【作者】 徐颖；

【导师】 张耀洲；

【作者基本信息】 浙江大学 ， 生物化学与分子生物学， 2004， 硕士

【摘要】 由家蚕核多角体病毒（BmNPV）引起的脓病是很常见的蚕病，常常引起蚕业经济上很大的损失，因此有效的防治由BmNPV引起的蚕病是一个急需解决的重要课题，同时也为研究杆状病毒提供了模式病毒。RNA干扰（RNA interfering，RNAi）是一种序列特异性的降解靶mRNA的基因调节机制，由于RNAi具有抵抗病毒侵入，抑制转座子活动，防止自私基因序列的过量增殖等作用，科学家已开始应用RNAi技术来进行人类疾病的探索。由dsRNA介导的基因沉默不仅为抑制病毒复制提供了一个潜在的有力的工具，也为探索蚕病防治提供了一条新的途径，通过设计合成针对病毒复制所必须基因的dsRNA或siRNA，使靶基因发生基因沉默，理论上可以有效的抑制病毒的复制。 本实验中我们在国际上首次应用BmNPV来研究RNAi，以BmNPV复制必需的DNA解旋酶基因和DNA聚合酶基因为靶序列，人工设计并合成了长度分别为435bp（Ap₁），300bp（Ap₂）和399bp（A_H）的三条dsRNAs，利用TransMessenger^TM transfection Reagent转染家蚕细胞，研究其对BmNPV复制的抑制效果。结果表明，在野生型病毒BmNPV感染家蚕细胞实验中，Ap₂和A_H这两个dsRNA能够有效的抑制病毒DNA的复制，并且在转染dsRNA后第四天抑制效果最佳，使病毒滴度（以空斑形成单位PFU／ml值表示）降低了10³～10⁴倍，而另外一个dsRNA（Ap₁）没有明显的抑制效果。DNA斑点杂交结果表明作为阳性对照的正常家蚕细胞BmN的胞内总DNA无杂交信号，而分别转染了A_p2和A_H的家蚕细胞其内病毒DNA的含量与阴性对照相比减少了72.3％和63.4％。RT-PCR结果也表明两种目的基因在转录水平上的表达量在dsRNA的用量为2μg／well时，两种目的基因（DNApolymerase和DNA helicase）的表达水下调了78.27％和86.70％，而其他用量时目的基因的表达水平只有略微的变化。 此外，本实验还对转染试剂的转染效率和细胞毒性；dsRNA在家蚕细胞内的分布进行了初步的研究。结果发现TransMessenger^TM transfection Reagen的转染效率为60一70%，当用量超过16pl时，对家蚕细胞有毒副作用。用荧光显微镜观察FAM标记的dsRNA在细胞内的定位，发现在转染24小时后，dsRNA开始越来越多的聚集在细胞核的周围，并呈不连续分布，细胞内的荧光强度也越来越强。dsRNA在家蚕细胞中可以维持5天的时间，但到第6天时细胞内已检测不出荧光信号。 综上所述，通过选择BmNPV复制最关键的必需基因作为靶分子，设计合成针对性的dsRNA可以有效的发挥RNAi作用，抑制病毒DNA的复制。dsRNA杭BmNPV的可行性为昆虫杆状病毒抑制研究提供了模式，也为将来RNAi在治疗人类病毒性疾病方面提供了参考。更多还原

【Abstract】 BmNPV often causes silkworm diseases and results in substantial economic losses for silkworm industry; thus it an important task to effectively prevent and cure silkworm diseases caused by BmNPV. RNA interference (RNAi) is a mechanism of gene regulation, which sequence-specifically degrades targeted mRNA. RNAi has been shown to protect against invading genetic elements such as transposons, transgenes and viruses, which potentially share a long dsRNA trigger. RNA-induced gene silencing offers a potentially useful method to inhibit viral replication. In theory, it can block viral replication effectively by synthetic dsRNAs or siRNAs, which target the indispensable genes of viral replication and thus silence the expression of target genes.To investigate the effect of dsRNA on silkworm cells, we transfected three kinds of synthetic dsRNAs of 435bp(Api), 300bp(Ap2) and 399bp(AM) in length against the various regions of BmNPV’s DNA polymerase gene and DNA helicase gene, which are indispensable for viral replication in silkworm cells by TransMessenger?transfection Reagent. Results indicated that in the experiment where silkworm cells were infected with wild-strain BmNPV of the three dsRNAs, Api and AH can effectively suppress the replication of virus, but Apt had no effect on the inhibition of viral replication. Api and AH can reduce the infective liter of BmNPV with a peak change of approximately 3-4 logs on day 4 post-infection. DNA dot blotting showed that total DNA of normal silkworm cells as a positive contrast had no hybrid signal; however, total DNA of infected cells, which transfected Ap2 or AH had a very weak hybrid Signal by contrast with the negative contrast (dot a). The quantity of viral DNA in dot c and dot d reduced 72.3% and 63.4% compared with negative contrast by gel imaging system. The results of RT-PCR also indicated that when the dosage of dsRNA was 2 u g/well, the expression level of the two target genes (Ap2 and AH) were all obviously reduced 78.27% and 86.70% compared with the interior contrast while in the other dosages their expression level changed very little.In addition, the transfection efficiency and Cytotoxicity of TransMessenger Reagent and the distribution of introduced dsRNA in cells had also been studied in this thesis. Using Flow cytometer (FCM) we found that the transfection efficiency of TransMessenger Reagent was 60-70%. From the test of MTT, we also found that the volume of Bm-N shrunk, the cells’ survival rate reduced, and there were many granules in cells when the dosage of transfection reagent exceeded 32 n L. FAM-labeled dsRNAs were transfected into silkworm cells and observed by fluorescence microscopy to determine their subcellular distribution pattern. We observed that a majority of dsRNA was localized in the nuclear periphery discontinuously after 24 hours of transfection. Time course showed that the labeled dsRNAs were maintained in cultured silkworm cells for up to 5 days. After six days, the signal was too weak to be detected.In conclution, we can inhibite the replication of viral DNA effectively by synthetic dsRNAs or siRNAs, which target the indispensable genes of viral replication. The feasibility of dsRNA inhibiting BmNPV replication can not only give a model in the research of insect Nucleopolyhedroviru inhibition, but also gives some references onRNAi’s application in the treatment of human viral diseases.更多还原