【摘要】 通过PCR扩增出猪传染性胃肠炎病毒(TGEV)聚合酶部分片段,反向插入逆转录病毒表达载体pLXSN中,用脂质体法将重组质粒plxas-pol转染PA317细胞,经抗生素G418筛选出稳定的产毒细胞克隆,分别扩大培养后,取其上清液感染小鼠成纤维细胞NIH3T3,细胞克隆产生的重组病毒效价达9.0×105CFU/mL。用高效价假病毒感染IBRS2细胞,再经抗生素G418筛选,提取细胞克隆总RNA,经RT-PCR证明plxas-pol整合入IBRS2细胞。以TGEV感染IBRS2细胞和具有抗性的IBRS2细胞所产生的细胞病变为指标,证明该反义RNA对病毒有明显抑制作用,抑制率约为80%。用2×103和4×102TCID50/mL剂量的TGEV感染时,引起细胞死亡的时间分别为21 h和27 h,与对照组相比,抗性细胞系可明显延迟因病毒感染引起细胞死亡的时间。更多还原

【Abstract】 To study the effect of antisense RNA on the development of transmissible gastroenteritis virus（TGEV）,the TGEV antisense construct,pLXSN-pol was engineered.The packaging cells line PA317 was transfected with the pLXSN-pol by lipofectAMINE.The viral supernatants of the clones selected with G418 were detected by NIH3T3 cell.Result showed that the highest viral titer among the clones was（9.0×）10⁵CFU/mL.Pseudovirus with the highest viral titer was used to infect IBRS₂ cells.Total cellular RNA from the IBRS₂ cells,and RT-PCR analysis indicated that the plxas-pol was inserted into the genome of IBRS₂ cells.The effect of the antisense RNA was evaluated by cytopathogenic effect assay using cultured IBRS₂ cells and anti-IBRS₂ cells infected by TGEV,respectively,and the inhibitory rate was appro-（ximately） 80%.Compared with that in control cells,the anti-IBRS₂ cells line was able to delay virus-induced cell death by 21 or 27h at an 50% tissue culture infective dose of 2×10³/mL or 4×10²/mL.更多还原