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

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  • ABSTRACT

    Recombinant proteins can be generated quickly and easily in large amounts and at low-cost in silkworm larvae by using Bombyx mori nuclear polyhedrosis virus (BmNPV). We searched for high-permissive silkworm strains that have high production levels of heterologous proteins and are thus suitable for use as biofactories. In this study, we performed the analysis using a BmNPV vector expressing luciferase as a marker, and we confirmed protein expression by evaluating luciferase activity, determined by western blotting and luciferase ELISA, and confirmed transcription expression by semi- and quantitative real time PCR. For the selection of host silkworm strains, we first chose 52 domestic BmNPV sensitive strains and then identified 10 high-permissive and 5 low-permissive strains. In addition, to determine which hybrid of the high-permissive strains would show heterosis, nine strains derived through three-way crossing were tested for luciferase activity by western blotting, and luciferase ELISA. We found a correlation between luciferase activity and luciferase protein expression, but not transcription. There was no noticeable difference in protein expression levels between Jam313 as the high-permissive control strain and the three-way hybrid strains; however, the three-way cross strains showed lower luciferase activity compared with Jam313. In this study, luciferase protein production in the larvae of 52 domestic silkworm strains was elucidated using BmNPV.


  • KEYWORD

    silkworm , luciferase , recombinant protein , BmNPV

  • Introduction

    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.

    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 (1.5×105 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.

      >  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 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.

      >  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 1,2- 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.

      >  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 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 1st 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.

      >  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 °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.

    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 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.

    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.

    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.

      >  Comparison of luciferase yields in purebred and hybrid strains

    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.

    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.

  • 1. Christensen T, Woeldike H, Boel E, Mortensen SB, Hjortshoej K, Thim L 1988 High level expression of recombinant genes in Aspergillus oryzae [Bio/Technology] Vol.6 P.1419-1422 google doi
  • 2. Cregg JM, Vedvick TS, Raschke WC 1993 Recent advances in the expression of foreign genes in Pichia pastoris [Bio/Technology] Vol.11 P.905-910 google doi
  • 3. Dartar RV, Carwright T, Rosen CG 1993 Process economics of animal cell and bacterial fermentations: a case study analysis of tissue plasminogen activator [Bio/Technology] Vol.11 P.349-357 google doi
  • 4. Demain AL, Vaishnav P 2009 Production of recombinant proteins by microbes and higher organisms [Biotechnology Advances] Vol.27 P.297-306 google doi
  • 5. Goodrick JC, Xu M, Finnegan R, Schilling BM, Schiavi S, Hoppe H, Wan NC 2001 High-level expression and stabilization of recombinant human chitinase produced in a continuous constitutive Pichia pastoris expression system [Biotech Bioeng] Vol.74 P.492-497 google doi
  • 6. Jarvis LM 2008 A technology bet. DSM's pharma product unit leverages its biotech strength to survive in a tough environment [Chem Eng News] Vol.86 P.30-31 google doi
  • 7. Kawakami N, Lee JM, Mon H, Kubo Y, Banno Y, Kawaguchi Y, Maenaka K, Park EY, Kusakabe t 2008 Efficient protein expression in Bombyx mori larvae of the strain d17 highly sensitive to B. mori nucleopolyhedrovirus [Mol Biotechnol] Vol.40 P.180-185 google doi
  • 8. Kost TA, Condreay JP 1999 Recombinant baculoviruses as expression vectors for insect and mammalian cells [Curr. Opin. Biotechnol] Vol.10 P.428-433 google doi
  • 9. Kusnadi AR, Nkolovv ZL, Howard JA 1997 Production of recombinant proteins in transgenic plants: practical considerations [Biotechnol Bioeng] Vol.56 P.473-484 google doi
  • 10. Lee JM, Mon H, Takahashi M, Kawakami N, Mitsunobu H, Banno Y, Koga K, Uchino K, Kawaguchi Y, Kusakabe T 2007 Screening of high-permissive silkworm strains for efficient recombinant protein production in Autographa califormoca nuclear polyhedrosis virus (AcNPV) [Journal of Insect Biotechnology and Sericology] Vol.76 P.101-105 google
  • 11. Lee JM, Mon H, Banno Y, Iiyama K, Kusakabe T 2012 Bombyx mori Strains Useful for Efficient Recombinant Protein Production Using a Baculovirus Vector [J Biotechnol Biomaterial] Vol.S9 P.003 google
  • 12. Lunn CA, Kathju S, Wallace BJ, Kushner SR, Pigiet V 1984 Amplification and purification of plasmid encoded thioredoxin from Escherichia coli K12 [J Biol Chem] Vol.259 P.10469-10474 google
  • 13. Maiorella B, Harano D 1988 Large scale insect cell culture for recombinant protein production [Bio/Technology] Vol.6 P.1406-1409 google doi
  • 14. Mathavan S, Gautvik VT, Rokkones E, Olstad OK, Kareem BN, Maeda S, Gautvik KM 1995 High-level production of human parathyroid hormone in Bombyx mori larvae and BmN cells using recombinant baculovirus [Gene] Vol.167 P.33-39 google doi
  • 15. Maeda S, Kwwai T, Obinata M, Fujiwara H, Horiuchi T, Saeki Y, Sato Y, Furusawa M 1985 Production of human a-interferon in silkworm using a baculovirus vector [Nature] Vol.315 P.592-594 google doi
  • 16. Morrow JK 2007 Improving protein production processes [Gen Eng News] Vol.27 P.41-50 google
  • 17. Mori H, Yamao M, Nakazawa H, Sugahara S, Shirai N, Matsubara F, Sumida M, Imamura T 1995 Tranovarian transmission of a foreigh gene in the silkworm Bombyx mori, by Autogrpha californica nuclear polyhedrosis virus [Biotechnology] Vol.13 P.1005-1007 google doi
  • 18. Motohashi T, Shimojima T, Fukagawa T, Maenaka K, Park EY 2005 Efficient large-scale protein production of larvae and pupae of silkworm by Bombyx mori nuclear polyhedrosis virus bacmid system [Biochem Biophys Res Commun 21] Vol.326 P.564-569 google doi
  • 19. Possee RD 1997 Baculoviruses as expression vectors [Curr. Opin, Biotechnol] Vol.8 P.569-572 google doi
  • 20. Sreekrishna K, Nelles L, Potenz R, Cruze J, Mazzaferro P 1989 High level expression, purification, and characterization of recombinant human tumor necrosis factor synthesized in the methylotrophic yeast Pichia pastoris [Biochemsitry] Vol.28 P.4117-4125 google doi
  • 21. Sterling J 1989 Genetic systems creates plants that produce peptides [Gen Eng News] Vol.9 P.1-39 google
  • 22. Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC 1985 Measurement of protein using bicinchoninic acid [Anal. Biochem] Vol.150 P.76-85 google doi
  • 23. Sumathy S, Palhan VB, Gopinathan KP 1996 Expression of human growth hormone in silkworm larvae through recombinant Bombyx mori nuclear polyhedrosis virus [Protein Expr. Purif] Vol.7 P.262-268 google doi
  • 24. Verdoes JC, Punt PJ, Burlingame R, Bartels J, Van Dijk R, Slump E, Meens M, Joosten R, Emalfarb M 2007 A dedicated vector for efficient library construction and highthroughput screening in the hyphal fungus Chrysosporium lucknowense [Ind Biotechnol] Vol.3 P.48-57 google doi
  • 25. Weikert C, Sauer U, Bailey JE 1997 Use of a glycerol-limited, long term chemostat for isolation of Escherichia coli mutants with improved physiological properties [Microbiology] Vol.143 P.1567-1574 google doi
  • 26. Wong MS, Wu S, Causey TB, Bennett GN, San K-Y 2008 Reduction of acetate accumulation in Escherichia coli cultures for increased recombinant protein production [Metab Eng] Vol.10 P.97-108 google doi
  • 27. Ward PP, Piddlington CS, Cunningham GA, Zhou X, Wyatt RD 1995 A system for production of commercial quantities of human lactoferrin: a broad spectrum natural antibiotic [Bio/Technology] Vol.13 P.498-503 google doi
  • [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.
    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.
  • [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
  • [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.
    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.
  • [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
    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