CN110256552B

CN110256552B - Duck lambda interferon mutant and preparation method and application thereof

Info

Publication number: CN110256552B
Application number: CN201910503363.XA
Authority: CN
Inventors: 胡小元; 张志芳; 刘兴健; 易咏竹; 李轶女; 王朋
Original assignee: Biotechnology Research Institute of CAAS
Current assignee: Biotechnology Research Institute of CAAS
Priority date: 2019-06-11
Filing date: 2019-06-11
Publication date: 2021-02-19
Anticipated expiration: 2039-06-11
Also published as: CN110256552A

Abstract

The invention discloses a duck lambda interferon mutant and a preparation method and application thereof. The invention firstly determines that the amino acid sequence shown in SEQ ID NO.1 is the duck lambda interferon original amino acid sequence, compares all amino acid sequences of duck lambda interferon to obtain a duck lambda interferon conserved sequence shown in SEQ ID NO.3, and performs codon optimization on a coding gene to obtain an optimized gene shown in SEQ ID NO. 5. The invention also carries out amino acid single site or multi-site mutation on the conserved sequence to obtain a plurality of duck lambda interferon mutants with improved antiviral activity. The duck lambda interferon or the mutant thereof is further expressed in a silkworm bioreactor by using a silkworm baculovirus expression system, the antiviral activity of the obtained mutant is greatly improved, the mutant can be used for preventing or treating duck viral diseases, and the mutant has obvious defense and inhibition effects on viral infectious diseases which have great threat in the duck breeding industry.

Description

Duck lambda interferon mutant and preparation method and application thereof

Technical Field

The invention relates to poultry interferon and a mutant thereof, in particular to duck lambda interferon and a mutant thereof, and also relates to a method for preparing the duck lambda interferon mutant by using a silkworm baculovirus expression system and application of the duck lambda interferon mutant in preventing or treating duck viral diseases, belonging to the field of preparation and application of the duck lambda interferon mutant.

Background

Interferon is a cytokine with broad spectrum of biological activities such as antiviral, anti-parasitic to intracellular bacteria, anti-tumor, and regulating immune function. The IFN protein family is classified into type I, type II and type III interferons according to the sequence of its coding gene, chromosomal localization and receptor specificity. The type I interferons include IFN-alpha, IFN-beta, IFN-omega, IFN-delta, IFN-epsilon, IFN-zeta, IFN-tau, and the like, and in mammals primarily IFN-alpha and IFN-beta. Wherein IFN-alpha has more than 23 subtypes, each subtype has similar structural characteristics, the molecular weight is between 19 and 26KDa, and the two disulfide bonds are contained and combined with IFNAR1 and IFNAR2 receptor systems. The I-type interferon has strong antiviral activity, mainly inhibits the multiplication of viruses by interfering the replication of the viruses, and also has the functions of resisting tumors and regulating immunity. Type II interferons have only one member of IFN-gamma, also known as immunointerferons, a molecular weight between 20 and 25kDa, no disulfide bonds, no monomer activity, an activated form of dimer or tetramer, and their receptors are IFNGR1 and IFNGR 2. The main function is to activate macrophages to kill microorganisms. Type III interferons are newly discovered cytokines including λ 1(IL-29), λ 2(IL-28a), λ 3(IL-28b) and λ 4, and the specific binding receptor is IFNLR, comprising two subunits IFNLR1(IL28RA) and IL10RB, where IL10RB is ubiquitously expressed and IFNLR1 is expressed primarily in epithelial cells, hepatocytes and certain immune cells, so these cells can respond to type III interferons. Type III interferons are closely related to type I interferons, but have specific physiological functions, such as stimulating the activation and expression of Major Histocompatibility Complex (MHC) molecules, modulating innate and acquired immunity, and the like. Because of its broad-spectrum antiviral and antitumor activities and strong immunoregulation action, interferon has become one of the research hotspots in the relevant fields of virology, cytology, molecular biology, clinical medicine, immunology, oncology, etc.

Interferons were first found in birds, but studies on duck interferon molecular levels began later compared to humans and other mammals, and were relatively rare compared to chickens. In recent years, the infectious diseases such as influenza, newcastle disease, infectious bronchitis, infectious laryngotracheitis, infectious bursal disease, duck plague, duck hepatitis and Marek, which are caused by poultry virus infection, are becoming more and more serious, which brings huge economic loss to the poultry industry every year, and seriously restricts the healthy development of the poultry industry, and how to effectively prevent and treat the viral infectious diseases of poultry is always the key point in the prevention and treatment research of poultry diseases. The duck interferon has the effects of preventing and inhibiting viral infectious diseases which have great threat in the duck breeding industry, brings a new hope for preventing and treating the viral diseases, provides favorable safety guarantee conditions for the duck breeding industry, and expands a wider space for developing the duck interferon through application research. Duck interferon has spectral antiviral effect, and can indirectly inhibit virus replication by directly activating immune cell nucleus to achieve antiviral effect; meanwhile, it also has the effects of resisting bacteria, rickettsia, chlamydia and protozoon parasitized in cells. Directly inhibiting intracellular bacteria and the like mainly by down-regulating a transferrin receptor; the immunity enhancing function is enhanced by stimulating NK cells and activated macrophages, directly promoting T, B cell differentiation and CTL maturation, and stimulating B cells to secrete antibodies, thereby enhancing the immune function of the organism.

The alpha interferon gene of duck was first obtained by screening clones by German researchers Schultz et al 1995 using the reported ChIFN-alpha gene as a probe. The alpha interferon gene of Beijing duck is reported in summer and spring 2000 by domestic researchers, the alpha interferon genes of various ducks such as Muscovy duck, Shaoxing duck, sheldrake and the like are subsequently reported by other researchers, in addition, the resistance effect of the recombinant duck alpha interferon to various viruses is successively reported by the researchers, and Schultz and the like prove that the recombinant interferon has the effect of resisting the virus of water-soaking stomatitis, influenza A virus, duck hepatitis virus and Newcastle disease virus; in 2007, Zhouyou and the like, after the cherry valley duck is injected with the plasmid with the duck alpha interferon gene, the duck shows a certain capability of resisting duck plague virulent infection; the resistance effect of 2-day-old ducks to highly pathogenic avian influenza virus H5N1 is researched by Pei Gao et al in 2018 by treating the 2-day-old ducks with the recombinant duck alpha interferon, and the result shows that the death rate of the ducks in the test group is reduced from 60% to 10% compared with that in the virus control group, which indicates that the recombinant duck alpha interferon has the resistance effect on the 2-day-old ducks infected with the avian influenza virus H5N 1. Yao et al cloned and expressed Peking duck type III interferon IFN-lambda in 293T cells in 2014, and research shows that recombinant duck lambda interferon can up-regulate mRNA levels of OASL and Mx-1 in primary duck liver cells, is closely related to antiviral defense and inflammatory reaction, but does not report activity of duck type III interferon IFN-lambda.

Disclosure of Invention

The invention aims to solve the first technical problem of providing duck lambda interferon and a mutant of duck lambda interferon, wherein the duck lambda 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 lambda interferon or the duck lambda interferon mutant by using a bombyx mori baculovirus expression system;

the third technical problem to be solved by the invention is to provide the duck lambda interferon or the duck lambda interferon mutant for preventing or treating duck viral diseases.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows:

the invention analyzes all duck lambda interferon amino acid sequences on NCBI, finally determines that the amino acid sequence shown by SEQ ID NO.1 is duck lambda interferon original amino acid sequence (namely DuIFN-lambda), and the polynucleotide sequence of the coding gene is shown as (a) or (b) or (c):

(a) the polynucleotide sequence shown in SEQ ID No. 2; or (b) a polynucleotide sequence which hybridizes to the complement of SEQ ID No.2 under stringent hybridization conditions, wherein the polynucleotide encodes a protein which still has the function or activity of an interferon; or (c) a polynucleotide sequence having at least more than 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 coded by the polynucleotide still has the function or activity of interferon; more preferably, the polynucleotide sequence has 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.

The invention further obtains the conserved sequence of the duck lambda interferon by comparing all amino acid sequences of the duck lambda interferon, wherein the amino acid sequence of the conserved sequence is shown as SEQ ID No.3, and the nucleotide sequence of the coding gene is shown as SEQ ID No. 4.

On the basis of a duck lambda interferon conserved amino acid sequence (shown as SEQ ID NO. 3), the gene sequence is optimized according to the codon preference of silkworm, multiple related parameters which influence the transcription efficiency, the translation efficiency and the GC content, the CpG dinucleotide content, the codon preference, the secondary structure of mRNA, the free energy stability of mRNA, the RNA unstable gene sequence, the repetitive sequence and the like of the gene are optimally designed, and the finally translated protein sequence is kept unchanged; in order to improve the translation initiation efficiency in a silkworm baculovirus eukaryotic expression system, a Kozak sequence ACGAAC is added in front of the gene ATG. In addition, restriction sites such as BamHI, EcoRI, SmaI and the like in the gene sequence are removed, BamHI is added at the upstream of the gene, an EcoRI restriction site is added at the downstream of the gene, and the optimized gene (namely DuIFN-lambda-C-O) is obtained under the premise of keeping the amino acid sequence (namely shown in SEQ ID NO. 3) unchanged, wherein the nucleotide sequence of the optimized gene is shown in SEQ ID NO. 5. The antiviral activity results showed that the DuIFN-lambda titer expressed in the silkworm larvae is 1.56X 10⁶U/mL, optimized gene (DuIFN-lambda-C-O) titer of 2.58X 10⁶U/mL。

The invention further designs a plurality of pairs of primers by taking the gene sequence of DuIFN-lambda-C-O as a template, and performs amino acid single-site mutation and amino acid multi-site mutation by utilizing a fusion PCR method to obtain a plurality of duck lambda interferon mutants; on the basis of DuIFN-lambda-C-O, after 5 site single site mutations of S30T, N56S, I95V, Q136K or V166L are respectively carried out, the titer of the expressed duck lambda interferon is higher than that measured by DuIFN-lambda-C-O expression, and the antiviral titer reaches 8.58 multiplied by 10⁴U/mL～3.65×10⁶U/mL, and the potency is unchanged or even reduced after mutation of the rest sites, which indicates that the mutation of the 5 sites is effective mutation and can achieve improvementThe purpose of high antiviral activity; among them, DuIFN-lambda-C-O-I95V mutant obtained by I95V single-site mutation has the strongest antiviral effect.

The amino acid single-site mutation 'S30T' of the invention means that the 30 th amino acid of the duck lambda interferon mutant with the amino acid sequence shown as SEQ ID NO.3 is mutated from serine (S) to threonine (T); "N56S" indicates that the 56 th amino acid is mutated from asparagine (N) to serine (S); the expression of the remaining single-site mutations is analogized.

The invention further combines single mutation sites S30T, N56S, I95V, Q136K or V166L with improved antiviral activity, and carries out multi-site mutation on DuIFN-lambda-C-O; concretely, the duck lambda interferon mutant with the amino acid sequence shown as SEQ ID NO.3 is subjected to S30-N56, S30-I95, S30-Q136, S30-V166, N56-I95, N56-Q136, N56-V166, I95-Q136, I95-V166, Q136-V166, S30-N56-I95, S30-N56-Q136, S30-N56-V166, S30-I95-Q136, S30-I95-V166, S30-Q136-V166, N56-I95-Q136, N56-I95-V166, N56-Q136-V166, I95-Q136-V166, S30-N56-I95-Q136, S30-N56-I95-V166, S30-N56-Q136-V166, S30-I95-V166, S30-N56-V136-V166, S30-N56-Q136-V166, S30-I95-V166, S30-V, S30-V136-V166, S30-V-166, S30-V, A mutant obtained by multi-site mutation of any one amino acid of S30T-N56S-I95V-Q136K-V166L; the detection result of antiviral activity shows that after N56S-I95V, I95V-Q136K, N56S-I95V-Q136K and N56S-I95V-Q136K-V166L are subjected to multiple mutations, the titer of the expressed duck lambda interferon is higher than the titer measured by expression of a conserved sequence and a single mutation sequence, and the antiviral titer reaches 8.16 multiplied by 10⁴U/mL～5.01×10⁶U/mL, and the potency is unchanged or even reduced after mutation of other groups of sites, which indicates that the mutation of the 4 combined sites is effective mutation, and the aim of improving antiviral activity can be achieved; the DuIFN-lambda-S-C-N56S-I95V-Q136K-V166L mutant obtained by multi-site mutation of N56S-I95V-Q136K-V166L has the strongest antiviral effect, the amino acid sequence of the mutant is shown as SEQ ID No.6, and the nucleotide sequence of the coding gene of the mutant is shown as SEQ ID No. 7.

The amino acid multi-site mutation 'N56S-I95V' refers to that the 56 th amino acid of the duck lambda interferon mutant with the amino acid sequence shown as SEQ ID NO.3 is mutated into serine (S) from asparagine (N) and the 95 th amino acid is mutated into valine (V) from isoleucine (I); the amino acid multiple site mutation "I95V-Q136K" means that the 95 th amino acid is mutated from isoleucine (I) to valine (V) and the 136 th amino acid is mutated from glutamine (Q) to lysine (K); the remainder of the multi-site mutations are expressed and so on.

The invention also discloses a recombinant vector or a recombinant host cell containing the encoding gene of the duck lambda interferon or the duck lambda 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) a vector pVL-DuIFN-lambda containing duck lambda interferon (DuIFN-lambda) gene;

(2) the carrier pVL-DuIFN-lambda-C-O contains a gene sequence optimized by a duck lambda interferon conserved amino acid sequence gene DuIFN-lambda-C;

(3) vector pVL-DuIFN-. lambda. -C-O-M1 containing the gene sequence of the mutant of DuIFN-. lambda. -C-O-M1 following a single-site mutation of the amino acid sequence of DuIFN-. lambda. -C-O (DuIFN-. lambda. -C-O-M1 mutant);

(4) vector pVL-DuIFN-. lambda. -C-O-M2 containing the gene sequence of the mutant of DuIFN-. lambda. -C-O-M2 after multiple amino acid mutations of DuIFN-. lambda. -C-O.

The recombinant baculovirus obtained by the invention comprises: recombinant bombyx mori nuclear polyhedrosis virus rBmBacmid (DuIFN-lambda), rBmBacmid (DuIFN-lambda-C-O, DuIFN-lambda-C-O-M1, DuIFN-lambda-C-O-M2).

The invention also discloses application of the duck lambda interferon or the duck lambda interferon mutant in preparing a medicament or a reagent for preventing or treating duck viral diseases.

Wherein the duck viral diseases comprise: one or more of duck virus hepatitis, duck plague (duck viral enteritis), gosling plague, muscovy duck parvovirus disease, duck muscovy duck gosling plague, duck epidemic hemorrhage (black feather disease), duck hepatopathy, 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.

The invention further discloses a method for preparing the duck lambda interferon or the duck lambda interferon mutant, which comprises the following steps: (1) respectively cloning the encoding genes of the duck lambda interferon or the duck lambda interferon mutant into a baculovirus transfer vector to construct a recombinant transfer vector; (2) co-transfecting the recombinant transfer vector and baculovirus DNA into an insect cell to obtain recombinant baculovirus; (3) infecting the recombinant baculovirus into insect cells or insect hosts, culturing the infected insect cells or insect hosts to express corresponding protein, and purifying to obtain the recombinant baculovirus.

Wherein the baculovirus transfer vector is preferably 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, pALF 2, pAcMLF 7, cMLLF 8, pAPLcM, pAcMP2, pAc58RP 24, pAcRP25, pAcRW4, pAMAG, pAcUWl, pAcUW21, pAcUW2A, pAcUW2B, pAcUW 6862, pAcUcUpYnw 56, pAcVECWv 13972, pAmYNCV 13972, pApYNCV 3611, pApYNcVpYNcVpV, pApYVC 9872, pApYVEpV, pApJV 369, pApYVEpJV 3611, pApJV 3611, pAcJpAcJpAcJpAcJpAcJpAcJpAcJpAcJpAcJpAcJV 2, pAcJpAcJpAcJpAcJpAcJV 2, pAcJpAcJLF 7, pAcJpAcJV 7, pAcJpAcJpAcJpAcJpAcJpAcJV 7, pAcUpIVF 8, pAcJV, pAcJpAcJV, pAcJV, pAcUpNV III, pAcJV;

preferably, the baculovirus is selected from the group consisting of bombyx mori baculovirus parent strain BmBacmid, BmNPV, AcMNPV, ApNPV, HaNPV, HzNPV, LdMNPV, MbMNPV, OpMNPV, SlMNPV, SeMNPV or SpltNPV;

preferably, the insect host is selected from the group consisting of Bombyx mori (Bombyx mori), Bombyx mori (Bombyx mandarina), Ricinus communis (Philosamia cynthia ricim), Bombyx mori (Dictyyoproca japonica), Ailanthus altissima (Philosamia cynthia pryeri), Antheraea pernyi (Antheraea pernyi), Antheraea japonicus (Antheraea yamamai), Bombyx mori (Antheraea polyphylla), Autographa californica (Atoga californica), Ectropis obliqua (Ectropis obliqua), Trichoplusia glauca (Mameria brassica), Trichoplusia Spodoptera (Spodoptera litura), Trichoplusia fortunei (Spodopterocarpus sorethopa), Trichoplusia ni (Heliotropoides), Heliothis virescens (Heliothis virescens), Heliothis virescens (Helicosa), Helicoverpa armigera (Helicosa), or tobacco (tobacco), Helicoverpa armigera (Helicosa);

most preferably, the baculovirus transfer vector is pVL 1393; the baculovirus is a parent strain BmBacmid of silkworm baculovirus; the insect host is silkworm (Bombyx mori).

The infection refers to that the recombinant baculovirus infects 1-5-year-old insect larvae or pupae bodies through swallowing or permeating epidermis; preferably, the recombinant silkworm baculovirus is used for infecting silkworm cells or inoculating silkworm larvae or pupae of 1-5 years old by puncture, and body fluid or tissue homogenate of the silkworm larvae or pupae containing various duck lambda interferon genes is collected after infection for 3-6 days; wherein, the pupa is preferably early young pupa of 1-2 days.

Compared with the prior art, the technical scheme of the invention has the following beneficial effects:

the invention analyzes all duck lambda interferon amino acid sequences on NCBI, performs sequence comparison and signal peptide analysis to generate a conserved sequence, performs codon optimization on the conserved sequence, designs a plurality of pairs of primers by using the sequence after codon optimization as a template, and performs amino acid single-site mutation and amino acid multi-site mutation by using a fusion PCR method to obtain a plurality of duck lambda interferon mutants. The duck lambda interferon mutant is expressed in a silkworm bioreactor by using a silkworm baculovirus expression system, and the duck III type interferon IFN-lambda is expressed in a silkworm (pupa) body by using the silkworm baculovirus expression system so as to obtain a large amount of high-quality and low-cost duck III type interferon protein; the antiviral activity of the expressed duck lambda interferon mutant is greatly improved, and the expressed duck lambda interferon mutant has obvious antiviral activity; the duck lambda interferon mutant provided by the invention can be used for preparing a medicine or a reagent for preventing or treating duck viral diseases, and has great significance for the development of duck breeding industry.

Definitions of terms to which the invention relates

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 the reference nucleic acid 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, phosphoramidates, and the like). 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 specified. 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 base and/or deoxyinosine residues (Batzer et al, Nucleic Acid Res.19:5081 (1991); Ohtsuka et al, J.biol.chem.260: 2605-S2608 (1985); and Cassol et al (1992); Rossolini et al, Molcell.Probes8:91-98 (1994)).

The term "homology" refers to sequence similarity to a native nucleic acid sequence. "homology" includes a nucleotide sequence 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 as a percentage (%), which can be used to assess 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 upon 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 reverse complementary to each other when the sequences are viewed in both 5 'to 3' directions. It is also known in the art that two sequences that hybridize to each other under a given set of conditions do not necessarily have to be 100% perfectly complementary.

The term "stringent hybridization conditions" means conditions of low ionic strength and high temperature as known in the art. Typically, a probe hybridizes to its target sequence to a greater extent (e.g., at least 2-fold over background) than to other sequences under stringent conditions. Stringent hybridization conditions are sequence dependent and will be different under different environmental conditions, with longer sequences specifically hybridizing 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 an exhaustive guidance of Nucleic acid Hybridization, reference is made to the literature (Tijssen, Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic acids Probes, "Overview of principles of Hybridization and the" protocol of Nucleic acid assays. 1993). More specifically, the stringent conditions are typically selected to be about 5-10 ℃ below the thermal melting point (Tm) of the specific sequence at a defined ionic strength pH. The 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 (because the target sequence is present in excess, 50% of the probes are occupied at Tm at equilibrium). Stringent conditions may be as follows: 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 by the addition of destabilizing agents such as formamide. For selective or specific hybridization, the positive signal can be at least two times background hybridization, optionally 10 times background hybridization. Exemplary stringent hybridization conditions may be as follows: 50% formamide, 5 XSSC and 1% SDS, incubated at 42 ℃; or 5 XSSC, 1% SDS, incubated at 65 ℃, washed in 0.2 XSSC and washed in 0.1% SDS at 65 ℃. The washing may be for 5, 15, 30, 60, 120 minutes or more.

The terms "mutation" and "mutant" have their usual meanings herein, and refer to a genetic, naturally occurring or introduced change in a nucleic acid or polypeptide sequence, which has the same meaning as is commonly known to those of skill 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 for insertion to produce the recombinant host cell, e.g., 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 a new genetic marker due to the incorporation of foreign DNA.

Drawings

FIG. 1 is a fluorescence plot corresponding to a cytopathic ratio; wherein, a, "-": (ii) cell-free lesions; b, "+/-": several cytopathies; c, "+": 5% -15% of cytopathic effect; d, "+ +": 20-30% of cytopathic effect.

FIG. 2 is a double restriction enzyme identification of recombinant plasmid pVL-DuIFN-lambda; wherein, M: DNA molecular mass standard; 1: negative control; 2: recombinant plasmid pVL-DuIFN-lambda double digestion product.

FIG. 3 shows the cells showing various ratios of fluorescence; wherein, A: inhibition of VSV virus replication in cells by interferon did not show fluorescence; b: VSV virus in cells failed to suppress the fluorescence exhibited by low doses of interferon; c: most cells failed to inhibit the fluorescence exhibited by VSV virus after replication by interferon.

Detailed Description

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. It is to be understood that the described embodiments are exemplary only and are not limiting upon the scope of the invention. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be within the scope of the invention.

1. Test materials and reagents

Coli strain TOP10, BmN cells, VSV-GFP virus, all preserved and provided by the institute of biotechnology of Chinese academy of agricultural sciences; the experimental silkworm variety of Qiufeng multiplied by white jade is provided by the institute of silkworm industry of Jiangsu science and technology university, and the parental virus BmBacmid DNA is constructed according to the method disclosed in the literature (patent number: ZL 201110142492.4, authorization date: 2013.01.23). SPF duck embryos purchased from Beijing Meiliya Winton laboratory animals technology Ltd, restriction enzyme, T₄DNA ligase was purchased from Promega, LA Taq DNA polymerase and other related reagents used in PCR were purchased from Takara, liposomes were purchased from Invitrogen, DMEM cell culture medium, and fetal bovine serum was produced by GIBCO. Reference is made to the relevant tool books on the formulation of solutions and media (Joseph et al, third edition of molecular cloning guidelines, 2002; Oseber, et al, eds. molecular biology guidelines, 1998; David L.Spector, cell experiments guidelines, 2001); unless otherwise indicated, percentages and parts are by weight.

2. General experimental method

The fusion PCR method for site-directed mutagenesis in the experimental methods was performed by referring to the method described in Kuang Jatin et al (a new method for vector construction: recombinant fusion PCR method, genomics and applied biology, 2012, Vol. 31, No.6, p. 634-639).

In the experimental method, the titer of interferon was calculated using DEF/VSV GFP system using the Reed-Muench method, and the specific procedures were performed by referring to Liuxing, et al (detection of expression and bioactivity of cat omega-like interferon in silkworms, biotechnological advances 2015, 5(6): 441) and Summers MD, et al (A manual of methods for bacterial and animal cell culture products [ R ]. Texas Agricultural Experiment Station, 1987), wherein the criteria for judging cytopathic effect was described in reference to FIG. 1.

The best improvement in each case served as a comparison criterion for the improvement in the next case.

Example 1 expression and detection of Duck Lambda interferon Gene in silkworm bioreactor

1. Experimental methods

1.1 Synthesis of the Gene of interest and construction of the recombinant plasmid

The invention analyzes all duck lambda interferon amino acid sequences on NCBI, carries out sequence comparison and signal peptide analysis, and finally determines that the amino acid sequence shown by the accession number NP-001297721.1 is taken as the original amino acid sequence of duck lambda interferon, 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.

Restriction sites were analyzed by DNAman software, and restriction sites not present in the sequence of the target gene were added to both ends of the target gene based on the analysis results of the restriction sites and the multiple cloning sites on the transfer vectors pVL1393 and pUC 57. According to the analysis result, a BamHI restriction site and a Kozak sequence are added to the 5 'end of the plasmid, an EcoRI restriction site is added to the 3' end of the plasmid, the determined gene sequence is synthesized by Nanjing Kingsry Biotech Co., Ltd, and the plasmid pUC 57-DuIFN-lambda is formed by inserting the plasmid pUC 57.

1.2 construction of recombinant baculovirus transfer vectors

And carrying out double enzyme digestion treatment on the synthesized pUC 57-DuIFN-lambda by using BamHI and EcoRI, recovering a target fragment by a glass milk method, and connecting the recovered DuIFN-lambda target fragment subjected to enzyme digestion with BamHI and EcoRI to a transfer vector pVL1393 subjected to double enzyme digestion treatment. By T₄DNA ligase, 16 ℃ and ligation overnight. Transforming the ligation product into escherichia coli competent cell TOP10, selecting a colony for culturing, upgrading the plasmid, carrying out double enzyme digestion with BamHI and EcoRI to identify positive clone, sending the correctly identified recombinant plasmid to Beijing engine biotechnology Limited for sequencing, and naming the plasmid with correct sequencing as pVL-DuIFN-lambda.

1.3 obtaining, purifying and amplifying recombinant silkworm baculovirus

The recovery and passage of the BmN cells and the screening of the recombinant viruses are carried out according to the methods reported in the literature. When the 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 mu g of bombyx mori baculovirus parent strain BmBacmid DNA, 2 mu g of recombinant transfer plasmid and 5 mu L of liposome into a sterilizing tube, complementing the volume to 60 mu L with sterile double distilled water, slightly mixing uniformly, standing for 15min, and then dropwise adding into a culture bottle for cotransfection. After 4h incubation at 27 ℃ 1.5mL serum free medium and 300. mu.L FBS were supplemented. Culturing at the constant temperature of 27 ℃ for 4-5 days until the cells shed and float, and collecting cell culture solution to obtain the recombinant virus rBmBacmid (DuIFN-lambda) containing the target gene.

The purification and amplification method of the recombinant silkworm baculovirus is as follows: inoculating a proper amount of cells (about 70-80%) in a small 35mm dish, sucking out the culture medium after the cells adhere to the wall, diluting the collected cell culture solution at different concentrations, adding 1mL of the diluted cell culture solution into the adherent cells, and uniformly distributing the cells. After infection for 1h at 27 ℃, absorbing infection liquid, melting 2% low melting point agarose gel in water bath at 60 ℃, cooling to 40 ℃, uniformly mixing with 2 XTC-100 culture medium (containing 20% FBS) preheated at 40 ℃, adding 4mL of gel into each dish, sealing with Parafilm after solidification, carrying out inverted culture at 27 ℃ for 3-5 d, and observing by using a microscope. And (3) selecting the plaques which do not contain the polyhedron, repeating the steps, and performing 2-3 rounds of purification to obtain the pure recombinant baculovirus rBmBacmid (DuIFN-lambda).

Infecting the recombinant silkworm baculovirus rBmBacmid (DuIFN-lambda) with the normally growing BmN cells, culturing for 3 days, and collecting supernatant, wherein the supernatant contains a large amount of the recombinant virus rBmBacmid (DuIFN-lambda).

1.4 Duck Lambda interferon is expressed in silkworm body

Recombinant virus culture solution is added according to the formula 10⁵PFU/head dose is injected into 5 th instar silkworm, and cultured at 27 deg.C and 70-80% humidity, and the DuIFN-lambda is expressed efficiently in the later growth stage of silkworm larva under the action of polyhedrin gene promoter. Inoculating infection for about 3.5-4 days, observing symptoms such as swelling of body node, abnormal behavior, and reduced appetite of silkworm larva, collecting haemolymph when observed that the larva volume is obviously reduced and stops feeding, and-20 deg.CAnd (5) storing for later use.

1.5 preparation of Duck Embryo Fibroblast (DEF) and detection of Duck lambda interferon antiviral activity

Taking 10-12 days old duck embryos, disinfecting the eggshells in an ultra-clean bench, breaking the eggshells from an air chamber, taking out embryo bodies, cutting heads and limbs, washing with HBSS for 3 times, putting the embryo bodies into a sterilized triangular flask and cutting the embryo bodies into the size of millet grains, washing with HBSS for 3 times, adding pancreatin, digesting for 40min at 37 ℃, removing the pancreatin, washing with HBSS for 3 times to remove residual pancreatin, adding 30mL of complete culture medium, blowing, beating and shaking the triangular flask to disperse cells, filtering with 8 layers of sterile gauze to obtain cell suspension, counting the cells, and diluting the cells to 10⁶one/mL.

The antiviral activity of duck lambda interferon expressed in silkworm haemolymph was examined on DEF/VSV GFP system using the microcytopathy inhibition method. DEF cells prepared at 3.0 × 10⁵The cells/mL were plated in 96-well plates. Preparing the silkworm hemolymph with ultrasonication and filter sterilization into solution with different dilution with M199 culture solution containing 50mL/L fetal bovine serum, inoculating the diluted sample into culture well with DEF cells at 100 μ L/well, setting at least 12 multiple wells for each dilution and control silkworm blood, setting cell control group without silkworm hemolymph and VSV GFP and virus control group with VSV GFP, and culturing at 37 deg.C and 5% CO₂Culturing for 18-24 h under the condition. Diluting to 100TCID₅₀The VSV GFP virus of (1) was added to the culture well from which the supernatant had been aspirated at 100. mu.L/well, and the mixture was incubated at 37 ℃ with 5% CO₂Culturing under the condition. When a large number of cells in each hole of the virus control group generate fluorescence and the cells in the cell control group still completely grow well and no fluorescence appears, the contrast system is completely qualified, and comprehensive observation can be carried out.

2. Results of the experiment

2.1 identification of recombinant transfer vectors

The recombinant transfer vector pVL-DuIFN-lambda is subjected to double digestion by BamHI and EcoRI, 2 fragments are separated by electrophoresis in 1% agarose gel, the small fragment is 500bp-750bp and is consistent with the size of a target gene fragment 558bp, and the large fragment is positioned above 8000bp and is consistent with the size of a pVL1393 fragment 9607 bp. The plasmid with correct enzyme restriction identification is sent to Beijing Optimalaceae New industry biotechnology Limited for nucleotide sequencing, and the MegaAlign comparison result shows that the sequence is consistent with the originally designed sequence, which shows that the duck lambda interferon gene is successfully inserted between BamHI and EcoRI in the pVL1393 transfer vector.

2.2 acquisition of Duck Lambda interferon recombinant virus and detection of recombinant product

And (3) detecting duck lambda interferon antiviral activity expressed by silkworm larvae on a DEF/VSV-GFP system by using a micro cytopathic inhibition method. The growth state of the cells in the cell control group is good and no fluorescence appears when the cells are observed under an inverted fluorescence microscope; cells infected with virus in a control group are diseased, most cells show fluorescence, and cells added with recombinant duck lambda interferon protein have the capacity of resisting virus infection. Observing the pathological degree of cells according to the protective effect of duck lambda interferon on DEF cells, marking the cells as "+" when green fluorescent cells appear, and calculating the titer of the interferon according to a Reed-Muench method. The results are shown in Table 1, and the DuIFN-lambda titer is 1.56X 10⁶U/mL。

TABLE 1 detection results of the antiviral activity of recombinant duck interferon lambda

Example 2 acquisition of DuIFN-Lambda amino acid conserved sequence and expression of the optimized sequence in silkworm bioreactor and detection of 1.1 acquisition of DuIFN-Lambda amino acid conserved sequence and construction of codon optimized mutant gene thereof

Analyzing all amino acid sequences of duck lambda interferon to generate a conserved sequence of duck lambda interferon, wherein the amino acid sequence is shown as SEQ ID NO.3, and the nucleotide sequence is shown as SEQ ID NO. 4.

The invention utilizes OptimumGene^TMThe technology optimizes the duck lambda interferon conserved sequence gene (SEQ ID NO.4), modifies the gene sequence according to the codon preference of the bioreactor silkworm, and influences the geneThe transcription efficiency, the translation efficiency, the GC content of protein folding, the CpG dinucleotide content, the codon preference, the secondary structure of mRNA, the stability of mRNA free energy, RNA instability motif, repetitive sequence and other related parameters are optimized and designed, so that the transcription efficiency and the 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 silkworm baculovirus eukaryotic expression system, a Kozak sequence ACGAAC is added in front of the gene ATG. In addition, restriction sites for BamHI, EcoRI, SmaI and the like in the gene sequence were removed, BamHI was added upstream of the gene, and EcoRI restriction sites were added downstream of the gene, for subsequent cloning into the eukaryotic transfer vector pVL 1393.

The designed optimized sequence of the duck lambda interferon gene is artificially synthesized by a biotechnology company and named as DuIFN-lambda-C-O mutant, the nucleotide sequence of the DuIFN-lambda-C-O mutant is shown as SEQ ID NO.5, and the synthesized gene fragment is inserted into a pUC57 vector to form a plasmid pUC57-DuIFN which is named as pUC 57-DuIFN-lambda-C-O.

1.2 construction of recombinant baculovirus transfer vectors

Specific procedures with reference to example 1, the correctly sequenced plasmid was designated pVL-DuIFN-. lambda. -C-O.

1.3 obtaining, purifying and amplifying recombinant silkworm baculovirus

Recovery and passage of BmN cells referring to example 1, recombinant virus rBmBacmid (DuIFN-. lambda. -C-O) containing the gene of interest was obtained by co-transfection. Purification and amplification method of recombinant Bombyx mori baculovirus referring to example 1, pure recombinant Bombyx mori baculovirus rBmBacmid (DuIFN-. lambda. -C-O) was obtained by purification.

Infecting the recombinant bombyx mori baculovirus rBmBacmid (DuIFN-lambda-C-O) with the normally growing BmN cells, culturing for 3 days, and collecting the supernatant, wherein the supernatant contains a large amount of the recombinant virus rBmBacmid (DuIFN-lambda-C-O).

1.4 Duck lambda interferon mutant is expressed in silkworm body

The same as in example 1.

2. Results of the experiment

2.1 identification of recombinant transfer vectors

The recombinant transfer vector pVL-DuIFN-lambda-C-O is subjected to double digestion by BamHI and EcoRI, 2 fragments are separated by electrophoresis in 1% agarose gel, the small fragment is between 500bp and 750bp and is consistent with the size of a target gene fragment 558bp, and the large fragment is positioned above 8000bp and is consistent with the size of a pVL1393 fragment 9607 bp. The plasmid with correct enzyme restriction identification is sent to Beijing Optimalaceae New industry biotechnology Limited for nucleotide sequencing, and the MegaAlign comparison result shows that the sequence is consistent with the originally designed sequence, which shows that the duck lambda interferon mutant gene is successfully inserted between BamHI and EcoRI in the pVL1393 transfer vector.

And (3) detecting duck lambda interferon antiviral activity expressed by silkworm larvae on a DEF/VSV-GFP system by using a micro cytopathic inhibition method. The growth state of the cells in the cell control group is good and no fluorescence appears when the cells are observed under an inverted fluorescence microscope; cells infected with virus in a control group are diseased, most cells show fluorescence, and cells added with recombinant duck lambda interferon protein have the capacity of resisting virus infection. Observing the pathological degree of cells according to the protective effect of duck lambda interferon on DEF cells, marking the cells as "+" when green fluorescent cells appear, and calculating the titer of the interferon according to a Reed-Muench method. The detection results are shown in Table 2, and the results of the antiviral activity determination show that DuIFN-lambda-C-O expressed in silkworm larvae has significant antiviral activity, and the potency reaches 2.58 multiplied by 10⁶U/mL, the expected effect is achieved, and the method for optimizing duck lambda interferon genes to improve DuIFN-lambda antiviral activity is feasible and effective.

TABLE 2 detection results of duck interferon-lambda mutant optimized antiviral activity

Example 3 expression and detection of DuIFN-. lambda.C-O in silkworm bioreactor after amino acid Single-site mutagenesis

1. Experimental methods

1.1 construction of Duck Lambda interferon mutant Gene

The invention takes DuIFN-lambda-C-O gene sequence as a template, designs a plurality of pairs of primers to carry out site-directed mutagenesis on the sequence, the site-directed mutagenesis is carried out by utilizing a fusion PCR method, the construction of a transfer vector of the mutant is carried out by adopting a homologous recombination method, and the specific method refers to Makai, and the like (comparison of constructing a pMIR-reporter vector by a double digestion and homologous recombination method [ J ]. China journal of pathogenic biology, 2015(6):495 and 499.) for carrying out sequencing verification.

The mutation sites are A19V, S30T, Y41S, L48Q, K51R, N56S, P62S, T74K, L84I, I95V, D104H, F117L, G126D, Q136K, H143Y, S158G, V166L and H173N respectively; the obtained duck lambda interferon mutant is named as DuIFN-lambda-C-O-M1 (A19V, S30T, Y41S, L48Q, K51R, N56S, P62S, T74K, L84I, I95V, D104H, F117L, G126D, Q136K, H143Y, S158G, V166L and H173N) mutant.

Primers required for amino acid single-site and multi-site mutation of the nucleotide sequence of DuIFN-lambda-C-O:

(1) primers for upstream and downstream on both sides:

F:TCATACCGTCCCACCATCGGGCCGGGATCCACGAACATGCTGTGCCCTGTG

R:GATCTGCAGCGGCCGCTCCGGAATTCTTAGGTGCAACGCTCAG

(2) intermediate upstream and downstream primers

1.A19V GCT-GTT

F1:TGGGTCTGGGACCACTGCTGGTTGGAGCCTTCCCACAGGCTG

R1:CAGCCTGTGGGAAGGCTCCAACCAGCAGTGGTCCCAGACCCA

2.S30T TCC-ACC

F2:CACAGGCTGCCCTGAAGAAGACCTGCCGCCTGAGCCAGTACG

R2:CGTACTGGCTCAGGCGGCTGGACTTCTTCAGGGCAGCCTGTG

3.Y41S TAC-TCC

F3:GCCAGTACGGATCTCCAGCTTCCTCAGAGCTGGCCGAAGTGCTG

R3:GCACTTCGGCCAGCTCTGAGGAAGCTGGAGATCCGTACTGGCTC

4.L48Q CTG-CAG

F4：TCAGAGCTGGCCGAAGTGCAGAAGTTCAAGAAGTACTACGA

R4：TCGTAGTACTTCTTGAACTTCTGCACTTCGGCCAGCTCTGA

5.K51R AAG-AGG

F5：GGCCGAAGTGCTGAAGTTCAGGAAGTACTACGAAAACATCAC

R5：GTGATGTTTTCGTAGTACTTCCTGAACTTCAGCACTTCGGCC

6.N56S AAC-AGC

F6:GTTCAAGAAGTACTACGAAAGCATCACCTCCAAGGACCCTAAG

R6:CTTAGGGTCCTTGGAGGTGATGCTTTCGTAGTACTTCTTGAAC

7.P62S CCT-TCT

F7:GAAAACATCACCTCCAAGGACTCTAAGTGCAGCACTCGCCTG

R7:CAGGCGAGTGCTGCACTTAGAGTCCTTGGAGGTGATGTTTTC

8.T74K ACC-AAG

F8:GCCTGTTCAACCGTAAGTGGAAGCCAAACGAGCTGTCCGTG

R8:CACGGACAGCTCGTTTGGCTTCCACTTACGGTTGAACAGGC

9.L84I CTG-ATC

F9:GCTGTCCGTGCCTGACCGTATCCTGCTGGTCGAGGCTGAAC

R9:GTTCAGCCTCGACCAGCAGGATACGGTCAGGCACGGACAGC

10.I95V ATC-GTC

F10:GGCTGAACTGGACCTGACTGTCGCTGTGCTGGCCCACCCTAC

R10:GTAGGGTGGGCCAGCACAGCGACAGTCAGGTCCAGTTCAGCC

11.D104H GAC-CAC

F11:GCTGGCCCACCCTACCGTGCACAAGCTGGCCGAAAAGTCTC

R11:GAGACTTTTCGGCCAGCTTGTGCACGGTAGGGTGGGCCAGC

12.F117L TTC-TTG

F12:CTCAGCAGCCCCTGGCTTTCTTGACTCAGGCCAGGGAAGACC

R12:GGTCTTCCCTGGCCTGAGTCAAGAAAGCCAGGGGCTGCTGAG

13.G126D GGA-GAC

F13:GGCCAGGGAAGACCTGAGAGACTGCGTGGCTGCCGAGGCTCC

R13:GGAGCCTCGGCAGCCACGCAGTCTCTCAGGTCTTCCCTGGCC

14.Q136K CAG-AAG

F14:GCCGAGGCTCCATCTCACAAGCCATCTGGCAAGCTGAGGCAC

R14:GCCTCAGCTTGCCAGATGGCTTGTGAGATGGAGCCTCGGCAGC

15.H143Y CAC-TAC

F15:CCATCTGGCAAGCTGAGGTACTGGCTGCAAAAGCTGGAGAC

R15:GTCTCCAGCTTTTGCAGCCAGTACCTCAGCTTGCCAGATGG

16.S158G TCT-GGT

F16:GCTAAGAAGACCGAAACTGCCGGTTGCCTGGAGTACTCAACCATC

R16:GATGGTTGAGTACTCCAGGCAACCGGCAGTTTCGGTCTTCTTAGC

17.V166L GTG-TTG

F17:CCTGGAGTACTCAACCATCTTGCACCTGTTCCAGGTCCTGC

R17:GCAGGACCTGGAACAGGTGCAAGATGGTTGAGTACTCCAGG

18.H173N CAC-AAC

F18:CACCTGTTCCAGGTCCTGAACGACCTGGGTTGCGTCGCC

R18:GGCGACGCAACCCAGGTCGTTCAGGACCTGGAACAGGTG

1.2 construction of recombinant baculovirus transfer vectors

The target fragment recovered by the glass milk method is homologously recombined with an inactivated baculovirus transfer vector pVL1393 which is subjected to double enzyme digestion by BamHI and EcoRI by using recombinase (pEASY-Uni Seamless Cloning and Assenbly Kit), the recombination product is transformed into an Escherichia coli competent cell TOP10, colony culture is selected, the plasmid is upgraded, positive clones are identified by double enzyme digestion by BamHI and EcoRI, the correctly sequenced recombinant plasmid is identified and named pVL-DuIFN-lambda-C-O-M1 (A19V, S30T, Y41S, L48Q, K51R, N56S, P62S, T74K, L84I, I95V, D104H, F117L, G126D, Q136K, H143Y, S158G, V166L, H173N).

1.3 obtaining, purifying and amplifying recombinant silkworm baculovirus

The same as in example 1.

The recombinant virus rBmBacmid (DuIFN-. lambda. -C-O-M1) containing the gene of interest was obtained by co-transfection.

Pure recombinant bombyx mori baculovirus rBmBacmid (DuIFN-lambda-C-O-M1) was obtained by purification.

The recombinant bombyx mori baculovirus rBmBacmid (DuIFN-lambda-C-O-M1) is infected with the normally growing BmN cells, and after 3 days of culture, the supernatant is collected, and the supernatant contains a large amount of the recombinant virus rBmBacmid (DuIFN-lambda-C-O-M1).

1.4 Duck lambda interferon mutant is expressed in silkworm body

The same as in example 1.

2. Results of the experiment

2.1 identification of recombinant transfer vectors

The recombinant transfer vector pVL-DuIFN-lambda-C-O-M1 is subjected to double enzyme digestion by BamHI and EcoRI, 2 fragments are separated by 1% agarose gel electrophoresis, the small fragment is between 500bp and 750bp and is consistent with the size of a target gene fragment 558bp, and the large fragment is above 8000bp and is consistent with the size of a pVL1393 fragment 9607 bp. The plasmid with correct enzyme restriction identification is sent to Beijing Optimalaceae New industry biotechnology Limited for nucleotide sequencing, and the MegaAlign comparison result shows that the sequence is consistent with the originally designed sequence, which shows that the duck lambda interferon mutant gene is successfully inserted between BamHI and EcoRI in the pVL1393 transfer vector.

And (3) detecting duck lambda interferon antiviral activity expressed by silkworm larvae on a DEF/VSV-GFP system by using a micro cytopathic inhibition method. The growth state of the cells in the cell control group is good and no fluorescence appears when the cells are observed under an inverted fluorescence microscope; cells infected with virus in a control group are diseased, most cells show fluorescence, and cells added with recombinant duck lambda interferon protein have the capacity of resisting virus infection. Observing the pathological change degree of cells according to the protective effect of duck lambda interferon on DEF cells, and waiting for green fluorescent cells to appearThe well cells were marked as "+", interferon titers were calculated according to the Reed-Muench method, the results are shown in Table 3, and the titers were measured at 8.58X 10 for all duck interferon lambda mutants⁴U/mL～3.65×10⁶U/mL, wherein after 5 sites of S30T, N56S, I95V, Q136K or V166L are mutated, the titer of the expressed duck lambda 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 rest sites, which indicates that the mutation of the 5 sites is effective mutation, and the aim of improving the DuIFN-lambda antiviral activity can be achieved. Among them, DuIFN-lambda-C-O-I95V mutant has the strongest antiviral effect.

TABLE 3 detection results of antiviral activity of single site mutation of recombinant duck interferon lambda

Example 4 expression and detection of DuIFN-. lambda.C-O-M1 mutant in silkworm bioreactor after amino acid Multi-site mutagenesis

1. Experimental methods

1.1 construction of Duck Lambda interferon mutant Gene

Considering the results of example 3, it was determined that partial site mutations were effective mutations for the purpose of increasing the antiviral activity of the DuIFN-. lambda. -C-O mutant. Considering that the sequence of amino acids is the primary structure of the protein and determines the higher order structure of the protein, and that the positions of the partial mutation sites in the single-site amino acid mutation performed in example 3 may be correlated with each other, multiple-site amino acid mutations were attempted. The invention combines single mutation sites S30T, N56S, I95V, Q136K and V166L with improved antiviral activity with each other to carry out multi-site mutation, the multi-site mutation is based on the single-site mutation sequence obtained in example 3, the DuIFN-lambda-C-O-M1 is used as a template, corresponding primers (see example 3 for details) are utilized to carry out site-directed mutation by a multi-round fusion PCR method, so as to obtain a target fragment of the multi-site mutation, the construction of a transfer vector of the mutant is carried out by a homologous recombination method, and the like (comparison of constructing a pMIR-reporter vector by a double enzyme digestion and homologous recombination method [ J ]. China journal of biology, 2015(6):495 and 499.) is referred to and sequence verification is carried out.

The multiple mutation sites are combined by S30-N56, S30-I95, S30-Q136, S30-V166, N56-I95, N56-Q136, N56-V166, I95-Q136, I95-V166, Q136-V166, S30-N56-I95, S30-N56-Q136, S30-N56-V166, S30-I95-Q136, S30-I95-V166, S30-Q136-V166, N56-I95-Q136, N56-I95-V166, N56-Q136-V166, I95-Q136-V166, S30-N56-I95-Q136, S30-N56-I95-V166, S30-N56-Q136-V166, S30-I95-Q166, N56-I95-Q136-V166, S30-V56-V136-V166 and S30-V95-V166, the obtained duck lambda interferon mutant is named as DuIFN-lambda-C-O-M (S30-N56, S30-I95, S30-Q136, S30-V166, N56-I95, N56-Q136, N56-V166, I95-Q136, I95-V166, Q136-V166, S30-N56-I95, S30-N56-Q136, S30-N56-V166, S30-I95-Q136, S30-I95-V166, S30-Q136-V166, N56-I95-Q136, N56-I95-V166, N56-Q136-V166, I95-Q136-V166, S30-N56-I95-Q136, S30-N56-I95-V166, S30-N56-Q136-V166, S30-I95-V166, S30-N56-V166, S30-N56-Q136-V166, S30-I95-V166, N56-V166, S30-V-166, S30-V-, S30T-N56S-I95V-Q136K-V166L).

1.2 construction of recombinant baculovirus transfer vectors

The procedure is as in example 3. The correctly sequenced plasmids were designated pVL-DuIFN-. lambda.C-O-M (S30-N56, S30-I95, S30-Q136, S30-V166, N56-I95, N56-Q136, N56-V166, I95-Q136, I95-V166, Q136-V166, S30-N56-I95, S30-N56-Q136, S30-N56-V166, S30-I95-Q136, S30-I95-V166, S30-Q136-V166, N56-I95-Q136, N56-I95-V166, N56-Q136-V166, I95-Q136-V166, S30-N56-I95-Q136, S30-N56-I95-V166, S30-N56-Q136-V166, S30-I95-V166, N56-V166, S30-N56-I95-V166, V-V166, S30-V-166, S30-V-166, S30T-N56S-I95V-Q136K-V166L).

1.3 obtaining, purifying and amplifying recombinant silkworm baculovirus

The same as in example 1.

The recombinant virus rBmBacmid (DuIFN-. lambda. -C-O-M2) containing the gene of interest was obtained by co-transfection.

Pure recombinant bombyx mori baculovirus rBmBacmid (DuIFN-lambda-C-O-M2) was obtained by purification.

The recombinant bombyx mori baculovirus rBmBacmid (DuIFN-lambda-C-O-M2) is infected with the normally growing BmN cells, and after 3 days of culture, the supernatant is collected, and the supernatant contains a large amount of the recombinant virus rBmBacmid (DuIFN-lambda-C-O-M2).

1.4 Duck lambda interferon mutant is expressed in silkworm body

The same as in example 1.

2. Results of the experiment

2.1 identification of recombinant transfer vectors

The recombinant transfer vector pVL-DuIFN-lambda-C-O-M2 is subjected to double enzyme digestion by BamHI and EcoRI, 2 fragments are separated by 1% agarose gel electrophoresis, the small fragment is located between 500bp and 750bp and is consistent with the size of a target gene fragment 558bp, and the large fragment is located above 8000bp and is consistent with the size of a pVL1393 fragment 9607 bp. And (3) carrying out nucleotide sequencing on the plasmid with correct restriction enzyme identification, and using a MegaAlign comparison result to indicate that the sequence is consistent with the originally designed sequence, thereby indicating that the duck lambda interferon mutant gene is successfully inserted between BamHI and EcoRI in the pVL1393 transfer vector.

And (3) detecting duck lambda interferon antiviral activity expressed by silkworm larvae on a DEF/VSV-GFP system by using a micro cytopathic inhibition method. The growth state of the cells in the cell control group is good and no fluorescence appears when the cells are observed under an inverted fluorescence microscope; cells infected with virus in a control group are diseased, most cells show fluorescence, and cells added with recombinant duck lambda interferon protein have the capacity of resisting virus infection. Observing the pathological change degree of cells according to the protective effect of duck lambda interferon on DEF cells, marking the cells as "+" when green fluorescent cells appear, calculating the interferon titer according to a Reed-Muench method, and obtaining the detection results shown in Table 4, wherein the titer of all duck lambda interferon mutants is measured according to the titer of the duck lambda interferon mutants8.16×10⁴U/mL～5.01×10⁶U/mL, wherein after four groups of N56S-I95V, I95V-Q136K, N56S-I95V-Q136K and N56S-I95V-Q136K-V166L are subjected to multiple mutations, the titer of the expressed duck lambda interferon is slightly higher than the titer measured by expression of a conserved sequence and a single mutation sequence, and the titer is unchanged or even reduced after mutation of other groups of sites, which indicates that the mutation of the 4 combined sites is effective mutation, and the aim of improving the antiviral activity of the DuIFN-lambda-C-O-M1 mutant can be achieved. Wherein, the DuIFN-lambda-C-O-N56S-I95V-Q136K-V166L mutant has the strongest antiviral effect, the amino acid sequence of the mutant is shown as SEQ ID NO.6, and the nucleotide sequence of the coding gene of the mutant is shown as SEQ ID NO. 7.

TABLE 4 detection results of antiviral activity of recombinant duck lambda interferon multi-site mutation

SEQUENCE LISTING

<110> institute of biotechnology of Chinese academy of agricultural sciences

<120> duck lambda interferon mutant and preparation method and application thereof

<130> BJ-2002-190529A

<160> 7

<170> PatentIn version 3.5

<210> 1

<211> 185

<212> PRT

<213> Duck

<400> 1

Met Leu Cys Pro Val Phe Ala Ala Val Leu Val Val Gly Leu Gly Pro

1 5 10 15

Leu Leu Ala Gly Ala Phe Pro Gln Ala Ala Leu Lys Lys Ser Cys Arg

20 25 30

Leu Ser Gln Tyr Gly Ser Pro Ala Tyr Ser Glu Leu Ala Glu Val Leu

35 40 45

Lys Phe Lys Lys Tyr Tyr Glu Asn Ile Thr Ser Lys Asp Pro Lys Cys

50 55 60

Ser Thr Arg Leu Phe Asn Arg Lys Trp Thr Pro Asn Glu Leu Ser Val

65 70 75 80

Pro Asp Arg Leu Leu Leu Val Glu Ala Glu Leu Asp Leu Thr Ile Ala

85 90 95

Val Leu Ala His Pro Thr Val Asp Lys Leu Ala Glu Lys Ser Gln Gln

100 105 110

Pro Leu Ala Phe Phe Thr Gln Ala Arg Glu Asp Leu Arg Gly Cys Val

115 120 125

Ala Ala Glu Ala Pro Ser His Gln Pro Ser Gly Lys Leu Arg His Trp

130 135 140

Leu Gln Lys Leu Glu Thr Ala Lys Lys Thr Glu Thr Ala Ser Cys Leu

145 150 155 160

Glu Tyr Ser Thr Ile Val His Leu Phe Gln Val Leu His Asp Leu Gly

165 170 175

Cys Val Ala Ile Pro Glu Arg Cys Thr

180 185

<210> 2

<211> 558

<212> DNA

<213> Duck

<400> 2

atgctgtgcc ccgtattcgc cgcggtgctg gtggtgggcc tggggcccct gctggcgggc 60

gccttccccc aggctgccct gaagaagagc tgccgcctct cccagtacgg atccccggcg 120

tattccgagc tggcggaggt gctgaagttt aagaagtact atgagaacat cacgtcgaag 180

gaccccaaat gcagcaccag gctcttcaat cggaagtgga cacccaacga gctgtcggtg 240

cctgaccgac tcctcctggt ggaagccgag ctggacctca ccatcgccgt gctggcacac 300

cccacagtcg acaagctggc cgagaagagc cagcagcccc tggccttctt cacccaagcc 360

cgggaggacc tgcgaggctg cgtggccgct gaggctcctt cgcatcagcc ctctgggaag 420

ctgaggcact ggctgcagaa gctggagacg gccaagaaga cggagaccgc cagctgcctg 480

gagtactcca ccatcgtcca cctcttccaa gtgctgcacg acctggggtg cgtggccatt 540

ccggagcggt gcacgtag 558

<210> 3

<211> 185

<212> PRT

<213> Duck

<400> 3

Met Leu Cys Pro Val Phe Ala Ala Val Leu Val Val Gly Leu Gly Pro

1 5 10 15

Leu Leu Ala Gly Ala Phe Pro Gln Ala Ala Leu Lys Lys Ser Cys Arg

20 25 30

Leu Ser Gln Tyr Gly Ser Pro Ala Tyr Ser Glu Leu Ala Glu Val Leu

35 40 45

Lys Phe Lys Lys Tyr Tyr Glu Asn Ile Thr Ser Lys Asp Pro Lys Cys

50 55 60

Ser Thr Arg Leu Phe Asn Arg Lys Trp Thr Pro Asn Glu Leu Ser Val

65 70 75 80

Pro Asp Arg Leu Leu Leu Val Glu Ala Glu Leu Asp Leu Thr Ile Ala

85 90 95

Val Leu Ala His Pro Thr Val Asp Lys Leu Ala Glu Lys Ser Gln Gln

100 105 110

Pro Leu Ala Phe Phe Thr Gln Ala Arg Glu Asp Leu Arg Gly Cys Val

115 120 125

Ala Ala Glu Ala Pro Ser His Gln Pro Ser Gly Lys Leu Arg His Trp

130 135 140

Leu Gln Lys Leu Glu Thr Ala Lys Lys Thr Glu Thr Ala Ser Cys Leu

145 150 155 160

Glu Tyr Ser Thr Ile Val His Leu Phe Gln Val Leu His Asp Leu Gly

165 170 175

Cys Val Ala Ile Pro Glu Arg Cys Thr

180 185

<210> 4

<211> 558

<212> DNA

<213> Duck

<400> 4

atgctgtgcc ccgtattcgc cgcggtgctg gtggtgggcc tggggcccct gctggcgggc 60

gccttccccc aggctgccct gaagaagagc tgccgcctct cccagtacgg atccccggcg 120

tattccgagc tggcggaggt gctgaagttt aagaagtact atgagaacat cacgtcgaag 180

gaccccaaat gcagcaccag gctcttcaat cggaagtgga cacccaacga gctgtcggtg 240

cctgaccgac tcctcctggt ggaagccgag ctggacctca ccatcgccgt gctggcacac 300

cccacagtcg acaagctggc cgagaagagc cagcagcccc tggccttctt cacccaagcc 360

cgggaggacc tgcgaggctg cgtggccgct gaggctcctt cgcatcagcc ctctgggaag 420

ctgaggcact ggctgcagaa gctggagacg gccaagaaga cggagaccgc cagctgcctg 480

gagtactcca ccatcgtcca cctcttccaa gtgctgcacg acctggggtg cgtggccatt 540

ccggagcggt gcacgtag 558

<210> 5

<211> 558

<212> DNA

<213> Artifical sequence

<400> 5

atgctgtgcc ctgtgttcgc tgccgtcctg gtggtgggtc tgggaccact gctggctgga 60

gccttcccac aggctgccct gaagaagtcc tgccgcctga gccagtacgg atctccagct 120

tactcagagc tggccgaagt gctgaagttc aagaagtact acgaaaacat cacctccaag 180

gaccctaagt gcagcactcg cctgttcaac cgtaagtgga ccccaaacga gctgtccgtg 240

cctgaccgtc tgctgctggt cgaggctgaa ctggacctga ctatcgctgt gctggcccac 300

cctaccgtgg acaagctggc cgaaaagtct cagcagcccc tggctttctt cactcaggcc 360

agggaagacc tgagaggatg cgtggctgcc gaggctccat ctcaccagcc atctggcaag 420

ctgaggcact ggctgcaaaa gctggagact gctaagaaga ccgaaactgc ctcttgcctg 480

gagtactcaa ccatcgtgca cctgttccag gtcctgcacg acctgggttg cgtcgccatc 540

cctgagcgtt gcacctaa 558

<210> 6

<211> 185

<212> PRT

<213> Artifical sequence

<400> 6

Met Leu Cys Pro Val Phe Ala Ala Val Leu Val Val Gly Leu Gly Pro

1 5 10 15

Leu Leu Ala Gly Ala Phe Pro Gln Ala Ala Leu Lys Lys Ser Cys Arg

20 25 30

Leu Ser Gln Tyr Gly Ser Pro Ala Tyr Ser Glu Leu Ala Glu Val Leu

35 40 45

Lys Phe Lys Lys Tyr Tyr Glu Ser Ile Thr Ser Lys Asp Pro Lys Cys

50 55 60

Ser Thr Arg Leu Phe Asn Arg Lys Trp Thr Pro Asn Glu Leu Ser Val

65 70 75 80

Pro Asp Arg Leu Leu Leu Val Glu Ala Glu Leu Asp Leu Thr Val Ala

85 90 95

Val Leu Ala His Pro Thr Val Asp Lys Leu Ala Glu Lys Ser Gln Gln

100 105 110

Pro Leu Ala Phe Phe Thr Gln Ala Arg Glu Asp Leu Arg Gly Cys Val

115 120 125

Ala Ala Glu Ala Pro Ser His Lys Pro Ser Gly Lys Leu Arg His Trp

130 135 140

Leu Gln Lys Leu Glu Thr Ala Lys Lys Thr Glu Thr Ala Ser Cys Leu

145 150 155 160

Glu Tyr Ser Thr Ile Leu His Leu Phe Gln Val Leu His Asp Leu Gly

165 170 175

Cys Val Ala Ile Pro Glu Arg Cys Thr

180 185

<210> 7

<211> 558

<212> DNA

<213> Artifical sequence

<400> 7

atgctgtgcc ctgtgttcgc tgccgtcctg gtggtgggtc tgggaccact gctggctgga 60

gccttcccac aggctgccct gaagaagtcc tgccgcctga gccagtacgg atctccagct 120

tactcagagc tggccgaagt gctgaagttc aagaagtact acgaaagcat cacctccaag 180

gaccctaagt gcagcactcg cctgttcaac cgtaagtgga ccccaaacga gctgtccgtg 240

cctgaccgtc tgctgctggt cgaggctgaa ctggacctga ctgtcgctgt gctggcccac 300

cctaccgtgg acaagctggc cgaaaagtct cagcagcccc tggctttctt cactcaggcc 360

agggaagacc tgagaggatg cgtggctgcc gaggctccat ctcacaagcc atctggcaag 420

ctgaggcact ggctgcaaaa gctggagact gctaagaaga ccgaaactgc ctcttgcctg 480

gagtactcaa ccatcttgca cctgttccag gtcctgcacg acctgggttg cgtcgccatc 540

cctgagcgtt gcacctaa 558

Claims

1. A duck lambda interferon mutant is characterized in that: the mutant is obtained by carrying out I95V amino acid single-site mutation on duck lambda interferon amino acid sequence shown in SEQ ID No. 3.

2. A duck lambda interferon mutant is characterized in that: the mutant is obtained by carrying out multi-site mutation on any one of amino acids of N56S-I95V, I95V-Q136K, N56S-I95V-Q136K or N56S-I95V-Q136K-V166L on the duck lambda interferon amino acid sequence shown in SEQ ID No. 3.

3. A recombinant vector or a recombinant host cell comprising a gene encoding a duck lambda interferon mutant according to claim 1 or 2.

4. Use of the duck lambda interferon mutant of claim 1 or 2 in the preparation of a medicament for preventing or treating duck viral diseases.

5. The use according to claim 4, wherein the duck viral diseases include: one or more of duck viral hepatitis, duck viral enteritis, gosling plague, muscovy duck parvovirus disease, duck epidemic hemorrhage, duck liver disease, duck viral encephalitis, duck reovirus infection, duck adenovirus infection, duck infectious bursal disease, duck paramyxovirus disease or duck newcastle disease, duck coronavirus infection or duck influenza.

6. The method for preparing duck lambda interferon mutant according to claim 1 or 2, which comprises the following steps:

(1) cloning coding genes of duck lambda interferon mutants of claim 1 or 2 into a baculovirus transfer vector to construct recombinant transfer vectors;

(2) co-transfecting the recombinant transfer vector and baculovirus DNA into an insect cell to obtain recombinant baculovirus;

(3) infecting the recombinant baculovirus into insect cells or insect hosts, culturing the infected insect cells or insect hosts to express corresponding protein, and purifying to obtain the recombinant baculovirus.

7. The method of claim 6, wherein the baculovirus transfer vector is selected from the group consisting of 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、pAcMLF 7、pAcMLF 8、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、pBlueBaScHs、pEV55、mXIV、pIEINeo、pJVETL、pJVNhel、pJVP10、pJVrsMAG. pMBac, pP10, pPAKl, pPBac, pSHONEX1.1, pSYN XIV VI +, pSYNVI + wp, pSYNXIV VI-, pVL1391, pVL 1392, pVL1393, pVL941, pVL 945, pVL 985, pVTBac, pBM030 or pUAC-5;

the baculovirus is selected from bombyx mori baculovirus parent strain BmBacmid, BmNPV, AcMNPV, ApNPV, HaNPV, HzNPV, LdMNPV, MbMNPV, OpMNPV, SlMNPV, SeMNPV or SpltNPV;

the insect host is selected from Bombyx moriBombyx mori) Wild silkworm (wild silkworm)Bombyx mandarina) Castor silkworm (1)Philosamia cynthia ricim) Antrodia camphorata (A, B, D)Dictyoploca japanica) Heaven silkworm (A. F.) (Philosamia cynthia pryeri) Tussah, tussah (Antheraea pernyi) Japanese tussah (A.pernyi)Antheraea yamamai) Wild silkworm (wild silkworm)Antheraea polyphymus) Alfalfa loopers (a) ((b))Atographa califorica) Tea geometrid inchworm (Ectropis obliqua) And Spodoptera glauca (L.) MoenchMamestra brassicae) (ii) Spodoptera lituraSpodoptera littoralis) Autumn armyworm (Spodoptera frugiperda) Powder looper (A, B)TricHoplusia ni) Marching insects (A)Thaumetopoea wilkinsoni) Cotton bollworm: (A)Heliothis armigera) American bollworm: (Heliothis zea) "YeqingchongHeliothis assulta) Tobacco budworm (2)Heliothis virescens) Oriental armyworm (Pseudaletia separata) Or gypsy mothLymantria dispar）；

The infection refers to the infection of 1-5-year-old insect larvae or pupae bodies by the recombinant baculovirus through ingestion or penetration of the epidermis.

8. The method of claim 7, wherein the baculovirus transfer vector is pVL 1393; the baculovirus is a parent strain BmBacmid of silkworm baculovirus; the insect host is silkworm.