CN105384828B - Long-acting interferon-α and its remodeling method - Google Patents
Long-acting interferon-α and its remodeling method Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/555—Interferons [IFN]
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- C07K2319/00—Fusion polypeptide
- C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
Abstract
The present invention relates to long-acting interferon-alpha fusion protein and its remodeling methods, connect interferon-' alpha ' molecule with Fc sections of mutant of IgG1 antibody molecule particular by link peptide, form the mode of fusion protein to increase the circulating half-life in vivo of interferon-' alpha '.The link peptide is the flexible polypeptide being made of glycine and serine；Fc sections of mutant of the IgG1 antibody molecule are human IgG1 the 224th (His) to the amino acid sequence of 447 positions (Lys) or human IgG1 the 231st (Ala) to the amino acid sequence of 447 positions (Lys), and 297 asparagines (Asn) are mutated into glutamine (Gln).Long-acting interferon-the alpha fusion protein is expressed by pichia yeast expression system and is prepared.Long-acting interferon of the invention-alpha fusion protein antiviral activity and commercialization long-acting interferon PEG-IFN alpha-2a are close, and to be considerably longer than PEG-IFN alpha-2a in rat Half-life in vivo.Long-acting interferon-α of the invention can be used for preparing the drug for treating virus hepatitis and other antiviral drugs.
Description
Technical field
The present invention relates to protein engineering fields, and in particular to fusion protein and its encoding gene and application especially relate to
And the expression side by transformation and optimization and the fusion protein with the active fusion protein of human interferon-alpha and its encoding gene
Method and the application in preparation treatment virus hepatitis and other antiviral drugs.
Background technique
Interferon-' alpha ' (IFN-α) is a kind of immunocompetence glycoprotein with multiple functions, is had good antiviral, anti-
The effects of tumour.Recombined human interferon-alpha is used for the treatment of virus hepatitis and tumour for a long time, has in hepatitis B and hepatitis treatment
It is not easy anti-medicine, the features such as effect is lasting, is the first-line drug in current various countries' antiviral therapy guide.Traditional recombined human interference
Plain α is easy to be degraded by glomerular filtration and haemocyanin enzyme, to cause in human body since molecular weight is smaller (18kDa)
Half-life period it is shorter, the disadvantages of leading to frequent drug administration by injection and larger side effect.In order to overcome this disadvantage, Schering Plough
(Schering-Plough is now purchased by Merck & Co., Inc.) and Roche Holding Ag (Roche) successively develop polyethylene glycol (PEG) change
Long-acting interferon (trade name be respectively as follows: dub in background music can, PEG-IFN alpha-2a), successfully by 10 times of its Increased Plasma Half-life or more.
Other than PEGylated, HAS or Fc fusion protein by interaction with FcRn and can increase protein point
The modes such as son amount extend half-life period of recombinant protein.Clinical research confirmation, the long-acting interferon-α Albufero of HAS fusion
(Human Genome Sciences) although half-life period is extended than PEG-IFN alpha-2a, its activity is too low, and dosage is too big, makes
At security risk.The prompt of this phenomenon, as can exploitation bioactive and PEG-IFN alpha-2a is suitable, half-life period be appropriately extended again it is novel
Long-acting interferon-α, then by with the stronger market competitiveness and wide prospect.There are several Fc fusion proteins at present
Depot drug product listing, such as whole world best-selling drugs Enbrel (TNFR-Fc).Nearest hundred Jian Ai enlightening company (Biogen Idec) exploitation
The fusion protein for recombinating VIII factor and Ⅸ factor and antibody Fc section successfully obtains FDA approval listing, greatly Fc has been inspired to melt
Conjunction technology is used to develop the confidence of long-acting fusion protein drug.
This seminar researcher accumulates by long-term research, is connected and composed using the Fc segment and IFN α -2b of IgG1
Fusion protein IFN-α/Fc, to improve the Half-life in vivo of IFN-α.Long-acting interferon IFN-α/Fc has tentatively been completed before this
Physicochemical property identification, the research of antiviral activity and the research of rat Half-life in vivo and have obtained related invention patent
(Xiao Weihua, Wang Lei, Authorization Notice No.: 1944463 B of CN).But Fc fragment structure is complicated, and there is disulfide bond and glycosylation to repair
Decorations generally require to be expressed using mammalian cell, and industrialization is expensive.In order to solve the problems, such as production cost, I
IFN-α/Fc fusion protein for being produced using pichia yeast expression system.But there are N- glycosylations for the Fc of antibody molecule section
Site, but this site has no effect on the combination of itself and FcRn.
Summary of the invention
In the present invention, in order to avoid the glycosylation modified potential antigenicity of yeast mannose type, the present invention is further
Site-directed point mutation (N is mutated into Q) is carried out to 297 glycosylation sites (N) of Fc segment, and in IFN-α and the junction Fc
The flexible peptide for introducing one section of immunologic inertia constructs glycosylation site mutation and the new long-acting interferon containing flexible peptide linker
α(IFN-α/Fc-MD).Recently some studies pointed out that (Tianlei Ying, et al., Soluble Monomeric IgG1Fc,
J.Biol.Chem.2012,287:19399-19408), the combination of Fc segment and FcRn are not rely on its duplex structure, and examine
Consider interferon molecule originally with monomeric form play biological action, therefore this research again construct single-stranded Fc segment and
The fusion protein (IFN-α/Fc-SC) of IFN α -2b.The present invention completes setting for above two novel IFN-α/Fc fusion protein
Meter, building, expression and purifying, and by itself and IFN-α/1 (IFN-α/Fc- of Fc fusion protein IFN α 2b-Fc γ for constructing before this
WT PEG-IFN alpha-2a (PEG-IFN- α)) and in the market carries out the comparison of pharmacodynamics and pharmacokinetics, with the albumen that determination is optimal
Design form (three kinds of IFN-α/Fc fusion protein structural schematic diagram is as shown in Figure 1).
In particular it relates to the following terms:
1. a kind of long-acting interferon-alpha fusion protein is connected by Fc sections of mutant of interferon-' alpha ' molecule and IgG1 antibody molecule
It connects, wherein described the 297th asparagine mutation of IgG1 that sport is at glutamine.
2. long-acting interferon-alpha fusion protein according to the 1st, wherein the hinge of Fc sections of the IgG1 antibody molecule
Area's amino acid is substituted by flexible polypeptide.
3. long-acting interferon-alpha fusion protein according to the 2nd, wherein
1) Fc sections of the amino acid EPKSCDKT of the end hinge area N- 8 (SEQ ID NO:1) is replaced by GGGGSGGG
(SEQ ID NO:2)；Or
2) Fc sections of the amino acid EPKSCDKTHTCPPCP of the end hinge area N- 15 (SEQ ID NO:3) is replaced by and exempts from
The soft peptide GGGGSGGGGS of epidemic disease inertia (SEQ ID NO:4).
4. long-acting interferon-alpha fusion protein according to the 3rd, wherein
In the case where 1), Fc sections of the IgG1 antibody molecule is amino of the human IgG1 the 231st (Ala) to 447 positions (Lys)
Acid sequence；
In the case where 2), Fc sections of the IgG1 antibody molecule is human IgG1 the 224th to 447 amino acid sequence.
5. long-acting interferon-alpha fusion protein according to the 1st, the interferon-' alpha ' are human interferon-alpha 2b.6. piece
According to long-acting interferon-alpha fusion protein described in the 3rd, amino acid sequence is one of the following terms:
1) SEQ ID NO:5；
2) SEQ ID NO:6.
7. long-acting interferon-alpha fusion protein gene described in any one of coding 1-6.
8. the gene according to the 7th, nucleic acid sequence is SEQ ID NO:7 or SEQ ID NO:8.
9. recombinant expression carrier, transgenic cell line and recombinant bacterium comprising gene described in the 7th, the cell line are excellent
It is selected as Pichia pastoris system.
10. 1-6 described in any item long-acting interferon-alpha fusion proteins are used to prepare the purposes of drug, the drug
It is antiviral or antitumor for improving immunity of organisms.
IFN-α/Fc-MD antiviral activity of the invention is slightly above IFN-α/Fc-WT, and long-acting interference is slightly below commercialized
Plain PEG-IFN alpha-2a PEG-IFN- α (66%~85% activity for being equivalent to PEG-IFN- α), but rat circulating half-life in vivo is significant
It is longer than PEG-IFN alpha-2a PEG-IFN- α, and can speculates that be will be more significant in human body is longer than PEG-IFN alpha-2a PEG-IFN- α；Meanwhile this hair
Bright IFN-α/Fc-SC antiviral activity is higher than commercialization long-acting interferon PEG-IFN alpha-2a PEG-IFN- α and (is equivalent to PEG-IFN-
158%~186% activity of α) and IFN-α/Fc-WT, although being slightly below PEG-IFN alpha-2a PEG- in rat circulating half-life in vivo
IFN-α, but can speculate will be longer than PEG-IFN alpha-2a PEG-IFN- α in the intracorporal circulating half-life of people.
Detailed description of the invention
The IFN-α of tri- kinds of different structure forms of Fig. 1 /Fc fusion protein schematic diagram；
Fig. 2 .IFN- α/Fc-WT expressing fusion protein colony screening；
(A) DB, Dot blot, dot blot；(B) WB:Western blot, immunoblotting analysis (anti-human Fc antibodies)；
Fig. 3 .IFN- α/Fc-WT fusion protein fermentation expression；
(A) thalli growth curve in fermentation process；
(B) destination protein accumulated time point detects；
Fig. 4 .IFN- α/Fc fusion protein purification and physical and chemical identification；
(A) IFN-α/Fc fusion protein purity SDS-PAGE detection；
(B) IFN-α/Fc fusion protein IFN-α segment and Fc segment Western blot detection；
(C) the glycosylation modified PAS dyeing detection of IFN-α/Fc fusion protein；
The detection of Fig. 5 .IFN- α/Fc fusion protein antiviral activity:
(A) WISH-VSV system measurement IFN-α/Fc fusion protein antiviral activity；
(B) MDBK-VSV system measurement IFN-α/Fc fusion protein antiviral activity；
Fig. 6 .IFN- α/Fc fusion protein stimulates PBMC to transcribe OAS1 vigour；
The detection of Fig. 7 .IFN- α/Fc fusion protein anti-tumour cell proliferative activity；
The detection of Fig. 8 .IFN- α/Fc fusion protein rat circulating half-life in vivo.
Specific embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples can obtain unless otherwise specified from the commercial sources of mark.
The synthesis of all primers and examining order in following embodiments are completed by the raw work in Shanghai；Pichia pastoris relevant carriers
Invitrogen (Life technologies) is derived from bacterial strain；Culture medium prescription is referring to Pichia Expression
Kit,A Manual of Methods for Expression of Recombinant Proteins in Pichia
Pastoris, Invitrogen).
Embodiment 1, the building of New-type long-acting interferon IFN-α/Fc fusion protein expression vector
One, IFN-α/Fc fusion amplification
With construct before this laboratory and save plasmid pPIC9K-Kex-IFN α 2b-Fc γ 1 (details referring to Wang Lei etc.,
Chin J Biotech 2008,January 25；24 (1): 53-62) it is template, it is (all to draw using primer α-Fw and Fc-Rv
1) object sequence is shown in Table, amplifying target genes.Amplified production equally use nucleic acid gel QIAquick Gel Extraction Kit (Axygen company, below
It recycles together).This gene product both ends has I restriction enzyme site of BamH I and EcoR, is named as IFN-α/Fc-WT.
The use of plasmid pPIC9K-Kex-IFN α 2b-Fc γ 1 is template, using three couples of primer α-Fw and SC-Rv, SC-Fw with
Three segments of M-Rv, M-Fv and Fc-Rv difference amplifying target genes.PCR program setting are as follows: 94 DEG C 2 minutes, 94 DEG C 30 seconds,
55 DEG C 40 seconds, 72 DEG C 60 seconds, procedure above 32 circulation, 72 DEG C 10 minutes.Amplified production is recycled using gel reclaims kit,
Operating procedure illustrates referring to kit.Recycling three sections of genes of gained are mixed and are used as template, are spliced with primer pair α-Fw and Fc-Rv
Complete genome.PCR program setting are as follows: 94 DEG C 5 minutes, 94 DEG C 30 seconds, 55 DEG C 40 seconds, 72 DEG C 120 seconds, procedure above 32 are followed
Ring, 72 DEG C 10 minutes.Amplified production equally uses nucleic acid gel QIAquick Gel Extraction Kit to recycle.This gene product both ends has BamH I
With I restriction enzyme site of EcoR, it is named as IFN-α/Fc-SC.
Using IFN-α/Fc-SC as template, two sections are expanded respectively using two couples of primer α-Fw and MD-Rv, MD-Fw and Fc-Rv
Gene, PCR program setting are as follows: 94 DEG C 2 minutes, 94 DEG C 30 seconds, 55 DEG C 40 seconds, 72 DEG C 60 seconds, procedure above 32 circulation, 72 DEG C
10 minutes.Amplified production is recycled using gel reclaims kit.Two sections of genes of gained are mixed and are used as template, with primer pair α-Fw
Splice complete genome with Fc-Rv.PCR program are as follows: 94 DEG C 5 minutes, 94 DEG C 30 seconds, 55 DEG C 40 seconds, 72 DEG C 120 seconds, procedure above
32 circulation, 72 DEG C 10 minutes.Amplified production equally uses nucleic acid gel QIAquick Gel Extraction Kit to recycle.This gene product both ends is same
With I restriction enzyme site of BamH I and EcoR, it is named as IFN-α/Fc-MD.
1. primer sequence table of table
Two, IFN-α/Fc fusion protein yeast expression vector constructs
Three antigen-4 fusion protein gene IFN-α/Fc-WT (SEQ ID NO:18) that upper step is obtained, IFN-α/Fc-SC (SEQ
ID NO:7) with IFN-α/Fc-MD (SEQ ID NO:8) and commercialization expression plasmid pPIC9 use restriction enzyme
I double digestion of BamH I and EcoR.Digestion products are using PCR product QIAquick Gel Extraction Kit recycling (Axygen company), operating procedure ginseng
See kit explanation.It recycles resulting digestion rear fusion protein gene and is connect with carrier after digestion using T4DNA ligase.By enzyme
Connect product object converts bacillus coli DH 5 alpha competent cell.Conversion operation step are as follows: take 5 μ L enzyme-linked products that 100 μ L large intestine bars are added
In bacterium competence, mix, on ice stand 15-30 minute, 42 DEG C heat shock 90 seconds, on ice standing 3 minutes, 37 DEG C of shaking table cultures 45 are divided
Clock is coated with LA resistant panel.Clone in resistant panel is selected after conversion and carries out PCR identification, and gained positive colony is respectively designated as
PPIC9-IFN- α/Fc-WT, pPIC9-IFN- α/Fc-SC and pPIC9-IFN- α/Fc-MD, and serve Hai Shenggong biological order-checking.
Three, result:
Always, clone's building is correct for sequencing result and theory expectation result.
The expression and purification of embodiment 2, New-type long-acting interferon IFN-α/Fc fusion protein
One, expressing fusion protein
Expanded using 50mL LA and cultivate above-mentioned positive colony, extracts matter respectively using amount in kit (Axygen company)
Grain pPIC9-IFN- α/Fc-WT, pPIC9-IFN- α/Fc-SC and pPIC9-IFN- α/Fc-MD.Expression plasmid prepared by upper step
PPIC9-IFN- α/Fc-WT, pPIC9-IFN- α/Fc-SC and pPIC9-IFN- α/Fc-MD are carried out linearly using restriction endonuclease Sal I
Change, and is recycled by ethanol precipitation.Methanol precipitation step: 1) into endonuclease reaction system be added two volumes dehydrated alcohol and
The 3M sodium acetate of 0.1 times of volume, -20 DEG C of precipitatings 2 hours or more；2) 12000g is centrifuged 10 minutes, reject supernatant；3)300μL
Precipitating is resuspended in 70% ethyl alcohol, and 12000g is centrifuged 10 minutes, reject supernatant；4) 37 DEG C of drying, are resuspended with deionized water and are measured dense
Degree adjusts concentration between 0.5-1.0 μ g/ μ L.
It prepares competent yeast: 1) choosing the pichia pastoris yeast GS115 bacterial strain frozen, line YPD plate, yeast
30 DEG C of constant temperature incubations of incubator.2) to clonal growth to diameter 1mm or so, picked clones are to 4mLYPD fluid nutrient medium, 30
Under the conditions of DEG C in shaking table constant temperature incubation.3) it cultivates 24-48 hours.Bacterium solution 1mL is inoculated into 50mL fresh YPD medium
In；4) to OD600Value reaches 1.0-1.5, bacterium solution is transferred to 50mL centrifuge tube, 4 DEG C, 1500g is centrifuged 5 minutes, abandons supernatant.5)
40mL ice water is resuspended, and 4 DEG C, 1500g is centrifuged 5 minutes, abandons supernatant, is repeated once；6) the cold sorbierite of 20mL1M is resuspended, and 4 DEG C,
1500g is centrifuged 5 minutes, abandons supernatant；7) the cold sorbierite of 500 μ L1M is resuspended spare.
Electrotransformation step: it takes above-mentioned 100 μ L of competent cell in the electric revolving cup of sterile pre-cooling, it is linear that 10 μ L is added thereto
Change plasmid (5-10 μ g), is uniformly mixed；Set electroporation parameter: 1500V, 200 Ω, 25 μ F；After electricity turn immediately into electric revolving cup
The cold sorbierite of 1mL1M is added；The above-mentioned bacterium solution coating MD plate of 100-1000 μ L is taken, plate is placed in 30 DEG C of constant temperature Yeast Cultivation case trainings
It supports.
Positive expression colony screening: after above-mentioned MD plate grows yeast clone, picking is several to be cloned into 4mLMGY culture
In base, 30 DEG C of constant temperature incubations of yeast shaking table to bacterium solution milky；Wherein 1mL bacterium solution conservation will be frozen, remaining 3mL bacterium solution 1500g
Centrifugation is replaced into 4mLBMMY culture medium after five minutes, continues to cultivate, final concentration of 0.5% methanol was added at interval of 24 hours；
After 48-72 hours, 12000g be centrifuged 1 minute, take supernatant using anti-human Fc antibodies (Beijing Zhong Shan Golden Bridge) carry out Dot blot and
Western Blot identification.
Two, fusion protein fermentation, purifying and identification
Express strain culturing: recombinant bacterial strain fermentation is carried out according to Invitrogen company fermentation Guide Book.It will screen
High expression strain inoculated shaking in tube to the MGY culture medium containing 4mL, 30 DEG C shaking table culture 24-48 hours, until OD600For
2-6, as fermentation primary seed solution.The 1mL seed liquor is taken to transfer in the conical flask containing 200mL BMGY culture medium, 30 DEG C
Shaking table culture 12-24 hours, as fermentation secondary seed solution.200mL secondary seed solution is all inoculated with and is trained into containing 6L BMGY
In the fermentor for supporting base.Setup parameter: 30 DEG C of cultivation temperature, pH 6.0, dissolved oxygen, revolving speed are monitored, starts fermented and cultured.Thallus is raw
The long stage uses BMGY culture medium, illustrates that basic glycerol is depleted in culture medium, starts to carry out glycerol when dissolved oxygen rapidly rises
Stream adds, and after reaching the left and right expected weight in wet base (180g/L), starts methanol induction, methanol induction process is according to the increased three-step approach of gradient
It carries out.
Culture solution clarification: after induction terminates, culture supernatant is adjusted to pH value and is centrifuged 20 minutes to 7-8,10000g, is received
Collect supernatant, and is clarified with 0.45 μm of filtering with microporous membrane.
Albumen capture: recombination fusion protein is captured using affinity chromatographic column Hitrap MabSelect.Specific steps are as follows: 1)
Deionized water rinses 5 column volumes of chromatographic column.2) PBS rinses 3 column volumes of chromatographic column.3) loading.4) after end of the sample, PBS
Rinse 3 column volumes of chromatographic column.5) destination protein is eluted, elutes formula of liquid are as follows: 20mM acetate/acetic, 100mM NaCl, pH
=3.0.After elution, eluent is adjusted to neutrality using 1M Tris-HCl.
Polishing purification: using AKTA Explorer 100System, balances Sephacryl-200HP molecular sieve with PBS,
After balance, is rinsed by loading after the filtering of sample concentration obtained by upper step, then with PBS, collect elution samples.
Recombinant protein identification: albumen progress SDS-PAGE, Western blot obtained by purifying (anti-human lgG Fc-HRP,
Purchased from Beijing Zhong Shan Golden Bridge；The anti-IFN-α monoclonal antibody of mouse, peace section biology give；The purchase of HPR-conjugated anti-mouse antibody
From Cell Signaling) and PAS dyeing identification.
IFN-α/Fc-WT, IFN-α/Fc-MD and IFN-α/Fc-SC amino acid sequence are respectively SEQ ID NO:17,
SEQ ID NO:6 and SEQ ID NO:5.
Three, result:
1. by taking IFN-α/Fc-WT expression bacterial strain screening as an example (other two kinds of albumen expressions are similar), as shown in Fig. 2,
It is screened by MD culture plate auxotroph, obtains the Yeast expression bacterial strain of fusion protein.Using Dot blot preliminary screening,
It is further confirmed that by Western Blot, the relatively high expression cloning of expression quantity can be filtered out, for next
Protein expression and purification；
2. fermentation termination thallus weight in wet base is about 240g/L, weight in wet base has downward trend at this time, judges fermentation termination with this, keeps away
Exempt from albumen excessive degradation (Fig. 3 A)；
3. destination protein persistently adds up from induction 0 hour to induction 18 hours, not to lower tank (induction 27 hours) later
Add up again, or even reduces (Fig. 3 B)；
4. identified through SDS-PAGE by centrifugal filtration and two-step purifying, purity of protein is up to 90% (Fig. 4 A), and IFN-
α/Fc-WT and IFN-α/Fc-MD exists with dimer, IFN-α/Fc-SC with monomeric form, consistent with expection；
5. identifying that IFN-α/Fc-WT, IFN-α/Fc-MD and IFN-α/Fc-SC contain by Western blot
IFN-α component and Fc component, gained recombination fusion protein are really target protein (Fig. 4 B)；
6. by PAS dyeing as it can be seen that fusion protein IFN-α/Fc-MD and IFN-α/Fc-SC degree of glycosylation compared to
IFN-α/Fc-WT significantly reduces (Fig. 4 C)；From the point of view of protein electrophoresis SDS-PAGE performance, IFN-α/Fc-MD is gone back in reduction with non-
Under old terms, molecular weight is more smaller than IFN-α/Fc-WT (Fig. 4 A, B and C), and also prompt is by mutation 297 site N of Fc segment, significantly
Reduce IFN-α/Fc-MD degree of glycosylation.
Embodiment 3, the detection of New-type long-acting interferon IFN-α/Fc fusion protein antiviral activity
This experiment measures fusion protein using WISH cell-VSV virus and MDBK cell-VSV two sets of experimental systems of virus
Antiviral activity.
One, using WISH cell-VSV virus system detection fusion albumen antiviral activity
1) amplification VSV virus: culture WISH cell (being purchased from ATCC) grows to confluent cultures ware to it in DMEM culture medium
60%-80%, the old culture medium of reject are diluted in the VSV virus of DMEM culture medium according to the inoculation of 1:200 ratio.It is small to cultivate 12-24
Shi Hou, WISH cell lesion reach 75% or more, and culture dish is placed in -80 DEG C of refrigerator multigelation 2-3 together with cell culture
It is secondary, discharge virion sufficiently.Pipette tips are blown and beaten repeatedly, collect culture, room temperature, 3000rpm is centrifuged 20 minutes, in collection
Clearly, it dispenses, freezes in -80 DEG C of refrigerators or liquid nitrogen.
2) VSV virus TCID is measured50: culture WISH cell in DMEM culture medium, it is to be grown it is good after, according to 3 × 105Carefully
Born of the same parents/mL density dilution, are inoculated in 96 porocyte culture plates, and every hole is inoculated with 100 μ L, cultivate 24 hours.Using containing 3% fetal calf serum
DMEM culture medium according on 10 times of gradient dilutions step gained VSV virus, with after dilution the base of Virus culture containing VSV displacement 96 holes
Old culture medium in culture plate, and be arranged and be free of virus control wells, continue to cultivate.Cytopathy situation is recorded, is recorded 5 days altogether.Root
The VSV virus TCID of amplification is calculated according to Reed-Muench Liang Shi method50。
3) measure fusion protein antiviral vitality: culture WISH cell in DMEM culture medium, it is to be grown it is good after, according to
2.5-3×105The dilution of/mL density is inoculated in 96 porocyte culture plates, and every hole is inoculated with 100 μ L, cultivates 6 hours.It will be to be detected small
Molecule IFN-α (peace section biology), PEGylated interferon PEG-IFN alpha-2a IFN-PEG (Roche Holding Ag), IFN-α/Fc-WT, IFN-α/Fc-
SC and IFN-α/Fc-MD uses the DMEM culture medium containing 7% fetal calf serum according to 10 times of gradient dilutions.With culture medium containing interferon
Old culture medium in 96 orifice plates is replaced, is cultivated 24 hours.VSV virus is diluted to containing 3% fetal calf serum DMEM culture medium
100TCID50Concentration is replaced old culture medium with the base of Virus culture containing VSV, is cultivated 24 hours.Mtt assay colour developing, microplate reader measurement are inhaled
Light value.
Two, MDBK cell-VSV virus system
Method and step are identical as WISH-VSV system, and MDBK cell is purchased from Wuhan Boster Biological Technology Co., Ltd..
Three, IFN-α/Fc fusion protein stimulation PBMC transcribes OAS1 vigour
Human PBMC is separated using Ficoll density-gradient centrifugation method.The PBMC being separated to is used containing 10% fetal calf serum
RPMI 1640 is diluted to 1.5-2 × 105/mL.Cell after 2mL dilution is added into each hole of 6 orifice plates.After culture 12 hours,
Different types of interferon is added into different holes with the final concentration of 10ng/mL.Total serum IgE, QRT-PCR are extracted in culture 20 hours
Measure target gene transcriptional level (bibliography: LaFleur D W, Nardelli B, Tsareva T, et
al.Interferon-κ,a novel type I interferon expressed in human keratinocytes
[J].Journal of Biological Chemistry,2001,276(43):39765-39771)。
Four, result
1. determining the antiviral vitality of recombination fusion protein using WISH cell-VSV virus system.First with WISH
Cell expands VSV virus, and the VSV virus TCID of amplification is calculated by Reed-Muench Liang Shi method50=107.5/0.1mL.Then
Use 100TCID50VSV virus attack poison in advance be added interferon-' alpha ' be incubated for WISH cell, using MTT colour developing judge interferon
Protective effect, and calculate the vigor of interferon.As shown in Figure 5A, three recombinant proteins and PEG-IFN alpha-2a PEG-IFN- α's is anti-
Viral viability is followed successively by IFN-α/Fc-SC > PEG-IFN- α > IFN-α/Fc-MD > IFN-α/Fc-WT.Under the system, IFN-α/
Fc-SC is equivalent to the 158% of PEG-IFN alpha-2a (PEG-IFN- α) vigor, is the 0.85% of small molecule interferon IFN-α vigor；IFN-
α/Fc-MD is equivalent to the 85.1% of PEG-IFN alpha-2a (PEG-IFN- α) vigor, is the 0.43% of small molecule interferon IFN-α vigor.
2. determining the antiviral vitality of recombination fusion protein using MDBK cell-VSV virus system.As shown in Figure 5 B,
The antiviral vitality of three recombinant proteins and PEG-IFN alpha-2a PEG-IFN- α be followed successively by IFN-α/Fc-SC > PEG-IFN- α > IFN-α/
Fc-MD > IFN-α/Fc-WT, it is more consistent with the above results.Under the system, IFN-α/Fc-SC is equivalent to PEG-IFN alpha-2a (PEG-
IFN-α) vigor 186%, be small molecule interferon IFN-α vigor 12.0%；IFN-α/Fc-MD is equivalent to PEG-IFN alpha-2a
The 66.1% of (PEG-IFN- α) vigor is the 4.3% of small molecule interferon IFN-α vigor.
3. as shown in fig. 6, transcribing OAS1 by comparing different IFN-α/Fc fusion protein and control sample stimulation PBMC
Vigor it is found that three recombination IFN-α/Fc fusion proteins and the stimulation vigor of PEG-IFN alpha-2a PEG-IFN- α are followed successively by PEG-
IFN-α > IFN-α/Fc-SC > IFN-α/Fc-MD > IFN-α/Fc-WT, substantially less than small molecule interferon IFN-α.
Embodiment 4, the detection of New-type long-acting interferon IFN-α/Fc fusion protein anti-tumor activity
One, experimental method
Daudi cell (Wuhan Boster Biological Technology Co., Ltd.) is cultivated in the RPMI 1640 for containing 10% fetal calf serum
Culture medium is diluted to 5 × 10 with culture medium after cell well-grown5/ mL is inoculated in 96 porocyte culture plates, every 50 μ of hole
L.By small molecule IFN-α to be detected (peace section biology), PEG-IFN alpha-2a, IFN-α/Fc-WT, IFN-α/Fc-SC and IFN-α/Fc-MD
With culture medium according to 10 times of gradient dilutions.Every hole adds 50 μ L gradient dilution interferon, cultivates 72 hours, mtt assay colour developing, enzyme
It marks instrument and measures light absorption value.
Two, result
As shown in fig. 7, small molecule IFN-α antiproliferative effect is most strong, three recombination fusion proteins and PEG-IFN alpha-2a PEG-
IFN-α vigor is followed successively by PEG-IFN- α > IFN-α/Fc-SC > IFN-α/Fc-WT > IFN-α/Fc-MD, wherein IFN-α/Fc-MD
It is closer to IFN-α/Fc-WT.
Embodiment 5, the detection of New-type long-acting interferon IFN-α/Fc fusion protein circulating half-life in vivo
One, experimental method
Experimental animal uses the healthy SD rat (Shanghai Slac Experimental Animal Co., Ltd.) of weight 180-220g,
It is randomly divided into 5 groups, every group 3.Using 20% urethane by Animal Anesthesia, injection dosage is 5ml/kg weight.After anesthesia, according to
30 μ g/kg dosage tail veins inject various interferon-' alpha 's (small molecule IFN-α, PEG-IFN alpha-2a PEG-IFN- α, IFN-α/Fc- respectively
WT, IFN-α/Fc-SC and IFN-α/Fc-MD).Blood is taken according to the docking of given time point, takes blood time point are as follows: 0.2,8,24,
48,72,96,120,144 hours, heparin sodium, centrifuging and taking supernatant is added after taking blood.Interferon concentration is measured using CBA in sample,
Medicine codes or data handles (bibliography: Zhang Y, Huo M, Zhou J, et according to non-compartment model using PKsolver software
al.PKSolver:An add-in program for pharmacokinetic and pharmacodynamic data
analysis in Microsoft Excel[J].Computer methods and programs in biomedicine,
2010,99(3):306-314)。
Two, result
The half-life period of the various interferon-' alpha 's of recombination fusion protein is determined using SD rat.As shown in figure 8, IFN-α/Fc-
WT, IFN-α/Fc-MD and IFN-α/Fc-SC are 38.2,68.3 and 18.6 hours in rat intracorporal half-life period point half；PEG-IFN alpha-2a
The half-life period of PEG-IFN- α is 20.2 hours；For small molecule IFN-α after injection 8 hours, plasma residence concentration has been lower than 1ng/
Ml, therefore do not count on Fig. 8.Therefore, aobvious by fusion protein IFN-α/Fc-MD rat circulating half-life in vivo of transformation
Work is longer than slightly longer than IFN-α/Fc-WT and PEG-IFN alpha-2a PEG-IFN- α, is longer than 2~5 hours that small molecule IFN-α is usually reported
Half-life period.And it is pointed out that antibody Fc fragment used in IFN-α/Fc-MD fusion protein is source of people Fc, with
The binding ability of people FcRn will be much stronger than SD rat FcRn.And according to TNFR-Fc (Enbrel) half in rat, monkey and human body
The phenomenon that phase of declining is respectively 45,77 and 102 hours speculates (ENBREL Product Monograph, Amgen, Date of
Approval:October 19,2015；Lon HK,et al.,Pharmacokinetic-Pharmacodynamic
Disease Progression Model for Effect of Etanercept in Lewis Rats with
Collagen-Induced Arthritis.Pharm Res-Dordr2011,28 (7): 1622-1630), fusion protein IFN-
α/Fc-MD and IFN-α/Fc-SC further may significantly extend in people's intracorporal half-life period.
Claims (9)
1. a kind of long-acting interferon-alpha fusion protein, connect by interferon-' alpha ' molecule with Fc sections of mutant of IgG1 antibody molecule and
At, wherein described the 297th asparagine mutation of IgG1 that sport is at glutamine；
Also, the amino acid sequence of the long-acting interferon-alpha fusion protein is one of the following terms:
1) SEQ ID NO:5；
2) SEQ ID NO:6.
2. long-acting interferon-alpha fusion protein according to claim 1, the interferon-' alpha ' is human interferon-alpha 2b.
3. encoding the gene of the described in any item long-acting interferon-alpha fusion proteins of claim 1-2.
4. gene according to claim 3, nucleic acid sequence is SEQ ID NO:7 or SEQ ID NO:8.
5. including the recombinant expression carrier of gene as claimed in claim 3.
6. the transgenic cell line comprising recombinant expression carrier described in claim 5.
7. the recombinant bacterium comprising recombinant expression carrier described in claim 5.
8. transgenic cell line according to claim 6, the cell line is Pichia pastoris system.
9. the described in any item long-acting interferon-alpha fusion proteins of claim 1-2 are used to prepare the purposes of drug, the drug
It is antiviral or antitumor for improving immunity of organisms.
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| Soluble Monomeric IgG1 Fc;Tianlei Ying等;《Journal Of Biological Chemistry》;20120601;第287卷(第23期);摘要，第19401-19404页，19407页 |
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