Preparation method of human vascular endothelial growth factor VEGF165 and freeze-dried powder

Abstract

The invention discloses a preparation method and freeze-dried powder of a human vascular endothelial growth factor VEGF165. The preparation method comprises the following steps: constructing a recombinant plasmid containing a VEGF165 gene, the recombinant plasmid comprises a vector and a VEGF165 gene segment inserted into a vector DNA, the recombinant plasmid has a plurality of resistance genes, and the VEGF165 gene segment has a purification tag; transfecting the recombinant plasmid into mammalian cells to obtain a transfection mixture, and culturing and screening the transfection mixture in a culture medium containing a first antibiotic to obtain a monoclonal cell strain; carrying out ELISA (Enzyme-Linked Immunosorbent Assay) detection on the monoclonal cell strain, calculating the expression quantity of VEGF165, and screening the cell strain with the expression quantity of VEGF165 greater than 10 mg / L to obtain a VEGF stably transfected cell strain; carrying out amplification culture on the VEGF stably transfected cell strain, filtering and concentrating to obtain a VEGF165 protein culture solution; the VEGF165 protein culture solution is subjected to multi-step chromatography purification treatment, VEGF165 protein is obtained, and chromatography columns of multi-step chromatography are different.

Description

Technical Field

[0001] This invention belongs to the field of biology, specifically relating to a method for preparing human vascular endothelial growth factor VEGF165 and its lyophilized powder. Background Technology

[0002] Vascular endothelial growth factor (VEGF) is a key signaling protein that significantly enhances the permeability of vascular endothelial cells. VEGF has been found to primarily promote endothelial cell proliferation, angiogenesis, and increased vascular permeability. Among these, VEGF165 is the most common and potent isoform, composed of 165 amino acids linked by disulfide bonds to form a homodimer, which significantly promotes angiogenesis and enhances vascular permeability.

[0003] Currently, the preparation of VEGF165 mainly relies on two major expression systems: prokaryotic expression systems (represented by Escherichia coli) and eukaryotic expression systems (represented by mammalian cells). However, the E. coli expression system cannot undergo glycosylation modification, easily forming inclusion bodies, and the refolding process leads to the loss of protein activity. On the other hand, the eukaryotic expression system mainly uses transient transfection technology, resulting in low cell expression levels and high costs, making it difficult to scale up production. Furthermore, the purity and yield obtained by existing purification methods are not ideal.

[0004] How to prepare VEGF165 to improve the purity and content of protein expression is a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0005] This invention aims to address, to a certain extent, one of the technical problems in related technologies. Therefore, this invention provides a method for preparing recombinant human vascular endothelial growth factor VEGF165 and a lyophilized powder.

[0006] As a first aspect of the present invention, a method for preparing human vascular endothelial growth factor VEGF165 is provided, the method comprising: A recombinant plasmid containing the VEGF165 gene was constructed. The recombinant plasmid includes a vector and a VEGF165 gene fragment inserted into the vector DNA. The recombinant plasmid has multiple resistance genes, and the VEGF165 gene fragment has a purification tag. The recombinant plasmid was transfected into mammalian cells to obtain a transfection mixture, wherein monoclonal cell lines were obtained by culturing and screening the transfection mixture in a medium containing a first antibiotic. The monoclonal cell lines were subjected to ELISA detection and the expression level of VEGF165 was calculated. Cell lines with VEGF165 expression level greater than 10 mg / L were screened to obtain VEGF stable cell lines. The VEGF-stable cell line was expanded and cultured, and then filtered and concentrated to obtain VEGF165 protein culture medium. The VEGF165 protein culture medium was purified by multi-step chromatography to obtain the VEGF165 protein, wherein different chromatography columns were used in the multi-step chromatography.

[0007] Furthermore, the VEGF165 protein culture medium is purified using a multi-step chromatography process, including: The VEGF165 protein culture medium was subjected to heparin affinity chromatography. The VEGF165 protein culture medium was subjected to nickel affinity chromatography.

[0008] Furthermore, the step of performing heparin affinity chromatography on the VEGF165 protein culture medium includes: The VEGF165 protein culture medium is passed through a chromatography column containing heparin affinity medium at a first flow rate between 0.25 mL / min and 0.5 mL / min. The chromatography column is eluted using a gradient with a salt buffer at a second flow rate between 0.5 mL / min and 1 mL / min. The salt concentration of the salt buffer is positively correlated with the elution time. The salt buffer comprises sodium chloride and tris(hydroxymethyl)aminomethane hydrochloride.

[0009] Further, the step of performing nickel affinity chromatography on the VEGF165 protein culture medium includes: The VEGF165 protein culture medium is passed through a chromatography column containing a nickel affinity medium at a first flow rate between 0.25 mL / min and 0.5 mL / min. The chromatography column was eluted using an imidazole-containing buffer at a second flow rate between 0.5 mL / min and 1 mL / min, wherein the imidazole content in the imidazole-containing buffer was positively correlated with the elution time.

[0010] Further, transfecting the recombinant plasmid into mammalian cells includes: The recombinant plasmid was mixed with serum-reduced culture medium, and then transfection reagent was added. The mixture was incubated at room temperature for 15 to 20 minutes to obtain the transfection complex. The complex to be transfected is mixed with a suspension of mammalian cells to obtain the transfection mixture; Monoclonal cell lines were obtained by culturing and screening the transfection mixture in a medium containing a first antibiotic, comprising: Add puromycin to the transfection mixture, and replace the culture medium containing puromycin every 2 to 3 days for several weeks; The surviving cells were diluted to 0.5 cell / mL to 1 cell / mL with puromycin-containing medium and seeded into the first well of the plate and cultured at 36°C to 38°C for 2 to 3 weeks. Monoclonal cells were picked from the first well plate and transferred for amplification culture. The culture was maintained in a medium containing puromycin to obtain the monoclonal cell line.

[0011] Further, the monoclonal cell line was subjected to ELISA detection and the VEGF165 expression level was calculated, including: The monoclonal cell line was centrifuged to collect the first supernatant; The second well plate is coated with a capture antibody, which includes an anti-human VEGF antibody. Add blocking buffer, the first supernatant, horseradish peroxidase-labeled detection antibody and tetramethylbenzidine colorimetric solution sequentially to the second well plate, incubate and develop color; The absorbance of the solution in the second well plate was measured at a set wavelength, and the expression level of VEGF165 was calculated.

[0012] Further, the VEGF-stabilized cell line was expanded and cultured, and then filtered and concentrated, including: The VEGF-stabilized cell line was transferred to serum-free medium for amplification culture, with an inoculation density of 0.5 × 10⁻⁶ cells / year. 6 cells / mL to 1×10 6 The cell / mL ratio should be between 110 rpm and 130 rpm, and the cells should be passaged every 2 to 3 days. When the cell density exceeds 2×10 6 At a cell / mL level, culture the cells in a medium containing puromycin and monitor feeding, then collect the cell supernatant. The cell supernatant was centrifuged, filtered, and concentrated sequentially to obtain the VEGF165 protein culture medium.

[0013] Furthermore, a recombinant plasmid containing the VEGF165 gene was constructed, including: The vector and VEGF165 gene were digested separately with enzymes and then mixed at a predetermined molar ratio to obtain the ligation product. The vector included a pHBLV vector, and the DNA of the vector contained ampicillin resistance genes and puromycin resistance genes. The restriction endonucleases used for digestion included EcoRI and XhoI, and the predetermined molar ratio was between 1:4 and 1:2. DNA ligase and ligation buffer were added, and the reaction was carried out at a temperature range of 15°C to 17°C. The ligation product was mixed with host cells using a heat shock method and then cultured in a first culture medium containing ampicillin to obtain multiple colonies. The colonies were subjected to plasmid extraction and verification to obtain the recombinant plasmid.

[0014] As a second aspect of this application, a freeze-dried powder is provided, the freeze-dried powder comprising freeze-dried protein, the freeze-dried protein comprising protein prepared using the preparation method described above.

[0015] Furthermore, the freeze-dried powder also includes protein protectants and stabilizers.

[0016] The preparation method of this application successfully established a stable VEGF cell line with an expression level greater than 10 mg / L by constructing a recombinant plasmid containing a gene containing multiple resistance genes and combining it with an antibiotic screening strategy. This cell line exhibits a higher expression level than products synthesized through traditional prokaryotic expression and transient transfection, significantly enhancing the expression level of VEGF165. Utilizing a mammalian cell expression system ensures that the VEGF165 protein has the correct spatial conformation and natural glycosylation modification, resulting in higher protein activity. A multi-step purification process using various chromatographic media with targeted elution further purifies the protein to higher purity. Furthermore, the method of this invention is stable with minimal batch-to-batch variation, enabling seamless transition from laboratory-scale to industrial production. This provides a high-quality, highly active, and economically feasible raw material guarantee for the application of VEGF165 in fields such as tumor diagnostic reagents and angiogenesis therapy drugs.

[0017] These features and advantages of the present invention will be disclosed in detail in the following specific embodiments and accompanying drawings. The preferred embodiments or means of the present invention will be shown in detail in conjunction with the accompanying drawings, but are not intended to limit the technical solutions of the present invention. In addition, each of these features, elements and components appearing in the following text and drawings is a plurality of, and different symbols or numbers are used for convenience of representation, but all represent parts with the same or similar construction or function. Attached Figure Description

[0018] The present invention will be further described below with reference to the accompanying drawings: Figure 1 This is a flowchart of a method for preparing human vascular endothelial growth factor VEGF165 provided in this application; Figure 2 This is the recombinant plasmid map provided in this application; Figure 3 This is a comparison chart of test data for different lanes provided in the test examples of this application; Figure 4 This is a graph of ELISA detection data provided in the embodiments of this application. Detailed Implementation

[0019] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described are intended to explain the present invention and should not be construed as limiting the invention.

[0020] The terms "an embodiment," "example," or "example" used in this specification refer to a particular feature, structure, or characteristic described in connection with the embodiment itself that may be included in at least one embodiment disclosed in this application. The phrase "in an embodiment" appearing in various places throughout the specification does not necessarily refer to the same embodiment.

[0021] The inventors of this application have discovered that the preparation of recombinant human vascular endothelial growth factor VEGF165 mainly relies on two major types of expression systems: prokaryotic expression systems (represented by *E. coli*) and eukaryotic expression systems (represented by mammalian cells). These two technical routes have different applications in industrial practice. In the *E. coli*-based prokaryotic expression system, the technical solution primarily uses *E. coli* as the expression host, cloning the VEGF165 gene into the pET-28a expression vector. By using specific restriction enzyme sites (NcoI / XhoI) and downstream primers with terminators, the expression of the 6×His tag of the vector itself is avoided, thus obtaining "tagless" VEGF165 protein. After the recombinant plasmid is transformed into the BL21(DE3) strain, the target protein is expressed in large quantities after IPTG induction. However, this protein mainly exists in the form of insoluble inclusion bodies, lacking the natural spatial structure and biological activity. The core challenge of this approach lies in the renaturation of the inclusion bodies. First, inclusion bodies are dissolved in high-concentration urea (4-8M), then refolded using a refolding buffer containing oxidized glutathione (GSSG) and reduced dithiothreitol (DTT). After refolding, purification is performed using a DEAE weak anion exchange column, ultimately yielding a protein with approximately 90% purity. In transient transfection systems based on mammalian cells, mammalian cell lines (such as HEK293) are often used for expression to obtain proteins with native activity. Transient transfection is typically employed. A plasmid carrying the VEGF165 gene is transfected into HEK293 cells, where the protein is synthesized intracellularly and secreted into the culture supernatant. Due to the eukaryotic system, the protein folds correctly and undergoes necessary post-translational modifications (such as glycosylation). To facilitate purification, purification tags are usually introduced into the gene sequence, most commonly histidine tags (His-Tag). Accordingly, purification primarily relies on nickel column affinity chromatography. After capture, the cell culture supernatant is directly loaded onto a nickel column, where it is washed with imidazole solutions of varying concentrations to separate the target protein. The advantage of this method is that it yields active proteins, but its main limitations lie in the low expression levels achieved through transient transfection, significant batch-to-batch variability, and high production costs, making it difficult to meet the demands of large-scale industrial production.

[0022] Therefore, the above preparation of VEGF165 protein has the following problems: (1) The E. coli expression system cannot be glycosylated and easily forms inclusion bodies, and the refolding process leads to loss of protein activity; (2) The expression level of transiently transfected HEK293 cells is low and the cost is high, making it difficult to produce on a large scale; (3) A single purification method is difficult to balance purity and recovery rate.

[0023] To address the aforementioned problems, as a first aspect of this invention, a method for preparing VEGF165 with high expression, high activity, and high purity is provided. Specifically, a method for preparing human vascular endothelial growth factor VEGF165 is provided, such as... Figure 1 As shown, the preparation method includes: S100. Construct a recombinant plasmid containing the VEGF165 gene. The recombinant plasmid includes a vector and a VEGF165 gene fragment inserted into the vector DNA. The recombinant plasmid has multiple resistance genes, and the VEGF165 gene fragment has a purification tag. S200. The recombinant plasmid is transfected into mammalian cells to obtain a transfection mixture. The transfection mixture is then cultured and screened in a medium containing the first antibiotic to obtain a monoclonal cell line. S300. Perform ELISA on single-clonal cell lines and calculate VEGF165 expression level. Screen cell lines with VEGF165 expression level greater than 10 mg / L to obtain VEGF stable cell lines. S400, the VEGF stable cell line was expanded and cultured, and then filtered and concentrated to obtain VEGF165 protein culture medium; S500, the VEGF165 protein culture medium was purified by multi-step chromatography to obtain VEGF165 protein, wherein the chromatography columns used in the multi-step chromatography were different.

[0024] The preparation method of this application successfully established a stable VEGF cell line with an expression level greater than 10 mg / L by constructing a recombinant plasmid containing a multi-resistance gene and combining it with an antibiotic screening strategy. This cell line exhibits a higher expression level than products synthesized through traditional prokaryotic expression and transient transfection, significantly enhancing the expression level of VEGF165. Utilizing a mammalian cell expression system ensures that the VEGF165 protein has the correct spatial conformation and natural glycosylation modification, resulting in higher protein activity. A multi-step purification process using various chromatographic media with targeted elution further purifies the protein to higher purity. Furthermore, the method of this invention is stable with minimal batch-to-batch variation, enabling seamless transition from laboratory-scale to industrial production. This provides a high-quality, highly active, and economically viable raw material guarantee for the application of VEGF165 in fields such as tumor diagnostic reagents and angiogenesis therapy drugs.

[0025] As an optional implementation, in step S500, the VEGF165 protein culture medium is purified by multi-step chromatography, including: Heparin affinity chromatography was used to treat the VEGF165 protein culture medium. The VEGF165 protein culture medium was subjected to nickel affinity chromatography.

[0026] Multi-step chromatographic elution using various binding mechanisms of VEGF165 protein achieves higher protein purification. Heparin affinity medium carries a negative charge, while VEGF165 protein carries a positive charge; their specific binding due to this charge difference allows the heparin affinity medium column to adsorb VEGF165 protein and elute it to varying degrees, thus achieving initial coarse purification of the VEGF165 protein culture medium. Nickel affinity medium utilizes the binding of nickel ions to proteins with His-tag purification labels to purify the protein, removing non-specifically bound proteins and further improving purification efficiency.

[0027] In some embodiments, the step of performing heparin affinity chromatography on the VEGF165 protein culture medium includes: The VEGF165 protein culture medium is passed through a chromatography column containing heparin affinity medium at a first flow rate between 0.25 mL / min and 0.5 mL / min. Within this flow rate range, the sample and medium are fully bound together, and the equilibration buffer is introduced to compact the protein binding to the chromatography column. The chromatography column was eluted using a gradient of a salt buffer at a second flow rate between 0.5 mL / min and 1 mL / min. The salt concentration of the salt buffer was positively correlated with the elution time. The salt buffer consisted of sodium chloride and tris(hydroxymethyl)aminomethane hydrochloride. Initially, a low-concentration salt buffer was used to wash away unbound or weakly bound proteins. Then, the salt concentration and elution content of the elution buffer were increased to elute the target protein bound to the heparin affinity medium. Finally, a high-concentration salt buffer was used to elute any remaining strongly bound proteins, thus separating the target protein from impurities.

[0028] In some embodiments, the step of performing nickel affinity chromatography on the VEGF165 protein culture medium includes: The VEGF165 protein culture medium was passed through a chromatography column containing a nickel affinity medium at a first flow rate between 0.25 mL / min and 0.5 mL / min. The chromatography column was eluted using a gradient of imidazole-containing buffer at a second flow rate between 0.5 mL / min and 1 mL / min. The imidazole content in the imidazole-containing buffer was positively correlated with the elution time.

[0029] Nickel affinity chromatography was performed using an elution process similar to heparin affinity chromatography. Imidazole can bind to nickel ions, causing it to compete with the target protein for binding. First, a low concentration of imidazole buffer was used to elute some impurities and weakly binding proteins. Then, the imidazole concentration was increased to elute a large amount of the target protein with the purification tag. Finally, a high concentration of imidazole buffer was used to elute the remaining strongly binding proteins.

[0030] In step S200, this application does not impose special limitations on transfection, as long as it meets the requirement of stable transfection, such as liposome transfection, virus-mediated transfection, etc. As an optional implementation, transfecting the recombinant plasmid into mammalian cells includes: The recombinant plasmid was mixed with serum-reduced culture medium, and then the transfection reagent was added. The mixture was incubated at room temperature for 15 to 20 minutes to obtain the transfection complex. The transfection complex was then mixed with a suspension of mammalian cells to obtain the transfection mixture. Monoclonal cell lines were obtained by culturing and screening the transfection mixture in a medium containing the first antibiotic, including: Add puromycin to the transfection mixture and replace the puromycin-containing culture medium every 2 to 3 days for several weeks; The surviving cells were diluted to 0.5 cells / mL to 1 cell / mL with puromycin-containing medium and seeded into the first well of the plate and cultured at 36°C to 38°C for 2 to 3 weeks. Single-clonal cells were picked from the first well of the plate and transferred for amplification culture. The culture was maintained in a medium containing puromycin to obtain a single-clonal cell line.

[0031] This application employs a stable transfection method, which involves transferring exogenous DNA into host cells and then integrating the exogenous target gene into the host chromosome for expression. Typically, the integration rate of exogenous DNA into the host cell chromosome is approximately 1%. This application utilizes puromycin to screen and culture cells. DNA that successfully integrates into the host cell chromosome carries the resistance gene of the recombinant plasmid and will not be killed during puromycin culture. Finally, single-clone cell lines are obtained by further culturing and screening from the surviving cells.

[0032] In step S300, the monoclonal cell lines obtained in the previous step are further screened. The screening criteria are based on detecting the expression level of VEGF165 using ELISA and screening for cell lines with high expression levels. Cell lines with VEGF165 expression levels meeting specific conditions are cultured as the basis for subsequent stable cell lines. As an optional implementation, the monoclonal cell lines are subjected to ELISA detection and VEGF165 expression level calculation, including: The monoclonal cell line was centrifuged, and the first supernatant was collected. The second well plate was coated with capture antibodies, including anti-human VEGF antibodies. Add blocking buffer, first supernatant, horseradish peroxidase-labeled detection antibody and tetramethylbenzidine colorimetric solution to the second well plate in sequence, and incubate and develop color. The absorbance of the solution in the second well was measured at a set wavelength, and the expression level of VEGF165 was calculated.

[0033] This application has undergone extensive experimental verification and comprehensive consideration of various factors, thus determining that cell lines with VEGF165 expression levels greater than 10 mg / L can be used as stable cell lines, producing more stable proteins with higher expression levels.

[0034] In step S400, the VEGF-stabilized cell line is expanded and concentrated by filtration, including: The VEGF-transfected cell line was transferred to serum-free medium for amplification culture, with an inoculation density of 0.5 × 10⁻⁶ cells / year. 6 cells / mL to 1×10 6 The cell / mL ratio should be between 110 rpm and 130 rpm, and the cells should be passaged every 2 to 3 days. When the cell density exceeds 2×10 6 At a cell / mL level, culturing with puromycin-containing medium and monitoring feeding, collecting cell supernatant, and culturing with puromycin-containing medium can induce the cell line to express the target gene.

[0035] The cell supernatant was centrifuged, filtered, and concentrated sequentially to obtain VEGF165 protein culture medium.

[0036] In step S100, a recombinant plasmid containing the VEGF165 gene is constructed, including: The vector and VEGF165 gene were digested separately with enzymes and then mixed at a predetermined molar ratio to obtain the ligation product. The vector included the pHBLV vector, and the DNA of the vector contained ampicillin resistance genes and puromycin resistance genes. The restriction endonucleases used for digestion included EcoRI and XhoI, with a molar ratio between 1:4 and 1:2. DNA ligase and ligation buffer were added, and the reaction was carried out at a temperature range of 15°C to 17°C. The ligation product was mixed with host cells using a heat shock method and then cultured in a first medium containing ampicillin to obtain multiple colonies. Plasmids were extracted and validated from the bacterial colonies to obtain recombinant plasmids.

[0037] The recombinant plasmid of this application is as follows: Figure 2 As shown, the recombinant plasmid DNA includes the VEGF gene segment, and 10 His-tags have been added to the C-terminus of the VEGF gene segment as purification tags. The recombinant plasmid also includes puro (purinolamine resistance gene) and AmpR (ampicillin resistance gene) for subsequent transfection screening.

[0038] As a second aspect of this application, a lyophilized powder is provided, the lyophilized powder comprising lyophilized protein, the lyophilized protein comprising protein prepared using the preparation method described above.

[0039] In some embodiments, the lyophilized powder also includes protein protectants and stabilizers.

[0040] This application addresses the problems of low protein activity, complex processes, and difficulties in large-scale production in existing technologies by constructing stable cell lines combined with a multi-step chromatographic purification strategy. Proteins prepared using the method described in this application are used to create lyophilized powders, which exhibit high chemical stability, and the recovered protein shows even higher activity.

[0041] The following explanation, in conjunction with preparation examples and embodiments, further clarifies this application. Preparation Example Preparation Example 1 This preparation example provides a method for preparing a recombinant plasmid, including: S1. Gene Synthesis: The human VEGF165 gene (NCBI reference sequence NP_001165097.1) was synthesized by a third party, and a 10×His tag sequence was added to the C-terminus of the gene. The accuracy of the sequence was verified by sequencing after synthesis.

[0042] S2. Vector Preparation: Extract the pHBLV vector (containing the CMV promoter and the puro resistance gene), and linearize the vector by digesting it at the multiple cloning site using restriction endonucleases (EcoRI and XhoI). After digestion, purify and recover the linearized vector by agarose gel electrophoresis.

[0043] S3. Fragment preparation: The synthesized VEGF165 gene fragment was digested with restriction endonucleases and then purified and recovered by gel electrophoresis.

[0044] S4. Ligation reaction: Mix the linearized vector and VEGF165 fragment at a molar ratio of 1:3, add T4 DNA ligase and ligation buffer, and react at 16°C for 1 to 2 hours or overnight (16 hours). The ligation volume is 20 μL, containing 50 to 100 ng of vector and 50 to 150 ng of insert fragment.

[0045] S5. Transformation: The ligation product was transformed into competent cells (E. coli DH5α). The ligation product was mixed with competent cells using the heat shock method. The cells were first placed on ice for 30 minutes, then heat-shocked at 42°C for 45 seconds, and then placed on ice for 2 minutes. LB medium was then added and the cells were incubated on a shaker at 37°C for 1 hour. The mixture was then spread on LB agar plates containing ampicillin (100 μg / mL) and incubated upside down at 37°C for 16 to 20 hours.

[0046] S6. Screening and Validation: Select a single colony, inoculate it in LB medium (containing ampicillin), extract the plasmid for restriction enzyme digestion identification and sequencing verification, and confirm that the correct recombinant plasmid is named pHBLV-VEGF165.

[0047] Preparation Example 2 This preparation example provides a method for preparing a monoclonal cell line, including: The recombinant plasmid from Preparation Example 1 was transfected into HEK-293F cells and single-clonal cell lines were obtained through screening. The detailed steps are as follows: S1. Cell Culture: HEK-293F cells were cultured in suspension in serum-free medium, maintaining a cell density of 0.5 × 10⁻⁶ cells / year. 6 Up to 2×10 6 The cells / mL were cultured at 37°C, 5% CO2, and a shaker speed of 110 to 130 rpm.

[0048] S2. Transfection preparation: When the cell density reaches 1×10⁶ cells / year... 6 Transfection was performed when the cell count / mL and cell viability >95%. An appropriate amount of cell suspension was taken, centrifuged, and resuspended in fresh serum-free medium, adjusting the density to 1×10⁶ cells / mL. 6 cells / mL.

[0049] S3. Transfection: The transfection complex was prepared using liposome transfection. The recombinant plasmid pHBLV-VEGF165 (1 μg / mL culture volume) was mixed with Opti-MEM medium, and then 3 μL / μg DNA of Lipofectamine 3000 reagent was added. The mixture was incubated at room temperature for 15 to 20 minutes. The complex was then slowly added to the cell suspension and gently mixed.

[0050] S4. Pressure screening: 48 hours after transfection, add puromycin to a final concentration of 1 to 2 μg / mL. Change the medium containing puro every 2 to 3 days for 2 weeks to kill untransfected cells.

[0051] S5. Limiting dilution method for screening single clones: Dilute surviving cells to 10 cells / mL with puro-containing medium, and then further dilute to 0.5 to 1 cell / mL. Seed into 96-well plates, 100 μL per well (approximately 0.05 to 0.1 cells / well), and incubate at 37°C and 5% CO2 for 2 to 3 weeks, observing clone formation periodically.

[0052] S6. Single clone amplification: Pick a single clone well, transfer it to a 24-well plate, and gradually amplify it to a 6-well plate and small shake flasks. Continue to maintain the culture with culture medium containing Puro (1 μg / mL).

[0053] Preparation Example 3 This preparation example provides a method for screening and preparing a high-expression cell line, including: The detailed steps for screening high-expressing cell lines using ELISA are as follows: S1. Supernatant collection: Centrifuge the monoclonal cell culture from Preparation Example 2 above to collect the supernatant, and store it at -20°C for later use.

[0054] S2, ELISA detection S20. Coat a 96-well plate with capture antibody (anti-human VEGF antibody) and incubate overnight at 4°C.

[0055] S21. Wash the plate 5 times with washing buffer (PBS containing 0.05% Tween-20).

[0056] S22. Add blocking buffer and incubate at 37°C for 1 hour.

[0057] S23. Add cell supernatant and incubate at 37°C for 1 hour.

[0058] S24. Add HRP-labeled detection antibody and incubate at 37°C for 1 hour.

[0059] S25. Add TMB substrate solution, incubate at 37°C in the dark for 10 minutes, and add 2NH2SO4 stop solution.

[0060] S26. Measure the absorbance at a wavelength of 450 nm and calculate the VEGF165 concentration.

[0061] S27. Compare the VEGF165 concentration in the supernatant of each monoclonal cell line, and select the cell line with an expression level greater than 10 mg / L, naming it the VEGF-293F stable cell line. Preserve the cells by cryopreservation in 90% FBS and 10% DMSO.

[0062] Preparation Example 4 This preparation example provides a method for expanding and culturing a stable cell line, including: The VEGF-293F stable cell line from Preparation Example 3 was expanded and cultured in serum-free medium. The detailed steps are as follows: S1. Cell thawing: Remove the VEGF-293F cryovial from liquid nitrogen and thaw rapidly in a 37°C water bath for 1 to 2 minutes. Transfer the cells to a 15 mL centrifuge tube, add 10 mL of pre-warmed serum-free medium, centrifuge to remove the cryopreservation solution, and resuspend in fresh serum-free medium.

[0063] S2. Amplification Culture: Start amplification by inoculating a 125 mL shake flask at an inoculation density of 0.5 × 10⁻⁶. 6 Up to 1×10 6 Cells / mL were cultured at 37°C, 5% CO2, and shaker speed of 110 to 130 rpm, and passaged every 2 to 3 days, maintaining a density of 0.5 to 2 × 10⁶ cells / mL. 6 cells / mL.

[0064] S3. Scale-up: Gradually scale up to 500 mL and 1 L shake flasks or 5 L stirred bioreactors, ensuring oxygen delivery through stirring and aeration control, until the cell density reaches 2 × 10⁻⁶ cells / year. 6 When the number of cells / mL reaches a certain level, the expression phase begins.

[0065] Expression phase: Change to culture medium supplemented with Puro for induction and feeding. Detailed steps are as follows: S4. Induction of expression: When cell density reaches 2×10⁻⁶ 6 When the cell count reaches 1000 cells / mL, switch to fresh serum-free medium (containing 1 μg / mL of Puro) to begin expression.

[0066] S5. 48 hours after expression, use a glucose meter or test strip to take a sample to detect the glucose concentration. If the glucose concentration is lower than 5 to 6 g / L, supplement the glucose solution to a final concentration of 6 to 8 g / L, and add 3 to 5% (v / v) of feeding medium at the same time.

[0067] S6. After 72 hours of expression, glucose was tested again, and glucose was supplemented to 6 to 8 g / L. 3 to 5% (v / v) feed medium was added, and sodium butyrate solution (100 mM stock) was added to a final concentration of 2 to 5 mM.

[0068] S7. After 96 hours of expression: End expression and collect cell supernatant.

[0069] Throughout the expression phase, maintain 37°C, 5% CO2, and a rotation speed of 110 to 130 rpm. At the same time, cell viability and pH need to be monitored regularly to ensure that cell viability is >80% and that the pH is maintained between 7.0 and 7.4.

[0070] Preparation Example 5 This preparation example provides a method for preparing protein culture medium using a stable cell line, including: S1. Centrifugation: Transfer the cell culture from Preparation Example 4 after 96 hours to a centrifuge bottle and centrifuge at 4°C to precipitate cell debris. Collect the supernatant.

[0071] S2. Filtration: The supernatant is filtered through a 0.22μm polyethersulfone membrane under vacuum or pressure to remove residual cells and particulate matter. The filtered supernatant is then stored on ice or at 4°C.

[0072] S3. Concentration: Concentrate using a centrifugal ultrafiltration tube. Add the supernatant to the centrifugal ultrafiltration tube and centrifuge at 4°C for 30 to 60 minutes. Repeat centrifugation until the desired volume is achieved.

[0073] S4. Sample preservation: The concentrated sample is aliquoted and stored at -80°C for subsequent purification.

[0074] Preparation Example 6 This preparation example provides a method for purifying protein culture medium, including: performing heparin affinity chromatography on the protein culture medium obtained in Preparation Example 5, with detailed steps as follows: S1. Column packing: Take a chromatography column (Bio-Rad Poly-Prep column, column bed volume 1 to 2 mL), close the bottom outlet valve, fully resuspend the heparin affinity medium slurry, slowly pour it into the column, allow it to settle naturally, open the outlet valve, and let the buffer solution flow out slowly to compact the medium in the column bed, ensuring that the column bed is uniform and free of cracks or bubbles.

[0075] S2. Equilibration: Rinse the chromatography column with at least 10 to 15 column volumes of equilibration buffer by connecting a buffer reservoir to the top of the column and allowing gravity flow at a rate between 0.5 mL / min and 1 mL / min. Collect the flow-through until the pH and conductivity of the flow-through match those of the equilibration buffer.

[0076] S3. Sample loading: Close the outlet valve, ensure there is a small amount of liquid at the top of the column bed, and slowly and evenly add the protein culture medium obtained in Preparation Example 5 to the surface of the column bed using a pipette to avoid damaging the column bed interface. Open the outlet valve and start collecting the flow-through liquid, controlling the flow rate at 0.25 to 0.5 mL / min to ensure that the sample and the medium are fully combined.

[0077] S4. Washing: After sample loading, wash the column with 5 to 10 column volumes of equilibration buffer. The equilibration buffer is 20 mM Tris-HCl buffer, pH 7.4. Collect this flow-through fraction, which can be used as a contaminating protein fraction. This step aims to wash away unbound or weakly bound proteins.

[0078] S5. Low-salt wash: Wash with 3 to 5 column volumes of Tris buffer containing 0.2 to 0.3 M NaCl to remove some heparin-weakly bound proteins. Collect this fraction.

[0079] S6. Elution of the target protein: Elute with 5 column volumes of elution buffer containing 0.6 M NaCl. Collect all eluent from this fraction, one tube per column volume or 0.5 mL. Immediately afterwards, elute with 5 column volumes of elution buffer containing 0.8 M NaCl, collecting the eluent. Finally, elute with 3 column volumes of elution buffer containing 1.0 M NaCl to remove any remaining strongly bound proteins, collecting this fraction.

[0080] S7. Column regeneration: Wash the column with 5 column volumes of Tris buffer containing 2 M NaCl, then equilibrate with 5 column volumes of equilibration buffer, and finally store at 4°C with equilibration buffer containing 0.02% NaN3.

[0081] After the heparin affinity chromatography was completed, the collected eluents were combined and subjected to nickel column affinity chromatography. The detailed steps are as follows: S8. Sample dialysis: The combined heparin column eluent was thoroughly dialyzed at 4°C using a buffer of 20 mM PBS and 20 mM imidazole to remove high salt content and introduce low concentrations of imidazole.

[0082] S9. Column Equilibration: Pack the Ni-NTA Agarose medium into the chromatography column and wash it with at least 10 column volumes of equilibration buffer, which consists of 20 mM imidazole and 20 mM PBS at pH 7.4.

[0083] S10. Sample loading: Slowly load the dialysis sample, controlling the flow rate at 0.25 to 0.5 mL / min, and collect the flow-through.

[0084] S11. Washing: Wash the column with 5 to 10 column volumes of washing buffer consisting of 20 mM Tris-HCl and 40 mM imidazole at pH 8.6. Collect this fraction.

[0085] S12, Stepwise Elution: Collect the eluent using 3 to 5 column volumes of elution buffer containing 50 mM imidazole (corresponding to lane 1 for subsequent tests). Collect the eluent using 3 to 5 column volumes of elution buffer containing 100 mM imidazole (corresponding to lane 2 for subsequent tests). Collect the eluent using 5 column volumes of elution buffer containing 250 mM imidazole (corresponding to lane 3 for subsequent tests). Collect the eluent stepwise at each step and label it immediately.

[0086] Test case Test Example 1 SDS-PAGE analysis was performed on the samples collected in different lanes from Preparation Example 6. Specifically, SDS-PAGE analysis was performed on the eluents of 50 mM (lane 1), 100 mM (lane 2), and 250 mM (lane 3) imidazole. The results are as follows. Figure 3As shown in the figure, the horizontal axis represents the collection solutions of different concentrations of elution (corresponding to lanes 1 to 3), and the vertical axis represents the molecular weight of the protein. The figure shows that lanes 1 to 3 exhibit a single, dense band at approximately 20 kDa to 31 kDa, with no other obvious bands, demonstrating that the target protein VEGF165 was eluted efficiently and with high purity. The protein purity, as measured by a protein gel imaging system, exceeded 95%. The high-purity eluent was dialyzed overnight at 4°C with imidazole-free PBS buffer to remove imidazole, yielding the finally purified VEGF165 protein, which was then stored at -80°C.

[0087] The following examples are used to further verify the technical effects of the preparation method of this application.

[0088] Example Example 1 The expression level comparison experiment aims to compare the differences in VEGF165 expression levels between the stable cell line constructed in this invention and the traditional transient transfection and E. coli system.

[0089] The following three experimental groups are provided: Experimental Group 1 (Stable Transfection of the Invention): The VEGF-293F stable transfected cell line constructed using the preparation methods of Preparation Examples 1 to 3 above was cultured according to the steps of Preparation Example 4, and the supernatant was collected after 96 hours.

[0090] Experimental Group 2 (Transient Transfection Control): The recombinant plasmid pHBLV-VEGF165 from Preparation Example 1 was transiently transfected into wild-type HEK-293F cells using the liposome method from Preparation Example 2. The supernatant was collected 72 hours after transfection.

[0091] Experimental group 3 (prokaryotic expression control): The VEGF165 gene (without His tag) was cloned into pET-28a according to the method described in patent CN103451220B, transformed into BL21(DE3) strain, and expressed by IPTG. The supernatant of cell lysis was collected (inclusion bodies need to be denatured and dissolved).

[0092] Detection method: The concentration of VEGF165 in each sample of the above experimental groups was quantitatively detected using a double-antibody sandwich ELISA kit. The test results are shown in Table 1.

[0093] Table 1 Comparison of VEGF165 yield in different expression systems

[0094] Note: The prokaryotic expression level is an estimated amount of VEGF165 in the total bacterial protein, in an insoluble form.

[0095] As shown in Table 1, the stable cell line constructed in this invention maintains an expression level of approximately 12.5 mg / L, significantly higher than that of the transient transfection system. This demonstrates the absolute advantage of the stable cell line in achieving high expression and high stability. Although the prokaryotic system yields slightly higher expression levels, the product is an inactive inclusion body, and the subsequent refolding process is complex and has an extremely low yield. From the perspective of efficiency in obtaining the final active protein, this invention is more advantageous.

[0096] Example 2 Methodological validation experiments (1) Sensitivity and standard curve Step 1: After determining the concentration of the purified VEGF165 protein, freeze-dry it to prepare a standard, perform double-antibody sandwich ELISA detection, and plot a standard curve, as shown below. Figure 4 As shown, the horizontal axis represents the known concentration of the standard, and the vertical axis represents its corresponding signal intensity (OD value). The "concentration-signal" curve, i.e., the standard curve, is plotted by fitting calculations.

[0097] according to Figure 4 The standard curve was obtained from curve fitting and ELISA detection data. The standard curve showed good linearity in the concentration range of 6.25 to 400 pg / mL. The fitting equation was Y = 3.17147 + ((0.101848 - 3.17147) / (1 + (x / 149.866)^1.26962)), R² = 1. Therefore, it can be concluded that the detection method established using the purified VEGF165 protein has high sensitivity, wide linear range, and accuracy that meets the quantitative requirements.

[0098] (2) Repeatability test Step 1: The same operator, under the same conditions, used the same VEGF-293F cell line to conduct three independent batches of expression and purification experiments (batch numbers L01, L02 and L03, respectively), and the results were tested. The test data are shown in Table 2.

[0099] Table 2 Protein detection data from different batches

[0100] As shown in Table 2, the expression levels and purity of the three batches of products were highly consistent, and the CV values ​​were all less than 6%, proving that the process of this invention has excellent reproducibility.

[0101] (3) Long-term stability and freeze-thaw test: Step 1: After aliquoting the protein, it was stored at -80°C, and samples were taken at 0, 1, 3, and 6 months. Three freeze-thaw cycles were also performed. The results showed that after 6 months of storage at -80°C, none of the indicators changed. After three freeze-thaw cycles, there was no significant protein aggregation, and the activity loss was <5%. This demonstrates that the product exhibits good long-term storage stability and freeze-thaw stability, further proving the robustness of the preparation process and the high quality of the product.

[0102] The above systematic embodiments and test data demonstrate that the method for preparing recombinant human vascular endothelial growth factor (VEGF)165 provided by this invention produces VEGF165 protein with high and stable expression efficiency. The expression level in stable cell lines is significantly higher than that in transient transfection. This application exhibits low batch-to-batch variability, solving the problems of capacity and consistency in large-scale production. Furthermore, this application employs a two-step purification strategy using heparin and nickel columns, achieving a high purity product (>95%), superior to single purification methods. The preparation method of this application is reliable, reproducible, and highly specific, with sensitive and accurate detection methods, high batch-to-batch repeatability, and meets quality control requirements. Simultaneously, the product quality is excellent and stable; the obtained VEGF165 protein has a natural glycosylated form and exhibits good stability under storage conditions.

[0103] The above are merely specific embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Those skilled in the art should understand that the present invention includes, but is not limited to, the contents described in the accompanying drawings and the specific embodiments above. Any modifications that do not depart from the functional and structural principles of the present invention will be included within the scope of the claims.

Claims

1. A method for preparing human vascular endothelial growth factor VEGF165, characterized in that, The preparation method includes: A recombinant plasmid containing the VEGF165 gene was constructed. The recombinant plasmid includes a vector and a VEGF165 gene fragment inserted into the vector DNA. The recombinant plasmid has multiple resistance genes, and the VEGF165 gene fragment has a purification tag. The recombinant plasmid was transfected into mammalian cells to obtain a transfection mixture, wherein monoclonal cell lines were obtained by culturing and screening the transfection mixture in a medium containing a first antibiotic. The monoclonal cell lines were subjected to ELISA detection and the expression level of VEGF165 was calculated. Cell lines with VEGF165 expression level greater than 10 mg / L were screened to obtain VEGF stable cell lines. The VEGF-stable cell line was expanded and cultured, and then filtered and concentrated to obtain VEGF165 protein culture medium. The VEGF165 protein culture medium was purified by multi-step chromatography to obtain the VEGF165 protein, wherein different chromatography columns were used in the multi-step chromatography.

2. The preparation method according to claim 1, characterized in that, The VEGF165 protein culture medium was purified by multi-step chromatography, including: The VEGF165 protein culture medium was subjected to heparin affinity chromatography. The VEGF165 protein culture medium was subjected to nickel affinity chromatography.

3. The preparation method according to claim 2, characterized in that, The step of performing heparin affinity chromatography on the VEGF165 protein culture medium includes: The VEGF165 protein culture medium is passed through a chromatography column containing heparin affinity medium at a first flow rate between 0.25 mL / min and 0.5 mL / min. The chromatography column is eluted using a gradient with a salt buffer at a second flow rate between 0.5 mL / min and 1 mL / min. The salt concentration of the salt buffer is positively correlated with the elution time. The salt buffer comprises sodium chloride and tris(hydroxymethyl)aminomethane hydrochloride.

4. The preparation method according to claim 2, characterized in that, The step of performing nickel affinity chromatography on the VEGF165 protein culture medium includes: The VEGF165 protein culture medium is passed through a chromatography column containing a nickel affinity medium at a first flow rate between 0.25 mL / min and 0.5 mL / min. The chromatography column was eluted using an imidazole-containing buffer at a second flow rate between 0.5 mL / min and 1 mL / min, wherein the imidazole content in the imidazole-containing buffer was positively correlated with the elution time.

5. The preparation method according to any one of claims 1 to 4, characterized in that, Transfecting the recombinant plasmid into mammalian cells includes: The recombinant plasmid was mixed with serum-reduced culture medium, and then transfection reagent was added. The mixture was incubated at room temperature for 15 to 20 minutes to obtain the transfection complex. The complex to be transfected is mixed with a suspension of mammalian cells to obtain the transfection mixture; Monoclonal cell lines were obtained by culturing and screening the transfection mixture in a medium containing a first antibiotic, including: Add puromycin to the transfection mixture, and replace the culture medium containing puromycin every 2 to 3 days for several weeks; The surviving cells were diluted to 0.5 cell / mL to 1 cell / mL with puromycin-containing medium and seeded into the first well of the plate and cultured at 36°C to 38°C for 2 to 3 weeks. Monoclonal cells were picked from the first well plate and transferred for amplification culture. The culture was maintained in a medium containing puromycin to obtain the monoclonal cell line.

6. The preparation method according to any one of claims 1 to 4, characterized in that, The monoclonal cell line was subjected to ELISA detection and VEGF165 expression level calculation, including: The monoclonal cell line was centrifuged to collect the first supernatant; The second well plate is coated with a capture antibody, which includes an anti-human VEGF antibody. Add blocking buffer, the first supernatant, horseradish peroxidase-labeled detection antibody and tetramethylbenzidine colorimetric solution sequentially to the second well plate, incubate and develop color; The absorbance of the solution in the second well plate was measured at a set wavelength, and the expression level of VEGF165 was calculated.

7. The preparation method according to any one of claims 1 to 4, characterized in that, The VEGF-stabilized cell line was expanded and cultured, then filtered and concentrated, including: The VEGF-stabilized cell line was transferred to serum-free medium for amplification culture, with an inoculation density of 0.5 × 10⁻⁶ cells / year. 6 cells / mL to 1×10 6 The cell / mL ratio should be between 110 rpm and 130 rpm, and the cells should be passaged every 2 to 3 days. When the cell density exceeds 2×10 6 At a cell / mL level, culture the cells in a medium containing puromycin and monitor feeding, then collect the cell supernatant. The cell supernatant was centrifuged, filtered, and concentrated sequentially to obtain the VEGF165 protein culture medium.

8. The preparation method according to any one of claims 1 to 4, characterized in that, Constructing a recombinant plasmid containing the VEGF165 gene includes: The vector and VEGF165 gene were digested separately with enzymes and then mixed at a predetermined molar ratio to obtain the ligation product. The vector included a pHBLV vector, and the DNA of the vector contained ampicillin resistance genes and puromycin resistance genes. The restriction endonucleases used for digestion included EcoRI and XhoI, and the predetermined molar ratio was between 1:4 and 1:

2. DNA ligase and ligation buffer were added, and the reaction was carried out at a temperature range of 15°C to 17°C. The ligation product was mixed with host cells using a heat shock method and then cultured in a first culture medium containing ampicillin to obtain multiple colonies. The colonies were subjected to plasmid extraction and verification to obtain the recombinant plasmid.

9. A freeze-dried powder, characterized in that, The lyophilized powder includes lyophilized protein, which includes protein prepared by any one of the preparation methods described in claims 1 to 8.

10. The freeze-dried powder according to claim 9, characterized in that, The freeze-dried powder also includes protein protectants and stabilizers.

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