日本血吸虫小热休克蛋白Sjp40的RNA干扰效应

摘要

Objective To study the effect of RNA interference (RNAi) on small heat shock protein (sHSP) Sjp40 of Schistosoma japonicum and its synergistic effect on the expression of SjHSP60,SjHSP70,and SjHSP90 mRNA,and observe the mRNA expression levels of Sjp40,SjHSP60,SjHSP70,and SjHSP90 in different stages of S.japonicum.Methods Double-stranded RNA (dsRNA) of Sjp40 (dsSjp40) and a control dsRNA of green fluorescent protein (dsGFP) were generated by in vitro transcription and transfected into adult worm by immersing the worm in dsRNA solution.The total RNA and proteins were isolated simultaneously from the adult worms using TRIzol reagent 7 days after transfection.The expression levels of Sjp40,SjHSP60,SjHSP70,and SjHSP90 mRNA and the expression level of Sjp40 protein were determined by quantitative real-time PCR (qPCR) and Western blotting,respectively.The mRNA expression of HSPs of S.japonicum in different stages was evaluated by qPCR.Results Compared with those in the control worms transfected with dsGFP,Sjp40 mRNA level was decreased by 80% in the worms transfected with dsSjp40,and the level of Sjp40 protein showed also a significant decrease.The mRNA expression levels of SjHSP60,SjHSP70,and SjHSP90 did not show an obvious synergism after Sjp40 RNAi.The expression profiles of Sjp40,SjHSP60,SjHSP70,and SjHSP90 showed significant differences in different stages of S.japonicum,and the expression level of Sjp40 mRNA in the egg stage was much higher than that of other HSP genes.Conclusion dsSjp40-RNAi can induce effective suppression of Sjp40 gene expression at both mRNA and protein levels,but no obvious synergism occurs in the mRNA expressions of SjHSP60,SjHSP70,and SjHSP90.%目的 研究日本血吸虫小热休克蛋白(sHSP)Sjp40基因的RNA干扰效应及其干扰后日本血吸虫HSP60、HSP70、HSP90基因在mRNA水平出现的协同效应,观察各期虫体Sjp40、SjHSP60、SjHSP70、SjHSP90基因mRNA表达水平.方法 以双链RNA(dsRNA)实现RNA干扰.体外转录合成dsRNA,将Sjp40的dsRNA(dsSjp40)及阴性对照GFP的dsRNA(dsGFP)通过浸泡法转染到日本血吸虫成虫中,体外培养7d后用TRIZOL法提取其总RNA及总蛋白,实时荧光定量PCR(qPCR)检测Sjp40基因及SjHSP60、SjHSP70、SjHSP90基因mRNA的表达,Western blot检测Sjp40蛋白表达产物.提取各期虫体总RNA,qPCR检测Sjp40、SjHSP60、SjHSP70、SjHSP90基因的mRNA表达.结果 转染后Sjp40 dsRNA转染组目的基因的转录水平较阴性对照GFP dsRNA转染组下降了80％左右；蛋白质水平较空白对照及阴性对照组(dsGFP)出现明显降低,qPCR结果显示在Sjp40基因RNA干扰后SjHSP60、SjHSP70、SjHSP90基因的mRNA表达水平未发现明显变化.此四种HSPs在日本血吸虫生活史不同时期mRNA表达存在明显差异,Sjp40基因在虫卵期mRAN水平表达相对其他HSPs基因高.结论 dsSjp40 RNAi可特异性抑制Sjp40的表达,但对SjHSP60、SjHSP70、SjHSP90基因mRNA的表达没有明显的影响.