TWI767433B - Cell strain having increased virus production ability and production method thereof - Google Patents

Abstract

The present invention relates to a cell line having an increased ability to produce virus and a method for preparing thereof, and more particularly, to a cell line that has an increased ability to produce virus, due to the overexpression of a CCCH-type zinc finger protein and to a production method thereof. According to the present invention, when the engineered cell line is used as a virus-producing cell line, the production yield of virus can be increased. In addition, the engineered cell line can be used for the production and research of vaccines for preventing viral diseases.

Description

Cell strain with increased virus production capacity and method for producing the same

The present invention relates to a cell line with increased virus production ability and a preparation method thereof, and more particularly, to a cell line with increased virus production ability due to overexpression of CCCH-type zinc finger protein and a preparation method thereof.

Vaccination with traditional vaccines, including live-attenuated vaccines and inactivated vaccines, has proven to be the most effective strategy for preventing and controlling viral diseases. Compared with traditional vaccine production methods (eg: chicken embryo culture for influenza virus and mouse brain culture for Japanese encephalitis vaccine), cell culture-based vaccine production has distinct advantages, including the following: (1) for use in The source of the cell line for producing the vaccine is easier and more convenient to obtain, and once a pandemic breaks out, the vaccine can be quickly produced; (2) it has a higher similarity with the influenza virus strain, which may improve the protective effect; (3) can control the production more process, speeding up the start of the vaccine production process; (4) avoiding the risk of severe allergic reactions caused by egg and mouse brain vaccines. However, production processes in cell culture may reduce viral yields as well as cost-effectiveness. Therefore, how to improve the virus yield has become a key issue for improving cell culture vaccines.

CCCH-type zinc finger proteins are RNA-binding molecules involved in RNA metabolic pathways such as RNA splicing, polyadenylation, and messenger RNA (mRNA) degradation. As a result of genome-wide search, a total of 57 CCCH genes were identified in humans, and they could be divided into 6 groups according to the amino acid sequence alignment (Liang, J et al. PLoS one 2008 , 3(8), e2880.).

Certain members of the CCCH-type zinc finger family, such as ZC3H12A (MCP-1-inducible protein 1), ZFP36 (Tristetraproline, TTP), ZC3H2 (Zinc Finger CCCH-type antiviral protein 1, also known as zinc finger antiviral protein (Zinc finger antiviral protein 1) antiviral protein, ZAP), which can directly bind to specific viral RNA sequences through its CCCH-type zinc finger motif (motif) and inhibit viral replication (Zhou, L et al. Circ Res 2006 , 98, 1–10; Taylor, GA et al. Immunity 1996 4, 445–454; Gao, G et al. Science 2002 , 297, 1703–1706.).

The functions of most CCCH-type zinc finger proteins remain unclear, and their interactions with viruses and their roles are rarely discussed. In addition to antiviral activity, some members may also possess proviral activities that enhance viral replication, which may be exploited to provide better viral yield and cost-effectiveness when using cell culture-based platforms for vaccine production.

Based on the above purpose, the present invention demonstrates that cells overexpressing ZC3HAV1L (Zinc finger CCCH-type, antiviral 1-like) protein can achieve higher yield of virus production.

Therefore, one aspect of the present invention relates to a virus-producing mutant cell by introducing the ZC3HAV1L gene into a cell with virus-producing ability, wherein the mutant cell increases the virus-producing ability by overexpressing the product of the gene.

In some embodiments of the invention, the mutant cell is infected with a virus.

In some embodiments of the invention, the virus is Flavivirus or Alphainfluenzavirus .

In some preferred embodiments of the present invention, the virus is selected from Japanese encephalitis virus (JEV), Dengue virus (DENV) and Influenza A virus (IAV).

In some preferred embodiments of the present invention, the mutant cell is an animal cell; preferably, the animal cell is a Vero cell.

In another aspect of the present invention, it relates to a method for preparing a virus-producing mutant cell, the method comprising introducing the ZC3HAV1L gene into a virus-producing cell.

In some embodiments of the invention, the method further comprises infecting the mutant cell with a virus.

In some embodiments of the invention, the mutant cells are infected with Flavivirus or Alphainfluenzavirus .

In some preferred embodiments of the present invention, the mutant cells are infected with Japanese encephalitis virus (JEV), Dengue virus (DENV) or Influenza A virus (IAV).

In some preferred embodiments of the present invention, the infected mutant cell is an animal cell; preferably, the animal cell is a Vero cell.

In yet another aspect of the present invention, it relates to a method for producing a desired virus, the method comprising the steps of: (1) infecting the virus-producing mutant cells with the desired virus; (2) culturing the infected cells of the desired virus Mutate the cells, and then collect the supernatant of the medium to recover the desired virus.

In some embodiments of the invention, the mutant cells are infected with the desired virus at a MOI value of 0.01 to 0.1.

In yet another aspect of the present invention, it relates to a method for producing a desired virus, the method comprising the steps of: (1) infecting the virus-producing mutant cells with the desired virus; (2) culturing the infected cells of the desired virus mutate cells, and then collect the supernatant of the medium to recover the desired virus; (3) attenuate or deactivate the recovered virus.

Other features and advantages of the present invention are further illustrated in the following examples, which are for illustration only and do not limit the scope of the invention.

Viruses, cell lines, chemicals and antibodies

JEV PR-9 strain (GenBank accession number: AF014161) (Chen, LK et al. Virology 1996 , 223, 79-88.) or DENV-2 PL046 virus strain (GenBank accession number: KJ734727) (Lin, YL et al. al. J Virol 1998 , 72, 9729-9737.) in C6/36 mosquito cells (ATCC, CRL-1660) in RPMI-1640 medium supplemented with 5% fetal bovine serum. JEV-EGFP (strain RP-9) and DENV-2-EGFP (strain PL046) were provided by Dr. Yiling Lin. JEV and DENV were quantified by measuring plaque formation in baby hamster kidney cells (Baby hamster kidney cell line; BHK-21 cells) cultured in supplemented 5% fetal bovine serum, 2 mM L-glutamine RPMI-1640 medium with L-glutamine and 1% penicillin-streptomycin. Influenza A/Puerto Rico/8/1934 (Influenza A/Puerto Rico/8/1934) (H1N1) (GenBank accession number: CY009444) used in this study was developed in a dog kidney epithelial cell line (Madin-Darby Canine Kidney cells; MDCK cells) grown in Dulbecco's modified Eagle's medium (DMEM; Gibico) containing 10% fetal bovine serum (FBS). Quantification of influenza A virus was performed by plaque formation assay on MDCK cells. Human non-small cell lung cancer cells A549 (ATCC, CCL-185) were cultured in F-12 medium supplemented with 10% FBS, 2 mM L-glutamine and 1% P/S. Human embryonic kidney cells 293T/17 (ATCC, CRL-11268) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS, 2 mM L-glutamine and 1% P/S. Lipofectamine 2000 reagent was purchased from Invitrogen. Anti-FLAG mouse monoclonal antibody was purchased from Sigma-Aldrich. Anti-ZC3HAV1L mouse monoclonal antibody (F4): sc-514959 was purchased from Santa Cruz Biotechnology. Mouse monoclonal anti-JEV NS3 antibodies have been previously described in Chen, LK et al. Virology 1996 , 217, 220-229.

Plasmid construction, lentivirus generation and establishment of stable cell line overexpressing ZC3HAV1L

ZC3HAV1L (NM_080660) Human Tagged ORF Clone (RC203208) was purchased from OriGene Technologies. The human ZC3HAV1L cDNA was combined with the FLAG-tag ZC3HAV1L Human Tagged ORF Clone, and subcloned into a self-inactivating lentiviral vector (pSIN), in which the inserted gene was expressed by a spleen focus-forming virus (Spleen focus-forming virus). , SFFV) promoter control (Godfrey, A et al. Blood 2005 , 105, 2510-2518.). The lentivirus was prepared by first co-transfecting human embryonic kidney cells 293 T/17 with the lentiviral expression construct (pSIN) and two helper plastids pMD.G and pCMVΔR8.91 with Lipofectamine 2000 reagent (Invitrogen). Transfected cells were incubated at 37°C for an additional 4-5 hours, then replaced with fresh medium. Viral particle-containing supernatants were then collected 24-60 hours post-transfection and stored at -80°C. The Vero cell line overexpressing ZC3HAV1L protein was prepared by transfecting A549 cells with a lentiviral vector expressing FLAG-tagged ZC3HAV1L protein or control EGFP protein for 72 hours.

Western blot analysis by western blot

A549 cells were transduced with lentiviral vectors expressing FLAG-tagged ZC3HAV1L protein or control mcherry protein for 72 hours, followed by infection with JEV-EGFP (MOI = 0.01, 0.1 or 1) for 16 hours. Sodium dodecyl sulfate (SDS) sample buffer (62.5 mM Tris-HCl [pH 6.8], 2% SDS, 10% glycerol, 50 mM dithiothreitol, 0.1% bromophenol blue) containing protease inhibitors ) to lyse the cell. Equal amounts of proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Hybond-C Super, Amersham). Nonspecific antibody binding sites were blocked with TBS-T (25 mM Tris, 0.8% NaCl, 2.68 mM KCl [pH 7.4], and 0.1% Tween 20) supplemented with 5% nonfat milk before adding the primary antibody. Blots were then treated with a secondary antibody (Amersham) with horseradish peroxidase and developed with the ECL system (Amersham).

Immunofluorescence assay

A549 cells were transduced with lentiviral vectors expressing FLAG-tagged ZC3HAV1L protein or control mcherry protein for 72 hours, then infected with JEV-EGFP (MOI = 0.01 or 0.1) for the indicated times, and the expression of EGFP protein was observed under a fluorescent microscope.

Virus growth curve assay

For JEV and DENV-2 infection, cell monolayers grown in 12-well dishes were adsorbed with virus at the indicated virus infectious dose (MOI) for 1 hour at 37°C. After 1 hour of adsorption, unbound virus was removed from the cells by gentle washing with RPMI 1640 medium. At the indicated post-infection time points (post-infection, p.i.), media was collected for plaque formation assays in BHK-21 cells.

For IAV (Influenza A virus) infection, cell monolayers grown in 12-well dishes were adsorbed at the indicated virus infection dose (MOI = 0.01) for 1 hour at 37°C. After 1 hour of adsorption, unbound virus was removed from the cells by gentle washing with PBS. At the indicated post-infection time points, supernatants were collected for plaque formation assays. Briefly, MDCK cells were plated at 6.0 x 105 cells/well in ⁶ -well dishes and then infected with virus for 24 hours at 37°C. Viruses to be quantified were serially diluted 10-fold in 6-well plates at 37°C. One hour after the infection ended, cells were washed twice with 1 mL of PBS and overlaid with 2 mL of DMEM/HG medium supplemented with 0.3% agarose. After 48 hours of incubation at 37°C, cells were fixed in 10% formalin for at least 1 hour before crystal violet staining. Virus quantification was calculated as plaque forming units (PFU) per milliliter.

Example 1 Overexpression of Zc3hav1L protein in cells

The pSIN vector was used to construct the ZC3HAV1L-Myc-Flag/pSIN plasmid. A549 cells (ATCC, CCL-185) overexpressing FLAG-tagged ZC3HAV1L protein were established by lentiviral transduction. Western blotting showed the expression of FLAG-tagged ZC3HAV1L protein or control EGFP protein in cells (Figure 1). Detected with anti-FLAG and anti-ZC3HAV1L monoclonal antibodies, the protein size was about 34 kDa.

Example 2 Effect of overexpression of Zc3hav1L protein on Japanese encephalitis virus (JEV) infection

A549 cells were transduced with lentiviral vectors expressing FLAG-tagged ZC3HAV1L protein or control group expressing mcherry protein, and then infected with JEV-EGFP. As shown in Figure 2, GFP expression was observed 6 hours after JEV-EGFP infection of ZC3HAV1L-overexpressing cells (MOI = 0.01) and 24 hours after infection of mcherry controls. In the JEV-EGFP infection at higher MOI (0.1), it was not until 16 hours before obvious GFP fluorescence was observed in the control group (Fig. 3).

Example 3 Western blot analysis of JEV protein NS3

A549 cells were transduced with lentiviral vectors expressing FLAG-tagged ZC3HAV1L protein or control group expressing mcherry protein, and then infected with JEV-EGFP (MOI = 0.01, 0.1, 1) for 16 hours. NS3, a nonstructural protein produced by JEV, was assayed by Western blotting, demonstrating that JEV replication can be efficiently performed in cells overexpressing the ZC3HAV1L protein (Figure 4).

Example 4 Establishment of stable cell lines overexpressing Zc3hav1L protein

Vero African green monkey kidney cells were transduced with a lentiviral vector expressing the FLAG-tagged ZC3HAV1L protein for 72 hours. The prepared transduced cells and parental cells were used for indirect immunofluorescence, and mouse anti-FLAG antibody and anti-mouse IgG antibody (Alexa-488) were used as primary and secondary antibodies, respectively (Figure 5). Almost all cells overexpressing the FLAG-tagged ZC3HAV1L could be stained compared to the parental cells.

Example 5 JEV Virus growth in infected Zc3hav1L protein-overexpressing cells

Vero cells with or without ZC3HAV1L overexpression were infected with JEV (MOI = 0.01) and the amount of virus replication was analyzed. Plaque assays were used to determine viral loads at 24, 48, 60, and 72 hours post-infection (Figure 6). Virus production was increased nearly 100-fold in cells overexpressing the ZC3HAV1L protein compared to parental cells as well as GFP-overexpressing cells.

Example 6 DENV-2 Virus growth in infected Zc3hav1L protein-overexpressing cells

Vero cells with or without ZC3HAV1L overexpression were infected with DENV-2 (MOI = 0.01) and the amount of virus replication was analyzed. Plaque assays were used to determine viral loads at 24, 48, 72, and 96 hours post-infection (Figure 7). Virus production in cells overexpressing the ZC3HAV1L protein was also increased by nearly 100-fold compared to parental cells as well as GFP-overexpressing cells.

Example 7 Virus growth in IAV infected Zc3hav1L protein overexpressing cells

Vero cells with or without ZC3HAV1L protein overexpression were infected with IAV (MOI = 0.01) and the amount of viral replication was analyzed. Plaque assays were used to determine viral loads at 24, 48, 64, and 80 hours post-infection (Figure 8). Virus production in cells overexpressing the ZC3HAV1L protein increased nearly 10-fold compared to parental cells as well as GFP-overexpressing cells.

As shown in the above examples, ZC3HAV1L, as a novel host cytokine, can significantly increase virus yield. Compared with parental cells, cells overexpressing ZC3HAV1L protein resulted in approximately a 100-fold increase in viral production of JEV and DENV and a more than 10-fold increase in IAV virus production. Compared to cell culture-based vaccine platforms in the art, the present invention provides higher virus yields and better cost-effectiveness in the production of attenuated and deactivated vaccines.

none

Figure 1. Western blot analysis of the expression of FLAG-tagged ZC3HAV1L protein in A549 cells transduced with lentivirus.

Figure 2 JEV-GFP (MOI = 0.01) was used to analyze JEV replication in A549 cells overexpressing ZC3HAV1L protein.

Figure 3. JEV-GFP (MOI = 0.1) was used to analyze JEV replication in A549 cells overexpressing ZC3HAV1L protein.

Figure 4 Immunofluorescence of a stable cell line overexpressing ZC3HAV1L-FLAG in Vero cells.

Figure 5. Production of the nonstructural viral protein NS3 in cells infected with JEV-EGFP by Western blot analysis.

Figure 6 Virus growth curve in JEV-infected cells.

Figure 7 Virus growth curve in DENV-2 infected cells.

Figure 8 Virus growth curve in IAV-infected cells.

none

Claims (9)

A virus-producing mutant cell, characterized in that a ZC3HA V1L gene has been introduced into a cell with virus-producing ability, and the mutant cell has increased virus-producing ability by overexpressing the product of the ZC3HA V1L gene; wherein the The mutant cell is an animal cell and is infected with a virus, either Flavivirus or Alphainfluenzavirus . The mutant cell of claim 1, wherein the virus is selected from Japanese encephalitis virus (JEV), Dengue virus (DENV) and Influenza A virus (IAV). The mutant cell of claim 1, wherein the animal cell is a Vero cell. A method for preparing a virus-producing mutant cell, comprising introducing the ZC3HA V1L gene into a cell having the ability to produce virus, and infecting the mutant cell with a virus; wherein the mutant cell is an animal cell, and the virus is a Flavivirus ) or influenza A ( Alphainfluenzavirus ). The method of claim 4, wherein the virus is selected from the group consisting of Japanese encephalitis virus (JEV), Dengue virus (DENV) and Influenza A virus (IAV). The method of claim 4, wherein the animal cell is a Vero cell. A method for producing a desired virus, comprising: (1) infecting a mutant cell with the virus-producing ability with the desired virus; wherein the mutant cell is an animal cell and ZC3HA V1L gene has been introduced, and the desired virus is a yellow Flavivirus or Alphainfluenzavirus ; (2) culturing the mutant cells infected with the desired virus, and then collecting the supernatant of the culture to recover the desired virus. The method of claim 7, wherein the mutant cells are infected with 0.01-0.1 MOI of the desired virus. A method for producing a desired virus, comprising: (1) infecting a mutant cell with the virus-producing ability with the desired virus; wherein the mutant cell is an animal cell and ZC3HA V1L gene has been introduced, and the desired virus is a yellow ( 2 ) culturing the mutant cells infected with the desired virus, and then collecting the supernatant of the culture to recover the desired virus; (3) recovering the recovered The desired virus is attenuated or deactivated.

Images