CN107217068A - A kind of method for improving canine recombinant interferon alpha fusion protein antiviral activity - Google Patents
A kind of method for improving canine recombinant interferon alpha fusion protein antiviral activity Download PDFInfo
- Publication number
- CN107217068A CN107217068A CN201710584157.7A CN201710584157A CN107217068A CN 107217068 A CN107217068 A CN 107217068A CN 201710584157 A CN201710584157 A CN 201710584157A CN 107217068 A CN107217068 A CN 107217068A
- Authority
- CN
- China
- Prior art keywords
- recombinant
- interferon
- alpha
- canine
- expression
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- 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]
- C07K14/56—IFN-alpha
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
- C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Gastroenterology & Hepatology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Toxicology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a kind of preparation method of canine recombinant interferon alpha fusion protein.Its nucleotides sequence is classified as shown in SEQ ID NO.1, and coded protein amino acid sequence is shown in SEQ ID NO.2.The present invention expresses dog interferon alpha gene and dog immunoglobulin CH3 segment compositions, not only it had been beneficial to the stability and activity but also the expression quantity for adding albumen of increase albumen, it the experiment proved that, the restructuring dog interferon alpha expressed by the present invention has compared with the bioactivity of natural dog interferon alpha to be significantly improved.
Description
Technical field
The present invention relates to the albumen of a kind of gene of restructuring, more particularly to canine recombinant interferon gene-α and its coding, sheet
Invention further relates to expression vector and engineered strain containing the gene, and dog interferon is being prepared the invention further relates to them
In purposes, belong to genetic engineering field.
Background technology
Canine occupies the status become more and more important as the companion animals of people in the life of the mankind, associated various therewith
Canine viral disease also repeats to occur in the life of people, and some turn into the cause of disease of zoonosis.But in veterinary clinic
Aspect, there is presently no a kind of specific drug for treating viral disease.Progress and the need of modern animal doctor's industry with science and technology
Will, the preventive and therapeutic effect of immunopotentiator is increasingly taken seriously, and characteristic is determined, efficient, preferable immunopotentiator stably, nontoxic
And curative drug will be the following main matter for preventing and treating animal viral disease.Wherein, topmost immunopotentiator is exactly
Interferon.Interferon（IFN）It is a kind of broad-spectrum disease resistance toxic agent, direct killing or suppression be not viral, and mainly passes through cell
Surface receptor effect makes cell produce antiviral protein, so as to suppress the duplication of virus；It can also strengthen NK simultaneously
（NK cells）, macrophage and T lymphocytes vigor, so as to play immunoregulation effect, and strengthen anti-virus ability.Interference
Element, which is one group, has the reactive protein of a variety of functions（Mainly glycoprotein）, it is that one kind is produced by monocyte and lymphocyte
Cell factor.Their antiviral, influence cell growths with wide spectrum on allogenic cell, and differentiation, the immune work(of regulation
The multiple biological activities such as energy.Due to interferon have it is nontoxic, have no side effect and antiviral advantage in extensive range, at the beginning of discovery
Just attract attention, by application development for many years, interferon have become a kind of extensive immunopotentiator be used for virosis and
The prevention and control field of the diseases such as tumour.
With increasing considerably for the canines such as domestic working dog, meat dog and pet dog, viral infectious turns into prestige
Coerce the immediate cause of canine life.The intimate contact of people and dog considerably increases the chance that people is infected, such as rabies viruses
Disease etc..And interferon has proved to be prevents the important preparation of viral infectious.Conventional interferon is given birth in the form of wild type
Production, although close with natural structure, but half-life period is shorter, causes raiser's use cost higher.Therefore, with genetic engineering skill
Original series are transformed by art, and it is current research emphasis to obtain effect and more useful recombinate dog interferon.
This method is operated with genetic engineering, interferon gene is connected into antibody constant region CH3 fragments and His labels, both
Half-life period can be extended, molecular weight of albumen is also controlled, make it possible to carry out expressing protein using prokaryotic expression system, while to original
Beginning sequence is transformed, and greatly improves expression quantity of the albumen in prokaryotic system, and obtaining effect, more useful canine recombinant is done
Element is disturbed, the prevention and control field that wild-type Interferon is applied to the diseases such as virosis and tumour is substituted.
The content of the invention
An object of the present invention is to provide a kind of canine recombinant interferon-' alpha ' gene, and the gene can be in prokaryotic expression system
Middle stabilization, efficient expression canine recombinant interferon-' alpha '.
The two of the object of the invention are to provide the expression vector containing above-mentioned canine recombinant interferon-' alpha ' gene.
The three of the object of the invention are to provide the engineered strain converted by above-mentioned canine recombinant interferon-' alpha ' gene.
The four of the object of the invention are to provide a kind of Prepare restructuring dog interferon-α method.
Above-mentioned purpose of the present invention is achieved through the following technical solutions：
A kind of canine recombinant interferon-' alpha ' gene, its nucleotides sequence is classified as shown in SEQ ID NO.1, and its amino acid sequence encoded is
Shown in SEQ ID NO.2.
The canine recombinant interferon-' alpha ' gene of the present invention is with dog interferon-α and dog immunoglobulin CH3 regions plus His marks
Label are formed by connecting, and its more control sequences is to be best suitable for the nucleotide sequence that prokaryotic expression host produces by Optimizing Reconstruction.
The structure of the canine recombinant interferon gene of the present invention is by dog interferon-α, dog immunoglobulin CH3 areas and 6 His
It is in series, the gene constitutes 310 amino acid of coding by 933 nucleotides.
The present invention is also constructed containing the recombinant expression plasmid of nucleotide sequence shown in SEQ ID NO.1 and with the restructuring table
Obtained engineered strain is converted up to plasmid.
The recombinant expression plasmid of the present invention can be built-up by the conventional method of this area, i.e., by SEQ ID NO.1 institutes
The nucleotide sequence shown is inserted between the suitable restriction enzyme site of expression vector, makes the nucleosides shown in SEQ ID NO.1
Acid sequence is exercisable to be connected with expression regulation sequence.As the embodiment of a reference, for example, canine recombinant can be done
Disturb element-α genes with escherichia coli prokaryotic expression carrier pET27b using restriction enzyme site BamHI and HindIII to be connected, be named as
pET-rCaIFN-α。
Recombinant expression plasmid constructed by the present invention can convert host cell by various conventional methods.As reference,
Recombinant plasmid pET-rCaIFN- α containing canine recombinant interferon-' alpha ' gene can be converted Escherichia coli Rosetta（Purchased from north
Jing Quanshijin Bioisystech Co., Ltd, article No. catalogue CD801）Obtained bacterial strain, is named asEscherichia coli
Rosetta/pET-rCaIFN- α, abbreviation Rosetta/pET-rCaIFN- α.
The specific method for producing recombinant protein using E. coli recombinant stain Rosetta/pET-rCaIFN- α is as follows：
1st, the preparation of seed liquor：Single bacterium colony will be obtained after strain line culture, picking single bacterium falls within 10mL LB fluid nutrient mediums
In, while 100 mg/L ampicillins are added, 37 DEG C of culture 12h.
2nd, ferment：The seed liquor of acquisition is pressed 1：100 are inoculated in fermentation medium, when culture to OD600For 0.4 or so
When, IPTG inductions are added, its final concentration of 0.5 mM cultivates 3h or so for 37 DEG C and receives bacterium.
The present invention also provides a kind of Prepare restructuring dog interferon-α method, comprises the following steps：
Build the recombinant expression plasmid containing the nucleotide sequence shown in SEQ ID NO.1；Cultivate with the recombinant expression plasmid institute
The host cell of conversion, obtains recombinant bacterial strain；Recombinant bacterial strain is cultivated, the expression of recombinant protein is induced, separates and purifies expressed
Recombinant protein.
In the method for above-mentioned Prepare restructuring albumen, described recombinant expression plasmid is preferably pET-rCaIFN- α；Described
Host cell is Escherichia coli（Escherichia coli）Rosetta (DE3), described recombinant bacterial strain is preferablyEscherichia coli Rosetta/pET-rCaIFN-α。
In the method for above-mentioned Prepare restructuring albumen, it is preferred that the separation and purifying of described recombinant protein include following step
Suddenly：
1st, the thalline of collection is suspended from phosphate buffer, low-temperature centrifugation collects supernatant after crushing, and utilizes Ni affinity columns and AKTA
Protein purification system carries out albumen and slightly purified.
2nd, recycle AKTA systems to carry out the purifying of molecular sieve in the albumen slightly purified, finally give purity reach 90% with
On albumen.
With natural dog interferon-α genes（The original series of dog interferon-α genes are shown in annex one）Disturbed instead of canine recombinant
Element-α genes carry out above step, obtain the natural dog interferon-α available for subsequent experimental as control.
Dog interferon-α and dog immunoglobulin CH3 areas amalgamation and expression are added stability and the work of albumen by the present invention
Property, while the Protein expression and purification method of the present invention has, the production time is short, expression efficiency is high, expression quantity is big, be easy to purifying
Advantage, and the experiment proved that, the canine recombinant interferon-' alpha ' expressed by the present invention is not only significantly better than naturally on half-life period
Dog interferon-α, and improve its antiviral activity.
Brief description of the drawings
The PAGE gel electrophoresis result of Fig. 1 canine recombinant interferon-' alpha 's.Wherein, M is Protein Marker
Marker, band for the purpose of 1.
The inhibitory action that Fig. 2 canine recombinants interferon-' alpha ' is bred to mdck cell.
Fig. 3 canine recombinants interferon-' alpha ' stimulates the time dependence that p53 is expressed.
Fig. 4 canine recombinants interferon-' alpha ' stimulates the dose dependent that p53 is expressed.
Embodiment
The invention will now be further described with reference to specific embodiments, advantages of the present invention and feature will be with description and
It is apparent.But these embodiments are only exemplary, do not constitute any limitation to the scope of the present invention.People in the art
Member to the details and form of technical solution of the present invention it should be understood that can enter without departing from the spirit and scope of the invention
Row modifications or substitutions, but these are changed and replacement is each fallen within protection scope of the present invention.Mdck cell：Purchased from ATCC, CCL-
34, by the culture of this laboratory passage, culture medium is the DMEM containing 10% hyclone.
The structure of the canine recombinant interferon-' alpha ' plasmid of embodiment 1.
1st, the canine recombinant interferon-' alpha ' gene needed for being synthesized by the method for chemical synthesis.
2nd, the structure of the recombinant plasmid of expression canine recombinant interferon-' alpha ' gene.
The canine recombinant interferon-' alpha ' gene that above-mentioned steps 1 are synthesized carries out enzyme with restriction enzyme BamHI and HindIII
Cut, and be connected with by the pET27b carriers after identical cleavage, convert bacillus coli DH 5 alpha competence, screening has ammonia benzyl
The transformant of resistance, proves that canine recombinant interferon-' alpha ' gene has been cloned into pET27b and carried after plasmid extraction, digestion identification, sequencing
On body, obtained recombinant plasmid is named as pET-rCaIFN- α.
The coli strain of the high efficient expression canine recombinant interferon-' alpha ' gene of embodiment 2E.coli Rosetta/pET-
RCaIFN- α structure.
With chemical conversion process by pET-rCaIFN- α convert toE.coliRosetta, in the LB containing ampicillin
Transformant is screened on flat board, the recon for proving to obtain through plasmid extraction, digestion identification, sequencing analysisE.coli Rosetta/
PET-rCaIFN- α are consistent with expection.
Embodiment 3 utilizes colibacillus engineeringE.coliRosetta/pET-rCaIFN- α production canine recombinant interference
Element-α.
1st, the cultivation and fermentation of strain
Picking colibacillus engineeringE.coliRosetta/pET-rCaIFN- α, LB is inoculated in by 1% inoculum concentration by engineering bacteria
In fluid nutrient medium, 37 DEG C of incubated 12-14h, next day 1：100 to expand culture to OD values be 0.4, plus IPTG is to final concentration
0.5 mM, continues to cultivate 3h, 4000 r/min, 30 min of centrifugation collect thalline.
2nd, the purifying of canine recombinant interferon-' alpha '
By the thalline ultrasonication collected after centrifugation supernatant of above-mentioned collection, entered after supernatant is filtered using AKTA protein purification systems
Row purifying, successively progress affinity chromatography and sieve chromatography obtain canine recombinant interferon-' alpha ' after purification.
Take a small amount of canine recombinant interferon-alpha proteins after purification to add 5 × SDS sample-loading buffers of 1/5 volume, boil 10
PAGE gel electrophoresis is carried out after minute.Electrophoresis result is shown in Fig. 1.
The inhibitory action that the mtt assay of embodiment 4 detection canine recombinant interferon-alpha proteins are bred to mdck cell.
The mdck cell of dog in exponential phase is collected after Trypsin Induced, 1 is prepared into104Individual/
ML cell suspension, 96 orifice plates of access after piping and druming is uniform, per the μ L cell suspensions of hole 200, in 37 DEG C, 5% CO2Incubator is relayed
After continuous overnight incubation, remove culture medium, washed with PBS once, be divided into 6 groups, every group of experimental port respectively adds 4,20,100,500,
1250th, the 6250 ng μ L of CaIFN- α protein 10s 0, control wells add 100 μ L DMEM.Respectively at 48 h, 72 h, added per hole
20 μ L MTT solution（5 mg/mL）, after 4 h, nutrient solution is discarded, is added per hole after 150 μ L DMSO, concussion 10min, uses enzyme
Linked immune analysis instrument is that 570nm determines OD values in determining wavelength.Growth inhibition ratio is calculated by following formula：
Inhibiting rate %=（Control group OD averages-treatment group's OD values）/ control group OD average × 100%.
As a result Fig. 2 is seen.From figure 2 it can be seen that with albumen buffer control group and natural dog interferon-α protein controls
Group is compared, and canine recombinant interferon-' alpha ' after purification has obvious inhibitory action to mdck cell growth, right with the increase of concentration
Mdck cell inhibiting rate gradually increases (* * p<0.01).
The Real-time PCR of embodiment 5 detection canine recombinant interferon-alpha proteins up-regulation mdck cell p53 mRNA expression
Level.
By the MDCK MDCKs in exponential phase, stimulated respectively with CaIFN- α albumen after purification, respectively
Albumen is subjected to 10 times, 100 times, 1 000 times of dilutions, under stimulating 4 h, detection CaIFN- α albumen to stimulate, p53 is with doses change
Relation and 100 times dilution after, 2 h, 4 h, 6 h stimulate under, the relation that p53 is changed over time.CaIFN- α albumen is stimulated
Afterwards, by cell dissociation, Trizol reagents are added, cell total rna is extracted, reverse transcription uses real-time fluorescence quantitative PCR into after cDNA
Technology for detection p53 mRNA relative expression's situation, using GAPDH expression quantity as internal reference, primer is closed by invitrogen companies
Into the primer is as follows：
P53 upstreams：5′ TTGCCAGCTGGCGAAGACCTG 3′；
P53 downstreams：5′ ACCTCGGGTGGCTCATAAGGCA 3′；
GAPDH upstreams：5′ TGCCGCCTGGAGAAAGCTGC 3′；
GAPDH downstreams：5′ TCCCAGGAAATGAGCTTGAC 3′.
Reacted respectively with two pairs of primers by template of cDNA, by kit（The SYBR GREEN PCR of ABI companies
Master Mix）Specification requires addition reagent, and every group of reaction is done three multiple holes, averaged.Real-time fluorescence quantitative PCR reacts
System is 20 μ L, cDNA is expanded using two-step method PCR response procedures, according to Thermal Cycler Dice TM Real
Time PCR（TaKaRa Code: TP800）Operation instructions require carry out experimental implementation.
As a result Fig. 3 is seen.As seen from Figure 3, under the stimulation of the dog interferon-α albumen of same dose, canine recombinant interferon-' alpha '
Albumen stimulates the endogenous p53 of mdck cell expression to be significantly higher than natural dog interferon-α albumen (* p<0.05), and restructuring
Dog interferon-α albumen stimulates the endogenous p53 of mdck cell expression to reach highest in 4h.
As a result Fig. 4 is seen.As seen from Figure 4, and with natural dog interferon-α and canine recombinant interferon-alpha proteins agent
Amount is continuously increased, and p53 mRNA level in-sites also have gradually increased trend, in dose-dependent relationship, significant difference (##p<
0.01,&&p<0.01).Under the stimulation of the dog interferon-α albumen of various dose, canine recombinant interferon-alpha proteins stimulate MDCK thin
The endogenous p53 of born of the same parents expression is significantly higher than natural dog interferon-α albumen（*p<0.05）.
Annex one
ATGGGATGCC ACCTGCCCGA CACCCACGGC CTGCGCAACT GGAGGGTCCT GACGCTCCTG 60
GGACAGATGA GGAGACTCTC CGCCGGCTCT TGTGACCACT ACACCAATGA CTTTGCCTTC 120
CCCAAGGAGC TGTTTGATGG CCAGCGGCTC CAGGAGGCGC AGGCCCTCTC TGTGGTCCAC 180
GTGATGACCC AGAAGGTCTT CCACCTCTTC TGCCCGGACA CGTCCTCTGC TCCTTGGAAC 240
ATGACTCTCC TGGAGGAACT GTGCTCGGGG CTCTCTGAGC AGCTGGATGA CCTGGAGGCC 300
TGTCCCCTGC AGGAGGCGGG GCTGGCCGAG ACCCCCCTCA TGCATGAGGA CTCCACCCTG 360
AGGACCTACT TCCAAAGGAT CTCCCTCTAC CTGCAAGACA GGAACCACAG CCCGTGTGCC 420
TGGGAGATGG TCCGAGCAGA AATCGGGAGA TCCTTCTTCT CCTCGACAAT CTTGCAAGAA 480
AGAATCAGGA GGAAGGAAGG TGGTGGTGGT AGCGGTGGTG GTGGTAGCGG TGGTGGTGGT 540
AGCCTCCCGT CCCCCATCGA GAGGACTATC TCCAAAGCCA GAGGGCAAGC CCATCAGCCC 600
AGTGTGTATG TCCTGCCACC ATCCCCAAAG GAGTTGTCAT CCAGTGACAC GGTCACCCTG 660
ACCTGCCTGA TCAAAGACTT CTTCCCACCT GAGATTGATG TGGAGTGGCA GAGCAATGGA 720
CAGCCGGAGC CCGAGAGCAA GTACCACACG ACTGCGCCCC AGCTGGACGA GGACGGGTCC 780
TACTTCCTGT ACAGCAAGCT CTCTGTGGAC AAGAGCCGCT GGCAGCAGGG AGACACCTTC 840
ACATGTGCGG TGATGCATGA AGCTCTACAG AACCACTACA CAGATCTATC CCTCTCCCAT 900
TCTCCGGGTA AACATCATCA TCATCATCAT TAAGGATCC 939
Sequence table
<110>Harbin purple light bio tech ltd
<120>A kind of method for improving canine recombinant interferon-' alpha ' fusion protein antiviral activity
<210> SEQ ID NO: 1
<211> 933
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> SEQ ID NO: 1
1 ATGGGCTGCC ACCTGCCGGA TACTCATGGT CTGCGTAATT GGCGCGTGCT GACCCTGTTA
61 GGCCAGATGC GTCGTCTGAG TGCCGGCAGC TGCGACCATT ACACCAATGA CTTCGCCTTT
121 CCGAAGGAGC TGTTTGATGG CCAGCGTCTG CAGGAAGCCC AGGCACTGAG CGTGGTGCAC
181 GTTATGACCC AGAAGGTGTT CCATCTGTTC TGCCCGGATA CCAGTAGCGC CCCTTGGAAT
241 ATGACCCTGC TGGAGGAACT GTGCAGCGGC CTGAGTGAGC AGCTGGATGA TCTGGAAGCA
301 TGTCCGCTGC AAGAAGCTGG CCTGGCAGAA ACCCCGCTGA TGCATGAAGA CAGCACCCTG
361 CGCACCTACT TTCAGCGCAT CAGCCTGTAC CTGCAGGATC GCAACCATAG CCCTTGTGCA
421 TGGGAAATGG TTCGTGCCGA AATCGGCCGC AGCTTCTTTA GCAGCACCAT TCTGCAGGAA
481 CGCATCCGTC GCAAAGAAGG TGGTGGTGGC AGTGGTGGTG GTGGTAGCGG CGGTGGTGGT
541 AGTCTGCCGA GCCCGATTGA ACGTACCATC AGTAAGGCAC GTGGCCAAGC ACATCAGCCT
601 AGTGTGTATG TGCTGCCGCC GAGCCCGAAA GAACTGAGCA GCAGCGATAC CGTGACCCTG
661 ACCTGCCTGA TCAAGGACTT TTTCCCGCCG GAGATTGACG TGGAGTGGCA GAGCAATGGC
721 CAGCCGGAGC CTGAAAGCAA ATACCATACC ACAGCCCCGC AGCTGGACGA GGATGGCAGC
781 TACTTTCTGT ACAGCAAGCT GAGCGTGGAC AAAAGTCGTT GGCAGCAGGG CGATACCTTT
841 ACCTGCGCCG TGATGCACGA AGCCCTGCAG AACCACTACA CCGACCTGAG CCTGAGCCAT
901 AGCCCGGGCA AGCATCATCA CCATCATCAT TAA
<210> SEQ ID NO: 2
<211> 310
<212> PRT
<213>Artificial sequence
<220>
<223>
<400> SEQ ID NO: 2
1 MET Gly Cys His Leu Pro Asp Thr His Gly Leu Arg Asn Trp Arg Val Leu Thr
Leu Leu
21 Gly Gln MET Arg Arg Leu Ser Ala Gly Ser Cys Asp His Tyr Thr Asn Asp
Phe Ala Phe
41 Pro Lys Glu Leu Phe Asp Gly Gln Arg Leu Gln Glu Ala Gln Ala Leu Ser
Val Val His
61 Val MET Thr Gln Lys Val Phe His Leu Phe Cys Pro Asp Thr Ser Ser Ala
Pro Trp Asn
81 MET Thr Leu Leu Glu Glu Leu Cys Ser Gly Leu Ser Glu Gln Leu Asp Asp
Leu Glu Ala
101 Cys Pro Leu Gln Glu Ala Gly Leu Ala Glu Thr Pro Leu MET His Glu Asp
Ser Thr Leu
121 Arg Thr Tyr Phe Gln Arg Ile Ser Leu Tyr Leu Gln Asp Arg Asn His Ser
Pro Cys Ala
141 Trp Glu MET Val Arg Ala Glu Ile Gly Arg Ser Phe Phe Ser Ser Thr Ile
Leu Gln Glu
161 Arg Ile Arg Arg Lys Glu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly
181 Ser Leu Pro Ser Pro Ile Glu Arg Thr Ile Ser Lys Ala Arg Gly Gln Ala
His Gln Pro
201 Ser Val Tyr Val Leu Pro Pro Ser Pro Lys Glu Leu Ser Ser Ser Asp Thr
Val Thr Leu
221 Thr Cys Leu Ile Lys Asp Phe Phe Pro Pro Glu Ile Asp Val Glu Trp Gln
Ser Asn Gly
241 Gln Pro Glu Pro Glu Ser Lys Tyr His Thr Thr Ala Pro Gln Leu Asp Glu
Asp Gly Ser
261 Tyr Phe Leu Tyr Ser Lys Leu Ser Val Asp Lys Ser Arg Trp Gln Gln Gly
Asp Thr Phe
281 Thr Cys Ala Val MET His Glu Ala Leu Gln Asn His Tyr Thr Asp Leu Ser
Leu Ser His
301 Ser Pro Gly Lys His His His His His His ***
Claims (7)
1. a kind of canine recombinant interference-α genes, it is characterised in that its nucleotides sequence is classified as shown in SEQ ID NO.1.
2. the protein of the canine recombinant interferon-' alpha ' coded by said gene of claim 1, it is characterised in that its amino acid sequence is
Shown in SEQ ID NO.2.
3. the recombinant expression carrier containing canine recombinant interferon-' alpha ' gene described in claim 1.
4. according to the recombinant expression carrier described in claim 3, it is characterised in that it is prokaryotic expression carrier.
5. obtained recombinant bacterial strain is converted by the recombinant expression carrier of claim 3 or 4.
6. a kind of Prepare restructuring dog interferon-α method, comprises the following steps：
Build the recombinant expression plasmid containing canine recombinant interferon-' alpha ' gene described in claim 1；Cultivate with the recombination expression matter
The host cell that grain is converted, obtains recombinant bacterial strain；Recombinant bacterial strain is cultivated, the expression of recombinant protein is induced, separates and purify institute's table
The recombinant protein reached.
7. in accordance with the method for claim 6, it is characterised in that the separation and purifying of described recombinant protein include following step
Suddenly：
（1）The thalline of collection is suspended with phosphate buffer, 0-4 DEG C of low-temperature centrifugation collects supernatant after crushing, affine using Ni
Post and AKTA protein purification systems carry out albumen and slightly purified；
（2）Recycle AKTA systems to carry out the purifying of molecular sieve in the albumen slightly purified, produce.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710584157.7A CN107217068A (en) | 2017-07-18 | 2017-07-18 | A kind of method for improving canine recombinant interferon alpha fusion protein antiviral activity |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710584157.7A CN107217068A (en) | 2017-07-18 | 2017-07-18 | A kind of method for improving canine recombinant interferon alpha fusion protein antiviral activity |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107217068A true CN107217068A (en) | 2017-09-29 |
Family
ID=59953342
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710584157.7A Pending CN107217068A (en) | 2017-07-18 | 2017-07-18 | A kind of method for improving canine recombinant interferon alpha fusion protein antiviral activity |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107217068A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108486127A (en) * | 2018-01-12 | 2018-09-04 | 中国农业科学院北京畜牧兽医研究所 | 6 α of dog interferon-α, 7 recombinant proteins and the preparation method and application thereof |
| CN108864292A (en) * | 2018-06-27 | 2018-11-23 | 军事科学院军事医学研究院军事兽医研究所 | A kind of interferon recombination fusion albumen and its application |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007018879A2 (en) * | 2005-07-08 | 2007-02-15 | Regeneron Pharmaceuticals, Inc. | Interferon-gamma antagonists and therapeutic uses thereof |
| CN102140476A (en) * | 2010-01-28 | 2011-08-03 | 哈尔滨市哈科隆生物制药研究所 | Gene of recombinant staphylococal protein A, expression vector containing gene and application thereof |
| CN102492714A (en) * | 2011-12-10 | 2012-06-13 | 江苏畜牧兽医职业技术学院 | Method for preparing recombinant Chinese rural dog interferon-alpha |
| CN102596235A (en) * | 2009-08-31 | 2012-07-18 | 免疫医疗公司 | Compositions and methods of use of immunotoxins comprising ranpirnase (RAP) show potent cytotoxic activity |
| US20130028867A1 (en) * | 2010-04-02 | 2013-01-31 | Hanmi Science Co., Ltd. | Long-acting interferon beta formulation using immunoglobulin fragment |
| CN103665166A (en) * | 2012-09-03 | 2014-03-26 | 福又达生物科技股份有限公司 | Dog fusion interferon |
| CN104447978A (en) * | 2014-12-10 | 2015-03-25 | 天津生机集团股份有限公司 | Recombinant chicken interferon alpha and preparation method thereof |
| CN104388452B (en) * | 2014-10-22 | 2017-01-04 | 华东师范大学 | A kind of dog interferon alpha fusion protein and its preparation method and application |
-
2017
- 2017-07-18 CN CN201710584157.7A patent/CN107217068A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007018879A2 (en) * | 2005-07-08 | 2007-02-15 | Regeneron Pharmaceuticals, Inc. | Interferon-gamma antagonists and therapeutic uses thereof |
| CN102596235A (en) * | 2009-08-31 | 2012-07-18 | 免疫医疗公司 | Compositions and methods of use of immunotoxins comprising ranpirnase (RAP) show potent cytotoxic activity |
| CN102140476A (en) * | 2010-01-28 | 2011-08-03 | 哈尔滨市哈科隆生物制药研究所 | Gene of recombinant staphylococal protein A, expression vector containing gene and application thereof |
| US20130028867A1 (en) * | 2010-04-02 | 2013-01-31 | Hanmi Science Co., Ltd. | Long-acting interferon beta formulation using immunoglobulin fragment |
| CN102492714A (en) * | 2011-12-10 | 2012-06-13 | 江苏畜牧兽医职业技术学院 | Method for preparing recombinant Chinese rural dog interferon-alpha |
| CN103665166A (en) * | 2012-09-03 | 2014-03-26 | 福又达生物科技股份有限公司 | Dog fusion interferon |
| CN104388452B (en) * | 2014-10-22 | 2017-01-04 | 华东师范大学 | A kind of dog interferon alpha fusion protein and its preparation method and application |
| CN104447978A (en) * | 2014-12-10 | 2015-03-25 | 天津生机集团股份有限公司 | Recombinant chicken interferon alpha and preparation method thereof |
Non-Patent Citations (4)
| Title |
|---|
| NCBI: "Canis familiaris immunoglobulin gamma heavy chain D mRNA，complete cds", 《GENBANK DATABASE》 * |
| NCBI: "interferon alpha-1/2 precursor [Canis lupus familiaris]", 《GENBANK DATABASE》 * |
| 王磊 等: "人干扰素α2b和IgG片段融合蛋白显著延长体内半衰期", 《生物工程学报》 * |
| 田硕 等: "长效干扰素研究进展", 《中国生物工程杂志》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108486127A (en) * | 2018-01-12 | 2018-09-04 | 中国农业科学院北京畜牧兽医研究所 | 6 α of dog interferon-α, 7 recombinant proteins and the preparation method and application thereof |
| CN108864292A (en) * | 2018-06-27 | 2018-11-23 | 军事科学院军事医学研究院军事兽医研究所 | A kind of interferon recombination fusion albumen and its application |
| CN108864292B (en) * | 2018-06-27 | 2021-08-17 | 军事科学院军事医学研究院军事兽医研究所 | Interferon recombinant fusion protein and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107254482A (en) | A kind of method for improving Recombinant Swine interferon alpha fusion protein antiviral activity | |
| CN106282216B (en) | A kind of preparation method of recombinant long-acting chicken interferon α | |
| CN111304181B (en) | Genetically engineered vibrio parahemolyticus phage lyase and preparation method and application thereof | |
| CN107217068A (en) | A kind of method for improving canine recombinant interferon alpha fusion protein antiviral activity | |
| CN108264548B (en) | Mandarin fish gamma interferon related factor, recombinant protein and application thereof | |
| CN109627316A (en) | Recombinant protein and the application of 2 gene of grass carp IFN-γ and its coding | |
| CN105367634B (en) | A kind of Bt PROTEIN C ry1Ie5, its encoding gene and application | |
| CN101638665B (en) | Construction and usage of lactobacillus single-plasmid Nisin inducible expression vector | |
| CN108840952A (en) | A kind of fusion protein and preparation method thereof being made of Chicken Albumin, chicken interferon gamma and chicken interferon α | |
| CN108794638A (en) | A kind of recombinant bovine long-acting interferon α and the fusion protein and preparation method thereof for preparing this long-acting interferon | |
| CN107177614A (en) | A kind of method for improving canine recombinant interferon gamma fusion protein antiviral activity | |
| CN107129994A (en) | A kind of method for improving canine recombinant interferon beta fusion protein antiviral activity | |
| CN101434958A (en) | Method for preparing porcine alpha-interferon | |
| CN108840951A (en) | A kind of fusion protein and preparation method thereof being made of pig albumin, Porcine interferon-gamma and porcine interferon alpha | |
| CN108676083B (en) | Sheep gamma interferon mutant and preparation method and application thereof | |
| CN113667005A (en) | Pacific codfish interferon-gamma protein, gene, recombinant plasmid, recombinant yeast engineering bacteria and application thereof | |
| CN107022552A (en) | Salt sward resistant gene of salt HgS2 and its application | |
| CN107253996A (en) | Sheep albumin interferon-tau interleukin-22 fusion protein, preparation method and its encoding gene, a kind of sheep long-acting interferon | |
| CN102899331A (en) | Complex duck interferon-alpha gene, and recombinant vector and application thereof | |
| CN107326037A (en) | A kind of method for improving Recombinant Swine interferon beta fusion protein antiviral activity | |
| CN104531691A (en) | Primers for obtaining genes of bovine interferon alpha and preparation method for recombinant bovine interferon alpha | |
| CN108794644A (en) | A kind of fusion protein and preparation method thereof being made of cattle interleukins-2 2, Bov IFN γ and Bov IFN α | |
| CN108794645A (en) | A kind of fusion protein and preparation method thereof being made of bovine albumin, Bov IFN γ and Bov IFN α | |
| CN107177613A (en) | A kind of method for improving restructuring Porcine interferon-gamma fusion protein antiviral activity | |
| CN107245110A (en) | A kind of fusion protein being made up of sheep albumin, sheep interferon gamma and sheep interferon-tau and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20170929 |