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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
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于晓龙1，2， 张海峰2， 李一经3
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(1. 黑龙江省农业科学院博士后工作站， 黑龙江 哈尔滨150086； 2.黑龙江省农业科学院 畜牧研究中心，
黑龙江 哈尔滨150086； 3. 东北农业大学 动物医学院， 黑龙江 哈尔滨150030)
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摘要：
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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plxaspol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90×105CFU/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RTPCR证明plxaspol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21h和27h，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。 |
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关键词：
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猪传染性胃肠炎病毒； 反义RNA； 逆转录病毒表达载体 |
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中图分类号：S 852.659.6 文献标识码：
A 文章编号：1673-4696(2006)02-0085-04 |
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Inhibition of TGEV in cells expressing antisense RNA targeted
against virus RNA polymerase gene |
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YU Xiaolong1，2, ZHANG Haifeng2， LI
Yijing3 |
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(1. Postdoctoral Scientific Research Station, Heilongjiang Academy of Agricultural Sciences, Harbin 150086, China;2. Research Center of Animal Science, Heilongjiang Academy of Agricultural Sciences, Harbin 150086, China；3. College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030, China)
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Abstract: To study the effect of antisense RNA on the development of transmissible gastroenteritis virus(TGEV), the TGEV antisense construct, pLXSNpol was engineered. The packaging cells line PA317 was transfected with the pLXSNpol 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 90×105CFU/mL. Pseudovirus with the highest viral titer was used to infect IBRS2 cells. Total cellular RNA from the IBRS2 cells, and RTPCR analysis indicated that the plxaspol 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 antiIBRS2 cells infected by TGEV, respectively, and the inhibitory rate was approximately 80%. Compared with that in control cells, the antiIBRS2 cells line was able to delay virusinduced cell death by 21 or 27h at an 50% tissue culture infective dose of 2×103/mL or 4×102/mL. |
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Key words: transmissible gastroenteritis virus； antisense RNA； retrovirus vector |
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