title: High-level expression of orange fluorescent protein in the silkworm larvae by the Bac-to-Bac system.
abstract: 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.
authors.affiliation: Institute of Sericulture and Apiculture, College of Animal Sciences, Zhejiang University, Hangzhou 310029, P.R. China.
authors.forename: Jian-Mei
authors.initials: JM
authors.lastname: Liu
authors.forename: Wan Chi Cheong
authors.initials: WC
authors.lastname: David
authors.forename: Denis Tsz-Ming
authors.initials: DT
authors.lastname: Ip
authors.forename: Xing-Hua
authors.initials: XH
authors.lastname: Li
authors.forename: Guang-Li
authors.initials: GL
authors.lastname: Li
authors.forename: Xiao-Feng
authors.initials: XF
authors.lastname: Wu
authors.forename: Wan-Fu
authors.initials: WF
authors.lastname: Yue
authors.forename: Chuan-Xi
authors.initials: CX
authors.lastname: Zhang
authors.forename: Yun-Gen
authors.initials: YG
authors.lastname: Miao
mesh_terms: 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*
chemical_list: D008164:Luminescent Proteins