CN107177614A - A kind of method for improving canine recombinant interferon gamma fusion protein antiviral activity - Google Patents
A kind of method for improving canine recombinant interferon gamma fusion protein antiviral activity Download PDFInfo
- Publication number
- CN107177614A CN107177614A CN201710584165.1A CN201710584165A CN107177614A CN 107177614 A CN107177614 A CN 107177614A CN 201710584165 A CN201710584165 A CN 201710584165A CN 107177614 A CN107177614 A CN 107177614A
- Authority
- CN
- China
- Prior art keywords
- recombinant
- interferon
- canine
- gene
- protein
- 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]
- C07K14/57—IFN-gamma
-
- 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)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a kind of preparation method of canine recombinant interferon gamma 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 γ genes 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 canine recombinant interferon gamma expressed by the present invention has compared with natural dog interferon γ bioactivity to be significantly improved.
Description
Technical field
The present invention relates to a kind of gene of rearrangement, more particularly to canine recombinant interferon-γ gene, the invention further relates to containing
The expression vector and engineered strain of the gene, the invention further relates to their purposes in dog interferon-γ is prepared, category
In interferon gene 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
It will be the main matter of following 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 is mainly made by cell surface receptor effect
Cell produces antiviral protein, so as to suppress the duplication of virus；It can also strengthen NK simultaneously（NK cells）, macrophage it is thin
Born of the same parents and the vigor of T lymphocytes, so as to play immunoregulation effect, and strengthen anti-virus ability.Interferon is one group with many
Plant the reactive protein of function（Mainly glycoprotein）, it is a kind of cell factor produced by monocyte and lymphocyte.It
On allogenic cell antiviral, influence cell growth with wide spectrum, and differentiation, regulation immunologic function etc. be a variety of biological living
Property.Due to interferon have it is nontoxic, have no side effect and antiviral advantage in extensive range, just attracted attention at the beginning of discovery, pass through
After application development for many years, interferon, which has become a kind of extensive immunopotentiator, is used for the anti-of the diseases such as virosis and tumour
Control field.
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-γ is 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
Original series are transformed by technology, and obtaining effect, more useful canine recombinant interferon-γ is current Research Thinking.
The content of the invention
An object of the present invention is to provide a kind of canine recombinant interferon-γ gene, and the gene can be in prokaryotic expression system
Stable in system, efficient expression restructuring dog interferon；
The two of the object of the invention are to provide the expression vector containing above-mentioned canine recombinant interferon-γ gene；
The three of the object of the invention are to provide the engineered strain converted by above-mentioned canine recombinant interferon-γ gene；
The four of the object of the invention are to provide a kind of Prepare restructuring dog interferon-γ method；
Above-mentioned purpose of the present invention is achieved through the following technical solutions：
A kind of canine recombinant interferon-γ gene, its nucleotides sequence is classified as shown in SEQ ID NO.1, its amino acid sequence encoded
Shown in SEQ ID NO.2.
The canine recombinant interferon-γ gene of the present invention is with dog interferon-γ, dog immunoglobulin CH3 regions and 6
His is 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-γ gene of the present invention is by dog interferon-γ, 6 His and dog immunoglobulin
CH3 areas are in series, and the gene constitutes 268 amino acid of coding by 807 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-γ genes with escherichia coli prokaryotic expression carrier pET27b using restriction enzyme site BamHI and HindIII to be connected, be named as
pET-rCaIFN-γ。
Recombinant expression plasmid constructed by the present invention can convert host cell by various conventional methods.As reference,
Recombinant plasmid pET-rCaIFN- γ containing canine recombinant interferon-γ gene can be converted Escherichia coli Rosetta（It is purchased from
Beijing Quanshijin Biotechnology Co., Ltd, article No. catalogue CD801）Obtained bacterial strain, is named asEscherichia coli
Rosetta/pET-rCaIFN- γ, abbreviation Rosetta/pET-rCaIFN- γ.
Can be with using the E. coli recombinant stain Rosetta/pET-rCaIFN- γ specific methods for producing recombinant protein：
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 12 h of culture.
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 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-γ 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- γ；Described
Host cell is Escherichia coli（Escherichia coli）Rosetta (DE3), described recombinant bacterial strain is preferablyEscherichia coli Rosetta/pET-rCaIFN-γ。
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 and reach more than 90%
Albumen.
With natural dog interferon gene-γ（Dog interferon gene-γ original series are shown in annex one）It is dry instead of canine recombinant
Disturb element-γ genes and carry out above step, obtain the natural dog interferon available for subsequent experimental as control.
Dog interferon-γ 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-γ expressed by the present invention is not only significantly better than naturally on half-life period
Dog interferon-γ, and improve its antiviral activity.
Brief description of the drawings
The PAGE gel electrophoresis result of Fig. 1 canine recombinants interferon-γ after purification.
Wherein, M is band for the purpose of Protein Marker Marker, 1.
The inhibitory action that Fig. 2 canine recombinants interferon-γ is bred to mdck cell.
Fig. 3 canine recombinants interferon-γ promotees the result of the test of mice spleen lymphocytes proliferation.
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 containing 10% hyclone
DMEM。
The structure of the canine recombinant interferon-γ gene plasmid of embodiment 1
1st, the canine recombinant interferon-γ gene needed for being synthesized by the method for chemical synthesis.
2nd, the structure of the recombinant plasmid of expression canine recombinant interferon-γ gene.
The canine recombinant interferon-γ gene that above-mentioned steps 1 are synthesized is carried out with restriction enzyme BamHI and HindIII
Digestion, and with being connected by the pET27b carriers after identical cleavage and converting bacillus coli DH 5 alpha competence, screening has
The transformant of ammonia benzyl resistance, proves that canine recombinant interferon-γ gene has been cloned into after plasmid extraction, digestion identification, sequencing
On pET27b carriers, obtained recombinant plasmid is named as pET-rCaIFN- γ.
The coli strain of the high efficient expression canine recombinant interferon-γ gene of embodiment 2E.coli Rosetta/pET-
RCaIFN- γ structure
With chemical conversion process by pET-rCaIFN- γ convert toE.coliRosetta, in the LB flat boards containing ampicillin
Upper screening transformant, the recon for proving to obtain through plasmid extraction, digestion identification, sequencing analysisE.coli Rosetta/pET-
RCaIFN- γ are consistent with expection.
Embodiment 3 utilizes colibacillus engineeringE.coliRosetta/pET-rCaIFN- γ production canine recombinant interference
Element-γ
1st, the cultivation and fermentation of strain
Picking colibacillus engineeringE.coliRosetta/pET-rCaIFN- γ, LB is inoculated in by 1% inoculum concentration by engineering bacteria
In fluid nutrient medium, 37 DEG C of incubated 12-14 h, 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-γ
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-γ after purification.
Take a small amount of canine recombinant interferon-γ albumen 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-γ albumen is bred to mdck cell
The mdck cell of dog in exponential phase is collected after Trypsin Induced, 1 is prepared into104Individual/mL's
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% CO2Continue to train in incubator
Support overnight, meanwhile, fresh pig blood 10ml is taken, isometric PBS is added, blood sample is diluted.Draw isometric lymphocyte separation medium
It is placed in 50ml centrifuge tubes.Blood sample after dilution is slowly added into lymphocyte separation medium by the amount of 2 times of volumes of separating liquid
Upper strata.Make separating liquid：Blood sample：The volume ratio of balanced salt solution is 1：1：1,2200r centrifugation 22min.It is quick draw tunica albuginea to from
In heart pipe, add at least 3 times volumes PBS into centrifuge tube, mix, 2200r centrifugation 8-10min, abandon supernatant, with 10ml without
Blood serum medium hangs（Big bottle）, counted.Trypan Blue counts living cells>When 95%, per hole in 96 well culture plates
200 μ L cell suspensions are added, 37 DEG C, 5%CO are put2Cultivated in incubator.
Remove culture medium, washed with PBS once, be divided into 6 groups, every group of experimental port respectively add 2,10,50,250,1250,6250ng
The μ L of CaIFN- γ protein 10s 0, control wells add 100 μ L DMEM.Respectively at 48 h, 72 h, 20 μ L MTT solution are added per hole（5
mg/mL）, after 4 h, nutrient solution is discarded, is added per hole after 150 μ L DMSO, concussion 10min, with ELISA instrument in survey
The a length of 570nm of standing wave determines OD values.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-γ protein controls group phase
Than canine recombinant interferon-γ 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 spleen lymphocyte proliferation of embodiment 5 is tested
1st, fresh dog blood 10ml is taken, isometric PBS is added, blood sample is diluted.Isometric lymphocyte separation medium is drawn to be placed in
In 50ml centrifuge tubes.
The 2nd, blood sample after dilution is slowly added into the upper strata of lymphocyte separation medium by the amount of 2 times of volumes of separating liquid.Make
Separating liquid：Blood sample：The volume ratio of balanced salt solution is 1：1：1,2200r centrifugation 22min.
3rd, quick tunica albuginea of drawing adds the PBS of at least 3 times volumes into centrifuge tube, mixed, 2200r into centrifuge tube
8-10min is centrifuged, supernatant is abandoned, is hanged with 10ml serum free mediums（Big bottle）, counted.Trypan Blue statistics is living thin
Born of the same parents>When 95%, 200 μ L cell suspensions are added per hole in 96 well culture plates, 37 DEG C, 5%CO are put2Cultivated in incubator.
4th, canine recombinant rIFN- γ albumen and natural canine IFN-γ albumen are separately added into after culture 1h in cell, and sets thin
Born of the same parents' control group.
5th, cultivating after 72h, then add MTT in every hole makes its final concentration of 5mg/mL, continues to cultivate 4h, takes out and cultivate
Plate, gently upset drains liquid in hole, is added per hole after 150 μ L DMSO vibrations 20min, with ELIASA test sample sample wells OD570
Value.As a result SPSS statistical softwares are used, statistical procedures are carried out to surveyed data, shown using one-factor analysis of variance method
Work property is examined.
As a result Fig. 3 is seen.From figure 3, it can be seen that adding restructuring rCaIFN- γ albumen than adding natural CaIFN- γ albumen
There is higher proliferation function to lymphocyte, and the cell for not adding CaIFN- γ albumen does not have proliferation function.
Annex one
1 ATGGATGTAT CGGACGGTGG GTCTCTTTTC GTAGATATTT TGAAGAAATG
51 GAGAGAGGAG AGTGACAAAA CAATCATTCA GAGCCAAATT GTCTCTTTCT
101 ACTTGAAACT GTTTGACAAC TTTAAAGATA ACCAGATCAT TCAAAGGAGC
151 ATGGATACCA TCAAGGAAGA CATGCTTGGC AAGTTCTTAA ATAGCAGCAC
201 CAGTAAGAGG GAGGACTTCC TTAAGCTGAT TCAAATTCCT GTGAACGATC
251 TGCAGGTCCA GCGCAAGGCG ATAAATGAAC TCATCAAAGT GATGAATGAT
301 CTCTCACCAA GATCCAACCT AAGGAAGCGG AAAAGGAGTC AGAATCTGTT
351 TCGAGGCCGC AGAGCATCGA AAGGTGGTGG TGGTAGCGGT GGTGGTGGTA
401 GCGGTGGTGG TGGTAGCCTC CCGTCCCCCA TCGAGAGGAC TATCTCCAAA
451 GCCAGAGGGC AAGCCCATCA GCCCAGTGTG TATGTCCTGC CACCATCCCC
501 AAAGGAGTTG TCATCCAGTG ACACGGTCAC CCTGACCTGC CTGATCAAAG
551 ACTTCTTCCC ACCTGAGATT GATGTGGAGT GGCAGAGCAA TGGACAGCCG
601 GAGCCCGAGA GCAAGTACCA CACGACTGCG CCCCAGCTGG ACGAGGACGG
651 GTCCTACTTC CTGTACAGCA AGCTCTCTGT GGACAAGAGC CGCTGGCAGC
701 AGGGAGACAC CTTCACATGT GCGGTGATGC ATGAAGCTCT ACAGAACCAC
751 TACACAGATC TATCCCTCTC CCATTCTCCG GGTAAACATC ATCATCATCA
801 TCATTAA
Sequence table
<110>Harbin purple light bio tech ltd
<120>A kind of method for improving canine recombinant interferon-γ fusion protein antiviral activity
<210> SEQ ID NO: 1
<211> 807
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> SEQ ID NO: 1
1 ATGGACGTTT CTGACGGTGG TTCTCTGTTC GTTGACATCC TGAAAAAATG GCGTGAAGAA
61 TCTGACAAAA CCATCATCCA GTCTCAGATC GTTTCTTTCT ACCTGAAACT GTTCGACAAC
121 TTCAAAGACA ACCAGATCAT CCAGCGTTCT ATGGACACCA TCAAAGAAGA CATGCTGGGT
181 AAATTCCTGA ACTCTTCTAC CTCTAAACGT GAAGACTTCC TGAAACTGAT CCAGATCCCG
241 GTTAACGACC TGCAGGTTCA GCGTAAAGCT ATCAACGAAC TGATCAAAGT TATGAACGAC
301 CTGTCTCCGC GTTCTAACCT GCGTAAACGT AAACGTTCTC AGAACCTGTT CCGTGGTCGT
361 CGTGCTTCTA AAGGTGGTGG TGGTTCTGGT GGTGGTGGTT CTGGTGGTGG TGGTTCTCTG
421 CCGTCTCCGA TCGAACGTAC CATCTCTAAA GCTCGTGGTC AGGCTCACCA GCCGTCTGTT
481 TACGTTCTGC CGCCGTCTCC GAAAGAACTG TCTTCTTCTG ACACCGTTAC CCTGACCTGC
541 CTGATCAAAG ACTTCTTCCC GCCGGAAATC GACGTTGAAT GGCAGTCTAA CGGTCAGCCG
601 GAACCGGAAT CTAAATACCA CACCACCGCT CCGCAGCTGG ACGAAGACGG TTCTTACTTC
661 CTGTACTCTA AACTGTCTGT TGACAAATCT CGTTGGCAGC AGGGTGACAC CTTCACCTGC
721 GCTGTTATGC ACGAAGCTCT GCAGAACCAC TACACCGACC TGTCTCTGTC TCACTCTCCG
781 GGTAAACACC ACCACCACCA CCACTAA
<210> SEQ ID NO: 2
<211> 268
<212> PRT
<213>Artificial sequence
<220>
<223>
<400> SEQ ID NO: 2
1 MET Asp Val Ser Asp Gly Gly Ser Leu Phe Val Asp Ile Leu Lys Lys Trp Arg Glu Glu
21 Ser Asp Lys Thr Ile Ile Gln Ser Gln Ile Val Ser Phe Tyr Leu Lys Leu Phe Asp Asn
41 Phe Lys Asp Asn Gln Ile Ile Gln Arg Ser MET Asp Thr Ile Lys Glu Asp MET Leu Gly
61 Lys Phe Leu Asn Ser Ser Thr Ser Lys Arg Glu Asp Phe Leu Lys Leu Ile Gln Ile Pro
81 Val Asn Asp Leu Gln Val Gln Arg Lys Ala Ile Asn Glu Leu Ile Lys Val MET Asn Asp
101 Leu Ser Pro Arg Ser Asn Leu Arg Lys Arg Lys Arg Ser Gln Asn Leu Phe Arg Gly Arg
121 Arg Ala Ser Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Leu
141 Pro Ser Pro Ile Glu Arg Thr Ile Ser Lys Ala Arg Gly Gln Ala His Gln Pro Ser Val
161 Tyr Val Leu Pro Pro Ser Pro Lys Glu Leu Ser Ser Ser Asp Thr Val Thr Leu Thr Cys
181 Leu Ile Lys Asp Phe Phe Pro Pro Glu Ile Asp Val Glu Trp Gln Ser Asn Gly Gln Pro
201 Glu Pro Glu Ser Lys Tyr His Thr Thr Ala Pro Gln Leu Asp Glu Asp Gly Ser Tyr Phe
221 Leu Tyr Ser Lys Leu Ser Val Asp Lys Ser Arg Trp Gln Gln Gly Asp Thr Phe Thr Cys
241 Ala Val MET His Glu Ala Leu Gln Asn His Tyr Thr Asp Leu Ser Leu Ser His Ser Pro
261 Gly Lys His His His His His His
Claims (7)
1. a kind of canine recombinant interferon-γ gene, it is characterised in that：Its nucleotides sequence is classified as shown in SEQ ID NO.1.
2. the protein coded by the canine recombinant interferon-γ 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-γ 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-γ method, comprises the following steps：
Build the recombinant expression plasmid containing canine recombinant interferon-γ 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 |
|---|---|---|---|
| CN201710584165.1A CN107177614A (en) | 2017-07-18 | 2017-07-18 | A kind of method for improving canine recombinant interferon gamma fusion protein antiviral activity |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710584165.1A CN107177614A (en) | 2017-07-18 | 2017-07-18 | A kind of method for improving canine recombinant interferon gamma fusion protein antiviral activity |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107177614A true CN107177614A (en) | 2017-09-19 |
Family
ID=59837790
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710584165.1A Pending CN107177614A (en) | 2017-07-18 | 2017-07-18 | A kind of method for improving canine recombinant interferon gamma fusion protein antiviral activity |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107177614A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108359004A (en) * | 2018-01-12 | 2018-08-03 | 中国农业科学院北京畜牧兽医研究所 | Dog recombinant interferon-λ 1 and the preparation method and application thereof |
| CN111233998A (en) * | 2018-08-02 | 2020-06-05 | 中国农业科学院北京畜牧兽医研究所 | Canine interferon CaIFN-lambda mutant and application thereof |
Citations (6)
| 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 |
| 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 |
-
2017
- 2017-07-18 CN CN201710584165.1A patent/CN107177614A/en active Pending
Patent Citations (6)
| 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 |
| 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 |
| 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 (3)
| Title |
|---|
| NASCIMENTO MS等: "interferon gamma precursor [Canis lupus familiaris]", 《GENBANK DATABASE》 * |
| TANG L等: "Canis familiaris immunoglobulin gamma heavy chain D mRNA，complete cds", 《GENBANK DATABASE》 * |
| 田硕等: "长效干扰素研究进展", 《中国生物工程杂志》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108359004A (en) * | 2018-01-12 | 2018-08-03 | 中国农业科学院北京畜牧兽医研究所 | Dog recombinant interferon-λ 1 and the preparation method and application thereof |
| CN111233998A (en) * | 2018-08-02 | 2020-06-05 | 中国农业科学院北京畜牧兽医研究所 | Canine interferon CaIFN-lambda mutant and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106282216B (en) | A kind of preparation method of recombinant long-acting chicken interferon α | |
| CN107254482A (en) | A kind of method for improving Recombinant Swine interferon alpha fusion protein antiviral activity | |
| CN101173004A (en) | Insect antimicrobial peptide Thanatin and method for producing deletion mutant thereof | |
| CN101892241B (en) | Grass carp interleukin 1 beta gene and protein and recombinant expression method thereof | |
| CN108424915A (en) | The preparation method of 2 recombinant proteins of dog interferon-α | |
| CN107177614A (en) | A kind of method for improving canine recombinant interferon gamma fusion protein antiviral activity | |
| CN109627316B (en) | Grass carp IFN-gamma 2 gene, recombinant protein coded by same and application of gene | |
| CN107217068A (en) | A kind of method for improving canine recombinant interferon alpha fusion protein antiviral activity | |
| CN107286255A (en) | It is a kind of by OVA, chicken interferon gamma and chicken interferon α fusion protein constituted and preparation method thereof | |
| CN101544693B (en) | Recombined extrasin alpha 1 two-strand body protein and preparation method thereof | |
| CN103012578B (en) | Recombinant porcine interleukin 2, and encoding gene and expression method thereof | |
| CN103232544B (en) | Recombination porcine interferon gamma-Fc fusion protein as well as coding gene and expression method thereof | |
| CN107435045B (en) | A kind of nucleotide sequence and solution expression with high efficiency method of optimum combination Human Inter Leukin-2 | |
| CN109438567A (en) | A kind of sturgeon anti pathologic immunity albumen and its preparation method and application | |
| CN107177613A (en) | A kind of method for improving restructuring Porcine interferon-gamma fusion protein antiviral activity | |
| CN107129994A (en) | A kind of method for improving canine recombinant interferon beta fusion protein antiviral activity | |
| CN102250239B (en) | Protein capable of combining with vp60 protein of rabbit hemorrhagic disease virus and use 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 | |
| CN100425697C (en) | Nucleotid sequence of human interleukin 21 and method and application of producing matured human interleukin 21 | |
| CN117304344B (en) | Fusion protein, recombinant strain expressing fusion protein and application of recombinant strain | |
| CN101089181A (en) | Production method of recombination human interleukin-4 | |
| CN1057564C (en) | Microbe capable of producing active recombinant phycobiliprotein and its preparing method | |
| CN113061176B (en) | Grass carp antimicrobial protein LECT2, and preparation method and application thereof | |
| 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: 20170919 |