CN108864298A - A kind of canine recombinant long-acting interferon and the fusion protein for preparing this long-acting interferon and preparation method thereof - Google Patents
A kind of canine recombinant long-acting interferon and the fusion protein for preparing this long-acting interferon and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a kind of canine recombinant long-acting interferon and the fusion proteins for preparing this long-acting interferon and preparation method thereof; the fusion protein is connected through flexible linker by canine leucocyte interleukin 2 and dog interferon alpha and is formed, and Hu Jing is freeze-dried to obtain canine recombinant long-acting interferon after fusion protein and freeze drying protectant mixture.The canine recombinant long-acting interferon is remarkably improved the half-life period of dog interferon, and the half-life period of more common dog interferon improves 10 times or more, and has broad-spectrum disease resistance toxic action and can improve the immune response of dog itself.
Description
Technical field
The invention belongs to technical field of biological genetic engineering, and in particular to a kind of canine recombinant long-acting interferon and prepare this length
Imitate the fusion protein and preparation method thereof of interferon.
Background technique
It is analyzed according to Chinese pet Industry, the market scale by the end of 2014 year end China pet industries has reached
105800000000 RMB, and Chinese Medical pet industry only accounts for about 20% at present, the city of the U.S. for the pet industry relative maturity that compares
, the output value ratio of Medical pet reaches 50%, and there are huge development spaces for Chinese Medical pet industry.
Dog is the friend of mankind's loyalty, with increasing considerably for the canines feeding quantity such as domestic pet dog, canine disease
The continuous raising of disease incidence, the death rate causes spirit and double loss economically to the mankind.Dog is led with mankind's intimate contact
It causes the mankind high by the probability of dog virus infection, such as rabies viruses, canine parvovirus and canine distemper virus, can all give the mankind
Health care belt greatly threatens.
Report interferon is studied as a kind of important cell factor, to canine distemper, canine parvovirus according to related science
Certain curative effect is all had in the treatments of diseases such as disease, canine infectious hepatitis and canine coronavirus disease.Therefore actively research interference
Effect of the element in canine viral disease treatment has broad application prospects.
IFN is that a kind of virus infection induction body generates and has broad-spectrum antiviral, antitumor and with immunoregulation effect
Protein, nineteen fifty-seven Issacs and Lindeman first discovery, it is a kind of multi-functional cell factor, with cell receptor
In conjunction with rear, it can induce body and generate a variety of specific proteins and enzyme, it is main by inhibiting viral gene transcription and degradation disease
Malicious RNA inhibits the growth and breeding of virus and plays antitumor etc. activity.According to the generation cell of IFN, biochemical character and
The difference to be played a role in terms of immunity of organism is divided into α, β, γ three classes.Now it is known that α type IFN in vivo can be selective
Ground acts on the infection cells such as virus, by inhibiting the biosynthesis of the virus protein in infected cell, plays wide spectrum and height
Imitate antivirus action.
IL-2, that is, interleukin 2 also known as t cell growth factor.Mainly had by what the T lymphocyte activated generated
The cell factor of extensive bioactivity, can both promote lymphopoiesis, enhance immune function, and can restricted T cells reaction
And enhance the immune tolerance of body, therefore can be used for treating tumour, infectious diseases and autoimmune disease.In medical domain
The recombinant human il-2 of upper commercialization has been used to the treatment of the diseases such as tumour, AIDS, hepatitis B.In animal doctor, due to
IL-2 can improve the immune response of vaccine and reduce the generation of disease, wide application prospect also occur.
The limitation of natural interferon and artificial recombination interferon generally existing half-life short at present, half-life period are generally 2-4
A hour.Half-life short brings great inconvenience to treatment, the increase for the treatment of number of times, corresponding time cost and it is economical at
This increases therewith, and the tenability limit of body is also possible to be broken the generation for leading to adverse reaction.The main original of half-life short
Because there are two:The too small tachytrophism in vivo of the molecular weight of interferon, the interferon especially poor quilt of recombinant interferon affinity
Immune system is removed.And common long-acting interferon is using polyethylene glycol fused interferon as representative on the market, only in molecular weight
Partially solve the problems, such as that interferon molecule amount is small and leads to half-life short in level, at the same polyethylene glycol fused interferon at
This is very high, is unfavorable for clinically applying in poultry.
Summary of the invention
In order to solve the above technical problems, the present invention provides a kind of canine recombinant long-acting interferon and preparing this long-acting interferon
Fusion protein and preparation method thereof, the canine recombinant long-acting interferon is remarkably improved the half-life period of dog interferon, more commonly
The half-life period of dog interferon improves 10 times or more, and has broad-spectrum disease resistance toxic action and can improve the immune response of dog itself.
The technical scheme adopted by the invention is as follows:
A kind of fusion protein being made of canine leucocyte interleukin 2 and dog interferon alpha, the amino acid sequence of the fusion protein
Table is as shown in 400 < of SEQUENCE LISTING, 1 >.
The present invention also provides the gene for encoding above-mentioned fusion protein, the nucleotides sequence list such as SEQUENCE of the gene
Shown in 400 < of LISTING, 2 >, it is denoted as genome 1;Or as shown in 400 < of SEQUENCE LISTING, 3 >, it is denoted as genome 2.
Fusion protein described in the genome 1 and the equal codified of the genome 2.Genome 2 is the nucleosides to genome 1
Acid sequence optimize after as a result, when usual codon adaptation indexI CAI=1.0 be considered the gene in the expression system
It is optimal high efficient expression state in system, CAI value is lower to show that expression is lower in host.G/C content is most managed in gene
Think that distribution is 30~70%, is more than that the range will affect translation and transcriptional efficiency in any region.It is examined using software
Surveying the codon for finding dog IL-2 and IFN-α original gene codon adaptation indexI (CAI) in Escherichia coli is respectively
0.19,0.27, GC percentage is 39.6%, 59.7%;And by obtained after dog IL-2 and IFN-α gene optimization recombination base
Because codon adaptation indexI (CAI) is 1.0,1.0, GC percentage 48.8%, 55.6% to genome 2 in Escherichia coli.Pass through
Gene optimization significantly reduces the utilization rate of low codon, avoids influence of the rare codon to protein expression, improves base
The G/C content of cause improves transcription and translation efficiency.
The present invention also provides the expression vectors containing genome 1 or genome 2.
Further, the expression vector is the pET-32a coli expression carrier containing genome 1 or genome 2.
The present invention also provides the genetic engineering bacteriums containing genome 1 or genome 2.
Further, the genetic engineering bacterium is pET-32a/rIL2-IFN α.
Host cell containing genome 1 or genome 2 also belongs to protection scope of the present invention, further, the place
Chief cell is e. coli host cell, and further, the e. coli host cell is that BL21 (DE3) competence is thin
Born of the same parents or BL21 (DE3) competent cell with pGro7 plasmid.
The present invention also provides a kind of canine recombinant long-acting interferon, the canine recombinant long-acting interferon is by the fusion egg
It is freeze-dried to form after the white mixture with freeze drying protectant.
The freeze drying protectant is glycerol, mannitol and sucrose, is buffer, the final concentration of three with 10mmol/L PBS
For glycerol 100mL/L, mannitol 0.12g/mL and sucrose 0.025g/mL.
The invention also discloses the preparation methods of the fusion protein, and the preparation method comprises the following steps:It will contain
The expression vector of genome 1 or genome 2 is imported into e. coli host cell, obtains genetic engineering bacterium, genetic engineering bacterium
The crude product of the fusion protein is obtained after IPTG inducing expression, and fusion protein can be obtained after purified.
The expression vector is the pET-32a coli expression carrier containing genome 1 or genome 2;
The genetic engineering bacterium is pET-32a/rIL2-IFN α, and preparation method is:
(1) design primer, is obtained or the canine leucocyte of the flexible linker sequence of artificial synthesized connection by reverse transcription
The target gene of interleukin 2 and dog interferon alpha;By flexible linker by the purpose base of canine leucocyte interleukin 2 and dog interferon alpha
Because connecting, the nucleotides sequence list of target gene is as shown in 400 < of SEQUENCE LISTING, 2 > or such as SEQUENCE
Shown in 400 < of LISTING, 3 >;
(2) target gene after connection is connected on pET-32a plasmid and obtains expression vector;
(3) expression vector is imported into e. coli host cell, genetic engineering bacterium pET-32a/rIL2- can be obtained
IFNα。
The e. coli host cell is BL21 (DE3) competent cell or BL21 (DE3) sense with pGro7 plasmid
By state cell.
The method of the purifying is:The crude product of fusion protein is successively through affinity chromatography, anion-exchange chromatography and molecular sieve
Chromatographic purifying.
BL21 (DE3) competent cell with pGro7 plasmid is purchased from Shanghai Jinan Technology Co., Ltd./glad hundred promise
Biology, article No. V205.
The acquisition methods of the genome 1 are:
A. design of primers
The primer sequence of canine leucocyte interleukin 2 (IL-2) is:
Upstream IL-2-F1:CGGGATCCATGTACAAAATGCAACTC has BamHI restriction enzyme site;
Downstream IL-2-R1:
ACCACCACCAGAACCACCACCACCAGTCAGTGTTGAGAAG, with flexible linker;
The primer sequence of dog interferon alpha (IFN-α) is:
Upstream IFN-α-F1:
GGTGGTTCTGGTGGTGGTGGTTCTATGGCCCTGCCC, with flexible linker;
Downstream IFN-α-R1:
CCCTCGAGTTTCCTCCTCCTTACT has XhoI restriction enzyme site;
B. RNA is extracted from dog liver, the target gene of IL-2 and IFN-α, the gene sequence of the two is obtained by reverse transcription
Column are respectively as shown in 400 < of SEQUENCE LISTING 400 <, 4 > and SEQUENCE LISTING, 5 >;
Respectively using IL-2 and the target gene of IFN-α as template, and be utilized respectively the upstream and downstream primer of IL-2 and IFN-α into
Row PCR amplification respectively obtains and connects the IL-2 of flexible linker and the target gene of IFN-α.
PCR reaction system and condition are:In the overall reaction system of 25 μ L, 1.5 μ L of template ribonucleic acid, upstream and downstream primer is each
0.5 μ L, 2.5 μ L, dNTP Mix of reverse transcriptase are 10 μ L, add RNase Free water to 25 μ L;The reaction of the RT-PCR reaction
Condition is:50 DEG C of reverse transcriptions 30min, 95 DEG C of initial denaturation 4min, into circulation;95 DEG C of denaturation 45s, 58 DEG C of annealing 45s, 72 DEG C are prolonged
1kb/min is stretched, is recycled 35 times, last 72 DEG C of extensions 10min.
C. IL-2 gene and IFN-α gene are connected using flexible linker
The PCR reaction system and reaction condition of connection be:In the overall reaction system of 25 μ L, connect flexible linker's
1 μ L of IL-2 template DNA, connects 1 μ L, IL-2 upstream primer of IFN-α template DNA, the 0.5 μ L of flexible linker, under IFN-α
Trip 0.5 μ L, Taq archaeal dna polymerase of primer, 2.5 μ L, dNTP Mix is 9 μ L, adds RNase Free water to 25 μ L;It is anti-to connect PCR
The condition is answered to be:95 DEG C of initial denaturation 4min, into circulation:94 DEG C of denaturation 45s;58 DEG C of annealing 45s, 72 DEG C of extension 1kb/min, altogether
35 circulations;Last 72 DEG C of extensions 10min.
Genome 2 is artificial synthesized gene after optimizing to genome 1, the acquisition methods of the genome 2
For:
A. design of primers
The primer sequence of canine leucocyte interleukin 2 (IL-2) is:
Upstream IL-2-F2:CGGGATCCATGTACAAAATGCAGC has BamHI restriction enzyme site;
Downstream IL-2-R2:
ACCACCACCAGAACCACCACCACCGGTCAGGGTAGAGAA, with flexible linker;
The primer sequence of dog interferon alpha (IFN-α) is:
Upstream IFN-α-F2:
GGTGGTTCTGGTGGTGGTGGTTCTATGGCTCTGCCGT, with flexible linker;
Downstream IFN-α-R2:
CCCTCGAGTTTACGACGACGAAC has XhoI restriction enzyme site;
B. the target gene of the IL-2 and IFN-α, the gene order of the two is respectively such as SEQUENCE LISTING 400
Shown in 6 > and SEQUENCE LISTING of <, 400 <, 7 >;
Respectively using IL-2 and the target gene of IFN-α as template, and be utilized respectively the upstream and downstream primer of IL-2 and IFN-α into
Row PCR amplification, the target gene of IL-2 and IFN-α after respectively obtaining the optimization for connecting flexible linker.
PCR reaction system and condition are:In the overall reaction system of 25 μ L, 1.0 μ L of genomic DNA, upstream and downstream primer is each
0.5 μ L, Taq DNA polymerase, 2.5 μ L, dNTP Mix is 10 μ L, adds RNase Free water to 25 μ L;The RT-PCR reaction
Reaction condition be:95 DEG C of initial denaturation 4min, into circulation;95 DEG C of denaturation 45S, 60 DEG C of annealing 45S, 72 DEG C of extension 1kb/
Min is recycled 35 times, last 72 DEG C of extensions 10min.
C. IL-2 gene and IFN-α gene are connected using flexible linker
The PCR reaction system and reaction condition of connection be:In the overall reaction system of 25 μ L, connect flexible linker's
1 μ L of IL-2 template DNA, connects 1 μ L, IL-2 upstream primer of IFN-α template DNA, the 0.5 μ L of flexible linker, under IFN-α
Trip 0.5 μ L, Taq archaeal dna polymerase of primer, 2.5 μ L, dNTP Mix is 9 μ L, adds RNase Free water to 25 μ L;It is anti-to connect PCR
The condition is answered to be:95 DEG C of initial denaturation 4min, into circulation:94 DEG C of denaturation 45s;60 DEG C of annealing 45s, 72 DEG C of extension 1kb/min, altogether
35 circulations;Last 72 DEG C of extensions 10min.
The present invention also provides the application of the canine recombinant long-acting interferon, long half time had up to 43 hours or more
Broad-spectrum disease resistance toxic action and the immune response that dog itself can be improved.
Compared with prior art, the present invention has the advantages that:
1. dog IL-2 and dog interferon alpha gene are realized fusion by flexibility linker, interferon half-life period is improved, with
Plain interferon is compared, and improves 10 times or more;Compared with common polyethylene glycol fused interferon, cost is significantly reduced.
2. improving dog IL-2 and dog interferon alpha fusion protein by optimizing to dog IL-2 and dog interferon alpha gene
Expression quantity.
3. using recombination bacillus coli BL21/pET-32a-IL2-IFN α as expression bacterial strain, by introducing molecular chaperones
PGro7 plasmid does not generate inclusion body in protein expression, forms soluble protein, avoids the mistake of inclusion body denaturation and renaturation
Journey substantially reduces the time of expressing fusion protein;
4. the wide spectrum that the fusion protein disclosed by the invention being made of dog IL-2 and dog interferon alpha not only has interferon-' alpha '
Antivirus action, while significantly improving the immune response of dog itself.
Detailed description of the invention
Fig. 1 is the result of 2 gene of canine leucocyte interleukin and dog interferon alpha gene RT-PCR amplification in embodiment 1;Swimming lane
M:DNA Marker DL2000;Swimming lane 1:Dog interleukin-22 RT-PCR amplified production;Swimming lane 2:Dog interferon alpha gene RT-
Pcr amplification product;
Fig. 2 is the result of the PCR amplification after the dog IL-2 in embodiment 1 is connected with the target gene of dog IFN-α;Swimming lane
M:DNA Marker DL2000;Swimming lane 1:Dog interferon alpha gene and dog interleukin-22 gene ligation amplification product;
Fig. 3 is the PCR amplification and double digestion qualification result of the positive colony plasmid in embodiment 1;Swimming lane M:DNA
Marker DL10000;Swimming lane 1:Recombinant plasmid double digestion result;Swimming lane 2:Plasmid PCR result;
Fig. 4 is the SDS-PAGE electrophoretic examinations result of the recombinant protein in embodiment 1;Swimming lane M:Albumen Marker;Swimming lane
1:Empty bacterium control;Swimming lane 2:It is precipitated after bacterial cell disruption after recombinant bacterium induction;Swimming lane 3:After bacterial cell disruption after recombinant bacterium induction
Supernatant;
Fig. 5 is the Western Blot qualification result for the fusion protein that embodiment 1 obtains;Swimming lane M:Albumen Marker;Swimming
Road 1:Empty bacterium control;Swimming lane 1:It is precipitated after recombinant bacterium induction is broken;Swimming lane 2:For the broken rear supernatant of recombinant bacterium induction;
Fig. 6 is that the canine recombinant long-acting interferon α as made from the fusion protein in embodiment 1 causes cell to VSV in embodiment 5
The inhibiting effect of lesion;1 is VSV virus control wells;2 be HEp-2 cell control well;A3-12 is gradient dilution (from right to left)
Human interferon standard items handle hole;B3-12 is that the recombinant long-acting dog interferon alpha of gradient dilution (from right to left) handles hole;
Fig. 7 is the canine recombinant long-acting interferon α intramuscular injection blood as made from the fusion protein in embodiment 1 in embodiment 8
Concentration-time changing curve;
Specific embodiment
Embodiment 1
A kind of fusion protein being made of canine leucocyte interleukin 2 and dog interferon alpha, preparation method are as follows:
1. the acquisition and amplification of canine leucocyte interleukin 2 (IL-2) and dog interferon alpha (IFN-α) target gene
Design of primers:
Synthetic primer is designed according to objective gene sequence reported in Genebank and is shown in Table 1, in the upstream of dog interleukin-22
BamHI restriction enzyme site and Linker sequence are introduced in primer and downstream primer respectively, dog interferon alpha upstream primer and under
Linker sequence and XhoI restriction enzyme site are introduced respectively in trip primer.
1 PCR amplification primer of table
RT-PCR obtains target gene:
RNA is extracted from dog liver organization, the target gene of IL-2 and IFN-α, the gene of the two are obtained by reverse transcription
Sequence is respectively as shown in 400 < of SEQUENCE LISTING 400 <, 4 > and SEQUENCE LISTING, 5 >;
RT-PCR reaction system (25 μ L) is shown in Table 2
2 RT-PCR reaction system of table
RNase Free water | 10μL |
dNTP Mix | 10μL |
Reverse transcriptase | 2.5μL |
Upstream and downstream primer | Each 0.5 μ L |
Geneome RNA | 1.5μL |
Response parameter is:95 DEG C of initial denaturation 4min, into circulation:95 DEG C of denaturation 45s;58 DEG C of annealing 45s, 72 DEG C of extensions
1kb/min, totally 35 recycle;Last 72 DEG C of extensions 10min.
There is specific band in 490bp and 590bp or so through agarose gel electrophoresis in RT-PCR amplified production, and result is such as
Shown in Fig. 1, illustrate to have respectively obtained the dog IL-2 of the flexible linker of connection and the target gene of IFN-α.
2. the connection of target gene
Target gene is diluted to 10ug/mL, connects even section target gene using over-lap PCR, 25 μ L reaction systems are such as
Shown in table 3:
3 PCR reaction system of table
Response parameter is:95 DEG C of initial denaturation 4min, into circulation:94 DEG C of denaturation 45s;58 DEG C of annealing 45s, 72 DEG C of extensions
1kb/min, totally 35 recycle;Last 72 DEG C of extensions 10min.
There is specific band in 1050bp or so through agarose gel electrophoresis in pcr amplification product, result as shown in Fig. 2,
Occurs the band of dog interleukin-22 amplified production and dog interferon alpha amplified production in Fig. 2, this is because in dog interleukin-22 base
During cause is connected with dog interferon alpha gene, there is non-specific responding.The nucleotide sequence of obtained target gene is such as
Shown in 400 < of SEQUENCE LISTING, 2 >.
3. expression vector establishment
The PCR glue recovery product for selecting the target gene after connecting errorless after being sequenced is used with pET-32a plasmid
BamHI and XhoI restriction enzyme carries out double digestion and recycling, does double digestion by 20 μ L systems in table 4:
4 double digestion system of table
General buffer | 2μL |
Restriction enzyme (a pair) | 1μL+1μL |
Carrier or recycling segment | 2ul |
RNase Free water | 14μL |
The digestion recovery product of target gene and pET-32a plasmid after connection is attached by the system in table 5,4
DEG C overnight connection:
Table 5
Target fragment DNA | 10μL |
Expression vector | 3μL |
Buffer | 2μL |
Ligase | 1μL |
RNase Free water | 4μL |
Connection product is converted into e. coli bl21 (DE3) competent cell, competence is coated on the mould of benzyl containing ammonia
The LB culture medium flat plate overnight incubation of element;The bacterium colony grown on LB plate is taken to identify target gene, positive colony mycoplasma through PCR
Grain identifies that being accredited as positive indicates expression vector establishment success through BamHI and XhoI double digestion, obtain engineering bacteria pET-
There is single band at the place 1050bp or so through agarose gel electrophoresis in 32a/rIL2-IFN α PCR amplification and double enzyme digestion product,
Its result is as shown in Figure 3.
4. the expression of recombinant protein
Picking engineering bacteria pET-32a/rIL2-IFN α shakes bacterium for 37 DEG C in the LB culture medium of the 100 μ g/ml containing ampicillin
1h recovery engineering bacteria activity;In LB culture medium (the 100 μ g/ml containing ampicillin) after amplification culture 4h, surveys OD value and reach
When 1.0;IPTG, 32 DEG C of inducing expression (100 μ g/ml of final concentration) 5h is added;Bacterium is collected, is examined through SDS-PAGE electrophoresis
Survey, result as shown in figure 4, it can be seen from the figure that recombinant bacterium induction 5h after bacterial cell disruption after supernatant be deposited in
Visible predominant expression band at 56.8KD illustrates in precipitating and successful expression equal in supernatant fusion protein.
Mass volume ratio 1 is added:Precipitating is resuspended in 1 PBS;- 20 DEG C precipitate 3 times in room temperature multigelation;4 DEG C of ultrasound cracking
Bacterial precipitation, work 10s, is spaced 3S, ultrasonic 6min, and whole process repeats 3~4 times;4 DEG C, 12000r/min is centrifuged 15min,
Supernatant, inclusion body (inclusion body is through dissolution, refolding strategy) are taken respectively, obtain crude fusion protein.
5. fusion protein purification
5.1His affinity chromatography
After membrane filtration of the crude fusion protein with 0.22 μm of aperture, loading is by being connected to AKTA explorer 100
On protein purification system, the His affinity column balanced with Binding Buffer I (PBS) is washed away not with PBS buffer solution
In conjunction with albumen, until A280nm stablizes, then with Elution buffer I (50mM trishydroxymethylaminomethane, 20~500mM
Imidazoles, PH8.0) elution, collect rIL2-IFN α protein peak.
5.2DEAE anion-exchange chromatography
By the albumen collected after His affinitive layer purification displacement to (the 50mM trihydroxy methyl of Binding Buffer II
Aminomethane, PH6.5) in after, loading by the DEAE anion exchange chromatography that has been balanced with Binding Buffer II,
After being stablized again with the column of Binding Buffer II to A280nm value, with (the 50mM trihydroxy methyl amino of Elution Buffer II
Methane, 1M NaCl, PH6.5) linear gradient elution, collect rIL2-IFN α protein peak.
5.3 sieve chromatography
Loading is by with Binding Buffer III after the sample concentration that ion-exchange chromatography is collected into
(50mMNa2HPO4,0.15M NaCl, PH7.4) has balanced 200 molecular sieve chromatography of Superdex, uses Binding
Buffer III is eluted, and collects rIL2-IFN α protein peak.
5.4 sample identification
Measure rIL2-IFN α potency and specific activity, specific activity >=1.0 × 105U/mg albumen is qualification;It is aseptic subpackaged, -80
DEG C save.The fusion protein being made of canine leucocyte interleukin 2 and dog interferon alpha can be obtained, amino acid sequence is such as
Shown in 400 < of SEQUENCE LISTING, 1 >.
Embodiment 2
A kind of fusion protein being made of canine leucocyte interleukin 2 and dog interferon alpha, other, only will wherein with embodiment 1
E. coli bl21 (DE3) competent cell replacement in order to have pGro7 plasmid BL21 (DE3) competent cell.It melts
The SDS-PAGE electrophoresis result of hop protein is compareed with embodiment 1, and 56.8KD or so place's predominant expression band is thicker in supernatant,
Illustrate after introducing molecular chaperones pGro7, expression of the destination protein in supernatant is more preferable, and obtained fusion protein amount is higher.Greatly
The albumen of enterobacteria expression is mostly present in inclusion body;By introducing molecular chaperones, coordinate expression egg in expression bacterial strain
White correct folding, reaches solubility expression of protein.
BL21 (DE3) competent cell with pGro7 plasmid is purchased from Shanghai Jinan Technology Co., Ltd./glad hundred promise
Biology, article No. V205.
Embodiment 3
A kind of fusion protein being made of canine leucocyte interleukin 2 and dog interferon alpha, preparation method are as follows:
1. the acquisition and amplification of canine leucocyte interleukin 2 (IL-2) and dog interferon alpha (IFN-α) target gene
To in embodiment 1 IL-2 and IFN-α optimize, artificial synthesized IL-2 and IFN-α target gene, after optimization,
The nucleotide sequence of the two is respectively as shown in 400 < of SEQUENCE LISTING 400 <, 6 > and SEQUENCE LISTING, 7 >.
1.1 codon optimization
Genetic codon has 64 kinds, but most biological tendencies are in utilizing a part in these codons.Those
By the most frequent referred to as best codon (optimal codons) utilized, what those were not frequently utilized that is known as rare or sharp
With the low codon of rate (rare or low-usage codons).In fact, common every kind of life for doing protein expression or production
Object (including Escherichia coli, yeast, mammalian cell, plant cell and insect cell) all shows password to a certain degree
The difference or preference that son utilizes.It is obviously high to the expression efficiency of the gene containing best codon in Escherichia coli, yeast and drosophila
In the expression efficiency of the gene of the codon containing poor efficiency.Therefore, in heterologous expression system, the preferences of codon are very
The expression of recombinant protein is affected in big degree.Using preference codon (preferred codons) and avoid using rare
Codon carries out gene chemical synthesis, and the redesign of this gene is codon optimization.Therefore, in the present embodiment to the IL-2 of dog and
IFN-α gene codon optimizes.
Interpretation of result after 1.2 codon optimizations
It is optimal efficient in the expression system to be considered the gene when usual codon adaptation indexI (CAI)=1.0
Expression status, CAI value is lower to show that expression is lower in host.In gene G/C content most ideal distribution range be 30~
70%, it is more than that the range will affect translation and transcriptional efficiency in any region.Using software detection discovery dog IL-2 and
The codon of IFN-α original gene codon adaptation indexI (CAI) in Escherichia coli is respectively 0.19,0.27, GC percentage
It is 39.6%, 59.7%;And by obtaining recombination genome 2 in Escherichia coli after dog IL-2 and IFN-α gene optimization
Middle codon adaptation indexI (CAI) is 1.0,1.0, GC percentage 48.8%, 55.6%.It is significantly reduced by gene optimization
The utilization rate of low codon avoids influence of the rare codon to protein expression, improves the G/C content of gene, improve
Transcription and translation efficiency, and then improve the expression quantity of recombinant protein.
1.3 design of primers:
6 PCR amplification primer of table
The genomic DNA of IL-2 and IFN-α after optimization are diluted to 0.05mg/mL respectively.Mesh is obtained using PCR amplification
Gene, 25 μ L reaction systems are as shown in table 7:
7 PCR reaction system of table
RNase Free water | 10.5μL |
dNTP Mix | 10.0μL |
Taq archaeal dna polymerase | 2.5μL |
Upstream and downstream primer | Each 0.5 μ L |
Genomic DNA | 1.0μL |
Response parameter is:95 DEG C of initial denaturation 4min, into circulation:95 DEG C of denaturation 45s;60 DEG C of annealing 45s, 72 DEG C of extensions
1kb/min, totally 35 recycle;Last 72 DEG C of extensions 10min.
IL-2 and the pcr amplification product of IFN-α occur in 490bp and 590bp or so special respectively through agarose gel electrophoresis
Different band illustrates the target gene for having respectively obtained the dog IL-2 after the optimization of the flexible linker of connection and IFN-α.
2. the connection of target gene
Target gene is diluted to 10ug/mL, connects target gene, 25 μ L reaction systems such as 8 institute of table using over-lap PCR
Show:
8 PCR reaction system of table
Response parameter is:95 DEG C of initial denaturation 4min, into circulation:94 DEG C of denaturation 45s;60 DEG C of annealing 45s, 72 DEG C of extensions
1kb/min, totally 35 recycle;Last 72 DEG C of extensions 10min.
There is specific band in 1050bp or so through agarose gel electrophoresis in pcr amplification product, illustrates successfully to have obtained IL-
2 connected with IFN-α after target gene.The nucleotide sequence of obtained target gene such as 400 < of SEQUENCE LISTING, 3 >
It is shown.
3. expression vector establishment
The glue recovery product for the PCR for selecting the target gene after connecting errorless after being sequenced is used with pET-32a plasmid
BamHI, XhoI restriction enzyme carry out double digestion and recycling, do double digestion by 20 μ L systems in table 9:
9 double digestion system of table
General buffer | 2μL |
Restriction enzyme (a pair) | 1μL+1μL |
Carrier or recycling segment | 2ul |
RNase Free water | 14μL |
The digestion recovery product of target gene and pET-32a plasmid after connection is attached by the system in table 10,4
DEG C overnight connection:
Table 10
Target fragment DNA | 10μL |
Expression vector | 3μL |
buffer | 2μL |
Ligase | 1μL |
RNase Free water | 4μL |
Connection product is converted into e. coli bl21 (DE3) competent cell, competence is coated on the mould of benzyl containing ammonia
The LB culture medium flat plate overnight incubation of element;The bacterium colony grown on LB plate is taken to identify target gene, positive colony mycoplasma through PCR
Grain identifies that being accredited as positive indicates expression vector establishment success, and PCR amplification and double digestion are produced through BamHI, XhoI double digestion
There is single band through agarose gel electrophoresis at 1050bp in object, illustrates the target gene after connecting containing IL-2 with IFN-α
Expression vector establishment success, obtained engineering bacteria pET-32a/rIL2-IFN α.
4. the expression of recombinant protein
Picking engineering bacteria pET-32a/rIL2-IFN α shakes bacterium for 37 DEG C in the LB culture medium of the 100 μ g/ml containing ampicillin
1h recovery engineering bacteria activity;In LB culture medium (the 100 μ g/ml containing ampicillin) after amplification culture 4h, surveys OD value and reach
When 1.0;IPTG, 32 DEG C of inducing expression (100 μ g/ml of final concentration) 5h is added;Bacterium is collected, is examined through SDS-PAGE electrophoresis
It surveys, supernatant is deposited in the visible predominant expression band in the place 56.8KD or so after the bacterial cell disruption after recombinant bacterium induction 5h, illustrates
Recombinant protein has been obtained in supernatant precipitating.
Mass volume ratio 1 is added:Precipitating is resuspended in 1 PBS;- 20 DEG C precipitate 3 times in room temperature multigelation;4 DEG C of ultrasound cracking
Bacterial precipitation, work 10s, is spaced 3S, ultrasonic 6min, and whole process repeats 3~4 times;4 DEG C, 12000r/min is centrifuged 15min,
Supernatant, inclusion body (inclusion body is through dissolution, refolding strategy) are taken respectively, obtain crude fusion protein.
5. fusion protein purification
5.1His affinity chromatography
After membrane filtration of the crude fusion protein with 0.22 μm of aperture, loading is by being connected to AKTA explorer 100
On protein purification system, the His affinity column balanced with Binding Buffer I (PBS) is washed away not with PBS buffer solution
In conjunction with albumen, until A280nm stablizes, then with Elution buffer I (50mM trishydroxymethylaminomethane, 20~500mM
Imidazoles, PH8.0) elution, collect rIL2-IFN α protein peak.
5.2DEAE anion-exchange chromatography
By the albumen collected after His affinitive layer purification displacement to (the 50mM trihydroxy methyl of Binding Buffer II
Aminomethane, PH6.5) in after, loading by the DEAE anion exchange chromatography that has been balanced with Binding Buffer II,
After being stablized again with the column of Binding Buffer II to A280nm value, with (the 50mM trihydroxy methyl amino of Elution Buffer II
Methane, 1M NaCl, PH6.5) linear gradient elution, collect rIL2-IFN α protein peak.
5.3 sieve chromatography
Loading is by with Binding Buffer III after the sample concentration that ion-exchange chromatography is collected into
(50mMNa2HPO4,0.15M NaCl, PH7.4) has balanced 200 molecular sieve chromatography of Superdex, uses Binding
Buffer III is eluted, and collects rIL2-IFN α protein peak.
5.4 sample identification
Measure rIL2-IFN α potency and specific activity, specific activity >=1.0 × 105U/mg albumen is qualification;It is aseptic subpackaged, -80
DEG C save.The fusion protein being made of canine leucocyte interleukin 2 and dog interferon alpha can be obtained, amino acid sequence is such as
Shown in 400 < of SEQUENCE LISTING, 1 >.
Embodiment 4
A kind of fusion protein being made of canine leucocyte interleukin 2 and dog interferon alpha, other, only will wherein with embodiment 3
E. coli bl21 (DE3) competent cell replacement in order to have pGro7 plasmid BL21 (DE3) competent cell.It melts
The SDS-PAGE electrophoresis result of hop protein is compareed with embodiment 3, and 56.8KD or so place's predominant expression band is thicker in supernatant,
Illustrate after introducing molecular chaperones pGro7, expression of the destination protein in supernatant is more preferable, and obtained fusion protein amount is higher.Greatly
The albumen of enterobacteria expression is mostly present in inclusion body;By introducing molecular chaperones, coordinate expression egg in expression bacterial strain
White correct folding, reaches solubility expression of protein.
BL21 (DE3) competent cell with pGro7 plasmid is purchased from Shanghai Jinan Technology Co., Ltd./glad hundred promise
Biology, article No. V205.
Embodiment 5
A kind of canine recombinant long-acting interferon α, it is mixed with freeze drying protectant respectively by the fusion protein in embodiment 1,2,3,4
Later, freeze-dried to form.The freeze drying protectant is glycerol, mannitol and sucrose, is buffering with 10mmol/L PBS
Liquid, final concentration of glycerol 100mL/L, mannitol 0.12g/mL and the sucrose 0.025g/mL of three.
Embodiment 6
Examples 1 to 4 obtains the identification for the fusion protein being made of canine leucocyte interleukin 2 and dog interferon alpha
The quantitative detection of 6.1 protein contents
With Lowry method, standard test is made with the standard protein of Chinese food pharmaceutical biological product calibrating institute, measures embodiment
1~4 obtained fusion protein concentration is respectively 0.5mg/ml 0.5mg/ml, 0.6mg/ml, 0.6mg/ml.
6.2SDS-PAGE electrophoresis detection
Compared with empty bacterium, fusion protein has the newly-increased protein band of a dense dye in 56.8KD or so, as shown in Figure 4.
6.3Western Blot result
Fusion protein in Examples 1 to 4 is detected, respectively with the anti-canine interferon alpha (1 of abcam company mouse:5000 dilutions)
For primary antibody, using goat anti-mouse IgG-HRP as secondary antibody (1:10000 dilutions).Recombinant long-acting dog interferon alpha sample can be with anti-dog
Specific reaction occurs for interferon-' alpha ' monoclonal antibody, and specific band occurs in the place 56.8KD or so, as shown in Figure 5.
Embodiment 7
Four parts of canine recombinant long-acting interferon α in embodiment 5 freeze-dried bioactivity
Inhibit method according to few cells lesion, Hep-2 cell is made into 5 × 10 with culture medium5Cell/ml cell suspends
Liquid, every hole inoculation 0.1ml move into 96 porocyte culture plates.37 DEG C, 5%CO2For 24 hours, the recombinant long-acting of various dose is added in culture
Dog interferon alpha is inhaled afterwards abandon for 24 hours, then is inoculated with 100TCID respectively50VSV virus.
Test result
The result shows that the canine recombinant long-acting interferon α obtained causes the lesion of HEp-2 cell to have apparent inhibit VSV
Effect.The lesions such as there is cell rounding, falls off, is disintegrated after untreated cell inoculation virus.And the canine recombinant obtained is long
After imitating interferon-' alpha ' treated cell inoculation virus, it is observed continuously under inverted microscope, cellular morphology is normal, not incumbent out
What lesion, measures potency >=1 × 105U/ml, as shown in Figure 6.
Embodiment 8
Being lyophilized respectively by four parts of canine recombinant long-acting interferon α that the fusion protein of Examples 1 to 4 obtains in embodiment 5
Measurement of the agent (being denoted as A, B, C, D respectively) in dog intracorporal half-life period
The blood concentration and time relationship of cytopathic-effect inhibition assay measurement rIL2-IFN α
The dog (half male and half female) that six weight are roughly the same is taken, 2mg/ml canine recombinant long-acting interferon α is subcutaneously injected in neck
Freeze-dried 2ml, respectively 1h, 2h, 4h, 8h, 16h, for 24 hours, 36h, 48h, 79h venous blood collection, 4 DEG C of blood sample solidification, 3500rpm
Low-temperature centrifugation 10min separates serum, and every dog blood sample of each time point is to be measured in -20 DEG C of preservations.It is measured using cytopathic-effect inhibition assay
The concentration of rIL2-IFN α in blood serum sample is carried out curve fitting and calculating parameter with DAS pharmacokinetics software.The matched curve of A
See Fig. 7, each parameter calculated result is shown in Table 11.
Dominant dynamic parameters in serum after 11 canine recombinant long-acting interferon α intramuscular injection of table
The result shows that canine recombinant long-acting interferon α has longer half-life period.Half-life period can reach 43h or so after measured, compared with
Plain interferon improves about 10 times.
Embodiment 9
The freeze-dried measurement that canine cells immune response is influenced of four parts of canine recombinant long-acting interferon α in embodiment 5
It takes six roughly the same dogs of weight to be divided into two groups, is denoted as experimental group and control group;The subcutaneous injection of experimental group neck
The PBS of 2mL is subcutaneously injected in the 2mg/ml canine recombinant long-acting interferon freeze-dried 2ml of α, control group neck, after taking injection 4 weeks outside dog
All blood takes weekly a blood later, separates lymphocyte using lymphocyte separation medium, lymphocyte passes through serum-free RPMI
It after 1640 culture mediums wash 2 times, is resuspended with complete medium, adjustment cell concentration is 2 × 106A/ml, 24 porocyte culture plates
Every hole addition 1ml lymphocyte, 37 DEG C, 5%CO272h is cultivated, supernatant when collecting lymphocyte culture.ELISA detection culture
IL-4, IFN-γ content in supernatant, are carried out by kit specification, and testing result is as shown in table 12:
It is horizontal that 12 ELISA of table detects each group canine cells immune response
The result shows that injection canine recombinant long-acting interferon α after, can significantly improve cell factor IL-4 in canine peripheral blood,
The content of IFN-γ enhances cellullar immunologic response level, significantly improves immunity level.
It is above-mentioned to canine recombinant long-acting interferon and to prepare fusion protein and its preparation of this long-acting interferon referring to embodiment
The detailed description that method carries out, is illustrative without being restrictive, can enumerate several implementations according to limited range
Example, therefore the change and modification in the case where not departing from present general inventive concept, should belong within protection scope of the present invention.
SEQUENCE LISTING
<110>Wuhu Co., Ltd of Ying Tefeier biological products industrial research institute
<120>A kind of canine recombinant long-acting interferon and the fusion protein for preparing this long-acting interferon and preparation method thereof
<130> 1
<160> 7
<170> PatentIn version 3.3
<210> 1
<211> 352
<212> PRT
<213>Canine recombinant long-acting interferon fusion protein
<400> 1
Met Tyr Lys Met Gln Leu Leu Ser Cys Ile Ala Leu Thr Leu Val Leu
1 5 10 15
Val Ala Asn Ser Ala Pro Ile Thr Ser Ser Ser Thr Lys Glu Thr Glu
20 25 30
Gln Gln Met Glu Gln Leu Leu Leu Asp Leu Gln Leu Leu Leu Asn Gly
35 40 45
Val Asn Asn Tyr Glu Asn Pro Gln Leu Ser Arg Met Leu Thr Phe Lys
50 55 60
Phe Tyr Thr Pro Lys Lys Ala Thr Glu Phe Thr His Leu Gln Cys Leu
65 70 75 80
Ala Glu Glu Leu Lys Asn Leu Glu Glu Val Leu Gly Leu Pro Gln Ser
85 90 95
Lys Asn Val His Leu Thr Asp Thr Lys Glu Leu Ile Ser Asn Met Asn
100 105 110
Val Thr Leu Leu Lys Leu Lys Gly Ser Glu Thr Ser Tyr Asn Cys Glu
115 120 125
Tyr Asp Asp Glu Thr Ala Thr Ile Thr Glu Phe Leu Asn Lys Trp Ile
130 135 140
Thr Phe Cys Gln Ser Ile Phe Ser Thr Leu Thr Gly Gly Gly Gly Ser
145 150 155 160
Gly Gly Gly Gly Ser Met Ala Leu Pro Cys Ser Phe Ser Val Ala Leu
165 170 175
Val Leu Leu Ser Cys His Ser Leu Cys Cys Leu Ala Cys Asp Leu Pro
180 185 190
Asp Thr His Ser Leu Arg Asn Trp Arg Val Leu Thr Leu Leu Gly Gln
195 200 205
Met Arg Arg Leu Ser Ala Ser Ser Cys Asp His Tyr Thr Thr Asp Phe
210 215 220
Ala Phe Pro Lys Glu Leu Phe Asp Gly Gln Arg Leu Gln Glu Ala Gln
225 230 235 240
Ala Leu Ser Val Val His Val Met Thr Gln Lys Val Phe His Leu Phe
245 250 255
Cys Thr Asn Met Ser Ser Ala Pro Trp Asn Met Thr Leu Leu Gly Glu
260 265 270
Leu Cys Ser Gly Leu Ser Glu Gln Leu Asp Asp Leu Asp Ala Cys Pro
275 280 285
Leu Gln Glu Ala Gly Leu Ala Glu Thr Pro Leu Met His Glu Asp Ser
290 295 300
Thr Leu Arg Thr Tyr Phe Gln Arg Ile Ser Leu Tyr Leu Gln Asp Arg
305 310 315 320
Asn His Ser Pro Cys Ala Trp Glu Met Val Arg Ala Glu Ile Gly Arg
325 330 335
Ser Phe Phe Ser Leu Thr Ile Leu Gln Glu Arg Val Arg Arg Arg Lys
340 345 350
<210> 2
<211> 1056
<212> DNA
<213>Long-acting dog interferon genome 1
<400> 2
atgtacaaaa tgcaactctt gtcttgcatc gcactgacgc ttgtacttgt cgcaaacagt 60
gcacctatta cttcaagctc tacaaaggaa acagagcaac agatggagca attactgctg 120
gatttacagt tgcttttgaa tggagttaat aattatgaga acccccaact ctccaggatg 180
ctcacattta agttttacac gcccaagaag gccacagaat ttacacacct tcaatgtcta 240
gcagaagaac tcaaaaacct ggaggaagtg ctaggtttac ctcaaagcaa aaacgttcac 300
ttgacagaca ccaaggaatt aatcagcaat atgaatgtaa cacttctgaa actaaaggga 360
tctgaaacaa gttacaactg tgaatatgat gacgagacag caaccattac agaatttctg 420
aacaaatgga ttaccttttg tcaaagcatc ttctcaacac tgactggtgg tggtggttct 480
ggtggtggtg gttctatggc cctgccctgc tccttctcgg tggccctggt gctgctcagc 540
tgccactccc tgtgctgtct ggcttgcgac ctgcccgaca cccacagcct gcgcaactgg 600
agggtcctga cgctcctggg acagatgagg agactctccg ccagctcttg tgaccactac 660
accactgact ttgccttccc caaggaactg tttgatggcc agcggctcca ggaggcgcaa 720
gccctctctg tggtccacgt gatgacccag aaggtcttcc acctcttctg cacgaacatg 780
tcctctgctc cttggaacat gaccctcctg ggggaattgt gctcggggct ctctgagcag 840
ctggatgacc tggatgcctg tcccctgcag gaggcagggc tggccgagac ccccctcatg 900
catgaagact ccaccctgag gacctacttc caaaggatct ccctctacct gcaagacagg 960
aaccacagcc cgtgtgcctg ggagatggtc cgagcagaaa tcgggagatc cttcttctcc 1020
ttgaccatct tgcaagaaag agtaaggagg aggaaa 1056
<210> 3
<211> 1056
<212> DNA
<213>Long-acting dog interferon genome 2
<400> 3
atgtacaaaa tgcagctgct gtcttgcatc gctctgaccc tggttctggt tgctaactct 60
gctccgatca cctcttcttc taccaaagaa accgaacagc agatggaaca gctgctgctg 120
gacctgcagc tgctgctgaa cggtgttaac aactacgaaa acccgcagct gtctcgtatg 180
ctgaccttca aattctacac cccgaaaaaa gctaccgaat tcacccacct gcagtgcctg 240
gctgaagaac tgaaaaacct ggaagaagtt ctgggtctgc cgcagtctaa aaacgttcac 300
ctgaccgaca ccaaagaact gatctctaac atgaacgtta ccctgctgaa actgaaaggt 360
tctgaaacct cttacaactg cgaatacgac gacgaaaccg ctaccatcac cgaattcctg 420
aacaaatgga tcaccttctg ccagtctatc ttctctaccc tgaccggtgg tggtggttct 480
ggtggtggtg gttctatggc tctgccgtgc tctttctctg ttgctctggt tctgctgtct 540
tgccactctc tgtgctgcct ggcttgcgac ctgccggaca cccactctct gcgtaactgg 600
cgtgttctga ccctgctggg tcagatgcgt cgtctgtctg cttcttcttg cgaccactac 660
accaccgact tcgctttccc gaaagaactg ttcgacggtc agcgtctgca ggaagctcag 720
gctctgtctg ttgttcacgt tatgacccag aaagttttcc acctgttctg caccaacatg 780
tcttctgctc cgtggaacat gaccctgctg ggtgaactgt gctctggtct gtctgaacag 840
ctggacgacc tggacgcttg cccgctgcag gaagctggtc tggctgaaac cccgctgatg 900
cacgaagact ctaccctgcg tacctacttc cagcgtatct ctctgtacct gcaggaccgt 960
aaccactctc cgtgcgcttg ggaaatggtt cgtgctgaaa tcggtcgttc tttcttctct 1020
ctgaccatcc tgcaggaacg tgttcgtcgt cgtaaa 1056
<210> 4
<211> 465
<212> DNA
<213>Dog IL-2
<400> 4
atgtacaaaa tgcaactctt gtcttgcatc gcactgacgc ttgtacttgt cgcaaacagt 60
gcacctatta cttcaagctc tacaaaggaa acagagcaac agatggagca attactgctg 120
gatttacagt tgcttttgaa tggagttaat aattatgaga acccccaact ctccaggatg 180
ctcacattta agttttacac gcccaagaag gccacagaat ttacacacct tcaatgtcta 240
gcagaagaac tcaaaaacct ggaggaagtg ctaggtttac ctcaaagcaa aaacgttcac 300
ttgacagaca ccaaggaatt aatcagcaat atgaatgtaa cacttctgaa actaaaggga 360
tctgaaacaa gttacaactg tgaatatgat gacgagacag caaccattac agaatttctg 420
aacaaatgga ttaccttttg tcaaagcatc ttctcaacac tgact 465
<210> 5
<211> 561
<212> DNA
<213>Dog IFN-α
<400> 5
atggccctgc cctgctcctt ctcggtggcc ctggtgctgc tcagctgcca ctccctgtgc 60
tgtctggctt gcgacctgcc cgacacccac agcctgcgca actggagggt cctgacgctc 120
ctgggacaga tgaggagact ctccgccagc tcttgtgacc actacaccac tgactttgcc 180
ttccccaagg aactgtttga tggccagcgg ctccaggagg cgcaagccct ctctgtggtc 240
cacgtgatga cccagaaggt cttccacctc ttctgcacga acatgtcctc tgctccttgg 300
aacatgaccc tcctggggga attgtgctcg gggctctctg agcagctgga tgacctggat 360
gcctgtcccc tgcaggaggc agggctggcc gagacccccc tcatgcatga agactccacc 420
ctgaggacct acttccaaag gatctccctc tacctgcaag acaggaacca cagcccgtgt 480
gcctgggaga tggtccgagc agaaatcggg agatccttct tctccttgac catcttgcaa 540
gaaagagtaa ggaggaggaa a 561
<210> 6
<211> 465
<212> DNA
<213>Dog IL-2
<400> 6
atgtacaaaa tgcagctgct gtcttgcatc gctctgaccc tggttctggt tgctaactct 60
gctccgatca cctcttcttc taccaaagaa accgaacagc agatggaaca gctgctgctg 120
gacctgcagc tgctgctgaa cggtgttaac aactacgaaa acccgcagct gtctcgtatg 180
ctgaccttca aattctacac cccgaaaaaa gctaccgaat tcacccacct gcagtgcctg 240
gctgaagaac tgaaaaacct ggaagaagtt ctgggtctgc cgcagtctaa aaacgttcac 300
ctgaccgaca ccaaagaact gatctctaac atgaacgtta ccctgctgaa actgaaaggt 360
tctgaaacct cttacaactg cgaatacgac gacgaaaccg ctaccatcac cgaattcctg 420
aacaaatgga tcaccttctg ccagtctatc ttctctaccc tgacc 465
<210> 7
<211> 561
<212> DNA
<213>Dog IFN-α
<400> 7
atggctctgc cgtgctcttt ctctgttgct ctggttctgc tgtcttgcca ctctctgtgc 60
tgcctggctt gcgacctgcc ggacacccac tctctgcgta actggcgtgt tctgaccctg 120
ctgggtcaga tgcgtcgtct gtctgcttct tcttgcgacc actacaccac cgacttcgct 180
ttcccgaaag aactgttcga cggtcagcgt ctgcaggaag ctcaggctct gtctgttgtt 240
cacgttatga cccagaaagt tttccacctg ttctgcacca acatgtcttc tgctccgtgg 300
aacatgaccc tgctgggtga actgtgctct ggtctgtctg aacagctgga cgacctggac 360
gcttgcccgc tgcaggaagc tggtctggct gaaaccccgc tgatgcacga agactctacc 420
ctgcgtacct acttccagcg tatctctctg tacctgcagg accgtaacca ctctccgtgc 480
gcttgggaaa tggttcgtgc tgaaatcggt cgttctttct tctctctgac catcctgcag 540
gaacgtgttc gtcgtcgtaa a 561
Claims (10)
1. a kind of fusion protein being made of canine leucocyte interleukin 2 and dog interferon alpha, it is characterised in that:The fusion protein
Amino acid sequence table is as shown in 400 < of SEQUENCE LISTING, 1 >.
2. a kind of gene for encoding fusion protein as described in claim 1, which is characterized in that the nucleotide sequence of the gene
Table is denoted as genome 1 as shown in 400 < of SEQUENCE LISTING, 2 >;Or as shown in 400 < of SEQUENCE LISTING, 3 >,
It is denoted as genome 2.
3. containing the expression vector of gene as claimed in claim 2.
4. containing the genetic engineering bacterium of gene as claimed in claim 2.
5. a kind of canine recombinant long-acting interferon, which is characterized in that the canine recombinant long-acting interferon is melted by described in claim 1
It is freeze-dried to form after hop protein and freeze drying protectant mixture.
6. the preparation method of fusion protein according to claim 1, which is characterized in that the preparation method includes following step
Suddenly:Expression vector as claimed in claim 3 is imported into e. coli host cell, genetic engineering bacterium, genetic engineering are obtained
Bacterium obtains the crude product of the fusion protein after IPTG inducing expression, and fusion protein can be obtained after purified.
7. preparation method according to claim 6, which is characterized in that the genetic engineering bacterium is pET-32a/rIL2-IFN
α, preparation method are:
(1) design primer, is obtained or the canine leucocyte interleukin 2 of the flexible linker sequence of artificial synthesized connection by reverse transcription
With the target gene of dog interferon alpha;Canine leucocyte interleukin 2 is connected with the target gene of dog interferon alpha by flexible linker
Get up, the nucleotides sequence list of target gene is as shown in 400 < of SEQUENCE LISTING, 2 > or such as SEQUENCE LISTING
Shown in 400 <, 3 >;
(2) target gene after connection is connected on pET-32a plasmid and obtains expression vector;
(3) expression vector is imported into e. coli host cell, genetic engineering bacterium pET-32a/rIL2-IFN can be obtained
α。
8. preparation method according to claim 6 or 7, which is characterized in that the e. coli host cell is BL21
(DE3) competent cell or BL21 (DE3) competent cell with pGro7 plasmid.
9. preparation method according to claim 6 or 7, which is characterized in that the method for the purifying is:Fusion protein it is thick
Product are successively through affinity chromatography, anion-exchange chromatography and molecular sieve chromatography purification.
10. the application of canine recombinant long-acting interferon according to claim 5, which is characterized in that the Recombinant Swine is long-acting dry
The long half time of element is disturbed up to 43 hours or more, there is spectrum antivirus action and the immune response of pig itself can be improved.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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CN2017106763289 | 2017-08-09 | ||
CN201710676328.9A CN107337738A (en) | 2017-08-09 | 2017-08-09 | A kind of canine recombinant long-acting interferon and prepare fusion protein of this long-acting interferon and preparation method thereof |
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