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Advanced Protein Expression Using Bombyx mori Nucleopolyhedrovirus (BmNPV) Bacmid in Silkworm

  • Tatsuya Kato
  • Enoch Y. Park
Chapter
Part of the Entomology in Focus book series (ENFO, volume 4)

Abstract

An expression system using a Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid has been developed and used for efficient expression of protein using silkworm. Silkworm can sustain large-scale production of recombinant proteins due to its ease of rearing and scaling-up. Our chapter focuses on the modification of a BmNPV bacmid for a more efficient protein expression system. For example, we discuss how to achieve construction of a stronger promoter, less proteolytic degradation of expressed proteins, and a chaperone-coexpressed expression system. We describe the application of functional BmNPV particles purified from silkworm hemolymph to vaccines, antibody production, and transmembrane protein analysis. For human use, the major problem of proteins produced in silkworm is contamination by adventitious agents and protein quality. Of special concern is that N-glycosylation in silkworms is of a high-mannose type in most cases, which is different from the complex type found in mammals. We end by looking to future prospects for integration/applications of protein expression systems with silkworm biotechnology.

Keywords

Recombinant Protein Rabies Virus Classical Swine Fever Virus Recombinant Protein Production Recombinant Protein Expression 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Abbreviations

α4GnT

Human α1,4-N-acetylglucosaminyltransferase

AcMNPV

Autographa californica multiple nucleopolyhedrovirus

ADCC

Antibody-dependent cell-mediated cytotoxicity

β3GnT

Human β1,3-N-acetylglucosaminyltransferase

BiP

Human heavy chain-binding protein

Bm

Bombyx mori

BmNPV

Bombyx mori nucleopolyhedrovirus

BmNPV CP (CP)

Cysteine protease-deficient BmNPV

BmNPV CP Chi (CPChi)

Cysteine protease- and chitinase-deficient BmNPV

CNX

Human calnexin

CRT

Human calreticulin

CSFV

Classical swine fever virus

DDS

Drug delivery system

ELISA

Enzyme-linked immunosorbent assay

ERp

Human ERp57

FMDV

Foot-and-mouth disease virus

GFP

Green fluorescent protein

HA

Hemagglutinin

Hsp70

Heat shock protein 70

IgG

Immunoglobulin G

IL-5

Interleukin-5

LUV

Large unilamellar vesicle

MSG

Middle silk gland

PIT, 7d

Post-injection time, 7 days

PSG

Posterior silk gland

RSV

Rous sarcoma virus

RV

Rabies virus

SAG1

Surface antigen 1

SRB

Sulforhodamine B

SRS2

Surface antigen 1-related sequence 2

ST6Gal1

α2,6-sialyltransferase 1

VLP

Virus-like particle

References

  1. 1.
    Maeda, S., Kawai, T., Obinata, M., Fujiwara, H., Horiuchi, T., Saeki, Y., Sato, Y., & Furusawa, M. (1985). Production of human α-interferon in silkworm using a baculovirus vector. Nature, 315, 592–594.CrossRefPubMedGoogle Scholar
  2. 2.
    Miyajima, A., Schreurs, J., Otsu, K., Kondo, A., Arai, K., & Maeda, S. (1987). Use of the silkworm, Bombyx mori, and an insect baculovirus vector for high-level expression and secretion of biologically active mouse interleukin-3. Gene, 58, 273–281.CrossRefPubMedGoogle Scholar
  3. 3.
    Motohashi, T., Shimojima, T., Fukagawa, T., Maenaka, K., & Park, E. Y. (2005). Efficient large-scale protein production of larvae and pupae of silkworm by Bombyx mori nuclear polyhedrosis virus bacmid system. Biochemistry Biophysics Research Communication, 326, 564–569.CrossRefGoogle Scholar
  4. 4.
    Mathavan, S., Gautvik, V. T., Rokkones, E., Olstad, O. K., Kareem, B. N., Maeda, S., & Guatvik, K. M. (1995). High-level production of human parathyroid hormone in Bombyx mori larvae and BmN cells using recombinant baculovirus. Gene, 167, 33–39.CrossRefPubMedGoogle Scholar
  5. 5.
    Higashihashi, N., Arai, Y., Enjo, T., Horiuchi, T., Saeki, Y., Sakano, K., Sato, Y., Takeda, K., Takashina, S., & Takahashi, T. (1991). High-level expression and characterization of hepatitis B virus surface antigen in silkworm using a baculovirus vector. Journal of Virological Methods, 35, 159–167.CrossRefPubMedGoogle Scholar
  6. 6.
    Qui, P., Qin, J., Ding, Y., & Zhu, D. (1995). Yeast-prepro-alpha-factor-leader-region-directed synthesis and secretion of truncated human macrophage colony-stimulating factor in the silkworm Bombyx mori. Biotechnology and Applied Biochemistry, 21, 67–75.Google Scholar
  7. 7.
    Wu, X., Kamei, K., Sato, H., Sato, S., Takano, R., Ichida, M., Mori, H., & Hara, S. (2001). High-level expression of human acidic fibroblast growth factor and basic fibroblast growth factor in silkworm (Bombyx mori L.) using recombinant baculovirus. Protein Expression and Purification, 21, 192–200.CrossRefPubMedGoogle Scholar
  8. 8.
    Park, E. Y., Kageshima, A., Kwon, M. S., & Kato, T. (2007). Enhanced production of secretory beta1,3-N-acetylglucosaminyltransferase 2 fusion protein into hemolymph of Bombyx mori larvae using recombinant BmNPV bacmid integrated signal peptide. Journal of Biotechnology, 129, 681–688.CrossRefPubMedGoogle Scholar
  9. 9.
    Suzuki, T., Kanaya, T., Okazaki, H., Ogawa, K., Usami, A., Watanabe, H., Kadono-Okuda, K., Yamakawa, M., Sato, H., Mori, H., Takahashi, S., & Oda, K. (1997). Efficient protein production using a Bombyx mori nuclear polyhedrosis virus lacking the cysteine proteinase gene. The Journal of General Virology, 78, 3073–3080.CrossRefPubMedGoogle Scholar
  10. 10.
    Ishihara, K., Satoh, I., Nittoh, T., Kanaya, T., Okazaki, H., Suzuki, T., Koyama, T., Sakamoto, T., Ide, T., & Ohuchi, K. (1999). Preparation of recombinant rat interleukin-5 by baculovirus expression system and analysis of its biological activities. Biochimica et Biophysica Acta, 1451, 48–58.CrossRefPubMedGoogle Scholar
  11. 11.
    Hiyoshi, M., Kageshima, A., Kato, T., & Park, E. Y. (2007). Construction of a cysteine protease deficient Bombyx mori multiple nucleopolyhedrovirus bacmid and its application to improve expression of a fusion protein. Journal of Virological Methods, 144, 91–97.CrossRefPubMedGoogle Scholar
  12. 12.
    Park, E. Y., Abe, T., & Kato, T. (2008). Improved expression of fusion protein using a cysteine-protease- and chitinase-deficient Bombyx mori (silkworm) multiple nucleopolyhedrovirus bacmid in silkworm larvae. Biotechnology and Applied Biochemistry, 49, 135–140.CrossRefPubMedGoogle Scholar
  13. 13.
    Manohar, S. L., Kanamasa, S., Nishina, T., Kato, T., & Park, E. Y. (2010) Enhanced gene expression in insect cells and silkworm larva by modified polyhedrin promoter using repeated burst sequence and very late transcriptional factor-1. Biotechnology and Bioengergy, 107.Google Scholar
  14. 14.
    Kato, T., Manohar, S. L., Kanamasa, S., Ogata, M., & Park, E. Y. (2012). Improvement of the transcriptional strength of baculovirus very late polyhedrin promoter by repeating its untranslated leader sequences and coexpression with the primary transactivator. Journal of Bioscience and Bioengineering, 113, 694–696.CrossRefPubMedGoogle Scholar
  15. 15.
    Ishiyama, S., & Ikada, M. (2010). High-level expression and improved folding of proteins by using the vp39 late promoter enhanced with homologous DNA regions. Biotechnological Letters, 32, 1637–1647.CrossRefGoogle Scholar
  16. 16.
    Nakajima, M., Kato, T., Kanamasa, S., & Park, E. Y. (2009). Molecular chaperone-assisted production of human alpha-1,4-N-acetylglucosaminyltransferase in silkworm larvae using recombinant BmNPV bacmids. Molecular Biotechnology, 43, 67–75.CrossRefPubMedGoogle Scholar
  17. 17.
    Dojima, T., Nishina, T., Kato, T., Uno, T., Yagi, H., Kato, K., Ueda, H., & Park, Y. (2010). Improved secretion of molecular chaperone-assisted human IgG in silkworm, and no alteration in their N-linked glycan structures. Biotechnology Progress, 26, 232–238.PubMedGoogle Scholar
  18. 18.
    Theilmann, D. A., & Stewart, S. (1992). Molecular analysis of the trans-activating IE-2 gene of Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus. Virology, 187, 84–96.CrossRefPubMedGoogle Scholar
  19. 19.
    Higgins, M. K., Demir, M., & Tate, C. G. (2003). Calnexin coexpression and the use of weaker promoters increase the expression of correctly assembled Shaker potassium channel in insect cells. Biochim Biophys Acta, 1610, 124–132.CrossRefPubMedGoogle Scholar
  20. 20.
    Hsu, T. A., & Betenbaugh, M. J. (1997). Coexpression of molecular chaperone BiP improves immunoglobulin solubility and IgG secretion from Trichoplusia ni insect cells. Biotechnology Progress, 13, 96–104.CrossRefPubMedGoogle Scholar
  21. 21.
    Kato, T., Murata, T., Usui, T., & Park, E. Y. (2005). Improvement of the production of GFPuv-beta1,3-N-acetylglucosaminyltransferase 2 fusion protein using a molecular chaperone-assisted insect-cell-based expression system. Biotechnology and Bioengineering, 89, 424–433.CrossRefPubMedGoogle Scholar
  22. 22.
    Zhang, L., Wu, G., Tate, C. G., Lookene, A., & Olivecrona, G. (2003). Calreticulin promotes folding/dimerization of human lipoprotein lipase expressed in insect cells (Sf21). The Journal of Biological Chemistry, 278, 29344–29351.CrossRefPubMedGoogle Scholar
  23. 23.
    Kajikawa, M., Sasaki, K., Wakimoto, Y., Toyooka, M., Motohashi, T., Shimojima, T., Takada, S., Park, E. Y., & Maenaka, K. (2009). Efficient silkworm expression of human GPCR (nociceptin receptor) by a Bombyx mori bacmid DNA system. Biochemical and Biophysical Research Communications, 385, 375–379.CrossRefPubMedGoogle Scholar
  24. 24.
    Muraki, M., & Honda, S. (2010). Efficient production of human Fas receptor extracellular domain-human IgG1 heavy chain Fc domain fusion protein using baculovirus/silkworm expression system. Protein Expression and Purification, 73, 209–216.CrossRefPubMedGoogle Scholar
  25. 25.
    Kato, T., Kajikawa, M., Maenaka, K., & Park, E. Y. (2010). Silkworm expression system as a platform technology in life science. Applied Microbiology and Biotechnology, 85, 459–470.CrossRefPubMedGoogle Scholar
  26. 26.
    Du, D., Kato, T., Nabi, A. H., Suzuki, F., & Park, E. Y. (2008). Expression of functional human (pro)renin receptor in silkworm (Bombyx mori) larvae using BmMNPV bacmid. Biotechnology and Applied Biochemistry, 49, 195–202.CrossRefPubMedGoogle Scholar
  27. 27.
    Deo, V. K., Yoshimatsu, K., Otsuki, T., Dong, J., Kato, T., & Park, E. Y. (2013). Display of Neospora caninum surface protein related sequence 2 on Rous sarcoma virus-derived gaga protein virus-like particles. Journal of Biotechnology, 165, 69–75.CrossRefPubMedGoogle Scholar
  28. 28.
    Chen, J., Wu, X. F., & Zhang, Y. Z. (2006). Expression, purification and characterization of human GM-CSF using silkworm pupae (Bombyx mori) as a bioreactor. Journal of Biotechnology, 123, 236–247.CrossRefPubMedGoogle Scholar
  29. 29.
    Kato, T., Thompson, J. R., & Park, E. Y. (2013). Construction of new ligation-independent cloning vectors for the expression and purification of recombinant proteins in silkworms using BmNPV bacmid system. PLoS One, 8, e64007.CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Wang, Y., Wu, X., Liu, G., Cao, C., Huang, H., Xu, Z., & Liu, J. (2005). Expression and porcine lactoferrin by using recombinant baculovirus in silkworm, Bombyx mori L., and its purification and characterization. Applied Microbiology and Biotechnology, 69, 385–389.CrossRefPubMedGoogle Scholar
  31. 31.
    Zhou, N., Zhang, Y., Jing, W., Li, Z., & Wu, X. (1995). High expression of HBV S gene in Bombyx mori cell culture and in silkworms. Chinese Journal of Biotechnology, 11, 149–156.PubMedGoogle Scholar
  32. 32.
    Dojima, T., Nishina, T., Kato, T., Uno, T., Yagi, H., Kato, K., & Park, E. Y. (2009). Comparison of the N-glycosylation of human beta1,3-N-acetylglucosaminyltransferase 2 expressed in insect cells and silkworm larvae. Journal of Biotechnology, 143, 27–33.CrossRefPubMedGoogle Scholar
  33. 33.
    Ogata, M., Nakajima, M., Kato, T., Obara, T., Yagi, H., Kato, K., Usui, T., & Park, E. Y. (2009). Synthesis of sialoglycopolypeptide for potentially blocking influenza virus infection using a rat alpha 2,6-sialyltransferase expressed in BmNPV bacmid-injected silkworm larvae. BMC Biotechnology, 9, 54.CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Berger, I., Fizgerald, D. J., & Richimond, T. J. (2004). Baculovirus expression system for heterologous multiprotein complexes. Nature Biotechnology, 22, 1583–1587.CrossRefPubMedGoogle Scholar
  35. 35.
    Yao, L., Wang, S., Su, S., Yao, N., He, J., Peng, L., & Sun, J. (2012). Construction of a baculovirus-silkworm multigene expression system and its application on producing virus-like particles. PLoS One, 7, e32510.CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Zhou, Y., Chen, H., Li, X., Wang, Y., Chen, K., Zhang, S., Meng, X., Lee, E. Y., & Lee, M. Y. (2011). Production of recombinant human DNA polymerase delta in Bombyx mori bioreactor. PLoS One, 6, e22224.CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Fraser, M. J., Ciszczon, T., Elick, T., & Bauser, C. (1996). Precise excision of TTAA-specific lepidopteran transposons piggybac (IFP2) and tagalong (TFP3) from the baculovirus genome in cell lines from two species of Lepidoptera. Insect Molecular Biology, 5, 141–151.CrossRefPubMedGoogle Scholar
  38. 38.
    Tamura, T., Thibert, C., Royer, C., Kanda, T., Abraham, E., Kamba, M., Komoto, N., Thomas, J. L., Mauchamp, B., Chavancy, G., Shirk, P., Fraser, M., Prudhomme, J. C., & Couble, P. (2000). Germline transformation of the silkworm Bombyx mori L. using a piggybac transposon-derived vector. Nature Biotechnology, 18, 81–84.CrossRefPubMedGoogle Scholar
  39. 39.
    Tomita, M. (2011). Transgenic silkworms that weave recombinant proteins into silk cocoons. Biotechnological Letters, 33, 645–654.CrossRefGoogle Scholar
  40. 40.
    Kurihara, H., Sezutsu, H., Tamura, T., & Yamada, K. (2007). Production of an active feline interferon in the cocoon of transgenic silkworms using the fibroin H-chain expression system. Biochemical and Biophysical Research Communications, 355, 976–980.CrossRefPubMedGoogle Scholar
  41. 41.
    Royer, C., Jalabert, A., Da Rocha, M., Grenier, A. M., Mauchamp, B., Couble, P., & Chavancy, G. (2005). Biosynthesis and cocoon-export of a recombinant globular protein in transgenic silkworms. Trangenic Research, 14, 463–472.CrossRefGoogle Scholar
  42. 42.
    Tatematsu, K., Sezutsu, H., & Tamura, T. (2012). Utilization of transgenic silkworms for recombinant protein production. Journal of Biotechnology and Biomaterials, S9, 004.Google Scholar
  43. 43.
    Tomita, M., Munetsuna, H., Sato, T., Adachi, T., Hino, R., Hayashi, M., Shimizu, K., Nakamura, N., Tamura, T., & Yoshizato, K. (2003). Transgenic silkworms produce recombinant human type II procollagen in cocoons. Nature Biotechnology, 21, 52–56.CrossRefPubMedGoogle Scholar
  44. 44.
    Ogawa, S., Tomita, M., Shimizu, K., & Yoshizato, K. (2007). Generation of a transgenic silkworm that secretes recombinant proteins in the sericin layer of cocoon: production human serum albumin. Journal of Biotechnology, 128, 531–544.CrossRefPubMedGoogle Scholar
  45. 45.
    Tomita, M., Hino, R., Ogawa, S., Iizuka, M., Adachi, T., Shimizu, K., Sotoshiro, H., & Yoshizato, K. (2007). A germline transgenic silkworm that secretes recombinant proteins in the sericin layer of cocoon. Trangenic Research, 16, 449–465.CrossRefGoogle Scholar
  46. 46.
    Iizuka, M., Ogawa, S., Takeuchi, A., Nakakita, S., Kubo, Y., Miyawaki, Y., Hirabayashi, J., & Tomita, M. (2009). Production of a recombinant mouse monoclonal antibody in transgenic silkworm cocoons. FEBS Journal, 276, 5806–5820.CrossRefPubMedGoogle Scholar
  47. 47.
    Sugiura, T., Sugita, S., Imagawa, H., Kanaya, T., Ishiyama, S., Saeki, N., Uchiyama, A., Tanigawa, M., & Kuwano, A. (2001). Serological diagnosis of equine influenza using the hemagglutinin protein produced in a baculovirus expression system. Journal of Virological Methods, 98, 1–8.CrossRefPubMedGoogle Scholar
  48. 48.
    Okuda, M., Taniguchi, T., & Takamiya, O. (2012). Properties of a recombinant bovine tissue factor expressed by silkworm pupae and its performance as an Owren-type prothrombin time reagent for warfarin monitoring. Thrombosis Research, 130, 520–527.CrossRefPubMedGoogle Scholar
  49. 49.
    Li, S., Ip, D. T., Lin, H. Q., Liu, J. M., Miao, Y. G., Ke, L. J., & Wan, D. C. (2010). High-level expression of functional recombinant human butyrylcholinesterase in silkworm larvae by Bac-to-Bac system. Chemico-Biological Interactions, 187, 101–105.CrossRefPubMedGoogle Scholar
  50. 50.
    Tanaka, T. (2012). Manufacturing pharmaceutical-grade interferons using silkworm-baculovirus system. Journal of Biotechnology and Biomaterials, S9, 002.CrossRefGoogle Scholar
  51. 51.
    Usami, A., Ishiyama, S., Enomoto, C., Okazaki, H., Higuchi, K., Ikeda, M., Yamamto, T., Sugai, M., Ishikawa, Y., Hosaka, Y., Koyama, T., Tobita, Y., Ebihara, S., Mochizuki, T., Asano, Y., & Nagaya, H. (2011). Comparison of recombinant protein expression in a baculovirus system in insect cells (Sf-9) and silkworm. Journal of Biochemistry, 149, 219–227.CrossRefPubMedGoogle Scholar
  52. 52.
    Lee, K. S., Sohn, M. R., Kim, B. Y., Choo, Y. M., Woo, S. D., Yoo, S. K., Je, Y. H., Choi, J. Y., Roh, J. Y., Koo, H. N., & Jin, B. R. (2012). Production of classical swine fever virus envelope glycoprotein E2 as recombinant polyhedra in baculovirus-infected silkworm larvae. Molecular Biotchnology, 50, 211–220.CrossRefGoogle Scholar
  53. 53.
    Li, Z., Yi, Y., Yin, X., Zhang, Z., & Liu, J. (2008). Expression of foot-and mouth disease virus capsid proteins in silkworm-baculovirus expression system and its utilization as a subunit vaccine. PLoS One, 3, e2273.CrossRefPubMedPubMedCentralGoogle Scholar
  54. 54.
    Li, Z., Yi, Y., Yin, X., Zhang, Y., Liu, M., Liu, H., Li, X., Li, Y., Zhang, Z., & Liu, J. (2012). Development of a foot-and-mouth disease virus serotype A empty capsid subunit vaccine using silkworm. PLoS One, 7, e43849.CrossRefPubMedPubMedCentralGoogle Scholar
  55. 55.
    Yin, X., Li, Z., Li, J., Yi, Y., Zhang, Y., Li, X., Li, B., Yang, B., Lan, X., Li, Y., Jiao, W., Zhang, Z., & Liu, J. (2013). Rabies virus nucleoprotein expressed in silkworm pupae at high-levels and evaluation of immune responses in mice. Journal of Biotechnology, 163, 333–338.CrossRefPubMedGoogle Scholar
  56. 56.
    Otsuki, T., Dong, J., Kato, T., & Park, E. Y. (2013). Expression, purification and antigenicity of Neospora caninum-antigens using silkworm larvae targeting for subunit vaccines. Veterinary Parasitology, 192, 284–287.CrossRefPubMedGoogle Scholar
  57. 57.
    Yoshimoto, M., Otsuki, T., Itagaki, K., Kato, T., Kohsaka, T., Matsumoto, Y., Ike, K., & Park, E. Y. (2015). Evaluation of recombinant Neospora caninum antigens purified from silkworm larvae for the protection of N. caninum infection in mice. Journal of Bioscience and Bioengergy (in press).Google Scholar
  58. 58.
    Xue, R., Liu, L., Cao, G., Xu, S., Li, J., Zou, Y., Chen, H., & Gong, C. (2013). Oral vaccination of BacFish-vp6 against grass carp reovirus evoking antibody response in grass carp. Fish & Shellfish Immunology, 34, 348–355.CrossRefGoogle Scholar
  59. 59.
    Lua, L. H. L., Connors, N. K., Sainsbury, F., Chuan, Y. P., Wibowo, N., & Middelberg, A. P. J. (2014). Bioengineering virus-like particles as vaccines. Biotechnology and Bioengineering, 111, 425–440.CrossRefPubMedGoogle Scholar
  60. 60.
    Feng, H., Liang, M., Wang, H. L., Zhang, T., Zhao, P. S., Shen, X. J., Zhang, R. Z., Hu, G. Q., Gao, Y. G., Wang, C. Y., Wang, T. C., Zhang, W., Yang, S. T., & Xia, X. Z. (2011). Recombinant canine parvovirus-like particles express foreign epitopes in silkworm pupae. Veterinary Microbiology, 154, 49–57.CrossRefPubMedGoogle Scholar
  61. 61.
    Deo, V. K., Yui, M., Alam, M. J., Yamazaki, M., Kato, T., & Park, E. Y. (2014). A model for targeting colon carcinoma cells using single-chain variable fragments anchored on virus-like particles via glycosyl phosphatidylinositol anchor. Pharmaceutical Research, 31, 2166–2177.Google Scholar
  62. 62.
    Tsuji, Y., Deo, V. K., Kato, T., & Park, E. Y. (2011). Park EY (2011) Production of Rous sarcoma virus-like particles displaying human transmembrane protein in silkworm larvae and its application to ligand-receptor binding assay. Journal of Biotechnology, 155, 185–192.CrossRefPubMedGoogle Scholar
  63. 63.
    Lu, H. Y., Chen, Y. H., & Liu, H. J. (2012). Baculovirus as a vaccine vector. Bioengineered, 3, 271–274.CrossRefPubMedPubMedCentralGoogle Scholar
  64. 64.
    Paul, A., Hasan, A., Rodes, L., & Sangaralingam, P. S. (2014). Bioengineering baculoviruses as new class of therapeutics using micro and nanotechnologies: Principles, prospects and challenges. Advanced Drug Delivery Reviews, 71, 115–130.CrossRefPubMedGoogle Scholar
  65. 65.
    Kato, T., Manoha, S. L., Tanaka, S., & Park, E. Y. (2009). High-titer preparation of Bombyx mori nucleopolyhedrovirus (BmNPV) displaying recombinant protein in silkworm larvae by size exclusion chromatography and its characterization. BMC Biotechnology, 9, 55.CrossRefPubMedPubMedCentralGoogle Scholar
  66. 66.
    Kato, T., Suzuki, F., & Park, E. Y. (2011). Purification of functional baculovirus particles from silkworm larval hemolymph and their use as nanoparticles for the detection of human prorenin receptor (PRR) binding. BMC Biotechnology, 11, 60.CrossRefPubMedPubMedCentralGoogle Scholar
  67. 67.
    Kato, T., Otsuki, T., Yoshimoto, M., Itagaki, K., Kohsaka, T., Matsumoto, Y., Ike, K., & Park, E. Y. (2015). Bombyx mori nucleopolyhedrovirus displaying Neospora caninum antigens as a vaccine candidate against N. caninum infection in mice. Molecular Biotechnology, 57, 145–154.CrossRefPubMedGoogle Scholar
  68. 68.
    Lee, J. M., Mori, H., Banno, Y., Iiyama, K., & Kusakabe, T. (2012). Bombyx mori strains useful for efficient recombinant protein production using a baculovirus vector. Journal of Biotechnology and Biomatrials, 120(6), 715–719.Google Scholar

Copyright information

© Springer International Publishing Switzerland 2016

Authors and Affiliations

  1. 1.Green Chemistry Research Division, Research Institute of Science and TechnologyShizuoka UniversityShizuokaJapan

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