CN108864302A - A kind of fusion protein and preparation method thereof being made of pig albumin, Porcine interferon-gamma and pig interleukin 2 and 6 - Google Patents
A kind of fusion protein and preparation method thereof being made of pig albumin, Porcine interferon-gamma and pig interleukin 2 and 6 Download PDFInfo
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Abstract
The fusion protein and preparation method thereof that the invention discloses a kind of to be made of pig albumin, Porcine interferon-gamma and pig interleukin 2 and 6; the fusion protein is connected through flexible linker by pig albumin, Porcine interferon-gamma and pig interleukin 2 and 6 and is formed, and is freeze-dried to obtain Recombinant Swine long-acting interferon after fusion protein and freeze drying protectant mixture.The Recombinant Swine long-acting interferon is remarkably improved the half-life period of pig interferon, and the half-life period of more common pig interferon improves 23 times or more, and has broad-spectrum disease resistance toxic action and can improve the immune response of pig itself.
Description
Technical field
The invention belongs to technical field of biological genetic engineering, and in particular to white thin by pig albumin, Porcine interferon-gamma and pig
The fusion protein and preparation method thereof that born of the same parents' interleukin 2 forms.
Background technique
Scale Compact Develop is rapidly growing in China in recent years, and China's live pig breeding stock and pork production are sure to occupy
First place in the world, traditional swine disease control method is far from the control for adapting to infectious disease in extensive intensive pig production production.I
Newly there are nearly 20 kinds of livestock and poultry infectious diseases in the past 20 years in state, in addition original animal epidemic, causes aquaculture industry of China huge
Economic loss.According to incompletely statistics, China is every year because various viral diseases cause mortality of livestock to be up to 15%-20%,
Economic loss reaches billions of members.
Mainly pass through vaccine inoculation to the prevention and treatment approach of porcine viral diseases at present and uses antibiotic, but by
It is not perfect in breeding environment, virus variation and Abwehrkraft des Koepers variation etc. reasons, make traditional prevention and treatment approach by
Huge challenge, most of antibiotics and traditional oral antiviral medicament give people due to medicament residue problem
Health is brought a negative impact;And traditional vaccine, high specific and side effect due to it, virus variation and new can not be resisted
Type virus, which continuously emerges, gives pig aquaculture bring significant damage.
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.
Cell factor IL-2, that is, interleukin 2 also known as t cell growth factor.Mainly generated by the T lymphocyte activated
The cell factor with extensive bioactivity, can both promote lymphopoiesis, enhance immune function, but can restricted T it is thin
Born of the same parents react and enhance the immune tolerance of body, therefore can be used for treating tumour, infectious diseases and autoimmune disease.In animal doctor
In, since IL-2 can improve the immune response of vaccine and reduce the generation of disease, also there is wide application prospect.IL-2 is because of energy
The immune level for enhancing body improves the disease resistance of body, thus exempts from for bacillary, viral and parasitic diseases
Epidemic disease treatment.In addition, IL-2 can also influence the metabolism of drug, extend the metabolism time of drug, action time increases, to improve
Curative effect of medication.IL-2, according to gene constructed, composition fusion protein, is generated and is mentioned to enhance the antibody of vaccine with other cell factors
High cellular immune level.
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.
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 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
It is situated between in order to solve the above technical problems, the present invention provides one kind by pig albumin, Porcine interferon-gamma and pig leucocyte
The fusion proteins and preparation method thereof of 2 composition of element, and thus after fusion protein and freeze drying protectant mixture, freeze-dried system
Standby to obtain a kind of Recombinant Swine long-acting interferon, the Recombinant Swine long-acting interferon is remarkably improved the half-life period of pig interferon, compared with
The half-life period of common pig interferon improves 23 times or more, and has broad-spectrum disease resistance toxic action and can improve the immune of pig itself and answer
It answers.
The technical scheme adopted by the invention is as follows:
A kind of fusion protein being made of pig albumin, Porcine interferon-gamma and pig interleukin 2 and 6, the fusion protein
Amino acid sequence 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
In be optimal high efficient expression state, CAI value is lower to show that expression is lower in host.Most ideal point of G/C content in gene
Cloth range is 30~70%, is more than that the range will affect translation and transcriptional efficiency in any region.It is sent out using software detection
The now codon of pig albumin, porcine IFN γ, pig IL-2 original gene codon adaptation indexI (CAI) difference in Escherichia coli
It is 43.7%, 39.2%, 38.7% for 0.24,0.24,0.23, GC percentage;And by pig albumin, porcine IFN γ, pig
It is 0.98,1.0,0.97, GC percentage that each gene codon adaptation indexI (CAI) in Escherichia coli is obtained after IL-2 gene optimization
Than 50.3%, 45.4%, 47.6%.The utilization rate of low codon is significantly reduced by gene optimization, avoids rare codon
Influence of the son to protein expression, improves the G/C content of gene, 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/rAlb-IFN γ-IL2.
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 Recombinant Swine long-acting interferon, the Recombinant Swine 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/rAlb-IFN γ-IL2, and preparation method is:
(1) design primer obtains by reverse transcription or is manually respectively synthesized the white egg of pig for connecting flexible linker sequence
The target gene of white, Porcine interferon-gamma, pig interleukin 2 and 6;By flexible linker by pig albumin, Porcine interferon-gamma, pig
The target gene of interleukin 2 connects, the nucleotides sequence list such as SEQUENCE of the target gene after connection
Shown in 400 < of LISTING, 2 > or as shown in 400 < of SEQUENCE 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/rAlb- can be obtained
IFNγ-IL2。
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 pig albumin (Alb) is:
Upstream Alb-F1:CATGCCATGGGATACATACA AGAGTGA has NcoI restriction enzyme site;
Downstream Alb-R1:
ACCACCACCAGAACCACCACCACCGGCTAAGATCCCTCG, with flexible linker;
The primer sequence of Porcine interferon-gamma (IFN-γ) is:
Upstream IFN-γ-F1:
GGTGGTTCTGGTGGTGGTGGTTCTATGAGTTATACAACTT, with flexible linker;
Downstream IFN-γ-R1:
ACCACCACCAGAACCACCACCACCTTTTGATGCTCTCTGGCC, with flexible linker;
The primer sequence of pig interleukin 2 and 6 (IL-2) is:
Upstream IL-2-F1:
GGTGGTTCTGGTGGTGGTGGTTCTATGTATAAGATGCAGC, with flexible linker;
Downstream IL-2-R1:CCCTCGAGAGTCAGTGTTGAGTAG has XhoI restriction enzyme site;
B. RNA is extracted from pig liver, by reverse transcription obtain pig Alb, porcine IFN γ and pig IL-2 target gene, three
The gene order of person respectively as shown in 400 < of SEQUENCE LISTING, 4 >, 400 < of SEQUENCE LISTING, 5 > and
Shown in 400 < of SEQUENCE LISTING, 6 >;
Respectively using the target gene of pig Alb, porcine IFN γ and pig IL-2 as template, and it is utilized respectively pig Alb, porcine IFN γ
PCR amplification is carried out with the upstream and downstream primer of pig IL-2.
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. rAlb-IFN γ gene is obtained using flexible linker connection pig Alb and porcine IFN γ target gene
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 connects 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 10 μ L, adds RNase Free water to 25 μ L;Connection
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, totally 35 recycle;Last 72 DEG C of extensions 10min.
D. rAlb-IFN γ-IL2 is obtained using flexible linker connection rAlb-IFN γ gene and pig IL-2 target gene
Gene
The PCR reaction system and reaction condition of connection be:In the overall reaction system of 25 μ L, rAlb-IFN γ gene template
1 μ L of DNA connects 1 μ L, Alb upstream primer of IL-2 template DNA, 0.5 μ L, IL-2 downstream primer, the 0.5 μ L of flexible linker,
2.5 μ L, dNTP Mix of Taq archaeal dna polymerase is 10 μ L, adds RNase Free water to 25 μ L;Connecting PCR reaction condition is:95℃
Initial denaturation 4min, into circulation:94 DEG C of denaturation 45s;58 DEG C of annealing 45s, 72 DEG C of extension 1kb/min, totally 35 recycle;Last 72
DEG C extend 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 pig albumin (Alb) is:
Upstream Alb-F2:CATGCCATGGTGACACCTACAAATCTG has NcoI restriction enzyme site;
Downstream Alb-R2:
ACCACCACCAGAACCACCACCACCAGCCAGGATACCACG, with flexible linker;
The primer sequence of Porcine interferon-gamma (IFN-γ) is:
Upstream IFN-γ-F2:
GGTGGTTCTGGTGGTGGTGGTTCTATGTCTTACACCACCT, with flexible linker;
Downstream IFN-γ-R2:
ACCACCACCAGAACCACCACCACCTTTAGAAGCACGCTG, with flexible linker;
Pig interleukin 2 and 6 (IL-2):
Upstream IL-2-F2:
GGTGGTTCTGGTGGTGGTGGTTCTATGTACAAAATGCAGC, with flexible linker;
Downstream IL-2-R2:
CCCTCGAGGGTCAGGGTAGAGTAG has III restriction enzyme site of Hind.
B. the target gene of the pig Alb, porcine IFN γ and pig IL-2, the gene order of three is respectively such as SEQUENCE
Shown in 400 < of LISTING, 7 >, 400 < of SEQUENCE LISTING 400 <, 8 > and SEQUENCE LISTING, 9 >;
Respectively using the target gene of pig Alb, porcine IFN γ and pig IL-2 as template, and it is utilized respectively pig Alb, porcine IFN γ
PCR amplification is carried out with the upstream and downstream primer of pig IL-2.
PCR reaction system and condition are:In the overall reaction system of 25 μ L, 1 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. rAlb-IFN γ gene is obtained using flexible linker connection pig Alb and porcine IFN γ target gene
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 connects 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 10 μ L, adds RNase Free water to 25 μ L;Connection
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 recycle;Last 72 DEG C of extensions 10min.
D. rAlb-IFN γ-IL2 is obtained using flexible linker connection rAlb-IFN γ gene and pig IL-2 target gene
Gene
The PCR reaction system and reaction condition of connection be:In the overall reaction system of 25 μ L, rAlb-IFN γ gene template
1 μ L of DNA connects 1 μ L, Alb upstream primer of IL-2 template DNA, 0.5 μ L, IL-2 downstream primer, the 0.5 μ L of flexible linker,
2.5 μ L, dNTP Mix of Taq archaeal dna polymerase is 10 μ L, adds RNase Free water to 25 μ L;Connecting PCR reaction condition is:95℃
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 recycle;Last 72
DEG C extend 10min.
The present invention also provides the application of the Recombinant Swine long-acting interferon, long half time had up to 73 hours or more
Broad-spectrum disease resistance toxic action and the immune response that pig itself can be improved.
Compared with prior art, the present invention has the advantages that:
1. pig Alb, porcine IFN γ and pig IL-2 gene are realized amalgamation and expression by flexibility linker, interferon is improved
Half-life period improves 23 times or more compared with plain interferon;Compared with common polyethylene glycol fused interferon, significantly reduce
Cost.
2. improving pig pig Alb, porcine IFN γ and pig by optimizing to pig Alb, porcine IFN γ and pig IL-2 gene
The expression quantity of IL-2 fusion protein.
3. using recombination bacillus coli pET-32a/rAlb-IFN γ-IL2 as expression bacterial strain, by introducing molecular chaperones
PGro7 plasmid, in protein expression, generation inclusion body is less, forms great amount of soluble albumen, avoids inclusion body denaturation and answers
The process of property, substantially reduces the time of expressing fusion protein;
4. the fusion protein disclosed by the invention being made of pig Alb, porcine IFN γ and pig IL-2 not only has IFN-γ
Broad-spectrum disease resistance toxic action, while significantly improving the immune response of pig itself.
Detailed description of the invention
Fig. 1 is that pig albumin gene, 2 gene of porcine interleukin and the Porcine interferon-gamma gene RT-PCR in embodiment 1 are expanded
Result;Swimming lane M:DNA Marker DL2000;Swimming lane 1:2 gene RT-PCR amplified production of porcine interleukin;Swimming lane 2:Pig interference
Plain γ gene RT-PCR amplified production;Swimming lane 3:Pig albumin gene RT-PCR amplified production;
Fig. 2 is the knot of the PCR amplification after pig Alb, the porcine IFN γ in embodiment 1 are connected with the target gene of pig IL-2
Fruit;Swimming lane M:DNA Marker DL10000;Swimming lane 1:Pig albumin gene, Porcine interferon-gamma gene and pig interleukin 2 and 6
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: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:It is unloaded
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:Supernatant after recombinant bacterium induction is broken;Swimming lane 2:It is precipitated after recombinant bacterium induction is broken;
Fig. 6 is that the Recombinant Swine long-acting interferon 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 Swine long-acting interferon of gradient dilution (from right to left) handles hole;
Fig. 7 is the Recombinant Swine long-acting interferon intramuscular injection blood medicine as made from the fusion protein in embodiment 1 in embodiment 8
Concentration-time change curve.
Specific embodiment
Embodiment 1
A kind of fusion protein being made of pig albumin, Porcine interferon-gamma and pig interleukin 2 and 6, preparation method is such as
Under:
1. the acquisition of pig albumin (Alb), Porcine interferon-gamma (IFN-γ) and pig interleukin 2 and 6 (IL-2) target gene
With amplification
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 pig albumin
NcoI restriction enzyme site and Linker sequence are introduced in primer and downstream primer respectively, upstream primer and downstream in Porcine interferon-gamma
Linker sequence is introduced in primer respectively, introduces Linker respectively in the upstream primer and downstream primer of pig interleukin 2 and 6
III restriction enzyme site of sequence and Hind.
1 PCR amplification primer of table
RT-PCR obtains target gene:
RNA is extracted from pig liver tissue, and the target gene of pig Alb, porcine IFN γ and pig IL-2 is obtained by reverse transcription,
The gene order of three is respectively such as 400 < of SEQUENCE LISTING, 4 >, 400 < of SEQUENCE LISTING, 5 > and SEQUENCE
Shown in 400 < of LISTING, 6 >;
RT-PCR reaction system (25 μ L) is shown in Table 2
2 RT-PCR reaction system of table
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 in 1780bp, 560bp and 490bp or so through agarose gel electrophoresis in RT-PCR amplified production,
Its result is as shown in Figure 1, the target gene of pig Alb, porcine IFN γ and pig IL-2 has been prepared in explanation.
2. the connection of target gene
Target gene is diluted to 10ug/mL, connects each section of target gene using over-lap PCR, 25 μ L reaction systems are such as
Shown in table 3, table 4:
3 rAlb-IFN γ PCR reaction system of table
4 rAlb-IFN γ-IL2 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 2770bp or so through agarose gel electrophoresis in pcr amplification product, result as shown in Fig. 2,
Occur rAlb-IFN γ and IL-2 amplified product band in Fig. 2, this is because connected in rAlb-IFN γ with IL-2 gene
In the process, there is non-specific responding.The nucleotide sequence of obtained target gene such as 400 < of SEQUENCE LISTING, 2 > institute
Show.
3. expression vector establishment
For target gene after selection connection after sequencing is errorless, PCR glue recovery product and pET-32a plasmid use NcoI
Double digestion and recycling are carried out with XhoI restriction enzyme, does double digestion by 20 μ L systems in table 5:
5 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 6,4
DEG C overnight connection:
6 enzyme disjunctor system of table
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, competent cell is coated on benzyl containing ammonia
The LB culture medium flat plate of penicillin is incubated overnight;Single colonie on picking LB plate carries out target gene PCR identification, positive colony
Bacteria plasmid identifies that being accredited as positive indicates that engineering bacteria constructs successfully, and PCR amplification and double digestion are produced through NcoI and XhoI double digestion
Object detects single band at the place 2770bp or so through agarose gel electrophoresis, and result is as shown in Figure 3.
4. the expression of recombinant protein
Picking engineering bacteria 37 DEG C of shaking table recovery 1h, engineering bacteria in the LB culture medium containing 100 μ g/ml ampicillins are denoted as
pET-32a/rAlb-IFNγ-IL2;Amplification culture 4h (OD ≈ 1.0) in LB culture medium (the 100 μ g/ml containing ampicillin),
Final concentration 100 μ g/ml IPTG, 32 DEG C of inducing expression 5h is added;Thallus is collected, through SDS-PAGE electrophoresis detection, result is as schemed
Shown in 4, it can be seen from the figure that be deposited in the place 119.8KD or so visible for supernatant after bacterial cell disruption after recombinant bacterium induction 5h
Predominant expression band 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
5.1 His affinity chromatographys
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 rAlb-IFN γ-IL2 protein peak.
5.2 DEAE anion-exchange chromatographies
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, then
After being stablized with the column of Binding Buffer II to A280nm value, with (the 50mM trihydroxy methyl amino first of Elution Buffer II
Alkane, 1M NaCl, PH6.5) linear gradient elution, collect rAlb-IFN γ-IL2 protein peak.
5.3 sieve chromatography
Loading is by with III (50mM of Binding Buffer after the sample concentration that ion-exchange chromatography is collected into
Na2HPO4,0.15M NaCl, PH7.4) 200 molecular sieve chromatography of Superdex has been balanced, it is washed with Binding Buffer III
It is de-, collect rAlb-IFN γ-IL2 protein peak.
5.4 sample identification
Measure rAlb-IFN γ-IL2 potency and specific activity, specific activity >=107U/mg, albumen are qualified;It is aseptic subpackaged, -80
DEG C save.The fusion protein being made of pig albumin, Porcine interferon-gamma and pig interleukin 2 and 6, amino acid sequence can be obtained
Column are as shown in 400 < of SEQUENCE LISTING, 1 >.
Embodiment 2
A kind of fusion protein being made of pig albumin, Porcine interferon-gamma and pig interleukin 2 and 6, other with embodiment 1,
Only e. coli bl21 therein (DE3) competent cell is replaced in order to have BL21 (DE3) competence of pGro7 plasmid
Cell.The SDS-PAGE electrophoresis result of its fusion protein is compareed with embodiment 1,119.8KD or so place's predominant expression in supernatant
Band is thicker, illustrates after introducing molecular chaperones pGro7, expression of the destination protein in supernatant is more preferable, obtained fusion protein
It measures higher.The albumen of Bacillus coli expression is mostly present in inclusion body;By introducing molecular chaperones in expression bacterial strain, assist
It is correctly folded with expression albumen, 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 pig albumin, Porcine interferon-gamma and pig interleukin 2 and 6, preparation method is such as
Under:
1. the acquisition of pig albumin (Alb), Porcine interferon-gamma (IFN-γ) and pig interleukin 2 and 6 (IL-2) target gene
With amplification
Pig Alb, porcine IFN γ and pig IL-2 in embodiment 1 is optimized, artificially synthesized pig Alb, porcine IFN γ and
Pig IL-2 target gene, after optimization, the nucleotide sequence of three is respectively such as 400 < of SEQUENCE LISTING 7 >, SEQUENCE
Shown in 400 < of LISTING 400 <, 8 > and SEQUENCE LISTING, 9 >.
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, in the present embodiment to pig Alb, porcine IFN γ and
Pig IL-2 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.Pig Alb, pig are found using software detection
The codon of IFN-γ and pig IL-2 original gene codon adaptation indexI (CAI) in Escherichia coli is respectively 0.24,0.24,
0.23, GC percentage is 43.7%, 39.2%, 38.7%;And by after to pig Alb, porcine IFN γ and pig IL-2 gene optimization
Obtaining recombination codon adaptation indexI (CAI) in Escherichia coli is respectively 0.98,1.0,0.97, GC percentage
50.3%, 45.4%, 47.6%.The utilization rate of low codon is significantly reduced by gene optimization, avoids rare codon
Influence to protein expression improves the G/C content of gene, improves transcription and translation efficiency, and then improves the table of recombinant protein
Up to amount.
1.3 design of primers:
7 PCR amplification primer of table
The genomic DNA of pig Alb, porcine IFN γ and pig IL-2 after optimization are diluted to 0.05mg/mL respectively.It utilizes
PCR amplification obtains target gene, and 25 μ L reaction systems are as shown in table 8:
8 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.
The pcr amplification product of pig Alb, porcine IFN γ and pig IL-2 are through agarose gel electrophoresis respectively in 1780bp, 560bp
There is specific band with 490bp or so, the purpose base of pig Alb, porcine IFN γ and pig IL-2 after illustrating to be prepared optimization
Cause.
2. the connection of target gene
Target gene is diluted to 10ug/mL, connects each section of target gene using over-lap PCR, 25 μ L reaction systems are such as
Shown in table 9, table 10:
9 rAlb-IFN γ PCR reaction system of table
10 rAlb-IFN γ-IL2 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 2770bp or so through agarose gel electrophoresis in pcr amplification product, illustrate successfully to be connected
RAlb-IFN γ-IL2 gene after connecing, 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
NcoI, XhoI restriction enzyme carry out double digestion and recycling, do double digestion by 20 μ L systems in table 11:
11 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 12,4
DEG C overnight connection:
Table 12
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
It is incubated overnight in the LB plate of element;The bacterium colony grown on LB plate is taken to identify target gene, positive colony bacteria plasmid warp through PCR
The identification of NcoI, XhoI double digestion, being accredited as positive indicates expression vector establishment success, and PCR amplification and double enzyme digestion product are through fine jade
There is single band at the place 2770bp or so in sepharose electrophoresis, illustrates that the expression containing rAlb-IFN γ-IL2 fusion carries
Body constructs successfully.
4. the expression of recombinant protein
Picking engineering bacteria 37 DEG C of shaking table recovery 1h, engineering bacteria in the LB culture medium containing 100 μ g/ml ampicillins are denoted as
pET-32a/rAlb-IFNγ-IL2;Amplification culture 4h (OD ≈ 1.0) in LB culture medium (the 100 μ g/ml containing ampicillin),
Final concentration 100 μ g/ml IPTG, 32 DEG C of inducing expression 5h is added;Thallus is collected, through SDS-PAGE electrophoresis detection, recombinant bacterium induction
Supernatant is deposited in the visible predominant expression band in the place 119.8KD or so after bacterial cell disruption after 5h, illustrates in supernatant precipitating
Recombinant protein is obtained.
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
5.1 His affinity chromatographys
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 rAlb-IFN γ-IL2 protein peak.
5.2 DEAE anion-exchange chromatographies
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, then
After being stablized with the column of Binding Buffer II to A280nm value, with (the 50mM trihydroxy methyl amino first of Elution Buffer II
Alkane, 1M NaCl, PH6.5) linear gradient elution, collect rAlb-IFN γ-IL2 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) 200 molecular sieve chromatography of Superdex has been balanced, with Binding Buffer
III elution, collects rAlb-IFN γ-IL2 protein peak.
5.4 sample identification
Measure rAlb-IFN γ-IL2 potency and specific activity, specific activity >=107U/mg, albumen are qualification;It is aseptic subpackaged ,-
80 DEG C of preservations.The fusion protein being made of pig albumin, Porcine interferon-gamma and pig interleukin 2 and 6, amino acid can be obtained
Sequence is as shown in 400 < of SEQUENCE LISTING, 1 >.
Embodiment 4
A kind of fusion protein being made of pig albumin, Porcine interferon-gamma and pig interleukin 2 and 6, other with embodiment 3,
Only e. coli bl21 therein (DE3) competent cell is replaced in order to have BL21 (DE3) competence of pGro7 plasmid
Cell.The SDS-PAGE electrophoresis result of its fusion protein is compareed with embodiment 3,119.8KD or so place's predominant expression in supernatant
Band is thicker, illustrates after introducing molecular chaperones pGro7, expression of the destination protein in supernatant is more preferable, obtained fusion protein
It measures higher.The albumen of Bacillus coli expression is mostly present in inclusion body;By introducing molecular chaperones in expression bacterial strain, assist
It is correctly folded with expression albumen, 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 Recombinant Swine long-acting interferon, 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 buffer with 10mmol/L PBS,
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 mirror for the fusion protein being made of pig albumin, Porcine interferon-gamma and pig interleukin 2 and 6
It is fixed
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 1.1mg/ml.
6.2 SDS-PAGE electrophoresis detections
Compared with empty bacterium, fusion protein has the newly-increased protein band of a dense dye in 119.8KD or so, as shown in Figure 4.
6.3 Western Blot results
Fusion protein in Examples 1 to 4 is detected, respectively with the anti-pig gamma interferon (1 of abcam company mouse:5000 dilutions) be
Primary antibody, using goat anti-mouse IgG-HRP as secondary antibody (1:10000 dilutions).Recombinant Swine long-acting interferon sample can be interfered with anti-pig
Specific reaction occurs for plain γ monoclonal antibody, and specific band occurs in the place 119.8KD or so, as shown in Figure 5.
Embodiment 7
The freeze-dried bioactivity of four parts of Recombinant Swine long-acting interferons in embodiment 5
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 Swine that various dose is added is long for culture
Interferon is imitated, inhales abandon afterwards for 24 hours, then inoculation 100TCID50VSV virus respectively.
Test result
The result shows that the Recombinant Swine long-acting interferon 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 Recombinant Swine obtained is long
After imitating interferon treated cell inoculation virus, it is observed continuously under inverted microscope, cellular morphology is normal, does not occur any
Lesion measures potency >=107U/ml, as shown in Figure 6.
Embodiment 8
The four parts of Recombinant Swine long-acting interferons obtained respectively by the fusion protein of Examples 1 to 4 in embodiment 5 are freeze-dried
The measurement of (being denoted as A, B, C, D respectively) in pig intracorporal half-life period
The blood concentration and time relationship of cytopathic-effect inhibition assay measurement rAlb-IFN γ-IL2
The pig (half male and half female) that six weight are roughly the same is taken, neck is subcutaneously injected 2mg/ml Recombinant Swine long-acting interferon and freezes
Dry agent 2ml, respectively 1h, 3h, 6h, 12h, for 24 hours, 48h, 72h, 96h, 120h venous blood collection, 4 DEG C of blood sample solidifications, 3500rpm is low
Temperature centrifugation 10min separates serum, and every pig blood sample of each time point is to be measured in -20 DEG C of preservations.Blood is measured using cytopathic-effect inhibition assay
The concentration of rAlb-IFN γ-IL2 in final proof product is carried out curve fitting and calculating parameter with DAS pharmacokinetics software.Parameter calculates knot
Fruit is shown in Table 13.
Dominant dynamic parameters in serum after 13 Recombinant Swine long-acting interferon intramuscular injection of table
The result shows that Recombinant Swine long-acting interferon has longer half-life period.Half-life period can reach 94h or so after measured, more general
Logical interferon improves about 23 times.
Embodiment 9
The freeze-dried measurement that pig cell immune response is influenced of four parts of Recombinant Swine long-acting interferons in embodiment 5
It takes six roughly the same pork pigs of weight to be divided into two groups, is denoted as experimental group and control group;Experimental group neck is subcutaneously infused
The 2mg/ml freeze-dried 2ml of Recombinant Swine long-acting interferon is penetrated, the PBS of 2mL is subcutaneously injected in control group neck, after taking injection 4 weeks outside pig
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-4 content, is carried out by kit specification, and testing result is as shown in table 14:
It is horizontal that 14 ELISA of table detects each group pig cell immune response
The result shows that containing for cell factor IL-4 in pig peripheral blood can be significantly improved after injection Recombinant Swine long-acting interferon
Amount enhances cellullar immunologic response level, significantly improves immunity level.
It is above-mentioned referring to embodiment to a kind of fusion egg being made of pig albumin, Porcine interferon-gamma and pig interleukin 2 and 6
The detailed description that bletilla preparation method carries out, is illustrative without being restrictive, can enumerate according to limited range
Several embodiments, therefore the change and modification in the case where not departing from present general inventive concept, should belong to protection scope of the present invention it
It is interior.
SEQUENCE LISTING
<110>Wuhu Co., Ltd of Ying Tefeier biological products industrial research institute
<120>A kind of fusion protein being made of pig albumin, Porcine interferon-gamma and pig interleukin 2 and 6 and its preparation side
Method
<130> 1
<160> 9
<170> PatentIn version 3.3
<210> 1
<211> 923
<212> PRT
<213>Fusion protein
<400> 1
Asp Thr Tyr Lys Ser Glu Ile Ala His Arg Phe Lys Asp Leu Gly Glu
1 5 10 15
Gln Tyr Phe Lys Gly Leu Val Leu Ile Ala Phe Ser Gln His Leu Gln
20 25 30
Gln Cys Pro Tyr Glu Glu His Val Lys Leu Val Arg Glu Val Thr Glu
35 40 45
Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys
50 55 60
Ser Ile His Thr Leu Phe Gly Asp Lys Leu Cys Ala Ile Pro Ser Leu
65 70 75 80
Arg Glu His Tyr Gly Asp Leu Ala Asp Cys Cys Glu Lys Glu Glu Pro
85 90 95
Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asn Asp Asn Pro Asp Ile
100 105 110
Pro Lys Leu Lys Pro Asp Pro Val Ala Leu Cys Ala Asp Phe Gln Glu
115 120 125
Asp Glu Gln Lys Phe Trp Gly Lys Tyr Leu Tyr Glu Ile Ala Arg Arg
130 135 140
His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Tyr Tyr Ala Ile Ile Tyr
145 150 155 160
Lys Asp Val Phe Ser Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala Cys
165 170 175
Leu Leu Pro Lys Ile Glu His Leu Arg Glu Lys Val Leu Thr Ser Ala
180 185 190
Ala Lys Gln Arg Leu Lys Cys Ala Ser Ile Gln Lys Phe Gly Glu Arg
195 200 205
Ala Phe Lys Ala Trp Ser Leu Ala Arg Leu Ser Gln Arg Phe Pro Lys
210 215 220
Ala Asp Phe Thr Glu Ile Ser Lys Ile Val Thr Asp Leu Ala Lys Val
225 230 235 240
His Lys Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp Arg
245 250 255
Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Thr Ile Ser Thr
260 265 270
Lys Leu Lys Glu Cys Cys Asp Lys Pro Leu Leu Glu Lys Ser His Cys
275 280 285
Ile Ala Glu Ala Lys Arg Asp Glu Leu Pro Ala Asp Leu Asn Pro Leu
290 295 300
Glu His Asp Phe Val Glu Asp Lys Glu Val Cys Lys Asn Tyr Lys Glu
305 310 315 320
Ala Lys His Val Phe Leu Gly Thr Phe Leu Tyr Glu Tyr Ser Arg Arg
325 330 335
His Pro Asp Tyr Ser Val Ser Leu Leu Leu Arg Ile Ala Lys Ile Tyr
340 345 350
Glu Ala Thr Leu Glu Asp Cys Cys Ala Lys Glu Asp Pro Pro Ala Cys
355 360 365
Tyr Ala Thr Val Phe Asp Lys Phe Gln Pro Leu Val Asp Glu Pro Lys
370 375 380
Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Lys Leu Gly Glu Tyr
385 390 395 400
Gly Phe Gln Asn Ala Leu Ile Val Arg Tyr Thr Lys Lys Val Pro Gln
405 410 415
Val Ser Thr Pro Thr Leu Val Glu Val Ala Arg Lys Leu Gly Leu Val
420 425 430
Gly Ser Arg Cys Cys Lys Arg Pro Glu Glu Glu Arg Leu Ser Cys Ala
435 440 445
Glu Asp Tyr Leu Ser Leu Val Leu Asn Arg Leu Cys Val Leu His Glu
450 455 460
Lys Thr Pro Val Ser Glu Lys Val Thr Lys Cys Cys Thr Glu Ser Leu
465 470 475 480
Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Thr Pro Asp Glu Thr Tyr
485 490 495
Lys Pro Lys Glu Phe Val Glu Gly Thr Phe Thr Phe His Ala Asp Leu
500 505 510
Cys Thr Leu Pro Glu Asp Glu Lys Gln Ile Lys Lys Gln Thr Ala Leu
515 520 525
Val Glu Leu Leu Lys His Lys Pro His Ala Thr Glu Glu Gln Leu Arg
530 535 540
Thr Val Leu Gly Asn Phe Ala Ala Phe Val Gln Lys Cys Cys Ala Ala
545 550 555 560
Pro Asp His Glu Ala Cys Phe Ala Val Glu Gly Pro Lys Phe Val Ile
565 570 575
Glu Ile Arg Gly Ile Leu Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly
580 585 590
Ser Met Ser Tyr Thr Thr Tyr Phe Leu Ala Phe Gln Leu Cys Val Thr
595 600 605
Leu Cys Phe Ser Gly Ser Tyr Cys Gln Ala Pro Phe Phe Lys Glu Ile
610 615 620
Thr Ile Leu Lys Asp Tyr Phe Asn Ala Ser Thr Ser Gly Val Pro Asn
625 630 635 640
Gly Gly Pro Leu Phe Leu Glu Ile Leu Glu Asn Trp Lys Glu Glu Ser
645 650 655
Asp Lys Lys Ile Ile Gln Ser Gln Ile Val Ser Phe Tyr Phe Lys Phe
660 665 670
Phe Glu Ile Phe Lys Asp Asn Gln Ala Ile Gln Arg Ser Met Asp Val
675 680 685
Ile Lys Gln Asp Met Phe Gln Arg Phe Leu Asn Gly Ser Ser Gly Lys
690 695 700
Leu Asn Asp Phe Glu Lys Leu Val Lys Ile Pro Val Asp Asn Leu Gln
705 710 715 720
Ile Gln Arg Lys Ala Ile Ser Glu Leu Ile Lys Val Met Asn Asp Leu
725 730 735
Ser Pro Arg Ser Asn Leu Arg Lys Arg Lys Arg Ser Gln Thr Met Phe
740 745 750
Gln Gly Gln Arg Ala Ser Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly
755 760 765
Ser Met Tyr Lys Met Gln Leu Leu Cys Cys Ile Ala Leu Thr Leu Ala
770 775 780
Leu Met Ala Asn Gly Ala Pro Thr Ser Ser Ser Thr Lys Asn Thr Lys
785 790 795 800
Lys Gln Leu Glu Pro Leu Leu Leu Asp Leu Gln Leu Leu Leu Lys Glu
805 810 815
Val Lys Asn Tyr Glu Asn Ala Asp Leu Ser Arg Met Leu Thr Phe Lys
820 825 830
Phe Tyr Met Pro Lys Gln Ala Thr Glu Leu Lys His Leu Gln Cys Leu
835 840 845
Val Glu Glu Leu Lys Ala Leu Glu Gly Val Leu Asn Leu Gly Gln Ser
850 855 860
Lys Asn Ser Asp Ser Ala Asn Ile Lys Glu Ser Met Asn Asn Ile Asn
865 870 875 880
Val Thr Val Leu Glu Leu Lys Gly Ser Glu Thr Ser Phe Lys Cys Glu
885 890 895
Tyr Asp Asp Glu Thr Val Thr Ala Val Glu Phe Leu Asn Lys Trp Ile
900 905 910
Thr Phe Cys Gln Ser Ile Tyr Ser Thr Leu Thr
915 920
<210> 2
<211> 2769
<212> DNA
<213>Genome 1
<400> 2
gatacataca agagtgaaat tgctcatcgg tttaaagatt tgggagaaca atatttcaaa 60
ggcctagtgc tgattgcctt ttctcagcat ctccagcaat gcccatatga agagcatgtg 120
aaattagtga gggaagtaac tgagtttgca aaaacatgtg ttgctgatga gtcagctgaa 180
aattgtgaca agtcaattca cactctcttt ggagataaat tatgtgcaat tccatccctt 240
cgtgaacact atggtgactt ggctgactgc tgtgaaaaag aagagcctga gagaaacgaa 300
tgcttcctcc aacacaaaaa tgataacccc gacatcccta aattgaaacc agaccctgtt 360
gctttatgcg ctgacttcca ggaagatgaa cagaagtttt ggggaaaata cctatatgaa 420
attgccagaa gacatcccta tttctacgcc ccagaactcc tttattatgc cattatatat 480
aaagatgttt tttcagaatg ctgccaagct gctgataaag ctgcctgcct gttaccaaag 540
attgagcatc tgagagaaaa agtactgact tccgccgcca aacagagact taagtgtgcc 600
agtatccaaa aattcggaga gagagctttc aaagcatggt cattagctcg cctgagccag 660
agatttccca aggctgactt tacagagatt tccaagatag tgacagatct tgcaaaagtc 720
cacaaggaat gctgccatgg tgacctgctt gaatgtgcag atgacagggc ggatcttgcc 780
aaatatatat gtgaaaatca agacacaatc tccactaaac tgaaggaatg ctgtgataag 840
cctctgttgg aaaaatccca ctgcattgct gaggcaaaaa gagatgaatt gcctgcagac 900
ctgaacccat tagaacatga ttttgttgaa gataaggaag tttgtaaaaa ctataaagaa 960
gcaaagcatg tcttcctggg cacgtttttg tatgagtatt caagaaggca cccagactac 1020
tctgtctcat tgctgctgag aattgccaag atatatgaag ccacactgga ggactgctgt 1080
gccaaagagg atcctccggc atgctatgcc acagtgtttg ataaatttca gcctcttgtg 1140
gatgagccta agaatttaat caaacaaaac tgtgaacttt ttgaaaaact tggagagtat 1200
ggattccaaa atgcgctcat agttcgttac accaagaaag taccccaagt gtcaactcca 1260
actcttgtgg aggtcgcaag aaaactagga ctagtgggct ctaggtgttg taagcgtcct 1320
gaagaagaaa gactgtcctg tgctgaagac tatctgtccc tggtcctgaa ccggttgtgc 1380
gtgttgcacg agaagacacc agtgagcgaa aaagttacca aatgctgcac agagtccttg 1440
gtgaacagac ggccttgctt ttctgctctg acaccagacg aaacatacaa acccaaagaa 1500
tttgttgagg gaaccttcac cttccatgca gacctatgca cacttcctga ggatgagaaa 1560
caaatcaaga agcaaactgc actcgttgag ttgttgaaac acaagcctca tgcaacagag 1620
gaacaactga gaactgtcct gggcaacttt gcagcctttg tacaaaagtg ctgcgccgct 1680
cctgaccatg aggcctgctt tgctgtggag ggtccgaaat ttgttattga aattcgaggg 1740
atcttagccg gtggtggtgg ttctggtggt ggtggttcta tgagttatac aacttatttc 1800
ttagcttttc agctttgcgt gactttgtgt ttttctggct cttactgcca ggcgcccttt 1860
tttaaagaaa taacgatcct aaaggactat tttaatgcaa gtacctcagg tgtacctaat 1920
ggtggacctc ttttcttaga aattttggag aattggaaag aggagagtga caaaaaaata 1980
attcagagcc aaattgtctc cttctacttc aaattctttg aaatcttcaa agataaccag 2040
gccattcaaa ggagcatgga tgtgatcaag caagacatgt ttcagaggtt cctaaatggt 2100
agctctggga aactgaatga cttcgaaaag ctggttaaaa ttccggtaga taatctgcag 2160
atccagcgca aagccatcag tgaactcatc aaagtgatga atgatctgtc accaagatct 2220
aacctaagaa agcggaagag aagtcagact atgttccaag gccagagagc atcaaaaggt 2280
ggtggtggtt ctggtggtgg tggttctatg tataagatgc agctcttgtg ttgcattgca 2340
ctaacccttg cactcatggc aaacggtgca cctacttcaa gctctacaaa gaacacaaag 2400
aaacaactgg agccattgct gctggattta cagttgcttt tgaaggaagt taagaattac 2460
gagaatgctg atctctccag gatgctcaca tttaaatttt acatgcccaa gcaggctaca 2520
gaattgaaac accttcagtg tttagtagaa gaactcaaag ctctggaggg agtgctaaat 2580
ttaggtcaaa gcaaaaactc tgactcagca aatatcaagg aatcaatgaa caatatcaac 2640
gtaacagttt tggaactaaa gggatctgaa acaagtttca aatgtgaata tgatgatgag 2700
acagtaactg ctgttgaatt tctgaacaaa tggattacct tttgtcaaag catctactca 2760
acactgact 2769
<210> 3
<211> 2769
<212> DNA
<213>Genome 2
<400> 3
gacacctaca aatctgaaat cgctcaccgt ttcaaagacc tgggtgaaca gtacttcaaa 60
ggtctggttc tgatcgcttt ctctcagcac ctgcagcagt gcccgtacga agaacacgtt 120
aaactggttc gtgaagttac cgaattcgct aaaacctgcg ttgctgacga atctgctgaa 180
aactgcgaca aatctatcca caccctgttc ggtgacaaac tgtgcgctat cccgtctctg 240
cgtgaacact acggtgacct ggctgactgc tgcgaaaaag aagaaccgga acgtaacgaa 300
tgcttcctgc agcacaaaaa cgacaacccg gacatcccga aactgaaacc ggacccggtt 360
gctctgtgcg ctgacttcca ggaagacgaa cagaaattct ggggtaaata cctgtacgaa 420
atcgctcgtc gtcacccgta cttctacgct ccggaactgc tgtactacgc tatcatctac 480
aaagacgttt tctctgaatg ctgccaggct gctgacaaag ctgcttgcct gctgccgaaa 540
atcgaacacc tgcgtgaaaa agttctgacc tctgctgcta aacagcgtct gaaatgcgct 600
tctatccaga aattcggtga acgtgctttc aaagcttggt ctctggctcg tctgtcgcag 660
aggttcccga aagctgactt caccgaaatc tctaaaatcg ttaccgacct ggctaaagtt 720
cacaaagaat gctgccacgg tgacctgctg gaatgcgctg acgaccgtgc tgacctggct 780
aaatacatct gcgaaaacca ggacaccatc tctaccaaac tgaaagaatg ctgcgacaaa 840
ccgctgctgg aaaaatctca ctgcatcgct gaagctaaac gtgacgaact gccggctgac 900
ctgaacccgc tggaacacga cttcgttgaa gataaggaag tgtgcaaaaa ctacaaagaa 960
gctaaacacg ttttcctggg taccttcctg tacgaatact ctcgtcgtca cccggactac 1020
tctgtttctc tgctgctgcg tatcgctaaa atctacgaag ctaccctgga agactgctgc 1080
gctaaagaag acccgccggc ttgctacgct accgttttcg acaaattcca gccgctggtt 1140
gacgaaccga aaaacctgat caaacagaac tgcgaactgt tcgaaaaact gggtgaatac 1200
ggtttccaga acgctctgat cgttcgttac accaaaaaag ttccgcaggt ttctaccccg 1260
accctggttg aagttgctcg taaactgggt ctggttggtt ctcgttgctg caaacgtccg 1320
gaagaagaac gtctgtcttg cgctgaagac tacctgtctc tggttctgaa ccgtctgtgc 1380
gttctgcacg aaaaaacccc ggtttctgaa aaagttacca aatgctgcac cgaatctctg 1440
gttaaccgtc gtccgtgctt ctctgctctg accccggacg aaacctacaa accgaaagaa 1500
ttcgttgaag gtaccttcac cttccacgct gacctgtgca ccctgccgga agacgaaaaa 1560
cagatcaaaa aacagaccgc tctggttgaa ctgctgaaac acaaaccgca cgctaccgaa 1620
gaacagctgc gtaccgttct gggtaacttc gctgctttcg ttcagaaatg ctgcgctgct 1680
ccggaccacg aagcttgctt cgctgttgaa ggtccgaaat tcgttatcga aatccgtggt 1740
atcctggctg gtggtggtgg ttctggtggt ggtggttcta tgtcttacac cacctacttc 1800
ctggctttcc agctgtgcgt taccctgtgc ttctctggtt cttactgcca ggctccgttc 1860
ttcaaagaaa tcaccatcct gaaagactac ttcaacgctt ctacctctgg tgttccgaac 1920
ggtggtccgc tgttcctgga aatcctggaa aactggaaag aagaatctga caaaaaaatc 1980
atccagtctc agatcgtttc tttctacttc aaattcttcg aaatcttcaa agacaaccag 2040
gctatccagc gttctatgga cgttatcaaa caggacatgt tccagcgttt cctgaacggt 2100
tcttctggta aactgaacga cttcgaaaaa ctggttaaaa tcccggttga caacctgcag 2160
atccagcgta aagctatctc tgaactgatc aaagttatga acgacctgtc tccgcgttct 2220
aacctgcgta aacgtaaacg ttctcagacc atgttccagg gtcagcgtgc ttctaaaggt 2280
ggtggtggtt ctggtggtgg tggttctatg tacaaaatgc agctgctgtg ctgcatcgct 2340
ctgaccctgg ctctgatggc taacggtgct ccgacctctt cttctaccaa aaacaccaaa 2400
aaacagctgg aaccgctgct gctggacctg cagctgctgc tgaaagaagt taaaaactac 2460
gaaaacgctg acctgtctcg tatgctgacc ttcaaattct acatgccgaa acaggctacc 2520
gaactgaaac acctgcagtg cctggttgaa gaactgaaag ctctggaagg tgttctgaac 2580
ctgggtcagt ctaaaaactc tgactctgct aacatcaaag aatctatgaa caacatcaac 2640
gttaccgttc tggaactgaa aggctcggaa accagcttca aatgcgaata cgacgacgaa 2700
accgttaccg ctgttgaatt cctgaacaaa tggatcacct tctgccagtc tatctactct 2760
accctgacc 2769
<210> 4
<211> 1749
<212> DNA
<213>Pig albumin
<400> 4
gatacataca agagtgaaat tgctcatcgg tttaaagatt tgggagaaca atatttcaaa 60
ggcctagtgc tgattgcctt ttctcagcat ctccagcaat gcccatatga agagcatgtg 120
aaattagtga gggaagtaac tgagtttgca aaaacatgtg ttgctgatga gtcagctgaa 180
aattgtgaca agtcaattca cactctcttt ggagataaat tatgtgcaat tccatccctt 240
cgtgaacact atggtgactt ggctgactgc tgtgaaaaag aagagcctga gagaaacgaa 300
tgcttcctcc aacacaaaaa tgataacccc gacatcccta aattgaaacc agaccctgtt 360
gctttatgcg ctgacttcca ggaagatgaa cagaagtttt ggggaaaata cctatatgaa 420
attgccagaa gacatcccta tttctacgcc ccagaactcc tttattatgc cattatatat 480
aaagatgttt tttcagaatg ctgccaagct gctgataaag ctgcctgcct gttaccaaag 540
attgagcatc tgagagaaaa agtactgact tccgccgcca aacagagact taagtgtgcc 600
agtatccaaa aattcggaga gagagctttc aaagcatggt cattagctcg cctgagccag 660
agatttccca aggctgactt tacagagatt tccaagatag tgacagatct tgcaaaagtc 720
cacaaggaat gctgccatgg tgacctgctt gaatgtgcag atgacagggc ggatcttgcc 780
aaatatatat gtgaaaatca agacacaatc tccactaaac tgaaggaatg ctgtgataag 840
cctctgttgg aaaaatccca ctgcattgct gaggcaaaaa gagatgaatt gcctgcagac 900
ctgaacccat tagaacatga ttttgttgaa gataaggaag tttgtaaaaa ctataaagaa 960
gcaaagcatg tcttcctggg cacgtttttg tatgagtatt caagaaggca cccagactac 1020
tctgtctcat tgctgctgag aattgccaag atatatgaag ccacactgga ggactgctgt 1080
gccaaagagg atcctccggc atgctatgcc acagtgtttg ataaatttca gcctcttgtg 1140
gatgagccta agaatttaat caaacaaaac tgtgaacttt ttgaaaaact tggagagtat 1200
ggattccaaa atgcgctcat agttcgttac accaagaaag taccccaagt gtcaactcca 1260
actcttgtgg aggtcgcaag aaaactagga ctagtgggct ctaggtgttg taagcgtcct 1320
gaagaagaaa gactgtcctg tgctgaagac tatctgtccc tggtcctgaa ccggttgtgc 1380
gtgttgcacg agaagacacc agtgagcgaa aaagttacca aatgctgcac agagtccttg 1440
gtgaacagac ggccttgctt ttctgctctg acaccagacg aaacatacaa acccaaagaa 1500
tttgttgagg gaaccttcac cttccatgca gacctatgca cacttcctga ggatgagaaa 1560
caaatcaaga agcaaactgc actcgttgag ttgttgaaac acaagcctca tgcaacagag 1620
gaacaactga gaactgtcct gggcaacttt gcagcctttg tacaaaagtg ctgcgccgct 1680
cctgaccatg aggcctgctt tgctgtggag ggtccgaaat ttgttattga aattcgaggg 1740
atcttagcc 1749
<210> 5
<211> 498
<212> DNA
<213>Porcine IFN γ
<400> 5
atgagttata caacttattt cttagctttt cagctttgcg tgactttgtg tttttctggc 60
tcttactgcc aggcgccctt ttttaaagaa ataacgatcc taaaggacta ttttaatgca 120
agtacctcag gtgtacctaa tggtggacct cttttcttag aaattttgga gaattggaaa 180
gaggagagtg acaaaaaaat aattcagagc caaattgtct ccttctactt caaattcttt 240
gaaatcttca aagataacca ggccattcaa aggagcatgg atgtgatcaa gcaagacatg 300
tttcagaggt tcctaaatgg tagctctggg aaactgaatg acttcgaaaa gctggttaaa 360
attccggtag ataatctgca gatccagcgc aaagccatca gtgaactcat caaagtgatg 420
aatgatctgt caccaagatc taacctaaga aagcggaaga gaagtcagac tatgttccaa 480
ggccagagag catcaaaa 498
<210> 6
<211> 462
<212> DNA
<213>Pig IL-2
<400> 6
atgtataaga tgcagctctt gtgttgcatt gcactaaccc ttgcactcat ggcaaacggt 60
gcacctactt caagctctac aaagaacaca aagaaacaac tggagccatt gctgctggat 120
ttacagttgc ttttgaagga agttaagaat tacgagaatg ctgatctctc caggatgctc 180
acatttaaat tttacatgcc caagcaggct acagaattga aacaccttca gtgtttagta 240
gaagaactca aagctctgga gggagtgcta aatttaggtc aaagcaaaaa ctctgactca 300
gcaaatatca aggaatcaat gaacaatatc aacgtaacag ttttggaact aaagggatct 360
gaaacaagtt tcaaatgtga atatgatgat gagacagtaa ctgctgttga atttctgaac 420
aaatggatta ccttttgtca aagcatctac tcaacactga ct 462
<210> 7
<211> 1749
<212> DNA
<213>Pig albumin
<400> 7
gacacctaca aatctgaaat cgctcaccgt ttcaaagacc tgggtgaaca gtacttcaaa 60
ggtctggttc tgatcgcttt ctctcagcac ctgcagcagt gcccgtacga agaacacgtt 120
aaactggttc gtgaagttac cgaattcgct aaaacctgcg ttgctgacga atctgctgaa 180
aactgcgaca aatctatcca caccctgttc ggtgacaaac tgtgcgctat cccgtctctg 240
cgtgaacact acggtgacct ggctgactgc tgcgaaaaag aagaaccgga acgtaacgaa 300
tgcttcctgc agcacaaaaa cgacaacccg gacatcccga aactgaaacc ggacccggtt 360
gctctgtgcg ctgacttcca ggaagacgaa cagaaattct ggggtaaata cctgtacgaa 420
atcgctcgtc gtcacccgta cttctacgct ccggaactgc tgtactacgc tatcatctac 480
aaagacgttt tctctgaatg ctgccaggct gctgacaaag ctgcttgcct gctgccgaaa 540
atcgaacacc tgcgtgaaaa agttctgacc tctgctgcta aacagcgtct gaaatgcgct 600
tctatccaga aattcggtga acgtgctttc aaagcttggt ctctggctcg tctgtcgcag 660
aggttcccga aagctgactt caccgaaatc tctaaaatcg ttaccgacct ggctaaagtt 720
cacaaagaat gctgccacgg tgacctgctg gaatgcgctg acgaccgtgc tgacctggct 780
aaatacatct gcgaaaacca ggacaccatc tctaccaaac tgaaagaatg ctgcgacaaa 840
ccgctgctgg aaaaatctca ctgcatcgct gaagctaaac gtgacgaact gccggctgac 900
ctgaacccgc tggaacacga cttcgttgaa gataaggaag tgtgcaaaaa ctacaaagaa 960
gctaaacacg ttttcctggg taccttcctg tacgaatact ctcgtcgtca cccggactac 1020
tctgtttctc tgctgctgcg tatcgctaaa atctacgaag ctaccctgga agactgctgc 1080
gctaaagaag acccgccggc ttgctacgct accgttttcg acaaattcca gccgctggtt 1140
gacgaaccga aaaacctgat caaacagaac tgcgaactgt tcgaaaaact gggtgaatac 1200
ggtttccaga acgctctgat cgttcgttac accaaaaaag ttccgcaggt ttctaccccg 1260
accctggttg aagttgctcg taaactgggt ctggttggtt ctcgttgctg caaacgtccg 1320
gaagaagaac gtctgtcttg cgctgaagac tacctgtctc tggttctgaa ccgtctgtgc 1380
gttctgcacg aaaaaacccc ggtttctgaa aaagttacca aatgctgcac cgaatctctg 1440
gttaaccgtc gtccgtgctt ctctgctctg accccggacg aaacctacaa accgaaagaa 1500
ttcgttgaag gtaccttcac cttccacgct gacctgtgca ccctgccgga agacgaaaaa 1560
cagatcaaaa aacagaccgc tctggttgaa ctgctgaaac acaaaccgca cgctaccgaa 1620
gaacagctgc gtaccgttct gggtaacttc gctgctttcg ttcagaaatg ctgcgctgct 1680
ccggaccacg aagcttgctt cgctgttgaa ggtccgaaat tcgttatcga aatccgtggt 1740
atcctggct 1749
<210> 8
<211> 498
<212> DNA
<213>Porcine IFN γ
<400> 8
atgtcttaca ccacctactt cctggctttc cagctgtgcg ttaccctgtg cttctctggt 60
tcttactgcc aggctccgtt cttcaaagaa atcaccatcc tgaaagacta cttcaacgct 120
tctacctctg gtgttccgaa cggtggtccg ctgttcctgg aaatcctgga aaactggaaa 180
gaagaatctg acaaaaaaat catccagtct cagatcgttt ctttctactt caaattcttc 240
gaaatcttca aagacaacca ggctatccag cgttctatgg acgttatcaa acaggacatg 300
ttccagcgtt tcctgaacgg ttcttctggt aaactgaacg acttcgaaaa actggttaaa 360
atcccggttg acaacctgca gatccagcgt aaagctatct ctgaactgat caaagttatg 420
aacgacctgt ctccgcgttc taacctgcgt aaacgtaaac gttctcagac catgttccag 480
ggtcagcgtg cttctaaa 498
<210> 9
<211> 462
<212> DNA
<213>Pig IL-2
<400> 9
atgtacaaaa tgcagctgct gtgctgcatc gctctgaccc tggctctgat ggctaacggt 60
gctccgacct cttcttctac caaaaacacc aaaaaacagc tggaaccgct gctgctggac 120
ctgcagctgc tgctgaaaga agttaaaaac tacgaaaacg ctgacctgtc tcgtatgctg 180
accttcaaat tctacatgcc gaaacaggct accgaactga aacacctgca gtgcctggtt 240
gaagaactga aagctctgga aggtgttctg aacctgggtc agtctaaaaa ctctgactct 300
gctaacatca aagaatctat gaacaacatc aacgttaccg ttctggaact gaaaggctcg 360
gaaaccagct tcaaatgcga atacgacgac gaaaccgtta ccgctgttga attcctgaac 420
aaatggatca ccttctgcca gtctatctac tctaccctga cc 462
Claims (10)
1. a kind of fusion protein being made of pig albumin, Porcine interferon-gamma and pig interleukin 2 and 6, it is characterised in that:It is described
The amino acid sequence table of fusion protein is denoted as fusion protein 1 as shown in 400 < of SEQUENCE LISTING, 1 >;Or such as
Shown in 400 < of SEQUENCE LISTING, 2 >, it is denoted as fusion protein 2.
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, 3 >;Or as shown in 400 < of SEQUENCE LISTING, 4 >,
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 Recombinant Swine long-acting interferon, which is characterized in that the Recombinant Swine 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/rAlb-IFN
γ-IL2, preparation method are:
(1) design primer obtains by reverse transcription or is manually respectively synthesized the pig albumin for connecting flexible linker sequence, pig
The target gene of interferon gamma, pig interleukin 2 and 6;By flexible linker by pig albumin, Porcine interferon-gamma, pig leucocyte
The target gene of interleukin 2 connects, the nucleotides sequence list of the target gene after connection such as SEQUENCE LISTING 400
Shown in 3 > of < or as shown in 400 < of SEQUENCE LISTING, 4 >;
(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-IL2。
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 Recombinant Swine 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 94 hours or more, there is broad-spectrum disease resistance toxic action and the immune response of pig itself can be improved.
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CN201710673412.5A CN107365390A (en) | 2017-08-09 | 2017-08-09 | A kind of fusion protein being made up of pig albumin, Porcine interferon-gamma and pig interleukin 2 and 6 and preparation method thereof |
CN2017106734125 | 2017-08-09 |
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Publication Number | Publication Date |
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CN108864302A true CN108864302A (en) | 2018-11-23 |
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CN201710673412.5A Pending CN107365390A (en) | 2017-08-09 | 2017-08-09 | A kind of fusion protein being made up of pig albumin, Porcine interferon-gamma and pig interleukin 2 and 6 and preparation method thereof |
CN201810752613.9A Withdrawn CN108864302A (en) | 2017-08-09 | 2018-07-10 | A kind of fusion protein and preparation method thereof being made of pig albumin, Porcine interferon-gamma and pig interleukin 2 and 6 |
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CN201710673412.5A Pending CN107365390A (en) | 2017-08-09 | 2017-08-09 | A kind of fusion protein being made up of pig albumin, Porcine interferon-gamma and pig interleukin 2 and 6 and preparation method thereof |
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CN108653194B (en) * | 2018-07-06 | 2020-09-29 | 中国科学院微生物研究所 | Chewing gum type medicine with fusion interferon as active component |
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2017
- 2017-08-09 CN CN201710673412.5A patent/CN107365390A/en active Pending
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Application publication date: 20181123 |