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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 the process of specific gene silencing induced by double-stranded RNA (dsRNA), and a large-scale preparation of double-stranded RNA can facilitate the application of RNAi technology. Taking the KPI gene of P. monodon as an example, a method for producing a large amount of dsRNA (> 300 nt) by in vivo vector expression is described in detail. Using commercial vectors pGEMT and pDRIVE, dsRNA was prepared by two-step cloning in vivo using transfection of E. coli HT115 (DE3) containing the hairpin loop dsRNA expression vector and RNAse III deficiency. The constructed hairpin RNA expression vector contains a forward targeting sequence of 494 bp and a reverse complementary target sequence of 403 bp, wherein an extra 91 bp of the forward target sequence can become a loop loop without the need for cloning again. One mL of purified dsRNA can be obtained by culturing 30 mL of bacteria at a cost that is only one-quarter that of a commercial in vitro transcription kit. To assess the effect of RNAi, 2 μg of dsRNA was injected intramuscularly per 1 g shrimp, haemolymph was harvested at 0, 6, 12 and 24 h after dsRNA injection, and the gene transcription level of KPI mRNA was determined by RT-PCR. Compared with the control GFP-dsRNA and NaCl injection groups, the KPI-dsRNA injection group could silence the KPI gene in the hemolymph within 24 hours. The results show that the method is a large-scale preparation of long dsRNA method.