Construction of a GFP-fused mouse PACRG baculovirus recombinant vector and expression of the fusion protein in Sf9 inset cells.

Jun-Pin LIU; Hong-Tao LI; Wei LI; Hong LIU; Ling ZHANG; Jie MIN; Ting ZHOU; Lei ZHOU; Zhi-Bing ZHANG

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Construction of a GFP-fused mouse PACRG baculovirus recombinant vector and expression of the fusion protein in Sf9 inset cells.

Author: Jun-Pin LIU ¹ ; Hong-Tao LI ² ; Wei LI ³ ; Hong LIU ¹ ; Ling ZHANG ¹ ; Jie MIN ¹ ; Ting ZHOU ¹ ; Lei ZHOU ² ; Zhi-Bing ZHANG ¹
Author Information

1. School of Public Health, Medical College, Wuhan University of Science and Technology, Wuhan, Hubei 430065, China.
2. Department of Physiology, Virginia Commonwealth University, Richmond, VA 23298, USA.
3. Department of Obstetrics and Gynecology, Virginia Commonwealth University, Richmond, VA 23298, USA.
Publication Type:Journal Article
Keywords: Parkin co-regulated gene (Pactg); baculovirus vector; eukaryotic expression; insect cell; spermatogenesis
MeSH: Animals; Baculoviridae; Blotting, Western; DNA, Complementary; Genetic Vectors; Green Fluorescent Proteins; biosynthesis; Mice; Plasmids; Polymerase Chain Reaction; Proteins; genetics; metabolism; Recombinant Fusion Proteins; biosynthesis; Sf9 Cells; Transfection
From: National Journal of Andrology 2016;22(7):591-595
CountryChina
Language:Chinese
Abstract:
ObjectiveTo construct a GFP-fused mouse Parkin co-regulated gene (PACRG) baculovirus recombinant PACRG/GFP-pFastBac1 vector and express the fusion protein in Sf9 insect cells.
METHODSFull-length mouse PACRG cDNA was amplified by PCR and cloned in frame to the vector pFastBac1 with eGFP (rpFBac-PACRG-GFP recombinant vector). The plasmid was transformed into DH10Bac cells to obtain the recombinant bacmid plasmid, the bacmid was transfected into Sf9 insect cells, and the expressed PACRG/GFP fusion protein was analyzed by Western blot and fluorescence microscopy.
RESULTSThe construction of the PACRG/GFP-pFastBac1 baculovirus plasmid was confirmed by sequencing and restriction enzyme digestion. Western blot showed the expression of the fusion protein carrying a green fluorescence in the Sf9 insect cells.
CONCLUSIONSConclusion: A PACRG/GFP-pFastBac1 recombinant baculovirus vector was successfully constructed and the fusion protein was highly expressed in the Sf9 insect cells. Our findings have provided a basis for further studies on the structure of the PACRG protein and regulation of spermatogenesis.