CN106674354A - Fusion protein of chicken interferon IFN-lambda and IFN-alpha - Google Patents
Fusion protein of chicken interferon IFN-lambda and IFN-alpha Download PDFInfo
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Abstract
The invention belongs to the technical field of biological engineering, and discloses fusion expression of chicken interferon genes lambda and alpha of an interferon fusion preparation, a production method and clinical application thereof. According to the interferon fusion preparation of biological engineering, total RNA (Ribonucleic Acid) of CEF cells is extracted, a specific primer is designed, the chicken interferon genes lambda and alpha are cloned and are fused by using a complementary hydrophobic flexible amino acid connector, and thus the complete chicken interferon fusion genes lambda and alpha can be obtained; the chicken interferon fusion genes lambda and alpha are cloned to a 19-T carrier, is massively cloned and expressed successfully, and is further connected with a pET-32a carrier for massive expression; a product is identified to ensure that the product is expressed in an inclusion body, and then modification, purification and renaturation are implemented; the anti-virus activity of the product is detected, and the clinical application effect of the product is evaluated. Prokaryotic expression plasmid pET32a-chIFN-lambda+alpha of the recombinant chicken interferon genes lambda and alpha is successfully established, and 1:1 fusion expression of the chicken interferon genes lambda and alpha on an escherichia coli prokaryotic expression system can be achieved.
Description
Technical field
The invention belongs to the gene fusion expression of technical field of bioengineering, and in particular to one kind fusion chicken λ, interferon-alpha
Gene and its preparation method and application.
Background technology
Interferon (interferon, IFN) is a kind of class tool that under the effect of specific derivant, host cell is produced
There are broad-spectrum antiviral, antitumor and strengthen the cytokine of immunologic function, it is in the nature the multi-functional glycoprotein of secreted.Nineteen fifty-seven
Find first when studying viral interphase interaction by British scientist lsaacs, it is in the nature protein, produced by virus induction,
Viral interference is replicated, limiting virus infection.Originated according to interferon, aminoacid sequence and biological activity are different, and interferon is divided
For three major types, respectively interferon type Ⅰ, including IFN-α, IFN-β, IFN- δ, IFN- κ, wherein IFN- ω and IFN- τ, IFN-α
With the research comparative maturity of IFN-β；Interferon type Ⅱ is IFN-γ, but its function predominantly adjusts immunologic function, disease-resistant toxic effect
Ying Buqiang；III type interferon includes IFN- λ 1 (IL-29), and IFN- λ 2 (IL-28A), IFN- λ 3 (IL-28B) are new with 2013 to be sent out
Existing IFN- λ 4.IFN-α belongs to interferon type Ⅰ, is made up of 582 nucleic acid, after removing 93 signal peptides, 162 ammonia is encoded altogether
Base acid.Guard relatively between different breeder IFN-αs, nucleotide homology is more than 99.3%.Can be sick in most cells
Poison induction is produced, although be not directly placed on virion, but because it can produce antiviral protein by inducing host cell quickly,
So all the time interferon type Ⅰ is all one of study hotspot.Research shows that chicken IFN-α can suppress H5N1 viruss to breed, together
When also have certain impact to newcastle disease vaccine immunity.IFN- λ (interferon λ), also known as III type interferon, be after interferon I,
The class new forms of interferon found after II.By tying with its specificity heterodimer coreceptor (IFN- λ R1/IL-10R2)
Close, JAK-STAT signal paths are activated, so as to play antiviral effect.Due to its receptor-specific, IFN- λ are caused to have difference
In other advantages of interferon type Ⅰ, the research of people IFN- λ is increasing, but still shallow to the research of chicken IFN- λ, and table is studied on a small quantity
Bright chicken IFN- λ can suppress the propagation of influenza virus in annulus trachealises experiment.There is no the two chicken interferon expressing in series at present
And detect the example of its antiviral activity.
Newcastle (Newcastle Disease, ND) is the urgency of the chicken and various birdss morbidity caused by Avian pneumo-encephalitis virus
Property highly contagious disease.The sick principal pathogenetic is characterized as dyspnea, neurological disorders, dysentery, mucosa and serous coat bleeding.
Because newcastle strain is different, the order of severity of newcastle infected animal has very big difference.The disease propagates rapid, and mortality rate is very
Height, serious harm aviculture, to World Economics massive losses are caused.
The content of the invention
In order to make up the defect of two kinds of interferon, the mutual supplement with each other's advantages of two kinds of interferon is realized.Chicken interference of the present invention
The fusion protein production of plain IFN- λ and IFN-α is easy, and low cost, activity is high, has to newcastle and influenza and good controls curative effect
Really.
The first object of the present invention is to provide above-mentioned chicken IFN- λ and IFN-α fusion protein.
It is a further object of the present invention to provide the production method of the fusion protein and prokaryotic expression plasmid pET32a-IFN λ-
The preparation method of linker-IFN α.
Another object of the present invention is to provide the fusion protein and suppresses virus breeding on cell and clinically treat dynamic
The application of thing viral blight and effect.
To reach above-mentioned purpose, the present invention is employed the following technical solutions：
The purpose of the present invention is achieved through the following technical solutions：One breeder IFN- λ and IFN-α fusion gene (IFN λ-
Linker-IFN α), its nucleotide sequence is as follows：
CAGGTCACCCCGAAGAAGAGCTGCAGCCTCTCCAAGTACCAGTTCCCTGCACCTTTGGAGTTGAAGGCA
GTGTGGAGGATGAAGGAGCAGTTTGAAGACATCATGCTGTTAACAAACAGAAAATGCAACACCAGACTCTTCCATCG
GAAGTGGGACATAGCTGAGCTGTCGGTACCTGACCGAATCACCCTGGTGGAGGCTGAGCTGGACCTCACCATCACCG
TGCTCACAAACCCCACAACCCAGAGACTGGCAGAGACGTGCCAACAGCCCCTGGCCTTCCTTACCCAAGTCCAGGAG
GACCTGCGAGACTGCTTGGCCCTCGAGGCACCTTCACATCAGCCCTCTGGGAAACTGAGGCACTGGCTGCAGAAGCT
GGAGACAGCCAAGAAGAAGGAGACCGCCGGCTGCCTGGAGGCCTCAGCCATCCTCCACATCTTCCAAGTACTGAACG
ACCTGCGGTGCGCAGCCCAGCGCGAGGATTGCACTTCTGGTGGAGGCGGTAGCGGAGGCGGAGGGTCGTGCAACCAC
CTTCGCCCCCAGGATGCCACCTTCTCTCACGACAGCCTCCAGCTCCTCCGGGACATGGCTCCCACACTACCCCAGCT
GTGCCCACAGCACAACGCGTCTTGCTCCTTCAACGACACCATCCTGGACACCAGCAACACCCGGCAAGCCGACAAAA
CCACCCACGACATCCTTCAGCACCTCTTCAAAATCCTCAGCAGCCCCAGCACTCCAGCCCACTGGAACGACAGCCAA
CGCCAAAGCCTCCTCAACCGGATCCACCGCTACACCCAGCACCTCGAGCAATGCTTGGACAGCAGCGACACGCGCTC
CCGGACGCGATGGCCTCGCAACCTTCACCTCACCATCAAAAAACACTTCAGCTGCCTCCACACCTTCCTCCAAGACA
ACGATTACAGCGCCTGCGCCTGGGAACACGTCCGCCTGCAAGCTCGTGCCTGGTTCCTGCACATCCACAACCTCACA
GGCAACACGCGCACTTAG
The above chicken IFN- λ and IFN-α fusion gene are by Overlap extension PCR method (gene splicing by
Overlapextension, SOE-PCR) fusion form, respectively by two interferon genes amplify come after, remove signal
Peptide, then (G4S) 3 be connected chicken IFN- λ with IFN-α by hydrophobicity flexible amino acid linker (linker), it is cloned into protokaryon table
Up to carrier pET32a (+), by BL21 system expressions and after purification, respectively in vivo with its antiviral activity of vitro detection.
Above-mentioned chicken IFN- λ are with IFN-α fusion gene through the following steps that obtaining：
(1) design of primers
Design of primers be according to Genbank provide chicken IFN- λ gene order (GenBank no.KF680102.1) with
The gene order (GenBank no.AM049251.1) of IFN-α, predicts and removes signal peptide by SignalP, and design two pairs is drawn
Thing, is IFN- λ-F, IFN- λ-R1, IFN-α-F1, IFN- respectively in upstream and downstream insertion EcoR1 and two restriction enzyme sites of Hind3
α-R.Then respectively in 2 drawing containing complementary hydrophobicity flexible amino acid linker of the downstream of IFN- λ and IFN-α upstream design
Thing IFN- λ-R2 and IFN-α-F2.Sequence is as follows：
IFN-λ-F：CCGGAATTCCAGGTCACCCCGAAGAA
IFN-λ-R1：CCCAAGCTTCTAAGTGCAATCCTCGCGCTGGGC
IFN-λ-R2：
CTCCGCTACCGCCTCCACCAGAGCCTCCTCCACCAGTGCAATCCTCGCGCTGGGC
IFN-α-F1：CCGGAATTCTGCAACCACCTTC
IFN-α-F2：
TCTGGTGGAGGCGGTAGCGGAGGCGGAGGGTCGTGCAACCACCTTC
IFN-α-R：CCCAAGCTTCTAAGTGCGCGTGTTGCC
(2) gene cloning and identification
With SPF Embryo Gallus domesticus make by oneself chick embryo fibroblast (CEF) cell, using newcastle GM strain counteracting toxic substances after, extracting RNA, Jing is anti-
Turn template cDNA for obtaining expanding chicken IFN- λ and IFN-α gene, using IFN- λ-F, IFN- λ-R1, IFN-α-F1, IFN-α-R
Primer amplifies chicken IFN- λ and goes the genetic fragment of signal peptide with IFN-α.
Next SOE-PCR methods are used by chicken IFN- λ and IFN-α gene fusion：The first step, respectively with IFN- λ-F, IFN-
λ-R2 and IFN-α-F2, IFN-α-R primers amplify containing linker two genetic fragments；Second step, is returned with first step glue
Receipts product is template, is not added with primer, and PCR expands 10-15 circulation；3rd step, with second step PCR primer as template, uses IFN-
λ-F and IFN-α-R amplification PCR, the product for obtaining i.e. chicken IFN- λ and IFN-α genetic fragment.
The structure of one breeder IFN- λ and IFN-α fusion protein (chIFN- λ+α) prokaryotic expression carrier
(1) by fusion gene cloning to pMD-19T carriers
Chicken IFN- λ and IFN-α fusion gene are connected to into pMD-19T carriers, 16 DEG C of connection more than 4h are converted to benzyl containing ammonia
Agar plate, picking monoclonal bacterium is put in LB culture medium and shakes bacterium, and bacterium solution PCR identifies positive colony, expands after sequencing is correct
Culture, extracts plasmid pMD-19T-chIFN- λ+α.
(2) pET32a-chIFN- λ+α construction of recombinant plasmid
Respectively by pMD-19T-chIFN- λ+α and pET32a empty carriers EcoR1 and the fast enzyme cutting double digestions of Hind3, glue is returned
T4 ligases, room temperature connection more than 30min is used to convert to the agar plate of benzyl containing ammonia after receipts, picking monoclonal bacterium is put into LB cultures
Bacterium is shaken in base, bacterium solution PCR identifies positive colony, amplification culture after being sequenced correctly, extraction recombiant plasmid pET32a-chIFN- λ+
α。
The present invention merges chicken IFN- λ and IFN-α using gene engineering method, there is following beneficial effect：
(1) present invention is melted both using gene engineering method using the receptor distributional difference rule of I, III type interferon
Close, be conducive to higher level comprehensively to play the effect of two interferon；
(2) chIFN- λ+alpha fusion protein is extracted in present invention success in inclusion body, and Jing degeneration, recovery, after purification can be carried
Higher concentration albumen is taken out, is that subsequent experimental is laid a good foundation.；
(3) the fusion protein chIFN- λ+α that the present invention is extracted are in vivo, have good disease-resistant toxic effect in experiment in vitro
Really.
Description of the drawings
Fig. 1 is the chicken IFN- λ gene agarose nucleic acid electrophoresis figures of PCR amplifications；Wherein, swimming lane M is DNA marker
DL2000, swimming lane 1 is negative control, and swimming lane 2 is chIFN- λ gene amplification results.
Fig. 2 is the chicken IFN-α gene agarose nucleic acid electrophoresis figure of PCR amplifications；Wherein, swimming lane M is DNA marker
DL5000, swimming lane 1,2 is chIFN- α gene amplification results, and swimming lane 3 is negative control.
Fig. 3 is the chIFN- λ+α gene agarose nucleic acid electrophoresis figures of SOE-PCR amplifications；Wherein, swimming lane M is DNA
Marker DL2000, swimming lane 1 is chIFN- λ+α gene amplification results, and swimming lane 2 is negative control.
Fig. 4 is that SDS-PAGE identifies chIFN- λ+α protein expression result figures；Wherein, swimming lane M is low molecular weight protein (LMWP)
Marker, swimming lane 1 is pET-32a empty vector controls, and swimming lane 2 is chIFN- λ+α supernatants, and swimming lane 3 is heavy for chIFN- λ+α inclusion bodys
Form sediment.
Fig. 5 is that Western blot identify chIFN- λ+α protein expression result figures；Wherein, swimming lane M is low molecular weight protein (LMWP)
Marker, swimming lane 1 is pET-32a empty vector controls, and swimming lane 2 is chIFN- λ+α supernatants, and swimming lane 3 is heavy for chIFN- λ+α inclusion bodys
Form sediment.
Specific embodiment
With reference to specific embodiment, the present invention is expanded on further.But the present invention is not limited to this.
The structure of the chicken IFN- λ of embodiment 1 and IFN-α (chIFN- λ+α) fusion gene pronucleus expression vector
(1) relevant primer design and synthesis
Design of primers be according to Genbank provide chicken IFN- λ gene order (GenBank no.KF680102.1) with
The gene order (GenBank no.AM049251.1) of IFN-α, predicts and removes signal peptide by SignalP, and design two pairs is drawn
Thing, is IFN- λ-F, IFN- λ-R1, IFN-α-F1, IFN- respectively in upstream and downstream insertion EcoR1 and two restriction enzyme sites of Hind3
α-R.Then respectively in 2 drawing containing complementary hydrophobicity flexible amino acid linker of the downstream of IFN- λ and IFN-α upstream design
Thing IFN- λ-R2 and IFN-α-F2.Sequence is as follows：
IFN-λ-F：CCGGAATTCCAGGTCACCCCGAAGAA
IFN-λ-R1：CCCAAGCTTCTAAGTGCAATCCTCGCGCTGGGC
IFN-λ-R2：
CTCCGCTACCGCCTCCACCAGAGCCTCCTCCACCAGTGCAATCCTCGCGCTGGGC
IFN-α-F1：CCGGAATTCTGCAACCACCTTC
IFN-α-F2：
TCTGGTGGAGGCGGTAGCGGAGGCGGAGGGTCGTGCAACCACCTTC
IFN-α-R：CCCAAGCTTCTAAGTGCGCGTGTTGCC
(2) gene cloning and identification
10 age in days SPF Embryo Gallus domesticus are taken, with tweezers head, extremity and internal organs are removed, Carnis Gallus domesticus are shredded into rear pancreatin digestion 4min, mistake
Filter, makes CEF cells, and after passing on once, using newcastle GM strain counteracting toxic substances, after 12h, extracting RNA, Jing reversions are expanded
Template cDNA of chicken IFN- λ and IFN-α gene, using IFN- λ-F, IFN- λ-R1, IFN-α-F1, IFN-α-R primers are expanded
Increase, reaction system is as follows：25 μ l Premix ExTaq enzymes, 2.5 μ l template DNAs (cDNA), 0.5 μ l forward primer, under 0.5 μ l
Trip primer, distilled water is mended to 50 μ l.Response procedures are as follows：94 DEG C of denaturations 4min, 94 DEG C of degeneration 1min, 55 DEG C of annealing 30s, 72
DEG C extend 40s, 72 DEG C of extension 7min, wherein the circulation of the 2nd to the 4th step 35.Nucleic acid electrophoresis stripe size respectively about 561bp with
582bp。
With SPF Embryo Gallus domesticus make by oneself chick embryo fibroblast (CEF) cell, using newcastle GM strain counteracting toxic substances after, extracting RNA, Jing is anti-
Turn template cDNA for obtaining expanding chicken IFN- λ and IFN-α gene, using IFN- λ-F, IFN- λ-R1, IFN-α-F1, IFN-α-R
Primer amplifies chicken IFN- λ and goes the genetic fragment of signal peptide with IFN-α.
Next SOE-PCR methods are used by chicken IFN- λ and IFN-α gene fusion：The first step, respectively with IFN- λ-F, IFN-
λ-R2 primers amplify the IFN- λ-linker fragments containing linker, with IFN-α-F2, IFN-α-R primers amplify containing
Both are distinguished glue reclaim by the linker-IFN- α fragments of linker；Second step, with first step glue reclaim product as template, etc.
Mole mixing, does not use primer, and PCR amplifications, reaction system is as follows：10 μ l Premix ExTaq enzymes, 8.4 μ l distilled water, first
Step glue reclaim product equimolar mixes totally 1.6 μ l, and response procedures are as follows：94 DEG C of denaturations 4min, 94 DEG C of degeneration 1min, 60 DEG C are moved back
Fiery 30s, 72 DEG C of extension 40s, 72 DEG C of extension 7min, wherein 15 circulations of the 2nd to the 4th step；3rd step, with second step PCR primer
For template, with IFN- λ-F and IFN-α-R PCR, the product for obtaining i.e. chIFN- λ+alpha gene fragment are expanded.
(3) by fusion gene cloning to pMD-19T carriers
ChIFN- λ+alpha fusion gene is connected to into pMD-19T carriers, reaction system is as follows：5.0μl Ligation
The μ l chIFN- λ+α PCR recovery products of Solution I, 0.5 μ l pMD 19-T vector, 4.5.16 DEG C of connection more than 4h, use
DH5 α competence is converted to the agar plate of benzyl containing ammonia, and 37 DEG C stand overnight, and picking monoclonal bacterium is put in the LB culture medium of benzyl containing ammonia
Bacterium 6-8h is shaken, bacterium solution PCR identifies positive colony, and reaction system is as follows：7.5 μ l Premix rTaq enzymes, 6.1 μ l ddH2O,
0.2 μ l forward primer IFN- λ-F, 0.2 μ l downstream primer IFN-α-R, 1.0 μ l bacterium solutions, response procedures are as follows：94 DEG C of denaturations
4min, 94 DEG C of degeneration 1min, 55 DEG C of annealing 30s, 72 DEG C of extension 40s, 72 DEG C of extension 7min, wherein the 2nd to the 4th step 30 is followed
Ring.Amplification culture after identification positive send sequencing, sequencing correct is selected, plasmid is extracted
pMD-19T-chIFN-λ+α。
(4) pET32a-chIFN- λ+α construction of recombinant plasmid
Respectively by pMD-19T-chIFN- λ+α and pET32a empty carriers EcoR1 and the fast enzyme cutting double digestions of Hind3, reaction
System is as follows：1.5 μ l EcoR1 enzymes, 1.5 μ l Hind3 enzymes, 5 μ 10 × Buffer of l, 2-5 μ g plasmids, ddH2O is mended to 50 μ l,
37 DEG C of water-bath 30min.T4 ligases are used after glue reclaim, digestion products are according to pMD-19T-chIFN- λ+α：PET32a empty carriers=
3:1 ratio adds 8 μ l, 1 μ l T4 ligases, 1 μ l T4 connection Buffer, room temperature to connect more than 30min, convert to benzyl containing ammonia
Agar plate, picking monoclonal bacterium is put in the LB culture medium of benzyl containing ammonia and shakes bacterium, and bacterium solution PCR identifies positive colony, send sequencing, surveys
Amplification culture after sequence is correct, extracts recombiant plasmid pET32a-chIFN- λ+α.
The protein expression of embodiment 2 and identification
Recombiant plasmid pET32a-chIFN- λ+α are converted with BL21 competence, after monoclonal bacterium amplification culture, according to 1:
100 ratios are inoculated with ammonia benzyl LB culture medium, 37 DEG C of shaking table cultures, until when OD600nm values are 0.6 or so, adding IPTG induction tables
After reaching, supernatant is abandoned in bacterium solution centrifugation, and PBS is resuspended, after eccentric cleaning 2 times, Ultrasonic Cell Disruptor 200W cracking 15min, 4 DEG C of centrifugations point
Precipitation is separated out, with pre-cooling PBS 2 times, with the dissolving inclusion bodys of the Lysis Equilibration Buffer containing 8M carbamide, room
Temperature incubation 60min, 4 DEG C are centrifuged away insoluble impurity, and supernatant is filtered with 0.45 μm of filter, it is combined with Ni posts, dense with difference
Degree imidazole elution eluting is 0 up to OD280 values, finally with the Elution Buffer eluting containing 250mmol/L imidazoles, is received
Collection filtrate.Filtrate is put in the bag filter processed with EDTA, respectively with 8M, 6M, 4M, 2M, 0M, PBS carries out gradient dialysis,
Per minor tick 12h, finally identified with SDS-PAGE.
The Anti-viral activity in vitro of embodiment 3 is identified
By DF-1 plating cells to 96 orifice plate cell plates, treat long to 80%-90%, by chIFN- λ+α with 4 times of gradient dilutions
100 μ l are added per hole, respectively from 4-1-4-10, 24 repetitions of each gradient are incubated 12h in 37 DEG C, respectively will
100TCID50VSV, NDV, AIV virus is added, and 100 μ l are incubated 1h per hole in 37 DEG C, and virus liquid is discarded, and are washed 2 times with PBS,
2% serum DMEM is changed, 37 DEG C of 48h are put in, viral level is detected.As a result show：ChIFN- λ+α produce good to DF-1 cells
Protective capability, have good VSV, NDV, AIV antiviral activity, respectively 3.2*10 on DF-1 cells in vitro4IU/mg,
4.6*10 4IU/mg, 1.5*105IU/mg。
The interior resisting virus activity identification of embodiment 4
ChIFN- λ+α antiviral activities are detected on 2 week old SPF chickens.Give the μ gchIFN- of 2 week old SPF chicken intravenous injection 100
λ+α, after 4h, with same amount intravenous injection are carried out again, then Avian pneumo-encephalitis virus is attacked in injection after 2h.Afterwards 1,3,5,7,9,11
Its collection throat swab and cloacal swab, common chicken embryonic breeding embryo detection toxin expelling；The feeding of daily observation chicken and mental status；Record is dead
Die situation.As a result show：ChIFN- λ+α groups can reach 25% to the protective rate of SPF chickens, and the survival rate of counteracting toxic substances matched group is only
8%；Additionally, throat swab shows with cloacal swab result, chIFN- λ+α groups compare with counteracting toxic substances matched group, can postpone 3-7d rows
Poison, and shedding virus substantially reduce；The SPF chickens feeding of chIFN- λ+α groups is normal, and spirit is good.
Above-described embodiment is the present invention preferably embodiment, but embodiments of the present invention not by above-described embodiment
Limit, other any spirit without departing from the present invention and the change, modification, replacement made under principle, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
SEQUENCE LISTING
Agricultural University Of South China
A kind of fusion protein of chicken interferon IFN- λ and IFN-α
（1）Chicken IFN- λ+α genes
CAGGTCACCC CGAAGAAGAG CTGCAGCCTC TCCAAGTACC AGTTCCCTGC ACCTTTGGAG 60
TTGAAGGCAG TGTGGAGGAT GAAGGAGCAG TTTGAAGACA TCATGCTGTT AACAAACAGA 120
AAATGCAACA CCAGACTCTT CCATCGGAAG TGGGACATAG CTGAGCTGTC GGTACCTGAC 180
CGAATCACCC TGGTGGAGGC TGAGCTGGAC CTCACCATCA CCGTGCTCAC AAACCCCACA 240
ACCCAGAGAC TGGCAGAGAC GTGCCAACAG CCCCTGGCCT TCCTTACCCA AGTCCAGGAG 300
GACCTGCGAG ACTGCTTGGC CCTCGAGGCA CCTTCACATC AGCCCTCTGG GAAACTGAGG 360
CACTGGCTGC AGAAGCTGGA GACAGCCAAG AAGAAGGAGA CCGCCGGCTG CCTGGAGGCC 420
TCAGCCATCC TCCACATCTT CCAAGTACTG AACGACCTGC GGTGCGCAGC CCAGCGCGAG 480
GATTGCACTg gtggaggagg cTCTGGTGGA GGCGGTAGCG GAGGCGGAGG GTCGTGCAAC 540
CACCTTCGCC CCCAGGATGC CACCTTCTCT CACGACAGCC TCCAGCTCCT CCGGGACATG 600
GCTCCCACAC TACCCCAGCT GTGCCCACAG CACAACGCGT CTTGCTCCTT CAACGACACC 660
ATCCTGGACA CCAGCAACAC CCGGCAAGCC GACAAAACCA CCCACGACAT CCTTCAGCAC 720
CTCTTCAAAA TCCTCAGCAG CCCCAGCACT CCAGCCCACT GGAACGACAG CCAACGCCAA 780
AGCCTCCTCA ACCGGATCCA CCGCTACACC CAGCACCTCG AGCAATGCTT GGACAGCAGC 840
GACACGCGCT CCCGGACGCG ATGGCCTCGC AACCTTCACC TCACCATCAA AAAACACTTC 900
AGCTGCCTCC ACACCTTCCT CCAAGACAAC GATTACAGCG CCTGCGCCTG GGAACACGTC 960
CGCCTGCAAG CTCGTGCCTG GTTCCTGCAC ATCCACAACC TCACAGGCAA CACGCGCACT 1020
TAG 1023
（2）Chicken IFN- λ genes
ATGGTATGCT ACGGGGTCAC AATTATTTTG GTGGGGACCC TGGGGTCCCT CCTGGTGGGT 60
GCCTTCCCCC AGGTCACCCC GAAGAAGAGC TGCAGCCTCT CCAAGTACCA GTTCCCTGCA 120
CCTTTGGAGT TGAAGGCAGT GTGGAGGATG AAGGAGCAGT TTGAAGACAT CATGCTGTTA 180
ACAAACAGAA AATGCAACAC CAGACTCTTC CATCGGAAGT GGGACATAGC TGAGCTGTCG 240
GTACCTGACC GAATCACCCT GGTGGAGGCT GAGCTGGACC TCACCATCAC CGTGCTCACA 300
AACCCCACAA CCCAGAGACT GGCAGAGACG TGCCAACAGC CCCTGGCCTT CCTTACCCAA 360
GTCCAGGAGG ACCTGCGAGA CTGCTTGGCC CTCGAGGCAC CTTCACATCA GCCCTCTGGG 420
AAACTGAGGC ACTGGCTGCA GAAGCTGGAG ACAGCCAAGA AGAAGGAGAC CGCCGGCTGC 480
CTGGAGGCCT CAGCCATCCT CCACATCTTC CAAGTACTGA ACGACCTGCG GTGCGCAGCC 540
CAGCGCGAGG ATTGCACTTA G 561
（3）Chicken IFN-α gene
ATGGCTGTGC CTGCAAGCCC ACAGCACCCA CGGGGGTACG GCATCCTGCT GCTCACGCTC 60
CTTCTGAAAG CTCTCGCCAC CACCGCCTCC GCCTGCAACC ACCTTCGCCC CCAGGATGCC 120
ACCTTCTCTC ACGACAGCCT CCAGCTCCTC CGGGACATGG CTCCCACACT ACCCCAGCTG 180
TGCCCACAGC ACAACGCGTC TTGCTCCTTC AACGACACCA TCCTGGACAC CAGCAACACC 240
CGGCAAGCCG ACAAAACCAC CCACGACATC CTTCAGCACC TCTTCAAAAT CCTCAGCAGC 300
CCCAGCACTC CAGCCCACTG GAACGACAGC CAACGCCAAA GCCTCCTCAA CCGGATCCAC 360
CGCTACACCC AGCACCTCGA GCAATGCTTG GACAGCAGCG ACACGCGCTC CCGGACGCGA 420
TGGCCTCGCA ACCTTCACCT CACCATCAAA AAACACTTCA GCTGCCTCCA CACCTTCCTC 480
CAAGACAACG ATTACAGCGC CTGCGCCTGG GAACACGTCC GCCTGCAAGC TCGTGCCTGG 540
TTCCTGCACA TCCACAACCT CACAGGCAAC ACGCGCACTT AG 582
（4）IFN-λ-F
CCGGAATTCC AGGTCACCCC GAAGAA 26
（5）IFN-λ-R1
CCCAAGCTTC TAAGTGCAAT CCTCGCGCTG GGC 33
（6）IFN-λ-R2
CTCCGCTACC GCCTCCACCA GAGCCTCCTC CACCAGTGCA ATCCTCGCGC TGGGC 55
（7）IFN-α-F1
CCGGAATTCT GCAACCACCT TC 22
（8）IFN-α-F2
TCTGGTGGAG GCGGTAGCGG AGGCGGAGGG TCGTGCAACC ACCTTC 46
（9）IFN-α-R
CCCAAGCTTC TAAGTGCGCG TGTTGCC 27
Claims (3)
1. a kind of chicken interferon λ and alpha fusion protein, it is characterised in that:Chicken interferon λ is with α genes with identical carrier in large intestine bar
Formed by complementary hydrophobicity flexible amino acid linker amalgamation and expression in bacterium, its nucleotide sequence such as SEQ ID NO:Shown in 1.
2. the fusion protein of chicken interferon λ as claimed in claim 1 and α, it is characterised in that：By following steps it is made and
Into：
A, design of primers
Gene order (its nucleotide sequence such as SEQ ID NO of the chicken IFN- λ provided according to Genbank:Shown in 2) and IFN-α
Gene order (its nucleotide sequence such as SEQ ID NO:Shown in 3), signal peptide, design two are predicted and removed by SignalP
It is IFN- λ-F, IFN- λ-R1, IFN-α-F1 respectively in upstream and downstream insertion EcoR1 and two restriction enzyme sites of Hind3 to primer,
IFN-α-R.Then respectively in the downstream of IFN- λ and IFN-α upstream design 2 containing complementary hydrophobicity flexible amino acid linker
Primer I FN- λ-R2 and IFN-α-F2.Sequence is as follows：
The sequence of IFN- λ-F such as SEQ ID NO:Shown in 4；
CCGGAATTCCAGGTCACCCCGAAGAA
The sequence of IFN- λ-R1 such as SEQ ID NO:Shown in 5；
CCCAAGCTTCTAAGTGCAATCCTCGCGCTGGGC
The sequence of IFN- λ-R2 such as SEQ ID NO:Shown in 6；
CTCCGCTACCGCCTCCACCAGAGCCTCCTCCACCAGTGCAATCCTCGCGCTGGGC
The sequence of IFN-α-F1 such as SEQ ID NO:Shown in 7；CCGGAATTCTGCAACCACCTTC
The sequence of IFN-α-F2 such as SEQ ID NO:Shown in 8；
TCTGGTGGAGGCGGTAGCGGAGGCGGAGGGTCGTGCAACCACCTTC
The sequence of IFN-α-R such as SEQ ID NO:Shown in 9；
CCCAAGCTTCTAAGTGCGCGTGTTGCC
B, gene cloning and identification
With SPF Embryo Gallus domesticus make by oneself chick embryo fibroblast (CEF) cell, using newcastle GM strain counteracting toxic substances after, extracting RNA, Jing is inverted
To amplification chicken IFN- λ and template cDNA of IFN-α gene, using IFN- λ-F, IFN- λ-R1, IFN-α-F1, IFN-α-R primers
Amplify chicken IFN- λ and go the genetic fragment of signal peptide with IFN-α.
Next SOE-PCR methods are used by chicken IFN- λ and IFN-α gene fusion：The first step, respectively with IFN- λ-F, IFN- λ-R2
With IFN-α-F2, IFN-α-R primers amplify containing linker two genetic fragments；Second step, with first step glue reclaim product
Thing is template, is not added with primer, and PCR expands 10-15 circulation；3rd step, with second step PCR primer as template, with IFN- λ-F and
IFN-α-R expands PCR, the product for obtaining i.e. chIFN- λ+alpha gene fragment.
The structure of C, a breeder IFN- λ and IFN-α fusion protein prokaryotic expression carrier
ChIFN- λ+alpha fusion gene is connected to into pMD-19T carriers, 16 DEG C of connection more than 4h are converted to the agar plate of benzyl containing ammonia,
Picking monoclonal bacterium is put in LB culture medium and shakes bacterium, and bacterium solution PCR identifies positive colony, and amplification culture after sequencing is correct is extracted
Plasmid pMD-19T-chIFN- λ+α.
Respectively by pMD-19T-chIFN- λ+α and pET32a empty carriers EcoR1 and the fast enzyme cutting double digestions of Hind3, after glue reclaim
T4 ligases, room temperature connection more than 30min is used to convert to the agar plate of benzyl containing ammonia, monoclonal bacterium is put in LB culture medium picking
Bacterium is shaken, bacterium solution PCR identifies positive colony, and amplification culture after sequencing is correct extracts recombiant plasmid pET32a-chIFN- λ+α.
3. the application in terms of the fusion protein of the chicken interferon λ described in claim 1 or 2 and α prepares the antiviral drugs of chicken.
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| CN108794637A (en) * | 2017-08-09 | 2018-11-13 | 芜湖英特菲尔生物制品产业研究院有限公司 | A kind of canine recombinant long-acting interferon α and the fusion protein and preparation method thereof for preparing this long-acting interferon |
| CN108840939A (en) * | 2017-08-09 | 2018-11-20 | 芜湖英特菲尔生物制品产业研究院有限公司 | A kind of recombination chicken long-acting interferon α and the fusion protein for preparing this long-acting interferon and preparation method thereof |
| CN108864293A (en) * | 2017-08-09 | 2018-11-23 | 芜湖英特菲尔生物制品产业研究院有限公司 | A kind of fusion protein being made of dog albumin and dog interferon γ and preparation method thereof and a kind of canine recombinant long-acting interferon γ |
| EP3618618A4 (en) * | 2017-05-01 | 2020-03-25 | Rutgers, The State University of New Jersey | Type i and type iii interferon fusion molecules and methods for use thereof |
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| CN108794638A (en) * | 2017-08-09 | 2018-11-13 | 芜湖英特菲尔生物制品产业研究院有限公司 | A kind of recombinant bovine long-acting interferon α and the fusion protein and preparation method thereof for preparing this long-acting interferon |
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| CN108864293A (en) * | 2017-08-09 | 2018-11-23 | 芜湖英特菲尔生物制品产业研究院有限公司 | A kind of fusion protein being made of dog albumin and dog interferon γ and preparation method thereof and a kind of canine recombinant long-acting interferon γ |
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