High-level expression of orange fluorescent protein in the silkworm larvae by the Bac-to-Bac system.
This novel orange fluorescent protein (OFP) emits brilliant orange fluorescent light. OFP has high fluorescence quantum yield, fast maturation rate, and stability, which imply this protein should be the most favorable biotechnological tools used to investigate the function of target gene by visualizing, monitoring, and quantifying in living cells. B. mori, silkworm has been used as an important bioreactor for the production of recombinant proteins through baculovirus expression system (BES). In this paper, we used infection technique which introduced the baculovirus DNA into silkworms using a cationic lipofectin reagent instead of directly injecting the virus, and demonstrated a high-level expression of the orange fluorescent protein (OFP) gene in the Bombyx mori, silkworm larvae. When recombinant rBacmid/BmNPV/OFP DNA ranging from 50-100 ng/larval was injected, a sufficient OFP expression in hemolymph was harvested. The recombinant viruses could be obtained from the hemolymph of infected larvae and stored as seed which could be used for the large-scale expression. This procedure omitted the costly and labor-consumed insect cell culture. Further investigation of OFP should provide us with more insight in unlocking the mystery of the mechanisms of autocatalytic bioluminescence and its utilization in biotechnology.
Liu
Jian-Mei
JM
Institute of Sericulture and Apiculture, College of Animal Sciences, Zhejiang University, Hangzhou 310029, P.R. China.
David
Wan Chi Cheong
WC
Ip
Denis Tsz-Ming
DT
Li
Xing-Hua
XH
Li
Guang-Li
GL
Wu
Xiao-Feng
XF
Yue
Wan-Fu
WF
Zhang
Chuan-Xi
CX
Miao
Yun-Gen
YG
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D012831:Bombyx; D006458:Hemolymph / Q000378:metabolism; D007814:Larva / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics*; D014162:Transfection / Q000379:methods*
D008164:Luminescent Proteins