Expression of non-structural protein 1 of Bombyx mori densovirus-3

Abstract:

We cloned the NS1 gene of BmDNV-3 into the prokaryotic expression vector pET-30a after we amplified the NS1 gene by PCR, and then we transformed the pET-30a-NS1 plasmid into BL21 star to express BmDNV-3 NS1. After we induced NS1 expression by IPTG, we used Western blot analysis to indentify the recombinant protein, the result indicated that the recombinant protein was BmDNV-3 NS1. After purification, we used NS1 to immunize New Zealand white rabbits following standard protocol to harvest anti-NS1 anti serum. On the other hand, we cloned the BmDNV-3 NS1 into pFastBacHTb-eGFP vector, and then transformed the pFastBacHTb-NS1-eGfp into BmDH10BAC to harvest recombinant bacmid genome. We obtained the recombinant virus from the cells, which was transfected by the recombinant bacmid genome using liposomes. We used the virus genome to infect Bombyx mori larvae. We observed the fluorescence in the cells and silkworm larvae at 2 days post infection, and then we used SDS-PAGE and fluorescence image analysis to identify the fusion protein. The result showed that the size of the fusion protein was not consistent with the expected size of NS1-eGFP, indicating the fusion protein was degraded randomly by the intrinsic digestive protease. To further confirm the fusion protein, we used Western blot with an anti-NS1 antibody.

Key words:Bombyx mori densovirus-3; non-structural protein 1; expression;baculovirus expression system; Western blot