RNA干扰对大鲵蛙病毒主要功能基因表达及其增殖的影响

Effect on main functional genes expression and replication of Chinese giant salamander ranavirus(CGSRV) by RNA interference

采用q PCR与病毒滴度测定观察RNA干扰(RNA interference,RNAi)对大鲵蛙病毒(Chinese giant salamander ranavirus,CGSRV)主要衣壳蛋白(major capsid protein,MCP)、甲基转移酶(methyltransferases,MTases)、DNA多聚酶(DNA polymerase)基因表达与病毒增殖的影响。结果表明,小干扰RNA(small interfering RNA,siRNA)能推迟鲤鱼上皮瘤细胞(epithelioma papillosum cyprini,EPC)出现病变,且病变程度也较对照组轻。在各功能基因表达量上,NC-FAM对照组与阴性对照组差异不显著(P>0.05);而干扰组的干扰率极显著高于阴性对照组,其中siR-DP-1组siRNA对MCP基因的干扰率为79%,极显著高于其余组(P<0.01);siR-MT-1、siR-MT-2组siRNA对MTases基因的干扰率为77%,极显著高于其余组(P<0.01);siR-DP-1组siRNA对DNA polymerase基因的干扰率为79%,极显著高于其余组(P<0.01)。干扰实验组病毒滴度与NC-FAM对照组和阴性对照组相比也有不同程度的降低。阴性对照组lg TCID50为8.362,NC-FAM对照组lg TCID50为7.848,siR-MCP、siR-MT、siR-DP实验组最低lg TCID50分别为5.764、5.317、5.362。证实RNAi能够抑制CGSRV主要功能基因的表达并影响病毒的复制。

We used qPCR and virus titer determination to investigate the effects on MCP, MTases and DNA polymerase gene expression, as well as replication of Chinese giant salamander ranavirus （CGSRV） by RNA interference （ RNAi ）. The results show that small interfering RNA （siRNA） delayed and reduced the cytopathic effects. Among the expression quantity of each functional genes, NC-FAM control group had no significant change （ P 〉 0. 05 ） but the interference rates of interferential experiment groups were higher than that of negative control group. The interference rate of siR-DP-1 group on MCP genes was 79% , very significantly higher than those of the other groups （ P 〈 0. 01 ） ; the interference rate of siR-MT-1 or siR-MT-2 group on MTases genes was 77% , very significantly higher than those of the other groups （P 〈0. 01 ） ; the interference rate of siR-DP-1 group on DNA polymerase genes was 79%, very significantly higher than those of the other groups （P 〈 0. 01 ）. The viral titers of interferential experiment groups inordinately decreased compared with those of the other groups. Furthermore, the lgTCID50 s of negative control group and NC-FAM control group were 8. 362 and 7. 848, respectively, and the lowest lgTCID50s of siR-MCP, siR-MT and siR-DP groups were 5. 764, 5. 317 and 5. 362, respectively. In conclusion, RNA interference inhibits the main functional genes expression and replication of CGSRV.