靶向基因shRNA干扰家蚕及猪若干病毒的复制和增殖研究
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摘要
RNA干扰(RNA interference, RNAi)是指在进化过程中高度保守的、由双链RNA(double-stranded RNA, dsRNA)诱发的、同源mRNA高效特异性降解的现象。由于使用RNAi技术可以特异性剔除或关闭特定基因的表达,所以该技术已被广泛用于探索基因功能和传染性疾病的基因治疗领域。
1猪圆环病毒(Porcine circovirus2, PVC2)病毒是我国猪场疫病复杂的重要原因,可以说它对养猪产业正常维持带来了极大的困难,对我国养猪产业的影响目前受到了仅次于高致病性蓝耳病的空前关注,且目前尚无有效疫苗与治疗方法。本文根据PVC2型病毒-Rep、Cap和ORF3/ORF4基因的结构序列和shRNA序列设计原则,进行地毯式搜索并设计合成了139对针对PVC2型病毒-Rep蛋白基因、134对针对PVC2型病毒-Cap蛋白基因、18对针对PVC2型病毒-ORF3/ORF4基因的shRNA序列,共计291对PVC2型病毒靶向shRNA。(?)设计合成的291对shRNAs连接到线性化的RNAi-Ready pSIREN-RetroQ-ZsGreen载体上,构建成RNA干扰载体。分别在转染试剂介导下导入无PCV2污染的猪肾PK15细胞,24小时后进行荧光观察,确定转染效率。并在转染24小时后对细胞接种PCV2病毒,接毒72小时后收集细胞,通过Real-Time PCR方法检测细胞中病毒的相对表达量,从而分析所设计的shRNA对PCV2病毒的抑制作用。结果表明,针对Rep基因序列设计的139对shRNAs中,pSIREN-REP-C, pSIREN-REP-4, pSIREN-REP-23', pSIREN-REP-30', pSIREN-REP-33', pSIREN-REP-67', pSIREN-REP-75', pSIREN-REP-80'共8个序列表现出对PVC2病毒复制与增殖的抑制作用；针对Cap基因序列所设计的134对shRNAs中,pSIREN-Cap-3, pSIREN-Cap-5, pSIREN-Cap-C11', pSIREN-Cap-14, pSIREN-Cap-80共5个序列表现出对PVC2病毒复制增殖的抑制作用,而针对ORF3和ORF4所设计的18对shRNAs并没有表现出较显著的对PVC2病毒复制增殖的抑制作用。综上所述,以上对PCV2具有显著抗病毒能力的shRNA靶向基因序列可作为转基因的候选shRNA序列。
2猪繁殖与呼吸综合症(Porcine Reproductive and Respiratory Syndrome, PRRS)俗称猪蓝耳病,是一种新的猪传染病,能够引起母猪的生殖障碍及仔猪的呼吸道症状,在世界各地危害很大。猪生殖和呼吸系统综合症病毒(PRRSV)由ORF5、ORF6分别编码的GP5、M蛋白是PRRSV病毒的主要结构蛋白,它们在病毒的致病性、病毒复制、病毒装配、病毒变异和保护性反应等方面具有重要意义。本文根据猪蓝耳病病毒PRRSV-ORF5及PRRSV-ORF6的基因结构序列和shRNA序列设计原则,分别设计和合成了5对针对PRRSV-ORF5,5对针对PRRSV-ORF6编码蛋白基因序列的靶向shRNA。(?)设计合成的shRNA连接到线性化的RNAi-Ready pSIREN-RetroQ-ZsGreen载体上,构建成RNA干扰载体。将上述质粒DNA,分别在转染试剂介导下导入Marc-145细胞,24小时后进行荧光观察,确定转染效率。同时,转染细胞接种PRRSV病毒,72小时后收集细胞,通过Real-Time PCR方法检测病毒感染后的相对表达量,分析所设计的shRNA对PRRSV病毒的抑制作用。结果表明,分别针对ORF5和ORF6设计的共10对shRNAs均表现出抑制PRRSV病毒增殖复制的作用,但不同shRNA的抑制效果存在差异(0.71%-67.52%)。其中以ORF6-6e的病毒相对表达量最低,细胞内病毒表达量仅为0.71%,即抗病毒能力最强,因此该序列可作为转基因的候选shRNA序列。
采用piggyBac转座子构建转座载体,为了便于筛选,以RNAi-Ready pSIREN-RetroQ-ZsGreen载体为模板进行PCR扩增,得到ZsGFP基因,连接到PXL-BACⅡ上形成重组载体PXL-BACⅡ-ZsGFP;再以pSilencer4.1-CMVneo载体为模板进行PCR扩增,得到Neomycinr基因,与PXL-BACⅡ-ZsGFP连接,形成重组载体PXL-BACⅡ-ZsGFP-Neo。为了后期能将这两个筛选基因给剔除掉,又在ZsGFP基因和Neomycinr基因的两端加入了FRT位点,形成最终的转座载体PXL-BACⅡ-FRT-ZsGFP-Neo.最后将在上述活性筛选试验中挑选的具有最佳抗病毒能力的shRNA (ORF6-6e)构建到该转座载体,命名为PXL-BACⅡ-FRT-ZsGFP-Neo-shRNA。该转基因载体同时具有筛选基因和标记基因,便于进行转基因细胞的建立。该细胞株可稳定传代,以细胞基因组和RNA反转录产物cDNA为模板,分别经PCR和RT-PCR确认,表明转基因细胞中外源基因shRNA转录框、标记基因GFP和筛选基因Neo均可正常转录和表达。该细胞株相对于正常Marc-145细胞具有较显著的抗病毒能力,病毒相对表达量仅为0.01%,表明病毒在该转基因细胞内无法复制增殖。该研究为抗PRRSV病毒转基因猪品种的培育与鉴定建立了方法,奠定了基础。
3家蚕核型多角体病毒(Bombyx mori nucleopolyhedrovirus, BmNPV)所引起的血液型脓病,是家蚕病毒病的主要类型,是在我国乃至世界养蚕业危害较为严重的一种病毒病。家蚕抗病能力除了作为宿主本身的家蚕抗性机理外,作为病原一方的复制增殖也同样重要,正所谓“无菌不成病”。本文正是从BmNPV在家蚕体内的复制繁殖的角度开展研究。以BmNPV病毒的结构序列和shRNA序列设计原则,分别针对病毒复制增殖所必需的极早期表达因子ie-1,晚期表达因子lef-1, lef-2和lef-3等靶基因设计合成32对shRNA,以BmN细胞基因组为模板,通过PCR方法扩增得到BmU6启动子,构建重组载体PXL-BACII-GFP-BmU6-shRNA.将上述质粒DNA转染家蚕BmN细胞后接种BmNPV病毒,72小时后收集细胞,采用Real-Time PCR方法检测病毒感染细胞后的相对表达量,以分析不同shRNA的抑制病毒复制与增殖活性。结果表明：shRNAs均表现出对BmNPV的抗病毒能力,但效果大有不同。其中,靶基因序列长度为21bp的shRNA-ie1c表现出对BmNPV复制增殖具有极显著的抑制作用,病毒相对表达量仅为5.5%,其次为Lef3e-2。与此同时,本试验还设计并比较了不同靶序列长度：19bp和21bp在抑制BmNPV病毒增殖复制时的作用和差别。结果表明,leflb(85%)和lef1b-2(29%), Ief2d (91%)和lef2d-2(38%)的抗病毒能力效果差异显著,靶序列长度为21bp的病毒相对表达量较19bp病毒相对表达量分别减少56%和53%,说明shRNA干扰靶序列长度为21bp的RNA干扰病毒复制增殖效果优于19bp。
经shRNA活性筛选32对特异干扰靶向shRNA,分别针对极早期表达因子ie-1和晚期表达因子lef3的ie1c和lef3e-2表现出良好的抑制BmNPV的作用,因此将iel c和leBe-2作为转基因的候选shRNA序列,构建到含有筛选基因Neo的转座重组载体上,并构建单联和双联重组转座子载体,分别命名为PXL-B ACII-GFP-BmU6-shRNA(ie1c)-Neo和PXL-B ACII-GFP-double(ie1c/lef3e2)-Neo.在细胞水平进行转基因研究,建立含抗BmNPV病毒shRNA的转基因细胞,评价其抑制病毒增殖活性。结果表明单联重组转座载体构建的转基因细胞相对于正常BmN细胞具有较显著的抗病毒能力：病毒感染0-24h,对照组细胞与转基因细胞内的病毒相对表达量均较低,分别为7.15%和0.45%,尽管在病毒感染初期,病毒DNA刚开始复制,仍可见转基因细胞已初步表现出抗病毒增殖的能力；24-48h,对照组细胞内病毒表达量呈指数增长趋势(56.23%),增加了49.08%,而转基因细胞组病毒相对表达量仍能维持较低水平(7.04%)；48-96h,对照组细胞与转基因细胞组的病毒相对表达量处于稳定期,对照组病毒表达量始终维持较高水平(56.23%-64.03%),高于转基因细胞组约47%-52%；病毒感染96h后,正常细胞组的病毒相对表达量又呈现增长趋势,约90%细胞的核内充满多角体,细胞死亡率上升,同时,随着病毒感染时间的延长,转基因细胞组抑制病毒复制增殖效果略有下降,但转基因细胞病毒相对表达量仍低于对照组细胞约30%-50%。因此,在感染BmNPV后0-96h内,转基因细胞表现出较好的抑制病毒增殖与复制的能力。
双联重组转座载体中含有上述活性检测选出的两条效果最好的shRNAs (ielc/lef3e2),其转基因细胞的病毒相对表达量为42.49%,虽然较正常BmN细胞内病毒表达量低,但是其抗病毒效果显著低于单联转座的转基因细胞(7.04%)的抗病毒能力。通过PCR、RT-PCR等方法确定转基因细胞中外源基因均能正常转录和表达,并采用Splinkerette-PCR方法经NCBI Blast检索分析外源基因插入位点。该研究结果为抗BmNPV转基因家蚕的培育以及鉴定奠定了基础。
Over the past decade RNA interference (RNAi) plays an important role in biology, especially for silencing gene expression. RNA interference (RNAi) is a natural process through which expression of a targeted gene can be knocked down with high specificity and selectivity. Methods of mediating the RNAi effect involve small interfering RNA (siRNA) and short hairpin RNA (shRNA). In various applications, RNAi has been used to create model systems, to identify novel molecular targets, to study gene function in a genome-wide fashion, and to create new avenues for clinical therapeutics. In this study, we developed a system of RNAi for the inhibition of replication and multiplication of some silkworm and swine viruses by targeting shRNAs. Our results suggest that RNAi technology might serve as an experimental basis and technical insight into the researches on virus disease therapeutics.
1Porcine circovirus type2(PCV2) is the primary causative agent of an emerging swine disease, it caused postweaning multisystemic wasting syndrome (PMWS) in pig farms in many swine-producing areas in the world in recent years, moreover, no antiviral treatment is available. To exploit the possibility of using RNA interference as a therapeutic approach against the PCV2, the291recombinant plasmid short hairpin RNAs (shRNAs) were constructed to target the Rep, Cap, ORF3and ORF4of PCV2genome. Transfection of these shRNAs into cultured PK15cells caused a different level of reduction in viral DNA replication. These results indicated that shRNAs are capable of inhibiting PCV2infection in vitro and thus may constitute an effective therapeutic strategy for PCV2infection.
2Porcine reproductive and respiratory syndrome (PRRS) is now considered one of the most important diseases in countries with intensive swine industries. However, at present there is no effective method to prevent and control the disease. Therefore it is needed to develop the new antiviral strategies. In this study, the recombinant plasmid expressing shRNAs, specifically targeting the GP5/M gene RNAs of PRRSV, were generated and used to inhibit the production of GP5/M gene RNAs. It was found that shRNAs could down-regulate effectively specific gene expression and inhibit viral replication in Marc-145cells when compared to the controls. The highest activity displayed in shRNAs of the ORF6e sequences, which the inhibition rate reached to99.09%. The results suggest that RNAi technology might serve as a potential molecular strategy for PRRSV therapy. Furthermore the transgenic Mac-145cell line of piggyBac transpo son-derived targeting shRNA interference against porcine reproductive and respiratory syndrome virus was established, which is necessary for research works on transformed pigs studies.
3The Bombyx mori nucleopolyhedrovirus (BmNPV) is one of the most destructive diseases in silkworm, which has caused the main damage to silk industry. In this study, we developed a system of RNAi for preventing the BmNPV infection using the piggyBac transpo son-derived targeting shRNA interference. The shRNAs targeting the genes of ie-1, lef1, lef2and lef3of.BmNPV were designed and used to inhibit the intracellular replication or multiplication of BmNPV in BmN cells. The highest activity was presented in the shRNA targeting the iel-c of BmNPV, which the inhibition rate reached94.5%. Further a stable BmN cell line of piggyBac transpo son-derived targeting shRNA interference against BmNPV was established, which presented highly efficacious suppression of virus proliferation. This efficient method of stable gene transformation opens a way for promising research and biotechnology application on silkworm lethal diseases.
1猪圆环病毒(Porcine circovirus2, PVC2)病毒是我国猪场疫病复杂的重要原因,可以说它对养猪产业正常维持带来了极大的困难,对我国养猪产业的影响目前受到了仅次于高致病性蓝耳病的空前关注,且目前尚无有效疫苗与治疗方法。本文根据PVC2型病毒-Rep、Cap和ORF3/ORF4基因的结构序列和shRNA序列设计原则,进行地毯式搜索并设计合成了139对针对PVC2型病毒-Rep蛋白基因、134对针对PVC2型病毒-Cap蛋白基因、18对针对PVC2型病毒-ORF3/ORF4基因的shRNA序列,共计291对PVC2型病毒靶向shRNA。(?)设计合成的291对shRNAs连接到线性化的RNAi-Ready pSIREN-RetroQ-ZsGreen载体上,构建成RNA干扰载体。分别在转染试剂介导下导入无PCV2污染的猪肾PK15细胞,24小时后进行荧光观察,确定转染效率。并在转染24小时后对细胞接种PCV2病毒,接毒72小时后收集细胞,通过Real-Time PCR方法检测细胞中病毒的相对表达量,从而分析所设计的shRNA对PCV2病毒的抑制作用。结果表明,针对Rep基因序列设计的139对shRNAs中,pSIREN-REP-C, pSIREN-REP-4, pSIREN-REP-23', pSIREN-REP-30', pSIREN-REP-33', pSIREN-REP-67', pSIREN-REP-75', pSIREN-REP-80'共8个序列表现出对PVC2病毒复制与增殖的抑制作用；针对Cap基因序列所设计的134对shRNAs中,pSIREN-Cap-3, pSIREN-Cap-5, pSIREN-Cap-C11', pSIREN-Cap-14, pSIREN-Cap-80共5个序列表现出对PVC2病毒复制增殖的抑制作用,而针对ORF3和ORF4所设计的18对shRNAs并没有表现出较显著的对PVC2病毒复制增殖的抑制作用。综上所述,以上对PCV2具有显著抗病毒能力的shRNA靶向基因序列可作为转基因的候选shRNA序列。
2猪繁殖与呼吸综合症(Porcine Reproductive and Respiratory Syndrome, PRRS)俗称猪蓝耳病,是一种新的猪传染病,能够引起母猪的生殖障碍及仔猪的呼吸道症状,在世界各地危害很大。猪生殖和呼吸系统综合症病毒(PRRSV)由ORF5、ORF6分别编码的GP5、M蛋白是PRRSV病毒的主要结构蛋白,它们在病毒的致病性、病毒复制、病毒装配、病毒变异和保护性反应等方面具有重要意义。本文根据猪蓝耳病病毒PRRSV-ORF5及PRRSV-ORF6的基因结构序列和shRNA序列设计原则,分别设计和合成了5对针对PRRSV-ORF5,5对针对PRRSV-ORF6编码蛋白基因序列的靶向shRNA。(?)设计合成的shRNA连接到线性化的RNAi-Ready pSIREN-RetroQ-ZsGreen载体上,构建成RNA干扰载体。将上述质粒DNA,分别在转染试剂介导下导入Marc-145细胞,24小时后进行荧光观察,确定转染效率。同时,转染细胞接种PRRSV病毒,72小时后收集细胞,通过Real-Time PCR方法检测病毒感染后的相对表达量,分析所设计的shRNA对PRRSV病毒的抑制作用。结果表明,分别针对ORF5和ORF6设计的共10对shRNAs均表现出抑制PRRSV病毒增殖复制的作用,但不同shRNA的抑制效果存在差异(0.71%-67.52%)。其中以ORF6-6e的病毒相对表达量最低,细胞内病毒表达量仅为0.71%,即抗病毒能力最强,因此该序列可作为转基因的候选shRNA序列。
采用piggyBac转座子构建转座载体,为了便于筛选,以RNAi-Ready pSIREN-RetroQ-ZsGreen载体为模板进行PCR扩增,得到ZsGFP基因,连接到PXL-BACⅡ上形成重组载体PXL-BACⅡ-ZsGFP;再以pSilencer4.1-CMVneo载体为模板进行PCR扩增,得到Neomycinr基因,与PXL-BACⅡ-ZsGFP连接,形成重组载体PXL-BACⅡ-ZsGFP-Neo。为了后期能将这两个筛选基因给剔除掉,又在ZsGFP基因和Neomycinr基因的两端加入了FRT位点,形成最终的转座载体PXL-BACⅡ-FRT-ZsGFP-Neo.最后将在上述活性筛选试验中挑选的具有最佳抗病毒能力的shRNA (ORF6-6e)构建到该转座载体,命名为PXL-BACⅡ-FRT-ZsGFP-Neo-shRNA。该转基因载体同时具有筛选基因和标记基因,便于进行转基因细胞的建立。该细胞株可稳定传代,以细胞基因组和RNA反转录产物cDNA为模板,分别经PCR和RT-PCR确认,表明转基因细胞中外源基因shRNA转录框、标记基因GFP和筛选基因Neo均可正常转录和表达。该细胞株相对于正常Marc-145细胞具有较显著的抗病毒能力,病毒相对表达量仅为0.01%,表明病毒在该转基因细胞内无法复制增殖。该研究为抗PRRSV病毒转基因猪品种的培育与鉴定建立了方法,奠定了基础。
3家蚕核型多角体病毒(Bombyx mori nucleopolyhedrovirus, BmNPV)所引起的血液型脓病,是家蚕病毒病的主要类型,是在我国乃至世界养蚕业危害较为严重的一种病毒病。家蚕抗病能力除了作为宿主本身的家蚕抗性机理外,作为病原一方的复制增殖也同样重要,正所谓“无菌不成病”。本文正是从BmNPV在家蚕体内的复制繁殖的角度开展研究。以BmNPV病毒的结构序列和shRNA序列设计原则,分别针对病毒复制增殖所必需的极早期表达因子ie-1,晚期表达因子lef-1, lef-2和lef-3等靶基因设计合成32对shRNA,以BmN细胞基因组为模板,通过PCR方法扩增得到BmU6启动子,构建重组载体PXL-BACII-GFP-BmU6-shRNA.将上述质粒DNA转染家蚕BmN细胞后接种BmNPV病毒,72小时后收集细胞,采用Real-Time PCR方法检测病毒感染细胞后的相对表达量,以分析不同shRNA的抑制病毒复制与增殖活性。结果表明：shRNAs均表现出对BmNPV的抗病毒能力,但效果大有不同。其中,靶基因序列长度为21bp的shRNA-ie1c表现出对BmNPV复制增殖具有极显著的抑制作用,病毒相对表达量仅为5.5%,其次为Lef3e-2。与此同时,本试验还设计并比较了不同靶序列长度：19bp和21bp在抑制BmNPV病毒增殖复制时的作用和差别。结果表明,leflb(85%)和lef1b-2(29%), Ief2d (91%)和lef2d-2(38%)的抗病毒能力效果差异显著,靶序列长度为21bp的病毒相对表达量较19bp病毒相对表达量分别减少56%和53%,说明shRNA干扰靶序列长度为21bp的RNA干扰病毒复制增殖效果优于19bp。
经shRNA活性筛选32对特异干扰靶向shRNA,分别针对极早期表达因子ie-1和晚期表达因子lef3的ie1c和lef3e-2表现出良好的抑制BmNPV的作用,因此将iel c和leBe-2作为转基因的候选shRNA序列,构建到含有筛选基因Neo的转座重组载体上,并构建单联和双联重组转座子载体,分别命名为PXL-B ACII-GFP-BmU6-shRNA(ie1c)-Neo和PXL-B ACII-GFP-double(ie1c/lef3e2)-Neo.在细胞水平进行转基因研究,建立含抗BmNPV病毒shRNA的转基因细胞,评价其抑制病毒增殖活性。结果表明单联重组转座载体构建的转基因细胞相对于正常BmN细胞具有较显著的抗病毒能力：病毒感染0-24h,对照组细胞与转基因细胞内的病毒相对表达量均较低,分别为7.15%和0.45%,尽管在病毒感染初期,病毒DNA刚开始复制,仍可见转基因细胞已初步表现出抗病毒增殖的能力；24-48h,对照组细胞内病毒表达量呈指数增长趋势(56.23%),增加了49.08%,而转基因细胞组病毒相对表达量仍能维持较低水平(7.04%)；48-96h,对照组细胞与转基因细胞组的病毒相对表达量处于稳定期,对照组病毒表达量始终维持较高水平(56.23%-64.03%),高于转基因细胞组约47%-52%；病毒感染96h后,正常细胞组的病毒相对表达量又呈现增长趋势,约90%细胞的核内充满多角体,细胞死亡率上升,同时,随着病毒感染时间的延长,转基因细胞组抑制病毒复制增殖效果略有下降,但转基因细胞病毒相对表达量仍低于对照组细胞约30%-50%。因此,在感染BmNPV后0-96h内,转基因细胞表现出较好的抑制病毒增殖与复制的能力。
双联重组转座载体中含有上述活性检测选出的两条效果最好的shRNAs (ielc/lef3e2),其转基因细胞的病毒相对表达量为42.49%,虽然较正常BmN细胞内病毒表达量低,但是其抗病毒效果显著低于单联转座的转基因细胞(7.04%)的抗病毒能力。通过PCR、RT-PCR等方法确定转基因细胞中外源基因均能正常转录和表达,并采用Splinkerette-PCR方法经NCBI Blast检索分析外源基因插入位点。该研究结果为抗BmNPV转基因家蚕的培育以及鉴定奠定了基础。
Over the past decade RNA interference (RNAi) plays an important role in biology, especially for silencing gene expression. RNA interference (RNAi) is a natural process through which expression of a targeted gene can be knocked down with high specificity and selectivity. Methods of mediating the RNAi effect involve small interfering RNA (siRNA) and short hairpin RNA (shRNA). In various applications, RNAi has been used to create model systems, to identify novel molecular targets, to study gene function in a genome-wide fashion, and to create new avenues for clinical therapeutics. In this study, we developed a system of RNAi for the inhibition of replication and multiplication of some silkworm and swine viruses by targeting shRNAs. Our results suggest that RNAi technology might serve as an experimental basis and technical insight into the researches on virus disease therapeutics.
1Porcine circovirus type2(PCV2) is the primary causative agent of an emerging swine disease, it caused postweaning multisystemic wasting syndrome (PMWS) in pig farms in many swine-producing areas in the world in recent years, moreover, no antiviral treatment is available. To exploit the possibility of using RNA interference as a therapeutic approach against the PCV2, the291recombinant plasmid short hairpin RNAs (shRNAs) were constructed to target the Rep, Cap, ORF3and ORF4of PCV2genome. Transfection of these shRNAs into cultured PK15cells caused a different level of reduction in viral DNA replication. These results indicated that shRNAs are capable of inhibiting PCV2infection in vitro and thus may constitute an effective therapeutic strategy for PCV2infection.
2Porcine reproductive and respiratory syndrome (PRRS) is now considered one of the most important diseases in countries with intensive swine industries. However, at present there is no effective method to prevent and control the disease. Therefore it is needed to develop the new antiviral strategies. In this study, the recombinant plasmid expressing shRNAs, specifically targeting the GP5/M gene RNAs of PRRSV, were generated and used to inhibit the production of GP5/M gene RNAs. It was found that shRNAs could down-regulate effectively specific gene expression and inhibit viral replication in Marc-145cells when compared to the controls. The highest activity displayed in shRNAs of the ORF6e sequences, which the inhibition rate reached to99.09%. The results suggest that RNAi technology might serve as a potential molecular strategy for PRRSV therapy. Furthermore the transgenic Mac-145cell line of piggyBac transpo son-derived targeting shRNA interference against porcine reproductive and respiratory syndrome virus was established, which is necessary for research works on transformed pigs studies.
3The Bombyx mori nucleopolyhedrovirus (BmNPV) is one of the most destructive diseases in silkworm, which has caused the main damage to silk industry. In this study, we developed a system of RNAi for preventing the BmNPV infection using the piggyBac transpo son-derived targeting shRNA interference. The shRNAs targeting the genes of ie-1, lef1, lef2and lef3of.BmNPV were designed and used to inhibit the intracellular replication or multiplication of BmNPV in BmN cells. The highest activity was presented in the shRNA targeting the iel-c of BmNPV, which the inhibition rate reached94.5%. Further a stable BmN cell line of piggyBac transpo son-derived targeting shRNA interference against BmNPV was established, which presented highly efficacious suppression of virus proliferation. This efficient method of stable gene transformation opens a way for promising research and biotechnology application on silkworm lethal diseases.
引文
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