1.Expression and assembly of rotavirus-like particles in insect cells mediated by recombinant Bombyx mori MultiBac.
Hu LONG ; Lun-guang YAO ; Shan-shan WANG ; Shin-xin CHEN ; Pei-chan TAN ; Jing-chen SUN
Journal of Southern Medical University 2010;30(7):1491-1495
OBJECTIVETo construct recombinant baculoviruses co-expressing three structural genes vp2, vp6 and vp7 of rotavirus, and assemble rotavirus-like particles (VLPs) in BmN cells.
METHODSHuman group A rotavirus was cultivated in MA104 cells, and the RNA was extracted and the three genes were obtained by RT-PCR. The PCR products were inserted into the transfer vectors pFBDM and pUCDM, respectively. A enhanced green fluorescent protein gene (egfp) driven by IE1 promoter was introduced into pFBDM to investigate the efficiency of infection. The expression baculoviruse was constructed by Tn7 and Cre-LoxP recombinant and transfected into BmN cells. The gene expression was determined by detecting 6-His tag fused into VP7 C-terminus, and the assembled VLPs were observed by transmission electron micrography.
RESULTSThree genes of rotavirus were cloned and BmMultiBac was constructed. The genes were expressed and the rotavirus-like particles assembled in BmN cells successfully as verified by ELISA and electron microscope.
CONCLUSIONWe have successfully constructed the recombinant baculovirus co-expressing the 3 structural genes of rotavirus, which provide the basis for producing protein complex containing multiple subunits and investigation of the structure of the macromolecules.
Animals ; Baculoviridae ; genetics ; metabolism ; Bombyx ; virology ; Capsid Proteins ; genetics ; Cell Line ; Gene Expression ; Genetic Vectors ; Rotavirus ; genetics ; metabolism