CN107383203A

CN107383203A - A kind of fusion protein being made up of recombinant chIL-2, chicken interferon gamma and chicken interferon α and preparation method thereof

Info

Publication number: CN107383203A
Application number: CN201710676329.3A
Authority: CN
Inventors: 赵俊; 李树启; 高耀辉; 戚仕梅; 王利利; 赖鹏飞; 周炜; 何志远
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: 2017-11-24
Also published as: CN109053897A

Abstract

The invention discloses a kind of fusion protein being made up of recombinant chIL-2, chicken interferon gamma and chicken interferon α and preparation method thereof; the fusion protein is connected by recombinant chIL-2, chicken interferon gamma with chicken interferon α and formed through flexible linker, through being freeze-dried to obtain recombination chicken long-acting interferon after fusion protein and freeze drying protectant mixture.The recombination chicken long-acting interferon is remarkably improved the half-life period of chicken interferon, and the half-life period of more common chicken interferon improves more than 18 times, and with broad-spectrum disease resistance toxic action and can improve the immune response of chicken itself.

Description

A kind of fusion being made up of recombinant chIL-2, chicken interferon gamma and chicken interferon α Albumen and preparation method thereof

Technical field

The invention belongs to technical field of biological genetic engineering, and in particular to by recombinant chIL-2, chicken interferon gamma and chicken Fusion protein of interferon-' alpha ' composition and preparation method thereof.

Background technology

In recent years, as the scale of aquaculture and livestock and poultry and products thereof circulation industry rapidly develop, China's domestic fowl farming takes Tremendous development is obtained, forms the huge industry that annual value of production exceedes hundred billion yuan.Yet with the animal epidemic prevention system that China is weak, poultry diease It is still the serious problems that China's poultry production faces, this has become an important factor for restricting the development of China's poultry cultivation industry. Poultry diease is more, loss is big, and the death rate is higher than 15%, and death and culling rate is every up to 20%~25% (developed country is less than 5%), China's poultry husbandry Chicken The dead quantity year caused by infectious disease is about 300,000,000, about 3,000,000,000 yuan of the economic loss directly contributed, caused by between Connect about 10,000,000,000 yuan of loss.

Prevention and treatment for chicken viral diseases at present are mainly using vaccine immunity and drug therapy, due to epidemic disease The immune serotype of seedling is single, and virus serotype is numerous and virus stain speed of mutation is fast, is lost so as to frequently result in vaccine immunity Lose.Although some antibiotic and the antiviral drugs of chemical synthesis have some effects to a small number of viruses, because medicament residue passes through Food chain negatively affects to mankind's health care belt, and China prohibited some antibiotic and antiseptic in aquaculture since 16 years In application.Therefore, be actively developed production have no toxic side effect, drug residue free and the interferon formulation for not producing drug resistance, it is right The predicament of chicken viral diseases prevention and treatment at present has important clinical meaning.

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 a kind of multi-functional cell factor, with cell receptor knot After conjunction, it can induce body and produce more species-specific proteins and enzyme, mainly by suppressing viral gene transcription and degraded virus RNA is come the activity that suppresses the growth and breeding of virus and play antitumor grade.Now, it is known that α types IFN in vivo can be selectively The infection cells such as virus are acted on, by suppressing the biosynthesis of the virus protein in infected cell, play wide spectrum and efficiently Antivirus action.It is but faint without acting on or acting on to normal host cell.IFN-α main physiological activity is with suppression virus Duplication, anti parasitic, the killing activity for suppressing various kinds of cell propagation, stimulating immunocyte.

γ types IFN is the T cell and the generation of NK cells by activating, and has relatively strong antiviral and immunoloregulation function.Largely 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- Should, 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.

Cell factor IL-2 is interleukin 2, also known as SCIF.Mainly produced by the T lymphocytes activated The cell factor with extensive bioactivity, can both promote lymphopoiesis, strengthen immunologic function, and can restricted T 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.

The limitation of natural interferon and the current generally existing half-life short of artificial recombination interferon, half-life period are 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 reason for half-life short There are two：The too small tachytrophism in vivo of molecular weight of interferon, interferon especially recombinant interferon affinity is poor to be exempted from Epidemic disease system is removed.

And common long-acting interferon is using polyethylene glycol fused interferon as representative on the market, only in the aspect of molecular weight Part solves the problems, such as that interferon molecule amount is small and causes half-life short, while polyethylene glycol fused interferon cost is very Height, it is unfavorable for clinically applying.

The content of the invention

In order to solve the above technical problems, disturbed the invention provides one kind by recombinant chIL-2, chicken interferon gamma and chicken Fusion protein of plain α composition and preparation method thereof, and thus fusion protein with after freeze drying protectant mixture, freeze-dried system Standby to obtain a kind of recombination chicken long-acting interferon, the recombination chicken long-acting interferon is remarkably improved the half-life period of chicken interferon, compared with The half-life period of common chicken interferon improves more than 18 times, and has broad-spectrum disease resistance toxic action and can improve the immune of chicken itself and answer Answer.

The technical scheme that the present invention takes is：

A kind of fusion protein being made up of recombinant chIL-2, chicken interferon gamma and chicken interferon α, the fusion protein Amino acid sequence table as shown in the ＞ of 400 ＜ of SEQUENCE LISTING 1.

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 2, genome 1 is designated as；Or as shown in the ＞ of 400 ＜ of SEQUENCE LISTING 3, it is designated as genome 2.

Fusion protein described in 2 equal codified of the genome 1 and the genome.Genome 2 is the nucleosides to genome 1 Acid sequence optimize after result, be considered as the gene in the expression system during usual codon adaptation indexI CAI=1.0 In be optimal high efficient expression state, CAI values are lower to show that expression is lower in host.Most preferable point of G/C content in gene Cloth scope is 30~70%, and the scope is exceeded in any region can influence translation and transcriptional efficiency.Sent out using software detection Existing recombinant chIL-2, chicken IFN-γ, the codon of chicken IFN-α original gene codon adaptation indexI in Escherichia coli (CAI) be respectively 0.27,0.25,0.31, GC percentages be 36.1%, 42.9%, 61.7%；And by chicken interleukin-2 2nd, obtained after chicken IFN-γ, chicken IFN-α gene optimization each gene in Escherichia coli codon adaptation indexI (CAI) be 1.0, 1.0th, 1.0, GC percentages 46.2%, 47.6%, 58.5%.The utilization rate of low codon is significantly reduced by gene optimization, Influence of the rare codon to protein expression is avoided, the G/C content of gene is improved, improves 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-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, and further, 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 recombination chicken long-acting interferon, the recombination chicken long-acting interferon is by described fusion egg In vain with after freeze drying protectant mixture, it is freeze-dried to form.

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

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

The genetic engineering bacterium is pET-32a/rIL2-IFN γ-IFN α, and its preparation method is：

(1) primer is designed, is obtained by reverse transcription or the chicken interleukin-2 of the flexible linker sequences of artificial synthesized band 2nd, chicken interferon gamma, chicken interferon α target gene；By flexible linker by recombinant chIL-2, chicken interferon gamma, chicken The target gene of interferon-' alpha ' connects, the nucleotides sequence list such as SEQUENCE LISTING of the target gene after connection Shown in the ＞ of 400 ＜ 2 or as shown in the ＞ of 400 ＜ of SEQUENCE LISTING 3；

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

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

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

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

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

The acquisition methods of the genome 1 are：

A. design of primers

The primer sequence of recombinant chIL-2 (IL-2) is：

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

Downstream IL-2-R1：

ACCACCACCAGAACCACCACCACCTTTTTGCAGATATCTCAC, with flexible linker；

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

Upstream IFN-γ-F1：

GGTGGTTCTGGTGGTGGTGGTTCTATGACTTGCCAGACTT, with flexible linker；

Downstream IFN-γ-R1：

ACCACCACCAGAACCACCACCACCGCAATTGCATCTCCTC, with flexible linker；

Chicken interferon α (IFN-α) primer sequence is：

Upstream IFN-α-F1：

GGTGGTTCTGGTGGTGGTGGTTCTCCCACCATGGCTGT, with flexible linker；

Downstream IFN-α-R1：CCCTCGAGAGTGCGCGTGTTG, with XhoI restriction enzyme sites；

B. RNA is extracted from chicken liver, the target gene of chicken IL-2 gene, chicken IFN-γ and chicken IFN-α is obtained by reverse transcription, The gene order of three respectively as shown in the ＞ of 400 ＜ of SEQUENCE LISTING 4, the ＞ of 400 ＜ of SEQUENCE LISTING 5 and Shown in the ＞ of 400 ＜ of SEQUENCE LISTING 6；

Respectively using the target gene of chicken IL-2 gene, chicken IFN-γ and chicken IFN-α as template, and it is utilized respectively chicken IL-2 gene, chicken The upstream and downstream primer of IFN-γ and chicken IFN-α enters performing PCR amplification, respectively obtains the chicken IL-2 gene for connecting flexible linker, chicken IFN-γ and chicken IFN-α gene.

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 is 10 μ L, adds RNase Free water to 25 μ L；The reaction of the RT-PCR reactions Condition is：50 DEG C of reverse transcriptions 30min, 95 DEG C of pre-degeneration 4min, into 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. rIL2-IFN γ genes are obtained using flexible linker connection chicken IL-2 genes and chicken IFN-γ target gene

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 10 μ L, adds RNase Free water to 25 μ L；Even Connecing 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 extension 1kb/ Min, totally 35 circulations；Last 72 DEG C of extensions 10min.

D. rIL2-IFN γ-IFN are obtained using flexible linker connections rIL2-IFN γ genes and chicken IFN-α target gene α genes

The PCR reaction systems and reaction condition of connection be：In 25 μ L overall reaction system, rIL2-IFN γ gene templates The μ L of DNA 1, connect the flexible linker μ L of 1 μ L, IL-2 sense primer of IFN-α template DNA 0.5, IFN-α anti-sense primer 0.5 μ L, Taq archaeal dna polymerase 2.5 μ L, dNTP Mix is 10 μ L, adds RNase Free water to 25 μ L；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 extension 1kb/min, totally 35 circulate；Most 72 DEG C of extension 10min afterwards.

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 recombinant chIL-2 (IL-2) is：

Upstream IL-2-F2：CGGGATCCATGATGTGCAAAGTTCT, with BamHI restriction enzyme sites；

Downstream IL-2-R2：

ACCACCACCAGAACCACCACCACCTTTCTGCAGGTAACG, with flexible linker；

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

Upstream IFN-γ-F2：

GGTGGTTCTGGTGGTGGTGGTTCTATGACCTGCCAGAC, with flexible linker；

Downstream IFN-γ-R2：

ACCACCACCAGAACCACCACCACCGCAGTTGCAACGAC, with flexible linker；

Chicken interferon α (IFN-α)：

Upstream IFN-α-F2：

GGTGGTTCTGGTGGTGGTGGTTCTCCGACCATGGCTG, with flexible linker；

Downstream IFN-α-R2：

CCCTCGAGGGTACGGGTGTTACC, with XhoI restriction enzyme sites.

B. the target gene of the chicken IL-2 gene, chicken IFN-γ and chicken IFN-α, the gene order of three is respectively such as SEQUENCE Shown in the ＞ of 400 ＜ of LISTING 7, the ＞ of 400 ＜ of SEQUENCE LISTING 400 ＜, 8 ＞ and SEQUENCE LISTING 9；

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, adds RNase Free water 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.

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 10 μ L, adds RNase Free water to 25 μ L；Even Connecing 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 extension 1kb/ Min, totally 35 circulations；Last 72 DEG C of extensions 10min.

The PCR reaction systems and reaction condition of connection be：In 25 μ L overall reaction system, rIL2-IFN γ gene templates The μ L of DNA 1, connect the flexible linker μ L of 1 μ L, IL-2 sense primer of IFN-α template DNA 0.5, IFN-α anti-sense primer 0.5 μ L, Taq archaeal dna polymerase 2.5 μ L, dNTP Mix is 10 μ L, adds RNase Free water to 25 μ L；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 extension 1kb/min, totally 35 circulate；Most 72 DEG C of extension 10min afterwards.

Present invention also offers the application of the recombination chicken long-acting interferon, its long half time had up to more than 73 hours Broad-spectrum disease resistance toxic action and the immune response that chicken itself can be improved.

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

1. chicken IL-2 gene, chicken IFN-γ and chicken IFN-α gene are realized into amalgamation and expression by flexible linker, interference is improved Plain half-life period, compared with plain interferon, improve more than 18 times；

2. by being optimized to chicken IL-2 gene, chicken IFN-γ and chicken IFN-α gene, improve chicken IL-2 gene, chicken IFN-γ and The expression quantity of chicken IFN-α fusion protein.

3. using recombination bacillus coli pET-32a/rIL2-IFN γ-IFN α as expression bacterial strain, by introducing molecular chaperones PGro7 plasmids, in protein expression, generation inclusion body is less, forms great amount of soluble albumen, avoids inclusion body denaturation and answers The process of property, substantially reduce the time of expressing fusion protein；

4. the fusion protein disclosed by the invention being made up of chicken IL-2 gene, chicken IFN-γ and chicken IFN-α not only has IFN-α Broad-spectrum disease resistance toxic action, while significantly improve the immune response of chicken itself.

Brief description of the drawings

Fig. 1 is recombinant chIL-2 gene, chicken interferon α genes and the chicken interferon gamma gene RT-PCR in embodiment 1 The result of amplification；Swimming lane M：DNA Marker DL2000；Swimming lane 1：The gene RT-PCR amplified productions of chicken interleukin-2 2；Swimming lane 2：Chicken Interferon-gamma gene RT-PCR amplified productions；Swimming lane 3：Chicken interferon α gene RT-PCR amplified productions；

Fig. 2 is the knot of the PCR amplifications after chicken IL-2 gene, the IFN-γ in embodiment 1 connect with the target gene of IFN-α Fruit；Swimming lane M：DNA Marker DL2000；Swimming lane 1：The gene of chicken interleukin-2 2, chicken interferon gamma gene and chicken interferon α genes Ligation amplification product；

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

Fig. 4 is the SDS-PAGE electrophoretic examinations results of the recombinant protein in embodiment 1；Swimming lane M：Albumen Marker；Swimming lane 1：Zero load control；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 obtains；Swimming lane M：Albumen Marker；Swimming Road 1：Supernatant after recombinant bacterium induction is broken；Swimming lane 2：Precipitated after being crushed for recombinant bacterium induction；

Fig. 6 is that the recombination chicken long-acting interferon α as made from the fusion protein in embodiment 1 causes cell to VSV in embodiment 5 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 is that the recombination chicken long-acting interferon α of gradient dilution (from right to left) handles hole；

Fig. 7 is the recombination chicken 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 recombinant chIL-2, chicken interferon gamma and chicken interferon α, its preparation method are as follows：

1. recombinant chIL-2 (IL-2), chicken interferon gamma (IFN-γ) and chicken interferon α (IFN-α) target gene Obtain and expand

Design of primers：

Objective gene sequence design synthetic primer according to having been reported in Genebank is shown in Table 1, in recombinant chIL-2 EcoRI restriction enzyme sites and Linker sequences are introduced in sense primer and anti-sense primer respectively, in the sense primer of chicken interferon gamma With Linker sequences are introduced in anti-sense primer respectively, introduced respectively in chicken interferon α sense primer and anti-sense primer Linker sequences and XhoI restriction enzyme sites.

The pcr amplification primer thing of table 1

RT-PCR obtains target gene：

RNA is extracted from chicken liver tissue, the purpose base of chicken IL-2 gene, chicken IFN-γ and chicken IFN-α is obtained by reverse transcription Cause, the gene order of three respectively such as the ＞ of 400 ＜ of SEQUENCE LISTING 4, the ＞ of 400 ＜ of SEQUENCE LISTING 5 and Shown in the ＞ of 400 ＜ of 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 460bp, 550bp and 610bp or so in RT-PCR amplified productions, its As a result as shown in figure 1, the chicken IL-2 gene for being connected to flexible linker sequences, chicken IFN-γ and chicken has been prepared in explanation respectively The target gene of IFN-α.

2. the connection of target gene

Target gene is diluted to 10ug/mL, connects each section of target gene using over-lap PCR, 25 μ L reaction systems are such as Shown in table 3, table 4：

The rIL2-IFN γ PCR reaction systems of table 3

The rIL2-IFN γ of table 4-IFN α PCR 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 1570bp or so in pcr amplification product, its result as shown in Fig. 2 Occur rIL2-IFN γ and IFN-α amplified production band in Fig. 2, because being connected in rIL2-IFN γ with IFN-α gene During, there is non-specific responding.The nucleotide sequence of the obtained target gene such as ＞ of 400 ＜ of SEQUENCE LISTING 2 It is shown.

3. expression vector establishment

After sequencing is errorless, PCR glue reclaims product uses target gene after selection connection with pET-32a plasmids EcoRI and XhoI restriction enzymes carry out double digestion and recovery, and double digestion is done by 20 μ L systems in table 5：

The double digestion system of table 5

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

The enzyme disjunctor system of table 6

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, competent cell is coated on benzyl containing ammonia The LB culture medium flat plates of penicillin are incubated overnight；Single bacterium colony on picking LB flat boards carries out target gene PCR identifications, positive colony Bacteria plasmid is identified through EcoRI and XhoI double digestions, is accredited as positive and is represented that engineering bacteria successfully constructs, PCR amplifications and double digestion Product detects single band through agarose gel electrophoresis at 1570bp or so places, and its result is as shown in figure 3, illustrate successfully to obtain PET-32a/rIL2-IFN γ-IFN α engineering bacteria.

4. the expression of recombinant protein

Picking engineering bacteria 37 DEG C of shaking table recovery 1h in the LB culture mediums containing 100 μ g/ml ampicillins, in LB culture mediums Amplification culture 4h (OD=1.0) in (the μ g/ml containing ampicillin 100), adds final concentration of 100 μ g/ml IPTG, 32 DEG C lure Lead expression 5h；Thalline is collected, through SDS-PAGE electrophoresis detections, its result is as shown in figure 4, it can be seen that recombinant bacterium lures Supernatant is deposited in the visible predominant expression band in 75.7KD or so places after leading the bacterial cell disruption after 5h, illustrates 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, work 10s, is spaced 3S, ultrasonic 6min, and whole process repeats 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 that have been 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-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, DEAE anion exchange chromatography that loading has balanced by using Binding Buffer II, then After crossing post to A280nm value stabilizations with Binding Buffer II, with (the 50mM trihydroxy methyl amino first of Elution Buffer II Alkane, 1M NaCl, PH6.5) linear gradient elution, collect rIL2-IFN γ-IFN α protein peak.

5.3 sieve chromatography

Loading is by using (the 50mM of Binding Buffer III after the sample concentration that ion-exchange chromatography is collected into Na₂HPO4,0.15M NaCl, PH7.4) molecular sieve chromatographies of Superdex 200 have been balanced, washed with Binding Buffer III It is de-, collect rIL2-IFN γ-IFN α protein peak.

5.4 sample identification

Determine rIL2-IFN γ-IFN α potency and specific activity, specific activity >=10⁷IU/mg, albumen are qualified；It is aseptic subpackaged ,- 80 DEG C of preservations.The i.e. available fusion protein being made up of recombinant chIL-2, chicken interferon gamma and chicken interferon α, 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 recombinant chIL-2, chicken interferon gamma and chicken interferon α, other same embodiments 1, simply e. coli bl21 therein (DE3) competent cell is replaced in order to which the BL21 (DE3) with pGro7 plasmids experiences State cell.The SDS-PAGE electrophoresis results of its fusion protein compare with embodiment 1,75.7KD or so places predominant expression in supernatant Band is thicker, illustrates after introducing molecular chaperones pGro7, expression of the destination protein in supernatant is more preferable, obtained fusion protein Amount is higher.The albumen of Bacillus coli expression is mostly present in inclusion body；By introducing molecular chaperones in bacterial strain is expressed, association Correctly folded with expressing protein, reach solubility expression of protein.

Embodiment 3

A kind of fusion protein being made up of recombinant chIL-2, chicken interferon gamma and chicken interferon α, its preparation method is such as Under：

Chicken IL-2 gene in embodiment 1, chicken IFN-γ and chicken IFN-α are optimized, artificial synthesized chicken IL-2 gene, chicken IFN-γ With chicken IFN-α target gene, after optimization, the nucleotide sequence of three respectively as the ＞ of 400 ＜ of SEQUENCE LISTING 7, Shown in the ＞ of 400 ＜ of SEQUENCE LISTING 400 ＜, 8 ＞ and SEQUENCE LISTING 9.

1.1 codon optimization

Genetic codon has 64 kinds, but most biological tendencies are in utilizing the part in these codons.Those By the most frequent referred to as 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 utilizing rare codon Gene chemical synthesis is carried out, the redesign of this gene is codon optimization.Therefore, the IL-2 in the present embodiment to chicken, chicken IFN- γ and chicken IFN-α gene codon optimize.

Interpretation of result after 1.2 codon optimizations

It is optimal efficient in the expression system to be considered as the gene during usual codon adaptation indexI (CAI)=1.0 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%, it can influence translation and transcriptional efficiency more than the scope in any region.Chicken IL-2 gene, chicken are found using software detection The codon of IFN-γ and chicken IFN-α original gene codon adaptation indexI (CAI) in Escherichia coli is respectively 0.27, 0.25th, 0.31, GC percentages are 36.1%, 42.9%, 61.7%；And by chicken IL-2 gene, chicken IFN-γ and chicken IFN-α gene Obtained after optimization recombination codon adaptation indexI (CAI) in Escherichia coli be respectively 1.0,1.0,1.0, GC percentages 46.2%th, 47.6%, 58.5%.The utilization rate of low codon is significantly reduced by gene optimization, avoids rare codon Influence to protein expression, the G/C content of gene is improved, improve transcription and translation efficiency.

1.3 design of primers：

The pcr amplification primer thing of table 7

The genomic DNA of chicken IL-2 gene after optimization, chicken IFN-γ and chicken IFN-α is diluted to 0.05mg/mL respectively.Utilize PCR amplifications obtain target gene, and 25 μ L reaction systems are as shown in table 8：

The PCR reaction systems of table 8

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.

Chicken IL-2 gene, chicken IFN-γ and chicken IFN-α pcr amplification product through agarose gel electrophoresis respectively 610bp, There is specific band in 550bp and 460bp or so, illustrate the chicken for being connected to flexible linker after optimization has been prepared IL-2, chicken IFN-γ and chicken IFN-α target gene.

2. the connection of target gene

Target gene is diluted to 10ug/mL, connects each section of target gene using over-lap PCR, 25 μ L reaction systems are such as Shown in table 9, table 10：

The rIL2-IFN γ PCR reaction systems of table 9

The rIL2-IFN γ of table 10-IFN-α PCR reaction systems

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 1570bp or so in pcr amplification product, illustrates successfully to be connected RIL2-IFN γ-IFN-α gene after connecing.The nucleotide sequence of obtained target gene such as SEQUENCE LISTING 400 Shown in the ＞ of ＜ 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 BamHI, XhoI restriction enzyme carry out double digestion and recovery, and double digestion is done by 20 μ L systems in table 11：

The double digestion system of table 11

General buffer	2μL
		Restriction enzyme (a pair)	1μL+1μL
Carrier or recovery 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 12,4 DEG C overnight connection：

Table 12

Connection product is converted into e. coli bl21 (DE3) competent cell, competence is coated on the mould of benzyl containing ammonia It is incubated overnight in the LB flat boards of element；The bacterium colony grown on LB flat boards is taken to identify target gene, positive colony bacteria plasmid warp through PCR BamHI, XhoI double digestion are identified, are accredited as positive and are represented expression vector establishment success, PCR amplifications and double digestion product are through fine jade There is single band at 1570bp or so places in sepharose electrophoresis, illustrates containing rIL2-IFN γ-IFN α fusion gene engineering bacterium PET-32a/rIL2-IFN γ-IFN α successfully constructs.

4. the expression of recombinant protein

Picking engineering bacteria 37 DEG C of shaking table recovery 1h in the LB culture mediums containing 100 μ g/ml ampicillins, in LB culture mediums Amplification culture 4h (OD=1.0) in (the μ g/ml containing ampicillin 100), adds final concentration of 100 μ g/ml IPTG, 32 DEG C lure Lead expression 5h；Thalline is collected, through SDS-PAGE electrophoresis detections, supernatant is deposited in after the bacterial cell disruption after recombinant bacterium induction 5h The visible predominant expression band in 75.7KD or so places, illustrate to have obtained recombinant protein in supernatant precipitates.

5. fusion protein purification

5.1His affinity chromatography

5.2DEAE anion-exchange chromatography

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) molecular sieve chromatographies of Superdex 200 have been balanced, with Binding Buffer III elution, collects rIL2-IFN γ-IFN α protein peak.

5.4 sample identification

Determine rIL2-IFN γ-IFN α potency and specific activity, specific activity >=10⁷IU/mg, albumen are qualified；Sterile point Dress, -80 DEG C of preservations.The i.e. available fusion protein being made up of recombinant chIL-2, chicken interferon gamma and chicken interferon α, its ammonia Base acid sequence is as shown in the ＞ of 400 ＜ of SEQUENCE LISTING 1.

Embodiment 4

A kind of fusion protein being made up of recombinant chIL-2, chicken interferon gamma and chicken interferon α, other same embodiments 3, simply e. coli bl21 therein (DE3) competent cell is replaced in order to which the BL21 (DE3) with pGro7 plasmids experiences State cell.The SDS-PAGE electrophoresis results of its fusion protein compare with embodiment 3,75.7KD or so places predominant expression in supernatant Band is thicker, illustrates after introducing molecular chaperones pGro7, expression of the destination protein in supernatant is more preferable, obtained fusion protein Amount is higher.The albumen of Bacillus coli expression is mostly present in inclusion body；By introducing molecular chaperones in bacterial strain is expressed, association Correctly folded with expressing protein, reach solubility expression of protein.

Embodiment 5

A kind of recombination chicken 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 is obtained by the mirror of recombinant chIL-2, chicken interferon gamma and chicken interferon the α fusion protein formed It is fixed

The quantitative detection of 6.1 protein contents

With Lowry methods, the standard protein that institute is examined and determine with Chinese food pharmaceutical biological product is made standard test, determines embodiment 1~4 obtained fusion protein concentration is all higher than 1.1mg/ml.

6.2SDS-PAGE electrophoresis detection

Compared with empty bacterium, fusion protein has the newly-increased protein band of a dense dye in 75.7KD or so, as shown in Figure 4.

6.3Western Blot results

Fusion protein in embodiment 1~4 is detected respectively, with the anti-chicken alpha interferon (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).Recombination chicken long-acting interferon sample can be with anti-chicken interferon α Monoclonal antibody occurs specific reaction, 75.7KD or so place and specific band occurs, as shown in Figure 5.

Embodiment 7

Bioactivity freeze-dried four parts of recombination chicken 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, adds the recombination chicken length of various dose Interferon-' alpha ' is imitated, inhales and abandons after 24h, then be inoculated with 100TCID respectively₅₀VSV viruses.

Result of the test

As a result the recombination chicken long-acting interferon α for showing to obtain causes the lesion of HEp-2 cells to have obvious suppress to VSV Effect.The lesion such as occurs cell rounding after undressed cell virus inoculation, comes off, is disintegrated.And the recombination chicken length obtained After imitating 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 >=10⁷IU/ml, as shown in Figure 6.

Embodiment 8

The four parts of recombination chicken 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

The blood concentration and time relationship of cytopathic-effect inhibition assay measure rIL2-IFN γ-IFN α

The broiler chicken (male and female half and half) that six body weight are roughly the same is taken, 2mg/ml recombination chicken long-acting interferons are subcutaneously injected in neck The freeze-dried 2ml of α, respectively in 1h, 2h, 4h, 8h, 16h, 32h, 64h, 96h venous blood collection, 4 DEG C of solidifications of blood sample, 3500rpm low temperature 10min separation serum is centrifuged, each every chicken blood sample of time point is to be measured in -20 DEG C of preservations.Serum is determined using cytopathic-effect inhibition assay The concentration of rIL2-IFN γ-IFN α in sample, carried out curve fitting and calculating parameter with DAS pharmacokinetics softwares.Parameter calculates knot Fruit is shown in Table 13.

Dominant dynamic parameters in serum after the recombination chicken long-acting interferon α intramuscular injection of table 13

As a result show that recombination chicken long-acting interferon α has longer half-life period.Half-life period can reach 73h or so after measured, compared with Plain interferon improves more than 18 times.

Embodiment 9

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

Take six roughly the same broiler chicken 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 recombination chicken long-acting interferon freeze-dried 2ml of α are penetrated, 2mL PBS is subcutaneously injected in control group neck, takes after injecting 4 weeks outside chicken All blood, take a blood weekly afterwards, separate lymphocyte using lymphocyte separation medium, lymphocyte passes through serum-free RPMI After 1640 culture mediums wash 2 times, it is resuspended with complete medium, adjusts cell concentration as 2 × 10⁶Individual/ml, 24 porocyte culture plates are every Hole addition 1ml lymphocytes, 37 DEG C, 5%CO₂72h is cultivated, supernatant when collecting lymphocyte culture.ELISA detects culture supernatant Middle IL-4 contents, are carried out, testing result is as shown in table 14 by kit specification：

The ELISA of table 14 detection each group chicken cell immune responses are horizontal

As a result show after injecting recombination chicken long-acting interferon α, can significantly improve chicken Evaluation of Cytokines in Peripheral Blood IL-4's Content, cellullar immunologic response level is enhanced, significantly improves immunity level.

It is above-mentioned with reference to embodiment to a kind of fusion egg being made up of recombinant chIL-2, chicken interferon gamma and chicken interferon α The detailed description that bletilla its preparation method is carried out, is illustrative rather than limited, can be included according to limited scope Several embodiments, therefore changing and modifications in the case where not departing from present general inventive concept, should belong to protection scope of the present invention it It is interior.

SEQUENCE LISTING

<110>Anhui Jiuchuan Biotechnology Co., Ltd.

<120>A kind of fusion protein being made up of recombinant chIL-2, chicken interferon gamma and chicken interferon α and its preparation

Method

<130> 1

<160> 9

<170> PatentIn version 3.3

<210> 1

<211> 522

<212> PRT

<213>Chicken IL2-IFN γ-IFN α fusion protein

<400> 1

Met Met Cys Lys Val Leu Ile Phe Gly Cys Ile Ser Val Ala Met Leu

1 5 10 15

Met Thr Thr Ala Tyr Gly Ala Ser Leu Ser Ser Glu Lys Trp Lys Thr

20 25 30

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

35 40 45

Lys Ile His Leu Glu Leu Tyr Thr Pro Thr Glu Thr Gln Glu Cys Thr

50 55 60

Gln Gln Thr Leu Gln Cys Tyr Leu Gly Glu Val Val Thr Leu Lys Lys

65 70 75 80

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

85 90 95

Gln Asn Ile Glu Lys Asn Leu Lys Ser Leu Thr Gly Leu Asn His Thr

100 105 110

Gly Ser Glu Cys Lys Ile Cys Glu Ala Asn Asn Lys Lys Lys Phe Pro

115 120 125

Asp Phe Leu His Glu Leu Thr Asn Phe Val Arg Tyr Leu Gln Lys Gly

130 135 140

Gly Gly Gly Ser Gly Gly Gly Gly Ser Met Thr Cys Gln Thr Tyr Asn

145 150 155 160

Leu Phe Val Leu Ser Val Ile Met Ile Tyr Tyr Gly His Thr Ala Ser

165 170 175

Ser Leu Ile Leu Val Gln Leu Gln Asp Asp Ile Ala Lys Leu Lys Ala

180 185 190

Asp Phe Asn Ser Ser His Ser Asp Val Ala Asp Gly Gly Pro Ile Ile

195 200 205

Ala Glu Lys Leu Lys Asn Trp Thr Glu Arg Asn Gln Lys Arg Ile Ile

210 215 220

Leu Ser Gln Ile Val Ser Met Tyr Leu Glu Met Leu Ala Asn Thr Asp

225 230 235 240

Lys Thr Lys Pro His Thr Lys His Ile Ser Glu Glu Leu Tyr Thr Leu

245 250 255

Lys Asn Asn Leu Pro Asp Gly Val Lys Lys Val Lys Asp Ile Met Asp

260 265 270

Leu Ala Lys Leu Pro Met Asn Asp Leu Arg Val Gln Leu Lys Ala Ala

275 280 285

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

290 295 300

Lys Arg Asn Met Ser Gln Ser Gln Arg Arg Cys Asn Cys Gly Gly Gly

305 310 315 320

Gly Ser Gly Gly Gly Gly Ser Pro Thr Met Ala Val Pro Ala Ser Pro

325 330 335

Gln His Pro Arg Gly Tyr Gly Ile Leu Leu Leu Thr Leu Leu Leu Lys

340 345 350

Ala Leu Ala Thr Thr Ala Ser Ala Cys Asn His Leu Arg Pro Gln Asp

355 360 365

Ala Thr Phe Ser His Asp Ser Leu Gln Leu Leu Arg Asp Met Ala Pro

370 375 380

Thr Leu Pro Gln Leu Cys Pro Gln His Asn Ala Ser Cys Ser Phe Asn

385 390 395 400

Asp Thr Ile Leu Asp Thr Ser Asn Thr Arg Gln Ala Asp Lys Thr Thr

405 410 415

His Asp Ile Leu Gln His Leu Phe Lys Ile Leu Ser Ser Pro Ser Thr

420 425 430

Pro Ala His Trp Asn Asp Ser Gln Arg Gln Ser Leu Leu Asn Arg Ile

435 440 445

His Arg Tyr Thr Gln His Leu Glu Gln Cys Leu Asp Ser Ser Asp Thr

450 455 460

Arg Ser Arg Thr Arg Trp Pro Arg Asn Leu His Leu Thr Ile Lys Lys

465 470 475 480

His Phe Ser Cys Leu His Thr Phe Leu Gln Asp Asn Asp Tyr Ser Ala

485 490 495

Cys Ala Trp Glu His Val Arg Leu Gln Ala Arg Ala Trp Phe Leu His

500 505 510

Ile His Asn Leu Thr Gly Asn Thr Arg Thr

515 520

<210> 2

<211> 1566

<212> DNA

<213>Genome 1

<400> 2

atgatgtgca aagtactgat ctttggctgt atttcggtag caatgctaat gactacagct 60

tatggagcat ctctatcatc agaaaaatgg aaaactcttc aaacattaat aaaggattta 120

gaaatattgg aaaatatcaa gaataagatt catctcgagc tctacacacc aactgagacc 180

caggagtgca cccagcaaac tctgcagtgt tacctgggag aagtggttac tctgaagaaa 240

gaaactgaag atgacactga aattaaagaa gaatttgtaa ctgctattca aaatatcgaa 300

aagaacctca agagtcttac gggtctaaat cacaccggaa gtgaatgcaa gatctgtgaa 360

gctaacaaca agaaaaaatt tcctgatttt ctccatgaac tgaccaactt tgtgagatat 420

ctgcaaaaag gtggtggtgg ttctggtggt ggtggttcta tgacttgcca gacttacaac 480

ttgtttgttc tgtccgtcat catgatttat tatggacata ctgcaagtag tctaattctt 540

gttcaacttc aagatgatat agccaaactg aaagctgact ttaactcaag tcattcagat 600

gtagctgacg gtggacctat tattgcagag aaactgaaga actggacaga gagaaatcag 660

aaaaggatca tactgagcca gattgtttcg atgtacttgg aaatgcttgc aaacactgac 720

aagacaaagc cgcacaccaa acacatatct gaggagctct atactctgaa aaacaacctt 780

cctgatggcg tgaagaaggt gaaagatatc atggacctgg ccaagctccc gatgaacgac 840

ttgagagtcc agctcaaagc cgcgaatgaa ctcttcagca tcttacagaa gctggtgaat 900

cctccgagtt tcaaaaggaa catgagccag tctcagagga gatgcaattg cggtggtggt 960

ggttctggtg gtggtggttc tcccaccatg gctgtgcctg caagcccaca gcacccacgg 1020

gggtacggca tcctgctgct cacgctcctt ctgaaagctc tcgccaccac cgcctccgcc 1080

tgcaaccacc ttcgccccca ggatgccacc ttctctcacg acagcctcca gctcctccgg 1140

gacatggctc ccacactacc ccagctgtgc ccacagcaca acgcgtcttg ctccttcaac 1200

gacaccatcc tggacaccag caacacccgg caagccgaca aaaccaccca cgacatcctt 1260

cagcacctct tcaaaatcct cagcagcccc agcactccag cccactggaa cgacagccaa 1320

cgccaaagcc tcctcaaccg gatccaccgc tacacccagc acctcgagca atgcttggac 1380

agcagcgaca cgcgctcccg gacgcgatgg cctcgcaacc ttcacctcac catcaaaaaa 1440

cacttcagct gcctccacac cttcctccaa gacaacgatt acagcgcctg cgcctgggaa 1500

cacgtccgcc tgcaagctcg tgcctggttc ctgcacatcc acaacctcac aggcaacacg 1560

cgcact 1566

<210> 3

<211> 1566

<212> DNA

<213>Genome 2

<400> 3

atgatgtgca aagtactgat ctttggctgt atttcggtag caatgctaat gactacagct 60

tatggagcat ctctatcatc agaaaaatgg aaaactcttc aaacattaat aaaggattta 120

gaaatattgg aaaatatcaa gaataagatt catctcgagc tctacacacc aactgagacc 180

caggagtgca cccagcaaac tctgcagtgt tacctgggag aagtggttac tctgaagaaa 240

gaaactgaag atgacactga aattaaagaa gaatttgtaa ctgctattca aaatatcgaa 300

aagaacctca agagtcttac gggtctaaat cacaccggaa gtgaatgcaa gatctgtgaa 360

gctaacaaca agaaaaaatt tcctgatttt ctccatgaac tgaccaactt tgtgagatat 420

ctgcaaaaag gtggtggtgg ttctggtggt ggtggttcta tgacctgcca gacctacaac 480

ctgttcgttc tgtctgttat catgatctac tacggtcaca ccgcttcttc tctgatcctg 540

gttcagctgc aggacgacat cgctaaactg aaagctgact tcaactcttc tcactctgac 600

gttgctgacg gtggtccgat catcgctgaa aaactgaaaa actggaccga acgtaaccag 660

aaacgtatca tcctgtctca gatcgtttct atgtacctgg aaatgctggc taacaccgac 720

aaaaccaaac cgcacaccaa acacatctct gaagaactgt acaccctgaa aaacaacctg 780

ccggacggtg ttaaaaaagt taaagacatc atggacctgg ctaaactgcc gatgaacgac 840

ctgcgtgttc agctgaaagc tgctaacgaa ctgttctcta tcctgcagaa actggttaac 900

ccgccgtctt tcaaacgtaa catgtctcag tctcagcgtc gttgcaactg cggtggtggt 960

ggttctggtg gtggtggttc tccgaccatg gctgttccgg cttctccgca gcacccgcgt 1020

ggttacggta tcctgctgct gaccctgctg ctgaaagctc tggctaccac cgcttctgct 1080

tgcaaccacc tgcgtccgca ggacgctacc ttctctcacg actctctgca gctgctgcgt 1140

gacatggctc cgaccctgcc gcagctgtgc ccgcagcaca acgcttcttg ctctttcaac 1200

gacaccatcc tggacacctc taacacccgt caggctgaca aaaccaccca cgacatcctg 1260

cagcacctgt tcaaaatcct gtcttctccg tctaccccgg ctcactggaa cgactctcag 1320

cgtcagtctc tgctgaaccg tatccaccgt tacacccagc acctggaaca gtgcctggac 1380

tcttctgaca cccgttctcg tacccgttgg ccgcgtaacc tgcacctgac catcaaaaaa 1440

cacttctctt gcctgcacac cttcctgcag gacaacgact actctgcttg cgcttgggaa 1500

cacgttcgtc tgcaggctcg tgcttggttc ctgcacatcc acaacctgac cggtaacacc 1560

cgtacc 1566

<210> 4

<211> 429

<212> DNA

<213>Chicken IL-2 gene

<400> 4

atgatgtgca aagtactgat ctttggctgt atttcggtag caatgctaat gactacagct 60

tatggagcat ctctatcatc agaaaaatgg aaaactcttc aaacattaat aaaggattta 120

gaaatattgg aaaatatcaa gaataagatt catctcgagc tctacacacc aactgagacc 180

caggagtgca cccagcaaac tctgcagtgt tacctgggag aagtggttac tctgaagaaa 240

gaaactgaag atgacactga aattaaagaa gaatttgtaa ctgctattca aaatatcgaa 300

aagaacctca agagtcttac gggtctaaat cacaccggaa gtgaatgcaa gatctgtgaa 360

gctaacaaca agaaaaaatt tcctgatttt ctccatgaac tgaccaactt tgtgagatat 420

ctgcaaaaa 429

<210> 5

<211> 492

<212> DNA

<213>Chicken IFN-γ

<400> 5

atgacttgcc agacttacaa cttgtttgtt ctgtccgtca tcatgattta ttatggacat 60

actgcaagta gtctaattct tgttcaactt caagatgata tagccaaact gaaagctgac 120

tttaactcaa gtcattcaga tgtagctgac ggtggaccta ttattgcaga gaaactgaag 180

aactggacag agagaaatca gaaaaggatc atactgagcc agattgtttc gatgtacttg 240

gaaatgcttg caaacactga caagacaaag ccgcacacca aacacatatc tgaggagctc 300

tatactctga aaaacaacct tcctgatggc gtgaagaagg tgaaagatat catggacctg 360

gccaagctcc cgatgaacga cttgagagtc cagctcaaag ccgcgaatga actcttcagc 420

atcttacaga agctggtgaa tcctccgagt ttcaaaagga acatgagcca gtctcagagg 480

agatgcaatt gc 492

<210> 6

<211> 585

<212> DNA

<213>Chicken IFN-α

<400> 6

cccaccatgg ctgtgcctgc aagcccacag cacccacggg ggtacggcat cctgctgctc 60

acgctccttc tgaaagctct cgccaccacc gcctccgcct gcaaccacct tcgcccccag 120

gatgccacct tctctcacga cagcctccag ctcctccggg acatggctcc cacactaccc 180

cagctgtgcc cacagcacaa cgcgtcttgc tccttcaacg acaccatcct ggacaccagc 240

aacacccggc aagccgacaa aaccacccac gacatccttc agcacctctt caaaatcctc 300

agcagcccca gcactccagc ccactggaac gacagccaac gccaaagcct cctcaaccgg 360

atccaccgct acacccagca cctcgagcaa tgcttggaca gcagcgacac gcgctcccgg 420

acgcgatggc ctcgcaacct tcacctcacc atcaaaaaac acttcagctg cctccacacc 480

ttcctccaag acaacgatta cagcgcctgc gcctgggaac acgtccgcct gcaagctcgt 540

gcctggttcc tgcacatcca caacctcaca ggcaacacgc gcact 585

<210> 7

<211> 429

<212> DNA

<213>Chicken IL-2 gene

<400> 7

atgatgtgca aagttctgat cttcggttgc atctctgttg ctatgctgat gaccaccgct 60

tacggtgctt ctctgtcttc tgaaaaatgg aaaaccctgc agaccctgat caaagacctg 120

gaaatcctgg aaaacatcaa aaacaaaatc cacctggaac tgtacacccc gaccgaaacc 180

caggaatgca cccagcagac cctgcagtgc tacctgggtg aagttgttac cctgaaaaaa 240

gaaaccgaag acgacaccga aatcaaagaa gaattcgtta ccgctatcca gaacatcgaa 300

aaaaacctga aatctctgac cggtctgaac cacaccggtt ctgaatgcaa aatctgcgaa 360

gctaacaaca aaaaaaaatt cccggacttc ctgcacgaac tgaccaactt cgttcgttac 420

ctgcagaaa 429

<210> 8

<211> 492

<212> DNA

<213>Chicken IFN-γ

<400> 8

atgacctgcc agacctacaa cctgttcgtt ctgtctgtta tcatgatcta ctacggtcac 60

accgcttctt ctctgatcct ggttcagctg caggacgaca tcgctaaact gaaagctgac 120

ttcaactctt ctcactctga cgttgctgac ggtggtccga tcatcgctga aaaactgaaa 180

aactggaccg aacgtaacca gaaacgtatc atcctgtctc agatcgtttc tatgtacctg 240

gaaatgctgg ctaacaccga caaaaccaaa ccgcacacca aacacatctc tgaagaactg 300

tacaccctga aaaacaacct gccggacggt gttaaaaaag ttaaagacat catggacctg 360

gctaaactgc cgatgaacga cctgcgtgtt cagctgaaag ctgctaacga actgttctct 420

atcctgcaga aactggttaa cccgccgtct ttcaaacgta acatgtctca gtctcagcgt 480

cgttgcaact gc 492

<210> 9

<211> 585

<212> DNA

<213>Chicken IFN-α

<400> 9

ccgaccatgg ctgttccggc ttctccgcag cacccgcgtg gttacggtat cctgctgctg 60

accctgctgc tgaaagctct ggctaccacc gcttctgctt gcaaccacct gcgtccgcag 120

gacgctacct tctctcacga ctctctgcag ctgctgcgtg acatggctcc gaccctgccg 180

cagctgtgcc cgcagcacaa cgcttcttgc tctttcaacg acaccatcct ggacacctct 240

aacacccgtc aggctgacaa aaccacccac gacatcctgc agcacctgtt caaaatcctg 300

tcttctccgt ctaccccggc tcactggaac gactctcagc gtcagtctct gctgaaccgt 360

atccaccgtt acacccagca cctggaacag tgcctggact cttctgacac ccgttctcgt 420

acccgttggc cgcgtaacct gcacctgacc atcaaaaaac acttctcttg cctgcacacc 480

ttcctgcagg acaacgacta ctctgcttgc gcttgggaac acgttcgtct gcaggctcgt 540

gcttggttcc tgcacatcca caacctgacc ggtaacaccc gtacc 585

Claims

A kind of 1. fusion protein being made up of recombinant chIL-2, chicken interferon gamma and chicken interferon α, it is characterised in that：It is described The amino acid sequence table of fusion protein is as shown in the ＞ of 400 ＜ of SEQUENCE LISTING 1.
A kind of 2. 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 2；Or as shown in the ＞ of 400 ＜ of SEQUENCE LISTING 3, 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 recombination chicken long-acting interferon, it is characterised in that the recombination chicken long-acting interferon is as melting described in claim 1 It is freeze-dried to form after hop protein and freeze drying protectant mixture.
6. the preparation method of fusion protein according to claim 1, 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, obtains genetic engineering bacterium, genetic engineering Bacterium obtains the crude product of the fusion protein after IPTG induced expressions, and fusion protein is can obtain after purified.
7. preparation method according to claim 6, it is characterised in that the genetic engineering bacterium is pET-32a/rIL2-IFN γ-IFN α, its preparation method are：

(1) primer is designed, is obtained by reverse transcription or the sheep interleukin 2 of the flexible linker sequences of artificial synthesized band, sheep The target gene of interferon gamma, sheep interferon-tau；Recombinant chIL-2, chicken interferon gamma, chicken are disturbed by flexible linker Plain α target gene connects, the nucleotides sequence list such as ＞ of 400 ＜ of SEQUENCE LISTING 2 of the target gene after connection It is shown or as shown in the ＞ of 400 ＜ of SEQUENCE LISTING 3；

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

(3) expression vector is imported into e. coli host cell, you can obtain genetic engineering bacterium pET-32a/rIL2-IFN γ-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 Product successively purify through affinity chromatography, anion-exchange chromatography and sieve chromatography.
10. the application of recombination chicken long-acting interferon according to claim 5, it is characterised in that the recombination chicken is long-acting dry The long half time of element is disturbed up to more than 73 hours, there is broad-spectrum disease resistance toxic action and the immune response of chicken itself can be improved.