CN102585013B - Fusion protein containing omega interferon and method for preparing same - Google Patents
Fusion protein containing omega interferon and method for preparing same Download PDFInfo
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- CN102585013B CN102585013B CN201110002132.4A CN201110002132A CN102585013B CN 102585013 B CN102585013 B CN 102585013B CN 201110002132 A CN201110002132 A CN 201110002132A CN 102585013 B CN102585013 B CN 102585013B
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Abstract
The invention relates to a fusion protein, in particular to a fusion protein containing recombinant human omega interferon and human immunoglobulin constant areas and a method for preparing the same. The fusion protein has a longer half-life period in the human body, and the drug use times can be obviously reduced.
Description
Technical field
The present invention relates to a kind of fusion rotein, particularly a kind of fusion rotein that contains omega interferon and human normal immunoglobulin constant region and preparation method thereof.
Background technology
Interferon, rabbit is the naturally occurring small protein of a class and glycoprotein, and they are produced and secrete the reaction of virus infection and other antigenic stimulation by most of karyocytes.Interferon, rabbit makes cell can resist virus infection and cell is shown to effect widely.They bring into play its activity by the specific membranes receptors bind with cell surface.
Interferon, rabbit (IFN) is divided into I type and II type, and people I type Interferon, rabbit mainly comprises IFN-α and IFN-β, and both share a kind of acceptor IFN-α/β acceptor, and II type Interferon, rabbit is IFN-γ.I type Interferon, rabbit (comprises IFN-α, IFN-β and IFN-ω) biological activity basic identical, mainly comprise antiviral, inhibition of cell proliferation and immunoregulation effect, can be used for clinically treating viral hepatitis (comprising hepatitis B, hepatitis C etc.), tumour, multiple sclerosis etc.; II type Interferon, rabbit (IFN-γ) inhibition of cell proliferation and immunoregulation effect are stronger, anti-virus ability a little less than.Can be used for clinically treating the diseases such as rheumatoid arthritis, chronic granuloma.
IFN-ω is the I type Interferon, rabbit of finding in 1985, because have antigenicity and the immunogenicity different from IFN-α, likely develops into a kind of new antiviral, and the recombinant human IFN-ω that CHO expresses shows obvious antiviral activity.But IFN molecular weight, easily by glomerular filtration, thereby during clinical application, plasma half-life is shorter, generally needs heavy dose of frequent medication, as 2～3 times weekly, and during the diseases such as IFN treatment hepatitis, tumour, multiple sclerosis, rheumatoid arthritis, treatment cycle usually reaches several years even several months, and the frequent medication of this long-term, high-dose has not only increased patient's misery and medical expense, and easily produces toxic side effects.
In order to solve the deficiency of common Interferon, rabbit on clinical treatment, Duo Jia biopharmaceutical company is devoted to develop long-acting interferon.Its means are by carrying out polyethyleneglycol modified to common Interferon, rabbit.Polyoxyethylene glycol (Poly ethleneglycol, pEG) is a kind of water-soluble high-molecular substance of nontoxic, non-immunogenicity, can pass through covalent bonds mode modifying protein.Polyethylene glycol modified protein medicine not only can reduce immunogenicity and toxicity but also can obviously extend plasma half-life, improves the stability of pharmaceutical grade protein.Clinically, the Peg-Intron of widespread use comprise Schering Plough company exploitation glycol interferon alpha-2b (trade(brand)name, wear happy can, PegIntron); Glycol interferon alpha-the 2a (trade(brand)name, Pai Luoxin, Pegasys) of Roche Holding Ag's exploitation, by combining the treatment that is widely used in chronic type b and hepatitis C with ribavirin.Extend Interferon, rabbit in animal body another method of transformation period be to make Interferon, rabbit form fusion rotein, for example form fusion rotein with human serum albumin, the increase of molecular weight, can reduce the filterability of renal glomerulus, reaches the object of prolong half-life.
Antiviral activity based on good, IFN-ω has wide potential applicability in clinical practice, so this area exist, high reactivity simple to preparation technology and have compared with long half-lift the demand of IFN-ω.
Summary of the invention
First object of the present invention provides a kind of be conducive to suitability for industrialized production and clinical application, active high and can obviously reduce the recombinant human omega interferon of medication number of times.
Based on above-mentioned purpose, first the present invention provides a kind of fusion rotein, and described fusion rotein contains omega interferon and human normal immunoglobulin constant region part.
In a preferred technical scheme, described human normal immunoglobulin is human IgG1.
In a preferred technical scheme, described human normal immunoglobulin constant region is partly Fc fragment.
In a preferred technical scheme, between described omega interferon and human normal immunoglobulin constant region, there is no connection peptides.
In a preferred technical scheme, described fusion rotein contains the aminoacid sequence shown in SEQ ID NO:2.
The present invention also provides the homodimer of above-mentioned fusion rotein, and in this dimer, two monomers are connected by disulfide linkage.
The present invention also provides the pharmaceutical preparation that contains above-mentioned fusion rotein.
The present invention further provides the nucleotide sequence that contains above-mentioned fusion rotein.
In a preferred technical scheme, described nucleotide sequence is selected from following sequence:
(1)SEQ?ID?NO：1；
(2) with the nucleotide sequence of nucleotide sequence SEQ ID NO:1 at least 95% homology;
(3) nucleotide sequence of hybridizing with nucleotide sequence SEQ ID NO:1 under rigorous condition;
(4) or with the nucleotide sequence of any nucleotide sequence complementation in (1)-(3).
" complementation " mentioned above, refers to the pair relationhip forming by hydrogen bond between the base of two strands in Nucleotide, that is, and and VITAMIN B4-thymus pyrimidine (A-T) pairing, guanine-cytosine pair (G-C) pairing.
Rigorous condition mentioned above refers to the strict degree of the required condition of nucleic acid sequence formation crossbred of avoiding non-homology or Homoeology in hybridization system.Generally that temperature of reaction is high, the low preciseness (while being conducive to strand formation, preciseness is high) that can increase reaction of ionic strength, it is suitable for, and base pairing is good, the nucleic acid hybridization reaction of the complete homology of base sequence, if temperature of reaction is low, the high hybridization of ionic strength preciseness is low, it is applicable to, and base pairing is poor, the nucleic acid reaction of the incomplete homology of base sequence.Rigorous condition of the present invention is, at 0.5M sodium phosphate, 7%SDS, hybridizes under the condition of 65 ℃, then uses 0.2 * SSC, and 1%SDS washes one or many at 65 ℃.
The present invention also provides the carrier that contains above-mentioned nucleotide sequence.
In a preferred technical scheme, described carrier is pMH3S.
The present invention further provides the cell that contains above-mentioned carrier.
Consider in people IFN-ω polypeptide chain and have glycosylation site, in a preferred technical scheme, described cell is mammalian cell, makes the glycosylation of IFN-ω and the glycosylation process after the interior protein translation of human body the most approaching.
In a preferred technical scheme, described cell is Chinese hamster ovary celI.
Another object of the present invention provides a kind of preparation technology of above-mentioned fusion rotein medicine.
Based on above-mentioned purpose, the invention provides a kind of preparation method of the fusion rotein that contains omega interferon and human normal immunoglobulin constant region, described method comprises:
(1) clone the encoding gene of omega interferon and human normal immunoglobulin constant region and merge;
(2) build the carrier that contains above-mentioned fusion gene;
(3) above-mentioned carrier be transformed in host cell and express described fusion rotein;
(4) fusion rotein described in separation and purifying.
In a preferred technical scheme, described carrier is pMH3S.
In step (3), the host of expressed fusion protein can be yeast, mammalian cell, bacterium, animal, plant etc.Fusion rotein or polypeptide may reside in host cell, can be also that secretion out, is preferably secreted out from host from host.Secrete signal peptide used, the signal peptide of preferred human tissue plasmin, the signal peptide of natural mouse source antibody, or the universal signal peptide of antibody, or the analogue of these three kinds of signal peptides.The preferred signal peptide with human tissue plasmin, during with this signal peptide, expressing fusion protein level is higher.
The host that can contain DNA construct of the present invention by cultivation, as recombination yeast, recombinant mammalian cells, recombinant bacteria, genetically modified animals and plants etc., produces fusion rotein of the present invention.Concrete cultural method, can, with shaking flask or bio-reactor etc., be preferably bio-reactor during production.Substratum should be able to provide thalline (or cell) growth and the required material of Product Expression, should comprise nitrogenous source, carbon source, pH buffer composition etc., and culture medium prescription generally should, according to different Objects of Development, obtain by test.Cultivation can divide two stages, and the first stage is mainly used in the growth of thalline (or cell), and subordinate phase is mainly used in synthetic product.
In step (4), can be with the method for various albumen sepn separated, purified fusion protein in the cell culture that contains DNA construct of the present invention.As saltout, the combination of the technology such as precipitation, ultrafiltration, liquid chromatography (LC) and these technology.Wherein liquid chromatography (LC) can be used gel exclusion, affine, ion-exchange, the chromatographic technique such as hydrophobic, anti-phase.In a preferred technical scheme, the purification process in step (4) is chromatography method, and described chromatography method can have ion-exchange, affinity chromatography, hydrophobic chromatography and sieve chromatography.
In a preferred technical scheme, described chromatography method is for take the affinity chromatography method that Protein A is medium.
The obtained technique effect of the present invention is mainly manifested in 3 aspects:
1. long half time
The fusion rotein of recombinant human interferon alpha 2 provided by the present invention and human normal immunoglobulin constant region has the blood level of longer time in vivo, the transformation period of rhIFN-ω-Fc in new zealand rabbit body is 53.3 hours, have significantly long-lastingly, and the transformation period of rhIFN-ω is 1.54 hours.Happy energy (PegIntron clinically wears
tM12K-polyoxyethylene glycol Interferon, rabbit alfa-2b) transformation period in human body is 40 hours, Pai Luoxin (Pegasys, 40K polyoxyethylene glycol Interferon, rabbit alfa-2a) transformation period in human body is 160 hours, and the long transformation period can reduce frequency injection and make patient acceptant.
2. specific activity is high
The fusion rotein of recombinant human interferon alpha 2 of the present invention and human normal immunoglobulin constant region is to adopt engineered method, use mammalian cell expression, make glycosylation and the glycosylation process after the interior protein translation of human body of Interferon, rabbit the most approaching, glycosylated restructuring IFN-ω has than other cell Interferon, rabbit that for example yeast cell to express goes out and has higher antiviral activity.The fusion rotein obtaining after high-density culture, purifying has higher biological activity, and specific activity is 3.7 * 10
7iU/mg, the specific activity 7 * 10 of the happy energy of wearing clinically
7iU/mg, the specific activity of Pai Luoxin is 1.4 * 10
7iU/mg.
3. preparation technology is simple
Recombinant human interferon alpha 2 of the present invention does not need complicated chemically modified with separated again with the fusion rotein of human normal immunoglobulin constant region, compare with the polyethyleneglycol modified long-acting product having gone on the market, preparation technology is simple, the sample that can obtain purity 95% by affinity chromatography single step purification, quality control is relatively easy.
Accompanying drawing explanation
The behave clone of IFN-ω gene of Fig. 1;
The behave clone of IgG1Fc gene of Fig. 2;
Fig. 3 is the clone of IFN-ω-Fc gene;
Fig. 4 is that the enzyme of pMD-IFN-ω-Fc is cut evaluation;
Fig. 5 is the structure schema of expression plasmid pMH3S;
Fig. 6 is that the enzyme of expression plasmid pMH3S-IFN-ω-Fc is cut evaluation;
Fig. 7 is the structure schema of expression plasmid pMH3S-IFN-ω-Fc;
Fig. 8 is that the SDS-PAGE (non-reduced condition) of the fusion rotein IFN-ω-Fc of purifying analyzes;
Fig. 9 is that the SDS-PAGE (reductive condition) of the fusion rotein IFN-ω-Fc of purifying analyzes;
Figure 10 is the de-glycosylation analysis of the fusion rotein IFN-ω-Fc of purifying;
Figure 11 is the Assay of Antiviral Activity of IFN-ω-Fc fusion rotein;
Figure 12 behave IFN-ω-Fc and the drug level time curve of people IFN-ω in rabbit body.
Embodiment
The clone of embodiment 1:IFN-ω and human IgG1 Fc gene
(1) clone of IFN-ω gene: the polysaccharase (Pyrobest Taq enzyme, TaKaRa company, the catalog number: DR0005A) that adopt high-fidelity.Use primer is
IFN-ω-up:5-AAT
gAATTCtCTGGGCTGTGATCTGCCTC-3, and,
IFN-ω-lower:5-AGATGAGCCCAGGTCT CTATC-3; (
gAATTCfor the restriction enzyme site of Eco RI, primer is synthetic by Beijing Ying Jun biotech company, is diluted to the final concentration of 50 μ M, the IFN-ω gene order Genbank accession number of design of primers institute foundation: NM_002177) with pure water.
The preparation of template: the normal human blood 5ml of aseptic extraction, with Ficoll-Paque Premium cellular segregation liquid (GE company, catalog number: 17-5442-02/3) separated normal people's peripheral blood leucocyte, use Trizol reagent (Invitrogen company, catalog number: 15596-026) extract total RNA, carry out reverse transcription (mRNA Selective PCR Kit Ver1.1, TaKaRa company, catalog number: DRR025A), obtain cDNA, as the template of PCR reaction.
PCR reaction system: contain in 50 μ l reaction systems: 10 * buffer 5ul; DNTP (0.2mM) 4ul; Each 0.5ul of Pyrobest Taq enzyme 0.25ulIFN-ω-up/IFN-ω-lower; 2ul cDNA makes template, with pure water, supplies 50 μ l.PCR reaction parameter: 94 ℃ of 5min; 94 ℃ of 30s, 58 ℃ of 30s, 72 ℃ of 1min20s; 25 circulations.72℃5min。PCR reaction product is carried out 1% agarose gel electrophoresis detection, and the recovery test kit of Qie Jiaobingyong BBI company reclaims DNA fragmentation, and the electrophoresis result of PCR product is shown in Fig. 1.In Fig. 1, M swimming lane is molecular weight marker, and 1 swimming lane is IFN-ω fragment.
(2) clone of Fc and recovery purifying: the polysaccharase (Pyrobest Taq enzyme, TaKaRa company, the catalog number: DR0005A) that adopt high-fidelity.Use primer is:
IFN-ω-Fc：5-
GTTGAGCCCAAATCTT GTG-3；
Fc?lower：5-AAT
GCGGCCGCTCATTAACCCGGAGACAGGGAGAG-3；
gCGGCCGCfor the restriction enzyme site of Not I,
The preparation of template: the normal human blood 5ml of aseptic extraction, with Ficoll-Paque Premium cellular segregation liquid (GE company, catalog number: 17-5442-02/3) separated normal people's peripheral blood leucocyte, use Trizol reagent (Invitrogen company, catalog number: 15596-026) extract total RNA, carry out reverse transcription (mRNA Selective PCR Kit Ver1.1, TaKaRa company, catalog number: DRR025A), obtain cDNA, as the template of PCR reaction.
PCR reaction system: contain in 50 μ l reaction systems: 10 * buffer 5ul; DNTP (0.2mM) 4ul; Each 0.5ul of Pyrobest Taq enzyme 0.25ul IFN-ω-Fc/Fc lower; 2ul cDNA makes template, with pure water, supplies 50 μ l.PCR reaction parameter: 94 ℃ of 5min; 94 ℃ of 30s, 58 ℃ of 30s, 72 ℃ of 1min20s; 25 circulations.72℃5min。PCR reaction product detects with 1% agarose gel electrophoresis, and Qie Jiaobingyong BBI company reclaims test kit and reclaims, and PCR product electrophoresis result is shown in Fig. 2.In Fig. 2, M swimming lane is molecular weight marker, and 1 swimming lane is Fc fragment.
(3) clone of IFN-ω-Fc fusion gene: the polysaccharase (Ex Taq enzyme, TaKaRa company, the catalog number: DRR001A), object is that the end at PCR product adds A, conveniently carries out T-A clone that adopt high amplification efficiency.PCR reaction system: contain in 50 μ l reaction systems: l0 * buffer 5ul; DNTP (0.2mM) 4ul; Ex Taq enzyme 0.25ul; Each 0.5ul of IFN-ω-up/Fc lower; The product of the product of 2ul step 1 and 2ul step 2 is made template, with pure water, supplies 50 μ l.PCR reaction parameter: 94 ℃ of 5min; 94 ℃ of 30s, 58 ℃ of 30s, 72 ℃ of 1min20s; 25 circulations.72℃5min。PCR reaction product detects with 1% agarose gel electrophoresis, and the recovery test kit of Qie Jiaobingyong BBI company reclaims, and PCR product electrophoresis result is shown in Fig. 3.In Fig. 3, M swimming lane is molecular weight marker, and 1 swimming lane is IFN-ω-Fc fragment.IFN-ω-Fc is (pMDl 8-T to specifications, TaKaRa company, catalog number: D101A) be cloned into (called after pMD-IFN-ω-Fc) on pMDl 8-T carrier, Song Ying fine horse biotech firm carries out nucleotide sequencing, and sequence is shown in SEQ ID NO:l.Enzyme is cut qualification result and is seen Fig. 4.In Fig. 4, M swimming lane is molecular weight marker, and 1 swimming lane is the plasmid after enzyme is cut, and 2 swimming lanes are the plasmid before enzyme is cut.
Embodiment 2:pMH3S-IFN-ω-Fc plasmid construction
(1) structure of pMH3S secretion type eukaryon expression vector
With carrier for expression of eukaryon pcDNA3.1 (+) (the Invitrogen company commonly using, catalog number V790-20) be basic framework, the multiple clone site district that Kozak sequence (GCCGCCACCATGA/G) and amended human tissue plasmin signal peptide sequence fusion gene (seeing SEQ ID NO:3) is cloned into expression vector, is built into pMH3S secretion type eukaryon expression vector.In amended gene, by by the TCGAACAGC (coded amino acid S-N-S) of fusion gene nucleotide sequence 5 ' end 70-78 position, be mutated into TCGAATTCT (coded amino acid S-N-S), guaranteeing that the restriction enzyme site (GAATTC) of introducing Eco RI under the immovable prerequisite of aminoacid sequence facilitates goal gene directly by Eco RI site, to be connected on pMH3S carrier, between signal peptide and goal gene, there is no unnecessary amino acid, target protein can be secreted in supernatant, facilitates the purifying in downstream.
Building process:
1) fusion gene of synthetic Kozak sequence (CCACCATG (A/G)) and amended human tissue plasmin signal peptide sequence first;
2) after pcDNA3.1 (+) is cut with Eco RI enzyme, by Klenow enzyme (TaKaRa, catalog number: D2140) fill, then use small intestine alkaline phosphatase CIP (NEB company, catalog number: M0290V) dephosphorylation is avoided from connecting;
3) T4DNA ligase enzyme (NEB company, the catalog number: M0202V) connect for carrier after synthetic gene and enzyme are cut;
4) transform escherichia coli DH5a competence, select the clone with amicillin resistance;
5) with OMEGA plasmid extraction kit extraction plasmid, and carry out determined dna sequence, order-checking obtains the pMH3S carrier of expecting after identifying, and (the structure flow process of pMH3S is shown in Fig. 5.)
(2) pMH3S-IFN-ω-Fc plasmid construction
With Eco RI and Not I (NEB company) double digestion pMD-IFN-ω-F
c, with glue, reclaim test kit (China, BBI company) and reclaim IFN-ω-F
cfragment (the about 1.2kb of length) is connected on the pMH3S with Eco RI and Not I double digestion, transform bacillus coli DH 5 alpha competence, on the penbritin LB of 100 μ g/ml flat board, cultivate, select resistance clone and carry out PCR detection (PCR condition is with the step in embodiment 1 (3)), after the PCR product electrophoresis detection positive, random choose positive colony is inoculated into overnight incubation in LB substratum, inferior daily plasmid extraction kit (China, OMEGA company) extract plasmid, the plasmid extracting is carried out to double digestion (Eco RI/Not I) and identify, qualification result is shown in Fig. 6.In Fig. 6, M swimming lane is molecular weight marker, and 1 swimming lane is the plasmid after enzyme is cut, and 2 swimming lanes are the plasmid before enzyme is cut.Expression vector establishment flow process is shown in Fig. 7.
Embodiment 3:IFN-ω-Fc high expressing cell clone's screening
From liquid nitrogen, get CHO-S cell (Invitrogen company) recovery, in T25 square vase, cultivate, substratum is DMEM/F12+10% foetal calf serum (FBS), is placed in 37 ℃, 5%CO
2in cell culture incubator (Forma), cultivate, after covering with, import in T75 square vase.After covering with, add 3ml trysinization 30s in T75 square vase, inhale and abandon pancreatin, with PBS, wash after cell two times centrifugal, get 1.5 * 10
7cell is resuspended with 0.5ml PBS (pH7.2), gets 10 μ l salmons essences, and linearizing expression plasmid 10 μ g, join in the middle of ready cell, joins ice bath in the 2mm electricity revolving cup of precooling after mixing, by electroporation 160V electric shock twice, between interval 1min.
Cell after electricity turns is divided into two parts of plastic culture dish that are placed in diameter 10em, add respectively 10ml contain 10%FBS, without dual anti-DMEM/F12 substratum.Be placed in 37 ℃, 5%CO
2in cell culture incubator, cultivate.Renew next day fresh 10ml contain 10%FBS, without dual anti-DMEM/F12 substratum.Until cell, grow to 50～60% when full, add the G418 of 1.8mg/ml, change every other day liquid, wash dead cell off, G418 concentration maintains 1.8mg/ml all the time, until cell is no longer dead, clone forms.The clone of formation is transferred in 96 orifice plates, and with containing 10%FBS, without dual anti-DMEM/F12 substratum, G418 concentration is 0.6mg/ml, is placed in 37 ℃, 5%CO
2in cell culture incubator, cultivate.Until substratum in 96 orifice plates, grow to over after half-full, change serum-free suspension medium, every hole 100 μ l.By the method for Dot-Blot, survey its 24h expression amount.Get cell conditioned medium 5 μ l points on nitrocellulose filter, after drying, be soaked in 5% skim-milk, be placed on shaking table and seal 1h, with HRP mark goat-anti people F
cbe two anti-, the skim-milk dilution in 1: 5000 with 5%, hatches with it, hatches 1h for 37 ℃, with TBST, washes film 3 times, each 10min, and chemoluminescence method develops the color.Select clone that expression amount is high as engineering cell strain.
Embodiment 4: the affinitive layer purification of fusion rotein IFN-ω-Fc
Utilize human interferon and human normal immunoglobulin fusion rotein Fc part can with the character of the special affine combination of Protein A, supernatant can carry out purifying by affinity chromatography.Affinity chromatography medium Mabselect sure (GE, cat. no: 17-5438-01) cell culture fluid process 12500rpm is centrifugal 10 minutes, gets supernatant and carries out purifying.First use 5 column volumes of level pad (20mM PBS) balance, after loading, with the albumen of level pad washing non-specific binding, finally use the citrate buffer solution wash-out target protein of pH3.50.1M, use immediately 1M Tris (pH8.5) neutralization of 1/10 volume.Purified product is carried out under reductive condition and non-reduced condition, carry out SDS-PAGE and detect analysis, the results are shown in Figure 8, Fig. 9 and Figure 10.In Fig. 9, the about 50kD of the molecular weight of target protein under reductive condition, the molecular weight of the target protein of inferring by amino acid is 46kD, apparent molecular weight is bigger.Analyzing reason is glycosylated result, by glycosylase PNGase (NEB company, catalog number (Cat.No.): P0704S) enzyme carries out electrophoresis after cutting and can confirm, the results are shown in Figure 10, under reductive condition, (in figure, represent that the sample after enzyme is cut is 1 swimming lane, the sample before enzyme is cut is 2 swimming lanes to big or small about 46kd after PNGase enzyme enzyme is cut.), consistent with the molecular size of inferring by aminoacid sequence.In Fig. 8 (under non-reduced condition), the about 100kD of molecular weight of target protein, infers that expression product forms dimer.
Embodiment 5: the antiviral activity of people IFN-ω-Fc fusion rotein
According to the method for bibliographical information, carry out the research of antiviral activity.In this research, select the appendix XC interferon biological activity assay method (cytopathic-effect inhibition assay) of three of Chinese Pharmacopoeias.According to human amniotic cell (Wish), avoid the effect that blister Stomatovirus (VSV) destroys.Wish cell dyeing by Viola crystallina to survival, measures its absorbancy in wavelength 570nm place, can obtain the protective effect curve of Interferon, rabbit to Wish cell, with this, measures the biologic activity of Interferon, rabbit.The specific activity of the IFN-ω-Fc measuring is 3.7 * 10
7iU/mg, is shown in Figure 11.
Embodiment 6:IFN-ω-Fc fusion rotein and the metabolism of people IFN-ω in plasma in rabbit
Select Bender MedSystems
tMcompany produces people IFN-ω ELISA medicine box (catalog number: the concentration of BMS233TEN) measuring recombinant human omega-Interferon, rabbit (rh INF-ω) in serum.Measuring principle, method, step and methodology reliability confirmation refer to test kit working instructions.Sensitivity reaches 5pg/ml.Selection new zealand rabbit carries out the preliminary study of pharmacokinetics, and 5 every group, back subcutaneous administration, dosage IFN-ω-Fc is 46 μ g/kg, and IFN-ω is 20 μ g/kg, and IFN-ω-Fc and IFN-ω are 1nmol/kg.Different time points 0.5h after administration, 1h, 2h, 4h, 8h, 12h, 24h, 48h, 72h, 96h, 120h, 144h gets blood, separation of serum carries out the mensuration of IFN-ω concentration, draws drug level time curve, sees Figure 12, X-coordinate represents it is different blood sampling time points, the concentration of the IFN-ω that ordinate zou represents, and solid line represents IFN-ω-Fc group; Dotted line represents IFN-ω group.IFN-ω group after 24h completely metabolism fall, IFN-ω is in background level.And IFN-ω-Fc group still has very high concentration after 144h, there is result for the treatment of.GraphPad Prism software analysis pharmacokinetic parameter, the results are shown in following table.
The rabbit drug disposition Pharmacokinetics Parameter (n=5) of table 1 single subcutaneous administration IFN-ω and IFN-ω-Fc
In table 1, t
maxrefer to time when drug level reaches maximum; c
maxrefer to the peak concentration that medicine reaches in rabbit body after administration; t
1/2refer to that medicine reaches the time that a half is fallen in metabolism after peak concentration.From table, can see, the transformation period of IFN-ω-Fc in new zealand rabbit body reaches 53.3 hours, be obviously longer than IFN-ω 1.54 hours.
Claims (13)
1. a fusion rotein for omega interferon and human normal immunoglobulin IgG1 constant region part Fc fragment, described fusion rotein aminoacid sequence, as shown in SEQ ID NO:2, does not have connection peptides between described omega interferon and human normal immunoglobulin constant region.
2. the homodimer of fusion rotein according to claim 1.
3. a pharmaceutical preparation that contains the fusion rotein described in claim 1 or 2.
4. the nucleotide sequence of fusion rotein described in the claim 1 of encoding.
5. nucleotide sequence according to claim 4, is characterized in that, described nucleotides sequence is classified SEQ ID NO:1 as.
6. a carrier that contains nucleotide sequence described in claim 5.
7. the carrier of nucleotide sequence according to claim 6, is characterized in that, described carrier is pMH3S-IFN-ω-Fc plasmid, and the construction step of described plasmid is:
(1) at the multiple clone site Eco of carrier for expression of eukaryon pcDNA3.1 RI place, insert the nucleotide sequence as shown in SEQ ID NO:3, obtain secretion type eukaryon expression vector pMH3S;
(2) nucleotide sequence orientation described in claim 5 is inserted between the multiple clone site Eco RI and Not I of described secretion type eukaryon expression vector pMH3S, obtains described pMH3S-IFN-ω-Fc plasmid.
8. a cell that contains carrier described in claim 7.
9. cell according to claim 8, is characterized in that, described cell is mammalian cell.
10. cell according to claim 9, is characterized in that, described cell is Chinese hamster ovary celI.
The preparation method of fusion rotein described in 11. 1 kinds of claims 1, described method comprises:
(1) clone the encoding gene of omega interferon and human normal immunoglobulin constant region and merge;
(2) build carrier claimed in claim 7;
(3) above-mentioned carrier be transformed in Chinese hamster ovary celI and express described fusion rotein;
(4) fusion rotein described in recovery and purifying.
12. methods according to claim 11, is characterized in that, the purification process in step (4) is chromatography method.
13. methods according to claim 12, is characterized in that, described chromatography method is for take the affinity chromatography method that Protein A is medium.
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| US5723125A (en) * | 1995-12-28 | 1998-03-03 | Tanox Biosystems, Inc. | Hybrid with interferon-alpha and an immunoglobulin Fc linked through a non-immunogenic peptide |
| AU777963B2 (en) * | 1999-05-19 | 2004-11-04 | Merck Patent Gmbh | Expression and export of interferon-alpha proteins as Fc fusion proteins |
| US7670595B2 (en) * | 2004-06-28 | 2010-03-02 | Merck Patent Gmbh | Fc-interferon-beta fusion proteins |
| KR20080021614A (en) * | 2005-05-26 | 2008-03-07 | 쉐링 코포레이션 | Interferon-igg fusion |
| CN1944463B (en) * | 2006-10-30 | 2010-05-12 | 中国科学技术大学 | Fusion protein with alpha-interferon activity and its coded gene and use |
| DK2662449T3 (en) * | 2007-05-30 | 2017-05-15 | Postech Academy-Industry- Found | immunoglobulin fusion proteins |
| ES2400107T3 (en) * | 2007-10-22 | 2013-04-05 | Merck Serono S.A. | Single IFN-beta fused to an Fc fragment of mutated lgG |
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2011
- 2011-01-07 CN CN201110002132.4A patent/CN102585013B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101146825A (en) * | 2005-02-14 | 2008-03-19 | 阿波罗生命科学有限公司 | A molecule and chimeric molecules thereof |
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