CN108794645A - A kind of fusion protein and preparation method thereof being made of bovine albumin, Bov IFN γ and Bov IFN α - Google Patents

Abstract

The invention discloses a kind of fusion proteins and preparation method thereof being made of bovine albumin, Bov IFN γ and Bov IFN α; the fusion protein is connected through flexible linker with Bov IFN α by bovine albumin, Bov IFN γ and is formed, through being freeze-dried to obtain recombinant bovine long-acting interferon after fusion protein and freeze drying protectant mixture.The recombinant bovine long-acting interferon is remarkably improved the half-life period of Bov IFN, and the half-life period of more common Bov IFN improves 18 times or more, and has broad-spectrum disease resistance toxic action and can improve the immune response of Niu Zishen.

Description

A kind of fusion protein being made of bovine albumin, Bov IFN γ and Bov IFN α and Preparation method

Technical field

The invention belongs to technical field of biological genetic engineering, and in particular to be interfered by bovine albumin, Bov IFN γ and ox The fusion protein and preparation method thereof of plain α compositions.

Background technology

Ox is important one of the herding type in China, with intensive and large-scale cultivation continuous development, bovine viral The incidence of communicable disease improves year by year.Large-scale pasture is popular for a long time at home for the communicable disease of many oxen, because Disease infects fast, the high sound development that seriously restrict China's ox aquaculture of incidence, the death rate；More seriously some Poultry suffers from the life and health that brucellosis, tuberculosis of infectious disease such as ox etc. directly threaten the mankind altogether.

By vaccine inoculation and antibiotic mainly is used to the prevention and treatment approach of ox communicable disease at present.Big portion Antibiotics and traditional oral antiviral medicament is divided to be brought a negative impact to health due to medicament residue problem；And Traditional vaccine, the high specific due to it and side effect, can not resist virus variation and new virus continuously emerges to pig The significant damage that aquaculture is brought.Interferon determines its tool with the antiviral activity of its wide spectrum and extensive immunoregulation capability There are huge clinical application potentiality, can be used for preventing and treating bovine viral bacterial infection disease.

IFN is that the infection induced body of a viroid is generated with broad-spectrum antiviral, antitumor and with immunoregulation effect Protein, nineteen fifty-seven, Issacs and Lindeman had found first, 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, mainly 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 types 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, the killing activity for anti parasitic, inhibiting various kinds of cell proliferation, stimulating immunocyte.

γ types IFN is the T cell and the generation of NK cells by activating, 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 virus infection it is anti- It answers, but the immunoregulatory activity of interferon gamma plays more in coordinating immune response and determining the long-term antiviral state of body Important role, therefore interferon gamma has particularly important clinical value.

Seralbumin is the important component of blood plasma, is not easy to penetrate glomerulus under normal circumstances, 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 linked in the cell by peptide bond through protein translation system in vain, is not required to additional extracorporeal treatment；The expression of albumin is higher, The expression of destination protein can be improved after being merged with it；Albumin is one stable " inert protein ", after being merged 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 the current generally existing half-life short of artificial recombination interferon, 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 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.

Invention content

In order to solve the above technical problems, the present invention provides one kind by bovine albumin, Bov IFN γ and Bov IFN α groups At fusion protein and preparation method thereof, and thus fusion protein with after freeze drying protectant mixture, it is freeze-dried to be prepared into To a kind of recombinant bovine long-acting interferon, the recombinant bovine long-acting interferon is remarkably improved the half-life period of Bov IFN, more commonly The half-life period of Bov IFN improves 18 times or more, and has broad-spectrum disease resistance toxic action and can improve the immune response of Niu Zishen.

The technical solution that the present invention takes is：

A kind of fusion protein being made of bovine albumin, Bov IFN γ and Bov IFN α, 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 2 equal codified of the genome 1 and the genome.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 values are lower to show that expression is lower in host.Most ideal point of G/C content in gene Cloth ranging from 30~70% can influence translation and transcriptional efficiency in any region more than the range.It is sent out using software detection Existing bovine albumin, ox IFN-γ, ox IFN-α original gene codon in Escherichia coli codon adaptation indexI (CAI) point Not Wei 0.22,0.24,0.25, GC percentages be 42.9%, 39.8%, 58.2%；And by bovine albumin, ox IFN-γ, It is 0.99,1.0,0.97, GC that each gene codon adaptation indexI (CAI) in Escherichia coli is obtained after ox IFN-α gene optimization Percentage 49.9%, 44.8%, 54.6%.The utilization rate that low codon is significantly reduced by gene optimization avoids rare Influence of the codon 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 carriers 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 plasmids.

The present invention also provides a kind of recombinant bovine long-acting interferon, the recombinant bovine long-acting interferon is by the fusion egg In vain with after freeze drying protectant mixture, it is freeze-dried to form.

The freeze drying protectant is glycerine, mannitol and sucrose, is buffer solution, the final concentration of three with 10mmol/L PBS For glycerine 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 induced expressions, can be obtained fusion protein after purified.

The expression vector is the pET-32a coli expression carriers 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 bovine albumin with flexible linker sequences, The target gene of Bov IFN γ, Bov IFN α；By flexible linker by bovine albumin, Bov IFN γ, Bov IFN α Target gene connect, nucleotides sequence list such as 400 < of SEQUENCE LISTING, the 2 > institutes of the target gene after connection Show or as shown in 400 < of SEQUENCE LISTING, 3 >；

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

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

The e. coli host cell is BL21 (DE3) competent cells or BL21 (DE3) senses with pGro7 plasmids 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.

Described BL21 (DE3) competent cell with pGro7 plasmids 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 bovine albumin (Alb) is：

Upstream Alb-F1：CCGGAATTCATGAAGTGGGTGACTT carries EcoRI restriction enzyme sites；

Downstream Alb-R1：

ACCACCACCAGAACCACCACCACCGGCTAAGGCTGTTTG, with flexible linker；

The primer sequence of Bov IFN γ (IFN-γ) is：

Upstream IFN-γ-F1：

GGTGGTTCTGGTGGTGGTGGTTCTATGAAATATACAAGCTATTT, with flexible linker；

Downstream IFN-γ-R1：

ACCACCACCAGAACCACCACCACCCGTTGATGCTCTCCG, with flexible linker；

The primer sequence of Bov IFN α (IFN-α) is：

Upstream IFN-α-F1：

GGTGGTTCTGGTGGTGGTGGTTCTTGCCACCTGCCTC, with flexible linker；

Downstream IFN-α-R1：CCCTCGAGGTCCTTTCTCCTGAAAC carries XhoI restriction enzyme sites；

B. RNA is extracted from cattle liver, by reverse transcription obtain ox Alb, ox IFN-γ and ox IFN-α target gene, 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 ox Alb, ox IFN-γ and ox IFN-α as template, and it is utilized respectively ox Alb, ox IFN- The upstream and downstream primer of γ and ox IFN-α carry out PCR amplification, respectively obtain the ox Alb for connecting flexible linker, ox IFN-γ and Ox IFN-α gene.

PCR reaction systems 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 reactions Condition is：50 DEG C of reverse transcriptions 30min, 95 DEG C of pre-degeneration 4min, into cycle；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 genes are obtained using flexible linker connection ox Alb and ox IFN-γ target gene

The PCR reaction systems and reaction condition of connection be：In the overall reaction system of 25 μ L, connect flexible linker's 1 μ L of Alb gene templates DNA connect 1 μ L, Alb sense primer of IFN-γ template DNA 0.5 the μ L, IFN- of flexible linker 0.5 μ L, Taq archaeal dna polymerase of γ downstream primers, 2.5 μ L, dNTP Mix is 10 μ L, adds RNase Free water to 25 μ L；Connection PCR reaction conditions are：95 DEG C of pre-degeneration 4min, into cycle：94 DEG C of denaturation 45s；58 DEG C of annealing 45s, 72 DEG C of extension 1kb/ Min, totally 35 recycle；Last 72 DEG C of extensions 10min.

D. rAlb-IFN γ-IFN are obtained using flexible linker connections rAlb-IFN γ genes and ox IFN-α target gene α genes

The PCR reaction systems and reaction condition of connection be：In the overall reaction system of 25 μ L, rAlb-IFN γ gene templates 1 μ L of DNA connect 1 μ L, Alb sense 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 conditions is：95 DEG C pre-degeneration 4min, into cycle：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 being optimized to genome 1, the acquisition methods of the genome 2 For：

A. design of primers

The primer sequence of bovine albumin (Alb) is：

Upstream Alb-F2：CGGGATCCATGAAATGGGTTACCTT carries BamHI restriction enzyme sites；

Downstream Alb-R2：

ACCACCACCAGAACCACCACCACCAGCCAGAGCGGTC, with flexible linker；

The primer sequence of Bov IFN γ (IFN-γ) is：

Upstream IFN-γ-F2：

GGTGGTTCTGGTGGTGGTGGTTCTATGAAATACACCTCTTAC, with flexible linker；

Downstream IFN-γ-R2：

ACCACCACCAGAACCACCACCACCGGTAGAAGCACGACG, with flexible linker；

Bov IFN α (IFN-α)：

Upstream IFN-α-F2：

GGTGGTTCTGGTGGTGGTGGTTCTTGCCACCTGCCG, with flexible linker；

Downstream IFN-α-R2：

CCCTCGAGGTCTTTACGACGGAAA carries XhoI restriction enzyme sites.

B. the ox Alb, ox IFN-γ and ox IFN-α target gene, the gene order of three is respectively such as SEQUENCE Shown in 400 < of LISTING, 7 >, 400 < of SEQUENCE LISTING 400 <, 8 > and SEQUENCE LISTING, 9 >；

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

PCR reaction systems 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 reactions Reaction condition is：95 DEG C of pre-degeneration 4min, into cycle；95 DEG C of denaturation 45s, 60 DEG C of annealing 45s, 72 DEG C extend 1kb/min, follow Ring 35 times, last 72 DEG C of extensions 10min.

C. rAlb-IFN γ genes are obtained using flexible linker connection ox Alb and ox IFN-γ target gene

The PCR reaction systems 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 connect 1 μ L, Alb sense 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 pre-degeneration 4min, into cycle：94 DEG C of denaturation 45s；60 DEG C of annealing 45s, 72 DEG C of extension 1kb/min, altogether 35 cycles；Last 72 DEG C of extensions 10min.

D. rAlb-IFN γ-are obtained using flexible linker connections rAlb-IFN γ genes and ox IFN-α target gene IFN-α gene

The PCR reaction systems and reaction condition of connection be：In the overall reaction system of 25 μ L, rAlb-IFN γ gene templates 1 μ L of DNA, 1 μ L of IFN-α template DNA connect Alb the sense primers 0.5 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 conditions is：95 DEG C pre-degeneration 4min, into cycle：94 DEG C of denaturation 45s；60 DEG C of annealing 45s, 72 DEG C of extension 1kb/min, totally 35 recycle；Finally 72 DEG C of extension 10min.

The present invention also provides the application of the recombinant bovine long-acting interferon, long half time had up to 72 hours or more Broad-spectrum disease resistance toxic action and the immune response that Niu Zishen can be improved.

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

1. ox Alb, ox IFN-γ and ox IFN-α gene are realized amalgamation and expression by flexible linker, interference is improved Plain half-life period improves 18 times or more compared with plain interferon；Compared with common polyethylene glycol fused interferon, significantly drop Low cost.

2. by being optimized to ox Alb, ox IFN-γ and ox IFN-α gene, ox Alb, ox IFN-γ and ox are improved 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 plasmids, 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 ox Alb, ox IFN-γ and ox IFN-α not only has IFN-α Broad-spectrum disease resistance toxic action, while significantly improving the immune response of Niu Zishen.

Description of the drawings

Fig. 1 is that bovine albumin gene, Bov IFN α genes and the Bov IFN γ genes RT-PCR in embodiment 1 are expanded Result；Swimming lane M：DNA Marker DL2000；Swimming lane 1：Bov IFN α gene RT-PCR amplified productions；Swimming lane 2：Ox interferes Plain γ genes RT-PCR amplified productions；Swimming lane 3：Bovine albumin gene RT-PCR amplified productions；

Fig. 2 be embodiment 1 in ox Alb, IFN-γ connected with the target gene of IFN-α after PCR amplification result； Swimming lane M：DNA Marker DL10000；Swimming lane 1：Bovine albumin gene, Bov IFN γ genes are connect with Bov IFN α genes Amplified production；

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 results 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 results for the fusion protein that embodiment 1 obtains；Swimming lane M：Albumen Marker；Swimming Road 1：Supernatant after recombinant bacterium induction is broken；Swimming lane 2：It is precipitated after being crushed for recombinant bacterium induction；

Fig. 6 is that the recombinant bovine long-acting interferon α 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 wells；A3-12 is gradient dilution (from right to left) Human interferon standard items handle hole；B3-12 is that the recombinant bovine long-acting interferon α of gradient dilution (from right to left) handles hole；

Fig. 7 is the recombinant bovine long-acting interferon α intramuscular injection blood made from the fusion protein in embodiment 1 in embodiment 8 Concentration-time changing curve.

Specific implementation mode

Embodiment 1

A kind of fusion protein being made of bovine albumin, Bov IFN γ and Bov IFN α, preparation method are as follows：

1. the acquisition of bovine albumin (Alb), Bov IFN γ (IFN-γ) and Bov IFN α (IFN-α) target gene with Amplification

Design of primers：

It is shown in Table 1 according to the objective gene sequence design synthetic primer reported in Genebank, in the upstream of bovine albumin EcoRI restriction enzyme sites and Linker sequences are introduced in primer and downstream primer respectively, Bov IFN γ sense primer and under Linker sequences are introduced respectively in trip primer, introduce Linker sequences respectively in the sense primer of Bov IFN α and downstream primer Row and XhoI restriction enzyme sites.

1 PCR amplification primer of table

RT-PCR obtains target gene：

RNA is extracted from cattle liver tissue, passes through reverse transcription acquisition ox Alb, the purpose base of ox IFN-γ and ox IFN-α 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 systems (25 μ L) are shown in Table 2

2 RT-PCR reaction systems 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 pre-degeneration 4min, into cycle：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 through agarose gel electrophoresis in 1850bp, 560bp and 530bp or so in RT-PCR amplified productions, The results are shown in Figure 1 for it, illustrate to be prepared respectively the ox Alb for being separately connected flexible linker sequences, ox IFN-γ and The target gene of ox 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 systems of table

4 rAlb-IFN γ of table-IFN α PCR reaction systems

Response parameter is：95 DEG C of pre-degeneration 4min, into cycle：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 through agarose gel electrophoresis in 2880bp or so in pcr amplification product, and the results are shown in Figure 2, Occur rAlb-IFN γ and IFN-α amplified production band in Fig. 2, this is because being connected with IFN-α gene in rAlb-IFN γ 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

After sequencing is errorless, PCR glue recovery product uses target gene after selection connection with pET-32a plasmids EcoRI and XhoI restriction enzymes carry out double digestion and recycling, and double digestion is done 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 plasmids 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

It converts in connection product to e. coli bl21 (DE3) competent cell, competent cell is coated on benzyl containing ammonia The LB culture medium flat plates of penicillin are incubated overnight；Single bacterium colony on picking LB tablets carries out target gene PCR identifications, positive colony Bacteria plasmid is identified through EcoRI and XhoI double digestions, is accredited as positive and is indicated that engineering bacteria is built successfully, PCR amplification and double digestion Product detects single band through agarose gel electrophoresis at 2880bp, and the results are shown in Figure 3, illustrates 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 mediums containing 100 μ g/ml ampicillins, in LB culture mediums 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；Thalline is collected, through SDS-PAGE electrophoresis detections, the results are 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 places 123.8KD or so after leading the bacterial cell disruption after 5h, illustrates in precipitation and supernatant In equal successful expression fusion proteins.

Mass volume ratio 1 is added:Precipitation 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 centrifuges 15min, Supernatant, inclusion body (inclusion body is through dissolving, 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 100 On protein purification system, the His affinity columns balanced with Binding Buffer I (PBS) are washed away not with PBS buffer solution In conjunction with albumen, until A280nm stablizes, then with Elution buffer I (50mM trishydroxymethylaminomethanes, 20~500mM Imidazoles, PH8.0) elution, collect rAlb-IFN γ-IFN α protein peak.

5.2DEAE anion-exchange chromatography

By the albumen collected after His affinitive layer purifications displacement to (the 50mM trihydroxy methyls 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 crossing column to A280nm value stabilizations with Binding Buffer II, 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 chromatographies of Superdex have 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 >=10⁷U/mg, albumen are qualified；It is aseptic subpackaged, -80 DEG C preserve.The fusion protein being made of bovine albumin, Bov IFN γ and Bov IFN α is 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 bovine albumin, Bov IFN γ and Bov IFN α, other are with embodiment 1, only E. coli bl21 therein (DE3) competent cell is replaced in order to which BL21 (DE3) competence with pGro7 plasmids is thin Born of the same parents.The SDS-PAGE electrophoresis results of its fusion protein are compareed with embodiment 1,123.8KD or so place's predominant expression items 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 Higher.The albumen of Bacillus coli expression is mostly present in inclusion body；By introducing molecular chaperones in expressing bacterial strain, cooperate with Expression albumen correctly folds, and reaches solubility expression of protein.

Described BL21 (DE3) competent cell with pGro7 plasmids 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 bovine albumin, Bov IFN γ and Bov IFN α, preparation method are as follows：

1. the acquisition of bovine albumin (Alb), Bov IFN γ (IFN-γ) and Bov IFN α (IFN-α) target gene with Amplification

Ox Alb, ox IFN-γ and ox IFN-α in embodiment 1 is optimized, artificial synthesized ox Alb, ox IFN-γ and Ox 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 the 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 each biology for doing protein expression or production (including Escherichia coli, yeast, mammalian cell, plant cell and insect cell) all shows the profit 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 ox Alb, ox IFN-γ and Ox 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 values are lower to show that expression is lower in host.G/C content most ideal distribution ranging from 30 in gene~ 70%, in any region translation and transcriptional efficiency can be influenced more than the range.Ox Alb, ox are found using software detection The codon of IFN-γ and ox IFN-α original gene codon adaptation indexI (CAI) in Escherichia coli is respectively 0.22, 0.24,0.25, GC percentages are 42.9%, 39.8%, 58.2%；And by ox Alb, ox IFN-γ and ox IFN-α gene Obtained after optimization recombination codon adaptation indexI (CAI) in Escherichia coli be respectively 0.99,1.0,0.97, GC percentages 49.9%, 44.8%, 54.6%.The utilization rate that low codon is significantly reduced by gene optimization, avoids rare codon Influence to protein expression improves the G/C content of gene, improves transcription and translation efficiency, and then improves the table of recombinant protein Up to amount.

1.3 design of primers：

7 PCR amplification primer of table

The genomic DNA of ox Alb, ox IFN-γ and ox 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 systems of table

Response parameter is：95 DEG C of pre-degeneration 4min, into cycle：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.

Ox Alb, ox IFN-γ and ox IFN-α pcr amplification product through agarose gel electrophoresis respectively 1850bp, There is specific band in 560bp and 530bp or so, illustrate that the ox for being separately connected flexible linker after optimization has been prepared The target gene of Alb, ox IFN-γ and ox 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 systems of table

10 rAlb-IFN γ of table-IFN-α PCR reaction systems

Response parameter is：95 DEG C of pre-degeneration 4min, into cycle：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 through agarose gel electrophoresis in 2880bp or so in pcr amplification product, illustrates successfully to be connected RAlb-IFN γ-IFN-α gene after connecing.The nucleotide sequence of obtained target gene such as SEQUENCE LISTING 400 Shown in 3 > of <.

3. expression vector establishment

The glue recovery product of target gene PCR errorless after sequencing after selection connection is used with pET-32a plasmids BamHI, XhoI restriction enzyme carry out double digestion and recycling, and double digestion is done by 20 μ L systems in table 11：

11 double digestion system of table

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

The digestion recovery product of target gene and pET-32a plasmids after connection is attached by the system in table 12,4 DEG C overnight connection：

Table 12

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

It converts in connection product to e. coli bl21 (DE3) competent cell, competence is coated on the mould of benzyl containing ammonia It is incubated overnight in the LB tablets of element；The bacterium colony grown on LB tablets is taken to identify target gene, positive colony bacteria plasmid warp through PCR BamHI, XhoI double digestion are identified, are accredited as positive and are indicated expression vector establishment success, PCR amplification and double digestion product are through fine jade There is single band at the places 2880bp or so in sepharose electrophoresis, illustrates containing rAlb-IFN γ-IFN α fusion gene engineering bacterium PET-32a/rAlb-IFN γ-IFN α is built successfully.

4. the expression of recombinant protein

Picking engineering bacteria 37 DEG C of shaking table recovery 1h in the LB culture mediums containing 100 μ g/ml ampicillins, in LB culture mediums 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；Thalline is collected, through SDS-PAGE electrophoresis detections, supernatant is deposited in after the bacterial cell disruption after recombinant bacterium induction 5h The visible predominant expression band in the places 123.8KD or so illustrates to have obtained recombinant protein in supernatant precipitates.

Mass volume ratio 1 is added:Precipitation 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 centrifuges 15min, Supernatant, inclusion body (inclusion body is through dissolving, 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 100 On protein purification system, the His affinity columns balanced with Binding Buffer I (PBS) are washed away not with PBS buffer solution In conjunction with albumen, until A280nm stablizes, then with Elution buffer I (50mM trishydroxymethylaminomethanes, 20~500mM Imidazoles, PH8.0) elution, collect rAlb-IFN γ-IFN α protein peak.

5.2DEAE anion-exchange chromatography

By the albumen collected after His affinitive layer purifications displacement to (the 50mM trihydroxy methyls 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 crossing column to A280nm value stabilizations with Binding Buffer II, 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 Binding Buffer III after the sample concentration that ion-exchange chromatography is collected into (50mMNa₂HPO4,0.15M NaCl, PH7.4) 200 molecular sieve chromatographies of Superdex have 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 >=10⁷U/mg, albumen are qualification；It is aseptic subpackaged ,- 80 DEG C of preservations.It can be obtained the fusion protein being made of bovine albumin, Bov IFN γ and Bov IFN α, amino acid sequence As shown in 400 < of SEQUENCE LISTING, 1 >.

Embodiment 4

A kind of fusion protein being made of bovine albumin, Bov IFN γ and Bov IFN α, other are with embodiment 3, only E. coli bl21 therein (DE3) competent cell is replaced in order to which BL21 (DE3) competence with pGro7 plasmids is thin Born of the same parents.The SDS-PAGE electrophoresis results of its fusion protein are compareed with embodiment 3,123.8KD or so place's predominant expression items 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 Higher.The albumen of Bacillus coli expression is mostly present in inclusion body；By introducing molecular chaperones in expressing bacterial strain, cooperate with Expression albumen correctly folds, and reaches solubility expression of protein.

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

Embodiment 5

A kind of recombinant bovine long-acting interferon α, by the fusion protein in embodiment 1,2,3,4 respectively with freeze drying protectant mixture Later, freeze-dried to form.The freeze drying protectant is glycerine, mannitol and sucrose, is buffer solution with 10mmol/L PBS, Final concentration of glycerine 100mL/L, mannitol 0.12g/mL and the sucrose 0.025g/mL of three.

Embodiment 6

Examples 1 to 4 is obtained by the identification of bovine albumin, Bov IFN γ and Bov IFN the α fusion protein formed

The quantitative detection of 6.1 protein contents

With Lowry methods, the standard protein that institute is examined and determine with Chinese food pharmaceutical biological product is made standard test, measures embodiment 1~4 obtained fusion protein concentration is all higher 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 123.8KD or so, as shown in Figure 4.

6.3Western Blot results

Fusion protein in Examples 1 to 4 is detected respectively, with the anti-ox alpha interferon of abcam companies mouse (1:5000 dilutions) it is one It is anti-, using goat anti-mouse IgG-HRP as secondary antibody (1:10000 dilutions).Recombinant bovine long-acting interferon sample can be with anti-Bov IFN α Specific reaction occurs for monoclonal antibody, and specific band occur in the places 123.8KD or so, as shown in Figure 5.

Embodiment 7

Bioactivity freeze-dried four parts of recombinant bovine long-acting interferon α in embodiment 5

Inhibit method according to few cells lesion, Hep-2 cells are made into 5 × 10 with culture medium⁵Cell/ml cells suspend Liquid, per hole, inoculation 0.1ml moves into 96 porocyte culture plates.37 DEG C, 5%CO₂For 24 hours, the recombinant bovine that various dose is added is long for culture Interferon-' alpha ' is imitated, inhales abandon afterwards for 24 hours, then inoculation 100TCID50VSV viruses respectively.

Test result

The result shows that the recombinant bovine long-acting interferon α obtained causes the lesion of HEp-2 cells to have apparent inhibit VSV Effect.The lesions such as occurs cell rounding after untreated cell inoculation virus, falls off, is disintegrated.And the recombinant bovine obtained is long After imitating interferon treated cell inoculation virus, it is observed continuously under inverted microscope, cellular morphology is normal, does not occur any Lesion measures potency >=10⁷U/ml, as shown in Figure 6.

Embodiment 8

The four parts of recombinant bovine long-acting interferon α freeze-dryings obtained respectively by the fusion protein of Examples 1 to 4 in embodiment 5 The measurement of half-life period of the agent (being denoted as A, B, C, D respectively) in ox body

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

Take the ox (half male and half female) that six weight are roughly the same, neck that 2mg/ml recombinant bovine long-acting interferons α is subcutaneously injected Freeze-dried 2ml, respectively in 1h, 2h, 4h, 8h, 16h, 26h, 44h, 79h venous blood collection, 4 DEG C of solidifications of blood sample, 3500rpm low temperature from Heart 10min detaches serum, and every ox blood sample of each time point is to be measured in -20 DEG C of preservations.Serum sample is measured using cytopathic-effect inhibition assay The concentration of rAlb-IFN γ-IFN α in product is carried out curve fitting and calculating parameter with DAS pharmacokinetics softwares.Parameter result of calculation It is shown in Table 13.

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

The result shows that recombinant bovine long-acting interferon α has longer half-life period.Half-life period can reach 72h or so after measured, compared with Plain interferon improves about 18 times.

Embodiment 9

The freeze-dried measurement that ox cellullar immunologic response is influenced of four parts of recombinant bovine long-acting interferon α in embodiment 5

It takes six roughly the same beef cattles of weight to be divided into two groups, is denoted as experimental group and control group；Experimental group neck is subcutaneously noted The 2mg/ml recombinant bovine long-acting interferon freeze-dried 2ml of α are penetrated, the PBS of 2mL is subcutaneously injected in control group neck, takes after injecting 4 weeks outside ox All blood takes weekly a blood later, detaches 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 lymphocytes, 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：

Table 14 ELISA detection each group ox cellullar immunologic responses are horizontal

The result shows that after injection recombinant bovine long-acting interferon α, can significantly improve ox 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 with reference to embodiment to a kind of fusion protein being made of bovine albumin, Bov IFN γ and Bov IFN α and The detailed description that preparation method carries out is illustrative without being restrictive, and can be enumerated 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 bovine albumin, Bov IFN γ and Bov IFN α

<130> 1

<160> 9

<170> PatentIn version 3.3

<210> 1

<211> 959

<212> PRT

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

<400> 1

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Leu Leu Phe Ser Ser Ala

1 5 10 15

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

20 25 30

His Arg Phe Lys Asp Leu Gly Glu Glu His Phe Lys Gly Leu Val Leu

35 40 45

Ile Ala Phe Ser Gln Tyr Leu Gln Gln Cys Pro Phe Asp Glu His Val

50 55 60

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

65 70 75 80

Glu Ser His Ala Gly Cys Glu Lys Ser Leu His Thr Leu Phe Gly Asp

85 90 95

Glu Leu Cys Lys Val Ala Ser Leu Arg Glu Thr Tyr Gly Asp Met Ala

100 105 110

Asp Cys Cys Glu Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Ser

115 120 125

His Lys Asp Asp Ser Pro Asp Leu Pro Lys Leu Lys Pro Asp Pro Asn

130 135 140

Thr Leu Cys Asp Glu Phe Lys Ala Asp Glu Lys Lys Phe Trp Gly Lys

145 150 155 160

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

165 170 175

Leu Leu Tyr Tyr Ala Asn Lys Tyr Asn Gly Val Phe Gln Glu Cys Cys

180 185 190

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

195 200 205

Arg Glu Lys Val Leu Ala Ser Ser Ala Arg Gln Arg Leu Arg Cys Ala

210 215 220

Ser Ile Gln Lys Phe Gly Glu Arg Ala Leu Lys Ala Trp Ser Val Ala

225 230 235 240

Arg Leu Ser Gln Lys Phe Pro Lys Ala Glu Phe Val Glu Val Thr Lys

245 250 255

Leu Val Thr Asp Leu Thr Lys Val His Lys Glu Cys Cys His Gly Asp

260 265 270

Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile Cys

275 280 285

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

290 295 300

Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu Val Glu Lys Asp Ala

305 310 315 320

Ile Pro Glu Asn Leu Pro Pro Leu Thr Ala Asp Phe Ala Glu Asp Lys

325 330 335

Asp Val Cys Lys Asn Tyr Gln Glu Ala Lys Asp Ala Phe Leu Gly Ser

340 345 350

Phe Leu Tyr Glu Tyr Ser Arg Arg His Pro Glu Tyr Ala Val Ser Val

355 360 365

Leu Leu Arg Leu Ala Lys Glu Tyr Glu Ala Thr Leu Glu Glu Cys Cys

370 375 380

Ala Lys Asp Asp Pro His Ala Cys Tyr Ser Thr Val Phe Asp Lys Leu

385 390 395 400

Lys His Leu Val Asp Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys Asp

405 410 415

Gln Phe Glu Lys Leu Gly Glu Tyr Gly Phe Gln Asn Ala Leu Ile Val

420 425 430

Arg Tyr Thr Arg Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val Glu

435 440 445

Val Ser Arg Ser Leu Gly Lys Val Gly Thr Arg Cys Cys Thr Lys Pro

450 455 460

Glu Ser Glu Arg Met Pro Cys Thr Glu Asp Tyr Leu Ser Leu Ile Leu

465 470 475 480

Asn Arg Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Glu Lys Val

485 490 495

Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser

500 505 510

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

515 520 525

Leu Phe Thr Phe His Ala Asp Ile Cys Thr Leu Pro Asp Thr Glu Lys

530 535 540

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

545 550 555 560

Lys Ala Thr Glu Glu Gln Leu Lys Thr Val Met Glu Asn Phe Val Ala

565 570 575

Phe Val Asp Lys Cys Cys Ala Ala Asp Asp Lys Glu Ala Cys Phe Ala

580 585 590

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

595 600 605

Gly Gly Gly Ser Gly Gly Gly Gly Ser Met Lys Tyr Thr Ser Tyr Phe

610 615 620

Leu Ala Leu Leu Leu Cys Gly Leu Leu Gly Phe Ser Gly Ser Tyr Gly

625 630 635 640

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

645 650 655

Ala Ser Ser Pro Asp Val Ala Lys Gly Gly Pro Leu Phe Ser Glu Ile

660 665 670

Leu Lys Asn Trp Lys Asp Glu Ser Asp Lys Lys Ile Ile Gln Ser Gln

675 680 685

Ile Val Ser Phe Tyr Phe Lys Leu Phe Glu Asn Leu Lys Asp Asn Gln

690 695 700

Val Ile Gln Arg Ser Met Asp Ile Ile Lys Gln Asp Met Phe Gln Lys

705 710 715 720

Phe Leu Asn Gly Ser Ser Glu Lys Leu Glu Asp Phe Lys Lys Leu Ile

725 730 735

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

740 745 750

Leu Ile Lys Val Met Asn Asp Leu Ser Pro Lys Ser Asn Leu Arg Lys

755 760 765

Arg Lys Arg Ser Gln Asn Leu Phe Arg Gly Arg Arg Ala Ser Thr Gly

770 775 780

Gly Gly Gly Ser Gly Gly Gly Gly Ser Cys His Leu Pro His Thr His

785 790 795 800

Ser Leu Ala Asn Arg Arg Val Leu Met Leu Leu Gly Gln Leu Arg Arg

805 810 815

Val Ser Pro Ser Ser Cys Leu Gln Asp Arg Asn Asp Phe Ala Phe Pro

820 825 830

Gln Glu Ala Leu Gly Gly Ser Gln Leu Gln Lys Ala Gln Ala Ile Ser

835 840 845

Val Leu His Glu Val Thr Gln His Thr Phe Gln Leu Phe Ser Thr Glu

850 855 860

Gly Ser Ala Thr Thr Trp Asp Glu Ser Leu Leu Asp Lys Leu Arg Ala

865 870 875 880

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

885 890 895

Glu Glu Leu Gln Gly Ala Pro Leu Leu Lys Glu Asp Ser Ser Leu Ala

900 905 910

Val Arg Lys Tyr Phe His Arg Leu Thr Leu Tyr Leu Gln Glu Lys Lys

915 920 925

His Ser Pro Cys Ala Trp Glu Val Val Arg Ala Gln Val Met Arg Ala

930 935 940

Phe Ser Ser Ser Thr Asn Leu Gln Glu Ser Phe Arg Arg Lys Asp

945 950 955

<210> 2

<211> 2877

<212> DNA

<213>Genome 1

<400> 2

atgaagtggg tgacttttat ttctcttctc cttctcttca gctctgctta ttccaggggt 60

gtgtttcgtc gagatacaca caagagtgag attgctcatc ggtttaaaga tttgggagaa 120

gaacatttta aaggcctggt actgattgcc ttttctcagt atctccagca gtgtccattt 180

gatgagcatg taaaattagt gaacgaacta actgagtttg caaaaacatg tgttgctgat 240

gagtcccatg ccggctgtga aaagtcactt cacactctct ttggagatga attgtgtaaa 300

gttgcatccc ttcgtgaaac ctatggtgac atggctgact gctgtgagaa acaagagcct 360

gaaagaaatg aatgcttcct gagccacaaa gatgatagcc cagacctccc taaattgaaa 420

ccagacccca atactttgtg tgatgagttt aaggcagatg aaaagaagtt ttggggaaaa 480

tacctatacg aaattgctag aagacatccc tacttttatg caccagaact cctttactat 540

gctaataaat ataatggagt ttttcaagaa tgctgccaag ctgaagataa aggtgcctgc 600

ctgctaccaa agattgaaac tatgagagaa aaggtactag cttcatctgc cagacagaga 660

ctcaggtgtg ccagtattca aaaatttgga gaaagagctt taaaagcatg gtcagtagct 720

cgcctgagcc agaaatttcc caaggctgag tttgtagaag ttaccaagct agtgacagat 780

ctcacaaaag tccacaagga atgctgccat ggtgacctac ttgaatgcgc agatgacagg 840

gcagatcttg ccaagtacat atgtgataat caagatacaa tctccagtaa actgaaggaa 900

tgctgtgata agcctttgtt ggaaaaatcc cactgcattg ctgaggtaga aaaagatgcc 960

atacctgaaa acctgccccc attaactgct gactttgctg aagataagga tgtttgcaaa 1020

aactatcagg aagcaaaaga tgccttcctg ggctcgtttt tgtatgaata ttcaagaagg 1080

catcctgaat atgctgtctc agtgctattg agacttgcca aggaatatga agccacactg 1140

gaggaatgct gtgccaaaga tgatccacat gcatgctatt ccacagtgtt tgacaaactt 1200

aagcatcttg tggatgagcc tcagaattta atcaaacaaa actgtgacca attcgaaaaa 1260

cttggagagt atggattcca aaatgcgctc atagttcgtt acaccaggaa agtaccccaa 1320

gtgtcaactc caactctcgt ggaggtttca agaagcctag gaaaagtggg tactaggtgt 1380

tgtacaaagc cggaatcaga aagaatgccc tgtactgaag actatctgag cttgatcctg 1440

aaccggttgt gcgtgctgca tgagaagaca ccagtgagtg aaaaagtcac caagtgctgc 1500

acagagtcat tggtgaacag acggccatgt ttctctgctc tgacacctga tgaaacatat 1560

gtacccaaag cctttgatga gaaattgttc accttccatg cagatatatg cacacttccc 1620

gatactgaga aacaaatcaa gaaacaaact gcacttgttg agctgttgaa acacaagccc 1680

aaggcaacag aggaacaact gaaaaccgtc atggagaatt ttgtggcttt tgtagacaag 1740

tgctgtgcag ctgatgacaa agaagcctgc tttgctgtgg agggtccaaa acttgttgtt 1800

tcaactcaaa cagccttagc cggtggtggt ggttctggtg gtggtggttc tatgaaatat 1860

acaagctatt tcttagcttt actgctctgt gggcttttgg gtttttctgg ttcttatggc 1920

cagggccaat tttttagaga aatagaaaac ttaaaggagt attttaatgc aagtagccca 1980

gatgtagcta agggtgggcc tctcttctca gaaattttga agaattggaa agatgaaagt 2040

gacaaaaaaa ttattcagag ccaaattgtc tccttctact tcaaactctt tgaaaacctc 2100

aaagataacc aggtcattca aaggagcatg gatatcatca agcaagacat gtttcagaag 2160

ttcttgaatg gcagctctga gaaactggag gacttcaaaa agctgattca aattccggtg 2220

gatgatctgc agatccagcg caaagccata aatgaactca tcaaagtgat gaatgacctg 2280

tcaccaaaat ctaacctcag aaagcggaag agaagtcaga atctctttcg aggccggaga 2340

gcatcaacgg gtggtggtgg ttctggtggt ggtggttctt gccacctgcc tcacacccac 2400

agcctggcca acaggagggt cctgatgctc ctgggacaac tgaggagggt ctccccttcc 2460

tcctgcctgc aggacagaaa tgacttcgca ttcccccagg aggcgctggg tggcagccag 2520

ttgcagaagg ctcaagccat ctctgtgctc cacgaggtga cccagcacac cttccagctt 2580

ttcagcacag agggctcggc cactacgtgg gatgagagcc tcctggacaa gctccgcgct 2640

gcactggatc agcagctcac tgacctgcaa gcctgtctga ggcaggagga ggagctgcaa 2700

ggagctcccc tgctcaagga ggactccagc ctggctgtga ggaaatactt ccacagactc 2760

actctctatc tgcaagagaa gaaacacagc ccttgtgcct gggaggttgt cagagcacaa 2820

gtcatgagag ccttctcttc ctcaacaaac ttgcaggaga gtttcaggag aaaggac 2877

<210> 3

<211> 2877

<212> DNA

<213>Genome 2

<400> 3

atgaaatggg ttaccttcat ctctctgctg ctgctgttct cttctgctta ctctcgtggt 60

gttttccgtc gtgacaccca caaatctgaa atcgctcacc gtttcaaaga cctgggtgaa 120

gaacacttca aaggtctggt tctgatcgct ttctctcagt acctgcagca gtgcccgttc 180

gacgaacacg ttaaactggt taacgaactg accgaattcg ctaaaacctg cgttgctgac 240

gaatctcacg ctggttgcga aaaatctctg cacaccctgt tcggtgacga actgtgcaaa 300

gttgcttctc tgcgtgaaac ctacggtgac atggctgact gctgcgaaaa acaggaaccg 360

gaacgtaacg aatgcttcct gtctcacaaa gacgactctc cggacctgcc gaaactgaaa 420

ccggacccga acaccctgtg cgacgaattc aaagctgacg aaaaaaaatt ctggggtaaa 480

tacctgtacg aaatcgctcg tcgtcacccg tacttctacg ctccggaact gctgtactac 540

gctaacaaat acaacggtgt tttccaggaa tgctgccagg ctgaagacaa aggtgcttgc 600

ctgctgccga aaatcgaaac catgcgtgaa aaagttctgg cttcttctgc tcgtcagcgt 660

ctgcgttgcg cttctatcca gaaattcggt gaacgtgctc tgaaagcttg gtctgttgct 720

cgtctgtctc agaaattccc gaaagctgaa ttcgttgaag ttaccaaact ggttaccgac 780

ctgaccaaag ttcacaaaga atgctgccac ggtgacctgc tggaatgcgc tgacgaccgt 840

gctgacctgg ctaaatacat ctgcgacaac caggacacca tctcttctaa actgaaagaa 900

tgctgcgaca aaccgctgct ggaaaaatct cactgcatcg ctgaagttga aaaagacgct 960

atcccggaaa acctgccgcc gctgaccgct gacttcgctg aagacaaaga cgtttgcaaa 1020

aactaccagg aagctaaaga cgcttttctc ggtagcttcc tgtacgaata ctctcgtcgt 1080

cacccggaat acgctgtttc tgttctgctg cgtctggcta aagaatacga agctaccctg 1140

gaagaatgct gcgctaaaga cgacccgcac gcttgctact ctaccgtttt cgacaaactg 1200

aaacacctgg ttgacgaacc gcagaacctg atcaaacaga actgcgacca gttcgaaaaa 1260

ctgggtgaat acggtttcca gaacgctctg atcgttcgtt acacccgtaa agttccgcag 1320

gtttctaccc cgaccctggt tgaagtttct cgttctctgg gtaaagttgg tacccgttgc 1380

tgcaccaaac cggaatctga acgtatgccg tgcaccgaag actacctgtc tctgatcctg 1440

aaccgtctgt gcgttctgca cgaaaaaacc ccggtttctg aaaaagttac caaatgctgc 1500

accgaatctc tggttaaccg tcgtccgtgc ttctctgctc tgaccccgga cgaaacctac 1560

gttccgaaag ctttcgacga aaaactgttc accttccacg ctgacatctg caccctgccg 1620

gacaccgaaa aacagatcaa aaaacagacc gctctggttg aactgctgaa acacaaaccg 1680

aaagctaccg aagaacagct gaaaaccgtt atggaaaact tcgttgcttt cgttgacaaa 1740

tgctgcgctg ctgacgacaa agaagcttgc ttcgctgttg aaggtccgaa actggttgtt 1800

tctacccaga ccgctctggc tggtggtggt ggttctggtg gtggtggttc tatgaaatac 1860

acctcttact tcctggctct gctgctgtgc ggtctgctgg gtttctctgg ttcttacggt 1920

cagggtcagt tcttccgtga aatcgaaaac ctgaaagaat acttcaacgc ttcttctccg 1980

gacgttgcta aaggtggtcc gctgttctct gaaatcctga aaaactggaa agacgaatct 2040

gacaaaaaaa tcatccagtc tcagatcgtt tctttctact tcaaactgtt cgaaaacctg 2100

aaagacaacc aggttatcca gcgttctatg gacatcatca aacaggacat gttccagaaa 2160

ttcctgaacg gttcttctga aaaactggaa gacttcaaaa aactgatcca gatcccggtt 2220

gacgacctgc agatccagcg taaagctatc aacgaactga tcaaagttat gaacgacctg 2280

tctccgaaat ctaacctgcg taaacgtaaa cgttctcaga acctgttccg tggtcgtcgt 2340

gcttctaccg gtggtggtgg ttctggtggt ggtggttctt gccacctgcc gcacacccac 2400

tctctggcta accgtcgtgt tctgatgctg ctgggtcagt tacgtcgtgt aagcccgtct 2460

tcttgcctgc aggaccgtaa cgacttcgct ttcccgcagg aagctctggg tggttctcag 2520

ctgcagaaag ctcaggctat ctctgttctg cacgaagtta cccagcacac cttccagctg 2580

ttctctaccg aaggttctgc taccacctgg gacgaatctc tgctggacaa actgcgtgct 2640

gctctggacc agcagctgac cgacctgcag gcttgcctgc gtcaggaaga agaactgcag 2700

ggtgctccgc tgctgaaaga agactcttct ctggctgttc gtaaatactt ccaccgtctg 2760

accctgtacc tgcaggaaaa aaaacactct ccgtgcgctt gggaagttgt tcgtgctcag 2820

gttatgcgtg ctttctcttc ttctaccaac ctgcaggaat ctttccgtcg taaagac 2877

<210> 4

<211> 1821

<212> DNA

<213>Bovine albumin

<400> 4

atgaagtggg tgacttttat ttctcttctc cttctcttca gctctgctta ttccaggggt 60

gtgtttcgtc gagatacaca caagagtgag attgctcatc ggtttaaaga tttgggagaa 120

gaacatttta aaggcctggt actgattgcc ttttctcagt atctccagca gtgtccattt 180

gatgagcatg taaaattagt gaacgaacta actgagtttg caaaaacatg tgttgctgat 240

gagtcccatg ccggctgtga aaagtcactt cacactctct ttggagatga attgtgtaaa 300

gttgcatccc ttcgtgaaac ctatggtgac atggctgact gctgtgagaa acaagagcct 360

gaaagaaatg aatgcttcct gagccacaaa gatgatagcc cagacctccc taaattgaaa 420

ccagacccca atactttgtg tgatgagttt aaggcagatg aaaagaagtt ttggggaaaa 480

tacctatacg aaattgctag aagacatccc tacttttatg caccagaact cctttactat 540

gctaataaat ataatggagt ttttcaagaa tgctgccaag ctgaagataa aggtgcctgc 600

ctgctaccaa agattgaaac tatgagagaa aaggtactag cttcatctgc cagacagaga 660

ctcaggtgtg ccagtattca aaaatttgga gaaagagctt taaaagcatg gtcagtagct 720

cgcctgagcc agaaatttcc caaggctgag tttgtagaag ttaccaagct agtgacagat 780

ctcacaaaag tccacaagga atgctgccat ggtgacctac ttgaatgcgc agatgacagg 840

gcagatcttg ccaagtacat atgtgataat caagatacaa tctccagtaa actgaaggaa 900

tgctgtgata agcctttgtt ggaaaaatcc cactgcattg ctgaggtaga aaaagatgcc 960

atacctgaaa acctgccccc attaactgct gactttgctg aagataagga tgtttgcaaa 1020

aactatcagg aagcaaaaga tgccttcctg ggctcgtttt tgtatgaata ttcaagaagg 1080

catcctgaat atgctgtctc agtgctattg agacttgcca aggaatatga agccacactg 1140

gaggaatgct gtgccaaaga tgatccacat gcatgctatt ccacagtgtt tgacaaactt 1200

aagcatcttg tggatgagcc tcagaattta atcaaacaaa actgtgacca attcgaaaaa 1260

cttggagagt atggattcca aaatgcgctc atagttcgtt acaccaggaa agtaccccaa 1320

gtgtcaactc caactctcgt ggaggtttca agaagcctag gaaaagtggg tactaggtgt 1380

tgtacaaagc cggaatcaga aagaatgccc tgtactgaag actatctgag cttgatcctg 1440

aaccggttgt gcgtgctgca tgagaagaca ccagtgagtg aaaaagtcac caagtgctgc 1500

acagagtcat tggtgaacag acggccatgt ttctctgctc tgacacctga tgaaacatat 1560

gtacccaaag cctttgatga gaaattgttc accttccatg cagatatatg cacacttccc 1620

gatactgaga aacaaatcaa gaaacaaact gcacttgttg agctgttgaa acacaagccc 1680

aaggcaacag aggaacaact gaaaaccgtc atggagaatt ttgtggcttt tgtagacaag 1740

tgctgtgcag ctgatgacaa agaagcctgc tttgctgtgg agggtccaaa acttgttgtt 1800

tcaactcaaa cagccttagc c 1821

<210> 5

<211> 498

<212> DNA

<213>Ox IFN-γ

<400> 5

atgaaatata caagctattt cttagcttta ctgctctgtg ggcttttggg tttttctggt 60

tcttatggcc agggccaatt ttttagagaa atagaaaact taaaggagta ttttaatgca 120

agtagcccag atgtagctaa gggtgggcct ctcttctcag aaattttgaa gaattggaaa 180

gatgaaagtg acaaaaaaat tattcagagc caaattgtct ccttctactt caaactcttt 240

gaaaacctca aagataacca ggtcattcaa aggagcatgg atatcatcaa gcaagacatg 300

tttcagaagt tcttgaatgg cagctctgag aaactggagg acttcaaaaa gctgattcaa 360

attccggtgg atgatctgca gatccagcgc aaagccataa atgaactcat caaagtgatg 420

aatgacctgt caccaaaatc taacctcaga aagcggaaga gaagtcagaa tctctttcga 480

ggccggagag catcaacg 498

<210> 6

<211> 498

<212> DNA

<213>Ox IFN-α

<400> 6

tgccacctgc ctcacaccca cagcctggcc aacaggaggg tcctgatgct cctgggacaa 60

ctgaggaggg tctccccttc ctcctgcctg caggacagaa atgacttcgc attcccccag 120

gaggcgctgg gtggcagcca gttgcagaag gctcaagcca tctctgtgct ccacgaggtg 180

acccagcaca ccttccagct tttcagcaca gagggctcgg ccactacgtg ggatgagagc 240

ctcctggaca agctccgcgc tgcactggat cagcagctca ctgacctgca agcctgtctg 300

aggcaggagg aggagctgca aggagctccc ctgctcaagg aggactccag cctggctgtg 360

aggaaatact tccacagact cactctctat ctgcaagaga agaaacacag cccttgtgcc 420

tgggaggttg tcagagcaca agtcatgaga gccttctctt cctcaacaaa cttgcaggag 480

agtttcagga gaaaggac 498

<210> 7

<211> 1821

<212> DNA

<213>Bovine albumin

<400> 7

atgaaatggg ttaccttcat ctctctgctg ctgctgttct cttctgctta ctctcgtggt 60

gttttccgtc gtgacaccca caaatctgaa atcgctcacc gtttcaaaga cctgggtgaa 120

gaacacttca aaggtctggt tctgatcgct ttctctcagt acctgcagca gtgcccgttc 180

gacgaacacg ttaaactggt taacgaactg accgaattcg ctaaaacctg cgttgctgac 240

gaatctcacg ctggttgcga aaaatctctg cacaccctgt tcggtgacga actgtgcaaa 300

gttgcttctc tgcgtgaaac ctacggtgac atggctgact gctgcgaaaa acaggaaccg 360

gaacgtaacg aatgcttcct gtctcacaaa gacgactctc cggacctgcc gaaactgaaa 420

ccggacccga acaccctgtg cgacgaattc aaagctgacg aaaaaaaatt ctggggtaaa 480

tacctgtacg aaatcgctcg tcgtcacccg tacttctacg ctccggaact gctgtactac 540

gctaacaaat acaacggtgt tttccaggaa tgctgccagg ctgaagacaa aggtgcttgc 600

ctgctgccga aaatcgaaac catgcgtgaa aaagttctgg cttcttctgc tcgtcagcgt 660

ctgcgttgcg cttctatcca gaaattcggt gaacgtgctc tgaaagcttg gtctgttgct 720

cgtctgtctc agaaattccc gaaagctgaa ttcgttgaag ttaccaaact ggttaccgac 780

ctgaccaaag ttcacaaaga atgctgccac ggtgacctgc tggaatgcgc tgacgaccgt 840

gctgacctgg ctaaatacat ctgcgacaac caggacacca tctcttctaa actgaaagaa 900

tgctgcgaca aaccgctgct ggaaaaatct cactgcatcg ctgaagttga aaaagacgct 960

atcccggaaa acctgccgcc gctgaccgct gacttcgctg aagacaaaga cgtttgcaaa 1020

aactaccagg aagctaaaga cgcttttctc ggtagcttcc tgtacgaata ctctcgtcgt 1080

cacccggaat acgctgtttc tgttctgctg cgtctggcta aagaatacga agctaccctg 1140

gaagaatgct gcgctaaaga cgacccgcac gcttgctact ctaccgtttt cgacaaactg 1200

aaacacctgg ttgacgaacc gcagaacctg atcaaacaga actgcgacca gttcgaaaaa 1260

ctgggtgaat acggtttcca gaacgctctg atcgttcgtt acacccgtaa agttccgcag 1320

gtttctaccc cgaccctggt tgaagtttct cgttctctgg gtaaagttgg tacccgttgc 1380

tgcaccaaac cggaatctga acgtatgccg tgcaccgaag actacctgtc tctgatcctg 1440

aaccgtctgt gcgttctgca cgaaaaaacc ccggtttctg aaaaagttac caaatgctgc 1500

accgaatctc tggttaaccg tcgtccgtgc ttctctgctc tgaccccgga cgaaacctac 1560

gttccgaaag ctttcgacga aaaactgttc accttccacg ctgacatctg caccctgccg 1620

gacaccgaaa aacagatcaa aaaacagacc gctctggttg aactgctgaa acacaaaccg 1680

aaagctaccg aagaacagct gaaaaccgtt atggaaaact tcgttgcttt cgttgacaaa 1740

tgctgcgctg ctgacgacaa agaagcttgc ttcgctgttg aaggtccgaa actggttgtt 1800

tctacccaga ccgctctggc t 1821

<210> 8

<211> 498

<212> DNA

<213>Ox IFN-γ

<400> 8

atgaaataca cctcttactt cctggctctg ctgctgtgcg gtctgctggg tttctctggt 60

tcttacggtc agggtcagtt cttccgtgaa atcgaaaacc tgaaagaata cttcaacgct 120

tcttctccgg acgttgctaa aggtggtccg ctgttctctg aaatcctgaa aaactggaaa 180

gacgaatctg acaaaaaaat catccagtct cagatcgttt ctttctactt caaactgttc 240

gaaaacctga aagacaacca ggttatccag cgttctatgg acatcatcaa acaggacatg 300

ttccagaaat tcctgaacgg ttcttctgaa aaactggaag acttcaaaaa actgatccag 360

atcccggttg acgacctgca gatccagcgt aaagctatca acgaactgat caaagttatg 420

aacgacctgt ctccgaaatc taacctgcgt aaacgtaaac gttctcagaa cctgttccgt 480

ggtcgtcgtg cttctacc 498

<210> 9

<211> 498

<212> DNA

<213>Ox IFN-α

<400> 9

tgccacctgc cgcacaccca ctctctggct aaccgtcgtg ttctgatgct gctgggtcag 60

ttacgtcgtg taagcccgtc ttcttgcctg caggaccgta acgacttcgc tttcccgcag 120

gaagctctgg gtggttctca gctgcagaaa gctcaggcta tctctgttct gcacgaagtt 180

acccagcaca ccttccagct gttctctacc gaaggttctg ctaccacctg ggacgaatct 240

ctgctggaca aactgcgtgc tgctctggac cagcagctga ccgacctgca ggcttgcctg 300

cgtcaggaag aagaactgca gggtgctccg ctgctgaaag aagactcttc tctggctgtt 360

cgtaaatact tccaccgtct gaccctgtac ctgcaggaaa aaaaacactc tccgtgcgct 420

tgggaagttg ttcgtgctca ggttatgcgt gctttctctt cttctaccaa cctgcaggaa 480

tctttccgtc gtaaagac 498

Claims (10)

1. a kind of fusion protein being made of bovine albumin, Bov IFN γ and Bov IFN α, 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 of coding 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. the expression vector containing gene as claimed in claim 2.

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

5. a kind of recombinant bovine long-acting interferon, which is characterized in that the recombinant bovine 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 described 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 induced expressions, can be obtained fusion protein 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) design primer obtains by reverse transcription or is manually respectively synthesized the bovine albumin with flexible linker sequences, Niu Gan Disturb the target gene of plain γ, Bov IFN α；By flexible linker by bovine albumin, the mesh of Bov IFN γ, Bov IFN α 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 >；

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

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

8. the preparation method described 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 plasmids.

9. the preparation method described 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 purified through affinity chromatography, anion-exchange chromatography and sieve chromatography.

10. the application of recombinant bovine long-acting interferon according to claim 5, which is characterized in that the recombinant bovine is long-acting dry The long half time of element is disturbed up to 72 hours or more, there is broad-spectrum disease resistance toxic action and the immune response of Niu Zishen can be improved.