应用RNA干扰技术抑制捻转血矛线虫H11基因研究

RNAi Effect of H11 Gene on L3 Larvae Developmen of Haemonchus contortus

【目的】为了检测RNAi沉默H11基因能否模拟H11疫苗免疫效果。【方法】针对H11基因序列设计并合成特异性dsRNA,通过浸泡方式分别将dsRNA导入捻转血矛线虫感染性L3期幼虫体内,利用实时荧光定量PCR分析H11基因在L3期幼虫中的转录抑制效果,同时,将H11-dsRNA浸泡L3期幼虫24 h后感染绵羊,定期采集绵羊粪便检查虫卵数,第30 d后剖检绵羊皱胃,检查荷虫数的变化。研究H11基因的沉默对捻转血矛线虫减虫率和减卵率的疫苗免疫效果。【结果】实时荧光定量PCR检测表明:试验组中H11基因的相对含量显著低于对照组(P<0.01),在浸泡72 h后,H11-dsRNA组基因的表达量仅为对照组的21.33%。绵羊体内干扰沉默效果显示,试验组H11干扰组虫卵减少率为41.02%,减虫率为39.6%。【结论】研究通过浸泡法导入的dsRNA能有效抑制H11基因的转录,同时H11基因的沉默与幼虫的发育密切相关,为捻转血矛线虫及其它寄生线虫的基因功能研究提供一种新的方法。

[ Objective ] The purpose of this project is to elucidate the function of Hl l gene from Haemonchus contortus and provide a scientific basis for the further research on this Hll gene of animal - parasitic nematodes, and also for developing new control strategies. [ Method] Hl l gene of Haemonchus contortus was cloned from L3 larvae of Haemonchus contortus by designing primers and it was used as the target gene for RNA silencing. The specific double - stranded RNA （dsRNA） was synthesized by using plasmid DNA containing target gene as template. The nematodes of L3 larvae of Haemonchus contortus were soaked with dsRNA of Hll and RNA interference （RNAi） effects were evaluated by detecting the gene transcription by RT -PCR and examining the faecal egg count （FEC） and worm burdens of Haemonchus contortus in vivo sheep. [ Result] RT - PCR revealed that the transcription of target gene was significantly degenerated after 72 h H11 -dsRNA, and compared with the control group, the experimental group was reduced by 21.33%. In vivo study of RNAi in an animal parasitic nematode, RNAi of the H11 gene in infective larvae prior to infection resulted in a 41.02% reduction in FEC, and 39.6% reduction in worm burden compared with pre - soaking in control. [ Conclusion] dsRNAs can inhibit the transcription of Hl l gene effectively, and their FEC and worm burdens can be reduced significantly. The function of Hll is probably related to the growth and development of Haernonchus contortus. It may provide a new way for gene function research Haemonchus contortus and other parasitic nematodes.