CN103102418A - Fusion protein of granulocyte colony-stimulating factor (G-CSF) as well as mutant of granulocyte colony-stimulating factor (mG-CSF) and human serum albumin 3rd domain (3DHSA) and application - Google Patents
Fusion protein of granulocyte colony-stimulating factor (G-CSF) as well as mutant of granulocyte colony-stimulating factor (mG-CSF) and human serum albumin 3rd domain (3DHSA) and application Download PDFInfo
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Abstract
The invention belongs to the technical field of a long-acting fusion protein medicine, and in particular relates to a fusion protein of a granulocyte colony-stimulating factor (G-CSF) as well as a mutant of the granulocyte colony-stimulating factor (mG-CSF) and a human serum albumin 3rd domain (3DHSA). The amino acid sequence of the human serum albumin 3rd domain of the fusion protein is expressed as SEQ NO: 1, and the amino acid sequences of the granulocyte colony-stimulating factor and mutant thereof are expressed as SEQ NO: 2 and SEQ NO: 3. The invention successfully constructs two fusion proteins for the first time, overcomes the shortcoming of short in vivo half-life period of the granulocyte colony-stimulating factor and provides a novel fusion protein molecule integrating characteristics of the human serum albumin 3rd domain and the granulocyte colony-stimulating factor. The fusion protein disclosed by the invention, on the basis of maintaining G-CSF bioactivity, has in vivo half-life periods of 3.3hours and 4.4hours which are about 1.6 and 2 times as much as the half-life period of the G-CSF, thereby providing an innovative drug molecule having a potential application value.
Description
Technical field
The invention belongs to long-acting fusion rotein technical field of pharmaceuticals, specifically relate to the fusion rotein of preparation granulocyte colony-stimulating factor and mutant and human serum albumin the 3rd structural domain.
Background technology
Filgrastim (G-CSF) is about the glycoprotein analog regulatory factor of 20KD by a kind of molecular weight that monocyte, fibroblast, endotheliocyte produce, its iso-electric point is about 6.0.It is hemopoietic forebody cell that G-CSF acts on neutrophilic granulocytes of bone marrow, promotes its propagation, differentiation, maturation and discharges.In ripe human body, G-CSF has 177 amino acid (a type) and two kinds of isomer of 174 amino acid (b) type, and the helical content of b type molecule is very high, and crystal configuration contains four α spirals, belongs to long-chain spiral cell factor family.In view of biologic activity and the stability of b type all is significantly higher than a type, b type molecule is subjected to investigator's high praise deeply.The 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 also is used to treat the diseases such as acute leukemia, aplastic anemia.G-CSF is mainly to increase the white corpuscle number in the effect aspect auxiliary oncotherapy, reduces the risk of chemotherapy and radiation.Existing multiple recombinant humangranulocyte preparation take the b type as the basis 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 non-glycosylated form of escherichia coli expression, and lenograstim be expressing cho cell with glycosylation modified G-CSF.The filgrastim aminoacid sequence is namely at the additional methionine(Met) of the N of natural G-CSF end, Neu-up 100 is the mutant of filgrastim, relate to altogether 5 amino acid whose rite-directed mutagenesises in natural G-CSF, concrete mutational site and mode are Thr 1 Ala, Leu 3 Thr, Gly 4 Tyr, Pro 5 Arg, Cys 17 Ser.
But, human G-CSF natural and gene recombination is easily degraded by glomerular filtration and body endoproteinase, and circulating half-life in vivo is shorter, and bioavailability is very low, need frequent or high dosage administration for keeping the drug effect concentration that plays a role, increased patient's pain and economical load.Therefore, the transformation period of prolongation granulocyte colony-stimulating factor is the emphasis of present various countries scholar's research.For extending the Half-life in vivo of granulocyte colony-stimulating factor, can carry out specific modification to it.A kind of approach is to increase the G-CSF apparent molecular weight by being coupled to reach with some specific components, thereby reduces the clearance rate of kidney.Another kind of approach is and inert protein merges, and stops contacting of proteolytic enzyme and albumen under the receptor mediated endocytosis protection, thereby reduces the risk that G-CSF body endoproteinase is degraded.Merge and Albumin fusion all can effectively improve the Half-life in vivo of G-CSF by polyethyleneglycol modified, antibody Fc fragment.But pegylation needs special chemical reagent, and the product homogeneity is poor, and the separation and purification difficulty causes the expensive of product.Need to select the very high mammalian expression system of cost for correct the folding that guarantees Fc fusion rotein disulfide linkage, in addition, the molecular weight of the Fc fusion rotein speed that easily causes fusion protein molecule to diffuse through mucous membrane bigger than normal is lower, and then affects drug effect.
Human serum albumin is the main component in human plasma, has the function of keeping plasma osmolarity, serves as simultaneously effect endogenous and external source many kinds of substance or pharmaceutical carrier.Because its molecular weight ratio is larger, be difficult for by glomerular filtration under normal circumstances on the one hand, on the other hand can with newborn Fc acceptor (FcRn) combination in body, tolerate the degraded of body endoproteinase by receptor mediated endocytosis, its Half-life in vivo is 19 days.Based on albuminous These characteristics, at first U.S. Human Genome Sciences has developed the Albumin fusion technology, and existing multiple albumin fusion protein medicine is in the clinical experiment stage at present.
Although the Albumin fusion technology has been successfully applied to the transformation period that extends multiple protein or polypeptide drug molecule, still has some to demand improved aspect urgently.At first, because the albumin molecular weight is about 66.5KD, can produces obvious sterically hindered effect and cause the active of fusion rotein obviously to reduce when merging other albumen or polypeptide.Secondly, signs of degradation easily occurs, and the degradation fragment physico-chemical property is very close with fusion rotein, has increased the difficulty of later separation purifying when yeast secreted expression albumin and fusion rotein thereof.At last, polymerism easily occurs in albumin fusion protein in the process that stores, easily bring out immune response in clinical application.
Have and studies confirm that the 3rd structural domain (3DHSA) of human serum albumin is the key position that human serum albumin is combined with FcRn, independently 3DHSA still has the ability of being 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 satisfying than Albumin fusion the needs that extend the differing molecular transformation period.Utilize overlapping extension PCR (Over lapping PCR) method, 3DHSA and effector molecule are directly merged, can effectively reduce the space steric effect that produces when merging on the one hand, avoid on the other hand causing the problems such as degraded and potential immunogenicity because of the introducing of connection peptides.
Can infer based on above-mentioned theory and practice, during with G-CSF and mutant and 3DHSA fusion, constructed albumen will have the dual biologic activity 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 the 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 makes its Half-life in vivo obtain proper extension keeping on G-CSF bioactive basis, and the molecule of the original new drug with potential using value is provided.
Technical scheme:
Two kinds of fusion roteins, namely the fusion rotein of granulocyte colony-stimulating factor and mutant thereof and human serum albumin the 3rd structural domain, is characterized in that its aminoacid sequence comprises respectively SEQ NO:1 and SEQ NO:2, SEQ NO:1 and SEQ NO:3.Described fusion rotein aminoacid sequence comprises SEQ NO:1 and SEQ NO:2, SEQ NO:1 and SEQ NO:3, under the principle of not damaging the fusion rotein characteristic, to the replacement of SEQ NO:2 and SEQ NO:3 partial amino-acid residue, lack or add the peptide molecule that obtains.
The granulocyte colony-stimulating factor of described fusion rotein and the aminoacid sequence of mutant thereof be the aminoacid sequence of homology more than at least 85% with SEQ NO:2 and SEQ NO:3 respectively.
The granulocyte colony-stimulating factor of described fusion rotein and the aminoacid sequence of mutant thereof be the aminoacid sequence of homology more than at least 95% with SEQ NO:2 and SEQ NO:3 respectively.
The aminoacid sequence of described fusion rotein is SEQ NO:14 and SEQ NO:15.
Described fusion rotein is in the application of preparation treatment granulocytopenia drugs with function.
Specifically:
Technical scheme of the present invention: increase respectively by round pcr and obtain 3DHSA-G10,3DHSA-mG10, H10-G-CSF and H10-mG-CSF gene fragment; Utilize overlap extension pcr to realize respectively 3DHSA-G10 and H10-G-CSF, the seamless link of 3DHSA-mG10 and H10-mG-CSF, agarose gel electrophoresis reclaims the PCR product and it is carried out successively enzyme is cut and glue reclaims, and the 3DHSA-G-CSF after enzyme cuts back to close is connected the pPICz α A carrier segments that cuts back to close with enzyme and is connected the structure recombinant expression vector with the 3DHSA-mG-CSF fusion gene; In the genome of the mode Insertion Into Host Cell Pichia pastoris GS115 that the sufficient recombinant expression vector of linearizing is transformed by electric shock, the clone that picking transforms out from the MD flat board carries out test tube and cultivates and induce, whether SDS-PAGE detects and induces supernatant, according to having the band of inducing of corresponding molecular weight positive recombinant bacterial strain to occur identifying.
The evaluation 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 see specific embodiment for details.
The invention provides the fusion rotein for preparing recombinant methionyl human G-CSF and mutant and human serum albumin the 3rd structural domain with aforesaid method, this fusion rotein comprise with the firstth district of human serum albumin the 3rd complete homology of structural domain and with the Second Region of Filgrastim and mutant at least 85% homology thereof, preferably comprise with the firstth district of human serum albumin the 3rd complete homology of structural domain sequence and with Filgrastim and the identical Second Region of mutant sequence thereof, or the derivative of 95% above homology and increased activity.
Described derivative refer under the condition of not damaging the fusion rotein characteristic to two district's partial amino-acid residues replacement, lack or add and a series of peptide molecules of obtaining.
In fusion rotein of the present invention, the 3rd structural domain of human serum albumin and the relative position of granulocyte colony-stimulating factor and mutant thereof can be both 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.Both select directly fusion, any connection peptides is not introduced in the centre, the problem of the proteolytic degradation that causes with the introducing of avoiding because of connection peptides and potential immunogenicity aspect.
The present invention also provides the DNA sequence dna of encoding human granulocyte colony-stimulating factor respectively and mutant and human serum albumin the 3rd structural domain fusion rotein (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 the identical Second Region of mutant (SEQNO:6) nucleotide sequence thereof.
SEQ NO:4 is the 3DHSA DNA sequence dna;
SEQNO:5 is the G-CSF DNA sequence dna;
SEQNO:6 is the mG-CSF DNA sequence dna;
SEQNO:4, SEQNO:5 and SEQNO:6 all hold to 3 ' end from 5 '
Another purpose of the present invention is to provide the recombinant expression vector that carries 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.Preferred pPICz α A carrier wherein, the molecular weight urine of this carrier own has the elements such as strong AOX1 promotor and MF α secreting signal peptide simultaneously in genetic manipulation.It is strong that the AOX1 promotor starts ability, can efficiently start the secreting, expressing of goal gene during as sole carbon source at methyl alcohol.
A further object of the present invention is to provide the host who expresses fusion rotein encoding gene of the present invention, this host is bacterium, yeast, insect cell, zooblast and the vegetable cell that can complete or linearizing fusion protein expression carrier be changed over to by the method that chemistry and electric shock transform, preferred yeast cell wherein, 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 the advantage such as industry amplification, have simultaneously Modifying Capability after eukaryotic protein translation, be easy to produce in a large number high-quality recombinant protein.Screening after the GS115 deficient strain is convenient to transform, and himself secretion foreign protein is convenient to purifying less, and degree of glycosylation is low, can effectively avoid modifying because of the yeast saccharomyces cerevisiae excessive glycosylation causing immunogenic problem.
The cell that success transforms, the cell that namely contains antigen-4 fusion protein gene of the present invention can carry out by extracting genomic dna the supernatant screening recombinant bacterial strain of inducing of PCR evaluation, bacterium colony Dot blotting, SDS-PAGE detection cell and maybe can select the antibody of mouse anti human G-CSF and HSA to carry out Western blotting evaluation to the culture supernatant of recombinant bacterial strain that aforesaid method screens.
Can contain fusion rotein in host's production the present invention of antigen-4 fusion protein gene of the present invention by cultivation.The host cultivates and both can select shaking flask also can select bio-reactor, preferred bio-reactor.Host's cultivation comprises two stages, and the fs is intended to host's raised growth, and subordinate phase is mainly used in the abduction delivering fusion rotein.Can fully utilize multiple means separates and purified fusion protein in the nutrient solution that contains antigen-4 fusion protein gene host of the present invention.As ultrafiltration, saltout, the optimum combination of the technology such as alcohol precipitation, preparation electrophoresis, chromatography and above-mentioned technology.
Beneficial effect: 1, the present invention utilize first the overlap extension pcr success with SEQ NO:4 and SEQ NO:5, SEQ NO:4 and SEQ NO:6 nucleotide sequence have carried out direct splicing, two kinds of fusion roteins have successfully been built, it overcomes the short shortcoming of granulocyte colony-stimulating factor Half-life in vivo, and it is the new and effective fusion protein molecule that integrates human serum albumin the 3rd structural domain and Filgrastim's characteristic.
2, the present invention has built the recombinant expression vector of suitable yeast secreted expression and has utilized the mode of electric shock conversion to be inserted in the pichia spp genome, the Pichia pastoris GS115 bacterium colony that comprehensive utilization bacterium colony trace and SDS-PAGE transform out electric shock has carried out screening fast, has not only reduced the screening operation amount but also guaranteed 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 the 3DHSA-mG-CSF of higher degree, purifying gained fusion rotein has all kept the biological activity of G-CSF, both in vivo transformation period is respectively 3.4h and 4.5h, be about 1.6 and 2.1 times of G-CSF, the molecule of the original new drug with potential using value is provided.
Description of drawings
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 evaluation 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 is cut qualification result, in figure A and B, M is Marker, the A1 swimming lane is recombinant plasmid pPICz α A-3DHSA-G-CSF, the A2 swimming lane is that the enzyme of recombinant plasmid pPICz α A-3DHSA-G-CSF is cut product, the B1 swimming lane is recombinant plasmid pPICz α A-3DHSA-mG-CSF, and the B2 swimming lane is that the enzyme of recombinant plasmid pPICz α A-3DHSA-mG-CSF is cut product.
The screening of the positive recombinant clone of Fig. 4 and evaluation, wherein A and B are respectively the bacterium colony trace result of pPICz α A-3DHSA-G-CSF and pPICz α A-3DHSA-mG-CSF, and C and D are respectively the SDS-PAGE detected result of pPICz α A-3DHSA-G-CSF and pPICz α A-3DHSA-mG-CSF.
Fig. 5 is that the Western blotting of fusion rotein identifies, 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 be induction time on the impact of fusion protein expression, figure A and figure B are respectively the SDS-PAGE result of 3DHSA-G-CSF and 3DHSA-mG-CSF, wherein M is Marker, 1～9 swimming lane is respectively that induction time is 24h, 36h, 48h, 60h, 72h, 84h, 96h, the result of 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 used, GS115 and pPICz α A are available from Invitrogen, and the structure of template plasmid pPICz α A-HSA-G-CSF (SEQ NO:7) is with reference to CN 01124114.4.
in each embodiment, plasmid extraction kit used and sepharose reclaim test kit available from the biochemical company limited of sky root (Beijing), human G-CSF Elisa test kit is (Shenzhen) Science and Technology Ltd. available from reaching section, various restriction enzymes, various archaeal dna polymerases, the T4DNA ligase enzyme, nucleic acid marker and small molecular weight protein marker are available from precious biotechnology (Dalian) company limited, primer is synthetic by the English Weihe River prompt base (Shanghai) trade Co., Ltd, Ji Saixin is available from North China Pharmacuetical Jintan Biotechnology Co., Ltd, restructuring HSA is available from the first biotech company of Wuhan standing grain, mouse anti human HSA and G-CSF antibody are available from U.S. Santa company, the anti-mouse IgG of HRP mark rabbit is available from biotech company of China fir Golden Bridge in Beijing, blue-sepharose filler and butyl dewatering filling are available from GE company, yeast powder and peptone are available from OXOID, YNB and bleomycin are available from Invitrogen, other reagent are analytical pure, available 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 ' holds with Xho I restriction enzyme site, CTCGAG), and primer SEQ NO:8.
P2:5 ' CCAGCGGGGTTAAGCCTAAGGCAGCTTGAC3 ', primer SEQ NO:9.
P3:5 ' ATGTGGGTGCTAAGCCTAAGGCAGCTTGAC3 ', primer SEQ NO: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 the upstream and downstream primer of 10 μ mol/L, the 3DHSA-mG10 the primer is P1 and P3), 2.5 the d NTP4 μ L of μ mol/L, 10 * Premistar buffer, 5 μ L, the Premistar archaeal dna polymerase 0.5 μ L of 5U/ μ L, pPICz α A-HSA-G-CSF plasmid (SEQ NO:7) 1 μ L, insufficient section is by sterilization distilled water polishing, reaction conditions is: 94 ℃ of denaturation 5min, 94 ℃ of sex change 1min, 56 ℃ of annealing 1min, 72 ℃ are extended 1min, repeat 25 circulations.
The amplification of H10-G-CSF and H10-mG-CSF, the primer is as follows:
P4:5 ' TTTGGGTTTGACCCCGCTGGGACCGGCAAG3 ', primer SEQ NO:11.
P5:5 ' CGT
CTCGAGTTAGGGCTGGGCAAGGTGGC3 ' (5 ' holds with Xho I restriction enzyme site, CTCGAG), and primer SEQ NO:12.
P6：5’TTTGGGTTTGGCACCCACATACAGAGCCAGCTCCCTGCCCCAGAGCTTCCTG
CTCAGAG 3 ', primer SEQ NO: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 the upstream and downstream primer of 10 μ mol/L, the H10-mG-CSF the primer is P6 and P5), 2.5 the d NTP4 μ L of μ mol/L, 10 * Premistar buffer5 μ L, the Premistar archaeal dna polymerase 0.5 μ L of 5U/ μ L, pPICz α A-HSA-G-CSF plasmid (SEQ NO:7) 1 μ L, insufficient section is by sterilization distilled water polishing, reaction conditions is: 94 ℃ of denaturation 5min, 94 ℃ of sex change 1min, 56 ℃ of annealing 1min, 72 ℃ are extended 1min, repeat 25 circulations.
Utilize overlapping extension PCR method respectively with 3DHSA-G10 and H10-G-CSF, 3DHSA-mG10 and H10-mG-CSF merge:
cut respectively glue and reclaim 3DHSA-G10, 3DHSA-mG10, the PCR product 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 the P1 of 10 μ mol/L and P5, 2.5 the d NTP4 μ L of μ mol/L, 10 * LA Taq buffer5 μ L, the LA Taq archaeal dna polymerase 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 be 3DHSA-mG10 and H10-mG-CSF) insufficient section is by the distilled water polishing of sterilizing, reaction conditions is: 94 ℃ of denaturation 5min, 94 ℃ of sex change 1min, 56 ℃ of annealing 1min, 72 ℃ are extended 1min30s, repeat 25 circulations.
By 1% agarose gel electrophoresis detection reaction product, the contrast DL2000, the application of sample swimming lane approximately the 1000bp size fusion gene band appears, as Fig. 2.Cut glue and reclaim the goal gene fragment, respectively goal gene fragment and the pPICz α A that reclaims carried out Xho I single endonuclease digestion, cut glue after agarose gel electrophoresis and reclaim fusion gene fragment and carrier segments, with the T4DNA ligase enzyme, above-mentioned recovery product is connected; Connect product and transform DH5 α competent cell, 37 ℃ of overnight incubation, single colony inoculation that picking transforms are 37 ℃ of cultivation 8h in 5mL LB liquid nutrient medium, and the extraction plasmid carries out enzyme and cuts evaluation, enzyme is cut gained size fragment and is coincide with carrier and fusion gene clip size respectively, as Fig. 3.Send Nanjing Si Pujin order-checking with above-mentioned clone, 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 strains
extract pPICz α A-3DHSA-G-CSF and pPICz α A-3DHSA-mG-CSF plasmid, carry out linearizing through Sal I single endonuclease digestion, agarose gel electrophoresis detects enzyme and cuts effect, utilize sepharose to reclaim test kit recovery enzyme and cut product, 1.5kV electric shock transforms the Pichia pastoris GS115 competent cell, 30 ℃ of standing 2h, conversion product is uniformly coated on 4 MD flat boards that contain the 1mol/L sorbyl alcohol, be inverted for 30 ℃ and cultivated 3 days, the clone's correspondence markings that transforms out is well transferred simultaneously on freshly prepd two MD flat boards, be inverted for 30 ℃ and cultivated 3 days, the pvdf membrane that uv irradiating is handled well is put down therein a MD flat-plate bacterial colony top gently, 2 of the filter paper that the placement uv irradiating is handled well on film simultaneously, with the 3mL anhydrous methanol, filter paper surface is all infiltrated, after 24h is cultivated in 30 ℃ of continuation, take out pvdf membrane and filter paper, according to Western blotting flow process, pvdf membrane is sealed successively, washing, hatch mouse anti human G-CSF antibody, washing, the anti-mouse Ig two of rabbit of hatching the HRP mark is anti-, washing and colour developing.(referring to Biotechnology122:181-184.1994 such as Scorer CA) is according to whether the positive recombinant bacterial strain of coloured bacterium colony circle preliminary judgement occurring, as Fig. 4 (A) and 4 (B).picking has corresponding single bacterium colony of bacterium colony circle formation and is inoculated in 10mLBMGY (30mg dipotassium hydrogen phosphate on another piece MD flat board, the 118mg potassium primary phosphate, 134mg is without amino substratum, the 100mg yeast powder, the 200mg peptone, 2 μ g vitamin Hs, 0.1mL glycerine, pH6.0) in liquid nutrient medium 30 ℃, 200r/min cultivates 24h, the centrifugal 5min of 2000r/min, abandon supernatant, with isopyknic BMMY (30mg dipotassium hydrogen phosphate, the 118mg potassium primary phosphate, 134mg is without amino substratum, the 100mg yeast powder, the 200mg peptone, 2 μ g vitamin Hs, 0.1mL anhydrous methanol, pH6.0) the resuspended thalline of liquid nutrient medium, 30 ℃, the every 12h of 200r/min adds the anhydrous methanol of 100 μ L to induce in BMMY, after cultivating 120h, the centrifugal 10min of 8000r/min, collect supernatant, carry out the SDS-PAGE checking, as Fig. 4 (C) and 4 (D).Contrast albumen Marker according to the thickness of protein electrophoresis band with bacterial strain called after GS115/pPICz α A-3DHSA-G-CSF and GS115/pPICz α A-3DHSA-mG-CSF that in positive colony, relative expression quantity is the highest.
The abduction delivering of embodiment 33DHSA-G-CSF and 3DHSA-mG-CSF fusion rotein and evaluation
With the GS115/pPICz α A-3DHSA-G-CSF that screens and the single colony inoculation of GS115/pPICz α A-3DHSA-mG-CSF to 100ml BMGY (0.3g dipotassium hydrogen phosphate is housed, 1.18g potassium primary phosphate, 1.34g be without amino substratum, the 1g yeast powder, the 2g peptone, 20 μ g vitamin Hs, 1mL glycerine is in 500ml Erlenmeyer flask pH6.0), 220r/min, cultivate 24h for 30 ℃, the centrifugal 5min of 2000r/min collects thalline.(0.3g dipotassium hydrogen phosphate, 1.18g potassium primary phosphate, 1.34g be without amino substratum, the 1g yeast powder with the BMMY of 100ml, the 2g peptone, 20 μ g vitamin Hs, 1mL anhydrous methanol, pH6.0) resuspended bacterial sediment, every 12h adds the 1mL anhydrous methanol, successive induction five days.The centrifugal 10min of 8000r/min collects supernatant, carries out SDS-PAGE and detects, and fusion protein expression improves constantly along with the prolongation of induction time, as Fig. 5 (A) and 5 (B).Adopt Western blotting to identify G-CSF and the HSA antigenicity of fusion rotein 3DHSA-G-CSF and 3DHSA-mG-CSF, set respectively G-CSF (referring to PNAS20:7292-7296 such as Yun Bai, 2005) and HSA as positive control (referring to 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
After crossing 0.45 μ m filter membrane, 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 dilutes three times with pure water, with the enrichment of blue-sepharose filler and purification of target albumen, the target protein peak of collecting is with butyl dewatering filling purified fusion protein, 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 are SEQ NO:14 and SEQ NO:15.Adopt the SDS-PAGE method that the purity of purifying gained fusion rotein is detected, as Fig. 7 (A) and 7 (B).
The short NFS-60 cell-proliferation activity of embodiment 53DHSA-G-CSF and 3DHSA-mG-CSF
Measure the short NFS-60 cell-proliferation activity of purifying gained fusion rotein 3DHSA-G-CSF and 3DHSA-mG-CSF with mtt assay.To go down to posterity and cultivate normal NFS-60 cell (U.S. ATCC) RPMI-1640 substratum centrifuge washing three times, cell will be pressed 1 * 10
5The density of individual/mL is suspended in the RPMI-1640 substratum that does not contain G-CSF that is rich in 5% horse serum, 5% foetal calf serum (V/V).Every hole adds 50 μ L cell suspensions in 96 orifice plates, establishes 6 multiple holes and cultivates, and except blank, adds the testing sample of different concns in every hole, chooses simultaneously the positive contrast medicine of Ji Saixin (recombinant human g-csf's trade(brand)name), and 37 ℃, 5%CO
2, after cultivating 48h under the 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, in wavelength 570nm place mensuration absorbancy, records measurement result.The activation analysis result shows: two kinds of fusion roteins all have the effect that dose-dependently stimulates NFS-60 cell proliferation, as Fig. 8.
The Half-life in vivo of embodiment 63DHSA-G-CSF and 3DHSA-mG-CSF is measured
Adopting the G-CSF final concentration is the body weight dosage of 1mg/kg, to subcutaneous Ji Saixin and the two kinds of fusion roteins injected respectively of ICR male mice in 6 ages in week, respectively at the 0.25h after administration, 0.5h, 1h, 2h, 4h, 8h, 12h, 24h and 36h carry out eye socket and get blood, after the standing 30min of normal temperature, 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 Concentration data input PKsolver is carried out data processing, the match plasma concentration curve.Result shows, the transformation period of 3DHSA-G-CSF and 3DHSA-mG-CSF is for being respectively 3.4h and 4.5h, is 1.6 times and 2.1 times of G-CSF, as Fig. 9.
Claims (6)
1. the fusion rotein of granulocyte colony-stimulating factor and mutant thereof and human serum albumin the 3rd structural domain is characterized in that it is the recombinant protein of being expressed by granulocyte colony-stimulating factor and mutant and human serum albumin the 3rd structural domain after gene level directly merges; The aminoacid sequence of described human serum albumin the 3rd structural domain is as shown in SEQ NO: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 thereof and human serum albumin the 3rd structural domain, it is characterized in that described fusion rotein aminoacid sequence comprises respectively SEQNO:1 and SEQNO:2, SEQNO:1 and SEQNO:3, or do not damage under the principle of fusion rotein characteristic, to the replacement of SEQ NO:2 and SEQ NO:3 partial amino-acid residue, lack or add the peptide molecule that obtains.
3. the fusion rotein of granulocyte colony-stimulating factor according to claim 1 and mutant thereof and human serum albumin the 3rd structural domain is characterized in that the aminoacid sequence of described granulocyte colony-stimulating factor and mutant aminoacid sequence thereof and SEQ NO:2 and SEQ NO:3 homology more than at least 85%.
4. the fusion rotein of granulocyte colony-stimulating factor according to claim 3 and mutant thereof and human serum albumin the 3rd structural domain is characterized in that the aminoacid sequence of described granulocyte colony-stimulating factor and mutant aminoacid sequence thereof and SEQ NO:2 and SEQ NO:3 homology more than at least 95%.
5. the fusion rotein of according to claim 1-4 described granulocyte colony-stimulating factors and mutant thereof 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 that the aminoacid sequence of the fusion rotein of the mutant of SEQNO:14, granulocyte colony-stimulating factor and human serum albumin the 3rd structural domain is SEQNO:15.
6. according to claim 1-5 any one described fusion rotein are in the application of preparation treatment granulocytopenia drugs with function.
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| CN106220724A (en) * | 2016-09-13 | 2016-12-14 | 河南师范大学 | Human fibroblastic growth factor 21 recombiant protein and its preparation method and application |
| CN106397607A (en) * | 2016-09-13 | 2017-02-15 | 河南师范大学 | Recombinant human fibroblast growth factor 21 fusion protein and application thereof in preparation of medicine for treating metabolic diseases |
| CN106432509A (en) * | 2016-09-13 | 2017-02-22 | 河南师范大学 | RhFGF-21(recombinant human fibroblast growth factor-21) fusion protein capable of treating metabolic diseases as well as preparation method and application of rhFGF-21 fusion protein |
| CN106432509B (en) * | 2016-09-13 | 2019-05-21 | 河南师范大学 | A kind of 21 fusion protein of recombinant human fibroblast growth factor and its preparation method and application for treating metabolic disease |
| CN106220724B (en) * | 2016-09-13 | 2019-10-11 | 河南师范大学 | 21 recombinant protein of human fibroblastic growth factor and its preparation method and application |
| CN109748969A (en) * | 2017-11-08 | 2019-05-14 | 兰州大学 | A kind of Dimerized fusion protein and its preparation method and application |
| CN109748969B (en) * | 2017-11-08 | 2023-08-08 | 兰州大学 | Dimerization fusion protein and preparation method and application thereof |
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| CN103102418B (en) | 2015-11-25 |
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