The baculovirus expression vector system (BEVS) is an effective and very popular used method for the production of recombinant proteins in insect cells. The baculovirus most often used as an expression vector is Autographa californica nucleopolyhedrovirus (AcNPV). Historically, the most widely used hosts for AcNPV were the established insect cell lines IPLB-Sf21-AE (Sf21) and Sf9, originally derived from Spodoptera frugiperda ovaries (Vaughn et al., 1977), and BTI-TN-5B1-4 (High-five), an insect cell line derived from Trichoplusia ni eggs, provided higher levels of foreign protein production (Wickham et al., 1992). However, levels of several recombinant protein expressions under the polyhedrin promoters are substantial, but still do not approach the levels of polyhedrin protein expression. A basic aspect of the BEVS which has been largely ignored is the importance of the baculovirus-cell culture systems in recombinant protein expression. Thus, protein production by baculovirus is affected by several factors, including the cell line (Wickham et al., 1992; McIntosh and Grasela, 1994; Hink et al., 1991), cell density (Reuveny et al., 1993), and medium replacement at the time of infection (Linday and Betenbaugh, 1992; Lazarte et al., 1992) or replacement and supplementation of medium with additional additives including serum (Reuveny et al., 1993; Bedard et al., 1994), the passage number of virus (Wickham et al., 1991), and multiplicity of infection (MOI) (Licari and Bailey, 1991). In addition to the above factors, the composition of the medium also greatly impacts culture productivity (Jesionowski et al., 1997).

Classical swine fever virus (CSFV), featuring high fever and hemorrhagic lesions, is a highly contagious disease threatening the pig industry of the world (Terpstra, 1991). CSFV is a member of the Pestivirus genus of the family of Flaviviridae (Francki et al., 1991). The genome is a single strand RNA of positive sense, approximately 12,300 nucleotides in length. It has a non-translated region at either end (5’NTR and 3’NTR), encompassing a single open reading frame encoding a large protein that is cleaved into smaller fragments (Paton et al, 2000). Structural components of the CSFV virion include the capsid (C) protein and glycoproteins E^rns (E0), E1, and E2 (Thiel et al., 1991). Glycoprotein E2 among them is the most immunogenic glycoprotein, inducing neutralizing antibodies and protection against lethal CSFV challenge (Risatti et al., 2007). Therefore, vaccines based on glycoprotein E2 protect swine from CSF and induce high titers of neutralizing Abs (van Rijn et al, 1999). Recently, cDNA sequences encoding different versions of glycoprotein E2 of CSFV have been inserted into the gX locus of pseudorabies virus (PRV) (van Ziji et al., 1991). The recombinant viruses contain the sequence of glycoprotein E2 without and with a C-terminal transmembrane region (TMR). This subunit vaccine suggested that pigs immunized with glycoprotein with TMR develop high levels of neutralizing antibodies against CSFV and are protected against challenge with a lethal dose of CSFV, whereas pig inoculated with glycoprotein E2 without TMR develop low levels of neutralizing antibodies and are partly protected upon challenge (van Ziji et al., 1991). However, subunit vaccines are capable of eliciting a protective immune response only when a large amount of antigen is applied.

In this study, with the aim of determining an effective cell line, p.i. time, MOI and cell culture media using BEVS, we analyzed the optimal expression conditions of recombinant glycoprotein E2. We constructed a recombinant AcNPV which contains a CSFV glycoprotein E2 with TMR gene. The glycoprotein E2 production in cells adapted to serum-free media and media with serum also was evaluated in three insect cell lines.

The basal insect cell line used was a strain of S. frugiperda cell line (Sf21) in SF900II (Gibco, USA) serum-free medium. Other cell lines were a strain of S. exigua cell line (Se301) in IPL-41 medium (Welgene, Republic of Korea) with 5% fetal bovine serum (FBS) and T. ni cell line (High-Five) was maintained in Express Five serum-free medium (Gibco, USA) with L-glutamine. All cells were maintained at 27˚C in T-flasks in a non-humidified incubator.

The insect media used were serum-free and serumsupplemented media. The serum-free media were SF900II (Gibco, USA), SF900III (Gibco, USA), Insect-Xpress (BioWhittaker, USA) and MAX-XP (BD, USA). The serum-supplemented media were TC-100 (Welgene, Republic of Korea), IPL-41 (Welgene, Republic of Korea), Grace’s insect medium (JBI, Republic of Korea) and Grace’s insect medium supplemented (Gibco, USA). 5% of FBS was added.

Viral RNA was extracted from CSFV-infected cell cultures with Viral Gene-spin^TM kit (iNtRON Biotechnology, Republic of Korea) as recommended by the manufacturer. The viral RNA was used as a template for cDNA synthesis using RNA LA PCR Kit (TaKaRa, Japan). The glycoprotein E2 gene was amplified from cDNA with primers CSFV E2-F (5’-GAATTCATAATGCGGCTAGCCTGCAAGGAAGATT-3’) and CSFV-E2-R (5’-CTGCAGACCAGCGGCGAGTTGTTCTGTTAGAA-3’). The purified PCR products were cloned into the T&A cloning vector (RBC Bioscience, Taiwan), and the plasmids were named as pTA-E2 (Fig. 1). The plasmids were digested with EcoR I (under lined) and Pst I (under lined), and then cloned into the transfer vector pBacPAK9 (Clontech, USA), to get the recombinant transfer plasmid pBacPAK9-E2. Sf21 cells were co-transfected with a mixture of purified transfer vector, bApGOZA DNA (Je et al., 2001), and Cellfectin II (Invitrogen, USA), according to the manufacturer’s instruction. Five days after adding the Cellfectin II-DNA complexes to the cells, the medium containing viruses released by the transfected cells was transferred to a sterile container and stored at 4˚C. A standard plaque assay procedure (O’Reilly et al., 1992) was used to obtain viral plaques from dilutions of the media harvested from the co-transfections. Individual plaques were examined under a microscope and scored for polyhedra production.

[Fig. 1.] Schematic representation of the recombinant virus generation.

Recombinant virus titer was determined by an end-point dilution assay. In this method, 96-well plates containing 100 μL of cellular suspension at a density of 2×10⁵ cells/mL were incubated for 1 h at 27˚C. Dilutions of the virus samples were performed serially in SF900II medium, from 1:10³ to 1:10⁸, and 100 μL were used to infect Sf21 cells monolayer after removing the medium from plate wells. Eight replicates for each dilution were performed in the same plate, and two independent plates were infected for each viral sample. SF900II medium was used as negative control. Plates were screened after seven days for occlusion bodies under a microscope. The 50% tissue-culture infectious dose (TCID₅₀), i.e., the dilution which is sufficient to infect half of the cells, was calculated using equations described elsewhere (King and Possee, 1992). This value was then converted to plaque-forming units (or number of infectious particles) by the relationship: PFU=0.69×TCID₅₀ (King and Possee, 1992).

Sf21 cells were infected with the wild-type AcNPV and recombinant virus in a 25 cm² flask (1×10⁶ cells) at various MOIs. After incubation at 27˚C, cells were harvested at every day for 5 days. For SDS-PAGE (Laemmli, 1970) of cell lysates, wild-type AcNPV and recombinant virus-infected cells were washed twice with phosphate buffered saline (PBS). The lysate was prepared by incubating the cells with lysis buffer (20 mM Tris-HCl pH 7.5, 50 mM NaCl, 5% Glycerol, 0.1% Triton X-100) containing protein inhibitor cocktail (Sigma-Aldrich, USA) for 30 min on ice followed by sonication and mixed with protein sample buffer and boiled. The total cellular lysates were subjected to 12% SDS-PAGE. For Coomassie stains, gels were washed with deionized water and stained with BioSafe Coomassie. For Western blot analysis, 12% SDS-PAGE was performed as described above. Proteins of cellular lysates were blotted on-to a nitrocellulose membrane (Pall Corp., USA). After transfer, the membrane was incubated for 60 min with gentle agitation in 5% (w/v) non-fat dry milk in TBST buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.05% Tween 20). The blot was incubated with anti-CSFV E2 monoclonal antibody (1:640) (JENO Biotech, Republic of Korea) in TBST for 1 h and washed. Subsequently, the membrane was incubated with anti-mouse horseradish peroxidase conjugate for 30 min at room temperature. After repeated washing, the immune reactive bands were visualized with the ECL Western Blotting Detection System (Elpis Biotech, Republic of Korea).

First, for the comparison of glycoprotein E2 productivity at different MOIs with Sf21 cells, 1×10⁶ cells/ml were infected with the recombinant baculovirus, rB9CSFV-E2, at a MOI of 1, 5 or 10. The effect of MOI on infectivity and yield is as important as the condition of cells and the time of infection (Kim et al, 2007). Expression of the glycoprotein E2 was analyzed by SDS-PAGE and Western blot with the specific CSFV-E2 monoclonal antibody. The highest amount of recombinant protein was expressed at a MOI of 5, with no further increase at a MOI of 10 (Fig. 2A). It had an apparent molecular weight around 35 kDa. To compare the productivity of glycoprotein E2 on the time of infection, Sf21 cells were harvested at every day for five days. The glycoprotein E2 appeared to start about three days post-infection, and reached a peak at 5 days. Presumably, the glycoprotein E2 seems to be accumulated during the expression time (Fig. 2B).

[Fig. 2.] The glycoprotein E2 expression in Sf21 cells by time course and MOIs .

The design of this study was to optimize the baculovirus mediated expression of the glycoprotein E2 as different levels of protein expression have been reported depending of the insect cell line used for infection (Davis et al., 1993). To compare the productivity of the glycoprotein E2 in three different insect cell lines, the protein expression of these cells were examined. Cells were infected with virus at MOI of 5 and harvested at five days post-infection. The highest amount of glycoprotein E2 was produced in High-Five cells, followed by Sf21 cells (Fig.3). However, the expression of the glycoprotein E2 was not detected in SDS-PAGE analysis (data not shown). Previous studies on the production of recombinant protein in various insect cell lines demonstrated cells derived from embryonic tissues of cabbage looper, T. ni (High-Five), were often a better choice for production of recombinant protein than Sf21 cells (Davis et al., 1993). These data corroborate previous studies which have shown that there are major differences between cell lines in their ability to produce recombinant protein in the BEVS (Hink et al, 1991; Wickham et al., 1992)

[Fig. 3.] Expression of glycoprotein E2 in Sf21, High-five and Se301 cells.

The productivity of the glycoprotein E2 in different culture media (four serum-free and four serum-supplemented media) was compared. The basal culture medium of Sf21 cells were a SF900II serum-free medium and different culture media were adapted before the experiment. However, Sf21 cells were not grown to IPL-41 and Grace’s insect medium; so, these used after direct transfer from basal media without adaptation. Eight different culture media-adapted cells were infected with virus at MOI of 5 and harvested at five days post-infection. As a result, Insect-Xpress insect medium in serum-free medium (Fig. 4A) and TC-100 insect medium in serum-supplement medium (Fig. 4B) were the best conditions for the expression of the glycoprotein E2. These results suggest that expression of glycoprotein E2 in Sf21 cells was influenced by media composition. Insect cell culture media contain sugars, salts, amino acids, vitamins, organic acids, lipids, and trace elements. The influence of various nutrients may be beneficial for increasing recombinant protein yields (Ikonomou et al., 2003). While the mechanism by which it promotes recombinant protein production remains unknown. This conclusion was further supported by the experiments where influence of nutrients on productivity in culture media. In addition, the glycoprotein E2 was expressed at the highest level in serum-supplemented media than serumfree media. These results indicate that FBS promote recombinant protein production in Sf21 cells (Nishikawa et al., 2003; Hink et al., 1991). Consequently, the recombinant protein expression was higher because a greater proportion of the culture media.

[Fig. 4.] Expression of glycoprotein E2 in various culture media.

The recombinant virus-infected Sf21 cells in eight different culture media are presented as budded virus (BV) titers in Table 1. Both initial infection rate and BV titers are correlated with the production of baculovirus-based recombinant proteins. To elucidate the relation between the virus and the recombinant protein productivities, BV productivity in various cell culture media was evaluated. The initial infection rate of virus in eight different insect cell culture media was determined at 1 hour post-infection. As can be seen from the results presented in Table 1, the initial infection rate of virus did not show any significant difference in serum-free or supplement media. At five days postinfection, however, as determined by titration on Sf21 cells in SF900II media, 750 times more budded virus was produced in Grace’s insect media supplemented with 5% FBS, suggesting that serum-supplement media accelerate a higher rate of BV production (9.2×10⁶ pfu/mL and 6.9×10⁹ pfu/mL, respectively). Both BV yield and recombinant protein production were apparently increased in serum-supplement media. This was corresponding to the previous results. Insect cells have been widely cultured in basal media supplemented with vertebrate serum, in particular with a concentration of 5% to 20% of FBS, which has been shown to support cell growth, high BV titers, and recombinant protein production (Wu et al., 1989; Matindoost et al., 2014). Most BV productivity had direct proportion with the recombinant protein production (Table 1, Fig. 4). However, the recombinant protein production was influenced not only the BV productivity but also the kind of used media.

[Table 1.] Baculovirus productivity in Sf21 cells under different culture media conditions

Baculovirus productivity in Sf21 cells under different culture media conditions

In conclusion, our study suggests that the High-Five cell line and serum-supplement TC-100 insect medium are optimal for the production of the CSFV glycoprotein E2 in Sf21 cells. These results would provide useful information on the determination of insect cells and insect media in production of recombinant proteins using the BEVS.