The flowchart of producing infective BmNPV expressing ten heterologous genes in silkworm larvae or pupae.

<p>The target genes are cloned into the three donor vector (pCTdual, pRADM and pUCDMIG) using usual method. The first two genes carried by pCTdual are inserted into BmBacmid through <i>I-Sce</i> I linearization and <i>red-gam</i> homologous recombination. Four genes in pRADM and the other four genes in pUCDMIG are then introduced into BmBacmid via Tn7 transposition and cre-loxp recombination, respectively. As a result, ten foreign expression cassettes and three antibiotic screening markers, as well as a GFP illumination marker are introduced into BmBacmid. The invasive and DAP auxotrophic <i>E. coli</i> carrying recombinant BmBacmid are injected into silkworm larvae at an appropriate dose. Consequently, recombinant BmNPV will be produced and multiple foreign genes will be expressed in green <i>B. mori</i> larvae or pupae.</p

SDS-PAGE analysis of purified VLPs of rotavirus from silkworm.

<p>5 µg VLPs was loaded on 12% SDS-PAGE gel. Lane Marker: standard protein marker. lane VLPs: purified VLPs from silkworm hemolymph. The three bands VP2, VP6 and VP7 were labelled.</p

Multiple genes expression and rotavirus-VLPs production in silkworm.

<p>(a) The larvae of six days post injection were observed using the fluorescence detection device. 1: the mock injected larvae; 2&3: the larvae injected with invasive and DAP auxotrophic <i>E. coli</i> carrying BmBacmid with six genes including <i>egfp</i>, <i>dsRed</i>, <i>eyfp</i>, <i>vp2</i>, <i>vp6</i> and <i>vp7</i> at a dose of 8.0×10⁸ cells per larva. (b) The hemolymph from a red larva was observed using laser confocal microscope. The images of hemocytes were taken at the bright (trans) channel (I), GFP (515 nm) detection channel (II), DsRed (590 nm) channel (III) and YFP (530 nm) channel (IV). (c) Western blot analysis of the hemolymph from the red larvae using anti-VP2, anti-VP6 and anti-VP7 rabbit antiserums. (d) The EM image of hemocytes collected from red larvae. R: the round Rotavirus-VLPs; B: the rod shape baculovirus particle, bar = 100 nm.</p