CN108840951A

CN108840951A - A kind of fusion protein and preparation method thereof being made of pig albumin, Porcine interferon-gamma and porcine interferon alpha

Info

Publication number: CN108840951A
Application number: CN201810768603.4A
Authority: CN
Inventors: 赵雨; 蒋敏之; 夏兵兵; 何志远; 刘家炉; 单雪芹; 李雅森
Original assignee: Wuhu Phil Biological Products Industry Research Institute Co Ltd
Current assignee: Wuhu Phil Biological Products Industry Research Institute Co Ltd
Priority date: 2017-08-09
Filing date: 2018-07-13
Publication date: 2018-11-20
Also published as: CN107353347A

Abstract

The fusion protein and preparation method thereof that the invention discloses a kind of to be made of pig albumin, Porcine interferon-gamma and porcine interferon alpha; the fusion protein is connected through flexible linker by pig albumin, Porcine interferon-gamma and porcine interferon alpha and is formed, and is freeze-dried to obtain Recombinant Swine long-acting interferon after fusion protein and freeze drying protectant mixture.The Recombinant Swine long-acting interferon is remarkably improved the half-life period of pig interferon, and the half-life period of more common pig interferon improves 24 times or more, and has broad-spectrum disease resistance toxic action and can improve the immune response of pig itself.

Description

A kind of fusion protein being made of pig albumin, Porcine interferon-gamma and porcine interferon alpha and Preparation method

Technical field

The invention belongs to technical field of biological genetic engineering, and in particular to be interfered by pig albumin, Porcine interferon-gamma and pig The fusion protein and preparation method thereof of plain α composition.

Background technique

Scale Compact Develop is rapidly growing in China in recent years, and China's live pig breeding stock and pork production are sure to occupy First place in the world, traditional swine disease control method is far from the control for adapting to infectious disease in extensive intensive pig production production.I Newly there are nearly 20 kinds of livestock and poultry infectious diseases in the past 20 years in state, in addition original animal epidemic, causes aquaculture industry of China huge Economic loss.According to incompletely statistics, China is every year because various viral diseases cause mortality of livestock to be up to 15%-20%, Economic loss reaches billions of members.

Mainly pass through vaccine inoculation to the prevention and treatment approach of porcine viral diseases at present and uses antibiotic, but by It is not perfect in breeding environment, virus variation and Abwehrkraft des Koepers variation etc. reasons, make traditional prevention and treatment approach by Huge challenge, most of antibiotics and traditional oral antiviral medicament give the mankind due to medicament residue problem Health is brought a negative impact；And traditional vaccine, high specific and side effect due to it, virus variation and new can not be resisted Type virus, which continuously emerges, gives pig aquaculture bring significant damage.

IFN is that a kind of virus infection induction body generates and has broad-spectrum antiviral, antitumor and with immunoregulation effect Protein, nineteen fifty-seven Issacs and Lindeman first discovery, it is a kind of multi-functional cell factor, with cell receptor knot After conjunction, it can induce body and generate a variety of specific proteins and enzyme, it is main by inhibiting viral gene transcription and degradation virus RNA inhibits the growth and breeding of virus and plays antitumor etc. activity.Now it is known that α type IFN in vivo can be selectively Act on the infection cells such as virus, by inhibit infected cell in virus protein biosynthesis, play wide spectrum and efficiently Antivirus action.But it is faint without acting on or acting on to normal host cell.IFN-α main physiological activity is with inhibition virus Duplication, anti parasitic, the killing activity for inhibiting various kinds of cell proliferation, stimulation immunocyte.

γ type IFN is that the T cell and NK cell by activating generate, and has relatively strong antiviral and immunoloregulation function.Largely Studies have shown that interferon gamma also plays crucial adjustment effect other than having the function of broad-spectrum antiviral, to immune system, so IFN-γ is also known as immunological regulation interferon.Although various types of interferon can mediated cell to the anti-of virus infection It answers, but the immunoregulatory activity of interferon gamma is coordinating immune response and determining in the long-term antiviral state of body to play more Important role, therefore interferon gamma has particularly important clinical value.

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 pig albumin, Porcine interferon-gamma and porcine interferon alpha group At fusion protein and preparation method thereof, it is freeze-dried to be prepared into and thus after fusion protein and freeze drying protectant mixture To a kind of Recombinant Swine long-acting interferon, the Recombinant Swine long-acting interferon is remarkably improved the half-life period of pig interferon, more commonly The half-life period of pig interferon improves 24 times or more, and has broad-spectrum disease resistance toxic action and can improve the immune response of pig itself.

The technical scheme adopted by the invention is as follows：

A kind of fusion protein being made of pig albumin, Porcine interferon-gamma and porcine interferon alpha, the amino of the fusion protein Acid sequence table is as shown in 400 < of SEQUENCE LISTING, 1 >.

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

Fusion protein described in the genome 1 and the equal codified of the genome 2.Genome 2 is the nucleosides to genome 1 Acid sequence optimize after as a result, when usual codon adaptation indexI CAI=1.0 be considered the gene in the expression system In be optimal high efficient expression state, CAI value is lower to show that expression is lower in host.Most ideal point of G/C content in gene Cloth range is 30~70%, is more than that the range will affect translation and transcriptional efficiency in any region.It is sent out using software detection The now codon of pig albumin, porcine IFN γ, pig IFN-α original gene codon adaptation indexI (CAI) point in Escherichia coli Not Wei 0.24,0.24,0.22, GC percentage be 43.7%, 39.2%, 59.1%；And by pig albumin, porcine IFN γ, It is 0.98,1.0,1.0, GC hundred that each gene codon adaptation indexI (CAI) in Escherichia coli is obtained after pig IFN-α gene optimization Divide ratio 50.3%, 45.4%, 55.6%.The utilization rate of low codon is significantly reduced by gene optimization, is avoided rare close Influence of the numeral 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-IFN α.

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

The present invention also provides a kind of Recombinant Swine long-acting interferon, the Recombinant Swine long-acting interferon is by the fusion egg It is freeze-dried to form after the white mixture with freeze drying protectant.

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

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

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

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

(1) design primer, obtained by reverse transcription or be manually respectively synthesized the pig albumin with flexible linker sequence, The target gene of Porcine interferon-gamma, porcine interferon alpha；By flexible linker by pig albumin, Porcine interferon-gamma, porcine interferon alpha Target gene connect, the nucleotides sequence list of the target gene after connection such as 400 < of SEQUENCE LISTING, 2 > institute Show 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-IFNα。

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

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

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

The acquisition methods of the genome 1 are：

A. design of primers

The primer sequence of pig albumin (Alb) is：

Upstream Alb-F1：CATGCCATGGGATACATACAAGAGTGA has NcoI restriction enzyme site；

Downstream Alb-R1：

ACCACCACCAGAACCACCACCACCGGCTAAGATCCCTCG, with flexible linker；

The primer sequence of Porcine interferon-gamma (IFN-γ) is：

Upstream IFN-γ-F1：

GGTGGTTCTGGTGGTGGTGGTTCTATGAGTTATACAACTTA, with flexible linker；

Downstream IFN-γ-R1：

ACCACCACCAGAACCACCACCACCTTTTGATGCTCTCTG, with flexible linker；

The primer sequence of porcine interferon alpha (IFN-α) is：

Upstream IFN-α-F1：

GGTGGTTCTGGTGGTGGTGGTTCTATGTGTTCCTATTTAAG, with flexible linker；

Downstream IFN-α-R1：CCCTCGAGCTCCTTCCTCCTG has XhoI restriction enzyme site；

B. RNA is extracted from pig liver, and the target gene of pig Alb, porcine IFN γ and pig IFN-α is obtained by reverse transcription, The gene order of three respectively as shown in 400 < of SEQUENCE LISTING, 4 >, 400 < of SEQUENCE LISTING, 5 > and Shown in 400 < of SEQUENCE LISTING, 6 >；

Respectively using the target gene of pig Alb, porcine IFN γ and pig IFN-α as template, and it is utilized respectively pig Alb, pig IFN- The upstream and downstream primer of γ and pig IFN-α carry out PCR amplification, respectively obtain the pig Alb for connecting flexible linker, porcine IFN γ and Pig IFN-α 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 pig Alb and porcine IFN γ target gene

The PCR reaction system and reaction condition of connection be：In the overall reaction system of 25 μ L, connect flexible linker's Alb gene template DNA1 μ L connects 1 μ L, Alb upstream primer of IFN-γ template DNA, the 0.5 μ L of flexible linker, IFN-γ 0.5 μ L, Taq archaeal dna polymerase of downstream primer, 2.5 μ L, dNTP Mix is 10 μ L, adds RNase Free water to 25 μ L；Connect PCR Reaction condition is：95 DEG C of initial denaturation 4min, into circulation：94 DEG C of denaturation 45s；58 DEG C of annealing 45s, 72 DEG C of extension 1kb/min, altogether 35 circulations；Last 72 DEG C of extensions 10min.

D. rAlb-IFN γ-IFN is obtained using flexible linker connection rAlb-IFN γ gene and pig IFN-α target gene α 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 IFN-α template DNA 0.5 the μ L, 0.5 μ of IFN-α downstream primer of flexible linker 2.5 μ L, dNTP Mix of L, Taq archaeal dna polymerase is 10 μ L, adds RNase Free water to 25 μ L；Connecting PCR reaction condition is：95 DEG C 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；Finally 72 DEG C of extension 10min.

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

A. design of primers

The primer sequence of pig albumin (Alb) is：

Upstream Alb-F2：CATGCCATGGTGACACCTACAAATCTG has NcoI restriction enzyme site；

Downstream Alb-R2：

ACCACCACCAGAACCACCACCACCAGCCAGGATACCACG, with flexible linker；

The primer sequence of Porcine interferon-gamma (IFN-γ) is：

Upstream IFN-γ-F2：

GGTGGTTCTGGTGGTGGTGGTTCTATGTCTTACACCACCT, with flexible linker；

Downstream IFN-γ-R2：

ACCACCACCAGAACCACCACCACCTTTAGAAGCACGCTG, with flexible linker；

Porcine interferon alpha (IFN-α)：

Upstream IFN-α-F2：

GGTGGTTCTGGTGGTGGTGGTTCTATGTGCTCTTACCTG, with flexible linker；

Downstream IFN-α-R2：

CCCTCGAGTTCTTTACGACGCAG has XhoI restriction enzyme site.

B. the target gene of the pig Alb, porcine IFN γ and pig IFN-α, 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 >；

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

The PCR reaction system and reaction condition of connection be：In the overall reaction system of 25 μ L, connect flexible linker's Alb gene template DNA1 μ L connects 1 μ L, Alb upstream primer of IFN-γ template DNA, the 0.5 μ L of flexible linker, IFN-γ 0.5 μ L, Taq archaeal dna polymerase of downstream primer, 2.5 μ L, dNTP Mix is 10 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, altogether 35 circulations；Last 72 DEG C of extensions 10min.

D. rAlb-IFN γ-is obtained using flexible linker connection rAlb-IFN γ gene and pig IFN-α target gene IFN-α 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 DNA1 μ L connects 1 μ L, Alb upstream primer of IFN-α template DNA 0.5 μ L, the 0.5 μ L of IFN-α downstream primer of flexible linker, 2.5 μ L, dNTP Mix of Taq archaeal dna polymerase is 10 μ L, adds RNase Free water to 25 μ L；Connecting PCR reaction condition is：95℃ Initial denaturation 4min, into circulation：94 DEG C of denaturation 45s；60 DEG C of annealing 45s, 72 DEG C of extension 1kb/min, totally 35 recycle；Last 72 DEG C extend 10min.

The present invention also provides the application of the Recombinant Swine long-acting interferon, long half time had up to 98 hours or more Broad-spectrum disease resistance toxic action and the immune response that pig itself can be improved.

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

1. pig Alb, porcine IFN γ and pig IFN-α gene are realized amalgamation and expression by flexibility linker, interference is improved Plain half-life period improves 24 times or more compared with plain interferon；It is significant to drop compared with common polyethylene glycol fused interferon Low cost.

2. improving pig Alb, porcine IFN γ and pig by optimizing to pig Alb, porcine IFN γ and pig IFN-α gene The expression quantity of IFN-α fusion protein.

3. using recombination bacillus coli pET-32a/rAlb-IFN γ-IFN α as expression bacterial strain, by introducing molecular chaperones PGro7 plasmid, in protein expression, generation inclusion body is less, forms great amount of soluble albumen, avoids inclusion body denaturation and answers The process of property, substantially reduces the time of expressing fusion protein；

4. the fusion protein disclosed by the invention being made of pig Alb, porcine IFN γ and pig IFN-α not only has IFN-α Broad-spectrum disease resistance toxic action, while significantly improving the immune response of pig itself.

Detailed description of the invention

Fig. 1 is that pig albumin gene, porcine interferon alpha gene and the Porcine interferon-gamma gene RT-PCR in embodiment 1 are expanded Result；Swimming lane M：DNAMarker DL2000；Swimming lane 1：Porcine interferon-gamma gene RT-PCR amplified production；Swimming lane 2：Pig interference Plain α gene RT-PCR amplified production；Swimming lane 3：Pig albumin gene RT-PCR amplified production；

Fig. 2 be embodiment 1 in pig Alb, IFN-γ connected with the target gene of IFN-α after PCR amplification result； Swimming lane M：DNA Marker DL10000；Swimming lane 1：Pig albumin gene, Porcine interferon-gamma gene are connect with porcine interferon alpha gene Amplified production；

Fig. 3 is the PCR amplification and double digestion qualification result of the positive colony plasmid in embodiment 1；Swimming lane M：DNAMarker 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：Supernatant after recombinant bacterium induction is broken；

Fig. 6 is that the Recombinant Swine long-acting interferon α as made from the fusion protein in embodiment 1 causes cell to VSV in embodiment 5 The inhibiting effect of lesion；1 is VSV virus control wells；2 be HEp-2 cell control well；A3-12 is gradient dilution (from right to left) Human interferon standard items handle hole；B3-12 is that the Recombinant Swine long-acting interferon α of gradient dilution (from right to left) handles hole；

Fig. 7 is the Recombinant Swine 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 pig albumin, Porcine interferon-gamma and porcine interferon alpha, preparation method are as follows：

1. the acquisition of pig albumin (Alb), Porcine interferon-gamma (IFN-γ) and porcine interferon alpha (IFN-α) target gene with Amplification

Design of primers：

Synthetic primer is designed according to objective gene sequence reported in Genebank and is shown in Table 1, in the upstream of pig albumin NcoI restriction enzyme site and Linker sequence are introduced in primer and downstream primer respectively, upstream primer and downstream in Porcine interferon-gamma Linker sequence is introduced in primer respectively, introduces Linker sequence respectively in the upstream primer and downstream primer of porcine interferon alpha With XhoI restriction enzyme site.

1 PCR amplification primer of table

RT-PCR obtains target gene：

RNA is extracted from pig liver tissue, and the purpose base of pig Alb, porcine IFN γ and pig IFN-α is obtained by reverse transcription Cause, the gene order of three respectively such as 400 < of SEQUENCE LISTING, 4 >, 400 < of SEQUENCE LISTING, 5 > and Shown in 400 < of SEQUENCE 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 1780bp, 560bp and 710bp or so through agarose gel electrophoresis in RT-PCR amplified production, Its result as shown in Figure 1, illustrate be prepared respectively the pig Alb for being separately connected flexible linker sequence, porcine IFN γ and The target gene of pig IFN-α.

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 of table-IFN α PCR reaction system

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 2990bp or so through agarose gel electrophoresis in pcr amplification product, result as shown in Fig. 2, Occur rAlb-IFN γ and IFN-α amplified product band in Fig. 2, this is because connecting in rAlb-IFN γ with IFN-α gene During, there is non-specific responding.The nucleotide sequence of obtained target gene such as 400 < of SEQUENCE LISTING, 2 > It is shown.

3. expression vector establishment

For target gene after selection connection after sequencing is errorless, PCR glue recovery product and pET-32a plasmid use NcoI Double digestion and recycling are carried out with XhoI restriction enzyme, does double digestion by 20 μ L systems in table 5：

5 double digestion system of table

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

The digestion recovery product of target gene and pET-32a plasmid after connection is attached by the system in table 6,4 DEG C overnight connection：

6 enzyme disjunctor system of table

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

Connection product is converted into e. coli bl21 (DE3) competent cell, competent cell is coated on benzyl containing ammonia The LB culture medium flat plate of penicillin is incubated overnight；Single colonie on picking LB plate carries out target gene PCR identification, positive colony Bacteria plasmid identifies that being accredited as positive indicates that engineering bacteria constructs successfully, and PCR amplification and double digestion are produced through NcoI and XhoI double digestion Object detects single band at the place 2990bp or so through agarose gel electrophoresis, and result is as shown in figure 3, illustrate successfully to obtain PET-32a/rAlb-IFN γ-IFN α engineering bacteria.

4. the expression of recombinant protein

Picking engineering bacteria 37 DEG C of shaking table recovery 1h in the LB culture medium containing 100 μ g/ml ampicillins, in LB culture medium Amplification culture 4h (OD=1.0) in (the 100 μ g/ml containing ampicillin), is added the IPTG of final concentration of 100 μ g/ml, 32 DEG C lure Lead expression 5h；Thallus is collected, through SDS-PAGE electrophoresis detection, result is as shown in figure 4, it can be seen from the figure that recombinant bacterium lures Supernatant is deposited in the visible predominant expression band in the place 127.8KD or so after bacterial cell disruption after leading 5h, illustrates in precipitating and supernatant In equal successful expression fusion proteins.

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 100 egg of AKTAexplorer On white purification system, the His affinity column balanced with Binding Buffer I (PBS) is washed away with PBS buffer solution and is not tied The albumen of conjunction, until A280nm stablizes, then with Elution buffer I (50mM trishydroxymethylaminomethane, 20~500mM miaow Azoles, PH8.0) elution, collect rAlb-IFN γ-IFN α protein peak.

5.2DEAE anion-exchange chromatography

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

5.4 sample identification

Measure rAlb-IFN γ-IFN α potency and specific activity, specific activity >=10⁵U/mg, albumen are qualified；It is aseptic subpackaged, -80 DEG C save.The fusion protein being made of pig albumin, Porcine interferon-gamma and porcine interferon alpha can be obtained, amino acid sequence is such as Shown in 400 < of SEQUENCE LISTING, 1 >.

Embodiment 2

A kind of fusion protein being made of pig albumin, Porcine interferon-gamma and porcine interferon alpha, other are with embodiment 1, only E. coli bl21 therein (DE3) competent cell is replaced in order to which BL21 (DE3) competence with pGro7 plasmid is thin Born of the same parents.The SDS-PAGE electrophoresis result of its fusion protein is compareed with embodiment 1,127.8KD or so place's predominant expression item in supernatant Band is thicker, illustrates after introducing molecular chaperones pGro7, expression of the destination protein in supernatant is more preferable, obtained fusion protein amount It is higher.The albumen of Bacillus coli expression is mostly present in inclusion body；By introducing molecular chaperones in expression bacterial strain, cooperate with Expression albumen correctly folds, and reaches solubility expression of protein.

Embodiment 3

Pig Alb, porcine IFN γ and pig IFN-α in embodiment 1 is optimized, artificially synthesized pig Alb, porcine IFN γ and Pig IFN-α target gene, after optimization, the nucleotide sequence of three is respectively such as 400 < of SEQUENCE LISTING 7 >, SEQUENCE Shown in 400 < of LISTING 400 <, 8 > and SEQUENCE LISTING, 9 >.

1.1 codon optimization

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

Interpretation of result after 1.2 codon optimizations

It is optimal efficient in the expression system to be considered the gene when usual codon adaptation indexI (CAI)=1.0 Expression status, CAI value is lower to show that expression is lower in host.In gene G/C content most ideal distribution range be 30~ 70%, it is more than that the range will affect translation and transcriptional efficiency in any region.Pig Alb, pig are found using software detection The codon of IFN-γ and pig IFN-α original gene codon adaptation indexI (CAI) in Escherichia coli is respectively 0.24, 0.24,0.22, GC percentage is 43.7%, 39.2%, 59.1%；And by pig Alb, porcine IFN γ and pig IFN-α gene It is respectively 0.98,1.0,1.0, GC percentage that recombination codon adaptation indexI (CAI) in Escherichia coli is obtained after optimization 50.3%, 45.4%, 55.6%.The utilization rate of low codon is significantly reduced by gene optimization, avoids rare codon Influence to protein expression improves the G/C content of gene, improves transcription and translation efficiency, and then improves the table of recombinant protein Up to amount.

1.3 design of primers：

Table 7PCR amplimer

The genomic DNA of pig Alb, porcine IFN γ and pig IFN-α 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

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

The pcr amplification product of pig Alb, porcine IFN γ and pig IFN-α through agarose gel electrophoresis respectively 1780bp, There is specific band in 560bp and 710bp or so, illustrate that the pig for being separately connected flexible linker after optimization has been prepared The target gene of Alb, porcine IFN γ and pig IFN-α.

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

Table 10rAlb-IFN γ-IFN-α PCR reaction system

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 2990bp or so through agarose gel electrophoresis and illustrates successfully to be connected in pcr amplification product RAlb-IFN γ-IFN-α gene afterwards.The nucleotide sequence of obtained target gene such as 400 < of SEQUENCE LISTING, 3 > It is shown.

3. expression vector establishment

The glue recovery product for the PCR for selecting the target gene after connecting errorless after being sequenced is used with pET-32a plasmid NcoI, XhoI restriction enzyme carry out double digestion and recycling, do double digestion by 20 μ L systems in table 11：

11 double digestion system of table

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 NcoI, XhoI double digestion, being accredited as positive indicates expression vector establishment success, and PCR amplification and double enzyme digestion product are through fine jade There is single band at the place 2990bp or so in sepharose electrophoresis, illustrates containing rAlb-IFN γ-IFN α fusion gene engineering bacterium PET-32a/rAlb-IFN γ-IFN α constructs successfully.

4. the expression of recombinant protein

Picking engineering bacteria 37 DEG C of shaking table recovery 1h in the LB culture medium containing 100 μ g/ml ampicillins, in LB culture medium Amplification culture 4h (OD=1.0) in (the 100 μ g/ml containing ampicillin), is added the IPTG of final concentration of 100 μ g/ml, 32 DEG C lure Lead expression 5h；Thallus is collected, through SDS-PAGE electrophoresis detection, supernatant is deposited in after the bacterial cell disruption after recombinant bacterium induction 5h The visible predominant expression band in the place 127.8KD or so illustrates to have obtained recombinant protein in supernatant precipitating.

5. fusion protein purification

5.1His affinity chromatography

5.2DEAE anion-exchange chromatography

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-IFN α protein peak.

5.4 sample identification

Measure rAlb-IFN γ-IFN α potency and specific activity, specific activity >=10⁵U/mg, albumen are qualification；It is aseptic subpackaged ,- 80 DEG C of preservations.The fusion protein being made of pig albumin, Porcine interferon-gamma and porcine interferon alpha, amino acid sequence can be obtained As shown in 400 < of SEQUENCE LISTING, 1 >.

Embodiment 4

A kind of fusion protein being made of pig albumin, Porcine interferon-gamma and porcine interferon alpha, other are with embodiment 3, only E. coli bl21 therein (DE3) competent cell is replaced in order to which BL21 (DE3) competence with pGro7 plasmid is thin Born of the same parents.The SDS-PAGE electrophoresis result of its fusion protein is compareed with embodiment 3,127.8KD or so place's predominant expression item in supernatant Band is thicker, illustrates after introducing molecular chaperones pGro7, expression of the destination protein in supernatant is more preferable, obtained fusion protein amount It is higher.The albumen of Bacillus coli expression is mostly present in inclusion body；By introducing molecular chaperones in expression bacterial strain, cooperate with Expression albumen correctly folds, and reaches solubility expression of protein.

Embodiment 5

A kind of Recombinant Swine long-acting interferon α, it is mixed with freeze drying protectant respectively by the fusion protein in embodiment 1,2,3,4 Later, freeze-dried to form.The freeze drying protectant is glycerol, mannitol and sucrose, is 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 identification for the fusion protein being made of pig albumin, Porcine interferon-gamma and porcine interferon alpha

The quantitative detection of 6.1 protein contents

With Lowry method, standard test is made with the standard protein of Chinese food pharmaceutical biological product calibrating institute, measures embodiment 1~4 obtained fusion protein concentration is all larger than 1.1mg/ml.

6.2SDS-PAGE electrophoresis detection

Compared with empty bacterium, fusion protein has the newly-increased protein band of a dense dye in 127.8KD or so, as shown in Figure 4.

6.3Western Blot result

Fusion protein in Examples 1 to 4 is detected, respectively with the anti-porcine alpha-IFN (1 of abcam company mouse:5000 dilutions) it is one It is anti-, using goat anti-mouse IgG-HRP as secondary antibody (1:10000 dilutions).Recombinant Swine long-acting interferon α sample can be with anti-pig interferon Specific reaction occurs for alpha monoclonal antibodies, and specific band occurs in the place 127.8KD or so, as shown in Figure 5.

Embodiment 7

Four parts of Recombinant Swine long-acting interferon α in embodiment 5 freeze-dried bioactivity

Inhibit method according to few cells lesion, Hep-2 cell is made into 5 × 10 with culture medium⁵Cell/ml cell suspends Liquid, every hole inoculation 0.1ml move into 96 porocyte culture plates.37 DEG C, 5%CO₂For 24 hours, the Recombinant Swine that various dose is added is long for culture Interferon-' alpha ' is imitated, inhales abandon afterwards for 24 hours, then inoculation 100TCID50VSV virus respectively.

Test result

The result shows that the Recombinant Swine long-acting interferon α obtained causes the lesion of HEp-2 cell to have apparent inhibit VSV Effect.The lesions such as there is cell rounding, falls off, is disintegrated after untreated cell inoculation virus.And the Recombinant Swine obtained is long After imitating interferon-' alpha ' treated cell inoculation virus, it is observed continuously under inverted microscope, cellular morphology is normal, not incumbent out What lesion, measures potency >=10⁵U/ml, as shown in Figure 6.

Embodiment 8

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

Cytopathic-effect inhibition assay measures rAlb-IFN γ-IFN α blood concentration and time relationship

The pig (half male and half female) that six weight are roughly the same is taken, 2mg/ml Recombinant Swine long-acting interferon α is subcutaneously injected in neck Freeze-dried 2ml, respectively in 1h, 2h, 4h, 8h, 16h, 26h, 44h, 79h, 144h venous blood collection, 4 DEG C of blood sample solidifications, 3500rpm Low-temperature centrifugation 10min separates serum, and every pig blood sample of each time point is to be measured in -20 DEG C of preservations.It is measured using cytopathic-effect inhibition assay RAlb-IFN γ-IFN α concentration in blood serum sample is carried out curve fitting and calculating parameter with DAS pharmacokinetics software.Parameter meter Calculation the results are shown in Table 13.

Dominant dynamic parameters in serum after 13 Recombinant Swine long-acting interferon α intramuscular injection of table

The result shows that Recombinant Swine long-acting interferon α has longer half-life period.Half-life period can reach 98h or so after measured, compared with Plain interferon improves about 24 times.

Embodiment 9

The freeze-dried measurement that pig cell immune response is influenced of four parts of Recombinant Swine long-acting interferon α in embodiment 5

It takes six roughly the same pork pigs of weight to be divided into two groups, is denoted as experimental group and control group；Experimental group neck is subcutaneously infused The 2mg/ml Recombinant Swine long-acting interferon freeze-dried 2ml of α is penetrated, the PBS of 2mL is subcutaneously injected in control group neck, after taking injection 4 weeks outside pig All blood takes weekly a blood later, separates lymphocyte using lymphocyte separation medium, lymphocyte passes through serum-free RPMI It after 1640 culture mediums wash 2 times, is resuspended with complete medium, adjustment cell concentration is 2 × 10⁶A/ml, 24 porocyte culture plates are every Hole addition 1ml lymphocyte, 37 DEG C, 5%CO₂72h is cultivated, supernatant when collecting lymphocyte culture.ELISA detects culture supernatant Middle IL-2, IL-4 content, is carried out by kit specification, and testing result is as shown in table 14：

It is horizontal that 14 ELISA of table detects each group pig cell immune response

The result shows that injection Recombinant Swine long-acting interferon α after, can significantly improve cell factor IL-2 in pig peripheral blood, 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 being made of pig albumin, Porcine interferon-gamma and porcine interferon alpha and The detailed description that preparation method carries out, is illustrative without being restrictive, can enumerate according to limited range several A embodiment, therefore the change and modification in the case where not departing from present general inventive concept, should belong within protection scope of the present invention.

SEQUENCE LISTING

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

<120>A kind of fusion protein and preparation method thereof being made of pig albumin, Porcine interferon-gamma and porcine interferon alpha

<130> 1

<160> 9

<170> PatentIn version 3.3

<210> 1

<211> 995

<212> PRT

<213>Pig albumin-interferon gamma-interferon alpha fusion protein

<400> 1

Asp Thr Tyr Lys Ser Glu Ile Ala His Arg Phe Lys Asp Leu Gly Glu

1 5 10 15

Gln Tyr Phe Lys Gly Leu Val Leu Ile Ala Phe Ser Gln His Leu Gln

20 25 30

Gln Cys Pro Tyr Glu Glu His Val Lys Leu Val Arg Glu Val Thr Glu

35 40 45

Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys

50 55 60

Ser Ile His Thr Leu Phe Gly Asp Lys Leu Cys Ala Ile Pro Ser Leu

65 70 75 80

Arg Glu His Tyr Gly Asp Leu Ala Asp Cys Cys Glu Lys Glu Glu Pro

85 90 95

Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asn Asp Asn Pro Asp Ile

100 105 110

Pro Lys Leu Lys Pro Asp Pro Val Ala Leu Cys Ala Asp Phe Gln Glu

115 120 125

Asp Glu Gln Lys Phe Trp Gly Lys Tyr Leu Tyr Glu Ile Ala Arg Arg

130 135 140

His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Tyr Tyr Ala Ile Ile Tyr

145 150 155 160

Lys Asp Val Phe Ser Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala Cys

165 170 175

Leu Leu Pro Lys Ile Glu His Leu Arg Glu Lys Val Leu Thr Ser Ala

180 185 190

Ala Lys Gln Arg Leu Lys Cys Ala Ser Ile Gln Lys Phe Gly Glu Arg

195 200 205

Ala Phe Lys Ala Trp Ser Leu Ala Arg Leu Ser Gln Arg Phe Pro Lys

210 215 220

Ala Asp Phe Thr Glu Ile Ser Lys Ile Val Thr Asp Leu Ala Lys Val

225 230 235 240

His Lys Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp Arg

245 250 255

Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Thr Ile Ser Thr

260 265 270

Lys Leu Lys Glu Cys Cys Asp Lys Pro Leu Leu Glu Lys Ser His Cys

275 280 285

Ile Ala Glu Ala Lys Arg Asp Glu Leu Pro Ala Asp Leu Asn Pro Leu

290 295 300

Glu His Asp Phe Val Glu Asp Lys Glu Val Cys Lys Asn Tyr Lys Glu

305 310 315 320

Ala Lys His Val Phe Leu Gly Thr Phe Leu Tyr Glu Tyr Ser Arg Arg

325 330 335

His Pro Asp Tyr Ser Val Ser Leu Leu Leu Arg Ile Ala Lys Ile Tyr

340 345 350

Glu Ala Thr Leu Glu Asp Cys Cys Ala Lys Glu Asp Pro Pro Ala Cys

355 360 365

Tyr Ala Thr Val Phe Asp Lys Phe Gln Pro Leu Val Asp Glu Pro Lys

370 375 380

Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Lys Leu Gly Glu Tyr

385 390 395 400

Gly Phe Gln Asn Ala Leu Ile Val Arg Tyr Thr Lys Lys Val Pro Gln

405 410 415

Val Ser Thr Pro Thr Leu Val Glu Val Ala Arg Lys Leu Gly Leu Val

420 425 430

Gly Ser Arg Cys Cys Lys Arg Pro Glu Glu Glu Arg Leu Ser Cys Ala

435 440 445

Glu Asp Tyr Leu Ser Leu Val Leu Asn Arg Leu Cys Val Leu His Glu

450 455 460

Lys Thr Pro Val Ser Glu Lys Val Thr Lys Cys Cys Thr Glu Ser Leu

465 470 475 480

Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Thr Pro Asp Glu Thr Tyr

485 490 495

Lys Pro Lys Glu Phe Val Glu Gly Thr Phe Thr Phe His Ala Asp Leu

500 505 510

Cys Thr Leu Pro Glu Asp Glu Lys Gln Ile Lys Lys Gln Thr Ala Leu

515 520 525

Val Glu Leu Leu Lys His Lys Pro His Ala Thr Glu Glu Gln Leu Arg

530 535 540

Thr Val Leu Gly Asn Phe Ala Ala Phe Val Gln Lys Cys Cys Ala Ala

545 550 555 560

Pro Asp His Glu Ala Cys Phe Ala Val Glu Gly Pro Lys Phe Val Ile

565 570 575

Glu Ile Arg Gly Ile Leu Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly

580 585 590

Ser Met Ser Tyr Thr Thr Tyr Phe Leu Ala Phe Gln Leu Cys Val Thr

595 600 605

Leu Cys Phe Ser Gly Ser Tyr Cys Gln Ala Pro Phe Phe Lys Glu Ile

610 615 620

Thr Ile Leu Lys Asp Tyr Phe Asn Ala Ser Thr Ser Gly Val Pro Asn

625 630 635 640

Gly Gly Pro Leu Phe Leu Glu Ile Leu Glu Asn Trp Lys Glu Glu Ser

645 650 655

Asp Lys Lys Ile Ile Gln Ser Gln Ile Val Ser Phe Tyr Phe Lys Phe

660 665 670

Phe Glu Ile Phe Lys Asp Asn Gln Ala Ile Gln Arg Ser Met Asp Val

675 680 685

Ile Lys Gln Asp Met Phe Gln Arg Phe Leu Asn Gly Ser Ser Gly Lys

690 695 700

Leu Asn Asp Phe Glu Lys Leu Val Lys Ile Pro Val Asp Asn Leu Gln

705 710 715 720

Ile Gln Arg Lys Ala Ile Ser Glu Leu Ile Lys Val Met Asn Asp Leu

725 730 735

Ser Pro Arg Ser Asn Leu Arg Lys Arg Lys Arg Ser Gln Thr Met Phe

740 745 750

Gln Gly Gln Arg Ala Ser Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly

755 760 765

Ser Met Cys Ser Tyr Leu Arg His Arg Pro Glu Gly Arg Ser Ser Asn

770 775 780

Ile Leu Glu Ser Arg Val Thr Glu Ser Pro Thr Ser Ala Arg Thr Ala

785 790 795 800

Ala Ser Ala Arg Ser Pro Met Ala Pro Thr Ser Ala Phe Leu Thr Ala

805 810 815

Leu Val Leu Leu Ser Cys Asn Ala Ile Cys Ser Leu Gly Cys Asp Leu

820 825 830

Pro Gln Thr His Ser Leu Ala His Thr Arg Ala Leu Arg Leu Leu Ala

835 840 845

Gln Met Arg Arg Ile Ser Pro Phe Ser Cys Leu Asp His Arg Arg Asp

850 855 860

Phe Gly Phe Pro Gln Glu Ala Leu Gly Gly Asn Gln Val Gln Lys Ala

865 870 875 880

Gln Ala Met Ala Leu Val His Glu Met Leu Gln Gln Thr Phe Gln Leu

885 890 895

Phe Ser Thr Glu Gly Ser Ala Ala Ala Trp Asp Glu Ser Leu Leu His

900 905 910

Gln Phe Cys Thr Gly Leu Asp Gln Gln Leu Arg Asp Leu Glu Ala Cys

915 920 925

Val Met Gln Glu Ala Gly Leu Glu Gly Thr Pro Leu Leu Glu Glu Asp

930 935 940

Ser Ile Leu Ala Val Arg Lys Tyr Phe His Arg Leu Thr Leu Tyr Leu

945 950 955 960

Gln Glu Lys Ser Tyr Ser Pro Cys Ala Trp Glu Ile Val Arg Ala Glu

965 970 975

Val Met Arg Ala Phe Ser Ser Ser Thr Asn Leu Gln Asp Arg Leu Arg

980 985 990

Arg Lys Glu

995

<210> 2

<211> 2985

<212> DNA

<213>Genome 1

<400> 2

gatacataca agagtgaaat tgctcatcgg tttaaagatt tgggagaaca atatttcaaa 60

ggcctagtgc tgattgcctt ttctcagcat ctccagcaat gcccatatga agagcatgtg 120

aaattagtga gggaagtaac tgagtttgca aaaacatgtg ttgctgatga gtcagctgaa 180

aattgtgaca agtcaattca cactctcttt ggagataaat tatgtgcaat tccatccctt 240

cgtgaacact atggtgactt ggctgactgc tgtgaaaaag aagagcctga gagaaacgaa 300

tgcttcctcc aacacaaaaa tgataacccc gacatcccta aattgaaacc agaccctgtt 360

gctttatgcg ctgacttcca ggaagatgaa cagaagtttt ggggaaaata cctatatgaa 420

attgccagaa gacatcccta tttctacgcc ccagaactcc tttattatgc cattatatat 480

aaagatgttt tttcagaatg ctgccaagct gctgataaag ctgcctgcct gttaccaaag 540

attgagcatc tgagagaaaa agtactgact tccgccgcca aacagagact taagtgtgcc 600

agtatccaaa aattcggaga gagagctttc aaagcatggt cattagctcg cctgagccag 660

agatttccca aggctgactt tacagagatt tccaagatag tgacagatct tgcaaaagtc 720

cacaaggaat gctgccatgg tgacctgctt gaatgtgcag atgacagggc ggatcttgcc 780

aaatatatat gtgaaaatca agacacaatc tccactaaac tgaaggaatg ctgtgataag 840

cctctgttgg aaaaatccca ctgcattgct gaggcaaaaa gagatgaatt gcctgcagac 900

ctgaacccat tagaacatga ttttgttgaa gataaggaag tttgtaaaaa ctataaagaa 960

gcaaagcatg tcttcctggg cacgtttttg tatgagtatt caagaaggca cccagactac 1020

tctgtctcat tgctgctgag aattgccaag atatatgaag ccacactgga ggactgctgt 1080

gccaaagagg atcctccggc atgctatgcc acagtgtttg ataaatttca gcctcttgtg 1140

gatgagccta agaatttaat caaacaaaac tgtgaacttt ttgaaaaact tggagagtat 1200

ggattccaaa atgcgctcat agttcgttac accaagaaag taccccaagt gtcaactcca 1260

actcttgtgg aggtcgcaag aaaactagga ctagtgggct ctaggtgttg taagcgtcct 1320

gaagaagaaa gactgtcctg tgctgaagac tatctgtccc tggtcctgaa ccggttgtgc 1380

gtgttgcacg agaagacacc agtgagcgaa aaagttacca aatgctgcac agagtccttg 1440

gtgaacagac ggccttgctt ttctgctctg acaccagacg aaacatacaa acccaaagaa 1500

tttgttgagg gaaccttcac cttccatgca gacctatgca cacttcctga ggatgagaaa 1560

caaatcaaga agcaaactgc actcgttgag ttgttgaaac acaagcctca tgcaacagag 1620

gaacaactga gaactgtcct gggcaacttt gcagcctttg tacaaaagtg ctgcgccgct 1680

cctgaccatg aggcctgctt tgctgtggag ggtccgaaat ttgttattga aattcgaggg 1740

atcttagccg gtggtggtgg ttctggtggt ggtggttcta tgagttatac aacttatttc 1800

ttagcttttc agctttgcgt gactttgtgt ttttctggct cttactgcca ggcgcccttt 1860

tttaaagaaa taacgatcct aaaggactat tttaatgcaa gtacctcagg tgtacctaat 1920

ggtggacctc ttttcttaga aattttggag aattggaaag aggagagtga caaaaaaata 1980

attcagagcc aaattgtctc cttctacttc aaattctttg aaatcttcaa agataaccag 2040

gccattcaaa ggagcatgga tgtgatcaag caagacatgt ttcagaggtt cctaaatggt 2100

agctctggga aactgaatga cttcgaaaag ctggttaaaa ttccggtaga taatctgcag 2160

atccagcgca aagccatcag tgaactcatc aaagtgatga atgatctgtc accaagatct 2220

aacctaagaa agcggaagag aagtcagact atgttccaag gccagagagc atcaaaaggt 2280

ggtggtggtt ctggtggtgg tggttctatg tgttcctatt taagacacag gcctgaggga 2340

aggtcttcaa acatcctaga gagcagggtc acagagtcac ccacctcagc caggacagca 2400

gcatctgcaa ggtccccaat ggccccaacc tcagccttcc tcacggccct ggtgctgctc 2460

agctgcaatg ccatctgctc tctgggctgc gacctgcctc agacccacag cctggctcac 2520

accagggccc tgaggctcct ggcacaaatg aggagaatct cccccttctc ctgcctggac 2580

cacagaaggg actttggatt cccccaagag gccttggggg gcaaccaggt ccagaaggct 2640

caagccatgg ctctggtgca tgagatgctc cagcagacct tccagctctt cagcacagag 2700

ggctcggctg ctgcctggga tgagagcctc ctgcaccagt tctgcactgg actggatcag 2760

cagctcaggg acctggaagc ctgtgtcatg caggaggccg ggctggaagg gacccccctg 2820

ctggaggagg actccatcct ggctgtgagg aaatacttcc acagactcac cctctatctg 2880

caagagaaga gctacagccc ctgtgcctgg gagatcgtca gggcagaagt catgagagcc 2940

ttctcttcct ccacaaacct gcaagacaga ctcaggagga aggag 2985

<210> 3

<211> 2985

<212> DNA

<213>Genome 2

<400> 3

gacacctaca aatctgaaat cgctcaccgt ttcaaagacc tgggtgaaca gtacttcaaa 60

ggtctggttc tgatcgcttt ctctcagcac ctgcagcagt gcccgtacga agaacacgtt 120

aaactggttc gtgaagttac cgaattcgct aaaacctgcg ttgctgacga atctgctgaa 180

aactgcgaca aatctatcca caccctgttc ggtgacaaac tgtgcgctat cccgtctctg 240

cgtgaacact acggtgacct ggctgactgc tgcgaaaaag aagaaccgga acgtaacgaa 300

tgcttcctgc agcacaaaaa cgacaacccg gacatcccga aactgaaacc ggacccggtt 360

gctctgtgcg ctgacttcca ggaagacgaa cagaaattct ggggtaaata cctgtacgaa 420

atcgctcgtc gtcacccgta cttctacgct ccggaactgc tgtactacgc tatcatctac 480

aaagacgttt tctctgaatg ctgccaggct gctgacaaag ctgcttgcct gctgccgaaa 540

atcgaacacc tgcgtgaaaa agttctgacc tctgctgcta aacagcgtct gaaatgcgct 600

tctatccaga aattcggtga acgtgctttc aaagcttggt ctctggctcg tctgtcgcag 660

aggttcccga aagctgactt caccgaaatc tctaaaatcg ttaccgacct ggctaaagtt 720

cacaaagaat gctgccacgg tgacctgctg gaatgcgctg acgaccgtgc tgacctggct 780

aaatacatct gcgaaaacca ggacaccatc tctaccaaac tgaaagaatg ctgcgacaaa 840

ccgctgctgg aaaaatctca ctgcatcgct gaagctaaac gtgacgaact gccggctgac 900

ctgaacccgc tggaacacga cttcgttgaa gataaggaag tgtgcaaaaa ctacaaagaa 960

gctaaacacg ttttcctggg taccttcctg tacgaatact ctcgtcgtca cccggactac 1020

tctgtttctc tgctgctgcg tatcgctaaa atctacgaag ctaccctgga agactgctgc 1080

gctaaagaag acccgccggc ttgctacgct accgttttcg acaaattcca gccgctggtt 1140

gacgaaccga aaaacctgat caaacagaac tgcgaactgt tcgaaaaact gggtgaatac 1200

ggtttccaga acgctctgat cgttcgttac accaaaaaag ttccgcaggt ttctaccccg 1260

accctggttg aagttgctcg taaactgggt ctggttggtt ctcgttgctg caaacgtccg 1320

gaagaagaac gtctgtcttg cgctgaagac tacctgtctc tggttctgaa ccgtctgtgc 1380

gttctgcacg aaaaaacccc ggtttctgaa aaagttacca aatgctgcac cgaatctctg 1440

gttaaccgtc gtccgtgctt ctctgctctg accccggacg aaacctacaa accgaaagaa 1500

ttcgttgaag gtaccttcac cttccacgct gacctgtgca ccctgccgga agacgaaaaa 1560

cagatcaaaa aacagaccgc tctggttgaa ctgctgaaac acaaaccgca cgctaccgaa 1620

gaacagctgc gtaccgttct gggtaacttc gctgctttcg ttcagaaatg ctgcgctgct 1680

ccggaccacg aagcttgctt cgctgttgaa ggtccgaaat tcgttatcga aatccgtggt 1740

atcctggctg gtggtggtgg ttctggtggt ggtggttcta tgtcttacac cacctacttc 1800

ctggctttcc agctgtgcgt taccctgtgc ttctctggtt cttactgcca ggctccgttc 1860

ttcaaagaaa tcaccatcct gaaagactac ttcaacgctt ctacctctgg tgttccgaac 1920

ggtggtccgc tgttcctgga aatcctggaa aactggaaag aagaatctga caaaaaaatc 1980

atccagtctc agatcgtttc tttctacttc aaattcttcg aaatcttcaa agacaaccag 2040

gctatccagc gttctatgga cgttatcaaa caggacatgt tccagcgttt cctgaacggt 2100

tcttctggta aactgaacga cttcgaaaaa ctggttaaaa tcccggttga caacctgcag 2160

atccagcgta aagctatctc tgaactgatc aaagttatga acgacctgtc tccgcgttct 2220

aacctgcgta aacgtaaacg ttctcagacc atgttccagg gtcagcgtgc ttctaaaggt 2280

ggtggtggtt ctggtggtgg tggttctatg tgctcttacc tgcgtcaccg tccggaaggt 2340

cgttcttcta acatcctgga atctcgtgtt accgaatctc cgacctctgc tcgtaccgct 2400

gcttctgctc gttctccgat ggctccgacc tctgctttcc tgaccgctct ggttctgctg 2460

tcttgcaacg ctatctgctc tctgggttgc gacctgccgc agacccactc tctggctcac 2520

acccgtgctc tgcgtctgct ggctcagatg cgtcgtatct ctccgttctc ttgcctggac 2580

caccgtcgtg acttcggttt cccgcaggaa gctctgggtg gtaaccaggt tcagaaagct 2640

caggctatgg ctctggttca cgaaatgctg cagcagacct tccagctgtt ctctaccgaa 2700

ggttctgctg ctgcttggga cgaatctctg ctgcaccagt tctgcaccgg tctggaccag 2760

cagctgcgtg acctggaagc ttgcgttatg caggaagctg gtctggaagg taccccgctg 2820

ctggaagaag actctatcct ggctgttcgt aaatacttcc accgtctgac cctgtacctg 2880

caggaaaaat cttactctcc gtgcgcttgg gaaatcgttc gtgctgaagt tatgcgtgct 2940

ttctcttctt ctaccaacct gcaggaccgt ctgcgtcgta aagaa 2985

<210> 4

<211> 1749

<212> DNA

<213>Pig albumin

<400> 4

gatacataca agagtgaaat tgctcatcgg tttaaagatt tgggagaaca atatttcaaa 60

ggcctagtgc tgattgcctt ttctcagcat ctccagcaat gcccatatga agagcatgtg 120

aaattagtga gggaagtaac tgagtttgca aaaacatgtg ttgctgatga gtcagctgaa 180

aattgtgaca agtcaattca cactctcttt ggagataaat tatgtgcaat tccatccctt 240

cgtgaacact atggtgactt ggctgactgc tgtgaaaaag aagagcctga gagaaacgaa 300

tgcttcctcc aacacaaaaa tgataacccc gacatcccta aattgaaacc agaccctgtt 360

gctttatgcg ctgacttcca ggaagatgaa cagaagtttt ggggaaaata cctatatgaa 420

attgccagaa gacatcccta tttctacgcc ccagaactcc tttattatgc cattatatat 480

aaagatgttt tttcagaatg ctgccaagct gctgataaag ctgcctgcct gttaccaaag 540

attgagcatc tgagagaaaa agtactgact tccgccgcca aacagagact taagtgtgcc 600

agtatccaaa aattcggaga gagagctttc aaagcatggt cattagctcg cctgagccag 660

agatttccca aggctgactt tacagagatt tccaagatag tgacagatct tgcaaaagtc 720

cacaaggaat gctgccatgg tgacctgctt gaatgtgcag atgacagggc ggatcttgcc 780

aaatatatat gtgaaaatca agacacaatc tccactaaac tgaaggaatg ctgtgataag 840

cctctgttgg aaaaatccca ctgcattgct gaggcaaaaa gagatgaatt gcctgcagac 900

ctgaacccat tagaacatga ttttgttgaa gataaggaag tttgtaaaaa ctataaagaa 960

gcaaagcatg tcttcctggg cacgtttttg tatgagtatt caagaaggca cccagactac 1020

tctgtctcat tgctgctgag aattgccaag atatatgaag ccacactgga ggactgctgt 1080

gccaaagagg atcctccggc atgctatgcc acagtgtttg ataaatttca gcctcttgtg 1140

gatgagccta agaatttaat caaacaaaac tgtgaacttt ttgaaaaact tggagagtat 1200

ggattccaaa atgcgctcat agttcgttac accaagaaag taccccaagt gtcaactcca 1260

actcttgtgg aggtcgcaag aaaactagga ctagtgggct ctaggtgttg taagcgtcct 1320

gaagaagaaa gactgtcctg tgctgaagac tatctgtccc tggtcctgaa ccggttgtgc 1380

gtgttgcacg agaagacacc agtgagcgaa aaagttacca aatgctgcac agagtccttg 1440

gtgaacagac ggccttgctt ttctgctctg acaccagacg aaacatacaa acccaaagaa 1500

tttgttgagg gaaccttcac cttccatgca gacctatgca cacttcctga ggatgagaaa 1560

caaatcaaga agcaaactgc actcgttgag ttgttgaaac acaagcctca tgcaacagag 1620

gaacaactga gaactgtcct gggcaacttt gcagcctttg tacaaaagtg ctgcgccgct 1680

cctgaccatg aggcctgctt tgctgtggag ggtccgaaat ttgttattga aattcgaggg 1740

atcttagcc 1749

<210> 5

<211> 498

<212> DNA

<213>Porcine IFN γ

<400> 5

atgagttata caacttattt cttagctttt cagctttgcg tgactttgtg tttttctggc 60

tcttactgcc aggcgccctt ttttaaagaa ataacgatcc taaaggacta ttttaatgca 120

agtacctcag gtgtacctaa tggtggacct cttttcttag aaattttgga gaattggaaa 180

gaggagagtg acaaaaaaat aattcagagc caaattgtct ccttctactt caaattcttt 240

gaaatcttca aagataacca ggccattcaa aggagcatgg atgtgatcaa gcaagacatg 300

tttcagaggt tcctaaatgg tagctctggg aaactgaatg acttcgaaaa gctggttaaa 360

attccggtag ataatctgca gatccagcgc aaagccatca gtgaactcat caaagtgatg 420

aatgatctgt caccaagatc taacctaaga aagcggaaga gaagtcagac tatgttccaa 480

ggccagagag catcaaaa 498

<210> 6

<211> 678

<212> DNA

<213>Pig IFN-α

<400> 6

atgtgttcct atttaagaca caggcctgag ggaaggtctt caaacatcct agagagcagg 60

gtcacagagt cacccacctc agccaggaca gcagcatctg caaggtcccc aatggcccca 120

acctcagcct tcctcacggc cctggtgctg ctcagctgca atgccatctg ctctctgggc 180

tgcgacctgc ctcagaccca cagcctggct cacaccaggg ccctgaggct cctggcacaa 240

atgaggagaa tctccccctt ctcctgcctg gaccacagaa gggactttgg attcccccaa 300

gaggccttgg ggggcaacca ggtccagaag gctcaagcca tggctctggt gcatgagatg 360

ctccagcaga ccttccagct cttcagcaca gagggctcgg ctgctgcctg ggatgagagc 420

ctcctgcacc agttctgcac tggactggat cagcagctca gggacctgga agcctgtgtc 480

atgcaggagg ccgggctgga agggaccccc ctgctggagg aggactccat cctggctgtg 540

aggaaatact tccacagact caccctctat ctgcaagaga agagctacag cccctgtgcc 600

tgggagatcg tcagggcaga agtcatgaga gccttctctt cctccacaaa cctgcaagac 660

agactcagga ggaaggag 678

<210> 7

<211> 1749

<212> DNA

<213>Pig albumin

<400> 7

gacacctaca aatctgaaat cgctcaccgt ttcaaagacc tgggtgaaca gtacttcaaa 60

ggtctggttc tgatcgcttt ctctcagcac ctgcagcagt gcccgtacga agaacacgtt 120

aaactggttc gtgaagttac cgaattcgct aaaacctgcg ttgctgacga atctgctgaa 180

aactgcgaca aatctatcca caccctgttc ggtgacaaac tgtgcgctat cccgtctctg 240

cgtgaacact acggtgacct ggctgactgc tgcgaaaaag aagaaccgga acgtaacgaa 300

tgcttcctgc agcacaaaaa cgacaacccg gacatcccga aactgaaacc ggacccggtt 360

gctctgtgcg ctgacttcca ggaagacgaa cagaaattct ggggtaaata cctgtacgaa 420

atcgctcgtc gtcacccgta cttctacgct ccggaactgc tgtactacgc tatcatctac 480

aaagacgttt tctctgaatg ctgccaggct gctgacaaag ctgcttgcct gctgccgaaa 540

atcgaacacc tgcgtgaaaa agttctgacc tctgctgcta aacagcgtct gaaatgcgct 600

tctatccaga aattcggtga acgtgctttc aaagcttggt ctctggctcg tctgtcgcag 660

aggttcccga aagctgactt caccgaaatc tctaaaatcg ttaccgacct ggctaaagtt 720

cacaaagaat gctgccacgg tgacctgctg gaatgcgctg acgaccgtgc tgacctggct 780

aaatacatct gcgaaaacca ggacaccatc tctaccaaac tgaaagaatg ctgcgacaaa 840

ccgctgctgg aaaaatctca ctgcatcgct gaagctaaac gtgacgaact gccggctgac 900

ctgaacccgc tggaacacga cttcgttgaa gataaggaag tgtgcaaaaa ctacaaagaa 960

gctaaacacg ttttcctggg taccttcctg tacgaatact ctcgtcgtca cccggactac 1020

tctgtttctc tgctgctgcg tatcgctaaa atctacgaag ctaccctgga agactgctgc 1080

gctaaagaag acccgccggc ttgctacgct accgttttcg acaaattcca gccgctggtt 1140

gacgaaccga aaaacctgat caaacagaac tgcgaactgt tcgaaaaact gggtgaatac 1200

ggtttccaga acgctctgat cgttcgttac accaaaaaag ttccgcaggt ttctaccccg 1260

accctggttg aagttgctcg taaactgggt ctggttggtt ctcgttgctg caaacgtccg 1320

gaagaagaac gtctgtcttg cgctgaagac tacctgtctc tggttctgaa ccgtctgtgc 1380

gttctgcacg aaaaaacccc ggtttctgaa aaagttacca aatgctgcac cgaatctctg 1440

gttaaccgtc gtccgtgctt ctctgctctg accccggacg aaacctacaa accgaaagaa 1500

ttcgttgaag gtaccttcac cttccacgct gacctgtgca ccctgccgga agacgaaaaa 1560

cagatcaaaa aacagaccgc tctggttgaa ctgctgaaac acaaaccgca cgctaccgaa 1620

gaacagctgc gtaccgttct gggtaacttc gctgctttcg ttcagaaatg ctgcgctgct 1680

ccggaccacg aagcttgctt cgctgttgaa ggtccgaaat tcgttatcga aatccgtggt 1740

atcctggct 1749

<210> 8

<211> 498

<212> DNA

<213>Porcine IFN γ

<400> 8

atgtcttaca ccacctactt cctggctttc cagctgtgcg ttaccctgtg cttctctggt 60

tcttactgcc aggctccgtt cttcaaagaa atcaccatcc tgaaagacta cttcaacgct 120

tctacctctg gtgttccgaa cggtggtccg ctgttcctgg aaatcctgga aaactggaaa 180

gaagaatctg acaaaaaaat catccagtct cagatcgttt ctttctactt caaattcttc 240

gaaatcttca aagacaacca ggctatccag cgttctatgg acgttatcaa acaggacatg 300

ttccagcgtt tcctgaacgg ttcttctggt aaactgaacg acttcgaaaa actggttaaa 360

atcccggttg acaacctgca gatccagcgt aaagctatct ctgaactgat caaagttatg 420

aacgacctgt ctccgcgttc taacctgcgt aaacgtaaac gttctcagac catgttccag 480

ggtcagcgtg cttctaaa 498

<210> 9

<211> 678

<212> DNA

<213>Pig IFN-α

<400> 9

atgtgctctt acctgcgtca ccgtccggaa ggtcgttctt ctaacatcct ggaatctcgt 60

gttaccgaat ctccgacctc tgctcgtacc gctgcttctg ctcgttctcc gatggctccg 120

acctctgctt tcctgaccgc tctggttctg ctgtcttgca acgctatctg ctctctgggt 180

tgcgacctgc cgcagaccca ctctctggct cacacccgtg ctctgcgtct gctggctcag 240

atgcgtcgta tctctccgtt ctcttgcctg gaccaccgtc gtgacttcgg tttcccgcag 300

gaagctctgg gtggtaacca ggttcagaaa gctcaggcta tggctctggt tcacgaaatg 360

ctgcagcaga ccttccagct gttctctacc gaaggttctg ctgctgcttg ggacgaatct 420

ctgctgcacc agttctgcac cggtctggac cagcagctgc gtgacctgga agcttgcgtt 480

atgcaggaag ctggtctgga aggtaccccg ctgctggaag aagactctat cctggctgtt 540

cgtaaatact tccaccgtct gaccctgtac ctgcaggaaa aatcttactc tccgtgcgct 600

tgggaaatcg ttcgtgctga agttatgcgt gctttctctt cttctaccaa cctgcaggac 660

cgtctgcgtc gtaaagaa 678

Claims

1. a kind of fusion protein being made of pig albumin, Porcine interferon-gamma and porcine interferon alpha, it is characterised in that：The fusion The amino acid sequence table of albumen 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 Recombinant Swine long-acting interferon, which is characterized in that the Recombinant Swine long-acting interferon is melted by described in claim 1 It is freeze-dried to form after hop protein and freeze drying protectant mixture.

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

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

(1) it is dry to obtain by reverse transcription or be manually respectively synthesized the pig albumin with flexible linker sequence, pig for design primer Disturb the target gene of plain γ, porcine interferon alpha；By flexible linker by pig albumin, Porcine interferon-gamma, porcine interferon alpha mesh Gene connect, the nucleotides sequence list of the target gene after connection as shown in 400 < of SEQUENCE LISTING, 2 > or As shown in 400 < of SEQUENCE LISTING, 3 >；

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

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

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

10. the application of Recombinant Swine long-acting interferon according to claim 5, which is characterized in that the Recombinant Swine is long-acting dry The long half time of element is disturbed up to 98 hours or more, there is broad-spectrum disease resistance toxic action and the immune response of pig itself can be improved.