化学合成小干扰RNA分子高效抑制草鱼呼肠孤病毒复制

Highly Efficient Inhibition on Replication of Grass Carp Reovirus Mediated by Chemically Synthesized Small Interfering RNAs

采用化学合成法分别合成靶向草鱼呼肠孤病毒(GCRV)RNA基因组节段L2片段上的RNA聚合酶基因(RdRp)和S4片段上的外衣壳蛋白基因(OCP)的小干扰RNA分子siRNA-RdRp1 286、siRNA-RdRp1 441和siRNA-OCP117,脂质体转染法转染草鱼肾脏组织细胞系CIK。转染6 h后用草鱼呼肠孤病毒感染细胞,48 h后收集感染细胞的培养液,微量培养法测定病毒lgTCID50/0.1mL值。以细胞管家基因-βactin mRNA水平为参照,RT-PCR法检测呼肠孤病毒siRNAs靶基因的mRNA水平。病毒滴度测定结果表明:转染siRNA-RdRp1 286、siRNA-RdRp1 441和siRNA-OCP117三种siRNAs的病毒滴度lgTCID50/0.1 mL分别为4.41±0.16、3.83±0.44和1.94±0.42,极显著低于阳性感染对照组的病毒滴度(7.92±0.52,P<0.01),转染siRNA阴性对照组的病毒滴度无明显变化(7.50±0.17,P>0.05)。RT-PCR检测结果显示:与阳性感染对照组比较,转染siRNA-RdRp1286、siRNA-RdRp1 441和siRNA-OCP117后的CIK细胞中GCRV mRNA水平显著下降,抑制率分别为82.08±2.15%、89.19±1.14%和92.62±0.17%,而转染阴性对照组对GCRV mRNA水平没有显著的影响(P>0.05)。

Two short interfering RNAs（siRNA-RdRp1286, siRNA-RdRp1441） and one short interfering RNA （siRNA-OCP117）, targeted to the RNA dependent RNA polymerase（RdRp） gene and outer capsid protein （OCP） gene of Grass carp reovirus （GCRV） respectively, were chemically synthesized and transfected into the CIK cells by lipofectamine 2000. 6 hours after transfection, the transfected CIK cells were challenged with GCRV. The culture media were collected at 48h post challenge and the virus was titrated in microculture system to evaluate the inhibition effect on GCRV replication mediated by siRNAs. Referring to the mRNA level of housekeeping gene 13-actin, RT-PCR was applied to detect the level of GCRV mRNA in transfected and challenged CIK cells. The results showed that the viral titer （lgTCID50/0. lmL） in siRNA-RdRp1286, siRNA-RdRp1441 and siRNA-OCPll7 transfected CIK cells were 4.41±0.16,3.83±0.44 and 1.94±0. 42 respectively, which were significantly lower than that in virus infection positive control （7. 92±0.52） （P〈0.01）. No significant change in viral titer was observed in the group transfected with siRNA negative control after challenged with GCRV（7.50±0.17, P〉0.05） . Compared with the mRNA transcriptional level of actin gene in virus infection positive control, the mRNA levels ofβ- GCRV in siR- NA-RdRpl 286,siRNA-RdRp1 441 and siRNA-OCP117 transfected and the inhibition rate reached to（82. 08±2.15）%, （89. 19±1.14）%. The mRNA level of GCRV in the siRNA negative control group had CIK cells were reduced significantly and （92. 62±0.17） %, respectively. no noticeable change（P〉0.05）.