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为方便筛选VP28特异性siRNAs,通过构建真核表达载体pEGFP-VP28,然后将设计的siRNA与pEGFP-VP28共转染BHK细胞,并采用Western blotting检测GFP-VP28融合蛋白的表达情况以及半定量RT-PCR检验siRNA抑制VP28转录的效果,建立了体外RNA干扰法筛选系统。结果表明,pEGFP-VP28能在BHK细胞正常表达,设计的3对siRNA对VP28的mRNA转录均有不同程度的干扰效果,其中siRNA2的干扰效果最为显著。研究结论为开展RNA干扰在对虾体内抑制WSSV的研究建立基础。
In order to facilitate the screening of VP28 specific siRNAs, we constructed the eukaryotic expression vector pEGFP-VP28 and then co-transfected the designed siRNA with pEGFP-VP28 into BHK cells. Western blotting was used to detect the expression of GFP-VP28 fusion protein and semi-quantitative RT -PCR to test the effect of siRNA on VP28 transcription, and to establish an in vitro RNA interference screening system. The results showed that pEGFP-VP28 could be expressed normally in BHK cells. Three pairs of siRNAs were designed to interfere with VP28 mRNA transcription to some extent. Among them, siRNA2 had the most significant interference effect. The conclusion of the study is to establish the basis of RNA interference in the inhibition of WSSV in shrimp.