Expression of the hepatitis B virus surface antigen in Drosophila S2 cells

Abstract

Drosophila melanogaster S2 cells were transfected with a plasmid vector (pAcHBsAgHy) containing the S gene, coding for the hepatitis B virus surface antigen (HBsAg), under control of the constitutive drosophila actin promoter (pAc), and the hygromycin B (Hy) selection gene. The vector was introduced into Schneider 2 (S2) Drosophila cells by DNA transfection and a cell population (S2AcHBsAgHy) was selected by its resistance to hygromycin B. The pAcHBsAgHy vector integrated in transfected S2 cell genome and approximately 1,000 copies per cell were found in a higher HBsAg producer cell subpopulation. The HBsAg production varied in different subpopulations, but did not when a given subpopulation was cultivated in different culture flasks. Higher HBsAg expression was found in S2AcHBsAgHy cells cultivated in Insect Xpress medium (13.5 mug/1E7 cells) and SFX medium (7 mug/1E7 cells) in comparison to SF900II medium (0.6 mug/1E7 cells). An increase of HBsAg was observed in culture maintained under hygromycin selection pressure. Data presented in the paper show that S2AcHBsAgHy cells produce efficiently the HBsAg which is mainly found in the cell supernatant, suggesting that HBsAg is secreted from the cells. The data also show that our approach using the Drosophila expression system is suitable for the preparation of other viral protein preparation.

Fig. 1

pAcHBsAgHy vector map. pAc represents the drosophila actin promoter; HBsAg, the gene of the surface antigen of Hepatitis B virus; SV40, the signal of polyadenylation of the SV40 virus; pCopia, the promoter of drosophila copia gene. Hygromycin, the hygromycin resistance gene; pUC ori and ampicillin, represent the replication origin and selection gene in bacteria, respectively

Fig. 2

Cell growth and HBsAg concentration cell supernatants and cell lysates. The S2AcHBsAgHy cells were cultivated in 100 mL shake flasks for 7 days with SF900II medium at 28 °C and 100 rpm. Periodically samples were taken, the cells counted and the HBsAg evaluated in the supernatant (μg/mL) and in cell fraction (μg/1E7 cells) after lysis with buffer. Data of at least 4 different experiments and standard deviation are shown

Fig. 3

Southern blotting analysis (a) and total HBsAg expression (b) of S2AcHBsAgHy cell populations. S2AcHBsAgHy cells were cultivated in shake flasks with SF900II for 6 days at 28 °C and 100 rpm, when cell supernatants and cell fractions were collected. Cell fraction was treated with lysis buffer for 10 min HBsAg DNA was evaluated by Southern blotting analysis (a) of DNA extracted from transfected cell populations or from pAcHBsAgHy. Arrows indicate the supercoiled pAcHBsAgHy DNA and the arrowhead the circular pAcHBsAgHy DNA. Total HBsAg content in cell supernatants and cell fractions was evaluated by ELISA (b) and expressed in μg/1E7 cells. The total HBsAg was obtained by the addition of values obtained for the cell supernatant and cell lysate. S2AcHBsAgHy is the initial cell population and 8A, 8C, 9C and 12E are selected subpopulations

Fig. 4

Cell growth (a), glucose (b) and glutamine (c) consumption, lactate production (d) and HBsAg expression (e, f) of S2AcHBsAgHy 9C cells cultivated in T-flasks or shake flasks. Cells were cultivated for 11 days with SF900II culture medium at 28 °C and 100 rpm. Periodically samples were taken and the cell growth, glucose, glutamine and lactate concentrations were evaluated in these cultures. HBsAg expression in S2AcHBsAgHy 9C cell supernatant and cell lysate were measured. Data of at least 3 different experiments and standard deviation are shown

Fig. 5

Kinetics of total HBsAg expression by S2HBsAgHy 9C cells cultivated in different media. S2AcHBsAgHy 9C cells were adapted and then cultivated in the indicated media, in shake flasks for 11 days at 28 °C and 100 rpm, when cells and cell supernatants were collected. Cells were then treated with lysis buffer for 10 min and the HBsAg content evaluated by ELISA. Total HBsAg expression, expressed in μg/1E7 cell, was obtained by the sum of values obtained for the cell supernatant and cell lysate. Data of a representative experiment are shown