关键词: PrV, Ea株, gD基因, 体外表达, 细胞系, 逆转录病毒介导

Abstract: The retrovirus mediated transfer gene method was applied to integrate PrV Ea gD gene into the chromosome of PK-15，and the cell line designated as PK^D expressing gD was established.The gD expressed by PK^D distributed in the cell membrane and demonstrate the same immunoreactivity as the original gD.It was also noticed that the gD was in apparent polar distribution.PCR technique and indirect immunofluorescence assay were applied to detect PK^D,the results demonstrates that the transferred gD gene is genetically stable.In the same culture condition,the growth speed and the shape of PK^D did not show apparent difference compared with PK-15.By lipofeltin mediated method,PrV Ea genome was transferred into PK^D,a typical cytopathic effect was observed after 30 hours which proved PK^D has the endurance to lipofeltin.The detection results of TCID₅₀ demonstrated that although PK^D showed apparent suppression to PrV Ea strain,PrV Ea strain can be amplified in it normally.All this research results suggest that PK^D can be used to establish PrV Ea strain gD deleted mutant and thus provides an essential tool for it.

Key words: pseudorabies virus, Ea strain, glycoprotein D gene, expression in vitro, cell line, retrovirus-mediated method