猪传染性胃肠炎病毒聚合酶基因反义RNA对病毒的抑制作用

Inhibition of TGEV in cells expressing antisense RNA targeted against virus RNA polymerase gene

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。

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× 10^5 CFU/mL. Pseudovirus with the highest viral titer was used to infect IBRSz cells. Total cellular RNA from the IBRSz cells, and RT-PCR analysis indicated that the plxas-pol was inserted into the genome of IBRSz cells. The effect of the antisense RNA was evaluated by cytopathogenic effect assay using cultured IBRSz cells and anti-IBRSz cells infected by TGEV, respectively, and the inhibitory rate was approximately 80%. Compared with that in control cells, the anti-IBRSz cells line was able to delay Virus-induced cell death by 21 or 27 h at an 50% tissue culture infective dose of 2× 10^3/mL or 4×10^2/mL.