Recombinant proteins benefit large sectors of the biopharmaceutical, enzyme, and agricultural industries. They are commercially produced in these industries with the aid of genetic and protein engineering. Various systems that produce recombinant proteins, such as bacterial (Lunn et al., 1984; Weikert et al., 1997; Wong et al., 2008), yeast (Sreekrishna et al., 1989; Cregg et al., 1993; Goodrick et al., 2001), mold (Christensen et al., 1988; Ward et al., 1995; Verdoes et al., 2007), mammalian (Dartar et al., 1993; Jarvis et al., 2008), plant (Sterling, 1989; Kusnadi et al., 1997), and insect systems (Maiorella et al., 1988; Morrow, 2007), are available. In these organisms, insect larvae expression systems (Mathavan et al., 1995; Sumathy et al., 1996) as well as insect cells (Possee, 1997; Kost et al., 1999) are among the most widely used systems for the routine production of recombinant proteins. The most commonly used vector systems for recombinant protein expression in insects are baculoviruses such as Autographa californica nuclear polyhedrosis virus (Ac- NPV) and Bombyx mori nuclear polyhedrosis virus (BmNPV), which display host specificity for infection (Mori et al., 1995; Motohashi et al., 2005). The use of insect larvae and pupae as biofactories for heterologous protein production has been touted as a substitute for cell culture. The advantages of using the silkworm for recombinant protein production include appropriate post-translation modification, manageable body size, a manually operable culture system, the possibility of maintaining diverse strains in the stock center, low-cost, high yield, and easy production scale-up.

China, Japan, Korea, and India in Asia, and France, Italy and Bulgaria in Europe retain silkworm bioresources in national resource stock centers. By screening silkworm strains maintained at Kyushu University in Japan, Lee et al.. (2007, 2012) and Kawakami et al. (2008) discovered and reported suitable highpermissive strains for AcNPV and BmNPV, respectively. They also identified the F1 hybrid or three-way cross of BmNPV-sensitive strains that produced an amount of recombinant proteins that was equivalent to that produced by the parental strains (Lee et al., 2012). In accordance with the Nagoya Protocol, research in each country should be aimed at the preservation and the application of the biological resources. This prompted us to investigate high-permissive silkworm strains in Korean silkworm stocks that produced high levels of recombinant proteins. In Korea, at the National Academy of Agricultural Sciences (Silkworm Breeding Lab), around 340 strains are bred, mainly for sericulture, and a certain number of strains are stored at the Foundation Original Seed Production Center of each province as a supply to sericultural households.

In this study, we screened several silkworm strains by introducing luciferase-expressing recombinant BmNPV, previously constructed by Kawakami et al. (2008), into 52 silkworm larvae. We then generated a three-way cross from a selected high-permissive strain. It was found that 10 of the 52 strains screened were highly permissive to BmNPV.

In this study, a previously constructed (Kawakami et al., 2008) recombinant BmNPV strain, expressing a luciferase reporter gene, was used (Fig. 1). In order to insert the luciferase gene downstream of the polyhedrin promoter of pFastBac1 (Invitrogen, Grand Island, NY, USA), the luciferase gene was amplified from pFastBac1BmActin3 (Lee et al., 2007) using a forward primer 5'-GAGCTCATGGAAGATGCCAAAAA-3' containing a SacI restriction site and a reverse primer 5'-CCGCCCTTCTTGGCCTTAATGAGA-3' and then digested with SacI (Takara Bio Inc., Japan). The pFastBac1 was digested with NotI (Takara Bio Inc., Japan), blunt-ended by T4 DNA polymerase (Takara Bio Inc., Japan), and further digested with SacI. The resultant pFastBac1 vector was ligated to the PCR fragment digested by SacI, using T4 DNA ligase (Takara Bio Inc., Japan). BmDH10Bac cells were transformed with the donor vector pFastBac1polH-Luc (Motohashi et al., 2005) by Tn7 transposase-mediated transposition. The recombinant BmNPV/BmA3-Luc bacmid was isolated with the alkaline lysis method and used for transfection of BmN4 cells (1.5×10⁵ cells) by using the FuGENE HD transfection reagent (Promega, Madison, WI, USA). BmN4 cells were then incubated at 25°C in TNM-FH insect medium (Wilgene, Korea) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA) for 4 days, after which the supernatant was collected and stored at 4°C after three iterations to achieve final viral titers of 0.5 × 10⁵ plaque-forming units (pfu) per milliliter.

[Fig. 1.] Screening of domestic silkworms using BmNPV. A, Selection of the high-and low-permissive strains for production of the firefly luciferase among 52 different silkworm strains. The luciferase activity of 10 high- (black), 5 low- (gray), and the medium-permissive strains (white) is expressed as relative light units (RLU) per μg cell protein. NC, Jam125 x Jam126 hybrid strain infected with PBS as negative control; Vertical bar is ± standard deviation. B, Schematic structure of BmNPV vector (BmNPV/polH·Luc) carrying the luciferase gene used in this study. Tn7R and L, right and left site for transposition site, respectively; polH, polyhedrin promoter sequences; B1 and B2, sitespecific recombinant sequences after LR reaction; polyA, simian virus 40 polyA sequences.

The experimental silkworm larvae comprised the larvae of 52 strains and, as a control strain, the F1 hybrid strain 125 × 126 (a certified strain used by farmers during summer and fall), supplied by the Gyeongsangbuk-do and the Kangwon-do Foundation Original Seed Production Center (Sericulture and Entomology Experiment Station), respectively. Details regarding these 52 silkworm strains are described in the National Academy of Agricultural Sciences ‘Genebank Information Center' (http:// www.genebank.go.kr/). The nine hybrids used in this study were produced by randomly crossing lines with high-permissive strains that were selected through the third screening in this study and were kept in the Kangwon-do Foundation Original Seed Production Center. Silkworms were reared on fresh mulberry leaves at 25°C–27°C and fifth-instar larvae were injected using a syringe (Hamilton Co., Reno, NV, USA) into the hemocoel, with 20 μL BmNPV/BmA3-Luc baculovirus (0.5 × 10⁵ pfu/mL). Hemolymph of the silkworm larvae was collected at 4 days post injection (d.p.i), and 1% phenylthiourea (Sigma-Aldrich, St. Louis, MO, USA) was added to prevent melanization. After the collection of hemolymph, the larvae were dissected and stored at −70°C prior to experimentation.

The collected hemolymph was centrifuged at 3,000 rpm for 10 min to remove the supernatant, and the hemocytes were washed in phosphate buffered saline (PBS, pH 7.3) and then resuspended for approximately 10 min in 120 μL lysis buffer containing 25 mM Tris phosphate (pH 7.8), 2 mM 1,2-diaminecyclohexane-N,N,N',N'-tetraacetic acid (DTT), 2 mM 1,2- cyclohexanediaminetetraacetic acid monohydrate (CDTA), 10% glycerol, and 1 % Triton X-100. In total, 100 μL of the lysed solution was added to an equal volume of a substrate solution containing beetle luciferin, and luciferase activity was measured with a GloMax Luminometer (Promega, USA). Data were expressed in relative light units (RLU) per microgram of total cell proteins.

Total RNA was isolated from the fat body of each silkworm strain after the luciferase assay, using TRIzol (Invitrogen, Grand Island, NY, USA), and eluted with RNase–free water. For semi- and quantitative reverse-transcription PCR (RT-PCR) analysis, 0.5 μg total RNA from each silkworm strain was reverse transcribed into cDNA using a first cDNA synthesis kit (Toyobo, Japan). The 20 μL reaction mixture, containing 0.5 μg total RNA, 4 μL 5X reverse transcriptase buffer, 2 μL 10 mM dNTPs, 1μL 10 U/μl RNase inhibitor, 1 μL 10 pmol/μL oligo (dT)₂₀ primer, and 1μL ReverTra Ace^® reverse transcriptase, was incubated at 42°C for 20 min. Subsequently, 1μL of each firststrand cDNA product was used for semi- and quantitative realtime RT-PCR. Each 50μL reaction mixture of semi-RT PCR contained 0.5 μL 1^st cDNA, 0.25 μL 5 U/μL TaKaRa Ex Taq polymerase (Takara Bio Inc., Japan), 5 μL 10X Ex Taq buffer, 4 μL dNTP mixture, and 10 pmol each 5′ and 3′ primers. The thermal cycling profile was as follows: 94°C for 5 min, 27 cycles of 94°C; for 30 s, 57°C for 30 s, and 72°C for 30 s, followed by a final extension at 72°C for 5 min (Takara Bio Inc., Japan). Each PCR product was electrophoresed in a 1.5% agarose gel in Tris acetate EDTA (TAE) buffer. Next, real-time PCR was performed according to SYBR Premix Ex Taq (Takara Bio Inc., Japan) in the Applied Biosystems 7500 Real-Time PCR system (Applied Biosystems, Grand Island, NY, USA). The thermal cycling profile was as follows: denaturation at 94°C for 30 s, followed by 40 cycles of 95°C for 5 s, 60°C for 34 s. The B. mori 18S rRNA gene was used as an internal control. The forward and reverse primers for semi- and quantitative real-time RT-PCR were 5'- GTACACCTTCGTGACTTCCCATTT-3′ and 5′-AATCCGGTACTGCCACTACTGTTC-3′, respectively, and were based on the luciferase sequence. The forward and reverse primers used for 18S rRNA (endogenous control) were 5′-GCCTGCGGCTTAATTTGACT-3′ and 5′-CAACTAAGAACGGCCATGCA-3′, respectively.

The recovered fat bodies (0.3 g fat body can be harvested from each silkworm larva) were suspended in 1 mL homogenization buffer (0.5 M Tris buffer containing 0.5 M NaCl, 1% NP40, 5% complete, pH 8.0) and homogenized using a Vibra-Cell homogenizer (Sonics, USA) for 5 min at °C. The homogenate was ultracentrifuged at 14,000 rpm for 20 min at 4 °C, the supernatant was re-centrifuged and used for western blotting and ELISA analysis. The concentrations of the purified proteins were determined using the bicinchoninic acid (BCA) method, using bovine serum albumin as a standard (Smith et al., 1985).

The fat body samples from silkworm larvae were denatured in 4x Laemmli sample buffer and resolved on 10% SDS-PAGE. After electrophoresis, proteins were transferred onto PVDF membranes for western blotting (Millipore, USA) and the membranes were blocked for 1 h at room temperature (RT) in TBS-0.1% Tween 20 (TBST) with 5 % skim milk, washed with TBST, and then incubated at room temperature for 1 h using antiluciferase antibody (Rochland, USA). The membranes were then washed five times with TBST, and incubated with anti-goat HRP conjugate (Rochland, USA) for 1 h. After extensive washing with TBST, protein bands were detected using the ECL western blotting detection system on Hyperfilm ECL Films (Amersham, USA).

The luciferase expressed in silkworm larvae was quantified using the ELISA indirect protocol modified (Abcam, USA) with luciferase antibody. First, the diluted proteins from silkworm fat body were added to a Nunc plate (Thermo Scientific, USA), incubated for 1 h at room temperature (RT) and washed three times with TBST. A total of 200 μL TBST with 1 % bovine serum albumin (BSA) as blocking solution was added to the protein-coated plate wells, followed by incubation for 30 min at RT. The wells were washed three times with TBST. Then, 100 μL per well of 10 μg/mL firefly anti-luciferase solution were added as a coating antibody, the wells were incubated for 1 h at RT and then washed with TBST. As a second antibody, 100 μL goat-HRP conjugate diluted 5,000 times in TBST were added to each well, incubated for 1 h and washed three times with TBST buffer. A total of 100 μL of 3,3′,5,5′- tetramethylbenzidine substrate (TMB in 100 mM citric acid with 0.02 % of hydrogen peroxidase) was added to each well and left at RT for blue color development (KPL, USA). The reaction was stopped by the addition of 100 μL of 2 M H₂SO₄ solution. The optical density (OD) of the samples was measured at a wavelength of 450 nm. Firefly luciferase reference serum (Sigma, USA, 1 mg/mL) was used as a standard for the calibration of luciferase.

In previous studies, Lee et al. (2007) and Kawakami et al. (2008) identified a d17 strain which was highly effective for foreign protein expression by AcNPV and BmNPV, respectively, in silkworm strains stocked at Kyushu University in Japan. In order to identify silkworm strains stored in Korea which are of use for the BmNPV expression system, 52 strains of the Gyeongsangbuk-do Foundation Original Seed Production Center (Sericulture and Entomology Experiment Station) were selected (Fig. 1A) and expression of luciferase as the target protein by BmNPV/polH·Luc was measured (Fig. 1B), as described by Kawakami et al. (2008). To express the luciferase in silkworm larvae, the recombinant BmNPV/polH·Luc was constructed as described in Materials and Methods (Fig. 1B). Recombinant baculovirus was successfully generated through transfection of BmN cells (1.5×10⁵) with BmNPV/polH·Luc DNA. Expression and activity of recombinant luciferase in BmN cells were confirmed by western blot and luciferase assay, respectively, at 4 d.p.i (data not shown). Using propagated BmNPV/polH·Luc in BmN cells, 52 silkworm strains were then screened for high permissiveness to BmNPV/polH·Luc (Fig. 1A). On day 1 of the fifth instar, larvae of the 52 strains were infected with 0.5 × 10⁵ pfu of BmNPV/polH·Luc each, and luciferase activity was measured 4 d.p.i. In accordance with the results obtained, we then divided all tested silkworm strains into 3 groups, namely the high-, medium-, and low-permissive strains. The high-permissive group had a highest RLU value of 46.9×10⁶ RLU and the low-permissive group a lowest value of 1.4×10⁶ RLU. Based on our results, we set a baseline value of >20×10⁶ RLU for the high-permissive group, of 5.0×10⁶ to 20×10⁶ RLU for the medium-permissive strains, and a cutoff at <5.0×10⁶ RLU for the low-permissive group. According to the above assumptions, the 52 silkworm strains tested were divided into 10 high-permissive strains, 37 medium-permissive strains, and 5 low-permissive strains (Fig. 1A). Strains included in the high-permissive group were Jam313, Rok1042, L864, JangSaJang, GalWon, Jam153, HanSungJeBeuRa, Jam143, C SuGang, and Lemon, and low-permissive strains were BY, Jam315, LY, Chung7, and Chung14 (Table 1). Among the 10 high-permissive strains, the Jam313 strain exhibited an up to about 40-fold higher luciferase activity than the low-permissive strains (Fig. 1A). Because of limited access to strains stocked in Gyeongsangbuk-do, this result may be insufficient to account for Jam313 as the most productive strain in Korea's silkworm stocks. These results indicate that the Jam313 strain, whose luciferase expression was the highest among the strains tested, was moderately suitable for the BmNPV expression system.

[Table 1.] Summary of the silkworm host showing high- and low-activity of BmNPV/polH·Luc in silkworm used in this study

Summary of the silkworm host showing high- and low-activity of BmNPV/polH·Luc in silkworm used in this study

To further confirm the luciferase activity in selected high- and low-permissive strains, crude preparations of individuals (n = 4) of each strain were analyzed via 10% SDS-PAGE and subjected to western blot (Fig. 2A) and ELISA (Fig. 2C) analysis using a firefly luciferase antibody. As shown in Fig. 2A, 10 high-permissive strains (left panels) showed higher expression levels of the luciferase protein, isolated from larval fat body, than 5 low-permissive strains (right panel). The expression of luciferase was confirmed through detection of a predicted band size of around 62 kDa. It was further confirmed by ELISA analysis by using the same luciferase antibody (Fig. 2C). The expression level of luciferase in all 10 high-permissive strains was higher than that in the low-permissive group, and, as expected, results derived from both luciferase activity and western blot analysis were found to be of a highly similar pattern (Fig. 2A, Fig. 2B, and Fig. 2C), while individuals of each strain exhibited slight variations in their level of luciferase expression.

[Fig. 2.] Comparison of luciferase protein and RNA expression levels between 10 high- and 5 low-permissive strains. Luciferase protein in supernatants of larvae fat body homogenates was detected by western blotting (A) and ELISA (C) using a luciferase antibody. Transcription expression, after cDNA synthesis and RNA isolation from fat body, was detected by semi- (B) and quantitative real-time PCR (D) using luciferase specific primer. 18s rRNA was used as an endogenous control. 1, 2, 3, and 4, individual samples of each strain.

To characterize the level of luciferase gene transcription, we employed semi-quantitative and quantitative real time PCR (Fig. 2B and 2D). The results showed no significant correlation between the level of luciferase gene transcription and the allocation of strains to the high- and low-permissive groups. Luciferase gene transcription levels of HanSungJeBeuRa and Lemon as high-permissive strains and of LY and Chung14 as low-permissive strains were much higher than that of Jam313, which had shown the highest luciferase activity (Fig. 2D). Whether levels of transcription and translation fluctuate in their correlation remains to be discovered. Further reciprocity studies using the recombinant counterparts may clarify the issue.

In Korea, the commercially available strains, such as Jam123 x Jam124, Jam125 x Jam126, Jam125 x Jam140, etc., are crossed at the Foundation Original Seed Production Center annually and then widely used by sericultural farm households. Because of the genetic weakness of pure breeds, we conducted F1 hybrid and three-way crossing using highpermissive strains for supply convenience. By three-way crossing, 9 combinations of high-permissive strains were prepared with the high-permissive strain Jam313 and the low-permissive strain Chung7 as control strains (Table 2). Among the 10 high-permissive strains, we hybridized using Jam313, Rok1042, L864, JangSaJang, HanSungJeBeuRa, and GlaWon with high levels in luciferase activity. Three-way combination strains were lower in luciferase activity than Jam313, tested as high-permissive control (HPC), and significantly higher in luciferase activity than Chung7, tested as low-permissive control (LPC, Fig. 3A). The luciferase expression was further investigated in 9 three-way combination strains, high-, and low-permissive controls, by western blot and ELISA (Fig. 3B and 3C). Both methods clearly demonstrated that the luciferase expression level was high in Jam313 and low in Chung7, and these results are in agreement with the respective luciferase activities (Fig. 3). Previous reports have shown that the F1 hybrid or three-way cross of the BmNPV sensitive strains have produced an amount of recombinant proteins equivalent to that of their parental strains (Lee et al., 2012). Because we did not perform luciferase activity assays on F1 hybrid strains, this result implied that three-way combination strains are less affected by heterosis of F1 hybrid strains.

[Table 2.] List of three ways cross produced by random combinations with high-permissive strains

List of three ways cross produced by random combinations with high-permissive strains

[Fig. 3.] Expression of the recombinant luciferase protein in Jam313 as a high-permissive control strain (HPC), Chung 7 as a low-permissive control strain (LPC), and 9 F1 hybrids as two-way cross hybrids of high-permissive strains. A, luciferase activity; B and C, ELISA and western blotting with a luciferase antibody, respectively; A-H, refer to the Table 2; HPC, Jam313 as high-permissive control; NC, 125x126 hybrid (certificated strain for farmers, used in summer and fall) injected PBS as negative control. *, strain highest in luciferase activity and expression among the strains tested

This study suggested that meticulous selection of a more suitable strain is required for more efficient production of heterologous protein, through the application of recombinant bacmid DNA derived from domestic BmNPV. In order to choose the right expression system for recombinant protein production, protein quality, functionality, production speed and yield are the most important factors to consider (Demain and Vaishnav, 2009). The silkworm expression system by BmNPV has been used as a bioreactor for recombinant protein production for two decades (Maeda et al., 1985) and will be abundantly available for the production of therapeutically important proteins, vaccines, and biomaterials in the future. We expect that more laboratories in Korea will be interested in its research and utilization.