Application of recombinant protein synthesized by bombyx mori in development of alternative serum-free medium and the medium

Abstract

The application discloses application of a recombinant protein synthesized by a silkworm in development of a basic type partial replacement serum formula medium or an induced type serum-free medium and a medium thereof. Various types of recombinant proteins synthesized by the silkworm are used to develop the basic type serum formula and the induced type serum formula medium, 50%-100% fetal bovine serum (FBS) is replaced, insect, mouse and human cell lines are cultured, the effect is consistent with that of a commercialized medium, FBS replacement can be realized, the use of FBS is greatly reduced, the cost of large-scale cell culture is reduced, and the application has a good application prospect.

Description

Technical Field

[0001] This invention relates to the field of culture media, specifically to the application of recombinant proteins synthesized by silkworms in the development of alternative culture media such as serum, and also to culture media containing recombinant proteins synthesized by silkworms. Background Technology

[0002] Since its establishment, cell culture technology has become an important tool in various fields of biological research, such as disease simulation, stem cell and tumor research, reproductive technology, biotechnology, and biopharmaceuticals. This has led to the widespread use of fetal bovine serum (FBS), rich in various macronutrients, micronutrients, and growth factors, as a primary component of cell culture media. Animal-derived cell culture media, with FBS as their nutrient component, have supported the development of modern molecular cell biology and are widely used in biopharmaceuticals, large-scale cell culture production, and other fields. However, over time, various drawbacks of animal-derived cell culture media have become increasingly apparent. Firstly, there is the impact of mad cow disease prions. The first case of mad cow disease was discovered in the UK in 1986, and it re-emerged 10 years later, causing thousands of cattle to become mentally unstable, demented, and soon die. When the culture medium carries viruses and animal foreign proteins, it not only threatens the health of the operator but also contaminates the entire laboratory or workshop. Secondly, animal-derived culture media are prone to performance instability, resulting in inconsistent results between batches, making test results incomparable.

[0003] Over the years, the increasing demand, high price, significant batch-to-batch variations in quality and composition of FBS, along with growing ethical concerns, have prompted efforts to reduce FBS consumption and seek alternatives. Currently, alternatives primarily include biological components such as pituitary extracts, chicken embryo extracts, platelet lysates, bovine eye drops, plant protein extracts, and silk sericin (SS), as well as industrial byproducts and combinations of different compounds.

[0004] Previous studies have shown that fetal bovine serum contains approximately 3.5%-5.0% protein, with serum albumin being the most abundant component at a concentration of 50 mg / mL, accounting for about half of the total protein content. Other components include total IgG, transferrin, and various growth factors. Therefore, developing low-cost, non-animal-derived cell culture media to replace existing FBS-based animal-derived cell culture media through genetic engineering techniques and utilizing E. coli, yeast, insect cells, mammalian cells, and animal-plant "bioreactor" platforms has significant application prospects and industrial value. Summary of the Invention

[0005] In view of this, one objective of the present invention is to provide an application of recombinant proteins synthesized by silkworms in the development of basic partially serum-replacement culture media or induced serum-free culture media; a second objective of the present invention is to provide a basic partially serum-replacement culture media developed based on recombinant proteins synthesized by silkworms; a third objective of the present invention is to provide an induced serum-free culture media developed based on recombinant proteins synthesized by silkworms; and a fourth objective of the present invention is to provide the application of the culture media in the culture of animal cell lines.

[0006] To achieve the above objectives, the present invention provides the following technical solution: 1. The application of recombinant proteins synthesized from silkworms in the development of basic partially serum-replacement culture media or induced serum-free culture media, characterized in that: the recombinant protein is recombinant human serum albumin, recombinant platelet-derived growth factor, or recombinant epidermal growth factor.

[0007] 2. A basic partially replaced serum-formulated culture medium developed based on recombinant proteins synthesized from silkworms, wherein the culture medium is supplemented with recombinant human serum albumin at a final concentration of 1 mg / mL and 5% FBS.

[0008] The preferred composition of this invention is as follows: 1 mg / mL recombinant human serum albumin, 5% FBS (volume fraction), and the remainder is DMEM culture medium.

[0009] Preferably, the culture medium is supplemented with recombinant human serum albumin at a final concentration of 1 mg / mL, sericin at a final concentration of 120 µg / mL, and FBS at a final concentration of 2.5%.

[0010] The preferred composition of this invention is as follows: 1 mg / mL recombinant human serum albumin, 120 µg / mL sericin, and 2.5% FBS (volume fraction), with the remainder being DMEM culture medium.

[0011] 3. An inducible serum-free culture medium developed based on recombinant proteins synthesized by silkworms, wherein the culture medium is supplemented with recombinant platelet-derived growth factor and recombinant epidermal growth factor at a final concentration of 5-50 ng / mL.

[0012] The preferred composition of this invention is as follows: 5-50 ng / mL of recombinant platelet-derived growth factor, 5-50 ng / mL of recombinant epidermal growth factor, and the remainder being DMEM culture medium.

[0013] The application of the culture medium in culturing animal cell lines.

[0014] Preferably, the animal cell line of the present invention is an insect, mouse, or human cell line.

[0015] The beneficial effects of this invention are as follows: This invention discloses the development of basic and inducible serum culture media using various recombinant proteins synthesized from silkworms, achieving a 50%-100% replacement of fetal bovine serum (FBS) for culturing insect, mouse, and human cell lines. Pilot-scale production of key raw materials such as sericin, recombinant human serum albumin (rHSA), and recombinant human growth factors (GFs) has been achieved using established processes. A core culture medium formulation with silkworm sericin, HSA, and rFGF2 as main components has been developed, demonstrating excellent performance in mouse and humanized cell culture, achieving results consistent with commercially available culture media, and has achieved FBS replacement, significantly reducing FBS usage and lowering the cost of large-scale cell culture. Attached Figure Description

[0016] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided for illustration: Figure 1 To facilitate the production of recombinant human serum albumin (rHSA) and human lactoferrin (rhLF) from silkworms (A: isolation and purification of rHSA from silkworm silk; B: determination of the secondary structure of rHSA by circular dichroism spectroscopy; C: isolation and purification of rhLF from silkworm silk; D: determination of the secondary structure of rhLF by circular dichroism spectroscopy).

[0017] Figure 2 To assess the potential of rHSA as a substitute for FBS as a component of cell culture medium (A: CCK-8 assay to detect the effect of rHSA on cell growth after replacing 50% FBS; B: EDU cell proliferation assay on day 4 after replacing 50% FBS with rHSA; C: LIVE & DEAD assay on 3 consecutive days after replacing 50% FBS with rHSA; data are presented as mean ± standard deviation (n=3), scale bar 400 µm).

[0018] Figure 3 The effect of replacing 50% FBS with rHSA on cell cycle and apoptosis (A: flow cytometry of cell cycle; B: flow cytometry of cell apoptosis; C: cell cycle distribution map; D: statistical graph of cell apoptosis. Statistical data are shown as mean ± standard deviation (n=3)).

[0019] Figure 4The cell culture effect of rHSA combined with sericin to replace FBS was analyzed (A: CCK-8 assay on day 5 of cells cultured with full sericin replacing FBS; B: CCK-8 assay on day 5 of cells cultured with full sericin replacing FBS; C: CCK-8 assay on day 5 of cells cultured with sericin under different concentrations of FBS; D: CCK-8 assay on day 3 of cells cultured with sericin under different concentrations of FBS; E: CCK-8 assay on day 5 of cells cultured with sericin-rHSA replacing FBS; SS: sericin; statistical data are shown as mean ± standard deviation (n=3)).

[0020] Figure 5 EDU detection for cells cultured using sericin-rHSA as a substitute for FBS; SS: sericin; scale bar: 400 µm.

[0021] Figure 6 Recombinant human cytokines synthesized by silkworms (A: Detection of PDGF-BB recombinant protein in silkworm silk; B: Detection of EGF recombinant protein in silkworm silk).

[0022] Figure 7 To detect the growth and proliferation of human mesenchymal stem cells (hMSCs) inducible culture medium.

[0023] Figure 8 To investigate the effect of EdU on the growth and proliferation of human mesenchymal stem cells (hMSCs) promoted by induced nutrient medium. Detailed Implementation

[0024] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0025] In this invention, pHSA is commercially available human serum albumin, purchased from Sigma-Aldrich.

[0026] Example 1: Synthesis of serum-free culture medium protein raw materials using a silkworm silk gland bioreactor Using the previously constructed silkworm bioreactor synthesis technology platform (see Chinese patents with publication numbers CN102492692A, CN102517301A, CN104513821A, CN103911383A, and CN108578771A), key nutritional proteins in multiple cell growth and proliferation processes were recombinantly expressed, and high-purity recombinant human serum albumin (rHSA) (see Chinese patent with publication number CN105861515A) and high-purity injection-grade sericin were obtained.

[0027] The recombinant protein purification method is as follows: ① Add the genetically modified silkworm cocoons to a self-made 50 L special stirring extraction tank, and after stirring and extraction at room temperature, obtain the crude extract.

[0028] ② The crude extract was passed through a membrane concentration system three times to obtain a protein extract after impurities were removed.

[0029] ③ Next, the protein extract is passed through a 5 L small concentrator to obtain a concentrate.

[0030] ④ Centrifuge the protein concentrate using a high-speed centrifuge at 18,000 rpm for 15 min. After centrifugation, collect the supernatant in the centrifuge tube and vacuum filter the supernatant through a 0.22 µm filter membrane.

[0031] ⑤ Connect the nickel ion affinity chromatography column protein purification device, add nickel to the column and equilibrate it, then load the solution obtained in the previous step onto the column at a flow rate of 0.45 mL. After the protein sample has been completely loaded, wash with equilibration buffer, then elute the column with 18 mM, 100 mM and 1 M imidazole solutions at a flow rate of 2 mL / min.

[0032] ⑥ Take 200 µL each of the protein extract, concentrate, and elution buffer of different concentrations from each of the above steps, perform SDS-PAGE electrophoresis, and then detect the results by Coomassie brilliant blue staining.

[0033] ⑦ Place the eluent containing protein into a dialysis bag, dialyze to remove imidazole, and then freeze-dry it in a freeze dryer to obtain protein powder.

[0034] BCA method for determining protein concentration ① Prepare a protein standard solution with a concentration of 0.5 mg / mL using PBS buffer.

[0035] ② According to the kit formula, prepare the working solution by mixing solution A and solution B in a volume ratio of 50:1, and shake thoroughly to make it turn light green.

[0036] ③ Add 0 µL, 1 µL, 2 µL, 4 µL, 8 µL, 12 µL, 16 µL, and 20 µL to a 96-well microplate, respectively. Then, use PBS buffer to bring the total volume of the solution to 20 µL in each well. Add another 20 µL of rHSA protein solution to another well. Repeat each sample 3 times.

[0037] ④ Add 200 µL of the protein working solution prepared in step 2 to each well, then put the entire plate into a disposable glove and place it in a 37 ℃ constant temperature incubator for 40 min.

[0038] ⑤ Use an ELISA reader to measure the absorbance in the wells of the ELISA plate at 560 nm, plot a standard curve using the absorbance values ​​of each group, and calculate the obtained protein concentration.

[0039] Protein purity analysis using ImageJ The protein gel was scanned, and the purity of the protein gel scan was analyzed using ImageJ software. The results are as follows: Figure 1 As shown.

[0040] The results showed that 9.21 grams of rhLF recombinant protein with a purity of 95.45% could be obtained by isolating and purifying 1 kg of rHSA recombinant protein with a purity of 99.69% after isolation and purification, providing a sufficient protein source for the development of serum-free cell culture medium.

[0041] Example 2: The rHSA synthesized by silkworms can replace 50% of fetal bovine serum (FBS) and promote normal cell growth. To evaluate whether recombinant rHSA could maintain cell growth and replace FBS as a component of cell culture medium, rHSA was first used to replace 50% of the original 10% FBS in DMEM medium, and the results were verified by culturing NIH-3T3 cells preserved in our laboratory. Four groups were set up: a 10% FBS DMEM medium group, a 5% FBS DMEM medium group, a 5% FBS + 1 mg / mL rHSA DMEM medium group, and a 5% FBS + 1 mg / mL pHSA DMEM medium group. CCK-8 assays were performed for 8 days, and the results are as follows: Figure 2 As shown in Figure A. The results showed that the test cells in DMEM medium containing 5% FBS + 1 mg / mL rHSA exhibited a good growth trend, significantly better than cells grown in DMEM medium containing 5% FBS, and comparable to cells grown in DMEM medium containing 10% FBS. Subsequently, EdU staining was performed on cells on day 5 of culture to reflect cell proliferation, and the results are shown in Figure A. Figure 2 As shown in Figure B. The results showed that the number of cells proliferating in DMEM medium containing 5% FBS + 1 mg / mL rHSA was significantly higher than that in DMEM medium containing 5% FBS, and comparable to the number of cells proliferating in DMEM cell culture medium containing 10% FBS or 5% FBS + 1 mg / mL pHSA. Cell viability was stained using Live&Dead staining for the first 3 days, and the results are shown in Figure B. Figure 2As shown in Figure C. The results showed that the total number of cells cultured in the DMEM sample group using 5% FBS + 1 mg / mL rHSA was significantly increased compared to the negative control group using 5% FBS, and was comparable to the total number of cells in the normal control group using 10% FBS or the positive control group using 5% FBS + 1 mg / mL pHSA, with no change in cell morphology. These results preliminarily indicate that rHSA purified from transgenic silkworm cocoons has the function of maintaining cell growth, and at a final concentration of 1 mg / mL, it can replace 50% of FBS and maintain normal cell growth.

[0042] Example 3: Effects of silkworm-synthesized rHSA replacing 50% FBS on cell cycle and apoptosis. Subsequently, flow cytometry was used to detect cell cycle and apoptosis in cells cultured after 50% FBS was replaced with rHSA. The results are as follows: Figure 3 As shown in AC, in cell culture medium containing 5% FBS and 1 mg / mL rHSA, the cell cycle distributions of G2 / M, S, and G0 / G1 were not significantly different from those in cell culture medium containing only 5% FBS and those containing 10% FBS or 5% FBS and 1 mg / mL pHSA. However, after apoptosis detection by flow cytometry, in... Figure 3 In the study, cells that were positive for Annexin V-FITC staining and negative for PI staining represented a percentage of early apoptosis within the range of 2%. These results indicate that using rHSA instead of 50% FBS to culture cells does not affect the cell cycle distribution and does not induce apoptosis.

[0043] Example 4: The rHSA synthesized by silkworms, combined with sericin, can achieve a 75% replacement of FBS. To further increase the replacement rate of FBS, an experiment was conducted to add silk sericin (SS), a cell growth promoter, to DMEM medium containing 1 mg / mL rHSA, thereby replacing more FBS. First, different concentrations of SS were added to FBS-free cell culture medium for NIH-3T3 cell culture. CCK-8 assay results showed that the cell growth trend at a concentration of 120 µg / mL was superior to other low-concentration sericin culture groups. Figure 4 The results (AB) indicate that this concentration of sericin significantly promotes cell growth. Therefore, different gradients of serum were added to a sericin concentration of 120 µg / mL, and cell growth trends were further monitored using CCK-8 assay. The results are as follows: Figure 4As shown in CD, cell culture effects varied under different serum concentrations. In the group treated with sericin replacing 50% FBS, cell growth trends were similar to those in the normal culture with 10% FBS. However, cell culture effects were weaker in media containing only 2.5% and 1% FBS when sericin was added. These results indicate that a concentration of 120 µg / mL of sericin can replace 50% of FBS to maintain the growth of NIH-3T3 cells.

[0044] Based on the above results, the effects of adding 1 mg / mL rHSA to the culture medium at a sericin concentration of 120 µg / mL were compared to those replacing 75% and 90% of FBS. Five experimental groups were set up: a normal culture group containing 10% FBS; a treatment group containing 1 mg / mL rHSA in a medium containing 2.5% FBS; a treatment group containing 120 µg / mL sericin in a medium containing 2.5% FBS; a treatment group containing 1 mg / mL rHSA and 120 µg / mL sericin in a medium containing 2.5% FBS; and a treatment group containing 1 mg / mL rHSA and 120 µg / mL sericin in a medium containing 1% FBS. Each group had three replicates. Data were collected from CCK-8 monitoring over five consecutive days. Figure 4 In the study (E), it was found that the total cell viability of the treatment group with 1 mg / mL rHSA and 120 µg / mL sericin added to the medium containing 2.5% FBS was almost the same as that of the normal culture group containing 10% FBS, while the cell culture effect of other treatment groups was poor. Moreover, the number of proliferating cells in the treatment group was similar to that in the normal group in EdU staining at 72 h. Figure 5 ).

[0045] In summary, this study found that adding rHSA at a final concentration of not less than 1 mg / mL and sericin at a final concentration of 120 µg / mL can replace 75% of the FBS in the cell culture medium. This formula can maintain the normal growth of NIH-3T3 cells, and after 5 days of culture, the cell confluence can reach more than 95%. Moreover, the cell growth status and various physiological indicators are consistent with those of commercial culture medium.

[0046] Example 5: Recombinant cytokine proteins synthesized by silkworms further enhance FBS replacement and promote stem cell proliferation and differentiation. To achieve a complete replacement of FBS and develop a cell culture medium free of animal serum, we further added recombinant humanized cytokine proteins synthesized in a silkworm silk gland bioreactor to the above-mentioned basic formulation. We focused on testing platelet-derived growth factor (PDGF-BB) and epidermal growth factor (EGF). Figure 6A formula for an induction culture medium for the synthesis of recombinant proteins in silkworms was developed, with the following specific formula: rHSA concentration not less than 1 mg / mL + sericin concentration not less than 120 µg / mL.

[0047] Recombinant PDGF-BB was expressed in a bioreactor with publication number CN108588083A. The purification steps are as follows: For recombinant EGF, see Wang, F., et al., Advanced silk material spun by a transgenic silkworm promotes cell proliferation for biomedical application. Acta Biomater, 2014.10(12): p.4947-55. Wang, F., et al., Large-scale production of bioactive recombinant human acidic fibroblast growth factor in transgenic Silkworm cocoons. Sci Rep, 2015.5:p.16323. The bioreactor disclosed is as follows: Meanwhile, commercial culture media were used as controls. The purchaser and catalog number of Gibco, 26010074 were referred to as commercial competitor 1, and the purchaser and catalog number of Gibco, 10099 were referred to as commercial competitor 2.

[0048] The results showed that the inducible culture medium supplemented with PDGF-BB and EGF could replace 100% fetal bovine serum (FBS) and effectively promote the growth and proliferation of human mesenchymal stem cells (hMSCs), achieving a cell confluence rate of over 96% after 14 days of culture. Figure 7 Furthermore, the growth indicators of stem cells are consistent with those of commercially available culture media. Figure 8 The results show that the induced serum-free cell culture medium developed in this study has the ability to promote the normal growth and proliferation of stem cells, effectively reducing the use of fetal bovine serum, and can be used for large-scale and low-cost stem cell culture in the future.

[0049] The above-described embodiments are merely preferred embodiments provided to fully illustrate the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention. The scope of protection of the present invention is defined by the claims.

Claims

1. The application of recombinant proteins synthesized by silkworms in the development of basic partially serum-replacement culture media, characterized by: The recombinant protein is recombinant human serum albumin; the culture medium includes recombinant human serum albumin at a final concentration of 1 mg / mL, sericin at a final concentration of 120 µg / mL and 2.5% FBS, and the culture medium is used to culture animal cell lines, the animal cell lines being mouse cell lines.

2. The application according to claim 1, characterized in that: The specific components of the culture medium are as follows: 1 mg / mL recombinant human serum albumin, 120 μg / mL sericin, and 2.5% FBS by volume, with the remainder being DMEM culture medium.

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