CN108840937A - A kind of fusion protein being made of Chicken Albumin and chicken interferon gamma and preparation method thereof and a kind of recombination chicken long-acting interferon γ - Google Patents

Abstract

The invention discloses a kind of fusion protein being made of Chicken Albumin and chicken interferon gamma and preparation method thereof and a kind of recombination chicken long-acting interferon γ; the fusion protein is connected through flexible linker by Chicken Albumin and chicken interferon gamma and is formed, and is freeze-dried to obtain recombination chicken long-acting interferon γ after fusion protein and freeze drying protectant mixture.The recombination chicken long-acting interferon γ is remarkably improved the half-life period of chicken interferon, and the half-life period of more common chicken interferon gamma improves 15 times or more, and has broad-spectrum disease resistance toxic action and can improve the immune response of chicken itself.

Description

A kind of fusion protein and preparation method thereof being made of Chicken Albumin and chicken interferon gamma With a kind of recombination chicken long-acting interferon γ

Technical field

The invention belongs to technical field of biological genetic engineering, and in particular to one kind is made of Chicken Albumin and chicken interferon gamma Fusion protein and preparation method thereof and a kind of recombination chicken long-acting interferon γ.

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.

The limitation of natural interferon and artificial recombination interferon generally existing half-life short at present, half-life period are generally 2-4 A hour.Half-life short brings great inconvenience, the increase for the treatment of number of times, corresponding time cost and economic cost to treatment Increase therewith, and the tenability limit of body is also possible to be broken the generation for leading to adverse reaction.The main reason for half-life short There are two：The too small tachytrophism in vivo of the molecular weight of interferon, interferon especially recombinant interferon affinity is poor to be exempted from Epidemic disease system is removed.And common long-acting interferon is using polyethylene glycol fused interferon as representative on the market, only in the layer of molecular weight Partially solve the problems, such as that interferon molecule amount is small and leads to half-life short on face.By being carried out to two kinds of different type interferon Fusion, not only can be improved the molecular weight of interferon, but also can cooperate with the effect for playing two kinds of interferon.

Seralbumin is the important component of blood plasma, is not easy under normal circumstances through glomerulus, internal distributed pole it is wide and There is no zymetology and immunologic competence, is ideal pharmaceutical carrier.Albumin fusion technology has the advantage that：Albumin and target egg It is white to be linked in the cell through protein translation system by peptide bond, it is not required to additional extracorporeal treatment；The expression of albumin is higher, The expression of destination protein can be improved after merging with it；Albumin is one stable " inert protein ", after merging with it The stability of destination protein can be improved；Albumin fusion protein has longer drug half-life, by small molecular protein drug It can be expected to improve half-life period in blood with Albumin fusion.Currently, in experimental animal after multiple protein and Albumin fusion The extension of Half-life in vivo is confirmed.

And common long-acting interferon is using polyethylene glycol fused interferon as representative on the market, only in the level of molecular weight Part solves the problems, such as that interferon molecule amount is small and leads to half-life short, while polyethylene glycol fused interferon cost is very Height is unfavorable for clinically applying.

Summary of the invention

In order to solve the above technical problems, merging egg with what chicken interferon gamma formed by Chicken Albumin the present invention provides a kind of Bletilla preparation method and a kind of recombination chicken long-acting interferon γ, the recombination chicken long-acting interferon γ are remarkably improved chicken interference The half-life period of element, the half-life period of more common chicken interferon gamma improve 15 times or more, and have broad-spectrum disease resistance toxic action and can improve The immune response of chicken itself.

The technical scheme adopted by the invention is as follows：

A kind of fusion protein being made of Chicken Albumin and chicken interferon gamma, the amino acid sequence table of the fusion protein is such as Shown in 400 < of SEQUENCE LISTING, 1 >.

The present invention also provides the gene for encoding above-mentioned fusion protein, the nucleotides sequence list such as SEQUENCE of the gene Shown in 400 < of LISTING, 2 >, it is denoted as genome 1；Or as shown in 400 < of SEQUENCE LISTING, 3 >, it is denoted as genome 2.

Fusion protein 1 described in 1 codified of genome；The 2 codified fusion protein 2 of genome.Genome 2 is pair The nucleotide sequence of genome 1 optimize after as a result, when usual codon adaptation indexI CAI=1.0 be considered the base Because being optimal high efficient expression state in the expression system, CAI value is lower to show that expression is lower in host.In gene G/C content most ideal distribution range is 30~70%, is more than that the range will affect translation and transcriptional efficiency in any region. The codon of the Chicken Albumin and IFN-γ original gene codon adaptation indexI in Escherichia coli is found using software detection (CAI) be respectively 0.27,0.25, GC percentage be 43.1%, 42.9%；And by Chicken Albumin and IFN-γ gene optimization After obtain recombination genome 2 in Escherichia coli codon adaptation indexI (CAI) be 0.99,1.0, GC percentage 49.2%, 47.6%.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.

The present invention also provides the expression vectors containing genome 1 or genome 2.

Further, the expression vector is the pET-32a coli expression carrier containing genome 1 or genome 2.

The present invention also provides the genetic engineering bacteriums containing genome 1 or genome 2.

Further, the genetic engineering bacterium is pET-32a/rAlb-IFN γ.

Host cell containing genome 1 or genome 2 also belongs to protection scope of the present invention, further, the place Chief cell is e. coli host cell, and further, the e. coli host cell is BL21 (DE3) competent cell Or BL21 (DE3) competent cell with pGro7 plasmid.

The present invention also provides a kind of recombination chicken long-acting interferon γ, the recombination chicken long-acting interferon γ is melted by described It is freeze-dried to form after hop protein and freeze drying protectant mixture.

The freeze drying protectant is glycerol, mannitol and sucrose, is buffer, the final concentration of three with 10mmol/L PBS For glycerol 100mL/L, mannitol 0.12g/mL and sucrose 0.025g/mL.

The invention also discloses the preparation methods of the fusion protein, and the preparation method comprises the following steps：It will contain The expression vector of genome 1 or genome 2 is imported into e. coli host cell, obtains genetic engineering bacterium, genetic engineering bacterium The crude product of the fusion protein is obtained after IPTG inducing expression, and fusion protein can be obtained after purified.

The expression vector is the pET-32a coli expression carrier containing genome 1 or genome 2；

The genetic engineering bacterium is pET-32a/rAlb-IFN γ, and preparation method is：

(1) design primer, is obtained or the Chicken Albumin of the flexible linker sequence of artificial synthesized connection by reverse transcription With the target gene of chicken interferon gamma；Chicken Albumin has been connected with the target gene of chicken interferon gamma by flexible linker Come, the nucleotides sequence list of target gene is as shown in 400 < of SEQUENCE LISTING, 2 > or such as SEQUENCE LISTING Shown in 400 <, 3 >；

(2) target gene after connection is connected on pET-32a plasmid and obtains expression vector；

(3) expression vector is imported into e. coli host cell, genetic engineering bacterium pET-32a/rAlb- can be obtained IFNγ。

The e. coli host cell is BL21 (DE3) competent cell or BL21 (DE3) sense with pGro7 plasmid By state cell.

The method of the purifying is：The crude product of fusion protein is successively through affinity chromatography, anion-exchange chromatography and molecular sieve Chromatographic purifying.

BL21 (DE3) competent cell with pGro7 plasmid is purchased from Shanghai Jinan Technology Co., Ltd./glad hundred promise Biology, article No. V205.

The acquisition methods of the genome 1 are：

A. design of primers

The primer sequence of Chicken Albumin (Alb) is：

Upstream Alb-F1：CCGGAATTCATGATGTGCAAAGTACT has EcoRI restriction enzyme site；

Downstream Alb-R1：

ACCACCACCAGAACCACCACCACCTTTTTGCAGATATCTCAC, 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：GCGTCGACGCAATTGCATCTCCTC has SalI restriction enzyme site；

B. RNA is extracted from chicken liver, and the target gene of Alb and IFN-γ, the gene sequence of the two are obtained by reverse transcription Column are respectively as shown in 400 < of SEQUENCE LISTING 400 <, 4 > and SEQUENCE LISTING, 5 >；

Respectively using Alb and the target gene of IFN-γ as template, and be utilized respectively the upstream and downstream primer of Alb and IFN-γ into Row PCR amplification respectively obtains and connects the Alb of flexible linker and the target gene of IFN-γ.

PCR reaction system and condition are：In the overall reaction system of 25 μ L, 1.5 μ L of template ribonucleic acid, upstream and downstream primer 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 condition of the RT-PCR reaction For：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 extend 1kb/min is recycled 35 times, last 72 DEG C of extensions 10min.

C. Alb gene and IFN-γ gene are connected using flexible linker

The PCR reaction system and reaction condition of connection be：In the overall reaction system of 25 μ L, connect flexible linker's 1 μ L of Alb template DNA connects 1 μ L, Alb upstream primer of IFN-γ template DNA, the 0.5 μ L of flexible linker, IFN-γ downstream 0.5 μ L, Taq archaeal dna polymerase of primer, 2.5 μ L, dNTP Mix is 9.0 μ L, adds RNase Free water to 25 μ L；Connect PCR reaction Condition is：95 DEG C of initial denaturation 4min, into circulation：94 DEG C of denaturation 45s；58 DEG C of annealing 45s, 72 DEG C of extension 1kb/min, totally 35 Circulation；Last 72 DEG C of extensions 10min.

Genome 2 is artificial synthesized gene after optimizing to genome 1, the acquisition methods of the genome 2 For：

A. design of primers

The primer sequence of 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：CCCTCGAGGCAGTTGCAACGAC has XhoI restriction enzyme site；

B. the target gene of the Alb and IFN-γ, the gene order of the two is respectively such as SEQUENCE LISTING 400 Shown in 6 > and SEQUENCE LISTING of <, 400 <, 7 >；

Respectively using Alb and the target gene of IFN-γ as template, and be utilized respectively the upstream and downstream primer of Alb and IFN-γ into Row PCR amplification, the target gene of Alb and IFN-γ after respectively obtaining the optimization for connecting flexible linker.

PCR reaction system and condition are：In the overall reaction system of 25 μ L, 1.0 μ L of genomic DNA, upstream and downstream primer is each 0.5 μ L, Taq archaeal dna polymerase, 2.5 μ L, dNTP Mix is 10 μ L, adds RNase Free water to 25 μ L；The RT-PCR reaction Reaction condition is：95 DEG C of initial denaturation 4min, into circulation；95 DEG C of denaturation 45s, 60 DEG C of annealing 45s, 72 DEG C of extension 1kb/min are followed Ring 35 times, last 72 DEG C of extensions 10min.

C. Alb gene and IFN-γ gene are connected using flexible linker

The PCR reaction system and reaction condition of connection be：In the overall reaction system of 25 μ L, connect flexible linker's 1 μ L of Alb template DNA connects 1 μ L, Alb upstream primer of IFN-γ template DNA, the 0.5 μ L of flexible linker, IFN-γ downstream 0.5 μ L, Taq archaeal dna polymerase of primer, 2.5 μ L, dNTP Mix is 9.0 μ L, adds RNase Free water to 25 μ L；Connect PCR reaction Condition is：95 DEG C of initial denaturation 4min, into circulation：94 DEG C of denaturation 45s；60 DEG C of annealing 45s, 72 DEG C of extension 1kb/min, totally 35 Circulation；Last 72 DEG C of extensions 10min.

The present invention also provides the application of the recombination chicken long-acting interferon γ, long half time was up to 61 hours or more, tool There is broad-spectrum disease resistance toxic action and the immune response of chicken itself can be improved.

Compared with prior art, the present invention has the advantages that：

1. Chicken Albumin and chicken interferon gamma gene are realized fusion by flexibility linker, improves interferon and partly decline Phase improves 15 times or more compared with plain interferon；Compared with common polyethylene glycol fused interferon, significantly reduce into This.

2. improving chicken Alb and chicken interferon gamma fusion by optimizing to Chicken Albumin and chicken interferon gamma gene The expression quantity of albumen.

3. using recombination bacillus coli BL21/pET-32a-Alb-IFN γ as expression bacterial strain, by introducing molecular chaperones PGro7 plasmid does not generate inclusion body in protein expression, forms soluble protein, avoids the mistake of inclusion body denaturation and renaturation Journey substantially reduces the time of expressing fusion protein；

4. the fusion protein disclosed by the invention being made of Chicken Albumin and chicken interferon gamma not only has interferon gamma Broad-spectrum disease resistance toxic action, while significantly improving the immune response of chicken itself.

Detailed description of the invention

Fig. 1 is the result of the Chicken Albumin gene and chicken interferon gamma gene RT-PCR amplification in embodiment 1；Swimming lane M： DNA Marker DL2000；Swimming lane 1：Chicken Albumin gene RT-PCR amplified production；Swimming lane 2：Chicken interferon gamma gene RT-PCR Amplified production；

Fig. 2 is the result of the PCR amplification after the Chicken Albumin in embodiment 1 is connected with the target gene of chicken IFN-γ； Swimming lane M：DNA Marker DL2000；Swimming lane 1：Chicken interferon gamma gene and Chicken Albumin gene ligation amplification product；

Fig. 3 is the PCR amplification and double digestion qualification result of the positive colony plasmid in embodiment 1；Swimming lane M：DNA Marker DL10000；Swimming lane 1：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：Empty bacterium Control；

Fig. 5 is the Western Blot qualification result for the fusion protein that embodiment 1 obtains；Swimming lane M：Albumen Marker；Swimming Road 1：It is precipitated after recombinant bacterium induction is broken；Swimming lane 2：For the broken rear supernatant of recombinant bacterium induction；

Fig. 6 is that the recombination chicken long-acting interferon γ as made from the fusion protein in embodiment 1 causes carefully VSV in embodiment 5 The inhibiting effect of born of the same parents' lesion；1 is VSV virus control wells；2 be HEp-2 cell control well；A3-12 is gradient dilution (from dextrad It is left) human interferon standard items handle hole；B3-12 is that the recombination chicken long-acting interferon γ of gradient dilution (from right to left) is handled Hole；

Fig. 7 is the recombination chicken long-acting interferon γ 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 and chicken interferon gamma, preparation method are as follows：

1. the acquisition and amplification of Chicken Albumin (Alb) and chicken interferon gamma (IFN-γ) target gene

Design of primers：

Synthetic primer is designed according to objective gene sequence reported in Genebank and is shown in Table 1, in the upstream of 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 and SalI restriction enzyme site are introduced respectively in trip primer.

1 PCR amplification primer of table

RT-PCR obtains target gene：

RNA is extracted from chicken liver tissue, the target gene of Alb and IFN-γ, the gene of the two are obtained by reverse transcription Sequence is respectively as shown in 400 < of SEQUENCE LISTING 400 <, 4 > and SEQUENCE LISTING, 5 >；

RT-PCR reaction system (25 μ L) is shown in Table 2

2 RT-PCR reaction system of table

RNase Free water	10μL
		dNTP Mix	10μL
Reverse transcriptase	2.5μL
		Upstream and downstream primer	Each 0.5 μ L
Geneome RNA	1.5μL

Response parameter is：50 DEG C of reverse transcriptions 30min, 95 DEG C of initial denaturation 4min, into circulation：95 DEG C of denaturation 45s；58 DEG C are moved back Fiery 45s, 72 DEG C of extension 1kb/min, totally 35 recycle；Last 72 DEG C of extensions 10min.

There is specific band, result in 1870bp and 520bp or so through agarose gel electrophoresis in RT-PCR amplified production As shown in Figure 1, explanation has respectively obtained the chicken Alb of the flexible linker of connection and the target gene of IFN-γ.

2. the connection of target gene

Target gene is diluted to 10ug/mL, connects even section target gene using over-lap PCR, 25 μ L reaction systems are such as Shown in table 3：

3 PCR reaction system of table

Response parameter is：95 DEG C of initial denaturation 4min, into circulation：94 DEG C of denaturation 45s；58 DEG C of annealing 45s, 72 DEG C of extensions 1kb/min, totally 35 recycle；Last 72 DEG C of extensions 10min.

There is specific band in 2370bp or so through agarose gel electrophoresis in pcr amplification product, result as shown in Fig. 2, Occurs the band of Chicken Albumin amplified production and chicken interferon gamma amplified production in Fig. 2, this is because in Chicken Albumin gene During connecting with chicken interferon gamma gene PCR, there is non-specific responding.The nucleotide sequence of obtained target gene is such as Shown in 400 < of SEQUENCE LISTING, 2 >.

3. expression vector establishment

The glue recovery product for the PCR for selecting the target gene after connecting errorless after being sequenced is used with pET-32a plasmid EcoRI and SalI restriction enzyme carries out double digestion and recycling, does double digestion by 20 μ L systems in table 4：

4 double digestion system of table

General buffer	2μL
		Restriction enzyme (a pair)	1μL+1μL
Carrier or recycling segment	2μL
		RNase Free water	14μL

The digestion recovery product of target gene and pET-32a plasmid after connection is attached by the system in table 5,4 DEG C overnight connection：

Table 5

Connection product is converted into e. coli bl21 (DE3) competent cell, competence is coated on the mould of benzyl containing ammonia The LB culture medium flat plate overnight incubation of element；The bacterium colony grown on LB plate is taken to identify target gene, positive colony bacteria plasmid through PCR It is identified through EcoRI and SalI double digestion, being accredited as positive indicates expression vector establishment success, has obtained engineering bacteria pET-32a/ rAlb-IFNγ；There is single band, knot at the place 2370bp or so through agarose gel electrophoresis in PCR amplification and double enzyme digestion product Fruit is as shown in Figure 3.

4. the expression of recombinant protein

Picking engineering bacteria pET-32a/rAlb-IFN γ shakes for 37 DEG C in the LB culture medium of the 100 μ g/ml containing ampicillin Bacterium 1h recovery engineering bacteria activity is surveyed OD value and is reached in LB culture medium (the 100 μ g/ml containing ampicillin) after amplification culture 4h When 1.0；IPTG, the final concentration of 100 μ g/mL, 32 DEG C of inducing expression 5h of IPTG is added；Bacterium is collected, through SDS-PAGE Electrophoresis detection, result as shown in figure 4, it can be seen from the figure that recombinant bacterium induction 5h after bacterial cell disruption after supernatant precipitate In the visible predominant expression band in the place 105KD or so, illustrate in precipitating and successful expression equal in supernatant fusion protein.

Mass volume ratio 1 is added:Precipitating is resuspended in 1 PBS；- 20 DEG C precipitate 3 times in room temperature multigelation；4 DEG C of ultrasound cracking Bacterial precipitation, work 10s, is spaced 3S, ultrasonic 6min, and whole process repeats 3~4 times；4 DEG C, 12000r/min is centrifuged 15min, Supernatant, inclusion body (inclusion body is through dissolution, refolding strategy) are taken respectively, obtain crude fusion protein.

5. fusion protein purification

5.1His affinity chromatography

After membrane filtration of the crude fusion protein with 0.22 μm of aperture, loading is by being connected to AKTA explorer On 100 protein purification systems, the His affinity column balanced with Binding Buffer I (PBS) is washed away with PBS buffer solution Unbonded albumen, until A280nm stablize, then with Elution buffer I (50mM trishydroxymethylaminomethane, 20~ 500mM imidazoles, PH8.0) elution, collect rAlb-IFN γ protein peak.

5.2DEAE anion-exchange chromatography

By the albumen collected after His affinitive layer purification displacement to (the 50mM trihydroxy methyl of Binding Buffer II Aminomethane, PH6.5) in after, loading by the DEAE anion exchange chromatography that has been balanced with Binding Buffer II, 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 protein peak.

5.3 sieve chromatography

Loading is by with Binding Buffer III after the sample concentration that ion-exchange chromatography is collected into (50mMNa2HPO4,0.15M NaCl, PH7.4) has balanced 200 molecular sieve chromatography of Superdex, with Binding Buffer III elution, collects rAlb-IFN γ protein peak.

5.4 sample identification

Measure rAlb-IFN γ potency and specific activity, specific activity >=1.0 × 10⁷U/mg albumen is qualification；It is aseptic subpackaged ,- 80 DEG C of preservations.The fusion protein being made of Chicken Albumin and chicken interferon gamma, amino acid sequence such as SEQUENCE can be obtained Shown in 400 < of LISTING, 1 >.

Embodiment 2

A kind of fusion protein being made of Chicken Albumin and chicken interferon gamma, other, only will be therein big with embodiment 1 The replacement of enterobacteria BL21 (DE3) competent cell is for BL21 (DE3) competent cell with pGro7 plasmid.It merges egg White SDS-PAGE electrophoresis result is compareed with embodiment 1, and 105KD or so place's predominant expression band is thicker in supernatant, and explanation is drawn After entering molecular chaperones pGro7, expression of the destination protein in supernatant is more preferable, and obtained fusion protein amount is higher.Escherichia coli The albumen of expression is mostly present in inclusion body；By introducing molecular chaperones in expression bacterial strain, coordinate expression albumen is correct It folds, reaches solubility expression of protein.

BL21 (DE3) competent cell with pGro7 plasmid is purchased from Shanghai Jinan Technology Co., Ltd./glad hundred promise Biology, article No. V205.

Embodiment 3

A kind of fusion protein being made of Chicken Albumin and chicken interferon gamma, preparation method are as follows：

1. the acquisition and amplification of Chicken Albumin (Alb) and chicken interferon gamma (IFN-γ) target gene

To in embodiment 1 Alb and IFN-γ optimize, artificial synthesized Alb and IFN-γ target gene, after optimization, The nucleotide sequence of the two is respectively as shown in 400 < of SEQUENCE LISTING 400 <, 6 > and SEQUENCE LISTING, 7 >.

1.1 codon optimization

Genetic codon has 64 kinds, but most biological tendencies are in utilizing a part in these codons.Those By the most frequent referred to as best codon (optimal codons) utilized, what those were not frequently utilized that is known as rare or utilizes The low codon of rate (rare or low-usage codons).In fact, common every kind of biology for doing protein expression or production (including Escherichia coli, yeast, mammalian cell, plant cell and insect cell) all shows the benefit of codon to a certain degree Difference or preference.The expression efficiency of the gene containing best codon is apparently higher than in Escherichia coli, yeast and drosophila and is contained The expression efficiency of the gene of the codon of poor efficiency.Therefore, in heterologous expression system, the preferences of codon are largely On affect the expression of recombinant protein.Using preference codon (preferred codons) and avoid utilizing rare codon Gene chemical synthesis is carried out, the redesign of this gene is codon optimization.Therefore, to the albumin of chicken and IFN- in the present embodiment γ gene codon optimizes.

Interpretation of result after 1.2 codon optimizations

It is optimal efficient in the expression system to be considered the gene when usual codon adaptation indexI (CAI)=1.0 Expression status, CAI value is lower to show that expression is lower in host.In gene G/C content most ideal distribution range be 30~ 70%, it is more than that the range will affect translation and transcriptional efficiency in any region.Using software detection discovery Chicken Albumin and The codon of IFN-γ original gene codon adaptation indexI (CAI) in Escherichia coli is respectively 0.27,0.25, GC percentage It is 43.1%, 42.9%；And by close in Escherichia coli to recombination is obtained after Chicken Albumin and IFN-γ gene optimization Numeral adaptation index (CAI) is 0.99,1.0, GC percentage 49.2%, 47.6%.It is significantly reduced by gene optimization low close The utilization rate of numeral avoids influence of the rare codon to protein expression, improves the G/C content of gene, improves transcription and turn over Efficiency is translated, and then improves the expression quantity of recombinant protein.

1.3 design of primers：

6 PCR amplification primer of table

The genomic DNA of Alb and IFN-γ after optimization are diluted to 0.05mg/mL respectively.Mesh is obtained using PCR amplification Gene, 25 μ L reaction systems are as shown in table 7：

7 PCR reaction system of table

RNase Free water	10.5μL
		dNTP Mix	10.0μL
Taq archaeal dna polymerase	2.5μL
		Upstream and downstream primer	Each 0.5 μ L
Genomic DNA	1.0μL

Response parameter is：95 DEG C of initial denaturation 4min, into circulation：95 DEG C of denaturation 45s；60 DEG C of annealing 45s, 72 DEG C of extensions 1kb/min, totally 35 recycle；Last 72 DEG C of extensions 10min.

Alb and the pcr amplification product of IFN-γ occur in 1870bp and 520bp or so special respectively through agarose gel electrophoresis Different band illustrates the target gene that the Alb after the optimization of the flexible linker of connection and IFN-γ have been prepared respectively.

2. the connection of target gene

Target gene is diluted to 10ug/mL, connects target gene, 25 μ L reaction systems such as 8 institute of table using over-lap PCR Show：

8 PCR reaction system of table

Response parameter is：95 DEG C of initial denaturation 4min, into circulation：94 DEG C of denaturation 45s；60 DEG C of annealing 45s, 72 DEG C of extensions 1kb/min, totally 35 recycle；Last 72 DEG C of extensions 10min.

There is specific band in 2370bp or so through agarose gel electrophoresis in pcr amplification product, illustrates successfully to have obtained Alb Target gene after being connected with IFN-γ, the nucleotide sequence of obtained target gene such as 400 < of SEQUENCE LISTING, 3 > It is shown.

3. expression vector establishment

The glue recovery product for the PCR for selecting the target gene after connecting errorless after being sequenced is used with pET-32a plasmid BamHI, XhoI restriction enzyme carry out double digestion and recycling, do double digestion by 20 μ L systems in table 9：

9 double digestion system of table

General buffer	2μL
		Restriction enzyme (a pair)	1μL+1μL
Carrier or recycling segment	2μL
		RNase Free water	14μL

The digestion recovery product of target gene and pET-32a plasmid after connection is attached by the system in table 10,4 DEG C overnight connection：

Table 10

Target fragment DNA	10μL
		Expression vector	3μL
buffer	2μL
		Ligase	1μL
RNase Free water	4μL

Connection product is converted into e. coli bl21 (DE3) competent cell, competence is coated on the mould of benzyl containing ammonia The LB culture medium flat plate overnight incubation of element；The bacterium colony grown on LB plate is taken to identify target gene, positive colony bacteria plasmid through PCR It is identified through BamHI, XhoI double digestion, being accredited as positive indicates expression vector establishment success, PCR amplification and double enzyme digestion product warp There is single band at the place 2370bp or so in agarose gel electrophoresis, illustrates the target gene after connecting containing Alb with IFN-γ Expression vector establishment success, obtain engineering bacteria pET-32a/rAlb-IFN γ.

4. the expression of recombinant protein

Picking engineering bacteria pET-32a/rAlb-IFN γ shakes for 37 DEG C in the LB culture medium of the 100 μ g/ml containing ampicillin Bacterium 1h recovery engineering bacteria activity is surveyed OD value and is reached in LB culture medium (the 100 μ g/ml containing ampicillin) after amplification culture 4h When 1.0；IPTG, the final concentration of 100 μ g/mL, 32 DEG C of inducing expression 5h of IPTG is added；Bacterium is collected, through SDS-PAGE Electrophoresis detection, recombinant bacterium induction 5h after bacterial cell disruption after supernatant be deposited in the visible predominant expression band in the place 105KD or so, say It is bright to have obtained recombinant protein in supernatant precipitating.

Mass volume ratio 1 is added:Precipitating is resuspended in 1 PBS；- 20 DEG C precipitate 3 times in room temperature multigelation；4 DEG C of ultrasound cracking Bacterial precipitation, work 10s, is spaced 3S, ultrasonic 6min, and whole process repeats 3~4 times；4 DEG C, 12000r/min is centrifuged 15min, Supernatant, inclusion body (inclusion body is through dissolution, refolding strategy) are taken respectively, obtain crude fusion protein.

5. fusion protein purification

5.1His affinity chromatography

After membrane filtration of the crude fusion protein with 0.22 μm of aperture, loading is by being connected to AKTA explorer On 100 protein purification systems, the His affinity column balanced with Binding Buffer I (PBS) is washed away with PBS buffer solution Unbonded albumen, until A280nm stablize, then with Elution buffer I (50mM trishydroxymethylaminomethane, 20~ 500mM imidazoles, PH8.0) elution, collect rAlb-IFN γ protein peak.

5.2DEAE anion-exchange chromatography

By the albumen collected after His affinitive layer purification displacement to (the 50mM trihydroxy methyl of Binding Buffer II Aminomethane, PH6.5) in after, loading by the DEAE anion exchange chromatography that has been balanced with Binding Buffer II, 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 protein peak.

5.3 sieve chromatography

Loading is by with Binding Buffer III after the sample concentration that ion-exchange chromatography is collected into (50mMNa2HPO4,0.15M NaCl, PH7.4) has balanced 200 molecular sieve chromatography of Superdex, with Binding Buffer III elution, collects rAlb-IFN γ protein peak.

5.4 sample identification

Measure rAlb-IFN γ potency and specific activity, specific activity >=1.0 × 10⁷U/mg albumen is qualification；It is aseptic subpackaged ,- 80 DEG C of preservations.The fusion protein being made of Chicken Albumin and chicken interferon gamma, amino acid sequence such as SEQUENCE can be obtained Shown in 400 < of LISTING, 1 >.

Embodiment 4

A kind of fusion protein being made of Chicken Albumin and chicken interferon gamma, other, only will be therein big with embodiment 3 The replacement of enterobacteria BL21 (DE3) competent cell is for BL21 (DE3) competent cell with pGro7 plasmid.It merges egg White SDS-PAGE electrophoresis result is compareed with embodiment 3, and 105KD or so place's predominant expression band is thicker in supernatant, and explanation is drawn After entering molecular chaperones pGro7, expression of the destination protein in supernatant is more preferable, and obtained fusion protein amount is higher.Escherichia coli The albumen of expression is mostly present in inclusion body；By introducing molecular chaperones in expression bacterial strain, coordinate expression albumen is correct It folds, reaches solubility expression of protein.BL21 (DE3) competent cell with pGro7 plasmid is purchased from Shanghai offshore section Skill Co., Ltd/glad hundred promise biology, article No. V205.

Embodiment 5

A kind of recombination chicken long-acting interferon γ is mixed with freeze drying protectant respectively by the fusion protein in embodiment 1,2,3,4 It is freeze-dried to form with later.The freeze drying protectant is glycerol, mannitol and sucrose, is buffering with 10mmol/L PBS Liquid, final concentration of glycerol 100mL/L, mannitol 0.12g/mL and the sucrose 0.025g/mL of three.

Embodiment 6

Examples 1 to 4 obtains the identification for the fusion protein being made of Chicken Albumin and chicken interferon gamma

The quantitative detection of 6.1 protein contents

With Lowry method, standard test is made with the standard protein of Chinese food pharmaceutical biological product calibrating institute, measures embodiment 1~4 obtained fusion protein concentration is all larger than 1.2mg/ml.

6.2SDS-PAGE electrophoresis detection

Compared with empty bacterium, fusion protein has the newly-increased protein band of a dense dye in 105KD or so, such as Fig. 4, shown.

6.3Western Blot result

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 γ sample can be dry with anti-chicken It disturbs plain γ monoclonal antibody and specific reaction occurs, specific band occurs in the place 105KD or so, as shown in Figure 5.

Embodiment 7

Four parts of recombination chicken long-acting interferon γ in embodiment 5 freeze-dried bioactivity

Inhibit method according to few cells lesion, Hep-2 cell is made into 5 × 10 with culture medium⁵Cell/ml cell suspends Liquid, every hole inoculation 0.1ml move into 96 porocyte culture plates.37 DEG C, 5%CO2 culture for 24 hours, the recombination chicken that various dose is added is long Interferon gamma is imitated, inhales abandon afterwards for 24 hours, then be inoculated with 100TCID respectively₅₀VSV virus.

Test result

The result shows that the recombination chicken long-acting interferon γ obtained causes the lesion of HEp-2 cell to have apparent suppression VSV Production is used.The lesions such as there is cell rounding, falls off, is disintegrated after untreated cell inoculation virus.And the recombination chicken obtained After long-acting interferon γ treated cell inoculation virus, it is observed continuously under inverted microscope, cellular morphology is normal, does not occur Any lesion, measures potency >=1.0 × 10⁷U/ml, as shown in Figure 6.

Embodiment 8

Being lyophilized respectively by four parts of recombination chicken long-acting interferon γ that the fusion protein of Examples 1 to 4 obtains in embodiment 5 Measurement of the agent (being denoted as A, B, C, D respectively) in chicken intracorporal half-life period

The blood concentration and time relationship of cytopathic-effect inhibition assay measurement rAlb-IFN γ

Take the broiler chicken (half male and half female) that six weight are roughly the same, the long-acting chicken interferon gamma freeze-drying of intramuscular injection 2mg/ml chicken Agent 2ml, respectively 1h, 2h, 4h, 8h, 16h, for 24 hours, 36h, 48h, 60h, 88h, 96h venous blood collection, 4 DEG C of blood sample solidification, 3500rpm 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.Using cytopathic effect inhibition Method measures the concentration of rAlb-IFN γ in blood serum sample, is carried out curve fitting and calculating parameter with DAS pharmacokinetics software.Fitting is bent Line is as shown in Figure 7；Parameter calculated result is shown in Table 11.

Dominant dynamic parameters in serum after 11 recombination chicken long-acting interferon γ intramuscular injection of table

The result shows that recombination chicken long-acting interferon γ has longer half-life period.Half-life period can reach 61h or so after measured, compared with Plain interferon improves about 15 times.

Embodiment 9

The freeze-dried measurement that chicken cell immune response is influenced of four parts of recombination chicken long-acting interferon γ 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 recombination chicken long-acting interferon freeze-dried 2ml of γ is penetrated, the PBS of 2mL is subcutaneously injected in control group neck, takes chicken after injection 4 weeks Peripheral blood takes weekly a blood later, separates lymphocyte using lymphocyte separation medium, lymphocyte passes through serum-free RPMI It after 1640 culture mediums wash 2 times, is resuspended with complete medium, adjustment cell concentration is 2 × 10⁶A/ml, 24 porocyte culture plates are every Hole addition 1ml lymphocyte, 37 DEG C, 5%CO₂72h is cultivated, supernatant when collecting lymphocyte culture.ELISA detects culture supernatant Middle IL-2, IL-4 content, is carried out by kit specification, and testing result is as shown in table 12：

It is horizontal that 12 ELISA of table detects each group chicken cell immune response

The result shows that injection recombination chicken long-acting interferon γ after, can significantly improve chicken Evaluation of Cytokines in Peripheral Blood IL-2, The content of IL-4 enhances cellullar immunologic response level, significantly improves immunity level.

It is above-mentioned referring to embodiment to a kind of fusion protein and preparation method thereof being made of Chicken Albumin and chicken interferon gamma The detailed description carried out with a kind of recombination chicken long-acting interferon γ, is illustrative without being restrictive, can be according to being limited Range enumerates several embodiments, therefore the change and modification in the case where not departing from present general inventive concept, should belong to of the invention Within protection scope.

SEQUENCE LISTING

<110>Wuhu Co., Ltd of Ying Tefeier biological products industrial research institute

<120>A kind of fusion protein being made of Chicken Albumin with chicken interferon gamma and preparation method thereof and a kind of recombination chicken are long

Imitate interferon gamma

<130> 1

<160> 7

<170> PatentIn version 3.3

<210> 1

<211> 789

<212> PRT

<213>Recombination chicken long-acting interferon γ 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

785

<210> 2

<211> 2367

<212> DNA

<213>Recombination chicken long-acting interferon γ 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 caattgc 2367

<210> 3

<211> 2367

<212> DNA

<213>Recombination chicken long-acting interferon γ 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 caactgc 2367

<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> 1845

<212> DNA

<213>Chicken Albumin

<400> 6

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> 7

<211> 492

<212> DNA

<213>Chicken IFN-γ

<400> 7

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

Claims (10)

1. a kind of fusion protein being made of Chicken Albumin and chicken interferon gamma, it is characterised in that：The amino of the fusion protein Acid sequence table is as shown in 400 < of SEQUENCE LISTING, 1 >.

2. a kind of gene for encoding fusion protein as described in claim 1, which is characterized in that the nucleotide sequence of the gene Table is denoted as genome 1 as shown in 400 < of SEQUENCE LISTING, 2 >；Or as shown in 400 < of SEQUENCE LISTING, 3 >, It is denoted as genome 2.

3. containing the expression vector of gene as claimed in claim 2.

4. containing the genetic engineering bacterium of gene as claimed in claim 2.

5. a kind of recombination chicken long-acting interferon γ, which is characterized in that the recombination chicken long-acting interferon γ is as described in claim 1 Fusion protein and freeze drying protectant mixture after, it is freeze-dried to form.

6. the preparation method of fusion protein according to claim 1, which is characterized in that the preparation method includes following step Suddenly：Expression vector as claimed in claim 3 is imported into e. coli host cell, genetic engineering bacterium, genetic engineering are obtained Bacterium obtains the crude product of the fusion protein after IPTG inducing expression, and fusion protein can be obtained after purified.

7. preparation method according to claim 6, which is characterized in that the genetic engineering bacterium is pET-32a/rAlb-IFN γ, preparation method are：

(1) design primer, is obtained or the Chicken Albumin of the flexible linker sequence of artificial synthesized connection and chicken by reverse transcription The target gene of interferon gamma；The target gene of Chicken Albumin and chicken interferon gamma is connected by flexible linker, mesh Gene nucleotides sequence list as shown in 400 < of SEQUENCE LISTING, 2 > or such as 400 < of SEQUENCE LISTING, 3 > It is shown；

(2) target gene after connection is connected on pET-32a plasmid and obtains expression vector；

(3) expression vector is imported into e. coli host cell, genetic engineering bacterium pET-32a/rAlb-IFN can be obtained γ。

8. preparation method according to claim 6 or 7, which is characterized in that the e. coli host cell is BL21 (DE3) competent cell or BL21 (DE3) competent cell with pGro7 plasmid.

9. preparation method according to claim 6 or 7, which is characterized in that the method for the purifying is：Fusion protein it is thick Product are successively through affinity chromatography, anion-exchange chromatography and molecular sieve chromatography purification.

10. the application of recombination chicken long-acting interferon γ according to claim 5, which is characterized in that the recombination chicken is long-acting The long half time of interferon gamma had broad-spectrum disease resistance toxic action and can improve the immune response of chicken itself up to 61 hours or more.