A method for increasing the titer of a baev envelope virus

Abstract

The application relates to the technical field of virus utilization, and specifically provides a method for improving BaEV envelope virus titer, step one: shRNA vector construction; step two: construction of a 293T stable cell line with ASCT receptor knockdown; step three: the 293T stable cell line with ASCT receptor knockdown is used for packaging BaEV GFP lentivirus. The lentivirus titer is improved by constructing the 293T cell line with ASCT2 receptor and ASCT1ASCT2 receptor knockdown, and the compounds used in the production process and the harvesting time are screened.

Description

Technical Field

[0001] This invention relates to the field of virus utilization technology, and in particular to a method for increasing the titer of BaEV enveloped virus. Background Technology

[0002] Currently, widely used lentiviral vectors all employ pseudotyped packaging, and their envelope proteins are mostly selected from the vesicular stomatitis virus glycoprotein VSV-G. This glycoprotein can achieve highly efficient infection of various cell types by binding to the low-density lipoprotein receptor LDLR on the cell surface. When using a four-plasmid system to package the pseudotyped VSV-G lentivirus VSV-G-LV, the viral infection titer is ≥1×10⁻⁶. 6 IU / mL can significantly reduce the preparation cost of cell therapy. VSV-G-LV has been successfully applied in the field of CAR-T cell therapy.

[0003] However, in the field of CAR-NK cell therapy, the transfection efficiency of VSV-G-LV to NK cells is usually below 20%, which is insufficient to meet the needs of CAR-NK cell therapy. Aurelien et al. replaced the envelope protein with BaEV, which significantly improved the transfection efficiency of NK cells to over 80%; Rafijul et al. also demonstrated that the expression levels of ASCT1 and ASCT2 on activated NK cells are much higher than LDLR, which is why BaEV-LV can efficiently infect NK cells. Although BaEV-LV can efficiently transfect a variety of cell types, the titer of BaEV-LV obtained by traditional packaging methods is much lower than that of VSV-G-LV. Due to its high production cost, BaEV-LV is difficult to use for the commercial production of cell therapy drugs.

[0004] Publicly available research has found that adding poly-L-lysine to immobilize 293T cells can reduce syncytial formation and double the viral titer, but the viral titer is still less than 1×10⁻⁶. 6 CN119020419A discloses a gene knockout method to eliminate the ASCT1 and ASCT2 genes in 293T cells, thereby reducing the expression of ASCT1 and ASCT2 receptors in 293T cells, thus reducing syncytial formation and increasing viral titer. Such methods mostly involve completely knocking out the ASCT1 and ASCT2 genes using CRISPR / Cas9 technology. ASCT1 / 2 are important glutamine transport proteins in cells. Knocking out ASCT1 and ASCT2 will seriously affect glutamine metabolism, inhibit cell proliferation and growth, and thus affect cell stability and viral yield.

[0005] Therefore, there is a need to develop a method to increase the titer of BaEV enveloped virus. Summary of the Invention

[0006] To address the aforementioned problems, the present invention aims to provide a method for increasing the titer of BaEV enveloped virus.

[0007] The method for increasing the BaEV envelope virus titer is as follows: Step 1: shRNA vector construction: The shRNA vector for knocking down the ASCT2 receptor is SEQ ID No. 2 driven by the H1 promoter, and the shRNA vectors for knocking down the ASCT1 and ASCT2 receptors are constructed in tandem by SEQ ID No. 1 driven by the U6 promoter and SEQ ID No. 2 driven by the H1 promoter. Step 2: Construct a stable 293T cell line with ASCT receptor knockdown, wherein the stable 293T cell line with ASCT receptor knockdown is any one of the following: (a) ASCT2 receptor knockdown stable 293T cell line: The shRNA vector of the knockdown ASCT2 receptor was packaged into lentivirus 1, and 293T cells were infected with MOI=10 and screened with puromycin to obtain the ASCT2 receptor knockdown stable 293T cell line. The method for packaging lentivirus 1 is as follows: Lentiviral packaging and transfection are performed using a 4-plasmid system (pLP1:pLP2:VSV-G:ASCT2 receptor knockdown shRNA vector) with a plasmid mass ratio of 0.25:0.25:0.125:0.375. Before transfection, the 293T cell density is (1.0~2.0)×10⁻⁶ cells / year. 5 / cm 2 Inoculation was performed, followed by transfection 24 hours later. The total plasmid concentration used during transfection was 0.25–0.35 μg / cm³. 2 The mass ratio of PEI to plasmid was 2:1. After 6 hours of transfection, the medium was replaced with DMEM complete medium. After 72 hours of transfection, virus harvesting was performed to obtain virus harvest solution 1. The virus harvest solution 1 was clarified through a 0.45 μm syringe filter and then centrifuged at 25,000 rpm for 2 hours. After centrifugation, the supernatant was discarded and the precipitate was re-dissolved in DMEM complete medium at 4°C for 18-24 hours to obtain the lentivirus 1. The cells infected with 293T cells and selected with puromycin were: 1.2 × 10⁻⁶. 5 / cm 2 293T cells were seeded and infected with lentivirus 1 at MOI=10 24 h later. After infection, the cells were replaced with DMEM complete medium 6 h later. After infection, the cells were screened and passaged at a concentration of 5 μg / ml puromycin 72 h later. Then, a second screening and amplification was performed using 10 μg / ml puromycin . (b) Stable 293T cell lines with ASCT1 and ASCT2 receptor knockdown: The shRNA vectors with ASCT1 and ASCT2 receptor knockdown were packaged into lentivirus 2, infected with 293T cells at MOI=10, and screened with puromycin to obtain the stable 293T cell lines with ASCT1 and ASCT2 receptor knockdown. The method for packaging lentivirus 2 is as follows: Lentiviral packaging and transfection are performed using a 4-plasmid system consisting of pLP1:pLP2:VSV-G:shRNA vector knocking down ASCT1 and ASCT2 receptors in a plasmid mass ratio of 0.25:0.25:0.125:0.375. Before transfection, the 293T cell density is (1.0~2.0)×10⁻⁶ cells / year. 5 / cm 2 Inoculation was performed, followed by transfection 24 hours later. The total plasmid concentration used during transfection was 0.25–0.35 μg / cm³. 2 The mass ratio of PEI to plasmid was 2:1. After 6 hours of transfection, the medium was replaced with DMEM complete medium. After 72 hours of transfection, virus harvesting was performed to obtain virus harvesting solution 2. The virus harvesting solution 2 was clarified through a 0.45 μm syringe filter and then centrifuged at 25,000 rpm for 2 hours. After centrifugation, the supernatant was discarded and the precipitate was re-dissolved in DMEM complete medium at 4°C for 18-24 hours to obtain the lentivirus 2. The cells infected with 293T cells and selected with puromycin were: 1.2 × 10⁻⁶. 5 / cm 2 293T cells were seeded and infected with lentivirus 2 at MOI=10 24 h later. After infection, the cells were replaced with DMEM complete medium 6 h later. After infection, the cells were screened and passaged at a concentration of 5 μg / ml puromycin 72 h later. Then, a second screening and amplification was performed using 10 μg / ml puromycin . Step 3: Packaging BaEV GFP lentivirus into the ASCT receptor knocked-down 293T stable cell line: According to (1.0~2.0)×10 5 / cm 2 The 293T stable cell line corresponding to ASCT receptor knockout was seeded at a specific density, and transfected 24 h later. The total plasmid concentration used during transfection was 0.25–0.35 μg / cm³. 2 The mass ratio of pLP1:pLP2:BaEV:GFP was 0.25:0.25:0.125:0.375, and the mass ratio of PEI to plasmid was 2:1. Heparin sodium was added to the culture dish when changing the medium 6 h after transfection, so that the final concentration of heparin sodium in DMEM complete medium was 10 U / ml. The BaEV enveloped virus was obtained 48 h to 72 h after transfection.

[0008] Furthermore, in step two (a), the density of 293T cells before transfection is 1.5 × 10⁻⁶. 5 / cm 2 Get vaccinated.

[0009] Furthermore, in step two (a), the total plasmid amount used during transfection is 0.3 μg / cm³. 2 .

[0010] Furthermore, in step two (a), the precipitate is re-dissolved for 21 hours.

[0011] Furthermore, in step two (b), the density of 293T cells before transfection is 1.5 × 10⁻⁶. 5 / cm 2 Get vaccinated.

[0012] Furthermore, in step two (b), the total plasmid amount used during transfection is 0.3 μg / cm³. 2 .

[0013] Furthermore, in step two (b), the precipitate is re-dissolved for 21 hours.

[0014] Furthermore, step three is performed according to 1.5 × 10 5 / cm 2 The density of seeding corresponds to the ASCT receptor knockdown of the 293T stable cell line.

[0015] Furthermore, the total plasmid amount used during transfection in step three is 0.3 μg / cm³. 2 .

[0016] Furthermore, in step three, virus harvesting is performed 60 hours after transfection.

[0017] Furthermore, in step two: constructing a stable 293T cell line with ASCT receptor knockdown, wherein the stable 293T cell line with ASCT receptor knockdown is (a) a stable 293T cell line with ASCT2 receptor knockdown.

[0018] The present invention can bring the following beneficial effects: This invention improves lentiviral titers by constructing a 293T cell line with knockdown of the ASCT2 and ASCT1-ASCT2 receptors, and screens the compounds and harvest time used in the production process. On one hand, the mechanism of this invention helps reduce "reinfection" of production cells by the produced lentivirus, thereby increasing viral titers. On the other hand, in the production process of this invention, the synergistic use of heparin sodium significantly reduces apoptosis after cell fusion, increases cell survival time, increases viral yield, reduces host protein and host DNA residues, and reduces the burden of subsequent purification.

[0019] The method of this invention reduces syncytial formation during viral packaging while preserving some ASCT2 and ASCT1 receptor functions, maintaining normal cell proliferation. Furthermore, the addition of heparin sodium after transfection can further increase viral titer, reduce host protein and nucleic acid residues in the feed solution, alleviate downstream purification pressure, and effectively improve the titer of BaEV enveloped lentivirus.

[0020] It is important to note that this invention systematically integrates two key elements: "cell engineering modification (construction of stable transgenic strains)" and "process parameter optimization (heparin sodium, plasmid ratio, cell density, and virus harvesting time)," forming a complete solution for BaEV pseudotyped lentivirus production. This method not only achieves a comprehensive improvement in viral titer but also boasts advantages such as ease of operation, good reproducibility, and easy scale-up. It overcomes the shortcomings of existing technologies where single improvements (such as simply adding poly-L-lysine or complete gene knockout) can lead to poor efficacy or cell damage. This provides a highly valuable technical pathway for the commercial production of highly effective cell therapy drugs such as CAR-NK that rely on BaEV-LV. Attached Figure Description

[0021] The accompanying drawings, which are included to provide a further understanding and demonstration of the invention, are provided in the drawings. In the drawings: Figure 1 Two shRNA vectors were constructed to knock down the ASCT2 receptors ASCT2 and ASCT1. Figure 2 To screen for 293T stable cell lines with ASCT2 receptor knockdown and ASCT1 ASCT2 receptor knockdown at 5 μg / ml puromycin; Figure 3 Cell states of the ASCT2 receptor knockdown 293T stable cell line and the ASCT1 ASCT2 receptor knockdown 293T stable cell line 24h, 48h and 72h after lentiviral transfection. Figure 4 For GFP lentivirus titers (*, p<0.05; **, p<0.01); Figure 5 Viral titers after adding heparin sodium to ASCT2 receptor knockdown 293T stable cell lines and ASCT1 ASCT2 receptor knockdown 293T stable cell lines (*, p<0.05; **, p<0.01). Detailed Implementation

[0022] To more clearly illustrate the overall concept of the present invention, the overall solution of the present invention will be described in detail below by way of embodiments; in the following description, numerous specific details are set forth in order to provide a more thorough understanding of the present invention; however, it will be apparent to those skilled in the art that the present invention may be practiced without one or more of these details; in other instances, some technical features known in the art have not been described in order to avoid confusion with the present invention.

[0023] If no culture medium is specified, DMEM complete medium is used by default.

[0024] Unless otherwise specified, all raw material components in the following examples can be purchased commercially, the experimental instruments used are all conventional laboratory instruments, and the performance testing methods are known in the art.

[0025] The implementation method and experimental verification are as follows: First, the preferred embodiment of the present invention is given: Embodiment 1: The following methods can be used to increase lentivirus titers: Step 1: shRNA vector construction: The shRNA vector for knocking down the ASCT2 receptor is SEQ ID No. 2 driven by the H1 promoter; Step 2: Construct a stable 293T cell line with ASCT receptor knockdown. The stable 293T cell line with ASCT receptor knockdown is as follows: (a) ASCT2 receptor knockdown stable 293T cell line: The shRNA vector of the knockdown ASCT2 receptor was packaged into lentivirus 1, and 293T cells were infected with MOI=10 and screened with puromycin to obtain the ASCT2 receptor knockdown stable 293T cell line. The method for packaging lentivirus 1 is as follows: A four-plasmid system (pLP1:pLP2:VSV-G:ASCT2 receptor knockdown shRNA vector) with a plasmid mass ratio of 0.25:0.25:0.125:0.375 is used for lentivirus packaging and transfection. Before transfection, the 293T cell density is 1.5 × 10⁻⁶ cells / year. 5 / cm 2 Inoculation was performed, followed by transfection 24 hours later. The total plasmid concentration used during transfection was 0.3 μg / cm³. 2 The mass ratio of PEI to plasmid was 2:1. After 6 hours of transfection, the medium was replaced with DMEM complete medium. After 72 hours of transfection, virus harvesting was performed to obtain virus harvesting solution 1. The virus harvesting solution 1 was clarified through a 0.45 μm syringe filter and then centrifuged at 25,000 rpm for 2 hours. After centrifugation, the supernatant was discarded and the precipitate was re-dissolved in DMEM complete medium at 4°C for 21 hours to obtain the lentivirus 1. The cells infected with 293T cells and selected with puromycin were: 1.2 × 10⁻⁶.5 / cm 2 293T cells were seeded and infected with lentivirus 1 at MOI=10 24 h later. After infection, the cells were replaced with DMEM complete medium 6 h later. After infection, the cells were screened and passaged at a concentration of 5 μg / ml puromycin 72 h later. Then, a second screening and amplification was performed using 10 μg / ml puromycin . Step 3: Packaging BaEV GFP lentivirus into the ASCT receptor knocked-down 293T stable cell line: According to 1.5×10 5 / cm 2 ASCT2 receptor knockout 293T stable transfected cell lines were seeded at a density of [missing information], and transfected 24 h later. The total plasmid concentration used during transfection was 0.3 μg / cm³. 2 The mass ratio of pLP1:pLP2:BaEV:GFP was 0.25:0.25:0.125:0.375, and the mass ratio of PEI to plasmid was 2:1. Heparin sodium was added to the culture dish when changing the medium 6 h after transfection, so that the final concentration of heparin sodium in DMEM complete medium was 10 U / ml. The BaEV enveloped virus was obtained 60 h after transfection.

[0026] Example 2: The difference between packaging BaEVGFP lentivirus in ASCT2 receptor knockdown 293T stable cell lines and packaging BaEVGFP lentivirus in conventional 293T cells: According to 1.5×10 5 / cm 2 ASCT2 receptor knockout 293T stable transfected cell lines were seeded at a specific density and transfected 24 h later; the total plasmid concentration used during transfection was 0.3 μg / cm³. 2 The pLP1:pLP2:BaEV:GFP ratio was 0.25:0.25:0.125:0.375, and the PEI to plasmid ratio was 2:1. The culture medium was changed 6 hours after transfection. Virus harvesting was performed 48 hours and 72 hours after transfection. The harvested fluid was filtered and clarified using a 0.45 μm syringe filter, and the titer of the clarified sample was determined. The clarified sample was treated with 1.2 × 10⁻⁶ ppm of the solution the day before infection. 5 / cm 2 The GFP positivity rate of inoculated 293T cells was measured and the titer was calculated at 24h, 48h, and 72h after infection. The states of ASCT2 receptor knockdown stable 293T cell lines and conventional 293T cells at 24h, 48h, and 72h during the viral loading process were compared as follows: Figure 3As shown, the GFP fluorescence intensity was stronger in the ASCT2 knockdown control group than in the untreated control group, indicating a reduction in syncytiotrophosomes after ASCT2 knockdown. The viral titers of BaEV GFP lentivirus packaged in ASCT2-knockdown 293T stable cell lines and conventional 293T cells are shown in the figure. Figure 4 As shown, the viral titers harvested from the ASCT2 receptor knockdown 293T stable cell lines were all higher than those of the control 293T group, with the highest viral titers harvested at 72 hours.

[0027] Example 3: The difference between packaging BaEV GFP lentivirus in 293T stable cell lines with ASCT1 and ASCT2 receptor knockdown and packaging BaEV GFP lentivirus in conventional 293T cells: According to 1.5×10 5 / cm 2 ASCT1 and ASCT2 receptor knockout 293T stable transfected cell lines were seeded at a density of [missing information], and transfection was performed 24 h after seeding; the total plasmid concentration used during transfection was 0.3 μg / cm³. 2 The pLP1:pLP2:BaEV:GFP ratio was 0.25:0.25:0.125:0.375, and the PEI to plasmid ratio was 2:1. The culture medium was changed 6 hours after transfection, and virus was harvested 48 and 72 hours after transfection. The harvested fluid was filtered and clarified using a 0.45 μm syringe filter, and the clarified sample was titered. The clarified sample was then treated with a 1.2 × 10⁻⁶ solution one day before infection. 5 / cm 2 The GFP positivity rate of inoculated 293T cells was measured and the titer was calculated at 24h, 48h, and 72h after infection. The states of ASCT1 and ASCT2 receptor knockdown-mediated stable 293T cell lines and conventional 293T cells at 24h, 48h, and 72h during the viral loading process were compared as follows: Figure 3 As shown, the GFP fluorescence intensity was stronger in the 293T stable cell line with ASCT1 and ASCT2 knockdown than in the untreated control group, indicating a reduction in syncytial counts after ASCT1 and ASCT2 knockdown. The viral titers of BaEV GFP lentivirus packaged in the ASCT1 and ASCT2 receptor knockdown 293T cell lines compared to those packaged in conventional 293T cells are shown in the figure. Figure 4 As shown, the 293T stable cell line with ASCT1 and ASCT2 receptor knockdown had a higher viral titer than the control 293T group, and the viral titer was higher at 72h. Further, combined with Example 2, it can be seen that the 293T stable cell line with ASCT2 receptor knockdown had the highest viral titer at 72h.

[0028] Example 4: Difference in viral titer between adding and not adding heparin sodium when packaging BaEV GFP lentivirus from ASCT receptor knockout 293T stable cell lines: According to 1.5×10 5 / cm 2 ASCT2 receptor knockout 293T stable cell lines and ASCT1 ASCT2 receptor knockout 293T stable cell lines were seeded at a density of [missing information]. Transfection was performed 24 h after seeding, with a total plasmid concentration of 0.3 μg / cm³ at transfection. 2 The ratio of pLP1:pLP2:BaEV:GFP was 0.25:0.25:0.125:0.375, and the ratio of PEI to plasmid was 2:1. Six hours after transfection, heparin sodium was added to the culture dish to achieve a final concentration of 10 U / ml, and heparin sodium was not added. Virus harvesting was performed 48 and 72 hours after transfection. The harvested fluid was filtered and clarified using a 0.45 μm syringe filter, and the clarified sample was titered. The clarified sample was then processed according to a 1.2 × 10⁻⁶ m² / day before infection. 5 / cm 2 The GFP positivity rate of inoculated 293T cells was measured and the titer was calculated 72 hours after infection. Results are as follows: Figure 5 As shown, 10 U / ml of heparin sodium can further increase the viral packaging titer of the ASCT2 receptor knockdown 293T stable cell line and the ASCT1 ASCT2 receptor knockdown 293T stable cell line. The BaEV viral packaging titer of the ASCT2 receptor knockdown 293T stable cell line with added 10 U / ml of heparin sodium is the highest.

[0029] As can be seen from Examples 2-4, Example 1 is indeed the best embodiment of the present invention. The method of constructing a 293T cell line that knocks down the ASCT2 receptor and the ASCT1ASCT2 receptor and screening the compounds used in the production process and the harvest time of the present invention does indeed improve the lentivirus titer.

[0030] The above description is merely an embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention can have various modifications and variations. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principle of the present invention should be included within the scope of the claims of the present invention.

Claims

1. A method for increasing the titer of BaEV enveloped virus, characterized in that, The method for increasing the BaEV envelope virus titer is as follows: Step 1: shRNA vector construction: The shRNA vector for knocking down the ASCT2 receptor is SEQ ID No. 2 driven by the H1 promoter, and the shRNA vectors for knocking down the ASCT1 and ASCT2 receptors are constructed in tandem by SEQ ID No. 1 driven by the U6 promoter and SEQ ID No. 2 driven by the H1 promoter. Step 2: Construct a stable 293T cell line with ASCT receptor knockdown, wherein the stable 293T cell line with ASCT receptor knockdown is any one of the following: (a) ASCT2 receptor knockdown stable 293T cell line: The shRNA vector of the knockdown ASCT2 receptor was packaged into lentivirus 1, and 293T cells were infected with MOI=10 and screened with puromycin to obtain the ASCT2 receptor knockdown stable 293T cell line. The method for packaging lentivirus 1 is as follows: Lentiviral packaging and transfection are performed using a 4-plasmid system (pLP1:pLP2:VSV-G:ASCT2 receptor knockdown shRNA vector) with a plasmid mass ratio of 0.25:0.25:0.125:0.

375. Before transfection, the 293T cell density is (1.0~2.0)×10⁻⁶ cells / year. 5 / cm 2 Inoculation was performed, followed by transfection 24 hours later. The total plasmid concentration used during transfection was 0.25–0.35 μg / cm³. 2 The mass ratio of PEI to plasmid was 2:

1. After 6 hours of transfection, the medium was replaced with DMEM complete medium. After 72 hours of transfection, virus harvesting was performed to obtain virus harvest solution 1. The virus harvest solution 1 was clarified through a 0.45 μm syringe filter and then centrifuged at 25,000 rpm for 2 hours. After centrifugation, the supernatant was discarded and the precipitate was re-dissolved in DMEM complete medium at 4°C for 18-24 hours to obtain the lentivirus 1. The cells infected with 293T cells and selected with puromycin were: 1.2 × 10⁻⁶. 5 / cm 2 293T cells were seeded and infected with lentivirus 1 at MOI=10 24 h later. After infection, the cells were replaced with DMEM complete medium 6 h later. After infection, the cells were screened and passaged at a concentration of 5 μg / ml puromycin 72 h later. Then, a second screening and amplification was performed using 10 μg / ml puromycin . (b) Stable 293T cell lines with ASCT1 and ASCT2 receptor knockdown: The shRNA vectors with ASCT1 and ASCT2 receptor knockdown were packaged into lentivirus 2, infected with 293T cells at MOI=10, and screened with puromycin to obtain the stable 293T cell lines with ASCT1 and ASCT2 receptor knockdown. The method for packaging lentivirus 2 is as follows: Lentiviral packaging and transfection are performed using a 4-plasmid system consisting of pLP1:pLP2:VSV-G:shRNA vector knocking down ASCT1 and ASCT2 receptors in a plasmid mass ratio of 0.25:0.25:0.125:0.

375. Before transfection, the 293T cell density is (1.0~2.0)×10⁻⁶ cells / year. 5 / cm 2 Inoculation was performed, followed by transfection 24 hours later. The total plasmid concentration used during transfection was 0.25–0.35 μg / cm³. 2 The mass ratio of PEI to plasmid was 2:

1. After 6 hours of transfection, the medium was replaced with DMEM complete medium. After 72 hours of transfection, virus harvesting was performed to obtain virus harvesting solution 2. The virus harvesting solution 2 was clarified through a 0.45 μm syringe filter and then centrifuged at 25,000 rpm for 2 hours. After centrifugation, the supernatant was discarded and the precipitate was re-dissolved in DMEM complete medium at 4°C for 18-24 hours to obtain the lentivirus 2. The cells infected with 293T cells and selected with puromycin were: 1.2 × 10⁻⁶. 5 / cm 2 293T cells were seeded and infected with lentivirus 2 at MOI=10 24 h later. After infection, the cells were replaced with DMEM complete medium 6 h later. After infection, the cells were screened and passaged at a concentration of 5 μg / ml puromycin 72 h later. Then, a second screening and amplification was performed using 10 μg / ml puromycin . Step 3: Packaging BaEV GFP lentivirus into the ASCT receptor knocked-down 293T stable cell line: According to (1.0~2.0)×10 5 / cm 2 The 293T stable cell line corresponding to ASCT receptor knockout was seeded at a specific density, and transfected 24 h later. The total plasmid concentration used during transfection was 0.25–0.35 μg / cm³. 2 The mass ratio of pLP1:pLP2:BaEV:GFP was 0.25:0.25:0.125:0.375, and the mass ratio of PEI to plasmid was 2:

1. Heparin sodium was added to the culture dish when changing the medium 6 h after transfection, so that the final concentration of heparin sodium in DMEM complete medium was 10 U / ml. The BaEV enveloped virus was obtained 48 h to 72 h after transfection.

2. The method for increasing the titer of BaEV enveloped virus according to claim 1, characterized in that, In step two (a), the density of 293T cells before transfection was 1.5 × 10⁻⁶. 5 / cm 2 Get vaccinated.

3. The method for increasing the titer of BaEV enveloped virus according to claim 1, characterized in that, In step two (a), the total plasmid amount used during transfection is 0.3 μg / cm³. 2 .

4. The method for increasing the titer of BaEV enveloped virus according to claim 1, characterized in that, In step two (a), the precipitation is re-dissolved for 21 hours.

5. The method for increasing the titer of BaEV enveloped virus according to claim 1, characterized in that, In step two (b), the density of 293T cells before transfection was 1.5 × 10⁻⁶. 5 / cm 2 Get vaccinated.

6. The method for increasing the titer of BaEV enveloped virus according to claim 1, characterized in that, In step two (b), the total plasmid amount used during transfection is 0.3 μg / cm³. 2 .

7. The method for increasing the titer of BaEV enveloped virus according to claim 1, characterized in that, In step two (b), the precipitation is re-dissolved for 21 hours.

8. The method for increasing the titer of BaEV enveloped virus according to claim 1, characterized in that, Step three is performed according to 1.5 × 10 5 / cm 2 The density of seeding corresponds to the ASCT receptor knockdown of the 293T stable cell line.

9. The method for increasing the titer of BaEV enveloped virus according to claim 1, characterized in that, The total plasmid amount used in step three, transfection, is 0.3 μg / cm³. 2 .

10. The method for increasing the titer of BaEV enveloped virus according to claim 1, characterized in that, Virus harvesting is performed 60 hours after transfection in step three.

11. The method for increasing the titer of BaEV enveloped virus according to claim 1, characterized in that, Step 2: Constructing a stable 293T cell line with ASCT receptor knockdown, wherein the stable 293T cell line with ASCT receptor knockdown is (a) a stable 293T cell line with ASCT2 receptor knockdown.

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