CN108794637A - A kind of canine recombinant long-acting interferon α and the fusion protein and preparation method thereof for preparing this long-acting interferon - Google Patents

Abstract

The invention discloses a kind of canine recombinant long-acting interferon α and the fusion proteins and preparation method thereof for preparing this long-acting interferon; the fusion protein is connect through flexible linker with dog interferon alpha by dog interferon γ and is formed, through being freeze-dried to obtain canine recombinant long-acting interferon α after fusion protein and freeze drying protectant mixture.The canine recombinant long-acting interferon α is remarkably improved the half-life period of dog interferon, and the half-life period of more common dog interferon improves 11 times or more, and has broad-spectrum disease resistance toxic action and can improve the immune response of dog itself.

Description

A kind of canine recombinant long-acting interferon α and prepare this long-acting interferon fusion protein and Preparation method

Technical field

The invention belongs to technical field of biological genetic engineering, and in particular to a kind of canine recombinant long-acting interferon α and prepare this Fusion protein of long-acting interferon and preparation method thereof.

Background technology

It is analyzed according to Chinese pet Industry, the market scale by the end of 2014 year end China pet industries has reached 105800000000 RMB, and Chinese Medical pet industry only accounts for about 20% at present, the city of the U.S. for the pet industry relative maturity that compares , the output value ratio of Medical pet reaches 50%, and there are huge development spaces for Chinese Medical pet industry.

Dog is the friend of mankind's loyalty, with increasing considerably for the canines feeding quantity such as domestic pet dog, canine disease The continuous raising of incidence, the death rate causes spirit and double loss economically to the mankind.Dog causes with mankind's intimate contact The probability that the mankind are infected by dog disease poison is high, such as rabies viruses, canine parvovirus and canine distemper virus, can all give human health Bring great threat.

Report interferon is studied as a kind of important cell factor, to canine distemper, canine parvovirus according to related science Certain curative effect is all had in the treatments of diseases such as disease, canine infectious hepatitis and canine coronavirus disease.Therefore actively research interference Element has broad application prospects in canine viral disease treatment.

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.According to the generation cell of IFN, biochemical character and The difference to be played a role in terms of immunity of organism, is divided into α, β, γ three classes.Now it is known that α types IFN can selectively make in vivo For infection cells such as viruses, by inhibiting the biosynthesis of the virus protein in infected cell, playing wide spectrum and efficiently resisting Virus function.IFN-α main physiological activity is with suppressing virus replication, anti parasitic, inhibits various kinds of cell proliferation, stimulation The killing activity of 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.

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 layer of molecular weight Partly solve the problems, such as that interferon molecule amount is small and leads to half-life short on face, while polyethylene glycol fused interferon cost is non- Chang Gao is unfavorable for clinically applying in domestic animal.

Invention content

In order to solve the above technical problems, the present invention provides a kind of canine recombinant long-acting interferon α and preparing this long-acting interference The fusion protein and preparation method thereof of element, the canine recombinant long-acting interferon is remarkably improved the half-life period of dog interferon, more general The half-life period of logical dog interferon improves 11 times or more, and has broad-spectrum disease resistance toxic action and can improve the immune response of dog itself.

The technical solution that the present invention takes is：

A kind of fusion protein being made of dog interferon γ and dog interferon alpha, the amino acid sequence table of the fusion protein 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 The codon of existing canine IFN-γ and IFN-α original gene codon adaptation indexI (CAI) in Escherichia coli is respectively 0.22, 0.27, GC percentage is 39.8%, 59.7%；And by existing to obtaining recombination after canine IFN-γ and IFN-α gene optimization Codon adaptation indexI (CAI) is 1.0,1.0, GC percentages 45.0%, 55.6% in Escherichia coli.It is notable by gene optimization The utilization rate for reducing low codon avoids influence of the rare codon to protein expression, improves the G/C content of gene, carries High transcription and translation efficiency.

The present invention also provides the expression vector 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/rIFN 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 canine recombinant long-acting interferon α, and the canine recombinant long-acting interferon α is by the fusion It is freeze-dried to form after albumen and freeze drying protectant mixture.

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/rIFN γ-IFN α, and preparation method is：

(1) design primer, is obtained or the dog interferon of the flexible linker sequences of artificial synthesized connection by reverse transcription The target gene of γ and dog interferon alpha；Dog interferon γ has been connected with the target gene of dog interferon alpha by flexible linker Come, the nucleotides sequence list of target gene is as shown in 400 < of SEQUENCE LISTING, 2 > or such as SEQUENCE LISTING Shown in 400 <, 3 >；

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

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

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

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 dog interferon γ (IFN-γ) is：

Upstream IFN-γ-F1：CGGGATCCATGAATTATACAAGCTATATC carries BamHI restriction enzyme sites；

Downstream IFN-γ-R1：ACCACCACCAGAACCACCACCACCTTTCGATGCTCTGC, with flexible linker；

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

Upstream IFN-α-F1：GGTGGTTCTGGTGGTGGTGGTTCTATGGCCCTGCCC, with flexible linker；

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

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

Respectively using the target gene of IFN-γ and IFN-α as template, and the upstream and downstream for being utilized respectively IFN-γ and IFN-α is drawn Object carries out PCR amplification, respectively obtains and connects the IFN-γ of flexible linker and the target gene of IFN-α.

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. IFN-γ gene and IFN-α gene are connected using flexible linker

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 IFN-γ template DNA connect the 1 μ L of IFN-α template DNA I of flexible linker, IFN-γ sense primer 0.5 μ L, IFN- 0.5 μ L, Taq archaeal dna polymerase of α downstream primers, 2.5 μ L, dNTP Mix is 9 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；58 DEG C of annealing 45s, 72 DEG C of extension 1kb/min, altogether 35 cycles；Last 72 DEG C of extensions 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 dog interferon γ (IFN-γ) is：

Upstream IFN-γ-F2：CGGGATCCATGAACTACACCTCTTAC carries BamHI restriction enzyme sites；

Downstream IFN-γ-R2：ACCACCACCAGAACCACCACCACCTTTAGAAGCACGACG；With flexible linker；

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

Upstream IFN-α-F2：GGTGGTTCTGGTGGTGGTGGTTCTATGGCTCTGCCGT, with flexible linker；

Downstream IFN-α-R2：CCCTCGAGTTTACGACGACGAAC carries XhoI restriction enzyme sites；

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

Respectively using the target gene of IFN-γ and IFN-α as template, and the upstream and downstream for being utilized respectively IFN-γ and IFN-α is drawn Object carries out PCR amplification, respectively obtains the target gene of the IFN-γ and IFN-α after the optimization for connecting flexible linker.

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. IFN-γ gene and IFN-α gene are connected using flexible linker

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 IFN-γ template DNA connect the 1 μ L of IFN-α template DNA of flexible linker, 0.5 μ L of IFN-γ sense primer, IFN-α 0.5 μ L, Taq archaeal dna polymerase of downstream primer, 2.5 μ L, dNTP Mix is 9 μ L, adds RNase Free water to 25 μ L；It is anti-to connect PCR The condition is answered to be：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, totally 35 A cycle；Last 72 DEG C of extensions 10min.

The present invention also provides the application of the canine recombinant long-acting interferon α, long half time had up to 46 hours or more Broad-spectrum disease resistance toxic action and the immune response that dog itself can be improved.

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

1. dog interferon γ and dog interferon alpha gene are realized fusion by flexible linker, improves interferon and partly decline Phase improves 11 times or more compared with plain interferon；Compared with common polyethylene glycol fused interferon, significantly reduce into This.

2. by being optimized to dog interferon γ and dog interferon alpha gene, improves interferon gamma and dog interferon alpha melts The expression quantity of hop protein；

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

4. the fusion protein disclosed by the invention being made of dog interferon γ and dog interferon alpha not only has interferon-' alpha ' Broad-spectrum disease resistance toxic action, while significantly improving the immune response of dog itself.

Description of the drawings

Fig. 1 is the result of the dog interferon γ genes and the RT-PCR amplifications of dog interferon alpha gene in embodiment 1；Swimming lane M： DNA Marker DL2000；Swimming lane 1：Dog interferon γ gene RT-PCR amplified productions；Swimming lane 2：Dog interferon alpha gene RT- Pcr amplification product；

Fig. 2 is the result of the PCR amplification after the canine IFN-γ in embodiment 1 is connected with the target gene of dog IFN-α；Swimming Road M：DNA Marker DL2000；Swimming lane 1：Dog interferon γ genes and dog interferon alpha gene ligation amplification product；

Fig. 3 is the PCR amplification and double digestion qualification result of the positive colony plasmid in embodiment 1；Swimming lane M：DNA Marker DL10000；Swimming lane 1：Recombinant plasmid double digestion result；Swimming lane 2：Plasmid PCR result；

Fig. 4 is the SDS-PAGE electrophoretic examinations 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：Empty bacterium 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：It is precipitated after recombinant bacterium induction is broken；Swimming lane 2：For the broken rear supernatant of recombinant bacterium induction；

Fig. 6 is that the canine recombinant 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 canine recombinant long-acting interferon α of gradient dilution (from right to left) handles hole；

Fig. 7 is the canine recombinant 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 dog interferon γ and dog interferon alpha, preparation method are as follows：

1. the acquisition and amplification of dog interferon γ (IFN-γ) and dog interferon alpha (IFN-α) target gene

Design of primers：

It is shown in Table 1 according to the objective gene sequence design synthetic primer reported in Genebank, in the upper of dog interferon γ Trip primer and downstream primer in introduce EcoRI restriction enzyme sites and Linker sequences respectively, dog interferon alpha sense primer and under Linker sequences and XhoI restriction enzyme sites are introduced respectively in trip primer.

1 PCR amplification primer of table

RT-PCR obtains target gene：

RNA is extracted from dog liver organization, the target gene of IFN-γ and IFN-α, the base of the two are obtained by reverse transcription Because sequence is respectively as shown in 400 < of SEQUENCE LISTING 400 <, 4 > and SEQUENCE LISTING, 5 >；

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 530bp and 590bp or so in RT-PCR amplified productions, and result is such as Shown in Fig. 1, illustrate that the target gene that the dog interferon γ for being separately connected flexible linker has successfully been prepared is interfered with dog The target gene of plain α.

2. the connection of target gene

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

3 PCR reaction systems of table

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 1090bp or so in pcr amplification product, and the results are shown in Figure 2, The nucleotide sequence of obtained target gene is as shown in 400 < of SEQUENCE LISTING, 2 >.

3. expression vector establishment

The PCR glue recovery product for selecting the target gene after connection errorless after sequencing is used with pET-32a plasmids BamHI and XhoI restriction enzymes carry out double digestion and recycling, and double digestion is done by 20 μ L systems in table 4：

4 double digestion system of table

General buffer	2μL
		Restriction enzyme (a pair)	1μL+1μL
Carrier or recycling segment	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 5,4 DEG C overnight connection：

Table 5

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 The LB culture medium flat plate overnight incubations of element；The bacterium colony grown on LB tablets is taken to identify target gene, positive colony bacteria plasmid through PCR It is identified through BamHI and XhoI double digestions, is accredited as positive and indicates expression vector establishment success, obtain engineering bacteria pET-32a/ rIFNγ-IFNα；There is single band through agarose gel electrophoresis at the places 1090bp or so in PCR amplification and double digestion product, The results are shown in Figure 3.

4. the expression of recombinant protein

Picking engineering bacteria pET-32a/rIFN γ-IFN α shakes for 37 DEG C in the LB culture mediums of the 100 μ g/ml containing ampicillin Bacterium 1h recoveries engineering bacteria activity is surveyed OD values and is reached in LB culture mediums (the 100 μ g/ml containing ampicillin) after amplification culture 4h When 1.0；IPTG, 32 DEG C of induced expression (100 μ g/ml of final concentration) 5h is added；Bacterium is collected, is examined through SDS-PAGE electrophoresis It surveys, supernatant is deposited in the visible predominant expression band in the places 58KD or so after the bacterial cell disruption after recombinant bacterium induction 5h, and result is such as Shown in Fig. 4, it can be seen from the figure that be deposited in the places 58KD or so visible excellent for supernatant after bacterial cell disruption after recombinant bacterium induction 5h Gesture band of expression illustrates precipitating and the fusion protein of equal successful expression in supernatant.

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

5.1 His affinity chromatographys

After membrane filtration of the crude fusion protein with 0.22 μm of aperture, loading is by being connected to AKTA explorer 100 On protein purification system, the His affinity 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 IFN γ-IFN α protein peak.

5.2 DEAE anion-exchange chromatographies

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 rIFN 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) has balanced 200 molecular sieve chromatographies of Superdex, with Binding Buffer III elution, collects rIFN γ-IFN α protein peak

5.4 sample identification：Measure rIFN γ-IFN α potency and specific activity, specific activity >=1.0 × 10⁵U/mg albumen is to close Lattice；It is aseptic subpackaged, -80 DEG C of preservations.It can be obtained the fusion protein being made of dog interferon γ and dog interferon alpha, amino acid Sequence is as shown in 400 < of SEQUENCE LISTING, 1 >.

Embodiment 2

A kind of fusion protein being made of dog interferon γ and dog interferon alpha, other, only will be therein with embodiment 1 E. coli bl21 (DE3) competent cell is replaced for BL21 (DE3) competent cell with pGro7 plasmids.It is merged The SDS-PAGE electrophoresis results of albumen are compareed with embodiment 1, and 58KD or so place's predominant expression bands are thicker in supernatant, and explanation is drawn After entering molecular chaperones pGro7, expression of the destination protein in supernatant is more preferable, obtained fusion protein amount higher.Escherichia coli The albumen of expression is mostly present in inclusion body；By introducing molecular chaperones in expressing bacterial strain, coordinate expression albumen is correct It folds, 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 dog interferon γ and dog interferon alpha, preparation method are as follows：

1. the acquisition and amplification of dog interferon γ (IFN-γ) and dog interferon alpha (IFN-α) target gene

To in embodiment 1 IFN-γ and IFN-α optimize, artificial synthesized IFN-γ and IFN-α target gene, optimization Afterwards, the nucleotide sequence of the two is respectively such as 400 < of SEQUENCE LISTING 400 <, 6 > and SEQUENCE LISTING, 7 > institutes Show.

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, to the IFN-γ of dog and IFN- in the present embodiment α gene codons optimize.

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.Using software detection find canine IFN-γ and The codon of IFN-α original gene codon adaptation indexI (CAI) in Escherichia coli is respectively 0.22,0.27, GC percentages It is 39.8%, 59.7%；And by obtaining recombination password in Escherichia coli after canine IFN-γ and IFN-α gene optimization Sub- adaptation index (CAI) is 1.0,1.0, GC percentages 45.0%, 55.6%.Low codon is significantly reduced by gene optimization Utilization rate, avoid influence of the rare codon to protein expression, improve the G/C content of gene, improve transcription and translation effect Rate, and then improve the expression quantity of recombinant protein.

1.3 design of primers：

6 PCR amplification primer of table

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

7 PCR reaction systems of table

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

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.

There is specific band through agarose gel electrophoresis in 530bp and 590bp or so in pcr amplification product, and explanation is prepared into To the target gene for being separately connected canine IFN-γ and dog IFN-α after the optimization of flexible linker.

2. the connection of target gene

Target gene is diluted to 10ug/mL, connects even section target gene using over-lap PCR, 25 μ L reaction systems are such as Shown in table 8：

8 PCR reaction systems of table

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 1090bp or so in pcr amplification product, illustrates successfully to obtain IFN-γ connected with IFN-α after target gene.The nucleotide sequence of obtained target gene such as SEQUENCE LISTING Shown in 400 <, 3 >.

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 9：

9 double digestion system of table

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

Table 10

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

It converts in connection product to e. coli bl21 (DE3) competent cell, competence is coated on the mould of benzyl containing ammonia The LB culture medium flat plate overnight incubations of element；The bacterium colony grown on LB tablets is taken to identify target gene, positive colony bacteria plasmid through PCR It is identified through BamHI, XhoI double digestion, is accredited as positive and indicates expression vector establishment success, obtain engineering bacteria pET-32a/ rIFNγ-IFNα；There is single band through agarose gel electrophoresis at the places 1090bp or so in PCR amplification and double digestion product, say It is bright connected with IFN-α containing IFN-γ after target gene expression vector establishment success.

4. the expression of recombinant protein

Picking engineering bacteria pET-32a/rIFN γ-IFN α shakes for 37 DEG C in the LB culture mediums of the 100 μ g/ml containing ampicillin Bacterium 1h recoveries engineering bacteria activity is surveyed OD values and is reached in LB culture mediums (the 100 μ g/ml containing ampicillin) after amplification culture 4h When 1.0；IPTG, 32 DEG C of induced expression (100 μ g/ml of final concentration) 5h is added；Bacterium is collected, is examined through SDS-PAGE electrophoresis It surveys, supernatant is deposited in the visible predominant expression band in the places 58KD or so after the bacterial cell disruption after recombinant bacterium induction 5h, illustrates upper Recombinant protein has been obtained in cleer and peaceful precipitation.

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 ultrasounds are split Bacterial precipitation is solved, work 10s, is spaced 3S, ultrasonic 6min, and whole process repeats 3~4 times；4 DEG C, 12000r/min centrifugations 15min takes supernatant, inclusion body (inclusion body is through dissolving, refolding strategy), obtains crude fusion protein respectively.

5. fusion protein purification

5.1 His affinity chromatographys

After membrane filtration of the crude fusion protein with 0.22 μm of aperture, loading is by being connected to AKTA explorer 100 On protein purification system, the His affinity 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 rIFN γ-IFN α protein peak.

5.2 DEAE anion-exchange chromatographies

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 rIFN 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) has balanced 200 molecular sieve chromatographies of Superdex, with Binding Buffer III elution, collects rIFN γ-IFN α protein peak.

5.4 sample identification

Measure rIFN γ-IFN α potency and specific activity, specific activity >=1 × 10⁵U/mg albumen is qualification；It is aseptic subpackaged, -80 DEG C preserve.It can be obtained the fusion protein being made of dog interferon γ and dog interferon alpha, amino acid sequence such as SEQUENCE Shown in 400 < of LISTING, 1 >.

Embodiment 4

A kind of fusion protein being made of dog interferon γ and dog interferon alpha, other, only will be therein with embodiment 3 E. coli bl21 (DE3) competent cell is replaced for BL21 (DE3) competent cell with pGro7 plasmids.It is merged The SDS-PAGE electrophoresis results of albumen are compareed with embodiment 3, and 58KD or so place's predominant expression bands are thicker in supernatant, and explanation is drawn After entering molecular chaperones pGro7, expression of the destination protein in supernatant is more preferable, obtained fusion protein amount higher.Escherichia coli The albumen of expression is mostly present in inclusion body；By introducing molecular chaperones in expressing bacterial strain, coordinate expression albumen is correct It folds, 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 canine recombinant 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 obtains the identification for the fusion protein being made of dog interferon γ and dog interferon alpha

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 1.2mg/ml.

6.2 SDS-PAGE electrophoresis detections

Compared with empty bacterium, fusion protein has the newly-increased protein band of a dense dye in 58KD or so, as shown in Figure 4.

6.3 Western Blot results

Fusion protein in Examples 1 to 4 is detected respectively, with the anti-canine interferon alpha 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).Canine recombinant long-acting interferon α samples can be with anti-dog interferon Specific reaction occurs for alpha monoclonal antibodies, and specific band occur in the places 58KD or so, as shown in Figure 5.

Embodiment 7

Bioactivity freeze-dried four parts of canine recombinant 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 canine recombinant 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 canine recombinant 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 canine recombinant obtained is long After imitating interferon-' alpha ' treated cell inoculation virus, it is observed continuously under inverted microscope, cellular morphology is normal, does not go out incumbent What lesion, measures potency >=1.0 × 10⁵U/ml, as shown in Figure 6.

Embodiment 8

The measurement of half-life period of the canine recombinant long-acting interferon α in dog body

The four parts of canine recombinant 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 dog body

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

Take the dog (half male and half female) that six weight are roughly the same, neck that 2mg/ml canine recombinant long-acting interferons α is subcutaneously injected Freeze-dried 2ml, respectively 1h, 2h, 4h, 8h, 16h, for 24 hours, 48h, 72h venous blood collection, the solidification of 4 DEG C of blood sample, 3500rpm low temperature from Heart 10min detaches serum, and every dog 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 rIFN γ-IFN α in product is carried out curve fitting and calculating parameter with DAS pharmacokinetics softwares.The matched curve of A such as Fig. 7 It is shown；Parameter result of calculation is shown in Table 11.

Dominant dynamic parameters in serum after 11 canine recombinant long-acting interferon α intramuscular injection of table

The result shows that canine recombinant long-acting interferon α has longer half-life period.Half-life period can reach 46h or so after measured, compared with Plain interferon improves 11 times.

Embodiment 9

The freeze-dried measurement that canine cells immune response is influenced of four parts of canine recombinant long-acting interferon α in embodiment 5

The young dog for taking six weight roughly the same is divided into two groups, is denoted as experimental group and control group；Experimental group neck is subcutaneously noted The 2mg/ml canine recombinant 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 dog 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 12：

Table 12 ELISA detection each group canine cells immune responses are horizontal

The result shows that after injection canine recombinant long-acting interferon α, can significantly improve cell factor IL-2 in canine peripheral blood, The content of IL-4 enhances cellullar immunologic response level, significantly improves immunity level.

It is above-mentioned to canine recombinant long-acting interferon α and to prepare fusion protein and its preparation of this long-acting interferon with reference to embodiment The detailed description that method carries out is illustrative without being restrictive, and can enumerate several implementations according to limited range Example, 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 canine recombinant long-acting interferon α and the fusion protein and preparation method thereof for preparing this long-acting interferon

<130> 1

<160> 7

<170> PatentIn version 3.3

<210> 1

<211> 363

<212> PRT

<213>Canine recombinant long-acting interferon alpha fusion protein

<400> 1

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

1 5 10 15

Cys Ser Ser Gly Cys Asn Cys Gln Ala Met Phe Phe Lys Glu Ile Glu

20 25 30

Asn Leu Lys Glu Tyr Phe Asn Ala Ser Asn Pro Asp Val Ser Asp Gly

35 40 45

Gly Ser Leu Phe Val Asp Ile Leu Lys Lys Trp Arg Glu Glu Ser Asp

50 55 60

Lys Thr Ile Ile Gln Ser Gln Ile Val Ser Phe Tyr Leu Lys Leu Phe

65 70 75 80

Asp Asn Phe Lys Asp Asn Gln Ile Ile Gln Arg Ser Met Asp Thr Ile

85 90 95

Lys Glu Asp Met Leu Gly Lys Phe Leu Asn Ser Ser Thr Ser Lys Arg

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

Met Ala Leu Pro Cys Ser Phe Ser Val Ala Leu Val Leu Leu Ser Cys

180 185 190

His Ser Leu Cys Cys Leu Ala Cys Asp Leu Pro Asp Thr His Ser Leu

195 200 205

Arg Asn Trp Arg Val Leu Thr Leu Leu Gly Gln Met Arg Arg Leu Ser

210 215 220

Ala Ser Ser Cys Asp His Tyr Thr Thr Asp Phe Ala Phe Pro Lys Glu

225 230 235 240

Leu Phe Asp Gly Gln Arg Leu Gln Glu Ala Gln Ala Leu Ser Val Val

245 250 255

His Val Met Thr Gln Lys Val Phe His Leu Phe Cys Thr Asn Met Ser

260 265 270

Ser Ala Pro Trp Asn Met Thr Leu Leu Gly Glu Leu Cys Ser Gly Leu

275 280 285

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

290 295 300

Leu Ala Glu Thr Pro Leu Met His Glu Asp Ser Thr Leu Arg Thr Tyr

305 310 315 320

Phe Gln Arg Ile Ser Leu Tyr Leu Gln Asp Arg Asn His Ser Pro Cys

325 330 335

Ala Trp Glu Met Val Arg Ala Glu Ile Gly Arg Ser Phe Phe Ser Leu

340 345 350

Thr Ile Leu Gln Glu Arg Val Arg Arg Arg Lys

355 360

<210> 2

<211> 1089

<212> DNA

<213>Canine recombinant long-acting interferon α genomes 1

<400> 2

atgaattata caagctatat cttagctttt cagctttgcg tgattttgtg ttcttctggc 60

tgtaactgtc aggccatgtt ttttaaagaa atagaaaacc taaaggaata ttttaatgca 120

agtaatccag atgtatcgga cggtgggtct cttttcgtag atattttgaa gaaatggaga 180

gaggagagtg acaaaacaat cattcagagc caaattgtct ctttctactt gaaactgttt 240

gacaacttta aagataacca gatcattcaa aggagcatgg ataccatcaa ggaagacatg 300

cttggcaagt tcttaaatag cagcaccagt aagagggagg acttccttaa gctgattcaa 360

attcctgtga acgatctgca ggtccagcgc aaggcgataa atgaactcat caaagtgatg 420

aatgatctct caccaagatc caacctaagg aagcggaaaa ggagtcagaa tctgtttcga 480

ggccgcagag catcgaaagg tggtggtggt tctggtggtg gtggttctat ggccctgccc 540

tgctccttct cggtggccct ggtgctgctc agctgccact ccctgtgctg tctggcttgc 600

gacctgcccg acacccacag cctgcgcaac tggagggtcc tgacgctcct gggacagatg 660

aggagactct ccgccagctc ttgtgaccac tacaccactg actttgcctt ccccaaggaa 720

ctgtttgatg gccagcggct ccaggaggcg caagccctct ctgtggtcca cgtgatgacc 780

cagaaggtct tccacctctt ctgcacgaac atgtcctctg ctccttggaa catgaccctc 840

ctgggggaat tgtgctcggg gctctctgag cagctggatg acctggatgc ctgtcccctg 900

caggaggcag ggctggccga gacccccctc atgcatgaag actccaccct gaggacctac 960

ttccaaagga tctccctcta cctgcaagac aggaaccaca gcccgtgtgc ctgggagatg 1020

gtccgagcag aaatcgggag atccttcttc tccttgacca tcttgcaaga aagagtaagg 1080

aggaggaaa 1089

<210> 3

<211> 1089

<212> DNA

<213>Canine recombinant long-acting interferon α genomes 2

<400> 3

atgaactaca cctcttacat cctggctttc cagctgtgcg ttatcctgtg ctcttctggt 60

tgcaactgcc aggctatgtt cttcaaagaa atcgaaaacc tgaaagaata cttcaacgct 120

tctaacccgg acgtttctga cggtggttct ctgttcgttg acatcctgaa aaaatggcgt 180

gaagaatctg acaaaaccat catccagtct cagatcgttt ctttctacct gaaactgttc 240

gacaacttca aagacaacca gatcatccag cgttctatgg acaccatcaa agaagacatg 300

ctgggtaaat tcctgaactc ttctacctct aaacgtgaag acttcctgaa actgatccag 360

atcccggtta acgacctgca ggttcagcgt aaagctatca acgaactgat caaagttatg 420

aacgacctgt ctccgcgttc taacctgcgt aaacgtaaac gttctcagaa cctgttccgt 480

ggtcgtcgtg cttctaaagg tggtggtggt tctggtggtg gtggttctat ggctctgccg 540

tgctctttct ctgttgctct ggttctgctg tcttgccact ctctgtgctg cctggcttgc 600

gacctgccgg acacccactc tctgcgtaac tggcgtgttc tgaccctgct gggtcagatg 660

cgtcgtctgt ctgcttcttc ttgcgaccac tacaccaccg acttcgcttt cccgaaagaa 720

ctgttcgacg gtcagcgtct gcaggaagct caggctctgt ctgttgttca cgttatgacc 780

cagaaagttt tccacctgtt ctgcaccaac atgtcttctg ctccgtggaa catgaccctg 840

ctgggtgaac tgtgctctgg tctgtctgaa cagctggacg acctggacgc ttgcccgctg 900

caggaagctg gtctggctga aaccccgctg atgcacgaag actctaccct gcgtacctac 960

ttccagcgta tctctctgta cctgcaggac cgtaaccact ctccgtgcgc ttgggaaatg 1020

gttcgtgctg aaatcggtcg ttctttcttc tctctgacca tcctgcagga acgtgttcgt 1080

cgtcgtaaa 1089

<210> 4

<211> 498

<212> DNA

<213>Dog interferon γ

<400> 4

atgaattata caagctatat cttagctttt cagctttgcg tgattttgtg ttcttctggc 60

tgtaactgtc aggccatgtt ttttaaagaa atagaaaacc taaaggaata ttttaatgca 120

agtaatccag atgtatcgga cggtgggtct cttttcgtag atattttgaa gaaatggaga 180

gaggagagtg acaaaacaat cattcagagc caaattgtct ctttctactt gaaactgttt 240

gacaacttta aagataacca gatcattcaa aggagcatgg ataccatcaa ggaagacatg 300

cttggcaagt tcttaaatag cagcaccagt aagagggagg acttccttaa gctgattcaa 360

attcctgtga acgatctgca ggtccagcgc aaggcgataa atgaactcat caaagtgatg 420

aatgatctct caccaagatc caacctaagg aagcggaaaa ggagtcagaa tctgtttcga 480

ggccgcagag catcgaaa 498

<210> 5

<211> 561

<212> DNA

<213>Dog interferon alpha

<400> 5

atggccctgc cctgctcctt ctcggtggcc ctggtgctgc tcagctgcca ctccctgtgc 60

tgtctggctt gcgacctgcc cgacacccac agcctgcgca actggagggt cctgacgctc 120

ctgggacaga tgaggagact ctccgccagc tcttgtgacc actacaccac tgactttgcc 180

ttccccaagg aactgtttga tggccagcgg ctccaggagg cgcaagccct ctctgtggtc 240

cacgtgatga cccagaaggt cttccacctc ttctgcacga acatgtcctc tgctccttgg 300

aacatgaccc tcctggggga attgtgctcg gggctctctg agcagctgga tgacctggat 360

gcctgtcccc tgcaggaggc agggctggcc gagacccccc tcatgcatga agactccacc 420

ctgaggacct acttccaaag gatctccctc tacctgcaag acaggaacca cagcccgtgt 480

gcctgggaga tggtccgagc agaaatcggg agatccttct tctccttgac catcttgcaa 540

gaaagagtaa ggaggaggaa a 561

<210> 6

<211> 498

<212> DNA

<213>Dog interferon γ

<400> 6

atgaactaca cctcttacat cctggctttc cagctgtgcg ttatcctgtg ctcttctggt 60

tgcaactgcc aggctatgtt cttcaaagaa atcgaaaacc tgaaagaata cttcaacgct 120

tctaacccgg acgtttctga cggtggttct ctgttcgttg acatcctgaa aaaatggcgt 180

gaagaatctg acaaaaccat catccagtct cagatcgttt ctttctacct gaaactgttc 240

gacaacttca aagacaacca gatcatccag cgttctatgg acaccatcaa agaagacatg 300

ctgggtaaat tcctgaactc ttctacctct aaacgtgaag acttcctgaa actgatccag 360

atcccggtta acgacctgca ggttcagcgt aaagctatca acgaactgat caaagttatg 420

aacgacctgt ctccgcgttc taacctgcgt aaacgtaaac gttctcagaa cctgttccgt 480

ggtcgtcgtg cttctaaa 498

<210> 7

<211> 561

<212> DNA

<213>Dog interferon alpha

<400> 7

atggctctgc cgtgctcttt ctctgttgct ctggttctgc tgtcttgcca ctctctgtgc 60

tgcctggctt gcgacctgcc ggacacccac tctctgcgta actggcgtgt tctgaccctg 120

ctgggtcaga tgcgtcgtct gtctgcttct tcttgcgacc actacaccac cgacttcgct 180

ttcccgaaag aactgttcga cggtcagcgt ctgcaggaag ctcaggctct gtctgttgtt 240

cacgttatga cccagaaagt tttccacctg ttctgcacca acatgtcttc tgctccgtgg 300

aacatgaccc tgctgggtga actgtgctct ggtctgtctg aacagctgga cgacctggac 360

gcttgcccgc tgcaggaagc tggtctggct gaaaccccgc tgatgcacga agactctacc 420

ctgcgtacct acttccagcg tatctctctg tacctgcagg accgtaacca ctctccgtgc 480

gcttgggaaa tggttcgtgc tgaaatcggt cgttctttct tctctctgac catcctgcag 540

gaacgtgttc gtcgtcgtaa a 561

Claims (10)

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

2. a kind of gene 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 canine recombinant long-acting interferon α, which is characterized in that the canine recombinant long-acting interferon α is by described in claim 1 It is freeze-dried to form after fusion 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/rIFN γ- IFN α, preparation method are：

(1) design primer, obtained by reverse transcription or the dog interferon γ of the flexible linker sequences of artificial synthesized connection and The target gene of dog interferon alpha；The target gene of dog interferon γ and dog interferon alpha are connected by flexible linker, The nucleotides sequence list of target gene is as shown in 400 < of SEQUENCE LISTING, 2 > or such as SEQUENCE LISTING 400 Shown in 3 > of <；

(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/r IFN γs- 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 canine recombinant long-acting interferon α according to claim 5, which is characterized in that the canine recombinant is long-acting dry The long half time of plain α is disturbed up to 46 hours or more, there is broad-spectrum disease resistance toxic action and the immune response of dog itself can be improved.