摘要
构建了重组逆转录病毒表达质粒plxas-N,该质粒能表达互补猪传染性胃肠炎病毒N基因全长的反义RNA序列。用质脂体方法将重组质粒plxas-N转染至PA317细胞中,经抗生素G418(500μg/ml)筛选出稳定的产毒细胞克隆。分别扩大培养细胞克隆,取其上清液感染小鼠成纤维细胞NIH3T3,测定细胞克隆产生的重组病毒滴度,并用高滴度重组病毒感染IBRS2细胞。提取被感染的IBRS2细胞的总RNA,RT-PCR证明plxas-N整合到IBRS2细胞基因组。通过TGEV分别感染IBRS2和IBRS2抗性细胞产生的病变,结果表明该反义RNA对病毒的复制有抑制作用,其抑制率近50%。
A recombinant expression plasmid, plxas-N, was designed to produce antisense RNA targeted to the N gene full length of Transmissible gastroenteritis virus of swine, TGEV. The plxas-N was transfected into packaging cells line PA317 with Lipofectamine. The viral supenatants of the clones selected with G418 （500μg/ml） were detected by murine fibre cell NIH3T3.The highest viral titer pseudovirus was used to infect IBRS2 cell. Total cellular RNA from the IBRS2 cell, and RT-PCR analysis indicated that the plxas-N had inserted into the genome of IBRS2 cell. Antiviral activitity of the antisense RNA was evaluated by cytopathic effect using cultured IBRS2 cell and anti- IBRS2 cell infected by TGEV respectively, and the inhibitory rate was about 50%.
出处
《中国农学通报》
CSCD
2005年第8期13-14,27,共3页
Chinese Agricultural Science Bulletin
