CHO cell transient transfection method and application thereof

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to a CHO cell transient transfection method and application thereof.The CHO cell transient transfection method comprises the following steps that subcultured CHO cells are resuspended through a transfection culture medium, the cell density in CHO cell suspension is 0.8 * 10 < 7 >-1.5 * 10 < 7 > cells / mL, a PEI transfection reagent containing 40 kDa linear PEI is used, plasmid DNA and the PEI transfection reagent with the mass ratio being 1: (1-6) are added into the CHO cell suspension for transfection, and transfection is conducted after the PEI transfection reagent is added into the CHO cell suspension; the addition amount of plasmid DNA in each milliliter of CHO cell suspension is 3.0-7.0 [mu] g, transfecting for 20-24 h, then adding a cell suspension culture medium, transfecting for 48-72 h, then repeatedly transfecting, and culturing to obtain the recombinant CHO cell. According to the method, the transfection efficiency can be improved, more cells for expressing the recombinant protein can be obtained, and the transient expression of the target protein can be remarkably improved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of genetic engineering, and specifically relates to a CHO cell transient transfection method and application thereof. BACKGROUND

[0002] Chinese hamster ovary (CHO) cells have become the dominant platform for the research and production of recombinant protein drugs due to their similar post-translational modification system to humanization and the characteristics of easy large-scale suspension culture. However, stable expression of recombinant proteins usually requires time-consuming antibiotic selection and monoclonal screening. In contrast, transient expression has the advantages of short time consumption and simple steps, but CHO cell transient expression faces the problems of low transfection efficiency and low expression level.

[0003] Therefore, it is of great significance to improve the transfection efficiency of target genes and the yield of recombinant proteins by modifying the CHO cell transient expression system. Existing technologies have attempted to modify CHO cell transfection through various ways: for example, patent CN118879779A discloses an improved CHO-S cell transient transfection method, which improves the transfection efficiency, but the cell transfection density is only 4×10 6 cells / mL~6×10 6 cells / mL; patent CN116769832B discloses a transient transfection method of mammalian cells, which improves the cell seeding density and directly transfects at about 48 hours after inoculation without medium change, but the transfection cell density is only 4.0×10 6 cells / mL. Patent CN112695056A discloses a transfection reagent and transfection method for improving transient transfection efficiency, which improves the transfection efficiency by adding 0.5v / v%~1v / v% DMSO in the transfection reagent, but the cell transfection density is only 1×10 6 cells / mL. Low cell density during transfection will directly limit the number of cells expressing target proteins and the expression level of recombinant proteins.

[0004] Transfection conditions are the key to affect the transfection efficiency of CHO cells and the transient expression of recombinant proteins. At present, the commonly used transfection methods include chemical transfection, electroporation and virus-mediated transfection. Among them, polyethyleneimine (PEI) is widely used in the transient transfection of CHO cells due to its high efficiency, low toxicity and low cost. However, the molecular weight, charge density and ratio of PEI to DNA all affect the transfection efficiency and cytotoxicity. At present, 25 kDa PEI is mostly used in transient transfection, but it has low transfection efficiency and batch instability. In addition, high-density suspension culture can be used to improve protein yield, but high-density suspension culture may lead to rapid consumption of nutrients and accumulation of metabolic waste, thereby affecting cell viability. Although there are currently methods for optimizing transient transfection and high-density suspension transfection conditions, the transfection reagent used by researchers in high-density transfection optimization is 25 kDa PEI, and the cells used in transfection method optimization are low-density culture (2×10 6 cells / mL~6×10 6 cells / mL), which limits the efficiency of the recombinant protein transient expression system and the potential of industrialized production. 25 kDa PEI cannot achieve the best effect under high-density transfection conditions, and the transfection method under low-density culture conditions is not completely suitable for high-density culture environment. Although the transfection efficiency of 40 kDa PEI is higher, its toxicity also increases with the increase of relative molecular mass. The invention patent with application number CN114561413A discloses a 40 kDa PEI transient transfection reagent prepared by using sodium chloride solution and magnesium chloride solution for adherent cell transfection, which has high transfection efficiency and low cytotoxicity, but for high-density suspension transfection, a new CHO cell transfection method needs to be developed. SUMMARY

[0005] In order to solve the technical problems of low transfection efficiency and low expression level of recombinant proteins in the transient transfection technology, the present application provides a CHO cell transient transfection method and its application, which can significantly improve the expression level of exogenous proteins in CHO cells and efficiently express target genes.

[0006] In order to achieve the above purpose, the first aspect of the present application provides a CHO cell transient transfection method, which comprises the following steps: Resuspend the CHO cells cultured by subculture with transfection medium to obtain a CHO cell suspension with a cell density of 0.8×10 7 cells / mL~1.5×10 7 cells / mL; Add plasmid DNA and PEI transfection reagent to the CHO cell suspension for cell transfection; The cell suspension culture medium is added 20h~24h after transfection, and the cells are collected 48h~72h after transfection; the CHO cells are suspended in the transfection culture medium, and repeated transfection is carried out, and then cell culture is carried out to obtain recombinant CHO cells; The amount of the plasmid DNA added in the CHO cell suspension is 3.0 μg~7.0 μg per milliliter, and the mass ratio of the plasmid DNA to the PEI transfection reagent is 1:1~6; the PEI is 40 kDa linear PEI.

[0007] Further, the cell suspension culture medium is based on the Pro® CHO cell suspension culture medium, and further includes 0.5 mM~1.0 mM valproic acid, 1.5 mM~2.5 mM sodium butyrate, 1 μM~3 μM soybean isoflavones and 0.5 g~1 g yeast extract; and the transfection culture medium is Pro® CHO cell transfection culture medium.

[0008] Further, the concentration of PEI in the PEI transfection reagent is 0.33 mg / mL; the pH value of the PEI transfection reagent is 7.2; and the PEI transfection reagent is composed of PEI, HEPES buffer solution, sodium chloride and magnesium chloride.

[0009] Further, the concentration of the PEI solution is 1.0 mg / mL, the concentration of the sodium chloride solution is 5 mol / L, and the concentration of the magnesium chloride solution is 1 mol / L.

[0010] Further, the volume ratio of the PEI solution, the sodium chloride solution and the magnesium chloride solution is 4:7.5:0.6.

[0011] Further, the temperature for mixing the plasmid DNA and the PEI transfection reagent is 37℃, and the mixing time is 15 min~20 min.

[0012] Further, the temperature for cell culture is 37℃, the volume fraction of CO2 is 5%, and the rotation speed is 110 rpm~130 rpm.

[0013] Further, the number of times of repeated transfection is 1~2 times.

[0014] Further, the CHO cells are CHO-S, CHO-K1, CHO-DXB11 or CHO-DG44.

[0015] The second aspect of the present application provides a recombinant CHO cell obtained by the method.

[0016] Compared with the prior art, the present application has the following beneficial effects: The application provides a CHO cell transient transfection method and application thereof, and comprises the following steps: resuspending the subcultured CHO cells by using a transfection culture medium, so that the cell density in the CHO cell suspension is 0.8*10 7 cells / mL~1.5*10 7 cells / mL, adding the plasmid DNA and PEI transfection reagent with a mass ratio of 1:1~6 into the CHO cell suspension for transfection, wherein the PEI transfection reagent comprises 40kDa linear PEI, HEPES buffer solution, sodium chloride and magnesium chloride; the added amount of the plasmid DNA in each milliliter of the CHO cell suspension is 3.0ug~7.0ug, the cell suspension culture medium is added after 20h~24h of transfection, repeated transfection is carried out after 48h~72h of transfection, and a recombinant CHO cell is obtained after cell culture.

[0017] (1) The application improves the cell transfection density, and the exogenous gene introduced into the host cell by transient gene expression is generally not integrated into the genome of the host cell, so that the recombinant protein expression can be realized in a short time (generally 96 hours to 7 days after transfection). The application improves the cell amount for expressing the recombinant protein by improving the cell transfection density, and higher expression of the target protein is obtained.

[0018] (2) The application adjusts the amount of the plasmid DNA, the ratio of the plasmid DNA and the PEI transfection reagent and the transfection times during the high-density cell transfection, improves the transfection efficiency, and obtains higher expression of the target protein.

[0019] (3) The application uses the PEI transfection reagent containing 40kDa linear PEI to improve the transient transfection efficiency, and the PEI transfection reagent comprises PEI, HEPES buffer solution, sodium chloride and magnesium chloride. The PEI transfection reagent can improve the cell transfection efficiency without affecting the cell viability. The cell suspension culture medium composed of Pro CHO cell suspension culture medium, valproic acid, sodium butyrate, soybean isoflavone and yeast extract is added after 20h~24h of transfection. On the one hand, the cell toxicity of the 40kDa PEI transfection reagent is reduced by supplementing the culture medium, and on the other hand, the additives such as yeast extract are added to supplement the nutrients required by the high-density cell culture, and the HEPES buffer solution, sodium chloride and magnesium chloride in the cell suspension culture medium have the effect of increasing the cell membrane permeability, so that the transient transfection has higher transfection efficiency. The cells are collected after 48h~72h of transfection, resuspended in fresh culture medium, and subjected to repeated transfection, so as to further improve the transfection efficiency. Compared with the traditional transfection method, higher cell amount for expressing the recombinant protein can be obtained by using the method provided by the application, and a new idea for improving the transient expression of the recombinant protein is provided. BRIEF DESCRIPTION OF DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the accompanying drawings required by the embodiments or the prior art description will be briefly introduced below. Obviously, the accompanying drawings in the following description only constitute some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained without creative labor based on these drawings.

[0021] Figure 1 Effect of transfection reagent prepared by different buffer solutions on transfection efficiency and cell viability, Figure 1 A graph in FIG. 1 is effect of transfection reagent prepared by different buffer solutions on transfection efficiency; Figure 1 B graph in FIG. 1 is effect of transfection reagent prepared by different buffer solutions on cell viability;*represents P <0.05.

[0022] Figure 2 Effect of different amounts of plasmid DNA on transfection efficiency, cell viability and recombinant protein expression; Figure 2 A graph in FIG. 2 is effect of different amounts of plasmid DNA on cell transfection efficiency and cell viability; Figure 2 B graph in FIG. 2 is fluorescence intensity analysis of recombinant green fluorescent protein (EGFP); Figure 2 C graph in FIG. 2 is effect on relative expression of recombinant secreted alkaline phosphatase (SEAP);*represents p <0.05.

[0023] Figure 3 Effect of different mass ratios of plasmid DNA and PEI transfection reagent on cell transfection efficiency, cell viability and recombinant protein expression; Figure 3 A graph in FIG. 3 is effect of different mass ratios of plasmid DNA and PEI transfection reagent on cell transfection efficiency and cell viability; Figure 3 B graph in FIG. 3 is fluorescence intensity analysis of recombinant green fluorescent protein (EGFP); Figure 3 C graph in FIG. 3 is effect on relative expression of recombinant secreted alkaline phosphatase (SEAP) activity;*represents p <0.05.

[0024] Figure 4 Effect of different repeated transfection times on cell transfection efficiency, cell viability and recombinant protein expression; Figure 4 A graph in FIG. 4 is effect of different repeated transfection times on cell transfection efficiency and cell viability; Figure 4 B graph in FIG. 4 is fluorescence intensity analysis of recombinant green fluorescent protein (EGFP) corresponding to different repeated transfection times; Figure 4 C graph in FIG. 4 is effect of different repeated transfection times on relative expression level of secreted alkaline phosphatase (SEAP);*represents p <0.05.

[0025] Figure 5 The Western blot detection results of the expression of the target protein are shown from left to right as follows: the expression level of recombinant human collagen type III (CollIII) in the control group, the expression level of human serum albumin (HSA) in the control group, the expression level of recombinant human collagen type III (CollIII) in the experimental group, and the expression level of human serum albumin (HSA) in the experimental group. DETAILED DESCRIPTION

[0026] The specific embodiments of the present application are described in detail below, but it should be understood that the scope of protection of the present application is not limited by the specific embodiments. Based on the examples in the present application, all other examples obtained by those of ordinary skill in the art without making creative efforts fall within the scope of protection of the present application. The experimental methods described in the embodiments of the present application are conventional methods, and the materials and reagents used in the following examples can be obtained from commercial channels unless otherwise specified.

[0027] The CHO cells used in the embodiments of the present application are domesticated serum-free medium cultured suspension cells, and the CHO cell line used is CHO-S. The Pro®CHO cell transfection medium is purchased from Henan Puno Yi Biological Product Research Institute Co., Ltd., valproic acid, sodium butyrate and yeast extract are purchased from Sigma, and soy isoflavones are purchased from Shanghai Yuan Ye Biological Technology Co., Ltd. The expression vector used in the present application is pWTY9.3 (purchased from Henan Puno Yi Biological Product Research Institute Co., Ltd.). First, the blasticidin resistance gene expression cassette in the pWTY9.3 vector is deleted to construct a pWTY-EGFP transient expression vector. The vectors used for expressing secreted alkaline phosphatase (SEAP), recombinant human collagen type III (CollIII) and human serum albumin (HAS) are pWTY-SEAP, pWTY-CollIII and pWTY-HAS, respectively. pWTY-SEAP, pWTY-CollIII and pWTY-HAS are obtained by replacing the EGFP sequence in the pWTY-EGFP vector with the SEAP, CollIII and HSA gene sequences, respectively, based on the pWTY-EGFP vector. 40kDa linear PEI is purchased from Polysciences Company, USA.

[0028] CHO cells have a similar post-translational modification system to humanization, are easy to culture in suspension on a large scale, and have become the main platform for the research and development and production of recombinant protein drugs. Stable expression of recombinant proteins usually requires antibiotic selection and monoclonal screening, which is time-consuming. Transient expression is time-saving and simple. However, CHO cell transient expression faces the problems of low transfection efficiency and low expression level.

[0029] The application provides a CHO cell transient transfection method and application thereof, and the method can realize high-density transfection of CHO cells expressing recombinant proteins, improves transfection efficiency, and thus higher expression of target proteins is obtained, and a new idea for improving transient expression of recombinant proteins is provided.

[0030] Embodiment 1: a CHO cell transient transfection method, comprising the following steps: 1. Cell culture CHO cells are inoculated in DMEM / F12 culture medium containing 10v / v% fetal bovine serum, and adherent culture is carried out at 37℃ and 5v / v% CO2; when the cell confluence is 80%-90%, the cells are trypsinized. Then, the CHO cells are inoculated in 125mL shake flasks at a cell density of 3×10 5 cells / mL-5×10 5 cells / mL, and are placed in a 37℃, 5v / v% CO2 cell incubator shaker at a rotation speed of 120rpm for suspension culture for 3 days.

[0031] 2. Cell transfection 2.1. CHO cells are collected, and the cell viability is detected to be higher than 98%; 1.0×10 7 cells / mL of the cells are added into 1.8mL of 37℃ preheated Pro® CHO transfection medium in a 6-well culture plate to obtain a CHO cell suspension.

[0032] 2.2. 1.5mL EP tubes are taken, DMEM / F12 base medium is added, 8.0μg of plasmid DNA is mixed, PEI transfection reagent with a concentration of 0.33mg / mL is added according to the mass ratio of plasmid DNA to PEI transfection reagent of 1:5 to obtain a DNA-PEI mixed solution (total volume 200μL), and 37℃ incubation is carried out for 20min.

[0033] 2.3. The DNA-PEI mixed solution is slowly added into the CHO cell suspension, and the transfected cells are placed in a 37℃, 5v / v% CO2 cell incubator at 120rpm for suspension culture.

[0034] 2.4. 2mL of Pro® CHO cell suspension culture medium is added 24h after transfection, and 0.75mM valproic acid, 2mM sodium butyrate, 2μM soybean isoflavone and 1g / L yeast extract are added; the cells are collected 48h after transfection, the CHO cells are suspended with Pro® CHO transfection medium, and repeated transfection is carried out; cell culture is carried out after repeated transfection to obtain recombinant CHO cells; the cell transfection efficiency and cell viability are detected 48h after transfection, and the recombinant protein expression level is detected 7d after transfection.

[0035] The present application further explores the influence of different conditions on the CHO cell transient transfection method, which is specifically as follows.

[0036] 1. Influence of transfection reagents prepared by different buffer solutions on transfection PEI was dissolved in distilled water and HEPES buffer (purchased from Biyun Tian) respectively to prepare PEI solution with a concentration of 1.0 mg / mL; then the PEI solution with a concentration of 1.0 mg / mL was mixed with 5 mol / L sodium chloride solution and 1 mol / L magnesium chloride solution according to a volume ratio of 4:7.5:0.6 to prepare PEI transfection reagent with a pH of 7.2 and a PEI concentration of 0.33 mg / mL.

[0037] The pWTY-EGFP plasmid was selected, and the mass ratio of plasmid DNA to PEI transfection reagent was 1:5 according to the volume of CHO cell suspension, and the PEI transfection reagent prepared by different buffer solutions (distilled water, HEPES, distilled water + sodium chloride + magnesium chloride, HEPES + sodium chloride + magnesium chloride) was added to obtain a DNA-PEI mixed solution, which was incubated at 37°C for 20 min.

[0038] According to the above CHO cell transient transfection method, the CHO cells were transiently transfected by using the transfection reagents prepared by different buffer solutions. The cell transfection efficiency and cell viability were detected.

[0039] The results are shown in Figure 1 The transfection efficiency of the PEI transfection reagent prepared by HEPES + sodium chloride + magnesium chloride buffer solution with a concentration of 0.33 mg / mL was the highest, and it had no significant effect on cell viability. Therefore, the PEI transfection reagent prepared by HEPES + sodium chloride + magnesium chloride buffer solution was used as the PEI transfection reagent in the subsequent experiment.

[0040] 2. Influence of different plasmid DNA addition amounts on transfection The pWTY-EGFP plasmid and the pWTY-SEAP plasmid were selected, and the concentration of plasmid DNA was 3 μg / mL, 4 μg / mL, 5 μg / mL, 6 μg / mL and 7 μg / mL according to the volume of CHO cell suspension, and the mass ratio of plasmid DNA to PEI transfection reagent was 1:5, and the PEI transfection reagent was added to obtain DNA-PEI mixed solution with different plasmid DNA amounts, which was incubated at 37°C for 20 min.

[0041] According to the above CHO cell transient transfection method, the CHO cells were transiently transfected by using the DNA-PEI mixed solution with different plasmid DNA amounts. The cell transfection efficiency, cell viability and recombinant protein expression level were detected.

[0042] The results are shown in Figure 2As shown, the transfection efficiency and expression levels of recombinant proteins EGFP and SEAP were highest in the 4 μg / mL plasmid DNA concentration group, while different plasmid DNA concentrations had no significant effect on cell viability. EGFP expression in the 4.0 µg / mL group was 1.61 times higher than that in the 5.0 µg / mL group (which had the lowest expression). p <0.05%, SEAP expression increased 1.48-fold ( p >0.05). Therefore, 4.0 µg / mL is the optimal plasmid DNA concentration.

[0043] 3. Effect of different plasmid DNA and PEI transfection reagent mass ratios on transfection pWTY-EGFP and pWTY-SEAP plasmids were selected respectively. PEI transfection reagent was added at a plasmid DNA concentration of 4.0 μg / mL and at a mass ratio of plasmid DNA to PEI transfection reagent of 1:1, 1:2, 1:3, 1:4, 1:5 and 1:6 to obtain DNA-PEI mixed solutions with different mass ratios of plasmid DNA to PEI transfection reagent.

[0044] Based on the transient transfection method for CHO cells described above, CHO cells were transiently transfected using DNA-PEI mixed solutions prepared above at different mass ratios. Cell transfection efficiency, cell viability, and recombinant protein expression levels were then assessed.

[0045] The results are as follows Figure 3 The results showed that when the mass ratio of plasmid DNA to PEI transfection reagent was 1:5, the cell transfection efficiency and recombinant protein expression were the highest, and EGFP expression increased by 3.96 times compared to the 1:1 group. p <0.05%, compared to the 1:2, 1:3, and 1:4 groups, the recombinant protein expression increased by 1.31-fold to 2.63-fold (%). p <0.05). Compared with the 1:1 group, SEAP activity was significantly increased in the 1:2, 1:3, 1:4, and 1:5 groups, increasing by 1.83-fold, 2.34-fold, 2.62-fold, and 3.43-fold respectively relative to the 1:1 group. p <0.05). When the mass ratio of plasmid DNA to PEI transfection reagent was 1:6, cell transfection efficiency, EGFP, and SEAP expression all showed a decreasing trend. Therefore, when the mass ratio of plasmid DNA to PEI transfection reagent was 1:5, the transient expression level of recombinant proteins in CHO cells was the highest. In addition, different mass ratios of DNA-PEI mixtures had no significant effect on cell viability.

[0046] 4. The effect of different transfection times on transfection Using pWTY-EGFP and pWTY-SEAP plasmids respectively, and following the transient transfection method for CHO cells described above, based on 1×10⁻⁶ cells / mL...7 The 1-time transfection group, the 2-time transfection group (repeated transfection once) and the 3-time transfection group (repeated transfection twice) were set under the conditions of a seeding cell density of 1×10

[0047] The results, as shown in Table 1, show that the transfection efficiency of the 2-time transfection group and the 3-time transfection group is significantly improved, and the EGFP fluorescence intensity is increased by 2.27 and 2.43 times respectively compared with the 1-time transfection group (P<0.05). Figure 4 p The SEAP activity is increased by 2.13 and 2.38 times respectively compared with the 1-time transfection group (P<0.05). p

[0048] According to the above optimal transfection conditions, the present application evaluates the influence of the optimal transfection conditions on the target protein, and the pWTY-CollIII plasmid and the pWTY-HSA plasmid are selected respectively. The experimental group: the CHO cell suspension with a cell density of 1×10 7 cells / mL is used for 2-time repeated transfection, the plasmid DNA is added in an amount of 4.0 μg per milliliter of the CHO cell suspension each time, and the plasmid DNA and the PEI transfection reagent are mixed in a mass ratio of 1:5 and then are used for suspension transfection.

[0049] The control group: the CHO cell suspension with a cell density of 2×10 6 cells / mL (the conventional cell transfection density) is used for 1-time transfection, the plasmid DNA is added in an amount of 3 μg per milliliter of the CHO cell suspension, and the plasmid DNA and the PEI transfection reagent are mixed in a mass ratio of 1:5 and then are used for suspension transfection.

[0050] The results, as shown in Table 2, show that compared with the control group, the transfection method of the experimental group can increase the expression amount of the recombinant human type III collagen and the human serum albumin by 4.86 times and 5.50 times respectively. Figure 5 From the above examples, it can be seen that the present application provides a CHO cell transient transfection method, which can significantly improve the expression level of the exogenous protein of the CHO cell and efficiently express the target gene.

[0051]

[0052] ​​​It is to be understood that every range of values disclosed herein is to be understood to encompass any and every sub-range of values within the range. Although the preferred embodiments of the invention have been described above, it will be appreciated that those skilled in the art, on consideration of this disclosure, will be able to devise additional embodiments that, although not explicitly described or shown herein, nonetheless fall within the scope of the present invention. Accordingly, the appended claims are intended to include within their scope all such alternatives, modifications and variations as fall within the scope of the present invention. Various features and aspects of the present invention will become apparent from the following examples, which are intended only to exemplify the invention. It should be understood, of course, that in the various examples of the present invention, the specific phrasing of the claims will depend on the exact nature of the claims sought.

[0053] Obviously, numerous modifications and variations of the present application are possible in light of the above teachings. It is therefore to be understood that within the scope of the appended claims and their equivalents, the present application can be practiced otherwise than as specifically described herein.

Claims

1. A method for transient transfection of CHO cells, characterized in that, Includes the following steps: CHO cells were resuspended in transfection medium to obtain a cell density of 0.8 × 10⁶ cells / year. 7 cells / mL ~1.5×10 7 CHO cell suspension with cells / mL; Plasmid DNA and PEI transfection reagent were added to the CHO cell suspension for cell transfection; 20-24 h after transfection, cell suspension medium was added. Cells were collected 48-72 h after transfection. CHO cells were suspended in transfection medium and transfected repeatedly. After repeated transfection, cells were cultured to obtain recombinant CHO cells. The amount of plasmid DNA added to each milliliter of the CHO cell suspension is 3.0 μg to 7.0 μg, and the mass ratio of the plasmid DNA to the PEI transfection reagent is 1:1 to 6; the PEI is a 40 kDa linear PEI.

2. The transfection method according to claim 1, characterized in that, Each liter of the cell suspension medium is based on Pro®CHO cell suspension medium and also includes 0.5mM~1.0mM valproic acid, 1.5mM~2.5mM sodium butyrate, 1μM~3μM soy isoflavones and 0.5g~1g yeast extract; the transfection medium is Pro®CHO cell transfection medium.

3. The transfection method according to claim 1, characterized in that, The PEI transfection reagent has a PEI concentration of 0.33 mg / mL; the PEI transfection reagent is composed of PEI, HEPES buffer solution, sodium chloride and magnesium chloride; the PEI solution has a concentration of 1.0 mg / mL, the sodium chloride solution has a concentration of 5 mol / L, and the magnesium chloride solution has a concentration of 1 mol / L; the volume ratio of the PEI solution, sodium chloride solution and magnesium chloride solution is 4:7.5:0.

6.

4. The transfection method according to claim 1, characterized in that, The plasmid DNA and PEI transfection reagent were mixed and incubated at 37°C for 15-20 minutes.

5. The transfection method according to claim 1, characterized in that, The cell culture temperature was 37°C, the CO2 volume fraction was 5%, and the rotation speed was 110 rpm to 130 rpm.

6. The transfection method according to claim 1, characterized in that, The number of repeated transfections is 1 to 2 times.

7. The transfection method according to claim 1, characterized in that, The CHO cells are CHO-S, CHO-K1, CHO-DXB11, or CHO-DG44.

8. A recombinant CHO cell obtained by the method according to any one of claims 1 to 6.

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