CN106892976A - A kind of recombination chicken interferon lambda（rChIFN‑λ）Clonal expression of gene and its preparation method and application - Google Patents
A kind of recombination chicken interferon lambda（rChIFN‑λ）Clonal expression of gene and its preparation method and application Download PDFInfo
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- CN106892976A CN106892976A CN201710078270.8A CN201710078270A CN106892976A CN 106892976 A CN106892976 A CN 106892976A CN 201710078270 A CN201710078270 A CN 201710078270A CN 106892976 A CN106892976 A CN 106892976A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/555—Interferons [IFN]
- C07K14/57—IFN-gamma
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Abstract
The invention belongs to the interferon field in technical field of bioengineering, and in particular to a kind of clonal expression of recombination chicken interferon lambda (rChIFN λ) gene and its preparation method and application.By recombination chicken interferon lambda (rChIFN λ) gene from POLY (I:C) extracted in the template obtained by the CEF cells for stimulating, be cloned into pET32a expression vectors, obtain recombinant plasmid pET32a rChIFN λ；By pET32a rChIFN λ recombinant plasmid transformeds to BL21 competence, Amplification Culture, after inclusion body modification, purifying and renaturation, recombination chicken interferon lambda (rChIFN λ) albumen needed for being obtained.
Description
Technical field
The invention belongs to the interferon field in technical field of bioengineering, and in particular to a kind of recombination chicken interferon lambda
Clonal expression of (rChIFN- λ) gene and its preparation method and application.
Background technology
IFN- λ are newfound type cytokines in recent years, are named as III type interferon, and its family member includes IFN-
λ 1, IFN- λ 2, IFN- λ 3 and IFN- λ 4, but chicken IFN- λ only have a kind of ChIFN- λ, and DNA fragmentation is 561bp, and albumen size is about
22kDa.Chicken IFN- λ have a pair special functional receptor complexs (IFN- λ R1/IL-10R2), when chicken IFN- λ and acceptor are answered
During zoarium combination, cause acceptor heterodimerisation, downstream JAK-STAT signal transductions can be activated, so as to play extensive biology
Effect, including antiviral effect, immunological regulation with it is antitumor etc..There are some researches show IFN- λ are played in virus once
Good antiviral effect, such as, and influenza virus, herpes simplex virus, hepatitis viruse etc..
Ewcastle disease is a kind of birds caused by avian paramyxovirus section NDV (Newcastle Disease, ND)
Acute, highly contagious disease, main infringement chicken and turkey.Sick major determinant alimentary canal, intestines and stomach and the nervous system.Newly
City epidemic disease not only serious harm aviculture, while also being brought about great losses to World Economics industry, is classified as by OIE
Statutory report disease, a class animal epidemic is classified as by China.
The content of the invention
Present invention aim at a kind of clone of highly efficient anti-virus active recombination chicken interferon lambda (rChIFN- λ) gene of invention
Expression and its preparation method and application.Chicken interferon λ of the present invention production is easy, and low cost, activity is high, to ewcastle disease and
Influenza has good therapeutic effect.
The first object of the present invention is to provide above-mentioned recombination chicken interferon lambda (rChIFN- λ) albumen, and its nucleotide sequence is such as
SEQ ID NO:Shown in 1.
It is a further object of the present invention to provide the production method and prokaryotic expression plasmid pET32a-rChIFN λ of the albumen
Preparation method.
Another object of the present invention is to provide the albumen and suppresses virus breeding on cell and clinically treat zoosis
The application of virus epidemic disease and effect.
To reach above-mentioned purpose, the present invention uses following technical scheme：
Step one：Design of primers and synthesis.Gene order (the GenBank of the chicken IFN- λ provided according to Genbank
No.KF680102.1), pair of primers IFN- λ-F1, IFN- λ-R1 are designed, signal peptide is predicted and removed by SignalP, designed
Primer, is IFN- λ-F, IFN- λ-R respectively in upstream and downstream insertion EcoR1 and two restriction enzyme sites of Hind3.Sequence is as follows：
IFN-λ-F1：ATGGTATGCTACGGGGTCAC
IFN-λ-R1：CTAAGTGCAATCCTCGCGCTG
IFN-λ-F：CCGGAATTCCAGGTCACCCCGAAGAA
IFN-λ-R：CCCAAGCTTCTAAGTGCAATCCTCGCGCTGGGC
Step 2：Gene cloning and identification.Chick embryo fibroblast (CEF) cell is made by oneself with SPF chicken embryos, use POLY (I:C)
After stimulating 2h, extracting RNA obtains expanding the template cDNA of chicken IFN- λ genes through reversion, uses IFN- λ-F, IFN- λ-R primers
Amplify the genetic fragment that chicken IFN- λ remove signal peptide.
Step 3：By gene cloning to pMD-19T carriers.Chicken IFN- λ genes are connected to pMD-19T carriers, 16 DEG C of companies
More than 4h is met, conversion to the agar plate of benzyl containing ammonia, picking monoclonal bacterium shakes bacterium in being put into LB culture mediums, and bacterium solution PCR identifies sun
Property clone, Amplification Culture after sequencing is correct extracts plasmid pMD-19T-rChIFN- λ.
Step 4：Build pET32a-rChIFN- λ recombinant plasmids.It is respectively that pMD-19T-rChIFN- λ and pET32a is unloaded
Body EcoR1 and the fast enzyme cutting double digestions of Hind3, use T4 ligases after glue reclaim, normal temperature connection more than 30min, conversion extremely contains ammonia
Benzyl agar plate, picking monoclonal bacterium shakes bacterium in being put into LB culture mediums, and bacterium solution PCR identifies positive colony, expands after sequencing is correct
Big culture, extracts recombinant plasmid pET32a-rChIFN- λ.
Beneficial effects of the present invention are as follows：
The present invention can extract the albumen of higher concentration and activity, be that subsequent experimental lays good basis, and in cellular layer
There is good antiviral activity in face, has significant curative effect in animal body for newcastle disease.
Brief 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 magnification results.
Fig. 2 is that SDS-PAGE identifies recombination chicken interferon lambda (rChIFN- λ) protein expression result figure；Wherein, swimming lane M is low
Molecular weight protein Marker, swimming lane 1 is that before pET-32a empty carriers inclusion body is denatured, swimming lane 2 is pET-32a empty carrier inclusion bodys
After renaturation, before swimming lane 3 is for restructuring chicken interferon λ (rChIFN- λ) inclusion body denaturation, swimming lane 4 is restructuring chicken interferon λ
After (rChIFN- λ) renaturing inclusion bodies.
Fig. 3 is that Western blot identify recombination chicken interferon lambda (rChIFN- λ) protein expression result figure；Wherein, swimming lane M
It is LMWP Marker, swimming lane 1 is compareed for pET-32a empty carriers supernatant, swimming lane 2 is pET-32a empty carrier inclusion bodys
Control, swimming lane 3 is restructuring chicken interferon λ (rChIFN- λ) supernatant, and swimming lane 4 is restructuring chicken interferon λ (rChIFN- λ) inclusion body
Precipitation.
Specific embodiment
With reference to specific embodiment, the present invention is expanded on further.But the present invention is not limited thereto.
The structure of the chicken IFN- λ prokaryotic expression vectors of embodiment 1
(1) relevant primer design and synthesis
Design of primers is the gene order (GenBank no.KF680102.1) of the chicken IFN- λ provided according to Genbank,
Design pair of primers IFN- λ-F1, IFN- λ-R1, predict and remove signal peptide by SignalP, primer are designed, respectively upper and lower
Trip insertion EcoR1 and two restriction enzyme sites of Hind3, are IFN- λ-F, IFN- λ-R.Sequence is as follows：
IFN-λ-F1：ATGGTATGCTACGGGGTCAC
IFN-λ-R1：CTAAGTGCAATCCTCGCGCTG
IFN-λ-F：CCGGAATTCCAGGTCACCCCGAAGAA
IFN-λ-R：CCCAAGCTTCTAAGTGCAATCCTCGCGCTGGGC
(2) gene cloning and identification
10 age in days SPF chicken embryos are taken, head, four limbs and internal organ are removed with tweezers, chicken is shredded into rear pancreatin digestion 4min, mistake
Filter, is made CEF cells, after passage once, uses POLY (I:C) stimulate, after 2h, abandon supernatant, take cell sample extracting RNA,
Obtain expanding the template cDNA of chicken IFN- λ genes through reversion, first amplify chicken IFN- λ genes using IFN- λ-F1, IFN- λ-R1,
Reuse IFN- λ-F, IFN- λ-R primers to be expanded, amplify chicken IFN- λ genes and remove signal fragments of peptides.Reaction system is as follows：
25 μ l Premix ExTaq enzymes, 2.5 μ l template DNAs (cDNA), 0.5 μ l sense primers, 0.5 μ l anti-sense primers, distilled water mend to
50μl.Response procedures are as follows：94 DEG C of predegenerations 4min, 94 DEG C of denaturation 1min, 55 DEG C of annealing 30s, 72 DEG C of extension 40s, 72 DEG C are prolonged
7min is stretched, wherein 35 circulations of the 2nd to the 4th step.Nucleic acid electrophoresis stripe size about 492bp.
(3) by gene cloning to pMD-19T carriers
RChIFN- λ genes are connected to pMD-19T carriers, reaction system is as follows：5.0μl Ligation Solution
I, 0.5 μ l pMD 19-T vector, 4.5 μ l chIFN- λ+α PCR recovery products.16 DEG C of connection more than 4h, are experienced with DH5 α
State is converted to the agar plate of benzyl containing ammonia, and 37 DEG C stand overnight, and picking monoclonal bacterium shakes bacterium 6-8h in being put into the LB culture mediums of benzyl containing ammonia,
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 upstreams
Primer I FN- λ-F, 0.2 μ l anti-sense primer IFN-αs-R, 1.0 μ l bacterium solutions, response procedures are as follows：94 DEG C of predegeneration 4min, 94 DEG C of changes
Property 1min, 55 DEG C of annealing 30s, 72 DEG C of extensions 40s, 72 DEG C of extension 7min, wherein the circulation of the 2nd to the 4th step 30.Select identification sun
Property sample send sequencing, Amplification Culture after sequencing is correct extracts plasmid pMD-19T-rChIFN- λ.
(4) pET32a-rChIFN- λ construction of recombinant plasmid
Respectively by pMD-19T-rChIFN- λ and pET32a empty carriers EcoR1 and the fast enzyme cutting double digestions of Hind3, reactant
System is as follows：1.5 μ l EcoR1 enzymes, 1.5 μ l Hind3 enzymes, 5 μ l 10 × Buffer, 2-5 μ g plasmids, ddH2O is mended to 50 μ l, 37
DEG C water-bath 30min.T4 ligases are used after glue reclaim, digestion products are according to pMD-19T-rChIFN- λ：PET32a empty carrier=3:1
Ratio add 8 μ l, 1 μ l T4 ligases, 1 μ l T4 connection Buffer, normal temperature connection more than 30min, conversion is to the fine jade of benzyl containing ammonia
Fat flat board, picking monoclonal bacterium shakes bacterium in being put into the LB culture mediums of benzyl containing ammonia, and bacterium solution PCR identifies positive colony, send sequencing, sequencing
Amplification Culture after correct, extracts recombinant plasmid pET32a-rChIFN- λ+α.
The protein expression of embodiment 2 and identification
Recombinant plasmid pET32a-rChIFN- λ are converted with BL21 competence, after monoclonal bacterium Amplification Culture, according to 1:
100 ratios are inoculated with ammonia benzyl LB culture mediums, 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 precooling PBS 2 times, inclusion body, room is dissolved with the Lysis Equilibration Buffer containing 8M urea
Temperature is incubated 60min, and 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 wash-out is 0 up to OD280 values, is finally eluted with the Elution Buffer containing 250mmol/L imidazoles, is received
Collection filtrate.Filtrate is put into the bag filter treated 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 rChIFN- λ with 4 times of gradient dilutions
100 μ l are added per hole, respectively from 4-1-4-10,, 24 repetitions of each gradient, in 37 DEG C be incubated 12h, respectively will
100TCID50VSV, NDV, AIV virus are 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.Result shows：RChIFN- λ produce good to DF-1 cells
Protective capability, have good VSV, respectively AIV antiviral activities, 1.87*10 on DF-1 cells in vitro3IU/mg, 7.8*
103IU/mg。
The interior resisting virus activity identification of embodiment 4
RChIFN- λ antiviral activities are detected on 2 week old SPF chickens.100 μ g are injected intravenously to 2 week old SPF chickens
RChIFN- λ, after 4h, are injected intravenously again with same amount, then attack NDV every injection after 2h.Afterwards 1,3,5,
7th, 9,11 days collections throat swab and cloacal swabs, common chicken embryonic breeding embryo detection toxin expelling；The feeding of daily observation chicken and mental status；
Record death condition.Result shows：RChIFN- λ groups can reach 33% to the protective rate of SPF chickens, and attack the survival of malicious control group
Rate only 8%；Additionally, throat swab shows that rChIFN- λ groups compare with malicious control group is attacked with cloacal swab result, 4-9d can be postponed
Toxin expelling, and shedding virus substantially reduce；The SPF chickens feeding of rChIFN- λ groups is normal, and spirit is good.
Above-described embodiment is the present invention preferably implementation method, but embodiments of the present invention are not by above-described embodiment
Limitation, it is other it is any without departing from Spirit Essence of the 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 recombination chicken interferon lambda（rChIFN-λ）Clonal expression of gene and its preparation method and application
（1）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 GTGGCAGCCC 540
AGCGCGAGGA TTGCACTTAG 560
（2） IFN-λ-F1
ATGGTATGCT ACGGGGTCAC 20
（3） IFN-λ-R1
CTAAGTGCAA TCCTCGCGCT G 21
（4） IFN-λ-F
CCGGAATTCC AGGTCACCCC GAAGAA 26
（5） IFN-λ-R
CCCAAGCTTC TAAGTGCAAT CCTCGCGCTG GGC 33
Claims (5)
1. a kind of high antiviral active recombination chicken interferon lambda (rChIFN- λ), it is characterised in that:Its nucleotide sequence such as SEQ ID
NO:Shown in 1.
2. the preparation method of a kind of high antiviral active recombination chicken interferon lambda (rChIFN- λ) according to claim 1, its
It is characterised by, is prepared from by following steps：
The gene order (GenBank no.KF680102.1) of the chicken IFN- λ for a. being provided according to Genbank designs signal peptide
Primer, make template needed for amplification by oneself with CEF, cDNA amplifies rChIFN- λ genetic fragments；
B. by gene cloning to pMD-19T carriers, cloning vector pMD-19T-rChIFN- λ are built, sequencing shows and Genbank
The chicken IFN- λ gene homologies of offer are up to 100%；
C. by pMD-19T-rChIFN- λ and pET32a empty carriers EcoR1 and the fast enzyme cutting double digestions of Hind3, genes of interest clone
To pET32a expression vectors, expressive host E.coli DH5 α are converted, obtain recombinant plasmid pET32a-rChIFN- λ；
D. recombinant plasmid pET32a-rChIFN- λ are converted with BL21 competence, after Amplification Culture, add IPTG induced expressions,
Inclusion body is taken after ultrasonication, recombination chicken interferon lambda (rChIFN- λ) albumen is collected after denaturation, purifying, renaturation.
3. a kind of preparation method of high antiviral active recombination chicken interferon lambda (rChIFN- λ) according to claim 2, it is special
Levy and be, in step d, IPTG induced concentrations are 1.0mmol/L, and inducing temperature is 37 DEG C, and induction time is 8h.
4. a kind of high antiviral active recombination chicken interferon lambda (rChIFN- λ) described in claim 1 is preparing suppression blister
Stomatovirus (VSV) is with influenza virus (H5N6) to the application in the pathogenic change drugs with function of DF-1.
5. a kind of high antiviral active recombination chicken interferon lambda (rChIFN- λ) described in claim 1 is reduced and postponed in preparation
NDV (GM) lethal effect in chicken body, suppresses the application in the medicine of shedding virus.
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Cited By (1)
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| CN108329390A (en) * | 2018-01-29 | 2018-07-27 | 中国农业科学院生物技术研究所 | 3 interferon of chicken λ, its mutant and its expression and application |
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