Insect expression method using cationic polymer as a vehicle and application thereof

Abstract

The application belongs to the technical field of biology and particularly relates to an insect expression method using cationic polymer as a medium and application. 7 ~3x10 7 Live cells / mL; linear polyethyleneimine with a molecular weight of 40 kDa is added to the cell suspension at an amount of 2-3 ug / 10 6 Live cells, and baculovirus DNA vector is added at an amount of 0.5-1.25 ug / 10 6 Live cells; the linear polyethyleneimine and baculovirus DNA vector are mixed immediately before transfection; and P0 generation baculovirus is obtained after culture after transfection. The application significantly improves the transfection efficiency of SF9 cells and the yield of P0 generation baculovirus by high-density transfection, optimization of the amount of transfection reagent and DNA-PEI complex incubation time, and in combination with auxiliary reagents, and the P0 generation baculovirus can be directly used for recombinant protein expression without virus amplification, effectively avoiding the problem of target gene loss caused by repeated amplification.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an insect expression method and its application using cationic polymers as a medium. Background Technology

[0002] Insect cell expression systems are important platforms for recombinant protein expression, with SF9 cells (Spodopterafrugiperda 9) being one of the most commonly used insect cell lines. SF9 cells exhibit excellent suspension culture characteristics, can grow in serum-free medium, and are widely used in baculovirus expression vector systems (BEVS) for recombinant protein expression. The baculovirus expression vector system achieves efficient expression of foreign proteins by inserting a foreign gene into the baculovirus genome, transfecting insect cells to generate recombinant baculoviruses, and then infecting the insect cells.

[0003] There are existing reports on the use of the cationic polymer polyethyleneimine (PEI) for insect cell transfection. For example, patent document CN106755093B discloses a process for transient transfection of Drosophila cells, which uses the cationic polymer PEI as a transfection reagent to perform transient transfection in Drosophila S2 cells, and optimizes parameters such as PEI dosage, DNA dosage and cell density to achieve low-cost and high-yield expression of target DNA. However, directly applying this technical solution to the baculovirus system in SF9 cells has significant shortcomings: First, SF9 cells differ from Drosophila S2 cells in terms of cell membrane structure and endocytosis pathways, making it difficult to achieve ideal transfection efficiency by directly applying existing parameters; second, this technical solution is designed for transient transfection of recombinant proteins, not for baculovirus preparation, and the requirements for cell state and transfection efficiency differ between the two; more importantly, the yield of P0 generation baculovirus in existing technologies is usually only 1-10 mL, which is low in yield and titer, requiring multiple rounds of virus amplification (P1, P2, etc.) to obtain sufficient virus for production, and repeated amplification can easily lead to the loss of target genes, affecting expression stability and protein yield.

[0004] Therefore, optimizing the transfection process for SF9 cells, increasing the yield and titer of P0 generation baculovirus, and reducing or avoiding viral amplification steps are technical problems that urgently need to be solved in this field. Summary of the Invention

[0005] The purpose of this invention is to provide an insect expression method using cationic polymers as a medium to solve the technical problems of low SF9 cell transfection efficiency, low yield and insufficient titer of P0 generation baculovirus in the prior art. Furthermore, it solves the technical problems of target gene loss, poor expression stability and long molecular screening cycle caused by multiple rounds of virus amplification due to insufficient P0 generation virus yield in the prior art.

[0006] The technical solution of the present invention is as follows: On one hand, it provides an insect expression method using cationic polymers as a medium, comprising the following steps: The density of SF9 cells in suspension culture was adjusted to 1.5 × 10⁶. 7 ~3×10 7 live cells / mL; Linearized polyethyleneimine (PEI) and baculovirus DNA vector were added to SF9 cell suspension for transfection. After transfection, P0 generation baculovirus was obtained through culture. The linearized polyethyleneimine had a molecular weight of 40 kDa and was added at an amount of 2–3 μg / 10⁻¹. 6 The amount of the baculovirus DNA vector added to each live cell was 0.8–1.25 μg / 10 cells. 6 One living cell.

[0007] Preferably, the amount of the baculovirus DNA vector added is 15–25 μg / mL of cell suspension.

[0008] Preferably, the transfection process further includes the step of adding an auxiliary reagent, wherein the auxiliary reagent is selected from at least one of filler DNA, sodium valproate, and dimethyl sulfoxide.

[0009] Preferably, the amount of filler DNA added is 10-20 μg / mL of cell suspension.

[0010] Preferably, the concentration of sodium valproate added is 0.5 to 1.0 mM.

[0011] Preferably, the volume percentage of added dimethyl sulfoxide (DMSO) is 1.0% to 2%.

[0012] Preferably, the baculovirus DNA vector is the AcMNPV baculovirus DNA vector system.

[0013] Preferably, during the process of obtaining P0 generation baculovirus through transfection, the cell density is adjusted to 1.5 × 10⁻⁶ cells / day one day after transfection. 6 ~2.5×10 6 live cells / mL.

[0014] Preferably, the method further includes a step of expressing recombinant proteins using the P0 generation baculovirus, adjusting the cell density to 2.5 × 10⁻⁶ cells one day after transfection. 6 ~3.5×10 6 live cells / mL.

[0015] On the other hand, an application of an insect expression method mediated by a cationic polymer is provided, the insect expression method being used to prepare P0 generation baculovirus or for transient expression of recombinant proteins.

[0016] Compared with the prior art, the advantages of the present invention are: (1) By increasing the SF9 cell transfection density to 1.5 × 10⁻⁶ 7 ~3×10 7 By optimizing the ratio of PEI to DNA vector and using live cells / mL, the transfection efficiency was significantly improved compared to conventional density transfection. Experiments showed that under high-density transfection conditions, the proportion of green fluorescent protein-positive cells and the fluorescence intensity were significantly better than under conventional density transfection.

[0017] (2) Using the method of the present invention, P0 generation baculovirus can be prepared with a yield of 100-200 mL and a viral gene copy number exceeding 1×10⁻⁶. 10 The yield is vg / mL, while the yield of P0 generation virus using traditional processes is typically only 1–10 mL. This breakthrough allows P0 generation virus to be directly used for subsequent recombinant protein expression without the need for multiple rounds of amplification such as P1 and P2.

[0018] (3) Since there is no need to repeatedly amplify the virus, the problem of target gene loss that is common in the repeated passage of baculoviruses is effectively avoided, and the stability of the expression system and the consistency of recombinant protein production are improved.

[0019] (4) The method of the present invention can directly obtain sufficient P0 generation baculovirus and transiently expressed recombinant protein products on a small to medium scale (50-200mL), without the time-consuming virus plaque selection, amplification and concentration steps in the traditional process, which significantly shortens the time of the early molecular screening.

[0020] (5) By optimizing the transfection conditions, the PEI dosage was reduced from 8 μg / 10 μg in the conventional process. 6 The number of viable cells decreased to 2–3 μg / 10 6 One live cell, DNA vector dosage from 2.7 μg / 10 6 The number of viable cells decreased to 0.8–1.25 μg / 102 6 Individual live cells reduce production costs. Attached Figure Description

[0021] The present invention will be further described below with reference to the accompanying drawings and embodiments: Figure 1 This is a comparative graph showing the effect of different cell transfection densities on the transfection efficiency of SF9 cells as described in this invention; Figure 2 This is a graph showing the effect of different DNA vector and PEI ratios on SF9 cell transfection efficiency as described in this invention. Figure 3 This is a graph showing the effect of different SF9 cell transfection densities on transfection efficiency as described in this invention; Figure 4This is a graph showing the effect of different incubation times of the DNA-PEI complex described in this invention on transfection efficiency; Figure 5 This is a graph showing the effect of different filler DNA addition amounts on SF9 cell transfection efficiency as described in this invention. Figure 6 This is a graph showing the effect of different amounts of Polybrene added on the transfection efficiency of SF9 cells as described in this invention. Figure 7 This is a graph showing the effect of different sodium valproate concentrations on SF9 cell transfection efficiency as described in this invention. Figure 8 This is a graph showing the effect of different DMSO addition amounts on SF9 cell transfection efficiency as described in this invention. Figure 9 This is a graph showing the effect of adjusting different cell densities on baculovirus yield one day after transfection, as described in this invention. Figure 10 This is a graph showing the effect of adjusting different cell densities one day after transfection on the expression efficiency of baculovirus-mediated recombinant proteins as described in this invention (based on green fluorescence intensity evaluation). Figure 11 This is a graph showing the effect of adjusting different cell densities one day after transfection on the expression level of recombinant proteins mediated by baculovirus (based on antibody protein expression level evaluation). Detailed Implementation

[0022] The present invention will be further described in detail below with reference to specific embodiments.

[0023] Example 1

[0024] SF9 cell culture and passage SF9 cells were purchased from Thermo Fisher Scientific. Cells were cultured in suspension in IncreaSect920 serum-free medium (Sinopharm Biotechnology (Suzhou) Co., Ltd.) at 27°C on a shaker at 100 rpm (amplitude 50 mm). Cells were passaged every 3–4 days at a seeding density of 0.5 × 10⁶ cells / year. 6 ~1×10 6 viable cells / mL. Cell density and viability were determined using a counter by trypan blue staining.

[0025] Example 2

[0026] Effect of different transfection densities on transfection efficiency To investigate the effect of transfection density on the transfection efficiency of SF9 cells, this example compares the differences between conventional density transfection and high-density transfection.

[0027] SF9 cells in the logarithmic growth phase were harvested and treated with 3 × 10⁻⁶ cells per cell. 6cells / mL (normal density) and 3×10 7 Cells were resuspended at a high density (cells / mL) in fresh IncreaSect920 medium. The reporter gene used for transfection was a green fluorescent protein (EGFP) expression vector driven by the OpIE-2 promoter. For transfection, linearized polyethyleneimine (PEI, molecular weight 40 kDa) and the DNA vector were added to the cell suspension in the amounts shown in Table 1. The PEI and DNA vector were added to the cell culture immediately after mixing, without incubation. Cells were cultured at 27°C and 100 rpm after transfection. Three days post-transfection, the proportion of EGFP-positive cells was assessed by flow cytometry, and the EGFP fluorescence intensity in the cell lysate was measured using a fluorometer to evaluate transfection efficiency.

[0028] Table 1: Transfection conditions for different transfection densities

[0029] Results analysis: such as Figure 1 As shown, the horizontal axis represents groups 1, 2, and 3, and the vertical axis represents fluorescence intensity (RFU). Group 1 is conventional density transfection (3 × 10⁻⁶). 6 Groups 1, 2, and 3 were both high-density transfections (3 × 10⁻⁶ cells / mL), with lower green fluorescence intensity after transfection; Groups 2 and 3 were both high-density transfections (3 × 10⁻⁶ cells / mL). 7 The green fluorescence intensity of group 3 (50 μg / mL PEI) was significantly higher than that of group 1, and the fluorescence intensity of group 3 (50 μg / mL PEI) was higher than that of group 2 (40 μg / mL PEI).

[0030] The above results indicate that increasing the transfection density to 3×10 7 PEI concentrations of 50 μg / mL significantly improved the transfection efficiency of SF9 cells, and the transfection efficiency was correlated with the amount of PEI used. Increasing the PEI concentration (50 μg / mL) further improved the efficiency.

[0031] Example 3

[0032] Optimization of the ratio of DNA vector to PEI dosage In this embodiment, the transfection density is fixed at 3×10⁻⁶. 7 The effect of different DNA vector amounts and PEI ratios on transfection efficiency was investigated under the condition of cells / mL.

[0033] The DNA vector dosage was set at two levels: 12 μg / mL and 24 μg / mL. The PEI dosage was set at four levels: 40, 60, 80, and 100 μg / mL. A total of 8 experimental groups were set up, and the specific ratios are shown in Table 2. The transfection procedure was the same as in Example 2, and the EGFP fluorescence intensity was detected 3 days after transfection.

[0034] Table 2: Ratio of different DNA vectors to PEI dosage

[0035] Results analysis: such as Figure 2 As shown, the horizontal axis represents groups (1–8), and the vertical axis represents fluorescence intensity (RFU). In groups with a DNA vector concentration of 12 μg / mL (groups 1–4), the fluorescence intensity gradually increased with increasing PEI concentration, but the overall level was lower than that of the corresponding group with a DNA vector concentration of 24 μg / mL. In groups with a DNA vector concentration of 24 μg / mL (groups 5–8), the highest fluorescence intensity was observed at PEI concentrations of 60 μg / mL (group 6) and 80 μg / mL (group 7), approximately 28,000 RFU and 27,000 RFU, respectively; the fluorescence intensity was approximately 18,000 RFU at PEI concentration of 40 μg / mL (group 5) and approximately 20,000 RFU at PEI concentration of 100 μg / mL (group 8).

[0036] The above results indicate that, in the high-density transfection system of the present invention, the optimal ratio of DNA vector dosage of 24 μg / mL and PEI dosage of 60–80 μg / mL is the optimal range.

[0037] Example 4

[0038] Optimization of different transfection density gradients This embodiment further investigates the impact of different transfection density gradients on transfection efficiency in order to determine the optimal range of transfection density.

[0039] Set four transfection density gradients: 1.5 × 10 7 2×10 7 2.5×10 7 3×10 7 cells / mL. Transfection conditions were performed using the optimal ratio optimized in Example 3: 24 μg / mL DNA vector and 60 μg / mL PEI. PEI was added to the cell suspension immediately after mixing with the DNA vector. EGFP fluorescence intensity was measured 3 days post-transfection.

[0040] Results analysis: such as Figure 3 As shown, the horizontal axis represents transfection density (cells / mL), and the vertical axis represents fluorescence intensity (RFU). At 1.5 × 10⁻⁶... 7 At a density of cells / mL, the fluorescence intensity is approximately 12000 RFU; at a density of 2×10⁻⁶ cells / mL, the fluorescence intensity is approximately 12000 RFU. 7 At a cell / mL density, the fluorescence intensity is approximately 20,000 RFU; at a density of 2.5 × 10⁻⁶ cells / mL, the fluorescence intensity is approximately 20,000 RFU. 7 At a density of cells / mL, the fluorescence intensity is approximately 26,000 RFU; at a density of 3 × 10⁻⁶ cells / mL, the fluorescence intensity is approximately 26,000 RFU. 7At a cell / mL density, the fluorescence intensity is approximately 27,000 RFU.

[0041] The above results indicate that a transfection density of 2×10⁻⁶ is suitable for this purpose. 7 ~3×10 7 The best transfection efficiency can be obtained within the range of cells / mL. Below this range, the transfection efficiency is insufficient, and above this range, the metabolic pressure on cells increases and the transfection efficiency is not significantly improved.

[0042] Example 5

[0043] Effect of DNA-PEI complex incubation time on transfection efficiency This example investigated the effect of different incubation times after mixing PEI with the DNA vector on transfection efficiency in order to determine the optimal conditions for complex formation.

[0044] Four incubation times were set: 0 minutes (no incubation, added to cells immediately after mixing), 5 minutes, 10 minutes, and 15 minutes. The transfection density was 3 × 10⁶ cells / day. 7 The concentration of cells / mL was 24 μg / mL for DNA vector and 60 μg / mL for PEI. Other procedures were the same as in Example 2. EGFP fluorescence intensity was detected 3 days after transfection.

[0045] Results analysis: such as Figure 4 As shown, the horizontal axis represents incubation time (minutes), and the vertical axis represents relative fluorescence intensity (the fluorescence intensity of the unincubated group is set to 100%). The relative fluorescence intensity of the unincubated group (0 minutes) is 100%; after 5 minutes of incubation, the relative fluorescence intensity decreases to approximately 85%; after 10 minutes of incubation, it decreases to approximately 70%; and after 15 minutes of incubation, it decreases to approximately 60%.

[0046] The unincubated group (transfected immediately after mixing) showed the highest transfection efficiency. As incubation time increased, the transfection efficiency gradually decreased, dropping to approximately 60% of that of the unincubated group after 15 minutes of incubation. Extended incubation time leads to an increase in the size of the PEI-DNA complex particles, which is detrimental to cellular uptake. Therefore, this invention preferably involves immediately transfecting the PEI-DNA vector after mixing, without incubation.

[0047] Example 6

[0048] Effect of auxiliary reagents on transfection efficiency This study investigated the effects of filler DNA, sodium valproate, polybrene, and dimethyl sulfoxide (DMSO) on transfection efficiency in order to screen for auxiliary components that could further improve transfection efficiency.

[0049] Transfection density is 3×10 7The concentration of EGFP cells / mL was 24 μg / mL, and the concentration of DNA vector was 60 μg / mL. EGFP fluorescence intensity was measured 3 days post-transfection, and results are expressed as relative fluorescence intensity (the untreated group was set as 100%).

[0050] The addition schemes for each auxiliary reagent are shown in Table 3.

[0051] Table 3: Auxiliary Reagent Addition Scheme

[0052] Analysis of the effect of adding auxiliary reagents on transfection efficiency: (1) filler DNA like Figure 5 As shown, the horizontal axis represents filler DNA concentration (μg / mL), and the vertical axis represents relative fluorescence intensity (%). The group without filler DNA was 100%; with 10 μg / mL, it was approximately 125%; with 15 μg / mL, it was approximately 140%; and with 20 μg / mL, it was approximately 130%. This indicates that adding filler DNA at a concentration of 10–20 μg / mL can improve transfection efficiency, with 15 μg / mL showing the best effect. As a non-specific DNA, filler DNA can form a complex with PEI, reducing the degradation of target DNA and contributing to the formation of more uniform transfection complex particles.

[0053] (2) Polyamine like Figure 6 As shown, the horizontal axis represents the concentration of polybrene (μg / mL), and the vertical axis represents the relative fluorescence intensity (%). The relative fluorescence intensity of each added group ranged from 95% to 105%, showing no significant difference from the unadded group. This indicates that polybrene did not significantly promote the transfection efficiency of this system.

[0054] (3) Sodium valproate like Figure 7 As shown, the horizontal axis represents sodium valproate concentration (mM), and the vertical axis represents relative fluorescence intensity (%). The group without sodium valproate was 100%; with 0.5 mM, it was approximately 135%; with 1 mM, it was approximately 150%; and with 2 mM, it was approximately 115%. This indicates that sodium valproate concentrations of 0.5–1 mM significantly improve transfection efficiency, with 1 mM showing the best effect. However, excessively high concentrations (2 mM) may cause cytotoxicity.

[0055] (4) Dimethyl sulfoxide (DMSO) like Figure 8As shown, the horizontal axis represents the volume percentage of DMSO (%), and the vertical axis represents the relative fluorescence intensity (%). The group without DMSO was 100%; with 1% DMSO, it was approximately 120%; with 2% DMSO, it was approximately 130%; and with 3% DMSO, it was approximately 108%. This indicates that adding 1.0%–2% DMSO can improve transfection efficiency, with 2% showing the best effect. Excessive concentrations (3%) may cause cytotoxicity.

[0056] Based on the above results, filler DNA, sodium valproate, and DMSO can all be used as preferred auxiliary reagents in this invention to further improve the transfection efficiency of SF9 cells within a specific concentration range.

[0057] Example 7

[0058] Effect of post-transfection cell density adjustment on P0 generation baculovirus yield This embodiment applies optimized transfection conditions to the transfection of AcMNPV baculovirus DNA vector (Bacmid) and examines the effect of cell density adjustment after transfection on the yield of P0 generation baculovirus in order to determine the optimal culture density in the virus preparation stage.

[0059] SF9 cells at 3×10 7 Transfection was performed at a cell / mL density under the following conditions: 24 μg / mL of DNA vector (AcMNPV Bacmid) and 60 μg / mL of PEI (40 kDa), mixed and transfected immediately. Simultaneously, filler DNA 15 μg / mL, sodium valproate 1 mM, and DMSO 2% were added. One day after transfection, the cell density was adjusted to 1.25 × 10⁶ cells / mL. 6 1.5×10 6 2×10 6 2.5×10 6 cells / mL. A control group was also set up using a standard density transfection process (transfection density 3 × 10⁻⁶ cells / mL). 6 (cells / mL, density not adjusted after transfection). After culturing for another 5 days, the baculovirus gene copy number in the culture supernatant was detected by qPCR.

[0060] Results analysis: such as Figure 9 As shown, the horizontal axis represents the adjusted cell density (cells / mL) or process type (conventional control group), and the vertical axis represents the viral gene copy number (vg / mL). The viral gene copy number in the conventional density transfection control group (group 5) was approximately 2 × 10⁻⁶. 8 vg / mL. Adjust cell density to 1.25 × 10⁻⁶. 6 The viral gene copy number per cell / mL (Group 1) was approximately 5 × 10⁻⁶. 8 vg / mL; adjust to 1.5×10 6The viral gene copy number in group 2 (cells / mL) was approximately 8 × 10⁻⁶. 9 vg / mL; adjust to 2×10 6 The viral gene copy number at cells / mL (group 3) was approximately 1.2 × 10⁻⁶. 10 vg / mL; adjust to 2.5×10 6 The viral gene copy number at cells / mL (group 4) was approximately 1.0 × 10⁻⁶. 10 vg / mL, with group 3 reaching approximately 1.2 × 10 10 vg / mL represents the highest level.

[0061] The above results indicate that adjusting cell density early after transfection has a significant impact on the packaging and release of baculovirus. Too low a density (1.25 × 10⁻⁶) results in adverse cell growth. 6 A density of 102.5×102.5 cells / mL is unfavorable for viral transmission and infection between cells; excessively high densities (>2.5×102.5) are also unfavorable. 6 The viral yield may be affected by the rapid proliferation of cells (cells / mL) consuming nutrients in the culture medium. In this invention, the post-transfection cell density is preferably adjusted to 1.5 × 10⁻⁶ cells / mL. 6 ~2.5×10 6 The highest viral yield can be obtained within this range of cells / mL. Simultaneously, the P0 generation baculovirus yield of this invention can reach 100–200 mL, with a viral gene copy number exceeding 1 × 10⁻⁶. 10 The yield is vg / mL, while the yield of P0 generation virus using conventional processes is usually only 1 to 10 mL.

[0062] Example 8

[0063] Effect of post-transfection cell density adjustment on transient expression of recombinant proteins In this embodiment, optimized transfection conditions were applied to the transfection of AcMNPV baculovirus DNA vector, and the effect of cell density adjustment after transfection on the transient expression of baculovirus-mediated recombinant protein was investigated to determine the optimal culture density for the protein expression stage.

[0064] The transfection conditions were the same as in Example 7. One day after transfection, the cell count was adjusted to 2 × 10⁶ cells / day. 6 2.5×10 6 3×10 6 cells / mL. Seven days after transfection, EGFP fluorescence intensity was measured (using the EGFP reporter gene) or antibody protein expression levels were measured by ELISA (using the antibody expression vector).

[0065] Evaluation results based on EGFP fluorescence intensity are as follows: Figure 10 As shown: the horizontal axis represents the adjusted cell density (cells / mL), and the vertical axis represents the fluorescence intensity (RFU). Adjusted to 2×10⁻⁶6 At 2.5 × 10⁻⁶ cells / mL, the fluorescence intensity was approximately 150,000 RFU; after adjustment to 2.5 × 10⁻⁶ cells / mL, the fluorescence intensity was approximately 150,000 RFU. 6 At cells / mL, the fluorescence intensity was approximately 250,000 RFU; adjusted to 3×10⁻⁶ cells / mL. 6 At cells / mL, the fluorescence intensity reaches its highest level at approximately 280,000 RFU.

[0066] Evaluation results based on antibody protein expression levels, such as Figure 11 As shown: the horizontal axis represents the adjusted cell density (cells / mL), and the vertical axis represents the antibody expression level (mg / L). Adjusted to 1.5 × 10⁻⁶ 6 In group 3, with cells / mL, the antibody expression level was approximately 80 mg / L; after adjusting to 2.5 × 10⁻⁶ cells / mL... 6 In group 2, with cells / mL, the antibody expression level was approximately 150 mg / L; after adjusting to 3×10⁻⁶ cells / mL... 6 In group 1, with cells / mL, the antibody expression level was approximately 180 mg / L.

[0067] In summary, the results indicate that post-transfection cell density adjustment significantly affects the recombinant protein expression efficiency. (2.5 × 10⁻⁶) 6 ~3×10 6 Higher protein expression levels can be achieved in the cells / mL range. Compared to the virus preparation stage, the protein expression stage requires a higher cell density, possibly because protein expression after baculovirus infection requires maintaining high cellular metabolic activity.

[0068] Example 9

[0069] Comparative Study of Different Dyeing Processes This embodiment compares the optimized complete process of the present invention with the traditional process, comparing transfection efficiency, P0 generation baculovirus yield, and subsequent protein expression effect.

[0070] This invention's process (high-density transfection process): SF9 cells are transfected at a density of 3 × 10⁻⁶. 7 Transfected at a density of 2 × 10⁶ cells / mL. Transfection conditions were: AcMNPV Bacmid DNA vector 24 μg / mL, PEI (40 kDa) 60 μg / mL, filler DNA 15 μg / mL, sodium valproate 1 mM, DMSO 2%. Transfected immediately after mixing. One day after transfection, the cell density was adjusted to 2 × 10⁶ cells / mL. 6 cells / mL (for virus preparation) or 3 × 10 6 cells / mL (for protein expression), continue culturing.

[0071] Traditional process 1 (low-density transfection process): SF9 cells at 3×106 Transfect at a cell / mL density using liposome transfection reagent according to the instructions. Do not adjust cell density after transfection; continue culturing.

[0072] Traditional Process 2 (Conventional PEI Transfection Process): SF9 cells were transfected at a rate of 3 × 10⁻⁶ cells / year. 6 Transfection was performed at a cell / mL density using PEI (40 kDa) as the transfection reagent at a dosage of 8 μg / 10. 6 One live cell, DNA vector dosage of 2.7 μg / 10 6 For each live cell, other operations were performed according to the method disclosed in CN106755093B, and the comparison results are shown in Table 4.

[0073] Table 4: Comparison Results of Different Transfection Processes

[0074] Traditional processes require P1 amplification before protein expression due to insufficient P0 virus yield and titer.

[0075] The above results demonstrate that the process of this invention is significantly superior to the traditional process in terms of transfection efficiency, P0 generation virus yield and titer, protein expression level, and overall cycle time. In particular, the P0 generation virus yield reaches 150 mL, and the titer reaches 1.2 × 10⁻⁶. 10 The concentration of vg / mL can be directly used for subsequent recombinant protein expression without the need for multiple rounds of viral amplification, effectively avoiding the problem of target gene loss.

[0076] Example 10

[0077] Application of the process of this invention in molecular screening This embodiment verifies the application effect of the process of the present invention in molecular screening, so as to evaluate whether it can meet the needs of rapid screening.

[0078] Using the process of this invention, P0 generation baculoviruses carrying different antibody candidate genes were prepared in a 50 mL culture system. A total of six viral samples with different antibody candidate genes were prepared. One day after transfection, the cell density was adjusted to 3 × 10⁻⁶ cells / day. 6 Cells / mL, culture supernatant was directly collected 7 days after transfection for antibody expression level detection. The entire process from cell transfection to obtaining results took 7 days. A control group using the traditional method (requiring P1 amplification) was also set up, where the entire process from transfection to obtaining results took 24 days.

[0079] Experimental results: All six P0 generation virus samples prepared by the process of this invention successfully expressed antibody proteins, with expression levels ranging from 120 to 200 mg / L. The traditional process control group required a virus amplification step, extending the time to 24 days, and due to potential gene loss during amplification, the expression levels of some samples were lower than expected.

[0080] The above results demonstrate that the process of this invention can directly obtain sufficient transient expression products on a small to medium scale (50–200 mL), and expression level detection can be completed without viral amplification, significantly shortening the time required for preliminary molecular screening. Furthermore, by avoiding the amplification step, the screening results can more accurately reflect the expression performance of the vector itself.

[0081] In summary, this invention establishes a highly efficient insect expression method mediated by cationic polymers by optimizing the transfection density of SF9 cells, the ratio of PEI to DNA vector, the incubation time of the DNA-PEI complex, and combining auxiliary reagents (filler DNA, sodium valproate, DMSO) and adjusting cell density after transfection. This method significantly improves the transfection efficiency of SF9 cells and the yield of P0 generation baculovirus, with P0 generation virus yield reaching 100–200 mL and viral gene copy number exceeding 1 × 10⁻⁶. 10 The concentration of vg / mL can be directly used for recombinant protein expression without the need for multiple rounds of viral amplification, effectively avoiding the problem of target gene loss. At the same time, it shortens the molecular screening cycle and reduces the amount of transfection reagents and DNA vectors used, which has important application value.

[0082] The above embodiments are merely illustrative of the technical concept and features of the present invention, intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly, and should not be construed as limiting the scope of protection of the present invention. It will be apparent to those skilled in the art that the present invention is not limited to the details of the above exemplary embodiments, and that the present invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the present invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects. The scope of the present invention is defined by the appended claims rather than the foregoing description, and thus all changes falling within the meaning and scope of the equivalents of the claims are intended to be included within the present invention.

Claims

1. A method for insect expression mediated by cationic polymers, comprising the following steps: The density of SF9 cells in suspension culture was adjusted to 1.5 × 10⁶. 7 ~3×10 7 live cells / mL; SF9 cell suspension was transfected with linearized polyethyleneimine and baculovirus DNA vector. After transfection, P0 generation baculovirus was obtained through culture. The linearized polyethyleneimine had a molecular weight of 40 kDa and was added at an amount of 2–3 μg / 10⁻¹². 6 The amount of the baculovirus DNA vector added to each live cell was 0.8–1.25 μg / 10 cells. 6 One living cell.

2. The insect expression method using cationic polymers as a medium according to claim 1, characterized in that, The amount of the baculovirus DNA vector added is 15–25 μg / mL of cell suspension.

3. The insect expression method using cationic polymers as a medium according to claim 1, characterized in that, It also includes the step of adding an auxiliary reagent during transfection, wherein the auxiliary reagent is selected from at least one of filler DNA, sodium valproate, and dimethyl sulfoxide.

4. The insect expression method using cationic polymers as a medium according to claim 3, characterized in that, The filler DNA was added at a rate of 10–20 μg / mL of cell suspension.

5. The insect expression method using cationic polymers as a medium according to claim 3, characterized in that, The concentration of sodium valproate added is 0.5–1.0 mM.

6. The insect expression method using cationic polymers as a medium according to claim 3, characterized in that, The volume percentage of the added dimethyl sulfoxide is 1.0% to 2%.

7. The insect expression method using cationic polymers as a medium according to claim 1, characterized in that, The baculovirus DNA vector is the AcMNPV baculovirus DNA vector.

8. The insect expression method using cationic polymers as a medium according to claim 1, characterized in that, During the process of obtaining P0 generation baculovirus through transfection, the cell density was adjusted to 1.5 × 10⁶ cells / day one day after transfection. 6 ~2.5×10 6 live cells / mL.

9. The insect expression method using cationic polymers as a medium according to claim 1, characterized in that, It also includes a step of expressing recombinant proteins using the P0 generation baculovirus, adjusting the cell density to 2.5 × 10⁻⁶ cells one day after transfection. 6 ~3.5×10 6 live cells / mL.

10. An application of an insect expression method mediated by a cationic polymer, characterized in that, The insect expression method is used to prepare generation P0 baculovirus or for transient expression of recombinant proteins.

Images