Human pluripotent stem cell culture medium free of heterogeneous components and limited in chemical components

Abstract

The invention discloses a human pluripotent stem cell culture medium free of heterogeneous components and limited in chemical components, the human pluripotent stem cell culture medium comprises a basic solution and an added mother solution, the added mother solution comprises a mother solution A, a mother solution B, a mother solution C, a mother solution D and a mother solution E; the invention provides the culture medium which can be used for human embryonic stem cells and induced pluripotent stem cells, has limited chemical components, does not contain heterogeneous components, can effectively amplify the pluripotent stem cells in vitro and maintain the differentiation capacity of the pluripotent stem cells, is suitable for the common scientific research and cell therapy industry direction, and has wide application prospects. The cell proliferation speed and the stability of storage at 4 DEG C have obvious advantages, and the maintenance of the pluripotency of the stem cells is not obviously different from that of a competitive product.

Description

Technical Field

[0001] The present invention relates to the fields of human embryonic stem cells, human induced pluripotent stem cells, pluripotent stem cell culture, regenerative medicine, biotechnology, cell culture and cell therapy technology, and in particular to a human pluripotent stem cell culture medium free of xenogeneic components and with limited chemical components. Background Art

[0002] Human embryonic stem cells (hES) and induced pluripotent stem cells (iPSC) were first successfully established by Thomson (Thomson et al., 1998) and Shinya Yamanaka (Takahashi et al., 2007) in 1998 and 2007, respectively. Both cells are pluripotent stem cells, which can complete self-renewal and differentiation into all cell types of adults through cell division. Therefore, pluripotent stem cells have a wide range of application value in research fields such as developmental biology, regenerative medicine, and the construction of in vitro drug screening models.

[0003] Cell culture medium not only provides nutrition and substances that promote cell proliferation by simulating the growth environment of cells in vivo, but also provides cells with a basic survival and proliferation environment, which is one of the key parts of cell in vitro culture technology. Traditional human pluripotent stem cells need to be cultured on a surface pretreated with gelatin and feeder cells made of mouse embryonic fibroblasts (MEF), and the culture medium also contains serum. Although the introduction of feeder cells and serum provides pluripotent stem cells with abundant growth factors, peptides, hormones and other substances, it has large batch differences, may be contaminated by viruses or mycoplasmas, unclear ingredients and contains heterologous ingredients, and is not conducive to the separation of target products. The maintenance and application prospects of pluripotent stem cells cultured in this system are seriously restricted. The chemically defined culture medium has clear components and small batch differences, which is conducive to improving the stability and standardization of pluripotent stem cell related products. However, the existing chemically defined culture medium still has many components, a short shelf life of the prepared complete culture medium, and contains heterologous components, which leads to a high cost of the culture medium and is not conducive to large-scale in vitro culture and clinical application of pluripotent stem cells.

[0004] Therefore, we proposed a xeno-free and chemically defined human pluripotent stem cell culture medium to address the above-mentioned issues. Summary of the invention

[0005] The object of the present invention is to provide a human pluripotent stem cell culture medium with no xenogeneic components and defined chemical composition, so as to solve the problems raised in the above-mentioned background technology.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] A human pluripotent stem cell culture medium without xenogeneic components and with limited chemical components comprises a basal solution and an additive mother solution, wherein the additive mother solution comprises mother solution A, mother solution B, mother solution C, mother solution D and mother solution E. The preparation method of the human pluripotent stem cell culture medium comprises the following steps: mixing the basal solution with the additive mother solution consisting of mother solution A, mother solution B, mother solution C, mother solution D and mother solution E in sequence to obtain a complete culture medium with a final concentration of 64 mg / L L-ascorbic acid-2-phosphate trisodium salt, 14 μg / L sodium selenite, 540 μg / mL sodium bicarbonate, 20 mg / L recombinant human insulin, 10 μg / L recombinant human transferrin, 100 μg / L human stable recombinant human fibroblast growth factor 2 and 2 μg / L recombinant human transforming growth factor β.

[0008] As a further solution of the present invention: the mother solution A comprises: weighing 320 mg of L-ascorbic acid-2-phosphate trisodium salt using an electronic balance, adding 1.75 mL of sterile water for injection and 250 μL of hydrochloric acid, filtering and sterilizing using a 0.22 μM filter membrane after fully dissolving, and storing at -20°C after aliquoting.

[0009] As a further solution of the present invention: the mother solution B comprises: using sterile water for injection added with 20 μL of hydrochloric acid to dissolve 100 mg of human recombinant insulin dry powder, adding 27.5 μL of NaOH solution after sufficient dissolution, filtering and sterilizing with a 0.22 μM filter membrane, and storing at -20°C after packaging.

[0010] As a further solution of the present invention: the mother solution C comprises: using 2.5 mL of sterile water for injection to dissolve 250 mg of recombinant human transferrin, 22.5 mg of heat-stable recombinant human fibroblast growth factor, and 350 μg of recombinant human transforming growth factor; after sufficient dissolution, filtering and sterilizing using a 0.22 μM filter membrane is performed, and the mother solution C is stored at -20°C after being divided and packaged.

[0011] As a further solution of the present invention: the mother solution D comprises: weighing 70 mg of sodium selenite using an electronic balance, adding 100 mL of sterile water for injection, filtering and sterilizing using a 0.22 μM filter membrane after fully dissolving, and storing at -20°C after aliquoting.

[0012] As a further solution of the present invention: the mother solution E comprises: 2.50 g of sodium bicarbonate is weighed using an electronic balance, 50 mL of sterile water for injection is added, after fully dissolving, the solution is filtered and sterilized using a 0.22 μM filter membrane, and the solution is stored at 4° C. after being divided and packaged.

[0013] As a further solution of the present invention: the human pluripotent stem cell culture medium adjusts the culture medium parameters as follows: pH 7.0-7.3, osmotic pressure 270-310 mOsm / kg; bacteria and fungi detection: negative; mycoplasma detection: negative; endotoxin: <0.5 EU / mL.

[0014] As a further scheme of the present invention: the human recombinant insulin dry powder includes human recombinant insulin, and the preparation method of human recombinant insulin includes the following steps: construction of a recombinant human proinsulin gene expression vector; induction expression of a recombinant human proinsulin fusion protein; purification and separation of recombinant human insulin analogs to obtain recombinant human insulin.

[0015] As a further scheme of the present invention: the construction of the recombinant human proinsulin gene expression vector includes: synthesizing the recombinant human proinsulin gene sequence having the gene sequences of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4 by a DNA synthesis company, connecting the synthesized recombinant human proinsulin target fragment with the prokaryotic expression vector pET30a(+), which has been connected to the SUMO gene, at 4°C overnight, and after enzyme digestion and identification, constructing the recombinant human proinsulin gene expression vector pET-30a-SUMO-Insulin.

[0016] As a further scheme of the present invention: the induced expression of the recombinant human proinsulin fusion protein includes: transforming the constructed recombinant human proinsulin gene expression vector pET-30a-SUMO-Insulin into the strain Rosseta (DE3), inoculating the transformed single colonies into 20mL LB culture medium containing ampicillin (Amp) (100ug / mL), culturing at 37°C for 8-10h, inoculating into another 20mL LB culture medium containing Amp (100μg / mL) at a ratio of 1:100, culturing at 37°C, and when A600 is 0.3-0.4, inducing for 6-10h under the conditions of temperature of 18-25°C, final concentration of isopropylthiogalactoside (IPTG) of 0.1-1mmol / L, and rotation speed of 40-120r / min, and then collecting the bacteria; the recombinant human insulin analog purification and separation to obtain recombinant human insulin includes: The cells are resuspended in buffer, ultrasonically disrupted and then centrifuged or filtered to obtain a bacterial supernatant, the supernatant is subjected to Ni column affinity chromatography to obtain a crude sample of SUMO-Insulin fusion protein, trypsin is added to the obtained crude sample of SUMO-Insulin fusion protein for enzymatic digestion, the enzymatic digestion product is intermediately purified by one or two steps of S or CM cation exchange chromatography, and finally purified by one or two steps of C8 or C18 high performance liquid chromatography preparation technology to obtain a recombinant human insulin with a purity greater than 98%.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] This xeno-free and chemically defined human pluripotent stem cell culture medium provides a chemically defined and xeno-free culture medium that can be used for human embryonic stem cells and induced pluripotent stem cells. The culture medium has simple and clear ingredients and does not contain xeno-free ingredients. It can effectively expand pluripotent stem cells in vitro and maintain their differentiation ability. It is suitable for general scientific research and cell therapy industry. It has obvious advantages in cell proliferation rate and stability when stored at 4°C, and there is no significant difference in maintaining stem cell pluripotency compared with competing products. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a comparison chart between stem cells obtained from the culture medium when hESCs are cultured to the 10th generation in the stem cell culture medium stability test of the present invention and commercially available competing products.

[0020] Figure 2 This is a comparison chart between stem cells obtained from the culture medium when hiPSCs are cultured to the 10th generation in the stem cell culture medium stability test of the present invention and commercially available competing products.

[0021] Figure 3 This is a comparison chart between stem cells obtained after hESC was continuously cultured for three days at 4° C. in the stem cell culture medium stability test of the present invention and commercially available competing products.

[0022] Figure 4 This is a comparison chart between the stem cells obtained after hiPSCs were continuously cultured at 4°C for three days in the stability test of the stem cell culture medium of the present invention and commercially available competing products.

[0023] Figure 5 This is a line graph showing the counting of cells in each group for three consecutive generations when the cells were cultured to the fifth generation in the present invention.

[0024] Figure 6 The figure is the result of immunofluorescence staining of pluripotency genes SOX2 and OCT4 after hESCs were cultured in the culture medium of the present invention for 10 generations.

[0025] Figure 7 The results of immunofluorescence staining of pluripotency genes SOX2 and OCT4 after hESCs were cultured in a commercial competitor culture medium for 10 generations.

[0026] Figure 8 The figure is the result of immunofluorescence staining of pluripotency genes SOX2 and OCT4 after hiPSCs were cultured in the culture medium of the present invention for 10 generations.

[0027] Fig. 9 The results of immunofluorescence staining of pluripotency genes SOX2 and OCT4 after hiPSCs were cultured in a commercial competitor culture medium for 10 generations. DETAILED DESCRIPTION

[0028] In one embodiment, Figure 1-Figure 5 As shown, a human pluripotent stem cell culture medium without xenogeneic components and with limited chemical components comprises a basal solution and an additive mother solution, wherein the additive mother solution comprises mother solution A, mother solution B, mother solution C, mother solution D and mother solution E, and a method for preparing the human pluripotent stem cell culture medium comprises mixing the basal solution with the additive mother solution consisting of mother solution A, mother solution B, mother solution C, mother solution D and mother solution E in sequence to obtain a complete culture medium with a final concentration of 64 mg / L L-ascorbic acid-2-phosphate trisodium salt, 14 μg / L sodium selenite, 540 μg / mL sodium bicarbonate, 20 mg / L recombinant human insulin, 10 μg / L recombinant human transferrin, 100 μg / L human stable recombinant human fibroblast growth factor 2, and 2 μg / L recombinant human transforming growth factor β;

[0029] Mother solution A includes: using an electronic balance to weigh 320 mg of L-ascorbic acid-2-phosphate trisodium salt, adding 1.75 mL of sterile water for injection and 250 μL of hydrochloric acid, filtering with a 0.22 μM filter membrane for sterilization after fully dissolving, and storing at -20°C after aliquoting; Mother solution B includes: using sterile water for injection with 20 μL of hydrochloric acid added to dissolve 100 mg of human recombinant insulin dry powder, adding 27.5 μL of NaOH solution, sterilized by filtration using a 0.22μM filter membrane, stored at -20°C after aliquoting; mother solution C includes: 250mg of recombinant human transferrin, 22.5mg of heat-stable recombinant human fibroblast growth factor, and 350μg of recombinant human transforming growth factor are dissolved in 2.5mL of sterile water for injection, sterilized by filtration using a 0.22μM filter membrane after full dissolution, stored at -20°C after aliquoting; mother solution D includes: 70mg of sodium selenite is weighed using an electronic balance, 100mL of sterile water for injection is added, sterilized by filtration using a 0.22μM filter membrane after full dissolution, stored at -20°C after aliquoting; mother solution E includes: 2.50g of sodium bicarbonate is weighed using an electronic balance, 50mL of sterile water for injection is added, sterilized by filtration using a 0.22μM filter membrane after full dissolution, stored at 4°C after aliquoting

[0030] The parameters of the human pluripotent stem cell culture medium were adjusted as follows: pH 7.0-7.3, osmotic pressure 270-310 mOsm / kg; bacteria and fungi detection: negative; mycoplasma detection: negative; endotoxin: <0.5 EU / mL;

[0031] Human pluripotent stem cell culture test:

[0032] 1. Take out the aliquoted vitronectin from -20℃, place it at 4℃ to melt overnight, dilute it in DMEM medium, and add it to a concentration of 1μg / cm 2Add the amount into a 6-well plate and pretreat the surface of the culture dish for more than 1 hour at room temperature.

[0033] 2. Resuscitated human embryonic stem cells H9 and human induced pluripotent stem cells hiPSC cells were inoculated at a density of 80,000 cells / well in a 6-well plate pretreated with vitronectin. The experimental group used the culture medium prepared with the above scheme, and the control group used the commercially available competitor culture medium. On the day of inoculation and passage, 5 μM Y-27632 was added to the culture medium of both groups to promote cell adhesion. The cells were cultured in a carbon dioxide incubator at 37°C, 5% CO2, and saturated humidity.

[0034] 3. After 24 hours of culture, the culture medium was replaced with the corresponding culture medium without Y27632. Thereafter, the cells were replaced with culture medium every day until the cell confluence reached about 80%.

[0035] 4. When the cell confluence reaches 80%, remove the original culture medium, rinse the cells with 1 mL of D-PBS, and then add 500 μL of TrypLE to digest the cells at 37°C. When the cells begin to fall off from the bottom of the culture dish in pieces, add 4 mL of D-PBS to stop digestion. After gently blowing and mixing, centrifuge at 200g for 5 minutes to collect the cells. After the cells are re-selected in the corresponding culture medium, take 20 μL of the cell suspension, use trypan blue with a final concentration of 0.04% for staining and cell counting, and compare the proliferation rate of each group of cells.

[0036] 5. Inoculate the collected cells again according to step "2".

[0037] 6. Observe the changes in cell morphology every day and compare the morphological changes of each group of cells.

[0038] Reference Figure 6-Figure 9 (The scale bar in the figure is 100μM). Immunofluorescence was used to detect the expression of pluripotency-related genes in pluripotent stem cells:

[0039] The experimental group was prepared using the above-mentioned scheme, and the control group was prepared using the commercially available competitive culture medium. Human embryonic stem cells and induced pluripotent stem cells were cultured continuously for 10 generations. After removing the culture medium, D-PBS was used to rinse once, and 4% paraformaldehyde was added to fix the cells at room temperature for 15 minutes, and then 0.5% Triton was used to treat the cells for 15 minutes. After blocking with 5% BSA at room temperature for 30 minutes, OCT4 and SOX2 antibodies diluted as required were added and incubated overnight at 4°C. After rinsing with D-PBS, fluorescent secondary antibodies and DAPI dye diluted as required were added and incubated at room temperature for 1 hour. Finally, after rinsing with D-PBS, the cells were observed under a fluorescence microscope and photographed and recorded. There was no significant difference in the staining results of each group. It can be seen that the culture medium in the present invention can promote the rapid proliferation of pluripotent stem cells while maintaining the pluripotency of the cells.

[0040] Stability test at 4°C:

[0041] 1. The experimental group used the culture medium prepared using the above scheme, and the control group used the commercially available competitor culture medium. After being prepared into complete culture medium, it was stored at 4°C for 3 weeks and then used for the culture of human embryonic stem cells and induced pluripotent stem cells.

[0042] 2. Use a microscope to take photos and record the changes in cell clone morphology during cell culture.

[0043] Depend on Figure 1-Figure 4 It can be seen that hESC and hiPSC were subcultured using the culture medium stored at 4°C for 3 weeks. After three days of continuous culture, it can be seen that the cell clones in the experimental group (patented culture medium) are dense, with clear and smooth edges, showing a typical pluripotent stem cell clone morphology; while the cell clones in the control group (commercial competitor) are loose and have rough edges, showing an early differentiation state; this shows that the culture medium in the experimental group has better stability at 4°C.

[0044] The human recombinant insulin dry powder includes human recombinant insulin. The preparation method of human recombinant insulin includes the following steps: constructing a recombinant human proinsulin gene expression vector; inducing expression of a recombinant human proinsulin fusion protein; purifying and separating recombinant human insulin analogs to obtain recombinant human insulin;

[0045] The construction of the recombinant human proinsulin gene expression vector includes: synthesizing the recombinant human proinsulin gene sequence with SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4 gene sequences by a DNA synthesis company, connecting the synthesized recombinant human proinsulin target fragment with the prokaryotic expression vector pET30a(+), which has been connected to the SUMO gene, at 4°C overnight, and constructing the recombinant human proinsulin gene expression vector pET-30a-SUMO-Insulin after enzyme digestion and identification; the induced expression of the recombinant human proinsulin fusion protein includes: transforming the constructed recombinant human proinsulin gene expression vector pET-30a-SUMO-Insulin into the strain Rosseta (DE3) for expression, and inoculating the transformed single colonies into 2 0mL of LB medium containing ampicillin (Amp) (100 μg / mL), cultured at 37°C for 8-10h, inoculated into another 20mL of LB medium containing Amp (100 μg / mL) at a ratio of 1:100, cultured at 37°C, when A600 is 0.3-0.4, induced at a temperature of 18-25°C, an isopropylthiogalactoside (IPTG) final concentration of 0.1-1mmol / L, and a rotation speed of 40-120r / min for 6-10h, and then collected the bacteria; recombinant human insulin analogs are purified and separated to obtain recombinant human insulin, including: the obtained bacteria, using lysis The cells are resuspended in buffer, ultrasonically disrupted, and then centrifuged or filtered to obtain a clear supernatant of the cells. The clear supernatant is subjected to Ni column affinity chromatography to obtain a crude sample of SUMO-Insulin fusion protein. Trypsin is added to the obtained crude sample of SUMO-Insulin fusion protein for enzymatic digestion. The enzymatic digestion product is intermediately purified by one or two steps of S or CM cation exchange chromatography, and finally purified by one or two steps of C8 or C18 high performance liquid chromatography preparation technology to obtain a recombinant human insulin with a purity greater than 98%; Figure 5 As shown, when the cells were cultured to the fifth generation, the cells in each group were counted for three consecutive generations, and the starting cell number of each group was 80,000 cells each time. It can be seen from the figure that the culture medium in the patent (experimental group) has a stronger ability to promote the proliferation of pluripotent stem cells than the commercially available competitor (control group).

[0046] The present invention provides a culture medium with defined chemical components and no xenobiotics that can be used for human embryonic stem cells and induced pluripotent stem cells. The culture medium has simple and clear components and does not contain xenobiotics. It can effectively amplify pluripotent stem cells in vitro and maintain their differentiation ability. It is suitable for general scientific research and cell therapy industry. It has obvious advantages in cell proliferation rate and stability when stored at 4°C, and has no significant difference from competing products in maintaining stem cell pluripotency.

[0047] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A xeno-free and chemically defined human pluripotent stem cell culture medium, characterized in that: The invention comprises a basal solution and an additive mother solution, wherein the additive mother solution comprises mother solution A, mother solution B, mother solution C, mother solution D and mother solution E. The preparation method of the human pluripotent stem cell culture medium comprises: mixing the basal solution with the additive mother solution composed of mother solution A, mother solution B, mother solution C, mother solution D and mother solution E in sequence to obtain a complete culture medium with a final concentration of 64 mg / L L-ascorbic acid-2-phosphate trisodium salt, 14 μg / L sodium selenite, 540 μg / mL sodium bicarbonate, 20 mg / L recombinant human insulin, 10 μg / L recombinant human transferrin, 100 μg / L human stable recombinant human fibroblast growth factor 2, and 2 μg / L recombinant human transforming growth factor β.

2. The xeno-free and chemically defined human pluripotent stem cell culture medium according to claim 1, characterized in that: The mother solution A comprises: weighing 320 mg of L-ascorbic acid-2-phosphate trisodium salt using an electronic balance, adding 1.75 mL of sterile water for injection and 250 μL of hydrochloric acid, filtering and sterilizing using a 0.22 μM filter membrane after fully dissolving, and storing at -20°C after aliquoting.

3. The xeno-free and chemically defined human pluripotent stem cell culture medium according to claim 2, characterized in that: The mother solution B comprises: using sterile injection water added with 20 μL of hydrochloric acid to dissolve 100 mg of human recombinant insulin dry powder, adding 27.5 μL of NaOH solution after sufficient dissolution, filtering and sterilizing with a 0.22 μM filter membrane, and storing at -20°C after aliquoting.

4. The xeno-free and chemically defined human pluripotent stem cell culture medium according to claim 1, characterized in that: The mother solution C comprises: using 2.5 mL of sterile water for injection to dissolve 250 mg of recombinant human transferrin, 22.5 mg of heat-stable recombinant human fibroblast growth factor, and 350 μg of recombinant human transforming growth factor; after being fully dissolved, filtering and sterilizing using a 0.22 μM filter membrane; and storing at -20°C after being divided and packaged.

5. The xeno-free and chemically defined human pluripotent stem cell culture medium according to claim 1, characterized in that: The mother solution D comprises: weighing 70 mg of sodium selenite using an electronic balance, adding 100 mL of sterile water for injection, filtering and sterilizing using a 0.22 μM filter membrane after fully dissolving, and storing at -20°C after aliquoting.

6. The xeno-free and chemically defined human pluripotent stem cell culture medium according to claim 1, characterized in that: The mother solution E comprises: weighing 2.50 g of sodium bicarbonate using an electronic balance, adding 50 mL of sterile water for injection, filtering and sterilizing using a 0.22 μM filter membrane after fully dissolving, and storing at 4° C. after aliquoting.

7. The xeno-free and chemically defined human pluripotent stem cell culture medium according to claim 1, characterized in that: The human pluripotent stem cell culture medium adjusts the culture medium parameters as follows: pH 7.0-7.3, osmotic pressure 270-310 mOsm / kg; bacteria and fungi detection: negative; mycoplasma detection: negative; Endotoxin: <0.5EU / mL.

8. The xeno-free and chemically defined human pluripotent stem cell culture medium according to claim 3, characterized in that: The human recombinant insulin dry powder includes human recombinant insulin. The preparation method of human recombinant insulin includes the following steps: construction of a recombinant human proinsulin gene expression vector; induction expression of a recombinant human proinsulin fusion protein; and purification and separation of recombinant human insulin analogs to obtain recombinant human insulin.

9. The xeno-free and chemically defined human pluripotent stem cell culture medium according to claim 8, characterized in that: The construction of the recombinant human proinsulin gene expression vector includes: synthesizing the recombinant human proinsulin gene sequence having the gene sequences of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 by a DNA synthesis company, connecting the synthesized recombinant human proinsulin target fragment with the prokaryotic expression vector pET30a(+), which has been connected to the SUMO gene, at 4°C overnight, and after enzyme digestion and identification, constructing the recombinant human proinsulin gene expression vector pET-30a-SUMO-Insulin.

10. The xeno-free and chemically defined human pluripotent stem cell culture medium according to claim 9, characterized in that: The inducible expression of the recombinant human proinsulin fusion protein includes: transforming the constructed recombinant human proinsulin gene expression vector pET-30a-SUMO-Insulin into the strain Rosseta (DE3), inoculating the transformed single colonies into 20 mL of LB culture medium containing ampicillin (Amp) (100 μg / mL), culturing at 37°C for 8-10 hours, inoculating into another 20 mL of LB culture medium containing Amp (100 μg / mL) at a ratio of 1:100, culturing at 37°C, and when A600 is 0.3-0.4, inducing for 6-10 hours under the conditions of a temperature of 18-25°C, a final concentration of isopropylthiogalactoside (IPTG) of 0.1-1 mmol / L, and a rotation speed of 40-120 r / min, and then collecting the bacteria; the recombinant human insulin analog purification and separation to obtain recombinant human insulin includes: The cells are resuspended in buffer, ultrasonically disrupted and then centrifuged or filtered to obtain a bacterial supernatant, the supernatant is subjected to Ni column affinity chromatography to obtain a crude sample of SUMO-Insulin fusion protein, trypsin is added to the obtained crude sample of SUMO-Insulin fusion protein for enzymatic digestion, the enzymatic digestion product is intermediately purified by one or two steps of S or CM cation exchange chromatography, and finally purified by one or two steps of C8 or C18 high performance liquid chromatography preparation technology to obtain a recombinant human insulin with a purity greater than 98%.

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