CN103102418B - The fusion rotein of granulocyte colony-stimulating factor (G-CSF) and mutant (mG-CSF) and human serum albumin the 3rd structural domain (3DHSA) and application - Google Patents
The fusion rotein of granulocyte colony-stimulating factor (G-CSF) and mutant (mG-CSF) and human serum albumin the 3rd structural domain (3DHSA) and application Download PDFInfo
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Abstract
The invention belongs to long-acting fusion rotein technical field of pharmaceuticals, specifically relate to the fusion rotein of granulocyte colony-stimulating factor (G-CSF) and mutant (mG-CSF) and human serum albumin the 3rd structural domain (3DHSA); Is the aminoacid sequence of the human serum albumin described in fusion rotein the 3rd structural domain as SEQ? NO:1, does is the aminoacid sequence of granulocyte colony-stimulating factor and mutant thereof SEQ? NO:2 and SEQ? NO:3; Described the present invention successfully constructs two kinds of fusion roteins first, it overcomes the short shortcoming of granulocyte colony-stimulating factor Half-life in vivo, and it is the new fusion protein molecule of the characteristic integrating human serum albumin the 3rd structural domain and Filgrastim.Fusion rotein of the present invention is on the reservation bioactive basis of G-CSF, and its transformation period is in vivo respectively 3.3h and 4.4h, is about 1.6 times and 2 times of G-CSF, provides the original new drug molecule with potential using value.
Description
Technical field
The invention belongs to long-acting fusion rotein technical field of pharmaceuticals, specifically relate to the fusion rotein preparing granulocyte colony-stimulating factor and mutant and human serum albumin the 3rd structural domain.
Background technology
Filgrastim (G-CSF) is the glycoprotein analog regulatory factor that a kind of molecular weight produced by monocyte, fibroblast, endotheliocyte is about 20KD, and its iso-electric point is about 6.0.G-CSF acts on neutrophilic granulocytes of bone marrow system hemopoietic forebody cell, promotes its propagation, differentiation, ripe and release.In ripe human body, G-CSF has 177 amino acid (a type) and 174 amino acid (b) types, two kinds of isomer, and the helical content of b type molecule is very high, and crystal configuration contains four α spirals, belongs to long-chain helical cytokine family.In view of the biologic activity of b type and stability are all significantly higher than a type, b type molecule is deeply by the high praise of investigator.Recombinant human g-csf has been widely used in the treatment of bone marrow transplantation and tumour, and in addition, recombinant methionyl human G-CSF is also used to disease such as treatment acute leukemia, aplastic anemia etc.The effect of G-CSF in adjuvant therapy treatment mainly increases number of white blood cells, reduces the risk of chemotherapy and radiation.Existing multiple recombinant humangranulocyte preparation based on b type goes through to go on the market at present, as filgrastim (filgrastim), and lenograstim (lenograstim), Neu-up 100 (nartograstim).Wherein filgrastim and Neu-up 100 are the recombinant human g-csf of the nonglycosylated form of escherichia coli expression, and the lenograstim G-CSF that to be the band of expressing cho cell glycosylation modified.Filgrastim aminoacid sequence is namely at the additional methionine(Met) of N end of natural G-CSF, and Neu-up 100 is the mutant of filgrastim, relates to 5 amino acid whose rite-directed mutagenesises in natural G-CSF altogether, concrete mutational site and mode are Thr1Ala, Leu3Thr, Gly4Tyr, Pro5Arg, Cys17Ser.
But, natural and human G-CSF that is gene recombination is easily degraded by glomerular filtration and body endoproteinase, and circulating half-life is in vivo shorter, and bioavailability is very low, needing frequent or high dosage administration for maintaining the drug effect concentration played a role, adding pain and the economical load of patient.Therefore, the transformation period extending granulocyte colony-stimulating factor is the emphasis of current scholars research.For extending the Half-life in vivo of granulocyte colony-stimulating factor, specific modification can be carried out to it.A kind of approach increases G-CSF apparent molecular weight by being coupled to reach with some specific components, thus reduce the clearance rate of kidney.Another kind of approach merges with inert protein, stops the contact of proteolytic enzyme and albumen under receptor mediated endocytosis protection, thus reduce the risk of G-CSF body endoproteinase degraded.To be merged by polyethyleneglycol modified, antibody Fc fragment and Albumin fusion all effectively can improve the Half-life in vivo of G-CSF.But pegylation needs special chemical reagent, and product uniformity is poor, separation and purification difficulty, causes the expensive of product.For ensureing the correct folding mammalian expression system needing to select cost very high of Fc fusion rotein disulfide linkage, in addition, the molecular weight speed easily causing fusion protein molecule to diffuse through mucous membrane bigger than normal of Fc fusion rotein is lower, and then affects drug effect.
Human serum albumin is the main component in human plasma, has the function maintaining plasma osmolarity, serves as endogenous and external source many kinds of substance or pharmaceutical carrier effect simultaneously.On the one hand because its molecular weight ratio is larger, under normal circumstances not easily by glomerular filtration, can combine with the Fc acceptor (FcRn) of new life in body on the other hand, by the degraded of the receptor mediated endocytosis endoproteinase of resistance to acceptor, its Half-life in vivo is 19 days.Based on albuminous These characteristics, Human Genome Sciences of the U.S. developed in the first albumin fusion technology, and existing multiple albumin fusion protein medicine is in clinical trial at present.
Although albumin fusion technology has been successfully applied to the transformation period extending multiple protein or polypeptide drug molecule, still have the aspect that some urgently improve.First, because albumin molecule amount is about 66.5KD, obvious sterically hindered effect can be produced when merging other albumen or polypeptide and cause the activity of fusion rotein obviously to reduce.Secondly, easily signs of degradation occurs when yeast secreted expression albumin and fusion rotein thereof, and degradation fragment physico-chemical property and fusion rotein are very close, add the difficulty of later separation purifying.Finally, easily there is polymerism in albumin fusion protein, easily bring out immune response in clinical application in the process stored.
Sero-abluminous 3rd structural domain (3DHSA) of existing research confirmer is the key position that human serum albumin is combined with FcRn, and independently 3DHSA still has the ability be combined with FcRn, and it can the prolong drug transformation period as mate molecule.In addition, have no signs of degradation when expressing human serum albumin the 3rd structural domain, and its molecular size range is moderate, is convenient to genetic manipulation, is more convenient for compared with Albumin fusion meeting the needs extending the differing molecular transformation period.Utilize Overlap extension PCR (OverlappingPCR) method, 3DHSA and effector molecule are directly merged, the space steric effect produced when effectively can reduce fusion on the one hand, avoids the introducing because of connection peptides to cause the problems such as degraded and potential immunogenicity on the other hand.
Can infer based on above-mentioned theory and practice, albumen constructed when G-CSF and mutant thereof and 3DHSA being merged will have the dual biological activities of G-CSF and 3DHSA simultaneously, can be used for the treatment of neutrophilic granulocytopenia, and extend the Half-life in vivo of G-CSF.
Summary of the invention
Problem to be solved by this invention is to overcome the short shortcoming of granulocyte colony-stimulating factor Half-life in vivo, and design preparation integrates the new fusion protein molecule of the characteristic of granulocyte colony-stimulating factor and human serum albumin the 3rd structural domain.Fusion rotein of the present invention, on the reservation bioactive basis of G-CSF, makes its Half-life in vivo obtain proper extension, provides the original new drug molecule with potential using value.
Technical scheme:
Two kinds of fusion roteins, namely the fusion rotein of granulocyte colony-stimulating factor and mutant and human serum albumin the 3rd structural domain, is characterized in that its aminoacid sequence comprises SEQNO:1 and SEQNO:2, SEQNO:1 and SEQNO:3 respectively.Described fusion rotein aminoacid sequence comprises SEQNO:1 and SEQNO:2, SEQNO:1 and SEQNO:3, under the principle not damaging fusion rotein characteristic, to the replacement of SEQNO:2 and SEQNO:3 partial amino-acid residue, lack or add the peptide molecule obtained.
The granulocyte colony-stimulating factor of described fusion rotein and the aminoacid sequence of mutant thereof be the aminoacid sequence of more than at least 85% homology with SEQNO:2 and SEQNO:3 respectively.
The granulocyte colony-stimulating factor of described fusion rotein and the aminoacid sequence of mutant thereof be the aminoacid sequence of more than at least 95% homology with SEQNO:2 and SEQNO:3 respectively.
The aminoacid sequence of described fusion rotein is SEQNO:14 and SEQNO:15.
Described fusion rotein is in the application of preparation treatment granulocytopenia drugs with function.
Specifically:
Technical scheme of the present invention: increased respectively by round pcr and obtain 3DHSA-G10,3DHSA-mG10, H10-G-CSF and H10-mG-CSF gene fragment; Overlap extension pcr is utilized to realize 3DHSA-G10 and H10-G-CSF respectively, the seamless link of 3DHSA-mG10 and H10-mG-CSF, agarose gel electrophoresis reclaims PCR primer and carries out enzyme successively to it and to cut and glue reclaims, and the pPICz α A carrier segments that 3DHSA-G-CSF with the 3DHSA-mG-CSF fusion gene after enzyme cuts back to close cuts back to close with enzyme is respectively connected and builds recombinant expression vector; By in the genome of sufficient for linearizing recombinant expression vector by electroporated mode Insertion Into Host Cell Pichia pastoris GS115, the clone that picking transforms out from MD flat board carries out Tube propagation and induces, whether SDS-PAGE detects induction supernatant, according to having the induction band of corresponding molecular weight to occur the positive recombinant bacterial strain of qualification.
The qualification of the acquisition of fusion gene, the structure of recombinant expression vector, recombinant bacterial strain, the abduction delivering of fusion rotein, purifying, biologic activity and pharmacokinetic properties research step refer to specific embodiment.
The invention provides the fusion rotein preparing recombinant methionyl human G-CSF and mutant and human serum albumin the 3rd structural domain with aforesaid method, this fusion rotein comprises and the firstth district of the complete homology of human serum albumin the 3rd structural domain and the secondth district with Filgrastim and mutant at least 85% homology thereof, preferably comprise with the firstth district of the complete homology of human serum albumin the 3rd domain sequence and with Filgrastim and identical the secondth district of mutant sequence thereof, or more than 95% homology and the derivative of increased activity.
Described derivative refers at the replacement of the condition Xia Dui bis-district partial amino-acid residue not damaging fusion rotein characteristic, a series of peptide molecules of lacking or adding and obtain.
In fusion rotein of the present invention, 3rd structural domain of human serum albumin and the relative position of granulocyte colony-stimulating factor and mutant thereof both can be 3DHSA-(m) G-CSF, can be again (m) G-CSF-3DHSA, be preferably 3DHSA-(m) G-CSF, namely the 3rd structural domain of human serum albumin is connected with the N-terminal of granulocyte colony-stimulating factor and mutant thereof.The two selects directly to merge, and any connection peptides is not introduced in centre, to avoid the problem of proteolytic degradation and the potential immunogenicity aspect caused because of the introducing of connection peptides.
The present invention also provides the DNA sequence dna of respectively encoding human granulocyte's G CFS and mutant and human serum albumin the 3rd domain fusion protein (3DHSA-G-CSF and 3DHSA-mG-CSF), this sequence comprise firstth district (SEQNO:4) identical with human serum albumin the 3rd structural domain nucleotide sequence and with granulocyte colony-stimulating factor (SEQNO:5) and identical the secondth district of mutant (SEQNO:6) nucleotide sequence thereof.
SEQNO:4 is 3DHSADNA sequence;
SEQNO:5 is G-CSFDNA sequence;
SEQNO:6 is mG-CSFDNA sequence;
SEQNO:4, SEQNO:5 and SEQNO:6 all hold to 3 ' from 5 ' end
Another object of the present invention is to provide the recombinant expression vector carrying the corresponding DNA sequence dna of code book invention fusion rotein.This recombinant expression vector comprises pPICz α A, pPIC9, pGAPz α B, preferred pPICz α A.Wherein preferred pPICz α A carrier, the molecular weight of this carrier own is little is convenient to genetic manipulation, has the element such as strong AOX1 promotor and MF α secreting signal peptide simultaneously.It is strong that AOX1 promotor starts ability, at methyl alcohol as the secreting, expressing that efficiently can start goal gene during sole carbon source.
Another object of the present invention is to provide the host expressing fusion rotein encoding gene of the present invention, this host be complete or linearizing fusion protein expression carrier is proceeded to by chemistry and electroporated method bacterium, yeast, insect cell, zooblast and vegetable cell, wherein preferred yeast cell, preferred yeast is pichia spp, and preferred pichia spp is GS115.
Similar prokaryotic expression system genetic background is clear, easy to operate, fecundity is strong, nutritional requirement is low except having for preferred yeast expression system, be easy to except the advantages such as industry amplification, there is Modifying Capability after eukaryotic protein translation simultaneously, be easy to produce high-quality recombinant protein in a large number.GS115 deficient strain is convenient to the screening after transforming, and himself secretion foreign protein is convenient to purifying less, and degree of glycosylation is low, can effectively avoid modifying because of yeast saccharomyces cerevisiae excessive glycosylation causing immunogenic problem.
The cell of successful conversion, namely containing the cell of antigen-4 fusion protein gene of the present invention, the induction supernatant screening recombinant bacterial strain that genomic dna carries out PCR qualification by extracting, bacterium colony Dotblotting, SDS-PAGE detect cell maybe can select the antibody of mouse anti human G-CSF and HSA to aforesaid method screen recombinant bacterial strain culture supernatant carry out Westernblotting qualification.
Can by cultivating containing the fusion rotein in host's production the present invention of antigen-4 fusion protein gene of the present invention.Host cultivates and shaking flask both can have been selected also can to select bio-reactor, preferred bio-reactor.The cultivation of host comprises two stages, and the first stage is intended to the raised growth of host, and subordinate phase is mainly used in abduction delivering fusion rotein.Multiple means can be fully utilized be separated and purified fusion protein from containing in the nutrient solution of antigen-4 fusion protein gene host of the present invention.As ultrafiltration, saltout, the optimum combination of the technology such as alcohol precipitation, PAGE production, chromatography and above-mentioned technology.
Beneficial effect: 1, the present invention utilizes overlap extension pcr successfully by SEQNO:4 and SEQNO:5 first, SEQNO:4 and SEQNO:6 nucleotide sequence has carried out direct splicing, successfully construct two kinds of fusion roteins, it overcomes the short shortcoming of granulocyte colony-stimulating factor Half-life in vivo, and it is the new and effective fusion protein molecule of the characteristic integrating human serum albumin the 3rd structural domain and Filgrastim.
2, the present invention constructs the recombinant expression vector of applicable yeast secreted expression and utilizes electroporated mode to be inserted in pichia spp genome, comprehensive utilization bacterium colony trace and SDS-PAGE to electroporated go out Pichia pastoris GS115 bacterium colony carried out screening fast, both reduced screening operation amount and in turn ensure that accuracy rate.
3, the present invention utilizes blue-sepharose gravity post and butyl drainage column purifying to obtain two kinds of fusion rotein: 3DHSA-G-CSF and 3DHSA-mG-CSF of higher degree, purifying gained fusion rotein all remains the biological activity of G-CSF, the two transformation period is in vivo respectively 3.4h and 4.5h, be about 1.6 and 2.1 times of G-CSF, the original new drug molecule with potential using value is provided.
Accompanying drawing explanation
Fig. 1 is the structure schematic diagram of plasmid pPICz α A-3DHSA-G-CSF and pPICz α A-3DHSA-mG-CSF.
Fig. 2 is the qualification of fusion gene 3DHSA-G-CSF and 3DHSA-mG-CSF
Fig. 3 is that the enzyme of recombinant plasmid pPICz α A-3DHSA-G-CSF and pPICz α A-3DHSA-mG-CSF cuts qualification result, in figure A and B, M is Marker, A1 swimming lane is recombinant plasmid pPICz α A-3DHSA-G-CSF, A2 swimming lane is the digestion products of recombinant plasmid pPICz α A-3DHSA-G-CSF, B1 swimming lane is recombinant plasmid pPICz α A-3DHSA-mG-CSF, and B2 swimming lane is the digestion products of recombinant plasmid pPICz α A-3DHSA-mG-CSF.
Fig. 4 is screening and the qualification of Positive recombinant clones, wherein A and B is respectively the bacterium colony Blot results result of pPICz α A-3DHSA-G-CSF and pPICz α A-3DHSA-mG-CSF, C and D is respectively the SDS-PAGE detected result of pPICz α A-3DHSA-G-CSF and pPICz α A-3DHSA-mG-CSF.
Fig. 5 is the Westernblotting qualification of fusion rotein, figure A is mouse anti human G-CSF antibody test result, wherein M is Marker, and 1 swimming lane is 3DHSA-G-CSF, and 2 swimming lanes are G-CSF, 3 swimming lanes are 3DHSA-mG-CSF, figure B is mouse anti human HSA antibody test result, and wherein M is Marker, and 1 swimming lane is 3DHSA-G-CSF, 2 swimming lanes are HSA, and 3 swimming lanes are 3DHSA-mG-CSF.
Fig. 6 is the impact of induction time on fusion protein expression, and figure A and figure B is respectively the SDS-PAGE result of 3DHSA-G-CSF and 3DHSA-mG-CSF, and wherein M is Marker, and 1 ~ 9 swimming lane is induction time is respectively 24h, 36h, 48h, 60h, 72h, the result of 84h, 96h, 108h and 120h.
Fig. 7 is the Purity of purifying rear fusion protein, and wherein M is Marker, and figure A1 is 3DHSA-G-CSF, and figure B1 is 3DHSA-mG-CSF.
Fig. 8 is the active determination in vitro of fusion rotein.
Fig. 9 measures the transformation period of fusion rotein.
Embodiment
In following examples, DH5 α, GS115 and pPICz α A used is purchased from Invitrogen, and the structure of template plasmid pPICz α A-HSA-G-CSF (SEQNO:7) is with reference to CN01124114.4.
In each embodiment, plasmid extraction kit used and sepharose reclaim test kit purchased from sky root (Beijing) biochemical company limited, human G-CSF Elisa test kit is (Shenzhen) Science and Technology Ltd. purchased from reaching section, various restriction enzyme, various archaeal dna polymerase, T4DNA ligase enzyme, nucleic acid marker and small molecular weight protein marker is purchased from precious biotechnology (Dalian) company limited, primer is synthesized by prompt base (Shanghai) trade Co., Ltd in the English Weihe River, Ji Saixin is purchased from North China Pharmacuetical Jintan Biotechnology Co., Ltd, HSA is purchased from Wuhan He Yuan biotech company in restructuring, mouse anti human HSA and G-CSF antibody purchased from American Santa company, HRP marks rabbit anti-mouse IgG purchased from Beijing biotech company of Zhong Shan Golden Bridge, blue-sepharose filler and butyl dewatering filling are purchased from GE company, yeast powder and peptone are purchased from OXOID, YNB and bleomycin are purchased from Invitrogen, other reagent are analytical pure, purchased from ancient cooking vessel state (Beijing) limited-liability company.
The clone of embodiment 13DHSA-G-CSF and 3DHSA-mG-CSF fusion gene
The amplification of 3DHSA-G10 and 3DHSA-mG10, the primer is as follows:
P1:5 ' TCT
cTCGAGaAGAGAGTGGAAGAGCCTCAGAATTTAAT3 ' (5 ' end band has XhoI restriction enzyme site, CTCGAG), primer SEQNO:8.
P2:5 ' CCAGCGGGGTTAAGCCTAAGGCAGCTTGAC3 ', primer SEQNO:9.
P3:5 ' ATGTGGGTGCTAAGCCTAAGGCAGCTTGAC3 ', primer SEQNO:10.
PCR method is as follows: add in 50 μ L systems: (amplification 3DHSA-G10 the primer is P1 and P2 to each 2 μ L of upstream and downstream primer of 10 μm of ol/L, 3DHSA-mG10 the primer is P1 and P3), the dNTP4 μ L of 2.5 μm of ol/L, 10 × Premistarbuffer5 μ L, the PremistarDNA polysaccharase 0.5 μ L of 5U/ μ L, pPICz α A-HSA-G-CSF plasmid (SEQNO:7) 1 μ L, insufficient section is by sterilizing distilled water polishing, reaction conditions is: 94 DEG C of denaturation 5min, 94 DEG C of sex change 1min, 56 DEG C of annealing 1min, 72 DEG C extend 1min, repeat 25 circulations.
The amplification of H10-G-CSF and H10-mG-CSF, the primer is as follows:
P4:5 ' TTTGGGTTTGACCCCGCTGGGACCGGCAAG3 ', primer SEQNO:11.
P5:5 ' CGT
cTCGAGtTAGGGCTGGGCAAGGTGGC3 ' (5 ' end band has XhoI restriction enzyme site, CTCGAG), primer SEQNO:12.
P6：5’TTTGGGTTTGGCACCCACATACAGAGCCAGCTCCCTGCCCCAGAGCTTCCTG
CTCAGAG3 ', primer SEQNO:13.
PCR method is as follows: add in 50 μ L systems: (amplification 3H10-G-CSF the primer is P4 and P5 to each 2 μ L of upstream and downstream primer of 10 μm of ol/L, H10-mG-CSF the primer is P6 and P5), the dNTP4 μ L of 2.5 μm of ol/L, 10 × Premistarbuffer5 μ L, the PremistarDNA polysaccharase 0.5 μ L of 5U/ μ L, pPICz α A-HSA-G-CSF plasmid (SEQNO:7) 1 μ L, insufficient section is by sterilizing distilled water polishing, reaction conditions is: 94 DEG C of denaturation 5min, 94 DEG C of sex change 1min, 56 DEG C of annealing 1min, 72 DEG C extend 1min, repeat 25 circulations.
Overlap extension PCR method is utilized 3DHSA-G10 and H10-G-CSF, 3DHSA-mG10 and H10-mG-CSF to be merged respectively:
Cut glue respectively and reclaim 3DHSA-G10, 3DHSA-mG10, the PCR primer of H10-G-CSF and H10-mG-CSF gene fragment also dilutes 30 times, add in 50 μ L systems: each 2 μ L of P1 and P5 of 10 μm of ol/L, the dNTP4 μ L of 2.5 μm of ol/L, 10 × LATaqbuffer5 μ L, the LATaqDNA polysaccharase 0.5 μ L of 5U/ μ L, (amplification 3DHSA-G-CSF is template used is 3DHSA-G10 and H10-G-CSF to each 1 μ L of recovery fragment after dilution, amplification 3DHSA-mG-CSF is template used is 3DHSA-mG10 and H10-mG-CSF) insufficient section is by sterilizing distilled water polishing, reaction conditions is: 94 DEG C of denaturation 5min, 94 DEG C of sex change 1min, 56 DEG C of annealing 1min, 72 DEG C extend 1min30s, repeat 25 circulations.
By 1% agarose gel electrophoresis detection reaction product, contrast DL2000, is about 1000bp size at application of sample swimming lane and occurs fusion gene band, as Fig. 2.Cut glue and reclaim goal gene fragment, respectively XhoI single endonuclease digestion is carried out to the goal gene fragment reclaimed and pPICz α A, cut glue after agarose gel electrophoresis and reclaim fusion gene fragment and carrier segments, with T4DNA ligase enzyme, above-mentioned recovery product is connected; Connect product conversion DH5 α competent cell, 37 DEG C of overnight incubation, single colony inoculation that picking transforms is 37 DEG C of cultivation 8h in 5mLLB liquid nutrient medium, and extraction plasmid carries out enzyme and cuts qualification, enzyme is cut gained size fragment and is coincide, as Fig. 3 with carrier and fusion gene clip size respectively.Sent by above-mentioned clone Nanjing Si Pujin to check order, sequencing result confirms that recombinant expression vector successfully constructs, called after pPICz α A-3DHSA-G-CSF and pPICz α A-3DHSA-mG-CSF.
The screening of embodiment 2 recombinant bacterial strain
Extract pPICz α A-3DHSA-G-CSF and pPICz α A-3DHSA-mG-CSF plasmid, linearizing is carried out through SalI single endonuclease digestion, agarose gel electrophoresis detects enzyme and cuts effect, utilize sepharose to reclaim test kit and reclaim digestion products, the electroporated Pichia pastoris GS115 competent cell of 1.5kV, 30 DEG C of standing 2h, conversion product is uniformly coated on 4 pieces containing on the MD flat board of 1mol/L sorbyl alcohol, be inverted cultivation 3 days for 30 DEG C, the clone's correspondence markings transformed out is well transferred simultaneously on freshly prepd two pieces of MD flat boards, be inverted cultivation 3 days for 30 DEG C, the pvdf membrane handled well by uv irradiating is put down gently wherein above one piece of MD flat-plate bacterial colony, on film, place the filter paper 2 that uv irradiating is handled well simultaneously, with 3mL anhydrous methanol, its surface is all infiltrated, after 24h is cultivated in 30 DEG C of continuation, take out pvdf membrane and filter paper, according to Westernblotting flow process, pvdf membrane is closed successively, washing, hatch mouse anti human G-CSF antibody, washing, the rabbit anti-mouse Ig bis-of hatching HRP mark resists, washing and colour developing.(see Biotechnology122:181-184.1994 such as ScorerCA) is according to whether occurring the positive recombinant bacterial strain of coloured bacterium colony circle preliminary judgement, as Fig. 4 (A) and 4 (B).The correspondence list bacterium colony that picking has bacterium colony circle to be formed on another block MD flat board is also inoculated in 10mLBMGY (30mg dipotassium hydrogen phosphate, 118mg potassium primary phosphate, 134mg is without amino substratum, 100mg yeast powder, 200mg peptone, 2 μ g vitamin Hs, 0.1mL glycerine, pH6.0) in liquid nutrient medium 30 DEG C, 200r/min cultivates 24h, the centrifugal 5min of 2000r/min, abandon supernatant, with isopyknic BMMY (30mg dipotassium hydrogen phosphate, 118mg potassium primary phosphate, 134mg is without amino substratum, 100mg yeast powder, 200mg peptone, 2 μ g vitamin Hs, 0.1mL anhydrous methanol, pH6.0) the resuspended thalline of liquid nutrient medium, 30 DEG C, the anhydrous methanol that the every 12h of 200r/min adds 100 μ L in BMMY is induced, after cultivating 120h, the centrifugal 10min of 8000r/min, collect supernatant, carry out SDS-PAGE checking, as Fig. 4 (C) and 4 (D).Strain Designation the highest for relative expression quantity in positive colony is GS115/pPICz α A-3DHSA-G-CSF and GS115/pPICz α A-3DHSA-mG-CSF according to the thickness of protein electrophoresis band by contrast albumen Marker.
The abduction delivering of embodiment 33DHSA-G-CSF and 3DHSA-mG-CSF fusion rotein and qualification
By the GS115/pPICz α A-3DHSA-G-CSF screened and the mono-colony inoculation of GS115/pPICz α A-3DHSA-mG-CSF to 100mlBMGY (0.3g dipotassium hydrogen phosphate is housed, 1.18g potassium primary phosphate, 1.34g without amino substratum, 1g yeast powder, 2g peptone, 20 μ g vitamin Hs, 1mL glycerine, pH6.0) 500ml Erlenmeyer flask in, 220r/min, cultivate the centrifugal 5min of 24h, 2000r/min for 30 DEG C, collect thalline.With the BMMY of 100ml (0.3g dipotassium hydrogen phosphate, 1.18g potassium primary phosphate, 1.34g without amino substratum, 1g yeast powder, 2g peptone, 20 μ g vitamin Hs, 1mL anhydrous methanol, pH6.0) resuspended bacterial sediment, every 12h adds 1mL anhydrous methanol, successive induction five days.The centrifugal 10min of 8000r/min, collect supernatant, carry out SDS-PAGE detection, fusion protein expression improves constantly along with the prolongation of induction time, as Fig. 5 (A) and 5 (B).Westernblotting is adopted to identify G-CSF and the HSA antigenicity of fusion rotein 3DHSA-G-CSF and 3DHSA-mG-CSF, set G-CSF respectively (see PNAS20:7292-7296 such as YunBai, 2005) and HSA as positive control (see PNAS47:19078-19083 such as YangHe, 2011) as Fig. 6 (A) and 6 (B).
The purifying of embodiment 43DHSA-G-CSF and 3DHSA-mG-CSF and Purity
Three times are diluted with pure water after the centrifugal gained supernatant of the positive recombinant bacterial strain fermented liquid of GS115/pPICz α A-3DHSA-G-CSF and GS115/pPICz α A-3DHSA-mG-CSF crosses 0.45 μm of filter membrane, with the enrichment of blue-sepharose filler and protein of interest, the target protein peak butyl dewatering filling purified fusion protein collected, the fusion rotein postlyophilization of 5mM sodium-acetate buffer dialysis purifying gained is preserved, called after 3DHSA-G-CSF and 3DHSA-mG-CSF, its aminoacid sequence is SEQNO:14 and SEQNO:15.The purity of SDS-PAGE method to purifying gained fusion rotein is adopted to detect, as Fig. 7 (A) and 7 (B).
The short NFS-60 cell-proliferation activity of embodiment 53DHSA-G-CSF and 3DHSA-mG-CSF
The short NFS-60 cell-proliferation activity of purifying gained fusion rotein 3DHSA-G-CSF and 3DHSA-mG-CSF is measured with mtt assay.Normal for Secondary Culture NFS-60 cell (U.S. ATCC) is used RPMI-1640 substratum centrifuge washing three times, by cell by 1 × 10
5the density suspension of individual/mL in be rich in 5% horse serum, 5% foetal calf serum not containing the RPMI-1640 substratum of G-CSF in (V/V).In 96 orifice plates, every hole adds 50 μ L cell suspensions, if 6 multiple holes are cultivated, except blank, add the testing sample of different concns in every hole, choose Ji Saixin (trade(brand)name of recombinant human g-csf) is positive control drug simultaneously, 37 DEG C, 5%CO
2, after cultivating 48h under saturated humidity condition, every hole adds MTT working fluid 20 μ L, continues to cultivate 4h, and every hole adds lysate 100 μ L, after mixing, puts into microplate reader, measures absorbancy in wavelength 570nm place, record measurement result.Activation analysis result shows: two kinds of fusion roteins all have the effect of dose-dependant sexual stimulus NFS-60 cell proliferation, as Fig. 8.
The Half-life in vivo of embodiment 63DHSA-G-CSF and 3DHSA-mG-CSF measures
Adopt G-CSF final concentration to be the body weight dose of 1mg/kg, inject Ji Saixin and two kind of fusion rotein respectively, respectively at the 0.25h after administration to the ICR male mice in 6 week age is subcutaneous, 0.5h, 1h, 2h, 4h, 8h, 12h, 24h and 36h carry out eye socket and get blood, after normal temperature leaves standstill 30min, the centrifugal 10min of 2000r/min, gets supernatant, utilizes the pre-coated Elisa test kit of human G-CSF to detect the concentration of G-CSF in serum.Plasma drug concentration data is inputted PKsolver and carry out data processing, matching plasma concentration curve.Result shows, and the transformation period of 3DHSA-G-CSF and 3DHSA-mG-CSF, for being respectively 3.4h and 4.5h, is 1.6 times and 2.1 times of G-CSF, as Fig. 9.
Claims (3)
1. the fusion rotein of granulocyte colony-stimulating factor and mutant and human serum albumin the 3rd structural domain, is characterized in that it is the recombinant protein of being expressed after gene level directly merges by granulocyte colony-stimulating factor or its mutant and human serum albumin the 3rd structural domain; The aminoacid sequence of described human serum albumin the 3rd structural domain is as shown in SEQNO:1; The aminoacid sequence of described granulocyte colony-stimulating factor and mutant thereof is SEQNO:2 and SEQNO:3.
2. the fusion rotein of granulocyte colony-stimulating factor according to claim 1 and mutant and human serum albumin the 3rd structural domain, is characterized in that: the aminoacid sequence of the fusion rotein of granulocyte colony-stimulating factor and human serum albumin the 3rd structural domain is SEQNO:14, the fusion rotein of the mutant of granulocyte colony-stimulating factor and human serum albumin the 3rd structural domain is SEQNO:15.
3. according to the fusion rotein application in preparation treatment granulocytopenia disease drug of claim 1-2 described in any one.
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