逆转录病毒介导的PrVEa株gD基因转基因细胞系的建立

Establishment of a Cell Line Transferred gD Gene of Pseudorabies Virus Ea Strain by Retrovirus-Mediated Method

采用逆转录病毒介导的方法,将PrVEa株gD基因整合进PK 15细胞染色体中,建立起表达gD蛋白的细胞系PKD。通过荧光观察,发现PKD表达的gD分布在细胞膜上,具备原有的免疫反应性;同时,还发现gD蛋白在PKD细胞膜上呈明显极性分布。PCR技术和间接免疫荧光染色法对PKD进行检测的结果表明该细胞系具有良好的遗传稳定性。在相同培养条件下,PKD细胞的生长速度及形态与正常PK 15细胞无明显差异。用脂质体介导的方法,将PrVEa株基因组转染PKD细胞,在转染后30h细胞发生典型病变,表明PKD细胞对脂质体的毒害具有耐受性。TCID50测定结果显示:PKD对PrVEa株增殖尽管有明显的抑制作用,但PrVEa株仍能在其上正常增殖。上述研究结果提示:所构建的细胞系PKD可用于构建PrVEa株gD基因缺失毒株,为PrVEa株gD基因缺失毒株的构建提供了必备的工具。

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 TCID_(50) 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.