CN108840935A - A kind of fusion protein being made of sheep albumin and sheep interferon gamma and preparation method thereof and a kind of recombination sheep long-acting interferon γ - Google Patents
A kind of fusion protein being made of sheep albumin and sheep interferon gamma and preparation method thereof and a kind of recombination sheep long-acting interferon γ Download PDFInfo
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Abstract
The invention discloses a kind of fusion protein being made of sheep albumin and sheep interferon gamma and preparation method thereof and a kind of recombination sheep long-acting interferon γ; the fusion protein is connect through flexible linker with sheep interferon gamma by sheep albumin and is formed, and is freeze-dried to obtain recombination sheep long-acting interferon γ after fusion protein and freeze drying protectant mixture.The recombination sheep long-acting interferon γ is remarkably improved the half-life period of sheep interferon, and the half-life period of more common sheep interferon gamma improves 12 times or more, and has broad-spectrum disease resistance toxic action and can improve the immune response of sheep itself.
Description
Technical field
The invention belongs to technical field of biological genetic engineering, and in particular to one kind is made of sheep albumin and sheep interferon gamma
Fusion protein and preparation method thereof and a kind of recombination sheep long-acting interferon γ.
Background technique
Animal infectious disease caused by virus seriously constrains the sound development of every country and regional aquaculture, in
State is the country that sheep breeding stock, the amount of delivering for sale, Mutton yield are most in the world, and Mutton Sheep Industry is also the mainstay of China's animal husbandry
One of industry.With the sustainable development of sheep aquaculture, inevitably will in face of virus caused by disease the problem of, domestic animals disease
Huge economic loss not only is caused to sheep culturist, more seriously, some Zoonosis communicable diseases return the mankind
Health care belt carrys out potential threat.
The prevention and treatment of sheep class communicable disease mainly uses vaccine immunity and drug therapy at present, due to the serum of vaccine immunity
Type is single, and the serotype of virus is complicated, and strain variation is fast, often results in vaccine immunity failure.Some virosis there is no epidemic disease at present
Seedling is available, some viruses may also directly jeopardize the health of the mankind.
Common drug therapy mainly uses antibiotic to be treated, but extensive and a large amount of due to antibiotic in recent years
It uses, causes endurance strain largely to generate, and be transmitted to people by food chain, bigger threat is brought to human health.It is existing
In some countries, oneself prohibites the application of some antibiotic and antibacterial agent in aquaculture.Therefore, positive using interferon
Treat and prevent domestic animal, the viral disease of poultry will be the problem of mankind pay close attention to the most.
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 knot
After conjunction, it can induce body and generate a variety of specific proteins and enzyme, it is main by inhibiting viral gene transcription and degradation virus
RNA inhibits the growth and breeding of virus and plays antitumor etc. activity.It is existing it is known that γ type IFN be T cell by activating and
NK cell generates, and has relatively strong antiviral and immunoloregulation function.A large number of studies show that interferon gamma is in addition to broad-spectrum disease resistance
Outside malicious function, crucial adjustment effect is also played to immune system, so IFN-γ is also known as immunological regulation interferon.Although each
The interferon of seed type can reaction of the mediated cell to virus infection, but the immunoregulatory activity of interferon gamma coordinate exempt from
Epidemic disease, which is reacted and determined, plays even more important effect in the long-term antiviral state of body, therefore interferon gamma is with particularly important
Clinical value.
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, the increase for the treatment of number of times, corresponding time cost and economic cost to treatment
Increase therewith, and the tenability limit of body is also possible to be broken the generation for leading to adverse reaction.The main reason for half-life short
There are two:The too small tachytrophism in vivo of the molecular weight of interferon, interferon especially recombinant interferon affinity is poor to be exempted from
Epidemic disease system is removed.And common long-acting interferon is using polyethylene glycol fused interferon as representative on the market, only in the layer of molecular weight
Partially solve the problems, such as that interferon molecule amount is small and leads to half-life short on face.By being carried out to two kinds of different type interferon
Fusion, not only can be improved the molecular weight of interferon, but also can cooperate with the effect for playing two kinds of interferon.
Seralbumin is the important component of blood plasma, is not easy under normal circumstances through glomerulus, internal distributed pole it is wide and
There is no zymetology and immunologic competence, is ideal pharmaceutical carrier.Albumin fusion technology has the advantage that:Albumin and target egg
It is white to be linked in the cell through protein translation system by peptide bond, it is not required to additional extracorporeal treatment;The expression of albumin is higher,
The expression of destination protein can be improved after merging with it;Albumin is one stable " inert protein ", after merging with it
The stability of destination protein can be improved;Albumin fusion protein has longer drug half-life, by small molecular protein drug
It can be expected to improve half-life period in blood with Albumin fusion.Currently, in experimental animal after multiple protein and Albumin fusion
The extension of Half-life in vivo is confirmed.
And common long-acting interferon is using polyethylene glycol fused interferon as representative on the market, only in the level of molecular weight
Part solves the problems, such as that interferon molecule amount is small and leads to half-life short, while polyethylene glycol fused interferon cost is very
Height is unfavorable for clinically applying.
Summary of the invention
In order to solve the above technical problems, merging egg with what sheep interferon gamma formed by sheep albumin the present invention provides a kind of
Bletilla preparation method and a kind of recombination sheep long-acting interferon γ, the recombination sheep long-acting interferon γ are remarkably improved sheep interference
The half-life period of element, the half-life period of more common sheep interferon gamma improve 12 times or more, and have broad-spectrum disease resistance toxic action and can improve
The immune response of sheep itself.
The technical scheme adopted by the invention is as follows:
A kind of fusion protein being made of sheep albumin and sheep interferon gamma, the amino acid sequence table of the fusion protein is such 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 1 described in 1 codified of genome;The 2 codified fusion protein 2 of genome.Genome 2 is pair
The nucleotide sequence of genome 1 optimize after as a result, when usual codon adaptation indexI CAI=1.0 be considered the base
Because being optimal high efficient expression state in the expression system, CAI value is lower to show that expression is lower in host.In gene
G/C content most ideal distribution range is 30~70%, is more than that the range will affect translation and transcriptional efficiency in any region.
The codon of the sheep albumin and IFN-γ original gene codon adaptation indexI in Escherichia coli is found using software detection
(CAI) be respectively 0.23,0.25, GC percentage be 44.0%, 40.9%;And by sheep albumin and IFN-γ gene optimization
After obtain recombination in Escherichia coli codon adaptation indexI (CAI) be 0.99,1.0, GC percentage 50.1%,
44.1%.The utilization rate of low codon is significantly reduced by gene optimization, avoids rare codon to the shadow of protein expression
It rings, improves the G/C content of gene, improve 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/rAlb-IFN γ.
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 BL21 (DE3) competent cell
Or BL21 (DE3) competent cell with pGro7 plasmid.
The present invention also provides a kind of recombination sheep long-acting interferon γ, the recombination sheep long-acting interferon γ is melted by described
It is freeze-dried to form after hop protein and freeze drying protectant mixture.
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/rAlb-IFN γ, and preparation method is:
(1) design primer, is obtained or the sheep albumin of the flexible linker sequence of artificial synthesized connection by reverse transcription
With the target gene of sheep interferon gamma;Sheep albumin has been connected with the target gene of sheep interferon gamma by flexible linker
Come, 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/rAlb- can be obtained
IFNγ。
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 sheep albumin (Alb) is:
Upstream Alb-F1CCGGAATTCATGAAGTGGGTGACT has EcoRI restriction enzyme site;
Downstream Alb-R1:
ACCACCACCAGAACCACCACCACCGGCTAAGGCTGCTT, with flexible linker;
The primer sequence of sheep interferon gamma (IFN-γ) is:
Upstream IFN-γ-F1:
GGTGGTTCTGGTGGTGGTGGTTCTATGAAATACACAAGCTC, with flexible linker;
Downstream IFN-γ-R1:CCCTCGAGTTACATTGATGCTCT has XhoI restriction enzyme site;
B. RNA is extracted from sheep liver, and the target gene of Alb and IFN-γ, the gene sequence of the two are obtained by reverse transcription
Column are respectively as shown in 400 < of SEQUENCE LISTING 400 <, 4 > and SEQUENCE LISTING, 5 >;
Respectively using Alb and the target gene of IFN-γ as template, and be utilized respectively the upstream and downstream primer of Alb and IFN-γ into
Row PCR amplification respectively obtains and connects the Alb of flexible linker and the target gene of IFN-γ.
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. Alb gene and IFN-γ 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 Alb gene template DNA, connect 1 μ L, Alb upstream primer of IFN-γ template DNA 0.5 the μ L, IFN- of flexible linker
0.5 μ L, Taq archaeal dna polymerase of γ downstream primer, 2.5 μ L, dNTP Mix is 9 μ L, adds RNase Free water to 25 μ L;Connect PCR
Reaction condition 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 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 sheep albumin (Alb) is:
Upstream Alb-F2:CCGGAATTCATGAAATGGGTTACCTT has EcoRI restriction enzyme site;
Downstream Alb-R2:
ACCACCACCAGAACCACCACCACCAGCCAGAGCAGCC, with flexible linker;
The primer sequence of sheep interferon gamma (IFN-γ) is:
Upstream IFN-γ-F2:
GGTGGTTCTGGTGGTGGTGGTTCTATGAAATACACCTCTTCT, with flexible linker;
Downstream IFN-γ-R2:
CCCTCGAGTTACATAGAAGCACG has XhoI restriction enzyme site;
B. the target gene of the Alb and IFN-γ, the gene order of the two is respectively such as SEQUENCE LISTING 400
Shown in 400 < of < 6 > and SEQUENCELISTING, 7 >;
Respectively using Alb and the target gene of IFN-γ as template, and be utilized respectively the upstream and downstream primer of Alb and IFN-γ into
Row PCR amplification, the target gene of Alb and IFN-γ 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 archaeal 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 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 extension 1kb/min are followed
Ring 35 times, last 72 DEG C of extensions 10min.
C. Alb gene and IFN-γ 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 Alb template DNA, connects 1 μ L, Alb upstream primer of IFN-γ template DNA, the 0.5 μ L of flexible linker, under IFN-γ
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;Connect PCR reaction
Condition 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 extension 1kb/min, totally 35
Circulation;Last 72 DEG C of extensions 10min.
The present invention also provides the applications of the recombination sheep long-acting interferon γ, and long half time was up to 49 hours or more, tool
There is broad-spectrum disease resistance toxic action and the immune response of sheep itself can be improved.
Compared with prior art, the present invention has the advantages that:
1. sheep albumin and sheep Interferon-gamma gene are realized fusion by flexibility linker, improves interferon and partly decline
Phase improves 12 times or more compared with plain interferon;Compared with common polyethylene glycol fused interferon, significantly reduce into
This.
2. improving sheep Alb and the fusion of sheep interferon gamma by optimizing to sheep albumin and sheep Interferon-gamma gene
The expression quantity of albumen.
3. using recombination bacillus coli BL21/pET-32a-Alb-IFN γ 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 fusion protein disclosed by the invention being made of sheep albumin and sheep interferon gamma not only has interferon gamma
Broad-spectrum disease resistance toxic action, while significantly improving the immune response of sheep itself.
Detailed description of the invention
Fig. 1 is the result of the sheep albumin gene and sheep Interferon-gamma gene RT-PCR amplification in embodiment 1;Swimming lane M:
DNA Marker DL2000;Swimming lane 1:Sheep Interferon-gamma gene RT-PCR amplified production;Swimming lane 2:Sheep albumin gene RT-PCR
Amplified production;
Fig. 2 is the result of the PCR amplification after the sheep albumin in embodiment 1 is connected with the target gene of sheep IFN-γ;
Swimming lane M:DNA Marker DL2000;Swimming lane 1:Sheep Interferon-gamma gene and sheep albumin 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:Plasmid PCR result;Swimming lane 2:Recombinant plasmid double digestion 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:It is precipitated after bacterial cell disruption after recombinant bacterium induction;Swimming lane 2:Supernatant after bacterial cell disruption after recombinant bacterium induction;Swimming lane 3:Empty bacterium
Control;
Fig. 5 is the Western Blot qualification result for the fusion protein that embodiment 1 obtains;Swimming lane M:Albumen Marker;Swimming
Road 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 recombination sheep long-acting interferon γ as made from the fusion protein in embodiment 1 causes carefully VSV in embodiment 5
The inhibiting effect of born of the same parents' lesion;1 is VSV virus control wells;2 be HEp-2 cell control well;A3-12 is gradient dilution (from dextrad
It is left) human interferon standard items handle hole;B3-12 is that the recombination sheep long-acting interferon γ of gradient dilution (from right to left) is handled
Hole;
Fig. 7 is to recombinate sheep long-acting interferon γ intramuscular injection blood in embodiment 8 as made from the fusion protein in embodiment 1
Concentration-time changing curve.
Specific embodiment
Embodiment 1
A kind of fusion protein being made of sheep albumin and sheep interferon gamma, preparation method are as follows:
1. the acquisition and amplification of sheep albumin (Alb) and sheep interferon gamma (IFN-γ) 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 sheep albumin
EcoRI restriction enzyme site and Linker sequence are introduced in primer and downstream primer respectively, sheep interferon gamma 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 sheep liver tissue, the target gene of Alb and IFN-γ, 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: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 are moved back
Fiery 45s, 72 DEG C of extension 1kb/min, totally 35 recycle;Last 72 DEG C of extensions 10min.
There is specific band, result in 1850bp and 520bp or so through agarose gel electrophoresis in RT-PCR amplified production
As shown in Figure 1, saying that bright Alto has not obtained the Alb of the flexible linker of connection and the target gene of IFN-γ.
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 2340bp or so through agarose gel electrophoresis in pcr amplification product, result as shown in Fig. 2,
Occurs the band of sheep albumin amplified production and sheep interferon gamma amplified production in Fig. 2, this is because in sheep albumin gene
During connecting with sheep Interferon-gamma gene PCR, 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 glue recovery product for the PCR for selecting the target gene after connecting errorless after being sequenced is used with pET-32a plasmid
EcoRI 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 | 2μL |
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
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 bacteria plasmid through PCR
It is identified through EcoRI and XhoI double digestion, being accredited as positive indicates expression vector establishment success, has obtained engineering bacteria pET-32a/
rAlb-IFNγ;There is single band, knot at the place 2340bp or so through agarose gel electrophoresis in PCR amplification and double enzyme digestion product
Fruit is as shown in Figure 3.
4. the expression of recombinant protein
Picking engineering bacteria pET-32a/rAlb-IFN γ shakes for 37 DEG C in the LB culture medium of the 100 μ g/ml containing ampicillin
Bacterium 1h recovery engineering bacteria activity is surveyed OD value and is reached in LB culture medium (the 100 μ g/ml containing ampicillin) after amplification culture 4h
When 1.0;IPTG, the final concentration of 100 μ g/mL, 32 DEG C of inducing expression 5h of IPTG is added;Bacterium is collected, through SDS-PAGE
Electrophoresis detection, result as shown in figure 4, it can be seen from the figure that recombinant bacterium induction 5h after bacterial cell disruption after supernatant precipitate
In the visible predominant expression band in the place 104.4KD or so, illustrate 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
On 100 protein purification systems, the His affinity column balanced with Binding Buffer I (PBS) is washed with PBS buffer solution
Remove unbonded albumen, until A280nm stablize, then with Elution buffer I (50mM trishydroxymethylaminomethane, 20~
500mM imidazoles, PH8.0) elution, collect rAlb-IFN γ 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 ammonia of Elution Buffer II
Methylmethane, 1M NaCl, PH6.5) linear gradient elution, collect rAlb-IFN γ 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, with Binding Buffer
III elution, collects rAlb-IFN γ protein peak.
5.4 sample identification
Measure rAlb-IFN γ potency and specific activity, specific activity >=1.0 × 106U/mg albumen is qualification;It is aseptic subpackaged ,-
80 DEG C of preservations.The fusion protein being made of sheep albumin and sheep interferon gamma, amino acid sequence such as SEQUENCE can be obtained
Shown in 400 < of LISTING, 1 >.
Embodiment 2
A kind of fusion protein being made of sheep albumin and sheep interferon gamma, other, only will be therein big with embodiment 1
The replacement of enterobacteria BL21 (DE3) competent cell is for BL21 (DE3) competent cell with pGro7 plasmid.It merges egg
White SDS-PAGE electrophoresis result is compareed with embodiment 1, and 104.4KD or so place's predominant expression band is thicker in supernatant, explanation
After introducing molecular chaperones pGro7, expression of the destination protein in supernatant is more preferable, and obtained fusion protein amount is higher.Large intestine bar
The albumen of bacterium expression is mostly present in inclusion body;By introducing molecular chaperones in expression bacterial strain, coordinate expression albumen is just
It really folds, 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 sheep albumin and sheep interferon gamma, preparation method are as follows:
1. the acquisition and amplification of sheep albumin (Alb) and sheep interferon gamma (IFN-γ) target gene
To in embodiment 1 Alb and IFN-γ optimize, artificial synthesized Alb and IFN-γ 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 utilizes
The low codon of rate (rare or low-usage codons).In fact, common every kind of biology for doing protein expression or production
(including Escherichia coli, yeast, mammalian cell, plant cell and insect cell) all shows the benefit of codon to a certain degree
Difference or preference.The expression efficiency of the gene containing best codon is apparently higher than in Escherichia coli, yeast and drosophila and is contained
The expression efficiency of the gene of the codon of poor efficiency.Therefore, in heterologous expression system, the preferences of codon are largely
On affect the expression of recombinant protein.Using preference codon (preferred codons) and avoid utilizing rare codon
Gene chemical synthesis is carried out, the redesign of this gene is codon optimization.Therefore, to the albumin of sheep and IFN- in the present embodiment
γ 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 sheep albumin and
The codon of IFN-γ original gene codon adaptation indexI (CAI) in Escherichia coli is respectively 0.23,0.25, GC percentage
It is 44.0%, 40.9%;And by close in Escherichia coli to recombination is obtained after sheep albumin and IFN-γ gene optimization
Numeral adaptation index (CAI) is 0.99,1.0, GC percentage 50.1%, 44.1%.It is significantly reduced by gene optimization low close
The utilization rate of numeral avoids influence of the rare codon to protein expression, improves the G/C content of gene, improves transcription and turn over
Efficiency is translated, and then improves the expression quantity of recombinant protein.
1.3 design of primers:
6 PCR amplification primer of table
The genomic DNA of Alb and IFN-γ 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.
Alb and the pcr amplification product of IFN-γ occur in 1850bp and 520bp or so special respectively through agarose gel electrophoresis
Different band illustrates the target gene that the Alb after the optimization of the flexible linker of connection and IFN-γ have been prepared respectively.
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 2340bp or so through agarose gel electrophoresis in pcr amplification product, illustrates successfully to have obtained Alb
Target gene after being connected with IFN-γ, 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
EcoRI, 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 | 2μL |
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 bacteria plasmid through PCR
It is identified through EcoRI, XhoI double digestion, being accredited as positive indicates expression vector establishment success, PCR amplification and double enzyme digestion product warp
There is single band at the place 2340bp or so in agarose gel electrophoresis, illustrates the target gene after connecting containing Alb with IFN-γ
Expression vector establishment success.
4. the expression of recombinant protein
Picking engineering bacteria shakes bacterium 1h recovery engineering bacteria activity for 37 DEG C in the LB culture medium of the 100 μ g/ml containing ampicillin,
Engineering bacteria is denoted as pET-32a/rAlb-IFN γ;In LB culture medium (the 100 μ g/ml containing ampicillin) after amplification culture 4h,
When survey OD value reaches 1.0;IPTG, the final concentration of 100 μ g/mL, 32 DEG C of inducing expression 5h of IPTG is added;Bacterium is collected,
Through SDS-PAGE electrophoresis detection, it is visible excellent to be deposited in the place 104.4KD or so for supernatant after the bacterial cell disruption after recombinant bacterium induction 5h
Gesture band of expression illustrates to have obtained recombinant protein 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
On 100 protein purification systems, the His affinity column balanced with Binding Buffer I (PBS) is washed with PBS buffer solution
Remove unbonded albumen, until A280nm stablize, then with Elution buffer I (50mM trishydroxymethylaminomethane, 20~
500mM imidazoles, PH8.0) elution, collect rAlb-IFN γ 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 ammonia of Elution Buffer II
Methylmethane, 1M NaCl, PH6.5) linear gradient elution, collect rAlb-IFN γ 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, with Binding Buffer
III elution, collects rAlb-IFN γ protein peak.
5.4 sample identification
Measure rAlb-IFN γ potency and specific activity, specific activity >=1.0 × 106U/mg albumen is qualification;It is aseptic subpackaged ,-
80 DEG C of preservations.The fusion protein being made of sheep albumin and sheep interferon gamma, amino acid sequence such as SEQUENCE can be obtained
Shown in 400 < of LISTING, 1 >.
Embodiment 4
A kind of fusion protein being made of sheep albumin and sheep interferon gamma, other, only will be therein big with embodiment 3
The replacement of enterobacteria BL21 (DE3) competent cell is for BL21 (DE3) competent cell with pGro7 plasmid.It merges egg
White SDS-PAGE electrophoresis result is compareed with embodiment 3, and 104.4KD or so place's predominant expression band is thicker in supernatant, explanation
After introducing molecular chaperones pGro7, expression of the destination protein in supernatant is more preferable, and obtained fusion protein amount is higher.Large intestine bar
The albumen of bacterium expression is mostly present in inclusion body;By introducing molecular chaperones in expression bacterial strain, coordinate expression albumen is just
It really folds, reaches solubility expression of protein.BL21 (DE3) competent cell with pGro7 plasmid is purchased from Shanghai offshore
Science and Technology Ltd./glad hundred promise biology, article No. V205.
Embodiment 5
A kind of recombination sheep long-acting interferon γ, is mixed with freeze drying protectant respectively by the fusion protein in embodiment 1,2,3,4
It is freeze-dried to form with later.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 sheep albumin and sheep interferon gamma
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 all larger than 1.2mg/ml.
6.2 SDS-PAGE electrophoresis detections
Compared with empty bacterium, fusion protein has the newly-increased protein band of a dense dye in 104.4KD or so, such as Fig. 4, shown.
6.3 Western Blot results
Fusion protein in Examples 1 to 4 is detected, respectively with the anti-sheep interferon (1 of abcam company mouse:5000 dilutions) be
Primary antibody, using goat anti-mouse IgG-HRP as secondary antibody (1:10000 dilutions).Recombinating sheep long-acting interferon γ sample can be dry with anti-sheep
It disturbs plain γ monoclonal antibody and specific reaction occurs, specific band occurs in the place 104.4KD or so, as shown in Figure 5.
Embodiment 7
Four parts in embodiment 5 recombinate the freeze-dried bioactivity of sheep long-acting interferon γ
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%CO2 culture for 24 hours, the recombination sheep that various dose is added is long
Interferon gamma is imitated, inhales abandon afterwards for 24 hours, then be inoculated with 100 TCID respectively50VSV virus.
Test result
The result shows that the recombination sheep long-acting interferon γ obtained causes the lesion of HEp-2 cell to have apparent suppression VSV
Production is used.The lesions such as there is cell rounding, falls off, is disintegrated after untreated cell inoculation virus.And the recombination sheep obtained
After long-acting interferon γ treated cell inoculation virus, it is observed continuously under inverted microscope, cellular morphology is normal, does not occur
Any lesion, measures potency >=1.0 × 106U/ml, as shown in Figure 6.
Embodiment 8
Being lyophilized respectively by four parts of recombination sheep long-acting interferon γ 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 sheep intracorporal half-life period
The blood concentration and time relationship of cytopathic-effect inhibition assay measurement rAlb-IFN γ
The sheep (half male and half female) that six weight are roughly the same is taken, the long-acting sheep interferon gamma of intramuscular injection 2mg/ml sheep is freeze-dried
2ml, respectively 1h, 2h, 4h, 8h, 16h, for 24 hours, 36h, 48h, 60h venous blood collection, 4 DEG C of blood sample solidification, 3500rpm low-temperature centrifugation
10min separates serum, and every sheep blood sample of each time point is to be measured in -20 DEG C of preservations.Blood serum sample is measured using cytopathic-effect inhibition assay
The concentration of middle rAlb-IFN γ is carried out curve fitting and calculating parameter with DAS pharmacokinetics software.Matched curve is as shown in Figure 7;Ginseng
Number calculated result is shown in Table 11.
Dominant dynamic parameters in serum after the recombination sheep long-acting interferon γ intramuscular injection of table 11
The result shows that recombination sheep long-acting interferon γ has longer half-life period.Half-life period can reach 49h or so after measured, compared with
Plain interferon improves about 12 times.
Embodiment 9
Four parts of freeze-dried measurements that sheep cellullar immunologic response is influenced of recombination sheep long-acting interferon γ in embodiment 5
It takes six roughly the same sheep of weight to be divided into two groups, is denoted as experimental group and control group;The subcutaneous injection of experimental group neck
2mg/ml recombinates the freeze-dried 2ml of sheep long-acting interferon γ, and the PBS of 2mL is subcutaneously injected in control group neck, after taking injection 4 weeks outside sheep
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 are every
Hole addition 1ml lymphocyte, 37 DEG C, 5%CO272h is cultivated, supernatant when collecting lymphocyte culture.ELISA detects culture supernatant
Middle IL-2, IL-4 content, is 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 sheep cellullar immunologic response
The result shows that injection recombination sheep long-acting interferon γ after, can significantly improve sheep Evaluation of Cytokines in Peripheral Blood IL-2,
The content of IL-4 enhances cellullar immunologic response level, significantly improves immunity level.
It is above-mentioned referring to embodiment to a kind of fusion protein and preparation method thereof being made of sheep albumin and sheep interferon gamma
It is illustrative without being restrictive with a kind of detailed description that recombination sheep long-acting interferon γ is carried out, it can be according to being limited
Range enumerates several embodiments, therefore the change and modification in the case where not departing from present general inventive concept, should belong to of the invention
Within protection scope.
SEQUENCE LISTING
<110>Wuhu Co., Ltd of Ying Tefeier biological products industrial research institute
<120>A kind of fusion protein being made of sheep albumin with sheep interferon gamma and preparation method thereof and a kind of recombination sheep are long
Imitate interferon gamma
<130> 1
<160> 7
<170> PatentIn version 3.3
<210> 1
<211> 783
<212> PRT
<213>Recombinate sheep long-acting interferon γ fusion protein
<400> 1
Met Lys Trp Val Thr Phe Ile Ser Leu Leu Leu Leu Phe Ser Ser Ala
1 5 10 15
Tyr Ser Arg Gly Val Phe Arg Arg Asp Thr His Lys Ser Glu Ile Ala
20 25 30
His Arg Phe Asn Asp Leu Gly Glu Glu Asn Phe Gln Gly Leu Val Leu
35 40 45
Ile Ala Phe Ser Gln Tyr Leu Gln Gln Cys Pro Phe Asp Glu His Val
50 55 60
Lys Leu Val Lys Glu Leu Thr Glu Phe Ala Lys Thr Cys Val Ala Asp
65 70 75 80
Glu Ser His Ala Gly Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp
85 90 95
Glu Leu Cys Lys Val Ala Thr Leu Arg Glu Thr Tyr Gly Asp Met Ala
100 105 110
Asp Cys Cys Glu Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Asn
115 120 125
His Lys Asp Asp Ser Pro Asp Leu Pro Lys Leu Lys Pro Glu Pro Asp
130 135 140
Thr Leu Cys Ala Glu Phe Lys Ala Asp Glu Lys Lys Phe Trp Gly Lys
145 150 155 160
Tyr Leu Tyr Glu Val Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu
165 170 175
Leu Leu Tyr Tyr Ala Asn Lys Tyr Asn Gly Val Phe Gln Glu Cys Cys
180 185 190
Gln Ala Glu Asp Lys Gly Ala Cys Leu Leu Pro Lys Ile Asp Ala Met
195 200 205
Arg Glu Lys Val Leu Ala Ser Ser Ala Arg Gln Arg Leu Arg Cys Ala
210 215 220
Ser Ile Gln Lys Phe Gly Glu Arg Ala Leu Lys Ala Trp Ser Val Ala
225 230 235 240
Arg Leu Ser Gln Lys Phe Pro Lys Ala Asp Phe Thr Asp Val Thr Lys
245 250 255
Ile Val Thr Asp Leu Thr Lys Val His Lys Glu Cys Cys His Gly Asp
260 265 270
Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile Cys
275 280 285
Asp His Gln Asp Ala Leu Ser Ser Lys Leu Lys Glu Cys Cys Asp Lys
290 295 300
Pro Val Leu Glu Lys Ser His Cys Ile Ala Glu Val Asp Lys Asp Ala
305 310 315 320
Val Pro Glu Asn Leu Pro Pro Leu Thr Ala Asp Phe Ala Glu Asp Lys
325 330 335
Glu Val Cys Lys Asn Tyr Gln Glu Ala Lys Asp Val Phe Leu Gly Ser
340 345 350
Phe Leu Tyr Glu Tyr Ser Arg Arg His Pro Glu Tyr Ala Val Ser Val
355 360 365
Leu Leu Arg Leu Ala Lys Glu Tyr Glu Ala Thr Leu Glu Asp Cys Cys
370 375 380
Ala Lys Glu Asp Pro His Ala Cys Tyr Ala Thr Val Phe Asp Lys Leu
385 390 395 400
Lys His Leu Val Asp Glu Pro Gln Asn Leu Ile Lys Lys Asn Cys Glu
405 410 415
Leu Phe Glu Lys His Gly Glu Tyr Gly Phe Gln Asn Ala Leu Ile Val
420 425 430
Arg Tyr Thr Arg Lys Ala Pro Gln Val Ser Thr Pro Thr Leu Val Glu
435 440 445
Ile Ser Arg Ser Leu Gly Lys Val Gly Thr Lys Cys Cys Ala Lys Pro
450 455 460
Glu Ser Glu Arg Met Pro Cys Thr Glu Asp Tyr Leu Ser Leu Ile Leu
465 470 475 480
Asn Arg Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Glu Lys Val
485 490 495
Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser
500 505 510
Asp Leu Thr Leu Asp Glu Thr Tyr Val Pro Lys Pro Phe Asp Glu Lys
515 520 525
Phe Phe Thr Phe His Ala Asp Ile Cys Thr Leu Pro Asp Thr Glu Lys
530 535 540
Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Leu Lys His Lys Pro
545 550 555 560
Lys Ala Thr Asp Glu Gln Leu Lys Thr Val Met Glu Asn Phe Val Ala
565 570 575
Phe Val Asp Lys Cys Cys Ala Ala Asp Asp Lys Glu Gly Cys Phe Val
580 585 590
Leu Glu Gly Pro Lys Leu Val Ala Ser Thr Gln Ala Ala Leu Ala Gly
595 600 605
Gly Gly Gly Ser Gly Gly Gly Gly Ser Met Lys Tyr Thr Ser Ser Phe
610 615 620
Leu Ala Leu Leu Leu Cys Val Leu Leu Gly Phe Ser Gly Ser Tyr Gly
625 630 635 640
Gln Gly Pro Phe Phe Lys Glu Ile Glu Asn Leu Lys Glu Tyr Phe Asn
645 650 655
Ala Ser Asn Pro Asp Val Ala Lys Gly Gly Pro Leu Phe Ser Glu Ile
660 665 670
Leu Lys Asn Trp Lys Glu Glu Ser Asp Lys Lys Ile Ile Gln Ser Gln
675 680 685
Ile Val Ser Phe Tyr Phe Lys Leu Phe Glu Asn Leu Lys Asp Asn Gln
690 695 700
Val Ile Gln Arg Ser Met Asp Ile Ile Lys Gln Asp Met Phe Gln Lys
705 710 715 720
Phe Leu Asn Gly Ser Ser Glu Lys Leu Glu Asp Phe Lys Arg Leu Ile
725 730 735
Gln Ile Pro Val Asp Asp Leu Gln Ile Gln Arg Lys Ala Ile Asn Glu
740 745 750
Leu Ile Lys Val Met Asn Asp Leu Ser Pro Lys Ser Asn Leu Arg Lys
755 760 765
Arg Lys Arg Ser Gln Asn Leu Phe Arg Gly Arg Arg Ala Ser Met
770 775 780
<210> 2
<211> 2352
<212> DNA
<213>Recombinate sheep long-acting interferon γ genome 1
<400> 2
atgaagtggg tgacttttat ttcccttctc cttctcttca gctctgctta ttccaggggt 60
gtgtttcgtc gagatacaca caagagtgag attgctcatc ggtttaatga tttgggagaa 120
gaaaattttc aaggcctggt gctgattgcc ttttctcagt atctccagca gtgtccattt 180
gacgaacatg taaaattagt gaaggagcta actgagtttg caaaaacatg tgttgctgat 240
gagtcacatg ccggttgtga taagtcactt cacactctct ttggagatga attgtgtaaa 300
gttgcaaccc ttcgcgaaac ctatggtgac atggccgact gctgtgagaa acaagagcct 360
gaaagaaatg aatgcttcct gaatcacaaa gatgatagcc cagacctccc taaactgaaa 420
ccagagcccg atactttgtg tgccgagttt aaggcagatg aaaagaagtt ttggggaaaa 480
tacctatacg aagttgccag aagacatccc tacttttatg caccagaact cctttactat 540
gctaataaat ataatggagt ttttcaagaa tgctgccaag ctgaagataa aggtgcctgc 600
ctactaccaa agattgacgc tatgagagaa aaagtactgg cttcatctgc cagacagaga 660
ctcaggtgtg ccagtattca aaaattcgga gaaagagctt taaaagcatg gtcagtagct 720
cgcctgagcc agaaatttcc caaggctgac tttacagatg ttaccaagat agtgacagat 780
ctcactaagg tccacaagga gtgttgccat ggtgacctgc ttgaatgcgc agacgacagg 840
gcagatcttg ccaagtacat atgtgatcat caagacgcac tctccagtaa actgaaggaa 900
tgctgtgata agcctgtgtt ggaaaaatcc cactgcattg ctgaggtaga taaagatgcc 960
gtgcctgaaa acctgccccc attaactgct gactttgctg aagataagga ggtttgcaaa 1020
aactatcagg aagcaaaaga cgtcttcctg ggctcgtttt tgtatgaata ttcaagaagg 1080
catcctgagt atgctgtctc agtgctattg agacttgcca aggaatatga agccacactg 1140
gaggactgct gtgccaaaga agatccacat gcctgctatg ccacagtgtt tgacaaactt 1200
aagcatcttg tggatgagcc tcagaattta atcaaaaaaa actgtgagct attcgaaaaa 1260
catggagagt atggattcca aaatgcgctc atagttcgtt acaccaggaa agcaccccaa 1320
gtgtcaactc caactctggt ggagatttca agaagcctag gaaaagtggg cactaagtgt 1380
tgtgcaaagc ctgaatcaga aagaatgccc tgtaccgaag actatctgag cttgatcctg 1440
aaccggttgt gcgtgttgca tgagaagaca ccagtgagtg aaaaagtcac caagtgctgc 1500
acggagtcat tggtgaacag acggccatgt ttctctgatc tgacacttga cgaaacatat 1560
gtacccaaac ccttcgatga gaaatttttc accttccatg cagatatatg cacacttcct 1620
gatactgaga aacaaatcaa gaaacaaact gcacttgttg agctgttgaa acacaagccc 1680
aaggcaacag atgaacaact gaaaaccgtt atggagaatt ttgtggcttt tgtagacaag 1740
tgctgtgcag ctgatgacaa agaaggctgc tttgttctgg agggtccaaa acttgttgct 1800
tcaactcaag cagccttagc cggtggtggt ggttctggtg gtggtggttc tatgaaatac 1860
acaagctcct tcttagcttt actgctctgt gtgcttttgg gtttttctgg ttcttatggc 1920
cagggcccat tttttaaaga aatagaaaac ttaaaggagt attttaatgc aagtaaccca 1980
gatgtagcta agggtgggcc tcttttctca gaaattttga agaattggaa agaggagagt 2040
gacaaaaaga ttattcagag ccaaattgtc tccttctact tcaaactctt tgaaaacctc 2100
aaagataacc aggtcattca aaggagcatg gatatcatca agcaagacat gtttcagaag 2160
ttcttgaacg gcagctctga gaaactggag gacttcaaaa ggctgattca aattccggtg 2220
gatgatctgc agatccagcg caaagccatc aatgaactca tcaaggtgat gaatgacctg 2280
tcgccaaaat ctaacctcag aaagcggaag agaagtcaga atctctttcg aggccggaga 2340
gcatcaatgt aa 2352
<210> 3
<211> 2352
<212> DNA
<213>Recombinate sheep long-acting interferon γ genome 2
<400> 3
atgaaatggg ttaccttcat ctctctgctg ctgctgttct cttctgctta ctctcgtggt 60
gttttccgtc gtgacaccca caaatctgaa atcgctcacc gtttcaacga cctgggtgaa 120
gaaaacttcc agggtctggt tctgatcgct ttctctcagt acctgcagca gtgcccgttc 180
gacgaacacg ttaaactggt taaagaactg accgaattcg ctaaaacctg cgttgctgac 240
gaatctcacg ctggttgcga caaatctctg cacaccctgt tcggtgacga actgtgcaaa 300
gttgctaccc tgcgtgaaac ctacggtgac atggctgact gctgcgaaaa acaggaaccg 360
gaacgtaacg aatgcttcct gaaccacaaa gacgactctc cggacctgcc gaaactgaaa 420
ccggaaccgg acaccctgtg cgctgaattc aaagctgacg aaaaaaaatt ctggggtaaa 480
tacctgtacg aagttgctcg tcgtcacccg tacttctacg ctccggaact gctgtactac 540
gctaacaaat acaacggtgt tttccaggaa tgctgccagg ctgaagacaa aggtgcttgc 600
ctgctgccga aaatcgacgc tatgcgtgaa aaagttctgg cttcttctgc tcgtcagcgt 660
ctgcgttgcg cttctatcca gaaattcggt gaacgtgctc tgaaagcttg gtctgttgct 720
cgtctgtctc agaaattccc gaaagctgac ttcaccgacg ttaccaaaat cgttaccgac 780
ctgaccaaag ttcacaaaga atgctgccac ggtgacctgc tggaatgcgc tgacgaccgt 840
gctgacctgg ctaaatacat ctgcgaccac caggacgctc tgtcttctaa actgaaagaa 900
tgctgcgaca aaccggttct ggaaaaatct cactgcatcg ctgaagttga caaagacgct 960
gttccggaaa acctgccgcc gctgaccgct gacttcgctg aagacaaaga agtttgcaaa 1020
aactaccagg aagctaaaga cgtttttctc ggtagcttcc tgtacgaata ctctcgtcgt 1080
cacccggaat acgctgtttc tgttctgctg cgtctggcta aagaatacga agctaccctg 1140
gaagactgct gcgctaaaga agacccgcac gcttgctacg ctaccgtttt cgacaaactg 1200
aaacacctgg ttgacgaacc gcagaacctg atcaaaaaaa actgcgaact gttcgaaaaa 1260
cacggtgaat acggtttcca gaacgctctg atcgttcgtt acacccgtaa agctccgcag 1320
gtttctaccc cgaccctggt tgaaatctct cgttctctgg gtaaagttgg taccaaatgc 1380
tgcgctaaac cggaatctga acgtatgccg tgcaccgaag actacctgtc tctgatcctg 1440
aaccgtctgt gcgttctgca cgaaaaaacc ccggtttctg aaaaagttac caaatgctgc 1500
accgaatctc tggttaaccg tcgtccgtgc ttctctgacc tgaccctgga cgaaacctac 1560
gttccgaaac cgttcgacga aaaattcttc accttccacg ctgacatctg caccctgccg 1620
gacaccgaaa aacagatcaa aaaacagacc gctctggttg aactgctgaa acacaaaccg 1680
aaagctaccg acgaacagct gaaaaccgtt atggaaaact tcgttgcttt cgttgacaaa 1740
tgctgcgctg ctgacgacaa agaaggttgc ttcgttctgg aaggtccgaa actggttgct 1800
tctacccagg ctgctctggc tggtggtggt ggttctggtg gtggtggttc tatgaaatac 1860
acctcttctt tcctggctct gctgctgtgc gttctgctgg gtttctctgg ttcttacggt 1920
cagggtccgt tcttcaaaga aatcgaaaac ctgaaagaat acttcaacgc ttctaacccg 1980
gacgttgcta aaggtggtcc gctgttctct gaaatcctga aaaactggaa agaagaatct 2040
gacaaaaaaa tcatccagtc tcagatcgtt tctttctact tcaaactgtt cgaaaacctg 2100
aaagacaacc aggttatcca gcgttctatg gacatcatca aacaggacat gttccagaaa 2160
ttcctgaacg gttcttctga aaaactggaa gacttcaaac gtctgatcca gatcccggtt 2220
gacgacctgc agatccagcg taaagctatc aacgaactga tcaaagttat gaacgacctg 2280
tctccgaaat ctaacctgcg taaacgtaaa cgttctcaga acctgttccg tggtcgtcgt 2340
gcttctatgt aa 2352
<210> 4
<211> 1821
<212> DNA
<213>Sheep albumin
<400> 4
atgaagtggg tgacttttat ttcccttctc cttctcttca gctctgctta ttccaggggt 60
gtgtttcgtc gagatacaca caagagtgag attgctcatc ggtttaatga tttgggagaa 120
gaaaattttc aaggcctggt gctgattgcc ttttctcagt atctccagca gtgtccattt 180
gacgaacatg taaaattagt gaaggagcta actgagtttg caaaaacatg tgttgctgat 240
gagtcacatg ccggttgtga taagtcactt cacactctct ttggagatga attgtgtaaa 300
gttgcaaccc ttcgcgaaac ctatggtgac atggccgact gctgtgagaa acaagagcct 360
gaaagaaatg aatgcttcct gaatcacaaa gatgatagcc cagacctccc taaactgaaa 420
ccagagcccg atactttgtg tgccgagttt aaggcagatg aaaagaagtt ttggggaaaa 480
tacctatacg aagttgccag aagacatccc tacttttatg caccagaact cctttactat 540
gctaataaat ataatggagt ttttcaagaa tgctgccaag ctgaagataa aggtgcctgc 600
ctactaccaa agattgacgc tatgagagaa aaagtactgg cttcatctgc cagacagaga 660
ctcaggtgtg ccagtattca aaaattcgga gaaagagctt taaaagcatg gtcagtagct 720
cgcctgagcc agaaatttcc caaggctgac tttacagatg ttaccaagat agtgacagat 780
ctcactaagg tccacaagga gtgttgccat ggtgacctgc ttgaatgcgc agacgacagg 840
gcagatcttg ccaagtacat atgtgatcat caagacgcac tctccagtaa actgaaggaa 900
tgctgtgata agcctgtgtt ggaaaaatcc cactgcattg ctgaggtaga taaagatgcc 960
gtgcctgaaa acctgccccc attaactgct gactttgctg aagataagga ggtttgcaaa 1020
aactatcagg aagcaaaaga cgtcttcctg ggctcgtttt tgtatgaata ttcaagaagg 1080
catcctgagt atgctgtctc agtgctattg agacttgcca aggaatatga agccacactg 1140
gaggactgct gtgccaaaga agatccacat gcctgctatg ccacagtgtt tgacaaactt 1200
aagcatcttg tggatgagcc tcagaattta atcaaaaaaa actgtgagct attcgaaaaa 1260
catggagagt atggattcca aaatgcgctc atagttcgtt acaccaggaa agcaccccaa 1320
gtgtcaactc caactctggt ggagatttca agaagcctag gaaaagtggg cactaagtgt 1380
tgtgcaaagc ctgaatcaga aagaatgccc tgtaccgaag actatctgag cttgatcctg 1440
aaccggttgt gcgtgttgca tgagaagaca ccagtgagtg aaaaagtcac caagtgctgc 1500
acggagtcat tggtgaacag acggccatgt ttctctgatc tgacacttga cgaaacatat 1560
gtacccaaac ccttcgatga gaaatttttc accttccatg cagatatatg cacacttcct 1620
gatactgaga aacaaatcaa gaaacaaact gcacttgttg agctgttgaa acacaagccc 1680
aaggcaacag atgaacaact gaaaaccgtt atggagaatt ttgtggcttt tgtagacaag 1740
tgctgtgcag ctgatgacaa agaaggctgc tttgttctgg agggtccaaa acttgttgct 1800
tcaactcaag cagccttagc c 1821
<210> 5
<211> 501
<212> DNA
<213>Sheep IFN-γ
<400> 5
atgaaataca caagctcctt cttagcttta ctgctctgtg tgcttttggg tttttctggt 60
tcttatggcc agggcccatt ttttaaagaa atagaaaact taaaggagta ttttaatgca 120
agtaacccag atgtagctaa gggtgggcct cttttctcag aaattttgaa gaattggaaa 180
gaggagagtg acaaaaagat tattcagagc caaattgtct ccttctactt caaactcttt 240
gaaaacctca aagataacca ggtcattcaa aggagcatgg atatcatcaa gcaagacatg 300
tttcagaagt tcttgaacgg cagctctgag aaactggagg acttcaaaag gctgattcaa 360
attccggtgg atgatctgca gatccagcgc aaagccatca atgaactcat caaggtgatg 420
aatgacctgt cgccaaaatc taacctcaga aagcggaaga gaagtcagaa tctctttcga 480
ggccggagag catcaatgta a 501
<210> 6
<211> 1821
<212> DNA
<213>Sheep albumin
<400> 6
atgaaatggg ttaccttcat ctctctgctg ctgctgttct cttctgctta ctctcgtggt 60
gttttccgtc gtgacaccca caaatctgaa atcgctcacc gtttcaacga cctgggtgaa 120
gaaaacttcc agggtctggt tctgatcgct ttctctcagt acctgcagca gtgcccgttc 180
gacgaacacg ttaaactggt taaagaactg accgaattcg ctaaaacctg cgttgctgac 240
gaatctcacg ctggttgcga caaatctctg cacaccctgt tcggtgacga actgtgcaaa 300
gttgctaccc tgcgtgaaac ctacggtgac atggctgact gctgcgaaaa acaggaaccg 360
gaacgtaacg aatgcttcct gaaccacaaa gacgactctc cggacctgcc gaaactgaaa 420
ccggaaccgg acaccctgtg cgctgaattc aaagctgacg aaaaaaaatt ctggggtaaa 480
tacctgtacg aagttgctcg tcgtcacccg tacttctacg ctccggaact gctgtactac 540
gctaacaaat acaacggtgt tttccaggaa tgctgccagg ctgaagacaa aggtgcttgc 600
ctgctgccga aaatcgacgc tatgcgtgaa aaagttctgg cttcttctgc tcgtcagcgt 660
ctgcgttgcg cttctatcca gaaattcggt gaacgtgctc tgaaagcttg gtctgttgct 720
cgtctgtctc agaaattccc gaaagctgac ttcaccgacg ttaccaaaat cgttaccgac 780
ctgaccaaag ttcacaaaga atgctgccac ggtgacctgc tggaatgcgc tgacgaccgt 840
gctgacctgg ctaaatacat ctgcgaccac caggacgctc tgtcttctaa actgaaagaa 900
tgctgcgaca aaccggttct ggaaaaatct cactgcatcg ctgaagttga caaagacgct 960
gttccggaaa acctgccgcc gctgaccgct gacttcgctg aagacaaaga agtttgcaaa 1020
aactaccagg aagctaaaga cgtttttctc ggtagcttcc tgtacgaata ctctcgtcgt 1080
cacccggaat acgctgtttc tgttctgctg cgtctggcta aagaatacga agctaccctg 1140
gaagactgct gcgctaaaga agacccgcac gcttgctacg ctaccgtttt cgacaaactg 1200
aaacacctgg ttgacgaacc gcagaacctg atcaaaaaaa actgcgaact gttcgaaaaa 1260
cacggtgaat acggtttcca gaacgctctg atcgttcgtt acacccgtaa agctccgcag 1320
gtttctaccc cgaccctggt tgaaatctct cgttctctgg gtaaagttgg taccaaatgc 1380
tgcgctaaac cggaatctga acgtatgccg tgcaccgaag actacctgtc tctgatcctg 1440
aaccgtctgt gcgttctgca cgaaaaaacc ccggtttctg aaaaagttac caaatgctgc 1500
accgaatctc tggttaaccg tcgtccgtgc ttctctgacc tgaccctgga cgaaacctac 1560
gttccgaaac cgttcgacga aaaattcttc accttccacg ctgacatctg caccctgccg 1620
gacaccgaaa aacagatcaa aaaacagacc gctctggttg aactgctgaa acacaaaccg 1680
aaagctaccg acgaacagct gaaaaccgtt atggaaaact tcgttgcttt cgttgacaaa 1740
tgctgcgctg ctgacgacaa agaaggttgc ttcgttctgg aaggtccgaa actggttgct 1800
tctacccagg ctgctctggc t 1821
<210> 7
<211> 501
<212> DNA
<213>Sheep IFN-γ
<400> 7
atgaaataca cctcttcttt cctggctctg ctgctgtgcg ttctgctggg tttctctggt 60
tcttacggtc agggtccgtt cttcaaagaa atcgaaaacc tgaaagaata cttcaacgct 120
tctaacccgg acgttgctaa aggtggtccg ctgttctctg aaatcctgaa aaactggaaa 180
gaagaatctg acaaaaaaat catccagtct cagatcgttt ctttctactt caaactgttc 240
gaaaacctga aagacaacca ggttatccag cgttctatgg acatcatcaa acaggacatg 300
ttccagaaat tcctgaacgg ttcttctgaa aaactggaag acttcaaacg tctgatccag 360
atcccggttg acgacctgca gatccagcgt aaagctatca acgaactgat caaagttatg 420
aacgacctgt ctccgaaatc taacctgcgt aaacgtaaac gttctcagaa cctgttccgt 480
ggtcgtcgtg cttctatgta a 501
Claims (10)
1. a kind of fusion protein being made of sheep albumin and sheep interferon gamma, it is characterised in that:The amino of the fusion protein
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 recombination sheep long-acting interferon γ, which is characterized in that the recombination sheep long-acting interferon γ is as described in claim 1
Fusion protein and freeze drying protectant mixture after, it is freeze-dried to form.
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/rAlb-IFN
γ, preparation method are:
(1) design primer, is obtained or the sheep albumin of the flexible linker sequence of artificial synthesized connection and sheep by reverse transcription
The target gene of interferon gamma;The target gene of sheep albumin and sheep interferon gamma is connected by flexible linker, mesh
Gene nucleotides sequence list as shown in 400 < of SEQUENCE LISTING, 2 > or such as 400 < of SEQUENCE LISTING, 3 >
It is shown;
(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/rAlb-IFN can be obtained
γ。
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 recombination sheep long-acting interferon γ according to claim 5, which is characterized in that the recombination sheep is long-acting
The long half time of interferon gamma had broad-spectrum disease resistance toxic action and can improve the immune response of sheep itself up to 49 hours or more.
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