WO2015172752A9 - 血小板衍生生长因子b突变体、其制备方法及用途 - Google Patents

血小板衍生生长因子b突变体、其制备方法及用途 Download PDF

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WO2015172752A9
WO2015172752A9 PCT/CN2015/084260 CN2015084260W WO2015172752A9 WO 2015172752 A9 WO2015172752 A9 WO 2015172752A9 CN 2015084260 W CN2015084260 W CN 2015084260W WO 2015172752 A9 WO2015172752 A9 WO 2015172752A9
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platelet
growth factor
derived growth
mutant
pdgf
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PCT/CN2015/084260
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French (fr)
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WO2015172752A3 (zh
WO2015172752A2 (zh
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陈薇
张晓鹏
于长明
付玲
戴萌萌
张军
徐俊杰
侯利华
李建民
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中国人民解放军军事医学科学院生物工程研究所
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Priority to AU2015258537A priority Critical patent/AU2015258537B2/en
Priority to KR1020167035429A priority patent/KR20170069177A/ko
Priority to KR1020197019647A priority patent/KR102155903B1/ko
Priority to EP15793254.2A priority patent/EP3178844B1/en
Priority to JP2017512094A priority patent/JP6643322B2/ja
Priority to US15/311,590 priority patent/US10450358B2/en
Publication of WO2015172752A2 publication Critical patent/WO2015172752A2/zh
Publication of WO2015172752A3 publication Critical patent/WO2015172752A3/zh
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    • C07K14/475—Growth factors; Growth regulators
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Definitions

  • the present invention relates to a platelet-derived growth factor B derivative, and in particular, to a platelet-derived growth factor B mutant, a nucleic acid molecule encoding the mutant, a vector containing the nucleic acid molecule, and a host cell.
  • the present invention also relates to a method for producing the mutant and a method for purifying the same, and the use of the mutant for the preparation of a medicament for promoting cell division, proliferation, promoting wound healing, skin regeneration, bone and tooth defect regeneration, and joint repair.
  • Platelet-derived growth factor is a polypeptide that can produce a variety of cells and stimulate the proliferation of mesenchymal-derived cells. It was first discovered by thrombocytes in the 1970s, hence the name ( 1). So far, four PDGF monomers PDGF-A, PDGF-B, PDGF-C and PDGF-D have been found. These monomers form five homo- or heterodimers through intrachain and interchain disulfide bonds: PDGF-AA, PDGF-BB, PDGF-AB, PDGF-CC, and PDGF-DD (2,3) .
  • PDGF genes and proteins belong to a family of structurally and functionally related growth factors, including vascular endothelial growth factor (VEGFs) and placental growth factor (PIGF) (4).
  • VEGFs vascular endothelial growth factor
  • PIGF placental growth factor
  • PDGF-R includes both PDGFR- ⁇ and PDGFR- ⁇ and belongs to the tyrosine kinase receptor. Binding of the ligand to the receptor initiates dimerization of the receptor monomer, which promotes autophosphorylation of the tyrosine residue in the intracellular region.
  • Two receptors can activate multiple signaling pathway key molecules, such as Ras-MAPK, PI3K and PLC- ⁇ (5), thereby activating the transcription of related genes, stimulating cell growth, inhibiting apoptosis, promoting differentiation, causing directional movement and migration. Etc., to play a variety of biological functions.
  • the PDGF-b gene is located on chromosome 22 and contains seven exon genes encoding a precursor protein of 241 amino acids.
  • the final mature product formed by protease hydrolysis is composed of 109 amino acids with a molecular weight of 12.3 kD. Peptide.
  • the active form of the PDGF-B protein is the formation of homologous PDGF-BB or heterodimeric PDGF-AB (6) by disulfide bonding of two monomers.
  • Each PDGF-B protein monomer contains eight highly conserved cysteine residues, of which six cysteines form an intrachain disulfide bond (Cys I-VI, III-VII, V-VIII) The other two intersect with the corresponding monomers to form an interchain disulfide bond (Cys II-IV) (7) to form a growth factor domain-cysteine knot characteristic of the PDGF protein family.
  • These intrachain and interchain disulfide bonds constitute the complex spatial structure of the PDGF-BB dimer protein (Fig. 1B).
  • PDGF proteins also have different splicing forms during expression synthesis, which makes the processing mature.
  • the PDGF protein exhibits a variety of structural forms. Purification of purified PDGF-BB from human platelet extracts revealed by N-terminal amino acid sequence analysis that at least three different splicing forms, 20% Ser1, 45% Thr6 and 35% Thr33, resulted in heterogeneity of these cleavage The proportion of proteins in various cut forms was uncontrollable when purifying PDGF-BB (8).
  • the inventors of the present invention have conducted extensive studies and found that protease degradation and/or glycosylation modification at a specific site is the main cause of the presence of various PDGF-B, and the PDGF-B mutant is obtained by site mutation, and the protein is uniform. The sex is greatly improved and the activity of the PDGF-B protein is retained.
  • a first aspect of the invention relates to a platelet-derived growth factor B mutant having a mutation at the 101st and 109th amino acid positions of wild-type platelet-derived growth factor B (herein the description of the position of the amino acid site is included
  • the mature PDGF-B of 109 amino acid residues is based on the same, and has the activity of platelet-derived growth factor B.
  • a platelet-derived growth factor B mutant according to any one of the first aspects of the present invention, which has a mutation at amino acid position 6 and has platelet-derived growth factor B activity.
  • a platelet-derived growth factor B mutant according to any one of the first aspects of the present invention, which has a mutation at the 32nd and/or 33rd amino acid position and has platelet-derived growth factor B activity.
  • the platelet-derived growth factor B mutant according to any one of the first aspects of the present invention has a N-terminal deletion of 5 amino acids and has platelet-derived growth factor B activity as compared with wild-type platelet-derived growth factor B.
  • the mutant is mutated to alanine at the 6th, 101st and 109th amino acid positions.
  • the mutant is mutated to alanine at amino acid positions 101 and 109.
  • a platelet-derived growth factor B mutant according to any one of the first aspects of the invention, which is mutated to a proline, a valine or an isoleucine at the 32nd and/or 33rd amino acid position.
  • valine in one embodiment, it is mutated to a valine, valine or isoleucine at position 32, preferably valine.
  • the platelet-derived growth factor B mutant has a N-terminal deletion of 5 amino acids, and the 6th, 101st, and 109th amino acids are mutated to alanine, and the 32nd The amino acid mutation is valine.
  • the platelet-derived growth factor B mutant is characterized in that the N-terminal deletion of 5 amino acids, the 101st and 109th amino acids are mutated to alanine, and the 32nd amino acid mutation is Proline.
  • the platelet-derived growth factor B mutant has a N-terminal deletion of 5 amino acids, and the 6th, 101st, and 109th amino acids are mutated to alanine, and the 32nd The amino acid mutation is valine.
  • the platelet-derived growth factor B mutant has a N-terminal deletion of 5 amino acids, and the 6th, 101st, and 109th amino acids are mutated to alanine, and the 32nd The amino acid is mutated to isoleucine.
  • the invention also includes combinations of the various technical solutions described above.
  • amino acid sequence of the mutant is the sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 5.
  • the platelet-derived growth factor B mutant according to any one of the first aspects of the present invention, wherein the amino acid sequence is substituted, deleted or added with one or several amino acids, and has platelet-derived growth factor B activity.
  • the platelet-derived growth factor B amino acid sequence is subjected to one or several amino acid substitutions, deletions and/or additions at other non-critical sites. Mutants that derive growth factor B activity are also within the scope of the invention.
  • a second aspect of the invention relates to a platelet-derived growth factor homologous or heterodimer comprising two intracellular and/or interchain disulfide bonds of the platelet-derived growth factor B mutant of any one of the first aspects of the invention
  • a combination of a platelet-derived growth factor B mutant according to any one of the first aspects of the invention and a platelet-derived growth factor A is formed by intrachain and/or interchain disulfide bonding.
  • the platelet-derived growth factor homologous or heterodimer is formed in the same manner as the wild-type platelet-derived growth factor.
  • the two platelet-derived growth factor B mutants of any of the first aspects of the invention are bound by intrachain and interchain disulfide bonds to form a PDGF-BB mutant.
  • a third aspect of the invention relates to a nucleic acid molecule encoding the platelet-derived growth factor B mutant of any of the first aspects of the invention.
  • nucleic acid molecule according to any one of the third aspects of the present invention, wherein the nucleotide sequence is selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6 to SEQ ID NO: 9.
  • a fourth aspect of the invention relates to a vector comprising the nucleic acid molecule of any of the third aspects of the invention.
  • an expression vector depending on the host cell used for expression, for example, a vector suitable for expression of yeast cells or mammalian cells can be selected.
  • the vector is pMEX9K.
  • a fifth aspect of the invention relates to a host cell comprising the vector of any of the fourth aspects of the invention.
  • a host cell according to any one of the fifth aspects of the invention which is a eukaryotic cell, such as a yeast cell, a mammalian cell or an insect cell.
  • the host cell according to any one of the fifth aspects of the present invention, which is, for example, Pichia pastoris (also called Pichia pastoris cell), Saccharomyces cerevisiae, Kluyveromyces ( Kluyveromyces lactis), Hansenula, Candida or Torulopsis.
  • Pichia pastoris also called Pichia pastoris cell
  • Saccharomyces cerevisiae Saccharomyces cerevisiae
  • Kluyveromyces Kluyveromyces lactis
  • Hansenula Candida or Torulopsis.
  • the Pichia cell is a GS115 cell.
  • the mammalian cell is, for example, a CHO cell, a BHK cell, an NSO cell, an SP2/0 cell, a HEK-293 cell, a COS cell or the like.
  • the present invention also relates to a method for producing a platelet-derived growth factor B mutant according to any one of the first aspects of the present invention, which comprises culturing, expressing (e.g., inducing expression), and optionally selecting a host cell according to any one of the fifth aspects of the present invention.
  • the purification step comprises culturing, expressing (e.g., inducing expression), and optionally selecting a host cell according to any one of the fifth aspects of the present invention.
  • a preparation method according to any one of the inventions comprising the steps of:
  • the host cell according to any one of the fifth aspects of the present invention is inoculated into a culture medium and subjected to stepwise amplification culture;
  • a preparation method according to any one of the inventions, characterized by one or more of the following:
  • the host cell in step 1) is a monoclonal cell strain
  • the stepwise amplification culture described in the step 1) refers to two-stage amplification culture, the culture temperature is 28-30 ° C, and each stage is cultured to an OD 600 of 1-12, for example 2-6;
  • step 2 The temperature induced in step 2) is about 28 ° C;
  • the final concentration of methanol in step 2) is 0.3-1.0% (v/v), such as 0.4-0.8% (v/v), such as 0.5% (v/v);
  • the time of induction of expression in step 2) is 48-96 h, for example 72 h;
  • the purification step in the step 3) includes hydrophobic chromatography, ion exchange chromatography, and gel chromatography in this order.
  • a sixth aspect of the present invention relates to a method for purifying a platelet-derived growth factor B or a mutant thereof, which comprises subjecting a culture supernatant or a cell lysate containing platelet-derived growth factor B or a mutant thereof to hydrophobic chromatography and ion exchange chromatography in sequence. And the step of gel chromatography.
  • the purification method according to any one of the sixth aspect of the invention wherein the platelet-derived growth factor B mutant is the platelet-derived growth factor B mutant according to any one of the first aspects of the invention.
  • the column medium for hydrophobic chromatography is Phenyl Sepharose 6 Fast Flow.
  • the chromatographic medium used for ion exchange chromatography is Source 30S.
  • the chromatographic medium for gel chromatography is Hiload Superdex 75 prep grad.
  • the hydrophobic chromatography comprises the following steps:
  • the culture supernatant or cell lysate containing platelet-derived growth factor B or a mutant thereof is adjusted with a regulatory buffer, and the final system after the addition of the adjustment buffer is 10-50 mM phosphate buffer, 0.8-1 M. (NH 4 ) 2 SO 4 , pH 6.8-7.5;
  • the equilibration buffer is formulated as 10-50 mM phosphate buffer, 0.8-1 M (NH 4 ) 2 SO 4 , pH 6.8-7.5;
  • the elution buffer is formulated as 10-50 mM phosphate buffer, 30%-50% ethylene glycol, pH 6.8-7.5;
  • the ion exchange chromatography comprises the following steps:
  • the equilibration buffer is formulated, 10-50 mM phosphate buffer, pH 6.8-7.5;
  • the elution buffer is formulated as 10-50 mM phosphate buffer, 0.8-1.2 M NaCl, pH 6.8-7.5;
  • the gel chromatography comprises the following steps:
  • the phosphate buffer is formulated as 10-50 mM phosphate buffer, 0.1-0.5 M NaCl, pH 6.8-7.5;
  • each loading volume does not exceed 0.3-4% (for example, 3%) of the column volume
  • the column is washed with the phosphate buffer in the step (1) to collect the protein of interest, thereby obtaining purified platelet-derived growth factor B or a mutant thereof.
  • the formulation of the phosphate buffer is well known in the art.
  • the phosphate buffer was 20mMPB solution containing 0.0144mol / L of Na 2 HPO 4 and 0.0056mol / L of NaH 2 PO 4, pH 6.8-7.5.
  • the formulation of the phosphate buffer is well known in the art.
  • the phosphate buffer is a PBS solution formulated as 10-50 mM PB solution, 0.15 M NaCl, pH 6.8-7.5.
  • the buffer of each step of chromatography has a pH of 7.2.
  • the concentration of the phosphate buffer in each step of chromatography is 20 mM.
  • the hydrophobic chromatography method is: (1) the yeast expression supernatant is adjusted for conductivity with 1/2 volume of conditioning buffer (60 mM PB, 3M (NH 4 ) 2 SO 4 , pH 7.2); (2) equilibrate the column with equilibration buffer (20 mM PB, 1 M (NH 4 ) 2 SO 4 , pH 7.2); (3) after the sample is applied to the column, wash the column with equilibration buffer to the baseline straight; (4) wash The target protein was collected by elution with a de-buffer (20 mM PB, 50% ethylene glycol, pH 7.2).
  • the ion exchange chromatography method is: (1) diluting the Phenyl HS elution peak with an equilibrium buffer (20 mM PB, pH 7.2) to a conductivity of 6 mS/cm or less; (2) using equilibrium The buffer equilibrates the column; (3) after the sample is applied to the column, the column is washed with equilibration buffer to a straight baseline; (4) The target protein is collected by elution with a gradient of elution buffer (20 mM PB, 1 M NaCl, pH 7.2).
  • the gel chromatography method is: (1) equilibrating the column with PBS buffer (20 mM PB, 0.15 M NaCl, pH 7.2); (2) loading the source 30S elution peak with loop , each injection volume does not exceed 3% of the column volume; (3) continue to wash the column with PBS buffer to collect the protein of interest.
  • the present invention also relates to the platelet-derived growth factor B mutant of any one of the first aspects of the present invention It is used for the preparation of a medicament for promoting cell division, proliferation, promoting wound healing, skin regeneration, bone and tooth defect regeneration, and joint repair.
  • the invention further relates to a method of promoting cell division, proliferation, promoting wound healing, skin regeneration, bone and tooth defect regeneration, joint repair, the method comprising administering to a subject in need thereof an effective amount of any of the first aspects of the invention The step of a platelet-derived growth factor B mutant.
  • the invention also relates to an antibody which is capable of specifically binding to a platelet-derived growth factor B mutant of any of the first aspects of the invention.
  • the present invention also relates to a method for more uniform expression of platelet-derived growth factor, comprising the step of modifying the amino acid sequence of a wild-type platelet-derived growth factor, the modification comprising one of the following a)-c) or Several items:
  • expression is carried out using a eukaryotic expression system, such as a yeast cell expression system, a mammalian cell expression system.
  • a eukaryotic expression system such as a yeast cell expression system, a mammalian cell expression system.
  • the modification refers to the deletion of 5 amino acids at the N-terminus, the mutation of the 6th, 101st and 109th amino acids to alanine, and the mutation of the 32nd amino acid to the ammonia acid.
  • the modification refers to the deletion of 5 amino acids at the N-terminus, the amino acid at positions 101 and 109 being mutated to alanine, and the amino acid at position 32 being mutated to valine.
  • the modification refers to the deletion of 5 amino acids at the N-terminus, the mutation of the 6th, 101st and 109th amino acids to alanine, and the mutation of the 32nd amino acid to the ammonia acid.
  • the modification refers to the deletion of 5 amino acids at the N-terminus,
  • the amino acids at positions 6, 101 and 109 are mutated to alanine, and the amino acid at position 32 is mutated to isoleucine.
  • the amino acid sequence of the engineered platelet-derived growth factor is the sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 5.
  • the invention also includes combinations of the various technical solutions described above.
  • the vector is, for example, a cloning vector or an expression vector.
  • the vector contains the nucleic acid molecule of the present invention, which can be obtained, for example, by inserting the above nucleic acid molecule into a cloning vector or an expression vector, or can be obtained by artificial synthesis.
  • the expression vector is, for example, a prokaryotic expression vector, a eukaryotic expression vector, a phage vector or a viral vector.
  • the prokaryotic expression vector is, for example, a pET vector, a pGEX vector
  • the eukaryotic expression vector is, for example, pcDNA3.1, pEGFP-C1, pPIC9K, pMEX9K, pPICZ, pPICZa, pFastBac, pPIC6aA, pPIC3.5K, pGAPZaA, pAO815,
  • the phage vector is, for example, a lambda phage vector ⁇ gt, ⁇ gt- ⁇ B
  • the viral vector is, for example, a retrovirus, a lentivirus, an adenovirus or an adeno-associated virus vector.
  • the vector is pMEX9K.
  • the host cell may be a prokaryotic cell (such as an E. coli cell) or a eukaryotic cell.
  • the eukaryotic cell is, for example, a yeast cell, a mammalian cell or an insect cell.
  • the host cell can be obtained by introducing/transfecting a nucleotide sequence or a vector of the above nucleic acid molecule into a prokaryotic cell or a eukaryotic cell.
  • the yeast cell strain is well known in the art and includes, but is not limited to, X33, KM71, KM71H, GS115, SMD1168, SMD1163, SMD1168H, SMD1163H.
  • a host cell transfected with a specific nucleic acid or vector can be obtained by any kind of transfection method known in the art, for example, a nucleic acid can be introduced into a cell by electroporation or microinjection; or, Lipid transfection reagents such as FuGENE 6, X-tremeGENE and LipofectAmine; alternatively, nucleic acids can be introduced into cells by appropriate viral viral vectors based on retroviruses, lentiviruses, adenoviruses and adeno-associated viruses.
  • the expression homogenization means that when the electrophoresis loading amount is not less than 10 ⁇ g, the target protein is stained by Coomassie brilliant blue into a single band by using reduced SDS-PAGE electrophoresis, and software such as Image J or Bandscan. Analytical purity >95%. Those skilled in the art can make the expression of the protein tend to be more uniform by exploring various techniques for different proteins.
  • the wild-type platelet-derived growth factor B refers to an unmutated platelet-derived growth factor B having a length of 109 amino acids; and for each point (eg, a mutation site, The positional description of glycosylation sites and restriction sites is also based on wild-type platelet-derived growth factor B.
  • the amino acid at position 6, 101, and 109 of the wild-type platelet-derived growth factor B is threonine (Thr); the amino acid at position 32 and the amino acid at position 33 are respectively arginine. (Arg); the first five amino acids of the N-terminus of wild-type platelet-derived growth factor B are serine (Ser), leucine (Leu), glycine (Gly), serine (Ser), and leucine (Leu).
  • the term "about” means in the range of from 90% to 110% of the value, for example, in the range of from 95% to 105%.
  • the present invention obtains a PDGF-B mutant by mutation, which has greatly improved protein homogeneity and still retains the activity of the PDGF-B protein.
  • FIG. 1 Schematic diagram of PDGF-B protein structure.
  • A the PDGF-B precursor protein hydrolyzes the N-terminal signal peptide, propeptide and C-terminal propeptide sequence by protease to become a mature protein.
  • ⁇ indicates protease hydrolysis site;
  • B two PDGF-B monomers form PDGF-BB homodimers through interchain disulfide bonds, each PDGF-B monomer contains 8 Cys, respectively forming 3 pairs of chains Disulfide bonds (C16-C60, C49-C97, C53-C99) and two pairs of interchain disulfide bonds (C43-C52, C52-C43).
  • SP signal peptide (PROP);
  • PRO the pro-sequence preceding the growth factor domain.
  • FIG. 1 SDS-PAGE electrophoresis analysis of PDGF-BB secreted by Pichia pastoris.
  • Three different batches of the purified recombinant PDGF-BB Thr6 were subjected to non-reducing conditions (left) and reducing conditions (right) SDS-PAGE analysis. Under non-reducing conditions, the protein is a single band; after DTT treatment, PDGF-B monomer exhibits multiple forms of molecular weight non-uniformity.
  • FIG. 3 the combination of proteolysis and glycosylation modification results in the formation of PDGF-B Thr6 multiple monomers.
  • A N-terminal amino acid sequence analysis of PDGF-B Thr6 protein by SDS-PAGE under reducing conditions. The first five amino acid residues at the N-terminus of the first, second, and third bands are all TIATP, and the first five amino acid residues at the N-terminus of the fourth and fifth bands are TNANF, indicating that protease cleavage occurs in Arg32-Thr33.
  • B Analysis of WB (middle) and glycoprotein staining (right) of PDGF-B Thr6 protein under reducing conditions.
  • the WB test strip corresponds to the 3rd and 5th bands of Coomassie blue staining (left); the glycoprotein stained strip corresponds to the first, second and fourth bands of the test.
  • C Treatment of PDGF-B Thr6 protein under reducing conditions with PNGase F and glycoprotein staining. There was no change in the banding type and molecular weight before and after treatment with PNGase F (left), and there was no difference in glycoprotein staining results; IFN- ⁇ was a positive control for glycosidase digestion and glycoprotein staining.
  • Figure 6 shows the post-translational modification site analysis of PDGF-B expressed by Pichia pastoris.
  • A Schematic diagram of PDGF-M1 and PDGF-M2 mutant site mutations.
  • B The WB assay showed that the PDGF-M1 and PDGF-M2 monomers were single bands, and the control PDGF-BThr6 was two.
  • C SDS-PAGE was used to detect PDGF-M1 and PDGF-M2 monomers. The results of Coomassie blue staining showed that PDGF-M2 was a single protein band, and PDGF-M1 was a two-protein band (as indicated by the arrow in the figure). .
  • D Glycoprotein staining almost no PDGF-M1 and PDGF-M2 protein monomers were detected.
  • Figure 8 Arg32 is the effect of mutation on expression.
  • A SDS-PAGE results of seven clones of the codon-optimized strains PDGF-IM-P, PDGF-IM-V and PDGF-IM-I. The protein expression of PDGF-IM-P was significantly higher than the other two strains.
  • B Screening for multi-insertion copies of codon-optimized strains PDGF-IM-P, PDGF-IM-V, and PDGF-IM-I by G418 resistance, respectively, and selecting G418 concentrations of 2.0 mg/ml and 4.0 mg/ Six clones of strains were screened for expression in ml under ml conditions. The results of SDS-PAGE showed that the expression of PDGF-IM-P high copy screening strain was significantly higher than the other two strains.
  • Figure 9 LC/MS map of PDGF-B wild type and PDGF-M2 mutants.
  • Genbank number of the wild type PDGF-B amino acid sequence is NM-002608.2.
  • the rhPDGF-BB Thr6 amino acid sequence is:
  • the rhPDGF-BB Thr6 nucleic acid sequence is:
  • the DNA sequences encoding various PDGF mutants were synthesized by Shanghai Biotech.
  • the gene fragment was cloned into the expression vector pMEX9K (see patent ZL02117906.9) by restriction enzyme sites XhoI and EcoRI, and confirmed by sequencing.
  • the recombinant plasmid was extracted and linearized by SalI digestion, and then transformed into Pichia pastoris expression strain GS115 competent cells by electroporation.
  • Yeast transformants were screened by histidine-deficient MD plates, and positive recombinant yeast strains were identified by PCR.
  • the recombinant yeast strain was inoculated into a medium containing 25 mL of BMGY medium (BMGY medium formula: weighing 10 g of yeast extract powder, 20 g of tryptone, dissolved in 700 ml of water, autoclaved at 121 ° C for 20 min; cooled to room temperature, and added with 100 ml of 1 M phosphoric acid. Potassium buffer, 100ml 10 ⁇ YNB, 100ml 10 ⁇ GY, stored at 4°C.
  • BMGY medium formula: weighing 10 g of yeast extract powder, 20 g of tryptone, dissolved in 700 ml of water, autoclaved at 121 ° C for 20 min; cooled to room temperature, and added with 100 ml of 1 M phosphoric acid.
  • Potassium buffer 100ml 10 ⁇ YNB, 100ml 10 ⁇ GY, stored at 4°C.
  • 10 ⁇ YNB 13.4% yeast nitrogen source
  • 10 ⁇ GY 10% glycerol
  • 1M potassium phosphate buffer 132ml 1M K 2 HPO 4
  • 868ml 1M KH 2 PO 4 adjusted to pH 6.0 ⁇ 0.1 with phosphoric acid or KOH, autoclaved at 121°C for 30min, stored at room temperature.
  • the induction temperature was 28 ° C and the shaking speed was 220 rpm; methanol was added every 24 hours to a final concentration of 0.5% for a total of 72 hours.
  • the supernatant containing the recombinant protein was collected by centrifugation at room temperature at 7000 rpm after the end of induction.
  • the supernatant of Pichia pastoris was centrifuged, filtered, and adjusted to a suitable buffer, followed by hydrophobic chromatography (Phenyl Sepharose 6 Fast Flow), ion exchange chromatography (Source 30S), and gel chromatography (Hiload Superdex 75 prep). Grad) A protein of >95% purity was obtained (Fig. 5).
  • the chromatographic media are products of GE Amersham Bioscience.
  • the hydrophobic chromatography method is: (1) the yeast expression supernatant is adjusted with 1/2 volume of conditioning buffer (60 mM PB, 3M (NH 4 ) 2 SO 4 , pH 7.2); (2) according to the method of the specification, with equilibrium The buffer (20 mM PB, 1 M (NH 4 ) 2 SO 4 , pH 7.2) equilibrates the column; (3) after the sample is applied to the column, the column is washed with equilibration buffer to the baseline; (4) with elution buffer (20 mM) PB, 50% ethylene glycol, pH 7.2) eluted to collect the protein of interest.
  • conditioning buffer 60 mM PB, 3M (NH 4 ) 2 SO 4 , pH 7.2
  • the ion exchange chromatography method is: (1) diluting the Phenyl HS elution peak with an equilibrium buffer (20 mM PB, pH 7.2) to a conductivity of 6 mS/cm or less; (2) equilibrating the column with an equilibration buffer according to the method described; (3) After the sample was applied to the column, the column was washed with an equilibration buffer to a baseline; (4) A gradient of elution buffer (20 mM PB, 1 M NaCl, pH 7.2) was used to collect the protein of interest.
  • the gel chromatography method is: (1) equilibrate the column with PBS buffer (20 mM PB, 0.15 M NaCl, pH 7.2); (2) load the source 30S elution peak with loop, each time the sample volume does not exceed the column 3% by volume; (3) continue to wash the column with PBS buffer to collect the protein of interest.
  • Sample preparation was the same as SDS-PAGE. 3 ⁇ l samples were taken for SDS-PAGE. After electrophoresis, the cells were transferred to a nitrocellulose membrane by a steady flow of 300 mA for 1 h, and 5% skim milk powder/TBST was blocked at room temperature for 1 h. 1:1000 diluted primary antibody PDGF-B (F-3) (Santa Cruz Biotechnology, SC-365805), coated at room temperature for 1 h, washed TBST multiple times, 1:10000 diluted HRP-labeled secondary antibody (Cell Signaling Technology, # 7076) Incubation for 1 h at room temperature, after TBST washing, substrate was added and imaged using the LAS400mini Gel Imaging System (GE).
  • F-3 primary antibody PDGF-B
  • HRP-labeled secondary antibody Cell Signaling Technology, # 7076
  • the sample was heated at 100 ° C to inactivate the enzyme, and then subjected to SDS-PAGE electrophoresis. After the end of the electrophoresis, staining was performed using a glycoprotein staining kit (Themo Scientific, #24562).
  • BALB/C 3T3 cells (purchased from Peking Union Cell Resource Center) were cultured in DMEM complete medium (Life Technology) containing 10% FBS at 37 ° C under 5% carbon dioxide. After digesting and collecting the cells, a cell suspension containing 5.0 ⁇ 10 4 cells per ml was prepared in a complete medium, inoculated into a 96-well cell culture plate, 100 ⁇ l per well, and culture was continued at 37° C. under 5% carbon dioxide. After 24 hours, the medium was replaced with maintenance medium (DMEM containing 0.4% FBS), and the culture was continued at 37 ° C under 5% carbon dioxide; after 24 hours of culture, the maintenance medium was discarded, and the recombinant PDGF-BB solution diluted in advance was added.
  • DMEM complete medium Life Technology
  • Example 1 The combination of proteolysis and glycosylation resulted in the formation of PDGF-BB Thr6 monomers.
  • PNGase F peptide N-glycosidase F
  • SDS-PAGE electrophoresis and glycoprotein staining analysis PNGase F
  • PNGaseF is an amidase that acts on almost all N-glycan chains in glycopeptides/glycoproteins, cleaves between the innermost GlcNAc and asparagine residues in the sugar chain portion, and asparagine It becomes aspartic acid (10), which is the most widely used enzyme for identifying N-glycoprotein in glycoproteomics research.
  • the recombinant IFN- ⁇ protein expressed by Pichia pastoris acts as a positive control for N-glycosylated protein.
  • Coomassie blue staining showed that the relative molecular weight of PDGF-B protein did not change before and after enzyme digestion, indicating that the PDGF-B protein did not undergo N-glycosylation (Fig. 3C, left).
  • the results of glycoprotein staining indicate that PDGF-B is indeed a glycoprotein (Fig. 3C, right). This means that PDGF-B secreted by Pichia pastoris has undergone O-glycosylation modification.
  • the complete PDGF in the table refers to PDGF-B Thr6 , which is PDGF-B with 5 amino acids deleted at the N-terminus.
  • the predicted potential glycosylation sites at positions 6, 101, and 109 are consistent with our results: the C-terminus of the PDGF-B Thr6 protein should have glycosylation modifications (Thr101, Thr109) because they Blocks binding to antibodies; there should be a glycosylation modification site before Thr33, as this will explain only one (Band 4) glycosylation-modified variant of the PDGF-B band that undergoes enzymatic hydrolysis, but not There are two glycosylated PDGF-B monomers that undergo enzymatic hydrolysis (Band 1, 2) (Fig. 3B; Table 1). To confirm the predicted results, we constructed two PDGF-B Thr6 mutants, PDGF-M1 and PDGF-M2.
  • the amino acid sequence of PDGF-M1 is:
  • the nucleotide sequence is:
  • the amino acid sequence of PDGF-M2 is:
  • the nucleotide sequence is:
  • the DNA sequences encoding PDGF-M1 and PDGF-M2 were inserted into the pMEX9K expression vector and integrated into the GS115 Pichia strain. Expression was induced by methanol and purified by chromatography. The purified PDGF-B protein was subjected to SDS-PAGE, glycoprotein staining and Western immunoblotting to determine the protein properties after the modification. The WB results showed that only two bands were detected after reduction of the two mutants, and the relative molecular mass was about 12 kDa, which was consistent with expectations (Fig. 6B). SDS-PAGE results showed that PDGF-M2 was a single band, but PDGF-M1 still had a weak band on the main band (Fig. 6C).
  • PDGF-M2 In order to increase the expression level of PDGF-M2, we used codon optimization of PDGF-M2 (ie PDGF-IM-P) using the online tool JAVA Condon Adaptation Tool, based on the preference of P. pastoris for codon selection during protein expression.
  • the optimized coding DNA sequence is as follows:
  • the protein sequence is identical to PDGF-M2.
  • Arg32 was mutated to Val (PDGF-IM-V, Val codon using GTT) and Ile (PDGF-IM-I, Ile codon using ATC), and PDGF-M2 expression. The analysis was performed horizontally to analyze the effect of Arg32 mutations on protein expression.
  • the nucleotide sequence of PDGF-IM-V is:
  • the nucleotide sequence of PDGF-IM-I is:
  • the DNA sequence encoding the PDGF-IM-P, PDGF-IM-V, PDGF-IM-I, ligase cleavage site, terminator and the like were cloned into the pMEX9K expression vector and integrated into the GS115 expression strain. After screening with histidine-deficient MD plates, 9 clones were randomly selected and subjected to methanol-induced expression in vitro. The culture supernatant was analyzed by SDS-PAGE and the PDGF-IM-P protein expression was significantly higher than the other two strains (Fig. 8A).
  • Recombinant PDGF-B wild-type and PDGF-M2 mutants were reduced with DTT (2.5 mM) for 30 min at 37 ° C, followed by dilution with buffer A (aqueous solution containing 0.1% formic acid), followed by liquid chromatography and mass spectrometry Analysis by (LC/MS).
  • Protein separation was performed on an Easy-spray column (15 cm x 75 ⁇ m ID, 3- ⁇ m C18 particles) using an EASY-nLC system (Thermo Fisher Scientific) using a linear gradient of Buffer B (0.1% formic acid in methanol; 0 -90%, 20 min) elution at a flow rate of 300 nl/min.
  • High resolution spectra were obtained using a Q Exactive Mass Spectrometer (Thermo Fisher Scientific) at a resolution of 60,000, m/z 350-1600, and deconvoluted using Xtract software (Thermo Scientific).

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Abstract

提供了血小板衍生生长因子B突变体、其编码的核酸分子、含有该核酸分子的载体和宿主细胞，还提供了所述突变体的制备方法及其用于制备促进细胞分裂、增殖、促进伤口愈合、皮肤再生、骨骼与牙齿缺损再生、关节修复的药物中的用途。

Description

血小板衍生生长因子B突变体、其制备方法及用途 技术领域
本发明涉及血小板衍生生长因子B衍生物，具体地，本发明涉及血小板衍生生长因子B突变体，编码该突变体的核酸分子，含有该核酸分子的载体和宿主细胞。本发明还涉及所述突变体的制备方法和纯化方法，以及所述突变体用于制备促进细胞分裂、增殖、促进伤口愈合、皮肤再生、骨骼与牙齿缺损再生、关节修复的药物中的用途。
背景技术
血小板衍生生长因子(Platelet-derived growth factor，PDGF)是一种能多种细胞产生的、能够刺激间质来源细胞增殖的多肽，上世纪70年代首先由Ross等从血小板中发现，因而得名(1)。到目前为止，共发现4种PDGF单体PDGF-A、PDGF-B、PDGF-C和PDGF-D。这些单体彼此通过链内及链间二硫键，形成5种同源或异源二聚体：PDGF-AA、PDGF-BB、PDGF-AB、PDGF-CC和PDGF-DD(2,3)。普遍认为PDGF基因和蛋白属于一类在结构与功能上相关的生长因子家族，这个家族还包括血管内皮生长因子(VEGFs)以及胎盘生长因子(PIGF)(4)。PDGF通过激活其受体PDGF-R发挥生理功能。PDGF-R包括PDGFR-α和PDGFR-β两种，属于酪氨酸激酶受体。配体与受体结合引发受体单体的二聚化，促使胞内区的酪氨酸残基自体磷酸化而激活。两种受体可以激活多条信号通路关键分子，如Ras-MAPK、PI3K和PLC-γ(5)，进而激活相关基因的转录，刺激细胞生长、抑制凋亡，促进分化，引起定向移动和迁移等，发挥多种多样的生物学功能。
PDGF-b基因定位于第22号染色体，含有7个外显子基因，编码241个氨基酸组成的前体蛋白，经蛋白酶水解加工形成的最终成熟产物是109个氨基酸组成的，分子量为12.3kD的多肽。在生物体内，PDGF-B蛋白的活性形式是由两条单体通过二硫键形成同源PDGF-BB或异源二聚体PDGF-AB(6)。每条PDGF-B蛋白单体含有8个高度保守的半胱氨酸残基，其中6个半胱氨酸两两形成链内二硫键(Cys Ⅰ-Ⅵ，Ⅲ-Ⅶ，Ⅴ-Ⅷ)，另外两个则与对应单体之间交叉形成链间二硫键(Cys Ⅱ-Ⅳ)(7)共同形成PDGF蛋白家族特征性的生长因子结构域—半胱氨酸结。这些链内及链间二硫键构成了PDGF-BB二聚体蛋白复杂的空间结构(图1B)。
另外，PDGF蛋白在表达合成时还存在不同剪切形式，使得加工成熟后 的PDGF蛋白呈现多种结构形式。从人血小板提取物中分离纯化的PDGF-BB，通过N端氨基酸序列分析表明存在至少三种不同的剪切形式，20％Ser1，45％Thr6及35％Thr33，这些切割的异质性，致使在纯化PDGF-BB时各种切割形式的蛋白比例不可控(8)。
发明内容
本发明的发明人经过大量研究，发现特定位点蛋白酶降解和/或糖基化修饰是造成多种PDGF-B存在的主要原因，进而通过位点突变获得了PDGF-B突变体，其蛋白均一性大大提高，并且仍保留PDGF-B蛋白的活性。
本发明第一方面涉及血小板衍生生长因子B突变体，其在野生型血小板衍生生长因子B的第101位和第109位氨基酸位点处具有突变(此处对氨基酸位点位置的描述均以含109个氨基酸残基的成熟PDGF-B为基础，以下同)，并且具有血小板衍生生长因子B的活性。
根据本发明第一方面任一项的血小板衍生生长因子B突变体，其在第6位氨基酸位点处具有突变，并且具有血小板衍生生长因子B的活性。
根据本发明第一方面任一项的血小板衍生生长因子B突变体，其在第32位和/或第33位氨基酸位点处具有突变，并且具有血小板衍生生长因子B的活性。
根据本发明第一方面任一项的血小板衍生生长因子B突变体，与野生型血小板衍生生长因子B相比，其N端缺失5个氨基酸，并且具有血小板衍生生长因子B的活性。
在本发明的一个实施方案中，所述突变体在第6位、第101位和第109位氨基酸位点处突变为丙氨酸。
在本发明的另一个实施方案中，所述突变体在第101位和第109位氨基酸位点处突变为丙氨酸。
根据本发明第一方面任一项的血小板衍生生长因子B突变体，其在第32位和/或第33位氨基酸位点处突变为脯氨酸、缬氨酸或异亮氨酸。
在本发明的一个实施方案中，其在第32位氨基酸位点处突变为脯氨酸、缬氨酸或异亮氨酸，优选为脯氨酸。
根据本发明第一方面任一项的血小板衍生生长因子B突变体，其中所述的血小板衍生生长因子B为哺乳动物来衍生的血小板衍生生长因子B，所述哺乳动物例如为人、小鼠。
在本发明的一个实施方案中，所述血小板衍生生长因子B突变体为，其N端缺失5个氨基酸，其第6位、第101位和第109位氨基酸突变为丙氨酸，其第32位氨基酸突变为脯氨酸。
在本发明的一个实施方案中，所述血小板衍生生长因子B突变体为，其N端缺失5个氨基酸，其第101位和第109位氨基酸突变为丙氨酸，其第32位氨基酸突变为脯氨酸。
在本发明的一个实施方案中，所述血小板衍生生长因子B突变体为，其N端缺失5个氨基酸，其第6位、第101位和第109位氨基酸突变为丙氨酸，其第32位氨基酸突变为缬氨酸。
在本发明的一个实施方案中，所述血小板衍生生长因子B突变体为，其N端缺失5个氨基酸，其第6位、第101位和第109位氨基酸突变为丙氨酸，其第32位氨基酸突变为异亮氨酸。
本发明还包括上述各种技术方案的组合。
在本发明的具体实施方案中，所述突变体的氨基酸序列为SEQ ID NO：3或SEQ ID NO：5所示的序列。
根据本发明第一方面任一项的血小板衍生生长因子B突变体，其氨基酸序列经过取代、缺失或添加一个或几个氨基酸，并且具有血小板衍生生长因子B的活性。
本领域技术人员公知，在蛋白的非关键位点可以进行一个或几个氨基酸的取代、缺失和/或添加，而不会影响该蛋白的活性。在本发明中，除了上面提到的氨基酸突变位点，在其它非关键位点对血小板衍生生长因子B的氨基酸序列进行一个或几个氨基酸的取代、缺失和/或添加后得到的仍保留血小板衍生生长因子B活性的突变体也在本发明的保护范围内。
本发明第二方面涉及血小板衍生生长因子同源或异源二聚体，其由两个本发明第一方面任一项的血小板衍生生长因子B突变体通过链内和/或链间二硫键结合而成，或者由一个本发明第一方面任一项的血小板衍生生长因子B突变体与一个血小板衍生生长因子A通过链内和/或链间二硫键结合而成。
在本发明中，所述血小板衍生生长因子同源或异源二聚体的形成方式与野生型血小板衍生生长因子相同。
在本发明的实施方案中，两个本发明第一方面任一项的血小板衍生生长因子B突变体通过链内及链间二硫键结合，形成PDGF-BB突变体。
本发明第三方面涉及核酸分子，其编码本发明第一方面任一项的血小板衍生生长因子B突变体。
根据本发明第三方面任一项的核酸分子，其核苷酸序列选自SEQ ID NO：4、SEQ ID NO：6～SEQ ID NO：9的序列。
本发明第四方面涉及载体，其含有本发明第三方面任一项的核酸分子。
在本发明中，本领域技术人员可以根据用于表达的宿主细胞的不同来选择表达载体，例如可以选择适合酵母细胞或哺乳动物细胞表达的载体。
在本发明的实施方案中，所述载体为pMEX9K。
本发明第五方面涉及宿主细胞，其含有本发明第四方面任一项的载体。
根据本发明第五方面任一项的宿主细胞，其为真核细胞，例如为酵母细胞、哺乳动物细胞或昆虫细胞。
根据本发明第五方面任一项的宿主细胞，所述酵母细胞例如为毕赤酵母细胞(Pichia pastoris，也叫巴斯德毕赤酵母细胞)、酿酒酵母(Saccharomyces cerevisiae)、克鲁维酵母(Kluyveromyces lactis)、汉逊酵母(Hansenula)、念珠酵母(Candida)或球拟酵母(Torulopsis)。
在本发明的实施方案中，所述毕赤酵母细胞为GS115细胞。
根据本发明第五方面任一项的宿主细胞，所述哺乳细胞例如为CHO细胞、BHK细胞、NS0细胞、SP2/0细胞、HEK-293细胞、COS细胞等。
本发明还涉及本发明第一方面任一项的血小板衍生生长因子B突变体的制备方法，其包括取本发明第五方面任一项的宿主细胞进行培养、表达(例如诱导表达)以及任选的纯化的步骤。
根据本发明任一项的制备方法，其包括以下步骤：
1)取本发明第五方面任一项的宿主细胞接种至培养基中，经过逐级放大培养；
2)收集宿主细胞，将重组细胞重悬于培养基中，添加甲醇开始诱导表达；
3)诱导表达结束后收集培养上清，经纯化获得血小板衍生生长因子B蛋白。
根据本发明任一项的制备方法，其特征在于以下几项中的一项或数项：
(1)步骤1)中的宿主细胞为单克隆细胞株；
(2)步骤1)中所述的逐级放大培养是指两级放大培养，培养温度为28-30℃，每级均培养至OD600为1-12、例如2-6；
(3)步骤2)中诱导表达的温度约为28℃；
(4)步骤2)中甲醇的终浓度为0.3-1.0％(v/v)，例如0.4-0.8％(v/v)，例如0.5％(v/v)；
(5)步骤2)中诱导表达的时间为48-96h，例如为72h；
(6)步骤3)中的纯化步骤依次包括疏水层析、离子交换层析、凝胶层析。
本发明第六方面涉及血小板衍生生长因子B或其突变体的纯化方法，其包括取含有血小板衍生生长因子B或其突变体的培养上清或细胞裂解液依次进行疏水层析、离子交换层析和凝胶层析的步骤。
根据本发明第六方面任一项的纯化方法，其中所述血小板衍生生长因子B突变体为本发明第一方面任一项的血小板衍生生长因子B突变体。
在本发明的实施方案中，用于疏水层析的层析柱介质为Phenyl Sepharose 6 Fast Flow。
在本发明的实施方案中，用于离子交换层析的层析介质为Source 30S。
在本发明的实施方案中，用于凝胶层析的层析介质为Hiload Superdex 75 prep grad。
根据本发明第六方面任一项的纯化方法，其中,
所述的疏水层析包括以下步骤：
(1)取含有血小板衍生生长因子B或其突变体的培养上清或细胞裂解液用调节缓冲液调节电导，所述调节缓冲液加入后的终体系为10-50mM磷酸缓冲液，0.8-1M(NH4)2SO4，pH 6.8-7.5；
(2)用平衡缓冲液平衡柱子，所述平衡缓冲液的配方为10-50mM磷酸缓冲液，0.8-1M(NH4)2SO4，pH 6.8-7.5；
(3)样品上柱后，用平衡缓冲液洗柱；
(4)用洗脱缓冲液洗脱，收集目的蛋白，所述洗脱缓冲液的配方为，10-50mM磷酸缓冲液，30％-50％乙二醇，pH 6.8-7.5；
所述离子交换层析包括以下步骤：
(1)用平衡缓冲液稀释疏水层析的洗脱峰至电导值为6mS/cm以下，所述平衡缓冲液的配方为，10-50mM磷酸缓冲液，pH 6.8-7.5；
(2)用平衡缓冲液平衡柱子；
(3)样品上柱后，用平衡缓冲液清洗柱子；
(4)用洗脱缓冲液梯度洗脱，收集目的蛋白，所述洗脱缓冲液的配方为10-50mM磷酸缓冲液，0.8-1.2M NaCl，pH 6.8-7.5；
所述凝胶层析包括以下步骤：
(1)用磷酸盐缓冲液平衡柱子，所述磷酸盐缓冲液的配方为，10-50mM磷酸缓冲液，0.1-0.5M NaCl，pH 6.8-7.5；
(2)将离子交换层析的洗脱峰上样，每次上样体积不超过柱体积的0.3-4％(例如3％)；
(3)继续用步骤(1)中的磷酸盐缓冲液清洗柱子，收集目的蛋白，即得到纯化的血小板衍生生长因子B或其突变体。在本发明中，所述磷酸缓冲液的配方为本领域所公知。在本发明的实施方案中，所述磷酸缓冲液为20mMPB溶液，其含有0.0144mol/L的Na2HPO4和0.0056mol/L的NaH2PO4，pH 6.8-7.5。
在本发明中，所述磷酸盐缓冲液的配方为本领域所公知。在本发明的实施方案中，所述磷酸盐缓冲液为PBS溶液，其配方为10-50mM PB溶液，0.15M NaCl，pH 6.8-7.5。
在本发明的实施方案中，各步层析的缓冲液的pH值为7.2。
在本发明的实施方案中，各步层析的磷酸缓冲液的浓度为20mM。
在本发明的具体实施方案中，疏水层析方法为：(1)酵母表达上清用1/2体积的调节缓冲液(60mM PB，3M(NH4)2SO4，pH 7.2)调节电导；(2)用平衡缓冲液(20mM PB，1M(NH4)2SO4，pH 7.2)平衡柱子；(3)样品上柱后，用平衡缓冲液清洗柱子至基线平直；(4)用洗脱缓冲液(20mM PB，50％乙二醇，pH 7.2)洗脱，收集目的蛋白。
在本发明的具体实施方案中，离子交换层析方法为：(1)用平衡缓冲液(20mM PB，pH 7.2)稀释Phenyl HS洗脱峰至电导值为6mS/cm以下；(2)用平衡缓冲液平衡柱子；(3)样品上柱后，用平衡缓冲液清洗柱子至基线平直；(4)用洗脱缓冲液(20mM PB，1M NaCl，pH 7.2)梯度洗脱，收集目的蛋白。
在本发明的具体实施方案中，凝胶层析方法为：(1)用PBS缓冲液(20mM PB，0.15M NaCl，pH 7.2)平衡柱子；(2)将Source 30S洗脱峰用loop上样，每次上样体积不超过柱体积的3％；(3)继续用PBS缓冲液清洗柱子，收集目的蛋白。
本发明还涉及本发明第一方面任一项的血小板衍生生长因子B突变体 用于制备促进细胞分裂、增殖、促进伤口愈合、皮肤再生、骨骼与牙齿缺损再生、关节修复的药物中的用途。
本发明还涉及促进细胞分裂、增殖、促进伤口愈合、皮肤再生、骨骼与牙齿缺损再生、关节修复的方法，所述方法包括给予有需要的受试者有效量的本发明第一方面任一项的血小板衍生生长因子B突变体的步骤。
本发明还涉及抗体，其能够与本发明第一方面任一项的血小板衍生生长因子B突变体特异性结合。
本发明还涉及一种使血小板衍生生长因子表达更均一化的方法，其包括对野生型血小板衍生生长因子的氨基酸序列进行改造的步骤，所述改造包括以下a)-c)中的一项或几项：
a)突变第101位和第109位氨基酸；
b)突变第6位氨基酸；
c)突变第32位和/或第33位氨基酸；
d)N端缺失5个氨基酸。
根据本发明任一项的方法，其特征在于以下i)-iii)中的一项或几项：
i)将第101位和第109位氨基酸突变为丙氨酸；
ii)将第6位氨基酸突变为丙氨酸；
iii)将第32位和/或第33位氨基酸突变为脯氨酸、缬氨酸或异亮氨酸。
根据本发明任一项的方法，利用真核表达系统进行表达，如酵母细胞表达系统、哺乳动物细胞表达系统。
在本发明的一个实施方案中，所述改造是指其N端缺失5个氨基酸，其第6位、第101位和第109位氨基酸突变为丙氨酸，其第32位氨基酸突变为脯氨酸。
在本发明的一个实施方案中，所述改造是指其N端缺失5个氨基酸，其第101位和第109位氨基酸突变为丙氨酸，其第32位氨基酸突变为脯氨酸。
在本发明的一个实施方案中，所述改造是指其N端缺失5个氨基酸，其第6位、第101位和第109位氨基酸突变为丙氨酸，其第32位氨基酸突变为缬氨酸。
在本发明的一个实施方案中，所述改造是指其N端缺失5个氨基酸， 其第6位、第101位和第109位氨基酸突变为丙氨酸，其第32位氨基酸突变为异亮氨酸。
在本发明的具体实施方案中，改造后的血小板衍生生长因子的氨基酸序列为SEQ ID NO：3或SEQ ID NO：5所示的序列。
本发明还包括上述各种技术方案的组合。
在本发明中，所述载体例如为克隆载体或表达载体。所述载体含有本发明所述的核酸分子，所述载体可以通过例如将上述核酸分子插入克隆载体或表达载体而得到，或者可以通过人工合成得到。
所述表达载体例如为原核表达载体，真核表达载体，噬菌体载体或病毒载体。其中所述原核表达载体例如为pET载体、pGEX载体，所述真核表达载体例如为pcDNA3.1、pEGFP-C1、pPIC9K、pMEX9K、pPICZ、pPICZa、pFastBac、pPIC6aA、pPIC3.5K、pGAPZaA、pAO815，所述噬菌体载体例如为λ噬菌体载体λgt、λgt-λB，所述病毒载体例如为逆转录病毒、慢病毒、腺病毒或腺相关病毒载体。在本发明的实施方案中，所述载体为pMEX9K。
在本发明中，所述宿主细胞可以为原核细胞(例如大肠杆菌细胞)或真核细胞。所述真核细胞例如为酵母细胞、哺乳动物细胞或昆虫细胞。所述宿主细胞可以通过向原核细胞或真核细胞中引入/转染上述的核酸分子的核苷酸序列或载体而得到。
在本发明中，所述酵母细胞株为本领域所公知，其包括但不限于X33，KM71，KM71H，GS115，SMD1168，SMD1163,SMD1168H，SMD1163H。
在本发明中，可以利用本领域所知的任何种类的转染方法获得转染有特定核酸或载体的宿主细胞，例如，可通过电穿孔或显微注射将核酸引入细胞中；或者，可使用脂转染试剂如FuGENE 6、X-tremeGENE和LipofectAmine；或者，可通过基于逆转录病毒、慢病毒、腺病毒和腺相关病毒的适当病毒病毒载体将核酸引入细胞中。
在本发明中，所述表达均一化是指在电泳上样量不低于10μg时，经还原型SDS-PAGE电泳分析，目的蛋白经考马斯亮蓝染色为单一条带，Image J或Bandscan等软件分析纯度>95％。本领域技术人员通过摸索，针对不同的蛋白，采取各种技术手段，可以使蛋白的表达趋向于更均一化。
在本发明中，所述野生型血小板衍生生长因子B是指长度为109个氨基酸的未经过突变的血小板衍生生长因子B；并且对各位点(例如突变位点、 糖基化位点、酶切位点)的位置描述也均是以野生型血小板衍生生长因子B为基准。
在本发明中，所述野生型血小板衍生生长因子B的第6位、第101位、第109位氨基酸均为苏氨酸(Thr)；第32位氨基酸、第33位氨基酸分别为精氨酸(Arg)；野生型血小板衍生生长因子B的N端前5个氨基酸依次为丝氨酸(Ser)、亮氨酸(Leu)、甘氨酸(Gly)、丝氨酸(Ser)、亮氨酸(Leu)。
在本发明中，所述“约”是指围绕该数值的90％～110％的范围内，例如95％～105％的范围内。
本发明通过突变获得了PDGF-B突变体，其蛋白均一性大大提高，并且仍保留了PDGF-B蛋白的活性。
附图说明
图1.PDGF-B蛋白结构示意图。A，PDGF-B前体蛋白通过蛋白酶水解掉N端信号肽、前肽及C端前肽序列，成为成熟蛋白。▲表示蛋白酶水解位点；B，两个PDGF-B单体通过链间二硫键形成PDGF-BB同源二聚体，每个PDGF-B单体含有8个Cys，分别形成3对链内二硫键(C16-C60、C49-C97、C53-C99)及2对链间二硫键(C43-C52、C52-C43)。SP:信号肽(signal peptide)；PRO:生长因子结构域前序列(the pro-sequence preceding the growth factor domain)。
图2.SDS-PAGE电泳分析毕赤酵母分泌表达的PDGF-BB。对3个不同批次发酵纯化的重组PDGF-BBThr6进行非还原条件(左)及还原条件(右)SDS-PAGE分析。非还原条件下蛋白为单一条带；经DTT处理后PDGF-B单体表现为分子量不均一的多种形式。
图3蛋白酶解及糖基化修饰共同作用致使PDGF-BThr6多种单体的形成。(A)对还原条件下SDS-PAGE分离PDGF-BThr6蛋白进行N-末端氨基酸序列分析。第1、2、3条带N端前5个氨基酸残基为均为TIATP，第4、5带N端前5个氨基酸残基为TNANF，表明在Arg32-Thr33发生蛋白酶切割。(B)对还原条件下PDGF-BThr6蛋白进行WB(中)及糖蛋白染色(右)分析。WB检测条带对应考马斯亮蓝染色(左)第3、5条带；糖蛋白染色条带对应考染第1、2、4条带。(C)利用PNGase F对还原条件下PDGF-BThr6蛋白进行处理并进行糖蛋白染色。蛋白经PNGase F处理前后带型、分子量无变化(左)，糖蛋白染色结果无差别；IFN-ω为糖苷酶酶切及糖蛋白染色 阳性对照。
图4.PDGF-BThr6蛋白序列O-连接糖基化位点预测结果。Thr6、Thr101、Thr109为可能的O-连接糖基化修饰位点。
图5 HPLC检测纯化后蛋白PDGF-M2纯度结果。
图6毕赤酵母表达PDGF-B发生翻译后修饰位点分析。(A)PDGF-M1及PDGF-M2突变体位点突变示意图。(B)WB检测显示PDGF-M1及PDGF-M2单体为单一条带，对照PDGF-BThr6为两条。(C)SDS-PAGE检测PDGF-M1及PDGF-M2单体，经考马斯亮蓝染色结果显示PDGF-M2为单一蛋白条带，PDGF-M1为两条蛋白条带(如图中箭头所示)。(D)糖蛋白染色几乎检测不到PDGF-M1及PDGF-M2蛋白单体。
图7 PDGF-M2的生物学活性高于PDGF-BBThr6，两者有统计学差异。实验重复3次，EC50表示为平均值±标准差，P值＝0.039。
图8 Arg32为突变对表达量的影响。(A)试管表达密码子优化株PDGF-IM-P、PDGF-IM-V、PDGF-IM-I各7个克隆的SDS-PAGE结果。PDGF-IM-P的蛋白表达量明显高于其他两株。(B)通过G418抗性对密码子优化株PDGF-IM-P、PDGF-IM-V、PDGF-IM-I分别进行多插入子拷贝筛选，分别选取G418浓度为2.0mg/ml及4.0mg/ml条件下筛选菌株6个克隆进行试管表达，SDS-PAGE结果显示，PDGF-IM-P高拷贝筛选株表达量明显高于其他两株。
图9 PDGF-B野生型和PDGF-M2突变体的LC/MS图。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
在前期研究中，我们成功利用毕赤酵母系统分泌表达了N端缺失5个氨基酸的rhPDGF-BBThr6，表达量可达100mg/L(参见专利：ZL200410068993.2)。选择PDGF-BThr6作为研究对象，是为了保证表达蛋白的均一性而生物活性又不受损。然而进一步研究发现，毕赤酵母所表达的rhPDGF-BThr6单体仍表现为分子量不均一的多种形式，分子量在10-15kDa之间(图2)。
以下实施例均是在rhPDGF-BThr6的基础上进行的改造，所有对位点位置 的描述均是以野生型PDGF-B(109个氨基酸)为基础。
其中野生型PDGF-B氨基酸序列的Genbank号为NM-002608.2。
rhPDGF-BBThr6氨基酸序列为：
TIAEPAMIAECKTRTEVFEISRRLIDRTNANFLVWPPCVEVQRCSGCCNNRNVQCRPTQVQLRPVQVRKIEIVRKKPIFKKATVTLEDHLACKCETVAAARPVT(SEQ ID NO：1)。
rhPDGF-BBThr6核酸序列为：
5’-ACCATTGCTGAGCCGGCCATGATCGCCGAGTGCAAGACGCGCACCGAGGTGTTCGAGATCTCCCGGCGCCTCATAGACCGCACCAACGCCAACTTCCTGGTGTGGCCGCCCTGTGTGGAGGTGCAGCGCTGCTCCGGCTGCTGCAACAACCGCAACGTGCAGTGCCGCCCCACCCAGGTGCAGCTGCGACCTGTCCAGGTGAGAAAGATCGAGATTGTGCGGAAGAAGCCAATCTTTAAGAAGGCCACGGTGACGCTGGAAGACCACCTGGCATGCAAGTGTGAGACAGTGGCAGCTGCACGGCCTGTGACC-3’(SEQ ID NO：2)。
材料和方法
重组表达克隆的构建
编码各种PDGF突变体DNA序列均由上海生工合成。基因片段通过酶切位点XhoI和EcoRI克隆至表达载体pMEX9K(参见专利ZL02117906.9)中，并经测序确认。提取重组质粒，经SalI酶切线性化后，利用电转化方法转化至毕赤酵母(Pichia pastoris)表达菌株GS115感受态细胞中。经组氨酸缺陷的MD平板筛选酵母转化子，并经PCR方法鉴定阳性重组酵母菌株。
重组蛋白的诱导表达
将重组酵母菌株单克隆接种至含有25mL BMGY培养基(BMGY培养基配方：称量酵母浸出粉10g，胰蛋白胨20g，溶于700ml水中，121℃高压灭菌20min；冷却至室温，加入100ml 1M磷酸钾缓冲液、100ml 10×YNB、100ml10×GY，4℃存放。其中：10×YNB(13.4％酵母氮源碱)、10×GY(10％甘油)、1M磷酸钾缓冲液：量取132ml 1M K2HPO4，868ml 1M KH2PO4，用磷酸或KOH调pH值至6.0±0.1，121℃高压灭菌30min，室温存放。Yeast Extract(LP0021)为OXOID公司产品，Peptone(211677)为B&D公司产品)的摇瓶中，28-30℃，220-250rpm培养增菌至OD600＝2-6(约16-18小时)；将25ml酵母培养物接种至含有1L BMGY的摇瓶中，继续28-30℃，220-250rpm 培养增菌至OD600＝2-6；室温1500-3000g离心5分钟收集酵母，去除上清后用1L BMMY培养基重悬酵母开始诱导表达。诱导温度为28℃，摇床转速为220rpm；每24小时添加甲醇至终浓度为0.5％，共诱导72小时。诱导结束后室温7000rpm离心收集含重组蛋白的上清。
重组蛋白的纯化
毕赤酵母表达上清经离心、过滤后调整至合适的缓冲液中，依次经过疏水层析(Phenyl Sepharose 6 Fast Flow)、离子交换层析(Source 30S)、凝胶层析(Hiload Superdex 75 prep grad)获得纯度>95％的目的蛋白(图5)。层析介质均为GE Amersham Bioscience公司产品。
疏水层析方法为：(1)酵母表达上清用1/2体积的调节缓冲液(60mM PB，3M(NH4)2SO4，pH 7.2)调节电导；(2)按照说明书方法，用平衡缓冲液(20mM PB，1M(NH4)2SO4，pH 7.2)平衡柱子；(3)样品上柱后，用平衡缓冲液清洗柱子至基线平直；(4)用洗脱缓冲液(20mM PB，50％乙二醇，pH 7.2)洗脱，收集目的蛋白。
离子交换层析方法为：(1)用平衡缓冲液(20mM PB，pH 7.2)稀释Phenyl HS洗脱峰至电导值为6mS/cm以下；(2)按照说明书方法，用平衡缓冲液平衡柱子；(3)样品上柱后，用平衡缓冲液清洗柱子至基线平直；(4)用洗脱缓冲液(20mM PB，1M NaCl，pH 7.2)梯度洗脱，收集目的蛋白。
凝胶层析方法为：(1)用PBS缓冲液(20mM PB，0.15M NaCl，pH 7.2)平衡柱子；(2)将Source 30S洗脱峰用loop上样，每次上样体积不超过柱体积的3％；(3)继续用PBS缓冲液清洗柱子，收集目的蛋白。
重组蛋白SDS-PAGE检测
取30μl合适浓度的纯化后蛋白分别加入10μl 4×SDS-PAGE上样缓冲液(不含及含20mM DTT)，100℃变性5分钟，离心后取30μl上清经SDS-PAGE电泳分析(分离胶为15％)。电泳后凝胶用考马斯亮蓝R250染色。
重组蛋白Western Blot(WB)检测
样品制备同SDS-PAGE，取3μl样品进行SDS-PAGE，电泳结束后，300mA稳流电转1h，将蛋白转移至硝酸纤维素膜上，5％脱脂奶粉/TBST室温封闭1h。1：1000稀释一抗PDGF-B(F-3)(Santa Cruz Biotechnology，SC-365805)，室温包被1h，TBST洗涤多次后，1：10000稀释HRP标记的二抗(Cell Signaling Technology，#7076)室温孵育1h，TBST洗涤后，加入底物并用LAS400mini凝胶成像系统(GE)成像。
N-末端氨基酸序列测序
样品制备及SDS-PAGE过程同前，电泳结束后，用CAPS电印迹缓冲液，300mA稳流电转1h，将蛋白转移至PVDF膜上，0.1％考马斯亮蓝R250染色后立即用50％甲醇充分脱色至蛋白条带清洗可见，剪下待测蛋白条带委托军事医学科学院生物医学分析中心色谱实验室测定。
蛋白糖基化修饰检测
取5μl样品及阳性对照IFN-ω，加入3μl 10×糖蛋白变性缓冲液(NEB PNGase F酶自带)及15μl水后100℃加热变性10min。冷却后加入3μl NP-40、3μl G7缓冲液(NEB PNGase F酶自带)及2μl肽N-糖苷酶F(PNGase F)(纽英伦生物技术有限公司(NEB)产品)，37℃酶切3h。酶切结束后100℃加热样品使酶失活后进行SDS-PAGE电泳。电泳结束后，利用糖蛋白染色试剂盒(Themo Scientific，#24562)进行染色。首先加入100ml 50％甲醇固定凝胶30min；3％乙酸清洗多次后将胶移入25ml Oxidizing Solution，轻摇15min；3％乙酸清洗多次后将胶移入25ml Glycoprotein Staining Reagent，轻摇15min后将胶移入25ml Reducing Solution，轻摇5min后用3％乙酸清洗并用去离子水漂洗。
PDGF-B生物学活性检测
BALB/C 3T3细胞(购自北京协和细胞资源中心)用含10％FBS的DMEM完全培养基(Life Technology)于37℃、5％二氧化碳条件下培养。消化和收集细胞后，用完全培养液配成每1ml含5.0×104个细胞的细胞悬液，接种于96孔细胞培养板中，每孔100μl，于37℃、5％二氧化碳条件下继续培养；24h后换成维持培养基(含0.4％FBS的DMEM)，于37℃、5％二氧化碳条件下继续培养；培养24h后弃去维持培养液，加入预先梯度稀释好的重组PDGF-BB溶液，每孔100μl；细胞在蛋白作用下继续培养64-72h后，采用WST-1法检测细胞增殖活性：每孔加入WST-1溶液(Roche，11644807001)10μl，于37℃、5％二氧化碳条件下继续培养3小时后用酶标仪在波长450nm处测定吸光度值(参考波长：630nm)。实验数据采用四参数回归计算法进行处理，分别计算出两种蛋白的EC50值，实验重复3次，两组差异统计分析采用t检验。
实施例1蛋白酶解及糖基化修饰共同作用致使PDGF-BBThr6多种单体的形成
我们首先怀疑蛋白酶解是造成多种PDGF-B单体形式的原因。通过SDS-PAGE还原电泳，分离PDGF-B的不同单体，考马斯亮蓝染色共检测出5条带(图3A)。将这5个蛋白条带分别进行N-末端氨基酸序列测序，结 果显示，第1、2、3条带的N末端前5个氨基酸序列为TIAEP，为PDGF-BThr6正确N端序列，而第4、5条带的N端序列为TNANF。经蛋白序列比对，确定4、5号蛋白片段为Arg32-Thr33发生蛋白酶切而产生的截短型蛋白(图3A)。
但是这并不能解释形成至少5种PDGF单体的原因。第1、2、3条带和第4、5条带之间的分子量的差异也许是由于C末端酶切导致的。为了回答这个问题，我们利用一株特异的抗PDGF-B C末端的单抗(F-3)(Santa Cruz Biotechnology，SC-365805)进行WB分析。检测结果表明，5条带中仅有2条带被检测出来。但是有趣的是，这两条与抗体结合的条带对应的似乎是第3和第5条蛋白片段(图3B)。如果第1、2、4条蛋白片段是由于发生了C末端酶切而导致不能被抗体检测到，那么它们的分子量应该更小，但是这显然与电泳检测的结果不符合。这意味着，还有其他原因有待进一步发现。
为了分析PDGF-B是否发生了糖基化修饰，我们利用肽N-糖苷酶F(PNGase F)对PDGF进行了酶切处理，并同时进行了SDS-PAGE电泳和糖蛋白染色分析。PNGaseF是一种酰胺酶，它几乎可以作用于糖肽/糖蛋白中所有的N-聚糖链，在糖链部分最内侧的GlcNAc和天冬酰胺残基之间进行切割，并将天冬酰胺变为天冬氨酸(10)，是目前糖蛋白组学研究中鉴定N-糖蛋白应用最为广泛的一种酶。同为毕赤酵母表达的重组IFN-ω蛋白作用为N-糖基化蛋白的阳性对照。考马斯亮蓝染色结果表明，酶切前后PDGF-B蛋白的相对分子量没有发生变化，说明PDGF-B蛋白并没有发生N-糖基化(图3C，左)。然而糖蛋白染色的结果表明，PDGF-B确实是糖蛋白(图3C，右)。这意味着，毕赤酵母分泌表达的PDGF-B发生了O-糖基化修饰。同时，进一步分析发现，糖染色仅检测到了3条蛋白片段，应该分别对应SDS-PAGE结果中的第1、2、4号条带(图3B)。这也与上面WB的结果相吻合：第1、2、4蛋白片段在C-末端发生了糖基化，从而影响了PDGF-BThr6与抗体的结合。
综合上述实验结果，我们推断PDGF-BThr6多种形式的单体是由于第27/28位氨基酸Arg32-Thr33之间发生了蛋白酶解和蛋白翻译后不同程度的糖基化修饰共同作用造成的(表1)。
表1 PDGF-BThr6修饰类型分析
Figure PCTCN2015084260-appb-000001
Figure PCTCN2015084260-appb-000002
注：表中完整PDGF是指PDGF-BThr6，即N端缺失5个氨基酸的PDGF-B。
实施例2 PDGF-M1及PDGF-M2改构体的构建及蛋白性质检测
更进一步，我们想确认上述的推断，并希望使PDGF-B在毕赤酵母中的表达均一化。首先我们要确定可能的O-糖基化位点。利用在线网站CBS(www.CBS.dtu.dk)(11)对PDGF-BThr6蛋白序列进行糖基化位点预测，结果显示第6位、101位、109位的Thr为可能的O-糖基化修饰位点(图4)。相对于N-糖基化而言，O-糖基化位点并没有明确的基序，因而其预测也相对困难。但是，预测的第6、101、109位的潜在糖基化修饰位点与我们上述的结果是相符的：PDGF-BThr6蛋白的C末端应该有糖基化修饰(Thr101，Thr109)，因为它们阻碍了与抗体的结合；在Thr33之前应该有糖基化修饰位点，因为这样才能解释发生酶解的PDGF-B条带中仅有一条(Band 4)糖基化修饰的变体，而未发生酶解的糖基化PDGF-B单体有两条(Band1，2)(图3B；表1)。为了证实预测的结果，我们构建了两个PDGF-BThr6的突变体PDGF-M1及PDGF-M2。PDGF-M1突变了C端的两个糖基化位点，而PDGF-M2突变了全部3个潜在糖基化位点(模式图见图6A)。同时，为了去除Arg32-Thr33这一蛋白酶切位点，我们将Arg32突变为Pro，突变为了Pro是考虑到进化对氨基酸的选择。我们注意到成熟PDGF-B蛋白有60％的氨基酸序列与PDGF-A具有同衍生性，并且两者在结构与功能上也具有很高的相似性，而PDGF-A蛋白序列在对应位置的氨基酸序列正是Pro。
PDGF-M1的氨基酸序列为：
TIAEPAMIAECKTRTEVFEISRRLIDPTNANFLVWPPCVEVQRCSGCCNNRNVQCRPTQVQLRPVQVRKIEIVRKKPIFKKATVTLEDHLACKCEAVAAARPVA(SEQ ID NO：3)；
核苷酸序列为：
ACCATTGCTGAGCCGGCCATGATCGCCGAGTGCAAGACGCGCACCGAGGTGTTCGAGATCTCCCGGCGCCTCATAGACCCCACCAACGCCAACTTCCTGGTGTGGCCGCCCTGTGTGGAGGTGCAGCGCTGCTCCGGCTGCTGCAACAACCGCAACGTGCAGTGCCGCCCCACCC AGGTGCAGCTGCGACCTGTCCAGGTGAGAAAGATCGAGATTGTGCGGAAGAAGCCAATCTTTAAGAAGGCCACGGTGACGCTGGAAGACCACCTGGCATGCAAGTGTGAGGCAGTGGCAGCTGCACGGCCTGTGGCC(SEQ ID NO：4)。
PDGF-M2的氨基酸序列为：
AIAEPAMIAECKTRTEVFEISRRLIDPTNANFLVWPPCVEVQRCSGCCNNRNVQCRPTQVQLRPVQVRKIEIVRKKPIFKKATVTLEDHLACKCEAVAAARPVA(SEQ ID NO：5)；
核苷酸序列为：
GCCATTGCTGAGCCGGCCATGATCGCCGAGTGCAAGACGCGCACCGAGGTGTTCGAGATCTCCCGGCGCCTCATAGACCCCACCAACGCCAACTTCCTGGTGTGGCCGCCCTGTGTGGAGGTGCAGCGCTGCTCCGGCTGCTGCAACAACCGCAACGTGCAGTGCCGCCCCACCCAGGTGCAGCTGCGACCTGTCCAGGTGAGAAAGATCGAGATTGTGCGGAAGAAGCCAATCTTTAAGAAGGCCACGGTGACGCTGGAAGACCACCTGGCATGCAAGTGTGAGGCAGTGGCAGCTGCACGGCCTGTGGCC(SEQ ID NO：6)。
将编码PDGF-M1和PDGF-M2的DNA序列插入到pMEX9K表达载体，并整合入GS115毕赤酵母菌株中。通过甲醇诱导表达，并经色谱纯化。对纯化后PDGF-B蛋白进行了SDS-PAGE、糖蛋白染色及Western免疫印迹检测，以确定改构后蛋白性质。WB结果显示，两种突变体还原后仅能检测到单一条带，相对分子质量约为12kDa，与预期一致(图6B)。SDS-PAGE结果显示，PDGF-M2为单一条带，但是PDGF-M1在主带上仍有一微弱条带(图6C)。推测此带是由于Thr6的糖基化导致的。糖蛋白染色结果显示，相对于突变前，两种突变体的糖基化水平非常低，已很难用糖蛋白染色的方法检测到(图6D)。上述结果表明，将第32位R突变为P，去除了潜在的Kex2蛋白酶切位点，阻止了Thr33截短型PDGF-B单体的形成，而将Thr6、101、109三个糖基化修饰位点的突变也去除了蛋白在翻译后形成不同程度糖基化修饰，成功使得PDGF-B蛋白在毕赤酵母中的表达均一化。
实施例3 PDGF-M2促细胞增殖活性检测
为了分析Thr6-Ala、Thr101-Ala、Thr109-Ala糖基化位点的突变和Arg32-Pro KEX酶切位点的突变会不会影响到PDGF-BB的生物学活性，我 们利用WST-1法检测了PDGF-BThr6和PDGF-M2对Balb/c 3T3细胞的增殖活性。结果显示，PDGF-BThr6的EC50为5.434±0.6475ng/ml，而PDGF-M2的EC50为3.492±0.4078ng/ml，经t检验，改构后蛋白活性高于改构前，P值为0.0117(图7)。
实施例4 PDGF-M2Arg32突变对表达水平的影响
为了提升PDGF-M2的表达水平，我们根据毕赤酵母在蛋白表达时对密码子选择的偏好性，利用在线工具JAVA Condon Adaptation Tool对PDGF-M2(即PDGF-IM-P)进行密码子优化，优化后编码DNA序列如下：
编码PDGF-IM-P的DNA序列：
5’GCTATCGCTGAACCAGCTATGATCGCTGAATGTAAGACTAGAACTGAAGTTTTCGAAATCTCTAGAAGATTGATCGACCCAACTAACGCTAACTTCTTGGTTTGGCCACCATGTGTTGAAGTTCAAAGATGTTCTGGTTGTTGTAACAACAGAAACGTTCAATGTAGACCAACTCAAGTTCAATTGAGACCAGTTCAAGTTAGAAAGATCGAAATCGTTAGAAAGAAGCCAATCTTCAAGAAGGCTACTGTTACTTTGGAAGACCACTTGGCTTGTAAGTGTGAAGCTGTTGCTGCTGCTAGACCAGTTGCT-3’(SEQ ID NO：7)。
蛋白序列同PDGF-M2。
在密码子优化的基础上，将Arg32分别突变为Val(PDGF-IM-V，Val密码子采用GTT，)和Ile(PDGF-IM-I，Ile密码子采用ATC)，与PDGF-M2的表达水平进行分析，以分析Arg32的突变对蛋白表达的影响。
PDGF-IM-V的核苷酸序列为：
GCTATCGCTGAACCAGCTATGATCGCTGAATGTAAGACTAGAACTGAAGTTTTCGAAATCTCTAGAAGATTGATCGACGTTACTAACGCTAACTTCTTGGTTTGGCCACCATGTGTTGAAGTTCAAAGATGTTCTGGTTGTTGTAACAACAGAAACGTTCAATGTAGACCAACTCAAGTTCAATTGAGACCAGTTCAAGTTAGAAAGATCGAAATCGTTAGAAAGAAGCCAATCTTCAAGAAGGCTACTGTTACTTTGGAAGACCACTTGGCTTGTAAGTGTGAAGCTGTTGCTGCTGCTAGACCAGTTGCT(SEQ ID NO：8)。
PDGF-IM-I的核苷酸序列为：
GCTATCGCTGAACCAGCTATGATCGCTGAATGTAAGACTAGAACTGAAGTTTTCGAAATCTCTAGAAGATTGATCGACATCACTAACGCTAACTTCTTGGTTTGGCCACCATGTGTTGAAGTTCAAAGATGTTCTGGTTGTTGTAACAACAGAAACGTTCAATGTAGACCAACTCAAGTTCAATTGAGACCAGTTCAAGTTAGAAAGATCGAAATCGTTAGAAAGAAGCCAATCTTCAAGAAGGCTACTGTTACTTTGGAAGACCACTTGGCTTGTAAGTGTGAAGCTGTTGCTGCTGCTAGACCAGTTGCT(SEQ ID NO：9)。
将编码PDGF-IM-P、PDGF-IM-V、PDGF-IM-I的DNA序列连接酶切位点、终止子等序列后克隆至pMEX9K表达载体，并整合至GS115表达菌株。经组氨酸缺陷MD平板筛选后，随机挑选9个克隆，在试管内进行甲醇诱导表达。培养上清经SDS-PAGE电泳分析，PDGF-IM-P蛋白表达量明显高于其他两株(图8A)。
通过G418对GS115/PDGF-IM-P、GS115/PDGF-IM-V、GS115/PDGF-IM-P进行了多拷贝插入子(Multiple Inserts)克隆的筛选。对分别在G418浓度为2.0mg/ml和4.0mg/ml平板上生长克隆进行了表达分析。结果仍然表明，PDGF-IM-P的平均表达水平高于其余两株(图8B)。这说明，Arg32位点的突变会影响PDGF在毕赤酵母中分泌表达水平，而将位点突变为Pro对表达相对有利。
实施例5 LC/MS方法检测PDGF-B和PDGF-M2突变体的糖基化方法
重组PDGF-B野生型和PDGF-M2突变体用DTT(2.5mM)在37℃条件下还原30min，接着用缓冲液A(含0.1％甲酸的水溶液)进行稀释，然后进行液相色谱和质谱联用(LC/MS)分析。利用EASY-nLC系统(Thermo Fisher Scientific)，在Easy-spray柱(15cm×75μm ID,3-μm C18particles)上进行蛋白的分离，使用线性梯度的缓冲液B(含0.1％甲酸的甲醇溶液；0–90％，20min)洗脱，流速300nl/min。使用Q Exactive Mass Spectrometer(Thermo Fisher Scientific)，在分辨率为60,000、m/z 350–1600的条件下获得高分辨率谱图，并用Xtract软件(Thermo Scientific)去卷积化。
结果
利用高分辨率LC/MS对PDGF-B野生型和PDGF-M2突变体的分析结果表明，在野生型PDGF中可检测到最多含有6个糖残基的不同异构体，其中含有三个糖残基的异构体含量最高。而同时，PDGF-M2(M2)突变体几乎检测不到糖基化(如图9所示)。
尽管本发明的具体实施方式已经得到详细的描述，本领域技术人员将会理解。根据已经公开的所有教导，可以对那些细节进行各种修改和替换，这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。
参考文献
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Claims (27)

  1. 血小板衍生生长因子B突变体，其在野生型血小板衍生生长因子B的第101位和第109位氨基酸位点处具有突变，并且具有血小板衍生生长因子B的活性。
  2. 权利要求1的血小板衍生生长因子B突变体，其在第6位氨基酸位点处具有突变，并且具有血小板衍生生长因子B的活性。
  3. 权利要求1或2的血小板衍生生长因子B突变体，其在第32位和/或第33位氨基酸位点处具有突变，并且具有血小板衍生生长因子B的活性。
  4. 权利要求1-3任一项的血小板衍生生长因子B突变体，与野生型血小板衍生生长因子B相比，其N端缺失5个氨基酸，并且具有血小板衍生生长因子B的活性。
  5. 权利要求1-4任一项的血小板衍生生长因子B突变体，其在第6位、第101位和第109位氨基酸位点处突变为丙氨酸。
  6. 权利要求1-4任一项的血小板衍生生长因子B突变体，其在第101位和第109位氨基酸位点处突变为丙氨酸。
  7. 权利要求1-6任一项的血小板衍生生长因子B突变体，其在第32位和/或第33位氨基酸位点处突变为脯氨酸、缬氨酸或异亮氨酸。
  8. 权利要求1-7任一项的血小板衍生生长因子B突变体，其中所述的血小板衍生生长因子B为哺乳动物来衍生的血小板衍生生长因子B，所述哺乳动物例如为人、小鼠。
  9. 权利要求1-8任一项的血小板衍生生长因子B突变体，其氨基酸序列为SEQ ID NO：3或SEQ ID NO：5所示的序列。
  10. 权利要求1-9任一项的血小板衍生生长因子B突变体，其氨基酸序列经过取代、缺失或添加一个或几个氨基酸，并且具有血小板衍生生长因子B的活性。
  11. 血小板衍生生长因子同源或异源二聚体，其由两个权利要求1-10任一项的血小板衍生生长因子B突变体通过链内和/或链间二硫键结合而成，或者由一个权利要求1-10任一项的血小板衍生生长因子B突变体与一个血小板衍生生长因子A通过链内和/或链间二硫键结合而成。
  12. 核酸分子，其编码权利要求1-10任一项的血小板衍生生长因子B突变体。
  13. 权利要求12的核酸分子，其核苷酸序列选自SEQ ID NO：4、SEQ ID NO：6～SEQ ID NO：9所示的序列。
  14. 载体，其含有权利要求12或13的核酸分子。
  15. 宿主细胞，其含有权利要求14的载体。
  16. 权利要求15的宿主细胞，其为真核细胞，例如为酵母细胞、哺乳动物细胞或昆虫细胞。
  17. 权利要求16的宿主细胞，其为毕赤酵母细胞。
  18. 权利要求1-10任一项的血小板衍生生长因子B突变体的制备方法，其包括取权利要求14或15的宿主细胞进行培养、表达(例如诱导表达)以及任选的纯化的步骤。
  19. 血小板衍生生长因子B或其突变体的纯化方法，其包括取含有血小板衍生生长因子B或其突变体的培养上清或细胞裂解液依次进行疏水层析、离子交换层析和凝胶层析的步骤。
  20. 权利要求19的纯化方法，其中所述血小板衍生生长因子B突变体为权利要求1-10任一项的血小板衍生生长因子B突变体。
  21. 权利要求19或20的纯化方法，其选自以下1)-3)项中的一项或几项：
    1)用于疏水层析的层析介质为Phenyl Sepharose 6Fast Flow；
    2)用于离子交换层析的层析介质为Source 30S；
    3)用于凝胶层析的层析介质为Hiload Superdex 75prep grad。
  22. 权利要求19-21任一项的纯化方法，其中,
    所述的疏水层析包括以下步骤：
    (1)取含有血小板衍生生长因子B或其突变体的培养上清或细胞裂解液用调节缓冲液调节电导，所述调节缓冲液加入后的终体系为10-50mM磷酸缓冲液，0.8-1M(NH4)2SO4，pH 6.8-7.5；
    (2)用平衡缓冲液平衡柱子，所述平衡缓冲液的配方为10-50mM磷酸缓冲液，0.8-1M(NH4)2SO4，pH 6.8-7.5；
    (3)样品上柱后，用平衡缓冲液洗柱；
    (4)用洗脱缓冲液洗脱，收集目的蛋白，所述洗脱缓冲液的配方为，10-50mM磷酸缓冲液，30％-50％乙二醇，pH 6.8-7.5；
    所述离子交换层析包括以下步骤：
    (1)用平衡缓冲液稀释疏水层析的洗脱峰至电导值为6mS/cm以 下，所述平衡缓冲液的配方为，10-50mM磷酸缓冲液，pH 6.8-7.5；
    (2)用平衡缓冲液平衡柱子；
    (3)样品上柱后，用平衡缓冲液清洗柱子；
    (4)用洗脱缓冲液梯度洗脱，收集目的蛋白，所述洗脱缓冲液的配方为10-50mM磷酸缓冲液，0.8-1.2M M NaCl，pH 6.8-7.5；
    所述凝胶层析包括以下步骤：
    (1)用磷酸盐缓冲液平衡柱子，所述磷酸盐缓冲液的配方为，10-50mM磷酸缓冲液，0.1-0.5M NaCl，pH 6.8-7.5；
    (2)将离子交换层析的洗脱峰上样，每次上样体积不超过柱体积的0.3-4％；
    (3)继续用步骤(1)中的磷酸盐缓冲液清洗柱子，收集目的蛋白，即得到纯化的血小板衍生生长因子B或其突变体。
  23. 权利要求1-10任一项的血小板衍生生长因子B突变体用于制备促进细胞分裂、增殖、促进伤口愈合、皮肤再生、骨骼与牙齿缺损再生、关节修复的药物中的用途。
  24. 抗体，其能够与权利要求1-10任一项的血小板衍生生长因子B突变体特异性结合。
  25. 一种使血小板衍生生长因子表达更均一化的方法，其包括对野生型血小板衍生生长因子的氨基酸序列进行改造的步骤，所述改造包括以下a)-c)中的一项或几项：
    a)突变第101位和第109位氨基酸；
    b)突变第6位氨基酸；
    c)突变第32位和/或第33位氨基酸；
    d)N端缺失5个氨基酸。
  26. 权利要求25的方法，其特征在于以下i)-iii)中的一项或几项：
    i)将第101位和第109位氨基酸突变为丙氨酸；
    ii)将第6位氨基酸突变为丙氨酸；
    iii)将第32位和/或第33位氨基酸突变为脯氨酸、缬氨酸或异亮氨酸。
  27. 促进细胞分裂、增殖、促进伤口愈合、皮肤再生、骨骼与牙齿缺损再生、关节修复的方法，所述方法包括给予有需要的受试者有效量的权利要求1-10任一项的的血小板衍生生长因子B突变体的步骤。
PCT/CN2015/084260 2014-05-16 2015-07-16 血小板衍生生长因子b突变体、其制备方法及用途 WO2015172752A2 (zh)

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