CN108383912A - Artificial fusion protein and application thereof - Google Patents

Artificial fusion protein and application thereof Download PDF

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CN108383912A
CN108383912A CN201810119171.4A CN201810119171A CN108383912A CN 108383912 A CN108383912 A CN 108383912A CN 201810119171 A CN201810119171 A CN 201810119171A CN 108383912 A CN108383912 A CN 108383912A
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fusion protein
hydrophilic
ifn
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artificial fusion
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高卫平
徐志坤
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Tsinghua University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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]
    • C07K14/56—IFN-alpha
    • A—HUMAN NECESSITIES
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    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
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    • A61K9/5169—Proteins, e.g. albumin, gelatin
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
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    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
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    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin

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Abstract

The present invention proposes a kind of method improving hydroaropic substance half-life period.This method includes that the hydroaropic substance is connected with self assembly polypeptide, and connection site is located at the active site non-interference area of the hydrophilic substance.Inventor has found, self assembly polypeptide is connected with the non-interference area of the active site of hydroaropic substance, nano particle can be formed under the self assembly of self assembly polypeptide mediation by being formed by artificial fusion protein, and, half-life period of the nano particle in body is compared to hydrophilic substance itself, its stability in body, pharmacokinetics and bio distribution have obtained very big improvement, and half-life period is significantly improved.

Description

Artificial fusion protein and application thereof
Technical field
The present invention relates to biomedicine field, in particular it relates to artificial fusion protein and application thereof, more specifically Ground, the present invention relates to improve the method for hydroaropic substance half-life period, artificial fusion protein, artificial nucleic acid, construct, recombinate carefully Born of the same parents, method, nano particle and the pharmaceutical composition for obtaining artificial fusion protein.
Background technology
Interferon-' alpha ' 2 (IFN-α 2) has antiviral duplication, antitumor proliferation and immunoregulation effect, has been employed successfully in Treat viral disease (such as hepatitis B, hepatitis C, condyloma acuminatum) and associated cancer (such as leukaemia, kidney, pernicious black Melanoma, multiple sclerosis etc.).But IFN is easy to be discharged by the degradation of vivo protein enzyme and kidney after systemic injection is administered, Circulating half-life is very short, needs frequent drug administration to maintain higher blood concentration, so as to cause serious toxic side effect, gives simultaneously Patient brings heavy financial burden.IFN is modified with polyethylene glycol (PEG), can effectively improve its pharmacokinetics, improves drug Distribution, improves its curative effect.However, current PEGylated interferon exists as reaction yield is low, binding site and conjugation chemistry measure The drawbacks such as uncontrollable, the serious reduction of bioactivity.Interferon cycle half can be effectively improved by merging human serum albumins Decline phase and effective controlled modification site, but activity holding only 1%, and clinical trial result is not obvious.
Therefore, how effectively to solve the problems, such as that difficult, therapeutic effect is undesirable as interferon-' alpha ' 2 is administered, be researcher Wipe critical issue to be solved.
Invention content
The application is made to the discovery of following facts and problem and understanding based on inventor：
Inventor has found, if it is hydrophilic that the polypeptide (self assembly polypeptide) with self assembly performance is connected to pharmaceutical protein etc. The active site non-interference area of polypeptide, being formed by fusion protein can form under the self assembly of self assembly polypeptide mediation Nano particle.What is more important is formed by the pharmaceutical protein of nano particle compared to the prior art, the stabilization in body Property, pharmacokinetics and bio distribution have obtained very big improvement.
For this purpose, in the first aspect of the present invention, the present invention proposes a kind of method improving hydroaropic substance half-life period.Root According to the embodiment of the present invention, the method includes the hydroaropic substance is connected with self assembly polypeptide, connection site is located at institute State the active site non-interference area of hydrophilic substance.Inventor has found, by the active site of self assembly polypeptide and hydroaropic substance Non-interference area is connected, and nano particle can be formed under the self assembly of self assembly polypeptide mediation by being formed by artificial fusion protein, Also, half-life period of the nano particle in body compared to hydrophilic substance itself, stability, drug metabolism in body Dynamics and bio distribution have obtained very big improvement, and half-life period is significantly improved.
According to an embodiment of the invention, the above method can further include at least one following additional technical feature：
According to an embodiment of the invention, the hydroaropic substance is hydrophilic medicament albumen.And then using according to the present invention The half-life period of the method for embodiment, hydrophilic medicament albumen is significantly improved, and overcomes in the prior art, hydrophilic medicament egg Circulating half-life is very short in vivo in vain, needs frequent drug administration to maintain higher blood concentration, makees so as to cause serious poison is secondary Defect.
According to an embodiment of the invention, the self assembly polypeptide is Elastin like polypeptides.Elastin like polypeptides from Assembling property is stronger, and then is further conducive to the formation of artificial fusion protein's nano particle.
According to an embodiment of the invention, the Elastin like polypeptides are amyloid peptide.
According to an embodiment of the invention, the amyloid peptide has SEQ ID NO:Amino acid sequence shown in 1.
Inventor has found there is SEQ ID NO by contrast experiment:The amyloid protein of amino acid sequence shown in 1 is more Peptide effect in terms of the formation for promoting artificial fusion protein's nano particle is more excellent.
In the second aspect of the present invention, the present invention proposes a kind of artificial fusion protein.According to an embodiment of the invention, institute Stating artificial fusion protein includes：Hydrophilic area, the hydrophilic area include hydrophilic substance；And hydrophobic region, the hydrophobic region include certainly Polypeptide is assembled, the self assembly polypeptide is connected with the active site non-interference area of the hydrophilic substance.According to embodiments of the present invention Artificial fusion protein can the self assembly of self assembly polypeptide mediation under form nano particle, be formed by nano particle not only compared with Remain Bioactivity well, and significantly improve the half-life period in vivo of hydrophilic substance in artificial fusion protein, Bio distribution and activity, to lay solid technical foundation to clinic conversion for long-acting hydrophilic substance.
According to an embodiment of the invention, above-mentioned artificial fusion protein can further include following additional technical feature extremely It is one of few：
According to an embodiment of the invention, the self assembly polypeptide is Elastin like polypeptides.Elastin like polypeptides from Assembling property is stronger, and then is further conducive to the formation of artificial fusion's polypeptide nano particle.
According to an embodiment of the invention, the Elastin like polypeptides are amyloid peptide.
Self assembly polypeptide can be amyloid peptide whole amino acid or any region, as long as selected areas has certainly Assemble function.According to a particular embodiment of the invention, the amyloid peptide has SEQ ID NO:Ammonia shown in 1 Base acid sequence comes from the N-terminal region of yeast sample amyloid proteins polypeptide.
SDSNQGNNQQNYQQYSQNGNQQQGNNRYQGYQAYNAQAQSFVPQGGYQQFQQFQPQQQQQQ(SEQ ID NO:1)。
Hydrophilic substance described herein includes but not limited to hydrophilic protein, polynucleotides and aptamer.
Wherein, hydrophilic protein includes medicine, agricultural, scientific research and the relevant albumen of other industrial circles, small peptide and antibody, Particularly including human cytokines such as interferon (interferon-' alpha ', interferon beta, interferon gamma, interferon lambda), insulin, monoclonal are anti- Body, blood factor, colony stimulating factor, growth hormone, interleukin, growth factor, therapeutic vaccine, calcitonin, neoplasm necrosis The factor (TNF) and enzyme etc..Specific example includes, but is not limited to：Asparaginase, glutamic acid enzyme, arginase, arginine Deaminase, adenosine deaminase ribalgilase, cytosine deaminase, trypsase, chymotrypsin, papain, table Skin growth factor (EGF), insulin-like growth factor (IGF), transforming growth factor (TGF), nerve growth factor (NGF), blood Growth factor derived from platelet (PDGF), bone morphogenetic protein (BMP), fibroblast growth factor, growth hormone release inhibiting hormone, growth Hormone, growth hormone, Somat, calcitonin, parathyroid hormone, colony stimulating factor (CSF), coagulation factor, Neoplasm necrosis factor, interferon, interleukins, gastrointestinal peptide, vasoactive intestinal peptide (VIP), cholecystokinin (CCK), stomach is secreted Element, secretin, hematopoietin, hormone, antidiuretic hormone, Octreotide, pancreas enzyme, superoxide dismutase, rush Thyroid hormone releasing hormone (TRH), thyrotropic hormone,；Luteinizing principle, luteinising hormone-releasing hormo (LHRH), tissue-type plasminogen activator, interleukin 1, interleukin-15, receptor antagonist (IL-1RA), pancreas Glucagon-like peptide -1 (GLP-1), leptin, auxin, G-GSF (GM-CSF), interleukin 2 (IL-2), adenosine deaminase, uricase, asparaginase, human growth hormone (HGH), asparaginase；Macrophage activation；Chorion Promoting sexual gland hormone, heparin, atrial natriuretic peptide, hemoglobin, retroviral vector, relaxin；Cyclosporin, oxytocins, epidemic disease Seedling, monoclonal antibody, single-chain antibody, ankyrin repeat protein, affine body etc..
The nucleotide includes natural or artificial synthesized, single-stranded or double-stranded polynucleotides or oligonucleotide, specifically Example includes but is not limited to：Antisense oligonucleotides, siRNA, anti-miR (target gene of the nucleotide such as Bcl-2, V2R, EphA2's, caveolin-1, TNF-α, MIF, GFPRAF-1, C-RAF, luciferase, vascular endothelial growth factor, SCV, FAS, INS2, Caspase-8 and HBsAg etc.).
The aptamer includes but is not limited to：Vascular endothelial growth factor (VEGF) aptamers, castor-oil plant Toxin aptamers, cucurbita pepo toxalbumin aptamers, thrombin aptamer, activated plasma PROTEIN C aptamers, HIV-1 reverse transcriptase Aptamers, HIV-1 integrases aptamers, protein kinase C aptamers, people's Neutrophilic elastase aptamers, L-selectin are suitable Ligand, palatelet-selectin aptamers, Yersinia ruckeri Protein-tyrosine-phosphatase aptamers, phospholipase A2 body angiogenin adaptation Body, rhinopathy virus capsid protein aptamers etc..
Wherein, the connection site of the self assembly polypeptide and the hydrophilic substance can be any separate hydrophilic substance activity The region in site or any region for not generating interference to hydrophilic substance active site.According to an embodiment of the invention, the parent Water substance is hydrophilic protein, and the self assembly polypeptide can be connected with the ends N- or C- of the hydrophilic substance.
In the third aspect of the present invention, the present invention proposes a kind of artificial fusion protein.According to an embodiment of the invention, institute Stating artificial fusion protein has SEQ ID NO:Amino acid sequence shown in 2.
CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWD ETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLS TNLQESLRSKEGSGGGGSLPETGG(SEQ ID NO:2)。
Above-mentioned artificial fusion protein according to the ... of the embodiment of the present invention is to merge egg in IFN-α 2 and self assembly polypeptide Sup35 In vain.The fusion protein can be self-assembled into protein nano particle, not only remain Bioactivity well, but also greatly change Half-life period, bio distribution and the antitumous effect of interferon in vivo have been apt to it, to be laid to clinic conversion for long-acting interferon Solid technical foundation.
In the fourth aspect of the present invention, this year invention proposes a kind of artificial nucleic acid.According to an embodiment of the invention, described Nucleic acid encode artificial fusion protein noted earlier.Artificial nucleic acid according to the ... of the embodiment of the present invention imports recipient cell, in suitable egg Under conditions of white expression, the artificial fusion protein obtained can be self-assembled into nano particle, which not only preferably protects Stayed Bioactivity, and significantly improved half-life period, bio distribution and the activity of hydrophilic substance in vivo, to for Long-acting hydrophilic substance lays solid technical foundation to clinic conversion.
According to an embodiment of the invention, above-mentioned artificial nucleic acid can further include following additional technical feature at least it One：
According to an embodiment of the invention, the nucleic acid has SEQ ID NO:Nucleotide sequence shown in 3.
TGTGATCTGCCTCAGACTCATTCTCTGGGTAGTCGTCGTACGCTGATGCTGCTGGCTCAAATGCGCCGTATTAGCCT GTTTTCTTGCCTGAAAGATCGCCACGATTTTGGGTTTCCACAGGAAGAATTTGGCAACCAGTTCCAGAAAGCCGAAA CAATTCCGGTACTGCACGAGATGATTCAACAAATCTTTAACCTGTTCAGCACCAAAGACTCTTCAGCTGCCTGGGAT GAAACACTGCTGGACAAATTCTATACCGAGCTGTATCAGCAACTGAACGATCTGGAGGCATGTGTTATTCAGGGTGT TGGTGTGACTGAAACTCCTCTGATGAAAGAGGATAGCATTCTGGCAGTCCGTAAATATTTTCAGCGTATCACACTGT ATCTGAAAGAGAAAAAATATAGCCCGTGTGCCTGGGAAGTTGTTCGTGCCGAAATCATGCGCAGCTTTAGTCTGTCT ACCAACCTGCAAGAGAGCCTGCGTTCTAAAGAAGGATCCGGCTCTTCGGATTCAAACCAAGGCAACAATCAGCAAAA CTACCAGCAATACAGCCAGAACGGTAACCAACAACAAGGTAACAACAGATACCAAGGTTATCAAGCTTACAATGCTC AAGCTCAATCTTTTGTTCCACAAGGTGGCTACCAACAGTTCCAACAGTTCCAGCCTCAACAGCAGCAACAGCAAGGA TCCGGTGGCGGTGGCTCTCTGCCGGAAACCGGTGGCCACCATCATCATCATCAT(SEQ ID NO:3)。
In the fifth aspect of the present invention, the present invention proposes a kind of construct.According to an embodiment of the invention, the structure Body carries foregoing artificial nucleic acid.Construct according to the ... of the embodiment of the present invention imports recipient cell, in suitable protein expression Under conditions of, the artificial fusion protein obtained can be self-assembled into nano particle, which not only preferably remains body Outer bioactivity, and half-life period, bio distribution and the activity of hydrophilic substance in vivo are significantly improved, to be long-acting parent Water substance lays solid technical foundation to clinic conversion.
According to an embodiment of the invention, above-mentioned construct can further include following additional technical feature at least it One：
According to an embodiment of the invention, the carrier of the construct is pET-25b (+).
In the sixth aspect of the present invention, the present invention proposes a kind of recombinant cell.According to an embodiment of the invention, described heavy Group cell carries foregoing artificial nucleic acid or foregoing construct.Under conditions of being suitble to protein expression, according to this The recombinant cell of inventive embodiments can express foregoing artificial fusion protein, which can be self-assembled into nanometer Grain, the nano particle not only preferably remain Bioactivity, but also significantly improve half of hydrophilic substance in vivo Decline phase, bio distribution and activity, to lay solid technical foundation to clinic conversion for long-acting hydrophilic substance.
In the seventh aspect of the present invention, the present invention proposes a kind of method obtaining artificial fusion protein noted earlier.Root According to the embodiment of the present invention, the method includes under conditions of being expressed suitable for the artificial fusion protein, culture is noted earlier Recombinant cell, to obtain the artificial fusion protein.Step is simple according to the method for the embodiment of the present invention, research and development reaction item Part is mild, bioactivity retains height, and the artificial fusion protein obtained is self-assembled into nano particle, and the nano particle is not only preferable Ground remains Bioactivity, and significantly improves the half-life period in vivo of hydrophilic substance in fusion protein, biology point Cloth and activity, to lay solid technical foundation to clinic conversion for long-acting hydrophilic substance.
In the eighth aspect of the present invention, the present invention proposes a kind of nano particle.According to an embodiment of the invention, described to receive Rice grain is self-assembly of by several foregoing artificial fusion proteins, including：Hydrophobic core, the hydrophobic core is by described The hydrophobic region of artificial fusion protein is constituted；And the outer surface of the hydrophobic core is arranged in hydrophilic outer shell, the hydrophilic outer shell, by The hydrophilic area of the artificial fusion protein is constituted.Nano particle according to the ... of the embodiment of the present invention preferably remains external biological work Property, and half-life period, bio distribution and the activity of hydrophilic substance in vivo in fusion protein are significantly improved, to be long-acting Hydrophilic substance lays solid technical foundation to clinic conversion.
According to an embodiment of the invention, above-mentioned nano particle can further include following additional technical feature at least it One：
According to an embodiment of the invention, the artificial fusion protein has SEQ ID NO:Amino acid sequence shown in 2.
By there is SEQ ID NO:The structure snd size for the self assembly protein nano particle that amino acid sequence shown in 2 mediates are not It is particularly limited, such as can be spherical shape, rodlike, the patterns such as threadiness.
According to a particular embodiment of the invention, the nano particle is spherical in shape, and a diameter of 48nm of the nano particle~ The thickness of 55nm, a diameter of 20nm of the hydrophobic core, the hydrophilic outer shell are 14~17.5nm.According to embodiments of the present invention Above-mentioned nano particle be 2 protein nano particle of IFN-α, there is good Bioactivity, interferon partly declining in vivo Phase, bio distribution and antitumous effect have obtained greatly improving.
In the ninth aspect of the present invention, the present invention proposes a kind of pharmaceutical composition.According to an embodiment of the invention, described Pharmaceutical composition is thin comprising artificial fusion protein noted earlier, foregoing nucleic acid, foregoing construct, front recombination Born of the same parents or nano particle noted earlier.Pharmaceutical composition microbic activity according to the ... of the embodiment of the present invention is high, target is strong, partly declines in body Phase length, pharmacokinetics have significant advantage.
Description of the drawings
Fig. 1 is interferon according to the ... of the embodiment of the present invention-self assembly polypeptide Sup35 fusion protein nano particle schematic diagrames；
Fig. 2 is the result according to the ... of the embodiment of the present invention for being purified by affinity chromatography and obtaining IFN α and IFN α-Sup35 Figure；
Fig. 3 is the result figure of the molecular weight of Q-TOF analysis IFN α-Sup35 and IFN α according to the ... of the embodiment of the present invention；
Fig. 4 is the result figure of the hydration radius of DLS analysis IFN α-Sup3NP and IFN α according to the ... of the embodiment of the present invention；
Fig. 5 is that DLS according to the ... of the embodiment of the present invention analyzes IFN α-Sup35 fusion protein nano particles in different solutions The result figure of performance；
Fig. 6 is the result figure of the secondary structure of IFN α-Sup35NP and IFN α according to the ... of the embodiment of the present invention；
Fig. 7 is the result figure of IFN α-Sup35NP maximum critical micelle concentrations according to the ... of the embodiment of the present invention；
Fig. 8 is the result figure of TEM observations IFN α-Sup35NP shape characteristics according to the ... of the embodiment of the present invention；
Fig. 9 is the knot for the Bioactivity that mtt assay according to the ... of the embodiment of the present invention measures IFN α-Sup35NP and IFN α Fruit is schemed；
Figure 10 is the external biological safety that mtt assay according to the ... of the embodiment of the present invention measures IFN α-Sup35NP and IFN α Result figure；
Figure 11 be the blood concentration of IFN α-Sup35NP and IFN α according to the ... of the embodiment of the present invention in nude mouse at any time Variation result figure；
Figure 12 is the distribution feelings of IFN α-Sup35NP and IFN α according to the ... of the embodiment of the present invention in tumour and its hetero-organization The result figure of condition；
Figure 13 is the result figure that IFN α-Sup35NP and IFN α according to the ... of the embodiment of the present invention inhibit tumour growth situation；
Figure 14 is the result figure of the survivorship curve in mouse therapeutic process according to the ... of the embodiment of the present invention；
Figure 15 be after mouse according to the ... of the embodiment of the present invention during tumour growth pictorial diagram；
Figure 16 is tumor locus histopathologic slide after treatment according to the ... of the embodiment of the present invention；
Figure 17 be the heart after treatment according to the ... of the embodiment of the present invention, liver and kidney histopathologic slide；
Figure 18 is that nude mice weight changes with time the result figure of situation after injection drug according to the ... of the embodiment of the present invention；
Figure 19 is heart, Liver and kidney function physiological indexes situation after mouse injection drug according to the ... of the embodiment of the present invention Result figure；
Figure 20 is the result figure of blood index situation after mouse injection drug according to the ... of the embodiment of the present invention；
Figure 21 is the plasmid construct figure according to the ... of the embodiment of the present invention for carrying IFN genes；And
Figure 22 is the plasmid construct figure according to the ... of the embodiment of the present invention for carrying IFN genes and Sup35 genes.
Specific implementation mode
The embodiment of the present invention is described below in detail, examples of the embodiments are shown in the accompanying drawings.Below with reference to The embodiment of attached drawing description is exemplary, it is intended to for explaining the present invention, and is not considered as limiting the invention.
According to an embodiment of the invention, protein-Elastin like polypeptides fusion protein is prepared the present invention provides a kind of Method, wherein the Elastin like polypeptides by being connected on the protein, the protein can be interferon, institute It states interferon and can be selected from interferon-' alpha ', interferon beta, interferon gamma, interferon lambda.The protein be selected from medicine, agricultural, scientific research with And the relevant albumen of other industrial circles, small peptide and antibody, such as granulocyte colony stimulating factor, leptin, glucagon Peptide -1 and the like or hirudin.
In embodiment, the subject fusion proteins IFN α-Sup35 of purifying is obtained by nickel (Ni) aggressive pathogenic.Profit With level four bars/ionization time of flight mass spectrometry (Q-TOF), microplate reader dynamic light scattering (DLS), circular dichroism spectra (CD), transmission electron microscope (TEM) molecular weight of analysis means characterization IFN α-Sup35NP and IFN α, phase transition temperature, the hydration objects such as radius and secondary structure such as Physicochemical performance.Rational cell line is selected, the Bioactivity of IFN α-Sup35NP and IFN α is tested, i.e., it is external anti- The ability of tumor cell proliferation, while testing its external biological safety；Using nude mice model, test IFN α-Sup35NP and The pharmacokinetics of IFN α in vivo；And nude mouse tumor model is established, test the antitumor of IFN α-Sup35NP and IFN α Effect and drug distribution.
According to an embodiment of the invention, detection means according to the present invention is as follows：
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) carries out preliminary analysis to sample.
Use dynamic light scattering (DLS) determination sample grain size.DLS tests use Malvern Zetasizer Nano-zs90.Data processing uses software Zetasizer software 6.32.
Q-TOF is selected to measure molecular weight.
Secondary structure is analyzed using circular dichroism spectrometer.
Using transmission electron microscope observing sample topography.
Antiproliferation and external biological safety are measured using MTT methods.
Using female athymic nude mice test drug metabolism dynamics, analyzed using 3.0 pharmacokinetics of DAS soft Part calculates pharmacokinetic parameter.
Establish antitumor activity and bio distribution in nude mice model test interferon-macromolecule combination body.
Tumor locus degree of necrosis and Main Tissues organ safety are led to using histopathologic slide's method analysis sample.
Using biochemical analysis and blood analysis method come verification sample biological safety.
Wherein,
IFN α gene order (NCBI GI 386795) is synthesized and is inserted by raw work biotechnology (Shanghai, China)In carrier T.Using round pcr, fromIFN α coded sequence is expanded in carrier T, passes through Eco RI/Bam HI restriction enzyme sites are inserted into pET-25b (+) carrier.
IFN gene orders such as SEQ ID NO:Shown in 4.
ATGTGTGATCTGCCTCAGACTCATTCTCTGGGTAGTCGTCGTACGCTGATGCTGCTGGCTCAAATGCGCCGTATTAG CCTGTTTTCTTGCCTGAAAGATCGCCACGATTTTGGGTTTCCACAGGAAGAATTTGGCAACCAGTTCCAGAAAGCCG AAACAATTCCGGTACTGCACGAGATGATTCAACAAATCTTTAACCTGTTCAGCACCAAAGACTCTTCAGCTGCCTGG GATGAAACACTGCTGGACAAATTCTATACCGAGCTGTATCAGCAACTGAACGATCTGGAGGCATGTGTTATTCAGGG TGTTGGTGTGACTGAAACTCCTCTGATGAAAGAGGATAGCATTCTGGCAGTCCGTAAATATTTTCAGCGTATCACAC TGTATCTGAAAGAGAAAAAATATAGCCCGTGTGCCTGGGAAGTTGTTCGTGCCGAAATCATGCGCAGCTTTAGTCTG TCTACCAACCTGCAAGAGAGCCTGCGTTCTAAAGAA(SEQ ID NO:4)。
Primer is as follows：
Sense primer：5’CGGAATTCATGTGTGATCTGCCTCAGAC3’(SEQ ID NO:5).
Downstream primer：5’CGGGATCCTTCTTTAGAACGCAGGCTCT3’(SEQ ID NO:6).
Plasmid construct such as Figure 21.
Sup35 gene orders are synthesized and are inserted by giving birth to work biotechnology (Shanghai, China)In carrier T.It utilizes Round pcr, fromSup35 sequences are expanded in carrier T, and pET-25b (+) carrier is inserted by Bam HI restriction enzyme sites In (above-mentioned structure containing IFN α have gene).
Sup35 gene orders such as SEQ ID NO:Shown in 7.
TCGGATTCAAACCAAGGCAACAATCAGCAAAACTACCAGcaaTACagcCAGAACGGTAACCAACAACAAGGTAACAA CAGATACCAAGGTTATCAAGCTTACAATGCTCAAGCTCAATCTTTTGTTCCACAAGGTGGCTACCAACAGTTCCAAC AGTTCCAGCCTCAACAGCAGCAACAGCAA(SEQ ID NO:7)。
Primer is as follows：
Sense primer：5’CGCGGATCCTCGGATTCAAACCAAGGCA3’(SEQ ID NO:8).
Sense primer：5’CCAGAACCGGATCCTTGCTGTTGCTGCT 3’(SEQ ID NO:9).
Plasmid construct is as shown in figure 22.
After plasmid construction success, it is transferred in Escherichia coli Rosetta-gami (DE3) pLysS expression bacterial strains, is expressed Detect the monoclonal bacterial strain that simultaneously picking includes pET-IFN α-Sup35 plasmids.The inoculation is being contained into ampicillin later 20ml LB culture mediums in, 37 DEG C, 250rpm is incubated overnight.Expand the TB that culture contains ampicillin to 1L later In (Terrific broth) culture medium, 37 DEG C, 250rpm cultivate to OD590=0.5, the different of final concentration of 0.5mM is added Propyl-β-D- thiogalactosides (Isopropyl- β-d-thiogalactoside, IPTG), 25 DEG C, 250rpm cultivated Night.
3000 × g centrifugations 15min collects the E. coli broth being incubated overnight, and is then resuspended to buffer solution A (10mM PBS, 5mM imidazoles, pH 7.4).Destroy bacteria cell wall with the method for ultrasonication at low temperature, later at 4 DEG C with 14000 × g centrifuges 20min and detaches broken rear product with supernatant.Then supernatant is collected, 2mL polyethyleneimines are added (Polyethyleneimine, PEI) (1%w/v) carrys out the substances such as precipitate nucleic acids, and 14000 × g centrifuges 15min to remove core later Acid precipitation, collects supernatant.
IFN α-Sup35 the recombinant proteins with His labels are purified with the method for Ni affinity chromatographys, are comprised the concrete steps that：It will After 0.22 μm of membrane filtration of supernatant, it is loaded in nickel affinity column, is then purified with 10 systems of AKTAPurifier, it Gradient washes are carried out with 0~100% buffer solution B (10mM PBS, 500mM imidazoles, pH 7.4) afterwards, collect each eluting peak, so It is analyzed afterwards with polyacrylamide gel electrophoresis (SDS-PAGE).After obtaining target protein, with 26/10 desalting columns of HiPrep Remove imidazoles, for juxtaposition by buffer exchange at 10mM PBS, pH 7.4 in solution, is stored in -80 DEG C.
SDS-PAGE analyzes sample and is prepared by the Laemmli sample buffers containing 5% beta -mercaptoethanol, a concentration of 1mg/ ML, at 95 DEG C after heating 5min, 10 μ L samples are loaded into prefabricated 10%SDS-PAGE gels, vertical electrophoresis 80~100V electricity (electrophoresis liquid is pressure operation 90min：25mM Tris, 250mM Glycine, 0.1%SDS).Gel Coomassie brilliant blue G-250 Pillar location is observed after dyeing processing.Fig. 2 shows the SDS-PAGE detections of IFN α-Sup35 and IFN α after purification.
The Physico-Chemical Characterization of 1 IFN α-Sup35 of embodiment
IFN α and IFN α-Sup35 molecular weight are measured with level four bars/ionization time of flight mass spectrometry (Q-TOF), first uses sample NanoACQUITY highly effective liquid phase chromatographic systems detach, and then use SYNAPT-G2-Si mass spectrometer separation samples, wherein mobile phase again A is made of 0.1% formic acid water, and Mobile phase B is made of 100% acetonitrile and 0.1% formic acid.Sample is put into autosampler later Electron spray ionisation is carried out, is analyzed in Q-TOF (SYNAPT G2-Si, Waters company) mass spectrometer.Fig. 3 is aobvious Q-TOF analysis results are shown.
On Malvern Zetasizer Nano-zs90 IFN α and IFN α-are measured with dynamic light scattering (DLS) method The hydration radius of Sup35.Sample is diluted in PBS buffer solution, through 0.22 μm of aperture membrane filtration processing before test.It is surveyed through DLS The hydrated diameter of examination, IFN α is 7nm, and the hydrated diameter of the IFN α-Sup35 synthesized is 55nm.By sample in 10%Tween and It is diluted in 10%SDS, to study the driving force of albumen self assembly.The results show that 10%Tween and 10%SDS can be by nanometer Grain de-assembly, particle size is reduced to 5.6 and 4.8nm by 55nm, in the same size with IFN α.Prove nano particle mainly by close and distant Water effect maintains its pattern.Fig. 4 shows the hydrated diameter of DLS analysis IFN α and IFN α-Sup35NP.Fig. 5 shows DLS points Analysis IFN α-Sup35 fusion protein nano particles show in different solutions.
The secondary structure of IFN α-Sup35NP is measured with circular dichroism spectrum analysis.Samples with water solution is diluted to 0.2mg/ ML carries out ultraviolet sweep with Pistar π -180 (Applied Photophysics Co., Ltds) in 200-250nm wave-length coverages Retouch analysis.Fig. 6 shows the secondary structure of circular dichroism chromatography IFN α and IFN α-Sup35, in 200-260nm wavelength Circular dichroism spectra in range all shows same 208/222nm bimodal curves, be typical αhelix, and with IFN curves Overlapping is good, shows that Sup35 has not significant impact with the secondary structure after IFN α amalgamation and expression.
IFN α-Sup35NP maximum micelle critical concentrations are measured with Nile red colouring method.Nile red dye is dissolved in PBS In, make its final concentration of 1.25 μM, then by sample and Nile red μM 1 from 8.3 μM to 0.03:1 doubling dilution.Later with sharp Shine 550nm, and light 630nm is in SpectraMax M3Microplate Reader (Molecular Devices) enzyme mark for transmitting Fluorescence intensity is measured on instrument.Fig. 7 shows IFN α-Sup35NP maximum micelle critical concentrations.
Protein nano granule-morphology is observed with transmission electron microscope (TEM).Albumen is diluted to 0.5mg/ml, is dripped to On the copper mesh of carbon covering, 5min is stood, draw solution, is dyed, room temperature is dried in the air with 2% (w/v) Salkowski's solution later It is dry, later with Hitachi H-7650B transmission electron microscopes come observing samples.TEM shows that IFN α-Sup35 nano particles are straight Diameter is 48nm, wherein hydrophilic outer shell about 12nm, hydrophobic inner core 20nm.Fig. 8 shows tem analysis result.
The Bioactivity of 2 IFN α-Sup35 of embodiment is surveyed and biological safety
The antiproliferation of IFN α-Sup35 and biological safety are measured using MTT methods in the present invention.We select IFN α-Sup35NP antiproliferations are tested with people's Burkitt ' s B lymphoma cells (Daudi B), because this is thin Born of the same parents have higher sensitivity to IFN-α 2.Select human mammary epithelial cell (L929) and l cell (MCF-10) Daudi B cells are in test IFN α-Sup35NP biological safeties.By cell containing 10%FBS, 50U/mL penicillin and In the RMPI-1640 of 50 μ g/mL streptomysins after culture a period of time (L929 and MCS-10 are cultivated in DMEM culture mediums), 96 Certain density cell suspending liquid (50 holes μ L/, 104 cells) is inoculated in orifice plate, by IFN α and IFN α-Sup35NP samples system Row dilution, each 50 μ L are added in 96 well culture plates, if negative control (being free of IFN-α 2) and blank control (containing only culture solution), 37 DEG C, 5%CO2 cultivates 72~96h, adds 20 holes μ L/ of MTT lysates (Promega), and each hole 490nm waves are measured with microplate reader after 3h Long absorption value, the degree of cell proliferation after more different sample treatments.Fig. 9 and table 1 show that MTT measures IFN α-Sup35NP Bioactivity, the IC50 of IFN α-Sup35NP and IFN α is respectively 289.1pg/mL and 49.28pg/mL, and activity is kept 17%, significantly larger than the active conservation rate of the IFN of human albumin modification (<1%).Figure 10 shows that MTT measures IFN α- Sup35NP external biological safety results, compared with the control group, IFN α-Sup35NP group cell growths are not affected.It should The result shows that IFN α-Sup35 is self-assembled into for there is no the serious activity for reducing IFN α after protein nano particle, and with good Biological safety, for internal antitumor activity test provide foundation.
Table 1：
Sample IC50(pg/mL) Relative activity (%)
IFNα 49.28 100
IFN-Sup35NP 289.1 17
The pharmacokinetics of 3 IFN α-Sup35NP of embodiment is tested
All zooperies are completed in the case where Tsinghua University is about every regulation guidance of zoopery below.In the present invention Using BABL/C athymic females nude mice model after tail vein injection IFN α or IFN α-Sup35NP, determines in blood and interfere The case where plain concentration changes with time, and carry out data analysis using DAS softwares.Before drug-treated, 68 week old weight are After male BABL/C observation a period of times of 20g or so, it is randomly divided into 2 groups.With 50 μ g/20g dosage tail vein injection IFN αs and Then IFN α-Sup35NP takes the mode of blood to acquire 10 μ L blood, 30min is stored at room temperature, 4 at the time point of setting with docking DEG C, upper serum is collected by centrifugation under 3000 × g, be stored in 80 DEG C of low temperature refrigerators of ﹣.With humanIFN-α's 2ELISA kits (PBL Interferon source) according to 2 content of IFN-α in specification measurement serum.Utilize 3.0 pharmacokinetics of DAS Analysis software calculates pharmacokinetic parameter.Using in DAS softwares chamber eliminate model analysis IFN-Sup35NP and The pharmacokinetic parameter of IFN α, IFN α are only 0.8h, after a few minutes are administered, the concentration of interferon in blood, that is, rapid Drop to the 50% of predose hereinafter, after for 24 hours residual concentration deficiency initial concentration 0.1%.And IFN-Sup35NP is in vivo Concentration gradually decrease, half-life period 17.9h is 21.5 times of IFN.To administration 48h after, still have 20% or more it is residual It stays.The internal area under the drug-time curve (AUC0- ∞) of IFN α-Sup35NP is 51.2 times of IFN α.The above result shows that with not The IFN α of modification is compared, and the half-life period of IFN α-Sup35NP and internal average residence time are obviously prolonged, and Drug-time curve area is aobvious It writes and increases, clearance rate significantly reduces.Figure 11 shows pharmacokinetics of the IFN α-Sup35NP in BABL/C nude mouses. Table 2 shows the pharmacokinetic data analysis of IFN α-Sup35NP.
Table 2：
Parameter IFNα IFN-Sup35NP
T1/2(h) 0.83±0.25 17.90±1.87
AUC(106pg/mL·h) 0.32±0.05 16.57±1.34
The distribution situations of 4 IFN-Sup35NP of embodiment in the tissue
It is determined using the nude mice for having transplanted ovarian cancer cell in the present invention and remaining interferon in respectively tissue is administered after 5h Concentration.6 BABL/C female athymic nude mices are divided into 2 groups, IFN α group and IFN α-Sup35NP groups, Proliferation of Human Ovarian Cell (OVCAR-3) containing 10%FBS, one is cultivated in the RMPI-1640 culture mediums of 50U/mL penicillin and 50 μ g/mL streptomysins After the section time, is removed with trypsin digestion, washed through PBS, be resuspended in the RMPI-1640 culture mediums without above-mentioned additive simultaneously With in the isometric mixtures of BD Matrigel Matrix, 0.2mL single cell suspensions (5 × 106A cell) it is inoculated in a nude mice left side Dorsal sc at hind leg femur, culture form 200mm after 30 days3The solid tumor lump of size.IFN α-Sup35 and IFN α are with tail Intravenous injection is squeezed into nude mouse, and dosage is 50 μ g/20g weight.Nude mice is put to death after administration 5h, collects major organs such as The heart, kidney, liver, spleen, lung, pancreas, muscle and tumour.Tissue Extraction buffer (PBS EDTA containing 1mM, 0.5%Triton X-100,0.5% NaTDC, 1mM PMSF, by 1:The protease inhibitor cocktail and phosphatase of 100 dilution proportions press down Preparation mixture (Sigma-Aldrich)) it is broken after, centrifuging and taking supernatant extracting solution.The concentration side ELISA of IFN α in tissue Standard measure measures.
Figure 12 shows that IFN α-Sup35 accumulates situation in each tissue.After injected sample 5h, IFN α-Sup35NP exists It can effectively be accumulated in each tissue.Especially in tumour, after sample injection 5h in IFN α-Sup35NP groups interferon concentration It is 11.3 times of distributed density in IFN α group tumour, this result, which fully demonstrates IFN α-Sup35NP protein nanos particle, to be had Effect enables albumen more to exist while circulating half-life extends in blood using enhancing penetrating and being detained (EPR) effect Assembled in tumour, to improve interferon bioavilability in vivo and antitumor efficacy.
5 nude mice model of embodiment tests antitumor activity in IFN α-Sup35NP bodies
It is lived in nude mice by subcutaneous tumor model to evaluate the vivo biodistribution of IFN α-Sup35NP with ovarian cancer cell in the present invention Property.As stated above, OVCAR-3 cell inoculations dorsal sc at nude mice left hind femur, culture form entity after~30 days Struma block (~30mm3), to establish nude mouse tumor model.30 nude mices are divided into 3 groups, IFN α-Sup35NP groups, IFN α group With PBS groups.It is squeezed into nude mouse with tail vein injection, dosage is 50 μ g/20g weight, once every four days, until control group Mouse is all dead.Observation nude mice survival condition and tumour growth situation when being administered every time, dynamic measure nude mice weight and swell Knurl product changes with time.When mice tumors grew is more than 500mm3Or weight loss is more than 15%, mouse, that is, euthanasia. Tumour, heart, liver and kidney are collected after treatment end, it is fixed, after embedding, H＆E dyeing, Nikon are carried out according to standard method Eclipse 90 is imaged.Blood is taken by eyeball simultaneously, blood is obtained and serum send to clinical laboratory of Hospital of Tsinghua University and measures breast Acidohydrogenase, creatine kinase isozyme, glutamic-pyruvic transaminase, glutamic-oxalacetic transaminease, creatinine, urea nitrogen, red blood cell, leucocyte, blood are small The basic physiologicals index such as plate, hemoglobin.
After starting administration, the tumour of PBS group nude mices increases rapidly, until at 24 days, all mouse tumor volumes are more than 500mm3More than, median survival is only 20 days.IFN α group mouse tumor gradually increases, until 28 days all mouse are condemned to death, it is raw It is 26 days to deposit intermediate value, does not show apparent antitumor activity.On the contrary, the tumour of IFN α-Sup35NP group nude mices is not obviously grown Greatly, until mouse tumor does not have significant change, median survival to extend to 62 days after terminating administration.This explanation, continuous injection modification Interferon can not effectively inhibit tumour growth.On the contrary, IFN α-Sup35 fusion protein the energy for passing through genetic engineering amalgamation and expression The growth of tumour is enough restrained effectively, there is extraordinary internal antitumor activity.Figure 13 shows that IFN α-Sup35 inhibits Tumour growth situation, Figure 14 show that the survivorship curve of mouse after injection drug, Figure 15 are mice tumors grew pictorial diagram.Figure 16 Show tumor locus pathological section after treating.
Nude mice weight within the test period slightly reduces, but no significant difference compared with the control group, shows IFN α-Sup35 There is no apparent side effect.The physical signs that mouse is monitored after administration in 28th day, without apparent area compared with not injecting drug Not, the Main Tissues slices of organs such as the heart, liver, kidney shows no significant change.Show that IFN α-Sup35NP will not make intracorporeal organ At overt toxicity, provide the foundation for Clinical practice for future.Nude mice weight is at any time after Figure 17 shows injection drug Situation of change.Figure 18 shows Main Tissues organ pathology slice map after treatment.Figure 19 shows heart after mouse injection drug (lactic dehydrogenase, creatine kinase isozyme), liver (glutamic-pyruvic transaminase, glutamic-oxalacetic transaminease), kidney (creatinine, urea nitrogen) function physiology Index situation of change.Figure 20 shows physiochemical indice (red blood cell, leucocyte, blood platelet, hemoglobin) after mouse injection drug Situation of change.
Interferon-Sup35 protein nano the particles prepared according to the method for the embodiment of the present invention have higher biology living Property storage rate, the interferon of Albumin fusion in notable extended half-life period and effective tumor inhibition effect, with document report In vitro and in vivo activity compared to showing more excellent effect.Therefore, the fusion protein self-assembling technique that Sup35 is mediated is expected to take Become for albumin fusion technology and improves medicine stability, improves pharmacokinetics and enhance the new method of therapeutic efficiency.
In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specifically show The description of example " or " some examples " etc. means specific features, structure, material or spy described in conjunction with this embodiment or example Point is included at least one embodiment or example of the invention.In the present specification, schematic expression of the above terms are not It must be directed to identical embodiment or example.Moreover, particular features, structures, materials, or characteristics described can be in office It can be combined in any suitable manner in one or more embodiments or example.In addition, without conflicting with each other, the skill of this field Art personnel can tie the feature of different embodiments or examples described in this specification and different embodiments or examples It closes and combines.
Although the embodiments of the present invention has been shown and described above, it is to be understood that above-described embodiment is example Property, it is not considered as limiting the invention, those skilled in the art within the scope of the invention can be to above-mentioned Embodiment is changed, changes, replacing and modification.
SEQUENCE LISTING
<110>Tsinghua University
<120>Artificial fusion protein and application thereof
<130> PIDC3180317
<160> 9
<170> PatentIn version 3.3
<210> 1
<211> 61
<212> PRT
<213> Artificial
<220>
<223>The amino acid sequence of amyloid peptide
<400> 1
Ser Asp Ser Asn Gln Gly Asn Asn Gln Gln Asn Tyr Gln Gln Tyr Ser
1 5 10 15
Gln Asn Gly Asn Gln Gln Gln Gly Asn Asn Arg Tyr Gln Gly Tyr Gln
20 25 30
Ala Tyr Asn Ala Gln Ala Gln Ser Phe Val Pro Gln Gly Gly Tyr Gln
35 40 45
Gln Phe Gln Gln Phe Gln Pro Gln Gln Gln Gln Gln Gln
50 55 60
<210> 2
<211> 178
<212> PRT
<213> Artificial
<220>
<223>The amino acid sequence of artificial fusion protein
<400> 2
Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met
1 5 10 15
Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp
20 25 30
Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln
35 40 45
Lys Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe
50 55 60
Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu
65 70 75 80
Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu
85 90 95
Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu Met Lys
100 105 110
Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu
115 120 125
Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg
130 135 140
Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser
145 150 155 160
Leu Arg Ser Lys Glu Gly Ser Gly Gly Gly Gly Ser Leu Pro Glu Thr
165 170 175
Gly Gly
<210> 3
<211> 747
<212> DNA
<213> Artificial
<220>
<223>Encode the nucleotide sequence of the nucleic acid of artificial fusion protein
<400> 3
tgtgatctgc ctcagactca ttctctgggt agtcgtcgta cgctgatgct gctggctcaa 60
atgcgccgta ttagcctgtt ttcttgcctg aaagatcgcc acgattttgg gtttccacag 120
gaagaatttg gcaaccagtt ccagaaagcc gaaacaattc cggtactgca cgagatgatt 180
caacaaatct ttaacctgtt cagcaccaaa gactcttcag ctgcctggga tgaaacactg 240
ctggacaaat tctataccga gctgtatcag caactgaacg atctggaggc atgtgttatt 300
cagggtgttg gtgtgactga aactcctctg atgaaagagg atagcattct ggcagtccgt 360
aaatattttc agcgtatcac actgtatctg aaagagaaaa aatatagccc gtgtgcctgg 420
gaagttgttc gtgccgaaat catgcgcagc tttagtctgt ctaccaacct gcaagagagc 480
ctgcgttcta aagaaggatc cggctcttcg gattcaaacc aaggcaacaa tcagcaaaac 540
taccagcaat acagccagaa cggtaaccaa caacaaggta acaacagata ccaaggttat 600
caagcttaca atgctcaagc tcaatctttt gttccacaag gtggctacca acagttccaa 660
cagttccagc ctcaacagca gcaacagcaa ggatccggtg gcggtggctc tctgccggaa 720
accggtggcc accatcatca tcatcat 747
<210> 4
<211> 498
<212> DNA
<213> Artificial
<220>
<223>IFN gene orders
<400> 4
atgtgtgatc tgcctcagac tcattctctg ggtagtcgtc gtacgctgat gctgctggct 60
caaatgcgcc gtattagcct gttttcttgc ctgaaagatc gccacgattt tgggtttcca 120
caggaagaat ttggcaacca gttccagaaa gccgaaacaa ttccggtact gcacgagatg 180
attcaacaaa tctttaacct gttcagcacc aaagactctt cagctgcctg ggatgaaaca 240
ctgctggaca aattctatac cgagctgtat cagcaactga acgatctgga ggcatgtgtt 300
attcagggtg ttggtgtgac tgaaactcct ctgatgaaag aggatagcat tctggcagtc 360
cgtaaatatt ttcagcgtat cacactgtat ctgaaagaga aaaaatatag cccgtgtgcc 420
tgggaagttg ttcgtgccga aatcatgcgc agctttagtc tgtctaccaa cctgcaagag 480
agcctgcgtt ctaaagaa 498
<210> 5
<211> 28
<212> DNA
<213> Artificial
<220>
<223>Upstream primer sequence for expanding IFN
<400> 5
cggaattcat gtgtgatctg cctcagac 28
<210> 6
<211> 28
<212> DNA
<213> Artificial
<220>
<223>Downstream primer sequence for expanding IFN
<400> 6
cgggatcctt ctttagaacg caggctct 28
<210> 7
<211> 183
<212> DNA
<213> Artificial
<220>
<223>Sup35 gene orders
<400> 7
tcggattcaa accaaggcaa caatcagcaa aactaccagc aatacagcca gaacggtaac 60
caacaacaag gtaacaacag ataccaaggt tatcaagctt acaatgctca agctcaatct 120
tttgttccac aaggtggcta ccaacagttc caacagttcc agcctcaaca gcagcaacag 180
caa 183
<210> 8
<211> 28
<212> DNA
<213> Artificial
<220>
<223>Upstream primer sequence for expanding Sup35
<400> 8
cgcggatcct cggattcaaa ccaaggca 28
<210> 9
<211> 28
<212> DNA
<213> Artificial
<220>
<223>Downstream primer sequence for expanding Sup35
<400> 9
ccagaaccgg atccttgctg ttgctgct 28

Claims (17)

1. a kind of method improving hydroaropic substance half-life period, which is characterized in that by the hydroaropic substance and self assembly polypeptide It is connected, connection site is located at the active site non-interference area of the hydrophilic substance.
2. according to the method described in claim 1, it is characterized in that, the hydroaropic substance is hydrophilic medicament albumen；
Optionally, the self assembly polypeptide is Elastin like polypeptides；
Optionally, the Elastin like polypeptides are amyloid peptide；
Optionally, the amyloid peptide has SEQ ID NO:Amino acid sequence shown in 1.
3. a kind of artificial fusion protein, which is characterized in that including：
Hydrophilic area, the hydrophilic area include hydrophilic substance；And
Hydrophobic region, the hydrophobic region include self assembly polypeptide, and the active site of the self assembly polypeptide and the hydrophilic substance is non- Interference range is connected.
4. artificial fusion protein according to claim 3, which is characterized in that the self assembly polypeptide is that elastin-like is more Peptide,
Optionally, the Elastin like polypeptides are amyloid peptide,
Optionally, the amyloid peptide has SEQ ID NO:Amino acid sequence shown in 1.
5. artificial fusion protein according to claim 3, which is characterized in that the hydrophilic substance includes being selected from hydrophilic egg In vain, at least one of polynucleotides and aptamer.
6. artificial fusion protein according to claim 3, which is characterized in that the hydrophilic substance is hydrophilic protein, described Self assembly polypeptide is connected with the ends N- or C- of the hydrophilic substance.
7. artificial fusion protein according to claim 3, which is characterized in that the hydrophilic protein packet is selected from interferon, pancreas Island element, monoclonal antibody, blood factor, colony stimulating factor, growth hormone, interleukin, growth factor, therapeutic vaccine, drop At least one of calcium element, tumor necrosis factor and enzyme.
8. a kind of artificial fusion protein, which is characterized in that have SEQ ID NO:Amino acid sequence shown in 2.
9. a kind of artificial nucleic acid, which is characterized in that any one of described nucleic acid encode claim 3~8 artificial fusion protein.
10. artificial nucleic acid according to claim 9, which is characterized in that the nucleic acid has SEQ ID NO:Ammonia shown in 3 Base acid sequence.
11. a kind of construct, which is characterized in that carry claim 9 or 10 any one of them artificial nucleic acids.
12. construct according to claim 11, which is characterized in that the carrier of the construct is pET-25b (+).
13. a kind of recombinant cell, which is characterized in that carry claim 9 or 10 any one of them artificial nucleic acids or right is wanted Seek 11 or 12 any one of them constructs.
14. a kind of method obtaining any one of claim 3~8 artificial fusion protein, which is characterized in that suitable for described Under conditions of artificial fusion protein's expression, the recombinant cell described in claim 11 is cultivated, to obtain artificial fusion's egg In vain.
15. a kind of nano particle, which is characterized in that by several claim 3~8 any one of them artificial fusion proteins from group Dress is formed, including：
Hydrophobic core, the hydrophobic core are made of the hydrophobic region of the artificial fusion protein；And
The outer surface of the hydrophobic core is arranged in hydrophilic outer shell, the hydrophilic outer shell, by the hydrophilic area of the artificial fusion protein It constitutes.
16. nano particle according to claim 15, which is characterized in that the artificial fusion protein has SEQ ID NO: Amino acid sequence shown in 2；
Optionally, the nano particle is spherical in shape, a diameter of 48nm~55nm of the nano particle, the hydrophobic core it is straight Diameter is 20nm, and the thickness of the hydrophilic outer shell is 14~17.5nm.
17. a kind of pharmaceutical composition, which is characterized in that include any one of claim 3~8 artificial fusion protein, right It is required that the weight described in 9~10 any one of them nucleic acid, claim 11~12 any one of them construct, claim 13 Nano particle described in group cell or claim 15 or 16.
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109627290A (en) * 2018-12-12 2019-04-16 华南理工大学 α spiral self-assembled short peptide and its application in protein purification
CN109627290B (en) * 2018-12-12 2022-03-29 华南理工大学 Alpha spiral self-assembly short peptide and application thereof in protein purification
CN110101868A (en) * 2019-05-24 2019-08-09 北京大学 A kind of environmental stimulus responsiveness protein high molecular conjugate self-assembly and the preparation method and application thereof
CN110101868B (en) * 2019-05-24 2021-03-23 北京大学 Environment stimulus responsive protein macromolecular conjugate self-assembly and preparation method and application thereof

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