Screening of Domestic Silkworm Strains for Efficient Heterologous Protein Expression by Bombyx mori Nuclear Polyhedrosis Virus (BmNPV)

Sun Jung Jo et al. Screening of Domestic Silkworm Strains for Efficient Heterologous Protein Expression by Bombyx mori Nuclear Polyhedrosis Virus (BmNPV) 186 187 strains screened were highly permissive to BmNPV. Materials and Methods Preparation of recombinant baculovirus 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 (1.5×10 5 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 × 10 5 plaque-forming units (pfu) per milliliter. Silkworms and inoculation 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 (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 (AcNPV) 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 Int. J. Indust. Entomol. Vol. 29, No. (2), pp. 185-192 (2014) 186 187 incubated at 42°C for 20 min. Subsequently, 1μL of each firststrand cDNA product was used for semiand 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 semiand 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. Translation analysis 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 4°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 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 × 10 5 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. Analysis of luciferase activity 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 1,2cyclohexanediaminetetraacetic 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. Transcription analysis 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 semiand quantitative reverse-transcription PCR (RTPCR) 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 Sun Jung Jo et al. Screening of Domestic Silkworm Strains for Efficient Heterologous Protein Expression by Bombyx mori Nuclear Polyhedrosis Virus (BmNPV) 188 189 (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. Results and Discussion Selection of a high-permissive silkworm strain as a bioreactor by use of BmNPV In previous studies, Lee et al. (2007) and Kawakami et al. (2008) identified a d17 strain which was highly ef