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目的研究旋毛虫蛋白酶体β7亚基(T.spiralis proteasome subunit beta type-7,Tspst)的基因功能。方法通过浸泡法将Tspst特异性的dsRNA(dsRNA-Tspst)导入旋毛虫肌幼虫体内,应用Real-time PCR和Western blot检测经dsRNA-Tspst浸泡处理后幼虫Tspst基因的转录和表达水平。体外培养观察Tspst基因沉默对幼虫存活的影响。将dsRNA浸泡后的幼虫经口接种小鼠,观察Tspst基因沉默对幼虫发育、存活及雌成虫生殖力的影响。结果Realtime PCR和Western blot检测显示,旋毛虫幼虫经40ng/μl dsRNA浸泡处理后Tspst基因的转录和表达分别下降71.87%和61.9%(P<0.05)。dsRNA浸泡处理组、Lip2000和PBS对照组幼虫的死亡率分别是33.4%、35%和35.4%(χ2=0.118,P>0.05)。将dsRNA浸泡的幼虫接种小鼠后6d,肠道成虫与35d肌幼虫的减虫率分别为26.51%和38.34%。dsRNA浸泡处理组、Lip2000和PBS对照组幼虫感染小鼠收集的雌虫体外培养72h新生幼虫产量分别是53.26±3.99、50.80±3.08和53.70±2.57条(P>0.05)。结论 dsRNA可明显抑制Tspst基因的表达及幼虫在小鼠体内的发育和存活。
Objective To investigate the gene function of T.spiralis proteasome subunit beta type-7 (Tspst). Methods Tspst-specific dsRNA (dsRNA-Tspst) was introduced into Trichinella spiralis larvae by immersion method. Real-time PCR and Western blot were used to detect the transcription and expression of Tspst after larvae were treated with dsRNA-Tspst. Effects of Tspst Gene Silencing on Larval Survival in vitro. The larvae soaked in dsRNA were orally inoculated with mice to observe the effects of Tspst gene silencing on larval development, survival and fecundity of female adults. Realtime PCR and Western blot showed that the transcription and expression of Tspst gene in Trichinella spiralis larvae decreased by 71.87% and 61.9%, respectively (P <0.05) after soaking with 40ng / μl dsRNA. The mortality rates of larvae in Liposome-treated and dsDNA-treated groups were 33.4%, 35% and 35.4%, respectively (χ2 = 0.118, P> 0.05). Six days after inoculation of dsRNA larvae into mice, the worm reduction rates of intestinal and 35d larvae were 26.51% and 38.34%, respectively. dsRNA immersion treatment group, Lip2000 and PBS control group of larvae infected mice collected in vitro 72 h neonatal larvae were 53.26 ± 3.99,50.80 ± 3.08 and 53.70 ± 2.57 (P> 0.05). Conclusion dsRNA can significantly inhibit the expression of Tspst gene and larval development and survival in mice.