Platelet-derived growth factor B mutant and application thereof

Abstract

The invention provides a PDGF-B mutant, and the amino acid sequence of the PDGF-B mutant is shown as SEQ ID NO.3. The mutant has higher inducible expression concentration, and the yield and the biological activity are remarkably improved.

Description

Technical Field

[0001] The present invention discloses a platelet-derived growth factor B mutant, belonging to the technical field of polypeptides. Background Art

[0002] Platelet-derived growth factor is an important component of platelets and is released during the blood clotting process at the site of injury. Platelet-derived growth factor is synthesized by platelets, smooth muscle cells, activated macrophages, and activated endothelial cells, and can promote cell mitosis and enhance chemotaxis to promote tissue repair and regeneration. Recombinant human platelet-derived growth factor is an important mitogen that can promote the proliferation of cells such as skin, cornea, osteoblasts, and periodontal ligament fibroblasts, exert chemotactic effects, promote cell migration, and promote the synthesis of blood vessels and matrix, achieving the effects of tissue regeneration and wound repair.

[0003] Platelet-derived growth factor (PDGF) is now considered a family of five heterodimeric and homodimeric proteins (PDGF-AB, PDGF-AA, PDGF-BB, PDGF-CC, and PDGF-DD). When PDGFs exist in monomeric form (PDGF-A, PDGF-B, PDGF-C, and PDGF-D), they are in an inactive state; when dimerized by disulfide bonds to form homodimeric subtypes (PDGF-AA, PDGF-BB, PDGF-CC, and PDGF-DD) or heterodimeric subtypes (PDGF-AB), they are in an active state. PDGFs exert their corresponding functions by binding to tyrosine kinase transmembrane receptors (PDGFRs) containing homodimeric and heterodimeric subtypes (PDGFR-αα, PDGFR-ββ, and PDGFR-αβ), where PDGFRs include an extracellular binding domain and an intracellular tyrosine kinase domain. Studies have shown that PDGF-BB can bind to three PDGFRs (PDGFR-αα, PDGFR-ββ, and PDGFR-αβ), while PDGF-AA can only bind to one PDGFR (PDGFR-αα). Therefore, the widely used one is PDGF-BB. When PDGFs bind to the extracellular binding domain of PDGFRs, it will cause autophosphorylation and activation of tyrosine residues in the cytoplasm, thereby triggering downstream reactions of the signaling pathway and promoting cell proliferation and chemotaxis.

[0004] The PDGF-B gene is located on chromosome 22 and contains 7 exons. It encodes a precursor protein composed of 241 amino acids. The final mature product formed by proteolytic hydrolysis and processing is a polypeptide composed of 109 amino acids with a molecular weight of 12.3 kD. PDGF-BB is a homodimer formed by two PDGF-B monomers through disulfide bonds. Each PDGF-B protein monomer contains 8 highly conserved cysteine residues. Among them, 6 cysteines form intramolecular disulfide bonds in pairs (Cys16-Cys60, Cys49-Cys97, and Cys53-Cys99), and the other 2 form intermolecular disulfide bonds with the corresponding monomers in a cross-linking manner (Cys43 and Cys52). These intramolecular and intermolecular disulfide bonds constitute the complex spatial structure of the PDGF-BB dimer protein. The intramolecular disulfide bonds are important for biological functions, and the intermolecular disulfide bonds are more important for the complete molecular structure. The PDGF-B protein monomer has three splicing forms: analysis by N-terminal aa shows that there are three splicing forms, 20% Ser1, 45% Thr6, and 35% Thr33. The heterogeneity of these cleavages leads to heterogeneity in the purified PDGF. A large number of studies have found that the platelet-derived growth factor has different splicing forms formed by the degradation of specific-site proteases, post-translational glycosylation modification, and complex dimer structures, etc., which affect the homogeneity, biological activity, stability, and physicochemical properties of the platelet-derived growth factor, and are the main reasons for the existence of various PDGF-Bs, posing a huge challenge to the druggability of the platelet-derived growth factor.

[0005] A large number of research data show that site-directed mutagenesis of some cleavage sites of the wild-type platelet-derived growth factor can improve the post-translational splicing and post-translational modification forms of the platelet growth factor dimer, and further affect the biological activity, homogeneity, and related physicochemical properties of the platelet-derived growth factor. In particular, the modification of the amino acid sequence in the binding region between the platelet-derived growth factor and its receptor directly affects the platelet-derived growth factor to activate its downstream signaling pathway and execute its biological functions. CN105085652A discloses a platelet-derived growth factor B mutant, aiming to improve the homogeneity of the PDGF-B protein on the premise of retaining protein activity. Both homogeneity and glycosylation are key factors affecting protein activity. Some specific sites of proteins are often related to protease degradation (homogeneity) and / or glycosylation modification at the same time. The ideal mutation site not only lies in maintaining the homogeneity of the protein, but also in not affecting the glycosylation site of the protein, and even optimizing the glycosylation site to improve the yield and biological activity of the mutant. The purpose of the present invention is to provide a platelet-derived growth factor B mutant with higher yield and improved activity. Summary of the Invention

[0006] For the above purposes, the present invention first provides a platelet-derived growth factor B (abbreviated as PDGF-B in the present invention) mutant, and the amino acid sequence of the PDGF-B mutant is shown in SEQ ID NO.3. In a specific embodiment of the present invention, the mutant is named "G1".

[0007] Secondly, the present invention provides a polynucleotide encoding the PDGF-B mutant. Based on the common knowledge of those skilled in the art and following the triplet codon rule of protein coding, the same amino acid has different triplet nucleotide codings. Therefore, any polynucleotide capable of encoding the PDGF-B mutant with the amino acid sequence shown in SEQ ID NO.3 is within the scope of the polynucleotide encoding the PDGF-B mutant defined by the present invention.

[0008] In a specific embodiment, the sequence of the polynucleotide is shown in SEQ ID NO.4.

[0009] Thirdly, the present invention provides an expression vector containing the above polynucleotide. In the art, any vector capable of expressing a coding gene with the polynucleotide sequence shown in SEQ ID NO.4 and producing the PDGF-B mutant with the amino acid sequence shown in SEQ ID NO.3 is within the protection scope of the present invention.

[0010] In a specific embodiment, the expression vector is pPICZαA. The pPICZαA is a commercially available Pichia pastoris protein secretion expression vector with a base length of 3.6 kb. The expressed recombinant protein is a fusion protein containing an N-terminal polypeptide encoding the α-factor secretion signal of Saccharomyces cerevisiae. The vector can use methanol induction to highly express the target protein in Pichia pastoris and can be used in any Pichia pastoris, including X33, GS115 strain, SMD1168H, KM71H. The pPICZαA vector contains the following elements: strict regulation with the AOX1 promoter at the 5'-end to express any gene of interest using methanol induction; the α-factor secretion signal can secrete and express the target protein; the Zeocin resistance gene can be used for screening in both Escherichia coli and Pichia pastoris; the C-terminal contains Myc and His tags, which can be used for detecting and purifying the recombinant protein.

[0011] Fourthly, the present invention provides a host cell containing the above expression vector. In a specific embodiment, the host cell is Pichia pastoris GS115.

[0012] Fifthly, the present invention provides a method for preparing the above PDGF-B mutant, and the method includes the following steps: (1)Construct Pichia pastoris GS115 containing the vector expressing the PDGF-B mutant as described above in the present invention; (2)Initial cell culture: Inoculate the seed solution of Pichia pastoris GS115 described in step (1) into a fermentation medium containing sodium chloride, with an inoculation ratio of 10 - 15 v / v%. The initial culture conditions are 30 - 32 °C, pH 4.5 - 5.5, and an aeration rate of 0.5 - 1.5 vvm. As the cells grow continuously, control the dissolved oxygen above 40% by increasing the rotation speed and aeration rate. When the dissolved oxygen value rapidly rebounds above 80%, proceed to step (3); (3)Supplementation of glycerol: After step (2) is completed, supplement a glycerol solution of about 50 v / v%. The supplementation rate starts at 8 - 12 ml / L / h and is supplemented in an exponential feeding manner. During the culture process, adjust the rotation speed and aeration rate to control the dissolved oxygen at 30 - 60%. When the wet cell weight reaches 145 - 155 g / l, stop supplementing glycerol; (4)Induction: After step (3) is completed, without starvation, lower the culture temperature to 24 - 28 °C, and start supplementing a 100% methanol solution for induction culture. During the induction process, adjust the rotation speed and aeration rate to control the dissolved oxygen at 35 - 55%. After 48 hours of induction, end the fermentation.

[0013] In a preferred technical solution, the above method further undergoes a purification step after step (4). The purification step includes cation exchange chromatography, gel chromatography, and strong anion composite mode chromatography steps.

[0014] In a preferred embodiment of the present invention, the packing material for the cation exchange chromatography is a strong cation exchange chromatography packing material of sulfopropyl, the packing material for the gel chromatography is a cross-linked agarose and dextran composite packing material, and the ligand for the strong anion composite mode chromatography is N-benzyl-n-methylethanolamine.

[0015] In a specific embodiment of the present invention, the cation exchange chromatography uses an SP Sepharose FF cation exchange chromatography column from Cytiva; the gel chromatography uses a Superdex™ 75 Increase gel chromatography column from Cytiva; the strong anion composite mode chromatography uses Capto adhere and Capto adhere ImpRes from Cytiva.

[0016] Finally, the present invention provides the application of the above PDGF-B mutant in the preparation of drugs for tissue regeneration and / or wound repair.

[0017] The PDGF-B mutant G1 provided by the present invention has a better induction expression concentration. The three PDGF-B mutants G0, G1, and G2 are fermented under the same conditions and the same scale, and are subjected to three-step chromatography under the same conditions. The volumes of the harvested crude target protein solutions are basically the same. However, the final protein concentration of the G1 mutant is the highest, reaching 1.85 mg / ml. Under the same conditions, the yield of the G1 mutant is about 20% higher than that of G0. Moreover, compared with the G0 mutant, the biological activity of G1 is increased by more than 2 times and is 5.2 times that of the international standard product, indicating that the biological activity of the PDGF-B mutant G1 provided by the present invention has been significantly improved. Description of the Drawings

[0018] Figure 1 . Schematic diagram of the PDGF-B protein structure, where the signal peptide (Signalpeptide) of amino acids 1-20, the pre-sequence of the growth factor domain (PRO) of amino acids 21-81, the mature PDGF-B peptide segment composed of 109 amino acids of amino acids 82-190, and the peptide segment that will also be cleaved during protein maturation of amino acids 191-241.

[0019] Figure 2 . Schematic diagram of the molecular construction process of the platelet-derived growth factor B mutant. The mutant of platelet-derived growth factor B is inserted into the vector PICZαA by the method of GIBSON assembly.

[0020] Figure 3 . SDS-PAGE electrophoresis diagram of different recombinant platelet-derived growth factor BB mutants after purification. Lane 5 is Marker, lane 6 is the electrophoresis diagram of the purified mutant G0, lane 7 is the electrophoresis diagram of the purified mutant G1, and lane 8 is the electrophoresis diagram of the purified mutant G2. It can be seen from the electrophoresis diagram that the purity of the three mutants after purification is 100%.

[0021] Figure 4 . Statistical chart of the biological activities of different platelet-derived growth factor B mutants after purification, where ST is the international standard product of platelet-derived growth factor BB (NIBSC, 94 / 728); G0 is the biological activity of platelet-derived growth factor BB with 5 amino acids deleted at the N-terminus; G1 is the biological activity of platelet-derived growth factor BB with 5 amino acids deleted at the N-terminus and the 32nd amino acid mutated to proline; G2 is the biological activity of platelet-derived growth factor BB with 5 amino acids deleted at the N-terminus, the 32nd amino acid mutated to proline, and the 6th, 101st, and 109th amino acids mutated to alanine. Detailed Embodiments

[0022] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are merely exemplary and do not constitute any limitation to the protection scope defined by the claims of the present invention.

[0023] Example 1. Mutation of PDGF-B protein and preparation of mutants

[0024] 1. Construction of recombinant expression clones Figure 1 Figure 1 is a schematic diagram of the PDGF-B protein structure. The precursor protein consists of 241 amino acids in total, including a signal peptide (Signal peptide) of amino acids 1-20, a pro-sequence preceding the growth factor domain (PRO) of amino acids 21-81, a mature PDGF-B peptide segment composed of 109 amino acids (Mature peptide) of amino acids 82-190, and a peptide segment that will also be cleaved during protein maturation of amino acids 191-241.

[0025] The present invention constructs 3 kinds of PDGF mutants respectively. Among them, a 104-amino acid sequence lacking the N-terminal 5 amino acids is G0 (CN105085652A); the one with the N-terminal 5 amino acids deleted and the arginine at position 32 mutated to proline is G1; the one with the N-terminal 5 amino acids deleted, the arginine at position 32 mutated to proline, and the threonine at positions 6, 101, and 109 mutated to alanine is G2.

[0026] The above mutant DNA sequences were all synthesized by Tsingke Biotechnology, and the synthesized sequences were inserted into the pPICZαA plasmid and named pPICZαA-G0, pPICZαA-G1, pPICZαA-G2 respectively. After the obtained plasmid sequences were confirmed to be correct by sequencing, they were amplified by Escherichia coli and the plasmids were extracted. Figure 2 Figure 2 shows a schematic diagram of the construction of inserting mutants of platelet-derived growth factor B into the vector PICZαA by GIBSON assembly. The construction process is briefly described as follows: The PICZαA vector was amplified from the two restriction enzyme sites (1271bp - 1195bp) of XhoⅠ and XbaⅠ by PCR, and then the synthesized platelet-derived growth factor mutant sequences were amplified by PCR to obtain target fragments with overlapping sequences of XhoⅠ and XbaⅠ restriction enzyme sites. Finally, the target fragments were inserted into the PICZαA vector by Giboson assembly to form three mutant vectors pPICZαA-G0, pPICZαA-G1, and pPICZαA-G2.

[0027] 2. Preparation of Genetically Engineered Bacteria The plasmids containing the PDGF-B mutant genes were linearized by digestion with SacⅠ enzyme to obtain linearized plasmids. The digestion system is shown in Table 1. The reaction was carried out in a water bath at 37°C for 2.0 h.

[0028] Table 1. SacⅠ Enzyme Digestion System of Expression Vector

[0029] After complete digestion, the obtained linearized plasmids were purified and stored at -20°C.

[0030] 2.1 Electroporation of Linearized Plasmids The linearized plasmids pPICZαA-G0, pPICZαA-G1, and pPICZαA-G2 were transformed into the competent cells of Pichia pastoris GS115 by electroporation. The specific steps are as follows: 2.1.1 Thaw the prepared competent cells and the purified linearized plasmids on ice. Pre-cool the YPDS medium and the electroporation cuvette on ice. 2.1.2 Put 900 ul of competent cells and 100 ul of linearized plasmid into a 1.5 ml sterile EP tube and mix well. Then transfer the mixture into a pre-cooled electroporation cuvette (0.2 cm). 2.1.3 Wipe the electroporation cuvette clean and place it in the electroporator. Select the electroporation program for Pichia pastoris (Pic-2kv 5ms) for electroporation. 2.1.4 Immediately after electroporation, add 900 ul of YPDS medium to the electroporation cuvette, mix well, and transfer it to a 1.5 ml sterile EP tube.

[0031] 2.2 Screening of Electroporated Strains Put the 1.5 ml sterile centrifuge tube containing the electroporated strains into a 30°C constant temperature incubator for 1.5 - 2 h; then transfer it to a constant temperature shaker and culture at 30°C and 220 rpm for 1.5 - 2 h. Then, perform primary screening of the strains on YPD (Yeast Extract Peptone Dextrose Medium) containing Zecion (bleomycin) resistance, and screen for monoclonal clones with high expression efficiency and identify positive recombinant yeast strains by PCR method.

[0032] 3. Induced Expression of Recombinant Protein 3.1 Initial Cultivation of Bacteria Inoculate the monoclonal of the screened recombinant yeast strain into YPD liquid medium for seed culture. After amplification culture, inoculate it into the fermentation medium (yeast extract: 10 g / L, tryptone: 20 g / L, potassium dihydrogen phosphate: 16 g / L, glycerol: 20 g / L, sodium chloride: 10 g / L, YNB (yeast nitrogen base medium): 12 g / L, pH value 5.5) at an inoculation ratio of 10 - 15 v / v%. The initial culture conditions are 30 - 32 °C and the aeration rate is 0.5 - 1.5 vvm. As the cells grow continuously, by increasing the rotation speed and aeration rate, control the dissolved oxygen above 40%. When the dissolved oxygen value quickly rebounds above 80%, enter the glycerol supplementation stage.

[0033] 3.2 Glycerol Supplementation After the initial culture of the cells, supplement about 50 v / v% glycerol solution. The supplementation rate is an exponential supplementation process starting from 8 - 12 ml / L / h. During the culture process, adjust the rotation speed and aeration rate to control the dissolved oxygen at 30 - 60%. When the wet cell weight is 150 ± 5 g / l, stop supplementing glycerol.

[0034] 3.3 Induced Expression After the glycerol supplementation stage, without starvation, lower the culture temperature to 24 - 28 °C, and start supplementing 100% methanol solution for induced culture. During the induction process, adjust the rotation speed and aeration rate to control the dissolved oxygen at 35 - 55%; After the induction ends, centrifuge the obtained bacterial liquid at 6000 rpm for 10 min, and then clarify the fermentation supernatant with a 0.22 - um filter membrane.

[0035] 4. Purification of Recombinant Protein The fermentation supernatant obtained after centrifugation and filtration is successively passed through cation exchange chromatography such as the SP Sepharose FF cation exchange chromatography column of Cytiva, whose packing material is sulfopropyl (SP) strong cation exchange chromatography packing material for rapid protein purification; gel filtration chromatography such as the Superdex™ 75 Increase gel filtration chromatography column of Cytiva, whose packing material is a cross - linked agarose and dextran complex; strong anion composite mode chromatography such as Capto adhere and Capto adhere ImpRes of Cytiva, whose ligand is N - benzyl - n - methylethanolamine, which can efficiently remove various negatively charged impurities such as endotoxin, HCP (host cell protein), and nucleic acid in the sample, and has higher purification efficiency and stronger impurity - removing ability than single anion exchange chromatography. The three chromatography modes endow the purification of platelet - derived growth factor mutants with advantages such as high throughput, high purity, easy operation, more economical, and safer.

[0036] 4.1 Ion Exchange Chromatography Includes the Following Steps: 4.1.1. Clarify the fermentation broth by centrifuging the culture supernatant containing the platelet-derived growth factor B mutant and then subjecting it to sterile filtration in series through a depth filter. 4.1.2. Connect the chromatography column to the chromatography system, set the linear flow rate at 300 cm / h, and equilibrate the chromatography column with 3 - 10 column volumes (CV) of equilibration buffer, which is 20 mmol / L PB, 0.15 mol / L sodium chloride, pH 7.2. 4.1.3. After loading the sample onto the column, wash the column with the equilibration buffer. 4.1.4. Elute the impurity proteins on the column using the wash buffer, which is 20 mmol / L PB, 0.5 mol / L sodium chloride, pH 7.2. 4.1.5. Elute the target protein using the elution buffer, collect the target protein, and the eluent is 20 mmol / L PB, 1 mol / L sodium chloride, pH 7.2.

[0037] 4.2 Gel filtration chromatography includes the following steps: 4.2.1. Connect the chromatography column to the chromatography system and set the linear flow rate at 30 cm / h. First, wash the chromatography column with 0.5 M NaOH for 1 - 2 column volumes (CV), and then soak it for at least 30 min. 4.2.2. Equilibrate the chromatography column with 2 - 3 column volumes (CV) of equilibration buffer, which is 20 mmol / L PB, 0.15 mol / L NaCl, pH 7.2. 4.2.3. Divide the target protein eluate from the previous step into two equal parts and load them onto the gel filtration chromatography column in two batches. The volume of each loading should not exceed 7% of the column volume (CV), and the interval between the two loadings should be at least 1 column volume (CV). 4.2.4. Continue to wash the column with the equilibration buffer and collect the target protein to obtain purified platelet-derived growth factor B or its mutant.

[0038] 4.3 Anion exchange chromatography in composite mode includes the following steps: 4.3.1. Connect the chromatography column to the chromatography system and set the linear flow rate at 300 cm / h. First, wash the chromatography column with 0.5 M NaOH for 5 column volumes (CV), and then soak it for at least 30 min. Then, regenerate the chromatography column with 1 M NaCl for 5 column volumes (CV). 4.3.2. Equilibrate the chromatography column with 10 column volumes (CV) of equilibration buffer, which is 20 mmol / L PB, 0.15 mol / L NaCl, pH 7.2. 4.3.3. Load the eluate obtained in the previous step onto the chromatography column. After starting the loading, collect the flow-through to obtain the purified mutant of platelet-derived growth factor B.

[0039] 5. SDS-PAGE Detection of Recombinant Protein Take 10 μg of the purified PDGF-B mutant stock solution and add it to the precast electrophoresis gel containing SDS (SurePAG, Bis-Tris, 15%, 12 wells). The loading buffer is 4X LDS Sample Buffer. Place the loaded electrophoresis gel into the Tris-MOPS-SDS Running Buffer for SDS-PAGE electrophoresis (180V, 50 min). Stain and decolorize the electrophoresed gel with an eStain L1 protein staining instrument, and finally perform electrophoretic purity analysis with a gel imager. Figure 3 This is the SDS-PAGE electrophoretogram of different purified recombinant platelet-derived growth factor BB mutants. Lane 5 is Marker, lane 6 is the electrophoretogram of the purified mutant G0, lane 7 is the electrophoretogram of the purified mutant G1, and lane 8 is the electrophoretogram of the purified mutant G2. It can be seen from the electrophoretogram that the purity of the three mutants after purification is 100%.

[0040] 6. Detection of Recombinant Protein Content 6.1 Preparation of Detection Reagents: Preparation of alkaline copper test solution: 4% anhydrous sodium carbonate and 0.2 mol / L NaOH solution as solution A; 2% potassium sodium tartrate hemihydrate as solution B1; 1% copper sulfate pentahydrate as solution B2.

[0041] 6.2 Preparation of Standard Product: Take bovine serum albumin (9048-46-8, National Institutes for Food and Drug Control) or the national standard product for protein content determination as the reference substance, dissolve it in water and prepare a 1 mg / ml solution.

[0042] 6.3 Preparation of Solutions: 6.3.1 Standard Product: Dilute the 1 mg / ml standard product to 200 μg / ml. Then add the corresponding volumes of reagents according to the content in Table 2.

[0043] Table 2. Standard Product Gradient Table

[0044] 6.3.2 Test Sample: Dilute the test sample to 100 μg / ml with a final volume of 1 ml.

[0045] 6.4 Detection Mix the solution A, solution B1, solution B2 of the alkaline copper reagent and the injection water in a volume ratio of 20:2.5:1.5:1. Then add 1 ml of the alkaline copper solution to each of the diluted standard product and the test sample, vortex and mix well, and let stand for 10 minutes. Mix the injection water and the Folin-Ciocalteu reagent (F9252, sigma) in a ratio of 15:1, and add 4 ml of the diluted Folin-Ciocalteu reagent to each of the test tubes of the standard product and the test sample that have already added the alkaline copper, vortex and mix well, and let stand for 30 minutes. Use a spectrophotometer to detect the absorbance value at a wavelength of 650 nm. Pour the prepared samples into the cuvettes in sequence for detection, and use the standard product of 0 μg / ml for blank zero adjustment. The experimental data is calculated by the linear regression calculation method, and the content of the protein sample is calculated according to the regression equation of the standard product. The parameters of the absorbance-protein content standard curve are shown in Table 3.

[0046] Table 3. Parameters of the absorbance-protein content standard curve

[0047] Table 4. Comparison table of mutant protein contents

[0048] Three PDGF-B mutants G0, G1, and G2 were fermented under the same conditions and the same scale, and subjected to three-step chromatography under the same conditions. The volumes of the harvested crude target protein solutions were basically the same. However, as shown in Table 4, the final protein concentration of the G1 mutant was the highest, and finally the yield of the G1 mutant was also the highest. It was found that the yield of the G1 mutant was about 20% higher than that of G0 and G1 under the same conditions.

[0049] Example 2. Detection of the biological activity of the PDGF-B protein mutant BALB / c 3T3-SRE-Luc cells (obtained by transfecting pGL4.33 plasmid into BALB / c 3T3 cells using the electroporation method) were cultured in a DMEM complete medium containing 10% FBS and 1% double antibody (penicillin-streptomycin) in an incubator at 37 °C and 5% carbon dioxide. After digesting the cells with 0.25% trypsin (0.02% EDTA), collecting and centrifuging, and counting with a cell counter, they were configured into 5×10 4A cell suspension of [[[number]]] cells / ml was inoculated into a 96-well cell culture plate with a white bottom and transparent walls. 100 μl of the cell suspension was added to each well and cultured in an incubator at 37 °C with 5% carbon dioxide for 8 h. Then, it was changed to DMEM basal medium and continued to be cultured in the incubator at 37 °C with 5% carbon dioxide overnight (15 h - 16 h). The original medium was discarded, and the pre-gradient-diluted recombinant PDGF-BB solution (100 μl per well) was added. Duplicate wells were set for each concentration. It was continued to be cultured in the incubator at 37 °C with 5% carbon dioxide for 4 h. The cell proliferation activity was detected by the reporter gene method. 100 μl of One-Lite Luciferase Assay System fluorescence enzyme activity detection reagent (DD1203, Novoprotein) was added to each well, and after incubating in the dark at room temperature for 20 min, the fluorescence signal was detected using SpectraMax Paradigm (Agilent Biotek). The experimental data was calculated using the four-parameter regression calculation method, and the EC50 value of the sample to be tested was calculated based on the EC50 fluorescence value of the standard protein sample. Figure 4 Figure [[[number]]] shows the statistical chart of the biological activities of different purified platelet-derived growth factor B mutants. The specific biological activity ratios are shown in Table 5. Compared with the G0 mutant, the biological activity of G1 was increased by more than 2-fold and was 5.2 times that of the international standard product.

[0050] Table 5. Detected values of the biological activities of samples Please note that the numbers in [[[number]]] need to be filled with the actual values in the original text.

Claims

1. A PDGF-B mutant, characterized in that, The amino acid sequence of the PDGF-B mutant is shown in SEQ ID NO.

3.

2. A polynucleotide encoding the PDGF-B mutant according to claim 1.

3. The polynucleotide according to claim 2, wherein, The sequence of the polynucleotide is shown in SEQ ID NO.

4.

4. An expression vector containing the polynucleotide according to claim 2 or 3.

5. The expression vector according to claim 4, wherein The expression vector is pPICZαA.

6. A host cell containing the expression vector according to claim 5, characterized in that, The host cell is Pichia pastoris GS115.

7. A method for preparing the PDGF-B mutant according to claim 1, characterized in that, The method comprises the following steps: (1) Construct Pichia pastoris GS115 according to claim 6; (2) Initial culture of the bacterial cells: Inoculate the seed solution of Pichia pastoris GS115 in step (1) into a fermentation medium containing sodium chloride, with an inoculation ratio of 10 - 15 v / v%, and the initial culture conditions are 30 - 32 °C, pH 4.5 - 5.5, aeration rate of 0.5 - 1.5 vvm. As the bacterial cells grow continuously, control the dissolved oxygen above 40% by increasing the rotation speed and aeration rate. When the dissolved oxygen value rebounds above 80%, enter step (3); (3) Supplement glycerol: After step (2) ends, supplement a 50 v / v% glycerol solution, with the supplementation rate starting from 8 - 12 ml / L / h and supplemented in an exponential feeding manner. During the culture process, adjust the rotation speed and aeration rate to control the dissolved oxygen at 30 - 60%. When the wet weight of the bacterial cells is 145 - 155 g / l, stop supplementing glycerol; (4) Induction: After step (3) ends, without starvation, lower the culture temperature to 24 - 28 °C, and start supplementing a 100% methanol solution for induction culture. During the induction process, adjust the rotation speed and aeration rate to control the dissolved oxygen at 35 - 55%. After 48 h of induction, end the fermentation.

8. The method according to claim 7, wherein The method further undergoes a purification step after step (4), and the purification step successively includes cation exchange chromatography, gel chromatography, and strong anion composite mode chromatography steps.

9. The method according to claim 8, wherein The packing material for the cation exchange chromatography is a strong cation exchange chromatography packing material of sulfopropyl, the packing material for the gel chromatography is a cross-linked agarose and dextran composite packing material, and the ligand for the strong anion composite mode chromatography is N-benzyl-n-methyl ethanolamine.

10. Use of the PDGF-B mutant according to claim 1 in the preparation of a drug for tissue regeneration and / or wound repair.

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