ABB  Vol.2 No.3 , June 2011
Molecular cloning, expression and characterization of a novel geneβ-N-acetylglucosaminidase from Bombyxmori
ABSTRACT
Previously, we have reported that a gene encoding Bombyxmoriβ-N-acetylglucosaminidase 2 (BmGlcNAcase2) has been identified differentially expressed in the midgut of Bombyxmori strain NB resistant to nucleopolyhedrovirus (BmNPV), strain 306 susceptible to NPV and a near isogenic line BC9 with similar genetic background to 306 but resistant to NPV by two-dimensional gel electrophoresis (2-DE). To get more knowledge about the relationship between β-N-acetylglucosaminidase and the resistance of NPV, in this study, the 1542 bp open reading frame of a putative bombyxmoriβ-N-acetylglucosaminidase 2 gene (BmGlcNAcase2) was amplified from a pool of bombyxmoricDNAs and inserted into the prokaryotic expression plasmid pET-30a(+).Western blotting analysis showed that BmGlcNAcase2 was expressed in hemolymph, ovary, testis, fat body, trachea, midgut and silk gland of fifth instar larvae respectively . Immunofluoresence analysis indicated that BmGlcNAcase2 was mainly located to the cytoplasm or some structure in cytoplasm.

Cite this paper
nullChang, C. , Liu, X. and Chen, K. (2011) Molecular cloning, expression and characterization of a novel geneβ-N-acetylglucosaminidase from Bombyxmori. Advances in Bioscience and Biotechnology, 2, 123-127. doi: 10.4236/abb.2011.23019.
References
[1]   Chen, H.Q., Chen, K.P., Yao, Q., Guo, Z.J. and Wang, L.L. (2007) Characterization of a late gene, ORF67 from bombyx mori nucleopolyhedrovirus. FEBS Letters, 581, 5836-5842. doi:10.1016/j.febslet.2007.11.059

[2]   Nakazawa, H., Tsuneishi, E., Ponnuvel, K.M., Furukawa, S., Asaoka, A., et al. (2004) Antiviral activity of a serine protease from the digestive juice of Bombyx mori larvae against nucleopolyhedrovirus. Virology, 321, 154-162. doi:10.1016/j.virol.2003.12.011

[3]   Ponnuvel, K.M., Nakazawa, H., Furukawa, S., Asaoka, A., Ishibashi, J., et al. (2003) A lipase isolated from the silkworm bombyx mori shows antiviral activity against nucleopolyhedrovirus. Journal of Virology, 77, 10725- 10729. doi:10.1128/JVI.77.19.10725-10729.2003

[4]   Aumiller, J.J., Hollister, J.R. and Jarvis, D.L. (2006) Molecular cloning and functional characterization of beta-N-acetylglucosaminidase genes from Sf9 cells. Protein Expression and Purification, 47, 571-590. doi:10.1016/j.pep.2005.11.026

[5]   Cattaneo, F., Pasini, M.E., Intra, J., Matsumoto, M., Briani, F., et al. (2006) Identification and expression analysis of Drosophila melanogaster genes encoding β-hexosaminidases of the sperm plasma membrane. Glycobiology, 16, 786-800. doi:10.1093/glycob/cwl007

[6]   Nagamatsu, Y., Yanagisawa, I., Kimoto, M., Okamoto, E. and Koga, D. (1995) Purification of a chitooligosaccharidolytic beta-N-acetylglucosaminidase from Bombyx mori larvae during metamorphosis and the nucleotide sequence of its cDNA. Bioscience, Biotechnology, and Biochemistry, 59, 219-225. doi:10.1271/bbb.59.219

[7]   Takahiro, O., Seiji, I., Hideki, S., Akihiro, U., Toshiki, T., et al. (2007) Molecular cloning and expression of two novel β-n-acetylglucosaminidases from silkworm bombyx mori. Bioscience, Biotechnology, and Biochemistry, 71, 1626-1635. doi:10.1271/bbb.60705

[8]   Jarvis, D.L., Wills, L., Burow, G. and Bohlmeyer, D.A. (1998) Mutational analysis of the N-linked glycans on autographa californica nucleopolyhedrovirus gp64. Journal of Virology, 72, 9459-9469.

[9]   Liu, X.Y., et al. (2010) Proteomic analysis of nucleopolyhedrovirus infection resistance in the silkworm, Bombyx mori (Lepidoptera: Bombycidae). Journal of Invertebrate Pathology, 105, 84-90.

[10]   Li, X.H., Wu, X.F., Yue, W.F., Liu, J.M., Li, G.L., et al. (2006) Proteomic analysis of the silkworm (Bombyx mori L.) hemolymph during developmental stage. Journal of Proteome Research, 5, 2809-2814. doi:10.1021/pr0603093

[11]   Kothari, H., Kumar, P. and Singh, N. (2006) Prokaryotic expression, purification, and polyclonal antibody production against a novel drug resistance gene of Leishmania donovani clinical isolate. Protein Expression and Purification, 45, 15-21. doi:10.1016/j.pep.2005.10.002

[12]   Bade, M.L. and Wyatt, G.R. (1962) Metabolic conversions during putation of the cecropia silkworm. 1. Deposition and utilization of nutrient. Biochemical Journal, 83, 470-478.

[13]   Zen, K.C., Choi, H.K., Krishnamachary, N., Muthukrishnan, S. and Kramer, K.J. (1996) Cloning, expression and hormonal regulation of an insect β-N-acetylglucosa- minidase gene. Insect Biochemistry and Molecular Biology, 26, 435-444. doi:10.1016/0965-1748(95)00111-5

 
 
Top