X. Hu, Y. Chen, C.O. Singh, S. Zhang, Y.G. Miao, Y. Zhou, J. Xu, B. Roy
Published: February 25, 2019
Genet. Mol. Res. 18(1): GMR18084
DOI: https://doi.org/10.4238/gmr18084
Cite this Article:
X. Hu, Y. Chen, C.O. Singh, S. Zhang, Y.G. Miao, Y. Zhou, J. Xu, B. Roy (2019). An improved Bac-to-Bac/BmNPV technology expressing envelope E2 glycoprotein of classical swine fever virus (CSFV) in the silkworm, Bombyx mori. Genet. Mol. Res. 18(1): GMR18084. https://doi.org/10.4238/gmr18084
About the Authors
X. Hu, Y. Chen, C.O. Singh, S. Zhang, Y.G. Miao, Y. Zhou, J. Xu, B. Roy
Corresponding Author
B. Roy
Email: roy@genomics.cn
ABSTRACT
The envelope E2 glycoprotein of classical swine fever virus (CSFV) is an immunodominant protein which neutralizes the virus. Previous trials to eradicate CSFV have been expensive, inefficient and time-consuming process without complete success. In contrast to DNA transfection into cultured cells, the efficacy of gene transduction in in vivo organ is very low because of the presence of cortical laminar structures and multilayered cells followed by quality and quantity of DNA. Therefore, to eradicate CSFs, developing an effective and inexpensive vaccine targeting E2 glycoprotein is important. Here, we reported using different quantity of DNA with lipofectamine-2000 reagents that could markedly enhance the effectiveness of gene transfer in particular experiment while we are looking for long term development of animal vaccine and an alternative strategy for large scale production of CSFV E2 glycoprotein using baculovirus (bac-to-bac) system in silkworm, Bombyx mori, L. Our results show successful expression of E2 glycoprotein in BmN cell lines and silkworm larvae. The direct injection of recombinant rBacmid/BmNPV/ E2 DNA with lipofectamine-2000 reagent infecting the silkworm larvae are varied in different groups and clear symptoms of infection were found and polyhedrons were counted by hemocytometer in individual and different batch. Confocal and electronic microscopy further revealed the expressed polyhedral, followed by SDS-PAGE and western blot further supporting our data. Our study provides an alternative strategy to produce large scale protein against CSFV. Current work to purify the E2 protein for elucidating its structure and development of vaccine is underway.
Key words: Baculovirus, Bombyx mori L, Classical swine fever virus (CSFV), E2 glycoprotein, Insect cell line.