CN107383202A

CN107383202A - A kind of fusion protein being made up of OVA, chicken interferon gamma and recombinant chIL-2 and preparation method thereof

Info

Publication number: CN107383202A
Application number: CN201710676089.7A
Authority: CN
Inventors: 王明丽; 符修乐; 王利利; 夏兵兵; 徐慕珍; 何志远; 徐文俊; 杨建伟
Original assignee: Wuhu Phil Biological Products Industry Research Institute Co Ltd
Current assignee: Wuhu Phil Biological Products Industry Research Institute Co Ltd
Priority date: 2017-08-09
Filing date: 2017-08-09
Publication date: 2017-11-24
Also published as: CN108822221A

Abstract

The invention discloses a kind of fusion protein being made up of OVA, chicken interferon gamma and recombinant chIL-2 and preparation method thereof; the fusion protein is connected by OVA, chicken interferon gamma and recombinant chIL-2 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 21 times, and with broad-spectrum disease resistance toxic action and can improve the immune response of chicken itself.

Description

A kind of fusion egg being made up of OVA, chicken interferon gamma and recombinant chIL-2 Bletilla its preparation method

Technical field

The invention belongs to technical field of biological genetic engineering, and in particular to thin in vain by OVA, chicken interferon gamma and chicken Fusion protein that born of the same parents' interleukin 2 forms 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.It is existing, it is known that γ types IFN be T cell by activating and NK cells produce, and have 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.It is although each The interferon of type can mediated cell to virus infection reaction, but the immunoregulatory activity of interferon gamma 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.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.IL-2 and other cell factors form fusion protein, produce and carry to strengthen the antibody of vaccine according to gene constructed High cellular immune level.

Seralbumin is the important component of blood plasma, is not easy to pass through glomerulus under normal circumstances, internal distributed pole it is wide and There is no zymetology and immunologic competence, be preferable pharmaceutical carrier.Albumin fusion technology has the advantage that：Albumin and target egg Linked in the cell through protein translation system by peptide bond in vain, be not required to extra extracorporeal treatment；The expression of albumin is higher, The expression of destination protein can be improved after being merged with it；Albumin is one stable " inert protein ", after being merged with it The stability of destination protein can be improved；Albumin fusion protein has longer drug half-life, by small molecular protein medicine It can be expected to improve half-life period in blood with Albumin fusion.At present, in experimental animal after multiple protein and Albumin fusion The extension of Half-life in vivo is confirmed.

The limitation of natural interferon and the current generally existing half-life short of artificial recombination interferon, half-life period are generally 2-4 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, the invention provides one kind by OVA, chicken interferon gamma and chicken interleukins Fusion proteins and preparation method thereof of the composition of element 2, 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 21 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 OVA, chicken interferon gamma and recombinant chIL-2, the fusion protein Amino acid sequence table is 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 SEQUENCE LISTING400 ＜ 3, it is designated as genome 2.

The equal codified fusion protein of genome 1 and the genome 2.Genome 2 is the nucleotides sequence to genome 1 Row optimize after result, be considered as the gene during usual codon adaptation indexI CAI=1.0 is in the expression system Optimal high efficient expression state, CAI values are lower to show that expression is lower in host.G/C content most ideal distribution model in gene Enclose for 30~70%, the scope is exceeded in any region can influence translation and transcriptional efficiency.Chicken is found using software detection Albumin, chicken IFN-γ, the codon of chicken IL-2 gene original gene in Escherichia coli codon adaptation indexI (CAI) be respectively 0.27th, 0.25,0.27, GC percentages are 43.1%, 42.9%, 36.1%；And by OVA, chicken IFN-γ, chicken IL- Obtained after 2 gene optimizations each gene in Escherichia coli codon adaptation indexI (CAI) be 0.99,1.0,1.0, GC percentages 49.2%th, 47.6%, 46.2%.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.

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/rAlb-IFN γ-IL2.

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/rAlb-IFN γ-IL2, and its preparation method is：

(1) primer is designed, the white egg of chicken for connecting flexible linker sequences is obtained or be manually respectively synthesized by reverse transcription In vain, the target gene of chicken interferon gamma, recombinant chIL-2；By flexible linker by OVA, chicken interferon gamma, chicken The target gene of interleukin 2 connects, the nucleotides sequence list such as SEQUENCE of the target gene after connection Shown in the ＞ of 400 ＜ of LISTING 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/rAlb- IFNγ-IL2。

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 OVA (Alb) is：

Upstream Alb-F1：CCGGAATTCATGAAGTGGGTAACATTA, with EcoRI restriction enzyme sites；

Downstream Alb-R1：

ACCACCACCAGAACCACCACCACCAGCACCAATTCCTAATG, 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；

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

Upstream IL-2-F1：

GGTGGTTCTGGTGGTGGTGGTTCTATGATGTGCAAAGTACT, with flexible linker；

Downstream IL-2-R1：CCCTCGAGAGTTTTTTGCAGATATC, with XhoI restriction enzyme sites；

B. RNA is extracted from chicken liver, obtains the target gene of chicken Alb, chicken IFN-γ and chicken IL-2 gene by reverse transcription, three The gene order of person 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 chicken Alb, chicken IFN-γ and chicken IL-2 gene target gene as template, and it is utilized respectively chicken Alb, chicken IFN-γ Enter performing PCR amplification with the upstream and downstream primer of chicken IL-2 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. rAlb-IFN γ genes are obtained using flexible linker connection chicken Alb 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 Alb gene templates DNA 1, connect flexible linker 1 μ L, Alb sense primer of IFN-γ template DNA 0.5 μ L, IFN- 0.5 μ L, Taq archaeal dna polymerase of γ anti-sense primers 2.5 μ L, dNTP Mix is 10 μ L, adds RNase Free water to 25 μ L；Connection PCR reaction conditions are：95 DEG C of pre-degeneration 4min, into 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. rAlb-IFN γ-IL2 bases are obtained using flexible linker connections rAlb-IFN γ genes and chicken IL2 target gene Cause

The PCR reaction systems and reaction condition of connection be：In 25 μ L overall reaction system, rAlb-IFN γ gene templates The μ L of DNA 1, the flexible linker μ L of 1 μ L, Alb sense primer of IL-2 template DNAs, 0.5 μ L, IL-2 anti-sense primer 0.5 are connected, Taq archaeal dna polymerases 2.5 μ L, dNTP Mix is 10 μ L, adds RNase Free water to 25 μ L；Connecting PCR reaction conditions is：95℃ 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；Last 72 DEG C extension 10min.

Genome 2 is artificial synthesized gene after being optimized to genome 1, the acquisition methods of the genome 2 For：

A. design of primers

The primer sequence of OVA (Alb) is：

Upstream Alb-F2：CGGGATCCATGAAATGGGTTACCC, with BamHI restriction enzyme sites；

Downstream Alb-R2：

ACCACCACCAGAACCACCACCACCAGCACCGATACCCA, 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；

Recombinant chIL-2 (IL-2)：

Upstream IL-2-F2：

GGTGGTTCTGGTGGTGGTGGTTCTATGATGTGCAAAGTTCT, with flexible linker；

Downstream IL-2-R2：

CCCTCGAGTTTCTGCAGGTAACG, with XhoI restriction enzyme sites.

B. the target gene of the chicken Alb, chicken IFN-γ and chicken IL-2 gene, 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 Alb gene templates DNA 1, connect flexible linker 1 μ L, Alb sense primer of IFN-γ template DNA 0.5 μ L, IFN- 0.5 μ L, Taq archaeal dna polymerase of γ anti-sense primers 2.5 μ L, dNTP Mix is 10 μ L, adds RNase Free water to 25 μ L；Connection PCR reaction conditions are：95 DEG C of pre-degeneration 4min, into 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.

D. rAlb-IFN γ-IL2 are obtained using flexible linker connections rAlb-IFN γ genes and chicken IL-2 gene target gene Gene

The PCR reaction systems and reaction condition of connection be：In 25 μ L overall reaction system, rAlb-IFN γ gene templates The μ L of DNA 1, the flexible linker μ L of 1 μ L, Alb sense primer of IL-2 template DNAs, 0.5 μ L, IL-2 anti-sense primer 0.5 are connected, Taq archaeal dna polymerases 2.5 μ L, dNTP Mix is 10 μ L, adds RNase Free water to 25 μ L；Connecting PCR reaction conditions is：95℃ 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；Last 72 DEG C extension 10min.

Present invention also offers the application of the recombination chicken long-acting interferon, its long half time had up to more than 84 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 Alb, chicken IFN-γ and chicken IL-2 gene gene are realized into amalgamation and expression by flexible linker, interferon is improved Half-life period, compared with plain interferon, improve more than 21 times；

2. by being optimized to chicken Alb, chicken IFN-γ and chicken IL-2 gene gene, chicken Alb, chicken IFN-γ and chicken are improved The expression quantity of IL-2 fusion proteins.

3. using recombination bacillus coli pET-32a/rAlb-IFN γ-IL2 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 Alb, chicken IFN-γ and chicken IL-2 gene not only has IFN-γ Broad-spectrum disease resistance toxic action, while significantly improve the immune response of chicken itself.

Brief description of the drawings

Fig. 1 is that OVA gene, the gene of chicken interleukin-2 2 and chicken interferon gamma gene RT-PCR in embodiment 1 are expanded Result；Swimming lane M：DNA Marker DL2000；Swimming lane 1：The gene RT-PCR amplified productions of chicken interleukin-2 2；Swimming lane 2：Chicken disturbs Plain γ genes RT-PCR amplified productions；Swimming lane 3：OVA gene RT-PCR amplified productions；

Fig. 2 is the result of the PCR amplifications after chicken Alb, the IFN γ in embodiment 1 connect with IL-2 target gene；Swimming Road M：DNA Marker DL10000；Swimming lane 1：OVA gene, chicken interferon gamma gene are connected expansion with the gene of chicken interleukin-2 2 Increase production thing；

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：Supernatant after bacterial cell disruption after recombinant bacterium induction；Swimming lane 2：Precipitated after bacterial cell disruption after recombinant bacterium induction；Swimming lane 3：It is unloaded Control；

Fig. 5 is the Western Blot qualification results for the fusion protein that embodiment 1 obtains；Swimming lane M：Albumen Marker；Swimming Road 1：Precipitated after recombinant bacterium induction is broken；Swimming lane 2：For the broken rear supernatant of 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 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 OVA, chicken interferon gamma and recombinant chIL-2, its preparation method is such as Under：

1. the acquisition of OVA (Alb), chicken interferon gamma (IFN-γ) and recombinant chIL-2 (IL-2) target gene With amplification

Design of primers：

Objective gene sequence design synthetic primer according to having been reported in Genebank is shown in Table 1, in the upstream of OVA EcoRI restriction enzyme sites and Linker sequences are introduced in primer and anti-sense primer respectively, chicken interferon gamma sense primer and under Linker sequences are introduced respectively in trip primer, are introduced respectively in the sense primer and anti-sense primer of recombinant chIL-2 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 target gene of chicken Alb, chicken IFN-γ and chicken IL-2 gene is obtained by reverse transcription, The gene order of three is respectively such as the ＞ of 400 ＜ of SEQUENCE LISTING 4, the ＞ and SEQUENCE of 400 ＜ of SEQUENCE LISTING 5 Shown in the ＞ of 400 ＜ of 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 1870bp, 550bp and 460bp or so in RT-PCR amplified productions, Its result is as shown in figure 1, chicken Alb, chicken IFN-γ and chicken IL-2 gene target gene has been prepared in explanation.

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 rAlb-IFN γ PCR reaction systems of table 3

RAlb-IFN γ-IL2PCR the reaction systems of table 4

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 2830bp or so in pcr amplification product, its result as shown in Fig. 2 Occur rAlb-IFN γ and IL-2 amplified production bands in Fig. 2, because connected in rAlb-IFN γ with IL-2 genes During, there is non-specific responding.The nucleotide sequence of the obtained target gene such as ＞ institutes of 400 ＜ of SEQUENCE LISTING 2 Show.

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

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 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 2830bp or so places, and its result is as shown in Figure 3.

4. the expression of recombinant protein

Picking engineering bacteria 37 DEG C of shaking table recovery 1h, engineering bacteria in the LB culture mediums containing 100 μ g/ml ampicillins are designated as pET-32a/rAlb-IFNγ-IL2；The amplification culture 4h (OD ≈ 1.0) in LB culture mediums (the μ g/ml containing ampicillin 100), Add final concentration 100 μ g/ml IPTG, 32 DEG C of induced expression 5h；Thalline is collected, through SDS-PAGE electrophoresis detections, its result is as schemed Shown in 4, it can be seen that to be deposited in 121.9KD or so places visible for supernatant after bacterial cell disruption after recombinant bacterium induction 5h Predominant expression band, illustrate precipitating with equal successful expression in supernatant 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

5.1 His affinity chromatographys

After membrane filtration of the rough fusion protein with 0.22 μm of aperture, loading is by being connected to AKTA explorer 100 On protein purification system, the His affinity columns 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 rAlb-IFN γ-IL2 protein peaks.

5.2 DEAE anion-exchange chromatographies

(the 50mM trihydroxy methyls of Binding Buffer II are arrived into the albumen collected after His affinitive layer purifications displacement Aminomethane, PH6.5) in after, 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 rAlb-IFN γ-IL2 protein peaks.

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 rAlb-IFN γ-IL2 protein peaks.

5.4 sample identification

Determine rAlb-IFN γ-IL2 potency and specific activity, specific activity >=10⁷IU/mg, albumen are qualified；It is aseptic subpackaged, -80 DEG C preserve.The i.e. available fusion protein being made up of OVA, chicken interferon gamma and recombinant chIL-2, its amino acid sequence Row are as shown in the ＞ of 400 ＜ of SEQUENCE LISTING 1.

Embodiment 2

A kind of fusion protein being made up of OVA, chicken interferon gamma and recombinant chIL-2, other with embodiment 1, Simply e. coli bl21 therein (DE3) competent cell is replaced for BL21 (DE3) competence with pGro7 plasmids Cell.The SDS-PAGE electrophoresis results of its fusion protein compare with embodiment 1,121.9KD 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

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

1.1 codon optimization

Genetic codon has 64 kinds, but most biological tendencies are in utilizing the part in these codons.Those By the most frequent referred to as 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, in the present embodiment to chicken Alb, chicken IFN-γ and Chicken IL-2 gene gene codon optimizes.

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 Alb, chicken are found using software detection The codon of IFN-γ and chicken IL-2 gene original gene codon adaptation indexI (CAI) in Escherichia coli is respectively 0.27,0.25, 0.27, GC percentage is 43.1%, 42.9%, 36.1%；And by after to chicken Alb, chicken IFN-γ and chicken IL-2 gene gene optimization Obtain recombination codon adaptation indexI (CAI) in Escherichia coli be respectively 0.99,1.0,1.0, GC percentages 49.2%, 47.6%th, 46.2%.The utilization rate of low codon is significantly reduced by gene optimization, avoids rare codon to albumen table The influence reached, 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 Alb after optimization, chicken IFN-γ and chicken IL-2 gene 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 Alb, chicken IFN-γ and chicken IL-2 gene pcr amplification product are through agarose gel electrophoresis respectively in 1870bp, 550bp There is specific band with 460bp or so, illustrate to be prepared the purpose base of chicken Alb after optimization, chicken IFN-γ and chicken IL-2 gene Cause.

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 rAlb-IFN γ PCR reaction systems of table 9

RAlb-IFN γ-IL2PCR the reaction systems of table 10

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 2830bp or so in pcr amplification product, illustrate successfully to be connected RAlb-IFN γ-IL2 genes after connecing, the nucleotide sequence such as ＞ of 400 ＜ of SEQUENCE LISTING 3 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 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

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 2830bp or so places in sepharose electrophoresis, illustrates that the expression containing rAlb-IFN γ-IL2 fusions carries Body successfully constructs.

4. the expression of recombinant protein

Picking engineering bacteria 37 DEG C of shaking table recovery 1h, engineering bacteria in the LB culture mediums containing 100 μ g/ml ampicillins are designated as pET-32a/rAlb-IFNγ-IL2；The amplification culture 4h (OD ≈ 1.0) in LB culture mediums (the μ g/ml containing ampicillin 100), Add final concentration 100 μ g/ml IPTG, 32 DEG C of induced expression 5h；Thalline is collected, through SDS-PAGE electrophoresis detections, recombinant bacterium induction Supernatant is deposited in the visible predominant expression band in 121.9KD or so places after bacterial cell disruption after 5h, illustrates in supernatant precipitates Recombinant protein is obtained.

5. fusion protein purification

5.1 His affinity chromatographys

5.2 DEAE anion-exchange chromatographies

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 rAlb-IFN γ-IL2 protein peaks.

5.4 sample identification

Determine rAlb-IFN γ-IL2 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 OVA, chicken interferon gamma and recombinant chIL-2, its amino acid Sequence is as shown in the ＞ of 400 ＜ of SEQUENCE LISTING 1.

Embodiment 4

A kind of fusion protein being made up of OVA, chicken interferon gamma and recombinant chIL-2, other with embodiment 3, Simply e. coli bl21 therein (DE3) competent cell is replaced for BL21 (DE3) competence with pGro7 plasmids Cell.The SDS-PAGE electrophoresis results of its fusion protein compare with embodiment 3,121.9KD 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, by the fusion protein in embodiment 1,2,3,4 respectively with freeze drying protectant mixture Afterwards, it is freeze-dried to form.The freeze drying protectant is glycerine, mannitol and sucrose, is buffer solution with 10mmol/L PBS, Final concentration of glycerine 100mL/L, mannitol 0.12g/mL and the sucrose 0.025g/mL of three.

Embodiment 6

Embodiment 1~4 obtains the mirror for the fusion protein being made up of OVA, chicken interferon gamma and recombinant chIL-2 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.

6.2 SDS-PAGE electrophoresis detections

Compared with empty bacterium, fusion protein has the newly-increased protein band of a dense dye in 121.9KD or so, as shown in Figure 4.

6.3 Western Blot results

Fusion protein in embodiment 1~4 is detected respectively, with the anti-chicken interferon-γ (1 of abcam companies mouse:5000 dilutions) be Primary antibody, using goat anti-mouse IgG-HRP as secondary antibody (1:10000 dilutions).Recombination chicken long-acting interferon sample can disturb with anti-chicken Plain γ monoclonal antibodies occur specific reaction, 121.9KD or so place and specific band occur, as shown in Figure 5.

Embodiment 7

The freeze-dried bioactivity of four parts of recombination chicken long-acting interferons 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 is imitated, inhales and abandons after 24h, then is inoculated with 100 TCID50 VSV viruses respectively.

Result of the test

As a result the recombination chicken long-acting interferon 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 processing, the Continuous Observation under inverted microscope, cellular morphology is normal, does not occur any Lesion, measure potency >=10⁷IU/ml, as shown in Figure 6.

Embodiment 8

The four parts of recombination chicken long-acting interferons obtained respectively by the fusion protein of embodiment 1~4 in embodiment 5 are freeze-dried The measure of the half-life period of (being designated as A, B, C, D respectively) in chicken body

Cytopathic-effect inhibition assay measure rAlb-IFN γ-IL2 blood concentration and time relationship

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 Freeze-dried 2ml is low in 1h, 2h, 4h, 8h, 16h, 32h, 48h, 72h, 96h venous blood collection, 4 DEG C of solidifications of blood sample, 3500rpm respectively Temperature centrifugation 10min separation serum, each every chicken blood sample of time point are to be measured in -20 DEG C of preservations.Blood is determined using cytopathic-effect inhibition assay RAlb-IFN γ-IL2 concentration in final proof product, 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 intramuscular injection of table 13

As a result show that recombination chicken long-acting interferon has longer half-life period.Half-life period can reach 84h or so after measured, more general Logical interferon improves about 21 times.

Embodiment 9

The freeze-dried measure influenceed on chicken cell immune response of four parts of recombination chicken long-acting interferons 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 freeze-dried 2ml of recombination chicken long-acting interferon 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, containing for chicken Evaluation of Cytokines in Peripheral Blood IL-4 can be significantly improved Amount, 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 OVA, chicken interferon gamma and recombinant chIL-2 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>Wuhu Co., Ltd of Ying Tefeier biological products industrial research institute

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

Method

<130> 1

<160> 9

<170> PatentIn version 3.3

<210> 1

<211> 942

<212> PRT

<213>Fusion protein

<400> 1

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

1 5 10 15

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

20 25 30

Glu Ile Ala His Arg Tyr Asn Asp Leu Lys Glu Glu Thr Phe Lys Ala

35 40 45

Val Ala Met Ile Thr Phe Ala Gln Tyr Leu Gln Arg Cys Ser Tyr Glu

50 55 60

Gly Leu Ser Lys Leu Val Lys Asp Val Val Asp Leu Ala Gln Lys Cys

65 70 75 80

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

85 90 95

Ile Leu Asp Glu Ile Cys Gln Val Glu Lys Leu Arg Asp Ser Tyr Gly

100 105 110

Ala Met Ala Asp Cys Cys Ser Lys Ala Asp Pro Glu Arg Asn Glu Cys

115 120 125

Phe Leu Ser Phe Lys Val Ser Gln Pro Asp Phe Val Gln Pro Tyr Gln

130 135 140

Arg Pro Ala Ser Asp Val Ile Cys Gln Glu Tyr Gln Asp Asn Arg Val

145 150 155 160

Ser Phe Leu Gly His Phe Ile Tyr Ser Val Ala Arg Arg His Pro Phe

165 170 175

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

180 185 190

Leu Gln Ser Cys Cys Lys Glu Ser Asp Val Gly Ala Cys Leu Asp Thr

195 200 205

Lys Glu Ile Val Met Arg Glu Lys Ala Lys Gly Val Ser Val Lys Gln

210 215 220

Gln Tyr Phe Cys Gly Ile Leu Lys Gln Phe Gly Asp Arg Val Phe Gln

225 230 235 240

Ala Arg Gln Leu Ile Tyr Leu Ser Gln Lys Tyr Pro Lys Ala Pro Phe

245 250 255

Ser Glu Val Ser Lys Phe Val His Asp Ser Ile Gly Val His Lys Glu

260 265 270

Cys Cys Glu Gly Asp Met Val Glu Cys Met Asp Asp Met Ala Arg Met

275 280 285

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

290 295 300

Asp Cys Cys Glu Lys Pro Ile Val Glu Arg Ser Gln Cys Ile Met Glu

305 310 315 320

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

325 330 335

Tyr Ile Glu Asp Lys Glu Val Cys Lys Ser Phe Glu Ala Gly His Asp

340 345 350

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

355 360 365

Phe Ser Ile Gln Leu Ile Met Arg Ile Ala Lys Gly Tyr Glu Ser Leu

370 375 380

Leu Glu Lys Cys Cys Lys Thr Asp Asn Pro Ala Glu Cys Tyr Ala Asn

385 390 395 400

Ala Gln Glu Gln Leu Asn Gln His Ile Lys Glu Thr Gln Asp Val Val

405 410 415

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

420 425 430

Lys Ser Ile Leu Ile Arg Tyr Thr Lys Lys Met Pro Gln Val Pro Thr

435 440 445

Asp Leu Leu Leu Glu Thr Gly Lys Lys Met Thr Thr Ile Gly Thr Lys

450 455 460

Cys Cys Gln Leu Pro Glu Asp Arg Arg Met Ala Cys Ser Glu Gly Tyr

465 470 475 480

Leu Ser Ile Val Ile His Asp Thr Cys Arg Lys Gln Glu Thr Thr Pro

485 490 495

Ile Asn Asp Asn Val Ser Gln Cys Cys Ser Ser Ser Tyr Ala Asn Arg

500 505 510

Arg Pro Cys Phe Thr Ala Met Gly Val Asp Thr Lys Tyr Val Pro Pro

515 520 525

Pro Phe Asn Pro Asp Met Phe Ser Phe Asp Glu Lys Leu Cys Ser Ala

530 535 540

Pro Ala Glu Glu Arg Glu Val Gly Gln Met Lys Leu Leu Ile Asn Leu

545 550 555 560

Ile Lys Arg Lys Pro Gln Met Thr Glu Glu Gln Ile Lys Thr Ile Ala

565 570 575

Asp Gly Phe Thr Ala Met Val Asp Lys Cys Cys Lys Gln Ser Asp Ile

580 585 590

Asn Thr Cys Phe Gly Glu Glu Gly Ala Asn Leu Ile Val Gln Ser Arg

595 600 605

Ala Thr Leu Gly Ile Gly Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly

610 615 620

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

625 630 635 640

Ile Tyr Tyr Gly His Thr Ala Ser Ser Leu Ile Leu Val Gln Leu Gln

645 650 655

Asp Asp Ile Ala Lys Leu Lys Ala Asp Phe Asn Ser Ser His Ser Asp

660 665 670

Val Ala Asp Gly Gly Pro Ile Ile Ala Glu Lys Leu Lys Asn Trp Thr

675 680 685

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

690 695 700

Leu Glu Met Leu Ala Asn Thr Asp Lys Thr Lys Pro His Thr Lys His

705 710 715 720

Ile Ser Glu Glu Leu Tyr Thr Leu Lys Asn Asn Leu Pro Asp Gly Val

725 730 735

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

740 745 750

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

755 760 765

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

770 775 780

Arg Arg Cys Asn Cys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Met

785 790 795 800

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

805 810 815

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

820 825 830

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

835 840 845

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

850 855 860

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

865 870 875 880

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

885 890 895

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

900 905 910

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

915 920 925

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

930 935 940

<210> 2

<211> 2826

<212> DNA

<213>Genome 1

<400> 2

atgaagtggg taacattaat ttcattcatt ttcctcttca gttcagcaac atccaggaat 60

ctgcaaagat ttgctcgtga tgcagagcac aagagtgaaa ttgcccatcg ctacaatgat 120

ttgaaagaag aaacatttaa ggcagttgcc atgatcacat ttgcccagta tctccagagg 180

tgctcttatg aaggactgtc taagcttgtg aaggatgttg ttgatctggc acaaaaatgt 240

gtagccaatg aagatgctcc tgaatgctca aaaccactgc cttccattat cctggatgaa 300

atctgccaag tggaaaagct ccgtgactct tatggtgcaa tggccgactg ctgtagcaaa 360

gctgatcctg aaagaaatga gtgtttcctg tcatttaaag tttcccaacc agacttcgtt 420

cagccatacc aaagaccagc ttctgatgtg atatgccagg aataccagga caacagagtg 480

tcatttctgg gacatttcat ctattctgtt gcaagaagac accccttctt gtatgcccct 540

gcaatcctta gttttgctgt tgattttgaa catgcacttc aaagctgttg caaagagagt 600

gatgtcggtg cttgcctgga caccaaggaa attgttatga gagaaaaagc caagggagta 660

agtgtgaagc agcagtattt ttgtggaatc ttgaagcagt tcggagatag agttttccaa 720

gcacgacaac ttatttacct aagccaaaaa taccccaagg ctccattctc agaggtttct 780

aaatttgtac atgattctat cggcgtccac aaagagtgct gtgaagggga catggtggag 840

tgcatggatg acatggcacg tatgatgagc aatctgtgct ctcaacaaga tgttttctca 900

ggtaaaatca aagactgctg tgagaagcct attgtggaac gaagccagtg cattatggag 960

gcagaatttg atgagaaacc tgcagatctt ccttcattag ttgaaaagta catagaagat 1020

aaggaagtgt gtaaaagttt tgaagcaggc cacgatgcat tcatggcaga gttcgtttat 1080

gaatactcac gaagacaccc tgagttctcc atacagctta ttatgagaat tgccaaagga 1140

tatgaatcac ttctggaaaa gtgctgcaaa actgataacc ctgctgagtg ctacgcaaat 1200

gctcaagagc aactgaacca acatatcaaa gaaactcagg atgttgtgaa gacaaactgt 1260

gatcttctcc atgaccatgg cgaggcagac ttcctcaagt ccatcctgat ccgctacact 1320

aagaaaatgc ctcaagtacc aactgatctc ctgcttgaaa ctggaaagaa aatgacaact 1380

attggtacta agtgctgcca gcttcctgaa gacagacgca tggcttgttc tgagggttat 1440

ctgagcattg tgattcatga tacgtgcagg aaacaggaga ccacacctat aaatgacaac 1500

gtttcacaat gctgcagcag ctcctatgct aacagaagac catgtttcac tgctatggga 1560

gtagatacca aatatgttcc tccaccattt aatcctgata tgttcagctt tgatgaaaaa 1620

ttgtgcagtg ctcctgctga agaacgagaa gtaggccaga tgaaattgct aatcaacctc 1680

attaaacgca agccccagat gacagaagaa caaataaaga caattgctga tggtttcact 1740

gccatggttg acaagtgctg caagcagtcg gacatcaata catgctttgg agaagagggt 1800

gccaacctaa tagtccaaag cagagccaca ttaggaattg gtgctggtgg tggtggttct 1860

ggtggtggtg gttctatgac ttgccagact tacaacttgt ttgttctgtc cgtcatcatg 1920

atttattatg gacatactgc aagtagtcta attcttgttc aacttcaaga tgatatagcc 1980

aaactgaaag ctgactttaa ctcaagtcat tcagatgtag ctgacggtgg acctattatt 2040

gcagagaaac tgaagaactg gacagagaga aatcagaaaa ggatcatact gagccagatt 2100

gtttcgatgt acttggaaat gcttgcaaac actgacaaga caaagccgca caccaaacac 2160

atatctgagg agctctatac tctgaaaaac aaccttcctg atggcgtgaa gaaggtgaaa 2220

gatatcatgg acctggccaa gctcccgatg aacgacttga gagtccagct caaagccgcg 2280

aatgaactct tcagcatctt acagaagctg gtgaatcctc cgagtttcaa aaggaacatg 2340

agccagtctc agaggagatg caattgcggt ggtggtggtt ctggtggtgg tggttctatg 2400

atgtgcaaag tactgatctt tggctgtatt tcggtagcaa tgctaatgac tacagcttat 2460

ggagcatctc tatcatcaga aaaatggaaa actcttcaaa cattaataaa ggatttagaa 2520

atattggaaa atatcaagaa taagattcat ctcgagctct acacaccaac tgagacccag 2580

gagtgcaccc agcaaactct gcagtgttac ctgggagaag tggttactct gaagaaagaa 2640

actgaagatg acactgaaat taaagaagaa tttgtaactg ctattcaaaa tatcgaaaag 2700

aacctcaaga gtcttacggg tctaaatcac accggaagtg aatgcaagat ctgtgaagct 2760

aacaacaaga aaaaatttcc tgattttctc catgaactga ccaactttgt gagatatctg 2820

caaaaa 2826

<210> 3

<211> 2826

<212> DNA

<213>Genome 2

<400> 3

atgaaatggg ttaccctgat ctctttcatc ttcctgttct cttctgctac ctctcgtaac 60

ctgcagcgtt tcgctcgtga cgctgaacac aaatctgaaa tcgctcaccg ttacaacgac 120

ctgaaagaag aaaccttcaa agctgttgct atgatcacct tcgctcagta cctgcagcgt 180

tgctcttacg aaggtctgtc taaactggtt aaagacgttg ttgacctggc tcagaaatgc 240

gttgctaacg aagacgctcc ggaatgctct aaaccgctgc cgtctatcat cctggacgaa 300

atctgccagg ttgaaaaact gcgtgactct tacggtgcta tggctgactg ctgctctaaa 360

gctgacccgg aacgtaacga atgcttcctg tctttcaaag tttctcagcc ggacttcgtt 420

cagccgtacc agcgtccggc ttctgacgtt atctgccagg aataccagga caaccgtgtt 480

tctttcctgg gtcacttcat ctactctgtt gctcgtcgtc acccgttcct gtacgctccg 540

gctatcctgt ctttcgctgt tgacttcgaa cacgctctgc agtcttgctg caaagaatct 600

gacgttggtg cttgcctgga caccaaagaa atcgttatgc gtgaaaaagc taaaggtgtt 660

tctgttaaac agcagtactt ctgcggtatc ctgaaacagt tcggtgaccg tgttttccag 720

gctcgtcagc tgatctacct gtctcagaaa tacccgaaag ctccgttctc tgaagtttct 780

aaattcgttc acgactctat cggtgttcac aaagaatgct gcgaaggtga catggttgaa 840

tgcatggacg acatggctcg tatgatgtct aacctgtgct ctcagcagga cgttttctct 900

ggtaaaatca aagactgctg cgaaaaaccg atcgttgaac gttctcagtg catcatggaa 960

gctgaattcg acgaaaaacc ggctgacctg ccgtctctgg ttgaaaaata catcgaagac 1020

aaagaagttt gcaaatcttt cgaagctggt cacgacgctt tcatggctga attcgtttac 1080

gaatactctc gtcgtcaccc ggaattctct atccagctga tcatgcgtat cgctaaaggt 1140

tacgaatctc tgctggaaaa atgctgcaaa accgacaacc cggctgaatg ctacgctaac 1200

gctcaggaac agctgaacca gcacatcaaa gaaacccagg acgttgttaa aaccaactgc 1260

gacctgctgc acgaccacgg tgaagctgac ttcctgaaat ctatcctgat ccgttacacc 1320

aaaaaaatgc cgcaggttcc gaccgacctg ctgctggaaa ccggtaaaaa aatgaccacc 1380

atcggtacca aatgctgcca gctgccggaa gaccgtcgta tggcttgctc tgaaggttac 1440

ctgtctatcg ttatccacga cacctgccgt aaacaggaaa ccaccccgat caacgacaac 1500

gtttctcagt gctgctcttc ttcttacgct aaccgtcgtc cgtgcttcac cgctatgggt 1560

gttgacacca aatacgttcc gccgccgttc aacccggaca tgttctcttt cgacgaaaaa 1620

ctgtgctctg ctccggctga agaacgtgaa gttggtcaga tgaaactgct gatcaacctg 1680

atcaaacgta aaccgcagat gaccgaagaa cagatcaaaa ccatagcgga cggcttcacc 1740

gctatggttg acaaatgctg caaacagtct gacatcaaca cctgcttcgg tgaagaaggt 1800

gctaacctga tcgttcagtc tcgtgctacc ctgggtatcg gtgctggtgg tggtggttct 1860

ggtggtggtg gttctatgac ctgccagacc tacaacctgt tcgttctgtc tgttatcatg 1920

atctactacg gtcacaccgc ttcttctctg atcctggttc agctgcagga cgacatcgct 1980

aaactgaaag ctgacttcaa ctcttctcac tctgacgttg ctgacggtgg tccgatcatc 2040

gctgaaaaac tgaaaaactg gaccgaacgt aaccagaaac gtatcatcct gtctcagatc 2100

gtttctatgt acctggaaat gctggctaac accgacaaaa ccaaaccgca caccaaacac 2160

atctctgaag aactgtacac cctgaaaaac aacctgccgg acggtgttaa aaaagttaaa 2220

gacatcatgg acctggctaa actgccgatg aacgacctgc gtgttcagct gaaagctgct 2280

aacgaactgt tctctatcct gcagaaactg gttaacccgc cgtctttcaa acgtaacatg 2340

tctcagtctc agcgtcgttg caactgcggt ggtggtggtt ctggtggtgg tggttctatg 2400

atgtgcaaag ttctgatctt cggttgcatc tctgttgcta tgctgatgac caccgcttac 2460

ggtgcttctc tgtcttctga aaaatggaaa accctgcaga ccctgatcaa agacctggaa 2520

atcctggaaa acatcaaaaa caaaatccac ctggaactgt acaccccgac cgaaacccag 2580

gaatgcaccc agcagaccct gcagtgctac ctgggtgaag ttgttaccct gaaaaaagaa 2640

accgaagacg acaccgaaat caaagaagaa ttcgttaccg ctatccagaa catcgaaaaa 2700

aacctgaaat ctctgaccgg tctgaaccac accggttctg aatgcaaaat ctgcgaagct 2760

aacaacaaaa aaaaattccc ggacttcctg cacgaactga ccaacttcgt tcgttacctg 2820

cagaaa 2826

<210> 4

<211> 1845

<212> DNA

<213>OVA

<400> 4

atgaagtggg taacattaat ttcattcatt ttcctcttca gttcagcaac atccaggaat 60

ctgcaaagat ttgctcgtga tgcagagcac aagagtgaaa ttgcccatcg ctacaatgat 120

ttgaaagaag aaacatttaa ggcagttgcc atgatcacat ttgcccagta tctccagagg 180

tgctcttatg aaggactgtc taagcttgtg aaggatgttg ttgatctggc acaaaaatgt 240

gtagccaatg aagatgctcc tgaatgctca aaaccactgc cttccattat cctggatgaa 300

atctgccaag tggaaaagct ccgtgactct tatggtgcaa tggccgactg ctgtagcaaa 360

gctgatcctg aaagaaatga gtgtttcctg tcatttaaag tttcccaacc agacttcgtt 420

cagccatacc aaagaccagc ttctgatgtg atatgccagg aataccagga caacagagtg 480

tcatttctgg gacatttcat ctattctgtt gcaagaagac accccttctt gtatgcccct 540

gcaatcctta gttttgctgt tgattttgaa catgcacttc aaagctgttg caaagagagt 600

gatgtcggtg cttgcctgga caccaaggaa attgttatga gagaaaaagc caagggagta 660

agtgtgaagc agcagtattt ttgtggaatc ttgaagcagt tcggagatag agttttccaa 720

gcacgacaac ttatttacct aagccaaaaa taccccaagg ctccattctc agaggtttct 780

aaatttgtac atgattctat cggcgtccac aaagagtgct gtgaagggga catggtggag 840

tgcatggatg acatggcacg tatgatgagc aatctgtgct ctcaacaaga tgttttctca 900

ggtaaaatca aagactgctg tgagaagcct attgtggaac gaagccagtg cattatggag 960

gcagaatttg atgagaaacc tgcagatctt ccttcattag ttgaaaagta catagaagat 1020

aaggaagtgt gtaaaagttt tgaagcaggc cacgatgcat tcatggcaga gttcgtttat 1080

gaatactcac gaagacaccc tgagttctcc atacagctta ttatgagaat tgccaaagga 1140

tatgaatcac ttctggaaaa gtgctgcaaa actgataacc ctgctgagtg ctacgcaaat 1200

gctcaagagc aactgaacca acatatcaaa gaaactcagg atgttgtgaa gacaaactgt 1260

gatcttctcc atgaccatgg cgaggcagac ttcctcaagt ccatcctgat ccgctacact 1320

aagaaaatgc ctcaagtacc aactgatctc ctgcttgaaa ctggaaagaa aatgacaact 1380

attggtacta agtgctgcca gcttcctgaa gacagacgca tggcttgttc tgagggttat 1440

ctgagcattg tgattcatga tacgtgcagg aaacaggaga ccacacctat aaatgacaac 1500

gtttcacaat gctgcagcag ctcctatgct aacagaagac catgtttcac tgctatggga 1560

gtagatacca aatatgttcc tccaccattt aatcctgata tgttcagctt tgatgaaaaa 1620

ttgtgcagtg ctcctgctga agaacgagaa gtaggccaga tgaaattgct aatcaacctc 1680

attaaacgca agccccagat gacagaagaa caaataaaga caattgctga tggtttcact 1740

gccatggttg acaagtgctg caagcagtcg gacatcaata catgctttgg agaagagggt 1800

gccaacctaa tagtccaaag cagagccaca ttaggaattg gtgct 1845

<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> 429

<212> DNA

<213>Chicken IL-2 gene

<400> 6

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> 7

<211> 1845

<212> DNA

<213>OVA

<400> 7

atgaaatggg ttaccctgat ctctttcatc ttcctgttct cttctgctac ctctcgtaac 60

ctgcagcgtt tcgctcgtga cgctgaacac aaatctgaaa tcgctcaccg ttacaacgac 120

ctgaaagaag aaaccttcaa agctgttgct atgatcacct tcgctcagta cctgcagcgt 180

tgctcttacg aaggtctgtc taaactggtt aaagacgttg ttgacctggc tcagaaatgc 240

gttgctaacg aagacgctcc ggaatgctct aaaccgctgc cgtctatcat cctggacgaa 300

atctgccagg ttgaaaaact gcgtgactct tacggtgcta tggctgactg ctgctctaaa 360

gctgacccgg aacgtaacga atgcttcctg tctttcaaag tttctcagcc ggacttcgtt 420

cagccgtacc agcgtccggc ttctgacgtt atctgccagg aataccagga caaccgtgtt 480

tctttcctgg gtcacttcat ctactctgtt gctcgtcgtc acccgttcct gtacgctccg 540

gctatcctgt ctttcgctgt tgacttcgaa cacgctctgc agtcttgctg caaagaatct 600

gacgttggtg cttgcctgga caccaaagaa atcgttatgc gtgaaaaagc taaaggtgtt 660

tctgttaaac agcagtactt ctgcggtatc ctgaaacagt tcggtgaccg tgttttccag 720

gctcgtcagc tgatctacct gtctcagaaa tacccgaaag ctccgttctc tgaagtttct 780

aaattcgttc acgactctat cggtgttcac aaagaatgct gcgaaggtga catggttgaa 840

tgcatggacg acatggctcg tatgatgtct aacctgtgct ctcagcagga cgttttctct 900

ggtaaaatca aagactgctg cgaaaaaccg atcgttgaac gttctcagtg catcatggaa 960

gctgaattcg acgaaaaacc ggctgacctg ccgtctctgg ttgaaaaata catcgaagac 1020

aaagaagttt gcaaatcttt cgaagctggt cacgacgctt tcatggctga attcgtttac 1080

gaatactctc gtcgtcaccc ggaattctct atccagctga tcatgcgtat cgctaaaggt 1140

tacgaatctc tgctggaaaa atgctgcaaa accgacaacc cggctgaatg ctacgctaac 1200

gctcaggaac agctgaacca gcacatcaaa gaaacccagg acgttgttaa aaccaactgc 1260

gacctgctgc acgaccacgg tgaagctgac ttcctgaaat ctatcctgat ccgttacacc 1320

aaaaaaatgc cgcaggttcc gaccgacctg ctgctggaaa ccggtaaaaa aatgaccacc 1380

atcggtacca aatgctgcca gctgccggaa gaccgtcgta tggcttgctc tgaaggttac 1440

ctgtctatcg ttatccacga cacctgccgt aaacaggaaa ccaccccgat caacgacaac 1500

gtttctcagt gctgctcttc ttcttacgct aaccgtcgtc cgtgcttcac cgctatgggt 1560

gttgacacca aatacgttcc gccgccgttc aacccggaca tgttctcttt cgacgaaaaa 1620

ctgtgctctg ctccggctga agaacgtgaa gttggtcaga tgaaactgct gatcaacctg 1680

atcaaacgta aaccgcagat gaccgaagaa cagatcaaaa ccatagcgga cggcttcacc 1740

gctatggttg acaaatgctg caaacagtct gacatcaaca cctgcttcgg tgaagaaggt 1800

gctaacctga tcgttcagtc tcgtgctacc ctgggtatcg gtgct 1845

<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> 429

<212> DNA

<213>Chicken IL-2 gene

<400> 9

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

Claims

A kind of 1. fusion protein being made up of OVA, chicken interferon gamma and recombinant chIL-2, 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/rAlb-IFN γ-IL2, its preparation method are：

(1) primer is designed, is obtained by reverse transcription or is manually respectively synthesized the OVA for connecting flexible linker sequences, chicken The target gene of interferon gamma, recombinant chIL-2；By flexible linker by OVA, chicken interferon gamma, chicken leucocyte The target gene of interleukin 2 connects, the nucleotides sequence list such as SEQUENCE LISTING 400 of the target gene after connection Shown in the ＞ of ＜ 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/rAlb-IFN γ-IL2。
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 84 hours, there is broad-spectrum disease resistance toxic action and the immune response of chicken itself can be improved.