CN107266586A - A kind of Recombinant Swine long-acting interferon γ and prepare this long-acting interferon γ fusion protein and preparation method thereof - Google Patents
A kind of Recombinant Swine long-acting interferon γ and prepare this long-acting interferon γ fusion protein and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a kind of Recombinant Swine long-acting interferon γ and the fusion protein for preparing this long-acting interferon γ and preparation method thereof; the fusion protein is connected by pig interleukin 2 and 6 with Porcine interferon-gamma and formed through flexible linker, through being freeze-dried to obtain Recombinant Swine long-acting interferon γ after fusion protein and freeze drying protectant mixture.The Recombinant Swine long-acting interferon γ is remarkably improved the half-life period of pig interferon, and the half-life period of more common Porcine interferon-gamma improves more than 13 times, and with broad-spectrum disease resistance toxic action and can improve the immune response of pig itself.
Description
Technical field
The invention belongs to technical field of biological genetic engineering, and in particular to a kind of Recombinant Swine long-acting interferon γ and prepare this
Long-acting interferon γ fusion protein and preparation method thereof.
Background technology
Scale Compact Develop is rapidly growing in China in recent years, and China's live pig breeding stock and pork production are sure to occupy
First place in the world, traditional swine disease prevention and controls are far from the control for adapting to infectious disease in extensive intensive pig production production.I
State newly occurs in that nearly 20 kinds of livestock and poultry infectious diseases over nearly 20 years, add original animal epidemic, China's aquaculture is caused huge
Economic loss.According to incompletely statistics, China is every year because various viral diseases cause mortality of livestock up to 15%-20%,
Economic loss reaches billions of members.
The prevention and treatment approach to porcine viral diseases mainly by vaccine inoculation and uses antibiotic at present, but by
In breeding environment imperfection, virus variation and Abwehrkraft des Koepers change etc. reason, make traditional prevention and treatment approach by
Huge challenge, most of antibiotics and traditional oral antiviral medicament, due to medicament residue problem, to health
It is negatively affected;And traditional vaccine, high specific and side effect due to it, it is impossible to resist virus variation and new disease
The significant damage brought to pig aquaculture continuously emerges in poison.
IFN is that the infection induced body of a viroid is produced with broad-spectrum antiviral, antitumor and with immunoregulation effect
Protein, nineteen fifty-seven, Issacs and Lindeman had found first, and it is the multi-functional cell factor of a class, with cell receptor knot
After conjunction, it can induce body and produce many species-specific proteins and enzyme, mainly by suppressing viral gene transcription and degraded virus
RNA suppresses the growth and breeding of virus and plays the activity of antitumor grade.According to IFN generation cell, biochemical character and
The difference played a role in terms of immunity of organism, is divided into the class of γ, β, γ tri-.It is existing, it is known that γ types IFN be T cell by activating and
NK cells are produced, with relatively strong antiviral and immunoloregulation function.Numerous studies show that interferon gamma is except with broad-spectrum disease resistance
Outside malicious function, the adjustment effect of key is also played to immune system, so IFN-γ is also known as immunological regulation interferon.Although each
The interferon of type can mediated cell to virus infection reaction, but interferon gamma immunoregulatory activity coordinate exempt from
Epidemic disease is reacted and determines to play even more important effect in the long-term antiviral state of body, thus interferon gamma have it is particularly important
Clinical value.
Cell factor IL-2 is interleukin 2, also known as SCIF.The main T lymphocytes by activating are produced
The cell factor with extensive bioactivity, can both promote lymphopoiesis, strengthen immunologic function, but can restricted T it is thin
Born of the same parents react and strengthen the immune tolerance of body, therefore available for treatment tumour, infectious diseases and autoimmune disease.In animal doctor
In, because IL-2 can improve the immune response of vaccine and reduce the generation of disease, also there is wide application prospect.IL-2 is because of energy
Strengthen the immune level of body, improve the resistance against diseases of body, thus exempt from for bacillary, viral and parasitic diseases
Epidemic disease is treated.In addition, IL-2 can also influence the metabolism of medicine, make the metabolism time lengthening of medicine, action time increase, so as to improve
Curative effect of medication.IL-2, according to gene constructed, constitutes fusion protein with other cell factors, is produced and is carried with the antibody for strengthening vaccine
High cellular immune level.
The limitation of natural interferon and the current generally existing half-life short of artificial recombination interferon, half-life period is generally 2-4
Individual hour.Half-life short brings great inconvenience, the increase for the treatment of number of times, corresponding time cost and financial cost to treatment
Increase therewith, and the tenability limit of body is also possible to be broken the generation for causing adverse reaction.The main cause of half-life short
There are two:The too small tachytrophism in vivo of molecular weight of interferon, interferon especially recombinant interferon affinity is poor to be exempted from
Epidemic disease system is removed.And common long-acting interferon is using polyethylene glycol fused interferon as representative on the market, the layer only in molecular weight
Partly solved on face interferon molecule amount it is small and the problem of cause half-life short, while polyethylene glycol fused interferon cost is non-
Chang Gao, is unfavorable for clinically applying.
The content of the invention
In order to solve the above technical problems, the invention provides a kind of Recombinant Swine long-acting interferon γ and preparing this long-acting interference
Plain γ fusion protein and preparation method thereof, the Recombinant Swine long-acting interferon γ is remarkably improved the half-life period of pig interferon,
The half-life period of more common Porcine interferon-gamma improves more than 13 times, and with broad-spectrum disease resistance toxic action and can improve the immune of pig itself
Response.
The technical scheme that the present invention takes is:
A kind of fusion protein being made up of pig interleukin 2 and 6 and Porcine interferon-gamma, the amino acid sequence of the fusion protein
List is designated as fusion protein 1 as shown in the > of 400 < of SEQUENCE LISTING 1;Or such as the > institutes of 400 < of SEQUENCE LISTING 2
Show, be designated as fusion protein 2.
Present invention also offers the gene for encoding above-mentioned fusion protein, the nucleotides sequence list such as SEQUENCE of the gene
Shown in the > of 400 < of LISTING 3, genome 1 is designated as;Or as shown in the > of 400 < of SEQUENCE LISTING 4, it is designated as genome 2.
Fusion protein 1 described in the codified of genome 1;The codified fusion protein 2 of genome 2.Genome 2 is pair
The nucleotide sequence of genome 1 optimize after result, be considered as the base during usual codon adaptation indexI CAI=1.0
Because being optimal high efficient expression state in the expression system, CAI values are lower to show that expression is lower in host.In gene
G/C content most ideal distribution scope is 30~70%, and the scope is exceeded in any region can influence translation and transcriptional efficiency.
The codon of pig IL-2 and IFN-γ original gene codon adaptation indexI (CAI) in Escherichia coli is found using software detection
Respectively 0.23,0.24, GC percentages are 38.7%, 39.2%;And by being obtained after pig IL-2 and IFN-γ gene optimization
Recombination genome 2 in Escherichia coli codon adaptation indexI (CAI) be 0.97,1.0, GC percentages 47.6%,
45.4%.The utilization rate of low codon is significantly reduced by gene optimization, it is to avoid shadow of the rare codon to protein expression
Ring, improve the G/C content of gene, improve transcription and translation efficiency.
Present invention also offers the expression vector containing genome 1 or genome 2.
Further, the expression vector is the pET-32a coli expression carriers containing genome 1 or genome 2.
Present invention also offers the genetic engineering bacterium containing genome 1 or genome 2.
Further, the genetic engineering bacterium is pET-32a/rIL2-IFN γ.
Host cell containing genome 1 or genome 2 falls within protection scope of the present invention, further, the place
Chief cell is e. coli host cell, further, and the e. coli host cell is BL21 (DE3) competent cell
Or BL21 (DE3) competent cell with pGro7 plasmids.
Present invention also offers a kind of Recombinant Swine long-acting interferon γ, the Recombinant Swine long-acting interferon γ is melted by described
It is freeze-dried to form after hop protein and freeze drying protectant mixture.
The freeze drying protectant is glycerine, mannitol and sucrose, is buffer solution, the final concentration of three with 10mmol/L PBS
For glycerine 100mL/L, mannitol 0.12g/mL and sucrose 0.025g/mL.
The invention also discloses the preparation method of the fusion protein, the preparation method comprises the following steps:It will contain
The expression vector of genome 1 or genome 2 is imported into e. coli host cell, obtains genetic engineering bacterium, genetic engineering bacterium
The crude product of the fusion protein is obtained after IPTG induced expressions, it is purified to can obtain fusion protein afterwards.
The expression vector is the pET-32a coli expression carriers containing genome 1 or genome 2;
The genetic engineering bacterium is pET-32a/rIL2-IFN γ, and its preparation method is:
(1) primer is designed, is obtained by reverse transcription or the artificial synthesized pig leucocyte for connecting flexible linker sequences
The target gene of interleukin 2 and Porcine interferon-gamma;By flexible linker by pig interleukin 2 and 6 and the purpose base of Porcine interferon-gamma
Because connecting, the nucleotides sequence list of target gene is as shown in the > of 400 < of SEQUENCE LISTING 3 or such as SEQUENCE
Shown in the > of 400 < of LISTING 4;
(2) target gene after connection is connected on pET-32a plasmids and obtains expression vector;
(3) expression vector is imported into e. coli host cell, you can obtain genetic engineering bacterium pET-32a/rIL2-
IFNγ。
The e. coli host cell is BL21 (DE3) competent cells or BL21 (DE3) senses with pGro7 plasmids
By state cell.
The method of the purifying is:Through affinity chromatography, anion-exchange chromatography and molecular sieve after the crude product elder generation of fusion protein
Chromatographic purifying.
Described BL21 (DE3) competent cell with pGro7 plasmids is purchased from Shanghai Jinan Technology Co., Ltd./glad hundred promise
Biology, article No. is V205.
The acquisition methods of the genome 1 are:
A. design of primers
The primer sequence of pig interleukin 2 and 6 (IL-2) is:
Upstream IL-2-F1:ATAGAATTCATGTATAAGATGCAGCTCTTGC, with EcoRI restriction enzyme sites;
Downstream IL-2-R1:
CCAGAACCACCTCCAGAACCTCCACCAGTCAGTGTTGAGTAGATGCTT, with flexible linker;
The primer sequence of Porcine interferon-gamma (IFN-γ) is:
Upstream IFN-γ-F1:
CTGGAGGTGGTTCTGGAGGTGGATCTATGAGTTATACAACTTATTTCTT, with flexible linker;
Downstream IFN-γ-R1:CCAAGCTTTTTTGATGCTCTCTGGCCTTG, with the restriction enzyme sites of Hind III;
B. RNA is extracted from pig liver, the target gene of IL-2 and IFN-γ, both gene sequences are obtained by reverse transcription
Row are respectively as shown in the > of 400 < of SEQUENCE LISTING 400 <, 5 > and SEQUENCE LISTING 6;
Respectively using the target gene of IL-2 and IFN-γ as template, and it is utilized respectively the upstream and downstream primer of IL-2 and IFN-γ
Enter performing PCR amplification, respectively obtain the flexible linker of connection IL-2 and the target gene of IFN-γ.
PCR reaction systems and condition are:In 25 μ L overall reaction system, the μ L of template ribonucleic acid 1.5, upstream and downstream primer is each
0.5 μ L, reverse transcriptase 2.5 μ L, dNTP Mix are 10 μ L, plus RNase Free water is to 25 μ L;The reaction of the RT-PCR reactions
Condition is:50 DEG C of reverse transcriptions 30min, 95 DEG C of pre-degeneration 4min, into circulation;95 DEG C of denaturation 45s, 58 DEG C of annealing 45s, 72 DEG C are prolonged
1kb/min is stretched, is circulated 35 times, last 72 DEG C of extensions 10min.
C. IL-2 genes are connected with IFN-γ gene using flexible linker
The PCR reaction systems and reaction condition of connection be:In 25 μ L overall reaction system, connect flexible linker's
The μ L of IL-2 gene templates DNA 1, the flexible linker μ L of 1 μ L, IL-2 sense primer of IFN-γ template DNA 0.5 are connected,
0.5 μ L, Taq archaeal dna polymerase of IFN-γ anti-sense primer 2.5 μ L, dNTP Mix is, 9.0 μ L, plus RNase Free water is to 25 μ L;
Connecting PCR reaction conditions is:95 DEG C of pre-degeneration 4min, into circulation:94 DEG C of denaturation 45s;58 DEG C of annealing 45s, 72 DEG C of extensions
1kb/min, totally 35 circulations;Last 72 DEG C of extensions 10min.
Genome 2 is artificial synthesized gene after being optimized to genome 1, the acquisition methods of the genome 2
For:
A. design of primers
The primer sequence of pig interleukin 2 and 6 (IL-2) is:
Upstream IL-2-F2:CATGCCATGGTATGTACAAAATGCAGC, with NcoI restriction enzyme sites;
Downstream IL-2-R2:
ACCACCACCAGAACCACCACCACCGGTCAGGGTAGAGTAG, with flexible linker;
The primer sequence of Porcine interferon-gamma (IFN-γ) is:
Upstream IFN-γ-F2:
GGTGGTTCTGGTGGTGGTGGTTCTATGTCTTACACCACCT, with flexible linker;
Downstream IFN-γ-R2:CCCTCGAGTTTAGAAGCACGCTG, with XhoI restriction enzyme sites;
B. the target gene of the IL-2 and IFN-γ, both gene orders are respectively such as SEQUENCE LISTING 400
Shown in the > of 7 > and SEQUENCE LISTING of <, 400 < 8;
Respectively using the target gene of IL-2 and IFN-γ as template, and it is utilized respectively the upstream and downstream primer of IL-2 and IFN-γ
Enter performing PCR amplification, respectively obtain the target gene of IL-2 and IFN-γ after the flexible linker of connection optimization.
PCR reaction systems and condition are:In 25 μ L overall reaction system, the μ L of genomic DNA 1.0, upstream and downstream primer is each
0.5 μ L, Taq archaeal dna polymerase 2.5 μ L, dNTP Mix is 10 μ L, plus RNase Free water is to 25 μ L;The RT-PCR reactions
Reaction condition is:95 DEG C of pre-degeneration 4min, into circulation;95 DEG C of denaturation 45S, 60 DEG C of annealing 45S, 72 DEG C of extension 1kb/min, are followed
Ring 35 times, last 72 DEG C of extensions 10min.
C. IL-2 genes are connected with IFN-γ gene using flexible linker
The PCR reaction systems and reaction condition of connection be:In 25 μ L overall reaction system, connect flexible linker's
The μ L of IL-2 gene templates DNA 1, the flexible linker μ L of 1 μ L, IL-2 sense primer of IFN-γ template DNA 0.5 are connected,
0.5 μ L, Taq archaeal dna polymerase of IFN-γ anti-sense primer 2.5 μ L, dNTP Mix is 9.0 μ L, plus RNase Free water is to 25 μ L;
Connecting PCR reaction conditions is:95 DEG C of pre-degeneration 4min, into circulation:94 DEG C of denaturation 45s;60 DEG C of annealing 45s, 72 DEG C of extensions
1kb/min, totally 35 circulations;Last 72 DEG C of extensions 10min.
Present invention also offers the application of the Recombinant Swine long-acting interferon γ, its long half time was up to more than 55 hours, tool
There is broad-spectrum disease resistance toxic action and the immune response of pig itself can be improved.
Compared with prior art, the present invention has the advantages that:
Merged 1. being realized pig IL-2 and Porcine interferon-gamma gene by flexible linker, improve interferon half-life period,
Compared with plain interferon, more than 13 times are improved;
2. by being optimized to pig IL-2 and Porcine interferon-gamma gene, improve IL-2 and Porcine interferon-gamma fusion protein
Expression quantity.
3. using recombination bacillus coli BL21/pET-32a-IL2-IFN γ as expression bacterial strain, by introducing molecular chaperones
PGro7 plasmids, do not produce inclusion body in protein expression, form soluble protein, it is to avoid the mistake of inclusion body denaturation and renaturation
Journey, substantially reduces the time of expressing fusion protein;
4. the fusion protein disclosed by the invention being made up of pig IL-2 and Porcine interferon-gamma is not only wide with interferon gamma
Antivirus action is composed, while significantly improving the immune response of pig itself.
Brief description of the drawings
The result that Fig. 1 expands for the pig IL-2 genes in embodiment 1 with Porcine interferon-gamma gene RT-PCR;Swimming lane M:DNA
Marker DL2000;Swimming lane 1:Porcine interferon-gamma gene RT-PCR amplified productions;Swimming lane 2:Pig IL2 genes RT-PCR amplification productions
Thing;
The result that Fig. 2 expands for the PCR after the pig IFN γ in embodiment 1 and pig IL-2 target gene connection;Swimming lane
M:DNA Marker DL2000;Swimming lane 1:Porcine interferon-gamma gene and the gene ligation amplification product of porcine interleukin 2;
PCR amplifications and double digestion qualification result of the Fig. 3 for the positive colony plasmid in embodiment 1;Swimming lane M:DNA
Marker DL10000;Swimming lane 1:Recombinant plasmid double digestion result;Swimming lane 2:Plasmid PCR result;
Fig. 4 be embodiment 1 in recombinant protein SDS-PAGE electrophoretic examinations results;Swimming lane M:Albumen Marker;Swimming lane
1:Supernatant after bacterial cell disruption after recombinant bacterium induction;Swimming lane 2:Precipitated after bacterial cell disruption after recombinant bacterium induction;Swimming lane 3:Empty bacterium
Control;
Fig. 5 is the Western Blot qualification results for the fusion protein that embodiment 1 is obtained;Swimming lane M:Albumen Marker;Swimming
Road 1:Precipitated after recombinant bacterium induction is broken;Swimming lane 2:For the broken rear supernatant of recombinant bacterium induction;
Fig. 6 causes thin for the Recombinant Swine long-acting interferon γ as made from the fusion protein in embodiment 1 in embodiment 5 to VSV
The inhibitory action of born of the same parents' lesion;1 is VSV virus control wells;2 be HEp-2 cell control wells;A3-12 is gradient dilution (from dextrad
It is left) human interferon standard items processing hole;B3-12 is the Recombinant Swine long-acting interferon γ processing of gradient dilution (from right to left)
Hole;
Fig. 7 is the Recombinant Swine long-acting interferon γ intramuscular injection blood as made from the fusion protein in embodiment 1 in embodiment 8
Concentration-time changing curve.
Embodiment
Embodiment 1
A kind of fusion protein being made up of pig interleukin 2 and 6 and Porcine interferon-gamma, its preparation method is as follows:
1. the acquisition of pig interleukin 2 and 6 (IL-2) and Porcine interferon-gamma (IFN-γ) target gene is set with amplimer
Meter:
Objective gene sequence design synthetic primer according to having been reported in Genebank is shown in Table 1, in the upstream of porcine interleukin 2
EcoRI restriction enzyme sites and Linker sequences are introduced in primer and anti-sense primer respectively, Porcine interferon-gamma sense primer and under
Linker sequences and the restriction enzyme sites of Hind III are introduced respectively in trip primer.
Table 1PCR amplimers
RT-PCR obtains target gene:
RNA is extracted from pig liver tissue, the target gene of IL-2 and IFN-γ, both genes are obtained by reverse transcription
Sequence is respectively as shown in the > of 400 < of SEQUENCE LISTING 400 <, 5 > and SEQUENCE LISTING 6;
RT-PCR reaction systems (25 μ L) are shown in Table 2
Table 2RT-PCR reaction systems
RNase Free water | 10μL |
dNTP Mix | 10μL |
Reverse transcriptase | 2.5μL |
Upstream and downstream primer | Each 0.5 μ L |
Geneome RNA | 1.5μL |
Response parameter is:50 DEG C of reverse transcriptions 30min, 95 DEG C of pre-degeneration 4min, into circulation:95 DEG C of denaturation 45s;58 DEG C are moved back
Fiery 45s, 72 DEG C of extension 1kb/min, totally 35 circulations;Last 72 DEG C of extensions 10min.
There is specific band through agarose gel electrophoresis in 490bp and 530bp or so in RT-PCR amplified productions, and its result is such as
Shown in Fig. 1.
2. the connection of target gene
Target gene is diluted to 10ug/mL, using over-lap PCR connection even section target gene, 25 μ L reaction systems are such as
Shown in table 3:
Table 3PCR reaction systems
Response parameter is:95 DEG C of pre-degeneration 4min, into circulation:94 DEG C of denaturation 45s;58 DEG C of annealing 45s, 72 DEG C of extensions
1kb/min, totally 35 circulations;Last 72 DEG C of extensions 10min.
There is specific band through agarose gel electrophoresis in 990bp or so in pcr amplification product, and its result is as shown in Fig. 2 figure
The band of the amplified production of porcine interleukin 2 and Porcine interferon-gamma amplified production is occurred in that in 2, because in the gene of porcine interleukin 2
During the connection of Porcine interferon-gamma gene PCR, non-specific responding is occurred in that.The nucleotide sequence of obtained target gene is such as
Shown in the > of 400 < of SEQUENCE LISTING 3.
3. expression vector establishment
The PCR errorless after sequencing of the target gene after connection glue reclaim product is selected to be used with pET-32a plasmids
EcoRI and the restriction enzymes of Hind III carry out double digestion and recovery, and double digestion is done by 20 μ L systems in table 4:
The double digestion system of table 4
The digestion recovery product of target gene after connection and pET-32a plasmids is attached by the system in table 5,4
DEG C overnight connect:
Table 5
Purpose fragment DNA | 10μL |
Expression vector | 3μL |
buffer | 2μL |
Ligase | 1μL |
RNase Free water | 4μL |
Connection product is converted into e. coli bl21 (DE3) competent cell, competence is coated on the mould of benzyl containing ammonia
The LB culture medium flat plate overnight incubations of element;The bacterium colony grown on LB flat boards is taken to identify target gene, positive colony bacteria plasmid through PCR
Identified through EcoRI and the double digestions of Hind III, be accredited as positive and represent expression vector establishment success, obtained engineering bacteria pET-
32a/rIL2-IFNγ;PCR is expanded and double digestion product single band occurs through agarose gel electrophoresis at 990bp or so places,
Its result is as shown in Figure 3.
4. the expression of recombinant protein
Picking engineering bacteria pET-32a/rIL2-IFN γ shake for 37 DEG C in the LB culture mediums of the μ g/ml containing ampicillin 100
Bacterium 1h recoveries engineering bacteria activity, in LB culture mediums (the μ g/ml containing ampicillin 100) after amplification culture 4h, surveys OD values and reaches
When 1.0;Add IPTG, IPTG final concentration of 100 μ g/mL, 32 DEG C of induced expression 5h;Bacterium is collected, through SDS-PAGE
Electrophoresis detection, its result are as shown in figure 4, it can be seen that recombinant bacterium induces supernatant after the bacterial cell disruption after 5h to precipitate
In the visible predominant expression band in 54.6KD or so places, illustrate in precipitation and supernatant equal successful expression fusion protein.
Add mass volume ratio 1:Precipitation is resuspended in 1 PBS;- 20 DEG C precipitate 3 times in room temperature multigelation;4 DEG C of ultrasonic degradations
Bacterial precipitation, worked 10s, is spaced 3S, and ultrasonic 6min, whole process is repeated 3~4 times;4 DEG C, 12000r/min centrifugation 15min,
Supernatant, precipitation are taken respectively, obtain rough fusion protein.
5. fusion protein purification
5.1His affinity chromatography
After membrane filtration of the rough fusion protein with 0.22 μm of aperture, loading is by being connected to AKTA explorer 100
On protein purification system, the His affinity columns balanced with Binding Buffer I (PBS) are washed away not with PBS
With reference to albumen, until A280nm is stable, then with Elution buffer I (50mM trishydroxymethylaminomethanes, 20~500mM
Imidazoles, PH8.0) elution, collect rIL2-IFN γ protein peaks.
5.2DEAE anion-exchange chromatography
(the 50mM trihydroxy methyls of Binding Buffer II are arrived into the albumen collected after His affinitive layer purifications displacement
Aminomethane, PH6.5) in after, the DEAE anion exchange chromatography that loading has been balanced by using Binding Buffer II,
Crossed again with Binding Buffer II after post to A280nm value stabilizations, with (the 50mM trihydroxy methyl ammonia of Elution Buffer II
Methylmethane, 1M NaCl, PH6.5) linear gradient elution, collect rIL2-IFN γ protein peaks.
5.3 sieve chromatography
Loading is by using Binding Buffer III after the sample concentration that ion-exchange chromatography is collected into
(50mMNa2HPO4,0.15M NaCl, PH7.4) has balanced the molecular sieve chromatographies of Superdex 200, uses Binding Buffer
III elution, collects rIL2-IFN γ protein peaks.
5.4 sample identification
Determine rIL2-IFN γ potency and specific activity, specific activity >=1.0 × 106IU/mg albumen is qualified;It is aseptic subpackaged ,-
80 DEG C of preservations.The fusion protein being made up of pig interleukin 2 and 6 and Porcine interferon-gamma is can obtain, its amino acid sequence is such as
Shown in the > of 400 < of SEQUENCE LISTING 1.
Embodiment 2
A kind of fusion protein being made up of pig interleukin 2 and 6 and Porcine interferon-gamma, other be the same as Examples 1, simply by it
In e. coli bl21 (DE3) competent cell replace for BL21 (DE3) competent cell with pGro7 plasmids.Its
The SDS-PAGE electrophoresis results be the same as Example 1 of fusion protein is compareed, and 54.6KD or so places predominant expression band is thicker in supernatant,
Illustrate to introduce after molecular chaperones pGro7, more preferably, obtained fusion protein amount is higher for expression of the destination protein in supernatant.Greatly
The albumen of enterobacteria expression is mostly present in inclusion body;By introducing molecular chaperones, coordinate expression egg in expression bacterial strain
White correct folding, reaches solubility expression of protein.
Described BL21 (DE3) competent cell with pGro7 plasmids is purchased from Shanghai Jinan Technology Co., Ltd./glad hundred promise
Biology, article No. V205.
Embodiment 3
A kind of fusion protein being made up of pig interleukin 2 and 6 and Porcine interferon-gamma, its preparation method is as follows:
1. the acquisition and amplification of pig interleukin 2 and 6 (IL-2) and Porcine interferon-gamma (IFN-γ) target gene
IL-2 and IFN-γ in embodiment 1 is optimized, artificial synthesized IL-2 and IFN-γ target gene, optimized
Afterwards, both nucleotide sequences are respectively such as the > institutes of 400 < of SEQUENCE LISTING 400 <, 7 > and SEQUENCE LISTING 8
Show.
1.1 codon optimization
Genetic codon has 64 kinds, but most biological tendencies are in utilizing the part in these codons.Those
By the most frequent referred to as optimal codon (optimal codons) utilized, what those were not frequently utilized that is referred to as rare or utilizes
The low codon of rate (rare or low-usage codons).In fact, conventional do protein expression or every kind of biology of production
(including Escherichia coli, yeast, mammalian cell, plant cell and insect cell) all shows codon profit to a certain degree
Difference or preference.In Escherichia coli, yeast and drosophila to the expression efficiency of the gene containing optimal codon apparently higher than containing
The expression efficiency of the gene of the codon of poor efficiency.Therefore, in heterologous expression system, the preferences of codon are largely
On have impact on the expression of recombinant protein.Using preference codon (preferred codons) and avoid utilize rare codon
Gene chemical synthesis is carried out, the redesign of this gene is codon optimization.Therefore, to the IL-2 and IFN-γ of pig in the present embodiment
Gene codon is optimized.
Interpretation of result after 1.2 codon optimizations
It is considered as the gene during usual codon adaptation indexI (CAI)=1.0 optimal efficient in the expression system
Expression status, CAI values are lower to show that expression is lower in host.In gene G/C content most ideal distribution scope be 30~
70%, the scope is exceeded in any region can influence to translate and transcriptional efficiency.Using software detection find pig IL-2 and
The codon of IFN-γ original gene codon adaptation indexI (CAI) in Escherichia coli is respectively 0.23,0.24, GC percentages
For 38.7%, 39.2%;And by obtaining recombination password in Escherichia coli after pig IL-2 and IFN-γ gene optimization
Sub- adaptation index (CAI) is 0.97,1.0, GC percentages 47.6%, 45.4%.Low password is significantly reduced by gene optimization
The utilization rate of son, it is to avoid influence of the rare codon to protein expression, improves the G/C content of gene, improves transcription and translation
Efficiency.
1.3 design of primers:
Table 6PCR amplimers
IL-2 and the genomic DNA of IFN-γ after optimization is diluted to 0.05mg/mL respectively.Expanded and obtained using PCR
Target gene, 25 μ L reaction systems are as shown in table 7:
Table 7PCR reaction systems
RNase Free water | 10.5μL |
dNTP Mix | 10.0μL |
Taq archaeal dna polymerases | 2.5μL |
Upstream and downstream primer | Each 0.5 μ L |
Genomic DNA | 1.0μL |
Response parameter is:95 DEG C of pre-degeneration 4min, into circulation:94 DEG C of denaturation 45s;60 DEG C of annealing 45s, 72 DEG C of extensions
1kb/min, totally 35 circulations;Last 72 DEG C of extensions 10min.
The pcr amplification product of IL-2 and IFN-γ occurs special in 490bp and 530bp or so respectively through agarose gel electrophoresis
Different band, illustrates to have prepared the target gene of the IL-2 and IFN-γ after the flexible linker of connection optimization.
2. the connection of target gene
Target gene is diluted to 10ug/mL, target gene, the 25 μ L reaction systems such as institute of table 8 are connected using over-lap PCR
Show:
Table 8PCR reaction systems
Response parameter is:95 DEG C of pre-degeneration 4min, into circulation:95 DEG C of denaturation 45s;60 DEG C of annealing 45s, 72 DEG C of extensions
1kb/min, totally 35 circulations;Last 72 DEG C of extensions 10min.
There is specific band through agarose gel electrophoresis in 990bp or so in pcr amplification product, illustrates successfully to have obtained IL-2
Target gene after being connected with IFN-γ, the nucleotide sequence such as > of 400 < of SEQUENCE LISTING 4 of obtained target gene
It is shown.
3. expression vector establishment
The PCR errorless after sequencing of the target gene after connection glue reclaim product is selected to be used with pET-32a plasmids
NcoI, XhoI restriction enzyme carry out double digestion and recovery, and double digestion is done by 20 μ L systems in table 9:
The double digestion system of table 9
General buffer | 2μL |
Restriction enzyme (a pair) | 1μL+1μL |
Carrier reclaims fragment | 2μL |
RNase Free water | 14μL |
The digestion recovery product of target gene after connection and pET-32a plasmids is attached by the system in table 10,4
DEG C overnight connect:
Table 10
Purpose fragment DNA | 10μL |
Expression vector | 3μL |
buffer | 2μL |
Ligase | 1μL |
RNase Free water | 4μL |
Connection product is converted into e. coli bl21 (DE3) competent cell, competence is coated on the mould of benzyl containing ammonia
The LB culture medium flat plate overnight incubations of element;The bacterium colony grown on LB flat boards is taken to identify target gene, positive colony bacteria plasmid through PCR
Identified through NcoI, XhoI double digestion, be accredited as positive and represent expression vector establishment success, PCR amplifications and double digestion product warp
There is single band at 990bp or so places in agarose gel electrophoresis, illustrates containing the target gene after IL-2 and IFN-γ connection
Expression vector establishment success, obtained engineering bacteria pET-32a/rIL2-IFN γ.
4. the expression of recombinant protein
Picking engineering bacteria pET-32a/rIL2-IFN γ shake for 37 DEG C in the LB culture mediums of the μ g/ml containing ampicillin 100
Bacterium 1h recoveries engineering bacteria activity;In LB culture mediums (the μ g/ml containing ampicillin 100) after amplification culture 4h, survey OD values and reach
When 1.0;Add IPTG, IPTG final concentration of 100 μ g/mL, 32 DEG C of induced expression 5h;Bacterium is collected, through SDS-PAGE
Supernatant is deposited in the visible predominant expression band in 54.6KD or so places after bacterial cell disruption after electrophoresis detection, recombinant bacterium induction 5h,
Illustrate to have obtained recombinant protein in supernatant is precipitated.
Add mass volume ratio 1:Precipitation is resuspended in 1 PBS;- 20 DEG C precipitate 3 times in room temperature multigelation;4 DEG C of ultrasounds are split
Bacterial precipitation is solved, work 10s, be spaced 3S, ultrasonic 6min, whole process is repeated 3~4 times;4 DEG C, 12000r/min centrifugations
15min, takes supernatant, precipitation, obtains rough fusion protein respectively.
5. fusion protein purification
5.1His affinity chromatography
After membrane filtration of the rough fusion protein with 0.22 μm of aperture, loading is by being connected to AKTA explorer 100
On protein purification system, the His affinity columns balanced with Binding Buffer I (PBS) are washed away not with PBS
With reference to albumen, until A280nm is stable, then with Elution buffer I (50mM trishydroxymethylaminomethanes, 20~500mM
Imidazoles, PH8.0) elution, collect rIL2-IFN γ protein peaks.
5.2DEAE anion-exchange chromatography
(the 50mM trihydroxy methyls of Binding Buffer II are arrived into the albumen collected after His affinitive layer purifications displacement
Aminomethane, PH6.5) in after, the DEAE anion exchange chromatography that loading has been balanced by using Binding Buffer II,
Crossed again with Binding Buffer II after post to A280nm value stabilizations, with (the 50mM trihydroxy methyl ammonia of Elution Buffer II
Methylmethane, 1M NaCl, PH6.5) linear gradient elution, collect rIL2-IFN γ protein peaks.
5.3 sieve chromatography
Loading is by using Binding Buffer III after the sample concentration that ion-exchange chromatography is collected into
(50mMNa2HPO4,0.15M NaCl, PH7.4) has balanced the molecular sieve chromatographies of Superdex 200, uses Binding Buffer
III elution, collects rIL2-IFN γ protein peaks.
5.4 sample identification
Determine rIL2-IFN γ potency and specific activity, specific activity >=1.0 × 106IU/mg albumen is qualified;It is aseptic subpackaged ,-
80 DEG C of preservations.The fusion protein being made up of pig interleukin 2 and 6 and Porcine interferon-gamma is can obtain, its amino acid sequence is such as
Shown in the > of 400 < of SEQUENCE LISTING 2.
Embodiment 4
A kind of fusion protein being made up of pig interleukin 2 and 6 and Porcine interferon-gamma, other be the same as Examples 3, simply by it
In e. coli bl21 (DE3) competent cell replace for BL21 (DE3) competent cell with pGro7 plasmids.Its
The SDS-PAGE electrophoresis results be the same as Example 3 of fusion protein is compareed, and 54.6KD or so places predominant expression band is thicker in supernatant,
Illustrate to introduce after molecular chaperones pGro7, more preferably, obtained fusion protein amount is higher for expression of the destination protein in supernatant.Greatly
The albumen of enterobacteria expression is mostly present in inclusion body;By introducing molecular chaperones, coordinate expression egg in expression bacterial strain
White correct folding, reaches solubility expression of protein.Described BL21 (DE3) competent cell with pGro7 plasmids is purchased from Shanghai
Offshore Science and Technology Ltd./glad hundred promise is biological, article No. V205.
Embodiment 5
A kind of Recombinant Swine long-acting interferon γ, is mixed with freeze drying protectant respectively by the fusion protein in embodiment 1,2,3,4
It is freeze-dried to form with after.The freeze drying protectant is glycerine, mannitol and sucrose, is buffering with 10mmol/L PBS
Liquid, final concentration of glycerine 100mL/L, mannitol 0.12g/mL and the sucrose 0.025g/mL of three.
Embodiment 6
Embodiment 1~4 obtains the identification for the fusion protein being made up of pig interleukin 2 and 6 and Porcine interferon-gamma
The quantitative detection of 6.1 protein contents
Lowry methods are used, the standard protein for examining and determine institute with Chinese food pharmaceutical biological product makees standard test, determine embodiment
1~4 obtained fusion protein concentration is all higher than 1.2mg/ml.
6.2SDS-PAGE electrophoresis detection
Compared with empty bacterium, fusion protein has the newly-increased protein band of a dense dye in 54.6KD or so, as Fig. 4, shown in.
6.3Western Blot results
Fusion protein in embodiment 1~4 is detected respectively, with the anti-pig gamma interferon (1 of abcam companies mouse:5000 dilutions) be
Primary antibody, using goat anti-mouse IgG-HRP as secondary antibody (1:10000 dilutions).Recombinant Swine long-acting interferon γ samples can be dry with anti-pig
Disturb plain γ monoclonal antibodies and occur specific reaction, specific band occur in 54.6KD or so places, as shown in Figure 5.
Embodiment 7
Bioactivity freeze-dried four parts of Recombinant Swine long-acting interferon γ in embodiment 5
Suppress method according to few cells lesion, Hep-2 cells are made into 5 × 10 with culture medium5Cell/ml cells suspend
Liquid, per hole, inoculation 0.1ml moves into 96 porocyte culture plates.37 DEG C, 5%CO2 culture 24h, the Recombinant Swine for adding various dose are long
Imitate to inhale after interferon gamma, 24h and abandon, then be inoculated with 100TCID respectively50VSV viruses.
Result of the test
As a result show that the Recombinant Swine long-acting interferon γ obtained causes the lesion of HEp-2 cells to have obvious suppression to VSV
Make and use.The lesion such as occurs cell rounding after undressed cell virus inoculation, comes off, is disintegrated.And the Recombinant Swine obtained
After cell virus inoculation after long-acting interferon γ processing, the Continuous Observation under inverted microscope, cellular morphology is normal, does not occur
Any lesion, measures potency >=1.0 × 106IU/ml, as shown in Figure 6.
Embodiment 8
The four parts of Recombinant Swine long-acting interferon γ obtained respectively by the fusion protein of embodiment 1~4 in embodiment 5 are freezed
The measure of half-life period of the agent (being designated as A, B, C, D respectively) in pig body
Cytopathic-effect inhibition assay determines rIL2-IFN γ blood concentration and time relationship
The piglet (male and female half and half) that six body weight are roughly the same is taken, the long-acting Porcine interferon-gamma of intramuscular injection 2mg/ml pigs is freezed
Agent 2ml, respectively in 1h, 2h, 4h, 8h, 16h, 24h, 36h, 48h, 60h, 88h venous blood collection, 4 DEG C of solidifications of blood sample, 3500rpm is low
Temperature centrifugation 10min separation serum, each every pig blood sample of time point is to be measured in -20 DEG C of preservations.Blood is determined using cytopathic-effect inhibition assay
RIL2-IFN γ concentration in final proof product, is carried out curve fitting and calculating parameter with DAS pharmacokinetics softwares.Matched curve such as Fig. 7
It is shown;Parameter result of calculation is shown in Table 11.
Dominant dynamic parameters in serum after the Recombinant Swine long-acting interferon γ intramuscular injection of table 11
As a result show that Recombinant Swine long-acting interferon γ has longer half-life period.Half-life period can reach 55h or so after measured, compared with
Plain interferon improves about 13 times.
Embodiment 9
The freeze-dried measure influenceed on pig cell immune response of four parts of Recombinant Swine long-acting interferon γ in embodiment 5
Take six roughly the same piglets of body weight to be divided into two groups, be designated as experimental group and control group;Experimental group neck is subcutaneously noted
The 2mg/ml Recombinant Swine long-acting interferon freeze-dried 2ml of γ are penetrated, 2mL PBS is subcutaneously injected in control group neck, takes pig after injecting 4 weeks
Peripheral blood, takes weekly a blood afterwards, and lymphocyte is separated using lymphocyte separation medium, and lymphocyte passes through serum-free RPMI
1640 culture mediums are washed after 2 times, and it is 2 × 10 to be resuspended with complete medium, adjust cell concentration6Individual/ml, 24 porocyte culture plates are every
Hole adds 1ml lymphocytes, 37 DEG C, 5%CO272h is cultivated, supernatant when collecting lymphocyte culture.ELISA detects culture supernatant
Middle IL-4 contents, are carried out, testing result is as shown in table 12 by kit specification:
Table 12ELISA detects each group pig cell immune response level
As a result show after injection Recombinant Swine long-acting interferon γ, cell factor IL-4 in pig peripheral blood can be significantly improved
Content, enhances cellullar immunologic response level, significantly improves immunity level.
It is above-mentioned with reference to embodiment to Recombinant Swine long-acting interferon γ and prepare this long-acting interferon γ fusion protein and its
The detailed description that preparation method is carried out, is illustrative rather than limited, can include several according to limited scope
Embodiment, therefore changing and modifications in the case where not departing from present general inventive concept, should belong within protection scope of the present invention.
SEQUENCE LISTING
<110>Anhui Jiuchuan Biotechnology Co., Ltd.
<120>A kind of Recombinant Swine long-acting interferon γ and prepare this long-acting interferon γ fusion protein and preparation method thereof
<130> 1
<160> 8
<170> PatentIn version 3.3
<210> 1
<211> 332
<212> PRT
<213>Recombinant Swine long-acting interferon γ fusion proteins 1
<400> 1
Met Tyr Lys Met Gln Leu Leu Cys Cys Ile Ala Leu Thr Leu Ala Leu
1 5 10 15
Met Ala Asn Gly Ala Pro Thr Ser Ser Ser Thr Lys Asn Thr Lys Lys
20 25 30
Gln Leu Glu Pro Leu Leu Leu Asp Leu Gln Leu Leu Leu Lys Glu Val
35 40 45
Lys Asn Tyr Glu Asn Ala Asp Leu Ser Arg Met Leu Thr Phe Lys Phe
50 55 60
Tyr Met Pro Lys Gln Ala Thr Glu Leu Lys His Leu Gln Cys Leu Val
65 70 75 80
Glu Glu Leu Lys Ala Leu Glu Gly Val Leu Asn Leu Gly Gln Ser Lys
85 90 95
Asn Ser Asp Ser Ala Asn Ile Lys Glu Ser Met Asn Asn Ile Asn Val
100 105 110
Thr Val Leu Glu Leu Lys Gly Ser Glu Thr Ser Phe Lys Cys Glu Tyr
115 120 125
Asp Asp Glu Thr Val Thr Ala Val Glu Phe Leu Asn Lys Trp Ile Thr
130 135 140
Phe Cys Gln Ser Ile Tyr Ser Thr Leu Thr Arg Ser Thr Ser Arg Thr
145 150 155 160
Thr Ser Arg Thr Ser Thr Met Ser Tyr Thr Thr Tyr Phe Leu Ala Phe
165 170 175
Gln Leu Cys Val Thr Leu Cys Phe Ser Gly Ser Tyr Cys Gln Ala Pro
180 185 190
Phe Phe Lys Glu Ile Thr Ile Leu Lys Asp Tyr Phe Asn Ala Ser Thr
195 200 205
Ser Gly Val Pro Asn Gly Gly Pro Leu Phe Leu Glu Ile Leu Glu Asn
210 215 220
Trp Lys Glu Glu Ser Asp Lys Lys Ile Ile Gln Ser Gln Ile Val Ser
225 230 235 240
Phe Tyr Phe Lys Phe Phe Glu Ile Phe Lys Asp Asn Gln Ala Ile Gln
245 250 255
Arg Ser Met Asp Val Ile Lys Gln Asp Met Phe Gln Arg Phe Leu Asn
260 265 270
Gly Ser Ser Gly Lys Leu Asn Asp Phe Glu Lys Leu Val Lys Ile Pro
275 280 285
Val Asp Asn Leu Gln Ile Gln Arg Lys Ala Ile Ser Glu Leu Ile Lys
290 295 300
Val Met Asn Asp Leu Ser Pro Arg Ser Asn Leu Arg Lys Arg Lys Arg
305 310 315 320
Ser Gln Thr Met Phe Gln Gly Gln Arg Ala Ser Lys
325 330
<210> 2
<211> 330
<212> PRT
<213>Recombinant Swine long-acting interferon γ fusion proteins 2
<400> 2
Met Tyr Lys Met Gln Leu Leu Cys Cys Ile Ala Leu Thr Leu Ala Leu
1 5 10 15
Met Ala Asn Gly Ala Pro Thr Ser Ser Ser Thr Lys Asn Thr Lys Lys
20 25 30
Gln Leu Glu Pro Leu Leu Leu Asp Leu Gln Leu Leu Leu Lys Glu Val
35 40 45
Lys Asn Tyr Glu Asn Ala Asp Leu Ser Arg Met Leu Thr Phe Lys Phe
50 55 60
Tyr Met Pro Lys Gln Ala Thr Glu Leu Lys His Leu Gln Cys Leu Val
65 70 75 80
Glu Glu Leu Lys Ala Leu Glu Gly Val Leu Asn Leu Gly Gln Ser Lys
85 90 95
Asn Ser Asp Ser Ala Asn Ile Lys Glu Ser Met Asn Asn Ile Asn Val
100 105 110
Thr Val Leu Glu Leu Lys Gly Ser Glu Thr Ser Phe Lys Cys Glu Tyr
115 120 125
Asp Asp Glu Thr Val Thr Ala Val Glu Phe Leu Asn Lys Trp Ile Thr
130 135 140
Phe Cys Gln Ser Ile Tyr Ser Thr Leu Thr Gly Gly Gly Gly Ser Gly
145 150 155 160
Gly Gly Gly Ser Met Ser Tyr Thr Thr Tyr Phe Leu Ala Phe Gln Leu
165 170 175
Cys Val Thr Leu Cys Phe Ser Gly Ser Tyr Cys Gln Ala Pro Phe Phe
180 185 190
Lys Glu Ile Thr Ile Leu Lys Asp Tyr Phe Asn Ala Ser Thr Ser Gly
195 200 205
Val Pro Asn Gly Gly Pro Leu Phe Leu Glu Ile Leu Glu Asn Trp Lys
210 215 220
Glu Glu Ser Asp Lys Lys Ile Ile Gln Ser Gln Ile Val Ser Phe Tyr
225 230 235 240
Phe Lys Phe Phe Glu Ile Phe Lys Asp Asn Gln Ala Ile Gln Arg Ser
245 250 255
Met Asp Val Ile Lys Gln Asp Met Phe Gln Arg Phe Leu Asn Gly Ser
260 265 270
Ser Gly Lys Leu Asn Asp Phe Glu Lys Leu Val Lys Ile Pro Val Asp
275 280 285
Asn Leu Gln Ile Gln Arg Lys Ala Ile Ser Glu Leu Ile Lys Val Met
290 295 300
Asn Asp Leu Ser Pro Arg Ser Asn Leu Arg Lys Arg Lys Arg Ser Gln
305 310 315 320
Thr Met Phe Gln Gly Gln Arg Ala Ser Lys
325 330
<210> 3
<211> 996
<212> DNA
<213>Recombinant Swine long-acting interferon γ genomes 1
<400> 3
atgtataaga tgcagctctt gtgttgcatt gcactaaccc ttgcactcat ggcaaacggt 60
gcacctactt caagctctac aaagaacaca aagaaacaac tggagccatt gctgctggat 120
ttacagttgc ttttgaagga agttaagaat tacgagaatg ctgatctctc caggatgctc 180
acatttaaat tttacatgcc caagcaggct acagaattga aacaccttca gtgtttagta 240
gaagaactca aagctctgga gggagtgcta aatttaggtc aaagcaaaaa ctctgactca 300
gcaaatatca aggaatcaat gaacaatatc aacgtaacag ttttggaact aaagggatct 360
gaaacaagtt tcaaatgtga atatgatgat gagacagtaa ctgctgttga atttctgaac 420
aaatggatta ccttttgtca aagcatctac tcaacactga ctagatccac ctccagaacc 480
acctccagaa cctccaccat gagttataca acttatttct tagcttttca gctttgcgtg 540
actttgtgtt tttctggctc ttactgccag gcgccctttt ttaaagaaat aacgatccta 600
aaggactatt ttaatgcaag tacctcaggt gtacctaatg gtggacctct tttcttagaa 660
attttggaga attggaaaga ggagagtgac aaaaaaataa ttcagagcca aattgtctcc 720
ttctacttca aattctttga aatcttcaaa gataaccagg ccattcaaag gagcatggat 780
gtgatcaagc aagacatgtt tcagaggttc ctaaatggta gctctgggaa actgaatgac 840
ttcgaaaagc tggttaaaat tccggtagat aatctgcaga tccagcgcaa agccatcagt 900
gaactcatca aagtgatgaa tgatctgtca ccaagatcta acctaagaaa gcggaagaga 960
agtcagacta tgttccaagg ccagagagca tcaaaa 996
<210> 4
<211> 990
<212> DNA
<213>Recombinant Swine long-acting interferon γ genomes 2
<400> 4
atgtacaaaa tgcagctgct gtgctgcatc gctctgaccc tggctctgat ggctaacggt 60
gctccgacct cttcttctac caaaaacacc aaaaaacagc tggaaccgct gctgctggac 120
ctgcagctgc tgctgaaaga agttaaaaac tacgaaaacg ctgacctgtc tcgtatgctg 180
accttcaaat tctacatgcc gaaacaggct accgaactga aacacctgca gtgcctggtt 240
gaagaactga aagctctgga aggtgttctg aacctgggtc agtctaaaaa ctctgactct 300
gctaacatca aagaatctat gaacaacatc aacgttaccg ttctggaact gaaaggctcg 360
gaaaccagct tcaaatgcga atacgacgac gaaaccgtta ccgctgttga attcctgaac 420
aaatggatca ccttctgcca gtctatctac tctaccctga ccggtggtgg tggttctggt 480
ggtggtggtt ctatgtctta caccacctac ttcctggctt tccagctgtg cgttaccctg 540
tgcttctctg gttcttactg ccaggctccg ttcttcaaag aaatcaccat cctgaaagac 600
tacttcaacg cttctacctc tggtgttccg aacggtggtc cgctgttcct ggaaatcctg 660
gaaaactgga aagaagaatc tgacaaaaaa atcatccagt ctcagatcgt ttctttctac 720
ttcaaattct tcgaaatctt caaagacaac caggctatcc agcgttctat ggacgttatc 780
aaacaggaca tgttccagcg tttcctgaac ggttcttctg gtaaactgaa cgacttcgaa 840
aaactggtta aaatcccggt tgacaacctg cagatccagc gtaaagctat ctctgaactg 900
atcaaagtta tgaacgacct gtctccgcgt tctaacctgc gtaaacgtaa acgttctcag 960
accatgttcc agggtcagcg tgcttctaaa 990
<210> 5
<211> 462
<212> DNA
<213>Pig IL-2
<400> 5
atgtataaga tgcagctctt gtgttgcatt gcactaaccc ttgcactcat ggcaaacggt 60
gcacctactt caagctctac aaagaacaca aagaaacaac tggagccatt gctgctggat 120
ttacagttgc ttttgaagga agttaagaat tacgagaatg ctgatctctc caggatgctc 180
acatttaaat tttacatgcc caagcaggct acagaattga aacaccttca gtgtttagta 240
gaagaactca aagctctgga gggagtgcta aatttaggtc aaagcaaaaa ctctgactca 300
gcaaatatca aggaatcaat gaacaatatc aacgtaacag ttttggaact aaagggatct 360
gaaacaagtt tcaaatgtga atatgatgat gagacagtaa ctgctgttga atttctgaac 420
aaatggatta ccttttgtca aagcatctac tcaacactga ct 462
<210> 6
<211> 498
<212> DNA
<213>Porcine IFN γ
<400> 6
atgagttata caacttattt cttagctttt cagctttgcg tgactttgtg tttttctggc 60
tcttactgcc aggcgccctt ttttaaagaa ataacgatcc taaaggacta ttttaatgca 120
agtacctcag gtgtacctaa tggtggacct cttttcttag aaattttgga gaattggaaa 180
gaggagagtg acaaaaaaat aattcagagc caaattgtct ccttctactt caaattcttt 240
gaaatcttca aagataacca ggccattcaa aggagcatgg atgtgatcaa gcaagacatg 300
tttcagaggt tcctaaatgg tagctctggg aaactgaatg acttcgaaaa gctggttaaa 360
attccggtag ataatctgca gatccagcgc aaagccatca gtgaactcat caaagtgatg 420
aatgatctgt caccaagatc taacctaaga aagcggaaga gaagtcagac tatgttccaa 480
ggccagagag catcaaaa 498
<210> 7
<211> 462
<212> DNA
<213>Pig IL-2
<400> 7
atgtacaaaa tgcagctgct gtgctgcatc gctctgaccc tggctctgat ggctaacggt 60
gctccgacct cttcttctac caaaaacacc aaaaaacagc tggaaccgct gctgctggac 120
ctgcagctgc tgctgaaaga agttaaaaac tacgaaaacg ctgacctgtc tcgtatgctg 180
accttcaaat tctacatgcc gaaacaggct accgaactga aacacctgca gtgcctggtt 240
gaagaactga aagctctgga aggtgttctg aacctgggtc agtctaaaaa ctctgactct 300
gctaacatca aagaatctat gaacaacatc aacgttaccg ttctggaact gaaaggctcg 360
gaaaccagct tcaaatgcga atacgacgac gaaaccgtta ccgctgttga attcctgaac 420
aaatggatca ccttctgcca gtctatctac tctaccctga cc 462
<210> 8
<211> 498
<212> DNA
<213>Porcine IFN γ
<400> 8
atgtcttaca ccacctactt cctggctttc cagctgtgcg ttaccctgtg cttctctggt 60
tcttactgcc aggctccgtt cttcaaagaa atcaccatcc tgaaagacta cttcaacgct 120
tctacctctg gtgttccgaa cggtggtccg ctgttcctgg aaatcctgga aaactggaaa 180
gaagaatctg acaaaaaaat catccagtct cagatcgttt ctttctactt caaattcttc 240
gaaatcttca aagacaacca ggctatccag cgttctatgg acgttatcaa acaggacatg 300
ttccagcgtt tcctgaacgg ttcttctggt aaactgaacg acttcgaaaa actggttaaa 360
atcccggttg acaacctgca gatccagcgt aaagctatct ctgaactgat caaagttatg 420
aacgacctgt ctccgcgttc taacctgcgt aaacgtaaac gttctcagac catgttccag 480
ggtcagcgtg cttctaaa 498
Claims (10)
1. a kind of fusion protein being made up of pig interleukin 2 and 6 and Porcine interferon-gamma, it is characterised in that:The fusion protein
Amino acid sequence table is designated as fusion protein 1 as shown in the > of 400 < of SEQUENCE LISTING 1;Or such as SEQUENCE LISTING
Shown in the > of 400 < 2, fusion protein 2 is designated as.
2. a kind of gene for encoding fusion protein as claimed in claim 1, it is characterised in that the nucleotide sequence of the gene
Table is designated as genome 1 as shown in the > of 400 < of SEQUENCE LISTING 3;Or as shown in the > of 400 < of SEQUENCE LISTING 4,
It is designated as genome 2.
3. the expression vector containing gene as claimed in claim 2.
4. the genetic engineering bacterium containing gene as claimed in claim 2.
5. a kind of Recombinant Swine long-acting interferon γ, it is characterised in that the Recombinant Swine long-acting interferon γ is as described in claim 1
Fusion protein and freeze drying protectant mixture after, it is freeze-dried to form.
6. the preparation method of fusion protein according to claim 1, it is characterised in that the preparation method includes following step
Suddenly:Expression vector described in claim 3 is imported into e. coli host cell, genetic engineering bacterium, genetic engineering is obtained
Bacterium obtains the crude product of the fusion protein after IPTG induced expressions, purified to can obtain fusion protein afterwards.
7. preparation method according to claim 6, it is characterised in that the genetic engineering bacterium is pET-32a/rIL2-IFN
γ, its preparation method is:
(1) primer is designed, is obtained by reverse transcription or the artificial synthesized pig interleukin 2 and 6 for connecting flexible linker sequences
With the target gene of Porcine interferon-gamma;The target gene of pig interleukin 2 and 6 and Porcine interferon-gamma is connected by flexible linker
Pick up and, the nucleotides sequence list of target gene is as shown in the > of 400 < of SEQUENCE LISTING 3 or such as SEQUENCE
Shown in the > of 400 < of LISTING 4;
(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。
8. the preparation method according to claim 6 or 7, it is characterised in that the e. coli host cell is BL21
(DE3) competent cell or BL21 (DE3) competent cell with pGro7 plasmids.
9. the preparation method according to claim 6 or 7, it is characterised in that the method for the purifying is:Fusion protein it is thick
Purified after product elder generation through affinity chromatography, anion-exchange chromatography and sieve chromatography.
10. Recombinant Swine long-acting interferon γ according to claim 5 application, it is characterised in that the Recombinant Swine is long-acting
The long half time of interferon gamma with broad-spectrum disease resistance toxic action and can improve the immune response of pig itself up to more than 55 hours.
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CN201810725703.9A CN108794640A (en) | 2017-08-09 | 2018-07-04 | A kind of Recombinant Swine long-acting interferon γ and the fusion protein and preparation method thereof for preparing this long-acting interferon γ |
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CN201810725703.9A Pending CN108794640A (en) | 2017-08-09 | 2018-07-04 | A kind of Recombinant Swine long-acting interferon γ and the fusion protein and preparation method thereof for preparing this long-acting interferon γ |
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