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High-level expression of orange fluorescent protein in the silkworm larvae by the Bac-to-Bac system

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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.

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Acknowledgments

We thank Prof. Wan Chi Cheong David, Lin huangquan, Denis Tsz-Ming Ip, Li xinghua and Li shuo for suggestions and helps in these experiments. The work was supported by the National Basic Research Program of China under grant no. 2005CB121003 and by the Hi-Tech Research and Development Program of China (No. 2006AA10A119).

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Authors and Affiliations

  1. Institute of Sericulture and Apiculture, College of Animal Sciences, Zhejiang University, Hangzhou, 310029, P.R. China

    Jian-mei Liu, Xing-hua Li, Guang-li Li, Xiao-feng Wu, Wan-fu Yue & Yun-gen Miao

  2. Department of Biochemistry, The Chinese University of Hong Kong, Hong Kong, P.R. China

    Wan Chi Cheong David & Denis Tsz-Ming Ip

  3. Institute of Insect Sciences, Zhejiang University, Hangzhou, 310029, P.R. China

    Chuan-xi Zhang

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  1. Jian-mei Liu
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  2. Wan Chi Cheong David
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  3. Denis Tsz-Ming Ip
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  4. Xing-hua Li
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  5. Guang-li Li
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  6. Xiao-feng Wu
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  7. Wan-fu Yue
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  8. Chuan-xi Zhang
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  9. Yun-gen Miao
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Correspondence to Yun-gen Miao.

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Liu, Jm., David, W.C.C., Ip, D.TM. et al. High-level expression of orange fluorescent protein in the silkworm larvae by the Bac-to-Bac system. Mol Biol Rep 36, 329–335 (2009). https://doi.org/10.1007/s11033-007-9183-2

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  • DOI: https://doi.org/10.1007/s11033-007-9183-2

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