应用dsRNA干扰技术对旋毛虫Kazal型丝氨酸蛋白酶抑制剂基因功能的初步研究

Functional identification of Kazal serine protease inhibitor gene of Trichinella spiralis using dsRNA interference

为研究旋毛虫Kazal型丝氨酸蛋白酶抑制剂基因的功能,通过电转法将dsRNA导入旋毛虫体内,应用实时荧光定量PCR和Western-blot检测经dsRNA-TsKaSPI电转法处理后肌幼虫TsKaSPI基因转录和表达水平。体外培养观察TsKaSPI基因沉默对肌幼虫存活的影响。将dsRNA导入后的幼虫经口接种小鼠,观察TsKaSPI基因沉默对幼虫存活、发育及雌虫生殖力的影响。实时荧光定量PCR和Western-blot检测结果显示,旋毛虫肌幼虫经30μg/mL dsRNA电转处理后TsKaSPI基因的转录和表达分别降低63.36%和69.24%(P<0.05)。TsKaSPI基因在dsRNA-TsKaSPI转入肌幼虫后第1天转录水平明显降低,第2和第3天仍处于较低的水平。PBS组、dseGFP对照组和dsRNA-TsKaSPI处理组的肌幼虫死亡率分别为61%、58%、52%(P>0.05)。将dsRNA电转处理的肌幼虫接种小鼠后6 d肠道成虫和35 d肌幼虫的减虫率分别为36.22%和31.87%。PBS组、dseGFP对照组和dsRNA-TsKaSPI处理组的感染小鼠收集到的雌虫新生幼虫量分别为49.20±5.93、49.40±4.62、50.80±4.32(P>0.05)。结果表明,dsRNA可明显抑制Kazal型丝氨酸蛋白酶抑制剂基因的转录和表达及幼虫在小鼠体内的存活和发育。

To investigate the role of Kazal serine protease inhibitor gene in Trichinella spiralis,the dsRNA was introduced into T.spiralis by electrophoretic transfer.The transcription and expression of TsKaSPI gene in muscle larvae treated by dsRNA-TsKaSPI electroporation were detected by real-time PCR and Western-blot. The effect of TsKaSPI gene silencing on the survival of muscle larvae was observed in vitro.The larvae after dsRNA introduction were infected orally into mice,and the effects of TsKaSPI gene silencing on larval survival,development and female fecundity were determined. In results,real-time PCR and Western-blot detection showed the mRNA and protein level of TsKaSPI in T.spiralis muscle larvae treated with 30 μg/mL dsRNA electroporation were decreased by 63.36% and 69.24%,respectively(P<0.05).The mRNA level of TsKaSPI gene decreased significantly on the 1st day after dsRNA-TsKaSPI was transferred into muscle larvae and remained this lower level on the 2nd and 3rd day.The mortality rates of muscle larvae in PBS group,dse GFP control group and dsRNA-TsKaSPI treatment group were 61%,58% and 52%,respectively(P>0.05).The worm reduction rates of 6-day intestinal adults and 35-day muscle larvae inoculated with dsRNA electroporation muscle larvae were 36.22% and 31.87%respectively.The amount of newborn female larvae collected from PBS group,dse GFP control group and dsRNA-TsKaSPI treated group was 49.20±5.93,49.40±4.62 and 50.80±4.32,respectively(P>0.05).In conclusion,dsRNA can significantly inhibit the transcription and expression level of Kazal serine protease inhibitor gene and the survival and development of larvae in mice.