CN107286257A - A kind of Recombinant Swine long-acting interferon α and prepare fusion protein of this long-acting interferon and preparation method thereof - Google Patents

Abstract

The invention discloses a kind of Recombinant Swine long-acting interferon α and the fusion protein for preparing this long-acting interferon and preparation method thereof; the fusion protein is connected by Porcine interferon-gamma with porcine interferon alpha and formed through flexible linker, through being freeze-dried to obtain Recombinant Swine long-acting interferon α after fusion protein and freeze drying protectant mixture.The Recombinant Swine long-acting interferon α is remarkably improved the half-life period of pig interferon, and the half-life period of more common pig interferon improves more than 16 times, and with broad-spectrum disease resistance toxic action and can improve the immune response of pig itself.

Description

A kind of Recombinant Swine long-acting interferon α and prepare this long-acting interferon fusion protein and Its preparation method

Technical field

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

Background technology

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

The prevention and treatment approach to porcine viral diseases mainly by vaccine inoculation and uses antibiotic at present, but by In breeding environment imperfection, virus variation and Abwehrkraft des Koepers change etc. reason, make traditional prevention and treatment approach by Huge challenge, most of antibiotics and traditional oral antiviral medicament, due to medicament residue problem, to health It is negatively affected；And traditional vaccine, high specific and side effect due to it, it is impossible to resist virus variation and new disease The significant damage brought to pig aquaculture continuously emerges in poison.

IFN is that the infection induced body of a viroid is produced with broad-spectrum antiviral, antitumor and with immunoregulation effect Protein, nineteen fifty-seven, Issacs and Lindeman had found first, and it is the multi-functional cell factor of a class, with cell receptor knot After conjunction, it can induce body and produce many species-specific proteins and enzyme, mainly by suppressing viral gene transcription and degraded virus RNA suppresses the growth and breeding of virus and plays the activity of antitumor grade.According to IFN generation cell, biochemical character and The difference played a role in terms of immunity of organism, is divided into the class of α, β, γ tri-.Now, it is known that α types IFN can selectively make in vivo For infection cells such as viruses, by suppressing the biosynthesis of the virus protein in infected cell, wide spectrum is played and efficiently anti- Virus function.IFN-α main physiological activity is with suppressing virus replication, anti parasitic, suppresses various kinds of cell propagation, stimulation The killing activity of immunocyte.

γ types IFN is that T cell and NK cells by activating are produced, with relatively strong antiviral and immunoloregulation function.Largely Research shows that interferon gamma also plays the adjustment effect of key in addition to broad-spectrum antiviral function, 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- Answer, but the immunoregulatory activity of interferon gamma is coordinating immune response and is determining to play more in the long-term antiviral state of body Important effect, 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 is generally 2-4 Individual hour.Half-life short brings great inconvenience, the increase for the treatment of number of times, corresponding time cost and financial cost to treatment Increase therewith, and the tenability limit of body is also possible to be broken the generation for causing adverse reaction.The main cause of 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, the layer only in molecular weight Partly solved on face interferon molecule amount it is small and the problem of cause half-life short, while polyethylene glycol fused interferon cost is non- Chang Gao, is unfavorable for clinically applying in domestic animal.

The content of the invention

In order to solve the above technical problems, the invention provides a kind of Recombinant Swine long-acting interferon α and preparing this long-acting interference Fusion protein of element and preparation method thereof, the Recombinant Swine long-acting interferon is remarkably improved the half-life period of pig interferon, more general The half-life period of logical pig interferon improves more than 16 times, and with broad-spectrum disease resistance toxic action and can improve the immune response of pig itself.

The technical scheme that the present invention takes is：

A kind of fusion protein being made up of Porcine interferon-gamma and porcine interferon alpha, the amino acid sequence table of the fusion protein As shown in the ＞ of 400 ＜ of SEQUENCE LISTING 1, fusion protein 1 is designated as；Or as shown in the ＞ of 400 ＜ of SEQUENCE LISTING 2, It is designated as fusion protein 2.

Present invention also offers the gene for encoding above-mentioned fusion protein, the nucleotides sequence list such as SEQUENCE of the gene Shown in the ＞ of 400 ＜ of LISTING 3, genome 1 is designated as；Or as shown in the ＞ of 400 ＜ of SEQUENCE LISTING 4, it is designated as genome 2.

Fusion protein 1 described in the codified of genome 1；The codified fusion protein 2 of genome 2.Genome 2 is pair The nucleotide sequence of genome 1 optimize after result, be considered as the base during usual codon adaptation indexI CAI=1.0 Because being optimal high efficient expression state in the expression system, CAI values are lower to show that expression is lower in host.In gene G/C content most ideal distribution scope is 30~70%, and the scope is exceeded in any region can influence translation and transcriptional efficiency. The codon of the porcine IFN γ and IFN-α original gene codon adaptation indexI in Escherichia coli is found using software detection (CAI) it is respectively that 0.24,0.22, GC percentages are 39.2%, 59.1%；And by porcine IFN γ and IFN-α gene optimization After obtain recombination in Escherichia coli codon adaptation indexI (CAI) be 1.0,1.0, GC percentages 45.4%, 55.6%. The utilization rate of low codon is significantly reduced by gene optimization, it is to avoid influence of the rare codon to protein expression, improved The G/C content of gene, improves 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.

Present invention also offers the genetic engineering bacterium 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 falls within protection scope of the present invention, further, the place Chief cell is e. coli host cell, further, and the e. coli host cell is BL21 (DE3) competent cell Or BL21 (DE3) competent cell with pGro7 plasmids.

Present invention also offers a kind of Recombinant Swine long-acting interferon α, the Recombinant Swine long-acting interferon α is by described 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 method of the fusion protein, 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, it is purified to can obtain fusion protein afterwards.

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 its preparation method is：

(1) primer is designed, is obtained by reverse transcription or the artificial synthesized pig interferon for connecting flexible linker sequences γ and porcine interferon alpha target gene；The target gene of Porcine interferon-gamma and porcine interferon alpha is connected by flexible linker Come, the nucleotides sequence list of target gene is as shown in the ＞ of 400 ＜ of SEQUENCE LISTING 3 or such as SEQUENCE LISTING Shown in the ＞ of 400 ＜ 4；

(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/rIFN 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. is V205.

The acquisition methods of the genome 1 are：

A. design of primers

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

Upstream IFN-γ-F1：ATAGAATTCATGAGTTATACAACTTATTTCTT, with EcoRI restriction enzyme sites；

Downstream IFN-γ-R1：

CCAGAACCACCTCCAGAACCTCCACCTTTTGATGCTCTCTGGCCTTG, with flexible linker；

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

Upstream IFN-α-F1：

CTGGAGGTGGTTCTGGAGGTGGATCTTGCGACCTGCCTCAGAC, with flexible linker；

Downstream IFN-α-R1：CCAAGCTTCTCCTTCTTCCTGAGTCTGT, with the restriction enzyme sites of Hind III；

B. RNA is extracted from pig liver, the target gene of IFN-γ and IFN-α, both genes are obtained by reverse transcription Sequence is respectively as shown in the ＞ of 400 ＜ of SEQUENCE LISTING 400 ＜, 5 ＞ and SEQUENCE LISTING 6；

Respectively using the target gene of IFN-γ and IFN-α as template, and it is utilized respectively the upstream and downstream of IFN-γ and IFN-α and draws Thing enters performing PCR amplification, respectively obtains the IFN-γ and the target gene of IFN-α for connecting flexible linker.

PCR reaction systems and condition are：In 25 μ L overall reaction system, the μ L of template ribonucleic acid 1.5, upstream and downstream primer is each 0.5 μ L, reverse transcriptase 2.5 μ L, dNTP Mix are 10 μ L, plus RNase Free water is 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 circulation；95 DEG C of denaturation 45s, 58 DEG C of annealing 45s, 72 DEG C are prolonged 1kb/min is stretched, is circulated 35 times, last 72 DEG C of extensions 10min.

C. IFN-γ gene is connected with IFN-α gene using flexible linker

The PCR reaction systems and reaction condition of connection be：In 25 μ L overall reaction system, connect flexible linker's The μ L of IFN-γ template DNA 1, connect the flexible linker μ L of IFN-α template DNA I 1, IFN-γ sense primer 0.5 μ L, IFN- 0.5 μ L, Taq archaeal dna polymerase of α anti-sense primers 2.5 μ L, dNTP Mix is 9 μ L, plus RNase Free water is to 25 μ L；Connect PCR Reaction condition is：95 DEG C of pre-degeneration 4min, into circulation：94 DEG C of denaturation 45s；58 DEG C of annealing 45s, 72 DEG C of extension 1kb/min, altogether 35 circulations；Last 72 DEG C of extensions 10min.

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 Porcine interferon-gamma (IFN-γ) is：

Upstream IFN-γ-F2：CATGCCATGGTATGTCTTACACCACCT, with NcoI restriction enzyme sites；

Downstream IFN-γ-R2：

ACCACCACCAGAACCACCACCACCTTTAGAAGCACGCTG is with flexible linker；

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

Upstream IFN-α-F2：

GGTGGTTCTGGTGGTGGTGGTTCTATGTGCTCTTACCTG, with flexible linker；

Downstream IFN-α-R2：CCCTCGAGTTCTTTACGACGCAG, with XhoI restriction enzyme sites；

B. the target gene of the IFN-γ and IFN-α, both gene orders are respectively such as SEQUENCE LISTING Shown in the ＞ of 400 ＜, 7 ＞ and SEQUENCE LISTING, 400 ＜ 8；

Respectively using the target gene of IFN-γ and IFN-α as template, and it is utilized respectively the upstream and downstream of IFN-γ and IFN-α and draws Thing enters performing PCR amplification, respectively obtains the target gene of IFN-γ and IFN-α after the optimization for connecting flexible linker.

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

C. IFN-γ gene is connected with IFN-α gene using flexible linker

The PCR reaction systems and reaction condition of connection be：In 25 μ L overall reaction system, connect flexible linker's The μ L of IFN-γ template DNA 1, connect the flexible linker μ L of 1 μ L, IIFN- γ sense primers of IFN-α template DNA 0.5, 0.5 μ L, Taq archaeal dna polymerase of IFN-α anti-sense primer 2.5 μ L, dNTP Mix is 9 μ L, plus RNase Free water is to 25 μ L；Connection PCR reaction conditions are：95 DEG C of pre-degeneration 4min, into circulation：94 DEG C of denaturation 45s；60 DEG C of annealing 45s, 72 DEG C of extension 1kb/ Min, totally 35 circulations；Last 72 DEG C of extensions 10min.

Present invention also offers the application of the Recombinant Swine long-acting interferon α, its long half time had up to more than 67 hours Broad-spectrum disease resistance toxic action and the immune response that pig itself can be improved.

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

Merged 1. being realized Porcine interferon-gamma and porcine interferon alpha gene by flexible linker, improve interferon and partly decline Phase, compared with plain interferon, improve more than 16 times；

2. by being optimized to Porcine interferon-gamma and porcine interferon alpha gene, improve interferon gamma and porcine interferon-α The expression quantity of fusion 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 produce inclusion body in protein expression, form soluble protein, it is to 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 up of Porcine interferon-gamma and porcine interferon alpha not only has interferon-' alpha ' Broad-spectrum disease resistance toxic action, while significantly improving the immune response of pig itself.

Brief description of the drawings

The result that Fig. 1 expands for the Porcine interferon-gamma gene in embodiment 1 with porcine interferon alpha gene RT-PCR；Swimming lane M： DNA Marker DL2000；Swimming lane 1：Porcine interferon alpha gene RT-PCR amplified productions；Swimming lane 2：Porcine interferon-gamma gene RT- Pcr amplification product；

The result that Fig. 2 expands for the PCR after the target gene connection of the pig IFN γ in embodiment 1 and pig IFN-α；Swimming Road M：DNA Marker DL2000；Swimming lane 1：Porcine interferon alpha gene and the gene ligation amplification product of porcine interleukin 2；

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

Fig. 4 be embodiment 1 in recombinant protein SDS-PAGE electrophoretic examinations results；Swimming lane M：Albumen Marker；Swimming lane 1：Empty bacterium is compareed；Swimming lane 2：Precipitated after bacterial cell disruption after recombinant bacterium induction；Swimming lane 3：After bacterial cell disruption after recombinant bacterium induction Supernatant；

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

Fig. 6 causes cell for the Recombinant Swine long-acting interferon α as made from the fusion protein in embodiment 1 in embodiment 5 to VSV The inhibitory action 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 processing hole；B3-12 handles hole for the Recombinant Swine long-acting interferon α of gradient dilution (from right to left)；

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

Embodiment

Embodiment 1

A kind of fusion protein being made up of Porcine interferon-gamma and porcine interferon alpha, its preparation method is as follows：

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

Design of primers：

Objective gene sequence design synthetic primer according to having been reported in Genebank is shown in Table 1, in the upper of Porcine interferon-gamma Trip primer and anti-sense primer in introduce EcoRI restriction enzyme sites and Linker sequences respectively, porcine interferon alpha sense primer and under Linker sequences and the restriction enzyme sites of Hind III are introduced respectively in trip primer.

The pcr amplification primer thing of table 1

RT-PCR obtains target gene：

RNA is extracted from pig liver tissue, the target gene of IFN-γ and IFN-α, both bases are obtained by reverse transcription Because sequence is respectively as shown in the ＞ of 400 ＜ of SEQUENCE LISTING 400 ＜, 5 ＞ and SEQUENCE LISTING 6；

RT-PCR reaction systems (25 μ L) are shown in Table 2

The RT-PCR reaction systems of table 2

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 circulation：95 DEG C of denaturation 45s；58 DEG C are moved back Fiery 45s, 72 DEG C of extension 1kb/min, totally 35 circulations；Last 72 DEG C of extensions 10min.

There is specific band through agarose gel electrophoresis in 530bp and 710bp or so in RT-PCR amplified productions, and its result is such as Shown in Fig. 1, illustrate to have obtained the Porcine interferon-gamma target gene for being connected to flexible linker and porcine interferon alpha purpose base Cause.

2. the connection of target gene

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

The PCR reaction systems of table 3

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

There is specific band through agarose gel electrophoresis in 1200bp or so in pcr amplification product, its result as shown in Fig. 2 The nucleotide sequence of obtained target gene is as shown in the ＞ of 400 ＜ of SEQUENCE LISTING 3.

3. expression vector establishment

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

The double digestion system of table 4

General buffer	2μL
		Restriction enzyme (a pair)	1μL+1μL
Carrier reclaims fragment	2uL
		RNase Free water	14μL

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

Table 5

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

Connection product is converted into e. coli bl21 (DE3) competent cell, competence is coated on the mould of benzyl containing ammonia The LB culture medium flat plate overnight incubations of element；The bacterium colony grown on LB flat boards is taken to identify target gene, positive colony bacteria plasmid through PCR Identified through EcoRI and the double digestions of Hind III, be accredited as positive and represent expression vector establishment success, obtain engineering bacteria pET-32a/ rIFNγ-IFNα；PCR is expanded and double digestion product single band occurs through agarose gel electrophoresis at 1200bp or so places, its As a result it is as 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 μ g/ml containing ampicillin 100 Bacterium 1h recoveries engineering bacteria activity, in LB culture mediums (the μ g/ml containing ampicillin 100) after amplification culture 4h, surveys OD values and reaches When 1.0；Add IPTG, 32 DEG C of induced expression (the μ g/ml of final concentration 100) 5h；Bacterium is collected, is examined through SDS-PAGE electrophoresis Survey, supernatant is deposited in the visible predominant expression band in 62.5KD or so places, its result after the bacterial cell disruption after recombinant bacterium induction 5h As shown in figure 4, it can be seen that recombinant bacterium induction 5h after bacterial cell disruption after supernatant be deposited in 62.5KD or so places can See predominant expression band, illustrate in precipitation and supernatant equal successful expression fusion protein.

Add mass volume ratio 1:Precipitation is resuspended in 1 PBS；- 20 DEG C precipitate 3 times in room temperature multigelation；4 DEG C of ultrasonic degradations Bacterial precipitation, worked 10s, is spaced 3S, and ultrasonic 6min, whole process is repeated 3~4 times；4 DEG C, 12000r/min centrifugation 15min, Supernatant, precipitation are taken respectively, obtain rough fusion protein.

5. fusion protein purification

5.1His affinity chromatography

After membrane filtration of the rough 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 With reference to albumen, until A280nm is stable, then with Elution buffer I (50mM trishydroxymethylaminomethanes, 20~500mM Imidazoles, PH8.0) elution, collect rIFN γ-IFN α protein peak.

5.2DEAE anion-exchange chromatography

(the 50mM trihydroxy methyls of Binding Buffer II are arrived into the albumen collected after His affinitive layer purifications displacement Aminomethane, PH6.5) in after, the DEAE anion exchange chromatography that loading has been balanced by using Binding Buffer II, then Crossed with Binding Buffer II after post to A280nm value stabilizations, 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 using Binding Buffer III after the sample concentration that ion-exchange chromatography is collected into (50mMNa2HPO4,0.15M NaCl, PH7.4) has balanced the molecular sieve chromatographies of Superdex 200, uses Binding Buffer III elution, collects rIFN γ-IFN α protein peak

5.4 sample identification：Determine rIFN γ-IFN α potency and specific activity, specific activity >=1.0 × 10⁶IU/mg albumen is conjunction Lattice；It is aseptic subpackaged, -80 DEG C of preservations.It can obtain the fusion protein being made up of Porcine interferon-gamma and porcine interferon alpha, its amino acid Sequence is as shown in the ＞ of 400 ＜ of SEQUENCE LISTING 1.

Embodiment 2

A kind of fusion protein being made up of Porcine interferon-gamma and porcine interferon alpha, other be the same as Examples 1 simply will be therein E. coli bl21 (DE3) competent cell is replaced for BL21 (DE3) competent cell with pGro7 plasmids.It is merged The SDS-PAGE electrophoresis results be the same as Example 1 of albumen is compareed, and 62.5KD or so places predominant expression band is thicker in supernatant, explanation Introduce after molecular chaperones pGro7, more preferably, obtained fusion protein amount is higher for expression of the destination protein in supernatant.Large intestine bar The albumen of bacterium expression is mostly present in inclusion body；By introducing molecular chaperones in expression bacterial strain, coordinate expression albumen is just Really fold, reach 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 up of Porcine interferon-gamma and porcine interferon alpha, its preparation method is as follows：

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

IFN-γ and IFN-α in embodiment 1 is optimized, artificial synthesized IFN-γ and IFN-α target gene, optimized Afterwards, both nucleotide sequences are respectively such as the ＞ institutes of 400 ＜ of SEQUENCE LISTING 400 ＜, 7 ＞ and SEQUENCE LISTING 8 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 optimal codon (optimal codons) utilized, what those were not frequently utilized that is referred to as rare or utilizes The low codon of rate (rare or low-usage codons).In fact, conventional do protein expression or every kind of biology of production (including Escherichia coli, yeast, mammalian cell, plant cell and insect cell) all shows codon profit to a certain degree Difference or preference.In Escherichia coli, yeast and drosophila to the expression efficiency of the gene containing optimal codon apparently higher than containing The expression efficiency of the gene of the codon of poor efficiency.Therefore, in heterologous expression system, the preferences of codon are largely On have impact on the expression of recombinant protein.Using preference codon (preferred codons) and avoid utilize rare codon Gene chemical synthesis is carried out, the redesign of this gene is codon optimization.Therefore, to the IFN-γ and IFN- of pig in the present embodiment α gene codons are optimized.

Interpretation of result after 1.2 codon optimizations

It is considered as the gene during usual codon adaptation indexI (CAI)=1.0 optimal efficient in the expression system Expression status, CAI values are lower to show that expression is lower in host.In gene G/C content most ideal distribution scope be 30~ 70%, the scope is exceeded in any region can influence to translate and transcriptional efficiency.Using software detection find porcine IFN γ and The codon of IFN-α original gene codon adaptation indexI (CAI) in Escherichia coli is respectively 0.24,0.22, GC percentages For 39.2%, 59.1%；And by obtaining recombination password in Escherichia coli after porcine IFN γ and IFN-α gene optimization Sub- adaptation index (CAI) is 1.0,1.0, GC percentages 45.4%, 55.6%.Low codon is significantly reduced by gene optimization Utilization rate, it is to avoid influence of the rare codon to protein expression, improve the G/C content of gene, improve transcription and translation effect Rate.

1.3 design of primers：

The pcr amplification primer thing of table 6

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

The PCR reaction systems of table 7

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 circulation：95 DEG C of denaturation 45s；60 DEG C of annealing 45s, 72 DEG C of extensions 1kb/min, totally 35 circulations；Last 72 DEG C of extensions 10min.

There is specific band through agarose gel electrophoresis in 530bp and 710bp or so in pcr amplification product, and explanation is prepared into To the porcine IFN γ and the target gene of pig IFN-α being connected to after flexible linker optimization.

2. the connection of target gene

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

The PCR reaction systems of table 8

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

There is specific band through agarose gel electrophoresis in 1200bp or so in pcr amplification product, illustrates successfully to obtain Target gene after IFN-γ and IFN-α connection.The nucleotide sequence of obtained target gene such as SEQUENCE LISTING Shown in the ＞ of 400 ＜ 3.

3. expression vector establishment

The PCR errorless after sequencing of the target gene after connection glue reclaim product is selected to be used with pET-32a plasmids NcoI, XhoI restriction enzyme carry out double digestion and recovery, and double digestion is done by 20 μ L systems in table 9：

The double digestion system of table 9

General buffer	2μL
		Restriction enzyme (a pair)	1μL+1μL
Carrier reclaims fragment	2ul
		RNase Free water	14μL

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

Table 10

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

Connection product is converted into e. coli bl21 (DE3) competent cell, competence is coated on the mould of benzyl containing ammonia The LB culture medium flat plate overnight incubations of element；The bacterium colony grown on LB flat boards is taken to identify target gene, positive colony bacteria plasmid through PCR Identified through NcoI, XhoI double digestion, be accredited as positive and represent expression vector establishment success, obtain engineering bacteria pET-32a/rIFN γ-IFNα；PCR is expanded and double digestion product single band occurs through agarose gel electrophoresis at 1200bp or so places, and explanation contains There is the expression vector establishment success of the target gene after IFN-γ and IFN-α connection.

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 μ g/ml containing ampicillin 100 Bacterium 1h recoveries engineering bacteria activity, in LB culture mediums (the μ g/ml containing ampicillin 100) after amplification culture 4h, surveys OD values and reaches When 1.0；Add IPTG, 32 DEG C of induced expression (the μ g/ml of final concentration 100) 5h；Bacterium is collected, is examined through SDS-PAGE electrophoresis Survey, supernatant is deposited in the visible predominant expression band in 62.5KD or so places after the bacterial cell disruption after recombinant bacterium induction 5h, illustrates Recombinant protein has been obtained in supernatant precipitation.

Add mass volume ratio 1:Precipitation is resuspended in 1 PBS；- 20 DEG C precipitate 3 times in room temperature multigelation；4 DEG C of ultrasonic degradations Bacterial precipitation, worked 10s, is spaced 3S, and ultrasonic 6min, whole process is repeated 3~4 times；4 DEG C, 12000r/min centrifugation 15min, Supernatant, precipitation are taken respectively, obtain rough fusion protein.

5. fusion protein purification

5.1 His affinity chromatographys

After membrane filtration of the rough 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 With reference to albumen, until A280nm is stable, 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

(the 50mM trihydroxy methyls of Binding Buffer II are arrived into the albumen collected after His affinitive layer purifications displacement Aminomethane, PH6.5) in after, the DEAE anion exchange chromatography that loading has been balanced by using Binding Buffer II, then Crossed with Binding Buffer II after post to A280nm value stabilizations, 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 using Binding Buffer III after the sample concentration that ion-exchange chromatography is collected into (50mMNa2HPO4,0.15M NaCl, PH7.4) has balanced the molecular sieve chromatographies of Superdex 200, uses Binding Buffer III elution, collects rIFN γ-IFN α protein peak.

5.4 sample identification

Determine rIFN γ-IFN α potency and specific activity, specific activity >=1 × 10⁶IU/mg albumen is qualified；It is aseptic subpackaged ,- 80 DEG C of preservations.It can obtain the fusion protein being made up of Porcine interferon-gamma and porcine interferon alpha, its amino acid sequence such as SEQUENCE Shown in the ＞ of 400 ＜ of LISTING 2.

Embodiment 4

A kind of fusion protein being made up of Porcine interferon-gamma and porcine interferon alpha, other be the same as Examples 3 simply will be therein E. coli bl21 (DE3) competent cell is replaced for BL21 (DE3) competent cell with pGro7 plasmids.It is merged The SDS-PAGE electrophoresis results be the same as Example 3 of albumen is compareed, and 62.5KD or so places predominant expression band is thicker in supernatant, explanation Introduce after molecular chaperones pGro7, more preferably, obtained fusion protein amount is higher for expression of the destination protein in supernatant.Large intestine bar The albumen of bacterium expression is mostly present in inclusion body；By introducing molecular chaperones in expression bacterial strain, coordinate expression albumen is just Really fold, reach 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 Swine long-acting interferon α, by the fusion protein in embodiment 1,2,3,4 respectively with freeze drying protectant mixture Afterwards, it is 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

Embodiment 1~4 obtains the identification for the fusion protein being made up of Porcine interferon-gamma and porcine interferon alpha

The quantitative detection of 6.1 protein contents

Lowry methods are used, the standard protein for examining and determine institute with Chinese food pharmaceutical biological product makees standard test, determine 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 62.5KD or so, as shown in Figure 4.

6.3 Western Blot results

Fusion protein in embodiment 1~4 is detected respectively, with the anti-porcine alpha-IFN (1 of abcam companies mouse:5000 dilutions) it is one It is anti-, using goat anti-mouse IgG-HRP as secondary antibody (1:10000 dilutions).Recombinant Swine long-acting interferon α samples can be with anti-pig interferon Specific reaction occurs for alpha monoclonal antibodies, and specific band occurs in 62.5KD or so place, as shown in Figure 5.

Embodiment 7

Bioactivity freeze-dried four parts of Recombinant Swine long-acting interferon α in embodiment 5

Suppress 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%CO₂24h is cultivated, the Recombinant Swine for adding various dose is long Imitate to inhale after interferon-' alpha ', 24h and abandon, then inoculation 100TCID50 VSV viruses respectively.

Result of the test

As a result show that the Recombinant Swine long-acting interferon α obtained causes the lesion of HEp-2 cells to have obvious suppression to VSV Effect.The lesion such as occurs cell rounding after undressed cell virus inoculation, comes off, is disintegrated.And the Recombinant Swine obtained is long Imitate after the cell virus inoculation after interferon-' alpha ' processing, the Continuous Observation under inverted microscope, cellular morphology is normal, does not go out incumbent What lesion, measures potency >=1.0 × 10⁶IU/ml, as shown in Figure 6.

Embodiment 8

The measure of half-life period of the Recombinant Swine long-acting interferon α in pig body

The four parts of Recombinant Swine long-acting interferon α obtained respectively by the fusion protein of embodiment 1~4 in embodiment 5 are freezed The measure of half-life period of the agent (being designated as A, B, C, D respectively) in chicken body

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

The piglet (male and female half and half) that six body weight are roughly the same is taken, 2mg/ml Recombinant Swine long-acting interferons are subcutaneously injected in neck The freeze-dried 2ml of α, respectively in 1h, 2h, 4h, 8h, 16h, 24h, 36h, 48h, 60h, 88h jugular vein blood collection, 4 DEG C of solidifications of blood sample, 3500rpm low-temperature centrifugations 10min separates serum, and each every pig blood sample of time point is to be measured in -20 DEG C of preservations.Using cytopathic effect inhibition Method determines the concentration of rIFN γ-IFN α in blood serum sample, is carried out curve fitting with DAS pharmacokinetics softwares and calculating parameter.A plan Close curve as shown in Figure 7；Parameter result of calculation is shown in Table 11.

Dominant dynamic parameters in serum after the Recombinant Swine long-acting interferon α intramuscular injection of table 11

As a result show that Recombinant Swine long-acting interferon α has longer half-life period.Half-life period can reach 67h or so after measured, compared with Plain interferon improves 16 times.

Embodiment 9

The freeze-dried measure influenceed on pig cell immune response of four parts of Recombinant Swine long-acting interferon α in embodiment 5

Take six roughly the same piglets of body weight to be divided into two groups, be designated as experimental group and control group；Experimental group neck is subcutaneously noted The 2mg/ml Recombinant Swine long-acting interferon freeze-dried 2ml of α are penetrated, 2mL PBS is subcutaneously injected in control group neck, taken after injecting 4 weeks outside pig All blood, takes weekly a blood afterwards, and lymphocyte is separated using lymphocyte separation medium, and lymphocyte passes through serum-free RPMI 1640 culture mediums are washed after 2 times, and it is 2 × 10 to be resuspended with complete medium, adjust cell concentration⁶Individual/ml, 24 porocyte culture plates are every Hole adds 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, testing result is as shown in table 12 by kit specification：

The ELISA of table 12 detects each group pig cell immune response level

As a result show after injection Recombinant Swine long-acting interferon α, can significantly improve cell factor IL-2 in pig peripheral blood, IL-4 content, enhances cellullar immunologic response level, significantly improves immunity level.

It is above-mentioned to Recombinant Swine 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 is carried out, is illustrative rather than limited, and several implementations can be included according to limited scope Example, therefore changing and modifications 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 Recombinant Swine long-acting interferon α and prepare fusion protein of this long-acting interferon and preparation method thereof

<130> 1

<160> 8

<170> PatentIn version 3.3

<210> 1

<211> 404

<212> PRT

<213>Recombinant Swine long-acting interferon alpha fusion protein 1

<400> 1

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Gln Arg Lys Ala Ile Ser 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 Thr Met Phe Gln

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

Arg Arg Lys Glu

<210> 2

<211> 402

<212> PRT

<213>Recombinant Swine long-acting interferon alpha fusion protein 2

<400> 2

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Gln Arg Lys Ala Ile Ser 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 Thr Met Phe Gln

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

Lys Glu

<210> 3

<211> 1212

<212> DNA

<213>Recombinant Swine long-acting interferon α genomes 1

<400> 3

atgagttata caacttattt cttagctttt cagctttgcg tgactttgtg tttttctggc 60

tcttactgcc aggcgccctt ttttaaagaa ataacgatcc taaaggacta ttttaatgca 120

agtacctcag gtgtacctaa tggtggacct cttttcttag aaattttgga gaattggaaa 180

gaggagagtg acaaaaaaat aattcagagc caaattgtct ccttctactt caaattcttt 240

gaaatcttca aagataacca ggccattcaa aggagcatgg atgtgatcaa gcaagacatg 300

tttcagaggt tcctaaatgg tagctctggg aaactgaatg acttcgaaaa gctggttaaa 360

attccggtag ataatctgca gatccagcgc aaagccatca gtgaactcat caaagtgatg 420

aatgatctgt caccaagatc taacctaaga aagcggaaga gaagtcagac tatgttccaa 480

ggccagagag catcaaaaag atccacctcc agaaccacct ccagaacctc caccatgtgt 540

tcctatttaa gacacaggcc tgagggaagg tcttcaaaca tcctagagag cagggtcaca 600

gagtcaccca cctcagccag gacagcagca tctgcaaggt ccccaatggc cccaacctca 660

gccttcctca cggccctggt gctgctcagc tgcaatgcca tctgctctct gggctgcgac 720

ctgcctcaga cccacagcct ggctcacacc agggccctga ggctcctggc acaaatgagg 780

agaatctccc ccttctcctg cctggaccac agaagggact ttggattccc ccaagaggcc 840

ttggggggca accaggtcca gaaggctcaa gccatggctc tggtgcatga gatgctccag 900

cagaccttcc agctcttcag cacagagggc tcggctgctg cctgggatga gagcctcctg 960

caccagttct gcactggact ggatcagcag ctcagggacc tggaagcctg tgtcatgcag 1020

gaggccgggc tggaagggac ccccctgctg gaggaggact ccatcctggc tgtgaggaaa 1080

tacttccaca gactcaccct ctatctgcaa gagaagagct acagcccctg tgcctgggag 1140

atcgtcaggg cagaagtcat gagagccttc tcttcctcca caaacctgca agacagactc 1200

aggaggaagg ag 1212

<210> 4

<211> 1206

<212> DNA

<213>Recombinant Swine long-acting interferon α genomes 2

<400> 4

atgtcttaca ccacctactt cctggctttc cagctgtgcg ttaccctgtg cttctctggt 60

tcttactgcc aggctccgtt cttcaaagaa atcaccatcc tgaaagacta cttcaacgct 120

tctacctctg gtgttccgaa cggtggtccg ctgttcctgg aaatcctgga aaactggaaa 180

gaagaatctg acaaaaaaat catccagtct cagatcgttt ctttctactt caaattcttc 240

gaaatcttca aagacaacca ggctatccag cgttctatgg acgttatcaa acaggacatg 300

ttccagcgtt tcctgaacgg ttcttctggt aaactgaacg acttcgaaaa actggttaaa 360

atcccggttg acaacctgca gatccagcgt aaagctatct ctgaactgat caaagttatg 420

aacgacctgt ctccgcgttc taacctgcgt aaacgtaaac gttctcagac catgttccag 480

ggtcagcgtg cttctaaagg tggtggtggt tctggtggtg gtggttctat gtgctcttac 540

ctgcgtcacc gtccggaagg tcgttcttct aacatcctgg aatctcgtgt taccgaatct 600

ccgacctctg ctcgtaccgc tgcttctgct cgttctccga tggctccgac ctctgctttc 660

ctgaccgctc tggttctgct gtcttgcaac gctatctgct ctctgggttg cgacctgccg 720

cagacccact ctctggctca cacccgtgct ctgcgtctgc tggctcagat gcgtcgtatc 780

tctccgttct cttgcctgga ccaccgtcgt gacttcggtt tcccgcagga agctctgggt 840

ggtaaccagg ttcagaaagc tcaggctatg gctctggttc acgaaatgct gcagcagacc 900

ttccagctgt tctctaccga aggttctgct gctgcttggg acgaatctct gctgcaccag 960

ttctgcaccg gtctggacca gcagctgcgt gacctggaag cttgcgttat gcaggaagct 1020

ggtctggaag gtaccccgct gctggaagaa gactctatcc tggctgttcg taaatacttc 1080

caccgtctga ccctgtacct gcaggaaaaa tcttactctc cgtgcgcttg ggaaatcgtt 1140

cgtgctgaag ttatgcgtgc tttctcttct tctaccaacc tgcaggaccg tctgcgtcgt 1200

aaagaa 1206

<210> 5

<211> 498

<212> DNA

<213>Porcine interferon-gamma

<400> 5

atgagttata caacttattt cttagctttt cagctttgcg tgactttgtg tttttctggc 60

tcttactgcc aggcgccctt ttttaaagaa ataacgatcc taaaggacta ttttaatgca 120

agtacctcag gtgtacctaa tggtggacct cttttcttag aaattttgga gaattggaaa 180

gaggagagtg acaaaaaaat aattcagagc caaattgtct ccttctactt caaattcttt 240

gaaatcttca aagataacca ggccattcaa aggagcatgg atgtgatcaa gcaagacatg 300

tttcagaggt tcctaaatgg tagctctggg aaactgaatg acttcgaaaa gctggttaaa 360

attccggtag ataatctgca gatccagcgc aaagccatca gtgaactcat caaagtgatg 420

aatgatctgt caccaagatc taacctaaga aagcggaaga gaagtcagac tatgttccaa 480

ggccagagag catcaaaa 498

<210> 6

<211> 678

<212> DNA

<213>Porcine interferon alpha

<400> 6

atgtgttcct atttaagaca caggcctgag ggaaggtctt caaacatcct agagagcagg 60

gtcacagagt cacccacctc agccaggaca gcagcatctg caaggtcccc aatggcccca 120

acctcagcct tcctcacggc cctggtgctg ctcagctgca atgccatctg ctctctgggc 180

tgcgacctgc ctcagaccca cagcctggct cacaccaggg ccctgaggct cctggcacaa 240

atgaggagaa tctccccctt ctcctgcctg gaccacagaa gggactttgg attcccccaa 300

gaggccttgg ggggcaacca ggtccagaag gctcaagcca tggctctggt gcatgagatg 360

ctccagcaga ccttccagct cttcagcaca gagggctcgg ctgctgcctg ggatgagagc 420

ctcctgcacc agttctgcac tggactggat cagcagctca gggacctgga agcctgtgtc 480

atgcaggagg ccgggctgga agggaccccc ctgctggagg aggactccat cctggctgtg 540

aggaaatact tccacagact caccctctat ctgcaagaga agagctacag cccctgtgcc 600

tgggagatcg tcagggcaga agtcatgaga gccttctctt cctccacaaa cctgcaagac 660

agactcagga ggaaggag 678

<210> 7

<211> 498

<212> DNA

<213>Porcine interferon-gamma

<400> 7

atgtcttaca ccacctactt cctggctttc cagctgtgcg ttaccctgtg cttctctggt 60

tcttactgcc aggctccgtt cttcaaagaa atcaccatcc tgaaagacta cttcaacgct 120

tctacctctg gtgttccgaa cggtggtccg ctgttcctgg aaatcctgga aaactggaaa 180

gaagaatctg acaaaaaaat catccagtct cagatcgttt ctttctactt caaattcttc 240

gaaatcttca aagacaacca ggctatccag cgttctatgg acgttatcaa acaggacatg 300

ttccagcgtt tcctgaacgg ttcttctggt aaactgaacg acttcgaaaa actggttaaa 360

atcccggttg acaacctgca gatccagcgt aaagctatct ctgaactgat caaagttatg 420

aacgacctgt ctccgcgttc taacctgcgt aaacgtaaac gttctcagac catgttccag 480

ggtcagcgtg cttctaaa 498

<210> 8

<211> 678

<212> DNA

<213>Porcine interferon alpha

<400> 8

atgtgctctt acctgcgtca ccgtccggaa ggtcgttctt ctaacatcct ggaatctcgt 60

gttaccgaat ctccgacctc tgctcgtacc gctgcttctg ctcgttctcc gatggctccg 120

acctctgctt tcctgaccgc tctggttctg ctgtcttgca acgctatctg ctctctgggt 180

tgcgacctgc cgcagaccca ctctctggct cacacccgtg ctctgcgtct gctggctcag 240

atgcgtcgta tctctccgtt ctcttgcctg gaccaccgtc gtgacttcgg tttcccgcag 300

gaagctctgg gtggtaacca ggttcagaaa gctcaggcta tggctctggt tcacgaaatg 360

ctgcagcaga ccttccagct gttctctacc gaaggttctg ctgctgcttg ggacgaatct 420

ctgctgcacc agttctgcac cggtctggac cagcagctgc gtgacctgga agcttgcgtt 480

atgcaggaag ctggtctgga aggtaccccg ctgctggaag aagactctat cctggctgtt 540

cgtaaatact tccaccgtct gaccctgtac ctgcaggaaa aatcttactc tccgtgcgct 600

tgggaaatcg ttcgtgctga agttatgcgt gctttctctt cttctaccaa cctgcaggac 660

cgtctgcgtc gtaaagaa 678

Claims (10)

1. a kind of fusion protein being made up of Porcine interferon-gamma and porcine interferon alpha, it is characterised in that：The amino of the fusion protein Acid sequence table is designated as fusion protein 1 as shown in the ＞ of 400 ＜ of SEQUENCE LISTING 1；Or such as SEQUENCE LISTING 400 Shown in the ＞ of ＜ 2, fusion protein 2 is designated as.

2. a kind of gene for encoding fusion protein as claimed in claim 1, it is characterised in that the nucleotide sequence of the gene Table is designated as genome 1 as shown in the ＞ of 400 ＜ of SEQUENCE LISTING 3；Or as shown in the ＞ of 400 ＜ of SEQUENCE LISTING 4, It is designated 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 Swine long-acting interferon α, it is characterised in that the Recombinant Swine long-acting interferon α is as 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, it is characterised 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 is obtained Bacterium obtains the crude product of the fusion protein after IPTG induced expressions, purified to can obtain fusion protein afterwards.

7. preparation method according to claim 6, it is characterised in that the genetic engineering bacterium be pET-32a/rIFN γ- IFN α, its preparation method is：

(1) design primer, obtained by reverse transcription or the flexible linker sequences of artificial synthesized connection Porcine interferon-gamma and The target gene of porcine interferon alpha；The target gene of Porcine interferon-gamma and porcine interferon alpha is connected by flexible linker, The nucleotides sequence list of target gene is as shown in the ＞ of 400 ＜ of SEQUENCE LISTING 3 or such as SEQUENCE LISTING 400 Shown in the ＞ of ＜ 4；

(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 according to claim 6 or 7, it is characterised in that the e. coli host cell is BL21 (DE3) competent cell or BL21 (DE3) competent cell with pGro7 plasmids.

9. the preparation method according to claim 6 or 7, it is characterised in that the method for the purifying is：Fusion protein it is thick Purified after product elder generation through affinity chromatography, anion-exchange chromatography and sieve chromatography.

10. Recombinant Swine long-acting interferon α according to claim 5 application, it is characterised in that the Recombinant Swine is long-acting dry Plain α long half time is disturbed up to more than 67 hours, with broad-spectrum disease resistance toxic action and the immune response of pig itself can be improved.