Expression and assembly of rotavirus-like particles in insect cells mediated by recombinant Bombyx mori MultiBac / 南方医科大学学报

ABSTRACT

<p><b>OBJECTIVE</b>To construct recombinant baculoviruses co-expressing three structural genes vp2, vp6 and vp7 of rotavirus, and assemble rotavirus-like particles (VLPs) in BmN cells.</p><p><b>METHODS</b>Human 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.</p><p><b>RESULTS</b>Three 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.</p><p><b>CONCLUSION</b>We 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.</p>