CN108822221A - A kind of fusion protein and preparation method thereof being made of Chicken Albumin, chicken interferon gamma and recombinant chIL-2 - Google Patents
A kind of fusion protein and preparation method thereof being made of Chicken Albumin, chicken interferon gamma and recombinant chIL-2 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 Chicken Albumin, chicken interferon gamma and recombinant chIL-2; the fusion protein is connected through flexible linker by Chicken Albumin, chicken interferon gamma and recombinant chIL-2 and is formed, and is freeze-dried to obtain recombination chicken long-acting interferon after fusion protein and freeze drying protectant mixture.The recombination chicken long-acting interferon is remarkably improved the half-life period of chicken interferon, and the half-life period of more common chicken interferon improves 21 times or more, and has broad-spectrum disease resistance toxic action and can improve the immune response of chicken itself.
Description
Technical field
The invention belongs to technical field of biological genetic engineering, and in particular to white thin by Chicken Albumin, chicken interferon gamma and chicken
The fusion protein and preparation method thereof that born of the same parents' interleukin 2 forms.
Background technique
In recent years, as the scale of aquaculture and livestock and poultry and products thereof circulation industry rapidly develop, China's domestic fowl farming is taken
Tremendous development is obtained, the huge industry that annual value of production exceedes hundred billion yuan is formd.However due to the animal epidemic prevention system of China's weakness, poultry diease
It is still the serious problems that China's poultry production faces, this has become an important factor for restricting the development of China's poultry cultivation industry.
Poultry diease is more, loss is big, and the death rate is higher than 15%, and for death and culling rate up to 20%~25% (developed country is less than 5%), China's poultry husbandry is every
Year chicken The dead quantity caused by infectious disease is about 300,000,000, about 3,000,000,000 yuan of the economic loss directly contributed, caused by between
Connect about 10,000,000,000 yuan of loss.
At present for the prevention and treatment of chicken viral diseases mainly using vaccine immunity and drug therapy, due to epidemic disease
The immune serotype of seedling is single, and virus serotype is numerous and virus stain speed of mutation is fast, to frequently result in vaccine immunity mistake
It loses.Although some antibiotic and chemically synthesized antiviral drugs have some effects to a small number of viruses, since medicament residue passes through
Food chain is brought a negative impact to human health, and China prohibited some antibiotic and antibacterial agent in aquaculture since 16 years
In application.Therefore, be actively developed production have no toxic side effect, drug residue free and the interferon formulation for not generating drug resistance, it is right
Chicken viral diseases, which prevent and treat predicament, at present important clinical meaning.
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
In order to solve the above technical problems, the present invention provides one kind by Chicken Albumin, chicken interferon gamma and chicken interleukins
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 recombination chicken long-acting interferon, the recombination chicken long-acting interferon is remarkably improved the half-life period of chicken interferon, compared with
The half-life period of common chicken interferon improves 21 times or more, and has broad-spectrum disease resistance toxic action and can improve the immune of chicken itself and answer
It answers.
The technical scheme adopted by the invention is as follows:
A kind of fusion protein being made of Chicken Albumin, chicken interferon gamma and recombinant chIL-2, 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.
The genome 1 and the equal codified fusion protein of the genome 2.Genome 2 is the nucleotides sequence to genome 1
Column optimize after as a result, when usual codon adaptation indexI CAI=1.0 be considered the gene and be in the expression system
Optimal high efficient expression state, CAI value is lower to show that expression is lower in host.G/C content most ideal distribution model in gene
Enclosing is 30~70%, is more than that the range will affect translation and transcriptional efficiency in any region.Chicken is found using software detection
Albumin, chicken IFN-γ, chicken IL-2 gene original gene codon in Escherichia coli codon adaptation indexI (CAI) be respectively
0.27,0.25,0.27, GC percentage is 43.1%, 42.9%, 36.1%;And by Chicken Albumin, chicken IFN-γ, chicken IL-
It is 0.99,1.0,1.0, GC percentage that each gene codon adaptation indexI (CAI) in Escherichia coli is obtained after 2 gene optimizations
49.2%, 47.6%, 46.2%.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.
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 recombination chicken long-acting interferon, the recombination chicken 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 chicken for connecting flexible linker sequence
The target gene of white, chicken interferon gamma, recombinant chIL-2;By flexible linker by Chicken Albumin, chicken interferon gamma, chicken
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 Chicken Albumin (Alb) is:
Upstream Alb-F1:CCGGAATTCATGAAGTGGGTAACATTA has EcoRI restriction enzyme site;
Downstream Alb-R1:
ACCACCACCAGAACCACCACCACCAGCACCAATTCCTAATG, with flexible linker;
The primer sequence of chicken interferon gamma (IFN-γ) is:
Upstream IFN-γ-F1:
GGTGGTTCTGGTGGTGGTGGTTCTATGACTTGCCAGACTT, with flexible linker;
Downstream IFN-γ-R1:
ACCACCACCAGAACCACCACCACCGCAATTGCATCTCCTC, with flexible linker;
The primer sequence of recombinant chIL-2 (IL-2) is:
Upstream IL-2-F1:
GGTGGTTCTGGTGGTGGTGGTTCTATGATGTGCAAAGTACT, with flexible linker;
Downstream IL-2-R1:CCCTCGAGAGTTTTTTGCAGATATC has XhoI restriction enzyme site;
B. RNA is extracted from chicken liver, by reverse transcription obtain chicken Alb, chicken IFN-γ and chicken IL-2 gene 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 chicken Alb, chicken IFN-γ and chicken IL-2 gene as template, and it is utilized respectively chicken Alb, chicken IFN-γ
PCR amplification is carried out with the upstream and downstream primer of chicken IL-2 gene.
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 chicken Alb and chicken 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 base is obtained using flexible linker connection rAlb-IFN γ gene and chicken IL2 target gene
Cause
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 Chicken Albumin (Alb) is:
Upstream Alb-F2:CGGGATCCATGAAATGGGTTACCC has BamHI restriction enzyme site;
Downstream Alb-R2:
ACCACCACCAGAACCACCACCACCAGCACCGATACCCA, with flexible linker;
The primer sequence of chicken interferon gamma (IFN-γ) is:
Upstream IFN-γ-F2:
GGTGGTTCTGGTGGTGGTGGTTCTATGACCTGCCAGAC, with flexible linker;
Downstream IFN-γ-R2:
ACCACCACCAGAACCACCACCACCGCAGTTGCAACGAC, with flexible linker;
Recombinant chIL-2 (IL-2):
Upstream IL-2-F2:
GGTGGTTCTGGTGGTGGTGGTTCTATGATGTGCAAAGTTCT, with flexible linker;
Downstream IL-2-R2:
CCCTCGAGTTTCTGCAGGTAACG has XhoI restriction enzyme site.
B. the chicken Alb, chicken IFN-γ and chicken IL-2 gene target gene, 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 chicken Alb, chicken IFN-γ and chicken IL-2 gene as template, and it is utilized respectively chicken Alb, chicken IFN-γ
PCR amplification is carried out with the upstream and downstream primer of chicken IL-2 gene.
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. rAlb-IFN γ gene is obtained using flexible linker connection chicken Alb and chicken 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 chicken IL-2 gene 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 recombination chicken long-acting interferon, long half time had up to 84 hours or more
Broad-spectrum disease resistance toxic action and the immune response that chicken itself can be improved.
Compared with prior art, the present invention has the advantages that:
1. chicken Alb, chicken IFN-γ and chicken IL-2 gene gene are realized amalgamation and expression by flexibility linker, interferon is improved
Half-life period improves 21 times or more compared with plain interferon;Compared with common polyethylene glycol fused interferon, significantly reduce
Cost.
2. improving chicken Alb, chicken IFN-γ and chicken by optimizing to chicken Alb, chicken IFN-γ and chicken IL-2 gene 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 chicken Alb, chicken IFN-γ and chicken IL-2 gene not only has IFN-γ
Broad-spectrum disease resistance toxic action, while significantly improving the immune response of chicken itself.
Detailed description of the invention
Fig. 1 is that Chicken Albumin gene, 2 gene of chicken interleukin-2 and the chicken 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 chicken interleukin-2;Swimming lane 2:Chicken interference
Plain γ gene RT-PCR amplified production;Swimming lane 3:Chicken Albumin gene RT-PCR amplified production;
Fig. 2 is the result of the PCR amplification after chicken Alb, the IFN γ in embodiment 1 are connected with the target gene of IL-2;Swimming
Road M:DNA Marker DL10000;Swimming lane 1:Chicken Albumin gene, chicken interferon gamma gene connect expansion with 2 gene of chicken interleukin-2
Increase production object;
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:Supernatant after bacterial cell disruption after recombinant bacterium induction;Swimming lane 2:It is precipitated 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: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 chicken long-acting interferon α as made from the fusion protein in embodiment 1 causes cell to VSV in embodiment 5
The inhibiting effect of lesion;1 is VSV virus control wells;2 be HEp-2 cell control well;A3-12 is gradient dilution (from right to left)
Human interferon standard items handle hole;B3-12 is that the recombination chicken long-acting interferon of gradient dilution (from right to left) handles hole;
Fig. 7 is the recombination chicken long-acting interferon α intramuscular injection blood as made from the fusion protein in embodiment 1 in embodiment 8
Concentration-time changing curve.
Specific embodiment
Embodiment 1
A kind of fusion protein being made of Chicken Albumin, chicken interferon gamma and recombinant chIL-2, preparation method is such as
Under:
1. the acquisition of Chicken Albumin (Alb), chicken interferon gamma (IFN-γ) and recombinant chIL-2 (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 Chicken Albumin
EcoRI restriction enzyme site and Linker sequence are introduced in primer and downstream primer respectively, chicken interferon gamma upstream primer and under
Linker sequence is introduced respectively in trip primer, is introduced respectively in the upstream primer and downstream primer of recombinant chIL-2
Linker sequence and XhoI restriction enzyme site.
1 PCR amplification primer of table
RT-PCR obtains target gene:
RNA is extracted from chicken liver tissue, the target gene of chicken Alb, chicken IFN-γ and chicken IL-2 gene are 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
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 in 1870bp, 550bp and 460bp or so through agarose gel electrophoresis in RT-PCR amplified production,
Its result is as shown in Figure 1, the target gene of chicken Alb, chicken IFN-γ and chicken IL-2 gene 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 2830bp 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
After sequencing is errorless, PCR glue recovery product uses target gene after selection connection 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 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, PCR amplification and double digestion through EcoRI and XhoI double digestion
Product detects single band at the place 2830bp 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 121.9KD 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,
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-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 Chicken Albumin, chicken interferon gamma and recombinant chIL-2, 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 Chicken Albumin, chicken interferon gamma and recombinant chIL-2, 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 1,121.9KD 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 Chicken Albumin, chicken interferon gamma and recombinant chIL-2, preparation method is such as
Under:
1. the acquisition of Chicken Albumin (Alb), chicken interferon gamma (IFN-γ) and recombinant chIL-2 (IL-2) target gene
With amplification
Chicken Alb, chicken IFN-γ and chicken IL-2 gene in embodiment 1 is optimized, artificial synthesized chicken Alb, chicken IFN-γ and
Chicken IL-2 gene 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 chicken Alb, chicken IFN-γ and
Chicken IL-2 gene 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.Chicken Alb, chicken are found using software detection
The codon of IFN-γ and chicken IL-2 gene original gene codon adaptation indexI (CAI) in Escherichia coli is respectively 0.27,0.25,
0.27, GC percentage is 43.1%, 42.9%, 36.1%;And by after to chicken Alb, chicken IFN-γ and chicken IL-2 gene gene optimization
Obtain recombination codon adaptation indexI (CAI) in Escherichia coli be respectively 0.99,1.0,1.0, GC percentage 49.2%,
47.6%, 46.2%.The utilization rate of low codon is significantly reduced by gene optimization, avoids rare codon to albumen table
The influence reached improves the G/C content of gene, improves transcription and translation efficiency, and then improves the expression quantity of recombinant protein.
1.3 design of primers:
7 PCR amplification primer of table
The genomic DNA of chicken Alb, chicken IFN-γ and chicken IL-2 gene 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 chicken Alb, chicken IFN-γ and chicken IL-2 gene are through agarose gel electrophoresis respectively in 1870bp, 550bp
There is specific band with 460bp or so, the purpose base of chicken Alb, chicken IFN-γ and chicken IL-2 gene 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 2830bp 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
BamHI, 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
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 BamHI, 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 2830bp 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 121.9KD 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,
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-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 Chicken Albumin, chicken interferon gamma and recombinant chIL-2, 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 Chicken Albumin, chicken interferon gamma and recombinant chIL-2, 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 3,121.9KD 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 recombination chicken 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 Chicken Albumin, chicken interferon gamma and recombinant chIL-2
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 121.9KD 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-chicken interferon-γ (1 of abcam company mouse:5000 dilutions) be
Primary antibody, using goat anti-mouse IgG-HRP as secondary antibody (1:10000 dilutions).Recombination chicken long-acting interferon sample can be interfered with anti-chicken
Specific reaction occurs for plain γ monoclonal antibody, and specific band occurs in the place 121.9KD or so, as shown in Figure 5.
Embodiment 7
The freeze-dried bioactivity of four parts of recombination chicken 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 recombination chicken that various dose is added is long for culture
Interferon is imitated, inhales abandon afterwards for 24 hours, then is inoculated with 100 TCID50 VSV viruses respectively.
Test result
The result shows that the recombination chicken 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 recombination chicken 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 recombination chicken 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 chicken intracorporal half-life period
The blood concentration and time relationship of cytopathic-effect inhibition assay measurement rAlb-IFN γ-IL2
The broiler chicken (half male and half female) that six weight are roughly the same is taken, 2mg/ml recombination chicken long-acting interferon is subcutaneously injected in neck
Freeze-dried 2ml, respectively in 1h, 2h, 4h, 8h, 16h, 32h, 48h, 72h, 96h venous blood collection, 4 DEG C of blood sample solidify, and 3500rpm is low
Temperature centrifugation 10min separates serum, and every chicken 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 recombination chicken long-acting interferon intramuscular injection of table
The result shows that recombination chicken long-acting interferon has longer half-life period.Half-life period can reach 84h or so after measured, more general
Logical interferon improves about 21 times.
Embodiment 9
The freeze-dried measurement that chicken cell immune response is influenced of four parts of recombination chicken long-acting interferons in embodiment 5
It takes six roughly the same broiler chicken 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 recombination chicken long-acting interferon is penetrated, the PBS of 2mL is subcutaneously injected in control group neck, after taking injection 4 weeks outside chicken
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 chicken cell immune response
The result shows that containing for chicken Evaluation of Cytokines in Peripheral Blood IL-4 can be significantly improved after injection recombination chicken 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 Chicken Albumin, chicken interferon gamma and recombinant chIL-2
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 Chicken Albumin, chicken interferon gamma and recombinant chIL-2 and its preparation side
Method
<130> 1
<160> 9
<170> PatentIn version 3.3
<210> 1
<211> 942
<212> PRT
<213>Fusion protein
<400> 1
Met Lys Trp Val Thr Leu Ile Ser Phe Ile Phe Leu Phe Ser Ser Ala
1 5 10 15
Thr Ser Arg Asn Leu Gln Arg Phe Ala Arg Asp Ala Glu His Lys Ser
20 25 30
Glu Ile Ala His Arg Tyr Asn Asp Leu Lys Glu Glu Thr Phe Lys Ala
35 40 45
Val Ala Met Ile Thr Phe Ala Gln Tyr Leu Gln Arg Cys Ser Tyr Glu
50 55 60
Gly Leu Ser Lys Leu Val Lys Asp Val Val Asp Leu Ala Gln Lys Cys
65 70 75 80
Val Ala Asn Glu Asp Ala Pro Glu Cys Ser Lys Pro Leu Pro Ser Ile
85 90 95
Ile Leu Asp Glu Ile Cys Gln Val Glu Lys Leu Arg Asp Ser Tyr Gly
100 105 110
Ala Met Ala Asp Cys Cys Ser Lys Ala Asp Pro Glu Arg Asn Glu Cys
115 120 125
Phe Leu Ser Phe Lys Val Ser Gln Pro Asp Phe Val Gln Pro Tyr Gln
130 135 140
Arg Pro Ala Ser Asp Val Ile Cys Gln Glu Tyr Gln Asp Asn Arg Val
145 150 155 160
Ser Phe Leu Gly His Phe Ile Tyr Ser Val Ala Arg Arg His Pro Phe
165 170 175
Leu Tyr Ala Pro Ala Ile Leu Ser Phe Ala Val Asp Phe Glu His Ala
180 185 190
Leu Gln Ser Cys Cys Lys Glu Ser Asp Val Gly Ala Cys Leu Asp Thr
195 200 205
Lys Glu Ile Val Met Arg Glu Lys Ala Lys Gly Val Ser Val Lys Gln
210 215 220
Gln Tyr Phe Cys Gly Ile Leu Lys Gln Phe Gly Asp Arg Val Phe Gln
225 230 235 240
Ala Arg Gln Leu Ile Tyr Leu Ser Gln Lys Tyr Pro Lys Ala Pro Phe
245 250 255
Ser Glu Val Ser Lys Phe Val His Asp Ser Ile Gly Val His Lys Glu
260 265 270
Cys Cys Glu Gly Asp Met Val Glu Cys Met Asp Asp Met Ala Arg Met
275 280 285
Met Ser Asn Leu Cys Ser Gln Gln Asp Val Phe Ser Gly Lys Ile Lys
290 295 300
Asp Cys Cys Glu Lys Pro Ile Val Glu Arg Ser Gln Cys Ile Met Glu
305 310 315 320
Ala Glu Phe Asp Glu Lys Pro Ala Asp Leu Pro Ser Leu Val Glu Lys
325 330 335
Tyr Ile Glu Asp Lys Glu Val Cys Lys Ser Phe Glu Ala Gly His Asp
340 345 350
Ala Phe Met Ala Glu Phe Val Tyr Glu Tyr Ser Arg Arg His Pro Glu
355 360 365
Phe Ser Ile Gln Leu Ile Met Arg Ile Ala Lys Gly Tyr Glu Ser Leu
370 375 380
Leu Glu Lys Cys Cys Lys Thr Asp Asn Pro Ala Glu Cys Tyr Ala Asn
385 390 395 400
Ala Gln Glu Gln Leu Asn Gln His Ile Lys Glu Thr Gln Asp Val Val
405 410 415
Lys Thr Asn Cys Asp Leu Leu His Asp His Gly Glu Ala Asp Phe Leu
420 425 430
Lys Ser Ile Leu Ile Arg Tyr Thr Lys Lys Met Pro Gln Val Pro Thr
435 440 445
Asp Leu Leu Leu Glu Thr Gly Lys Lys Met Thr Thr Ile Gly Thr Lys
450 455 460
Cys Cys Gln Leu Pro Glu Asp Arg Arg Met Ala Cys Ser Glu Gly Tyr
465 470 475 480
Leu Ser Ile Val Ile His Asp Thr Cys Arg Lys Gln Glu Thr Thr Pro
485 490 495
Ile Asn Asp Asn Val Ser Gln Cys Cys Ser Ser Ser Tyr Ala Asn Arg
500 505 510
Arg Pro Cys Phe Thr Ala Met Gly Val Asp Thr Lys Tyr Val Pro Pro
515 520 525
Pro Phe Asn Pro Asp Met Phe Ser Phe Asp Glu Lys Leu Cys Ser Ala
530 535 540
Pro Ala Glu Glu Arg Glu Val Gly Gln Met Lys Leu Leu Ile Asn Leu
545 550 555 560
Ile Lys Arg Lys Pro Gln Met Thr Glu Glu Gln Ile Lys Thr Ile Ala
565 570 575
Asp Gly Phe Thr Ala Met Val Asp Lys Cys Cys Lys Gln Ser Asp Ile
580 585 590
Asn Thr Cys Phe Gly Glu Glu Gly Ala Asn Leu Ile Val Gln Ser Arg
595 600 605
Ala Thr Leu Gly Ile Gly Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly
610 615 620
Ser Met Thr Cys Gln Thr Tyr Asn Leu Phe Val Leu Ser Val Ile Met
625 630 635 640
Ile Tyr Tyr Gly His Thr Ala Ser Ser Leu Ile Leu Val Gln Leu Gln
645 650 655
Asp Asp Ile Ala Lys Leu Lys Ala Asp Phe Asn Ser Ser His Ser Asp
660 665 670
Val Ala Asp Gly Gly Pro Ile Ile Ala Glu Lys Leu Lys Asn Trp Thr
675 680 685
Glu Arg Asn Gln Lys Arg Ile Ile Leu Ser Gln Ile Val Ser Met Tyr
690 695 700
Leu Glu Met Leu Ala Asn Thr Asp Lys Thr Lys Pro His Thr Lys His
705 710 715 720
Ile Ser Glu Glu Leu Tyr Thr Leu Lys Asn Asn Leu Pro Asp Gly Val
725 730 735
Lys Lys Val Lys Asp Ile Met Asp Leu Ala Lys Leu Pro Met Asn Asp
740 745 750
Leu Arg Val Gln Leu Lys Ala Ala Asn Glu Leu Phe Ser Ile Leu Gln
755 760 765
Lys Leu Val Asn Pro Pro Ser Phe Lys Arg Asn Met Ser Gln Ser Gln
770 775 780
Arg Arg Cys Asn Cys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Met
785 790 795 800
Met Cys Lys Val Leu Ile Phe Gly Cys Ile Ser Val Ala Met Leu Met
805 810 815
Thr Thr Ala Tyr Gly Ala Ser Leu Ser Ser Glu Lys Trp Lys Thr Leu
820 825 830
Gln Thr Leu Ile Lys Asp Leu Glu Ile Leu Glu Asn Ile Lys Asn Lys
835 840 845
Ile His Leu Glu Leu Tyr Thr Pro Thr Glu Thr Gln Glu Cys Thr Gln
850 855 860
Gln Thr Leu Gln Cys Tyr Leu Gly Glu Val Val Thr Leu Lys Lys Glu
865 870 875 880
Thr Glu Asp Asp Thr Glu Ile Lys Glu Glu Phe Val Thr Ala Ile Gln
885 890 895
Asn Ile Glu Lys Asn Leu Lys Ser Leu Thr Gly Leu Asn His Thr Gly
900 905 910
Ser Glu Cys Lys Ile Cys Glu Ala Asn Asn Lys Lys Lys Phe Pro Asp
915 920 925
Phe Leu His Glu Leu Thr Asn Phe Val Arg Tyr Leu Gln Lys
930 935 940
<210> 2
<211> 2826
<212> DNA
<213>Genome 1
<400> 2
atgaagtggg taacattaat ttcattcatt ttcctcttca gttcagcaac atccaggaat 60
ctgcaaagat ttgctcgtga tgcagagcac aagagtgaaa ttgcccatcg ctacaatgat 120
ttgaaagaag aaacatttaa ggcagttgcc atgatcacat ttgcccagta tctccagagg 180
tgctcttatg aaggactgtc taagcttgtg aaggatgttg ttgatctggc acaaaaatgt 240
gtagccaatg aagatgctcc tgaatgctca aaaccactgc cttccattat cctggatgaa 300
atctgccaag tggaaaagct ccgtgactct tatggtgcaa tggccgactg ctgtagcaaa 360
gctgatcctg aaagaaatga gtgtttcctg tcatttaaag tttcccaacc agacttcgtt 420
cagccatacc aaagaccagc ttctgatgtg atatgccagg aataccagga caacagagtg 480
tcatttctgg gacatttcat ctattctgtt gcaagaagac accccttctt gtatgcccct 540
gcaatcctta gttttgctgt tgattttgaa catgcacttc aaagctgttg caaagagagt 600
gatgtcggtg cttgcctgga caccaaggaa attgttatga gagaaaaagc caagggagta 660
agtgtgaagc agcagtattt ttgtggaatc ttgaagcagt tcggagatag agttttccaa 720
gcacgacaac ttatttacct aagccaaaaa taccccaagg ctccattctc agaggtttct 780
aaatttgtac atgattctat cggcgtccac aaagagtgct gtgaagggga catggtggag 840
tgcatggatg acatggcacg tatgatgagc aatctgtgct ctcaacaaga tgttttctca 900
ggtaaaatca aagactgctg tgagaagcct attgtggaac gaagccagtg cattatggag 960
gcagaatttg atgagaaacc tgcagatctt ccttcattag ttgaaaagta catagaagat 1020
aaggaagtgt gtaaaagttt tgaagcaggc cacgatgcat tcatggcaga gttcgtttat 1080
gaatactcac gaagacaccc tgagttctcc atacagctta ttatgagaat tgccaaagga 1140
tatgaatcac ttctggaaaa gtgctgcaaa actgataacc ctgctgagtg ctacgcaaat 1200
gctcaagagc aactgaacca acatatcaaa gaaactcagg atgttgtgaa gacaaactgt 1260
gatcttctcc atgaccatgg cgaggcagac ttcctcaagt ccatcctgat ccgctacact 1320
aagaaaatgc ctcaagtacc aactgatctc ctgcttgaaa ctggaaagaa aatgacaact 1380
attggtacta agtgctgcca gcttcctgaa gacagacgca tggcttgttc tgagggttat 1440
ctgagcattg tgattcatga tacgtgcagg aaacaggaga ccacacctat aaatgacaac 1500
gtttcacaat gctgcagcag ctcctatgct aacagaagac catgtttcac tgctatggga 1560
gtagatacca aatatgttcc tccaccattt aatcctgata tgttcagctt tgatgaaaaa 1620
ttgtgcagtg ctcctgctga agaacgagaa gtaggccaga tgaaattgct aatcaacctc 1680
attaaacgca agccccagat gacagaagaa caaataaaga caattgctga tggtttcact 1740
gccatggttg acaagtgctg caagcagtcg gacatcaata catgctttgg agaagagggt 1800
gccaacctaa tagtccaaag cagagccaca ttaggaattg gtgctggtgg tggtggttct 1860
ggtggtggtg gttctatgac ttgccagact tacaacttgt ttgttctgtc cgtcatcatg 1920
atttattatg gacatactgc aagtagtcta attcttgttc aacttcaaga tgatatagcc 1980
aaactgaaag ctgactttaa ctcaagtcat tcagatgtag ctgacggtgg acctattatt 2040
gcagagaaac tgaagaactg gacagagaga aatcagaaaa ggatcatact gagccagatt 2100
gtttcgatgt acttggaaat gcttgcaaac actgacaaga caaagccgca caccaaacac 2160
atatctgagg agctctatac tctgaaaaac aaccttcctg atggcgtgaa gaaggtgaaa 2220
gatatcatgg acctggccaa gctcccgatg aacgacttga gagtccagct caaagccgcg 2280
aatgaactct tcagcatctt acagaagctg gtgaatcctc cgagtttcaa aaggaacatg 2340
agccagtctc agaggagatg caattgcggt ggtggtggtt ctggtggtgg tggttctatg 2400
atgtgcaaag tactgatctt tggctgtatt tcggtagcaa tgctaatgac tacagcttat 2460
ggagcatctc tatcatcaga aaaatggaaa actcttcaaa cattaataaa ggatttagaa 2520
atattggaaa atatcaagaa taagattcat ctcgagctct acacaccaac tgagacccag 2580
gagtgcaccc agcaaactct gcagtgttac ctgggagaag tggttactct gaagaaagaa 2640
actgaagatg acactgaaat taaagaagaa tttgtaactg ctattcaaaa tatcgaaaag 2700
aacctcaaga gtcttacggg tctaaatcac accggaagtg aatgcaagat ctgtgaagct 2760
aacaacaaga aaaaatttcc tgattttctc catgaactga ccaactttgt gagatatctg 2820
caaaaa 2826
<210> 3
<211> 2826
<212> DNA
<213>Genome 2
<400> 3
atgaaatggg ttaccctgat ctctttcatc ttcctgttct cttctgctac ctctcgtaac 60
ctgcagcgtt tcgctcgtga cgctgaacac aaatctgaaa tcgctcaccg ttacaacgac 120
ctgaaagaag aaaccttcaa agctgttgct atgatcacct tcgctcagta cctgcagcgt 180
tgctcttacg aaggtctgtc taaactggtt aaagacgttg ttgacctggc tcagaaatgc 240
gttgctaacg aagacgctcc ggaatgctct aaaccgctgc cgtctatcat cctggacgaa 300
atctgccagg ttgaaaaact gcgtgactct tacggtgcta tggctgactg ctgctctaaa 360
gctgacccgg aacgtaacga atgcttcctg tctttcaaag tttctcagcc ggacttcgtt 420
cagccgtacc agcgtccggc ttctgacgtt atctgccagg aataccagga caaccgtgtt 480
tctttcctgg gtcacttcat ctactctgtt gctcgtcgtc acccgttcct gtacgctccg 540
gctatcctgt ctttcgctgt tgacttcgaa cacgctctgc agtcttgctg caaagaatct 600
gacgttggtg cttgcctgga caccaaagaa atcgttatgc gtgaaaaagc taaaggtgtt 660
tctgttaaac agcagtactt ctgcggtatc ctgaaacagt tcggtgaccg tgttttccag 720
gctcgtcagc tgatctacct gtctcagaaa tacccgaaag ctccgttctc tgaagtttct 780
aaattcgttc acgactctat cggtgttcac aaagaatgct gcgaaggtga catggttgaa 840
tgcatggacg acatggctcg tatgatgtct aacctgtgct ctcagcagga cgttttctct 900
ggtaaaatca aagactgctg cgaaaaaccg atcgttgaac gttctcagtg catcatggaa 960
gctgaattcg acgaaaaacc ggctgacctg ccgtctctgg ttgaaaaata catcgaagac 1020
aaagaagttt gcaaatcttt cgaagctggt cacgacgctt tcatggctga attcgtttac 1080
gaatactctc gtcgtcaccc ggaattctct atccagctga tcatgcgtat cgctaaaggt 1140
tacgaatctc tgctggaaaa atgctgcaaa accgacaacc cggctgaatg ctacgctaac 1200
gctcaggaac agctgaacca gcacatcaaa gaaacccagg acgttgttaa aaccaactgc 1260
gacctgctgc acgaccacgg tgaagctgac ttcctgaaat ctatcctgat ccgttacacc 1320
aaaaaaatgc cgcaggttcc gaccgacctg ctgctggaaa ccggtaaaaa aatgaccacc 1380
atcggtacca aatgctgcca gctgccggaa gaccgtcgta tggcttgctc tgaaggttac 1440
ctgtctatcg ttatccacga cacctgccgt aaacaggaaa ccaccccgat caacgacaac 1500
gtttctcagt gctgctcttc ttcttacgct aaccgtcgtc cgtgcttcac cgctatgggt 1560
gttgacacca aatacgttcc gccgccgttc aacccggaca tgttctcttt cgacgaaaaa 1620
ctgtgctctg ctccggctga agaacgtgaa gttggtcaga tgaaactgct gatcaacctg 1680
atcaaacgta aaccgcagat gaccgaagaa cagatcaaaa ccatagcgga cggcttcacc 1740
gctatggttg acaaatgctg caaacagtct gacatcaaca cctgcttcgg tgaagaaggt 1800
gctaacctga tcgttcagtc tcgtgctacc ctgggtatcg gtgctggtgg tggtggttct 1860
ggtggtggtg gttctatgac ctgccagacc tacaacctgt tcgttctgtc tgttatcatg 1920
atctactacg gtcacaccgc ttcttctctg atcctggttc agctgcagga cgacatcgct 1980
aaactgaaag ctgacttcaa ctcttctcac tctgacgttg ctgacggtgg tccgatcatc 2040
gctgaaaaac tgaaaaactg gaccgaacgt aaccagaaac gtatcatcct gtctcagatc 2100
gtttctatgt acctggaaat gctggctaac accgacaaaa ccaaaccgca caccaaacac 2160
atctctgaag aactgtacac cctgaaaaac aacctgccgg acggtgttaa aaaagttaaa 2220
gacatcatgg acctggctaa actgccgatg aacgacctgc gtgttcagct gaaagctgct 2280
aacgaactgt tctctatcct gcagaaactg gttaacccgc cgtctttcaa acgtaacatg 2340
tctcagtctc agcgtcgttg caactgcggt ggtggtggtt ctggtggtgg tggttctatg 2400
atgtgcaaag ttctgatctt cggttgcatc tctgttgcta tgctgatgac caccgcttac 2460
ggtgcttctc tgtcttctga aaaatggaaa accctgcaga ccctgatcaa agacctggaa 2520
atcctggaaa acatcaaaaa caaaatccac ctggaactgt acaccccgac cgaaacccag 2580
gaatgcaccc agcagaccct gcagtgctac ctgggtgaag ttgttaccct gaaaaaagaa 2640
accgaagacg acaccgaaat caaagaagaa ttcgttaccg ctatccagaa catcgaaaaa 2700
aacctgaaat ctctgaccgg tctgaaccac accggttctg aatgcaaaat ctgcgaagct 2760
aacaacaaaa aaaaattccc ggacttcctg cacgaactga ccaacttcgt tcgttacctg 2820
cagaaa 2826
<210> 4
<211> 1845
<212> DNA
<213>Chicken Albumin
<400> 4
atgaagtggg taacattaat ttcattcatt ttcctcttca gttcagcaac atccaggaat 60
ctgcaaagat ttgctcgtga tgcagagcac aagagtgaaa ttgcccatcg ctacaatgat 120
ttgaaagaag aaacatttaa ggcagttgcc atgatcacat ttgcccagta tctccagagg 180
tgctcttatg aaggactgtc taagcttgtg aaggatgttg ttgatctggc acaaaaatgt 240
gtagccaatg aagatgctcc tgaatgctca aaaccactgc cttccattat cctggatgaa 300
atctgccaag tggaaaagct ccgtgactct tatggtgcaa tggccgactg ctgtagcaaa 360
gctgatcctg aaagaaatga gtgtttcctg tcatttaaag tttcccaacc agacttcgtt 420
cagccatacc aaagaccagc ttctgatgtg atatgccagg aataccagga caacagagtg 480
tcatttctgg gacatttcat ctattctgtt gcaagaagac accccttctt gtatgcccct 540
gcaatcctta gttttgctgt tgattttgaa catgcacttc aaagctgttg caaagagagt 600
gatgtcggtg cttgcctgga caccaaggaa attgttatga gagaaaaagc caagggagta 660
agtgtgaagc agcagtattt ttgtggaatc ttgaagcagt tcggagatag agttttccaa 720
gcacgacaac ttatttacct aagccaaaaa taccccaagg ctccattctc agaggtttct 780
aaatttgtac atgattctat cggcgtccac aaagagtgct gtgaagggga catggtggag 840
tgcatggatg acatggcacg tatgatgagc aatctgtgct ctcaacaaga tgttttctca 900
ggtaaaatca aagactgctg tgagaagcct attgtggaac gaagccagtg cattatggag 960
gcagaatttg atgagaaacc tgcagatctt ccttcattag ttgaaaagta catagaagat 1020
aaggaagtgt gtaaaagttt tgaagcaggc cacgatgcat tcatggcaga gttcgtttat 1080
gaatactcac gaagacaccc tgagttctcc atacagctta ttatgagaat tgccaaagga 1140
tatgaatcac ttctggaaaa gtgctgcaaa actgataacc ctgctgagtg ctacgcaaat 1200
gctcaagagc aactgaacca acatatcaaa gaaactcagg atgttgtgaa gacaaactgt 1260
gatcttctcc atgaccatgg cgaggcagac ttcctcaagt ccatcctgat ccgctacact 1320
aagaaaatgc ctcaagtacc aactgatctc ctgcttgaaa ctggaaagaa aatgacaact 1380
attggtacta agtgctgcca gcttcctgaa gacagacgca tggcttgttc tgagggttat 1440
ctgagcattg tgattcatga tacgtgcagg aaacaggaga ccacacctat aaatgacaac 1500
gtttcacaat gctgcagcag ctcctatgct aacagaagac catgtttcac tgctatggga 1560
gtagatacca aatatgttcc tccaccattt aatcctgata tgttcagctt tgatgaaaaa 1620
ttgtgcagtg ctcctgctga agaacgagaa gtaggccaga tgaaattgct aatcaacctc 1680
attaaacgca agccccagat gacagaagaa caaataaaga caattgctga tggtttcact 1740
gccatggttg acaagtgctg caagcagtcg gacatcaata catgctttgg agaagagggt 1800
gccaacctaa tagtccaaag cagagccaca ttaggaattg gtgct 1845
<210> 5
<211> 492
<212> DNA
<213>Chicken IFN-γ
<400> 5
atgacttgcc agacttacaa cttgtttgtt ctgtccgtca tcatgattta ttatggacat 60
actgcaagta gtctaattct tgttcaactt caagatgata tagccaaact gaaagctgac 120
tttaactcaa gtcattcaga tgtagctgac ggtggaccta ttattgcaga gaaactgaag 180
aactggacag agagaaatca gaaaaggatc atactgagcc agattgtttc gatgtacttg 240
gaaatgcttg caaacactga caagacaaag ccgcacacca aacacatatc tgaggagctc 300
tatactctga aaaacaacct tcctgatggc gtgaagaagg tgaaagatat catggacctg 360
gccaagctcc cgatgaacga cttgagagtc cagctcaaag ccgcgaatga actcttcagc 420
atcttacaga agctggtgaa tcctccgagt ttcaaaagga acatgagcca gtctcagagg 480
agatgcaatt gc 492
<210> 6
<211> 429
<212> DNA
<213>Chicken IL-2 gene
<400> 6
atgatgtgca aagtactgat ctttggctgt atttcggtag caatgctaat gactacagct 60
tatggagcat ctctatcatc agaaaaatgg aaaactcttc aaacattaat aaaggattta 120
gaaatattgg aaaatatcaa gaataagatt catctcgagc tctacacacc aactgagacc 180
caggagtgca cccagcaaac tctgcagtgt tacctgggag aagtggttac tctgaagaaa 240
gaaactgaag atgacactga aattaaagaa gaatttgtaa ctgctattca aaatatcgaa 300
aagaacctca agagtcttac gggtctaaat cacaccggaa gtgaatgcaa gatctgtgaa 360
gctaacaaca agaaaaaatt tcctgatttt ctccatgaac tgaccaactt tgtgagatat 420
ctgcaaaaa 429
<210> 7
<211> 1845
<212> DNA
<213>Chicken Albumin
<400> 7
atgaaatggg ttaccctgat ctctttcatc ttcctgttct cttctgctac ctctcgtaac 60
ctgcagcgtt tcgctcgtga cgctgaacac aaatctgaaa tcgctcaccg ttacaacgac 120
ctgaaagaag aaaccttcaa agctgttgct atgatcacct tcgctcagta cctgcagcgt 180
tgctcttacg aaggtctgtc taaactggtt aaagacgttg ttgacctggc tcagaaatgc 240
gttgctaacg aagacgctcc ggaatgctct aaaccgctgc cgtctatcat cctggacgaa 300
atctgccagg ttgaaaaact gcgtgactct tacggtgcta tggctgactg ctgctctaaa 360
gctgacccgg aacgtaacga atgcttcctg tctttcaaag tttctcagcc ggacttcgtt 420
cagccgtacc agcgtccggc ttctgacgtt atctgccagg aataccagga caaccgtgtt 480
tctttcctgg gtcacttcat ctactctgtt gctcgtcgtc acccgttcct gtacgctccg 540
gctatcctgt ctttcgctgt tgacttcgaa cacgctctgc agtcttgctg caaagaatct 600
gacgttggtg cttgcctgga caccaaagaa atcgttatgc gtgaaaaagc taaaggtgtt 660
tctgttaaac agcagtactt ctgcggtatc ctgaaacagt tcggtgaccg tgttttccag 720
gctcgtcagc tgatctacct gtctcagaaa tacccgaaag ctccgttctc tgaagtttct 780
aaattcgttc acgactctat cggtgttcac aaagaatgct gcgaaggtga catggttgaa 840
tgcatggacg acatggctcg tatgatgtct aacctgtgct ctcagcagga cgttttctct 900
ggtaaaatca aagactgctg cgaaaaaccg atcgttgaac gttctcagtg catcatggaa 960
gctgaattcg acgaaaaacc ggctgacctg ccgtctctgg ttgaaaaata catcgaagac 1020
aaagaagttt gcaaatcttt cgaagctggt cacgacgctt tcatggctga attcgtttac 1080
gaatactctc gtcgtcaccc ggaattctct atccagctga tcatgcgtat cgctaaaggt 1140
tacgaatctc tgctggaaaa atgctgcaaa accgacaacc cggctgaatg ctacgctaac 1200
gctcaggaac agctgaacca gcacatcaaa gaaacccagg acgttgttaa aaccaactgc 1260
gacctgctgc acgaccacgg tgaagctgac ttcctgaaat ctatcctgat ccgttacacc 1320
aaaaaaatgc cgcaggttcc gaccgacctg ctgctggaaa ccggtaaaaa aatgaccacc 1380
atcggtacca aatgctgcca gctgccggaa gaccgtcgta tggcttgctc tgaaggttac 1440
ctgtctatcg ttatccacga cacctgccgt aaacaggaaa ccaccccgat caacgacaac 1500
gtttctcagt gctgctcttc ttcttacgct aaccgtcgtc cgtgcttcac cgctatgggt 1560
gttgacacca aatacgttcc gccgccgttc aacccggaca tgttctcttt cgacgaaaaa 1620
ctgtgctctg ctccggctga agaacgtgaa gttggtcaga tgaaactgct gatcaacctg 1680
atcaaacgta aaccgcagat gaccgaagaa cagatcaaaa ccatagcgga cggcttcacc 1740
gctatggttg acaaatgctg caaacagtct gacatcaaca cctgcttcgg tgaagaaggt 1800
gctaacctga tcgttcagtc tcgtgctacc ctgggtatcg gtgct 1845
<210> 8
<211> 492
<212> DNA
<213>Chicken IFN-γ
<400> 8
atgacctgcc agacctacaa cctgttcgtt ctgtctgtta tcatgatcta ctacggtcac 60
accgcttctt ctctgatcct ggttcagctg caggacgaca tcgctaaact gaaagctgac 120
ttcaactctt ctcactctga cgttgctgac ggtggtccga tcatcgctga aaaactgaaa 180
aactggaccg aacgtaacca gaaacgtatc atcctgtctc agatcgtttc tatgtacctg 240
gaaatgctgg ctaacaccga caaaaccaaa ccgcacacca aacacatctc tgaagaactg 300
tacaccctga aaaacaacct gccggacggt gttaaaaaag ttaaagacat catggacctg 360
gctaaactgc cgatgaacga cctgcgtgtt cagctgaaag ctgctaacga actgttctct 420
atcctgcaga aactggttaa cccgccgtct ttcaaacgta acatgtctca gtctcagcgt 480
cgttgcaact gc 492
<210> 9
<211> 429
<212> DNA
<213>Chicken IL-2 gene
<400> 9
atgatgtgca aagttctgat cttcggttgc atctctgttg ctatgctgat gaccaccgct 60
tacggtgctt ctctgtcttc tgaaaaatgg aaaaccctgc agaccctgat caaagacctg 120
gaaatcctgg aaaacatcaa aaacaaaatc cacctggaac tgtacacccc gaccgaaacc 180
caggaatgca cccagcagac cctgcagtgc tacctgggtg aagttgttac cctgaaaaaa 240
gaaaccgaag acgacaccga aatcaaagaa gaattcgtta ccgctatcca gaacatcgaa 300
aaaaacctga aatctctgac cggtctgaac cacaccggtt ctgaatgcaa aatctgcgaa 360
gctaacaaca aaaaaaaatt cccggacttc ctgcacgaac tgaccaactt cgttcgttac 420
ctgcagaaa 429
Claims (10)
1. a kind of fusion protein being made of Chicken Albumin, chicken interferon gamma and recombinant chIL-2, it is characterised in that:It is described
The amino acid sequence table of fusion protein 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 chicken long-acting interferon, which is characterized in that the recombination chicken 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 Chicken Albumin for connecting flexible linker sequence, chicken
The target gene of interferon gamma, recombinant chIL-2;By flexible linker by Chicken Albumin, chicken interferon gamma, chicken 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 2 > of < 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-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 recombination chicken long-acting interferon according to claim 5, which is characterized in that the recombination chicken is long-acting dry
The long half time of element is disturbed up to 84 hours or more, there is broad-spectrum disease resistance toxic action and the immune response of chicken itself can be improved.
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CN2017106760897 | 2017-08-09 | ||
CN201710676089.7A CN107383202A (en) | 2017-08-09 | 2017-08-09 | A kind of fusion protein being made up of OVA, chicken interferon gamma and recombinant chIL-2 and preparation method thereof |
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Publication Number | Publication Date |
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CN108822221A true CN108822221A (en) | 2018-11-16 |
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CN201710676089.7A Pending CN107383202A (en) | 2017-08-09 | 2017-08-09 | A kind of fusion protein being made up of OVA, chicken interferon gamma and recombinant chIL-2 and preparation method thereof |
CN201810752509.XA Withdrawn CN108822221A (en) | 2017-08-09 | 2018-07-10 | A kind of fusion protein and preparation method thereof being made of Chicken Albumin, chicken interferon gamma and recombinant chIL-2 |
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CN201710676089.7A Pending CN107383202A (en) | 2017-08-09 | 2017-08-09 | A kind of fusion protein being made up of OVA, chicken interferon gamma and recombinant chIL-2 and preparation method thereof |
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CN114224851A (en) * | 2021-11-19 | 2022-03-25 | 北京东方百泰生物科技股份有限公司 | Freeze-dried powder preparation of human interleukin 10-Fc fusion protein and preparation method thereof |
-
2017
- 2017-08-09 CN CN201710676089.7A patent/CN107383202A/en active Pending
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2018
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Application publication date: 20181116 |
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