Screening of silkworm strains for efficient recombinant protein production by Autographa californica nucleopolyhedrosis virus (AcNPV)

  • cc icon
  • ABSTRACT

    Baculoviruses base vectors come to be regarded as methods for in vivo gene delivery and transient expression to the silkworm. In the case of silkworm, B. mori, two types of baculoviruses, AcNPV (Autographa californica nuclear polyhedrosis virus) and BmNPV (Bombyx mori nuclear polyhedrosis virus), are potentially applicable as vectors. Recently, AcNPV showed promising results with some silkworm strains despite different hostspecificities. We searched for a highly-permissive silkworm strain in the B. mori stocks of Kyungpook National University that could produce high levels of recombinant protein. Seventy strains were screened using the recombinant AcNPV/BmA3-Luc virus. Based on the measured luciferase activity, the strains could be divided into three groups, high-, middle-, and low-permissive strains, according to their relative recombinant protein expression levels. At 48 hours post-injection, the luciferase activity in the high-permissive strains was 500-fold greater than that of the low-permissive strains. At 72 hours post-injection, a significant elevation in luciferase activity was observed in the hemocytes of all strains. Then, based on the above results, the High Permissive Strain (HPS) S10 and the Low Permissive Strain (LPS) S39 were pick up and was carried out Dot blotting, RT-PCR and Real time PCR.


  • KEYWORD

    silkworm , Autographa californica nucleopolyhedrosis virus , Bombyx mori nuclearpolyhedrosis virus

  • 1. Guo T. Q., Wang J. Y., Guo X. Y., Wang S. P., Lu C. D 2005 Transient in vivo gene delivery to the silkworm B. mori by EGT-null recombinant AcNPV using EGFP as a reporter [Arch. Virol.] Vol.150 P.93-105 google
  • 2. Guo T. Q., Wang S. P., Guo X. Y., Lu C. D 2005 Productive infection of Autographa californica nucleopolyhedrovirus in silkworm B. mori strain Haoyue due to the absence of a host antiviral factor [Virology.] Vol.341 P.231-237 google
  • 3. Hiyoshi M., Kageshima A., Kato T., Park E. Y 2007 Construction of a cysteine protease deficient B. mori multiple nucleo bacmid and its application to improve expression of a fusion protein [J. Virol. Methods] Vol.144 P.91-97 google
  • 4. Ikeda M., Katou Y., Yamada Y., Chaeychomsri S., Kobayashi M 2001 Characterization of Autographa californica nucleopolyhedrovirus infection in cell lines from B. mori [J. Insect Biotechnol. Sericol.] Vol.70 P.49-58 google
  • 5. Ikonomou L., Schneider Y. J., Agathos S. N 2003 Insect cell culture for industrial production of recombinant proteins [Appl. Microbiol .Biotechnol.] Vol.62 P.1-20 google
  • 6. Kondo A., Maeda S 1991 Host range expansion by recombination of the baculovirus B. mori nuclear polyhedrosis virus and Autographa californica nuclear polyhedrosis virus [J. Virol.] Vol.65 P.3625-3632 google
  • 7. Lee J. M 2003 Characterization and construction of constitutive gene expression systems in B. mori, PhD thesis google
  • 8. Lee J. M., 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 californica Nuclear Polyhedrosis Virus (AcNPV) [J. Insect Biotechnol. Sericol.] Vol.76 P.101-105 google
  • 9. Luckow V. A., Summers M. D 1988 Trends in the development of baculovirus expression vectors [Bio/technology] Vol.6 P.47-55 google
  • 10. Luckow V. A., Lee S. C., Barry G. F., Olins P. O 1993 Efficient generation of infectious recombinant baculoviruses by site-specific transposon-mediated insertion of foreign genes into a baculovirus genome propagated in Escherichia coli [J. Virol.] Vol.67 P.4566-4579 google
  • 11. 1985 Production of human α-interferon in silkworm using a baculovirus vector [Nature] Vol.315 P.592-594 google
  • 12. Maeda S 1989 Expression of foreign genes in insects using baculovirus vectors [Annu. Rev. Entomol.] Vol.34 P.351-372 google
  • 13. Maeda S., Kamita S. G., Kondo A 1993 Host range expansion of Autographa californica nuclear polyhedrosis virus (NPV) following recombination of a 0.6-kilobase-pair DNA fragment originating from B. mori NPV [J. Virol.] Vol.67 P.6234-6238 google
  • 14. Miller L. K 1995 Genetically engineered insect pesticides: present and future [J. Invertebr. Pathol.] Vol.65 P.211-216 google
  • 15. Mishra S. 1998 Baculoviruses as biopesticides [Curr. Sci.] Vol.75 P.1015-1022 google
  • 16. Morris T. D., Miller L. K 1993 Characterization of productive and non-productive AcMNPV infection in selected insect cell lines [Virology] Vol.197 P.339-348 google
  • 17. Motohashi T., Shimojima T., Fukagawa T., Maenaka K., Park E. Y 2005 Efficient large-scale protein production of larvae and pupae of silkworm by B. mori nuclear polyhedrosis virus bacmid system [Biochem. Biophys. Res. Commun.] Vol.326 P.564-569 google
  • 18. O'Reilly D. R., Miller L. K., Luckow V. A 1992 Baculovirus Expression Vectors. A Laboratory Manual google
  • 19. Rahman M. M., Gopinathan K. P 2002 Analysis of host specificity of two closely related baculoviruses in permissive and nonpermissive cell lines [Virus Res.] Vol.93 P.13-23 google
  • 20. Shikata M., Shibata H., Sakurai M., Sano Y., Hashimoto Y., Matsumoto T 1998 The cdysteroid UDP-glucosyltransferase gene of Autographa californica nucleopolyhedrovirus alters the moulting and metamorphosis of a non-target insect, the silkworm, B. mori (Lepidoptera, Bombycidae) [J. Gen. Virol.] Vol.79 P.1547-1551 google
  • 21. Smith G. E., Summers M. D., Fraser M. J 1983 Production of human beta interferon in insect cells infected with a baculovirus expression vector [Mol. Cell. Biol.] Vol.3 P.2156-2165 google
  • 22. Tanaka Y 1913 A study of Mendelian factors in the silkworm, B. mori [J. Coll. Agric. Tohoku Imp] Vol.5 P.91-113 google
  • 23. Tamura T., Thibert C., Royer C., Kanda T., Abraham E., Kamba M., Komoto N., Thomas J. L., Kauchamp B., Chavancy G., Shirk P., Fraser M., Prudhomme J. C., Couble P 2000 Germline transformation of the silkworm B. mori L. using a piggybac transposon-derived vector [Nat Biotechnol.] Vol.18 P.81-84 google
  • 24. Yamao M., Katayama N., Nakazawa H., Yamakawa M., Hayashi Y., Hara S., Kamei K., Mori H 1999 Gene targeting in the silkworm by use of a baculovirus [Genes Dev.] Vol.13 P.511-516 google
  • [Table 1] This study used 70 kinds of silkworm strain and features(ft.)
    This study used 70 kinds of silkworm strain and features(ft.)
  • [Fig. 1] Construction of AcNPV/BmA3-Luc bacmid using the Bac-to-Bac baculovirus expression system.
    Construction of AcNPV/BmA3-Luc bacmid using the Bac-to-Bac baculovirus expression system.
  • [Fig. 2] Screening for the AcNPV high-permissive host range strain in 70 different silkworm. The luciferase activity achieved by the infection of the recombinant AcNPV in larval hemocyte was assayed at 48 hrs post- infection.
    Screening for the AcNPV high-permissive host range strain in 70 different silkworm. The luciferase activity achieved by the infection of the recombinant AcNPV in larval hemocyte was assayed at 48 hrs post- infection.
  • [Table 2] The results of silkworm host range against the propagation of recombinant AcNPV.
    The results of silkworm host range against the propagation of recombinant AcNPV.
  • [Fig. 3] Time-course of luciferase activity in 5th instar larva of HPS (S37) infected with 1X105 AcNPV/BmA3-Luc. A~D represents the virus injection time after 4th molting, just molting. 24, 48, 72hr, respectively.
    Time-course of luciferase activity in 5th instar larva of HPS (S37) infected with 1X105 AcNPV/BmA3-Luc. A~D represents the virus injection time after 4th molting, just molting. 24, 48, 72hr, respectively.
  • [Fig. 4] Time-course of luciferase activity in 5th instar larva of LPS (S39) infected with 1X105 AcNPV/BmA3-Luc. A~D represents the virus injection time after 4th molting, just molting, 24, 48, 72hr, respectively.
    Time-course of luciferase activity in 5th instar larva of LPS (S39) infected with 1X105 AcNPV/BmA3-Luc. A~D represents the virus injection time after 4th molting, just molting, 24, 48, 72hr, respectively.
  • [Fig. 5] Time-course of luciferase activity in 5th instar larva of HPS (S37) infected with 2X103 AcNPV/BmA3-Luc. A~D represents the virus injection time after 4th molting, just molting, 24, 48, 72hr, respectively.
    Time-course of luciferase activity in 5th instar larva of HPS (S37) infected with 2X103 AcNPV/BmA3-Luc. A~D represents the virus injection time after 4th molting, just molting, 24, 48, 72hr, respectively.
  • [Fig. 6] Time-course of luciferase activity in 5th instar larva of LPS (S39) infected with 2X103 AcNPV/BmA3-Luc. A~D represents the virus injection time after 4th molting, just molting, 24h, 48h, 72h, respectively.
    Time-course of luciferase activity in 5th instar larva of LPS (S39) infected with 2X103 AcNPV/BmA3-Luc. A~D represents the virus injection time after 4th molting, just molting, 24h, 48h, 72h, respectively.
  • [Fig. 7] Viral DNA in tissues of AcNPV/BmA3-LUC infected silkworm larvae. Total DNA extracted at the indicated times of post infection (shown on the right) from tissues, hemocyte(hemo) and fatbody(fat), of silkworm larvae. HSP and LPS in the upper indicates the high and low permissive strain, respectiviely. The amount of 0.5ug DNA was dotted and hybridized with 32P-dCTP labeled luciferase DNA. Dots on the first lane, M, indicated luciferase DNA (amounts are shown on the left) used as a standard.
    Viral DNA in tissues of AcNPV/BmA3-LUC infected silkworm larvae. Total DNA extracted at the indicated times of post infection (shown on the right) from tissues, hemocyte(hemo) and fatbody(fat), of silkworm larvae. HSP and LPS in the upper indicates the high and low permissive strain, respectiviely. The amount of 0.5ug DNA was dotted and hybridized with 32P-dCTP labeled luciferase DNA. Dots on the first lane, M, indicated luciferase DNA (amounts are shown on the left) used as a standard.
  • [Fig. 8] Expression profile of luciferase gene monitored at tissues of AcNPV/BmA3-Luc infected (A) HSP (high-permissive strain), (B) LSP (low-permissive strain) larvae by RT-PCR. Total RNA extracted at the indicated times of post infection (shown on the upper) from tissues. B. mori cytoplasmic actin3 was used as a control.
    Expression profile of luciferase gene monitored at tissues of AcNPV/BmA3-Luc infected (A) HSP (high-permissive strain), (B) LSP (low-permissive strain) larvae by RT-PCR. Total RNA extracted at the indicated times of post infection (shown on the upper) from tissues. B. mori cytoplasmic actin3 was used as a control.
  • [Fig. 9] Expression profile of luciferase gene monitored at tissues of silkworm larvae by Real-time PCR. the white box indicate the high-permissive strain (HSP) and grey box indicate the low-permissive stain (LPS). , Hemocyte; , fat body; , mid-gut; , gonad; , gonad; , silk glad. 18s RNA wre quantified by densitometric scanning.
    Expression profile of luciferase gene monitored at tissues of silkworm larvae by Real-time PCR. the white box indicate the high-permissive strain (HSP) and grey box indicate the low-permissive stain (LPS). , Hemocyte; , fat body; , mid-gut; , gonad; , gonad; , silk glad. 18s RNA wre quantified by densitometric scanning.