acid unit point mutation on the duck alpha interferon mutant with the amino acid sequence shown as SEQ ID NO. 3; preferably, the duck alpha interferon mutant with the amino acid sequence shown in SEQ ID NO.3 is subjected to any one of amino acid unit point mutation of P25L, N51D, Q80R, H D, L136S and D170E. Wherein, the amino acid sequence of the mutant obtained by carrying out N51D amino acid unit point mutation on the duck alpha interferon mutant with the amino acid sequence shown as SEQ ID NO.3 is shown as SEQ ID NO.5, and the nucleotide sequence of the encoding gene is shown as SEQ ID NO. 6.
The amino acid unit point mutation P2A of the invention shows that the 2 nd amino acid of the duck alpha interferon mutant with the amino acid sequence shown as SEQ ID NO.3 is mutated from proline (P) to alanine (A); L19T represents a mutation of amino acid 19 from leucine (L) to threonine (T); and so on.
The invention also discloses a duck alpha interferon mutant, which is obtained by carrying out double-site mutation on any one of amino acids of P25L-N51D, P L-Q80R, P25L-H108D, P L-L136S, P L-D170E, N D-Q80R, N D-H108D, N D-L136S, N D170E, Q80R-H108D, Q80R-L136S, Q R-D170E, H D-L136S, H D-D170E or L136S-D170E on the duck alpha interferon mutant with the amino acid sequence shown as SEQ ID NO. 3; preferably, the duck alpha interferon mutant with the amino acid sequence shown in SEQ ID NO.3 is subjected to double-site mutation of any one of amino acids P25L-Q80R, N D-H108D, N D-D170E. Wherein, the amino acid sequence of the mutant obtained by carrying out double-site mutation on the duck alpha interferon mutant with the amino acid sequence shown as SEQ ID NO.3 and N51D-H108D amino acid is shown as SEQ ID NO.7, and the nucleotide sequence of the encoding gene is shown as SEQ ID NO. 8.
Wherein the amino acid double-site mutation P25L-N51D disclosed by the invention is characterized in that the 25 th amino acid of a duck alpha interferon mutant with an amino acid sequence shown as SEQ ID NO.3 is mutated from proline (P) to leucine (L), and the 51 st amino acid is mutated from asparagine (N) to aspartic acid (D); the amino acid double site mutation P25L-Q80R means that the 25 th amino acid is mutated from proline (P) to leucine (L) and the 80 th amino acid is mutated from glutamine (Q) to arginine (R); and so on.
The invention also discloses a duck alpha interferon mutant which is obtained by carrying out any amino acid multisite mutation on the duck alpha interferon mutant with the amino acid sequence shown as SEQ ID NO.3, namely P25L-N51D-Q80R, P L-Q80R-H108D, P L-Q80R-D170E, P L-N51D-H108D, N D-Q80R-H108D, N D-H108D-D170E, P L-N51D-D170E, N D-Q80R-D170E, P L-N51D-Q80R-H108D, N D-Q80R-H108D-D170E, P L-N51D-H108D-D170E or P25L-N51D-Q80R-H108D-D170E; preferably, the duck alpha interferon mutant with the amino acid sequence shown in SEQ ID NO.3 is subjected to multi-site mutation of P25L-N51D-Q80R-H108D-D170E amino acid. Wherein, the amino acid sequence of the mutant obtained by carrying out P25L-N51D-Q80R-H108D-D170E amino acid multisite mutation on the duck alpha interferon mutant with the amino acid sequence shown as SEQ ID NO.3 is shown as SEQ ID NO.9, and the nucleotide sequence of the encoding gene is shown as SEQ ID NO. 10.
Wherein the amino acid multi-site mutation P25L-N51D-Q80R of the invention represents that the 25 th site of a duck alpha interferon mutant with an amino acid sequence shown as SEQ ID NO.3 is mutated from proline (P) to leucine (L), the 51 st site of amino acid is mutated from asparagine (N) to aspartic acid (D), and the 80 th site of amino acid is mutated from glutamine (Q) to arginine (R); and so on.
The invention analyzes all duck alpha interferon amino acid sequences on NCBI to generate a consensus sequence, and takes the amino acid sequence shown in the consensus sequence as the original amino acid sequence of duck alpha interferon (the amino acid sequence is shown as SEQ ID NO.1, and the nucleotide sequence of the encoding gene is shown as SEQ ID NO. 2). The antiviral activity results show that the DuIFN-alpha titer expressed in the bodies of silkworm larvae is 8.70X10 5 U/mL。
On the basis of the original sequence of duck alpha interferon, the gene sequence is optimized according to the codon preference of silkworm, and various related parameters affecting the gene transcription efficiency, translation efficiency, GC content of protein folding, cpG dinucleotide content, codon preference, secondary structure of mRNA, free energy stability of mRNA, RNA instability gene sequence, repeated sequence and the like are optimally designed, so that the finally translated protein sequence is kept unchanged; in order to improve the translation initiation efficiency in a eukaryotic expression system of the silkworm baculovirus, a Kozak sequence GCCAAC is added in front of the gene ATG. In addition, the restriction enzyme sites such as BamHI, ecoRI, smaI and the like in the gene sequence are removed, bamHI is added at the upstream of the gene, ecoRI restriction enzyme sites are added at the downstream of the gene, and a novel DuIFN-alpha-O mutant of the duck alpha interferon mutant is obtained, the amino acid sequence of the DuIFN-alpha-O mutant is shown as SEQ ID NO.3, and the optimized nucleotide sequence of the gene is shown as SEQ ID NO. 4. The antiviral activity results show that the DuIFN-alpha titer expressed in the bodies of silkworm larvae is 1.14X10 6 U/mL。
The invention designs a plurality of pairs of primers by taking the optimized gene sequence of DuIFN-alpha as a template, and utilizes a fusion PCR method to carry out amino acid single-site mutation, amino acid double-site mutation and amino acid multi-site mutation, thereby obtaining a plurality of duck alpha interferon mutants. Wherein, after the single site mutation of the 6 sites P25L, N D, Q80R, H108D, L136S and D170E is respectively carried out on the basis of DuIFN-alpha-O, the expressed duck alpha interferon titer is higher than the titer measured by DuIFN-alpha-O expression, and the antiviral titer reaches 7.80 multiplied by 10 4 U/mL～3.16×10 6 U/mL; while the titers of the other sites are unchanged even after mutationThe reduction indicates that the mutation of the 6 sites is effective mutation, and the aim of improving the antiviral activity can be achieved. Wherein, the DuIFN-alpha-O-N51D mutant obtained by single-site mutation of N51D has the strongest antiviral effect.
The invention further combines the single mutation sites P25L, N51D, Q80R, H D, L136S and D170E with each other, which have improved antiviral activity, and performs double-site mutation on DuIFN-alpha-O. The detection result of antiviral activity shows that after three groups of P25L-Q80R, N D-H108D, N D-D170E double mutation, the expressed duck alpha interferon titer is higher than that measured by the conserved sequence and single mutation sequence expression, and the antiviral titer reaches 8.01X10 4 U/mL～4.50×10 6 U/mL, and the potency of the other groups of sites is unchanged or even reduced after mutation, which shows that the mutation of the 3 combined sites is effective mutation, and the purpose of improving the antiviral activity can be achieved. Wherein, the DuIFN-alpha-O-N51D-H108D mutant obtained by carrying out double-site mutation of N51D-H108D has the strongest antiviral effect.
The invention further combines the obtained double mutation sites with high titer, and makes the DuIFN-alpha-O mutant undergo amino acid multi-site mutation. The detection result of the antiviral activity of the mutant shows that after the P25L-N51D-Q80R-H108D-D170E multi-site mutation, the titer of the expressed duck alpha interferon is far higher than that measured by the expression of a conserved sequence, a single mutation sequence and a double mutation sequence, and is 5.78X10 6 U/mL; and the titers of the other groups of sites are unchanged or even reduced after mutation. The mutation of the combined site is effective mutation, and the aim of improving the antiviral activity can be achieved.
The invention also discloses a recombinant vector or a recombinant host cell containing the coding gene of the duck alpha interferon or the duck alpha interferon mutant. Wherein the recombinant vector is a recombinant expression vector or a recombinant cloning vector.
The transfer vector constructed by the invention comprises:
(1) Vector pVL-DuIFN-alpha-O-M1 comprising the gene sequence of a mutant (DuIFN-alpha-O-M1 mutant) after amino acid unit point mutation of DuIFN-alpha-O;
(2) Vector pVL-DuIFN-alpha-O-M1-M2 comprising the gene sequence of a mutant (DuIFN-alpha-O-M1-M2 mutant) after double site mutation of an amino acid by DuIFN-alpha-O;
(3) Vectors pVL-DuIFN-alpha-O-M1-M2-M3, pVL-DuIFN-alpha-O-M1-M2-M3-M4-M5 comprising DuIFN-alpha-O-M1-M2-M3, duIFN-alpha-M1-M2-M3-M4, duIFN-alpha-O-M1-M2-M3-M4-M5, which contain the DuIFN-alpha-O-DuIFN-alpha-M1-M2-M3-M4.
The recombinant baculovirus obtained by the invention comprises: recombinant silkworm nuclear polyhedrosis virus rBmBacmid (DuIFN-alpha), rBmBacmid (DuIFN-alpha-O, duIFN-alpha-O-M1, duIFN-alpha-O-M1-M2-M3-M4-M5).
The invention also discloses application of the duck alpha interferon or the duck alpha interferon mutant in preparing medicines or reagents for preventing or treating duck viral diseases.
Wherein the duck viral disease comprises: at least one of a duckling viral hepatitis, a duck plague (duck viral enteritis), a gosling plague, a Muscovy duck parvovirus disease, a duckling gosling plague, a duck epidemic hemorrhagic disease (black feather disease), a duck liver disease, a duck viral encephalitis, a duck reovirus infection, a duck adenovirus infection, a duck infectious bursal disease, a duck paramyxovirus disease (duck newcastle disease), a duck coronavirus infection, and a duck influenza.
The invention also discloses a method for preparing the duck alpha interferon or the duck alpha interferon mutant, which comprises the following steps: (1) Cloning the coding genes of the duck alpha interferon or the duck alpha interferon mutant into a baculovirus transfer vector respectively, and constructing to obtain a recombinant transfer vector; (2) Co-transfecting insect cells with the recombinant transfer vector and baculovirus DNA to obtain recombinant baculovirus; (3) And (3) infecting the insect cells or insect hosts with the recombinant baculovirus, culturing the infected insect cells or insect hosts to express the corresponding proteins, and purifying to obtain the recombinant baculovirus.
Wherein, the liquid crystal display device comprises a liquid crystal display device, 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, pAcLF 8, pAcPlP 2, pAcRP23, pAcRP 25, pAcRW4, pAcsMAG, pAcUWl, pAcUW21, pAcUW2A, pAcUW2B, pAcUW3, pAcUW31, pAcUW41, pAcUW42, pAcUW43, pAcUW51, pAcVC2, pAcVC 3, pAcYMl, pAcJcC, pBacl 2, pBlueBacIII, pBlueBaSWis, pEV, mXIV, pIEINeo, pJVETL, pJVNhel, pJVP, pJVrsMAG, pMBac, pP, pPAKl, pPBac, pSHONEX.1, pSYNVL 92, pSVL 13, pVL 91, pVL13, pVL 5, pVL94, pVL 91, pVL 5, pVL94, pVL13, pVL 5, pVL 93, pVL 5;
The baculovirus is selected from silkworm baculovirus parent strain BmBacmid, bmNPV, acMNPV, apNPV, haNPV, hzNPV, ldMNPV, mbMNPV, opMNPV, slMNPV, seMNPV or SpltNPV;
the insect host is selected from the group consisting of Bombyx mori (Bombyx mori), wild silkworm (Bombyx mandarina), castor silkworm (Philosamia cynthia ricim), camphor (Dictyoploca japanica), ailanthus (Philosamia cynthia pryeri), tussah (Antheraea pernyi), japanese tussah (Antheraea yamamai), wild silkworm (Antheraea polyphymus), alfalfa geometrid (Atographa califorica), tea geometrid (Ectropis) obliqua, cabbage looper (Mamestra brassicae), prodenia litura (Spodoptera littoralis), fall armyworm (Spodoptera frugiperda), cabbage looper (Triswopsis ni), armyworm (Thaumetopoea wilkinsoni), cotton bollworm (Heliothis armigera), american cotton bollworm (Heliotis zea), tobacco budworm (Heliothis assulta), tobacco looper (Heliothis virescens), oriental armyworm (Pseudaletia separata) and gymantria dispar;
preferably, the baculovirus transfer vector is pVL1393; the baculovirus is silkworm baculovirus parent strain BmBacmid; the insect host is Bombyx mori (Bombyx mori).
The infection refers to that the recombinant baculovirus infects 1-5-year-old insect larvae or pupae by swallowing or penetrating epidermis; preferably, the recombinant silkworm baculovirus is used for infecting silkworm cells or puncture inoculating silkworm larvae or pupae of 1-5 years old, and body fluid or tissue homogenate of the silkworm larvae or pupae containing various duck alpha interferon genes is collected after 3-6 days of infection; wherein the pupae is the early tender pupae of 1-2 days.
Wherein, SEQ ID NO.1: duck alpha interferon original amino acid sequence;
MPGPSAPPPPAIYSALALLLLLTPPANAFSCSPLRLHDSAFAWDSLQLLRNMAPSPTQPCPQQHAPCSFPDTLLDTNDTQQAAHTALHLLQHLFDTLSSPSTPAHWLHTARHDLLNQLQHHIHHLERCFPADAARLHRRGPRNLHLSINKYFGCIQHFLQNHTYSPCAWDHVRLEAHACFQRIHRLTRTMR*
SEQ ID NO.2: duck alpha interferon original nucleotide sequence;
ATGCCTGGGCCATCAGCCCCACCACCACCAGCCATCTACAGCGCCCTGGCCCTCCTGCTCCTCCTCACGCCTCCCGCCAACGCCTTCTCCTGCAGCCCCCTGCGCCTCCACGACAGCGCCTTCGCCTGGGACAGCCTCCAGCTCCTCCGCAACATGGCTCCCAGCCCCACACAGCCCTGCCCGCAGCAACACGCGCCTTGCTCCTTCCCGGACACCCTCCTGGACACCAACGACACGCAGCAAGCCGCACACACCGCCCTCCACCTCCTCCAACACCTCTTCGACACCCTCAGCAGCCCCAGCACCCCCGCGCACTGGCTCCACACCGCACGCCACGACCTCCTCAACCAGCTTCAGCACCACATCCACCACCTCGAGCGCTGCTTCCCAGCCGACGCCGCGCGCCTCCACAGGCGAGGGCCCCGCAACCTTCACCTCAGCATCAACAAGTACTTCGGCTGCATCCAACACTTCCTCCAGAACCACACCTACAGCCCCTGCGCATGGGACCACGTCCGCCTCGAGGCTCACGCCTGCTTCCAGCGCATCCACCGCCTCACCCGCACCATGCGCTAA
SEQ ID NO.3: a DuIFN-alpha-O amino acid sequence;
MPGPSAPPPPAIYSALALLLLLTPPANAFSCSPLRLHDSAFAWDSLQLLRNMAPSPTQPCPQQHAPCSFPDTLLDTNDTQQAAHTALHLLQHLFDTLSSPSTPAHWLHTARHDLLNQLQHHIHHLERCFPADAARLHRRGPRNLHLSINKYFGCIQHFLQNHTYSPCAWDHVRLEAHACFQRIHRLTRTMR*
SEQ ID NO.4: duIFN-alpha-O nucleotide sequence;
ATGCCTGGACCATCAGCTCCTCCACCGCCCGCCATCTACTCTGCTTTGGCCCTGTTGCTCCTGTTGACCCCTCCAGCTAACGCCTTCAGCTGCTCCCCGTTGAGACTCCACGACTCGGCTTTCGCCTGGGATAGTCTGCAACTCCTGAGAAATATGGCTCCTAGCCCAACTCAACCTTGCCCACAACAGCATGCTCCGTGTTCCTTCCCCGACACATTGCTCGACACCAACGATACACAACAGGCTGCCCACACTGCTCTCCACCTGTTGCAACACCTCTTCGACACTCTGTCATCTCCTTCAACCCCAGCTCACTGGCTGCACACTGCCAGACACGATCTCCTGAATCAATTGCAGCACCACATACACCACCTCGAAAGATGCTTCCCTGCTGACGCTGCCAGATTGCACAGAAGAGGTCCAAGAAACCTGCACTTGTCAATCAACAAATACTTCGGATGTATTCAACACTTCCTCCAGAACCACACCTACTCTCCATGCGCCTGGGATCACGTGAGACTGGAAGCTCACGCCTGTTTCCAGAGAATCCACAGATTGACAAGAACTATGAGATAA
SEQ ID NO.5: duIFN-alpha-O-N51D mutant amino acid sequence;
MPGPSAPPPPAIYSALALLLLLTPPANAFSCSPLRLHDSAFAWDSLQLLRDMAPSPTQPCPQQHAPCSFPDTLLDTNDTQQAAHTALHLLQHLFDTLSSPSTPAHWLHTARHDLLNQLQHHIHHLERCFPADAARLHRRGPRNLHLSINKYFGCIQHFLQNHTYSPCAWDHVRLEAHACFQRIHRLTRTMR*
SEQ ID NO.6: duIFN-alpha-O-N51D mutant nucleotide sequence;
ATGCCTGGACCATCAGCTCCTCCACCGCCCGCCATCTACTCTGCTTTGGCCCTGTTGCTCCTGTTGACCCCTCCAGCTAACGCCTTCAGCTGCTCCCCGTTGAGACTCCACGACTCGGCTTTCGCCTGGGATAGTCTGCAACTCCTGAGAGATATGGCTCCTAGCCCAACTCAACCTTGCCCACAACAGCATGCTCCGTGTTCCTTCCCCGACACATTGCTCGACACCAACGATACACAACAGGCTGCCCACACTGCTCTCCACCTGTTGCAACACCTCTTCGACACTCTGTCATCTCCTTCAACCCCAGCTCACTGGCTGCACACTGCCAGACACGATCTCCTGAATCAATTGCAGCACCACATACACCACCTCGAAAGATGCTTCCCTGCTGACGCTGCCAGATTGCACAGAAGAGGTCCAAGAAACCTGCACTTGTCAATCAACAAATACTTCGGATGTATTCAACACTTCCTCCAGAACCACACCTACTCTCCATGCGCCTGGGATCACGTGAGACTGGAAGCTCACGCCTGTTTCCAGAGAATCCACAGATTGACAAGAACTATGAGATAA
SEQ ID NO.7: duIFN-alpha-O-N51D-H108D mutant amino acid sequence;
MPGPSAPPPPAIYSALALLLLLTPPANAFSCSPLRLHDSAFAWDSLQLLRDMAPSPTQPCPQQHAPCSFPDTLLDTNDTQQAAHTALHLLQHLFDTLSSPSTPAHWLDTARHDLLNQLQHHIHHLERCFPADAARLHRRGPRNLHLSINKYFGCIQHFLQNHTYSPCAWDHVRLEAHACFQRIHRLTRTMR*
SEQ ID NO.8: duIFN-alpha-O-N51D-H108D mutant nucleotide sequence;
ATGCCTGGACCATCAGCTCCTCCACCGCCCGCCATCTACTCTGCTTTGGCCCTGTTGCTCCTGTTGACCCCTCCAGCTAACGCCTTCAGCTGCTCCCCGTTGAGACTCCACGACTCGGCTTTCGCCTGGGATAGTCTGCAACTCCTGAGAGATATGGCTCCTAGCCCAACTCAACCTTGCCCACAACAGCATGCTCCGTGTTCCTTCCCCGACACATTGCTCGACACCAACGATACACAACAGGCTGCCCACACTGCTCTCCACCTGTTGCAACACCTCTTCGACACTCTGTCATCTCCTTCAACCCCAGCTCACTGGCTGGACACTGCCAGACACGATCTCCTGAATCAATTGCAGCACCACATACACCACCTCGAAAGATGCTTCCCTGCTGACGCTGCCAGATTGCACAGAAGAGGTCCAAGAAACCTGCACTTGTCAATCAACAAATACTTCGGATGTATTCAACACTTCCTCCAGAACCACACCTACTCTCCATGCGCCTGGGATCACGTGAGACTGGAAGCTCACGCCTGTTTCCAGAGAATCCACAGATTGACAAGAACTATGAGATAA
SEQ ID NO.9: duIFN-alpha-O-P25L-N51D-Q80R-H108D-D170E mutant amino acid sequence;
MPGPSAPPPPAIYSALALLLLLTPLANAFSCSPLRLHDSAFAWDSLQLLRDMAPSPTQPCPQQHAPCSFPDTLLDTNDTRQAAHTALHLLQHLFDTLSSPSTPAHWLDTARHDLLNQLQHHIHHLERCFPADAARLHRRGPRNLHLSINKYFGCIQHFLQNHTYSPCAWEHVRLEAHACFQRIHRLTRTMR*
SEQ ID NO.10: duIFN- α -O-P25L-N51D-Q80R-H108D-D170E mutant nucleotide sequence:
ATGCCTGGACCATCAGCTCCTCCACCGCCCGCCATCTACTCTGCTTTGGCCCTGTTGCTCCTGTTGACCCCTCTAGCTAACGCCTTCAGCTGCTCCCCGTTGAGACTCCACGACTCGGCTTTCGCCTGGGATAGTCTGCAACTCCTGAGAGATATGGCTCCTAGCCCAACTCAACCTTGCCCACAACAGCATGCTCCGTGTTCCTTCCCCGACACATTGCTCGACACCAACGATACACGACAGGCTGCCCACACTGCTCTCCACCTGTTGCAACACCTCTTCGACACTCTGTCATCTCCTTCAACCCCAGCTCACTGGCTGGACACTGCCAGACACGATCTCCTGAATCAATTGCAGCACCACATACACCACCTCGAAAGATGCTTCCCTGCTGACGCTGCCAGATTGCACAGAAGAGGTCCAAGAAACCTGCACTTGTCAATCAACAAATACTTCGGATGTATTCAACACTTCCTCCAGAACCACACCTACTCTCCATGCGCCTGGGAACACGTGAGACTGGAAGCTCACGCCTGTTTCCAGAGAATCCACAGATTGACAAGAACTATGAGATAA
compared with the prior art, the technical scheme of the invention has the following beneficial effects:
According to the invention, through analyzing all duck alpha interferon amino acid sequences on NCBI, performing sequence comparison and signal peptide analysis, generating a consensus sequence as an original sequence, performing codon optimization on the original sequence, designing a plurality of pairs of primers by taking the sequence after the codon optimization as a template, and performing amino acid single-site mutation, amino acid double-site mutation and amino acid multi-site mutation by using a fusion PCR method, thereby obtaining a plurality of duck alpha interferon mutants. The invention expresses the duck alpha interferon mutant in a silkworm bioreactor by utilizing a silkworm baculovirus expression system, and the expressed duck alpha interferon mutant has greatly improved antiviral activity and obvious antiviral activity. The method has simple process and can rapidly obtain a large amount of safe and reliable duck alpha interferon. The duck alpha interferon mutant can be used for preparing medicines or reagents for preventing or treating duck viral diseases, and has great significance for development of duck breeding industry.
Definition of terms in connection with the present invention:
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.
The term "polynucleotide" or "nucleotide" means deoxyribonucleotides, deoxyribonucleosides, ribonucleosides, or ribonucleotides and polymers thereof in either single-or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that 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 PNAs (peptide nucleic acids), DNA analogs used in antisense technology (phosphorothioates, phosphoroamidites, etc.). Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (including, but not limited to, degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated. In particular, degenerate codon substitutions may be achieved by generating sequences in which the 3 rd position of one or more selected (or all) codons is substituted with mixed bases 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, molcell. Probe8:91-98 (1994)).
The term "homology" refers to sequence similarity to a native nucleic acid sequence. "homology" includes nucleotide sequences having preferably 85% or more, more preferably 90% or more, and most preferably 95% or more identity to the nucleotide sequence of the regulatory fragment of the present invention. Homology can be assessed visually or by computer software. Using computer software, homology between two or more sequences can be expressed in percent (%), which can be used to evaluate homology between related sequences.
The term "complementary" as used herein refers to two nucleotide sequences comprising antiparallel nucleotide sequences capable of pairing with each other after hydrogen bonding between complementary base residues of the antiparallel nucleotide sequences. It is known in the art that the nucleotide sequences of two complementary strands are complementary to each other in reverse when the sequences are all seen in the 5 'to 3' direction. It is also known in the art that two sequences which hybridize to each other under a given set of conditions do not necessarily have to be 100% completely complementary.
The term "stringent hybridization conditions" means conditions of low ionic strength and high temperature as known in the art. Typically, the probe hybridizes to its target sequence to a greater degree of detectability (e.g., at least 2-fold over background) under stringent conditions than to other sequences. Stringent hybridization conditions are sequence dependent and will be different under different environmental conditions, longer sequences hybridizing specifically at higher temperatures. Target sequences that are 100% complementary to the probe can be identified by controlling the stringency of hybridization or wash conditions. For a detailed guidance on nucleic acid hybridization, reference is made to the 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 typically selected to be about 5-10℃below the thermal melting point (Tm) for the specific sequence at the defined ionic strength pH. Tm is the temperature (at a given ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (50% of the probes at equilibrium are occupied at Tm because the target sequence is present in excess). Stringent conditions may be the following conditions: wherein the salt concentration is less than about 1.0M sodium ion concentration, typically about 0.01 to 1.0M sodium ion concentration (or other salt) at pH 7.0 to 8.3, and the temperature is at least about 30℃for short probes, including but not limited to 10 to 50 nucleotides, and at least about 60℃for long probes, including but not limited to greater than 50 nucleotides. Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, the positive signal may be at least twice background hybridization, optionally 10 times background hybridization. Exemplary stringent hybridization conditions can be as follows: 50% formamide, 5 XSSC and 1% SDS, at 42 ℃; or 5 XSSC, 1% SDS, at 65℃in 0.2 XSSC and at 65℃in 0.1% SDS. The washing may be performed for 5, 15, 30, 60, 120 minutes or more.
The terms "mutation" and "mutant" have their usual meaning herein, referring to genetic, naturally occurring or introduced changes in a nucleic acid or polypeptide sequence, which are in the same sense as commonly known to those skilled in the art.
The term "host cell" or "recombinant host cell" means a cell comprising a polynucleotide of the invention, regardless of the method used to insert to produce a recombinant host cell, such as direct uptake, transduction, f-pairing, or other methods known in the art. The exogenous polynucleotide may remain as a non-integrating vector, such as a plasmid, or may integrate into the host genome.
The term "transfection" refers to the process by which eukaryotic cells acquire new genetic markers due to the incorporation of exogenous DNA.
Drawings
FIG. 1 is a fluorescence plot corresponding to cytopathic ratio; wherein, A, "-": no cytopathy; b, "±": several cytopathic disorders; c, "+":20% -30% cytopathy; d, the step of setting the position of the base plate, "+". ". 50% -60% cytopathy;
FIG. 2 is a double restriction identification of recombinant plasmid pVL-DuIFN- α; wherein M: a DNA molecular mass standard; 1: recombinant plasmid pVL-DuIFN-alpha double enzyme cutting products; 2: a negative control;
FIG. 3 shows the presence of cells in various proportions of fluorescence; wherein A: cells do not exhibit fluorescence after high concentrations of interferon completely inhibit VSV virus; b: a number of cells in the cell control group showed fluorescence upon VSV virus infection; c: the low concentration of interferon does not completely inhibit the fluorescence exhibited by a portion of cells infected with VSV virus.
Detailed Description
The invention will be further described with reference to specific embodiments, and advantages and features of the invention will become apparent from the description. It should be understood that the embodiments described are exemplary only and should not be construed as limiting the scope of the invention in any way. It will be understood by those skilled in the art that various changes and substitutions can be made in the details and form of the technical solution of the present invention without departing from the spirit and scope of the invention, but these changes and substitutions fall within the scope of the present invention.
1. Test materials and reagents
The transfer vectors pVL1393, E.coli strain TOP10, bmN cells and VSV-GFP virus are all saved and provided by the institute of biotechnology of the national academy of agricultural sciences; the test silkworm variety Qiufeng Xbaiyu is provided by the silkworm industry research institute of Jiangsu university of science and technology, and the parent virus BmBacmid DNA is constructed according to the method disclosed in the literature (patent number: ZL 201110142492.4, authorized date: 2013.01.23). SPF duck embryo is purchased from Beijing Meili Asian Vietnam laboratory animal technology Co., ltd., restriction endonuclease, T 4 The DNA ligase is purchased from Promega company, the LA Taq DNA polymerase and other related reagents used in PCR reaction are all purchased from TakaRa company, the liposome is purchased from Invitrogen company, and DMEM cell culture medium and fetal bovine serum are manufactured by GIBCO company. Methods for the preparation of solutions and media are described in the relevant literature (Joseph et al, third edition of molecular cloning protocols, 2002; osbo, et al, fine programming protocols for molecular biology, 1998;David L.Spector, cell protocols, 2001); percentages and parts are by weight unless otherwise indicated.
2. Experimental method
PHA induction of duck peripheral blood mononuclear cells was performed in the experimental procedure as described in Huang Ailong et al (Duck. Alpha. -interferon gene expression and diversity analysis [ J ]. J.Chinese J.Immunol., 2000,16 (12): 644-647).
Fusion PCR methods for site-directed mutagenesis in experimental methods were performed as described in , U.S. Pat. No. 5,000 (novel method for vector construction: recombinant fusion PCR method, genomics and applied biology, 2012, 31, 6, 634-639).
The titers of interferon were calculated in the experimental procedure, using the DEF/VSV GFP system, using the Reed-Muench method, specific procedures reference Liu Xingjian, et al (expression and biological activity detection of feline omega-like interferon in silkworms, biotechnology development 2015,5 (6): 441-445) and the method described by Summers MD et al (A manual of methods for baculovirus vectors and insect cell culture procedures [ R ]. Texas Agricultural Experiment Station, 1987), wherein criteria for determining cytopathic effects are described with reference to fig. 1.
The best improvement in each embodiment serves as a comparison standard for the improvement in the next embodiment.
EXAMPLE 1 expression and detection of DuIFN-alpha original sequence in silkworm bioreactor
1. Experimental method
1.1 Duck interferon alpha original sequence acquisition
1.1.1 in vitro stimulation of Duck Peripheral Blood Mononuclear Cells (PBMC)
The peripheral anticoagulation of the sterile picked ducks adopts lymphocyte separation liquid to separate peripheral blood mononuclear cells and erythrocytes. And further separating duck T cells by utilizing the specific adsorption effect of nylon wool. 1mL of 8X 10 cells were added to 24-well cell culture plates 6 mL -1 The PBMC were incubated at 37℃for 1-2 h to allow antigen presenting somatic cells (APCs) to adhere. The isolated T lymphocytes (8X 10) were added to the above-described APC-adsorbed 24-well plate 6 mL/well) and erythrocytes (4X 10) 6 mL/well) and final concentration of Phytohemagglutinin (PHA) at 5 μg/mL, incubated at 37 ℃ for 4h, and cells were collected.
1.1.2 extraction of Total RNA from cells
Taking a certain amount of collected cells in 1mL of Trizol reagent, fully blowing and mixing uniformly, and standing at room temperature for 10min; 200 mu L of chloroform is added, vortex oscillation is carried out for 15s, and standing is carried out for 5min at room temperature; centrifuging at 12000rpm at 4deg.C for 15min; taking the upper water phase into a new EP pipe, adding 500 mu L of isopropanol, and carrying out ice bath for 10min; centrifuging at 12000rpm for 10min at 4 ℃; washing the precipitate with 70% ethanol once, and centrifuging at 12000rpm at 4deg.C for 5min; and (3) airing the precipitate at room temperature, adding the precipitate into a proper amount of DEPC treated double distilled water or deionized formamide, and preserving at-20 ℃ for later use.
1.1.3 RT-PCR reaction
Synthesis of first strand cDNA. Taking 2 mu L of RNA (less than or equal to 1 mu g), adding 2 mu L of DuIFN-alpha-RT, adding 13.75 mu L of DEPC treated water, fully mixing, reacting at 70 ℃ for 5min, and rapidly carrying out ice bath for 5min; mu.L of 5 XM-MLV buffer,1.25 mu.L of 10mM dNTPs and 1 mu L M-MLV-RT (200U) were added to make the final volume 25. Mu.L, and the mixture was left at room temperature for 10 minutes after mixing; reacting for 1h at 42 ℃; inactivating RTase at 70 deg.C for 2min, and storing at-20 deg.C for use. Specific primers used for reverse transcription are as follows.
DuIFN-α-RT：5’-TTATCTCATAGTTCTTGTCA-3’(SEQ ID NO.11)
And amplifying the target fragment by PCR. The BamHI and EcoRI recognition sites were introduced into the upstream and downstream primers, respectively, and a Kozak sequence GCCAAC was introduced before the initiation codon, and the target fragment was amplified by conventional PCR methods. The primers were as follows:
F:5’-CGGGATCCGCCAACATGCCTGGACCATCAGCTC-3’(SEQ ID NO.12)
R:5’-CGGAATTCTTATCTCATAGTTCTTGTCAATC-3’(SEQ ID NO.13)
1.1.4 purification of glass milk and recovery of DNA fragments
Preparing 1% (w/v) agarose gel, and carrying out electrophoresis on PCR amplified products; placing agarose gel under ultraviolet lamp, rapidly cutting off gel containing single target nucleic acid band, placing into 1.5mL centrifuge tube, weighing, adding 6M NaI with triple volume, and melting in 37 deg.C constant temperature incubator; adding 8 mu L of Glassmik into the completely melted solution, uniformly mixing, ice-bathing for 5min, and shaking in the middle for two times; centrifuging at 8000rpm for 10s, and discarding the supernatant; adding 800 mu L of New Wash for washing, slightly bouncing, centrifuging, and repeating for 2 times; discarding the supernatant, and placing the centrifuge tube in a constant temperature incubator at 37 ℃ for drying for 2-3 min; after drying, 20. Mu.L of 0.1 XTE was added for dissolution, and the mixture was mixed to dissolve DNA sufficiently, and centrifuged at 12000rpm for 5 minutes, and the supernatant was immediately used for ligation, and the rest was kept at-20 ℃.
1.2 construction of recombinant baculovirus transfer vectors
The DuIFN-alpha target fragment recovered by digestion was ligated with the BamHI, ecoRI double digested transfer vector pVL 1393. By T 4 DNA ligase, 16℃overnight. Transforming the ligation product into competent cell TOP10 of Escherichia coli, selecting colony for culturing, extracting plasmid, identifying positive clone by BamHI and EcoRI double enzyme digestion, and delivering the identified correct recombinant plasmid to Beijing qing family biological technologySequencing by the company Limited gave the results shown in SEQ ID NO. 1. The recombinant plasmid obtained was named: pVL-DuIFN- α.
1.3 obtaining recombinant silkworm baculovirus
Resuscitation of BmN cells, passaging and screening of recombinant viruses were performed according to methods reported in the literature. When BmN cells were cultured until the cell monolayer reached about 80%, the old medium was poured off, washed three times with serum-free TC-100 medium, and 1.5mL of FBS-free medium was added. Sequentially adding 1 mug of silkworm baculovirus parent strain BmBacmid DNA,2 mug of recombinant transfer plasmid pVL-DuIFN-alpha and 5 mug of liposome into a sterilizing tube, supplementing the volume to 60 mug by using sterile double distilled water, gently mixing, standing for 15min, and then dropwise adding into a culture flask for cotransfection. After 4h incubation at 27℃1.5mL of serum-free medium and 300. Mu.L of FBS were added. Culturing at 27 deg.c for 4-5 days until the cell is dropped and floated, and collecting cell culture liquid to obtain recombinant virus rBmBacmid (DuIFN-alpha) containing target gene.
The purification and amplification method of the recombinant silkworm baculovirus comprises the following steps: inoculating appropriate amount of cells (about 70-80%) in a small 35mm dish, sucking the culture medium after the cells are attached, diluting the collected cell culture solution with different concentrations, adding 1mL into the attached cells, and uniformly distributing. After 1h of infection at 27 ℃, the infection liquid is sucked, 2% low melting point agarose gel is melted in a water bath at 60 ℃, cooled to 40 ℃ and evenly mixed with 2 xTC-100 culture medium (containing 20% FBS) preheated at 40 ℃, 4mL of glue is added to each plate, after solidification, parafilm is used for sealing, inverted culture is carried out at 27 ℃ for 3-5 d, and microscopic observation is carried out. Selecting the plaque without polyhedron, repeating the steps, and purifying for 2-3 rounds to obtain the pure recombinant silkworm baculovirus rBmBacmid (DuIFN-alpha).
Recombinant silkworm baculovirus rBmBacmid (DuIFN-alpha) is infected with BmN cells which normally grow, and after 3 days of culture, supernatant is collected, and the supernatant contains a large amount of recombinant virus rBmBacmid (DuIFN-alpha).
1.4 Duck interferon alpha expression in vivo in silkworm
10 portions of recombinant virus culture solution 5 PFU/head dosage injection for 5-year-old silkworm raising, culturing at 27 deg.C under 70-80% humidity, and growing silkworm larvaLate DuIFN-alpha is expressed with high efficiency under the action of polyhedrin gene promoter. The symptoms of swelling, abnormal behaviors, appetite decrease and the like of silkworm larva can be observed after inoculation infection for about 3.5-4 d, when the larva volume is observed to be obviously reduced, haemolymph is collected and stored at-20 ℃ for standby.
1.5 preparation of Duck Embryo Fibroblast (DEF) and Duck interferon alpha antiviral Activity detection
Taking 10-12 day old duck embryo, sterilizing eggshell in ultra clean bench, knocking eggshell from air chamber, taking out embryo body, cutting off head and limbs, flushing with HBSS for 3 times, placing embryo body into sterilized triangular flask and cutting into small pieces, flushing with HBSS for 3 times, adding pancreatin, digesting at 37deg.C for 40min, discarding pancreatin and flushing with HBSS for 3 times to remove residual pancreatin, adding 30mL complete culture medium, blowing and shaking triangular flask to disperse cells, filtering with 8 layers of sterilized gauze to obtain cell suspension, counting cell, and diluting cells to 10 6 And each mL.
The antiviral activity of duck alpha interferon expressed in silkworm haemolymph is detected on a DEF/VSV GFP system by adopting a micro cytopathic inhibition method. The prepared DEF cells were cultured at 3.0X10 times 5 The density of individual/mL was inoculated into 96-well plates. Preparing solution of different dilutions of silkworm hemolymph after ultrasonic disruption and filtration sterilization with M199 culture solution containing 50mL/L fetal bovine serum, inoculating diluted sample into culture well filled with DEF cells according to 100 μL/well, arranging at least 12 compound wells for each dilution and control silkworm blood, and simultaneously arranging cell control group without silkworm hemolymph and VSV-GFP and virus control group with VSV-GFP at 37deg.C and 5% CO 2 Culturing for 18-24 h under the condition. Will be diluted to 100TCID 50 GFP virus was added to the culture well from which the supernatant had been aspirated at 100. Mu.L/well, and placed at 37℃in 5% CO 2 Culturing under the condition. When the cells in the cell control group still grow well completely and no fluorescence appears, the control system is completely qualified and can be comprehensively observed.
2. Experimental results
2.1 identification of recombinant transfer vectors
The recombinant transfer vector pVL-DuIFN-alpha is digested with BamHI and EcoRI, 2 fragments are separated by 1% agarose gel electrophoresis, the small fragment is positioned between 500 and 750bp, the size of the fragment corresponds to 576bp of the target gene, the large fragment is positioned above 8000bp, and the size of the fragment 9607bp corresponds to pVL 1393. The plasmid with correct enzyme digestion identification is sent to Beijing qing new industry biotechnology Co-Ltd for nucleotide sequencing, and the MegaAlign comparison result shows that the sequence is consistent with the originally designed sequence, and the duck alpha interferon gene is successfully inserted between BamHI and EcoRI in the pVL1393 transfer vector.
2.2 Duck interferon-alpha recombinant virus acquisition and detection of recombinant products
The antiviral activity of duck alpha interferon expressed by silkworm larvae is detected on a DEF/VSV GFP system by using a micro cytopathic inhibition method. Observed under an inverted fluorescence microscope, the cell in the cell control group has good growth state and no fluorescence; cells in the infected virus control group are diseased, most cells fluoresce, and cells added with the recombinant duck alpha interferon protein have the capability of resisting virus infection. According to the protection effect of duck alpha interferon on DEF cells, observing the pathological change degree of the cells, and calculating the titer of the interferon according to a Reed-Muench method when green fluorescent cells appear, wherein the hole cells are marked as "+". The detection result shows that the potency reaches 8.70 multiplied by 10 5 U/mL。
EXAMPLE 2 expression and detection of DuIFN-alpha optimized sequence in silkworm bioreactor
1.1 construction of Duck interferon-alpha codon-optimized mutant Gene
The amino acid sequence of the consensus sequence is identical with the original amino acid sequence of the duck alpha interferon obtained by cloning in example 1 by analyzing all the duck alpha interferon amino acid sequences on NCBI, the amino acid sequence is shown as SEQ ID NO.1, and the nucleotide sequence of the coding gene is shown as SEQ ID NO. 2.
The invention utilizes OptimumGene TM The technology optimizes the duck alpha interferon consensus sequence gene,the gene sequence is modified according to codon preference of the silkworm of a bioreactor, and various related parameters such as GC content, cpG dinucleotide content, codon preference, secondary structure of mRNA, free energy stability of mRNA, RNA instability motif, repeated sequence and the like which influence the transcription efficiency, translation efficiency and protein folding of the gene are optimally designed, so that the transcription efficiency and translation efficiency of the optimized gene in the silkworm are improved, and the finally translated protein sequence is kept unchanged.
In order to improve the translation initiation efficiency in a eukaryotic expression system of the silkworm baculovirus, a Kozak sequence GCCAAC is added in front of the gene ATG. In addition, a restriction enzyme site such as BamHI, ecoRI, smaI in the gene sequence was removed, bamHI was added upstream of the gene, and EcoRI restriction enzyme site was added downstream of the gene, so that the gene was subsequently cloned into eukaryotic transfer vector pVL 1393.
The designed duck alpha interferon gene is synthesized artificially by biotechnology company, named as DuIFN-alpha-O, and the nucleotide sequence is shown in SEQ ID NO.4, and the synthesized gene fragment is inserted into pUC57 vector to form plasmid pUC57-DuIFN, named pUC 57-DuIFN-alpha-O.
1.2 construction of recombinant baculovirus transfer vectors
Specific procedures referring to example 1, the plasmid with the correct sequencing was designated pVL-DuIFN-. Alpha. -O.
1.3 obtaining, purifying and amplifying recombinant silkworm baculovirus
Resuscitating and passaging of BmN cells referring to example 1, recombinant virus rBmBacmid (DuIFN-. Alpha. -O) containing the target gene was obtained by cotransfection.
Purification and amplification method of recombinant silkworm baculovirus referring to example 1, pure recombinant silkworm baculovirus rbmmbacmid (DuIFN- α -O) was obtained by purification.
Recombinant silkworm baculovirus rBmBacmid (DuIFN-alpha-O) is infected with BmN cells which normally grow, the supernatant is collected after 3 days of culture, and the supernatant contains a large amount of recombinant virus rBmBacmid (DuIFN-alpha-O).
1.4 Duck interferon alpha mutant expression in vivo in silkworm
As in example 1.
1.5 preparation of Duck Embryo Fibroblast (DEF) and Duck interferon alpha antiviral Activity detection
As in example 1.
2. Experimental results
2.1 identification of recombinant transfer vectors
The recombinant transfer vector pVL-DuIFN-alpha-O is digested with BamHI and EcoRI, 2 fragments are separated by 1% agarose gel electrophoresis, the small fragment is located between 500 and 750bp, the size of the fragment corresponds to 576bp of the target gene, the large fragment is located above 8000bp, and the size of the fragment corresponds to 9607bp of pVL 1393. The plasmid with correct enzyme digestion identification is sent to Beijing qing new industry biotechnology Co-Ltd for nucleotide sequencing, and the MegaAlign comparison result shows that the sequence is consistent with the originally designed sequence, and the duck alpha interferon mutant gene is successfully inserted between BamHI and EcoRI in the pVL1393 transfer vector.
2.2 Duck interferon-alpha recombinant virus acquisition and detection of recombinant products
The antiviral activity of duck alpha interferon expressed by silkworm larvae is detected on a DEF/VSV GFP system by using a micro cytopathic inhibition method. Observed under an inverted fluorescence microscope, the cell in the cell control group has good growth state and no fluorescence; cells in the infected virus control group are diseased, most cells fluoresce, and cells added with the recombinant duck alpha interferon protein have the capability of resisting virus infection. According to the protection effect of duck alpha interferon on DEF cells, observing the pathological change degree of the cells, and calculating the titer of the interferon according to a Reed-Muench method when green fluorescent cells appear, wherein the hole cells are marked as "+". The detection results are shown in Table 1, and the antiviral activity measurement results show that the DuIFN-alpha-O expressed in silkworm larvae has more remarkable antiviral activity, and the potency reaches 1.14X10 6 U/mL, achieves the desired effect, indicating that an optimized approach to DuIFN-alpha antiviral activity on the DuIFN-alpha gene is feasible and effective.
Table 1 detection results of antiviral activity after optimization of duck interferon alpha mutant
EXAMPLE 3 expression and detection in a silkworm bioreactor after DuIFN-alpha-O was subjected to amino acid unit point mutation
1. Experimental method
1.1 construction of Duck interferon-alpha mutant Gene
The invention designs a plurality of pairs of primer pair sequences to carry out site-directed mutagenesis by taking the gene sequence of DuIFN-alpha-O as a template, wherein the site-directed mutagenesis is carried out by utilizing a fusion PCR method, and the fusion PCR method is shown in the '2' and the experimental method.
The mutation sites were P2A, L19T, P25L, R35P, D38N, N D, H64N, L I, Q80R, D95K, H D, Q117R, H123Y, R127Q, L S, R C, I148T, L F, D E or R182C respectively; the resulting duck interferon alpha mutant was designated as DuIFN-alpha-O-M1 (P2A, L19T, P25L, R35P, D38N, N D, H64N, L I, Q80R, D95K, H D, Q117R, H123Y, R127Q, L S, R C, I148T, L159F, D E or R182C) mutant.
Primers required for single-site, double-site and multi-site mutation of amino acid by DuIFN-alpha-O nucleotide sequence:
(1) Two side upstream and downstream primers:
F:TCATACCGTCCCACCATCGGGCGCGGATCCGCCAACATGCCTGGACCATCAG(SEQ ID NO.14)
R:GATCTGCAGCGGCCGCTCCGGAATTCTTATCTCATAGTTCTTGTCAATC(SEQ ID NO.15)
(2) Intermediate upstream and downstream primers
P2A primers are both ends of the entire strand
F1：TCATACCGTCCCACCATCGGGCGCGGATCCGCCAACATGGCTGGACCATCAG(SEQ ID NO.16)
R1：GATCTGCAGCGGCCGCTCCGGAATTCTTATCTCATAGTTCTTGTCAATC(SEQ ID NO.17)
2.
F2：GCTTTGGCCCTGACGCTCCTGTTGAC(SEQ ID NO.18)
R2：GTCAACAGGAGCGTCAGGGCCAAAGC(SEQ ID NO.19)
3.
F3：GTTGACCCCTCTAGCTAACGCCTTCAGC(SEQ ID NO.20)
R3：GCTGAAGGCGTTAGCTAGAGGGGTCAAC(SEQ ID NO.21)
4.
F4：GCTGCTCCCCGTTGCCACTCCACGACTC(SEQ ID NO.22)
R4：GAGTCGTGGAGTGGCAACGGGGAGCAGC(SEQ ID NO.23)
5.
F5：GAGACTCCACAACTCGGCTTTCGCCTGG(SEQ ID NO.24)
R5：CAGGCGAAAGCCGAGTTGTGGAGTCTCAACG(SEQ ID NO.25)
6.
F6：GCAACTCCTGAGAGATATGGCTCCTAGC(SEQ ID NO.26)
R6：GCTAGGAGCCATATCTCTCAGGAGTTGC(SEQ ID NO.27)
7.
F7：CACAACAGAATGCTCCGTGTTCCTTCCCCGAC(SEQ ID NO.28)
R7：GAACACGGAGCATTCTGTTGTGGGCAAGGTTG(SEQ ID NO.29)
8.
F8：CGACACAATACTCGACACCAACGATACACAAC(SEQ ID NO.30)
R8：CGTTGGTGTCGAGTATTGTGTCGGGGAAGGAAC(SEQ ID NO.31)
9.
F9：CAACGATACACGACAGGCTGCCCACACTGC(SEQ ID NO.32)
R9：GTGGGCAGCCTGTCGTGTATCGTTGGTGTCG(SEQ ID NO.33)
10.
F10：GCAACACCTCTTCAAGACTCTGTCATCTC(SEQ ID NO.34)
R10：GAGATGACAGAGTCTTGAAGAGGTGTTGC(SEQ ID NO.35)
11.
F11：GCTCACTGGCTGGACACTGCCAGACACG(SEQ ID NO.36)
R11：CGTGTCTGGCAGTGTCCAGCCAGTGAGC(SEQ ID NO.37)
12.
F12：CGATCTCCTGAATCGATTGCAGCACCACA(SEQ ID NO.38)
R12：GCTGCAATCGATTCAGGAGATCGTGTCTG(SEQ ID NO.39)
13.
F13：GCAGCACCACATATACCACCTCGAAAGATGC(SEQ ID NO.40)
R13：GCATCTTTCGAGGTGGTATATGTGGTGCTGC(SEQ ID NO.41)
14.
F14：CATACACCACCTCGAACAATGCTTCCCTGC(SEQ ID NO.42)
R14：GCGTCAGCAGGGAAGCATTGTTCGAGGTGG(SEQ ID NO.43)
15.
F15：CGCTGCCAGATCGCACAGAAGAGGTCCAAG(SEQ ID NO.44)
R15：CTCTTCTGTGCGATCTGGCAGCGTCAGCAG(SEQ ID NO.45)
16.
F16：GAGGTCCATGCAACCTGCACTTGTCAATC(SEQ ID NO.46)
R16：GTGCAGGTTGCATGGACCTCTTCTGTGC(SEQ ID NO.47)
17.
F17：GCACTTGTCAACCAACAAATACTTCGGATG(SEQ ID NO.48)
R17：CGAAGTATTTGTTGGTTGACAAGTGCAGG(SEQ ID NO.49)
18.
F18：GTATTCAACACTTCTTCCAGAACCACAC(SEQ ID NO.50)
R18：GTGGTTCTGGAAGAAGTGTTGAATACATC(SEQ ID NO.51)
19.
F19：CATGCGCCTGGGAACACGTGAGACTGGAAG(SEQ ID NO.52)
R19：CTCACGTGTTCCCAGGCGCATGGAGAGTAG(SEQ ID NO.53)
20.
F20：GCTCACGCCTGTTTCCAGTGCATCCACAG(SEQ ID NO.54)
R20：CTGTGGATGCACTGGAAACAGGCGTGAGC(SEQ ID NO.55)
1.2 construction of recombinant baculovirus transfer vectors
The specific operation was the same as in example 1. The plasmid sequenced correctly was designated pVL-DuIFN-. Alpha. -O-M1 (P2A, L19T, P L, R35P, D38N, N D, H64N, L I, Q80R, D95K, H108D, Q117R, H123Y, R127Q, L S, R142 148T, L159F, D E or R182C).
1.3 obtaining, purifying and amplifying recombinant silkworm baculovirus
As in example 1.
Recombinant virus rBmBacmid (DuIFN-. Alpha. -O-M1) containing the target gene was obtained by cotransfection.
The pure recombinant silkworm baculovirus rBmBacmid (DuIFN-alpha-O-M1) is obtained by purification.
Recombinant silkworm baculovirus rBmBacmid (DuIFN-alpha-O-M1) is infected with BmN cells which normally grow, the supernatant is collected after 3 days of culture, and the supernatant contains a large amount of recombinant virus rBmBacmid (DuIFN-alpha-O-M1).
1.4 Duck interferon alpha mutant expression in vivo in silkworm
As in example 1.
1.5 preparation of Duck Embryo Fibroblast (DEF) and Duck interferon alpha antiviral Activity detection
As in example 1.
2. Experimental results
2.1 identification of recombinant transfer vectors
The recombinant transfer vector pVL-DuIFN-alpha-O-M1 is digested with BamHI and EcoRI, 2 fragments are separated by 1% agarose gel electrophoresis, the small fragment is located between 500 and 750bp, the size of the fragment corresponds to 576bp of the target gene, the large fragment is located above 8000bp, and the size of the fragment corresponds to 9607bp of pVL 1393. The plasmid with correct enzyme digestion identification is sent to Beijing qing new industry biotechnology Co-Ltd for nucleotide sequencing, and the MegaAlign comparison result shows that the sequence is consistent with the originally designed sequence, and the duck alpha interferon mutant gene is successfully inserted between BamHI and EcoRI in the pVL1393 transfer vector.
2.2 Duck interferon-alpha recombinant virus acquisition and detection of recombinant products
The antiviral activity of duck alpha interferon expressed by silkworm larvae is detected on a DEF/VSV GFP system by using a micro cytopathic inhibition method. Observed under an inverted fluorescence microscope, the cell in the cell control group has good growth state and no fluorescence; cells in the infected virus control group are diseased, most cells fluoresce, and cells added with the recombinant duck alpha interferon protein have the capability of resisting virus infection. Protection of DEF cells according to duck interferon alphaThe effect was observed, the pathological changes of the cells were observed, the cells were counted as "+" when green fluorescent cells appeared, the interferon titers were calculated according to the Reed-Muench method, the detection results are shown in Table 2, and the titers were measured at 7.80X 10 for all duck interferon alpha mutants 4 U/mL～3.16×10 6 U/mL, wherein after the mutation of the 6 sites P25L, N51D, Q80R, H108D, L136S and D170E, the titer of the expressed duck alpha interferon is slightly higher than the titer measured by the expression of a conserved sequence, and the titer is unchanged or even reduced after the mutation of the other sites, which indicates that the mutation of the 6 sites is effective mutation, and the aim of improving the antiviral activity of DuIFN-alpha can be achieved. Wherein the DuIFN-alpha-O-N51D mutant has the strongest antiviral effect, the amino acid sequence of the DuIFN-alpha-O-N51D mutant is shown as SEQ ID NO.5, and the nucleotide sequence of the coding gene is shown as SEQ ID NO. 6.
TABLE 2 detection results of antiviral Activity of recombinant Duck interferon alpha Single Point mutations
EXAMPLE 4 expression and detection of DuIFN-alpha-O-M1 mutant in silkworm bioreactor after amino acid double site mutation
1. Experimental method
1.1 construction of Duck interferon-alpha mutant Gene
In view of the results of example 3, it was determined that partial site mutations were effective mutations, and the objective of improving the antiviral activity of the DuIFN-. Alpha. -O mutant could be achieved. In view of the fact that the amino acid sequence is the primary structure of the protein and determines the higher structure of the protein, and that the amino acid unit site mutation performed in example 3 may have positions of partial mutation sites associated with each other, an amino acid double site mutation was attempted. The invention combines single mutation sites P25L, N D, Q80R, H108D, L136S and D170E with improved antiviral activity two by two to carry out double site mutation, wherein the double site mutation is based on the single site mutation sequence obtained in the example 3, the double site mutation (DuIFN-alpha-O-M1) is used as a template, the corresponding primer (see the example 3 for details) is utilized to carry out second site mutation by a fusion PCR method, thus obtaining a target fragment of the double site mutation, and the fusion PCR method is shown in the previous "2 and experimental method".
The double mutation site is 15 combinations of P25L-N51D, P L-Q80 5625L-H108D, P L-L136S, P L-D170E, N D-Q80R, N D-H108D, N D-L136S, N D-D170E, Q R-H108D, Q R-L136S, Q R-D170E, H D-L136S, H108D-D170E or L136S-D170E, and the obtained duck interferon alpha mutant is named as DuIFN-alpha-O-M1-M2 (P25L-N51D, P L-Q80R, P25L-H108D, P L-L136S, P L-D170E, N D-Q80R, N D-H108D, N D-L136S, N D-D170R-D170E, Q R-L E, Q R-D170E, Q D-D170E 170D 170E) or DuIFN alpha-O-M1-M170D 170E.
1.2 construction of recombinant baculovirus transfer vectors
The procedure is as in example 1. The plasmid sequenced correctly was designated pVL-DuIFN-. Alpha. -O-M1-M2 (P25L-N51D, P L-Q80R, P L-H108D, P L-L136S, P L-D170E, N D-Q80R, N D-H108D, N D-L136S, N D-D170E, Q R-H108D, Q R-L136S, Q R-D170E, H D-L136S, H D-D170E or L136S-D170E).
1.3 obtaining, purifying and amplifying recombinant silkworm baculovirus
As in example 1.
Recombinant virus rBmBacmid (DuIFN-. Alpha. -O-M1-M2) containing the target gene was obtained by cotransfection.
The pure recombinant silkworm baculovirus rBmBacmid (DuIFN-alpha-O-M1-M2) is obtained by purification.
Recombinant silkworm baculovirus rBmBacmid (DuIFN-alpha-O-M1-M2) is infected with BmN cells which grow normally, the supernatant is collected after 3 days of culture, and the supernatant contains a large amount of recombinant virus rBmBacmid (DuIFN-alpha-O-M1-M2).
1.4 Duck interferon alpha mutant expression in vivo in silkworm
As in example 1.
1.5 preparation of Duck Embryo Fibroblast (DEF) and Duck interferon alpha antiviral Activity detection
As in example 1.
2. Experimental results
2.1 identification of recombinant transfer vectors
The recombinant transfer vector pVL-DuIFN-alpha-O-M1-M2 is digested with BamHI and EcoRI, 2 fragments are separated by 1% agarose gel electrophoresis, the small fragment is located between 500 and 750bp, the size of the fragment corresponds to 576bp of the target gene, the large fragment is located above 8000bp, and the size of the fragment corresponds to 9607bp of pVL 1393. The plasmid with correct enzyme digestion identification is sent to Beijing qing new industry biotechnology Co-Ltd for nucleotide sequencing, and the MegaAlign comparison result shows that the sequence is consistent with the originally designed sequence, and the duck alpha interferon mutant gene is successfully inserted between BamHI and EcoRI in the pVL1393 transfer vector.
2.2 Duck interferon-alpha recombinant virus acquisition and detection of recombinant products
The antiviral activity of duck alpha interferon expressed by silkworm larvae is detected on a DEF/VSV GFP system by using a micro cytopathic inhibition method. Observed under an inverted fluorescence microscope, the cell in the cell control group has good growth state and no fluorescence; cells in the infected virus control group are diseased, most cells fluoresce, and cells added with the recombinant duck alpha interferon protein have the capability of resisting virus infection. According to the protection effect of duck interferon alpha on DEF cells, observing the pathological changes of the cells, and when green fluorescent cells appear, marking the cell as "+", calculating the interferon titer according to a Reed-Muench method, wherein the detection result is shown in Table 3, and the measured titers of all duck interferon alpha mutants are 8.01X10 4 U/mL～4.50×10 6 U/mL, wherein after three groups of double mutations of P25L-Q80R, N D-H108D, N D-D170E, the titer of the expressed duck alpha interferon is slightly higher than that measured by the expression of a conserved sequence and a single mutation sequence, and the titers of the other groups of sites are unchanged or even reduced after mutation, which indicates that the mutation of the 3 combined sites is effective mutation, and the aim of improving the antiviral activity of the DuIFN-alpha-O-M1 mutant can be achieved. Wherein the DuIFN-alpha-O-N51D-H108D mutant has the strongest antiviral effect, the amino acid sequence is shown as SEQ ID NO.7, and the nucleotide sequence of the encoding gene isSEQ ID NO. 8.
Table 3 detection results of antiviral Activity of recombinant Duck interferon alpha double site mutation
EXAMPLE 5 expression and detection in a silkworm bioreactor after amino acid multiple site mutation of DuIFN-. Alpha. -O-M1-M2 mutant
1. Experimental method
1.1 construction of Duck interferon-alpha mutant Gene
In view of the results of example 4, considering that the order of amino acids is the primary structure of a protein and determining the higher structure of a protein, it is assumed that the amino acid multiple site mutation is attempted because the amino acid single site mutation is performed with the positions of the partial mutation sites being closely related to each other. The invention combines the obtained double mutation sites with high titer to determine a third mutation site, the multi-site mutation is based on the double mutation sequence obtained in the example 4, the (DuIFN-alpha-O-M1-M2) is used as a template, the corresponding primer (see the example 3 for details) is used for carrying out the site-directed mutation of the third site by a fusion PCR method, thus obtaining the target fragment of the multi-site mutation, and the fusion PCR method is shown in the previous "2 and experimental method".
The following combinations were obtained: P25L-N51D-Q80R, P L-Q80R-H108D, P L-Q80R-D170E, P L-N51D-H108D, N D-Q80R-H108D, N D-H108D-D170E, P L-N51D-D170E, N D-Q80R-D170E, P L-N51D-Q80R-H108D, N D-Q80R-H108D-D170E, P L-N51D-H108D-D170E or P25L-N51D-Q80R-H108D-D170E. The obtained duck interferon-alpha mutant was named DuIFN-alpha-O-M1-M2-M3 (P25L-N51D-Q80R-R, P L-Q80R-H108D, P L-Q80R-D170E, P L-N51D-H108D, N D-Q80R-H108D, N D-H108D-D170E, P25L-N51D-D170E, N D-Q80R-D170E) or DuIFN-alpha-O-M1-M2-M3-M4 (P25L-N51D-Q80R-H108D, N D-Q80R-H108D-D170E, P L-N51D-H108D-D170E) or DuIFN-alpha-O-M1-M2-M3-M4-M5 (P25L-N51D-Q80R-M108D-D170E).
1.2 construction of recombinant baculovirus transfer vectors
As in example 1.
The plasmids which were sequenced correctly were designated pVL-DuIFN-. Alpha. -O-M1-M2-M3 (P25L-N51D-Q80R, P L-Q80R-H108D, P L-Q80R-D170E, P L-N51D-H108D, N D-Q80R-H108D, N D-H108D-D170E, P25L-N51D-D170E, N D-Q80R-D170E) or pVL-DuIFN-. Alpha. -O-M1-M2-M3-M4 (P25L-N51D-Q80R-H108D, N D-Q80R-H108D 170E, P L-N51D-H108D-D170E) or pVL-DuIFN-. Alpha. -O-M1-M2-M3-M4-M5 (P25L-N51D-Q80R-H108D 170E).
1.3 obtaining, purifying and amplifying recombinant silkworm baculovirus
See example 1 for specific operation.
Recombinant viruses rBm-Bacmid (DuIFN-. Alpha. -O-M1-M2-M3 or DuIFN-. Alpha. -O-M1-M2-M3-M4-M5) containing the target gene were obtained by cotransfection.
The recombinant silkworm baculovirus rBm-Bacmid (DuIFN-alpha-O-M1-M2-M3 or DuIFN-alpha-O-M1-M2-M3-M4-M5) was purified.
Recombinant silkworm baculovirus rBm-Bacmid (DuIFN-alpha-O-M1-M2-M3 or DuIFN-alpha-O-M1-M2-M3-M4-M5) was infected with BmN cells which grew normally, and after 3 days of culture, the supernatant was collected and contained a large amount of recombinant virus rBm-Bacmid (DuIFN-alpha-O-M1-M2-M3 or DuIFN-alpha-O-M3-M4 or DuIFN-alpha-O-M1-M2-M3-M4-M5).
1.4 Duck interferon alpha mutant expression in vivo in silkworm
As in example 1.
1.5 preparation of Duck Embryo Fibroblast (DEF) and Duck interferon alpha antiviral Activity detection
As in example 1.
2. Experimental results
2.1 identification of recombinant transfer vectors
The recombinant transfer vector pVL-DuIFN-alpha-O-M1-M2-M3 or pVL-DuIFN-alpha-O-M1-M2-M3-M4-M5 is digested with BamHI and EcoRI, 2 fragments are separated by 1% agarose gel electrophoresis, the small fragment is between 500 and 750bp, the size of the small fragment is consistent with the size of 576bp of the target gene fragment, the large fragment is above 8000bp, and the size of the large fragment is consistent with the size of 9607bp of the pVL1393 fragment. The plasmid with correct enzyme digestion identification is sent to Beijing qing new industry biotechnology Co-Ltd for nucleotide sequencing, and the MegaAlign comparison result shows that the sequence is consistent with the originally designed sequence, and the duck alpha interferon mutant gene is successfully inserted between BamHI and EcoRI in the pVL1393 transfer vector.
2.2 Duck interferon-alpha recombinant virus acquisition and detection of recombinant products
The antiviral activity of duck alpha interferon expressed by silkworm larvae is detected on a DEF/VSV GFP system by using a micro cytopathic inhibition method. Observed under an inverted fluorescence microscope, the cell in the cell control group has good growth state and no fluorescence; cells in the infected virus control group are diseased, most cells fluoresce, and cells added with the recombinant duck alpha interferon protein have the capability of resisting virus infection. According to the protection effect of duck alpha interferon on DEF cells, observing the pathological changes of the cells, and when green fluorescent cells appear, marking the cell as "+", calculating the interferon titer according to a Reed-Muench method, wherein the detection result is shown in Table 4, and the measured titers of all duck alpha interferon mutants are 2.51X10 5 U/mL～5.78×10 6 U/mL, wherein after five-site P25L-N51D-Q80R-H108D-D170E mutation, the titer of the expressed duck alpha interferon is far higher than that measured by the expression of a conserved sequence, a single mutation sequence and a double mutation sequence, and is 5.78X10 6 U/mL. The potency of the mutant of the rest groups of sites is unchanged or even reduced, which indicates that the mutation of the combined sites is effective mutation, and the aim of improving the antiviral activity of the DuIFN-alpha-O-M1-M2 mutant can be achieved. The amino acid sequence of the DuIFN-alpha-O-P25L-N51D-Q80R-H108D-D170E mutant is shown as SEQ ID NO.9, and the nucleotide sequence of the encoding gene is shown as SEQ ID NO. 10.
TABLE 4 detection results of antiviral Activity of recombinant Duck interferon alpha multiple site mutations
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Sequence listing
<110> Jiangsu university of science and technology
<120> duck alpha interferon, mutant thereof, preparation method and application
<160> 55
<170> SIPOSequenceListing 1.0
<210> 1
<211> 191
<212> PRT
<213> Duck (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (Artificial Sequence)
<400> 11
ttatctcata gttcttgtca 20
<210> 12
<211> 33
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 12
cgggatccgc caacatgcct ggaccatcag ctc 33
<210> 13
<211> 31
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 13
cggaattctt atctcatagt tcttgtcaat c 31
<210> 14
<211> 52
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 14
tcataccgtc ccaccatcgg gcgcggatcc gccaacatgc ctggaccatc ag 52
<210> 15
<211> 49
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 15
gatctgcagc ggccgctccg gaattcttat ctcatagttc ttgtcaatc 49
<210> 16
<211> 52
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 16
tcataccgtc ccaccatcgg gcgcggatcc gccaacatgg ctggaccatc ag 52
<210> 17
<211> 49
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 17
gatctgcagc ggccgctccg gaattcttat ctcatagttc ttgtcaatc 49
<210> 18
<211> 26
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 18
gctttggccc tgacgctcct gttgac 26
<210> 19
<211> 26
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 19
gtcaacagga gcgtcagggc caaagc 26
<210> 20
<211> 28
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 20
gttgacccct ctagctaacg ccttcagc 28
<210> 21
<211> 28
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 21
gctgaaggcg ttagctagag gggtcaac 28
<210> 22
<211> 28
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 22
gctgctcccc gttgccactc cacgactc 28
<210> 23
<211> 28
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 23
gagtcgtgga gtggcaacgg ggagcagc 28
<210> 24
<211> 28
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 24
gagactccac aactcggctt tcgcctgg 28
<210> 25
<211> 31
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 25
caggcgaaag ccgagttgtg gagtctcaac g 31
<210> 26
<211> 28
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 26
gcaactcctg agagatatgg ctcctagc 28
<210> 27
<211> 28
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 27
gctaggagcc atatctctca ggagttgc 28
<210> 28
<211> 32
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 28
cacaacagaa tgctccgtgt tccttccccg ac 32
<210> 29
<211> 32
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 29
gaacacggag cattctgttg tgggcaaggt tg 32
<210> 30
<211> 32
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 30
cgacacaata ctcgacacca acgatacaca ac 32
<210> 31
<211> 33
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 31
cgttggtgtc gagtattgtg tcggggaagg aac 33
<210> 32
<211> 30
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 32
caacgataca cgacaggctg cccacactgc 30
<210> 33
<211> 31
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 33
gtgggcagcc tgtcgtgtat cgttggtgtc g 31
<210> 34
<211> 29
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 34
gcaacacctc ttcaagactc tgtcatctc 29
<210> 35
<211> 29
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 35
gagatgacag agtcttgaag aggtgttgc 29
<210> 36
<211> 28
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 36
gctcactggc tggacactgc cagacacg 28
<210> 37
<211> 28
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 37
cgtgtctggc agtgtccagc cagtgagc 28
<210> 38
<211> 29
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 38
cgatctcctg aatcgattgc agcaccaca 29
<210> 39
<211> 29
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 39
gctgcaatcg attcaggaga tcgtgtctg 29
<210> 40
<211> 31
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 40
gcagcaccac atataccacc tcgaaagatg c 31
<210> 41
<211> 31
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 41
gcatctttcg aggtggtata tgtggtgctg c 31
<210> 42
<211> 30
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 42
catacaccac ctcgaacaat gcttccctgc 30
<210> 43
<211> 30
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 43
gcgtcagcag ggaagcattg ttcgaggtgg 30
<210> 44
<211> 30
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 44
cgctgccaga tcgcacagaa gaggtccaag 30
<210> 45
<211> 30
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 45
ctcttctgtg cgatctggca gcgtcagcag 30
<210> 46
<211> 29
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 46
gaggtccatg caacctgcac ttgtcaatc 29
<210> 47
<211> 28
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 47
gtgcaggttg catggacctc ttctgtgc 28
<210> 48
<211> 30
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 48
gcacttgtca accaacaaat acttcggatg 30
<210> 49
<211> 29
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 49
cgaagtattt gttggttgac aagtgcagg 29
<210> 50
<211> 28
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 50
gtattcaaca cttcttccag aaccacac 28
<210> 51
<211> 29
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 51
gtggttctgg aagaagtgtt gaatacatc 29
<210> 52
<211> 30
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 52
catgcgcctg ggaacacgtg agactggaag 30
<210> 53
<211> 30
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 53
ctcacgtgtt cccaggcgca tggagagtag 30
<210> 54
<211> 29
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 54
gctcacgcct gtttccagtg catccacag 29
<210> 55
<211> 29
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 55
ctgtggatgc actggaaaca ggcgtgagc 29

Claims (4)

1. The duck alpha interferon mutant is characterized in that the amino acid sequence of the duck alpha interferon is subjected to L136S amino acid unit point mutation to obtain the mutant; wherein the amino acid sequence of the duck alpha interferon is shown as SEQ ID NO. 1.
2. A recombinant vector or recombinant host cell comprising the gene encoding the duck interferon alpha mutant of claim 1.
3. Use of the coding gene of the duck interferon alpha mutant as claimed in claim 1 in the preparation of a medicament or a reagent for preventing or treating duck viral diseases.
4. The use according to claim 3, wherein the duck viral disease comprises: the duck virus hepatitis, duck plague, gosling plague, muscovy duck parvovirus disease, young muscovy duck gosling plague, duck epidemic hemorrhagic disease, duck liver disease, duck virus encephalitis, duck reovirus infection, duck adenovirus infection, infectious bursal disease of duck, duck paramyxovirus disease, duck coronavirus infection, and duck influenza.
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Publication number Priority date Publication date Assignee Title
CN1861796A (en) * 2005-05-13 2006-11-15 东北农业大学 Process of preparing duck alpha-interferon expressing gene in bacillus coli and insect cell
CN101209345A (en) * 2006-12-26 2008-07-02 河南农业大学 Animal genetic engineering interferon alpha and gamma composite preparations and production method and clinical application thereof

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US5885567A (en) * 1993-10-22 1999-03-23 University Of Connecticut Treatment of infection in fowl by oral administration of avian interferon proteins
CN101870976B (en) * 2010-03-16 2012-05-09 南京农业大学 Gene synthesis duck alpha interferon gene and protein expression
CN102899331B (en) * 2012-09-25 2015-04-22 广东省农业科学院兽医研究所 Complex duck interferon-alpha gene, and recombinant vector and application thereof
CN109517779A (en) * 2019-01-28 2019-03-26 大连三仪动物药品有限公司 The building and its application of one plant weight group duck interferon-' alpha ' lactobacillus

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1861796A (en) * 2005-05-13 2006-11-15 东北农业大学 Process of preparing duck alpha-interferon expressing gene in bacillus coli and insect cell
CN101209345A (en) * 2006-12-26 2008-07-02 河南农业大学 Animal genetic engineering interferon alpha and gamma composite preparations and production method and clinical application thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
鸭α-干扰素的真核与原核表达及生物活性的比较;邹欢等;兽医科学(第1期);第118-120页 *

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