Expression of Dendrolimus puntatus Cytoplasmic Polyhedrosis Virus(DpCPV1)Non-structural Protein p44 in Bac-to-Bac System and Localization in Infected Cells

doi:10.13560/j.cnki.biotech.bull.1985.2016.03.020

Abstract

Abstract: In order to study the function of the Dendrolimus puntatus cytoplasmic polyhedrosis virus(DpCPV1)protein p44, PCR primers were designed according to the sequence of genome segment S8, the prokaryotic expression vector for genome segment 8 of DpCPV1 was constructed, and the polyclonal antibodies were prepared by immunizing rabbits with the purified expressed protein. Three recombinant plasmids(Bacmid-p44, Bacmid-p44-eGFP and Bacmid-eGFP)were constructed using Bac-to-Bac Baculovirus expression system., and they were transfected into insect cell Sf9 for the expression. The detection and subcellular localization of the protein were determined by Western blot. The results from Western blot showed that the actual expressed protein of Bacmid-S8 in Sf9 was 35 kD, smaller than the one(44 kD)expressed in the prokaryotic expression vector. The subcellular localization of fusion protein of p44-eGFP was determined by laser scanning confocal microscope, and the fused green fluorescent protein(eGFP)of p44 concentrated in the cytoplasm of the cells, while infused eGFP distributed throughout the whole cell, indicating that p44 protein of DpCPV1 was localized mainly in the cytoplasm of the cell. This work was the first study of having the eukaryotic expression of DpCPV1 S8 and subcellular location of it in insect cells,

Key words: Dendrolimus puntatus cytoplasmic polyhedrosis virus, polyclonal antibody, eukaryotic expression, cellular localization

PENG Han, WANG Hong-xiu, WANG Jin-chang, GUAN Li-mei, JIN Liang, WAN Cui-xiang. Expression of Dendrolimus puntatus Cytoplasmic Polyhedrosis Virus(DpCPV1)Non-structural Protein p44 in Bac-to-Bac System and Localization in Infected Cells[J]. Biotechnology Bulletin, 2016, 32(3): 122-128.

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