表达短lef-1 dsRNA的转化细胞对家蚕核型多角体病毒的抗性

Resistance to BmNPV of Transformation Cells Expressing Short lef-1 dsRNA

为了探讨RNAi抑制家蚕核型多角体病毒(BmNPV)增殖的效果,用带有lef-1dsRNA表达盒的转基因载体pigA3-LEF-Neo转染家蚕BmN培养细胞,通过G418(750～800mg/L)筛选,获得了稳定转化细胞系.病毒感染试验显示,稳定转化细胞的病毒感染率比正常细胞低53%、转化细胞形成的多角体数量为普通细胞中的2/3、细胞培养上清中的游离病毒减少了90%以上,表明病毒在转化细胞中的增殖受到明显抑制;半定量RT-PCR结果显示,转化细胞中病毒lef-1的转录水平仅为正常细胞的2/5～3/5,表明转化细胞表达的lef-1dsRNA抑制了病毒lef-1基因的表达.通过反向PCR分析外源DNA片段插入基因组位点,结果表明,在转化细胞中外源DNA可通过随机整合或按照piggyBac特定的转座位点TTAA插入细胞基因组.

In order to investigate the inhibiting effect of RNAi on the proliferation of Bombyx mori nucleopolyhedrovirus （BmNPV）, BmN cells were transfected with transgenic vector pigA3-LEF-Neo containing lef-1 dsRNA expression cassette, to produce stable transformation cell line screened with G418（750 -800 mg/L）. Virus infection test showed that （1） the infection ratio of the stable transformation cells was lower than that of normal cells by 53%, （2） the quantity of polyhedron from transformation cells were 2/3 of that of normal cells and （3） the number of liberation virus from cell culture＇s supernatant decreased more than 90%, suggesting that the virus＇ proliferation in the transformation cells was depressed significantly. The result underlying the transcriptional level of the lef-1 gene in the transformation cells was only 2/5 -3/5 of that in the normal cells suggested that the transcribed lef-1 dsRNA in transformation cells inhibited the expression of the lef-1 gene. Inverse PCR was used to locate the site of exogenous DNA fragment insertion in genome, of which the result showed that in the transformation cells, exogenous DNA could be inserted into the cell genome randomly or at TTAA target sequence specifically for p iggyBac element transposition.