CN107266586A

CN107266586A - A kind of Recombinant Swine long-acting interferon γ and prepare this long-acting interferon γ fusion protein and preparation method thereof

Info

Publication number: CN107266586A
Application number: CN201710675967.3A
Authority: CN
Inventors: 赵俊; 韩国祥; 赵雨; 高耀辉; 戚仕梅; 马腾飞; 周炜; 程正阳
Original assignee: ANHUI JIUCHUAN BIOTECHNOLOGY Co Ltd
Current assignee: ANHUI JIUCHUAN BIOTECHNOLOGY Co Ltd
Priority date: 2017-08-09
Filing date: 2017-08-09
Publication date: 2017-10-20
Also published as: CN108794640A

Abstract

The invention discloses a kind of Recombinant Swine long-acting interferon γ and the fusion protein for preparing this long-acting interferon γ and preparation method thereof; the fusion protein is connected by pig interleukin 2 and 6 with Porcine interferon-gamma and formed through flexible linker, through being freeze-dried to obtain Recombinant Swine long-acting interferon γ after fusion protein and freeze drying protectant mixture.The Recombinant Swine long-acting interferon γ is remarkably improved the half-life period of pig interferon, and the half-life period of more common Porcine interferon-gamma improves more than 13 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 γ and the fusion protein for preparing this long-acting interferon γ And preparation method thereof

Technical field

The invention belongs to technical field of biological genetic engineering, and in particular to a kind of Recombinant Swine long-acting interferon γ and prepare this Long-acting interferon γ fusion protein 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 γ, β, γ tri-.It is existing, it is known that γ types IFN be T cell by activating and NK cells are produced, with relatively strong antiviral and immunoloregulation function.Numerous studies show that interferon gamma is except with broad-spectrum disease resistance Outside malicious function, the adjustment effect of key is also played to immune system, so IFN-γ is also known as immunological regulation interferon.Although each The interferon of type can mediated cell to virus infection reaction, but interferon gamma immunoregulatory activity coordinate exempt from Epidemic disease is reacted and determines to play even more important effect in the long-term antiviral state of body, thus interferon gamma have it is particularly important Clinical value.

Cell factor IL-2 is interleukin 2, also known as SCIF.The main T lymphocytes by activating are produced The cell factor with extensive bioactivity, can both promote lymphopoiesis, strengthen immunologic function, but can restricted T it is thin Born of the same parents react and strengthen the immune tolerance of body, therefore available for treatment tumour, infectious diseases and autoimmune disease.In animal doctor In, because IL-2 can improve the immune response of vaccine and reduce the generation of disease, also there is wide application prospect.IL-2 is because of energy Strengthen the immune level of body, improve the resistance against diseases of body, thus exempt from for bacillary, viral and parasitic diseases Epidemic disease is treated.In addition, IL-2 can also influence the metabolism of medicine, make the metabolism time lengthening of medicine, action time increase, so as to improve Curative effect of medication.IL-2, according to gene constructed, constitutes fusion protein with other cell factors, is produced and is carried with the antibody for strengthening vaccine High cellular immune level.

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.

The content of the invention

In order to solve the above technical problems, the invention provides a kind of Recombinant Swine long-acting interferon γ and preparing this long-acting interference Plain γ fusion protein and preparation method thereof, the Recombinant Swine long-acting interferon γ is remarkably improved the half-life period of pig interferon, The half-life period of more common Porcine interferon-gamma improves more than 13 times, and with broad-spectrum disease resistance toxic action and can improve the immune of pig itself Response.

The technical scheme that the present invention takes is：

A kind of fusion protein being made up of pig interleukin 2 and 6 and Porcine interferon-gamma, the amino acid sequence of the fusion protein List is designated as fusion protein 1 as shown in the ＞ of 400 ＜ of SEQUENCE LISTING 1；Or such as the ＞ institutes of 400 ＜ of SEQUENCE LISTING 2 Show, be 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 pig IL-2 and IFN-γ original gene codon adaptation indexI (CAI) in Escherichia coli is found using software detection Respectively 0.23,0.24, GC percentages are 38.7%, 39.2%；And by being obtained after pig IL-2 and IFN-γ gene optimization Recombination genome 2 in Escherichia coli codon adaptation indexI (CAI) be 0.97,1.0, GC percentages 47.6%, 45.4%.The utilization rate of low codon is significantly reduced by gene optimization, it is to avoid shadow of the rare codon to protein expression Ring, improve the G/C content of gene, improve transcription and translation efficiency.

Present invention also offers 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/rIL2-IFN γ.

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 γ, the Recombinant Swine long-acting interferon γ is melted by described It is freeze-dried to form after hop protein and freeze drying protectant mixture.

The freeze drying protectant is 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/rIL2-IFN γ, and its preparation method is：

(1) primer is designed, is obtained by reverse transcription or the artificial synthesized pig leucocyte for connecting flexible linker sequences The target gene of interleukin 2 and Porcine interferon-gamma；By flexible linker by pig interleukin 2 and 6 and the purpose base of Porcine interferon-gamma Because connecting, the nucleotides sequence list of target gene is as shown in the ＞ of 400 ＜ of SEQUENCE LISTING 3 or such as SEQUENCE Shown in the ＞ of 400 ＜ of LISTING 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/rIL2- IFNγ。

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

The method of the purifying is：Through affinity chromatography, anion-exchange chromatography and molecular sieve after the crude product elder generation of fusion protein Chromatographic purifying.

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 pig interleukin 2 and 6 (IL-2) is：

Upstream IL-2-F1：ATAGAATTCATGTATAAGATGCAGCTCTTGC, with EcoRI restriction enzyme sites；

Downstream IL-2-R1：

CCAGAACCACCTCCAGAACCTCCACCAGTCAGTGTTGAGTAGATGCTT, with flexible linker；

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

Upstream IFN-γ-F1：

CTGGAGGTGGTTCTGGAGGTGGATCTATGAGTTATACAACTTATTTCTT, with flexible linker；

Downstream IFN-γ-R1：CCAAGCTTTTTTGATGCTCTCTGGCCTTG, with the restriction enzyme sites of Hind III；

B. RNA is extracted from pig liver, the target gene of IL-2 and IFN-γ, both gene sequences are obtained by reverse transcription Row are respectively as shown in the ＞ of 400 ＜ of SEQUENCE LISTING 400 ＜, 5 ＞ and SEQUENCE LISTING 6；

Respectively using the target gene of IL-2 and IFN-γ as template, and it is utilized respectively the upstream and downstream primer of IL-2 and IFN-γ Enter performing PCR amplification, respectively obtain the flexible linker of connection IL-2 and the target gene of IFN-γ.

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. IL-2 genes are connected with IFN-γ 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 IL-2 gene templates DNA 1, the flexible linker μ L of 1 μ L, IL-2 sense primer of IFN-γ template DNA 0.5 are connected, 0.5 μ L, Taq archaeal dna polymerase of IFN-γ anti-sense primer 2.5 μ L, dNTP Mix is, 9.0 μ L, plus RNase Free water is to 25 μ L； Connecting PCR reaction conditions 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.

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 pig interleukin 2 and 6 (IL-2) is：

Upstream IL-2-F2：CATGCCATGGTATGTACAAAATGCAGC, with NcoI restriction enzyme sites；

Downstream IL-2-R2：

ACCACCACCAGAACCACCACCACCGGTCAGGGTAGAGTAG, with flexible linker；

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

Upstream IFN-γ-F2：

GGTGGTTCTGGTGGTGGTGGTTCTATGTCTTACACCACCT, with flexible linker；

Downstream IFN-γ-R2：CCCTCGAGTTTAGAAGCACGCTG, with XhoI restriction enzyme sites；

B. the target gene of the IL-2 and IFN-γ, both gene orders are respectively such as SEQUENCE LISTING 400 Shown in the ＞ of 7 ＞ and SEQUENCE LISTING of ＜, 400 ＜ 8；

Respectively using the target gene of IL-2 and IFN-γ as template, and it is utilized respectively the upstream and downstream primer of IL-2 and IFN-γ Enter performing PCR amplification, respectively obtain the target gene of IL-2 and IFN-γ after the flexible linker of connection optimization.

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. IL-2 genes are connected with IFN-γ 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 IL-2 gene templates DNA 1, the flexible linker μ L of 1 μ L, IL-2 sense primer of IFN-γ template DNA 0.5 are connected, 0.5 μ L, Taq archaeal dna polymerase of IFN-γ anti-sense primer 2.5 μ L, dNTP Mix is 9.0 μ L, plus RNase Free water is to 25 μ L； Connecting PCR reaction conditions 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.

Present invention also offers the application of the Recombinant Swine long-acting interferon γ, its long half time was up to more than 55 hours, tool There is broad-spectrum disease resistance toxic action and the immune response of pig itself can be improved.

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

Merged 1. being realized pig IL-2 and Porcine interferon-gamma gene by flexible linker, improve interferon half-life period, Compared with plain interferon, more than 13 times are improved；

2. by being optimized to pig IL-2 and Porcine interferon-gamma gene, improve IL-2 and Porcine interferon-gamma fusion protein Expression quantity.

3. using recombination bacillus coli BL21/pET-32a-IL2-IFN γ 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 pig IL-2 and Porcine interferon-gamma is not only wide with interferon gamma Antivirus action is composed, while significantly improving the immune response of pig itself.

Brief description of the drawings

The result that Fig. 1 expands for the pig IL-2 genes in embodiment 1 with Porcine interferon-gamma gene RT-PCR；Swimming lane M：DNA Marker DL2000；Swimming lane 1：Porcine interferon-gamma gene RT-PCR amplified productions；Swimming lane 2：Pig IL2 genes RT-PCR amplification productions Thing；

The result that Fig. 2 expands for the PCR after the pig IFN γ in embodiment 1 and pig IL-2 target gene connection；Swimming lane M：DNA Marker DL2000；Swimming lane 1：Porcine interferon-gamma 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：Recombinant plasmid double digestion result；Swimming lane 2：Plasmid PCR result；

Fig. 4 be embodiment 1 in recombinant protein SDS-PAGE electrophoretic examinations results；Swimming lane M：Albumen Marker；Swimming lane 1：Supernatant after bacterial cell disruption after recombinant bacterium induction；Swimming lane 2：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 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 thin for the Recombinant Swine long-acting interferon γ as made from the fusion protein in embodiment 1 in embodiment 5 to VSV The inhibitory action of born of the same parents' lesion；1 is VSV virus control wells；2 be HEp-2 cell control wells；A3-12 is gradient dilution (from dextrad It is left) human interferon standard items processing hole；B3-12 is the Recombinant Swine long-acting interferon γ processing of gradient dilution (from right to left) Hole；

Fig. 7 is the Recombinant Swine long-acting interferon γ 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 pig interleukin 2 and 6 and Porcine interferon-gamma, its preparation method is as follows：

1. the acquisition of pig interleukin 2 and 6 (IL-2) and Porcine interferon-gamma (IFN-γ) target gene is set with amplimer Meter：

Objective gene sequence design synthetic primer according to having been reported in Genebank is shown in Table 1, in the upstream of porcine interleukin 2 EcoRI restriction enzyme sites and Linker sequences are introduced in primer and anti-sense primer respectively, Porcine interferon-gamma sense primer and under Linker sequences and the restriction enzyme sites of Hind III are introduced respectively in trip primer.

Table 1PCR amplimers

RT-PCR obtains target gene：

RNA is extracted from pig liver tissue, the target gene of IL-2 and IFN-γ, 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；

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

Table 2RT-PCR reaction systems

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 490bp and 530bp or so in RT-PCR amplified productions, and its result is such as Shown in Fig. 1.

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：

Table 3PCR reaction systems

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 990bp or so in pcr amplification product, and its result is as shown in Fig. 2 figure The band of the amplified production of porcine interleukin 2 and Porcine interferon-gamma amplified production is occurred in that in 2, because in the gene of porcine interleukin 2 During the connection of Porcine interferon-gamma gene PCR, non-specific responding is occurred in that.The nucleotide sequence of obtained target gene is such as Shown in the ＞ of 400 ＜ of SEQUENCE LISTING 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 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

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, obtained engineering bacteria pET- 32a/rIL2-IFNγ；PCR is expanded and double digestion product single band occurs through agarose gel electrophoresis at 990bp or so places, Its result is as shown in Figure 3.

4. the expression of recombinant protein

Picking engineering bacteria pET-32a/rIL2-IFN γ shake 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, IPTG final concentration of 100 μ g/mL, 32 DEG C of induced expression 5h；Bacterium is collected, through SDS-PAGE Electrophoresis detection, its result are as shown in figure 4, it can be seen that recombinant bacterium induces supernatant after the bacterial cell disruption after 5h to precipitate In the visible predominant expression band in 54.6KD or so places, 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 rIL2-IFN γ protein peaks.

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, Crossed again with Binding Buffer II after post to A280nm value stabilizations, with (the 50mM trihydroxy methyl ammonia of Elution Buffer II Methylmethane, 1M NaCl, PH6.5) linear gradient elution, collect rIL2-IFN γ protein peaks.

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 rIL2-IFN γ protein peaks.

5.4 sample identification

Determine rIL2-IFN γ potency and specific activity, specific activity >=1.0 × 10⁶IU/mg albumen is qualified；It is aseptic subpackaged ,- 80 DEG C of preservations.The fusion protein being made up of pig interleukin 2 and 6 and Porcine interferon-gamma is can obtain, its amino acid sequence is such as Shown in the ＞ of 400 ＜ of SEQUENCE LISTING 1.

Embodiment 2

A kind of fusion protein being made up of pig interleukin 2 and 6 and Porcine interferon-gamma, other be the same as Examples 1, simply by it In e. coli bl21 (DE3) competent cell replace for BL21 (DE3) competent cell with pGro7 plasmids.Its The SDS-PAGE electrophoresis results be the same as Example 1 of fusion protein is compareed, and 54.6KD or so places predominant expression band is thicker in supernatant, Illustrate to introduce after molecular chaperones pGro7, more preferably, obtained fusion protein amount is higher for expression of the destination protein in supernatant.Greatly The albumen of enterobacteria expression is mostly present in inclusion body；By introducing molecular chaperones, coordinate expression egg in expression bacterial strain White correct folding, 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

1. the acquisition and amplification of pig interleukin 2 and 6 (IL-2) and Porcine interferon-gamma (IFN-γ) target gene

IL-2 and IFN-γ in embodiment 1 is optimized, artificial synthesized IL-2 and IFN-γ 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 IL-2 and IFN-γ of pig in the present embodiment Gene codon is 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 pig IL-2 and The codon of IFN-γ original gene codon adaptation indexI (CAI) in Escherichia coli is respectively 0.23,0.24, GC percentages For 38.7%, 39.2%；And by obtaining recombination password in Escherichia coli after pig IL-2 and IFN-γ gene optimization Sub- adaptation index (CAI) is 0.97,1.0, GC percentages 47.6%, 45.4%.Low password is significantly reduced by gene optimization The utilization rate of son, it is to avoid influence of the rare codon to protein expression, improves the G/C content of gene, improves transcription and translation Efficiency.

1.3 design of primers：

Table 6PCR amplimers

IL-2 and the genomic DNA of IFN-γ 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：

Table 7PCR reaction systems

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：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.

The pcr amplification product of IL-2 and IFN-γ occurs special in 490bp and 530bp or so respectively through agarose gel electrophoresis Different band, illustrates to have prepared the target gene of the IL-2 and IFN-γ after the flexible linker of connection optimization.

2. the connection of target gene

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

Table 8PCR reaction systems

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 990bp or so in pcr amplification product, illustrates successfully to have obtained IL-2 Target gene after being connected with IFN-γ, the nucleotide sequence such as ＞ of 400 ＜ of SEQUENCE LISTING 4 of obtained target gene It is shown.

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	2μL
		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

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, PCR amplifications and double digestion product warp There is single band at 990bp or so places in agarose gel electrophoresis, illustrates containing the target gene after IL-2 and IFN-γ connection Expression vector establishment success, obtained engineering bacteria pET-32a/rIL2-IFN γ.

4. the expression of recombinant protein

Picking engineering bacteria pET-32a/rIL2-IFN γ shake 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, survey OD values and reach When 1.0；Add IPTG, IPTG final concentration of 100 μ g/mL, 32 DEG C of induced expression 5h；Bacterium is collected, through SDS-PAGE Supernatant is deposited in the visible predominant expression band in 54.6KD or so places after bacterial cell disruption after electrophoresis detection, recombinant bacterium induction 5h, Illustrate to have obtained recombinant protein in supernatant is precipitated.

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 ultrasounds are split Bacterial precipitation is solved, work 10s, be spaced 3S, ultrasonic 6min, whole process is repeated 3~4 times；4 DEG C, 12000r/min centrifugations 15min, takes supernatant, precipitation, obtains rough fusion protein respectively.

5. fusion protein purification

5.1His affinity chromatography

5.2DEAE anion-exchange chromatography

5.3 sieve chromatography

5.4 sample identification

Determine rIL2-IFN γ potency and specific activity, specific activity >=1.0 × 10⁶IU/mg albumen is qualified；It is aseptic subpackaged ,- 80 DEG C of preservations.The fusion protein being made up of pig interleukin 2 and 6 and Porcine interferon-gamma is can obtain, its amino acid sequence is such as Shown in the ＞ of 400 ＜ of SEQUENCE LISTING 2.

Embodiment 4

A kind of fusion protein being made up of pig interleukin 2 and 6 and Porcine interferon-gamma, other be the same as Examples 3, simply by it In e. coli bl21 (DE3) competent cell replace for BL21 (DE3) competent cell with pGro7 plasmids.Its The SDS-PAGE electrophoresis results be the same as Example 3 of fusion protein is compareed, and 54.6KD or so places predominant expression band is thicker in supernatant, Illustrate to introduce after molecular chaperones pGro7, more preferably, obtained fusion protein amount is higher for expression of the destination protein in supernatant.Greatly The albumen of enterobacteria expression is mostly present in inclusion body；By introducing molecular chaperones, coordinate expression egg in expression bacterial strain White correct folding, reaches solubility expression of protein.Described BL21 (DE3) competent cell with pGro7 plasmids is purchased from Shanghai Offshore Science and Technology Ltd./glad hundred promise is biological, article No. V205.

Embodiment 5

A kind of Recombinant Swine long-acting interferon γ, is mixed with freeze drying protectant respectively by the fusion protein in embodiment 1,2,3,4 It is freeze-dried to form with after.The freeze drying protectant is glycerine, mannitol and sucrose, is buffering with 10mmol/L PBS Liquid, 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 pig interleukin 2 and 6 and Porcine interferon-gamma

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.

6.2SDS-PAGE electrophoresis detection

Compared with empty bacterium, fusion protein has the newly-increased protein band of a dense dye in 54.6KD or so, as Fig. 4, shown in.

6.3Western Blot results

Fusion protein in embodiment 1~4 is detected respectively, with the anti-pig gamma interferon (1 of abcam companies mouse:5000 dilutions) be Primary antibody, using goat anti-mouse IgG-HRP as secondary antibody (1:10000 dilutions).Recombinant Swine long-acting interferon γ samples can be dry with anti-pig Disturb plain γ monoclonal antibodies and occur specific reaction, specific band occur in 54.6KD or so places, as shown in Figure 5.

Embodiment 7

Bioactivity freeze-dried four parts of Recombinant Swine long-acting interferon γ 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%CO2 culture 24h, the Recombinant Swine for adding various dose are long Imitate to inhale after interferon gamma, 24h and abandon, then be inoculated with 100TCID respectively₅₀VSV viruses.

Result of the test

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

Embodiment 8

The four parts of Recombinant Swine long-acting interferon γ 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 pig body

Cytopathic-effect inhibition assay determines rIL2-IFN γ blood concentration and time relationship

The piglet (male and female half and half) that six body weight are roughly the same is taken, the long-acting Porcine interferon-gamma of intramuscular injection 2mg/ml pigs is freezed Agent 2ml, respectively in 1h, 2h, 4h, 8h, 16h, 24h, 36h, 48h, 60h, 88h venous blood collection, 4 DEG C of solidifications of blood sample, 3500rpm is low Temperature centrifugation 10min separation serum, each every pig blood sample of time point is to be measured in -20 DEG C of preservations.Blood is determined using cytopathic-effect inhibition assay RIL2-IFN γ concentration in final proof product, is carried out curve fitting and calculating parameter with DAS pharmacokinetics softwares.Matched curve such as Fig. 7 It is shown；Parameter result of calculation is shown in Table 11.

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

As a result show that Recombinant Swine long-acting interferon γ has longer half-life period.Half-life period can reach 55h or so after measured, compared with Plain interferon improves about 13 times.

Embodiment 9

The freeze-dried measure influenceed on pig cell immune response of four parts of Recombinant Swine long-acting interferon γ 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 γ are penetrated, 2mL PBS is subcutaneously injected in control group neck, takes pig after injecting 4 weeks Peripheral 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-4 contents, are carried out, testing result is as shown in table 12 by kit specification：

Table 12ELISA detects each group pig cell immune response level

As a result show after injection Recombinant Swine long-acting interferon γ, cell factor IL-4 in pig peripheral blood can be significantly improved Content, enhances cellullar immunologic response level, significantly improves immunity level.

It is above-mentioned with reference to embodiment to Recombinant Swine long-acting interferon γ and prepare this long-acting interferon γ fusion protein and its The detailed description that preparation method is carried out, is illustrative rather than limited, can include several according to limited scope Embodiment, 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>Anhui Jiuchuan Biotechnology Co., Ltd.

<120>A kind of Recombinant Swine long-acting interferon γ and prepare this long-acting interferon γ fusion protein and preparation method thereof

<130> 1

<160> 8

<170> PatentIn version 3.3

<210> 1

<211> 332

<212> PRT

<213>Recombinant Swine long-acting interferon γ fusion proteins 1

<400> 1

Met Tyr Lys Met Gln Leu Leu Cys Cys Ile Ala Leu Thr Leu Ala Leu

1 5 10 15

Met Ala Asn Gly Ala Pro Thr Ser Ser Ser Thr Lys Asn Thr Lys Lys

20 25 30

Gln Leu Glu Pro Leu Leu Leu Asp Leu Gln Leu Leu Leu Lys Glu Val

35 40 45

Lys Asn Tyr Glu Asn Ala Asp Leu Ser Arg Met Leu Thr Phe Lys Phe

50 55 60

Tyr Met Pro Lys Gln Ala Thr Glu Leu Lys His Leu Gln Cys Leu Val

65 70 75 80

Glu Glu Leu Lys Ala Leu Glu Gly Val Leu Asn Leu Gly Gln Ser Lys

85 90 95

Asn Ser Asp Ser Ala Asn Ile Lys Glu Ser Met Asn Asn Ile Asn Val

100 105 110

Thr Val Leu Glu Leu Lys Gly Ser Glu Thr Ser Phe Lys Cys Glu Tyr

115 120 125

Asp Asp Glu Thr Val Thr Ala Val Glu Phe Leu Asn Lys Trp Ile Thr

130 135 140

Phe Cys Gln Ser Ile Tyr Ser Thr Leu Thr Arg Ser Thr Ser Arg Thr

145 150 155 160

Thr Ser Arg Thr Ser Thr Met Ser Tyr Thr Thr Tyr Phe Leu Ala Phe

165 170 175

Gln Leu Cys Val Thr Leu Cys Phe Ser Gly Ser Tyr Cys Gln Ala Pro

180 185 190

Phe Phe Lys Glu Ile Thr Ile Leu Lys Asp Tyr Phe Asn Ala Ser Thr

195 200 205

Ser Gly Val Pro Asn Gly Gly Pro Leu Phe Leu Glu Ile Leu Glu Asn

210 215 220

Trp Lys Glu Glu Ser Asp Lys Lys Ile Ile Gln Ser Gln Ile Val Ser

225 230 235 240

Phe Tyr Phe Lys Phe Phe Glu Ile Phe Lys Asp Asn Gln Ala Ile Gln

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

Ser Gln Thr Met Phe Gln Gly Gln Arg Ala Ser Lys

325 330

<210> 2

<211> 330

<212> PRT

<213>Recombinant Swine long-acting interferon γ fusion proteins 2

<400> 2

Met Tyr Lys Met Gln Leu Leu Cys Cys Ile Ala Leu Thr Leu Ala Leu

1 5 10 15

Met Ala Asn Gly Ala Pro Thr Ser Ser Ser Thr Lys Asn Thr Lys Lys

20 25 30

Gln Leu Glu Pro Leu Leu Leu Asp Leu Gln Leu Leu Leu Lys Glu Val

35 40 45

Lys Asn Tyr Glu Asn Ala Asp Leu Ser Arg Met Leu Thr Phe Lys Phe

50 55 60

Tyr Met Pro Lys Gln Ala Thr Glu Leu Lys His Leu Gln Cys Leu Val

65 70 75 80

Glu Glu Leu Lys Ala Leu Glu Gly Val Leu Asn Leu Gly Gln Ser Lys

85 90 95

Asn Ser Asp Ser Ala Asn Ile Lys Glu Ser Met Asn Asn Ile Asn Val

100 105 110

Thr Val Leu Glu Leu Lys Gly Ser Glu Thr Ser Phe Lys Cys Glu Tyr

115 120 125

Asp Asp Glu Thr Val Thr Ala Val Glu Phe Leu Asn Lys Trp Ile Thr

130 135 140

Phe Cys Gln Ser Ile Tyr Ser Thr Leu Thr Gly Gly Gly Gly Ser Gly

145 150 155 160

Gly Gly Gly Ser Met Ser Tyr Thr Thr Tyr Phe Leu Ala Phe Gln Leu

165 170 175

Cys Val Thr Leu Cys Phe Ser Gly Ser Tyr Cys Gln Ala Pro Phe Phe

180 185 190

Lys Glu Ile Thr Ile Leu Lys Asp Tyr Phe Asn Ala Ser Thr Ser Gly

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

Asn Asp Leu Ser Pro Arg Ser Asn Leu Arg Lys Arg Lys Arg Ser Gln

305 310 315 320

Thr Met Phe Gln Gly Gln Arg Ala Ser Lys

325 330

<210> 3

<211> 996

<212> DNA

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

<400> 3

atgtataaga tgcagctctt gtgttgcatt gcactaaccc ttgcactcat ggcaaacggt 60

gcacctactt caagctctac aaagaacaca aagaaacaac tggagccatt gctgctggat 120

ttacagttgc ttttgaagga agttaagaat tacgagaatg ctgatctctc caggatgctc 180

acatttaaat tttacatgcc caagcaggct acagaattga aacaccttca gtgtttagta 240

gaagaactca aagctctgga gggagtgcta aatttaggtc aaagcaaaaa ctctgactca 300

gcaaatatca aggaatcaat gaacaatatc aacgtaacag ttttggaact aaagggatct 360

gaaacaagtt tcaaatgtga atatgatgat gagacagtaa ctgctgttga atttctgaac 420

aaatggatta ccttttgtca aagcatctac tcaacactga ctagatccac ctccagaacc 480

acctccagaa cctccaccat gagttataca acttatttct tagcttttca gctttgcgtg 540

actttgtgtt tttctggctc ttactgccag gcgccctttt ttaaagaaat aacgatccta 600

aaggactatt ttaatgcaag tacctcaggt gtacctaatg gtggacctct tttcttagaa 660

attttggaga attggaaaga ggagagtgac aaaaaaataa ttcagagcca aattgtctcc 720

ttctacttca aattctttga aatcttcaaa gataaccagg ccattcaaag gagcatggat 780

gtgatcaagc aagacatgtt tcagaggttc ctaaatggta gctctgggaa actgaatgac 840

ttcgaaaagc tggttaaaat tccggtagat aatctgcaga tccagcgcaa agccatcagt 900

gaactcatca aagtgatgaa tgatctgtca ccaagatcta acctaagaaa gcggaagaga 960

agtcagacta tgttccaagg ccagagagca tcaaaa 996

<210> 4

<211> 990

<212> DNA

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

<400> 4

atgtacaaaa tgcagctgct gtgctgcatc gctctgaccc tggctctgat ggctaacggt 60

gctccgacct cttcttctac caaaaacacc aaaaaacagc tggaaccgct gctgctggac 120

ctgcagctgc tgctgaaaga agttaaaaac tacgaaaacg ctgacctgtc tcgtatgctg 180

accttcaaat tctacatgcc gaaacaggct accgaactga aacacctgca gtgcctggtt 240

gaagaactga aagctctgga aggtgttctg aacctgggtc agtctaaaaa ctctgactct 300

gctaacatca aagaatctat gaacaacatc aacgttaccg ttctggaact gaaaggctcg 360

gaaaccagct tcaaatgcga atacgacgac gaaaccgtta ccgctgttga attcctgaac 420

aaatggatca ccttctgcca gtctatctac tctaccctga ccggtggtgg tggttctggt 480

ggtggtggtt ctatgtctta caccacctac ttcctggctt tccagctgtg cgttaccctg 540

tgcttctctg gttcttactg ccaggctccg ttcttcaaag aaatcaccat cctgaaagac 600

tacttcaacg cttctacctc tggtgttccg aacggtggtc cgctgttcct ggaaatcctg 660

gaaaactgga aagaagaatc tgacaaaaaa atcatccagt ctcagatcgt ttctttctac 720

ttcaaattct tcgaaatctt caaagacaac caggctatcc agcgttctat ggacgttatc 780

aaacaggaca tgttccagcg tttcctgaac ggttcttctg gtaaactgaa cgacttcgaa 840

aaactggtta aaatcccggt tgacaacctg cagatccagc gtaaagctat ctctgaactg 900

atcaaagtta tgaacgacct gtctccgcgt tctaacctgc gtaaacgtaa acgttctcag 960

accatgttcc agggtcagcg tgcttctaaa 990

<210> 5

<211> 462

<212> DNA

<213>Pig IL-2

<400> 5

atgtataaga tgcagctctt gtgttgcatt gcactaaccc ttgcactcat ggcaaacggt 60

gcacctactt caagctctac aaagaacaca aagaaacaac tggagccatt gctgctggat 120

ttacagttgc ttttgaagga agttaagaat tacgagaatg ctgatctctc caggatgctc 180

acatttaaat tttacatgcc caagcaggct acagaattga aacaccttca gtgtttagta 240

gaagaactca aagctctgga gggagtgcta aatttaggtc aaagcaaaaa ctctgactca 300

gcaaatatca aggaatcaat gaacaatatc aacgtaacag ttttggaact aaagggatct 360

gaaacaagtt tcaaatgtga atatgatgat gagacagtaa ctgctgttga atttctgaac 420

aaatggatta ccttttgtca aagcatctac tcaacactga ct 462

<210> 6

<211> 498

<212> DNA

<213>Porcine IFN γ

<400> 6

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> 7

<211> 462

<212> DNA

<213>Pig IL-2

<400> 7

atgtacaaaa tgcagctgct gtgctgcatc gctctgaccc tggctctgat ggctaacggt 60

gctccgacct cttcttctac caaaaacacc aaaaaacagc tggaaccgct gctgctggac 120

ctgcagctgc tgctgaaaga agttaaaaac tacgaaaacg ctgacctgtc tcgtatgctg 180

accttcaaat tctacatgcc gaaacaggct accgaactga aacacctgca gtgcctggtt 240

gaagaactga aagctctgga aggtgttctg aacctgggtc agtctaaaaa ctctgactct 300

gctaacatca aagaatctat gaacaacatc aacgttaccg ttctggaact gaaaggctcg 360

gaaaccagct tcaaatgcga atacgacgac gaaaccgtta ccgctgttga attcctgaac 420

aaatggatca ccttctgcca gtctatctac tctaccctga cc 462

<210> 8

<211> 498

<212> DNA

<213>Porcine IFN γ

<400> 8

atgtcttaca ccacctactt cctggctttc cagctgtgcg ttaccctgtg cttctctggt 60

tcttactgcc aggctccgtt cttcaaagaa atcaccatcc tgaaagacta cttcaacgct 120

tctacctctg gtgttccgaa cggtggtccg ctgttcctgg aaatcctgga aaactggaaa 180

gaagaatctg acaaaaaaat catccagtct cagatcgttt ctttctactt caaattcttc 240

gaaatcttca aagacaacca ggctatccag cgttctatgg acgttatcaa acaggacatg 300

ttccagcgtt tcctgaacgg ttcttctggt aaactgaacg acttcgaaaa actggttaaa 360

atcccggttg acaacctgca gatccagcgt aaagctatct ctgaactgat caaagttatg 420

aacgacctgt ctccgcgttc taacctgcgt aaacgtaaac gttctcagac catgttccag 480

ggtcagcgtg cttctaaa 498

Claims

1. a kind of fusion protein being made up of pig interleukin 2 and 6 and Porcine interferon-gamma, it is characterised in that：The fusion protein Amino acid sequence table is designated as fusion protein 1 as shown in the ＞ of 400 ＜ of SEQUENCE LISTING 1；Or such as SEQUENCE LISTING Shown in the ＞ of 400 ＜ 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 γ, it is characterised in that the Recombinant Swine long-acting interferon γ is as described in claim 1 Fusion protein and freeze drying protectant mixture after, it is freeze-dried to form.

6. the preparation method of fusion protein according to claim 1, 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 is pET-32a/rIL2-IFN γ, its preparation method is：

(1) primer is designed, is obtained by reverse transcription or the artificial synthesized pig interleukin 2 and 6 for connecting flexible linker sequences With the target gene of Porcine interferon-gamma；The target gene of pig interleukin 2 and 6 and Porcine interferon-gamma is connected by flexible linker Pick up and, the nucleotides sequence list of target gene is as shown in the ＞ of 400 ＜ of SEQUENCE LISTING 3 or such as SEQUENCE Shown in the ＞ of 400 ＜ of LISTING 4；

(3) expression vector is imported into e. coli host cell, you can obtain genetic engineering bacterium pET-32a/rIL2-IFN γ。

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 γ according to claim 5 application, it is characterised in that the Recombinant Swine is long-acting The long half time of interferon gamma with broad-spectrum disease resistance toxic action and can improve the immune response of pig itself up to more than 55 hours.