猪蛔虫感染相关基因07E12的RNAi研究

Primary Studies on the Functions of Differentially Expressed Gene(07E12) in Infective Larvae of Ascaris suum by RNA Interference

为寻找猪蛔虫免疫防治的"关键"靶分子,从已构建的猪蛔虫感染期幼虫消减cDNA文库中筛选了一感染相关基因(EST编号为07E12),采用RNA干扰(RNAi)技术对该基因的功能进行了初步探讨。针对其EST序列设计了1对特异引物,体外转录合成双链RNA(dsRNA)后,通过浸泡的方式将dsRNA导入猪蛔虫感染期幼虫中,在浸泡后的4个不同时间点采用RT-PCR方法检测虫体浸泡后靶基因被干扰的效果。试验结果表明,在浸泡后24、48、72 h都检测到了相对应基因表达的存在,但表达量随时间依次减少,96 h之后试验组没有检测到07E12的相应RNA的表达,提示dsRNA进入虫体并逐渐发挥了干扰作用,说明该基因在猪蛔虫幼虫感染宿主的过程中可能扮演重要角色。

In order to elucidate the function of a key developmental gene using double-stranded RNA mediated interference（RNAi）,the EST 07E12 was selected from an Ascaris.suum infective larvae-specific cDNA library.A pair of specific primers was designed to amplify the 07E12 and the dsRNA was synthesized using MEGAscript RNAi kit.The dsRNA was delivered into infective larvae by soaking,and RT-PCR was used to detect the target mRNA after different lengths of soaking time.The results showed that in RNA-treated worms,soaking for 24 h did not result in decreased target mRNA levels;the reduction of 07E12 transcript levels began from 24 to 72 h,and continued to decrease until they were no longer detectable at 96 h.The results indicated that RNAi effect was evident.