一种大规模制备双链RNA的简单方法及其在斑节对虾中的应用

Synthesis double-stranded RNA on a large scale and its application in Penaeus monodon

RNA干扰(RNAi)是由双链RNA(dsRNA)诱发的特异性沉默目的基因表达的过程,一种大规模制备双链RNA的方法可以便利RNAi技术的应用。以斑节对虾kazal型蛋白激酶抑制剂(KPI)基因为例,详细介绍了一种以体内载体表达大量制备dsRNA(>300nt)的方法。使用商业载体pGEMT和pDRIVE,以2步克隆法构建含有发夹环(hairpin loop)dsRNA表达载体,转化RNA酶Ⅲ缺陷的大肠杆菌HT115(DE3)进行体内转录制备dsRNA。构建的发夹RNA表达载体含有494bp的正向靶序列和403bp的反向互补靶序列,其中正向靶序列多出的91bp即可成为loop环,而无需再次克隆加入。培养30mL的细菌,即可得到1mg纯化的dsRNA,而其成本仅为使用商业化体外转录试剂盒的四分之一。为评估RNAi效果,按照每1克虾体肌肉注射2μg dsRNA的剂量,在dsRNA注射后0,6,12和24h采集血淋巴,RT-PCR检测KPI mRNA的基因转录水平。与对照组GFP-dsRNA和NaCl注射组相比,KPI-dsRNA注射组可以在24h内沉默血淋巴中的KPI基因。结果表明该方法是可大规模制备长的dsRNA的方法。

RNA interference（RNAi）is initiated by double-stranded RNA（dsRNA）and has been used to improve our knowledge of the shrimp immune system.RNAi has also been used on a therapeutic approach for shrimp virus control.Method for dsRNA synthesis on a large scale will facilitate the application of RNAi in farmed shrimp.Kazal proteinase inhibitor（KPI） gene of shrimp Penaeus monodon was used as an example to show a large-scale preparation of long double-stranded RNA（300 nt） in detail by a 2-step cloning method.Two commercial vectors pGEMT and pDRIVE were used to construct a dsRNA-expression system,which transformed into RNase-deficient Escherichia coli HT115（DE3）.The hairpin-RNA consisted of the target forward sequence（494 bp）and a 91-base shortened version of its inverted repeat（403 bp）to introduce a loop,no more need for direct cloning of the loop segment.The hairpin-expression vector resulted in large-scale dsRNA synthesis,the yield of dsRNA was about 1 mg dsRNA/30 mL bacterial culture,and its cost was approximately one fourth of the cost of same production by using a commercial in vitro transcription kit.A test group of shrimp Penaeus monodon（8 g,12 shrimp each group）was injected intramuscularly in the fourth or fifth abdominal segment with 16 μg dsRNA to investigate the sequence specific RNAi effect on endogenous KPI mRNA expression.The NaCl injected group and GFP-dsRNA injected group were used as control.Hemolymph（200 μL）was collected from 3 shrimp at 0 h,6 h,12 h,and 24 h.RT-PCR test showed that KPI expression was completely knocked-down at 24 h.It should be possible to produce industrial-scale dsRNA production for shrimp farms by this in vivo transcription method.