RNA干扰南美白对虾白斑病毒VP28基因的体外研究

In vitro Study of RNA Interference on VP28 Gene in White Spot Syndrome Virus(WSSV) of Penaeus vannamei Boone

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 screen out specific siRNA for VP28,a vitro RNA interference screening system was constructed by constructing expression vector pEGFP-VP28,and co-transfection of designed siRNA and pEGFP-VP28 into BHK cell.The expression of pEGFP-VP28 fusion protein was detected by Western blotting and the interference effect of siRNA to VP28 in transcription was detected by semi-quantitative reverse transcription and polymerase chain reaction.The results demonstrated that pEGFP-VP28 could be expressed in BHK cell normally,the mRNA transcription of VP28 was inhibited by three pairs of siRNA specifically,and the siRNA2 had the most remarkable effect.These results would provide a foundation on studying the RNA interference to inhibit the WSSV in shrimp.