CONSTRUCTION AND PRELIMINARY FUNCTION STUDY OFMURINE RECOMBINANT PHOSPHOLIPASE D2 ADENOVIRUS
鼠重组磷脂酶D2腺病毒的构建及功能的初步研究

To construct the murine recombinant phospholipase D2 adenovirus and study preliminarily its function in mammalian cells,Murine phosphatidylcholine-specific phospholipase D2 gene and its catalytically inactive mutant (K758R) was firstly sublconed from the eukaryotic expression vector pCGN into the shuttle vector pAdTrack-CMV encoding GFP (green fluorescent protein). The PmeI-linearized pAdTrack-mPLD2 was cotransformed with supercoiled adenoviral backbone pAdEasy-1 vector into BJ5183 E.coli for homologous recombination. Subsequently the recombinant mPLD2 adenoviral particles was successfully constructed. These particles infected human embryonic kidney 293 cells stably expressing M 3 muscarinic acetylcholine receptor (M 3 mAChR) with high efficiency detected by GFP. Meantime, significant PLD2 protein expression in 293 cells examined by immunoblotting has no effect on M3 mAChR-mediated PLD stimulation. However, infection by recombinant mPLD2 K758R adenoviruses inhibited the PLD stimulation induced by PMA, which was increased by its counterpart of wild-type mPLD2 adenoviruses. Our results demonstrated that homologous recombinant mPLD2 adenoviruses may be a powerful tool for studying further the important physiological role of PLD2 and protein kinase C might be involved specifically in PLD2 stimulation pathway.