Construction of an Enhanced Green Fluorescent (eGFP) Tagged Dinsect -baculovirus Transference Vector

doi:10.11924/j.issn.1000-6850.2012-1539

Abstract

Abstract: To construct an enhanced green fluorescent protein (eGFP) tagged insect-baculovirus transference system and expressed eGFP by Sf-9 cells. After gp64SP and TM/CTD gene had been amplified from bacmid by PCR, and eGFP gene had been amplified from pEGFP-C3 plasmid by PCR, the three fragments were connected together by SOE-PCR. The recombinant gene digested by Xba I/Hind Ⅲ, it was recombined into baculovirus transference vector pFastBac[PH] to create a novel pFB/LM-PH vector. E.coli DH10Bac was transformed with the pFB/LM-PH vector, a recombinant baculovirus Bv-LM-PH, was obtained with the action of Tn7 transposon. The insect Sf-9cells were infected with the recombinant baculovirus Bv-LM-PH and expressed eGFP. Meanwhile, the Sf9cells infected by the recombinant baculovirus were observed under inverted fluorescence microscope. The eGFP expression in infected Sf9cells was proved by inverted fluorescence microscope. The bright green fluorescence was demonstrated in plasma and nuclei of infected Sf-9cells. The novel pFB/LM-PH baculovirus expression system could expressed eGFP efficiently and could became a useful EGFP tagged expression system for detection of the interaction of proteins.

ping jingge. Construction of an Enhanced Green Fluorescent (eGFP) Tagged Dinsect -baculovirus Transference Vector[J]. Chinese Agricultural Science Bulletin, 2013, 29(8): 26-30.

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