A method for producing BaEV enveloped lentivirus

Abstract

The present invention belongs to the field of microbial fermentation technology, and specifically relates to a method for producing BaEV enveloped lentivirus. It includes: inoculating 293T cells into a culture dish for culture; mixing plasmids pGFP, pLP1, pLP2 and plp-BaEV with DMEM complete medium, incubating at room temperature to obtain a plasmid culture medium mixture; mixing PEI solution with DMEM complete medium to obtain a PEI culture medium mixture; adding the PEI culture medium mixture to the plasmid culture medium mixture to obtain a plasmid PEI mixture; adding the plasmid PEI mixture to a culture dish for culture, and transfecting 293T cells; after transfection for 5 to 6 hours, replacing the culture medium with an equal amount of DMEM complete medium for continued culture; harvesting 293T cell culture fluid 36 to 72 hours after transfection, filtering the 293T cell culture fluid, and harvesting lentivirus. The lentivirus yield is effectively improved.

Description

Technical Field

[0001] The present invention belongs to the field of cell and gene therapy, and in particular relates to a method for producing BaEV enveloped lentivirus. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Lentivirus is a type of retrovirus with a particle size of 80-120 nm. It consists of a single-stranded RNA sequence that is transcribed into DNA and integrated into the host genome, resulting in persistent infection. Lentiviral vectors (LV) are commonly used in cell and gene therapy to transfer and integrate target genes into recipient cells for therapeutic effects. Most lentiviral vectors are derived from HIV-1 and are modified to retain the ability to integrate into the cellular genome. LV is usually pseudotyped, that is, the envelope protein of the vector is exchanged with the envelope protein of another virus, and the virus particles are actually coated with proteins from other viruses, which provide different properties to the vector, affecting its tropism and the intended target cells. Many viral envelope proteins can be pseudotyped onto LV particles. However, the most commonly pseudotyped envelope protein is the VSV-G glycoprotein from the Indiana vesicular stomatitis virus. The receptor of the VSV-G enveloped lentivirus (VSVG-LV) is LDL-R, but this virus has poor efficiency in infecting NK cells. Baboon enveloped pseudotyped lentiviral vector (BaEV-LV) can significantly improve the transduction efficiency of NK cells.

[0004] Although BaEV-LV is suitable for infecting NK cells, the production of BaEV-LV is very low. It is reported that the titer of BaEV-Rless-LV produced under the same conditions is about 100 times lower than that of VSVG-LV. The specific source is https: / / doi.org / 10.1016 / S1525-0016(16)33339-1. During the packaging process of BaEV-LV, 293T cells will form syncytia, leading to cell apoptosis and reducing the virus titer. The researchers optimized the method of fixing 293T cells to culture flasks using polylysine, which can increase the virus titer by 10 times, but the titer only reached 1×10 4 TU / ml, still failed to reach the titer level of VSVG-LV (1×10 6 TU / ml). The prior art still does not provide a method for producing BaEV enveloped lentivirus. Summary of the invention

[0005] In order to solve the technical problems existing in the prior art, the present invention provides a method for producing BaEV enveloped lentivirus. The present invention optimizes the production method in terms of cell density before transfection, total plasmid dosage, envelope plasmid dosage, etc., and increases the packaging virus titer to 6×10 6 TU / ml, which is 600 times higher than the currently reported titer. The virus yield can reach 99.92% and the titer can reach 1×10 8 TU / ml, reaching the titer level of VSVG-LV, significantly reducing the cost of viral vectors for cell therapy.

[0006] Specifically, the technical solution of the present invention is as follows:

[0007] A first aspect of the present invention provides a method for producing a BaEV enveloped lentivirus, the method comprising:

[0008] The cells used in the examples are 293T cells, and the virus packaging of the present invention is carried out in a 10 cm cell culture dish. The virus titer detection in the examples is carried out using 293 cells. The 293 cells are plated one day before infection, using a 24-well plate, and the number of cells per well is 1.2×10 5 On the second day, the virus solution was diluted 10 times, 100 times, and 1000 times in three gradients. The culture medium in the wells was aspirated, and 0.5 ml of the diluted virus sample was added to each well. After culturing in the incubator for 72 h, the virus titer was detected using a flow cytometer.

[0009] (1) inoculating 293T cells into a culture dish for culture, wherein the culture medium in the culture dish is DMEM complete medium;

[0010] (2) Preparing a plasmid PEI mixture: mixing four plasmids with DMEM complete medium, incubating at room temperature, and obtaining a plasmid medium mixture; wherein the four plasmids are pGFP, pLP1, pLP2, and plp-BaEV; mixing a polyethyleneimine (PEI) solution with a DMEM complete medium, incubating at room temperature, and obtaining a PEI medium mixture; adding the PEI medium mixture to the plasmid medium mixture, mixing, and incubating at room temperature, and obtaining a plasmid PEI mixture;

[0011] (3) adding the plasmid PEI mixture to the culture dish in step (1), mixing well and placing in a carbon dioxide incubator for culture to transfect the 293T cells in step (1);

[0012] (4) 5-6 hours after transfection, the culture medium in the culture dish was replaced with an equal amount of DMEM complete medium and the culture dish was placed in a carbon dioxide incubator for further culture;

[0013] (5) 36 to 72 hours after transfection, the 293T cell culture fluid is harvested, the 293T cell culture fluid is filtered, and the lentivirus is harvested.

[0014] In a specific embodiment, in step (1), 0.8 × 10 5 ~4.0×10 5 / cm 2 293T cells were seeded into culture dishes at a seeding density of

[0015] Preferably, the seeding density is: 0.8 × 10 5 / cm 2 , 1.2 × 10 5 / cm 2 , 1.5 × 10 5 / cm 2 , 2.0 × 10 5 / cm 2 or 4.0 × 10 5 / cm 2 ; More preferably, 1.2 × 10 5 / cm 2 ; Inoculated cell suspension volume = inoculation density × culture dish area ÷ live cell density; Use DMEM complete medium to make up to the minimum volume required for culture dish culture.

[0016] In a specific embodiment, in step (1), the culture dish is placed in a 37° C., 5% carbon dioxide incubator for static culture.

[0017] In a specific embodiment, in step (2), the mass ratio of the four plasmids pGFP, pLP1, pLP2 and plp-BaEV is 3:2:2:0.2~1; preferably, the mass ratio of the four plasmids pGFP, pLP1, pLP2 and plp-BaEV is 3:2:2:0.3.

[0018] In a specific embodiment, the total dosage of the four plasmids in step (2) is 0.25-0.35 μg / cm 2 Preferably, the total dosage of the four plasmids is 0.35 μg / cm 2 .

[0019] In a specific embodiment, in step (2), the PEI culture medium mixture is added to the plasmid culture medium mixture, mixed evenly, and incubated at room temperature for 20 to 60 minutes to obtain a plasmid PEI mixture.

[0020] In a specific embodiment, in step (2), the PEI solution is mixed with DMEM complete medium, wherein the amount of PEI solution added is three times the total amount of plasmid divided by the concentration of the PEI solution. Preferably, the concentration of the PEI solution is 0.5-1.5 mg / ml, more preferably 1 mg / ml.

[0021] In a second aspect of the present invention, there is provided a method for producing BaEV enveloped lentivirus according to the first aspect, and its application in the preparation of lentivirus.

[0022] The present invention has the following beneficial effects:

[0023] Based on the existing 293T cell packaging lentivirus and ultracentrifugation purification lentivirus process, the present invention optimizes the cell density before transfection, the total plasmid dosage, the envelope plasmid dosage and other aspects for the purpose of increasing the virus yield, and forms a transfection condition suitable for BaEV-LV. The optimized conditions are used for virus packaging and purification, and the crude virus titer can reach 4×10 6 TU / ml, the virus titer can reach 1×10 8 TU / ml and above. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0025] Figure 1 This is a graph of virus titers at different cell transfection densities in Example 1 of the present invention;

[0026] Figure 2 This is a graph showing the virus titer at different total plasmid dosages in Example 2 of the present invention;

[0027] Figure 3 This is a graph of virus titers at different harvest times in Example 3 of the present invention;

[0028] Figure 4 This is a graph of virus titers with different envelope plasmid ratios in Example 4 of the present invention;

[0029] Figure 5 This is a graph showing virus resolubilization yields under different ultracentrifugation conditions in Example 5 of the present invention;

[0030] Figure 6 This is a titer chart of lentiviral packaging and ultracentrifugation purification in Example 5 of the present invention. DETAILED DESCRIPTION

[0031] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0032] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof. If the experimental methods of specific conditions are not indicated in the following specific embodiments, they are generally in accordance with conventional methods and conditions in the art, and such techniques and conditions are fully explained in the literature.

[0033] The present invention is further explained by the following examples, but they are not intended to limit the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0034] The cells used in the examples of the present invention are 293T cells, and virus packaging is performed in a 10 cm cell culture dish. The virus titer detection in the examples is performed using 293 cells. The 293 cells are plated one day before infection, using a 24-well plate, and the number of cells per well is 1.2×10 5 On the second day, the virus solution was diluted 10 times, 100 times, and 1000 times in three gradients, the culture medium in the wells was aspirated, and 0.5 ml of the diluted virus sample was added to each well. After culturing in the incubator for 72 h, the virus titer was detected by flow cytometry.

[0035] Example 1

[0036] 1. Cell preparation and plating:

[0037] 1. Observe cells: Observe the cultured 293T cells under an inverted biological microscope. Subculture when the cell coverage rate is above 80%.

[0038] 2. Equipment and reagent preparation: Open the biosafety cabinet in advance, perform UV disinfection and self-cleaning; open the water bath in advance, set the temperature to 37°C, and preheat DMEM complete medium. DPBS and TrypLE™ Select do not need to be preheated when stored at room temperature, but need to be preheated in advance if stored at 2~8°C.

[0039] 3. Material transfer: 293T cells, preheated DMEM complete medium, TrypLE™ Select, DPBS, cell culture dishes, 50 ml centrifuge tubes, pipettes, etc. are wiped with 75% alcohol disinfectant and placed in the biosafety cabinet.

[0040] 4. Wash and digest cells: Open the lid of the 293T cell culture container, discard the supernatant, add DPBS and shake gently to wash once. The amount of DPBS added should at least cover the bottom of the container. Aspirate and discard the DPBS, add an appropriate amount of TrypLE™ Select to cover the bottom of the container. Gently shake the container to allow TrypLE™ Select to cover the entire bottom of the culture container, and digest at room temperature until most of the cells fall off the bottom. Add at least 2 times the volume of TrypLE™ Select in DMEM complete medium to terminate the digestion.

[0041] 5. Pipette and take samples for counting: Use a pipette to suck up the cell suspension and pipette the bottom of the culture container to make the cells completely fall off the bottom of the culture container. Pipette the cell suspension to make a single cell suspension, transfer it to a 50 ml centrifuge tube, mix well, and take a sample of 0.5 ml for cell counting and viability detection.

[0042] 6. Inoculate cells: 0.8 × 10 5 / cm 2 , 1.2 × 10 5 / cm 2 , 1.5 × 10 5 / cm 2 , 2.0 ×10 5 / cm 2 , 4.0 ×10 5 / cm 2 Inoculation density: Inoculate the above cells into the culture dish; the volume of inoculated cell suspension = inoculation density × culture dish area ÷ live cell density; use DMEM complete medium to make up to the minimum volume required for culture dish culture.

[0043] 7. Label and culture: Label the cell name, cell generation, inoculation cell density, culture time and other information on the culture container, and place it in a 37°C, 5% carbon dioxide incubator for static culture.

[0044] 2. Transfection

[0045] 1. Prepare the plasmid culture medium mixture: according to the total amount of plasmid 0.35 μg / cm 2 The ratio of the four plasmids was pGFP:pLP1:pLP2:plp-BaEV = 3:2:2:1. The plasmids were mixed with 1 ml of DMEM complete medium and incubated at room temperature for 5 min.

[0046] 2. Prepare PEI medium mixture: Mix PEI solution with 1 ml DMEM complete medium and incubate at room temperature for 5 minutes. The amount of PEI solution added = total amount of plasmid × 3 ÷ concentration of PEI solution.

[0047] 3. Prepare plasmid PEI mixture: Add the above PEI medium mixture to the above plasmid medium mixture, mix well, and incubate at room temperature for 40 min.

[0048] 4. Take out the 293T cells inoculated the day before, carefully add the incubated plasmid PEI mixture into the culture dish, shake gently to mix, and place the culture dish in a carbon dioxide incubator for culture after marking.

[0049] 5. 5.5 h after transfection, discard the culture medium in the bottle, replace it with an equal amount of fresh DMEM complete medium, and continue culturing in a carbon dioxide incubator.

[0050] 6. The lentivirus can be harvested 72 hours after transfection.

[0051] The cell culture medium was harvested 72 h after transfection and filtered using a 0.45 μm syringe filter.

[0052] 3. Titer detection

[0053] 1. Plate 293 cells one day before harvesting in a 24-well plate at 1.2 × 10 5 / hole;

[0054] 2. Dilute the filtered cell culture fluid sample with DMEM complete medium in three gradients of 10 times, 100 times, and 1000 times;

[0055] 3. Discard the culture medium in the 24-well plate plated the day before, add 500 μl of diluted cell culture sample, and culture in the incubator for 72 h;

[0056] 4. After 72 h of culture, the cells were digested from the 24-well plate and the virus titer was detected using flow cytometry.

[0057] Figure 1 This is a graph of virus titers at different cell transfection densities in Example 1 of the present invention. The cell density was calculated to be 1.2×10 5 / cm 2 The virus titer reached the highest level at 2.07×10 6 TU / ml.

[0058] Example 2

[0059] 1. Cell preparation and plating:

[0060] This step is the same as in Example 1.

[0061] 2. Transfection

[0062] 1. Prepare the plasmid culture medium mixture: according to the total amount of plasmid 0.35 μg / cm 2 , 0.33 μg / cm 2 , 0.30 μg / cm 2 , 0.25 μg / cm 2 The ratio of the four plasmids was pGFP:pLP1:pLP2:plp-BaEV=3:2:2:1. The plasmids were mixed with DMEM complete medium and incubated at room temperature for more than 5 minutes.

[0063] 2. Prepare PEI culture medium mixture: Mix PEI solution and DMEM complete culture medium and incubate at room temperature for 5 min. The amount of PEI solution added = total amount of plasmid × 3 ÷ concentration of PEI solution.

[0064] 3. Prepare the plasmid PEI mixture: Add the PEI medium mixture to the plasmid medium mixture, mix well, and incubate at room temperature for 20 min.

[0065] 4. Take out the 293T cells inoculated the day before, carefully add the incubated plasmid PEI mixture into the culture flask, shake gently to mix, mark and place in a CO2 incubator for culture.

[0066] 5. 5.5 h after transfection, discard the culture medium in the bottle, replace it with an equal amount of fresh DMEM complete medium, and continue culturing in a carbon dioxide incubator.

[0067] 6. The lentivirus can be harvested 72 hours after transfection.

[0068] The cell culture medium was harvested 72 h after transfection and filtered using a 0.45 μm syringe filter.

[0069] Titer detection

[0070] This step is the same as in Example 1.

[0071] Figure 2 This is a graph of virus titers at different total plasmid dosages in Example 2 of the present invention. The total plasmid dosage was calculated to be 0.35

[0072] μg / cm 2 The virus titer was the highest at 3.13×10 6 TU / ml.

[0073] Example 3

[0074] 1. Cell preparation and plating:

[0075] This step is the same as in Example 1.

[0076] 2. Transfection

[0077] 1. Prepare the plasmid culture medium mixture: according to the total amount of plasmid 0.35 μg / cm 2 The ratio of the four plasmids was pGFP:pLP1:pLP2:plp-BaEV=3:2:2:1. The plasmids were mixed with DMEM complete medium and incubated at room temperature for more than 5 min.

[0078] 2. Prepare PEI culture medium mixture: Mix PEI solution and DMEM complete culture medium and incubate at room temperature for 5 min. The amount of PEI solution added = total amount of plasmid × 3 ÷ concentration of PEI solution.

[0079] 3. Prepare the plasmid PEI mixture: Add the PEI medium mixture to the plasmid medium mixture, mix well, and incubate at room temperature for 40 min.

[0080] 4. Take out the 293T cells inoculated the day before, carefully add the incubated plasmid PEI mixture into the culture flask, shake gently to mix, mark and place in a CO2 incubator for culture.

[0081] 5. 5.5 h after transfection, discard the culture medium in the bottle, replace it with an equal amount of fresh DMEM complete medium, and continue culturing in a carbon dioxide incubator.

[0082] 6. Harvest lentivirus 24 h, 36 h, 48 h, 60 h, and 72 h after transfection.

[0083] The cell culture medium was harvested and filtered using a 0.45 μm syringe filter.

[0084] 3. Titer detection

[0085] This step is the same as in Example 1.

[0086] Figure 3 The figure is a graph of virus titers at different harvest times in Example 3 of the present invention. It was detected and calculated that the virus titer was the highest when the harvest time was 48 h, reaching 3.01×10 6 TU / ml.

[0087] Example 4

[0088] 1. Cell preparation and plating:

[0089] This step is the same as in Example 1.

[0090] 2. Transfection

[0091] 1. Prepare the plasmid culture medium mixture: according to the total amount of plasmid 0.33 μg / cm 2 The ratios of the four plasmids were pGFP:pLP1:pLP2:plp-BaEV=3:2:2:1, 3:2:2:0.75, 3:2:2:0.5, 3:2:2:0.3, and 3:2:2:0.2. The plasmids were mixed with DMEM complete medium and incubated at room temperature for 5 min.

[0092] 2. Prepare PEI culture medium mixture: Mix PEI solution and DMEM complete culture medium and incubate at room temperature for 5 min. The amount of PEI solution added = total amount of plasmid × 3 ÷ concentration of PEI solution.

[0093] 3. Prepare the plasmid PEI mixture: Add the PEI medium mixture to the plasmid medium mixture, mix well, and incubate at room temperature for 60 min.

[0094] 4. Take out the 293T cells inoculated the day before, carefully add the incubated plasmid PEI mixture into the culture dish, shake gently to mix, mark and place in a carbon dioxide incubator for culture.

[0095] 5. 5.5 h after transfection, discard the culture medium in the bottle, replace it with an equal amount of fresh DMEM complete medium, and continue culturing in a carbon dioxide incubator.

[0096] 6. Harvest lentivirus 48 h after transfection.

[0097] The cell culture medium was harvested 48 h after transfection and filtered using a 0.45 μm syringe filter.

[0098] 3. Titer detection

[0099] This step is the same as in Example 1.

[0100] Figure 4 The figure is a graph of virus titers of different envelope plasmid ratios in Example 4 of the present invention. The virus titer of the filtered virus sample was the highest when the plasmid ratio was 3:2:2:0.3, reaching 4.43×10 6 TU / ml.

[0101] Example 5

[0102] 1. Cell preparation and plating:

[0103] This step is the same as in Example 1.

[0104] 2. Transfection

[0105] 1. Prepare the plasmid culture medium mixture: according to the total amount of plasmid 0.33 μg / cm 2The ratio of the four plasmids was pGFP:pLP1:pLP2:plp-BaEV=3:2:2:0.5. The plasmids were mixed with DMEM and incubated at room temperature for more than 5 minutes.

[0106] 2. Prepare PEI culture medium mixture: Mix PEI solution and DMEM complete culture medium and incubate at room temperature for 5 min. The amount of PEI solution added = total amount of plasmid × 3 ÷ concentration of PEI solution.

[0107] 3. Prepare the plasmid PEI mixture: Add the PEI medium mixture to the plasmid medium mixture, mix well, and incubate at room temperature for 20-60 minutes.

[0108] 4. Take out the 293T cells inoculated the day before, carefully add the incubated plasmid PEI mixture into the culture flask, shake gently to mix, mark and place in a CO2 incubator for culture.

[0109] 5. 5.5 h after transfection, discard the culture medium in the bottle, replace it with an equal amount of fresh DMEM complete medium, and continue culturing in a carbon dioxide incubator.

[0110] 6. Harvest lentivirus 48 h after transfection.

[0111] 3. Ultracentrifugation

[0112] 1. Wash the ultracentrifuge tubes in advance and sterilize them by high pressure.

[0113] 2. 48 h after transfection, collect the lentiviral supernatant into a 50 ml centrifuge tube and centrifuge at 2000 g for 10 min.

[0114] 3. Transfer the supernatant from the centrifugation to a new 50 ml centrifuge tube, filter using a 0.45 μm PP filter, and collect the clarified liquid in a 50 ml centrifuge tube.

[0115] 4. Transfer the clarified liquid to a sterilized ultracentrifuge tube, weigh the ultracentrifuge tube with a 100% balance, balance it with the clarified liquid and place it in an ultracentrifuge, set the parameters: 30,000 rpm, 4°C, and centrifuge for 2.5 h.

[0116] 5. Prepare lentivirus storage solution according to the volume ratio of basal culture medium: serum-free cell freezing solution = 1:1.

[0117] 6. After ultracentrifugation, use tweezers to remove the ultracentrifuge tube, discard the supernatant, and then invert the ultracentrifuge tube on sterilized dust-free paper to absorb the remaining centrifuge supernatant. Then add 0.75 ml of lentivirus freezing solution to each tube and blow the lentivirus precipitate.

[0118] 7. Place the ultracentrifuge tube in a 50 ml centrifuge tube, tighten the lid, and place at 4°C for re-dissolution to obtain the cell culture fluid sample.

[0119] 4. Titer detection

[0120] This step is the same as in Example 1.

[0121] Figure 5 The figure is a graph of virus recovery rate under different ultracentrifugation conditions in Example 5 of the present invention, wherein the residual virus is the least under the centrifugation condition of 30000 rpm for 2.5 h, and the recovery rate reaches 99.92%. Figure 6 This is a titer diagram of lentiviral packaging and ultracentrifugation purification in Example 5 of the present invention. Figure 6 It can be seen that the titer of the harvested cell culture supernatant, i.e., the crude virus, reached 1.29 × 10 6 TU / ml. After filtration and clarification with a 0.45 μm filter membrane, the virus was lost and the titer dropped to 8.81×10 5 TU / ml. The virus was purified by ultracentrifugation method, and the final virus titer was 1.24×10 8 TU / ml.

[0122] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for producing BaEV enveloped lentivirus, characterized in that: The method comprises: (1) inoculating 293T cells into a culture dish for culture, wherein the culture medium in the culture dish is DMEM complete medium; (2) Preparing a plasmid PEI mixture: mixing four plasmids with DMEM complete medium, incubating at room temperature, and obtaining a plasmid medium mixture; wherein the four plasmids are pGFP, pLP1, pLP2, and plp-BaEV; mixing PEI solution with DMEM complete medium, incubating at room temperature, and obtaining a PEI medium mixture; adding the PEI medium mixture to the plasmid medium mixture, mixing, and incubating at room temperature, and obtaining a plasmid PEI mixture; (3) adding the plasmid PEI mixture to the culture dish in step (1), mixing well and placing in a carbon dioxide incubator for culture to transfect the 293T cells; (4) 5-6 h after transfection, replace the culture medium in the culture dish with an equal amount of DMEM complete medium and continue culturing in a carbon dioxide incubator; (5) 36 to 72 hours after transfection, the 293T cell culture fluid is harvested, the 293T cell culture fluid is filtered, and the lentivirus is harvested; The mass ratio of the four plasmids pGFP, pLP1, pLP2 and plp-BaEV in step (2) is: 3:2:2:0.3~1; The total dosage of the four plasmids in step (2) is 0.3~0.35 μg / cm 2 .

2. The method according to claim 1, characterized in that In step (1), 8 × 10 4 ~4.0×10 5 / cm 2 293T cells were seeded into culture dishes at a seeding density of 3. The method according to claim 2, characterized in that The seeding density in step (1) is: 8 × 10 4 / cm 2 , 1.2 × 10 5 / cm 2 , 1.5 × 10 5 / cm 2 , 2.0 × 10 5 / cm 2 or 4.0 × 10 5 / cm 2 .

4. The method according to claim 1, characterized in that In step (1), the culture dish is placed in a 37° C., 5% carbon dioxide incubator for static culture.

5. The method according to claim 1, characterized in that In step (2), the PEI culture medium mixture is added to the plasmid culture medium mixture and mixed evenly, and incubated at room temperature for 20 to 60 minutes to obtain a plasmid PEI mixture.

6. The method according to claim 1, characterized in that In step (2), the PEI solution is mixed with DMEM complete medium, wherein the amount of PEI solution added is three times the total amount of plasmid divided by the concentration of the PEI solution.

7. The method according to claim 1, characterized in that Harvest the 293T cell culture medium 44 to 52 hours after transfection in step (5).

8. Use of the method for producing BaEV enveloped lentivirus according to any one of claims 1 to 7 in the preparation of lentivirus.

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