Ao38, a new cell line from eggs of the black witch moth, Ascalapha odorata (Lepidoptera: Noctuidae), is permissive for AcMNPV infection and produces high levels of recombinant proteins

Abstract

Background: The insect cell line is a critical component in the production of recombinant proteins in the baculovirus expression system and new cell lines hold the promise of increasing both quantity and quality of protein production.

Results: Seventy cell lines were established by single-cell cloning from a primary culture of cells derived from eggs of the black witch moth (Ascalapha odorata; Lepidoptera, Noctuidae). Among 8 rapidly growing lines, cell line 38 (Ao38) was selected for further analysis, based on susceptibility to AcMNPV infection and production of secreted alkaline phosphatase (SEAP) from a baculovirus expression vector. In comparisons with low-passage High Five (BTI-Tn-5B1-4) cells, infected Ao38 cells produced beta-galactosidase and SEAP at levels higher (153% and 150%, respectively) than those measured from High Five cells. Analysis of N-glycans of SEAP produced in Ao38 cells revealed two N-glycosylation sites and glycosylation patterns similar to those reported for High Five and Sf9 cells. Glycopeptide isoforms consisted of pauci- or oligomannose, with and without fucose on N-acetylglucosamine(s) linked to asparagine residues. Estimates of Ao38 cell volume suggest that Ao38 cells are approximately 2.5x larger than Sf9 cells but only approximately 74% of the size of High Five cells. Ao38 cells were highly susceptible to AcMNPV infection, similar to infectivity of Sf9 cells. Production of infectious AcMNPV budded virions from Ao38 cells peaked at approximately 4.5 x 10(7) IU/ml, exceeding that from High Five cells while lower than that from Sf9 cells. Ao38 cells grew rapidly in stationary culture with a population doubling time of 20.2 hr, and Ao38 cells were readily adapted to serum-free medium (Sf-900III) and to a suspension culture system. Analysis of Ao38 and a parental Ascalapha odorata cell line indicated that these lines were free of the alphanodavirus that was recently identified as an adventitious agent in High Five cell lines.

Conclusions: Ao38 cells represent a highly productive new insect cell line that will be useful for heterologous protein expression and other applications in biotechnology.

Figure 1

Generation of Ascalapha odorata cell lines. Cell line development from a primary culture of Ascalapha odorata cells. Seventy-seven days after initiating primary cultures from A. odorata eggs, the Ao 2.0AA primary culture showed three cell foci containing proliferating cells. Left and right panels show Foci 1 and 2, respectively, which generated cells with distinct morphologies. The black bar represents 100 μm.

Figure 2

Clonal Ascalapha odorata cell lines and AcMNPV infection. (A) Clonal cell lines Ao11, Ao26, Ao38, Ao47, Ao53, and Ao56, are shown in the panels. The black bar represents 50 μm. (B) Comparison of uninfected and infected High Five and Ao38 cell lines. Left panels show uninfected High Five cells and Ao38 cells. Right panels show corresponding AcMNPV-infected High Five and Ao38 cells at 48 h pi. The black bar represents 50 μm.

Figure 3

Growth of Ao38 cells in culture. (A) The graph shows a one-step cell culture growth curve (solid line) of adherent cells. In addition, measurements of cell viability (dashed lines) are also included. Cell lines Sf9 (closed triangle), High Five (closed squares), and Ao38 (closed circles) grown in TNMFH medium were also compared with Ao38 cells grown in Sf-900III medium (open circles). All data points represent averages of triplicate samples and standard deviations are indicated by bars. (B) The graph shows a one step (dashed line) and a multi-step step (solid line) growth curve (open boxes) and viability (closed boxes) of suspension-culture Ao38 cells that were grown in Sf-900III medium in a shaker culture vessel at 100 rpm. Cells were diluted to approximately 0.25 × 10⁶ cells/ml in fresh media on days 5 and 8. Data points represent the average of triplicate experiments and standard deviations are plotted.

Figure 4

Ao38 cell volumes, AcMNPV susceptibility, and virus yields. (A) Measurements of packed cell volumes (PCV, μl per 3 × 10⁶ cells) of Ao38, Sf9 and High Five cells are compared in a bar graph. Gray bars represent averages and standard deviations from 3 replicate measurements are indicated. (B) Susceptibility of Ao38, Sf9, and High Five cells to AcMNPV infection. The relative susceptibilities of Ao38, Sf9, and High Five cells to AcMNPV infection was measured by infecting each cell line with an AcMNPV virus (βgal-AcMNPV, 1 × 10⁷ IU/ml) that was initially titred on Sf9 cells. The same virus preparation (βgal-AcMNPV) was then titred on each cell line. Gray bars represent averages of virus titers determined on each cell line at 4 days p.i. and standard deviations of triplicate infections are indicated. (C) Comparisons of WT AcMNPV production from Sf9, High Five, and Ao38 cells in TNMFH medium, and from Ao38 cells in Sf-900III serum-free medium. Virus titres were determined from the supernatants of triplicate infections of each cell line with WT AcMNPV at various times post infection (Y-axis).

Figure 5

β-gal and SEAP production of Sf9, High Five, and Ao38 cells. (A) Beta-galactosidase production was measured from Ao38, High Five, and Sf9 cells at various time post infection (Days post infection) by virus βgal-AcMNPV. Ao38.SFM represents Ao38 cells grown in serum-free medium (Sf-900III medium) and all other samples were produced in TNMFH containing 10% FBS. Data points and bars represent averages and standard deviations, respectively, from triplicate infections. (B) Secreted human placental alkaline phosphatase (SEAP) was measured from Ao38, High Five, and Sf9 cells at various time post infection (Days post infection) by virus SEAP-AcMNPV. Ao38.SFM represents Ao38 cells grown in serum-free medium (Sf-900III medium) and all other samples were produced in TNMFH containing 10% FBS. Data points and bars represent averages and standard deviations, respectively, from triplicate infections.

Figure 6

SDS-PAGE analysis of SEAP production in SEAP-AcMNPV infected Ao38 cells. Samples of 20 μl from the supernatant of SEAP-AcMNPV infected Ao38 cells grown in Sf-900III medium were electrophoresed and major protein bands identified by Coomassie blue staining. Lane 1; protein marker sizes in kDa are indicated on the left. Lanes 2-3 represent two independent preparations of SEAP-AcMNPV infected culture medium (20 μl). Lane 4, SEAP-AcMNPV infected culture medium (20 μl) after centrifugation (80,520 × g for 60 min) to remove baculovirus virions. Lane 5, Control from WT AcMNPV infected culture medium (20 μl). An arrow indicates the position of the protein band that was excised and subjected to N-glycan analysis by LC-MS/MS.

Figure 7

RT-PCR analysis and TNCL virus detection. (A) To examine cell lines for the presence of a TNCL virus, five primer pairs were used for RT-PCR analysis of total RNA from cell lines. Ao38, AoP (passage 5), Sf9, and High Five cells were infected with AcMNPV 48 h prior to RNA isolation. The relative locations of primer pairs on TNCL Virus RNAs 1 and 2 are indicated in the upper diagram, and primer sequences are listed in Table 2. Primer sets were labeled as follows: Set 1, Noda-R1-190F and Noda-R1-636R; Set 2, Noda-R1-1093F and Noda-R1-1531R, Set 3; Noda-R1-2368F and Noda-R1-2933R; Set 4, Noda-R2-269F and Noda-R2-810R; Set 5, Noda-D4 and Noda-U4. Primer sets and cell lines are indicated above each lane, and sizes (Kbp) of marker DNAs are indicated on the left of each gel. (B). RT-PCR analysis of gp64 transcript RNA control. RT-PCR (left) and PCR (right) were used to analyze total RNA from Ao38, Ao-P (passage 5), Sf9, and High Five cells infected with WT AcMNPV, AcMNPV gp64 primers are listed in Table 2. Samples treated with DNaseI (+) or not treated with DNaseI (-) are indicated above lanes. Sizes (kbp) of marker DNAs are indicated on the left.