CN110272479A

CN110272479A - 3 interferon mutant of ox λ and its preparation method and application

Info

Publication number: CN110272479A
Application number: CN201810207481.1A
Authority: CN
Inventors: 易咏竹; 沈兴家; 唐顺明; 赵璐璐; 赵泽; 邵丽萍; 王朋; 魏珍珍
Original assignee: Jiangsu University of Science and Technology
Current assignee: Jiangsu University of Science and Technology
Priority date: 2018-03-14
Filing date: 2018-03-14
Publication date: 2019-09-24
Anticipated expiration: 2038-03-14
Also published as: CN110272479B

Abstract

The invention discloses a kind of 3 interferon mutants of ox λ and its preparation method and application.Belong to field of biotechnology.Be using accession number for XP_010847513.1 3 interferon amino acid sequence of ox λ as the 5th amino acids of original series by cysteine mutation at arginine, the 19th amino acids by mutant serine at proline, obtained 3 interferon mutant of ox λ.Mutant is further subjected to amino acid unit point mutation, double-site mutant and multisite mutation, obtains 3 interferon mutant of ox λ that multiple antiviral activities improve.The antiviral activity for 3 interferon mutant of ox λ that the present invention is expressed in silkworm biological reactor using baculovirus expression vector system increases substantially.3 interferon mutant of ox λ provided by the invention can be used in the drug or reagent of preparation prevention or treatment bovine viral disease.

Description

3 interferon mutant of ox λ and its preparation method and application

Technical field

The present invention relates to 3 interferon mutants of ox λ and its preparation method and application, belong to field of biotechnology.

Background technique

Interferon is a kind of protein on allogenic cell with antiviral activity, active to play again by cell base Because of the regulation and control of group, it is related to the synthesis of RNA and protein.Sequence of the IFN protein family according to its encoding gene, chromosome Positioning and receptor-specific are divided into I type, II type and III type interferon.Interferon type Ⅰ includes IFN-α, IFN-β, IFN- ω, IFN- δ, IFN- ε, IFN- ζ, IFN- τ etc., are mainly IFN-α and IFN-β in mammal, and interferon type Ⅰ has stronger antiviral Activity, its proliferation of the main inhibition of DNA replication by viral interference, also has antitumor and immunoregulatory function.Interferon type Ⅱ Only one member of IFN-γ, also known as immune interferon, main function are that activating macrophage kills microorganism.The interference of III type Element is newfound cell factor, including λ 1 (IL-29), λ 2 (IL-28a) and λ 3 (IL-28b).III type interferon and I type interfere Element is in close relations, but has special physiologic function, such as stimulates major histocompatibility complex (major histocom- Patibility complex, MHC) molecule activation and expression, adjust congenital immunity and acquired immunity etc..Due to interferon With broad-spectrum antiviral, antitumor active and powerful immunoregulation effect, virology, cytology, molecule are become One of research hotspots of related fieldss such as biology, clinical medicine, immunology, oncology.

The cultivation of ox occupies critically important status in animal husbandry development, and ox gallbladder, cow-bezoar can be used as medicine；Beef, ox-hide, dairy Product have become indispensable product in people's lives,.But ox is subjected to the influence of some viral diseases in the breeding process, The survival and development for affecting ox class, also affect daily life, huge loss are brought to raiser, so cattle disease Prevention and control be an important job in breeding process.Therefore, it is necessary to high-quality, cheap ox Lambda interferon products to answer To this problem.

Summary of the invention

Technical problem

The purpose of the present invention is to provide and 3 interferon mutant of ox λ, 3 interferon mutant of ox λ have high disease-resistant Cytotoxic activity；3 interferon mutant of ox λ is prepared using baculovirus expression vector system, and 3 interferon mutant of ox λ can be with Application in the drug or reagent of preparation prevention or treatment bovine viral disease.

Technical solution

A kind of 3 interferon of ox λ is preparing the application in interferon mutant, it is characterised in that: the 3 interferon ammonia of ox λ Base acid sequence is shown in SEQ ID No.1, and the polynucleotide sequence of encoding gene is shown for (a) or (b) or (c):

(a) polynucleotide sequence shown in SEQ ID No.2；Or

(b) polynucleotide sequence hybridized is able to carry out in stringent hybridisation conditions with the complementary series of SEQ ID No.2, it should The albumen of polynucleotide encoding still has the function of interferon or activity；Or

(c) with the polynucleotide sequence of the polynucleotide sequence of SEQ ID No.2 at least 80% or more homology, and should The albumen of polynucleotide encoding still has the function of interferon or activity；Preferably, with the polynucleotide sequence of SEQ ID No.2 At least polynucleotide sequence of 85% or more homology, and the albumen of the polynucleotide encoding still have the function of interferon or Activity；It is furthermore preferred that the polynucleotide sequence with polynucleotide sequence at least 90% or more the homology of SEQ ID No.2, And the albumen of the polynucleotide encoding still has the function of interferon or activity.

It is with its amino acid sequence for SEQ ID No.1 the present invention further discloses a kind of 3 interferon mutant of ox λ 3 interferon amino acid sequence of ox λ be that the 5th amino acids of original series are mutated into arginine (R) by cysteine (C), the 19 amino acids are mutated into the mutant that proline (P) obtains by serine (S)；The amino acid sequence of the mutant is SEQ Shown in ID NO.3, the nucleotides sequence of encoding gene is classified as shown in SEQ ID NO.4.

It is by amino acid sequence is SEQ ID NO.3 institute the present invention further discloses a kind of 3 interferon mutant of ox λ 125th amino acids of 3 interferon mutant of ox λ shown are mutated into threonine (T) by methionine (M), the 164th amino acids The mutant that glutamic acid (E) obtains is mutated by lysine (K)；The amino acid sequence of the mutant is SEQ ID NO.5 institute Show, the nucleotides sequence of encoding gene is classified as shown in SEQ ID NO.6.

It is by amino acid sequence is SEQ ID NO.5 institute the present invention further discloses a kind of 3 interferon mutant of ox λ 3 interferon mutant of ox λ that shows carry out E51A, T57R, F66R, H76R, T81S, H86Q, A109S, H117R, L124H, Q127R, I129V, S131A, T142M, S144G, S145P, S165P or D167S any one amino acid unit point mutation obtain Mutant；Be preferably, the progress of 3 interferon mutant of ox λ shown in SEQ ID NO.5 F66R by amino acid sequence, The mutant that A109S, L124H, S131A, S145P or S165P any one amino acid unit point mutation obtain.Wherein, by ammonia Base acid sequence is the mutation that 3 interferon mutant of ox λ shown in SEQ ID NO.5 carries out that the point mutation of F66R amino acid unit obtains The amino acid sequence of body is shown in SEQ ID NO.7, and the nucleotides sequence of encoding gene is classified as shown in SEQ ID NO.8.

Wherein, amino acid unit point mutation E51A of the present invention indicates amino acid sequence to be shown in SEQ ID NO.5 The 51st amino acids of 3 interferon mutant of ox λ alanine (A) is mutated by glutamic acid (E)；F66R is indicated the 66th ammonia Base acid is mutated into arginine (R) by phenylalanine (F)；The rest may be inferred.

It is by amino acid sequence for shown in SEQ ID NO.5 the invention also discloses a kind of 3 interferon mutant of ox λ 3 interferon mutant of ox λ carry out F66R-A109S, F66R-L124H, F66R-S131A, F66R-S145P, F66R-S165P, A109S-L124H、A109S-S131A、A109S-S145P、A109S-S165P、L124H-S131A、L124H-S145、L124H- The mutation that S165P, S131A-S145P, S131A-S165P or S145P-S165P any one amino acid double-site mutant obtain Body；It preferably, is that 3 interferon mutant of ox λ shown in SEQ ID NO.5 carries out F66R-L124H, F66R- by amino acid sequence The mutant that S145P or A109S-S131A any one amino acid double-site mutant obtains.It wherein, is SEQ by amino acid sequence 3 interferon mutant of ox λ shown in ID NO.5 carries out the amino for the mutant that F66R-L124H amino acid double-site mutant obtains Acid sequence is shown in SEQ ID NO.9, and the nucleotides sequence of encoding gene is classified as shown in SEQ ID NO.10.

Wherein, amino acid double-site mutant F66R-A109S of the present invention indicates amino acid sequence to be SEQ ID 66th amino acids of 3 interferon mutant of ox λ shown in NO.5 are mutated into arginine (R) by phenylalanine (F), and by 109 amino acids are mutated into serine (S) by alanine (A)；Amino acid double-site mutant F66R-L124H is indicated the 66th Amino acids are mutated into arginine (R) by phenylalanine (F), and the 109th amino acids are mutated into ammonia by alanine (A) Sour (S)；The rest may be inferred.

It is by amino acid sequence for shown in SEQ ID NO.5 the invention also discloses a kind of 3 interferon mutant of ox λ 3 interferon mutant of ox λ carries out F66R-A109S-L124H, F66R-A109S-S131A, F66R-A109S-S145P, F66R- L124H-S131A, F66R-L124H-S145P, F66R-S131A-S145P, A109S-L124H-S131A or A109S-S131A- The mutant that S145P any one amino acid multisite mutation obtains；It preferably, is SEQ ID NO.5 institute by amino acid sequence 3 interferon mutant of ox λ shown carries out the mutant that F66R-L124H-S145P amino acid multisite mutation obtains.Wherein, will Amino acid sequence is that 3 interferon mutant of ox λ shown in SEQ ID NO.5 carries out F66R-L124H-S145P amino acid multidigit point The amino acid sequence for being mutated the mutant obtained is shown in SEQ ID NO.11, and the nucleotides sequence of encoding gene is classified as SEQ ID Shown in NO.12.

Wherein, amino acid multisite mutation F66R-L124H-S145P of the present invention indicates amino acid sequence to be SEQ 66th amino acids of 3 interferon mutant of ox λ shown in ID NO.5 are mutated into arginine (R) by phenylalanine (F), and 124th amino acids are mutated into histidine (H) by leucine (L), while the 145th amino acids being mutated by serine (S) At proline (P)；The rest may be inferred.

The invention also discloses the recombinations containing 3 interferon of ox λ or the encoding gene of 3 interferon mutant of ox λ Carrier or recombinant host cell.

The invention also discloses 3 interferon of ox λ or 3 interferon mutant of ox λ in preparation prevention or treatment ox The drug of viral disease or the purposes in reagent.

Wherein, the bovine viral disease includes: aftosa FMD, bovine viral diarrhea BVD and ox infectious rhinotracheitis Scorching IBR etc. can pass through the epidemic disease of alimentary canal, respiratory infectious.

The invention also discloses a kind of methods for preparing 3 interferon of ox λ or 3 interferon mutant of ox λ, including with The encoding gene of 3 interferon mutant of ox λ: (1) being cloned into baculovirus transfer vector by lower step, and building obtains weight Group transfer vector；(2) by recombinant transfer vector and baculovirus DNA cotransfection insect cell, recombinant baculovirus is obtained；(3) By recombinate shape virus infection insect cell or insect host, cultivates infected insect cell or insect host expression is corresponding Albumen, purifying to get.

Wherein, the baculovirus transfer vector be selected from AcRP23-lacZ, AcRP6-SC, AcUWl-lacZ, BacPAK6, Bac to Pac、Bacmid、BlucBacII(pETL)、p2Bac、p2Blue、p89B310、pAc360、pAc373、pAcAB3、 pAcAB4、PAcAS3、pAcC129、pAcC4、DZI、pAcGP67、pAcIEl、pAcJPl、pAcMLF2、pAcMLF 7、 pAcMLF8、pAcMPl、pAcMP2、pAcRP23、pAcRP 25、pAcRW4、pAcsMAG、pAcUWl、pAcUW21、pAcUW2A、 pAcUW2B、pAcUW3、pAcUW31、pAcUW41、pAcUW42、pAcUW43、pAcUW51、pAcVC2、pAcVC 3、pAcYMl、 pAcJcC5、pBacl、pBac2、pBlueBacIII、pBlueBacHis、pEV55、mXIV、pIEINeo、pJVETL、 pJVNhel、pJVP10、pJVrsMAG、pMBac、pP10、pPAKl、pPBac、pSHONEX 1.1、pSYN XIV VI+、 pSYNVI+wp、pSYNXIV VI-、pVL1391、pVL 1392、pVL 1393、pVL941、pVL 945、pVL 985、 PVTBac, pBM030 or pUAC-5；

The baculoviral be selected from silkworm baculovirus parent plant BmBacmid, BmNPV, AcMNPV, ApNPV, HaNPV, HzNPV, LdMNPV, MbMNPV, OpMNPV, SlMNPV, SeMNPV or SpltNPV；

The insect host is selected from silkworm (Bombyx mori), wild silkworm (Bombyx mandarina), castor silkworm (Philosamia cynthia ricim), wild silkworm (Dictyoploca japanica), Philosamia cynthia (Philosamia cynthia Pryeri), tussah (Antheraea pernyi), yamama (Antheraea yamamai), wild giant silkworm (Antheraea Polyphymus), autographa california (Atographa califorica), tea geometrid (Ectropis obliqua), cabbage army worm (Mamestra brassicae), prodenia litura (Spodoptera littoralis), autumn armyworm (Spodoptera Frugiperda), cabbage looper (Trichoplusia ni), armyworm (Thaumetopoea wilkinsoni), bollworm (Heliothis armigera), U.S. bollworm (Heliothis zea), oriental tobacco budworm (Heliothis assulta), tobacco Noctuid (Heliothis virescens), oriental armyworm (Pseudaletia separata) or gypsymoth (Lymantria dispar)；

Preferably, the baculovirus transfer vector is pVL1393；The baculoviral is silkworm baculovirus parent plant BmBacmid；The insect host is silkworm (Bombyx mori).

It is described to infect the insect larvae or pupa for referring to recombinant baculovirus by eating or infecting through epidermis 1-5 age Body；Preferably, it by recombinant Bombyx mori baculovirus infected silkworm cell or the silkworm larva or pupa in percutaneous puncture-inoculation 1-5 age, is infecting Body fluid or the tissue homogenate of silkworm larva or pupa containing various 3 interferon genes of ox λ are collected after 3-6 days；Wherein, the pupa is most Excellent is 1-2 days early stage tender pupas.

The utility model has the advantages that

The rule of amino acid sequence point mutation does not have rule that can follow, and mutant and mutation effect do not correspond, success Mutant ratio be very low.The present invention is carried out by the 3 interferon amino acid sequence of ox λ announced on analysis NCBI The signal peptide analysis of sequence alignment and profession, final determine with accession number is the ammonia of XP_010847513.1 (SEQ ID NO.1) Base acid sequence is main reference sequences, is first become the 5th cysteine (C) of original amino acid arginine (R), the 19th Position serine (S) becomes proline (P), i.e. C5R, S19P, obtains the 3 interferon mutant of signal peptide of ox λ of IFN, is named as BoIFN- λ 3-S mutant.The BoIFN- λ 3-S antiviral activity expressed in silkworm larva body is 6.31 × 10⁵U/mL, than ox λ 3 The original sequence B oIFN- λ 3 of interferon improves 81%.

The general amino acid for becoming similar properties, conserved structure is generally constant, and the present invention is further attempted to 3 interferon of ox λ Mutant of signal peptide BoIFN- λ 3-S carries out conserved sequence mutation, i.e., by the 125th first sulphur of BoIFN- λ 3-S amino acid sequence Propylhomoserin (M) becomes threonine (T), and the 164th lysine (K) is mutated into glutamic acid (E), i.e. M125T, K164E, and according to silkworm Codon preference carries out codon optimization to its gene order, obtains new 3 interferon mutant BoIFN- λ 3-S-C-O of ox λ Mutant, the BoIFN- λ 3-S-C-O expressed in silkworm larva body have a more significant antiviral activity, potency up to 1.45 × 10⁶U/mL.Than 3 interferon original series potency 3.48 × 10 of ox λ⁵U/mL improves 3.2 times.

The present invention further on the basis of BoIFN- λ 3-S-C-O mutant, carry out respectively F66R, A109S, L124H, After this 6 site single-site mutants of S131A, S145P, S165P, the 3 interferon potency of ox λ of expression is higher than the guarantor of signal polypeptide mutant It keeps sequence and expresses the potency measured, antiviral activity reaches 1.58 × 10⁶-3.16×10⁶U/mL；And remaining site mutation aftereffect Valence is constant or even reduces, and illustrates that the mutation in this 6 sites is effectively to be mutated, and can achieve the purpose for improving antiviral activity.Its In, carry out the acquisition of F66R single-site mutant BoIFN- λ 3-S-C-O-F66R mutant have most strong antivirus action 3.16 × 10⁶U/mL.Than 3 interferon original series potency 3.48 × 10 of ox λ⁵U/mL improves 8 times.

The present invention further by antiviral activity improve single mutation site F66R, A109S, L124H, S131A, S145P, BoIFN- λ 3-S-C-O mutant is carried out double-site mutant by S165P combination of two.The testing result of antiviral activity shows After tri- groups of F66R-L124H, F66R-S145P, A109S-S131A double mutation, the 3 interferon potency of ox λ of expression is higher than conservative sequence Column, single mutation sequence express the potency measured, and antiviral activity reaches 3.72 × 10⁶-3.98×10⁶U/mL, and remaining several groups of position Potency is constant after point mutation or even reduces, and illustrates that the mutation in this 3 combination sites is effectively to be mutated, it is antiviral to can achieve raising Active purpose.Wherein, the BoIFN- λ 3-S-C-O-F66R-L124H mutant of F66R-L124H double-site mutant acquisition is carried out With most strong antivirus action, than 3 interferon original series potency 3.48 × 10 of ox λ⁵U/mL improves 10.4 times.

The present invention further combines double mutational sites with high-titer of acquisition, by BoIFN- λ 3-S-C-O mutant Carry out amino acid multisite mutation.The testing result of mutant antiviral activity shows that tri- site F66R-L124H-S145P is prominent After change, the 3 interferon potency of ox λ of expression expresses the potency measured much higher than conserved sequence, single mutation sequence, double mutant nucleotide sequences, It is 4.68 × 10⁶U/mL, than 3 interferon original series potency 3.48 × 10 of ox λ⁵U/mL improves 12.5 times；And remaining several groups of site Potency is constant after mutation or even reduces.The mutation for illustrating this combination site is effectively to be mutated, and can achieve raising antiviral activity Purpose.

The method of the present invention simple process can be quickly obtained a large amount of safe and reliable 3 interferon of ox λ.Ox λ of the present invention 3 interferon mutants can be used in the drug or reagent of preparation prevention or treatment bovine viral disease, have to the development of animal husbandry It is of great importance.

Detailed description of the invention

Fig. 1 is the corresponding fluorogram of cytopathy ratio；Wherein, A, "-": cell-free lesion；B, " ± ": several cytopathies Become；C, "+": 20%~30% cytopathy；D, " ++ ": 50%~60% cytopathy；

The double digestion that Fig. 2 is recombinant plasmid pVL-BoIFN- λ 3 is identified；Wherein, M:DNA molecular mass standard；1: recombination matter 3 double enzyme digestion product of grain pVL-BoIFN- λ；2: recombinant plasmid pVL-BoIFN- λ 3-S double enzyme digestion product；It is negative control；

Fig. 3 is that cell the case where various ratios of fluorescence occurs；Wherein, A: the fluorescence that interferon inhibits VSV virus to show； The fluorescence that B:VSV virus-infected controls group is shown；C: the fluorescence that part cell infection VSV virus is shown.

Specific embodiment

Transfer vector pVL1393, E.coli bacterial strain TOP10, BmN cell, MDBK cell, VSV-GFP virus are purchased from Promega company；It tests cultivated silkworm breed variety JY1 and is purchased from Zhenjiang seed station, restriction enzyme, T₄DNA ligase is purchased from Promega Company, PCR reaction LA Taq archaeal dna polymerase used and other related reagents are purchased from TakaRa company, and liposome is purchased from Invitrogen company, DMEM cell culture medium, fetal calf serum are GIBCO Products.

The expression and detection of 1 N of 3 interferon of λ of embodiment and its mutant of signal peptide gene in silkworm biological reactor

The building of 1.1 target gene synthesis and recombinant plasmid

The present invention analyzes the 3 interferon amino acid sequence of ox λ announced on NCBI, and (accession number is respectively as follows: XP_ 010847513.1, NP_001268830.1, DAA19870.1, ADP05157.1), carry out the signal peptide of sequence alignment and profession Analysis, it is final determine using accession number for XP_010847513.1 amino acid sequence as original series, sent out in sequencing procedure Now the signal peptide of the sequence has multiple cutting peaks, and decision is mutated 3 interferon amino acid sequence of ox λ.It is interfered with reference to pig λ 3 The 5th cysteine of original amino acid is dashed forward in plain amino acid sequence design, influence of the comparison signal peptide to antiviral activity Become arginine, the 19th mutant serine is proline, obtains the 3 interferon mutant of signal peptide of ox λ of IFN, is named as BoIFN- λ 3-S mutant, amino acid sequence is as shown in SEQ ID NO.3, and gene order is as shown in SEQ ID NO.4.According to Restriction enzyme site analyzes the multiple cloning sites on result and transfer vector pVL1393 and pUC57, and BamHI enzyme is added at its end 5' EcoRI restriction enzyme site and its protection base, the gene determined is added in enzyme site and its protection base and Kozak sequence, the end 3' Sequence transfers to Nanjing Genscript Biotechnology Co., Ltd. to synthesize, and is inserted into pUC57 carrier, forms plasmid pUC57-BoIFN- λ 3。

1.2 building recombinant baculovirus transfer vectors

Double digestion processing is carried out with plasmid pUC57-BoIFN- λ 3 of the BamHI and EcoRI to synthesis, glass milk method recycles mesh Segment, T₄DNA ligase connects target fragment and carries out the baculovirus transfer vector after double digestion processing and inactivation PVL1393,16 DEG C, connection is overnight.Connection product is converted into competent escherichia coli cell TOP10, choosing colony culture, upgrading Grain identifies positive colony with BamHI and EcoRI double digestion, will identify that correct recombinant plasmid send Beijing to hold up section's biotechnology and has The sequencing of limit company, is sequenced correct plasmid and is named as pVL-BoIFN- λ 3.

Plasmid pVL-BoIFN- λ 3 is subjected to double digestion processing with BamHI and EcoRI, uses glass after agarose gel electrophoresis Glass milk method recycle target fragment, reapply fusion DNA vaccine Technology design primer pair target fragment carry out rite-directed mutagenesis (rite-directed mutagenesis Fusion DNA vaccine method, a kind of new method of vector construction graceful et al. referring to Kuang bird with red feathers: recombination fusion DNA vaccine method, genomics and application Biology, 2012, volume 31, the 6th phase, the 634-639 pages) described in method carry out), use T later₄DNA ligase connects Baculovirus transfer vector pVL1393 (16 DEG C, connect overnight) after connecing target fragment and carrying out double digestion processing and inactivate.It will Connection product converts competent escherichia coli cell TOP10, choosing colony culture, upgrading grain, with BamHI and EcoRI double digestion It identifies positive colony, will identify that correct recombinant plasmid send Beijing Qing Ke Bioisystech Co., Ltd to be sequenced, correct matter is sequenced Grain is named as pVL-BoIFN- λ 3-S.

As the original nucleotide sequence mutation of 3 interferon of ox λ at needed for 3 interferon mutant of signal peptide nucleotide sequence of ox λ Primer:

(1) two sides upstream and downstream primer:

P1-F:cgggatccaacatggccccgggcagaacgctggtgctg

P1-R:cggaattctcagacacactggtctccgctggcaacacat

(2) intermediate upstream and downstream primer:

P2-F:acaaccgtggcgctgccaaggacaggagcagttcct

P2-R:aggaactgctcctgtccttggcagcgccacggttgt

Acquisition, purifying and the amplification of 1.3 recombinant Bombyx mori baculovirus

Parental virus BmBacmid DNA according to document (patent No.: ZL 201110142492.4, authorization date: 2013.01.23) disclosed in method building.1 μ g silkworm baculovirus parent plant is sequentially added into a sterile tube BmBacmid DNA, 1 μ g recombinant transfer plasmid pVL-BoIFN- λ 3 or 1 μ g recombinant transfer plasmid pVL-BoIFN- λ 3-S, 5 μ L rouge Plastid is supplied volume to 60 μ L with aseptic double-distilled water, is mixed gently, and after standing 15min, is added dropwise in culture bottle and is total to Transfection.1.5mL serum free medium and 300 μ L FBS are added after 27 DEG C of culture 4h.27 DEG C constant temperature incubation 4~5 days, until cell is de- It falls and floats, collect cell culture fluid, obtain recombinant virus rBmBacmid (BoIFN- λ 3), rBmBacmid containing target gene (BoIFN-λ3-S)。

The purifying of recombinant Bombyx mori baculovirus and amplification method are as follows: it is small in 35mm to be inoculated with appropriate cell (about 70~80%) In plate, after cell is adherent, culture medium is sucked, the cell culture fluid of collection is subjected to various concentration dilution, takes 1mL to be added to adherent In cell, it is evenly distributed.After 27 DEG C of infection 1h, infection liquid is sucked, 2% low fusion agarose gel is melted in 60 DEG C of water-baths To change, is cooled to 40 DEG C and is uniformly mixed with 40 DEG C of 2 × TC-100 culture mediums preheated (containing 20%FBS), each plate adds 4mL glue, to It is sealed after solidification with Parafilm, 3~5d, micro- sem observation are cultivated in 27 DEG C of inversions.Virus plaque is picked out, more than repetition Step obtains pure recombinant Bombyx mori baculovirus rBmBacmid (BoIFN- λ 3), rBmBacmid by the purifying of 2~3 wheels (BoIFN-λ3-S)。

Recombinant Bombyx mori baculovirus rBmBacmid (BoIFN- λ 3), rBmBacmid (BoIFN- λ 3-S) are infected normal raw Long BmN cell, culture collect supernatant, contain a large amount of recombinant virus rBmBacmid (BoIFN- λ in supernatant after 3 days 3)、rBmBacmid(BoIFN-λ3-S)。

1.4 Ns of 3 type interferon of λ and its mutant are expressed in silkworm body

Recombinant virus culture solution is pressed 10⁵PFU/ dosage injects silkworm from 5 ages, in 27 DEG C, 70%~80% humidity Under the conditions of cultivate, silkworm (kind JY1) larva grows advanced stage, and BoIFN- λ 3 obtains height under the action of polyhedrosis gene promoter Effect expression.Inoculation 3.5d~4d or so, it can be observed that the diseases such as the swelling of silkworm larva body segment, abnormal behavior, loss of appetite Shape when stopping feed, collects hemolymph, -20 DEG C save backup when observing that larva volume is obviously reduced.

1.5 Ns of 3 type interferon of λ and its detection of mutant protein antiviral activity

Method is inhibited to detect 3 type of ox λ expressed in silkworm hemolymph in MDBK/VSV*GFP system using few cells lesion The antiviral activity of interferon.By MDBK cell in good condition with 3.0 × 10⁵The density of a/mL is inoculated in 96 well culture plates In.The DMEM culture solution of silkworm hemolymph fetal calf serum containing 70mL/L of ultrasonication and filtration sterilization is configured to different dilute The sample diluted is inoculated in the culture hole for being paved with MDBK cell, each dilution by the solution for degree of releasing by 100 holes μ L/ Degree and control silkworm blood at least set 12 multiple holes, while setting the cell controls group that silkworm hemolymph and VSV*GFP is not added and addition VSV* The virus control group of GFP, in 37 DEG C, 5%CO₂Under the conditions of culture 18~for 24 hours.100TCID will be diluted to₅₀VSV*GFP virus, It is added to have inhaled by 100 holes μ L/ and abandon in the culture hole of supernatant, be placed in 37 DEG C, 5%CO₂Under the conditions of cultivate.It is aobvious being inverted fluorescence Micro- microscopic observation, when fluorescence occur in cells a large amount of in each hole of virus control group, and the cell in cell controls group is still grown completely Well, when unstressed configuration occurs, then show that contradistinction system is completely qualified, complete observation can be made.

2, experimental result

The identification of 2.1 recombinant transfer vectors

Recombinant transfer vector pVL-BoIFN- λ 3, pVL-BoIFN- λ 3-S are through BamHI and EcoRI double digestion, in 1% agar 2 segments are isolated in sugared gel electrophoresis, and small fragment is consistent between 500-750bp with target gene fragment 588bp size, Large fragment is located above 8000bp, is consistent with pVL1393 segment 9607bp size.Electrophoresis result is as shown in Figure 2.Digestion is identified Correct plasmid send Beijing Qing Kexin industry Bioisystech Co., Ltd to carry out nucleotide sequencing, with MegaAlign comparison result table Bright sequence is consistent with the sequence originally designed, shows that 3 type interferon of ox λ and its mutant of signal peptide gene have been successfully plugged into In pVL1393 transfer vector between BamHI and EcoRI.

The acquisition of 2.2 Bov IFN recombinant viruses and the detection of recombinant products

3 type of ox λ for inhibiting method to detect silkworm larva expression in MDBK/VSV*GFP system using few cells lesion is dry Disturb plain antiviral activity.It is observed under inverted fluorescence microscope, the cell growth state in cell controls group is good, unstressed configuration Occur；Lesion occurs for the cell in virus infection control group, and fluorescence occur in most cells, adds 3 interferon protein of recombinant bovine λ Cell have resist virus infection ability (Fig. 3).Protective effect according to 3 type interferon of ox λ to MDBK cell, observation are thin The lesion degree of born of the same parents, green cells appearance to be had, this hole cell are just denoted as "+", calculate according to Reed-Muench method The potency of interferon.Testing result is listed in table 1, Assay of Antiviral Activity the result shows that, the BoIFN- λ expressed in silkworm larva body 3 have a more significant antiviral activity, and potency is up to 3.48 × 10⁵U/mL, mutant nucleotide sequence BoIFN- λ 3-S antiviral activity are higher than BoIFN- λ 3 is 6.31 × 10⁵U/mL, sequence B oIFN- λ 3 more original than 3 interferon of ox λ improve 81%, illustrate through mutation letter The method of number peptide specific cleavage site is come to improve 3 antiviral activity of BoIFN- λ be feasible and effective.

The testing result of 1 recombinant bovine λ of table, 3 antiviral activity of interferon

Table after the progress conserved sequence mutation of embodiment 2BoIFN- λ 3-S mutant and optimization in silkworm biological reactor Up to detection

The general amino acid for becoming similar properties, conserved structure is generally constant, and the present invention is further attempted to referring to 1 side of embodiment 3 interferon mutant of signal peptide of ox λ (BoIFN- λ 3-S mutant) is carried out conserved sequence mutation and optimized, to BoIFN- λ by method 125th methionine of the amino acid sequence of 3-S mutant sports threonine, and the 164th lysine mutation is glutamic acid, New 3 interferon mutant of ox λ is obtained, BoIFN- λ 3-S-C mutant, amino acid sequence such as SEQ ID NO.5 institute are named as Show.

In addition, the present invention utilizes OptimumGene^TMTechnology optimizes above-mentioned 3 interferon mutant gene of ox λ, root Gene order is transformed according to the codon preference of bioreactor silkworm, to influence genetic transcription efficiency, translation efficiency Stablize with G/C content, CpG dinucleotides content, codon preference, the secondary structure of mRNA, the mRNA free energy of protein folding Property, a variety of relevant parameters such as RNA unstability motif, repetitive sequence optimize, be conducive to improve institute's optimization gene and exist Transfer efficient record and translation efficiency in silkworm, and keep the protein sequence finally translated into constant.

In order to improve the translation initiation efficiency in silkworm baculovirus eukaryotic expression system, add before gene Kozak sequence AAC, in order to improve translation termination efficiency, terminator codon is changed to TAA.In addition, also removing inside gene order The restriction enzyme sites such as BamHI, EcoRI, SmaI, added BamHI in upstream region of gene, added in downstream of gene EcoRI restriction enzyme site is cloned into eukaryon transfer vector pVL1393 so as to subsequent.

Sequence after designed 3 type interferon mutant gene of λ optimizes is artificial synthesized by biotechnology company, life Entitled BoIFN- λ 3-S-C-O mutant, nucleotide sequence as shown in SEQ ID NO.6, be inserted by the genetic fragment of synthesis PUC57 carrier forms plasmid pUC57-BoIFN, is named as pUC57-BoIFN- λ 3-S-C-O.Construct recombinant baculovirus transfer Carrier is named as pVL-BoIFN- λ 3-S-C-O.Preparation and reorganization virus rBmBacmid (BoIFN- λ 3-S-C-O).Antiviral work Property measurement result shows that the BoIFN- λ 3-S-C-O expressed in silkworm larva body has more significant antiviral activity, and potency reaches 1.45×10⁶U/mL (table 2), than 3 interferon original series potency 3.48 × 10 of ox λ⁵U/mL improves 3.2 times, illustrates dry in ox λ 3 It disturbs and carries out conserved sequence mutation on plain mutant of signal peptide and the method that optimizes is come to improve 3 antiviral activity of BoIFN- λ be feasible With it is effective.

The testing result of antiviral activity after 2 Ns of 3 interferon mutant of λ optimizations of table

3 BoIFN- λ 3-S-C-O mutant of embodiment optimizes and carries out anti-in silkworm biology after amino acid unit point mutation Answer the expression and detection in device

Referring to Examples 1 and 2 method, using the gene order of the BoIFN- λ 3-S-C-O mutant after codon optimization as mould Plate further designs multipair primer pair conserved sequence and carries out rite-directed mutagenesis, the interferon potency ratio that will be expressed after mutation here Higher point mutation is listed in herein, as the basis that further mutation improves, and the mutant interferon potency expressed after being mutated Very poor mutational site is not just listed one by one herein, because successfully mutant ratio is very low, subsequent embodiment In also like this principle carry out.Mutational site be respectively E51A, T57R, F66R, H76R, T81S, H86Q, A109S, H117R, L124H,Q127R,I129V,S131A,T142M,S144G,S145P,S165P,D167S；Resulting 3 interferon mutant of ox λ Be named as BoIFN- λ 3-S-C-O-M1 (E51A, T57R, F66R, H76R, T81S, H86Q, A109S, H117R, L124H, Q127R, I129V, S131A, T142M, S144G, S145P, S165P, D167S) mutant.

BoIFN- λ 3-S-C-O mutant nucleotide sequence carries out needed for amino acid unit point, double site and multisite mutation Primer:

(1) two sides upstream and downstream primer:

P3-F:tcataccgtcccaccatcgggcgcggatccaacatggctccggga

P3-R:gatctgcagcggccgctccggaattcttaaacacattgatcg

(2) intermediate upstream and downstream primer

Construct recombinant baculovirus transfer vector, be named as pVL-BoIFN- λ 3-S-C-O-M1 (E51A, T57R, F66R, H76R、T81S、H86Q、A109S、H117R、L124H、Q127R、I129V、S131A、T142M、S144G、S145P、S165P、 D167S).Preparation and reorganization virus rBmBacmid (BoIFN- λ 3-S-C-O-M1).

Testing result is listed in table 3, and all 3 interferon mutants of ox λ measure potency 3.72 × 10⁵U/mL~3.16 × 10⁶U/mL, wherein after this 6 site mutations of F66R, A109S, L124H, S131A, S145P, S165P, the ox λ 3 of expression is interfered The conserved sequence that plain potency is slightly above signal polypeptide mutant expresses the potency measured, and potency is constant after remaining site mutation or even drops It is low, illustrate that the mutation in this 6 sites is effectively to be mutated, can achieve the mesh for improving BoIFN- λ 3-S-C mutant antiviral activity 's.Wherein, BoIFN- λ 3-S-C-O-F66R mutant has most strong antivirus action, than 3 interferon original series potency of ox λ 3.48×10⁵U/mL improves 8 times, and amino acid sequence is shown in SEQ ID NO.7, and the nucleotides sequence of encoding gene is classified as SEQ Shown in ID NO.8.

The testing result of 3 recombinant bovine λ of table, 3 interferon single-site mutant antiviral activity

In silkworm biological reactor after embodiment 4BoIFN- λ 3-S-C-O-M1 mutant progress amino acid double-site mutant In expression and detection

In view of embodiment 3 as a result, determine moiety site mutation be effectively to be mutated, can achieve improve BoIFN- λ 3- The antiviral activity purpose of S-C mutant.In view of putting in order for amino acid is the primary structure of protein, and decide egg The higher structure of white matter, and the position that the amino acid unit point mutation carried out in embodiment 3 has fractional mutations site may be phase Mutual correlation, therefore attempt to carry out amino acid double-site mutant.Single mutation site F66R that the present invention improves antiviral activity, A109S, L124H, S131A, S145P, S165P combination of two carry out double-site mutant, and double-site mutant is obtained in embodiment 3 On the basis of the single-site mutant sequence obtained, using its (BoIFN- λ 3-S-C-O-M1) as template, corresponding primer is utilized (detailed in Example 3) carries out deputy rite-directed mutagenesis by the method for fusion DNA vaccine, to obtain the purpose of double-site mutant Segment.

Double mutational sites be F66R-A109S, F66R-L124H, F66R-S131A, F66R-S145P, F66R-S165P, A109S-L124H、A109S-S131A、A109S-S145P、A109S-S165P、L124H-S131A、L124H-S145、L124H- S165P, S131A-S145P, S131A-S165P, S145P-S165P totally 15 kinds of combinations, 3 interferon mutant of ox λ obtained It is named as BoIFN- λ 3-S-C-O-M1-M2 (F66R-A109S, F66R-L124H, F66R-S131A, F66R-S145P, F66R- S165P、A109S-L124H、A109S-S131A、A109S-S145P、A109S-S165P、L124H-S131A、L124H-S145、 L124H-S165P, S131A-S145P, S131A-S165P, S145P-S165P) mutant.

Construct recombinant baculovirus transfer vector, be named as pVL-BoIFN- λ 3-S-C-O-M1-M2 (F66R-A109S, F66R-L124H、F66R-S131A、F66R-S145P、F66R-S165P、A109S-L124H、A109S-S131A、A109S- S145P、A109S-S165P、L124H-S131A、L124H-S145、L124H-S165P、S131A-S145P、S131A-S165P Or S145P-S165P).

Recombinant virus rBmBacmid (BoIFN- λ 3-S-C-O-M1-M2) testing result is listed in table 4, all 3 interferon of ox λ Mutant measures potency 6.31 × 10⁵U/mL~3.98 × 10⁶U/mL, wherein F66R-L124H, F66R-S145P, A109S- After tri- groups of S131A double mutation, the 3 interferon potency of ox λ of expression is slightly above conserved sequence, single mutation sequence expresses the effect measured Valence, and potency is constant after remaining several groups of site mutation or even reduces, and illustrates that the mutation in this 3 combination sites is effectively to be mutated, it can To achieve the purpose that improve BoIFN- λ 3-S-C-O-M1 mutant antiviral activity.Wherein, BoIFN- λ 3-S-C-O-F66R- L124H mutant has most strong antivirus action, than 3 interferon original series potency 3.48 × 10 of ox λ⁵U/mL improves 10.4 Times, amino acid sequence is shown in SEQ ID NO.9, and the nucleotides sequence of encoding gene is classified as shown in SEQ ID NO.10.

The testing result of 4 recombinant bovine λ of table, 3 interferon double-site mutant antiviral activity

It is anti-in silkworm biology after 5 BoIFN- λ 3-S-C-O-M1-M2 mutant of embodiment progress amino acid multisite mutation Answer the expression and detection in device

In view of embodiment 4 as a result, in view of putting in order for amino acid be the primary structure of protein, and decide egg The higher structure of white matter, thus it is speculated that may be since the amino acid unit point mutation of progress has the position in fractional mutations site mutually to lean on It is close associated with each other, therefore attempt to carry out amino acid multisite mutation.The present invention is by double mutational sites with high-titer of acquisition Combination, determines third mutational site, multisite mutation be on the basis of the double-site mutant sequence of the acquisition of embodiment 4, Pass through fusion DNA vaccine using corresponding primer (detailed in Example 3) using its (BoIFN- λ 3-S-C-O-M1-M2) as template Method carries out the rite-directed mutagenesis of third position, obtains following 8 kinds and combines: F66R-A109S-L124H, F66R-A109S-S131A, F66R-A109S-S145P、F66R-L124H-S131A、F66R-L124H-S145P、F66R-S131A-S145P、A109S- L124H-S131A,A109S-S131A-S145P.3 interferon mutant of ox λ obtained is named as BoIFN- λ 3-S-C-O- M1-M2-M3(F66R-A109S-L124H、F66R-A109S-S131A、F66R-A109S-S145P、F66R-L124H-S131A、 F66R-L124H-S145P, F66R-S131A-S145P, A109S-L124H-S131A, A109S-S131A-S145P) mutant.

Recombinant baculovirus transfer vector is constructed, pVL-BoIFN- λ 3-S-C-O-M1-M2-M3 (F66R- is named as A109S-L124H、F66R-A109S-S131A、F66R-A109S-S145P、F66R-L124H-S131A、F66R-L124H- S145P、F66R-S131A-S145P、A109S-L124H-S131A、A109S-S131A-S145P)。

Recombinant virus rBm-Bacmid (BoIFN- λ 3-S-C-O-M1-M2-M3) testing result is listed in table 5, and all ox λ 3 are dry It disturbs plain mutant and measures potency 1.78 × 10⁶U/mL~4.68 × 10⁶U/mL, wherein tri- site F66R-L124H-S145P is prominent After change, the 3 interferon potency of ox λ of expression expresses the potency measured much higher than conserved sequence, single mutation sequence, double mutant nucleotide sequences, It is 4.68 × 10⁶U/mL, than 3 interferon original series potency 3.48 × 10 of ox λ⁵12.5 times of U/mL raising, and remaining several groups of site Potency is constant after mutation or even reduces, and illustrates that the mutation in this combination site is effectively to be mutated, can achieve and improve BoIFN- λ 3-S- The purpose of C-O-M1-M2 mutant antiviral activity.BoIFN-λ3-S-C-O-F66R-L124H-S145P

The amino acid sequence of mutant is shown in SEQ ID NO.11, and the nucleotides sequence of encoding gene is classified as SEQ ID Shown in NO.12.

The testing result of 5 recombinant bovine λ of table, 3 interferon multisite mutation antiviral activity

Research finds that ox IFN- λ 3 can significantly reduce the sense of foot and mouth disease virus (FMDV) on bovine embryo kidney cell (EBK) Dye, effect has been more than the rejection ability of ox IFN-a and pig IFN-a to FMDV, stimulates interferon to stimulate while antiviral Gene (ISGs), especially in upper respiratory tract mucous membrane and skin histology, be proved so far most possibly develop into Choice drug (Diaz-San Segundo F., Weiss M., the Perez-Martin E., et of aftosa biological therapy al.Antiviral activity of bovine type III interferon against foot-and-mouth disease virus.[J].Virology,2011,413(2):283-292.；Perez-Martin E.,Weiss M., Diaz-San Segundo F.,et al.Bovine type III interferon significantly delays and reduces the severity of foot-and-mouth disease in cattle.[J].J Virol,2012,86 (8): 4477-4487.) all 3 interferon mutants of ox λ that the present invention obtains correspondingly can be in preparation prevention or treatment cattle disease The drug of viral disease or the application in reagent, the bovine viral disease include: aftosa (FMD), bovine viral diarrhea (BVD) and infectious bovine rhinotrachetis (IBR) etc. can pass through the epidemic disease of alimentary canal, respiratory infectious.

In the preparation method of above-mentioned 3 interferon mutant of ox λ, with baculovirus transfer vector AcRP23-lacZ, AcRP6- SC、AcUWl-lacZ、BacPAK6、Bac to Pac、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、pBlueBacHis、pEV55、mXIV、 pIEINeo、pJVETL、pJVNhel、pJVP10、pJVrsMAG、pMBac、pP10、pPAKl、pPBac、pSHONEX 1.1、 pSYN XIV VI+、pSYNVI+wp、pSYNXIV VI-、pVL1391、pVL 1392、pVL941、pVL 945、pVL 985、 It is common sense well known in the art that pVTBac, pBM030 or pUAC-5, which replace pVL1393,.

With silkworm baculovirus parent plant, BmNPV, AcMNPV, ApNPV, HaNPV, HzNPV, LdMNPV, MbMNPV, It is also common sense well known in the art that OpMNPV, SlMNPV, SeMNPV or SpltNPV, which replace BmBacmid,.

With insect host silkworm (Bombyx mori), wild silkworm (Bombyx mandarina), castor silkworm (Philosamia Cynthia ricim), wild silkworm (Dictyoploca japanica), Philosamia cynthia (Philosamia cynthia pryeri), toothed oak Silkworm (Antheraea pernyi), yamama (Antheraea yamamai), wild giant silkworm (Antheraea Polyphymus), autographa california (Atographa califorica), tea geometrid (Ectropis obliqua), cabbage army worm (Mamestra brassicae), prodenia litura (Spodoptera littoralis), autumn armyworm (Spodoptera Frugiperda), cabbage looper (Trichoplusia ni), armyworm (Thaumetopoea wilkinsoni), bollworm (Heliothis armigera), U.S. bollworm (Heliothis zea), oriental tobacco budworm (Heliothis assulta), tobacco Noctuid (Heliothis virescens), oriental armyworm (Pseudaletia separata) or gypsymoth (Lymantria Dispar) replacing cultivated silkworm breed variety JY1 is also common sense well known in the art.

Amino acid sequence and nucleic acid sequence

SEQ ID NO.1: ox λ 3 interferon original amino acid；

MAPGCTLVLVLMLTTVALSRTGAVPVPSAPRALPPARGCHMAQFKSLSPQELQAFKTARDAFEDSFLPKDWDCSTHL FPRTRDLKHLQVWERPVALEAELALTLTVLEAMANSSLGHSLEQPLLMLQNIHSKLQACVPAQPTASSRPRGRLHHW LHRLQEARKKSQDCLEASVMFNLLRLLTRDLKCVASGDQCV.

SEQ ID NO.2: ox λ 3 interferon original nucleotide sequences；

ATGGCCCCGGGCTGCACGCTGGTGCTGGTGCTGATGCTGACAACCGTGGCGCTGAGCAGGACAGGAGCA GTTCCTGTGCCCTCTGCCCCCAGGGCCCTCCCACCTGCCAGGGGCTGCCACATGGCCCAGTTCAAGTCTCTGTCCCC TCAAGAGCTGCAGGCCTTCAAGACGGCCAGGGATGCCTTTGAAGACTCGTTCTTGCCAAAGGACTGGGACTGCAGCA CCCACCTTTTCCCCAGGACCCGGGACCTGAAGCACCTGCAGGTGTGGGAGCGCCCTGTAGCTCTGGAGGCAGAGCTG GCCCTGACACTGACGGTCCTGGAGGCCATGGCTAACTCATCCCTGGGCCACAGCCTGGAGCAGCCCCTTCTCATGTT GCAGAACATCCACTCCAAGCTCCAGGCCTGTGTCCCAGCTCAGCCCACAGCAAGCTCCAGGCCCCGGGGCCGCCTCC ACCACTGGCTGCACCGCCTCCAGGAGGCCCGGAAGAAGTCCCAGGACTGCCTCGAAGCCTCTGTGATGTTCAACCTC CTCCGCCTTCTCACCCGGGACCTGAAATGTGTTGCCAGCGGAGACCAGTGTGTCTGA

SEQ ID NO.3:BoIFN- λ 3-S variant amino acid sequence

MAPGRTLVLVLMLTTVALPRTGAVPVPSAPRALPPARGCHMAQFKSLSPQELQAFKTARDAFEDSFLPKDWDCSTHL FPRTRDLKHLQVWERPVALEAELALTLTVLEAMANSSLGHSLEQPLLMLQNIHSKLQACVPAQPTASSRPRGRLHHW LHRLQEARKKSQDCLEASVMFNLLRLLTRDLKCVASGDQCV.

SEQ ID NO.4:BoIFN- λ 3-S mutant nucleotide sequence；

ATGGCCCCGGGCAGAACGCTGGTGCTGGTGCTGATGCTGACAACCGTGGCGCTGCCAAGGACAGGAGCA GTTCCTGTGCCCTCTGCCCCCAGGGCCCTCCCACCTGCCAGGGGCTGCCACATGGCCCAGTTCAAGTCTCTGTCCCC TCAAGAGCTGCAGGCCTTCAAGACGGCCAGGGATGCCTTTGAAGACTCGTTCTTGCCAAAGGACTGGGACTGCAGCA CCCACCTTTTCCCCAGGACCCGGGACCTGAAGCACCTGCAGGTGTGGGAGCGCCCTGTAGCTCTGGAGGCAGAGCTG GCCCTGACACTGACGGTCCTGGAGGCCATGGCTAACTCATCCCTGGGCCACAGCCTGGAGCAGCCCCTTCTCATGTT GCAGAACATCCACTCCAAGCTCCAGGCCTGTGTCCCAGCTCAGCCCACAGCAAGCTCCAGGCCCCGGGGCCGCCTCC ACCACTGGCTGCACCGCCTCCAGGAGGCCCGGAAGAAGTCCCAGGACTGCCTCGAAGCCTCTGTGATGTTCAACCTC CTCCGCCTTCTCACCCGGGACCTGAAATGTGTTGCCAGCGGAGACCAGTGTGTCTGA

SEQ ID NO.5:BoIFN- λ 3-S-C-O variant amino acid sequence；

MAPGRTLVLVLMLTTVALPRTGAVPVPSAPRALPPARGCHMAQFKSLSPQELQAFKTARDAFEDSFLPKDWDCSTHL FPRTRDLKHLQVWERPVALEAELALTLTVLEAMANSSLGHSLEQPLLTLQNIHSKLQACVPAQPTASSRPRGRLHHW LHRLQEARKESQDCLEASVMFNLLRLLTRDLKCVASGDQCV.

SEQ ID NO.6:BoIFN- λ 3-S-C-O mutant nucleotide sequence；

ATGGCTCCGGGAAGAACACTGGTGTTGGTTCTCATGCTGACAACTGTTGCTCTGCCAAGAACTGGTGCT GTCCCAGTGCCATCAGCTCCAAGAGCTTTGCCTCCAGCTAGAGGATGCCACATGGCCCAATTCAAATCGTTGAGTCC TCAAGAACTCCAGGCCTTCAAGACTGCTAGAGACGCCTTCGAAGATAGCTTCCTCCCTAAAGACTGGGATTGTTCCA CTCACCTGTTCCCAAGAACCAGAGACTTGAAGCACCTCCAGGTTTGGGAAAGACCAGTCGCTTTGGAAGCCGAATTG GCTCTCACCCTGACAGTCCTCGAAGCTATGGCCAACTCATCTCTCGGCCACTCGCTGGAACAACCGCTGTTGACCCT GCAAAATATCCACAGTAAATTGCAAGCCTGCGTGCCTGCTCAGCCAACAGCCAGCTCCAGACCCAGAGGTAGATTGC ACCACTGGCTGCACAGATTGCAAGAAGCTAGAAAAGAATCACAGGATTGTCTCGAAGCCTCTGTGATGTTCAACCTC CTGAGATTGCTCACCAGAGACCTGAAGTGCGTGGCTTCCGGCGATCAATGTGTTTAA

SEQ ID NO.7:BoIFN- λ 3-S-C-O-F66R variant amino acid sequence；

MAPGRTLVLVLMLTTVALPRTGAVPVPSAPRALPPARGCHMAQFKSLSPQELQAFKTARDAFEDSRLPKDWDCSTHL FPRTRDLKHLQVWERPVALEAELALTLTVLEAMANSSLGHSLEQPLLTLQNIHSKLQACVPAQPTASSRPRGRLHHW LHRLQEARKESQDCLEASVMFNLLRLLTRDLKCVASGDQCV.

SEQ ID NO.8:BoIFN- λ 3-S-C-O-F66R mutant nucleotide sequence；

ATGGCTCCGGGAAGAACACTGGTGTTGGTTCTCATGCTGACAACTGTTGCTCTGCCAAGAACTGGTGCT GTCCCAGTGCCATCAGCTCCAAGAGCTTTGCCTCCAGCTAGAGGATGCCACATGGCCCAATTCAAATCGTTGAGTCC TCAAGAACTCCAGGCCTTCAAGACTGCTAGAGACGCCTTCGAAGATAGCAGACTCCCTAAAGACTGGGATTGTTCCA CTCACCTGTTCCCAAGAACCAGAGACTTGAAGCACCTCCAGGTTTGGGAAAGACCAGTCGCTTTGGAAGCCGAATTG GCTCTCACCCTGACAGTCCTCGAAGCTATGGCCAACTCATCTCTCGGCCACTCGCTGGAACAACCGCTGTTGACCCT GCAAAATATCCACAGTAAATTGCAAGCCTGCGTGCCTGCTCAGCCAACAGCCAGCTCCAGACCCAGAGGTAGATTGC ACCACTGGCTGCACAGATTGCAAGAAGCTAGAAAAGAATCACAGGATTGTCTCGAAGCCTCTGTGATGTTCAACCTC CTGAGATTGCTCACCAGAGACCTGAAGTGCGTGGCTTCCGGCGATCAATGTGTTTAA

SEQ ID NO.9:BoIFN- λ 3-S-C-O-F66R-L124H variant amino acid sequence；

MAPGRTLVLVLMLTTVALPRTGAVPVPSAPRALPPARGCHMAQFKSLSPQELQAFKTARDAFEDSRLPKDWDCSTHL FPRTRDLKHLQVWERPVALEAELALTLTVLEAMANSSLGHSLEQPLHTLQNIHSKLQACVPAQPTASSRPRGRLHHW LHRLQEARKESQDCLEASVMFNLLRLLTRDLKCVASGDQCV.

SEQ ID NO.10:BoIFN- λ 3-S-C-O-F66R-L124H mutant nucleotide sequence；

ATGGCTCCGGGAAGAACACTGGTGTTGGTTCTCATGCTGACAACTGTTGCTCTGCCAAGAACTGGTGCT GTCCCAGTGCCATCAGCTCCAAGAGCTTTGCCTCCAGCTAGAGGATGCCACATGGCCCAATTCAAATCGTTGAGTCC TCAAGAACTCCAGGCCTTCAAGACTGCTAGAGACGCCTTCGAAGATAGCAGACTCCCTAAAGACTGGGATTGTTCCA CTCACCTGTTCCCAAGAACCAGAGACTTGAAGCACCTCCAGGTTTGGGAAAGACCAGTCGCTTTGGAAGCCGAATTG GCTCTCACCCTGACAGTCCTCGAAGCTATGGCCAACTCATCTCTCGGCCACTCGCTGGAACAACCGCTGCACACCCT GCAAAATATCCACAGTAAATTGCAAGCCTGCGTGCCTGCTCAGCCAACAGCCAGCTCCAGACCCAGAGGTAGATTGC ACCACTGGCTGCACAGATTGCAAGAAGCTAGAAAAGAATCACAGGATTGTCTCGAAGCCTCTGTGATGTTCAACCTC CTGAGATTGCTCACCAGAGACCTGAAGTGCGTGGCTTCCGGCGATCAATGTGTTTAA

SEQ ID NO.11:BoIFN- λ 3-S-C-O-F66R-L124H-S145P variant amino acid sequence；

MAPGRTLVLVLMLTTVALPRTGAVPVPSAPRALPPARGCHMAQFKSLSPQELQAFKTARDAFEDSRLPKDWDCSTHL FPRTRDLKHLQVWERPVALEAELALTLTVLEAMANSSLGHSLEQPLHTLQNIHSKLQACVPAQPTASPRPRGRLHHW LHRLQEARKESQDCLEASVMFNLLRLLTRDLKCVASGDQCV.

SEQ ID NO.12:BoIFN- λ 3-S-C-O-F66R-L124H-S145P mutant nucleotide sequence；

ATGGCTCCGGGAAGAACACTGGTGTTGGTTCTCATGCTGACAACTGTTGCTCTGCCAAGAACTGGTGCTGTCCCAGT GCCATCAGCTCCAAGAGCTTTGCCTCCAGCTAGAGGATGCCACATGGCCCAATTCAAATCGTTGAGTCCTCAAGAAC TCCAGGCCTTCAAGACTGCTAGAGACGCCTTCGAAGATAGCAGACTCCCTAAAGACTGGGATTGTTCCACTCACCTG TTCCCAAGAACCAGAGACTTGAAGCACCTCCAGGTTTGGGAAAGACCAGTCGCTTTGGAAGCCGAATTGGCTCTCAC CCTGACAGTCCTCGAAGCTATGGCCAACTCATCTCTCGGCCACTCGCTGGAACAACCGCTGCACACCCTGCAAAATA TCCACAGTAAATTGCAAGCCTGCGTGCCTGCTCAGCCAACAGCCAGCCCAAGACCCAGAGGTAGATTGCACCACTGG CTGCACAGATTGCAAGAAGCTAGAAAAGAATCACAGGATTGTCTCGAAGCCTCTGTGATGTTCAACCTCCTGAGATT GCTCACCAGAGACCTGAAGTGCGTGGCTTCCGGCGATCAATGTGTTTAA。

Sequence table

<110>Jiangsu University of Science and Technology

<120>3 interferon mutant of ox λ and its preparation method and application

<160> 12

<170> SIPOSequenceListing 1.0

<210> 1

<211> 195

<212> PRT

<213> bovine

<400> 1

Met Ala Pro Gly Cys Thr Leu Val Leu Val Leu Met Leu Thr Thr Val

1 5 10 15

Ala Leu Ser Arg Thr Gly Ala Val Pro Val Pro Ser Ala Pro Arg Ala

20 25 30

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

35 40 45

Pro Gln Glu Leu Gln Ala Phe Lys Thr Ala Arg Asp Ala Phe Glu Asp

50 55 60

Ser Phe Leu Pro Lys Asp Trp Asp Cys Ser Thr His Leu Phe Pro Arg

65 70 75 80

Thr Arg Asp Leu Lys His Leu Gln Val Trp Glu Arg Pro Val Ala Leu

85 90 95

Glu Ala Glu Leu Ala Leu Thr Leu Thr Val Leu Glu Ala Met Ala Asn

100 105 110

Ser Ser Leu Gly His Ser Leu Glu Gln Pro Leu Leu Met Leu Gln Asn

115 120 125

Ile His Ser Lys Leu Gln Ala Cys Val Pro Ala Gln Pro Thr Ala Ser

130 135 140

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

145 150 155 160

Ala Arg Lys Lys Ser Gln Asp Cys Leu Glu Ala Ser Val Met Phe Asn

165 170 175

Leu Leu Arg Leu Leu Thr Arg Asp Leu Lys Cys Val Ala Ser Gly Asp

180 185 190

Gln Cys Val

195

<210> 2

<211> 588

<212> DNA

<213> bovine

<400> 2

atggccccgg gctgcacgct ggtgctggtg ctgatgctga caaccgtggc gctgagcagg 60

acaggagcag ttcctgtgcc ctctgccccc agggccctcc cacctgccag gggctgccac 120

atggcccagt tcaagtctct gtcccctcaa gagctgcagg ccttcaagac ggccagggat 180

gcctttgaag actcgttctt gccaaaggac tgggactgca gcacccacct tttccccagg 240

acccgggacc tgaagcacct gcaggtgtgg gagcgccctg tagctctgga ggcagagctg 300

gccctgacac tgacggtcct ggaggccatg gctaactcat ccctgggcca cagcctggag 360

cagccccttc tcatgttgca gaacatccac tccaagctcc aggcctgtgt cccagctcag 420

cccacagcaa gctccaggcc ccggggccgc ctccaccact ggctgcaccg cctccaggag 480

gcccggaaga agtcccagga ctgcctcgaa gcctctgtga tgttcaacct cctccgcctt 540

ctcacccggg acctgaaatg tgttgccagc ggagaccagt gtgtctga 588

<210> 3

<211> 195

<212> PRT

<213> artifical sequence

<400> 3

Met Ala Pro Gly Arg Thr Leu Val Leu Val Leu Met Leu Thr Thr Val

1 5 10 15

Ala Leu Pro Arg Thr Gly Ala Val Pro Val Pro Ser Ala Pro Arg Ala

20 25 30

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

35 40 45

Pro Gln Glu Leu Gln Ala Phe Lys Thr Ala Arg Asp Ala Phe Glu Asp

50 55 60

Ser Phe Leu Pro Lys Asp Trp Asp Cys Ser Thr His Leu Phe Pro Arg

65 70 75 80

Thr Arg Asp Leu Lys His Leu Gln Val Trp Glu Arg Pro Val Ala Leu

85 90 95

Glu Ala Glu Leu Ala Leu Thr Leu Thr Val Leu Glu Ala Met Ala Asn

100 105 110

Ser Ser Leu Gly His Ser Leu Glu Gln Pro Leu Leu Met Leu Gln Asn

115 120 125

Ile His Ser Lys Leu Gln Ala Cys Val Pro Ala Gln Pro Thr Ala Ser

130 135 140

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

145 150 155 160

Ala Arg Lys Lys Ser Gln Asp Cys Leu Glu Ala Ser Val Met Phe Asn

165 170 175

Leu Leu Arg Leu Leu Thr Arg Asp Leu Lys Cys Val Ala Ser Gly Asp

180 185 190

Gln Cys Val

195

<210> 4

<211> 588

<212> DNA

<213> artifical sequence

<400> 4

atggccccgg gcagaacgct ggtgctggtg ctgatgctga caaccgtggc gctgccaagg 60

acaggagcag ttcctgtgcc ctctgccccc agggccctcc cacctgccag gggctgccac 120

atggcccagt tcaagtctct gtcccctcaa gagctgcagg ccttcaagac ggccagggat 180

gcctttgaag actcgttctt gccaaaggac tgggactgca gcacccacct tttccccagg 240

acccgggacc tgaagcacct gcaggtgtgg gagcgccctg tagctctgga ggcagagctg 300

gccctgacac tgacggtcct ggaggccatg gctaactcat ccctgggcca cagcctggag 360

cagccccttc tcatgttgca gaacatccac tccaagctcc aggcctgtgt cccagctcag 420

cccacagcaa gctccaggcc ccggggccgc ctccaccact ggctgcaccg cctccaggag 480

gcccggaaga agtcccagga ctgcctcgaa gcctctgtga tgttcaacct cctccgcctt 540

ctcacccggg acctgaaatg tgttgccagc ggagaccagt gtgtctga 588

<210> 5

<211> 195

<212> PRT

<213> artifical sequence

<400> 5

Met Ala Pro Gly Arg Thr Leu Val Leu Val Leu Met Leu Thr Thr Val

1 5 10 15

Ala Leu Pro Arg Thr Gly Ala Val Pro Val Pro Ser Ala Pro Arg Ala

20 25 30

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

35 40 45

Pro Gln Glu Leu Gln Ala Phe Lys Thr Ala Arg Asp Ala Phe Glu Asp

50 55 60

Ser Phe Leu Pro Lys Asp Trp Asp Cys Ser Thr His Leu Phe Pro Arg

65 70 75 80

Thr Arg Asp Leu Lys His Leu Gln Val Trp Glu Arg Pro Val Ala Leu

85 90 95

Glu Ala Glu Leu Ala Leu Thr Leu Thr Val Leu Glu Ala Met Ala Asn

100 105 110

Ser Ser Leu Gly His Ser Leu Glu Gln Pro Leu Leu Thr Leu Gln Asn

115 120 125

Ile His Ser Lys Leu Gln Ala Cys Val Pro Ala Gln Pro Thr Ala Ser

130 135 140

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

145 150 155 160

Ala Arg Lys Glu Ser Gln Asp Cys Leu Glu Ala Ser Val Met Phe Asn

165 170 175

Leu Leu Arg Leu Leu Thr Arg Asp Leu Lys Cys Val Ala Ser Gly Asp

180 185 190

Gln Cys Val

195

<210> 6

<211> 588

<212> DNA

<213> artifical sequence

<400> 6

atggctccgg gaagaacact ggtgttggtt ctcatgctga caactgttgc tctgccaaga 60

actggtgctg tcccagtgcc atcagctcca agagctttgc ctccagctag aggatgccac 120

atggcccaat tcaaatcgtt gagtcctcaa gaactccagg ccttcaagac tgctagagac 180

gccttcgaag atagcttcct ccctaaagac tgggattgtt ccactcacct gttcccaaga 240

accagagact tgaagcacct ccaggtttgg gaaagaccag tcgctttgga agccgaattg 300

gctctcaccc tgacagtcct cgaagctatg gccaactcat ctctcggcca ctcgctggaa 360

caaccgctgt tgaccctgca aaatatccac agtaaattgc aagcctgcgt gcctgctcag 420

ccaacagcca gctccagacc cagaggtaga ttgcaccact ggctgcacag attgcaagaa 480

gctagaaaag aatcacagga ttgtctcgaa gcctctgtga tgttcaacct cctgagattg 540

ctcaccagag acctgaagtg cgtggcttcc ggcgatcaat gtgtttaa 588

<210> 7

<211> 195

<212> PRT

<213> artifical sequence

<400> 7

Met Ala Pro Gly Arg Thr Leu Val Leu Val Leu Met Leu Thr Thr Val

1 5 10 15

Ala Leu Pro Arg Thr Gly Ala Val Pro Val Pro Ser Ala Pro Arg Ala

20 25 30

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

35 40 45

Pro Gln Glu Leu Gln Ala Phe Lys Thr Ala Arg Asp Ala Phe Glu Asp

50 55 60

Ser Arg Leu Pro Lys Asp Trp Asp Cys Ser Thr His Leu Phe Pro Arg

65 70 75 80

Thr Arg Asp Leu Lys His Leu Gln Val Trp Glu Arg Pro Val Ala Leu

85 90 95

Glu Ala Glu Leu Ala Leu Thr Leu Thr Val Leu Glu Ala Met Ala Asn

100 105 110

Ser Ser Leu Gly His Ser Leu Glu Gln Pro Leu Leu Thr Leu Gln Asn

115 120 125

Ile His Ser Lys Leu Gln Ala Cys Val Pro Ala Gln Pro Thr Ala Ser

130 135 140

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

145 150 155 160

Ala Arg Lys Glu Ser Gln Asp Cys Leu Glu Ala Ser Val Met Phe Asn

165 170 175

Leu Leu Arg Leu Leu Thr Arg Asp Leu Lys Cys Val Ala Ser Gly Asp

180 185 190

Gln Cys Val

195

<210> 8

<211> 588

<212> DNA

<213> artifical sequence

<400> 8

atggctccgg gaagaacact ggtgttggtt ctcatgctga caactgttgc tctgccaaga 60

actggtgctg tcccagtgcc atcagctcca agagctttgc ctccagctag aggatgccac 120

atggcccaat tcaaatcgtt gagtcctcaa gaactccagg ccttcaagac tgctagagac 180

gccttcgaag atagcagact ccctaaagac tgggattgtt ccactcacct gttcccaaga 240

accagagact tgaagcacct ccaggtttgg gaaagaccag tcgctttgga agccgaattg 300

gctctcaccc tgacagtcct cgaagctatg gccaactcat ctctcggcca ctcgctggaa 360

caaccgctgt tgaccctgca aaatatccac agtaaattgc aagcctgcgt gcctgctcag 420

ccaacagcca gctccagacc cagaggtaga ttgcaccact ggctgcacag attgcaagaa 480

gctagaaaag aatcacagga ttgtctcgaa gcctctgtga tgttcaacct cctgagattg 540

ctcaccagag acctgaagtg cgtggcttcc ggcgatcaat gtgtttaa 588

<210> 9

<211> 195

<212> PRT

<213> artifical sequence

<400> 9

Met Ala Pro Gly Arg Thr Leu Val Leu Val Leu Met Leu Thr Thr Val

1 5 10 15

Ala Leu Pro Arg Thr Gly Ala Val Pro Val Pro Ser Ala Pro Arg Ala

20 25 30

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

35 40 45

Pro Gln Glu Leu Gln Ala Phe Lys Thr Ala Arg Asp Ala Phe Glu Asp

50 55 60

Ser Arg Leu Pro Lys Asp Trp Asp Cys Ser Thr His Leu Phe Pro Arg

65 70 75 80

Thr Arg Asp Leu Lys His Leu Gln Val Trp Glu Arg Pro Val Ala Leu

85 90 95

Glu Ala Glu Leu Ala Leu Thr Leu Thr Val Leu Glu Ala Met Ala Asn

100 105 110

Ser Ser Leu Gly His Ser Leu Glu Gln Pro Leu His Thr Leu Gln Asn

115 120 125

Ile His Ser Lys Leu Gln Ala Cys Val Pro Ala Gln Pro Thr Ala Ser

130 135 140

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

145 150 155 160

Ala Arg Lys Glu Ser Gln Asp Cys Leu Glu Ala Ser Val Met Phe Asn

165 170 175

Leu Leu Arg Leu Leu Thr Arg Asp Leu Lys Cys Val Ala Ser Gly Asp

180 185 190

Gln Cys Val

195

<210> 10

<211> 588

<212> DNA

<213> artifical sequence

<400> 10

atggctccgg gaagaacact ggtgttggtt ctcatgctga caactgttgc tctgccaaga 60

actggtgctg tcccagtgcc atcagctcca agagctttgc ctccagctag aggatgccac 120

atggcccaat tcaaatcgtt gagtcctcaa gaactccagg ccttcaagac tgctagagac 180

gccttcgaag atagcagact ccctaaagac tgggattgtt ccactcacct gttcccaaga 240

accagagact tgaagcacct ccaggtttgg gaaagaccag tcgctttgga agccgaattg 300

gctctcaccc tgacagtcct cgaagctatg gccaactcat ctctcggcca ctcgctggaa 360

caaccgctgc acaccctgca aaatatccac agtaaattgc aagcctgcgt gcctgctcag 420

ccaacagcca gctccagacc cagaggtaga ttgcaccact ggctgcacag attgcaagaa 480

gctagaaaag aatcacagga ttgtctcgaa gcctctgtga tgttcaacct cctgagattg 540

ctcaccagag acctgaagtg cgtggcttcc ggcgatcaat gtgtttaa 588

<210> 11

<211> 195

<212> PRT

<213> artifical sequence

<400> 11

Met Ala Pro Gly Arg Thr Leu Val Leu Val Leu Met Leu Thr Thr Val

1 5 10 15

Ala Leu Pro Arg Thr Gly Ala Val Pro Val Pro Ser Ala Pro Arg Ala

20 25 30

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

35 40 45

Pro Gln Glu Leu Gln Ala Phe Lys Thr Ala Arg Asp Ala Phe Glu Asp

50 55 60

Ser Arg Leu Pro Lys Asp Trp Asp Cys Ser Thr His Leu Phe Pro Arg

65 70 75 80

Thr Arg Asp Leu Lys His Leu Gln Val Trp Glu Arg Pro Val Ala Leu

85 90 95

Glu Ala Glu Leu Ala Leu Thr Leu Thr Val Leu Glu Ala Met Ala Asn

100 105 110

Ser Ser Leu Gly His Ser Leu Glu Gln Pro Leu His Thr Leu Gln Asn

115 120 125

Ile His Ser Lys Leu Gln Ala Cys Val Pro Ala Gln Pro Thr Ala Ser

130 135 140

Pro Arg Pro Arg Gly Arg Leu His His Trp Leu His Arg Leu Gln Glu

145 150 155 160

Ala Arg Lys Glu Ser Gln Asp Cys Leu Glu Ala Ser Val Met Phe Asn

165 170 175

Leu Leu Arg Leu Leu Thr Arg Asp Leu Lys Cys Val Ala Ser Gly Asp

180 185 190

Gln Cys Val

195

<210> 12

<211> 588

<212> DNA

<213> artifical sequence

<400> 12

atggctccgg gaagaacact ggtgttggtt ctcatgctga caactgttgc tctgccaaga 60

actggtgctg tcccagtgcc atcagctcca agagctttgc ctccagctag aggatgccac 120

atggcccaat tcaaatcgtt gagtcctcaa gaactccagg ccttcaagac tgctagagac 180

gccttcgaag atagcagact ccctaaagac tgggattgtt ccactcacct gttcccaaga 240

accagagact tgaagcacct ccaggtttgg gaaagaccag tcgctttgga agccgaattg 300

gctctcaccc tgacagtcct cgaagctatg gccaactcat ctctcggcca ctcgctggaa 360

caaccgctgc acaccctgca aaatatccac agtaaattgc aagcctgcgt gcctgctcag 420

ccaacagcca gcccaagacc cagaggtaga ttgcaccact ggctgcacag attgcaagaa 480

gctagaaaag aatcacagga ttgtctcgaa gcctctgtga tgttcaacct cctgagattg 540

ctcaccagag acctgaagtg cgtggcttcc ggcgatcaat gtgtttaa 588

Claims

1. a kind of 3 interferon of ox λ is preparing the application in interferon mutant, it is characterised in that: the ox λ 3 interferes amino acid Sequence is shown in SEQ ID NO.1, and the polynucleotide sequence of encoding gene is shown for (a) or (b) or (c):

(a) polynucleotide sequence shown in SEQ ID No.2；Or

(b) polynucleotide sequence hybridized is able to carry out in stringent hybridisation conditions with the complementary series of SEQ ID No.2, the multicore The albumen of thuja acid coding still has the function of interferon or activity；Or

(c) with the polynucleotide sequence of the polynucleotide sequence of SEQ ID No.2 at least 80% or more homology, and the multicore The albumen of thuja acid coding still has the function of interferon or activity；Preferably, at least with the polynucleotide sequence of SEQ ID No.2 There is the polynucleotide sequence of 85% or more homology, and the albumen of the polynucleotide encoding still has the function of interferon or work Property；It is furthermore preferred that the polynucleotide sequence with polynucleotide sequence at least 90% or more the homology of SEQ ID No.2, and The albumen of the polynucleotide encoding still has the function of interferon or activity.

2. a kind of 3 interferon mutant of ox λ, it is characterised in that: be 3 interferon amino acid sequence of ox λ described in claim 1 SEQ ID NO.1 be original series the 5th amino acids by cysteine mutation at arginine, the 19th amino acids are by serine It is mutated into proline, obtained 3 interferon mutant of ox λ；The amino acid sequence of the mutant be SEQ ID NO.3 shown in, The nucleotides sequence of its encoding gene is classified as shown in SEQ ID NO.4.

3. a kind of 3 interferon mutant of ox λ, it is characterised in that: be by the of 3 interferon mutant of ox λ as claimed in claim 2 125 amino acids are mutated into threonine by methionine, 164 amino acids by lysine mutation at glutamic acid, obtained ox λ 3 Interferon mutant；The amino acid sequence of the mutant is shown in SEQ ID NO.5, and the nucleotides sequence of encoding gene is classified as Shown in SEQ ID NO.6.

4. a kind of 3 interferon mutant of ox λ, it is characterised in that: be to carry out 3 interferon mutant of ox λ as claimed in claim 3 E51A、T57R、F66R、H76R、T81S、H86Q、A109S、H117R、L124H、Q127R、I129V、S131A、T142M、 3 interferon mutant of ox λ that any one of S144G, S145P, S165P or D167S amino acid unit point mutation obtain；

Preferably, 3 interferon mutant of ox λ as claimed in claim 3 is subjected to F66R, A109S, L124H, S131A, S145P Or 3 interferon mutant of ox λ that any one of S165P amino acid unit point mutation obtains；

Wherein, 3 interferon mutant of ox λ as claimed in claim 3 is subjected to the ox λ 3 that the point mutation of F66R amino acid unit obtains The amino acid sequence of interferon mutant is shown in SEQ ID NO.7, and the nucleotides sequence of encoding gene is classified as SEQ ID NO.8 It is shown.

5. a kind of 3 interferon mutant of ox λ, it is characterised in that: carry out 3 interferon mutant of ox λ as claimed in claim 3 F66R-A109S、F66R-L124H、F66R-S131A、F66R-S145P、F66R-S165P、A109S-L124H、A109S- S131A、A109S-S145P、A109S-S165P、L124H-S131A、L124H-S145、L124H-S165P、S131A-S145P、 3 interferon mutant of ox λ that any one of S131A-S165P or S145P-S165P amino acid double-site mutant obtain；

Preferably, 3 interferon mutant of ox λ as claimed in claim 3 is subjected to F66R-L124H, F66R-S145P or A109S- 3 interferon mutant of ox λ that S131A any one amino acid double-site mutant obtains；

Wherein, 3 interferon mutant of ox λ as claimed in claim 3 F66R-L124H amino acid double-site mutant is carried out to obtain 3 interferon mutant of ox λ amino acid sequence be SEQ ID NO.9 shown in, the nucleotides sequence of encoding gene is classified as SEQ Shown in ID NO.10.

6. a kind of 3 interferon mutant of ox λ, it is characterised in that: carry out 3 interferon mutant of ox λ as claimed in claim 3 F66R-A109S-L124H、F66R-A109S-S131A、F66R-A109S-S145P、F66R-L124H-S131A、F66R- L124H-S145P, F66R-S131A-S145P, A109S-L124H-S131A or A109S-S131A-S145P any one amino 3 interferon mutant of ox λ that sour multisite mutation obtains；

Preferably, 3 interferon mutant of ox λ as claimed in claim 3 is subjected to F66R-L124H-S145P amino acid multidigit point It is mutated 3 interferon mutant of ox λ obtained；

Wherein, 3 interferon mutant of ox λ as claimed in claim 3 progress F66R-L124H-S145P amino acid multidigit point is dashed forward Become the amino acid sequence of 3 interferon mutant of ox λ obtained as shown in SEQ ID NO.11, the nucleotide sequence of encoding gene For shown in SEQ ID NO.12.

7. recombinant vector or the recombination place of the encoding gene containing any one of claim 1-6 3 interferon mutant of ox λ Chief cell.

8. 3 interferon mutant of ox λ described in claim 1-6 any one is in preparation prevention or treats bovine viral disease Application in drug or reagent.

9. applying according to claim 7, which is characterized in that the bovine viral disease includes: aftosa FMD, cattle disease Diarrhea virus BVD and infectious bovine rhinotrachetis IBR etc. can pass through the epidemic disease of alimentary canal, respiratory infectious.

10. a kind of method for preparing any one of claim 1-6 3 interferon mutant of ox λ, which is characterized in that including Following steps:

(1) encoding gene of any one of claim 1-6 3 interferon mutant of ox λ is cloned into baculoviral respectively In transfer vector, building obtains recombinant transfer vector；

(2) by recombinant transfer vector and baculovirus DNA cotransfection insect cell, recombinant baculovirus is obtained；

(3) by recombinate shape virus infection insect cell or insect host, infected insect cell or insect host table are cultivated Up to corresponding albumen, purifying to get；

Wherein, the baculovirus transfer vector is selected from AcRP23-lacZ, AcRP6-SC, AcUWl-lacZ, BacPAK6, Bac to Pac、Bacmid、BlucBacII(pETL)、p2Bac、p2Blue、p89B310、pAc360、pAc373、pAcAB3、 pAcAB4、PAcAS3、pAcC129、pAcC4、DZI、pAcGP67、pAcIEl、pAcJPl、pAcMLF2、pAcMLF 7、 pAcMLF8、pAcMPl、pAcMP2、pAcRP23、pAcRP 25、pAcRW4、pAcsMAG、pAcUWl、pAcUW21、pAcUW2A、 pAcUW2B、pAcUW3、pAcUW31、pAcUW41、pAcUW42、pAcUW43、pAcUW51、pAcVC2、pAcVC3、pAcYMl、 pAcJcC5、pBacl、pBac2、pBlueBacIII、pBlueBacHis、pEV55、mXIV、pIEINeo、pJVETL、 pJVNhel、pJVP10、pJVrsMAG、pMBac、pP10、pPAKl、pPBac、pSHONEX 1.1、pSYN XIV VI+、 pSYNVI+wp、pSYNXIV VI-、pVL1391、pVL 1392、pVL 1393、pVL941、pVL 945、pVL 985、 PVTBac, pBM030 or pUAC-5；

Preferably, the baculovirus transfer vector is pVL1393；The baculoviral is silkworm baculovirus parent plant BmBacmid；The insect host is silkworm (Bombyx mori)；

It is described to infect the insect larvae or pupal cell for referring to recombinant baculovirus by eating or infecting through epidermis 1-5 age.